The Collaboration for AIDS Vaccine Discovery Profiles 2018/2018-CAVD...The goal of the...

The Collaboration for AIDS Vaccine Discovery

13th Annual Meeting December 4 - 6, 2018

Seattle, Washington

Grantee Profiles

Ackerman/Alter: Leveraging Antibody Effector Function

PrincipalInvestigatorMargaret Ackerman, PhDGalit Alter, PhD

GranteeInstitutionMassachusetts General Hospital, Boston, USA

Project TitleHigh-throughput Technology for Assessing Antibody Effector Function

OPPID1032817

Grant AwardUp to $8 Million, awarded in October, 2011

Collaborating Institutions◊ Beth Israel Deaconess Medical

Center

Grant at a GlanceOVERVIEWNeutralizing antibodies are the holy grail of HIV vaccine development but attempts to elicit them with vaccines have yielded little in the way of success. The fi eld is gradually opening up to a broader view, with more attention paid to antibodies that protect by mechanisms other than neutralization. The successes of the RV144 trial and non-human primate (NHP) studies of passively transferred antibodies have provided new enthusiasm for the extra-neutralizing antiviral properties of antibodies.

Two obstacles are encountered in attempting to monitor and improve upon these non-traditional effector functions of antibodies elicited by new vaccine regimens. First, the methods currently used to determine the innate immune-recruiting properties of antibodies are not compatible with the scale and standardization of analysis necessary to properly evaluate the potential role of this mechanism of protection in pre-clinical and clinical vaccine trials. Second, the signals responsible for driving B cells to produce potent innate immune-recruiting antibodies are not known. New technologies coupled to a better understanding of underlying signals that induce these types of immune responses are critically needed to improve upon the current vaccine approaches.

The consortium led by Drs. Margaret Ackerman of Thayer School of Engineering at Dartmouth and Galit Alter of Massachusetts General Hospital seeks to defi ne, induce, and evaluate protection afforded by potent innate immune-recruiting antibodies. These are antibodies that form a bridge between the adaptive and innate immune systems. This may be particularly important in the case of HIV, where a narrow window of time soon after the virus enters the body may represent the best opportunity to prevent infection. Innate effector mechanisms are designed in part to contain pathogens until the adaptive immune system can respond, and this proposal will attempt to harness the capacity of antibodies to recruit innate immunity in the crucial early days following HIV transmission.

These studies will develop enabling technology for monitoring of Ab effector functions, as well as defi ne the signals required to induce such protective Abs in vivo, providing new approaches aimed at harnessing the antiviral activity of the Ab-Fc domain to provide sterilizing protection from HIV infection.

RESEARCH OBJECTIVES1.) To develop a high-throughput proteomic-based microarray approach to quantify the spectrum of innate

immune-recruiting antibody effector functions.

2.) To develop a high-throughput in vitro system to defi ne the innate immune infl ammatory signals required for the induction of innate immune-recruiting antibody effector functions.

3.) To develop a robust computational prediction model and scoring system to apply to microarray output.

4.) To apply the proteomic microarray and in vitro screening system to defi ne the top innate immune signals that result in the induction of the most potent innate immune-recruiting antibody functions.

5.) To defi ne whether in vitro innate immune signals coupled to gp140 clade C trimers induce innate immune-recruiting antibodies in vivo, and to defi ne their protective effi cacy in a SHIV challenge model.

6.) To determine if specifi c stimulatory signals can durably programmed antibody glycosylation and if antibody glycosylation can be recalled following subsequent antigenic exposure.

PROGRESSThe goal of the Ackerman/Alter CAVD is to defi ne, induce, and evaluate protection afforded by potent innate immune-recruiting antibodies through the development of (a) robust, high-throughput array technology to predict Fc-effector function and (b) an in vitro–screening approach to defi ne the signals in B cells that result in the targeted production of innate immune–recruiting antibodies. Objectives 1 and 3 have generated a remarkable platform to defi ne the spectrum of biophysical antibody features that reliably predict specifi c effector functions. This platform now provides a compre-hensive set of standardized (and some qualifi ed) tools that objectively and deeply interrogate the polyclonal humoral immune response following infection and/or vaccination in both humans and non-human primates. Moreover, linked to unsupervised and supervised systems level machine learning analyses, vaccine profi les and correlates analyses can be defi ned for HIV and beyond.

In parallel, in Objective 2, we developed a robust in vitro B cell-screening approach, using single cell RNA sequencing and ATAC sequencing, to specifi cally defi ne the molecular programs that control Fc-effector function. Specifi cally, a set of innate immune infl ammatory signals have been identifi ed that specifi cally and selectively skew antibody function via glycosylation. Through studies performed in vitro, in mice, and in non-human primates, it has become clear that adjuvants can selectively skew Fc-antibody profi les. Interestingly, profound shifts towards highly cytolytic, low-fu-cosylated glycans were observed with viral TLR agonists including TLR7/8/9, whereas more phagocytic antibodies were observed with bacterial TLR agonists including TLR2/4/5. These data point to the evolutionary programming of distinct effector functions based on pathogen-type. However, how these particular adjuvants mediate these differences and/or how vaccine-regimens may be customized to drive enhanced protective functional antibody profi les remains unknown. Thus in the fi nal phase of this grant, we propose to take 2 of our most potent adjuvants: a bacterial and a viral adjuvant and examine their capacity to: 1) induce potent and distinct functional antibody profi les, 2) program long-lived functional B cell responses, and 3) provide protection from SHIV challenge in NHPs through distinct functional mechanisms. This experiment will test the hypothesis whether ADCC or phagocytic antibodies provide enhanced protection from SHIV infection and will provide the fi rst proof-of-concept that vaccines may selec-tively program long-lived innate immune– recruiting antibody activity that may prevent SHIV infection.

Alt: Physiologically Relevant and Rapidly Generated HIV-1 Vaccine Mouse Models

PrincipalInvestigatorFrederick W. Alt, PhD

GranteeInstitutionBoston Children’s Hospital; Boston, USA

Project TitlePhysiologically Relevant and Rapidly Generated HIV-1 Vaccine Mouse Models

OPPID1175860

Grant AwardUp to $2.5 Million, awarded in August 2017

Grant at a GlanceOVERVIEWMouse models expressing bNAbs or their precursors are commonly used as assay systems to test and opti-mize immunogens at the preclinical stage. The Alt laboratory developed a new mouse model that allows the precursor human immunoglobulin heavy (IgH) chain variable region exon (VH) for a bNAb to be developmen-tally assembled by V(D)J recombination and to dominate the IgH repertoire of the mice, and demonstrated the technique for the potent VRC01 class of HIV-1 bNAbs. In this VRC01-rearranging model, most individual B cells express one of a multitude of different variations of the potential VRC01 precursor IgH chain, provid-ing a much more human-like precursor VRC01 repertoire. Indeed, sequential immunization in this model induced affi nity maturation of VRC01-type HIV-1 neutralizing antibodies, although it did not achieve fully ma-ture VRC01-class bNAbs. The diverse repertoire of naïve B cells in this VRC01-rearranging model stands in contrast to models that “knock-in” the pre-rearranged variable region exons of the bNAb unmutated common ancestor (UCA). This latter case results in an essentially monoclonal naïve B cell population that expresses the installed elements of the UCA, and may be poorly refl ective of the true physiological circumstances of priming very rare precursors in a diverse repertoire. At the other extreme are mouse models with fully human Ig variable region loci; while these models can generate more complex antibody repertoires, mice have far fewer B cells than humans, which substantially diminishes the probability that such an engineered mouse will harbor the rare, specifi c precursor that must be engaged to prime the bNAb lineage. Here again, the VH re-arranging mouse model from the Alt group offers the advantage of higher frequency for such precursors while maintaining B cell receptor diversity.

Under this award, the investigator’s laboratory is making signifi cant progress in the development of even more physiologically relevant mouse models for testing candidate HIV-1 vaccine strategies. The fi rst aim is engineering a mouse model that generates highly diverse IgL chain repertoires of potential VRC01 precursors. Combining this IgL rearranging capacity with the VRC01 IgH rearranging model will generate an extremely di-verse primary BCR repertoire of VRC01 precursors in mice for testing immunization strategies to elicit VRC01-class bNAbs. Other refi nements of IgL rearranging strategy seek to generate a more “human-like” IgL repertoire in mice by enhancing the junctional diversity during V(D)J recombination. The second aim is to make a model in which expression of bNAb affi nity maturation intermediates is targeted specifi cally to mouse germinal center B cells. Expressing such bNAb intermediates at this physiologically relevant stage can avoid potential central or peripheral tolerance checkpoints and will be especially important for testing boost immunogens in sequential vaccination strategies. Notably, the bNAb intermediates evaluated in the model can include those identifi ed from human subjects in clinical trials of immunogens designed to elicit bNAbs. A third aim involves making these models readily available on an ongoing basis to major colleagues in the fi eld for vaccination experiments. The Alt lab has provided different versions of the existing VRC01 models to Dr. Facundo Batista’s lab to test immunogens developed by Dr. Rogier Sanders and John Moore’s groups. Newly developed mouse models, once established, will continue to be made available to collaborators.

The grant is led by Frederick W. Alt, at the Boston Children’s Hospital.

RESEARCH OBJECTIVES1.) Generation of VCR01 mouse models with diverse bNAb IgH and IgL precursor repertoires.

2.) Evaluate mouse models that express bNAb intermediates directly in germinal center B cells.

3.) Provide of cohorts of pertinent mouse models to CAVD collaborators.

Alter: A Genetic Approach to Optimizing the Antigenicity of HIV-1 Envelope

PrincipalInvestigatorGalit Alter, PhD


Project TitleA Generic Approach to Optimizing the Antigenicity of HIV-1 Envelope Immunogens

OPPID1097381

Grant AwardUp to $2.6 Million, awarded in November, 2013

Collaborating Institutions◊ Complex Carbohydrate Research

Center, University of Georgia Center

◊ University of Massachusetts Medical School

Grant at a GlanceOVERVIEWOver the past decade, a number of novel neutralizing Abs (nAbs) have been defi ned that target the V2 and neighboring loops that are highly exposed and may contribute to viral neutralization due to their involvement in trimerization. Many of these novel Abs show unique antigen-binding specifi cities, involving glycan-dependent specifi cities. Moreover, given that sugars consist of approximately half the mass of the HIV envelope, mout-ing evidence suggests that glycans interact with bNAbs against every target on the viral envelope. However, while previous efforts have manipulated individual glycans or groups of neighboring glycan to de-emphasize/emphasize particular Ab targets, to date, few immunogens have attempted to actively manipulate the overall HI envelope glycan composition, rather than target glycans. Thus, this project proposes to actively manipulate and exploit the overall composition, quality, and landscape of the HIV glycans to enhance protein antigenicity. Thus using cutting edge mass spectrometric glycomic analytical tools, genome screening technologies, and systems level machine learning tools, the HIV envelope glycome will be actively re-engineered to enhance the generation of higher quality vaccine anitgens to promote more effective humoal immunity.

RESEARCH OBJECTIVESPhase 1:

1.) Objective identifi cation of genetic modifi cations that improve gp120-1086 production

2.) Unbiased identifi cation of genetic modifi cations that improve gp120-1086 antigenicity

3.) Cross-validation of “hits” in a CHO cell line for ultimate ENV-producer cell line development

Phase 2:

1.) Objective identifi cation of gene modifi ers that improve high quality SOSIP secretion

2.) Unbiased identifi cation of gene modifi ers that improve SOSIP antigenicitiy

3.) Identifi cation of gene modifi ers that drive enhanced In vivo immunogenicity

PROGRESSThis project brings together the genome-wide screening expertise of Dr. Abraham Brass (University of Massachusetts), the glycobiology expertise of Drs. Lance Wells and Mike Tiemeyer (Complex Carbohydrate Research Center), and the HIV immunology expertise of Dr. Alter (Ragon Institute/ Harvard) to develop an approach to enhance the antigenicity of HIV ENV immunogens. In the fi rst phase of the study, signifi cant headway has been achieved in the following areas:

1.) Glycan composition, site occupancy, and site specifi c glycan analysis was performed on 94 individual HIV ENV proteins linked to antigenicity profi ling by nAb and non-nAb binding. These reveal both the conservation as well as heterogeneity of glycosylation at specifi c glycan sites within the ENV protein. Machine learning algorithms used to interrogate glycan landscape profi les linked to antibody binding profi les across various proteins, clades, and tiers of viruses provides a novel predictive framework for the identifi cation of glycan-dependent footprints of both previously characterized and uncharacterized monoclonals or polyclonal antibody populations. These proof of concept studies on recombinant gp120 monomers have led to the production of antigenically superior recombinant gp120s, able to evade non-nAb recognition, but bind potently to nAbs. These principles are now being applied to SOSIP molecules.

2.) The fi rst genome-wide screen using 2 siRNA libraries (Ambion and Dharmacon) are now complete -identifying a number of hits that improved both ENV secretion and antigenicity.

- Remarkably, both ER and non-ER (transcriptional) hits were identifi ed for enhancing protein production levels, suggesting a complex regulatory pathway that may be easily manipulated to improve therapeutic target protein production.

- No difference was observed in PGT121 and VRC01 antigenicity, but signifi cant hits were observed for PGT128 antigenicity. A unique set of genes, involved in Golgi organization, vesicular transport, and metabolism were identifi ed as enhancers of bnAb antigenicity.

However given the growing interest in native like trimers, confi rmation/validation exercises were strategically transferred to a SOSIP screen. Within this validation screen, CRISPR/CAS9 will be used directly on CHO cells, and gene modifi ers that improve the level of:

1.) high quality SOSIP (ratio),

2.) the antigenic profi le of SOSIP, and

3.) the levels of high quality SOSIP will be defi ned.

Ultimately, collective, validated, converging hits will be computationally assembled and SOSIP proteins will be produced and tested in vivo for improved antigenicity and immunogenicity.

Alter: Signatures of Antibody Responses that Correlate with Protection to Develop Down-Selection Criteria to Guide Vaccine Candidate Selection

PrincipalInvestigatorGalit Alter, PhD

GranteeInstitutionRagon Institute of MHG, MIT, and Harvard, USA

Project TitleSignatures of Antibody Responses that Correlate with Protection to Develop Down-Selection Criteria to Guide Vaccine Candidate Selection

OPPID1114729

Grant AwardUp to $3.1 Million, awarded in November 2014

Collaborating Institutions◊ Dartmouth College

Grant at a Glance

Ragon Institute of Ragon Institute of

OVERVIEWThe goal of this investment is to standardize non-human primate (NHP) assays and to defi ne the non-neutral-izing humoral immune correlates of protection from SIV/SHIV infection in NHP vaccine sample sets that have shown a range of protective signals. These results will be used to develop a predictive framework to guide the down-selection of future vaccine candidates that elicit potent non-neutralizing antibody responses.

Since partial protection observed in the RV144 trial was mediated by the induction on non-neutralizing anti-bodies (nNAbs) and a moderate T cell response, it seems that other immune mechanisms in addition to classi-cal neutralizing antibody responses are required to achieve protection against HIV infection. The functional ac-tivity of antibodies extends beyond their variable (Fv) domains. Indeed, the constant domains (Fc) orchestrate a variety of effector functions through the recruitment of an array of innate immune cells. Given that innate immune effector cells are abundantly present at mucosal sites and are armed and prepared to act without the need for prior antigen sensitization, Abs that could recruit these antiviral effectors could provide a robust level of control that could dramatically enhance protection from HIV infection. Non-neutralizing antibodies function in multiple ways, including antibody-dependent cell-mediated cytotoxicity, complement-mediated cytotoxici-ty, and antibody-dependent cell-mediated phagocytosis. Several NHP challenge studies support the RV144 fi ndings. Namely, the potential role of Fc-FcγR-mediated innate and adaptive immune functions in HIV-1 pro-tection indicates that the quality rather than the quantity of the antibody response is critical. Therefore, it will be key for HIV-1 vaccine development to understand the Fc-FcγR interaction effector function, including the sequences and functions of the receptors in NHP (which are used readily as a translational HIV/SHIV model).

We have systematically developed high-throughput tools that enable the dissection of humoral immune re-sponses at unprecedented depth. Using these high-throughput ‘systems serology’ approaches, we have begun to defi ne protective non-neutralizing humoral immune response signatures among vaccinees and naturally infected cohorts. Interestingly, RV144 vaccinees and spontaneous controllers of HIV both elicit poly-functional Ab responses able to recruit multiple innate immune effector activities, with a critical role for the IgG3 responses and specifi c antibody glycoprofi les. This points to specifi c non-neutralizing Ab signatures that may be associated with robust antiviral control if induced with adequate durability.

Ultimately, these studies will (1) defi ne shared and disparate correlates of protection from infection, (2) identify mechanisms of resistance from in-fection, (3) develop a framework for the down-selection of future vaccine strategies that elicit ‘protective humoral signatures,’ and (4) develop highly standardized systems to vet novel immunogenicity/protection studies. Together, these fi ndings will lead to the development of a predictive modeling system to identify the most promising new vaccine concepts that warrant accelerated development to clinical testing.

Galit Alter (Ragon Institute) is the principal investigator for this grant. The effort includes extensive collaborative interactions with the laboratories of Margaret Ackerman and Chris Bailey Kellogg (Dartmouth College).

RESEARCH OBJECTIVES1.) Standardization of Fc effector profi ling assays for NHP (including the multiplexed Fc effector array).

2.) Testing of NHP samples to defi ne the signatures of protective immunity against SIV/SHIV infection.

3.) Development and validation of a predictive model by testing samples from emerging NHP effi cacy and immunogenicity studies.

4.) Enhancement of the capacity for standardized, high-throughput analyses of vaccine-induced humoral immune profi les, including the estab-lishment of a GCLP laboratory environment.

Baker: Testing the Nearest Neighbor Approach to Active Vaccination for HIV-1 bNAbs

PrincipalInvestigatorDavidBaker, PhD

GranteeInstitutionUniversity of Washington, Seattle, USA

Project TitleTesting the Nearest Neighbor Approach to Active Vaccination for HIV-1 bNAbs

OPPID1111923

Grant AwardUp to $7.5 million, awarded in November, 2014

Collaborating Institutions◊ Weill Cornell Medical College, New

York, USA◊ Academic Medical Center,

Amsterdam, The Netherlands◊ Duke University, Durham, USA◊ The Scripps Research Institute

(funded under a separate analytics grant), La Jolla, USA

◊ Fred Hutchinson Cancer Research Center, Seattle, WA

◊ Garvan Institute of Medical Research, Darlinghurst, Australia

Grant at a GlanceOVERVIEWThis program seeks to develop improved HIV-1 envelope glycoprotein (Env) immunogens based on the engineered SOSIP HIV glycoprotein trimer constructs developed by the Moore/Sanders/Ward labs. The original premise of the approach is the observation by Dr. Garnett Kelsoe’s group at Duke that the generation of some broadly neutralizing antibodies (bNAbs) to Env appears to be restricted by host epitope mimicry. Hence deletion of many responsive B cell clones, either during B cell development or in germinal centers, may be the basis for sub-optimal immune responses to current HIV-1 Env vaccines. Therefore the goal of this grant is to arrive at next generation “Nearest Neighbor (NN)” SOSIP Env immunogens that avoid restriction by such tolerance mechanisms that may underlie the sub-optimal immune responses to current Env vaccines. NN immunogens may be produced either as soluble antigens or via multivalent display on designed protein nanoparticles based on technology developed in the King and Baker labs.

This goal remains the same but now follows a more broadly-defi ned hypothesis that the diffi culty in generating a broadly neutralizing immune response to the virus arises not from mimicry of specifi c host proteins but from a more general similarity in properties of the HIV Env surface to those of host proteins (e.g., the surface of Env is nearly completely covered by glycans, so antibodies against Env often interact with glycan and hence may cross-react with host proteins). To test this hypothesis, the team is testing variants of Env with substitu-tions that alter residues at which there is strong selective pressure in strains isolated from patients that (1) cannot be explained by computational modeling based on the structure and (2) are not conserved in laboratory virus passaging experiments. To increase the extent to which these NN variants can rescue anergic B cells producing precursors to HIV Nabs that weakly cross-react with self and drive them toward germinal centers, the designed NN immunogens will be displayed with high multivalency on nanoparticles.

In the fi nal year of the grant, the PI team will be pursuing two parallel tracks. Track 1 (King, Moore, Sanders, Ward) focuses on a systematic exploration of the Env antigen-nanoparticle display platform including epitope accessibility of Env trimers on nanoparticles and the relationship between nanoparticle geometry/valency and immunogenicity. Track 2 (Baker, Bloom, Sanders, Goodnow) will focus on the design of NN immunogens as described above.

The work is a collaborative effort between the academic research labs of David Baker, PhD (University of Washington - Seattle), Neil King, PhD (University of Washington – Seattle), Jesse Bloom, PhD (Fred Hutchinson Cancer Research Center), John Moore, PhD (Weill Cornell Medical College), Rogier Sanders (University of Amsterdam), Andrew Ward, PhD (The Scripps Research Institute), and Christopher Goodnow, PhD (Garvan Institute of Medical Research).

RESEARCH OBJECTIVES1.) Systematic investigation of the relationship between particulate antigen presentation and immunogenicity (King, Moore, Sanders, Ward).

ο Computational design of protein nanoparticles (King)

ο Expression, purifi cation and biophysical characterization of SOSIP trimer on protein nanoparticles (King, Ward, Sanders, Moore)

ο In vitro antigenicity studies, rabbit immunogenicity studies and serological analysis (Moore, Sanders)

2.) Identifi cation of naïve B cell bNAb precursors using designed NN immunogens (Baker, Bloom, Sanders, Goodnow). Sub efforts include:

ο Computational design and viral fi tness studies/deep mutational scanning to identify NN immunogens (Baker/Bloom)

ο Expression and structural validation of NN-mutant trimers and NN-mutant trimers expressed on self-assembling protein nanoparticles. The NN Env mutants will be elected based on the NN design protocol of the Bloom and Baker labs (Sanders)

ο Identify NN Env constructs with conserved epitopes specifi cally bound to cell surface antibodies carried by circulating B cells present in most healthy people (via multi-color fl ow cytometry). Each NN immunogen will be tested by two complementary fl ow cytometric platforms to cover the full range of possible BCR affi nities and the likelihood that cross-reactivity with self-antigens will downregulate surface IgM resulting in low binding (Goodnow)

Barouch: Therapeutic Effi cacy of Potent Broadly Neutralizing mAbs for HIV-1 Eradication

PrincipalInvestigatorDan H. Barouch, MD, PhD

GranteeInstitutionBeth Israel Deaconess Medical Center, Boston, USA

Project TitleTherapeutic Effi cacy of Potent Broadly Neutralizing mAbs for HIV-1 Eradication

OPPID1107669

Grant AwardUp to $25 million, awarded in July, 2014

Collaborating Institutions◊ Ragon Institute◊ The Scripps Research Institute◊ Theraclone Sciences◊ Gilead Sciences◊ IAVI

Grant at a GlanceOVERVIEWWe have recently demonstrated the profound therapeutic effi cacy of the potent broadly neutralizing mAb PGT121 in rhesus monkeys chronically infected with SHIV-SF162P3. In particular, we showed that PGT121, both alone and in combination with other mAbs, resulted in a rapid and precipitous decline of plasma viremia, as well as reduced proviral DNA in lymph nodes and gastrointestinal mucosa.

Based on these data, we propose to evaluate the therapeutic effi cacy of PGT121 in HIV-1-infected humans and to conduct proof-of-concept studies in rhesus monkeys. We hypothesize that PGT121, either alone or in combination with other mAbs, will result in virologic suppression in both peripheral blood and tissues. We further hypothesize that PGT121 in conjunction with antiretroviral therapy (ART) and reservoir activators may reduce viral reservoirs that persist with ART alone and may prove useful in HIV-1 eradication strategies.

The Primary Outcome of this project is to determine the proof-of-concept therapeutic effi cacy of PGT121 in HIV-1-infected humans and to defi ne an optimal mAb cocktail for further clinical development.

This consortium is led by Dr. Dan Barouch and includes researchers from Beth Israel Deaconess Medical Center (BIDMC), the Ragon Institute, the Scripps Research Institute, Theraclone Sciences, Gilead Sciences, and IAVI. The origin award was in July, 2014, with a subsequent supplement to manufacture PGDM1400 for clincal testing.

RESEARCH OBJECTIVES1.) To conduct key preclinical studies in nonhuman primates to inform the clinical development program;

2.) To manufacture clinical-grade PGT121 and to conduct preclinical toxicology studies;

3.) To conduct phase I clinical trials to assess the therapeutic effi cacy of PGT121 in humans; and

4.) To provide administrative and programmatic support for this program.

Barouch: Epitope-Modifi ed Env Trimers for Induction of Heterologous Tier 2 NAbs

PrincipalInvestigatorDan H. Barouch, MD, PhD

GranteeInstitutionBeth Israel Deaconess Medical Center, Boston, USA

Project TitleEpitope-Modifi ed Env Trimers for Induction of Heterologous Tier 2 NAbs

OPPID1169339

Grant AwardUp to $4.1 Million, awarded in May, 2017

Collaborating Institutions◊ Los Alamos National Laboratories,

Los Alamos, USA◊ Children’s Hospital of Boston,

Boston, USA

Grant at a GlanceOVERVIEWWe have recently developed a novel strategy for the generation of HIV-1 Env immunogens aimed at eliciting neutralizing antibodies (NAbs). Signature-based Epitope Targeted (SET) vaccines are designed to incorpo-rate bNAb signatures to optimize key HIV-1 Env epitopes. In our initial studies, the computational approach defi ned elements associated with bNAb neutralization sensitivity and resistance and resolved common pat-terns across bNAbs with shared epitope specifi cities. The bNAb classes evaluated included those that target the V2 region, the V3 region, and the CD4 binding site.

In this proposal, we will evaluate the hypothesis that bioinformatic optimization of HIV-1 Env immunogens will improve the induction of heterologous tier 2 NAb responses. This work will include developing improved immunization regimens with our V2 SET immunogens to increase both the titers and breadth observed in the guinea pig model and increase the fraction of vaccinated animals that develop heterologous tier 2 NAbs. We will examine differing boosting schemes and adjuvants using our existing protein immunogens, as well as examining SOSIP trimers containing the V2 SET sequences of our current vaccines. Simultaneously, we will be generating a second generation of SET designs that will be informed by the recent expansion of data for neutralizing antibodies. We are also exploring new molecular engineering methods to produce the SET immunogens, either for mulitmeric presentation as nanoparticles and nanodiscs, or as improved soluble mi-metics of the native Env trimer. These immunogens will also be evaluated in the guinea pig model. All these efforts are slated for the fi rst two years of the program, leading to a Go/No-Go decision for the initiation of a rhesus monkey study in year three.

RESEARCH OBJECTIVESOutcome 1: Development of Improved Epitope-Modifi ed Env Trimers in Guinea Pigs

1.A.1. Defi nition of the optimal vaccine regimen in guinea pigsTo manufacture clinical-grade PGT121 and to conduct preclinical toxicology studies;

1.A.2. Comparison of SOSIP vs foldon versions of the WT+Opt+Alt 459C and BG505 gp140 cocktails in guinea pigs

1.A.3. Evaluation of next generation immunogens and immunization strategies in guinea pigs

1.B.1. Second generation bioinformatic design of V2, V3, and CD4bs SET vaccines

1.B.2. Iterative cycle of vaccine design, if warranted, based on outcomes in guinea pig vaccination studies

1.B.3. Statistical analysis of immunological data and modeling to compare vaccine effi cacy and the relative importance of different factors and interpretation of serological reactivity patterns

1.C.1. Production and antigenicity of multimerized HIV-1 Env immunogens and soluble Env trimers with the current V2 SET sequences

1.C.2. Production and antigenicity of multimerized HIV-1 Env immunogens and soluble Env trimers with the next generation V2 SET sequences

Outcome 2: Evaluation of Optimized Epitope-Modifi ed Env Trimers in Rhesus Monkeys

2.1. Immunogenicity of epitope-modifi ed vaccines in rhesus monkeys

2.2. Protective effi cacy of epitope-modifi ed vaccines in rhesus monkeys

Batista: Accelerating Vaccine Design through Interrogation of Physiologically Relevant Mouse Models

Principal InvestigatorFacundo Batista, Ph.D.

Grantee InstitutionMassachusetts General Hospital Corporation – Ragon Institute of MGH, MIT, and Harvard (Cambridge, MA) USA

Project TitleAccelerating Vaccine Design through Interrogation of Physiologically Relevant Mouse Modelsr

OPPID1185467

Grant AwardUp to $2.9 Million, awarded in February 2018

Grant at a GlanceOVERVIEWTransgenic mice expressing human immunoglobulin genes encoding inferred germline precursors to known broadly neutralizing antibodies (bNAbs) will be used to evaluate the ability of novel HIV immunogens to elicit protective immune responses. The premise of vaccine strategies to induce bNAbs in humans is the deter-mination that a small percentage of HIV-infected patients naturally produce high titers of bNAbs capable of neutralizing several strains of HIV. These bNAbs also protect macaques against simian/human immunode-ficiency virus (SHIV) when used for passive immunization prior to challenge. These observations suggest that engineered immunogens against specific HIV epitopes could potentially stimulate the generation of bNAbs in the HIV-free human population for protection against HIV infection. However, at present, testing of immunogens on human subjects is both impractical and unsafe. Therefore, to drive HIV vaccine develop-ment, animal studies using sophisticated knock-in (KI) mouse models are essential in order to determine: (i) how B cell precursors corresponding to the most promising bNAbs respond to various HIV immunogens; (ii) whether these B cell precursors mature into B cells with desirable qualities, such as adequate somatic hypermutation; and (iii) whether these immunogens induce a rapid and robust memory response capable of neutralizing a wide number of HIV strains. Our laboratory has developed a highly efficient technique for generating such KI mouse models using a one-step CRISPR/Cas9-induced homology-directed recombina-tion approach, via direct injection of a donor plasmid, guide RNA, and Cas9 protein into mouse oocytes. The goal of this grant is to (i) use germline-targeting KI mouse models to determine whether a given immunogen will be able to bind to precursor B cells and elicit a desired immune response (thereby making the immuno-gen a viable clinical trial vaccine candidate); and (ii) use KI models to advance the clinical testing of eOD-GT8 and BG505.SOSIP-derived (GT1.1 and GT1.2) germline-targeting immunogens.

