RESUMEN
INTRODUCTION: Pigs without intestinal receptors for F4 fimbriae are congenitally resistant to F4 fimbriae-bearing enterotoxigenic Escherichia coli (ETEC F4). In general, 50 % and 100 % of piglets born to resistant (RR) sows crossed with hetero- or homozygous susceptible (SR, SS) boars, respectively, are susceptible but do not receive colostral antibodies against F4 fimbriae unless the sows have been vaccinated. The question arises as to whether resistant sows produce protective amounts of F4 antifimbrial antibodies after vaccination. The serum and colostrum antibody titres of 12 resistant and 12 susceptible vaccinated gilts were compared. The effect of the receptor status of the dam and sire on the preweaning performance of 5027 piglets was evaluated using Agroscope's recordings. The sows of the experimental herd, where ETEC F4 was circulating, were vaccinated against ETEC twice during the first pregnancy and once during each following pregnancy. The log2 transformed F4 antibody titres in the serum obtained after the second vaccine injection as well as in the colostrum of the 12 resistant animals were lower than the titres of the susceptible animals (serum: F4ab 11,19 ± 1,44 vs. 12,18 ± 1,33, P = 0,096; F4ac 10,03 ± 1,58 vs. 11,59 ± 1,43, P = 0,019; colostrum: F4ab 12,20 ± 2,41 vs. 14,02 ± 1,31, P = 0,033; F4ac 10,93 ± 2,46 vs. 13,03 ± 5,21, P = 0,006). The heat labile enterotoxin (LT) antibody titres after vaccination did not differ between susceptible and resistant animals (p > 0,10). Preweaning mortality in the offspring of RR sows × SS boars was slightly lower than in the offspring of SS sows × RR boars (P = 0,04), suggesting that the disease risk of susceptible piglets born to vaccinated resistant sows was not increased, even though they received colostrum with a slightly reduced content of antibody against F4 fimbriae.
INTRODUCTION: Les porcs dépourvus de récepteurs intestinaux pour les fimbriae F4 sont congénitalement résistants aux Escherichia coli entérotoxinogènes porteurs de fimbriae F4 (ETEC F4). En général, 50 % et 100 % des porcelets nés de truies résistantes (RR) croisées avec des verrats hétéro- ou homozygotes sensibles (SR, SS), respectivement, sont sensibles mais ne reçoivent pas d'anticorps colostraux contre les fimbriae F4, à moins que les truies n'aient été vaccinées. La question se pose de savoir si les truies résistantes produisent des quantités protectrices d'anticorps antifimbriae F4 après la vaccination. Les titres d'anticorps dans le sérum et le colostrum de 12 truies reproductrices vaccinées résistantes et de 12 truies reproductrices vaccinées sensibles ont été comparés et l'effet du statut récepteur de la mère et du père sur les performances avant sevrage de 5027 porcelets a été évalué. Les truies du troupeau expérimental, où circulait ETEC F4, ont été vaccinées deux fois au cours de la première gestation et une fois au cours de chaque gestation suivante contre ETEC. Les titres d'anticorps F4 transformés en log2 dans le sérum obtenu après la deuxième injection de vaccin ainsi que dans le colostrum des 12 animaux résistants étaient inférieurs aux titres des animaux sensibles (sérum : F4ab 11,19 ± 1,44 vs. 12,18 ± 1,33, P = 0,096 ; F4ac 10,03 ± 1,58 vs. 11,59 ± 1,43, P = 0,019 ; colostrum : F4ab 12,20 ± 2,41 vs. 14,02 ± 1,31, P = 0,033 ; F4ac 10,93 ± 2,46 vs. 13,03 ± 5,21, P = 0,006). Les titres d'anticorps contre l'entérotoxine thermolabile (LT) après la vaccination ne différaient pas entre les animaux sensibles et résistants (p > 0,10). La mortalité avant sevrage dans la progéniture des truies RR × verrats SS était légèrement inférieure à celle de la progéniture des truies SS × verrats RR (P = 0,04), ce qui suggère que le risque de maladie des porcelets sensibles nés de truies résistantes vaccinées n'a pas été augmenté, même s'ils ont reçu du colostrum avec une teneur légèrement réduite en anticorps contre les fimbriae F4.
Asunto(s)
Escherichia coli Enterotoxigénica , Infecciones por Escherichia coli , Vacunas contra Escherichia coli , Fimbrias Bacterianas , Enfermedades de los Porcinos , Animales , Porcinos , Infecciones por Escherichia coli/veterinaria , Infecciones por Escherichia coli/prevención & control , Infecciones por Escherichia coli/inmunología , Enfermedades de los Porcinos/prevención & control , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/microbiología , Femenino , Vacunas contra Escherichia coli/inmunología , Vacunas contra Escherichia coli/administración & dosificación , Escherichia coli Enterotoxigénica/inmunología , Fimbrias Bacterianas/inmunología , Fimbrias Bacterianas/genética , Embarazo , Calostro/inmunología , Anticuerpos Antibacterianos/sangre , DesteteRESUMEN
Background: Escherichia coli is one of the serious pathogens causing various infections in the animal field, such as neonatal calf diarrhea, which is responsible for mortality associated with diarrhea during the first days of life. Aim: Current work is aimed at designing an effective and safe multiepitope vaccine candidate against E. coli infection in calves based on the fimbrial protein K99 of Enterotoxigenic E. coli (ETEC) and Immuno-informatics. Methods: A conserved sequence of K99 protein was generated, and then highly antigenic, nonallergic, and overlapped epitopes were used to construct a multiepitope vaccine. Five THL, six MHC II, and four beta cell epitopes were targeted to create the candidate. The candidate vaccine was produced utilizing 15 epitopes and three types of linkers, two types of untranslated region (UTR) human hemoglobin subunit beta (HBB), UTR beta-globin (Rabb), and RpfE protein as an immunomodulation adjuvant. Results: Immuno-informatics analysis of the constructed protein showed that the protein was antigenic (antigenic score of 0.8841), stable, nonallergen, and soluble. Furthermore, the Immuno-informatics and physiochemical analysis of the constructed protein showed a stable, nonallergic, soluble, hydrophilic, and acidic PI (isoelectric point). of 9.34. Docking of the candidate vaccine with the toll-like receptor TLR3 was performed, and results showed a strong interaction between the immune receptor and the vaccine. Finally, the expression efficiency of the construct in E. coli was estimated via computational cloning of the vaccine sequence into Pet28a. Conclusion: Results of immunoinformatics and in silico approaches reveal that the designed vaccine is antigenic, stable, and able to bind to the immune cell receptors. Our results interpret the proposed multiepitope mRNA vaccine as a good preventive option against E. coli infection in calves.
