Abstract
The hemagglutinin (HA) of influenza A viruses has been classified into sixteen distinct subtypes (H1–H16) to date. The HA subtypes of influenza A viruses are principally defined as serotypes determined by neutralization or hemagglutination inhibition tests using polyclonal antisera to the respective HA subtypes, which have little cross-reactivity to the other HA subtypes. Thus, it is generally believed that the neutralizing antibodies are not broadly cross-reactive among HA subtypes. In this study, we generated a novel monoclonal antibody (MAb) specific to HA, designated MAb S139/1, which showed heterosubtypic cross-reactive neutralization and hemagglutination inhibition of influenza A viruses. This MAb was found to have broad reactivity to many other viruses (H1, H2, H3, H5, H9, and H13 subtypes) in enzyme-linked immunosorbent assays. We further found that MAb S139/1 showed neutralization and hemagglutination-inhibition activities against particular strains of H1, H2, H3, and H13 subtypes of influenza A viruses. Mutant viruses that escaped neutralization by MAb S139/1 were selected from the A/Aichi/2/68 (H3N2), A/Adachi/2/57 (H2N2), and A/WSN/33 (H1N1) strains, and sequence analysis of the HA genes of these escape mutants revealed amino acid substitutions at positions 156, 158, and 193 (H3 numbering). A molecular modeling study showed that these amino acids were located on the globular head of the HA and formed a novel conformational epitope adjacent to the receptor-binding domain of HA. Furthermore, passive immunization of mice with MAb S139/1 provided heterosubtypic protection. These results demonstrate that MAb S139/1 binds to a common antigenic site shared among a variety of HA subtypes and neutralizes viral infectivity in vitro and in vivo by affecting viral attachment to cells. The present study supports the notion that cross-reactive antibodies play some roles in heterosubtypic immunity against influenza A virus infection, and underscores the potential therapeutic utility of cross-reactive antibodies against influenza.
Highlights
Neutralizing antibodies play a critical role in protection from influenza virus infection
Since HA subtypes of influenza A viruses are principally defined as serotypes determined by neutralization or hemagglutination inhibition tests using polyclonal antisera to the respective HA subtypes, which have little crossreactivity to the other HA subtypes, it is generally believed that the neutralizing antibodies are not broadly crossreactive among HA subtypes
The cross-reactivity of monoclonal antibody (MAb) S139/1 to multiple subtypes of influenza A virus HAs was tested by enzymelinked immunosorbent assay (ELISA) using several H1, H2, H3, H5, H9, and H13 subtypes (Fig. 1)
Summary
Neutralizing antibodies play a critical role in protection from influenza virus infection. Most neutralizing antibodies recognize hemagglutinin (HA), which is the major surface glycoprotein of influenza viruses. HA is responsible for virus entry into target cells, virus binding to the host receptor, internalization of the virus, and subsequent membrane-fusion events. It is initially synthesized as a precursor polypeptide, HA0, that requires proteolytic cleavage into disulfidelinked HA1 and HA2 before it is functional and virus particles are infectious. The major part of HA1 forms the ‘‘globular head’’ region, which contains the necessary structure for binding to the sialic acid receptors. It has been recognized that there is considerable amino acid variability (antigenic difference) in the globular head region among HA subtypes, whereas the structure of the stem region is relatively conserved
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