Virology 406 (2010) 48–55

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Recombinant vector-induced HIV/SIV-specific CD4+ T lymphocyte responses inrhesus monkeys

Yue Sun a, Sampa Santra a, Adam P. Buzby a, John R. Mascola b, Gary J. Nabel b, Norman L. Letvin a,b,⁎a Division of Viral Pathogenesis, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USAb Vaccine Research Center, National Institutes of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20892, USA

⁎ Corresponding author. Division of Viral PathogeneBeth Israel Deaconess Medical Center, Harvard Medical330 Brookline Avenue, Boston, MA 02115, USA. Fax: +1

E-mail address: nletvin@bidmc.harvard.edu (N.L. Le

0042-6822/$ – see front matter. Published by Elsevierdoi:10.1016/j.virol.2010.07.004

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 May 2010Returned to author for revision 14 June 2010Accepted 2 July 2010Available online 27 July 2010

Keywords:HIV vaccineCD4+ T cell responsesRhesus monkeySHIV-89.6P

The recently reported modest success of the RV144 Thai trial vaccine regimen in preventing HIV-1 acquisitionhas focused interest on the potential contribution to that protection of vaccine-elicited CD4+ T cell responses.We evaluated the induction of virus-specific CD4+ T cell responses in rhesus monkeys using a series of diversevaccine vectors. We assessed both the magnitudes and functional profiles of the antigen-specific CD4+ T cellsby measuring cytokine production, memory differentiation, and the expression of mucosal homingmolecules.We found that DNAprime/recombinantMVAboost immunizations induced particularly high-frequency virus-specific CD4+ T cell responses with polyfunctional repertoires, and these responses were partially preservedfollowing SHIV-89.6P challenge. The majority of the vaccine-elicited CD4+ T cells were CD28+memory T cellsthat expressed low levels of β7. Neither themagnitudes nor the functional profiles of the virus-specific CD4+ Tcells generated by vaccinationwere associatedwith a preservation of CD4+T cells or control of viral replicationfollowing SHIV-89.6P challenge. Interestingly, monkeys primedwith recombinant Ad5 immunogens showed adramatic expansion of both the magnitude and polyfunctionality of the vaccine-elicited CD4+ T cell responsesfollowing envelope protein boost. These results demonstrate that vaccine strategies that include recombinantMVA or recombinant Ad5 vectors can elicit robust CD4+ T cell responses.

sis, Department of Medicine,School, Center for Life Science,617 735 4527.

tvin).

Inc.

Published by Elsevier Inc.

Introduction

The findings in the recently reported HIV-1 vaccine trial in Thailand(RV144) have refocused interest on vaccine-elicited CD4+ T cell andantibody responses. In this human vaccine trial, a recombinantcanarypox priming immunization followed by an envelope proteinboosting immunization generated modest protection against theacquisition of an HIV-1 infection (Rerks-Ngarm et al., 2009). Since thisvaccine regimen elicited CD4+ T cell and antibody responses but notCD8+ T cell responses, attention is turning to the evaluation of vaccinestrategies that induce robust and durable responses of these types.

Virus-specific CD4+ T cells play a central role in the immunecontainment of HIV-1. They contribute to HIV-1 clearance both byproviding help for B cell responses and by maintaining effectivecytotoxic T lymphocytes (CTLs) (Kalams and Walker, 1998). Func-tional CD4+ T cells are also required at the time of immune primingfor the normal development of memory CD8+ T cells (Janssen et al.,2003; Shedlock and Shen, 2003). It is therefore important to explorevaccine strategies for eliciting antigen-specific CD4+ T cell responses.

While the evaluation of HIV-1 vaccine candidates has, for the mostpart, relied on determining the magnitude of vaccine-elicited Tlymphocyte IFN-γ responses, emerging data are indicating thatdifferent vaccine modalities can induce cellular immune responsesthat are qualitatively very different (Casimiro et al., 2003; Cox et al.,2008; Hansen et al., 2009; Hovav et al., 2007; Sun et al., 2008). Forexample, some vaccine modalities bias T cells to central memory andothers to effector memory responses. Moreover, different vaccineplatforms induce T lymphocyte responses with different functionalprofiles. It will be important to evaluate diverse characteristics ofvaccine-induced CD4+ T cell responses.

