A polyvalent multiepitope protein cross-protects against Vibrio cholerae infection in rabbit colonization and passive protection models.

Using epitope- and structure-based multiepitope fusion antigen vaccinology platform, we constructed a polyvalent protein immunogen that presents antigenic domains (epitopes) of Vibrio cholerae toxin-coregulated pilus A, cholera toxin (CT), sialidase, hemolysin A, flagellins (B, C, and D), and peptides mimicking lipopolysaccharide O-antigen on a flagellin B backbone. Mice and rabbits immunized intramuscularly with this polyvalent protein immunogen developed antibodies to all of the virulence factors targeted by the immunogen except lipopolysaccharide. Mouse and rabbit antibodies exhibited functional activities against CT enterotoxicity, CT binding to GM1 ganglioside, bacterial motility, and in vitro adherence of V. cholerae O1, O139, and non-O1/non-O139 serogroup strains. When challenged orogastrically with V. cholerae O1 El Tor N16961 or a non-O1/non-O139 strain, rabbits IM immunized with the immunogen showed a 2-log (99%) reduction in V. cholerae colonization of small intestines. Moreover, infant rabbits born to the mother immunized with the protein immunogen acquired antibodies passively and were protected from bacterial intestinal colonization (>2-log reduction), severe diarrhea (100%), and mild diarrhea (88%) after infection with V. cholerae O1 El Tor (N16961), O1 classical (O395), O139 (Bengal), or a non-O1/non-O139 strain. This study demonstrated that this polyvalent cholera protein is broadly immunogenic and cross-protective, and an adult rabbit colonization model and an infant rabbit passive protection model fill a gap in preclinical efficacy assessment in cholera vaccine development.

Evaluation of Multivalent Enterotoxigenic Escherichia coli Vaccine Candidate MecVax Antigen Dose-Dependent Effect in a Murine Model.

There are no licensed vaccines against enterotoxigenic Escherichia coli (ETEC), a leading cause of children's diarrhea and travelers' diarrhea. Recently, protein-based vaccine candidate MecVax was demonstrated to induce functional antibodies against both ETEC toxins (heat-stable toxin [STa] and heat-labile toxin [LT]) and seven ETEC adhesins (CFA/I and CS1 to CS6) and to protect against ETEC clinical diarrhea or intestinal colonization preclinically. Those studies used intraperitoneal, intramuscular, and intradermal routes, and a dose range for MecVax protein antigens, toxoid fusion 3xSTaN12S-mnLTR192G/L211A, and adhesin CFA/I/II/IV MEFA has not been investigated. Here, we further characterized MecVax broad immunogenicity, utilizing a subcutaneous route, and examined vaccine dose-dependent antibody response effects and also antibody functional activities against ETEC enterotoxicity and bacterial adherence. Data showed that mice immunized subcutaneously with MecVax developed robust IgG responses to seven ETEC adhesins (CFA/I, as well as CS1 to CS6) and two toxins (STa and LT). At a subcutaneous dose of 25, 20, or 10 µg or at an intramuscular dose of 12, 6, or 3 µg, MecVax induced similar levels IgG responses to the targeted toxins and adhesins, and these antibodies exhibited equivalent functional activities against ETEC toxin enterotoxicity and bacterial adherence. Once the intramuscular dose was decreased to 1 µg, vaccine-induced antibodies were significantly reduced and no longer neutralized STa enterotoxicity. The results indicated that MecVax administered subcutaneously is broadly immunogenic and, at an intramuscular dose of 3 µg, can induce functional antitoxin and anti-adhesin antibodies in mice, providing instructive information for future vaccine dose studies in humans and accelerating MecVax vaccine development. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) is a leading cause of children's diarrhea and the most common cause of travelers' diarrhea. ETEC infections are responsible for >200 million diarrhea clinical cases and near 100,000 deaths annually. Currently, there are no licensed vaccines for ETEC diarrhea. The protein-based vaccine candidate MecVax unprecedentedly targets two ETEC toxins (STa and LT, produced by all ETEC strains) and seven ETEC adhesins (CFA/I, as well as CS1 to CS6, associated with >60% of ETEC clinical diarrhea cases) and has been demonstrated to be broadly immunogenic and cross protective; as such, it represents a potentially effective multivalent vaccine against ETEC-associated children's and travelers' diarrhea. This study further confirmed MecVax broad immunogenicity and evaluated the vaccine antigen dose effect on the induction of antigen-specific antibody responses in mice and on antibody functional activities against ETEC toxin enterotoxicity and bacterial adherence, yielding useful information for future human volunteer studies and the development of MecVax as an effective ETEC vaccine.

