of April 17, 2017.This information is current as

Persistent Viremia Vaginal Acquisition andmac251from SIV

T Cell Responses in Protection+ and CD8+Antibody to the gp120 V1/V2 Loops and CD4

Genoveffa FranchiniBarney S. Graham, Douglas R. Lowy, John T. Schiller andM. Xenophontos, David Venzon, Marjorie Robert-Guroff, Montefiori, Michael Piatak, Jr., Jeffrey D. Lifson, AnastasiaNicolas Cuburu, Christopher B. Buck, Guido Ferrari, David Poonam Pegu, Namal P. M. Liyanage, Monica Vaccari,Brandon F. Keele, Egidio Brocca-Cofano, Yongjun Guan, Shari N. Gordon, Melvin N. Doster, Rhonda C. Kines,

http://www.jimmunol.org/content/193/12/6172doi: 10.4049/jimmunol.1401504November 2014;

2014; 193:6172-6183; Prepublished online 14J Immunol 

MaterialSupplementary

4.DCSupplementalhttp://www.jimmunol.org/content/suppl/2014/11/14/jimmunol.140150

Referenceshttp://www.jimmunol.org/content/193/12/6172.full#ref-list-1

, 27 of which you can access for free at: cites 54 articlesThis article

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The Journal of Immunology

Antibody to the gp120 V1/V2 Loops and CD4+ and CD8+

T Cell Responses in Protection from SIVmac251 VaginalAcquisition and Persistent Viremia

Shari N. Gordon,* Melvin N. Doster,* Rhonda C. Kines,† Brandon F. Keele,‡

Egidio Brocca-Cofano,x Yongjun Guan,{ Poonam Pegu,* Namal P. M. Liyanage,*

Monica Vaccari,* Nicolas Cuburu,† Christopher B. Buck,† Guido Ferrari,‖

David Montefiori,‖ Michael Piatak, Jr.,‡ Jeffrey D. Lifson,‡ Anastasia M. Xenophontos,*

David Venzon,# Marjorie Robert-Guroff,x Barney S. Graham,** Douglas R. Lowy,†

John T. Schiller,† and Genoveffa Franchini*

The human papillomavirus pseudovirions (HPV-PsVs) approach is an effective gene-delivery system that can prime or boost an

immune response in the vaginal tract of nonhuman primates and mice. Intravaginal vaccination with HPV-PsVs expressing SIV

genes, combined with an i.m. gp120 protein injection, induced humoral and cellular SIV-specific responses in macaques. Priming

systemic immune responses with i.m. immunization with ALVAC-SIV vaccines, followed by intravaginal HPV-PsV–SIV/gp120

boosting, expanded and/or recruited T cells in the female genital tract. Using a stringent repeated low-dose intravaginal challenge

with the highly pathogenic SIVmac251, we show that although these regimens did not demonstrate significant protection from virus

acquisition, they provided control of viremia in a number of animals. High-avidity Ab responses to the envelope gp120 V1/V2

region correlated with delayed SIVmac251 acquisition, whereas virus levels in mucosal tissues were inversely correlated with

antienvelope CD4+ T cell responses. CD8+ T cell depletion in animals with controlled viremia caused an increase in tissue virus

load in some animals, suggesting a role for CD8+ T cells in virus control. This study highlights the importance of CD8+ cells and

antienvelope CD4+ T cells in curtailing virus replication and antienvelope V1/V2 Abs in preventing SIVmac251 acquisition. The

Journal of Immunology, 2014, 193: 6172–6183.

Thedevelopment of a vaccine that prevents HIVacquisitionremains a formidable challenge. Most currently licensedprotective viral vaccines induce neutralizing Abs that

mediate long-lasting immunity. However, broadly neutralizing Abstake an average of 2.5 y to develop during natural HIV infection (1)and often have extensive somatic hypermutation (2), a property

likely to be difficult to induce via vaccination. In addition, clinicaltrials using a protein vaccine that primarily induced Ab responses

failed to prevent HIV infection (3, 4) and led to increased em-

phasis on vaccines that induce HIV-specific T cell responses.

However, vaccines that induced robust T cell responses failed to

prevent HIV infection in clinical efficacy trials (Merck STEP trial-

HVTN 502, HVTN 503, and HVTN 505) (5–7). In addition, in

some of the trials, a higher number of infections occurred in

vaccinated individuals than in the placebo arms. All three trials

included systemically administered adenovirus vectors, and al-

though the role of vector-specific responses remains unclear, the

results suggest that systemic CD8 T cells alone are not sufficient

to prevent HIV acquisition.The RV144 Thai trial was the first HIV vaccine clinical trial to

demonstrate measurable protective efficacy. Vaccination signifi-

cantly reduced the risk of HIV infection, with an estimated efficacy

of 31.2% (8). The vaccine regimen consisted of an i.m. injection of

the canarypox vector ALVAC expressing HIV genes, paired with

a bivalent envelope protein gp120 boost. This regimen induced

mainly nonneutralizing Abs and CD4+ T cell responses (8, 9). Abs

directed to the V1/V2 region of gp120 were found to be a primary

correlate of a reduced risk of HIVacquisition, whereas Ab-dependent

cellular cytotoxicity (ADCC) was a secondary correlate (9). These

findings highlighted the potential of vaccine-induced Abs in pre-

venting HIV acquisition. Binding nonneutralizing functional Abs

could prevent virus entry and dissemination by impairing virus

mobility at the portal of entry, or by destroying newly infected

cells by activating the complement pathway, and/or coordinating

with macrophages or NK cells (10).

*Animal Models and Retroviral Vaccines Section, National Cancer Institute, Bethesda,MD 20892; †Laboratory of Cellular Oncology, Center for Cancer Research, NationalCancer Institute, Bethesda, MD 20982; ‡AIDS and Cancer Virus Program, Leidos Bio-medical Research, Frederick National Laboratory for Cancer Research, Frederick, MD21702; xVaccine Branch, National Cancer Institute, Bethesda, MD 20892; {Division ofBasic Science and Vaccine Research, Institute of Human Virology, University of Mary-land School of Medicine, Baltimore, MD 21201; ‖Department of Surgery, Duke Univer-sity Medical Center, Durham, NC 27710; #Biostatistics and Data Management Section,National Cancer Institute, Bethesda, MD 20892; and **Vaccine Research Center, Na-tional Institute of Allergy and Infectious Diseases, Bethesda, MD 20892

Received for publication June 26, 2014. Accepted for publication October 18, 2014.

This work was supported by the intramural budget (to G.F. and J.T.S.), by an Officeof AIDS award, and in part by federal funds from the National Cancer Institute underContracts HHSN261200800001E and HHSN266200400088C.

The sequences presented in this article have been submitted to GenBank (http://www.ncbi.nlm.nih.gov/genbank) under accession numbers KF646830–KF647217.

Address correspondence and reprint requests to Dr. Genoveffa Franchini, AnimalModels and Retroviral Vaccines Section, National Cancer Institute, 9000 RockvillePike, Building 41, Room D804, Bethesda, MD 20892. E-mail address: franchig@mail.nih.gov

The online version of this article contains supplemental material.

Abbreviations used in this article: ADCC, Ab-dependent cellular cytotoxicity; HPV,human papillomavirus; MPL, monophosphoryl lipid A; N9, nonoxynol 9; PsV, pseu-dovirion.

