Immunogenicity of chimeric haemagglutinin-based, universal influenza virus vaccine candidates: interim results of a randomised, placebo-controlled, phase 1 clinical trial.

BACKGROUND: Influenza viruses cause substantial annual morbidity and mortality globally. Current vaccines protect against influenza only when well matched to the circulating strains. However, antigenic drift can cause considerable mismatches between vaccine and circulating strains, substantially reducing vaccine effectiveness. Moreover, current seasonal vaccines are ineffective against pandemic influenza, and production of a vaccine matched to a newly emerging virus strain takes months. Therefore, there is an unmet medical need for a broadly protective influenza virus vaccine. We aimed to test the ability of chimeric H1 haemagglutinin-based universal influenza virus vaccine candidates to induce broadly cross-reactive antibodies targeting the stalk domain of group 1 haemagglutinin-expressing influenza viruses. METHODS: We did a randomised, observer-blinded, phase 1 study in healthy adults in two centres in the USA. Participants were randomly assigned to one of three prime-boost, chimeric haemagglutinin-based vaccine regimens or one of two placebo groups. The vaccine regimens included a chimeric H8/1, intranasal, live-attenuated vaccine on day 1 followed by a non-adjuvanted, chimeric H5/1, intramuscular, inactivated vaccine on day 85; the same regimen but with the inactivated vaccine being adjuvanted with AS03; and an AS03-adjuvanted, chimeric H8/1, intramuscular, inactivated vaccine followed by an AS03-adjuvanted, chimeric H5/1, intramuscular, inactivated vaccine. In this planned interim analysis, the primary endpoints of reactogenicity and safety were assessed by blinded study group. We also assessed anti-H1 haemagglutinin stalk, anti-H2, anti-H9, and anti-H18 IgG antibody titres and plasmablast and memory B-cell responses in peripheral blood. This trial is registered with ClinicalTrials.gov, number NCT03300050. FINDINGS: Between Oct 10, 2017, and Nov 27, 2017, 65 participants were enrolled and randomly assigned. The adjuvanted inactivated vaccine, but not the live-attenuated vaccine, induced a substantial serum IgG antibody response after the prime immunisation, with a seven times increase in anti-H1 stalk antibody titres on day 29. After boost immunisation, all vaccine regimens induced detectable anti-H1 stalk antibody (2·2-5·6 times induction over baseline), cross-reactive serum IgG antibody, and peripheral blood plasmablast responses. An unsolicited adverse event was reported for 29 (48%) of 61 participants. Solicited local adverse events were reported in 12 (48%) of 25 participants following prime vaccination with intramuscular study product or placebo, in 12 (33%) of 36 after prime immunisation with intranasal study product or placebo, and in 18 (32%) of 56 following booster doses of study product or placebo. Solicited systemic adverse events were reported in 14 (56%) of 25 after prime immunisation with intramuscular study product or placebo, in 22 (61%) of 36 after immunisation with intranasal study product or placebo, and in 21 (38%) of 56 after booster doses of study product or placebo. Disaggregated safety data were not available at the time of this interim analysis. INTERPRETATION: The tested chimeric haemagglutinin-based, universal influenza virus vaccine regimens elicited cross-reactive serum IgG antibodies that targeted the conserved haemagglutinin stalk domain. This is the first proof-of-principle study to show that high anti-stalk titres can be induced by a rationally designed vaccine in humans and opens up avenues for further development of universal influenza virus vaccines. On the basis of the blinded study group, the vaccine regimens were tolerable and no safety concerns were observed. FUNDING: Bill & Melinda Gates Foundation.

Broadly Cross-Reactive, Nonneutralizing Antibodies against Influenza B Virus Hemagglutinin Demonstrate Effector Function-Dependent Protection against Lethal Viral Challenge in Mice.

