The biological characteristics of SARS-CoV-2 spike protein Pro330-Leu650.

SARS-CoV-2 is the cause of the worldwide outbreak of COVID-19 that has been characterized as a pandemic by the WHO. Since the first report of COVID-19 on December 31, 2019, 179,111 cases were confirmed in 160 countries/regions with 7426 deaths as of March 17, 2020. However, there have been no vaccines approved in the world to date. In this study, we analyzed the biological characteristics of the SARS-CoV-2 Spike protein, Pro330-Leu650 (SARS-CoV-2-SPL), using biostatistical methods. SARS-CoV-2-SPL possesses a receptor-binding region (RBD) and important B (Ser438-Gln506, Thr553-Glu583, Gly404-Aps427, Thr345-Ala352, and Lys529-Lys535) and T (9 CD4 and 11 CD8 T cell antigenic determinants) cell epitopes. High homology in this region between SARS-CoV-2 and SARS-CoV amounted to 87.7%, after taking the biological similarity of the amino acids into account and eliminating the receptor-binding motif (RBM). The overall topology indicated that the complete structure of SARS-CoV-2-SPL was with RBM as the head, and RBD as the trunk and the tail region. SARS-CoV-2-SPL was found to have the potential to elicit effective B and T cell responses. Our findings may provide meaningful guidance for SARS-CoV-2 vaccine design.

Intranasal Immunization Using CTA1-DD as a Mucosal Adjuvant for an Inactivated Influenza Vaccine.

OBJECTIVE: To evaluate the effect of intranasal immunization with CTA1-DD as mucosal adjuvant combined with H3N2 split vaccine. METHODS: Mice were immunized intranasally with PBS (negative control), or H3N2 split vaccine (3 µg/mouse) alone, or CTA1-DD (5 µg/mouse) alone, or H3N2 split vaccine (3 µg/mouse) plus CTA1-DD (5 µg/mouse). Positive control mice were immunized intramuscularly with H3N2 split vaccine (3 µg/mouse) and alum adjuvant. All the mice were immunized twice, two weeks apart. Then sera and mucosal lavages were collected. The specific HI titers, IgM, IgG, IgA, and IgG subtypes were examined by ELISA. IFN-γ and IL-4 were test by ELISpot. In addition, two weeks after the last immunization, surivival after H3N2 virus lethal challenge was measured. RESULTS: H3N2 split vaccine formulated with CTA1-DD could elicit higher IgM, IgG and hemagglutination inhibition titers in sera. Furthermore, using CTA1-DD as adjuvant significantly improved mucosal secretory IgA titers in bronchoalveolar lavages and vaginal lavages. Meanwhile this mucosal adjuvant could enhance Th-1-type responses and induce protective hemagglutination inhibition titers. Notably, the addition of CTA1-DD to split vaccine provided 100% protection against lethal infection by the H3N2 virus. CONCLUSION: CTA1-DD could promote mucosal, humoral and cell-mediated immune responses, which supports the further development of CTA1-DD as a mucosal adjuvant for mucosal vaccines.

Intranasal vaccination with ebola virus GP amino acids 258-601 protects mice against lethal challenge.

Ebola virus (EBOV) disease (EVD) leads to lethal hemorrhagic fever with a case fatality rate as high as 90%, thus posing a serious global public health concern. However, while several vaccines based on the EBOV glycoprotein have been confirmed to be effective in animal experiments, no licensed vaccines or effective treatments have been approved since the first outbreak was reported in 1976. In this study, we prepared the extracellular domain of the EBOV GP protein (designated as N20) by prokaryotic expression and purification via chromatography. Using CTA1-DD (designated as H45) as a mucosal adjuvant, we evaluated the immunogenicity of N20 by intranasal administration and the associated protective efficacy against mouse-adapted EBOV challenge in mice. We found that intranasal vaccination with H45-adjuvanted N20 could stimulate humoral immunity, as supported by GP-specific IgG titers; Th1 cellular immunity, based on IgG subclasses and IFN-γ/IL-4 secreting cells; and mucosal immunity, based on the presence of anti-EBOV IgA in vaginal lavages. We also confirmed that the vaccine could completely protect mice against a lethal mouse-adapted EBOV (MA-EBOV) challenge with few side effects (based on weight loss). In comparison, mice that received N20 or H45 alone succumbed to lethal MA-EBOV challenge. Therefore, mucosal vaccination with H45-adjuvanted N20 represents a potential vaccine candidate for the prevention of EBOV in an effective, safe, and convenient manner.

[Prokaryotic soluble expression of protein D of Haemophilus influenzae type b].

