Glycosylation of Residue 141 of Subtype H7 Influenza A Hemagglutinin (HA) Affects HA-Pseudovirus Infectivity and Sensitivity to Site A Neutralizing Antibodies

Human infections with H7 subtype influenza virus have been reported, including an H7N7 outbreak in Netherlands in 2003 and H7N9 infections in China in 2013. Previously, we reported murine monoclonal antibodies (mAbs) that recognize the antigenic site A of H7 hemagglutinin (HA). To better understand protective immunity of H7 vaccines and vaccine candidate selection, we used these mAbs to assess the antigenic relatedness among two H7 HA isolated from past human infections and determine residues that affect susceptibility to neutralization. We found that these mAbs neutralize pseudoviruses bearing HA of A/Shanghai/02/2013(H7N9), but not A/Netherlands/219/2003(H7N7). Glycosylation of the asparagine residue at position 141 (N141) (N133, H3 HA numbering) in the HA of A/Netherlands/219/2003 HA is responsible for this resistance, and it affects the infectivity of HA-pseudoviruses. The presence of threonine at position 143 (T135, H3 HA numbering) in the HA of A/Netherlands/219/2003, rather than an alanine found in the HA of A/Shanghai/02/2013(H7N9), accounts for these differences. These results demonstrate a key role for glycosylation of residue N141 in affecting H7 influenza HA-mediated entry and sensitivity to neutralizing antibodies, which have implications for candidate vaccine design.


Introduction
Avian influenza viruses with potential to acquired human-to-human transmission are a public health concern. Human infections with H7 subtype influenza virus have been documented in people who had direct contact with infected birds or humans with influenza infection [1,2]. More recently, a novel avian influenza virus A (H7N9) emerged in China in 2013 with about 36% fatality rate [3], although there is no evidence of sustained person-to-person spread [4].
Because of the concerns of potential future H7 pandemics, several candidate vaccine strains for the H7 subtype were developed before the 2013 H7N9 outbreak [5][6][7]. The immunogenicity of these earlier H7 vaccines was poor [8,9]. More recently, candidate H7N9 vaccines derived from A/Shanghai/02/2013 have been prepared [10] and tested for immunogenicity in clinical trials [11][12][13][14]. However the protective immunity induced by H7 vaccines is not completely understood. The glycosylation of HA can influence its antigenicity and thus could play an important role in candidate vaccine strain selection and vaccination effectiveness. Different cell substrates can also affect glycosylation [15,16]. It has been demonstrated that gain of a sidechain, resulting in an antigenic change by preventing antibody binding, can have a selective advantage [15,16].
To better understand immunity to H7 influenza and vaccine candidate selection, we assessed antigenic relatedness among H7 HA. We found that glycosylation of an asparagine residue at position 141 (N133, H3 HA numbering) in hemagglutinin plays an important role in modifying the infectivity and neutralization sensitivity of pseudoviruses bearing HA from A/ Shanghai/02/2013 and A/Netherlands/219/2003.

Neutralization assay
HA-pseudoviruses containing approximately 15 ng/ml of p24 antigen and 12 ng/ml of HA were incubated with mAb samples for 1 hr at 37°C, prior to inoculating mixtures onto 293T cells, as described previously [20]. The mAb dilution causing a 95% reduction of luciferase activity compared to control (IC95), calculated using Graphpad Prism software, was used as the neutralization titer [20].

Hemagglutination inhibition and ELISA assays
The hemagglutination inhibition assay (HI) was performed in 96-well plates (V-bottom) by a standard method, essentially as described in the WHO Manual on Animal Influenza Diagnosis and Surveillance (http://www.who.int/csr/resources/publications/influenza/en/ whocdscsrncs20025rev.pdf) using 0.5% turkey red blood cells (Lampire Biological Laboratories, Pipersville, PA) suspended in PBS (pH 7.2). Standard ELISA assay was applied to detect the binding of H7 mAb to HA-pseudovirus. Briefly, HA-pseudoviruses were coated onto ELISA plates (10 μg/ml, 100 μl/well). The virus-coated plates were blocked and then incubated with mAb at different concentrations. After an additional incubation with peroxidase-conjugated goat anti-mouse IgG (KPL, Gaithersburg, MD), the signal was developed using TMB as substrate. The reaction was stopped with 1N H 2 SO 4 and OD450 values were recorded.

