A Preliminary Study On Human Adenovirus Type 3 And 7 Inactivated Vaccines

[Objective]To obtain a cell substrate of human adenovirus type 3 and 7 for vaccine production,and to isolate,culture and perform plaque purification for human adenovirus type 3 and 7 vaccine candidate strains.[Methods]1.Preliminary screening of cell substrates:The existing human adenovirus types 3and 7 were infected with five cell lines,HEK293,AD293,Vero,MRC-5,and WI-38,and CPE changes and viral infection titers were observed.A susceptible cell line was screened out.Recombinant adenovirus 3?Ad3EGFP?carrying the GFP?Green Fluorescent Protein?gene was cultured by AD293 and MRC-5 cells,respectively.A549cells were infected by recombinant adenovirus 3 which was purified by CsCl gradient centrifugation.Thus,a cell substrate for human adenoviruses type 3 and type 7 vaccine production was selected.2.Human adenovirus type 3 and 7 virus strains isolation and immunization:153samples of throat swabs from Guangzhou and Beijing were isolated and cultured in MRC-5 cells.Hexon gene,fiber gene and penton base gene of adenovirus were amplified by PCR and were identified by sequencing.Viral strains were carried through five passages in MRC-5 cells and determined viral infection titer.Adenovirus strains with an infection titer?4.0 lg CCID₅₀/mL were selected to subsequent assay.These virus strains were cultured by MRC-5 cells and repeatedly frozen and thawed three times,and the supernatant was removed by centrifugation.The virus supernatant was inactivated by adding?-propiolactone at a ratio of 1:4000 and allowed to stand at 4°C for 24 hours and37°C for 2 hours.The mice were injected intraperitoneally with an immunization dose of500 ul/mouse with adenovirus strains inactivated.Control mice were injected with phosphate-buffered saline?PBS?.21 and 44 days after the finalimmunization,sera were collected,heat-inactivated and kept frozen for neutralization tests.3.Plaque purification:Adenovirus strains which neutralizing antibodies titer were 1:8above were plaque purified by Vero,A549 and MRC-5 cells,respectively.The types of adenovirus strains were turn out to be true by PCR amplification and sequencing of the three major protein genes,and serum identification tests.The adenovirus strains of infection titer was measured using three cell lines,A549,HEK293,and MRC-5 cells,respectively.4.Adenovirus culture conditions were initially established:human adenovirus type 3BJ-50 strain and human adenovirus type 7 BJ-70274 strain were used for virus inoculation of 0.006 MOI,0.012 MOI,and 0.018 MOI,and the medium contained serum concentration 2%,5%and 10%,respectively,were cultured at 35°C and 37°C in a 5%CO2 incubator,and observed the cells of CPE and virus infected titer at 12,24,36,48,60,72,84,96,and 108 h post-infection in MRC-5 cells.[Results]1.Among the five cell lines,human adenovirus type 3 was most sensitive in HEK293 cells,followed by MRC-5 and AD293 cells,and insensitive cells were Vero and WI-38 cells.Human adenovirus type 7 was most sensitive in HEK293 and MRC-5 cells,followed by AD293 cells,insensitive cells were Vero and WI-38 cells.Under the same culture conditions,recombinant adenovirus type 3 Ad3EGFP was cultured to obtain total virus particles of 3.0×10¹¹,2.04×10¹² by MRC-5 and AD293 cells,respectively.However,Ad3EGFP virus obtained from MRC-5 cells was observed green fluorescence with 1×10⁷virus particles inoculation at 48 h post-infection A549 cells,while nothing was observed in AD293 cells.2.31 viral strains were isolated from 153 throat swabs,of which 21 strains were human adenovirus type 3 and 10 strains were human adenovirus type 7.Eleven strains of virus with a titer of 4.0 lg CCID₅₀/ml above were selected,of which 6 strains were human adenoviruses type 3 and 5 strains were human adenoviruses type 7.Eleven strains of adenoviruses were inactivated by?