Preparation of novel trivalent vaccine against enterotoxigenic Escherichia coli for preventing newborn piglet diarrhea.

OBJECTIVE: To develop a trivalent genetically engineered inactivated Escherichia coli vaccine (K88ac-3STa-LTB) that neutralizes the STa toxin by targeting fimbriae and entertoxins for the treatment of enterotoxigenic E coli. ANIMALS: 18- to 22-g mice, rabbits, pregnant sows. PROCEDURES: Using PCR, the K88ac gene and LTB gene were cloned separately from the template C83902 plasmid. At the same time, the 3 STa mutant genes were also amplified by using the gene-directed mutation technology. Immune protection experiments were performed, and the minimum immune dose was determined in mice and pregnant sows. RESULTS: The ELISA test could be recognized by the STa, LTB, and K88ac antibodies. Intragastric administration in the suckling mouse confirmed that the protein had lost the toxicity of the natural STa enterotoxin. The results of the immune experiments showed that K88ac-3STa-LTB protein could stimulate rabbits to produce serum antibodies and neutralize the toxicity of natural STa enterotoxin. The efficacy test of the K88ac-3STa-LTB-inactivated vaccine showed that the immune protection rate of the newborn piglets could reach 85% on the first day after suckling. At the same time, it was determined that the minimum immunization doses for mice and pregnant sows were 0.2 and 2.5 mL, respectively. CLINICAL RELEVANCE: This research indicates that the K88ac-3STa-LTB trivalent genetically engineered inactivated vaccine provides a broad immune spectrum for E coli diarrhea in newborn piglets and prepares a new genetically engineered vaccine candidate strain for prevention of E coli diarrhea in piglets.

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.

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.

Formaldehyde effects on kanamycin resistance gene of inactivated recombinant Escherichia coli vaccines.

OBJECTIVES: Earlier studies have demonstrated the use of inactivated recombinant E. coli (bacterins), to protect against Clostridium spp. in vaccinated animals. These bacterins have a simpler, safer, and faster production process. However, these bacterins carry expression plasmids, containing antibiotic resistance gene, which could be assimilate accidentally by environmental microorganisms. Considering this, we aimed to impair this plasmids using formaldehyde at different concentrations. RESULTS: This compound inactivated the highest density of cells in 24 h. KanR cassette amplification was found to be impaired with 0.8% for 24 h or 0.4% for 72 h. Upon electroporation, E. coli DH5α ultracompetent cells were unable to acquire the plasmids extracted from the bacterins after inactivation procedure. Formaldehyde-treated bacterins were incubated with other viable strains of E. coli, leading to no detectable gene transfer. CONCLUSIONS: We found that this compound is effective as an inactivation agent. Here we demonstrate the biosafety involving antibiotic resistance gene of recombinant E. coli vaccines allowing to industrial production and animal application.

Immunoinformatics and Epitope Prediction.

With advancements in sequencing technologies, vast amount of experimental data has accumulated. Due to rapid progress in the development of bioinformatics tools and the accumulation of data, immunoinformatics or computational immunology emerged as a special branch of bioinformatics which utilizes bioinformatics approaches for understanding and interpreting immunological data. One extensively studied aspect of applied immunology involves using available databases and tools for prediction of B- and T-cell epitopes. B and T cells comprise two arms of adaptive immunity.This chapter first reviews the methodology we used for computational identification of B- and T-cell epitopes against enterotoxigenic Escherichia coli (ETEC). Then we discuss other databases of epitopes and analysis tools for T-cell and B-cell epitope prediction and vaccine design. The predicted peptides were analyzed for conservation and population coverage. HLA distribution analysis for predicted epitopes identified efficient MHC binders. Epitopes were further tested using computational docking studies to bind in MHC-I molecule cleft. The predicted epitopes were conserved and covered more than 80% of the world population.

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.

Recombinant HcpA-EspA-Tir-Stx2B chimeric protein induces immunity against attachment and toxicity of Escherichia coli O157:H7.

