Immune Deficient Mosquitoes for Improved Malaria Sporozoite Vaccine Manufacture

Abstract An ideal tool for eliminating Plasmodium falciparumPfthe causative agent ofof all malaria deathswould be a highly effective vaccine that prevents blood stage infection and thereby prevents both disease and transmissionSanaria has developed two effective vaccines that are phaseclinical trialsPf sporozoiteSPZvaccine that prevents Pf blood stage infection in andgtof recipients and PfSPZ Challengeinfectious PfSPZused in PfSPZ CVaca vaccination approach utilizing PfSPZ Challenge administered in the presence of an anti malarial drug such as chloroquine which prevents blood stage infection inof the volunteersBoth these vaccines are on an aggressive timeline to commercializationPfSPZ used in these products are extracted from aseptically reared mosquitoesIncreasing the number of PfSPZ mosquito will reduce the cost of goodsThis project will develop a line of immune deficient Anopheles stephensi mosquitoes highly susceptible to PfSPZ infectionsSanaria and the Univof Maryland Institute for Bioscience and Biotechnology Research used transgenic technologies to introduce and express genes that resulted in the down regulation of RELimmune pathway in Astephensiincluding Casparnegative regulator of RelReltranscription factorand LRIMeffectorReduction of LRIMexpression using transgenic RNA interference resulted in increased Pf infections of Pf oocysts and PfSPZfold increasein non aseptic AstephensiThese results are encouraging because transgenic RNA interference results in hypomorphic phenotypesNull alleles are expected to have stronger phenotypes with even more intense infectionsWe will use CRISPR Castechnologies to create in Astephensi null alleles through mutagenesis of LRIMand three additional immune genesTEPLLand JNKthat are implicated in limiting Pf infections in Anopheles mosquitoesThese immune deficient Astephensi lineswill provide a stable and much more efficient PfSPZ production platform for Sanariaandapos s manufacturing processWe willGenerate mosquito lines null for LRIMTEPLLand JNK using CRISPR Casdriven gene specific mutagenesisTest the mutant lines and select at least one line that producefold higher PfSPZ infection intensities compared to wild typeadapt this line to aseptic PfSPZ manufacturing process and test the potency of PfSPZ manufactured in this selected linetest the prevalence and intensity of Pf infection in Astephensi mutant lines when infected with other strains of PfSubmit a Biologics Master File to the FDA describing the strain and its incorporation into Sanariaandapos s manufacturing process PROJECT NARRATIVE Sanaria has developed two highly effective vaccines to protect individuals against malaria and to eradicate malaria from defined geographical areasthe first is a live attenuated whole organism vaccine called SanariaPfSPZ Vaccine and the second is a live infectious vaccine administered with an antimalarial druga vaccine called SanariaPfSPZ CVacBoth these vaccines require the production of PfSPZ in aseptic Anopheles stephensi mosquitoesThis proposal aims to increase the yields of PfSPZ by genetically modifying the mosquito to suppress its immune systemSuccessful implementation of this project will greatly reduce cost of vaccine manufacturethereby making the vaccine available more cheaply for travelers and developing world populations