RESUMO
Bacterial O-SP-core antigens can be conjugated to proteins in the same, simple way as synthetic, linker-equipped carbohydrates by applying squaric acid chemistry. Introduction of spacers (linkers) to either O-SP-core antigens or protein carriers, which is involved in commonly applied protocols, is not required. The newly developed method described here consists of preparation of a squaric acid monoester derivative of O-SP-core antigen, utilizing the amino group inherent in the core, and reaction of the monoester with the carrier protein. The intermediate monoester can be easily purified; its conjugation can be monitored by SELDI-TOF mass spectrometry and, thus, readily controlled, since the conjugation can be terminated when the desired carbohydrate-protein ratio is reached. Here, we describe production of conjugates containing the O-SP-core antigen of Vibrio cholerae O1, the major cause of cholera, a severe dehydrating diarrheal disease of humans. The resultant products are recognized by convalescent phase sera from patients recovering from cholera in Bangladesh, and anti-O-SP-core-protein responses correlate with plasma antilipopolysaccharide and vibriocidal responses, which are the primary markers of protection from cholera. The results suggest that such conjugates have potential as vaccines for cholera and other bacterial diseases.
Assuntos
Vacinas contra Cólera/química , Cólera/prevenção & controle , Antígenos O/química , Vibrio cholerae O1/química , Cólera/imunologia , Vacinas contra Cólera/imunologia , Humanos , Antígenos O/imunologia , Vacinas Conjugadas/química , Vacinas Conjugadas/imunologia , Vibrio cholerae O1/imunologiaRESUMO
Transcutaneous vaccination can induce both mucosal and systemic immune responses. However, there are few data on anti-polysaccharide responses following transcutaneous vaccination of polysaccharides, despite the role that anti-polysaccharide responses play in protecting against intestinal mucosal and respiratory pathogens. Whether transcutaneous vaccination with a conjugate polysaccharide vaccine would be able to induce memory responses is also unknown. To address this, we transcutaneously vaccinated mice with virulence antigen (Vi) polysaccharide of Salmonella enterica serovar Typhi (the cause of typhoid fever), either in unconjugated or conjugated form (the latter as a Vi-DT conjugate). We also assessed the ability of the immunoadjuvant cholera toxin to impact responses following vaccination. We found that presenting Vi in a conjugate versus nonconjugate form transcutaneously resulted in comparable serum IgG responses but higher serum and lamina propria lymphocyte IgA anti-Vi responses, as well as increased IgG memory responses. The addition of immunoadjuvant did not further increase these responses; however, it boosted fecal IgA and serum IgG anti-Vi responses. Our results suggest that transcutaneous vaccination of a conjugate vaccine can induce systemic as well as enhanced mucosal and memory B-cell anti-polysaccharide responses.
Assuntos
Anticorpos Antibacterianos/sangue , Imunidade Humoral/efeitos dos fármacos , Imunidade nas Mucosas/efeitos dos fármacos , Salmonella typhi/imunologia , Febre Tifoide/prevenção & controle , Vacinas Tíficas-Paratíficas/administração & dosagem , Vacinação/métodos , Administração Cutânea , Animais , Modelos Animais de Doenças , Feminino , Humanos , Esquemas de Imunização , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Memória Imunológica/efeitos dos fármacos , Camundongos , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/imunologia , Salmonella typhi/efeitos dos fármacos , Salmonella typhi/patogenicidade , Febre Tifoide/imunologia , Febre Tifoide/microbiologia , Vacinas Tíficas-Paratíficas/biossíntese , Vacinas ConjugadasRESUMO
Although mechanisms of acquired resistance of epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancers to EGFR inhibitors have been identified, little is known about how resistant clones evolve during drug therapy. Here we observe that acquired resistance caused by the EGFR(T790M) gatekeeper mutation can occur either by selection of pre-existing EGFR(T790M)-positive clones or via genetic evolution of initially EGFR(T790M)-negative drug-tolerant cells. The path to resistance impacts the biology of the resistant clone, as those that evolved from drug-tolerant cells had a diminished apoptotic response to third-generation EGFR inhibitors that target EGFR(T790M); treatment with navitoclax, an inhibitor of the anti-apoptotic factors BCL-xL and BCL-2 restored sensitivity. We corroborated these findings using cultures derived directly from EGFR inhibitor-resistant patient tumors. These findings provide evidence that clinically relevant drug-resistant cancer cells can both pre-exist and evolve from drug-tolerant cells, and they point to therapeutic opportunities to prevent or overcome resistance in the clinic.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , RNA Mensageiro/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Gefitinibe , Humanos , Técnicas In Vitro , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Mutação , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
