Consequences of cytotoxic T-lymphocyte escape: common escape mutations in simian immunodeficiency virus are poorly recognized in naive hosts

Cytotoxic T lymphocytes (CTL) are associated with control of immunodeficiency virus infection but also select for variants that escape immune recognition. Declining frequencies of epitope-specific CTL frequencies have been correlated with viral escape in individual hosts. However, escape mutations may give rise to new epitopes that could be recognized by CTL expressing appropriate T-cell receptors and thus still be immunogenic when escape variants are passed to individuals expressing the appropriate major histocompatibility complex class I molecules. To determine whether peptide ligands that have been altered through escape can be immunogenic in new hosts, we challenged naïve, immunocompetent macaques with a molecularly cloned simian immunodeficiency virus (SIV) bearing common escape mutations in three immunodominant CTL epitopes. Responses to the altered peptides were barely detectable in fresh samples at any time after infection. Surprisingly, CTL specific for two of three escaped epitopes could be expanded by in vitro stimulation with synthetic peptides. Our results suggest that some escape variant epitopes evolving in infected individuals do not efficiently stimulate new populations of CTL, either in that individual or upon passage to new hosts. Nevertheless, escape variation may not completely abolish an epitope''s immunogenicity. Moreover, since the mutant epitope sequences did not revert to wild type during the study period, it is possible that low-frequency CTL exerted enough selective pressure to preserve epitope mutations in viruses replicating in vivo.In recent years, there has been increasing interest in AIDS vaccine approaches that elicit cytotoxic T lymphocytes (CTL), which recognize and eliminate cells infected with human immunodeficiency virus (HIV) (26). Unlike antibodies, effective CTL responses can be directed against epitopes derived from any viral protein, raising the possibility that CTL can be targeted to regions that are more conserved than the viral envelope. Current vaccine modalities can elicit potent CTL responses against multiple viral epitopes (25). Indeed, many lines of evidence indicate that cell-mediated immunity plays a major role in control of virus replication. Several studies have suggested an association between certain major histocompatibility complex (MHC) class I and class II alleles and control of viral replication or susceptibility to disease (6, 7, 11, 12, 15-17, 28, 36, 38, 39). CTL are also implicated in the initial control of immunodeficiency virus infection, since they appear in close temporal association with the reduction in peak viremia in both HIV-infected humans (5, 22) and simian immunodeficiency virus (SIV)-infected macaques (23). Antibody-mediated depletion of CD8⁺ cells in infected macaques resulted in dramatically increased virus loads in both acute and chronic infection (14, 27, 37).However, the plasticity of the viral genome also allows the generation of mutants that escape CTL recognition. Certain high-frequency CTL exert intense selective pressure on virus sequences, as revealed by the nearly total extinction of CTL-susceptible sequences from the actively replicating virus population within a few weeks of infection (2, 32). Escape from CTL has been observed in several studies of infected humans (12, 18, 21, 34, 35, 41) and macaques (2, 8, 30, 32, 40). Moreover, one report has shown that an HIV-1 escape mutant can be transmitted vertically (11), while other studies in vaccinated macaques have suggested that the evolution of escape mutants may be associated with a loss of containment of viral replication (4, 31). It therefore seems likely that escape from CTL responses occurs in most infected individuals (32).The apparent ubiquity of CTL escape may greatly complicate the design of CTL-based vaccines. The evolution of escape variants during infection of a single host may play a key part in viral persistence and therefore in the ultimate failure of immune containment and progression to AIDS. However, some investigators have suggested that T-cell receptor repertoires can recognize multiple epitope variants, so that CTL responses can coevolve along with viral escape variants in infected individuals (13). If T-cell receptor populations can recognize new variant epitopes arising within a single host, it seems plausible that variant epitope sequences could also be recognized efficiently in new hosts. Escape could also create “neo-epitopes,” novel sequences that are immunogenic to naïve T cells in individuals expressing the appropriate MHC class I molecules.The most rigorous test of the immunogenicity of epitopes altered through escape is to challenge a fully intact immune system with an escape mutant virus. Therefore, we identified common escape mutations that accumulated in immunodominant epitopes of SIVmac239 in infected macaques expressing the high-frequency MHC class I molecules Mamu-A*01 and Mamu-B*17. Together, these molecules bind three immunodominant CTL epitopes in SIVmac239: Gag_181-189CM9 (CTPYDINQM, Gag CM9) and Tat_28-35SL8 (STPESANL, Tat SL8) are bound by Mamu-A*01, and Nef_165-173IW9 (IRYPKTFGW, Nef IW9) is bound by Mamu-B*17. We have previously shown that the acute-phase response in Mamu-A*01 Mamu-B*17 double-positive macaques is dominated by CTL that recognize these three epitopes (33). We introduced common escape mutations into the SIVmac239 molecular clone and challenged macaques expressing both Mamu-A*01 and Mamu-B*17 with the mutant virus. CTL responses directed against the mutant epitopes were extremely low frequency or undetectable in fresh samples from each of the infected animals. In the absence of these responses, a completely new immunodominance hierarchy was established. Our results suggest it is unlikely that “escaped” epitopes will be recognized in newly infected individuals expressing appropriate MHC class I molecules.     

Gag-Protease-Mediated Replication Capacity in HIV-1 Subtype C Chronic Infection: Associations with HLA Type and Clinical Parameters

The mechanisms underlying HIV-1 control by protective HLA class I alleles are not fully understood and could involve selection of escape mutations in functionally important Gag epitopes resulting in fitness costs. This study was undertaken to investigate, at the population level, the impact of HLA-mediated immune pressure in Gag on viral fitness and its influence on HIV-1 pathogenesis. Replication capacities of 406 recombinant viruses encoding plasma-derived Gag-protease from patients chronically infected with HIV-1 subtype C were assayed in an HIV-1-inducible green fluorescent protein reporter cell line. Viral replication capacities varied significantly with respect to the specific HLA-B alleles expressed by the patient, and protective HLA-B alleles, most notably HLA-B*81, were associated with lower replication capacities. HLA-associated mutations at low-entropy sites, especially the HLA-B*81-associated 186S mutation in the TL9 epitope, were associated with lower replication capacities. Most mutations linked to alterations in replication capacity in the conserved p24 region decreased replication capacity, while most in the highly variable p17 region increased replication capacity. Replication capacity also correlated positively with baseline viral load and negatively with baseline CD4 count but did not correlate with the subsequent rate of CD4 decline. In conclusion, there is evidence that protective HLA alleles, in particular HLA-B*81, significantly influence Gag-protease function by driving sequence changes in Gag and that conserved regions of Gag should be included in a vaccine aiming to drive HIV-1 toward a less fit state. However, the long-term clinical benefit of immune-driven fitness costs is uncertain given the lack of correlation with longitudinal markers of disease progression.There is broad heterogeneity in the ability of HIV-infected individuals to control virus replication, ranging from elite controllers, who maintain undetectable viral loads without treatment, to rapid progressors, who progress to AIDS within 2 years of infection (9, 22, 32). Many interrelated factors, including host and viral genetic factors involved in antiviral immunity and the viral life cycle, may partially account for the differences in the course of disease progression (10, 11, 30, 41). The complex interplay between host genetic factors and viral factors is exemplified by human leukocyte antigen (HLA) class I-restricted cytotoxic T-lymphocyte (CTL) responses, which exert considerable immune pressure on the virus, resulting in escape mutations that affect the interaction of viral and host proteins, thereby influencing infection outcome.The exact mechanisms by which some HLA class I alleles, such as HLA-B*57 and HLA-B*27, are associated with slower progression to AIDS, while others, such as B*5802 and B*18, are associated with accelerated disease progression (6, 20, 42), are unclear. The magnitude and/or breadth of HLA-restricted CTL responses to the conserved Gag protein has been correlated inversely with disease progression or markers of disease progression in several studies (12, 21, 28, 31, 35, 43, 46), although there are some exceptions (4, 16, 37), while preferential targeting of the highly variable envelope protein (as occurs in HLA-B*5802-positive individuals) correlates with higher viral loads (21, 29). Protective HLA alleles restrict CTL responses that impose a strong selection pressure on a few specific Gag p24 epitopes, resulting in escape mutations (14) for which fitness costs have been demonstrated either through site-directed mutations introduced into a reference strain background (2, 8, 25, 38) or through in vivo reversion of these mutations after transmission to an HLA-mismatched individual (8, 24). Recent evidence suggests that Gag escape mutations with a fitness cost, particularly those in p24, are a significant determinant of disease progression: the transmitted number of HLA-B-associated polymorphisms in Gag was found to significantly impact the viral set point in recipients (although an associated fitness cost was not shown) (7, 15), and in a small number of infants, decreased fitness of the transmitted virus with HLA-B*5703/5801-selected mutations in Gag p24 epitopes resulted in slower disease progression (33, 39). Also, the number of reverting Gag mutations (thought to revert as a consequence of fitness costs) associated with individual HLA-B alleles was strongly correlated with the HLA-linked viral set point in chronically infected patients (26). A recent in vitro study showed that HLA-associated variation in Gag-protease, with resulting reduced replication capacity, may contribute to viral control in HIV-1 subtype B-infected elite controllers (27). Taken together, these studies suggest that CTL responses restricted by favorable HLA alleles select for escape mutations in conserved epitopes, particularly those in Gag, resulting in a fitness cost to HIV and therefore at least partly explaining the slower disease progression in individuals carrying these alleles.To date, many of the studies investigating the fitness cost of Gag escape mutations and their clinical relevance have concentrated on escape mutations associated with protective HLA alleles, have not assessed fitness consequences in the natural sequence background (in the presence of other escape and compensatory mutations), and/or have focused on a limited number of patients. Most importantly, the majority of studies have focused on HIV-1 subtype B. The present study is the first to use a large population-based approach and clinically derived Gag-protease sequences to investigate comprehensively the relationships between immune-driven sequence variation in Gag, viral replication capacity, and markers of disease progression in chronic infection with HIV-1 subtype C, the most predominant subtype in the epidemic. We assayed the replication capacity of recombinant viruses encoding patient Gag-protease in an HIV-1-inducible green fluorescent protein (GFP) reporter cell line and found associations between lower replication capacities, protective HLA alleles, protective HLA-associated mutations, lower baseline viral loads, and higher baseline CD4 counts. However, Gag-protease replication capacity did not correlate with the subsequent rate of CD4 decline.     