The present study was initiated to explore quantitative andqualitative differences in the virus-specific CD4+ T cell responsesgenerated in rhesus monkeys using different vaccine modalities. Wehad the opportunity to carry out an evaluation of these immuneresponses elicited by different vaccine vectors expressing the sameHIV and SIV gene inserts.

Results

Study design

The immunization and challenge schedule for each experimentalgroup of monkeys is summarized in Table 1 (Letvin et al., 2004; Letvinet al., 2006; Santra et al., 2004, 2007, 2009; Seaman et al., 2005). In the

49Y. Sun et al. / Virology 406 (2010) 48–55

homologous prime–boost experimental groups, 6 monkeys received1012 particles of rAd5 as a priming immunization at weeks 0 and 8,followed by a homologous rAd5 boost at week 26. Six monkeysreceived 109 pfu of recombinant MVA at weeks 0 and 8, followed byhomologous boosting immunizations at weeks 26 and 43. Another 4monkeys received 5mg plasmid DNA at weeks 0, 4, and 8, followed bya homologous DNA plasmid boosting immunization at week 42. In theheterologous DNA-rAd5 experimental group, 17 monkeys received4 mg inoculations of plasmid DNA at weeks 0, 4, and 8 followed by aheterologous rAd5 boosting immunization atweek 26. Twelvemonkeysin the DNA–rMVA experimental group received 5 mg inoculations ofplasmid DNA at weeks 0, 4, and 8 followed by a heterologousrecombinant MVA boosting immunization at week 42. Six monkeys inthe rAd5-protein group received 1011 particles of rAd5 as a primingimmunization at weeks 0 and 3, followed by an HIV-1 envelope proteinboost (SF162 gp140 delta V2) at week 39. At 12 weeks (DNA-rAd5),18 weeks (DNA–rMVA, DNA–DNA), 21 weeks (rMVA–rMVA), or22 weeks (rAd5–rAd5) after the final immunizations, five groups ofexperimental and 14 control monkeys were challenged intravenouslywith fifty 50% monkey infectious doses of pathogenic SHIV-89.6P fromthe same virus stock.

Virus-specific CD4+ T cell responses following vaccination and followingSHIV-89.6P challenge

We first sought to characterize the virus-specific CD4+ T cellresponses elicited by various well-established vaccine vectors. PBMCsfrom monkeys receiving homologous or heterologous vaccinationregimens were isolated at 2 weeks following priming immunization(prime, peak), 12 weeks following boosting immunization (boost,plateau), 2 weeks following challenge (challenge, peak), and 12 weeksfollowing challenge (challenge, plateau). PBMCs were exposed topools of overlapping peptides spanning the SIV Gag or HIV-1 Envprotein, and the fractions of CD4+ T cells producing IFN-γ, TNF-α, orIL-2 were determined by intracellular cytokine staining (Fig. 1).

Very few virus-specific CD4+ T cellswere detected inmonkeys thatreceived a rAd5 or rMVA priming immunization. In contrast, monkeysprimedwith plasmidDNAdeveloped substantial virus-specific CD4+ Tcell responses that could be measured by IFN-γ, TNF-α, or IL-2production. Homologous rAd5, rMVA, or DNA boosting elicited nofurther increase in the magnitude of the virus-specific CD4+ T cellresponses. However, the monkeys that received DNA immunizationsdeveloped a dramatic expansion of their virus-specific CD4+ T cellresponses following a heterologous rMVA boosting. A substantialexpansion of virus-specific CD4+ T cell responses was also seen afterheterologous rAd5 boosting. Of the vaccine regimens that weevaluated, DNA prime, heterologous rMVA boost immunizationelicited the highest frequency virus-specific CD4+ T cell responses.

We then sought to evaluate whether these different vaccinationregimens affected the magnitude of the CD4+ T cell responses thatdeveloped in these monkeys following a SHIV challenge (Fig. 1, lowertwo panels). The virus-specific CD4+ T cell responses of these groupsof vaccinated monkeys fell 2 weeks after challenge, consistent with

Table 1Immunization and challenge schedule.

