A rapid and flexible microneutralization assay for serological assessment of influenza viruses

Background Serological responses from influenza vaccination or infection are typically measured by Hemagglutinin Inhibition (HAI) or Microneutralization (MN). Both methods are limited in feasibility, standardization, and generalizability. We describe a luciferase MN (LMN) assay that combines the advantages of the conventional MN assay with the ease of the HAI assay Methods Sera were obtained from the HIVE study. Plasmid transfection was used to generate recombinant influenza viruses expressing the hemagglutinin and neuraminidase of test strains, all other viral proteins from A/WSN33, and a NanoLuc reporter. Serum neutralization of luciferase-expressing targets was quantified as a reduction in Relative Light Unit (RLU) emission from infected cells. Neutralization titers were measured for cell- and egg-adapted versions of A/H3N2/Hong Kong/4801/2014 and A/H3N2/Singapore/INFIMH-16-0019/2016 and compared to HAI titers against egg-grown antigens. Results 333 sera were collected from 259 participants between May 2016 and July 2018. Sampled participants ranged from 7 to 68 years of age and >80% were vaccinated against influenza. HAI and LMN titers were correlated for A/H3N2/Hong Kong/4801/2014 (ρ=0.45, p≤0.01) and A/H3N2/Singapore/INFIMH-16-0019/2016 (ρ=0.75, p≤0.01). LMN titers were lower for cell strains compared to egg strains (A/H3N2/Hong Kong/4801/2014 mean log2 fold change = −2.66, p≤0.01 and A/H3N2/Singapore/INFIMH-16-0019/2016 mean log2 fold change= −3.15, p≤0.01). Conclusions The LMN assay was feasible using limited sample volumes and was able to differentiate small antigenic differences, specifically between egg-adapted and cell-derived strains. The correspondence of these results with the commonly-used HAI confirms the potential utility of this assay for high-throughput studies of correlates of protection and vaccine response. ### Competing Interest Statement The authors have declared no competing interest.