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Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of diarrheal illnesses annually ranging from mildly symptomatic cases to severe, life-threatening cholera-like diarrhea. Although ETEC are associated with long-term sequelae including malnutrition, the acute diarrheal illness is largely self-limited. Recent studies indicate that in addition to causing diarrhea, the ETEC heat-labile toxin (LT) modulates the expression of many genes in intestinal epithelia, including carcinoembryonic cell adhesion molecules (CEACAMs) which ETEC exploit as receptors, enabling toxin delivery. Here however, we demonstrate that LT also enhances the expression of CEACAMs on extracellular vesicles (EV) shed by intestinal epithelia and that CEACAM-laden EV increase in abundance during human infections, mitigate pathogen-host interactions, scavenge free ETEC toxins, and accelerate ETEC clearance from the gastrointestinal tract. Collectively, these findings indicate that CEACAMs play a multifaceted role in ETEC pathogen-host interactions, transiently favoring the pathogen, but ultimately contributing to innate responses that extinguish these common infections.
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BACKGROUND: Enterotoxigenic E. coli (ETEC) is a principal cause of diarrhea in travelers, deployed military personnel, and children living in low to middle-income countries. ETEC expresses a variety of virulence factors including colonization factors (CF) that facilitate adherence to the intestinal mucosa. We assessed the protective efficacy of a tip-localized subunit of CF antigen I (CFA/I), CfaE, delivered intradermally with the mutant E. coli heat-labile enterotoxin, LTR192G, in a controlled human infection model (CHIM). METHODS: Three cohorts of healthy adult subjects were enrolled and given three doses of 25 µg CfaE + 100 ng LTR192G vaccine intradermally at 3-week intervals. Approximately 28 days after the last vaccination, vaccinated and unvaccinated subjects were admitted as inpatients and challenged with approximately 2 × 107 cfu of CFA/I+ ETEC strain H10407 following an overnight fast. Subjects were assessed for moderate-to-severe diarrhea for 5 days post-challenge. RESULTS: A total of 52 volunteers received all three vaccinations; 41 vaccinated and 43 unvaccinated subjects were challenged and assessed for moderate-to-severe diarrhea. Naïve attack rates varied from 45.5% to 64.7% across the cohorts yielding an overall efficacy estimate of 27.8% (95% confidence intervals: -7.5-51.6%). In addition to reducing moderate-severe diarrhea rates, the vaccine significantly reduced loose stool output and overall ETEC disease severity. CONCLUSIONS: This is the first study to demonstrate protection against ETEC challenge after intradermal vaccination with an ETEC adhesin. Further examination of the challenge methodology is necessary to address the variability in naïve attack rate observed among the three cohorts in the present study.
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Campylobacter jejuni infection is a leading cause of foodborne disease, common to children, adult travelers, and military populations in low- to middle-income countries. In the absence of a licensed vaccine, efforts to evaluate prophylactic agents are underway. The prophylactic efficacy of a twice-daily, 550 mg dose of the antibiotic rifaximin demonstrated no efficacy against campylobacteriosis in a controlled human infection model (CHIM); however, samples from the CHIM study were utilized to assess how the human gut microbiome responds to C. jejuni infection, and if a 'protective' microbiota exists in study participants not developing campylobacteriosis. Statistically significant, but minor, differences in study participant beta diversity were identified during the challenge period (p = 0.002, R2 = 0.042), but no significant differences were otherwise observed. Pre-challenge alpha diversity was elevated in study participants who did not develop campylobacteriosis compared to those who did (p < 0.001), but alpha diversity declined in all study participants from the pre-challenge period to post-discharge. Our work provides insight into gut microbiome shifts observed during a C. jejuni CHIM and following antibiotic treatment. This study utilized a high dose of 1.7 x 105 colony-forming units of C. jejuni; future work could include CHIM studies performed with inocula more closely mimicking natural exposure as well as field studies involving naturally-occurring enteric infections.
