Vaccine-induced CD8+ T cells control AIDS virus replication.

Developing a vaccine for human immunodeficiency virus (HIV) may be aided by a complete understanding of those rare cases in which some HIV-infected individuals control replication of the virus. Most of these elite controllers express the histocompatibility alleles HLA-B*57 or HLA-B*27 (ref. 3). These alleles remain by far the most robust associations with low concentrations of plasma virus, yet the mechanism of control in these individuals is not entirely clear. Here we vaccinate Indian rhesus macaques that express Mamu-B*08, an animal model for HLA-B*27-mediated elite control, with three Mamu-B*08-restricted CD8(+) T-cell epitopes, and demonstrate that these vaccinated animals control replication of the highly pathogenic clonal simian immunodeficiency virus (SIV) mac239 virus. High frequencies of CD8(+) T cells against these Vif and Nef epitopes in the blood, lymph nodes and colon were associated with viral control. Moreover, the frequency of the CD8(+) T-cell response against the Nef RL10 epitope (Nef amino acids 137-146) correlated significantly with reduced acute phase viraemia. Finally, two of the eight vaccinees lost control of viral replication in the chronic phase, concomitant with escape in all three targeted epitopes, further implicating these three CD8(+) T-cell responses in the control of viral replication. Our findings indicate that narrowly targeted vaccine-induced virus-specific CD8(+) T-cell responses can control replication of the AIDS virus.

Vaccination with Gag, Vif, and Nef gene fragments affords partial control of viral replication after mucosal challenge with SIVmac239.

UNLABELLED: Broadly targeted cellular immune responses are thought to be important for controlling replication of human and simian immunodeficiency viruses (HIV and SIV). However, eliciting such responses by vaccination is complicated by immunodominance, the preferential targeting of only a few of the many possible epitopes of a given antigen. This phenomenon may be due to the coexpression of dominant and subdominant epitopes by the same antigen-presenting cell and may be overcome by distributing these sequences among several different vaccine constructs. Accordingly, we tested whether vaccinating rhesus macaques with "minigenes" encoding fragments of Gag, Vif, and Nef resulted in broadened cellular responses capable of controlling SIV replication. We delivered these minigenes through combinations of recombinant Mycobacterium bovis BCG (rBCG), electroporated recombinant DNA (rDNA) along with an interleukin-12 (IL-12)-expressing plasmid (EP rDNA plus pIL-12), yellow fever vaccine virus 17D (rYF17D), and recombinant adenovirus serotype 5 (rAd5). Although priming with EP rDNA plus pIL-12 increased the breadth of vaccine-induced T-cell responses, this effect was likely due to the improved antigen delivery afforded by electroporation rather than modulation of immunodominance. Indeed, Mamu-A*01(+) vaccinees mounted CD8(+) T cells directed against only one subdominant epitope, regardless of the vaccination regimen. After challenge with SIVmac239, vaccine efficacy was limited to a modest reduction in set point in some of the groups and did not correlate with standard T-cell measurements. These findings suggest that broad T-cell responses elicited by conventional vectors may not be sufficient to substantially contain AIDS virus replication. IMPORTANCE: Immunodominance poses a major obstacle to the generation of broadly targeted, HIV-specific cellular responses by vaccination. Here we attempted to circumvent this phenomenon and thereby broaden the repertoire of SIV-specific cellular responses by vaccinating rhesus macaques with minigenes encoding fragments of Gag, Vif, and Nef. In contrast to previous mouse studies, this strategy appeared to minimally affect monkey CD8(+) T-cell immundominance hierarchies, as seen by the detection of only one subdominant epitope in Mamu-A*01(+) vaccinees. This finding underscores the difficulty of inducing subdominant CD8(+) T cells by vaccination and demonstrates that strategies other than gene fragmentation may be required to significantly alter immunodominance in primates. Although some of the regimens tested here were extremely immunogenic, vaccine efficacy was limited to a modest reduction in set point viremia after challenge with SIVmac239. No correlates of protection were identified. These results reinforce the notion that vaccine immunogenicity does not predict control of AIDS virus replication.

