Anti-neuraminidase immunity in the combat against influenza.

INTRODUCTION: Anti-neuraminidase (NA) immunity correlates with the protection against influenza virus infection in both human and animal models. The aim of this review is to better understand the mechanism of anti-NA immunity, and also to evaluate the approaches on developing NA-based influenza vaccines or enhancing immune responses against NA for current influenza vaccines. AREAS COVERED: In this review, the structure of influenza neuraminidase, the contribution of anti-NA immunity to protection, as well as the efforts and challenges of targeting the immune responses to NA were discussed. We also listed some of the newly discovered anti-NA monoclonal antibodies and discussed their contribution in therapeutic as well as the antigen design of a broadly protective NA vaccine. EXPERT OPINION: Targeting the immune response to both HA and NA may be critical for achieving the optimal protection since there are different mechanisms of HA and NA elicited protective immunity. Monoclonal antibodies (mAbs) that target the conserved protective lateral face or catalytic sites are effective therapeutics. The epitope discovery using monoclonal antibodies may benefit NA-based vaccine elicited broadly reactive antibody responses. Therefore, the potential for a vaccine that elicits cross-reactive antibodies against neuraminidase is a high priority for next-generation influenza vaccines.

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.

A pan-influenza antibody inhibiting neuraminidase via receptor mimicry.

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift1 and suboptimal immune responses2. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs. FNI9 broadly neutralizes seasonal IAVs and IBVs, including the immune-evading H3N2 strains bearing an N-glycan at position 245, and shows synergistic activity when combined with anti-haemagglutinin stem-directed antibodies. Structural analysis reveals that D107 in the FNI9 heavy chain complementarity-determinant region 3 mimics the interaction of the sialic acid carboxyl group with the three highly conserved arginine residues (R118, R292 and R371) of the neuraminidase catalytic site. FNI9 demonstrates potent prophylactic activity against lethal IAV and IBV infections in mice. The unprecedented breadth and potency of the FNI9 monoclonal antibody supports its development for the prevention of influenza illness by seasonal and pandemic viruses.

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.

Novel Epitopes of the Influenza Virus N1 Neuraminidase Targeted by Human Monoclonal Antibodies.

Influenza virus neuraminidase (NA)-targeting antibodies are an independent correlate of protection against influenza. Antibodies against the NA act by blocking enzymatic activity, preventing virus release and transmission. As we advance the development of improved influenza virus vaccines that incorporate standard amounts of NA antigen, it is important to identify the antigenic targets of human monoclonal antibodies (mAbs). Here, we describe escape mutants generated by serial passage of A/Netherlands/602/2009 (H1N1)pdm09 in the presence of human anti-N1 mAbs. We observed escape mutations on the head domain of the N1 protein around the enzymatic site (S364N, N369T, and R430Q) and also detected escape mutations located on the sides and bottom of the NA (N88D, N270D, and Q313K/R). This work increases our understanding of how human antibody responses target the N1 protein. IMPORTANCE As improved influenza virus vaccines are being developed, the influenza virus neuraminidase (NA) is becoming an important new target for immune responses. By identifying novel epitopes of anti-NA antibodies, we can improve vaccine design. Additionally, characterizing escape mutations in these epitopes aids in identifying NA antigenic drift in circulating viruses.

Multiple Neuraminidase Containing Influenza Virus-like Particle Vaccines Protect Mice from Avian and Human Influenza Virus Infection.

Avian influenza virus remains a threat for humans, and vaccines preventing both avian and human influenza virus infections are needed. Since virus-like particles (VLPs) expressing single neuraminidase (NA) subtype elicited limited heterosubtypic protection, VLPs expressing multiple NA subtypes would enhance the extent of heterosubtypic immunity. Here, we generated avian influenza VLP vaccines displaying H5 hemagglutinin (HA) antigen with or without avian NA subtypes (N1, N6, N8) in different combinations. BALB/c mice were intramuscularly immunized with the VLPs to evaluate the resulting homologous and heterosubtypic immunity upon challenge infections with the avian and human influenza viruses (A/H5N1, A/H3N2, A/H1N1). VLPs expressing H5 alone conferred homologous protection but not heterosubtypic protection, whereas VLPs co-expressing H5 and NA subtypes elicited both homologous and heterosubtypic protection against human influenza viruses in mice. We observed that VLP induced neuraminidase inhibitory activities (NAI), virus-neutralizing activity, and virus-specific antibody (IgG, IgA) responses were strongly correlated with the number of different NA subtype expressions on the VLPs. VLPs expressing all 3 NA subtypes resulted in the highest protection, indicated by the lowest lung titer, negligible body weight changes, and survival in immunized mice. These results suggest that expressing multiple neuraminidases in avian HA VLPs is a promising approach for developing a universal influenza A vaccine against avian and human influenza virus infections.

