A Bayesian approach to incorporate structural data into the mapping of genotype to antigenic phenotype of influenza A(H3N2) viruses.

Surface antigens of pathogens are commonly targeted by vaccine-elicited antibodies but antigenic variability, notably in RNA viruses such as influenza, HIV and SARS-CoV-2, pose challenges for control by vaccination. For example, influenza A(H3N2) entered the human population in 1968 causing a pandemic and has since been monitored, along with other seasonal influenza viruses, for the emergence of antigenic drift variants through intensive global surveillance and laboratory characterisation. Statistical models of the relationship between genetic differences among viruses and their antigenic similarity provide useful information to inform vaccine development, though accurate identification of causative mutations is complicated by highly correlated genetic signals that arise due to the evolutionary process. Here, using a sparse hierarchical Bayesian analogue of an experimentally validated model for integrating genetic and antigenic data, we identify the genetic changes in influenza A(H3N2) virus that underpin antigenic drift. We show that incorporating protein structural data into variable selection helps resolve ambiguities arising due to correlated signals, with the proportion of variables representing haemagglutinin positions decisively included, or excluded, increased from 59.8% to 72.4%. The accuracy of variable selection judged by proximity to experimentally determined antigenic sites was improved simultaneously. Structure-guided variable selection thus improves confidence in the identification of genetic explanations of antigenic variation and we also show that prioritising the identification of causative mutations is not detrimental to the predictive capability of the analysis. Indeed, incorporating structural information into variable selection resulted in a model that could more accurately predict antigenic assay titres for phenotypically-uncharacterised virus from genetic sequence. Combined, these analyses have the potential to inform choices of reference viruses, the targeting of laboratory assays, and predictions of the evolutionary success of different genotypes, and can therefore be used to inform vaccine selection processes.

The health of influenza surveillance and pandemic preparedness in the wake of the COVID-19 pandemic.

The COVID-19 pandemic is the first to have emerged when Next Generation Sequencing was readily available and it has played the major role in following evolution of the causative agent, Severe Acute Respiratory Syndrome Coronavirus 2. Response to the pandemic was greatly facilitated though use of existing influenza surveillance networks: World Health Organization (WHO) Global Influenza Surveillance and Response System (GISRS), focussing largely on human influenza, and the OFFLU network of expertise on avian influenza established by the Food and Agricultural Organization of the United Nations (FAO) and the World Organization for Animal Health (WOAH). Data collection/deposition platforms associated with these networks, notably WHO's FluNet and the Global Initiative on Sharing All Influenza Data (GISAID) were/are being used intensely. Measures introduced to combat COVID-19 resulted in greatly decreased circulation of human seasonal influenza viruses for approximately 2 years, but circulation continued in the animal sector with an upsurge in the spread of highly pathogenic avian influenza subtype H5N1 with large numbers of wild bird deaths, culling of many poultry flocks and sporadic spill over into mammalian species, including humans, thereby increasing pandemic risk potential. While there are proposals/implementations to extend use of GISRS and GISAID to other infectious disease agents (e.g. Respiratory Syncytial Virus and Monkeypox), there is need to ensure that influenza surveillance is maintained and improved in both human and animal sectors in a sustainable manner to be truly prepared (early detection) for the next influenza pandemic.

Temporal and Gene Reassortment Analysis of Influenza C Virus Outbreaks in Hong Kong, SAR, China.

From 2014 to week 07/2020 the Centre for Health Protection in Hong Kong conducted screening for influenza C virus (ICV). A retrospective analysis of ICV detections to week 26/2019 revealed persistent low-level circulation with outbreaks occurring biennially in the winters of 2015 to 2016 and 2017 to 2018 (R. S. Daniels et al., J Virol 94:e01051-20, 2020, https://doi.org/10.1128/JVI.01051-20). Here, we report on an outbreak occurring in 2019 to 2020, reinforcing the observation of biennial seasonality in Hong Kong. All three outbreaks occurred in similar time frames, were subsequently dwarfed by seasonal epidemics of influenza types A and B, and were caused by similar proportions of C/Kanagawa/1/76 (K)-lineage and C/São Paulo/378/82 S1- and S2-sublineage viruses. Ongoing genetic drift was observed in all genes, with some evidence of amino acid substitution in the hemagglutinin-esterase-fusion (HEF) glycoprotein possibly associated with antigenic drift. A total of 61 ICV genomes covering the three outbreaks were analyzed for reassortment, and 9 different reassortant constellations were identified, 1 K-lineage, 4 S1-sublineage, and 4 S2-sublineage, with 6 of these being identified first in the 2019-1920 outbreak (2 S2-lineage and 4 S1-lineage). The roles that virus interference/enhancement, ICV persistent infection, genome evolution, and reassortment might play in the observed seasonality of ICV in Hong Kong are discussed. IMPORTANCE Influenza C virus (ICV) infection of humans is common, with the great majority of people being infected during childhood, though reinfection can occur throughout life. While infection normally results in "cold-like" symptoms, severe disease cases have been reported in recent years. However, knowledge of ICV is limited due to poor systematic surveillance and an inability to propagate the virus in large amounts in the laboratory. Following recent systematic surveillance in Hong Kong SAR, China, and direct ICV gene sequencing from clinical specimens, a 2-year cycle of disease outbreaks (epidemics) has been identified, with gene mixing playing a significant role in ICV evolution. Studies like those reported here are key to developing an understanding of the impact of influenza C virus infection in humans, notably where comorbidities exist and severe respiratory disease can develop.

Correction: Breadth and function of antibody response to acute SARS-CoV-2 infection in humans.

