Assessing the stability-indicating properties of alternative potency assays for inactivated influenza vaccine.

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.

Neutralizing and Neuraminidase Antibodies Correlate With Protection Against Influenza During a Late Season A/H3N2 Outbreak Among Unvaccinated Military Recruits.

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.

The neuraminidase of A(H3N2) influenza viruses circulating since 2016 is antigenically distinct from the A/Hong Kong/4801/2014 vaccine strain.

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.

Use of lentiviral pseudotypes as an alternative to reassortant or Triton X-100-treated wild-type Influenza viruses in the neuraminidase inhibition enzyme-linked lectin assay.

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.

Antigenic Drift of the Influenza A(H1N1)pdm09 Virus Neuraminidase Results in Reduced Effectiveness of A/California/7/2009 (H1N1pdm09)-Specific Antibodies.

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.

VaxArray for hemagglutinin and neuraminidase potency testing of influenza vaccines.

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.

Comparison of the Efficacy of N9 Neuraminidase-Specific Monoclonal Antibodies against Influenza A(H7N9) Virus Infection.

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.

Assessment of influenza A neuraminidase (subtype N1) potency by ELISA.

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.

Amino Acids in Hemagglutinin Antigenic Site B Determine Antigenic and Receptor Binding Differences between A(H3N2)v and Ancestral Seasonal H3N2 Influenza Viruses.

Influenza A H3N2 variant [A(H3N2)v] viruses, which have caused human infections in the United States in recent years, originated from human seasonal H3N2 viruses that were introduced into North American swine in the mid-1990s, but they are antigenically distinct from both the ancestral and current circulating H3N2 strains. A reference A(H3N2)v virus, A/Minnesota/11/2010 (MN/10), and a seasonal H3N2 strain, A/Beijing/32/1992 (BJ/92), were chosen to determine the molecular basis for the antigenic difference between A(H3N2)v and the ancestral viruses. Viruses containing wild-type and mutant MN/10 or BJ/92 hemagglutinins (HAs) were constructed and probed for reactivity with ferret antisera against MN/10 and BJ/92 in hemagglutination inhibition assays. Among the amino acids that differ between the MN/10 and BJ/92 HAs, those in antigenic site A had little impact on the antigenic phenotype. Within antigenic site B, mutations at residues 156, 158, 189, and 193 of MN/10 HA to those in BJ/92 switched the MN/10 antigenic phenotype to that of BJ/92. Mutations at residues 156, 157, 158, 189, and 193 of BJ/92 HA to amino acids present in MN/10 were necessary for BJ/92 to become antigenically similar to MN/10. The HA amino acid substitutions responsible for switching the antigenic phenotype also impacted HA binding to sialyl receptors that are usually present in the human respiratory tract. Our study demonstrates that antigenic site B residues play a critical role in determining both the unique antigenic phenotype and receptor specificity of A(H3N2)v viruses, a finding that may facilitate future surveillance and risk assessment of novel influenza viruses. IMPORTANCE: Influenza A H3N2 variant [A(H3N2)v] viruses have caused hundreds of human infections in multiple states in the United States since 2009. Most cases have been children who had contact with swine in agricultural fairs. These viruses originated from human seasonal H3N2 viruses that were introduced into the U.S. swine population in the mid-1990s, but they are different from both these ancestral viruses and current circulating human seasonal H3N2 strains in terms of their antigenic characteristics as measured by hemagglutination inhibition (HI) assay. In this study, we identified amino acids in antigenic site B of the surface glycoprotein hemagglutinin (HA) that explain the antigenic difference between A(H3N2)v and the ancestral H3N2 strains. These amino acid mutations also alter binding to minor human-type glycans, suggesting that host adaptation may contribute to the selection of antigenically distinct H3N2 variants which pose a threat to public health.

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 °C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in ≥50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

Comparative Efficacy of Monoclonal Antibodies That Bind to Different Epitopes of the 2009 Pandemic H1N1 Influenza Virus Neuraminidase.