Intradermally Administered Enterotoxigenic Escherichia coli Vaccine Candidate MecVax Induces Functional Serum Immunoglobulin G Antibodies against Seven Adhesins (CFA/I and CS1 through CS6) and Both Toxins (STa and LT).

There are no vaccines licensed for enterotoxigenic Escherichia coli (ETEC), a leading bacterial cause of children's diarrhea and travelers' diarrhea. MecVax, a multivalent E. coli vaccine candidate composed of two epitope- and structure-based polyvalent proteins (toxoid fusion 3xSTaN12S-mnLTR192G/L211A and colonization factor antigen [CFA]/I/II/IV multiepitope fusion antigen [MEFA]), is designed to induce broad antiadhesin and antitoxin antibodies against heterogeneous ETEC pathovars. When administered intraperitoneally or intramuscularly, MecVax was shown to induce antibodies against seven ETEC adhesins (CFA/I and CS1 to CS6) produced by ETEC pathovars that cause over 60% of ETEC-associated diarrheal cases and moderate-to-severe cases and both toxins (heat-labile toxin [LT] and heat-stable toxin [STa]) expressed by all ETEC strains. To further characterize the immunogenicity of this protein-based injectable subunit vaccine candidate and to explore other parenteral administration routes for the product, in this study we immunized mice intradermally (i.d.) with MecVax and measured antigen-specific antibody responses and further antibody functional activities against the adhesins and toxins targeted by the vaccine. Data showed that mice immunized i.d. with MecVax developed robust anti-CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, LT and anti-STa IgG responses. Furthermore, antibodies derived from MecVax administered via the i.d. route inhibited the adherence of ETEC or E. coli strains expressing any of the seven target adhesins (CFA/I and CS1 to CS6) and neutralized the enterotoxicity of LT and STa. These results confirmed broad immunogenicity of MecVax and suggested that this multivalent ETEC subunit vaccine candidate can be effectively delivered via the i.d. route. IMPORTANCE ETEC is a leading bacterial cause of diarrhea in children living in developing countries and international travelers. Developing an effective vaccine for ETEC diarrhea has been hampered because of the challenges of virulence heterogeneity and the difficulties of inducing neutralizing antibodies against the key toxin STa. MecVax, a subunit vaccine candidate carrying two polyvalent protein antigens, for the first time induces functional antibodies against the most important ETEC adhesins, which are associated with a majority of diarrheal cases and moderate-to-severe cases, and also against the enterotoxicity of LT and more importantly STa, which plays a key role in children's diarrhea and travelers' diarrhea, potentially leading to the development of a truly effective ETEC vaccine. Data from this study may also indicate that this ETEC subunit vaccine can be administered effectively via the i.d. route, expanding clinical administration options for this vaccine product.

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.

Neutralizing Anti-Heat-Stable Toxin (STa) Antibodies Derived from Enterotoxigenic Escherichia coli Toxoid Fusions with STa Proteins Containing N12S, L9A/N12S, or N12S/A14T Mutations Show Little Cross-Reactivity with Guanylin or Uroguanylin.