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Infecções por Campylobacter , Campylobacter jejuni , Microbioma Gastrointestinal , Adulto , Assistência ao Convalescente , Criança , Humanos , Alta do PacienteRESUMO
Recent studies have gained a better appreciation of the potential impacts of enteric infections beyond symptomatic diarrhea. It is recognized that infections by several enteropathogens could be associated with growth deficits in children and intestinal and systemic inflammation may play an important underlying role. With enterotoxigenic E. coli (ETEC) being one of the leading causes of diarrhea among children in the developing world and important contributor to stunting, a better understanding of the impact of ETEC infection beyond diarrhea is timely and greatly needed. To address this, we evaluated if ETEC infection induces intestinal and systemic inflammation and its impact on colonization and immune responses to ETEC vaccine-specific antigens in a dose descending experimental human challenge model using ETEC strain H10407. This study demonstrates that the concentrations of myeloperoxidase (MPO) in stool and intestinal fatty acid-binding protein (an indicator of compromised intestinal epithelial integrity) in serum, significantly increased following ETEC infection in both diarrhea and asymptomatic cases and the magnitudes and kinetics of MPO are dose and clinical outcome dependent. Cytokines IL-17A and IFN-γ were significantly increased in serum post-ETEC challenge. In addition, higher pre-challenge concentrations of cytokines IL-10 and GM-CSF were associated with protection from ETEC diarrhea. Interestingly, higher MPO concentrations were associated with higher intestinal colonization of ETEC and lower seroconversions of colonization factor I antigen, but the reverse was noted for seroconversions to heat-labile toxin B-subunit. Together this study has important implications for understanding the acute and long-term negative health outcomes associated with ETEC infection.
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Escherichia coli Enterotoxigênica/crescimento & desenvolvimento , Escherichia coli Enterotoxigênica/imunologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Inflamação/microbiologia , Intestinos/microbiologia , Anticorpos Antibacterianos/sangue , Infecções Assintomáticas , Toxinas Bacterianas/imunologia , Citocinas/sangue , Diarreia/microbiologia , Enterotoxinas/imunologia , Proteínas de Escherichia coli/imunologia , Proteínas de Ligação a Ácido Graxo/sangue , Fezes/química , Proteínas de Fímbrias/imunologia , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Inflamação/imunologia , Peroxidase/análiseRESUMO
Enterotoxigenic Escherichia coli (ETEC) infections are a common cause of severe diarrheal illness in low- and middle-income countries. The live-attenuated ACE527 ETEC vaccine, adjuvanted with double mutant heat-labile toxin (dmLT), affords clear but partial protection against ETEC challenge in human volunteers. Comparatively, initial wild-type ETEC challenge completely protects against severe diarrhea on homologous re-challenge. To investigate determinants of protection, vaccine antigen content was compared to wild-type ETEC, and proteome microarrays were used to assess immune responses following vaccination and ETEC challenge. Although molecular interrogation of the vaccine confirmed expression of targeted canonical antigens, relative to wild-type ETEC, vaccine strains were deficient in production of flagellar antigens, immotile, and lacked production of the EtpA adhesin. Similarly, vaccination ± dmLT elicited responses to targeted canonical antigens, but relative to wild-type challenge, vaccine responses to some potentially protective non-canonical antigens including EtpA and the YghJ metalloprotease were diminished or absent. These studies highlight important differences in vaccine and wild-type ETEC antigen content and call attention to distinct immunologic signatures that could inform investigation of correlates of protection, and guide vaccine antigen selection for these pathogens of global importance.
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Enterotoxigenic Escherichia coli (ETEC) is a leading cause of bacterial diarrhea both among children in low and middle income countries and in travelers to these regions. Although there are several approaches to develop an effective vaccine for ETEC, no licensed vaccines are currently available. The most advanced ETEC vaccine candidates include multiple colonization factors along with the heat labile toxin B subunit. In the absence of known correlates of protection, and to understand the mechanism of protection, monitoring immune responses to a majority of the vaccine associated antigens using various types of samples is needed. Unfortunately, a traditional ELISA is time consuming, labor intensive and requires substantial amounts of antigens and sample volumes. To address these constraints, we developed and validated a novel high throughput electrochemiluminescent (ECL) - based multiplex immunoassay using Meso Scale Discovery (MSD) platform for analyzing immune responses to ETEC antigens. The ETEC multiplex ECL assay is an 8-plex assay which includes the ETEC colonization factor antigens (CFA/I, CS1, CS2, CS3, CS5 and CS6) along with the two subunits of heat labile toxin (LTA and LTB). Our data suggested that a single dilution of sample provides a quantifiable result for a wide range of sample titers. To compare ETEC multiplex ECL with ELISA, we carried out assays using the same antigens with the two immunoassay platforms using a common sample set of serum and ALS (antibodies in lymphocyte supernatant) specimens. The MSD platform achieved excellent correlations with ELISA for the antigens tested, consistently detecting comparable antibody levels in the samples. The ETEC multiplex ECL can serve as a fundamental platform in evaluating performances of candidate ETEC vaccines in future field trials.
