BACKGROUND:Antineuraminidase antibodies have been identified as a correlate of protection for influenza virus infection. We evaluated the immunogenicity of enhanced influenza vaccines vs standard-dose vaccine in inducing neuraminidase inhibition (NAI) antibodies in older adults in a 2-year randomized trial. METHODS:In 2017-2018, older adults aged 65 to 82 years in Hong Kong were randomly allocated to receive standard-dose quadrivalent (SD-IIV4), high-dose trivalent (HD-IIV3), MF59-adjuvanted trivalent (aIIV3), or recombinant quadrivalent (RIV4) influenza vaccines of 2017-2018 northern hemisphere formations; HD-IIV3, aIIV3, and RIV4 are enhanced vaccines. NAI antibodies to the 2017-2018 A(H1N1)pdm09 and A(H3N2) vaccine strains were determined from 400 recipients (100 per vaccine group). In 2018-2019, participants were rerandomized to receive the same or a different type of vaccine of northern hemisphere formations. NAI antibodies to the 2018-2019 A(H1N1)pdm09 and A(H3N2) vaccine strains were determined from SD-IIV4 (n = 45), HD-IIV3 (n = 64), aIIV3 (n = 75), or RIV4 (n = 29) recipients. NAI antibody titers on the day of vaccination and 30 days postvaccination were used to compare the geometric mean fold rise (GMFR) of titers and the seroconversion rates of enhanced influenza vaccines vs SD-IIV4. RESULTS:SD-IIV4, HD-IIV3, and aIIV3 induced detectable NAI antibodies to N1 and N2 antigens with GMFR significantly greater than 1. In both years, aIIV3 induced significantly higher GMFR and seroconversion rates to N1 and N2 antigens than SD-IIV4. Notably, individual baseline NAI antibody titers were inversely associated with the postvaccination antibody titer fold rises in all vaccine groups. CONCLUSIONS:MF-59-adjuvanted aIIV3 induced a superior NAI antibody response in older adults than SD-IIV4 in a 2-year randomized trial. CLINICAL TRIALS REGISTRATION:ClinicalTrials.gov: NCT03330132.

Neuraminidase (NA)-specific antibodies contribute to immunity against influenza. While studies have demonstrated increased NA inhibiting (NAI) antibody titers after vaccination with egg-derived inactivated influenza vaccines (eIIV), the response to cell culture-derived (c) IIV has not been reported. METHODS:An immunogenicity sub-study was performed within a clinical trial comparing the effectiveness of egg, cell, and recombinant hemagglutinin (HA)-derived influenza vaccines during the 2018-2019 and 2019-2020 influenza seasons. NAI and neutralizing antibody titers against the A(H1N1)pdm09 and A(H3N2) components of the vaccines were measured in pre- and post-vaccination sera. RESULTS:Responses to the N1 component of eIIV and cIIV were different in both study years 1 and 2 whereas response rate and antibody titers to the N2 component of egg and cell culture-derived vaccines were similar. For example, 43.5 % of eIIV and no cIIV recipients had four-fold NAI titer increases in year 1. There was a weak positive correlation between responses to N1 and N2 for both vaccine types but no correlation between NAI and HA-specific neutralizing antibody responses. Recombinant HA vaccine that does not contain NA served as a specificity control; NAI antibody titers did not increase in recipients except in two individuals presumed to have subclinical infection. CONCLUSION:Antibody responses to NA following vaccination with eIIV and cIIV were not the same; although the responses to the N1 and N2 components of eIIV were similar, there were fewer responders to N1 than N2 of cIIV. Studies to determine the impact of NA immunity on influenza vaccine effectiveness are warranted.

Determination of the potency of a vaccine is critical to ensuring that an appropriate dose is delivered, lot-to-lot consistency is maintained, and that the formulation is stable over the life of the vaccine. The potency of inactivated influenza vaccines is determined routinely by the Single Radial Immunodiffusion (SRID) assay. A number of alternative potency assays have been proposed and have been under evaluation in recent years. The aim of this study was to compare a surface plasmon resonance-based assay and two different enzyme linked immunoassays against the current potency assay, SRID, and against mouse immunogenicity when haemagglutinin antigen of the A(H1N1)pdm09 component of an inactivated influenza vaccine is stressed by elevated temperature, low pH and freezing. This analysis demonstrated that the alternative assays had good correspondence with SRID for samples from most stress conditions and that the immunogenicity in mice corresponded with potency in SRID for all stress samples. Subject to further analysis, the assays have been shown to have the potential to possibly replace, and at least complement, SRID.

