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Journal of Virology, December 2005, p. 15556-15566, Vol. 79, No. 24
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.24.15556-15566.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Cytotoxic T-Lymphocyte Escape Does Not Always Explain the Transient Control of Simian Immunodeficiency Virus SIVmac239 Viremia in Adenovirus-Boosted and DNA-Primed Mamu-A*01-Positive Rhesus Macaques

Adrian B. McDermott,1 David H. O'Connor,1 Sarah Fuenger,1 Shari Piaskowski,1 Sarah Martin,1 John Loffredo,1 Matthew Reynolds,1 Jason Reed,1 Jessica Furlott,1 Timothy Jacoby,1 Cara Riek,1 Elizabeth Dodds,1 Kendall Krebs,1 Mary-Ellen Davies,2 William A. Schleif,2 Danilo R. Casimiro,2 John W. Shiver,2 and D. I. Watkins1*

Wisconsin National Primate Research Center and Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School, University of Wisconsin, Madison, Wisconsin,1 Department of Vaccine and Biologics Research, Merck Research Laboratories, Merck & Co., 770 Sumneytown Pike, West Point, Pennsylvania2

Received 14 April 2005/ Accepted 20 August 2005


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ABSTRACT
 
Adenovirus 5 (Ad5) vectors show promise as human immunodeficiency virus vaccine candidates. Indian rhesus macaques vaccinated with Ad5-gag controlled simian-human immunodeficiency virus SHIV89.6P viral replication in the absence of Env immunogens that might elicit humoral immunity. Here we immunized 15 macaques using either a homologous Ad5-gag/Ad5-gag (Ad5/Ad5) or a heterologous DNA-gag/Ad5-gag (DNA/Ad5) prime-boost regimen and challenged them with a high dose of simian immunodeficiency virus SIVmac239. Macaques vaccinated with the DNA/Ad5 regimen experienced a brief viral load nadir of less than 10,000 viral copies per ml blood plasma that was not seen in Mamu-A*01-negative DNA/Ad5 vaccinees, Mamu-A*01-positive Ad5/Ad5 vaccinees, or vaccine-naive controls. Interestingly, most of these animals were not durably protected from disease progression when challenged with SIVmac239. To investigate the reasons underlying this short-lived vaccine effect, we investigated breadth of the T-cell response, immunogenetic background, and viral escape from CD8+ lymphocytes that recognize immunodominant T-cell epitopes. We show that these animals do not mount unusually broad cellular immune response, nor do they express unusual major histocompatibility complex class I alleles. Viral recrudescence occurred in four of the five Mamu-A*01-positive vaccinated macaques. However, only a single animal in this group demonstrated viral escape in the immunodominant Gag181-189CM9 response. These results suggest that viral "breakthrough" in vaccinated animals and viral escape are not inextricably linked and underscore the need for additional research into the mechanisms of vaccine failure.


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INTRODUCTION
 
Despite intense research, there are no effective vaccines against human immunodeficiency virus (HIV). Control of virus replication is associated with an increase in antigen-specific CD8+ T cells, while depletion of these cells results in increased virus replication (23, 28, 30, 40). Consequently, a variety of immunization strategies aimed at stimulating CD8+ T-cell immunity have been assessed in Indian rhesus macaques challenged with different simian immunodeficiency viruses (SIVs) and simian-human immunodeficiency viruses (SHIVs) (4, 8, 13, 19, 20, 27).

Among the most promising vaccine regimens are those that utilize nonreplicating adenovirus immunization vectors. An adenovirus type 5 (Ad5) recombinant expressing SIV-env elicited cellular and humoral immune responses and decreased acute-phase viral load following intravaginal SIVmac251 challenge (11). In addition, Ad5 vectors became one of the leading candidates in the HIV vaccine pipeline for eliciting robust cellular immunity after the demonstration of vaccine protection from SHIV89.6P disease progression using non-Env immunogens (41). However, a high frequency of preexisting immunity directed at target populations has stymied Ad5 as the vector of choice for future vaccine development (35, 42). This has prompted investigation into alternative adenovirus serotypes, such as Ad35 or Ad11, and additional viral vectors with enhanced delivery capacities (7, 21, 24, 45).

