C. Bronke

Dynamics of cytomegalovirus (CMV)-specific T cells in HIV-1-infected individuals progressing to AIDS with CMV end-organ disease

BACKGROUND Since cytomegalovirus (CMV) infection can cause serious clinical complications in immunocompromised individuals, we assessed cellular immune requirements for protection against CMV end-organ disease (CMV-EOD) in human immunodeficiency virus type 1 (HIV-1) infection. METHODS Longitudinal samples from HIV-1-infected patients in the Amsterdam cohort were analyzed. Dynamics of CMV-specific CD8(+) and CD4(+) T cell responses were analyzed by 4-color fluorescence analysis using major histocompatibility class I CMV peptide-tetramers and by intracellular staining for perforin, granzyme B, and interferon (IFN)- gamma after stimulation with CMV-specific stimuli. CMV load was measured in parallel. RESULTS In individuals progressing to acquired immunodeficiency syndrome with CMV-EOD, CMV-specific IFN- gamma -producing CD4(+) T cells disappeared during the year before onset of CMV-EOD. This disappearance was accompanied by a sharp increase in CMV load before onset of disease. Despite increasing CMV-specific CD8(+) T cell counts, decreasing CMV-specific IFN- gamma -producing CD8(+) T cell counts were found over time. In contrast, the percentage of CMV-specific perforin- and granzyme B-expressing CD8(+) T cells increased. CONCLUSIONS Our data indicate that insufficient help of CD4(+) T cells may cause loss of IFN- gamma -producing CD8(+) T cells and loss of control of CMV dissemination. Increasing CMV-infected cell counts in the face of high CMV-specific perforin- and granzyme B-expressing CD8(+) T cell counts may explain the immune pathological characteristics of CMV disease.

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.

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.

Cytomegalovirus rather than HIV triggers the outgrowth of effector CD8+CD45RA+CD27-T cells in HIV-1-infected children

Objective:To analyse the effect of viral coinfections on immune reconstitution in HIV-1-infected children (< 18 years) taking highly active antiretroviral therapy (HAART). Methods:Absolute lymphocyte numbers of various subsets of CD8 T cells were measured. Results:Prior cytomegalovirus (CMV) infection correlated with an increased number of CD8 effector T cells (i.e., CD45RA+CD27−) at baseline (CMV-seropositive versus CMV-seronegative patients; P = 0.009), as well as an increased state of T cell activation as defined by HLA-DR and CD38 expression. The expansion of effector CD8 T cells persisted over time, independent of the HIV response to HAART. Numbers of CD8 effector T cells were significantly higher in patients with CMV replication as reflected by persistent urinary CMV shedding and periodic CMV DNAaemia (P = 0.02). These patients also showed an increase in CMV-specific antibodies compared with those without CMV shedding (P = 0.007). The number of CMV-specific interferon-γ (IFN-γ)-producing CD8 T cells was lower in children who persistently shed CMV compared with those who did not (P = 0.02). In contrast, CMV-specific CD4 T cell responses were detected at similar levels in both groups. Conclusions:In HIV-1-infected children, CMV infection correlated with the outgrowth of CD8+CD45RA+CD27− effector T cells. Activation of the immune system by persistent CMV secretion resulted in increasing CMV-specific IgG and higher numbers of CD8 effector T cells. Despite these increases, the CMV-specific IFN-γ-producing CD8 T cell response was diminished, which could explain the inability to suppress CMV completely in 41% of HIV-1-infected children.

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.