Recipient Organization
SOUTH DAKOTA STATE UNIVERSITY
PO BOX 2275A
BROOKINGS,SD 57007
Performing Department
Veterinary & Biomedical Sciences
Non Technical Summary
Porcine diarrhea disease causes significant economic loss to the swine industry. The principal sign in diarrhea is a fluid, yellow or grey diarrhea, lasting for a week or more and resulting in slow growth or death. This diarrhea is caused by infection of ETEC strains that typically express virulence factors known as fimbriae or colonization factors (CFA) and enterotoxins (LT and ST). The vast majority of ETEC diarrhea in porcine are caused by E. coli strains express K88 or F18 fimbriae. These fimbriae mediate the attachment of bacteria to the epithelium cells in porcine small intestine and initiate E. coli colonization. Purified fimbriae or subunit proteins, as well as truncated fimbriae have been used to develop vaccines. However, most of these vaccines resulted in only partial protection against porcine ETEC infection. LT enterotoxin consists of one A subunit (28 kDa) and five B subunits (12.8 kDa each). The five B subunits arrange in a circle and anchore the A subunit in the center to form a structure called holotoxin. The B subunits bind the ganglioside GM1 in the host cell membrane, and the enzymatic active A subunit stimulates fluid secretion. LT toxins from the human and porcine ETEC strains are highly homologous. Differing from the large LT toxin, ST toxins are small. STa is an 18- (in porcine) or 19- (in human) amino-acid peptide with a molecular mass of about 2 kDa (Fig. 1). STa toxin binds to an intestinal epithelial receptor, guanylate cyclase type C (GC-C), then utilizes the GC-C to alter ion transport in gut, and results in increased levels of intracellular cyclic GMP. The activation of cyclic GMP stimulates chloride secretion and inhabits NaCl absorption, and causes net intestinal fluid secretion In contrast to LT, STa has not much been targeted as an antigen for vaccine development, simply because STa is poorly immunogenic. However, studies demonstrated that genetic fusions between STa and several carrier proteins, such as LTA or LTB subunit, facilitated the immunogenicity and antigenicity of STa. The STa-fusion strategy overcomes the poor immunogenicity of STa. However, fusion of a native STa maintains a fully activated STa and is expected to retain its toxicity in fusion products. The possession of its toxicity makes fusion STa an undesirable antigen for vaccine development. Thus, it is a must to reduce or eliminate the toxicity of STa before we can apply STa as an antigen in vaccine development.
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Goals / Objectives
Enterotoxigenic Escherichia coli (ETEC)-associated diarrhea is economically one of the most important diseases for the swine industry, with an estimate of nearly $ 100 million loss each year to swine producers in the U. S. alone. ETEC strains produce fimbriae for their attachment to the porcine small intestine and bacterial colonization, and secrete enterotoxins, heat-labile (LT) and heat-stable (pSTa and pSTb in porcine ETEC, whereas hSTa in human) toxins, into the small intestines to stimulate fluid hypersecretion. ETEC diarrhea is characterized by severe diarrhea, dehydration, weight reduction, and death. Currently, there are no commercially available vaccines to effectively protect the human and pigs from ETEC diarrheal disease. Therefore, there is a great need for vaccine development against ETEC diarrhea. Efforts have been made in past to develop vaccines against ETEC diarrhea. However, conventional vaccine strategies emphasize more on LT as antigens since the LT is strongly immunogenic and antigenic. The poorly immunogenic STs, especially STa, have not much been included. Consequently, vaccines developed from the LT antigen provide protection against infection only caused by LT-producing ETEC strains, but could not extend their protection against ETEC strains which produce ST toxins. Since vast majority of the diarrheagenic ETEC strains produce LT/STa or STa toxin, STa antigen must be included for developing effective vaccines against ETEC diarrhea. Two key technique obstacles must be overcome to include STa as a vaccine component. First, STa is a small molecule (18 amino acids in porcine STa, and 19-AA in human STa) and is poorly immunogenic. The poor immunogenicity prevents STa antigens from producing antibodies, thus cannot be a vaccine component. Second, STa itself is sufficiently toxic to cause diarrhea. Therefore, native STa is not safe to be used directly as antigens. However, with current molecular techniques, we have been resolved both problems in our laboratory. First, with construction of genetic fusions between pSTa and a pLT mutant carrier protein, we successfully enhanced the immunogenicity of pSTa and made it suitable for antibody stimulation. Second, by genetic manipulation of the pSTa toxin gene, we generated pSTa mutants with substantially reduced toxicity. Such a low-toxic pSTa mutant is a desirable antigen for developing safe vaccines. By overcoming two key technique challenges, we are at excellent position to develop effective vaccines against ETEC. The purposes of this research project are: 1) To conduct anti-STa antibody neutralization assay to assess protection from anti STa antibodies against ETEC strains; 2) To apply pLT:pSTa fusion strategy in vaccine development against human ETEC diarrhea. In projective 1, we will use anti-pLT:pSTa antibodies from rabbit sera to neutralize native STa toxin, and assess protection from these antibodies against infection of STa-producing ETEC strains. In projective 2, we will generate a a genetic a fusion of the hLT and hSTa proteins, test anti-STa antigenicity, anti-STa antibody neutralization and protection against infection of human ETEC strains.
Project Methods
To conduct anti STa antibody neutralization assay to assess protection from anti STa antibodies against STa-positive ETEC strains. Rabbits immunized with the purified "pLT192:pSTa12" and "pLT192:pSTa13" fusion proteins produced a significantly high level of anti STa antibodies. Now the question is whether these antibodies are protective, because only protective antibodies can protect against infection. To test if anti STa antibodies that we produced from the fusion antigen are protective is to demonstrate if they can neutralize native STa toxin. Therefore, we propose to conduct in vitro and in vivo assays: 1) a cyclic GMP ELISA, we hypothesize that these antibodies will neutralize native STa and thus prevent STa toxin from stimulate cGMP level in host cells; and 2) a gut loop assay, we hypothesize that these antibodies will neutralize STa toxicity and thus prevent STa toxin from stimulating fluid secretion in porcine gut loops. to apply this porcine "LT:STa" fusion strategy in vaccine development against human ETEC diarrhea in a piglet model: Up to three quarters of human ETEC strains produce hSTa alone or together with hLT toxin. Anti STa antibodies must be included in human ETEC vaccines. However, hSTa is also non-immunogenic and thus itself cannot stimulate anti-STa antibody production. We hypothesize that genetic fusions of hLT mutant (hLT192) with a nontoxic hSTa mutant (hSTa13 and hSTa14, corresponding to pSTa12 and pSTa13) will significantly increase STa immunogenicity and antigenicity, and produce high titer of anti STa antibodies. The human LT192 mutant has been explored as an antigen and/or immunogen. In this aim, we will isolate human eltAB and estA genes from a human ETEC prototype H10407, and mutate the eltAB gene at nucleotides coding amino acid #192, and the estB gene at nucleotides coding amino acid #13 and #14, respectively. The mutated genes will be genetically fused for chimeric gene to produce "hLT192:hSTa13" and "hLT192:hSTa14" fusion proteins. Each fusion protein will be purified and be used to immunize adult rabbits for anti STa antibody production.