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Anticorpos Antibacterianos/sangue , Técnicas Eletroquímicas/métodos , Escherichia coli Enterotoxigênica/imunologia , Vacinas contra Escherichia coli/análise , Imunoensaio/métodos , Medições Luminescentes/métodos , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/química , Toxinas Bacterianas/imunologia , Técnicas Eletroquímicas/normas , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/imunologia , Humanos , Soros Imunes/química , Imunoensaio/normas , Luminescência , Medições Luminescentes/normas , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: There is no licensed vaccine against enterotoxigenic Escherichia coli (ETEC), a major cause of diarrhea-associated morbidity and mortality among infants and children in low-income countries and travelers. The results of this vaccination/challenge study demonstrate strong protection by an attenuated ETEC vaccine candidate, ACE527, when co-administered with a mucosal adjuvant, the double-mutant heat-labile toxin (dmLT) of ETEC. METHODS: Sixty healthy adults participated in a randomized, placebo-controlled, double-blind study with three doses of lyophilized ACE527 (â¼3â¯×â¯109 of each strain per dose) administered orally with or without dmLT adjuvant (25⯵g/dose). Six months later, 36 of these volunteers and a control group of 21 unvaccinated volunteers were challenged with virulent ETEC strain H10407. The primary outcome was severe diarrhea, defined as passing >800â¯g of unformed stools during the inpatient period following challenge. FINDINGS: The vaccine was well tolerated and induced robust immune responses to key antigens. The protective efficacy (PE) against the primary outcome of severe diarrhea was 65.9% (95% confidence interval [CI] 5.4-87.7, pâ¯=â¯0.003). Among subjects receiving the adjuvanted vaccine, the attack rate of severe diarrhea was 23.1, while in unimmunized controls it was 67.7%. The PE against diarrhea of any severity was 58.5% (95% CI 3.8- 82.1, pâ¯=â¯0.016). There was a strong inverse correlation between shedding of the vaccine strain after either of the first two doses and absence of severe diarrhea upon challenge (RRâ¯=â¯0.29, 95% CI 0.08-1.05, pâ¯=â¯0.041). Challenge strain shedding was 10-fold lower in those receiving the adjuvant than in those receiving vaccine alone. The unadjuvanted vaccine was not protective (PEâ¯=â¯23.1%). INTERPRETATION: The results of this study support further development of ACE527â¯+â¯dmLT as a vaccine for children in endemic countries and travelers. This is the first clinical demonstration that dmLT can contribute significantly to vaccine efficacy and may warrant testing with other oral vaccines. (ClinicalTrials.gov registration: NCT01739231).
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Adjuvantes Imunológicos , Escherichia coli Enterotoxigênica/imunologia , Infecções por Escherichia coli/prevenção & controle , Vacinas contra Escherichia coli/imunologia , Vacinas Atenuadas/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adulto , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Vacinas contra Escherichia coli/administração & dosagem , Voluntários Saudáveis , Humanos , Pessoa de Meia-Idade , Adulto JovemRESUMO
Enterotoxigenic Escherichia coli (ETEC) infections are highly prevalent in developing countries, where clinical presentations range from asymptomatic colonization to severe cholera-like illness. The molecular basis for these varied presentations, which may involve strain-specific virulence features as well as host factors, has not been elucidated. We demonstrate that, when challenged with ETEC strain H10407, originally isolated from a case of cholera-like illness, blood group A human volunteers developed severe diarrhea more frequently than individuals from other blood groups. Interestingly, a diverse population of ETEC strains, including H10407, secrete the EtpA adhesin molecule. As many bacterial adhesins also agglutinate red blood cells, we combined the use of glycan arrays, biolayer inferometry, and noncanonical amino acid labeling with hemagglutination studies to demonstrate that EtpA is a dominant ETEC blood group A-specific lectin/hemagglutinin. Importantly, we have also shown that EtpA interacts specifically with glycans expressed on intestinal epithelial cells from blood group A individuals and that EtpA-mediated bacterial-host interactions accelerate bacterial adhesion and effective delivery of both the heat-labile and heat-stable toxins of ETEC. Collectively, these data provide additional insight into the complex molecular basis of severe ETEC diarrheal illness that may inform rational design of vaccines to protect those at highest risk.
