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. Author manuscript; available in PMC: 2019 Aug 1.
Published in final edited form as: Curr Opin Immunol. 2018 Apr 16;53:38–44. doi: 10.1016/j.coi.2018.03.025

Neuraminidase as an influenza vaccine antigen: a low hanging fruit, ready for picking to improve vaccine effectiveness

Maryna C Eichelberger a, David M Morens b, Jeffery K Taubenberger c
PMCID: PMC6141346  NIHMSID: NIHMS960572  PMID: 29674167

Abstract

Neuraminidase (NA) plays an essential role in influenza virus replication, facilitating multicycle infection predominantly by releasing virions from infected cells. NA-inhibiting antibodies provide resistance to disease and NA-specific antibodies contribute to vaccine efficacy. The primary reason NA vaccine content and immunogenicity was not routinely measured in the past, was the lack of suitable assays to quantify NA and NA-specific antibodies. These are now available and with recent appreciation of its contribution to immunity, NA content of seasonal and pandemic vaccines is being considered. An added benefit of NA as a vaccine antigen is that many NA-specific antibodies bind to domains that are well conserved within a subtype, protecting against heterologous viruses. This suggests NA may be a good choice for inclusion in universal influenza vaccines.

Introduction

The effectiveness of influenza vaccines can vary from year-to-year, usually due to differences in antigenicity between vaccine hemagglutinins (HAs) and those of the circulating viruses and waning immunity [1–3]. For these reasons, development of more broadly-protective “universal” vaccines has become an important goal. While HA is the component on which vaccine potency is based, neuraminidase (NA), the second most abundant glycoprotein on the surface of influenza A and B viruses, is also an important antigen. NA removes decoy receptors from mucins that trap inhaled virus particles [4,5] and removes sialic acids from the surface of the infected cell, allowing the release and spread of newly formed virus particles [6,7]. Antibodies that inhibit NA therefore reduce plaque size and limit virus replication [8]. Naturally-acquired NA-inhibiting (NI) antibodies protect against disease [9,10] and NI antibody titers correlate with live, attenuated and inactivated influenza vaccine effectiveness [11,12]. These observations suggest NA would be an ideal target for vaccine development, but there have been significant hurdles to overcome, including lack of a practical assay to measure the antibody response to NA, and the inability to accurately quantify the potency of NA in multivalent influenza vaccines. This review of recent advances describes newer assays to quantify NA and NA-specific antibodies, as well as recent findings which explain the contribution of NA-specific antibodies to immunity (Figure 1).

Figure 1.

Figure 1

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Reasons why NA is a low hanging fruit: (1) NA is immunogenic, (2) Many studies demonstrate that NA antibodies contribute to immunity, and (3) Practical assays have been developed to measure antibody responses to NA and to measure NA content of influenza vaccines.

NA is immunogenic

NA is quite immunogenic, inducing antibody responses in mice [13–15], guinea pigs [16], ferrets [17–19] and humans [12,20,21]. A significant proportion of individuals vaccinated with either live or inactivated, split seasonal vaccines exhibit increases in NI antibody titers [21,22]. Increased NI antibody titers are also observed in clinical studies of pandemic vaccines; these include whole, cell-grown H5N1 virus [20] and H7N9 virus-like particle (VLP) vaccines [23], where the HA component is novel to humans. In the H7N9 VLP study, NA immunogenicity was increased substantially by the addition of ISCOMATRIX adjuvant [23].

The NA ‘head’ contains overlapping antigenic domains that were identified by selecting influenza virus escape variants in the presence of monoclonal antibodies (mAbs) [24,25]. While some NA-specific mouse mAbs bind only to the immunizing antigen, many bind conserved antigenic domains, allowing reactivity with viruses of the same NA subtype. For example, mAbs that bind conserved sites of A/Brisbane/59/2007 (H1N1) NA inhibit the NAs of A/California/7/2009 (A/H1N1pdm09) and A/Vietnam/1203/2004 (H5N1) [24]. mAbs that bind conserved NA domains of A/H1N1pdm [26], A/H7N9 [27] and influenza B [28] viruses have also been characterized. The presence of conserved epitopes has consequently garnered a lot of interest from investigators seeking to develop vaccines that induce greater breadth of immunity.

There are multiple mechanisms by which NA-specific antibodies contribute to protection against disease. Antibodies that inhibit NA activity block virus egress from decoy receptors on mucins and thereby limit infection. They also inhibit release of newly formed virus particles, reducing virus spread to neighboring epithelial cells. Strain-specific antibodies are most effective at inhibiting NA and virus release, while broadly-reactive antibodies usually inhibit NA activity at higher concentrations [29]. Antibodies that bind, but do not inhibit NA may also contribute to immunity by directing the activity of complement and FcR-expressing cytolytic cells to infected cells (Fig. 2).

Figure 2.