Contribution of Neuraminidase to the Efficacy of Seasonal Split Influenza Vaccines in the Ferret Model.

Seasonal influenza vaccination takes into account primarily hemagglutinin (HA)-specific neutralizing antibody responses. However, the accumulation of substitutions in the antigenic regions of HA (i.e., antigenic drift) occasionally results in a mismatch between the vaccine and circulating strains. To prevent poor vaccine performance, we investigated whether an antigenically matched neuraminidase (NA) may compensate for reduced vaccine efficacy due to a mismatched HA. Ferrets were vaccinated twice with adjuvanted split inactivated influenza vaccines containing homologous HA and NA (vacH3N2), only homologous HA (vacH3N1), only homologous NA (vacH1N2), heterologous HA and NA (vacH1N1), or phosphate-buffered saline (vacPBS), followed by challenge with H3N2 virus (A/Netherlands/16190/1968). Ferrets vaccinated with homologous HA (vacH3N2 and vacH3N1) displayed minimum fever and weight loss compared to vacH1N1 and vacPBS ferrets, while ferrets vaccinated with NA-matched vacH1N2 displayed intermediate fever and weight loss. Vaccination with vacH1N2 further led to a reduction in virus shedding from the nose and undetectable virus titers in the lower respiratory tract, similarly to when the homologous vacH3N2 was used. Some protection was observed upon vacH1N1 vaccination, but this was not comparable to that observed for vacH1N2, again highlighting the important role of NA in vaccine-induced protection. These results illustrate that NA antibodies can prevent severe disease caused by influenza virus infection and that an antigenically matched NA in seasonal vaccines might prevent lower respiratory tract complications. This underlines the importance of considering NA during the yearly vaccine strain selection process, which may be particularly beneficial in seasons when the HA component of the vaccine is mismatched. IMPORTANCE Despite the availability of vaccines, influenza virus infections continue to cause substantial morbidity and mortality in humans. Currently available influenza vaccines take primarily the hemagglutinin (HA) into account, but the highly variable nature of this protein as a result of antigenic drift has led to a recurrent decline in vaccine effectiveness. While the protective effect of neuraminidase (NA) antibodies has been highlighted by several studies, there are no requirements with regard to quantity or quality of NA in licensed vaccines, and NA immunity remains largely unexploited. Since antigenic changes in HA and NA are thought to occur asynchronously, NA immunity could compensate for reduced vaccine efficacy when drift in HA occurs. By matching and mismatching the HA and NA components of monovalent split inactivated vaccines, we demonstrated the potential of NA immunity to protect against disease, virus replication in the lower respiratory tract, and virus shedding in the ferret model.

Antibodies directed towards neuraminidase restrict influenza virus replication in primary human bronchial epithelial cells.

Influenza neuraminidase (NA) is implicated in various aspects of the virus replication cycle and therefore is an attractive target for vaccination and antiviral strategies. Here we investigated the potential for NA-specific antibodies to interfere with A(H1N1)pdm09 replication in primary human airway epithelial (HAE) cells. Mouse polyclonal anti-NA sera and a monoclonal antibody could block initial viral entry into HAE cells as well as egress from the cell surface. NA-specific polyclonal serum also reduced virus replication across multiple rounds of infection. Restriction of virus entry correlated with the ability of the serum or monoclonal antibody to mediate neuraminidase inhibition (NI). Finally, human sera with NI activity against the N1 of A(H1N1)pdm09 could decrease H6N1 virus infection of HAE cells, highlighting the potential contribution of anti-NA antibodies in the control of influenza virus infection in humans.

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.

Identification of Hemagglutinin Mutations Caused by Neuraminidase Antibody Pressure.

