A Single-Cycle Influenza A Virus-Based SARS-CoV-2 Vaccine Elicits Potent Immune Responses in a Mouse Model.

Ratchanont Viriyakitkosol,Kanjana Srisutthisamphan,Surapong Koonpaew,Sukontip Poonsuk,Challika Kaewborisuth,Asawin Wanitchang,Juggragarn Jengarn,Anan Jongkaewwattana,Theeradej Thaweerattanasinp,Jarin Kramyu,Janya Saenboonrueng

doi:10.3390/vaccines9080850

Abstract

The use of virus-vectored platforms has increasingly gained attention in vaccine development as a means for delivering antigenic genes of interest into target hosts. Here, we describe a single-cycle influenza virus-based SARS-CoV-2 vaccine designated as scPR8-RBD-M2. The vaccine utilizes the chimeric gene encoding 2A peptide-based bicistronic protein cassette of the SARS-CoV-2 receptor-binding domain (RBD) and influenza matrix 2 (M2) protein. The C-terminus of the RBD was designed to link with the cytoplasmic domain of the influenza virus hemagglutinin (HA) to anchor the RBD on the surface of producing cells and virus envelope. The chimeric RBD-M2 gene was incorporated in place of the HA open-reading frame (ORF) between the 3′ and 5′ UTR of HA gene for the virus rescue in MDCK cells stably expressing HA. The virus was also constructed with the disrupted M2 ORF in segment seven to ensure that M2 from the RBD-M2 was utilized. The chimeric gene was intact and strongly expressed in infected cells upon several passages, suggesting that the antigen was stably maintained in the vaccine candidate. Mice inoculated with scPR8-RBD-M2 via two alternative prime-boost regimens (intranasal-intranasal or intranasal-intramuscular routes) elicited robust mucosal and systemic humoral immune responses and cell-mediated immunity. Notably, we demonstrated that immunized mouse sera exhibited neutralizing activity against pseudotyped viruses bearing SARS-CoV-2 spikes from various variants, albeit with varying potency. Our study warrants further development of a replication-deficient influenza virus as a promising SARS-CoV-2 vaccine candidate.

Highlights

Severe acute respiratory syndrome coronavirus (SARS-CoV-2), a newly emerged human coronavirus, has been identified as a causative agent of COVID-19 and has led to a global pandemic
In order to generate the recombinant influenza gene that encodes the membraneanchored receptor-binding domain (RBD) of SARS-CoV-2 spike presented on the influenza virus envelope, the RBD region fused with the transmembrane domain and cytoplasmic tail of PR8 HA (RBD-HAcyt) was constructed to replace the HA open-reading frame (ORF) of PR8 HA segment
We showed that RBD-matrix 2 (M2) protein was expressed as shown by immunofluorescence assay (IFA) against α-RBD and α-Spike antibodies (Figure 1B) and Western blot analysis (Figure 1C) and maintained in the viral genome after multiple passages (Figure 1D)

Summary

Introduction

Severe acute respiratory syndrome coronavirus (SARS-CoV-2), a newly emerged human coronavirus, has been identified as a causative agent of COVID-19 and has led to a global pandemic. Influenza A virus (IAV) vectors harboring foreign antigens have proven successful in inducing humoral immune responses against pathogen-derived proteins, including West Nile virus, Bacillus anthracis, HIV, and botulinum neurotoxin in several preclinical animal models [4,5,6,7]. These studies demonstrated that the insertion of foreign genes into IAV gene segments encoding viral surface proteins, such as hemagglutinin (HA) or neuraminidase (NA), could provide protective immunity against relevant antigens. IAV-based vector vaccines developed against various pathogens could be administered via several routes, including intramuscularly and intranasally to broaden their utilization in inducing protective immunity

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