Immunity induced by vaccination with recombinant influenza B virus neuraminidase protein breaks viral transmission chains in guinea pigs in an exposure intensity-dependent manner.

IMPORTANCE: Vaccines that can slow respiratory virus transmission in the population are urgently needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus. Here, we describe how a recombinant neuraminidase-based influenza virus vaccine reduces transmission in vaccinated guinea pigs in an exposure intensity-based manner.

Antibodies targeting the neuraminidase active site inhibit influenza H3N2 viruses with an S245N glycosylation site.

Contemporary influenza A H3N2 viruses circulating since 2016 have acquired a glycosylation site in the neuraminidase in close proximity to the enzymatic active site. Here, we investigate if this S245N glycosylation site, as a result of antigenic evolution, can impact binding and function of human monoclonal antibodies that target the conserved active site. While we find that a reduction in the inhibitory ability of neuraminidase active site binders is measurable, this class of broadly reactive monoclonal antibodies maintains protective efficacy in vivo.

Assessment of a quadrivalent nucleoside-modified mRNA vaccine that protects against group 2 influenza viruses.

Combined vaccine formulations targeting not only hemagglutinin but also other influenza virus antigens could form the basis for a universal influenza virus vaccine that has the potential to elicit long-lasting, broadly cross-reactive immune responses. Lipid nanoparticle (LNP)-encapsulated messenger RNA (mRNA) vaccines can be utilized to efficiently target multiple antigens with a single vaccine. Here, we assessed the immunogenicity and protective efficacy of nucleoside-modified mRNA-LNP vaccines that contain four influenza A group 2 virus antigens (hemagglutinin stalk, neuraminidase, matrix protein 2, and nucleoprotein) in mice. We found that all vaccine components induced antigen-specific cellular and humoral immune responses after administration of a single dose. While the monovalent formulations were not exclusively protective, the combined quadrivalent formulation protected mice from all challenge viruses, including a relevant H1N1 influenza virus group 1 strain, with minimal weight loss. Importantly, the combined vaccine protected from morbidity at a dose of 125 ng per antigen after a single vaccination in mice. With these findings, we confidently conclude that the nucleoside-modified mRNA-LNP platform can be used to elicit protection against a large panel of influenza viruses.

Immunity induced by vaccination with recombinant influenza B virus neuraminidase protein breaks viral transmission chains in guinea pigs in an exposure intensity-dependent manner.

Mucosal vaccines and vaccines that block pathogen transmission are under-appreciated in vaccine development. However, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has shown that blocking viral transmission is an important attribute of efficient vaccines. Here, we investigated if recombinant influenza virus neuraminidase (NA) vaccines delivered at a mucosal site could protect from onward transmission of influenza B viruses in the guinea pig model. We tested four different scenarios in which sequential transmission was investigated in chains of four guinea pigs. The variables tested included a low and a high viral inoculum (104 vs 105 plaque forming units) in the initial donor guinea pig and variation of exposure/cohousing time (1 day vs 6 days). In three out of four scenarios - low inoculum-long exposure, low inoculum-short exposure and high inoculum-short exposure - transmission chains were efficiently blocked. Based on this data we believe an intranasal recombinant NA vaccine could be used to efficiently curtail influenza virus spread in the human population during influenza epidemics.

Murine Broadly Reactive Antineuraminidase Monoclonal Antibodies Protect Mice from Recent Influenza B Virus Isolates and Partially Inhibit Virus Transmission in the Guinea Pig Model.

Current influenza virus vaccines and antivirals have limitations, some of which disproportionately affect their utilization against influenza B viruses. To inform ongoing efforts to address the considerable global burden of influenza B viruses, we previously described five murine monoclonal antibodies that broadly bind conserved epitopes on the neuraminidase of influenza B viruses and protect against lethal challenge in a mouse model when delivered via intraperitoneal injection. Here, we validate the continued relevance of these antibodies by demonstrating that their protective effects extend to lethal challenge with mouse-adapted influenza B viruses recently isolated from humans. We also found that humanization of murine antibodies 1F2 and 4F11 resulted in molecules that retain the ability to protect mice from lethal challenge when administered prophylactically. Intranasal administration as an alternative route of 1F2 delivery revealed no differences in the mouse challenge model compared to intraperitoneal injection, supporting further assessment of this more targeted and convenient administration method. Lastly, we evaluated the potential for intranasal 1F2 administration initiated 1 day after infection to prevent transmission of an influenza B virus between cocaged guinea pigs. Here, we observed a 40% rate of transmission with the 1F2 antibody administered to the infected donor compared to 100% transmission with administration of an irrelevant control antibody. These data suggest that intranasal administration could be a viable route of administration for antibody therapeutics. Collectively, these findings demonstrate the potential of broad antineuraminidase antibodies as therapeutics to prevent and treat infections caused by influenza B viruses. IMPORTANCE The global health burden of influenza B viruses, especially in children, has long been underappreciated. Although two antigenically distinct influenza B virus lineages cocirculated before the coronavirus disease 2019 (COVID-19) pandemic, the commonly used trivalent seasonal vaccines contain antigens from only one influenza B virus, providing limited cross-protection against viruses of the other lineage. Additionally, studies have called into question the clinical effectiveness of the neuraminidase inhibitors that comprise the majority of available antivirals in treating influenza B virus infections. We previously described antibodies that bind broadly to neuraminidases of influenza B viruses across decades of antigenic evolution and potently protect mice against lethal challenge. Here we appraise additional factors to develop these antineuraminidase antibodies as antivirals to prevent and treat infections caused by an extensive range of influenza B viruses. In addition this work assesses recent clinical isolates belonging to the two influenza B virus lineages, finding evidence supporting the development of these antibodies for prophylactic and therapeutic use.