Protection from influenza virus infection is canonically associated with antibodies that neutralize the virus by blocking the interaction between the viral hemagglutinin and host cell receptors. However, protection can also be conferred by other mechanisms, including antibody-mediated effector functions. Here, we report the characterization of 22 broadly cross-reactive, nonneutralizing antibodies specific for influenza B virus hemagglutinin. The majority of these antibodies recognized influenza B viruses isolated over the period of 73 years and bind the conserved stalk domain of the hemagglutinin. A proportion of the characterized antibodies protected mice from both morbidity and mortality after challenge with a lethal dose of influenza B virus. Activity in an antibody-dependent cell-mediated cytotoxicity reporter assay correlated strongly with protection, suggesting that Fc-dependent effector function determines protective efficacy. The information regarding mechanism of action and epitope location stemming from our characterization of these antibodies will inform the design of urgently needed vaccines that could induce broad protection against influenza B viruses.IMPORTANCE While broadly protective antibodies against the influenza A virus hemagglutinin have been well studied, very limited information is available for antibodies that broadly recognize influenza B viruses. Similarly, the development of a universal or broadly protective influenza B virus vaccine lags behind the development of such a vaccine for influenza A virus. More information about epitope location and mechanism of action of broadly protective influenza B virus antibodies is required to inform vaccine development. In addition, protective antibodies could be a useful tool to treat or prevent influenza B virus infection in pediatric cohorts or in a therapeutic setting in immunocompromised individuals in conjugation with existing treatment avenues.

An immuno-assay to quantify influenza virus hemagglutinin with correctly folded stalk domains in vaccine preparations.

The standard method to quantify the hemagglutinin content of influenza virus vaccines is the single radial immunodiffusion assay. This assay primarily relies on polyclonal antibodies against the head domain of the influenza virus hemagglutinin, which is the main target antigen of influenza virus vaccines. Novel influenza virus vaccine candidates that redirect the immune response towards the evolutionary more conserved hemagglutinin stalk, including chimeric hemagglutinin and headless hemagglutinin constructs, are highly dependent on the structural integrity of the protein to present conformational epitopes for neutralizing antibodies. In this study, we describe a novel enzyme-linked immunosorbent assay that allows quantifying the amount of hemagglutinin with correctly folded stalk domains and which could be further developed into a potency assay for stalk-based influenza virus vaccines.

Influenza Infection in Humans Induces Broadly Cross-Reactive and Protective Neuraminidase-Reactive Antibodies.

Antibodies to the hemagglutinin (HA) and neuraminidase (NA) glycoproteins are the major mediators of protection against influenza virus infection. Here, we report that current influenza vaccines poorly display key NA epitopes and rarely induce NA-reactive B cells. Conversely, influenza virus infection induces NA-reactive B cells at a frequency that approaches (H1N1) or exceeds (H3N2) that of HA-reactive B cells. NA-reactive antibodies display broad binding activity spanning the entire history of influenza A virus circulation in humans, including the original pandemic strains of both H1N1 and H3N2 subtypes. The antibodies robustly inhibit the enzymatic activity of NA, including oseltamivir-resistant variants, and provide robust prophylactic protection, including against avian H5N1 viruses, in vivo. When used therapeutically, NA-reactive antibodies protected mice from lethal influenza virus challenge even 48 hr post infection. These findings strongly suggest that influenza vaccines should be optimized to improve targeting of NA for durable and broad protection against divergent influenza strains.

A universal influenza virus vaccine candidate confers protection against pandemic H1N1 infection in preclinical ferret studies.

Influenza viruses evade human adaptive immune responses due to continuing antigenic changes. This makes it necessary to re-formulate and re-administer current seasonal influenza vaccines on an annual basis. Our pan-influenza vaccination approach attempts to redirect antibody responses from the variable, immuno-dominant hemagglutinin head towards the conserved-but immuno-subdominant-hemagglutinin stalk. The strategy utilizes sequential immunization with chimeric hemagglutinin-based vaccines expressing exotic head domains, and a conserved hemagglutinin stalk. We compared a live-attenuated influenza virus prime followed by an inactivated split-virus boost to two doses of split-virus vaccines and assessed the impact of adjuvant on protection against challenge with pandemic H1N1 virus in ferrets. All tested immunization regimens successfully induced broadly cross-reactive antibody responses. The combined live-attenuated/split virus vaccination conferred superior protection against pandemic H1N1 infection compared to two doses of split-virus vaccination. Our data support advancement of this chimeric hemagglutinin-based vaccine approach to clinical trials in humans.

