Genetically Modified Conditional Sexing A. stephensi Line for PfSPZ Manufacture

There is an the urgent need for new tools for prevention, control, and elimination of malaria: “..after an unprecedented period of success in global malaria control, progress has stalled. In 2016, there were an estimated 216 million cases of malaria, an increase of about 5 million cases over 2015. Deaths reached 445 000”, 15,000 more than in 2015. Plasmodium falciparum (Pf) sporozoites (SPZ) are the only immunogens that have prevented Pf infection in andgt;90% of human recipients. Sanaria® PfSPZ Vaccine, composed of radiation attenuated, aseptic, purified, cryopreserved PfSPZ has repeatedly conferred high level protection against controlled human malaria infection (CHMI) and naturally transmitted heterogeneous Pf. PfSPZ are grown in aseptically reared Anopheles stephensi mosquitoes. In this project we plan to make the aseptic mosquito rearing process up to 100% more efficient at no additional cost by removing male mosquitoes from the system at the embryonic stage using a lethal gene insert that can be controlled by application of the drug, doxycycline. In our four specific aims we will: 1) Establish a Y-linked docking strain in A. stephensi. Using CRISPR/Cas9 gene insertion we will create a site in the Y (male) chromosome into which larger gene drive cassettes can be inserted; 2) Establish A. stephensi carrying Y-linked dominant-negative form of relish2 (dnRel2) under the control of Tet-on transactivator. Expression of dnRel2 in A. stephensi causes embryonic lethality. The Y-linked docking strain will be used to integrate a gene drive cassette containing a Tet-response element (TRE), a minimal promoter and dnRel2. Here, dnRel2 will be expressed only when both doxycycline and the Tet-On transactivator are available; 3) Establish a transgenic A. stephensi line carrying Tet-On transactivator. Expression of dnRel2 in the transgenic line requires the binding of Tet-On transactivator to TRE. Therefore, another transgenic line will be established by inserting Tet-On transactivator under the control of bZP1, an A. stephensi early embryonic gene promoter. In this line, the expression of the transactivator will be tightly controlled by the bZP1 promoter which is expressed until 36 h after egg laying. A homozygous line for this transgene will be established; and 4) Generate and test a transgenic conditional male-lethal sexing strain of A. stephensi. dnRel2 and the transactivator will be brought together by crossing dnRel2 males with Tet-On transactivator females. In the progenitor male eggs, the Tet-On transactivator will bind to TRE in the presence of doxycycline, driving the expression of dnRel2, thus causing lethality to the transgenic males. In the absence of doxycycline both sexes will survive because the Tet-On transactivator is not able to bind to TRE. Female mosquitoes from this strain will be assessed for their capacity to harbor high intensity infections of Pf. Eggs from all lines and strains generated in this project will be cryopreserved.The ideal tool for eliminating Plasmodium falciparum malaria from defined geographical areas would be a highly effective, long-acting vaccine that prevents infection, disease and parasite transmission: both Plasmodium falciparum (Pf) sporozoite (SPZ) Vaccine (PfSPZ Vaccine) and PfSPZ chemoprophylaxis vaccine (PfSPZ-CVac) offer durable 100% protective immunity against controlled human malaria infection (CHMI) and are manufactured in aseptically reared mosquitoes. This proposal aims to develop a conditional male-lethal sexing A. stephensi line using the Tet-On system in which male mosquito embryos will be selectively killed by conditional induction of a dominant-negative form of relish2. Success in this project will increase by ~100% the numbers of female mosquitoes reared per unit space thereby greatly reducing cost of goods for Sanaria’s vaccines.