RESUMEN

Typical enteropathogenic Escherichia coli (tEPEC) infection is a major cause of diarrhoea and contributor to mortality in children <5 years old in developing countries. Data were analysed from the Global Enteric Multicenter Study examining children <5 years old seeking care for moderate-to-severe diarrhoea (MSD) in Kenya. Stool specimens were tested for enteric pathogens, including by multiplex polymerase chain reaction for gene targets of tEPEC. Demographic, clinical and anthropometric data were collected at enrolment and ~60-days later; multivariable logistic regressions were constructed. Of 1778 MSD cases enrolled from 2008 to 2012, 135 (7.6%) children tested positive for tEPEC. In a case-to-case comparison among MSD cases, tEPEC was independently associated with presentation at enrolment with a loss of skin turgor (adjusted odds ratio (aOR) 2.08, 95% confidence interval (CI) 1.37-3.17), and convulsions (aOR 2.83, 95% CI 1.12-7.14). At follow-up, infants with tEPEC compared to those without were associated with being underweight (OR 2.2, 95% CI 1.3-3.6) and wasted (OR 2.5, 95% CI 1.3-4.6). Among MSD cases, tEPEC was associated with mortality (aOR 2.85, 95% CI 1.47-5.55). This study suggests that tEPEC contributes to morbidity and mortality in children. Interventions aimed at defining and reducing the burden of tEPEC and its sequelae should be urgently investigated, prioritised and implemented.

Asunto(s)

Diarrea/microbiología , Infecciones por Escherichia coli/microbiología , Estudios de Casos y Controles , Trastornos de la Nutrición del Niño , Preescolar , Diarrea/epidemiología , Escherichia coli Enteropatógena , Infecciones por Escherichia coli/epidemiología , Infecciones por Escherichia coli/mortalidad , Femenino , Humanos , Lactante , Recién Nacido , Kenia/epidemiología , Masculino

RESUMEN

A prospective, randomised, controlled observer-blind trial measuring the efficacy and immunogenicity of trivalent influenza vaccine (TIV) and the immunogenicity of quadrivalent meningococcal conjugate vaccine (MCV) in pregnant women and their infants up to 6 months of age was conducted in Mali. Here we reported the immunogenicity of MCV, which was used as a comparator vaccine to TIV, in this population. Third-trimester pregnant Malian women were randomized to receive TIV or MCV. Blood samples were collected from women prior to vaccination, 28 days post-vaccination, at delivery and 3 and 6 months post-delivery and from infants at birth and 3 and 6 months of age. Meningococcal-specific serogroup (Men) A, C, Y and W-specific antibodies were measured by enzyme linked immunosorbent assay in a randomly selected subset of 50 mother-infant pairs where the mother had received MCV. At birth, 94.0% (47/50) of infants had MenA specific IgG levels ≥ 2 µg/mL decreasing to 72.9% and 30.4% at 3 and 6 months of age. For MenC, 81.3% (39/48) of infants had MenC specific IgG levels ≥ 2 µg/mL at birth decreasing to 29.4% and 17.8% at 3 and 6 months of age. For MenY, 89.6% (43/48) of infants had MenY specific IgG levels ≥ 2 µg/mL at birth decreasing to 64.6% and 62.5% at 3 and 6 months of age. For MenW, 89.6% (43/48) of infants had MenW specific IgG levels ≥ 2 µg/ml at birth decreasing to 62.5% and 41.7% at 3 and 6 months of age. Maternal immunization with MCV conveyed protective levels of IgG at birth through to 3 months of age in the majority of infants.

Asunto(s)

Anticuerpos Antibacterianos/sangre , Inmunidad Materno-Adquirida , Inmunoglobulina G/sangre , Vacunas Meningococicas/inmunología , Adulto , Femenino , Humanos , Lactante , Recién Nacido , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/inmunología , Cinética , Masculino , Malí , Infecciones Meningocócicas/prevención & control , Vacunas Meningococicas/administración & dosificación , Embarazo , Estudios Prospectivos , Serogrupo , Vacunación , Vacunas Conjugadas/inmunología , Adulto Joven

