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Published in final edited form as: Trends Immunol. 2023 Dec 15;45(1):11–19. doi: 10.1016/j.it.2023.11.001

Targeting neuraminidase: the next frontier for broadly protective influenza vaccines

Nicholas C Wu 1,2,3,4,§, Ali H Ellebedy 5,6,7,§
PMCID: PMC10841738  NIHMSID: NIHMS1947174  PMID: 38103991

Abstract

Current seasonal influenza vaccines, which mainly target hemagglutinin (HA), require annual updates due to the continuous antigenic drift of the influenza virus. Developing an influenza vaccine with increased breadth of protection will have significant public health benefits. The recent discovery of broadly protective antibodies to neuraminidase (NA) has provided important insights into developing a universal influenza vaccine, either by improving seasonal influenza vaccines or designing novel immunogens. However, further in-depth molecular characterizations of NA antibody responses are warranted to fully leverage broadly protective NA antibodies for influenza vaccine designs. Overall, we posit that focusing on NA for influenza vaccine development is synergistic with existing efforts targeting HA, and may represent a cost-effective approach to generating a broadly protective influenza vaccine.

A recent focus on NA antibody responses

Several broadly protective human antibodies to influenza virus neuraminidase (NA) have been discovered in the past few years [1–6], including five that were identified and characterized in-depth in recent studies from our group [7, 8]. These antibodies have revealed that NA has the potential of becoming a target for generating broadly protective influenza vaccines. In this Opinion article, we review the history of influenza vaccine development and our current knowledge of NA antibody responses. We also discuss how NA antibody responses might be leveraged for the development of a broadly protective influenza vaccine, including the technical challenges and areas of future focus. We postulate that induction of robust NA antibody responses by vaccination is not only highly feasible, but also represents a crucial step towards designing broadly protective influenza vaccines.

A brief history of the influenza vaccine

Human influenza viruses were first isolated and identified in 1933 [9] (Figure 1). Subsequently, embryonated eggs were used to propagate influenza viruses, facilitating influenza vaccine development [10]. In 1942, an inactivated bivalent influenza vaccine against human H1N1 and influenza B virus entered a large-scale clinical trial [11]. Three years later, an inactivated influenza vaccine was licensed in the USA [12]. The egg-based system has also enabled the development of a live attenuated influenza vaccine that was approved by the USA Food and Drug Administration (FDA) in 2003 [13]. Alternative vaccine production methods have also been developed because inactivated and live attenuated influenza vaccines often contain egg-adaptive mutations that can change their antigenicity [14, 15]. Establishing the use of Madin-Darby Canine Kidney (MDCK) cells to culture influenza virus in the early 1960s [16] facilitated the development of a cell-based inactivated influenza vaccine that was approved by the USA FDA in 2012 [17]. Similarly, establishing a baculovirus-insect cell protein expression system in the early 1980s [18] facilitated the development of a recombinant influenza vaccine that was approved by the USA FDA in 2013 [19]. Nevertheless, all currently available seasonal influenza vaccines require annual updates due to antigenic drift of the circulating strains. In 2018, the National Institute of Allergy and Infectious Diseases (NIAID) outlined a strategic plan for developing a universal influenza vaccine [20].

Figure 1. Key events in the history of influenza vaccine development.

Figure 1.

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The years of influenza pandemics since 1918 and the re-emergence of H1N1 (red) are indicated. The years when different types of influenza vaccines were first licensed (blue) are indicated [12, 13, 17]. The years of major discoveries and technological advancements that have facilitated influenza vaccine development (orange) are indicated [9, 10, 16, 18, 19]. In 2018, NIAID outlined a strategic plan for developing a universal influenza vaccine [20].

Development of a “universal” influenza vaccine

Influenza A has two major antigens: hemagglutinin (HA) and NA. HA mediates viral entry by binding sialic acid on host cells (e.g. respiratory epithelial cells) and promotes virus-host membrane fusion, whereas NA cleaves sialic acid to facilitate the release of progeny virions [21]. Historically, HA has been the focus of influenza vaccine development, partly because of assay availability for measuring HA antibody responses. While the HA inhibition assay was established in 1942 [22], the NA inhibition assay was available 20 years later [23, 24]. Additionally, reagents for the NA inhibition assay are not as readily available as those for HA [25]. Subsequently, the discovery of broadly neutralizing human antibodies to the conserved HA stem domain in the late 2000s further sparked interest in developing HA-based universal influenza vaccines [26].

