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While SARS-CoV-2-specific T cells have been characterized to play essential roles in host immune protection in COVID-19 patients, few researches focus on the functional validation of T cell epitopes and development of vaccines inducing specific T cell responses. In this study, 120 CD8+ T cell epitopes from E, M, N, S and RdRp proteins were validated. Among them, 110 epitopes have not been reported previously; 110, 15, 6, 14 and 12 epitopes were highly homologous with SARS-CoV, OC43, NL63, HKU1, and 229E, respectively; 4 epitopes from S protein displayed one amino acid distinct from the current variants of SARS-CoV-2. Thirty-one epitopes restricted by HLA-A2 molecule were used to generate peptide cocktail vaccines in combination with Poly(I:C), R848 or polylactic-co-glycolic acid nanoparticles, which elicited robust specific CD8+ T cell responses in wild-type and HLA-A2/DR1 transgenic mice. Seven of the 31 epitopes were found to be cross-presented by HLA-A2 and H-2K/Db molecules. Unlike previous researches, this study established a modified cell co-culture system of DC-peptide-PBL using healthy donors PBMCs to validate the CD8+ T cell epitope on-silicon predicted; provided a library of CD8+ T cell epitopes restricted by a series of high-frequency HLA-A allotypes which covering broad Asian populations; identified the HLA-A cross-restrictions of these CD8+ T cell epitopes using competitive binding experiments with HMy2.CIR cell lines expressing indicated HLA-A molecules; and initially confirmed the in vivo feasibility of 9 or 10-mer peptide cocktail vaccines of SARS-CoV2. These data will facilitate the development of vaccines inducing antiviral CD8+ T cell responses.
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The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to infect people globally. The increased COVID-19 cases and no licensed vaccines highlight the need to develop safe and effective vaccines against SARS-CoV-2 infection. Multiple vaccines candidates are under pre-clinical or clinical trails with different strengths and weaknesses. Here we developed a pilot scale production of a recombinant subunit vaccine (RBD-Fc Vacc) with the Receptor Binding Domain of SARS-CoV-2 S protein fused with the Fc domain of human IgG1. RBD-Fc Vacc induced SARS-CoV-2 specific neutralizing antibodies in non-human primates and human ACE2 transgenic mice. The antibodies induced in macaca fascicularis neutralized three divergent SARS-CoV2 strains, suggesting a broader neutralizing ability. Three times immunizations protected Macaca fascicularis (20ug or 40ug per dose) and mice (10ug or 20ug per dose) from SARS-CoV-2 infection respectively. These data support clinical development of SARS-CoV-2 vaccines for humans. RBD-Fc Vacc is currently being assessed in randomized controlled phase 1/II human clinical trails. SummaryThis study confirms protective efficacy of a SARS-CoV-2 RBD-Fc subunit vaccine.
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The ongoing SARS-CoV-2 pandemic has brought an urgent need for animal models to study the pathogenicity of the virus. Herein, we generated and characterized a novel mouse-adapted SARS-CoV-2 strain, named MASCp36, that causes severe acute respiratory symptoms and mortality in standard laboratory mice. Particularly, this model exhibits age and gender related skewed distribution of mortality akin to severe COVID-19, and the 50% lethal dose (LD50) of MASCp36 was 58 PFU in 9-month-old, male BALB/c mice. Deep sequencing identified three amino acid substitutions, N501Y, Q493H, and K417N, subsequently emerged at the receptor binding domain (RBD) of MASCp36, during in vivo passaging. All three mutations in RBD significantly enhanced the binding affinity to its endogenous receptor, mouse ACE2 (mACE2). Cryo-electron microscopy (cryo-EM) analysis of human ACE2 (hACE2) or mACE2 in complex with the RBD of MASCp36 at 3.1 to 3.7 angstrom resolution elucidates molecular basis for the receptor-binding switch driven by specific amino acid substitutions. Interestingly, N501Y and Q493H enhanced the binding affinity to human ACE2 (hACE2); while triple mutations N501Y/Q493H/K417N decreased affinity to hACE2, thus led to the reduced infectivity of MASCp36 to human cells. Our study not only provides a robust platform for studying the pathogenesis of severe COVID-19 and rapid evaluation of coutermeasures against SARS-CoV-2, but also unveils the molecular mechanism for the rapid adaption and evolution of SARS-CoV-2 in human and animals. One sentence summaryA mouse adapted SARS-CoV-2 strain that harbored specific amino acid substitutions in the RBD of S protein showed 100% mortality in aged, male BALB/c mice.
