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1.
Nat Commun ; 11(1): 851, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32051408

RESUMO

Lipopolysaccharide (LPS) O-antigen (O-Ag) is known to limit antibody binding to surface antigens, although the relationship between antibody, O-Ag and other outer-membrane antigens is poorly understood. Here we report, immunization with the trimeric porin OmpD from Salmonella Typhimurium (STmOmpD) protects against infection. Atomistic molecular dynamics simulations indicate this is because OmpD trimers generate footprints within the O-Ag layer sufficiently sized for a single IgG Fab to access. While STmOmpD differs from its orthologue in S. Enteritidis (SEn) by a single amino-acid residue, immunization with STmOmpD confers minimal protection to SEn. This is due to the OmpD-O-Ag interplay restricting IgG binding, with the pairing of OmpD with its native O-Ag being essential for optimal protection after immunization. Thus, both the chemical and physical structure of O-Ag are key for the presentation of specific epitopes within proteinaceous surface-antigens. This enhances combinatorial antigenic diversity in Gram-negative bacteria, while reducing associated fitness costs.


Assuntos
Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Imunização , Antígenos O/imunologia , Salmonella typhimurium/imunologia , Animais , Anticorpos Antibacterianos/sangue , Formação de Anticorpos , Especificidade de Anticorpos , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteção Cruzada , Modelos Animais de Doenças , Epitopos/química , Epitopos/imunologia , Imunoglobulina G/sangue , Camundongos , Modelos Moleculares , Antígenos O/química , Antígenos O/genética , Porinas/química , Porinas/genética , Porinas/imunologia , Conformação Proteica , Salmonelose Animal/imunologia , Salmonelose Animal/prevenção & controle , Análise de Sequência de Proteína
2.
J Infect Dis ; 221(2): 191-200, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31504647

RESUMO

BACKGROUND: Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Here, we determined the ability of a C. trachomatis recombinant major outer membrane protein (rMOMP) vaccine to elicit cross-serogroup protection. METHODS: Female C3H/HeN mice were vaccinated by mucosal and systemic routes with C. trachomatis serovar D (UW-3/Cx) rMOMP and challenged in the ovarian bursa with serovars D (UW-3/Cx), D (UCI-96/Cx), E (IOL-43), or F (N.I.1). CpG-1826 and Montanide ISA 720 were used as adjuvants. RESULTS: Immune responses following vaccination were more robust against the most closely related serovars. Following a genital challenge (as determined by number of mice with positive vaginal cultures, number of positive cultures, number of inclusion forming units recovered, and number of days with positive cultures) mice challenged with C. trachomatis serovars of the same complex were protected but not those challenged with serovar F (N.I.1) from a different subcomplex. Females were caged with male mice. Based on fertility rates, number of embryos, and hydrosalpinx formation, vaccinated mice were protected against challenges with serovars D (UW-3/Cx), D (UCI-96/Cx), and E (IOL-43) but not F (N.I.1). CONCLUSIONS: This is the first subunit vaccine shown to protect mice against infection, pathology, and infertility caused by different C. trachomatis serovars.


Assuntos
Infecções por Chlamydia/prevenção & controle , Proteção Cruzada/imunologia , Infertilidade Feminina/prevenção & controle , Porinas/imunologia , Vacinas Sintéticas/imunologia , Vagina/microbiologia , Animais , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/uso terapêutico , Infecções por Chlamydia/imunologia , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/imunologia , Chlamydia trachomatis/isolamento & purificação , Feminino , Imunoglobulina G , Infertilidade Feminina/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Gravidez , Sorogrupo , Vagina/imunologia
3.
Biologicals ; 62: 22-26, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31668855

RESUMO

Salmonella is found to be a major causes of food borne diseases globally. Poultry products contaminated with this pathogen is one of the major sources of infections in humans. Outer membrane protein C (OmpC) of Salmonella Typhimurium is a promising DNA vaccine candidate to mitigate Salmonella infection in poultry. However, the large-scale production of bioactive recombinant OmpC (rOmpC) protein is hindered due to the formation of inclusion bodies in Escherichia coli. The objective of this work was to attain high level expression of rOmpC protein, purify and evaluate its functional properties. The ompC gene was optimized and fused with small ubiquitin-related modifier (SUMO) gene for high level expression as soluble protein. The fusion protein with ~58 kDa molecular weight was observed on SDS-PAGE gel. The expression levels of rOmpC fusion protein reached maximum of 38% of total soluble protein (TSP) after 8 h of 0.2% rhamnose induction. Protein purification was carried out using nickel nitrilotriacetic acid (Ni-NTA) purification column. Western blot were performed to analyse expression and immunoreactivity of rOmpC fusion protein. The results indicate that SUMO fusion system is ideal for large scale production of functional rOmpC fusion protein expression in E. coli.


