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@#Objective To express the capsid proteins VP1 and VP3 of hepatitis A virus(HAV) in prokaryotic cells and evaluate their immunogenicity.Methods VP1 and VP3 gene fragments were amplified by PCR,cloned into vector pETG28a to construct recombinant expression plasmids pET-G28a-VP1 and pET-G28a-VP3,which were transformed into competent E.coli BL21(DE3),induced by IPTG,and then analyzed by 12% SDS-PAGE.The target protein was purified by ion exchange chromatography,renatured and combined with several adjuvants to immunize mice.The mice were divided into VP1-MF59,VP1-AH,VP1-AP,VP1-AP-10CpG,VP1-AP-50CpG,VP3-AP,VP3-VP1-AP and PBS control groups,five for each group.Serum IgG antibody titers of mice in various groups were detected by ELISA,and serum neutralizing antibody titers of mice in VP1-AP,VP3-VP1-AP and PBS control groups were detected by rapid fluorescence focus immunosuppression experiment.Results Colony PCR and sequencing showed that the recombinant plasmids pET-G28a-VP1 and pET-G28a-VP3were constructed correctly.The recombinant proteins VP1 and VP3,with relative molecular masses of about 37 000 and26 000 respectively,mainly existed in the form of inclusion bodies,the expression levels were 18.6% and 32.4%,and the purity was 86.3% and 84.7%,respectively.The recombinant proteins VP1 and VP3 reacted specifically with rabbit anti-HAV antiserum and mouse anti-HAV-VP3 antiserum respectively.The serum IgG antibody titer of mice in VP1-AP-50CpG group was significantly higher than that in VP1-AP group(q=22.05,P <0.01),and the serum IgG antibody titer of mice in VP3-VP1-AP group was significantly higher than that in VP1-AP group and VP3-AP group(q=22.05 and 22.49 respectively,each P <0.01).Compared with PBS control group,the serum neutralizing antibody titer of mice in VP1-AP and VP3-VP1-AP group increased significantly(q=7.79 and 25.11 respectively,P<0.01) Conclusion Prokaryotic HAV capsid proteins VP1 and VP3 showed high purity and good immunogenicity,and the addition of CpG in the preparation was beneficial to enhance the immunogenicity of antigens.This study laid a foundation of the development of HAV recombinant subunit vaccine.
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@#Objective To optimize the adsorption condition of hepatitis A virus(HAV)antigen by aluminum hydroxide[Al(OH)_3]adjuvant and evaluate the immunogenicity of the adsorbed products. Methods The final molar concentration of buffer(0. 01,0. 02 and 0. 04 mol/L),pH value(5. 5,6. 0,6. 5,7. 0,7. 5 and 8. 0),final molar concentration of sodium chloride(NaCl)(0. 075,0. 13,0. 15,0. 17,0. 45,0. 75 and 1. 0 mol/L),mixing mode(mixing after dilution and mixing directly without dilution)and dropping order[HAV stock solution into Al(OH)_3 solution,and Al(OH)_3 solution into HAV stock solution]were selected for single factor screening. Taking Al(OH)_3 adsorption rate as the evaluation index,the selected factors were optimized by orthogonal test. NIH mice were immunized intraperitoneally with adsorbed products prepared under optimal and suboptimal combination conditions with 0. 5 mL/mouse,10 mice in each group with half male and half female. After 28 d of immunization,the blood samples were collected from eyeball or heart,and the serum was separated. The HAV antibody level was detected by ELISA,and the immunogenicity of adsorbed products was evaluated by the seroconversion rate of mouse serum antibody. Results The results of single factor screening test showed that the final molar concentration of buffer,pH value and final molar concentration of NaCl had significant influence on the adsorption effect(F = 6. 582,14. 007 and 8. 612,respectively,each P < 0. 05),while the mixing mode and dropping order had no effect(t =-0. 696 and 1. 153,respectively,each P > 0. 05). The final molar concentration of buffer was 0. 01 and 0. 02 mol/L,the pH value was 5. 5 ～ 7. 0,and the final molar concentration of NaCl was 0. 13 ～ 0. 17 mol/L. The orthogonal test showed that the order of the influence on adsorption effect was the final molar concentration of buffer > pH value > NaCl final molar concentration. The optimal combination was pH 6. 3 + final molar concentration of buffer 0. 01 mol/L + NaCl molar final concentration 0. 15 mol/L,and the suboptimal combination was pH 5. 8 + final molar concentration of buffer 0. 02 mol/L +NaCl final molar concentration 0. 17 mol/L. The seroconversion rate of serum antibody in mice immunized with the adsorbed products prepared by the optimal and suboptimal combination was both 100%. Conclusion The adsorbed products prepared under the optimized conditions have good immunogenicity,which lays a foundation of the research of inactivated hepatitis A vaccine
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@#ObjectiveTo explore the purification effect of hepatitis A virus(HAV)with a new type of composite medium Capto Core 400 chromatography packing.MethodsHAV crude liquid cultured with human embryonic lung diploid cells(2BS strain)was purified using Capto Core 400 chromatography media. The optimum conditions for purification of HAV were determined from flow rates(60,150,300 and 450 cm/h),pH of loading buffer(6. 6,7. 0 and 7. 4),salt ion concentration of loading buffer(0,0. 15,0. 3 and 0. 45 mol/L)and capacity[0. 5,1,2 and 4 column volume(CV)],respectively. The purification effect was compared with that of traditional Sepharose 4FF chromatography media.Results The optimum purification conditions of Capto Core 400 were preliminarily determined as follows:flow rate of 150 cm/h,loading buffer pH of 7. 0 ～ 7. 4,salt ion concentration of 0. 15 mol/L,loading volume of 2 CV. The average recovery rate of virus was 92. 61%,and the average removal rate of protein was 84. 25%. The average recovery rate of HAV antigen purified by Sepharose 4FF chromatography media was 76. 03% and the average protein removal rate was 73. 21%. The purification effect of Capto Core 400 chromatography media was better than that of Sepharose 4FF chromatography media.ConclusionThe Capto Core 400 composite chromatography media had a high recovery rate for HAV purification and a relatively simple operation,which was suitable for HAV purification.
