Generation And Immunoreactivity Of Recombinant Rabies Virus Expressing Echinococcus Granulosus EG95 Protein

BackroundHydatidosis is a zoonosis caused by the parasitic host organs of the larva of Echinococcus.The disease is prevalent in China,Central Asia,the Middle East,South America and parts of Europe.Among them,cystic hydatidosis(CE)caused by Echinococcus granulosus(E.g)is widely prevalent in China,leading to serious disease and economical burden in livestock industry.Sheep,cattle and other domestic animals act as intermediate hosts for the parasite,while canids are the definitive hosts.Human beings are occasionally infected as the intermediate hosts.In this disease,clinical symptoms are due to space-occupying lesions that can obstruct ducts,impair organ function,and rupture,causing anaphylactic shock or secondary bacterial infections.CE causes 19,300 deaths and 871,000 disable-adjusted life-years(DALYs)global each year,and annual costs associated with the disease are estimated to be US$3 billion for treating cases and losses to the livestock industry.In order to effective control of E.g,it is necessary to stop the parasite’s development at different stages of its life cycle by vaccinating the intermediate host.The subunit vaccine made from EG95 protein has been proved safe and effective in CE epidemic areas.However,its vaccination procedure is complicated,multiple inoculation and adjuvant is needed to achieve the expected immune protection effect,and the social cost is high.Therefore,it is urgent to develop a cheaper and more effiective vaccine against E.g.Meanwhile,rabies is a serious zoonosis caused by rabies virus(RABV).All warm-blooded animals can be infected with almost 100%case fatality rate.The disease is a global epidemic,mainly occurring in developing countries and posing a serious threat to their public health.Due to insufficient control of canine rabies and the spread of wild animal rabies,rabies of sheep,cattle and other domestic animals in pastoral areas of China has been increasing rapidly in recent years.It not only causes economic losses of livestock industry,but also brings potential infection risk to farmers and veterinarians.The Office International Des Epizooties(OIE)recommends vaccinating cattle and sheep against rabies in endemic areas and has been successfully implemented in some countries.As a good vector for expressing exogenous genes,RABV has unique advantages.Many researchers have developed recombinant vaccines expressing various kinds of exogenous virus antigen proteins using RABV as the vector.To sum up,in order to avoid the severe economic losses caused by the double epidemic of CE and rabies to cattle and sheep in pastoral areas,it is urgent to develop a safe and effective bivalent vaccine for cattle and sheep which can simultaneously prevent E.g and RABV.Objectives1.The rabies virus strain LBNSE was used as the vector to construct recombinant rabies virus expressing EG95 protein of E.g,which can become a bivalent candidate vaccine against CE and rabies.2.The biological characteristics of LBNSE-EG95 were analyzed in vitro cell experiments.3.Pathogenicity,protection and immunological responses of LBNSE-EG95 were evaluated in animal experiments.Methods1.Construction and identification of recombinant virus LBNSE-EG95.The recombinant virus LBNSE-EG95 was constructed by inserting the EG95 gene into the pseudogene region of LBNSE.The expression of the target protein of the recombinant virus was identified by RT-PCR,immunofluorescence assay(IFA),flow cytometry and Western blot.2.Pathogenicity analysis of recombinant virus LBNSE-EG95.Female BALB/c mice(6-8weeks)were randomly divided into the experimental group,the control group and the mock group.The mice were intramuscularly inoculated with LBNSE-EG95,LBNSE and DMEM.The body weight,diet and mental status of the mice were observed and monitored in 21 days after vaccination.3.Detection of the ability of recombinant virus LBNSE-EG95 to induce specific protective antibodies in mice.Serum of mice were collected at 2,4,and 8 weeks after immunization.The antibody titer of RABV neutralizing antibody was detected by the Fluorescence