The balance in the functions of hemagglutinin (HA) and neuraminidase (NA) plays an important role in influenza virus genesis. However, whether and how N2 neuraminidase-specific antibodies may affect the attributes of HA remains to be investigated. In this study, we examined the presence of amino acid mutations in the HA of mutants selected by incubation with N2-specific monoclonal antibodies (MAbs) and compared the HA properties to those of the wild-type (WT) A/Chicken/Jiangsu/XXM/1999 (XXM) H9N2 virus. The higher NA inhibition (NI) ability of N2-specific MAbs was found to result in greater proportions of mutations in the HA head. The HA mutations affected the thermal stability, switched the binding preferences from α2,6-linked sialic acid receptor to α2,3-linked sialic acid receptor, and promoted viral growth in mouse lungs. These mutations also caused significant HA antigenic drift as they decreased hemagglutination inhibition (HI) titers. The evolutionary analysis also proved that some HA mutations were highly correlated with NA antibody pressure. Our data demonstrate that HA mutations caused by NA-specific antibodies affect HA properties and may contribute to HA evolution. IMPORTANCE HA binds with the sialic acid receptor on the host cell and initiates the infection mode of influenza virus. NA cleaves the connection between receptor and HA of newborn virus at the end of viral production. The HA-NA functional balance is crucial for viral production and interspecies transmission. Here, we identified mutations in the HA head of H9N2 virus caused by NA antibody pressure. These HA mutations changed the thermal stability and switched the receptor-binding preference of the mutant virus. The HI results indicated that these mutations resulted in significant antigenic drift in mutant HA. The evolutionary analysis also shows that some mutations in HA of H9N2 virus may be caused by NA antibody pressure and may correlate with the increase in H9N2 infections in humans. Our results provide new evidence for HA-NA balance and an effect of NA antibody pressure on HA evolution.

Matrix M Adjuvanted H5N1 Vaccine Elicits Broadly Neutralizing Antibodies and Neuraminidase Inhibiting Antibodies in Humans That Correlate With In Vivo Protection.

The highly pathogenic avian influenza H5N1 viruses constantly evolve and give rise to novel variants that have caused widespread zoonotic outbreaks and sporadic human infections. Therefore, vaccines capable of eliciting broadly protective antibody responses are desired and under development. We here investigated the magnitude, kinetics and protective efficacy of the multi-faceted humoral immunity induced by vaccination in healthy adult volunteers with a Matrix M adjuvanted virosomal H5N1 vaccine. Vaccinees were given escalating doses of adjuvanted vaccine (1.5µg, 7.5µg, or 30µg), or a non-adjuvanted vaccine (30µg). An evaluation of sera from vaccinees against pseudotyped viruses covering all (sub)clades isolated from human H5N1 infections demonstrated that the adjuvanted vaccines (7.5µg and 30µg) could elicit rapid and robust increases of broadly cross-neutralizing antibodies against all clades. In addition, the adjuvanted vaccines also induced multifaceted antibody responses including hemagglutinin stalk domain specific, neuraminidase inhibiting, and antibody-dependent cellular cytotoxicity inducing antibodies. The lower adjuvanted dose (1.5µg) showed delayed kinetics, whilst the non-adjuvanted vaccine induced overall lower levels of antibody responses. Importantly, we demonstrate that human sera post vaccination with the adjuvanted (30µg) vaccine provided full protection against a lethal homologous virus challenge in mice. Of note, when combining our data from mice and humans we identified the neutralizing and neuraminidase inhibiting antibody titers as correlates of in vivo protection.

Influenza Neuraminidase Characteristics and Potential as a Vaccine Target.

Neuraminidase of influenza A and B viruses plays a critical role in the virus life cycle and is an important target of the host immune system. Here, we highlight the current understanding of influenza neuraminidase structure, function, antigenicity, immunogenicity, and immune protective potential. Neuraminidase inhibiting antibodies have been recognized as correlates of protection against disease caused by natural or experimental influenza A virus infection in humans. In the past years, we have witnessed an increasing interest in the use of influenza neuraminidase to improve the protective potential of currently used influenza vaccines. A number of well-characterized influenza neuraminidase-specific monoclonal antibodies have been described recently, most of which can protect in experimental challenge models by inhibiting the neuraminidase activity or by Fc receptor-dependent mechanisms. The relative instability of the neuraminidase poses a challenge for protein-based antigen design. We critically review the different solutions that have been proposed to solve this problem, ranging from the inclusion of stabilizing heterologous tetramerizing zippers to the introduction of inter-protomer stabilizing mutations. Computationally engineered neuraminidase antigens have been generated that offer broad, within subtype protection in animal challenge models. We also provide an overview of modern vaccine technology platforms that are compatible with the induction of robust neuraminidase-specific immune responses. In the near future, we will likely see the implementation of influenza vaccines that confront the influenza virus with a double punch: targeting both the hemagglutinin and the neuraminidase.

Antigenic evolution of human influenza H3N2 neuraminidase is constrained by charge balancing.