Figure 2

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NA-specific antibodies use a variety of mechanisms to control infection, including inhibition of enzyme activity by strain-specific antibodies to prevent virus spread and inhibition of activity by antibodies that bind conserved antigenic domains. The latter may not be as effective as strain-specific antibodies but nevertheless, reduce virus release. NA-specific antibodies may also function by limiting release of virus particles from mucins or other glycosylated proteins (not shown in the figure) or binding to NA expressed on infected cells, establishing a target for ADCC or complement (C′) activity.

NA-specific antibodies contribute to immunity

The contribution of NA-specific antibodies in reducing the severity of infection during the 1968 A/H3N2 pandemic was recognized almost five decades ago [30], shortly after the in vitro effect of NA-inhibition was described [8]. In addition, investigators demonstrated that NA-inhibiting antibodies contributed to immunity by challenging A/H2N2 vaccine recipients with the new A/H3N2 virus that had a novel HA, but had the same NA subtype [9]. A study in which vaccinated volunteers were challenged with wild type influenza is particularly intriguing because it suggests NA-specific antibody titers correlate with effectiveness of live, attenuated vaccines [11], raising the question of whether NA content is more important for some vaccine types and whether NA-specific antibodies at the site of infection (mucosal surface) should be a critical consideration when evaluating NA-based vaccines. A recent clinical study in which young, healthy adults were challenged with A/California/7/2009 (H1N1pdm09) demonstrated clear differences in the role of HA and NA-specific antibodies in immunity [31]. While reduction in virus shedding correlated with HA inhibition (HI) titers, NI antibody titers were associated with fewer symptoms, reduced symptom severity score and reduced duration of symptoms and shedding. The differences in clinical impact by HA and NA-specific antibodies is likely due to their unique mechanisms of action (antibodies that obstruct hemagglutination are likely to block infection, while antibodies that inhibit NA activity, limit virus spread) and explain why NA and HA-specific antibodies are independent correlates of protection from influenza [10,12,32].

The benefit of NA immunity is also evident in animal studies. This includes studies of recombinant NA [33,34], NA-expressing vectors [35,36], and NA-only VLPs [37] in mice. Most of these studies challenged control and immunized mice with mouse-adapted seasonal influenza A viruses or in some cases wild-type A/H5N1 or A/H7N9 viruses. These studies as well as prophylactic or therapeutic treatment of mice with NA-specific monoclonal antibodies [24,27–29,38–40], demonstrate that NA-specific antibodies protect against weight loss and death.

Both clinical and animal studies suggest that NA vaccines provide immunity against a broad range of viruses within the same NA subtype as the vaccine strain. Such heterologous protection was evident in early experiments carried out by the Kilbourne laboratory [41] and is also demonstrated in recent experiments where mice immunized with the NA of an A/H1N1pdm09 virus, were protected against H5N1 virus challenge [37,42]. In addition, antibodies against the NA of influenza B viruses protect against heterologous B viruses [33]. Heterologous NA-based protection is also evident in ferret challenge studies [18] and is thought to be one reason the elderly were largely protected against A/H1N1 infection during the 2009 H1N1 pandemic [43]. Antibodies that target conserved regions of the HA head or stem may also have contributed to this protection [44].

Despite the potential benefit of broadly-reactive antibodies, it should be kept in mind that strain-specific antibodies are usually the most effective at limiting virus spread in vitro and preventing death in mice [29]. The changes in antigenicity that have been observed for NA occur less frequently than HA antigenic drift and can be the result of a single amino acid change [45,46], making it difficult to predict the emergence of serologically-distinct NA variants. There is therefore a need to monitor NA antigenic drift so that viruses recommended for vaccine production have NAs antigenically similar to viruses in circulation.

Assays to quantify NA-specific antibodies

The immunogenicity of NA can be measured by determining the titer of antibodies that bind or inhibit NA. The thiobarbituric acid (TBA) assay that was originally used to measure NI antibody titers [47] is cumbersome to perform and requires hazardous chemicals. An optimized enzyme linked lectin assay (ELLA) [48,49] is suitable for performing serology and has excellent intra- as well as inter-laboratory reproducibility [50]. This assay can also be used to evaluate antigenic drift [51]. A caveat is that when the assay uses wild-type viruses, antibodies which bind HA may non-specifically inhibit NA activity by steric hindrance of the enzyme’s active site. Antibodies that block HA binding to fetuin, the substrate that is coated to the ELLA plate, also interfere with the assay by limiting access of NA to its sialylated substrate [52]. For this reason, reassortant viruses that express a novel HA subtype are generally used as antigen, but alternate sources of antigen, for example recombinant NA [53], pseudotyped viruses [54], or detergent-disrupted wild-type viruses [21] can also be employed.

