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.     

T-Cell Tolerance for Variability in an HLA Class I-Presented Influenza A Virus Epitope

To escape immune recognition, viruses acquire amino acid substitutions in class I human leukocyte antigen (HLA)-presented cytotoxic T-lymphocyte (CTL) epitopes. Such viral escape mutations may (i) prevent peptide processing, (ii) diminish class I HLA binding, or (iii) alter T-cell recognition. Because residues 418 to 426 of the hypervariable influenza A virus nucleoprotein (NP_418-426) epitope are consistently bound by class I HLA and presented to CTL, we assessed the impact that intraepitope sequence variability has upon T-cell recognition. CTL elicited by intranasal influenza virus infection were tested for their cross-recognition of 20 natural NP_418-426 epitope variants. Six of the variant epitopes, of both H1N1 and H3N2 origin, were cross-recognized by CTL while the remaining NP_418-426 epitope variants escaped targeting. A pattern emerged whereby variability at position 5 (P5) within the epitope reduced T-cell recognition, changes at P4 or P6 enabled CTL escape, and a mutation at P8 enhanced T-cell recognition. These data demonstrate that substitutions at P4 and/or P6 facilitate influenza virus escape from T-cell recognition and provide a model for the number, nature, and location of viral mutations that influence T-cell cross-recognition.Cytotoxic T-lymphocytes (CTL) kill virus-infected cells and release antiviral cytokines upon recognition of short viral peptides displayed on the cell surface by the class I HLA molecule (36). Virus-derived peptides are processed in the cytoplasm by proteasome degradation of viral proteins (25), shuttled into the lumen of the endoplasmic reticulum (ER) by the transporter-associated protein, and loaded into the basket-like groove of the class I molecule. Class I HLA molecules await peptide loading in the ER and demonstrate specificity for viral peptides with particular anchor residues representing a good fit for the class I HLA binding groove. Once stable class I HLA-peptide complexes are formed, the class I molecule and its peptide cargo are transported via the Golgi apparatus to the cell surface, where the complex is anchored to the plasma membrane (21, 36-38). CTL then survey class I HLA-presented peptides on the cell surface. Viral peptides must therefore be processed, specifically bound by class I HLA, and presented at the plasma membrane for CTL to distinguish infected cells from uninfected tissue.A high mutation rate is one of many mechanisms utilized by viruses to escape detection by the immune system. Mutations within the genome allow viruses to accumulate and select for amino acid substitutions that (i) inhibit proteasome processing and viral peptide generation (2, 23), (ii) alter anchor residues within viral peptides to diminish class I HLA binding specificity (3, 14, 24, 32), or (iii) reduce immune recognition of the class I HLA-peptide complex by varying amino acids that come in contact with the T-cell receptor (6, 10, 27, 30, 35). While viral mutations might be advantageous for escaping immune detection, such flexibility can cost the virus in terms of replicative fitness. In order to maintain reproductive fitness and structural integrity, viruses must temper their use of genetic flexibility as a means of immune escape.Influenza viruses have the well-documented ability to escape detection by various immune epitopes (3, 10, 27). A priori, investigators often assume that variable regions of the virus represent poor immune targets because such regions will not be consistently processed, presented, or recognized (15, 20). However, we along with others continue to find that a hypervariable stretch of the influenza virus nucleoprotein consisting of residues 418 to 426 (NP_418-426) is presented to CTL by different HLA-B alleles (B*0702 and B*3501) in spite of extensive viral variability within this epitope (8, 10, 27, 34). Moreover, NP_418-426 is a dominant immune epitope (8, 10, 27, 34). The consistent processing and presentation of NP_418-426 by class I HLA can be explained by the finding that different influenza virus isolates cannot mutate the proline located at position 2 (P2) within the epitope because elimination of this proline reduces viral fitness (4, 5). Little to no variability is found at the methionine P9 anchor as well. These facts lead to the unique observation that strain-to-strain variability does not abrogate class I HLA presentation of the influenza virus NP_418-426 epitope and that CTL respond to this consistently presented viral epitope in an immunodominant fashion.In this study we took advantage of the anchor residue conservation that prompts the NP_418-426 epitope to be consistently presented to CTL by investigating the functional impact that influenza virus intraepitope variability has on CTL recognition. The amino acid alignment of human influenza A (H1N1 and H3N2) virus nucleoprotein molecules identifies 20 unique NP_418-426 peptide sequences which demonstrate amino acid diversity between the anchors. We infected HLA-transgenic mice intranasally with influenza virus and tested CTL from these animals for their ability to recognize each of the 20 NP_418-426 variants. These 20 NP_418-426 sequences represent a natural “recombinant library” of viral epitopes that the immune system has and will face. The resulting data demonstrate a gradient of viral substitutions whereby CTL recognition diminishes depending upon the number of viral substitutions and their location within the epitope. Understanding how intraepitope variability impacts CTL recognition is discussed in terms of eliciting immune responses to variants of influenza.     

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.     

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.     

