Timing Constraints of In Vivo Gag Mutations during Primary HIV-1 Subtype C Infection

Background: Aiming to answer the broad question “When does mutation occur?” this study examined the time of appearance, dominance, and completeness of in vivo Gag mutations in primary HIV-1 subtype C infection. Methods: A primary HIV-1C infection cohort comprised of 8 acutely and 34 recently infected subjects were followed frequently up to 500 days post-seroconversion (p/s). Gag mutations were analyzed by employing single-genome amplification and direct sequencing. Gag mutations were determined in relation to the estimated time of seroconversion. Time of appearance, dominance, and completeness was compared for different types of in vivo Gag mutations. Results: Reverse mutations to the wild type appeared at a median (IQR) of 62 (44;139) days p/s, while escape mutations from the wild type appeared at 234 (169;326) days p/s (p<0.001). Within the subset of mutations that became dominant, reverse and escape mutations appeared at 54 (30;78) days p/s and 104 (47;198) days p/s, respectively (p<0.001). Among the mutations that reached completeness, reverse and escape mutations appeared at 54 (30;78) days p/s and 90 (44;196) days p/s, respectively (p = 0.006). Time of dominance for reverse mutations to and escape mutations from the wild type was 58 (44;105) days p/s and 219 (90;326) days p/s, respectively (p<0.001). Time of completeness for reverse and escape mutations was 152 (100;176) days p/s and 243 (101;370) days p/s, respectively (p = 0.001). Fitting a Cox proportional hazards model with frailties confirmed a significantly earlier time of appearance (hazard ratio (HR): 2.6; 95% CI: 2.3–3.0), dominance (4.8 (3.4–6.8)), and completeness (3.6 (2.3–5.5)) of reverse mutations to the wild type Gag than escape mutations from the wild type. Some complex mutational pathways in Gag included sequential series of reversions and escapes. Conclusions: The study identified the timing of different types of in vivo Gag mutations in primary HIV-1 subtype C infection in relation to the estimated time of seroconversion. Overall, the in vivo reverse mutations to the wild type occurred significantly earlier than escape mutations from the wild type. This shorter time to incidence of reverse mutations remained in the subsets of in vivo Gag mutations that reached dominance or completeness

Dynamic correlation between CTL response and viral load in primary human immunodeficiency virus-1 infected Koreans

<p>Abstract</p> <p>Background</p> <p>HIV-1 specific cytotoxic T lymphocytes (CTLs) have an important role as antiviral effector cells for controlling HIV-1 infection.</p> <p>Methods</p> <p>To investigate CTL response during the early stage of HIV infection, we measured immunity-related factors including CD4⁺T cell counts, CD8⁺T cell counts, HIV-1 RNA viral loads and IFN-γ secretion according to CTL response in 78 selected primary HIV-1-infected Koreans.</p> <p>Results</p> <p>The CTL response was strongly induced by HIV-1 specific Gag and Nef peptides (p = 0.016) compared with induction by Tat or Env peptides. These results suggest that the major antiviral factors inducing strong HIV-specific CTL responses are associated with the Gag and Nef viral regions in primary HIV-1 infected Koreans. The relationship between viral load and CTL response showed varying correlations with time following HIV infection. CTL response was inversely correlated with viral loads at preseroconversion stage I (r = -0.224 to -0.33) and changed to a positive correlation at the preseroconversion stage II (r = 0.132 to 0.854). Finally, it changed to an inverse correlation again after seroconversion until a viral set point was established on serological profiling (r = -0.195 to -0.407).</p> <p>Conclusions</p> <p>These findings demonstrate a dynamic correlation between viral load and subsequent CTL responses during early HIV infection.</p

Unique CRF01_AE Gag CTL Epitopes Associated with Lower HIV-Viral Load and Delayed Disease Progression in a Cohort of HIV-Infected Thais

Cytotoxic T Lymphocytes (CTLs) play a central role in controlling HIV-replication. Although numerous CTL epitopes have been described, most are in subtype B or C infection. Little is known about CTL responses in CRF01_AE infection. Gag CTL responses were investigated in a cohort of 137 treatment-naïve HIV-1 infected Thai patients with high CD4+ T cell counts, using gIFN Enzyme-Linked Immunospot (ELISpot) assays with 15-mer overlapping peptides (OLPs) derived from locally dominant CRF01_AE Gag sequences. 44 OLPs were recognized in 112 (81.8%) individuals. Both the breadth and magnitude of the CTL response, particularly against the p24 region, positively correlated with CD4+ T cell count and inversely correlated with HIV viral load. The breadth of OLP response was also associated with slower progression to antiretroviral therapy initiation. Statistical analysis and single peptide ELISpot assay identified at least 17 significant associations between reactive OLP and HLA in 12 OLP regions; 6 OLP-HLA associations (35.3%) were not compatible with previously reported CTL epitopes, suggesting that these contained new CTL Gag epitopes. A substantial proportion of CTL epitopes in CRF01_AE infection differ from subtype B or C. However, the pattern of protective CTL responses is similar; Gag CTL responses, particularly against p24, control viral replication and slow clinical progression