ELISA can be a suitable alternative to ELLA because it allows large numbers of sera to be analyzed. To obtain antibody titers that correspond with functional activity, it is imperative to use the native form of NA as antigen in ELISAs. Whole virus can be used to coat the plates if there are no HA or nucleoprotein antibodies present in the serum samples, but given the advances in expressing multimeric proteins, recombinant NA is used more commonly as the coating antigen. ELISA antibody titers from assays in which active recombinant NA is used as antigen correlate well with functional NI antibody titers [55]. A cell-based ELISA in which NA is transiently expressed offers as alternative approach to laboratories unable to obtain recombinant NA for serologic analysis [24].

Assays to quantify NA content of vaccines

An accurate mass spectrometry (MS)-based method has been developed to quantify both NA and HA in influenza vaccines [56]. This isotope dilution MS (IDMS) method uses isotopically-labeled peptide standards which are specific for each influenza type and subtype to determine the molar amount of each NA. While the assay is not necessarily reflective of NA immunogenicity, it provides a way to measure the absolute concentration of each NA type/subtype in seasonal and pandemic vaccines [57].

An assay that is predictive of immunogenicity, i.e., a potency assay, is needed to evaluate the stability of NA and to establish a dose that will consistently induce protective antibody levels. The ability of NA to induce functional antibodies is dependent on its structure – only its native, tetrameric form will induce antibodies that bind to epitopes near the active site and inhibit enzyme activity [14]. Enzyme activity is an excellent indicator of native structure and correlates well with immunogenicity [14,58]. Enzyme activity can therefore be used as a measure to assess the potency of NA in a monovalent vaccine, but since this assay cannot differentiate between NA types/subtypes, it is not suitable for measuring NA content of current trivalent and quadrivalent seasonal influenza vaccines. For this reason, capture ELISAs have been developed to quantify the native form of NA in monovalent or multivalent vaccines. Monoclonal antibodies specific to the NA subtype of interest are used to capture NA and also to detect bound NA. The amount of NA is than determined by comparison with a standard containing a known concentration of NA. The amount of NA in seasonal influenza vaccines measured by capture ELISA corresponds well with its immunogenicity [57], suggesting that this type of assay can be used for routine determination of NA potency in trivalent and quadrivalent influenza vaccines. It is anticipated that mouse monoclonal antibodies against other NA subtypes will be used to develop similar capture ELISAs that quantify the immunogenic form of NA in the near future.

The amount of NA in current vaccines is likely to differ between strains because of its dependence on the proportions of HA and NA in each virus. It is also likely to differ between licensed products because manufacturing processes are not the same. Nevertheless, since there is an increase in NI antibody titer following immunization with most vaccines [59], it seems prudent to measure the potency (immunogenic form) of NA in vaccines under development to ensure consistent antibody responses are elicited throughout the product shelf life.

Picking the low hanging fruit

The contribution of NA-specific antibodies to immunity was recognized many decades ago [9], and now that practical methods have been established to measure the NA content and serologic response, it is possible to consider designing vaccines which target the induction of NA-specific antibodies. Studies show that NA inhibition antibody titers are an independent correlate of protection [12] and since substantial numbers of individuals have increased NA-specific antibody titers following vaccination, it follows that most seasonal inactivated influenza vaccines already include NA that might contribute to immunity. The lowest ‘fruit’ to pick is therefore to confirm that the NA content of current vaccines is stable throughout its shelf life. These tests can be conducted without the need for clinical studies, and if needed, changes to formulations that improve NA stability could be introduced. The next low hanging fruits are vaccines designed to induce protective levels of NA specific antibodies. These may be standard vaccines with an increased NA dose. This could be achieved by addition of recombinant NA. Alternatively, the vaccine could be formulated at a higher overall dose. Such a “high dose” vaccine is licensed for older adults, and results in higher NI antibody titers post-vaccination [21]. New types of vaccines that use DNA or RNA vectors, virus-like particles or stand-alone recombinant NA are also platforms that could be used to induce protective levels of NI antibodies. The immunogenicity of NA in any of these vaccines could be increased by addition of adjuvants. The serum NI antibody titer that correlates with protection has not been established. NI antibody titers ≥40 were associated with reduced illness following challenge with A/California/7/2009 (H1N1pdm) [31], but additional studies are needed to establish the applicability of this titer as a surrogate marker of protection against other influenza A and B strains. It is also important to establish whether this surrogate is the same across different age groups and vaccine types. As for all vaccines, clinical studies will be required to demonstrate the safety and efficacy of NA-based vaccines.

Conclusions

This review highlights advances that support the development of influenza vaccines targeting induction of NA-inhibiting antibodies. Immune responses to NA correlate with vaccine effectiveness, likely by limiting viral replication and disease severity rather than preventing infection [31]. Therefore there has been interest in evaluating NA content during the development and manufacture of new seasonal and pandemic vaccines. Recent technical advances include the development of capture ELISAs to measure NA potency and ELLA and ELISAs to measure NA antibody responses. The availability of these assays makes it feasible for vaccine developers and manufacturers to consider measuring NA content and immunogenicity of products. This puts development of NA-based vaccines in reach, however in order to bring this fruit to market, clinical studies are needed. These studies should be designed to identify the NA-specific antibody titer associated with protection against disease and to establish the dose of NA which elicits this protective titer. The development of vaccines that aim to induce NA-specific immunity would most certainly benefit from this additional information.

