Multiplex evaluation of influenza neutralizing antibodies with potential applicability to in-field serological studies

Abstract

The increased number of outbreaks of H5 and H7 LPAI and HPAI viruses in poultry has major public and animal health implications. The continuous rapid evolution of these subtypes and the emergence of new variants influence the ability to undertake effective surveillance. Retroviral pseudotypes bearing influenza haemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins represent a flexible platform for sensitive, readily standardized influenza serological assays. We describe a multiplex assay for the study of neutralizing antibodies that are directed against both influenza H5 and H7 HA. This assay permits the measurement of neutralizing antibody responses against two antigenically distinct HAs in the same serum/plasma sample thus increasing the amount and quality of serological data that can be acquired from valuable sera. Sera obtained from chickens vaccinated with a monovalent H5N2 vaccine, chickens vaccinated with a bivalent H7N1/H5N9 vaccine, or turkeys naturally infected with an H7N3 virus were evaluated in this assay and the results correlated strongly with data obtained by HI assay. We show that pseudotypes are highly stable under basic cold-chain storage conditions and following multiple rounds of freeze-thaw. We propose that this robust assay may have practical utility for in-field serosurveillance and vaccine studies in resource-limited regions worldwide.

Figure 1

Radial phylogenetic tree showing the relationship between the full-length HA genes of vaccine and serological antigens (MATLAB Software). MV: monovalent vaccine strain, BV: bivalent vaccine strain. Viruses encircled in blue represent vaccine strains, in red represent pseudotype antigen strains, and in green represent HI antigen strains. The amino acid identity of these strains is shown in Figure 4.

Figure 2

The influence of freeze-thaw cycles on pseudotype virus titre. The stability of H5N1 lacZ pseudotypes was evaluated by subjecting aliquots of virus to 5 cycles of freeze-thaw. Results for pseudotypes bearing the rabies virus (CVS-11) and HIV-1 envelope proteins are shown for comparison. One biological replicate of each pseudotype virus was used to generate three technical replicates. Error bars represent standard deviation.

Figure 3

The influence of different storage temperatures on pseudotype virus titre. The stability of H5N1 lacZ pseudotypes was evaluated by storing aliquots of virus at different temperatures for up to 6 months. Pseudotype titres for the time course are given relative to the titre of virus stocks maintained at −80°C which is considered the optimal storage temperature for retroviral pseudotypes. One biological replicate pseudotype virus was used to generate three technical replicates. Error bars represent standard deviation.

Figure 4

Amino acid identity grid for pseudotype, HI, and vaccine antigen strains. MV: monovalent vaccine antigen, BV: bivalent vaccine antigen, pp: pseudotype antigen, and HI: haemagglutination inhibition assay antigen.

Figure 5

(a) Comparison of pp-NT with HI titers. Scatterplots showing the correlation of antibody logarithmic titers measured by pp-NT versus HI. For the pp-NT assay, HPAI H5 from A/Vietnam/1194/04 VN04 and A/Indonesia/5/05 (IND05) were tested. Pearson's correlation analysis was carried out and the coefficient of determination (r ²) for each strain reported on the graph. (b) Paired t-test performed by using GraphPad.

Figure 6

Comparison of pp-NT with HI titers. Scatterplots showing the correlation of antibody logarithmic titers measured by pp-NT (using HPAI H7 A/chicken/Italy/13474/99 pp) versus HI (HI antigen: H7N3 A/ty/Italy/9289/V02). The total number of sera was 51. Graph shows the linear regression fitted to the data using GraphPad.

Figure 7

(a) Correlation of monoplex versus multiplex IC₅₀ pseudotype neutralization titres for sera collected from chickens vaccinated with an inactivated bivalent vaccine produced with the AI strains H7N1 (A/ck/Italy/1067/99, LPAI) and H5N9 (A/ck/Italy/22A/98, LPAI). Antibody titres between monoplex and multiplex assays correlate when tested with both H5 and H7 pseudotypes. Neutralizing titres against H5 A/Vietnam/1194/2004 (grey dots) and H7 A/chicken/Italy/13474/1999 (empty circles) were determined in separate wells (single) or in the same well (multiplex). Correlation coefficient and P values were calculated using Pearson's correlation. Plot drawn with GraphPad. (b) IC₅₀ values for each sera tested by monoplex and multiplex pp-NT assays using H5 A/Vietnam/1194/04 (firefly luciferase gene) and H7 A/chicken/Itlay/13474/1999 (carrying firefly luciferase and Renilla luciferase gene) were calculated and plotted (the wide horizontal bar represents the means of IC₅₀ titres). Results were subsequently analyzed by performing Student's t-test on the paired dataset.