Publications

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    ABSTRACT: A long-standing challenge in developing vaccines against enterotoxigenic Escherichia coli (ETEC), the most common bacteria causing diarrhea in children of developing countries and travelers to these countries, is to protect against heat-stable toxin type Ib (STa or hSTa). STa and heat-labile toxin (LT) are virulence determinants in ETEC diarrhea. LT antigens are often used in vaccine development, but STa has not been included because of its poor immunogenicity and potent toxicity. Toxic STa is not safe for vaccines, but only STa possessing toxicity is believed to be able to induce neutralizing antibodies. However, recent studies demonstrated that nontoxic STa derivatives (toxoids), after being fused to an LT protein, induced neutralizing antibodies, and suggested that different STa toxoids fused to an LT protein might exhibit different STa antigenic propensity. In this study, we selected 14 STa toxoids from a mini STa toxoid library based on toxicity reduction and reactivity to anti-native STa antibodies, and genetically fused each toxoid to a monomeric double mutant LT (dmLT) peptide for 14 STa-toxoid-dmLT toxoid fusions. These toxoid fusions were used to immunize mice, and were characterized for induction of anti-STa antibody response. Results showed different STa toxoids (in fusions) varied greatly in anti-STa antigenicity. Among them, STaN12S, STaN12T and STaA14H were the top toxoids in inducing anti-STa antibodies. In vitro neutralization assays indicated antibodies induced by fusion 3xSTaN12S-dmLT exhibited the greatest neutralizing activity against STa toxin. These results suggested 3xSTaN12S-dmLT is a preferred fusion antigen to induce anti-STa antibody response, and provided long-awaited information for effective ETEC vaccine development.
    Infection and immunity 02/2014; · 4.21 Impact Factor
  • Weiping Zhang
    Pathogenic Escherichia coi: Molecular and Cellular Microbiology, Edited by S. Morabito, 01/2014: chapter 15: pages 273-302; Caister Academic Press.
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    ABSTRACT: Diarrhea is the second leading cause of death to children younger than five years and continues to be a major threat to global health. Enterotoxigenic Escherichia coli (ETEC) are the most common bacteria causing diarrhea in developing countries. ETEC are able to attach to host small intestinal epithelial cells using bacterial colonization factor antigen (CFA) adhesins. This attachment helps to initiate the diarrheal disease. Vaccines inducing anti-adhesin immunity to block adherence of ETEC strains that express immunologically heterogeneous CFA adhesins are expected to protect against ETEC diarrhea. In this study, we created a CFA multiepitope fusion antigen (MEFA) carrying representative epitopes of CFA/I, CFA/II (CS1, CS2, CS3) and CFA/IV (CS4, CS5, CS6), examined its immunogenicity in mice, and assessed the potential of this MEFA as an anti-adhesin vaccine against ETEC. Mice intraperitoneally immunized with this CFA MEFA exhibited no adverse effects, and developed immune responses to CFA/I, CFA/II and CFA/IV adhesins. Moreover, after incubation with serum of the immunized mice, ETEC or E. coli strains expressing CFA/I, CFA/II or CFA/IV adhesins were significantly inhibited in adherence to Caco-2 cells. Our results indicated this CFA MEFA elicited antibodies that not only cross reacted to CFA/I, CFA/II and CFA/IV adhesins but also broadly inhibited adherence of E. coli strains expressing these seven adhesins, and suggested that this CFA MEFA could be a candidate to induce broad-spectrum anti-adhesin protection against ETEC diarrhea. Additionally, this antigen construction approach (creating a MEFA) may be generally used in vaccine development against heterogenic pathogens.
