An effective vaccine against group A streptococcus(GAS)is highly desirable for definitive control of GAS infections.In the present study,two variants of amphiphilic chitosan nanoparticles-based GAS vaccines were devel...An effective vaccine against group A streptococcus(GAS)is highly desirable for definitive control of GAS infections.In the present study,two variants of amphiphilic chitosan nanoparticles-based GAS vaccines were developed.The vaccines were primarily composed of encapsulated KLH protein(a source of T helper cell epitopes)and lipidated M-protein derived B cell peptide epitope(lipoJ14)within the amphiphilic structure of nanoparticles.The only difference between themwas one of the nanoparticles vaccines received additional surface coating with poly(I:C).The formulated vaccines exhibited nanosized particles within the range of 220–240 nm.Cellular uptake study showed that nanoparticles vaccine without additional poly(I:C)coating has greater uptake by dendritic cells and macrophages compared to nanoparticles vaccine that was functionalized with poly(I:C).Both vaccines were found to be safe in mice and showed negligible cytotoxicity against HEK293 cells.Upon immunization in mice,both nanoparticle vaccines produced high antigen-specific antibodies titres that were regulated by a balanced Th1 and Th2 response compared to physical mixture.These antibodies elicited high opsonic activity against the tested GAS strains.Overall,our data demonstrated that amphiphilic chitosan nanoparticles platform induced a potent immune response even without additional inclusion of poly(I:C).展开更多
Some pathogens can use host suppressor of cytokine signaling I (SOCS-1), an important negative-feedback molecule, as the main mode of immune evasion. Here we found that group A Streptococcus (GAS) is capable of in...Some pathogens can use host suppressor of cytokine signaling I (SOCS-1), an important negative-feedback molecule, as the main mode of immune evasion. Here we found that group A Streptococcus (GAS) is capable of inducing SOCS-1 expression in RAW264.7 and BMDM macrophages. IFN-p plays a role in GAS-induced SOCS-1 expression in macrophages following the induction of cytokine expression by GAS, representing the classical pathway of SOCS-1 expression. However, GAS also induced STAT1 activation and SOCS-1 expression when GAS-infected cells were incubated with anti-IFN-p monoclonal antibody in this study. Moreover, upon comparing TLR4-/- BMDM macrophages with wild-type (WT) cells, we found that TLR4 also plays an essential role in the induction of SOCS-1. MyD88, which is an adaptor protein for TLR4, contributes to STAT1 activation and phosphorylation by forming a complex with Janus kinase 1 (JAK1) and signal transducer and activator of transcription 1 (STAT1) in macrophages. GAS-stimulated expression of STAT1 was severely impaired in MyD88-/- macrophages, whereas expression of JAK1 was unaffected, suggesting that MyD88 was involved in STAT1 expression and phosphorylation. Together, these data demonstrated that in addition to IFN-p signaling and MyD88 complex formation, JAK1 and STAT1 act in a novel pathway to directly induce SOCS-1 expression in GAS-infected macrophages, which may be more conducive to rapid bacterial infection.展开更多
Group A streptococcus (GAS), an important human pathogen, can cause various kinds of infections including superficial infections and potentially lethal infections, and the search for an effective vaccine to prevent ...Group A streptococcus (GAS), an important human pathogen, can cause various kinds of infections including superficial infections and potentially lethal infections, and the search for an effective vaccine to prevent GAS infections has been ongoing for many years. This paper compares the immunogenicity and immunoprotection of FbaA (an Fn-binding protein expressed on the surface of GAS) with that of M protein, the best immunogen of GAS. Assay for immune response showed that FbaA, similar to M protein, could induce protein-specific high IgG titer in BALB/c mice. Furthermore, following GAS challenge, the mice immunized with FbaA showed the same protective rate as those with M protein. These results indicate that FbaA is similar in ability to M protein in inducing protective immunity against GAS challenge in mice. Cellular & Molecular Immunology.展开更多