BACKGROUND: It is about 4 decades from the identification of Enterohemorrhagic Escherichia coli (EHEC) as a food-borne pathogen. There are many shreds of evidence that the bacteria are significant sources of intestinal infections and outbreaks even in developed countries. Developing an effective vaccine against O157 and non-O157 serotypes of EHEC is a good strategy to combat the bacteria. Raising antibody against main toxicity, adhering and colonizing apparatus of the bacteria using a multi-antigenic protein can be hopefully applicable. MATERIAL AND METHODS: A synthetic cassette consists of HcpA-EspA-Tir-Stx2B (HETS) subunit proteins were constructed and sub-cloned in pET32a (+). The protein was expressed and purified with Ni-NTA column and the BALB/c mice were immunized by the purified protein. HETS protein efficacy to elicit immune responses, O157 fecal shedding and immunity against Stx toxin were assessed. In addition, the cellular assays were performed to investigate the immune sera capability for neutralizing of Stx toxin and bacterial attachment apparatus. RESULTS: The HETS protein (611 amino acid length) was expressed and validated by Western blotting. Exerted EHEC bacteria ratio in the immunized mice was reduced close to 60% in shedding test. Cellular assays revealed that the sera of the immunized animals were able to neutralize Stx holotoxin in an extent of 70%; also, immunized mice were able to tolerate up to 200 LD50 of the active toxin. Moreover, toxin neutralization assay showed the capability of the immunized sera to block the cell adhesion. CONCLUSION: Regarding a lack of an efficient vaccine against EHEC, the proposed candidate immunogen, which consists of main adhesion and invasion factors, can overcome the lack of a vaccine against the bacteria.

Direct Cloning of Bacterial Surface Polysaccharide Gene Cluster for One-Step Production of Glycoconjugate Vaccine.

Bacterial pathogen infections are fast-growing public health threats and worldwide problems. Glycoconjugate vaccines are among the most effective means in combating such infections. Recent advances in bacterial protein glycan coupling technology (PGCT) have revolutionized the production of glycoconjugate vaccines and drawn enormous attention from both researchers and pharmaceutical companies. Cloning of bacterial surface polysaccharide gene cluster is a prerequisite for the application of PGCT. In this study, we applied the RecET direct cloning strategy for rapid and efficient cloning of O-antigen polysaccharide gene clusters from Escherichia coli serotypes O25b, O26, and O55 in a high-fidelity manner. Then, these gene clusters were applied in PGCT to produce corresponding glycoconjugates. Subsequent immunological studies verified the abilities of glycoconjugate vaccine candidates O25-maltose-binding protein (MBP), O26-MBP, and O55-MBP to generate serotype-specific antibodies and confer protection against E. coli infections. The combination of RecET direct cloning and PGCT makes the rapid production of glycoconjugate vaccines against fast-expanding bacterial pathogens possible.

Rational Design and Evaluation of an Artificial Escherichia coli K1 Protein Vaccine Candidate Based on the Structure of OmpA.

Escherichia coli (E. coli) K1 causes meningitis and remains an unsolved problem in neonates, despite the application of antibiotics and supportive care. The cross-reactivity of bacterial capsular polysaccharides with human antigens hinders their application as vaccine candidates. Thus, protein antigens could be an alternative strategy for the development of an E. coli K1 vaccine. Outer membrane protein A (OmpA) of E. coli K1 is a potential vaccine candidate because of its predominant contribution to bacterial pathogenesis and sub-cellular localization. However, little progress has been made regarding the use of OmpA for this purpose due to difficulties in OmpA production. In the present study, we first investigated the immunogenicity of the four extracellular loops of OmpA. Using the structure of OmpA, we rationally designed and successfully generated the artificial protein OmpAVac, composed of connected loops from OmpA. Recombinant OmpAVac was successfully produced in E. coli BL21 and behaved as a soluble homogenous monomer in the aqueous phase. Vaccination with OmpAVac induced Th1, Th2, and Th17 immune responses and conferred effective protection in mice. In addition, OmpAVac-specific antibodies were able to mediate opsonophagocytosis and inhibit bacterial invasion, thereby conferring prophylactic protection in E. coli K1-challenged adult mice and neonatal mice. These results suggest that OmpAVac could be a good vaccine candidate for the control of E. coli K1 infection and provide an additional example of structure-based vaccine design.