Group Priming immunization Boosting immunization

rAd5–rAd5 (n=6) rAd5, 1012 particles; weeks 0 and 8 rAd5, 1012 particles; werMVA–rMVA (n=6) rMVA, 109 pfu; weeks 0 and 8 rMVA, 109 pfu; weeks 2DNA–DNA (n=4) DNA, 5 mg; weeks 0, 4 and 8

IL-2/Ig plasmid, 5 mg; weeks 0 and 4DNA, 5 mg; week 42

DNA–rAd5 (n=17) DNA, 4 mg; weeks 0, 4 and 8 rAd5, 1012 particles; we

DNA–rMVA (n=12) DNA, 5 mg; weeks 0, 4 and 8IL-2/Ig plasmid, 5 mg; weeks 0 and 4

rMVA, 109 pfu; week 42

rAd5–protein (n=6) rAd5, 1011 particles; weeks 0 and 3 HIV-1 SF162 gp140, 100

the well-described loss of naïve CD4+ T cells during the first 2 weeksafter SHIV-89.6P infection (Nishimura et al., 2005). However, thesevirus-specific CD4+ T cell responses returned to baseline levels12 weeks following SHIV-89.6P challenge. As expected, the controlmonkeys only generated low frequency virus-specific CD4+ T cellresponses following challenge. Twelve weeks following SHIV-89.6Pchallenge, highly significant differences were found between themagnitudes of the CD4+ T cell responses in the control monkeys andthe groups of monkeys receiving DNA prime/recombinant viral vectorboost vaccinations (Kruskal–Wallis tests, Pb0.01).

Functional profiles of virus-specific CD4+ T cells following vaccinationand following SHIV-89.6P challenge

We previously showed that heterologous prime–boost vaccineregimens induced high-frequency virus-specific CD8+ T cell responseswith polyfunctional repertoires. Moreover, the functional profiles of thevaccine-induced virus-specific CD8+T cellswere associatedwith controlof viral replication following SHIV-89.6P challenge (Sun et al., 2008).Wewanted to determinewhether these different vaccine strategies inducedqualitatively different virus-specific CD4+ T cell responses. Total virus-specific cytokine-producing CD4+ T cells were divided into sevendistinct populations based on their production of IFN-γ, TNF-α, and IL-2,either individually or in any combination. The functional profiles of thevaccine-induced CD4+ T cells are shown by expressing each type ofcytokine response as a proportion of the total response. Themean valuesfor the animals in each experimentally vaccinated group are shown in aseries of pie charts (Fig. 2).

Monkeys primed with rAd5 or DNA developed polyfunctionalvirus-specific CD4+ T cell responses that were predominantlycytokine triple positive or cytokine double positive (IFN-γ+TNF-α+IL-2− and IFN-γ−TNF-α+IL-2+). Homologous or heterologousboosting did not further expand the representation of polyfunctionalvirus-specific CD4+ T cell responses. In addition, the cytokine profilesof the virus-specific CD4+ T cells of these groups of vaccinatedmonkeys were indistinguishable 12 weeks following SHIV-89.6Pchallenge, with most cells being IL-2−IFN-γ+TNF-α+ (Fig. 2, lowertwo panels). Moreover, no significant differences were found betweenthe control and vaccinated groups in the representation of polyfunc-tional virus-specific CD4+ T cells observed following SHIV-89.6Pchallenge.

Expression of memory- and mucosal homing-associated molecules onvirus-specific CD4+ T cells following vaccination and following challenge

Having failed to detect a qualitative difference in the cytokineprofiles of the virus-specific CD4+T cell elicitedby these various vaccineregimens, we sought to determine whether these different vaccineregimens induced qualitatively different virus-specific CD4+ T cellresponses asmeasured byβ7 and CD28 expression (Fig. 3).β7-Integrinsare expressed on mucosal lymphocytes and mediate lymphocytetrafficking to and retention in mucosal tissues (Gorfu et al., 2009). Wetherefore evaluated the expression of β7 on virus-specific CD4+ T cells