RESUMEN

Given the challenges in accurately identifying unexposed controls in case-control studies of diarrhoea, we examined diarrhoea incidence, subclinical enteric infections and growth stunting within a reference population in the Global Enteric Multicenter Study, Kenya site. Within 'control' children (0-59 months old without diarrhoea in the 7 days before enrolment, n = 2384), we examined surveys at enrolment and 60-day follow-up, stool at enrolment and a 14-day post-enrolment memory aid for diarrhoea incidence. At enrolment, 19% of controls had ⩾1 enteric pathogen associated with moderate-to-severe diarrhoea ('MSD pathogens') in stool; following enrolment, many reported diarrhoea (27% in 7 days, 39% in 14 days). Controls with and without reported diarrhoea had similar carriage of MSD pathogens at enrolment; however, controls reporting diarrhoea were more likely to report visiting a health facility for diarrhoea (27% vs. 7%) or fever (23% vs. 16%) at follow-up than controls without diarrhoea. Odds of stunting differed by both MSD and 'any' (including non-MSD pathogens) enteric pathogen carriage, but not diarrhoea, suggesting control classification may warrant modification when assessing long-term outcomes. High diarrhoea incidence following enrolment and prevalent carriage of enteric pathogens have implications for sequelae associated with subclinical enteric infections and for design and interpretation of case-control studies examining diarrhoea.

RESUMEN

AIMS: O-polysaccharide (OPS) molecules are protective antigens for several bacterial pathogens, and have broad utility as components of glycoconjugate vaccines. Variability in the OPS chain length is one obstacle towards further development of these vaccines. Introduction of sizing steps during purification of OPS molecules of suboptimal or of mixed lengths introduces additional costs and complexity while decreasing the final yield. The overall goal of this study was to demonstrate the utility of engineering Gram-negative bacteria to produce homogenous O-polysaccharide populations that can be used as the basis of carbohydrate vaccines by overexpressing O-polysaccharide chain length regulators of the Wzx-/Wzy-dependent pathway. METHOD AND RESULTS: The O-polysaccharide chain length regulators wzzB and fepE from Salmonella Typhimurium I77 and wzz2 from Pseudomonas aeruginosa PAO1 were cloned and expressed in the homologous organism or in other Gram-negative bacteria. Overexpression of these Wzz proteins in the homologous organism significantly increased the proportion of long or very long chain O-polysaccharides. The same observation was made when wzzB was overexpressed in Salmonella Paratyphi A and Shigella flexneri, and wzz2 was overexpressed in two other strains of P. aeruginosa. CONCLUSIONS: Overexpression of Wzz proteins in Gram-negative bacteria using the Wzx/Wzy-dependant pathway for lipopolysaccharide synthesis provides a genetic method to increase the production of an O-polysaccharide population of a defined size. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods presented herein represent a cost-effective and improved strategy for isolating preferred OPS vaccine haptens, and could facilitate the further use of O-polysaccharides in glycoconjugate vaccine development.

Asunto(s)

Proteínas Bacterianas , Glicosiltransferasas , Bacterias Gramnegativas , Proteínas de Transporte de Membrana , Antígenos O , Vacunas Conjugadas , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Glicoconjugados , Glicosiltransferasas/análisis , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/metabolismo , Haptenos , Proteínas de Transporte de Membrana/análisis , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Antígenos O/análisis , Antígenos O/genética , Antígenos O/metabolismo

RESUMEN

Previously we have extensively characterized Salmonella enterica serovar Typhi (S. Typhi)-specific cell-mediated immune (CMI) responses in volunteers orally immunized with the licensed Ty21a typhoid vaccine. In this study we measured Salmonella-specific multifunctional (MF) CD8+ T-cell responses to further investigate whether Ty21a elicits crossreactive CMI against S. Paratyphi A and S. Paratyphi B that also cause enteric fever. Ty21a-elicited crossreactive CMI responses against all three Salmonella serotypes were predominantly observed in CD8+ T effector/memory (T(EM)) and, to a lesser extent, in CD8+CD45RA+ T(EM) (T(EMRA)) subsets. These CD8+ T-cell responses were largely mediated by MF cells coproducing interferon-γ and macrophage inflammatory protein-1ß and expressing CD107a with or without tumor necrosis factor-α. Significant proportions of Salmonella-specific MF cells expressed the gut-homing molecule integrin α4ß7. In most subjects, similar MF responses were observed to S. Typhi and S. Paratyphi B, but not to S. Paratyphi A. These results suggest that Ty21a elicits MF CMI responses against Salmonella that could be critical in clearing the infection. Moreover, because S. Paratyphi A is a major public concern and Ty21a was shown in field studies not to afford cross-protection to S. Paratyphi A, these results will be important in developing a S. Typhi/S. Paratyphi A bivalent vaccine against enteric fevers.

