Serological Epidemiological Study And Immunological Evaluation Of Candidate Vaccine For Human Adenovirus Type 7 Vaccine

Human adenoviruses?HAdVs?infection can cause a variety of clinical syndromes,including respiratory diseases,conjunctivitis,cystitis,gastroenteritis,and nervous system diseases.More than 90 types of adenovirus serotypes have been discovered,and the main infected populations include infants and young children,immunocompromised patients,and training camp recruits.However,there is no effective therapeutic drugs to control adenovirus infection,the live,oral adenovirus type 4 and type 7 vaccine jointly developed by the United States Department of Defense?USDOD?and Teva Pharmaceutical Industries Limited?TVEA?have shown remarkable results in preventing the outbreak of adenovirus in recruits.However,there is no related vaccine listed or a special vaccine for military immunization in China.In recent years,some outbreaks of adenovirus infection emerged in some regions and military camps in China,HAdV7 infection is more popular,and the damage is the most serious.Developing HAdV-7 vaccine is an urgent need.Neutralizing antibody?nAb?is an importantindex in the epidemiological study of HAdV-7,and is also a key indicator for evaluating the immune response of HAdV-7vaccine.The traditional detection method for HAdV-7 nAb is based on the cytopathic effect?CPE?,which is time-consuming?4⁸ days?,lowly sensitive and unstable^[1].Here a novel quantitive HAdV-7 nAb assay was established based on the recombinant HAdV-7 expressing luciferase.Briefly,the pAd7-Luc recombinant plasmid was constructed with E1 and E3 region partially deleted by homologous recombination,and the expression cassette with firefly luciferase was placed in E1 deleted region.The linearized pAd7-Luc plasmids were transfected into HEK293 cells and the recombinant Ad7-Luc expressing luciferase was rescued.The assay for HAdV-7 nAb by luciferase detection was established based on Ad7-Luc recombinant virus,and the assay validation was carried out.The results showed that the recombinant Ad7-Luc virus expressing luciferase was prepared,and virus titer was up to 4.85×10⁸ IFU/mL.There was a good linear correlation in HAdV-7 nAb titers measured by luciferase detection and the traditional CPE assay?R=0.8612,P<0.0001?.The intra-and inter-batch variation were within 10%and 20%.Compared with the traditional CPE method,the HAdV-7nAb assay by luciferase detection is more rapid?one day?,sensitive and accurate,and is suitable for the HAdV-7 serum epidemiological investigation and the nAb detection of HAdV-7 vaccine.The novel assay for HAd7 nAb was applied on serum investigation for 375samples from Beijing and Taizhou,Jiangsu Province.The results showed that the seroprevalence of HAdV-7 in Beijing and Taizhou was 56.47%?144/255?and 57.50%?69/120?,respectively,the low titer level?12²00?positive serum accounted for70.14%?101/144?and 85.51%?59/69?of the total positive serum samples.Statistical analysis of the HAdV-4 and HAdV-5 data in this lab showed that the seroprevalence of HAdV-7 in both places was significantly lower than that of HAdV-5?p<0.001?,but no significant difference from HAdV-4?p>0.05?.At the same time,we found that there was no gender difference in the seroprevalence rate of HAdV-7,and it gradually increased with age in two regions.Next,the immune responses for different HAdV-7 live vaccines were evaluated on several animal models.Firstly,the live HAdV-7 was administrated in BALB/c mice,C57 BL/6 mice and Mesocricetus auratus by intramuscular injection?IM?and intraperitoneal injection?IP?.The results showed that:both of deliveries by IM and IP could produce high levels of anti-HAdV-7 nAb,peaking at 4 weeks post immunization,then gradually decreased,but still maintained a certain level for a long time.Secondly,the dose-dependent relationship and cellular immune response by IM was evaluated in BALB/c mice.The results showed that low dose of HAdV-7?1×105 IFU/mouse?could produce nAb in 50%of mice,and the titer of nAb increased with the increasing dose.The specific CD4⁺T cell response was up-regulated,and no specific CD8⁺T cell response was detected in BALB/c mice.Finally,the live HAdV-7 vaccine's immune responses by different delivery were evaluated on Rhesus monkeys.Humoral and cellular immune responses were investigated after Rhesus monkeys were immunized by IM and oral delivery.The results showed that nAb could be rapidly produced by IM,peaking at day 14,then decreased significantly but still maintained a certain level for a long time,while nAb was not detected in the oral administration group.The specific CD8⁺T cell responses?IFN-?,TNF-?,IL-2 and CD107a?and CD4⁺T cell responses?IFN-?,IL-2 and CD154?on day7 post immunization by IM were significantly up-regulated,followed by a significant decrease at day 14.However,no specific cellular immune response was detected in the oral immunization group.We speculate that the live HAdV-7 vaccine did not reach the intestine of the monkeys and did not infected the mucosal epithelial cells,no immune responses were further stimulated with live HAd7 infection.In summary,the novel HAdV-7 nAbassay based on luciferase detection is rapid,sensitive and accurate,and is suitable for HAdV-7 sero-epidemiological investigation and nAb assay of the related vaccines.The immunological evaluation of HAdV-7vaccine found that intramuscular immunization can rapidly stimulate humoral i and cellular immune response to HAdV-7.However,oral live vaccines still need to be further investigated.