Assuntos
Proteínas de Bactérias , Escherichia coli , Imunoglobulinas/imunologia , Porinas , Proteínas Recombinantes de Fusão , Proteína SUMO-1 , Salmonella typhimurium/genética , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/isolamento & purificação , Galinhas , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Porinas/biossíntese , Porinas/genética , Porinas/imunologia , Porinas/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteína SUMO-1/biossíntese , Proteína SUMO-1/genética , Proteína SUMO-1/imunologia , Proteína SUMO-1/isolamento & purificação , Salmonella typhimurium/metabolismo
4.
Biologicals ; 62: 50-56, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31606267

RESUMO

Salmonella typhi is a causative organism for typhoid fever. Free Vi capsular polysaccharide (Vi) is licensed for use as vaccine for typhoid fever in individuals 2 years of age and older, which has limited memory response. There is dire need of protein or peptide as conjugate partner with Vi polysaccharide to improve shortcomings of Vi vaccine. Prediction of immunogenic peptide was deduced by program T sites. Carbodiimide mediated conjugation of Vi polysaccharide with OmpCp was performed utilizing ADH as linker. Immune response of Vi-conjugates along with control group was tested in mice. Ig and IgG antibodies against Vi polysaccharide was measured by ELISA. Two immunodominant regions (loop number 3a and 7) with high content of T-cell epitopes from OmpC was selected and synthesized. Vi poly/OmpCp ratios in Vi-conjugates were ~0.43-0.65. Vi polysaccharide alone elicited very low levels of Vi antibody without any booster effect. Vi-conjugate evoked 20-fold higher immune response compared to free Vi. Further, adequate levels of IgG antibodies were induced only by the Vi-conjugate suggesting that T-helper cells had been induced. Our data suggest that selected short peptide (OmpCp)as a carrier with Vi polysaccharide is assumed to be a promising molecule for candidate vaccine for typhoid fever.


Assuntos
Proteínas de Bactérias/imunologia , Polissacarídeos Bacterianos/imunologia , Porinas/imunologia , Vacinas contra Salmonella/imunologia , Salmonella typhi/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Feminino , Imunoglobulina G/imunologia , Camundongos , Febre Tifoide/imunologia , Febre Tifoide/prevenção & controle
5.
J Appl Microbiol ; 127(6): 1646-1655, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31529560

RESUMO

AIMS: The outer membrane porin protein (OMPP) of Bordetella bronchiseptica is an important adhesion factor and protective immunogen. The aim of this study was to verify the immunogenicity of recombinant OMPP and its protective efficacy against a lethal challenge with B. bronchiseptica in rabbits. METHODS AND RESULTS: Soluble rOMPP was successfully expressed in Escherichia coli, and the purified recombinant protein was mixed with the ISA 201 VG adjuvant to prepare a subunit vaccine for B. bronchiseptica. Rabbits were immunized with the rOMPP subunit vaccine and then infected with the virulent B. bronchiseptica strain QDBb01. Rabbits immunized with the subunit vaccine were completely protected compared to the control group, and the protective effect was obviously better than that of the inactivated whole-cell vaccine. Moreover, analysis of the immunization duration showed that the rOMPP subunit vaccine provided immune protection for at least 4 months after the second immunization. CONCLUSIONS: The rOMPP subunit vaccine completely protected rabbits from a subsequent B. bronchiseptica challenge. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will provide key information for the development of a safe and effective recombinant subunit vaccine against B. bronchiseptica in rabbits.


Assuntos
Vacinas Bacterianas/imunologia , Infecções por Bordetella/prevenção & controle , Bordetella bronchiseptica/imunologia , Porinas/imunologia , Adjuvantes Imunológicos , Animais , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/isolamento & purificação , Infecções por Bordetella/imunologia , Bordetella bronchiseptica/patogenicidade , Imunização , Porinas/genética , Porinas/isolamento & purificação , Porinas/metabolismo , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Vacinas de Subunidades
6.
Mol Immunol ; 114: 651-660, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31557626

RESUMO

Brucella poses a serious threat to human health. High quality vaccines for Brucella are urgently needed to effectively reduce the incidence of brucellosis. OMP2b and BCSP31 are important component proteins of the Brucella outer membrane and are highly immunogenic. Here, we used the bioinformatics software ProtParam, SOPMA, SWISS-MODEL, Rasmol, BepiPred, SYFPEITHI and IEDB to analyze the structure of these two proteins and predict the epitopes of T cells and B cells. Through analysis, we predicted three Th cell epitopes, seven CTL epitopes, eight B cell epitopes, and one T-B combined epitope of OMP2b protein. Subsequently, we also obtained three Th cell epitopes, six CTL epitopes, nine B cell epitopes and one T-B combined epitope of BCSP31 protein. The T-B combined epitopes and CTL epitopes of OMP2b and those of BCSP31 were synthesized to detect their immunogenicity. The IFN-γ ELISPOT assay showed that the T-B combined epitope peptides of OMP2b and BCSP31 activated Th cell immune responses. ELISA analysis detected the specific antibodies against the T-B combined epitope peptide of OMP2b and BCSP31 in the serum of Brucellosis patients. Additionally, CTL epitope peptide of OMP2b and BCSP31 proteins promoted the secretion of soluble perforin and granzyme B in the culture supernatant. In conclusion, our study shows that the T-B combined epitopes and CTL epitopes of OMP2b and BCSP31 have immunogenicity and immunoreactivity. Our results may lay a theoretical foundation for the development of vaccines against Brucella.


