Universal type/subtype-specific antibodies for quantitative analyses of neuraminidase in trivalent influenza vaccines

Abstract

Both influenza viral hemagglutinin and neuraminidase can induce protective immune responses in humans. Although the viral hemagglutinin antigens have been quantified in influenza vaccines, the amounts of neuraminidase remain undetermined. Using comprehensive bioinformatics analyses of all neuraminidase sequences, we identified highly conserved and subtype-specific peptide epitopes within each of N1, N2 and type B neuraminidase groups. Mono-specific antibodies generated against these peptides bound to their respective subtype/type only while demonstrating remarkable specificity against the viral neuraminidase sequences without any cross-reactivity with allantoic and cellular proteins. Moreover, the subtype/type-specific antibodies were found not to interfere with one another when a mixture of vaccine samples was analysed. Importantly, immunoassay based on these antibodies can quantitatively determine neuraminidase in commercial trivalent vaccine samples. Analyses of vaccines from eight manufacturers using the same vaccine seeds revealed significant differences in neuraminidase levels. Specifically, while the ratio between neuraminidase and hemagglutinin in some products are found to be close 1/5, other products have a ratio of approximately 1/100, a level which is far below the theoretical ratio between neuraminidase and hemagglutinin in a virus. The antibody-based assays reported here could be of great value for better quality control of both monovalent and trivalent vaccines.

Broadly Inhibiting Antineuraminidase Monoclonal Antibodies Induced by Trivalent Influenza Vaccine and H7N9 Infection in Humans

“…The authors found that 14 to 35% of influenza A virus-specific MAbs induced by natural infection bound NA, whereas only 0 to 2% did so after vaccination. They confirmed that the NA antigen is poorly represented in many subunit vaccines and that the quality and quantity of NA in different vaccines varies (30,31).…”

A neuraminidase potency assay for quantitative assessment of neuraminidase in influenza vaccines

“…The NA from H3N2 viruses has been reported to be more stable than N1 and thus the relatively constant signal observed for N2 NA could be due to the protein’s better stability. 7 In any case, this analysis appears to support the notion that NA content in commercial vaccines is highly variable, 30 although such a conclusion cannot be drawn without quantification using a calibration standard. Future studies using VXI-sNA to assess fresh lots of vaccine are needed to address the magnitude of lot-to-lot and year-to-year NA content variation.…”

“…28 Two alternative methods utilizing immunochemistry have been described recently. 29,30 However, one of these methods was designed solely for the quantification of NA from H1N1 strains 29 and another, while resistant to antigenic change due to the probing of a conserved, linear epitope, is not sensitive to changes in protein stability due to the requirement of degrading the NA protein before quantification. 30…”

Design of peptide epitope from the neuraminidase protein of influenza A and influenza B towards short peptide vaccine development

“…Xu et al [ 32 ] identified highly conserved and subtype-specific peptide epitopes within each of N1, N2 and type B neuraminidase proteins using Geneious 7.0.6 software program. These peptides were shown to generate mono-specific antibodies against their respective subtype/type in experimental rabbits.…”

“…Immune response to peptides is influenced by the way they are presented to the immune system, and therefore a multifunctional delivery systems coupling the antigen with adjuvant is needed [ 34 ]. This approach has been addressed subsequently by many researchers and has shown similar effects [ 32 ]. Gold nanoparticles mediated OVA peptide delivery [ 35 ], peptides adsorbed on poly (D, L-lactide-co-glycolide) (PLGA) particles as a controlled-release vaccine delivery system [ 36 ].…”