Helicobacter ISSN 1523-5378 © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd, Helicobacter 11: 517–522 517 Blackwell Publishing Ltd Oxford, UK HEL Helicobacter 1083-4389 Blackwell Publishing Ltd, 2006 8 Original Article Helicobacter pylori urease B DNA vaccine Zavala-Spinetti et al. Development and Evaluation of a DNA Vaccine Based on Helicobacter pylori urease B: Failure to Prevent Experimental Infection in the Mouse Model Livania Zavala-Spinetti, * Mary B. Breslin, † Hernán Correa ‡ and Rodolfo E. Bégué *† * Division of Infectious Diseases and † Division of Research, Department of Pediatrics; ‡ Department of Pathology, Louisiana State University Health Sciences Center, New Orleans, Louisiana, USA Abstract Background: The development of a vaccine against Helicobacter pylori has become a priority to prevent major morbidity and mortality associated with this infection. Our goal was to prepare and evaluate a DNA vaccine based on the urease B gene ( ure B). Methods: The ure B gene of H. pylori was amplified and cloned into the eukaryotic expression vector pcDNA3.1/TOPO. Plasmid DNA was purified from transformed Escherichia coli cells and used to immunize mice by the intragastric, intramuscular, intrarectal (40 μg each) and intranasal (16 μg) route, three doses every 2 weeks, with CpG oligodeoxynucleotide (ODN) as adjuvant. Four weeks after the third dose, animals were orally challenged with Helicobacter felis and were sacrificed 6 weeks later. The stomach was stained to detect the presence of infection. Results: Despite in vitro confirmation of successful cloning and functionality of the ure B gene with expression of a protein morphologically and antigenically identical to urease B, the DNA vaccine did not perform well in vivo . Immunization of mice produced a weak immune response. Overall, intrarectal and intranasal administration seemed more immunogenic than other routes. Protection against challenge was modest and nonsignificant, and slightly better on animals immunized by the intramuscular and intranasal route. Conclusion: A DNA vaccine based on H. pylori urease B was poorly immunogenic and nonprotective at the conditions evaluated. Higher doses, better adjuvants or a prime-boost approach may circumvent these limitations. Keywords Helicobacter pylori, Helicobacter felis, DNA vaccines, animal experimentation. Reprint requests to: Rodolfo E Bégué, MD, Children’s Hospital, Infectious Diseases, 200 Henry Clay Avenue, New Orleans, LA, USA. Tel.: (504) 896-9583; E-mail: rbegue@lsuhsc.edu The bacterium Helicobacter pylori infects at least 50% of the world’s population, causing gastric inflammation, peptic ulcer disease and gastric carcinoma [1]. Because of the large toll of the disease and the difficulties in treatment – such as antibiotic resistance – experts agree that the best preventive measure is the development of a vaccine. It is uncertain, however, whether protective immunity can be developed for H. pylori as chronic infection is the norm despite the presence of specific antibodies [2]. Of the many H. pylori antigens investigated as potential vaccine candidates, the protein urease is the most pro- mising [3]. This protein counters the acidic environment of the stomach, a function that is crucial for the survival of the organism as evidenced by the fact that urease-deficient mutants are unable to colonize the gastric mucosa [4]. Since urease is present on the cell surface of the bacterium, it is accessible to the immune system and, as a result, natural infection frequently results in systemic and gastric immunoglobulin (Ig) G and IgA-specific antibodies [5]. H. pylori urease is comprised of two polypeptides, A (30 kDa) and B (62 kDa), of which the B subunit (urease B) is more important for protection. In 1994, Michetti et al. [6] immunized BALB/c mice intragastrically with recombinant H. pylori urease A or B subunit or both and reported 80% protection against challenge with H. felis for those immunized with urease B, as compared to 70% for both and 59% for urease A. Similarly, Ferrero et al. [7] reported that orogastric immunization with recombinant urease B resulted in 25–60% protection against H. felis challenge, as compared to 0% protection for urease A. One problem with orally administered protein-based vaccines is their poor immunogenicity and need for a strong