Antibody Virus Neutralization Test(FAVN),and the specific antibody level of EG95 was detected by enzyme linked immunosorbent assay(ELISA).4.Protective evaluation of recombinant virus LBNSE-EG95.At 4 weeks after vaccination,mice were intramuscularly challenged with RABV in their hind limbs.The survival rate,changes of body weight,diet and clinical symptoms of mice were observed and monitored in 21 days after challenge to evaluate the protective effect of LBNSE-EG95 on mice.5.Detection of the ability of recombinant virus LBNSE-EG95 to recruit/activate DC cells in mouse lymph nodes.Inguinal lymph nodes were collected from mice on day 3,6,and 9 after immunization.The ability of LBNSE-EG95 to induce recruitment/activation of DC cells in inguinal lymph nodes of mice was detected by flow cytometry.6.Detection of the ability of recombinant virus LBNSE-EG95 to recruit/activate B lymphocytes in lymph nodes and peripheral blood of mice.Lymphocytes from inguinal lymph nodes were collected at 3,6,and 9 days after immunization,and peripheral blood was collected at 7,14,and 28 days after immunization.The ability of LBNSE-EG95 to recruit/activate B lymphocytes in mice was detected by flow cytometry.7.Detection of the ability of recombinant virus LBNSE-EG95 to induce the production of IFN-y and IL-4 antigen-specific T cells.Splenic lymphocytes of mice were collected at 7 and 14 days after immunization,and the ability of LBNSE-EG95 to induce the production of specific CD4+and CD8+T cells secreting IFN-y and IL-4 was detected by flow cytometry under the specific stimulation of RABV and EG95 proteins.At 28 days after immunization,spleen lymphocytes were isolated from mice,and the ability of mice to produce IFN-y and IL-4 specific T cells was detected by enzyme-linked immunodot assay(ELISpot).8.Detection of the ability of LBNSE-EG95 to induce specific antibody subtypes in mice.The serum was collected at 4 weeks after immunization,and the specific IgG2a/IgGl titer under specific stimuulation of RABV and EG95 was tested by ELISA.Results1.The recombinant virus LBNSE-EG95 was rescued successfully,and identified by IFA,RT-PCR,flow cytometry and Western blot,which indicated that the recombinant virus could correctly express the EG95 protein.2.During the 21-day observation period,there was no significant difference in the body weight of mice in the LBNSE-EG95 group compared with the Mock group and the LBNSE group,and the body weight of mice among three groups showed a steady upward tendency,with good growth state and no abnormal behavior.3.The result of FAVN assay showed that LBNSE-EG95 could induce the production of high levels of neutralizing antibody against RABV and specific antibody against EG95 in mice.4.The result of RABV infection test showed that LBNSE-EG95 and LBNSE group could provide complete protection against the fatal attack of RABV,while the mock group all occurred clinical symptoms.5.The results of flow cytometry showed that LBNSE-EG95 could induce the recruitment/activation of more DC cells in inguinal lymph nodes of the mice compared with the mock group.6.The results of flow cytometry showed that LBNSE-EG95 could induce the recruitment/activation of more B cells in mouse lymph nodes and continued recruitment/activation B cells in peripheral blood compared with the mock group.7.The results of flow cytometry showed that LBNSE-EG95 induced the production of more CD4+and CD8+T cells secreting IFN-y in mice.The results of ELISpot showed that with the stimulation of EG95 protein and RABV G protein,LBNSE-EG95 group could induce more specific T cells secreting IFN-y and IL-4,which was significantly increased more than those in the mock group and the control group.8.The results of ELISA showed that the immune response of mice immunized with LBNSE-EG95 was more inclined to Th1 type immune response.ConclusionIn this study,the recombinant rabies virus LBNSE-EG95 expressing EG95 protein was successfully generated.The recombinant virus showed high safe,and could induce mice to produce strong specific humoral and cellular immunity.It indicateds that LBNSE-EG95 is an ideal bivalent candidate vaccine for against CE and rabies.