Background. Low vaccine effectiveness against A(H3N2) influenza in seasons with little antigenic drift has been attributed to substitutions in hemagglutinin (HA) acquired during vaccine virus propagation in eggs. Clinical trials comparing recombinant HA vaccine (rHA) and cell-derived inactivated influenza vaccine (IIV) to egg-derived IIVs provide opportunities to assess how egg-adaptive substitutions influence HA immunogenicity. Methods. Neutralization titers in pre- and postimmunization sera from 133 adults immunized with 1 of 3 types of influenza vaccines in a randomized, open-label trial during the 2018-2019 influenza season were measured against egg- and cell-derived A/Singapore/INFIMH-16-0019/2016-like and circulating A(H3N2) influenza viruses using HA pseudoviruses. Results. All vaccines elicited neutralizing antibodies to all H3 vaccine antigens, but the rHA vaccine elicited the highest titers and seroconversion rates against all strains tested. Egg- and cell-derived IIVs elicited responses similar to each other. Preimmunization titers against H3 HA pseudoviruses containing egg-adaptive substitutions T160K and L194P were high, but lower against H3 HA pseudoviruses without those substitutions. All vaccines boosted neutralization titers against HA pseudoviruses with egg-adaptive substitutions, but poorly neutralized wild-type 2019-2020 A/Kansas/14/2017 (H3N2) HA pseudoviruses. Conclusion. Egg- and cell-derived 2018-2019 season influenza vaccines elicited similar neutralization titers and response rates, indicating that the cell-derived vaccine did not improve immunogenicity against the A(H3N2) viruses. The higher responses after rHA vaccination may be due to its higher HA content. All vaccines boosted titers to HA with egg-adaptive substitutions, suggesting boosting from past antigens or better exposure of HA epitopes. Studies comparing immunogenicity and effectiveness of different influenza vaccines across many seasons are needed.

Influenza virus infection elicits antibodies against the receptor-binding protein hemagglutinin (HA) and the receptor-cleaving protein neuraminidase (NA). Because HA is essential for viral entry, antibodies targeting HA often potently neutralize the virus in single-cycle infection assays. However, antibodies against NA are not potently neutralizing in such assays, since NA is dispensable for single-cycle infection. Here we show that a modified influenza virus that depends on NA for receptor binding is much more sensitive than a virus with receptor-binding HA to neutralization by some anti-NA antibodies. Specifically, a virus with a receptor-binding G147R N1 NA and a binding-deficient HA is completely neutralized in single-cycle infections by an antibody that binds near the NA active site. Infection is also substantially inhibited by antibodies that bind NA epitopes distant from the active site. Finally, we demonstrate that this modified virus can be used to efficiently select mutations in NA that escape antibody binding, a task that can be laborious with typical influenza viruses that are not well neutralized by anti-NA antibodies. Thus, viruses dependent on NA for receptor binding allow for sensitive in vitro detection of antibodies binding near the catalytic site of NA and enable the selection of viral escape mutants.