Highly potent HIV-specific antibody neutralization in vitro translates into effective protection against mucosal SHIV challenge in vivo.

Most animal studies using passive administration of HIV broadly neutralizing monoclonal antibodies (bnMAbs) have associated protection against high-dose mucosal viral challenge with relatively high serum concentrations of antibody. We recently identified several bnMAbs remarkable for their in vitro potency against HIV. Of these bnMAbs, PGT121 is one of the most broad and potent antibodies isolated to date and shows 10- to 100-fold higher neutralizing activity than previously characterized bnMAbs. To evaluate the protective potency of PGT121 in vivo, we performed a protection study in rhesus macaques. Animals were i.v. administered 5 mg/kg, 1 mg/kg, or 0.2 mg/kg PGT121 24 h before being vaginally challenged with a single high dose of chimeric simian-human immunodeficiency virus (SHIV)(SF162P3). Sterilizing immunity was achieved in all animals administered 5 mg/kg and 1 mg/kg and three of five animals administered 0.2 mg/kg PGT121, with corresponding average antibody serum concentrations of 95 µg/mL, 15 µg/mL, and 1.8 µg/mL, respectively. The results suggest that a protective serum concentration for PGT121 is in the single-digit µg/mL for SHIV(SF162P3), showing that PGT121 can mediate sterilizing immunity at serum concentrations that are significantly lower than those observed in previous studies and that may be achievable through vaccination with the development of a suitable immunogen.

Vaccination with cancer- and HIV infection-associated endogenous retrotransposable elements is safe and immunogenic.

The expression of endogenous retrotransposable elements, including long interspersed nuclear element 1 (LINE-1 or L1) and human endogenous retrovirus, accompanies neoplastic transformation and infection with viruses such as HIV. The ability to engender immunity safely against such self-antigens would facilitate the development of novel vaccines and immunotherapies. In this article, we address the safety and immunogenicity of vaccination with these elements. We used immunohistochemical analysis and literature precedent to identify potential off-target tissues in humans and establish their translatability in preclinical species to guide safety assessments. Immunization of mice with murine L1 open reading frame 2 induced strong CD8 T cell responses without detectable tissue damage. Similarly, immunization of rhesus macaques with human LINE-1 open reading frame 2 (96% identity with macaque), as well as simian endogenous retrovirus-K Gag and Env, induced polyfunctional T cell responses to all Ags, and Ab responses to simian endogenous retrovirus-K Env. There were no adverse safety or pathological findings related to vaccination. These studies provide the first evidence, to our knowledge, that immune responses can be induced safely against this class of self-antigens and pave the way for investigation of them as HIV- or tumor-associated targets.

Dengue virus-specific CD4+ and CD8+ T lymphocytes target NS1, NS3 and NS5 in infected Indian rhesus macaques.

Every year, Dengue virus (DENV) infects approximately 100 million people. There are currently several vaccines undergoing clinical studies, but most target the induction of neutralizing antibodies. Unfortunately, DENV infection can be enhanced by subneutralizing levels of antibodies that bind virions and deliver them to cells of the myeloid lineage, thereby increasing viral replication (termed antibody-dependent enhancement [ADE]). T lymphocyte-based vaccines may offer an alternative that avoids ADE. The goal of our study was to describe the cellular immune response generated after primary DENV infection in Indian rhesus macaques. We infected eight rhesus macaques with 105 plaque-forming units (PFU) of DENV serotype 2 (DENV2) New Guinea C (NGC) strain, and monitored viral load and the cellular immune response to the virus. Viral replication peaked at day 4 post-infection and was resolved by day 10. DENV-specific CD4+ and CD8+ T lymphocytes targeted nonstructural (NS) 1, NS3 and NS5 proteins after resolution of peak viremia. DENV-specific CD4+ cells expressed interferon-gamma (IFN-γ) along with tumor necrosis factor-alpha (TNF-α), interleukin-2 (IL-2), and macrophage inflammatory protein-1 beta (MIP-1ß). In comparison, DENV-specific CD8+ cells expressed IFN-γ in addition to MIP-1ß and TNF-α and were positive for the degranulation marker CD107a. Interestingly, a fraction of the DENV-specific CD4+ cells also stained for CD107a, suggesting that they might be cytotoxic. Our results provide a more complete understanding of the cellular immune response during DENV infection in rhesus macaques and contribute to the development of rhesus macaques as an animal model for DENV vaccine and pathogenicity studies.