Significance of Enterotoxigenic Escherichia coli (ETEC) Heat-Labile Toxin (LT) Enzymatic Subunit Epitopes in LT Enterotoxicity and Immunogenicity.

Enterotoxigenic Escherichia coli (ETEC) strains producing heat-labile toxin (LT) and/or heat-stable toxin (STa) are a top cause of children's diarrhea and travelers' diarrhea. Holotoxin-structured GM1-binding LT is a strong immunogen and an effective adjuvant, and can serve a carrier or a platform for multivalent vaccine development. However, the significance of peptide domains or epitopes of LT particularly enzymatic LTA subunit in association with LT enterotoxicity and immunogenicity has not been characterized. In this study, we identified B-cell epitopes in silico from LTA subunit and examined epitopes for immunogenicity and association with LT enterotoxicity. Epitopes identified from LTA subunit were individually fused to a modified chicken ovalbumin carrier protein, and each epitope-ovalbumin fusion was used to immunize mice. Data showed all 11 LTA epitopes were immunogenic; epitope 7 (105SPHPYEQEVSA115) induced greater titers of anti-LT antibodies which neutralized LT enterotoxicity more effectively. To examine these epitopes for the significance in LT enterotoxicity, we constructed LT mutants by substituting each of 10 epitopes at the toxic A1 domain of LTA subunit with a foreign epitope and examined LT mutants for enterotoxicity and GM1-binding activity. Data showed that LT mutants exhibited no enterotoxicity but retained GM1-binding activity. The results from this study indicated that while not all immunodominant LTA epitopes were neutralizing, LT mutants with an individual epitope substituted lost enterotoxicity but retained GM1-binding activity. These results provided additional information to understand LT immunogenicity and enterotoxicity and suggested the potential application of LT platform for multivalent vaccines against ETEC diarrhea and other diseases.IMPORTANCE No vaccine is licensed for enterotoxigenic Escherichia coli (ETEC) strains, which remain a leading cause of diarrhea in children from developing countries and international travelers. GM1-binding heat-labile toxin (LT) which is a key virulence factor of ETEC diarrhea is a strong vaccine antigen and a self-adjuvant. LT can also serve a backbone or platform for MEFA (multiepitope fusion antigen), a newly developed structural vaccinology technology, to present heterogeneous epitopes (by replacing LT epitopes) and to mimic epitope antigenicity for development of broadly protective vaccines. Data from this study identified neutralizing LT epitopes and demonstrated that substitution of LT epitopes eliminated LT enterotoxicity without altering GM1-binding activity, suggesting LT is potentially a versatile MEFA platform to present heterogeneous epitopes for multivalent vaccines against ETEC and other pathogens.

Immunization With Skp Delivered on Outer Membrane Vesicles Protects Mice Against Enterotoxigenic Escherichia coli Challenge.

Outer membrane vesicles (OMVs) are promising vaccine components because they combine antigen and adjuvant in a single formulation. Detoxified Salmonella enterica strains that express penta-acylated lipid A retain OMV immunogenicity but with reduced reactogenicity. We have previously shown that a recombinant form of the enterotoxigenic Escherichia coli (ETEC) 17 kilodalton protein (Skp) protects mice in a pulmonary challenge model, when fused to the glutathione-S-transferase (GST) epitope and combined with cholera toxin. Here we compared directly the efficacy of expressing Skp in detoxified Salmonella OMVs to GST-Skp for their ability to protect mice against ETEC challenge. We observed that the display of Skp on OMVs, in the absence of exogenous adjuvant, protects the mice as well as the recombinant GST-Skp with adjuvant, showing that we can achieve protection when antigen and adjuvant are administered as a single formulation. Collectively, these data demonstrate the utility of using OMVs for the expression and display of antigens for use in vaccine development and validate previously published work demonstrating that immunization with Skp is efficacious in protecting mice against ETEC challenge.