UNLABELLED: Antibodies against the neuraminidase (NA) of influenza virus correlate with resistance against disease, but the effectiveness of antibodies against different NA epitopes has not been compared. In the present study, we evaluated the in vitro and in vivo efficacies of four monoclonal antibodies (MAbs): HF5 and CD6, which are specific to two different epitopes in the NA of 2009 pandemic H1N1 (pH1N1) virus, and 4E9 and 1H5, which are specific to a conserved epitope in the NA of both H1N1 and H5N1 viruses. In the in vitro assays, HF5 and CD6 inhibited virus spread and growth more effectively than 4E9 and 1H5, with HF5 being the most effective inhibitor. When administered prophylactically at 5 mg/kg of body weight, HF5 and CD6 protected ~90 to 100% of DBA/2 mice against lethal wild-type pH1N1 virus challenge; however, at a lower dose (1 mg/kg), HF5 protected ~90% of mice, whereas CD6 protected only 25% of mice. 4E9 and 1H5 were less effective than HF5 and CD6, as indicated by the partial protection achieved even at doses as high as 15 mg/kg. When administered therapeutically, HF5 protected a greater proportion of mice against lethal pH1N1 challenge than CD6. However, HF5 quickly selected pH1N1 virus escape mutants in both prophylactic and therapeutic treatments, while CD6 did not. Our findings confirm the important role of NA-specific antibodies in immunity to influenza virus and provide insight into the properties of NA antibodies that may serve as good candidates for therapeutics against influenza. IMPORTANCE: Neuraminidase (NA) is one of the major surface proteins of influenza virus, serving as an important target for antivirals and therapeutic antibodies. The impact of NA-specific antibodies on NA activity and virus replication is likely to depend on where the antibody binds. Using in vitro assays and the mouse model, we compared the inhibitory/protective efficacy of four mouse monoclonal antibodies (MAbs) that bind to different sites within the 2009 pandemic H1N1 (pH1N1) virus NA. The ability of each MAb to protect mice against lethal pH1N1 infection corresponded to its ability to inhibit NA activity in vitro; however, the MAb that was the most effective inhibitor of NA activity selected pH1N1 escape variants in vivo. One of the tested MAbs, which binds to a conserved region in the NA of pH1N1 virus, inhibited NA activity but did not result in escape variants, highlighting its suitability for development as a therapeutic agent.

Comparability of neuraminidase inhibition antibody titers measured by enzyme-linked lectin assay (ELLA) for the analysis of influenza vaccine immunogenicity.

Neuraminidase-inhibition (NI) antibody titers can be used to evaluate the immunogenicity of inactivated influenza vaccines and have provided evidence of serologic cross-reactivity between seasonal and pandemic H1N1 viruses. The traditional thiobarbituric acid assay is impractical for large serologic analyses, and therefore many laboratories use an enzyme-linked lectin assay (ELLA) to determine serum NI antibody titers. The comparability of ELLA NI antibody titers when measured in different laboratories was unknown. Here we report a study conducted through the Consortium for the Standardisation of Influenza SeroEpidemiology (CONSISE) to evaluate the variability of the ELLA. NI antibody titers of a set of 12 samples were measured against both N1 and N2 neuraminidase antigens in 3 independent assays by each of 23 laboratories. For a sample repeated in the same assay, ≥96% of N1 and N2 assays had less than a 4-fold difference in titer. Comparison of the titers measured in assays conducted on 3 different days in the same laboratory showed that a four-fold difference in titer was uncommon. Titers of the same sera measured in different laboratories spanned 3 to 6 two-fold dilutions (i.e., 8-64 fold difference in titer), with an average percent geometric coefficient of variation (%GCV) of 112 and 82% against N1 and N2 antigens, respectively. The difference in titer as indicated by fold range and %GCV was improved by normalizing the NI titers to a standard that was included in each assay. This study identified background signal and the amount of antigen in the assay as critical factors that influence titer, providing important information toward development of a consensus ELLA protocol.

