HLA-A*23 Is Associated With Lower Odds of Acute Retroviral Syndrome in Human Immunodeficiency Virus Type 1 Infection: A Multicenter Sub-Saharan African Study.

The role of human leukocyte antigen (HLA) class I and killer immunoglobulin-like receptor molecules in mediating acute retroviral syndrome (ARS) during human immunodeficiency virus type 1 (HIV-1) infection is unclear. Among 72 sub-Saharan African adults, HLA-A*23 was associated with lower odds of ARS (adjusted odds ratio, 0.10 [95% confidence interval, .01-.48]; P = .009), which warrants further studies to explore its role on HIV-1-specific immunopathogenesis.

Mosaic HIV-1 vaccination induces anti-viral CD8+ T cell functionality in the phase 1/2a clinical trial APPROACH.

IMPORTANCE: The functionality of CD8+ T cells against human immunodeficiency virus-1 (HIV-1) antigens is indicative of HIV-progression in both animal models and people living with HIV. It is, therefore, of interest to assess CD8+ T cell responses in a prophylactic vaccination setting, as this may be an important component of the immune system that inhibits HIV-1 replication. T cell responses induced by the adenovirus serotype 26 (Ad26) mosaic vaccine regimen were assessed previously by IFN-γ ELISpot and flow cytometric assays, yet these assays only measure cytokine production but not the capacity of CD8+ T cells to inhibit replication of HIV-1. In this study, we demonstrate direct anti-viral function of the clinical Ad26 mosaic vaccine regimen through ex vivo inhibition of replication of diverse clades of HIV-1 isolates in the participant's own CD4+ T cells.

Cohort-Specific Peptide Reagents Broaden Depth and Breadth Estimates of the CD8 T Cell Response to HIV-1 Gag Potential T Cell Epitopes.

An effective HIV vaccine will need to stimulate immune responses against the sequence diversity presented in circulating virus strains. In this study, we evaluate breadth and depth estimates of potential T-cell epitopes (PTEs) in transmitted founder virus sequence-derived cohort-specific peptide reagents against reagents representative of consensus and global sequences. CD8 T-cells from twenty-six HIV-1+ PBMC donor samples, obtained at 1-year post estimated date of infection, were evaluated. ELISpot assays compared responses to 15mer consensus (n = 121), multivalent-global (n = 320), and 10mer multivalent cohort-specific (n = 300) PTE peptides, all mapping to the Gag antigen. Responses to 38 consensus, 71 global, and 62 cohort-specific PTEs were confirmed, with sixty percent of common global and cohort-specific PTEs corresponding to consensus sequences. Both global and cohort-specific peptides exhibited broader epitope coverage compared to commonly used consensus reagents, with mean breadth estimates of 3.2 (global), 3.4 (cohort) and 2.2 (consensus) epitopes. Global or cohort peptides each identified unique epitope responses that would not be detected if these peptide pools were used alone. A peptide set designed around specific virologic and immunogenetic characteristics of a target cohort can expand the detection of CD8 T-cell responses to epitopes in circulating viruses, providing a novel way to better define the host response to HIV-1 with implications for vaccine development.

Assessment of a diverse panel of transmitted/founder HIV-1 infectious molecular clones in a luciferase based CD8 T-cell mediated viral inhibition assay.

Introduction: Immunological protection against human immunodeficiency virus-1 (HIV-1) infection is likely to require both humoral and cell-mediated immune responses, the latter involving cytotoxic CD8 T-cells. Characterisation of CD8 T-cell mediated direct anti-viral activity would provide understanding of potential correlates of immune protection and identification of critical epitopes associated with HIV-1 control. Methods: The present report describes a functional viral inhibition assay (VIA) to assess CD8 T-cell-mediated inhibition of replication of a large and diverse panel of 45 HIV-1 infectious molecular clones (IMC) engineered with a Renilla reniformis luciferase reporter gene (LucR), referred to as IMC-LucR. HIV-1 IMC replication in CD4 T-cells and CD8 T-cell mediated inhibition was characterised in both ART naive subjects living with HIV-1 covering a broad human leukocyte antigen (HLA) distribution and compared with uninfected subjects. Results & discussion: CD4 and CD8 T-cell lines were established from subjects vaccinated with a candidate HIV-1 vaccine and provided standard positive controls for both assay quality control and facilitating training and technology transfer. The assay was successfully established across 3 clinical research centres in Kenya, Uganda and the United Kingdom and shown to be reproducible. This IMC-LucR VIA enables characterisation of functional CD8 T-cell responses providing a tool for rational T-cell immunogen design of HIV-1 vaccine candidates and evaluation of vaccine-induced T-cell responses in HIV-1 clinical trials.