Human Immunodeficiency Virus Type 1-Specific CD8+ T-Cell Responses during Primary Infection Are Major Determinants of the Viral Set Point and Loss of CD4+ T Cells

Primary HIV-1 infection (PHI) is marked by a flu-like syndrome and high levels of viremia that decrease to a viral set point with the first emergence of virus-specific CD8⁺ T-cell responses. Here, we investigated in a large cohort of 527 subjects the immunodominance pattern of the first virus-specific cytotoxic T-lymphocyte (CTL) responses developed during PHI in comparison to CTL responses in chronic infection and demonstrated a distinct relationship between the early virus-specific CTL responses and the viral set point, as well as the slope of CD4⁺ T-cell decline. CTL responses during PHI followed clear hierarchical immunodominance patterns that were lost during the transition to chronic infection. Importantly, the immunodominance patterns of human immunodeficiency virus type 1 (HIV-1)-specific CTL responses detected in primary, but not in chronic, HIV-1 infection were significantly associated with the subsequent set point of viral replication. Moreover, the preservation of the initial CD8⁺ T-cell immunodominance patterns from the acute into the chronic phase of infection was significantly associated with slower CD4⁺ T-cell decline. Taken together, these data show that the specificity of the initial CTL response to HIV is critical for the subsequent control of viremia and have important implications for the rational selection of antigens for future HIV-1 vaccines.In the first weeks after human immunodeficiency virus type 1 (HIV-1) acquisition, viral loads peak at high levels, accompanied by a flu-like syndrome (15). A rapid depletion of the CD4⁺ T-cell population occurs during this acute infection, in particular, within the gastrointestinal tract-associated lymphoid tissue (6, 19, 20), marking a nonrecoverable scar on the immune system. With the resolution of the clinical syndromes, viral loads decrease to a set point, which persists at this level for months to years until progressive CD4⁺ T-cell decline results in the onset of AIDS. It has been shown that the initial viral set point following primary infection is a very strong predictor of the disease-free period until the onset of AIDS (18, 21, 22).The initial decrease in the viral load during primary HIV-1 infection (PHI) is temporally associated with the first emergence of virus-specific CD8⁺ T-cell responses, and several studies have provided strong evidence that HIV-1-specific CD8⁺ T-cell responses are capable of controlling viral replication (5, 16, 24, 25, 27, 31, 33). However, significant numbers of virus-specific CD8⁺ T cells are detectable both in chronically infected individuals who progress rapidly to AIDS and in those who do not experience HIV-1 disease progression for decades (1, 11), and the characteristics that define a protective HIV-1-specific CD8⁺ T-cell response are not known. In particular, the level of control over viral replication is not predicted by the overall breadth, magnitude, or function of virus-specific CD8⁺ T-cell responses in chronic HIV-1 infection (1, 4, 11, 26, 28).Here, we demonstrate in a large cohort of individuals identified during PHI that immunodominance patterns of virus-specific CD8⁺ T-cell responses detected in PHI, but not in chronic HIV-1 infection, are strongly associated with the subsequent set point of viral replication. These data show that the specificity of the initial CD8⁺ T-cell response to HIV is critical for the subsequent control of viremia and have important implications for the rational selection of antigens for future HIV-1 vaccines.     

HLA-B57/B*5801 Human Immunodeficiency Virus Type 1 Elite Controllers Select for Rare Gag Variants Associated with Reduced Viral Replication Capacity and Strong Cytotoxic T-Lymphotye Recognition

Human immunodeficiency virus type 1 (HIV-1) elite controllers (EC) maintain viremia below the limit of commercial assay detection (<50 RNA copies/ml) in the absence of antiviral therapy, but the mechanisms of control remain unclear. HLA-B57 and the closely related allele B*5801 are particularly associated with enhanced control and recognize the same Gag_240-249 TW10 epitope. The typical escape mutation (T242N) within this epitope diminishes viral replication capacity in chronically infected persons; however, little is known about TW10 epitope sequences in residual replicating viruses in B57/B*5801 EC and the extent to which mutations within this epitope may influence steady-state viremia. Here we analyzed TW10 in a total of 50 B57/B*5801-positive subjects (23 EC and 27 viremic subjects). Autologous plasma viral sequences from both EC and viremic subjects frequently harbored the typical cytotoxic T-lymphocyte (CTL)-selected mutation T242N (15/23 sequences [65.2%] versus 23/27 sequences [85.1%], respectively; P = 0.18). However, other unique mutants were identified in HIV controllers, both within and flanking TW10, that were associated with an even greater reduction in viral replication capacity in vitro. In addition, strong CTL responses to many of these unique TW10 variants were detected by gamma interferon-specific enzyme-linked immunospot assay. These data suggest a dual mechanism for durable control of HIV replication, consisting of viral fitness loss resulting from CTL escape mutations together with strong CD8 T-cell immune responses to the arising variant epitopes.A subset of human immunodeficiency virus type 1 (HIV-1)-infected persons who control viremia to below the limit of detection (<50 RNA copies/ml plasma) without antiviral therapy has been termed elite controllers/suppressors (EC) (2, 3, 6, 13, 32). Some of these individuals have been infected in excess of 30 years, indicating prolonged containment of HIV replication, but the mechanisms associated with this extreme viremia control remain elusive (13). Among EC, certain HLA class I alleles are overrepresented, in particular HLA-B57, strongly suggesting that HIV-1-specific cytotoxic T-lymphocyte (CTL) responses restricted by these alleles may be crucial for viremia control (16, 29, 32). However, to date, there has been no clear explanation as to why some subjects can control viremia but others cannot, even when carrying the same allegedly protective HLA alleles. Moreover, the characteristics of virus-specific immune responses as well as the impact of viral escape mutations on in vitro replicative fitness in persons with different disease outcomes remain unclear.Growing numbers of studies suggest that CTL targeting Gag, particularly the p24 capsid protein, play an important role in controlling viremia (7, 15, 22, 26, 32, 33, 38). Indeed, the most protective HLA class I allele, B57, which is present in over 40% of EC (32), restricts four immunodominant CTL epitopes in the p24 capsid protein. Previous studies have failed to find differences in the recognition of Gag epitopes or in gamma interferon (IFN-γ) responses to HIV proteins between B57-positive (B57⁺) long-term nonprogressors and B57⁺ progressors (28). Other studies have shown differences in the frequency of polyfunctional CD8⁺ T cells between B57⁺ EC and B57⁺ progressors (5); likewise, differences in the frequency of IFN-γ/interleukin-2-producing CD8⁺ T cells between controllers and progressors with protective HLA alleles were reported (16). Recently, Bailey et al. reported that plasma viruses in B57⁺ EC can harbor CTL escape mutations in the Gag protein, and in some cases these autologous variants were recognized by CTL (3). However, since there were no comparisons to progressors, it is unclear whether the viral variants that were detected or the apparent de novo CTL responses to the variant viruses are characteristic features among B57⁺ persons who maintain persistent control.Of the four immunodominant Gag CTL epitopes restricted by HLA-B57, TW10 (TSTLQEQIGW [Gag residues 240 to 249]) is known to be the earliest target in acute infection (1, 11, 36), therefore likely playing an important role in defining the plasma viral load set point. This epitope is also known to be presented by the closely related B*5801 allele, which is also associated with viral control (21). One of the most frequently detected mutations within this epitope, T242N, is known to occur rapidly and almost universally after acute infection in persons expressing HLA-B57/B*5801 (11, 17, 23). The same mutation has been shown to have a negative impact on viral replication capacity (VRC) by both clinical observation and in vitro experiments (8, 23, 25). Moreover, as plasma viral load increases, compensatory mutations accumulate, restoring VRC to some extent (8). Additional studies, predominantly with children, indicated that some TW10 escape variants may be targeted by specific immune responses (17). Together, these data suggest a hypothesis to explain the diverse disease courses among B57⁺ subjects, namely, that a combination of fitness cost by CTL escape from the TW10 response, variable accumulation of compensatory mutations, and variable generation of specific CTL responses to the new variant influence plasma viral loads.In this study, we investigated plasma viral sequences and IFN-γ-specific enzyme-linked immunospot (ELISPOT) assay responses to autologous Gag TW10 sequences in HLA-B57/B*5801-positive EC and compared these data to those obtained from persons with detectable viremia. Our results indicate that the TW10 T242N mutation does not differentiate HLA-B57/B*5801 EC from those with viremia and that CTL responses to this variant epitope are frequently detected in both viremic and aviremic subjects. However, some rare variants within and flanking this epitope were observed exclusively in HIV controllers, most of which not only reduced VRC but also were recognized by specific CTL at a high magnitude. These data suggest that the additive effects of both CTL-mediated selection for less fit viral variants and CD8 T-cell responses to the variant viruses contribute to strict viremia control in HLA-B57/B*5801-positive controllers.     

