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

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

The Effect of HLA Polymorphisms on the Recognition of Gag Epitopes in HIV-1 CRF01_AE Infection

Introduction

The design of a globally effective vaccine rests on the identification of epitopes capable of eliciting effective cytotoxic T lymphocyte (CTL) responses across multiple HIV clades in different populations. This study aims to discern the effect of HLA polymorphisms and the cross-clade reactivity or clade-specificity of epitopes in Thailand where HIV-1 CRF01_AE is circulating.

Materials and Methods

14 peptides based on consensus HIV-1 CRF01_AE amino acid sequences were designed for use in IFN-γ ELISpot assays and ⁵¹Cr release assays among 66 HIV-1 CRF01_AE-infected Thai patients. For ELISpot responders carrying HLA alleles currently unknown to restrict CRF01_AE epitopes, in silico epitope-HLA prediction was performed.

Results

29/66 (43.9%) patients recognized at least one peptide. In total 79 responses were seen against all 14 peptides. 28/79 (35.4%) of the responses were in patients with HLA alleles previously reported to restrict CRF01_AE epitopes, 24/79 (30.4%) responses were in individuals with HLA alleles previously reported to restrict epitopes of HIV clades other than CRF01_AE, and the remaining 27/79 (34.2%) responses were not associated with HLA alleles previously known to restrict HIV epitopes. In silico epitope prediction detected 19 novel, epitope-HLA combinations, and 11/19 (57.9%) were associated with HLA-C alleles. We further confirmed a novel HLA restriction of a previously identified HIV-1 Gag epitope [p24_122–130: PPIPVGDIY (PY9)] by HLA-B*40:01 with a standard ⁵¹Cr release assay.

Discussion

CTL recognition sites in HIV-1 Gag were similar among different clades but the HLA restriction differed in Thai patients. This disparity in HLA restriction along different populations illustrated the importance of clade- and population-specific HLA analysis prior to CTL vaccine design.     

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.     

HIV-1 Replication Fitness of HLA-B*57/58:01 CTL Escape Variants Is Restored by the Accumulation of Compensatory Mutations in Gag

Expression of HLA-B*57 and the closely related HLA-B*58:01 are associated with prolonged survival after HIV-1 infection. However, large differences in disease course are observed among HLA-B*57/58:01 patients. Escape mutations in CTL epitopes restricted by these HLA alleles come at a fitness cost and particularly the T242N mutation in the TW10 CTL epitope in Gag has been demonstrated to decrease the viral replication capacity. Additional mutations within or flanking this CTL epitope can partially restore replication fitness of CTL escape variants. Five HLA-B*57/58:01 progressors and 5 HLA-B*57/58:01 long-term nonprogressors (LTNPs) were followed longitudinally and we studied which compensatory mutations were involved in the restoration of the viral fitness of variants that escaped from HLA-B*57/58:01-restricted CTL pressure. The Sequence Harmony algorithm was used to detect homology in amino acid composition by comparing longitudinal Gag sequences obtained from HIV-1 patients positive and negative for HLA-B*57/58:01 and from HLA-B*57/58:01 progressors and LTNPs. Although virus isolates from HLA-B*57/58:01 individuals contained multiple CTL escape mutations, these escape mutations were not associated with disease progression. In sequences from HLA-B*57/58:01 progressors, 5 additional mutations in Gag were observed: S126N, L215T, H219Q, M228I and N252H. The combination of these mutations restored the replication fitness of CTL escape HIV-1 variants. Furthermore, we observed a positive correlation between the number of escape and compensatory mutations in Gag and the replication fitness of biological HIV-1 variants isolated from HLA-B*57/58:01 patients, suggesting that the replication fitness of HLA-B*57/58:01 escape variants is restored by accumulation of compensatory mutations.     