Heat-stable toxin (STa)-producing enterotoxigenic Escherichia coli (ETEC) strains are a top cause of moderate-to-severe diarrhea in children from developing countries and a common cause of travelers' diarrhea. Recent progress in using STa toxoids and toxoid fusions to induce neutralizing anti-STa antibodies has accelerated ETEC vaccine development. However, concern remains regarding whether the derived anti-STa antibodies cross-react with STa-like guanylin and uroguanylin, two guanylate cyclase C (GC-C) ligands regulating fluid and electrolyte transportation in human intestinal and renal epithelial cells. To further divert STa from guanylin and uroguanylin structurally and antigenically and to eliminate anti-STa antibody cross-reactivity with guanylin and uroguanylin, we mutated STa at the 9th (leucine), 12th (asparagine), and 14th (alanine) residues for the double and triple mutants STaL9A/N12S, STaL9A/A14H, STaN12S/A14T, and STaL9A/N12S/A14H We then fused each STa mutant (three copies) to a monomeric heat-labile toxin (LT) mutant (mnLTR192G/L211A) for the toxoid fusions 3×STaL9A/N12S-mnLTR192G/L211A, 3×STaL9A/A14H-mnLTR192G/L211A, 3×STaN12S/A14T-mnLTR192G/L211A, and 3×STaL9A/N12S/A14H-mnLTR192G/L211A; examined each fusion for anti-STa immunogenicity; and assessed the derived antibodies for in vitro neutralization activity against STa toxicity and for cross-reactivity with guanylin and uroguanylin. Mice subcutaneously immunized with each fusion protein developed anti-STa antibodies, and the antibodies derived from 3×STaN12S-mnLTR192G/L211A, 3×STaL9A/N12S-mnLTR192G/L211A, or 3×STaN12S/A14T-mnLTR192G/L211A prevented STa from the stimulation of intracellular cGMP in T-84 cells. Competitive enzyme-linked immunosorbent assays (ELISAs) showed that guanylin and uroguanylin hardly blocked the binding of anti-STa antibodies to the coated STa-ovalbumin conjugate. These results indicated that antibodies derived from 3×STaN12S-mnLTR192G/L211A, 3×STaL9A/N12S-mnLTR192G/L211A, or 3×STaN12S/A14T-mnLTR192G/L211A neutralized STa and had little cross-reactivity with guanylin and uroguanylin, suggesting that these toxoid fusions are suitable antigens for ETEC vaccines.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children's diarrhea and travelers' diarrhea. Currently, there is no licensed vaccine against ETEC diarrhea. One key challenge is to identify safe antigens to induce antibodies neutralizing the key STa without cross-reacting with guanylin and uroguanylin, two important ligands controlling homeostasis in human intestinal and renal epithelial cells. In this study, we generated nontoxic fusion antigens that induced antibodies that neutralize STa enterotoxicity in vitro and do not cross-react with guanylin or uroguanylin. These fusions have become the preferred antigens for the development of ETEC vaccines to potentially prevent the deaths of hundreds of thousands of young children and hundreds of millions of diarrheal cases each year.

Antimicrobial susceptibility profiles and molecular typing of Campylobacter jejuni and Campylobacter coli isolates from ducks in South Korea.

Campylobacter is a food-borne zoonotic pathogen that causes human gastroenteritis worldwide. Campylobacter bacteria are commensal in the intestines of many food production animals, including ducks and chickens. The objective of the study was to determine the prevalence of Campylobacter species in domestic ducks, and the agar dilution method was used to determine resistance of the isolates to eight antibiotics. In addition, multilocus sequence typing (MLST) was performed to determine the sequence types (STs) of selected Campylobacter isolates. Between May and September 2012, 58 duck farms were analyzed, and 56 (96.6%) were positive for Campylobacter. Among the isolates, 82.1% were Campylobacter jejuni, 16.1% were C. coli, and one was unidentified by PCR. Of the 46 C. jejuni isolates, 87.0%, 10.9%, and 21.7% were resistant to ciprofloxacin, erythromycin, and azithromycin, respectively. Among the C. coli isolates, all 9 strains were resistant to ampicillin, and 77.8% and 33.3% were resistant to ciprofloxacin and azithromycin, respectively. The majority of the Campylobacter isolates were classified as multidrug resistant. Twenty-eight STs were identified, including 20 STs for C. jejuni and 8 STs for C. coli. The most common clonal complexes in C. jejuni were the ST-21 complex and the ST-45 complex, while the ST-828 complex predominated in C. coli. The majority of isolates were of STs noted in ducks and humans from earlier studies, along with seven STs previously associated only with human disease. These STs overlapped between duck and human isolates, indicating that Campylobacter isolates from ducks should be considered potential sources of human infection.

