Andrea M. Harris

Construction of genetically defined double aro mutants of Salmonella typhi

The construction of genetically defined, double aro mutant strains CVD906 and CVD908, which were derived from Salmonella typhi strain ISP1820 (a recent isolate of S. typhi from Chile) and from laboratory strain Ty2, respectively, is described. Strains CVD906 and CVD908 differ from previously described aro mutants of S. typhi as their aro deletion mutations do not extend beyond the limits of the mutated aro genes, and no antibiotic-resistance genes, plasmid sequences or S. typhimurium DNA sequences remain in the mutant strains. In minimal medium the aro mutants of S. typhi are unable to replicate whereas the wild type parent strains grow well in minimal medium. Using intraperitoneal inoculation of mice with S. typhi strains suspended in hog gastric mucin as a virulence assay, it is shown that the single aro mutants and the double aro mutants of Ty2 and ISP1820 are attenuated in mice. Trans complementation of the aro mutants with the aroC gene or aroD gene, or both, results in strains that are phenotypically identical to that of the wild type parents indicating that no measurable additional changes other than loss of the aro gene function occurred during strain construction.

Evaluation in volunteers of a candidate live oral attenuated Salmonella typhi vector vaccine.

Candidate vector vaccine strain CVD 906 (aroC- and aroD- derivative of virulent Salmonella typhi strain ISP1820) was evaluated in phase 1 clinical trials. The first nine volunteers ingested a single dose of 5 x 10(7) CVD 906 bacilli. At this dose CVD 906 stimulates remarkable systemic and mucosal immune responses, inasmuch as 89% of volunteers developed marked serum antibody levels to S. typhi antigens and high numbers of antigen-specific gut-derived antibody-secreting cells. Four (44%) volunteers developed asymptomatic vaccinemia 4-10 d after immunization and all volunteers excreted CVD 906 on at least one occasion. However, two volunteers developed febrile adverse reactions, one on the day of vaccination and the other on day 4. Of 11 volunteers who ingested a single dose of 5 x 10(3) CVD 906 bacilli, none displayed side effects but 27% developed significant serum responses to S. typhi LPS. In vitro, CVD 906 replicates for only nine generations in pooled human serum, indicating that CVD 906 growth is limited in this physiologically relevant medium. In phorbol myristate acetate-induced U937 human macrophage-like cells, CVD 906 replicates intracellularly to a lesser extent than parent strain ISP1820. Although, strain CVD 906 is attenuated and highly immunogenic, the occasional febrile reactions at high doses indicate that further attenuation of this strain is necessary.

Toward the development of a stable, freeze-dried formulation of Helicobacter pylori killed whole cell vaccine adjuvanted with a novel mutant of Escherichia coli heat-labile toxin

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 H. pylori whole cell (HWC) vaccine, given orally in combination with the mucosal adjuvant mLT(R192G), a mutant of Escherichia 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 degrees C the lyophilized vaccine remained equally as protective as vaccine stored at 2-8 degrees C. The formulation selected for clinical development consisted of 2.5 x 10(10) formalin-inactivated cells per ml in 6.5% trehalose, 0.5% mannitol, and 10mM citrate buffer at pH 6.8.

Adaptive acid tolerance response by Salmonella typhi and candidate live oral typhoid vaccine strains

The data presented here demonstrate that Salmonella typhi is capable of expressing an acid tolerance response (ATR) and that effective induction of this response (in nutrient-rich medium) occurs at pH 5.0 in anaerobic conditions. The candidate live oral S. typhi vaccine strains made by precise genetic methods and which carry auxotrophic mutations were CVD 906 (carries defined attenuating deletion mutations: delta aroC, delta aroD), CVD 908 (carries defined attenuating deletion mutations: delta aroC, delta aroD), 541Ty (carries attenuating deletion mutations: aroA, purA), and galE, Vi-negative (via) strain EX462. All generate an effective ATR. In contrast, nitrosoguanidine-derived live oral typhoid vaccine strain Ty21a only weakly expresses acid tolerance. This further demonstrates that the non-specific mutagenesis process used to produce Ty21a affects genetic loci outside the intended target genes for mutagenesis, galE and via, and further emphasizes the importance of using precise genetic techniques when developing live oral S. typhi vaccines.