Balancing Reversion of Cytotoxic T-Lymphocyte and Neutralizing Antibody Escape Mutations within Human Immunodeficiency Virus Type 1 Env upon Transmission

Human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is subject to both neutralizing antibody (NAb) and CD8 T-cell (cytotoxic T-lymphocyte [CTL]) immune pressure. We studied the reversion of the Env CTL escape mutant virus to the wild type and the relationship between the reversion of CTL mutations with N-linked glycosylation site (NLGS)-driven NAb escape in pigtailed macaques. Env CTL mutations either did not revert to the wild type or only transiently reverted 5 to 7 weeks after infection. The CTL escape mutant reversion was coincident, for the same viral clones, with the loss of NLGS mutations. At one site studied, both CTL and NLGS mutations were needed to confer NAb escape. We conclude that CTL and NAb escape within Env can be tightly linked, suggesting opportunities to induce effective multicomponent anti-Env immunity.CD8 T-cell responses against human immunodeficiency virus (HIV) have long been observed to select for viral variants that avoid cytotoxic T-lymphocyte (CTL) recognition (2, 5, 15, 18, 27). These immune escape mutations may, however, result in reduced replication competence (“fitness cost”) (11, 20, 26). CTL escape variants have been shown to revert to the wild type (WT) upon passage to major histocompatibility complex-mismatched hosts, both in macaques with simian immunodeficiency virus (SIV) or chimeric SIV/HIV (SHIV) infection (11, 12) and in humans with HIV type 1 (HIV-1) infection (1, 19).Most analyses of CTL escape and reversion have studied Gag CTL epitopes known to facilitate control of viremia (7, 14, 21, 30). Fewer analyses have studied Env-specific CTL epitopes. Recent sequencing studies suggest the potential for mutations within predicted HIV-1 Env-specific CTL epitopes to undergo reversion to the WT (16, 23). Env-specific CTL responses may, however, have less impact on viral control of both HIV-1 and SIV/SHIV than do Gag CTL responses (17, 24, 25), presumably reflecting either less-potent inhibition of viral replication or minimal fitness cost of escape (9).Serial viral escape from antibody pressure also occurs in both macaques and humans (3, 13, 28). Env is extensively glycosylated, and this “evolving glycan shield” can sterically block antibody binding without mutation at the antibody-binding site (8, 16, 31). Mutations at glycosylation sites, as well as other mutations, are associated with escape from neutralizing antibody (NAb) responses (4, 13, 29). Mutations in the amino acid sequences of N-linked glycosylation sites (NLGS) can alter the packing of the glycan cloud that surrounds the virion, by a loss, gain, or shift of an NLGS (32), thus facilitating NAb escape.Env is the only viral protein targeted by both CTL and NAb responses. The serial viral escape from both Env-specific CTL and NAb responses could have implications for viral fitness and the reversion of multiple mutations upon transmission to naïve hosts.We previously identified three common HIV-1 Env-specific CD8 T cell epitopes, RY8_788-795, SP9_110-118, and NL9_671-679, and their immune escape patterns in pigtail macaques (Macaca nemestrina) infected with SHIV_mn229 (25). SHIV_mn229 is a chimeric virus constructed from an SIV_mac239 backbone and an HIV-1_HXB2 env fragment that was passaged through macaques to become pathogenic (11). This earlier work provided an opportunity for detailed studies of how viruses with Env-specific CTL escape mutations, as well as mutations in adjacent NLGS, evolve when transmitted to naïve pigtail macaques.     

Protective HLA Class I Alleles That Restrict Acute-Phase CD8+ T-Cell Responses Are Associated with Viral Escape Mutations Located in Highly Conserved Regions of Human Immunodeficiency Virus Type 1

The control of human immunodeficiency virus type 1 (HIV-1) associated with particular HLA class I alleles suggests that some CD8⁺ T-cell responses may be more effective than others at containing HIV-1. Unfortunately, substantial diversities in the breadth, magnitude, and function of these responses have impaired our ability to identify responses most critical to this control. It has been proposed that CD8 responses targeting conserved regions of the virus may be particularly effective, since the development of cytotoxic T-lymphocyte (CTL) escape mutations in these regions may significantly impair viral replication. To address this hypothesis at the population level, we derived near-full-length viral genomes from 98 chronically infected individuals and identified a total of 76 HLA class I-associated mutations across the genome, reflective of CD8 responses capable of selecting for sequence evolution. The majority of HLA-associated mutations were found in p24 Gag, Pol, and Nef. Reversion of HLA-associated mutations in the absence of the selecting HLA allele was also commonly observed, suggesting an impact of most CTL escape mutations on viral replication. Although no correlations were observed between the number or location of HLA-associated mutations and protective HLA alleles, limiting the analysis to mutations selected by acute-phase immunodominant responses revealed a strong positive correlation between mutations at conserved residues and protective HLA alleles. These data suggest that control of HIV-1 may be associated with acute-phase CD8 responses capable of selecting for viral escape mutations in highly conserved regions of the virus, supporting the inclusion of these regions in the design of an effective vaccine.Despite substantial advances in antiretroviral therapies, development of an effective human immunodeficiency virus type 1 (HIV-1) vaccine remains a critical goal (6, 39, 82). Unfortunately, current vaccine efforts have failed to reduce infection rates in humans (9, 75) and have only achieved modest decreases in viral loads in the simian immunodeficiency virus (SIV)/SHIV macaque model (21, 44, 81). A majority of these vaccine approaches have focused on inducing T-cell responses, utilizing large regions of the virus in an attempt to induce a broad array of immune responses (6, 34, 44, 81). While it is well established that CD8⁺ T-cell responses play a critical role in the containment of HIV-1 (45, 49, 67), supported in part by the strong association of particular HLA class I alleles with control of HIV (20, 33, 42, 61), it remains unclear which particular CD8⁺ T-cell responses are best able to control the virus and thus should be preferentially targeted by a vaccine. Studies comparing the magnitude, breadth, and function of CD8⁺ T-cell responses in subjects exhibiting either enhanced or poor control of HIV-1 have yielded few clues as to the specific factors associated with an effective CD8⁺ T-cell response (2, 28, 64, 67). Various differences in the functional capacity of T-cell responses have been observed in long-term nonprogressors (1, 26, 64), although it is possible that these differences may be reflective of an intact immune response, as opposed to having had directly enhanced immune control. As such, efforts are needed to identify factors or phenotypes associated with protective CD8⁺ T-cell responses in order to enable vaccines to induce the most effective responses.Recent studies have begun to suggest that the specificity of the CD8⁺ T-cell response, or the targeting of specific regions of the virus, may be associated with control of HIV-1. Preferential targeting of Gag, a structurally conserved viral protein responsible for multiple functions, has been associated with lower viral loads (25, 43, 56, 60, 77, 85). Furthermore, Kiepiela et al. (43) recently illustrated in a large cohort of 578 clade C-infected subjects that Gag-specific responses were associated with lowered viremia, in contrast to Env-specific responses, which were associated with higher viremia. These data are in line with previous observations that many of the major histocompatibility complex (MHC) class I alleles most strongly associated with control of HIV-1 and SIV, namely, HLA-B57, HLA-B27, and Mamu-A*01, restrict immunodominant CD8⁺ T-cell responses against the Gag protein (8, 10, 24, 63, 68, 83). However, other alleles associated with slower disease progression, such as HLA-B51 in humans and Mamu-B08 and B-17 in the rhesus macaque, do not immunodominantly target Gag, suggesting that targeting of some other regions of the virus may also be capable of eliciting control (8, 52-54). In addition, recent studies investigating the pattern of HIV-1-specific CD8⁺ T-cell responses during acute infection reveal that only a small subset of CD8⁺ T-cell responses restricted by any given HLA allele arise during acute infection and that there exist clear immunodominance patterns to these responses (8, 77, 85). Since control of HIV-1 is likely to be established or lost during the first few weeks of infection, these data suggest that potentially only a few key CD8⁺ T-cell responses may be needed to adequately establish early control of HIV-1.One of the major factors limiting the effectiveness of CD8⁺ T-cell responses is the propensity for HIV-1 to evade these responses through sequence evolution or viral escape (3, 13, 66). Even single point mutations within a targeted CD8 epitope can effectively abrogate recognition by either the HLA allele or the T-cell receptor. However, recent studies have begun to highlight that many sequence polymorphisms will revert to more common consensus residues upon transmission of HIV-1 to a new host, including many cytotoxic T-lymphocyte (CTL) escape mutations (4, 30, 33, 48, 50). Notably, the more rapidly reverting mutations have been observed to preferentially occur at conserved residues, indicating that structurally conserved regions of the virus may be particularly refractory to sequence changes (50). In support of these data, many CTL escape mutations have now been observed to directly impair viral replication (15, 23, 55, 74), in particular those known to either revert or require the presence of secondary compensatory mutations (15, 23, 73, 74). Taken together, these data suggest that, whereas CTL escape mutations provide a benefit to the virus to enable the evasion of host immune pressures, some of these mutations may come at a substantial cost to viral replication. These data may also imply that the association between Gag-specific responses and control of HIV-1 may be due to the targeting of highly conserved regions of the virus that are difficult to evade through sequence evolution.The propensity by which HIV-1 escapes CD8⁺ T-cell responses, and the reproducibility by which mutations arise at precise residues in targeted CD8 epitopes (3, 48), also enables the utilization of sequence data to predict which responses may be most capable of exerting immune selection pressure on the virus. Studies in HIV-1, SIV, and hepatitis C virus (16, 58, 65, 78) are now rapidly identifying immune-driven CTL escape mutations across these highly variable pathogens at the population level by correlating sequence polymorphisms in these viruses with the expression of particular HLA alleles. We provide here an analysis of HLA-associated mutations across the entire HIV-1 genome using a set of sequences derived from clade B chronically infected individuals. Through full-length viral genome coverage, these data provide an unbiased analysis of the location of these mutations and suggest that the control of HIV-1 by particular HLA alleles correlates with their ability to preferentially restrict early CD8⁺ T-cell responses capable of selecting for viral escape mutations at highly conserved residues of the virus. These data provide support for the inclusion of specific highly conserved regions of HIV-1 into vaccine antigens.     