UNLABELLED: Rociletinib is a third-generation EGFR inhibitor active in lung cancers with T790M, the gatekeeper mutation underlying most first-generation EGFR drug resistance. We biopsied patients at rociletinib progression to explore resistance mechanisms. Among 12 patients with T790M-positive cancers at rociletinib initiation, six had T790-wild-type rociletinib-resistant biopsies. Two T790-wild-type cancers underwent small cell lung cancer transformation; three T790M-positive cancers acquired EGFR amplification. We documented T790-wild-type and T790M-positive clones coexisting within a single pre-rociletinib biopsy. The pretreatment fraction of T790M-positive cells affected response to rociletinib. Longitudinal circulating tumor DNA (ctDNA) analysis revealed an increase in plasma EGFR-activating mutation, and T790M heralded rociletinib resistance in some patients, whereas in others the activating mutation increased but T790M remained suppressed. Together, these findings demonstrate the role of tumor heterogeneity when therapies targeting a singular resistance mechanism are used. To further improve outcomes, combination regimens that also target T790-wild-type clones are required. SIGNIFICANCE: This report documents that half of T790M-positive EGFR-mutant lung cancers treated with rociletinib are T790-wild-type upon progression, suggesting that T790-wild-type clones can emerge as the dominant source of resistance. We show that tumor heterogeneity has important clinical implications and that plasma ctDNA analyses can sometimes predict emerging resistance mechanisms.
Assuntos
Acrilamidas/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/genética , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Pirimidinas/administração & dosagem , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Acrilamidas/farmacologia , Linhagem Celular Tumoral , DNA de Neoplasias/sangue , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/sangue , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Mutação , Estudos Prospectivos , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Carcinoma de Pequenas Células do Pulmão/genéticaRESUMO
UNLABELLED: BRAF mutations occur in approximately 10% of colorectal cancers. Although RAF inhibitor monotherapy is highly effective in BRAF-mutant melanoma, response rates in BRAF-mutant colorectal cancer are poor. Recent clinical trials of combined RAF/EGFR or RAF/MEK inhibition have produced improved efficacy, but patients ultimately develop resistance. To identify molecular alterations driving clinical acquired resistance, we performed whole-exome sequencing on paired pretreatment and postprogression tumor biopsies from patients with BRAF-mutant colorectal cancer treated with RAF inhibitor combinations. We identified alterations in MAPK pathway genes in resistant tumors not present in matched pretreatment tumors, including KRAS amplification, BRAF amplification, and a MEK1 mutation. These alterations conferred resistance to RAF/EGFR or RAF/MEK combinations through sustained MAPK pathway activity, but an ERK inhibitor could suppress MAPK activity and overcome resistance. Identification of MAPK pathway reactivating alterations upon clinical acquired resistance underscores the MAPK pathway as a critical target in BRAF-mutant colorectal cancer and suggests therapeutic options to overcome resistance. SIGNIFICANCE: RAF inhibitor combinations represent promising approaches in clinical development for BRAF-mutant colorectal cancer. Initial characterization of clinical acquired resistance mechanisms to these regimens identified several MAPK pathway alterations driving resistance by reactivating MAPK signaling, highlighting the critical dependence of BRAF-mutant colorectal cancers on MAPK signaling and offering potential strategies to overcome resistance.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Amplificação de Genes , Humanos , MAP Quinase Quinase 1/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteína Oncogênica p21(ras)/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Ativação TranscricionalRESUMO
BACKGROUND: Protective immunity against cholera is serogroup specific. Serogroup specificity in Vibrio cholerae is determined by the O-specific polysaccharide (OSP) of lipopolysaccharide (LPS). Generally, polysaccharides are poorly immunogenic, especially in young children. METHODOLOGY: Here we report the evaluation in mice of a conjugate vaccine for cholera (OSP:TThc) made from V. cholerae O1 Ogawa O-Specific Polysaccharide-core (OSP) and recombinant tetanus toxoid heavy chain fragment (TThc). We immunized mice intramuscularly on days 0, 21, and 42 with OSP:TThc or OSP only, with or without dmLT, a non-toxigenic immunoadjuvant derived from heat labile toxin of Escherichia coli. PRINCIPAL FINDINGS: We detected significant serum IgG antibody responses targeting OSP following a single immunization in mice receiving OSP:TThc with or without adjuvant. Anti-LPS IgG responses were detected following a second immunization in these cohorts. No anti-OSP or anti-LPS IgG responses were detected at any time in animals receiving un-conjugated OSP with or without immunoadjuvant, and in animals receiving immunoadjuvant alone. Responses were highest following immunization with adjuvant. Serum anti-OSP IgM responses were detected in mice receiving OSP:TThc with or without immunoadjuvant, and in mice receiving unconjugated OSP. Serum anti-LPS IgM and vibriocidal responses were detected in all vaccine cohorts except in mice receiving immunoadjuvant alone. No significant IgA anti-OSP or anti-LPS responses developed in any group. Administration of OSP:TThc and adjuvant also induced memory B cell responses targeting OSP and resulted in 95% protective efficacy in a mouse lethality cholera challenge model. CONCLUSION: We describe a protectively immunogenic cholera conjugate in mice. Development of a cholera conjugate vaccine could assist in inducing long-term protective immunity, especially in young children who respond poorly to polysaccharide antigens.
