The most common medications for the treatment of zoonotic toxoplasmosis are pyrimethamine and sulfadiazine,which may cause serious undesirable side effects.Thus,there is an urgent need to develop novel therapeutics.Ba...The most common medications for the treatment of zoonotic toxoplasmosis are pyrimethamine and sulfadiazine,which may cause serious undesirable side effects.Thus,there is an urgent need to develop novel therapeutics.Baicalein(BAI,C_(15)H_(10)O_(5))has been shown to perform well against protozoan parasites including Leishmania and Cryptosporidium.In this study,the inhibition efficacy of BAI on Toxoplasma gondii was evaluated using plaque,invasion,and intracellular proliferation assays.BAI effectively inhibited T.gondii(half-maximum inhibitory concentration(IC_(50))=6.457×10^(−5)mol/L),with a reduced invasion rate(33.56%)and intracellular proliferation,and exhibited low cytotoxicity(half-maximum toxicity concentration(TC_(50))=5.929×10^(−4)mol/L).Further investigation using a mouse model shed light on the inhibitory efficacy of BAI against T.gondii,as well as the potential mechanisms underlying its anti-parasitic effects.The survival time of T.gondii-infected ICR mice treated with BAI was remarkably extended,and their parasite burdens in the liver and spleen were greatly reduced compared with those of the negative control group.Histopathological examination of live sections revealed effective therapeutic outcomes in the treatment groups,with no notable pathological alterations observed.Furthermore,alterations in cytokine levels indicated that BAI not only effectively suppressed the growth of T.gondii but also prevented excessive inflammation in mice.Collectively,these findings underscore the significant inhibitory efficacy of BAI against T.gondii,positioning it as a promising alternative therapeutic agent for toxoplasmosis.展开更多
Background Toxoplasma gondii,an intracellular parasitic protozoan,which infects almost all warm-blooded animals,including humans,causes toxoplasmosis.However,we lack effective drugs and vaccines to control toxoplasmos...Background Toxoplasma gondii,an intracellular parasitic protozoan,which infects almost all warm-blooded animals,including humans,causes toxoplasmosis.However,we lack effective drugs and vaccines to control toxoplasmosis,representing a clinical challenge.Therefore,safe and effective vaccines are urgently needed.In this study,a self-replicating mRNA vaccine comprising four T.gondii antigens:ROP18,TGME49_237490,TGME49_268230,and MIC13,named 4x-mRNA-LNP(lipid nanoparticle),was developed,and its protective efficacy was evaluated in mice.Methods The expression of this vaccine in eukaryotic Human embryonic kidney 293 T(HEK-293 T)cells and mouse myoblast(C2C12)cells were analyzed,followed by enzyme-linked immunosorbent assay(ELISA)evaluation of the elicited humoral immune response.Subsequently,the vaccine-triggered immune responses in mice were detected,including antibody titers,T lymphocyte subsets,and cytokine levels.Finally,its immunoprotective effects were evaluated after challenging mice with T.gondii PRU oocysts or tachyzoites of different strains and analyzing the pathological changes,parasite loads,and mouse survival time.Western blotting and ELISA confirmed the successful eukaryotic expression and immunogenicity of 4x-mRNA,respectively.Statistical analyses,including the log-rank(Mantel–Cox)test,Student’s t-test,and one-way ANOVA,were performed using GraphPad Prism software.Results Mice vaccinated with 4x-mRNA-LNP generated higher levels of IgG1 and IgG2a antibodies(P<0.05)and cytokines(IL-2,IL-4,IL-10,IL-12,IFN-γ)(P<0.05)compared with the control group.The high specific IgG titer was maintained for at least 10 weeks after the last vaccination.The proportion of CD3^(+)CD4^(+)T cells and CD3^(+)CD8^(+)T cells also increased significantly(P<0.05),along with increased spleen cell proliferation in 4x-mRNA-LNP-vaccinated mice.Notably,limited pathological changes and<10 fg of parasites/mg were found in the immunized mice tissues post-pathogen challenge.During observation for 30 days,4x-mRNA-LNP-immunized mice survived significantly longer under challenge with lethal doses of RH,ME49,or WH6 tachyzoites(survival rates=60%,80%,and 60%,respectively).Following PRU oocyst challenge,vaccinated mice had notably decreased cyst burdens(72.5%,P<0.05)compared with control mice.Conclusions The 4x-mRNA-LNP vaccine triggered effective long-term antibody levels in mice,thus representing a promising candidate to further develop anti-toxoplasmosis vaccines.展开更多
基金supported by the National Natural Science Foundation of China(No.32370997)the Key Projects Jointly Constructed by the Ministry and the Province of Zhejiang Medical and Health Science and Technology Project(No.WKJ-ZJ-2545)+2 种基金the Chinese Medicine Science and Technology Program of Zhejiang Province(No.2024ZL367)the Health Commission of Zhejiang Province(No.2024KY923)the Foundation of GuoTai(Taizhou)Center of Technology Innovation for Veterinary Biologicals(No.GTKF(23)001),China.