Highlights.

  • Neuraminidase (NA)-inhibiting antibodies are an independent correlate of vaccine efficacy

  • Many NA-specific antibodies bind to conserved epitopes and protect against heterologous viruses

  • Assays to measure NA content and immunogenicity of influenza vaccines have been developed

Acknowledgments

This work was supported by CBER, US-FDA and by the intramural research program of the NIAID, NIH.

Footnotes

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References and Recommended Reading

  • 1.Belongia EA, Simpson MD, King JP, Sundaram ME, Kelley NS, Osterholm MT, McLean HQ. Variable influenza vaccine effectiveness by subtype: a systematic review and meta-analysis of test-negative design studies. Lancet Infect Dis. 2016;16:942–951. doi: 10.1016/S1473-3099(16)00129-8. [DOI] [PubMed] [Google Scholar]
  • 2.Ferdinands JM, Fry AM, Reynolds S, Petrie J, Flannery B, Jackson ML, Belongia EA. Intraseason waning of influenza vaccine protection: Evidence from the US Influenza Vaccine Effectiveness Network, 2011–12 through 2014–15. Clin Infect Dis. 2017;64:544–550. doi: 10.1093/cid/ciw816. [DOI] [PubMed] [Google Scholar]
  • 3.Bragstad K, Emborg H, Fischer TK, Voldstedlund M, Gubbels S, Andersen B, Molbak K, Krause T. Low vaccine effectiveness against influenza A(H3N2) virus among elderly people in Denmark in 2012/13--a rapid epidemiological and virological assessment. Euro Surveill. 2013:18. [PubMed] [Google Scholar]
  • 4.Matrosovich MN, Matrosovich TY, Gray T, Roberts NA, Klenk HD. Neuraminidase is important for the initiation of influenza virus infection in human airway epithelium. J Virol. 2004;78:12665–12667. doi: 10.1128/JVI.78.22.12665-12667.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Yang X, Steukers L, Forier K, Xiong R, Braeckmans K, Van Reeth K, Nauwynck H. A beneficiary role for neuraminidase in influenza virus penetration through the respiratory mucus. PLoS One. 2014;9:e110026. doi: 10.1371/journal.pone.0110026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Palese P, Tobita K, Ueda M, Compans RW. Characterization of temperature sensitive influenza virus mutants defective in neuraminidase. Virology. 1974;61:397–410. doi: 10.1016/0042-6822(74)90276-1. [DOI] [PubMed] [Google Scholar]
  • 7.Griffin JA, Basak S, Compans RW. Effects of hexose starvation and the role of sialic acid in influenza virus release. Virology. 1983;125:324–334. doi: 10.1016/0042-6822(83)90205-2. [DOI] [PubMed] [Google Scholar]
  • 8.Kilbourne ED, Laver WG, Schulman JL, Webster RG. Antiviral activity of antiserum specific for an influenza virus neuraminidase. J Virol. 1968;2:281–288. doi: 10.1128/jvi.2.4.281-288.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Murphy BR, Kasel JA, Chanock RM. Association of serum anti-neuraminidase antibody with resistance to influenza in man. N Engl J Med. 1972;286:1329–1332. doi: 10.1056/NEJM197206222862502. [DOI] [PubMed] [Google Scholar]
  • 10•.Couch RB, Atmar RL, Franco LM, Quarles JM, Wells J, Arden N, Nino D, Belmont JW. Antibody correlates and predictors of immunity to naturally occurring influenza in humans and the importance of antibody to the neuraminidase. J Infect Dis. 2013;207:974–981. doi: 10.1093/infdis/jis935. HI and NI antibody titers were measured in serum and nasal secretions (NS) of volunteers who were monitored for acute respiratory illness. Multivariate analyses indicated serum HI and NI each independently predicted immunity to infection and infection-associated illness. Only serum NI independently predicted reduced illness among infected subjects. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Clements ML, Betts RF, Tierney EL, Murphy BR. Serum and nasal wash antibodies associated with resistance to experimental challenge with influenza A wild-type virus. J Clin Microbiol. 1986;24:157–160. doi: 10.1128/jcm.24.1.157-160.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12•.Monto AS, Petrie JG, Cross RT, Johnson E, Liu M, Zhong W, Levine M, Katz JM, Ohmit SE. Antibody to Influenza Virus Neuraminidase: An Independent Correlate of Protection. J Infect Dis. 2015;212:1191–1199. doi: 10.1093/infdis/jiv195. NI antibody titers were measured in sera of individuals vaccinated with either live, attenuated or inactivated influenza vaccines. Analyses suggested an independent role for NI antibodies in protection, which was similar in the IIV, LAIV, and placebo groups. [DOI] [PubMed] [Google Scholar]