Magnitude and Complexity of Rectal Mucosa HIV-1-Specific CD8+ T-Cell Responses during Chronic Infection Reflect Clinical Status

The intestinal mucosa displays robust virus replication and pronounced CD4+ T-cell loss during acute human immunodeficiency virus type 1 (HIV-1) infection. The ability of HIV-specific CD8+ T-cells to modulate disease course has prompted intensive study, yet the significance of virus-specific CD8+ T-cells in mucosal sites remains unclear.We evaluated five distinct effector functions of HIVgag-specific CD8+ T-cells in rectal mucosa and blood, individually and in combination, in relationship to clinical status and antiretroviral therapy (ART). In subjects not on ART, the percentage of rectal Gag-specific CD8+ T-cells capable of 3, 4 or 5 simultaneous effector functions was significantly related to blood CD4 count and inversely related to plasma viral load (PVL) (p<0.05). Polyfunctional rectal CD8+ T-cells expressed higher levels of MIP-1beta and CD107a on a per cell basis than mono- or bifunctional cells. The production of TNFalpha, IFN-gamma, and CD107a by Gag-specific rectal CD8+ T-cells each correlated inversely (p<0.05) with PVL, and MIP-1beta expression revealed a similar trend. CD107a and IFN-gamma production were positively related to blood CD4 count (p<0.05), with MIP-1beta showing a similar trend. IL-2 production by rectal CD8+ T-cells was highly variable and generally low, and showed no relationship to viral load or blood CD4 count.The polyfunctionality of rectal Gag-specific CD8+ T-cells appears to be related to blood CD4 count and inversely related to PVL. The extent to which these associations reflect causality remains to be determined; nevertheless, our data suggest a potentially important role for mucosal T-cells in limiting virus replication during chronic infection

Multiplex RT-PCR Amplification of HIV Genes to Create a Completely Autologous DC-Based Immunotherapy for the Treatment of HIV Infection

BACKGROUND: Effective therapy for HIV-infected individuals remains an unmet medical need. Promising clinical trials with dendritic cell (DC)-based immunotherapy consisting of autologous DC loaded with autologous virus have been reported, however, these approaches depend on large numbers of HIV virions to generate sufficient doses for even limited treatment regimens. METHODOLOGY/PRINCIPAL FINDINGS: The present study describes a novel approach for RT-PCR amplification of HIV antigens. Previously, RT-PCR amplification of autologous viral sequences has been confounded by the high mutation rate of the virus which results in unreliable primer-template binding. To resolve this problem we developed a multiplex RT-PCR strategy that allows reliable strain-independent amplification of highly polymorphic target antigens from any patient and requires neither viral sequence data nor custom-designed PCR primers for each individual. We demonstrate the application of our RT-PCR process to amplify translationally-competent RNA encoding regions of Gag, Vpr, Rev and Nef. The products amplified using this method represent a complex mixture of autologous antigens encoded by viral quasispecies. We further demonstrate that DCs electroporated with in vitro-transcribed HIV RNAs are capable of stimulating poly-antigen-specific CD8+ T cell responses in vitro. CONCLUSION/SIGNIFICANCE: This study describes a strategy to overcome patient to patient viral diversity enabling strain-independent RT-PCR amplification of RNAs encoding sequence divergent quasispecies of Gag, Vpr, Rev and Nef from small volumes of infectious plasma. The approach allows creation of a completely autologous therapy that does not require advance knowledge of the HIV genomic sequences, does not have yield limitations and has no intact virus in the final product. The simultaneous use of autologous viral antigens and DCs may provoke broad patient-specific immune responses that could potentially induce effective control of viral loads in the absence of conventional antiretroviral drug therapy

Epitope Mapping of HIV-Specific CD8+ T cells in a Cohort Dominated by Clade A1 Infection

CD8+ T cell responses are often detected at large magnitudes in HIV-infected subjects, and eliciting these responses is the central aim of many HIV-1 vaccine strategies. Population differences in CD8+ T cell epitope specificity will need to be understood if vaccines are to be effective in multiple geographic regions.In a large Kenyan cohort, we compared responsive CD8+ T cell HIV-1 Env overlapping peptides (OLPs) to Best Defined Epitopes (BDEs), many of which have been defined in clade B infection. While the majority of BDEs (69%) were recognized in this population, nearly half of responsive OLPs (47%) did not contain described epitopes. Recognition frequencies of BDEs were inversely correlated to epitopic sequence differences between clade A1 and BDE (P = 0.019), and positively selected residues were more frequent in "new" OLPs (without BDEs). We assessed the impact of HLA and TAP binding on epitope recognition frequencies, focusing on predicted and actual epitopes in the HLA B7 supertype.Although many previously described CD8 epitopes were recognized, several novel CD8 epitopes were defined in this population, implying that epitope mapping efforts have not been completely exhausted. Expansion of these studies will be critical to understand population differences in CD8 epitope recognition

Quantifying the impact of human immunodeficiency virus-1 escape from cytotoxic T-lymphocytes.

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