The immune response of pregnant sow after vaccination with crude fimbriae (F4) extracts vaccine and immunoprotection of nursery pig against pathogenic E. coli (F4+ETEC).

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.

Aerosol vaccination of chicken pullets with irradiated avian pathogenic Escherichia coli induces a local immunostimulatory effect.

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.

A multi-epitope fusion antigen candidate vaccine for Enterotoxigenic Escherichia coli is protective against strain B7A colonization in a rabbit model.

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.

Mucosal Immune Responses Against an Oral Enterotoxigenic Escherichia coli Vaccine Evaluated in Clinical Trials.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of mortality and morbidity in children in low-income countries. We have tested an oral ETEC vaccine, ETVAX, consisting of inactivated E coli overexpressing the most prevalent colonization factors and a toxoid, LCTBA, administered together with a mucosal adjuvant, double-mutant heat-labile toxin (dmLT), for capacity to induce mucosal immune responses and immunological memory against the primary vaccine antigens, ie, colonization factors, heat-labile toxin B-subunit and O antigen. The studies show that ETVAX could induce strong intestine-derived and/or fecal immune responses in a majority of vaccinated Swedish adults and in different age groups, including infants, in Bangladesh.

A Vaccine to Prevent Egg Layer Peritonitis in Chickens.

A series of studies was undertaken in specific-pathogen-free white leghorn chickens for the development of a chicken model of avian pathogenic Escherichia coli (APEC) peritonitis. Once established, this model was then used to measure the effectiveness of a siderophore receptor and porin proteins (SRP®) APEC vaccine. Initially, five pilot studies were performed to compare the E. coli serotype, challenge route, and dose of inoculum that resulted in pathologies characteristic of the peritonitis observed in commercial layer facilities, such as widespread organ infection, atrophy, discoloration, corrugation of yolk sacs, and the presence of caseous exudate. Isolates of serotypes O1, O2, and O78 were tested by intravenous, intravaginal, intratracheal, and intraperitoneal routes and were compared at various levels of challenge inoculum. Daily observations of mortality and morbidity were made, and at necropsy, gross lesion scores were collected and bacterial colonization of internal organs determined. Outcomes varied from a complete lack of mortality or detectable pathology and low, or no, organ colonization in the case of intravaginal and intratracheal routes with each E. coli serotype to moderate to high levels of mortality, pathology, and colonization after challenge via the intravenous and intraperitoneal routes with O2 and O78 serotypes, respectively. The O78 serotype was found to result in pathologies consistent with field observations of peritonitis, and therefore, subsequent studies were performed only with O78. In addition to the relative failure with both the intratracheal and intravaginal routes of challenge, the intravenous route was found to be inconsistent and often resulted in lameness not observed with the intraperitoneal route. A final pilot study confirmed that the dose (∼ 8 log 10 CFU) administered by the intraperitoneal route replicated peritonitis, and therefore, all vaccination/challenge studies were conducted in this manner. Five vaccination/challenge studies are reported here in which variables of chicken age, vaccination interval, and vaccination to challenge interval were examined. In all studies, vaccine effectiveness was dramatic and was shown to completely protect against mortality and substantially against tissue colonization and pathology typical of APEC infections. The vaccine elicited a rapid onset of immunity with both narrow and broad vaccination intervals and in both young and mature chickens. Additionally, the vaccine was demonstrated to sustain robust effectiveness against mortality over 3 months. The SRP APEC vaccine should provide effective protection of young and mature chickens from E. coli under broadly flexible conditions of use in commercial operations.