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@#Abstract：Objective To investigate the adaptability and genetic stability of hepatitis A virus（HAV）SYX1 strain in human diploid cell MRC⁃5. Methods HAV SYX1 strain isolated from feces of patients with hepatitis A was continuously propagated in MRC⁃5 cells for 28 passages，of which the 1st ~ 26th passages were determined for antigen contents and virus titers，the 6th passage was observed for the morphology under microscope and detected for physicochemical properties，and the 13th ~ 15th passages were studied for virus proliferation dynamics to determine the peak yield of virus proliferation. Genomic RNA was extracted from the 8th，12th，18th，20th，22nd，25th，26th and 28th passages and sequenced to analyze the genetic stability. The main seed batch and working seed batch of HAV SYX1 strain were established and verified according to the requirements of Chinese Pharmacopoeia（VolumeⅢ，2020 edition）. Results The antigen content of HAV SYX1 was stable at 160 ~ 320 EU／mL and the titer was maintained at 7. 3 ~ 8. 3 lgCCID50／mL after the 8th passages in MRC 5 cells；Virus particles showed two types：hollow and solid，with a diameter of 27 ~ 32 nm，spherical，without envelope and protrusions on the surface，which tolerated low pH value and ether. The peak period of virus proliferation was 10 d with an antigen content of more than 160 EU／mL and a virus titer of more than 7. 0 lgCCID50／mL. HAV SYX1 was a subtype of HAV IB，and no mutation in the coding region of all structural proteins during passage was observed. The verification results of main seed batch and working seed batch of HAV all met the relevant requirements. Conclusion HAV SYX1 strain showed good adapt⁃ ability and genetic stability in MRC⁃5，which might be used for the development and production of inactivated hepatitis A vaccine.
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Background: HAV infection is endemic in many developing countries like India, Pakistan, Nepal etc. Several seroprevalence studies show high rates of sero-positivity among children by sub-clinical infection. Therefore mass vaccination against HAV has not been recommended in endemic countries. Objective: To determine whether routine hepatitis A vaccination is indicated for all Bangladeshi children & also to know whether pre-vaccination screening is necessary. Materials & Methods: Serum samples from 254 children aged between 1-15 years were tested for antibody (IgM & IgG) against hepatitis A virus (HAV) to determine the seroprevalence of HAV antibody and do a cost-benefit analysis for decision making about vaccination against HAV among the children of Bangladesh. Results: Hepatitis A virus antibody was positive in 141 (55.5%) of 254 children. Age-specific sero-prevalence was 13 (23.2%) of 56 in 1-3 year,64 (55.2%) of 116 in 3-5 year, 39 (70.9%) of 55 in 5-10 year & 25 (92.6%) of 27 in 10-15 year age group. Cost benefit analysis showed that the total cost of screening followed by vaccination was almost 1.8 times less than the total cost of vaccination of all children without screening. Conclusions: Majority of the children were found sero-positive against HAV around 15 year of age. Therefore mass vaccination against HAV may not be required for Bangladeshi children.
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Background: Viral hepatitis is one of the major causes of mortality & morbidity in developing countries. Present study was aimed to know the seroprevalence and co infection of the acute viral hepatitis caused by hepatitis A, hepatitis B, hepatitis C and hepatitis E virus from patient attending one of the largest tertiary care hospitals. Material and Methods: Study was carried out from May 2009 to June 2010 at our hospital. A total of 556 serum samples were tested for HBsAg, antibody for HCV, IgM antibody of HAV and HEV by ELISA method from the patients having clinical signs & symptoms of acute viral hepatitis. Statistical analysis: Chi square test was done and the association was considered to be statistically significant if p < 0.05. Results: Out of 556 suspected cases of acute hepatitis 281 were positive. Among the total positive cases, IgM anti HAV antibodies was 70 (24.91%), IgM anti HEV antibodies was 141 (50.18 %), HBsAg was 65 (23.13%) and HCV was 5 (1.78 %). Dual infection was seen in 14 (4.98 %) cases with 6 children cases. Among the HAV positive patients, 80% were children, In contrast to that HEV infection (63.1%) and HBsAg infection (61.5%) was common in young adult. HCV infection was found common in adults more than 20 years of age. Overall male was affected more than female. Conclusion: Sporadic HEV infection constitutes significant cause of the acute viral hepatitis. In the light of this result a nationwide survey is recommended to confirm this pattern in the other areas. As vaccine for HEV is not available, prevention in form of improvement of socio - economic and hygienic standards of the population is the better option.