Mathematical Modeling of Ultradeep Sequencing Data Reveals that Acute CD8+ T-Lymphocyte Responses Exert Strong Selective Pressure in Simian Immunodeficiency Virus-Infected Macaques but Still Fail To Clear Founder Epitope Sequences

The prominent role of antiviral cytotoxic CD8⁺ T-lymphocytes (CD8-TL) in containing the acute viremia of human and simian immunodeficiency viruses (HIV-1 and SIV) has rationalized the development of T-cell-based vaccines. However, the presence of escape mutations in the acute stage of infection has raised a concern that accelerated escape from vaccine-induced CD8-TL responses might undermine vaccine efficacy. We reanalyzed previously published data of 101,822 viral genomes of three CD8-TL epitopes, Nef_103-111RM9 (RM9), Tat_28-35SL8 (SL8), and Gag_181-189CM9 (CM9), sampled by ultradeep pyrosequencing from eight macaques. Multiple epitope variants appeared during the resolution of acute viremia, followed by the predominance of a single mutant epitope. By fitting a mathematical model, we estimated the first acute escape rate as 0.36 day⁻¹ within escape-prone epitopes, RM9 and SL8, and the chronic escape rate as 0.014 day⁻¹ within the CM9 epitope. Our estimate of SIV acute escape rates was found to be comparable to very early HIV-1 escape rates. The timing of the first escape was more highly correlated with the timing of the peak CD8-TL response than with the magnitude of the CD8-TL response. The transmitted epitope decayed more than 400 times faster during the acute viral decline stage than predicted by a neutral evolution model. However, the founder epitope persisted as a minor population even at the viral set point; in contrast, the majority of acute escape epitopes were completely cleared. Our results suggest that a reservoir of SIV infection is preferentially formed by virus with the transmitted epitope.A critical role of CD8⁺ T-lymphocytes (CD8-TL) in controlling the peak of acute viral replication has been demonstrated both in HIV-1 (10, 31, 57) and experimental SIV infections (51). HIV-1-infected patients with strong HIV-1-specific CD8-TL responses early after the onset of the acute retroviral syndrome showed more effective control of primary viremia than patients with low or undetectable virus-specific CD8-TL activity (10). Delayed HIV-1-specific CD8-TL responses within an acutely infected individual was found to be one factor contributing to the patient''s persistent viremia, symptoms, and low CD4⁺ T-cell counts (31). A close temporal association between the magnitude of immunodominant B57-restrcited HIV-1-specific CD8 T cells and viral load was observed (57). In nonhuman primate models, the effect of CD8⁺ T cells on acute viral containment has been more directly probed by administering an anti-CD8 antibody to transiently deplete CD8⁺ lymphocytes from the peripheral blood (51). The resolution of peak viremia was much slower in the CD8⁺ lymphocyte-depleted rhesus macaques than in the untreated control animals (51).CT8-TL responses provide selective pressure within human leukocyte antigen (HLA)-restricted regions of the viral genome, which can select for escape variants. Understanding the kinetics of viral escape has important implications for the development of T-cell-based vaccines. Recently, in acutely infected HIV-1 subjects, single-genome amplification (SGA) and sequencing have shown that while only random mutations were observed prior to peak viremia (50), CD8-TL escape mutations were prominent as early as 20 to 30 days after the acute peak of viremia (24), well before the establishment of the viral set point. Indeed, it was observed that the emergence of viral escape mutants occurred coincidently with the expansion of the epitope-specific CD8-TL population in the acutely infected host, and that it resulted in amino acid substitutions in the transmitted/founder virus that diminished recognition by CT8-TL specific for the original (transmitted) epitope (24).Quantitatively, the average rate of CD8-TL escape mutation within 20 days of HIV-1 infection since the first screening has been estimated as 0.33 day⁻¹ (24). This early escape rate is substantially greater than the chronic escape rate, which has been estimated as 0.04 day⁻¹ (6). However, these prior estimates (6, 24) have been based on Sanger sequencing data from a limited number of virus clones. The availability of ultradeep pyrosequencing methods provides the opportunity to revisit these estimates using much richer data sets, which can detect mutations with a frequency of as little as 1% (8). The quantification of the rate of CD8-TL escape in SIV and HIV-1 is important, since it can serve as a surrogate measure of the magnitude and effectiveness of the host CD8-TL response. Mathematical models have been developed to quantify the process of viral CD8-TL escape (6, 7, 23), which framed the escape phenomenon as a synergetic outcome of the differences of wild and mutant epitopes in terms of susceptibility to cytotoxic T-lymphocyte (CTL) killing versus their intrinsic viral fitness.The goal of the present study was to quantify escape dynamics within three well-defined CD8-TL epitopes by rigorously analyzing both previously published and newly generated ultradeep pyrosequencing data from a set of eight SIV-infected macaques (8). Bimber and colleagues (8) previously demonstrated multifarious patterns of CTL escape in these SIV-infected macaques, and a recently published analysis of the same data set by Hughes et al. revealed that the persistence of low levels of inoculum sequence and its consistent loss kinetics enable the reliable inference of the wild-type sequence when only samples from later in infection are available for study (26). Here, we used the same extensive sequence data set, in combination with newly generated data, to quantify viral escape dynamics for three well-defined CD8-TL epitopes relative to the transmitted (wild-type) epitope sequence. By fitting a mathematical model of CD8-TL escape (6) to the experimentally determined CT8-TL escape kinetics, we compared the rate of the first CD8-TL escape of the escape-prone epitopes, Nef_103-111RM9 and Tat_28-35SL8, to that of the escape-resistant epitope, Gag_181-189CM9. For this purpose, we define the time to first CD8-TL escape as the time when the first CD8-TL escape mutant comprises 50% of the combined population of the transmitted (wild) sequence and the first escape mutant clone. This definition is different from the timing of the first emergence of amino acid variants within an epitope. Our definition can be used when individual clones are obtained either by single-genome amplification (42, 49) or pyrosequencing (32, 48).In this study, by employing a rich data set from ultradeep pyrosequencing, we tested the hypothesis that the transmitted epitope contributes to the formation of a reservoir of infection. Our results suggest that this is indeed the case, and they also suggest that viral reversion (13, 21, 34, 37) is complicated in some cases by the unexpected persistence of wild-type, transmitted virus strains long after initial infection.     

