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

Source of this paper is: Benitez AJ, McNair N, Mead JR. Clinical and vaccine immunology. 2009. 16(9):1272-1278.

The live oral Salmonella vaccines have been successfully used in delivering heterologous antigens and in generating a mucosal immune response against a number of organisms including bacteria, viruses and parasites. Among the parasitic species Toxoplasma gondii and Eimeria tenella are two good eaxmples. The advantages of attenuated Salmonella vaccines include:

• They induce both cell-mediated and humoral responses
  
• They elicit both systemic and local response
  
• They are easy to administer
  
• They are affordable
  

In this paper from the laboratory of Dr. Jan R Mead, Emory University School of Medicine, Atlanta, Georgia, the authors assessed the use of an attenuated strain of Salmonella carrying specific C. parvum antigens as a vaccine receptor and the protection that it offers against Cryptosporidium parvum infection.

Cryptosporidium parvum is an obligate intracellular protozoan parasite that causes gastrointestinal symptoms when ingested by humans. Infection with C. parvum can be severe and even life threatening in immuno-compromised individuals. Treatment is available but it could it could be less efficient in immuno-deficient individuals, thus, development of a vaccine that is capable of at least inducing partial immunity in such individuals would be beneficial.

The experiments performed and results obtained are:

1. Expression of Cp23 and Cp40 S. Typhimurium constructs: Cp23 and Cp40 genes from C. parvum were PCR amplified and sub-cloned into suitable expression vectors. The resulting constructs were transformed into Salmonella vaccine strain SL3261 (this strain contains a mutation in the aromatic amino acid biosynthetic pathway and has been previously reported to be the safest vaccine strain) and the expression of proteins was tested by SDS-PAGE and Western Blotting using mouse C. parvum serum. Both proteins were stably expressed in both aerobic and anaerobic conditions and in vaccine strain SL3261 and remained soluble.
   
2. Plasmid stability and Salmonella persistence in vivo: SL3261 containing the Cp23 protein was passaged in antibiotic free medium for a period of nearly five days. The plasmid was stably maintained when plated on to medium with or without antibiotics. Furthermore, three groups of mice were inoculated orally with SL3261 and euthanized at days 3, 10 and 25 post-immunization and bacterial counts per time point were performed on homogenates of spleen, liver and intestines. These counts showed that the recombinant construct grew and persisted in the reticulo-endothelial system.
   
3. Immunogenicity of Salmonella derived Cp23 and Cp40 antigens: Mice were immunized with SL3261 without Cp23 or Cp40 and with SL3261 with Cp23 and SL3261 with Cp40. Cp23 and Cp40 antibody production was determined in vaccinated mice at seven weeks after immunization. Specific serum Ig G levels were enhanced in mice immunized with Sl3261/Cp23 or SL3261/Cp40 in comparison to mice immunized with the control. Furthermore, the oral immunization with SL3261/Cp23 or SL3261/Cp40 induced only an IgG1 antibody response and no IgG2a was detected. These findings suggested that in response to immunization with S. Typhimurium encoding C. parvum Cp23 and Cp40 antigens, a Th2 type response is generated. It was also observed that a prior immunization with a recombinant DNA vaccine resulted in significant increase in titres of IgG specific anti-Cp23 and anti-Cp40 antibody response.
   
4. In vitro detection of neutralizing antibodies: To check whether antibodies from orally immunized mice can inhibit parasite from infecting host cells, sera from immunized mice were incubated with sporozoites and they were then allowed to infect HCT-8 cells. It was observed that sera from mice immunized with either Cp23 or Cp40 were able to reduce infection by sporozoites by 30%.
   

Conclusions:

The study demonstrates the immunogenicity of a single oral immunization with Salmonella vaccine strain SL3261 that expresses recombinant C. parvum antigens (Cp23 and Cp40) in mice. The study also shows the stability and safety of the recombinant Salmonella vector and a partial inhibition of infection by sporozoites incubated with immune sera. Although with some optimizations are still required, the use of Salmonella vaccine vector to deliver C. parvum antigens appears to be a feasible way to elicit humoral and mucosal immune response in the immunized host,