Poliovirus replicons encoding the B subunit of Helicobacter pylori urease elicit a Th1 associated immune response

Abstract

The development of a vaccine for Helicobacter pylori is a key strategy for reducing the worldwide prevalence of H. pylori infection. Although immunization with recombinant B subunit of H. pylori urease (ureB) has yielded promising results, for the most part, these studies relied on the use of strong adjuvant, cholera toxin, precluding the use in humans. Thus, the development of new vaccine strategies for H. pylori is essential. Previous studies from our laboratory have described a vaccine vector based on poliovirus in which foreign genes are substituted for the poliovirus capsid genes. The genomes encoding foreign proteins (replicons) are encapsidated into authentic poliovirions by providing the capsids in trans. To test the utility of replicons as a vaccine vector for H. pylori, a replicon was constructed which encodes ureB. Expression of ureB in cells from the replicon was demonstrated by metabolic labeling followed by immunoprecipitation with anti-urease antibodies. To investigate the immunogenicity of the replicons, mice containing the transgene for the receptor for poliovirus were immunized via the intramuscular route. Mice given three doses of replicons did not develop substantial antibodies to ureB as determined by Western blot analysis using lysates from H. pylori. In contrast, mice given two doses of replicon followed by a single injection of recombinant ureB developed serum antibodies to ureB which were predominately IgG2a. Splenic lymphocytes from mice immunized with replicons alone, or replicons plus recombinant ureB produced abundant interferon-γ and no detectable interleukin-4 upon stimulation with recombinant ureB. These results establish that poliovirus replicons encoding H. pylori ureB are immunogenic and induce primarily a T helper 1 associated immune response.

Introduction

Helicobacter pylori is a non-invasive, Gram-negative bacterium that causes chronic superficial gastritis and peptic ulcer disease and is associated with gastric carcinoma [4], [29]. Although antibiotic treatment for H. pylori infection has resulted in the eradication in the infected host, the economics of treatment in developing countries coupled with the potential for emergence of resistance has necessitated the development of alternative strategies. A vaccine against H. pylori would be a cost effective way to prevent the late and possible life threatening disease manifestations. There have been considerable efforts toward the development of a vaccine using H. pylori urease, a major protein constituent of H. pylori which is involved in the pathogenesis of H. pylori infection [6], [8], [11], [12]. A great challenge in the development of vaccine strategies for H. pylori is to stimulate a mucosal immune response to this pathogen. Towards this goal, recombinant urease produced in E. coli protects mice from challenge with Helicobacter spp. when administered orally with cholera toxin [7], [9], [13], [14], [25]. Although results of these studies serve as an important proof of concept, in humans, the oral administration of mucosal adjuvants such as cholera toxin or heat labile E. coli enterotoxin might result in an unacceptable level of diarrhea [10], [15]. To circumvent this problem, recently genetically engineered mutants of heat labile E. coli enterotoxin [2] or cholera toxin [30], [31] have been made to eliminate toxicity. Oral administration of recombinant H. pylori urease in combination with E. coli heat-labile enterotoxin has recently been shown to protect against H. pylori challenge in non-human primates [3].

Genetically engineered poliovirus offers many attractive features for development as a vaccine vector for mucosal immunization. Poliovirus is transmitted orally and enters immunoreactive mucosal sites. Moreover, the virus is available in an attenuated form that is both safe and effective as an oral vaccine [24]. To exploit the potential of poliovirus as a vaccine vector, our laboratory has developed recombinant genomes (replicons) in which a foreign gene is substituted for the genes encoding the capsid. Replicons undergo a process of RNA amplification and express foreign protein upon introduction into cells. Importantly, since replicons do not encode capsids, they cannot spread from cell to cell and therefore do not cause disease. To encapsidate these replicons, a recombinant vaccinia virus that expresses the capsid proteins of poliovirus (VV-P1) is used to provide the capsid protein in trans [20]. Previous studies from this laboratory have described immunogenicity of replicons encoding the C fragment of tetanus toxin [19] or HIV antigens [20]. In both studies, replicons were given to transgenic mice which contain the receptor for poliovirus and resulted in production of serum antibodies to the foreign protein.

To determine if poliovirus based vectors would be suitable as a vector for H. pylori proteins, we have constructed poliovirus replicons that encode the H. pylori urease B subunit (ureB). Expression of the ureB protein from cells infected with replicons encoding ureB was confirmed using immunoprecipitation with specific antibodies. Immunization of transgenic mice susceptible to poliovirus with ureB was used to investigate the immune response. The results of this study point to the continued development of poliovirus replicons as a vaccine vector for H. pylori.