Development and use of Lentiviral Vectors Pseudotyped with Influenza B Haemagglutinins: application to vaccine immunogenicity, mAb potency and sero-surveillance studies

Abstract

Influenza B viruses cause respiratory disease epidemics in human populations and are included in seasonal influenza vaccines. Serological methods are employed to evaluate vaccine immunogenicity prior to licensure. However, the haemagglutination inhibition assay, which represents the gold standard for assessing the immunogenicity of influenza vaccines, has been shown to be relatively insensitive for the detection of antibodies against influenza B viruses. Furthermore, this assay, and the serial radial haemolysis assay are not able to detect stalk-directed cross-reactive antibodies. For these reasons there is a need to develop new assays that can overcome these limitations. The use of replication-defective viruses, such as lentiviral vectors pseudotyped with influenza A haemagglutinins, in microneutralization assays is a safe and sensitive alternative to study antibody responses elicited by natural infection or vaccination. We have produced Influenza B haemagglutininpseudotypes using plasmid-directed transfection. To activate influenza B haemagglutinin, we have explored the use of proteases by adding relevant encoding plasmids to the transfection mixture. When tested for their ability to transduce target cells, the newly produced influenza B pseudotypes exhibit tropism for different cell lines. Subsequently the pseudotypes were evaluated as surrogate antigens in microneutralization assays using reference sera, monoclonal antibodies, human sera collected during a vaccine immunogenicity study and surveillance sera from seals. The influenza B pseudotype virus neutralization assay was found to effectively detect neutralizing and cross-reactive responses despite lack of significant correlation with the haemagglutinin inhibition assay.

Correlation of Influenza B Haemagglutination Inhibiton, Single-Radial Haemolysis and Pseudotype-Based Microneutralisation Assays for Immunogenicity Testing of Seasonal Vaccines

“…Anti-B/Brisbane/60/2008 HA serum (11/136) was obtained from the NIBSC and employed against all strains of influenza B as a serum positive control. This antiserum had previously been tested in our laboratory, showing that it was capable of neutralising all available strains of influenza B to varying degrees, with the highest potency against the matched B/Brisbane/60/2008 strain [ 54 ].…”

Cross-Reactive and Lineage-Specific Single Domain Antibodies against Influenza B Hemagglutinin

“…The pseudotype-based microneutralisation assay (pMN) was carried out in Nunc TM F96 microplates (Thermo Fisher Scientific) [24,25]; 1:2 serial dilutions were performed with the relevant sdAbs, across the 96-well plate in a total of 50 µL DMEM (10% foetal bovine serum, 1% penicillin/streptomycin). HIV-1-derived lentiviral pseudotypes, bearing the influenza B HA of choice, was then added to yield a relative luminescence unit (RLU) input of 1.5 × 10 6 per well, in a total volume of 50 µL [26]. The plates were then incubated in a humidified incubator at 37 °C, 5% CO 2 , for one hour, after which 1 × 10 4 HEK293T/17 cells were added, per well, in a total volume of 50 µL.…”

Influenza D pseudotyped lentiviruses: production, neutralisation assay and serological surveillance

“…Aims: The VPU has been successful in producing lentiviral pseudotypes and microneutralisation assays for influenza A and B viruses [6,7]. This platform can be used to produce influenza D pseudotyped lentiviruses (ID-PVs) to establish a microneutralisation (MN) assay for serological surveillance.…”