Age-related changes in magnitude and diversity of cross-reactive CD4+ T-cell responses to the novel pandemic H1N1 influenza hemagglutinin

Comparison of antibody and T-cell responses elicited by licensed inactivated- and live-attenuated influenza vaccines against H3N2 hemagglutinin

“…Overlapping peptides were predominantly 17 to 16 amino acid long with 11 to 12 amino acid overlaps and spanned the entirety of the H3N2 HA protein (566 aa) in 94 peptides. To assess responses elicited by the variant or the conserved regions of HA, we performed interferon-␥ (IFN-␥) ELISPOT with 2 peptide pools that spanned these regions individually, as we have described in detail earlier [25]. Briefly, the variant peptide pool used in this study spanned the N-terminal half of the H3N2 HA protein (aa 1 to 279 of A/Wisconsin/67/05 primary sequence, in 47 overlapping peptides), and comprised the variant globular region of HA.…”

“…The secretion of IFN-␥ was detected by ELISPOT assays [25,27]. Briefly, multiscreen 96-well plates (Millipore, Bedford, MA) were coated overnight at 4ЊC with 1 g/ml anti-human IFN-␥ capture antibody (Mabtech, Mariemont, OH), washed with PBS and blocked for 4 hours with complete RPMI medium (Invitrogen, Carlsbad, CA).…”

“…SFU were enumerated using an automated ELISPOT reader (Cellular Technologies, Shaker Heights, OH), and mean SFU was calculated for each stimulation after subtracting values from negative control wells. Data are presented as mean SFU per 10 6 PBMC using standard procedures that we and others have described earlier [25,27].…”

“…We have previously described methods that assess domain-specific T-cell responses by using overlapping peptide pools that individually span the variant and conserved regions of the HA primary sequence [25]. Using this approach, we compared variant and conserved region specific HA responses elicited among the TIV, LAIV, and the combined vaccine groups.…”

“…Also, it is unclear whether the two influenza vaccines currently licensed for use in humans, the trivalent inactivated influenza vaccine (TIV), and the live-attenuated influenza vaccine (LAIV), vary in their ability to elicit T-cell responses to the conserved and the antigenically drifting variant region of the HA antigens. We recently reported that though antibody responses to highly variant HA antigens are lacking or limited, significant cross-reactive T-cell responses that target the variant globular region of the antigen are prevalent among naturally infected individuals [25]. Further elucidating such region-specific T-cell responses following vaccination could shed light on the ability of T cells to provide protection against variant viruses that may escape antibody-mediated neutralization.…”