Protein microarray analysis of the specificity and cross-reactivity of influenza virus hemagglutinin-specific antibodies

Rie Nakajima, Medalyn Supnet, Algis Jasinskas, Aarti Jain, Omid Taghavian, Joshua Obiero, Donald K. Milton, Wilbur H. Chen, Michael Grantham, Richard Webby, Florian Krammer, Darrick Carter, Philip L. Felgner, D. Huw Davies

Research output: Contribution to journalArticlepeer-review

36 Scopus citations


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 crossreactivity. 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.

Original languageEnglish
Article numbere00592-18
Issue number6
StatePublished - 1 Nov 2018


  • Hemagglutinin
  • Influenza
  • Protein microarrays


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