Macaque long-term nonprogressors resist superinfection with multiple CD8+ T cell escape variants of simian immunodeficiency virus.

Human immunodeficiency virus (HIV)-positive individuals can be superinfected with different virus strains. Individuals who control an initial HIV infection are therefore still at risk for subsequent infection with divergent viruses, but the barriers to such superinfection remain unclear. Here we tested long-term nonprogressors' (LTNPs') susceptibility to superinfection using Indian rhesus macaques that express the major histocompatibility complex class I (MHC-I) allele Mamu-B 17, which is associated with control of the pathogenic AIDS virus SIVmac239. The Mamu-B 17-restricted CD8(+) T cell repertoire is focused almost entirely on 5 epitopes. We engineered a series of SIVmac239 variants bearing mutations in 3, 4, or all 5 of these epitopes and used them to serially challenge 2 Mamu-B 17-positive LTNPs. None of the escape variants caused breakthrough replication in LTNPs, although they readily infected Mamu-B 17-negative naive macaques. In vitro competing coculture assays and examination of viral evolution in hosts lacking Mamu-B 17 suggested that the mutant viruses had negligible defects in replicative fitness. Both LTNPs maintained robust immune responses, including simian immunodeficiency virus (SIV)-specific CD8(+) and CD4(+) T cells and neutralizing antibodies. Our results suggest that escape mutations in epitopes bound by "protective" MHC-I molecules may not be sufficient to establish superinfection in LTNPs.

Robust, vaccine-induced CD8(+) T lymphocyte response against an out-of-frame epitope.

Rational vaccines designed to engender T cell responses require intimate knowledge of how epitopes are generated and presented. Recently, we vaccinated 8 Mamu-A*02(+) rhesus macaques with every SIV protein except Envelope (Env). Surprisingly, one of the strongest T cell responses engendered was against the Env protein, the Mamu-A*02-restricted epitope, Env(788-795)RY8. In this paper, we show that translation from an alternate reading frame of both the Rev-encoding DNA plasmid and the rAd5 vector engendered Env(788-795)RY8-specific CD8(+) T cells of greater magnitude than "normal" SIV infection. Our data demonstrate both that the pathway from vaccination to immune response is not well understood and that products of alternate reading frames may be rich and untapped sources of T cell epitopes.

Chinese origin rhesus macaque major histocompatibility complex class I molecules promiscuously present epitopes from SIV associated with molecules of Indian origin; implications for immunodominance and viral escape.

The presentation of identical peptides by different major histocompatibility complex class I (MHC-I) molecules, termed promiscuity, is a controversial feature of T cell-mediated immunity to pathogens. The astounding diversity of MHC-I molecules in human populations, presumably to enable binding of equally diverse peptides, implies promiscuity would be a rare phenomenon. However, if it occurs, it would have important implications for immunity. We screened 77 animals for responses to peptides known to bind MHC-I molecules that were not expressed by these animals. Some cases of supposed promiscuity were determined to be the result of either non-identical optimal peptides or were simply not mapped to the correct MHC-I molecule in previous studies. Cases of promiscuity, however, were associated with alterations of immunodominance hierarchies, either in terms of the repertoire of peptides presented by the different MHC-I molecules or in the magnitude of the responses directed against the epitopes themselves. Specifically, we found that the Mamu-B*017:01-restricted peptides Vif HW8 and cRW9 were also presented by Mamu-A2*05:26 and targeted by an animal expressing that allele. We also found that the normally subdominant Mamu-A1*001:01 presented peptide Gag QI9 was also presented by Mamu-B*056:01. Both A2*05:26 and B*056:01 are molecules typically or exclusively expressed by animals of Chinese origin. These data clearly demonstrate that MHC-I epitope promiscuity, though rare, might have important implications for immunodominance and for the transmission of escape mutations, depending on the relative frequencies of the given alleles in a population.