Infection with HIV-1 subtype D among acutely infected Ugandans is associated with higher median concentration of cytokines compared to subtype A.

Objective: The observation that HIV-1 subtype D progresses faster to disease than subtype A prompted us to examine cytokine levels early after infection within the predominant viral subtypes that circulate in Uganda and address the following research questions: (1) Do cytokine levels vary between subtypes A1 and D? (2) Do cytokine profiles correlate with disease outcomes? Methods: To address these questions, HIV-1 subtypes were determined by population sequencing of the HIV-1 pol gene and 37 plasma cytokine concentrations were evaluated using V-Plex kits on Meso Scale Discovery platform in 65 recent sero-converters. Results: HIV-1 subtype D (pol) infections exhibited significantly higher median plasma concentrations of IL-5, IL-16, IL-1α, IL-7, IL-17A, CCL11 (Eotaxin-1), CXCL10 (IP-10), CCL13 (MCP-4) and VEGF-D compared to subtype A1 (pol) infections. We also found that IL-12/23p40 and IL-1α were associated with faster CD4+T cell count decline, while bFGF was associated with maintenance of CD4+ counts above 350 cells/microliter. Conclusion: Our results suggest that increased production of cytokines in early HIV infection may trigger a disruption of the immune environment and contribute to pathogenic mechanisms underlying the accelerated disease progression seen in individuals infected with HIV-1 subtype D in Uganda.

Characterization of Near Full-Length Transmitted/Founder HIV-1 Subtype D and A/D Recombinant Genomes in a Heterosexual Ugandan Population (2006-2011).

Detailed characterization of transmitted HIV-1 variants in Uganda is fundamentally important to inform vaccine design, yet studies on the transmitted full-length strains of subtype D viruses are limited. Here, we amplified single genomes and characterized viruses, some of which were previously classified as subtype D by sub-genomic pol sequencing that were transmitted in Uganda between December 2006 to June 2011. Analysis of 5' and 3' half genome sequences showed 73% (19/26) of infections involved single virus transmissions, whereas 27% (7/26) of infections involved multiple variant transmissions based on predictions of a model of random virus evolution. Subtype analysis of inferred transmitted/founder viruses showed a high transmission rate of inter-subtype recombinants (69%, 20/29) involving mainly A1/D, while pure subtype D variants accounted for one-third of infections (31%, 9/29). Recombination patterns included a predominance of subtype D in the gag/pol region and a highly recombinogenic envelope gene. The signal peptide-C1 region and gp41 transmembrane domain (Tat2/Rev2 flanking region) were hotspots for A1/D recombination events. Analysis of a panel of 14 transmitted/founder molecular clones showed no difference in replication capacity between subtype D viruses (n = 3) and inter-subtype mosaic recombinants (n = 11). However, individuals infected with high replication capacity viruses had a faster CD4 T cell loss. The high transmission rate of unique inter-subtype recombinants is striking and emphasizes the extraordinary challenge for vaccine design and, in particular, for the highly variable and recombinogenic envelope gene, which is targeted by rational designs aimed to elicit broadly neutralizing antibodies.

Breadth of CD8 T-cell mediated inhibition of replication of diverse HIV-1 transmitted-founder isolates correlates with the breadth of recognition within a comprehensive HIV-1 Gag, Nef, Env and Pol potential T-cell epitope (PTE) peptide set.