The Ad5-mediated protection from SHIV89.6P disease progression, however, may not accurately reflect the difficulty of protecting against HIV disease progression. Unlike most transmitted HIV isolates and many SIVs, SHIV89.6P utilizes CXCR4 as a coreceptor for viral entry (34, 46). SHIV89.6P infection is also characterized by a profound and immediate CD4+ T-lymphocyte depletion in the first few weeks of infection (34). Animals that resist this initial CD4+ T-lymphocyte decline frequently manifest low plasma viral loads and delayed disease progression. Therefore, there is a pressing need to evaluate the outcome of Ad5 vaccination in nonhuman primates challenged with SIV strains that more closely approximate the disease course of HIV (15). Recently, Indian rhesus macaques were challenged with highly pathogenic SIVmac239 following the DNA-gag/Ad5-gag immunization regimen that conferred protection from disease progression in SHIV89.6P infection. The remarkable control observed in SHIV89.6P infection (41) did not occur in the SIVmac239-challenged macaques (12).

Nonetheless, vaccinated animals that expressed the high-frequency major histocompatibility complex (MHC) class I allele Mamu-A*01 experienced a short-lived viral load reduction between 100 and 200 days postchallenge. Certain MHC genotypes have a strong influence on SIV survivorship and plasma viral loads (32, 33, 47). Mamu-A*01-positive animals have delayed disease progression following SHIV89.6P infection as well as lower plasma and lymph node viral loads (47). The reduced lymph node viral loads are associated with improved lymph node architecture, possibly explaining the atypical outcomes in these animals. The effect of MHC genotype is even more striking in animals positive for both Mamu-A*01 and Mamu-B*17 (36).

These results illustrate some confounding issues that complicate interpretation of SIV challenge studies. First, existing knowledge about linkages between nonhuman primate MHC class I alleles and disease progression is largely based on a subset of well-studied MHC class I alleles known to bind T-cell epitopes. Many MHC class I alleles likely remain undiscovered, and genotyping tools are not widely available even for those alleles that have been characterized. Groups of vaccinated animals may contain significant numbers of animals that are predisposed towards a particular outcome. Second, Indian rhesus macaques can express more than 10 MHC class I alleles, although the contribution and cell surface expression of each of these alleles to T-cell immunity is unclear (10, 14). Immunization strategies that include entire SIV genes are likely to elicit broadly directed T-cell reactivity, though the magnitude and efficacy of individual responses will differ. Evaluating the immunogenicity of such vaccines requires analysis of T-cell responses against the entire immunogen. Focusing on known dominant, T-cell epitopes (such as the Mamu-A*01-restricted Gag181-189CM9 epitope) may underestimate the contribution of other partially effective subdominant epitopes.

Escape from T-cell responses may have multiple consequences on viral replication. Barouch et al. reported that viral escape from the Gag181-189CM9 response preceded viral replication breakthrough in both SIVsmE660- and SHIV89.6P-infected macaques (5, 6). Escape, however, may also have beneficial effects in the setting of vaccination. Burmese rhesus macaques vaccinated with a Sendai virus vector expressing SIV gag developed strong T-cell responses; even though viral escape from these vaccine-induced responses occurred rapidly after SIVmac239 challenge, plasma viral loads in these animals remained low (28). An intriguing possibility is that T-cell escape from the dominant response compromised viral replicative fitness, resulting in the emergence of a cytotoxic T-lymphocyte (CTL)-resistant but poorly replicating SIV that the remaining immune response effectively controlled.

In this study, we examined several potential causes for the unusually low viral load nadir that occurred in SIVmac239-infected, Mamu-A*01-positive animals vaccinated using a heterologous DNA-gag/Ad5-gag prime-boost regimen (DNA/Ad5). MHC class I genotyping of these animals for 60 alleles did not reveal any clear differences between these animals and Mamu-A*01-negative animals that received the same vaccine, unvaccinated Mamu-A*01-positive controls, or Mamu-A*01-positive macaques that received Ad5/Ad5 immunization but did not exhibit control of viremia. We then hypothesized that an unusually broad T-cell response, elicited by the potent DNA/Ad5 vaccination regimen and including Mamu-A*01-restricted epitopes, may explain the viral load depression. Neither the total number of peptide pools recognized per animal nor the number of pools recognized per SIV protein appeared to differ markedly among the vaccinees, though robust responses against the Mamu-A*01-restricted Gag181-189CM9 and Gag372-379LF8 epitopes were detected in the Mamu-A*01-positive animals. Finally, we examined whether viral escape from immunodominant Gag-specific responses accounted for the precipitous decline in plasma viral load and discovered that there was comparatively little amino acid substitution in virus from these animals at 1 year postinfection. Additionally, viral rebound correlated with a decline in circulating Gag181-189CM9-specific T cells and probable viral escape in only one of five macaques.