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Sistema ABO de Grupos Sanguíneos/metabolismo , Diarreia , Escherichia coli Enterotoxigênica , Células Epiteliais/metabolismo , Infecções por Escherichia coli/metabolismo , Mucosa Intestinal/metabolismo , Adesinas de Escherichia coli/metabolismo , Diarreia/metabolismo , Diarreia/microbiologia , Diarreia/patologia , Escherichia coli Enterotoxigênica/metabolismo , Escherichia coli Enterotoxigênica/patogenicidade , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Infecções por Escherichia coli/patologia , Feminino , Humanos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Índice de Gravidade de DoençaRESUMO
Background: Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal illness in the developing world. Enterotoxigenic E coli vaccinology has been challenged by genetic diversity and heterogeneity of canonical antigens. Examination of the antigenic breadth of immune responses associated with protective immunity could afford new avenues for vaccine development. Methods: Antibody lymphocyte supernatants (ALS) and sera from 20 naive human volunteers challenged with ETEC strain H10407 and from 10 volunteers rechallenged 4-6 weeks later with the same strain (9 of whom were completely protected on rechallenge) were tested against ETEC proteome microarrays containing 957 antigens. Results: Enterotoxigenic E coli challenge stimulated robust serum and mucosal (ALS) responses to canonical vaccine antigens (CFA/I, and the B subunit of LT) as well as a small number of antigens not presently targeted in ETEC vaccines. These included pathovar-specific secreted proteins (EtpA, EatA) as well as highly conserved E coli antigens including YghJ, flagellin, and pertactin-like autotransporter proteins, all of which have previously afforded protection against ETEC infection in preclinical studies. Conclusions: Taken together, studies reported here suggest that immune responses after ETEC infection involve traditional vaccine targets as well as a select number of more recently identified protein antigens that could offer additional avenues for vaccine development for these pathogens.
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Antígenos de Bactérias/imunologia , Escherichia coli Enterotoxigênica/imunologia , Infecções por Escherichia coli/imunologia , Vacinas contra Escherichia coli/imunologia , Anticorpos Antibacterianos/imunologia , Proteínas de Transporte/imunologia , Proteínas de Escherichia coli/imunologia , Humanos , Glicoproteínas de Membrana/imunologia , Peptídeo HidrolasesRESUMO
A reliable and effective human challenge model is needed to help down-select the most promising ETEC vaccines currently under development. Such a model would need to reliably induce diarrhea in a high proportion of volunteers using the lowest possible inoculum to maximize safety and sensitivity. Previously we validated a challenge model that utilized a dose of 2x107 CFU of ETEC strain H10407 (LT+, ST+, CFA/I+ and O78+) to induce attack rates for moderate to severe diarrhea (MSD) of ~60-70%. Here we detail efforts to further refine the model in an attempt to determine if a lower challenge dose of H10407 can be used. Thirty subjects were randomized 1:1 to receive an oral administration of H10407 at doses of 106 or 105 CFU in bicarbonate buffer. After challenge, subjects were monitored for signs and symptoms of enteric illness and stool samples were collected to detect shedding of the challenge strain. Systemic and mucosal immune responses were measured using serum, antibody in lymphocyte supernatant and fecal samples. The attack rate was 13.3% (2/15) and 26.7% (4/15) for MSD in the 105 and 106 groups, respectively. Four MSD cases met criteria for early antibiotic treatment. All subjects but one shed the challenge strain in fecal samples. The frequency and magnitude of anti-LT toxin, CFA/I and LPS O78 immune responses were antigen, dose, severity of diarrhea and shedding levels dependent. Notably, although of lower magnitude, there were considerable immune responses in the subjects with no diarrhea. This may indicate that immune responses to asymptomatic infections of ETEC in children in the endemic countries may contribute to protection. Based on this and our prior studies, we conclude that a dose of 2x107 H10407 remains the lowest practical dose for use in future volunteer studies evaluating candidate vaccines and other preventive or therapeutic ETEC interventions. TRIAL REGISTRATION: ClinicalTrials.gov NCT00844493.
