Attenuated Shigella flexneri 2a Delta guaBA strain CVD 1204 expressing enterotoxigenic Escherichia coli (ETEC) CS2 and CS3 fimbriae as a live mucosal vaccine against Shigella and ETEC infection.

Two bacterial enteric pathogens that have been identified by the World Health Organization as constituting important targets for the development of vaccines are enterotoxigenic Escherichia coli (ETEC) and Shigella (35, 38). In developing countries, ETEC is a major cause of diarrheal dehydration in infants (4), whereas Shigella is the main agent of bacillary dysentery in young children (35). Both pathogens contribute in a major way to the mortality burden attributable to enteric pathogens (4, 35). ETEC is also the most frequent etiologic agent associated with traveler's diarrhea (3, 14, 29, 51), whereas in many studies Shigella is often the second most incriminated pathogen (14, 29). Traveler's diarrhea caused by Shigella tends to be clinically more severe and debilitating than that caused by ETEC. Both ETEC and Shigella are deemed to be worthy targets for immunoprophylaxis of travelers from industrialized countries who visit developing regions of the world (45). Among the promising candidate vaccines against Shigella are parenteral O polysaccharide-carrier protein conjugates (7, 8), intranasally administered proteosomes consisting of outer membrane protein vesicles of group B Neisseria meningitidis to which Shigella lipopolysaccharide is noncovalently bound (47, 48), and attenuated strains of Shigella used as live oral vaccines (9, 34). Within the four Shigella species (also referred to as groups), 39 main serotypes and subtypes are recognized (15, 35), and epidemiologic and experimental observations indicate that immunity is group-specific and, in many instances, serotype-specific (21, 22). Consequently, initial success with prototype vaccines will have to be followed by the development of a final vaccine formulation that incorporates a strategy for conferring broad-spectrum protection against the epidemiologically most important Shigella serotypes (35, 53). In recent years, candidate human vaccines against ETEC have been prepared that are based on stimulating intestinal antibodies against the colonization factor fimbriae by which ETEC attaches to enterocytes and on stimulating antitoxin to neutralize heat-labile enterotoxin (LT) (1, 19, 41, 43, 61, 66, 67). Antigens to stimulate anticolonization immunity have included inactivated fimbriated ETEC whole bacteria (1, 16, 19, 60), purified ETEC fimbriae administered in native form (18, 43) or contained within polylactide-polyglycolide microspheres (67), and live oral vaccines consisting of either fimbriated nontoxigenic ETEC strains (36, 37) or of attenuated Shigella or Salmonella enterica serovars Typhi or Typhimurium live vectors expressing ETEC fimbriae and mutant LT or the LT B subunit (26, 31, 42, 54, 55). ETEC vaccines must also address the considerable antigenic heterogeneity among ETEC strains that cause human diarrheal disease (24, 39, 42). It is widely agreed that an ETEC vaccine should include colonization factor antigen I (CFA/I) and coli surface antigens 1 to 6 (CS1 to CS6) fimbrial antigens (42). The candidate ETEC vaccine that is furthest along in clinical trials consists of an oral formulation containing a mixture of inactivated, fimbriated ETEC strains that express CFA/I and CS1-6, coadministered in combination with the cholera toxin B subunit (CT-BS) (60, 61). CT and CT-BS elicit cross-reacting antibodies that can neutralize the LT variant found in ETEC strains in humans (LTh) (46, 65); CT-BS, by itself, has conferred short-term protection (for several months) against diarrhea caused by LT-producing ETEC (6, 56). We have embarked on a long-term project to develop a multivalent hybrid vaccine to prevent both Shigella dysentery and ETEC diarrhea caused by the epidemiologically most important serotypes and antigenic types (34, 39, 53). The approach consists of engineering five attenuated Shigella strains (representing five epidemiologically and immunologically critical serotypes), each expressing two separate ETEC fimbrial antigens and an antigen to elicit antibodies against LTh (31, 39, 55). Towards this goal, we have prepared improved Shigella vaccine candidates by introducing a deletion mutation in the guaBA operon (which encodes two enzymes involved in the synthesis of guanine nucleotides) in wild-type S. flexneri 2a, resulting in vaccine strain CVD 1204 as a basis of further derivatives (34, 54). This serotype is used as a model because of its epidemiologic importance, the presence in its chromosome of a pathogenicity island that includes Shigella enterotoxin 1 (20, 52), and extensive experience with this serotype in experimental challenge studies in volunteers (11–13, 32, 33). The effect of introducing additional attenuating mutations into CVD 1204, such as deletions in virG (also referred to as icsA, encoding a protein involved with intracellular and intercellular spread of Shigella), resulting in CVD 1205 (54), and in the genes encoding Shigella enterotoxins 1 and 2 (Δset1A, Δsen), resulting in CVD 1207, have been evaluated (34). We have previously reported cloning the genes necessary for the expression of CFA/I by CVD 1204 and the ability of that live vector to elicit both anti-S. flexneri 2a and anti-CFA/I antibodies (31). The research reported herein describes the expression of rigid CS2 fimbriae and flexible CS3 fibrillae by attenuated S. flexneri 2a strain CVD 1204; an estimation of the stability of the expression plasmids in CVD 1204; the suitability of the osmolarity-activated ompC promoter in promoting fimbrial expression; the ability of the live vector to elicit antibodies to each fimbrial antigen individually and to both fimbriae simultaneously, in addition to S. flexneri O antigen; and, finally, a demonstration that expression of ETEC fimbriae by the live vector does not diminish its ability to protect against virulent S. flexneri 2a in a challenge model.