Copyright� 2014 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/14/$16.00

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Repeated low-dose mucosal challenges with simian HIV or SIVviruses in macaques are reasonable models of HIV sexual transmis-sion (11). The SIVmac251 challenge used in this study is a pathogenicCCR5 user that is resistant to neutralization, similar to most HIVprimary isolates. To date, HIV vaccine candidates tested in this ma-caque model, using mucosal repeated low doses of SIV, have reca-pitulated the results of HIV clinical trials in humans (12–14).Preventing HIV transmission remains the primary goal of HIV

vaccines; however, once infection has occurred, the reduction ofchronic-phase viremia and the slowing or halting of disease pro-gression are also important objectives. Increasing evidence suggeststhat although a vaccine-induced humoral response is important forprotection from virus acquisition (15–17), CD8+ T cell responsescontribute to virus control after lentiviral transmission (16, 18–20).In the RV144 Thai trial, the ALVAC-HIV/gp120 regimen inducednegligible CD8+ T cell responses, and vaccinees that becameinfected had virus levels and CD4+ T cell counts similar to those ofthe placebo group, requiring the initiation of antiretroviral therapy(21). Multiple lines of evidence implicate CD8+ T cells in thecontrol of HIV/SIV replication; for example, CD8+ T cell depletionof macaques during SIV infection causes a rapid increase in viralburden (22, 23). In addition, during primary HIV infection, thepostpeak decline in viremia is temporally associated with the in-duction of CD8+ T cell responses (24, 25). The immunologicpressure imposed by CD8+ T cells on HIV is evidenced by theemergence of MHC I–restricted escape mutations (26–28). In-triguingly, recent studies have demonstrated potent control of SIVinfection by broadly distributed T cell responses, induced by rhesusCMV vaccine vectors that generate unusual MHC class II–restrictedCD8+ T cells targeting promiscuous SIV epitopes (29, 30).Our goal was to develop a novel vaccine regimen that induces

mucosal CD8+ T cells together with binding functional Abs and toask whether this vaccine regimen alone could protect, or whetherprior priming with systemic immunization could further improveprotection. Human papillomaviruses (HPVs) are small non-enveloped DNA viruses that naturally infect epithelial cells withinthe genital tract; we used HPV-based vectors as a delivery systemto specifically target Ag expression to the vaginal epithelium. Wecreated HPV-pseudovirions (PsVs) that express SIV genes anddelivered them to basal epithelial cells in the female genital tract.Infection in the vaginal tract is facilitated by microtrauma thatallows access to the basal epithelial layers (31). HPV-PsV–me-diated gene expression in the female genital tract has been shownto be transient, lasting for ∼5 d, during which priming of T cells inthe genital draining lymph nodes and Ag recall in the genitalmucosa have been reported in murine models (32, 33). Initiationof an adaptive response is likely enhanced by the adjuvant-likepotential of the HPV capsid, with its ordered protein arrangement(34). HPV-like particles have been shown to induce maturation ofdendritic cells, resulting in the production of IL-6, IL-12, andTNF-a, and may be recognized by TLRs on mucosal cells en-gaging pathogen-associated molecular patterns on the HPV capsid(33, 35, 36). In previous studies, we demonstrated the feasibilityof this vaccine approach using model Ags and the ability of HPV-PsVs to express foreign genes in the vaginal tract and induce HPVcapsid–specific Abs in serum to each HPV serotype (37).In this study, we evaluated whether the local mucosal immune

responses, induced by HPV-PsV vaccines paired with a gp120 proteinboost, might prevent SIVmac251 intravaginal transmission. In addition,because ALVAC/gp120 regimens demonstrate limited but significantprotection from infection in humans as well as in nonhuman primates(8, 13, 20, 38, 39), we examined whether an ALVAC-SIV systemicprime, paired with an HPV-PsV-SIV/gp120 boost, by inducing alsohigher systemic response, could increase vaccine efficacy.

Materials and MethodsAnimals, immunization, and SIV challenge

This study used 36 female rhesus macaques of Indian origin, aged 3.5–7 y.All animals were housed and cared for under the guidelines of the Asso-ciation for the Assessment and Accreditation of Laboratory Animal Care,and the study was conducted with the approval of the Institutional AnimalCare and Use Committee at Advanced BioSciences Laboratories inRockville, MD. The animals were divided into 3 groups of 12 animals,each based on their MHC alleles. In one group, the 12 animals werevaccinated with 108 PFU of ALVAC-SIVencoding SIVmac251 gag, pol, andenv (gp160) by i.m. injection in the thigh at weeks 0 and 4. The ALVAC-SIV vector was made as previously described (40). The 12 ALVAC-SIV–vaccinated animals and 12 additional macaques were vaccinated intra-vaginally with HPV-PsVs expressing SIVmac251 genes (HPV-PsV-SIV) atweeks 6, 10, and 24. HPV-PsVs were produced as previously described(37, 41). Briefly, DNA constructs encoding the capsids of HPV serotypes16, 45, and 58 and DNA constructs encoding SIV gag-pro, gp120, or thereassortant genes rev, tat, and nef, were cotransfected into 293T cells. Theresulting PsVs were purified, propagated, and titered. At 28 d prior to eachHPV-PsV-SIV vaccination, macaques were given 30 mg/kg Depo-Proverai.m. to thin the vaginal epithelia, and 1 wk prior to vaccination, macaqueswere treated with antibiotics to prevent vaginosis. At 6 and 24 h beforevaccination, a vaginal application of nonoxynol 9 (N9), a nonionic de-tergent, was administered as a 10% gel mixed with 4% carboxymethylcellulose (Sigma-Aldrich, St. Louis, MO). N9 induces microtrauma in theepithelia, which facilitates HPV-PsV vaccination. At 6 h after the last N9treatment, a standard 500 ml inoculum, consisting of 1010 IU (infectiousunits) of HPV-PsVs and carboxymethyl cellulose, was instilled into thevaginal vault, using a positive displacement pipette. In addition, all 24vaccinated macaques were given 2 i.m. injections with 200 mg gp120protein, as done previously (39). The protein was mixed with the adjuvantsalum and monophosphoryl lipid A (MPL) and administered in the thighmuscle at weeks 10 and 24. A total of 12 macaques were used as controls.Control animals were given the ALVAC vector that did not express SIVgenes, HPV-PsVs that expressed luciferase, and the adjuvants alum andMPL at similar doses and times as the vaccinated animals.

At week 28, 4 wk after the last HPV-PsV vaccination, all 36 rhesusmacaques were challenged intravaginally with 250 tissue culture–infectivedoses, 50%, of SIVmac251. The virus was kindly provided by Nancy Miller inthe Division of AIDS, National Institutes of Health. Blood was collected, andSIV RNA was quantified in plasma 7 d after challenge; animals with virusloads ,50 copies per milliliter were rechallenged. Animals with two suc-cessive viral determinations.104 were considered persistently SIV infected;repeated SIV challenges were stopped, and virus loads were monitoredweekly in the acute phase and monthly in the chronic phase. Animals withvirus loads between 50 and 104 copies were retested at day 10. If the virusload increased at day 10 to.104, the animal was considered persistently SIVinfected; the challenge phase was stopped, and virus load in plasma wasmonitored thereafter. If, however, the virus load at day 10 was ,50 copiesper milliliter, the animal was considered transiently infected, and repeatedlow-dose challenges were resumed. A maximum of nine repeated low dosesof SIVmac251 were administered at 10-d intervals.

Mucosal Abs

Vaginal secretions were collected using absorbent cotton sponges. To elutesecretions, the sponges were incubated for 10 min in elution buffer, on ice;transferred into a Salivette column (Sarstedt); and then centrifuged at 3000rpm for 30 min at 4˚C. For SIV-specific IgA and IgG, serially dilutedvaginal secretions were applied to 96-well half-area plates (Greiner Bio-One), previously coated with 50 ml (10 mg/ml) SIVmac251 gp120 (Ad-vanced Bioscience Laboratories), and blocked with 1% BSA BlockingSolution (KPL). After overnight incubation at 4˚C, plates were washedwith PBS-Tween, reacted with peroxidase-conjugated anti-monkey IgA orIgG Ab (Alpha Diagnostic), and incubated for another hour at roomtemperature. After washing, 50 ml TMB peroxidase substrate solution wasadded to each well and incubated for 20 min at room temperature. Reac-tions were stopped by adding 50 ml 2 M H2SO4, and plates were read at450 nm within 30 min. Titer was defined as the reciprocal of the dilution atwhich the absorbance of the test sample was twice that of the negativecontrol sample diluted 1:5. Total IgA and IgG concentrations were simi-larly determined by incubating serially diluted mucosal samples and a di-lution series of a standard normal rhesus macaque serum with knownconcentrations of IgG and IgA on microplates coated with 1 mg/ml purifiedgoat anti-monkey IgA or IgG Ab. The Env-specific IgA or IgG titer wasdivided by the corresponding total IgA or IgG concentration in each se-cretion and reported as titer per microgram of total IgG or IgA.