Chicken embryo lethality assay for determining the virulence of Riemerella anatipestifer isolates.

Riemerella anatipestifer is the causative agent of polyserositis and septicaemia in waterfowl. Twenty-one serotypes have been reported, and there is a strong variation in virulence between strains according to serotype or strain. However, little information is available to assess virulence, such as virulence-associated genes; thus, it is difficult to estimate the risk from field strains. Hence, we established a chicken embryo lethality assay (ELA) model to determine the virulence of R. anatipestifer strains. Three virulent strains (RA T1, RA T7, and V-1) and three avirulent strains (Av-1, Av-2, and Av-3), which were confirmed by duck challenge, were used to perform the ELA. Inoculating 10(2) to 10(4) colony-forming units into the allantoic cavity of 10-day-old embryos discriminated between virulent and avirulent strains based on mortality. Differences in invasion rates into embryonic tissues were found between the RA T1 and Av-1 strains. The maximum colony-forming units of the RA T1 strain were about 1000 times higher than those of the Av-1 strain in the tissue invasion rate for 4 days. We found that the virulent strains killed embryos at mortality rates ≥ 50% during the first 3 days after inoculation and that the avirulent strains had death rates of ≤ 20% over 5 days. These results obtained by repeated testing suggest that the ELA could be used as a first-line screening method to determine the virulence of R. anatipestifer strains.

A Polyvalent Adhesin-Toxoid Multiepitope-Fusion-Antigen-Induced Functional Antibodies against Five Enterotoxigenic Escherichia coli Adhesins (CS7, CS12, CS14, CS17, and CS21) but Not Enterotoxins (LT and STa).

The increasing prevalence and association with moderate-to-severe diarrhea make enterotoxigenic Escherichia coli (ETEC) adhesins CS7, CS12, CS14, CS17, and CS21 potential targets of ETEC vaccines. Currently, there are no vaccines licensed to protect against ETEC, a top cause of children's diarrhea and travelers' diarrhea. Recently, a polyvalent adhesin protein (adhesin MEFA-II) was demonstrated to induce antibodies that inhibited adherence from these five ETEC adhesins and reduced the enterotoxicity of ETEC heat-stable toxin (STa), which plays a key role in causing ETEC-associated diarrhea. To improve adhesin MEFA-II for functional antibodies against STa toxin and the other ETEC toxin, heat-labile toxin (LT), we modified adhesin MEFA-II by adding another STa toxoid and an LT epitope; we examined the new antigen immunogenicity (to five adhesins and two toxins) and more importantly antibody functions against ETEC adherence and STa and LT enterotoxicity. Data show that mice intramuscularly immunized with the new antigen (adhesin MEFA-IIb) developed robust IgG responses to the targeted adhesins (CS7, CS12, CS14, CS17, and CS21) and toxins (STa and LT). Mouse antibodies inhibited the adherence of ETEC strains expressing any of these five adhesins but failed to neutralize STa or LT enterotoxicity. In further studies, rabbits intramuscularly immunized with adhesin MEFA-IIb developed robust antigen-specific antibodies; when challenged with an ETEC isolate expressing CS21 adhesin (JF2101, CS21, and STa), the immunized rabbits showed a significant reduction in intestinal colonization by ETEC bacteria. These data indicate that adhesin MEFA-IIb is broadly immunogenic and induces functional antibodies against the targeted ETEC adhesins but not the toxins.

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.

Feasibility of avian antibodies as prophylaxis against enterotoxigenic escherichia coli colonization.