Asunto(s)

Linfocitos T CD8-positivos/inmunología , Fiebre Paratifoidea/prevención & control , Polisacáridos Bacterianos/inmunología , Fiebre Tifoidea/prevención & control , Vacunas Tifoides-Paratifoides/inmunología , Adulto , Reacciones Cruzadas/inmunología , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Fiebre Paratifoidea/inmunología , Salmonella paratyphi A/inmunología , Salmonella paratyphi B/inmunología , Salmonella typhi/inmunología , Fiebre Tifoidea/inmunología , Adulto Joven

RESUMEN

AIMS: Isolation of Salmonella Typhi from blood culture is the standard diagnostic for confirming typhoid fever but it is unavailable in many developing countries. We previously described a Microwave Accelerated Metal Enhanced Fluorescence (MAMEF)-based assay to detect Salmonella in medium. Attempts to detect Salmonella in blood were unsuccessful, presumably due to the interference of erythrocytes. The objective of this study was to evaluate various blood treatment methods that could be used prior to PCR, real-time PCR or MAMEF to increase sensitivity of detection of Salmonella. METHODS AND RESULTS: We tested ammonium chloride and erythrocyte lysis buffer, water, Lymphocyte Separation Medium, BD Vacutainer(®) CPT(™) Tubes and dextran. Erythrocyte lysis buffer was the best isolation method as it is fast, inexpensive and works with either fresh or stored blood. The sensitivity of PCR- and real-time PCR detection of Salmonella in spiked blood was improved when whole blood was first lysed using erythrocyte lysis buffer prior to DNA extraction. Removal of erythrocytes and clotting factors also enabled reproducible lysis of Salmonella and fragmentation of DNA, which are necessary for MAMEF sensing. CONCLUSIONS: Use of the erythrocyte lysis procedure prior to DNA extraction has enabled improved sensitivity of Salmonella detection by PCR and real-time PCR and has allowed lysis and fragmentation of Salmonella using microwave radiation (for future detection by MAMEF). SIGNIFICANCE AND IMPACT OF THE STUDY: Adaptation of the blood lysis method represents a fundamental breakthrough that improves the sensitivity of DNA-based detection of Salmonella in blood.

Asunto(s)

Métodos Analíticos de la Preparación de la Muestra/métodos , Eritrocitos/química , Salmonella typhi/aislamiento & purificación , Fiebre Tifoidea/microbiología , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Salmonella typhi/genética , Sensibilidad y Especificidad , Fiebre Tifoidea/sangre

RESUMEN

We studied the induction of antigen-specific IgA memory B cells (B(M)) in volunteers who received live attenuated Shigella flexneri 2a vaccines. Subjects ingested a single oral dose of 10(7), 10(8) or 10(9) CFU of S. flexneri 2a with deletions in guaBA (CVD 1204) or in guaBA, set and sen (CVD 1208). Antigen-specific serum and stool antibody responses to LPS and Ipa B were measured on days 0, 7, 14, 28 and 42. IgA B(M) cells specific to LPS, Ipa B and total IgA were assessed on days 0 and 28. We show the induction of significant LPS-specific IgA B(M) cells in anti-LPS IgA seroresponders. Positive correlations were found between anti-LPS IgA B(M) cells and anti-LPS IgA in serum and stool; IgA B(M) cell responses to IpaB were also observed. These B(M) cell responses are likely play an important role in modulating the magnitude and longevity of the humoral response.

Asunto(s)

Antígenos/inmunología , Subgrupos de Linfocitos B/inmunología , Inmunoglobulina A/inmunología , Vacunas contra la Shigella/inmunología , Shigella flexneri/inmunología , Vacunación/métodos , Vacunas Atenuadas/inmunología , Administración Oral , Adolescente , Adulto , Formación de Anticuerpos/inmunología , Antígenos CD/metabolismo , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/metabolismo , Proteínas Bacterianas/inmunología , Heces/química , Humanos , Inmunidad Mucosa/inmunología , Inmunoglobulina A/análisis , Inmunoglobulina A/sangre , Inmunoglobulina A/metabolismo , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Integrinas/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Lipopolisacáridos/inmunología , Recuento de Linfocitos , Persona de Mediana Edad , Eliminación de Secuencia , Vacunas contra la Shigella/administración & dosificación , Shigella flexneri/genética , Vacunas Atenuadas/administración & dosificación , Adulto Joven

