Epitope mapping of the 2009 pandemic and the A/Brisbane/59/2007 seasonal (H1N1) influenza virus haemagglutinins using mAbs and escape mutants

Abstract

Research Center in Infectious Diseases of the CHUQ-CHUL and Laval University, Qué bec City, QC, Canada mAbs constitute an important biological tool for influenza virus haemagglutinin (HA) epitope mapping through the generation of escape mutants, which could provide insights into immune evasion mechanisms and may benefit the future development of vaccines. Several influenza A (H1N1) pandemic 2009 (pdm09) HA escape mutants have been recently described. However, the HA antigenic sites of the previous seasonal A/Brisbane/59/2007 (H1N1) (Bris07) virus remain poorly documented. Here, we produced mAbs against pdm09 and Bris07 HA proteins expressed in human HEK293 cells. Escape mutants were generated using mAbs that exhibited HA inhibition and neutralizing activities. The resulting epitope mapping of the pdm09 HA protein revealed 11 escape mutations including three that were previously described (G172E, N173D and K256E) and eight novel ones (T89R, F128L, G157E, K180E, A212E, R269K, N311T and G478E). Among the six HA mutations that were part of predicted antigenic sites (Ca1, Ca2, Cb, Sa or Sb), three (G172E, N173D and K180E) were within the Sa site. Eight escape mutations (H54N, N55D, N55K, L60H, N203D, A231T, V314I and K464E) were obtained for Bris07 HA, and all but one (N203D, Sb site) were outside the predicted antigenic sites. Our results suggest that the Sa antigenic site is immunodominant in pdm09 HA, whereas the N203D mutation (Sb site), present in three different Bris07 escape mutants, appears as the immunodominant epitope in that strain. The fact that some mutations were not part of predicted antigenic sites reinforces the necessity of further characterizing the HA of additional H1N1 strains.

Diverse antigenic site targeting of influenza hemagglutinin in the murine antibody recall response to A(H1N1)pdm09 virus

“…Although our study, and those of others (Rudneva et al, 2012; Chen et al, 2013; Retamal et al, 2014), did not observe this epitope, this highlights the need to further expand our understanding of the antigenicity of H1N1pdm09 HA, beyond that of the classical antigenic mapping of the HA of PR/8 (Caton et al, 1982). This will not only aid our understanding of the protective antibody response against this virus, but will also support surveillance and vaccine candidate selection efforts that monitor viral drift in the human population.…”

Antigenic sites in influenza H1 hemagglutinin display species-specific immunodominance

“…To ensure the loss of antigenicity for an individual antigenic site, each mutant virus in our panel contained 5 or more amino acid substitutions within 1 antigenic site. Additionally, several amino acid substitutions that were included in our panel were consistent with previously described escape mutations (18). While our study focused on the classically defined epitopes of pandemic H1 hemag glutinin, several nonclassical epitopes have been described as being HI sensitive (11,19,20).…”

Antigenic characterization of novel H1 influenza A viruses in swine

“…These low-impact substitutions are common in the evolution of IAVs and are useful for tracking antigenic evolution 50 . The amino acid substitution G155E has been previously reported as causing a high-impact antigenic change in A(H1N1)pdm09 IAVs 44,45,55,56 . This substitution is analogous to the substitution G158E, which is responsible for major antigenic changes during evolution of H3N2 IAVs 51 .…”

“…Nevertheless, we found a high antigenic variability within the Chilean A(H1N1)pdm09-like cluster and some Chilean strains had additional glycosylation sites in or near the antigenic site Sa. This site is one of the most variable antigenic sites among Chilean A(H1N1)pdm09-like viruses, which has been shown to be important for the antigenic evolution of A(H1N1)pdm09-like viruses [42][43][44][45] . Of interest, previously it has been shown that most of glycosylation sites acquired in H1 viruses from 1918 to 2009 have been located in or near antigenic site Sa 20 .…”