Assuntos
Vacinas contra Cólera/imunologia , Cólera/prevenção & controle , Antígenos O/imunologia , Vacinas Conjugadas/imunologia , Animais , Anticorpos Antibacterianos/sangue , Cólera/imunologia , Cólera/mortalidade , Vacinas contra Cólera/química , Vacinas contra Cólera/metabolismo , Modelos Animais de Doenças , Feminino , Camundongos , Antígenos O/química , Antígenos O/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Vacinas Conjugadas/química , Vacinas Conjugadas/metabolismoRESUMO
Targeted cancer therapies have produced substantial clinical responses, but most tumors develop resistance to these drugs. Here, we describe a pharmacogenomic platform that facilitates rapid discovery of drug combinations that can overcome resistance. We established cell culture models derived from biopsy samples of lung cancer patients whose disease had progressed while on treatment with epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors and then subjected these cells to genetic analyses and a pharmacological screen. Multiple effective drug combinations were identified. For example, the combination of ALK and MAPK kinase (MEK) inhibitors was active in an ALK-positive resistant tumor that had developed a MAP2K1 activating mutation, and the combination of EGFR and fibroblast growth factor receptor (FGFR) inhibitors was active in an EGFR mutant resistant cancer with a mutation in FGFR3. Combined ALK and SRC (pp60c-src) inhibition was effective in several ALK-driven patient-derived models, a result not predicted by genetic analysis alone. With further refinements, this strategy could help direct therapeutic choices for individual patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/tratamento farmacológico , Terapia de Alvo Molecular/métodos , Modelagem Computacional Específica para o Paciente , Inibidores de Proteínas Quinases/uso terapêutico , Quinase do Linfoma Anaplásico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/genética , Análise Mutacional de DNA , Ensaios de Seleção de Medicamentos Antitumorais , Ativação Enzimática/genética , Receptores ErbB/antagonistas & inibidores , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Mutação , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , Pirimidinas/uso terapêutico , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Sulfonas/uso terapêutico , Células Tumorais CultivadasRESUMO
Salmonella enterica serotype Paratyphi A is a human-restricted pathogen and the cause of paratyphoid A fever. Using a high-throughput immunoscreening technique, in vivo-induced antigen technology (IVIAT), we identified 20 immunogenic bacterial proteins expressed in humans who were bacteremic with S. Paratyphi A but not those expressed in S. Paratyphi A grown under standard laboratory conditions. The majority of these proteins have known or potential roles in the pathogenesis of S. enterica. These include proteins implicated in cell adhesion, fimbrial structure, adaptation to atypical conditions, oxidoreductase activity, proteolysis, antimicrobial resistance, and ion transport. Of particular interest among these in vivo-expressed proteins were S. Paratyphi A (SPA)2397, SPA2612, and SPA1604. SPA2397 and SPA2612 are prophage related, and SPA1604 is in Salmonella pathogenicity island 11 (SPI-11). Using real-time quantitative PCR (RT-qPCR), we confirmed increased levels of mRNA expressed by genes identified by IVIAT in a comparison of mRNA levels in organisms in the blood of bacteremic patients to those in in vitro cultures. Comparing convalescent- to acute-phase samples, we also detected a significant increase in the reaction of convalescent-phase antibodies with two proteins identified by IVIAT: SPA2397 and SPA0489. SPA2397 is a phage-related lysozyme, Gp19, and SPA0489 encodes a protein containing NlpC/P60 and cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domains. In a previous study utilizing a different approach, we found that transcripts for 11 and 7 of the genes identified by IVIAT were detectable in organisms in the blood of humans in Bangladesh who were bacteremic with S. Paratyphi A and Salmonella enterica serovar Typhi, respectively. S. Paratyphi A antigens identified by IVIAT warrant further evaluation for their contributions to pathogenesis and might have diagnostic, therapeutic, or preventive relevance.
