Maintenance of the STa structure, especially the epitope topology in fusions or conjugates is believed to be important in eliciting neutralizing anti-STa antibodies (communication with Dr. elicited protective ABT-639 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. ABT-639 Based on structure similarity and toxic activity, three modified STa peptides: STa(E8A), STa(T16Q) and STa(G17S), were selected to construct LT192-STa-toxoidfusions. Constructed fusions were used to immunize mice, and JAK1 immunized mice developed anti-STa antibodies. Results from this study provide useful information in developing toxoid vaccines against ETEC diarrhea. Keywords:STa toxin, ETEC, toxoid, LT-STa toxoid fusion, vaccine, diarrhea == 1. Introduction == A recent systematic survey indicated that diarrhea kills up to 2 million children under the age of five, more than AIDS, malaria and measles combined [1,2]. EnterotoxigenicEscherichia coli(ETEC) strains that colonize host small intestines and produce one or two enterotoxins are the major bacterial cause of childrens diarrhea, and are responsible for approximately 200 million episodes of diarrhea and 380, 000 deaths annually [3]. In addition, ETEC strains are also the most common cause of travellers diarrhea [4,5]. Although experimental vaccines currently under development show promising, no broadly effective vaccines are available to protect humans and animals against ETEC diarrhea. The key virulence factors of ETEC in diarrhea are colonization factors and enterotoxins [6,7,8,9,10,11,12]. Colonization factors mediate initial attachment of ETEC bacteria to host small intestinal enterocytes and subsequent colonization. Enterotoxins known as heat-stable (STa) and heat-labile (LT) toxins disrupt fluid homeostasis and cause fluid and electrolyte hyper-secretion through activation of adenyl cyclase (by LT) or guanylate cyclase (by STa) in small intestinal epithelial cells that leads to diarrhea [13,14]. LT and STa toxins, in addition to colonization factors are proven the virulence determinants in ETEC diarrhea. LT is a typical 1A:5B toxin that consists of one enzymatic A subunit (25.5 KDa) and five identical GM1-binding B subunits (12 KDa), and is strongly immunogenic. In contrast, STa associated with human diarrhea (hSTa; STa used in following context is referred as hSTa) is a small peptide of only 19 amino acids (2 KDa) and is poorly immunogenic [10]. Epidemiological and clinical studies showed that one half of human diarrheal cases associated with ETEC are caused by strains that produce STa toxin only, a quarter express LT only, and another quarter produce both toxins [15,16]. Human volunteers and animal challenge studies demonstrated that an enterotoxigenicE. colistrain expressing either STa or LT toxin is sufficiently virulent to cause diarrhea [10,11,12,17]. Therefore, both STa and LT toxins must be targeted in vaccine development against ETEC diarrhea [18,19,20,21]. Indeed, experimental vaccine studies indicated that anti-LT immunity alone is not sufficiently effective in protection against ETEC. Vaccine candidates carrying LT antigens provided protection against only LT-producing ETEC strains, but not against ETEC strains expressing STa toxin [22,23]. It becomes commonly acknowledged that anti-STa immunity must also ABT-639 be induced by vaccines for effective protection against ETEC diarrhea. The potent toxicity of LT and STa, however, must be attenuated, and the poor immunogenicity of STa must be enhanced before LT and STa can be used as safe and effective vaccine antigens. LT toxoids, especially LT(R192G) that has the Arg192substituted with Gly, have toxicity much reduced but LT immunogenicity (and ABT-639 adjuvanticity) maintained and were used as antigens in ETEC vaccine development [24,25,26]. This LT(R192G) has also been used as an adjuvant to enhance immunogenicity of otherwise poorly immunogenic antigens [27,28,29,30,31,32]. In contrast, although early studies demonstrated a few modified STa peptides including STa12, STa13and STa14showed toxicity reduction [33,34], STa toxoids have not been included in ETEC vaccine development until recently [35,36]. Recent studies showed that porcine-type STa toxoids, pSTa(P12F) and pSTa(A13Q) which are the analogues of human-type STa13and STa14, had anti-STa immunogenicity enhanced and elicited protective anti-STa antibodies after being genetically fused to toxoid LT(R192G) [35] orE. coliK88ac fimbriae [36]. Candidacy of other STa toxoids in vaccine application, however, has not been evaluated. In addition, studies showed that different STa toxoids exhibited differences in toxicity reduction [34] and likely structure alteration [35]. Further studies.