Asunto(s)
Enfermedades de los Bovinos , Biología Computacional , Escherichia coli Enterotoxigénica , Infecciones por Escherichia coli , Vacunas contra Escherichia coli , Animales , Bovinos , Escherichia coli Enterotoxigénica/inmunología , Infecciones por Escherichia coli/veterinaria , Infecciones por Escherichia coli/prevención & control , Infecciones por Escherichia coli/inmunología , Vacunas contra Escherichia coli/inmunología , Enfermedades de los Bovinos/prevención & control , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/microbiología , Epítopos/inmunología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Modelos Moleculares , InmunoinformáticaRESUMEN
OBJECTIVES: The pathogenic microorganisms that cause intestinal diseases can significantly jeopardize people's health. Currently, there are no authorized treatments or vaccinations available to combat the germs responsible for intestinal disease. METHODS: Using immunoinformatics, we developed a potent multi-epitope Combination (combo) vaccine versus Salmonella and enterohemorrhagic E. coli. The B and T cell epitopes were identified by performing a conservancy assessment, population coverage analysis, physicochemical attributes assessment, and secondary and tertiary structure assessment of the chosen antigenic polypeptide. The selection process for vaccine development included using several bioinformatics tools and approaches to finally choose two linear B-cell epitopes, five CTL epitopes, and two HTL epitopes. RESULTS: The vaccine had strong immunogenicity, cytokine production, immunological properties, non-toxicity, non-allergenicity, stability, and potential efficacy against infections. Disulfide bonding, codon modification, and computational cloning were also used to enhance the stability and efficacy of expression in the host E. coli. The vaccine's structure has a strong affinity for the TLR4 ligand and is very durable, as shown by molecular docking and molecular modeling. The results of the immunological simulation demonstrated that both B and T cells had a heightened response to the vaccination component. CONCLUSIONS: The comprehensive in silico analysis reveals that the proposed vaccine will likely elicit a robust immune response against pathogenic bacteria that cause intestinal diseases. Therefore, it is a promising option for further experimental testing.
Asunto(s)
Epítopos de Linfocito B , Epítopos de Linfocito T , Vacunología , Humanos , Epítopos de Linfocito T/inmunología , Vacunología/métodos , Epítopos de Linfocito B/inmunología , Vacunas Combinadas/inmunología , Genómica/métodos , Escherichia coli Enterohemorrágica/inmunología , Salmonella/inmunología , Animales , Biología Computacional/métodos , Simulación del Acoplamiento Molecular , Vacunas contra Escherichia coli/inmunología , Infecciones por Escherichia coli/prevención & control , Infecciones por Escherichia coli/inmunología , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/prevención & control , Antígenos Bacterianos/inmunología , Desarrollo de Vacunas/métodos , Vacunas Bacterianas/inmunologíaRESUMEN
The emergence of antimicrobial resistance (AMR) is a principal global health crisis projected to cause 10 million deaths annually worldwide by 2050. While the Gram-negative bacteria Escherichia coli is commonly found as a commensal microbe in the human gut, some strains are dangerously pathogenic, contributing to the highest AMR-associated mortality. Strains of E. coli that can translocate from the gastrointestinal tract to distal sites, called extraintestinal E. coli (ExPEC), are particularly problematic and predominantly afflict women, the elderly, and immunocompromised populations. Despite nearly 40 years of clinical trials, there is still no vaccine against ExPEC. One reason for this is the remarkable diversity in the ExPEC pangenome across pathotypes, clades, and strains, with hundreds of genes associated with pathogenesis including toxins, adhesins, and nutrient acquisition systems. Further, ExPEC is intimately associated with human mucosal surfaces and has evolved creative strategies to avoid the immune system. This review summarizes previous and ongoing preclinical and clinical ExPEC vaccine research efforts to help identify key gaps in knowledge and remaining challenges.
Asunto(s)
Infecciones por Escherichia coli , Vacunas contra Escherichia coli , Escherichia coli Patógena Extraintestinal , Humanos , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/inmunología , Escherichia coli Patógena Extraintestinal/genética , AnimalesRESUMEN
A US collection of invasive Escherichia coli serotype O1 bloodstream infection (BSI) isolates were assessed for genotypic and phenotypic diversity as the basis for designing a broadly protective O-antigen vaccine. Eighty percent of the BSI isolate serotype O1 strains were genotypically ST95 O1:K1:H7. The carbohydrate repeat unit structure of the O1a subtype was conserved in the three strains tested representing core genome multi-locus sequence types (MLST) sequence types ST95, ST38, and ST59. A long-chain O1a CRM197 lattice glycoconjugate antigen was generated using oxidized polysaccharide and reductive amination chemistry. Two ST95 strains were investigated for use in opsonophagocytic assays (OPA) with immune sera from vaccinated animals and in murine lethal challenge models. Both strains were susceptible to OPA killing with O1a glycoconjugate post-immune sera. One of these, a neonatal sepsis strain, was found to be highly lethal in the murine challenge model for which virulence was shown to be dependent on the presence of the K1 capsule. Mice immunized with the O1a glycoconjugate were protected from challenges with this strain or a second, genotypically related, and similarly virulent neonatal isolate. This long-chain O1a CRM197 lattice glycoconjugate shows promise as a component of a multi-valent vaccine to prevent invasive E. coli infections. IMPORTANCE: The Escherichia coli serotype O1 O-antigen serogroup is a common cause of invasive bloodstream infections (BSI) in populations at risk such as newborns and the elderly. Sequencing of US BSI isolates and structural analysis of O polysaccharide antigens purified from strains that are representative of genotypic sub-groups confirmed the relevance of the O1a subtype as a vaccine antigen. O polysaccharide was purified from a strain engineered to produce long-chain O1a O-antigen and was chemically conjugated to CRM197 carrier protein. The resulting glycoconjugate elicited functional antibodies and was protective in mice against lethal challenges with virulent K1-encapsulated O1a isolates.