Artíclo regular­Vacuna para prevenir la peritonitis de gallina de postura. Se llevó a cabo una serie de estudios en aves tipo Leghorn blancas libres de patógenos específicos para el desarrollo de un modelo en pollo para la peritonitis causada por Escherichia coli patógena aviar (APEC). Una vez establecido, este modelo se utilizó para medir la eficacia de una vacuna con proteínas del receptor de sideróforo y de porina (SRP®) de E. coli patógena aviar. Inicialmente, se realizaron cinco estudios piloto para comparar el serotipo de E. coli, la ruta de desafío y la dosis de inóculo que resultaron en patologías características de las peritonitis observadas en instalaciones comerciales de ponedoras, como la infección generalizada de órganos, atrofia, decoloración, ondulación de saco vitelino y la presencia de exudado caseoso. Los aislamientos de los serotipos O1, O2 y O78 se analizaron por vías intravenosa, intravaginal, intratraqueal e intraperitoneal y se compararon a varios niveles de inóculo de desafío. Se realizaron observaciones diarias de mortalidad y morbilidad, en la necropsia, se registraron puntuaciones de lesiones macroscópicas y se determinó la colonización bacteriana de los órganos internos. Los resultados variaron desde una ausencia total de mortalidad o patología detectable y una colonización de órganos baja o nula en el caso de las rutas intravaginal e intratraqueal con cada serotipo de E. coli hasta niveles de mortalidad, patología y colonización de moderados a altos después del desafío por vía intravenosa e intraperitoneal con los serotipos O2 y O78, respectivamente. Se encontró que el serotipo O78 dio como resultado patologías consistentes con las observaciones de campo de la peritonitis y por lo tanto, los estudios posteriores se realizaron solo con el serotipo O78. Además del fracaso relativo con las rutas de desafío intratraqueal e intravaginal, se descubrió que la vía intravenosa era inconsistente y a menudo, provocaba cojera que no se observaba con la vía intraperitoneal. Un estudio piloto final confirmó que la dosis (∼8 log10 UFC) administrada por vía intraperitoneal reproducía la peritonitis y por lo tanto, todos los estudios de vacunación/desafío se realizaron de esta manera. En este estudio se reportan cinco estudios de vacunación/desafío en los que se examinaron las variables de edad del pollo, intervalo de vacunación e intervalo de vacunación al desafío. En todos los estudios, la eficacia de la vacuna fue muy evidente y se demostró que protege completamente contra la mortalidad y sustancialmente contra la colonización de tejidos y la patología típica de las infecciones por E. coli patógena aviar. La vacuna provocó un rápido inicio de la inmunidad con intervalos de vacunación tanto estrechos como amplios y tanto en aves jóvenes como maduras. Además, se demostró que la vacuna mantiene una sólida eficacia contra la mortalidad durante tres meses. La vacuna con proteínas del receptor de sideróforo y de porina de E. coli patógena aviar debería proporcionar una protección eficaz de las aves jóvenes y maduras contra E. coli en condiciones de uso ampliamente flexibles en operaciones comerciales.

A Systems Biology Approach Identifies B Cell Maturation Antigen (BCMA) as a Biomarker Reflecting Oral Vaccine Induced IgA Antibody Responses in Humans.

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.

Preclinical Characterization of Immunogenicity and Efficacy against Diarrhea from MecVax, a Multivalent Enterotoxigenic E. coli Vaccine Candidate.

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.

Anti-CfaE nanobodies provide broad cross-protection against major pathogenic enterotoxigenic Escherichia coli strains, with implications for vaccine design.

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.

High efficiency biosynthesis of O-polysaccharide-based vaccines against extraintestinal pathogenic Escherichia coli.

Extraintestinal pathogenic Escherichia coli (ExPEC) has presented a major clinical infection emerged in the past decades. O-polysaccharide (OPS)-based glycoconjugate vaccines produced using the bacterial glycosylation machinery can be utilized to confer protection against such infection. However, constructing a low-cost microbial cell factory for high-efficient production of OPS-based glycoconjugate vaccines remains challenging. Here, we engineered a glyco-optimized chassis strain by reprogramming metabolic network. The yield was enhanced to 38.6 mg L-1, the highest level reported so far. MS analysis showed that designed glycosylation sequon was modified by target polysaccharide with high glycosylation efficiency of 90.7 % and 76.7 % for CTB-O5 and CTB-O7, respectively. The glycoconjugate vaccines purified from this biosystem elicited a marked increase in protection against ExPEC infection in mouse model, compared to a non-optimized system. The glyco-optimized platform established here is broadly suitable for polysaccharide-based conjugate production against ExPEC and other surface-polysaccharide-producing pathogens.