    Clinical and vaccine Immunology: CVI 12/2013; · 2.60 Impact Factor
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    ABSTRACT: Diarrhea is one of the most important bovine diseases. Enterotoxigenic Escherichia coli (ETEC) and bovine viral diarrhea virus (BVDV) are the major causes of diarrhea in calves and cattle. ETEC expressing K99 (F5) fimbriae and heat-stable type Ia (STa) toxin are the leading bacteria causing calf diarrhea, and BVDV causes diarrhea and other clinical illnesses in cattle of all ages. It is reported that maternal immunization with K99 fimbrial antigens provides passive protection to calves against K99-fimbrial ETEC, and that BVDB major structural protein E2 elicits antibodies neutralizing against BVDV viral infection. Vaccines inducing anti-K99 and anti-STa immunity would protect calves more effectively against ETEC diarrhea, and those also inducing anti-E2 neutralizing antibodies would protect calves and cattle against diarrhea caused by both ETEC and BVDV. In this study, we used ETEC K99 major subunit FanC as a backbone, genetically embedded STa toxoid STaP12F and the most antigenic B cell epitope and T cell epitope predicted from the BVDV E2 glycoprotein into FanC for multivalent antigen FanC-STa-E2, and examined immunogenicity of this multivalent antigen to assess vaccine potential against bovine diarrhea. Mice IP immunized with this multivalent antigen developed anti-K99, anti-STa, and anti-BVDV antibodies. Moreover, elicited antibodies showed neutralization activities, as they inhibited adherence of K99 fimbrial E. coli, neutralized STa toxin, and prevented homologous BVDV viral infection in vitro. Results from this study suggest this multiepitope fusion antigen can potentially be developed as vaccines for broad protection against bovine diarrhea, and the multiepitope fusion strategy may be generally applied for multivalent vaccine development against heterogeneous pathogens.
    Clinical and vaccine Immunology: CVI 05/2013; · 2.60 Impact Factor
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    ABSTRACT: Previous studies in piglets indicate that heat labile enterotoxin (LT) expression enhances intestinal colonization by K88 adhesin-producing enterotoxigenic Escherichia coli (ETEC) as wild-type ETEC adhered to intestinal epithelium in substantially greater numbers than did non-toxigenic constructs. Enzymatic activity of the toxin was also shown to contribute to the adhesion of ETEC and non-ETEC bacteria to epithelial cells in culture. To further characterize the contribution of LT to host cell adhesion, a nontoxigenic, K88-producing E. coli was transformed with either the gene encoding for LT holotoxin, a catalytically-attenuated form of the toxin [LT(R192G)], or LTB subunits, and resultant changes in bacterial adherence to IPEC-J2 porcine intestinal epithelial cells were measured. Strains expressing LT holotoxin or mutants were able to adhere in significantly higher numbers to IPEC-J2 cells than was an isogenic, toxin-negative construct. LT+ strains were also able to significantly block binding of a wild-type LT+ ETEC strain to IPEC-J2 cells. Adherence of isogenic strains to IPEC-J2 cells was unaltered by cycloheximide treatment, suggesting that LT enhances ETEC adherence to IPEC-J2 cells independent of host cell protein synthesis. However, pretreating IPEC-J2 cells with LT promoted adherence of negatively charged latex beads (a surrogate for bacteria which carry a negative change), which adherence was inhibited by cycloheximide, suggesting LT may induce a change in epithelial cell membrane potential. Overall, these data suggest that LT may enhance ETEC adherence by promoting an association between LTB and epithelial cells, and by altering the surface charge of the host plasma membrane to promote non-specific adherence.
    Veterinary Microbiology 02/2013; · 3.13 Impact Factor
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    ABSTRACT: Influenza A virus causes a highly contagious respiratory disease in a variety of avian and mammalian hosts, including humans and pigs. The primary means for preventing influenza epidemics is vaccination. Epitope-based vaccine represents a new approach to achieve protective immunity. The objective of this study was to construct and evaluate the immunogenicity of an epitope-based antigen for its potential application in future influenza vaccine development. The antigen, comprised of a set of consensus influenza A virus epitopes (IAVe), was genetically linked to a subunit of the bacterial heat-labile enterotoxin (LTB) as an adjuvant. Immunogenicity of this LTB-IAVe antigen was evaluated in a pig model. Despite an inability to detect neutralizing antibodies directed toward the whole virus, humoral immunity against the IAVe was demonstrated in both serum (IgA and IgG) and mucosal secretions (IgG) of immunized pigs. Specific cellular immunity was also induced after LTB-IAVe immunization, as evidenced by up-regulating of IL-1β, IL-8, and IL-4 expression in peripheral blood mononuclear cells (PBMCs) of vaccinated pigs. In comparison to the non-immunized pigs, pigs immunized with the LTB-IAVe showed improved protection against a pathogenic H1N1 swine influenza virus challenge, with about 50% decrease of pneumonic lesions and 10-fold reduction of the viral load in lung and nasal secretion at five days post challenge. This study establishes a platform for future construction of epitope-based vaccines against influenza A virus infection.
