Immunoassays for identifying and evaluating vaccine candidates that target pre-erythrocytic stages of Plasmodium vivax

Evaluating pre-erythrocytic (PE) vaccine candidates is challenging due to the biological complexity of Plasmodium parasites and their host interactions. In particular, identifying and characterizing new PE vaccine candidates has been hampered by the inefficiency of functional assays to evaluate targets in the PE stages of Plasmodium vivax. To overcome this obstacle, we developed novel functional assays to immunologically assess potential PE targets of Plasmodium sporozoites and early liver stages. These highly efficient, quantitative functional assays recapitulate the pivotal period of sporozoite transition from mosquito to human byoptimization of the in vitro microenvironment and exposure to biological stimulatory factors. Liver-stage growth is measured by sporozoite inoculation into an in vitro human liver model, achieving infection rates of 3-7%, where high-content image analysis is used to quantify parasite invasion and development in primary human hepatocytes. An inhibition of liver-stage development assay (ILSDA) was first optimized with species-specific, anti-CSP monoclonal antibodies to the P. vivax circumsporozoite (CSP) protein. The modified ILSDA is sensitive and efficient, showing blocking of early PE stages of P. vivax in a concentration-dependent manner with developmental delays on parasite growth. The ILSDA experiments have now been extended using monoclonal antibodies and immune sera to additional target antigens. To refine functional analysis of PE targets in early infection phases, two additional high-content imaging assays were created that analyze sporozoite migration in a live motility assay and a live hepatocyte cell traversal assa. Altogether, these novel functional assays simulate key development and transition phases that enable us to evaluate potential PE vaccine candidates and analyze complex Plasmodium sporozoite phenotypes.