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MATERIALS AND METHODS
 
Animals and SIVmac239 challenge. Fifteen Mamu-A*01-positive Indian rhesus macaques were divided into three groups (1, 3, and 5), and 10 Mamu-A*01-negative animals were divided into two groups (2 and 4). Groups 1 and 2 (DNA/Ad5) received 5 mg of V1Jns/SIV Gag formulated in the CRL1005/BAK adjuvant at 0, 4, and 8 weeks followed by a single boost of 1011 virus particles Ad5/SIV gag booster at week 24. Group 3 (Ad5/Ad5) received 1011 virus particles of Ad5/SIV Gag three times at 0, 4, and 24 weeks. Groups 4 and 5 were not vaccinated and served as our naive control animals. Details of the administration are described elsewhere (12). Plasma and peripheral blood mononuclear cell (PBMC) samples were collected during immunization at 16, 18, 26, 30, and 32 weeks and then at 4.4, 6.4, 16.4, 18.4, 20.4, 23.4, 29.6, 33.7, 59.4, 60.4, and 61.4 weeks after SIVmac239 challenge as standard animal protocols allowed.

Animals were challenged with SIVmac239 nef/open virus stock (provided by Ronald Desrosiers, Harvard Medical School, Southborough, MA). Animals were challenged intrarectally with 1 x 104 50% tissue culture infectious doses of the virus. Viral loads were monitored by kinetic reverse transcription-PCR and are published in detail elsewhere (12). Animal care and treatment were in accordance with standards approved by the Institutional Animal Care and Use Committee according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council.

MHC genotyping. Each animal in this study was screened with allele-specific primers for Mamu-A*01, -A*02, and -B*17 as previously described (references 3 and 26 and data not shown). Class I MHC alleles were also assessed in these macaques by using reference strand-mediated conformational analysis (RSCA). Briefly, RNA was extracted from 5 x 106 PBMC using a kit according to the manufacturer's protocol (RNAeasy; QIAGEN). To perform RSCA, reference strands were synthesized by PCR amplification using the following primer sequences: sense primer (5'Refstrand), [Cy5]GCTACGTGGACGACACGC; antisense primer (3' Refstrand), CAGAAGGCACCACCACAGC. Primers for amplifying cDNA from the macaques were not labeled with Cy5. Reference strands were synthesized from the following Mamu class I alleles: A*15, A*20, B*05, B*07, and B*60. The reference and unknown amplicons were mixed by adding 1 µl of reference to 3 µl of amplified unknown MHC class I PCR product at 92°C in a PCR tube for 5 min, 55°C for 5 min, 15°C for 5 min, and then at 4°C until ready to use. Heteroduplexes are preferably run within 2 h of being made. RSCA gels were run using the Amersham Pharmacia Alfexpress II using standard conditions described by Pel-Freez Clinical Systems Inc. (Milwaukee, WI). Peaks corresponding to MHC class I alleles were determined manually. Mobility values for each peak were determined by comparison to known standards run in parallel with unknowns on each gel. Known MHC class I clones were run at least three times with each reference strand to obtain an average mobility value. The standard deviation from each run was calculated, and peaks that fell within three standard deviations of known values for all five reference strands were considered possible allele matches.

Peptides. Overlapping 15-mer pools, individual 15-mers, or previously defined minimal optimal CTL epitope peptides were synthesized by the National Institute of Health or the Natural and Medical Science Institute (University of Tubingen, Tubingen, Germany) based on SIVmac239 protein sequences. Consecutive 15-mers overlapping by 11 amino acids were pooled into groups of 10 spanning the whole proteome (83 pools in total). All peptides were at a 1 mM stock concentration and were used at 10 µM final concentrations.

IFN-{gamma} enzyme-linked immunospot (ELISPOT) assay. Peptide-specific gamma interferon (IFN-{gamma}) secretion was measured by ELISPOT as described previously (29). Briefly, 96-well flat-bottom plate ELISPOT kits (U-CyTech-BV, Utrecht, The Netherlands) were used to detect the presence of IFN-{gamma}. The plates were coated with 5 µg of anti-IFN-{gamma} monoclonal antibody MD-1 (U-CyTech-BV) and then stored overnight at 4°C. Following the incubation, the plates were washed with PBST (phosphate-buffered saline [Gibco-BRL] containing 0.05% Tween 20 [Sigma, St. Louis, MO]) and blocked using 2% PBSA (PBS containing 2% bovine serum albumin [Sigma]) overnight at 4°C. Fresh PBMCs were resuspended at 2.0 x106/ml in R10. The cell suspension was added to duplicate wells containing 10 µM of peptide. Positive controls for the assay were concanavalin-A (Sigma) and a known immunodominant Mamu-A*01 peptide.

