Universal anti-neuraminidase antibody inhibiting all influenza A subtypes
Introduction
Current seasonal influenza vaccines are designed to provide strain-specific protection against two circulating subtypes of influenza A virus (H1N1 and H3N2) and one influenza B virus. The main target in these vaccines is the hemagglutinin (HA) for which there are at least 16 subtypes. HA has been identified to primarily mediate entry into host cells. Thus, neutralizing antibodies induced by vaccines can block viral entry by either preventing the attachment of the virus to sialic acid receptors on host cells or by interfering with HA-mediated viral fusion (Kida et al., 1983, Yoden et al., 1986).
Neuraminidase (NA) is another major glycoprotein on the surface of the virus of which there are at least 9 known subtypes (Baker et al., 1987, Webster and Bean, 1978). Recently, a highly divergent influenza A virus was isolated from little yellow shouldered bats (designated as H17N10). However, the N10 is a NA-like protein which does not possess sialidase activity (Tong et al., 2012). NA exerts its primary functional role during viral exit from the infected cells by cleaving the sialic acid receptors to facilitate the release of viral particles (Skehel and Wiley, 2000, Air and Laver, 1989, Palese et al., 1974). This role is supported by reports that NA-defective virus, or wild type viruses in the presence of NA inhibitors, form aggregations of viruses on the apical surface of the cells (Griffin et al., 1983, Liu et al., 1995, Hashem et al., 2009, Govorkova and McCullers, 2013). NA may also promote the breakdown of mucus in the respiratory tracts and the release of virus from sialic acid containing inhibitors, thus facilitating viral diffusion and access to the respiratory tract during infection (Matrosovich et al., 2004) and contributing to the entry and fusion of the influenza virus into host cells (Su et al., 2009). In contrast to HA, NA antibodies are not known to neutralize viral infectivity, but they have been shown to reduce viral yield by inhibiting NA enzymatic activity and thus to contribute to protection against influenza infection (Johansson et al., 1989, Qiu et al., 2006, Chen et al., 2000, Chen et al., 2005).
Both HA and NA proteins are highly variable being subject to antigenic drift and shift (Johansson and Brett, 2007, Air, 2011, Hashem et al., 2011, Ilyushina et al., 2012). Therefore, heterosubtypic protection against influenza A viruses is largely believed to be mediated by cross-reactive cytotoxic T cells against the highly conserved internal proteins rather than by antibodies against HA or NA (Epstein et al., 2005, Takada et al., 2003, Rimmelzwaan et al., 2007). However, recent reports have identified various broadly neutralizing antibodies targeting conserved epitopes in HA (Thosby et al., 2008, Thomson et al., 2012, Yoshida et al., 2009, Ekiert et al., 2009, Ekiert et al., 2011). Moreover, Sandbulte et al. (2007) and Marcelin et al. (2011) have shown that cross-reactive anti-neuraminidase antibodies elicited by seasonal H1N1 infection could not only contribute to protection against H1N1 but also provide cross-protection against H5N1 and H1N1pdm09 viruses, respectively. However, an antigenically conserved sequence affording heterosubtypic inhibition across all subtypes of NA has not been reported (Marcelin et al., 2012, Johansson and Brett, 2007).
We recently identified a universally conserved peptide located between amino acids (a.a.) 222–230 (N2 numbering). This epitope, comprised of “ILRTQESEC”, constitutes part of the enzymatic active site (Gravel et al., 2010, Varghese et al., 1983), and plays critical roles in viral replication (Doyle et al., 2013). Using a monoclonal antibody (MAb) generated against this peptide, we found that this peptide epitope could be an attractive antiviral target.
Section snippets
Antibodies against the universally conserved epitope in NA
A rabbit MAb against the highly conserved sequence was generated (Gravel et al., 2011, Gravel et al., 2010, Chun et al., 2008). The MAb, denoted as HCA-2 MAb, was used in all experiments reported here.
Viruses
Influenza A strains used to investigate the heterosubtypic inhibitory effect of HCA-2 MAb were propagated at 37 °C in the allantoic cavities of 10-day-old embryonated hen eggs for 24 h (hrs). Allantoic fluid was clarified by centrifugation, aliquoted and stored at −80 °C until used. Viruses were
HCA-2 MAb inhibits diverse strains of influenza viruses
The MAb was determined for its inhibitory activities against all 9 NA subtypes of influenza viruses. As shown in Fig. 1, the MAb resulted in a significant decrease in NA enzymatic activity of all NA subtypes in ELLA assay. Specifically, HCA-2 MAb inhibited NA activity in a dose response manner in which enzymatic activity of all NA subtypes were reduced by 80–100% at the highest concentration of the MAb in comparison to the IgG control which did not cause any reduction in NA activity. This
Discussion
The importance of NA in inducing protective antibodies has been well documented (Marcelin et al., 2012). Recent publications suggest that polyclonal antibodies against NA can induce cross-protection against diverse strains within the same subtype (Marcelin et al., 2011, Sandbulte et al., 2007), but NA heterosubtypic protection, i.e., antibody generated by immunization of one NA subtype capable of protecting against a different NA subtypes, has not yet been reported (Johansson and Cox, 2011).
Conclusions
In conclusion, during the course of infection, this universal peptide could be sufficiently exposed to allow access by macromolecules as large as an antibody, making it worthwhile being considered for future antiviral intervention and vaccine development. This notion could be substantiated by the recent findings that the epitope is crucial for effective virus replication (Doyle et al., 2013).
Competing financial interests statement
The authors declare no conflict of interest.
Acknowledgements
Jianjun Jia and Suzie Wang are acknowledged for their assistance with the animal study; Monika Tocchi is thanked for editing the manuscript. The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health and Ageing. AMH is supported by King Abdulaziz University. This work was supported by the Canadian Regulatory Strategy for Biotechnology.
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These two authors contributed to this work equally.