RESUMEN
Enterotoxigenic Escherichia coli (ETEC) is the most prevalent bacterial pathogen causing young children to suffer acute watery diarrhea in Low- and Middle-Income Countries (LMICs) [...].
RESUMEN
An oral, microencapsulated anti-colonization factor 6 antigen (meCS6) vaccine, with or without heat-labile enterotoxin with mutation R192G (LT(R192G)) (mucosal adjuvant), against enterotoxigenic Escherichia coli (ETEC) was evaluated for regimen and adjuvant effects on safety and immunogenicity. Sixty subjects were enrolled into a three-dose, 2-week interval or four-dose, 2-day interval regimen. Each regimen was randomized into two equal groups of meCS6 alone (1 mg) or meCS6 with adjuvant (2 microg of LT(R192G)). The vaccine was well tolerated and no serious adverse events were reported. Serologic response to CS6 was low in all regimens (0 to 27%). CS6-immunoglobulin A (IgA) antibody-secreting cell (ASC) responses ranged from 36 to 86%, with the highest level in the three-dose adjuvanted regimen; however, the magnitude was low. As expected, serologic and ASC LT responses were limited to adjuvanted regimens, with the exception of fecal IgA, which appeared to be nonspecific to LT administration. Further modifications to the delivery strategy and CS6 and adjuvant dose optimization will be needed before conducting further clinical trials with this epidemiologically important class of ETEC.
Asunto(s)
Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Diarrea/prevención & control , Enterotoxinas/inmunología , Infecciones por Escherichia coli/prevención & control , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/efectos adversos , Vacunas contra Escherichia coli/inmunología , Adyuvantes Inmunológicos , Administración Oral , Adolescente , Adulto , Toxinas Bacterianas/genética , Diarrea/inmunología , Diarrea/microbiología , Enterotoxinas/genética , Escherichia coli/inmunología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Vacunas contra Escherichia coli/administración & dosificación , Femenino , Humanos , Inmunoglobulina A/análisis , Masculino , Persona de Mediana Edad , Mutación , Resultado del TratamientoRESUMEN
Colonization factor CS6 expressed by enterotoxigenic Escherichia coli (ETEC) is a nonfimbrial polymeric protein. A substantial proportion of ETEC strains isolated from patients in endemic settings and in people who travel to regions where ETEC is endemic are ETEC strains expressing CS6, either alone or in combination with fimbrial colonization factor CS5 or CS4. However, relatively little is known about the natural immune responses elicited against CS6 expressed by ETEC strains causing disease. We studied patients who were hospitalized with diarrhea (n = 46) caused by CS6-expressing ETEC (ETEC expressing CS6 or CS5 plus CS6) and had a disease spectrum ranging from severe dehydration (27%) to moderate or mild dehydration (73%). Using recombinant CS6 antigen, we found that more than 90% of the patients had mucosal immune responses to CS6 expressed as immunoglobulin (IgA) antibody-secreting cells (ASC) or antibody in lymphocyte supernatant (ALS) and that about 57% responded with CS6-specific IgA antibodies in feces. More than 80% of the patients showed IgA seroconversion to CS6. Significant increases in the levels of anti-CS6 antibodies of the IgG isotype were also observed in assays for ASC (75%), ALS (100%), and serum (70%). These studies demonstrated that patients hospitalized with the noninvasive enteric pathogen CS6-expressing ETEC responded with both mucosal and systemic antibodies against CS6. Studies are needed to determine if the anti-CS6 responses protect against reinfection and if protective levels of CS6 immunity are induced by vaccination.
