| The corona virus disease 2019(COVID-19)pandemic caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection presents a significant threat to human life and property.Although vaccination efforts have been effective in curbing the spread of SARS-CoV-2,the emergence of mutant strains has reduced the efficacy of existing vaccines and increased the likelihood of breakthrough infections.Therefore,it is crucial to develop novel strategies for COVID-19 vaccine development to effectively respond to outbreaks and protect human health and public health security.In this study,we developed two types of COVID-19 vaccines:a recombinant ALVAC vaccine utilizing the canarypox virus vector(CNPV),and a nanoparticle vaccine utilizing self-assembled ferritin as a vector.We evaluated the immunogenicity and protective effects of these vaccines,aiming to provide technical support for the development of a novel COVID-19 vaccine.In this study,we constructed three different forms of recombinant ALVAC carrying SARS-CoV-2(Wuhan-Hu-1)genes:ALVAC-2S(double-copy S gene),ALVAC-2RBD(double-copy RBD gene)and ALVAC-VLP(S,M and E gene),by using ALVAC as a vector with CRISPR/Cas9 gene editing technology.Western blot and indirect immunofluorescence assays showed that the three recombinant ALVAC strains successful expression of SARS-CoV-2 target proteins after infecting chicken embryonic fibroblasts(CEF)cells.Furthermore,we purified the supernatant of ALVAC-VLP-infected CEF cells using ultracentrifugation.Under a transmission electron microscope,we observed typical features of SARS-CoV-2 virus-like particles(VLPs).Additionally,viral growth curves demonstrated that the in vitro proliferation efficiency of the three recombinant viruses was comparable to that of the parent virus.In conclusion,we successfully obtained three recombinant ALVAC strains expressing SARS-CoV-2 target proteins.These strains can serve as promising COVID-19 vaccine candidates for further experimental immunization studies.BALB/c mice were immunized with three recombinant viruses,and the subsequent serological analysis showed that the antibody titer induced by ALVAC-VLP was significantly higher than that of ALVAC-2S and ALVAC-2RBD.Therefore,we conducted further studies on the immunogenicity and efficacy of ALVAC-VLP.ALVAC-VLP induced robust antibody responses in 6-8-week-old BALB/c mice,9-month-old BALB/c mice and 4-6-week-old golden hamsters,indicating that the ALVAC-VLP had good immunogenicity in different rodent models.In terms of innate immune response,the results of in vitro and in vitro studies showed that ALVAC-VLP efficiently recruited and activated dendritic cells(DCs),and at the same times,it stimulated the expression of IL-12p70,IL-10,and IL-6 in serum at the early stage.Regarding the synergistic stimulation of the adaptive immune response,ALVAC-VLP significantly triggered a substantial accumulation of humoral immune-related cells,including follicular helper T(Tfh)cells,germinal center B(GCB)cells,and plasma cells.Additionally,ALVAC-VLP induced a strong antigen-specific cytotoxicity(CD8~+IFN-γ~+)T cells and promoted a more balanced CD4~+TH1 and CD4~+TH2 immune responses.Furthermore,ALVAC-VLP stimulated a significant increase in the proportion of central memory T cells(CD4~+/CD8~+&CD44~+&CD62L~+).In protective efficiency assays,we inoculated the lethal SARS-CoV-2 mouse adaptation strain C57MA14 in immunized 9-month-old BALB/c mice.The control mice died within 7 days,in contrast,all of the mice in the ALVAC-VLP-immunized group survived the challenge without any obvious clinical symptoms.The results of viral load analysis revealed that ALVAC-VLP significantly inhibited SARS-CoV-2 replication in the upper and lower respiratory tracts,while reducing the pathological lung damage caused by SARS-CoV-2 infection.Moreover,the results of attack protection in the golden hamster model demonstrated that ALVAC-VLP induced a broad-spectrum immune response that 100%protect golden hamsters against SARS-CoV-2 Wuhan-Hu-1,Beta,Delta,Omicron BA.1,and BA.2infections.In conclusion,ALVAC-VLP immunization provided 100%protection against the lethal challenge of the SARS-CoV-2 mouse adaptation strain in mice while