Autophagy serves an innate immune function in defending the host against invading bacteria,including group A Streptococcus(GAS).Autophagy is regulated by numerous host proteins,including the endogenous negative regula...Autophagy serves an innate immune function in defending the host against invading bacteria,including group A Streptococcus(GAS).Autophagy is regulated by numerous host proteins,including the endogenous negative regulator calpain,a cytosolic protease.Globally disseminated serotypeM1T1 GAS strains associated with high invasive disease potential express numerous virulence factors and resist autophagic clearance.Upon in vitro infection of human epithelial cell lines with representative wild-type GAS M1T1 strain 5448(M1.5448),we observed increased calpain activation linked to a specific GAS virulence factor,the interleukin-8 protease SpyCEP.Calpain activation inhibited autophagy and decreased capture of cytosolic GAS in autophagosomes.In contrast,the serotype M6 GAS strain JRS4(M6.JRS4),which is highly susceptible to host autophagy-mediated killing,expresses low levels of SpyCEP and does not activate calpain.Overexpression of SpyCEP in M6.JRS4 stimulated calpain activation,inhibited autophagy,and significantly decreased bacterial capture in autophagosomes.These paired loss-and gain-of-function studies reveal a novel role for the bacterial protease SpyCEP in enabling GAS M1 evasion of autophagy and host innate immune clearance.展开更多
Background:The neuropsychiatric disorders due to post-streptococcal autoimmune complications such as Sydenham's chorea(SC)are associated with acute rheumatic fever and rheumatic heart disease(ARF/RHD).An animal mo...Background:The neuropsychiatric disorders due to post-streptococcal autoimmune complications such as Sydenham's chorea(SC)are associated with acute rheumatic fever and rheumatic heart disease(ARF/RHD).An animal model that exhibits char-acteristics of both cardiac and neurobehavioral defects in ARF/RHD would be an important adjunct for future studies.Since age,gender,strain differences,and geno-types impact on the development of autoimmunity,we investigated the behavior of male and female Wistar and Lewis rat strains in two age cohorts(6 weeks and 12 weeks)under normal husbandry conditions and following exposure to group A streptococcus(GAS).Methods:Standard behavioral assessments were performed to determine the impair-ments in fine motor control(food manipulation test),gait and balance(beam walk-ing test),and obsessive-compulsive behavior(grooming and marble burying tests).Furthermore,electrocardiography,histology,and behavioral assessments were per-formed on male and female Lewis rats injected with GAS antigens.Results:For control Lewis rats there were no significant age and gender dependent differences in marble burying,food manipulation,beam walking and grooming be-haviors.In contrast significant age-dependent differences were observed in Wistar rats in all the behavioral tests except for food manipulation.Therefore,Lewis rats were selected for further experiments to determine the effect of GAS.After ex-posure to GAS,Lewis rats demonstrated neurobehavioral abnormalities and cardiac pathology akin to SC and ARF/RHD,respectively.Conclusion:We have characterised a new model that provides longitudinal stability of age-dependent behavior,to simultaneously investigate both neurobehavioral and cardiac abnormalities associated with post-streptococcal complications.展开更多
Development of an effective vaccine against the leading human bacterial pathogen group A Streptococcus(GAS)is a public health priority.The species defining group A cell wall carbohydrate(GAC,Lancefield antigen)can be ...Development of an effective vaccine against the leading human bacterial pathogen group A Streptococcus(GAS)is a public health priority.The species defining group A cell wall carbohydrate(GAC,Lancefield antigen)can be engineered to remove its immunodominant N-acetylglucosamine(GlcNAc)side chain,implicated in provoking autoimmune cross-reactivity in rheumatic heart disease,leaving its polyrhamnose core(GACPR).Here we generate a novel protein conjugate of the GACPR and test the utility of this conjugate antigen in active immunization.Instead of conjugation to a standard carrier protein,we selected SpyAD,a highly conserved GAS surface protein containing both B-cell and T-cell epitopes relevant to the bacterium that itself shows promise as a vaccine antigen.SpyAD was synthesized using the XpressTM cell-free protein expression system,incorporating a non-natural amino acid to which GACpr was conjugated by site-specific click chemistry to yield high molecular mass SpyAD-GACPR conjugates and avoid disruption of important T-cell and B-cell immunological epitopes.The conjugated SpyAD-GACPR elicited antibodies that bound the surface of multiple GAS strains of diverse M types and promoted opsonophagocytic killing by human neutrophils.Active immunization of mice with a multivalent vaccine consisting of SpyAD-GACPR,together with candidate vaccine antigens streptolysin O and C5a peptidase,protected against GAS challenge in a systemic infection model and localized skin infection model,without evidence of cross reactivity to human heart or brain tissue epitopes.This general approach may allow GAC to be safely and effectively included in future GAS subunit vaccine formulations with the goal of broad protection without autoreactivity.展开更多
基金supported financially by Universiti Kebangsaan Malaysia(UKM),Malaysia[DCP-2017-003/2].
