Environmental enteric dysfunction induces regulatory T cells that inhibit local CD4+ T cell responses and impair oral vaccine efficacy.

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.

Progress toward a vaccine for extraintestinal pathogenic E. coli (ExPEC) II: efficacy of a toxin-autotransporter dual antigen approach.

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.

A cynomolgus monkey E. coli urinary tract infection model confirms efficacy of new FimH vaccine candidates.

The increase in urinary tract infections (UTI) caused by antibiotic-resistant Escherichia coli requires the development of new therapeutic agents and prophylactic vaccines. To evaluate the efficacy of new lead candidates, we implemented a cynomolgus macaque UTI challenge model that mimics human uncomplicated cystitis in response to transurethral challenge with a multidrug-resistant (MDR) E. coli serotype O25b ST131 isolate. E. coli fimbrial adhesin FimH and O-antigens are separately under clinical evaluation by others as vaccine candidates to prevent UTI and invasive urosepsis disease, respectively. Accordingly, we assessed the protective efficacy of three 50-µg intramuscular doses of a novel recombinant FimH antigen adjuvanted with liposomal QS21/MPLA compared with saline placebo in groups of nine animals. A third group was vaccinated with this FimH formulation in combination with 1 µg each of a four-valent mixture of serotype O1a, O2, O6, and O25b O-antigen CRM197 lattice glycoconjugates. Both vaccines elicited high levels of serum FimH IgG and adhesin blocking antibodies at the time of bacterial challenge and, for the combination group, O-antigen-specific antibodies. Following bacterial challenge, both vaccinated groups showed >200- and >700-fold reduction in bacteriuria at day 2 and day 7 post-infection compared with placebo, respectively. In parallel, both vaccines significantly reduced levels of inflammatory biomarkers IL-8 and myeloperoxidase in the urine at day 2 post-infection relative to placebo. Results provide preclinical proof-of-concept for the prevention of an MDR UTI infection by these new vaccine formulations.

Putative new combination vaccine candidates identified by reverse vaccinology and genomic approaches to control enteric pathogens.

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.

Local induction of bladder Th1 responses to combat urinary tract infections.

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.

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.

The development and characterization of an E. coli O25B bioconjugate vaccine.

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.

Evaluation of the Immunogenicity and Protective Efficacy of an Enterotoxigenic Escherichia coli CFA/I Adhesin-Heat-Labile Toxin Chimera.

Recent efforts to develop an enterotoxigenic Escherichia coli (ETEC) vaccine have focused on the antigenically conserved tip adhesins of colonization factors. We showed previously that intranasal immunization with dsc19CfaE, a soluble variant of the in cis donor strand-complemented tip adhesin of a colonization factor of the class 5 family (CFA/I) fimbria, is highly immunogenic and protects against oral challenge with CFA/I-positive (CFA/I+) ETEC strain H10407 in the Aotus nancymaae nonhuman primate. We also reported a cholera toxin (CT)-like chimera (called dsc19CfaE-CTA2/CTB) in which the CTA1 domain of CT was replaced by dsc19CfaE that was strongly immunogenic when administered intranasally or orogastrically in mice. Here, we evaluate the immunogenicity and protective efficacy (PE) of a refined and more stable chimera comprised of a pentameric B subunit of ETEC heat-labile toxin (LTB) in lieu of the CTB pentamer and a donor strand truncation (dsc14) of CfaE. The refined chimera, dsc14CfaE-sCTA2/LTB, was highly immunogenic in mice when administered intranasally or intradermally, eliciting serum and fecal antibody responses against CfaE and LTB, as well as strong hemagglutination inhibition titers, a surrogate for neutralization of intestinal adhesion mediated by CfaE. Moreover, the chimera was safe and highly immunogenic when administered intradermally to guinea pigs. In A. nancymaae, intradermal (i.d.) immunization with chimera plus single-mutant heat-labile toxin [LT(R192G)] elicited strong serum anti-CfaE and anti-LTB antibody responses and conferred significant reduction of diarrhea compared to phosphate-buffered saline (PBS) controls (PE = 84.1%; P < 0.02). These data support the further evaluation of dsc14CfaE-sCTA2/LTB as an ETEC vaccine in humans.