Assuntos
Proteínas de Bactérias/imunologia , Brucella/imunologia , Brucelose/imunologia , Epitopos/imunologia , Porinas/imunologia , Vacinas/imunologia , Adolescente , Adulto , Antígenos de Bactérias/imunologia , Biologia Computacional/métodos , ELISPOT/métodos , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
7.
Sci Rep ; 9(1): 12024, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31427597

RESUMO

There is no information on cytokine profiles for use as markers of protection in Campylobacter jejuni infection. To study this, we used outer membrane protein (MOMP [PorA]) as the vaccine for protection and spleen cell cytokines as markers of protection. We cloned and expressed porA from C. jejuni111 and immunized mice by the intraperitoneal route. Subsequently, mice were orally challenged with live C. jejuni 111. The vaccine induced protection as evidenced by reduced fecal excretion of C. jejuni111. Cytokines were measured in vitro after stimulation of spleen cells with MOMP. The levels of pro-inflammatory cytokines, IL-12, TNF-α, IL-17A and IL-17F were similar in control and test mice. The levels of pro-inflammatory cytokines, IL-2 and IFN-γ were higher in control mice than in test mice, and the levels of pro-inflammatory cytokines, IL-8 and IL-1ß were higher in test mice than in control mice. Among the two anti-inflammatory cytokines, the levels were similar for IL-10 but higher for IL-4 in test mice than in control mice. Ratios of pro-inflammatory to anti-inflammatory cytokines showed a bias towards an anti-inflammatory response in favor of antibody production reflecting the role of antibodies in immunity. Cytokine production patterns by spleen cells may be used as markers of protection in the mouse model.


Assuntos
Proteínas de Bactérias/imunologia , Infecções por Campylobacter/imunologia , Infecções por Campylobacter/metabolismo , Campylobacter jejuni/imunologia , Citocinas/metabolismo , Porinas/imunologia , Proteínas Recombinantes , Animais , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/genética , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/genética , Clonagem Molecular , Citocinas/sangue , Citocinas/genética , Expressão Gênica , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Porinas/genética , Baço/imunologia , Baço/metabolismo
8.
Braz J Microbiol ; 50(4): 979-984, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31352634

RESUMO

Control of brucellosis as a worldwide zoonotic disease is based on vaccination of animals and diagnosis of infected cases to be eradicated. Accurate and rapid detection of infected animals is of critical importance for preventing the spread of disease. Current detection of brucellosis is based on whole-cell antigens and investigating serum antibodies against Brucella lipopolysaccharide (LPS). The critical disadvantage is misdiagnosis of vaccinated animals as infected ones and also cross-reactions with other Gram-negative bacteria. Recombinant outer membrane protein 2b (Omp2b) of Brucella abortus was evaluated as a novel serodiagnostic target in comparison to conventional tests which are based on LPS. Recombinant Omp2b (rOmp2b) was expressed in Escherichia coli BL21 and purified by Ni2+-based chromatography. rOmp2b was evaluated in an indirect enzyme-linked immunosorbent assay (ELISA) system for diagnosis of brucellosis, with sera from Brucella-infected mice along with negative sera and sera from mice which were inoculated with other Gram-negative species for assurance of specificity. Thereafter, cattle sera collected from different regions were assessed along with known negative and known positive serum samples. We found that Omp2b can discriminate between Brucella-infected animals and non-infected ones. Results for assessment of two hundred and fifty cattle sera by Omp2b-based indirect ELISA which were compared to Rose Bengal plate agglutination test (RBPT) and serum tube agglutination test (SAT) showed that our proposed procedure has the sensitivity of 88.5%, specificity of 100%, and accuracy of 90.8%. We suggest that recombinant Omp2b could be used as a protein antigen for diagnosis of brucellosis in domestic animals and can be evaluated for detection of human brucellosis.


Assuntos
Antígenos de Bactérias/análise , Proteínas de Bactérias/análise , Brucella abortus/isolamento & purificação , Brucelose/veterinária , Doenças dos Bovinos/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Porinas/análise , Testes Sorológicos/métodos , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Brucella abortus/genética , Brucella abortus/imunologia , Brucelose/diagnóstico , Brucelose/microbiologia , Bovinos , Doenças dos Bovinos/microbiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Porinas/genética , Porinas/imunologia
9.
Front Immunol ; 10: 1577, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31333682