The 96-well plates were incubated overnight (16 to 18 h) at 37°C in 5% CO2. Cells were removed from the wells and washed, and 1 µg of rabbit polyclonal biotinylated detector (U-CyTech-BV) antibody solution was added and incubated for a further 60 min at 37°C. Following an additional wash, gold-labeled anti-biotin-immunoglobulin G solution (U-CyTech-BV) was added per the manufacturer's protocol. The plates were developed with an activator mixture of a silver salt solution, which precipitated at the sites of gold clusters (from the gold-labeled antibiotin solution) and appeared in 10 to 15 min as black spots. All test wells were imaged using an AID ELISPOT reader (Strassberg, Germany). A peptide-specific IFN-{gamma} response was considered significant if the adjusted mean number of spot-forming cells (SFCs) of duplicate sample wells exceeded background plus two standard deviations.

Sequencing of SIVmac239 gag. Full-length SIVmac239 gag was directly sequenced as described previously (36), with slight modifications. Three overlapping PCR primer pairs span Gag: (i) 1295-F and 1960-R; (ii) NLS5'EGag (1569-F) and NLS3'EGag (2288-R); and (iii) 2205-F and 2900-R. The sequences were run on an ABI 3730 Automated DNA Sequencer (Applied Biosystems, Foster City, CA). Sequences were edited with Sequencher 4.2 (Genecodes, Ann Arbor, MI), and mixed bases were identified automatically by Sequencher. Nucleotide sequences were aligned to wild-type SIVmac239 in MacVector 7.2.2 trial version (Accelrys, San Diego, CA). These nucleotide alignments were conceptually translated into amino acid alignments that distinguish mixed-base substitutions from complete substitutions.


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RESULTS
 
Resolution of acute viremia in DNA-gag/Ad5-gag-vaccinated Mamu-A*01-positive macaques. Vaccination did not have a durable effect on viral load or survivorship, but Mamu-A*01-positive DNA/Ad5 vaccinees had unusually low viral load profiles (12). After resolution of peak viremia, viral loads dropped below 1.1 x 104 copies/ml blood plasma in all five Mamu-A*01-positive DNA/Ad5-vaccinated animals. In contrast, only one of five Mamu-A*01-positive vaccine-naive controls and one of five Mamu-A*01-positive Ad5/Ad5-vaccinated macaques exhibited viral loads of less than 1.1 x 104 (Fig. 1).



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  		FIG. 1. Control of viral replication in the DNA/Ad5 Mamu-A*01-positive vaccinees challenged with SIVmac239. Viral loads fell below 10,000 copies/ml during the first 200 days of infection in all five Mamu-A*01-positive DNA/Ad5 vaccinees. The viral loads of these animals are shown in light blue. The geometric mean (Geomean) viral load of the Mamu-A*01-positive DNA/Ad5 animals, the Mamu-A*01-positive Ad5/Ad5 animals, and the Mamu-A*01-positive vaccine-naive animals are indicated with heavy blue, green, and magenta lines, respectively. The inset shows viral loads during the first 600 days of infection. Each blue dot represents a single data point from one Mamu-A*01-positive DNA/Ad5 vaccinee, while the heavier lines are the geometric mean viral loads of the three Mamu-A*01-positive groups as indicated in the main figure.

Plasma viral concentrations varied among the vaccinated Mamu-A*01-positive animals. The timing and stability of the low viral loads varied from animal to animal. Animal CC6V brought viral loads below 1.1 x 104 by 24 days postinfection; however, this was the viral load nadir. One week later, viral loads rebounded to 2.2 x 104, with a further increase to 1.1 x 105 by day 53. Animals 99C005 and 99C010 also exhibited early control, with viral loads of less than 1 x 104 occurring by day 40. In contrast to CC6V, viral loads stabilized in these animals and did not exceed 1.0 x 105 for the first year of infection. Viral loads increased in animal 99C010 and remained consistently greater than 1.0 x 105 until the completion of this study at day 686. Macaque 99C005 is still controlling viral replication as of this writing, with viral loads at day 686 of 1.9 x 103. The remaining two Mamu-A*01-positive DNA/Ad5 vaccinees experienced a delayed viral load nadir. Animal 99C013 did not have a viral load of less than 1.1 x 104 until 87 days postinfection, while animal 99C014 crossed this threshold at 75 days postinfection. Complex relationships between host immunogenetics, vaccine-elicited immunity, challenge-elicited immunity, and viral variation likely determine the timing and durability of virus suppression.