Impact of HLA in Mother and Child on Disease Progression of Pediatric Human Immunodeficiency Virus Type 1 Infection

A broad Gag-specific CD8⁺ T-cell response is associated with effective control of adult human immunodeficiency virus (HIV) infection. The association of certain HLA class I molecules, such as HLA-B*57, -B*5801, and -B*8101, with immune control is linked to mutations within Gag epitopes presented by these alleles that allow HIV to evade the immune response but that also reduce viral replicative capacity. Transmission of such viruses containing mutations within Gag epitopes results in lower viral loads in adult recipients. In this study of pediatric infection, we tested the hypothesis that children may tend to progress relatively slowly if either they themselves possess one of the protective HLA-B alleles or the mother possesses one of these alleles, thereby transmitting a low-fitness virus to the child. We analyzed HLA type, CD8⁺ T-cell responses, and viral sequence changes for 61 mother-child pairs from Durban, South Africa, who were monitored from birth. Slow progression was significantly associated with the mother or child possessing one of the protective HLA-B alleles, and more significantly so when the protective allele was not shared by mother and child (P = 0.007). Slow progressors tended to make CD8⁺ T-cell responses to Gag epitopes presented by the protective HLA-B alleles, in contrast to progressors expressing the same alleles (P = 0.07; Fisher''s exact test). Mothers expressing the protective alleles were significantly more likely to transmit escape variants within the Gag epitopes presented by those alleles than mothers not expressing those alleles (75% versus 21%; P = 0.001). Reversion of transmitted escape mutations was observed in all slow-progressing children whose mothers possessed protective HLA-B alleles. These data show that HLA class I alleles influence disease progression in pediatric as well as adult infection, both as a result of the CD8⁺ T-cell responses generated in the child and through the transmission of low-fitness viruses by the mother.Human immunodeficiency virus (HIV)-specific CD8⁺ T cells play a central role in controlling viral replication (12). It is the specificity of the CD8⁺ T-cell response, particularly the response to Gag, that is associated with low viral loads in HIV infection (7, 17, 34). Although immune control is undermined by the selection of viral mutations that prevent recognition by the CD8⁺ T cells, evasion of Gag-specific responses mediated by protective class I HLA-B alleles typically brings a reduction in viral replicative capacity, facilitating subsequent immune control of HIV (2, 20, 21). The same principle has been demonstrated in studies of simian immunodeficiency virus infection (18, 22).Recent studies showed that the class I HLA-B alleles that protect against disease progression present more Gag-specific CD8⁺ T-cell epitopes and drive the selection of more Gag-specific escape mutations than those alleles that are associated with high viral loads (23). These protective HLA-B alleles not only are beneficial to infected individuals expressing those alleles but also benefit a recipient following transmission, since the transmitted virus carrying multiple Gag escape mutations may have substantially reduced fitness (3, 4, 8). However, there is no benefit to the recipient if he or she shares the same protective allele as the donor because the transmitted virus carries escape mutations in the Gag epitopes that would otherwise be expected to mediate successful immune control in the recipient (8, 11).The sharing of HLA alleles between donor and recipient occurs frequently in mother-to-child transmission (MTCT). The risk of MTCT is related to viral load in the mother, and a high viral load is associated with nonprotective alleles, such as HLA-B*18 and -B*5802. This may contribute in two distinct ways to the more rapid progression observed in pediatric HIV infection (24, 26, 27). First, because infected children share 50% or more of their HLA alleles with the transmitting mother, they are less likely than adults to carry protective HLA alleles (16). Thus, infected children as a group carry fewer protective HLA alleles and more nonprotective HLA alleles. Second, even when the child has a protective allele, such as HLA-B*27, this allele does not offer protection if the maternally transmitted virus carries escape mutations within the key Gag epitopes that are presented by the protective allele (11, 19).However, it is clear that infected children who possess protective alleles, such as HLA-B*27 or HLA-B*57, can achieve durable immune control of HIV infection if the virus transmitted from the mother is not preadapted to those alleles (6, 10). HIV-specific CD8⁺ T-cell responses are detectable from birth in infected infants (32). Furthermore, as in adult infection (3, 8), HIV-infected children have the potential to benefit from transmission of low-fitness viruses in the situation where the mother possesses protective HLA alleles and the child does not share those protective alleles. MTCT of low-fitness viruses carrying CD8⁺ T-cell escape mutations was recently documented (28; J. Prado et al., unpublished data).In this study, undertaken in Durban, South Africa, we set out to test the hypothesis that HIV-infected children are less likely to progress rapidly to disease if either the infected child or the transmitting mother possesses a protective HLA allele that is not shared. The HLA alleles most strongly associated with low viral loads and high CD4 counts in a cohort of >1,200 HIV-infected adults in Durban are HLA-B*57 (-B*5702 and -B*5703), HLA-B*5801, and HLA-B*8101 (16; A. Leslie et al., unpublished data). These four alleles all present Gag-specific CD8⁺ T-cell epitopes, and in each case the escape mutations selected in these epitopes reduce viral replicative capacity (2-4, 8, 21, 23).Analyzing a previously described cohort of 61 HIV-infected children in Durban (24, 26, 32), South Africa, who were all monitored from birth, we first addressed the question of whether possession of any of these four alleles by either mother or child is associated with slower disease progression in the child and then determined whether sharing of protective alleles by mother and child affects the ability of the child to make the Gag-specific CD8⁺ T-cell responses restricted by the shared allele.     