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Statistical analysis

Comparisons between groups were performed using the Mann–Whitney–Wilcoxon test for continuous factors, and paired comparisons between twotimes were assessed by the Wilcoxon signed rank test. Correlations wereperformed using the Spearman rank correlation method. The differencebetween groups in the binding of each of the overlapping gp120 peptideswas tested using the exact Mann–Whitney–Wilcoxon test, and the p valueswere corrected for multiple comparisons by the Hochberg method.Graphical analysis was performed using GraphPad Prism, and error bars ongraphs represent the SEMs.

IFN-g ELISPOT

SIV-specific T cells were assessed using an IFN-g ELISpot kit fromMabtech, as previously described (39). Cryopreserved PBMCs werethawed, rested, and stimulated with either SIVmac251 Gag or gp120 (Env)overlapping 15-mer peptides, Con A, or were left unstimulated. PBMCsand stimulants were added to IFN-g–coated plates and incubated for 24 h.The plates were developed, and the frequency of IFN-g–positive spot-forming cells per 106 PBMCs was determined after background subtrac-tion.

Pentamer staining and intracellular cytokine assays

Ten-color flow cytometric analysis was performed on mononuclear cells fromblood and from cervicovaginal and rectal biopsy specimens. Pinch biopsyspecimens obtained from the cervix, vaginal tract, or rectum were washed andincubated for 1 h with collagenase D at a concentration of 2 mg/ml in Iscove’smedium with antibiotics and amphotericin. Following incubation, the re-maining tissue was mechanically disrupted to obtain a mononuclear cell sus-pension. Filtered single-cell suspensions of mononuclear cells were used inan intracellular cytokine assay performed as previously described (37). Cellswere stimulated with either Env peptides at a concentration of 2 mg/ml or withPMA and ionomycin, or were left unstimulated in the presence of Golgitransport inhibitors, CD107a, clone H4A3; anti-CD28ECD, clone CD28.2(eBiosciences); and CD49D, clone 9F10 (BD Biosciences) for 6 h. Cells werethen surface stained with CD3 (cloneSP34-2), CD4 (clone L200), CD8 (cloneRPA-T8), CD95 (clone DX2), and the LIVE/DEAD yellow fixable amine dyefrom Invitrogen. Surface-stained samples were washed, permeabilized withCytofix/Cytoperm, and stained intracellularly with IFN-g (clone B27), TNF-a(clone MAB11), and IL-2 (clone MQ1-17H12). Staining reagents were ob-tained from BD Biosciences unless otherwise stated. Cytokine production afterbackground subtraction from memory (CD95+), CD4+, and CD8+ T cells andthe proportion of monofunctional and polyfunctional (simultaneous productionof multiple cytokines) responses were determined. For Gag CM9 pentamerdetection (obtained from ProImmune), cells were stained for 15 min with theGag CM9 PE pentamer, washed, and then stained with the amine dye andCD3, CD4, CD8, CD28, and CD95, using the same clones as above. Sampleswere washed, permeabilized with Cytofix/Cytoperm, and stained intracellularlywith Ki67 (clone B56, BD Biosciences). All cells were fixed with 1% para-formaldehyde and acquired on an LSR II (BD Biosciences). Data analysis wasperformed with FlowJo (TreeStar) and with SPICE (National Institute of Al-lergy and Infectious Diseases) (42).

CFSE proliferation assay

The lymphoproliferation assay was performed as previously described (39).Cells were briefly incubated with 5 mM CFSE (Invitrogen), washed,enumerated, and stimulated with 5 mg/ml SIV Env or Con A, or were leftunstimulated for 5 d. Cells were then harvested and stained with CD3,CD4, CD8, CD28, CD95, and the amine dye, as described above. Sampleswere acquired on an LSR II flow cytometer, and the frequency of CD3+

CD4+CD95+ or CD3+CD8+CD95+ T cells with diminished expression ofCFSE (proliferated) after 5 d of culture was determined and the back-ground subtracted (%CFSE dim in unstimulated cells).

Binding Abs and pepscans

An ELISA was used to detect SIVmac251–gp120 binding Abs in blood, aspreviously described (40), and to detect binding to overlapping peptidesspanning gp120. A serial dilution of plasma was added to microtiter platescoated with native purified gp120 Env protein of SIVmac251 or individualpeptides, and the Ab titer determined. The absorbance at OD 450 nm wasreported for peptide mapping. For binding Abs to gp120, the endpointtiters were defined as 23 the OD 450 of the negative control serum.

B cell ELISPOT

SIV Env-specific or total IgG or IgA Ab-secreting cells were analyzed bya B cell ELISPOT, as described previously (39). Briefly, MultiScreen 96-

well plates (Millipore MAIPS4510) were incubated with 70% ethanol,rinsed, and coated with SIVmac251 gp120 protein or goat anti-monkey IgGor IgA (KPL). Coated plates were incubated at 4˚C overnight, washed, andblocked for 2 h at 37˚C. PBMCs were stimulated with CpG (ODN-2006;Operon), CD40L, and IL-21 (PeproTech) for 3 d at 37˚C in 24-well plates.Stimulated PBMCs were next harvested and washed, and 3 3 105 cellswere plated and incubated overnight at 37˚C. Plates were then washed andincubated with biotinylated goat anti-monkey IgG or IgA (Rockland), andHRP–avidin D conjugate (Vector Laboratories) was added. After severalwashes, plates were developed using 3-amino-9-ethyl-carbazole (Sigma-Aldrich). Spot quantitation was performed with an ELISPOT reader.

ADCC

ADCC activity mediated by Abs in plasma samples was detected by theGranToxiLux (GTL) procedure, as previously described (20, 43). Briefly,CEM.NKRCCR5 target T cells were coated with recombinant SIVmac251

gp120 and labeled with a fluorescent target-cell marker and a viabilitymarker. Labeled target cells were washed and plated. Cryopreserved hu-man PBMCs from an HIV-seronegative donor served as effectors and wereadded to the assay wells at an E:T ratio of 30:1. Fluorogenic granzyme Bsubstrate (OncoImmunin) was added to each well. After incubation, seri-ally diluted plasma samples were added to the assay wells. The plates wereincubated for 15 min at room temperature, centrifuged, and incubated for1 h at 37˚C. The plates were then washed, cells were resuspended in PBS,and$2500 events representing viable target T cells were acquired for eachwell, using an LSR II flow cytometer (BD Biosciences). Data analysis wasperformed using the FlowJo 8.8.4 software (TreeStar). The final results areexpressed as ADCC titer and maximum granzyme B activity.

Neutralization assays

Neutralization was measured as a reduction in luciferase reporter geneexpression after a single round of infection in TZM-bl cells, as describedpreviously (20, 44). TZM-bl cells were obtained from the NIH AIDSResearch and Reference Reagent Program. Virus was incubated with serial3-fold dilutions of samples in duplicate. Freshly trypsinized cells wereadded to each well. One set of control wells received cells and virus (viruscontrol), and another set received cells only (background control). Aftera 48-h incubation, cells were transferred to 96-well black solid plates(Costar) for measurements of luminescence. Neutralization titers are thedilution at which relative luminescence units were reduced by 50% com-pared with that in virus control wells after subtraction of backgroundrelative luminescence units. Assay stocks of molecularly cloned Env-pseudotyped viruses, SIVmac251.6, SIVmac251.30, were prepared by trans-fection in 293T cells and titrated in TZM-bl cells. The SIVmac251 challengestock was obtained from Nancy Miller in the Division of AIDS, NationalInstitutes of Health, expanded on rhesus PBMCs, titered, and used.