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The COVID-19 pandemic caused by the SARS-CoV-2 virus has resulted in an unprecedented public health crisis. There are no approved vaccines or therapeutics for treating COVID-19. Here we reported a humanized monoclonal antibody, H014, efficiently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2 at nM level by engaging the S receptor binding domain (RBD). Importantly, H014 administration reduced SARS-CoV-2 titers in the infected lungs and prevented pulmonary pathology in hACE2 mouse model. Cryo-EM characterization of the SARS-CoV-2 S trimer in complex with the H014 Fab fragment unveiled a novel conformational epitope, which is only accessible when the RBD is in open conformation. Biochemical, cellular, virological and structural studies demonstrated that H014 prevents attachment of SARS-CoV-2 to its host cell receptors. Epitope analysis of available neutralizing antibodies against SARS-CoV and SARS-CoV-2 uncover broad cross-protective epitopes. Our results highlight a key role for antibody-based therapeutic interventions in the treatment of COVID-19. One sentence summaryA potent neutralizing antibody conferred protection against SARS-CoV-2 in an hACE2 humanized mouse model by sterically blocking the interaction of the virus with its receptor.
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Coronavirus disease 2019 (COVID-19) threatens global public health and economy. In order to develop safe and effective vaccines, suitable animal models must be established. Here we report the rapid adaption of SARS-CoV-2 in BALB/c mice, based on which a convenient, economical and effective animal model was developed. Specifically, we found that mouse-adapted SARS-CoV-2 at passage 6 (MACSp6) efficiently infected both aged and young wild-type BALB/c mice, resulting in moderate pneumonia as well as inflammatory responses. The elevated infectivity of MACSp6 in mice could be attributed to the substitution of a key residue (N501Y) in the receptorbinding domain (RBD). Using this novel animal model, we further evaluated the in vivo protective efficacy of an RBD-based SARS-CoV-2 subunit vaccine, which elicited highly potent neutralizing antibodies and conferred full protection against SARS-CoV-2 MACSp6 challenge. This novel mouse model is convenient and effective in evaluating the in vivo protective efficacy of SARS-CoV-2 vaccine. SummaryThis study describes a unique mouse model for SARS-CoV-2 infection and confirms protective efficacy of a SARS-CoV-2 RBD subunit vaccine.
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Objective To establish a mouse model of acute liver failure induced by lipopolysaccharide /D-galac-tosamine ( LPS/D-GalN) .Methods The optimum dose of LPS/D-GalN was determined by i .p.injection of eight differ-ent doses of LPS and D-GalN into 40 female C57BL/6 mice and observation of their survival time .Then, 32 female C57BL/6 mice were i.p.injected with the optimal dose of LPS/D-GalN and sacrificed at 0, 1, 4, 8 hours after the injec-tion, 8 mice in each group.The control mice received saline injection .Hepatic changes were observed by pathology and se-rum ALT, IL-6, MCP-1 and TNF-αwere measured by biochemistry or flow cytometry .Results LPS (2.5 mg/kg) and D-GalN (0.3 g/kg) were determined as the optimal dose for the establishment of mouse model of acute liver injury .Com-pared with the control group , the hepatocellular damages were progressing in a positive correlation with the time course after LPS/D-GalN administration .The level of serum ALT was significantly increased after LPS/D-GalN administration ( P <0.001).The levels of inflammatory cytokines IL-6, MCP-1 and TNF-αwere increased and reached a peak at one hour after LPS/D-GalN administration and then decreased almost to that of the control group 8 hours later(P<0.001).Conclusions The mouse model of acute liver injury is successfully established by LPS /D-GalN administration , and provide an effective animal model for the study of pathogenic mechanisms of acute liver failure and evaluation of therapeutic drugs .
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Objective To analyze and compare the pathological changes of lung tissue in mice infected with the novel H7N9 influenza virus and 2009 pandemic H1N1 influenza virus, respectively, and to preliminarily study the mecha-nisms of acute lung injury induced by those virus infection .Methods SPF 6-week old BALB/c mice ( body weight 18-20 g, male∶female=1∶1) (n=3 in each subgroup) were intranasally infected with H7N9 virus and H1N1 virus, respec-tively.The behavior and survival time of mice after virus infection were observed and the survival rates were analyzed .The heart, liver, spleen, lung, kidney, intestines, and brain were collected at indicated time points for histopathological exami-nation using H&E staining .The distribution of virus antigen was detected by immunohistochemistry .The neutrophil infiltra-tion was also observed .The correlation of lung injury with virus replication and host immune responses was analyzed .Re-sults The lung and spleen injury of mice infected with H 7N9 virus was slighter and their survival rate (100%) was high-er than those of mice infected with H1N1 virus.The damages of the lung and spleen in H1N1virus-infected mice were more severe than that in H7N9 virus-infected mice, and all the 10 mice in this group died within 9 days after virus inoculation . The distributions of both the virus antigens were mainly in the bronchial epithelial cells , a few stromal cells and alveolar ep-ithelial cells .The levels of virus replication in the two groups were not significantly different .There were more intense neu-trophil infiltration in the lung and inflammatory response in the H 1N1 virus-infected mice than those in the H7N9 virus-in-fected mice .Conclusions There are some differences of the pathological characteristics and extent of lung injury in the mice infected with H7N9 virus and H1N1 virus, respectively.The virus replication is a precipitating factor but not the deci-sive factor of the lung injury , and there is a close relationship between the host immune responses and acute lung injury .