Asunto(s)
Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Diarrea/inmunología , Proteínas de Escherichia coli/inmunología , Proteínas de Escherichia coli/metabolismo , Escherichia coli/patogenicidad , Inmunoglobulina A Secretora , Inmunoglobulina G , Adolescente , Adulto , Anticuerpos Antibacterianos/análisis , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Especificidad de Anticuerpos , Antígenos Bacterianos/genética , Preescolar , Diarrea/microbiología , Escherichia coli/inmunología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Heces/química , Femenino , Hospitalización , Humanos , Inmunidad Mucosa , Inmunoglobulina A Secretora/análisis , Inmunoglobulina A Secretora/sangre , Inmunoglobulina A Secretora/inmunología , Inmunoglobulina G/sangre , Lactante , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
The aim of this study was to measure serum and mucosal antibody responses following intranasal administration of biodegradable poly(DL-lactide-co-glycolide) (PLGA) microspheres loaded with the CS3 colonization factor isolated from enterotoxigenic Escherichia coli (ETEC). The response was compared against that measured in mice similarly administered the native CS3 antigen and in mice co-administered, along with the CS3 antigen, a known mucosal adjuvant, the R192G mutant heat-labile enterotoxin (mLT). The integrity of the CS3 antigen released from the microspheres was maintained as determined by SDS-PAGE and immunoblotting. Native CS3 induced serum and mucosal (bronchoalveolar, small intestinal and faecal) IgG and IgA responses. The co-administration of the mLT mucosal adjuvant significantly enhanced (P<0.001) serum and mucosal antibody responses to the CS3 protein. Likewise, the CS3-loaded PLGA microspheres induced significantly greater (P<0.001) serum and mucosal antibody responses than native CS3, as well as inducing antibody responses superior to those of the CS3 plus mLT formulation. Following administration of CS3 plus mLT, the mice became distressed (loss of activity, increased huddling, ruffled fur), a situation not seen following administration of the CS3-loaded PLGA microspheres. The results in this trial show that the CS3-loaded PLGA microspheres when administered intranasally to mice caused no observable distress to the mice and significantly (P<0.001) enhanced the immunogenicity of the CS3 protein.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/inmunología , Escherichia coli/inmunología , Proteínas Fimbrias/química , Proteínas Fimbrias/inmunología , Microesferas , Adyuvantes Inmunológicos , Administración Intranasal , Animales , Anticuerpos Antibacterianos/análisis , Proteínas de Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/inmunología , Femenino , Proteínas Fimbrias/administración & dosificación , Inmunoglobulina E/análisis , Inmunoglobulina E/sangre , Ácido Láctico , Ratones , Ratones Endogámicos BALB C , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , PolímerosRESUMEN
The immunogenicity induced in BALB/c mice following intranasal challenge with a viable nonlethal dose (1.2 x 10(8) CFU) of enterotoxigenic Escherichia coli (ETEC) strain E23477A (O139:H28:CS1:CS3:LT+:ST+) was studied over a 140-day period. Serum IgG and IgM antibodies against coli surface antigen 3 (CS3), O139 lipopolysaccharide and heat-labile enterotoxin were measured by day 14 and remained at elevated levels out to day 140. The serum IgG response to the somatic antigens (CS3 and O139 lipopolysaccharide) was significantly greater (P < 0.05) than the IgG response to heat-labile enterotoxin, and the serum IgG response to CS3 was significantly greater (P < 0.05) than the IgG response to O139 lipopolysaccharide. The predominant serum IgG subclasses to CS3 were IgG1 and IgG2a, and they were significantly greater (P < 0.05) than IgG2b and IgG3. The predominant serum IgG subclass response to O139 lipopolysaccharide was initially IgG3 until day 56, after which IgG1 was predominant. The serum subclass response to CS3 indicated a mixed T helper 1/2 (Th1/Th2) profile, whereas the response to O139 lipopolysaccharide was primarily that of a Th2-type, at least over time. Fecal IgG and IgA responses to CS3 and O139 lipopolysaccharide were detected by day 14 and were measured out to day 140, with the CS3 fecal antibody responses being significantly greater (P < 0.05) than the O139 lipopolysaccharide and heat-labile enterotoxin fecal antibody responses. The aim of this study is the development of the intranasal mouse model that can aid in better understanding the immunopathology of ETEC infection and in screening of vaccine candidates prior to volunteer trials.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Toxinas Bacterianas/inmunología , Enterotoxinas/inmunología , Infecciones por Escherichia coli/inmunología , Proteínas de Escherichia coli/inmunología , Escherichia coli/patogenicidad , Membrana Mucosa/inmunología , Administración Intranasal , Animales , Anticuerpos Antibacterianos/análisis , Toxinas Bacterianas/metabolismo , Enterotoxinas/metabolismo , Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/metabolismo , Heces , Femenino , Proteínas Fimbrias/inmunología , Humanos , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos BALB C , Factores de TiempoRESUMEN