Potential improvement of the immune response of chickens against E. coli vaccine by using two forms of chitosan nanoparticles.

Colibacillosis disease has an important economic impact on poultry production worldwide. It is one of the most common causes of mortality in commercial layer and breeder chickens. Avian pathogenic Escherichia coli (APEC) is the main cause of this disease. Nanoparticles have been widely used in vaccine design as both adjuvants and antigen delivery vehicles. The present study aimed to produce an efficient vaccine from E. coli serogroups O1 and O78 to help in controlling colibacillosis in chicken using two forms of chitosan (CS) and ascorbate chitosan (AsCS) nanoparticles. Nanovaccines has been prepared through loading and encapsulation of outer membrane and flagellar antigen on CS and AsCS nanoparticles with loading efficiency 86, 63,55, 48% for CS-loaded-, Cs-capsulated-, AsCS-loaded- and AsCS-capsulated-E. coli Antigen, respectively. Two hundred specific pathogens free (SPF) 3-weeks old broiler chickens were used and divided into four groups to investigate the immune response of nanovaccines. The immune response was measured by the microagglutination, ELISA, and challenge test. From results, it could be concluded that generally adding chitosan NPs is capable of improving vaccine efficacy via the induction of strong immunity. Moreover, we recommend the production of the nanovaccine CS-capsulated -antigen from E. coli O1 and O78 serotypes to be used as a potent vaccine to aid in controlling colibacillosis. Also, the ascorbate chitosan is a great alternate for the initiation of a potent immune response in critical infection cases.

Short communication: J-5 Escherichia coli vaccination does not influence severity of an Escherichia coli intramammary challenge in primiparous cows.

The objective of the present study was to evaluate the efficacy of a J-5 Escherichia coli vaccine in a mild to moderate inflammatory challenge model using primiparous dairy cows for inoculation only. We hypothesized a clinical difference between placebo and J-5 E. coli vaccinated animals with the mild to moderate inflammatory challenge model. In case the null hypothesis could not be confirmed, the alternate hypothesis was no clinical difference between both treatment groups. Therefore, 23 primiparous cows in mo 7 of pregnancy were randomly assigned to 1 of 2 treatment groups: J-5 E. coli vaccine (n = 12) or placebo (n = 11). Animals were vaccinated 3 times at 56 (±7) and 28 (±7) d before expected calving date and within 14 d postcalving (DIM 5 ± 3). All cows were challenged by infusion with E. coli P4:O32 into 2 left mammary quarters between 14 and 28 d postparturition, at least 10 d after the 3rd vaccination, immediately after the morning milking. Clinical observations and blood and milk samples were collected at several time points from 7 d pre-challenge until 13 d post-challenge. Primiparous cows responded mild to moderately to intramammary E. coli challenge with little clinical difference between treatment groups. Rectal temperature increased earlier in the vaccinated animals, which also eliminated bacteria faster during the early hours after intramammary E. coli challenge. At post-infusion hour 9, the bacterial population was significantly lower in the infected quarters of the vaccinated animals. Blood leukocyte number was only numerically higher in the vaccinated animals, in combination with a numerically higher percentage of late immature polymorphonuclear leukocytes (band cells) in circulation. Even in the nonvaccinated animals, the E. coli challenge in the primiparous cows elicited only a mild to moderate response. The absence of a pronounced clinical difference between vaccinated and nonvaccinated animals was therefore not surprising.

Immunogenicity noninferiority study of 2 doses and 3 doses of an Escherichia coli-produced HPV bivalent vaccine in girls vs. 3 doses in young women.