RESUMO

We tested the hypothesis that the impact of the Fms-like tyrosine kinase 3-ligand (Flt3L; FL) on recombinant Vibrio cholerae ghost (rVCG) vaccine-induced chlamydial immunity is influenced by route of vaccine delivery. Female C57BL/6J mice were immunized rectally (IR) or intramuscularly (IM) with rVCG co-expressing the Chlamydia trachomatis PmpD and PorB proteins (rVCG- PmpD/PorB) with and without FL or glycoprotein D of HSV-2 (rVCG-gD2) as antigen control. Vaccine evaluation was based on measurement of T cell proliferation, Th1/Th2 cytokine, and humoral responses at systemic and mucosal compartments, and protection against intravaginal challenge infection. Results revealed that high levels of CD4+ T cell-mediated and humoral immune responses, were elicited in mice as a function of both IR and IM immunization. Unexpectedly, co-administration of vaccine with FL enhanced specific Th1-type cytokine levels and T cell proliferative responses following IR but not IM immunization. While administration of vaccine with FL enhanced the specific mucosal and systemic IgA antibody responses following both immunization routes, IgG2c responses were not enhanced following IR delivery. The vaccine-induced immune effectors protected mice against live heterologous C. muridarum infection irrespective of route of vaccine administration, with the regimen incorporating FL having a protective advantage. Further evaluation showed that protection afforded by the FL adjuvanted vaccine was facilitated by CD4+ T cells, as indicated by reduction in the intensity and duration of genital chlamydial shedding by naïve mice following adoptive transfer of immune CD4+ T cells. Taken together, the results indicate that comparable protective immunity, which is enhanced by co-delivery with FL, is elicited in the female genital tract against Chlamydia infection after mucosal and systemic administration, highlighting the ability of FL to function as an effective immunostimulator at both mucosal and systemic sites. The differential modulation of humoral and cellular immune responses, and protective immunity afforded by the FL adjuvanted vaccine following IR administration indicates that the immunomodulatory impact of FL on chlamydial-specific immunity is influenced by the route of vaccine administration. Thus, targeting of VCG-based vaccines to antigen presenting cells by co-delivery with FL is a feasible immunization approach for inducing effective chlamydial immunity in the female genital tract.


Assuntos
Vacinas Bacterianas , Chlamydia trachomatis/imunologia , Imunidade nas Mucosas , Tirosina Quinase 3 Semelhante a fms/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/farmacologia , Chlamydia trachomatis/genética , Feminino , Imunoglobulina A/imunologia , Linfogranuloma Venéreo/genética , Linfogranuloma Venéreo/imunologia , Linfogranuloma Venéreo/prevenção & controle , Camundongos , Porinas/genética , Porinas/imunologia , Células Th1/imunologia , Células Th2/imunologia , Vibrio cholerae/genética , Vibrio cholerae/imunologia , Tirosina Quinase 3 Semelhante a fms/genética
10.
Sci Rep ; 9(1): 6843, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31048732

RESUMO

Outer membrane vesicle (OMV)- based vaccines have been used to provide strain-specific protection against capsular group B Neisseria meningitidis infections, but the full breadth of the immune response against the components of the OMV has not been established. Sera from adults vaccinated with an OMV vaccine were used to screen 91 outer membrane proteins (OMPs) incorporated in an antigen microarray panel. Antigen-specific IgG levels were quantified pre-vaccination, and after 12 and 18 weeks. These results were compared with IgG levels from mice vaccinated with the same OMV vaccine. The repertoires of highly responding antigens in humans and mice overlapped, but were not identical. The highest responding antigens to human IgG comprised four integral OMPs (PorA, PorB, OpcA and PilQ), a protein which promotes the stability of PorA and PorB (RmpM) and two lipoproteins (BamC and GNA1162). These observations will assist in evaluating the role of minor antigen components within OMVs in providing protection against meningococcal infection. In addition, the relative dominance of responses to integral OMPs in humans emphasizes the importance of this subclass and points to the value of maintaining conformational epitopes from integral membrane proteins in vaccine formulations.


Assuntos
Antígenos de Bactérias/imunologia , Vacinas Bacterianas/uso terapêutico , Neisseria meningitidis Sorogrupo B/imunologia , Adolescente , Adulto , Animais , Vacinas Bacterianas/imunologia , Cromatografia em Gel , Feminino , Humanos , Imunoglobulina G/imunologia , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Porinas/imunologia , Porinas/metabolismo , Adulto Jovem
11.
Hum Vaccin Immunother ; 15(11): 2778-2781, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31112447

RESUMO

Our laboratory has focused on Porin B (PorB), an outer membrane protein from Neisseria meningitidis and TLR2 ligand-based adjuvant, to characterize specific molecular and cellular pathways involved in improved immune responses induced by vaccine adjuvants. PorB's ability to form micellar nanoparticular multi-molecular organized structures and its interaction with Toll-like receptor 2/1 complexes likely accounts for its potent adjuvant activity. Downstream from this stimulation, we have observed enhanced antigen uptake in antigen presenting cells (APC), greater antigen deposition in secondary lymphoid organs, and promotion of germinal center reactions. In mice, antigen-specific IgGs were increased after PorB adjuvanted vaccination using the model antigen ovalbumin (OVA). Likewise, this formulation resulted in more IL-4 and IFN-γ positive T cells. Mice that received PorB adjuvanted vaccinations benefitted from lower bacterial burdens when challenged with recombinant Listeria monocytogenes expressing OVA. Mouse models lacking MyD88 signaling in various APC types helped identify macrophages as an essential cell type for the adjuvant activity of PorB. We believe the work presented here provides examples of the mechanistic studies required to understand how vaccine adjuvants are contributing to the establishment of protective immunity.


