Cellular immune correlates analysis of an HIV-1 preexposure prophylaxis trial.

HIV-1-specific T-cell responses in exposed seronegative subjects suggest that a viral breach of the exposure site is more common than current transmission rates would suggest and that host immunity can extinguish subsequent infection foci. The Preexposure Prophylaxis Initiative (iPrEx) chemoprophylaxis trial provided an opportunity to rigorously investigate these responses in a case-control immunology study; 84 preinfection peripheral blood mononuclear cell samples from individuals enrolled in the iPrEx trial who later seroconverted were matched with 480 samples from enrolled subjects who remained seronegative from both the placebo and active treatment arms. T-cell responses to HIV-1 Gag, Protease, Integrase, Reverse Transcriptase, Vif, and Nef antigens were quantified for all subjects in an IFN-γ enzyme-linked immunospot (ELISpot) assay. IFN-γ responses varied in magnitude and frequency across subjects. A positive response was more prevalent in those who remained persistently HIV-1-negative for Gag (P = 0.007), Integrase (P < 0.001), Vif (P < 0.001), and Nef (P < 0.001). When correlated with outcomes in the iPrEx trial, Vif- and Integrase-specific T-cell responses were associated with reduced HIV-1 infection risk [hazard ratio (HR) = 0.36, 95% confidence interval (95% CI) = 0.19-0.66 and HR = 0.52, 95% CI = 0.28-0.96, respectively]. Antigen-specific responses were independent of emtricitabine/tenofovir disoproxil fumarate use. IFN-γ secretion in the ELISpot was confirmed using multiparametric flow cytometry and largely attributed to effector memory CD4+ or CD8+ T cells. Our results show that HIV-1-specific T-cell immunity can be detected in exposed but uninfected individuals and that these T-cell responses can differentiate individuals according to infection outcomes.

Whole-genome characterization of human and simian immunodeficiency virus intrahost diversity by ultradeep pyrosequencing.

Rapid evolution and high intrahost sequence diversity are hallmarks of human and simian immunodeficiency virus (HIV/SIV) infection. Minor viral variants have important implications for drug resistance, receptor tropism, and immune evasion. Here, we used ultradeep pyrosequencing to sequence complete HIV/SIV genomes, detecting variants present at a frequency as low as 1%. This approach provides a more complete characterization of the viral population than is possible with conventional methods, revealing low-level drug resistance and detecting previously hidden changes in the viral population. While this work applies pyrosequencing to immunodeficiency viruses, this approach could be applied to virtually any viral pathogen.

Conserved HIV-1 Gag p24 Epitopes Elicit Cellular Immune Responses That Impact Disease Outcome.

Although the breadth of the human immunodeficiency virus type 1 (HIV-1)-specific cellular immune response and its impact on the control of viral replication have already been addressed, reported data have proven controversial. We hypothesize that the nature of targeted epitopes, rather than the simple breadth or magnitude of responses, correlates with disease outcome. In this study, we explore the occurrence of patterns of Gag p24 recognition among untreated HIV-1-infected patients by identifying the epitopes that compose such patterns and how they distinctly associate with disease progression. Utilizing enzyme-linked immunospot (ELISPOT) interferon gamma (IFN-γ), we screened cellular responses of 27 HIV-1-infected subjects against 15-mer peptides encompassing the whole Gag p24 protein. Obtained data were used to develop a clustering analysis that allowed definition of two groups of individuals with totally distinct patterns of recognition. Although targeted Gag p24 peptides were completely different between the two groups, the breadth and magnitude of the responses were not. Interestingly, viral control and preservation of CD4+ T cells were increased in one group. In addition, we compared genetic conservation of amino acid sequences of the recognized peptides, as well as of the human leucocyte antigen class I (HLA-I)-restricted epitopes within them. Subjects presenting higher control of HIV-1 replication targeted more conserved epitopes, and higher genetic variation was present mainly in anchor residues for HLA-I molecules. We strengthen the existing evidence from cases of HIV-1 infection in humans that, cellular immune responses targeting conserved epitopes, rather than the magnitude and breadth of responses, associate with a better control of viral replication and maintenance of peripheral CD4+ T cell counts.

Short communication: HIV type 1 subtype BF leads to faster CD4+ T cell loss compared to subtype B.

Although it has been suggested that biological differences among HIV-1 subtypes exist, their possible influence on disease progression has not been fully revealed. In particular, the increasing emergence of recombinants stresses the need to characterize disease presentation in persons infected by these diverse HIV-1 forms. We explored this issue among 83 Brazilian subjects infected with either HIV-1 subtype B or recombinant subtype BF, all followed since incident infection in a cohort study. Viral subtypes were assigned by full length sequencing of HIV-1 genomes. We observed that the baseline measures for CD4(+) T cells and viral load did not differ between the groups. However, longitudinal analysis revealed that subtype BF was clearly associated with a faster CD4(+) T cell decline compared to infection with subtype B, in spite of a similar plasma HIV-1 load. While subtype B-infected subjects presented a loss of 3.6 CD4(+) T cells/µl per month, subtype BF-infected individuals showed a monthly decay of 6.3 CD4(+) T cells/µl (p<0.01). The time to reach 350 CD4(+) T cells/µl and the time to start antiretroviral treatment were also shorter in subtype BF-infected persons. The elucidation of an accelerated CD4(+) T cell loss associated with subtype BF suggests that this HIV-1 genetic form could be more pathogenic than subtype B.