SHIV-89.6P challenge Reference

ek 26 week 48 Santra et al. (2009)6 and 43 week 64 Santra et al. (2007)

week 60 Santra et al. (2004)

ek 26 week 38 Letvin et al. (2004)Letvin et al. (2006)

week 60 Santra et al. (2004)

μg adjuvanted with MF59; week 39 2 2

Fig. 1. Antigen-specific CD4+ T cell responses following vaccination and following SHIV-89.6P challenge. Peripheral blood lymphocytes from monkeys receiving homologous (rAd5/rAd5,rMVA/rMVA, DNA/DNA) or heterologous vaccination regimens (DNA/rAd5, DNA/rMVA)were exposed to pools of overlapping peptides spanning the SIV Gag or HIV-1 Env protein and thefractions of CD4+T cells producing IFN-γ, TNF-α, or IL-2 are shown in theupper twopanels. All experimental and14 controlmonkeyswere then challengedwith SHIV-89.6P, and the antigen-specific CD4+ T cell responses that developed in these monkeys following challenge are shown in the lower two panels.

Fig. 2. Cytokine profiles of antigen-specific CD4+ T cells following vaccination and following SHIV-89.6P challenge. PBL isolated from the different cohorts of rhesus monkeys at theindicated times following vaccination and following virus challenge were exposed to pools of overlapping peptides spanning the Gag or Env proteins and cytokine production wasmeasured by intracellular cytokine staining. Antigen-specific cytokine-producing CD4+ T cellswere divided into sevendistinct populations based on their production of IFN-γ, TNF-α, andIL-2 either individually or in any combination. Cytokine profiles were determined by expressing each cytokine response as a proportion of the total antigen-specific cytokine-producingCD4+ T cell response, and data are presented as the mean values from each experimental group in a pie chart.

50 Y. Sun et al. / Virology 406 (2010) 48–55

Fig. 3. Expression of memory- and mucosal homing-associated molecules on antigen-specific CD4+ T cells following vaccination and following challenge. PBL isolated from thedifferent cohorts of rhesus monkeys at the indicated times following vaccination or challenge were exposed to pools of overlapping peptides spanning the Gag or Env proteins, andthe fractions of CD4+ T cells producing IFN-γ, TNF-α, or IL-2 were determined by intracellular cytokine staining. The sum total production of all three cytokines was determined byflow cytometric analysis. Expression of CD28 (memory-associated molecule) and β7 (mucosal homing-associated molecule) on these antigen-specific cytokine-producing CD4+ Tcells is presented as mean±SEM.

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following vaccination and following challenge in these cohorts ofmonkeys. Few virus-specific CD4+ T cells expressed β7 after rAd5 orDNA priming. Homologous or heterologous boost immunization did notexpand the proportion of β7+ virus-specific CD4+ T cells. Therefore,virus-specific CD4+ T cells elicited by vaccination expressed low levelsof this mucosal homing molecule. However, approximately 20%–30% ofthe virus-specific CD4+ T cells began to express β7 after SHIV-89.6Pchallenge, and no significant differences were detected between thecontrol and vaccinated animals. A detailed phenotypic analysis was alsodone to assess thememory differentiation of thesevirus-specific CD4+Tcells. Virus-specific CD4+ T cells were divided into central and effectormemory cells based on their expression of CD28 (Pitcher et al., 2002).Interestingly, virus-specific CD4+ T cells were predominantly CD28+

memory cells, and no significant differences were detected in therelative representation of these CD28+CD4+ T cells between thedifferent groups of animals following vaccination and followingchallenge.

Magnitude and quality of vaccine-induced virus-specific CD4+ T cellresponses were not associated with preservation of CD4+ T cells andcontrol of viral replication following SHIV-89.6P challenge