Mice were intranasally administered enterotoxigenic Escherichia coli colonization factor CS6 encapsulated in poly(DL-lactide-co-glycolide) microspheres (CS6-PLG), with immune response measured and compared to that of similarly administered native CS6 and CS6 plus mutant heat-labile enterotoxin mucosal adjuvant (CS6+mLT). Native CS6 and the CS6-PLG microspheres administered intranasally to mice induced serum IgG responses, with the CS6-PLG microspheres inducing a significantly greater (P<0.001) response than native CS6. Following intranasal administration of native CS6, no fecal IgG and IgA responses were measured; however, the CS6-PLG microspheres induced significantly greater (P<0.001) fecal IgG and IgA responses than native CS6. The coadministration of the mLT mucosal adjuvant with CS6 induced significantly greater serum (P<0.001) and fecal (P<0.01) responses than the CS6-PLG microspheres. However, following intranasal administration of the mLT adjuvant, the mice showed definite signs of distress, indicating an adverse reaction to the mLT. Thus, this brings into question the safety of the mLT and its use as an intranasal adjuvant. In contrast, the PLG-microspheres administered intranasally caused no noticeable distress to the mice. The results obtained in this study indicate that the encapsulation of CS6 in PLG-microspheres administered intranasally to mice acted in an adjuvant capacity to enhance the CS6 immune response.
Asunto(s)
Antígenos Bacterianos/inmunología , Infecciones por Escherichia coli/prevención & control , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/inmunología , Ácido Láctico , Microesferas , Ácido Poliglicólico , Polímeros , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Administración Intranasal , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/administración & dosificación , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/farmacología , Enterotoxinas/administración & dosificación , Enterotoxinas/farmacología , Escherichia coli/inmunología , Proteínas de Escherichia coli/administración & dosificación , Proteínas de Escherichia coli/farmacología , Vacunas contra Escherichia coli/administración & dosificación , Heces/química , Femenino , Inmunoglobulina A/análisis , Inmunoglobulina G/análisis , Ácido Láctico/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Modelos Animales , Vehículos Farmacéuticos , Ácido Poliglicólico/administración & dosificación , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Polímeros/administración & dosificaciónRESUMEN
The colonization factors (CF) of enterotoxigenic Escherichia coli (ETEC) are being targeted for inclusion in a multi-subunit ETEC vaccine. This study was designed to examine the preclinical safety and immunogenicity of CF CS6, encapsulated in a biodegradable poly(DL-lactide-co-glycolide) (meCS6), and administered in the presence or absence of a mutated heat-labile enterotoxin, LT(R192G), in the non-human primate, Aotus nancymae. A. nancymae were inoculated intranasally (IN) with meCS6 (200 microg; positive control), or intragastrically (IG) with meCS6 (200 or 1000 microg) with or without 2 microg LT(R192G) in three doses given at 2-week intervals. In a second experiment, A. nancymae were inoculated IG with 950 microg of meCS6 with or without 2 microg LT(R192G) in four doses given every 48 h. Blood was collected to assess anti-CS6 and -LT serum immunoglobulin G (IgG) and IgA responses and safety variables (complete blood count and chemistry). Safety parameters were unchanged from baseline following all vaccinations. In Experiment 1, a dose-related serologic response to CS6 was observed; 78.6 and 57.1% of monkeys given 1000 microg meCS6 (n = 14) had a serum IgG and IgA response, respectively, compared to only 28.6% of monkeys given 200 microg meCS6 (n = 14) with a serum IgG and IgA response. No significant effect on the number of responders or the magnitude of responses was observed with the addition of LT(R192G). The three-dose, 2-week regimen with 1000 microg meCS6 was more effective at eliciting an immune response than the four-dose, 48-h regimen with 950 microg meCS6. Results from this study indicate that A. nancymae provide a useful ETEC preclinical safety and immunogenicity model.