Generation And Immunoreactivity Of Recombinant Rabies Virus Expressing IL-33 Protein

BackgroundRabies,an ancient zoonotic disease,is caused by the rabies virus(RABV),which is distributed on all continents except Antarctica.The disease is one of the diseases with the highest case fatality rate(nearly 100%).,and its infection spectrum is wide,and warm-blooded animals are easily infected,which can cause severe encephalitis.About 59,000 people worldwide die of rabies every year,that is,one person dies of the disease every 9 minutes.About 35,000 people in Asia die from dog-mediated rabies each year,accounting for about 59.6%of global deaths.Currently there is no effective treatment and antiviral drugs,vaccination is the most effective way to prevent.Rabies vaccine is widely used,and its immune protection mechanism relies on the traditional "immune-boost" principle,and the immune characteristics have not been fully elucidated,especially cellular immunity.In the early stage,some scholars enhanced the immunogenicity of rabies vaccine by enhancing the humoral immune response,such as expressing double or triple copies of G protein,macrophage inflammatory protein 1-α(MIP1α),flagellin,etc.The lack of insights of cellular immunity leads to low-level or non-protective cellular immune responses.Thus,this study aimed to learn the role of cellular immunity for rabies vaccine by expressing cytokine to enhance cellular immunity.Many kinds of cytokine are involved in cellular immunity,such as interleukin,interferon,tumor necrosis factor,etc.IL-33 belongs to the IL-1 family and is a nuclear alarm protein with a wide range of sources.IL-33 is expressed in a variety of cells and organs.Studies have confirmed that IL-33 could play a role in respiratory virus infection-related diseases,coxsackievirus B5(CVB5)infection,mouse cytomegalovirus(MCMV)infection and lymphocytic choriomeningitis virus(LCMV)infection.IL-33 C-terminal part of protein shown that IL-1 domain,is the ligand of ST2 receptor,when virus invaded the body,cells or tissue damaged and released IL-33,which could induce Thl and Th2 immune responses,and coordinated protective antiviral cytotoxic T lymphocyte(CTL)response,involved in cellular immunity.Therefore,the purpose of this study was to construct recombinant rabies virus expressing IL-3 3,and to analyze the effect of IL-33 overexpression on the biological characteristics,pathogenicity and the adaptive immune response of RABV.To provide a theoretical basis for studying the relevant mechanism of RABV cellular immunity.Objective1)Recombinant virus LBNSE-IL-33 expressing IL-33 was constructed using RABV attenuated strain LBNSE strain as the vector;2)In vitro cell experiments to study whether LBNSE-IL-33 correctly expresses IL-33 protein and its effect on the growth kinetics of RABV;3)The effects of IL-33 on the pathogenicity,humoral immunity,cellular immunity and protection of RABV were analyzed by animal immune experiments,which provided theoretical basis for studying the related mechanism of humoral immunity and cellular immunity of RABV.Methods1)Construction and identification of recombinant virus LBNSE-IL-33:a full-length infectious clone was constructed by using two restriction sites of Pfl23II and NheI,and T4 DNA ligase,and transfected into BHK-21 cells to obtain recombinant virus.The genome was identified by RT-PCR assay,and the protein expression was identified by immunofluorescence assay(IFA),Western blot and flow cytometry(FACS).Growth curves were plotted to learn the growth kinetics of the recombinant virus.2)Pathogenicity analysis of recombinant virus LBNSE-IL-33:Female BALB/c mice aged 6-8 weeks were randomly divided into three groups,immunized with LBNSE-IL-33,LBNSE,and DMEM respectively.The diet,mental state and body weight of the mice were observed within 3 weeks after immunization.3)Evaluation of the protective ability of recombinant virus LBNSE-IL-33 against RABV infection:4 weeks after immunization,RABV infection was performed,and the state of mice were continuously observed for 3 weeks after infection,and the survival curve and clinical symptom score were drawn.Mice brain