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies.

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

Broadly protective murine monoclonal antibodies against influenza B virus target highly conserved neuraminidase epitopes.

A substantial proportion of influenza-related childhood deaths are due to infection with influenza B viruses, which co-circulate in the human population as two antigenically distinct lineages defined by the immunodominant receptor binding protein, haemagglutinin. While broadly cross-reactive, protective monoclonal antibodies against the haemagglutinin of influenza B viruses have been described, none targeting the neuraminidase, the second most abundant viral glycoprotein, have been reported. Here, we analyse a panel of five murine anti-neuraminidase monoclonal antibodies that demonstrate broad binding, neuraminidase inhibition, in vitro antibody-dependent cell-mediated cytotoxicity and in vivo protection against influenza B viruses belonging to both haemagglutinin lineages and spanning over 70 years of antigenic drift. Electron microscopic analysis of two neuraminidase-antibody complexes shows that the conserved neuraminidase epitopes are located on the head of the molecule and that they are distinct from the enzymatic active site. In the mouse model, one therapeutic dose of antibody 1F2 was more protective than the current standard of treatment, oseltamivir, given twice daily for six days.

Novel Cross-Reactive Monoclonal Antibodies against Ebolavirus Glycoproteins Show Protection in a Murine Challenge Model.

Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (∼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2-/- mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro, suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity.IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies, including two antibodies with broad cross-reactivity to all known ebolavirus species. The antibodies were raised using a heterologous DNA-viral vector prime-boost regimen, resulting in a high proportion of cross-reactive antibodies (25%). Similar vaccination regimens have been used successfully to induce broad protection against influenza viruses in humans, and our limited data indicate that this might be a useful strategy for filovirus vaccines as well. Several of our antibodies showed protective efficacy when tested in a novel murine challenge model and may be developed into future therapeutics.

A cross-clade H5N1 influenza A virus neutralizing monoclonal antibody binds to a novel epitope within the vestigial esterase domain of hemagglutinin.

The sporadic outbreaks of highly pathogenic H5N1 avian influenza virus have raised public health concerns. Monoclonal antibodies (MAbs) against hemagglutinin (HA) have been increasingly used successfully for therapeutic purposes. Previously, MAb 9F4, generated against clade 1 H5N1 HA, was observed to have cross-clade neutralizing efficacy and inhibited viral entry by preventing the pH-mediated conformational change of HA. Furthermore, mouse-human chimeric MAb 9F4 was found to retain high degrees of neutralizing activity. In this study, through escape mutant generation and in-silico prediction, it was revealed that MAb 9F4 binds to a novel epitope in the vestigial esterase sub-domain of HA comprising at least three non-continuous amino acid residues, arginine (R) at position 62, tryptophan (W) at position 69 and phenylalanine (F) at position 79, which interacted with MAb 9F4 in a conformation-dependent manner. Binding and neutralization studies suggested that R62 is the critical residue for MAb 9F4 binding whereas W69 and F79 seem to cooperate with R62 to stabilize the epitope. Mutation of either R62 or W69 did not affect replicative fitness of the virus in vitro. Interestingly, MAb 9F4 retained neutralizing efficacy against a clade 2.3.2.1a H5N1 virus consisting of an arginine to lysine substitution at position 62 in HA.

Globular Head-Displayed Conserved Influenza H1 Hemagglutinin Stalk Epitopes Confer Protection against Heterologous H1N1 Virus.

