This information is current as of August 20, Nina Malkevitch, L. Jean Patterson, Kristine Aldrich, Ersell

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1 This information is current as of August 20, References Subscription Permissions Alerts A Replication Competent Adenovirus 5 Host Range Mutant-Simian Immunodeficiency Virus (SIV) Recombinant Priming/Subunit Protein Boosting Vaccine Regimen Induces Broad, Persistent SIV-Specific Cellular Immunity to Dominant and Subdominant Epitopes in Mamu-A*01 Rhesus Macaques Nina Malkevitch, L. Jean Patterson, Kristine Aldrich, Ersell Richardson, W. Gregory Alvord and Marjorie Robert-Guroff J Immunol 2003; 170: ; ; doi: /jimmunol This article cites 51 articles, 28 of which you can access for free at: Why The JI? Submit online. Rapid Reviews! 30 days* from submission to initial decision No Triage! Every submission reviewed by practicing scientists Fast Publication! 4 weeks from acceptance to publication *average Information about subscribing to The Journal of Immunology is online at: Submit copyright permission requests at: Receive free -alerts when new articles cite this article. Sign up at: Downloaded from by guest on August 20, 2018 The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD Copyright 2003 by The American Association of Immunologists All rights reserved. Print ISSN: Online ISSN:

2 The Journal of Immunology A Replication Competent Adenovirus 5 Host Range Mutant-Simian Immunodeficiency Virus (SIV) Recombinant Priming/Subunit Protein Boosting Vaccine Regimen Induces Broad, Persistent SIV-Specific Cellular Immunity to Dominant and Subdominant Epitopes in Mamu-A*01 Rhesus Macaques Nina Malkevitch,* L. Jean Patterson,* Kristine Aldrich,* Ersell Richardson,* W. Gregory Alvord, and Marjorie Robert-Guroff 1 * CTL are important in controlling HIV and SIV infection. To quantify cellular immune responses induced by immunization, CD8 T cells specific for the subdominant Env p15m and p54m epitopes and/or the dominant Gag p11c epitope were evaluated by tetramer staining in nine macaques immunized with an adenovirus (Ad) 5 host range mutant (Ad5hr)-SIVenv/rev recombinant and in four of nine which also received an Ad5hr-SIVgag recombinant. Two Ad5hr-SIV recombinant priming immunizations were followed by two boosts with gp120 protein or an envelope polypeptide representing the CD4 binding domain. Two mock-immunized macaques served as controls. IFN- -secreting cells were also assessed by ELISPOT assay using p11c, p15m, and p54m peptide stimuli and overlapping pooled Gag and Env peptides. As shown by tetramer staining, Ad-recombinant priming elicited a high frequency of persistent CD8 T cells able to recognize p11c, p15m, and p54m epitopes. The presence of memory cells 38 wk postinitial immunization was confirmed by expansion of tetramer-positive CD8 T cells following in vitro stimulation. The SIV-specific CD8 T cells elicited were functional and secreted IFN- in response to SIV peptide stimuli. Although the level and frequency of response of peripheral blood CD8 T cells to the subdominant Env epitopes were not as great as those to the dominant p11c epitope, elevated responses were observed when lymph node CD8 T cells were evaluated. Our data confirm the potency and persistence of functional cellular immune responses elicited by replication competent Ad-recombinant priming. The cellular immunity elicited is broad and extends to subdominant epitopes. The Journal of Immunology, 2003, 170: The prevalence of HIV-1 continues to increase, especially in developing countries. In Africa, for example, it is estimated that over 30 million individuals are infected (1). An effective vaccine for HIV, believed to be the only way to prevent HIV transmission and control the epidemic, is thus urgently needed. The immunodeficiency induced by SIV in rhesus macaques is quite similar to the disease caused by HIV-1 in humans (2), thus providing a suitable model to evaluate immune responses and protective efficacy elicited by candidate vaccines. Both neutralizing Abs and CTL responses have been shown to contribute to protection against SIV infection in the macaque model. Passive administration of neutralizing Abs has protected against both i.v. and mucosal challenge with chimeric simian-human immunodeficiency viruses and SIV (3 7). As for CTL, their emergence has correlated with viral clearance in rhesus macaques (8). More convincingly, depletion of CD8 T lymphocytes in SIV-infected macaques has resulted in increased viral loads during both acute and *Basic Research Laboratory, National Cancer Institute, Bethesda, MD 20892; and Data Management Services, National Cancer Institute-Frederick, Frederick, MD Received for publication November 19, Accepted for publication February 13, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Address correspondence and reprint requests to Dr. Marjorie Robert-Guroff, National Cancer Institute, National Institutes of Health, 41 Library Drive, Building 41 Room D804, Bethesda, MD address: guroffm@exchange.nih.gov chronic phases of