Ann-Mari Svennerholm

Protection against Cholera in Breast-Fed Children by Antibodies in Breast Milk

We performed a prospective study to examine whether the IgA antibodies against cholera that are present in breast milk protect breast-fed infants and children against colonization with Vibrio cholerae 01 and disease. Among families of patients with cholera, we collected breast milk from mothers who had not had diarrhea in the previous week and monitored them and their breast-fed children for cholera colonization and diarrhea for 10 days. Breast milk was assayed for IgA antibodies to cholera toxin and lipopolysaccharide. Ninety-three mother--child pairs were studied; 30 infants became colonized with V. cholerae 01 and disease developed in 19. There were no differences between the antibody levels in milk fed to children who became colonized and in milk fed to children who did not. However, among the children who became colonized, those who had diarrhea drank breast milk containing significantly lower levels of both kinds of cholera antibodies than were present in the milk consumed by children who had no symptoms. We conclude that breast-milk antibodies against cholera do not appear to protect children from colonization with V. cholerae 01 but do protect against disease in those who are colonized.

Breast milk reduces the risk of illness in children of mothers with cholera: observations from an epidemic of cholera in Guinea-Bissau

Background: A protective effect of breastfeeding against cholera has been demonstrated in areas endemic of cholera. To assess the protection offered by breast milk from mothers living in an area that had been free from cholera for 7 years, we investigated mothers with cholera and their children during an epidemic with Vibrio cholerae El Tor in the capital of Guinea-Bissau. Methods: Eighty mothers with clinical cholera and their children were identified, and interviewed. Blood samples for vibriocidal and antitoxin antibodies were collected from mother-and-child pairs. Breast milk samples were collected from lactating mothers. Cholera was defined as acute watery diarrhea during the epidemic and a vibriocidal reciprocal titer of 20 or above. Results: Three (7%) of 42 breastfed children had cholera as defined above compared with 9 (24%) of 38 nonbreastfed children (RR for breastfed children, 0.19; 95% CI, 0.04–0.91, adjusted for age). The 3 breastfed children who developed cholera received milk containing lower concentrations of anticholera toxin IgA/total IgA (median, 2.0 units/mL) than 14 children who had serologic signs of colonization but did not develop the disease (median, 17.4 units/mL). Conclusions: The protective effect of breast milk against cholera is not confined to endemic areas. Lactating mothers with cholera should receive supportive care to continue breastfeeding.

Monoclonal Antibodies and Immunodetection Methods for Vibrio cholerae and Escherichia coli Enterotoxins

Publisher Summary This chapter discusses the preparation of the monoclonal antibodies (MAbs) against cholera toxin (CT), and E. coli heat-labile enterotoxin (LT) of human (LTh) and porcine (LTp). Most of the MAbs produced has been directed against the B-subunit portion of the toxin molecules and were of the IgG or IgG 2 isotypes. Both MAbs against the B-subunit portion of the CT and LT had toxin-neutralizing capacity, whereas none of nine MAbs against the cholera A subunit had any detectable neutralizing ability. Of 70 anti-CT MAbs, no less than 44 reacted specifically with CT, the remaining ones showing full or partial cross-reactivity with LTs. Seven of the 25 MAbs against LTh reacted only with LTh, and five of 20 MAbs against LTp were LTp-specific. One MAb, which showed full cross-reactivity between CT, LT, and LTp, and one MAb that is specific for each of these toxins, are exploited for development of diagnostic LT detection methods based on the GM1-ELISA principle. These methods fulfilled the criteria of excellent sensitivity and specificity for the detection of any heat-labile toxin using the cross-reactive MAb, or for the detection of species-specific toxin using the unique epitope MAbs. By these methods, heat-labile enterotoxins are demonstrated in overnight cultures of fecal isolates; the sensitivity of the methods is further increased by inoculation of bacterial isolates or stools directly in GM1-coated microtiter plates.

Resistance Pattern and Molecular Characterization of Enterotoxigenic Escherichia coli (ETEC) Strains Isolated in Bangladesh.