Significant genetic variability in the head region of the influenza A hemagglutinin, the main target of current vaccines, makes it challenging to develop a long-lived seasonal influenza prophylaxis. Vaccines based on the conserved hemagglutinin stalk domain might provide broader cross-reactive immunity. However, this region of the hemagglutinin is immunosubdominant to the head region. Peptide-based vaccines have gained much interest as they allow the immune system to focus on relevant but less immunogenic epitopes. We developed a novel influenza A hemagglutinin-based display platform for H1 hemagglutinin stalk peptides that we identified in an epitope mapping assay using human immune sera and synthetic HA peptides. Flow cytometry and competition assays suggest that the identified stalk sequences do not recapitulate the epitopes of already described broadly neutralizing stalk antibodies. Vaccine constructs displaying 25-mer stalk sequences provided up to 75% protection from lethal heterologous virus challenge in BALB/c mice and induced antibody responses against the H1 hemagglutinin. The developed platform based on a vaccine antigen has the potential to be either used as stand-alone or as prime-vaccine in combination with conventional seasonal or pandemic vaccines for the amplification of stalk-based cross-reactive immunity in humans or as platform to evaluate the relevance of viral peptides/epitopes for protection against influenza virus infection.

Cryo-electron Microscopy Structures of Chimeric Hemagglutinin Displayed on a Universal Influenza Vaccine Candidate.

UNLABELLED: Influenza viruses expressing chimeric hemagglutinins (HAs) are important tools in the quest for a universal vaccine. Using cryo-electron tomography, we have determined the structures of a chimeric HA variant that comprises an H1 stalk and an H5 globular head domain (cH5/1 HA) in native and antibody-bound states. We show that cH5/1 HA is structurally different from native HA, displaying a 60° rotation between the stalk and head groups, leading to a novel and unexpected "open" arrangement of HA trimers. cH5/1N1 viruses also display higher glycoprotein density than pH1N1 or H5N1 viruses, but despite these differences, antibodies that target either the stalk or head domains of hemagglutinins still bind to cH5/1 HA with the same consequences as those observed with native H1 or H5 HA. Our results show that a large range of structural plasticity can be tolerated in the chimeric spike scaffold without disrupting structural and geometric aspects of antibody binding. IMPORTANCE: Chimeric hemagglutinin proteins are set to undergo human clinical trials as a universal influenza vaccine candidate, yet no structural information for these proteins is available. Using cryo-electron tomography, we report the first three-dimensional (3D) visualization of chimeric hemagglutinin proteins displayed on the surface of the influenza virus. We show that, unexpectedly, the chimeric hemagglutinin structure differs from those of naturally occurring hemagglutinins by displaying a more open head domain and a dramatically twisted head/stalk arrangement. Despite this unusual spatial relationship between head and stalk regions, virus preparations expressing the chimeric hemagglutinin are fully infectious and display a high glycoprotein density, which likely helps induction of a broadly protective immune response.

Hemagglutinin Stalk- and Neuraminidase-Specific Monoclonal Antibodies Protect against Lethal H10N8 Influenza Virus Infection in Mice.

UNLABELLED: Between November 2013 and February 2014, China reported three human cases of H10N8 influenza virus infection in the Jiangxi province, two of which were fatal. Using hybridoma technology, we isolated a panel of H10- and N8-directed monoclonal antibodies (MAbs) and further characterized the binding reactivity of these antibodies (via enzyme-linked immunosorbent assay) to a range of purified virus and recombinant protein substrates. The H10-directed MAbs displayed functional hemagglutination inhibition (HI) and neutralization activity, and the N8-directed antibodies displayed functional neuraminidase inhibition (NI) activity against H10N8. Surprisingly, the HI-reactive H10 antibodies, as well as a previously generated, group 2 hemagglutinin (HA) stalk-reactive antibody, demonstrated NI activity against H10N8 and an H10N7 strain; this phenomenon was absent when virus was treated with detergent, suggesting the anti-HA antibodies inhibited neuraminidase enzymatic activity through steric hindrance. We tested the prophylactic efficacy of one representative H10-reactive, N8-reactive, and group 2 HA stalk-reactive antibody in vivo using a BALB/c challenge model. All three antibodies were protective at a high dose (5 mg/kg). At a low dose (0.5 mg/kg), only the anti-N8 antibody prevented weight loss. Together, these data suggest that antibody targets other than the globular head domain of the HA may be efficacious in preventing influenza virus-induced morbidity and mortality. IMPORTANCE: Avian H10N8 and H10N7 viruses have recently crossed the species barrier, causing morbidity and mortality in humans and other mammals. Although these reports are likely isolated incidents, it is possible that more cases may emerge in future winter seasons, similar to H7N9. Furthermore, regular transmission of avian influenza viruses to humans increases the risk of adaptive mutations and reassortment events, which may result in a novel virus with pandemic potential. Currently, no specific therapeutics or vaccines are available against the H10N8 influenza virus subtype. We generated a panel of H10- and N8-reactive MAbs. Although these antibodies may practically be developed into therapeutic agents, characterizing the protective potential of MAbs that have targets other than the HA globular head domain will provide insight into novel antibody-mediated mechanisms of protection and help to better understand correlates of protection for influenza A virus infection.