The work includes ad hoc collaborations with the academic research labs of Rogier Sanders, Ph.D. (University of Amsterdam and Weill Cornell Medical College, New York), William Schief, Ph.D. (The Scripps Research Institute, La Jolla), and Fred Alt, Ph.D. (Children’s Hospital, Boston).

RESEARCH OBJECTIVES1.) Generation of new KI mouse models bearing human germline-reverted sequences of three heavy chain and three light chain bNAb sequences.

Sub-efforts include:

◊ Perform immunocharacterization experiments on KI mice

◊ Immunization of KI mice using eOD-GT8 and BG505.SOSIP-derived immunogens

2.) Assess the potential of human Ig precursors to mature into bNAbs in KI mice by rigorously testing the effects of antigen design, dose, timing, adjuvant, and whether sequential or combinatorial challenge with immunogens results in the best immune outcome.

3.) Test the capacity of verified immunogens to stimulate B cell responses in vivo by immunizing KI mice. Sub-efforts include:

◊ Before antigen challenge, the B cell development and maturation of KI mice will be compared with wild-type mice by examining key B cell markers

◊ Conduct similar immunization experiments in wild-type mice into which low numbers of KI B cells will be adoptively transferred prior to immunization

◊ Examine the functionality of antibodies that are produced upon immunogen stimulation

Busch: Validation of Existing and Ultra-Sensitive Assays for Quantifying HIV Persistence

PrincipalInvestigatorMichaelBusch, MD, PhD

GranteeInstitutionVitalant (formerly Blood Systems, Inc., San Francisco, USA

Project TitleValidation of Existing and Ultra-Sensitive Assays for Quantifying HIV Persistence

OPPID1115400

Grant AwardUp to $4.6 million, awarded in October, 2014

Collaborating Institutions◊ University of California, San

Francisco, USA◊ South African National Blood

Service, South Africa◊ University of Pittsburgh, Pittsburgh,

USA◊ University of California, San Diego,

USA

Grant at a Glance

Inc., San Francisco, USAInc., San Francisco, USA

OVERVIEWThe HIV Reservoir Assay Validation and Evaluation Network (RAVEN) project will facilitate the development and rigorous evaluation of performance characteristics (sensitivity, specificity, limits of detection and quantitative dynamic ranges) for blood-based assays to detect and quantify HIV persistence, by establishing a large and well-characterized repository of samples from HIV-infected participants on suppressive ART therapy, initiated either early or late following viral acquisition.

A major barrier to the discovery and development of curative interventions for HIV is the lack of validated, high-throughput assays that reliably quantify the size of the viral reservoir (total body burden) of replication-competent HIV. Multiple assays are in development, but there is an immediate need for a rigorous head-to-head comparative evaluation to establish which HIV reservoir assays have the best performance characteristics, and hence should be “scaled up”, optimally by commercial manufacturers, and employed in prospective cure research protocols. To do this, academic and commercial laboratories require high volume plasma/leukocyte sample procurement, processing to capture or concentrate virus/infected cells, and ultimately HIV RNA/DNA/ protein detection and characterization. The present project is an international collaboration that seeks to validate established and novel molecular methods for quantifying HIV persistence that are blood-based, high-throughput, and applicable for use in cure research protocols and eventually routine clinical application.

Apheresis collections were performed serially on 50 ART-suppressed participants in San Francisco (HIV-1 clade B) and 25 in South Africa (HIV-1 clade C) and the HIV reservoirs characterized with respect to low level plasma viremia, cell-associated HIV DNA and RNA and quantitative viral outgrowth assays (QVOA). Cryopre-served PBMC, CD4+ T cells and plasma aliquots were coded andassembled into “qualification” and “evalu-ation” panels that are being distributed to academic and commercial labs focused on developing or performing HIV reservoir assays. These panels are being used to evaluate induced viral outgrowth assays, molecular assays for plasma and cell-associated HIV DNA and RNA, HIV sequence based assays and immunological assays that indirectly reflect reservoir size. Blinded panels of analytical and clinical samples are provided to laboratories, and the RAVEN team is responsible for decoding results and final data analysis.

RAVEN administration originates under Vitalent Research Institute (formerly Blood Systems Research Institute) in San Francisco; the RAVEN team includes two international clinical sites (University of California, San Francisco; and South African NationalBlood Service), a Steering Committee, senior laboratory scientists and staff, apheresis technicians, data management specialists, regulatory and

fiscal oversight, and numerous collaborating laboratories. As a collaboratory network, RAVEN includes an evolving roster of both academic and industry laboratories with expertise in viral quantification, assay development, and/or active discovery pipelines in support of HIV cure research.

The work is a collaborative effort led by Michael Busch, MD, PhD (Vitalant Research Institute in San Francisco), Steven Deeks, MD (University of California, San Francisco), John W. Mellors, MD (University of Pittsburgh), Douglas Richman, MD (University of California, San Diego) and Charlotte Ingram, MD (South African National Blood Service), The award was received in October, 2014 with an initial agreement length of 4 years.

RESEARCH OBJECTIVES1.) Conduct a validation study of the current and next generation of viral outgrowth assays;

2.) Rigorously evaluate the performance characteristics (e.g., sensitivity, specifi city, reproducibility and precision) of currently established and novel molecular and immunological assays for HIV reservoir detection and quantitation using coded panels of well characterized analytical control samples and low-level HIV positive clinical samples for which large volumes of plasma and leukocytes will be procured

3.) Compare performance of these assays when applied to clinical samples from representative cohorts of ART-suppressed patients with clade B and C HIV-1 infections.

Corey: Support for HVTN 702

PrincipalInvestigatorLarry Corey, MD

Co-PI’sJim Kublin, MDGlenda Gray, MD

GranteeInstitutionFred Hutchinson Cancer Research Center, Seattle, WA USA

Project TitleHVTN 702: “A pivotal phase 2b/3 multi-site, randomized, double-blind, placebo-controlled clinical trial to evaluate the safety and effi cacy of ALVAC-HIV (vCP2438) and Bivalent Subtype C gp120/MF59 in preventing HIV-1 infection in adults in South Africa”

OPPID1148133

Grant AwardUp to $64 million, awarded in August 2016, over an expected duration of 64 months

Grant at a GlanceOVERVIEWHVTN 702 is a pivotal phase 2b/3 multi-site, randomized, double-blind, placebo-controlled clinical trial to evaluate the safety and effi cacy of ALVAC-HIV (vCP2438) and Bivalent Subtype C gp120/MF59 in preventing HIV-1 infection in adults in South Africa. It is co-funded by the NIAID’s Division of AIDS (DAIDS), and conducted by the HIV Vaccine Trials Network (HVTN). The trial is designed to provide data that might verify and extend the fi ndings of the modest effi cacy and the correlates of risk observed in the RV144 trial conducted with a similar pox/protein HIV vaccine regimen in Thailand. HVTN 702 is a culmination of collaborative efforts by the Pox Protein Public Private Partnership (P5) whose members include BMGF, NIAID, the Fred Hutchinson Cancer Research Center, Sanofi -Pasteur, Glaxo-Smith Kline, and the Medical Research Council of the Republic of South Africa. P5 efforts have included the development of clade C vaccine products adapted to match the circulating HIV clade most prevalent in this region as well as other vaccine regimen modifi cations designed to enhance the magnitude and durability of immune responses that were found to correlate with a decreased risk of HIV infection risk in RV144. HVTN 100, the antecedent phase 1-2 trial of this vaccine regimen fulfi lled specifi c pre-determined immunogenicity criteria predicated on these immunological correlates from RV144, leading to the decision to proceed with the HVTN 702 trial.

This phase 2b/3 trial of the ALVAC-HIV (vCP2438) / Bivalent Subtype C gp120/MF59 prime-boost vaccine regimen will enroll a total of 5400 healthy, HIV-1–uninfected adults who are aged 18 to 35 years and who are at risk for HIV infection, across 15 research sites in South Africa. Participants will be enrolled over 20–22 months and randomized with equal probability to the placebo or vaccine regimen with n = 2700 in each group. The study is designed to enroll approximately 60% women. Participants will receive ALVAC-HIV (vCP2438) or placebo at months 0, 1, 3, 6, 12 and 18 and will receive Bivalent Subtype C gp120/MF59 or placebo at months 3, 6, 12 and 18. There are 2 co-primary objectives:

• To evaluate the preventive vaccine effi cacy (VE) of ALVAC-HIV (vCP2438) + Bivalent Subtype C gp120/MF59 for the prevention of HIV infection in HIV-seronegative South African adults over 24 months from enrollment

• To evaluate the safety and tolerability of ALVAC-HIV (vCP2438) + Bivalent Subtype C gp120/MF59 in adults in South Africa

HIV diagnostic tests will be administered at Month 0 and every 3 months thereafter, with all participants followed to 24 months (Stage 1). Overall, the study design provides approximately 90% power to detect VE from enrollment through 24 months [VE(0-24)] of at least 50% (versus the null hypothesis of H0: VE(0-24) ≤ 25%). If the lower bound of the 95% confi dence interval for VE(0-24) is > 0% at the end of Stage 1, all participants will continue follow-up to 36 months (through Stage 2); if Stage 2 occurs, evaluation of the durability of the vaccine effi cacy from enrollment through 36 months is included as a secondary objective.

Other secondary objectives include evaluation of: VE from Month 6.5 (Week 26) through 24 months post enrollment; immunogenicity of the vaccine regimen; immunogenicity and immune response biomarkers among vaccine recipients at Month 6.5 as correlates of risk of subsequent HIV acqui-sition between Month 6.5 and Month 24, with an expanded scope of analysis if Stage 2 occurs; VE by various demographic characteristics; if and how VE depends on genotypic characteristics of HIV, such as signature mutations; genomic sequences of viral isolates from HIV-1– infected vaccine and placebo recipients; and whether there is evidence of vaccine-induced immune pressure on the viral sequences through sieve analysis methods.

The trial will include monitoring by NIAID’s independent DSMB for harm, futility/non-effi cacy and high effi cacy. Early stopping of the vaccine regimen will be recommended if one of the potential harm or non-effi cacy boundaries is met at a pre-specifi ed analysis time. Other contingency plans address trial procedures if the high-effi cacy boundary is met at a planned interim analysis, and the aforementioned refi nement of continuing the trial to Stage 2.

If effi cacy is demonstrated at Stage 1 analysis, then this is expected to trigger further product process development and additional activities needed to support an application for market authorization in RSA of an HIV vaccine regimen with an indication to prevent HIV acquisition in adults in South Africa. Additional trials are anticipated in this endeavor as well, the details of which are still under development, but include adolescent trials, evalu-ation of phase 3 clinical trial material and lot-to-lot consistency trials, among others.

This grant is led by Larry Corey, MD (FHCRC), Glenda Gray, MD (Medical Research Council of South Africa), and Jim Kublin, MD (FHCRC). The HVTN 702 protocol involves engagement of many biomedical research organizations and as well as the 15 South African clinical sites and a multitude of community representatives and in-country partners. The award was made in August 2016, with an original agreement length of 64 months; the BMGF grant is primarily directed to the support of the clinical research sites.

Crotty: Improving the identifi cation of epitope-specifi c precursor naive B cells in the human B cell repertoire

PrincipalInvestigatorShane Crotty, Ph.D.

GranteeInstitutionLa Jolla Institute for Immunology,USA

Project TitleImproving the effi ciency of identifi cation of epitope-specifi c precursor naive B cells in the human B cell repertoire

OPPID1203211

Grant AwardUp to $599,999.00 awarded in August 2018

Grant at a GlanceOVERVIEWHIV is a worldwide problem impacting people in all countries, but particularly Southern Africa and other de-veloping regions. An HIV vaccine represents one of our best hopes in combatting this epidemic. Current immunization strategies, including those supported by the CAVD, are increasingly targeting precise B cell specifi cities to mimic neutralizing antibody responses generated during natural infection, in an effort to max-imize the potency of the vaccine-elicited Ab response. An understanding of the frequencies and affi nities of human naive B cell specifi cities capable of immunogen recognition in unimmunized individuals can aid in immunogen design and inform decision-making for advancement of promising immunogens to clinical trials. Immunogen-specifi c human naive B cell repertoire analysis is a platform for accelerating iterative immunogen design and immunogen advancement along the clinical development pipeline. This investment will be used to improve the effi ciency of a platform to accelerate HIV vaccine immunogen design.

This investment is primarily to support equipment purchase and personnel in activities that underlay BMGF efforts to elicit broadly neutralizing antibodies using germline targeting (GT) approaches. Briefl y, the premise of these efforts is that investigators can design immunogens that will target rare precursor B cells, activate and expand them, and then use additional immunogens to shepherd these responses via germinal center somatic hypermutation and affi nity maturation to elicit broadly neutralizing antibodies. An understanding of the human B cell specifi cities capable of immunogen recognition in unimmunized individuals can aid in immunogen de-sign and inform decision making for clinical advancement to vaccine trials. Identifying and isolating these rare human naive B cells by cell sorting is key to determine their frequencies and affi nities for CAVD-supported potential vaccine candidates that depend on germline-targeting or rational protein design vaccine strategies for HIV, or other pathogens.

The Crotty lab was the fi rst in the world to succeed in sorting human epitope-specifi c naive B cells specifi c for a candidate HIV vaccine immunogen, which helped support the decision to advance eOD-GT8 60mers to Phase 1 clinical trials. Identifying human epitope-specifi c naive B cells specifi c for new candidate HIV vaccine immunogens with conventional fl ow cytometry instruments is extraordinarily diffi cult, due to the rarity and low affi nity of the cells, resulting in major signal-to-noise challenges. The S6 Symphony instrument helps overcome signal-to-noise limitations of conventional cell sorters, allowing for more accurate identifi cation and isolation of epitope-specifi c precursor naive B cells, which has important implications for vaccine immunogen design and the selection of immunogens for advancement to clinical trials. In summary, immunogen-specifi c human naive B cell repertoire analysis will accel-erate the immunogen design of CD4-binding site and V2-apex immunogens and will provide information useful for decision making on immunogen advancement along the clinical development pipeline.

RESEARCH OBJECTIVES1.) Obtain a BD S6 Symphony instrument at LJI and validate its use for improved characterization of the human naive B cell repertoire specifi c for

the eOD-GT8 immunogen.

2.) Characterize the human naive B cell repertoire to novel CD4-binding site and V2-apex candidate immunogens to feed back into the iterative immunogen design cycle.

Farzan: Preventing HIV-1 transmission with eCD4-Ig

PrincipalInvestigatorMichael Farzan, PhD

GranteeInstitutionThe Scripps Research Institute, Jupiter, USA

Project TitlePreventing HIV-1 transmission with eCD4-Ig

OPPID1132169

Grant AwardUp to $5 million, awarded September, 2015

Collaborating Institutions◊ Wisconsin National Primate

Research Center, Madison, USA

Grant at a GlanceOVERVIEWThere are approximately 35 million people living with HIV-1, 25 million of whom live in sub-Saharan Africa. There are more than 2 million new infections annually. To eradicate HIV-1, we must first stop these new infections, but prophylaxis strategies based on conventional vaccines have largely or wholly failed. These failures are a consequence of two properties of the virus. First, HIV-1 has been selected for generations in the presence of very active immune responses and has developed effective strategies for evading these responses. It is especially adept at evading human antibody responses, integral to most current vaccine approaches. Second, HIV-1 thrives when the immune system is most active. As a result, efforts to enhance an inadequate immune response can in some instances make infection more likely. Effective prophylaxis may therefore require new approaches that do not rely on, and are not limited by, the human immune system. Here we develop one such approach based on a novel entry inhibitor, eCD4-Ig, and an established gene-therapy vector system.

eCD4-Ig is an exceptional HIV-1 entry inhibitor that is on average more potent, and much broader, than the best broadly neutralizing antibodies (bnAbs). eCD4-Ig is potent because it avidly binds both the CD4- and the coreceptor-binding sites of the HIV-1 envelope glycoprotein (Env) trimer. It is broad because it binds only these two conserved, functionally important Env regions. As a result, in laboratory studies, the virus cannot escape eCD4-Ig under conditions where escape from bnAbs is readily observed. Moreover, eCD4-Ig works well in vivo. When an adeno-associated viral (AAV) vector was used to express a rhesus form of eCD4-Ig in four rhesus macaques, these macaques were protected for up to one year from a series of robust intra- venous SHIV and SIV challenges. Moreover, AAV-expressed effectively suppressed rebound of an estab-lished SHIV infection after cessation of ART. These data raise the possibility that eCD4-Ig can be used to prevent new HIV-1 infections and treat established ones. Here we lay the preclinical foundations for human trials of passively administered eCD4-Ig, and further develop AAV-eCD4-Ig as a candidate HIV-1 vaccine alternative. Work to date has improved the half-life of eCD4-Ig as a protein, improved the potency of eCD4-Ig, and reduced its already low immunogenicity. It has as also shown that AAV-mediate prophylaxis extends to a Tier 3 SIV isolate, SIVmac239, confi rming in vivo the exceptional breadth and potency of eCD4-Ig observed in vitro. Control of viral infection after ART cessation further undescores the diffi culty of escape from eCD4-Ig in vivo. Studies in the fi nal year will lead to more bioavailable forms of eCD4-Ig, and to more effi cient AAV vectors and protocols for expressing eCD4-Ig.

The work is a collaborative effort, led by Michael Farzan, PhD (The Scripps Research Institute); Nancy Schultz-Darken, PhD will oversee the performance of the nonhuman primate studies at the Wisconsin National Primate Research Center. This project will also have extensive engagement by the IAVI Vaccine Product Development Center. The award was received in September, 2015 with an original agreement length of 4 years.

RESEARCH OBJECTIVES1.) Foundations for a clinical evaluation of the safety and effi cacy of eCD4-Ig administered as a protein (“foundations for clinical studies”). Sub-aims

include:

a.) Development of a product development plan for eCD4-Ig

b.) Production of research-grade IgG1 and IgG2 forms of human eCD4-Ig for non-human primate studies

c.) An assessment in macaques of the pharmacokinetics (PK) and safety of human eCD4-IgG1 and -IgG2, and of their abilities to protect from a SHIV challenge

d.) An assessment of the PK, safety, and anti-HIV activity of human eCD4-Ig in SHIV infected macaques

e.) GMP protocols for production of human eCD4-Ig

2.) A comprehensive preclinical evaluation of the feasibility of using AAV-delivered eCD4-Ig as an alternative to an HIV-1 vaccine (“preclinical studies”)

a.) Characterization of the off-target effects of tyrosine-protein sulfotransferase 2, used to enhance eCD4-Ig sulfation

b.) Development and testing in nonhuman primates of AAV vectors expressing improved eCD4-Ig variants, TPST2, and regulatory elements that regulate or enhance eCD4-Ig expression

c.) An assessment of AAV-rh-eCD4-Ig-mediated protection from a clade C SHIV and from SIVmac239

Feinberg: IAVI Neutralizing Antibody Consortium (NAC)

PrincipalInvestigatorMark Feinberg, MD, PhD

Co-PrincipalInvestigatorsDennis Burton, PhDTom Hassell, PhD

GranteeInstitutionIAVI, New York, USA

Project TitleIAVI Neutralizing Antibody

OPPID1084519

Grant AwardUp to $50 Million, awarded November, 2013

Grant at a GlanceOVERVIEWThirty years since the identification of HIV as the cause of AIDS, the development of a safe and effective HIV vaccine remains a global health priority. There is a growing body of literature to suggest that a broadly neutralizing antibodies (bnAb)-based vaccine against HIV may be achievable. The PRIMARY OUTCOME of this proposal is the demonstration that an HIV ENV based immunogen(s) can be designed to elicit bnAbs in humans.

Classical approaches to vaccine discovery have thus far failed to identify immunogen(s) capable of eliciting HIV bnAbs. Using rational vaccine design, this CAVD grant investigates the interaction of HIV and bnAbs at the molecular level, takes this information for immunogen design and advances candidate immunogens through an iterative pipeline towards human clinical testing. We aim to develop at least one vaccine approach (immunogen(s), immunization strategy) that induces in humans, nAbs against 50% of HIV primary isolates at a titer of at least 1:100. We employ detailed antibody response analysis in our efforts to iteratively design, improve and evaluate immunogen candidates.

RESEARCH OBJECTIVES / PROGRESSWe have achieved significant progress during the first four years of this CAVD program, including but not limited to:• Development of the germline-targeting concept and design of the fi rst germline-targeting immunogen

(eOD-GT8), evaluated in knock-in mice, transgenic mice and entering Phase I clinical trials this year (2018). In addition to eOD-GT8, we have designed germline-targeting immunogens for HCDR3-depen-dent bnAbs to three major sites of vulnerability on the HIV trimer: V3-glycan, V2-apex, and MPER, that are in various stages of pre-clinical testing. Our germline-targeting immunogen for the V3-glycan (N332-GT5) has been selected for manufacture and clinical testing.

• Development of an immunofocusing strategy to initiate V2 apex bnAb responses using the V1V2 epi-tope transplanted onto different trimer backgrounds.

• Improvements in the yield, antigenic profile, thermal and structural stability of well-ordered trimer immunogens. In addition to selecting BG505 SOIP.664 for clinical manufacturing, we developed a stabilization platform for the HIV trimer (MD39 and descendants) that improves signifi -cantly in several dimensions on the original native-like Env trimer BG505 SOSIP. Capitalizing on the MD39 improvements, we also developed trimer-nanoparticles being used for various prime and boosting immunogens. We also developed an alternative to SOSIP as a platform for native-like trimers; the NFL (native fl exibly linked) trimer platform.

• Establishment of an antibody response analysis core for evaluation of antibody responses to candidate immunogens and vaccine regimens.

• Establishment and application of approaches for site-specifi c analysis of glycosylation to a number of immunogens, including eOD-GT8, CD4 binding site shepherding immunogens and V2 apex germline-targeting and immunofocusing immunogens.

• The first high-resolution structure of a V2 apex bnAb bound to a native-like HIV-1 Env trimer and the structures of a series of antibodies from longitudinal studies on bnAb evolution.

Gallo: FLSC Phase I & II Clinical Trials

PrincipalInvestigatorRobertGallo, MD

GranteeInstitutionUniversity of Maryland, Institute of Human Virology, Baltimore, USA

Project TitleGallo: FLSC Phase I & II Clinical Trials

OPPID1017606

Grant AwardUp to $16.8 million, awarded April, 2011

Collaborating Institutions◊ The Institute for Human Virology◊ Profectus BioSciences◊ Sanofi Pasteur◊ The Military HIV Research Program

Grant at a GlanceOVERVIEWPartial effi cacy observed in the Phase IIb clinical trial (RV144) in Thailand underscores the need to develop “next generation” regimens that elicit broader and more potent arrays of humoral effector mechanisms against HIV. Protection in RV144 likely involved antiviral antibodies against conserved/functional domains on the HIV envelope glycoprotein, gp120. Accordingly, attempts to expand and enhance such anti-gp120 antibody responses as well as antibodies linked with reduced risk of infection in RV144 are warranted. One practical strategy towards this goal is to build on RV144 via rationally modifi ed ALVAC prime/envelope protein boost regimens. The Institute of Human Virology (IHV) group postulates that the monomeric gp120 protein used in RV144 was likely the most limited component of the vaccine. Such monomers typically elicited only type-specifi c antibody responses and did not protect against HIV infection in earlier trials. An alternative envelope protein component capable of (1) boosting humoral specifi cities linked with reduced risk in RV144, and (2) concurrently raising antibodies to conserved gp120 domains linked with protection elsewhere, should boost the protective effi cacy of ALVAC prime/protein boost regimens.

The IHV group proposes that a conformationally constrained and stabilized gp120, embodied by a full-length single chain gp120-CD4 complex (FLSC), has properties that will focus immune responses on conserved/ functional gp120 domains; thus, optimizing the chances for broad protection against HIV. Five IHV studies in macaques support this concept. In the earliest study, a single chain complex containing rhesus CD4 (rhFLSC, engineered specifi cally for macaque studies) elicited antibody responses against highly conserved and functional gp120 epitopes and afforded non-sterilizing control of both plasma and tissue viremia following a single high-dose heterologous mucosal challenge with SHIV162P3. A second study showed that such protection depended on vaccine dose. A third study, using multiple low dose virus challenges with SHIV162P3, showed rhFLSC immunization provided an interval of sterilizing protection. A fourth study confi rmed the third study. Both studies showed that non-selective T-cell activation diminished the effi cacy of FLSC but that ADCC activity against CD4i domains, implicated as affording reduced risk in RV144, increased protective effi cacy. A fi fth study compared immunization of macaques with the RV144 vaccine versus a regimen that primed with a poxvirus encoding rhFLSC and boosted with rhFLSC protein. This study showed that compared to the RV144-based protocol, the rhFLSC-based strategy elicited statistically superior titers of anti-V1V2 antibodies (a correlate of decreased risk in RV144) and neutralizing antibodies. Overall, these experiments show that vaccination regimens using FLSC (1) will help improve humoral responses linked to reduced risk in human trials; (2) raise responses to highly conserved and functional gp120 epitopes, including ones termed CD4-induced (CD4i), that mediate humoral effector functions correlating with reduced risk in nonhuman primate and human vaccine trials.

The research team seeks to develop the FLSC as a new immunogen that can be used alone or in combination with vCP2438 (due to current unavail-ability of vCP1521) or related pox vectors to improve upon the effi cacy observed in RV144. This program includes production of GMP-grade FLSC; preclinical safety studies; evaluation of safety and immunogenicity for FLSC in a Phase 1 clinical trial; and ancillary studies in rhesus macaques to defi ne how humoral response magnitude, quality and durability elicited by rhFLSC is influenced by combinations with vCP2438 and/or different adjuvant formulations on. In addition, basic studies will compare the antigenicity of vCP2438 versus new poxvirus constructs encoding FLSC. Proposed Phase II clinical studies will be performed in collaboration with investigators at Sanofi -Pasteur and the Military HIV Research Program. These studies will test whether an ALVAC prime/ FLSC boost immunization strategy: (1) elicits a novel combination of humoral immune responses that have been linked with protective effi cacy in previous studies; (2) substantially improves the response rates, magnitudes and breadth of humoral responses that correlated with reduced risk in RV144. These characteristics will be established in part by comparisons of Objective 2 with RV144 and with ongoing Phase II/Phase III trials of other envelope-based vaccines. Based on these studies, follow-on Phase 2B and/or Phase 3 clinical trials will be designed to further evaluate the effi cacy of vaccine strategies using vCP2438/FLSC prime/boost or FLSC protein alone.

RESEARCH OBJECTIVES1.) Preclinical development and Phase I clinical testing of FLSC to identify a safe and immunogenic dose of FLSC/Alum that will be carried into

Phase II studies.

2.) Evaluate FLSC in Phase IIa clinical trials in combination with vCP2438.

3.) Determine whether the envelope encoded by ALVAC vCP2438 fortuitously encodes a constrained gp120 immunogen resembling the antigenic character of FLSC. Develop and test versions of ALVAC that express either soluble or membrane-anchored FLSC.

4.) Non-Human Primate studies -

a.) Establish the immune profi les elicited by a vCP1521 prime, rhFLSC boost vaccine regimen in the rhesus macaque model.

b.) Evaluate in rhesus macaques the humoral response profi les elicited by rhFLSC formulated in four different adjuvants, including Alum.

PROGRESSThe main aim of this program is to establish that FLSC can be combined with ALVAC to produce a vaccine regimen that (1) elicits a novel combination of humoral immune responses that have been linked with protective effi cacy in previous studies; (2) substantially improves the response rates, magnitudes and breadth of humoral responses that correlated with reduced risk in RV144. (Objective 2). Other Objectives were designed to support this goal.

Objective 1 was to prepare and release FLSC clinical trial material, perform the supportive preclinical studies, and demonstrate safety and immunogenicity of the product when formulated in Alum in a Phase 1 clinical trial. Objective 1 accomplishments include; (1) FLSC Drug Substance has been cGMP manufactured and released, yield ~ 1g/L; (2) Al(PO4) formulation (Alum) identifi ed that yields >95% adsorption and has shown stability

(Cont.)

Gallo: FLSC Phase I & II Clinical Trials

>1 month at 37oC; (3) Alum formulated FLSC drug product has been vialed; (4) ~ 3400 vials at 300 µg/mL of drug product will be available for Phase I clinical testing; (5) ~ 125 g of cGMP manufactured unformulated FLSC [Clinical Grade] has been aliquoted and stored for future studies; (6)

Toxicology/immunotoxicology studies are complete; (7) IND has been filed; (7) IRB approval for the Phase I trial has been obtained; and (8) clinical trial enrollment is complete (60 volunteers), with final dosing to occur January 2018 and final follow up to occur July 2018.

The final configuration (trial design) of Objective 2 has been developed, with strategies and trial sites identified.

Studies in Objective 3 indicate that the CD4-induced A32 domain, a key ADCC target in RV144 and natural HIV infection, is constitutively exposed on the 92TH023 envelope and on the corresponding vCP1521 vaccine construct. This property is shared by FLSC. ALVAC constructs that express either soluble or membrane bound FLSC have been developed and are being characterized immunochemically. Rhesus macaques were immunized with a regimen in which the animals were primed with the ALVAC construct encoding secreted rhFLSC and boosted with rhFLSC. This regimen exhibited superior responses against V1V2 compared to macaques given the RV144 regimen.

Objective 4 to test the immunogenicity of FLSC formulated in different adjuvants has been completed. Overall, Alum was not inferior to alternative adjuvants with respect to quality, quantity or durability of humoral responses. Given such data and established safety profiles, Alum remains the lead adjuvant for Phase II testing.