Targeting peptide-enhanced antibody and CD11c+ dendritic cells to inclusion bodies expressing protective antigen against ETEC in mice.

Enterotoxigenic Escherichia coli (ETEC) remains a massive burden in developing countries with increasing morbidity and mortality rates; it is also an important pathogen in the farming industry and is a leading cause of bacterial diarrhea. Our previous study showed that nanometer-sized inclusion bodies (IBs) of the fimbrial adhesin subunit protein (FaeG), mutation heat-stable enterotoxin a (mSTa), heat-labile enterotoxin b (LTb), and STb (nontargeting) fusion protein as an oral vaccine induced both systemic and mucosal immune responses. In this study, to enhance the protective efficacy to ETEC, we used Yersinia enterocolitica adhesive and M-cell-targeting peptides to analyze high-efficiency antigen-specific immune presentation in the gut. Here, we showed that immunization with the IBs of ETEC-FaeG-mSTa-LTb-STb-induced a specific systemic and mucosal immune response in the gut, whereas the combination of both targeting peptides resulted in the highest titer, protective immune response against ETEC. A lymphocyte proliferation assay has shown that the IBs induced immunologic memory. The specific antibody of the targeting groups could effectively neutralize toxins, thereby protecting the cells of the small intestine and reducing the level of cAMP and cGMP, and the groups with double targeting showed the best effect. The most important finding was that the targeting peptides stimulate the T helper (Th) cells through Th17 and Th1 and that Th1 cells dominated the cellular immune response. We found that the targeting peptide could also activate CD11c+ on lymphoid dendritic cells, which processed and presented antigens to T cells through Th1-mediated IFN-γ and IL-12, thereby enhancing the antibody titers. The double-targeting peptide had a better effect on stimulating the immune cells to enhance the antibody titers.-Jiang, X., Xia, S., He, X., Ma, H., Feng, Y., Liu, Z., Wang, W., Tian, M., Chen, H., Peng, F., Wang, L., Zhao, P., Ge, J., Liu, D. Targeting peptide-enhanced antibody and CD11c+ dendritic cells to inclusion bodies expressing protective antigen against ETEC in mice.

A tale of two bacterial enteropathogens and one multivalent vaccine.

Shigella and enterotoxigenic Escherichia coli (ETEC) are among the top four enteric pathogens that cause diarrheal illness in young children in developing countries and are major etiologic agents of travellers' diarrhoea. A single vaccine that could target both of these pathogens would have significant public health impact. In this review, we highlight the many pivotal contributions of Phillippe Sansonetti to the identification of molecular mechanisms of pathogenesis of Shigella that paved the way for the development of rationally designed, novel vaccines candidates. The CVD developed a series of live attenuated Shigella vaccine strains based on the most prevalent serotypes associated with disease. Shigella vaccine strains were engineered to express critical ETEC antigens to form a broadly protective Shigella-ETEC multivalent vaccine.

Design, development, and evaluation of the efficacy of a nucleic acid-free version of a bacterial ghost candidate vaccine against avian pathogenic E. coli (APEC) O78:K80 serotype.

One of the major bacterial infectious diseases in the poultry industry is avian pathogenic Escherichia coli (APEC), which causes colibacillosis in chickens. To develop a novel nucleic acid-free bacterial ghost (BG) vaccine against the O78:K80 serotype of APEC, in this study we constructed a plasmid that harbored E-lysis and S nuclease (SNUC). Following the expression, the O78:K80 bacteria lost all of their cytoplasmic content and nucleic acids by enzymatic digestion. The functionality of these two proteins in the production procedure of bacterial ghosts was confirmed by monitoring the number of colonies, scanning electron microscopy imaging, gel electrophoresis of genomic DNA, and qPCR on the plasmid content of bacterial ghosts. The protective efficacy of the ghost vaccine generated from O78:K80 serotype of APEC was tested in chickens by injection and inhalation routes and compared with that in chickens that received the injection of a killed vaccine. The O78:K80 BG vaccine candidate, used as injection and inhalation, in comparison with the killed vaccine, triggered higher proinflammatory cytokine expression including IL-6, IL-1ß, and TNFSF15; a higher level of antibody-dependent humoral (IgY and IgA) and cellular immune responses (IFNγ and lymphocyte proliferation); and lower lesion scores. According to the results of this study, we suggest that the bacterial ghost technology has the potential to be applied for the development of novel vaccines against avian colibacillosis. This technology provides an effective and reliable approach to make multivalent vaccines for more prevalent APEC strains involved in the establishment of this infectious disease in the poultry industry.