As one of the main influenza antigens, neuraminidase (NA) in H3N2 virus has evolved extensively for more than 50 years due to continuous immune pressure. While NA has recently emerged as an effective vaccine target, biophysical constraints on the antigenic evolution of NA remain largely elusive. Here, we apply combinatorial mutagenesis and next-generation sequencing to characterize the local fitness landscape in an antigenic region of NA in six different human H3N2 strains that were isolated around 10 years apart. The local fitness landscape correlates well among strains and the pairwise epistasis is highly conserved. Our analysis further demonstrates that local net charge governs the pairwise epistasis in this antigenic region. In addition, we show that residue coevolution in this antigenic region is correlated with the pairwise epistasis between charge states. Overall, this study demonstrates the importance of quantifying epistasis and the underlying biophysical constraint for building a model of influenza evolution.

Designing a multi-epitope vaccine to provoke the robust immune response against influenza A H7N9.

A new strain of Influenza A Virus (IAV), so-called "H7N9 Avian Influenza", is the first strain of this virus in which a human is infected by transmitting the N9 of influenza virus. Although continuous human-to-human transmission has not been reported, the occurrence of various H7N9-associated epidemics and the lack of production of strong antibodies against H7N9 in humans warn of the potential for H7N9 to become a new pandemic. Therefore, the need for effective vaccination against H7N9 as a life-threatening viral pathogen has become a major concern. The current study reports the design of a multi-epitope vaccine against Hemagglutinin (HA) and Neuraminidase (NA) proteins of H7N9 Influenza A virus by prediction of Cytotoxic T lymphocyte (CTL), Helper T lymphocyte (HTL), IFN-γ and B-cell epitopes. Human ß-defensin-3 (HßD-3) and pan HLA DR-binding epitope (PADRE) sequence were considered as adjuvant. EAAAK, AAY, GPGPG, HEYGAEALERAG, KK and RVRR linkers were used as a connector for epitopes. The final construct contained 777 amino acids that are expected to be a recombinant protein of about ~ 86.38 kDa with antigenic and non-allergenic properties after expression. Modeled protein analysis based on the tertiary structure validation, docking studies, and molecular dynamics simulations results like Root-mean-square deviation (RMSD), Gyration, Root-mean-square fluctuation (RMSF) and Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) showed that this protein has a stable construct and capable of being in interaction with Toll-like receptor 7 (TLR7), TLR8 and m826 antibody. Analysis of the obtained data the demonstrates that suggested vaccine has the potential to induce the immune response by stimulating T and Bcells, and may be utilizable for prevention purposes against Avian Influenza A (H7N9).

Repeated Influenza Vaccination Boosts and Maintains H1N1pdm09 Neuraminidase Antibody Titers.

Antibodies to influenza surface protein neuraminidase (NA) have been found to reduce disease severity and may be an independent correlate of protection. Despite this, current influenza vaccines have no regulatory requirements for the quality or quantity of the NA antigen and are not optimized for induction of NA-specific antibodies. Here we investigate the induction and durability of NA-specific antibody titers after pandemic AS03-adjuvanted monovalent H1N1 vaccination and subsequent annual vaccination in health care workers in a five-year longitudinal study. NA-specific antibodies were measured by endpoint ELISA and functional antibodies measured by enzyme-linked lectin assay (ELLA) and plaque reduction naturalisation assay. We found robust induction of NA inhibition (NAI) titers with a 53% seroconversion rate (>4-fold) after pandemic vaccination in 2009. Furthermore, the endpoint and NAI geometric mean titers persisted above pre-vaccination levels up to five years after vaccination in HCWs that only received the pandemic vaccine, which demonstrates considerable durability. Vaccination with non-adjuvanted trivalent influenza vaccines (TIV) in subsequent influenza seasons 2010/2011 - 2013/2014 further boosted NA-specific antibody responses. We found that each subsequent vaccination increased durable endpoint titers and contributed to maintaining the durability of functional antibody titers. Although the trivalent influenza vaccines boosted NA-specific antibodies, the magnitude of fold-increase at day 21 declined with repeated vaccination, particularly for functional antibody titers. High levels of pre-existing antibodies were associated with lower fold-induction in repeatedly vaccinated HCWs. In summary, our results show that durable NA-specific antibody responses can be induced by an adjuvanted influenza vaccine, which can be maintained and further boosted by TIVs. Although NA-specific antibody responses are boosted by annual influenza vaccines, high pre-existing titers may negatively affect the magnitude of fold-increase in repeatedly vaccinated individuals. Our results support continued development and standardization of the NA antigen to supplement current influenza vaccines and reduce the burden of morbidity and mortality.