The role of MHC class I allele Mamu-A*07 during SIV(mac)239 infection.

Virus-specific CD8(+) T cells play an important role in controlling HIV/SIV replication. These T cells recognize intracellular pathogen-derived peptides displayed on the cell surface by individual MHC class I molecules. In the SIV-infected rhesus macaque model, five Mamu class I alleles have been thoroughly characterized with regard to peptide binding, and a sixth was shown to be uninvolved. In this study, we describe the peptide binding of Mamu-A1*007:01 (formerly Mamu-A*07), an allele present in roughly 5.08% of Indian-origin rhesus macaques (n = 63 of 1,240). We determined a preliminary binding motif by eluting and sequencing endogenously bound ligands. Subsequently, we used a positional scanning combinatorial library and panels of single amino acid substitution analogs to further characterize peptide binding of this allele and derive a quantitative motif. Using this motif, we selected and tested 200 peptides derived from SIV(mac)239 for their capacity to bind Mamu-A1*007:01; 33 were found to bind with an affinity of 500 nM or better. We then used PBMC from SIV-infected or vaccinated but uninfected, A1*007:01-positive rhesus macaques in IFN-γ Elispot assays to screen the peptides for T-cell reactivity. In all, 11 of the peptides elicited IFN-γ(+) T-cell responses. Six represent novel A1*007:01-restricted epitopes. Furthermore, both Sanger and ultradeep pyrosequencing demonstrated the accumulation of amino acid substitutions within four of these six regions, suggestive of selective pressure on the virus by antigen-specific CD8(+) T cells. Thus, it appears that Mamu-A1*007:01 presents SIV-derived peptides to antigen-specific CD8(+) T cells and is part of the immune response to SIV(mac)239.

Effective simian immunodeficiency virus-specific CD8+ T cells lack an easily detectable, shared characteristic.

The immune correlates of human/simian immunodeficiency virus control remain elusive. While CD8(+) T lymphocytes likely play a major role in reducing peak viremia and maintaining viral control in the chronic phase, the relative antiviral efficacy of individual virus-specific effector populations is unknown. Conventional assays measure cytokine secretion of virus-specific CD8(+) T cells after cognate peptide recognition. Cytokine secretion, however, does not always directly translate into antiviral efficacy. Recently developed suppression assays assess the efficiency of virus-specific CD8(+) T cells to control viral replication, but these assays often use cell lines or clones. We therefore designed a novel virus production assay to test the ability of freshly ex vivo-sorted simian immunodeficiency virus (SIV)-specific CD8(+) T cells to suppress viral replication from SIVmac239-infected CD4(+) T cells. Using this assay, we established an antiviral hierarchy when we compared CD8(+) T cells specific for 12 different epitopes. Antiviral efficacy was unrelated to the disease status of each animal, the protein from which the tested epitopes were derived, or the major histocompatibility complex (MHC) class I restriction of the tested epitopes. Additionally, there was no correlation with the ability to suppress viral replication and epitope avidity, epitope affinity, CD8(+) T-cell cytokine multifunctionality, the percentage of central and effector memory cell populations, or the expression of PD-1. The ability of virus-specific CD8(+) T cells to suppress viral replication therefore cannot be determined using conventional assays. Our results suggest that a single definitive correlate of immune control may not exist; rather, a successful CD8(+) T-cell response may be comprised of several factors.