Viral ImmunologyVol. 33, No. 3 Opinions and ReviewsOpen AccessCreative Commons licensePeter's Paradigm and Pandemic PreparednessMaryna C. EichelbergerMaryna C. EichelbergerAddress correspondence to: Dr. Maryna C. Eichelberger, Division of Biological Standards and Quality Control, Office of Compliance and Biologics Quality, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20982 E-mail Address: maryna.eichelberger@fda.hhs.govDivision of Biological Standards and Quality Control, Office of Compliance and Biologics Quality, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, Maryland.Search for more papers by this authorPublished Online:15 Apr 2020https://doi.org/10.1089/vim.2019.0155AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Peter Doherty and Rolf Zinkernagel's discovery of how the body's immune system distinguishes virus-infected cells from normal cells is an essential element to curbing influenza infection and preventing severe disease. The recognition that CD8+ T cells kill virus-infected cells by engaging with an “altered self” (20), a complex of self major histocompatibility complex (MHC) molecule and the foreign antigen, enabled others to identify peptides of conserved influenza proteins as the primary targets of these killer cells (12, 17), providing a mechanism to explain protection against influenza A/H1N1 (or other subtypes) after influenza A/H3N2 infection. Vaccines that target the induction of CD8+ T cells and heterosubtypic immunity have, therefore, become an important strategy in the development of universal vaccines capable of broad protection and preventing severe disease during a pandemic (9, 3).During my postdoctoral training in the Doherty laboratory at St. Jude Children's Research Hospital, we investigated several aspects of CD8+ T cell immunity. “Tea time” each morning in the old St Jude cafeteria is where ideas were mulled and experimental plans developed. The ideas were big, leaving lots of room for alternatives and testable hypotheses. We discussed how the immune response was initiated and argued about whether antigen persisted. Conversations continued throughout the day, with Peter always available to answer questions or discuss a problem. Peter's love for learning, openness to sharing ideas, and generosity in spending time with his staff in discussions not only of work but also life, provided a foundation for my career period. His example has been a model that I still try to follow and encourage others to aspire to.We worked as a team in the Doherty laboratory; it was common for us to contribute to one another's experiments even if only to infect mice or harvest lymph nodes. In Peter's laboratory there were many opportunities to collaborate with molecular immunologists who were creating knockout or transgenic mice; an incredible time to show the in vivo function of single genes and to demonstrate the contribution of CD8+ T cells to influenza immunity. These opportunities to collaborate and Peter's amazing ability to communicate clearly provided me with a good number of quality publications (2, 4–8, 18) that advanced my career.Although most questions during my time working with Peter were targeted at understanding specific immune mechanisms, some of his work was translational, including several projects to identify and understand the human T cell response to viruses. From this came my interest to apply paradigms established in the laboratory to the human immune response, and for that reason, I joined the Center for Immunization Research at Johns Hopkins School of Public Health.The first DNA vaccine trial, a naked plasmid expressing hemagglutinin (HA), was conducted soon after I arrived at Hopkins. My group was responsible for establishing tests to measure antibody and T cell responses. Unfortunately, the vaccine was not immunogenic at any of the doses tested; the antibody responses were undetectable, and T cell responses were negligible (unpublished). Since HA does not have well-characterized class I human leukocyte antigen (HLA)-restricted epitopes, this study missed an opportunity to examine the ability of this novel vaccine to induce CD8+ T cell responses. DNA vaccines can indeed activate CD8+ T cells; a later DNA vaccine expressing a known target of human CD8+ T cells nucleoprotein (NP) increased the number of γ-interferon-producing T cells (16).Unfortunately, many prelicensure clinical studies have shortcomings due to designs that do not consider findings from basic research. This may include discoveries related to vaccine immunogenicity or improvements that have been made to measure the immune response. Although research in mice should not be used as a substitute for human studies, incorporation of lessons learnt from mouse studies will improve the chance of success of a universal vaccine. For example, vaccines that target the induction of cytotoxic T cells should be formulated or designed to express a known T cell target antigen in dendritic cells or to allow cross-presentation. This is easily achieved by live viruses, recombinant vectors that express the targeted antigen, or messenger RNA vaccines, whereas inactivated or peptide vaccines require the use of delivery vehicles such as liposomes, or adjuvants to deposit the antigen appropriately.The long history of studies by the Doherty laboratory and others demonstrating protection against influenza by T cell responses is finally being followed by clinical trials addressing that form of protection (9, 11). When human vaccine studies are planned, they should also consider findings from other clinical or epidemiologic studies. Despite strong evidence from studies conducted during the 1968 influenza pandemic that antibodies to neuraminidase (NA) contribute to immunity against influenza (14), responses to NA have only recently been considered more routinely as a secondary immunogenicity end point.NA inhibiting antibodies do not prevent infection but limit virus release from infected cells, resulting in “infection-permissive” immunity (13, 1). It is highly likely that NA-specific antibodies also provide a frontline defense against influenza infection by preventing virus release from mucins, thereby reducing the number of infectious particles that are available to infect mucosal epithelial cells. Although we have a good understanding of the mechanism of NA-specific antibody action, clinical studies of most vaccines containing both HA and NA are designed in such a way that they cannot evaluate the contribution of NA immunity. For example, vaccine efficacy studies routinely use PCR-confirmed influenza as an end point. Considering NA immunity does not prevent infection but rather reduces disease by limiting. Virus spread, the contribution of NA immunity would require clinical measures of illness severity or duration of virus replication.Test negative postlicensure observational studies are typically used to evaluate influenza vaccine effectiveness. In these studies, all subjects have symptoms of acute respiratory illness (i.e., influenza-like illness); the vaccine status of subjects positive for influenza by PCR testing is compared with the status of subjects who had a negative PCR test result. This type of study is very different from earlier observational studies of vaccine effectiveness in which cases were patients with influenza-like illness and controls were individuals without symptoms. With an understanding that NA immunity does not prevent infection but reduces clinical