From another angle, NA antibodies have been known to confer protection to humans since the 1970s [27, 28]. Shortly after the 1968 H3N2 pandemic, pre-existing antibodies to H2N2 NA helped to protect against H3N2 infection in a cohort of close to 300 adults [28]. Yet, the importance of NA in influenza vaccine development did not gain additional attention until the past decade, when several clinical studies showed that NA antibodies constituted an independent correlate of protection [29–32]. Furthermore, several human antibodies to the highly conserved NA active site have been identified [1–6]. These NA antibodies are protective and cross-react with antigenically diverse strains within a given NA subtype [1, 2] or across multiple NA subtypes [4–6]. Our research group also recently identified other broadly protective epitopes on NA that are distal from the active site [7, 8]. Collectively, these findings suggest that NA antibody responses can contribute to developing a universal influenza vaccine.

Leveraging NA antibody responses in influenza vaccine development

A simple step towards developing a universal influenza vaccine is to improve seasonal influenza vaccines by increasing their antibody response to NA, regardless of whether broadly protective NA epitopes can be targeted or not [25, 33, 34]. It is estimated that current inactivated influenza vaccines result in only approximately 30% seroconversion against NA in humans [25, 35–40]. Accordingly, a previous study showed that two inactivated influenza vaccines, Fluarix and Fluzone, were poorly recognized by NA antibodies in humans, suggesting that most conformational epitopes on NA may be disrupted during vaccine production [41]. Of note, the recombinant influenza vaccine Flublok does not contain NA [42]. Therefore, current seasonal influenza vaccines mainly rely on HA antibody responses for protection with little emphasis on NA antibody responses. However, mouse immunization studies have demonstrated that spiking-in purified NA can quickly improve the NA antibody response of seasonal influenza vaccines [43, 44].

The effectiveness of seasonal influenza vaccines is influenced by antigenic matching between the HAs of the vaccine and circulating strains [45]. Since the production of seasonal influenza vaccines takes up to 6 months, vaccine strain selection requires a prediction of circulating strains early before the onset of each influenza season [46]. If HA antigenicity differs between the selected vaccine strains and the circulating ones during the influenza season, vaccine mismatch occurs [46]. Nevertheless, HA antigenic drift is not necessarily accompanied by NA antigenic drift because the antigenic drifts of HA and NA are asynchronous [36, 47, 48]. In addition, NA antigenic drift is slower than HA [47]. Consequently, seasonal influenza vaccines that elicit a robust NA antibody response might help mitigate the decrease in vaccine effectiveness due to HA antigenic drift (Figure 2).

Figure 2. Model for Improving the protection breadth of seasonal influenza vaccines via hemagglutinin (HA) plus neuraminidase (NA) antibody responses.

Figure 2.

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The schematic shows the change in vaccine effectiveness when HA antigenic drift occurs before NA antigenic drift. Seasonal influenza vaccines that elicit an antibody response to only HA poorly protect against a strain bearing an antigenically drifted HA (top) [46]. In contrast, seasonal influenza vaccines that elicit an antibody response to both HA and NA should offer some protection against a strain with antigenically drifted HA as long as its NA is not antigenically drifted (bottom).

If seasonal influenza vaccines can achieve high seroconversion against NA, ideally, they should also help protect against certain zoonotic strains with pandemic potential. For example, mouse studies have shown that antibodies generated against human H1N1 NA can cross-protect against H5N1 infection [49, 50]. Furthermore, as mentioned above, human antibody responses to H2N2 NA cross-protect against H3N2 infection [28]. However, we acknowledge that there are 11 known NA subtypes (N1 to N11) specific to influenza A virus [51], whereas seasonal influenza vaccines only contain N1 and N2, as well as type B NA [46]. Consequently, although improving antibody titers against NA would likely increase the protection breadth of a seasonal influenza vaccine, additional efforts are needed to develop a robust global influenza vaccine.