Asunto(s)
Infecciones por Escherichia coli , Escherichia coli , Glicoconjugados , Antígenos O , Animales , Antígenos O/inmunología , Antígenos O/genética , Ratones , Infecciones por Escherichia coli/prevención & control , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/inmunología , Escherichia coli/genética , Escherichia coli/inmunología , Glicoconjugados/inmunología , Humanos , Serogrupo , Vacunas contra Escherichia coli/inmunología , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Femenino , Virulencia , Vacunas Conjugadas/inmunología , Tipificación de Secuencias Multilocus , Modelos Animales de Enfermedad , Bacteriemia/prevención & control , Bacteriemia/microbiología , Bacteriemia/inmunología , Proteínas BacterianasRESUMEN
Extraintestinal pathogenic Escherichia coli (ExPEC) is a leading cause of worldwide morbidity and mortality, the top cause of antimicrobial-resistant (AMR) infections, and the most frequent cause of life-threatening sepsis and urinary tract infections (UTI) in adults. The development of an effective and universal vaccine is complicated by this pathogen's pan-genome, its ability to mix and match virulence factors and AMR genes via horizontal gene transfer, an inability to decipher commensal from pathogens, and its intimate association and co-evolution with mammals. Using a pan virulome analysis of >20,000 sequenced E. coli strains, we identified the secreted cytolysin α-hemolysin (HlyA) as a high priority target for vaccine exploration studies. We demonstrate that a catalytically inactive pure form of HlyA, expressed in an autologous host using its own secretion system, is highly immunogenic in a murine host, protects against several forms of ExPEC infection (including lethal bacteremia), and significantly lowers bacterial burdens in multiple organ systems. Interestingly, the combination of a previously reported autotransporter (SinH) with HlyA was notably effective, inducing near complete protection against lethal challenge, including commonly used infection strains ST73 (CFT073) and ST95 (UTI89), as well as a mixture of 10 of the most highly virulent sequence types and strains from our clinical collection. Both HlyA and HlyA-SinH combinations also afforded some protection against UTI89 colonization in a murine UTI model. These findings suggest recombinant, inactive hemolysin and/or its combination with SinH warrant investigation in the development of an E. coli vaccine against invasive disease.
Asunto(s)
Infecciones por Escherichia coli , Proteínas de Escherichia coli , Vacunas contra Escherichia coli , Escherichia coli Patógena Extraintestinal , Proteínas Hemolisinas , Animales , Escherichia coli Patógena Extraintestinal/genética , Escherichia coli Patógena Extraintestinal/inmunología , Infecciones por Escherichia coli/prevención & control , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/inmunología , Ratones , Proteínas Hemolisinas/inmunología , Proteínas Hemolisinas/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/inmunología , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/genética , Femenino , Factores de Virulencia/genética , Factores de Virulencia/inmunología , Sistemas de Secreción Tipo V/inmunología , Sistemas de Secreción Tipo V/genética , Modelos Animales de Enfermedad , HumanosRESUMEN
BACKGROUND: Neonatal and post-weaning diarrhea is a concern disease caused by enterotoxigenic Escherichia coli fimbriae F4 (F4+ETEC) in pig farms. Diarrhea outbreaks are often severe and costly due to the high prevalence and spread of the disease within the same herd. Vaccine is one of strategic solution in protecting pig against F4+ETEC infection in particular pig farm. In present study, we conducted two trials of vaccination with crude F4 fimbriae extract vaccine in pregnant sow and nursery pigs. METHODS: In experiment 1 (20 sows; non-vaccinated control, n=10), we vaccinated pregnant sows (n=10) twice at 4 wk and 2 wk before farrowing and evaluated impact of vaccination on maternal immunity. The sow serum and colostrum were collected before vaccination, 2 and 4 weeks after vaccination, 6 hours after farrowing, respectively, and the piglet's serum from both groups (2 piglet/sow, 10 piglets from each group) were also collected on 3 days old to measure F4 specific IgG, F4 specific IgA using in house ELISA kit. In experiment 2, to optimize doses and dosage of candidate vaccine in piglets, 18 piglets (3 piglets/group) were allocated into five immunized groups and one control group (unimmunized group), we immunized piglets twice at 4 and 6 weeks old with difference doses (i.e., 0, 50, 100, 150, 200 µg), and for a dose 150 µg, we immunized with two dosages at 1 ml and 2 ml. Piglets were challenged with a 3 ml dose of 3 × 109 CFU/ml bacterial culture of enterotoxigenic Escherichia coli (F4+ETEC) in order to evaluate the efficacy of vaccine. After challenging, the clinical sign of the piglets was daily observed and the rectal swab was performed every day for investigation of the fecal shedding of Escherichia coli (F4+ETEC) by using PCR technique. Serum were collected before, 2 and 4 weeks after vaccination and 1 week after challenge to measure F4 specific IgG, F4 specific IgA using in house ELISA kit and cytokines levels (i.e., IL-1 beta, IL-6, IL-8 and TNF alpha) before and 1 week after challenge using commercial ELISA kit. RESULTS: The levels of antibody results showed that in experiment 1, the anti-F4 antibody levels both F4 specific IgG and F4 specific IgA in serum and colostrum of vaccinated sow increased significantly after vaccination. The piglets of immunized sows have antibody level both F4 specific IgG and F4 specific IgA in their serum higher than those piglets of unimmunized sows significantly (p < 0.01). In experiment 2, irrespective of different doses and dosage, there is no difference in term of F4 specific IgG and F4 specific IgA levels among immunized groups. However, all of vaccinated piglets showed F4 specific IgG and F4 specific IgA levels higher and the elimination of Escherichia coli (F4+ETEC) in feces post challenge faster (< 3 days) than unvaccinated group (> 5 days). For cytokines levels, a higher level of IL-1 beta, IL-6, IL-8 and TNF alpha at 1 week after challenge in vaccinated groups was found when compared with the levels in non-vaccinated group. CONCLUSIONS: Our results suggest that crude F4 fimbriae extract autogenous vaccine is a candidate vaccine for protecting piglets against diarrhea disease caused by enterotoxigenic Escherichia coli (F4+ETEC) and vaccination the pregnant sow twice before farrowing is one of strategies to provide maternal derived antibody to the newborn piglets for against enterotoxigenic Escherichia coli (F4+ETEC) during early life.