Assuntos
Adjuvantes Imunológicos , Células Apresentadoras de Antígenos/imunologia , Infecções Meningocócicas/prevenção & controle , Porinas/imunologia , Animais , Animais Geneticamente Modificados , Anticorpos Antibacterianos/sangue , Citocinas/imunologia , Feminino , Imunoglobulina G/sangue , Camundongos , Fator 88 de Diferenciação Mieloide , Neisseria meningitidis , Porinas/administração & dosagem , Linfócitos T/imunologia
12.
Methods Mol Biol ; 1997: 1-27, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31119614

RESUMO

Neisseria gonorrhoeae infection is a major public health problem worldwide. The increasing incidence of gonorrhea coupled with global spread of multidrug-resistant isolates of gonococci has ushered in an era of potentially untreatable infection. Gonococcal disease elicits limited immunity, and individuals are susceptible to repeated infections. In this chapter, we describe gonococcal disease and epidemiology and the structure and function of major surface components involved in pathogenesis. We also discuss the mechanisms that gonococci use to evade host immune responses and the immune responses following immunization with selected bacterial components that may overcome evasion. Understanding the biology of the gonococcus may aid in preventing the spread of gonorrhea and also facilitate the development of gonococcal vaccines and treatments.


Assuntos
Proteínas de Bactérias/metabolismo , Gonorreia/imunologia , Evasão da Resposta Imune , Neisseria gonorrhoeae/patogenicidade , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Fímbrias Bacterianas/imunologia , Fímbrias Bacterianas/metabolismo , Carga Global da Doença , Gonorreia/epidemiologia , Gonorreia/microbiologia , Humanos , Incidência , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Neisseria gonorrhoeae/citologia , Neisseria gonorrhoeae/imunologia , Porinas/imunologia , Porinas/metabolismo
13.
J Pediatr Gastroenterol Nutr ; 69(2): 176-181, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30964819

RESUMO

OBJECTIVE: Hirschsprung-associated enterocolitis (HAEC) is the most frequent complication in Hirschsprung disease (HSCR) patients. Currently HAEC is diagnosed clinically, leaving uncertainty in the diagnosis thereby potentially leading to over- or undertreatment of patients. The aim of this study was to identify immune biomarkers to aid in the diagnosis of HAEC. METHODS: From 2012 to 2017, 43 children with HSCR enrolled in a multicenter study, underwent retrospective evaluation of their medical records, and questionnaire-directed parent interviews. HAEC status was determined using HAEC score with cutoff ≥4. Plasma was collected and analyzed by ELISA for the inflammatory bowel disease-associated antibodies: anti-Saccharomyces cerevisiae mannan antibodies (ASCA), outer membrane porin C (OmpC), CBir1, antineutrophil cytoplasmic antibodies. Data were analyzed using t test, univariate, multivariable, and binomial regression models. RESULTS: Eighteen patients had at least 1 episode of HAEC, 25 had no history of HAEC. The HAEC and NO HAEC groups had similar median ages (3 years) and family histories of HSCR. The HAEC group showed markedly elevated ASCA IgA and OmpC antibody levels compared with the NO HAEC group, whereas CBir1 and antineutrophil cytoplasmic antibodies were similar between the groups. Both univariate and multivariable analysis revealed higher OmpC antibody levels associated with HAEC (odds ratio 1.39, confidence interval 1-1.92, P = 0.048), whereas univariate analysis identified a trend toward elevated IgA and immunoglobulin G ASCA levels with HAEC. CONCLUSIONS: We identified elevated OmpC and ASCA serum antibody levels in HAEC patients, and that increased OmpC antibody levels correlated with HAEC occurrence, suggesting HAEC and Crohn disease share gut microbial-host immune responses. These antibodies may serve as potential biomarkers for HAEC, although prospective study with larger sample size is needed.


Assuntos
Biomarcadores/sangue , Enterocolite/diagnóstico , Doença de Hirschsprung/diagnóstico , Adolescente , Adulto , Anticorpos Anticitoplasma de Neutrófilos/sangue , Anticorpos Antifúngicos/sangue , Proteínas da Membrana Bacteriana Externa/imunologia , Criança , Pré-Escolar , Enterocolite/sangue , Proteínas de Escherichia coli/imunologia , Feminino , Flagelina/imunologia , Doença de Hirschsprung/sangue , Humanos , Lactente , Recém-Nascido , Doenças Inflamatórias Intestinais/imunologia , Masculino , Mananas/imunologia , Registros Médicos , Porinas/imunologia , Valor Preditivo dos Testes , Estudos Retrospectivos , Adulto Jovem
14.
Microb Pathog ; 132: 87-99, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31029716