    Veterinary Microbiology 02/2013; · 3.13 Impact Factor
  • Xiaosai Ruan, Weiping Zhang
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    ABSTRACT: ETEC strains expressing K88 (F4) or F18 fimbriae and enterotoxins are the predominant cause of porcine post-weaning diarrhea (PWD). PWD continues causing significant economic losses to swine producers worldwide. Vaccines effectively protecting against PWD are needed. Our recent study revealed that a tripartite adhesin-toxin monomer (FaeG-FedF-LT(A2-B)) elicited protective antibodies. In this study, we constructed a new adhesin-toxoid fusion, expressed it as a 1A:5B holotoxin-structured antigen (1FaeG-FedF-LT(192A2):5LT(B)) in an avirulent Escherichia coli strain, and evaluated its vaccine potential in pig challenge studies. Piglets orally inoculated with this live strain showed no adverse effects but developed systemic and mucosal antibodies that neutralized cholera toxin and inhibited adherence of K88 and F18 fimbriae in vitro. Moreover, the immunized piglets, when were challenged with ETEC strain 3030-2 (K88ac/LT/STb), had significant fewer bacteria colonized at small intestines and did not develop diarrhea; whereas the control piglets developed severe diarrhea and died. These results indicated the 1FaeG-FedF-LT(192A2):5LT(B) fusion antigen induced protective antiadhesin and antitoxin immunity in pigs, and suggested a live attenuated vaccine can be potentially developed against porcine ETEC diarrhea. Additionally, presenting antigens in a holotoxin structure to target host local mucosal immunity can be used in vaccine development against other enteric diseases.
    Vaccine 01/2013; · 3.77 Impact Factor
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    ABSTRACT: Diarrhea is the second leading cause of death to young children. Enterotoxigenic Escherichia coli (ETEC) are the most common bacteria causing diarrhea. Adhesins and enterotoxins are the virulence determinants in ETEC diarrhea. Adhesins mediate bacterial attachment and colonization, and enterotoxins including heat-labile (LT) and heat-stable type Ib toxin (STa) disrupt fluid homeostasis in host cells that leads to fluid hyper-secretion and diarrhea. Thus, adhesins and enterotoxins have been primarily targeted in ETEC vaccine development. A recent study reported toxoid fusions with STa toxoid (STaP13F) fused at the N- or C-terminus, or inside the A subunit of LTR192G elicited neutralizing antitoxin antibodies, and suggested application of toxoid fusions in ETEC vaccine development (Liu et al., Infect. Immun. 79:4002-4009, 2011). In this study, we generated a different STa toxoid (STaA14Q) and a triple-mutant LT toxoid (LTS63K/R192G/L211A, tmLT), constructed a toxoid fusion (3xSTaA14Q-tmLT) that carried 3 copies of STaA14Q for further facilitation of anti-STa immunogenicity, and assessed antigen safety and immunogenicity in a murine model to explore its potential for ETEC vaccine development. Mice immunized with this fusion antigen showed no adverse effects, and developed antitoxin antibodies particularly through the IP route. Anti-LT antibodies were detected and were shown neutralizing against CT in vitro. Anti-STa antibodies were also detected in the immunized mice, and serum from the IP immunized mice neutralized STa toxin in vitro. Data from this study indicated that toxoid fusion 3xSTaA14Q-tmLT is safe and can induce neutralizing antitoxin antibodies, and provided helpful information for vaccine development against ETEC diarrhea.
    PLoS ONE 01/2013; 8(10):e77386. · 3.53 Impact Factor
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    ABSTRACT: F18 fimbriae and toxins produced by F18 fimbriae-carrying Escherichia coli (E. coli) strains are known virulence factors responsible for post-weaning diarrhea (PWD) and edema disease (ED). In this study, we showed that fliC isogenic mutants constructed in two reference wild-type F18 fimbriae (F18+) E. coli were markedly impaired in adherence in vitro cell models (p < 0.05). Flagella purified from F18+E. coli could directly bind to cultured piglet epithelial cells and block adherence of F18+E. coli to cells when pre-incubated. In addition, the F18+E. coli fliC deletion mutants up-regulated the expression of type I fimbriae produced by F18+E. coli strains. These results demonstrated that expression of flagella is essential for the adherence of F18+E. coli in vitro.
