No abstract

Three attenuated Salmonella typhi strains have been constructed by introducing deletions in aroC and aroD or deletions in cya and crp into one of two wild-type parent strains, Ty2 or ISP1820. These mutant strains were designated CVD 906 (ISP1820 AaroC AaroD), CVD 908 (Ty2 AaroC AaroD), and X3927 (Ty2 Acya Acrp). Two studies were conducted with 36 healthy adult inpatient volunteers to determine in a double-blind fashion the safety and immunogenicity of approximately 5 x 104 and 5 x 105 CFU of each of these three vaccine candidates given as a single dose. No statistically significant difference in the incidence of reactions among vaccinees was observed. Fever (oral temperature 2 38.2°C) occurred in 2 of 12 volunteers who received CVD 906, in 0 of 12 who received CVD 908, and in 1 of 12 who received x3927. Vaccine bacteremia without symptoms occurred in 1 of 12 vaccinees who received CVD 906, in 0 of 12 who received CVD 908, and in 2 of 12 who received x3927. Overall, 19 (53%) of 36 vaccinees developed immunoglobulin G antibody to S. typhi lipopolysaccharide after vaccination, with no statistically significant differences in the rate of seroconversion among volunteers in the three groups. We conclude that defined mutations in the aromatic biosynthetic pathway and in the cyclic AMP global regulatory system attenuate S. typhi. Mutant strains CVD 906, CVD 908, and X3927 are highly (and approximately equally) immunogenic but possibly differ in their propensity to induce fever. Further studies are needed to document the apparent relative safety of CVD 908 as a typhoid vaccine and as a vaccine carrier of foreign antigens. Live oral attenuated Salmonella typhi vaccines are likely to replace the parenteral killed whole-cell vaccine as safer and possibly more effective vaccines against typhoid fever for use in healthy adults and children (19). Ingestion of attenuated bacteria mimics natural enteric infection and potentially improves the intensity and duration of the immune response compared with parenterally administered vaccines. These strains may also serve as vaccine carriers of cloned protective antigens of other pathogens. A prototype attenuated S. typhi vaccine strain, Ty21a, developed in the early 1970s by chemical mutagenesis before the advent of recombinant DNA technology (7), has been licensed in the United States and used to prevent typhoid fever. Ty2la has also been used as a carrier of foreign antigens, with variable success (1, 26). However, Ty21a requires multiple doses to achieve acceptable immunogenicity and is a difficult background in which to perform genetic manipulations. Therefore, new oral typhoid vaccines have been sought to serve as immunoprophylaxis against typhoid and as a recombinant vaccine carrier.

No vaccine exists for the prevention of infection with the ubiquitous gastric pathogen Helicobacter pylori, and drug therapy for the infection is complicated by poor patient compliance, the high cost of treatment, and ineffectiveness against drug resistant strains. A new medical advancement is required to reduce the incidence of peptic ulcer disease and stomach cancer, two conditions caused by infection with H. pylori. Clinical trials have been performed with a formalin-inactivated Helicobacter pylori Whole Cell (HWC) vaccine, given orally in combination with the mucosal adjuvant mLT(R192G), a mutant of E. coli heat-labile toxin. Following the initial dose of this vaccine, some subjects experienced gastrointestinal side effects. To reduce side effects and potentially further increase the amount of adjuvant that can safely be administered with the HWC vaccine, experiments were performed with a form of LT that carried two mutations in the A subunit, a substitution of G for R at position 192, and A for L at position 211. The double-mutant LT (dmLT) adjuvant stimulated immune responses as effectively as the single mutant LT in mice. Additionally, following a challenge infection, the dmLT-adjuvanted vaccine was as effective as single mutant LT in reducing gastric urease levels (diagnostic for H. pylori infection), and H. pylori colonization in the stomach as assessed by quantitative analysis of stomach homogenates. A lyophilized formulation of HWC was developed to improve stability and to potentially reduce reliance on cold chain maintenance. It was observed that a dmLT-adjuvanted lyophilized vaccine was equally as protective in the mouse model as the liquid formulation as assessed by gastric urease analysis and analysis of stomach homogenates for viable H. pylori. No readily detectable effect of tonicity or moisture content was observed for the lyophilized vaccine within the formulation limits evaluated. In an accelerated stability study performed at 37°C the lyophilized vaccine remained equally as protective as vaccine stored at 2-8°C. The formulation selected for clinical development consisted of 2.5×10 10 formalin-inactivated cells per ml in 6.5% trehalose, 0.5% mannitol, and 10 mM citrate buffer at pH 6.8.

Clin. Invest. 1992. 90:412-420.)