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.     

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.     

Vaccination reduces simian-human immunodeficiency virus sequence reversion through enhanced viral control

It has been suggested that vaccination prior to infection may direct the mutational evolution of human immunodeficiency virus type 1 (HIV-1) to a less fit virus, resulting in an attenuated course of disease. The present study was initiated to explore whether prior immunization might prevent the reversion of the virus to the wild-type form. Mamu-A*01 monkeys were vaccinated to generate a cytotoxic T-lymphocyte response to the immunodominant Gag p11C epitope and were then challenged with a cloned pathogenic CXCR4-tropic simian-human immunodeficiency virus (SHIV) expressing a mutant Gag p11C sequence (Δp11C SHIV). The epitopic and extraepitopic compensatory mutations introduced into gag of Δp11C SHIV resulted in attenuated replicative capacity and eventual reversions to the wild-type Gag p11C sequence in naïve rhesus monkeys. However, in vaccinated rhesus monkeys, no reversions of the challenge virus were observed, an effect that may have been a consequence of significantly decreased viral replication rather than a redirection of the mutational evolution of the virus. These findings highlight the multifactorial pressures that affect the evolution of primate immunodeficiency viruses.CD8⁺ cytotoxic T-lymphocyte (CTL) responses are important for controlling replication of human immunodeficiency virus type 1 (HIV-1) in humans and simian immunodeficiency virus (SIV) in rhesus monkeys (6, 15, 19, 25, 32, 37, 39-41). However, the accumulation of mutations in dominant epitopes of these viruses can undermine this immune control (1, 8, 13, 18, 28). It has been proposed that a preexisting memory-specific CTL response elicited by vaccination prior to HIV-1/SIV infection might change the epitope specificity or the mutational pattern of the infecting virus (9). It is also possible that vaccine-induced cellular immunity might diminish the level of virus replication in individuals following infection and in doing so decrease the rate of accumulation of viral mutations and the likelihood of emergence of viruses that can escape CTL recognition.Our laboratory has previously described a rare SHIV-89.6P escape virus that contains a mutation in the dominant Mamu-A*01-restricted Gag p11C C-M (CTPYDINQM) epitope (3, 4). The emergence of this viral variant was associated with an increase in viral load and the eventual death of the previously vaccinated rhesus monkey 798. Analysis of the escape virus demonstrated a threonine-to-isoleucine mutation at amino acid position 47 (T47I) of the SIV capsid protein, which corresponds to position 2 of the Gag p11C epitope. This T47I mutation abrogated binding to the Mamu-A*01 class I molecule, allowing the virus to escape from recognition by the dominant epitope-specific CTL population (4). In addition to the T47I mutation, a downstream isoleucine-to-valine (I71V) substitution was found to be required for the viability of the escape virus in vitro (12, 29, 30, 42).The present studies were initiated to study the effects of prior vaccination on Gag p11C sequence reversion by infecting monkeys with a simian-human immunodeficiency virus (SHIV) clone containing the gag mutations found in the escape virus that evolved in monkey 798. We first explored the effects of these mutations in vivo by infecting naïve Mamu-A*01⁺ rhesus monkeys with a cloned SHIV (Δp11C SHIV) containing both the Gag p11C T47I mutation and the downstream I71V compensatory substitutions. We then determined whether vaccination prior to infection could generate a cellular immune response that might alter the expected pattern of virus mutation in the immunodominant Mamu-A*01-restricted Gag p11C epitope of Δp11C SHIV.     

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.     

Ultradeep Pyrosequencing Detects Complex Patterns of CD8+ T-Lymphocyte Escape in Simian Immunodeficiency Virus-Infected Macaques

Human and simian immunodeficiency viruses (HIV/SIV) exhibit enormous sequence heterogeneity within each infected host. Here, we use ultradeep pyrosequencing to create a comprehensive picture of CD8⁺ T-lymphocyte (CD8-TL) escape in SIV-infected macaques, revealing a previously undetected complex pattern of viral variants. This increased sensitivity enabled the detection of acute CD8-TL escape as early as 17 days postinfection, representing the earliest published example of CD8-TL escape in intrarectally infected macaques. These data demonstrate that pyrosequencing can be used to study the evolution of CD8-TL escape during immunodeficiency virus infection with an unprecedented degree of sensitivity.Rapid sequence evolution is a hallmark of immunodeficiency virus infection and represents a major obstacle toward the development of a successful human immunodeficiency virus (HIV) vaccine (2, 3). Viral evolution has implications for HIV treatment and provides critical information about host immune responses. Although the viral population contains an enormous amount of sequence diversity, standard sequencing methods are limited to the detection of high-frequency variants. Techniques that permit characterization of rare variants, such as molecular cloning, single-genome amplification, or quantitative RT-PCR, are either labor intensive or restricted to the detection of a single variant, limiting their widespread use (9, 11, 12, 18). As a result, the functional consequences of low-frequency variants and subtle differences in the kinetics of viral evolution are not well understood.CD8⁺ T lymphocytes (CD8-TL) play a critical role in the suppression of immunodeficiency viruses and are a driving force in HIV/SIV (simian immunodeficiency virus) viral evolution (7, 8, 15, 20). Because the emergence of escape mutations within CD8-TL epitopes alters the recognition of infected cells, monitoring viral variation within epitopes has important implications (10, 16). Due to the sequencing limitations noted above, studies of CD8-TL escape are generally limited to the detection of high-frequency variants. As a result, CD8-TL escape is frequently viewed as a binary event: an epitope is either wild type or escaped.In this study, we applied ultradeep pyrosequencing to evaluate acute CD8-TL escape in SIV-infected macaques. We validated this method by sequencing the Tat_28-35SL8 (SL8) epitope in eight Indian rhesus macaques, demonstrating the ability to detect amino acid variants with a frequency as low as 1%. We then examined Nef_103-111RM9 (RM9) viral escape in four Mauritian cynomolgus macaques (MCMs), demonstrating that viral escape within RM9 occurs as early as 17 days postinfection. Pyrosequencing detected a considerable heterogeneity in the diversity, frequency, and kinetics of viral variation between animals that was undetectable by conventional methods. This exceptional variability is present in the viral population until at least 20 weeks postinfection. These studies demonstrate that ultradeep pyrosequencing is a high-throughput method that can be used to sensitively detect and characterize CD8-TL escape variants in any given epitope.     