Role of Natural Killer Cells in a Cohort of Elite Suppressors: Low Frequency of the Protective KIR3DS1 Allele and Limited Inhibition of Human Immunodeficiency Virus Type 1 Replication In Vitro

Natural killer (NK) cells are associated with the innate immune response and are important in many viral infections. Recent studies indicate that NK cells can control human immunodeficiency virus type 1 (HIV-1) replication. We studied the effect of NK cells on HIV-1 replication in a subpopulation of HIV-1-infected individuals termed elite suppressors (ES) or elite controllers. These patients maintain a clinically undetectable viral load without treatment and thus provide a fascinating cohort in which to study the immunological response to HIV-1. Using an autologous system, we analyzed the effects of NK cells and CD8⁺ T cells on viral replication in CD4⁺ T lymphoblasts. Although we had postulated that NK cells of ES would be highly effective at controlling viral replication, we found that NK cells from some, but not all, ES were capable of inhibiting replication in the presence of interleukin-2, and the inhibition was less robust than that mediated by CD8⁺ T cells. Additionally, we examined whether particular alleles of the KIR receptors, specifically KIR3DS1 and KIR3DL1, or allele-ligand combinations correlated with the control of HIV-1 replication by NK cells and whether any specific KIR alleles were overrepresented in ES. Our ES cohort did not differ from the general population with respect to the frequency of individual KIR. However, of the eight ES studied, the four exhibiting the most NK cell-mediated control of viral replication also had the fewest activating KIR and were haplotype A. Thus, the strong NK cell-mediated inhibition of viral replication is not necessary for the immunological control of HIV-1 in all ES.A small subset of untreated, human immunodeficiency virus type 1 (HIV-1)-infected individuals referred to as elite suppressors (ES) control viremia to levels that are undetectable by ultrasensitive commercial assays while maintaining high CD4⁺ T-cell counts (13, 37). While defective virus has been shown to account for the control of virus in some patients, examining multiple host factors in ES with replication-competent virus (9) already has provided critical information on the immune response to HIV-1 and may yield important insights into future therapies and vaccine development.Research on ES suggests that CD8⁺ T cells play a crucial role in an effective response to HIV-1. CD8⁺ T cells from ES are capable of controlling viral replication in autologous CD4⁺ T cells significantly better than CD8⁺ T cells from progressors (36), and only the former proliferate (29) and secrete multiple cytokines (8) in response to HIV-1 antigens. Furthermore, certain class I HLA alleles, such as HLA-B*27 and HLA-B*57, which appear to be important in the cytotoxic T-lymphocyte (CTL) response, are overrepresented in ES (15, 19, 21, 30, 32). A second, less well studied cytotoxic cell also may play a role in the control of HIV-1. Natural killer (NK) cells are part of the innate immune system and are an important component of the host response to many viral infections. They act on target cells via cytokine release and cytolysis in response to the integration of signals from inhibitory and activating receptors.The striking propensity of HIV-1 to evolve rapidly in response to immunologic or pharmacologic pressure suggests that the virus has the capability to evade the NK cell response, and indeed selection for evasive measures seems to have occurred. The virus-induced downregulation of HLA-A and -B molecules on infected cells provides some protection against the CTL response; at the same time, however, HLA-C molecules are not downregulated upon infection (12). NK cell interaction with HLA-C can inhibit NK cytotoxic effects, and thus the retention of HLA-C on infected cells can provide some protection against the NK cell response. Additionally, a variety of alterations in NK cell function have been observed during HIV-1 infection. NK cells of patients with chronic HIV-1 have altered phenotypes and effector capabilities: NK cells from viremic patients have an increased expression of inhibitory receptors, and there is an expansion of the defective CD56⁻ NK cells compared to the levels in patients on highly active antiretroviral therapy or in ES (7, 27). These changes may be due to alterations in the cytokine environment during infection, which can affect the activation of the NK cells (39); they also may be due to direct interactions between HIV-1 gene products and the NK cells (20). Although the precise cause is unknown, the result is the development of defective NK cells that express an altered receptor and NK cell marker phenotype.Studies specifically examining a role for NK cells in the response to HIV-1 have yielded conflicting results. During acute HIV-1 infection, the NK cell population is activated and expands, particularly the cytotoxic CD56^dim population (2, 3). This activation declines in the chronic phase, and at least one study suggests that the drop in the viral load (VL) of patients during acute infection occurs before the CD8⁺ T-cell response is fully activated; this could be attributed to the effect of NK cells (2). At the same time, the study of exposed, uninfected individuals shows a correlation between resistance to acquiring HIV-1 infection and NK cell activation levels, cytokine release, and cytotoxicity to NK cell-sensitive cell lines (33, 38). Additionally, a recent whole-genome association study identified three single-nucleotide polymorphisms that appear to be important for the host control of HIV-1 (16). Two of these may have an impact on NK cell function, one that is associated with HLA-B*57 and a second that correlates with higher HLA-C mRNA expression. Taken together, such data suggest that NK cells are important for preventing HIV-1 infection and/or reducing the magnitude of viral replication in acute infection, thereby contributing to the ability of ES to control viremia.In this study, we provide the first characterization of NK cells in patients who naturally control HIV-1 infection. Considering that the effectiveness of CD8⁺ T cells against viral replication is well documented, we directly compared the effect of NK cells to that of CD8⁺ T cells from ES on viral replication to put the effect of NK cells in perspective. We studied the NK cell response by measuring the change in p24 production when autologous effector cell populations were coincubated with infected CD4⁺ lymphoblasts with and without the addition of interleukin-2 (IL-2). Additionally, we examined the killer immunoglobulin-like receptors (KIR) and KIR ligand genotype of ES patients to determine whether any KIR are overrepresented in ES and whether KIR-ligand combinations correlated with the HIV-1 inhibitory activity of the NK cells from specific patients. Previous studies have identified correlations between the expression of certain KIR and progression to AIDS in chronic progressors (25, 26); however, a connection between KIR, KIR ligands, and the control of HIV-1 has yet to be identified in ES. The results of these studies significantly advance the understanding of the nature of NK cells and of their potential role in reducing HIV-1 replication.     

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 cells may play a critical role in blunting peak viremia and controlling human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. The transient depletion of CD8⁺ cells in SIV-infected macaques results in increased viral replication (26, 31, 51, 70). The emergence of virus-specific CD8⁺ T cells coincides with the reduction of peak viremia (12, 39, 42, 63), and CD8⁺ T-cell pressure selects for escape mutants (6, 9, 13, 28, 29, 38, 60, 61, 85). Furthermore, particular major histocompatibility complex (MHC) class I alleles are overrepresented in SIV- and HIV-infected elite controllers (15, 29, 33, 34, 46, 56, 88).Because it has been difficult to induce broadly neutralizing antibodies (Abs), the AIDS vaccine field is currently focused on developing a vaccine designed to elicit HIV-specific CD8⁺ T cells (8, 52, 53, 82). Investigators have tried to define the immune correlates of HIV control. Neither the magnitude nor the breadth of epitopes recognized by virus-specific CD8⁺ T-cell responses correlates with the control of viral replication (1). The quality of the immune response may, however, contribute to the antiviral efficacy of the effector cells. It has been suggested that the number of cytokines that virus-specific CD8⁺ T cells secrete may correlate with viral control, since HIV-infected nonprogressors appear to maintain CD8⁺ T cells that secrete several cytokines, compared to HIV-infected progressors (11, 27). An increased amount of perforin secretion may also be related to the proliferation of HIV-specific CD8⁺ T cells in HIV-infected nonprogressors (55). While those studies offer insight into the different immune systems of progressors and nonprogressors, they did not address the mechanism of viral control. Previously, we found no association between the ability of SIV-specific CD8⁺ T-cell clones to suppress viral replication in vitro and their ability to secrete gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), or interleukin-2 (IL-2) (18).Evidence suggests that some HIV/SIV proteins may be better vaccine targets than others. CD8⁺ T cells recognize epitopes derived from Gag as early as 2 h postinfection, whereas CD8⁺ T cells specific for epitopes in Env recognize infected cells only at 18 h postinfection (68). Additionally, a previously reported study of HIV-infected individuals showed that an increased breadth of Gag-specific responses was associated with lower viral loads (35, 59, 65, 66). CD8⁺ T-cell responses specific for Env, Rev, Tat, Vif, Vpr, Vpu, and Nef were associated with higher viral loads, with increased breadth of Env in particular being significantly associated with a higher chronic-phase viral set point.None of the many sophisticated methods employed for analyzing the characteristics of HIV- or SIV-specific immune responses clearly demarcate the critical qualities of an effective antiviral response. In an attempt to address these questions, we developed a new assay to measure the antiviral efficacy of individual SIV-specific CD8⁺ T-cell responses sorted directly from fresh peripheral blood mononuclear cells (PBMC). Using MHC class I tetramers specific for the epitope of interest, we sorted freshly isolated virus-specific CD8⁺ T cells and determined their ability to suppress virus production from SIV-infected CD4⁺ T cells. We then looked for a common characteristic of efficacious epitope-specific CD8⁺ T cells using traditional methods.     