A suboptimal molecular understanding of broadly protective NA epitopes

A key to developing HA-based global influenza vaccine candidates is a detailed molecular understanding of broadly protective epitopes of HA [26]. For example, the highly conserved HA stem domain is known to be targeted by multidonor antibodies, which are antibodies from different individuals but with similar sequence features [52], substantiating that HA stem antibodies can be elicited across individuals. In addition, the HA stem domain is known to be immunosubdominant to the HA head domain [53], which has motivated the design of HA stem-based immunogens [54, 55]. However, comparatively, our understanding of broadly protective epitopes on NA is suboptimal and must be improved. Our knowledge of broadly protective NA epitopes is primarily limited by the small number of known human monoclonal antibodies to NA. To date, over 5,000 human monoclonal antibodies against HA have been isolated [56]. In contrast, the number of known monoclonal antibodies to NA is considerably less. Therefore, there is a need to continue isolating and characterizing NA-specific human monoclonal antibodies from different individuals. These efforts can facilitate the characterization of the immunodominance hierarchy of NA epitopes, the identification of recurring molecular features of NA antibody responses from different individuals, and the discovery of additional broadly protective epitopes, if any.

We acknowledge that the role of mouse monoclonal antibodies in epitope discovery should not be ignored. The first broadly neutralizing antibody against the HA stem, C179, was isolated from mice and reported in 1993 [57]. However, the importance of the HA stem as yielding broadly neutralizing epitopes was not fully recognized until the late 2000s, when multiple HA stem-specific human monoclonal antibodies were discovered [58–61]. Thus, overlooking the significance of C179 may have set back the pursuit of a universal influenza vaccine by almost two decades. However, there are major differences between human and mouse antibody responses. For example, these species-specific HA responses exhibit different immunodominance hierarchies [62]. In addition, the third complementarity-determining region of the heavy chain (CDR H3) sequences of human antibodies are more hydrophobic and longer than those of mouse antibodies [63]. As a result, while we should not neglect the potential for uncovering broadly protective NA epitopes using mouse monoclonal antibodies, caution is needed when extrapolating findings from murine responses to humans.

By systematically analyzing existing NA-antibody complex structures, a recent study from our group showed that most of the NA head domain surface is targeted by antibodies [8] (Figure 3). This observation made us wonder if antibodies could also target the NA protomer-protomer interface, which is buried between protomers when NA is properly tetramerized, unlike known NA epitopes. Previous studies demonstrated that the conserved HA protomer-protomer interface is transiently exposed to solvent due to the breathing motions of the HA head domain [64] and hence, is targeted by broadly protective antibodies [65]. Similarly, an “open” conformation can be observed in recombinant NA [66], suggesting that the NA protomer-protomer interface is accessible to antibodies, although this remains to be directly demonstrated. Antibodies that disrupt NA tetramerization, if they exist, are expected to inhibit viral growth since NA tetramerization is required for its enzymatic activity [67]. Furthermore, the NA protomer-protomer interface is highly conserved and has a low mutational tolerance [68]. Thus, antibodies to the NA protomer-protomer interface are expected to be broadly protective. We propose that a potentially high-risk, high-reward focus for future antibody discovery studies is to isolate antibodies that target the NA protomer-protomer interface, which can have important implications for therapeutic development and vaccine design.

Figure 3. Most of the NA head domain surface is immunogenic.

Figure 3.

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(A) Protein surface representation of NA with one protomer colored in white and the other three colored in dark gray (PDB 8DWB [68]). Indicated are the locations of the active site and the highly conserved calcium-binding site in one of the protomers. Calcium ion is represented by a red sphere. Sialic acid in the active site is represented by yellow sticks. (B) Shown are structures of representative NA antibodies that bind to distinct epitopes, namely NC41 (PDB 1NCA [94]), 1G01 (PDB 6Q23 [5]), 3A10 (PDB 8EZ3 [8]), 2H08 (PDB 8E6K [7]), NA-63 (PDB 6PZF [1]), and NA-22 (PDB 6PZW [1]). In addition to NC41, which is a mouse antibody, other represented antibodies shown are from human.

Our recent study also identified three protective antibodies to the conserved underside of the NA head domain [8]. These antibodies cross-reacted with NAs from human H3N2 strains isolated during a span of multiple decades. The underside of the NA head domain on the virus surface may be immunosubdominant, since its accessibility to the antibody and the B cell receptor (BCR) requires a transient tilting motion [64]. However, the accessibility of the underside of the NA head domain might increase dramatically in recombinant soluble NA due to the absence of membrane attachment [64]. Consequently, the immunodominance hierarchy of NA epitopes may differ between its membrane-bound and soluble forms; this is relevant because immunodominance hierarchy can inform strategies for immunogen design. Thus, in-depth molecular characterization of antibody responses to broadly protective NA epitopes may help leverage NA-specific influenza vaccine development.

