Attenuated Salmonella enterica serovar Typhi has been studied as an oral vaccine vector. Despite success with attenuated S. enterica serovar Typhimurium vectors in animals, early clinical trials of S. enterica serovar Typhi expressing heterologous antigens have shown that few subjects have detectable immune responses to vectored antigens. A previous clinical study of phoP/phoQ-deleted S. enterica serovar Typhi expressing Helicobacter pylori urease from a multicopy plasmid showed that none of eight subjects had detectable immune responses to the vectored antigen. In an attempt to further define the variables important for engendering immune responses to vectored antigens in humans, six volunteers were inoculated with 5 ؋ 10 7 to 8 ؋ 10 7 CFU of phoP/phoQdeleted S. enterica serovar Typhimurium expressing the same antigen. Two of the six volunteers had fever; none had diarrhea, bacteremia, or other serious side effects. The volunteers were more durably colonized than in previous studies of phoP/phoQ-deleted S. enterica serovar Typhi. Five of the six volunteers seroconverted to S. enterica serovar Typhimurium antigens and had strong evidence of anti-Salmonella mucosal immune responses by enzyme-linked immunospot studies. Three of six (three of five who seroconverted to Salmonella) had immune responses in the most sensitive assay of urease-specific immunoglobulin production by blood mononuclear cells in vitro. One of these had a fourfold or greater increase in end-point immunoglobulin titer in serum versus urease. Attenuated S. enterica serovar Typhimurium appears to be more effective than S. enterica serovar Typhi for engendering immune responses to urease. Data suggest that this may be related to a greater stability of antigen-expressing plasmid in S. enterica serovar Typhimurium and/or prolonged intestinal colonization. Specific factors unique to nontyphoidal salmonellae may also be important for stimulation of the gastrointestinal immune system. Attenuated salmonellae have been extensively studied as live bacterial vectors for delivery of heterologous antigens because these intracellular microorganisms stimulate humoral, mucosal, and cellular immune responses in humans and animals. A goal of these studies has been to develop multivalent oral vaccines based upon Salmonella enterica serovar Typhi for human use. The existence of the safe live attenuated vaccine strain Ty21a, an effective vaccine against typhoid fever, and the species specificity of S. enterica serovar Typhi for humans has focused investigative attention on this serotype. Most human studies of attenuated S. enterica serovar Typhi have been based on preclinical data obtained in the murine model of systemic salmonellosis in which BALB/c mice are infected orally with attenuated Salmonella enterica serovar Typhimurium. More recently, mice have been infected nasally with S. enterica serovar Typhi strains (8). Although murine experiments are useful screening studies, their predictive value in forecasting immunogenicity and safety in clinical trials of atte...

Listeria monocytogenes is an intracellular bacterial pathogen which causes bacteremia and has a tropism for the central nervous system and a propensity to cause maternofetal infection. L. monocytogenes has been shown to be an effective prophylactic and a therapeutic vaccine vector for viral and tumor antigens in animal models. L. monocytogenes mutants lacking the ActA protein, which is essential for intracellular movement, are attenuated but retain immunogenicity in mice. Given the pathogenic potential of L. monocytogenes, we created an attenuated mutant strain bearing double deletions in the actA and plcB virulence genes for an initial clinical safety study of a prototype L. monocytogenes vector in adults. Twenty healthy volunteers received single escalating oral doses (10 6 to 10 9 CFU, 4 volunteers per dose cohort) of this attenuated L. monocytogenes, designated LH1169. Volunteers were monitored in the hospital for 14 days with frequent clinical checks and daily blood and stool cultures, and they were monitored for six additional weeks as outpatients. There were no positive blood cultures and no fevers attributable to the investigational inoculation. Most volunteers shed vaccine bacteria for 4 days or less, without diarrhea. One volunteer had a late positive stool culture during outpatient follow-up. Three volunteers had abnormal liver function test results temporally associated with inoculation; one could be reasonably attributed to another cause. In the highest-dose cohort, humoral, mucosal, and cellular immune responses to the investigational organism were detected in individual volunteers. Attenuated L. monocytogenes can be studied in adult volunteers without serious long-term health sequelae.Listeria monocytogenes is a gram-positive bacterium which has long been studied in mice to elucidate mechanisms of cellular immune responses to intracellular pathogens (29, 35). Schafer and colleagues initially proposed that escape of L. monocytogenes from the phagocytic vacuole into the eukaryotic cytoplasm might make these organisms efficient vectors for delivery of antigens to the major histocompatibility complex class I-restricted antigen processing pathways (48). L. monocytogenes vectors have been used to deliver lymphocytic choriomeningitis virus (LCMV) nucleoprotein antigens to mice, with subsequent protection against fatal challenge with LCMV (21, 52, 53). Recombinant L. monocytogenes expressing cottontail rabbit papillomavirus E1 antigen has been successfully used as a therapeutic immunogen in animals bearing papillomavirus-induced cutaneous tumors (28). L. monocytogenes is being pursued as a vector for antigens derived from human papillomaviruses (23) and human immunodeficiency virus type 1 (HIV-1) (40,41). A murine study showed that oral inoculation of L. monocytogenes expressing HIV-1-gag induced mucosal and systemic immunity to this viral antigen (45). After careful review of available data on oral inoculation of wild-type L. monocytogenes in primates (17) and farm animals, (34,42,43) clinical data avail...