| Toxoplasma gondii is an important medical pathogen that infects approximately 30%of the global population.Generally,toxoplasmosis is asymptomatic in immune-competent hosts,however,it can result in severe symptoms in immunocompromised individuals due to cerebral cyst reactivation.Another potentially fatal presentation is vertical transmission in the fetus,which can result in encephalitis,neonatal malformations,or spontaneous abortion.Current drug treatment cannot control this disease completely because of the inability of drugs to kill bradyzoites.Therefore,the advantages of a preemptive vaccine for preventing toxoplasmosis are obvious.Traditional vaccine development strategies against T.gondii mainly focused on subunit and DNA vaccines.Their use raises several issues,since subunit vaccines have poor stability and may cause undesired immune responses and DNA vaccines have the theoretical risk of genomic integration into host cells.Peptide-based vaccines could overcome these weaknesses.They use minimal antigenic epitopes to induce desired immune responses,and are less likely to trigger allergic or autoimmune responses.In recent years,there has been increasing interest in the study of peptide-based vaccines.T.gondii is an intracellular parasite with a complex life cycle,so synthetic multiple antigenic peptide(MAP)vaccines containing different epitopes may prove a highly efficacious strategy in the development of T.gondii vaccines.For vaccines to be effective against toxoplasmosis,they should include antigen epitopes that can elicit a protective Th1 immune response,characterized by the generation of long-lived CD8 T cells that can secrete IFN-y and develop cytotoxic activity against infected cells.CD8 cytotoxic T lymphocyte(CTL)-mediated resistance to T.gondii cysts in the brain is absolutely correlated with the major histocompatibility complex(MHC)class I Ld allele in mice.The secreted proteins of T.gondii,such as dense granules(GRAs)and rhoptry proteins(ROPs),are antigens that are recognized by murine T lymphocytes and are vaccine candidates against toxoplasmosis.The peptide(HPGSVNEFDF)(HF10),derived from GRA6,and the peptide(IPAAAGRFF),derived from ROP7,are the protective immunodominant Ld-restricted epitopes in mice.Antibodies also play an important role in host immunity against T.gondii,they can directly block tachyzoites and impair their attachment to host cells.SAG1 protein is the major immunogenic surface antigen that is involved in attachment to host cells during T.gondii invasion,which makes it a suitable source of B cell epitopes.SAG 182-102 is a highly immunogenic conformational B cell epitope but was not considered as a vaccine candidate because of the lack of a suitable carrier to maintain the native spatial conformation until date SAG1301-320 is a linear B cell epitope that can protect mice against a lethal challenge and is strongly recognized by sera from toxoplasmosis patients.Moreover,CD4+T cells constitute an important component of the immune response to T.gondii,and AS 15 is a CD4+T cell-stimulating peptide that can confer protection against toxoplasmosis.Most of these molecules are conserved between type I and type II strains of T.gondii.Although all three strains of the parasite have been isolated from humans,type I and type II strains are more often associated with human disease and the type III strain seems to be more common in animalsThe life cycle of T.gondii is very complex,so we selected a B cell epitope(SAG 182-102 or SAG 1301-320),a CD8+T cell epitope(HF10 or ROP7)and a CD4+T cell epitope(AS 15)as candidate molecules for multiple antigenic peptide vaccines against T.gondii.However,the above MAP vaccines are not without disadvantages For example,peptides themselves are poor immunogens and require the help of delivery systems or adjuvants.Moreover,they are highly susceptible to enzymatic degradation and lack stability compared to natively folded proteins.Therefore,it is particularly important to find suitable vectors for MAP vaccinesVirus-like particles(VLPs)are diverse nanoparticles(size 20-100nm)that are formed by structural viral proteins,such as capsids,and can self-assemble in vitro They resemble viruses but are noninfectious because they lack viral genetic materials VLPs mimic the 3D conformation of native viruses and display a high density of repetitive effective antigenic epitopes on their surface,which stimulate the desired humoral and cellular responses in humans.In recent years,VLPs have provided an excellent option as vectors for producing vaccines against infectious diseases and some VLPs vaccines are available commercially.However,not much research has been performed on VLPs vaccines against T.gondii,and more work is required to fill the gaps in this promising field.Hepatitis B virus(HBV)core antigen(HBc)has been extensively used as a VLPs platform for many years.It is