Study On A Recombinant Trivalent Vaccine Candidate Against Human Adenovirus Types 3,7,and 55 And Fiber Knob Chimeric Human Adenovirus Types 3

Objective Human adenoviruses(HAdV)3,7 and 55 can cause acute respiratory disease epidemics and outbreaks.1.We constructed a recombinant trivalent vaccine candidate against human adenovirus types 3,7,and 55 to development of a new adenovirus vaccine candidate.2.Identification of neutralizing antigens is vital for surveillance and vaccine development.We generated the recombinant capsid-chimeric human adenoviruses rAd3E-Fk7,containing the Ad3 E backbone and the HAdV-7 fiber knob,and rAd3E-H7Fk7,which contain an Ad3 E backbone but recombinant HAdV-7 hexon and fiber knob to identification of major neutralizing antigens.3.We generated HAdV-5 fiber knob capsid-chimeric recombinant human adenoviruses base on HAdV-3 vector to study its infectivity.Methods 1.After restriction digestion of p SKE3LR-h55 and p Ad MHE3,the h55 fragment of p SKE3LR-h55 was cloned into p Ad MHE3,and the trivalent virus vector p Ad MHE3-h55 was generated by homologous recombination.When we used p Ad MHE3-h55 to transfect AD293 cells,the cells developed a typical CPE of adenovirus infection.Furthermore,the transfected cell suspension could infect the fresh cultures of AD293 cells.These results indicated that the trivalent virus rAd MHE3-h55 was successfully rescued and possessed infectivity.The growth curve of the recombinant adenovirus was measured to analyze its growth characteristics,and the in vitro neutralization test of the recombinant virus.The mice were immunized with rAd MHE3-h55 and challenged with HAdV-3,7,55 drops of the nose respectively.The lung tissues were subjected to Q-PCR and HE staining to detect the protective effect of recombinant adenovirus on mice.2.We generated the recombinant capsid-chimeric human adenoviruses rAd3E-Fk7,containing the Ad3 E backbone and the HAdV-7 fiber knob,and rAd3E-H7Fk7,which contain an Ad3 E backbone but recombinant HAdV-7 hexon and fiber knob.In vitro neutralization tests with these chimeric adenoviruses using both mouse and human antisera.3.We generated the recombinant capsid-chimeric human adenoviruses rAd3E-Fk5,containing the Ad3 E backbone and the HAdV-5 fiber knob.Recombinant human adenovirus rAd3E-Fk5 infects both mouse and gold hamster primary renal epithelial cells.And study the expression the late gene in gold hamster primary renal epithelial cells.Results 1.We constructed a new recombinant trivalent human adenovirus vaccine(rAd MHE3-h55),which expresses the hexon protein of HAdV-55 in the E3 region of rAd MHE3,a previously prepared bivalent vaccine candidate against HAdV-3 and HAdV-7.The results of in vitro neutralization assays indicate that rAd MHE3-h55 can induce the production of neutralizing antibodies against HAdV-3,HAdV-7,and HAdV-55 in mice.Furthermore,immunization with the recombinant trivalent vaccine candidate completely protected the mice challenged with HAdV-3,HAdV-7,or HAdV-55,respectively,showing lower lung viral loads and less lung Pathological changes was compared with those in unvaccinated mice.The current findings contribute to the development of a new adenovirus vaccine candidate and also advance this construction method for the generation of recombinant adenovirus vaccines.In conclusion,our recombinant trivalent vaccine rAd MHE3-h55 can provides protection against challenge with HAdV-3,HAdV-7,or HAdV-55 in mice.2.We generated the recombinant capsid-chimeric human adenoviruses rAd3E-Fk7 and rAd3E-H7Fk7.In vitro neutralization tests with these chimeric adenoviruses using both mouse and human antisera.indicated that hexon and fiber knob are the major targets recognized by neutralizing antibodies against HAdV-3 or HAdV-7,and other capsid proteins including the penton base and fiber shaft may not contribute to neutralizing antibody responses.In conclusion,both hexon and fiber knob structures in HAdV-3 and HAdV-7 may be the proteins which induce neutralizing antibody responses.3.We constructed a recombinant human adenovirus ciliary rAd3E-Fk5.As a result,it was found that the rAd3E-Fk5 virus significantly proliferated in primary hamster cells.The rAd3 E virus did not significantly proliferate in primary hamster cells.The human type 3 adenovirus rAd3 E had low ability to infect mouse primary cells,while the rAd3E-Fk5 had high infectivity,similar to rAd5 E.The rAd3E-Fk5 virus gives late gene expression in primary hamster cells.Conclusions 1.In vivo and in vitro experiments show that recombinant adenovirus rAd MHE3-h55 can induce immune responses in mice and produce antibodies against HAdV-3,HAdV-7 and HAdV-55.It’s important for the development of recombinant adenovirus vaccines.2.Both hexon and the fiber knob contain the major neutralizing antigens.It may be important for adenovirus vaccine and drug development.3.Fiber knob protein chimeric recombinant adenovirus rAd3E-Fk5 could infect mouse primary epithelial cells,primary hamster lung and primary kidney cells,its infection efficiency is similar to HAdV-5,the infection efficiency is much higher compared to rAd3 E.The rAd3E-Fk5 has a significant replication in the gold hamster cells.It can be used for animal model of human type 3 adenovirus vaccine evaluation,antiviral drug evaluation,pathogenic mechanism.