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Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Diarreia/imunologia , Escherichia coli Enterotoxigênica/imunologia , Infecções por Escherichia coli/imunologia , Administração Oral , Adulto , Diarreia/microbiologia , Infecções por Escherichia coli/microbiologia , Fezes/microbiologia , Feminino , Humanos , Imunidade nas Mucosas/imunologia , Intestinos/microbiologia , Masculino , Pessoa de Meia-Idade , Voluntários , Adulto JovemRESUMO
Campylobacter jejuni infections are a leading cause of bacterial food-borne diarrhoeal illness worldwide, and Campylobacter infections in children are associated with stunted growth and therefore long-term deficits into adulthood. Despite this global impact on health and human capital, how zoonotic C. jejuni responds to the human host remains unclear. Unlike other intestinal pathogens, C. jejuni does not harbour pathogen-defining toxins that explicitly contribute to disease in humans. This makes understanding Campylobacter pathogenesis challenging and supports a broad examination of bacterial factors that contribute to C. jejuni infection. Here, we use a controlled human infection model to characterize C. jejuni transcriptional and genetic adaptations in vivo, along with a non-human primate infection model to validate our approach. We found that variation in 11 genes is associated with either acute or persistent human infections and includes products involved in host cell invasion, bile sensing and flagella modification, plus additional potential therapeutic targets. In particular, a functional version of the cell invasion protein A (cipA) gene product is strongly associated with persistently infecting bacteria and we identified its biochemical role in flagella modification. These data characterize the adaptive C. jejuni response to primate infections and suggest therapy design should consider the intrinsic differences between acute and persistently infecting bacteria. In addition, RNA sequencing revealed conserved responses during natural host commensalism and human infections. Thirty-nine genes were differentially regulated in vivo across hosts, lifestyles and C. jejuni strains. This conserved in vivo response highlights important C. jejuni survival mechanisms such as iron acquisition and evasion of the host mucosal immune response. These advances highlight pathogen adaptability across host species and demonstrate the utility of multidisciplinary collaborations in future clinical trials to study pathogens in vivo.
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Proteínas de Bactérias/genética , Infecções por Campylobacter/patologia , Campylobacter jejuni/genética , Campylobacter jejuni/patogenicidade , Flagelos/genética , Doenças Transmitidas por Alimentos/patologia , Proteínas de Membrana/genética , Animais , Azitromicina/uso terapêutico , Infecções por Campylobacter/tratamento farmacológico , Infecções por Campylobacter/microbiologia , Galinhas/microbiologia , Ciprofloxacina/uso terapêutico , Doenças Transmitidas por Alimentos/tratamento farmacológico , Doenças Transmitidas por Alimentos/microbiologia , Regulação Bacteriana da Expressão Gênica/genética , Variação Genética/genética , Humanos , Intestinos/microbiologia , Intestinos/patologia , Rifaximina/uso terapêuticoRESUMO
Background: Campylobacter species are a leading cause of diarrheal disease globally with significant morbidity. Primary prevention efforts have yielded limited results. Rifaximin chemoprophylaxis decreases rates of travelers' diarrhea and may be suitable for high-risk persons. We assessed the efficacy of rifaximin in the controlled human infection model for Campylobacter jejuni. Methods: Twenty-eight subjects were admitted to an inpatient facility and randomized to a twice-daily dose of 550 mg rifaximin or placebo. The following day, subjects ingested 1.7 × 105 colony-forming units of C. jejuni strain CG8421. Subjects continued prophylaxis for 3 additional days, were followed for campylobacteriosis for 144 hours, and were subsequently treated with azithromycin and ciprofloxacin. Samples were collected to assess immunologic responses to CG8421. Results: There was no difference (P = 1.0) in the frequency of campylobacteriosis in those receiving rifaximin (86.7%) or placebo (84.6%). Additionally, there were no differences in the clinical signs and symptoms of C. jejuni infection to include abdominal pain/cramps (P = 1.0), nausea (P = 1.0), vomiting (P = .2), or fever (P = 1.0) across study groups. Immune responses to the CG8421 strain were comparable across treatment groups. Conclusions: Rifaximin did not prevent campylobacteriosis in this controlled human infection model. Given the morbidity associated with Campylobacter infection, primary prevention efforts remain a significant need. Clinical Trials Registration: NCT02280044.
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Antibacterianos/uso terapêutico , Infecções por Campylobacter/prevenção & controle , Quimioprevenção , Rifaximina/uso terapêutico , Adulto , Antibacterianos/administração & dosagem , Azitromicina/uso terapêutico , Campylobacter jejuni , Ciprofloxacina/uso terapêutico , Diarreia/prevenção & controle , Método Duplo-Cego , Feminino , Voluntários Saudáveis , Experimentação Humana , Humanos , Masculino , Rifaximina/administração & dosagem , Adulto JovemRESUMO
BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in inhabitants from low-income countries and in visitors to these countries. The impact of the human intestinal microbiota on the initiation and progression of ETEC diarrhea is not yet well understood. RESULTS: We used 16S rRNA (ribosomal RNA) gene sequencing to study changes in the fecal microbiota of 12 volunteers during a human challenge study with ETEC (H10407) and subsequent treatment with ciprofloxacin. Five subjects developed severe diarrhea and seven experienced few or no symptoms. Diarrheal symptoms were associated with high concentrations of fecal E. coli as measured by quantitative culture, quantitative PCR, and normalized number of 16S rRNA gene sequences. Large changes in other members of the microbiota varied greatly from individual to individual, whether or not diarrhea occurred. Nonetheless the variation within an individual was small compared to variation between individuals. Ciprofloxacin treatment reorganized microbiota populations; however, the original structure was largely restored at one and three month follow-up visits. CONCLUSION: Symptomatic ETEC infections, but not asymptomatic infections, were associated with high fecal concentrations of E. coli. Both infection and ciprofloxacin treatment caused variable changes in other bacteria that generally reverted to baseline levels after three months.