infection, implicating Ag-specific CTL in control of viral replication (9 11). Further, vaccine regimens that elicited only CTL have conferred significant protection of rhesus macaques against simian-human immunodeficiency virus challenge (12 14), further supporting a role for cellular immunity in vaccine strategies. The fact that CTL exert significant selective pressure against SIV (15) also provides evidence of their impact on viral replication in vivo. As both arms of the immune system contribute to protection against infection and disease progression, an optimal vaccine should induce both broad neutralizing Ab and potent cellular immune responses. In the absence of achieving sterilizing immunity, the former would lessen the initial infectious virus load, providing a more manageable viral burden to be controlled by induced cellular immunity. To this end, we are pursuing a vaccine strategy using E3 regiondeleted ( E3), replication competent adenovirus (Ad) 2 vectors to prime immune responses. Ad vectors were selected because they replicate in epithelial cells of the upper respiratory tract and gut, thereby eliciting mucosal as well as humoral and cellular immune responses (16, 17). In fact, using the SIV-macaque model we have demonstrated the induction of SIV-specific Abs in serum and secretory fluids, T cell proliferative responses, and CTL activity 2 Abbreviations used in this paper: Ad, adenovirus; Ad5hr, adenovirus 5 host range mutant; LN, lymph node; SFC, spot forming cell; SI, stimulation index; MVA, modified vaccinia Ankara. Copyright 2003 by The American Association of Immunologists, Inc /03/$02.00

3 4282 CELLULAR IMMUNITY INDUCED BY Ad-SIV RECOMBINANT VACCINES (18 21). 3 A similar strategy in which chimpanzees were sequentially vaccinated with Ad4-, Ad5-, and Ad7-HIV-1 MN gp160 recombinants followed by boosting with gp120 successfully elicited potent neutralizing Abs and CTL activity, and resulted in protection against low- and high-dose HIV challenges, including a heterologous primary isolate challenge (22 24). Our earlier studies in macaques used only an Ad 5 host range mutant (Ad5hr)-SIVenv/rev recombinant-based vaccine as a model system to allow evaluation of both neutralizing Ab and cellular immune responses. Recombinants with additional gene inserts, encoding SIV Rev-independent Gag, and SIV Nef, have now been constructed to increase the breadth of the induced cellular immune response, and are now under investigation in rhesus macaques. In this study, we focus on induced cellular immune responses to Gag and Env, using new techniques that have made it possible to readily quantify vaccine or infection-induced CD8 T cell responses. These include the enumeration of IFN- -secreting cells by the ELISPOT technique, and of epitope-specific cells by staining with MHC-I-peptide-tetrameric complexes (25). The latter technique has been extensively exploited in the SIV macaque model, taking advantage of the 25% of rhesus macaques that express the class I MHC-I molecule, Mamu-A*01 (26). Tetramer staining has been primarily applied to the study of the dominant SIV Gag epitope, p11c, C-M (27 29), which we will refer to in this study as p11c. However, many other CD8 T cell epitopes bound by Mamu-A*01 have been identified (30, 31). Although priming with replication competent Ad5hr-SIV recombinants was previously shown to induce CTL activity (18 21), in this study our goal was to evaluate vaccine-induced cellular responses in greater depth, by precisely quantifying SIV-specific CD8 T cells in Mamu-A*01 rhesus macaques. This would facilitate comparison of the immune responses elicited by our vaccine regimen with other approaches. Further, we wanted to determine the ability of the replication competent Ad-recombinant approach to elicit immune responses not only to dominant T cell epitopes, but also to subdominant epitopes, thereby expanding the breadth of vaccine-induced immunity. Therefore, we have analyzed CD8 T cells from peripheral blood and lymph nodes of nine Mamu-A*01 rhesus macaques immunized with Ad5hr-SIVenv/rev and -SIVgag recombinants for their recognition of the dominant Gag epitope p11c, and the subdominant Env epitopes, p15m and p54m. The functionality of these viral-specific CD8 T cells was also assessed by measuring their secretion of IFN- in response to specific SIV Gag and Env stimuli. Materials and Methods Recombinant and subunit immunogens 3 J. Zhao, Y. Lou, J. Pinczewski, N. Malkevitch, K. Aldrich, V. S. Kalyanaraman, D. Venzon, B. Peng, L. J. Patterson, Y. Edghill-Smith, et al. Boosting of SIV-specific immune responses in rhesus macaques by repeated administration of Ad5hr-SIV env/ rev and SIV gag recombinants. Submitted for publication. 