RESUMEN

Enterotoxigenic Escherichia coli (ETEC) use colonization factors to attach to the human intestinal mucosa, followed by enterotoxin expression that induces net secretion and diarrhoeal illness. ETEC strain H10407 expresses CFA/I fimbriae, which are composed of multiple CfaB structural subunits and a CfaE tip subunit. Currently, the contribution of these individual fimbrial subunits in intestinal binding remains incompletely defined. To identify the role of CfaE in attachment in the native ETEC background, an R181A single-amino-acid substitution was introduced by recombination into the H10407 genome. The substitution of R181A eliminated haemagglutination and binding of intestinal mucosa biopsies in in vitro organ culture assays, without loss of CFA/I fimbriae expression. Wild-type in trans plasmid-expressed cfaE restored the binding phenotype. In contrast, in trans expression of cfaE containing amino acid 181 substitutions with similar amino acids, lysine, methionine and glutamine did not restore the binding phenotype, indicating that the loss of the binding phenotype was due to localized areas of epitope disruption. R181 appears to have an irreplaceable role in the formation of a receptor-binding feature on CFA/I fimbriae. The results specifically indicate that the CfaE tip protein is a required binding factor in CFA/I-mediated ETEC colonization, making it a potentially important vaccine antigen.

Asunto(s)

Escherichia coli Enterotoxigénica/genética , Proteínas de Escherichia coli/genética , Proteínas Fimbrias/genética , Fimbrias Bacterianas/genética , Mucosa Intestinal/metabolismo , Sustitución de Aminoácidos , Adhesión Bacteriana/genética , Sitios de Unión/genética , Escherichia coli Enterotoxigénica/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/fisiología , Proteínas Fimbrias/química , Proteínas Fimbrias/fisiología , Fimbrias Bacterianas/química , Fimbrias Bacterianas/metabolismo , Humanos , Mucosa Intestinal/microbiología , Recombinación Genética

RESUMEN

We evaluated B memory responses in healthy adult volunteers who received one oral dose of live-attenuated Shigella flexneri 2a vaccine. LPS-specific B(M) cells increased from a median of 0 at baseline to 20 spot forming cells (SFC)/10(6) expanded cells following vaccination (p=0.008). A strong correlation was found between post-vaccination anti-LPS B(M) cell counts and peak serum anti-LPS IgG titers (rs=0.95, p=0.0003). Increases in B(M) specific for IpaB approaching significance were also observed. In sum, oral vaccination with live-attenuated S. flexneri 2a elicits B(M) cells to LPS and IpaB, suggesting that B(M) responses to Shigella antigens should be further studied as a suitable surrogate of protection in shigellosis.

Asunto(s)

Anticuerpos Antibacterianos/biosíntesis , Linfocitos B/inmunología , Vacunas Bacterianas/inmunología , Memoria Inmunológica , Lipopolisacáridos/inmunología , Shigella flexneri/inmunología , Administración Oral , Adolescente , Adulto , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Especificidad de Anticuerpos , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Vacunas Bacterianas/administración & dosificación , Disentería Bacilar/prevención & control , Femenino , Humanos , Inmunización , Masculino , Persona de Mediana Edad , Plásmidos , Shigella flexneri/genética , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Voluntarios , Adulto Joven

RESUMEN

Induction of effective memory T cells is likely to be critical to the level and duration of protection elicited by novel live oral typhoid vaccines. Using cells from volunteers who ingested Salmonella Typhi vaccine strain CVD 909, we characterized the induction of interferon (IFN)-gamma-secreting central (T(CM), CD45RO(+)CD62L(+)) and effector (T(EM), CD45RO(+)CD62L(-)) memory T populations, and their gut-homing potential based on integrin alpha4/beta7 expression. Both CD4(+) T(EM) and T(CM) populations secreted IFN-gamma. However, although CD4(+) T(EM) expressed, or not, integrin alpha(4)/beta(7), CD4(+) T(CM) cells were predominantly integrin alpha(4)/beta(7)(+). In contrast, IFN-gamma-secreting CD8(+) cells were predominantly classical T(EM) and CD45RA(+) T(EM) (T(EMRA), CD45RO(-)CD62L(-)) subsets. However, although CD8(+) T(EM) expressed, or not, integrin alpha(4)/beta(7), CD8(+) T(EMRA) were predominantly integrin alpha(4)/beta(7)(+). This is the first demonstration that oral immunization of humans with S. Typhi elicits diverse IFN-gamma-secreting CD4(+) and CD8(+) T(CM) and T(EM) subsets able to migrate to the gut and other lymphoid tissues.