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Aotidae , Vacunas contra Escherichia coli/inmunología , Escherichia coli/inmunología , Modelos Animales , Vacunas de Subunidad/inmunología , Adyuvantes Inmunológicos , Administración Intranasal , Animales , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/efectos adversos , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Recuento de Células Sanguíneas , Análisis Químico de la Sangre , Enterotoxinas/administración & dosificación , Enterotoxinas/genética , Enterotoxinas/inmunología , Infecciones por Escherichia coli/prevención & control , Proteínas de Escherichia coli/administración & dosificación , Proteínas de Escherichia coli/efectos adversos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/efectos adversos , Femenino , Lavado Gástrico , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Ácido Láctico , Masculino , Mutación , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Polímeros , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/efectos adversosRESUMEN
Infections of the intestinal, urogenital, and respiratory tracts are serious health problems worldwide from both a morbidity and mortality perspective. Mucosal pathogens attach to surfaces of mucosa as a prerequisite for colonization and subsequent pathogenesis. By expressing various surface adhesins (colonization factors, CF) they are able to bind to specific mucosal receptors. Enterotoxigenic Escherichia coli (ETEC) can express numerous CF that allow them to attach to a variety of hosts. Mucosal immunity directed against pathogenic microorganisms is critical in host protection with secretory IgA being particularly important in preventing microoganisms from colonizing host cells. M cells likewise have an important immunological function in the small intestines by binding and transporting antigens to lymphocytes and macrophages thus enhancing the immune response. The use of subunit vaccines, such as antigen encapsulated microspheres, can act to effectively deliver specific antigens so as to optimize their immunological response. With the threat of bioterrorism becoming a reality in recent years, the miroencapsulation of antigens from potential bioterrorist agents may be an effective method of delivery so as to induce a level of protection in at risk individuals. The encapsulation of ETEC colonization factors in microspheres and their subsequent administration in small animals and humans has been conducted for many years. Evidence suggests that this type of delivery system for ETEC antigens may enhance their immunogenicity and provide protection against this microorganism.
Asunto(s)
Adhesinas de Escherichia coli , Composición de Medicamentos/métodos , Escherichia coli , Mucosa Intestinal , Vacunas Virales/inmunología , Adhesinas de Escherichia coli/inmunología , Adhesinas de Escherichia coli/fisiología , Animales , Escherichia coli/efectos de los fármacos , Escherichia coli/inmunología , Escherichia coli/patogenicidad , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , MicroesferasRESUMEN
Colonization factor antigen I (CFA/I) is the archetype of eight genetically related fimbriae of enterotoxigenic Escherichia coli (ETEC) designated class 5 fimbriae. Assembled by the alternate chaperone pathway, these organelles comprise a rigid stalk of polymerized major subunits and an apparently tip-localized minor adhesive subunit. We examined the evolutionary relationships of class 5-specific structural proteins and correlated these with functional properties. We sequenced the gene clusters encoding coli surface antigen 4 (CS4), CS14, CS17, CS19, and putative colonization factor antigen O71 (PCFO71) and analyzed the deduced proteins and the published homologs of CFA/I, CS1, and CS2. Multiple alignment and phylogenetic analysis of the proteins encoded by each operon define three subclasses, 5a (CFA/I, CS4, and CS14), 5b (CS1, CS17, CS19, and PCFO71), and 5c (CS2). These share distant evolutionary relatedness to fimbrial systems of three other genera. Subclass divisions generally correlate with distinguishing in vitro adherence phenotypes of strains bearing the ETEC fimbriae. Phylogenetic comparisons of the individual structural proteins demonstrated greater intrasubclass conservation among the minor subunits than the major subunits. To correlate this with functional attributes, we made antibodies against CFA/I and CS17 whole fimbriae and maltose-binding protein fusions with the amino-terminal half of the corresponding minor subunits. Anti-minor subunit Fab preparations showed hemagglutination inhibition (HAI) of ETEC expressing homologous and intrasubclass heterologous colonization factors while anti-fimbrial Fab fractions showed HAI activity limited to colonization factor-homologous ETEC. These results were corroborated with similar results from the Caco-2 cell adherence assay. Our findings suggest that the minor subunits of class 5 fimbriae may be superior to whole fimbriae in inducing antiadhesive immunity.