4 L. J. Patterson, N. Malkevich, J. Pinczewski, D. Venzon, Y. Lou, B. Peng, C. Munch, M. Leonard, E. Richardson, K. Aldrich, et al. Potent, persistent induction and modulation of cellular immune responses in rhesus macaques primed with Ad5hr-SIV env/rev, gag, and/or nef vaccines and boosted with SIV gp120. Submitted for publication. A replication competent Ad5hr-SIV recombinant carrying the SIV smh4 env and rev genes in the deleted E3 region and expressing the entire SIV envelope and Rev proteins was used in this study. It replicates in monkey cells in vitro (32) and in vivo (18, 19). A replication competent Ad5hr- SIVnef recombinant lacking Nef amino acids 1 13 including the myristoylation signal was similarly constructed. 4 An additional Ad5hr-SIVgag recombinant carrying a rev-independent SIV mac239 gag gene (33) has been described elsewhere. 3 Native gp120 was purified from a productive tissue culture medium as previously described for HIV-1 gp120 (34). An SIV polypeptide representing covalently joined repeating 18-mer amino acid segments homologous to the CD4 binding region of SIV mac251 gp120 was synthesized as previously described (20, 35, 36). The polypeptide, termed a peptomer, forms an -helix in solution and binds CD4. Animals and immunization regimen The 11 Mamu-A*01 rhesus macaques used in this study were negative for SIV, simian retrovirus type D, and simian T cell leukemia virus. The immunogens administered to each macaque are listed in Table I. The dose of each recombinant, by each route of administration, was PFU in 500 l of PBS. Macaques that received only one or two recombinants received additional doses of Ad5hr- E3 vector so that the total amount of Ad administered to each macaque was PFU. The oral immunizations were given by stomach tube following administration of bicarbonate solution. The protein boosts (100 g/dose) in monophosphoryl A-stable emulsion adjuvant (Wyeth-Lederle Vaccines, Pearl River, NY) were administered i.m. The control Mamu-A*01 macaques received the Ad5hr- E3 vector and adjuvant only. Sample collection PBMC, obtained routinely over the course of immunization, were separated from whole blood on lymphocyte separation medium (ICN Pharmaceuticals, Aurora, OH), washed twice with PBS, and used immediately for ELISPOT assay and flow cytometry. The remaining PBMC were divided into aliquots and viably frozen for further use. Thirty-eight weeks postinitial immunization, an inguinal lymph node (LN) biopsy was obtained from immunized and control macaques. The LNs were minced, and the released lymphocytes were washed two times in PBS containing 2% FCS and stained for flow cytometry. ELISPOT assay for secretion of IFN- IFN- secretion by PBMC in response to SIV Gag and Env peptide pools and known peptide epitopes was evaluated by ELISPOT assay. Fifty Gag 20-mer peptides with 10 amino acid overlap spanned the entire SIV mac239 Gag protein and were obtained from the AIDS Research and Reference Reagent Program (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD). The peptides were dissolved in water or DMSO according to instructions provided. SIV Env peptides (Advanced BioScience Laboratories, Kensington, MD), representing the SIV smh4 gp160 protein minus the signal peptide, were 15-mers with an 11 amino acid overlap. They were dissolved in DMSO and diluted to the appropriate concentration in the tissue culture medium described below. The 214 Env peptides were divided into three pools of peptides each for PBMC stimulation. The Gag peptides were used in two pools of 25 peptides each. Three peptides representing Mamu-A*01-restricted CTL epitopes, Gag p11c (CTPYDINQM) (26, 27) and Env p15m (CAPPGY ALL) and p54m (TVPWPNETL) (29), were synthesized by Peptide Technologies (Gaithersburg, MD). The frequency of SIV-specific IFN- -secreting cells was determined using an ELISPOT kit (U-Cytech, Utrecht, The Netherlands) according to the manufacturer s manual with slight modifications. 3 Serial dilutions of PBMC were tested in triplicate wells at concentrations of cells per 100 l of R-5 medium (RPMI 1640 containing 5% human AB serum, 2 mm L-glutamine, 25 mm HEPES, 100 U/ml penicillin, and 100 mg/ml streptomycin) together with 1 g/ml of each peptide in the Gag or Env peptide pools, or the individual p11c, p15m, and p54m peptides. Con A(5 g/ml; Sigma-Aldrich, St. Louis, MO), R5 medium, or R5 medium containing an appropriate concentration of DMSO were used as positive and negative controls. Following completion of the assay, spots were counted visually using an inverted microscope. After subtraction of spot forming cells (SFC) attributable to medium alone, a positive ELISPOT response was defined as a level of SFC/10 6 PBMC at least three times the mean SFC of the control macaques and 50 SFC over the mean control value. SFC resulting from stimulation with the three Env and two Gag peptide pools were summed and reported as single values. Tetramer staining Fresh PBMC ( cells) were stained in the dark for 40 min at room temperature with a mixture of Abs including: CD3-FITC (20 l; BD PharMingen, San Diego, CA), CD8-PerCP (20 l; BD Biosciences, San Jose, CA) and p11c-, p15 m-, or p54 m-mamu-a*01-pe (1 l of5 g/ml in 100 l of FACS buffer consisting of PBS containing 1% FCS and 1% BSA). The tetramer reagents were provided by the National Institute of Allergy and Infectious Diseases Tetramer Core Facility (Atlanta, GA).

