New assays for P. vivax drug and vaccine development

Plasmodium vivax is the most widespread and, except in equatorial Africa, the most prevalent malaria parasite of man. Although generally not lethal, vivax malaria is a highly debilitating disease affecting millions of people every year thereby significantly contributing to the poverty in malaria endemic countries. Vivax malaria research, drug, and vaccine development have never drawn the same attention as that of P. falciparum resulting in substantial deficiencies in P. vivax research. There is an urgent need for in vitro study models to expedite and facilitate the development of new P. vivax drugs as well as a vaccine. Only in vitro assays reliably simulating clinical conditions can provide the means for assessing drug and vaccine efficacy in a reproducible, well controlled environment. We recently demonstrated that a simple, semi-automated, ELISA-based procedure can be employed to measure parasite growth and its inhibition in P. falciparum in vitro. It is therefore the principal aim of this project to establish new culture models and to develop a semiautomated, nonisotopic growth inhibition assay for P. vivax. Initially a suitable target for the new P. vivax growth inhibition assay will be identified. In preliminary studies Plasmodium aldolase has been established as a promising target molecule. Based on the experience acquired during the development of the HRP2 drug sensitivity assay a new ELISA for measuring P. vivax biomass (and therefore growth and its inhibition) will be designed. A double-site sandwich ELISA based on two monoclonal antibodies specific to the target molecule combines high sensitivity and specificity with simplicity of use. High sensitivity will be essential as P. vivax parasite densities are generally much lower than those of P. falciparum. Due to the selective preference of P. vivax for invading reticulocytes a particular challenge in the development of a growth inhibition assay for P. vivax that reliably simulates in vivo conditions (i.e. beyond the first 48 hour cycle) will be to optimize growth conditions for P. vivax short term culture up to 72 hours. The test and plate layout for the P. vivax growth inhibition assay will be based on the design of the ELISA-based HRP2 drug sensitivity and bioassay for P. falciparum. After establishing culture models and setting up the growth inhibition assay the new test will be validated with fresh P. vivax isolates in field studies in Thailand and Bangladesh against the morphological assessment of parasite growth. New tools for P. vivax research will be essential to be able to control malaria in endemic regions of the world. The new assay is expected to provide a vital tool for malaria research and to have major implications for drug and vaccine development for P. vivax.

Plasmodium vivax is the most widespread and, except in equatorial Africa, the most prevalent malaria infection of man. Although generally not lethal, vivax malaria is a highly debilitating disease affecting millions of people every year. Due to its inherent tendency to cause chronic (relapsing) disease that leads to repeated attacks even after treating the primary infection, P. vivax is one of the main factors leading to economic constrains and poverty in endemic countries. P. vivax in vitro growth inhibition assays for assessing the spread of antimalarial drug resistance as well as for drug and vaccine development. They provide the means for assessing drug and vaccine efficacy on blood stage parasites in a reproducible, well controlled environment. A P. vivax in vitro growth inhibition assay will allow for the identification of suitable target molecules and the testing of ex vivo samples. We have developed a highly sensitive tool for assessing the presence, parasite density, and parasite growth of P. vivax as well as its inhibition by antimalarial drugs or vaccines. The assay is based on an enzyme-linked immunosorbent assay (ELISA), is highly sensitive, relatively inexpensive, semiautomated, nonisotopic and can easily be performed under field conditions. The use of antibodies specific to the target molecule combines high sensitivity and specificity with simplicity of use. The ELISA results produced an excellent, highly reproducible correlation with parasite density both in vivo as well as in vitro and allowed for the detection of P. vivax field isolates down to 0.0005% parasite density. Due to the selective preference of P. vivax for invading immature red blood cells the development of a growth inhibition assay for P. vivax that reliably simulates in vivo conditions has been a particular challenge. In the course of this project culture models for P. vivax have been developed based on a total number 94 different media compositions to define optimal culture conditions for short and medium term in vitro culture. The newly developed assays provide a highly sensitive tool for antigen detection (e.g. in diagnostic or screening settings), to quantify parasite biomass in growth inhibition assays (as used in drug and vaccine development), as well as for field research.