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Ciprofloxacina/uso terapêutico , Escherichia coli Enterotoxigênica/efeitos dos fármacos , Escherichia coli Enterotoxigênica/fisiologia , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Adulto , Ciprofloxacina/farmacologia , Diarreia/tratamento farmacológico , Diarreia/microbiologia , Fezes/microbiologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Metagenoma , Metagenômica/métodos , Pessoa de Meia-Idade , RNA Ribossômico 16S , Curva ROC , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Experimental human challenge models have played a major role in enhancing our understanding of infectious diseases. Primary outcomes have typically utilized overly simplistic outcomes that fail to entirely account for complex illness syndromes. We sought to characterize clinical outcomes associated with experimental infection with enterotoxigenic Escherichia coli (ETEC) and to develop a disease score. METHODS: Data were obtained from prior controlled human ETEC infection studies. Correlation and univariate regression across sign and symptom severity was performed. A multiple correspondence analysis was conducted. A 3-parameter disease score with construct validity was developed in an iterative fashion, compared to standard outcome definitions and applied to prior vaccine challenge trials. RESULTS: Data on 264 subjects receiving seven ETEC strains at doses from 1x105 to 1x1010 cfu were used to construct a standardized dataset. The strongest observed correlation was between vomiting and nausea (r = 0.65); however, stool output was poorly correlated with subjective activity-impacting outcomes. Multiple correspondence analyses showed covariability in multiple signs and symptoms, with severity being the strongest factor corresponding across outcomes. The developed disease score performed well compared to standard outcome definitions and differentiated disease in vaccinated and unvaccinated subjects. CONCLUSION: Frequency and volumetric definitions of diarrhea severity poorly characterize ETEC disease. These data support a disease severity score accounting for stool output and other clinical signs and symptoms. Such a score could serve as the basis for better field trial outcomes and gives an additional outcome measure to help select future vaccines that warrant expanded testing in pivotal pre-licensure trials.
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Diarreia/fisiopatologia , Escherichia coli Enterotoxigênica/patogenicidade , Infecções por Escherichia coli/fisiopatologia , Vacinas contra Escherichia coli/uso terapêutico , Adulto , Diarreia/microbiologia , Infecções por Escherichia coli/microbiologia , Feminino , Febre/microbiologia , Febre/fisiopatologia , Cefaleia/microbiologia , Cefaleia/fisiopatologia , Humanos , Masculino , Náusea/microbiologia , Náusea/fisiopatologia , Índice de Gravidade de Doença , Vômito/microbiologia , Vômito/fisiopatologiaRESUMO
Shigella causes high morbidity and mortality worldwide, but there is no licensed vaccine for shigellosis yet. We evaluated the safety and immunogenicity of a formalin-inactivated whole-cell Shigella flexneri2a vaccine, Sf2aWC, given orally to adult volunteers. In a double-blind, placebo-controlled trial, 82 subjects were randomized to receive three doses of vaccine in dose escalation (2.6 ± 0.8 × 10(8), × 10(9), × 10(10), and × 10(11)vaccine particles/ml). Vaccine safety was actively monitored, and antigen-specific systemic and mucosal immune responses were determined in serum, antibody in lymphocyte supernatant (ALS), and fecal samples. Cytokines were measured in the serum. Sf2aWC was well tolerated and generally safe at all four dose levels. The vaccine resulted in a dose-dependent immune response. At the highest dose, the vaccine induced robust responses to lipopolysaccharide (LPS) in both serum and ALS samples. The highest magnitude and frequency of responses occurred after the first dose in almost all samples but was delayed for IgG in serum. Fifty percent of the vaccinees had a >4-fold increase in anti-LPS fecal antibody titers. Responses to invasion plasmid antigens (Ipa) were low. The levels of interleukin-17 (IL-17), IL-2, gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-10 were increased, and IL-8 was decreased immediately after first dose, but these changes were very transient. This phase I trial demonstrated that the Sf2aWC vaccine, a relatively simple vaccine concept, was safe and immunogenic. The vaccine elicited immune responses which were comparable to those induced by a live, attenuated Shigella vaccine that was protective in prior human challenge studies.