文摘An effective vaccine against group A streptococcus(GAS)is highly desirable for definitive control of GAS infections.In the present study,two variants of amphiphilic chitosan nanoparticles-based GAS vaccines were developed.The vaccines were primarily composed of encapsulated KLH protein(a source of T helper cell epitopes)and lipidated M-protein derived B cell peptide epitope(lipoJ14)within the amphiphilic structure of nanoparticles.The only difference between themwas one of the nanoparticles vaccines received additional surface coating with poly(I:C).The formulated vaccines exhibited nanosized particles within the range of 220–240 nm.Cellular uptake study showed that nanoparticles vaccine without additional poly(I:C)coating has greater uptake by dendritic cells and macrophages compared to nanoparticles vaccine that was functionalized with poly(I:C).Both vaccines were found to be safe in mice and showed negligible cytotoxicity against HEK293 cells.Upon immunization in mice,both nanoparticle vaccines produced high antigen-specific antibodies titres that were regulated by a balanced Th1 and Th2 response compared to physical mixture.These antibodies elicited high opsonic activity against the tested GAS strains.Overall,our data demonstrated that amphiphilic chitosan nanoparticles platform induced a potent immune response even without additional inclusion of poly(I:C).
文摘Some pathogens can use host suppressor of cytokine signaling I (SOCS-1), an important negative-feedback molecule, as the main mode of immune evasion. Here we found that group A Streptococcus (GAS) is capable of inducing SOCS-1 expression in RAW264.7 and BMDM macrophages. IFN-p plays a role in GAS-induced SOCS-1 expression in macrophages following the induction of cytokine expression by GAS, representing the classical pathway of SOCS-1 expression. However, GAS also induced STAT1 activation and SOCS-1 expression when GAS-infected cells were incubated with anti-IFN-p monoclonal antibody in this study. Moreover, upon comparing TLR4-/- BMDM macrophages with wild-type (WT) cells, we found that TLR4 also plays an essential role in the induction of SOCS-1. MyD88, which is an adaptor protein for TLR4, contributes to STAT1 activation and phosphorylation by forming a complex with Janus kinase 1 (JAK1) and signal transducer and activator of transcription 1 (STAT1) in macrophages. GAS-stimulated expression of STAT1 was severely impaired in MyD88-/- macrophages, whereas expression of JAK1 was unaffected, suggesting that MyD88 was involved in STAT1 expression and phosphorylation. Together, these data demonstrated that in addition to IFN-p signaling and MyD88 complex formation, JAK1 and STAT1 act in a novel pathway to directly induce SOCS-1 expression in GAS-infected macrophages, which may be more conducive to rapid bacterial infection.
基金supported by National Natural Science Foundation of China (30771970 and 30872399)Scientific Research Foundation of Health Bureau of Hebei Province (08054).
文摘Group A streptococcus (GAS), an important human pathogen, can cause various kinds of infections including superficial infections and potentially lethal infections, and the search for an effective vaccine to prevent GAS infections has been ongoing for many years. This paper compares the immunogenicity and immunoprotection of FbaA (an Fn-binding protein expressed on the surface of GAS) with that of M protein, the best immunogen of GAS. Assay for immune response showed that FbaA, similar to M protein, could induce protein-specific high IgG titer in BALB/c mice. Furthermore, following GAS challenge, the mice immunized with FbaA showed the same protective rate as those with M protein. These results indicate that FbaA is similar in ability to M protein in inducing protective immunity against GAS challenge in mice. Cellular & Molecular Immunology.