(Cont.)

Gottardo: Vaccine Immunology Statistical Center

PrincipalInvestigatorRaphaelGottardo, PhD

GranteeInstitutionFred Hutchinson Cancer Research Center, Seattle, USA

Project TitleVaccine Immunology Statistical Center

OPPID1151646

Grant AwardUp to $15.1 million, awarded October, 2016

External Scientifi c Advisory Board◊ Dean Follmann, National Institute

of Allergy and Infectious Diseases◊ Anthony (Tony) Rossini, Novartis

Pharma AG◊ Mark Segal, University of California

– San Francisco◊ Vince Carey, Harvard University

Grant at a GlanceOVERVIEWThe Vaccine Immunology Statistical Center (VISC) is organized within the CAVD to provide services in the areas of statistical design and data analysis, a central repository for data from key CAVD studies, and support for laboratory data management. The diverse staff at VISC has expertise in the areas of biostatistics, immunologic assays, bioinformatics, data management, and information systems.

VISC ensures that CAVD studies are effi cient by providing state-of-the-art statistical methods and, when required, develop novel methods. Specifi c areas of novel methodological development are the design of repeated low-dose challenge non-human primate studies, the analysis of immunologic checkerboard data, signal processing, normalization and analysis of fl ow cytometry-based assays and HIV antigen microarrays, and network models for the analysis of multivariate outcomes in mouse immunogenicity experiments. The VISC team also designs data models for novel immunologic assays and engineers integrated data pipelines from lab instruments to the CAVD central data repository. Finally, VISC staff support data access, data sharing, and collaboration through a customized web portal available to the large community of CAVD investigators.

The VISC assembles interdisciplinary teams to work on specifi c CAVD collaborative research projects. Through its work on each project, VISC identifi es best practices for study designs and data analysis, develops new and improved existing online tools, and settles upon standard nomenclatures and data formats to facil-itate data sharing and communication. In this way, project-specifi c solutions to research problems become platforms on which the larger research program can be based.

RESEARCH OBJECTIVES1.) Increased knowledge of potential immune correlates obtained from active/passive immunization

strategies (Study designs and data analyses).

2.) Improved tools and approaches to generating, processing and interpreting assay data (Data pipelines and methods).

3.) Improved assay data quality and the effectiveness of data operations services (Data ops and study management).

4.) Increased viewing and utilization of CAVD data for independent and cross-study analyses (Data base and web portal).

PROGRESSThe VISC continues to devote a large portion of its efforts towards assisting the CAVD Vaccine Immune Monitoring Consortia (VIMCs) with assay development and testing and data operations support for specifi c CAVD projects. These activities include improved data management, information exchange with investigators and lab personnel, study design, and statistical analysis.

The primary focus of VISC has naturally shifted from infrastructure development and data operations to scientifi c collaboration. Recent biostatistical collaboration projects include:

● Correlates analysis for non-human primate vaccine study featuring a VSV vector.

● Analyze data generated by the Fc Core, a new addition to the CAVIMC.

● Characterize antigen-specifi c B cells by analyzing data generated from transcriptomics, fl ow cytometry, and B-cell receptor sequencing.

● Conduct of analysis of non-human primate studies to help inform regimen dose and scheduling as the candidate vaccine components move forward into Phase I clinical trials.

● Conduct of analysis of data derived from novel vaccine candidates in human clinical trial to aid in the characterization of the vaccine elicited immune responses.

● Conduct of analysis of several antibody-effector function assays to characterize and compare their correlations and utilities in differentiating clinical samples.

● Development of methods for ICS polyfunctionality, peptide microarray and Fluidigm single-cell data to improve the effi ciency, standardization and throughput of the analyses of these data.

● Development of methods for the quality control, normalization and analysis of peptide antigen microarray data; analyses of these data to identify signatures predictive of a broad neutralization phenotype.

Hassell: Vaccine Product Development Center: GMP Manufacturing, Toxicology, and Clinical Data Management

PrincipalInvestigatorThomas Hassell, PhD

GranteeInstitutionInternational AIDS Vaccine, Initiative, New York, USA

Project TitleVaccine Product Development Center: GMP Manufacturing, Toxicology, and Clinical Data Management

OPPID1147661

Grant AwardUp to $57.6 million, awarded in May 2016

Grant at a GlanceOVERVIEWTo further product advancement within the CAVD, the Foundation has worked with the International AIDS Vaccine Initiative (IAVI) to create a specialized Central Service Facility titled the Vaccine Product Development Center (VxPDC) that acts to support a series of CAVD Principal Investigators (PIs). The VxPDC works with PIs to progress their preventative projects (vaccines and antibodies) through a translational stage and into clinical studies. An appreciable fraction of this work is funded through the VxPDC Core Grant, whereas this specifi c grant is a companion module. Specifi cally, it became apparent that a more effi cient and effective support of investigators could be achieved by further centralizing that support with funds to cover:

• the costs associated with the cGMP manufacture of the vaccine through a variety of Contract Manufac-turing Organizations (CMOs);

• the associated toxicology study(ies) necessary for regulatory fi lings typically carried out at specialist Clinical Research Organizations (CROs);

• and the subsequent clinical data analyses on completion of clinical studies.

Giving IAVI the fl exibility to contract directly with third-party service providers will allow IAVI to utilize both the expertise of their specialists and their portfolio of development projects, both considerable advantages vs. having individual CAVD PI institutions pursue negotiations where they may lack such expertise or the leverage of larger portfolios. In such a centralized context, a number of advantages should be realized. The costs and time involved in setting up contracts with these companies will be reduced, again as specialists will be used to set up the contracts and since IAVI has been in a position to negotiate favorable rates with multiple suppliers. Similarly, savings in cost and time can be realized by having IAVI implement master service agreements with key CMO and CRO’s. The number of errors (setbacks for time and costs of a project) will be reduced. When errors do occur with a particular project, management by a single team ensures that lessons from the events furnish expertise that can be applied to other projects, thereby improving performance and quality.

One key point to highlight is that the overall governance of the process has been designed to ensure the CAVD PI (and colleagues) are kept fully aware of the work conducted with the CMOs and CROs in support of their program, and to provide clarity over the PI’s key decision making role in the fi nal selection of the vendors/suppliers.

This grant is led by Thomas Hassell, PhD at the International AIDS Vaccine Initiative (IAVI). The majority of costs are allocated to contracted services provided by third parties. The CMOs and CROs selected will primarily be based in the U.S.A. However, in some instances selection of overseas groups will be considered, and these are most likely to be based in Europe to be proximal to particular CAVD grantees. The award was made in May, 2016 and then further supplemented through an amendment in October of 2017 with an anticipated end date of 12/31/2021.

RESEARCH OBJECTIVESInitial objectives include:

1.) Production of MPER peptides in liposomes

2.) Production of BG505 SOSIP.664

3.) Production of selected monoclonal antibodies

4.) Production of eCD4-Ig

5.) Production of a CMV vectored HIV/AIDS vaccine.

Hassell: Vaccine Product Development Center

PrincipalInvestigatorThomas Hassell, PhD

GranteeInstitutionInternational AIDS Vaccine Initiative, New York, USA

Project TitleVaccine Product Development Center

OPPID1153692

Grant AwardUp to $29.4 Million, awarded August, 2016

Grant at a GlanceOVERVIEWThe Vaccine Product Development Center at IAVI is designed to serve the Collaboration for AIDS Vaccine Discovery (CAVD) and the broader fi eld, with the primary goal of accelerating the development and testing of novel HIV vaccine candidates.

In essence, this program provides a methodology by which Principal Investigators, currently supported through the CAVD, could rapidly and confidently access additional HIV vaccine development expertise that helps to ensure the success of their project in reaching fi rst in human studies. Done in a timely fashion, this results in an efficient way of taking the vaccine candidate(s) forward to clinical development with full regulatory support. Potential issues with the development of the candidate product will be identified and expertise provided in ways to address such issues.

RESEARCH OBJECTIVES1.) Provide an expert range of development services from which the CAVD PI can select appropriate support

(depending on their particular needs), specifi cally:

a.) Product development planning, including supporting development of Product Development Plan and Target Product Profi le and provision of tools for monitoring of objectives vs. deliverables.

b.) Development of regulatory strategy and preparation of related fi lings, e.g. pre-IND, IND (USA and International).

c.) Design and management of preclinical studies, e.g. Toxicology.

d.) Identifi cation, commissioning, and management of manufacturing partners and distribution of clinical materials.

e.) Clinical development planning and execution through Phase I/II.

f.) Continuous project management assistance/support.

g.) Quality assurance support across entire process.

2.) Work with the PI to identify their specifi c needs and then provide a proposal on how the grantee’s needs may be met, including specifi c work recommended, internal and external resources that would be utilized, deliverables and timelines.

3.) Grantee determines which of the recommended services they would like to accept and liaise with CAVD Program Offi cers on overall plans to discuss likely impacts of any suggested modifi cations to the project (e.g. additional activities and associated costs).

4.) Assuming agreement, specifi c projects will commence with agreed upon plan, timing, milestones, and monitoring.

5.) Request feed-back from the CAVD PI on the success of their project, lessons learned and implement process of continuous improvement.

PROGRESSA total of 15 CAVD PIs have been supported to date through this mechanism, with 18 individual projects, multiple cGMP campaigns, tox studies, and regulatory fi lings and 17 Phase I clinical studies launched through December of 2018.

Haynes: Centralized Envelope Phase I Study

PrincipalInvestigatorBarton Haynes, MD

GranteeInstitutionDuke University,Durham, USA

Project TitleCentralized Envelope Comparative Immunogenicity (CECI) Study

OPPID52282

Grant AwardUp to $7.4 Million, awarded November, 2009


Center, USA◊ Fred Hutchinson Cancer Research

Center, USA◊ IPPOX Foundation, Switzerland◊ Los Alamos National Laboratory,

USA◊ National Institute of Allergy and

Infectious Diseases, USA

Grant at a GlanceOVERVIEWThis project was aimed at to solving a central problem blocking the development of a successful HIV-1 vaccine—how to design vaccine immunogens from a T-cell epitope perspective to successfully address the broad genetic diversity of HIV-1. The initial goal was to enable acceleration of vaccine development by ‘validating’ an in silico approach for optimizing HIV-1 T cell immunogens for testing in clinical trials. Over the past two decades, extensive viral sequencing has enabled an in silico approach to immunogen design to be developed, and substantial preclinical progress has been made. Centralized genes have now been identified which, as predicted in silico, have been proven to substantially improve the breadth of T cell reactivity, particu-larly CD8+ T-cells, in both small animals and non-human primates. This clinical trial, named HVTN 106, sought to determine if the observations in small animals and non-human primates extend to humans.

To test this, the arms of the trial (HVTN 106) included:

1.) Single wildtype transmitted/founder Env (30 subjects + 5 controls)

2.) Single group M consensus Env (30 subjects + 5 controls)

3.) Trivalent mosaic Env (30 subjects + 5 controls)

The vaccines were Env gp160s given as DNAs for priming and boosted with the MVA-CMDR recombinant Env. Three injections of DNA (4 mg) were given at weeks 0, 4 and 8 followed by two injections of MVA-CMDR (1x108 pfu) at weeks 16 and 32. Placebo controls were given as empty vaccine diluent (saline).

RESEARCH OBJECTIVESHVTN106 was a phase I proof-of-concept study initiated to test two centralized approaches (consensus and mosaic Envs) versus single wildtype Env, as a means of enhancing the breadth and coverage of T cell responses to HIV. The critical scientific questions addressed by the clinical trial include:

1.) Is consensus Env superior to wildtype transmitted/founder Env for induction of T cell breadth?

2.) Is consensus Env superior to trivalent mosaic Env for induction of T cell breadth?

3.) Can in silico predictions of superiority of induced breadth of T cell responses by consensus and mosaic immunogens be validated in humans in vivo?

Data analysis to determine the breadth and depth of T cell of responses induced by priming with WT vs. consensus vs. trivalent mosaic Env DNAs is almost complete.

PROGRESSThe HVTN106 Phase I clinical trial concluded in September 2016, and the primary CAVD award ended in March 2018. Data analyses are close to completion for all study endpoints. Dr. Nicole Frahm at the FHCRC and Dr. Bette Korber at LANL have been working to measure the magnitude, depth, and breadth of T-cell responses elicited by the HVTN106 vaccine regimens, and Dr. Kevin Saunders at the DHVI has been characterizing the corresponding antibody repertoire.

T cell response rates and response magnitude to the different DNA primes were measured by intracellular cytokine staining (ICS), and the analysis was completed in August 2017. Epitope mapping by ELISpot assay was also performed to characterize the breadth and depth of T cell responses to the different DNA priming immunogens and to assess the impact of the MVA-CMDR boost. Early results indicated that the mosaic DNA induces higher magnitude Env-specifi c T cell responses compared with natural T/F and could potentially serve as a universal prime. The last round of epitope mapping to quantify response breadth was completed in November 2017. Each DNA prime appeared to target different regions of the HIV-1 gp160. Interestingly, several CD4+ T cell responses clustered the same V2 region as the immunodominant responses seen in the RV144 trial. Dr. Bette Korber has been working with the SCHARP team to extend the analysis to correlate T cell and IgG responses.

Dr. Saunders and team isolated a total of 120 monoclonal antibodies from the fi nal vaccinee samples collected after the last MVA-CMDR boost. Through selective screening for Env-targeted antibodies, they selected 31 mAbs for thorough characterization of epitope specifi cities and antibody effector functions (ADCC, ADCP, virus capture). Similar to previous studies, they observed a bias toward gp41 reactivity in the HVTN106 vaccinee antibody responses. Several of the gp41-reactive mAbs also exhibited cross-reactivity to human intestine microbiota antigens, and the LLRAIE epitope was required for gp140-reactivity. Additionally, two new V2-specifi c antibodies, similar to the CH58 and CH59 mAbs from RV144, were isolated and characterized.

A collaborative study with Dr. Suzanne Campion and Dr. Andrew McMichael at Oxford University has been done in parallel with other analyses to assess how the pre-immune CD4+ T-cell repertoire shapes the post-vaccination T cell responses to the HVTN 106 vaccine regimens. This study also aimed to determine whether post-vaccination responses from the naïve or memory precursors pool, how ontogeny affects the persistence and immunodominance of the response, and the infl uence of the microbiome on post-vaccination T cell responses. The Oxford team has completed epitope mapping for 20 donors against the Con-S peptide set at both the pre-vaccination (visit 2) and post-DNA primes (visit 7) timepoints. Prelim-inary results suggest that the pre-immune repertoire were often immunodominant in the fi rst responses after vaccination.

Analysis is wrapping up and several manuscripts are in preparation from all of the teams at the HVTN, Duke University and Oxford University.

Haynes: MPER-Peptide Liposome Immunogen Testing


GranteeInstitutionDuke University,Durham, USA

Project TitleMPER-Peptide Liposome Immunogen Testing

OPPID1094352


Collaborating Institutions◊ Infectious Diseases Research

Institute (IDRI), Seattle, USA◊ International AIDS Vaccine Initiative

VxPDC, New York City, USA◊ Human Vaccine Trials Network,

Seattle, USA◊ NIAID/ Division of AIDS,

Washington D.C., USA

Grant at a GlanceOVERVIEWOne key strategy of immunogen design for HIV vaccine development is to identify immunogens that selectively bind neutralizing versus non-neutralizing antibodies. This approach circumvents the induction of dominant non-neutralizing responses frequently observed during natural infection and with current vaccine protein immunogens. We previously demonstrated that the virion lipid is an important component of the binding immunogen for the gp41 membrane proximal external region (MPER) neutralizing epitope. Moreover, we have shown that antibodies that target this MPER neutralizing site are frequently polyreactive and negatively controlled by immune tolerance mechanisms. To overcome the disfavored status of MPER neutralizing antibodies, the Haynes team designed an immunogen to recreate this neutralizing epitope that includes the gp41 MPER associated with lipid. It retains the same binding properties for the MPER neutralizing antibodies as seen with virions. Specifi cally, MPER-peptide liposomes selectively bind the 2F5 and 4E10 mAbs and their unmutated ancestor antibodies (the putative naïve B cell receptors), but do not bind non-neutralizing gp41 MPER antibodies. Additionally, in a 2F5 MPER bnAb variable Heavy and variable light chain (VHVL) mature knock-in mouse model, the team has demonstrated that the MPER-peptide liposome immunogen induces 2F5 bnAbs when formulated with the TLR4 agonist monophosphoryl lipid A.

The overall goal of this project is to develop a GMP manufacturing process for production of MPER peptide-li-posomes (a.k.a MPER-656 Liposomes) at the appropriate quality and scale for use in a fi rst-in-human phase I clinical trial to evaluate safety and immunogenicity. Specifi cally, we will determine if the MPER peptide-lipo-somes can expand gp41 proximal MPER broadly neutralizing precursors after vaccination.

RESEARCH OBJECTIVES1.) Formulation of research grade MPER peptide-liposomes and immunological evaluation in murine and

NHP animal models.

2.) GMP vaccine production of MPER peptide-liposomes by IDRI in Seattle, WA in collaboration with the IAVI VxPDC team.

3.) Carry out a Phase I safety and immunogenicity trial with the HVTN.

PROGRESS• An adjuvant selection trial was performed in non-human primates (NHPs), and Alum was selected as the

adjuvant for the GMP MPER-peptide liposome formulation.

• A preclinical study in 2F5 mature knock-in mice confirmed the immunogenicity of MPER-656 Liposomes after several months of frozen storage. Additionally, continuous stability testing of early R&D lots has demonstrated that MPER-656 Liposomes are stable for at least 24 months when stored at -20°C.

• Process development for the GMP manufacturing process was completed at IDRI in 2017 with the support of the IAVI VxPDC team, and material was produced for use in the toxicology study.

• An ID50/ potency study was performed in mice using the toxicology study batch (engineering run) of MPER-peptide liposomes. These data will provide a baseline by which to compare potency of subsequent MPER-peptide liposome lots. An identical study will be done with the GMP clinical trial material (CTM) after production.

• A preclinical GLP toxicology study in rabbits was conducted by Charles River Laboratories (CRL) this year (2018) to verify safety of vaccination with MPER-peptide liposomes plus Alum adjuvant. There were no unscheduled deaths, no evidence of systemic toxicity, and no local irritation at the administration sites that were related to treatment with MPER-656 Liposomes. The in-life phase of the study ran from April to July 2018, and the full fi nal report is almost complete.

• The HVTN face-to-face meeting for clinical protocol development was held in October 2018, and the protocol is entering into the regulatory review phase. The team is targeting late 1Q/ early 2Q 2019 for IND submission.

• Manufacture of the GMP grade clinical trial material (CTM) will be completed by the end of this year (2018).

On the current track, start of enrollment for the Phase I clinical study is expected to begin in summer 2019.

Haynes: Protein Production Facility


Frederick Porter, PhD Senior Director of Product Development

James Peacock, PhD, Director of Protein Production Facility

GranteeInstitutionDuke University, Durham, USA

Project TitleProtein Production Facility

OPPID1154951

Grant AwardUp to $2.4 Million, awarded July 2016

Grant at a GlanceOVERVIEWThe Protein Production Facility at the Duke Human Vaccine Institute (DHVI) is equipped to produce HIV-1 Env proteins, HIV-1 Env SOSIP trimers, Fc-gamma receptors, and antibodies for the field to study, as standardized, quality in vitro reagents. The hope is that these reagents will both optimize clinical trial immune monitoring, as well as facilitate Env immunogen discovery. The proteins and antibodies produced in the Protein Production Facility are not intended for use in humans or clinical trials. All proteins and antibodies are produced under standardized conditions in that procedures, equipment, operator training, and documentation are governed by Standard Operating Procedures with oversight by Duke’s Quality Assurance for Duke Vaccine Immunoge-nicity Programs (QADVIP) unit. Documentation of all procedures is retained, and the facility is monitored for compliance to ensure that consistent, reproducible, auditable, and reliable results and products are delivered.

Quality control is performed on the proteins produced in the PPF. This includes testing for bioburden (endotoxin, sterility, and mycoplasma) as well as sequence confi rmation analysis by LC/MS (for proteins only). SDS-PAGE under reduced and non-reduced conditions is also performed. Size Exclusion Chromatography by FPLC may be performed as a secondary purifi cation step if required. All protein characterization, bioburden, and QC results are reported on the certifi cate of analysis upon delivery. QC for SOSIP trimers also includes negative stain scanning electron microscopy and binding analysis (BLI).

The facility currently has resources provided by the Foundation to produce recombinant proteins and antibodies for CAVD members on a first-come, first-serve basis. Application for protein production requires the approval of the CAVD member’s program offi cer and the approval of the PPF’s program offi cer. Please submit protein order requests through the Protein Production Facility website on the CAVD Portal.

For any questions or inquiries, please contact Jamie Peacock ([email protected]).

Ho: Engineered Bispecifi c bNAbs for Prevention

PrincipalInvestigatorDavid Ho, MD

GranteeInstitutionAaron Diamond AIDS Research Center, New York, USA

Project TitleIbalizumab-based Bispecifi c Antibod-ies for HIV Prevention

OPPID1040731

Grant AwardUp to $12.1 Million, awarded April, 2012

Collaborating Institutions◊ Tulane National Primate Research

Center◊ WuXi Biologics

Grant at a GlanceOVERVIEWThe overall aim of this grant is to accelerate the development of a broadly neutralizing antibody (bNAb) product for passive immunization against HIV. Passive administration of an HIV-neutralizing monoclonal antibody on an infrequent basis has the potential, like long-acting anti-retrovirals (ARVs), to fi ll a critical gap between now and the launch of an effective, active vaccine against HIV.

The key obstacle for development of bNAbs for HIV prevention has been the discovery of an antibody that possesses suitable breadth, potency, and pharmacokinetics (PK). Next generation antibody-like molecules that could achieve greater potency and breadth would provide a more practical, low cost product generally more feasible for most of the countries hit hardest by HIV/AIDS.

This grant to Dr. Ho seeks to capture promising new results based on bispecifi c antibodies. Bispecifi c antibodies targeting both an epitope on HIV and the host cell receptor on HIV’s target cells can have a greater than 100-fold increase in potency and increased breadth of coverage, over either antibody alone or the combi-nation, in laboratory studies. If fully developed, such next generation engineered bNAbs would meet the requirements for the low cost products for populations in developing countries.

RESEARCH OBJECTIVES1.) Develop a lead bispecifi c antibody-like molecule with much enhanced anti-HIV breadth and potency that

could be administered on a bimonthly basis

2.) Assess the “developability” of the best candidate bispecifi c antibodies and advance one lead molecule into clinical development

3.) Express a bispecifi c antibody in vivo by gene transfer using mini-circle DNA delivered into muscle via electroporation

PROGRESSThe Ho research consortium has generated a panel of over 200 antibody-like molecules, most of which are bispecific with one arm directed to one of the viral receptors and one arm directed to an element on the viral envelope glycoprotein. In collaboration with Dr. Michael Seaman at the Beth Israel Deaconess Medical Center, part of the Montefiore Antibody Vaccine Immune Monitoring Consortium (Ab VIMC), a select pool of these antibodies were identified to be extremely potent and broad at neutralizing a large panel of HIV isolates. While the Ho consortium continues to expand this antibody library in order to identify additional potent and broad antibody-like molecules, a largely empirical process, they in parallel are advancing the top antibody candidates already identified through preclinical development activities. The Ho consortium has characterized the developability, manufacturability, and in vivo properties of the top antibody candidates identified and selected a lead candidate, 10E8.4/iMab, for further clinical development as passive immunization for HIV prevention. In addition, the Ho consortium is developing an antibody gene-transfer method to deliver DNA encoding for potent bispecific antibodies by electroporation. This technology has the potential to express physiologically relevant levels of a bispecific antibody for an extended period after administration while circumventing many of the cost and production barriers that may limit traditional monoclonal antibody therapeutic approaches. The ultimate goal of the consortium is to create a novel bispecific antibody that has dramatically improved anti-HIV breadth and potency as well as a pharmacokinetic profile suitable for bimonthly administration to humans.

Ho: First in human clinical evaluation of 10E8.4/iMab, a potent and broad bispecifi c antibody against HIV

PrincipalInvestigatorDavid Ho, MD

GranteeInstitutionAaron Diamond AIDS Research Center, New York, USA

Project TitleFirst in human clinical evaluation of 10E8.4/iMab, a potent and broad bispecifi c antibody against HIV

OPPID1169162

Grant AwardUp to $7.9 Million, awarded July, 2017

Grant at a GlanceOVERVIEWProtection against HIV infection by the infrequent, passive administration of a potent HIV-neutralizing antibody could be a better alternative to current pre-exposure prophylaxis regimens which are based on daily dosing with antiviral drugs. This option requires that the chosen antibody possesses suitable antiviral breadth, potency, and pharmacokinetic properties.

Background: Prior in vitro and preclinical studies at the Aaron Diamond AIDS Research Center (ADARC), performed with Foundation funding, identifi ed 10E8.4/iMab as a potent and broad bispecifi c antibody against HIV. It neutralized a global panel of 118 HIV-1 pseudotyped viruses with a geometic mean IC50 of 0.001 µg/mL. An earlier variant of 10E8.4/iMab also potently neutralized 98% of viruses in a second panel of 200 HIV-1 isolates belonging to clade C, the dominant subtype accounting for 50% of new infections worldwide, and the most prevalent clade in sub-Saharan Africa. 10E8.4/iMab also reduced virus load substantially in HIV-1-infected humanized mice, and an earlier variant of 10E8.4/iMab provided complete protection when administered prior to systemic HIV challenge.

The activities under this award will advance 10E8.4/iMab into clinical development in order to further evaluate its potential as a novel prophylactic agent against HIV-1. Investigators at ADARC will conduct a fi rst-in-human clinical trial evaluating the safety, pharmacokinetics, and antiviral activity of 10E8.4/iMab. A dose escalation study will be conducted in a phased approach and will evaluate IV administration in HIV-negative participants, IV administration in HIV-positive participants with viremia, and subcutaneous admnistration in HIV-negative subjects. In addition, a master cell bank of an earlier variant of 10E8.4/iMab, known as 10E8.2/iMab, will be generated as a back-up molecule for potential GMP manufacture.

The grant is led by David D. Ho at the Aaron Diamond AIDS Research Center.

RESEARCH OBJECTIVES1.) Phase 1 clinical trial to determine the safety, tolerability, pharmacokinetics and antiviral activity of 10E8.4/iMab for HIV-1 prevention

2.) 10E8.2/iMab master cell bank creation for potential GMP manufacture

Johnson: Single cell transcriptional profi ling of latently infected CD4+ T cells

PrincipalInvestigatorR. PaulJohnson, MD

GranteeInstitutionEmory University, Yerkes National Primate Research Center, Atlanta, USA

Project TitleSingle cell transcriptional profi ling of latently infected CD4+ T cells

OPPID1110661


Grant at a GlanceOVERVIEWHIV-1 rapidly establishes a latent infection of long-lived quiescent CD4+ T cells, which carry an integrated provirus that is largely transcriptionally silent, functionally invisible to immune surveillance and impervious to the activity of antiretroviral drugs. The long half-life of these cells and their capacity to be reactivated and produce infectious virions remain the primary obstacles to viral eradication. Currently research techniques have numerous limitations, because the relative rarity of latently infected cells severely constrains efforts to dissect molecular mechanisms involved in latency. Furthermore, the predominant reservoirs of latently infected cells are found in secondary lymphoid and mucosal tissues, not in peripheral blood, the primary site that is readily available for studies in HIV-infected human subjects. We propose a novel approach using a highly sensitive, high throughput single cell qPCR technique to analyze CD4+ T cells isolated from lymphoid tissues from SIV-infected macaques treated with potent antiretroviral therapy (ART), coupled with the identifi cation of subsets of memory CD4+ T cells that are highly enriched for latently-infected cells. By analyzing expression of a panel of molecules that are differentially expressed on the surface of CD4+ T cells, we will identify novel cell surface biomarkers, that either alone or in combination, represent a distinct signature of latently infected cells. The use of single cell transcriptional profi ling represents a unique approach to address the fundamental challenges in characterizing latently-infected reservoirs and should provide essential information that could be then used for design of therapies to selectively target latently-infected cells in HIV-infected patients.

This grant is led by R. Paul Johnson, MD (Emory University, Yerkes National Primate Research Center). The award was received in November, 2014, with an initial agreement length of 2 years.

RESEARCH OBJECTIVES1.) To establish potent suppression of viral replication in SIV-infected macaques using optimized ART regimens. From such treated animals, we

will collect and cryopreserve a comprehensive set of tissues that includes, inter alia, secondary lymphoid and mucosal tissues, and CNS tissues

2.) To apply recent advances in the characterization of memory CD4+ T cells to identify enriched reservoirs of latent SIV-infected cells. In addition, we will include monocytic cell populations in the analysis

3.) To apply single cell transcriptional profi ling to identify novel biomarkers present on the surface of latently infected cells. Our primary method for such profi ling is real-time PCR, and we will evaluate RNAseq as an alternative means for such characterization

Jolicoeur: A new class of immunogens for raising broadly neutralizing antibodies

Principal InvestigatorPaul Jolicoeur, MD, PhD

GranteeInstitutionInstitut de Recherches Cliniques de Montréal, Montréal, Québec CA

Project TitleDevelopment of a new class of immunogens for raising broadly neutralizing antibodies

OPPID1146356

Grant AwardUp to $698,000, awarded in May 2016

Grant at a GlanceOVERVIEWThis grant is based on the hypothesis that it should be possible to exploit anti-idiotype monoclonal antibodies (mAbs) as a tool to obtain mirror image representations of the HIV-1 Env neutralizing paratopes on anti-Env broadly neutralizing Abs (bnAbs), and then to use these anti-idiotypic Abs as immunogens. Such an anti-id-iotypic Abs should mimic the Env conserved region targeted by the cognate anti-Env bnAb and, when used for immunization would be expected to generate a bnAb against HIV-1 Env in a single step. This approach could overcome the long maturation process and other problems when Env is used as an immunogen. The application originated from the Foundation’s Grand Challenges program.