Whole Genome Deep Sequencing of HIV-1 Reveals the Impact of Early Minor Variants Upon Immune Recognition During Acute Infection

Deep sequencing technologies have the potential to transform the study of highly variable viral pathogens by providing a rapid and cost-effective approach to sensitively characterize rapidly evolving viral quasispecies. Here, we report on a high-throughput whole HIV-1 genome deep sequencing platform that combines 454 pyrosequencing with novel assembly and variant detection algorithms. In one subject we combined these genetic data with detailed immunological analyses to comprehensively evaluate viral evolution and immune escape during the acute phase of HIV-1 infection. The majority of early, low frequency mutations represented viral adaptation to host CD8+ T cell responses, evidence of strong immune selection pressure occurring during the early decline from peak viremia. CD8+ T cell responses capable of recognizing these low frequency escape variants coincided with the selection and evolution of more effective secondary HLA-anchor escape mutations. Frequent, and in some cases rapid, reversion of transmitted mutations was also observed across the viral genome. When located within restricted CD8 epitopes these low frequency reverting mutations were sufficient to prime de novo responses to these epitopes, again illustrating the capacity of the immune response to recognize and respond to low frequency variants. More importantly, rapid viral escape from the most immunodominant CD8+ T cell responses coincided with plateauing of the initial viral load decline in this subject, suggestive of a potential link between maintenance of effective, dominant CD8 responses and the degree of early viremia reduction. We conclude that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation. These data support the critical need for vaccine-induced CD8+ T cell responses to target more highly constrained regions of the virus in order to ensure the maintenance of immunodominant CD8 responses and the sustained decline of early viremia.     

Compartmentalization and Clonal Amplification of HIV-1 Variants in the Cerebrospinal Fluid during Primary Infection

Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) is a severe neurological disease that affects a subset of HIV-1-infected individuals. Increased compartmentalization has been reported between blood and cerebrospinal fluid (CSF) HIV-1 populations in subjects with HAD, but it is still not known when compartmentalization arises during the course of infection. To assess HIV-1 genetic compartmentalization early during infection, we compared HIV-1 populations in the peripheral blood and CSF in 11 primary infection subjects, with analysis of longitudinal samples over the first 18 months for a subset of subjects. We used heteroduplex tracking assays targeting the variable regions of env and single-genome amplification and sequence analysis of the full-length env gene to identify CSF-compartmentalized variants and to examine viral genotypes within the compartmentalized populations. For most subjects, HIV-1 populations were equilibrated between the blood and CSF compartments. However, compartmentalized HIV-1 populations were detected in the CSF of three primary infection subjects, and longitudinal analysis of one subject revealed that compartmentalization during primary HIV-1 infection was resolved. Clonal amplification of specific HIV-1 variants was identified in the CSF population of one primary infection subject. Our data show that compartmentalization can occur in the central nervous system (CNS) of subjects in primary HIV-1 infection in part through persistence of the putative transmitted parental variant or via viral genetic adaptation to the CNS environment. The presence of distinct HIV-1 populations in the CSF indicates that independent HIV-1 replication can occur in the CNS, even early after HIV-1 transmission.Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) can lead to neurological disease in a subset of HIV-infected individuals and may include the development of HIV-1-associated dementia (HAD) (2, 18). HAD is characterized by severe neurological dysfunction, and affected individuals generally have impaired cognitive and motor functions. HIV-1 enters the CNS during primary infection, most likely via the migration of infected monocytes and lymphocytes across the blood-brain barrier (33, 37, 42). The main cell types in the CNS that HIV-1 can productively infect are the perivascular macrophages and microglial cells, which express low receptor densities of CD4, CCR5, and CXCR4 (7, 18, 60, 63). Previous studies have also reported that neurotropic HIV-1 variants are generally macrophage tropic (19, 20, 32, 45, 52, 61). Although cells in the CNS may be infected with HIV-1 during the course of disease, it is still unclear whether productive HIV-1 replication occurs in the CNS early during infection.Genetically compartmentalized HIV-1 variants have been detected in the brains of HAD subjects at autopsy (13, 14, 43, 48, 52) and in the cerebrospinal fluid (CSF) of HAD subjects sampled over the course of infection (26, 46, 51, 59). Extensive compartmentalization between the periphery and the CNS has been reported in subjects with HAD; however, it is not yet known when compartmentalization occurs during the course of HIV-1 infection. Primary HIV-1 infection refers to the acute and early phases of infection, during which peak plasma viremia often occurs and a viral “set point” may be reached (8, 34), within the first year after HIV exposure (64). Studies examining compartmentalization between the blood plasma and CSF during primary infection have been limited, and extensive compartmentalization has not been detected in primary infection subjects (26, 50).In this study, we examined HIV-1 genetic compartmentalization between the peripheral blood and CSF during primary HIV-1 infection. Cross-sectional and longitudinal blood plasma and CSF samples were analyzed for viral compartmentalization using the heteroduplex tracking assay (HTA) and single genome amplification (SGA). We used the HTA to differentiate between HIV-1 variants in the CSF that were either compartmentalized to the CSF or equilibrated with the peripheral blood. Previous studies have used the HTA to separate HIV-1 genetic variants in different anatomical compartments (10, 24, 27, 51) and to follow HIV-1 evolutionary variants over the course of infection (9, 25, 31, 41, 49, 50). We also conducted SGA on a subset of subjects to further examine viral genetic compartmentalization during primary infection. Here we report the detection of compartmentalized and clonally amplified HIV-1 variants in the CSF of subjects in the primary stage of HIV-1 infection. Our results suggest that minor to extensive HIV-1 genetic compartmentalization can occur between the periphery and the CNS during primary HIV-1 infection and that viral compartmentalization, as measured in the CSF, is transient in some subjects.     