Adjuvant selection regulates gut migration and phenotypic diversity of antigen-specific CD4+ T cells following parenteral immunization.

Infectious diarrheal diseases are the second leading cause of death in children under 5 years, making vaccines against these diseases a high priority. It is known that certain vaccine adjuvants, chiefly bacterial ADP-ribosylating enterotoxins, can induce mucosal antibodies when delivered parenterally. Based on this, we reasoned vaccine-specific mucosal cellular immunity could be induced via parenteral immunization with these adjuvants. Here, we show that, in contrast to the Toll-like receptor-9 agonist CpG, intradermal immunization with non-toxic double-mutant heat-labile toxin (dmLT) from enterotoxigenic Escherichia coli drove endogenous, antigen-specific CD4+ T cells to expand and upregulate the gut-homing integrin α4ß7. This was followed by T-cell migration into gut-draining lymph nodes and both small and large intestines. We also found that dmLT produces a balanced T-helper 1 and 17 (Th1 and Th17) response, whereas T cells from CpG immunized mice were predominantly Th1. Immunization with dmLT preferentially engaged CD103+ dendritic cells (DCs) compared with CpG, and mice deficient in CD103+ DCs were unable to fully license antigen-specific T-cell migration to the intestinal mucosae following parenteral immunization. This work has the potential to redirect the design of existing and next generation vaccines to elicit pathogen-specific immunity in the intestinal tract with non-mucosal immunization.

Enterotoxigenic Escherichia coli Adhesin-Toxoid Multiepitope Fusion Antigen CFA/I/II/IV-3xSTaN12S-mnLTG192G/L211A-Derived Antibodies Inhibit Adherence of Seven Adhesins, Neutralize Enterotoxicity of LT and STa Toxins, and Protect Piglets against Diarrhea.

Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children's diarrhea and travelers' diarrhea. Vaccines inducing antibodies to broadly inhibit bacterial adherence and to neutralize toxin enterotoxicity are expected to be effective against ETEC-associated diarrhea. 6×His-tagged adhesin-toxoid fusion proteins were shown to induce neutralizing antibodies to several adhesins and LT and STa toxins (X. Ruan, D. A. Sack, W. Zhang, PLoS One 10:e0121623, 2015, https://doi.org/10.1371/journal.pone.0121623). However, antibodies derived from His-tagged CFA/I/II/IV-2xSTaA14Q-dmLT or CFA/I/II/IV-2xSTaN12S-dmLT protein were less effective in neutralizing STa enterotoxicity and were not evaluated in vivo for efficacy against ETEC diarrhea. Additionally, His-tagged proteins are considered less desirable for human vaccines. In this study, we produced a tagless adhesin-toxoid MEFA (multiepitope fusion antigen) protein, enhanced anti-STa immunogenicity by including a third copy of STa toxoid STaN12S, and examined antigen immunogenicity in a murine model. Moreover, we immunized pregnant pigs with the tagless adhesin-toxoid MEFA protein and evaluated passive antibody protection against STa+ or LT+ ETEC infection in a pig challenge model. Results showed that tagless adhesin-toxoid MEFA CFA/I/II/IV-3xSTaN12S-mnLTR192G/L211A induced broad antiadhesin and antitoxin antibody responses in the intraperitoneally immunized mice and the intramuscularly immunized pigs. Mouse and pig serum antibodies significantly inhibited adherence of seven colonization factor antigen (CFA) adhesins (CFA/I and CS1 to CS6) and effectively neutralized both toxins. More importantly, suckling piglets born to the immunized mothers acquired antibodies and were protected against STa+ ETEC and LT+ ETEC diarrhea. These results indicated that tagless CFA/I/II/IV-3xSTaN12S-mnLTR192G/L211A induced broadly protective antiadhesin and antitoxin antibodies and demonstrate that this adhesin-toxoid MEFA is a potential antigen for developing broadly protective ETEC vaccines.

