Comparison of the Efficacy of N9 Neuraminidase-Specific Monoclonal Antibodies against Influenza A(H7N9) Virus Infection

Abstract

The fifth wave of A(H7N9) virus infection in China from 2016 to 2017 caused great concern due to the large number of individuals infected, the isolation of drug-resistant viruses, and the emergence of highly pathogenic strains. Antibodies against neuraminidase (NA) provide added benefit to hemagglutinin-specific immunity and may be important contributors to the effectiveness of A(H7N9) vaccines. We generated a panel of mouse monoclonal antibodies (MAbs) to identify antigenic domains on NA of the novel A(H7N9) virus and compared their functional properties. The loop formed in the region of residue 250 (250 loop) and the domain formed by the loops containing residues 370, 400, and 430 were identified as major antigenic regions. MAbs 1E8, 2F6, 10F4, and 11B2, which recognize these two antigenic domains, were characterized in depth. These four MAbs differ in their abilities to inhibit cleavage of small and large substrates (methyl-umbelliferylacetyl neuraminic acid [MU-NANA] and fetuin, respectively) in NA inhibition assays. 1E8 and 11B2 did not inhibit NA cleavage of either MU-NANA or fetuin, and 2F6 inhibited cleavage of fetuin alone, whereas 10F4 inhibited cleavage of both substrates. All four MAbs reduced the in vitro spread of viruses carrying either the wild-type N9 or N9 with antiviral-resistant mutations but to different degrees. These MAbs have different in vivo levels of effectiveness: 10F4 was the most effective in protecting mice against challenge with A(H7N9) virus, 2F6 was less effective, and 11B2 failed to protect BALB/c mice at the doses tested. Our study confirms that NA-specific antibodies can protect against A(H7N9) infection and suggests that in vitro properties can be used to rank antibodies with therapeutic potential. IMPORTANCEThe novel A(H7N9) viruses that emerged in China in 2013 continue to infect humans, with a high fatality rate. The most recent outbreak resulted in a larger number of human cases than previous epidemic waves. Due to the absence of a licensed vaccine and the emergence of drug-resistant viruses, there is a need to develop alternative approaches to prevent or treat A(H7N9) infection. We have made a panel of mouse monoclonal antibodies (MAbs) specific for neuraminidase (NA) of A(H7N9) viruses; some of these MAbs are effective in inhibiting viruses that are resistant to antivirals used to treat A(H7N9) patients. Binding avidity, inhibition of NA activity, and plaque formation correlated with the effectiveness of these MAbs to protect mice against lethal A(H7N9) virus challenge. This study identifies in vitro measures that can be used to predict the in vivo efficacy of NA-specific antibodies, providing a way to select MAbs for further therapeutic development.

The neuraminidase of A(H3N2) influenza viruses circulating since 2016 is antigenically distinct from the A/Hong Kong/4801/2014 vaccine strain

“…SGP/16, the A(H3N2) vaccine virus for the 2018–19 Northern Hemisphere and 2018 Southern Hemisphere influenza seasons, is typical of recently circulating A(H3N2) viruses that possessed the potential NA245 glycosylation site motif (N245/A246/T247). We used reassortant A(H6N2) viruses in Western blot and other assays in the present study to be consistent with using such viruses in NA inhibition (NI) assays 17,18 and in our mouse challenge experiments. As shown in Fig.…”

The Protective Effects of the A/ZJU01/ PR8/2013 Split H7N9 Avian Influenza Vaccine Against Highly Pathogenic H7N9 in BALB/c Mice

“…Secondly, the NA is another significant antigen. The antibodies induced by NA provide additional benefit to hemagglutinin-specific immunity and may be an important contributor to the effectiveness of H7N9 vaccines [19]. Two major antigenic regions of NA, the 250-loop and 370/400/430-loop domains were found the same between A/Zhejiang/DTID-ZJU01/2013 (H7N9) and A/Guangdong/HP001/2017(H7N9).…”

“…Moreover, a fifth H7N9 epidemic began earlier in the season, and had spread to more districts and counties in affected provinces. Worryingly, it involved more confirmed cases than previous epidemics [18, 19]. The isolation of drug-resistant viruses and emergence of highly pathogenic strains in chickens and humans has become a domestic and international concern [7, 20, 21].…”

Aerosol Transmission from Infected Swine to Ferrets of an H3N2 Virus Collected from an Agricultural Fair and Associated with Human Variant Infections

“…Additional studies assessing the balance of 2010.1 H3 with NA of each 2002 NA clade are warranted given the fact that these combinations represent the majority of recent variant H3N2 spillover into humans (22). Antibodies inhibiting NA activity are known to reduce the severity and duration of IAV-mediated disease in humans (52,53) and protect mice from lethal IAV challenge (54,55). The presence of neuraminidase antibodies in humans following vaccination or natural infection (56,57) suggests that NA-mediated immunity may provide some protection against zoonotic infection, though a more comprehensive understanding will be required to assess the contributions of NA-inhibiting antibodies in protection, particularly N2 genes that have been evolving in swine for more than 15 years.…”