Background: This research aims to evaluate the feasibility of using avian immunoglobulins (IgY) raised against adhesion factors of enterotoxigenic Escherichia coli (ETEC) as prophylaxis of diarrheal illness caused by these pathogens. ETEC requires adhesion to human intestinal epithelial cells as a primary step in establishing enteric infection. Therefore, inhibition of adhesion may prevent such infections and reduce clinical burdens of diarrheal illness. Methods: IgY samples were prepared from eggs of hens immunized with an adhesin-tip multiepitope fusion antigen (MEFA), developed against nine adhesin tip epitopes derived from clinically relevant ETEC strains. The resulting IgY was evaluated for its ability to inhibit adhesion of ETEC to cell-surface targets. Potential impacts of anti-MEFA IgY on growth of both pathogenic and commensal E. coli isolates were also evaluated. Results: Enzyme linked immunosorbent assay (ELISA) titers were achieved for IgY targeting each of the nine individual epitopes included in the adhesin-tip MEFA. Furthermore, anti-MEFA titers exceeding 1:219 were sustained for at least 23 weeks. All ETEC strains used in design of the adhesin-tip MEFA, and five of five clinical ETEC strains were significantly (P < 0.05) inhibited from adhesion to mammalian cells in culture. Conclusions: These findings demonstrate that IgY targeting ETEC adhesin-tip MEFA have the potential to disrupt in vitro adherence of ETEC. A formulation containing adhesin-tip MEFA IgY can be considered a potential candidate for in vivo evaluation as prophylaxis of diarrheal diseases. Animal studies of this formulation are planned.

Vaccines against gastroenteritis, current progress and challenges.

Enteric viral and bacterial infections continue to be a leading cause of mortality and morbidity in young children in low-income and middle-income countries, the elderly, and immunocompromised individuals. Vaccines are considered an effective and practical preventive approach against the predominantly fecal-to-oral transmitted gastroenteritis particularly in the resource-limited countries or regions where implementation of sanitation systems and supply of safe drinking water are not quickly achievable. While vaccines are available for a few enteric pathogens including rotavirus and cholera, there are no vaccines licensed for many other enteric viral and bacterial pathogens. Challenges in enteric vaccine development include immunological heterogeneity among pathogen strains or isolates, a lack of animal challenge models to evaluate vaccine candidacy, undefined host immune correlates to protection, and a low protective efficacy among young children in endemic regions. In this article, we briefly updated the progress and challenges in vaccines and vaccine development for the leading enteric viral and bacterial pathogens including rotavirus, human calicivirus, Shigella, enterotoxigenic Escherichia coli (ETEC), cholera, nontyphoidal Salmonella, and Campylobacter, and introduced a novel epitope- and structure-based vaccinology platform known as MEFA (multiepitope fusion antigen) and the application of MEFA for developing broadly protective multivalent vaccines against heterogenous pathogens.

Adjuvant effect of enterotoxigenic Escherichia coli (ETEC) double-mutant heat-labile toxin (dmLT) on systemic immunogenicity induced by the CFA/I/II/IV MEFA ETEC vaccine: Dose-related enhancement of antibody responses to seven ETEC adhesins (CFA/I, CS1-CS6).

Double-mutant heat-labile toxin (dmLT, LTR192G/L211A) of enterotoxigenic Escherichia coli (ETEC) is an effective mucosal adjuvant. Recent studies have shown that dmLT also exhibits adjuvanticity for antigens administered parenterally. In this study, we subcutaneously (SC) immunized mice with the ETEC adhesin-based vaccine, CFA/I/II/IV MEFA (multiepitope fusion antigen), adjuvanted with dmLT and examined the impact of dmLT on antibody responses specific to the seven adhesins in the vaccine construction [CFA/I, CFA/II (CS1, CS2, CS3) and CFA/IV (CS4, CS5, CS6)]. Mice were immunized with a fixed dose of CFA/I/II/IV MEFA and ascending doses of dmLT adjuvant (0, 0.05, 0.1, 0.5 or 1.0 µg) to assess the potential dmLT dose response relationship. Data showed that dmLT enhanced systemic antibody responses to all seven antigens (CFA/I, CS1-CS6) targeted by MEFA in a dose-dependent way. The adjuvant effect of dmLT on the MEFA construct plateaued at a dose of 0.1 µg. Results also indicated that dmLT is an effective parenteral adjuvant when given by the SC route with the ETEC adhesin MEFA vaccine and that antibody enhancement was achieved with relatively low doses. These observations suggest the potential usefulness of dmLT for parenteral ETEC vaccine candidates and also perhaps for vaccines against other pathogens.

