Current seasonal
influenza virus vaccines engender antibody-mediated protection that is
hemagglutinin (HA) subtype specific and relatively short-lived. Coverage for other subtypes or even variants within a subtype could be improved from a better understanding of the factors that promote HA-specific antibody cross-reactivity. Current assays to evaluate cross-reactivity, such as the ELISA, require a separate test for each
antigen and are neither high-throughput nor sample-sparing. To address this need, we produced an array of 283 purified HA
proteins from influenza A virus subtypes H1 to
H16 and H18 and influenza B virus. To evaluate performance, arrays were probed with sera from individuals before and after a booster dose of inactivated heterologous H5N1
vaccine and naturally infected cases at presentation and follow-up during the 2010 to 2011
influenza season, when H3N2 was prevalent. The response to the H5
vaccine boost was
IgG only and confined to H5 variants. The response to natural H3N2
infection consisted of
IgG and
IgA and was reactive with all H3 variants displayed, as well as against other group 2 HA subtypes. In both groups, responses to HA1
proteins were subtype specific. In contrast, baseline signals were higher, and responses broader, against full-length HA
proteins (HA1+HA2) compared to HA1 alone. We propose that these elevated baseline signals and breadth come from the recognition of conserved
epitopes in the stalk domain by cross-reactive
antibodies accumulated from previous exposure(s) to seasonal influenza virus. This array is a valuable high-throughput alternative to the ELISA for monitoring specificity and cross-reactivity of HA
antibodies and has many applications in
vaccine development.IMPORTANCE Seasonal
influenza is a serious public health problem because the
viral infection spreads easily from person to person and because of antigenic drift in neutralizing
epitopes.
Influenza vaccination is the most effective way to prevent the disease, although challenging because of the constant evolution of influenza virus subtypes. Our high-throughput
protein microarrays allow for interrogation of subunit-specific
IgG and
IgA responses to 283 different HA
proteins comprised of HA1 and HA2 domains as well as full-length HA
proteins. This provides a tool that allows for novel insights into the response to exposure to influenza virus
antigens. Data generated with our technology will enhance our understanding of the factors that improve the strength, breadth, and durability of
vaccine-mediated immune responses and develop more effective
vaccines.