Ab Avidity

Three recombinant SIV envelope proteins—full-length gp120, gp120 de-leted of the V1V2 region, and the V1V2 mini protein—were made fromcodon-optimized SIVmac239 gp120. The V1V2 mini protein was fused tothe C-terminal tag of HIV-1 gp120. These proteins were used as an Ag forthe capture ELISA to detect SIV Abs against conformational epitopes, aspreviously described (39). Parallel ELISAs were used to determine Abavidity. Heat-inactivated plasma samples were serially diluted and appliedto a 96-well plate capturing SIVmac239 gp120 proteins in parallel dupli-cates. After 1 h of incubation, the plate was washed, and half the sampleswere treated with TBS, whereas the paired samples were treated with 1.5M sodium thiocyanate (Sigma-Aldrich) for 10 min at room temperature.The plate was washed, and a goat anti-monkey IgG-detecting Ab (Fitz-gerald) was used. The avidity index (%) was calculated by taking the ratioof the sodium thiocyanate–treated plasma dilution, giving an OD of 0.5 tothe TBS-treated plasma dilution giving an OD of 0.5, and multiplying by100. Plasma of uninfected normal macaques served as negative controls. Ahigh-avidity monkey mAb of 3.11H was included on every plate as thestandard.

Viral load and transmitted founder variants

Plasma SIV RNA was quantified by nucleic acid sequence–based ampli-fication, as previously described (45, 46). SIV DNA was quantified inmucosal tissues 3 wk after SIV infection by a real-time quantitative PCRassay with sensitivity #10 copies per 106 cells, as previously described(45). Briefly, genomic DNAwas extracted from the rectal biopsy specimenwith the DNeasy Blood & Tissue Kit (QIAGEN), according to the man-ufacturer’s protocol, except the DNA elution step. The quantity and qualityof the DNA were assessed by OD 260 measurements using an ND-1000

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spectrophotometer (NanoDrop). The TaqMan probe and PCR primersfor the real-time PCR were designed within the conserved gag gene ofSIVmac239, and probe and primer sequences were used for the monkey al-bumin gene detection. The reaction conditions are as follows: the 25 ml PCRmixture consisted of 500 ng genomic DNA extracted from tissues; 200 nMprimers; 100 nM probe; 23 TaqMan Universal PCR Mastermix (AppliedBiosystems) consisting of 10 mM Tris–HCl (pH 8.3); 50 mM KCl; 5 mMMgCl2; 300 mM each of 2’-deoxyadenosine triphosphate, deoxycytidinetriphosphate, and deoxyguanosine triphosphate; 600 mM 2’-deoxyuridine5’-triphosphate; 0.625 U AmpliTaq Gold DNA polymerase; and 0.25 Uuracil N-glycosylase. Amplification was performed using one cycle at50˚C for 2 min and one cycle at 95˚C for 10 min, followed by a two-stepPCR procedure consisting of 50 cycles of 15 s at 95˚C and 1 min at 60˚C.PCR amplification was performed using the ABI Prism 7500 SequenceDetector System (Applied Biosystems). The normalized value of the SIVproviral DNA load was calculated as SIV DNA copy number/Mac albumingene copy number 3 2 3 106, and expressed as the number of SIV proviralDNA copies per 106 PBMCs or cells. In addition, an ultrasensitive nestedquantitative real-time PCR and RT-PCR approach was also used to identifySIV RNA or DNA in vaginal and rectal tissues before and after CD8+ T celldepletion, as previously described (18).

Transmitted/founder viruses and their progeny were identified by single-genome amplification of SIV RNA from plasma or rectal pinches. SIV RNAwas extracted, and limiting-dilution PCR of newly synthesized cDNAwasperformed. Reverse transcription of RNA to single-stranded cDNA was

performed using SuperScript III reverse transcriptase according to themanufacturer’s recommendations (Invitrogen), using gene specific prim-ing: SIVEnvR1, 59-TGTAATAAATCC CTT CCA GTC CCC CC-39. Theenvelope gene was then amplified via limiting-dilution PCR in which onlyone amplifiable molecule was present in each reaction, using a 13 PCRbuffer consisting of 2 mM MgCl2, 0.2 mM of each deoxynucleoside tri-phosphate, 0.2 mM of each primer, and 0.025 U/ml Platinum Taq Poly-merase (Invitrogen) in a 20-ml reaction. First-round PCR was performedwith sense primer SIVEnvF1 59-CCT CCC CCT CCA GGA CTA GC-39and antisense primer SIVEnvR1 under the following conditions: 1 cycle of94˚C for 2 min, 35 cycles at 94˚C for 15 s, 55˚C for 30 s, and 72˚C for 4min, followed by a final extension of 72˚C for 10 min. Nested PCR wasperformed with the following primers: SIVEnvF2, 59-TATAATAGA CATGGA GAC ACC CTT GAG GGA GC-39; and SIVEnvR2, 59-ATG AGACAT RTC TAT TGC CAA TTT GTA-39 under the same conditions usedfor first-round PCR, but with a total of 45 cycles. Transmitted/foundervirus lineages were determined phylogenetically by identifying all dis-tinct, low-diversity lineages, as described previously (20, 39, 47–49). All388 sequences are deposited in GenBank under accession numbersKF646830–KF647217 (www.ncbi.nlm.nih.gov/genbank).

CD8+ cell depletion

CD8+ lymphocyte depletion was performed in 11 macaques. Animals wereinfused with the aCD8-depleting rhesus recombinant Ab M-T807R1, ob-

FIGURE 1. HPV-PsV-SIV vaccines induce cell-mediated responses in the blood and female genital tract. (A) Overview of the vaccination regimen, which

includes 36 macaques. Group 1 was given HPV-PsV-SIV + gp120 vaccines; group 2 was given ALVAC-SIV, followed by HPV-PsV-SIV + gp120. The third

group comprises the controls, which were given the ALVAC-mock vector and HPV-PsV-luciferase. All animals were given alum and MPL adjuvants,

represented as adj. Vaccine-induced immune responses were measured in blood at various time points throughout the study and in vaginal biopsy specimens

at weeks 11 and 17, indicated by white arrows below the regimen. (B) Cell-mediated responses in blood, measured using IFN-g ELISPOT at week 17 after

vaccination. Shown is the number of spot-forming units (SFU) per 106 PBMCs after Gag peptide stimulation (left) or Env peptide stimulation (right). Gag/

Env-specific responses are shown after background subtraction of unstimulated cells. Circles represent animals in the ALVAC/HPV group, squares the HPV

group, and triangles the control animals. (C) The frequency of proliferating CD4+ and CD8+ memory (CD95+) T cells in blood is shown 1 wk after the last

vaccination, week 25. Proliferating cells are calculated as the percentage of CFSE dim cells after 5 d of culture with SIVmac251 Gag protein (left) or

SIVmac251 Env protein (right). Data presented are after background subtraction of unstimulated cells. A significant difference in CD8+ T cell Env pro-

liferation was observed between the ALVAC/HPV group and the HPV group, represented by an asterisk using the Mann–Whitney–Wilcoxon test, with p =

0.039. (D) The frequency of memory CD95+ Gag CM9+ CD8+ T cells in the blood of Mamu-A*01–positive animals over the course of the vaccination.

White arrows indicate weeks 11 and 17 after vaccination (E) Memory CD95+ Gag CM9+ CD8+ T cells in the vaginal tract measured after the second HPV-

PsV vaccination at weeks 11 and 17. The ALVAC/HPV group is represented by white bars, the HPV group by hatched bars, and the controls by black bars.

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tained from the Non-Human Primate Reagent Resource. Animals weregiven three doses of aCD8 Abs on days 0, 3, and 7. The first dose wasadministered at a concentration of 10 mg/kg, whereas the other two doseswere given at 5 mg/kg. The number or frequency of CD3 (clone SP34-2),CD4 (clone L200), CD8 (clone DK25 Dako), and CD20 (clone B9E9Beckman Coulter) expressing cells was monitored in the blood and in thevaginal and rectal biopsy specimens. The CD8 Ab used for flow cytometrywas chosen, as it has been shown not to compete with or mask the epitopeof the CD8-depleting Ab (50).

