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Bacterial carriers for the mucosal delivery of antigens, particularly those capable of colonizing or invading through the mucosal epithelia, have been intensively investigated (1). Live bacterial vectors can be generated with attenuated pathogens, such as Salmonella and Listeria, or nonpathogenic commensal species, such as Lactococcus and Lactobacillus. Bacteria in the former group of are capable of inducing strong and long-lasting immune responses to passenger antigens but present serious safety concerns, whereas bacteria in the latter group are safer but typically induce much lower immune responses to the passenger antigens (1-3). As a safe nonpathogenic live bacterial vector, Bacillus subtilis, a spore-forming soil Gram-positive bacterial species, has been engineered to express antigens as either vegetative cells or spores (4, 5). As antigen carriers, B. subtilis spores have several attractive features, including a safe record of human and animal use as both probiotic and food additives, remarkable heat resistance, and rather easy genetic and bacteriological manipulation.Currently, two major genetic approaches have been proposed to generate recombinant B. subtilis spores as vaccine delivery vectors. The first approach relies on the expression of a heterologous protein genetically fused to surface-exposed spore coat proteins, such as CotB, CotC, or CotG (6, 7). Such a strategy would allow a better presentation of the passenger antigen to the mucosa-associated lymphoid tissue (MALT) afferent sites, leading to the induction of adaptive immune responses, such as mucosal secretory (IgA) or systemic (IgG) antigen-specific antibody responses (6-8). The second approach is based on a distinct rationale and has employed episomal vectors in which the target antigen is expressed under the control of a promoter (PgsiB) active only at the vegetative cell stage, which means immediately after spore germination (9-11). This antigen delivery approach relies on the fact that B. subtilis spores germinate during transit through the gastrointestinal tract and produce the target antigen at the intestinal lumen or inside the phagocytes of antigen-presenting cells (APCs), leading to the induction of antibody responses in the serum (IgG) and mucosa (fecal IgA) (9-11).However, in both cases, the administration of recombinant spores via mucosal routes typically confers immune responses to the passenger antigen lower than those achieved with delivery systems based on attenuated bacterial strains capable of colonizing the mammalian gastrointestinal tract. The reduced mucosal adjuvant effects of B. subtilis spores have been attributed to several factors, such as a previously established immunity generated by the frequent ingestion of spores, the reduced amount of expressed antigens, and the rapid transit through the gastrointestinal tract, which reduces the chances of a productive interaction with the gut-associated lymphoid tissue (GALT), such as M cells and APCs at Peyer's patches (PPs) (12, 13).In an attempt to improve the performance o...

e Globally, enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood and travelers' diarrhea, for which an effective vaccine is needed. Prevalent intestinal colonization factors (CFs) such as CFA/I fimbriae and heat-labile enterotoxin (LT) are important virulence factors and protective antigens. We tested the hypothesis that donor strand-complemented CfaE (dscCfaE), a stabilized form of the CFA/I fimbrial tip adhesin, is a protective antigen, using a lethal neonatal mouse ETEC challenge model and passive dam vaccination. For CFA/I-ETEC strain H10407, which has been extensively studied in volunteers, an inoculum of 2 ؋ 10 7 bacteria resulted in 50% lethal doses (LD 50 ) in neonatal DBA/2 mice. Vaccination of female DBA/2 mice with CFA/I fimbriae or dscCfaE, each given with a genetically attenuated LT adjuvant (LTK63) by intranasal or orogastric delivery, induced high antigen-specific serum IgG and fecal IgA titers and detectable milk IgA responses. Neonates born to and suckled by dams antenatally vaccinated with each of these four regimens showed 78 to 93% survival after a 20؋ LD 50 challenge with H10407, compared to 100% mortality in pups from dams vaccinated with sham vaccine or LTK63 only. Crossover experiments showed that high pup survival rates after ETEC challenge were associated with suckling but not birthing from vaccinated dams, suggesting that vaccine-specific milk antibodies are protective. In corroboration, preincubation of the ETEC inoculum with antiadhesin and antifimbrial bovine colostral antibodies conferred a dose-dependent increase in pup survival after challenge. These findings indicate that the dscCfaE fimbrial tip adhesin serves as a protective passive vaccine antigen in this small animal model and merits further evaluation.