  • 13.Easterbrook JD, Kash JC, Sheng ZM, Qi L, Gao J, Kilbourne ED, Eichelberger MC, Taubenberger JK. Immunization with 1976 swine H1N1- or 2009 pandemic H1N1-inactivated vaccines protects mice from a lethal 1918 influenza infection. Influenza Other Respir Viruses. 2011;5:198–205. doi: 10.1111/j.1750-2659.2010.00191.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Sultana I, Gao J, Markoff L, Eichelberger MC. Influenza neuraminidase-inhibiting antibodies are induced in the presence of zanamivir. Vaccine. 2011;29:2601–2606. doi: 10.1016/j.vaccine.2011.01.047. [DOI] [PubMed] [Google Scholar]
  • 15.Johansson BE, Bucher DJ, Kilbourne ED. Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection. J Virol. 1989;63:1239–1246. doi: 10.1128/jvi.63.3.1239-1246.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Wodal W, Falkner FG, Kerschbaum A, Gaiswinkler C, Fritz R, Kiermayr S, Portsmouth D, Savidis-Dacho H, Coulibaly S, Piskernik C, et al. A cell culture-derived whole-virus H9N2 vaccine induces high titer antibodies against hemagglutinin and neuraminidase and protects mice from severe lung pathology and weight loss after challenge with a highly virulent H9N2 isolate. Vaccine. 2012;30:4625–4631. doi: 10.1016/j.vaccine.2012.04.102. [DOI] [PubMed] [Google Scholar]
  • 17.Chen Z, Kim L, Subbarao K, Jin H. The 2009 pandemic H1N1 virus induces anti-neuraminidase (NA) antibodies that cross-react with the NA of H5N1 viruses in ferrets. Vaccine. 2012;30:2516–2522. doi: 10.1016/j.vaccine.2012.01.090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Rockman S, Brown LE, Barr IG, Gilbertson B, Lowther S, Kachurin A, Kachurina O, Klippel J, Bodle J, Pearse M, et al. Neuraminidase-inhibiting antibody is a correlate of cross-protection against lethal H5N1 influenza virus in ferrets immunized with seasonal influenza vaccine. J Virol. 2013;87:3053–3061. doi: 10.1128/JVI.02434-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Bosch BJ, Bodewes R, de Vries RP, Kreijtz JH, Bartelink W, van Amerongen G, Rimmelzwaan GF, de Haan CA, Osterhaus AD, Rottier PJ. Recombinant soluble, multimeric HA and NA exhibit distinctive types of protection against pandemic swine-origin 2009 A(H1N1) influenza virus infection in ferrets. J Virol. 2010;84:10366–10374. doi: 10.1128/JVI.01035-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Fritz R, Sabarth N, Kiermayr S, Hohenadl C, Howard MK, Ilk R, Kistner O, Ehrlich HJ, Barrett PN, Kreil TR. A vero cell-derived whole-virus H5N1 vaccine effectively induces neuraminidase-inhibiting antibodies. J Infect Dis. 2012;205:28–34. doi: 10.1093/infdis/jir711. [DOI] [PubMed] [Google Scholar]
  • 21.Cate TR, Rayford Y, Nino D, Winokur P, Brady R, Belshe R, Chen W, Atmar RL, Couch RB. A high dosage influenza vaccine induced significantly more neuraminidase antibody than standard vaccine among elderly subjects. Vaccine. 2010;28:2076–2079. doi: 10.1016/j.vaccine.2009.12.041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Hassantoufighi A, Zhang H, Sandbulte M, Gao J, Manischewitz J, King L, Golding H, Straight TM, Eichelberger MC. A practical influenza neutralization assay to simultaneously quantify hemagglutinin and neuraminidase-inhibiting antibody responses. Vaccine. 2010;28:790–797. doi: 10.1016/j.vaccine.2009.10.066. [DOI] [PubMed] [Google Scholar]
  • 23.Fries LF, Smith GE, Glenn GM. A recombinant viruslike particle influenza A (H7N9) vaccine. N Engl J Med. 2013;369:2564–2566. doi: 10.1056/NEJMc1313186. [DOI] [PubMed] [Google Scholar]
  • 24•.Wan H, Gao J, Xu K, Chen H, Couzens LK, Rivers KH, Easterbrook JD, Yang K, Zhong L, Rajabi M, et al. Molecular basis for broad neuraminidase immunity: conserved epitopes in seasonal and pandemic H1N1 as well as H5N1 influenza viruses. J Virol. 2013;87:9290–9300. doi: 10.1128/JVI.01203-13. A panel of mouse mAbs was selected against the NA of a seasonal H1N1 virus. The mAbs reacted differently with H1N1 and H5N1 viruses, suggesting a number of epitopes. The mAbs were used to select escape mutants; these were sequenced to map the antigenic domains. The changes in escape mutants identified strain-specific amino acids critical for binding of mAbs that reacted only with the immunizing virus, and conserved amino acids that were essential for binding broadly-reactive mAbs. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Webster RG, Brown LE, Laver WG. Antigenic and biological characterization of influenza virus neuraminidase (N2) with monoclonal antibodies. Virology. 1984;135:30–42. doi: 10.1016/0042-6822(84)90114-4. [DOI] [PubMed] [Google Scholar]