Asunto(s)

Movimiento Celular/inmunología , Memoria Inmunológica/inmunología , Intestinos/inmunología , Tejido Linfoide/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Vacunas Tifoides-Paratifoides/inmunología , Administración Oral , Adulto , Antígenos Bacterianos/inmunología , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Intestinos/citología , Tejido Linfoide/citología , Salmonella typhi/inmunología , Solubilidad , Linfocitos T/metabolismo , Vacunas Tifoides-Paratifoides/administración & dosificación

RESUMEN

Introduction: Acute bacterial meningitis (ABM) is one of the most severe diseases in Sub-Saharan Africa. Although data for the continent is very limited, more than one million cases are estimated per year, with mortality and life-long sequelae occurring in 50% of these cases. Methods: As part of the clinical management of children admitted to the Manhiça District Hospital, information on cases of ABM was recorded. We analysed data from June 1998 to November 2003. Results: During the study period, 475 cerebrospinal-fluid (CSF) samples were collected from 20,173 children <15 years of age admitted to hospital. Culture results confirmed 71 (15%) cases of ABM. The most prevalent bacterial aetiologies were Streptotoccus pneumoniae (pneumococcus, n=31), Haemophilus influenzae (n=13) and Neisseria meningitis (n=8). Other important bacteria were Streptococcus sp. (n=7), Salmonella sp. (n=4) and Staphylococcus aureus (n=3). Crude incidence rates of ABM and pneumococcal meningitis were 20/100,000 and 10/100,000 children-year-at-risk, respectively. Incidences were more than three times higher in the <1 year age group. Overall case fatality rate was 36%, and was highest for H. influenzae and pneumococcal meningitis (55% and 45%, respectively, p=0.044). Pneumococcal susceptibility was 81% for oxacillin and 93% for chloramphenicol. For H. influenzae isolates, susceptibility was 54% for ampicillin and 62% for chloramphenicol. Conclusions: S. pneumoniae and H. influenzae are the main aetiologies responsible for the high burden of morbidity and mortality associated with ABM in rural Mozambique. These findings are important to evaluate treatment guidelines and potential impact of control measures

Asunto(s)

Humanos , Masculino , Femenino , Preescolar , Niño , Infecciones por Bacterias Gramnegativas/microbiología , Bacterias Gramnegativas/aislamiento & purificación , Meningitis Bacterianas/microbiología , Meningitis Bacterianas/mortalidad , Meningitis Bacterianas/epidemiología , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/mortalidad , Infecciones por Bacterias Grampositivas/epidemiología , Infecciones por Bacterias Gramnegativas/mortalidad , Infecciones por Bacterias Gramnegativas/epidemiología , Antibacterianos/farmacología

RESUMEN

INTRODUCTION: Acute bacterial meningitis (ABM) is one of the most severe diseases in Sub-Saharan Africa. Although data for the continent is very limited, more than one million cases are estimated per year, with mortality and life-long sequelae occurring in 50% of these cases. METHODS: As part of the clinical management of children admitted to the Manhiça District Hospital, information on cases of ABM was recorded. We analysed data from June 1998 to November 2003. RESULTS: During the study period, 475 cerebrospinal-fluid (CSF) samples were collected from 20,173 children <15 years of age admitted to hospital. Culture results confirmed 71 (15%) cases of ABM. The most prevalent bacterial aetiologies were Streptotoccus pneumoniae (pneumococcus, n=31), Haemophilus influenzae (n=13) and Neisseria meningitis (n=8). Other important bacteria were Streptococcus sp. (n=7), Salmonella sp. (n=4) and Staphylococcus aureus (n=3). Crude incidence rates of ABM and pneumococcal meningitis were 20/100,000 and 10/100,000 children-year-at-risk, respectively. Incidences were more than three times higher in the <1 year age group. Overall case fatality rate was 36%, and was highest for H. influenzae and pneumococcal meningitis (55% and 45%, respectively, p=0.044). Pneumococcal susceptibility was 81% for oxacillin and 93% for chloramphenicol. For H. influenzae isolates, susceptibility was 54% for ampicillin and 62% for chloramphenicol. CONCLUSIONS: S. pneumoniae and H. influenzae are the main aetiologies responsible for the high burden of morbidity and mortality associated with ABM in rural Mozambique. These findings are important to evaluate treatment guidelines and potential impact of control measures.