4 The Journal of Immunology 4283 Table I. Immunization regimen a Macaques Priming Recombinants Administered Protein Boosts 26, 790 Ad5hr-SIVenv/rev SIV gp120 2, 28 Ad5hr-SIVenv/rev Ad5hr-SIVgag SIV gp120 4, 32 Ad5hr-SIVenv/rev Ad5hr-SIVnef SIV gp120 15, 33 Ad5hr-SIVenv/rev Ad5hr-SIVgag Ad5hr-SIVnef SIV gp Ad5hr-SIVenv/rev SIV peptomer 39, 44 Ad5hr- E3 vector MPL-SE adjuvant a Ad5hr-SIV recombinants were administered orally and intranasally at week 0 and intratracheally at week 12. Proteins and/or adjuvant were given i.m. at weeks 24 and 36. Cells were washed twice with FACS buffer and fixed with 3.7% paraformaldehyde in PBS. PBMC from Mamu-A*01-positive macaques, uninfected or chronically infected with SIV mac251, were used as negative and positive controls, respectively. Sample data were acquired on a FACSCalibur (BD Biosciences) and analyzed using CellQuest software (BD Immunocytometry Systems, San Jose, CA). Positive tetramer staining was defined as a percentage of stained cells at least three times the mean percentage observed in control cells and at least 0.1%. In vitro stimulation of tetramer-positive CD8 T lymphocytes PBMC ( cells/ml) were placed in flasks containing R-10 medium (RPMI 1640 supplemented with 10% FCS, 2 mm L-glutamine, 25 mm HEPES, and penicillin/streptomycin), and peptides p11c, p15m, and p54m were added at a final concentration of 10 g/ml. Following culture at 37 C, 5% CO 2 for 72 h, the medium was replaced with fresh R-10 containing 10% IL-2 and cultured for an additional 72 h. Subsequently, the lymphocytes were cocultured for 5 days with autologous B cells which had been pulsed with peptides (35 g/ml) in R-10 medium for 2hat37 C and washed in R-10 before transferring to the flasks containing lymphocytes. The medium was changed every 2 days. The cells were then washed two times in PBS, and stained with tetramers as described above. Proliferation assay PBMCs were thawed, washed twice, and resuspended in R-10 medium. The cells were plated in triplicate, per 100 l, together with 1 g/ml native endotoxin-free SIV gp120 or SIV p27 (Advanced BioScience Laboratories, Inc.), or aldrithiol-2-inactivated SIV mac239 (AIDS Vaccine Program, Science Applications International Corporation, National Cancer Institute-Frederick, Frederick, MD). Con A (5 g/ml), R-10 medium alone, or microvesicles harvested from aldrithiol-2-treated Supt-1 cells served as positive and negative controls, respectively. Following 5 days of incubation at 37 C, 5% CO 2, the cells were pulsed with [ 3 H]thymidine (1 Ci/ well) and further incubated overnight. Cells were harvested using a Mach IIM (Tomtec, Hamden, CT) cell harvester and were counted on a Pharmacia (Wallac, Gaithersburg, MD) betaplate counter. Stimulation indexes (SI) were calculated by dividing the mean cpm with Ag by the mean cpm with medium alone. A positive response was defined as being two times greater than the mean response of controls, and having an SI of at least 2. The value of the microvesicle control was subtracted from the SI resulting from aldrithiol-2-inactivated SIV before making this determination. Statistical analysis Data in this study were evaluated using standard descriptive techniques and graphical methods, regression analyses, area-under-the-curve analyses, one-sample and two-sample repeated measure ANOVA, and follow-up Student s t tests. ELISPOT means and SE are depicted on a (raw) scale of counts (SFC/10 6 PBMC); however, within-subject and between-subject analyses were performed on log-transformed scores. Of prime interest to us were increases in immune responses of immunized macaques. For simplicity in interpreting immunologically significant results at specific time points, we report probabilities obtained from onesample, one-tailed post hoc Student s t tests. One-sample Student s t tests were performed for measures of immunized macaques adjusted for background measures obtained from two control macaques. Therefore, probability measures reported are conservative. We used one-tailed, paired Student s t tests to assess differences in percentages of tetramer-positive cells in paired LN and peripheral blood samples on the hypothesis that greater numbers of lymph node CD8 T cells would stain positive compared with CD8 T cells in peripheral blood (37, 38). Data in this study arose from small samples. In particular, p11c tetramer data were available for only four macaques. We considered p values 0.05 to be statistically significant, though we report p 0.10 as marginally significant. Results Tetramer staining of PBMC We systematically evaluated elicitation of SIV-specific cellular immune responses in 11 Mamu-A*01 rhesus macaques over the course of immunization with two sequential Ad5hr-SIV recombinant administrations followed by two protein subunit boosts. Representative tetramer staining of both fresh and stimulated immunized and control macaque PBMC is illustrated in Fig. 1. Shown are fresh cells from immunized macaque 33 obtained 38 wk postinitial immunization. At this late time point, CD8 T cells stained positively with p11c and p15m tetramers, but were negative with the p54m tetramer. A recall response was still present, however, as macaque 33-stimulated cells exhibited positive staining with all three tetramers. Tetramer staining of both fresh and stimulated cells from control macaque 39 was negative with all three tetramers. Overall, the percentage of positive tetramer staining in fresh cells obtained from immunized macaques over the 38 weeks of monitoring ranged from 0.1 to 1.98%. The range of positive responses increased in stimulated cells to as much as 83.1%. SIV Gag-specific CD8 T cell responses elicited in freshly isolated PBMCs from four macaques which had been immunized with the Ad5hr-SIVgag recombinant and from the two control FIGURE 1. Representative tetramer staining of nonstimulated and stimulated PBMC. PBMC obtained from control macaque no. 39 and immunized macaque no. 33 were obtained at weeks 26 and 38 postinitial immunization, respectively, and stained with p11c, p15m, and p54m tetramers as described in Materials and Methods. Cells were gated on CD8 /CD3 T lymphocytes.