OBJECTIVE: To express the recombinant D protein in prokaryotic expression system solubly and make preparation for producing D-carrier conjugate vaccine next step. METHODS: The hpd gene fragment removed of signal peptide from genomic DNA of Hib CMCC was inserted into pET43. 1a. The recombinant plasmid was transformed to competent E. coli BL21 (DE3) for expression under induction of IPTG. The expressed recombination protein was precipitated with ammonium sulfate, purified by DEAE anion exchange column chromatography and identified for reactogenicity by Western Blot. RESULTS: The expressed recombination protein, in a soluble form, constained about 50% of total somatic protein and showed specific reaction with the HIB antisera after preliminary purification. CONCLUSION: The D protein recombined expression plasmid was constructed successfully and expressed D protein in prokaryotic cells in a solube form.

[The preliminary study of HBV genotype distribution and the relationship with clinical manifestation].

OBJECTIVE: To explore the relationship between HBV genotypes distribution in hepatitis B patients and clinical manifestations in Beijing area. METHODS: 200 HBsAg positive serum were choosen from 1148 serum samples. useing of nested PCR amplification method, HBV DNA S genes were Sequencing and typing; genotype distribution and the relationship with the clinical manifestation were Statistical analysis. RESULTS: In Beijing area, HBV has B, C, D three genotypes, including B gene type 17 cases (12. 5%), C genotype 116 cases (85.2%), D genotype 3 cases (0.02%); in the carriers, patients with chronic hepatitis B, cirrhosis, liver cancer, B type have been gradually falling, C type have been gradually rising trend. CONCLUSION: Serious liver disease relate with C type, B type patients with HBV infection is better than C type patients in clinical prognosis and antivirus treatment response.

[Construction of recombinant CHO cell strain for high expression of HBsAg].

OBJECTIVE: To overexpress hepatitis B virus S gene in CHO cells cultured in serum-free media. METHOD: Plasmid was constructed by cloning of HBV S gene and then it was transfected into CHO cells. After cell screen, the positive clones were identified and isolated into a serum-free media followed by the serological and morphological characterization of the expression product. RESULT: CHO cell strains which can express HBsAg efficiently and stably were obtained. Spherical and filamentous HBsAg could be detected under electronic microscope. The titer of the expression product was up to 1:5000. CONCLUSION: Serum-free media cultured CHO cell strain for overexpression of HBsAg was successfully constructed and the expression product was high antigenic.

[Antigenic analysis of two chimeric hepatitis B core particles presenting the preS1 neutralizing epitopes].

OBJECTIVE: To construct full-length hepatitis B core particles presenting preS1 aa 21-47 epitope and truncated core particles presenting preS1 aa 37-45 epitope on their surface and compare their antigenicity. METHODS: PreS1 aa21-47 epitope and aa 37-45 epitope were inserted respectively into full-length hepatitis B core (aa 1-183) and truncated HBcAg (aa 1-144), between the 78th (Asp) and 79th (Pro). The genes synthesized after the codon optimization were ligated to the pET43. 1a vector with the same cohesive terminal (NdeI and XhoI) and expressed in the E. coli expression system. The morphology of the proteins of interest were observed by electron microscope and characterized by ELISA and Western Blotting. RESULTS: The morphology of the virus-like particles were confirmed by electron microscope. H2 were solid particles with a diameter of (31.61 +/- 1.27) nm, while H3 were hollow particles with a diameter of (28.46 +/- 1.16) nm. Statistical analysis showed that H2 is larger than H3 in the diameter (P < 0.01). The antigenicity of the inserted epitopes and carrier protein were identified by ELISA and Western Blotting. CONCLUSION: Chimeric hepatitis B core particles presenting the preS1 neutralizing epitopes on their surface have been expressed, purified and identified, which lays the foundation for its application in vaccine research.

[The assemblage, purification and characterization of EV71 VLPs expressed in baculovirus].

To construct a recombinant expression plasmid Bacmid-P1-3CD containing the P1 and 3CD genes of enterovirus 71(EV71), the P1 and 3CD genes were cloned into the same baculovirus shuttle vector (Bacmid). Recombinant AcMNPV-P1-3CD was obtained by transfecting the Bacmid-P1-3CD into the insect cell line of S f9. With the IFA and Western-blot methods for identification of expression products confirmed that the target protein was expressed in interior of infected S f9 cells. Electron microscopy showed that the structural protein capsid P1 was cut by virus-encoded protease 3CD and assembled into EV71 virus like particles (VLPs) about 27nm diameter. Different values of MOI and time points of expression were compared to explore the optimal expression condition, and the results showed that the time point could be a more important factor. Then we used S f9 cells with serum-free medium in CellSTACK-10 Culture Chambers to produce EV71 VLPs in the confirmed condition. After purification of VLPs by density gradient centrifugation, we observed on SDS-PAGE profile the purified sample contained three major proteins whose molecular masses corresponded to those of VP1 (39kD), VP0 (34kD) and VP3 (26kD) as well as the intact structure, which can be greatly used for further study in protein structure and genetic engineering vaccine research.