    Microbial Pathogenesis 10/2012; · 1.97 Impact Factor
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    Weiping Zhang, David A Sack
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    ABSTRACT: Diarrhea is the second leading cause of death in children younger than 5 years. Enterotoxigenic Escherichia coli (ETEC) strains are the most common bacterial cause of diarrhea in young children living in endemic countries and children and adults traveling to these areas. Pathogenesis of ETEC diarrhea has been well studied, and the key virulence factors are bacterial colonization factor antigens and enterotoxins produced by ETEC strains. Colonization factor antigens mediate bacteria attachment to host small intestinal epithelial cells and subsequent colonization, whereas enterotoxins including heat-labile and heat-stable toxins disrupt fluid homeostasis in host epithelial cells, which leads to fluid and electrolyte hypersecretion and diarrhea. Vaccines stimulating host anti-adhesin immunity to block ETEC attachment and colonization and also antitoxin immunity to neutralize enterotoxicity are considered optimal for prevention of ETEC diarrhea. Vaccines under development have been designed to stimulate local intestinal immunity and are either oral vaccines or transcutaneous vaccines. A cholera vaccine (Dukoral®) does stimulate anti-heat-labile toxin immunity and is licensed for short-term protection of ETEC diarrhea in travelers in some countries. Newer experimental ETEC vaccine candidates are being developed with hope to provide long-lasting and more broad-based protection against ETEC. Some have shown promising results in safety and immunogenicity studies and are approaching field trials for efficacy. A key problem is the development of a vaccine that is both practical and inexpensive so that it can be affordable for use in poor countries where it is needed.
    Expert Review of Vaccines 06/2012; 11(6):677-94. · 4.22 Impact Factor
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    ABSTRACT: Bacterial flagella contribute to pathogen virulence; however, the role of flagella in the pathogenesis of F18ab E. coli-mediated swine edema disease (ED) is not currently known. We therefore evaluated the role of flagella in F18ab E. coli adhesion, invasion, biofilm formation, and IL-8 production using an in vitro cell infection model approach with gene-deletion mutant and complemented bacterial strains. We demonstrated that the flagellin-deficient fliC mutant had a marked decrease in the ability to adhere to and invade porcine epithelial IPEC-J2 cells. Surprisingly, there was no difference in adhesion between the F18 fimbriae-deficient ΔfedA mutant and its parent strain. In addition, both the ΔfedA and double ΔfliCΔfedA mutants exhibited an increased ability to invade IPEC-J2 cells compared to the wild-type strain, although this may be due to increased expression of other adhesins following the loss of F18ab fimbriae and flagella. Compared to the wild-type strain, the ΔfliC mutant showed significantly reduced ability to form biofilm, whereas the ΔfedA mutant increased biofilm formation. Although ΔfliC, ΔfedA, and ΔfliCΔfedA mutants had a reduced ability to stimulate IL-8 production from infected Caco-2 cells, the ΔfliC mutant impaired this ability to a greater extent than the ΔfedA mutant. The results from this study clearly demonstrate that flagella are required for efficient F18ab E. coli adhesion, invasion, biofilm formation, and IL-8 production in vitro.
    Veterinary Microbiology 05/2012; 160(1-2):132-40. · 3.13 Impact Factor
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    ABSTRACT: Virulence of enterotoxigenic Escherichia coli (ETEC) is associated with fimbrial adhesins and enterotoxins such as heat-labile (LT) and/or heat-stable (ST) enterotoxins. Previous studies using a cell culture model suggest that exclusion of ETEC from attachment to epithelial cells requires expression of both an adhesin such as K88 (F4) fimbriae, and LT. To test the ability of non-pathogenic E. coli constructs to exclude virulent ETEC sufficiently to prevent clinical disease, we utilized a piglet ETEC challenge model. Thirty-nine 5-day-old piglets were inoculated with a placebo (control), or with either of the three K88(+)E. coli strains isogenic with regard to modified LT expression: 8017 (pBR322 plasmid vector control), non-toxigenic mutant 8221 (LT(R192G)) in pBR322, or 8488, with the LT gene fused to the STb gene in pBR322 (LT(R192G)-STb). Piglets were challenged with virulent ETEC Strain 3030-2 (K88(+)/LT/STb) 24h post-inoculation. K88ac receptor-positive piglets in the control group developed diarrhea and became dehydrated 12-24h post-challenge. Piglets inoculated with 8221 or 8488 did not exhibit clinical signs of ETEC disease; most piglets inoculated with 8017 showed diarrhea. Control pigs exhibited significant weight loss, increased blood total protein, and higher numbers of colony-forming units of 3030-2 E. coli in washed ileum and jejunum than treated pigs. This study shows for the first time that pre-inoculation with an avirulent strain expressing adhesive fimbriae and a non-toxic form of LT provides significant short term protection from challenge with a virulent ETEC strain that expresses the same fimbrial adhesion and enterotoxin.