5 4284 CELLULAR IMMUNITY INDUCED BY Ad-SIV RECOMBINANT VACCINES Table II. Frequency of tetramer binding by SIV-specific CD8 T cells of immunized macaques a Unstimulated PBMC Stimulated PBMC Tetramer Specificity Post-Ad5hr-SIV immunizations Postbooster immunizations Post-Ad5hr-SIV immunizations Postbooster immunizations p11c 4/4 (100) 4/4 (100) 4/4 (100) 4/4 (100) p15m 3/9 (33) 5/9 (56) 5/9 (56) 5/9 (56) p54m 1/9 (11) 4/9 (44) 4/9 (44) 5/9 (56) a Frequency is reported as the number of macaques exhibiting a positive response divided by the number of macaques tested, followed by the percent of positive responders in parentheses. monkeys were evaluated by staining with p11c-tetramer complexes. All four macaques developed positive responses to the p11c epitope in both freshly stained PBMC, as well as following stimulation with the p11c peptide (Table II). In fresh PBMC the mean number of CD8 T cells binding p11c tetramer reached a peak following the second Ad-recombinant immunization ( p 0.012) and subsequently declined over the ensuing 24 wk (Fig. 2A). Responses of individual immunized macaques are shown in Fig. 2C to facilitate comparison with results of other studies. Overall, p11c tetramer-positive cells persisted 38 wk past the first Ad5hr-SIV immunization. The presence of these cells was readily confirmed following in vitro stimulation of PBMC with p11c peptide. Sixty-six percent of the stimulated CD8 T cells were able to bind p11c tetramer at week 14 ( p ), 54% at week 26 ( p ), and 53% at week 38 (PBMC from only two immunized macaques were available for stimulation at week 38) (Fig. 2B). Responses of individual immunized macaques are shown in Fig. 2D. Elicitation of SIV Env-specific CD8 T cell responses against two nondominant epitopes, p15m and p54m, was evaluated in nine Mamu-A*01 macaques immunized with the Ad5hr-SIVenv/rev recombinant. As summarized in Table II, responses to these peptide epitopes were less frequent than those observed in response to the Gag epitope p11c. Following the two Ad5hr-recombinant immunizations, less than half of the macaques exhibited positive tetramer binding responses by fresh PBMC, although in vitro stimulation with the p15m or p54m peptides led to an increased ability to detect positive cells. Although the percentage of p15m and p54m tetramer-positive cells did not reach the levels seen with p11c, CD8 T cells specific for these Env epitopes also persisted 38 wk past initial immunization ( p values 0.066, 0.060, 0.089, and 0.044, respectively, for Figs. 3, A and B, and 4, A and B). Of interest is the somewhat higher frequency of detection of positive p15m and p54m responses in fresh cells obtained following the protein booster immunizations (Table II). This may reflect expansion of the SIV-specific cells in vivo as a result of induced CD4 FIGURE 2. p11c tetramer staining of peripheral blood CD8 T lymphocytes of immunized and control macaques. Filled arrows indicate immunization at weeks 0 and 12 with Ad5hr-SIV recombinants, and open arrows indicate boosting at weeks 24 and 36 with subunit protein. A and B, Mean values at each time point were calculated for all immunized macaques that exhibited positive staining at one or more time points and were plotted together with mean values for the control macaques. Tetramer staining values of the individual macaques immunized with Ad5hr-SIVgag are shown in C and D.

