Preparation Of Bovine Parainfluenza Virus 3 Type A Virus-like Particle Vaccine

Bovine parainfluenza virus type 3(BPIV3)is a highly contagious respiratory pathogen of cattle that often causes calf disease.Traditional inactivated and attenuated vaccines have certain limitations in terms of immunogenicity and safety.Developing a new,safe,and effective BPIV3 vaccine to control the epidemic is of great significance.Virus-Like Particle(VLPs)vaccines offer improved immunogenicity,enhanced safety,and the ability to induce stronger and more durable immune responses.However,there is relatively limited research on BPIV3 VLPs vaccines.In this study,we present a method for preparing BPIV3 Type A VLPs and evaluate their immunogenicity and protective efficacy.The c DNA of BPIV3 type A was used as a template to design primers for PCR amplification of M,HN,and F nucleic acid fragments.These fragments were seamlessly cloned into the p Fast Bac plasmid.Following three rounds of blue-white screening,a positive baculovirus plasmid was extracted and transfected into insect cells to generate the P1 recombinant baculovirus,which was then propagated to the P3 generation.Western blot and sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)analyses confirmed the expression of M,HN,and F proteins.Sf9 cells were infected with a mixture of P3 baculovirus containing r BV-M,r BV-HN,and r BV-F at an equal ratio(MOI 1:1:1),or a combination of r BV-M and r BV-HN at a ratio of 1:2.The resulting proteins were concentrated and analyzed by SDS-PAGE,Western blot,and transmission electron microscopy(TEM).VLPs of approximately 200 nm were observed in the r BV-M and r BV-HN combination.These VLPs were purified using sucrose gradient centrifugation and characterized by TEM and dynamic light scattering.TEM revealed surface features on the VLPs resembling the spinous processes found on authentic BPIV3 type A virions,exhibiting similar size and morphology(210.2 nm cumulative particle size for VLPs and 281.5 nm for the BPIV3 type A wild strain).The hemagglutination test demonstrated a hemagglutination titer of1:25 for the VLPs.Taken together,these results confirm the successful preparation of BPIV3 type A VLPs.Animal experiments showed that BPIV3 type A VLPs had good immunogenicity and could induce significant humoral and cellular immune responses in immunized animals.Specifically,the Ig G specific antibody in the serum of immunized guinea pigs increased gradually over time and reached the highest level at day 42.The average titer of hemagglutination inhibition antibody was 25.3 on day 14,27.2 on day 28 and 28.7 on day 42.The average titer of serum neutralizing antibody was 24 on day 14,27.2 on day 28,and 29.8on day 42,indicating that the vaccine successfully induced specific humoral immune response.At the same time,cytokines were upregulated in the immune group,and IL-4was about 59.2 pg/m L.IL-10 is about 84.3 pg/m L;INF-γ was about 270.9 pg/m L,indicating that the vaccine also induced a cellular immune response.Clinical symptoms and body temperature data showed that only 1 guinea pig in the immune group had runny nose and fever,while all guinea pigs except 1 asymptomatic guinea pig in the challenge group had runny nose and fever,which further proved the protective effect of the vaccine.The results showed that the average virus titer in lung tissue was 102 TCID50/0.1 m L in the immune group and 104 TCID50/0.1 m L in the challenge group.Histopathology showed no obvious abnormality in the immune group and interstitial pneumonia in the challenge group.It was confirmed that the BPIV3 type A VLPs vaccine had a protective effect against BPIV3 type A infection.These results indicate that BPIV3 type A VLPs vaccine is a promising candidate vaccine for preventing BPIV3 type A infection in cattle.