The program will generate anti-idiotypic Abs in llama immunized with anti-Env bnAbs. The choice of llama was based on the unique class of natural antibodies found in camelids. These antibodies have no light chain and are made of a single heavy chain whose short variable domain, designated VHH, is suffi cient for antigenic recognition. VHH are very small and are capable of penetrating into small pockets of molecules, thus enhancing the chance to recognize the paratope 3D structure of the human anti-Env bnAb.

Anti-idiotypic llama VHH Ab will fi rst be screened for the capacity to inhibit the neutralization of their cognate bnAb. For this initial screen, the program will take advantage of robust, established methods to generate libraries of VHH cDNA clones using RNA from PBMCs harvested from bnAb vaccinated animals. The cDNA clones are constructed in a GST expression context, with the VHH-GST protein readily produced in a semi-au-tomated manner for ensuing assessment in a standardized in vitro neutralization competition assay against their cognate bnAb. VHH proteins passing this fi rst screen will then be assessed for function in vivo (i.e. for their capacity as immunogen to generate anti-HIV neutralizing antibodies). For this latter stage, mice and guinea pigs will be immunized with anti-idiotypic VHH mAb proteins to generate anti-HIV bnAbs. Overall, the approach apprehends a broad diversity in the VHH libraries and funnels the anti-idiotype antibodies through a stringent screen.

The program is led by Paul Jolicoeur, MD, PhD at the Institut de Recherches Cliniques de Montréal, an affi liate of the Université de Montréal. The award was made in May 2016 with an estimated duration of 23 months.

RESEARCH OBJECTIVES1.) Generating cDNA libraries of VHH clones, and producing VHH proteins from two cDNA libraries.

2.) Screening VHH candidates capable of competing the neutralizing activity of their cognate bnAb in vitro.

3.) In vivo screening of pre-selected VHH candidates for their capacity to elicit an anti-HIV nAb activity.

Kamarck: Clinical Assessment of hCMV-HIV Prototype Vector

Principal InvestigatorMichael Kamarck, Ph.D.

Grantee InstitutionVir Biotechnology Inc., San Francisco, USA

Project TitleClinical Assessment of Live Attenuated hCMV-HIV Prototype Vector

OPPID1187970

Grant AwardUp to $12.1 Million, awarded in February 2018

Collaborating Institutions◊ Oregon Health and Science

University

Grant at a GlanceOVERVIEWVir Biotechnology Inc. (Vir), in collaboration with Oregon Health and Sciences University (OHSU), is develop-ingCMV-VIR2-HIV-001,anovel,first-in-class,prophylacticvaccineforhumanimmunodeficiencyvirus(HIV).The potential of a cytomegalovirus (CMV)-based vaccine to elicit broad and durable cellular responses, and the central importance of unconventional (MHC-E-restricted and MHC-II-restricted) CD8+ T-cell effector re-sponses, have been shown in published and unpublished work by the Picker group. The concept of immune programming (i.e. the use of CMV-based vectors to elicit CD8+ T-cell responses that are distinct in their epi-toperecognitionandMHCrestriction,dependingonthegeneticmodificationofthevector)representsanewparadigm in vaccine development. Based on this new paradigm, Vir intends to develop an hCMV vector-based antigen delivery and immune programming platform which can be used as a vaccine for multiple diseases, including HIV.

The prototype of the vaccine contains a live attenuated hCMV derived from clinical isolate TR (Smith 1997). ThisTRisolatewascloned(Murphy2003)andgeneticallymodified,introducingtheUL97andUS2-7genesfrom strain AD169 to provide ganciclovir sensitivity and MHC-I inhibitory activity, respectively. This clone was then attenuated by replacing the gene encoding the tegument pp71 with a genetic sequence encoding the HIV-1 clade A gag transgene derived from the GRIN plasmid (Keefer 2012, Keefer 2010). The CMV-VIR2-HIV-001 vaccine also contains deletions of CMV genes UL128, UL130, UL146, and UL147.

BecausethevaccinethatVirproposestomanufactureandtestinafirst-in-human(FIH)clinicalstudycarriesan HIV clade A gag insert only, it is not meant to be a clinical product. This initial vaccine is intended to be used in a proof-of-concept experimental medicine study to determine the safety of the vector backbone and whether the unconventional responses observed in nonhuman primates (NHP) translate to humans. If successful, a vaccine containing an epitope-optimized version of HIV inserts will be used in the next-generation vectors. These new inserts will be sequence-optimized to best match global circulating HIV strains, will include gag, pol,andnefsequences,andwilllikelyfocusonuniversalMHC-E-restrictedepitopes(supertopes).Thefinaldesign of these inserts will be based on ongoing studies at OHSU.

ThegrantisledbyMichaelKamarckatVirBiotechnologyInc.(SanFrancisco,CA).TheprimaryscientificcollaborationiswiththeteamatOHSU,led by Louis Picker. IDT Biologika (Rockville, MD) is the CRO that will conduct the engineering run, formulation, and stability program, as well as a clinical study material production run, all conducted using Good Manufacturing Practice (GMP). The IAVI VxPDC (New York, NY) assisted in estab-lishing the IDT contract.

RESEARCH OBJECTIVES1.) Completethelastpreclinicalpharmacologystudies(confirmatoryinvitrostudiesandaGLPtoxicologystudyinrabbits)neededtofileanInves-

tigational New Drug (IND) application.

2.) Manufacture clinical study materials.

3.) Conduct an FIH clinical study that will inform whether the vector backbone is safe to use in humans, based on results of physical examinations, clinical laboratory analyses, tolerability assessments, and shedding evaluations.

◊ Dose-escalation phase: initially evaluate 4 dose escalation levels of the vaccine in a total of 32 healthy (HIV- hCMV+) adult subjects (24 in the active group and 8 in the placebo group).

◊ Dose-expansion phase: gather additional data from 20 subjects (16 in the active group and 4 in the placebo group) on 2 of the dose levels.

4.) Demonstrate proof-of-concept for the CMV platform by evaluating whether the unconventional T-cell response (MHC-II and MHC-E restriction and long-term effector levels) can be elicited in humans.

Kay: Comparative NHP Study to Test New Capside Against AAV1

PrincipalInvestigatorMark Kay, PhD

GranteeInstitutionStanford University, CA, USA

Project TitleComparative NHP Study to Test New Capsids Against AAV1

OPPID1154293

Grant AwardUp to $717,000, awarded in September, 2016

Collaborating Institutions◊ UC Davis – California National

Primate Research Center, Davis, CA USA

Grant at a GlanceOVERVIEWThis grant was awarded to conduct a nonhuman primate (NHP) experiment, to evaluate the in vivo perfor-mance of improved adeno-associated virus (AAV) vectors recently developed in the Kay laboratory under a prior CAVD grant. AAV vectors are of interest as gene transduction systems for the endogenous production of anti-HIV broadly neutralizing antibodies, with the caveat that current AAV vectors have seen limited success in achieving high expression levels of secreted proteins. The improved AAV vectors from the Kay laboratory (AAV-NP22 and AAV-NP66) have been engineered to have superior human skeletal muscle tropism and, when assessed in vitro using human muscle explant models, have provided much higher transduction levels than previously possible. For pre-clinical in vivo testing of the new AAV vectors, murine models are a poor choice as they have historically been poorly predictive of the cellular tropisms and transduction effi ciencies of AAV serotypes in humans. The Kay laboratory has already shown via explant models of NHP muscle tissues that the AAV-NP22 and NP66 achieve the same signifi cant improvement in gene transduction as observed with the human tissue model, an important prelude to proceeding with the in vivo NHP evaluation.

The NHP model will establish the relative transduction effi cacy of AAV-NP22 and AAV-NP66 against a standard AAV-1 vector in the skeletal muscle of non-human primates to predict future passive vaccine expression levels in human patients. Translating these high transduction levels to a clinical setting would enable lower dosing of this passive antibody vaccine while still achieving therapeutic levels of antibody expression, thereby reducing the cost of vaccine per patient and reducing the probability host T cell responses against transduced muscle fi bers.

The grant is led by Mark Kay, PhD (Stanford University), and the animal model will be run at the University of California – Davis California National Primate Research Center, where the protocol will be directed by Christopher J. Miller, DVM, PhD. Phil Johnson, MD (IAVI) serves as a consultant. The award was made in September, 2016 with an anticipated duration of 18 months.

RESEARCH OBJECTIVES

1.) Construction and functional verifi cation of rhesus AAV vector plasmids, with ensuing GMP-like production of 3 AAV vectors.

2.) Animal selection after excluding animals with neutralizing anti-AAV antibodies against the 3 AAV serotypes.

3.) Administration of the vectors and monitoring the animals for six months, using routine veterinary clinical laboratory assessments as well as evaluating transgene expression levels. The study will conclude with tissue analysis of the subject animals.

Koup: Comprehensive Cellular Vaccine Immune Monitoring Consortium

PrincipalInvestigatorRichard Koup, MD,NIAID, Vaccine Research Center

GranteeInstitutionFoundation for the NIH, USA

Project TitleComprehensive Cellular Vaccine Immune Monitoring Consortium

OPPID1147555

Grant AwardUp to $11.5 Million, awarded in July 2016

Collaborating Institutions◊ Case Western Reserve University,

USA◊ Duke University, USA ◊ Fred Hutchinson Cancer Research

Center, USA◊ National Institute of Allergy and

Infectious Diseases, NIH, USA

External Scientifi c Advisory Board◊ Jaap Goudsmit, Harvard◊ Danilo Casimiro, Sanofi Pasteur◊ Quentin Sattentau, Oxford

University◊ Nelson Michael, Walter Reed Army

Institute of Research◊ Louis Picker, Vaccine Gene

Therapy Institute, Oregon Health & Science University

◊ Susan Barnett, Bill & Melinda Gates Foundation (ex offi cio)

◊ David Montefi ori, Duke University Medical Center (ex offi cio)

◊ Patricia D’Souza, National Institutes of Health (ex offi cio)

◊ Raphael Gottardo, Fred Hutchinson Cancer Research Center (ex offi cio)

◊ Jean Patterson, National Institutes of Health (ex offi cio)

Grant at a GlanceOVERVIEWThe Comprehensive Cellular Vaccine Immune Monitoring Consortium (CCVIMC) is a Central Service Facility (CSF) within the CAVD providing a variety of cellular assays in support of pre-clinical and clinical HIV vaccine trials. Beyond providing exploratory, qualifi ed, standardized and GCLP-compliant cellular assays, the CCVIMC consults with the Vaccine Discovery Consortia (VDCs) on both pre-trial design and as well as post-trial/assay assessments. The goal of these efforts is to facilitate the development and licensure of a safe and effective HIV vaccine.

RESEARCH OBJECTIVES1.) Maintain a Clinical Trial Testing Core (CTTC) with GCLP-compliant laboratories at the Fred Hutchinson

Cancer Research Center (FHCRC) and the Vaccine Immunology Testing Laboratory (VITL) in the Vaccine Research Center (VRC) at the National Institute of Allergy and Infectious Diseases (NIAID).

2.) Maintain a Nonhuman Primate Core (NHPC) providing standardized and exploratory assays at the VRC.

3.) Maintain a Transcriptomic Core (TC) provide enabling transcriptomic technology, systems biology expertise, and bioinformatics consultation in order to generate biomarkers of vaccine protection and testable hypotheses for improving vaccine regimens.

4.) Develop and optimize exploratory cellular assays and technologies for monitoring cellular immune responses in clinical and pre-clinical trials.

5.) Enable effi cient technology transfer and deployment of new and improved exploratory assays for avail-ability to CAVD community as standardized, qualifi ed, validated, or GCLP-compliant assays.

6.) Supply the CAVD community with quality-controlled, well characterized PBMC samples for use in the assay development and standardization.

7.) Investigate vaccine-related scientifi c questions that are not being addressed by individual VDCs.

8.) Maintain an Administration and Management Core (AMC) responsible for grants management, compliance oversight, and initiating and facilitating inter- and intra-consortium communications within the CAVD.

SERVICESThe CCVIMC provides both standardized, and a variety of research, assays—including an expanding reper-toire of B cell, tissue imaging, and immune complex assays—to CAVD investigators for both human clinical trials and NHP studies. Our preference is to begin working with VDC investigators early in the study design process so that we can provide expertise and experience in assay and sampling selection (both time points and tissue selection), perform QA/QC for sample processing (collecting, storing and shipping), and assure the timely procurement and validation of assay reagents. Our partnership with each VDC continues throughout the study, convening interim calls as necessary, and lending our collective expertise to investi-gators as they interpret assay data.

Our standardized assays include:

● Intracellular cytokine staining (ICS), with several panels available to measure both CD8 and CD4 responses and a variety of other markers in monkeys and humans.

● A standardized IFN-g ELISpot assay is also available from our core of clinical and NHP labs.

● B cell phenotyping is a standardized assay for NHP trial.

● Transcriptomic analysis – RNAseq (or microarray for historic comparisons) is available including systems biology analysis.

● Secreted cytokine assay to measure factors linked to variety of immune functions, such as Th1, Th2, pro-infl ammatory, regulatory, and chemotactic responses;

● Clinical Virus Inhibition Assay for measuring CD8-mediated virus replication inhibition.

Our research assays, which serve as secondary endpoints, include:

● Epitope mapping, using either ELISpot or ICS platforms to defi ne which epitopes are targeted by a T cell response;

● B cell, T cell, and NK phenotyping using multi-parameter fl ow cytometry to defi ne representation and activation state of lymphocyte subsets;

● Fluidigm assays (multiplexed qPCR) for T and B cell interrogation, both nanoarray and single cell, to quantify gene expression profi les;

● Transcriptomics to identify gene expression profi le signatures of cell/tissue subsets;

● TCR clonotyping to defi ne the repertoire of epitope (peptide) specifi c responses.

(Cont.)

Koup: Comprehensive Cellular Vaccine Immune Monitoring Consortium

Additional assay development efforts include multiplexed confocal imaging to assess the anatomic and structural features of germinal centers in response to vaccine strategies or SIV/SHIV infections.

The CCVIMC also provides CAVD investigators with a variety of clinical and NHP PBMC specimens, and some NHP tissue specimens to support assay development initiatives.

PROGRESSStandardized assays as well as research assays have been provided for both clinical and pre-clinical (NHP) vaccine trials within the CAVD. The data generated from these assays are provided to the Vaccine Immunology Statistical Center (VISC) for statistical analysis and reported directly to the Vaccine Discovery Consortia (VDC) for the evaluation of the impact of data on the results of the trial.

The CCVIMC continues to expand services to include more advanced research assays and broaden the role of data analysis and interpretation. We continue to adapt our services to meet and anticipate the changing landscape of HIV vaccine design.

High-throughput processes have been established to interrogate antigen-specific B cell populations. The sensitivity and throughput capabilities in our laboratories have opened the door for exploring novel vaccine strategies based on targeting specific naïve B cell populations, stimulating the expansion and maturation of genotypic lineages that serve as precursors to broadly neutralize antibodies.

High parameter flow cytometers (BD Symphony) are established at the VRC and the FHCRC. New panels developed for these instruments are available for use by the CAVD through the CCVIMC with ongoing expanded-panel development underway to offer the capability to interrogate a wide array of immune responses.

Our integrative systems biology approach combines, plasma cytokine/chemokines levels with RNA-Seq analysis, FCM phenotypic and other functional measurements to identify correlates of protection (including pre-vaccination markers of vaccine conferred immunogenicity and protection).

The CCVIMC provides the analysis of tissue immunodynamics by advanced confocal microscopy and quantitative image analysis. We engage in the development, optimization and application of cutting-edge imaging assays that can be applied as part of a comprehensive analysis of tissues of interest for further understanding of T and B cell dynamics in secondary lymphoid organs in natural infections (HIV/SIV) and after vaccination. This type of analysis provides critical information for the cellular and molecular mechanisms underlying i) the vaccine or pathogen-induced B cell responses and ii) the formation of “persistent viral reservoirs” at the tissue level.

We work in concert with our VISC and CAVIMC collaborators, convening joint VIMC Scientific Advisory Board annual meetings to showcase our collaborative efforts, enabling combined assessments of cellular and antibody responses observed in CAVD sponsored studies.

(Cont.)

Kublin: HIV Vaccine Trials Site Development for the P5 Program

PrincipalInvestigatorJamesKublin, MD, MPH


Project TitleHIV Vaccine Trials Site Development for the P5 Program

OPPID1088039

Grant AwardUp to $7.1 Million, awarded in November 2013

Grant at a GlanceOVERVIEWThe Pox-Protein Public-Private Partnership (P5) was established in 2010 to build on results from the RV144 trials. The partnership seeks to advance and ultimately license HIV pox-protein vaccine candidates that have the potential to achieve a broad public health impact. The P5 assembled a collaborative team across four continents, including representation from academia, private industry, government agencies, the HIV Vaccine Trials Network (HVTN), and the Bill and Melinda Gates Foundation. The goal of this project is to expand the HVTN’s clinical trials capacity in Africa to 18 active sites, through site identifi cation, selection, development, and training. Specifi cally, the need existed to increase capacity to conduct HIV clinical trials in humans in the clade C region of southern Africa for the P5 program. The outcome of this investment is 11 new active sites to support three early phase and one effi cacy study conducted concurrently. These sites will serve an estimated 5,996 participants. The specifi c studies/trials are as follows: 1) the Phase 1 HVTN 100 (n=240), which led to the Phase 3 development trial HVTN 702 (n=5,400); and 2) three phase 1 trials: HVTN 108 (n=132 at African sites), HVTN 111 (n=132), HVTN 120 (n=92 at African sites).

Site development to prepare for the capacity required to conduct the P5 program occurred in stages. The fi rst round of site development prepared 4 new sites to conduct the smaller early phase trials. The second round prepared 7 additional sites. The clinical trials network in the clade C region for the P5 program consists of 18 sites: 3 sites active at the start of this award in the conduct of HVTN trials, 2 sites operated and funded by the U.S. Military HIV Research Program, 2 sites developed with funding from the US NIH, National institute of Allergy and Infectious Diseases (NIAID), and 11 sites developed as a result of this request.

Development and training activities at each site included equipment acquisition; renovations for clinics, pharmacies, and laboratories; development of site management documentation (e.g. standard operating procedures and clinical quality management plans); and development of a small community engagement program. The HVTN has a structured process for site preparation, including standardized assessment checklists. These checklists were uniquely tailored to clinic facilities, site management, participant management, data management, community education, community advising, pharmacies, and laboratories. The pre-activation training period focused on capacity building: local institution training, Good Clinical Practices, human subject protection, HVTN core competencies, and protocol-specifi c guidelines. Laboratory training included specimen processing and Good Clinical Laboratory Practice guidance. Clinical site training incorporated skill-building for talking about HIV research with regulatory bodies, the media, and the community. Workshops ensured that site personnel (at all levels) were trained in the standards necessary to conduct HIV vaccine trials. These trainings were developed into online tools on clinical and non-clinical topics relevant to protocol implementation. The curriculum also included such topics as pregnancy prevention counseling, mucosal sample collection, and data management. These tools can be used for sites to orient and train new staff as turnover occurs.

RESEARCH OBJECTIVES

1.) Completion of structured site preparation visits for selected sites.

2.) Completion of action items identifi ed during the site preparation visits (unique to each site).

3.) NIAID approval to conduct HVTN trials at all newly-developed sites.

4.) Completion of clinic, pharmacy, and laboratory training by all site staff prior to the opening of each site’s fi rst trial.

Kublin: HVTN 100



Project TitleHVTN 100 A phase 1-2 randomized, double-blind, placebo-controlled clinical trial of clade C ALVAC-HIV® (vCP2438) and Bivalent Subtype C gp120/MF59® in HIV-uninfected adults at low risk of HIV infection

OPPID1110792


Collaborating Institutions◊ Hutchinson Center Research In-

stitute of South Africa, Cape Town, South Africa

◊ Centre Hospitalier de l’Universite de Montreal, Montreal, Canada

Grant at a Glance

Cancer Research Center, Cancer Research Center,

OVERVIEWThe need for a preventive HIV-1 vaccine is acute, especially in southern Africa, the world region most heavily burdened by the HIV epidemic. This grant provides funding for initial testing in South Africa of a vaccine regimen based on the regimen shown to be modestly efficacious in preventing HIV infection by the RV144 clinical trial in Thailand. Under the aegis of the Pox-Protein Public Private Partnership (P5), a vaccine regimen has been developed that is similar to the regimen in the RV144 trial, but adapted to the clade C strains of HIV circulating in southern Africa and modified (in vaccination schedule and certain product components) to enhance the magnitude and durability of the immune responses elicited by this regimen. This investment will support the implementation of HVTN 100, a phase 1-2 randomized, double-blind, placebo-controlled clinical trial of clade C ALVAC-HIV (vCP2438) and Bivalent Subtype C gp120/MF59® in HIV-uninfected adults in South Africa, who are at low risk of HIV infection. Results from this trial will determine whether the ALVAC-HIV (vCP2438) / Bivalent Subtype C gp120/MF59® prime-boost vaccine regimen qualifies for advancement to phase 3 efficacy testing in the Republic of South Africa. This determination will be based on evaluation of vaccine safety/tolerability and peak immunogenicity following the primary immunization schedule (i.e., Month 6.5). Rates and magnitudes of vaccine-generated immune responses will be evaluated against preset criteria, designed to ensure immunogenicity sufficient to support testing the hypothesis that the presence of binding antibodies to the V1V2 region of HIV Env is a vaccine correlate of reduced risk of HIV-1 infection.

Based on interim safety and immunogenicity data for the primary vaccination series (i.e. Months 0, 1, 3, and 6), a decision has been made to advance the HVTN 100 vaccine regimen to a pivotal efficacy trial in South Africa (HVTN 702). In this context, understanding the durability of vaccine-elicited immune responses and the extent to which those responses can be maintained or enhanced by subsequent booster vaccinations has assumed great importance. Part B of HVTN 100 seeks to provide this understanding by characterizing the immune responses elicited by different booster vaccinations at Month 24 and also to explore differences between boosting at 1 year and 2 year intervals.

Participants in Part B will receive vaccinations at Month 24 with ALVAC-HIV (vCP2438) plus Bivalent Subtype C gp120/MF59, with Bivalent Subtype C gp120/MF59 alone, or placebo. Participants in the three subgroups will also receive booster vaccinations at Month 36. Participants in subgroups that received Month 24 booster vaccinations with ALVAC-HIV (vCP2438) plus Bivalent Subtype C gp120/MF59 or with Bivalent Subtype C gp120/MF59 alone will receive the same booster vaccinations at Month 36. Part A vaccinees who received placebo injections at Month 24 will receive boosts of ALVAC-HIV (vCP2438) plus Bivalent Subtype C gp120/MF59 at Month 36.

Part B of HVTN 100 thus provides an opportunity to characterize the immune responses elicited by booster vaccinations at Months 24 and/or 36 and, importantly, in the event that vaccine-induced immune correlates of risk or protection are identified in the planned HVTN 702 trial, samples from Part B of HVTN 100 can be evaluated to determine whether booster vaccinations can maintain or even enhance those correlates. This information may inform investigation of boost strategies should the vaccine regimen be advanced toward an application for marketing authorization.

The program also includes development of next-generation CD4+ T cell laboratory evaluations that will be conducted through collaboration with Dr. Daniel Kaufmann at McGill University using microfluidic qRT-PCR arrays (90 gene panels, Fluidigm platform) on sorted HIV-specific CD4+ T cells expressing CD40L and through evaluation of single-cell expression of 40 immunological markers using the CyTOF technology. These data sets will be analyzed and compared with the validated ICS and cytokine bead array HVTN endpoint assays from the same participants to extend the functional profile of CD4+ T cell responses and to determine if the exploratory studies provide additional signatures that may merit analysis in the future phase 3 trial.

This grant is a collaborative effort led by James Kublin, MD, MPH (HIV Vaccine Trials Network (HVTN), Fred Hutchinson Cancer Research Center (FHCRC)), with the participation of M. Juliana McElrath, PhD (FHCRC, HVTN, and the Hutchinson Center Research Institute of South Africa (HCRISA)), and of Daniel Kaufmann, MD (Centre Hospitalier de l’Universite de Montreal). The CAVD award was received in November 2014, with an initial agreement length of 2.4 years; HVTN 100 derives additional support from NIAID.

RESEARCH OBJECTIVES1.) To evaluate the safety and tolerability of 2 doses of ALVAC-HIV (vCP2438) followed by 2 doses of ALVAC-HIV (vCP2438) + Bivalent Subtype C

gp120/MF59® in HIV-seronegative low risk South African adults

2.) To evaluate the immunogenicity of ALVAC-HIV® (vCP2438) followed by 2 doses of ALVAC-HIV (vCP2438) + Bivalent Subtype C gp120/MF59® in HIV-seronegative South African adults at the month 6.5 time point (2 weeks after completion of the primary immunization series)

Additional Objectives for Part B include:

3.) To evaluate the safety and tolerability of Bivalent Subtype C gp120/MF59 and of ALVAC-HIV (vCP2438) + Bivalent Subtype C gp120/MF59 when given as boosts in participants previously vaccinated in Part A

4.) To evaluate the immunogenicity of Bivalent Subtype C gp120/MF59 and of ALVAC-HIV (vCP2438) + Bivalent Subtype C gp120/MF59 when given as boosts at Month 30 in participants previously vaccinated in Part A at 2 weeks following each vaccination

Kublin: HVTN 705: A phase 2b clinical trial to evaluate the effi cacy of Ad26 recombinant HIV vaccine + gp140



Project TitleHVTN 705: A phase 2b clinical trial to evaluate the effi cacy of Ad26 recombinant HIV vaccine + gp140

OPPID1165951

Grant AwardUp to $34.8 Million, awarded in May, 2017

Collaborating Institutions◊ Janssen Vaccines and Prevention

B.V., Leiden, NL◊ National Institute of Allergy and

Infectious Diseases, Bethesda, US◊ Hutchinson Center Research In-

stitute of South Africa, Cape Town, South Africa

◊ Affi liated trial sites in South Africa, Malawi, Mozambique, Zambia, and Zimbabwe

Grant at a GlanceOVERVIEWThis grant, under the leadership of Larry Corey, Glenda Gray, and Jim Kublin, and awarded to the Fred Hutchinson Cancer Research Center, will support the HVTN 705 trial (also referred to as HPX2008). The trial is entitled “Multicenter, Randomized, Double-blind, Placebo-controlled Phase 2b Effi cacy Study of a Heterologous Prime/Boost Vaccine Regimen of Ad26.Mos4.HIV and Aluminum Phosphate-adjuvanted Clade C gp140 in Preventing HIV-1 Infection in Adult Women”.

The vaccine regimen is comprised of 2 administrations of tetravalent Ad26 vaccine (4 recombinant Ad26 vectors expressing mosaic inserts of HIV gag-pol or env genes) at months 0 and 3, followed by 2 admin-istrations of the same tetravalent rAd26 vaccine with soluble trimeric Clade C gp140 formulated in alum at months 6, and 12. The potential effi cacy of this approach has been demonstrated through rather stringent non-human primate (NHP) studies, where the per exposure risk against a mucosal SHIV challenge was reduced by 94%. Earlier phase 1-2 trials showed the vaccine regimen to be well-tolerated, with a very good response rate among vaccine recipients. Importantly, initial vaccine immunogenicity data in humans showed humoral and cellular responses that recapitulate those observed as correlates of protection in the NHP model.

As described at clinicaltrials.gov , the primary purpose of the phase 2b study is to assess the preventive vaccine effi cacy (VE), safety and tolerability of the aforementioned heterologous prime/boost regimen for the prevention of HIV infection in HIV-seronegative women residing in sub-Saharan Africa. The estimated en-rollment is 2600 participants, equally divided between the vaccine recipients and a placebo comparator. The vaccine effi cacy primary endpoint will be gauged from confi rmed infections diagnosed between the Month 7 and Month 24 visits. The award commenced in May 2017, enrollment will be completed in 1-2Q 2019, and the study is anticipated to run into 4Q2022.

Jim Kublin is the executive director of the HVTN, at the Fred Hutchinson Cancer Research Center. Larry Co-rey (PI:HVTN, FHCRC) and Glenda Gray (Co-PI:HVTN, South African Medical Research Council) are also in leadership roles for the study. Janssen Vaccines & Prevention B.V. is the study sponsor, with co-funding by NIAID.

RESEARCH OBJECTIVES1.) To evaluate the vaccine effi cacy as derived from the confi rmed HIV-1 infections diagnosed between the

month 7 and month 24 visits. Secondary endpoints assess vaccine effi cacy over different intervals, or by various baseline and demographic characteristics.

2.) To determine the safety and tolerability of the vaccine regimen, based on percentages of participants experiencing reactogenicity signs or symptoms, or participants with adverse events.Secondary study endpoints include measuring the immunogenicity of the vaccine regimen, assessed by vaccine-induced antibody and T cell responses.

Lewis: Ab Specifi city & Fc-Mediated Protection

PrincipalInvestigatorGeorge K. Lewis, PhD

GranteeInstitutionInstitute of Human Virology, University of Maryland, Baltimore, USA

Project TitleAntibody Specifi city, Fc-Mediated Effector Function, and HIV-1 Vaccines

OPPID1033109

Grant AwardUp to $7 Million, awarded in October, 2011

Collaborating Institutions◊ Advanced Biosciences Laboratory

Grant at a GlanceOVERVIEWIt is well accepted that direct virus neutralization, in which the binding of the variable domains of antibodies to epitopes on the envelope glycoprotein prevent viral entry into target cells, is an important element of antibody-mediated protection against HIV-1. Less is known about the role of Fc-mediated effector functions, i.e. those functions that depend on the nature of the constant regions of antibodies, in the control of HIV-1. Epidemiological studies of HIV infected individuals have shown an inverse relationship between disease progression and antibody-dependent cell mediated cytotoxicity (ADCC), which is supported by a correlation between Fc-receptor genotype and reduced risk of progression. More recently, results from the Vax004 vaccine effi cacy trial and the RV144 vaccine trial in Thailand provide further evidence that vaccine-elicited non-neutralizing antibodies might play a role in protection from infection.