Novel Immunodominant Peptide Presentation Strategy: a Featured HLA-A*2402-Restricted Cytotoxic T-Lymphocyte Epitope Stabilized by Intrachain Hydrogen Bonds from Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein

Antigenic peptides recognized by virus-specific cytotoxic T lymphocytes (CTLs) are presented by major histocompatibility complex (MHC; or human leukocyte antigen [HLA] in humans) molecules, and the peptide selection and presentation strategy of the host has been studied to guide our understanding of cellular immunity and vaccine development. Here, a severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid (N) protein-derived CTL epitope, N1 (QFKDNVILL), restricted by HLA-A*2402 was identified by a series of in vitro studies, including a computer-assisted algorithm for prediction, stabilization of the peptide by co-refolding with HLA-A*2402 heavy chain and β₂-microglobulin (β₂m), and T2-A24 cell binding. Consequently, the antigenicity of the peptide was confirmed by enzyme-linked immunospot (ELISPOT), proliferation assays, and HLA-peptide complex tetramer staining using peripheral blood mononuclear cells (PBMCs) from donors who had recovered from SARS donors. Furthermore, the crystal structure of HLA-A*2402 complexed with peptide N1 was determined, and the featured peptide was characterized with two unexpected intrachain hydrogen bonds which augment the central residues to bulge out of the binding groove. This may contribute to the T-cell receptor (TCR) interaction, showing a host immunodominant peptide presentation strategy. Meanwhile, a rapid and efficient strategy is presented for the determination of naturally presented CTL epitopes in the context of given HLA alleles of interest from long immunogenic overlapping peptides.In 2003, severe and acute respiratory syndrome (SARS), emerging from China, caused a global outbreak, affecting 29 countries, with over 8,000 human cases and greater than 800 deaths (5, 9, 24, 33, 37). Thanks to the unprecedented global collaboration coordinated by the WHO, SARS coronavirus (SARS-CoV), a novel member of Coronaviridae family, was rapidly confirmed to be the etiological agent for the SARS epidemic (36). Soon after the identification of the causative agent, SARS was controlled and then quickly announced to be conquered through international cooperation on epidemiological processes (9). However, the role that human immunity played in the clearance of SARS-CoV and whether the memory immunity will persist for the potential reemergence of SARS are not yet well understood.In viral infections, CD8⁺ cytotoxic T lymphocytes (CTLs) are essential to the control of infectious disease. Virus-specific CD8⁺ T cells recognize peptides which have 8 to 11 amino acids, in most cases presented by major histocompatibility complex (MHC) class I molecules. However, identification of virus-specific CD8⁺ T-cell epitopes remains a complicated and time-consuming process. Various strategies have been developed to define CTL epitopes so far. One of the most common practices to determine immunodominant CTL epitopes on a large scale is based on screening and functional analysis of overlapping 15- to 20-mer peptides covering an entire viral proteome or a given set of immunogenic proteins (19, 23, 32). However, peptides identified through this method are too long to be naturally processed CTL epitopes, and the definition of MHC class I restriction of these peptides still requires further analysis. Rapid and efficient strategies should be developed for the determination of naturally presented CTL epitopes in the context of any given HLA allele of interest. Furthermore, no other HLA alleles except HLA-A2-restricted CTL epitopes have been reported for SARS-CoV-derived proteins (16, 22, 31, 43, 46, 47, 49). This is primarily because of the limitation of the experimental methods for the other HLA alleles. HLA-A24 is one of the most common HLA-A alleles throughout the world, especially in East Asia, where SARS-CoV emerged, second only to HLA-A2 (30). The development of a fast and valid method to screen and identify HLA-A24-restricted epitopes would greatly contribute to the understanding of the specific CTL epitope-stimulated response and widen the application of the epitope-based vaccine among a more universal population (17). A genomewide scanning of HLA binding peptides from SARS-CoV has been performed by Sylvester-Hvid and colleagues, through which dozens of peptides with major HLA supertypes, including HLA-A24 binding capability, have been identified (41).There are strong indications that different peptide ligands, such as peptides with distinct immunodominance, can elicit a diverse specific T-cell repertoire, and even subtle changes in the same peptide can have a profound effect on the response (25, 44). Furthermore, a broader T-cell receptor (TCR) repertoire to a virus-specific peptide-MHC complex can keep the host resistant to the virus and limit the emergence of virus immune-escape mutants (29, 34, 38). Recent studies have demonstrated that the diversity of the selected TCR repertoire (designated as T-cell receptor bias) is clearly influenced by the conformational characteristics of the bound peptide in the MHC groove. Peptides with a flat, featureless surface when presented by MHC generate only limited TCR diversity in a mature repertoire, while featured peptides with exposed residues (without extreme bulges) protruding outside the pMHC landscapes are rather associated with the more diverse T-cell repertoire (15, 28, 39, 44, 45). Therefore, being able to determine the binding features of a peptide to MHC and describe the peptide-MHC topology will help us understand the immunodominance of a given peptide and demonstrate the peptide presentation strategy of the host.Structural proteins of SARS-CoV, such as spike, membrane, and nucleocapsid (N), have been demonstrated as factors of the antigenicity of the virus, as compared with the nonstructural proteins (12, 20). Coronavirus nucleocapsid (N) protein is a highly phosphorylated protein which not only is responsible for construction of the ribonucleoprotein complex by interacting with the viral genome and regulating the synthesis of viral RNA and protein, but also serves as a potent immunogen that induces humoral and cellular immunity (13, 14, 26, 48). The CD8⁺ T-cell epitopes derived from SARS-CoV N protein defined so far mainly cluster in two major immunogenic regions (4, 21, 23, 31, 32, 43). One of them, residues 219 to 235, comprises most of the N protein-derived minimal CTL epitopes identified so far—N220-228, N223-231, N227-235, etc.—all of which are HLA-A*0201 restricted (4, 43). The other region, residues 331 to 365, also includes high-immunogenicity peptides that can induce memory T-lymphocyte responses against SARS-CoV (21, 23, 32). However, until now, no minimal CTL epitope with a given HLA allele restriction has been investigated in this region.Here, based on previously defined immunogenic regions derived from SARS-CoV N protein (21), we identified an HLA-A*2402-restricted epitope, N1 (residues 346 to 354), in the region through a distinct strategy using structural and functional approaches. The binding affinity with HLA-A*2402 molecules and the cellular immunogenicity of the peptide were demonstrated in a series of assays. The X-ray crystal structure of HLA-A*2402 complexed with peptide N1 has shown a novel host strategy to present an immunodominant CTL epitope by intrachain hydrogen bond as a featured epitope.     