RESUMO

Treponema is a diverse bacterial genus, the species of which can be pathogenic, symbiotic, or free living. These treponemes can cause various diseases in humans and other animals, such as periodontal disease, bovine digital dermatitis and animal skin lesions. However, the most important and well-studied disease of treponemes that affects humans is 'syphilis'. This disease is caused by Treponema pallidum subspecie pallidum with 11-12 million new cases around the globe on an annual basis. In this study we analyze the transportome of ten Treponema species, with emphasis on the types of encoded transport proteins and their substrates. Of the ten species examined, two (T. primitia and T. azonutricium) reside as symbionts in the guts of termites; six (T. pallidum, T. paraluiscuniculi, T. pedis, T. denticola, T. putidum and T. brennaborense) are pathogens of either humans or animals, and T. caldarium and T. succinifaciens are avirulent species, the former being thermophilic. All ten species have a repertoire of transport proteins that assists them in residing in their respective ecological niches. For instance, oral pathogens use transport proteins that take up nutrients uniquely present in their ecosystem; they also encode multiple multidrug/macromolecule exporters that protect against antimicrobials and aid in biofilm formation. Proteins of termite gut symbionts convert cellulose into other sugars that can be metabolized by the host. As often observed for pathogens and symbionts, several of these treponemes have reduced genome sizes, and their small genomes correlate with their dependencies on the host. Overall, the transportomes of T. pallidum and other pathogens have a conglomerate of parasitic lifestyle-assisting proteins. For example, a T. pallidum repeat protein (TprK) mediates immune evasion; outer membrane proteins (OMPs) allow nutrient uptake and end product export, and several ABC transporters catalyze sugar uptake, considered pivotal to parasitic lifestyles. Taken together, the results of this study yield new information that may help open new avenues of treponeme research.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Genômica/métodos , Treponema/classificação , Treponema/genética , Treponema/fisiologia , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Proteínas de Transporte/classificação , Farmacorresistência Bacteriana/genética , Microbioma Gastrointestinal , Tamanho do Genoma , Interações Hospedeiro-Patógeno , Humanos , Evasão da Resposta Imune , Porinas/genética , Porinas/imunologia , Proteoma , Especificidade da Espécie , Especificidade por Substrato , Simbiose , Sífilis/microbiologia , Treponema/patogenicidade , Treponema pallidum/genética
15.
Bull Exp Biol Med ; 166(6): 751-753, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31028578

RESUMO

In vivo experiments showed that antibodies to OmpC and OmpF porins of Yersinia pseudotuberculosis increased thyroxine (T4) level in the blood of experimental animals. The mice were immunized with different antigens: recombinant OmpF porin in a soluble monomeric form, trimers of OmpC and OmpF porins isolated from the outer membrane, or antibodies to them. The level of thyroxine in the blood of mice immunized with OmpF and OmpC porins increased by 5.47 and 22.3 times, respectively; after immunization with antibodies to these proteins, blood thyroxine increased by 9.28 and 14.29 times. Immunization with recombinant OmpF porin induced no reliable increase in thyroxine level. Hence, the serum to recombinant OmpF porin contains no antibodies specific to conformational antigenic determinants that are present in the protein trimer and, according to our previous findings from molecular docking studies, determine cross-reactions between OmpF porin of Y. pseudotuberculosis and thyroidstimulating hormone receptor.


Assuntos
Antígenos de Bactérias/imunologia , Hipertireoidismo/induzido quimicamente , Porinas/imunologia , Yersinia pseudotuberculosis/química , Animais , Anticorpos Antibacterianos/administração & dosagem , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/química , Feminino , Hipertireoidismo/imunologia , Hipertireoidismo/metabolismo , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Porinas/administração & dosagem , Porinas/química , Multimerização Proteica , Receptores da Tireotropina/imunologia , Receptores da Tireotropina/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Tiroxina/biossíntese , Yersinia pseudotuberculosis/imunologia
16.
Vaccine ; 37(13): 1807-1818, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30797635

RESUMO

BACKGROUND: Syphilis is resurgent in many developed countries and still prevalent in developing nations. Current and future control campaigns would benefit from the development of a vaccine, but although promising vaccine candidates were identified among the putative surface-exposed integral outer membrane proteins of the syphilis spirochete, immunization experiments in the rabbit model using recombinant antigens have failed to fully protect animals upon infectious challenge. We speculated that such recombinant immunogens, purified under denaturing conditions from Escherichia coli prior to immunization might not necessarily harbor their original structure, and hypothesized that enhanced protection would result from performing similar immunization/challenge experiments with native antigens. METHODS: To test our hypothesis, we engineered non-infectious Borrelia burgdorferi strains to express the tp0897 (tprK) and tp0435 genes of Treponema pallidum subsp. pallidum and immunized two groups of rabbits by injecting recombinant strains intramuscularly with no adjuvant. TprK is a putative integral outer membrane protein of the syphilis agent, while tp0435 encodes the highly immunogenic T. pallidum 17-kDa lipoprotein, a periplasmic antigen that was also shown on the pathogen surface. Following development of a specific host immune response to these antigens as the result of immunization, animals were challenged by intradermal inoculation of T. pallidum. Cutaneous lesion development was monitored and treponemal burden within lesions were assessed by dark-field microscopy and RT-qPCR, in comparison to control rabbits. RESULTS: Partial protection was observed in rabbits immunized with B. burgdorferi expressing TprK while immunity to Tp0435 was not protective. Analysis of the humoral response to TprK antigen suggested reactivity to conformational epitopes. CONCLUSIONS: Immunization with native antigens might not be sufficient to obtain complete protection to infection. Nonetheless we showed that non-infectious B. burgdorferi can be an effective carrier to deliver and elicit a specific host response to T. pallidum antigens to assess the efficacy of syphilis vaccine candidates.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Borrelia burgdorferi/genética , Expressão Gênica , Porinas/imunologia , Sífilis/prevenção & controle , Treponema pallidum/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Vacinas Bacterianas/genética , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Epitopos/imunologia , Imunofluorescência , Imunidade Celular , Imunização , Espectrometria de Massas , Camundongos , Peptídeos/síntese química , Peptídeos/imunologia , Porinas/química , Porinas/genética , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sífilis/imunologia , Sífilis/patologia , Treponema pallidum/genética
17.
Clin Gastroenterol Hepatol ; 17(9): 1799-1806, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30213581