tissue were identified by RT-PCR.4)Detection of specific antibody level induced by recombinant virus LBNSE-IL-33:1,2,and 4 weeks after immunization,the neutralizing antibody titer in mice serum was detected by fluorescent antibody virus neutralization assay(FAVN).Enzyme-linked immunosorbent assay(ELISA)was used to detect the titer of RABV-specific antibody subtypes IgG1 and IgG2a,and their ratios.5)Detection of the ability of the recombinant virus LBNSE-IL-33 to induce the recruitment of activated dendritic cells(DC)and B cells in mice:3,6,and 9 days after immunization,the inguinal lymph nodes and peripheral blood of the mice were collected.The activated DCs and B cells in the lymph nodes were detected.And B cells in peripheral blood were detected too.6)Detection of the ability of recombinant virus LBNSE-IL-33 to induce the production of specific T cells in mice:At 1,2,and 4 weeks after immunization,the mice spleens were collected,stimulated with RABV antigen,and flow cytometry was used to detect proportion of CD8+T cells,CD4+Thl and Th2 cells.1,2,4 and 8 weeks after immunization,the spleen lymphocytes of mice were isolated,and the number of RABV-specific T cells was detected by enzyme-linked immunospot assay(ELISpot).Results1)The recombinant virus LBNSE-IL-33 was successfully rescued.RT-PCR results showed that the virus genome was constructed correctly.IFA,western blot and flow cytometry experiments confirmed that the recombinant virus expressed both RABV G protein and IL-33 protein,and the size was the same as expected.The growth curve showed that the peak titer of the recombinant virus on BHK-21 cells was no different from that of parental strain LBNSE,but slightly higher on Neuro-2a cells(P<0.05),and the insertion of exogenous gene IL-33 did not affect the recombination virus replication.2)The body weight of the mice in three groups showed an increasing trend within 3 weeks after immunization,and there was no significant difference in body weight compared with the blank group and LBNSE group(P>0.05).The mice had smooth hair,no abnormal changes in mental state,indicating that LBNSE-IL-33 had no obvious pathogenicity.3)Within 3 weeks after infection,the mice in LBNSE-IL-33 group did not show any abnormal symptoms,and could provide protection against RABV infection in mice.4)Antibody detection showed that the mean values of neutralizing antibodies at 1,2,and 4 weeks after immunization were 2.55IU/mL,6.86IU/mL,and 5.24IU/mL,which were higher than the level of 0.5IU/mL,which was the titer of antibodies with effective protection recognized by WHO.And at 2 weeks after immunization,the neutralizing antibody titer of LBNSE-IL-33 group was significantly higher than that of LBNSE group,with a significant difference(P<0.01).The ELISAresults showed that LBNSE-IL-33 could induce the production of specific IgGl and IgG2a antibodies similar to LBNSE.At 1 week after immunization,the average titers of the two antibodies in the LBNSE-IL-33 group were 1:459.48 and 1:475.48,both higher than those in the LBNSE group(P<0.05),and at 4 and 8 weeks after immunization,the IgG2a titer in the LBNSE-IL-33 group was still higher than those in the LBNSE group(P<0.05).5)FACS results showed that compared with the blank group and LBNSE group,LBNSEIL-33 could induce the recruitment and activation of more DCs and B cells in mice(P<0.05).6)The results of FACS showed that the proportion of CD8+T cells,CD4+Th1 and Th2 cells was higher in the LBNSE-IL-33 group(P<0.05).The results of ELISpot experiment showed that compared with blank group and LBNSE group,LBNSE-IL-33 could induce a higher number of RABV-specific T cells(P<0.05).ConclusionsIn this study,the recombinant rabies virus LBNSE-IL-33 expressing IL-33 was constructed.The expression of IL-33 promotes the rapid induction of humoral and cellular immunity by RABV.A single immunization of mice can rapidly induce good immunogenicity and protection,to provide a theoretical basis for the research on the mechanism of action of IL33 to promote the cellular immunity of RABV vaccines,and provides new ideas for the development of new,safe and efficient rabies vaccines.