Engineered Recombinant Escherichia coli Probiotic Strains Integrated with F4 and F18 Fimbriae Cluster Genes in the Chromosome and Their Assessment of Immunogenic Efficacy in Vivo.

F4 (K88) and F18 fimbriaed enterotoxigenic Escherichia coli (ETEC) are the predominant causes of porcine postweaning diarrhea (PWD), and vaccines are considered the most effective preventive approach against PWD. Since heterologous DNA integrated into bacterial chromosomes could be effectively expressed with stable inheritance, we chose probiotic EcNc (E. coli Nissle 1917 prototype cured of cryptic plasmids) as a delivery vector to express the heterologous F4 or both F4 and F18 fimbriae and sequentially assessed their immune efficacy of anti-F4 and F18 fimbriae in both murine and piglet models. Employing the CRISPR-cas9 technology, yjcS, pcadA, lacZ, yieN/trkD, maeB, and nth/tppB sites in the chromosome of an EcNc strain were targeted as integration sites to integrate F4 or F18 fimbriae cluster genes under the Ptet promotor to construct two recombinant integration probiotic strains (RIPSs), i.e., nth integration strain (EcNcΔnth/tppB::PtetF4) and multiple integration strain (EcNc::PtetF18x4::PtetF4x2). Expression of F4, both F4 and F18 fimbriae on the surfaces of two RIPSs, was verified with combined methods of agglutination assay, Western blot, and immunofluorescence microscopy. The recombinant strains have improved adherence to porcine intestinal epithelial cell lines. Mice and piglets immunized with the nth integration strain and multiple integration strain through gavage developed anti-F4 and both anti-F4 and anti-F18 IgG immune responses. Moreover, the serum antibodies from the immunized mice and piglets significantly inhibited the adherence of F4+ or both F4+ and F18+ ETEC wild-type strains to porcine intestinal cell lines in vitro, indicating the potential of RIPSs as promising probiotic strains plus vaccine candidates against F4+/F18+ ETEC infection.

Mapping the neutralizing epitopes of F18 fimbrial adhesin subunit FedF of enterotoxigenic Escherichia coli (ETEC).

K88 and F18 fimbrial enterotoxigenic Escherichia coli (ETEC) are the major causes of post-weaning diarrhea (PWD) in pigs. A vaccine that induces broad immunity to prevent K88 and F18 fimbrial ETEC bacterial attachment and colonization in pig small intestines and to neutralize enterotoxin enterotoxicity would be effective for PWD. Structure-based multiepitope-fusion-antigen (MEFA) technology using a backbone immunogen to present neutralizing epitopes of representing virulence factors capacitates development of broadly protective ETEC vaccines. Neutralizing epitopes have been identified from K88 fimbrial adhesin (FaeG) and enterotoxins but not F18 fimbrial adhesin. In this study, we in silico identified immunodominant epitopes from F18ac fimbrial subunit FedF which plays a critical role in F18 fimbrial adherence, genetically fused each epitope to a carrier, examined immunogenicity of each epitope fusion, and determined epitope-derived antibodies neutralizing activities against F18 fimbrial adherence. Data showed that seven immune-dominant epitopes were identified from FedF subunit. Fused to heterologous human ETEC adhesin subunit CfaB, epitope fusions induced anti-F18 antibodies in subcutaneously immunized mice. Moreover, antibodies derived from each fusion significantly blocked adherence of a F18-fimbrial E. coli bacteria to pig intestinal cell line IPEC-J2. While all seven epitopes exhibited neutralizing activity, results from this study identified FedF epitopes #3 (IPSSSGTLTCQAGT) and #7 (QPDATGSWYD) the most effective for antibodies against F18 fimbrial adherence, and suggested their future application in PWD vaccine development.

