Development of a T Cell-Based COVID-19 Vaccine Using a Live Attenuated Influenza Vaccine Viral Vector

Abstract

The COVID-19 pandemic emerged in 2020 and has caused an unprecedented burden to all countries in the world. SARS-CoV-2 continues to circulate and antigenically evolve, enabling multiple reinfections. To address the issue of the virus antigenic variability, T cell-based vaccines are being developed, which are directed to more conserved viral epitopes. We used live attenuated influenza vaccine (LAIV) virus vector to generate recombinant influenza viruses expressing various T-cell epitopes of SARS-CoV-2 from either neuraminidase (NA) or non-structural (NS1) genes, via the P2A self-cleavage site. Intranasal immunization of human leukocyte antigen-A*0201 (HLA-A2.1) transgenic mice with these recombinant viruses did not result in significant SARS-CoV-2-specific T-cell responses, due to the immunodominance of NP366 influenza T-cell epitope. However, side-by-side stimulation of peripheral blood mononuclear cells (PBMCs) of COVID-19 convalescents with recombinant viruses and LAIV vector demonstrated activation of memory T cells in samples stimulated with LAIV/SARS-CoV-2, but not LAIV alone. Hamsters immunized with a selected LAIV/SARS-CoV-2 prototype were protected against challenge with influenza virus and a high dose of SARS-CoV-2 of Wuhan and Delta lineages, which was confirmed by reduced weight loss, milder clinical symptoms and less pronounced histopathological signs of SARS-CoV-2 infection in the lungs, compared to LAIV- and mock-immunized animals. Overall, LAIV is a promising platform for the development of a bivalent vaccine against influenza and SARS-CoV-2.

Expression of the SARS-CoV-2 receptor-binding domain by live attenuated influenza vaccine virus as a strategy for designing a bivalent vaccine against COVID-19 and influenza

“…In the modified versions of the A/Leningrad/134/17/57 NS gene, the ORF of the NS1 protein was truncated to 126 amino acid residues, as described previously [ 21 , 22 ]. The SARS-CoV-2 genetic material was inserted after the 126 th codon of NS1 in several modifications: (1) after the P2A-encoding sequence, (2) connected to the NS1 through the GGGGS linker, and (3) after the Stop-Start pentanucleotide of influenza B virus TAATG [ 23 ].…”

“…The rescue of recombinant viruses was performed with the same procedure as described previously [ 21 ]. In brief, purified plasmids encoding all influenza genes (PB2, PB1, PA, NP, M of A/Leningrad/134/17/57, the NA of A/Anhui/1/2013, modified or non-modified NS from A/Leningrad/134/17/57, and modified or non-modified HA from A/Anhui/1/2013) were mixed (2 μg of each plasmid) for transfection of Vero cells.…”

“…The IFN-γ response of isolated splenocytes and lung cells of Syrian hamsters on the 5th day after challenge was measured using an IFN-γ ELISPOT Plus kit (Mabtech, Sweden) according to the manufacturer's protocol and as described in [ 21 ]. Briefly, a pre-coated with monoclonal antibody ELISPOT plate was washed 4 times with sterile PBS (200 µl/well) and then incubated with the CR-10 medium (RPMI supplemented with 10% FBS, 5 mM HEPES, 1× antibiotic-antimycotic, and 50 μM β-mercaptoethanol) for 30 minutes at room temperature.…”

“…Lung tissues of Syrian hamsters immunized with tested vaccines or mock-immunized (PBS) and challenged with SARS-CoV-2 were subjected to histopathological evaluation using previously described methodology [ 21 ]. Briefly, the lungs ( n =4 per group) were fixed in 10% neutral buffered formalin for at least 48 hours.…”

“…Morphometric measurements included semiquantitative assessment of airway damage (comprised of % airway affected, airway severity and bronchiolar epithelial hyperplasia), lung/alveolar damage (comprising of % alveoli affected, alveolar severity and type II pneumocyte hyperplasia), and vascular damage (comprising of % vessels affected, vascular/perivascular lesions and necrotizing vasculitis/thrombi) according to [ 28 ]. The detailed information of the scoring criteria was published earlier [ 21 ].…”