Research On Immunological Evaluation And Combined Immunization Strategy Of CCHFV DNA Vaccine And VLP Vaccine

1.BackgroundCrimean-Congo hemorrhagic Fever(CCHF)is a tick-borne infectious disease.CCHF is caused by Crimean-Congo hemorrhagic fever virus(CCHFV).Because of the wide distribution of CCHF and its high fatality rate in humans,the development of the vaccines is of great concern.Currently,CCHF has no licensed vaccine and urgently needs to develop a new vaccine that is safe and effective.DNA vaccine and VLP vaccine are two hot spots in the research of CCHFV vaccine at present.The two kinds of vaccines have their own advantages,but also have their own disadvantages.Combined with the current research status of CCHFV vaccine and the previous research basis of our research group,this study intends to prepare CCHFV DNA vaccine and Virus-like Particle(VLP)vaccine respectively.Through sequential immunization,mixed immunization and chimeric immunization,the immune effects of different combined immunization strategies of the two vaccines are studied,to achieve the purpose of complementary advantages between different types of vaccines,and to provide a new research idea for the development of CCHFV vaccine.2.Methods2.1 Construction,identification and immunological characterization of CCHFV recombinant DNA expression vectorThe gene fragments of the envelope glycoprotein Gc,Gn and nucleocapsid protein NP of CCHFV IbAr10200 strain were cloned into pVAX1 and pVAX-LAMP1 vector which contain the gene of lysosome-associated membrane protein1(helpful for antigen presentation).Recombinant DNA expression vectors pVAX-Gc,pVAX-Gn,pVAX-NP,pVAX-LAMP 1-Gc,pVAX-LAMP 1-Gn,pVAX-LAMP 1-NP were constructed,and the recombinant vectors were identified by PCR and sequencing.The correct recombinant DNA expression vectors were transfected into cells.The expressions of corresponding proteins were identified by Western-blot and immunofluorescence assay.HLA-A11/DR1 humanized-transgenic mice were immunized by intramuscular injection of the recombinant DNA expression vectors.The immunological characteristics of the recombinant DNA expression vectors were analyzed.Specific antibodies and neutralizing antibody titers in sera of immunized mice were detected by Enzyme Linked Immunosorbent assay(ELISA)and in vitro neutralization assay.Secreted cytokines in immunized mice were detected by Enzyme Linked Immunospot assay(ELISPOT).Cytotoxic T lymphocyte(CTL)killing test was used to determine CTL activity of spleen cells.Flow cytometry was used to determine types of splenic lymphocytes.Immunized mice were attacked with tecVLPs(transcription and entry-competent virus-like particle),a non-infectious substitute for CCHFV.The contents of CCHFV tecVLPs in mice were detected to evaluate the protective effects of recombinant DNA expression vectors on animals.2.2 Preparation and identification of CCHFV VLP and CCHFV VLP/DNA chimeras The GPC gene fragment of CCHFV IbAr10200 strain was cloned into the pFastBacDual transfer vector to construct the pFastBacDual-CCHFV-GPC recombinant vector.The recombinant vector was identified by PCR and sequencing.The recombinant vectors were transformed into Escherichia coli DH10Bac and the recombinant Bacmid was extracted.The recombinant vector was identified by PCR.The recombinant baculovirus rBV-CCHFVGPC which express CCHFV envelope glycoprotein was packaged by transfecting the recombinant Bacmid into Sf9 cells.The expression of VLP by recombinant baculovirus was identified by Western-blot and immunofluorescence assay.The identified rBV-CCHFVGPC was used to infect Sf9 cells.The multiplicity of infection and time of infection were screened.A mass of VLP was prepared.CCHFV VLP was purified by iodoxanol density gradient centrifugation.The purified CCHFV VLP was identified by SDS-PAGE and Western-blot.The morphology of CCHFV VLP was observed by electron microscopy.The recombinant expression vectors(pVAX-NP,pVAX-LAMP 1-NP)were packaged by CCHFV VLP to prepare VLP/DNA chimeras.VLP/DNA chimeras were id-entified by immunofluorescence assay and PCR.2.3 Comparative study on the immune effect of multiple combined immunization strategies of DNA vaccines and VLP vaccineCCHFV VLP and recombinant DNA expression vectors(pVAX-NP,pVAX-LAMP1-NP)are