Optimization of an enzyme-linked lectin assay suitable for rapid antigenic characterization of the neuraminidase of human influenza A(H3N2) viruses.

Antibodies to neuraminidase (NA), the second most abundant surface protein of the influenza virus, contribute to protection against influenza virus infection. Although traditional and miniaturized thiobarbituric acid (TBA) neuraminidase inhibition (NI) assays have been successfully used to characterize the antigenic properties of NA, these methods are cumbersome and not easily amendable to rapid screening. An additional difficulty of the NI assay is the interference by hemagglutinin (HA)-specific antibodies. To prevent interference of HA-specific antibodies, most NI assays are performed with recombinant viruses containing a mismatched HA. However, generation of these viruses is time consuming and unsuitable for large-scale surveillance. The feasibility of using the recently developed enzyme-linked lectin assay (ELLA) to evaluate the antigenic relatedness of NA of wild type A(H3N2) viruses was assessed. Rather than using recombinant viruses, wild type A(H3N2) viruses were used as antigen with ferret sera elicited against recombinant viruses with a mismatched HA. In this study, details of the critical steps that are needed to modify and optimize the NI ELLA in a format that is reproducible, highly sensitive, and useful for influenza virus surveillance to monitor antigenic drift of NA are provided.

Structural characterization of a protective epitope spanning A(H1N1)pdm09 influenza virus neuraminidase monomers.

A(H1N1)pdm09 influenza A viruses predominated in the 2013-2014 USA influenza season, and although most of these viruses remain sensitive to Food and Drug Administration-approved neuraminidase (NA) inhibitors, alternative therapies are needed. Here we show that monoclonal antibody CD6, selected for binding to the NA of the prototypic A(H1N1)pdm09 virus, A/California/07/2009, protects mice against lethal virus challenge. The crystal structure of NA in complex with CD6 Fab reveals a unique epitope, where the heavy-chain complementarity determining regions (HCDRs) 1 and 2 bind one NA monomer, the light-chain CDR2 binds the neighbouring monomer, whereas HCDR3 interacts with both monomers. This 30-amino-acid epitope spans the lateral face of an NA dimer and is conserved among circulating A(H1N1)pdm09 viruses. These results suggest that the large, lateral CD6 epitope may be an effective target of antibodies selected for development as therapeutic agents against circulating H1N1 influenza viruses.

An optimized enzyme-linked lectin assay to measure influenza A virus neuraminidase inhibition antibody titers in human sera.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. The traditional thiobarbituric acid (TBA) method to quantify NA inhibiting antibodies is cumbersome and not suitable for routine serology. An enzyme-linked lectin assay (ELLA) described by Lambre et al. (1990) is a practical alternative method for measuring NA inhibition (NI) titers. This report describes optimization of the ELLA for measuring NI titers in human sera against influenza A viruses, using H6N1 and H6N2 viruses as antigens. The optimized ELLA is subtype-specific and reproducible. While the titers measured by ELLA are somewhat greater than those measured by a miniaturized TBA method, seroconversion rates are the same, suggesting similarity in assay sensitivity under these optimized conditions. The ELLA described in this report provides a practical format for routine evaluation of human antibody responses to NA.

Stability of neuraminidase in inactivated influenza vaccines.

