Malaria kills about 445,000 people and infects over 200 million people despite current control methods, including bed nets, insecticides, insect repellants, and antimalarial drugs. A safe and effective vaccine against Plasmodium falciparum, the parasite responsible for most of these deaths, would represent a major public health milestone and save millions of lives. Development of such a vaccine will require reliable and cost-efficient production of Plasmodium falciparum sporozoites. Unfortunately, it is not feasible either technically or economically to isolate these sporozoites in sufficient numbers for widespread vaccination efforts from infected mosquitos. An alternative method of making these sporozoites is therefore essential to vaccine development and production. MalarVx, Inc. has demonstrated the capability of growing sporozoites in a practical, cost-effective culture system. These sporozoites closely recapitulate the properties of mosquito-derived sporozoites, according to comprehensive in vitro tests. In collaboration with Dr. Brandon Wilder at the U.S. Naval Medical Research Unit- 6 (NAMRU6) in Lima, Peru, we propose to demonstrate that Plasmodium falciparum sporozoites produced in vitro are biologically equivalent to Plasmodium falciparum sporozoites isolated from the salivary glands of infected mosquitoes. We will test the in vitro-produced sporozoites for 1) infectivity and development in vivo in a humanized mouse model, and 2) immunogenicity in non-human primates. These studies will use both the wild- type NF54 GFP-Luciferase (GFP-Luc) reporter strain and NF54 GAP3KO, a genetically attenuated parasite strain currently undergoing clinical development as a vaccine candidate. To test equivalence, we will study protein markers expressed in sporozoites, including the major surface protein CSP as well as TRAP and mTIP. We will also analyze sporozoite functions, including gliding motility and hepatocyte infection. We will utilize cell culture approaches and a humanized-liver chimeric mouse model to test hepatocyte invasion, traversal, and infection. Finally, Dr. Wilder at the NAMRU6 will supervise testing of the immunogenicity of cultured sporozoites in Aotus monkeys. Aotus are the only immune-competent animals (other than humans) that can be fully infected with both Plasmodium falciparum and Plasmodium vivax, the two major species of human malaria parasites. This work will support the further development of the Aotus model for testing of WSV safety, immunogenicity, and efficacy against multiple strains and species of Plasmodium. In Phase II of this SBIR, we will 1) increase the scale of our culturing system and the SPZ purification process, 2) optimize cryopreservation, and 3) perform additional pre-IND studies.
The only effective vaccines against Plasmodium falciparum (Pf) malaria are based on whole sporozoites that are attenuated by irradiation or genetic modification or that are administered in combination with chemoprophylaxis. Studies proposed here will test equivalence of in vitro sporozoites (IVS) produced using cell culturing methods to sporozoites obtained from mosquitoes by studying protein expression and function of IVS. Scalable production of Pf sporozoites would provide sufficient material for vaccine commercialization.