A Plant-Produced Candidate Subunit Vaccine Reduces Shedding of Enterohemorrhagic Escherichia coli in Ruminants.

Enterohemorrhagic Escherichia coli (EHEC) are commonly present in the gastrointestinal tract of cattle and cause serious infectious disease in humans. Immunizing cattle against EHEC is a promising strategy to decrease the risk of food contamination; however, veterinary vaccines against EHEC such as Econiche have not been widely adopted by the agricultural industry, and have been discontinued, prompting the need for more cost-effective EHEC vaccines. The objective of this project is to develop a platform to produce plant-made antigens for oral vaccination of ruminants against EHEC. Five recombinant proteins were designed as vaccine candidates and expressed transiently in Nicotiana benthamiana and transplastomically in Nicotiana tabacum. Three of these EHEC proteins, NleA, Stx2b, and a fusion of EspA accumulated when transiently expressed. Transient protein accumulation was the highest when EHEC proteins were fused to an elastin-like polypeptide (ELP) tag. In the transplastomic lines, EspA accumulated up to 479 mg kg-1 in lyophilized leaf material. Sheep that were administered leaf tissue containing recombinant EspA shed less E. coli O157:H7 when challenged, as compared to control animals. These results suggest that plant-made, transgenic EspA has the potential to reduce EHEC shedding in ruminants.

A Chimeric protein of CFA/I, CS6 subunits and LTB/STa toxoid protects immunized mice against enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia Coli (ETEC) strains are the commonest bacteria causing diarrhea in children in developing countries and travelers to these areas. Colonization factors (CFs) and enterotoxins are the main virulence determinants in ETEC pathogenesis. Heterogeneity of CFs is commonly considered the bottleneck to developing an effective vaccine. It is believed that broad spectrum protection against ETEC would be achieved by induced anti-CF and anti-enterotoxin immunity simultaneously. Here, a fusion antigen strategy was used to construct a quadrivalent recombinant protein called 3CL and composed of CfaB, a structural subunit of CFA/I, and CS6 structural subunits, LTB and STa toxoid of ETEC. Its anti-CF and antitoxin immunogenicity was then assessed. To achieve high-level expression, the 3CL gene was synthesized using E. coli codon bias. Female BALB/C mice were immunized with purified recombinant 3CL. Immunized mice developed antibodies that were capable of detecting each recombinant subunit in addition to native CS6 protein and also protected the mice against ETEC challenge. Moreover, sera from immunized mice also neutralized STa toxin in a suckling mouse assay. These results indicate that 3CL can induce anti-CF and neutralizing antitoxin antibodies along with introducing CFA/I as a platform for epitope insertion.

A novel Salmonella strain inactivated by a regulated autolysis system and expressing the B subunit of Shiga toxin 2e efficiently elicits immune responses and confers protection against virulent Stx2e-producing Escherichia coli.

BACKGROUND: Salmonella Typhimurium (S. Typhimurium) inactivated by a regulated autolysis system was genetically engineered to express the homo-pentameric B subunit of Shiga toxin 2e (Stx2eB) on its surface. To prepare a strain able to yield autolyzed Salmonella bearing Stx2eB, the plasmid pJHL184 harboring stx 2eB gene was transformed into the attenuated S. Typhimurium strain, JOL1454. Stx2eB subcloned into the antigen delivery cassette of the plasmid was expressed as fusion protein with the outer membrane protein RESULTS: The expression of Stx2eB fused to the signal peptide in JOL1454 was validated by immunoblot analysis. To determine the immunogenicity of JOL1454, female BALB/c mice were intramuscularly injected with 1 × 108 CFU of the inactivated cells at weeks 0 and 2. Significantly elevated levels of IgG and IgA specific to Stx2eB was observed at weeks 4 and 6 post-immunization (PI) (P <0.05). Proportion of CD3+CD4+ T lymphocyte subpopulation was also significantly augmented in in vivo stimulated splenocytes relative to that in the control group. The increased titers of IgG1 and IgG2a, and of immunomodulatory cytokines indicated that the immunization elicited Th1 and Th2 immune responses. Further, immunomodulatory cytokine genes (IL-6, IL-17A, IL21 and JOL1454) efficiently upregulated in naïve porcine peripheral blood mononuclear cells (PBMCs) pulsed with JOL1454. At week 6 PI, following the challenge with a virulent Stx2e-producing Escherichia coli in the mice, all immunized mice survived whereas approximately 30% of the mice in the control group died. CONCLUSIONS: JOL1454 provided superior immunogenicity and effective protection against challenge with a sublethal dose, which demonstrates its potential as a candidate vaccine against edema disease.

