Adaptability and genetic stability of hepatitis A virus SYX_1 strain in human diploid cell MRC-5 / 中国生物制品学杂志

@#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.

Total serum IgE in people immunized with human diploid cell rabies vaccines with different production processes / 中华微生物学和免疫学杂志

Objective:To investigate the effects of different production processes on sensitization responses to human diploid cell rabies vaccines.Methods:This study randomly collected 360 serum samples in clinical trials of four rabies vaccines with different production processes. Total IgE levels at different time points were detected by ELISA. SPSS19.0 software was used for statistical analysis.Results:Total IgE test results showed that the seropositive rate was 20% (6/30) for all four vaccines. The lowest mean value of total IgE was 9 IU/ml and the highest was 210 IU/ml. Repeated measures analysis of variance showed that there was no significant difference in total IgE levels at different sampling time points ( P=0.284), and the total IgE level in people injected with multistep concentrated human diploid cell rabies vaccine was significantly below that in people immunized with Vero cell rabies vaccine ( P=0.024). Conclusions:Increasing the immune dosage of human rabies vaccine could not result in a rise in total IgE. Human diploid cell rabies vaccines had good safety as the production process could remove most of allergenic impurities.

Acute motor axonal neuropathy following anti-rabies human diploid cell vaccine: A rare case and review

Rationale: Guillain Barre syndrome (GBS) is an acute neurological illness leading to quadriparesis with respiratory involvement. It can be triggered by infections, vaccinations, surgery, trauma, transplantation and drugs. Anti-rabies cell culture vaccines introduced to overcome the high rate of neurological complications associated with tissue based rabies vaccine, can be very rarely associated with GBS. Patient concerns: A 50-year-old female presented with acute severe upper back pain evolving into pure motor quadriparesis following administration of human diploid cell vaccine for rabies. Diagnosis: Acute motor axonal neuropathy variant of GBS following anti-rabies human diploid cell vaccine. Interventions: Intravenous high dose steroids. Outcomes: Patient recovered completely within 1 month. Lessons: Although anti-rabies cell culture vaccines are highly immunogenic and safe, they are rarely associated with GBS. Clinicians should be aware of this link because prompt diagnosis and treatment can result in complete recovery and avoid complications.

Investigation on the effects of booster immunization of human diploid cell rabies vaccine after eight years of primary vaccination / 中华实验和临床病毒学杂志

Objective@#To disclose the effects of booster immunization of human diploid cell rabies vaccine (HDCV) after eight years of primary vaccination.@*Methods@#Sixty subjects who had participated the phase Ⅲ clinical trial of freeze-dried HDCV were selected and gaven booster immunization after eight years of primary vaccination. The side effects of booster immunization were observed. The serum before and after 14 days of booster immunization were collected and detected the rabies virus neutralizing antibody (RVNA) by rapid fluorescent focus inhibition test (RFFIT). The positive rate and geometric mean titer (GMT) of RVNA before and after booster immunization were made statistical analysis.@*Results@#Total 54 subjects finished the follow-up and RVNA detection. No sever side-effects were observed in 30 min or 15 days of follow-up after booster immunization. The positive rate of RVNA before and after booster immunization were 51.85% (28/54) and 96.30% (52/54). The GMT of RVNA before and after booster immunization were 1.42 IU/ml and 30.61 IU/ml.@*Conclusions@#The freeze-dried HDCV has good immune effects with one-dose of booster immunization after eight years of primary vaccination.

Dynamic monitoring of anti-poliovirus neutralizing antibody and anti-hepatitis A virus IgG antibody in healthy children immunized with inactivated enterovirus 71 vaccine (human diploid cell) / 中华微生物学和免疫学杂志

