| Malaria remains one of the most serious diseases of mankind. The annual incidence of the disease is estimated to be 300-500 million, and it causes about 1-3 million deaths each year. Traditional methods to controlling malaria have met great challenges due to emergence and rapid spread of the drug-resistant strains of the parasite and insecticide-resistant mosquito vectors. Development of an effective vaccine against malaria would be an important alternative approach. Since only asexual blood stage of the parasite's life cycle causes the disease, development of malaria vaccine against this stage should reduce the mortality and morbidity of the disease. In the past decade, several vaccine candidates derived from the blood-stage of the parasite have been identified. Of them, two antigens, the 19kDa carboxyl-terminal domain of Merozoite Surface Protein 1 (MSP1-19) and the domain III of Apical Membrane Antigen 1[AMA-1 (III)] have been considered a leading vaccine candidates because of experimental evidences showing that they can induce inhibitory antibodies. A chimeric protein named PfCP-2.9 was constructed in this laboratory, which is composed of MSP1-19 and AMA-1 (III) of Plasmodium falciparum via a hinge sequence. The chimeric protein has been produced in Pichia pastoris at a yield of 2.6g/ liter. Immunization of animals with PfCP-2.9 formulated with ISA720 Elicited high levels of antibodies. Moreover, immune sera against the antigen in rabbit as well as monkey almost completely inhibited the parasite growth in vitro. The PfCP-2.9 vaccine candidate has been approved for clinical trial by Chinese State Drug Administration. In this study, we investigated immunogenicity of the vaccine candidate in various strains of mice as well as various adjuvants. In addition, the potency and stability of the vaccine formulated with ISA720 was studied.Five strains of mice, including four inbred mice and one outbred mice, were immunized subcutaneously with the fusion protein formulated with ISA720. Specific antibodies responses were analyzed by using ELISA and IFA. The result showed that strong immune responses to the antigen were induced in all strains, and specificantibody titer reached 105 in all immune sera after the third immunization. Moreover, immune sera can significantly recognize the cultured malaria parasite. The prevailing isotype of antibody responses associated with immunization were IgGl and IgG2a, whereas IgG2b and IgG3 were minor antibody response. However, there were clear differences in antibody levels, the isotype distribution of the IgG response, and recognition to nature antigens of the parasite among five strains mice because of distinct genetic backgrounds. The much stronger antibody responses were observed in Kunming and BALB/ca strains of mice comparing with that in other strains studied. However the highest IFA titer was still detected in BALB/ca mice but Kunming strain of mice generated the lowest IFA antibody titer.Based on the above experiments, BALB/ca and C57BL/6J mice as well as rabbits were selected to further investigate the immunogenicity of the antigen formulated with various adjuvants. Animals were immunized with PfCP-2.9 formulated by Freund, alum and ISA 720 adjuvants. Specific antibodies and isotypes of antibodies responses were analyzed by using ELISA and IFA, and cellular immune responses were determined by lymphocyte proliferation assay and cytokine measurement. Immune sera were detected for their ability to inhibit the parasite growth in vitro. The results indicated that adjuvants obviously influenced the immune responses in the animals on antibody levels, IgG subclass, invasion-inhibitory rate, stimulation index (SI) of lymphocyte and production of cytokine.Compared homologically with C57BL/6 immunized groups, immune effects of BALB/c groups were stronger, which is similar with the result of different strains mice. However, antibody and IgG subclass levels of all the immunized groups of ISA720 and freund adjuvant are notably higher than those of Alum adjuvant. In BALB/c mice...
|
PDF Full Text Request