signs of disease, there is a good chance that the apparent poor effectiveness reported from studies using a test negative design may be the result of not counting individuals with subclinical or mild disease as benefiting from vaccination. The same issue applies to T cell vaccines.In my opinion, a universal vaccine that targets CD8+ T cells may be somewhat effective when CD8+ T cell memory is established in lymph nodes; however, there is a delay when the T cells are recalled to the site of infection (10). Therefore, the most effective vaccine may be one that induces local T cell immunity and results in memory T cells in the lungs. The benefit of having such CD8+ T cell memory located in the lungs in reducing virus load and recovery from infection is evident in a mouse model (19). Vaccines that target the induction of local immunity would need to be administered intranasally. This idea is verified by the rapid and robust protection observed in mice that were immunized with universal vaccine candidates intranasally (15).Although animal studies can demonstrate that influenza-specific CD8+ T cells have been induced and are present in the nasal or bronchial-associated lymphoid tissue or lungs, this would be difficult to evaluate during a human vaccine study. Evaluation of the benefit of vaccination is also difficult; as for NA, influenza-specific cytolytic T cells do not protect from infection and, therefore, clinical benefit such as shortened duration of infection or reduced signs of disease would need to be demonstrated by daily monitoring of clinical signs and samples collected at several time points to determine virus titer or duration of infection. This is not possible to achieve in a typical observational study of vaccine efficacy.Clinical challenge studies may be essential to establish overall benefit NA or CD8+ T cell-inducing vaccines in reducing symptoms and/or duration of influenza-like illness, and some such studies have been carried out. Although CD8+ T cell immunity may have minimal impact on seasonal influenza in a background of robust antibody responses to vaccines well matched to the virus, it is likely to be critical during a pandemic or an outbreak of an unexpected strain.Given the current emphasis and need for development of a universal influenza vaccine, it would serve funding bodies and regulators well to make sure Peter's discoveries are considered in the development of universal influenza vaccines that target induction of CD8+ T cell responses. This type of vaccine could save millions of lives during a pandemic.Author DisclaimerMy comments are an informal communication and represent my own best judgment. These comments do not bind or obligate the Food and Drug Administration.Author Disclosure StatementNo competing financial interests exist.References1. Couch RB, Kasel JA, Gerin JL, Schulman JL, and Kilbourne ED. Induction of partial immunity to influenza by a neuraminidase-specific vaccine. J Infect Dis 1974;129:411–420. Crossref, Medline, Google Scholar2. Deckhut AM, Allan W, McMickle A, et al. Prominent usage of V beta 8.3 T cells in the H-2Db-restricted response to an influenza A virus nucleoprotein epitope. J Immunol 1993;151:2658–2666. Medline, Google Scholar3. Dutton RW, Swain SL, and Woodland DL. Vaccines against pandemic influenza. Viral Immunol 2007;20:326–327. Link, Google Scholar4. Eichelberger M, Allan W, Carding SR, Bottomly K, and Doherty PC. Activation status of the CD4-8-gamma delta-T cells recovered from mice with influenza pneumonia. J Immunol 1991;147:2069–2074. Medline, Google Scholar5. Eichelberger M, Allan W, Zijlstra M, Jaenisch R, and Doherty PC. Clearance of influenza virus respiratory infection in mice lacking class I major histocompatibility complex-restricted CD8+ T cells. J Exp Med 1991;174:875–880. Crossref, Medline, Google Scholar6. Eichelberger M, and Doherty PC. Gamma delta T cells from influenza-infected mice develop a natural killer cell phenotype following culture. Cell Immunol 1994;159:94–102. Crossref, Medline, Google Scholar7. Eichelberger M, McMickle A, Blackman M, et al. Functional analysis of the TCR alpha- beta+ cells that accumulate in the pneumonic lung of influenza virus-infected TCR-alpha−/− mice. J Immunol 1995;154:1569–1576. Medline, Google Scholar8. Eichelberger MC, Wang ML, Allan W, Webster RG, and Doherty PC. Influenza virus RNA in the lung and lymphoid tissue of immunologically intact and CD4-depleted mice. J Gen Virol 1991;72(Pt. 7):1695–1698. Crossref, Medline, Google Scholar9. Epstein SL. Universal influenza vaccines: progress in achieving broad cross-protection in vivo. Am J Epidemiol 2018;187:2603–2614. Crossref, Medline, Google Scholar10. Flynn KJ, Belz GT, Altman JD, et al. Virus-specific CD8+ T cells in primary and secondary influenza pneumonia. Immunity 1998;8:683–691. Crossref, Medline, Google Scholar11. Gilbert SC. Advances in the development of universal influenza vaccines. Influenza Other Respir Viruses 2013;7:750–758. Crossref, Medline, Google Scholar12. Gotch F, Rothbard J, Howland K, Townsend A, and McMichael A. Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2. Nature 1987;326:881–882. Crossref, Medline, Google Scholar13. Kilbourne ED. Comparative efficacy of neuraminidase-specific and conventional influenza virus vaccines in induction of antibody to neuraminidase in humans. J Infect Dis 1976;134:384–394. Crossref, Medline, Google Scholar14. Murphy BR, Kasel JA, and Chanock RM. Association of serum anti-neuraminidase antibody with resistance to influenza in man. N Engl J Med 1972;286:1329–1332. Crossref, Medline, Google Scholar15. Price GE, Soboleski MR, Lo CY, et al. Single-dose mucosal immunization with a candidate universal influenza vaccine provides rapid protection from virulent H5N1, H3N2 and H1N1 viruses. PLoS One 2010;5:e13162. Crossref, Medline, Google Scholar16. Smith LR, Wloch MK, Ye M, et al. Phase 1 clinical trials of the safety and immunogenicity of adjuvanted plasmid DNA vaccines encoding influenza A virus H5 hemagglutinin. Vaccine 2010;28:2565–2572. Crossref, Medline, Google Scholar17. Townsend AR, Rothbard J, Gotch FM, et al. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell 1986;44:959–968. Crossref, Medline, Google Scholar18. Van Kaer L, Ashton-Rickardt PG, Eichelberger M, et al. Altered peptidase and viral-specific T cell response in LMP2 mutant mice. Immunity 1994;1:533–541. Crossref, Medline, Google Scholar19. Wiley JA, Hogan RJ, Woodland DL, and Harmsen AG. Antigen-specific CD8(+) T cells persist in the upper respiratory tract following influenza virus infection. J Immunol 2001;167:3293–3299. Crossref, Medline, Google Scholar20. Zinkernagel RM, and Doherty PC. Immunological surveillance against altered self components by sensitised T lymphocytes in lymphocytic choriomeningitis. Nature 1974;251:547–548. Crossref, Medline, Google ScholarFiguresReferencesRelatedDetails Volume 33Issue 3Apr 2020 Information© Maryna C. Eichelberger, 2020; Published by Mary Ann Liebert, Inc.To cite this article:Maryna C. Eichelberger.Peter's Paradigm and Pandemic Preparedness.Viral Immunology.Apr 2020.208-210.http://doi.org/10.1089/vim.2019.0155creative commons licensePublished in Volume: 33 Issue 3: April 15, 2020Open accessThis Open Access article is distributed under the terms of the Creative Commons Attribution Noncommercial License ( http://creativecommons.org/licenses/by-nc/4.0/) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are cited.PDF download

