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

Abstract

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 to recent strains. We developed 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, a Michigan household cohort. Reverse genetics was used to generate recombinant influenza viruses expressing the hemagglutinin and neuraminidase of test strains, all other viral proteins from an A/WSN/1933 backbone, and a NanoLuc reporter. Serum neutralization of luciferase-expressing targets was quantified as a reduction in light emission from infected cells. Neutralization titers were measured for cell- and egg-adapted versions of A/Hong Kong/4801/2014 and A/Singapore/INFIMH-16-0019/2016 and compared to HAI titers against egg-grown antigens.

Results: Three hundred thirty-three sera were collected from 259 participants between May 2016 and July 2018. Sampled participants were 7-68 years of age, and >80% were vaccinated against influenza. HAI and LMN titers were correlated for A/Hong Kong/4801/2014 (ρ = 0.52, p ≤ 0.01) and A/Singapore/INFIMH-16-0019/2016 (ρ = 0.79, p ≤ 0.01). LMN titers were lower for cell strains compared to egg strains (A/Hong Kong/4801/2014 mean log₂ fold change = -2.66, p ≤ 0.01 and A/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 able to differentiate small antigenic differences between egg-adapted and cell-derived strains. The correspondence of these results with the commonly used HAI confirms the utility of this assay for high-throughput studies of correlates of protection and vaccine response.

Keywords: hemagglutination; influenza virus; neutralization; serology.

FIGURE 1

Design of the luciferase microneutralization assay (LMN). (A) Plate layout: Sera wells (rows A‐H, columns 1–10) are considered positive for 75% neutralization of virus if the relative light unit (RLU) output is above the virus control RLU average minus the cell control RLU average divided by 4. Numbers in back titer wells indicate fold dilution of the input virus (100 infectious units). A back titer well is considered positive if the RLU output was greater than twice the cell only control RLU average. A plate was considered “passing” if the first negative well fell between the fifth and eighth dilution (1:32 to 1:256). Positive sera and back titer wells are colored. (B) Example of back titer with RLU (y‐axis) for each virus dilution (x‐axis). A visible leveling off in back titer between the fifth and eighth dilution was required for a plate to pass quality control. The red line denotes the cutoff of the cell only control average multiplied by two. (C) Example of positive (human) neutralization control (circles) and negative (sheep) neutralization control (squares). The neutralization cutoff, based on 75% reduction in RLU, is denoted by a red line. (D) Example of titrations for six sera, each denoted by a different shape. The 75% cutoff for neutralization is denoted by a red line.

FIGURE 2

Correlation between HAI and LMN NT₇₅ for (A) A/Hong Kong/4801/2014 and (B) A/Singapore/INFIMH‐16‐0019/2016; n = 333 *Spearman's Rank Correlation Coefficients with a p value ≤0.01.

FIGURE 3

Difference in paired profile of LMN NT₇₅ across egg‐ and cell‐ adapted influenza viruses. (A,B) Mean log₂ fold change in LMN NT₇₅ from cell‐ to egg‐adapted viral targets is denoted by a blue line. Both the Hong Kong and Singapore strains showed a statistically significant different LMN NT₇₅ for cell‐ and egg‐adapted virus strains (Hong Kong t‐value = −34.36, dF = 258, p value ≤0.01; Singapore t value = −36.26, dF = 258, p value ≤0.01). (C) and (D) Mean log₂ fold difference in LMN NT₇₅ across cell‐ and egg‐adapted virus strains (Hong Kong mean log₂ fold change = −2.66 [−2.82, −2.51]; Singapore mean log₂ fold change = −3.15 [−3.33, −2.98]). All individuals had higher LMN NT₇₅ for the egg‐adapted strains except two individuals with a onefold difference for the A/Hong Kong/4801/2014 and one individual with a threefold difference for A/Singapore/INFIMH‐16‐0019/2016.