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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 ...

Food and Drug Administration

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.

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

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.

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

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.

Stability of neuraminidase in inactivated influenza vaccines.

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.

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

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.

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

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.

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

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.

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

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.

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

Article	Year
Protection against a lethal H5N1 influenza challenge by intranasal immunization with virus-like particles containing 2009 pandemic H1N1 neuraminidase in mice. Virology\| PubMed ID: 22727831	2012
Molecular basis for broad neuraminidase immunity: conserved epitopes in seasonal and pandemic H1N1 as well as H5N1 influenza viruses. Journal of virology\| PubMed ID: 23785204	2013
Stability of neuraminidase in inactivated influenza vaccines. Vaccine\| PubMed ID: 24613525	2014
An optimized enzyme-linked lectin assay to measure influenza A virus neuraminidase inhibition antibody titers in human sera. Journal of virological methods\| PubMed ID: 25233882	2014
Structural characterization of a protective epitope spanning A(H1N1)pdm09 influenza virus neuraminidase monomers. Nature communications\| PubMed ID: 25668439	2015
Optimization of an enzyme-linked lectin assay suitable for rapid antigenic characterization of the neuraminidase of human influenza A(H3N2) viruses. Journal of virological methods\| PubMed ID: 25712563	2015
Comparative Efficacy of Monoclonal Antibodies That Bind to Different Epitopes of the 2009 Pandemic H1N1 Influenza Virus Neuraminidase. Journal of virology\| PubMed ID: 26468531	2016
Comparability of neuraminidase inhibition antibody titers measured by enzyme-linked lectin assay (ELLA) for the analysis of influenza vaccine immunogenicity. Vaccine\| PubMed ID: 26707221	2016
Seasonal Influenza Vaccination in Patients With Chronic Lymphocytic Leukemia Treated With Ibrutinib. JAMA oncology\| PubMed ID: 27533065	2016

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