[Detection of hepatitis A virus RNA with an improved loop-mediated isothermal amplification assay].

OBJECTIVE: To establish a novel improved loop-mediated isothermal amplification (LAMP) technique to detect hepatitis A virus (HAV). METHODS: A novel improved LAMP assay based on the addition of an acceleration primer was developed for hepatitis A virus nucleotide acid detection. RESULTS: Precision and reproducibility analysis proved its high stability and reliability. Comparison between the improved and conventional LAMP assays revealed that the former was more sensitive with a detection limit of 5 TCID50/ml. The novel detection method displayed 100% consistency with the TaqMan real-time PCR assay when applied to clinical specimens collected from patients with confirmed acute HAV infection. CONCLUSION: This novel technique is widely applicable as a simple diagnostic tool in the clinical field as well as for the surveillance and investigation of the infectious disease in developing countries where HAV is endemic.

[The investigation of hepatitis D virus infection situation in the human with HBsAg in Foshan of Guangdong province].

OBJECTIVE: To investigate the seroprevalence of hepatitis D virus in Foshan of Guangdong province, to provide the data for the study about it in China. METHODS: ELISA kits from two different companies were used for detecting anti-HDV IgG of all the serum samples, and then RT-PCR was carried out about the selected serum to ensure the results. All the serum samples were collected in 2011 in The First People's Hospital of Foshan. RESULTS: The results from two ELISA kits and RT-PCR were identical. Eight samples were positive. CONCLUSIONS: The seroprevalence rate of HDV in Foshan is higher than that in China. It has no statistically significant difference between female and male. Morever, the older with HBsAg are susceptible to HDV.

[Screening of the target genes transactivated by PS1TP1 protein with suppression subtractive hybridization technique].

OBJECTIVE: To construct a subtractive cDNA library of genes transactivated by PS1TP1 protein with suppression subtractive hybridization (SSH) technique. METHODS: Suppression subtractive hybridization technique and bioinformatics technique were used, the mRNA from HepG2 cells transfected with pcDNA3.1 (-) -PS1TP1 and pcDNA3. 1 (-) empty vector was isolated, respectively; cDNA underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. RESULTS: The subtractive library of genes transactivated by PS1TP1 was constructed successfully. Sequence analysis was performed in 43 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 12 coding sequences were gotten, which consisted of 10 known and 2 unknown ones. CONCLUSION: The obtained sequences may be target genes transactivated by PS1TP1 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity and cell apoptosis. This finding brought some clues for studying the biological functions of PS1T1.

[Relationship between viral genotype and specific and nonspecific CTL of patients with cirrhotic hepatitis B and its significance].

OBJECTIVE: To explore relationship between different HBV genotypes and peripheral blood HBV specific and nonspecific CTL of patients with cirrhotic hepatitis B and its significance. METHODS: HBV genotypes were tested in 91 patients with cirrhotic hepatitis B, differences of HBV specific and nonspecific CTL between patients infected with genotype B and C were compared and its significance was explored. RESULTS: In 91 cases of cirrhotic hepatitis B, 55 cases (60.44%) belong to genotype C, 35 cases (38.46%) belong to genotype B, 1 case (1.1%) belongs to mixture genotype B and C. In genotype C, 27 cases (49.09%) had positive (HLA)-A2, HBV specific CTL was 0.18% +/- 0.03%. In genotype B, 18 cases (51.43%) had positive HLA-A2, HBV specific CTL was 0.38% +/- 0.04%, higher than that in genotype C, t = 5.01, P < 0.01. Nonspecific CTL: genotype C (11.87% +/- 1.50%); genotype B (11.90% +/- 1.51%), t = 0.14, P < 0.05. HBV DNA level: genotype C (6.01 +/- 0.81) log10 copy/ml, higher than that in genotype B (5.01 +/- 0.54) log10 copy/ml, t = 5.01, P < 0.01. ALT: genotype C (251.13 +/- 131.11) U/L, higher than that in genotype B (121.25 +/- 63.21) U/L, t = 3.61, P < 0.01. TBil (45.61 +/- 15.11) micromol/L, higher than that in genotype B (28.11 +/- 6.25) micromol/L, t = 3.05, P < 0.01. CONCLUSION: Compared with patients infected with genotype B of cirrhotic hepatitis B, HBV specific CTL of patients infected with genotype C was lower, resulting in higher level of HBV DNA and more severe damage of liver function.

