Judith Greenwood

Attenuated Salmonella enterica Serovar Typhi Expressing Urease Effectively Immunizes Mice against Helicobacter pylori Challenge as Part of a Heterologous Mucosal Priming-Parenteral Boosting Vaccination Regimen

ABSTRACT Recombinant vaccine strains of Salmonella enterica serovar Typhi capable of expressing Helicobacter pylori urease were generated by transforming strains CVD908 and CVD908-htrA with a plasmid harboring the ureAB genes under the control of an in vivo-inducible promoter. The plasmid did not interfere with the ability of either strain to replicate and persist in human monocytic cells or with their transient colonization of mouse lungs. When administered to mice intranasally, both recombinant strains elicited antiurease immune responses skewed towards a Th1 phenotype. Vaccinated mice exhibited strong immunoglobulin G2a (IgG2a)-biased antiurease antibody responses as well as splenocyte populations capable of proliferation and gamma interferon (IFNγ) secretion in response to urease stimulation. Boosting of mice with subcutaneous injection of urease plus alum enhanced immune responses and led them to a more balanced Th1/Th2 phenotype. Following parenteral boost, IgG1 and IgG2a antiurease antibody titers were raised significantly, and strong urease-specific splenocyte proliferative responses, accompanied by IFNγ as well as interleukin-4 (IL-4), IL-5, and IL-10 secretion, were detected. Neither immunization with urease-expressing S. enterica serovar Typhi alone nor immunization with urease plus alum alone conferred protection against challenge with a mouse-adapted strain of H. pylori; however, a vaccination protocol combining both immunization regimens was protective. This is the first report of effective vaccination against H. pylori with a combined mucosal prime-parenteral boost regimen in which serovar Typhi vaccine strains are used as antigen carriers. The significance of these findings with regard to development of a human vaccine against H. pylori and modulation of immune responses by heterologous prime-boost immunization regimens is discussed.

Generation and characterization of a live attenuated enterotoxigenic Escherichia coli combination vaccine expressing six colonization factors and heat-labile toxin subunit B.

ABSTRACT Live attenuated oral enterotoxigenic Escherichia coli (ETEC) vaccines have been demonstrated to be safe and immunogenic in human volunteers and to provide a viable approach to provide protection against this important pathogen. This report describes the construction of new ETEC vaccine candidate strains from recent clinical isolates and their characterization. All known genes for ETEC toxins were removed, and attenuating deletion mutations were made in the aroC, ompC, and ompF chromosomal genes. An isolate expressing coli surface antigen 2 (CS2), CS3, heat-labile toxin (LT), heat-stable toxin (ST), and enteroaggregative Escherichia coli heat-stable toxin 1 (EAST1) was attenuated to generate ACAM2007. The subsequent insertion of the operon encoding CS1 created ACAM2017, and this was further modified by the addition of an expression cassette containing the eltB gene, encoding a pentamer of B subunits of LT (LTB), to generate ACAM2027. Another isolate expressing CS5, CS6, LT, ST, and EAST1 was attenuated to generate ACAM2006, from which a lysogenic prophage was deleted to create ACAM2012 and an LTB gene was introduced to form ACAM2022. Finally, a previously described vaccine strain, ACAM2010, had the eltB gene incorporated to generate ACAM2025. All recombinant genes were incorporated into the chromosomal sites of the attenuating mutations to ensure maximal genetic stability. The expression of the recombinant antigens and the changes in plasmids accompanying the deletion of toxin genes are described. Strains ACAM2025, ACAM2022, and ACAM2027 have been combined to create the ETEC vaccine formulation ACE527, which has recently successfully completed a randomized, double-blind, placebo-controlled phase I trial and is currently undergoing a randomized, double-blind placebo-controlled phase II challenge trial, both in healthy adult volunteers.

Construction and Phase I Clinical Evaluation of the Safety and Immunogenicity of a Candidate Enterotoxigenic Escherichia coli Vaccine Strain Expressing Colonization Factor Antigen CFA/I

ABSTRACT Oral delivery of toxin-negative derivatives of enterotoxigenic Escherichia coli (ETEC) that express colonization factor antigens (CFA) with deletions of the aroC, ompC, ompF, and toxin genes may be an effective approach to vaccination against ETEC-associated diarrhea. We describe the creation and characterization of an attenuated CFA/I-expressing ETEC vaccine candidate, ACAM2010, from a virulent isolate in which the heat-stable enterotoxin (ST) and CFA/I genes were closely linked and on the same virulence plasmid as the enteroaggregative E. coli heat-stable toxin (EAST1) gene. A new suicide vector (pJCB12) was constructed and used to delete the ST and EAST1 genes and to introduce defined deletion mutations into the aroC, ompC, and ompF chromosomal genes. A phase I trial, consisting of an open-label dose escalation phase in 18 adult outpatient volunteers followed by a placebo-controlled double-blind phase in an additional 31 volunteers, was conducted. The vaccine was administered in two formulations, fresh culture and frozen suspension. These were both well tolerated, with no evidence of significant adverse events related to vaccination. Immunoglobulin A (IgA) and IgG antibody-secreting cells specific for CFA/I were assayed by ELISPOT. Positive responses (greater than twofold increase) were seen in 27 of 37 (73%) subjects who received the highest dose level of vaccine (nominally 5 × 109 CFU). Twenty-nine of these volunteers were secreting culturable vaccine organisms at day 3 following vaccination; five were still positive on day 7, with a single isolation on day 13. This live attenuated bacterial vaccine is safe and immunogenic in healthy adult volunteers.