Progress in SARS and MERS vaccines: lessons for the development of COVID-19 vaccine / 中华微生物学和免疫学杂志

Safe and effective vaccines are urgently needed to prevent and control the outbreak of COVID-19. SARS-CoV-2 belongs to the genus Betacoronavirus like severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). Here we summarized the current progress and problems in the development of vaccines against SARS-CoV and MERS-CoV in order to provide reference for COVID-19 vaccine development.

Progress in SARS and MERS vaccines: lessons for the development of COVID-19 vaccine / 中华微生物学和免疫学杂志

Safe and effective vaccines are urgently needed to prevent and control the outbreak of COVID-19. 2019-nCoV belongs to the genus Betacoronavirus like severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). Here we summarized the current progress and problems in the development of vaccines against SARS-CoV and MERS-CoV in order to provide reference for COVID-19 vaccine development.

Recombinase-mediated in vitro rapid construction of replication-competent human adenovirus type 14 vector encoding EGFP / 中华微生物学和免疫学杂志

Objective@#To rapidly and efficiently construct a replication-competent human recombinant adenovirus type 14 vector expressing enhanced green fluorescence protein (rAd14-EGFP) using in vitro homologous recombination.@*Methods@#The skeleton plasmid pBRAd14 was constructed using homologous recombination in Escherichia coli (E.coli) BJ5183 competent cells. The plamid was linearized and transfected into AD293 cells to rescue Ad14. Exnase, a recombinase, was used to construct the shuttle plasmid pSK14-EGFP in vitro using homologous recombination among four fragments. The overlapping sequence was 15-20 bp. Three exogenous fragments generated with PCR including Ad14 E3L fragment, EGFP gene and Ad14 E3R fragment were cloned into the plasmid pBluescript Ⅱ SK(-) simultaneously. Recombinant plasmid pBRAd14-EGFP was constructed by in vitro homologous recombination between 27 kb fragment of plasmid pBRAd14 obtained through double digestion and Ad14 E3L-EGFP-Ad14 E3R fragment amplified by PCR using the shuttle plasmid pSK14-EGFP as template. The plasmid pBRAd14-EGFP was linearized and transfected into cells to obtain the viral vector rAd14-EGFP, which was then used to immunize mice to detect the induced immune responses.@*Results@#A replication-competent E3-deleted adenovirus vector rAd14-EGFP expressing EGFP was successfully constructed. Intracellular proliferation properties and immunogenicity of the vector were no significantly differences compared with those of Ad14.@*Conclusions@#In vitro homologous recombination using the commercial recombinase Exnase can be a rapid, efficient and accurate method to construct adenoviral vector.

Recombinase-mediated in vitro rapid construction of replication-competent human adenovirus type 14 vector encoding EGFP / 中华微生物学和免疫学杂志

Objective To rapidly and efficiently construct a replication-competent human recombi-nant adenovirus type 14 vector expressing enhanced green fluorescence protein ( rAd14-EGFP) using in vitro homologous recombination. Methods The skeleton plasmid pBRAd14 was constructed using homologous re-combination in Escherichia coli ( E. coli) BJ5183 competent cells. The plamid was linearized and transfected into AD293 cells to rescue Ad14. Exnase, a recombinase, was used to construct the shuttle plasmid pSK14-EGFP in vitro using homologous recombination among four fragments. The overlapping sequence was 15-20 bp. Three exogenous fragments generated with PCR including Ad14 E3L fragment, EGFP gene and Ad14 E3R fragment were cloned into the plasmid pBluescript Ⅱ SK (-) simultaneously. Recombinant plasmid pBRAd14-EGFP was constructed by in vitro homologous recombination between 27 kb fragment of plasmid pBRAd14 obtained through double digestion and Ad14 E3L-EGFP-Ad14 E3R fragment amplified by PCR using the shuttle plasmid pSK14-EGFP as template. The plasmid pBRAd14-EGFP was linearized and trans-fected into cells to obtain the viral vector rAd14-EGFP, which was then used to immunize mice to detect the induced immune responses. Results A replication-competent E3-deleted adenovirus vector rAd14-EGFP expressing EGFP was successfully constructed. Intracellular proliferation properties and immunogenicity of the vector were no significantly differences compared with those of Ad14. Conclusions In vitro homologous recombination using the commercial recombinase Exnase can be a rapid, efficient and accurate method to construct adenoviral vector.

