Inhibition of viral replication by icIgA antibodies has only been observed with in vitro studies using epithelial cell lines in transwell cultures. This effect appears to involve an interaction between polymeric immunoglobulin A (pIgA) and viral particles within an intracellular compartment, since IgA is transported across polarized cells. Polyclonal guinea pig antisera against purified influenza A virus and mouse antisera prepared against Influenza A/H3N2 hemagglutinin (HA0) cleavage loop peptides, were used in confocal fluorescence microscopy to show specific staining of wild-type influenza H1N1 and H3N2 viruses in clinical specimens. The HA0 cleavage loop peptides used for intranasal immunization of mice were designed and synthesized from specific conserved regions of influenza A/H1N1 & A/H3N2 viruses. Anti-human secretory IgA antibodies were used to show co-localisation of influenza A virus and icIgA. The results showed specific immunofluorescent staining of influenza A/H3N2 (X31) (HA0 uncleaved)-infected MDCK cells and the presence of icIgA in respiratory exudate cells of infected patients. Both results confirm specific co-localisation and suggest interaction between influenza A virus and icIgA in patients´ respiratory exudate cells. Importantly, antisera to the mouse anti-HA0 cleavage site were specific for wild-type virus in clinical specimens, indicating that the conserved region of HA0 was present in the uncleaved form. Similar staining and colocalization patterns between icIgA and virus were observed with polyclonal guinea pig antisera against influenza A virus. These are the first observations of co-localization of influenza A virus and intracellular IgA in clinical specimens. Role of icIgA: This report shows the co-localization of influenza A virus HA0 and icIgA antibodies in respiratory exudate cells of patients who were culture and viral RNA positive, suggesting that icIgA directed against the conserved HA0 site may have a privileged and unique opportunity to act on immature virus and thus prevent HA0 cleavage, maturation and subsequent cycles of viral replication. The precise mechanism by which icIgA mediates intracellular viral neutralization remains to be fully elucidated.

SIGNIFICANCE: The above findings in clinical specimens would contribute strongly to our understanding of the mechanisms and kinetics of icIgA neutralization in relation to viral entry and early replication steps of mucosal viral infections. A rapid, objective and sensitive assay - by ex vivo enumeration of respiratory epithelial cells that have co-localized influenza virus and icIgA - would contribute to further mucosal immunity studies and inform the design of more effective vaccines against influenza and other viral infections transmitted via the mucosal route e.g. respiratory syncytial virus, rotavirus.