Production And Immunogenicity Of Canine Parvovirus-like Particles

Canine parvovirus infection is caused by canine parvovirus type2(CPV-2) with high mortality and morbilty, it is highly contagious and the most common clinical presentation include acute hemorrhagic gastroenteritis, vomiting and severe leukopenia. CPV-2was first identified in1978from dog with gastroenteritis. Subsequently, CPV-2underwent a rapid evolution which produced new antigen variants named CPV-2a, CPV-2b and CPV-2c respectively. Substitution of key amino acid in VP2sequence can change the antigen characters influencing the cell and tissue tropisms. Compared with the original type CPV-2, the new variants get more virulent and acquired a larger host range allowing it to infect cats and dogs. Research show that the new viarant CPV-2c can also cause some adult dogs infected. Currently the new variants of CPV (CPV-2a, CPV-2b and CPV-2c) have completely replaced the original type CPV-2and co-circulate in varying proportions in different parts.The development and widly use of CPV-2live attenuated vaccine made the CPV infection control. However, Traditional vaccine including live attenuated vaccine and inactived CPV strain had disadvantage, there were many cases of CPV infection after immunization in many countries. As we got more insights in immunology, the disadvantage of traditional vaccine exposed. In recent years, vaccine based on VP2of canine parvovirus including multi-peptide vaccine, subunit vaccine, DNA vaccine, recombinant live vector vaccine had got serial progress. These vaccines would not be interfered with maternal-derived antibody and were potential vaccine.Due to the high immunogenicity of VP2protein, it could be used as subunit vaccine or DNA vaccine. When VP2protein were expressed via baculovirus/insect expression system, it could auto-accumulated into virus-like particle which similar to the naive CPV in size and morphology. Based on this character, VP2protein was expressed via baculovirus/insect expression system and auto-accumulated into virus-like particles. Subsequently, the immunogenicity of CPV virus-like particle were evaluated in guinea pig and domestic dogs two animal modle. Our research provide a theoretical basis for production of subunit vaccine.Our research include two chapter as followed.1. Development and identification of CPV virus-like particles. First of all we amplified the complete VP2sequence via PCR using a DNA of CPV as template with two pairs of primers with different enzyme sites respectively, then gel extract these two PCR products and insert them under two different promoters on vector of pFastBacDual seperately. The recombinant plasmid was transferred into Esherichia coli DH10Bac/BmNPV according to the manufactures’ instruction (Invitrogen). The expression of VP2protein would auto-accumulated into virus-like particles.2. Evaluated the immunnogenicity of CPV virus-like particles. The recombinant baculovirus named P1, The recombinant baculovirus was propagated on BmN cells, about three days later, harvested the virus, subsequently named P2, then test the titer which was2.5×108pfu/ml. About5×106pfu of the recombinant baculovirus were injected into silkworm pupae at day5with a needle and syringe. The infected pupae were collected120h post-infection, ground in PBS (pH=7.2) at a proportion of4ml for each gramer. Subsequently, the hemagglutination titers of homogenates containing VLPs were tested. The homogenates were used as vaccine which was intramuscularly and orally administrated to guinea pigs with different doses in two weeks interval. Then the homogenates were administrated to dogs via oral route, and a booster two weeks later. Serum samples were collected pre-immunization and each week post-immunization, then recovered from centrifuge and underwent HI test to evaluate the antibody titer.In this study, VP2protein was expressed in BmN cells and auto-accumulated into virus-like particles. VP2protein expressed in silkworm pupae were found self-assembled into virus-like particles with hemagglutinin titer as high as1:210after recombinant baculovirus infection, this result showed that the silkworm pupae can be used as an expression systerm for virus-like particles production. Subsequently, genuine pigs were immunized with canine parvovirus-like particles intramuscularly. The result showed that canine parvovirus-like particles were high immunogenic which elicted immune responses and the hemagglutinition inhibition antibody titer could reached as high as1:29.7. Genuine pigs and dogs were immunized with canine parvovirus-like particles orally. The result showed that canine parvovirus-like particles could evoke humoral immune response via oral route. Animal immune research showed canine parvovirus-like particles were high immunogenic and can provoked humoral immune response wether via intramuscular route or oral route with fewer doses. This research supplied a support for the development of new type subunit vaccine for canine parvovirus prevention.