  • 26.Wan H, Yang H, Shore DA, Garten RJ, Couzens L, Gao J, Jiang L, Carney PJ, Villanueva J, Stevens J, et al. Structural characterization of a protective epitope spanning A(H1N1)pdm09 influenza virus neuraminidase monomers. Nat Commun. 2015;6:6114. doi: 10.1038/ncomms7114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27•.Wan H, Qi L, Gao J, Couzens LK, Jiang L, Gao Y, Sheng ZM, Fong S, Hahn M, Khurana S, et al. Comparison of the Efficacy of N9 Neuraminidase-Specific Monoclonal Antibodies against Influenza A(H7N9) Virus Infection. J Virol. 2018:92. doi: 10.1128/JVI.01588-17. Of outstanding interest: The ability of mAbs specific for the NA of an A/H7N9 virus were used prophylactically to protect mice from homologous lethal A/H7N9 virus challenge. The most effect mAb was one that inhibited cleavage of both small and large substrates in vitro, suggesting that in vitro characterization of mAb activity could be used to select mAbs for further development as therapeutics. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Wohlbold TJ, Podolsky KA, Chromikova V, Kirkpatrick E, Falconieri V, Meade P, Amanat F, Tan J, tenOever BR, Tan GS, et al. Broadly protective murine monoclonal antibodies against influenza B virus target highly conserved neuraminidase epitopes. Nat Microbiol. 2017;2:1415–1424. doi: 10.1038/s41564-017-0011-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Jiang L, Fantoni G, Couzens L, Gao J, Plant E, Ye Z, Eichelberger MC, Wan H. Comparative Efficacy of Monoclonal Antibodies That Bind to Different Epitopes of the 2009 Pandemic H1N1 Influenza Virus Neuraminidase. J Virol. 2015;90:117–128. doi: 10.1128/JVI.01756-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Monto AS, Kendal AP. Effect of neuraminidase antibody on Hong Kong influenza. Lancet. 1973;1:623–625. doi: 10.1016/s0140-6736(73)92196-x. [DOI] [PubMed] [Google Scholar]
  • 31•.Memoli MJ, Shaw PA, Han A, Czajkowski L, Reed S, Athota R, Bristol T, Fargis S, Risos K, Powers JH, et al. Evaluation of Antihemagglutinin and Antineuraminidase Antibodies as Correlates of Protection in an Influenza A/H1N1 Virus Healthy Human Challenge Model. MBio. 2016;7:e00417–00416. doi: 10.1128/mBio.00417-16. Of outstanding interest: A healthy volunteer challenge study that evaluated the correlation between anti-HA/anti-NA antibodies and disease severity. The study showed that NA inhibition antibody titers can be an independent predictor of reduction of all aspects of influenza disease. Importantly, the study demonstrated that although HAI titers >1:40 are predictive of some protection, there is strong evidence to suggest that NI titers are more predictive of protection and reduced disease. This is the first time NI titer has been clearly identified as an independent predictor of a reduction in all aspects of influenza. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Dunning AJ, DiazGranados CA, Voloshen T, Hu B, Landolfi VA, Talbot HK. Correlates of Protection against Influenza in the Elderly: Results from an Influenza Vaccine Efficacy Trial. Clin Vaccine Immunol. 2016;23:228–235. doi: 10.1128/CVI.00604-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33•.Wohlbold TJ, Nachbagauer R, Xu H, Tan GS, Hirsh A, Brokstad KA, Cox RJ, Palese P, Krammer F. Vaccination with adjuvanted recombinant neuraminidase induces broad heterologous, but not heterosubtypic, cross-protection against influenza virus infection in mice. MBio. 2015;6:e02556. doi: 10.1128/mBio.02556-14. Of outstanding interest: Studies were conducted using recombinant NAs of different subtypes to vaccinate mice that were subsequently challenged with homologous and heterologous viruses. Of particular importance is the finding that mice challenged with homologous viruses were protected against morbidity and mortality, and although there was some morbidity, all mice were protected against death when challenged with heterologous virus of the same NA subtype. This study demonstrates the cross-protective potential of influenza virus NA and suggests vaccines with targeted NA content will have greater breadth of protection. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Kilbourne ED, Pokorny BA, Johansson B, Brett I, Milev Y, Matthews JT. Protection of mice with recombinant influenza virus neuraminidase. J Infect Dis. 2004;189:459–461. doi: 10.1086/381123. [DOI] [PubMed] [Google Scholar]