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Disenteria Bacilar/prevenção & controle , Vacinas contra Shigella/imunologia , Shigella flexneri/imunologia , Administração Oral , Adolescente , Adulto , Anticorpos Antibacterianos/análise , Anticorpos Antibacterianos/sangue , Citocinas/sangue , Método Duplo-Cego , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Fezes/química , Feminino , Voluntários Saudáveis , Humanos , Linfócitos/imunologia , Masculino , Pessoa de Meia-Idade , Placebos/administração & dosagem , Plasmídeos , Soro/química , Vacinas contra Shigella/administração & dosagem , Vacinas contra Shigella/efeitos adversos , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/efeitos adversos , Vacinas de Produtos Inativados/imunologia , Adulto JovemRESUMO
BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a globally prevalent cause of diarrhea. Though usually self-limited, it can be severe and debilitating. Little is known about the host transcriptional response to infection. We report the first gene expression analysis of the human host response to experimental challenge with ETEC. METHODS: We challenged 30 healthy adults with an unattenuated ETEC strain, and collected serial blood samples shortly after inoculation and daily for 8 days. We performed gene expression analysis on whole peripheral blood RNA samples from subjects in whom severe symptoms developed (n = 6) and a subset of those who remained asymptomatic (n = 6) despite shedding. RESULTS: Compared with baseline, symptomatic subjects demonstrated significantly different expression of 406 genes highlighting increased immune response and decreased protein synthesis. Compared with asymptomatic subjects, symptomatic subjects differentially expressed 254 genes primarily associated with immune response. This comparison also revealed 29 genes differentially expressed between groups at baseline, suggesting innate resilience to infection. Drug repositioning analysis identified several drug classes with potential utility in augmenting immune response or mitigating symptoms. CONCLUSIONS: There are statistically significant and biologically plausible differences in host gene expression induced by ETEC infection. Differential baseline expression of some genes may indicate resilience to infection.
Assuntos
Escherichia coli Enterotoxigênica/imunologia , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/imunologia , Transcriptoma/imunologia , Adulto , Infecções por Escherichia coli/sangue , Infecções por Escherichia coli/microbiologia , Feminino , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/genética , Imunidade Inata/imunologia , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , RNA/sangue , RNA/genética , Adulto JovemRESUMO
Enterotoxigenic Escherichia coli (ETEC) bacteria are the most common bacterial cause of diarrhea in children in resource-poor settings as well as in travelers. Although there are several approaches to develop an effective vaccine for ETEC, no licensed vaccines are currently available. A significant challenge to successful vaccine development is our poor understanding of the immune responses that correlate best with protection against ETEC illness. In this study, ETEC-specific mucosal immune responses were characterized and compared in subjects challenged with ETEC strain H10407 and in subjects rechallenged with the homologous organism. IgA responses to lipopolysaccharide (LPS), heat-labile toxin B subunit (LTB), and colonization factor antigen I (CFA/I) in antibody in lymphocyte supernatant (ALS), feces, lavage fluid, and saliva samples were evaluated. In all assay comparisons, ALS was the most sensitive indicator of a local immune response, but serum IgA was also a useful indirect marker of immune response to oral antigens. Volunteers challenged and then rechallenged with strain H10407 were protected from illness following rechallenge. Comparing mucosal antibody responses after primary and homologous rechallenge, protection against disease was reflected in reduced antibody responses to key ETEC antigens and in reduced fecal shedding of the H10407 challenge strain. Subjects challenged with strain H10407 mounted stronger antibody responses to LPS and LTB than subjects in the rechallenge group, while responses to CFA/I in the rechallenge group were higher than in the challenge group. We anticipate that this study will help provide an immunological benchmark for the evaluation of ETEC vaccines and immunization regimens in the future.