RESUMO

BACKGROUND & AIMS: There are few serum biomarkers to identify patients with Crohn's disease (CD) who are at risk for stricture development. The extracellular matrix components, collagen type III alpha 1 chain (COL3A1) and cartilage oligomeric matrix protein (COMP), could contribute to intestinal fibrosis. We investigated whether children with inflammatory CD (B1) who later develop strictures (B2) have increased plasma levels of COL3A1 or COMP at diagnosis, compared with children who remain B1. We compared results with previously studied biomarkers, including autoantibodies against colony-stimulating factor 2 (CSF2). METHODS: We selected 161 subjects (mean age, 12.2 y; 62% male) from the Risk Stratification and Identification of Immunogenic and Microbial Markers of Rapid Disease Progression in Children with Crohn's cohort, completed at 28 sites in the United States and Canada from 2008 through 2012. The children underwent colonoscopy and upper endoscopy at diagnosis and were followed up every 6 months for 36 months; plasma samples were collected at baseline. Based on CD phenotype, children were separated to group 1 (B1 phenotype at diagnosis and follow-up evaluation), group 2 (B2 phenotype at diagnosis), or group 3 (B1 phenotype at diagnosis who developed strictures during follow-up evaluation). Plasma samples were collected from patients and 40 children without inflammatory bowel disease (controls) at baseline and analyzed by enzyme-linked immunosorbent assay to measure COL3A1 and COMP. These results were compared with those from a previous biomarker study. The Kruskal-Wallis test and the pairwise Dunn test with Bonferroni correction were used to compare differences among groups. RESULTS: The median baseline concentration of COL3A1 was significantly higher in plasma from group 3 vs group 1 (P < .01) and controls (P = .01). Median baseline plasma concentrations of COMP did not differ significantly among groups. A model comprising baseline concentrations of COL3A1 and anti-CSF2 identified patients with B2 vs B1 CD with an area under the curve of 0.80 (95% CI, 0.71-0.89); the combined concentration identified patients with strictures with a sensitivity value of 0.70 (95% CI, 0.55-0.83) and a specificity value of 0.83 (95% CI, 0.67-0.93). CONCLUSIONS: We found median plasma concentrations of COL3A1, measured by enzyme-linked immunosorbent assay at diagnosis, to be significantly higher in patients with CD who later developed strictures than in patients without strictures. The combination of concentrations of COL3A1 and anti-CSF2 might be used to identify pediatric patients at CD diagnosis who are at risk for future strictures. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00790543.


Assuntos
Proteína de Matriz Oligomérica de Cartilagem/sangue , Colágeno Tipo III/sangue , Doença de Crohn/sangue , Adolescente , Anticorpos Anticitoplasma de Neutrófilos , Anticorpos Antifúngicos , Autoanticorpos/imunologia , Criança , Constrição Patológica , Doença de Crohn/classificação , Doença de Crohn/patologia , Doença de Crohn/fisiopatologia , Feminino , Flagelina , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Masculino , Porinas/imunologia
18.
Microb Pathog ; 126: 185-192, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30408491

RESUMO

Vibrio ichthyoenteri is a marine pathogen that primarily causes bacterial enteritis of flatfish. In order to screen out the effective vaccine candidate proteins, five outer membrane proteins (OMPs) of V. ichthyoenteri, including OmpV, OmpU, OmpT, Omp1 and Omp2, were selected and recombinantly expressed in Escherichia coli. The result of western blotting showed that all of these recombinant proteins could be recognized by flounder anti-V. ichthyoenteri antibodies. The immune protective effect of the five rOMPs were also investigated, and the relative percent survival (RPS) of rOmpV, rOmpU, rOmpT, rOmp1 and rOmp2 was 69.2%, 46.2%, 76.9%, 65.4% and 30.8%, respectively. The RPS of rOmpT was significantly higher than inactivated V. ichthyoenteri (57.7%) and other rOMPs, so the immune responses of flounder induced by rOmpT were further investigated. The results showed that both the production of specific serum antibodies and the proliferation of sIg + lymphocytes could be significantly induced by rOmpT. Meanwhile, the MHCIIα, INF-γ, CD4-1, and IL-1ß genes were significantly up-regulated after immunization with rOmpT. These results demonstrated that rOmpT could induce strong innate and humoral immune response in flounder and provided effective immune protection against V. ichthyoenteri challenge, which indicated that OmpT is a promising vaccine candidate against V. ichthyoenteri infection.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Linguado/imunologia , Imunização , Porinas/genética , Porinas/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vibrioses/prevenção & controle , Vibrio/genética , Animais , Anticorpos Antibacterianos/sangue , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Modelos Animais de Doenças , Escherichia coli/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Doenças dos Peixes/prevenção & controle , Linguado/microbiologia , Regulação Bacteriana da Expressão Gênica , Imunidade Humoral , Imunidade Inata , Linfócitos/imunologia , Mortalidade , Taxa de Sobrevida , Vacinação , Vibrio/patogenicidade , Vibrioses/veterinária
19.
Front Immunol ; 9: 2003, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30271401