Immuno-epidemiological Modeling of HIV-1 Predicts High Heritability of the Set-Point Virus Load,while Selection for CTL Escape Dominates Virulence Evolution

It has been suggested that HIV-1 has evolved its set-point virus load to be optimized for transmission. Previous epidemiological models and studies into the heritability of set-point virus load confirm that this mode of adaptation within the human population is feasible. However, during the many cycles of replication between infection of a host and transmission to the next host, HIV-1 is under selection for escape from immune responses, and not transmission. Here we investigate with computational and mathematical models how these two levels of selection, within-host and between-host, are intertwined. We find that when the rate of immune escape is comparable to what has been observed in patients, immune selection within hosts is dominant over selection for transmission. Surprisingly, we do find high values for set-point virus load heritability, and argue that high heritability estimates can be caused by the ‘footprints’ left by differing hosts'' immune systems on the virus.     

HLA-Cw*03-Restricted CD8+ T-Cell Responses Targeting the HIV-1 Gag Major Homology Region Drive Virus Immune Escape and Fitness Constraints Compensated for by Intracodon Variation

The potential importance of HLA-C-restricted CD8⁺ cytotoxic T lymphocytes (CTL) in HIV infection remains undetermined. We studied the dominant HLA-Cw*03-restricted CTL response to YVDRFFKTL_296-304 (YL9), within the conserved major homology region (MHR) of the Gag protein, in 80 HLA-Cw*03-positive individuals with chronic HIV infection to better define the efficacy of the YL9 HLA-C-restricted response. The HLA-Cw*03 allele is strongly associated with HIV sequence changes from Thr-303 to Val, Ile, or Ala at position 8 within the YL9 epitope (P = 1.62 × 10⁻¹⁰). In vitro studies revealed that introduction of the changes T303I and T303A into the YL9 epitope both significantly reduced CTL recognition and substantially reduced the viral replicative capacity. However, subsequent selection of the Val-303 variant, via intracodon variation from Ile-303 (I303V) or Ala-303 (A303V), restored both viral fitness and CTL recognition, as supported by our in vivo data. These results illustrate that HLA-C-restricted CTL responses are capable of driving viral immune escape within Gag, but in contrast to what was previously described for HLA-B-restricted Gag escape mutants, the common Cw*03-Gag-303V variant selected resulted in no detectable benefit to the host.Human leukocyte antigen (HLA) class I is the most polymorphic region of the human genome. HLA class I genes are found at the A, B, and C loci of chromosome 6 and have been shown to play an important role in control of infections by intracellular pathogens (3). Of these three loci, HLA-B has had many more unique molecules identified than has HLA-A or -C (http://www.anthonynolan.org.uk/HIG/index.html). This locus variability is likely to reflect functional differences among HLA-B alleles and disease progression (20). In the context of HIV infection, several studies have demonstrated that HLA-B alleles have the greatest impact on HIV-1 replication control (7, 22). The reason why particular HLA-B, and not HLA-A or -C, alleles have been associated with improved outcome in HIV is unknown but may be explained by the numbers and regions of viral proteomes presented in the context of HLA-B molecules (19, 23, 46).Regardless of the importance of HLA-B-restricted responses in HIV control, the roles of the HLA-A and -C class I alleles have not been fully investigated. A genomewide analysis investigating single-nucleotide polymorphisms (SNP) has associated both the presence of certain HLA-B alleles and a dimorphism upstream of the HLA-C gene (−35C/T) as two of the strongest predictors of the plasma viral load in HIV infection (14). In addition, the −35 SNP has recently been associated with levels of