Assuntos
Antígenos de Bactérias/sangue , Bacteriemia/microbiologia , Proteínas de Bactérias/sangue , Proteínas de Bactérias/imunologia , Febre Paratifoide/microbiologia , Salmonella paratyphi A/imunologia , Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/imunologia , Bacteriemia/imunologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Humanos , Febre Paratifoide/diagnóstico , Febre Paratifoide/imunologia , Prófagos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Salmonella paratyphi A/genética , Salmonella paratyphi A/virologiaRESUMO
BACKGROUND: Salmonella enterica serotype Typhi is a human-restricted intracellular pathogen and the cause of typhoid fever. Cellular immune responses are required to control and clear Salmonella infection. Despite this, there are limited data on cellular immune responses in humans infected with wild type S. Typhi. METHODOLOGY/PRINCIPAL FINDINGS: For this work, we used an automated approach to purify a subset of S. Typhi proteins identified in previous antibody-based immuno-affinity screens and antigens known to be expressed in vivo, including StaF-putative fimbrial protein-STY0202, StbB-fimbrial chaperone-STY0372, CsgF-involved in curli production-STY1177, CsgD- putative regulatory protein-STY1179, OppA-periplasmic oligopeptide binding protein precursor-STY1304, PagC-outer membrane invasion protein-STY1878, and conserved hypothetical protein-STY2195; we also generated and analyzed a crude membrane preparation of S. Typhi (MP). In comparison to samples collected from uninfected Bangladeshi and North American participants, we detected significant interferon-γ responses in PBMCs stimulated with MP, StaF, StbB, CsgF, CsgD, OppA, STY2195, and PagC in patients bacteremic with S. Typhi in Bangladesh. The majority of interferon-γ expressing T cells were CD4 cells, although CD8 responses also occurred. We also assessed cellular proliferation responses in bacteremic patients, and confirmed increased responses in infected individuals to MP, StaF, STY2195, and PagC in convalescent compared to acute phase samples and compared to controls. StaF is a fimbrial protein homologous to E. coli YadK, and contains a Pfam motif thought to be involved in cellular adhesion. PagC is expressed in vivo under the control of the virulence-associated PhoP-regulon required for intra-macrophage survival of Salmonella. STY2195 is a conserved hypothetical protein of unknown function. CONCLUSION/SIGNIFICANCE: This is the first analysis of cellular immune responses to purified S. Typhi antigens in patients with typhoid fever. These results indicate that patients generate significant CD4 and CD8 interferon-γ responses to specific S. Typhi antigens during typhoid fever, and that these responses are elevated at the time of clinical presentation. These observations suggest that an interferon-γ based detection system could be used to diagnose individuals with typhoid fever during the acute stage of illness.