RESUMO

Outer membrane porins, as the major components of Gram-negative bacterial membrane proteins, have been proven to be involved in interactions with the host immune system and potent protective antigen candidates against bacterial infection in fish. Outer membrane porin F (OmpF) is one of the major porins of Yersinia ruckeri (Y. ruckeri), the causative agent of enteric red mouth disease of salmonid and non-salmonid fish. In the present study, the molecular characterization and phylogenetic analysis of OmpF gene was studied, heterogenous expression, immunogenicity and protective immunity of OmpF were systemically evaluated as a subunit vaccine for channel catfish against Y. ruckeri infection. The results showed that OmpF gene was highly conserved among 15 known Yersinia species based on the analysis of conserved motifs, sequences alignment and phylogenetic tree, and was subjected to negative/purifying selection with global dN/dS ratios value of 0.649 throughout the evolution. Besides, OmpF was also identified to have immunogenicity by western blotting and was verified to be located on the surface of Y. ruckeri using cell surface staining and indirect immunofluorescence assays. Moreover, recombinant OmpF (rtOmpF) as a subunit vaccine was injected with commercial adjuvant ISA763, significantly enhanced the immune response by increasing serum antibody levels, lysozyme activity, complement C3 activity, total protein content, SOD activity, immune-related genes expression in the head kidney and spleen, and survival percent of channel catfish against Y. ruckeri infection. Thus, our present results not only enriched the information of molecular characterization and phylogenetics of OmpF, but also demonstrated that OmpF holds promise to be used as a potential antigen against Y. ruckeri infection in fish.


Assuntos
Vacinas Bacterianas/imunologia , Doenças dos Peixes/imunologia , Rim Cefálico/fisiologia , Ictaluridae/imunologia , Porinas/genética , Yersiniose/imunologia , Yersinia ruckeri/fisiologia , Animais , Anticorpos Antibacterianos/sangue , Complemento C3/metabolismo , Proteínas de Peixes/metabolismo , Imunidade Inata , Estrutura Molecular , Filogenia , Porinas/imunologia , Transcriptoma , Vacinas de Subunidades
20.
Am J Reprod Immunol ; 80(5): e13051, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30281189

RESUMO

PROBLEM: The accuracy of Chlamydia trachomatis antibody test in predicting tubal factor infertility (TFI) is limited, and more accurate methods are needed. Cell-mediated immune response (CMI) is crucial in the resolution of pathogen, but it may play an important role in the pathogenesis of C trachomatis-associated tubal damage. We studied whether combining the markers of C trachomatis-induced CMI to humoral immune response improves the accuracy of serology in TFI prediction. METHOD OF STUDY: Our prospective study consists of 258 subfertile women, of whom 22 (8.5%) had TFI. Women with other causes for subfertility served as a reference group. Serum C trachomatis major outer membrane protein (MOMP) and chlamydial heat-shock protein 60 (cHSP60) IgG antibodies were measured by ELISA. CMI was studied by lymphocyte proliferation assay in vitro. RESULTS: Serological markers were more prevalent in women with TFI than in other subfertile women (40.9% vs 12.3% for MOMP IgG and 27.3% vs 10.2% for cHSP60 IgG). The best test combination for TFI was C. trachomatis MOMP and cHSP60 antibody with an accuracy of 90.3%, sensitivity of 22.7% and specificity of 96.6%. Positive post-test probability of this combination was 54.2%, and negative post-test probability was 12.4%. Adding of the markers of CMI did not significantly improve the accuracy of serology in TFI prediction. CONCLUSION: The accuracy of TFI prediction increases when the combination of C trachomatis MOMP and cHSP60 antibody tests is used. C trachomatis-induced CMI was common in our study population, but the markers of CMI did not predict TFI.


Assuntos
Infecções por Chlamydia/imunologia , Chlamydia trachomatis/fisiologia , Tubas Uterinas/patologia , Imunidade Humoral , Infertilidade Feminina/imunologia , Linfócitos/imunologia , Adulto , Anticorpos Antibacterianos/sangue , Biomarcadores/sangue , Proliferação de Células , Células Cultivadas , Chaperonina 60/imunologia , Infecções por Chlamydia/diagnóstico , Feminino , Humanos , Imunidade Celular , Infertilidade Feminina/diagnóstico , Porinas/imunologia , Valor Preditivo dos Testes , Gravidez , Prognóstico , Estudos Prospectivos , Sensibilidade e Especificidade
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