CD8+ T cell recognition of cryptic epitopes is a ubiquitous feature of AIDS virus infection.

Vaccines designed to elicit AIDS virus-specific CD8+ T cells should engender broad responses. Emerging data indicate that alternate reading frames (ARFs) of both human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) encode CD8+ T cell epitopes, termed cryptic epitopes. Here, we show that SIV-specific CD8+ T cells from SIV-infected rhesus macaques target 14 epitopes in eight ARFs during SIV infection. Animals recognized up to five epitopes, totaling nearly one-quarter of the anti-SIV responses. The epitopes were targeted by high-frequency responses as early as 2 weeks postinfection and in the chronic phase. Hence, previously overlooked ARF-encoded epitopes could be important components of AIDS vaccines.

Recombinant yellow fever vaccine virus 17D expressing simian immunodeficiency virus SIVmac239 gag induces SIV-specific CD8+ T-cell responses in rhesus macaques.

Here we describe a novel vaccine vector for expressing human immunodeficiency virus (HIV) antigens. We show that recombinant attenuated yellow fever vaccine virus 17D expressing simian immunodeficiency virus SIVmac239 Gag sequences can be used as a vector to generate SIV-specific CD8(+) T-cell responses in the rhesus macaque. Priming with recombinant BCG expressing SIV antigens increased the frequency of these SIV-specific CD8(+) T-cell responses after recombinant YF17D boosting. These recombinant YF17D-induced SIV-specific CD8(+) T cells secreted several cytokines, were largely effector memory T cells, and suppressed viral replication in CD4(+) T cells.

High viremia is associated with high levels of in vivo major histocompatibility complex class I Downregulation in rhesus macaques infected with simian immunodeficiency virus SIVmac239.

Human and simian immunodeficiency viruses (HIV and SIV) downregulate major histocompatibility complex class I (MHC-I) molecules from the surface of infected cells. Although this activity is conserved across viral isolates, its importance in AIDS pathogenesis is not clear. We therefore developed an assay to detect the level of MHC-I expression of SIV-infected cells directly ex vivo. Here we show that the extent of MHC-I downregulation is greatest in SIVmac239-infected macaques that never effectively control virus replication. Our results suggest that a high level of MHC-I downregulation is a hallmark of fast disease progression in SIV infection.

Macaques vaccinated with simian immunodeficiency virus SIVmac239Delta nef delay acquisition and control replication after repeated low-dose heterologous SIV challenge.

An effective human immunodeficiency virus (HIV) vaccine will likely need to reduce mucosal transmission and, if infection occurs, control virus replication. To determine whether our best simian immunodeficiency virus (SIV) vaccine can achieve these lofty goals, we vaccinated eight Indian rhesus macaques with SIVmac239Delta nef and challenged them intrarectally (i.r.) with repeated low doses of the pathogenic heterologous swarm isolate SIVsmE660. We detected a significant reduction in acquisition of SIVsmE660 in comparison to that for naïve controls (log rank test; P = 0.023). After 10 mucosal challenges, we detected replication of the challenge strain in only five of the eight vaccinated animals. In contrast, seven of the eight control animals became infected with SIVsmE660 after these 10 challenges. Additionally, the SIVsmE660-infected vaccinated animals controlled peak acute virus replication significantly better than did the naïve controls (Mann-Whitney U test; P = 0.038). Four of the five SIVsmE660 vaccinees rapidly brought virus replication under control by week 4 postinfection. Unfortunately, two of these four vaccinated animals lost control of virus replication during the chronic phase of infection. Bulk sequence analysis of the circulating viruses in these animals indicated that recombination had occurred between the vaccine and challenge strains and likely contributed to the increased virus replication in these animals. Overall, our results suggest that a well-designed HIV vaccine might both reduce the rate of acquisition and control viral replication.