Asunto(s)
Escherichia coli/patogenicidad , Proteínas Fimbrias/fisiología , Fimbrias Bacterianas/fisiología , Adhesión Bacteriana , Sitios de Unión , Células CACO-2 , Proteínas Fimbrias/química , Fimbrias Bacterianas/clasificación , Fimbrias Bacterianas/genética , Humanos , Operón , Fenotipo , Filogenia , Subunidades de ProteínaRESUMEN
Enterotoxigenic Escherichia coli (ETEC) is the most common cause of bacterial diarrhea worldwide and is an important cause of infant morbidity and mortality in developing nations. ETEC colonization factors (CF) are virulence determinants that appear to be protective antigens in humans and are the major target of vaccine efforts. One of the most prevalent CF, CS6, is expressed by about 30% of ETEC worldwide. This study was designed to compare the immunogenicity between encapsulated CS6 (CS6-PLG) and unencapsulated CS6. Recombinant CS6 was purified and encapsulated in biodegradable poly(DL-lactide-co-glycolide) (PLG) microspheres using current Good Manufacturing Practices (cGMP). CS6-PLG and CS6 were administered intranasally (IN) to BALB/c mice in three vaccinations 4 weeks apart. Enzyme linked immunosorbent assay (ELISA) was used to measure the anti-CS6 response in serum and mucosal secretions following each of the three inoculations. Mice vaccinated with two or three doses of CS6-PLG demonstrated a significantly greater rise in serum anti-CS6 IgG and mucosal IgA titer values than those immunized with two or three doses of CS6 alone. Three doses of CS6-PLG led to anti-CS6 serum IgG and mucosal IgA titer values 14-fold and 4.4-fold greater, respectively, than three doses of CS6 (P<0.02). IN administered CS6 to mice is safe and highly immunogenic either alone or when encapsulated in microspheres. PLG microsphere encapsulation of CS6 significantly augments the antibody response to that antigen when administered to a mucosal surface.
Asunto(s)
Vacunas Bacterianas/inmunología , Escherichia coli/inmunología , Administración Intranasal , Animales , Formación de Anticuerpos , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/aislamiento & purificación , Peso Corporal , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/prevención & control , Proteínas Fimbrias/inmunología , Inmunoglobulina G/sangre , RatonesRESUMEN
Mice (BALB/c) were intranasally (IN) and intragastrically (IG) administered the ETEC colonization factors (CF), CFA/I and CS6, with and without the R192G mutant heat-labile enterotoxin (mLT), and immunogenicity and efficacy measured. The IN administration of CFA/I to mice induced strong serum and fecal IgG and IgA responses. The IG administration of CFA/I to mice induced serum IgG and fecal IgA responses, but only when mLT was co-administered with CFA/I were serum IgA titers detected. The IN administration of CS6 to mice induced serum IgG antibodies, and mLT, when co-administered with CS6, enhanced the serum IgG response. Only when the mLT was co-administered with CS6, were serum and fecal IgA responses detected. The IG administration of CS6 plus mLT induced serum IgG and fecal IgA responses. Partial protection against lethal challenge with ETEC strain H10407 was seen in the mice IN administered the CFA/I plus mLT (P<0.01), and H10407 was cleared from the lungs of CFA/I plus mLT-immunized mice at a significantly greater rate than from the control mice (P<0.05). CFA/I and CS6 administered IN and IG induced mixed Th1/Th2 immune responses with the Th2 type being predominant as evidenced by IgG1>IgG2a. The administration of colonization factors to mice, particularly by the IN route, potentially serves as a useful way to measure the serum and mucosal immune responses to these antigens prior to their use in volunteers.
Asunto(s)
Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Enterotoxinas/inmunología , Infecciones por Escherichia coli/inmunología , Proteínas de Escherichia coli/inmunología , Escherichia coli/inmunología , Proteínas Fimbrias/inmunología , Mucosa Gástrica/inmunología , Inmunidad Mucosa , Animales , Anticuerpos Antibacterianos/sangre , Formación de Anticuerpos , Femenino , Ratones , Ratones Endogámicos BALB CRESUMEN
As a step in the development of an oral vaccine against ETEC, we evaluated the safety and immunogenicity of CS6, a polymeric protein commonly found on the surface of ETEC. Formulations included 1 and 5mg doses of CS6, either encapsulated in biodegradable polymer poly(D, L)-lactide-co-glycolide (PLG), or as free protein, administered orally in a solution of either normal saline or a rice-based buffer. Three doses of CS6 were given at 2-week intervals. Blood was collected immediately before and 7 days after each dose. All formulations were well tolerated. Four of five volunteers who received 1mg CS6 in PLG microspheres with buffer had significant IgA ASC responses (median=30 ASC per 10(6) PBMC) and significant serum IgG responses (median=3.5-fold increase). Oral administration of these prototype ETEC vaccine formulations are safe and can elicit immune responses. The ASC, serum IgA, and serum IgG responses to CS6 are similar in magnitude to the responses after challenge with wild-type ETEC [Coster et al., unpublished data]. Further studies are underway to determine whether these immune responses are sufficient for protection.