Dr. George Lewis and colleagues at the University of Maryland are pursuing the hypothesis that anti-en-velope antibodies directed against conserved domains, exposed upon virus entry/attachment, are capable of providing protective humoral immunity through Fc-dependent effector functions, even in the absence of a direct neutralizing function. In this scenario, diverse effector mechanisms need not be mutually exclusive, might be complimentary, and might apply differentially according to the mode of exposure.

RESEARCH OBJECTIVESResolution of the role of Fc-mediated effector function, particularly antibody-dependent cellular cytotoxicity (ADCC), is being pursued through fi ve objectives. Objective 1 is to streamline and standardize an ADCC assay such that it can be used for high throughput use. Objective 2 is to isolate and characterize new monoclonal antibodies (mAbs) based on specifi city, neutralization, and Fc-mediated effector function. Objective 3 deter-mines epitope exposure during viral entry. Objective 4 maps epitopes recognized by new mAbs using X-ray crystallography. Objective 5- synthesizes information from the preceding objectives to design passive immunization studies against SHIV in animal model to resolve the relationships between neutralization and Fc-mediated effector function in protective immunity against HIV-1.

PROGRESSTheir team has characterized a large panel of mAbs for neutralization breadth and potency as well as Fc-mediated effector functions, in particular ADCC. The epitopes recognized by these antibodies have been characterized by mutagenesis and X-ray crystallography. Further, their exposure during viral entry or virus budding has been determined using single particle imaging. Two signifi cant observations have emerged. First, a broadly neutralizing antibody has isolated that recognizes a new glycan shield epitope involving elements of the V1/V2 and V3 regions as well as a glycan at residue 301, which distal to the glycan at 332 recognized by other glycan shield monoclonal antibodies. Further, this mAb employs a moder-ately mutated heavy chain variable region and a near germline light chain variable region. This mAb protects rhesus macaques completely from a high-dose challenge with SHIV. It also decreases viral loads in SHIV infected Rhesus Macaques. Based on these observations, we are now deter-mining the role of Fc-mediated effector function for protection in rhesus macaques.

Second, two highly conserved ADCC hotspots recognized by non-neutralizing mAbs have been mapped in gp120 and gp41. An epitope group in the gp120 hotspot has been implicated as a target for potentially protective antibodies in both HIV-1 infected people and in the RV144 vaccine trial. This epitope group has been characterized by both mutagenesis and X-ray crystallography, providing the fi rst picture of this site at atomic resolution, which was published recently in the Journal of Virology. This information is being used to design a new vaccine candidate as well as to determine the protective role of antibodies against this region via both active and passive immunization models in animals. In addition, this information is being used to evaluate immunologic pressure against this epitope region in HIV-1 infected people. Collectively, these two lines of investigation are leading to new knowledge about the mechanisms of antibody-mediated protection against HIV as well as new vaccine candidates and new antibodies for therapy.

Masopust: Vaccination for prevention of HIV

Principal InvestigatorDavid Masopust, PhD

GranteeInstitutionUniversity of Minnesota, Minneapolis, MN USA

Project TitleVaccination for prevention of HIV

OPPID1116224


Collaborating Institutions◊ Oregon Health and Sciences

University, Oregon National Primate Research Center, Beaverton, OR USA

Grant at a GlanceOVERVIEWThe proposed work will test in a NHP SIV vaginal challenge model whether a T cell vaccine could prevent HIV. The vaccination regimen is an alternative vaccination approach that elicits abundant tissue resident virus specifi c memory CD8 T cells in cervical vaginal tissues. It is a heterologous prime-boost-boost (HPBB) vaccine strategy, employing sequential immunizations with an identical antigen insert expressed by serologi-cally distinct virus vectors, thereby avoiding neutralization of the boosts by the response to the previous vector. Specifi cally, adult female rhesus macaques will be immunized with recombinant vesicular stomatitis virus (New Jersey serotype, VSV-NJ) and vaccinia virus (VV) vectors that expressed the full gag coding sequence obtained from pCMVgagDX, a high level protein expression construct engineered by codon optimization and inactivation/removal of instability sequences for Rev-independent expression, and human adenovirus serotype 5 also expressing full length Gag.

Preliminary data in Mamu-A*001+ rhesus macaques indicates that this regimen establishes an unprece-dented magnitude of Gag-specifi c memory CD8 T cells (2-12% of CD8+ PBMC remained CM9-specifi c 16 weeks after the fi nal immunization with the RhAd5 construct. Gag-specifi c memory CD8 T cells were highly polyfunctional with potent degranulation behavior and were broadly distributed, present in all 29 anatomic compartments sampled. Flow cytometric analyses of Gag-specifi c memory CD8 T cells in cervix and vagina at 294 days post vaccination showed the induced mucosal memory cells were actually more abundant than the primary effector response during acute infection of unvaccinated animals. The initial Mamu-A*001+ cohort then received a single dose vaginal challenge with SIVmac251, using either 2000 or 8000 TCID50. While all 6 naïve controls were viremic within 7 days after challenge, 5 out of 8 vaccinated animals did not exhibit evidence of systemic infection measured by plasma viremia out to 100 days.

The major goal of this grant is to perform a repeat test of the immunization, measurements of immunogenicity, and a more highly powered assessment of protection in a repeat vaginal low dose SIVmac239 challenge model. A secondary objective for the study is to distinguish if protection occurs by eliminating a detactable transmission, vs. preventing detectable transmission.

Demonstration of this principle could accelerate our prospects for developing a human HIV vaccine by indicating the effi cacy of a new class of CD8 T cell vaccines that would complement approaches that attempt to elicit neutralizing antibody.

The grant is led by David Masopust, from the Department of Microbiology and Immunology at the University of Minnesota. The nonhuman primate model will be conducted at the Oregon Nation Primate Research Center, affi liated with the Oregon Health and Sciences University. The award was made in November 2015, with an anticipated duration of 24 months.

McElrath: Durability of HIV-specific protective Ab responses in human immunology-based experimental medicine trials

PrincipalInvestigatorM. JulianaMcElrath, PhD


Project TitleDurability of HIV-specifi c protective Ab responses in human immunology-based experimental medicine trials

OPPID1107954


Collaborating Institutions◊ Infectious Disease Research

Institute, USA◊ The Scripps Research Institute,

USA◊ Weill Cornell Medical College, USA◊ International AIDS Vaccine

Initiative, USA◊ National Institute of Allergy and

Infectious Diseases, NIH, USA◊ Dynavax Technologies, USA◊ 3M, USA

Grant at a Glance

Cancer Research Center, Cancer Research Center,

OVERVIEWDevelopment of an HIV vaccine that can elicit long-lived protective immunity against new HIV infections offers the best promise to end the AIDS epidemic. The low-level (31%) efficacy observed in the RV144 Thai trial suggests that a preventive HIV vaccine may be possible and that anti-Env antibodies, including those lacking classical neutralizing activities, may play some role in human protection. To advance the field and improve efficacy, new HIV vaccine designs are needed, and particularly those that retain native trimer structures; express conserved, cross-reactive epitopes; and that induce potent, durable anti-viral effector antibodies that can access and bind these epitopes.

This grant will employ experimental phase 1 human studies led by Julie McElrath (Fred Hutchinson Cancer Research Center [FHCRC]) with in-depth immunologic analyses in blood and draining lymph nodes to determine if the novel HIV-1 Env native trimer BG505 SOSIP.664 glycoprotein with adjuvant formulations can induce greater antibody potency and durability than seen with alum-formulated envelope proteins more commonly used in clinical studies. The overall experimental design will determine if one or more Env/adjuvant formulation(s) can induce anti-Env antibodies capable of autologous and/or broadly reactive tier 2 neutralizing antibodies, and can heighten the peak and extend the duration of antibody (Ab) responses at year one and thereafter. The planned adjuvants are 3M-052+Alum, GLA-LSQ, CpG 1018+Alum, and Alum, based upon their ability to trigger diverse innate pathways, the feasibility of obtaining and formulating stable, clinical grade material in a timely way, and the agreement among adjuvant developers to provide and compare products in head-to-head studies.

This grant is a collaborative effort led by M. Juliana McElrath, MD, PhD (FHCRC), John Moore, PhD (Cornell Weill Medical College), Darrick Carter, PhD and Chris Fox, PhD (Infectious Disease Research Institute), Michael Pensiero, PhD (NIH NIAID, DAIDS), and Mingchao Shen, PhD (FHCRC). It includes collaborative interactions with Andrew Ward, PhD (The Scripps Research Institute), Robert Coffman, PhD (Dynavax), and Mark Tomai, PhD (3M). It will also leverage CAVD Central Services Facilities, including Vaccine Product Development Center led by Tom Hassell, PhD (IAVI), and Comprehensive Antibody and Cellular Vaccine Immune Monitoring Cores. The award was received in November, 2014 with an initial agreement length of 5 years.

RESEARCH OBJECTIVESAssess the immunogenicity of an HIV-1 subunit vaccine when formulated with several novel adjuvants, with alum as an adjuvant comparator. Initial target responses include:

1.) 5-10 fold greater peak binding Ab titers to the homologous Env antigen and significantly greater peak response rates after 3 doses.

2.) 5-10 fold greater binding Ab titers to the homologous Env antigen and significantly greater response rates one year after the last immunization.

3.) Neutralization of autologous and a panel of Tier 2 virus strains.

4.) 2-fold greater frequency of circulating Env-specific CD4+ T cells with significantly greater polyfunctionality at the peak response.

5.) Significantly improved qualitative Ab responses, such as avidity, anti-viral effector function, and/or somatic hypermutation/affinity maturation.

6.) Distinct innate immune profi les for each adjuvant.

PROGRESSWe have conducted formulation compatibility studies and evaluated the effects of adjuvants on BG505 SOSIP.664 trimer structures by electron microscopy, BLI, ELISA, and other assays. The native trimer structures were retained in most cases, but were affected by some adjuvants in dose dependent and time dependent manners.

We are conducting a guinea pig study to assess the immunogenicity of BG505 SOSIP.664 with various adjuvants. Autologous tier 2 neutralizing antibodies were induced in most of the study groups. We will measure the durability of neutralizing and binding antibody responses. We are also conducting a rabbit study to assess the immunogenicity of BG505 SOSIP.664 with various adjuvants.

We conducted a pilot rat study of BG505 SOSIP.664 with adjuvants and showed that the vaccines are immunogenic in rats. We have started a GLP toxicity study in rats to demonstrate the safety and tolerability of BG505 SOSIP.664 with these adjuvants. Upon completion of the toxicity study, we will prepare to start a clinical trial in collaboration with HVTN.

McMichael: HLA-E restricted CD8 responses induced by CMV vaccines

PrincipalInvestigatorAndrewMcMichael, MD

GranteeInstitutionUniversity of Oxford, Oxford, UK

Project TitleHLA-E restricted CD8 responses induced by CMV vaccines

OPPID11133649

Grant AwardUp to $556,000, awarded in July, 2014

Grant at a GlanceOVERVIEWDespite very signifi cant but partial success of drug based approaches and public health measures, there are still over 2 million new HIV-1 infections per year. Therefore an effective prophylactic vaccine is very badly needed. Attempts have also been made to design vaccines that stimulate HIV-1 specifi c CD8+ T cells, but no protection has been seen in three trials, STEP, Phambili, and HVTN505, using adenovirus-5 vectors to deliver antigen. The level and breadth of CD8+ T cell responses stimulated were not high and, in each case, much of the response was focused on variable epitopes in Env, Gag, and Pol. Recent experiments from Louis Picker’s group have raised the possibility that the right kind of vaccine-induced CD8+ T cells could protect. In rhesus monkeys he used a Rhesus CMV vector RhCMV68-1 to deliver SIV genes as a vaccine. When these animals were challenged mucosally a year after vaccination, 53% of those infected were able to clear virus completely. The virus was eradicated by CD8+ T cells; however the T cell responses are very atypical with the CD8+ T cell responses against multiple epitopes that are even in magnitude and are restricted by either MHC-II or MHC-E. These results are strikingly different from all previous monkey-SIV protection experiments even in the presence of strong and broad classical CD8+ T cell responses. Therefore it seems likely that these unique T cell responses are important to the clearance of virus by CD8+ T cells.

These results from the Picker group raise the following important questions:

1.) Can these T cell responses be elicited in humans?

2.) What atypical antigen processing pathways are important for priming these T cells and why are the T cell responses so broad?

3.) Have they played a part in elite control and could they account for apparent lack of infection in some very highly HIV-exposed individuals?

4.) Are similar responses elicited by immunogens other than RhCMV68.1?

Our laboratory has extensive experience in characterizing the biological functions of HLA-E, for example demonstrating that the normal function of HLA-E is to present an HLA-Ia signal peptide to the NKG2A/CD94 and NKG2C/CD94 receptors on natural killer (NK) cells. We also determined the fi rst structure of HLA-E bound to the signal peptide VL9 (15) and showed that the identical peptide sequence in the signal peptide of HCMV UL40 subverted NK cell recognition of HCMV infected cells. We are therefore well placed to explore the role of HLA-E in HIV-1 infection. The studies proposed here will complement the vaccine program that is ongoing in Louis Picker’s laboratory.

This grant is led by Andrew McMichael, MD (University of Oxford), in a collaborative association Louis Picker, MD (Oregon Health and Sciences University). The award was received in July, 2015 with an initial agreement length of 3 years.

RESEARCH OBJECTIVES1.) Identifi cation of HLA-E binding peptides in HIV-1.

2.) Identifi cation of peptides presented with HLA-E in HIV-1 infected cells.

3.) Analysis of whether the two allotypes of HLA-E differ in their peptide presentation and level of expression.

4.) Identifi cation of HLA-E restricted CD8+ T cells in HIV seronegative and infected donors and vaccine recipients. This will span the following subject types:

a.) Naïve CD8+ T cells in healthy HIV-1 negative blood donors

b.) HIV-infected individuals

c.) Donors who have been highly exposed to HIV-1 but have not become chronically infected

d.) Volunteers who have received HIV-1 vaccines including BCG based vaccines

5.) Analysis of the phenotype of HLA-E restricted CD8+ T cells.

6.) Analysis of how these fi ndings relate to Rhesus monkeys.

PROGRESS1.) We have developed HLA-E-peptide binding and expression assays that have led to identifi cation of 3 HIV derived binding peptides.

2.) We have eluted HIV-1 peptides from HLA-E on PBMC and cell lines and identifi ed putative epitopes.

3.) HLA-E*0103 is expressed at higher levels than HLA-E*0103 and appears to bind more peptides.

4.) We have established a CD8 T cell repertoire assay that detects primary CD8 T cell responses in HIV-negative donors, including responses to (non-HIV) epitopes restricted by HLA-E.

5.) We have determined the crystal structure of HLA-E bound to a non-canonical Mycobacterial epitope peptide.

6.) We have shown that HCMV UL40 and RhCMV Rh67 serve similar functions in expressing MHC-E on CMV infected cells when TAP is blocked..

Montefi ori: Antibody Vaccine Immune Monitoring Consortium

PrincipalInvestigatorDavid Montefi ori, PhD

GranteeInstitutionDuke University, Durham, USA

Project TitleComprehensive Antibody Vaccine Immune Monitoring Consortium

OPPID1146996

Grant AwardUp to $33.8 Million, awarded June, 2006Up to $32.4 Million, awarded June 2011Up to $32.6 million , awarded July 2016


Center, USA◊ Fraunhofer – Institut fur

Biomedizinische Technik, Germany◊ Massachusetts General Hospital,

USA◊ National Institute for

Communicable Diseases, South Africa

◊ New Mexico Consortium, USA◊ Thayer School of Engineering at

Dartmouth◊ University of Alabama at

Birmingham, USA◊ University of Cape Town, South

Africa

External Scientifi c Advisory Board◊ James Hoxie, University of

Pennsylvania (Chair)◊ Patricia D’Souza, National

Institutes of Allergy and Infectious Diseases

◊ Shiu-Lok Hu, University of Washington

◊ Quentin Sattentau, The Sir William Dunn School of Pathology

Grant at a GlanceOVERVIEWThe Comprehensive Antibody Vaccine Immune Monitoring Consortium (CAVIMC) utilizes valid laboratory criteria and Good Clinical Laboratory Practices (GCLP) to monitor systemic and mucosal antibodies in preclinical and clinical testing of candidate HIV vaccines and passively delivered antibodies. We also seek to identify correlates of protective immunity and to generate new scientific findings that will help bridge the gap between preclinical research and human clinical trials. These combined efforts aim to facilitate the discovery and licensure of a safe, effective and practical HIV vaccine, or long-acting pharmacologic intervention, to reduce the acquisition of HIV infection.

RESEARCH OBJECTIVES1.) Obtain increased knowledge of potential immune correlates from active immunization strategies to guide

the design and identification of an effective HIV vaccine.

2.) Obtain increased knowledge of potential immune correlates from passive immunization strategies to guide the design and identification of effective long-acting pharmacologic interventions to reduce the acquisition of HIV infection.

3.) Obtain enhanced understanding of what antibody activities to measure and how best to measure them to obtain valid results through development and improvement in reagents and assay protocols.

4.) Obtain new scientific findings to help bridge the gap between preclinical vaccine discovery and human clinical trials.

5.) Assure best in class assay services and reagents in order to satisfy external regulatory agencies.

6.) Facilitate collaboration and integration of the consortium within the CAVD, ensure effective and efficient operation of consortium and provision of CSF services, and the ability to quickly adapt to the changing needs of the CAVD.

PROGRESS• The CAVIMC offers best in class assays for assessing neutralization, antigen binding, ADCC, phagocy-

tosis, infecting cell binding, FcR binding and IgG glycan profi les in preclinical and clinical studies as part of their Central Service Facility. Antibodies are assessed in terms of their magnitude, breadth, kinetics, durability, epitope specifi cities, isotype/subclass and affi nity/avidity.

• The CAVIMC provides customized, quantitative binding and neutralization measures of serum bnAb concentrations for pharmacokinetic (PK) assessments of passively transferred bnAbs. The CAVIMC also provides measurements of anti-drug antibodies (ADA) in preclinical and clinical studies of passive bnAbs.

• The CAVIMC provides critical assay support for bnAb optimization for manufacturability, GMP product stability and extended plasma half-life of engineered bnAbs for clinical development.

• The CAVIMC tailors new qualifi ed assays to meet the evolving needs of the CAVD.

• The CAVIMC maintains a strong Central Quality Assurance Unit that oversees all quality aspects of Good Clinical Laboratory Practice (GCLP) compliant CAVIMC Cores and enables them to provide best in class assay services and reagents.

• The CAVIMC has developed novel reagents and approaches to enable monitoring of the earliest stages of bnAb induction in preclinical and clinical studies.

• The CAVIMC provides detailed characterization of neutralizing antibody responses induced by a wide range of engineered envelope immunogens, including different SOSIP confi gurations and adjuvants

• The CAVIMC continues to develop new computational methods and conduct analyses. The CAVIMC acquired a deeper understanding of which bnAbs and their combinations are most favorable for clinical development based on an expanded analysis of recent bnAbs, bispecifi c bnAbs and a wider range of viral subtypes.

• The CAVIMC evaluates and identifi es non-neutralizing antibody correlates of protection against SHIV challenge in nonhuman primates.

• The CAVIMC maintains a strong program in molecular virology needed to overcome the genetic variability of HIV-1 as a barrier to vaccine discovery.

• The CAVIMC developed a novel assay approach to generate env quasispecies viruses of infectious molecular clones (Env.QS-IMC) from HIV-in-fected subjects for rapidly testing their neutralization sensitivity to bnAbs being passively infused and virus escape studies.

Comprehensive Antibody Vaccine Comprehensive Antibody Vaccine

Moore: Next generation BG505 SOSIP.664-trimer based lineage HIV vaccines

PrincipalInvestigatorJohn Moore, PhD

GranteeInstitutionWeill Cornell Medical College, New York, USA

Project TitleNext generation BG505 SOSIP.664-trimer based lineage HIV vaccines

OPPID1132237

Grant AwardUp to $8,723,473, awarded July, 2015

Collaborating Institutions◊ Academic Medical Center –

University of Amsterdam, Amsterdam, The Netherlands

◊ Kymab Ltd, Cambridge, UK (funded under separate contract)

Grant at a GlanceOVERVIEWThe central goal of the application is to facilitate the development of trimer-based immunogens that are being designed and developed to induce broadly neutralizing antibodies (bNAbs). Recent results from passive antibody transfer studies indicate that virus neutralization assays yield a reasonable measure of a protective response. Our goal, then, is to generate appropriate titers of serum antibodies in vivo that are capable of neutralizing relatively resistant (Tier 2 and 3) viruses in vitro. We believe that the native pre-fusion conformation of the Env trimer is a suitable basis for a vaccine intended to induce bNAbs, particularly now that we and other groups have generated high-resolution structural data to guide the design of trimer immunogens. Native trimers are unique in presenting almost all bNAb epitopes, and do so in an appropriate quaternary context.

We will at present restrict our program to the SOSIP configuration of recombinant trimers that we developed and are highly familiar with, and that we and others have characterized extensively to demonstrate their excellent mimicry of native Env. We are now able to make multiple native-like SOSIP.664 trimers from clades A, B and C, as well as A/C hybrids, by combining protein-engineering techniques with bNAb-purification columns that allow isolation of native structures. We are also using structure-guided design techniques to create improvements to existing trimers, as well as to generate new ones. For example, our overall understanding of structure-function relationships within the trimer has advanced such that we are capable of making native trimers from most env sequences. Thus, we are acquiring the ability to design a large number of hypothesis-based immunization experiments involving multiple variants of native-like SOSIP trimers.

To advance the aforementioned immunization experiments, we have made four different GMP-grade CHO cell lines that express SOSIP.664 trimers of various designs, using a GMP facility available at the Weill Cornell Medical College (WCMC). The lines express next-generation variants of SOSIP.664 trimers that are designed to increase the probability of inducing bNAbs when used as immunogens. Cell lines can be appropriately banked for future GMP production, as well as used for non-GMP, pre-clinical studies of the produced trimers at WCMC and/or the Academic Medical Center (AMC). We will also conduct immunogenicity studies in rabbits and macaques, the goal being to assess which trimers are best suited to the goals of inducing bNAbs by Env vaccination. Those studies will include experiments with the germline targeting GT1.1 trimer developed at the Academic Medical Center (AMC), Amsterdam. This trimer is now in the GMP development program, after the appropriate CHO cell line was made at WCM in this project.

This grant is a collaborative effort led by John Moore, PhD (Weill Cornell Medical College) with the participation of Rogier Sanders (University of Amsterdam) Bali Pulendran (Stanford University) and Francois Villinger (University of Louisiana). The award was received in July, 2015 with an initial agreement length of 2 years.

RESEARCH OBJECTIVES1.) Develop GMP-grade CHO cell lines expressing SOSIP.664 trimers, down-selected to a choice of four cell lines.

2.) Implement rabbit and macaque studies of next-generation SOSIP.664 trimers.

Nussenzweig: Clinical Trial of Broadly Neutralizing anti-HIV Antibodies

PrincipalInvestigatorMichel C. Nussenzweig, MD,PhD

GranteeInstitutionRockefeller University, New York, USA

Project TitleClinical Trial of Broadly Neutralizing anti-HIV Antibodies

OPPID1092074

Grant AwardUp to $19.1 million, awarded October, 2013

Collaborating Institutions◊ Brigham and Women’s Hospital,

USA◊ Beth Israel Deaconess Medical

Center, USA◊ Weill Cornell Medical Center, USA◊ University of Cologne, Germany◊ Military HIV Research Program,

USA◊ National Cancer Institute, USA◊ Celldex Therapeutics, USA ◊ Vaccine Research Center, USA

Grant at a GlanceOVERVIEWRecent evidence lends support to the idea that broadly neutralizing antibodies might play an important role in vaccine design because they can protect macaques from infection, and because anti-HIV antibodies were the only significant, positive correlate of protection in the recent RV144 trial.

Despite their potential importance to vaccine design and development, little was known about the molecular composition of the human anti-HIV antibody response until single cell antibody cloning techniques were de-veloped to characterize IgGs from the sera of HIV-infected individuals with broadly neutralizing activity. Initial studies revealed that neutralization breadth can be accounted for by combinations of antibodies and by a few highly potent antibody clones that target different viral envelope epitopes, such as the CD4 binding site (3BNC117) and the base of the V3 loop (10-1074).

When tested in in vitro TZM-bl assays, both 3BNC117 and 10-1074 showed neutralizing activity against > 90% of all HIV-1 isolates tested, and combinations of antibodies could achieve greater than 99% coverage of all strains at IC80 concentrations below 50 ng/ml. In vivo neutralizing activity at low antibody concentrations has been demonstrated in humanized mice, using HIV-1, and in rhesus macaques, using 2 different tier 2 SHIVs. 3BNC117 and 10-1074 are 10-50X better at protecting against SHIV infection than VRC01, the most advanced clinical candidate to date. These results suggest that 3BNC117 and 10-1074, alone or in combination, might have utility in immune prophylaxis of HIV acquisition in humans. Further potential of these antibodies has re-cently been demonstrated for therapy of HIV infection. Passively administered combinations of potent human monoclonal antibodies controlled established infection in humanized mice for prolonged periods of time. More-over, during chronic infection in humanized mice, even a single monoclonal antibody could control infection when the viral load was initially suppressed by anti-retroviral therapy. Similar results have been obtained in ma-caques chronically infected with two different tier 2 SHIVs (SHIVAd8 and SHIV162P3). To date, 19 macaques infected for 1-3 years have been treated with 3BNC117 and or 10-1074. Viral loads ranged from 103-105, and in all but one case, viremia became undetectable within 7 days. Viremia remained controlled for an average of 4-6 weeks or as long as antibody levels were maintained, however 20% of the animals remained aviremic even after antibody levels became undetectable. The 1 animal that did not respond had high levels of endogenous broadly neutralizing antibodies before therapy and likely harbored resistant HIV-1 variants before treatment.

These experiments suggest that broadly neutralizing monoclonal antibodies may be powerful tools for both the prevention of HIV-1 acquisition and the treatment of established infection. The proposed clinical trials are designed to support both clinical indications for each antibody alone, or in combination.

The objectives of the proposed research are to evaluate the safety, tolerability, pharmacokinetics/pharma-codynamics and in vivo antiretroviral effects of two highly neutralizing anti-HIV monoclonal antibodies when administered to HIV-uninfected and HIV-infected individuals in three proof-of-concept phase 1 studies: (1) A dose-escalating single infusion study of 3BNC117 alone, (2) A dose-escalating single infusion study of 10-1074 alone, and (3) A single infusion study of the combination of 3BNC117 and 10-1074, at the dose level selected in (1) and (2). The long-term goal is to develop the antibodies for two potential clinical indications: prevention of HIV acquisition in healthy HIV-1-uninfected individuals at risk for HIV infection, and adjunct therapy of HIV-1-infected individuals.

RESEARCH OBJECTIVES1.) Conduct a phase 1 study with 3BNC117 mAb in three target populations (HIV-uninfected, HIV-infected who are viremic, and HIV-infected on

ART with suppressed viremia).

2.) Conduct a phase 1 study with 10-1074 mAb in three target populations, as in Objective 1.

3.) Conduct a phase 1 study with the combination of 3BNC117 and 10-1074 mAbs in three target populations, as in Objectives 1 and 2.

PROGRESS1.) The fi rst subject in the the Phase 1 study of 3BNC117 (objective 1) was enrolled in February of 2014. As of October of 2016 dose escalation

to 30mg/kg in both HIV-uninfected and HIV-infected subjects has been completed with 30 subjects enrolled. To date the antibody is safe and well tolerated. Safety data as well as virologic data continues was collected. A single injection of 3BNC117 reduced viral loads transiently. The antibody treatment not only blocked free virus from infecting new cells, it also accelerated the clearance of infected cells. We were able to demonstrate that therapeutic antibody treatment enhanced infected individuals’ humoral response against the virus. Thus, neutralizing antibodies may be a promising therapy as well as prophylaxis for HIV-1 because of their potential to reduce the viral reservoir.

2.) As of October 2016 the FIH clinical trial of 10-1074 (Objective 2) in HIV negative and positive participants is fully enrolled with 33 participants. Dosing is complete in all participants. Clinical activities are complete in all US sites with follow up continuing at the University of Cologne Hospital in Germany. To date the antibody is safe and well tolerated.

3.) As of October 2016, 6 participants are enrolled in the combination trial of 3BNC117 and 10-1074. The trial is continuing enrollment and dosing in the US and Germany.

Nussenzweig: Development of optimized broadly neutralizing antibodies for HIV-1 prevention

PrincipalInvestigatorMichelNussenzweig, MD, PhD

GranteeInstitutionThe Rockefeller University, New York, USA

Project TitleDevelopment of optimized broadly neutralizing antibodies for HIV-1 prevention

OPPID1124068

Grant AwardUp to $13 million, awarded in June, 2015

Collaborating Institutions◊ California Institute of Technology,

Pasadena, USA◊ National Institute of Allergy and

Infectious Diseases – Viral Patho-genesis and Vaccine Section, Bethesda, USA

Grant at a Glance

The Rockefeller University, The Rockefeller University,

OVERVIEWDespite development of excellent therapeutics and strategies for HIV-1 prevention, HIV/AIDS infection remains a major problem in both the developed and developing world. Vaccines do not exist and there are signifi cant barriers to their development. Although not considered practical until very recently, the discovery of highly potent broadly neutralizing antibodies has brought new impetus to the passive antibody approach to HIV-1 prevention. Importantly these antibodies have been shown to prevent infection in mice and macaque models and have shown signifi cant effi cacy against HIV-1 in a phase 1 clinical trial. A key objective of this proposal is to improve the activity of the naturally occurring anti-HIV-1 antibodies by altering the mechanism of antigen recognition and/or by enhancing their effector functions. The majority of the research activities run in parallel, as the main aim of the proposal is to continuously generate optimized anti-HIV-1 antibodies and evaluate their activity in in vivo infection models using humanized mice and NHP. These activities are expected to occur throughout the entire 4-year period and it is anticipated that every year new anti-HIV-1 antibodies will be isolated, optimized and eventually tested in vivo. By the end of the 4-year period, new classes of anti-HIV-1 antibodies with substantially improved neutralization activity and effector function will be available for pre-clinical evaluation in humans. The 4-year period for the grant refl ects the requirement that the majority of the described in vivo experiments, and particularly those involving NHP, require a minimum of least a year to complete.