Intranasal immunization with novel EspA-Tir-M fusion protein induces protective immunity against enterohemorrhagic Escherichia coli O157:H7 challenge in mice.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and hemolytic uremic syndrome in humans. Due to the risks associated with antibiotic treatment against EHEC O157:H7 infection, vaccines represent a promising method for prevention of EHEC O157:H7 infection. Therefore, we constructed the novel bivalent antigen EspA-Tir-M as a candidate EHEC O157:H7 subunit vaccine. We then evaluated the immunogenicity of this novel EHEC O157:H7 subunit vaccine. Immune responses to the fusion protein administered by intranasal and subcutaneous routes were compared in mice. Results showed higher levels of specific mucosal and systemic antibody responses induced by intranasal as compared to subcutaneous immunization. Intranasal immunization enhanced the concentration of interleukin-4, interleukin-10, and interferon-γ, while subcutaneous immunization enhanced only the latter two. In addition, intranasal immunization protected against EHEC O157:H7 colonization and infection in mice at a rate of 90%.Histopathological analysis revealed that vaccination reduced colon damage, especially when administered intranasally. In contrast, subcutaneous immunization elicited a weak immune response and exhibited a low protection rate. These findings demonstrate that intranasal immunization with the fusion protein induces both humoral and cellular immune (Th1/Th2) responses in mice. The novel EspA-Tir-M novel fusion protein therefore represents a promising subunit vaccine against EHEC O157:H7 infection.

Dimeric and Trimeric Fusion Proteins Generated with Fimbrial Adhesins of Uropathogenic Escherichia coli.

Urinary tract infections (UTIs) are associated with high rates of morbidity and mortality worldwide, and uropathogenic Escherichia coli (UPEC) is the main etiologic agent. Fimbriae assembled on the bacterial surface are essential for adhesion to the urinary tract epithelium. In this study, the FimH, CsgA, and PapG adhesins were fused to generate biomolecules for use as potential target vaccines against UTIs. The fusion protein design was generated using bioinformatics tools, and template fusion gene sequences were synthesized by GenScript in the following order fimH-csgA-papG-fimH-csgA (fcpfc) linked to the nucleotide sequence encoding the [EAAAK]5 peptide. Monomeric (fimH, csgA, and papG), dimeric (fimH-csgA), and trimeric (fimH-csgA-papG) genes were cloned into the pLATE31 expression vector and generated products of 1040, 539, 1139, 1442, and 2444 bp, respectively. Fusion protein expression in BL21 E. coli was induced with 1 mM IPTG, and His-tagged proteins were purified under denaturing conditions and refolded by dialysis using C-buffer. Coomassie blue-stained SDS-PAGE gels and Western blot analysis revealed bands of 29.5, 11.9, 33.9, 44.9, and 82.1 kDa, corresponding to FimH, CsgA, PapG, FC, and FCP proteins, respectively. Mass spectrometry analysis by MALDI-TOF/TOF revealed specific peptides that confirmed the fusion protein structures. Dynamic light scattering analysis revealed the polydispersed state of the fusion proteins. FimH, CsgA, and PapG stimulated the release of 372-398 pg/mL IL-6; interestingly, FC and FCP stimulated the release of 464.79 pg/mL (p ≤ 0.018) and 521.24 pg/mL (p ≤ 0.002) IL-6, respectively. In addition, FC and FCP stimulated the release of 398.52 pg/mL (p ≤ 0.001) and 450.40 pg/mL (p ≤ 0.002) IL-8, respectively. High levels of IgA and IgG antibodies in human sera reacted against the fusion proteins, and under identical conditions, low levels of IgA and IgG antibodies were detected in human urine. Rabbit polyclonal antibodies generated against FimH, CsgA, PapG, FC, and FCP blocked the adhesion of E. coli strain CFT073 to HTB5 bladder cells. In conclusion, the FC and FCP proteins were highly stable, demonstrated antigenic properties, and induced cytokine release (IL-6 and IL-8); furthermore, antibodies generated against these proteins showed protection against bacterial adhesion.

