This Small Business Innovation Research Phase I project aims to develop a bivalent monoclonal antibody using novel Plasmodium falciparum (Pf) and P. vivax (Pv) proteins shed in the urine of febrile malaria patients. Using a bioinformatics approach, we will create, score, and select in silico-generated recombinants of natural antigen variants designed to express mosaics that maximize the inclusion of common potential in one structure and minimize the inclusion of rare epitopes. Antigenic properties of the recombinant antigens are validated by indirect ELISA using panels of well-characterized sera from apparently healthy individuals and clinical Pf and Pv malaria patients. The utility of the recombinant antibodies are evaluated in a dipstick test using a range of clinical urine samples from Pf/Pv endemic regions. Our overall objective is to develop a simple one-step urine-based dipstick test for the simultaneous but specific diagnosis of clinical Pf and/or Pv malaria.

The broader impact/commercial potential of this project relates to the development of a broad-based one step urine test for home-based or point-of-care diagnosis of clinical Pf and/or Pv malaria in persons with fever. The test will potentially provide a reliable tool to differentiate between symptomatic and asymptomatic disease, and provide better treatment options to those in need. It will potentially offer wider acceptability of clinical malaria diagnosis in endemic regions, especially in remote settings and in places where mixed infections frequently occur by obviating the occupational hazards involved with phlebotomy, thus helping to reduce the unnecessary use of antimalarial drugs. Overall, it has the potential to markedly impact the way over 95% of all clinical malaria is diagnosed and treated in endemic regions, and drive the current global effort towards home-based or point-of-care testing for malaria mandated by the World Health Organization prior to treatment in all cases of fever. Since it is based on the same platform as current RDTs, this test can be easily integrated into current healthcare structures to provide significant benefits to public health in most endemic countries. No such test is currently available.

Project Report

Malaria diagnostic dipstick tests have recently become the mainstay of current global malaria control efforts. However, these rapid tests are not amenable for use at home or in point-of-need settings where most cases of clinical malaria are managed. Public health experts have always known the greater value of urine over blood as source material for clinical malaria testing in patients with fever. The major limitation has been that specific parasite proteins that could be used as targets in a urine test are not available. This is due in part to the fact that, due to its acidity, urine tends to denature urine-excreted Plasmodium parasite target proteins that could serve as targets for antibody and urine dipstick test developement. In our approach, a finescale tool termed "mass spectrometry" was used to analyze urine samples from malaria patients and controls, resulting in the identification of unique malaria parasite protein fragments that withstood denaturation in patient urine. In the Phase 1 project, we have successfully applied a complement of bioinformatics, recombinant DNA technology and protein engineering of the identified urine-excreted parasite protein fragments to generate novel "mosaic" antibody reagents. These were shown to be specifically reactive to parasite proteins in the urine of clinical malaria patients infected with the two parasite species that cause over 98% of all malaria cases. In the Phase 2 project, the antibody reagents are being deployed in the development of single-step non-invasive urine-based tests. Our ultimate goal is to develop a non-invasive, one-step alternative test that can be performed by anyone, anywhere, to facilitate prompt diagnosis and immediate treatment in home or community settings.

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Fyodor Biotechnologies, Inc
United States
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