HLA-C expression and may indicate a new role for HLA-C alleles in HIV-1 control (40).One reason that has been postulated to explain the lack of HLA-C association with immune pressure is the lower expression of HLA-C on the cell surface (18, 36, 37). However, HLA-C, unlike HLA-A and HLA-B, is not downregulated by the Nef protein, and this factor, therefore, may explain the lower expression level (9).Analyses of large numbers of HIV sequences have identified associations between particular HLA-C polymorphisms in the HIV pol gene and the set point viral load (27). These polymorphisms were within or in close proximity to defined HLA-C-restricted epitopes, suggesting that HLA-C-restricted responses could be driving HIV evolution (22, 27). Confirmation that all HLA-C-restricted epitopes are not fundamentally ineffective was recently provided by a study of an HLA-Cw*01-restricted epitope targeted to p15 Gag, which was found to drive selection of a 3-amino-acid insertion in a HIV-infected patient (6). Additionally, in vitro studies have demonstrated antiviral activities of HLA-C-restricted cytotoxic T lymphocytes (CTL) clones against HIV comparable to other HLA-B-restricted responses (2). In spite of all these data, the contribution of HLA-C responses to HIV control is still unknown.Our study was undertaken to address the role of an HLA-Cw*0303/0304-restricted response to YVDRFFKTL_296-304 (YL9) in HIV infection. This response was of particular interest due to its dominance and because it targets a highly conserved region of the Gag protein among different retroviruses, namely, the major homology region (MHR). In the study cohort of 778 individuals with chronic HIV infection, 80 were found to carry Cw*0303/0304. Gag viral sequences were obtained from HLA-Cw*0303/0304 subjects and used to identify and define HIV immune escape driven by the YL9-especific responses. We then studied the impacts of these mutations on viral recognition by CD8⁺ T cells and on virus replicative capacity (RC).     

Variations in the Biological Functions of HIV-1 Clade C Envelope in a SHIV-Infected Rhesus Macaque during Disease Progression

A better understanding of how the biological functions of the HIV-1 envelope (Env) changes during disease progression may aid the design of an efficacious anti-HIV-1 vaccine. Although studies from patient had provided some insights on this issue, the differences in the study cohorts and methodology had make it difficult to reach a consensus of the variations in the HIV-1 Env functions during disease progression. To this end, an animal model that can be infected under controlled environment and reflect the disease course of HIV-1 infection in human will be beneficial. Such an animal model was previously demonstrated by the infection of macaque with SHIV, expressing HIV-1 clade C Env V1-V5 region. By using this model, we examined the changes in biological functions of Env in the infected animal over the entire disease course. Our data showed an increase in the neutralization resistance phenotype over time and coincided with the decrease in the net charges of the V1-V5 region. Infection of PBMC with provirus expressing various Env clones, isolated from the infected animal over time, showed a surprisingly better replicative fitness for viruses expressing the Env from early time point. Biotinylation and ELISA data also indicated a decrease of cell-surface-associated Env and virion-associated gp120 content with disease progression. This decrease did not affect the CD4-binding capability of Env, but were positively correlated with the decrease of Env fusion ability. Interestingly, some of these changes in biological functions reverted to the pre-AIDS level during advance AIDS. These data suggested a dynamic relationship between the Env V1-V5 region with the host immune pressure. The observed changes of biological functions in this setting might reflect and predict those occurring during natural disease progression in human.     