The live-attenuated yellow fever vaccine 17D induces broad and potent T cell responses against several viral proteins in Indian rhesus macaques--implications for recombinant vaccine design.

The yellow fever vaccine 17D (YF17D) is one of the most effective vaccines. Its wide use and favorable safety profile make it a prime candidate for recombinant vaccines. It is believed that neutralizing antibodies account for a large measure of the protection afforded to YF17D-vaccinated individuals, however cytotoxic T lymphocyte (CTL) responses have been described in the setting of YF17D vaccination. YF17D is an ssRNA flavivirus that is translated as a full-length polyprotein, several domains of which pass into the lumen of the endoplasmic reticulum (ER). The processing and presentation machinery for MHC class I-restricted CTL responses favor cytoplasmic peptides that are transported into the ER by the transporter associated with antigen presentation proteins. In order to inform recombinant vaccine design, we sought to determine if YF17D-induced CTL responses preferentially targeted viral domains that remain within the cytoplasm. We performed whole YF17D proteome mapping of CTL responses in six Indian rhesus macaques vaccinated with YF17D using overlapping YF17D peptides. We found that the ER luminal E protein was the most immunogenic viral protein followed closely by the cytoplasmic NS3 and NS5 proteins. These results suggest that antigen processing and presentation in this model system is not preferentially affected by the subcellular location of the viral proteins that are the source of CTL epitopes. The data also suggest potential immunogenic regions of YF17D that could serve as the focus of recombinant T cell vaccine development.

Novel translation products from simian immunodeficiency virus SIVmac239 Env-encoding mRNA contain both Rev and cryptic T-cell epitopes.

Understanding the correlates of immune protection against human immunodeficiency virus and simian immunodeficiency virus (SIV) will require defining the entire cellular immune response against the viruses. Here, we define two novel translation products from the SIV env mRNA that are targeted by the T-cell response in SIV-infected rhesus macaques. The shorter product is a subset of the larger product, which contains both the first exon of the Rev protein and a translated portion of the rev intron. Our data suggest that the translation of viral alternate reading frames may be an important source of T-cell epitopes, including epitopes normally derived from functional proteins.

Mauritian cynomolgus macaques share two exceptionally common major histocompatibility complex class I alleles that restrict simian immunodeficiency virus-specific CD8+ T cells.

Vaccines that elicit CD8(+) T-cell responses are routinely tested for immunogenicity in nonhuman primates before advancement to clinical trials. Unfortunately, the magnitude and specificity of vaccine-elicited T-cell responses are variable in currently utilized nonhuman primate populations, owing to heterogeneity in major histocompatibility (MHC) class I genetics. We recently showed that Mauritian cynomolgus macaques (MCM) have unusually simple MHC genetics, with three common haplotypes encoding a shared pair of MHC class IA alleles, Mafa-A*25 and Mafa-A*29. Based on haplotype frequency, we hypothesized that CD8(+) T-cell responses restricted by these MHC class I alleles would be detected in nearly all MCM. We examine here the frequency and functionality of these two alleles, showing that 88% of MCM express Mafa-A*25 and Mafa-A*29 and that animals carrying these alleles mount three newly defined simian immunodeficiency virus-specific CD8(+) T-cell responses. The epitopes recognized by each of these responses accumulated substitutions consistent with immunologic escape, suggesting these responses exert antiviral selective pressure. The demonstration that Mafa-A*25 and Mafa-A*29 restrict CD8(+) T-cell responses that are shared among nearly all MCM indicates that these animals are an advantageous nonhuman primate model for comparing the immunogenicity of vaccines that elicit CD8(+) T-cell responses.

Infection with "escaped" virus variants impairs control of simian immunodeficiency virus SIVmac239 replication in Mamu-B*08-positive macaques.