-propiolactone and tested for immunogenicity.The initial neutralizing titers of 11 strains were 1:8 above,and the rate of immunopositive reactions were 100%.After the initial immunization,there were no significant differences in the neutralizing antibody titers induced by strains of adenovirus type 3strains BJ-35,BJ-50,BJ-57,BJ-70302,and BJ-70248.The neutralizing antibody titer of BJ-70327 strain was lower than that of the other 4 strains?BJ-35,BJ-50,BJ-57,BJ-70302?.There was no significant difference in the neutralizing antibody titer induced by the adenovirus type 7 strains.There was no significant difference in neutralizing antibody titer induced by different 3 and 7 adenovirus strains after the second immunization,but the neutralizing antibody titer induced by the secondary immunization was higher than that of the primary immunization.3.Try to use Vero,A549,MRC-5 cells for plaque purification of 11 strains adenovirus strains,only Vero cells successfully plaque purified.The PCR amplification and sequencing of the three major protein genes of the purified virus strains and serum identification experiments together confirmed that the adenovirus types were correct.The A549,HEK293,and MRC-5 cell lines were used to measure the titer of infection.The virus infected titers of 11 strains adenoviruses in MRC-5 cells and HEK293 cells were the same.Of which 6 strains were human adenovirus type 3,3 strains were human adenovirus type 7.The virus infected titers of 9 adenovirus strains were 5.0 lg CCID₅₀/ml,but the other two human adenovirus type 7 strains was lg 4.0 lg CCID₅₀/ml.The virus infected titers in A549 cells were 1 lg higher than those in the other two cell lines,MRC-5 and HEK293 cells.5.When the human adenovirus type 3 BJ-50 strain and human adenovirus type 7BJ-70274 strain were cultured in MEM medium containing 10%serum,the inoculated virus amounts were 0.006,0.012,and 0.018 MOI,respectively,exhibiting quickly CPE.Viruses were harvested at 48⁶0 h post-infection,but no significant difference in virus infected titers.When cultured in medium containing 2%and 5%serum,the virus inoculation amount was 0.006,0.012,and 0.018 MOI respectively,and there was no significant difference in virus infected titer.But the cells overlaid with medium containing 5%FBS exhibiting faster CPE than incubated with 2%FBS,and virus could be collected 24 h in advance.Both the 35°C and 37°C incubations with 5%CO2incubator did not affect the titer of the virus,but 37°C under 5%CO2 incubator showed faster CPE and the virus were harvested 24⁴8 hours earlier.Human adenovirus type 3BJ-50 strain and human adenovirus type 7 BJ-70274 strain were inoculated into MRC-5cells with a virus dose of 0.012 MOI,using a medium containing 5%serum,at 37°C,5%CO2 incubator The virus began to proliferate after 12 h of culture,and the virus rapidly proliferated within 12³6 h.All the cells were observed CPE at 72 h post-infection.The virus infected titer remained unchanged at 48¹08 h at 5.2 lg CCID₅₀/ml.[Conclusion]1.MRC-5 is a suitability cell substrate for adenovirus types 3 and type 7 vaccine production from five cell lines,HEK293,AD293,Vero,MRC-5,WI-38 cells.2.Six strains human adenovirus type 3 vaccine candidate strains and five strains human adenovirus type 7 vaccine strains were isolated and purified from clinically adenovirus-infected samples from 2015 to 2017.3.Determine the suitable conditions for vaccination candidate human adenovirus type 3 BJ-50 strain and human adenovirus type 7 BJ-70274 strain for inoculation:use0.012 MOI virus to inoculate MRC-5 cells overlaid with MEM medium containing 5%serum and incubated at 37°C under 5%CO2 incubator for 48-72 h.4.This study may contribute to the development of human adenovirus type 3 and type 7 inactivated vaccines.