Cytolysins expressing liver stage parasites as novel live attenuated malaria vaccines

Malaria is amongst the deadliest of infectious diseases globally. It is a vector-borne disease transmitted by the bite of female Anopheles mosquitos. Causative agents are Plasmodium species protozoan parasites. There are 5 species that are known to cause disease in humans. Over 3 billion people are at risk of malaria transmission. Prevention methods such as vector control and seasonal mass drug administration are effective tools for malaria control but with increasing resistance to frontline drugs, efficient vaccine development becomes imperative for malaria elimination. Ideal malaria vaccine should protect against the most common Plasmodium species and should have more than 75% efficacy, and a long-lasting protection. Only available vaccine so far is RTS,S which underperforms the expectations. Complex biology and various immune evasion strategies of Plasmodium parasites makes vaccine development challenging. Pre-erythrocytic stages are the bottleneck of development in vertebrate hosts. Vaccine strategies against this stage are so far the most promising. Live-attenuated whole sporozoite vaccines have been proven to confer sterile protection. Various genetic modification strategies allowed precise attenuation profiles which led to development of genetically attenuated parasites (GAP). In this thesis study, we designed two alternative attenuation strategy to be evaluated as GAP vaccines that aimed for late liver stages arrest in rodent malaria model. Both candidates had similar designs: knockout of an essential liver stages gene, and expression of a bacterial cytolysin protein. Selected genes, LISP1 and LISP2, are only expressed during liver stages. Bacterial cytolysins sequences of Streptolysin O (SLO) from Streptococcus pyogenes and Listeriolysin O (LLO) from Listeria monocytogenes were replaced with coding sequences of target genes. Resulting strains, ΔLISP1::SLO and ΔLISP2::LLO, were phenotypically analyzed for all life cycle stages of the parasite. To understand the effect of cytolysins, we replaced the cytolysin sequences with fluorescent proteins that would also work as reporter genes for liver stages expression (ΔLISP1::mNeonGreen and ΔLISP2::mTurquoise2). Cytolysin expressions significantly improved attenuation profiles of both strains. BALB/c mice were immunized with both strains and immunized mice were challenged against wild type sporozoites. As a result, ΔLISP1::SLO strain were completely attenuated. ΔLISP1::SLO strain-immunization of mice could protect against lower numbers of sporozoites but could not protect against higher numbers of sporozoites. In contrast, attenuation profile of ΔLISP2::LLO strain were strong, but there were occasional breakthrough infections. ΔLISP2::LLO strain-immunization of mice conferred sterile protection against extremely high numbers of sporozoites. Attenuation profile of ΔLISP2::LLO can be improved with various modifications, and this strategy can easily be applied to human malaria as a promising vaccine candidate in future studies.