Exploiting bacterial outer membrane vesicles as a cross-protective vaccine candidate against avian pathogenic Escherichia coli (APEC).

BACKGROUND: The well-known fact that avian pathogenic Escherichia coli (APEC) is harder to prevent due to its numerous serogroups has promoted the development of biological immunostimulatory materials as new vaccine candidates in poultry farms. Bacterial outer membrane vesicles (OMVs), known as spherical nanovesicles enriched with various immunostimulants, are naturally secreted by Gram-negative bacteria, and have gained much attention for developing effective vaccine candidates. Recent report has demonstrated that OMVs of APEC O78 can induce protective immunity in chickens. Here, a novel multi-serogroup OMVs (MOMVs) vaccine was developed to achieve cross-protection against APEC infection in broiler chickens. RESULTS: In this study, OMVs produced by three APEC strains were isolated, purified and prepared into MOMVs by mixing these three OMVs. By using SDS-PAGE and LC-MS/MS, 159 proteins were identified in MOMVs and the subcellular location and biological functions of 20 most abundant proteins were analyzed. The immunogenicity of MOMVs was evaluated, and the results showed that MOMVs could elicit innate immune responses, including internalization by chicken macrophage and production of immunomodulatory cytokines. Vaccination with MOMVs induced specific broad-spectrum antibodies as well as Th1 and Th17 immune responses. The animal experiment has confirmed that immunization with an appropriate dose of MOMVs could not cause any adverse effect and was able to reduce bacteria loads and pro-inflammatory cytokines production, thus providing effective cross-protection against lethal infections induced by multi-serogroup APEC strains in chickens. Further experiments indicated that, although vesicular proteins were able to induce stronger protective efficiency than lipopolysaccharide, both vesicular proteins and lipopolysaccharide are crucial in MOMVs-mediated protection. CONCLUSIONS: The multi-serogroup nanovesicles produced by APEC strains will open up a new way for the development of next generation vaccines with low toxicity and broad protection in the treatment and control of APEC infection.

A multivalent vaccine candidate targeting enterotoxigenic Escherichia coli fimbriae for broadly protecting against porcine post-weaning diarrhea.

Fimbriae-mediated initial adherence is the initial and critical step required for enterotoxigenic Escherichia coli (ETEC) infection. Therefore, vaccine candidates have been developed that target these fimbriae and induce specific anti-fimbriae antibodies to block initial ETEC attachment. While this vaccine effectively protects against ETEC-associated post-weaning diarrhea (PWD), developing a broadly effective vaccine against initial ETEC attachment remains a challenging problem, owing to the immunological heterogeneity among these antigens. Here, we applied multi-epitope fusion antigen (MEFA) technology to construct a FaeG-FedF-FanC-FasA-Fim41a MEFA using the adhesive subunits of predominant fimbriae K88 and F18 as the backbone, which also integrated epitopes from adhesive subunits of the rare fimbriae K99, 987P, and F41; we then generated a MEFA computational model and tested the immunogenicity of this MEFA protein in immunized mice. We next evaluated the potential of the fimbriae-targeted MEFA as a vaccine candidate to effectively prevent PWD using in vitro assessment of its anti-fimbriae, antibody-directed inhibition of bacterial adherence. Computational modeling showed that all relevant epitopes were exposed on the MEFA surface and mice subcutaneously immunized with the MEFA protein developed IgG antibodies to all five fimbriae. Moreover, anti-fimbriae antibodies induced by the MEFA protein significantly inhibited the adhesion of K88+, F18+, K99+, 987P+, and F41+ ETEC strains to piglet small intestinal IPEC-1 and IPEC-J2 cell lines. Taken together, these results indicate that FaeG-FedF-FanC-FasA-Fim41a MEFA protein induced specific anti-fimbriae neutralizing antibodies against the five targeted fimbriae. Critically, these results show the potential of fimbriae-targeted MEFA and indicate their promise as a broad, effective vaccine against PWD.