Multifaceted immune responses and protective efficacy elicited by a recombinant autolyzed Salmonella expressing FliC flagellar antigen of F18+Escherichia coli.

Porcine edema disease (ED) caused by F18+ Shiga toxin 2e-producing Escherichia coli (STEC) has imposed significant economic losses in the swine industry worldwide, resulting in sudden deaths in post-weaned piglets. The flagellin protein of F18+ STEC, a structural component of the flagellar filament, is a known virulence factor that mediates adhesion and invasion to porcine epithelial cells. In this study, Salmonella inactivated by the E lysis gene and expressing the flagellin (fliC) antigen was genetically engineered utilizing a plasmid (pMMP184) carrying an efficient heterologous antigen delivery system. The resulting strain JOL1485 producing FliC was successfully inactivated by the E lysis gene cassette. Following the lysis procedure, FliC secretion and production of JOL1485 was validated by immunoblot analysis. To evaluate protective immunogenicity elicited by the constructed strain, BALB/c mice were injected with 1×108 lysed cells via the intramuscular route. The markedly elevated titers of FliC-specific IgG, IgG1 and sIgA antibodies were observed, indicating a robust Th2-associated humoral immune response was raised in the immunized mice. The proportion of CD3+ CD4+ splenic T cells and proliferative activity were also elevated in in vivo and in vitro stimulated mice splenocytes. Further, JOL1485 successfully elicited upregulated gene expression of cytokines IL-6, IL-8, IL17, IL-21, IFN-γ and TNF-α in naïve porcine peripheral blood mononuclear cells (PBMCs). The overall immune response elicited by JOL1485 conferred a significant rise of protection against a lethal virulent F18+ STEC challenge whereas all non-immunized mice died following the challenge. Our results demonstrate that fliC efficiently expressed in the genetically inactivated Salmonella strain has immunostimulatory and protective effects against a F18+ STEC lethal challenge, and may be promising as a potential vaccine candidate against ED infection.

Construction and immunogenic properties of a chimeric protein comprising CfaE, CfaB and LTB against Enterotoxigenic Escherichia coli.

ETEC (Enterotoxigenic Escherichia coli) is a major cause of diarrhea in developing countries and children. ETEC has two virulence factors including colonization factors antigen (CFA) and labile enterotoxins (LTs). CFA/I consists the major pilin subunit CfaB and a minor adhesive subunit, CfaE. In this study a tripartite fusion protein containing CfaB, CfaE and LTB was designed. In silico analysis of the tertiary structure of the chimeric protein showed a protein with three main domains linked together with linkers. Linear and conformational B-cell epitopes were identified. A chimera consisting cfaB, cfaE and ltB(BET)was then synthesized with E. coli codon bias in pUC57 and sub cloned into pET32 vector. Recombinant protein was expressed and purified by affinity chromatography and confirmed by western blotting. Mice were immunized with recombinant protein and the antibody titer and specificity of the sera were analyzed by ELISA. The efficiency of the immune sera against ETEC was evaluated by binding assay and GM1-ELISA. VaxiJen analysis of the protein showed high antigenicity. Post-immune sera contained high titers of anti-BET IgG. Pretreatment of ETEC cells with sera from immunized mice decreased their ability to adhere to cells of the human colon adenocarcinoma cell line HT29.