MHC class I alleles in the cohort of rhesus macaques. The viral load trajectories in Mamu-A*01-positive DNA/Ad5 vaccinees was surprising but not unique. One of the five Mamu-A*01-negative DNA/Ad5 vaccinees, two of the five Mamu-A*01-negative vaccine-naive controls, and one of the five Mamu-A*01-positive vaccine-naive controls had viral load nadirs of less than 1.0 x 104. Since animals that controlled virus replication were distributed throughout the vaccine and naive groups, we reasoned that MHC class I alleles other than Mamu-A*01 might explain the unusual control. MHC typing of all 25 animals was carried out by PCR-sequence-specific primers (PCR-SSP), a technique that reliably determines the presence or absence of eight common MHC class I alleles (Fig. 2). In agreement with our previous findings (36), Mamu-B*17-positive macaques fared well. Three of the five Mamu-B*17-positive macaques controlled viral replication (Fig. 2) irrespective of vaccine status. The Mamu-A*01-positive DNA/Ad5 vaccinees shared Mamu-A*01 but none of the other PCR-SSP testable MHC class I alleles. If the atypical control in these animals is mediated by an MHC class I effect, it is most likely due to vaccine-elicited Gag-specific Mamu-A*01-restricted CD8+ T-cell responses or an MHC class I allele that cannot be typed by PCR-SSP.



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  		FIG. 2. Control of viral replication is not correlated with the presence of any individual MHC class I alleles. MHC class I genotyping of 25 animals by PCR-SSP. "X" indicates the presence of a particular allele in a given animal. Animals highlighted in yellow experienced a preterminal viral load nadir of <10,000 copies/ml blood plasma.

To examine the latter possibility, we performed RSCA-based MHC class I typing on the group 1 animals and on the nongroup 1 SIV controllers. RSCA detects conformational differences between heteroduplexes formed between unknown MHC alleles and a single MHC-derived reference strand. In principle, RSCA can be used to identify all of the MHC class I alleles in a particular animal; however, we have not yet validated the technique for this use. We have, however, used RSCA to identify matching peaks that are shared between animals. RSCA on the Mamu-A*01-positive DNA/Ad5 vaccinees verified that only Mamu-A*01 is shared among all of the animals (data not shown). Interestingly, two of the three Mamu-B*17-negative controllers not in group 1 appear to share an MHC class I allele that has not yet been characterized (data not shown). This allele, like Mamu-B*17, may predispose animals to a favorable outcome following SIV challenge.

Breadth of Gag-specific CD8+ T-cell responses after immunization. We expected the Ad5/SIV gag immunization to elicit strong CD8+ T-cell responses. T-cell breadth following immunization was measured by IFN-{gamma} ELISPOT assays using fresh PBMC. One-hundred twenty-five 15-mer peptides (that overlapped by 11 amino acids) spanning the length of the SIVmac239-Gag protein were used for stimulation of the PBMCs either in 13 pools or individually to define "epitopic regions." All animals immunized using either a DNA/Ad5 or Ad5/Ad5 prime-boost regimen had detectable IFN-{gamma}-producing CD8+ T cells after priming (Fig. 3A). Following three priming immunizations with DNA-gag, PBMC from animals in the Mamu-A*01-positive and Mamu-A*01-negative groups recognized an average of 3 (range, 2 to 4) and 1.6 (range, 0 to 3) pools each, respectively. Similarly, Ad5/SIV gag-primed Mamu-A*01-positive animals recognized an average of 3.6 (range, 1 to 7) pools. Ad5/SIV gag boosts increased the number of SIVmac239 pools recognized in all groups (Fig. 3A, Table 1). Most notably, PBMC from Mamu-A*01-positive animals primed with DNA/Ad5 vaccine recognized an average of 9.4 SIVmac239-gag epitopes, whereas Ad5/Ad5-vaccinated Mamu-A*01-positive animals recognized an average of 5.4 epitopes following Ad5 boosting. Overall, despite the low numbers of SIVmac239-gag-reactive pools or mapped epitopes detected by IFN-{gamma} ELISPOT postimmunization, Mamu-A*01-positive rhesus macaques, which received DNA/Ad5, recognized more epitopes, on average, than either Mamu-A*01-negative or Mamu-A*01 Ad5/Ad5-immunized animals (Table 1).