Asunto(s)
Anticuerpos Antibacterianos , Escherichia coli Enterotoxigénica , Infecciones por Escherichia coli , Vacunas contra Escherichia coli , Enfermedades de los Porcinos , Animales , Porcinos , Femenino , Infecciones por Escherichia coli/prevención & control , Infecciones por Escherichia coli/veterinaria , Infecciones por Escherichia coli/inmunología , Enfermedades de los Porcinos/prevención & control , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/microbiología , Escherichia coli Enterotoxigénica/inmunología , Vacunas contra Escherichia coli/inmunología , Vacunas contra Escherichia coli/administración & dosificación , Embarazo , Anticuerpos Antibacterianos/sangre , Calostro/inmunología , Inmunoglobulina A/sangre , Vacunación/veterinaria , Inmunoglobulina G/sangre , Fimbrias Bacterianas/inmunología , Diarrea/prevención & control , Diarrea/veterinaria , Diarrea/microbiología , Diarrea/inmunología , Animales Recién Nacidos/inmunología , Inmunidad Materno-AdquiridaRESUMEN
Colibacillosis, a disease caused by Escherichia coli in broiler chickens has serious implications on food safety, security, and economic sustainability. Antibiotics are required for treating the disease, while vaccination and biosecurity are used for its prevention. This systematic review and meta-analysis, conducted under the COST Action CA18217-European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT), aimed to assess the efficacy of E. coli vaccination in broiler production and provide evidence-based recommendations. A comprehensive search of bibliographic databases, including, PubMed, CAB Abstracts, Web of Science and Agricola, yielded 2,722 articles. Following a defined protocol, 39 studies were selected for data extraction. Most of the studies were experimental infection trials, with only three field studies identified, underscoring the need for more field-based research. The selected studies reported various types of vaccines, including killed (n = 5), subunit (n = 8), outer membrane vesicles/protein-based (n = 4), live/live-attenuated (n = 16), and CpG oligodeoxynucleotides (ODN) (n = 6) vaccines. The risk of bias assessment revealed that a significant proportion of studies reporting mortality (92.3%) or feed conversion ratio (94.8%) as outcomes, had "unclear" regarding bias. The meta-analysis, focused on live-attenuated and CpG ODN vaccines, demonstrated a significant trend favoring both vaccination types in reducing mortality. However, the review also highlighted the challenges in reproducing colibacillosis in experimental setups, due to considerable variation in challenge models involving different routes of infection, predisposing factors, and challenge doses. This highlights the need for standardizing the challenge model to facilitate comparisons between studies and ensure consistent evaluation of vaccine candidates. While progress has been made in the development of E. coli vaccines for broilers, further research is needed to address concerns such as limited heterologous protection, practicability for application, evaluation of efficacy in field conditions and adoption of novel approaches.
Asunto(s)
Pollos , Infecciones por Escherichia coli , Enfermedades de las Aves de Corral , Vacunación , Animales , Pollos/microbiología , Infecciones por Escherichia coli/prevención & control , Infecciones por Escherichia coli/veterinaria , Enfermedades de las Aves de Corral/prevención & control , Enfermedades de las Aves de Corral/microbiología , Vacunación/veterinaria , Escherichia coli/inmunología , Vacunas contra Escherichia coli/inmunología , Vacunas contra Escherichia coli/administración & dosificaciónRESUMEN
The present study investigated the expression of cytokines and cellular changes in chickens following vaccination with irradiated avian pathogenic Escherichia coli (APEC) and/or challenge. Four groups of 11-week-old pullets, each consisting of 16 birds were kept separately in isolators before they were sham inoculated (N), challenged only (C), vaccinated (V) or vaccinated and challenged (V+C). Vaccination was performed using irradiated APEC applied via aerosol. For challenge, the homologous strain was administered intratracheally. Birds were sacrificed on 3, 7, 14 and 21 days post challenge (dpc) to examine lesions, organ to body weight ratios and bacterial colonization. Lung and spleen were sampled for investigating gene expression of cytokines mediating inflammation by RT-qPCR and changes in the phenotype of subsets of mononuclear cells by flow cytometry. After re-stimulation of immune cells by co-cultivation with the pathogen, APEC-specific IFN-γ producing cells were determined. Challenged only birds showed more severe pathological and histopathological lesions, a higher probability of bacterial re-isolation and higher organ to body weight ratios compared to vaccinated and challenged birds. In the lung, an upregulation of IL-1ß and IL-6 following vaccination and/or challenge at 3 dpc was observed, whereas in the spleen IL-1ß was elevated. Changes were observed in macrophages and TCR-γδ+ cells within 7 dpc in spleen and lung of challenged birds. Furthermore, an increase of CD4+ cells in spleen and a rise of Bu-1+ cells in lung were present in vaccinated and challenged birds at 3 dpc. APEC re-stimulated lung and spleen mononuclear cells from only challenged pullets showed a significant increase of IFN-γ+CD8α+ and IFN-γ+TCR-γδ+ cells. Vaccinated and challenged chickens responded with a significant increase of IFN-γ+CD8α+ T cells in the lung and IFN-γ+TCR-γδ+ cells in the spleen. Re-stimulation of lung mononuclear cells from vaccinated birds resulted in a significant increase of both IFN-γ+CD8α+ and IFN-γ+TCR-γδ+ cells. In conclusion, vaccination with irradiated APEC caused enhanced pro-inflammatory response as well as the production of APEC-specific IFN-γ-producing γδ and CD8α T cells, which underlines the immunostimulatory effect of the vaccine in the lung. Hence, our study provides insights into the underlying immune mechanisms that account for the defense against APEC.