[A sero-epidemiological study on hepatitis C in China].

OBJECTIVE: To better understand and measure the status of hepatitis C virus (HCV) infection, we conducted a sero-epidemiological study using the remaining blood samples and data of the nationwide survey of hepatitis B in Chinese residents which was carried out in 2006. METHODS: The anti-HCV reagent was screened out from the reagents by the HCV infection blood serum plate with anti-HCV positives or negatives. This plate recognized the Murex 3.0 and Ortho 3.0 reagents as gold standards. Anti-HCV in the blood samples were tested using this reagent and confirmed by Chiron HCV RIBA 3.0 reagents. RESULTS: Among the population aged 1 year to 59 year-olds, the overall prevalence rate of anti-HCV was 0.43% (95%CI: 0.33% - 0.53%), with the rates of anti-HCV among males and females as 0.46% and 0.40%, respectively. The prevalence rate of anti-HCV in urban area was 0.43%, and in rural area it was 0.43%. The prevalence rate of anti-HCV in the Eastern, Middle and Western areas were 0.37% (95%CI: 0.21% - 0.53%), 0.67% (95%CI: 0.40% - 0.94%) and 0.31% (95%CI: 0.20% - 0.42%) respectively. The prevalence rates of anti-HCV for the three areas did not show significant differences, statistically. The prevalence rate of anti-HCV in the South and North areas were 0.29% (95%CI: 0.21% - 0.52%) and 0.53% (95%CI: 0.38% - 0.64%) respectively. CONCLUSION: Our data revealed that China was in the low prevalence area for hepatitis C infection and the results also suggested that the comprehensive measures for HCV control and prevention had been successfully achieved in the country.

[Expression of hepatitis C virus subunit fusion protein and analysis of its immunogenicity].

OBJECTIVE: Obtain the hepatitis C virus high purified subunit fusion protein and detect its immunogenicity. METHODS: With the vector of pET-11d, fusion protein was expressed in Escherichia coli BL21 (DE3) after induced by IPTG. The protein was then purified by DEAE negative ion exchange chromatography and Ni2+ affinity chromatography. Western Blot analysis was used to detect the antigenicity of the fusion protein. At the same time, the sera were collected and prepared from the immunized experimental animals in order to investigate the immunogenicity of the protein by EIA. RESULTS: High purified hepatitis C virus subunit fusion protein was obtained successfully. The EIA indicated that the fusion protein could elicit specific antibodies in the animals with very high titers. CONCLUSION: The hepatitis C virus subunit fusion protein expressed in prokaryotic system was proved to have strong immunogenicity. It could provide some helpful and useful information to the hepatitis C virus prophylactic and therapeutic vaccine development.

[Construction and characterization of hepatitis B surface antigen "a" epitope virus-like particles].

OBJECTIVE: To construct virus-like particles of hepatitis B core antigen, with HBsAg "a" epitope exposed on the surface. METHODS: Hepatitis B surface antigen "a" epitope were inserted into the Hepatitis B core antigen, between the 78th (Asp) and the 79th (Pro) amino acids. The gene was synthesized after the codon optimized, then it was ligated to the express vector after been enzyme digest. The virus-like particles were observed by electron microscope and detected by ELISA after been expressed and purified. Immune the rabbits by the VLPs, then detect the antibody. RESULT: The virus-like particles were confirmed by electron microscope. Its antigenicity and immunogenicity were identified by ELISA. CONCLUSION: The prokaryotic express plasmid with the fusion gene has been constructed successfully. The virus-like particles have been expressed, purified and identified, which lays the foundation for its application in the further.

[Preparation and identification of the monoclonal antibodies against VP1 capsid protein of Enterovius 71].

OBJECTIVE: To prepare monoclonal antibodies (McAbs) against VP1 capsid protein of Enterovirus 71. METHODS: Two peptides, SP55 and SP70, containing amino acid 163-177 and 208-222 of VP1, were synthesized respectively. Immunized BALB/c mice with the synthetic peptides to establish the hybridoma cell strains secreting specific McAb to VP1. After the specific McAbs were prepared, identified and analyzed the titer by indirect ELISA assay. The positive clones were selected and their neutralization titer were determined by neutralization test. RESULTS: Two high titered anti-VP1 antibodies secreted by the hybridoma cells showed good neutralization reaction with enterovirus 71 on RD cells, and the neutralization titer were 1:8 and 1:16 respectively. CONCLUSION: Two high titered anti-VP1 antibodies, with good neutralization activity, secreted by the hybridoma cells, which lays the foundation for further study.