We have previously shown that both the magnitude andfunctional profile of the virus-specific CD8+ T cells generated byvaccination were associated with control of viral replication followingSHIV-89.6P challenge (Sun et al., 2008). In the current study, weevaluated whether an association also exists between the magnitudeand quality of vaccine-elicited virus-specific CD4+ T cell responsesand disease progression following viral infection. To evaluate thispossibility, we divided all 33 animals that received experimentalvaccines into halves, low and high, on the basis of the magnitudes of

their CD4+ T cell counts, as well as the magnitudes of their peak andtheir plateau plasma viral RNA levels. Both the quantity and quality ofvirus-specific CD4+ T cell responses after the boost immunizationswere then compared in these cohorts (Fig. 4). The vaccine-elicitedvirus-specific CD4+ T cells were of comparable magnitudes and hadcomparable functional profiles in animals that demonstrated low orhigh set point CD4+ T cell counts post-challenge. Furthermore, thevaccine-elicited virus-specific CD4+ T cells were also comparable inthe monkeys with low or high peak and set point viral RNA levels.Thus, neither the magnitudes nor the functional profiles of the virus-specific CD4+ T cells generated by vaccination were associated with apreservation of CD4+ T cells or control of viral replication followingSHIV-89.6P challenge.

rAd5 prime, envelope protein boost induced high-frequency envelope-specific CD4+ T cell responses with polyfunctional repertoires

In the recently reported RV144 HIV-1 vaccine trial in Thailand, arecombinant canarypox priming immunization followed by anenvelope protein boosting immunization generated modest protec-tion against acquiring an HIV-1 infection (Rerks-Ngarm et al., 2009).We were therefore interested to evaluate both the magnitude andfunctional profile of the vaccine-elicited envelope-specific CD4+ T cellresponses in six rhesus monkeys that received a recombinant vectorpriming immunization followed by an HIV-1 envelope boostingimmunization.

Monkeys primed with rAd5 developed envelope-specific T cellresponses that included both CD4+ and CD8+ T cells (Fig. 5A).However, a dramatic CD4+ T cell-biased expansion of envelope-specific T cell responses was seen 2 weeks after the envelope proteinboost. We then analyzed the functional profile of these envelope-

Fig. 4.Magnitude and functionality of the vaccine-elicited antigen-specific CD4+ T cell responses were not associated with preserved CD4+ T cells or reduced plasma viral RNA levelsfollowing SHIV-89.6P challenge. The 33 animals that received different vaccine regimens were divided into halves, low and high, based on themagnitudes of their plateau CD4+ T cellcounts (A), peak plasma viral RNA levels (B), or plateau plasma viral RNA levels (C). PBL isolated following the boost immunizations were exposed to pools of overlapping peptidesspanning the Gag or Env proteins, and the fractions of CD4+ T cells producing IFN-γ, TNF-α, or IL-2 were determined by intracellular cytokine staining. In the left panels, themagnitudes of the vaccine-elicited antigen-specific CD4+ T cell responses are presented as means±SEM in bar charts. In the right panels, functional profiles of the vaccine-elicitedantigen-specific CD4+ T cells are shown as the mean values from each group of monkeys in pie charts.

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specific CD4+ T cells. Monkeys primed with rAd5 developedpolyfunctional envelope-specific CD4+ T cell responses that werepredominantly IFN-γ+TNF-α+IL-2+ or IFN-γ+TNF-α+IL-2−. Enve-lope protein boosting induced a further expansion of the polyfunc-tional envelope-specific CD4+ T cell responses, with 75% of theresponses made up of either cytokine double-positive or triple-positive CD4+ T cells. Further, the cytokine profiles of these cells wereunchanged 12 weeks following the boost immunization. A detailedphenotypic analysis was also done to assess the memory differenti-ation of these envelope-specific CD4+ T cells. Consistent withprevious results, the envelope-specific CD4+ T cells elicited by theprotein boosting immunization were predominantly CD28+ memorycells (Fig. 5B). These results indicate that rAd5 prime/envelopeprotein boost immunization induced high-frequency envelope-spe-cific CD28+ memory CD4+ T cell responses with polyfunctionalrepertoires.

Discussion

In the wake of the recently reported modest success of the RV144Thai vaccine trial, interest has turned to the potential contribution ofvaccine-elicited CD4+ T cell and antibody responses to protectionagainst HIV-1 acquisition. In the current study, we found that differentvectors generated virus-specific CD4+ T cell responses of differentmagnitudes and with different functional profiles. Of the vaccineregimens that we evaluated, DNA prime/rMVA boost immunizationelicited the highest-frequency virus-specific CD4+ T cell responses,and these cells had polyfunctional repertoires. A substantial expan-

sion of the virus-specific CD4+ T cell responses was also seen inplasmid DNA primed monkeys after rAd5 boosting.