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The effect of DMSO and sodium butyrate on the production of recombinant hepatitis A virus (HAV) capsid protein VP1 was evaluated and optimized in the culture of stably transfected Drosophila melanogaster S2 cells using culture plates and spinner flasks. The effect of DMSO and sodium butyrate was also evaluated to improve the recombinant VP1 production in stably transfected Drosophila S2 cells. A production level of 0.88 mg of recombinant VP1/liter was obtained in the culture-plate culture of stably transfected S2 cells at 6 days after induction with 0.5 mM CuSO4. The supplements of 2% DMSO and 10 mM sodium butyrate at 4 days post-inoculation increased recombinant VP1 accumulation by 141 and 104%, respectively, resulting in 2.17 and 1.7 mg/liter of recombinant VP1 production. In spinner flasks, recombinant VP1 production reached maximum level at 9 days after induction with 0.5 mM CuSO4, with approximately 4.96 mg/liter of recombinant VP1 production level. When 2% DMSO or 10 mM sodium butyrate was added at 5 days post-inoculation, the recombinant VP1 production was increased to 8.35 and 5.85 mg/liter, respectively. However, the synergistic effects of DMSO and sodium butyrate were not observed. These results indicate that DMSO and/or sodium butyrate can be successfully used to improve the recombinant HAV VP1 production in culture plates and spinner flasks.
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Butyrates , Capsid Proteins , Dimethyl Sulfoxide , Drosophila , Drosophila melanogaster , Efficiency , Hepatitis , Hepatitis A , Hepatitis A virus , SodiumABSTRACT
Fulminant hepatic failure (FHF) is characterized by massive hepatocellular injury, whose physiopathology is still unclear. Hepatitis B (HBV) is probably the most common viral cause of FHF, while hepatitis A (HAV) virus seem occurs less frequently. However, the host and viral factors that determine the outcome of these infections are poorly understood. In the present study, viral load and genotyping determining regions of HAV and HBV genomes were sequenced. Eight FHF patients and one patient with severe acute hepatitis (SAH) were included. Liver and blood samples were collected during liver transplantation or necropsy procedures. HAV-RNA and HBV-DNA were extracted from serum, biopsy and paraffin liver. Nucleotide sequencing of HAV-RNA was performed from VP1/2A and HBV-DNA from PreS/S region. The amplified samples were quantified by Real-Time PCR. The cases of HAV infection were due to subgenotype IA. The cases of HBV infection were due to genotype A2 and D4. The case of HAV/HBV coinfection was infected by genotype IA and D3. Hepatitis A and B infection were associated with genotypes most prevalent in Brazil. In hepatitis A infection the mean of period evolution was 13 days. In hepatitis B, FHF patients infected by genotype D have a shorter period of evolution than FHF patients infected by genotype A (mean 15 v. 53 days). There was no association with genotype-determining region with the severity of hepatitis, however nucleotide differences and high viral load could be observed among FHF.
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Adult , Aged , Child , Child, Preschool , Female , Humans , Male , Middle Aged , Hepatitis A Virus, Human/genetics , Hepatitis A/virology , Hepatitis B virus/genetics , Hepatitis B/virology , Liver Failure, Acute/virology , Acute Disease , Base Sequence , Brazil , DNA, Viral/analysis , Genotype , Hepatitis A Virus, Human/immunology , Hepatitis A/complications , Hepatitis B virus/immunology , Hepatitis B/complications , Molecular Sequence Data , Mutation , Phylogeny , Polymerase Chain Reaction , RNA, Viral/analysis , Viral LoadABSTRACT
OBJECTIVE: This study was aimed to characterize the clinical features and course of acute hepatitis A in Korean adults. METHODS: One-hundred and thirteen cases of acute hepatitis A, diagnosed between Jan. 1995 to July 1998 at 6 medical centers in Korea, were reviewed retrospectively. The clinical course of 94 cases with follow-up duration longer than 3 months were analyzed. RESULTS: The median age was 26 (16-65) years and 97.3% of the patients were under 40 years. The presumed sources of infection were identifed in 62 cases (54.9%). Among those, the leading source was ingestion of raw food. All patients showed normalization of bilirubin level within 8 weeks. The ALT levels normalized within 8 weeks in all patients except three patients (3.2%). Three patients with prolonged elevation of ALT showed second rise of ALT, suggesting a possibility of relapsing hepatitis. Prolonged fever (>38 degree C) more than 10 days was observed in 3 patients (3.2%). One case showed prolonged elevation of alkaline phosphatase (> x3 upper normal limit). No case of fulminant hepatic failure or death was observed. CONCLUSION: The majority of cases with acute hepatitis A in Korean adults showed self-limited course with full recovery.