The vaccine was safe and broadly immunogenic. dmLT further enhanced mucosal immune responses to CF antigens present in low amounts in the vaccine. Based on these encouraging results, the vaccine will be tested for safety and immunogenicity in different age groups including infants in Bangladesh and for protective efficacy in travelers.

A first-generation oral inactivated whole-cell enterotoxigenic Escherichia coli (ETEC) vaccine, comprising formalin-killed ETEC bacteria expressing different colonization factor (CF) antigens combined with cholera toxin B subunit (CTB), when tested in phase III studies did not significantly reduce overall (generally mild) ETEC diarrhea in travelers or children although it reduced more severe ETEC diarrhea in travelers by almost 80%. We have now developed a novel more immunogenic ETEC vaccine based on recombinant non-toxigenic E. coli strains engineered to express increased amounts of CF antigens, including CS6 as well as an ETEC-based B subunit protein (LCTBA), and the optional combination with a nontoxic double-mutant heat-labile toxin (LT) molecule (dmLT) as an adjuvant. Two test vaccines were prepared under GMP: (1) A prototype E. coli CFA/I-only formalin-killed whole-cell+LCTBA vaccine, and (2) A "complete" inactivated multivalent ETEC-CF (CFA/I, CS3, CS5 and CS6 antigens) whole-cell+LCTBA vaccine. These vaccines, when given intragastrically alone or together with dmLT in mice, were well tolerated and induced strong intestinal-mucosal IgA antibody responses as well as serum IgG and IgA responses to each of the vaccine CF antigens as well as to LT B subunit (LTB). Both mucosal and serum responses were further enhanced (adjuvanted) when the vaccines were co-administered with dmLT. We conclude that the new multivalent oral ETEC vaccine, both alone and especially in combination with the dmLT adjuvant, shows great promise for further testing in humans.

No abstract

Mouse peritoneal macrophages were infected with varying numbers of Nocardia asteroides 10905, and the fate of the ingested organisms was determined by viable plate count (VPC), light microscopy (LM), immunofluorescent microscopy (IM), and electron microscopy (EM). The results obtained with these methods differed. VPC indicated that intracellular Nocardia decreased in numbers during the first 12 days, followed by significant increases after 16 days. LM suggested that N. asteroides 10905 was slowly degraded by macrophages with no subsequent increases observed. In contrast, IM demonstrated large numbers of intracellular Nocardia throughout the experiment. EM studies of' infected macrophages failed to demonstrate intact bacteria after 8 days; however, wall-less and spheroplast-like organisms were seen. These results suggested that N. asteroides 10905 was present within the macrophages in an altered form. By using hypertonic culture medium, we were able to isolate, from infected macrophages, organisms which exhibited many of the properties of' bacterial L-forms. IM demonstrated these variants to be of nocardial origin. These altered forms also reverted to typical nocardial cells either spontaneously or upon transfer into broth. These findings indicate that N. asteroides 10905 is capable of existing within macrophages in an altered state. Further investigation is in progress to determine whether these altered forms represent L-forms or transitional-phase variants.

Antibody-secreting cell (ASC) and antibodies in lymphocyte supernatant (ALS) assays are used to assess intestinal mucosal responses to enteric infections and vaccines. The ALS assay, performed on cell supernatants, may represent a convenient alternative to the more established ASC assay. The two methods, measuring immunoglobulin A to Salmonella enterica serovar Typhi lipopolysaccharide, were compared in volunteers vaccinated with a live-attenuated typhoid vaccine M01ZH09. The specificity of the ALS assay compared to the ASC assay was excellent (100%), as was sensitivity (82%). The ALS assay was less sensitive than the ASC assay at <42 spots/10 6 peripheral blood lymphocytes.