| African swine fever(ASF)is a virulent animal disease caused by the African swine fever virus(ASF).Since its discovery,ASF has put the pig industry and food safety at great risk in most countries around the world.Studies have shown that inactivated vaccines,attenuated vaccines,genetically deficient live attenuated vaccines and antigenic vaccines are not effective against ASFV.The lack of a safe and effective vaccine to prevent and treat ASF has made it difficult to control the epidemic situation,so the development of a safe and effective ASF vaccine is urgent.In the present study,20 ASFV antigenic proteins were systematically screened for the use of recombinant adeno-associated virus as a vector to construct a viral vector vaccine and perform preliminary validation and virus neutralization evaluation.1.Construction of rAAV virus vector vaccineASFV 061R(P12),D117L(P11),E120R(P14),B438L(P49),B646L(p72),E183L(p54),E248R,E199L(p E199L),A104R(P10),I215L,C257L,S273R,A179L A238L,C475L,NP419L,B407L,B354L,D345L,B475L antigen of viral vector vaccine.Using the vector rAAV-Flag,the antigenic sequence of ASFV was inserted into the vector by molecular cloning techniques to successfully construct the rAAV-ASFV-Flag plasmid.Next,rAAV was packaged to obtain a recombinant adeno-associated virus expressing the ASFV antigen.The successful expression of the antigen protein was verified by purification and concentration to increase the virus titer,q RT-PCR to determine the virus titer,and Western blot.2.Immunization of mice with ASF virus vector vaccine and preliminary assessment of its immunogenicityThe successful ASF virus vector vaccine was immunized in mice and its immunogenicity was initially assessed.Firstly,20 vaccines were divided into four groups according to the function of the antigenic proteins they expressed.The sera from the immunized mice were tested by ELISA for the production of specific antibodies to the various antigens and the results showed that specific antibodies were produced in the sera of the immunized mice.The immune response of the animals was analyzed by q RT-PCR and FASC.The results showed that the levels of IFN-γ,TNF-α,IL-2,IL-4and IL-6 cytokines were up-regulated in rAAV-ASFV-Flag immunized mice compared to rAAV-GFP-Flag immunized mice.T-cell subpopulation analysis showed that the ratio of CD4~+T cells and CD8~+T cells was upregulated,indicating that immunization with the vaccine could effectively activate specific immune responses in mice.Based on these results,the five antigens that produced high levels of specific antibodies were screened as P11,P14,B354L,B475L,C475L and recombined for further evaluation and validation.3.Preliminary validation of P11,P14,B354L,B475L,C475L vaccines in immunized mice and pigsThe above-mentioned vector vaccines expressing antigenic proteins P11,P14,B354L,B475L and C475L were recombined and immunized in mice and pigs,and sera were collected at 0d,14d,28d and 42d,respectively.The results showed that the expression levels of various cytokines and the proportion of T-cell populations in rAAV-ASFV-Flag mice were down-regulated compared to rAAV-GFP-Flag mice,while the proportion of B-cells in immunized mice and pigs increased significantly,indicating that after stimulation with antigens P11,P14,B354L,B475L and C475L,the animals’ability to produce antibodies in vivo increased significantly.The antibody production capacity in the body increased significantly after stimulation with antigens P11,P14,B354L,B475L and C475L.4.Serum neutralization of ASFV infection in vitro after immunization with P11,P14,B354L,B475L,C475L vector vaccinesIn order to test whether the antibodies produced in the sera of the pigs after the above immunization with antigens P11,P14,B354L,B475L and C475L could neutralize ASFV.The sera to be tested,the negative sera and the positive sera were first incubated with the virus solution and then added to the PAM cells,and the results of the experiments were detected by indirect immunofluorescence using the P72 antibody,and the fluorescence intensity values of the cells were counted.The results showed that at a certain ratio of serum and virus,the ASFV antigen immunized group was extremely significantly different from the negative and positive control groups respectively(P<0.001),indicating that the antibodies produced in the immunized pigs could neutralize some of the ASFV.In summary,the vaccine constructed with rAAV as the vector could effectively activate the immune system of mice when they were initially screened for immunization,and the combinations obtained from the screening showed a significant increase in the proportion of B cells after further validation,and in vitro neutralization tests with immune sera from pigs showed that the experimental ASFV antigens P11,P14,B354L,B475L and C475L immunization had some neutralizing ability against ASFV.This study provides a new idea for the development of a safe and effective vaccine for ASF. |
PDF Full Text Request