Full characterisation of functional HIV-1-specific T-cell responses, including identification of recognised epitopes linked with functional antiviral responses, would aid development of effective vaccines but is hampered by HIV-1 sequence diversity. Typical approaches to identify T-cell epitopes utilising extensive peptide sets require subjects' cell numbers that exceed feasible sample volumes. To address this, CD8 T-cells were polyclonally expanded from PBMC from 13 anti-retroviral naïve subjects living with HIV using CD3/CD4 bi-specific antibody. Assessment of recognition of individual peptides within a set of 1408 HIV-1 Gag, Nef, Pol and Env potential T-cell epitope peptides was achieved by sequential IFNγ ELISpot assays using peptides pooled in 3-D matrices followed by confirmation with single peptides. A Renilla reniformis luciferase viral inhibition assay assessed CD8 T-cell-mediated inhibition of replication of a cross-clade panel of 10 HIV-1 isolates, including 9 transmitted-founder isolates. Polyclonal expansion from one frozen PBMC vial provided sufficient CD8 T-cells for both ELISpot steps in 12 of 13 subjects. A median of 33 peptides in 16 epitope regions were recognised including peptides located in previously characterised HIV-1 epitope-rich regions. There was no significant difference between ELISpot magnitudes for in vitro expanded CD8 T-cells and CD8 T-cells directly isolated from PBMCs. CD8 T-cells from all subjects inhibited a median of 7 HIV-1 isolates (range 4 to 10). The breadth of CD8 T-cell mediated HIV-1 inhibition was significantly positively correlated with CD8 T-cell breadth of peptide recognition. Polyclonal CD8 T-cell expansion allowed identification of HIV-1 isolates inhibited and peptides recognised within a large peptide set spanning the major HIV-1 proteins. This approach overcomes limitations associated with obtaining sufficient cell numbers to fully characterise HIV-1-specific CD8 T-cell responses by different functional readouts within the context of extreme HIV-1 diversity. Such an approach will have useful applications in clinical development for HIV-1 and other diseases.

Increased Frequency of Inter-Subtype HIV-1 Recombinants Identified by Near Full-Length Virus Sequencing in Rwandan Acute Transmission Cohorts.

Most studies of HIV-1 transmission have focused on subtypes B and C. In this study, we determined the genomic sequences of the transmitted founder (TF) viruses from acutely infected individuals enrolled between 2005 and 2011 into IAVI protocol C in Rwanda and have compared these isolates to viruses from more recent (2016-2019) acute/early infections in three at risk populations - MSM, high risk women (HRW), and discordant couples (DC). For the Protocol C samples, we utilized near full-length single genome (NFLG) amplification to generate 288 HIV-1 amplicons from 26 acutely infected seroconverters (SC), while for the 21 recent seroconverter samples (13 from HRW, two from DC, and six from MSM), we PCR amplified overlapping half-genomes. Using PacBio SMRT technology combined with the MDPseq workflow, we performed multiplex sequencing to obtain high accuracy sequences for each amplicon. Phylogenetic analyses indicated that the majority of recent transmitted viruses from DC and HRW clustered within those of the earlier Protocol C cohort. However, five of six sequences from the MSM cohort branched together and were greater than 97% identical. Recombination analyses revealed a high frequency (6/26; 23%) of unique inter-subtype recombination in Protocol C with 19% AC and 4% CD recombinant viruses, which contrasted with only 6.5% of recombinants defined by sequencing of the pol gene previously. The frequency of recombinants was significantly higher (12/21; 57%) in the more recent isolates, although, the five related viruses from the MSM cohort had identical recombination break points. While major drug resistance mutations were absent from Protocol C viruses, 4/21 of recent isolates exhibited transmitted nevirapine resistance. These results demonstrate the ongoing evolution and increased prevalence of recombinant and drug resistant transmitted viruses in Rwanda and highlight the importance of defining NFLG sequences to fully understand the nature of TF viruses and in particular the prevalence of unique recombinant forms (URFs) in transmission cohorts.