For 50 years it has been known that antibodies to neuraminidase (NA) protect against infection during seasonal and pandemic influenza outbreaks. However, NA is largely ignored in the formulation and standardization of our current influenza vaccines. There are a number of factors that contributed to this antigen being forgotten, including the lack of an easily performed test to measure NA antibody. With the availability of that test, it has been possible to show its independent contribution to protection in various situations. The challenge now is to make it possible to include known amounts of NA in investigational vaccines or to routinely measure NA content in licensed vaccines. Vaccines containing optimal amounts of NA may be particularly useful when there are antigenic changes, either drift or shift, in the hemagglutinin because NA immunity offers broad protection. It is now time to remember the NA as we work toward improved influenza vaccines.

The effectiveness of influenza vaccines against circulating A(H1N1)pdm09 viruses was modest for several seasons despite the absence of antigenic drift of hemagglutinin (HA), the primary vaccine component. Since antibodies against HA and neuraminidase (NA) contribute independently to protection against disease, antigenic changes in NA may allow A(H1N1)pdm09 viruses to escape from vaccine-induced immunity. In this study, analysis of the specificities of human NA-specific monoclonal antibodies identified antigenic sites that have changed over time. The impact of these differences on in vitro inhibition of enzyme activity was not evident for polyclonal antisera until viruses emerged in 2013 without a predicted glycosylation site at amino acid 386 in NA. Phylogenetic and antigenic cartography demonstrated significant antigenic changes that in most cases aligned with genetic differences. Typical of NA drift, the antigenic difference is observed in one direction, with antibodies against conserved antigenic domains in A/California/7/2009 (CA/09) continuing to inhibit NA of recent A(H1N1)pdm09 viruses reasonably well. However, ferret CA/09-specific antiserum that inhibited the NA of A/Michigan/45/2015 (MI/15) very well in vitro, protected mice against lethal MI/15 infection poorly. These data show that antiserum against the homologous antigen is most effective and suggest the antigenic properties of NA should not be overlooked when selecting viruses for vaccine production.IMPORTANCE The effectiveness of seasonal influenza vaccines against circulating A(H1N1)pdm09 viruses has been modest in recent years, despite the absence of antigenic drift of HA, the primary vaccine component. Human monoclonal antibodies identified antigenic sites in NA that changed early after the new pandemic virus emerged. The reactivity of ferret antisera demonstrated antigenic drift of A(H1N1)pdm09 NA from 2013 onward. Passive transfer of serum raised against A/California/7/2009 was less effective than ferret serum against the homologous virus in protecting mice against a virus with the NA of more recent virus, A/Michigan/45/2015. Given the long-standing observation that NA-inhibiting antibodies are associated with resistance against disease in humans, these data demonstrate the importance of evaluating NA drift and suggest that vaccine effectiveness might be improved by selecting viruses for vaccine production that have NAs antigenically similar to those of circulating influenza viruses.

A(H3N2) virus predominated recent influenza seasons, which has resulted in the rigorous investigation of haemagglutinin, but whether neuraminidase (NA) has undergone antigenic change and contributed to the predominance of A(H3N2) virus is unknown. Here, we show that the NA of the circulating A(H3N2) viruses has experienced significant antigenic drift since 2016 compared with the A/Hong Kong/4801/2014 vaccine strain. This antigenic drift was mainly caused by amino acid mutations at NA residues 245, 247 (S245N/S247T; introducing an N -linked glycosylation site at residue 245) and 468. As a result, the binding of the NA of A(H3N2) virus by some human monoclonal antibodies, including those that have broad reactivity to the NA of the 1957 A(H2N2) and 1968 A(H3N2) reference pandemic viruses as well as contemporary A(H3N2) strains, was reduced or abolished. This antigenic drift also reduced NA-antibody-based protection against in vivo virus challenge. X-ray crystallography showed that the glycosylation site at residue 245 is within a conserved epitope that overlaps the NA active site, explaining why it impacts antibody binding. Our findings suggest that NA antigenic drift impacts protection against influenza virus infection, thus highlighting the importance of including NA antigenicity for consideration in the optimization of influenza vaccines.