Unique challenges for NA-based immunogen design

Several vaccine candidates that aim to elicit high antibody titers against NA have been tested in clinical trials, including inactivated vaccines with recombinant NA proteins spiked-in [69] and mRNA vaccines that encode NA as a component (NCT05333289i). While these efforts are important steps towards achieving a universal influenza vaccine, the quality and protection breadth of NA antibody responses can likely be improved by immunogen design. A relatively straightforward strategy for NA-based immunogen design is to engineer mutations that stabilize its “closed” conformation [1, 66]. Recombinant soluble NAs from some human influenza strains exhibit a predominantly “open” conformation, resembling four monomers linked by flexible linkers [66]. Mouse immunization studies have shown that the NA tetramer induces a much stronger protective antibody response than the NA monomer [70, 71]. Thust, stabilizing the “closed” NA conformation, minimizing its transition to a monomer-like conformation, might benefit the development of recombinant soluble NA as a vaccine component. Of note, the “open” conformation has only been observed in the context of recombinant soluble NA, and seems uncommon in the native membrane-bound form of NA [66]; this suggests that the “open” conformation might be absent on the virus surface. Native membrane-bound NA can be expressed as an immunogen as an mRNA vaccine. We argue that this might be a better NA-specific vaccination strategy compared to the recombinant NA protein because it might better mimic the NA conformation on the virus surface, although this remains conjectural.

Nevertheless, we posit that the ultimate goal is to design an NA-based immunogen that elicits a broadly protective antibody response. Common immunogen design strategies for developing broadly protective vaccines include epitope removal, domain chimera, consensus sequence, glycan masking, and mosaic nanoparticle display [72] (Figure 4). Examples of epitope removal include two HA stem-based immunogens, namely mini-HA [54] and headless HA [55], in which the HA head domain was removed. These immunogens allowed antibodies specific to the highly conserved HA stem domain to be elicited without being outcompeted by antibodies against the hypervariable HA head domain [54, 55]. We reason that applying epitope removal to NA is very challenging because all of its known epitopes, either conserved or hypervariable, exist within a single folded domain (i.e., NA head domain) [8]. Consequently, removing NA hypervariable epitopes can almost inevitably disrupt the protein’s conserved conformational epitopes. From another angle, considering an antigen with multiple immunogenic domains is a prerequisite for designing domain chimera. It is possible that the use of domain chimera, which have been applied to influenza HA and SARS-CoV-2 spike proteins [73, 74], might not apply to NA.

Figure 4. Feasibility of different NA immunogen design strategies.

Figure 4.

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Previous studies on broadly protective HA-based immunogen design have involved the consensus sequence approach [76–78], glycan masking [80, 81], mosaic nanoparticle display [95], epitope removal [54, 55], and generating domain chimera [73]. Our proposed ranking of their feasibility for NA-based immunogen design is indicated via the decreasing black triangle gradient. This ranking is arbitrarily based on the degree of technical challenges that must be overcome.

In contrast, approaches such as consensus sequence generation, glycan masking, and mosaic nanoparticle display, apply to single protein domains. Using the consensus sequence approach for immunogen design is best exemplified by studies using the computationally optimized broadly reactive antigen (COBRA) technology, which involves multiple layers of consensus building [75]. COBRA has been applied to different HA subtypes to elicit antibody responses against antigenically distinct strains within a given subtype [76–78]. As a lot of NA sequences are available on Global Initiative on Sharing All Influenza Data (GISAID) [79], applying COBRA to NA should be highly feasible. Glycan masking, which has been applied to various viral glycoproteins including influenza HA [80, 81], involves the introduction of N-glycosylation sites to mask undesired immunodominant epitopes and focus antibody responses to desired, potentially immunosubdominant, epitopes [72]. However, understanding NA epitopes and their immunodominance hierarchy prior to glycan masking must be achieved to optimally apply this approach to NA. Another strategy, mosaic nanoparticle display, is relatively new in vaccine design, and involves the multivalent display of different antigen variants, thereby promoting an antibody response against conserved epitopes [82, 83]. Mosaic nanoparticle display has been applied to influenza HA and the SARS-CoV-2 spike [84, 85], using a two-component nanoparticle designed to display trimeric antigen [86]. Since NA is a tetramer, displaying NA on a mosaic nanoparticle will evidently require a different nanoparticle design with a tetrameric component [87]. Given the established strategies on immunogen design, engineering a broadly protective NA-based immunogen seems like a reachable goal and that can contribute to the development of a global influenza vaccine.