Asunto(s)

Infecciones por Bacterias Gramnegativas/epidemiología , Infecciones por Bacterias Gramnegativas/microbiología , Infecciones por Bacterias Grampositivas/epidemiología , Infecciones por Bacterias Grampositivas/microbiología , Meningitis Bacterianas/epidemiología , Meningitis Bacterianas/microbiología , Adolescente , Factores de Edad , Antibacterianos/farmacología , Líquido Cefalorraquídeo/microbiología , Niño , Preescolar , Femenino , Bacterias Gramnegativas/aislamiento & purificación , Infecciones por Bacterias Gramnegativas/mortalidad , Bacterias Grampositivas/aislamiento & purificación , Infecciones por Bacterias Grampositivas/mortalidad , Humanos , Incidencia , Lactante , Recién Nacido , Masculino , Meningitis Bacterianas/mortalidad , Pruebas de Sensibilidad Microbiana , Mozambique/epidemiología , Población Rural

RESUMEN

Infants in developing countries are at high risk of developing severe clinical measles if they become infected during the "window of vulnerability" (age 4-9 months), when declining maternal antibodies do not protect against wild virus, yet impede successful immunization by attenuated measles vaccine. We developed two Sindbis replicon-based DNA vaccines expressing measles virus hemagglutinin and fusion protein with the goal of priming young infants to respond safely and effectively to subsequent boosting with attenuated measles vaccine. Intradermal prime with DNA vaccines by needle-free injection followed by aerosol or parenteral boost with licensed measles vaccine was well tolerated by juvenile and young infant rhesus macaques, and protected against clinical measles and viremia on wild-type virus challenge. A proteosome-measles vaccine administered alone (three doses) or as a boost following DNA vaccine priming was also safe and protective. These promising results pave the way for clinical trials to assess this prime-boost strategy.

Asunto(s)

Hemaglutininas Virales , Inmunización Secundaria , Inmunización/métodos , Vacuna Antisarampión/síntesis química , Virus del Sarampión/inmunología , Sarampión/prevención & control , Vacunas de ADN/síntesis química , Aerosoles , Animales , Inyecciones Intradérmicas/instrumentación , Macaca mulatta , Sarampión/inmunología , Vacuna Antisarampión/administración & dosificación , Vacuna Antisarampión/inmunología , Replicón , Virus Sindbis , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/síntesis química , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología

Asunto(s)

Vacunación Masiva , Fiebre Tifoidea/prevención & control , Vacunas Tifoides-Paratifoides , Humanos , Fiebre Tifoidea/epidemiología , Fiebre Tifoidea/inmunología , Vacunas Tifoides-Paratifoides/administración & dosificación , Vacunas Tifoides-Paratifoides/inmunología

RESUMEN

The efficiency of any live bacterial vector vaccine hinges on its ability to present sufficient foreign antigen to the human immune system to initiate the desired protective immune response(s). However, synthesis of sufficient levels of heterologous antigen can result in an increase in metabolic burden with an accompanying decrease in the fitness of the live vector, which can ultimately lower desired immune responses to both live vector and heterologous antigen. Here, we explore the underlying mechanisms of metabolic load and propose ways of minimizing such burdens to enhance the fitness and immunogenicity of Salmonella-based live vector vaccines.

Asunto(s)

Antígenos Bacterianos/inmunología , Vacunas Bacterianas/inmunología , Salmonella typhi/inmunología , Salmonella typhimurium/inmunología , Vacunas Sintéticas/inmunología , Antígenos Bacterianos/genética , Infecciones Bacterianas/prevención & control , Vacunas Bacterianas/genética , Vectores Genéticos , Humanos , Salmonella typhi/genética , Salmonella typhi/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Sintéticas/genética