Background. Antibodies that inhibit hemagglutination have long been considered a correlate of protection against influenza, but these antibodies are only a subset of potentially protective antibodies. Neutralizing and neuraminidase antibodies may also contribute to protection, but data on their associations with protection are limited. Methods. We measured preoutbreak hemagglutinin pseudovirus neutralization (PVN) and neuraminidase inhibition (NAI) antibody titers in unvaccinated military recruits who experienced an H3N2 influenza outbreak during training. We conducted a case-control study to investigate the association between titers and protection against influenza illness or H3N2-associated pneumonia using logistic regression. Results. With every 2-fold increase in PVN titer, the odds of medically attended polymerase chain reaction-confirmed H3N2 infection (H3N2(+)) decreased by 41% (odds ratio [OR], 0.59; 95% confidence interval [CI], .45 to .77; P < .001). Among those who were H3N2(+), the odds for pneumonia decreased by 52% (OR, 0.48; CI, .25 to .91; P = .0249). With every 2-fold increase in NAI titer, the odds of medically attended H3N2 infection decreased by 32% (OR, 0.68; 95% CI, .53 to .87; P = .0028), but there was no association between NAI titers and H3N2-associated pneumonia. There was also no synergistic effect of PVN and NAI antibodies. Conclusions. PVN and NAI titers were independently associated with reduced risk of influenza illness. NAI titers associated with protection had greater breadth of reactivity to drifted strains than PVN titers. These findings show that PVN and NAI titers are valuable biomarkers for assessing the odds of influenza infection.

ABSTRACTBackgroundInfluenza vaccines are important for prevention of influenza-associated hospitalization. Assessments of serologic correlates of protection can support interpretation of influenza vaccine effectiveness evaluations in hospitalized populations.MethodsSerum specimens collected at admission from adults hospitalized for treatment of acute respiratory illnesses during two influenza seasons were tested in hemagglutination-inhibition (HAI) and neuraminidase-inhibition (NAI) assays. We evaluated the suitability of these specimens as proxies for pre-infection immune status, and measured associations between antibody titers and influenza vaccination and infectionResultsSpecimens were collected within 3 days of illness onset from 65% of participants; geometric mean titers (GMTs) did not vary by day of collection. In both seasons, vaccinated participants had higher HAI and NAI GMTs than unvaccinated participants. HAI titers against the 2014-2015 A(H3N2) vaccine strain did not correlate with protection from infection with antigenically-drifted A(H3N2) viruses that circulated that season. In contrast, higher HAI titers against the A(H1N1)pdm09 vaccine strain were associated with reduced odds of A(H1N1)pdm09 infection in 2015-2016.ConclusionsSerum collected after hospital admission can be used to assess correlates of protection against influenza infection. Broader implementation of similar studies would provide an opportunity to understand the successes and shortcomings of current influenza vaccines.

BACKGROUND:Formulation of neuraminidase (NA) within influenza vaccines is gaining importance in light of recent human studies. The enzyme-linked lectin assay (ELLA) is considered a reliable assay to evaluate human anti-NA antibodies.OBJECTIVES:To overcome interference by hemagglutinin (HA)-specific antibodies and detect neuraminidase inhibitory (NI) antibodies only, two different sources of antigen have been studied in ELLA: reassortant viruses with a mismatched avian origin-HA or Triton X-100 (Tx)-treated wild-type viruses. Pseudotypes or pseudovirus (PV), characterized by a lentivirus core bearing human influenza NA and avian influenza HA, were investigated as an alternative source of antigen and compared to HA-mismatched and Tx-treated viruses, since represent a safer product to be handled.METHODS:Two independent panels of sera were analyzed by ELLA to evaluate the anti-NA response against N1 (A/California/07/2009 (H1N1pdm)) and N2 (A/Hong Kong/4801/2014 (H3N2)). The NA inhibition (NI) antibody titers measured as either the 50% end point or 50% inhibitory concentration (IC50 ) were compared for every source of antigen.RESULTS:The ELLA assay performed well with all three sources of antigen. NI titers measured using each antigen type correlated well when reported either as end point titers or as the IC50 .CONCLUSIONS:This study suggests that HA-mismatched whole virus, Triton-treated wild-type virus or PV can be used to measure NI antibody titers of human sera, but further comparability/validation assays should be performed to assess statistical differences. The data support the use of PV as an attractive alternative source of antigen and justify further investigation to improve stability of this antigen source.