6 The Journal of Immunology 4285 FIGURE 3. p15m tetramer staining of peripheral blood CD8 T lymphocytes of immunized and control macaques. Data are presented as in the legend to Fig. 2. Th cell responses resulting from the protein and/or adjuvant administration. A similar increase in frequency was not observed for p11c, as all four macaques immunized with the Ad5hr-SIVgag recombinant responded to this dominant epitope following the two Ad-recombinant immunizations. Macaques that were immunized with Ad5hr-SIVgag, in addition to Ad5hr-SIVenv/rev, exhibited fewer positive responses to the subdominant epitopes, p15m or p54m, as shown by tetramer staining (Table III). This was the case for both freshly assayed PBMC as well as in vitro-stimulated cells, but was not an unexpected response following immunization with Ad recombinants expressing both dominant and subdominant epitopes (39). FIGURE 4. p54m tetramer staining of peripheral blood CD8 T lymphocytes of immunized and control macaques. Data are presented as in the legend to Fig. 2. Ag-specific immune response measured by ELISPOT The functional state of SIV-specific CD8 T cells in immunized monkey PBMCs was assessed using an IFN- ELISPOT assay. First, fresh cells secreting IFN- in response to overnight stimulation with p11c (CTPYDINQM), p15m (CAPPGYALL), or p54m (TVPWPNETL) peptides were enumerated. A single recombinant Ad5hr-SIVgag immunization induced IFN- -secreting cells to p11c in all four immunized macaques (Fig. 5A), with a mean level of 281 SFC/10 6 PBMC (range of positive responses ). Following the second Ad-recombinant immunization, the number of IFN- -secreting cells increased, although the kinetics were delayed, with mean SFC/10 6 PBMC at week 22 reaching 1604 (range of positive responses ) ( p 0.028). Surprisingly, with each protein boost at weeks 24 and 36, there was a subsequent drop in the level of IFN- -secreting cells (Fig. 5A), and also in the number of macaques responding, with only two of four macaques exhibiting positive responses at weeks 26 and 38. This may reflect a transient shift in immune response toward Th2-type responses. In fact, SIV Env-specific Ab titers increased dramatically 2 wk following each protein immunization administered at weeks 24 and 36 (data not shown). Nevertheless, the mean level of IFN- -secreting cells rebounded as shown by the increased values at week 34 postimmunization ( p ). At this time point, all four macaques exhibited positive responses. Thus, overall, the vaccine regimen elicited potent, functional CD8 T cells that persisted for 38 wk past initial immunization, mirroring the results of p11c tetramer staining. Similar results were obtained for IFN- secretion in response to the subdominant Env epitope peptides, p15m and p54m, although the overall levels of responses were lower (Fig. 5, B and C). All nine immunized macaques exhibited positive responses to p15m at one or more time points following the Ad-recombinant immunizations as well as the subunit protein boosts. Delayed kinetics of the immune response was again observed (Fig. 5B), with the mean peak response following Ad-recombinant priming occurring at 22 wk postimmunization ( p 0.027) (585 SFC/10 6 PBMC; range of positive responses ). A drop in IFN- -secreting cells and in frequency of responder macaques after each subunit boost was again observed. Only two of nine macaques exhibited positive responses at week 26, although by week 34, positive responses were again observed in eight of nine macaques ( p ) (mean level of 721 and range of SFC/10 6 PBMC). As with responses to the dominant epitope, p11c, high and persistent ELISPOT responses to p15m were maintained 38 wk postinitial immunization. The pattern of IFN- secretion in response to the subdominant epitope p54m (Fig. 5C) was similar to that obtained with p15m. All nine immunized macaques exhibited positive responses, and delayed kinetics, with response levels peaking 10 wk past each Ad-recombinant immunization ( p ). The mean peak level of IFN- -secreting cells at week 22 post immunization was 866, with a range of SFC/10 6 PBMC. The similarity to the p15m response was reflected in the drop in number of macaques responding following the first subunit boost, with the same two of nine macaques exhibiting positive responses. By week 34, the

7 4286 CELLULAR IMMUNITY INDUCED BY Ad-SIV RECOMBINANT VACCINES Table III. Distribution of positive tetramer responses to dominant and subdominant epitopes according to immunization group PBMC Immunogen Number Positive Responders/Number Immunized (%) p11c a p15m p54m Post-Ad-SIV Postsubunit Post-Ad-SIV Postsubunit Post-Ad-SIV Postsubunit Fresh Ad5hr-SIVgag -SIV env/rev 4/4 (100) 4/4 (100) 0/4 (0) 1/4 (25) 0/4 (0) 1/4 (25) Ad5hr-SIV env/rev only NA NA 3/5 (60) 4/5 (80) 1/5 (20) 3/5 (60) Stimulated Ad5hr-SIVgag -SIV env/rev 4/4 (100) 4/4 (100) 1/4 (25) 2/4 (50) 0/4 (0) 1/4 (25) Ad5hr-SIV env/rev only NA NA 4/5 (80) 3/5 (60) 4/5 (80) 4/5 (80) a NA, not applicable. mean response had returned to 770, with a range of SFC/ 10 6 PBMC for the eight of nine responders ( p ). To further explore the functionality of the SIV-specific CD8 T cells, IFN- secretion in response to Gag peptide pools was examined by ELISPOT in fresh PBMC. As shown in Fig. 6A, potent and persistent responses ( p , week 34) were observed in macaques out to 38 wk postinitial immunization with four of four animals responding. However, the level of immune response elicited was no greater than that observed in assays using the p11c peptide alone (Fig. 5A). In addition, only three of the four macaques immunized with Ad5hr-SIVgag recombinant exhibited positive responses against the Gag peptide pools following the two Ad immunizations, in contrast to four of four macaques exhibiting positive responses to p11c. Although the p11c sequence was included in the Gag-pooled peptides, these were 20-mers, not optimized for recognition by the TCR. This may explain the better response seen to the p11c peptide itself. Potent immune responses to Env peptide pools were observed after two Ad-recombinant immunizations, at weeks 22, 26, 34, and 38 ( p values 0.071, 0.016, , and 0.037, respectively) (Fig. 6B). The level of IFN- -secreting cells was comparable to that observed with p15m and p54m (Fig. 5, B and C). Overall, nine of nine macaques developed positive responses over the immunization course, however, no greater sensitivity was obtained by assaying with the Env peptide pools, which were composed of 15- mers. The apparent increase in level of IFN- -secreting cells, seen at week 34 following the first envelope protein boost, may be in FIGURE 5. Enumeration of IFN- -secreting cells in immunized and control macaques by ELISPOT assay. Mean values at each time point were calculated for all immunized macaques that exhibited positive staining at one or more time points and plotted. PBMC were stimulated with p11c, p15m, or p54m peptides as indicated. Note the different y-axis scales for A C. FIGURE 6. Enumeration of IFN- -secreting cells in immunized and control macaques by ELISPOT assay. Mean values at each time point were calculated for all immunized macaques that exhibited positive staining at one or more time points and were plotted. PBMC were stimulated with overlapping Gag or overlapping Env peptides as indicated.