Low-cost ultra-wide genotyping using Roche/454 pyrosequencing for surveillance of HIV drug resistance.

BACKGROUND: Great efforts have been made to increase accessibility of HIV antiretroviral therapy (ART) in low and middle-income countries. The threat of wide-scale emergence of drug resistance could severely hamper ART scale-up efforts. Population-based surveillance of transmitted HIV drug resistance ensures the use of appropriate first-line regimens to maximize efficacy of ART programs where drug options are limited. However, traditional HIV genotyping is extremely expensive, providing a cost barrier to wide-scale and frequent HIV drug resistance surveillance. METHODS/RESULTS: We have developed a low-cost laboratory-scale next-generation sequencing-based genotyping method to monitor drug resistance. We designed primers specifically to amplify protease and reverse transcriptase from Brazilian HIV subtypes and developed a multiplexing scheme using multiplex identifier tags to minimize cost while providing more robust data than traditional genotyping techniques. Using this approach, we characterized drug resistance from plasma in 81 HIV infected individuals collected in São Paulo, Brazil. We describe the complexities of analyzing next-generation sequencing data and present a simplified open-source workflow to analyze drug resistance data. From this data, we identified drug resistance mutations in 20% of treatment naïve individuals in our cohort, which is similar to frequencies identified using traditional genotyping in Brazilian patient samples. CONCLUSION: The developed ultra-wide sequencing approach described here allows multiplexing of at least 48 patient samples per sequencing run, 4 times more than the current genotyping method. This method is also 4-fold more sensitive (5% minimal detection frequency vs. 20%) at a cost 3-5× less than the traditional Sanger-based genotyping method. Lastly, by using a benchtop next-generation sequencer (Roche/454 GS Junior), this approach can be more easily implemented in low-resource settings. This data provides proof-of-concept that next-generation HIV drug resistance genotyping is a feasible and low-cost alternative to current genotyping methods and may be particularly beneficial for in-country surveillance of transmitted drug resistance.

Decay kinetics of HIV-1 specific T cell responses in vertically HIV-1 exposed seronegative infants.

OBJECTIVE: The majority of infants born, in developed countries, to HIV-1 positive women are exposed to the HIV-1 virus in utero or peri/post-partum, but are born uninfected. We, and others, have previously shown HIV-1 specific T cell responses in HIV-1 exposed seronegative (HESN) neonates/infants. Our objective in this study was to examine the rate of decay in their HIV-1 specific T cell response over time from birth. DESIGN: Cross-sectional and longitudinal studies of HIV-1 specific T cell responses in HESN infants were performed. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from 18 HIV-1 DNA PCR negative infants born to HIV-1 infected mothers receiving care at the Jacobi Medical Center, Bronx, NY, USA. PBMC were examined for T cell responses to HIV-1 antigens by interferon-gamma (IFN-γ) ELISPOT. RESULTS: PBMC from 15 HESN neonates/infants were analyzed. We observed a decay of HIV-1 specific T cell responses from birth at a rate of -0.599 spot forming unit/106 cells per day, with a median half-life decay rate of 21.38 weeks (13.39-115.8). CONCLUSION: Our results support the dynamic nature of T cell immunity in the context of a developing immune system. The disparate rate of decay with studies of adults placed on antiretroviral drugs suggests that antigen specific T cell responses are driven by the natural rate of decay of the T cell sub-populations themselves.

Unexpected diversity of cellular immune responses against Nef and Vif in HIV-1-infected patients who spontaneously control viral replication.

BACKGROUND: HIV-1-infected individuals who spontaneously control viral replication represent an example of successful containment of the AIDS virus. Understanding the anti-viral immune responses in these individuals may help in vaccine design. However, immune responses against HIV-1 are normally analyzed using HIV-1 consensus B 15-mers that overlap by 11 amino acids. Unfortunately, this method may underestimate the real breadth of the cellular immune responses against the autologous sequence of the infecting virus. METHODOLOGY AND PRINCIPAL FINDINGS: Here we compared cellular immune responses against nef and vif-encoded consensus B 15-mer peptides to responses against HLA class I-predicted minimal optimal epitopes from consensus B and autologous sequences in six patients who have controlled HIV-1 replication. Interestingly, our analysis revealed that three of our patients had broader cellular immune responses against HLA class I-predicted minimal optimal epitopes from either autologous viruses or from the HIV-1 consensus B sequence, when compared to responses against the 15-mer HIV-1 type B consensus peptides. CONCLUSION AND SIGNIFICANCE: This suggests that the cellular immune responses against HIV-1 in controller patients may be broader than we had previously anticipated.