Construction And Immunological Evaluation Of HCV Vaccine Adnsmut Based On Adenoviral Vector

Objective:Hepatitis C virus(HCV)is an important global pathogen which can induce infectious disease.Chronic infection of HCV always causes hepatitis,liver fibrosis,liver cirrhosis and even liver cancer.The traditional treatment for HCV infection was combination of ribavirin and interferon,with SVR is only 50%-60%,associated with some toxic side effects.Currently,Direct-acting antivirus drugs(DAAs)were available and more effective than the traditional therapy.In some areas the SVR could be more than 90%with DAAs.However,there are also some disadvantages and shortcomings:All of the drugs were disable to reverse the progression of liver disease in the late-stage;the DAAs were so expensive that people in the developing countries and poor area were unable to afford them;Also DAAs drugs showed less effect for some HCV serotypes,and virus could mutant to resist the drugs;Most importantly,for the absence of screening system of diseases in the developing countries,a large proportion of individuals infected with HCV were unaware of their sickness and didn't ask for treatment,thus transmitting the HCV potentially and constantly.In addition,in October 2016,FDA warned that patients had been treated with DAAs drugs,would be more likely to get infection with HBV again,thus leading to severe liver injury or even death,once the patients have exposed in HBV before.In conclusion,developing the HCV vaccine was essential for preventing the virus from spreading.The main obstacle to develop a promising HCV vaccine was the high viral variability.HCV strains were classified into 7 genotypes(numbered 1-7),the nucleotides of which differed from 31 to 34%.However,modeling studies estimated that even a vaccine based on one genotype had substantial impact on HCV prevalence and incidence,especially in highrisk populations,in some certain area.So far,there were several kinds of HCV vaccines,such as recombinant protein vaccine,peptide vaccine,DNA vaccine,viral vector vaccine,DC cells vaccine,HCVcc and so on.Among those,the adenoviral vector vaccine was known to be safe,effective,with long duration.It always induces robust T cell response.Studies had shown that the most effective immune response to HCV infection was the cellular immune response.Therefore,we choose the antigen capable of eliciting significant cellular immune response,HCV lb NS3-5Bmut,to construct a HCV candidate vaccine AdNSmut based on adenoviral vector system,AdMAX and evaluate the immunogen on mices.Method:HCV NS3-NS4A and NS4B-NS5B fragments were amplified by PCR from the gene of HCV,serotype 1b,acquried from a HCV infected donor sample Z14.And NS5B RNA polymerase was inactivated by site-directed mutagenesis.The NS3-NS5Bmut fragment was then obtained by ligating NS3-4A with NS4B-NS5Bmut using overlapping extension PCR.NS3-5Bmut fregment was inserted into the adenoviral shuttle plasmid pDC315 by In-Fusion clone,and the plasmid was confirmed by sequencing.Transfecting the plasmid pDC315-NSmut into 293A cell,the expression of NS3 and NS5B protein was identified by immunofluorescence and western-blot.After that,plasmid pDC315-NSmut and adenoviral bone plasmid pBHGlox(delta)E1,3Cre were co-transfected into 293A cell to produce the replication-defective recombinant adenovirus,AdNSmut.In order to make sure of the gene integrity of the AdNSmut,NS3-5Bmut DNA was amplified from AdNSmut by PCR.NS3-5Bmut RNA was detected in the 293A cell infected with AdNSmut by RT-PCR.The expression of NS3 and NS5B protein was detected by western blot and mass spectrometry.After propagation and purification by CsCl gradient density centrifugation,a large number of adenoviruses were collected,the titer was measured by TCID50 assay.To do the animal assays,C57BL/6 mice were vaccinated intramuscular with different doses of AdNSmut,from 10gPFU to 1011PFU.3 weeks after the prime,the mices were boosted with the same dose.2 weeks after boost,all of the mices were sacrificed and the spleen lymphocytes were separated for Elispot and inner cell staining to detect cellular immune response,blood was collected to detect humoral immune response by ELISA.Results:The HCV lb NS3-5Bmut sequence was amplified by PCR and the pDC315-NSmut shuttle plasmid was successfully constructed.The expression of NS3 NS5B protein by 293A cell transfected with pDC315-NSmut was identified by immunofluorescence and western-blot.Recombinant shuttle plasmid and backbone plasmid were co-transfected into 293A cells,which produced infectious replication-deficient recombinant adenovirus AdNSmut expressing foreign gene appropriately.After proliferation and purification,we collected a large number of high purity adenovirus,the titer of which was 2.74 × 1013PFU/ml.C57BL/6 mices were immunized according to regimen,and a significant cellular immune response was detected afterpriming and boosting.However,no specific antibody response can be detected.Conclusion:A replication-deficient recombinant adenovirus AdNSmut expressing HCV lb non-structural protein NS3-5Bmut was successfully constructed as an HCV vaccine candidate in this study.The high purity adenovirus can be achieved rapidly and plentifully,which can induce significant cellular immune response in C57BL/6 mice,without obvious toxic or side effects.It was safe and effective.The immunogen was promising to be a great potential one in area with high HCV 1b infection incidence.