Studies On The Generation Of COVID-19 Mouse Model And The Role Of Virus Specific T Cells

BackgroundCoronavirus Disease 2019（COVID-19）is an acute respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2（SARS-CoV-2）.SARS-CoV-2 broke out in Wuhan,China in December 2019,and then spread rapidly,causing a global outbreak.As of April 11,2022,there have been about 500 million confirmed cases of COVID-19 globally,including more than 6 million deaths.Respiratory infectious viruses,including SARS-CoV-2,have triggered many public health emergencies,posing a major threat to people’s health and the national economy around the world.An ideal mouse model could be used to evaluate therapies,vaccines and uncover the pathogenesis of SARS-CoV-2 et al.,helping the prevention and control of COVID-19 pandemic.The receptor of SARS-CoV-2 is human angiotensin-converting enzyme 2（hACE2）,while mouse homologous receptor（mouse ACE2,m ACE2）cannot mediate virus invasion due to the differences of key amino acid sites,which must be overcome in generation of a COVID-19 mouse model.Host antiviral immune response is a double-edged sword.Analyzing the immune response and immune regulation mechanism against SARS-CoV-2 is helpful to clinical diagnosis,therapy,disease recovery and vaccine development.Anti-virus cellular immune response is an essential component of immune defense.Studies have shown that T cells play a protective role in SARS-CoV and MERS-CoV infection.Therefore,dissect the role of T cell immune response in SARS-CoV-2 infection is of great significance.ObjectiveThis study aims to quickly generate a COVID-19 mouse model,and use this model to map the epitopes and elucidate the function of SARS-CoV-2 specific T cells in infected mice.Method1.We generated recombinant adenovirus expressing hACE2（Ad5-hACE2）by the Ad Easy system.Then Ad5-hACE2 was purified by Cs Cl gradient centrifugation,and titer was determined by focus forming assay.The proper expression and function of Ad5-hACE2 was confirmed in vitro and in vivo.2.For rapid generation of SARS-CoV-2 susceptible mouse model,mouse was transduced with Ad5-hACE2 intranasally（i.n.）,inducing high expression of hACE2 in the lung,and infected with SARS-CoV-2 at day 5 post transduction.3.In Ad5-hACE2-transduced SARS-CoV-2-infected mice,the role of innate immune response was evaluated by comparing the phenotypic differences in weight loss,lung viral load and pathological changes between gene knockout mice and WT mice after infection,as well as the effect of Poly I:C treatment on the disease;the role of T cells was evaluated by the phenotypic changes after deleting T cells;the therapeutic effect of plasma/serum and Remdesivir was evaluated by the phenotypic changes of infected mice after adoptive transfer of plasma/serum and Remdesivir treatment;the protective effect of vaccine was evaluated by immunizing mice with VRP expressing SARS-CoV-2-S protein（VRP-S）.The anti-SARS-CoV-2 innate and adaptive immune responses and the effects of drug,plasma/immunized serum and vaccines were comprehensively analyzed to evaluate the application value of the mouse model.4.SARS-CoV-2-specific CD4⁺and CD8⁺T cell epitopes were mapped in Ad5-hACE2-transduced SARS-CoV-2-infected mice by ex vivo peptide stimulation of lymphocytes,ICS and flow cytometry.5.SARS-CoV-2-specific CD4⁺and CD8⁺T cells were characterized by expression of phenotypic makers and multi-cytokines,avidity to epitopes and in vivo cytotoxicity.6.To evaluate the role of SARS-CoV-2-specific T cells,mice were immunized with VRPs expressing single dominant CD4⁺or CD8⁺T cell epitope.After infection,viral titers and histopathological changes were examined.7.The effect of IFN-I pathway on T cell responses were analyzed by comparing the T cell response and function between IFNAR^-/-and WT mice.8.Candidate conserved cross-reactive T cell epitopes were found by alignment in epitopes of three high pathogenic human coronaviruses and confirmed by T cell responses ex vivo.Result1.Ad5-hACE2-transduced SARS-CoV-2-infected BALB/c mice lost up to～20%of their body weight in the first 4–6 days of infection,and C57BL/6 mice lost about 15%body weight.High viral titers（up to 10⁷ PFU/gm）and a variety of lesions including perivascular to interstitial inflammatory cell infiltrates,necrotic cell debris,and alveolar edema in lung tissue were found in both mouse strains.These results show that the Ad5-hACE2-transduced SARS-CoV-2-infected mouse model is successfully developed.2.Using this model,we found that compared with WT mice,IFNAR^-/-mice had delayed virus clearance（2 d.p.i.,p=0.0104;4 d.p.i.,p=0.0003;6 d.p.i.,p=0.0023）and diminished inflammation,although this did not translate into a significant difference in weight loss.IFN-（?）