Background Enterotoxigenic Escherichia coli (ETEC) is a common cause of bacterial infection leading to acute watery diarrhea in infants and young children as well as in travellers to ETEC endemic countries. Ciprofloxacin is a broad-spectrum antimicrobial agent nowadays used for the treatment of diarrhea. This study aimed to characterize ciprofloxacin resistant ETEC strains isolated from diarrheal patients in Bangladesh. Methods A total of 8580 stool specimens from diarrheal patients attending the icddr,b Dhaka hospital was screened for ETEC between 2005 and 2009. PCR and Ganglioside GM1- Enzyme Linked Immuno sorbent Assay (ELISA) was used for detection of Heat labile (LT) and Heat stable (ST) toxins of ETEC. Antimicrobial susceptibilities for commonly used antibiotics and the minimum inhibitory concentration (MIC) of nalidixic acid, ciprofloxacin and azithromycin were examined. DNA sequencing of representative ciprofloxacin resistant strains was performed to analyze mutations of the quinolone resistance-determining region of gyrA, gyrB, parC and parE. PCR was used for the detection of qnr, a plasmid mediated ciprofloxacin resistance gene. Clonal variations among ciprofloxacin resistant (CipR) and ciprofloxacin susceptible (CipS) strains were determined by Pulsed-field gel electrophoresis (PFGE). Results Among 1067 (12%) ETEC isolates identified, 42% produced LT/ST, 28% ST and 30% LT alone. Forty nine percent (n = 523) of the ETEC strains expressed one or more of the 13 tested colonization factors (CFs) as determined by dot blot immunoassay. Antibiotic resistance of the ETEC strains was observed as follows: ampicillin 66%, azithromycin 27%, ciprofloxacin 27%, ceftriazone 13%, cotrimaxazole 46%, doxycycline 44%, erythromycin 96%, nalidixic acid 83%, norfloxacin 27%, streptomycin 48% and tetracycline 42%. Resistance to ciprofloxacin increased from 13% in 2005 to 34% in 2009. None of the strains was resistant to mecillinam. The MIC of the nalidixic acid and ciprofloxacin of representative CipR strains were 256 μg/ml and 32μg/ml respectively. A single mutation (Ser83-Leu) in gyrA was observed in the nalidixic acid resistant ETEC strains. In contrast, double mutation in gyrA (Ser83-Leu, Asp87-Asn) and a single mutation in parC (Glu84-Ly) were found in ciprofloxacin resistant strains. Mutation of gyrB was not found in either the nalidixic acid or ciprofloxacin resistant strains. None of the ciprofloxacin resistant strains was found to be positive for the qnr gene. Diverse clones were identified from all ciprofloxacin resistant strains by PFGE analysis in both CF positive and CF negative ETEC strains. Conclusion Emergence of ciprofloxacin resistant ETEC strains results in a major challenge in current treatment strategies of ETEC diarrhea.

Déterminants des réponses aux vaccins oraux dans les pays en développement

Les vaccins oraux conçus pour une utilisation globale n’induisent pas nécessairement les mêmes réponses immunitaires chez tous les enfants du monde entier. En effet, plusieurs vaccins induisent souvent des réponses anticorps moyennes moins fréquentes et moins intenses chez les enfants des pays en développement, suggérant qu’ils pourraient moins les protéger. Les causes de cette différence ne sont pas totalement élucidées, mais il apparaît que des facteurs liés à la nutrition, dont une malnutrition tant en protéines et en calories qu’en micronutriments, sont importants pour expliquer ce problème. Des domaines voisins dont l’allaitement maternel, l’interférence d’anticorps placentaires maternels, des infections parasitaires intestinales, des lésions de la muqueuse intestinale et, peut-être, une malnutrition maternelle au cours de la grossesse paraissent également importants. Les vaccins conçus pour la voie orale doivent être adaptés à ces problèmes potentiels afin d’optimiser leurs bénéfices pour tous les enfants. Les vaccins oraux administrés aux enfants des pays en développement peuvent nécessiter une dose plus élevée, des doses de rappel, une supplémentation en calories, micronutriments et vitamines, un arrêt de l’allaitement maternel avant la vaccination, des médicaments contre les parasites intestinaux ou d’autres mesures afin d’obtenir leur bénéfice complet.

The Binding of Bacteria Carrying CFAs and Putative CFAs to Rabbit Intestinal Brush Border Membranes

Enterotoxigenic Escherichia coli, ETEC, colonize the human intestine by means of fimbrial structures called colonization factor antigens, CFAs. On human ETEC strains three different types of CFAs have been described, i.e. the colonization factor antigens I, (CFA/I), CFA/II and CFA/IV. In addition a number of “new” putative colonization factor antigens have been identified, e.g. PCF0166, CS7, CS17 and CFA/III [M.M McConnell, this volume] although their prevalence and role in colonization have not been fully assessed.