Fluidity of HIV-1-Specific T-Cell Responses during Acute and Early Subtype C HIV-1 Infection and Associations with Early Disease Progression

Deciphering immune events during early stages of human immunodeficiency virus type 1 (HIV-1) infection is critical for understanding the course of disease. We characterized the hierarchy of HIV-1-specific T-cell gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay responses during acute subtype C infection in 53 individuals and associated temporal patterns of responses with disease progression in the first 12 months. There was a diverse pattern of T-cell recognition across the proteome, with the recognition of Nef being immunodominant as early as 3 weeks postinfection. Over the first 6 months, we found that there was a 23% chance of an increased response to Nef for every week postinfection (P = 0.0024), followed by a nonsignificant increase to Pol (4.6%) and Gag (3.2%). Responses to Env and regulatory proteins appeared to remain stable. Three temporal patterns of HIV-specific T-cell responses could be distinguished: persistent, lost, or new. The proportion of persistent T-cell responses was significantly lower (P = 0.0037) in individuals defined as rapid progressors than in those progressing slowly and who controlled viremia. Almost 90% of lost T-cell responses were coincidental with autologous viral epitope escape. Regression analysis between the time to fixed viral escape and lost T-cell responses (r = 0.61; P = 0.019) showed a mean delay of 14 weeks after viral escape. Collectively, T-cell epitope recognition is not a static event, and temporal patterns of IFN-γ-based responses exist. This is due partly to viral sequence variation but also to the recognition of invariant viral epitopes that leads to waves of persistent T-cell immunity, which appears to associate with slower disease progression in the first year of infection.For more than a decade, there has been a wealth of evidence to show that human immunodeficiency virus (HIV)-specific cytotoxic T-cell (CTL) responses play a role in the control of HIV-1 and simian immunodeficiency virus (SIV) infection. In humans, the first appearance of CTL in primary HIV-1 infection coincides with the decline of peak viremia (7, 27), while depletion of CD8⁺ T cells in SIV infection resulted in elevated viremia (45). Additionally, polymorphisms in HLA class I-restricted CTL responses are associated with differential HIV-1 disease outcomes (25), and the emergence of viral escape within CTL epitopes during acute and chronic SIV or HIV-1 infection demonstrates the effectiveness of CD8⁺ T cells to exert viral selection pressure (21). Dissecting the specificity of HIV-1-specific CD8⁺ T-cell responses that associate with the control of viral replication during acute/early infection is thought to be critical for the design of vaccines and potential immunotherapeutic strategies aimed at stimulating these responses.Preferential targeting of class I-restricted CTL epitopes in Gag during early and chronic HIV-1 infection has been associated with lower viral loads (15, 25, 34, 48, 55), whereas Env- and Nef-specific CD8⁺ T-cell responses have been associated with higher viremia (15, 34, 55). Increasing evidence suggests that patterns of immunodominant HIV-specific CD8⁺ T-cell responses restricted by specific HLA alleles are major determinants of the viral set point (47). In addition, Goonetilleke et al. (17) have provided insight into the rapidity of early escape and the contribution of the first HIV-specific CD8⁺ T-cell responses to the transmitted/founder virus in control of acute viremia. The restriction of CTL epitopes by HLA-B*5801, for example, has also been associated with better viral control (16, 24). However, the temporal nature of epitope-specific responses that associate with viral control has not been explored. Recently, we found no association between the magnitude and breadth of gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay responses at a static 3-month time point with the viral set point at 12 months (22). The unpredictability of early T-cell responses with later viral control could be a result of HIV variability resulting in epitope escape from humoral and T-cell pressure (1, 8). For example, the impact of CTL pressure on shaping viral diversity at a human population level has been observed through HLA imprinting (6, 9, 44), and several studies have shown that certain selected escape mutations can compromise viral fitness (10, 29, 33, 39). Other studies have also demonstrated that the selection of escape variants in chronic HIV-1 and SIV infection can result in the loss of immune control and disease progression (3, 20). Assessing the nature of T-cell responses longitudinally and relating the patterns of contemporaneous viral recognition with viral diversity may represent alternative insights into factors associated with set point and disease progression.As the global AIDS epidemic continues to expand in sub-Saharan Africa, and South Africa in particular, the need to implement a preventive vaccine through the public health sector remains paramount. To date, several prototype antibody and T-cell-based candidate vaccine trials have been completed worldwide (37), and the recent failure of a phase IIb Ad5-Gag-Pol-Nef HIV-1 vaccine trial has emphasized the challenge of producing an effective T-cell-based vaccine against HIV. Data from the recent ALVAC and AIDSVAX (RV144) trials in Thailand have provided modest efficacy of a vaccine regimen in reducing HIV infection (42), and while the immune mechanisms for this are as yet unclear, these findings have created a platform for identifying immune responses that correlate with protection.The identification of the earliest targets of T cells during acute HIV-1 infection would be helpful in understanding the evolution of immunity when a host first encounters the virus and also would provide insight into the host-pathogen interplay when there is a rapidly changing target. We describe some of the earliest T-cell responses that occur during acute subtype C HIV-1 infection, how these change over time and associate with early disease progression, as well as the kinetics of these changes in relation to autologous viral escape.     