Direct identification of HLA-presented CD8 T cell epitopes from transmitted founder HIV-1 variants.

Cytotoxic T lymphocytes (CTLs) are a critical arm of the immune response to viral infections. The activation and expansion of antigen specific CTL requires recognition of peptide antigens presented on class I major histocompatibility complex molecules (MHC-1) of infected cells. Methods to identify presented peptide antigens that do not rely on the pre-existence of antigen specific CTL are critical to the development of new vaccines. We infected activated CD4+ T cells with two HIV-1 transmitted founder (TF) isolates and used high-resolution mass spectrometry (MS) to identify HIV peptides bound on MHC-1. Using this approach, we identified 14 MHC-1 bound peptides from across the two TF isolates. Assessment of predicted binding thresholds revealed good association of the identified peptides to the shared HLA alleles between the HIV+ donors and the naïve PBMC sample with three peptides identified through peptide sequencing inducing a CD8 T-cell response (p < 0.05). Direct infection of naïve CD4 cells by HIV TF isolates and sequencing of MHC-I presented peptides by HPLC-MS/MS enables identification of novel peptides that may be missed by alternative epitope mapping strategies and can provide valuable insight in to the first peptides presented by an HIV-infected CD4 cell in the first few days post infection.

Selective HLA restriction enables the evaluation and interpretation of immunogenic breadth at comparable levels to that observed with broader HLA distribution.

Existing approaches to identifying predictive T-cell epitopes have traditionally utilized either 2-digit HLA super-families or more commonly utilizing autologous HLA alleles to facilitate the predictions. However, the use of these criteria may not consider the HLA representation within any target population. Here we propose a modification to concept of utilizing autologous HLA whereby subsets of individuals are selected for their specific HLA allele profiles and the representation they provide within a given population. Using this selective approach to HLA selection and the linkages to specific individuals may enable the design of more targeted experimentalstrategies.

Subtype-specific differences in Gag-protease replication capacity of HIV-1 isolates from East and West Africa.

BACKGROUND: The HIV-1 epidemic in sub-Saharan Africa is heterogeneous with diverse unevenly distributed subtypes and regional differences in prevalence. Subtype-specific differences in disease progression rate and transmission efficiency have been reported, but the underlying biological mechanisms have not been fully characterized. Here, we tested the hypothesis that the subtypes prevalent in the East Africa, where adult prevalence rate is higher, have lower viral replication capacity (VRC) than their West African counterparts where adult prevalence rates are lower. RESULTS: Gag-protease sequencing was performed on 213 and 160 antiretroviral-naïve chronically infected participants from West and East Africa respectively and bioinformatic tools were used to infer subtypes and recombination patterns. VRC of patient-derived gag-protease chimeric viruses from West (n = 178) and East (n = 114) Africa were determined using a green fluorescent protein reporter-based cell assay. Subtype and regional differences in VRC and amino acid variants impacting VRC were identified by statistical methods. CRF02_AG (65%, n = 139), other recombinants (14%, n = 30) and pure subtypes (21%, n = 44) were identified in West Africa. Subtypes A1 (64%, n = 103), D (22%, n = 35), or recombinants (14%, n = 22) were identified in East Africa. Viruses from West Africa had significantly higher VRC compared to those from East Africa (p < 0.0001), with subtype-specific differences found among strains within West and East Africa (p < 0.0001). Recombination patterns showed a preference for subtypes D, G or J rather than subtype A in the p6 region of gag, with evidence that subtype-specific differences in this region impact VRC. Furthermore, the Gag A83V polymorphism was associated with reduced VRC in CRF02_AG. HLA-A*23:01 (p = 0.0014) and HLA-C*07:01 (p = 0.002) were associated with lower VRC in subtype A infected individuals from East Africa. CONCLUSIONS: Although prevalent viruses from West Africa displayed higher VRC than those from East Africa consistent with the hypothesis that lower VRC is associated with higher population prevalence, the predominant CRF02_AG strain in West Africa displayed higher VRC than other prevalent strains suggesting that VRC alone does not explain population prevalence. The study identified viral and host genetic determinants of virus replication capacity for HIV-1 CRF02_AG and subtype A respectively, which may have relevance for vaccine strategies.