Immediately after a SHIV-89.6P challenge, the virus-specific CD4+

T cell populations in these vaccinated monkeys decreased, likely as aconsequence of the well-described loss of CD4+ T cells during the firstweeks following a pathogenic CXCR4-tropic SHIV infection (Nishimuraet al., 2005). Although these virus-specific CD4+ T cell responsesdecreased followingSHIV-89.6P challenge, highly significantdifferenceswere observed between the magnitudes of these responses in thecontrolmonkeys and groups ofmonkeys receiving heterologous prime–boost vaccinations. These results demonstrate that immunization byDNA prime, heterologous vector boost induced high frequency HIV-1-and SIV-specific CD4+ T cell responses that are partially preservedfollowing an intravenous infection with a pathogenic CXCR4-tropicSHIV.

We also evaluated the expression of memory- and mucosalhoming-associated molecules on SIV-specific CD4+ T cells followingvaccination and following SHIV-89.6P challenge.We found that after apriming immunization very few of the virus-specific CD4+ T cellsexpressed β7, the mucosal homing-associated molecule. Moreover, ahomologous or heterologous boosting immunization did not expandthe proportion of β7+ virus-specific CD4+ T cells. Therefore, virus-specific CD4+ T cells elicited by vaccination expressed low levels ofthis mucosal homing molecule. A detailed memory phenotypicanalysis showed that majority of the virus-specific CD4+ T cellselicited by vaccination were CD28+ central memory T cells, and nosignificant differences were detected in the relative representation ofthese CD28+CD4+ T cells in the various groups of animals following

Fig. 5. HIV-1 envelope-specific CD4+ T cell responses following rAd5 prime and envelope protein boost. (A) PBL from six animals that received a rAd5 priming immunizationfollowed by an HIV-1 envelope boosting immunization were exposed to pools of overlapping peptides spanning the HIV-1 envelope protein and the fractions of CD4+ or CD8+ T cellproducing IFN-γ, TNF-α, or IL-2 are shown in the left panel. Functional profiles of the vaccine-elicited envelope-specific CD4+ T cells are shown as themean values in pie charts in theright panel. (B) Expression of IFN-γ, TNF-α, or IL-2 by CD4 + T cells from a representative rhesusmonkey following rAd5 prime and envelope protein boost immunization is shown inthe left panels. Expression of CD28 on envelope-specific cytokine-producing CD4+ T cells from a representative rhesus monkey is shown in the middle panel. Expression of CD28 onthese envelope-specific cytokine-producing CD4+ T cells is presented as mean±SEM in the right panel.

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vaccination and following challenge. Therefore, these results suggestthat these T cell vaccine strategies generated CD28+ memory CD4+ Tcell populations that home to the lymphoid tissue.

We have previously shown that both the magnitude andfunctional profile of the virus-specific CD8+ T cells generated byvaccination were associated with control of viral replication following

SHIV-89.6P challenge (Sun et al., 2008). In the current study, we foundthat neither the magnitudes nor the functional profiles of the virus-specific CD4+ T cells generated by vaccination were associated with apreservation of CD4+ T cells or control of viral replication followingSHIV-89.6P challenge. It is now clear that CD8+ T cell responses areassociated with the containment of HIV/SIV replication in acute

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infection, and they are also important for the maintenance of viralcontrol during chronic infection (Borrow et al., 1997; Schmitz et al.,1999). CD4+ T cells provide help for B cell responses and maintaineffective cytotoxic T lymphocytes (CTL) (Kalams and Walker, 1998).Functional CD4+ T cells are also required at the time of immunepriming for the development of long-term memory CD8+ T cells(Janssen et al., 2003; Shedlock and Shen, 2003). Therefore, althoughwe did not document a direct association between the magnitudes ofthe virus-specific CD4+ T cell population generated by vaccinationand the control of viral replication after challenge, we previouslyreported that a survival advantagewas associatedwith themagnitudeof the virus-specific CD4+ T cell responses generated by vaccinationand also associated with the preservation of the virus-specific CD4+ Tcell responses following SIVmac251 infection (Sun et al., 2006). Thedifference between these findings in the setting of a SHIV-89.6P andan SIVmac251 infection may reflect differences in the pathogenicconsequences of a CXCR4-tropic and a CCR5-tropic lentivirus.