Asunto(s)
Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/inmunología , Enterotoxinas/administración & dosificación , Enterotoxinas/inmunología , Proteínas de Escherichia coli/administración & dosificación , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/inmunología , Escherichia coli/inmunología , Administración Oral , Adolescente , Adulto , Anticuerpos Antibacterianos/análisis , Antígenos Bacterianos/efectos adversos , Cápsulas , Escherichia coli/metabolismo , Proteínas de Escherichia coli/efectos adversos , Vacunas contra Escherichia coli/efectos adversos , Femenino , Humanos , Inmunidad Celular , Inmunización , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Masculino , Persona de Mediana Edad , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/uso terapéuticoRESUMEN
Upon activation with various noncytokine stimuli, polymorphonuclear leukocytes (PMNs) mobilize intracellular sialidase to the plasma membrane, where the sialidase releases sialic acid from the cell surface. This desialylation enhances PMN adherence, spreading, deformability, and motility, functions critical to diapedesis. We now have examined the role of sialidase activity in PMN adhesion to and migration across the endothelium in vivo. A polyclonal antibody prepared against Clostridium perfringens neuraminidase 1) detected surface expression of sialidase on human PMNs stimulated with IL-8 in vitro and on murine PMNs stimulated in vivo, but not on that of unstimulated cells, 2) recognized proteins in human PMN lysates and granule preparations that were not detected by preimmune antibody, 3) inhibited bacterial neuraminidase and human PMN sialidase activities in vitro, and 4) inhibited both pulmonary leukostasis in mice systemically infused with cobra venom factor and intrapulmonary transendothelial migration of PMNs into the bronchoalveolar compartment of mice intranasally challenged with interleukin-8. We conclude that the chemokine interleukin-8, like other PMN agonists, induces the translocation of sialidase to the PMN surface and that surface expression of this sialidase is a prerequisite to PMN recruitment in vivo. The ability of antibodies raised against a prokaryotic neuraminidase to recognize eukaryotic sialidase extends the concept of the neuraminidase superfamily to mammalian enzymes. Inhibition of mobilized endogenous sialidase may provide a novel strategy for limiting the inflammatory response.
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Neuraminidasa/metabolismo , Neutrófilos/citología , Animales , Western Blotting , Bronquios/citología , Bronquios/metabolismo , Calcimicina/farmacología , Clostridium perfringens/enzimología , Humanos , Inmunoglobulina G/inmunología , Interleucina-8/farmacología , Ratones , Modelos Animales , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Conejos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The pathogenicity and immunogenicity induced in BALB/c mice by intranasal (i.n.) inoculation of enterotoxigenic Escherichia coli (ETEC) strains H10407 (O78:H11:CFA/I:LT(+):ST(+)) and B7A (O148:H28:CS6:LT(+):ST(+)) (two ETEC strains previously used in human challenge trials) were studied. The i.n. inoculation of BALB/c mice with large doses of ETEC strains H10407 and B7A caused illness and death. The H10407 strain was found to be consistently more virulent than the B7A strain. Following i.n. challenge with nonlethal doses of H10407 and B7A, the bacteria were cleared from the lungs of the mice at a steady rate over a 2-week period. Macrophages and neutrophils were observed in the alveoli and bronchioles, and lymphocytes were observed in the septa, around vessels, and in the pleura of the lungs in mice challenged with H10407 and B7A. In mice i.n. challenged with H10407, serum immunoglobulin G (IgG) and IgM antibodies were measured at high titers to the CFA/I and O78 lipopolysaccharide (LPS) antigens. In mice i.n. challenged with B7A, low serum IgG antibody titers were detected against CS6, and low serum IgG and IgM antibody titers were detected against O148 LPS. The serum IgG and IgM antibody titers against the heat-labile enterotoxin were equivalent in the H10407- and B7A-challenged mice. The CFA/I and O78 LPS antigens gave mixed T-helper cell 1-T-helper cell 2 (Th1-Th2) responses in which the Th2 response was greater than the Th1 response (i.e., stimulated primarily an antibody response). These studies indicate that the i.n. challenge of BALB/c mice with ETEC strains may provide a useful animal model to better understand the immunogenicity and pathogenicity of ETEC and its virulence determinants. This model may also be useful in providing selection criteria for vaccine candidates for use in primate and human trials.