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  		FIG. 3. Breadth of T-cell responses pre- and postchallenge detected by IFN-{gamma} ELISPOT using autologous 15-mer proteome pools (10 peptides/pool) either spanning Gag or all the SIVmac239 proteins. Panel A compares the number of SIVmac239-Gag peptide pools recognized after prime, boost, and challenge for each immunization group. Panel B shows the total number of SIVmac239 whole-proteome peptide pools detected for each animal after challenge, including the naive control groups. Panel C represents the total number of pools detected by IFN-{gamma} ELISPOT for each protein in the whole group.


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  		TABLE 1. Number of CD8 T-cell epitopic regions recognized following immunization

To assess the contribution of Mamu-A*01-restricted responses to overall breadth of CD8+ T-cell responses prior to SIVmac239 challenge, we also screened fresh PBMC with known Mamu-A*01-restricted 9-mer epitopes by IFN-{gamma} ELISPOT. The immunodominant Mamu-A*01-Gag181-189CM9 epitope was recognized in all vaccinated Mamu-A*01-positive animals at mean frequencies of 2,060 and 1,631 spot-forming cells (SFCs) per 106 PBMC following immunization. The subdominant Mamu-A*01-Gag372-379LF8 epitope was also recognized in all Mamu-A*01 animals prior to challenge. Other Mamu-A*01 epitopes, Gag149-157LW9, Gag340-349VT10, and Gag372-380LA9, were also detected in some of the Mamu-A*01-positive macaques prior to challenge (data not shown).

Breadth of CD8 T-cell responses after SIVmac239 challenge. Following SIVmac239 challenge, we analyzed CD8+ T-cell responses by IFN-{gamma} ELISPOT in all groups with autologous 15-mer peptide pools that spanned the whole proteome of SIVmac239. Lymphocytes from all macaques recognized peptide pools in all of the SIV proteins following challenge (Fig. 3B and C). We determined that a similar number of peptide pools were recognized postinfection by vaccinated Mamu-A*01-positive macaques as naive controls. Therefore, the low virus load control in the Mamu-A*01-positive DNA/Ad5 vaccinees occurs in the presence of a CD8+ T-cell response that is not substantially broader than that observed in Mamu-A*01-positive vaccine-naive controls. This would suggest a qualitative difference between the CD8+ T-cell responses in the vaccinated Mamu-A*01 animals compared to the responses in the Mamu-A*01-positive controls.

We then asked whether CD8+ T-cell response breadth against other SIV proteins could explain the virus control in the Mamu-A*01-positive DNA/Ad5 vaccinees. The number of peptide pools recognized varied among animals, possibly obfuscating increased T-cell response breadth in the Mamu-A*01-positive vaccinees. Interestingly, strong reactivity to 15-mer peptide pools spanning SIVmac239-nef protein was observed, particularly in the immunized Mamu-A*01 animals, compared to the naive controls (Fig. 3C). This association between Nef response breadth and vaccine status is surprising, since neither the DNA nor Ad5 vaccines contained Nef immunogen.

Amino acid variation of virus sequences 59 weeks postinfection. It has previously been suggested that viral escape in a single CTL epitope can presage a burst of viral replication, disease progression, and death (5, 6). We therefore speculated that the viral rebound from the postacute phase nadir might be the result of escape from Gag-specific T-cell responses in Mamu-A*01-positive animals vaccinated with DNA-gag/Ad5-gag. To examine this possibility, we sequenced full-length Gag at approximately 400 days postinfection (Fig. 4 and 5).



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  		FIG. 4. Full length of the SIVmac239 gag gene was sequenced from immunized Mamu-A*01 animals and representatives from the non-Mamu-A*01 groups, as described in Materials and Methods, at 59 weeks postchallenge. Epitopes recognized by IFN-{gamma} ELISPOT are highlighted in yellow and 15-mers are highlighted in blue. Residues in capital letters represent complete substitutions at the position indicated, whereas lowercase letters represent mixed or partial substitutions.



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  		FIG. 5. The longitudinal virus loads and contemporaneous Mamu-A*01 Gag181-189CM9 tetramer frequencies are shown for each of the Mamu-A*01 groups. Panel A represents four animals from group 1 immunized with a DNA/Ad5 regimen. Panel B represents group 3 immunized with an Ad5/Ad5 regimen. Panel C represents the naive control group 4. The dotted line indicates the sampling time for gag sequence. Asterisks denote termination of the animals due to complications from the infection. Longitudinal Gag181-189CM9 tetramer data was not collected for 99C010.