A new HPV-16/18 bivalent vaccine expressed by the Escherichia coli has been proven to be efficacious in adult women. A randomized, immunogenicity noninferiority study of this candidate vaccine was conducted in December 2015 in China. Girls aged 9-14 years were randomized to receive 2 doses at months 0 and 6 (n=301) or 3 doses at months 0, 1 and 6 (n=304). Girls aged 15-17 years (n=149) and women aged 18-26 years (n=225) received 3 doses. The objectives included noninferiority analysis of the IgG geometric mean concentration (GMC) ratio (95% CI, lower bound>0.5) to HPV-16 and HPV-18 at month 7 in girls compared with women. In the per-protocol set, the GMC ratio of IgG was noninferior for girls aged 9-17 years receiving 3 doses compared with women (1.76 (95% CI, 1.56, 1.99) for HPV-16 and 1.93 (95% CI, 1.69, 2.21) for HPV-18) and noninferior for girls aged 9-14 years receiving 2 doses compared with women (1.45 (95% CI, 1.25, 1.62) for HPV-16 and 1.17 (95% CI, 1.02, 1.33) for HPV-18). Noninferiority was also demonstrated for neutralizing antibodies. The immunogenicity of the HPV vaccine in girls receiving 3 or 2 doses was noninferior compared with that in young adult women.

Preformulation Characterization and Stability Assessments of Secretory IgA Monoclonal Antibodies as Potential Candidates for Passive Immunization by Oral Administration.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal disease among children in developing countries, and there are no licensed vaccines to protect against ETEC. Passive immunization by oral delivery of ETEC-specific secretory IgAs (sIgAs) could potentially provide an alternative approach for protection in targeted populations. In this study, a series of physiochemical techniques and an in vitro gastric digestion model were used to characterize and compare key structural attributes and stability profiles of 3 anti-heat-labile enterotoxin mAbs (sIgA1, sIgA2, and IgG1 produced in CHO cells). The mAbs were evaluated in terms of primary structure, N-linked glycan profiles, size and aggregate content, relative apparent solubility, conformational stability, and in vitro antigen binding. Compared to IgG1 mAb, sIgA1 and sIgA2 mAbs showed increased sample heterogeneity, especially in terms of N-glycan composition and the presence of higher molecular weight species. The sIgA mAbs showed overall better physical stability and were more resistant to loss of antigen binding activity during incubation at low pH, 37°C with pepsin. These results are discussed in terms of future challenges to design stable, low-cost formulations of sIgA mAbs as an oral supplement for passive immunization to protect against enteric diseases in the developing world.

Effects of the Probiotic Candidate E. faecalis-1, the Poulvac E. coli Vaccine, and their Combination on Growth Performance, Caecal Microbial Composition, Immune Response, and Protection against E. coli O78 Challenge in Broiler Chickens.

The present study was performed on 180-day-old commercial Cobb chicks to assess the effects of the probiotic candidate Enterococcus faecalis-1, the Poulvac Escherichia coli vaccine, and their combination on growth parameters, intestinal microbial composition, immune response, and protection against challenge with the avian pathogen E. coli O78. The experimental groups were as follows: G1, basal diet; G2, basal diet and challenge with O78 at 28 days of growth; G3, basal diet, vaccination with Poulvac (1 and 15 days), and challenge with O78 at 28 days of growth; G4, basal diet, E. faecalis-1 supplementation for the first 3 days of growth, and challenge with O78 at 28 days of growth; G5, basal diet, E. faecalis-1 supplementation for the first 3 days of growth, vaccination with Poulvac (1 and 15 days), and challenge with O78 at 28 days of growth; G6, basal diet and E. faecalis-1 supplementation for the first 3 days of growth. The results showed that E. faecalis-1 in drinking water significantly improved the growth performance and immune response, increased the total Enterococcus counts, reduced the mortality, and decreased the visceral invasion by O78 in challenged broilers. While the effect of the Poulvac vaccine alone or with E. faecalis-1 was not significant compared with that of the E. faecalis-1 supplement, the vaccine improved the growth rate and decreased the mortality and visceral invasion by APEC O78 in challenged broilers. These results showed that E. faecalis-1 supplementation and routine vaccination with the Poulvac vaccine could improve the growth performance and immune response of broiler chickens and protect against challenge with APEC O78.

A new human challenge model for testing heat-stable toxin-based vaccine candidates for enterotoxigenic Escherichia coli diarrhea - dose optimization, clinical outcomes, and CD4+ T cell responses.

