Person:

Pereyra, F

Background: Spontaneous control of HIV to <50 copies RNA/ml is observed in rare individuals. An improved understanding of this phenomenon may provide insight into host mechanisms that can be modulated for therapeutic benefit by vaccines. Recent studies of these 'elite' controllers (EC) revealed HLA-associated changes in Gag-Protease that resulted in reduced replication capacity. This project assessed the possibility of immune-mediated defects in the Pol gene (RT-Integrase) of EC. Methods: Chimeric NL4-3 mutants encoding patient plasmaderived RT-Integrase sequences from EC (N = 58) and chronic progressors (N = 50) were constructed by homologous recombination. Replication capacity (RC) for each variant strain was assessed using a GFP-reporter T cell assay. Results were correlated with clinical and host genetic factors, including CD4 count, plasma viral load (pVL), and patient HLA. Results: Viruses encoding Pol sequences from EC replicated significantly less well than those from individuals with progressive disease (Mann-Whitney, p < 0.0001). No association was observed between RC and CD4 T cell count in EC or progressors, nor between RC and pVL in progressors (Spearman, all p > 0.05). Viruses derived from HLA-B57+ EC (N = 20) appeared to replicate slower than those from B57+ progressors (N = 8) (p = 0.004). Similar results were observed between B51+ EC (N = 4) and B51+ progressors (N = 10) (p = 0.024), but not between B27+ EC (N = 9) and B27+ progressors (N = 5) (p = 0.437). Conclusion: This study extends previous observations for Gag and demonstrates that Pol variants from EC also display reduced function. The association between fitness and expression of certain HLA that present Pol epitopes suggests that immune-mediated mutations impairing viral fitness may play a key role in spontaneous control of HIV. Results indicate that HLA alleles responsible for such defects in protein function may differ among viral genes. Further identification of HLA-associated changes in HIV may allow design of vaccines targeting the most vulnerable regions of the virus.

Many immune correlates of CD8+ T-cell-mediated control of HIV replication, including polyfunctionality, proliferative ability, and inhibitory receptor expression, have been discovered. However, no functional correlates using ex vivo cells have been identified with the known ability to cause the direct elimination of HIV-infected cells. We have recently discovered the ability of human CD8+ T-cells to rapidly upregulate perforin—an essential molecule for cell-mediated cytotoxicity—following antigen-specific stimulation. Here, we examined perforin expression capability in a large cross-sectional cohort of chronically HIV-infected individuals with varying levels of viral load: elite controllers (n = 35), viremic controllers (n = 29), chronic progressors (n = 27), and viremic nonprogressors (n = 6). Using polychromatic flow cytometry and standard intracellular cytokine staining assays, we measured perforin upregulation, cytokine production, and degranulation following stimulation with overlapping peptide pools encompassing all proteins of HIV. We observed that HIV-specific CD8+ T-cells from elite controllers consistently display an enhanced ability to express perforin directly ex vivo compared to all other groups. This ability is not restricted to protective HLA-B haplotypes, does not require proliferation or the addition of exogenous factors, is not restored by HAART, and primarily originates from effector CD8+ T-cells with otherwise limited functional capability. Notably, we found an inverse relationship between HIV-specific perforin expression and viral load. Thus, the capability of HIV-specific CD8+ T-cells to rapidly express perforin defines a novel correlate of control in HIV infection.

Background: HIV-1 infection is associated with profound dysfunction of myeloid dendritic cells, for reasons that remain ill-defined. Soluble HLA class I molecules can have important inhibitory effects on T cells and NK cells, but may also contribute to reduced functional properties of professional antigen-presenting cells. Here, we investigated the expression of soluble HLA class I isoforms during HIV-1 infection and assessed their functional impact on antigen-presenting characteristics of dendritic cells. Results: Soluble HLA class I molecules were highly upregulated in progressive HIV-1 infection as determined by quantitative Western blots. This was associated with strong increases of intracellular expression of HLA class I isoforms in dendritic cells and monocytes. Using mixed lymphocyte reactions, we found that soluble HLA class I molecules effectively inhibited the antigen-presenting properties of dendritic cells, however, there was no significant influence of HLA class I molecules on the cytokine-secretion properties of these cells. The immunomodulatory effects of soluble HLA class I molecules were mediated by interactions with inhibitory myelomonocytic MHC class I receptors from the Leukocyte Immunoglobulin Like Receptor (LILR) family. Conclusions: During progressive HIV-1 infection, soluble HLA class I molecules can contribute to systemic immune dysfunction by inhibiting the antigen-presenting properties of myeloid dendritic cells through interactions with inhibitory myelomonocytic HLA class I receptors.

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.

Cytotoxic T lymphocyte (CTL) responses targeting specific HIV proteins, in particular Gag, have been associated with relative control of viral replication (in) (vivo). However, Gag-specific CTL can also be detected in individuals who do not control the virus and it remains thus unclear how Gag-specific CTL may mediate the beneficial effects in some individuals but not in others. Here, we used a 10mer peptide set spanning HIV Gag-p24 to determine immunogen-specific T-cell responses and to assess functional properties including functional avidity and cross-reactivity in 25 HIV-1 controllers and 25 non-controllers without protective HLA class I alleles. Our data challenge the common belief that Gag-specific T cell responses dominate the virus-specific immunity exclusively in HIV-1 controllers as both groups mounted responses of comparable breadths and magnitudes against the p24 sequence. However, responses in controllers reacted to lower antigen concentrations and recognized more epitope variants than responses in non-controllers. These cross-sectional data, largely independent of particular HLA genetics and generated using direct (ex-vivo) samples thus identify T cell responses of high functional avidity and with broad variant reactivity as potential functional immune correlates of relative HIV control.

Cellular HIV-1 reservoirs that persist despite antiretroviral treatment are incompletely defined. We show that during suppressive antiretroviral therapy, CD4+ T memory stem cells (TSCM) harbor high per-cell levels of HIV-1 DNA, and make increasing contributions to the total viral CD4+ T cell reservoir over time. Moreover, phylogenetic studies suggested long-term persistence of viral quasispecies in CD4+ TSCM cells. Thus, HIV-1 may exploit stem cell characteristics of cellular immune memory to promote long-term viral persistence.