Genetic Characterization of Hepatitis B Virus in Peripheral Blood Leukocytes: Evidence for Selection and Compartmentalization of Viral Variants with the Immune Escape G145R Mutation

The compartmentalization of viral variants in distinct host tissues is a frequent event in many viral infections. Although hepatitis B virus (HBV) classically is considered hepatotropic, it has strong lymphotropic properties as well. However, unlike other viruses, molecular evolutionary studies to characterize HBV variants in compartments other than hepatocytes or sera have not been performed. The present work attempted to characterize HBV sequences from the peripheral blood leukocytes (PBL) of a large set of subjects, using advanced molecular biology and computational methods. The results of this study revealed the exclusive compartmentalization of HBV subgenotype Ae/A2-specific sequences with a potent immune escape G145R mutation in the PBL of the majority of the subjects. Interestingly, entirely different HBV genotypes/subgenotypes (C, D, or Aa/A1) were found to predominate in the sera of the same study populations. These results suggest that subgenotype Ae/A2 is selectively archived in the PBL, and the high prevalence of G145R indicates high immune pressure and high evolutionary rates of HBV DNA in the PBL. The results are analogous to available literature on the compartmentalization of other viruses. The present work thus provides evidence in favor of the compartment-specific abundance, evolution, and emergence of the potent immune escape mutant. These findings have important implications in the field of HBV molecular epidemiology, transmission, transfusion medicine, organ transplantation, and vaccination strategies.Hepatitis B virus (HBV) is the prototype member of the Hepadnaviridae family and classically has been described to be hepatotropic, causing a wide range of clinical and subclinical manifestations of liver disease (57). Nevertheless, studies of HBV-infected human subjects and woodchucks infected with Woodchuck hepatitis virus (WHV; an animal model of hepadnaviral infection) have reported different molecular forms of replicative intermediates in the lymphatic cells and have established that hepadnaviruses are strongly lymphotropic in nature (29). Moreover, the results of studies of human subjects as well as with animal models have revealed that the life-long occult persistence of replication- and transmission-competent viruses in lymphatic cells is a strict consequence of hepadnaviral infections (29).More interestingly, in animal models, lymphatic system-restricted occult hepadnaviral infection has been found to be transmissible vertically as an asymptomatic, serologically occult infection exclusively confined to the lymphatic system (29). Earlier we provided evidence that occult HBV persisting in the lymphatic cells are transmissible, specifically to the PBL through horizontal intrafamilial modes (9). These observations clearly indicate important immunological, pathogenic, and epidemiological implications of lymphatic system-restricted hepadnaviral infections. Although the involvement of specific viral variants has been suggested to explain this lymphatic system-restricted hepadnaviral infection and transmission (29), the classical belief that hepatocytes are the primary target and only reservoir of HBV has precluded the genetic characterization of hepadnaviruses from extrahepatic sites.Fascinatingly, despite being classically considered a hepatotropic virus, hepatitis C virus (HCV), belonging to the family Flaviviridae, also shows occult persistence and lymphotropism very similar to that of hepadnaviruses (37). Similarly to WHV, HBV, and HCV, other viruses, including HIV (human immunodeficiency virus), small ruminant lentivirus, and Epstein-Barr virus, also have been shown to infect and persist in different anatomical compartments of the body in addition to their classical target cells (38, 40, 43, 45, 50). Furthermore, recent molecular evolutionary analyses based on envelope sequences of these viruses (e.g., HIV, HCV, small ruminant lentivirus, Epstein-Barr virus, etc.) have established clearly that these viruses undergo selection and independent evolution in diverse tissues, leading to the tissue-specific compartmentalization of viral populations (38, 40, 43, 45, 50). In contrast to other viruses, to the best of our knowledge, methodical molecular evolutionary studies to characterize HBV sequences isolated from extrahepatic sites of HBV-infected subjects have not been reported in the literature.We hypothesized that similar to other viruses, HBV also undergo independent evolution in different compartments of the body under the influence of differential immune pressure. To examine our hypothesis, we used the most easily available lymphatic cells, the peripheral blood leukocytes (PBL), determined the HBV envelope sequences from HBV DNA isolated from these cells, and performed advanced genetic, phylogenetic, and mutational analysis. The results of this work demonstrate a highly compartment-specific preponderance of HBV genetic variants in serum and PBL of the same study population, providing evidence in favor of the compartmentalization of HBV genetic variants. The results and important implications of these findings are discussed in this work.     

