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

Abstract

We developed an influenza hemagglutinin (HA) pseudotype library encompassing Influenza A subtypes HA1-18 and Influenza B subtypes (both lineages) to be employed in influenza pseudotype microneutralization (pMN) assays. The pMN is highly sensitive and specific for detecting virus-specific neutralizing antibodies against influenza viruses and can be used to assess antibody functionality in vitro. Here we show the production of these viral HA pseudotypes and their employment as substitutes for wildtype viruses in influenza neutralization assays. We demonstrate their utility in detecting serum responses to vaccination with the ability to evaluate cross-subtype neutralizing responses elicited by specific vaccinating antigens. Our findings may inform further preclinical studies involving immunization dosing regimens in mice and may help in the creation and selection of better antigens for vaccine design. These HA pseudotypes can be harnessed to meet strategic objectives that contribute to the strengthening of global influenza surveillance, expansion of seasonal influenza prevention and control policies, and strengthening pandemic preparedness and response.

Influenza A (N1-N9) and Influenza B (B/Vic and B/Yam) Neuraminidase Pseudotypes as Tools for Pandemic Preparedness and Improved Influenza Vaccine design

“… (A) Schematic representation of the production of influenza NA pseudotypes (PV) by quadruple plasmid transfection to produce pseudotypes expressing NA only or HA and NA on the PV surface. Protease is only required when HA is expressed, as per previous optimization (41). Images created using Biorender.…”

“…To assess the ability of our NA to release HA, we produced PV with IAV H5 and IAV NA (N1-N9) and IBV NA (B/Vic and B/Yam) in place of exogenous NA and titrated the PV via a luciferase reporter assay (41). N10 and N11 have previously been shown to not be required for HA release (41,55) and were therefore not tested herein. All the NA PV tested were capable of releasing H5 as evidenced by the titers observed ( Figure 2D ).…”

“…Taking this into account, we tested representative N1 PV (A/England/195/2009) inhibition sensitivity to a variety of monoclonal antibodies (mAb) in pELLA inhibition. As the PV created herein express H11, we tested the ability of HA stem-directed mAb CR9114 (44), which was previously shown to neutralize IAV and IBV HA PV (41), to inhibit NA enzymatic activity in a traditional ELLA (60). CR9114 inhibited the N1 PV tested ( Figure 3C ) with half maximal inhibitory concentration IC 50 of 31.52 ng/mL ( Figure 3E ) as determined by non-linear regression.…”

“…The function of NA and interactions in vivo with HA are increasingly being explored especially in relation to the design and efficacy of novel vaccine or drug candidates. The NA PV library we have constructed can be utilized to this end in a similar fashion to the HA PV library we previously described (41). Our comprehensive library has representative strains of IAV N1-11 and IBV B/Vic NA and B/Yam NA ( Supplementary Table 1) and the methods used to produce these PV can easily be employed to include additional NA strains as required.…”

“…For production of H11-NA(X) PV, human embryonic kidney 293T/17 (HEK293T/17, ATCC: CRL-11268 a ) were maintained in complete medium, (Dulbecco’s Modified Essential Medium (DMEM) (PANBiotech P04-04510) with high glucose and GlutaMAX supplemented with 10% (v/v) heat-inactivated Fetal Bovine Serum (PANBiotech P30-8500), and 1% (v/v) Penicillin-Streptomycin (PenStrep) (Sigma P4333)) at 37°C and 5% CO 2 . Transfection was done as previously described (41). On the day prior to transfection, 4×10 5 HEK293T/17 cells in complete DMEM were seeded per well of a 6-well plate.…”