Enhanced Early Innate and T Cell-mediated Responses in Subjects Immunized with Anthrax Vaccine Adsorbed Plus CPG 7909 (AV7909)

NuThrax™ (Anthrax Vaccine Adsorbed with CPG 7909 Adjuvant) (AV7909) is in development. Samples obtained in a phase Ib clinical trial were tested to confirm biomarkers of innate immunity and evaluate effects of CPG 7909 (PF-03512676) on adaptive immunity. Subjects received two intramuscular doses of commercial BioThrax(®) (Anthrax Vaccine Adsorbed, AVA), or two intramuscular doses of one of four formulations of AV7909. IP-10, IL-6, and C-reactive protein (CRP) levels were elevated 24-48 h after administration of AV7909 formulations, returning to baseline by Day 7. AVA (no CPG 7909) resulted in elevated IL-6 and CRP, but not IP-10. Another marker of CpG, transiently decreased absolute lymphocyte counts (ALCs), correlated with transiently increased IP-10. Cellular recall responses to anthrax protective antigen (PA) or PA peptides were assessed by IFN-γ ELISpot assay performed on cryopreserved PBMCs obtained from subjects prior to immunization and 7 days following the second immunization (study day 21). One-half of subjects that received AV7909 with low-dose (0.25mg/dose) CPG 7909 possessed positive Day 21 T cell responses to PA. In contrast, positive T cell responses occurred at an 11% average rate (1/9) for AVA-treated subjects. Differences in cellular responses due to dose level of CPG 7909 were not associated with differences in humoral anti-PA IgG responses, which were elevated for recipients of AV7909 compared to recipients of AVA. Serum markers at 24 or 48 h (i.e. % ALC decrease, or increase in IL-6, IP-10, or CRP) correlated with the humoral (antibody) responses 1 month later, but did not correlate with cellular ELISpot responses. In summary, biomarkers of early responses to CPG 7909 were confirmed, and adding a CpG adjuvant to a vaccine administered twice resulted in increased T cell effects relative to vaccine alone. Changes in early biomarkers correlated with subsequent adaptive humoral immunity but not cellular immunity.

Construction and characterization of isogenic O-antigen variants of Salmonella typhi

A 7.5 kb Kpnl‐generated fragment, from within the rfb cluster of Salmonella typhimurium LT2 that encodes abequose synthase (the rfbJ gene) which is necessary for O4 antigen synthesis, and flanking sequences, was inserted into a suicide vector. Using allelic exchange techniques, these rfb sequences of S. typhimurium were integrated into the rfb clusters of wild‐type Salmonella typhi Vi‐positive strain ISP 1820 (i.e. serotype 09,12; Vi+ H‐d), S. typhi Vi‐negative strain H400 (i.e. serotype 09,12; Vi−; H‐d), and a double aro mutant of S. typhi ISP 1820, strain CVD 906, resulting in the isolation of strains H325, H404 and CVD 906‐O4, respectively. Immunoblot analysis of lipopolysaccharide (LPS) purified from H325, H404 and CVD 906‐O4 demonstrated that these 8trains express the 04 antigen (an abequose residue) in place of the O9 antigen (a tyvelose residue) in the LPS molecule. Hence, the serotype of H325 is O4,12; Vi+; H‐d and the serotype of H404 is O4,12; Vi−; H‐d. DNA hybridization analysis of chromosomal DNA from H325, H404 and CVD 906‐O4 confirmed that a precise recombination event within sequences flanking rfbSE of S. typhi (which encodes the enzymes necessary for cytidine diphosphate‐tyvelose synthesis) resulted in replacement of rfbSE with rfbJ (which encodes abequose synthase and is necessary for O4 synthesis) of S. typhimurium in strains H325, H404 and CVD 906‐O4. The resistance of each strain to the bactericidal effects of guinea‐pig serum (GPC) was assessed. Whereas ISP 1820, H325 and H404 exhibit similar resistance patterns in GPC, strain H400 is sensitive to the bactericidal effects of GPC. The results suggest that the development of the O‐antigen serotype diversity of Salmonella is probably the result of both sequence divergence and recombination