Deglycosylation of HA
To determine the occupancy by a glycan of aspartic acid in the predicted glycosylation site in HA, HA-pseudoviruses were digested with Peptide: N-Glycosidase F (PNGase F) kit (New England Biolabs, Ipswich, MA) according to the manufactured instructions. Briefly, HA-pseudoviruses made in serum-free media were pelleted by centrifugation for 3 hr at 13,000 x g, then re-suspended in phosphate-buffered saline (PBS) containing 1% NP40. HA-pseudoviruses were denatured with 1X Glycoprotein Denaturing Buffer at 100°C for 10 min. After the addition of G7 Reaction Buffer and PNGase F, the mixtures were incubated for 1hr at 37°C. Samples were resolved on SDS-PAGE, and detected by Western blot using Goat antisera against A/ Netherlands/219/2003 HA.

Data analysis
Infectivity data reported were from at least three independent experiments. T test for paired data comparison and corresponding P value were analyzed using GraphPad Prism software. P values <0.05 were considered statistically significant.

5A6, 4A2 and 2C4 mAbs neutralize A/Shanghai/02/2013, but not A/ Netherlands/219/2003
We previously described a panel of H7 monoclonal antibodies (mAbs) (5A6, 4A2 and 2C4) that recognize antigenic site A in the HA of A/Shanghai/02/2013(H7N9) [21], which is structurally similar to H3 HA. Because this site is conserved across H7 strains, it is a potentially important protective epitope for cross-reactive antibodies. We compared the neutralizing activity of these mAbs against pseudoviruses bearing HA of A/Netherlands/219/2003 and A/ Shanghai/02/2013, two H7 viruses isolated from human infections of H7N7 in 2003 [26] and H7N9 in 2013 [27]. Both viruses are Eurasian lineage viruses and are similar in sequence, differing at only 11 amino acids in the mature HA1 portion of the hemagglutinin (excluding the polybasic cleavage site at the junction of HA1 and HA2 in H7N7). Despite the conservation of amino acids in site A, we found that these mAbs (5A6, 4A2 and 2C4) only neutralized A/ Shanghai/02/2013(H7N9) HA-pseudoviruses, and not A/Netherlands/219/2003(H7N7) HApseudoviruses. As expected, the A/Puerto Rico/8/1934(H1N1) HA-pseudovirus was also not neutralized by the H7 mAbs (Table 1).
To determine whether T143 or G195 is responsible for A/Netherlands/219/2003 resistance to neutralizing mAbs that target antigenic site A, we introduced mutations at these positions into A/Shanghai/02/2013 HA and A/Netherlands/219/2003 HA. In both neutralization and hemagglutination inhibition assays, we found that A/Shanghai/02/2013 HA-pseudoviruses with an alanine to threonine substitution at position 143 (A143T) in HA became resistant to all mAbs in our panel, while A/Netherlands/219/2003 HA-pseudoviruses bearing a threonine to alanine substitution at position 143 (T143A) in HA gained sensitivity to these neutralizing mAbs (Tables 1 and 2). Notably, neutralization sensitivity was not changed by other substitutions, including a serine to aspartic acid substitution at position 183 (S183D), a valine to glycine substitution at position 195 (V195G), and an alanine to threonine substitution at position 198   (Tables 1 and 2). These results indicate that T143 alone is responsible for resistance of A/Netherlands/219/2003 to neutralizing mAbs that target antigenic site A. Previously, we generated an escape mutant virus with an arginine to glycine substitution at position 149 (R149G) in A/Shanghai/02/2013 HA that confers resistance to these neutralizing mAbs [21]

Glycosylation of asparagine 141 is occupied in A/Netherlands/219/2003 HA
To understand the resistance mechanism conferred by T143, we analyzed the HA sequence and found that T143 in A/Netherlands/219/2003 HA creates a glycosylation motif (N-X-T/S) involving asparagine 141 (N141) (N133, H3 numbering). Western blot analysis showed that A/ Shanghai/02/2013 HA containing A143T has a higher molecular weight than wild type A/ Shanghai/02/2013 HA (Fig 1C). Similarly, the wild-type A/Netherlands/219/2003 HA with T143 has a higher molecular weight than A/Netherlands/219/2003 HA with T143A. Moreover, PNGase F treatment, which removes the N-linked glycans, resulted in a decrease of the molecular weight of both A/Shanghai/02/2013 HA with A143T and wild type A/Netherlands/219/ 2003 HA that contains T143 (Fig 1C). These results suggest that N141 is glycosylated when a threonine is present at position 143 (T143) in the H7 HA, and thus glycosylation of N141 is responsible for the lack of sensitivity of A/Netherlands/219/2003 to neutralizing mAbs that target to antigenic site A. The more efficient processing of HA0 in A/Shanghai/02/2013 HA-pseudoviruses compared to A/Netherlands/219/2003 HA-pseudoviruses (Fig 1C) is due to the addition a protease (HAT) expression vector during the preparation of A/Shanghai/02/2013 HA-pseudoviruses. Because A/Netherlands/219/2003 HA contains a polybasic sequence that can be cleaved during biogenesis by cellular proteases such as furin, a protease expression vector is not used to produce HA-pseudoviruses for strains with the polybasic cleavage site.