Preinfection Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T Lymphocytes Failed To Prevent HIV Type 1 Infection from Strains Genetically Unrelated to Viruses in Long-Term Exposed Partners

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.Understanding the mechanisms of altered susceptibility or control of human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) persons may provide invaluable information aiding the design of HIV-1 vaccines and therapy (9, 14, 15, 33, 45, 57, 58). In a cohort of female commercial sex workers in Nairobi, Kenya, a small proportion of individuals remained seronegative for over 3 years despite the continued practice of unprotected sex (12, 28, 55, 56). Similarly, resistance to HIV-1 infection has been reported in homosexual men who frequently practiced unprotected sex with infected partners (1, 15, 17, 21, 61). Multiple factors have been associated with the resistance to HIV-1 infection in ES individuals (32), including host genetic factors (8, 16, 20, 37-39, 44, 46, 47, 49, 59, 63), such as certain HLA class I and II alleles (41), as well as cellular (1, 15, 26, 55, 56), humoral (25, 29), and innate immune responses (22, 35).Seroconversion in previously HIV-resistant Nairobi female commercial sex workers, despite preexisting HIV-specific cytotoxic T-lymphocyte (CTL) responses, has been reported (27). Similarly, 13 of 125 ES enrollees in our Seattle ES cohort (1, 15, 17) have become late seroconverters (H. Zhu, T. Andrus, Y. Liu, and T. Zhu, unpublished observations). Here, we analyze the virology, genetics, and immune responses of HIV-1 infection in one of the later seroconverting subjects, LSC63, who had developed broad CTL responses before seroconversion.     

Effect of B-Cell Depletion on Viral Replication and Clinical Outcome of Simian Immunodeficiency Virus Infection in a Natural Host

Simian immunodeficiency virus (SIV)-infected African nonhuman primates do not progress to AIDS in spite of high and persistent viral loads (VLs). Some authors consider the high viral replication observed in chronic natural SIV infections to be due to lower anti-SIV antibody titers than those in rhesus macaques, suggesting a role of antibodies in controlling viral replication. We therefore investigated the impact of antibody responses on the outcome of acute and chronic SIVagm replication in African green monkeys (AGMs). Nine AGMs were infected with SIVagm.sab. Four AGMs were infused with 50 mg/kg of body weight anti-CD20 (rituximab; a gift from Genentech) every 21 days, starting from day −7 postinfection up to 184 days. The remaining AGMs were used as controls and received SIVagm only. Rituximab-treated AGMs were successfully depleted of CD20 cells in peripheral blood, lymph nodes (LNs), and intestine, as shown by the dynamics of CD20⁺ and CD79a⁺ cells. There was no significant difference in VLs between CD20-depleted AGMs and control monkeys: peak VLs ranged from 10⁷ to 10⁸ copies/ml; set-point values were 10⁴ to 10⁵ SIV RNA copies/ml. Levels of acute mucosal CD4⁺ T-cell depletion were similar for treated and nontreated animals. SIVagm seroconversion was delayed for the CD20-depleted AGMs compared to results for the controls. There was a significant difference in both the timing and magnitude of neutralizing antibody responses for CD20-depleted AGMs compared to results for controls. CD20 depletion significantly altered the histological structure of the germinal centers in the LNs and Peyer''s patches. Our results, although obtained with a limited number of animals, suggest that humoral immune responses play only a minor role in the control of SIV viral replication during acute and chronic SIV infection in natural hosts.In marked contrast to pathogenic human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections of humans and macaques, which are characterized by the constant progression to AIDS in a variable time frame (26), African monkey species naturally infected with SIV are generally spared from any signs of disease (reviewed in references 53 and 71).There are currently three animal models of SIV infection in natural hosts: SIVagm infection of African green monkeys (AGMs), SIVsmm infection of sooty mangabeys, and SIVmnd-1 and SIVmnd-2 infection of mandrills (53, 71). SIV infection in natural hosts is characterized by the following: (i) active viral replication, with set-point viral loads (VLs) similar to or even higher than those found in pathogenic infections (44-46, 49, 50, 52, 61-63); (ii) transient depletion of peripheral CD4⁺ T cells during primary infection, which rebound to preinfection levels during chronic infection (12, 30, 44-46, 49, 62); (iii) significant CD4⁺ T-cell depletion in the intestine, which can be partially restored during chronic infection in spite of significant viral replication (21, 48); (iv) low levels of CD4⁺ CCR5⁺ cells in blood and tissues (47); (v) transient and moderate increases in immune activation and T-cell proliferation during acute infection, with a return to baseline levels during the chronic phase (44-46, 49, 50, 52, 61-63), as a result of an anti-inflammatory milieu which is rapidly established after infection (14, 30); and (vi) no significant increase in CD4⁺ T-cell apoptosis during either acute or chronic infection (37, 48), thus avoiding enteropathy and microbial translocation, which control excessive immune activation and prevent disease progression by allowing CD4⁺ T-cell recovery in the presence of high VLs (21, 48). Hence, the current view is that the main reason behind the lack of disease progression in natural African hosts lies in a better adaptation of the host in response to the highly replicating virus. A better understanding of the mechanisms underlying the lack of disease in natural hosts for SIV infection may provide important clues for understanding the pathogenesis of HIV infection (53, 71).To date, it is still unknown whether or not immune responses are responsible for the lack of disease progression in natural hosts, since data are scarce. Studies of cellular immune responses are significantly more limited than is the case with pathogenic infection, and although not always in agreement (3, 13, 28, 29, 73, 76), their convergence point is that cellular immune responses are not essentially superior to those observed in pathogenic infections (3, 13, 28, 29, 73, 76). This observation is not surprising in the context of the high viral replication in natural hosts. Data are even scarcer on the role of humoral immune responses in the control of disease progression in natural hosts. However, several studies reported that anti-SIV antibody titers are lower in SIV infections of natural hosts, with a lack of anti-Gag responses being characteristic of natural SIV infections in African nonhuman primates (1, 6, 24, 25, 42, 43, 71). Because the viral replication in SIVagm-infected AGMs is of the same magnitude or higher than that in pathogenic infections of rhesus macaques (RMs), it has been hypothesized that these high VLs may be a consequence of the lower antibody titers. Moreover, a recent study has also shown that B cells in lymph nodes (LNs) of AGMs are activated at an earlier time point than is the case for SIVmac251-infected RMs, which implies that humoral immune responses may be important in controlling SIV replication in the natural hosts (9). Conversely, it has been shown that passively transferring immunoglobulins from animals naturally infected with SIVagm prior to infection with a low dose of SIVagm did not prevent infection in AGMs (42, 60), which is in striking contrast to results in studies of pathogenic infections, which convincingly demonstrated with animal models that intravenously administered or topically applied antibodies can protect macaques against intravenous or mucosal simian-human immunodeficiency virus challenge (34-36, 54, 72).Previous CD20⁺ B-cell-depletion studies during pathogenic RM infections have indicated that humoral immune responses may be important for controlling both the postpeak VL and disease progression (38, 57). However, these studies used strains that are highly resistant to neutralization (SIVmac251 and SIVmac239), making it difficult to assess the role that antibodies have in controlling SIV replication and disease progression. Moreover, our recent results suggested a limited impact of humoral immune responses in controlling replication of a neutralization-sensitive SIVsmm strain in rhesus macaques (18).To investigate the effect that CD20⁺ B cells and antibodies have on SIV replication in natural hosts, we have depleted CD20⁺ B cells in vivo in AGMs infected with SIVagm.sab92018. We assessed the impact of humoral immune responses on the control of viral replication and other immunological parameters, and we report that ablating humoral immune responses in SIVagm-infected AGMs does not significantly alter the course of virus replication or disease progression.     