Performance of International AIDS Vaccine Initiative African clinical research laboratories in standardised ELISpot and peripheral blood mononuclear cell processing in support of HIV vaccine clinical trials.

BACKGROUND: Standardisation of procedures for performing cellular functional assays across laboratories participating in multicentre clinical trials is key for generating comparable and reliable data. OBJECTIVE: This article describes the performance of accredited laboratories in Africa and Europe on testing done in support of clinical trials. METHODS: For enzyme-linked immunospot assay (ELISpot) proficiency, characterised peripheral blood mononuclear cells (PBMCs) obtained from 48 HIV-negative blood donors in Johannesburg, South Africa, were sent to participating laboratories between February 2010 and February 2014. The PBMCs were tested for responses against cytomegalovirus, Epstein Barr and influenza peptide pools in a total of 1751 assays. In a separate study, a total of 1297 PBMC samples isolated from healthy HIV-negative participants in clinical trials of two prophylactic HIV vaccine candidates in Kenya, Uganda, Rwanda and Zambia were analysed for cell viability, cell yield and cell recovery from frozen PBMCs. RESULTS: Most (99%) of the 1751 ELISpot proficiency assays had data within acceptable ranges with low responses to mock stimuli. No significant statistical difference were observed in ELISpot responses at the five laboratories actively conducting immunological analyses. Of the 1297 clinical trial PBMCs processed, 94% had cell viability above 90% and 96% had cell yield above 0.7 million per mL of blood in freshly isolated cells. All parameters were within the predefined acceptance criteria. CONCLUSION: We demonstrate that multiple laboratories can generate reliable, accurate and comparable data by using standardised procedures, having regular training, having regular equipment maintenance and using centrally sourced reagents.

A Novel Sample Selection Approach to Aid the Identification of Factors That Correlate With the Control of HIV-1 Infection.

Individuals infected with HIV display varying rates of viral control and disease progression, with a small percentage of individuals being able to spontaneously control infection in the absence of treatment. In attempting to define the correlates associated with natural protection against HIV, extreme heterogeneity in the datasets generated from systems methodologies can be further complicated by the inherent variability encountered at the population, individual, cellular and molecular levels. Furthermore, such studies have been limited by the paucity of well-characterised samples and linked epidemiological data, including duration of infection and clinical outcomes. To address this, we selected 10 volunteers who rapidly and persistently controlled HIV, and 10 volunteers each, from two control groups who failed to control (based on set point viral loads) from an acute and early HIV prospective cohort from East and Southern Africa. A propensity score matching approach was applied to control for the influence of five factors (age, risk group, virus subtype, gender, and country) known to influence disease progression on causal observations. Fifty-two plasma proteins were assessed at two timepoints in the 1st year of infection. We independently confirmed factors known to influence disease progression such as the B*57 HLA Class I allele, and infecting virus Subtype. We demonstrated associations between circulating levels of MIP-1α and IL-17C, and the ability to control infection. IL-17C has not been described previously within the context of HIV control, making it an interesting target for future studies to understand HIV infection and transmission. An in-depth systems analysis is now underway to fully characterise host, viral and immunological factors contributing to control.

Utilizing Computational Machine Learning Tools to Understand Immunogenic Breadth in the Context of a CD8 T-Cell Mediated HIV Response.

Predictive models are becoming more and more commonplace as tools for candidate antigen discovery to meet the challenges of enabling epitope mapping of cohorts with diverse HLA properties. Here we build on the concept of using two key parameters, diversity metric of the HLA profile of individuals within a population and consideration of sequence diversity in the context of an individual's CD8 T-cell immune repertoire to assess the HIV proteome for defined regions of immunogenicity. Using this approach, analysis of HLA adaptation and functional immunogenicity data enabled the identification of regions within the proteome that offer significant conservation, HLA recognition within a population, low prevalence of HLA adaptation and demonstrated immunogenicity. We believe this unique and novel approach to vaccine design as a supplement to vitro functional assays, offers a bespoke pipeline for expedited and rational CD8 T-cell vaccine design for HIV and potentially other pathogens with the potential for both global and local coverage.