Respiratory and Intramuscular Immunization With ChAdOx2-NPM1-NA Induces Distinct Immune Responses in H1N1pdm09 Pre-Exposed Pigs.

There is a critical need to develop superior influenza vaccines that provide broader protection. Influenza vaccines are traditionally tested in naive animals, although humans are exposed to influenza in the first years of their lives, but the impact of prior influenza exposure on vaccine immune responses has not been well studied. Pigs are an important natural host for influenza, are a source of pandemic viruses, and are an excellent model for human influenza. Here, we investigated the immunogenicity of the ChAdOx2 viral vectored vaccine, expressing influenza nucleoprotein, matrix protein 1, and neuraminidase in H1N1pdm09 pre-exposed pigs. We evaluated the importance of the route of administration by comparing intranasal, aerosol, and intramuscular immunizations. Aerosol delivery boosted the local lung T-cell and antibody responses, while intramuscular immunization boosted peripheral blood immunity. These results will inform how best to deliver vaccines in order to harness optimal protective immunity.

A Novel Recombinant Influenza Virus Neuraminidase Vaccine Candidate Stabilized by a Measles Virus Phosphoprotein Tetramerization Domain Provides Robust Protection from Virus Challenge in the Mouse Model.

Current seasonal influenza virus vaccines do not induce robust immune responses to neuraminidase. Several factors, including immunodominance of hemagglutinin over neuraminidase, instability of neuraminidase in vaccine formulations, and variable, nonstandardized amounts of neuraminidase in the vaccines, may contribute to this effect. However, vaccines that induce strong antineuraminidase immune responses would be beneficial, as they are highly protective. Furthermore, antigenic drift is slower for neuraminidase than for hemagglutinin, potentially providing broader coverage. Here, we designed stabilized recombinant versions of neuraminidase by replacing the N-terminal cytoplasmic domain, transmembrane, and extracellular stalk with tetramerization domains from the measles or Sendai virus phosphoprotein or from an Arabidopsis thaliana transcription factor. The measles virus tetramerization domain-based construct, termed N1-MPP, was chosen for further evaluation, as it retained antigenicity, neuraminidase activity, and structural integrity and provided robust protection in vivo against lethal virus challenge in the mouse model. We tested N1-MPP as a standalone vaccine, admixed with seasonal influenza virus vaccines, or given with seasonal influenza virus vaccines but in the other leg of the mouse. Admixture with different formulations of seasonal vaccines led to a weak neuraminidase response, suggesting a dominant effect of hemagglutinin over neuraminidase when administered in the same formulation. However, administration of neuraminidase alone or with seasonal vaccine administered in the alternate leg of the mouse induced robust antibody responses. Thus, this recombinant neuraminidase construct is a promising vaccine antigen that may enhance and broaden protection against seasonal influenza viruses. IMPORTANCE Influenza virus infections remain a high risk to human health, causing up to 650,000 deaths worldwide every year, with an enormous burden on the health care system. Since currently available seasonal vaccines are only partially effective and often mismatched to the circulating strains, a broader protective influenza virus vaccine is needed. Here, we generated a recombinant influenza virus vaccine candidate based on the more conserved neuraminidase surface glycoprotein in order to induce a robust and broader protective immune response against a variety of circulating influenza virus strains.

Functionality of the putative surface glycoproteins of the Wuhan spiny eel influenza virus.

A panel of influenza virus-like sequences were recently documented in fish and amphibians. Of these, the Wuhan spiny eel influenza virus (WSEIV) was found to phylogenetically cluster with influenza B viruses as a sister clade. Influenza B viruses have been documented to circulate only in humans, with certain virus isolates found in harbor seals. It is therefore interesting that a similar virus was potentially found in fish. Here we characterize the putative hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins of the WSEIV. Functionally, we show that the WSEIV NA-like protein has sialidase activity comparable to B/Malaysia/2506/2004 influenza B virus NA, making it a bona fide neuraminidase that is sensitive to NA inhibitors. We tested the functionality of the HA by addressing the receptor specificity, stability, preferential airway protease cleavage, and fusogenicity. We show highly specific binding to monosialic ganglioside 2 (GM2) and fusogenicity at a range of different pH conditions. In addition, we found limited antigenic conservation of the WSEIV HA and NA relative to the B/Malaysia/2506/2004 virus HA and NA. In summary, we perform a functional and antigenic characterization of the glycoproteins of WSEIV to assess if it is indeed a bona fide influenza virus potentially circulating in ray-finned fish.