Asunto(s)
Infecciones por Escherichia coli , Vacunas contra Escherichia coli , Animales , Pollos , Femenino , Vacunas contra Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/inmunología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/prevención & control , Infecciones por Escherichia coli/veterinaria , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/prevención & control , AerosolesRESUMEN
Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children's and travelers' diarrhea. Developing effective vaccines against this heterologous group has proven difficult due to the varied nature of toxins and adhesins that determine their pathology. A multivalent candidate vaccine was developed using a multi-epitope fusion antigen (MEFA) vaccinology platform and shown to effectively elicit broad protective antibody responses in mice and pigs. However, direct protection against ETEC colonization of the small intestine was not measured in these systems. Colonization of ETEC strains is known to be a determining factor in disease outcomes and is adhesin-dependent. In this study, we developed a non-surgical rabbit colonization model to study immune protection against ETEC colonization in rabbits. We tested the ability for the MEFA-based vaccine adhesin antigen, in combination with dmLT adjuvant, to induce broad immune responses and to protect from ETEC colonization of the rabbit small intestine. Our results indicate that the candidate vaccine MEFA antigen elicits antibodies in rabbits that react to seven adhesins included in its construction and protects against colonization of a challenge strain that consistently colonized naïve rabbits.
Asunto(s)
Antígenos Bacterianos/administración & dosificación , Diarrea/prevención & control , Escherichia coli Enterotoxigénica/crecimiento & desarrollo , Escherichia coli Enterotoxigénica/inmunología , Epítopos/inmunología , Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/administración & dosificación , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Diarrea/sangre , Diarrea/microbiología , Modelos Animales de Enfermedad , Escherichia coli Enterotoxigénica/genética , Epítopos/genética , Infecciones por Escherichia coli/sangre , Infecciones por Escherichia coli/microbiología , Vacunas contra Escherichia coli/genética , Vacunas contra Escherichia coli/inmunología , Humanos , Inmunización , Intestino Delgado/inmunología , Intestino Delgado/microbiología , ConejosRESUMEN
BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) infection is a primary cause of livestock diarrhea. Therefore, effective vaccines are needed to reduce the incidence of ETEC infection. OBJECTIVES: Our study aimed to develop a multivalent ETEC vaccine targeting major virulence factors of ETEC, including enterotoxins and fimbriae. METHODS: SLS (STa-LTB-STb) recombinant enterotoxin and fimbriae proteins (F4, F5, F6, F18, and F41) were prepared to develop a multivalent vaccine. A total of 65 mice were immunized subcutaneously by vaccines and phosphate-buffered saline (PBS). The levels of specific immunoglobulin G (IgG) and pro-inflammatory cytokines were determined at 0, 7, 14 and 21 days post-vaccination (dpv). A challenge test with a lethal dose of ETEC was performed, and the survival rate of the mice in each group was recorded. Feces and intestine washes were collected to measure the concentrations of secretory immunoglobulin A (sIgA). RESULTS: Anti-SLS and anti-fimbriae-specific IgG in serums of antigen-vaccinated mice were significantly higher than those of the control group. Immunization with the SLS enterotoxin and multivalent vaccine increased interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) concentrations. Compared to diarrheal symptoms and 100% death of mice in the control group, mice inoculated with the multivalent vaccine showed an 80% survival rate without any symptom of diarrhea, while SLS and fimbriae vaccinated groups showed 60 and 70% survival rates, respectively. CONCLUSIONS: Both SLS and fimbriae proteins can serve as vaccine antigens, and the combination of these two antigens can elicit stronger immune responses. The results suggest that the multivalent vaccine can be successfully used for preventing ETEC in important livestock.
Asunto(s)
Diarrea , Infecciones por Escherichia coli , Vacunas contra Escherichia coli/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Diarrea/prevención & control , Diarrea/veterinaria , Modelos Animales de Enfermedad , Escherichia coli Enterotoxigénica , Enterotoxinas/inmunología , Infecciones por Escherichia coli/prevención & control , Infecciones por Escherichia coli/veterinaria , Proteínas de Escherichia coli/inmunología , Proteínas Fimbrias/inmunología , Inmunoglobulina A/análisis , Inmunoglobulina G/sangre , Ratones , Vacunas Combinadas/inmunologíaRESUMEN
Environmental enteric dysfunction (EED) is a gastrointestinal inflammatory disease caused by malnutrition and chronic infection. EED is associated with stunting in children and reduced efficacy of oral vaccines. To study the mechanisms of oral vaccine failure during EED, we developed a microbiota- and diet-dependent mouse EED model. Analysis of E. coli-labile toxin vaccine-specific CD4+ T cells in these mice revealed impaired CD4+ T cell responses in the small intestine and but not the lymph nodes. EED mice exhibited increased frequencies of small intestine-resident RORγT+FOXP3+ regulatory T (Treg) cells. Targeted deletion of RORγT from Treg cells restored small intestinal vaccine-specific CD4 T cell responses and vaccine-mediated protection upon challenge. However, ablation of RORγT+FOXP3+ Treg cells made mice more susceptible to EED-induced stunting. Our findings provide insight into the poor efficacy of oral vaccines in EED and highlight how RORγT+FOXP3+ Treg cells can regulate intestinal immunity while leaving systemic responses intact.
