Construction And Immunogenicity Evaluation Of A SARS-CoV-2 Recombinant Vesicular Stomatitis Virus-Vectored Vaccine

Background:The pandemic of COVID-19 poses a serious threat to public health safety and the global economy.As of March 12,2022,COVID-19 has caused more than450 million confirmed cases and more than 6.02 million deaths worldwide.Besides,the infection of SARS-CoV-2 in animals is also rising.According to OIE,35 countries and regions reported 645 cases of SARS-CoV-2 infection in 19 species of animals including minks,cats,hamsters,white tailed deers,lions,gorilla,etc by January 31,2022.Among them,SARS-CoV-2 mink variants appear in minks.Since the transmission of SARS-CoV-2 mink variants from minks to humans was confirmed,millions of minks in the Netherlands,Denmark and other countries were culled.At present,closer attention has being paid to SARS-CoV-2 infection in animals.Correspondingly,veterinary vaccine research and vaccination plans are also advancing in an orderly manner.In this situation,the development of veterinary COVID-19 vaccine is of great significance to the protection of SARS-CoV-2 susceptible companion animals,fur animals,livestock and rare wild animals as well as the reduction of the risk of SARS-CoV-2 transmission from animals to humans.Object:As a vaccine vector,vesicular stomatitis virus has the advantages of high growth titer,preeminent immunogenicity,and is free from anti vector immunity.This study intends to construct a replication-competent VSV-vectored recombinant COVID-19 vaccine.Immunogenicity evaluations were conducted in mice and golden hamsters.We explored the differences in immune response and protective efficacy when recombinant vaccine was delivered by intramuscular injection or intranasal inoculation.We aim to provide a safe and effective veterinary COVID-19 vaccine candidate.Methods:1 Construction and identification of a SARS-CoV-2 recombinant VSV-vectored vaccineThe open reading frames of the spike gene of SARS-CoV-2 mink variant cluster 5were cloned into the full-length genome of VSV,replacing the VSV glycoprotein gene.Two SARS-CoV-2 recombinant VSV infectious clone full-length plasmids were named p3.1-VSVΔG-S and p3.1-VSVΔG-S-e GFP,respectively.The full-length plasmid and helper plasmids including p3.1-VSV-N,p3.1-VSV-P,p3.1-VSV-L and p3.1-VSV-G were co transfected into BSR-T7 cells to rescue the recombinant virus r VSVΔG-S and e GFP tag-carrying recombinant pseudovirus r VSVΔG-S-e GFP.Subsequently,the recombinant viruses were serial passaged and identified by RT-PCR,indirect immunofluorescence,western blot,transmission electron microscopy and growth kinetics.2.Immunogenicity evaluation of SARS-CoV-2 recombinant VSV-vectored vaccine in miceOn day 0 and day 21,BALB/c mice were inoculated with r VSV-ΔG-S through intramuscular injection or intranasal injection at a dose of 10⁶TCID₅₀/animal.Virus specific antibody,antibody subtype and neutralizing antibody were detected on 14,28and 42 dpv.Alveolar lavage fluid was collected and SARS-CoV-2 specific SIg A was detected on 35 dpv.IL-4 and IFN-γin splenocytes were detected by ELISpot on 28 dpv.CD4⁺and CD8⁺T cells were detected by flow cytometry on 28 dpv.3.Immunogenicity evaluation of SARS-CoV-2 recombinant VSV vectored-vaccine and SARS-CoV-2 challenge in golden hamstersThe immune programme in golden hamsters was similar to mice.Blood samples were collected on 14 and 28 dpv for n Abs and virus specific Ig G detection.On 35 dpv,animals were challenged with 10⁵TCID₅₀SARS-CoV-2.Clinical symptoms and body weight of these animals were recorded continuously within one week after the challenge.On 3 dpi,turbinate and lung tissues were taken for TCID₅₀titration and RNA copys detection.Meanwhile,pathological section of lung tissue was analyzed by HE staining and immunohistochemistry.Results:1.The