Novel biotechnological approaches for monitoring and immunization against resistant to antibiotics Escherichia coli and other pathogenic bacteria.

The application of next-generation molecular, biochemical and immunological methods for developing new vaccines, antimicrobial compounds, probiotics and prebiotics for zoonotic infection control has been fundamental to the understanding and preservation of the symbiotic relationship between animals and humans. With increasing rates of antibiotic use, resistant bacterial infections have become more difficult to diagnose, treat, and eradicate, thereby elevating the importance of surveillance and prevention programs. Effective surveillance relies on the availability of rapid, cost-effective methods to monitor pathogenic bacterial isolates. In this opinion article, we summarize the results of some research program initiatives for the improvement of live vaccines against avian enterotoxigenic Escherichia coli using virulence factor gene deletion and engineered vaccine vectors based on probiotics. We also describe methods for the detection of pathogenic bacterial strains in eco-environmental headspace and aerosols, as well as samples of animal and human breath, based on the composition of volatile organic compounds and fatty acid methyl esters. We explain how the introduction of these low-cost biotechnologies and protocols will provide the opportunity to enhance co-operation between networks of resistance surveillance programs and integrated routine workflows of veterinary and clinical public health microbiology laboratories.

Hyperimmune Bovine Colostral Anti-CS17 Antibodies Protect Against Enterotoxigenic Escherichia coli Diarrhea in a Randomized, Doubled-Blind, Placebo-Controlled Human Infection Model.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) commonly cause diarrhea in children living in developing countries and in travelers to those regions. ETEC are characterized by colonization factors (CFs) that mediate intestinal adherence. We assessed if bovine colostral IgG (bIgG) antibodies against a CF, CS17, or antibodies against CsbD, the minor tip subunit of CS17, would protect subjects against diarrhea following challenge with a CS17-expressing ETEC strain. METHODS: Adult subjects were randomized (1:1:1) to receive oral bIgG against CS17, CsbD, or placebo. Two days prior to challenge, subjects began dosing 3 times daily with the bIgG products (or placebo). On day 3, subjects ingested 5 × 109 cfu ETEC strain LSN03-016011/A in buffer. Subjects were assessed for diarrhea for 120 hours postchallenge. RESULTS: A total of 36 subjects began oral prophylaxis and 35 were challenged with ETEC. While 50.0% of the placebo recipients had watery diarrhea, none of the subjects receiving anti-CS17 had diarrhea (P = .01). In contrast, diarrhea rates between placebo and anti-CsbD recipients (41.7%) were comparable (P = 1.0). CONCLUSIONS: This is the first study to demonstrate anti-CS17 antibodies provide significant protection against ETEC expressing CS17. More research is needed to better understand why anti-CsbD was not comparably efficacious. Clinical Trials Registration. NCT00524004.

Intradermal or Sublingual Delivery and Heat-Labile Enterotoxin Proteins Shape Immunologic Responses to a CFA/I Fimbria-Derived Subunit Antigen Vaccine against Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of infectious diarrhea in children, travelers, and deployed military personnel. As such, development of a vaccine would be advantageous for public health. One strategy is to use subunits of colonization factors combined with antigen/adjuvant toxoids as an ETEC vaccine. Here, we investigated the intradermal (i.d.) or sublingual (s.l.) delivery of CFA/I fimbrial antigens, including CfaEB and a CfaE-heat-labile toxin B subunit (LTB) chimera admixed with double mutant heat-labile toxin (LT) LT-R192G/L211A (dmLT). In addition, we compared dmLT with other LT proteins to better understand the generation of adjuvanted fimbrial and toxoid immunity as well as the influence on any local skin reactogenicity. We demonstrate that immunization with dmLT admixed with CfaEB induces robust serum and fecal antibody responses to CFA/I fimbriae and LT but that i.d. formulations are not optimal for s.l. delivery. Improved s.l. vaccination outcomes were observed when higher doses of dmLT (1 to 5 µg) were admixed with CfaEB or, even better, when a CfaE-LTB chimera antigen was used instead. Serum anti-CFA/I total antibodies, detected by enzyme-linked immunosorbent assay, were the best predictor of functional antibodies, based on the inhibition of red blood cell agglutination by ETEC. Immunization with other LT proteins or formulations with altered B-subunit binding during i.d. immunization (e.g., by addition of 5% lactose, LTA1, or LT-G33D) minimally altered the development of antibody responses and cytokine recall responses but reduced skin reactogenicity at the injection site. These results reveal how formulations and delivery parameters shape the adaptive immune responses to a toxoid and fimbria-derived subunit vaccine against ETEC.