ResultsInduction of T cell responses by intravaginal HPV-PsVvaccination

The 36 rhesus macaques were distributed into three groups(Fig. 1A): HPV, group 1; ALVAC/HPV, group 2; and controls,group 3. Each group contained 3 Mamu-A*01–positive animals.The HPV vaccine group was given three intravaginal vaccinationswith HPV-PsVs that expressed the SIV genes gag-pro, gp120, andrev, tat, nef (HPV-PsV-SIV) at weeks 6, 10, and 24. At weeks 10and 24, animals were also given monomeric gp120 protein adju-vanted in alum and MPL. In the second cohort of animals(ALVAC/HPV, group 2), the systemic immune system was primedwith ALVAC expressing SIV genes gag, pol, and gp160 (40) atweeks 0 and 4 and then boosted intravaginally with HPV-PsV-SIVand i.m. with gp120 protein in alum and MPL, similar to the group1 animals. Twelve control macaques (group 3) were vaccinatedwith the ALVAC empty vector, HPV-PsVs that expressed lucif-erase and were given the adjuvants at the same time and dose asthe other groups.Previously, we demonstrated that intravaginal delivery of HPV-

PsVs expressing SIV Gag recruited CD4+ and CD8+ T cells to thefemale genital tract, and resulted in SIV-specific responses in thecervicovaginal lamina propria (37). In this study, we extend and

confirm those findings and measured T cell responses in the bloodthroughout the study; however, cervicovaginal biopsies werelimited to baseline (before vaccination) and weeks 11 and 17 (1and 6 wk after the second HPV vaccination) (Fig. 1A). This wasdone to allow sufficient time for healing before the intravaginalSIV challenge at week 28. Vaccination induced similar SIV Gag-and Env-specific immune responses in the blood of both vaccinegroups measured by IFN-g ELISPOT at 17 wk after vaccination(Fig. 1B). Proliferative responses were measured 1 wk after thelast vaccination (Fig. 1C). ALVAC-primed HPV + gp120–boostedanimals had higher levels of CD4+ Gag and Env T cell prolifer-ation compared with the HPV group, although the difference wasnot significant (Fig. 1C). Env-specific CD8 T cell proliferativeresponses were higher in the ALVAC/HPV group compared withthe HPV group (p = 0.039). Gag CM9 staining in Mamu-A*01–positive animals revealed no Gag-specific CD8+ T cells in theblood of animals from the HPV or control groups (Fig. 1D). Asexpected, the ALVAC/HPV group developed a systemic CD8+

Gag response that was boosted by intravaginal HPV-PsV-SIVvaccinations. The frequency of GagCM9 CD8+ T cells was alsodetermined in the vaginal tract after the second HPV vaccinationat weeks 11 and 17. Similar to the blood, Gag-specific CD8+

T cells were detected in the ALVAC/HPV group at week 11, butnot in the HPV group (Fig. 1E). HPV-PsV entry in woundedkeratinocytes is a slow process taking many hours, with peakexpression on days 2–3 (31). Thus, 7 d after vaccination may nothave been sufficient time for Ag presentation and T cell expan-sion. However, 6 wk later, T cell responses were detected in theHPV group and were expanded in the ALVAC/HPV group. Thefrequency of Gag-specific T cells was 14- to 16-fold greater in thevaginal tract compared with the blood, and 2- to 3-fold higher inthe vaginal tract of the ALVAC/HPV group compared with the

FIGURE 2. Vaccination induced mainly monofunctional responses in the blood and vaginal tract. (A) Cytokine production following Env peptide

stimulation of mononuclear cells from vaginal biopsies obtained at week 17. Shown is the sum of IFN-g, TNF-a, IL-2, and CD107 production after

background subtraction in CD95+CD4+ and CD95+CD8+ T cells. (B) The functional capacity of the SIV-specific response is represented by the pie charts;

they show the proportion of cells that responded to stimulation by producing either IFN-g, TNF-a, IL-2, or CD107, or a combination thereof. The fraction

of cells that responded to stimulation by producing one cytokine is shown in gray; two cytokines, black; three cytokines, green; or four cytokines, orange.

CD4 responses are in the top pie panel and CD8 responses are in the lower pie panel (C) Total Env-specific cytokine production in blood after the last

vaccination, week 26, in CD95+CD4+ and CD95+CD8+ T cells. Shown is the sum of IFN-g, TNF-a, IL-2, and CD107 production. (D) Pies show the fraction

of cells that responded to stimulation by producing one cytokine (gray), two cytokines (black), three cytokines (green), or four cytokines (orange). CD4

responses are in the top pie panel and CD8 responses are in the lower pie panel.

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HPV group. Gag-specific CD8+ T cells were not detected inthe rectum of vaccinated animals at week 17 (data not shown).The increased T cell response, observed in the vaginal tract ofthe ALVAC/HPV group, and the absence of Gag-specific T cellsin the rectum of ALVAC-SIV–primed animals suggest that sys-temic priming followed by intravaginal boosting recruits and/orexpands cell-mediated responses in the female genital tract.Pinch biopsies of cervicovaginal tissues yield a limited number of

mononuclear cells. Mononuclear cells isolated from theMamu-A*01–positive animals were used to measure the frequency of GagCM9+

CD8+ T cells, whereas cells from the remaining 27 Mamu-A*01–negative animals were used to measure functional mucosal responsesto envelope peptides at week 17 (Fig. 2A). Intracellular cytokinestaining for IFN-g, TNF-a, IL-2, as well as the expression ofCD107, was determined following 6-h stimulation with overlapp-ing Env peptides. Vaccination induced mainly monofunctionalCD4+ and CD8+ T cell responses that secreted IFN-g, TNF-a, orCD107 (Fig. 2B). The frequency of Env-specific T cells was sim-ilar in the two vaccination regimens. At 2 wk before the first SIVchallenge (week 26), a similar analysis of cytokine profile was per-formed in the blood of all vaccinated animals (Fig. 2C). ALVAC/HPV-vaccinated animals had a greater frequency of blood CD4+ T cellresponses compared with the HPV group. Similar to the vaginal tract,primarily monofunctional memory responses were induced in blood(Fig. 2D); however, TNF-a was the dominating cytokine responsein the ALVAC/HPV group, whereas either TNF-a, IFN-g, or IL2was produced in the HPV group.

Systemic and mucosal gp120-specific Abs induced byvaccination

ALVAC-SIV priming induced gp120-specific IgG in the blood, butby the end of the vaccination regimen, both groups had similar

levels of high-titer binding Abs (Fig. 3A). To assess Abs in mu-cosal secretions, we collected vaginal swabs after the last vacci-nation. Equivalent levels of gp120-specific IgG were found in theALVAC/HPVand HPV groups presented as titer per microgram oftotal IgG to normalize for the levels of total IgG isolated fromeach animal (Fig. 3B). Before normalization by total IgG, wedirectly compared the gp120-specific titers in each animal’s bloodand vaginal mucosa and observed that, on average, Env-specificIgG was approximately one log lower in the vaginal mucosa thanin blood. Low levels of gp120-specific IgA were detected in thevaginal secretions of both vaccinated groups (Fig. 3B). A simi-lar frequency of Env-specific memory B cells was measured inboth groups 1 wk prior to SIV challenge (Fig. 3C). Althoughboth vaccine regimens induced measurable IgG+ gp120-specificB cells, no IgA+ gp120-specific B cells were detected in blood(data not shown).The functional capacity of Abs induced by the two vaccine

regimens was determined in the blood, owing to the limitedquantity of protein extracted from vaginal swabs. The two vaccineregimens induced serum Abs that mediated similar levels ofADCC, measured as % maximum granzyme activity and ADCCtiter (Fig. 3D). In contrast, the ALVAC/HPV group had signifi-cantly greater neutralization titers for the tier-1–like SIVmac251.6

virus compared with the HPV group p = 0.0023 (Fig. 3E). Neithervaccine regimen induced Abs that neutralized the tier-2–likeSIVmac251.30 isolate (data not shown) or the SIVmac251 challengestock (Fig. 3E).Abs to the V1/V2 loop of gp120 were found to be a correlate of

reduced HIV risk in the RV144 Thai trial, using ALVAC-HIV andgp120 immunogens (9). In another study involving ALVAC-SIV/gp120 vaccination, we found that animals that resisted SIVmac251

infection had high-avidity Abs directed to the V1/V2 region (39).