文摘The most common medications for the treatment of zoonotic toxoplasmosis are pyrimethamine and sulfadiazine,which may cause serious undesirable side effects.Thus,there is an urgent need to develop novel therapeutics.Baicalein(BAI,C_(15)H_(10)O_(5))has been shown to perform well against protozoan parasites including Leishmania and Cryptosporidium.In this study,the inhibition efficacy of BAI on Toxoplasma gondii was evaluated using plaque,invasion,and intracellular proliferation assays.BAI effectively inhibited T.gondii(half-maximum inhibitory concentration(IC_(50))=6.457×10^(−5)mol/L),with a reduced invasion rate(33.56%)and intracellular proliferation,and exhibited low cytotoxicity(half-maximum toxicity concentration(TC_(50))=5.929×10^(−4)mol/L).Further investigation using a mouse model shed light on the inhibitory efficacy of BAI against T.gondii,as well as the potential mechanisms underlying its anti-parasitic effects.The survival time of T.gondii-infected ICR mice treated with BAI was remarkably extended,and their parasite burdens in the liver and spleen were greatly reduced compared with those of the negative control group.Histopathological examination of live sections revealed effective therapeutic outcomes in the treatment groups,with no notable pathological alterations observed.Furthermore,alterations in cytokine levels indicated that BAI not only effectively suppressed the growth of T.gondii but also prevented excessive inflammation in mice.Collectively,these findings underscore the significant inhibitory efficacy of BAI against T.gondii,positioning it as a promising alternative therapeutic agent for toxoplasmosis.
基金supported by the National Natural Science Foundation of China[Grant Numbers 32370997,81871684]the Provincial Key R&D program of Zhejiang Department of Science and Technology[Grant Number 2022C03109]+3 种基金the Central Leading Local Science and Technology Development Fund Project[Grant Number 2023ZY1019]the Key Projects Jointly Constructed by the Ministry and the Province of Zhejiang Medical and Health Science and Technology Project[Grant Number WKJ-ZJ-2545]the Foundation of GuoTai(Taizhou)Center of Technology Innovation for Veterinary Biologicals[Grant Number GTKF(23)001]the Key Discipline of Zhejiang Province in Public Health and Preventive Medicine(First Class,Category A),Hangzhou Medical College.
文摘Background Toxoplasma gondii,an intracellular parasitic protozoan,which infects almost all warm-blooded animals,including humans,causes toxoplasmosis.However,we lack effective drugs and vaccines to control toxoplasmosis,representing a clinical challenge.Therefore,safe and effective vaccines are urgently needed.In this study,a self-replicating mRNA vaccine comprising four T.gondii antigens:ROP18,TGME49_237490,TGME49_268230,and MIC13,named 4x-mRNA-LNP(lipid nanoparticle),was developed,and its protective efficacy was evaluated in mice.Methods The expression of this vaccine in eukaryotic Human embryonic kidney 293 T(HEK-293 T)cells and mouse myoblast(C2C12)cells were analyzed,followed by enzyme-linked immunosorbent assay(ELISA)evaluation of the elicited humoral immune response.Subsequently,the vaccine-triggered immune responses in mice were detected,including antibody titers,T lymphocyte subsets,and cytokine levels.Finally,its immunoprotective effects were evaluated after challenging mice with T.gondii PRU oocysts or tachyzoites of different strains and analyzing the pathological changes,parasite loads,and mouse survival time.Western blotting and ELISA confirmed the successful eukaryotic expression and immunogenicity of 4x-mRNA,respectively.Statistical analyses,including the log-rank(Mantel–Cox)test,Student’s t-test,and one-way ANOVA,were performed using GraphPad Prism software.Results Mice vaccinated with 4x-mRNA-LNP generated higher levels of IgG1 and IgG2a antibodies(P<0.05)and cytokines(IL-2,IL-4,IL-10,IL-12,IFN-γ)(P<0.05)compared with the control group.The high specific IgG titer was maintained for at least 10 weeks after the last vaccination.The proportion of CD3^(+)CD4^(+)T cells and CD3^(+)CD8^(+)T cells also increased significantly(P<0.05),along with increased spleen cell proliferation in 4x-mRNA-LNP-vaccinated mice.Notably,limited pathological changes and<10 fg of parasites/mg were found in the immunized mice tissues post-pathogen challenge.During observation for 30 days,4x-mRNA-LNP-immunized mice survived significantly longer under challenge with lethal doses of RH,ME49,or WH6 tachyzoites(survival rates=60%,80%,and 60%,respectively).Following PRU oocyst challenge,vaccinated mice had notably decreased cyst burdens(72.5%,P<0.05)compared with control mice.Conclusions The 4x-mRNA-LNP vaccine triggered effective long-term antibody levels in mice,thus representing a promising candidate to further develop anti-toxoplasmosis vaccines.