High Frequency of Herpesvirus-Specific Clonotypes in the Human T Cell Repertoire Can Remain Stable over Decades with Minimal Turnover

High-throughput T cell receptor sequencing on sequentially banked blood samples from healthy individuals has shown that high-frequency clonotypes can remain relatively stable for up to 18 years, with minimal inflation, deflation, or turnover. These populations included T cell expansions specific for Epstein-Barr virus. Thus, in spite of exposure to a barrage of microorganisms over the course of life, the dominant clonotypes in the mature peripheral T cell repertoire can alter surprisingly little.     

Dynamic Regulation of Host Restriction Factor Expression over the Course of HIV-1 Infection In Vivo

In this study, we investigated the expression levels of host restriction factors in six untreated HIV-1-positive patients over the course of infection. We found that the host restriction factor gene expression profile consistently increased over time and was significantly associated with CD4⁺ T cell activation and viral load. Our data are among the first to demonstrate the dynamic nature of host restriction factors in vivo over time.     

A Human Antibody to the CD4 Binding Site of gp120 Capable of Highly Potent but Sporadic Cross Clade Neutralization of Primary HIV-1

Primary isolates of HIV-1 resist neutralization by most antibodies to the CD4 binding site (CD4bs) on gp120 due to occlusion of this site on the trimeric spike. We describe 1F7, a human CD4bs monoclonal antibody that was found to be exceptionally potent against the HIV-1 primary isolate JR-FL. However, 1F7 failed to neutralize a patient-matched primary isolate, JR-CSF even though the two isolates differ by <10% in gp120 at the protein level. In an HIV-1 cross clade panel (n = 157), 1F7 exhibited moderate breadth, but occasionally achieved considerable potency. In binding experiments using monomeric gp120s of select resistant isolates and domain-swap chimeras between JR-FL and JR-CSF, recognition by 1F7 was limited by sequence polymorphisms involving at least the C2 region of Env. Putative N-linked glycosylation site (PNGS) mutations, notably at position 197, allowed 1F7 to neutralize JR-CSF potently without improving binding to the cognate, monomeric gp120. In contrast, flow cytometry experiments using the same PNGS mutants revealed that 1F7 binding is enhanced on cognate trimeric Env. BN-PAGE mobility shift experiments revealed that 1F7 is sensitive to the diagnostic mutation D368R in the CD4 binding loop of gp120. Our data on 1F7 reinforce how exquisitely targeted CD4bs antibodies must be to achieve cross neutralization of two closely related primary isolates. High-resolution analyses of trimeric Env that show the orientation of glycans and polymorphic elements of the CD4bs that affect binding to antibodies like 1F7 are desirable to understand how to promote immunogenicity of more conserved elements of the CD4bs.     

Longitudinal Changes in Plasma Caspase-1 and Caspase-3 during the First 2 Years of HIV-1 Infection in CD4Low and CD4High Patient Groups

Over 95% of CD4 cell death occurs by Caspase-1-mediated pyroptosis during HIV infection. Caspase-3-mediated apoptosis accounts for the death in a small proportion of infected CD4 cells. To date, there have been no reports on the dynamics of Caspase-1 and Caspase-3 and their relationship with disease progression in acute HIV-1 infection. In this study, two distinct HIV-1 patient groups were enrolled. The CD4_High group maintained a CD4 level above450 cells/μl while CD4 levels in the CD4_Low group dropped below 250 cells/μl within 2 years after infection. Blood samples were collected at 1, 2, 3, 4, 6, 12 and 24 months after HIV infection. Plasma Caspase-1 and Caspase-3 levels in the two patients groups were determined by a single-step ELISA using commercially available monoclonal antibodies. The results showed that Caspase-1 and Caspase-3 levels in the CD4_High group increased rapidly and then decreased within a short time during early HIV-1 infection. In contrast, Caspase-1 and Caspase-3 levels in the CD4_Low group were obviously increased after 1 year of HIV-1 infection.