Practical methods to measure the potency of influenza vaccines are needed as alternatives for the standard single radial immunodiffusion (SRID) assay. VaxArray assays for influenza hemagglutinin (HA) and neuraminidase (NA) have been developed to address this need. In this report, we evaluate the use of these assays to assess the potency of HA and NA of an A/H3N2 subunit vaccine by determining the correlation between the amounts measured by VaxArray and the immunogenicity in mice. The antibody response after one and two doses of five formulations of the vaccine ranging from 5 µg/mL to 80 µg/mL of HA, was measured by hemagglutination inhibition (HAI) and neuraminidase inhibition (NAI) assays. For hemagglutinin, vaccine potency determined by VaxArray was equivalent to potency measured SRID and these amounts were predictive of immunogenicity, with excellent correlation between potency measured by VaxArray and the HAI geometric mean titers (GMT). Likewise, the amount of NA measured by VaxArray was predictive of the NAI GMT. The VaxArray NA assay reported non-detectable levels of intact NA for a sample that had been heat degraded at 56 °C for 20 h, demonstrating that the assay measures the native, active form of NA. Similarly, the HA potency measured by VaxArray in this heat-treated sample was very low when a monoclonal antibody was used to detect the amount of antigen bound. Importantly, the force degraded sample induced low HAI titers and the NAI titers were not measurable, supporting the conclusion that the VaxArray HA and NA assays measure the immunogenic forms of these A/H3N2 antigens. This study indicates that VaxArray assays can be used to assess the potency of HA and NA components in influenza vaccines as a proxy for immunogenicity.

Influenza subtypes such as H7 have pandemic potential since they are able to infect humans with severe consequences, as evidenced by the ongoing H7N9 infections in China that began in 2013. The diversity of H7 viruses calls for a broadly cross-protective vaccine for protection. We describe the construction of recombinant modified vaccinia virus Ankara (MVA) vectors expressing the hemagglutinin (HA) or neuraminidase (NA) from three H7 viruses representing both Eurasian and North American H7 lineages – A/mallard/Netherlands/12/2000 (H7N3), A/Canada/rv444/2004 (H7N3), and A/Shanghai/02/2013 (H7N9). These vectors were evaluated for immunogenicity and protective efficacy against H7N3 virus in a murine model of intranasal challenge. High levels of H7-, N3-, and N9-specific antibodies, including neutralizing antibodies, were induced by the MVA-HA and MVA-NA vectors. Mice vaccinated with MVA vectors expressing any of the H7 antigens were protected, suggesting cross-protection among H7 viruses. In addition, MVA vectors expressing N3 but not N9 elicited protection against H7N3 virus challenge. Similar outcomes were obtained when immune sera from MVA vector-immunized mice were passively transferred to naïve mice prior to challenge with the H7N3 virus. The results support the further development of an MVA vector platform as a candidate vaccine for influenza strains with pandemic potential.

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.

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.

ABSTRACT The fifth wave of A(H7N9) virus infection in China from 2016 to 2017 caused great concern due to the large number of individuals infected, the isolation of drug-resistant viruses, and the emergence of highly pathogenic strains. Antibodies against neuraminidase (NA) provide added benefit to hemagglutinin-specific immunity and may be important contributors to the effectiveness of A(H7N9) vaccines. We generated a panel of mouse monoclonal antibodies (MAbs) to identify antigenic domains on NA of the novel A(H7N9) virus and compared their functional properties. The loop formed in the region of residue 250 (250 loop) and the domain formed by the loops containing residues 370, 400, and 430 were identified as major antigenic regions. MAbs 1E8, 2F6, 10F4, and 11B2, which recognize these two antigenic domains, were characterized in depth. These four MAbs differ in their abilities to inhibit cleavage of small and large substrates (methyl-umbelliferyl-acetyl neuraminic acid [MU-NANA] and fetuin, respectively) in NA inhibition assays. 1E8 and 11B2 did not inhibit NA cleavage of either MU-NANA or fetuin, and 2F6 inhibited cleavage of fetuin alone, whereas 10F4 inhibited cleavage of both substrates. All four MAbs reduced the in vitro spread of viruses carrying either the wild-type N9 or N9 with antiviral-resistant mutations but to different degrees. These MAbs have different in vivo levels of effectiveness: 10F4 was the most effective in protecting mice against challenge with A(H7N9) virus, 2F6 was less effective, and 11B2 failed to protect BALB/c mice at the doses tested. Our study confirms that NA-specific antibodies can protect against A(H7N9) infection and suggests that in vitro properties can be used to rank antibodies with therapeutic potential. IMPORTANCE The novel A(H7N9) viruses that emerged in China in 2013 continue to infect humans, with a high fatality rate. The most recent outbreak resulted in a larger number of human cases than previous epidemic waves. Due to the absence of a licensed vaccine and the emergence of drug-resistant viruses, there is a need to develop alternative approaches to prevent or treat A(H7N9) infection. We have made a panel of mouse monoclonal antibodies (MAbs) specific for neuraminidase (NA) of A(H7N9) viruses; some of these MAbs are effective in inhibiting viruses that are resistant to antivirals used to treat A(H7N9) patients. Binding avidity, inhibition of NA activity, and plaque formation correlated with the effectiveness of these MAbs to protect mice against lethal A(H7N9) virus challenge. This study identifies in vitro measures that can be used to predict the in vivo efficacy of NA-specific antibodies, providing a way to select MAbs for further therapeutic development.