Elevated levels of inflammatory plasma biomarkers are associated with risk of HIV infection.

BACKGROUND: To determine if individuals, from HIV-1 serodiscordant couple cohorts from Rwanda and Zambia, who become HIV-positive have a distinct inflammatory biomarker profile compared to individuals who remain HIV-negative, we compared levels of biomarkers in plasma of HIV-negative individuals who either seroconverted (pre-infection) and became HIV-positive or remained HIV-negative (uninfected). RESULTS: We observed that individuals in the combined cohort, as well as those in the individual country cohorts, who later became HIV-1 infected had significantly higher baseline levels of multiple inflammatory cytokines/chemokines compared to individuals who remained HIV-negative. Genital inflammation/ulceration or schistosome infections were not associated with this elevated profile. Defined levels of ITAC and IL-7 were significant predictors of later HIV acquisition in ROC predictive analyses, whereas the classical Th1 and Th2 inflammatory cytokines such as IL-12 and interferon-γ or IL-4, IL-5 and Il-13 were not. CONCLUSIONS: Overall, the data show a significant association between increased plasma biomarkers linked to inflammation and immune activation and HIV acquisition and suggests that pre-existing conditions that increase systemic biomarkers represent a factor for increased risk of HIV infection.

HIV-1 infection and the lack of viral control are associated with greater expression of interleukin-21 receptor on CD8+ T cells.

OBJECTIVES: Interleukin-21 (IL-21) has been linked with the generation of virus-specific memory CD8+ T cells following acute infection with HIV-1 and reduced exhaustion of CD8+ T cells. IL-21 has also been implicated in the promotion of CD8+ T-cell effector functions during viral infection. Little is known about the expression of interleukin-21 receptor (IL-21R) during HIV-1 infection or its role in HIV-1-specific CD8+ T-cell maintenance and subsequent viral control. METHODS: We compared levels of IL-21R expression on total and memory subsets of CD8+ T cells from HIV-1-negative and HIV-1-positive donors. We also measured IL-21R on antigen-specific CD8+ T cells in volunteers who were positive for HIV-1 and had cytomegalovirus-responding T cells. Finally, we quantified plasma IL-21 in treatment-naive HIV-1-positive individuals and compared this with IL-21R expression. RESULTS: IL-21R expression was significantly higher on CD8+ T cells (P = 0.0256), and on central memory (P = 0.0055) and effector memory (P = 0.0487) CD8+ T-cell subsets from HIV-1-positive individuals relative to HIV-1-negative individuals. For those infected with HIV-1, the levels of IL-21R expression on HIV-1-specific CD8+ T cells correlated significantly with visit viral load (r = 0.6667, P = 0.0152, n = 13) and inversely correlated with plasma IL-21 (r = -0.6273, P = 0.0440, n = 11). Lastly, CD8+ T cells from individuals with lower set point viral load who demonstrated better viral control had the lowest levels of IL-21R expression and highest levels of plasma IL-21. CONCLUSION: Our data demonstrates significant associations between IL-21R expression on peripheral CD8+ T cells and viral load, as well as disease trajectory. This suggests that the IL-21 receptor could be a novel marker of CD8+ T-cell dysfunction during HIV-1 infection.

A Stronger Innate Immune Response During Hyperacute Human Immunodeficiency Virus Type 1 (HIV-1) Infection Is Associated With Acute Retroviral Syndrome.