Additive Contribution of HLA Class I Alleles in the Immune Control of HIV-1 Infection

Previous studies have identified a central role for HLA-B alleles in influencing control of HIV infection. An alternative possibility is that a small number of HLA-B alleles may have a very strong impact on HIV disease outcome, dominating the contribution of other HLA alleles. Here, we find that even following the exclusion of subjects expressing any of the HLA-B class I alleles (B*57, B*58, and B*18) identified to have the strongest influence on control, the dominant impact of HLA-B alleles on virus set point and absolute CD4 count variation remains significant. However, we also find that the influence of HLA on HIV control in this C-clade-infected cohort from South Africa extends beyond HLA-B as HLA-Cw type remains a significant predictor of virus and CD4 count following exclusion of the strongest HLA-B associations. Furthermore, there is evidence of interdependent protective effects of the HLA-Cw*0401-B*8101, HLA-Cw*1203-B*3910, and HLA-A*7401-B*5703 haplotypes that cannot be explained solely by linkage to a protective HLA-B allele. Analysis of individuals expressing both protective and detrimental alleles shows that even the strongest HLA alleles appear to have an additive rather than dominant effect on HIV control at the individual level. Finally, weak but significant frequency-dependent effects in this cohort can be detected only by looking at an individual''s combined HLA allele frequencies. Taken together, these data suggest that although individual HLA alleles, particularly HLA-B, can have a strong impact, HIV control overall is likely to be influenced by the additive effect of some or all of the other HLA alleles present.HIV-specific CD8⁺ T cells play a central role in resolution of primary viremia and the long-term suppression of viral replication (13). Supporting this notion is the observed correlation between possession of particular human leukocyte antigen (HLA) class I alleles and control of HIV, measured both directly by time-to-AIDS (5, 6) and indirectly via clinical markers of disease progression (viral load [VL] and CD4 count) (15, 26, 28). Specific HLA class I alleles have been associated with relatively successful control of viral replication and slow disease progression, most notably, alleles HLA-B*57 and HLA-B*27 (1, 7, 12, 15, 21, 23), and also with relatively ineffective control of viral replication and rapid disease progression [B*35(Px), B*5802, and B*18] (5, 15, 17, 23). In addition, general trends suggesting an HLA class I heterozygote advantage (5) and rare allele advantage (28) and, most recently, a correlation between levels of surface expression linked to certain HLA-Cw alleles (11, 27) and HIV control has also been described.Among the different HLA class I loci, the HIV-specific CD8⁺ T-cell responses restricted by HLA-B alleles are thought to play the central role in determining disease outcome: the majority of detectable HIV-specific CD8⁺ T-cell responses are restricted by HLA-B alleles (3, 15, 16), HLA-B-restricted responses typically express a more effective “polyfunctional” phenotype (14), the strongest HLA-associations with either slow or rapid progression are with HLA-B alleles (5, 10, 11, 15), and HLA-B-restricted CD8⁺ T cells exert the strongest selection pressure on the virus (15, 19, 24). However, whether this apparent association between HIV immune control and HLA-B is a general and causal trend or, rather, is biased by the coincidence that the strongest HLA associations with either extreme of disease control happen, by chance, to involve HLA-B alleles remains uncertain.In order to further investigate the correlation between HLA type and HIV infection control, we here examine a cohort now comprising >1,200 chronically HIV C-clade-infected, treatment-naïve subjects from Durban, South Africa, in an extended analysis following from our previous studies of a smaller cohort (15). We first address the question of whether the dominant role of HLA-B in this population compared to the roles of HLA-A or HLA-C results from the influence of HLA-B alleles in general or is dependent on a few known strong associations, such as that between HLA-B*57 alleles and low viremia. Second, in light of recent data (11, 27), we assess the impact of HLA-C alleles on HIV disease outcome and examine the effect of HLA haplotypes on observed HLA associations with disease control. Third, we investigate the question of whether the impact of certain HLA-B alleles on HIV outcome dominates that of other HLA-B alleles to negate the contribution of the latter or whether the impact of individual HLA alleles can be additive. Finally, we compare the impact of individual HLA alleles on HIV on immune control to the impact of heterozygote and rare allele advantage in this cohort.     

Analysis of Human Immunodeficiency Virus Type 1 Viremia and Provirus in Resting CD4+ T Cells Reveals a Novel Source of Residual Viremia in Patients on Antiretroviral Therapy

Highly active antiretroviral therapy (HAART) can reduce human immunodeficiency virus type 1 (HIV-1) viremia to clinically undetectable levels. Despite this dramatic reduction, some virus is present in the blood. In addition, a long-lived latent reservoir for HIV-1 exists in resting memory CD4⁺ T cells. This reservoir is believed to be a source of the residual viremia and is the focus of eradication efforts. Here, we use two measures of population structure—analysis of molecular variance and the Slatkin-Maddison test—to demonstrate that the residual viremia is genetically distinct from proviruses in resting CD4⁺ T cells but that proviruses in resting and activated CD4⁺ T cells belong to a single population. Residual viremia is genetically distinct from proviruses in activated CD4⁺ T cells, monocytes, and unfractionated peripheral blood mononuclear cells. The finding that some of the residual viremia in patients on HAART stems from an unidentified cellular source other than CD4⁺ T cells has implications for eradication efforts.Successful treatment of human immunodeficiency virus type 1 (HIV-1) infection with highly active antiretroviral therapy (HAART) reduces free virus in the blood to levels undetectable by the most sensitive clinical assays (18, 36). However, HIV-1 persists as a latent provirus in resting, memory CD4⁺ T lymphocytes (6, 9, 12, 16, 48) and perhaps in other cell types (45, 52). The latent reservoir in resting CD4⁺ T cells represents a barrier to eradication because of its long half-life (15, 37, 40-42) and because specifically targeting and purging this reservoir is inherently difficult (8, 25, 27).In addition to the latent reservoir in resting CD4⁺ T cells, patients on HAART also have a low amount of free virus in the plasma, typically at levels below the limit of detection of current clinical assays (13, 19, 35, 37). Because free virus has a short half-life (20, 47), residual viremia is indicative of active virus production. The continued presence of free virus in the plasma of patients on HAART indicates either ongoing replication (10, 13, 17, 19), release of virus after reactivation of latently infected CD4⁺ T cells (22, 24, 31, 50), release from other cellular reservoirs (7, 45, 52), or some combination of these mechanisms. Finding the cellular source of residual viremia is important because it will identify the cells that are still capable of producing virus in patients on HAART, cells that must be targeted in any eradication effort.Detailed analysis of this residual viremia has been hindered by technical challenges involved in working with very low concentrations of virus (13, 19, 35). Recently, new insights into the nature of residual viremia have been obtained through intensive patient sampling and enhanced ultrasensitive sequencing methods (1). In a subset of patients, most of the residual viremia consisted of a small number of viral clones (1, 46) produced by a cell type severely underrepresented in the peripheral circulation (1). These unique viral clones, termed predominant plasma clones (PPCs), persist unchanged for extended periods of time (1). The persistence of PPCs indicates that in some patients there may be another major cellular source of residual viremia (1). However, PPCs were observed in a small group of patients who started HAART with very low CD4 counts, and it has been unclear whether the PPC phenomenon extends beyond this group of patients. More importantly, it has been unclear whether the residual viremia generally consists of distinct virus populations produced by different cell types.Since the HIV-1 infection in most patients is initially established by a single viral clone (23, 51), with subsequent diversification (29), the presence of genetically distinct populations of virus in a single individual can reflect entry of viruses into compartments where replication occurs with limited subsequent intercompartmental mixing (32). Sophisticated genetic tests can detect such population structure in a sample of viral sequences (4, 39, 49). Using two complementary tests of population structure (14, 43), we analyzed viral sequences from multiple sources within individual patients in order to determine whether a source other than circulating resting CD4⁺ T cells contributes to residual viremia and viral persistence. Our results have important clinical implications for understanding HIV-1 persistence and treatment failure and for improving eradication strategies, which are currently focusing only on the latent CD4⁺ T-cell reservoir.     

Different In Vivo Effects of HIV-1 Immunodominant Epitope-Specific Cytotoxic T Lymphocytes on Selection of Escape Mutant Viruses