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.A few individuals spontaneously control the replication of human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) to very low levels. The precise mechanisms underlying this control are of great interest, as a clear understanding of what constitutes a successful immune response may aid in developing an AIDS vaccine. Particularly pressing questions for vaccine design include which proteins to use as immunogens, the extent to which increasing the breadth and magnitude of responses is advantageous, how immunodomination affects T-cell responses, and if biasing the immune response toward particular effector profiles is beneficial. Characterization of immune responses made by elite controllers (ECs) may reveal patterns that can then be applied to vaccine formulation and evaluation.HIV ECs are generally not infected with grossly unfit viruses (6, 42). Instead, elite control of immunodeficiency virus replication is correlated with the presence of particular major histocompatibility complex class I (MHC-I) alleles (11, 12, 18, 32, 41, 55). The association of MHC-I alleles with the control of viremia implicates CD8⁺ T cells as being mediators of this immune containment. Several lines of evidence support this hypothesis. These lines of evidence include the correlation between the appearance of CD8⁺ T-cell responses and the resolution of peak viremia during acute infection (7, 29), the finding that alleles associated with viral control restrict dominant acute-phase CD8⁺ T-cell responses (3), and the finding that responses directed against epitopes restricted by these alleles frequently select for viral escape variants (4, 27, 38). Perhaps most compelling is the observation that for a few HIV-infected individuals, the selection of escape variants by an immunodominant HLA-B27-restricted T-cell response temporally preceded substantial increases in viremia (17, 21, 53). While viruses exhibiting escape variants in epitopes restricted by protective alleles are often detectably less fit in vitro (10, 38, 43, 51), recent data have found normal, high levels of replication in vivo upon the transmission of some of these variants (15).The association of control with MHC-I alleles does not, of course, implicate solely CD8⁺ T cells. MHC-I molecules are also ligands for killer immunoglobulin receptors (KIRs), which are predominantly expressed on natural killer (NK) cells. Genetic studies of HIV-infected humans suggest a model in which individuals with particular KIR/HLA combinations are predisposed to control HIV replication more readily than those with other KIR/HLA combinations (36, 37). These data were supported by functional studies of this KIR/HLA pairing in vitro, which demonstrated an inhibition of HIV replication by such NK cells (2). The relative contributions of NK and CD8⁺ T-cell responses to control have yet to be elucidated and may be closely intertwined.Previously, the experimental depletion of circulating CD8⁺ cells from SIVmac239-infected ECs resulted in a sharp spike in viremia, which resolved as CD8⁺ cells repopulated the periphery (19). During the reestablishment of control of SIV replication, CD8⁺ T cells targeting multiple epitopes restricted by alleles associated with elite control expanded in frequency, providing strong circumstantial evidence for their role in maintaining elite control (19, 31). However, CD8 depletion antibodies used in macaques also remove NK cells, which, at least in vitro, also inhibit SIV replication (19). It was therefore difficult to make definitive conclusions regarding the separate contributions of these subsets to maintaining the control of SIV replication in vivo.Here we investigate elite control in the rhesus macaque model for AIDS. We focused on the macaque MHC-I allele most tightly associated with the control of SIVmac239, Mamu-B*08. Approximately 50% of Mamu-B*08-positive (Mamu-B*08⁺) animals infected with SIVmac239 become ECs (32). Peptides presented by Mamu-B*08 share a binding motif with peptides presented by HLA-B27. Although these two MHC-I genes are dissimilar in domains that are important for peptide binding, each molecule can bind peptides that are presented by the other molecule (33). This striking similarity suggests that the elite control of SIVmac239 in Mamu-B*08⁺ animals is a good model for the elite control of HIV.Seven SIVmac239 epitopes restricted by Mamu-B*08 accrue variation in Mamu-B*08⁺ rhesus macaques (30, 31). For an eighth Mamu-B*08-restricted epitope, which is also restricted by Mamu-B*03 (Mamu-B*03 differs from Mamu-B*08 by 2 amino acids in the α1 and α2 domains [9, 32]), escape has been documented only for SIV-infected Mamu-B*03⁺ macaques (16). Variation in these CD8⁺ T-cell epitopes accumulates with different kinetics, starting during acute infection for those targeted by high-magnitude responses.In this study, we addressed the question of whether the elite control of SIVmac239 in Mamu-B*08⁺ animals is mediated by the known high-frequency CD8⁺ T-cell responses targeting Mamu-B*08-restricted epitopes. To this end, we introduced point mutations into eight epitopes, with the goal of reducing or abrogating immune responses directed against these epitopes during acute infection. We hypothesized that Mamu-B*08⁺ macaques would be unable to control SIV replication without these Mamu-B*08-restricted T-cell responses.     