FIGURE 3. Vaccine-induced binding Abs and their functional capacity. (A) gp120-specific IgG titer in blood during the vaccination phase, with the

ALVAC/HPV group represented as circles and with a large dashed line; the HPV group, squares with a small dashed line; and controls, triangles with a solid

line. Vertical lines indicate the time when a vaccine was given: ALVAC, weeks 0 and 4; HPV-PsVs, weeks 6, 10, and 24; and gp120/adjuvant, weeks 10 and

24. (B) Gp120 binding Ab titers in mucosal secretions per microgram of total IgG/IgA measured after the last vaccination, week 25. IgG is shown on the left

and IgA is on the right. The ALVAC/HPV group is represented by white bars and the HPV group by hatched bars. (C) Percent gp120-specific IgG memory

B cells measured by B cell ELISPOT in PBMCs at week 27. (D) ADCC in the blood shown as % granzyme B activity (left) or ADCC titer (right) measured

at week 26. Circles represent animals in the ALVAC/HPV group; squares, the HPV group; and triangles, the control group. (E) Neutralization of an easy-to-

neutralize tier-1–like virus SIVmac251.6 (left) and the SIVmac251 challenge stock (right) measured at week 26 after the last vaccination. A significantly higher

level of neutralization was observed in the ALVAC/HPV group, indicated by the asterisk, using the Mann–Whitney–Wilcoxon test, with p = 0.0023.

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We therefore measured vaccine-induced Ab binding to over-lapping linear peptides that spanned gp120, including the V1/V2region, and Ab avidity. Both regimens had an overall similarrecognition of overlapping peptides spanning the constant andvariable regions of gp120 (Fig. 4A). We compared the averagebinding of each peptide in the two vaccination regimens, using theZ statistic of the Mann–Whitney–Wilcoxon test (Fig. 4B). Aftercorrection for multiple comparisons by the Hochberg method, twopeptides, 24 and 29, in the V2 loops had significantly greater Abrecognition in the HPV group (Fig. 4B). The ALVAC/HPV groupshowed increased binding to peptides 16 and 17 within the C1/V1region and to peptide 40 in the C2 regions of gp120, but thedifference was not statistically significant. The avidity of Abs tothe whole gp120 protein of SIVmac239 was evaluated after sodiumthiocyanate treatment. On average, Abs from both vaccine regi-mens had a similar avidity index (Fig. 4C).To determine the contribution of the V1/V2 region of gp120, the

avidity index of vaccine-induced Abs was assessed using a gp120protein in which the V1/V2 loop was deleted (DV1/V2) anda conformational protein containing the entire V1/V2 stem loop ofSIVmac239, linked to a tag from the C-terminal of HIV gp120. Asignificant reduction in the avidity index was observed when the

V1/V2 region of gp120 was deleted (p , 0.0001) (Fig. 4C). Theaverage avidity to the entire gp120 was 20.1, whereas the DV1/V2avidity index was 6.4. Furthermore, when the avidity index of Absto the V1/V2 mini protein was assessed, an average avidity of 30.9was observed, a significant increase when compared with gp120protein (p = 0.0011) (Fig. 4C). In some animals, the V1/V2 aviditywas greater than 40. Of interest, an avidity index of 35–44 hasbeen observed in other vaccination regimens in macaques pro-tected from SIVmac251 and SIVsmE660 infection (15, 39).

Vaccination with ALVAC-SIV/HPV-PsV-SIV/gp120 influencespersistent viremia

The efficacy of each vaccination regimen was assessed by chal-lenging animals with up to nine intravaginal low doses of SIVmac251

(250 tissue culture–infective doses, 50%,), given every 10 d, be-ginning 4 wk after the last vaccination. The level of SIV RNAwasdetermined in plasma by nucleic acid sequence–based amplifica-tion, 7 d after each challenge; animals that tested negative (,50copies per milliliter) were rechallenged. All three groups acquiredSIV at a similar rate (Fig. 5A), and at the end of the challengephase five vaccinated animals, two in the ALVAC/HPV groupand three in the HPV group, remained SIV negative in plasma,

FIGURE 4. Both vaccination regimens induce Abs that target the V1/V2 region of gp120. (A) Average Ab binding to 89 overlapping peptides that span

gp120 with the ALVAC/HPV group on the left and the HPV group on the right. Variable regions are represented by colored bars, and dashed vertical lines

designate the boundaries of the constant and variable regions. Blue arrows indicate peptides 24 and 29 in the V2 loop. (B) Comparative recognition of

overlapping peptides presented as the difference between the median absorbance in the ALVAC/HPV group, relative to the HPV group, shown in red on the

right y-axis. The statistical significance of the differences is shown by the Z statistic of the Mann–Whitney–Wilcoxon test in black, on the left y-axis, with

the dashed lines marking significance at the p = 0.05 level after the correction for multiple comparisons by the Hochberg method. Increased recognition by

the ALVAC group is presented as positive (0 to 5) on the top half of the graph, and increased recognition in the HPV group is presented as negative (0 to25)

on the lower half of the graph. Two peptides in the V2 loop, 24 and 29, demonstrated significantly greater Ab recognition in the HPV group relative to the

ALVAC group, denoted by asterisks. (C) Avidity, shown as the avidity index of Abs to the entire gp120 of SIVmac239 on the left. The ALVAC/HPV group is

represented by white bars and the HPV group by hatched bars. On the right is the comparison of the avidity of Abs induced by both vaccine regimens to the

entire gp120 of SIVmac239, a gp120 protein deleted of the V1/V2 loop (DV1/V2), and a conformational mini protein containing the entire V1/V2 step loop

of SIVmac239. A significant difference in the avidity index is observed between gp120 and DV1/V2-gp120, with p , 0.0001, and between gp120 and the

V1/V2 mini protein, with p = 0.0011, indicated by the asterisk, using the Wilcoxon signed rank test.

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whereas one control animal remained negative. Because most HIVinfections are initiated with a single or few viral variants, weaimed to model this outcome in our mucosal challenge experimentin macaques. The number of transmitted viral variants is an in-dependent analysis of a limiting dose challenge (47). Thus, wequantified the number of variants in all SIV-infected animals thathad at least two viral load measurements .104 RNA copies permilliliter and created neighbor joining trees for each group(Supplemental Fig. 1). No difference in the number of transmittedvariants was observed between the three groups. Each group hada median of one viral variant and a maximum of three (Figure 5B,Supplemental Fig. 1). This finding suggests that our intravaginalSIVmac251 was given at a dose that models HIV heterosexualtransmission. Furthermore, neither the intravaginal vaccinationnor the progesterone/N9 treatment caused a significant increase inthe number of transmitted variants. A similar number of variants(median 1) were also observed in naive macaques that were givena low-dose challenge by the vaginal or rectal route (39) (N. Miller,unpublished observations). No significant associations were ob-served between vaccine-induced immune responses and thenumber of transmitted variants.

