Development of Lentiviral Vectors Pseudotyped With Influenza B Hemagglutinins: Application in Vaccine Immunogenicity, mAb Potency, and Sero-Surveillance Studies.

Francesca Ferrara,Kelly A S Da Costa,Sasan Fereidouni,Craig Thompson,Simon Scott,Rebecca Kinsley,Nigel Temperton,Joanne Marie M Del Rosario,George Carnell,Sarah Gilbert,Paul Kellam

doi:10.3389/fimmu.2021.661379

Abstract

Influenza B viruses (IBV) cause respiratory disease epidemics in humans and are therefore components of seasonal influenza vaccines. Serological methods are employed to evaluate vaccine immunogenicity prior to licensure. However, classical methods to assess influenza vaccine immunogenicity such as the hemagglutination inhibition assay (HI) and the serial radial hemolysis assay (SRH), have been proven to have many limitations. As such, there is a need to develop innovative methods that can improve on these traditional assays and provide advantages such as ease of production and access, safety, reproducibility, and specificity. It has been previously demonstrated that the use of replication-defective viruses, such as lentiviral vectors pseudotyped with influenza A hemagglutinins in microneutralization assays (pMN) is a safe and sensitive alternative to study antibody responses elicited by natural influenza infection or vaccination. Consequently, we have produced Influenza B hemagglutinin-pseudotypes (IBV PV) using plasmid-directed transfection. To activate influenza B hemagglutinin, we have explored the use of proteases in increasing PV titers via their co-transfection during pseudotype virus production. When tested for their ability to transduce target cells, the influenza B pseudotypes produced exhibit tropism for different cell lines. The pseudotypes were evaluated as alternatives to live virus in microneutralization assays using reference sera standards, mouse and human sera collected during vaccine immunogenicity studies, surveillance sera from seals, and monoclonal antibodies (mAbs) against IBV. The influenza B pseudotype pMN was found to effectively detect neutralizing and cross-reactive responses in all assays and shows promise as an effective and versatile tool in influenza research.

Highlights

Seasonal influenza causes severe illness and mortality in highrisk human populations such as children, the elderly, and individuals with underlying morbidity
For Influenza B viruses (IBV) HA in pI.18, the highest titers of around 109 relative luminescence units (RLU)/mL were achieved using HAT as protease, except for B/Brisbane/60/08 which achieved its highest titer of ~108 RLU/mL with TMPRSS4 (Figure 2A)
Knowledge of the life cycle of the influenza B virus is essential for production of high titer IBV pseudotypes

Summary

Introduction

Seasonal influenza causes severe illness and mortality in highrisk human populations such as children, the elderly, and individuals with underlying morbidity. If infection was not limited by other factors controlling viral replication, the HA would allow broad viral tropism, since sialic acids are present on almost all cell types regardless of species. These limiting factors are mainly represented by restriction of the influenza polymerase complex activity and activation of influenza HA [10, 13,14,15,16]. This permits envelope-endosome membrane fusion and the release of the viral core into the cell cytosol [14] These factors do not completely limit the ability of influenza viruses to infect different animal species and as is the case with IAV, IBVs cause human infections and have zoonotic and pandemic potential due to their ability to infect non-human mammalian species. IBV has a lower infection and mortality rate than influenza A and this has resulted in influenza B being relatively understudied [15]

Methods

Results

Conclusion