8 The Journal of Immunology 4287 FIGURE 7. T cell proliferative responses of PBMC to SIV gp120 and aldrithiol-2-inactivated SIV. Positive proliferative responses are depicted as follows for immunized macaques: u, post-ad-recombinant immunizations; f, postsubunit boosts; and for control macaques: s, post-ad-vector administrations;, postadjuvant administrations. part attributable to IFN- secretion by SIV envelope-specific CD4 T cells. One macaque (no. 19, Table I) was boosted with the SIV peptomer rather than gp120. Although statistical analysis is not possible, it is worth pointing out that patterns of tetramer binding and ELISPOT responses over the immunization course were not noticeably different in macaque 19 from those of the other eight immunized macaques boosted with SIV gp120 (data not shown). Fluctuations in response profiles seen following the booster immunizations could occur in response to peptomer immunization by similar mechanisms as for gp120 immunization, as the peptomer not only is a B cell immunogen, but also contains a Th epitope (40). Proliferative T cell responses To pursue the question of whether the Ad5hr-SIV recombinant vaccine regimen elicited CD4 Th cell responses in the Mamu- A*01 macaques, we performed proliferation assays using aldrithiol-2-inactivated SIV and SIV gp120 as stimulating Ags. Three of nine immunized macaques exhibited positive proliferative responses to the inactivated SIV preparation, one following the Adrecombinant immunization and three following the boosts with subunit protein. The frequency of positive responses was a bit higher against SIV gp120, with six of nine macaques positive overall: four of nine after the two Ad recombinant immunizations and four of nine, including two different macaques, following the protein boosts. In general, the level of the proliferative responses observed was low (Fig. 7). This result suggests the immunization regimen elicits primarily CD8 T cell responses. Tetramer staining of LN cells Previous macaque studies have shown that lymphoid tissues may exhibit greater levels of HIV-2-specific CTL activity when compared with PBMC (37), although others have reported no difference in levels of viral-specific CD8 T cells in LNs and peripheral blood from SIV-infected animals (41). In HIV-1-infected individuals, HIV-specific CD8 T cells have been shown to be preferentially located in LNs in comparison to peripheral blood (38). Further, it is known that the route of immunization can influence the tissue distribution of Ag-specific CD8 T lymphocytes (42). PBMC and inguinal LN cells from immunized Mamu-A*01 monkeys were therefore examined using the tetramer technique to determine whether LNs exhibited a greater frequency and level of Gag- and Env-specific CD8 T cell responses. The results, presented in Table IV, show that the frequency of responder macaques tended to be higher when LN cells were examined, although this did not reach statistical significance. The levels of tetramer staining for p15m and p11c were marginally statistically higher ( p and p 0.054, respectively) compared with peripheral blood, and significantly higher ( p 0.017) for p54m. These results reconfirm the observation that specific cellular immune responses may be more readily detected in LN cells than in peripheral blood. More importantly, the data demonstrate a more potent immune response to the nondominant Env epitopes than would have been detected had only PBMC been evaluated. It is again noteworthy that these responses to p15m and p54m were obtained 38 wk past the initial immunization, further indicating the persistence of the immune response to the subdominant epitopes as well as the dominant p11c. Discussion Evaluation of replication competent Ad5hr-SIV recombinant priming and subunit boosting of Mamu-A*01 macaques by the quantitative techniques of tetramer staining and ELISPOT analysis has shown that the vaccine regimen elicits potent SIV-specific cellular immunity which persists for 38 wk past the initial priming immunization. This was the case with tetramer staining, in which persistent responses to both dominant and subdominant CD8 T cell epitopes seen in fresh PBMC were confirmed by measurement of memory responses in stimulated cells. The potent and persistent SIV-specific CD8 T cells were also functional as demonstrated by their secretion of IFN- in response to SIV peptide epitopes. The kinetics of responses measured by the tetramer and ELISPOT techniques varied, reflecting the difference in detecting SIV epitope-specific binding and functional responses. Yet both methods were effective and reliable in assessing immune responses over the entire immunization period. The delayed kinetics observed in appearance of positive responses by both tetramer staining and ELISPOT assay suggests that the time chosen for sampling PBMC can be critical in evaluating immune responses. The more frequent the sampling, the greater the likelihood of correctly evaluating the induced immune response. Table IV. SIV tetramer-positive CD8 T cells in inguinal LN and peripheral blood a Tetramer Tissue Number Positive/Number Tested (%) Mean % Positive SEM PBMC vs LN p11c PBMC 1/4 (25) p LN 3/4 (75) p15m PBMC 5/9 (56) p LN 5/7 (71) p54m PBMC 3/9 (33) p LN 6/7 (86) a Inguinal LN cells were obtained from seven of nine immunized macaques and the two control macaques at 38 weeks postinitial immunization. Tetramer staining of these cells was compared with staining of PBMC obtained from all 11 macaques at the same time.

