| Contagious pustular dermatitis,commonly known as "Orf",is an acute,highly contagious disease caused by Orf virus(ORFV)infection.The formation of blisters,pustules,ulcers,and verrucous thick crusts on the skin or mucous membranes of damaged parts of the mouth,lips,nose,and tongue of infected sheep,goats and other small and medium-sized ruminant animals mainly characterizes it.ORFV mainly infects lambs aged 3-6 months,while adult sheep are less affected.However,once infected,it is difficult to clear the virus in the flock,which seriously endangers the healthy development of the sheep industry in China and even in the world.In addition,humans can also be infected through contact with diseased sheep,especially those engaged in slaughter,breeding,veterinary.Therefore,the Orf disease is also a zoonotic infectious disease that affects public health and safety.Vaccination is an essential tool for effectively preventing the Orf disease.However,there is a lack of specific antiviral drugs and efficient commercial vaccines to prevent and control the disease.Therefore,there is an urgent need to develop safe and efficient vaccines to prevent and control of Orf.Genetically engineered vaccines(recombinant subunit vaccine,DNA vaccine,adenovirus vaccine)have the advantages of high safety and strong induction of immune response,and have become an important direction for developing of new,safe and efficient vaccines for Orf.It has been shown that the B2 L and F1 L proteins encoded by ORFV have good immunogenicity and are candidate antigens for preparing of genetically engineered vaccines.Therefore,in this study,we propose to use the above candidate proteins to carry out the study of Orf vaccine.We obtained recombinant B2 L and c F1 L proteins as subunit vaccine candidates through gene structure prediction,construction of prokaryotic expression vector and purification of prokaryotic expression.The recombinant proteins were mixed with Freund’s adjuvant and immunized BABL/c mice to prepare mouse polyclonal antibodies against B2 L and c F1 L proteins,respectively.The Polyclonal antibodies detected by Western blot can specifically bind to the recombinant protein,which has good immunogenicity.The candidate subunit vaccine was prepared by mixing the recombinant protein with Freund’s adjuvant,and BABL/c mice were immunized on day 0 and day 21.The specific antibody,cytokine level,lymphocyte proliferation reaction and T lymphocyte subsets were detected,respectively.The results showed that two-proteins immunization had advantages in inducing specific antibodies and cellular immunity compared to single-protein immunization.This result suggests that the combination of two proteins can help improve the immune effect of the vaccine.This result suggests that the combination of two proteins can help to improve the immune effect of the vaccine.By using overlapping extension PCR,the B2 L and F1 L genes and viral 2A peptide(P2A)DNA sequences were linked to form a B2L-P2A-F1 L fusion gene fragment,which was cloned into eukaryotic expression vectors to obtain candidate DNA vaccine.Western blot and immunofluorescence detection results showed that B2 L and F1 L proteins could be independently expressed under the action of P2 A selfcleaving peptide.The candidate DNA vaccine plasmid was mixed with Freund’s adjuvant,and BABL/c mice were immunized on day 0 and 21.The specific antibody,cytokine level,lymphocyte proliferation reaction and T lymphocyte subsets were detected,respectively.The results showed specific antibodies production in all immunized groups.Of note,B2L-P2A-F1 L group could induce stronger cellular immunity than the B2 L and F1 L single gene groups.B2L-P2A-F1 L was further cloned into an adenovirus expression vector,and packaged a recombinant adenovirus r Ad V-B2L-P2A-F1 L that can express B2 L and F1 L proteins and can stably inherit.BABL/c mice were immunized with candidate adenovirus vaccine r Ad V-B2L-P2A-F1 L,specific antibody,cytokine level,lymphocyte proliferation responses and T lymphocyte subsets were detected,respectively.The results showed that the specific antibody levels in mice immunized with recombinant adenovirus carrying the B2L-P2A-F1 L rapidly increased,reaching the highest level on day 28;meanwhile,it also showed significant T-lymphocyte proliferation ability against ORFV-specific antigens,with significantly higher levels of CD3+CD4+and CD3+CD8+T lymphocytes compared to the PBS group and adenovirus control group(P<0.01 and P<0.001).The above results indicate that B2 L and F1 L recombinant adenovirus can induce high level of humoral and cellular immune response.To optimize the immune effects of Orf vaccines and to explore the best vaccine combinations,we immunized mice with the above vaccines in either homologous or heterologous prime-boost immunization,and with PBS,pc DNA3.1 empty vector,and p Adeno G as controls.Comprehensive evaluation of immune efficacy was then performed to provide technical support for developing new Orf disease immunization strategies.Indirect ELISA,MTT,and flow cytometry were used to measure the humoral and cellular immune levels of all animals in different groups,and the pathological damage after immunization,to evaluate the immune effects of different immune strategies.The results showed that DNA vaccine prime-Subunit vaccine boost immunization strategy induced higher levels of humoral and cellular immune responses in mice.The results indicated that the Th1-type immune response was predominant,but no ORFV-neutralizing antibody was detected,suggesting that cellular immunity-based immune responses were mainly induced.H&E staining was used to detect histological changes in the heart,liver,lung and kidney from the mice after vaccine immunization.The results showed that there were no obvious pathological changes in various tissues,indicating that the vaccine would not cause adverse reactions in the mice.Based on the above data,this study further evaluated the immune effects of DNA vaccine prime-adenovirus vaccine boost and DNA vaccine prime-subunit vaccine boost by using native animals.Fifteen ORFV antibody-negative sheep were randomly divided into three groups: the DNA/adenovirus group,DNA/subunit group,and PBS control group,and were immunized on day 0 and 21.The humoral and cellular immune responses induced by the above immune groups in the native animals,and the pathological damage after the challenge,were evaluated.The results showed that the DNA vaccine prime-subunit vaccine boost strategy can induce higher levels of specific antibodies,more significant T-lymphocyte proliferation ability,and partially resist the attack of ORFV in sheep.The above results indicate that the DNA vaccine prime-subunit boost immune strategy can display a degree of immune protection in native animal sheep.In conclusion,this study successfully constructed ORFV recombinant subunit vaccine,DNA vaccine and recombinant adenovirus vaccine,which provided an essential material basis for the clinical prevention and control of Orf disease in sheep;Based on the above data,the immune protection effects under different immune strategies were systematically compared,and DNA prime-subunit boost immunity was determined as the best vaccine combination strategy,which could induce a stronger Th1-based immune response in mice/sheep,and displays relative good protection effect,and it is expected to provide critical technical means for clinical prevention and control of this disease. |
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