    Veterinary Microbiology 04/2012; 159(3-4):337-42. · 3.13 Impact Factor
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    ABSTRACT: Enterotoxigenic Escherichia coli (ETEC) strains are the leading bacterial cause of diarrhea to humans and farm animals. These ETEC strains produce heat-labile toxin (LT) and/or heat-stable toxins that include type I (STa), type II (STb), and enteroaggregative heat-stable toxin 1 (EAST1). LT, STa, and STb (in pigs) are proven the virulence determinants in ETEC diarrhea. However, significance of EAST1 in ETEC-associated diarrheal has not been determined, even though EAST1 is highly prevalent among ETEC strains. In this study, we constructed E. coli strains to express EAST1 toxin as the only toxin and studied them in cell lines and five-day old gnotobiotic piglets to determine significance of EAST1 toxin. Data from in vitro studies indicated that EAST1 did not stimulate an increase of intracellular cyclic AMP or GMP levels in T-84 cells or porcine cell line IPEC-J2, nor did it enhance LT or STa toxin of ETEC strains in stimulation of cAMP or cGMP in T-84 cells. In addition, 5-day old gnotobiotic pigs challenged with E. coli strains expressing EAST1 as the only toxin did not developed diarrhea or signs of clinical disease during 72 h post-inoculation. Results from this study indicated that EAST1 alone is not sufficient to cause diarrhea in five-day old gnotobiotic pigs, and suggest that EAST1 likely is not a virulence determinant in ETEC-associated diarrhea.
    PLoS ONE 01/2012; 7(8):e43203. · 3.53 Impact Factor
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    ABSTRACT: Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrhea in humans and animals. Heat-stable (STa) and heat-labile (LT) enterotoxins produced by ETEC disrupt fluid homeostasis in host small intestinal epithelial cells and cause fluid and electrolyte hyper-secretion that leads to diarrhea. ETEC strains producing STa or LT are sufficiently virulent to cause diarrhea, therefore STa and LT antigens must be included in ETEC vaccines. However, potent toxicity and poor immunogenicity (of STa) prevent them from being directly applied as vaccine components. While LT toxoids, especially LT(R192G), being used in vaccine development, STa toxoids have not been included. A recent study (IAI, 78:316-325) demonstrated porcine-type STa toxoids [pSTa(P12F) and pSTa(A13Q)] elicited protective anti-STa antibodies after being fused to a porcine-type LT toxoid [pLT(R192G)]. In this study, we substituted the 8th, 9th, 16th, or the 17th amino acid of a human-type STa (hSTa) and generated 28 modified STa peptides. We tested each STa peptide for toxicity and structure integrity, and found nearly all modified STa proteins showed structure alteration and toxicity reduction. Based on structure similarity and toxic activity, three modified STa peptides: STa(E8A), STa(T16Q) and STa(G17S), were selected to construct LT(192)-STa(-toxoid) fusions. Constructed fusions were used to immunize mice, and immunized mice developed anti-STa antibodies. Results from this study provide useful information in developing toxoid vaccines against ETEC diarrhea.