基金supported in part by Marie Skłodowska-Curie actions Intra-European Fellowships(MC-IEF-329520)The Naito Foundation,The Takeda Science Foundation,and the US National Institutes of Health(R01-AI145325)。
文摘Autophagy serves an innate immune function in defending the host against invading bacteria,including group A Streptococcus(GAS).Autophagy is regulated by numerous host proteins,including the endogenous negative regulator calpain,a cytosolic protease.Globally disseminated serotypeM1T1 GAS strains associated with high invasive disease potential express numerous virulence factors and resist autophagic clearance.Upon in vitro infection of human epithelial cell lines with representative wild-type GAS M1T1 strain 5448(M1.5448),we observed increased calpain activation linked to a specific GAS virulence factor,the interleukin-8 protease SpyCEP.Calpain activation inhibited autophagy and decreased capture of cytosolic GAS in autophagosomes.In contrast,the serotype M6 GAS strain JRS4(M6.JRS4),which is highly susceptible to host autophagy-mediated killing,expresses low levels of SpyCEP and does not activate calpain.Overexpression of SpyCEP in M6.JRS4 stimulated calpain activation,inhibited autophagy,and significantly decreased bacterial capture in autophagosomes.These paired loss-and gain-of-function studies reveal a novel role for the bacterial protease SpyCEP in enabling GAS M1 evasion of autophagy and host innate immune clearance.
基金RAM Rafeek is recipient of International Postgraduate Research Award(IPRA)from University of New England.CM Lobbe and E.Wilkinson are recipients of student scholarship from the Royal College of Pathologists of Australasia(RCPA).
文摘Background:The neuropsychiatric disorders due to post-streptococcal autoimmune complications such as Sydenham's chorea(SC)are associated with acute rheumatic fever and rheumatic heart disease(ARF/RHD).An animal model that exhibits char-acteristics of both cardiac and neurobehavioral defects in ARF/RHD would be an important adjunct for future studies.Since age,gender,strain differences,and geno-types impact on the development of autoimmunity,we investigated the behavior of male and female Wistar and Lewis rat strains in two age cohorts(6 weeks and 12 weeks)under normal husbandry conditions and following exposure to group A streptococcus(GAS).Methods:Standard behavioral assessments were performed to determine the impair-ments in fine motor control(food manipulation test),gait and balance(beam walk-ing test),and obsessive-compulsive behavior(grooming and marble burying tests).Furthermore,electrocardiography,histology,and behavioral assessments were per-formed on male and female Lewis rats injected with GAS antigens.Results:For control Lewis rats there were no significant age and gender dependent differences in marble burying,food manipulation,beam walking and grooming be-haviors.In contrast significant age-dependent differences were observed in Wistar rats in all the behavioral tests except for food manipulation.Therefore,Lewis rats were selected for further experiments to determine the effect of GAS.After ex-posure to GAS,Lewis rats demonstrated neurobehavioral abnormalities and cardiac pathology akin to SC and ARF/RHD,respectively.Conclusion:We have characterised a new model that provides longitudinal stability of age-dependent behavior,to simultaneously investigate both neurobehavioral and cardiac abnormalities associated with post-streptococcal complications.
基金supported by the CARB-X,Vaxcyte,Inc.,and NIH Grant(AI077780)NJG was supported in part by a Ruth L.Kirschstein Institutional National Research Award from the National Institute for General Medical Sciences(T32 GM008666)。
文摘Development of an effective vaccine against the leading human bacterial pathogen group A Streptococcus(GAS)is a public health priority.The species defining group A cell wall carbohydrate(GAC,Lancefield antigen)can be engineered to remove its immunodominant N-acetylglucosamine(GlcNAc)side chain,implicated in provoking autoimmune cross-reactivity in rheumatic heart disease,leaving its polyrhamnose core(GACPR).Here we generate a novel protein conjugate of the GACPR and test the utility of this conjugate antigen in active immunization.Instead of conjugation to a standard carrier protein,we selected SpyAD,a highly conserved GAS surface protein containing both B-cell and T-cell epitopes relevant to the bacterium that itself shows promise as a vaccine antigen.SpyAD was synthesized using the XpressTM cell-free protein expression system,incorporating a non-natural amino acid to which GACpr was conjugated by site-specific click chemistry to yield high molecular mass SpyAD-GACPR conjugates and avoid disruption of important T-cell and B-cell immunological epitopes.The conjugated SpyAD-GACPR elicited antibodies that bound the surface of multiple GAS strains of diverse M types and promoted opsonophagocytic killing by human neutrophils.Active immunization of mice with a multivalent vaccine consisting of SpyAD-GACPR,together with candidate vaccine antigens streptolysin O and C5a peptidase,protected against GAS challenge in a systemic infection model and localized skin infection model,without evidence of cross reactivity to human heart or brain tissue epitopes.This general approach may allow GAC to be safely and effectively included in future GAS subunit vaccine formulations with the goal of broad protection without autoreactivity.