ABSTRACTNeuraminidase is one of the two surface glycoproteins of influenza A and B viruses. It has enzymatic activity that cleaves terminal sialic acid from glycans, and that activity is essential at several points in the virus life cycle. While neuraminidase is a major target for influenza antivirals, it is largely ignored in vaccine development. Current inactivated influenza virus vaccines might contain neuraminidase, but the antigen quantity and quality are varied and not standardized. While there are data that show a protective role of anti-neuraminidase immunity, many questions remain unanswered. These questions, among others, concern the targeted epitopes or antigenic sites, the potential for antigenic drift, and, connected to that, the breadth of protection, differences in induction of immune responses by vaccination versus infection, mechanisms of protection, the role of mucosal antineuraminidase antibodies, stability, and the immunogenicity of neuraminidase in vaccine formulations. Reagents for analysis of neuraminidase-based immunity are scarce, and assays are not widely used for clinical studies evaluating vaccines. However, efforts to better understand neuraminidase-based immunity have been made recently. A neuraminidase focus group, NAction!, was formed at a Centers of Excellence for Influenza Research and Surveillance meeting at the National Institutes of Health in Bethesda, MD, to promote research that helps to understand neuraminidase-based immunity and how it can contribute to the design of better and broadly protective influenza virus vaccines. Here, we review open questions and knowledge gaps that have been identified by this group and discuss how the gaps can be addressed, with the ultimate goal of designing better influenza virus vaccines.

Antibodies that inhibit neuraminidase (NA) activity of influenza virus provide resistance against disease and have been associated with milder epidemics. Although studies have demonstrated a correlation between NA inhibition antibody titers and vaccine efficacy, neither the quantity nor form of NA is measured in seasonal and pandemic influenza vaccines. In this report, we describe development of enzyme-linked immunosorbent assays (ELISAs) that are suitable for quantitation of the native form of NA of subtype N1. The assays use mouse monoclonal antibodies (mAbs) 1H5 and CD6 to capture NAs of viruses, and a different mAb 4E9 to detect bound antigen. The 1H5-capture ELISA detects NAs of seasonal and pandemic H1N1 viruses as well as H5N1 viruses and has a limit of quantitation (LOQ) of 5.5ng/mL for seasonal H1N1A/Brisbane/59/2007 NA. The CD6-capture ELISA is specific for NA of the 2009 pandemic viruses with a LOQ of 67ng/mL for A/California/07/2009 NA. The ELISA signals in both assays are proportional to NA enzymatic activity and correlate with NA immunogenicity. The ELISAs we describe may expedite the development of NA-based influenza vaccines by providing a practical assay to measure NA potency.

ABSTRACT Antibody responses to influenza virus hemagglutinin provide protection against infection and are well studied. Less is known about the human antibody responses to the second surface glycoprotein, neuraminidase. Here, we assessed human antibody reactivity to a panel of N1, N2, and influenza B virus neuraminidases in different age groups, including children, adults, and the elderly. Using enzyme-linked immunosorbent assays (ELISA), we determined the breadth, magnitude, and isotype distribution of neuraminidase antibody responses to historic, current, and avian strains, as well as to recent isolates to which these individuals have not been exposed. It appears that antibody levels against N1 neuraminidases were lower than those against N2 or B neuraminidases. The anti-neuraminidase 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.” Titers measured by ELISA correlated well with titers measured by the neuraminidase inhibition assays. However, in the case of the 2009 pandemic H1N1 virus, we found evidence of interference from antibodies binding to the conserved stalk domain of the hemagglutinin. In conclusion, we found that antibodies against the neuraminidase differ in magnitude and breadth between subtypes and age groups in the human population. (This study has been registered at ClinicalTrials.gov under registration no. NCT00336453, NCT00539981, and NCT00395174.) IMPORTANCE Anti-neuraminidase antibodies can afford broad protection from influenza virus infection in animal models and humans. However, little is known about the breadth and magnitude of the anti-neuraminidase response in the human population. Here we assessed antibody levels of children, adults, and the elderly against a panel of N1, N2, and type B influenza virus neuraminidases. We demonstrated that antibody levels measured by ELISA correlate well with functional neuraminidase inhibition titers. This is an important finding since ELISA is a simpler method than functional assays and can be implemented in high-throughput settings to analyze large numbers of samples. Furthermore, we showed that low titers of broadly cross-reactive antibodies against neuraminidase are prevalent in humans. By the use of an appropriate vaccination strategy, these titers could potentially be boosted to levels that might provide broad protection from influenza virus infection.