Vaccination with soluble headless hemagglutinin protects mice from challenge with divergent influenza viruses.

Current influenza virus vaccines provide solid protection from infection with viruses that are well matched with the vaccine strains. However, they do not protect efficiently against drifted or shifted strains. We developed an antigen based on the conserved stalk domain of the influenza virus hemagglutinin and tested its efficacy as a vaccine in a mouse virus challenge model. Although the antigen lacked the correct conformation of the native stalk domain and was not recognized by a panel of neutralizing stalk-reactive antibodies, it did induce considerable protection against H1N1, H5N1 and H6N1 challenge strains. Protection was enhanced when mice had pre-existing immunity against the stalk domain. Since pre-existing immunity is also present in the human population, we hypothesize that a similar antigen could show efficacy in humans as well.

Vaccination with adjuvanted recombinant neuraminidase induces broad heterologous, but not heterosubtypic, cross-protection against influenza virus infection in mice.

UNLABELLED: In an attempt to assess the cross-protective potential of the influenza virus neuraminidase (NA) as a vaccine antigen, different subtypes of recombinant NA were expressed in a baculovirus system and used to vaccinate mice prior to lethal challenge with homologous, heterologous, or heterosubtypic viruses. Mice immunized with NA of subtype N2 were completely protected from morbidity and mortality in a homologous challenge and displayed significantly reduced viral lung titers. Heterologous challenge with a drifted strain resulted in morbidity but no mortality. Similar results were obtained for challenge experiments with N1 NA. Mice immunized with influenza B virus NA (from B/Yamagata/16/88) displayed no morbidity when sublethally infected with the homologous strain and, importantly, were completely protected from morbidity and mortality when lethally challenged with the prototype Victoria lineage strain or a more recent Victoria lineage isolate. Upon analyzing the NA content in 4 different inactivated-virus vaccine formulations from the 2013-2014 season via Western blot assay and enzyme-linked immunosorbent assay quantification, we found that the amount of NA does indeed vary across vaccine brands. We also measured hemagglutinin (HA) and NA endpoint titers in pre- and postvaccination human serum samples from individuals who received a trivalent inactivated seasonal influenza vaccine from the 2004-2005 season; the induction of NA titers was statistically less pronounced than the induction of HA titers. The demonstrated homologous and heterologous protective capacity of recombinant NA suggests that supplementing vaccine formulations with a standard amount of NA may offer increased protection against influenza virus infection. IMPORTANCE: Despite the existence of vaccine prophylaxis and antiviral therapeutics, the influenza virus continues to cause morbidity and mortality in the human population, emphasizing the continued need for research in the field. While the majority of influenza vaccine strategies target the viral hemagglutinin, the immunodominant antigen on the surface of the influenza virion, antibodies against the viral neuraminidase (NA) have been correlated with less severe disease and decreased viral shedding in humans. Nevertheless, the amount of NA is not standardized in current seasonal vaccines, and the exact breadth of NA-based protection is unknown. Greater insight into the cross-protective potential of influenza virus NA as a vaccine antigen may pave the way for the development of influenza vaccines of greater breadth and efficacy.

An H10N8 influenza virus vaccine strain and mouse challenge model based on the human isolate A/Jiangxi-Donghu/346/13.