Immunogenicity characterization of genetically fused or chemically conjugated heat-stable toxin toxoids of enterotoxigenic Escherichia coli in mice and pigs.

Enterotoxigenic Escherichia coli (ETEC) producing type Ib heat-stable toxin (STa) are a main cause of children's diarrhea and travelers' diarrhea, thus STa needs to be targeted in ETEC vaccine development. However, because this 19-amino acid STa is poorly immunogenic, attempts to genetically fuse or chemically couple it to carrier proteins have been made to enhance STa immunogenicity. In this study, we selected one genetic fusion and one chemical conjugate to comparatively evaluate STa immunogenicity. The genetic fusion is 3xSTaN12S-mnLTR192G/L211A carrying three toxoid (STaN12S) genetically fused to a double mutant LT monomer (mnLTR192G/L211A); the chemical conjugate is BSA-STaA14T, which has toxoid STaA14T chemically coupled to bovine serum albumin (BSA). We immunized mice with the STa toxoid fusion and chemical conjugates, and examined antibody responses. Furthermore, we immunized pigs and evaluated derived antibodies for efficacy to passively provide protection against ETEC diarrhea using a piglet model. Data showed that mice subcutaneously immunized with BSA-STaA14T or 3xSTaN12S-mnLTR192G/L211A developed a strong anti-STa antibody, and the induced antibodies exhibited equivalent toxin-neutralizing activities. Pigs immunized with 3xSTaN12S-mnLTR192G/L211A or BSA-STaA14T developed similar levels of anti-STa antibodies; piglets with passively acquired antibodies induced by the genetic fusion appeared better protected against STa + ETEC. Results from the current study indicate that the fusion and conjugate approaches are viable options for facilitating STa immunogenicity and developing ETEC vaccines.

Antibodies induced by enterotoxigenic Escherichia coli (ETEC) adhesin major structural subunit and minor tip adhesin subunit equivalently inhibit bacteria adherence in vitro.

Antibodies that block the adherence of enterotoxigenic Escherichia coli (ETEC) to host intestinal epithelial cells are protective. Multiepitope-fusion-antigens (MEFAs) carrying epitopes of ETEC adhesin major subunits or tip minor subunits induced antibodies against ETEC adherence. Adherence inhibition effectiveness of antibodies induced by major subunit epitopes versus minor tip subunit epitopes, however, has not been comparatively characterized. In this study, we immunized mice with a major subunit MEFA or a tip MEFA, evaluated MEFA anti-adhesin immunogenicity, and examined induced-antibodies against bacteria in vitro adherence or in vivo colonization in mice. Mice subcutaneously immunized with major subunit MEFA CFA/I/II/IV or tip MEFA showed no adverse effects and developed strong antigen-specific antibody responses. Data showed that antibodies derived from two MEFAs were equally effective against adherence of the bacteria expressing CS1, CS2, CS3, CS4/CS6, CS5/CS6, or CS6 adhesin in vitro. Subsequently, we immunized mice with CFA/I fimbriae, major subunit CfaB, or minor tip adhesin subunit CfaE. We found that antibodies induced by CFA/I, CfaB and CfaE equally inhibited in vitro adherence of ETEC strain H10407. Furthermore, we immunized mice with CFA/I fimbriae, CfaB, or CfaE, and then challenged the mice with H10407. Data showed that although not significantly, fewer H10407 bacteria colonized the immunized mice. These results suggest that ETEC adhesin major subunit and minor tip subunit should be equally effective in inducing neutralizing anti-adhesin antibodies, and that major subunit CFA/I/II/IV MEFA or tip MEFA, perhaps combined with toxoid fusion 3xSTaN12S-mnLTR192G/L211A, can be used for development of broadly protective vaccines against ETEC diarrhea.