This collaborative effort is led by Michel Nussenzweig, MD, PhD (The Rockefeller University) with Jeffery Ravetch, MD, PhD (The Rockefeller University) as Co-Principal Investigator, and the participation of Pamela Bjorkman, PhD (California Institute of Technology) and Malcolm Martin, MD (National Institute of Allergy and Infectious Diseases). The award was received in June, 2015 with an initial agreement length of 4 years.

RESEARCH OBJECTIVES1.) Optimized antibody based therapeutic interventions for HIV-1 prevention in pre-clinical models for trans-

lation to humans

1.1.) Produce humanized mice for the studies proposed by all investigators in the consortium

1.2.) Optimize antibody-based interventions for prevention in preclinical models

2.) Develop optimized anti-HIV-1 bnAbs with increased potency/breadth and resistance to viral mutation

2.1.) Improve conventional format human IgG antibodies using structure-based design

2.2.) Create intra-spike crosslinking reagents that utilize avidity effects to neutralize with synergy

2.3.) Produce most potent/most synergistic bispecifi c reagents for in vivo evaluation

3.) Generation and pre-clinical evaluation of Fc-optimized anti-HIV-1 antibodies with improved effector activity

3.1.) Assess in vivo activity of Fc optimized anti-HIV-1 bnAbs in humanized mouse models of HIV-1 infection

3.2.) Evaluate in vivo protective activity of Fc optimized bnAbs in non-human primates

4.) Blocking the establishment of virus infection with anti-HIV-1 bnAbs using the SHIV macaque model of HIV AIDS

4.1.) Assess in vivo potency and durability of optimized anti-HIV Nabs in pre-exposure prophylaxis experiments

4.2.) Suppress virus replication in SHIV infected macaques

PROGRESS1.) This sub-objective focuses on producing mice for testing bNAb-based prevention strategies. It requires breeding NOD/SCID γc-/- mice, and

identifying newborn mice to use as recipients, production of CD34+ fetal liver cells for transfer, irradiation and injection of recipients and tracking the level of reconstitution by fl ow cytometry. The mice have been used to assay antibody activity including prevention and therapy in our own laboratory and in collaborative experiments with Dr. Bjorkman and Dr. Ravetch. To date the mouse experiments have been predictive of antibody activity in humans. In addition we have developed new assays for antibody clearance of infected cells in humanized mice. These experiments, recently published in Science, revealed that Fc effector functions contribute to antibody activity in humans by accelerating the clearance of infected cells.

2.) We solved a 3.5Å crystal structure of a native-like Env trimer with fully-processed native glycosylation, revealing an extensive glycan shield of untrimmed high-mannose and complex-type N-glycans on an Env crystal structure for the fi rst time. The trimer was complexed with two glycan-interacting bNAbs from the Nussenzweig lab: 10-1074 against the V3-loop (in clinical trials), and IOMA, a new CD4bs antibody. Although IOMA evolved from the VH1-2*02 germline gene of CD4bs-targeting VRC01-class bNAbs, its light chain lacks the short (5 aa) CDRL3 loop that defi nes VRC01-class bNAbs, and its binding resembles orientations of VH1-46–derived CD4bs bNAbs. The existence of bNAbs that mix features of VRC01-class and VH1-46-class antibodies has implications for immunization strategies targeting VRC01-like bNAbs; namely, researchers should search for VH1-2–derived Abs with normal-length CDRL3s in vaccine results (instead of focusing only on VH1-2 derived Abs with 5-residue CDRL3s) because these IOMA-like bNAbs would be easier to elicit than VRC01-class bNAbs since they are less heavily mutated.

3.) A panel of Fc domain variants of human IgG1 has been generated with differential binding capacity to the different classes of human and mouse FcγRs. Out of these variants, GASDALIE (G236A/S239D/A330L/I332E) exhibited the best enhancement in the affi nity for activating FcγRs, without any impairment for FcRn binding. This variant was selected and bNAb GASDALIE Fc variants were generated for 3BNC117,

(Cont.)

Nussenzweig: Development of optimized broadly neutralizing antibodies for HIV-1 prevention

10-1074 and PG16 and their activity assessed in in vivo experiments using HIV-1-infected humanized mice. In preliminary experiments, we have assessed the activity of Fc-optimized bNAbs (3BNC117, 10-1074 and PG16) in HIV-1-infected humanized mice. Compared to wild-type hIgG1 or FcRnull binding bNAbs, Fc-optimized (GASDALIE) bNAbs revealed improved in vivo therapeutic activity.

We have assessed the capacity of wild-type and Fc-optimized anti-HIV-1 bNAbs to engage rhesus macaque FcγRs. Macaque FcγR were cloned and their extracellular, IgG binding domain was expressed recombinantly. Their binding affinity for wild-type hIgG1 and Fc domain variants was measured by surface plasmon resonance (SPR). Additionally, genetic variants of rhesus macaque FcγRs were analyzed and their impact on human IgG1 binding was characterized. No significant impact on their binding capacity for hIgG was observed for the most common genetic variants of rhesus FcRs.

4.) Based on the relatively long term protection conferred by Hepatitis A immune globulin, we tested the efficacy of a single injection (20mg/kg) of four anti-HIV-1 neutralizing monoclonal antibodies (MAbs) (VRC01, VRC01-LS, 3BNC117, and 10-10749-12) in blocking repeated weekly low dose virus challenges of the clade B SHIVAD8. Compared to control animals, which required 2 to 6 challenges (median=3 weeks) for infection, a single bNAb infusion prevented virus acquisition for up to 23 weeks. This effect depended on antibody potency and half-life. The highest levels of plasma neutralizing activity and correspondingly, the longest protection, were found in monkeys administered the more potent antibodies, 3BNC117 and 10 1074 (median=13 and 12.5 weeks respectively). VRC01, which showed lower plasma-neutralizing activity, protected for a shorter time (median=8 weeks). The introduction of a mutation that extends antibody half-life into the Fc domain of VRC01 increased median protection from 8 to 14.5 weeks.

(Cont.)

Nussenzweig: Isolation of Human Monoclonal Antibodies for HIV Prevention

PrincipalInvestigatorMichel C. Nussenzweig, MD, PhD


Project TitleIsolation of human monoclonal anti-bodies for HIV prevention

OPPID1033115

Grant AwardUp to $6.4 Million, awarded Septem-ber, 2011

Collaborating Institutions◊ Massachusetts Institute of

Technology, USA◊ University fuer Bodenkultur, Austria◊ University of Cologne, Germany◊ University Medicine Berlin,

Germany◊ The General Hospital Corporation

(MGH, Ragon Institute), USA

Grant at a GlanceOVERVIEWAn effective vaccine against HIV remains elusive and AIDS continues to be a major source of morbidity and mortality worldwide. The failure of many HIV vaccine products has elevated the importance of pursuing an alternative form of prevention, namely the concept of using anti-HIV monoclonal antibodies as a passive form of prevention. Recent studies in macaques have shown that antibodies can prevent HIV infection. Moreover, a significant fraction of HIV-infected individuals develop serum antibodies that neutralize a broad spectrum of viruses even at low concentration. Until two years ago, only a very small number of such antibodies had been characterized and the most potent of these, b12, was not a naturally occurring product.

The consortium assembled and led by Dr. Michel Nussenzweig seeks to push the passive prevention avenue forward by discovering and characterizing additional neutralizing antibodies. They plan on accomplishing this by building upon techniques developed by Dr. Nussenzweig for the cloning of anti-HIV human antibodies from single cells. An additional step for the development of broadly neutralizing antibodies for passive prevention will be the optimization of in vivo effector functions such as antibody dependent cytotoxicity or antibody dependent anti-viral activity. Dr. Jeffrey Ravetch at Rockefeller University discovered and characterized the Fc receptors responsible for antibody effector function and developed methods to optimize antibody effector function in vivo.

RESEARCH OBJECTIVES1.) Isolate broadly neutralizing antibodies from the memory cells and plasma cells from a large group of

patients with high titers of serum neutralizing activity,

2.) Engineer a scalable, highly cost-effective platform to rapidly identify natural human mAbs capable of neutralizing HIV-1,

3.) Determine the optimal Fc effector function in novel in vitro and in vivo systems and combine broadly neutralizing antibodies with optimal Fc function and validate their efficacy in relevant in vivo systems

Select clones for manufacturing antibodies for preclinical and clinical trials.

PROGRESSResearch objectives 1-4 above were accomplished and extended to carry out the following clinical trial. A phase IIa open-label clinical trial evaluating 3BNC117, a broad and potent neutralizing antibody (bNAb) against the CD4 binding site of HIV-ENV, was initiated in the setting of analytical treatment interruption (ATI) in 13 HIV-1-infected individuals. Participants with 3BNC117-sensitive virus outgrowth cultures were enrolled.

Infusions (30mg/kg) of 3BNC117 were generally well tolerated. The infusions were associated with a delay in viral rebound for 5-9 weeks after 2 infusions, and up to 19 weeks after 4 infusions, or an average of 6.7 and 9.9 weeks, respectively (compared with 2.6 weeks for historical controls). Rebound viruses arose predominantly from a single provirus. In most individuals, emerging viruses showed increased resistance indicating escape. However, 30% of participants remained suppressed until antibody concentrations waned below 20 �g/ml, and the viruses emerging in all but one of these individuals showed no apparent resistance to 3BNC117, suggesting failure to escape over a period of 9-19 weeks. These results demonstrate that administration of 3BNC117 exerts strong selective pressure on HIV-1 emerging from latent reservoirs during ATI in humans.

Nussenzweig: Phase 1 studies of the pharmacokinetics, safety, and bioactivity of 3BNC117-LS and10-1074-LS among adults in the US and sub-Saharan Africa

PrincipalInvestigatorMichelNussenzweig, MD, PhD


Project TitlePhase 1 studies of the pharmacokinetics, safety, and bioactivity of 3BNC117-LS and 10-1074-LS among adults in the US and sub-Saharan Africa

OPPID1168933


Collaborating Institutions◊ International AIDS Vaccine

Initiative, New York◊ University of Washington, Seattle◊ Fred Hutchinson Cancer Research

Center, Seattle

Grant at a Glance

The Rockefeller University, The Rockefeller University,

OVERVIEWDespite signifi cant efforts over the last 30 years, and exceptional recent scientifi c advances, it seems highly unlikely that we will be able to develop an effective active anti-HIV vaccine in the near future. In the interim, there is a need for HIV prophylaxis methods that are effective in controlling new infections, deliverable in areas most in need (such as sub-Saharan Africa), and economically feasible in these regions. Passive immuni-zation with broadly neutralizing monoclonal antibodies (bNAbs) is being considered in this context. Passive bNAb immunization has been shown to prevent SHIV infection in rhesus macaque models, can be delivered subcutaneously (enabling self-administration in some settings), and the antibodies have a long half-life. This is particularly true of antibodies such as the two that will be investigated in this grant, 3BNC117-LS and 10-1074-LS, which have been specifi cally modifi ed to have increased half-lives in vivo. This grant will be used to evaluate the safety, tolerability, pharmacokinetic profi le, and antiretroviral effects of two human anti-HIV neutralizing antibodies given in combination in humans, with the long-term goal of achieving high levels of effi cacy in preventing HIV acquisition.

3BNC117-LS and 10-1074-LS are broad and potent anti-HIV neutralizing antibodies recognizing the HIV-1 envelope CD4 binding site and the V3 loop of the HIV-1 envelope, respectively. Two amino acid substitutions (the LS mutation) were introduced in the Fc region of the parental antibodies to extend their half-lives. Both the parental and LS-modifi ed antibodies provide broad coverage of diverse HIV-1 strains, and the parental antibodies showed favorable safety profi les in phase I trials. The LS mutation alters the binding properties of the antibodies for the neonatal Fc receptor (FcRN). It thereby enhances antibody re-cycling and prolongs the half-lives by 3- to 4-fold. The increased half-lives should be dose sparing and allow for quarterly or biannual dosing.

This proposal builds on the research performed under OPP1092074 (awarded to M. Nussenzweig, Rockefeller University), and enables the development and evaluation of a combination of 3NBC117-LS and 10-1074-LS for passive immunoprophylaxis in sub-Saharan Africa. It includes phase 1 studies in the US and in sub-Sa-haran Africa, as well as overall management and oversight of product development. Data generated from these studies will support phase 2b effi cacy studies in sub-Saharan Africa. Two studies will be conducted at the Rockefeller University, and the other will be conducted in four sub-Saharan African sites. The ultimate goal is to develop a commercially viable product for sub-Saharan African countries that can be delivered subcuta-neously once every 3-6 months.

The grant is led by Michel Nussenzweig at Rockefeller University. Drs. Connie Celum (University of Washington, Seattle) and Julie McElrath (Fred Hutchinson Cancer Research Center, Seattle) are partner investigators for the proposed phase 1b study in Africa. The Product Development Center at the International AIDS Vaccine Initiative (IAVI) will manage the production of 10-1074-LS and will assist with regulatory submissions in the US and Africa; these activities are funded under separate awards to IAVI.

RESEARCH OBJECTIVES1.) Phase 1a: First-in-Human Studies in the US. Two phase 1, dose-escalation studies to assess the safety, pharmacokinetics, and antiretro-

viral activity of 10-1074-LS alone and in combination with 3BNC117-LS in HIV-uninfected and HIV-infected individuals will be conducted. The antibodies will be administered intravenously or subcutaneously.

2.) Phase 1b: Study in sub-Saharan African Sites. This will be a phase 1b, placebo-controlled study of the safety, tolerability, and pharmacoki-netics/pharmacodynamics of 10-1074-LS and 3BNC117-LS administered in combination subcutaneously to HIV-uninfected individuals in Kenya, Uganda, and South Africa.

Pantaleo: Generation/Isolation of Novel bnAbs from Lymph Node B cells

PrincipalInvestigatorGiuseppe Pantaleo, MD

GranteeInstitutionCentre Hospitalier Universitaire Vaudois, Lausanne, Switzerland

Project TitleGeneration/Isolation of Novel bnAbs from Lymph Node B cells

OPPIDOPP1190237 & OPP1114725


Collaborating Institutions◊ Institute for Research in Biomedi-

cine, Bellinzona, CH◊ Fred Hutchinson Cancer Research

Center, Seattle, USA◊ University of Regensburg, Regens-

burg, DE◊ Sacco Hospital – University of

Milan, Milan, IT

Funded under separate contract:◊ Atreca, Redwood City, USA

Grant at a GlanceOVERVIEWGeneration of potent broad neutralizing antibodies (bnAbs) occurs only in a minority (<5%) of HIV infected subjects. The mechanisms preventing the generation of bnAbs remain mostly unknown. Recent advances in the development of methods for supporting proliferation and differentiation of single Env-specific B-cells have led to the isolation of potent bnAbs targeting different regions of HIV envelope. The currently avail-able bnAbs have been derived from cloning memory B cells isolated from the blood, however it is unknown whether circulating B cells are quantitatively and qualitatively reflective of the overall B cell repertoire. Studies of the B cell repertoire in humans have been limited to blood B cells and no information is available on the B cell repertoire in B cells isolated from lymphoid tissues. The differences in the composition of B cell populations isolated from blood and lymph nodes (LNs) in humans are substantial. In particular, the germinal center (GC) B cell population is virtually absent in blood. The study of this population may be particularly relevant in HIV infection since >50% of total B cells in LNs of viremic subjects are composed of GC B cells. It is also highly likely that GC B cells are the precursors of memory B cells and/or plasma cells producing the type of antibodies with features typical of bnAbs, i.e. marked somatic hypermutation or long CDR H3.

The analysis of Env-specific B cells isolated from LNs will allow us to address a series of unresolved ques-tions regarding the generation of bnAbs. These include:

a.) The frequency of Env-specific B cells in blood and LNs,

b.) The distribution/enrichment of Env-specific B cells within the different B cell populations in blood and LNs,

c.) The identification of the B cell population enriched in precursors of B cells producing bnAbs,

d.) The diversity of the B cell repertoire of Env-specific B cells,

e.) Block in the maturation of the B cells producing bnAbs and

f.) Defective retention for B cells producing high affinity antibodies. If the central hypothesis of the proposed research project is correct and the frequency of B cells producing bnAbs is much higher in LN as compared to the blood and the quality of B cells (GC B cells) is crucial, the isolation and cloning of LN B cells will lead to more efficient generation of novel bnAbs.

Based on the initial results, Pantaleo/CHUV was awarded with the 2nd award focusing on the optimization of the LN02 bNabs.

RESEARCH OBJECTIVESThe overall primary goal of the project is to generate novel broadly neutralizing antibodies. The specific objectives include:1.) Isolation and characterization of novel bnAbs from lymph nodes B-cells

2.) Determination if generation of rare bnAbs is due to a block of B cell maturation or defective retention of B-cell producing high affinity antibody

3.) Development of a computational framework to analyze the phenotype, function, signaling, gene expression profile of different B cell populations and B cell repertoire in lymph node B cells, the functional profile of Tfh cells and their association with bnAbs production

4.) Improve the potency and breadth of LN02 and facilitate the advancement for preclinical and clinical evaluation

PROGRESSMajor advances have been made in all the different areas of the project and include:

1.) Identifi ed two lead candidates LN01 (an MPER antibody) and LN02 (an gp120-gp41 interface Ab), both demonstrating great neutralization potency in terms of breadth and magnitude;

2.) Demonstration that the frequency of HIV-specific B cells is significantly higher in lymph nodes and particularly in germinal centers B cells;

3.) Identification of distinct phenotypic and functional profiles in lymph nodes versus blood B cells;

4.) Identification of unique phenotypic and functional profiles of Tfh and B cells associated with HIV infection.

Pantaleo: Poxvirus-Based Vaccine Development



Project TitlePoxvirus Vaccine Regimen Design

OPPID38599 & 52845

Grant AwardUp to $21.1 Million, awarded August, 2006Up to $5.7 Million, awarded November, 2009

Collaborating Institutions◊ Arizona State University, USA◊ Biomedical Primate Research

Centre, The Netherlands ◊ CHU Henri Mondor, University

Paris 12, France◊ Consejo Superior de Investiga-

ciones Cientifi cas, Spain◊ Fred Hutchinson Cancer Research

Center, USA ◊ Imperial College London, UK ◊ Institute for Research in Biomedi-

cine, Switzerland ◊ IPPOX Foundation, Switzerland◊ Leiden University Medical Centre,

The Netherlands◊ Murdoch University, Australia◊ Oregon Health Sciences University,

VGTI, USA◊ Sanofi Pasteur, Canada◊ University of Cambridge, UK◊ University of Montreal, Canada◊ University of Regensburg, Ger-

many◊ University of Washington, USA

External Scientifi c Advisory Board◊ Rafi Ahmed, Emory Vaccine Centre◊ Andrew McMichael, Weatherall In-

stitute of Molecular Medicine, John Radcliffe Hospital

◊ Stanley Plotkin, Sanofi Pasteur

Grant at a GlanceOVERVIEWThe primary goal of the Pantaleo-led research consortium is to generate highly attenuated replication-competent poxvirus vectors that would substantially improve the breadth of HIV-1-specific vaccine induced immune responses. This strategy is also combined with the deletions of certain poxvirus genes known to interfere with the induction of the immune response. These newly generated vectors, in addition to im-proving the magnitude and the quality of the vaccine-induced HIV-1-specific T cell response, may serve as potent priming strategies for envelope protein-based vaccines, and thus for the induction of potent antibody responses.

Furthermore, Pantaleo/CHUV was awarded with a 2nd grant to develop the DNA/NYVAC platform. The DNA/ NYVAC platform has been tested in multiple phase I and II clinical trials in Europe and has shown to be highly immunogenic. This part of the project represents further development of the DNA/NYVAC platform targeted to improve both the magnitude of the T-cell response (particularly the CD8 T cell response) and breadth of the response. The primary goal of the project is to investigate whether the 2nd generation DNA-C/NYVAC-C vaccine combination, together with novel immunization strategies, is able to increase response magnitude and breadth and induce balanced Env (versus Gag, Pol and Nef) HIV-1-specific T cell responses in humans.

RESEARCH OBJECTIVES1.) Development of poxvirus-based vaccine candidate(s) with at least a 10-fold increase in immunogenicity,

as measured by the frequency of vaccine-induced T-cells when compared to the current poxvirus vectors; development of novel formulation/delivery strategies.

2.) Conduct NHP studies as well as a phase I safety and immunogenicity study with the 2nd generation DNA and NYVAC vaccine candidates, to generate data for the phase IIB study in sub-Saharan Africa.

PROGRESSAt present, the research consortium has:

1.) Completed several NHP studies:

a.) Demonstrated that the new NYVAC and DNA vaccines developed within PTVDC are highly immunogenic, both in terms of T-cell and B-cell responses

b.) Evaluated and compared different vaccine combinations, inserts, and regimens which have provided pivotal data for the design of future clinical studies

2.) 2. In collaboration with HVTN, a number of phase I/II trials have been completed evaluating the safety and immunogenicity of different vaccination regimens combining DNA, NYVAC, and protein vaccines in the US and sub-Saharan Africa. The more recent effort has been focused on the evaluation of a DNA and protein combination, and results have demonstrated that the combination elicits robust antibody and T-cell responses. A comparative analysis with the ALVAC/protein combination has shown that the DNA/protein regimen induces potent V1V2 responses, greater than the ALVAC/protein regimen. These responses were shown to be immune correlates of reduced risk from HIV infection in the RV144 trial. The DNA/protein is now planned for a phase IIB effi cacy trial in Africa, supported by a European program.

3.) 3. In collaboration with the Haynes and Felber groups, a NHP SHIV challenge study has been initiated, evaluating the protective effi cacy of different homologous prime-boost regimens combining NYVAC-KC, DNA-HIV-PT123, and sequential CH505 DNA gp145s co-administered with gp120 proteins (the EnvSeq-2 vaccine).

Pantaleo: RepliVax Vaccine Platform



Project TitleA novel replication competent fl avivi-rus-based HIV vaccine platform, i.e. RepliVax, as a priming component for improving antibody response

OPPID1040705

Grant AwardUp to $8.8 Million, awarded Septem-ber, 2012

Collaborating Institutions◊ Arizona State University, USA ◊ Baylor Research Institute, USA◊ Consejo Superior de

Investigaciones Cientifi cas◊ Fred Hutchinson Cancer ◊ Institut Nationaal de la Sante et de

la Recherche Medicale, France◊ Sanofi Pasteur, Canada◊ University of Regensburg,

Germany

Grant at a GlanceOVERVIEWPantaleo’s RepliVax VDC focuses on the development of novel replication competent platforms for optimal priming of antibody responses. The overarching goal of the work performed for this grant is to develop a replication competent self-limiting single cycle flavivirus vector based HIV vaccine candidate, ie RepliVax®, for use in a prime-protein boost regimen with a strong emphasis on eliciting optimal, robust and durable/boostable antibody responses. In parallel to the RepliVax® development, the VDC will also focus on the development of novel delivery systems for Env proteins and generation of novel Env immunogens. Two main approaches to this end are planned: (i) target ENV antigen(s) to dendritic cells (DCs) using a DC-specific antibody and (ii) use bioinformatics to generate ENV immunogens (ENV) with improved epitopes selected basd on binding to broadly neutralizing antibodies.

RESEARCH OBJECTIVES1.) To develop a RepliVax® based viral vector platform for delivering HIV antigens

2.) To generate novel Env immunogens and delivery systems for Env proteins

3.) Immunological characterization of the RepliVax® vectors and prime/boost immunization strategies

PROGRESSThe VDC has completed a series of in vitro and in vivo (mice and NHP) immunogenicity studies with RepliVax in different prime-boost regimens combining with DNA, and/or NYVAC, and/or protein, and have demonstrated that:

● RepliVax is highly attenuated in sensitive 2-3 day old suckling mouse neurovirulence studies and has shown to be safe and well tolerated in NHP studies

● RepliVax vectors can be used in combination with other vectors to elicit HIV specific cellular and humoral immune responses as a potentially effective vaccination strategy.

In collaboration with Inserm, the VDC has also conducted several NHP studies with DC-targeting based vaccines. Meta-analysis across several NHP studies has been performed by VISC comparing DC-targeting vaccines bearing Env gp140 with the gp120 protein formulated with MF59 adjuvant, and the outcome indicated portential advantages for DC-targeting via CD40 for more durable cellular and humoral responses.

Last but not least the VDC has successfully generated a number of novel Env proteins which have exhibited potentially improved antigenic properties. Results from the rabbit immunogenicity study has shown that these variants induced robust binding antibody responses and high neutralization against the Tier 1A isolate.

Parks: HIV Vaccine Clinical Candidates based on Replication-Competent Viral Vectors that Preferentially Replicate in Lymphoid Tissues

PrincipalInvestigatorChris Parks, PhD


Project TitleHIV Vaccine Clinical Candidates based on Replication-Competent Viral Vectors that Preferentially Replicate in Lymphoid Tissues

OPPID1148476

Grant AwardUp to $5.8 Million, awarded March, 2016

Grant at a GlanceOVERVIEWViral diseases such as measles, mumps, rubella, and yellow fever are controlled by immunization with live attenuated viral vaccines. The mild or asymptomatic infections caused by the attenuated vaccine viruses generates immunity that prevents disease after later natural exposure to the viral disease agents. Similarly, immunity induced by experimental live attenuated SIV vaccines has been shown to protect macaques from progressive SIV infection caused by highly pathogenic challenge viruses. Unfortunately, a vaccination strat-egy based on live attenuated strains of HIV for use in people is too risky, thus the objective of the program led by Dr. Chris Parks at The International AIDS Vaccine Initiative (IAVI) is to use viruses that do not cause serious human illness to generate replication-competent viral vectors to deliver HIV vaccine immunogens.

Vectors based on vesicular stomatitis virus (VSV) have been the primary focus of recent research because VSV can be used to generate chimeric viruses in which the natural VSV glycoprotein (G) is replaced with HIV Env. VSV-HIV chimeric viruses can be developed that express Env that incorporates in the infected cell membrane and VSV particle where it performs functions needed to support viral replication specifically in T lymphocytes that express the HIV coreceptors CD4 and CCR5. Therefore, vaccination with a live VSV-HIV chimera has the potential to mimic multiple important aspects of Env presentation that would occur during an HIV infection.

A lead chimeric VSV-HIV vaccine candidate (VSVG-Env.BG505) has been developed based on clade A HIV Env from strain BG505. In the first preclinical vaccine efficacy study conducted in Indian rhesus ma-caques, 7 of 10 animals vaccinated with VSVG-Env.BG505 resisted infection following repetitive rectal challenge with heterologous clade B SHIV SF162p3 while the 3 macaques that became infected were shown to have substantially lower Env antibody responses induced by vaccination. Efficacy also was shown to be associated with the chimeric virus design, as a group of macaques vaccinated with a more typical VSV vector that expressed both VSV G and Env developed Env antibodies but failed to resist SHIV infection.

The efficacy of VSVG-Env.BG505 as well as the association between antibodies and protection support additional investigation and develop-ment of the vaccine platform and the immune responses it elicits. A grant to further develop the VSVG-Env.BG505 vector was awarded to sup-port a second preclinical efficacy study and to initiate activities required to advance a VSVG-Env.BG505 vector for future evaluation in a phase 1 exploratory clinical trial.

RESEARCH OBJECTIVES1.) 1.) Evaluate the VSVG-Env.BG505 vector in a non-human primate study to confirm efficacy observed previously and investigate variables

affecting vaccine immunogenicity.

2.) Develop methods for further assessment of the immune response elicited in the non-human primates and detailed analysis of the composition of the vaccine vector

3.) Construct Vero CD4+/CCR5+ cell line, required to propagate the VSVG-Env.BG505 vector, which complies with cGMP vaccine manufacturing

4.) Prepare VSVG-Env.BG505 pre-Master Virus Seed that complies with cGMP vaccine manufacturing

Parks: Live Attenuated VSV-HIV-Env Vaccine Candidate for Evaluation in a Clinical Trial

PrincipalInvestigatorChris Parks, PhD


Project TitleLive Attenuated VSV-HIV-Env Vaccine Candidate for Evaluation in a Clinical Trial

OPPID1152832

Grant AwardUp to $21.8 Million, awarded October, 2016

Grant at a GlanceOVERVIEWViral diseases such as measles, mumps, rubella, and yellow fever are controlled by immunizationwith live attenuated viral vaccines. The mild or asymptomatic infections caused by the attenuated vaccine viruses generates immunity that prevents disease after later natural exposure to the viral disease agents. Similarly, immunity induced by experimental live attenuated SIV vaccines has been shown to protect macaques from progressive SIV infection caused by highly pathogenic challenge viruses. Unfortunately, a vaccination strategy based on live attenuated strains of HIV for use in people is too risky, thus the objective of the program led by Dr. Chris Parks at The International AIDS Vaccine Initiative (IAVI) is to use viruses that do not cause serious human illness to generate replication-competent viral vectors to deliver HIV vaccine immunogens.