HIV-1 escape mutants are well known to be selected by immune pressure via HIV-1-specific cytotoxic T lymphocytes (CTLs) and neutralizing antibodies. The ability of the CTLs to suppress HIV-1 replication is assumed to be associated with the selection of escape mutants from the CTLs. Therefore, we first investigated the correlation between the ability of HLA-A*1101-restricted CTLs recognizing immunodominant epitopes in vitro and the selection of escape mutants. The result showed that there was no correlation between the ability of these CTLs to suppress HIV-1 replication in vitro and the appearance of escape mutants. The CTLs that had a strong ability to suppress HIV-1 replication in vitro but failed to select escape mutants expressed a higher level of PD-1 in vivo, whereas those that had a strong ability to suppress HIV-1 replication in vitro and selected escape mutants expressed a low level of PD-1. Ex vivo analysis of these CTLs revealed that the latter CTLs had a significantly stronger ability to recognize the epitope than the former ones. These results suggest that escape mutations are selected by HIV-1-specific CTLs that have a stronger ability to recognize HIV-1 in vivo but not in vitro.HIV-1-specific cytotoxic T lymphocytes (CTLs) have an important role in the control of HIV-1 replication during acute and chronic phases of an HIV-1 infection (5, 28, 33). On the other hand, HIV-1 can escape from the host immune system by various mechanisms. These may include the appearance of HIV-1 carrying escape mutations in its immunodominant CTL epitopes as well as Nef-mediated downregulation of HLA class I molecules. There is a growing body of evidence for the former mechanism, i.e., that CTLs targeting immunodominant HIV-1 epitopes select escape mutants in chronically HIV-1-infected individuals (18, 20, 36), whereas the latter mechanism was proved by demonstrating that HIV-1-specific CTLs fail to kill Nef-positive-HIV-1-infected CD4⁺ T cells but effectively kill Nef-defective-HIV-1-infected ones or that they suppress the replication of Nef-defective HIV-1 much more than that of Nef-positive HIV-1 (12, 13, 42, 45).It is speculated that HIV-1 immunodominant epitope-specific CTLs have the ability to suppress HIV-1 replication and effectively select escape mutants. However, the correlation between this ability of the CTLs and the appearance of escape mutants is still unclear, because it is not easy to evaluate the ability of HIV-1-specific CTLs to exert a strong immune pressure in vivo. To examine this ability, most previous studies measured the number of HIV-1-specific CTLs or CD8⁺ T cells and the CTL activity against target cells prepulsed with the epitope peptide or those infected with HIV-1 recombinant vaccinia virus (6, 7, 23, 46). However, the results obtained from such experiments do not reflect the ability of the CTLs to exert immune pressure in vivo. We and other groups previously utilized an assay to directly evaluate the ability of the CTLs to suppress HIV-1 replication in vitro (1, 17, 18, 42, 43). This assay may be better for evaluation of immune pressure by HIV-1-specific CTLs than other assays, because the ability of the CTLs to suppress HIV-1 replication is directly measured in cultures of HIV-1-infected CD4⁺ T cells incubated with HIV-1-specific CTL clones. But it still remains unknown whether this assay reflects immune pressure in vivo.In the present study, we investigated whether HIV-1-specific CTLs having a strong ability to suppress HIV-1 replication could positively select escape mutants. Since HLA-A*1101 is known to be an HLA allele relatively associated with a slow progression to AIDS (32), it is speculated that some HLA-A*1101-restricted CTLs would have a strong ability to suppress HIV-1 replication in vitro. Therefore, we first focused on 4 well-known HLA-A*1101-restricted CTL epitopes in the present study. We investigated the frequency of CTLs specific for these epitopes in chronically HIV-1-infected individuals, the ability of these CTLs to suppress HIV-1 replication in vitro, and whether the escape mutants were selected by the CTLs. Furthermore, we analyzed the expression of Programmed Death-1 (PD-1) on these CTLs ex vivo and antigen recognition of them.     

Enhancement of human immunodeficiency virus (HIV)-specific CD8+ T cells in cerebrospinal fluid compared to those in blood among antiretroviral therapy-naive HIV-positive subjects

During untreated human immunodeficiency virus type 1 (HIV-1) infection, virus-specific CD8⁺ T cells partially control HIV replication in peripheral lymphoid tissues, but host mechanisms of HIV control in the central nervous system (CNS) are incompletely understood. We characterized HIV-specific CD8⁺ T cells in cerebrospinal fluid (CSF) and peripheral blood among seven HIV-positive antiretroviral therapy-naïve subjects. All had grossly normal brain magnetic resonance imaging and spectroscopy and normal neuropsychometric testing. Frequencies of epitope-specific CD8⁺ T cells by direct tetramer staining were on average 2.4-fold higher in CSF than in blood (P = 0.0004), while HIV RNA concentrations were lower. Cells from CSF were readily expanded ex vivo and responded to a broader range of HIV-specific human leukocyte antigen class I restricted optimal peptides than did expanded cells from blood. HIV-specific CD8⁺ T cells, in contrast to total CD8⁺ T cells, in CSF and blood were at comparable maturation states, as assessed by CD45RO and CCR7 staining. The strong relationship between higher T-cell frequencies and lower levels of viral antigen in CSF could be the result of increased migration to and/or preferential expansion of HIV-specific T cells within the CNS. This suggests an important role for HIV-specific CD8⁺ T cells in control of intrathecal viral replication.Human immunodeficiency virus type 1 (HIV-1) invades the central nervous system (CNS) early during primary infection (21, 30, 35), and proviral DNA persists in the brain throughout the course of HIV-1 disease (7, 25, 29, 47, 77, 83). Limited data from human and nonhuman primate studies suggest that little or no viral replication occurs in the brain during chronic, asymptomatic infection, based on the absence of demonstrable viral RNA or proteins (8, 85). In contrast, cognitive impairment affects approximately 40% of patients who progress to advanced AIDS without highly active antiretroviral therapy (21, 30, 35, 65). During HIV-associated dementia, there is active HIV-1 replication in the brain (23, 52, 61, 81), and viral sequence differences between cerebrospinal fluid (CSF) and peripheral tissues suggest distinct anatomic compartments of replication (18, 19, 22, 53, 75, 76, 78). Host mechanisms that control viral replication in the CNS during chronic, asymptomatic HIV-1 infection are incompletely understood.Anti-HIV CD8⁺ T cells are present in blood and peripheral tissues throughout the course of chronic HIV-1 infection (2, 14). Multiple lines of evidence support a critical role for these cells in controlling HIV-1 replication. During acute HIV-1 infection, the appearance of CD8⁺ T-cell responses correlates temporally with a decline in viremia (11, 43), and a greater proliferative capacity of peripheral blood HIV-specific CD8⁺ T cells correlates with better control of viremia (36, 54). In addition, the presence of certain major histocompatibility complex class I human leukocyte antigen (HLA) alleles, notably HLA-B*57, predicts slower progression to AIDS and death during chronic, untreated HIV-1 infection (55, 62). Finally, in the simian immunodeficiency virus (SIV) model, macaques depleted of CD8⁺ T cells experience increased viremia and rapid disease progression (39, 51, 67).Little is known regarding the role of intrathecal anti-HIV CD8⁺ T cells in HIV neuropathogenesis. Nonhuman primate studies have identified SIV-specific CD8⁺ T cells in the CNS early after infection (16, 80). Increased infiltration of SIV antigen-specific CD8⁺ T cells and cytotoxic T lymphocytes has been detected only in CSF of slow progressors without neurological symptoms (72). In chronically infected macaques with little or no SIV replication in the brain, the frequency of HIV-specific T cells was higher in CSF than in peripheral blood but did not correlate with the level of plasma viremia or CD4⁺ T-cell counts (56). Although intrathecal anti-HIV CD8⁺ T cells may help control viral replication, a detrimental role in the neuropathogenesis of HIV-1 has also been postulated (38). Immune responses contribute to neuropathogenesis in models of other infectious diseases, and during other viral infections cytotoxic T lymphocytes can worsen disease through direct cytotoxicity or release of inflammatory cytokines such as gamma interferon (IFN-γ) (3, 17, 31, 37, 42, 44, 71).We tested the hypothesis that quantitative and/or qualitative differences in HIV-specific CD8⁺ T-cell responses are present in CSF compared to blood during chronic, untreated HIV-1 infection. We characterized HIV-specific CD8⁺ T-cell responses in CSF among seven antiretroviral therapy-naïve adults with chronic HIV-1 infection, relatively high peripheral blood CD4⁺ T-cell counts, and low plasma HIV-1 RNA concentrations. We show that among these HIV-positive individuals with no neurological symptoms and with little or no HIV-1 RNA in CSF, frequencies of HIV-specific T cells are significantly higher in CSF than in blood. These CSF cells are at a state of differentiation similar to that of T cells in blood and are functionally competent for expansion and IFN-γ production. The higher frequency of functional HIV-specific CD8⁺ T cells in CSF, in the context of low or undetectable virus in CSF, suggests that these cells play a role in the control of intrathecal viral replication.     

Early Selection in Gag by Protective HLA Alleles Contributes to Reduced HIV-1 Replication Capacity That May Be Largely Compensated for in Chronic Infection