Assuntos
Antígenos de Bactérias/imunologia , Bacteriemia/imunologia , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Salmonella typhi/imunologia , Febre Tifoide/imunologia , Adolescente , Adulto , Bacteriemia/microbiologia , Bangladesh , Proliferação de Células , Células Cultivadas , Criança , Pré-Escolar , Humanos , Lactente , Pessoa de Meia-Idade , Febre Tifoide/microbiologia , Adulto JovemRESUMO
BACKGROUND: Salmonella enterica serotype Typhi is the cause of typhoid fever. It is a human-restricted pathogen, and few data exist on S. Typhi gene expression in humans. METHODOLOGY/PRINCIPAL FINDINGS: We applied an RNA capture and amplification technique, Selective Capture of Transcribed Sequences (SCOTS), and microarray hybridization to identify S. Typhi transcripts expressed in the blood of five humans infected with S. Typhi in Bangladesh. In total, we detected the expression of mRNAs for 2,046 S. Typhi genes (44% of the S. Typhi genome) in human blood; expression of 912 genes was detected in all 5 patients, and expression of 1,100 genes was detected in 4 or more patients. Identified transcripts were associated with the virulence-associated PhoP regulon, Salmonella pathogenicity islands, the use of alternative carbon and energy sources, synthesis and transport of iron, thiamine, and biotin, and resistance to antimicrobial peptides and oxidative stress. The most highly represented group were genes currently annotated as encoding proteins designated as hypothetical, unknown, or unclassified. Of the 2,046 detected transcripts, 1,320 (29% of the S. Typhi genome) had significantly different levels of detection in human blood compared to in vitro cultures; detection of 141 transcripts was significantly different in all 5 patients, and detection of 331 transcripts varied in at least 4 patients. These mRNAs encode proteins of unknown function, those involved in energy metabolism, transport and binding, cell envelope, cellular processes, and pathogenesis. We confirmed increased expression of a subset of identified mRNAs by quantitative-PCR. CONCLUSIONS/SIGNIFICANCE: We report the first characterization of bacterial transcriptional profiles in the blood of patients with typhoid fever. S. Typhi is an important global pathogen whose restricted host range has greatly inhibited laboratory studies. Our results suggest that S. Typhi uses a largely uncharacterized genetic repertoire to survive within cells and utilize alternate energy sources during infection.
Assuntos
RNA Bacteriano/sangue , Salmonella typhi/genética , Febre Tifoide/microbiologia , Adolescente , Adulto , Bacteriemia/microbiologia , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Bangladesh , Criança , Pré-Escolar , Perfilação da Expressão Gênica , Humanos , Lactente , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , RNA Bacteriano/química , RNA Bacteriano/classificação , RNA Mensageiro/sangue , RNA Mensageiro/química , RNA Mensageiro/classificação , Reação em Cadeia da Polimerase em Tempo Real , Salmonella typhi/isolamento & purificação , Febre Tifoide/sangueRESUMO
BACKGROUND: Salmonella enterica serotype Paratyphi A is a human-restricted cause of paratyphoid fever, accounting for up to a fifth of all cases of enteric fever in Asia. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we applied an RNA analysis method, Selective Capture of Transcribed Sequences (SCOTS), and cDNA hybridization-microarray technology to identify S. Paratyphi A transcripts expressed by bacteria in the blood of three patients in Bangladesh. In total, we detected 1,798 S. Paratyphi A mRNAs expressed in the blood of infected humans (43.9% of the ORFeome). Of these, we identified 868 in at least two patients, and 315 in all three patients. S. Paratyphi A transcripts identified in at least two patients encode proteins involved in energy metabolism, nutrient and iron acquisition, vitamin biosynthesis, stress responses, oxidative stress resistance, and pathogenesis. A number of detected transcripts are expressed from PhoP and SlyA-regulated genes associated with intra-macrophage survival, genes contained within Salmonella Pathogenicity Islands (SPIs) 1-4, 6, 10, 13, and 16, as well as RpoS-regulated genes. The largest category of identified transcripts is that of encoding proteins with unknown function. When comparing levels of bacterial mRNA using in vivo samples collected from infected patients to samples from in vitro grown organisms, we found significant differences for 347, 391, and 456 S. Paratyphi A transcripts in each of three individual patients (approximately 9.7% of the ORFeome). Of these, expression of 194 transcripts (4.7% of ORFs) was concordant in two or more patients, and 41 in all patients. Genes encoding these transcripts are contained within SPI-1, 3, 6 and 10, PhoP-regulated genes, involved in energy metabolism, nutrient acquisition, drug resistance, or uncharacterized genes. Using quantitative RT-PCR, we confirmed increased gene expression in vivo for a subset of these genes. CONCLUSION/SIGNIFICANCE: To our knowledge, we describe the first microarray-based transcriptional analysis of a pathogen in the blood of naturally infected humans.