Determinanten für Reaktionen auf orale Impfstoffe in Entwicklungsländern

Orale, zur globalen Anwendung vorgesehene Impfstoffe bewirken nicht unbedingt bei allen Kindern auf der ganzen Welt die selbe Immunantwort. Tatsächlich sind die durchschnittlichen Antikörperreaktionen auf mehrere Vakzine bei Kindern in Entwicklungsländern oft seltener und weniger stark ausgeprägt, was darauf schliessen lässt, dass diese Impfstoffe bei Kindern in diesen Regionen eine geringere Schutzwirkung entfalten. Obwohl noch nicht vollständig geklärt ist, welche Gründe für diese schwächer ausgeprägten Immunreaktionen verantwortlich sind, scheinen ernährungsbedingte Faktoren, u.a. die Proteinmangelernährung sowie ein Mikronährstoffmangel, eine wichtige Rolle zu spielen, um die bei diesen Kindern verminderte Immunantwort zu verstehen. Damit verbundene Faktoren, wie z.B. Stillen, Beeinträchtigung durch maternale, über die Plazenta übertragene Antikörper, parasitäre Darminfektionen, Schädigung der Darmschleimhaut und eine mögliche Mangelernährung der Mutter während der Schwangerschaft, scheinen ebenfalls von Bedeutung zu sein. Die zur oralen Applikation vorgesehenen Impfstoffe müssen bezüglich dieser potenziellen Probleme angepasst werden, um bei allen Kindern einen maximalen Nutzen zu erzielen. Wird Kindern in Entwicklungsländern ein oraler Impfstoff verabreicht, so sind möglicherweise eine höhere Impfstoffdosierung, Auffrischimpfungen, eine Supplementierung von Kalorien, Mikronährstoffen und Vitaminen, eine Stillpause vor der Impfung, Entwurmungsmedikamente oder andere Massnahmen erforderlich, um die Nutzwirkung der Impfung voll auszuschöpfen.

Mucosal Immunity in the Gastrointestinal Tract in Relation to ETEC Vaccine Development

Enteric infection with enterotoxinogenic Escherichia coli (ETEC) is one of the most important causes of diarrhea in developing countries and is also the most frequent cause of diarrhea among travelers. Illness resulting from ETEC infection is characterized by watery diarrhea, often accompanied by low grade fever, abdominal cramps, malaise, and vomiting. In its most severe form ETEC infection may result in cholera-like disease leading to severe dehydration and sometimes death.

Determinantes de las respuestas a vacunas orales en países en vías de desarrollo

Las vacunas orales que se han diseñado para uso global no inducen necesariamente las mismas respuestas inmunitarias en todos los niños de todo el mundo. De hecho, varias vacunas inducen a menudo respuestas medias de anticuerpos menos frecuentes y menores en niños en países en vías de desarrollo, lo que permite suponer que las vacunas pueden producir un efecto menos protector en los niños de estas zonas. Aunque los motivos de esta respuesta menos enérgica no se conocen por completo, parece que factores relacionados con la nutrición, incluyendo la desnutrición de proteínas y calorías y la desnutrición de micronutrientes, son aspectos importantes para comprender la hiporreactividad observada en estos niños. También parecen importantes cuestiones relacionadas, entre las que destacan la lactancia materna, la interferencia a partir de anticuerpos placentarios maternos, infecciones parasitarias intestinales, lesión de la mucosa intestinal y, posiblemente, malnutrición materna durante el embarazo. Las vacunas diseñadas para administración oral tendrán que ser ajustadas a estos problemas potenciales con objeto de incrementar al máximo sus beneficios para todos los niños. Las vacunas orales que se administran en niños de países en vías de desarrollo pueden requerir dosis mayores de vacunas, dosis de refuerzo, complementos de calorías, micronutrientes y vitaminas, retirada de la leche materna antes de la administración de las vacunas, medicamentos antihelmínticos u otras medidas para desarrollar sus beneficios totales.

CHAPTER 61 – Engineered bacterial toxin vaccines and adjuvants

There has been remarkable progress in the understanding of the structure-function relationship of several bacterial toxins with an advanced understanding of toxin genes and the regulation of their expression. This has resulted in completely new possibilities to prepare detoxified yet immunologically active toxin derivates. Recently, engineered detoxified toxins or non-toxic subunits have also attracted much interest as vaccine carrier proteins, adjuvants, and/or inmiunomodulators, in some cases for parenteral use but especially for mucosal administration. This chapter describes the properties and clinical potential of various types of genetically engineered bacterial toxins and subunits in these different areas of vaccination, adjuvanting, and immunomodulation. Much less well appreciated than vaccination against infections, mucosal immunization may also be used to induce peripheral tolerance to prevent or suppress the development of harmful immune responses to foreign proteins derived from ingested food or from commensal microorganisms, in case these antigens would reach the body interior in an undegraded, immunogenic form. For both types of applications, it has been found that bacterial toxin derivatives can substantially influence the outcome in ways that give hope for further development toward practical medical use. Likewise, engineered bacterial toxin proteins have found a useful place as carrier proteins for various capsular polysaccharides in the development of conjugate vaccines against important infections with encapsulated bacteria.