  • 35•.Kingstad-Bakke B, Kamlangdee A, Osorio JE. Mucosal administration of raccoonpox virus expressing highly pathogenic avian H5N1 influenza neuraminidase is highly protective against H5N1 and seasonal influenza virus challenge. Vaccine. 2015;33:5155–5162. doi: 10.1016/j.vaccine.2015.08.005. Mouse studies examining efficacy of poxvirus vaccines against H5N1 influenza challenge. Raccoon poxvirus expressing HA provided strong protection when administered by parenteral routes, however raccoon poxvirus-NA was only effective when administered intranasally. This study suggests NA-specific antibodies may be most effective in the upper respiratory tract where local secretory IgA and not transudated IgG is effective. [DOI] [PubMed] [Google Scholar]
  • 36.Lei H, Peng X, Zhao D, Ouyang J, Jiao H, Shu H, Ge X. Lactococcus lactis displayed neuraminidase confers cross protective immunity against influenza A viruses in mice. Virology. 2015;476:189–195. doi: 10.1016/j.virol.2014.12.017. [DOI] [PubMed] [Google Scholar]
  • 37.Easterbrook JD, Schwartzman LM, Gao J, Kash JC, Morens DM, Couzens L, Wan H, Eichelberger MC, Taubenberger JK. Protection against a lethal H5N1 influenza challenge by intranasal immunization with virus-like particles containing 2009 pandemic H1N1 neuraminidase in mice. Virology. 2012;432:39–44. doi: 10.1016/j.virol.2012.06.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Wohlbold TJ, Chromikova V, Tan GS, Meade P, Amanat F, Comella P, Hirsh A, Krammer F. Hemagglutinin Stalk- and Neuraminidase-Specific Monoclonal Antibodies Protect against Lethal H10N8 Influenza Virus Infection in Mice. J Virol. 2015;90:851–861. doi: 10.1128/JVI.02275-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Wilson JR, Belser JA, DaSilva J, Guo Z, Sun X, Gansebom S, Bai Y, Stark TJ, Chang J, Carney P, et al. An influenza A virus (H7N9) anti-neuraminidase monoclonal antibody protects mice from morbidity without interfering with the development of protective immunity to subsequent homologous challenge. Virology. 2017;511:214–221. doi: 10.1016/j.virol.2017.08.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Shoji Y, Chichester JA, Palmer GA, Farrance CE, Stevens R, Stewart M, Goldschmidt L, Deyde V, Gubareva L, Klimov A, et al. An influenza N1 neuraminidase-specific monoclonal antibody with broad neuraminidase inhibition activity against H5N1 HPAI viruses. Hum Vaccin. 2011;7(Suppl):199–204. doi: 10.4161/hv.7.0.14595. [DOI] [PubMed] [Google Scholar]
  • 41.Schulman JL, Khakpour M, Kilbourne ED. Protective effects of specific immunity to viral neuraminidase on influenza virus infection of mice. J Virol. 1968;2:778–786. doi: 10.1128/jvi.2.8.778-786.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Liu WC, Lin CY, Tsou YT, Jan JT, Wu SC. Cross-Reactive Neuraminidase-Inhibiting Antibodies Elicited by Immunization with Recombinant Neuraminidase Proteins of H5N1 and Pandemic H1N1 Influenza A Viruses. J Virol. 2015;89:7224–7234. doi: 10.1128/JVI.00585-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Marcelin G, Bland HM, Negovetich NJ, Sandbulte MR, Ellebedy AH, Webb AD, Griffin YS, DeBeauchamp JL, McElhaney JE, Webby RJ. Inactivated seasonal influenza vaccines increase serum antibodies to the neuraminidase of pandemic influenza A(H1N1) 2009 virus in an age-dependent manner. J Infect Dis. 2010;202:1634–1638. doi: 10.1086/657084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Wrammert J, Koutsonanos D, Li GM, Edupuganti S, Sui J, Morrissey M, McCausland M, Skountzou I, Hornig M, Lipkin WI, et al. Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection. J Exp Med. 2011;208:181–193. doi: 10.1084/jem.20101352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Kilbourne ED, Johansson BE, Grajower B. Independent and disparate evolution in nature of influenza A virus hemagglutinin and neuraminidase glycoproteins. Proc Natl Acad Sci U S A. 1990;87:786–790. doi: 10.1073/pnas.87.2.786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Sandbulte MR, Westgeest KB, Gao J, Xu X, Klimov AI, Russell CA, Burke DF, Smith DJ, Fouchier RA, Eichelberger MC. Discordant antigenic drift of neuraminidase and hemagglutinin in H1N1 and H3N2 influenza viruses. Proc Natl Acad Sci U S A. 2011;108:20748–20753. doi: 10.1073/pnas.1113801108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Webster RG, Laver WG. Preparation and properties of antibody directed specifically against the neuraminidase of influenza virus. J Immunol. 1967;99:49–55. [PubMed] [Google Scholar]