    Toxins 09/2011; 3(9):1146-62. · 2.13 Impact Factor
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    ABSTRACT: Enterotoxigenic Escherichia coli (ETEC) strains expressing K88 (F4) or F18 fimbriae and heat-labile (LT) and/or heat-stable (ST) toxins are the major cause of diarrhea in young pigs. Effective vaccines inducing antiadhesin (anti-K88 and anti-F18) and antitoxin (anti-LT and anti-ST) immunity would provide broad protection to young pigs against ETEC. In this study, we genetically fused nucleotides coding for peptides from K88ac major subunit FaeG, F18 minor subunit FedF, and LT toxoid (LT(192)) A2 and B subunits for a tripartite adhesin-adhesin-toxoid fusion (FaeG-FedF-LT(192)A2:B). This fusion was used for immunizations in mice and pigs to assess the induction of antiadhesin and antitoxin antibodies. In addition, protection by the elicited antiadhesin and antitoxin antibodies against a porcine ETEC strain was evaluated in a gnotobiotic piglet challenge model. The data showed that this FaeG-FedF-LT(192)A2:B fusion elicited anti-K88, anti-F18, and anti-LT antibodies in immunized mice and pigs. In addition, the anti-porcine antibodies elicited neutralized cholera toxin and inhibited adherence against both K88 and F18 fimbriae. Moreover, immunized piglets were protected when challenged with ETEC strain 30302 (K88ac/LT/STb) and did not develop clinical disease. In contrast, all control nonvaccinated piglets developed severe diarrhea and dehydration after being challenged with the same ETEC strain. This study clearly demonstrated that this FaeG-FedF-LT(192)A2:B fusion antigen elicited antibodies that neutralized LT toxin and inhibited the adherence of K88 and F18 fimbrial E. coli strains and that this fusion could serve as an antigen for vaccines against porcine ETEC diarrhea. In addition, the adhesin-toxoid fusion approach used in this study may provide important information for developing effective vaccines against human ETEC diarrhea.
    Clinical and vaccine Immunology: CVI 08/2011; 18(10):1593-9. · 2.60 Impact Factor
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    ABSTRACT: Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals. Adhesins and enterotoxins, including heat-labile (LT) and heat-stable (STa) toxins, are the key virulence factors. Antigenic adhesin and LT antigens have been used in developing vaccines against ETEC diarrhea. However, STa has not been included because of its poor immunogenicity and potent toxicity. Our recent study showed that porcine-type STa toxoids became immunogenic and elicited neutralizing anti-STa antibodies after being genetically fused to a full-length porcine-type LT toxoid, LT(R₁₉₂G) (W. Zhang et al., Infect. Immun. 78:316-325, 2010). In this study, we mutated human-type LT and STa genes, which are highly homologous to porcine-type toxin genes, for a full-length LT toxoid (LT(R₁₉₂)) and a full-length STa toxoid (STa(P₁₃F)) and genetically fused them to produce LT₁₉₂-STa₁₃ toxoid fusions. Mice immunized with LT₁₉₂-STa₁₃ fusion antigens developed anti-LT and anti-STa IgG (in serum and feces) and IgA antibodies (in feces). Moreover, secretory IgA antibodies from immunized mice were shown to neutralize STa and cholera toxins in T-84 cells. In addition, we fused the STa₁₃ toxoid at the N terminus and C terminus, between the A1 and A2 peptides, and between the A and B subunits of LT₁₉₂ to obtain different fusions in order to explore strategies for enhancing STa immunogenicity. This study demonstrated that human-type LT₁₉₂-STa₁₃ fusions induce neutralizing antitoxin antibodies and provided important information for developing toxoid vaccines against human ETEC diarrhea.
    Infection and immunity 07/2011; 79(10):4002-9. · 4.21 Impact Factor
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    Chengxian Zhang, Weiping Zhang
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    ABSTRACT: Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals. Bacterial adhesins and heat-labile (LT) and heat-stable (ST) enterotoxins are the virulence determinants in ETEC diarrhea. It is believed that vaccines inducing anti-adhesin immunity to inhibit bacterial adherence and anti-toxin immunity to eliminate toxin activity would provide broad-spectrum protection against ETEC. In this study, an ETEC fimbrial adhesin was used as a platform to express LT and STa for adhesin-toxin fusion antigens to induce anti-toxin and anti-adhesin immunity. An epitope from the B subunit of LT toxin (LTP1, (8)LCSEYRNTQIYTIN(21)) and an STa toxoid epitope ((5)CCELCCNPQCAGCY(18)) were embedded in the FaeG major subunit of E. coli K88ac fimbriae. Constructed K88ac-toxin chimeric fimbriae were harvested and used for rabbit immunization. Immunized rabbits developed anti-K88ac, anti-LT, and anti-STa antibodies. Moreover, induced antibodies not only inhibited adherence of K88ac fimbrial E. coli to porcine small intestinal enterocytes but also neutralized cholera toxin and STa toxin. Data from this study demonstrated that K88ac fimbriae expressing LT and STa epitope antigens elicited neutralizing anti-toxin antibodies and anti-adhesin antibodies and suggested that E. coli fimbriae could serve as a platform for the development of broad-spectrum vaccines against ETEC.