Assuntos
Escherichia coli Enterotoxigênica/imunologia , Infecções por Escherichia coli/imunologia , Vacinas contra Escherichia coli/imunologia , Imunidade nas Mucosas , Anticorpos Antibacterianos/sangue , Formação de Anticorpos , Antígenos de Bactérias/imunologia , Diarreia/imunologia , Diarreia/microbiologia , Diarreia/prevenção & controle , Escherichia coli Enterotoxigênica/química , Escherichia coli Enterotoxigênica/patogenicidade , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/imunologia , Vacinas contra Escherichia coli/administração & dosagem , Fezes/microbiologia , Proteínas de Fímbrias/imunologia , Humanos , Imunoglobulina A/sangue , Saliva/imunologiaRESUMO
BACKGROUND: The P2-VP8 subunit vaccine for the prevention of rotavirus gastroenteritis is comprised of a truncated VP8 subunit protein from the rotavirus Wa strain (G1[P8]) fused to the tetanus toxin P2 epitope, and adsorbed on aluminum hydroxide for intramuscular administration. METHODS: Three groups of 16 adults were randomized to receive three injections of P2-VP8 (12) or placebo (4) at doses of 10, 30 or 60 µg of vaccine. IgG and IgA antibodies to P2-VP8 were assessed by ELISA in serum and lymphocyte supernatant (ALS). Serum samples were tested for neutralizing antibodies to homologous and heterologous strains of rotavirus. RESULTS: The vaccine was well-tolerated. All vaccine recipients demonstrated significant IgA responses and all but one demonstrated IgG responses; in the 60 µg cohort, geometric mean titers (GMTs) rose 70- and 80-fold for IgA and IgG, respectively. Homologous neutralizing antibody responses were observed in about half of participants in all three dose cohorts; in the 60 µg cohort, GMTs against Wa rose from 128 to 992. Neutralizing antibody responses were robust to P[8] strains, moderate to P[4] strains and negligible to P[6] strains. ALS IgA responses were dose dependent. CONCLUSIONS: The P2-VP8 subunit vaccine was well tolerated and evoked promising immune responses. CLINICAL TRIALS REGISTRATION: NCT01764256.
Assuntos
Gastroenterite/prevenção & controle , Proteínas de Ligação a RNA/imunologia , Infecções por Rotavirus/prevenção & controle , Vacinas contra Rotavirus/efeitos adversos , Vacinas contra Rotavirus/imunologia , Proteínas não Estruturais Virais/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adolescente , Adulto , Hidróxido de Alumínio/administração & dosagem , Anticorpos Neutralizantes/sangue , Método Duplo-Cego , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Voluntários Saudáveis , Humanos , Imunoglobulina A/análise , Imunoglobulina A/sangue , Imunoglobulina G/análise , Imunoglobulina G/sangue , Injeções Intramusculares , Linfócitos/imunologia , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Placebos/administração & dosagem , Vacinas contra Rotavirus/administração & dosagem , Resultado do Tratamento , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/efeitos adversos , Vacinas de Subunidades Antigênicas/imunologia , Adulto JovemRESUMO
Finding an effective human immunodeficiency virus type 1 (HIV-1) vaccine remains a major global health priority. In a phase I/II, placebo-controlled trial, healthy, HIV-1-negative adults were randomized to receive one of 5 vaccine regimens: LIPO-5 (combination of 5 lipopeptides) alone (250 µg), ALVAC-HIV (vCP1452) alone, or 3 groups of ALVAC-HIV (vCP1452) followed by ALVAC-HIV (vCP1452) plus LIPO-5 (250, 750, and 2,500 µg). Only 73/174 participants (42%) received all four vaccinations due to a study halt related to myelitis. There were no significant differences in systemic reactions between groups or in local reactogenicity between groups receiving ALVAC-HIV (vCP1452). Significant differences in local reactogenicity occurred between groups receiving LIPO-5 (P ≤ 0.05). Gag and Env antibodies were undetectable by ELISA 2 weeks after the fourth vaccination for all but one recipient. Antibodies to Gag and Env were present in 32% and 24% of recipients of ALVAC-HIV (vCP1452) alone and in 47% and 35% of ALVAC-HIV (vCP1452)+LIPO recipients, respectively. Coadministration of LIPO-5 did not significantly increase the response rate compared to ALVAC-HIV (vCP1452) alone, nor was there a significant relationship between dose and antibody responses among ALVAC-HIV (vCP1452)+LIPO groups. Over 90% of study participants had no positive gamma interferon (IFN-γ) enzyme-linked immunosorbent spot assay (ELISpot) responses to any peptide pool at any time point. The study was halted due to a case of myelitis possibly related to the LIPO-5 vaccine; this case of myelitis remains an isolated event. In general, there was no appreciable cell-mediated immunity detected in response to the vaccines used in this study, and antibody responses were limited. The clinical trial is registered on ClinicalTrials.gov with registry number NCT00076063.