highly expressed in many recombinant gene expression systems,including prokaryotic expression systems,and is easy to self-assemble in vitro.It can greatly improve the immunogenicity of foreign antigenic epitopes presented on its surface,especially those inserted into the major immune dominant region(MIR)of the capsid located at the tips of the surface "spikes".Various foreign antigens from bacteria,viruses,and protozoa have been genetically inserted into the particles,and some of these HBc-antigen fusions have reached the clinical testing stage.In view of this,the chimeric VLPs vaccines constructed by loading multiple antigenic peptide derived from T.gondii onto HBc particles are expected to develop into a highly effective vaccine against T.gondii infection.Objectives1.The conformational B cell epitope,td-restricted CD8+T cell epitope and Ab-restricted CD4+ T cell epitope were loaded onto HBc virus like particles,and the feasibility of this new T.gondii vaccine formulation were explore in this study.2.As a vaccine against T.gondii,the efficacy of chimeric HBc virus like particles was evaluated using acute and chronic T.gondii infection model in mice.Methods1.Construction,purification and identification of chimeric HBc VLPsIn this study,the CD8 cell epitope(HF10 or ROP7)and the B cell epitope(SAG182-102 or SAG1301-320)of T.gondii were inserted between amino acids 78 and 79 of truncated HBc particles(HBc?),with a Gln(Q)and Asp(D)linker at both ends.A CD4+cell epitope(AS 15)was fused into the C-terminal of HBc? particles.The final recombinant plasmids were named pET-30a(+)/HBc?,HBc?H82,HBC?H301,HBc?R82 and HBc?R301.After amplification and identification,the five recombinant plasmids were transformed into E.coli BL21(DE3)cells.After induction by IPTG,chimeric HBc VLPs(HBc?,HBc?H82,HBc?H301,HBc?R82 and HBc?R301)were collected.These target proteins were identified by SDS-PAGE gel electrophoresis.After induction of large expression of the chimeric HBc VLPs,the HBc? protein present in a soluble state was directly purified by Ni-NTA agarose gel,and the protein was collected after dialysis concentration.And the chimeric proteins(HBc?H82,HBc?H301,HBc?R82 and HBc?R301)present in the precipitated state of inclusion bodies are first dissolved in a lysate containing urea,and purified by Ni-NTA agarose gel,and then subjected to gradient dialysis renaturation,finally getting the correct folded proteinSince the chimeric HBc VLPs contain a His-tagged protein,they can be bound to the His-Tag monoclonal antibody,and thus can be identified by Western Blot assay Finally,the chimeric HBc VLPs were analyzed by transmission electron microscopy to determine whether them formed the icosahedral three-dimensional structure of the virus2.Evaluation of immunogenicity of the chimeric HBc VLPs vaccinesEndotoxin contamination in the chimeric HBc VLPs was analyzed using Limulus amebocyte lysate(LAL).Four multiepitope peptides,i.e.,H82,H301,R82,and R301,containing a CD8+cell epitope(HF10 or ROP7),a B cell epitope(SAG182-102 or SAG1301-320),and a CD4+cell epitope(AS 15)were synthesizedThe mice were segregated into groups of 23 each and immunized subcutaneously with 50 ?g of the recombinant protein,i.e.,HBc?,H82,HBc?H82,H301,HBc?H301,R82,HBc?R82,R301,or HBc?R301)or 50 ?l PBS(blank control)on days 0,14,and 28 For monitoring of the humoral response,serum samples were collected by retro-orbital bleeding at 0,2,4,and 6 weeks after the first immunization and were stored at-80? until analysis.For monitoring of the cellular response,lymphocyte proliferation and cytokine production tests were performed.The T.gondii-specific IgG,IgGl and IgG2a antibody levels in the serum samples were measured using enzyme-linked immunosorbent assay(ELISA).At 2 weeks after the last immunization,three mice per group were sacrificed and their spleens were obtained.The percentages of the CD4 and CD8 T lymphocytes subsets of immunized mice were analyzed by flow cytometry.The cell-free supernatants from cultured splenocytes were collected and assayed for IL-2 and IL-4 activities at 24 h,for IL-10 activity at 72 h,and for IFN-y activity at 96 h using ELISA assay.To investigate the immune protection of the chimeric HBc VLPs against acute T.gondii infection,10 mice per group were infected intraperitoneally with a lethal dose(200 tachyzoites)of the RH strain and were monitored over 20 days.To evaluate the effect of vaccination in mice with chronic toxoplasmosis,the remaining 10 mice in each group were challenged orally with a sublethal dose(30 cysts)of the PRU strain.All the mice were euthanized at 45 days after the challenge and their brains were removed and homogenized in 1 ml PBS.The T.gondii cyst burden in the mouse brains was confirmed by analyzing 10 ?l samples of each cerebral homogenate with a light microscope.Results1.The chimeric HBc VLPs vaccines were successfully