Early Intervention With Antiviral Therapy And DNA Vaccine Prevents Chinese Woodchucks From The Primary And Secondary Woodchuck Hepatitis Virus (WHV) Infection

BackgroundAmerican woodchuck infected with woodchuck hepatitis virus (WHV) is an excellentmodel for studying acute and chronic hepadnaviral infections. Recently Chinesewoodchucks have been proved to be susceptible to WHV infection, and may serve asexcellent animal model for HBV study. Recently study indicates that antiviral treatmentcombination with DNA vaccination can protect newborn from persistent hepadnavialinfection. But whether antiviral treatment combination with DNA vaccination can preventadults from the primary and secondary hepadnavial infection is unknown.Objective1. To establish and improve the WHV experimental infection model in Chinese Marmots,for further study of the susceptibility to WHV of different region, and evaluation of theeffectiveness of different stranis or different doses of WHV infection on the ChineseMarmots.2. To establish the approach of T lymphocyte immune response in Chinese Marmots.3. To investigate early intervention with antiviral therapy and DNA vaccine, for evaluatingthe possibility of protecting the Chinese Marmots from primary and secondary woodchuckhepatitis virus (WHV) infection Methods1. Chinese Marmots(2-3kg) were captured from four areas in Northwest of China. ChineseMarmots were inoculated with the sera of WHV persistant infected woodchck,and studyof the susceptibility to WHV of different region, and evaluation of the effectiveness ofdifferent stranis or different doses of WHV infection on the Chinese Marmots.2. Separation peripheral blood mononuclear cells(PBMC) of Chinese Marmots, andestablish the approach of T lymphocyte immune response,including T lymphocyteproliferation by CFSE staining, CTL response by CD107a staining, establish flowcytometry to detect PD1, PDL1and FOXP3expression of Chinese Marmots.In addition,CD molecules and cytokines mRNAs related to adaptive immune response in PBMCwere detected by real-time RT-PCR.3. Twenty four adults Chinese woodchucks were inoculated intravenously with WHVstock. Then eighteen were treated with antiviral drug (ETV,0.5mg/kg/day, for8weeks)alone, DNA vaccination (pWHcIm,1mg, at week1,4,7) alone, or antiviral drug plusDNA vaccination, respectively. Six animals were untreated and served as control.Twenty weeks later, animals were rechallenged with similar WHV stock. The other twoanimals were inoculated with WHV stock and served as rechallenge control.WHsAg, WHcAb, and WHV DNA in serum were measured by ELISA and Real-timePCR. In addition, CD molecules and cytokines mRNAs related to innate and adaptiveimmune response in peripheral blood mononuclear cells(PBMC) were detected byreal-time RT-PCR.Results1. The Himalayan marmot from1,3region susceptible to WHV, showed high replication.After inoculated with WHV59strains,100%animals derived from1,3area showacute infection, After inoculated with the sera of WHV infected Chinese Marmots(stock0908and stock0925), Chinese Marmots show the comparable infectionhistory.which have higher virus titers and longer duration, but appeared the time of virus infection later than WHV59; After inoculated with medium dose of WHV59,animals show delay of viremia, while the low dose of WHV does not arouse WHVinfection of Chinese Marmots.2. Freshly isolated PBMC of the marmot, after5μg/ml the ConA stimulated5days, thepercentage of CD4+T cells were up to92.15%by CFSE staining. PBMC freshlyisolated from acute WHV infection, after WHc96-110peptide-specific stimulation for3days, show the expression of CD3+CD4-CD107a+cells were3.98%, compared toCMV unrelated peptide stimulation (0.85%) and control (0.67%) were significantlyincreased. The peak of viremia, the expression of CD4-PD-1+cells reached a peak, thepercentage of positive cells reached13.77%, and the expression of CD4+FOXP3+cellswere4.44%, However,the expression of PD-L1in acute WHV infection was low, in thelate viremia, the expression of PD-L1of CD4+and CD4-T cells were transientlyincreased, the percentages were2.17%and6.05%respectively,and reduced to normallevels after viral clearance.3. After the primary infection, all animals treated with pWHcIm alone developed typicalviremia and anti-WHc antibody similar to those of untreated control, and all animalsexcept two treated with ETV alone or ETV plus pWHcIm only developed anti-WHcantibody, the two animals treated with ETV alone didn’t show any sign of viralinfection. After rechallenged, all animals untreated, treated with pWHcIm alone, ortreated with ETV plus pWHcIm were protected from the secondary infection. The twoanimals treated with ETV alone but not developed anti-WHc antibody developedtypical viramia.ConclusionEarly intervention with antiviral drug can protect adults from the primary hepadnaviralinfection, but antiviral treatment combined with DNA vaccination can protect adults fromboth the primary and the secondary viral infection, which provides opportunity for explorethe immune response in the primary and secondary protection. It seems likely that antiviral treatment combined with DNA vaccination may be useful in post-exposure therapy forHBV infection.