Assuntos
Bacteriemia/microbiologia , Sangue/microbiologia , Perfilação da Expressão Gênica , Febre Paratifoide/microbiologia , Salmonella paratyphi A/genética , Adolescente , Adulto , Bangladesh , Criança , Pré-Escolar , DNA Complementar/genética , Humanos , Análise em Microsséries , Pessoa de Meia-Idade , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salmonella paratyphi A/isolamento & purificação , Adulto JovemRESUMO
Salmonella enterica serotype Typhi is the cause of typhoid fever and a human-restricted pathogen. Currently available typhoid vaccines provide 50 to 90% protection for 2 to 5 years, and available practical diagnostic assays to identify individuals with typhoid fever lack sensitivity and/or specificity. Identifying immunogenic S. Typhi antigens expressed during human infection could lead to improved diagnostic assays and vaccines. Here we describe a platform immunoaffinity proteomics-based technology (IPT) that involves the use of columns charged with IgG, IgM, or IgA antibody fractions recovered from humans bacteremic with S. Typhi to capture S. Typhi proteins that were subsequently identified by mass spectrometry. This screening tool identifies immunogenic proteins recognized by antibodies from infected hosts. Using this technology and the plasma of patients with S. Typhi bacteremia in Bangladesh, we identified 57 proteins of S. Typhi, including proteins known to be immunogenic (PagC, HlyE, OmpA, and GroEL) and a number of proteins present in the human-restricted serotypes S. Typhi and S. Paratyphi A but rarely found in broader-host-range Salmonella spp. (HlyE, CdtB, PltA, and STY1364). We categorized identified proteins into a number of major groupings, including those involved in energy metabolism, protein synthesis, iron homeostasis, and biosynthetic and metabolic functions and those predicted to localize to the outer membrane. We assessed systemic and mucosal anti-HlyE responses in S. Typhi-infected patients and detected anti-HlyE responses at the time of clinical presentation in patients but not in controls. These findings could assist in the development of improved diagnostic assays.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Bacteriemia/imunologia , Proteínas de Bactérias/imunologia , Salmonella typhi/imunologia , Febre Tifoide/imunologia , Adolescente , Adulto , Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Bangladesh , Criança , Pré-Escolar , Cromatografia de Afinidade , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Espectrometria de Massas , Pessoa de Meia-Idade , Proteômica/métodos , Adulto JovemRESUMO
Antibodies specific for Vibrio cholerae lipopolysaccaride (LPS) are common in humans recovering from cholera, and constitute a primary component of the vibriocidal response, a serum complement-mediated bacteriocidal response correlated with protection against cholera. In order to determine whether transcutaneous immunization (TCI) with a V. cholerae neoglycoconjugate (CHO-BSA) comprised of a synthetic terminal hexasaccharide of the O-specific polysaccharide of V. cholerae O1 (Ogawa) conjugated with bovine serum albumin (BSA) could induce anti-V. cholerae LPS and vibriocidal responses, we applied CHO-BSA transcutaneously in the presence or absence of the immune adjuvant cholera toxin (CT) to mice. Transcutaneously applied neoglycoconjugate elicited prominent V. cholerae specific LPS IgG responses in the presence of CT, but not IgM or IgA responses. CT applied on the skin induced strong IgG and IgA serum responses. TCI with neoglycoconjugate did not elicit detectable vibriocidal responses, protection in a mouse challenge assay, or stool anti-V. cholerae IgA responses, irrespective of the presence or absence of CT. Our results suggest that transcutaneously applied synthetic V. cholerae neoglycoconjugate is safe and immunogenic, but predominantly induces systemic LPS responses of the IgG isotype.
Assuntos
Anticorpos Antibacterianos/sangue , Vacinas contra Cólera/administração & dosagem , Vacinas contra Cólera/imunologia , Vibrio cholerae/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Administração Cutânea , Animais , Toxina da Cólera/administração & dosagem , Toxina da Cólera/farmacologia , Vacinas contra Cólera/efeitos adversos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana , Antígenos O/química , Antígenos O/imunologia , Soroalbumina Bovina/química , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/imunologia , Vibrio cholerae O1/imunologiaRESUMO
Clostridium difficile is the leading cause of nosocomial infectious diarrhea. C. difficile produces two toxins (A and B), and systemic and mucosal anti-toxin A antibodies prevent or limit C. difficile-associated diarrhea. To evaluate whether transcutaneous immunization with formalin-treated C. difficile toxin A (CDA) induces systemic and mucosal anti-CDA immune responses, we transcutaneously immunized three cohorts of mice with CDA with or without immunoadjuvantative cholera toxin (CT) on days 0, 14, 28, and 42. Mice transcutaneously immunized with CDA and CT developed prominent anti-CDA and anti-CT immunoglobulin G (IgG) and IgA responses in serum and anti-CDA and anti-CT IgA responses in stool. Sera from immunized mice were able to neutralize C. difficile toxin A activity in an in vitro cell culture assay. CDA itself demonstrated adjuvant activity and enhanced both serum and stool anti-CT IgA responses. Our results suggest that transcutaneous immunization with CDA toxoid may be a feasible immunization strategy against C. difficile, an important cause of morbidity and mortality against which current preventative strategies are failing.