Most infected animals demonstrated high peak (106–108) and setpoint (105–107) plasma virus (Fig. 5C). However, of the vacci-nated animals, 8 had no detectable plasma virus or transientplasma viremia that remained below the limit of assay detection:50 SIV RNA copies per milliliter (Fig. 5C). In total, 16 of 24vaccinated animals and 10 of 12 controls demonstrated persistentSIV viremia. Persistent viremia was defined as at least two suc-cessive plasma viral load measurements .104 SIV RNA copiesper milliliter. We next compared the viremia over the 16 wk offollow-up in the persistently SIV-infected animals. No significantdifferences in either peak or set point viremia in vaccinated ani-mals or controls were observed (Fig. 5D). In addition, a similarloss of CD4+ T cells was observed in the blood of all persistentlySIV-infected macaques (Fig. 5E). To assess virus levels in mu-cosal tissues, pinch biopsies were collected from the vagina andrectum during acute SIV infection, and the levels of SIV DNAwere determined (Fig. 6A, 6B). Unlike our findings for plasmaviremia, significantly less SIV DNA was measured in the vaginaland rectal mucosa during the acute phase in the ALVAC/HPV-vaccinated animals in comparison with controls (p = 0.014 andp = 0.022) (Fig. 6A, 6B). Reduced viral DNA in the mucosa

FIGURE 5. Vaccine efficacy and plasma viral loads after SIV infection. (A) The rate of SIV infection is shown by the percentage of uninfected animals at

each challenge in the control group (solid black line), the ALVAC/HPV group (large dashes), and the HPV group (small dashes). (B) The number of

transmitted founder viral variants is shown for each vaccine group, with the ALVAC/HPV group represented by open bars, the HPV group by hatched bars,

and the control group by black bars. The number of variants was determined during the first 2 wk of infection in animals that had two successive positive

tests for SIV RNA in plasma, with .104 copies per milliliter. (C) Plasma viral load over time in the ALVAC/HPV group is represented by open circles, in

the HPV group by squares, and in the control group by triangles. The animal codes of animals with transient plasma viremia, or those that tested negative

for SIV RNA in plasma, are shown to the right of each graph. (D) Geometric mean of plasma viral load in animals that were persistently SIV infected.

Persistent infection was defined as two successive positive tests for SIV RNA in plasma, with.104 copies per milliliter. (E) The average absolute number of

CD4+ T cells in the blood per cubic millimeter is shown for persistently SIV-infected animals in the ALVAC/HPV group (open circles), the HPV group

(squares), and the control group (triangles).

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during the acute phase of SIV infection was also temporally as-sociated with the expansion of GagCM9+CD8+ T cells measured10 d after SIV infection (Fig. 6C), with the vaginal tract having thehighest frequency of SIV-specific CD8+ T cells and the lowestvirus DNA levels in the acute phase of infection.

CD4+T cells and Abs to V1/V2 correlate with protection frompersistent viremia

We investigated potential associations between mucosal SIV DNAlevels in the acute phase and vaccine-induced responses. We ob-served an inverse correlation between the level of Env-specificCD4+ T cell proliferation in blood measured 2 wk after the lastvaccination and SIV DNA in the vaginal and rectal tract after SIVinfection (r =20.5 and20.47 and p = 0.014 and 0.027 for vaginaland rectal tissues, respectively) (Fig. 6D). We did not observea correlation between vaccine-induced CD8 T cell responses andSIV viral loads. However, the temporal expansion of vaginal CD8T cells during the acute phase and association of CD4 helperresponses with reduced viremia may indicate that the increasedCD4+ T cell responses induced by the ALVAC/HPV may havehelped the development of a secondary CD8+ T cell response,which in turn affected virus replication in the mucosa.Next, we investigated the four animals with transient viremia;

these animals had virus loads ranging from 50 to 104 copies permilliliter and then controlled viremia during the remaining re-peated low-dose challenges and for 14 wk of follow-up. Wequestioned whether their SIV-specific immune responses wereboosted during the successive SIV challenges. We observed a re-duction in gp120 binding Abs and IFN-g ELISPOT responses toGag in the vaccinated animals, comparing responses before thefirst SIV challenge with samples collected after the fifth challenge(Supplemental Fig. 2A). In addition, we did not detect a Vif-specific response in the cervicovaginal tract at the end of the

challenge phase (Supplemental Fig. 2B). Vif is not in any of thevaccines administered but is abundant in the challenge virus.Furthermore, the levels of gp120 binding Ab titers in the vaginalsecretions had also declined following the ninth and final SIVchallenge, when compared with pre-SIV levels, consistent withthe findings in blood (Supplemental Fig. 2C).The two distinct outcomes observed in this study, that is, per-

sistent SIV infection versus protection from infection or high virusreplication, gave us the opportunity to investigate any associationsbetween the measured immune responses and outcome. A com-parison of immune responses in protected animals and persistentlySIVmac251-infected macaques yielded a significant difference inthe levels of Abs with high avidity to the V1/V2 region (Fig. 7A).Furthermore, we found a significant correlation between the num-ber of challenges to attain persistent infection and the avidityindex of V1/V2 Abs in blood (Fig. 7B). In all, these data highlightthe importance of Env-specific CD4+ T cells in the containment ofvirus replication at mucosal sites and of high-avidity Abs targetedto the V1/V2 region of gp120 in the prevention of SIVmac251 ac-quisition.

CD8 T cells contribute to protection from persistent viremia

At 3 wk after the ninth SIV challenge, SIV-negative animals andanimals with transient viremia had ,50 copies of SIV RNA inplasma and were tested for SIV DNA in the mucosa (Fig. 6A, 6B,arrows). With the exception of one of the control animals that had24 copies of SIV DNA per 106 cells in the rectum, the remainingnine animals tested negative for SIV DNA in the vaginal and rectaltract, using an assay that detects .10 SIV DNA copies per 106

cells (45). We followed these 10 animals for 14 wk, testing forSIV RNA in blood, and all remained SIV negative.To investigate in more detail the immune control of viremia, we

performed CD8 depletion in nine of the ten animals (one animal

FIGURE 6. Reduced SIV DNA in mucosal tissues of ALVAC/HPV-vaccinated animals. (A) Vaginal SIV DNA levels per 106 cells. Circles represent the

ALVAC/HPV group, squares the HPV group, and triangles the controls. Significantly less SIV DNAwas present in the vaginal tissues of the ALVAC/HPV

group compared with the control group, denoted by an asterisk using the Mann–Whitney–Wilcoxon test, with p = 0.014. Arrows indicate animals that had

either transient plasma viremia or tested negative for SIV RNA in plasma. (B) Rectal SIV DNA per 106 cells in vaccinated macaques and controls.

Significantly less SIV DNA was present in the rectal tissues of the ALVAC/HPV group compared with controls, denoted by an asterisk using the Mann–

Whitney–Wilcoxon test, with p = 0.022. (C) Mononuclear cells from the blood and from vaginal and rectal biopsy specimens were obtained 10 d after SIV

infection. The frequency of memory (CD95+) GagCM9-specific CD8+ T cells is shown in the vaccinated animals (white bars) and controls (black bars). (D)

An inverse correlation was observed between the levels of SIV DNA in the vaginal tract (left) and the rectum (right), and the Env-specific proliferating

memory (CD95+) CD4+ T cells presented as the square root of the data. The correlation was assessed using a nonparametric Spearman test with r values of

20.5 and 20.47 and p values of 0.014 and 0.027 for the vaginal and rectal tracts, respectively.

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was excluded owing to surgical complications), as well as in twopersistently viremic macaques, as a control. Treatment with anti-CD8 Ab rapidly depleted CD8+ cells in the blood (SupplementalFig. 2D) and caused a significant decline in the frequency of CD8+

cells in the lymph nodes (57%) and rectum (62%) (SupplementalFig. 2E). As expected, in the two animals with persistent viremia,plasma virus levels further increased following CD8 depletion(Supplemental Fig. 2F). We collected vaginal and rectal biopsiesbefore CD8 depletion (pre), 9 d after depletion (during) whenCD8+ T cells were undetectable in the blood, and 42 d post-treatment (post) when CD8+ T cells had rebounded, and quantifiedSIV DNA and RNA using a low-copy ultrasensitive assay (18).Both unimmunized animals tested positive at mucosal sites eitherpre–, during, or post–CD8+ T cell depletion (Table I). Of thevaccinated animals, four remained negative, and three of thembelonged to the HPV/gp120 group (Table I), suggesting early andlong-term control of virus replication. Collectively, these resultsindicate that undetectable plasma virus does not exclude localinfection and highlight the importance of CD8+ T cells in the localcontrol of virus.