Vectors based on vesicular stomatitis virus (VSV) have been the primary focus of recent research because VSV can be used to generate chimeric viruses in which the natural VSV glycoprotein (G) is replaced with HIV Env. VSV-HIV chimeric viruses can be developed that express Env that incorporates in the infected cell membrane and VSV particle where it performs functions needed to support viral replication specifi cally in T lymphocytes that express the HIV co-receptors CD4 and CCR5. Therefore, vaccination with a live VSV-HIV chimera has the potential to mimic multiple important aspects of Env presentation that would occur during an HIV infection.

A lead chimeric VSV-HIV vaccine candidate (VSVG-Env.BG505) has been developed based on clade A HIV Env from strain BG505. In the fi rst preclinical vaccine effi cacy study conducted in Indian rhesus ma-caques, 7 of 10 animals vaccinated with VSVG-Env.BG505 resisted infection following repetitive rectal challenge with heterologous clade B SHIV SF162p3 while the 3 macaques that became infected were shown to have substantially lower Env antibody responses induced by vaccination. Effi cacy also was shown to be associated with the chimeric virus design, as a group of macaques vaccinated with a more typical VSV vector that expressed both VSV G and Env developed Env antibodies but failed to resist SHIV infection.

The effi cacy of VSVG-Env.BG505 as well as the association between antibodies and protection support additional investigation and develop-ment of the vaccine platform. A grant was awarded to further develop the VSVG-Env.BG505 vector, including advancement of processes to support vaccine manufacturing and analytical assessment of the vaccine product, manufacturing of the VSVG-Env.BG505 vaccine material, preclinical safety assessment, and a phase 1 exploratory clinical trial.

RESEARCH OBJECTIVES1.) Conduct a non-human primate study to further evaluate variable affecting VSVG-Env.BG505 vector immunogenicity and effi cacy that will

inform the design of the exploratory phase 1 clinical trial

2.) Complete construction of the Vero CD4+/CCR5+ cell line and establish a qualifi ed cell bank.

3.) Complete rescue and characterization of a VSVG-Env.BG505 vector and establish a virus seed that will support vaccine manufacturing

4.) Conduct preclinical safety / toxicology assessment of the VSVG-Env.BG505 vector

5.) Develop and transfer processes and analytical testing methods to support vaccine manufacturing a contract manufacturing organization. Manufacture VSVG-Env.BG505 clinical trial material.

6.) Communicate with regulatory authorities and fi le an IND and other document as required to enable a phase 1 exploratory clinical trial

7.) Conduct an exploratory phase 1 clinical trial including immunologic assessment to determine if Env antibody responses associated with preclinical effi cacy are observed in clinical trial volunteers.

Pettit: Just Biotherapeutics Support for HIV bNAbs

PrincipalInvestigatorsDeanPetttit, PhDBruceKerwin, PhD

GranteeInstitutionJustBiotherapeuticsSeattle, USA

Project TitleJust Biotherapeutics Support for HIV bNAbs

OPPID1138851


Collaborating InstitutionsThe following institutions receive support under separate grants:◊ Beth Israel Deaconess Medical

Center, Boston, USA◊ The Rockefeller University, New

York, USA◊ Aaron Diamond AIDS Research

Center, New York, USA◊ The Scripps Research Institute,

Jupiter, USA◊ University of Kansas, Lawrence,

USA

Grant at a GlanceOVERVIEWDuring the past few years the Bill & Melinda Gates Foundation has engaged with universities developing broadly neutralizing monoclonal antibodies (bnAbs) against HIV. Molecules under investigation by CAVD grantees include bnAbs 3BNC117, 10-1074, PGT121, PGDM 1400, bi-specific antibody constructs, and the HIV-1 entry inhibitor eCD4-Ig. While the molecules mentioned here show good in vitro neutralization activity and are being tested in clinical trials they have not been sequence optimized for commercialization, which may lead to significant late stage commercialization problems during scale-up, formulation and fill/finish opera-tions. Just Biotherapeutics, Inc., has developed a proprietary set of in silico tools for sequence optimization of antibodies and related proteins to significantly increase success during commercialization.

The focus of the current grant proposal from Just is 3-fold: 1) delivery of 2 commercially optimized HIV bnAbs from the set 3BNC117, 10-1074, PGT121 and PGDM 1400 for First-In-Human (FIH) clinical trials, which, if successful could be taken through to commercialization; 2) process economic modeling of the drug substance manufacturing; and 3) sequence optimization for non-traditional bnAbs and other proteins. The HIV bnAbs, from the set including 3BNC117, 10-1074, PGT 121 and PGDM 1400 will be sequence optimized using Just’s Abacus in silico design, experimentally validated in vitro, and tested by academic PI’s in non-human primate PK studies. In concert with the optimization, it is important to know if and how cost targets for manufacturing of the drug substance can be met. This will be accomplished through modeling, focusing only on the most cost effective manufacturing option. In addition to optimization of the parental HIV bnAbs, the bi-specific bnAbs will be sequence optimized through in silico modeling only, and technical expertise provided for the anti-HIV Fc-fusion eCD4-Ig, with additional scale-up directed through the development laboratories. Specific sequence improvements for the molecules include addition of mutations for increasing half-life, increasing expression, decreasing self-interaction, and limiting the effects of multiple potential degradative modifications. Together, the sequence optimization of the antibodies will significantly improve our ability to enhance expression and intensify the purification process (important for reducing manufacturing costs), while limiting phase separation, aggregation, and high viscosity associated with poor sequences. The sequence optimization should allow for improved properties leading to lower cost biotherapeutics.

This grant is led by Dean Pettit, PhD and Bruce Kerwin, PhD (Just Biotheraputics Inc.) with the collaborative engagement of Dan Barouch, MD, PhD (Beth Israel Deaconess Medical Center) and Michel Nussenzweig MD, PhD (The Rockefeller University) for in vivo evaluations of bnAbs in non-human primate models, David Ho (Aaron Diamond AIDS Research Center) for in vitro characterization of bi-specific bnAbs, Michael Farzan (The Scripps Research Institute) for scientific issues concerning eDC4-Ig, and David Volkin (University of Kansas) for structural analytics on purified constructs. In addition, the grant will leverage the following Central Services Facilities: The Vaccine Product Development Center for bnAb toxicology studies in rodents and non-human primates; and the Comprehensive Antibody Vaccine Immune Monitoring Consortium for in vitro neutralization measurements. The award was made in October 2015 with an original agreement length of 50 months.

RESEARCH OBJECTIVES

1.) Delivery of 2 commercially optimized HIV bnAbs from the set 3BNC117, 10-1074, PGT121, and PGDM 1400

a.) Develop optimized bnAb cell lines that allow for maximizing specific productivity for efficient utilization within a J.POD facility, and

b.) Increase bnAb stability allowing for time outside of the cold chain for greater distribution within Sub-Saharan Africa

2.) Process economic modeling for manufacturing the bNAbs

3.) Sequence optimization for bi-specific bnAbs and the eCD4-Ig entry inhibitor

PROGRESSSignificant progress has been made toward optimization of 3BNC117, 10-1074, PGT121, and PGDM1400 with optimization sites identified by in-silico modeling and variants produced and tested for retention of neutralization and increases in desired biophysical parameters. For 3BNC117, the data showed that sites which provided signifi cant biophysical optimization interfered with neutralization activity leading to continued use of the parental molecule. In contrast, 10-1074 could be signifi cantly modifi ed with no loss of activity allowing for optimization resulting in increased thermodynamic stabilization which manifested as increased low pH stability and severely reduced aggregation during 40°C storage. Mouse PK studies also indicated that the sequence optimized 10-1074 bnAb had longer elimination half-live as compared to the parental sequence, and the LS modifi ed parental sequence. Cell line development has been completed for both 3BNC117 and 10-1074 and scale-up for GMP production has been initiated to produce material for clinical trials. Optimization of PGT121 and PGDM1400 has also been completed. For PGT121 optimization resulted in increased thermal stability and resistance to chemical unfolding. Additionally, residues were identifi ed that were responsible for low pH instability such that the bnAb can now be incubated at acidic pH with little to no aggregation while the parent shows up to 90% aggregation. Importantly, for room temperature stability the accelerated stability at 40°C demonstrated negligible aggregation during a 2-month incubation while the parental molecules show approximately 2% over the same timeframe. For PGDM1400 signifi cant increases in thermal stability and resistance to chemical unfolding were achieved with retention of neutralization activity against a select set of viruses. Mouse PK studies in Tg276 human FcRn knock-in mice have been completed for the optimized PGT121 variants. The results showed that the optimized variants which include the LS mutation showed a 2.2-fold increase over the parental PGT121-LS molecule.

In addition to the optimization programs, process economic modeling has been conducted to identify processes that significantly affect the manufac-tured cost of goods. Based on the analysis, specific productivity provides the greatest impact on initially lowering the cost. Modeling of the bnAbs shows that costs in the $60 - $80 per gram range can be achieved based on volumetric productivity with further reductions achieved through facility scheduling, bioreactor size, chemically defi ned media and purifi cation procedures.

Picker: Development of Attenuated CMV Vectors for an HIV/AIDS Vaccine

PrincipalInvestigatorLouisPicker, MD

GranteeInstitutionOregon Health & Science University (OHSU)Vaccine and Gene Therapy Institute,Portland, USA

Project TitleDevelopment of Attenuated CMV Vectors for an HIV/AIDS Vaccine

OPPID1107409

Grant AwardUp to $25 million, awarded in July, 2014

Collaborating Institutions◊ University of California - Davis,

Davis, USA◊ Los Alamos National Laboratory,

Los Alamos, USA

Grant at a GlanceOVERVIEWIn the past decade, we have demonstrated in rhesus models that persistent SIV vaccine vectors based on the ubiquitous β-herpesvirus cytomegalovirus (RhCMV/SIV vectors) elicit and indefi nitely maintain systemic high frequency, circulating and tissue-resident effector memory T cell (TEM) responses that can intercept and strin-gently control a highly pathogenic, AIDS-causing SIV very early, if not immediately, after exposure. Moreover, these responses not only maintain this control over the long term, but actually clear the infection to the degree that protected animals are not distinguishable from non-SIV-exposed animals by state-of-the-art analysis at necropsy.

In addition, we have shown that Rhesus (Rh) CMV vectors can be: 1) used repeatedly in RhCMV+ RM without any inhibition of immunogenicity by pre-existing immunity (a critical feature since the vast majority of people worldwide are naturally CMV-infected in infancy or childhood); 2) programmed to elicit unusually broad CD8+ T cell responses that recognize conventional and/or unconventional epitopes; 3) modifi ed to express multiple vaccine inserts totaling 6 kb or more of exogenous sequence, using endogenous promoters to control insert expression; and 4) signifi cantly attenuated without loss of immunogenicity or effi cacy. Our understanding of this system has matured such that we know which CMV genes to manipulate for optimal attenuation (pp71 deletion), or for tuning epitope responses to conventional vs. unconventional presentation (UL128/UL30-mediated “epitope switch mechanism”). Human CMV (HCMV) infection of humans closely resembles RhCMV infection of monkeys, raising the prospect of developing an HCMV-based vaccine platform that recapitulates the biologic properties of our effective RhCMV/SIV vectors. In other funded efforts, we have developed human (HCMV/HIV) homologues of our validated RhCMV/SIV designs as candidate vectors for a prophylactic HIV/AIDS vaccine.

This grant extends these efforts, in two steps. In the fi rst step, we will construct and manufacture (using existing GMP-qualifi ed cell lines) a prototype pp71-defi cient HCMV/HIVgag vector for a fi rst-in-man phase 1 clinical trial of an attenuated HCMV vector. This trial is intended to address: 1) confi rmation of the safety of pp71-defi cient HCMV vector design in humans, 2) confi rmation that HCMV-seropositive humans can be super-infected with HCMV-based vectors, 3) determination of the relationship between pp71-defi cient HCMV vector dose and immunogenicity, and 4) evaluation of the quantity, quality (effector memory differentiation and function) and durability of the vector-elicited HIVgag-specifi c T cell responses. The second step, which will occur in parallel with step 1, is the development, validation and production of a 2nd generation vector (or vector set) that is optimized for safety (this vector design will include genetic changes in addition to pp71 deletion that will serve as secondary attenuation), effi cacy (this vector design will have 1 or more vectors with multiple HIV inserts encoding Gag, Pol, and Nef that will be sequence-optimized for cross-reactivity against global M group HIVs and will have optimized CD8+ T cell epitope programming – either conventional or unconventional CD8+ T cell epitopes, as dictated by the presence or absence of UL128-131 genes, or potentially, constitute a vector set with both vector types), and manufacture (will have a custom developed GMP-qualifi ed pp71-complementing cell line for effi cient manufacture of fully complemented vector). This 2nd generation vector will undergo stringent safety and stability evaluation in the rhesus macaque model.

This collaborative effort is lead by Louis Picker, MD (Oregon Health and Sciences University/Vaccine and Gene Therapy Institute), with the partici-pation of Peter Barry, PhD (University of California, Davis, for fetal macaque studies), and Betty Korber, PhD (Los Alamos National Laboratory, for insert designs). It will also leverage the Vaccine Product Development Center. The grant was awarded in July, 2014, with an original agreement length of 5 years.

RESEARCH OBJECTIVES1.) Performance of a phase 1 clinical trial with a prototype ∆UL82/pp71 HCMV/HIVgag vector [with or without deletion of UL128-130]

2.) Development of a 2nd generation, safety- and effi cacy-optimized HCMV/HIV vector set suitable for clinical development as a global prophylactic HIV vaccine

Picker: MHC II and MHC E-Restricted CD8+ T Cells and Control of HIV

PrincipalInvestigatorLouisPicker, MD

GranteeInstitutionOregon Health & Science University (OHSU)Vaccine and Gene Therapy Institute,Portland, USA

Project TitleMHC II and MHC E-Restricted CD8+ T Cells and Control of HIV

OPPID1108533

Grant AwardUp to $4.9 million, awarded in September, 2014

Collaborating Institutions◊ Emory University, Atlanta, USA◊ Ragon Institute, Boston, USASupported under separate contract:◊ Stanford University, Stanford, USA

Grant at a GlanceOVERVIEWIn the past decade, we have demonstrated in rhesus models that persistent SIV vaccine vectors based on the ubiquitous β-herpesvirus cytomegalovirus (RhCMV/SIV vectors) elicit and indefi nitely maintain systemic high frequency, circulating and tissue-resident effector memory T cell (TEM) responses that can intercept and stringently control a highly pathogenic, AIDS-causing SIV very early, if not immediately, after exposure. Moreover, these responses not only maintain this control over the long term, but actually clear the infection to the degree that protected animals are not distinguishable from non-SIV-exposed animals by state-of-the-art analysis at necropsy. During the course of these investigations, it was determined that these TEM-type responses are associated with unprecedented array of SIV epitope-specifi c CD8+ T cell responses (3-fold the breadth of conventional responses) that were restricted by MHC class II (MHC II; two-thirds of responses) and non-classical MHC E (one-third of responses) proteins, rather than the conventional polymorphic MHC class I (MHC I) presentation. Also in contrast to conventional CD8+ T cell responses, many of the unconven-tional viral epitopes targeted by these responses were common to most or even all monkeys, despite the fact that these monkeys were out-bred and quite genetically heterogeneous. These highly unusual CD8+ T cell responses were found to be due to deletion of 2 genes, UL128 and UL130, in the fi broblast-adapted CMV vector compared to wildtype CMV, and repair of these 2 genes completely reversed the recognition properties back to conventional characteristics.

Given that HIV and SIV have evolved to evade conventional CD8+ T cell responses, it’s possible that the unconventionally targeted CD8+ T cell responses elicited by the UL128/UL130-deleted CMV vectors account for their demonstrated effi cacy against SIV challenge. It is also noteworthy that since the discovery of these responses in UL128/UL130-deleted CMV vector-vaccinated monkeys, careful and specifi c scrutiny of CD8+ T cell responses in humans and monkeys with spontaneous (e.g., not vaccine-related) HIV or SIV control has led to the identifi cation of minor, but unequivocal, MHC II-restricted, HIV- or SIV-specifi c CD8+ T cell populations. This fi nding raises the question of whether these spontaneous (e.g., non-CMV vector-elicited) unconventional CD8+ T cell responses might have contributed to the viral control in these subjects. Evidence of the importance of these unconventional responses would have implications for HIV vaccine development, both to inform the clinical development of CMV vectors and to develop alternative methods to generate these responses. Consequently this consortium effort was established to determine 1) the mechanisms responsible for generation of unconventionally targeted CD8+ T cell responses, 2) the functional implications of unconven-

tional epitope recognition by CD8+ T cells, and most importantly, 3) whether unconventionally targeted SIV- or HIV-specifi c CD8+ T cells manifest enhanced in vivo viral control compared to conventionally targeted CD8+ T cell response.

This grant is led by Louis Picker, MD (Oregon Health & Science University (OHSU)/Vaccine and Gene Therapy Institute), with participation of John Altman, PhD (Emory University, tetramer development), Bruce Walker MD (Ragon Institute of MGH, MIT, and Harvard), and Mark Davis (Stanford University, systems immunology). The award was received in September 2014 with an initial agreement length of 3 years.

RESEARCH OBJECTIVES1.) Determine the contribution of unconventionally targeted, SIV-specifi c CD8+ T cells (MHC II- and/or MHC E-restricted) to RhCMV/SIV vector

effi cacy

2.) Compare the phenotypic and functional differentiation of unconventional vs. conventional epitope-specifi c CD8+ T cells elicited by strain 68-1 and strain 68-1.2 RhCMV/SIV vectors vs. controlled SIV infection

3.) Characterize the TCR repertoire and developmental origin of unconventionally targeted CD8+ T cells elicited by strain 68-1 RhCMV/SIVgag vectors

4.) Determine the strain 68-1 RhCMV-mediated mechanism(s) that direct the development of unconventionally targeted CD8+ T cells and prevent conventional epitope recognition

5.) Identify and characterize MHC II-restricted CD8+ T cells in HIV-infected individuals

Pulendran: Protective humoral responses against HIV

PrincipalInvestigatorBali Pulendran, PhD

GranteeInstitutionStanford University, California, USA

Project TitleProgramming the magnitude and persistence of protective humoral responses against HIV

OPPID1040768

Grant AwardUp to $5.9 Million, awarded in August, 2012

Collaborating Institutions◊ École polytechnique fédérale de

Lausanne◊ La Jolla Institute of Allergy and

Infectious Diseases

Grant at a Glance

Stanford University, Stanford University,

OVERVIEWDeveloping a vaccine that generates an effective immune response to HIV is a signifi cant challenge. Key issues for current pre-clinical and clinical vaccines include insuffi cient breadth of neutralizing antibodies and B cell memory responses. The results of the RV144 “Thai trial” are encouraging since the trial is the fi rst demon-stration that an immune response can provide some degree of protection against acquisition of HIV infection. The protection, however, declined from approximately 60% after one year to ~31.5% after 3.5 years of the study. Dr. Bali Pulendran hypothesizes that improvements in persistent and broad antibody responses can be generated through the synergistic activation of TLR4 and TLR7/8 on antigen presenting cells using TLR agonists in combination with an appropriate delivery system. Such improvements could make a signifi cant contribution to producing effi cacious HIV vaccines.

Dr. Pulendran’s laboratory previously published that PLGA nanoparticle delivery of a vaccine antigen (inacti-vated infl uenza) in combination with 2 independent adjuvants that activate TLR4 and TLR7/8, respectively, rather than in combination with a single TLR agonist, enhances virus specifi c neutralizing antibody titers in mice. Similarly, enhanced neutralizing antibody titers were observed in non-human primates (NHP) for the immunogenic compositions containing both TLR activating adjuvants in combination with PLGA. Studies in mice indicate that a combination of long lived B cell memory responses, T cell help and enhanced germinal center follicle formation contributed to the higher quality and quantity of neutralizing antibodies. For this grant, Dr. Pulendran will seek to recapitulate and extend these fi ndings using HIV envelope (env) antigens in NHP. He will also evaluate an alternative delivery vehicle (PPS) that can be used to delivery antigen and/or TLR ligands and offers different advantages from PLGA with respect to inducing cellular immune responses in mice. Optimal formulation will be selected for NHP studies, including a repeat low dose rectal challenge model to assess effi cacy.

The product of this project will be a vaccine platform that incorporates nanoparticle delivery systems with novel adjuvants (GLA and 3M-052) and a relevant clade C HIV env (1086C) together in a realistic immunogenic composition suitable for pre-clinical trials and with a potential for translational Phase I human clinical trials.

The studies outlined in this proposal will be performed at Emory University, La Jolla Institute for Allergy and Infectious Diseases, and École polytechnique fédérale de Lausanne (EPFL) by a highly integrated and experienced team of immunologists, virolo-gists, and veterinarians that have been working collaboratively on this project under the overall direction of Dr. Bali Pulendran.

RESEARCH OBJECTIVES1.) Determine if Env (1086C/PLGA/TLR ligand formulations are superior to MF-59 with respect to the magnitude, quality, persistence, and/or

breadth of HIV-1 Env specifi c humoral responses in NHPs.

2.) Defi ne the most optimal delivery vehicle for antigen/TLR ligand based adjuvants for induction of high magnitude and persistent Env specifi c neutralizing antibodies, systemically and at mucosa in mice and NHPs.

3.) Evaluate the protective effi cacy of a HIV vaccine in NHPs, designed from the best formulated TLR ligand based adjuvant, HIV Env immunogen and delivery vehicle, selected from objectives 1 and 2.

Reinherz: HIV clade C MPER immunogens eliciting 10E8-like specifi cities

PrincipalInvestigatorEllis Reinherz, MD

GranteeInstitutionDana-Farber Cancer Institute, Boston, USA

Project TitleHIV-1 clade C MPER immunogens eliciting 10E8-like specifi cities

OPPID1108179

Grant AwardUp to $2 Million, awarded in July, 2014

Collaborating Institutions◊ Harvard University, USA ◊ Massachusetts Institute of

Technology, USA

Grant at a GlanceOVERVIEWA vaccine capable of eliciting antibodies neutralizing a broad range of HIV-1 strains must target a region of the HIV-1 virus that is highly conserved across strains. The Reinherz consortium is aimed at eliciting antibodies directed against a region of the viral gp41 envelope protein known as the membrane proximal external region (MPER). Structural information on this lipid-embedded region will be exploited for vaccine immunogen design and delivery to the immune system in the form of lipid-coated nanoparticles. This vehicle system has the advantage of concomitantly facilitating delivery of adjuvants including Toll-like receptor (TLR) ligands. In addition, nanodisc wafers shall be used to restrict antibody access to the MPER segment, thereby selecting desired anti-MPER specifi cities.

RESEARCH OBJECTIVES1.) Structural analysis of HIV-1 MPER from clade C viruses with and without the appended transmembrane

(TM) gp41 segment in lipid environments.

2.) Creation of MPER nanoparticles consisting of a polymer core particle encapsulated by an outer lipid vesicle or “skin” for MPER presentation to the immune system.

3.) Elicitation of humoral immune responses to MPER immunogens, assessment of antibody titer, specifi city and HIV-1 neutralizing activity in serum IgG as well as characterization of individual bone marrow plasma cells.

PROGRESSAlthough infrequently elicited during the course of natural infection and rarely, if at all, upon conventional vaccination, the membrane-proximal external region (MPER) of the HIV-1 gp41 glycoprotein is the target of several human broadly neutralizing antibodies (bNabs): 4E10, 2F5, Z13e1 and more recently, 10E8. How these bNabs bind to their lipid-embedded epitopes and mediate antiviral activity is unclear, but this infor-mation may offer important insight into a worldwide health imperative. The Reinherz research team, including Gerhard Wagner, Jim Sun, Likai Song and Mikyung Kim, has utilized EPR and NMR techniques to defi ne the manner in which these bNabs differentially recognize viral membrane-encrypted residues confi gured within the L-shaped helix–hinge–helix MPER segment. Two distinct modes of antibody-mediated interference of viral infection were identifi ed. 2F5, like 4E10, induces large conformational changes in the MPER relative to the membrane. However, although 4E10 straddles the hinge and extracts residues W672 and F673, 2F5 lifts up residues N-terminal to the hinge region, exposing L669 and W670. Detailed analysis of 2F5 extraction using the above techniques in conjunction with hydrogen exchange mass spectrometry (HX-MS) with John Engen demon-strates that 2F5 recognition is stepwise, involving a paratope more extensive than the core binding site contacts alone, and dynamically rearranging via an apparent CDRH3 scoop-like movement essential for MPER extraction from the viral membrane. Core epitope recognition on the virus requires induction of conformational changes in both the MPER and paratope. Hence, target neutralization through this lipid-embedded viral segment places stringent requirements on antibody combining-site plasticity. In contrast, Z13e1 affects little change in membrane orientation or conformation, but rather immobilizes the MPER hinge through extensive rigidifying surface contacts. Thus, bNabs disrupt HIV-1 MPER fusogenic functions critical for virus entry into human CD4 T cells and macrophages, either by preventing hinge motion or by perturbing MPER orientation. HIV-1 MPER features, important for targeted vaccine design, have been revealed, with implications extending to bNab targets on other viral fusion proteins. 10E8 also binds to W672 and F673, but with a different extraction profi le to that of 4E10.

Progress was made in NMR spectroscopic studies to characterize three clade C MPER segments in a lipid environment. These clade C peptides all share the typical helix-hinge-helix motif fi rst observed in a HxB2 peptide from clade B. We showed that the MPER consists of a structurally conserved pair of viral lipid-immersed helices separated by a hinge with tandem joints that can be locked by capping residues between helices. This design fosters effi cient HIV-1 fusion via inter-converting structures while at the same time affording immune escape. Disruption of both joints by double alanine mutations at Env positions 671 and 674 (AA) results in attenuation of Env-mediated cell-cell fusion and hemifusion as well as viral infectivity mediated by both CD4-dependent and CD4-independent viruses. The potential mechanism of disruption was revealed by structural analysis of MPER conformational changes induced by AA mutation. A deeper acyl chain-buried MPER middle section and the elimination of cross- hinge rigid-body motion almost certainly impede requisite structural rearrangements during the fusion process, explaining the absence of MPER AA variants among all known naturally occurring HIV-1 viral sequences. Furthermore, those broadly neutralizing antibodies directed against the HIV-1 MPER, including 10E8, exploit the tandem joint architecture involving helix-capping, thereby disrupting hinge function.

In addition, the Irvine group’s work on phospholipid-enveloped biodegradable microparticles and nanoparticles includes multilamellar vesicles, polymer nanoparticles and stealth liposomes as vaccines displaying MPER segments on lipid. In collaborative efforts within the group, specifi c anti-MPER antibodies have been generated with broad specifi city for clade B and C sequences. Improvement in affi nity through more optimal T follicular helper cell elicitation, and other particle and immunogen tuning efforts, are ongoing to generate useful neutralizing antibodies. The impact of surface MPER density and adjuvant choice are key elements of this investigation that are now well studied. Activators of the STING pathway, such as cyclic dinucleotides, are being used to effectively enhance antibody titer ten-fold. By incorporating microengraving methods of the Love laboratory at MIT, individual bone marrow plasma cells elicited by such immunization can be interrogated and monoclonal antibodies rescued using single cell PCR cloning for repertoire analysis and characterization. In this regard, we recently demonstrated that several immunizations with MPER/liposomes induce high levels of bone marrow long-lived plasma cell (LLPC) antibody production. Single-cell immunoglobulin gene retrieval analysis shows that these plasma cells are derived from a germ line repertoire of B cells with a diverse representation of immunoglobulin genes, exhibiting antigen- driven positive selection. Characterization of LLPC recombinant monoclonal antibodies (rMAbs) indicates that antigen recognition is achieved through convergence on a common epitopic focus by utilizing various complementarity-determining region H3 (CDRH3) lengths. Importantly, the vast majority of rMAbs produced from these cells lack polyreactivity yet manifest antigen specifi city in the context of lipids, shaping MPER-specifi c paratopes through selective pressure. Taken together, these fi ndings demonstrate that the MPER is a vaccine target with minimal risk of generating

(Cont.)

Reinherz: HIV clade C MPER immunogens eliciting 10E8-like specificities

off-target autoimmunity.

Inclusion of the TM segment of gp41 with the MPER in microparticle display and its impact on immunogenicity is a focus of intense interest for the Consortium. While structural characterization of epitope-paratope pairs has contributed to the understanding of antigenicity, by contrast, few structural studies relate to immunogenicity, the process of antigen-induced immune responses in vivo. Using a lipid-arrayed MPER as a model antigen, we investigated the influence of physicochemical properties on immunogenicity in relation to structural modifications of MPER/liposome vaccines. Anchoring the MPER to the membrane via an alkyl tail or transmembrane domain retained the MPER on liposomes in vivo, while pre-serving MPER secondary structure. However, structural modifications that affected MPER membrane orientation and antigenic residue accessibility strongly impacted induced antibody responses. The solvent exposed MPER tryptophan residue (W680) was immunodominant, focusing immune responses despite sequence variability elsewhere. Nonetheless, immunogenicity could be readily manipulated using site-directed mutagenesis or structural constraints to modulate amino acid surface display. These studies provide fundamental insights for future immunogen design aimed at targeting B cell antibody responses, including induction of bNabs.

Although we have generated antibodies with high specificity against the MPER that lack polyreactivity, no BNAbs emerged. Antibodies elicited to the MPER in liposomes do not react with gp160 trimers expressing the same MPER sequence as arrayed on the liposome vaccines, unlike 4E10, 2F5 or 10E8. This striking result emphasizes a limitation of the current immunization strategy, namely that antibody can vector onto the lipid bilayer to bind MPER in myriad orientations. However, on the virion, the MPER is largely occluded by the three outer blades of the trimer. To the contrary, 2F5, 4E10 and 10E8 all approach the MPER at a tilted/flat angle relative to the membrane We reason that there will be a much greater chance to generate BNAbs if we recapitulate this restricted approach angle. To achieve this goal, advanced nanodisc technologies developed in the Wagner lab are being exploited. We shall utilize the spacing between two opposing nanodiscs to restrict antibody accessibility. In these immunogen designs, we can leave the MPER flat or, alternatively, lift the N-helix of the MPER off the lipid nanodisc surface by attaching it to an opposing nanodisc via cysteine coupled thiol chemistry, further tunable by DNA double-stranded oligonucleotides to form an MPERTM embedded nanodisc wafer. The distance between the two opposing nanodiscs is adjusted by DNA pillar length to approximate the space between gp160 trimer blades and the viral membrane.