^-/-mice showed no differences.STAT1^-/-mice exhibited greater weight loss,enhanced inflammatory cell infiltration into the lungs,and delayed virus clearance（2 d.p.i.,p=0.0289;4 d.p.i.,p=0.0806）.These results suggest that IFN-I and STAT1 but not IFN-（?）signaling is protective in SARS-CoV-2 infection.Poly I:C treatment resulted in significantly diminished clinical disease and induced more rapid kinetics of initial virus clearance（2 d.p.i.,p=0.0005）.This result confirmed the protective role of IFN-I signaling.The results of RNA-seq showed that the upregulated genes in the lungs of transduced/infected mice were associated with inflammation pathways,innate and adaptive immune response pathways.These results indicate an antiviral immune state in the infected mice.3.In transduced/infected mice,SARS-CoV-2 induced neutralizing antibodies and virus-specific T cell responses.Neutralizing antibodies peaked at 10 d.p.i.,and virus-specific CD4⁺and CD8⁺T cells peaked at 8 d.p.i..SARS-CoV-2 clearance was delayed post deletion of CD4⁺（5 d.p.i.,p<0.0001）or CD8⁺T（5 d.p.i.,p<0.0001）cells.These results suggest that T cells are protective in SARS-CoV-2 infection.4.Adaptive transfer of VRP-S immunized sera（1 d.p.i.,p<0.0001）or convalescence patient plasma（1 d.p.i.,p=0.0021）accelerated SARS-CoV-2 clearance,and Remdesivir treatment accelerated virus clearance（1 d.p.i.,p<0.0001）and reduced pathological damage,suggesting that antibodies and remdesivir were protective in SARS-CoV-2infection.5.VRP-S vaccination accelerated SARS-CoV-2 clearance（BALB/c,1 d.p.i.,p=0.0005;C57BL/6,1 d.p.i.,p<0.0001）,indicating that VRP-S vaccination was protective in SARS-CoV-2 infection.6.SARS-CoV-2-specific T cell epitopes were mapped in SARS-CoV-2 infected BALB/c and C57BL/6 mice.The dominant CD4⁺and CD8⁺T cell epitopes were SARS-CoV-2-N351-365（I-A^d）and S535-543（H2-D^d）in BALB/c mice,and ORF3a266-280（I-A^b）and S538-546（H2-D^b）in C57BL/6 mice,respectively.7.SARS-CoV-2-specific CD4⁺and CD8⁺T cell responses peaked at 8 d.p.i.,and were stronger in the virus-infected site（airway）than in the secondary lymphoid organs.They expressed many functional phenotype makers as well as multi-cytokines,were sensitive to epitope peptide stimulation,and were cytotoxic in vivo.8.Vaccination with VRPs expressing a single dominant CD4⁺or CD8⁺T cell epitope accelerated virus clearance and reduced lung pathological damage in SARS-CoV-2infected BALB/c mice,indicating that virus-specific CD4⁺（2 d.p.i.,p=0.0051）and CD8⁺T（2 d.p.i.,p=0.0026）cells are protective in SARS-CoV-2 infection.9.The frequency and cell number,the ability to express multi-cytokines,and the avidity to epitopes of SARS-CoV-2-specific T cells were all lower in IFNAR^-/-mice than in WT mice,suggesting that IFN-I deficiency can result in compromised virus-specific T cell responses.10.In BALB/c mice,conserved T cell epitopes between SARS-CoV and SARS-CoV-2,but not MERS-CoV,could induce SARS-CoV-2-specific CD4⁺and CD8⁺T cell responses ex vivo,showing that cross-reactive CD4⁺and CD8⁺T cell responses exist in BALB/c mice between SARS-CoV and SARS-CoV-2,but not MERS-CoV.Conclusion1.Ad5-hACE2-transduced SARS-CoV-2-infected COVID-19 mouse model was successfully and rapidly developed in this study.2.In this transduced/infected mouse model,IFN-I signaling,T cells,immunized sera/plasma of convalescent patients,Remdesivir and VRP-S vaccination were protective.3.SARS-CoV-2-specific T cell epitopes in BALB/c and C57BL/6 mice were successfully mapped.The virus-specific T cells were multi-functional,and single epitope-specific T cells could achieve protection in SARS-CoV-2 infected mice.4.IFN-I signaling deficiency resulted in compromised virus-specific T cell responses.5.Cross-reactive T cell responses exist between SARS-CoV and SARS-CoV-2 in BALB/c mice,but not MERS-CoV.Significance1.In this study,COVID-19 mouse model was successfully developed by transduction of Ad5-hACE2 i.n..Compared with hACE2 transgenic mouse,this model has a short generation cycle（2-3 weeks）,does not need special breeding,and can be used for the generation of a variety of gene modified animal models.The technical method is simple and easy to repeat,which is suitable for large-scale promotion.This animal model can be used for the emergency evaluation of antiviral drugs,antibodies and vaccines and studying the pathogenesis,which effectively alleviates the problem of the lack of COVID-19 pneumonia animal model.2.This is the first study to analyze the characteristics and function of virus-specific T cell in SARS-CoV-2-infected BALB/c and C57BL/6 mice.This study will greatly promote the study of cellular immune response against SARS-CoV-2 infection,and provide useful information and support for the study of SARS-CoV-2 pathogenesis,the development of novel vaccines,and regional immune characteristics of lung tissues and diseases.