Biochemical and Immunological Evaluation of Recombinant CS6-Derived Subunit Enterotoxigenic Escherichia coli Vaccine Candidates.

CS6, a prevalent surface antigen expressed in nearly 20% of clinical enterotoxigenic Escherichia coli (ETEC) isolates, is comprised of two major subunit proteins, CssA and CssB. Using donor strand complementation, we constructed a panel of recombinant proteins of 1 to 3 subunits that contained combinations of CssA and/or CssB subunits and a donor strand, a C-terminal extension of 16 amino acids that was derived from the N terminus of either CssA or CssB. While the entire panel of recombinant proteins could be obtained as soluble, folded proteins, it was observed that the proteins possessing a heterologous donor strand, derived from the CS6 subunit different from the C-terminal subunit, had the highest degree of physical and thermal stability. Immunological characterization of the proteins, using a murine model, demonstrated that robust anti-CS6 immune responses were generated from fusions containing both CssA and CssB. Proteins containing only CssA were weakly immunogenic. Heterodimers, i.e., CssBA and CssAB, were sufficient to recapitulate the anti-CS6 immune response elicited by immunization with CS6, including the generation of functional neutralizing antibodies, as no further enhancement of the response was obtained with the addition of a third CS6 subunit. Our findings here demonstrate the feasibility of including a recombinant CS6 subunit protein in a subunit vaccine strategy against ETEC.

Immunizations with Enterotoxigenic Escherichia coli Heat-Stable Toxin Conjugates Engender Toxin-Neutralizing Antibodies in Mice That Also Cross-React with Guanylin and Uroguanylin.

Infection with enterotoxigenic Escherichia coli (ETEC) is a common cause of childhood diarrhea in low- and middle-income countries, as well as of diarrhea among travelers to these countries. In children, ETEC strains secreting the heat-stable toxin (ST) are the most pathogenic, and there are ongoing efforts to develop vaccines that target ST. One important challenge for ST vaccine development is to construct immunogens that do not elicit antibodies that cross-react with guanylin and uroguanylin, which are endogenous peptides involved in regulating the activity of the guanylate cyclase-C (GC-C) receptor. We immunized mice with both human ST (STh) and porcine ST (STp) chemically coupled to bovine serum albumin, and the resulting sera neutralized the toxic activities of both STh and STp. This suggests that a vaccine based on either ST variant can confer cross-protection. However, several anti-STh and anti-STp sera cross-reacted with the endogenous peptides, suggesting that the ST sequence must be altered to reduce the risk of unwanted cross-reactivity. Epitope mapping of four monoclonal anti-STh and six anti-STp antibodies, all of which neutralized both STh and STp, revealed that most epitopes appear to have at least one amino acid residue shared with guanylin or uroguanylin. Despite this, only one monoclonal antibody displayed demonstrable cross-reactivity to the endogenous peptides, suggesting that targeted mutations of a limited number of ST residues may be sufficient to obtain a safe ST-based vaccine.

Mapping the Neutralizing Epitopes of Enterotoxigenic Escherichia coli K88 (F4) Fimbrial Adhesin and Major Subunit FaeG.

