Identification of H5N1-specific T-cell responses in a high-risk cohort in vietnam indicates the existence of potential asymptomatic infections

Abstract

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-γ 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 cross-reactivity 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.

Figure 1.

Enzyme-linked immunospot spot (ELISPOT) assay results in cohort volunteers with H5 peptide-specific responses. Peripheral blood mononuclear cells were stimulated with overlapping peptides from H1, H3, and H5 proteins to the first half (A) and second half (B) of the protein. Asterisks denote donors who were also positive for H5 antibody. C, Percentage of each cohort who had H5-specific T-cell responses by interferon-γ ELISPOT assays. Number of donors in each cohort is shown underneath each bar. Abbreviations: HA, hemagglutinin; SFU, spot-forming units.

Figure 2.

Hemagglutinin (HA)–specific short-term T-cell lines show specificity for H5 pools, but not H3 or H1 pools, and are specific to single H5 HA peptides. A, Peripheral blood mononuclear cells (PBMCs) were expanded in vitro using HA5-1 or HA5-2 peptide pools and interleukin (IL) 2. Ten days later, cells were rested overnight in IL-2–free media and then tested in an enzyme-linked immunospot spot (ELISPOT) assay using H5 pools, HA5-1 or HA5-2, depending on the specificity of the line, or a mixture of H3/H1 pools from the same corresponding region of the HA protein. B, C, STLs were restimulated with antigen pulsed autologous B cells. After 10-day stimulation and 30-hour rest, cells were tested in their response to specific peptides and the release of interferon-γ was measured by ELISPOT assay. A, B, C, Results shown are spot-forming units (SFU) per 10⁵ cells of duplicate or triplicate wells (± standard deviations).

Figure 3.

Short-term T-cell lines derived from H3N2 internal peptide pools show cross-reactive responses to both H3N2 and H5N1 peptide pools. Short-term lines were rested overnight in H10 media and then tested in a cultured enzyme-linked immunospot spot assay against peptide pools from internal genes of H3N2 and H5N1 peptide pools. Line specificity is shown on the axis, and duplicate values with background subtracted are shown ± standard deviations. Abbreviations: NP, nucleoprotein; NS, non-structural protein; PB1 and 2, polymerase basic protein 1 and 2; PA, polymerase acidic protein; M, matrix protein; SFU, spot-forming units.