RESUMEN

A promising live attenuated typhoid vaccine candidate strain for mucosal immunization was developed by introducing a deletion in the guaBA locus of pathogenic Salmonella enterica serovar Typhi strain Ty2. The resultant DeltaguaBA mutant, serovar Typhi CVD 915, has a gene encoding resistance to arsenite replacing the deleted sequence within guaBA, thereby providing a marker to readily identify the vaccine strain. CVD 915 was compared in in vitro and in vivo assays with wild-type strain Ty2, licensed live oral typhoid vaccine strain Ty21a, or attenuated serovar Typhi vaccine strain CVD 908-htrA (harboring mutations in aroC, aroD, and htrA). CVD 915 was less invasive than CVD 908-htrA in tissue culture and was more crippled in its ability to proliferate after invasion. In mice inoculated intraperitoneally with serovar Typhi and hog gastric mucin (to estimate the relative degree of attenuation), the 50% lethal dose of CVD 915 (7.7 x 10(7) CFU) was significantly higher than that of wild-type Ty2 (1.4 x 10(2) CFU) and was only slightly lower than that of Ty21a (1.9 x 10(8) CFU). Strong serum O and H antibody responses were recorded in mice inoculated intranasally with CVD 915, which were higher than those elicited by Ty21a and similar to those stimulated by CVD 908-htrA. CVD 915 also elicited potent proliferative responses in splenocytes from immunized mice stimulated with serovar Typhi antigens. Used as a live vector, CVD 915(pTETlpp) elicited high titers of serum immunoglobulin G anti-fragment C. These encouraging preclinical data pave the way for phase 1 clinical trials with CVD 915.

Asunto(s)

Vacunas contra la Salmonella/inmunología , Salmonella typhi/inmunología , Vacunas Sintéticas/inmunología , Animales , División Celular , Seguridad de Productos para el Consumidor , Medios de Cultivo , Femenino , Ingeniería Genética , Genotipo , Guanina/metabolismo , Ratones , Ratones Endogámicos BALB C , Mutagénesis , Fenotipo , Vacunas contra la Salmonella/genética , Salmonella typhi/genética , Salmonella typhi/crecimiento & desarrollo , Salmonella typhi/fisiología , Vacunas Atenuadas/inmunología , Vacunas Sintéticas/genética

RESUMEN

To construct a prototype hybrid vaccine against Shigella and enterotoxigenic Escherichia coli (ETEC), the genes encoding the production of ETEC CS2 and CS3 fimbriae were isolated and expressed in attenuated Shigella flexneri 2a guaBA strain CVD 1204. The CS2 cotA to -D genes, isolated from ETEC strain C91F, and the CS3 cstA to -H genes, subcloned from plasmid pCS100, were cloned into ~15-copy-number-stabilized pGA1 behind the osmotically regulated ompC promoter, resulting in high expression of both fimbriae. Under nonselective in vitro growth conditions, pGA1-CS2 and pGA1-CS3 were stable in CVD 1204, exhibiting a plasmid loss of only approximately 1% per duplication. Expression of CS2 and CS3 reduced the invasiveness of Shigella for HeLa cells and slowed the intracellular growth rate. Guinea pigs immunized intranasally with CVD 1204(pGA1-CS2) or CVD 1204(pGA1-CS3), or with a mixture of these strains, developed secretory immunoglobulin A (IgA) in tears and serum IgG antibodies against Shigella lipopolysaccharide, CS2, and CS3 antigens. Moreover, the animals were protected against keratoconjunctivitis following conjunctival challenge with virulent S. flexneri 2a strain 2457T. Animals immunized with Shigella expressing CS2 or CS3 developed serum antibodies that agglutinated Shigella as well as an ETEC strain bearing the homologous fimbriae, whereas animals immunized with combined CVD 1204(pGA1-CS2) and CVD 1204(pGA1-CS3) developed antibodies that agglutinated all three test strains. These observations support the feasibility of a multivalent vaccine against shigellosis and ETEC diarrhea consisting of multiple Shigella live vectors expressing relevant ETEC antigens.

Asunto(s)

Vacunas Bacterianas/inmunología , Disentería Bacilar/prevención & control , Infecciones por Escherichia coli/prevención & control , Escherichia coli/inmunología , Fimbrias Bacterianas/inmunología , Shigella flexneri/inmunología , Vacunas Sintéticas/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Clonación Molecular , Diarrea/prevención & control , Cobayas , Humanos , Inmunización , Concentración Osmolar , Shigella flexneri/genética