An understanding of the mechanism(s) by which some individuals spontaneously control human immunodeficiency virus (HIV)/simian immunodeficiency virus replication may aid vaccine design. Approximately 50% of Indian rhesus macaques that express the major histocompatibility complex (MHC) class I allele Mamu-B*08 become elite controllers after infection with simian immunodeficiency virus SIVmac239. Mamu-B*08 has a binding motif that is very similar to that of HLA-B27, a human MHC class I allele associated with the elite control of HIV, suggesting that SIVmac239-infected Mamu-B*08-positive (Mamu-B*08+) animals may be a good model for the elite control of HIV. The association with MHC class I alleles implicates CD8+ T cells and/or natural killer cells in the control of viral replication. We therefore introduced point mutations into eight Mamu-B*08-restricted CD8+ T-cell epitopes to investigate the contribution of epitope-specific CD8+ T-cell responses to the development of the control of viral replication. Ten Mamu-B*08+ macaques were infected with this mutant virus, 8X-SIVmac239. We compared immune responses and viral loads of these animals to those of wild-type SIVmac239-infected Mamu-B*08+ macaques. The five most immunodominant Mamu-B*08-restricted CD8+ T-cell responses were barely detectable in 8X-SIVmac239-infected animals. By 48 weeks postinfection, 2 of 10 8X-SIVmac239-infected Mamu-B*08+ animals controlled viral replication to <20,000 viral RNA (vRNA) copy equivalents (eq)/ml plasma, while 10 of 15 wild-type-infected Mamu-B*08+ animals had viral loads of <20,000 vRNA copy eq/ml (P = 0.04). Our results suggest that these epitope-specific CD8+ T-cell responses may play a role in establishing the control of viral replication in Mamu-B*08+ macaques.

Vaccine-induced cellular responses control simian immunodeficiency virus replication after heterologous challenge.

All human immunodeficiency virus (HIV) vaccine efficacy trials to date have ended in failure. Structural features of the Env glycoprotein and its enormous variability have frustrated efforts to induce broadly reactive neutralizing antibodies. To explore the extent to which vaccine-induced cellular immune responses, in the absence of neutralizing antibodies, can control replication of a heterologous, mucosal viral challenge, we vaccinated eight macaques with a DNA/Ad5 regimen expressing all of the proteins of SIVmac239 except Env. Vaccinees mounted high-frequency T-cell responses against 11 to 34 epitopes. We challenged the vaccinees and eight naïve animals with the heterologous biological isolate SIVsmE660, using a regimen intended to mimic typical HIV exposures resulting in infection. Viral loads in the vaccinees were significantly less at both the peak (1.9-log reduction; P < 0.03) and at the set point (2.6-log reduction; P < 0.006) than those in control naïve animals. Five of eight vaccinated macaques controlled acute peak viral replication to less than 80,000 viral RNA (vRNA) copy eq/ml and to less than 100 vRNA copy eq/ml in the chronic phase. Our results demonstrate that broad vaccine-induced cellular immune responses can effectively control replication of a pathogenic, heterologous AIDS virus, suggesting that T-cell-based vaccines may have greater potential than previously appreciated.

Ex vivo analysis of SIV-infected cells by flow cytometry.

Deciphering the complex interactions between human and simian immunodeficiency viruses (HIV/SIV) and their host cells is crucial to the development of improved therapies and vaccines. Investigating these relationships has been complicated by the inability to directly analyze infected cells among freshly isolated peripheral blood lymphocytes. Here, we describe a method to detect cells productively infected with SIVmac239 ex vivo from the blood or lymph nodes by flow cytometry. Using this method, we show a close correlation between the frequency of productively infected cells in both sample type and the plasma viral load. We define that the minimum threshold for detecting productively infected cells in lymph nodes by flow cytometry requires a plasma virus concentration of ∼2.5 × 10(4) vRNA copy Equivalents (Eq)/ml. Conversely, an approximately 2 logs higher plasma viral load is needed to detect productively infected cells in the peripheral blood. This novel protocol provides a direct analytical tool to assess interactions between SIV and host cells, which is of key importance to investigators in AIDS research.