Glycosylation prevents binding of neutralizing mAbs to antigenic site A
We next investigated the mechanism of resistance to neutralization conferred by glycosylation of N141. In immunoprecipitation assays using HA-pseudoviruses, we found that the A143T substitution reduced mAb 5A6 binding to A/Shanghai/02/2013 HA approximately four fold, while the S183D, V195G and A198T substitutions did not decrease binding (Fig 2A). As a specificity control, we did not observe mAb 5A6 binding to H1, H3 and H5 HAs (data not shown). Similarly, the T143A substitution, but not D183S, G195V, or T198A substitutions, enhanced A/Netherlands/219/2003 HA binding to mAb 5A6 approximately ten fold (Fig 2B). These results demonstrate that glycosylation of N141 directly blocks the interaction of the neutralizing mAbs with H7 HA.
To further confirm this finding, the binding of H7 mAbs to H7 HA was also evaluated using an ELISA assy. The ELISA results showed that the A143T substitution reduced binding of mAbs 5A6, 4A2 and 2C4 to A/Shanghai/02/2013 HA-pseudoviruses approximately ten fold, while the S183D, V195G and A198T substitutions did not decrease binding ( Table 3). As a specificity control, these mAbs did not bind to the A/Puerto Rico/8/1934 (H1N1) HA-pseudovirus, while the mAb H9-A22, which is specific for this strain, did bind this HA-pseudovirus. Similarly, the T143A substitution, but not D183S, G195V, or T198A substitutions, enhanced binding of mAbs 5A6, 4A2 and 2C4 to A/Netherlands/219/2003 HA-pseudoviruses approximately ten to twenty fold (Table 3).

Discussion
Candidate H7 vaccines have been made to prepare for potential future H7 pandemics, but the immunogenicity of these vaccines was generally poor [8,9], and the protective immunity induced by the H7 vaccines is not completely understood. To better understand immune responses to H7 subtypes, we assessed antigenic relatedness of two H7 HA isolated from human outbreaks and found that glycosylation of N141 (N133, H3 HA numbering) has a significant impact on H7 HA-pseudovirus infectivity and sensitivity to neutralizing antibodies that target antigenic site A on HA.
The effects of HA glycosylation on HA antigenicity have been well documented. Gain of a side-chain can prevent antibody binding and lead to antigenic changes [15,16]. HA glycosylation may provide a selective advantage for influenza virus to escape immune-protection [15,16,[32][33][34][35]. Thus, understanding HA glycosylation may be important for selection of candidate vaccine strains, as well as how propagation of vaccine viruses in different cell substrates may affect immunogenicity [15,16]. We previously found that the conserved antigenic site A in H7 HA is a potentially important protective epitope for cross-reactive antibody responses [21]. Our finding that glycosylation of N141, which occurs naturally in the A/Netherlands/219/2003 virus, protects H7 virus from neutralization by site A antibodies again suggests the importance of antigenic site A. The candidate H7 vaccine strains available from WHO [10,36], including recent H7N9 candidate vaccines derived from A/Shanghai/2/213 and A/Anhui/1/2013, as well as older Eurasian H7 viruses derived from A/mallard/Netherlands/12/2000, do not have the glycosylation site at N141. This suggests that an antibody response to antigenic site A elicited by vaccines developed from these candidate vaccine viruses may not contribute to protection against H7 viruses that are glycosylated at N141. While the protective antibody response likely involves multiple HA epitopes, this observation emphasizes the importance of a better understanding of the nature of a protective antibody response to H7 influenza viruses. Similarly, a more complete understanding of the antibody response to candidate vaccines is needed. Interestingly, T143A in A/ Netherlands/219/2003 HA was also recently shown to improve the immunogenicity of A/Netherlands/219/2003 HA [37], further highlighting the role of N141 glycosylation in masking site A of H7 HA.
Unexpectedly, we also found that N141 glycosylation affects pseudovirus infectivity. Both A/Netherlands/219/2003 and A/Shanghai/02/2013 HA-pseudoviruses displayed higher infectivity when residue at 143 was glycosylated. This finding is consistent with previous reports of T143 in A/Netherlands/219/2003 HA being associated with enhanced viral replication [38] and of an A143T substitution in A/Anhui/1/2013 (H7N9) HA emerging during transmission in ferret [39]. Altogether, our data, along with other reports, indicate that glycosylation of N141 in HA plays an important role in H7 viral infectivity and sensitivity to neutralization and should be taken into consideration when selecting strains for H7 vaccines.