Establishment Of Canine Lyme Disease ELISA Method,Construction And Immunogenicity Study Of OspA Chimeric VLPs

Lyme disease is a natural epidemic zoonosis transmitted by ticks.The typical clinical symptom of the disease is erythema migrans,which mainly violates the heart,joints,and nerves.Once infected,it can cause irreversible damage to the body.Small mammals are the main hosts of ticks.With the development of economy,the frequent contact between people and pets increased the potential risk of Lyme disease in humans.The diagnosis of Lyme disease may not in time due to the complex clinical manifestations,difficult and time-consuming pathogen isolation.Therefore,many patients with Lyme disease missed the optimal treatment time.At present,the test method often selects the bacterial proteins with early appearance,strong antigenicity and long duration as the detection antigen,but,these bacteria proteins have certain heterogeneity,which can easily lead to poor detection effect,which is not conducive to the prevention and control of Lyme disease in China.Therefore,according to the actual epidemic situation in China,it is urgent to establish a detection method matching the epidemic pathogenic genotype.Vaccination is the most effective way to prevent Lyme disease,but there is no commercial vaccine to prevent the disease in humans in the world.Currently,the development of Lyme disease vaccines is mainly focused on whole-bacteria and subunit vaccines,which have poor safety and low immunogenicity.Therefore,it is urgent to develop an efficient and safe new-type Lyme disease vaccine based on the concept of green environmental protection.Aiming at the above problems,this study aims to establish an indirect ELISA method for Lyme disease in canines,so as to provide a rapid and effective clinical method for detection of canine Lyme disease in China.Secondly,the outer surface protein A(OspA)of Borrelia burgdorferi was chimed into the membrane surface of Newcastle disease virus-like particles(NDV VLPs)using different construction strategies.By comparing the different chimeric virus-like particles(cVLPs)we constructed,cVLPs carrying the most OspA proteins were screened out.Finally,the immunogenicity of optimal cVLPs was also studied to explore its potential for further application and provide new ideas for the research of new Lyme disease vaccines.The main research contents are as follows:1.Establish of an indirect ELISA method for detection of Lyme disease in caninesIn this study,the outer membrane surface protein C(OspC)of Borrelia garinii(B.g),a prevalent pathogenic genotype in China,and Borrelia burgdorferi sensu stricto(B.b.s.s),an internationally prevailing pathogenic genotype were selected to be expressed through the prokaryotic expression system and these two proteins were further purified after identification by SDS-PAGE and Western Blot.The two proteins were used as antigens to coat ELISA plate.Antigen coating concentration,serum dilution ratio,enzyme-labeled antibody working concentration and other reagents were optimized and a feasible ELISA detection method was established,which provided technical support for the clinical diagnosis of Lyme disease antibody samples in canines in China.2.Construction of canine Lyme disease cVLPs-rdHN OspAIn this study,three different cVLPs containing the OspA protein were constructed based on the NDV VLPs vectors.The details are as follows: GPI OspA(GPI anchored OspA),rdHN OspA(HN linked OspA that has deleted HN ectodomains),and HN OspA(HN linked OspA)were assembled to NDV VLPs vectors respectively.cVLPs-GPI OspA,cVLPs-rdHN OspA,cVLPs-HN OspA were expressed through the insect baculovirus expression system.Western Blot and transmission electron microscope(TEM)were used to test whether these three cVLPs were constructed successfully.Subsequently,the optimal cVLPs were selected by comparing the protein content of the chimeric OspA on the cVLPs.The results showed that regular surface spikes could be observed on the cVLPs-GPI OspA and cVLPs-rdHN OspA through TEM,and chimeric OspA was correct on the membrane surface of NDV VLPs.Meanwhile,OspA proteins were the most abundant in cVLPs-rdHN OspA.In conclusion,the cVLPs-rdHN OspA is the optimal cVLPs.3.Study on the immunogenicity of canine Lyme disease cVLPs-rdHN OspAIn this study,the cVLPs-rdHN OspA was used as an experimental group,while soluble rOspA group(prokaryotic expression),VLPs-M group(empty NDV VLPs),and PBS group were set for analysis and evaluation.BALB/c mice were immunized with the above substances by intraperitoneal injection respectively.On the one hand,serum antibodies were obtained from tail vein and the humoral immune response of the mice in each group was compared by ELISA.On the other hand,the isolated spleen lymphocytes in mice from each group were marked with CD3,CD4 and CD8 T cell markers and then detected by flow cytometry.To characterize cellular immune responses of mice in each group,the ratio of CD3+CD4+ to CD3+CD8+ T cells was also compared.The results showed that the cVLPs-rdHN OspA group revealed faster and higher antibody levels than the soluble rOspA group,VLPs-M group,and PBS group.Moreover,the ratio of CD3+CD4+ to CD3+CD8+ T cells in the cVLPs-rdHN OspA group was significantly higher than other groups.Based on the results,the cVLPs-rdHN OspA group can stimulate higher levels of humoral and cellular immune responses.In summary,the cVLPs-rdHN OspA group had better immunogenicity than the prokaryotic expression OspA,providing a new idea for a novel vaccine against Lyme disease in canines.