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Background. Most reported human H5N1 viral infections have been severe and were detected after hospital admission. A case ascertainment bias may therefore exist, with mild cases or asymptomatic infections going undetected. We sought evidence of mild or asymptomatic H5N1 infection by examining H5N1-specific T-cell and antibody responses in a high-risk cohort in Vietnam. Methods. Peripheral blood mononuclear cells were tested using interferon-&ggr; enzyme-linked immunospot T assays measuring the response to peptides of influenza H5, H3, and H1 hemagglutinin (HA), N1 and N2 neuraminidase, and the internal proteins of H3N2. Horse erythrocyte hemagglutination inhibition assay was performed to detect antibodies against H5N1. Results. Twenty-four of 747 individuals demonstrated H5-specific T-cell responses but little or no crossreactivity with H3 or H1 HA peptides. H5N1 peptide-specific T-cell lines that did not cross-react with H1 or H3 influenza virus HA peptides were generated. Four individuals also had antibodies against H5N1. Conclusions. This is the first report of ex vivo H5 HA-specific T-cell responses in a healthy but H5N1-exposed population. Our results indicate that the presence of H5N1-specific T cells could be an additional diagnostic tool for asymptomatic H5N1 infection. [ABSTRACT FROM AUTHOR]

Abstract: In the absence of replication of wells, empirical criteria for enzyme-linked immunospot (ELISpot) positivity use fixed differences or ratios between spot forming units (SFU) counts between test and control. We propose an alternative approach which first identifies the optimally variance-stabilizing transformation of the SFU counts, based on the Bland–Altman plot of the test and control wells. The second step is to derive a positivity threshold from the difference in between-plate distribution functions of the transformed test and control SFU counts. This method is illustrated using 1309 assay results from a cohort study of influenza in Vietnam in which some, but not all, of the peptide pools have clear tendencies for SFU counts to be higher in test than control wells. [Copyright &y& Elsevier]

The threat of avian influenza A (H5N1) infection in humans remains a global health concern. Current influenza vaccines stimulate antibody responses against the surface glycoproteins but are ineffective against strains that have undergone significant antigenic variation. An alternative approach is to stimulate pre-existing memory T cells established by seasonal human influenza A infection that could cross-react with H5N1 by targeting highly conserved internal proteins. To determine how common cross-reactive T cells are, we performed a comprehensive ex vivo analysis of cross-reactive CD4+ and CD8+ memory T cell responses to overlapping peptides spanning the full proteome of influenza A/Viet Nam/CL26/2005 (H5N1) and influenza A/New York/232/2004 (H3N2) in healthy individuals from the United Kingdom and Viet Nam. Memory CD4+ and CD8+ T cells isolated from the majority of participants exhibited human influenza-specific responses and showed cross-recognition of at least one H5N1 internal protein. Participant CD4+ and CD8+ T cells recognized multiple synthesized influenza peptides, including peptides from the H5N1 strain. Matrix protein 1 (M1) and nucleoprotein (NP) were the immunodominant targets of cross-recognition. In addition, cross-reactive CD4+ and CD8+ T cells recognized target cells infected with recombinant vaccinia viruses expressing either H5N1 M1 or NP. Thus, vaccine formulas inducing heterosubtypic T cell-mediated immunity may confer broad protection against avian and human influenza A viruses. [ABSTRACT FROM AUTHOR]

Dengue virus infection is an increasingly important tropical disease, causing 100 million cases each year. Symptoms range from mild febrile illness to severe hemorrhagic fever. The pathogenesis is incompletely understood, but immunopathology is thought to play a part, with antibody-dependent enhancement and massive immune activation of T cells and monocytes/macrophages leading to a disproportionate production of proinflammatory cytokines. We sought to investigate whether a defective population of regulatory T cells (T reg cells) could be contributing to immunopathology in severe dengue disease. CD4(+)CD25(high)FoxP3(+) T reg cells of patients with acute dengue infection of different severities showed a conventional phenotype. Unexpectedly, their capacity to suppress T cell proliferation and to secrete interleukin-10 was not altered. Moreover, T reg cells suppressed the production of vasoactive cytokines after dengue-specific stimulation. Furthermore, T reg cell frequencies and also T reg cell/effector T cell ratios were increased in patients with acute infection. A strong indication that a relative rise of T reg cell/effector T cell ratios is beneficial for disease outcome comes from patients with mild disease in which this ratio is significantly increased (P < 0.0001) in contrast to severe cases (P = 0.2145). We conclude that although T reg cells expand and function normally in acute dengue infection, their relative frequencies are insufficient to control the immunopathology of severe disease.

T-cell responses to dengue viruses may be important in both protective immunity and pathogenesis. This study of 48 Vietnamese adults with secondary dengue virus infections defined the breadth and magnitude of peripheral T-cell responses to 260 overlapping peptide antigens derived from a dengue virus serotype 2 (DV2) isolate. Forty-seven different peptides evoked significant gamma interferon enzyme-linked immunospot (ELISPOT) assay responses in 39 patients; of these, 34 peptides contained potentially novel T-cell epitopes. NS3 and particularly NS3200-324 were important T-cell targets. The breadth and magnitude of ELISPOT responses to DV2 peptides were independent of the infecting dengue virus serotype, suggesting that cross-reactive T cells dominate the acute response during secondary infection. Acute ELISPOT responses were weakly correlated with the extent of hemoconcentration in individual patients but not with the nadir of thrombocytopenia or overall clinical disease grade. NS3556-564 and Env414-422 were identified as novel HLA-A*24 and B*07-restricted CD8+ T-cell epitopes, respectively. Acute T-cell responses to natural variants of Env414-422 and NS3556-564 were largely cross-reactive and peaked during disease convalescence. The results highlight the importance of NS3 and cross-reactive T cells during acute secondary infection but suggest that the overall breadth and magnitude of the T-cell response is not significantly related to clinical disease grade.