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Toxinas Bacterianas/metabolismo , Enterotoxinas/metabolismo , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/mortalidad , Proteínas de Escherichia coli , Escherichia coli/patogenicidad , Administración Intranasal , Animales , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Modelos Animales de Enfermedad , Enterotoxinas/inmunología , Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Cinética , Pulmón/inmunología , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , VirulenciaRESUMEN
Transcutaneous immunization (TCI) is a new method for vaccine delivery that has been shown to induce immunity relevant to enteric disease vaccines. We evaluated the clinical safety and immunogenicity of a recombinant subunit vaccine against enterotoxigenic Escherichia coli (ETEC) delivered by TCI. Adult volunteers received patches containing the recombinant ETEC colonization factor CS6, either with heat-labile enterotoxin (LT) or patches containing CS6 alone. The vaccine was administered at 0, 1, and 3 months, and serum antibodies and antibody-secreting cells (ASCs) were assessed. Among the 26 volunteers that completed the trial, there were no responses to CS6 in the absence of LT. In the groups receiving both CS6 and LT, 68 and 53% were found to have serum anti-CS6 immunoglobulin G (IgG) and IgA, respectively; 37 and 42% had IgG and IgA anti-CS6 ASCs. All of the volunteers receiving LT had anti-LT IgG, and 90% had serum anti-LT IgA; 79 and 37% had anti-LT IgG and IgA ASCs. Delayed-type hypersensitivity (DTH), suggesting T-cell responses, was seen in 14 of 19 volunteers receiving LT and CS6; no DTH was seen in subjects receiving CS6 alone. This study demonstrated that protein antigens delivered by a simple patch could induce significant systemic immune responses but only in the presence of an adjuvant such as LT. The data suggest that an ETEC vaccine for travelers delivered by a patch may be a viable approach worthy of further evaluation.
Asunto(s)
Antígenos Bacterianos , Antígenos de Superficie/inmunología , Proteínas Bacterianas/inmunología , Toxinas Bacterianas/inmunología , Enterotoxinas/inmunología , Proteínas de Escherichia coli , Vacunas contra Escherichia coli/inmunología , Administración Cutánea , Adolescente , Adulto , Anticuerpos Antibacterianos/sangre , Células Productoras de Anticuerpos/fisiología , Diarrea/prevención & control , Vacunas contra Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/efectos adversos , Femenino , Humanos , Hipersensibilidad Tardía , Masculino , Persona de Mediana EdadRESUMEN
Virulence of enterotoxicogenic Escherichia coli is mediated by rodlike, rigid, highly hydrophobic proteins designated fimbriae or colonization factors (CFs). More than 20 different colonization factors have been described so far using predominantly immunological and genetic methods. To characterize these hydrophobic proteins by liquid chromatography-mass spectrometry (LC-MS), different methodologies were explored. A novel LC-MS method was developed using hexafluoroisopropanol to maintain the hydrophobic proteins in solution. In addition, these proteins were digested with cyanogen bromide and peptide mapping by LC-MS was established. This technique was particularly useful in identification of closely related CFs. Both LC-MS and peptide mapping methodologies were found to be useful in characterizing highly hydrophobic CFs of E. coli. To search for molecular weights of mature proteins in the National Center for Biotechnology Information (NCBI) database, a new feature was developed and its applicability tested. The identification of a class of pathogenic virulence proteins, either intact or digested, is possible with molecular weight database searching.