[This corrects the article DOI: 10.1371/journal.ppat.1009352.].

Correction: Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans.

[This corrects the article DOI: 10.1371/journal.ppat.1009330.].

Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans.

Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations.

Breadth and function of antibody response to acute SARS-CoV-2 infection in humans.

Serological and plasmablast responses and plasmablast-derived IgG monoclonal antibodies (MAbs) have been analysed in three COVID-19 patients with different clinical severities. Potent humoral responses were detected within 3 weeks of onset of illness in all patients and the serological titre was elicited soon after or concomitantly with peripheral plasmablast response. An average of 13.7% and 3.5% of plasmablast-derived MAbs were reactive with virus spike glycoprotein or nucleocapsid, respectively. A subset of anti-spike (10 of 32) antibodies cross-reacted with other betacoronaviruses tested and harboured extensive somatic mutations, indicative of an expansion of memory B cells upon SARS-CoV-2 infection. Fourteen of 32 anti-spike MAbs, including five anti-receptor-binding domain (RBD), three anti-non-RBD S1 and six anti-S2, neutralised wild-type SARS-CoV-2 in independent assays. Anti-RBD MAbs were further grouped into four cross-inhibiting clusters, of which six antibodies from three separate clusters blocked the binding of RBD to ACE2 and five were neutralising. All ACE2-blocking anti-RBD antibodies were isolated from two recovered patients with prolonged fever, which is compatible with substantial ACE2-blocking response in their sera. Finally, the identification of non-competing pairs of neutralising antibodies would offer potential templates for the development of prophylactic and therapeutic agents against SARS-CoV-2.

Influenza returns with a season dominated by clade 3C.2a1b.2a.2 A(H3N2) viruses, WHO European Region, 2021/22.

In the WHO European Region, COVID-19 non-pharmaceutical interventions continued slowing influenza circulation in the 2021/22 season, with reduced characterisation data. A(H3) predominated and, in some countries, co-circulated with A(H1)pdm09 and B/Victoria viruses. No B/Yamagata virus detections were confirmed. Substantial proportions of characterised circulating virus subtypes or lineages differed antigenically from their respective northern hemisphere vaccine components. Appropriate levels of influenza virus characterisations should be maintained until the season end and in future seasons, when surveillance is adapted to integrate SARS-CoV-2.

Reduced sialidase activity of influenza A(H3N2) neuraminidase associated with positively charged amino acid substitutions.

Neuraminidase (NA) inhibitors (NAI), oseltamivir and zanamivir, are the main antiviral medications for influenza and monitoring of susceptibility to these antivirals is routinely done by determining 50 % inhibitory concentrations (IC50) with MUNANA substrate. During 2010-2019, levels of A(H3N2) viruses presenting reduced NAI inhibition (RI) were low (~0.75 %) but varied year-on-year. The highest proportions of viruses showing RI were observed during the 2013-2014, 2016-2017 and 2017-2018 Northern Hemisphere seasons. The majority of RI viruses were found to contain positively charged NA amino acid substitutions of N329K, K/S329R, S331R or S334R, being notably higher during the 2016-2017 season. Sialidase activity kinetics were determined for viruses of RI phenotype and contemporary wild-type (WT) viruses showing close genetic relatedness and displaying normal inhibition (NI). RI phenotypes resulted from reduced sialidase activity compared to relevant WT viruses. Those containing S329R or N329K or S331R showed markedly higher Km for the substrate and Ki values for NAIs, while those with S334R showed smaller effects. Substitutions at N329 and S331 disrupt a glycosylation sequon (NDS), confirmed to be utilised by mass spectrometry. However, gain of positive charge at all three positions was the major factor influencing the kinetic effects, not loss of glycosylation. Because of the altered enzyme characteristics NAs carrying these substitutions cannot be assessed reliably for susceptibility to NAIs using standard MUNANA-based assays due to reductions in the affinity of the enzyme for its substrate and the concentration of the substrate usually used.

Broadly Inhibiting Antineuraminidase Monoclonal Antibodies Induced by Trivalent Influenza Vaccine and H7N9 Infection in Humans.

The majority of antibodies induced by influenza neuraminidase (NA), like those against hemagglutinin (HA), are relatively specific to viruses isolated within a limited time window, as seen in serological studies and the analysis of many murine monoclonal antibodies (MAbs). We report three broadly reactive human MAbs targeting N1 NA. Two were isolated from a young adult vaccinated with trivalent influenza vaccine (TIV), which inhibited N1 NA from viruses isolated from humans over a period of a hundred years. The third antibody, isolated from a child with acute mild H7N9 infection, inhibited both group 1 N1 and group 2 N9 NAs. In addition, the antibodies cross-inhibited the N1 NAs of highly pathogenic avian H5N1 influenza viruses. These antibodies are protective in prophylaxis against seasonal H1N1 viruses in mice. This study demonstrates that human antibodies to N1 NA with exceptional cross-reactivity can be recalled by vaccination and highlights the importance of standardizing the NA antigen in seasonal vaccines to offer optimal protection.IMPORTANCE Antibodies to the influenza virus NA can provide protection against influenza disease. Analysis of human antibodies to NA lags behind that of antibodies to HA. We show that human monoclonal antibodies against NA induced by vaccination and infection can be very broadly reactive, with the ability to inhibit a wide spectrum of N1 NAs on viruses isolated between 1918 and 2018. This suggests that antibodies to NA may be a useful therapy and that the efficacy of influenza vaccines could be enhanced by ensuring the appropriate content of NA antigen.