Construction of real-time polymerase chain reaction detection for infection-related cytokines of tree shrew / 生物医学工程学杂志

Tree shrew is a novel and high-quality experimental animal model. In this study, the real-time polymerase chain reaction methods were established to detect infection-related cytokines interleukin-6 (IL-6), IL-8, IL-10, IL-17A, interferon-γ (IFN-γ) and housekeeping gene glyceraldehyde-phosphate dehydrogenase ( ) of tree shrew. The results indicated that the establised methods had good specificity. The high point of the linear range of these reagents reached 1 × 10 copies, and the low points ranged from 10 copies (IL-6, IL-17A), 100 copies (IL-10, ) to 1 000 copies (IL-8, IFN-γ). In this interval, the linear correlation coefficient of each reagent was greater than 0.99. The lowest detectable values of IL-6, IL-8, IL-10, IL-17A, IFN-γ and were 8, 8, 4, 8, 128 and 4 copies, respectively. The results showed that the established detection methods had good specificity, sensitivity and wide linear range. The methods were suitable for detection of multiple concentration range samples, and could be used for the subsequent studies of tree shrew cytokines.

Construction of human adenovirus type 4 vector expressing enhanced green fluorescence protein / 中华微生物学和免疫学杂志

Objective To prepare human adenovirus type 4 (Ad4) vector expressing enhanced green fluorescence protein (EGFP). Methods This study used a previously prepared plasmid pBRAd4 containing the whole genome DNA of Ad4-GZ01 strain. The Ad4 genome E3 region of pBRAd4 was deleted and replaced with the EGFP expression frame by conventional molecular cloning method. Then the recombi-nant plasmid was transfected into AD293 cells to rescue recombinant virus which was identified by sequen-cing,SDS-PAGE and ELISA. The purified virions were injected to mice and the induced immune responses were detected by ELISA and microneutralization test. Results The recombinant Ad4 vector rAd4EGFP ex-pressing EGFP was obtained and could be recognized and neutralized by monoclonal antibody MN4b and an-tisera against Ad4. The Ad4-specific and EGFP-specific antibodies with high titers could be detected in mice immunized with rAd4EGFP. Conclusion Human Ad4 vector expressing EGFP was successfully obtained and could be used in research on vaccine development,drug evaluation and transgene vector.

Molecular epidemiology of HFMD in mainland China and discussion on prevention and control measures / 中华微生物学和免疫学杂志

Objective To summarize the epidemiological data of hand, foot and mouth disease (HFMD) in China from 2013 to 2017 and to analyze its molecular epidemiological characteristics in order to provide scientific basis for improving prevention and control measures. Methods China National Knowledge In-frastructure,Wanfang Database of China,Chinese VIP Journal Net and Pubmed were used to search epidemio-logical data of HFMD published in recent years. National notification data and surveillance data of HFMD in ma-inland China were obtained from the National Health and Family Planning Commission of China and the World Health Organization. Basic statistic tools were used for data analysis. Results From 2013 to 2016, the inci-dence rates of HFMD were 134. 37/100 000, 203. 16/100 000, 145. 30/100 000, 176. 62/100 000 and 140.46/100 000,respectively. Enterovirus 71(EV71),coxsackievirus A16(CA16),CA6 and CA10 were the predominant pathogens causing HFMD in the first half of 2013-2017. CA6 was the main epidemic strain in most areas of China. EV71 remained the predominant pathogen causing severe HFMD, but CA6, CA16 and CA10 were also critical pathogens of concern. The predominant strains of enteroviruses varied with year and region. Conclusion Although the EV71 vaccine has been approved since 2016, HFMD has not been controlled com-pletely in China. It is badly in need of more comprehensive surveillance of other types of enteroviruses and HFMD polyvaccine to improve the prevention and control of HFMD.

Construction of a hexon-chimeric human adenovirus type 3 vector expressing two neutralizing epitopes of hepatitis B surface antigen preS1 and analysis of antigenicity of the chimeric epitopes / 中华微生物学和免疫学杂志

Objective To construct a hexon-chimeric human adenovirus type 3 ( HAd3 ) vector expressing two neutralizing epitopes of hepatitis B surface antigen preS 1 (HBsAg-preS1) and to analyze the antigenicity of the chimeric epitopes .Methods Two neutralizing epitopes of HBsAg-preS1 including KR359 and KR127 were inserted into hypervariable region 1 ( HVR1) and hypervariable region 2 ( HVR2) of HAd3 hexon .Chimeric hexon gene encoding the two epitopes was amplified by overlap PCR and then subcloned in -to shuttle plasmid pBR322-L/R containing the homologous recombination regions .The digested shuttle plas-mid containing chimeric hexon gene was co-transfected into Escherichia coli BJ5183 cells together with back-bone plasmid pBRAdΔE3GFP to construct pBRAdΔE3GFP-preS1 vector.Then pBRAdΔE3GFP-preS1 vector was digested with AsiSⅠand transfected into AD293 cells to construct recombinant virus (rAD3E-preS1). CsCl gradient centrifugation was used for purification .Glutathione S-transferase ( GST ) fusion protein KR359KR127 ( GST-KR359KR127 ) was expressed in Escherichia coli BL21 by using expression vector pGEX-4T3.Female BALB/c mice at age 6-8 weeks were intraperitoneally injected with 1010 virus particles or 80 μg GST fusion protein .Serum samples were collected to analyze the antigenicity of two epitopes by ELISA and Western blot .Results ELISA showed that KR 359 and KR127 were successfully exposed on viral sur-faces by using hexon-chimeric HAd3 vector .The induced polyclonal antibodies in serum samples could rec-ognize GST fusion protein and native HBsAg from patients infected with hepatitis B virus .Conclusion The antigen capsid-incorporation strategy could be used to display epitopes on viral surface .Enhanced antigen-specific responses could be achieved through inserting multiple foreign epitopes into hexon HVRs .This study provided evidence for further application of hexon -chimeric human adenovirus type 3 vector in the developmentof vaccine, especially for the development of multivalent hepatitis B vaccine .