CONCLUDING REMARKS

While influenza viruses have two major surface antigens, HA and NA, influenza vaccine development has historically focused on HA. When the first HA structure was determined in 1981 [88], five major antigenic sites were identified [89]. Additional HA epitopes, including several targeted by broadly neutralizing antibodies, have been subsequently discovered and characterized [65]. These efforts eventually led to the development of broadly protective HA-based vaccine candidates [26], some of which have entered clinical trials [90, 91]. Similarly, when the first NA structure was determined in 1983 [92], seven major antigenic sites at the rim of the catalytic site were identified [93]. However, although additional NA epitopes were discovered in the past few years, including several broadly protective ones [1–8], there is yet to be a commercially available influenza vaccine that elicits a robust NA antibody response. In this opinion article, we argued that increasing NA antibody responses from seasonal influenza vaccination might be achieved in the near future, thus representing a first step towards constructing a more universal influenza vaccine. Nevertheless, the long-term goal is to develop broadly protective NA-based immunogens, which requires a robust molecular understanding of NA epitopes and antibody responses (see Outstanding questions). We posit that including NA in future influenza vaccine endeavors can synergize with existing efforts to develop broadly protective HA-based vaccines. We also argue that focusing on the NA protein as an approach to vaccine development might result in a cost-effective strategy to achieving a global influenza vaccine. Thus, future endeavors in this realm should constitute a fruitful area of investigation.

OUTSTANDING QUESTIONS.

  • What is the immunodominance hierarchy of NA epitopes?

  • Is the immunodominance hierarchy different between membrane-bound and soluble NAs?

  • Does the NA protomer-protomer interface contain any epitope?

  • What are the recurring molecular features of NA antibody responses across different individuals?

  • Are there additional yet-to-be discovered broadly protective epitopes?

  • What is the best approach for engineering a broadly protective NA-based immunogen?

HIGHLIGHTS.

  • Recent studies have identified multiple broadly protective epitopes on influenza virus neuraminidase (NA).

  • While hemagglutinin (HA) and NA are major surface antigens of the influenza virus, molecular features of antibody responses to NA are much less well characterized than those to HA.

  • The protection breadth of seasonal influenza vaccines can be improved by increasing their ability to induce antibody responses against NA.

  • We propose that engineering a broadly protective NA-based immunogen for vaccine development is feasible.

SIGNIFICANCE.

  • Although hemagglutinin (HA) has been the main target of influenza virus vaccine development, neuraminidase (NA) has gained increasing attention in the past few years. Several broadly protective NA antibodies have been recently identified, providing important insights into the potential for developing universal influenza vaccines.

ACKNOWLEDGMENTS

N.C.W. was partially supported with funding from the US National Institutes of Health (NIH) R01 AI165475 and DP2 AT011966. A.H.E was supported in part with funding from US NIH U01 AI141990, U01 AI150747, U01 AI144616 and R01 AI168178. We thank Huibin Lv for the helpful discussion.

DECLARATION OF INTERESTS

N.C.W. is a consultant for HeliXon. The Ellebedy laboratory received funding under sponsored research agreements from Moderna, Emergent BioSolutions, and AbbVie. AHE has received consulting and speaking fees from InBios International, Inc., Fimbrion Therapeutics, RGAX, Mubadala Investment Company, Moderna, Pfizer, GSK, Danaher, Third Rock Ventures, Goldman Sachs, and Morgan Stanley and is the founder of ImmuneBio Consulting.

GLOSSARY

Antigenic drift

Accumulation of mutations that leads to immune escape

Protection breadth

Diversity of strains that the host can be protected against

Multidonor antibodies

Antibodies from different individuals but with similar sequence features

Epitope removal

Removing regions that contain undesired epitopes from an antigen

Domain chimera

One domain of a multidomain antigen variant is replaced by that of another antigen variant

Consensus sequence

A calculated sequence of the most frequent amino acid variant at each position in a sequence alignment

Glycan masking

Introducing N-glycosylation sites to an antigen to shield undesired epitopes

Mosaic nanoparticle display

Multivalent display of different antigen variants on a nanoparticle

Protomer-protomer interface

Surface buried between protomers, hence, solvent inaccessible when the protein multimerizes

Breathing motions

Protomers of a multimerized protein transiently dissociate

Low mutational tolerance

A large fraction of possible mutations hampers viral replication fitness

Footnotes

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RESOURCES

This trial is listed in https://clinicaltrials.gov/study/NCT05333289

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