We were unable to correlate viral escape from the immunodominant Gag181-189CM9 epitope with viral rebound in four of the five DNA/Ad5-vaccinated animals (longitudinal Gag181-189CM9 was not collected for 99C010) that experienced greater than 10-fold increases from the viral nadir. The trajectory of viral loads in animal 99C014 is consistent with escape from Gag181-189CM9 preceding viral breakthrough, as suggested by previous studies (5, 6). In animal 99C005, where viral loads continue to hover near the limit of detection, virus variation may be constrained by low levels of viral replication. In the remaining three animals, however, viral load increases occur in spite of the wild-type epitope sequence in Gag181-189CM9. Of the five animals that received homologous Ad5 immunization, four demonstrated characteristic escape mutations in immunodominant Mamu-A*01 Gag181-189CM9 (Fig. 4). However, animal 99C093 has controlled viral replication to less than 100,000 copies/ml for 2 years despite escape in Gag181-189CM9. Furthermore, animal 99C230 died at approximately 500 days postinfection, with no evidence of escape in Gag181-189CM9.

An alternate role for viral escape in modulating the effect of CD8+ T-cell vaccines was recently postulated (28). If the vaccination regimen prompts selection of viral variants with an attenuated replicative capacity, the immune response may be more likely to control viral replication. This type of vaccine-elicited selection may occur by the elicitation of CD8+ T-cell responses or other, uncharacterized mechanisms. Interestingly, viruses from 10 of 15 vaccinated animals, including 4 of the 5 Mamu-A*01-positive DNA/Ad5-vaccinated macaques, harbored amino acid replacements in the Mamu-A*01-restricted Gag372-379LF8 epitope. Whether this effect is mediated by Mamu-A*01-specific CD8+ T cells is uncertain, as virus from all three Mamu-A*01-negative DNA/Ad5 vaccinees that we sequenced also had variation in this epitope region. In contrast, only 1 of 9 vaccine-naive controls and 2 of 35 animals in a separate viral evolution study had variation in this region (36). We conclude that the Ad5 vaccination promoted viral evolution within this region of gag, though the significance of this observation is unknown.


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DISCUSSION
 
It has been exceedingly difficult to ameliorate disease progression in vaccinated Indian rhesus macaques challenged with highly pathogenic SIVs (1, 22, 44). The most promising of the current candidates for immunization are the nonreplicating adenovirus 5 (Ad5) vectors, which are currently in clinical trials. A companion paper shows that DNA-gag prime-Ad5-gag boost, which was previously reported to be protective against challenge with SHIV89.6P, failed to protect against a high-dose challenge with pathogenic SIVmac239 (12). Importantly, the immunization regimen lowered virus loads by approximately 1 log for approximately 200 days in Mamu-A*01-positive macaques compared to naive controls, prompting our thorough investigation into the immunological parameters of this transient control.

This distinct relationship between the virus load and the MHC class I immunogenetic background of the animals in this study suggests a role for MHC class I-restricted immune responses. However, despite an increased breadth and magnitude of IFN-{gamma} ELISPOT responses in Mamu-A*01-positive vaccinees postimmunization, the animals ultimately failed to control virus replication following high-dose SIVmac239 challenge. Interestingly, two vaccine-naive Mamu-B*17-positive control animals controlled replication of SIVmac239 to <100,000 copies/ml following challenge, providing additional evidence that this allele is linked to low viral loads in SIVmac239-infected Indian rhesus macaques (36). Thus, the immunogenetics of the macaques in this cohort played a pivotal role in the outcome of the trial and should be considered when designing future nonhuman primate vaccine studies.

The lack of correlation between the breadth of IFN-{gamma}-reactive pools and control of virus replication has led us to reassess our current measures of immunogenicity in the context of vaccination. Following SIVmac239 challenge, fewer potentially important subdominant epitopes were detected by our IFN-{gamma} ELISPOT screening compared to the frequency observed postvaccination (data not shown) (43). The epitopic breadth in animals with lower virus loads may have been underestimated by the use of peptide pools for initial screening or the high dose of virus initially used in the challenge inoculum. It is possible that detection of subdominant responses, using pools of 15-mers that overlap by 11 amino acids, may not be optimal for IFN-{gamma} ELISPOT (9 and A. B. McDermott, unpublished observations). In addition, the use of multiparameter flow cytometry for functional characterization of CD4+ and CD8+ T-cell subsets would aid in future characterization of vector-generated immune responses compared to those of acute HIV infection (39 and M. Betts, personal communication). Lastly, a repeated low-dose model should be considered for the assessment of potential vaccine regimens (29).