Mutations that allow escape from CD8 T-cell responses are common in HIV-1 and may attenuate pathogenesis by reducing viral fitness. While this has been demonstrated for individual cases, a systematic investigation of the consequence of HLA class I-mediated selection on HIV-1 in vitro replication capacity (RC) has not been undertaken. We examined this question by generating recombinant viruses expressing plasma HIV-1 RNA-derived Gag-Protease sequences from 66 acute/early and 803 chronic untreated subtype B-infected individuals in an NL4-3 background and measuring their RCs using a green fluorescent protein (GFP) reporter CD4 T-cell assay. In acute/early infection, viruses derived from individuals expressing the protective alleles HLA-B*57, -B*5801, and/or -B*13 displayed significantly lower RCs than did viruses from individuals lacking these alleles (P < 0.05). Furthermore, acute/early RC inversely correlated with the presence of HLA-B-associated Gag polymorphisms (R = −0.27; P = 0.03), suggesting a cumulative effect of primary escape mutations on fitness during the first months of infection. At the chronic stage of infection, no strong correlations were observed between RC and protective HLA-B alleles or with the presence of HLA-B-associated polymorphisms restricted by protective alleles despite increased statistical power to detect these associations. However, RC correlated positively with the presence of known compensatory mutations in chronic viruses from B*57-expressing individuals harboring the Gag T242N mutation (n = 50; R = 0.36; P = 0.01), suggesting that the rescue of fitness defects occurred through mutations at secondary sites. Additional mutations in Gag that may modulate the impact of the T242N mutation on RC were identified. A modest inverse correlation was observed between RC and CD4 cell count in chronic infection (R = −0.17; P < 0.0001), suggesting that Gag-Protease RC could increase over the disease course. Notably, this association was stronger for individuals who expressed B*57, B*58, or B*13 (R = −0.27; P = 0.004). Taken together, these data indicate that certain protective HLA alleles contribute to early defects in HIV-1 fitness through the selection of detrimental mutations in Gag; however, these effects wane as compensatory mutations accumulate in chronic infection. The long-term control of HIV-1 in some persons who express protective alleles suggests that early fitness hits may provide lasting benefits.The host immune response elicited by CD8⁺ cytotoxic T lymphocytes (CTLs) is a major contributor to viral control following human immunodeficiency virus type 1 (HIV-1) infection (6, 39), but antiviral pressure exerted by CTLs is diminished by the selection of escape mutations in targeted regions throughout the viral proteome (7, 18, 29, 35, 41, 45, 57). A comprehensive identification of HLA-associated viral polymorphisms has recently been achieved through population-based analyses of HIV-1 sequences and HLA class I types from different cohorts worldwide (3, 8, 13-15, 34, 43, 50, 56, 63). However, despite improved characterization of the sites and pathways of immune escape, effective ways to incorporate these findings into immunogen design remain an area of debate. A better understanding of the impact of escape mutations on viral fitness may provide novel directions for HIV-1 vaccines that are designed to attenuate pathogenesis.The development of innovative vaccine strategies that can overcome the extreme diversity of HIV is a key priority (4). One proposed approach is to target the most conserved T-cell epitopes, which presumably cannot escape from CTL pressure easily due to structural or functional constraints on the viral protein (55). Complementary approaches include the design of polyvalent and/or mosaic immunogens that incorporate commonly observed viral diversity (4, 38) or the specific targeting of vulnerable regions of the viral proteome that do escape but only at a substantial cost to viral replication capacity (RC) (1, 40). A chief target of such vaccine approaches is the major HIV-1 structural protein Gag, which is known to be highly immunogenic and to elicit CTL responses that correlate with the natural control of infection (22, 36, 66). Indeed, several lines of evidence support a relationship between the selection of CTL escape mutations and reduced HIV-1 fitness. These include the reversion of escape mutations following transmission to an HLA-mismatched recipient who cannot target the epitope (19, 24, 41) as well as reduced plasma viral load (pVL) set point following the transmission of certain escape variants from donors who expressed protective HLA alleles (17, 27). Notably, these in vivo observations have been made most often for variations within Gag that are attributed to CTL responses restricted by the protective alleles HLA-B*57 and -B*5801 (17, 19, 27, 41). Most recently, reduced in vitro RCs of clinical isolates and/or engineered strains encoding single or multiple escape mutations in Gag selected in the context of certain protective HLA alleles, including B*57, B*5801, B*27, and B*13, have been demonstrated (9, 10, 42, 53, 59, 62). Despite these efforts, the goal of a T-cell vaccine that targets highly conserved and attenuation-inducing sites is hampered by a lack of knowledge concerning the contribution of most escape mutations to HIV-1 fitness as well as a poor understanding of the relative influence of HLA on the viral RC at different stages of infection.The mutability of HIV-1 permits the generation of progeny viruses encoding compensatory mutations that restore normal protein function and/or viral fitness. Detailed studies have demonstrated that the in vitro RC of escape variants in human and primate immunodeficiency viruses can be enhanced by the addition of secondary mutations outside the targeted epitope (10, 20, 52, 59, 65). Thus, vaccine strategies aimed at attenuating HIV-1 must also consider, among other factors, the frequency, time course, and extent to which compensation might overcome attenuation mediated by CTL-induced escape. Despite its anticipated utility for HIV-1 vaccine design, systematic studies to examine the consequences of naturally occurring CTL escape and compensatory mutations on viral RC have not been undertaken.We have described previously an in vitro recombinant viral assay to examine the impact of Gag-Protease mutations on HIV-1 RC (47, 49). Gag and protease have been included in each virus to minimize the impact of sequence polymorphisms at Gag cleavage sites, which coevolve with changes in protease (5, 37). Using this approach, we have demonstrated that viruses derived from HIV-1 controllers replicated significantly less well than those derived from noncontrollers and that these differences were detectable at both the acute/early (49) and chronic (47) stages. Escape mutations in Gag associated with the protective HLA-B*57 allele, as well as putative compensatory mutations outside known CTL epitopes, contributed to this difference in RC (47). However, substantial variability was observed for viruses from controllers and noncontrollers, indicating that additional factors were likely to be involved. Benefits of this assay include its relatively high-throughput capacity as well as the fact that clinically derived HIV-1 sequences are used in their entirety. Thus, it is possible to examine a large number of “real-world” Gag-Protease sequences, to define an RC value for each one, and to identify sequences within the population of recombinant strains that are responsible for RC differences.Here, we use this recombinant virus approach to examine the contribution of HLA-associated immune pressure on Gag-Protease RC during acute/early (n = 66) and chronic (n = 803) infections in the context of naturally occurring HIV-1 subtype B isolates from untreated individuals. In a recent report (64), we employed this system to examine the Gag-Protease RC in a similar cohort of chronic HIV-1 subtype C-infected individuals. The results of these studies provide important insights into the roles of immune pressure and fitness constraints on HIV-1 evolution that may contribute to the rational design of an effective vaccine.     

Impact of Cytotoxic-T-Lymphocyte Memory Induction without Virus-Specific CD4+ T-Cell Help on Control of a Simian Immunodeficiency Virus Challenge in Rhesus Macaques

Despite many efforts to develop AIDS vaccines eliciting virus-specific T-cell responses, whether induction of these memory T cells by vaccination before human immunodeficiency virus (HIV) exposure can actually contribute to effective T-cell responses postinfection remains unclear. In particular, induction of HIV-specific memory CD4⁺ T cells may increase the target cell pool for HIV infection because the virus preferentially infects HIV-specific CD4⁺ T cells. However, virus-specific CD4⁺ helper T-cell responses are thought to be important for functional CD8⁺ cytotoxic-T-lymphocyte (CTL) induction in HIV infection, and it has remained unknown whether HIV-specific memory CD8⁺ T cells induced by vaccination without HIV-specific CD4⁺ T-cell help can exert effective responses after virus exposure. Here we show the impact of CD8⁺ T-cell memory induction without virus-specific CD4⁺ T-cell help on the control of a simian immunodeficiency virus (SIV) challenge in rhesus macaques. We developed a prophylactic vaccine by using a Sendai virus (SeV) vector expressing a single SIV Gag_241-249 CTL epitope fused with enhanced green fluorescent protein (EGFP). Vaccination resulted in induction of SeV-EGFP-specific CD4⁺ T-cell and Gag_241-249-specific CD8⁺ T-cell responses. After a SIV challenge, the vaccinees showed dominant Gag_241-249-specific CD8⁺ T-cell responses with higher effector memory frequencies in the acute phase and exhibited significantly reduced viral loads. These results demonstrate that virus-specific memory CD8⁺ T cells induced by vaccination without virus-specific CD4⁺ T-cell help could indeed facilitate SIV control after virus exposure, indicating the benefit of prophylactic vaccination eliciting virus-specific CTL memory with non-virus-specific CD4⁺ T-cell responses for HIV control.Virus-specific T-cell responses are crucial for controlling human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication (3, 4, 12, 20, 28, 36, 37). Therefore, a great deal of effort has been exerted to develop AIDS vaccines eliciting virus-specific T-cell responses (23, 27, 30, 47), but whether this approach actually results in HIV control remains unclear (1, 6). It is important to determine which T-cell responses need to be induced by prophylactic vaccination for HIV control after virus exposure.Because HIV preferentially infects HIV-specific CD4⁺ T cells (5), induction of HIV-specific memory CD4⁺ T cells by vaccination may increase the target cell pool for HIV infection and could enhance viral replication (42). However, CD4⁺ helper T-cell responses are important for functional CD8⁺ cytotoxic-T-lymphocyte (CTL) induction (11, 40, 43, 46), and it has remained unknown whether HIV-specific memory CD8⁺ T cells induced by vaccination with non-virus-specific CD4⁺ T-cell help (but without HIV-specific CD4⁺ T-cell help) can exert effective responses after virus exposure. Indeed, the real impact of prophylactic induction of CTL memory itself on HIV replication has not been well documented thus far.We previously developed a prophylactic AIDS vaccine consisting of DNA priming followed by boosting with a recombinant Sendai virus (SeV) vector expressing SIVmac239 Gag (26). Evaluation of this vaccine''s efficacy against a SIVmac239 challenge in Burmese rhesus macaques showed that some vaccinees contained SIV replication whereas unvaccinated animals developed AIDS (15, 27). In particular, vaccination consistently resulted in control of SIV replication in those animals possessing the major histocompatibility complex class I (MHC-I) haplotype 90-120-Ia. Gag_206-216 (IINEEAADWDL) and Gag_241-249 (SSVDEQIQW) epitope-specific CD8⁺ T-cell responses were shown to be involved in SIV control in these vaccinated macaques (14, 16).In the present study, focusing on CD8⁺ T-cell responses directed against one of these epitopes, we have evaluated the efficacy of a vaccine expressing the Gag_241-249 epitope fused with enhanced green fluorescent protein (EGFP) against a SIVmac239 challenge in 90-120-Ia-positive rhesus macaques. The animals exhibited this single-epitope-specific CD8⁺ T-cell response and SeV-EGFP-specific CD4⁺ T-cell responses after vaccination and showed rapid, dominant induction of potent secondary Gag_241-249-specific CD8⁺ T-cell responses after a SIV challenge. Plasma viral loads in these vaccinees were significantly reduced compared to those of naive controls. These results indicate that induction of CD8⁺ T-cell memory without virus-specific CD4⁺ T-cell help by prophylactic vaccination can result in effective CD8⁺ T-cell responses after virus exposure.