Cloning and expression of hexon protein of human respiratory adenovirus of three serotypes and analysis of antigenicity of the recombinant proteins / 中华微生物学和免疫学杂志

Objective To clone and express the hexon protein of three prevalent human adenovi -rus strains causing respiratory disease and analyze the antigenic characteristics of the recombinant proteins . Methods The full length genes encoding hexon protein of human adenovirus serotype 3(HAdV3), serotype 4(HAdV4) and serotype 7(HAdV7) were cloned by PCR and sequenced , respectively.The alignment anal-ysis was performed by using hexon gene sequences from GenBank .The major antigenic regions of hexon pro-tein of the three serotypes were expressed in E.coli and purified.The antigenicity, immunogenicity and cross reactivity of the recombinant proteins were determined by ELISA and Western blot assay .Results The full length gene sequence encoding hexon protein of human adenovirus serotype 4 was firstly reported in China , which showed more than 99%homology in both nucleotide and amino acid sequences with the human adeno-virus type 4 NHRC3 strain.The partial hexon protein sequence of HAdV 3, HAdV4 and HAdV7 containing all of the 7 hyperviriable regions ( HVRs) were expressed in E.coli, respectively .The purified recombinant proteins could be recognized by antiserum of the three serotypes of adenovirus .The antiserum samples against the three recombinant proteins could cross-react with particles of the three serotypes of adenovirus . The possible type-and species-specific epitopes were predicted .Conclusion The major antigenic regions of hexon protein of the three serotypes were successfully expressed .The purified recombinant proteins contai-ning both intertypes and type-specific epitopes showed a strong immunogenicity .

Screening and research of homologous peptide with adenovirus receptor / 中国免疫学杂志

Objective:To screen the binding peptide against adenovirus type 7(Ad7) and evaluate the relevance with the ade-novirus receptor .Methods:Binding peptide against Ad 7 was screened by panning the phage display 12 peptides library .The antibody against the selected peptide was prepared and was used to study the binding to the membrane by immunofluorescence technique .Re-sults:Using Ad7 as the target protein , GTS09 peptide was selected from the phage display 12 peptides library by biopanning .GTS09-phage complex could significantly bind Ad 7, with the affinity constant up to 1.93 ×1010 L/mol;at the same time, immunofluorescence showed that antibody of GTS09 could specifically bind to membrane of 293 cell.Conclusion: Antibody against GTS09 peptide could specifically bind to membrane of 293 cell,which shows that the peptide may presumably have homology with the cell receptors of Ad 7.

Construction of the recombinant human adenovirus type 3 expressing Norovirus capsid protein gene / 中华微生物学和免疫学杂志

Objective To prepare recombinant human adenovirus type 3 expressing Norovirus cap-sid protein gene(Noro-orf2). Methods The cDNA for Noro-orf2 was amplifed by RT-PCR from stool of in-fantile gastroenteritis and cloned into the adenovirus shuttle vector pBSE3CMV-egfp. The vector pBSE3CMV-Nor was linearized with EeoR Ⅴ and Not Ⅰ, and transformed into E. coil BJ5183 with lined edenovirus ge-nomic DNA pLasmid pBRAdv3 by Rsr Ⅱ. The identification of recombinant adenovirus plasmid pBRAdv3E3dNor was performed by PCR, enzyme digestion and DNA sequencing. Then pBRAdv3E3dNor was digested with AsiS Ⅰ and transfeeted into Hep-2 cells with LipofectAMINETM 2000 to package recombi-nant adenovirus particles. Results Noro-orf2 was successfully inserted into the shuttle vector. The recombi-nant adenoviral plasmid pBRAdv3E3dNor was generated by homologous recombination in E. coil BJ5183 and confirmed by PCR and enzyme digestion. The recombinant adenovirus was successfully packaged and puri-fied. Norovirus eapsid protein gene expression was confirmed in Hep-2 cells by immunecytochemistry assay. Conclusion The recombinant type 3 adenovirus expressing Norovirus eapsid protein gene was successfully constructed. This study laid a foundation for developing vaccine against Norovirus.