9 4288 CELLULAR IMMUNITY INDUCED BY Ad-SIV RECOMBINANT VACCINES The level of immune response elicited by the vaccine regimen compared very favorably with other vaccine approaches. For example, the level of p11c tetramer staining exhibited was comparable to that reported following vaccination with DNA/modified vaccinia Ankara (MVA) combinations (13) and multiple DNA and/or DNA/IL-2Ig protein or plasmid combinations (12). Somewhat higher levels of p11c tetramer binding were obtained by the Ad-recombinant approach in comparison to immunization with canarypox (43) and MVA (44) recombinants. In contrast, the level of tetramer binding was less than that reported following two highdose immunizations with a replication defective Ad5-SIVgag recombinant (14). Similar results of comparisons of vaccine-induced IFN- -secreting cells in ELISPOT assays reveal comparable responses to those following DNA/MVA immunization (45) and a higher level of IFN- -secreting cells than seen with a vaccine regimen based on vesicular stomatitis virus recombinants, although in this case a different method of Ag presentation was used in the assay (46). Although the availability of quantitative methods of evaluating CD8 T cell responses makes comparative assessments easier, the most effective comparisons in the future will be headto-head, with vaccine approaches using the same HIV or SIV genes, and the assays done in an identical fashion. The ultimate comparison will be correlation of cellular immune response(s) with protective efficacy. It will be of interest to determine whether a threshold level of cellular immune response to one or more epitopes confers protection. Fewer studies have been reported in which nondominant epitopes were examined. In this study, we observed a strong response of high frequency in peripheral blood, in particular when the functionality of IFN- secretion was assessed. In contrast to these results, a canarypox vaccine regimen elicited very little immunity to a subdominant pol epitope, p68a (43) as assessed by tetramer staining. However, a DNA/IL-2Ig vaccine regimen elicited positive tetramer staining to an HIV subdominant Env epitope, p41a (12) comparable to the level of staining observed in this study with the subdominant SIV Env epitopes p15m and p54m. Of interest, the level of tetramer positivity and IFN- secretion observed with p15m following immunization with the Ad5hr-SIV recombinants was even greater than that reported for SIV-infected macaques in which recognition of the same epitope, called Env_CL9, was studied (31). The level of p54m tetramer staining and IFN- secretion achieved with our vaccine regimen was more comparable to that of SIV-infected macaques examined for recognition of the Env_TL9 epitope, with a slightly different amino acid sequence at the C-terminal end (TVPWPNASL) (31). Although as expected, we observed a clear immunodominant response to the Gag p11c epitope, the responses observed to the nondominant epitopes p15m and p54m were significant. Several factors can contribute to immunodominance, including binding affinity of the specific peptide epitope for the class I molecule (47, 48), Ag processing and presentation (49), and competition in the TCR-APC interaction (50). A recent review discusses immunodominance extensively (39). The pattern of response to immunodominant and subdominant epitopes can differ with regard to the nature of the immunogen, as shown by good cellular immune responses elicited to a subdominant epitope following vaccination with a plasmid DNA in comparison to immunization with an MVA recombinant (51). In the case of our replication competent Ad recombinants, it may be that deletion of the E3 region genes which modulate the host immune response, prevent down-modulation of MHC class I molecules, leading to better presentation of subdominant as well as dominant CD8 T cell epitopes. We noted a tendency toward higher response frequency to CTL epitopes in LN compared with PBMC. Further, we observed a significantly increased immune response to the subdominant epitope p54m in LN cells compared with peripheral blood, and similarly, marginally higher response levels to both the p15m and p11c epitopes in LN cells. 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