combined to immunize HLA-A11/DR humanized-transgenic mice by sequential immunization(DNA immunization followed by VLP immunization),mixed immunization(immunization with mixture of recombinant DNA expression vectors and VLP),and chimeric immunization(immunization with VLP/DNA chimeras).The immune effects of combined immunization strategies were evaluated.ELISA and in vitro neutralization test were used to detect the specific antibodies and neutralizing antibody titers in the sera of immunized mice.ELISPOT was used to detect the secreted cytokines of immunized mice.CTL activity of spleen cells was detected by CTL test.Splenic lymphocyte types were detected by flow cytometry.Immunized mice were attacked with CCHFV tecVLPs.The contents of CCHFV tecVLPs in mice were detected to evaluate the protective effects of various combined immunization strategies.3.Results3.1 Multiple recombinant DNA expression vectors of CCHFV were successfully constructed,and two immune-dominant candidate vaccines pVAX-NP and pVAXLAMP1-NP were obtainedThe target gene fragments were cloned into pVAX1 and pVAX-LAMP1 vectors.PCR identification results showed that the corresponding target bands could be amplified from recombinant DNA expression vectors.The sequencing results showed that all recombinant DNA expression vectors had no mutation and the sequences were correct.The results of Western-blot and immunofluorescence assay showed that the recombinant DNA expression vectors could express the corresponding target proteins after transfection.The protein sizes were consistent with expectations.The detection results of immunological characteristics of each recombinant DNA expression vector showed that,in terms of humoral immune response,each recombinant DNA expression vector immunized mice could produce corresponding specific antibodies.After associating with LAMP1,each recombinant plasmid could enhance the specific antibody response to corresponding antigens(p<0.05).In terms of the detection of neutralizing antibody,the neutralizing antibody could be detected in the sera of mice immunized with pVAX-LAMP1-Gc and pVAX-Gc.The neutralizing antibody response could be significantly enhanced by associating with LAMP1(p=0.033).While the titers of neutralizing antibody in the sera of the mice of other groups was extremely low(GMT<10).In terms of cellular immune response,pVAX-LAMP 1-NP immunization can more effectively stimulate spleen cells to secrete a variety of cytokines,especially IFN-γ(p<0.001)and IL-2(p<0.05).The average levels of IFN-γ and IL-4 production in CD4+T cells of pVAX-LAMP 1-NP immunized mice were significantly higher than those in other groups(p<0.001).In addition,the average level of intracellular IFN-γ production in CD8+T cells of pVAX-LAMP 1-NP immunized mice was significantly higher than that in the other groups(p<0.001).In the aspect of CTL activity detection,the CTL activities of spleen cells were enhanced with the increase of E/T ratio after pVAX-LAMP 1-NP immunization,and was the strongest at 100:1.Compared with the pVAX-NP group,spleen cells immunized by pVAXLAMP1-NP showed higher CTL activity(p<0.05).And no obvious CTL activity was shown in other groups.In animal protection experiments,pVAX-LAMP 1-NP can provide stronger immune protection than other groups against the attack of CCHFV tecVLPs(p<0.05).Compared with PBS group,NanoLuc activity in liver,spleen and kidney of pVAX-LAMP 1-Gc immunized mice was significantly decreased(liver:p=0.0005,spleen:p=0.0001,kidney:p=0.0003),suggesting that pVAX-LAMP1-Gc immunization also provided protective effect against CCHFV tecVLP attack.3.2 CCHFV VLP was successfully prepared and two VLP/DNA chimeras were obtainedThe CCHFV GPC gene was cloned into the pFastBacDual transfer vector.PCR results showed that the target bands were successfully amplified.The sequencing results showed that there was no base mutation and the sequence was correct.PCR results of rBacmid showed that the target bands with the same size as expected were successfully amplified.The recombinant baculovirus was transfected into Sf9 cells.Western-blot analysis showed that recombinant baculovirus could express target proteins with the same sizes as expected after the infection with Sf9 cells.Immunofluorescence assay results