Three human cases of H10N8 viruses were reported in China in late 2013 and early 2014, two of which were fatal. This was the first time the H10N8 subtype has been detected in humans and no vaccine candidates or antibody therapy has been developed for these viruses so far. We developed an H10N8 vaccine candidate virus based on A/Jiangxi-Donghu/346/13 that can also be used in a murine challenge model for vaccine and monoclonal antibody research. The vaccine virus is a 6:2 re-assortant virus expressing the surface glycoproteins of A/Jiangxi-Donghu/346/13 on an A/Puerto Rico/8/34 backbone. Vaccination with inactivated challenge virus or recombinant hemagglutinin or neuraminidase derived from this strain protected mice from viral challenge.

Induction of broadly reactive anti-hemagglutinin stalk antibodies by an H5N1 vaccine in humans.

UNLABELLED: Influenza virus infections are a major public health concern and cause significant morbidity and mortality worldwide. Current vaccines are effective but strain specific due to their focus on the immunodominant globular head domain of the hemagglutinin (HA). It has been hypothesized that sequential exposure of humans to hemagglutinins with divergent globular head domains but conserved stalk domains could refocus the immune response to broadly neutralizing epitopes in the stalk. Humans have preexisting immunity against H1 (group 1 hemagglutinin), and vaccination with H5 HA (also group 1)--which has a divergent globular head domain but a similar stalk domain--represents one such sequential-exposure scenario. To test this hypothesis, we used novel reagents based on chimeric hemagglutinins to screen sera from an H5N1 clinical trial for induction of stalk-specific antibodies by quantitative enzyme-linked immunosorbent assay (ELISA) and neutralization assays. Importantly, we also investigated the biological activity of these antibodies in a passive transfer in a mouse challenge model. We found that the H5N1 vaccine induced high titers of stalk-reactive antibodies which were biologically active and protective in the passive-transfer experiment. The induced response showed exceptional breadth toward divergent group 1 hemagglutinins but did not extend to group 2 hemagglutinins. These data provide evidence for the hypothesis that sequential exposure to hemagglutinins with divergent globular head domains but conserved stalk domains can refocus the immune response toward the conserved stalk domain. Furthermore, the results support the concept of a chimeric hemagglutinin universal influenza virus vaccine strategy that is based on the same principle. IMPORTANCE: Influenza virus vaccines have to be reformulated and readministered on an annual basis. The development of a universal influenza virus vaccine could abolish the need for this cumbersome and costly process and would also enhance our pandemic preparedness. This study addressed the following questions, which are essential for the development of a hemagglutinin stalk-based universal influenza virus vaccine. (i) Can stalk-reactive antibodies be boosted by vaccination with divergent HAs that share conserved epitopes? (ii) How long-lived are these vaccine-induced stalk-reactive antibody responses? (iii) What is the breadth of this reactivity? (iv) Are these antibodies functional and protective? Our results further strengthen the concept of induction of stalk-reactive antibodies by sequential exposure to hemagglutinin immunogens with conserved stalk and divergent head domains. A universal influenza virus vaccine based on the same principles seems possible and might have a significant impact on global human health.

In the shadow of hemagglutinin: a growing interest in influenza viral neuraminidase and its role as a vaccine antigen.

Despite the availability of vaccine prophylaxis and antiviral therapeutics, the influenza virus continues to have a significant, annual impact on the morbidity and mortality of human beings, highlighting the continued need for research in the field. Current vaccine strategies predominantly focus on raising a humoral response against hemagglutinin (HA)-the more abundant, immunodominant glycoprotein on the surface of the influenza virus. In fact, anti-HA antibodies are often neutralizing, and are used routinely to assess vaccine immunogenicity. Neuraminidase (NA), the other major glycoprotein on the surface of the influenza virus, has historically served as the target for antiviral drug therapy and is much less studied in the context of humoral immunity. Yet, the quest to discern the exact importance of NA-based protection is decades old. Also, while antibodies against the NA glycoprotein fail to prevent infection of the influenza virus, anti-NA immunity has been shown to lessen the severity of disease, decrease viral lung titers in animal models, and reduce viral shedding. Growing evidence is intimating the possible gains of including the NA antigen in vaccine design, such as expanded strain coverage and increased overall immunogenicity of the vaccine. After giving a tour of general influenza virology, this review aims to discuss the influenza A virus neuraminidase while focusing on both the historical and present literature on the use of NA as a possible vaccine antigen.