Asunto(s)
Toxinas Bacterianas/inmunología , Vacunas contra Escherichia coli/inmunología , Enfermedades Gastrointestinales/inmunología , Intestino Delgado/inmunología , Linfocitos T Reguladores/inmunología , Administración Oral , Animales , Línea Celular , Modelos Animales de Enfermedad , Drosophila , Escherichia coli/inmunología , Femenino , Factores de Transcripción Forkhead/metabolismo , Enfermedades Gastrointestinales/microbiología , Enfermedades Gastrointestinales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , VacunaciónRESUMEN
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain is known as one of the major human foodborne pathogens. Lack of effective clinical treatment for human diarrheal diseases confirms the need for vaccine production against enteric bacteria such as E.coli O157:H7. Shiga-like toxin (Stx), EscC, and Intimin are the main important virulent factors of this enteric pathogen. In the present study, a comparative Omics analysis was conducted to identify most invasion EHEC antigenic factors as a potential immunogen. SEI (Stx-EscC-Intimin) trivalent chimeric protein was designed from the exposed and epitope rich part of these virulence factors. Sequence optimization, physicochemical properties, mRNA folding, three-dimensional structure and immunoinformatics data were investigated. The chimeric gene was synthesized with codon bias of E. coli. Recombinant protein was expressed and confirmed by western blot analysis. To evaluate the immunogenicity of the designed protein, the protein was administered to BALB/c mice and the serum IgG was determined by ELISA. Based on the Ramachandran plot, the validation data showed that 90.1 % of residues lie in the favored region. The high antigenicity of the multimeric protein was predicted by the immunoinformatic analysis. Epitope prediction had shown the proper distribution of linear and conformational B-cell epitopes and the competition of T-cell epitopes to bind MHC molecules too. Recombinant ESI Protein with 74.5 kDa was expressed in E. coli. Western blot analysis by anti-Stx antibody, confirmed a single band of chimeric protein. Consequently, the chimeric gene was designed and constructed after assessments. From in silico approach, the protein deduced from this cassette can be an immunogen candidate, and act against toxicity and adherence of EHEC.
Asunto(s)
Adhesinas Bacterianas/inmunología , Infecciones por Escherichia coli , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/inmunología , Proteínas Recombinantes de Fusión/inmunología , Toxinas Shiga/inmunología , Sistemas de Secreción Tipo III/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Adhesión Bacteriana , Biología Computacional , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/prevención & control , Escherichia coli O157/inmunología , Femenino , Genes Bacterianos/inmunología , Ratones , Ratones Endogámicos BALB CRESUMEN
There are no vaccines licensed for enterotoxigenic Escherichia coli (ETEC), a leading cause of diarrhea for children in developing countries and international travelers. Virulence heterogeneity among strains and difficulties identifying safe antigens for protective antibodies against STa, a potent but poorly immunogenic heat-stable toxin which plays a key role in ETEC diarrhea, are challenges in ETEC vaccine development. To overcome these challenges, we applied a toxoid fusion strategy and a novel epitope- and structure-based multiepitope fusion antigen (MEFA) vaccinology platform to construct two chimeric multivalent proteins, toxoid fusion 3xSTaN12S-mnLTR192G/L211A and adhesin CFA/I/II/IV MEFA, and demonstrated that the proteins induced protective antibodies against STa and heat-labile toxin (LT) produced by all ETEC strains or the seven most important ETEC adhesins (CFA/I and CS1 to CS6) expressed by the ETEC strains causing 60 to 70% of diarrheal cases and moderate to severe cases. Combining two proteins, we prepared a protein-based multivalent ETEC vaccine, MecVax. MecVax was broadly immunogenic; mice and pigs intramuscularly immunized with MecVax developed no apparent adverse effects but had robust antibody responses to the target toxins and adhesins. Importantly, MecVax-induced antibodies were broadly protective, demonstrated by significant adherence inhibition against E. coli bacteria producing any of the seven adhesins and neutralization of STa and cholera toxin (CT) enterotoxicity. Moreover, MecVax protected against watery diarrhea and provided over 70% and 90% protection against any diarrhea from an STa-positive or an LT-positive ETEC strain in a pig challenge model. These results indicated that MecVax induces broadly protective antibodies and prevents diarrhea preclinically, signifying that MecVax is potentially an effective injectable vaccine for ETEC. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) bacteria are a top cause of children's diarrhea and travelers' diarrhea and are responsible for over 220 million diarrheal cases and more than 100,000 deaths annually. A safe and effective ETEC vaccine can significantly improve public health, particularly in developing countries. Data from this preclinical study showed that MecVax induces broadly protective antiadhesin and antitoxin antibodies, becoming the first ETEC vaccine candidate to induce protective antibodies inhibiting adherence of the seven most important ETEC adhesins and neutralizing the enterotoxicity of not only LT but also STa toxin. More importantly, MecVax is shown to protect against clinical diarrhea from STa-positive or LT-positive ETEC infection in a pig challenge model, recording protection from antibodies induced by the protein-based, injectable, subunit vaccine MecVax against ETEC diarrhea and perhaps the possibility of intramuscularly administered protein vaccines for protection against intestinal mucosal infection.