Analysis of full-length Gag sequences at 400 days postchallenge revealed variation in several epitopes consistent with selective pressure by specific T-cell responses (5, 18, 25, 36, 37). DNA prime-Ad5 boost immunization elicited strong T-cell responses against the Gag372-379LF8 Mamu-A*01 epitope that is subdominant in infection of naive SIVmac239 (2, 31). Viral sequences from Mamu-A*01-positive macaques contained mutations in this epitope that are infrequently observed during natural infection (36). Interestingly, several of the same changes were also observed in Mamu-A*01-negative animals. Whether these changes represent overlapping CTL escape from multiple clustered epitopes or nonspecific selection of viral variants by the vaccine itself (unrelated to the SIV-specific CTL response) is unknown. Further analysis of the pattern of escape in subdominant epitopes elicited by immunization may provide clues to the types of subdominant epitopes that should be included in future vaccine candidates.

The immunodominant Gag181-189CM9 epitope is conserved, typically only escaping after 1 year of SIVmac239 infection. This epitope induces specific T-cell responses that utilize a number of T-cell receptor Vß rearrangements and will revert if escaped virus is transmitted to Mamu-A*01-naive hosts (16, 36, 38). It has also previously been reported that escape in the Gag181-189CM9 epitope can presage viral breakthrough in vaccinated macaques (5, 6). As expected, all Mamu-A*01-positive macaques recognized the immunodominant Gag181-189CM9 epitope following immunization, and vaccine-naive animals mounted responses only after challenge. Mamu-A*01-positive macaques vaccinated with a DNA prime-Ad5 boost regimen controlled SIVmac239 viral replication for approximately 200 days postchallenge; virus load levels then increased and became similar to that of the remaining animals in the study. Only one of these macaques showed evidence of escape in the Gag181-189CM9 epitope at approximately 400 days postinfection. In contrast, Mamu-A*01 animals vaccinated with Ad5 prime-boost all escaped from immunodominant vaccine-elicited Gag181-189CM9 T-cell responses with characteristic compensatory mutations (17). The presence of escape mutations in the replicating virus did not provide an adequate explanation for viral recrudescence and vaccine failure in the Mamu-A*01-positive DNA prime-Ad5 boost-vaccinated macaques as had previously been described for SHIV89.6P and SIV E660 (5, 6). Virus escape in the Gag181-189CM9 immunodominant epitope is, therefore, unlikely to account for the poor control of viral replication in these animals 200 days postchallenge. In addition, without any evidence of escape in the immunodominant Gag181-189CM9 epitope recognized by immunodominant vaccine-elicited T-cell responses, the plasma viral concentrations increased in two of the five Mamu-A*01-positive macaques vaccinated using the DNA prime-Ad5 boost regimen. Furthermore, increases in plasma viral concentration correlated with escape in this epitope in only four of the seven other macaques. Finally, the eventual demise of macaques in this study occurred more than a year after escape occurred, making it very difficult to claim that escape in the immunodominant Gag181-189CM9 epitope played a significant role in disease progression.

In summary, the DNA prime-Ad5 boost vaccine regimen is the most immunogenic of any known vaccine strategy in the rhesus macaque model. Frequencies of the immunodominant T-cell response to the Gag181-189CM9 epitope exceeded 1% of CD8+ and CD3+ PBMC in the Mamu-A*01-positive vaccinees after boosting and dominated the vaccine-induced response. DNA primed-Ad5 boosted Mamu-A*01-positive macaques controlled viral replication to <100,000 copies/ml for approximately 200 days postchallenge, but comparison of plasma viral concentrations between these vaccinees and the naive Mamu-A*01-positive animals was not statistically significant at days 250 to 360 postchallenge. gag sequences from these five immunized macaques at approximately 400 days postinfection found evidence for escape from the immunodominant response in only one of the five macaques. Understanding the basis for increased viral replication in these vaccinated macaques might aid us in our ability to design effective vaccines.


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ACKNOWLEDGMENTS
 
This work was supported by NIH grants RO1-AI-49120 and RO1-AI-52056 to D.I.W. and P51 RR000167 to the Wisconsin National Primate Research Center.


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FOOTNOTES
 
* Corresponding author. Mailing address: Wisconsin National Primate Center, 1220 Capitol Court, Madison, WI 53715. Phone: (608) 265-3380. Fax: (608) 265-8084. E-mail: watkins{at}primate.wisc.edu. Back


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Journal of Virology, December 2005, p. 15556-15566, Vol. 79, No. 24
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.24.15556-15566.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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