demonstrating effective broad-spectrum protection in the golden hamster model.Additionally,we also construct a nanoparticle vaccine for SARS-CoV-2 Omicron BA.5 using a self-assembled ferritin(Fe)backbone.First,we added a human Ig G1 Fc tag to the C-terminus of the Omicron BA.5 RBD protein,and the resulting RBD FC protein was transiently expressed in HEK293F suspension cells and purified using a protein A column.Simultaneously,a protein A tag was added to the N-terminal of the Fe protein gene,and protein A-Fe protein was obtained through a prokaryotic expression system.To assemble the vaccine,the purified RBD-Fc protein was incubated with the protein A-Fe protein at a molar ratio of 24:1 for 1 hour at room temperature.The resulting complex,called protein A-Fe+RBD-Fc(PAFFA),was obtained through size exclusion chromatography.We confirmed the composition of PAFFA through reductive and non-reducing western blot analyses.Reductive western blotting showed the presence of a 30 k Da protein A-Fe and a 57 k Da RBD-Fc protein in the PAFFA complex.Non-reducing western blotting revealed that the size of the PAFFA complex was above 250 k Da.Furthermore,dynamic light scattering analysis and transmission electron microscopy results revealed that protein A-Fe monomers formed smooth spherical skeleton particles of 10.0±2.5 nm,and PAFFA exhibited protein spikes on the surface of the spherical core,with a size of 41±3.6 nm.In conclusion,we successfully developed a nanoparticle vaccine for SARS-CoV-2Omicron BA.5 using self-assembled ferritin as the backbone.The nanoparticles and RBD-Fc proteins were supplemented with addavax adjuvant to immunize BALB/c mice,respectively.The results demonstrated that the nanoparticle vaccine could rapidly and efficiently induce high levels of broad-spectrum neutralizing antibodies,especially in golden hamsters.Compared with the neutralizing antibody titer against Omicron BA.4,the potency against Delta,Gamma,Beta,Alpha,and Wuhan 01 decreased by 1.5-,1.7-,2.2-,3.1-,and 4.2-fold,respectively.Moreover,the results of RAW264.7 cell uptake and DCs activation in vivo indicated that the antigen modified by the Fe protein significantly enhanced the uptake and activation efficiency of antigen-presenting cells.Furthermore,the nanoparticle vaccine exhibited a significant advantage in inducing the proliferation and activation of immune cells associated with humoral immunity.On the other hand,although the RBD-Fc protein supplemented with addavax adjuvant upregulated the proportion of IFN-γ-secreting T cells,the nanoparticle vaccine induced a higher proportion of CD4~+/CD8~+IFN-γ~+T cells.In the attack-protection assay,the nanoparticle vaccine elicited a broad-spectrum immune and provided 100%protection for golden hamsters against clinical diseases caused by multiple SARS-CoV-2 strains.In conclusion,the nanoparticle vaccine exhibited significant superiority over the RBD-Fc monomer in inducing innate immunity,humoral immunity,and cellular immunity.It generated a broad-spectrum immune response capable of effectively countering infection and replication by multiple SARS-CoV-2 strains in golden hamsters.In summary,we constructed recombinant ALVAC vector vaccines expressing SARS-CoV-2 VLPs and Omicron BA.5 NPs with Fe as the backbone,respectively.Both vaccine candidates effectively stimulated the generation of humoral and cellular immune responses.Immunization with ALVAC-VLP provided 100%protection against lethal challenge by SARS-CoV-2 mouse adaptation strain.Additionally,it significantly reduced infections in golden hamsters with SARS-CoV-2 Wuhan-Hu-1,Beta,Delta,and Omicron BA.1 and BA.2,as evidenced by lower upper and lower respiratory viral loads.The nanoparticle vaccine exhibited accelerated and enhanced uptake and activation efficiency of antigen-presenting cells,resulting in synergistic stimulation of adaptive immune responses.Furthermore,the broad-spectrum protective immune response induced by nanoparticle vaccine provides 100%protection against clinical diseases caused by various SARS-CoV-2 infections in golden hamsters.In conclusion,this study provides both data support and technical support for the development of a novel COVID-19 vaccine. |