  • 48.Couzens L, Gao J, Westgeest K, Sandbulte M, Lugovtsev V, Fouchier R, Eichelberger M. An optimized enzyme-linked lectin assay to measure influenza A virus neuraminidase inhibition antibody titers in human sera. J Virol Methods. 2014;210:7–14. doi: 10.1016/j.jviromet.2014.09.003. [DOI] [PubMed] [Google Scholar]
  • 49•.Gao J, Couzens L, Eichelberger MC. Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay. J Vis Exp. 2016 doi: 10.3791/54573. A video accompanied by a step-by-step standard operating procedure to measure NA inhibition antibody titers. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Eichelberger MC, Couzens L, Gao Y, Levine M, Katz J, Wagner R, Thompson CI, Hoschler K, Laurie K, Bai T, et al. Comparability of neuraminidase inhibition antibody titers measured by enzyme-linked lectin assay (ELLA) for the analysis of influenza vaccine immunogenicity. Vaccine. 2016;34:458–465. doi: 10.1016/j.vaccine.2015.12.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Westgeest KB, Bestebroer TM, Spronken MI, Gao J, Couzens L, Osterhaus AD, Eichelberger M, Fouchier RA, de Graaf M. Optimization of an enzyme-linked lectin assay suitable for rapid antigenic characterization of the neuraminidase of human influenza A(H3N2) viruses. J Virol Methods. 2015;217:55–63. doi: 10.1016/j.jviromet.2015.02.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Kosik I, Yewdell JW. Influenza A virus hemagglutinin specific antibodies interfere with virion neuraminidase activity via two distinct mechanisms. Virology. 2017;500:178–183. doi: 10.1016/j.virol.2016.10.024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Prevato M, Ferlenghi I, Bonci A, Uematsu Y, Anselmi G, Giusti F, Bertholet S, Legay F, Telford JL, Settembre EC, et al. Expression and Characterization of Recombinant, Tetrameric and Enzymatically Active Influenza Neuraminidase for the Setup of an Enzyme-Linked Lectin-Based Assay. PLoS One. 2015;10:e0135474. doi: 10.1371/journal.pone.0135474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Prevato M, Cozzi R, Pezzicoli A, Taddei AR, Ferlenghi I, Nandi A, Montomoli E, Settembre EC, Bertholet S, Bonci A, et al. An Innovative Pseudotypes-Based Enzyme-Linked Lectin Assay for the Measurement of Functional Anti-Neuraminidase Antibodies. PLoS One. 2015;10:e0135383. doi: 10.1371/journal.pone.0135383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55•.Rajendran M, Nachbagauer R, Ermler ME, Bunduc P, Amanat F, Izikson R, Cox M, Palese P, Eichelberger M, Krammer F. Analysis of Anti-Influenza Virus Neuraminidase Antibodies in Children, Adults, and the Elderly by ELISA and Enzyme Inhibition: Evidence for Original Antigenic Sin. MBio. 2017:8. doi: 10.1128/mBio.02281-16. Analysis of human antibody reactivity to a panel of N1, N2, and influenza B virus NAs in different age groups. The anti-NA antibody levels increased with age and were, in general, highest against strains that circulated during the childhood of the tested individuals, providing evidence for original antigenic sin (or back-boosting of the response). Importantly, titers measured by ELISA correlated well with NA-inhibition antibody titers. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Williams TL, Pirkle JL, Barr JR. Simultaneous quantification of hemagglutinin and neuraminidase of influenza virus using isotope dilution mass spectrometry. Vaccine. 2012;30:2475–2482. doi: 10.1016/j.vaccine.2011.12.056. [DOI] [PubMed] [Google Scholar]
  • 57.Wan H, Sultana I, Couzens LK, Mindaye S, Eichelberger MC. Assessment of influenza A neuraminidase (subtype N1) potency by ELISA. J Virol Methods. 2017;244:23–28. doi: 10.1016/j.jviromet.2017.02.015. [DOI] [PubMed] [Google Scholar]
  • 58.Sultana I, Yang K, Getie-Kebtie M, Couzens L, Markoff L, Alterman M, Eichelberger MC. Stability of neuraminidase in inactivated influenza vaccines. Vaccine. 2014;32:2225–2230. doi: 10.1016/j.vaccine.2014.01.078. [DOI] [PubMed] [Google Scholar]
  • 59.Couch RB, Atmar RL, Keitel WA, Quarles JM, Wells J, Arden N, Nino D. Randomized comparative study of the serum antihemagglutinin and antineuraminidase antibody responses to six licensed trivalent influenza vaccines. Vaccine. 2012;31:190–195. doi: 10.1016/j.vaccine.2012.10.065. [DOI] [PMC free article] [PubMed] [Google Scholar]

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