TRIAL REGISTRATION: ClinicalTrials.gov ClinicalTrials.gov, Project ID: NCT02870751.

The effect of J5 bacterins on clinical, behavioral, and antibody response following an Escherichia coli intramammary challenge in dairy cows at peak lactation.

Vaccination against coliform mastitis has become part of mastitis control programs in the past 3 decades, as a means of reducing the severity of clinical mastitis. Our study objective was to evaluate the effect of 2 commercially available vaccines on clinical, behavioral, and antibody response following Escherichia coli intramammary challenge in cows near peak lactation. Cows (n = 12 per group) were vaccinated with vaccine 1 (V1) or vaccine 2 (V2) at dry-off, 21 d pre-calving, and 14 d post-calving. Twelve cows served as unvaccinated controls (CTL). Cows were challenged with E. coli in a rear quarter at approximately 100 d in milk. Milk samples were collected pre- and post-challenge to enumerate E. coli and determine somatic cell count. Serum was collected before each vaccination and at d 0, 1, 2, 3, 6, 30, and 60 relative to challenge, to study antibody response. Milk IgA and tumor necrosis factor-α concentrations were determined in whey. Vaginal temperature, cow activity, and milk yield and components were monitored post-challenge. Bacterial count, somatic cell score, milk yield and component decline, vaginal temperature, activity measures, and antibody and cytokine response were analyzed for treatment differences. The effects of parity, breed, and a repeated measure of time were also tested. Seven cows had to be removed from the study post-challenge for antibiotic treatment (CTL and V1, n = 3 each; V2, n = 1), 2 of which were euthanized (both CTL). Vaccinated cows exhibited fever (vaginal temperature ≥39.4°C) 3 h earlier than CTL cows, but we found no differences between treatments for bacterial count, somatic cell score, or milk yield reduction. Vaccinated cows spent more time lying per rest bout 2 d post-challenge, but total daily lying time was not different from CTL cows during the 7 d post-challenge. The vaccines differed in antibody response: V1 cows had greater serum IgG1 and IgG2 post-challenge. A parity effect was also evident: primiparous cows had lower bacterial counts, somatic cell score and a smaller milk yield decline than multiparous cows, but also had lower antibody production. Immunization with either J5 bacterin did not reduce clinical signs of mastitis in cows challenged at 100 d in milk, demonstrating that the effects of J5 vaccination had diminished at peak lactation.

Virus-like particle-display of the enterotoxigenic Escherichia coli heat-stable toxoid STh-A14T elicits neutralizing antibodies in mice.

Enterotoxigenic Escherichia coli (ETEC) causes diarrhoea by secreting enterotoxins into the small intestine. Human ETEC strains may secrete any combination of three enterotoxins: the heat-labile toxin (LT) and the heat-stable toxins (ST), of which there are two variants, called human ST (STh) and porcine ST (STp). Strains expressing STh, either alone or in combination with LT and/or STp, are among the four most important diarrhoea-causing pathogens affecting children in low- and middle-income countries. ST is therefore an attractive target for ETEC vaccine development. To produce a safe ST-based vaccine, several challenges must be solved. ST must be rendered immunogenic and non-toxic, and antibodies elicited by an ST vaccine should neutralize ST but not cross-react with the endogenous ligands uroguanylin and guanylin. Virus-like particles (VLPs) tend to be highly immunogenic and are increasingly being used as carriers for presenting heterologous antigens in new vaccines. In this study, we have coupled native STh and the STh-A14T toxoid to the coat protein of Acinetobacter phage AP205 by using the SpyCatcher system and immunized mice with these VLPs without the use of adjuvants. We found that both STs were efficiently coupled to the VLP, that both the STh and STh-A14T VLPs were immunogenic in mice, and that the resulting serum antibodies could completely neutralize the toxic activities of native STh. The serum antibodies showed a high degree of immunological cross-reaction to STp, while there was little or no unwanted cross-reaction to uroguanylin and guanylin. Moreover, compared to native STh, the STh-A14T mutation did not seem to negatively impact the immunogenicity of the construct or the neutralizing ability of the resulting sera. Taken together, these findings demonstrate that VLPs are suitable carriers for making STs immunogenic, and that the STh-A14T-coupled AP205 VLP represents a promising ETEC vaccine candidate.

