S. Roberts

Recovery of CD4+ T Cells in HIV Patients With a Stable Virologic Response to Antiretroviral Therapy Is Associated With Polymorphisms of Interleukin-6 and Central Major Histocompatibility Complex Genes

We investigated whether polymorphisms in genes associated with HIV disease progression and/or immune activation affect CD4+ T-cell recovery in HIV patients who began combination antiretroviral therapy (ART) with advanced immunodeficiency and achieved stable control of plasma viremia. Patients with CD4+ T-cell counts <300 cells/μL (n = 33) and >400 cells/μL (n = 37) on ART were compared. A multiple case-control logistic regression associated carriage of BAT1(1,2) or interleukin (IL)6-174(2,2) with low CD4+ T-cell counts (P = 0.012). BAT1*2 uniquely marks the central major histocompatibility complex region of a conserved haplotype (HLA-A1,B8,BAT1*2,TNFA-308*2,DR3,DQ2). There was no association between alleles carried at CCR5Δ32, CCR5 59029, CCR5 59353, CCR2+190 (V64I), SDF1 3′UTR, IL1A+4845, IL1B+3953, IL4-589, IL10-592, IL10-R1+536, IL10-R1+1112, IL12B 3′UTR, TNFA-308, or TNFA-1031 and CD4+ T-cell counts. We suggest that immune activation and/or CD4+ T-cell apoptosis in HIV patients on effective ART is influenced by genetic factors.

Translation of HLA–HIV Associations to the Cellular Level: HIV Adapts To Inflate CD8 T Cell Responses against Nef and HLA-Adapted Variant Epitopes

Strong statistical associations between polymorphisms in HIV-1 population sequences and carriage of HLA class I alleles have been widely used to identify possible sites of CD8 T cell immune selection in vivo. However, there have been few attempts to prospectively and systematically test these genetic hypotheses arising from population-based studies at a cellular, functional level. We assayed CD8 T cell epitope-specific IFN-γ responses in 290 individuals from the same cohort, which gave rise to 874 HLA–HIV associations in genetic analyses, taking into account autologous viral sequences and individual HLA genotypes. We found immunological evidence for 58% of 374 associations tested as sites of primary immune selection and identified up to 50 novel HIV-1 epitopes using this reverse-genomics approach. Many HLA-adapted epitopes elicited equivalent or higher-magnitude IFN-γ responses than did the nonadapted epitopes, particularly in Nef. At a population level, inclusion of all of the immunoreactive variant CD8 T cell epitopes in Gag, Pol, Nef, and Env suggested that HIV adaptation leads to an inflation of Nef-directed immune responses relative to other proteins. We concluded that HLA–HIV associations mark viral epitopes subject to CD8 T cell selection. These results can be used to guide functional studies of specific epitopes and escape mutations, as well as to test, train, and evaluate analytical models of viral escape and fitness. The inflation of Nef and HLA-adapted variant responses may have negative effects on natural and vaccine immunity against HIV and, therefore, has implications for diversity coverage approaches in HIV vaccine design.

High-avidity, high-IFNγ-producing CD8 T-cell responses following immune selection during HIV-1 infection

HIV‐1 mutations, which reduce or abolish CTL responses against virus‐infected cells, are frequently selected in acute and chronic HIV infection. Among population HIV‐1 sequences, immune selection is evident as human leukocyte antigen (HLA) allele‐associated substitutions of amino acids within or near CD8 T‐cell epitopes. In these cases, the non‐adapted epitope is susceptible to immune recognition until an escape mutation renders the epitope less immunogenic. However, several population‐based studies have independently identified HLA‐associated viral changes, which lead to the formation of a new T‐cell epitope, suggesting that the immune responses that these variants or ‘neo‐epitopes’ elicit provide an evolutionary advantage to the virus rather than the host. Here, we examined the functional characteristics of eight CD8 T‐cell responses that result from viral adaptation in 125 HLA‐genotyped individuals with chronic HIV‐1 infection. Neo‐epitopes included well‐characterized immunodominant epitopes restricted by common HLA alleles, and in most cases the T‐cell responses against the neo‐epitope showed significantly greater functional avidity and higher IFNγ production than T cells for non‐adapted epitopes, but were not more cytotoxic. Neo‐epitope formation and emergence of cognate T‐cell response coincident with a rise in viral load was then observed in vivo in an acutely infected individual. These findings show that HIV‐1 adaptation not only abrogates the immune recognition of early targeted epitopes, but may also increase immune recognition to other epitopes, which elicit immunodominant but non‐protective T‐cell responses. These data have implications for immunodominance associated with polyvalent vaccines based on the diversity of chronic HIV‐1 sequences.

Automation of the ELISpot assay for high-throughput detection of antigen-specific T-cell responses.

The enzyme linked immunospot (ELISpot) assay is a fundamental tool in cellular immunology, providing both quantitative and qualitative information on cellular cytokine responses to defined antigens. It enables the comprehensive screening of patient derived peripheral blood mononuclear cells to reveal the antigenic restriction of T-cell responses and is an emerging technique in clinical laboratory investigation of certain infectious diseases. As with all cellular-based assays, the final results of the assay are dependent on a number of technical variables that may impact precision if not highly standardised between operators. When studies that are large scale or using multiple antigens are set up manually, these assays may be labour intensive, have many manual handling steps, are subject to data and sample integrity failure and may show large inter-operator variability. Here we describe the successful automated performance of the interferon (IFN)-gamma ELISpot assay from cell counting through to electronic capture of cytokine quantitation and present the results of a comparison between automated and manual performance of the ELISpot assay. The mean number of spot forming units enumerated by both methods for limiting dilutions of CMV, EBV and influenza (CEF)-derived peptides in six healthy individuals were highly correlated (r>0.83, p<0.05). The precision results from the automated system compared favourably with the manual ELISpot and further ensured electronic tracking, increased through-put and reduced turnaround time.