Objective To investigate the dynamic changes of three types of anti-poliovirus neutralizing antibodies and anti-hepatitis A virus (HAV) IgG antibody in children who were immunized with inactivated enterovirus 71 (EV71) vaccine (human diploid cell).Methods Serum samples were collected from the subjects immunized with inactivated EV71 vaccine.Neutralizing antibodies against EV71 and poliovirus were detected by micro-cytopathic effect neutralization test.Enzyme linked immunosorbent assay (ELISA) was used to detect IgG antibody against HAV.Results The geometric mean titers (GMTs) of anti-EV71 neutralizing antibody increased to 4.85 following the first-dose injection of inactivated EV71 vaccine.A significant increase of GMTs (up to 64.37) could be observed 28 days after the second-dose vaccination.Meanwhile, results of the dynamic monitor showed that there were slight fluctuations in the neutralizing antibodies against three types of poliovirus on day 28 (28 days after the first-dose vaccination) compared with those on day 0 (before vaccination) (P0.05).The level of anti-HAV IgG antibody was stable and no significant difference was found during the observation period (P>0.05).Conclusion This study shows that inactivated EV71 vaccine has no impact on anti-HAV IgG antibody in Children during the two-dose vaccination and in anti-EV71 antibody-producing period, but has slight influence on the anti-poliovirus antibodies.In general, changes in antibody profile do not affect the clinical efficacy of immune response.

The optimization of culture conditions of MRC -5 human diploid cell / 预防医学

Objective To optimize the culture conditions of MRC -5 human diploid cell.Methods To compare the growth status of MRC -5 cells,three kinds of culture medium with T25 bottles and Spinner cultivation system Cytodex1 micro carrier were used.Morphology,cell counting,growth curve,glucose -lactic acid value were observed and detected daily for screening a kind of suitable medium.Cell proliferation was compared with different levels of the bovine serum.Results There were no significant differences among the three kinds of culture medium.There were significant differences among MEM((43.25 ±0.60)×104 cells/mL,(12.98 ±1.27)×105 cells /mL),M199 ((35.40 ±1.41 )×104 cells/mL, (10.76 ±1.31)×105 cells /mL)and DMEM/F12 ((36.75 ±1.59)×104 cells/mL,(11.22 ±1.42)×105 cells /mL)(P<0.01).The cell proliferation of MEM cultures was 5.17 and 6.49 times better than those of M199 and DMEM/F12 cultures.Imported fetal bovine serum cell proliferation ((4.55 ±0.51)×105 cells /mL)was better than the other three bovine serum ((4.12 ±1.03,3.59 ±0.48,3.53 ±0.52)×105 cells /mL).Conclusion Tree kinds of culture medium can be used to culture MRC -5 human diploid cell.The MEMculture is better.Imported fetal bovine serum is better than other kinds of serum.

STR profiling on authentication of human cell lines for production / 中华微生物学和免疫学杂志

Objective To study the application of short tandem repeat (STR) profiling in quality control of human cell lines used for biological production. Methods The methods detecting 9 and 16 STR loci to identify human cell lines by PCR-capiilary electrophoresis were established respectively. Human cell lines, which were derived from many corporations and including diploid cell strains used for virus-vaccine production and 293 cell lines used for gene therapy products, were analyzed and compared by these two methods. Results The STR profiling methods used for authentication of human cell lines were established. Most of human diploid cell strains(20/21 ) used for virus-vaccine production from 13 corporations were iden-tiffed as the intended cells and no cross-contamination was found. However, one MRC-5 cells was identified as a false cell line and one MRC-5 had 3 alleles in D13S317 locus. For 12 strains of 293 cell lines, there were significant differences in STR profiling from different manufactures, which was likely be explained that the sources and gene modifications of these 293 cell lines are not well known and their genes are unstable during passage. Conclusion The STR profiling method has the advantages of high sensitivity and specifici-ty, and can be used for authentication of each of human cell lines for biological production.

AN OBSERVATION OF A HUMAN DIPLOID FIBROBLAST CELL LINE UNDER LONG TERM PRESERVATION IN VITRO / 解剖学报

A human diploid fibroblast cell line derived from normal abdominal skin of a 17-year-old female patient with ovary carcinoma was established and designated as HSF 79-2. The cultivation of skin fragments was employed in the primary explantation. The medium used was McCoy 5A supplemented with 15～25% calf serum. The grown fibroblasts were succesively subcultured at 1 to 2 split and stopped at the 45th to 54th passage.Having reached confluence the cells still had the ability to divide during 16 months observation if the medium was changed periodically. They displayed the property of dense growth and forming heavy, multilayer sheets, which became a visible membrane to the naked eyes. Under histological examination the membrane had the appearance of connective tissue. Part of the cells were subcultured again after 3,6, and 12 months maintenance in the same culture flask. Their growth character, ploidy, and the generation time were similar to that of the cells passaged normally.The characteristics of this cell line mentioned above appear to be shared by other diploid fibroblasts. It might be used for preserving cell lines and as a model for studying cell motility and differentiation.