Assuntos
Anticorpos Antibacterianos/imunologia , Toxinas Bacterianas/administração & dosagem , Clostridioides difficile/química , Enterotoxinas/administração & dosagem , Imunidade nas Mucosas , Administração Cutânea , Animais , Anticorpos Antibacterianos/análise , Toxinas Bacterianas/imunologia , Enterotoxinas/imunologia , Imunização , Camundongos , Mucosa/imunologia , Testes de Neutralização , Toxoides/administração & dosagem , Toxoides/imunologiaRESUMO
Toxin-coregulated pilin A (TcpA) is the main structural subunit of a type IV bundle-forming pilus of Vibrio cholerae, the cause of cholera. Toxin-coregulated pilus is involved in formation of microcolonies of V. cholerae at the intestinal surface, and strains of V. cholerae deficient in TcpA are attenuated and unable to colonize intestinal surfaces. Anti-TcpA immunity is common in humans recovering from cholera in Bangladesh, and immunization against TcpA is protective in murine V. cholerae models. To evaluate whether transcutaneously applied TcpA is immunogenic, we transcutaneously immunized mice with 100 mug of TcpA or TcpA with an immunoadjuvant (cholera toxin [CT], 50 mug) on days 0, 19, and 40. Mice immunized with TcpA alone did not develop anti-TcpA responses. Mice that received transcutaneously applied TcpA and CT developed prominent anti-TcpA immunoglobulin G (IgG) serum responses but minimal anti-TcpA IgA. Transcutaneous immunization with CT induced prominent IgG and IgA anti-CT serum responses. In an infant mouse model, offspring born to dams transcutaneously immunized either with TcpA and CT or with CT alone were challenged with 10(6) CFU (one 50% lethal dose) wild-type V. cholerae O1 El Tor strain N16961. At 48 h, mice born to females transcutaneously immunized with CT alone had 36% +/- 10% (mean +/- standard error of the mean) survival, while mice born to females transcutaneously immunized with TcpA and CT had 69% +/- 6% survival (P < 0.001). Our results suggest that transcutaneous immunization with TcpA and an immunoadjuvant induces protective anti-TcpA immune responses. Anti-TcpA responses may contribute to an optimal cholera vaccine.
Assuntos
Cólera/prevenção & controle , Proteínas de Fímbrias/administração & dosagem , Proteínas de Fímbrias/imunologia , Vibrio cholerae O1/imunologia , Administração Cutânea , Animais , Anticorpos Antibacterianos/análise , Anticorpos Antibacterianos/sangue , Vacinas contra Cólera/imunologia , Feminino , Imunização , Imunoglobulina A/análise , Imunoglobulina A/sangue , Imunoglobulina G/análise , Imunoglobulina G/sangue , Camundongos , Mucosa/imunologiaRESUMO
More than half of patients with X-linked lympho-proliferative disease, which is caused by a defect in the intracellular adapter protein SH2D1A, suffer from an extreme susceptibility to Epstein-Barr virus. One-third of these patients, however, develop dysgammaglobulenemia without an episode of severe mononucleosis. Here we show that in SH2D1A(-/-) mice, both primary and secondary responses of all Ig subclasses are severely impaired in response to specific antigens. Because germinal centers were absent in SH2D1A(-/-) mice upon primary immunization, and because SH2D1A was detectable in wt germinal center B cells, we examined whether SH2D1A(-/-) B cell functions were impaired. Using the adoptive cotransfer of B lymphocytes from hapten-primed SH2D1A(-/-) mice with CD4(+) T cells from primed wt mice into irradiated wt mice provided evidence that signal transduction events controlled by SH2D1A are essential for B cell activities resulting in antigen specific IgG production. Defects in naive SH2D1A(-/-) B cells became evident upon cotransfer with non-primed wt CD4(+) cells into Rag2(-/-) recipients. Thus, both defective T and B cells exist in the absence of SH2D1A, which may explain the progressive dysgammaglobulinemia in a subset of X-linked lympho-proliferative disease patients without involvement of Epstein-Barr virus.