Evaluation Of Immune Response Of Rhesus Macaque To Novel HCV Vaccines Based On Three Viral Vectors

Hepatitis C virus (HCV) can create acute or chronic hepatitis, and evenhepatocellular carcinoma (HCC) after being transmitted through blood, vaccine aretherefore critical for the prevention and treatment of HCV. However, there is currentlyno safe and effective anti-HCV vaccine, though part of HCV infected patients canclear the virus themselves without treatment, developing HCV vaccine is still full ofhopes and challenges. Previous researches indicated that the strength of humoralimmune response, the ability of antibody neutralization as well as the level of specificcellular immune response are key factors for successful immunization, and virusvector vaccine just have advantages in those respects.The immunity system of rhesus macaque is close to human being and easier tobe obtained compared to chimpanzee. Based on the preliminary studies, we testedthree novel vaccines in rhesus based on three viral vectors, which including the HCVvaccine derived from replication-defective adenovirus vector of serotype5(rAd-HCV), pseudotyped virus particles based on the integration-defective lentivirus(HCVpp), and HCV vaccine derived from replication-defective vaccinia vector(Tiantan strain)(rNTV). Vaccines containing the HCV1b envelope protein E1E2geneas well as non-structural proteins NS3gene of HCV were constructed by the threevectors. Rhesus monkeys were divided into four groups of four and immunized withthe following four different immune combinations separately: rAd-HCV+HCVpp+rNTV-HCV, HCVpp+rNTV-HCV, HCVpp+rAd-HCV and rNTV-HCV+rAd-HCV.Four monkeys were immunized with DNA and rNTV vector containing HBV CS1and SS1antigen genes as control group. With a long-term (76weeks) observing and sampling, the immune effect of thosevaccines were analyzed and assessed. Our researches focused on detection ofHCV-specific humoral and cellular immune responses induced by vaccines, the HCVneutralization ability of serum as well as the changes of cytokines associated with avariety of immune responses, thus to provide theory basis for the selection of vaccinefor clinical use and immunization programs.Our main results of our study are as follows:1) Primary immune with rAd-HCV could induce HCV E1specific IgGantibodies in rhesus monkeys at titer range of102.3-3.5, but titer of antibody to E2andNS3are relatively lower. Obvious neutralizing capacity was detected in the obtainedserum, which could neutralize36.6%of1a and48.7%of1b subtype HCVpseudovirus. Weak antigen-specific cellular immune response can be detected, weakspecific reaction to peptide pool of E2and NS3was detected in only one sample.2) As primary antigen, rNTV-HCV could induce obvious humoral and cellularimmune responses in rhesus monkey. After single injection, antibody titers ofvaccinated monkeys rose to E1103.35±0.39and E2103.01±0.35, better than HCVpp.Moreover, their neutralizing capacity was also superior to HCVpp in strength (1b,56.7%) and cross (1a,1b). Moreover, rNTV-HCV primarily induced strong cellularimmune responses (median597;311-1210SFC/Million PBMC) in rhesus monkeys.It can be concluded that the rNTV-HCV has strong dual inducibilities inantigen-specific cellular and humoral immunity.3) HCVpp could induce obvious humoral immune response in rhesus monkeys.After two-pin injection, serum HCV-specific antibody titers of the HCVpp primaryimmune group were enhanced to the level of E1102.34-2.55E2102.73-2.81, and NS3102.24-2.37, and the serums had nice HCV neutralizing capacity (1b43.1%), but had noobvious positive results in the detection of cell-mediated immunity (except forabnormal reaction). Immunogenicity of HCVpp is weaker than rNTV-HCV bycomparing. 4) Effect comparison of the four immune combinations：Only one group primarily immunized by rAd-HCV was boosted with HCVpp(rAd-HCV+HCVpp), the E1antibody titer increased to103.8±0.12after the first boostinjection, but no obvious enhancement was detected by the second injection withHCVpp. Neutralization ability enhanced10%to1b by HCVpp boost. Cellularimmune response increased significantly after first boost with HCVpp, whereas notfurther strengthened in the second needle.However, the rNTV-HCV boosting to HCVpp primary immune group andrAd+HCVpp preimmune group (HCVpp+rNTV-HCV and rAd+HCVpp+rNTV-HCV)had not obviously induced enhancement in antibody titers and neutralization. In thecellular immune response, the spot of rAd+HCVpp preimmune group enhancedobviously (median324;10-1518SFC/Million PBMC) by rNTV-HCV boost.When the rAd-HCV was used as boost antigen, in groups primarily immunizedwith HCVpp and rNTV (HCVpp+rAd-HCV and rNTV-HCV+rAd-HCV), antibodytiters of HCV E1could be enhanced up to103.05±0.15and103.88±0.18and E2to102.77±0.35and103.48±0.22. It also enhanced the ability of neutralization, which can effectivelyblock three type of HCV pseudovirus from1a,1b and5a. Nevertheless, boosting withrAd-HCV could induce weak T cell response in the HCVpp primary immune group,with a low background. Monkeys primarily immunized with rNTV-HCV could beeffectively enhanced by rAd-HCV because its dot number reached to the highestvalue (median1545; range:818-2210SFC/Million PBMC). In addition, Th1andTh2cytokines, especially for Th1, both can be enhanced by this immune strategy.In conclusion, for the rhesus, HCVpp and rAd-HCV vaccines have significantimmune response inducibility in humoral but weak in cellular, only rNTV-HCVvaccine shows dual induction in cellular and humoral immunity. The HCV vaccine candidate immune program of rNTV-HCV+rAd-HCV is excellent compared to otherstrategies and suitable for further research and development.