Development of a pentavalent broadly protective nucleoside-modified mRNA vaccine against influenza B viruses.

Messenger RNA (mRNA) vaccines represent a new, effective vaccine platform with high capacity for rapid development. Generation of a universal influenza virus vaccine with the potential to elicit long-lasting, broadly cross-reactive immune responses is a necessity for reducing influenza-associated morbidity and mortality. Here we focus on the development of a universal influenza B virus vaccine based on the lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) platform. We evaluate vaccine candidates based on different target antigens that afford protection against challenge with ancestral and recent influenza B viruses from both antigenic lineages. A pentavalent vaccine combining all tested antigens protects mice from morbidity at a very low dose of 50 ng per antigen after a single vaccination. These findings support the further advancement of nucleoside-modified mRNA-LNPs expressing multiple conserved antigens as universal influenza virus vaccine candidates.

Human Anti-neuraminidase Antibodies Reduce Airborne Transmission of Clinical Influenza Virus Isolates in the Guinea Pig Model.

The public health burden caused by influenza virus infections is not adequately addressed with existing vaccines and antivirals. Identifying approaches that interfere with human-to-human transmission of influenza viruses remains a pressing need. The importance of neuraminidase (NA) activity for the replication and spread of influenza viruses led us to investigate whether broadly reactive human anti-NA monoclonal antibodies (MAbs) could affect airborne transmission of the virus using the guinea pig model. In that model, infection with recent influenza virus clinical isolates resulted in 100% transmission from inoculated donors to recipients in an airborne transmission setting. Anti-NA MAbs were administered either to the inoculated animals on days 1, 2, and 4 after infection or to the naive contacts on days 2 and 4 after donor infection. Administration of NA-1G01, a broadly cross-reactive anti-NA MAb, to either the donor or recipient reduced transmission of the A/New York City/PV02669/2019 (H1N1) and A/New York City/PV01148/2018 (H3N2) viruses. Administration of 1000-3C05, an anti-N1 MAb, to either the donor or recipient reduced transmission of A/New York City/PV02669/2019 (H1N1) virus but did not reduce transmission of A/New York City/PV01148 (H3N2) virus. Conversely, 229-2C06, an anti-N2 MAb, reduced transmission of A/New York City/PV01148 (H3N2) but did not impact transmission of A/New York City/PV02669/2019 (H1N1) virus. Our work demonstrates that anti-NA MAbs could be further developed into prophylactic or therapeutic agents to prevent influenza virus transmission to control viral spread. IMPORTANCE The burden of influenza remains substantial despite unremitting efforts to reduce the magnitude of seasonal influenza epidemics and prepare for pandemics. Although vaccination remains the mainstay of these efforts, current vaccines are designed to stimulate an immune response against the viral hemagglutinin. Interest in the role immunity against neuraminidase plays in influenza virus infection and transmission has recently surged. Human antibodies that bind broadly to neuraminidases of diverse influenza viruses and protect mice against lethal viral challenge have previously been characterized. Here, we show that three such antibodies inhibit the neuraminidase activity of recent isolates and reduce their airborne transmission in a guinea pig model. In addition to contributing to the accumulating support for incorporating neuraminidase as a vaccine antigen, these findings also demonstrate the potential of direct administration of anti-neuraminidase antibodies to individuals infected with influenza virus and to individuals for postexposure prophylaxis to prevent the spread of influenza virus.

Broadly neutralizing antibodies target a haemagglutinin anchor epitope.

Broadly neutralizing antibodies that target epitopes of haemagglutinin on the influenza virus have the potential to provide near universal protection against influenza virus infection1. However, viral mutants that escape broadly neutralizing antibodies have been reported2,3. The identification of broadly neutralizing antibody classes that can neutralize viral escape mutants is critical for universal influenza virus vaccine design. Here we report a distinct class of broadly neutralizing antibodies that target a discrete membrane-proximal anchor epitope of the haemagglutinin stalk domain. Anchor epitope-targeting antibodies are broadly neutralizing across H1 viruses and can cross-react with H2 and H5 viruses that are a pandemic threat. Antibodies that target this anchor epitope utilize a highly restricted repertoire, which encodes two public binding motifs that make extensive contacts with conserved residues in the fusion peptide. Moreover, anchor epitope-targeting B cells are common in the human memory B cell repertoire and were recalled in humans by an oil-in-water adjuvanted chimeric haemagglutinin vaccine4,5, which is a potential universal influenza virus vaccine. To maximize protection against seasonal and pandemic influenza viruses, vaccines should aim to boost this previously untapped source of broadly neutralizing antibodies that are widespread in the human memory B cell pool.