    Clinical and vaccine Immunology: CVI 10/2010; 17(12):1859-67. · 2.60 Impact Factor
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    Weiping Zhang, David H Francis
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    ABSTRACT: Enterotoxigenic Escherichia coli (ETEC)-associated diarrhea causes a substantial economic loss to swine producers worldwide. The majority of ETEC strains causing porcine diarrhea, especially postweaning diarrhea (PWD), produce heat-labile toxin (LT) and heat-stable toxin b (STb). LT is commonly used in vaccine development, but STb has not been included because of its poor immunogenicity. As a virulence factor in porcine diarrhea, STb needs to be included as an antigen for development of broad-spectrum vaccines. In this study, we used an LT toxoid (LT(R192G) [hereafter, LT(192)]) derived from porcine ETEC to carry a mature STb peptide for LT(192)-STb fusions to enhance STb immunogenicity for potential vaccine application. Anti-LT and anti-STb antibodies were detected in immunized rabbits and pigs. In addition, when challenged with an STb-positive ETEC strain, all 10 suckling piglets borne by immunized gilts remained healthy, whereas 7 out 9 piglets borne by unimmunized gilts developed moderate diarrhea. This study indicates that the LT(192)-STb fusion enhanced anti-STb immunogenicity and suggests the LT(192)-STb fusion antigen can be used in future vaccine development against porcine ETEC diarrhea.
    Clinical and vaccine Immunology: CVI 08/2010; 17(8):1223-31. · 2.60 Impact Factor
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    ABSTRACT: Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and farm animals. E. coli fimbriae, or colonization factor antigens (CFAs), and enterotoxins, including heat-labile enterotoxins (LT) and heat-stable enterotoxins (ST), are the key virulence factors in ETEC diarrhea. Unlike fimbriae or LT, STa has not often been included as an antigen in development of vaccines against ETEC diarrhea because of its poor immunogenicity. STa becomes immunogenic only after being coupled with a strongly immunogenic carrier protein. However, native or shorter STa antigens either had to retain toxic activity in order to become antigenic or elicited anti-STa antibodies that were not sufficiently protective. In this study, we genetically mutated the porcine LT (pLT) gene for a pLT(192(R-->G)) toxoid and the porcine STa (pSTa) gene for three full-length pSTa toxoids [STa(11(N-->K)), STa(12(P-->F)), and STa(13(A-->Q))] and used the full-length pLT(192) as an adjuvant to carry the pSTa toxoid for pLT(192):pSTa-toxoid fusion antigens. Rabbits immunized with pLT(192):pSTa(12) or pLT(192):pSTa(13) fusion protein developed high titers of anti-LT and anti-STa antibodies. Furthermore, rabbit antiserum and antifecal antibodies were able to neutralize purified cholera toxin (CT) and STa toxin. In addition, preliminary data suggested that suckling piglets born by a sow immunized with the pLT(192):pSTa(13) fusion antigen were protected when challenged with an STa-positive ETEC strain. This study demonstrated that pSTa toxoids are antigenic when fused with a pLT toxoid and that the elicited anti-LT and anti-STa antibodies were protective. This fusion strategy could provide instructive information to develop effective toxoid vaccines against ETEC-associated diarrhea in animals and humans.
    Infection and immunity 10/2009; 78(1):316-25. · 4.21 Impact Factor
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    Chengxian Zhang, Dana Rausch, Weiping Zhang
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    ABSTRACT: To examine whether the heat-labile enterotoxin gene in porcine enterotoxigenic Escherichia coli (ETEC) strains is as divergent as in human ETEC strains, we sequenced the heat-labile and heat-stable toxin genes from 52 and 33 porcine ETEC strains, respectively. We found that the STa gene is identical, that the LT gene has only two mutations in 4 (of 52) strains, and that both mutations cause a reduction in GM1 binding and toxicity.
    Applied and Environmental Microbiology 09/2009; 75(19):6402-5. · 3.95 Impact Factor

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