We used archived PBMCs from 51 rhesus monkeys from previouslyreported studies that received different homologous or heterologousprime–boost immunizations. There are therefore caveats that should beacknowledged when interpreting these findings. The differences in theimmunization and challenge schedules for each experimental group ofmonkeys in these studies might have influenced the functional T celldata. In addition, the administration of the IL-2/Ig plasmid during DNAvaccine priming in the DNA/DNA and DNA/rMVA groups may have ledto augmented immune responses that were capable of controllingviremia and preventing disease progression following a SHIV-89.6Pchallenge. Nevertheless, the findings in the present study demonstratethat vaccine strategies that include recombinant MVA or recombinantAd5 vectors can elicit robust CD4+ T cell responses.

In the current study, we found that rhesus monkeys primed withrAd5 showed a dramatic expansion of virus-specific CD4+ T cellresponses following an envelope protein boost. In addition, boostingwith MF59-adjuvanted protein elicited a further expansion of thepolyfunctional Env-specific CD4+ T cell responses, with 3/4 of theresponses made up of either cytokine double-positive or triple-positive CD4+ T cells. Further, these polyfunctional Env-specific CD4+

T cell responses were well preserved 12 weeks following the proteinboost immunization. These results indicate that a protein boostimmunization induced durable, high-frequency envelope-specificCD4+ T cell responses with polyfunctional repertoires.

Materials and methods

Selection of rhesus monkeys

Heparinizedblood sampleswere obtained fromMamu-A*01− rhesusmonkeys (Macacamulatta). All animals were maintained in accordancewith the NIH Guide to the Care and Use of Laboratory Animals (NationalResearch Council, 1996) and with the approval of the InstitutionalAnimal Care and Use Committee of Harvard Medical School and theNational Institutes of Health.

Immunization and challenge of rhesus monkeys

Archived PBL from 51 rhesus monkeys from previously reportedstudieswere assigned to six experimental groups that received differenthomologous or heterologous prime–boost immunizations (Letvin et al.,2004, 2006; Santra et al., 2004, 2007,2009; Seamanet al., 2005). PlasmidDNA, rAd5 and rMVA vaccine vectors were constructed as previouslydescribed and all vaccine regimenswere administered by intramuscularinjection using a needle-free Biojector system. In the rAd5-proteingroup, six monkeys received 100 μg of SF162 (delta V2) gp140 proteinadministered with MF59 adjuvant as a boosting immunization (Burkeet al., 2009). The immunization and challenge schedules for eachexperimental group ofmonkeys are summarized in Table 1. Five groups

of experimentally vaccinated and 14 Mamu-A*01− control monkeyswere challenged intravenously with fifty 50% monkey infectious dosesof pathogenic SHIV-89.6P from the same virus stock.

CD4+ T lymphocyte counts and plasma viral RNA levels

Peripheral blood CD4+ T lymphocyte counts were calculated bymultiplying the total lymphocyte countby thepercentage of CD3+CD4+

Tcells determinedbymAb stainingandflowcytometric analysis. Plasmaviral RNA levels were measured by an ultra-sensitive branched DNAamplification assaywith a detection limit of 125 copies perml (SiemensDiagnostics, Berkeley, CA).