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<p><b>OBJECTIVE</b>To construct a dual-reporter gene system that will be applicable for use as a tool to screen and evaluate therapeutic drug compounds that inhibit transcription of the gene encoding collagen I, chain at1 (COL1A1).</p><p><b>METHODS</b>The full-length eDNA of transforming growth factor beta1 (TGFbeta1) was cloned by RT-PCR and inserted into two vectors, pcDNA3.1 and pJW4303, for construction of two eukaryotic expression vectors, pcDNA3.1-TGFbeta1 and pJW4303-TGFbeta1.Next, the promoter region of COL1A1, cloned by PCR using human genome DNA as template, was inserted into the vector pGL4.29 to construct the reporter gene vector, pGL4.29-COL1A1 promoter.All three recombinant vectors were verified by restriction enzyme digestion and DNA sequencing.Either the pcDNA3.1-TGFbeta1 or pJW4303-TGFbeta1 vector along with the pGL4.29-COL1A1 promoter vector or a pRL-null, control reporter, vector were co-transfected into the LX-2 human hepatic stellate cells to establish the transcription-activated dualreporter gene system.This system was used as a cell model for screening anti-liver fibrosis compounds that inhibit the transcription of COL1A1.Dexamethasone, a model drug that is known to inhibit the expression of COL1A1, was used as a control to validate the dual-reporter gene system.</p><p><b>RESULTS</b>The two TGFbeta1-expressing vectors and the reporter gene vector containing the promoter region of COL1A1 were successfully constructed.The results of a dual-reporter gene assay showed that TGFbeta1 co-expression increased the activity of the COL1A1 promoter by above 200-fold (t =21.78, P =0.0001), whereas in the absence of TGF31 co-expression the activity was below 2-fold (t =3.396, P =0.0274).The transcriptionactivated dual-reporter gene system was successfully established.The model drug, dexamethasone, effectively inhibited the activity of the COL 1A1 promoter in dose-dependent manner; the activity decreased 29.6% with 10 mumol/L dexamethasone (t =4.140, P =0.0144) and 53.9% with 100 mumol/L (t =6.193, P =0.0035).</p><p><b>CONCLUSION</b>The dual-luciferase reporter system of TGFbeta1 and COL1A1 co-expression developed here can be used as a cell model to screen and evaluate anti-liver fibrosis compounds that inhibit activity of the COL1A1.</p>
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Humanos , Secuencia de Bases , Colágeno Tipo I , Genética , Evaluación Preclínica de Medicamentos , Genes Reporteros , Vectores Genéticos , Cirrosis Hepática , Quimioterapia , Luciferasas , Regiones Promotoras Genéticas , Activación Transcripcional , Transfección , Factor de Crecimiento Transformador beta1RESUMEN
Objective To obtain Chinese hamster ovary (CHO) cell lines that stably express a targeting complement inhibitor CR2-CD59.Methods The recombinant plasmid PEE14.1-CR2-CD59 was constru-cted by cloning the DNA fragment CR2-CD59 into plasmid PEE14.1,and the obtained plasmid was transfected into CHO cells by FuGENE 6.The clones with stable high expression of target fragment were selected by methionine sulfoximine (MSX),the expression of CR2-CD59 was analyzed by ELISA,SDS-PAGE and Western blotting analysis.Results Several stable expression clones were obtained,and CR2-CD59 was highly expressed in the secret form in CHO cells.SDS-PAGE analysis showed that the molecular weight of the recombined protein CR2-CD59 was consistent with the predicted one.ELISA and Western blotting results revealed that the CR2-CD59 could react with both anti-human CR2 and anti-human CD59 polyclonal antibodies.Compared with serum-containing medium,the protein was highly expressed in serum-free medium (P
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Objective To study the rule of distribution and evol vement of TCM syn dromes in HIV/AIDS patients. Methods Totally 177 HIV/AI DS patients were investigated with questionnaire in the epide miological study to collect the data of diagnosis, and the rou tine test of CD4+T lymphocyte count was carried out. Results The young and mid dle-aged females were the most involved group with an increasing incidence in G u angdong Province and with a strong relation of sex and infection route. The main symptoms of the 177 HIV/AIDS patients were fatigue, cough and loss of appetite, the secondary ones were night sweating, skin itching, headache, and hair loss, and the most common sign was skin rash. The leading TCM syndromes were qi and yi n deficiency and lung and kidney deficiency. The distribution of symptoms and si gns, syndromes and various indices was significantly different in gender, age an d infection route (P
