Loss of HIV-1-specific CD8+ T cell proliferation after acute HIV-1 infection and restoration by vaccine-induced HIV-1-specific CD4+ T cells.

Virus-specific CD8(+) T cells are associated with declining viremia in acute human immunodeficiency virus (HIV)1 infection, but do not correlate with control of viremia in chronic infection, suggesting a progressive functional defect not measured by interferon gamma assays presently used. Here, we demonstrate that HIV-1-specific CD8(+) T cells proliferate rapidly upon encounter with cognate antigen in acute infection, but lose this capacity with ongoing viral replication. This functional defect can be induced in vitro by depletion of CD4(+) T cells or addition of interleukin 2-neutralizing antibodies, and can be corrected in chronic infection in vitro by addition of autologous CD4(+) T cells isolated during acute infection and in vivo by vaccine-mediated induction of HIV-1-specific CD4(+) T helper cell responses. These data demonstrate a loss of HIV-1-specific CD8(+) T cell function that not only correlates with progressive infection, but also can be restored in chronic infection by augmentation of HIV-1-specific T helper cell function. This identification of a reversible defect in cell-mediated immunity in chronic HIV-1 infection has important implications for immunotherapeutic interventions.

Longitudinal analysis of clinical markers following antiretroviral therapy initiated during acute or early HIV type 1 infection.

BACKGROUND: Treatment of acute human immunodeficiency virus type 1 (HIV-1) infection may have unique immunologic, virological, and clinical benefits. However, the timing of treatment, optimal starting regimens, and expected response to therapy have not been defined.Methods. One hundred two subjects treated during acute and early HIV-1 infection were observed prospectively to determine the effect of time elapsed before initiation of therapy on time to virological suppression and absolute CD4+ cell count. Subjects were divided into pre- and postseroconversion groups on the basis of HIV-1 antibody status at the time of initiation of treatment. Absolute CD4+ cell counts were compared between these groups and with those of historical untreated persons who had experienced seroconversion. Potential predictors of time to virological suppression and CD4+ cell count at > or =12 months were assessed. RESULTS: Ninety-nine (97%) of 102 subjects achieved virological suppression. The median time to suppression was 11.1 weeks (95% confidence interval, 9.4-14.9) and was independent of initial regimen. The mean CD4+ cell count at 12 months was 702 cells/mm3 (95% confidence interval, 654-750 cells/mm3) and showed an increasing trend over 60 months. Treated subjects demonstrated a statistically significant gain in the CD4+ cell count, compared with untreated historical control subjects, at > or =12 months. Comparable virological and immunologic outcomes were seen in the pre- and postseroconversion groups. Baseline virus load and nadir CD4+ cell count predicted time to virological suppression and CD4+ cell count at > or =12 months, respectively. CONCLUSIONS: Early treatment of HIV-1 infection is well tolerated and results in rapid and sustained virological suppression. Preservation of CD4+ cell counts may be achieved with early therapy, independent of seroconversion status. Protease inhibitor-based and nonnucleoside reverse-transcriptase inhibitor-based regimens show comparable performance in tolerability, time to virological suppression, and CD4+ cell count when used as a first regimen.

HIV-1 Nef is preferentially recognized by CD8 T cells in primary HIV-1 infection despite a relatively high degree of genetic diversity.

OBJECTIVE: To compare the magnitude, breadth and protein specificity of HIV-1-specific CD8 T-cell responses against the clade B consensus sequence during primary and chronic HIV-1 infection and to analyze the impact of viral diversity on the localization of detected responses. METHODS: HIV-1-specific CD8 T-cell responses against the clade B consensus sequence in individuals with acute (n = 10), early (n = 19) and chronic (n = 10) infection were longitudinally assessed using an interferon-gamma EliSpot assay. RESULTS: CD8 T-cell responses against clade B consensus sequences were preferentially directed against central regions of Nef during primary HIV-1 infection, despite a relatively higher degree of genetic diversity compared with other subsequently targeted regions. In subjects with acute and early infection, Nef-specific CD8 T-cell responses against the consensus Nef sequence represented 94 and 46% of the total magnitude of HIV-1-specific CD8 T-cell responses, respectively. Subjects with untreated chronic infection exhibited broadly diversified CD8 T-cell responses against more conserved viral regions, with only 17% of virus-specific T-cell responses targeting Nef. The initial immunodominance of Nef persisted in individuals with treated acute infection, but shifted rapidly to Gag, Env and Pol in subjects with continuous antigen exposure. CONCLUSION: These data show that despite relatively high sequence variability, viral regions within the clade B consensus sequence of Nef are preferentially recognized during primary HIV-1 infection. Later diversification of responses to other proteins during prolonged antigen exposure provides evidence of the initial preferential immunogenicity of Nef epitopes compared to similarly conserved regions within other viral proteins.

TPCK inhibits AGC kinases by direct activation loop adduction at phenylalanine-directed cysteine residues.

N-alpha-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) has anti-tumorigenic properties, but its direct cellular targets are unknown. Previously, we showed TPCK inhibited the PDKl-dependent AGC kinases RSK, Akt and S6K1 without inhibiting PKA, ERK1/2, PI3K, and PDK1 itself. Here we show TPCK-inhibition of the RSK-related kinases MSK1 and 2, which can be activated independently of PDK1. Mass spectrometry analysis of RSK1, Aktl, S6K1 and MSK1 immunopurified from TPCK-treated cells identified TPCK adducts on cysteines located in conserved activation loop Phenylalanine-Cysteine (Phe-Cys) motifs. Mutational analysis of the Phe-Cys residues conferred partial TPCK resistance. These studies elucidate a primary mechanism by which TPCK inhibits several AGC kinases, inviting consideration of TPCK-like compounds in chemotherapy given their potential for broad control of cellular growth, proliferation and survival.