(Cont.)

Shattock: DNA Vaccination for HIV Immunogen Discovery

PrincipalInvestigatorRobin Shattock, PhD

GranteeInstitutionImperial College, London, UK

Project TitleDNA Vaccination for HIV Immunogen Discovery

OPPID1084580


Grant at a GlanceOVERVIEWA globally effective HIV-1 vaccine will likely need to induce broadly neutralizing antibodies (bNAbs) against HIV-1. This proposal recognizes the strategic importance of performing iterative small human studies to accel-erate HIV-1 immunogen discovery, where DNA vaccines offer the fastest and most cost effective approach for rapid screening of multiple immunogens. In contrast to previous approaches that aimed to only develop DNA immunization as a practical product development platform, this project aims to leverage DNA immunization as a platform to accelerate the discovery of HIV-1 immunogens capable of inducing bNAbs in humans. Additional benefi t will be provided by the potential establishment of a platform for rapid generation of human monoclonal antibodies to antigens per se that could be used for passive immunization not only against HIV but also for other neglected diseases.

To meet this aim this project seeks to apply recent state of the art technological advances in DNA vaccination and immune monitoring, both at the single cell and molecular level, to enable detailed probing of developing vaccine induced antibody responses. In this respect it represents the fi rst attempt in humans to use a DNA vaccine approach to investigate the development and focusing of B cell responses to vaccination. Should this approach be successful it will provide an important new strategy for rapidly conducting systematic clinical research studies in humans aimed at moving the HIV-1 vaccine fi eld closer to achieving the key objective of identifi cation of immunogens and vaccine strategies required for induction of bNAbs in humans.

Success of this project will be indicated on the basis of meeting the critical milestone: induction of suffi cient B cell responses by DNA vaccination to facilitate antibody repertoire analysis. Successful achievement of the “go” criteria will trigger an expanded program of experimental iterative clinical DNA vaccine studies to identify and refi ne immunogens capable of driving B cells along rare but desirable maturation pathways towards the development of bNAbs. This would allow rapid up-selection of promising new approaches reducing the risk of failure in advanced clinical development.

Prof Shattock (PI) and Imperial College (IC) have considerable experience in running human vaccine studies and coordinating collaborative partner-ships. Phase I clinical trials will be conducted at the IC and St Thomas’ clinical research facilities under the joint direction of Profs Sheena McCormack, Julie Fox and Shattock. This project will engage all CAVD central services facilities including the CTVIMC to ensure full T cell evaluation, the CAVIMC to ensure comprehensive analysis of antibody function, the CVISC to ensure appropriate biostatistical analyses of clinical study data and IAVI’s VxPDC for product development support. In addition this project will aim to work through the Foundation to engage ATRECA in parallel genomic and systems-level bioinformatics to capture a deeper genetic record of the evolving vaccine induced B cell responses to DNA vaccination.

Wayne Koff (IAVI), Barton Haynes (Duke CHAVI-ID), Dennis Burton (Scrips CHAVI-ID) and John Mascola (NIH VRC) will serve on the scientifi c advisory board of this project. If this project is successful in meeting its “go” criteria and were an expanded project funded, the members of the advisory board would become active partners in any expanded program. Engagement of this larger consortium will maximize the potential impact of a human DNA vaccine program focused on identifi cation of immunogens and immunization strategies that aim to build a comprehensive roadmap to drive B cells along rare but desirable maturation pathways towards the development of bNAbs. This would provide an essential springboard to accelerate HIV vaccine research and development, a key strategic component of the Foundation’s HIV/AIDS program.

RESEARCH OBJECTIVES1.) Optimization of DNA vaccination to maximize B cell responses

2.) Characterization of evolving B cell repertoire in response to DNA vaccination

PROGRESSAll regulatory and ethical approvals are in place. All vaccinations and follow-up visits have been completed at the Trial Site. The Trial Site will be closed as soon as fi nal monitoring has been performed. Research analysis has been completed and sent through to EMMES, who have drafted the CSR. This document will be fi nalized soon. Research analysis to be written up for publication and all trial documentation archived by Dec 18.

Shaw: Novel SHIV Design to Elicit Broadly Neutralizing Antibodies and Guide Iterative Vaccine Development

Principal InvestigatorGeorge Shaw, MD, PhD

GranteeInstitutionUniversity of Pennsylvania, Philadelphia, PA USA

Project TitleNovel SHIV Design to Elicit Broadly Neutralizing Antibodies and Guide Iterative Vaccine Development

OPPID1145046

Grant AwardUp to $3.6 million, awarded in January, 2016

Grant at a GlanceOVERVIEWThe goal of this investment is to develop simian-human immunodeficiency viruses (SHIVs) that elicit strain-specific and broadly neutralizing antibodies in rhesus macaques (RMs) as a guide to iterative HIV-1 vaccine development. Historically, models using rhesus macaques and employing challenges with SHIVs have had disappointing outcomes. SHIVs generally failed to elicit broadly neutralizing antibodies (bNAbs) directed against the envelope protein (Env) of HIV-1 and failed to recapitulate the course of antibody evolution seen in naturally infected humans. The hypothesis underlying this grant is that this discrepancy stems from properties of the HIV-1 Envs used in creating the SHIVs, as these have frequently been chosen from strains containing fortuitous mutations that provide for binding to rhesus CD4 (rhCD4), but often at the cost of reduced confor-mational masking and protection from antibody neutralization.

The approach here is to engineer SHIVs from an SIVmac251 vector backbone by inserting the complete tat-rev- vpu-env (gp160) cassettes of transmitted/founder (T/F) or otherwise desirable primary HIV-1 strains, and to modify the Env residue 375 which lines the Phe43 binding cavity and thereby facilitate rhCD4 engagement. Substitutions at Env 375 result in enhanced binding to rhCD4, and SHIVs expressing these modified Envs replicate consistently and persistently in Indian-origin rhesus macaques, eliciting strain-specific, autologous tier 2 neutralizing antibodies similar to those found in humans. The new SHIV design further leads (in some animals) to CD4 T cell decline, immunopathology and AIDS-related death. Importantly, the Envs of all SHIVs tested retained effective conformational masking, tier 2 antibody sensitivity, and antigenicity virtually indis- tinguishable from their wild type S375 counterparts. This strategy has been elaborated to develop SHIVs containing the BG505 Env gp140, where allelic variants of BG505 Env at codons S375Y/W/H and T332N support efficient virus infection and replication in RM CD4 T cells in vitro and in vivo.

This project will test 12 allelic variants of SHIV BG505 for replication in RMs in vivo and will down-select the variants to two: one with a T332N substitution like the variant used to make BG505-SOSIP, and one without a T332N substitution like the variant that infected the human subject who subsequently developed bNAbs. The two SHIV BG505 variants will then be used to infect RMs; then virus-Ab coevolution will be analyzed, including determining strain-specific and bNAb responses, cloning the neutralizing mAbs, and inferring the unmutated common ancestor immunoglobulin receptors (UCA-IgRs). This same experimental strategy will be extended to make additional novel SHIVs including those bearing the Envs of HIV-1 B41 and others that target human V2 bNAb lineage germ line (GL) Ig receptors, including a novel SIVcpz variant MT145. The variants generated under this aim will be down-selected on the basis of in vitro experiments, with three SHIVs advancing to evaluation in the rhesus model.

Grant Leadership

This grant is led by George Shaw, MD, PhD (University of Pennsylvania), with consultation by other CAVD investigators working in the research space centered on anti-Env bNAb development. The award was made in January 2016, with an anticipated duration of 36 months.

RESEARCH OBJECTIVES1.) Initial evidence of persistent SHIV replication and strain-specific and heterologous nAb induction in rhesus macaques by new BG505 SHIVs.

2.) Generation of a large volume, high titer BG505-SHIV challenge stock, titrated in rhesus macaques for preclinical challenges.

3.) Optimization of heterologous bnAb induction in the SHIV BG505 infection model, using down-selected variants assessed under objective 1.

4.) Construction and evaluation of additional SHIVs including those bearing Envs of HIV-1 B41 and others that have been shown to bind to human V2 targeted bnAb lineage germline Ig receptors, including a novel SIVcpz variant MT145.

5.) Description of SHIV/Env-Ab co-evolution that leads to bnAb induction in rhesus macaques as a guide for iterative HIV-1 vaccine design.

RESEARCH OUTCOMES1.) BG505 SHIVs with or without an N332 glycan replicated effi ciently in 18 Indian RMs. In each case, strain-specifi c NAbs developed targeting

glycan holes at residues 241/289 or C3/V5. In two RMs, potent bNAbs targeting the V3 high mannose patch, N332 glycan and GDIR motifs developed, thus demonstrated the immunogenic potential of BG505 Env trimer. Rhesus V3 glycan bNAb mAbs are being cloned for use in HIV vaccine design and animal model testing.

2.) BG505.N332 SHIV was grown in primary rhesus CD4 T cells as a challenge stock and used to demonstrate, for the fi rst time, SOSIP-mediated protection from mucosal infection of a primary transmitted/founder Env containing SHIV. “Threshold” titers of serum NAbs required for 50% or 90% clinical protection from mucosal infection of a primary (T/F) virus strain were established in the RM model.

3.) Six primary HIV-1 Envs that bind the UCAs of human V2 apex bNAbs were constructed into SHIVs using the Δ375 strategy. All replicated effi ciently in RMs. All six SHIVs elicited V2 apex NAbs with a subset of animals achieving neutralization breadth. bNAbs targeted the V2 apex C strand and were variably depend on N160 and/or N156 interactions, with escape patterns mirroring those observed in humans. Cloning of rhesus V2 apex bNAb mAbs is underway.

4.) The Env of a novel SIVcpz variant MT145 that shows antigenic cross-reactivity to human V2 apex bNAbs and their respective UCAs was constructed into a SHIV and shown to replicate effi ciently in RMs, resulting in V2 apex C strand targeted NAbs. This Env has been advanced for human phase 1 testing and preclinical testing in human Ig knockin mice and RMs.

Philadelphia, PA USAPhiladelphia, PA USA

Überla: Antibodies without HIV T helper cells

PrincipalInvestigatorKlaus Überla, MD

GranteeInstitutionUniversity Hospital Erlangen, Germany

Project TitleInduction of affi nity matured HIV Env antibodies in the absence of HIV specifi c T helper cells

OPPID1040727

Grant AwardUp to $2.4 Million, awarded September, 2012

Collaborating Institutions◊ German Primate Center, Göttingen,

DE

Grant at a GlanceOVERVIEWThe HIV vaccine field has shifted over the years from focusing on vaccines that primarily induce neutral-lizing antibodies to those focusing on T cell responses (i.e. Merck STEP trial) to ones that attempt to elicit both humoral and cellular immune responses. The failed Merck T-cell vaccine was both disappointing and surprising in that not only did the vaccine fail to protect but an ad hoc analysis showed that there was an increase in the risk of acquisition of infection in some subjects. The favored hypothesis is that the vaccine may have increased the risk of acquisition of infection in some individuals by increasing the number of HIV specific activated CD4 T cells, which are the target of HIV replication, at the time of natural infection or during the early stages of viral infection in the face of non-sterilizing immunity. Understanding the mechanism(s) of increased risk of infection in the STEP study is of the utmost importance as several new HIV vaccine candidates are presently moving into the clinic in the form of “adeno vector prime/boost” vaccines. Identifying approaches that avoid the issues observed in the STEP trail has the potential to significantly impact vaccine design.

The experimental vaccine proposed by Dr. Klaus Überla from University Hospital Erlangen employs a prime/boost strategy of vaccination - Ad5 expressing a heterologous (non-HIV or non-SIV in the case of a SIV or SHIV challenge model) retroviral Gag (intrastructural) antigen to prime CD4 T cells followed by boosting with the same heterologous Gag protein encapsulated in an Env virus like particle (VLP). Since the Gag proteins in the VLP are not physically linked to Env it is expected that the native Env structure is preserved. Immunization against the non-HIV Gag proteins provided heterologous “intra-structural” T cell help for Env-specific antibody responses improving the magnitude and the quality of anti-Env antibodies in mice. Non-human primate studies will reveal, whether avoiding HIV specific CD4 T cells that may have contributed to the findings of the STEP trial indeed improve vaccine efficacy.

These experiments will generate information that addresses two key issues in HIV vaccinology: 1) does boosting HIV specific CD4 T helper cell responses required for producing protective antibodies detract from vaccine efficacy and 2) what immunological parameters are associated with the risk of acquisition of infection observed after immunization in humans with an adenoviral vector 5 (Ad5) non-structural protein T cell vaccine (STEP trial). The outcome of this work will be to inform more rational HIV vaccine design.

The studies will be performed at Virology Institute of the University Hospital Erlangen in close collaboration with Dr. Christiane Stahl-Hennig´s team from the German Primate Center.

RESEARCH OBJECTIVES1.) Induce affi nity-matured antibodies to the Env trimer by recruitment of T-helper cells specifi c for heterologous vaccine antigens

2.) Assess the effi cacy of the heterologous intra-structural help vaccine in a low dose NHP challenge model

3.) Characterize the type of vaccine-induced immune responses that are associated with enhanced acquisition of infection

Walker: bNAbs After Infection and Immunization

PrincipalInvestigatorBruce Walker, MD


Project TitleDevelopment of broadly neutralizing antibodies in HIV infection and following immunization

OPPID1066973

Grant AwardUp to $12.9 Million, awarded in November, 2012

Collaborating Institutions◊ Atreca, USA◊ Jessen-Jessen Medical Practice,

Germany◊ University of KwaZulu-Natal, South

Africa◊ Umkhuseli Innovation and

Research Management

Grant at a GlanceOVERVIEWThe development of a safe and effective HIV vaccine is the fi nal missing piece in the complex puzzle of how to attain affordable, durable control of the HIV/AIDS pandemic. At present there are promising leads, but no clear and unequivocal path, to the development of an HIV vaccine. It is likely that the fi eld will have to overcome the key challenge of eliciting a broad neutralizing antibody response in the majority of vaccinated individuals, but so far, candidate HIV vaccines have not elicited any signifi cant levels of broadly neutralizing antibodies (bNAbs). This grant directly addresses the neutralizing antibody problem using innovative technology that may be transformative in the quest for an HIV vaccine.

By stepping back from the problem and taking a comprehensive and unbiased look at how broad neutralizing activity arises in the approximately 25% of HIV-infected individuals who do develop it, this grant proposes to effi ciently learn the circumstances under which bNAbs are elicited, and, correspondingly, what vaccines need to do to elicit them. The project will be anchored by technology developed by Atreca, Inc., which represents a powerful, high-throughput, unbiased approach to understanding the antibody response to infection and vaccination. The earliest B-cell responses in HIV infection, including those active just before infection, will be studied and followed prospectively for up to three years in human volunteers who face an exceptionally high rate of HIV infection in KwaZulu-NatalZN, South Africa, along with other cohorts with more immediately available samples. The study in South Africa (called “FRESH”) will be unique in its capacity to enroll and follow volunteers twice-weekly with a fi nger-stick blood sample, permitting ascertainment of HIV infection as soon as any trace of the virus is detectable in the bloodstream. Evaluation of the status of the B-cell response before infection, during the fi rst days and weeks of infection, and for the next 2-3 years will be enabled, and the follow-up period will allow identifi cation of those who do and do not go on to develop broad neutralizing activity. For this work the researchers have defi ned broad neutralizing activity as a continuum, ranging from neutralization of only the infecting virus strain (called autologous neutralization) at one end, and equivalent to the very best broadly neutralizing antibodies (bNAbs) at the other, in order to capture all stages of bNAb development. Moreover, the study will include a broader assessment of innate and adaptive immunity, building up a complex of immune data that will provide important context for understanding the factors that support bNAb development. It represents a signifi cant step away from a purely empirical approach to HIV vaccines.

This consortium is led by Bruce Walker, a leading HIV researcher who excels at assembling informative cohorts for studies of immune protection against HIV. A team of investigators from the Ragon Institute of MGH, MIT, and Harvard, will join with investigators from the University of KwaZulu- Natal (SA) and Atreca, to conduct different aspects of the study.

RESEARCH OBJECTIVES1.) To collect longitudinal samples from individuals with acute HIV-1 infection to investigate the development of broad neutralizing responses

2.) To defi ne the B-cell signatures associated with the induction of HIV-specifi c neutralizing antibodies using the Atreca technology and other measures of B-cell development and maturation

3.) To defi ne the HIV-specifi c CD4+ T-cell responses and their relationship to the establishment of broad neutralizing responses

4.) To identify transcriptional signatures of HIV-specifi c broad neutralizing activity using a systems biology approach applied to relevant cell subsets of the immune system

5.) To obtain large volume blood samples for characterizing the parameters associated with breadth of neutralizing antibodies in HIV controllers with different degrees of antigen load and diversity, and with relatively good preservation of immune function

PROGRESSThe FRESH Cohort (Females Rising through Education, Support and Health) of women at high risk for HIV infection was established in KwaZu- lu-Natal Province, South Africa, where infection rates of around 9% per year have been documented. The study was originally intended to follow persons with untreated infection, but soon after the study was initiated evidence suggested that early treatment with antiretroviral therapy could improve outcomes. Prior to changes in the South African treatment policies we were given permission to initiate therapy at the initial diagnosis of infection, allowing us to implement therapy before peak viremia. The clinic site is at a shopping mall where each participant is seen twice weekly and given classes designed to empower them by teaching life skills, job skills, and help with obtaining a high school degree. Each time the partici-pants come in they also have a fi nger prick blood draw looking for acute HIV infection and pre and post-infection blood samples are being obtained for studies of the ontogeny of neutralizing antibodies. The seroconversion rate remained approximately 8% per year, and new infections continue to occure despite implemental of PrEP.

Studies of the earliest immune responses induced, prior to peak viremia, and longitudinal responses in these have been conducted. The immunology studies are being done at the Doris Duke Medical Research Center in Durban, as well as the adjacent Africa Health Research Institute (AHRI, formerly KwaZulu-Natal Research Institute for TB and HIV (K-RITH)). Because of the implementation of ART, we are not able to follow the devel-opment of broadly neutralizing antibodies, but instead have turned our attention to assessing the impact of limited antigen exposure on the devel-opment of adaptive cellular and humoral immune responses.

Longitudinal and cross-sectional studies of the CD4 T cell and B cell signatures associated with the development of broadly neutralizing antibodies (Cont.)

Walker: bNAbs After Infection and Immunization

have been performed in the HIV Controller cohort, and cell subsets have been isolated for evaluation of transcriptional signatures of HIV specific broadly neutralizing antibodies. The BCR repertoire of both antigen-specific memory B cells and plasmablasts has already been generated by Atreca on eight spontaneous controllers who exhibit broad neutralizing antibody responses. More than 2500 sequences have now been collected, and phylogenetic tress have been constructed per individual as well as for the whole cohort. Preliminary studies on 16 monoclonal antibodies generated from the first three sequenced subjects showed antigen specificity and neutralizing capacity. Seventy antibodies have now been selected from the second batch of five controllers, based on high levels of somatic hypermutation and long CDRH3 lengths, to maximize the likelihood of selecting antibodies with potent antiviral activity. Interestingly, unique patterns of clonal diversification are observable among all sequenced subjects; however, massive diversification of single clonal families appears to be a hallmark of the most potent neutralizers. Additional analyses using sophisticated computational approaches developed by Atreca are underway to gain deeper insights into the pathways and mechanisms by which neutralizing B cell responses are selected.

(Cont.)

Walker: Implications for vaccine design from T and B cell mechanisms of HIV control

PrincipalInvestigatorBruce Walker, MD


Project TitleIdentifi cation of new bNAbs in Elite Controllers and implications of early ARV on acute infection in a clade C cohort

OPPID1146433

Grant AwardUp to $4.8 million, awarded in June 2016

Collaborating Institutions◊ KwaZulu Natal Research Institute

for TB and HIV, Durban ZA◊ The Doris Duke Medical Research

Institute, Durban ZA

Grant at a GlanceOVERVIEWEfforts under this grant will characterize the ontogeny and immunologic signatures of neutralization breadth in unique cohorts of HIV infected persons, thematic elements initiated under a prior CAVD award. One of the main outcomes from this branch of the program will be the identification and characterization of new bnAbs in HIV controllers, for potential clinical development. A secondary objective for this new grant is to define the phenotype, specificity, and function of early T and B cell responses generated in peripheral blood and lymph nodes following early treatment in acute infection.

For bnAb discovery, the program will make use of the HIV Controllers cohort, individuals who maintain viral loads of less than 2000 RNA copies/ml plasma without the need for combination antiretroviral therapy (cART). The HIV Controllers cohort was used to previously identify 3BNC117, a broad and potent CD4-binding site bnAb where proof of concept studies have established clinical anti-viral activity. In collaboration with the CAVIMC, the neutralization breadth has already been characterized in over 600 HIV controllers, and the ongoing work will define the antibody repertoires generated in those with the most potent neutralization breadth. The latter effort includes the expression of a refined set of B cell receptors (BCR) selected from the 17,000 BCRs already sequenced from this controller cohort. Selection of this set will use an evolutionary profiling analytical algorithm developed by Tom Kepler. In addition, next-generation native-like trimers that omit binding of non-neutralizing antibodies (e.g. SOSIP, fold-on trimer, etc.) will be used to pull out additional rare, memory B cells from samples of the top broadly neutralizing controllers, utilizing multiple tissues, including lymph nodes and bone marrow.

Under the secondary objective, investigations will endeavor to define the phenotype, specificity, and function of early T and B cell responses generated in peripheral blood and lymph nodes following early treatment in acute infection. By focusing on this period of infection before peak viremia, and by initiating immediate therapy in these persons, it will be possible to define the phenotype, function and specificity of the earliest T and B cell responses that occur in blood and lymph nodes in the absence of ongoing viremia, CD4+ T cell depletion and T cell exhaustion. This research component will engage the FRESH cohort (“Females Rising through Education, Support and Health”, Umzali Township, Durban, see https://youtu.be/D8SY98CZiYY), a cohort of South African women identified at the time of “hyperacute infection”, defined as the period from first detectable viremia to peak viremia.

Grant LeadershipThe grant is led by Bruce Walker, MD at the Massachusetts General Hospital. The investigative team includes participants from the Ragon Institute of MGH, MIT and Harvard, from the Doris Duke Medical Research Institute, and the Africa Health Research Institute (AHRI, formerly K-RITH). The work will include extensive engagement of the CAVIMC for viral neutralization assays, and BCR characterization will be performed in collaboration via Atreca, to be secured via the GH-VAP. The award was made in June 2016 with an anticipated duration of 24 months, and is now in a no-cost extension.

RESEARCH OBJECTIVES1.) To identify and clone novel broadly neutralizing antibodies (bNAbs) of unique specifi city in HIV controllers for clinical development.

2.) To defi ne the phenotype, specifi city and function of the earliest T and B cell responses generated in the peripheral blood and in lymph nodes following treatment-limited antigen exposure in acute HIV infection.

Ward: Establishment of an Analytics Network to Support Qualifi ed/Validated Assays Required for Characterization of HIV Env Immunogens

PrincipalInvestigatorAndrewWard

GranteeInstitutionThe Scripps Research Institute, La Jolla, USA

Project TitleEstablishment of an Analytics Network to Support Qualifi ed /Validated Assays Required for Characterization of HIV Env immunogens

OPPID1115782

Grant AwardUp to $3.6 million, awarded in June, 2015

Grant at a GlanceOVERVIEWEliciting broadly neutralizing antibodies (bnAbs) that target the envelope glycoprotein (Env) spike on the surface of HIV-1 is thought to be required for a successful HIV vaccine. Env is a highly glycosylated trimer of heterodimers composed of gp120 and gp41 subunits. While a large number of bnAbs from naturally infected patients have been isolated and characterized, little is known regarding how to elicit such antibodies with a vaccine. A number of recent efforts have been placed on developing trimeric Env proteins that preferentially expose bnAb epitopes while masking non-nAb epitopes, similar to the native spike. This task has proven quite difficult because Env is a meta-stable type I viral fusion machine that has a large number of disulfide bonds that must be correctly formed during the folding process.

We have demonstrated that a well-folded, native-like trimer has implications for antigenicity, immunogenicity, glycosylation pattern, and thermostability. It requires several assays to accurately characterize these qualities of Env trimers. These include i) differential scanning calorimetry (DSC) to assess stability, (ii) bio-layer inter-ferometry (Octet) to assess antigenic profile, (iii) antibody capture and size exclusion chromatography to assess purity, (iv) negative stain EM to assess conformational and compositional heterogeneity and antige-nicity, and (v) high-resolution cryoEM to assess the molecular details of Env trimers. Moreover, we conduct such assays with rigorous controls/comparators in order to avoid misinterpretation and flawed conclusions. Overall, we have developed a pipeline with these methods to rapidly analyze and compare Env trimer candi-dates, furnishing results in a timely manner as Env Immunogens are devised and produced for immunization programs. These data can then be directly compared to the outcomes from animal immunization experiments, thereby informing rational vaccine design and facilitating iteration between structure-based Env design and empirical vaccination. We have adapted our imaging pipeline and leveraged our considerable database of trimer-antibody images to map polyclonal antibody responses during vaccine trials. We and others are using this information to guide immunogen redesign with the goal of guiding immune responses toward development of broadly neutralizing antibodies.

We also conduct the above assays using adjuvant formulated immunogens, conditions that we refer to as stress tests, in order to ensure high quality reagents are delivered to animal and human immunization experiments. This includes a recently produced cGMP batch of BG505 SOSIP.664 trimers suitable for human clinical trials. Finally, we have begun to construct and characterize Env trimer presenting nanoparticles to potentially improve immunogenicity.

This grant is led by Andrew Ward, PhD (The Scripps Research Institute). The award was received in June, 2015 with an initial agreement length of 5 years.

RESEARCH OBJECTIVES1.) To perform structural and biophysical characterization on up to 1000 Env trimers and/or trimer antibody complexes per year.

2.) To produce a variety of full-length Env trimers for structural, antigenic, and glycan profile characterization that will serve as important compar-ators to the soluble, native-like Env trimers being developed as immunogens.

3.) To provide structural information and support for CAVD vaccine design efforts, e.g. the “Nearest Neighbor” consortium.

RESEARCH PROGRESS1.) In the first three years of the award we have analyzed almost 2300 Env immunogens and Env-antibody complexes from an international network

of over 30 collaborating labs from academic and industrial partners. ~100 of these samples have been from the “Nearest Neighbor” consortium and include Env trimer presenting nanoparticles.

2.) This work has resulted in nearly 60 publications and nearly 200 EMDB structure depositions ranging in resolution from 3.3 Å to ~20 Å.

3.) We have supported production and characterization of Env trimer immunogens for many animal immunization experiments and are involved in developing the GMP process for production of trimer immunogens for human vaccine trials.

4.) We have also developed and implemented a real-time EM based analysis of the polyclonal response in ongoing vaccine trials.

Research Institute, Research Institute,

Watson: Messenger RNA Encoded Abs for HIV Prophylaxis

Principal InvestigatorMike Watson, PhD

Grantee InstitutionValera, LLC, Cambridge, MA USA

Project TitleMessenger RNA encoded antibodies for HIV prophylaxis

OPPID1147797

Grant AwardUp to $20 million over 48 months

Grant at a GlanceOVERVIEWThis grant is focused on the development of mRNA-encoded antibodies to be used as prophylactic treatment against HIV. The research will be performed by Valera, LLC; Valera is an affiliate of Moderna Therapeutics, focused exclusively on the advancement of vaccines and therapeutics for the prevention and treatment of viral, bacterial and parasitic infectious diseases. It will utilize Moderna’s modified messenger RNA (mRNA) platform, a technology that Valera has already utilized in preclinical settings to generate safe and effective prophylactic vaccines against multiple viruses. The enabling advantages of this technology are the ability to generate cocktails of antibodies as a single product, cost of production, and potentially increased duration of protein expression.

The program has a multi-stage framework.

• Stage One will involve production/formulation of mRNA constructs for both heavy and light chain of a selected set of broadly neutralizing anti-Env antibodies, for assessment of protein expression levels in vitro and in vivo (rodent models), allowing for sequence optimization of variants resulting in higher expression of antibodies.

• Stage Two proceeds by engineering the top candidates from Stage One into single chain versions that retain both variable and constant regions (scFv-Fc antibodies). Expressed versions of these scFv-Fc antibodies will be evaluated by affinity measurements, neutralization behavior, glycosylation, other biophysical properties, as well as other functional characteristics. The mRNA constructs for the ScFv-Fc antibodies, spanning a variety of sequence variants, will evaluated for expression levels in murine models.

• Stage Three advances optimization in a research context, with selection of final mRNA constructs, and initial preclinical studies in rat and rhesus, testing ScFv-Fc’s individually and as cocktails of antibodies. Evaluation in rhesus will have a challenge arm, as well as study arms for PK data capture.

• Stage Four is primarily focused on completing GLP toxicology studies in mouse and rhesus, with the prerequisites steps of Active Pharma- ceutical Ingredient (API) and Drug Product (DP) manufacturing, with related analytics.

• Stage Five requires the GMP manufacturing of the API and DP for a Phase I study, using a CRO as subcontractor for the execution of the study.

This grant is led by Mike Watson, PhD, in the role of Chief Scientific Officer for Vallera, LLC. Stages of the work will involve service contracts with a number of entities yet to be determined. The Central Services Facilities will be engaged during multiple stages to furnish functional assays such as neutralization testing and binding assays, and discrete analytical evaluations such as glycosylation features. The award began in January 2016 and has an anticipated duration of 48 months.

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