identification results of recombinant virus-vectored vaccine showed that a specific band was observed at 3816 bp after amplification with SARS-CoV-2-S specific primers,which was correct according to sequencing results.Indirect immunofluorescence and WB identification proved that the recombinant virus was recognized by SARS-CoV-2-S specific antibody,the target protein band was about 180k Da.A typical coronal shape of the spike of the recombinant virus was exhibited through transmission electron microscopy,indicating that the S protein was successfully assembled to the surface of virus particles.The growth kinetics suggested that r VSVΔG-S and r VSVΔG-S-e GFP exhibited similar growth kinetic characteristics.They reached the highest growth titer of 10^6.8TCID₅₀/m L at 72 h.The above results indicated that recombinant viruses r VSVΔG-S and r VSVΔG-S-e GFP were rescued successfully.2.In mice immunization experiment,the GMT of n Abs in i.m.group reached 143on 56 dpv while the GMT of virus specific Ig G titer reached 25267 on 42 dpv,Ig G2a/Ig G1<1.IL-4 and IFN-γincreased significantly in splenic lymphocytes.In the i.n.group,the GMT of n Abs was 13 on 56 dpv while the GMT of virus specific Ig G titer was 3158 on 42 dpv,Ig G2a/Ig G1>1.Alveolar SIg A titer was between 1:64 and 1:512.IFN-γwas induced in splenocytes.These results showed that r VSV-ΔG-S induced higher n Abs and virus specific Ig G levels in the i.m.group while certain SIg A and Th1-basied cellular immune responses were detected in the i.n.group.3.In golden hamsters immunization and challenge experiment,n Abs and virus specific antibody were relatively low in the i.m.group,virus load and RNA copys were reduced post SARS-CoV-2 challenge while infectious SARS-CoV-2 was not effectively eliminated.Moderate injury was observed in the lung tissue,accompanied by antigen residue.In the i.n.group,the GMT of neutralizing antibody titer was 1878 whilst the GMT of virus specific Ig G response was 96647 on 28 dpv.After the challenge of SARS-CoV-2,no infectious virus was detected in turbinate and lung,viral RNA copies decreased significantly.The above results indicated that the i.n.group was better than the i.m.group in terms of humoral immune response and protective efficacy.Conclusion:1.The replication-competent SARS-CoV-2 recombinant VSV vectored-vaccine r VSVΔG-S as well as recombinant pseudovirus r VSVΔG-S-e GFP were constructed successfully.2.In mice,r VSVΔG-S induced a strong humoral immune response by i.m.vaccination.NAbs and virus specific antibody induced by i.m.vaccination were significantly higher than those in the i.n.vaccination group.3.In golden hamsters,r VSVΔG-S induced a strong humoral immune response through i.n.vaccination,virus titer in turbinate and lung decreased significantly post SARS-CoV-2 challenge,lung tissue was effectively protected.The humoral immunity and the protective efficacy induced by i.n.vaccination were better than that of i.m.vaccination.4.The humoral immunity induced by i.n.vaccination in mice was limited,but in golden hamsters,i.n.vaccination accomplished strong humoral immune response and prominent protective efficacy.The reason for this difference is that golden hamsters are more susceptible to SARS-CoV-2 than mice.Therefore,the recombinant virus-vectored vaccine can effectively replicate in golden hamsters and trigger an immune response after i.n.vaccination whilst the replication efficiency of recombinant virus is limited in mice.Therefore,the immunogenicity and protective efficacy of recombinant virus-vectored vaccine delivered by i.n.in golden hamsters can better reflect the real situation in other SARS-CoV-2 susceptible animals.This study provides a safe,effective and convenient COVID-19 vaccine candidate for SARS-CoV-2 susceptible companion animals,fur animals,livestock and rare wild animals.