showed that recombinant baculovirus could successfully express the target proteins after infection with Sf9 cells.The results of screening the multiplicity of infection and the time of infection of recombinant baculovirus showed that the optimal multiplicity of infection was 1 and the time of infection was 3 d.Density gradient centrifugation was used to purify the massprepared VLP.The purification result showed that there was an obvious band at the position of sample No.6.SDS-PAGE and Western-blot identification results showed that protein bands with the expected sizes can be detected in the centrifuge band sample,indicating that VLP was concentrated in this centrifuge band area.The purified VLP was observed by electron microscopy.The results showed that spherical particles with a diameter of 80-100 nm could be observed.The results of immunoelectron microscopy showed that CCHFV antibody could specifically label VLP.The above results indicated that CCHFV VLP was successfully obtained.The screened recombinant DNA expression vectors were packaged by CCHFV VLP respectively.And the chimeras were identified by immunofluorescence assay.The immunofluorescence assay results showed that the VLP/DNA chimeras were successfully packaged.The best package result was achieved when the ratio of VLP and DNA was 60:15.PCR identification results showed that the target bands were successfully detected.The packaging ratio was consistent with the IFA results.3.3 Comparing the immunological effects of multiple combined immunization strategies,chimeric immunization was determined to be superior to other combined immunization strategiesThe results of the comparison of immune effects of the three combined immunization strategies showed that,in terms of humoral immune response,the chimeric immunization group containing LAMP1 vector had a significantly stronger effect on the induction of CCHFV antigen(Gc,Gn,NP)specific antibodies than the DNA recombinant vectors(p<0.01),VLP alone(p<0.05)and the sequential immunization group with vector containing LAMP1 gene(p<0.001).In addition,the chimeric immunization group containing vector with LAMP1 significantly increased the titer of neutralizing antibody compared with the CCHFV VLP group(p=0.046),the mixed immunization group containing vector with LAMP1(p=0.046)and the sequential immunization group containing vector with LAMP1(p=0.0001).In terms of cellular immune response,the chimeric immunization group containing vector with LAMP1 had a stronger ability to induce cytokine secretion from spleen cells than other immune strategies(p<0.05).In terms of splenic lymphocyte types detection,the average levels of IFN-y and IL-4 production in CD4+T cells of mice in chimeric immunization group containing vector with LAMP1 were significantly higher than those in other groups(p<0.01).In addition,the average level of IFN-y production in CD8+T cells in chimeric immunization group containing vector with LAMP1 was significantly higher than that in other groups(p<0.001).In terms of CTL activity detection in spleen cells,the CTL activity of spleen cells in chimeric immunization group containing vector with LAMP1 enhanced with the increase of E/T ratio,and was the strongest at 100:1.Compared with other groups,spleen cells of the mice of chimeric immunization group containing vector with LAMP1 also showed higher CTL activity(p<0.01).In animal protection experiments,the chimeric immunization group containing vector with LAMP1 could provide stronger immune protection against the attack of CCHFV tecVLPs than other combined immunization strategies(p<0.05).4.ConclusionIn this study,several recombinant DNA expression vectors were successfully constructed.The immune effect of pVAX-LAMP1-NP was superior to other recombinant vectors.CCHFV VLP was successfully expressed by baculovirus expression vector system.VLP/DNA chimeras were successfully packaged with CCHFV VLP and pVAX-NP or pVAX-LAMP1-NP,respectively.By comparing the different immunization strategies of CCHFV VLP combined with pVAX-NP or pVAX-LAMP1-NP,chimeric immunization was superior to sequential and mixed immunization.This study provides a new idea for the development of novel CCHFV vaccines and combined immunization strategies with more comprehensive immune protection.