Influenza vaccines are effective in protecting against illness and death caused by this seasonal pathogen. Antibodies that block the function of either hemagglutinin (HA) or neuraminidase (NA) contribute to vaccine efficacy, however vaccine potency is based only on HA content. NA protein content in vaccines varies from season to season due to differences in the relative amounts of HA and NA in influenza A, H1N1 and H3N2, and influenza B viruses that are selected for each manufacturing campaign. This, as well as potential inherent differences in NA immunogenicity, may result in varying responses from year to year. Moreover, the antigenic stability of NA is likely to dictate whether similar antibody responses will be obtained to this antigen throughout the shelf-life of the vaccine. To address this factor, we subjected NAs of influenza A (subtypes N1 and N2) and B viruses to denaturing conditions to evaluate the stability of enzyme activity. Each NA type/subtype had unique sensitivity to denaturing conditions. The N2 enzyme activity was more thermostable than that of N1 or influenza B, while the NA activity of influenza B was most resistant to detergent. N1 enzyme activity was most resistant of the three NAs to freeze-thaw cycling. In these experiments, enzyme activity was indicative of the immunogenicity of NA, but was strain-dependent, with greater neuraminidase inhibiting (NI) antibody titers elicited following immunization with the 2009 H1N1 pandemic virus A/California/7/2009, than the previously circulating seasonal H1N1 strain, A/Brisbane/59/2007. Robust NI antibody titers against both N1 and N2 components were induced following vaccination of mice with a trivalent inactivated influenza vaccine. When stored under recommended conditions, the NA of both N1 and N2 subtypes remained immunogenic well after the vaccine expiry date.

Molecular basis for broad neuraminidase immunity: conserved epitopes in seasonal and pandemic H1N1 as well as H5N1 influenza viruses.

Influenza A viruses, including H1N1 and H5N1 subtypes, pose a serious threat to public health. Neuraminidase (NA)-related immunity contributes to protection against influenza virus infection. Antibodies to the N1 subtype provide protection against homologous and heterologous H1N1 as well as H5N1 virus challenge. Since neither the strain-specific nor conserved epitopes of N1 have been identified, we generated a panel of mouse monoclonal antibodies (MAbs) that exhibit different reactivity spectra with H1N1 and H5N1 viruses and used these MAbs to map N1 antigenic domains. We identified 12 amino acids essential for MAb binding to the NA of a recent seasonal H1N1 virus, A/Brisbane/59/2007. Of these, residues 248, 249, 250, 341, and 343 are recognized by strain-specific group A MAbs, while residues 273, 338, and 339 are within conserved epitope(s), which allows cross-reactive group B MAbs to bind the NAs of seasonal H1N1 and the 1918 and 2009 pandemic (09pdm) H1N1 as well as H5N1 viruses. A single dose of group B MAbs administered prophylactically fully protected mice against lethal challenge with seasonal and 09pdm H1N1 viruses and resulted in significant protection against the highly pathogenic wild-type H5N1 virus. Another three N1 residues (at positions 396, 397, and 456) are essential for binding of cross-reactive group E MAbs, which differ from group B MAbs in that they do not bind 09pdm H1N1 viruses. The identification of conserved N1 epitopes reveals the molecular basis for NA-mediated immunity between H1N1 and H5N1 viruses and demonstrates the potential for developing broadly protective NA-specific antibody treatments for influenza.

Protection against a lethal H5N1 influenza challenge by intranasal immunization with virus-like particles containing 2009 pandemic H1N1 neuraminidase in mice.

Highly pathogenic H5N1 influenza shares the same neuraminidase (NA) subtype with the 2009 pandemic (H1N1pdm09), and cross-reactive NA immunity might protect against or mitigate lethal H5N1 infection. In this study, mice were either infected with a sublethal dose of H1N1pdm09 or were vaccinated and boosted with virus-like particles (VLP) consisting of the NA and matrix proteins, standardized by NA activity and administered intranasally, and were then challenged with a lethal dose of HPAI H5N1 virus. Mice previously infected with H1N1pdm09 survived H5N1 challenge with no detectable virus or respiratory tract pathology on day 4. Mice immunized with H5N1 or H1N1pdm09 NA VLPs were also fully protected from death, with a 100-fold and 10-fold reduction in infectious virus, respectively, and reduced pathology in the lungs. Human influenza vaccines that elicit not only HA, but also NA immunity may provide enhanced protection against the emergence of seasonal and pandemic viruses.