Immunogenicity and safety of a live Riemerella anatipestifer vaccine and the contribution of IgA to protective efficacy in Pekin ducks.

Riemerella anatipestifer (RA) infections cause major economic losses in the duck industry. In this study, we developed an RA vaccine to control virulent serotype 1 and 2 RA, which predominate in worldwide prevalence. We established a strategy for vaccine candidate screening, and selected strains D15-RDA-92 (serotype 1) and D14-RDA-8 (serotype 2). These strains were characterized by ≤50% embryo mortality and <3.0 serum resistance assay values in in vitro screening. We evaluated the protective efficacy of live bivalent RA vaccines against virulent homologous serotype RA. Ducklings received two oral immunizations with the bivalent vaccine and showed significant protection against two virulent strains (serotypes 1 and 2) at 21 days post-immunization. No death or clinical signs of diarrhea, tremors, or limb swelling were observed in the immunized ducks. In a safety evaluation, ducks immunized with 100 times higher doses showed no clinical signs, mortality, gross lesions, or histological lesions, and body weight of the ducks showed no significant difference compared to that of negative controls. In addition, IgA analysis showed a significant increase in secretory IgA antibodies generated in the trachea and duodenum of orally immunized ducks at 28 days of age. The IgA might be involved in one of the major immune responses to RA and contributes to protecting against virulent RA. In this study, we developed monovalent and bivalent RA vaccines that were safe in ducks and provided significant protective efficacy against virulent homologous RA strains.

Effects of Various LED Light Colors on Growth and Immune Response in Broilers.

We evaluated the effects of different light-emitting diode (LED) colors between blue and green on growth performance and the immune response in broilers. A total of 1,200 1-day-old Ross broilers were divided randomly into six groups and exposed to pure blue (PB), bright blue (BB), sky blue (SB), greenish blue (GB), pure green (PG), or white (W) using LEDs for 6 weeks. Consequently, body weights were higher in chickens reared under PB and GB on day (d) 7 and SB on d 21 than the other groups. Chickens in the PB group on d 42 were the heaviest among the groups, followed by the BB group and were significantly heavier than the W group. Splenocyte proliferation was significantly enhanced in chickens reared under PB followed by BB on d 42 and proliferation of peripheral blood mononuclear cells was significantly enhanced in chickens reared under BB on d 42. In addition, chickens in the BB group showed significantly elevated nitric oxide production on d 42, indicating activation of macrophages. These results suggest that immune function and growth of broilers can be improved at the later stage by rearing under shorter wavelength LEDs such as PB and BB.

Surveillance and characterization of Riemerella anatipestifer from wild birds in South Korea.

We conducted surveillance for Riemerella anatipestifer (RA) in wild birds along the East Asian-Australasian flyway in South Korea. Detected RA were characterized by serotype, antibiotic susceptibility, and sequence analysis of the 16S rRNA gene. We collected 944 wild birds of 34 species from 19 of South Korea's major migratory wild bird habitats between 2011 and 2012. We identified RA by PCR and rRNA gene sequence in 71/102 (69.6%) pharyngeal swabs and 19/944 (2.0%) cloacal swabs of wild birds. Most RA positives (71/75 [95%] pharyngeal and 19/704 [(2.6%] cloacal) were from three duck species (family Anatidae): Mallard Duck (Anas platyrhynchos), Northern Pintail (Anas acuta), and Spot-billed Duck (Anas poecilorhyncha). Thirty-three RA isolates obtained and examined were highly resistant to aminoglycosides: kanamycin (100%), gentamicin (94%), amikacin (91%), neomycin (88%), and streptomycin (82%). Six isolates were identified as serotype 4 by agar gel precipitation. Serotypes 1 and 7, which are known virulent serotypes, were also identified in three isolates from wild duck species.