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.     

HLA-Associated Clinical Progression Correlates with Epitope Reversion Rates in Early Human Immunodeficiency Virus Infection

Human immunodeficiency virus type 1 (HIV-1) can evade immunity shortly after transmission to a new host but the clinical significance of this early viral adaptation in HIV infection is not clear. We present an analysis of sequence variation from a longitudinal cohort study of HIV adaptation in 189 acute seroconverters followed for up to 3 years. We measured the rates of variation within well-defined epitopes to determine associations with the HLA-linked hazard of disease progression. We found early reversion across both the gag and pol genes, with a 10-fold faster rate of escape in gag (2.2 versus 0.27 forward mutations/1,000 amino acid sites). For most epitopes (23/34), variation in the HLA-matched and HLA-unmatched controls was similar. For a minority of epitopes (8/34, and generally associated with HLA class I alleles that confer clinical benefit), new variants appeared early and consistently over the first 3 years of infection. Reversion occurred early at a rate which was HLA-dependent and correlated with the HLA class 1-associated relative hazard of disease progression and death (P = 0.0008), reinforcing the association between strong cytotoxic T-lymphocyte responses, viral fitness, and disease status. These data provide a comprehensive overview of viral adaptation in the first 3 years of infection. Our findings of HLA-dependent reversion suggest that costs are borne by some escape variants which may benefit the host, a finding contrary to a simple immune evasion paradigm. These epitopes, which are both strongly and frequently recognized, and for which escape involves a high cost to the virus, have the potential to optimize vaccine design.The dynamics of viral replication in acute and early human immunodeficiency virus (HIV) infection are not well understood as longitudinal data from large cohorts of seroconverters are hard to assemble. Recent studies have shown that new HIV infections may be the result of a single transmitted variant, that new env gene mutations can be detected within a few weeks (25), and that early immune escape can be detected at sites across the HIV genome (9). These data add to a body of work showing that cytotoxic T cells act early, contributing to the early reduction in viremia (8, 30).Whether early cytotoxic T-lymphocyte (CTL) immune responses influence longer-term clinical outcome is not clear. Antigen-specific CTLs capable of producing gamma interferon and other cytokines are detectable at all stages of HIV infection (1, 3, 24, 41). Much weight is placed on the macaque/simian immunodeficiency virus model in which nearly total peripheral blood CD8⁺ T-cell elimination using monoclonal antibodies results in rising viremia (42). The role of other forms of host immunity (e.g., neutralizing antibodies, natural killer cells, and macrophages) has, to some extent, been pursued with less intensity in light of persuasive evidence that CTLs can control retrovirus infection (46). The extent to which the simian model mirrors HIV infection has been questioned (5) and, despite exhaustive cellular assays of T-cell function—from gamma interferon enzyme-linked immunospot assays(1, 27, 38) to polyfunctional cytokine matrices (2, 6)—no CTL function correlates robustly with HIV plasma viral load or viral dynamics. Moreover, analyses of evolutionary data suggest that CTLs are inefficient at killing HIV-infected cells (4).However, statistical analysis of data from large cross-sectional studies link HLA class I alleles with specific genome-wide HIV polymorphisms, suggestive of a pervasive selection pressure enacted by CTLs (7, 10, 18, 36, 40). It is clear that associations between some HLA class I alleles and particular amino acid polymorphisms are robust although it is disputed whether immune escape influences disease progression. The viral fitness costs resulting from immune escape may even contribute to better clinical outcomes associated with the possession of HLA class I alleles such as B*27, B*57, and B*58 (18).Evolutionary studies of HIV require longitudinal data from large cohorts of patients sampled since seroconversion to detect adaptation in new hosts as it accrues. HIV is one of the few pathogens where it is possible to do this within individuals because of the high viral turnover and rapid intrahost evolution. Here, we investigate a cohort of 189 acute seroconverters—the largest cohort reported to date—followed for up to 3 years to study the rates of viral mutation in individual epitopes within internal HIV proteins and to determine the association between HLA class I alleles and rates of immune escape and reversion.