Asunto(s)
Diarrea/microbiología , Diarrea/prevención & control , Escherichia coli Enterotoxigénica/inmunología , Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Anticuerpos Neutralizantes/inmunología , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Diarrea/inmunología , Modelos Animales de Enfermedad , Epítopos/inmunología , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/efectos adversos , Ratones , Proteínas Recombinantes de Fusión/inmunología , Porcinos , Vacunas Combinadas/genética , Vacunas Combinadas/inmunologíaRESUMEN
Vaccines against enteric diseases could improve global health. Despite this, only a few oral vaccines are currently available for human use. One way to facilitate such vaccine development could be to identify a practical and relatively low cost biomarker assay to assess oral vaccine induced primary and memory IgA immune responses in humans. Such an IgA biomarker assay could complement antigen-specific immune response measurements, enabling more oral vaccine candidates to be tested, whilst also reducing the work and costs associated with early oral vaccine development. With this in mind, we take a holistic systems biology approach to compare the transcriptional signatures of peripheral blood mononuclear cells isolated from volunteers, who following two oral priming doses with the oral cholera vaccine Dukoral®, had either strong or no vaccine specific IgA responses. Using this bioinformatical method, we identify TNFRSF17, a gene encoding the B cell maturation antigen (BCMA), as a candidate biomarker of oral vaccine induced IgA immune responses. We then assess the ability of BCMA to reflect oral vaccine induced primary and memory IgA responses using an ELISA BCMA assay on a larger number of samples collected in clinical trials with Dukoral® and the oral enterotoxigenic Escherichia coli vaccine candidate ETVAX. We find significant correlations between levels of BCMA and vaccine antigen-specific IgA in antibodies in lymphocyte secretion (ALS) specimens, as well as with proportions of circulating plasmablasts detected by flow cytometry. Importantly, our results suggest that levels of BCMA detected early after primary mucosal vaccination may be a biomarker for induction of long-lived vaccine specific memory B cell responses, which are otherwise difficult to measure in clinical vaccine trials. In addition, we find that ALS-BCMA responses in individuals vaccinated with ETVAX plus the adjuvant double mutant heat-labile toxin (dmLT) are significantly higher than in subjects given ETVAX only. We therefore propose that as ALS-BCMA responses may reflect the total vaccine induced IgA responses to oral vaccination, this BCMA ELISA assay could also be used to estimate the total adjuvant effect on vaccine induced-antibody responses, independently of antigen specificity, further supporting the usefulness of the assay.
Asunto(s)
Antígeno de Maduración de Linfocitos B/genética , Vacunas contra el Cólera/administración & dosificación , Cólera/prevención & control , Escherichia coli Enterotoxigénica/inmunología , Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/administración & dosificación , Inmunidad Humoral/genética , Inmunoglobulina A/inmunología , Biología de Sistemas/métodos , Vacunación/métodos , Vibrio cholerae/inmunología , Administración Oral , Adulto , Linfocitos B/inmunología , Biomarcadores , Células Cultivadas , Cólera/microbiología , Vacunas contra el Cólera/inmunología , Infecciones por Escherichia coli/microbiología , Vacunas contra Escherichia coli/inmunología , Voluntarios Sanos , Humanos , Memoria Inmunológica , TranscriptomaRESUMEN
Extraintestinal pathogenic Escherichia coli (ExPEC) cause a wide range of clinical diseases such as bacteremia and urinary tract infections. The increase of multidrug resistant ExPEC strains is becoming a major concern for the treatment of these infections and E. coli has been identified as a critical priority pathogen by the WHO. Therefore, the development of vaccines has become increasingly important, with the surface lipopolysaccharide constituting a promising vaccine target. This study presents genetic and structural analysis of clinical urine isolates from Switzerland belonging to the serotype O25. Approximately 75% of these isolates were shown to correspond to the substructure O25B only recently described in an emerging clone of E. coli sequence type 131. To address the high occurrence of O25B in clinical isolates, an O25B glycoconjugate vaccine was prepared using an E. coli glycosylation system. The O antigen cluster was integrated into the genome of E. coli W3110, thereby generating an E. coli strain able to synthesize the O25B polysaccharide on a carrier lipid. The polysaccharide was enzymatically conjugated to specific asparagine side chains of the carrier protein exotoxin A (EPA) of Pseudomonas aeruginosa by the PglB oligosaccharyltransferase from Campylobacter jejuni. Detailed characterization of the O25B-EPA conjugate by use of physicochemical methods including NMR and GC-MS confirmed the O25B polysaccharide structure in the conjugate, opening up the possibility to develop a multivalent E. coli conjugate vaccine containing O25B-EPA.
Asunto(s)
Vacunas contra Escherichia coli/inmunología , Escherichia coli/clasificación , Glicoconjugados , Vacunas ConjugadasRESUMEN
Given the high frequency of urinary tract infections (UTIs) and their recurrence, there is keen interest in developing effective UTI vaccines. Currently, most vaccine studies, including those in humans, involve parenteral vaccination aimed at evoking and sustaining elevated levels of systemic antibody directed at the uropathogens. In view of recent reports of aberrant Th2-biased bladder immune responses to infection, we hypothesized that immunizing mice intravesically with antigens from uropathogenic Escherichia coli (UPEC) combined with a Th1-skewing adjuvant could correct this defect and promote protection against UTIs. Here we report that compared with mice immunized subcutaneously with this vaccine combination, intravesically immunized mice were markedly more protected from UTIs because of their distinctive ability to recruit Th1 cells into the bladder. This mode of vaccination was effective even in mice that experienced multiple UTIs and displayed pronounced aberrant bladder immune responses. Thus, intravesical vaccination with one or more UPEC antigens to induce bladder Th1 responses represents a superior strategy to combat UTIs, especially in UTI-prone subjects.
Asunto(s)
Infecciones por Escherichia coli , Vacunas contra Escherichia coli/farmacología , Células TH1/inmunología , Vejiga Urinaria/inmunología , Infecciones Urinarias , Escherichia coli Uropatógena/inmunología , Animales , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/inmunología , Ratones , Ratones Noqueados , Infecciones Urinarias/inmunología , Infecciones Urinarias/prevención & controlRESUMEN
Enterotoxigenic Escherichia coli (ETEC) is estimated to cause approximately 380,000 deaths annually during sporadic or epidemic outbreaks worldwide. Development of vaccines against ETEC is very challenging due to the vast heterogeneity of the ETEC strains. An effective vaccines would have to be multicomponent to provide coverage of over ten ETEC strains with genetic variabilities. There is currently no vaccine licensed to prevent ETEC. Nanobodies are successful new biologics in treating mucosal infectious disease as they recognize conserved epitopes on hypervariable pathogens. Cocktails consisting of multiple nanobodies could provide even broader epitope coverage at a lower cost compared to monoclonal antibodies. Identification of conserved epitopes by nanobodies can also assist reverse engineering of an effective vaccine against ETEC. By screening nanobodies from immunized llamas and a naïve yeast display library against adhesins of colonization factors, we identified single nanobodies that show cross-protective potency against eleven major pathogenic ETEC strains in vitro. Oral administration of nanobodies led to a significant reduction of bacterial colonization in animals. Moreover, nanobody-IgA fusion showed extended inhibitory activity in mouse colonization compared to commercial hyperimmune bovine colostrum product used for prevention of ETEC-induced diarrhea. Structural analysis revealed that nanobodies recognized a highly-conserved epitope within the putative receptor binding region of ETEC adhesins. Our findings support further rational design of a pan-ETEC vaccine to elicit robust immune responses targeting this conserved epitope.