[Preliminary study on genotype of hepatitis B virus detected from Xinjiang Uygur Autonomous Region in China].

OBJECTIVE: To determine the main genotype of hepatitis B virus (HBV) in Xinjiang. METHODS: 200 HBsAg positive serum specimens were detected from more than 2000 serum of Xinjiang inhabitants, and HBV S gene was detected by using nPCR amplifying, and compared with the standard S region HBV nucleotide sequences of genotypes A-H retrieved from GenBank, then analyzed and drawn the polygenetic tree by MEGA3 software. RESULT: Gene in 127 (63.5%) serum specimens was detected from 200 samples. Among 127 serum specimens, 10 (7.8%) was genotype B, 58 (45.7%) was genotype C, and 59 (46.5%) was genotype D. CONCLUSION: Genotype B, C and D have been found in Xinjiang.

[Cloning of PD-1 gene and its prokaryotic expression in Escherichia coli].

OBJECTIVE: To clone human PD-1 gene, construct a prokaryotic expression plasmid and express in E. coli. METHODS: The human PD-1 cDNA was cloned by RT-PCR from the total RNA, which was extracted from peripheral blood lymphocyte cell of the patient with chronic hepatitis B. Recombinant PD-1 protein was been expressed and purified after the prokaryotic expression plasmid had been constructed. It was identified by SDS-PAGE, DNA sequencing and amino acid sequencing. RESULTS: The PD-1 gene was cloned and confirmed by DNA sequencing. The recombinant protein was expressed in E. coli. The purified protein was obtained, then been confirmed by amino acid sequencing. CONCLUSION: The human PD-1 gene was successfully cloned and expressed in E. coli, which lays the foundation for further study on the function and application of PD-1.

[A serological survey of Epstein-Barr virus infection in children in Beijing].

OBJECTIVE: To understand the prevalence of Epstein-Barr virus (EBV) infection in urban and rural areas of Beijing using the serological method. METHODS: Totally 589 serum samples were collected from children in Beijing urban and rural areas who were 0--14 years old and tested with Viron-Seron ELISA classic EBV virus capsid antigen IgG antibody (EBV VCA IgG) kit. Optical density of serum samples was obtained at the wavelength of 405 nanometers. Sero-positive or negative samples were determined according to standard curve and cut-off attached in ELISA classic EBV VCA IgG kits. The activity of EBV VCA IgG was calculated by using special formula. The percentage and activity of EBV VCA IgG from Beijing children were compared with SPSS 13.0 between the urban and rural areas. RESULTS: The percentage of EBV VCA IgG seropositive samples was 83.6%, and 80.8% in those from urban and 86.2% in those from rural areas. The peak value of EBV infection was 71% seen among children under the age of 3 years, and in urban area the rate was 67.7%, which was lower than that in the rural area (75.3%), and was 82.5% by the age of 6, which was lower than the data (up to 90%) reported 30 years ago. There was a significant difference in EBV infection rate and VCA IgG activities in children at different ages between urban and rural areas (P < 0.05). CONCLUSION: The rate of EBV infection in children living in urban area was lower by the age of 6 years. The primary infection of EBV occurred late in part of children lived in urban area.

[Antigenic properties of mutant hepatitis B virus surface antigen].

OBJECTIVE: To study the antigenic properties of mutant hepatitis B virus surface antigen, to understand the sensitivity of the commercially available HBsAg assays to the variants and to reduce the undetectability of the variants. METHODS: Recombinant eukaryotic expression plasmids for HBsAg. The recombinant eukaryotic expression plasmids pSS1adr, pSS1adw2, pSS1adw2- 145Arg, pSS1adr-126 Asn and pSS1adr-126Ser were transfected into COS-7 cells. HBsAg in the supernatants of transfected cells was detected by using different commercial ELISA kits. RESULTS: The absorbance value of pSS1adr-126 Asn and pSS1adr-126Ser plasmids were similar to that of the wild type HBsAg, the absorbance value of pSS1adw2-145Arg plasmids was lower than that of the wild type HBsAg. CONCLUSION: It is estimated that the antigenicity of HBsAg mainly depended on the amino acid sequence of "a" antigen determinant and its conformation, so 145 amino acid substitutions led to the change of conformation and the antigenicity of variant HBsAg was lower than that of the wild type.