Development of a Gold Nanoparticle Vaccine against Enterohemorrhagic Escherichia coli O157:H7.

Here we exploit the natural properties of a synthetic nanoparticle (NP) scaffold as a subunit vaccine against enterohemorrhagic Escherichiacoli (EHEC). Two EHEC-specific immunogenic antigens, namely, LomW and EscC, either alone or in combination, were covalently linked on the surface of gold nanoparticles (AuNPs) and used to immunize mice prior to challenge with EHEC O157:H7 strain 86-24. LomW is a putative outer membrane protein encoded in bacteriophage BP-933W, while EscC is a structural type III secretion system protein which forms a ring in the outer membrane. The resulting AuNP preparations, AuNP-LomW and AuNP-EscC, showed that the nanoparticles were able to incorporate the antigens, forming stable formulations that retained robust immunogenicity in vivo after subcutaneous immunization. When administered subcutaneously, AuNP-LomW or AuNP-EscC or a combination containing equivalent amounts of both candidates resulted in higher IgG titers in serum and secretory IgA titers in feces. The serum IgG titers correlated with a significant reduction in EHEC intestinal colonization after 3 days postinoculation. In addition, we showed that serum from antigen-coated AuNP-immunized mice resulted in a reduction of adherence to human intestinal epithelial cells for EHEC, as well as for two other E. coli pathotypes (enteropathogenic E. coli [EPEC], encoding EscC, and enteroaggregative E. coli [EAEC], encoding LomW). Further, the serum had antigen-specific bactericidal properties, engaging the classical complement pathway. Overall, our results demonstrate the immunogenicity and stability of a novel nanovaccine against EHEC. These results also strengthen the prospect of development of a synthetic nanoparticle vaccine conjugated to E. coli antigens as a promising platform against other enteric pathogens.IMPORTANCE Enterohemorrhagic E. coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis, which can progress to hemolytic uremic syndrome. These complications represent a serious global public health problem that requires laborious public health interventions and safety control measures to combat recurrent outbreaks worldwide. Today, there are no effective interventions for the control of EHEC infections, and, in fact, the use of antibiotics is counterindicated for EHEC disease. Therefore, a viable alternative for the prevention of human infections is the development of vaccines; however, no such vaccines are approved for human use. In this study, we developed a novel gold nanoparticle platform which acts as a scaffold for the delivery of various antigens, representing a nanovaccine technology which can be applied to several disease models.

Vaccines Against Shigella and Enterotoxigenic Escherichia coli: A summary of the 2018 VASE Conference.

PATH hosted the second Vaccines Against Shigella and Enterotoxigenic Escherichia coli (VASE) Conference in Mexico City in June 2018, again providing experts from around the world an opportunity to participate in a highly collaborative forum to discuss progress in the development of new enteric vaccines. Through a combination of plenary sessions and posters, keynote presentations, and workshops, the 2018 VASE Conference aimed to accelerate communication and progress among those working to achieve the goal of licensed vaccines against these two bacterial pathogens. Many presentations recognized the importance of diarrheal disease and long-term sequelae caused by infections with Shigella and enterotoxigenic E. coli (ETEC). Other presentations explored new strategies for vaccine development, including the search for novel, possibly conserved, antigens for more effective vaccines. Much progress is being made as some vaccine candidates are now moving through clinical trials. Research presented in oral and poster presentations at the VASE Conference covered a range of topics, including: the global burden of disease, epidemiology, and health economics; host parameters and genomics that predict responses to infection and disease; preclinical evaluations of vaccine antigens and models of enteric diseases; and vaccine candidates in clinical trials and human challenge studies. This article reviews key points and highlighted research presented in each of the plenary conference sessions and poster presentations at the 2018 VASE Conference.