DiscussionSIV infection of rhesus macaques models key aspects of HIVinfection. To date, HIV vaccine candidates tested in the macaquemodel, using repeated low doses of SIV given across mucosalsurfaces, have recapitulated the results of HIV clinical trials (12–14). Furthermore, a low-dose mucosal challenge with an unclonedSIV swarm can be titered to transmit a single or few virus variants

in macaques (49), similar to the bottleneck described during mostHIV infections (48). Thus, in this study, we used a repeatedintravaginal challenge with a SIVmac251 swarm to test the efficacyof a novel mucosal vaccination regimen HPV-PsV-SIV, with andwithout ALVAC-SIV priming. Neither vaccine regimen signifi-cantly altered the rate of SIV acquisition compared with controls,although several animals were either protected from SIV infectionor had transient viremia. In all, 16 of 24 (∼67%) vaccinatedanimals, and 10 of 12 (∼83%) of controls, developed persistentinfection. Vaccinated animals that became persistently viremichad similar peak and set-point plasma virus levels, which was notsurprising, given the low levels of systemic CD8+ T cell responsesinduced by these vaccines. However, we observed a reduction inviral burden in mucosal tissues in vaccinated animals comparedwith controls, and this reduction was significant in the ALVAC/HPV group, which had the highest levels of vaginal Gag-specificCD8+ T cell responses. The limited number of mononuclear cellsisolated from the vaginal tract precluded our assessment of vaginalGag-specific responses in all animals and of rev, tat, and nefresponses. In addition, we observed an inverse correlation betweenproliferating Env-specific CD4+ T cells in blood and the levels ofmucosal SIV DNA. A number of vaccinated animals had a long-lasting control of viremia. Altogether, these data suggest thatT cell responses induced by HPV-PsV vaccination exerted earlymucosal virus control, but virus expansion likely outpaced T cellexpansion, leading to systemic dissemination and uncontrolledviremia. Surprisingly, the mucosal T cell response induced by theHPV-PsVs/gp120 regimen did not curtail local virus levels orprovide sustained virus control, whereas priming with ALVAC-SIV induced an overall higher Gag and Env proliferative T cellresponse (Fig. 1C) and better control of mucosal virus levels(Fig. 6A, 6B). This suggests a role for SIV-specific T cells in earlyprotection from virus replication. In a murine model, intravaginalHPV-PsV vaccination induces long-lived CD103+ CD8+ T cellsthat home to the site of vaccination and intercalate throughout theepithelial layers of the vaginal tract (32). If HPV-PsV vaccinationsimilarly induces tissue-resident CD8+ T cells in humans as it doesin mice, these CD8+ T cells would be well positioned to combatHIV at the site of virus entry. The exposed columnar epithelium inthe cervix of young women is potentially susceptible to HPVvaccination and a likely site of HIV transmission (51).Progesterone treatment was used to facilitate vaccine delivery

and may have influenced the vaccine-induced immune response, asprogesterone has been shown to reduce antiviral responses (52).Intravaginal delivery of vaccines and the disruption of the epi-thelium used to facilitate delivery are cumbersome, and they in-troduce several challenges for clinical applications. However,vaccination in the secretory phase may eliminate the need for

FIGURE 7. High-avidity Abs to the V1/V2 region associated with

delayed persistent SIV infection. (A) Avidity to V1/V2 in the plasma of

vaccinated animals grouped by infection status. The animals with persis-

tent SIV infection were compared with the animals that were protected

either from SIV infection or from high viremia. A significant difference

was observed using a Mann–Whitney–Wilcoxon test with p = 0.024 (B) A

direct correlation was observed between the V1V2 Ab avidity and the

number of SIV challenges needed to attain persistent SIV infection,

assessed by a Spearman test, with r = 0.49 and p = 0.014. Animals that

either were protected from infection or had only transient viremia are

represented at challenge 9.

Table I. Virus in mucosal tissues before, during, or after CD8 depletion

Vaginal Biopsies Rectal Biopsies

Animal Group SIV Infected Pre– aCD8+ tx During aCD8+ tx Post– aCD8+ tx Pre– aCD8+ tx During aCD8+ tx Post–aCD8+ tx

P448 HPV No – – – – – –P449 HPV No – – – – – –P779 HPV No – – – – – –P793 HPV Yes – – – – pos –P781 ALVAC/HPV No – – – – – –P454 ALVAC/HPV Yes – – – – – posP786 ALVAC/HPV Yes – – – pos pos –P788 Control Yes pos – – pos pos –P468 Control Yes – – pos – – –

Bold text denotes SIV infected animals. –, no SIV DNA or RNA detected; pos, positive for SIV RNA or DNA or both; tx, treatment.

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hormonal treatment, and the collection of a cytology specimenas routinely done during a Papanicolaou test causes sufficientmicrotrauma to facilitate HPV vaccination (53). Thus, intravaginalHPV vaccination could be easily incorporated into a routine gy-necologic visit and would potentially confer protection againstboth HPV and HIV.Both regimens (ALVAC/HPV and HPV) elicited high-titer

gp120-specific IgG in the blood and vaginal secretions, but nei-ther regimen induced appreciable levels of IgA in the vaginalsecretions, nor detectable gp120-specific IgA+ memory B cells inthe blood. Thus, IgA was unlikely to have played a role in theoutcome of these studies. We found similar levels of ADCC, andneither vaccine regimen induced Abs capable of neutralizing theSIVmac251 challenge stock.A theme is emerging from nonhuman primate studies that are

consistent with the results of the RV144 Thai trial: nonneutraliz-ing Abs mediate protection from lentiviral infection. Indeed, ina vaccine regimen that efficiently primes CD8+ T cells using gp96Ig to express SIV peptides, protection from SIV infection wasachieved only when an Ab-inducing protein boost was added tothe vaccination regimen (54). Our results similarly support thisconcept, as delayed persistent SIV infection was associated withthe avidity of Abs directed to the V1/V2 region of gp120 and notwith T cells. The functional role of Abs to the V1/V2 region in theefficacy of HIV vaccines remains to be clearly defined, and recentdata suggest that mAbs to V2 synergize with other enveloperegions to neutralize the virus (55). The studies described under-score the importance of this immunologic target, as we demon-strated that Abs targeting the V1/V2 region were associated withdelayed virus acquisition, as in the case of HIV in RV144. Thesedata suggest the relevance of this animal model for the preclinicalevaluation of HIV vaccine candidates.

AcknowledgmentsWe thank Dr. Nancy Miller and the Division of AIDS, National Institutes of

Health, for the SIVmac251 virus stock and for supporting the measurement

of several Ab responses; Dr. Jean Charles Grivel for a processing protocol

for vaginal biopsies; Robyn Parks for help in processing samples; Teresa

Habina for editing the manuscript; Cynthia Thompson for helping with

HPV-PsV expansion; Kathy McKinnon for flow cytometric support; Ad-

vanced Bioscience Laboratories, specifically Debora Weiss, Jim Treece,

Maria Grazia Ferrari, Hye-kyung Chung, and Eun Mi Lee, for animal care,

sample collection, and quantification of SIV RNA and DNA; and Keith

Reimann and the National Institutes of Health reagent resource for the

CD8 depleting reagent.

DisclosuresJ.T.S. and B.S.G. are named inventors on United States patent application

12/863,572 filed July 19, 2010, HPV virus-like particles for delivery of

gene-based vaccines. G.F. is named inventor on United States patent appli-

cation PCT/US1992/005107 filed June 12, 1992, immunodeficiency virus

recombinant poxvirus vaccine.

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