BACKGROUND: Acute retroviral syndrome (ARS) is associated with human immunodeficiency virus type 1 (HIV-1) subtype and disease progression, but the underlying immunopathological pathways are poorly understood. We aimed to elucidate associations between innate immune responses during hyperacute HIV-1 infection (hAHI) and ARS. METHODS: Plasma samples obtained from volunteers (≥18.0 years) before and during hAHI, defined as HIV-1 antibody negative and RNA or p24 antigen positive, from Kenya, Rwanda, Uganda, Zambia, and Sweden were analyzed. Forty soluble innate immune markers were measured using multiplexed assays. Immune responses were differentiated into volunteers with stronger and comparatively weaker responses using principal component analysis. Presence or absence of ARS was defined based on 11 symptoms using latent class analysis. Logistic regression was used to determine associations between immune responses and ARS. RESULTS: Of 55 volunteers, 31 (56%) had ARS. Volunteers with stronger immune responses (n = 36 [65%]) had increased odds of ARS which was independent of HIV-1 subtype, age, and risk group (adjusted odds ratio, 7.1 [95% confidence interval {CI}: 1.7-28.8], P = .003). Interferon gamma-induced protein (IP)-10 was 14-fold higher during hAHI, elevated in 7 of the 11 symptoms and independently associated with ARS. IP-10 threshold >466.0 pg/mL differentiated stronger immune responses with a sensitivity of 84.2% (95% CI: 60.4-96.6) and specificity of 100.0% (95% CI]: 90.3-100.0). CONCLUSIONS: A stronger innate immune response during hAHI was associated with ARS. Plasma IP-10 may be a candidate biomarker of stronger innate immunity. Our findings provide further insights on innate immune responses in regulating ARS and may inform the design of vaccine candidates harnessing innate immunity.

Comprehensive epitope mapping using polyclonally expanded human CD8 T cells and a two-step ELISpot assay for testing large peptide libraries.

The genetic diversity of circulating HIV-1 strains poses a major barrier to the design, development and evaluation of HIV-1 vaccines. The assessment of both vaccine- and natural infection-elicited T cell responses is commonly done with multivalent peptides that are designed to maximally capture the diversity of potential T cell epitopes (PTEs) observed in natural circulating sequences. However, depending on the sequence diversity of viral subtypes and number of the HIV immunogens under investigation, PTE estimates, including HLA-guided computational methods, can easily generate enormous peptide libraries. Evaluation of T cell epitope specificity using such extensive peptide libraries is usually limited by sample availability, even for high-throughput and robust epitope mapping techniques like ELISpot assays. Here we describe a novel, two-step protocol for in-vitro polyclonal expansion of CD8 T cells from a single vial of frozen PBMC, which facilitated the screening 441 HIV-1 Gag peptides for immune responses among 32 HIV-1 positive subjects and 40 HIV-1 negative subjects for peptide qualification. Using a pooled-peptide mapping strategy, epitopes were mapped in two sequential ELISpot assays; the first ELISpot screened 33 large peptide pools using CD8 T cells expanded for 7 days, while the second step tested pool-matrix peptides to identify individual peptides using CD8 T cells expanded for 10 days. This comprehensive epitope screening established the breadth and magnitude of HIV-1 Gag-specific CD8 T cells and further revealed the extent of immune responses to variable/polymorphic epitopes.

HIV-1 Gag-Pol Sequences from Ugandan Early Infections Reveal Sequence Variants Associated with Elevated Replication Capacity.

The ability to efficiently establish a new infection is a critical property for human immunodeficiency virus type 1 (HIV-1). Although the envelope protein of the virus plays an essential role in receptor binding and internalization of the infecting virus, the structural proteins, the polymerase and the assembly of new virions may also play a role in establishing and spreading viral infection in a new host. We examined Ugandan viruses from newly infected patients and focused on the contribution of the Gag-Pol genes to replication capacity. A panel of Gag-Pol sequences generated using single genome amplification from incident HIV-1 infections were cloned into a common HIV-1 NL4.3 pol/env backbone and the influence of Gag-Pol changes on replication capacity was monitored. Using a novel protein domain approach, we then documented diversity in the functional protein domains across the Gag-Pol region and identified differences in the Gag-p6 domain that were frequently associated with higher in vitro replication.