Extensive HLA class I allele promiscuity among viral CTL epitopes.

Promiscuous binding of T helper epitopes to MHC class II molecules has been well established, but few examples of promiscuous class I-restricted epitopes exist. To address the extent of promiscuity of HLA class I peptides, responses to 242 well-defined viral epitopes were tested in 100 subjects regardless of the individuals' HLA type. Surprisingly, half of all detected responses were seen in the absence of the originally reported restricting HLA class I allele, and only 3% of epitopes were recognized exclusively in the presence of their original allele. Functional assays confirmed the frequent recognition of HLA class I-restricted T cell epitopes on several alternative alleles across HLA class I supertypes and encoded on different class I loci. These data have significant implications for the understanding of MHC class I-restricted antigen presentation and vaccine development.

Impact of HLA-B alleles, epitope binding affinity, functional avidity, and viral coinfection on the immunodominance of virus-specific CTL responses.

Immunodominance is variably used to describe either the most frequently detectable response among tested individuals or the strongest response within a single individual, yet factors determining either inter- or intraindividual immunodominance are still poorly understood. More than 90 individuals were tested against 184 HIV- and 92 EBV-derived, previously defined CTL epitopes. The data show that HLA-B-restricted epitopes were significantly more frequently recognized than HLA-A- or HLA-C-restricted epitopes. HLA-B-restricted epitopes also induced responses of higher magnitude than did either HLA-A- or HLA-C-restricted epitopes, although this comparison only reached statistical significance for EBV epitopes. For both viruses, the magnitude and frequency of recognition were correlated with each other, but not with the epitope binding affinity to the restricting HLA allele. The presence or absence of HIV coinfection did not impact EBV epitope immunodominance patterns significantly. Peptide titration studies showed that the magnitude of responses was associated with high functional avidity, requiring low concentration of cognate peptide to respond in in vitro assays. The data support the important role of HLA-B alleles in antiviral immunity and afford a better understanding of the factors contributing to inter- and intraindividual immunodominance.

Control of human immunodeficiency virus replication by cytotoxic T lymphocytes targeting subdominant epitopes.

Despite limited data supporting the superiority of dominant over subdominant responses, immunodominant epitopes represent the preferred vaccine candidates. To address the function of subdominant responses in human immunodeficiency virus infection, we analyzed cytotoxic T lymphocyte responses restricted by HLA-B*1503, a rare allele in a cohort infected with clade B, although common in one infected with clade C. HLA-B*1503 was associated with reduced viral loads in the clade B cohort but not the clade C cohort, although both shared the immunodominant response. Clade B viral control was associated with responses to several subdominant cytotoxic T lymphocyte epitopes, whereas their clade C variants were less well recognized. These data suggest that subdominant responses can contribute to in vivo viral control and that high HLA allele frequencies may drive the elimination of subdominant yet effective epitopes from circulating viral populations.

HLA-B63 presents HLA-B57/B58-restricted cytotoxic T-lymphocyte epitopes and is associated with low human immunodeficiency virus load.

Several HLA class I alleles have been associated with slow human immunodeficiency virus (HIV) disease progression, supporting the important role HLA class I-restricted cytotoxic T lymphocytes (CTL) play in controlling HIV infection. HLA-B63, the serological marker for the closely related HLA-B*1516 and HLA-B*1517 alleles, shares the epitope binding motif of HLA-B57 and HLA-B58, two alleles that have been associated with slow HIV disease progression. We investigated whether HIV-infected individuals who express HLA-B63 generate CTL responses that are comparable in breadth and specificity to those of HLA-B57/58-positive subjects and whether HLA-B63-positive individuals would also present with lower viral set points than the general population. The data show that HLA-B63-positive individuals indeed mounted responses to previously identified HLA-B57-restricted epitopes as well as towards novel, HLA-B63-restricted CTL targets that, in turn, can be presented by HLA-B57 and HLA-B58. HLA-B63-positive subjects generated these responses early in acute HIV infection and were able to control HIV replication in the absence of antiretroviral treatment with a median viral load of 3,280 RNA copies/ml. The data support an important role of the presented epitope in mediating relative control of HIV replication and help to better define immune correlates of controlled HIV infection.

HIV-1-specific cytotoxicity is preferentially mediated by a subset of CD8(+) T cells producing both interferon-gamma and tumor necrosis factor-alpha.

CD8(+) T cells play a crucial role in the control of viral infections by direct elimination of infected cells and secretion of a number of soluble factors. Recent data suggest that HIV-1-specific CD8(+) T cell subsets may differ in their ability to exert these effector functions. Here, we directly compared the cytokine secretion patterns and cytotoxic capacity of HIV-1-specific CD8(+) T cells, using a flow-cytometric cytotoxicity assay based on caspase-3 activation in dying target cells. These experiments revealed considerable intraindividual and interindividual differences among epitope-specific T-cell effector functions: while the frequency of HIV-1-specific CD8(+) T cells secreting interferon-gamma but no tumor necrosis factor-alpha (TNF-alpha) following antigenic stimulation was only weakly correlated to their cytotoxic activity (R = 0.05, P =.57), a subset of CD8(+) T cells secreting both inter-feron-gamma and TNF-alpha was substantially more strongly associated with cytotoxicity (R = 0.67, P <.001). This subset of CD8(+) T cells also exhibited stronger intracellular perforin expression and more pronounced direct ex vivo HIV-1-specific cytoxicity than CD8(+) T cells secreting solely interferon-gamma following sorting of these subpopulations according to their cytokine profile. These results suggest that HIV-1-specific cytotoxicity of CD8(+) T cells is preferentially mediated by a subset of CD8(+) T cells secreting both interferon-gamma and TNF-alpha.