Enterotoxigenic Escherichia coli (ETEC) strains that produce immunologically heterogeneous fimbriae and enterotoxins are the primary cause of neonatal diarrhea and postweaning diarrhea in young pigs. A multivalent vaccine inducing protective immunity against ideally all ETEC fimbriae and enterotoxins could be effective against diarrhea in young pigs. However, developing a vaccine to broadly protect against various ETEC virulence determinants has proven challenging. Recently developed structure- and epitope-based multiepitope fusion antigen (MEFA) technology that presents neutralizing epitopes of various virulence determinants at a backbone immunogen and that mimics epitope native immunogenicity suggests the feasibility of developing multivalent vaccines. With neutralizing epitopes from ETEC fimbria F18 and enterotoxins being identified, it becomes urgent to identify protective epitopes of K88 (F4) fimbriae, which play a major role in pig neonatal and postweaning diarrhea. In this study, we identified B-cell immunodominant epitopes in silico from the K88ac fimbrial major subunit (also adhesin) FaeG and embedded each epitope in a heterogeneous carrier for epitope fusions. We then immunized mice with each epitope fusion protein and examined epitope antigenicity and also neutralizing activities of epitope-induced antibodies. Data showed that while all nine FaeG epitope fusions induced antibodies to K88ac fimbria, anti-K88 IgG antibodies derived from epitopes MTGDFNGSVD (ep1), LNDLTNGGTK (ep2), GRTKEAFATP (ep3), ELRKPDGGTN (ep4), PMKNAGGTKVGAVKVN (ep5), and RENMEYTDGT (ep8) significantly inhibited adherence of K88ac fimbrial bacteria to porcine intestinal cell line IPEC-J2, indicating that these peptides were the neutralizing epitopes of K88ac fimbrial major subunit FaeG and suggesting the future application of FaeG epitopes in ETEC vaccine development.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) strains producing K88ac fimbriae and enterotoxins are a major cause of porcine neonatal diarrhea and postweaning diarrhea in the United States. Currently, there is no vaccine to induce broadly protective antiadhesin and antitoxin immunity against ETEC-associated diarrhea. To develop a broadly effective ETEC vaccine, we need to target the most important if not all ETEC virulence determinants. While conventional vaccinology approaches encounter difficulties at integrating or including heterogeneous ETEC fimbria and toxin antigens into a vaccine product, multiepitope fusion antigen (MEFA) structural vaccinology provides a new platform to combine neutralizing antigenic elements or epitopes from various heterogeneous virulence factors for broad immunity and protection. Identification of the neutralizing epitopes of K88ac fimbria from this study added the last antigens to an MEFA-based multivalent vaccine against ETEC-associated diarrhea in pigs. An effective vaccine against pig diarrhea can significantly improve swine health and well-being and reduce economic losses to the swine industry worldwide.

Preparation of porcine enterotoxigenic Escherichia coli (ETEC) ghosts and immunogenic analysis in a mouse model.

Enterotoxignenic Escherichia coli (ETEC)-associated colibacillosis causes high levels of morbidity and mortality in neonatal piglets. Vaccination is among effective strategy to fight against ETEC-related diseases. Bacterial ghosts (BGs) are empty bacterial envelopes, which substain subtle antigenic comformation in bacterial outer membrane. In this study, a BG vaccine was generated using porcine ETEC isolated strain DQ061 and evaluated its safety and immunogenicity in a mouse model. The recombinant bacteria were constructed by transformation of lysis plasmid pHH43 and generation of BGs was conducted in a lysis rate of 99.93% by incubation of the recombinant bacteria at 42 °C for 2 h. Mice were immunized subcutaneously twice in 2-week intervals with BGs, BGs emulsified with ISA 206 adjuvant, or formalin-inactivated ETEC vaccine after safety test. Mice with either of two BG vaccines developed higher titer of antibodies, secreted higher titer of interleukin 4, gamma interferon and alpha tumor necrosis factor after 2 doses than those with formalin-inactivated ETEC vaccine or those with adjuvant placebo (P < 0.01). The quantity of CD4+ and CD8+ T lymphocyte in spleen was higher in both BG groups than that in the inactivated vaccine group or adjuvant group 2 weeks post boost immunization (P < 0.05). The vaccinated mice were challenged intraperitoneally with 10 × LD50 dose of DQ061. Mice with the BGs plus adjuvant were completely protected against challenge, compared to 60% protection of mice with the inactivated vaccine. Mice exhibited decreased tissue lesion and reduced bacterial loads in the BGs groups by comparison with those with the inactivated vaccine or adjuvant only. Our results validated that the ETEC BGs bear high safety and immunogenicity in a mouse model, suggesting a potential of further evaluation in a pig model.