HIV escape mutations occur preferentially at HLA-binding sites of CD8 T-cell epitopes

Objective:To define the relative frequencies of different mechanisms of viral escape. Design:A population-based approach to examine the distribution of HIV polymorphism associated with diverse population human leucocyte antigens (HLAs) at sites within and flanking CD8 T-cell epitopes as a correlate of likely mechanisms of viral escape. Methods:Sequence windows surrounding 874 HLA allele-specific polymorphisms across the full HIV-1 proteomic consensus sequence were scanned by an epitope-prediction programme. Either already known or probable CD8 T-cell epitopes with HLA restriction matching that of the proximal HLA association were identified and synthesized. These peptides were used as stimulating antigens in automated enzyme-linked immunospot (ELISpot) assays. Peptide arrays were customized to each individual based on their HLA genotype. Results:Among HLA-associated HIV polymorphisms detected in the viral sequences of a cohort of 800 individuals with chronic subtype B HIV infection, those which were likely to affect HLA peptide binding were significantly more common than polymorphisms at nonanchor HLA binding sites. HIV epitopes with such polymorphisms were associated with reduced IFN&ggr; responses in ELISpot assays. HIV escape at sites affecting T-cell receptor (TCR) engagement and epitope processing were also evident. Conclusion:HIV escape from HLA-peptide binding predominates as an effective viral evasion strategy and therefore has implications for inclusion of HLA-adapted epitopes in vaccine immunogens.

Evaluation of the dual IFNγ/IL-2 fluorospot-assay with flow cytometry for detection of HLA-restricted HIV-specific T-cell responses in HIV controllers

Background: The IFNγ ELISpot assay is used widely for high through-put screening of HIV-specific responses in studies of HIV infection and vaccine studies. However, dual production of IFN/IL-2 and increased proliferative capacity may be associated with better natural control of HIV infection. We evaluated a novel fluorospot assay enabling the identification of dual IFNγ/ IL-2 producing antigen-specific cells and compared it with intracellular cytokine staining by standard flow cytometry in individuals with natural control of HIV-infection. Methods: PBMC from five untreated HIV-infected individuals were stimulated overnight with HIV peptides or controls in dual IFN/IL-2 pre-coated plates. Peptide arrays were specifically selected based on the individual HLA type. Secreted IFNγ and IL-2 were detected using fluorescent-conjugated antibodies. Fluorescent spots were enumerated on the iSpot AID reader. Responses were considered positive if >50 spots/million cells SFU after background subtraction.Positive responses were then evaluated by flow cytometry using the Gallios flow cytometer. Results: Dual IL-2/IFNγ producing cells were detected to anti-CD3- stimulated PBMC from all patients. IFNγ responses alone were detected to 35 of 136 HLA-restricted peptides tested (median =73, 52-4190 SFU) across the 5 patients (1/19, 5/19, 7/28, 9/15 and 13/55 for each patient), while IL-2 responses were either low grade or undetectable for the majority of HIV peptides tested. Dual IFNγ/IL-2 producing HIV-specific T cells were not detected using the fluorospot assay. 24/35 peptides induced CD8 T cell- IFNγ production by flow cytometry. Conclusion: HIV-specific mono-IFNγ responses were detected using the novel fluorospot assay. However limited HIV-specific dual IFNγ/IL-2 responses were detected in this patient group. A greater number of epitope-specific positive responses were detected in the fluorospot compared with flow cytometry suggesting the fluorospot may be more sensitive in detecting a greater breadth of epitope-specific T cell responses, and therefore better for screening purposes than flow cytometric methods.

P09-15. Selection of higher avidity HLA-restricted T cell responses as a viral adaptation strategy

Background Loss of immune reactivity due to HIV mutational escape is well described. Data generated from a large population- based study (n>800) suggested that certain CD8 T cell epitopes are created as a result of HIV adaptation and are associated with enhanced viral replication. Here we sought to investigate the HLA-restricted T-cell responses associated with seven such adaptations. Methods 180 cryopreserved PBMC samples from 112 patients were assayed for IFN- γ production and functional avidity to HIV peptides in ELISpot assays. CTL lines generated from short term PBMC cultures were confirmed and phenotyped by flow cytometry and cytotoxicity was assessed by chromium release. Results Responses were detected to non-adapted peptides in 48 samples (327 median [68–3067] range spot forming units (SFU)/10 6 cells) and adapted peptides in 54 samples (317 [63–2332] SFU/10 6 ). Responses to both non- and adapted peptides were detected in 19% (35/180) of samples (480[68–2225], 638[74–2332] SFU/10 6 respectively). Overall, responses to adapted epitopes were significantly greater than responses to non-adapted epitopes in patients with detectable viral load in screening assays (n = 14, p = 0.0413, Wilcoxon Rank Sum Test) and when half maximal peptide concentration data was analysed (p < 0.05 paired t-test). The frequency of IFN- γ producing cells from cultured CTL restricted by HLA-C*0702- KY11 (n = 7) was higher in central memory (p = 0.03) and effector memory CD8 T cell (p = 0.02) populations from adapted epitope (KRQEILDLWVY) stimulated cultures compared with non-adapted epitope (KRQDILDLWVYY) stimulated T cells. No difference in central or effector memory population size or IL-2 production was detected and preliminary data from chromium release assays suggests that CTL cultured with adapted peptides have differential killing against adapted versus non-adapted epitopes. Conclusion These data suggest that some high avidity and high IFN- γ - producing CD8 T cell responses are the result, rather than the cause, of viral adaptation. These data have implications for vaccine development.