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

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.

Exploiting Pan Influenza A and Pan Influenza B Pseudotype Libraries for Efficient Vaccine Antigen Selection

“…Influenza HA pseudotypes were produced as described previously [13,60]. Briefly, 4 × 10 5 HEK 293T/17 cells in complete DMEM were seeded per well of a 6-well plate and incubated at 37 • C, 5% CO 2 overnight.…”

“…It is desirable to have antibodies that will elicit a broad, cross-subtype specific response in order to address a pandemic threat. The influenza pseudotype microneutralization (pMN) assay is highly sensitive and specific for detecting neutralizing antibodies against influenza viruses regardless of whether they are HA-head specific or are targeted against the HA stem, making it an excellent test of antibody functionality in vitro [13,47,57,84]. Several broadly reactive monoclonal antibodies have been developed for use in immunotherapy against influenza.…”

“…From its wild bird reservoir, IAV is able to transmit from domestic poultry [9], which is the gateway to infection of mammals, most notably, swine and humans [10]. IBV's natural reservoir is humans, however there have been reports of infection in seals [11][12][13], alluding to its potential to cause disease in other species.…”

“…To improve current vaccination strategies and to aid the development of a universal influenza vaccine, additional reliable tools are necessary to identify and progress promising candidates targeting both the hemagglutinin head and stem domains [49,[56][57][58]. The advent of pseudotyped lentiviral vectors have enabled the study of HA interactions with antibodies, drugs, and host cell receptors with ease [13,[59][60][61]. These pseudotypes (PV) undergo abortive replication and do not give rise to replication-competent progeny [62,63].…”

Antibody Responsiveness to Influenza: What Drives It?

“…This enables the detection of antibodies targeting these epitopes that are normally inaccessible on wild-type viruses. The challenge remains in validating and standardizing these assays to current serological standards as they have shown variable performance in several comparative studies [218,230].…”