Antibodies

The antibodies used in this study were directly coupled tofluorescein isothiocyanate (FITC), phycoerythrin (PE), phycoerythrin–Texas red (ECD), peridinium chlorophyll protein-Cy5.5 (PerCP-Cy5.5),phycoerythrin-Cy7 (PE-Cy7), AmCyan, Pacific Blue®, allophycocyanin(APC), Alexa Fluor® 700 and Quantum-Dot 605. All reagents werevalidated and titrated using rhesus monkey PBL. The following mAbswere used: anti-TNF-α-FITC (MAb11; BD Biosciences, San Jose, CA),anti-β7-PE (FIB504; BD Biosciences), anti-CD95-ECD (DX2; BD Bios-ciences), anti-CD28-PerCP-Cy5.5 (L293; BDBiosciences), anti-IFN-γ-PE-Cy7 (B27; BD Biosciences), anti-CD3-Pacific Blue® (SP34-2; BDBiosciences), anti-IL-2-APC (MQ1-17H12; BD Biosciences), anti-CD8α-Alexa Fluor® 700 (RPA-T8; BD Biosciences), and anti-CD4-Qantum-Dot605 (unconjugated anti-CD4antibodywasobtained fromBDBioscience,Qantum-Dot 605 was obtained from Invitrogen, Carlsbad, CA). A violetfluorescent reactive dye (ViViD; Invitrogen) was also used as a viabilitymarker to exclude dead cells in the analysis.

PBL stimulation and intracellular cytokine staining

Purified PBL were isolated from EDTA-anticoagulated blood andfrozen in the vapor phase of liquid nitrogen. Cells were later thawed andallowed to rest for 6 h at 37 °C in a 5% CO2 environment. The viability ofthese cells was N90%. PBL were then incubated at 37 °C in a 5% CO2

environment for 6 h in the presence of RPMI/10% fetal calf serum alone(unstimulated), a pool of 15-mer Gag or Env peptides (2 μg/ml of eachpeptide; AIDS Research and Reference Reagent Program, Division ofAIDS, NIAID, NIH, Germantown, MD), or staphylococcal enterotoxin B(SEB) (5 μg/ml, Sigma-Aldrich, St. Louis, MO) as a positive control. Allcultures contained Monensin (GolgiStop; BD Biosciences) as well as1 μg/ml anti-CD49d (BD Biosciences). Anti-CD28 and anti-CD49dmAbsare usually used to costimulate T cell activation in intracellular cytokinestaining assays. Since we included anti-CD28 antibody in our stainingpanel ofmAbs,weexcluded the anti-CD28mAb in the stimulation phaseof the assay to avoid the down-regulation of this molecule. The culturedcells were stained with mAbs specific for cell surface moleculesincluding CD3, CD4, CD8, CD28, CD95, and β7. After fixing withCytofix/Cytoperm solution (BD Biosciences), cells were permeabilizedand stained with antibodies specific for IFN-γ, TNF-α, and IL-2. Labeledcells were fixed in 1% formaldehyde-PBS.

Flow cytometric analysis

Sampleswere collected on a BD LSRIIflow cytometer (BDBiosciences)and analyzed using FlowJo software (Tree Star, Ashland, OR). Approx-imately 500,000 to 1,000,000 events were collected per sample. Doubletswereexcludedby forwardscatter (FSC)-areaversusFSC-height.Deadcellswere excluded by their stainingwith amine reactive dye. CD4+ and CD8+

T cells were determined by their expression of CD3, CD4, or CD8.Functional analysesweredonebyplotting the expressionof each cytokinemolecule against another, and a boolean combination of single functionalgates was generated using FlowJo software. The frequency of cells

55Y. Sun et al. / Virology 406 (2010) 48–55

producing IFN-γ, TNF-α, and IL-2, either individually or in anycombination, was determined from the FlowJo, formatted in PESTLE,and analyzed using SPICE software (both PESTLE and SPICE softwarewere provided by M. Roederer, Bethesda, NIH). All values used foranalysis are background subtracted. Responses were consideredpositive when the percentage of total cytokine-producing cells was atleast twice that of the background.

Statistical analyses

Statistical analyses and graphical presentationswere computedwithGraphPad Prism. The Kruskal–Wallis test for multiple groups (or theMann–Whitney test for two groups) was used to compare the cellularimmune responses of the different groups of experimental animals.

Acknowledgments

We are grateful to Mario Roederer for helpful conversations andMichelle Lifton for technical assistance. The protein and adjuvantwere provided by Dr. Susan Barnett (Novartis). This work wassupported in part by funds from the intramural research program ofthe Vaccine Research Center, NIAID, NIH, the Harvard Medical SchoolCFAR grant AI060354, and the NIH grant N01-AI30033.

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