constructedAfter digestion with restriction endonucleases,the recombinant plasmids(pET-30a(+)/HBc?,HBc?H82,HBc?H301,HBc?R82,and HBc?R301)were confirmed by agarose gel electrophoresis.The gene fragment of the chimeric HBc VLPs has been correctly inserted into the prokaryotic expression plasmid pET-30a(+),and the recombinant plasmids were successfully constructed.Whether the soluble protein(HBc?)or inclusion body proteins(HBc?H82,HBc?H301,HBc?R82,and HBc?R301),higher purity chimeric HBc VLPs were obtained by Ni-NTA agarose gel.Western blot and transmission electron microscopy analysis showed that His-tagged the chimeric HBc VLPs(HBc?H82,HBc?H301,HBc?R82,and HBc?R301)were successfully expressed in prokaryotic expression system and demonstrated good capability for self-assembly and had icosahedral morphology similar to the original corresponding non-chimeric VLPs(HBc?),which ensured that the B cell epitope of T.gondii maintained the correct spatial conformation,densely and uniformly presented the epitopes derived from this parasite on the surface of the particles.It lays a solid material basis for the subsequent induction of T.gondii specific humoral and cellular immune responses in mice.2.The chimeric HBc VLPs had strong immunogenicityTotal IgG antibodies were detectable as early as 4 weeks after the first vaccination in the mice immunized with HBc?H82 and HBc?R82 protein compared with the mice immunized with PBS.And these proteins induced the highest IgG antibody levels 6 weeks after the first vaccination in all vaccinated mice(p<0.001).Compared with the control group,only mice immunized with HBc?H82 protein showed an increase in antibody titers of IgG1 and IgG2a(p<0.05),and the ratio of IgGl/IgG2a was less than 1 in all immunized mice.This indicated that the chimeric HBc VLPs vaccine induced a specific immune response based on Thl type in mice.The percentages of CD4+T cells in the mice immunized with the recombinant proteins containing the CD4+T cell epitope(AS 15),especially the chimeric HBc particles(HBc?H82,HBc?H301,HBc?R82 and HBc?R301),were higher than those for the PBS group(p<0.01).Apart for the finding for the mice immunized with HBc? and PBS,the percentages of CD8+T cells in mice immunized with other recombinant proteins all increased,especially for the chimeric HBc VLPs containing HF10 epitope(HBc?H82 and HBc?H301;p<0.001).The IFN-y levels in the splenocyte supernatants from the mice immunized with HBc?H82 and HBc?H301(p<0.001),HBc?R82 and HBc?R301(p<0.01),and with H82,H301,R82 and R301(p<0.05)proteins were significantly higher than those of the mice immunized with PBS.The HBc?H82(p<0.001)and HBc?H301(p<0.01)vaccinated mice had higher IL-2 levels than the control group did.However,a slight increase in IL-4 and IL-10 production was observed only in the mice immunized with HBc?H82 protein compared with those of PBS.The mice vaccinated with the chimeric HBc VLPs:HBc?H82(15.6±3.8 days),HBc?H301(8.1 ± 1.5 days),HBc?R82(8.8±1.3 days),and HBc?R301(6.6±1.2 days)were partially protected compared with the control group(PBS).At 45 days after the challenge,the mice vaccinated with HBc?H82(1454±239;p<0.01)and HBc?H301(1730 ± 230;p<0.05)proteins had fewer cysts than the PBS immunized mice did(2091±263).These results suggested that immunization of the mice with HBc?H82,which contained the conformational B cell epitopes(SAG 182-102),CD8 T cell epitope(HF10),and CD4 T cell epitope(AS 15),induced substantial resistance to acute and chronic infection with T gondii.Conclusions1.Chimeric HBc VLPs(HBc?H82,HBc?H301,HBc?R82,and HBc?R301)can be successfully expressed in E.coli and had icosahedral morphology similar to the original VLPs(HBc?).It is possible to correctly and densely display the B cell conformational epitope and T cell linear epitopes of T.gondi in the particles.2.The chimeric HBc VLPs vaccines(HBc?H82,HBc?H301,HBc?R82,and HBc?R301)have strong immunogenicity and can induce strong T.gondii specific humoral and cellular immune responses in mice.It is a new and effective vaccine formulation against acute and chronic toxoplasmosis in mice.Innovation and significanceInnovation:This study was the first to construct a vaccine containing a B cell conformational epitope derived from T.gondii SAG1.For the first time,a multiple antigenic peptide vaccine containing a conformation B cell epitope,an Ld-restricted CD8+T cell epitope and an Ab-restricted CD4+T cell epitope was loaded onto HBc VLPs.This new vaccine formulation of T.gondii has proven to be an effective strategy for the prevention of toxoplasmosis.Significance:This study provided a new and effective vaccine formulation against T.gondii infection.It gave an important reference for the development of safe,effective,economical T.gondii vaccine,and lay the foundation for the prevention and control of toxoplasmosis. |
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