Design of the Recombinant Influenza Neuraminidase Antigen Is Crucial for Its Biochemical Properties and Protective Efficacy.

Supplementing influenza vaccines with recombinant neuraminidase (rNA) antigens remains a promising approach for improving suboptimal vaccine efficacy. However, correlations among rNA designs, properties, and protection have not been systematically investigated. Here, we performed a comparative analysis of several rNAs produced by the baculovirus/insect cell system. The rNAs were designed with different tetramerization motifs and NA domains from a recent H1N1 vaccine strain (A/Brisbane/02/2018) and compared for enzymatic properties, antigenicity, stability, and protection in mice. We found that the enzymatic properties differ between rNAs containing the NA head domain versus the full ectodomain, the formation of higher-order rNA oligomers is tetramerization domain dependent, whereas the protective efficacy is more contingent on the combination of the tetramerization and NA domains. Following single-dose immunizations, an rNA possessing the full ectodomain and the tetramerization motif from the human vasodilator-stimulated phosphoprotein provided much better protection than an rNA with ∼10-fold more enzymatically active molecules that is comprised of the head domain and the same tetramerization motif. In contrast, these two rNA designs provided comparable protection when the tetramerization motif from the tetrabrachion protein was used instead. These findings demonstrate that individual rNAs should be thoroughly evaluated for vaccine development, as the heterologous domain combination can result in rNAs with similar key attributes that vastly differ in protection. IMPORTANCE For several decades, it has been proposed that influenza vaccines could be supplemented with recombinant neuraminidase (rNA) to improve efficacy. However, some key questions for manufacturing stable and immunogenic rNAs remain to be answered. We show here that the tetramerization motifs and NA domains included in the rNA construct design can have a profound impact on the biochemical, immunogenic, and protective properties. We also show that the single-dose immunization regimen is more informative for assessing the rNA immune response and protective efficacy, which is surprisingly more dependent on the specific combination of NA and tetramerization domains than common attributes for evaluating NA. Our findings may help to optimize the design of rNAs that can be used to improve or develop influenza vaccines.

The Potential of Neuraminidase as an Antigen for Nasal Vaccines To Increase Cross-Protection against Influenza Viruses.

Despite the availability of vaccines that efficiently reduce the severity of clinical symptoms, influenza viruses still cause substantial morbidity and mortality worldwide. In this regard, nasal influenza vaccines-because they induce virus-specific IgA-may be more effective than traditional parenteral formulations in preventing infection of the upper respiratory tract. In addition, the neuraminidase (NA) of influenza virus has shown promise as a vaccine antigen to confer broad cross-protection, in contrast to hemagglutinin (HA), the target of most current vaccines, which undergoes frequent antigenic changes, leading to vaccine ineffectiveness against mismatched heterologous strains. However, the usefulness of NA as an antigen for nasal vaccines is unclear. Here, we compared NA and HA as antigens for nasal vaccines in mice. Intranasal immunization with recombinant NA (rNA) plus adjuvant protected mice against not only homologous but also heterologous virus challenge in the upper respiratory tract, whereas intranasal immunization with rHA failed to protect against heterologous challenge. In addition, intranasal immunization with rNA, but not rHA, conferred cross-protection even in the absence of adjuvant in virus infection-experienced mice; this strong cross-protection was due to the broader capacity of NA-specific antibodies to bind to heterologous virus. Furthermore, the NA-specific IgA in the upper respiratory tract that was induced through rNA intranasal immunization recognized more epitopes than did the NA-specific IgG and IgA in plasma, again increasing cross-protection. Together, our findings suggest the potential of NA as an antigen for nasal vaccines to provide broad cross-protection against both homologous and heterologous influenza viruses. IMPORTANCE Because mismatch between vaccine strains and epidemic strains cannot always be avoided, the development of influenza vaccines that induce broad cross-protection against antigenically mismatched heterologous strains is needed. Although the importance of NA-specific antibodies to cross-protection in humans and experimental animals is becoming clear, the potential of NA as an antigen for providing cross-protection through nasal vaccines is unknown. We show here that intranasal immunization with NA confers broad cross-protection in the upper respiratory tract, where virus transmission is initiated, by inducing NA-specific IgA that recognizes a wide range of epitopes. These data shed new light on NA-based nasal vaccines as powerful anti-influenza tools that confer broad cross-protection.