Oral Immunization with Attenuated Salmonella enterica Serovar Typhimurium Encoding Cryptosporidium parvum Cp23 and Cp40 Antigens Induces a Specific Immune Response in Mice

Attenuated Salmonella enterica serovar Typhimurium vaccine strain SL3261 was used as an antigen delivery system for the oral immunization of mice against two Cryptosporidium parvum antigens, Cp23 and Cp40. Each antigen was subcloned into the pTECH1 vector system, which allows them to be expressed as fusion proteins with highly immunogenic fragment C of tetanus toxin under the control of the anaerobically inducible nirB promoter. The recombinant vector was introduced into Salmonella Typhimurium vaccine strain SL3261, and the stable soluble expression of the chimeric protein was evaluated and confirmed by Western blotting with polyclonal C. parvum antisera. Mice were inoculated orally with a single dose of SL3261/pTECH-Cp23 or Cp40, respectively, and plasmid stability was demonstrated both in vitro and in vivo. Specific serum immunoglobulin G (IgG) antibodies against the Cp23 or Cp40 antigen were detected by enzyme-linked immunosorbent assay 35 days after immunization. Also, serum IgA and mucosal (feces) IgA antibodies were detected in 30% of the mice immunized with Cp23. In addition, prime-boosting with Cp23 and Cp40 DNA vaccine vectors followed by Salmonella immunization significantly increased antibody responses to both antigens. Our data show that a single oral inoculation with recombinant S. Typhimurium SL3261 can induce specific antibody responses to the Cp23 or Cp40 antigen from C. parvum in mice, suggesting that recombinant Salmonella is a feasible delivery system for a vaccine against C. parvum infection.Cryptosporidium parvum is an obligate intracellular parasite that infects intestinal epithelial cells and has been identified as being a significant cause of diarrheal disease in a variety of mammalian species including rodents, livestock, and humans (24). Infection is usually self-limiting in immunocompetent individuals but can be severe and even life-threatening for those that have compromised immune systems, such as human immunodeficiency virus-infected individuals, transplant recipients, children, and the elderly (30). The incidence of cryptosporidiosis has been reported to be in the range of 1 to 10% (34) but has been reported to be as high as 30% in children in India and Saudi Arabia (1, 10, 14). In light of the fact that chemotherapeutic agents for the treatment of infections of immunodeficient individuals are limited and not always efficacious, the development of a vaccine that is capable of inducing at least partial protection would be beneficial to specific high-risk populations. Data from human volunteer studies have suggested that at least partial immunity develops, as subsequent exposures with the parasite resulted in less-severe clinical signs (26).Since all life cycle stages occur in the host epithelium, the mucosal immune response is paramount to providing resistance and protection. The use of live oral Salmonella vaccines has been successful at delivering heterologous antigens and at generating a mucosal immune response against a number of organisms including intestinal parasitic species such as Toxoplasma gondii and Eimeria tenella (18, 29). Advantages of attenuated Salmonella vaccines include the fact that they induce both cell-mediated and humoral responses, elicit a systemic and local response, are easy to administer, and are affordable (13). To date, reports of the use of attenuated Salmonella as a vaccine vector in C. parvum are not available. Through this study, we assessed the use of an attenuated Salmonella strain carrying specific C. parvum antigens as a vaccine vector and the potential that it offers against C. parvum infection.In this study, we compared the abilities of attenuated strains of Salmonella to express the immunodominant antigens Cp23 and Cp40. These surface antigens of C. parvum are considered to be immunodominant since they are recognized by serum antibodies of humans and many other animals (25, 31, 36). Moreover, the level of oocyst secretion was reduced following the administration of colostrum directed against the Cp23 antigen (26). T-cell responses to Cp23 from infected mice (3), calves (36), and human peripheral blood mononuclear cells (33) with C. parvum infection have been reported, indicating its role in the immune response to C. parvum. Recombinant Cp40 antigen was previously shown to generate a T-cell proliferation response in mice (31). Also, monoclonal antibodies against Cp40 antigen have been shown to neutralize C. parvum infection and inhibit attachment in vitro (4). We also report the safety and plasmid stability of the Salmonella vaccine vector in mice as well as the ability to induce an antibody response against the expressed antigens.