RESUMEN

The anaerobically induced promoter dmsA (PdmsA) was adapted to optimize in vivo expression of foreign antigens in attenuated Salmonella enterica serovar Typhi live vector vaccines CVD 908-htrA. PdmsA from Escherichia coli and two derivatives, PdmsA2 and PdmsA3 were cloned into a plasmid driving the expression of a gene encoding tetanus toxin fragment C. Expression of fragment C varied from a low level induced by pTETdmsA, to moderate and high levels induced, respectively, by pTETdmsA2 and pTETdmsA3. Mice were immunized intranasally with CVD 908-htrA harboring pTETdmsA2 or pTETdmsA3, and the serum antitoxin response was compared to that elicited by CVD 908-htrA(pTETnir15) (Pnir15 is a benchmark anaerobically activated promoter). S. Typhi carrying pTETdmsA2 elicited modest tetanus antitoxin titers while S. Typhi harboring pTETdmsA3 generated elevated titers (GMT=55384) that were higher than elicited by pTETnir15 (GMT=4354) (P=0.007). Mice immunized with CVD 908-htrA carrying pTETdmsA3 and pTETnir15 survived tetanus toxin challenge. P(dmsA) derivatives are attractive promoters for in vivo expression of foreign genes in attenuated live vector vaccines.

Asunto(s)

Genes Bacterianos/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Regiones Promotoras Genéticas/genética , Vacunas contra la Salmonella/genética , Toxina Tetánica/genética , Toxina Tetánica/inmunología , Vacunas Atenuadas/genética , Vacunas Sintéticas/genética , Anaerobiosis , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/toxicidad , Secuencia de Bases , Western Blotting , Clonación Molecular , Secuencia de Consenso/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Nitratos/farmacología , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/toxicidad , Vacunas contra la Salmonella/inmunología , Salmonella typhi/genética , Salmonella typhi/inmunología , Tasa de Supervivencia , Toxina Tetánica/biosíntesis , Toxina Tetánica/toxicidad , Vacunas Atenuadas/inmunología , Vacunas Sintéticas/inmunología

RESUMEN

To investigate the potential immunomodulatory effects of concurrent ascariasis on the cytokine response to a live oral vaccine, we measured cytokine responses to cholera toxin B subunit (CT-B) following vaccination with the live oral cholera vaccine CVD 103-HgR in Ascaris lumbricoides-infected subjects randomized in a double-blind study to receive two doses of either albendazole or placebo prior to vaccination and in a group of healthy U.S. controls. Postvaccination cytokine responses to CT-B were characterized by transient increases in the production of interleukin-2 (IL-2; P = 0.02) and gamma interferon (IFN-gamma; P = 0.001) in the three study groups combined; however, postvaccination increases in IFN-gamma were significant only in the albendazole-treated A. lumbricoides infection group (P = 0.008). Postvaccination levels of IL-2 were significantly greater in the albendazole-treated group compared with the placebo group (P = 0.03). No changes in levels of Th1 and Th2 cytokines in response to control ascaris antigens were observed over the same period. These findings indicate that vaccination with CVD 103-HgR is associated with a Th1 cytokine response (IL-2 and IFN-gamma) to CT-B, that infection with A. lumbricoides diminishes the magnitude of this response, and that albendazole treatment prior to vaccination was able to partially reverse the deficit in IL-2. The potential modulation of the immune response to oral vaccines by geohelminth parasites has important implications for the design of vaccination campaigns in geohelminth-endemic areas.

Asunto(s)

Ascariasis/inmunología , Ascaris lumbricoides/inmunología , Toxina del Cólera/inmunología , Vacunas contra el Cólera/uso terapéutico , Cólera/prevención & control , Interleucina-2/sangre , Adulto , Albendazol/uso terapéutico , Animales , Antihelmínticos/uso terapéutico , Antígenos Helmínticos/inmunología , Ascariasis/tratamiento farmacológico , Ascaris lumbricoides/efectos de los fármacos , Vacunas contra el Cólera/inmunología , Método Doble Ciego , Femenino , Humanos , Interferón gamma/sangre , Leucocitos Mononucleares/inmunología , Masculino , Vacunación

RESUMEN

Deleting transmembrane alpha-helix motifs from Plasmodium falciparum sporozoite surface protein (SSP-2) allowed its secretion from Salmonella enterica serovar Typhimurium SL3261 and S. enterica serovar Typhi CVD 908-htrA by the Hly type I secretion system. In mice immunized intranasally, serovar Typhimurium constructs secreting SSP-2 stimulated greater gamma interferon splenocyte responses than did nonsecreting constructs (P = 0.04).

Asunto(s)

Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Salmonella/genética , Animales , Femenino , Inmunización , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Peso Molecular , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Linfocitos T Citotóxicos/inmunología , Vacunas de ADN/inmunología