Asunto(s)
Diarrea/prevención & control , Escherichia coli Enterotoxigénica/inmunología , Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/administración & dosificación , Anticuerpos de Dominio Único/administración & dosificación , Animales , Anticuerpos Antibacterianos/administración & dosificación , Anticuerpos Antibacterianos/inmunología , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/inmunología , Células CACO-2 , Camélidos del Nuevo Mundo , Protección Cruzada , Diarrea/inmunología , Diarrea/microbiología , Modelos Animales de Enfermedad , Diseño de Fármacos , Mapeo Epitopo , Epítopos/inmunología , Infecciones por Escherichia coli/inmunología , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/inmunología , Proteínas Fimbrias/antagonistas & inhibidores , Proteínas Fimbrias/inmunología , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/inmunología , Masculino , Ratones , Anticuerpos de Dominio Único/inmunologíaRESUMEN
Avian pathogenic Escherichia coli (APEC) O78 and Salmonella typhimurium (S. Typhimurium) are two leading bacterial pathogens that cause significant economic loss in the poultry industry. O-antigen is an important immunogen of these two bacteria to induce host protective immune responses during infection. To develop a bivalent vaccine against APEC O78 and S. Typhimurium, the attenuated Salmonella ST01 (Δasd ΔrfbP Δcrp) was genetically constructed to deliver APEC O78 O-antigen polysaccharide (OPS), which stably expresses OPS with asd+ balanced-lethal system in vitro and in vivo. After oral immunization, the recombinant attenuated Salmonella vaccine (RASV) strain ST01 (pSS26-O78) provided insufficient protection against the APEC O78 challenge. Therefore, the regulated delayed attenuation strain ST02 (Δasd ΔrfbP ΔPcrp::TTaraC PBADcrp) was further constructed by regulating cyclic AMP receptor protein (crp) with araC PBAD cassette to better present the heterologous O-antigen to the host immune system. The innovative recombinant strain ST02 (pSS26-O78) stimulated robust antibody responses against APEC O78 and S. Typhimurium OPS, with serum titers over 1:800 for both IgG and IgA, thereby providing the complement-mediated bactericidal activity and stronger protection against APEC O78 and S. Typhimurium infection. Collectively, this study demonstrates a biologically-conjugated polysaccharide vaccine candidate that can enhance homologous protection against APEC O78 and S. Typhimurium.
Asunto(s)
Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/inmunología , Escherichia coli/inmunología , Enfermedades de las Aves de Corral/prevención & control , Salmonelosis Animal/prevención & control , Vacunas contra la Salmonella/inmunología , Salmonella typhimurium/inmunología , Animales , Pollos/inmunología , Escherichia coli/patogenicidad , Inmunización , Enfermedades de las Aves de Corral/microbiología , Salmonella typhimurium/genética , Vacunas Atenuadas/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
Escherichia coli is the leading cause of severe mastitis in dairy farms. As E. coli mastitis is refractory to the hygienic control measures adapted to contagious mastitis, efficient vaccines are in demand. Existing mastitis vaccines, based on the use of killed rough E. coli J5 as the antigen, aim at inducing phagocytosis by neutrophils. We assessed the binding of J5-induced antibodies to isogenic rough and smooth strains along with a panel of mastitis-associated E. coli Analysis by enzyme-linked immunosorbent assay revealed that antibodies to OmpA or killed J5 bind readily to rough E. coli but poorly to smooth strains. Flow cytometry analysis indicated that immunization with J5 induced antibodies that cross-reacted with rough E. coli strains but with only a small subpopulation of smooth strains. We identified type 1 fimbriae as the target of most antibodies cross-reacting with the smooth strains. These results suggest that the O-polysaccharide of lipopolysaccharide shields the outer membrane antigens and that only fiber antigens protruding at the bacterial surface can elicit antibodies reacting with mastitis-associated E. coli We evaluated J5-induced antibodies in an opsonophagocytic killing assay with bovine neutrophils. J5 immune serum was not more efficient than preimmune serum, showing that immunization did not improve on the already high efficiency of naturally acquired antibodies to E. coli In conclusion, it is unlikely that the efficiency of J5 vaccines is related to the induction of opsonic antibodies. Consequently, other research directions, such as cell-mediated immunity, should be explored to improve E. coli mastitis vaccines.IMPORTANCE Despite intensive research, mastitis remains an important disease in dairy cattle with a significant impact on animal welfare, use of antibiotics, and, in the end, the economy of dairy farms. Although vaccines available so far have shown limited efficacy against coliform mastitis, vaccination is considered one of the measures that could limit the consequences of mastitis. One reason for the lack of efficiency of current vaccines likely stems from the current evaluation of vaccines that relies mostly on measuring antibody production against vaccine antigens. This report clearly shows that vaccine-induced antibodies fail to bind to most mastitis-associated E. coli strains because of the presence of an O-antigen and, thus, do not allow for improved phagocytosis of pathogens. As a consequence, this report calls for revised criteria for the evaluation of vaccines and suggests that cell-mediated immunity should be targeted by new vaccinal strategies. More generally, these results could be extended to other vaccine development strategies targeting coliform bacteria.