Introduction and Background of the Project

Introduction

Plasmodium knowlesi is the leading cause of malaria in Malaysia, and cases across Southeast Asia, particularly Indonesia and Thailand, are rapidly increasing. This zoonotic species is naturally carried by macaque monkeys and transmitted by the Anopheles Leucosphyrus Complex. Deforestation and land-use change are contributing to the rise in human cases by increasing mosquito abundance in forest fringes and human settlements, and disruption of macaques’ natural habitat is increasing monkey-human interactions.

Rising zoonotic malaria has significant implications for achieving elimination status. Recent WHO advice indicates that elimination certification can only be granted if all human malaria species are eliminated and cases of zoonotic malaria are negligible. There is an urgent need for P. knowlesi-specific tools for patient diagnosis, and for surveillance to guide species-specific interventions.

Project objective

There are no P. knowlesi-specific point-of-care (PoC) tests. Rapid diagnostic tests based on the pLDH biomarker show high cross-reactivity between P. vivax and P. knowlesi making them impossible to distinguish. Current diagnostic practices take time and delay patient access to treatment. Simple, accessible PoC tools are urgently required.

Identification of P. knowlesi-specific diagnostic markers has been largely neglected. The serine repeat antigen (sera) multigene family has been extensively studied in P. falciparum and rodent parasite lines and plays critical roles across the parasite life cycle. The P. knowlesi Serine Repeat Antigen 3 (PkSERA3) antigen 2 has been identified as a P. knowlesi-specific exposure marker, with laboratory and population-level evaluations showing no cross-reactivity with P. vivax, a phylogenetically closely related species.

We will use this antigen to develop reagents for a P. knowlesi PoC diagnostic test.

Project design

Overall aim: Validate novel biomarker(s) and associated monoclonal antibodies for lateral flow assay development for the diagnosis of acute infections. 

Objective 1: Reagent optimisation: the optimised PkSERA3 ag 2 protein plus two variants will be used in the generation of monoclonal antibodies (mAbs). 

Objective 2: Analytical and clinical validation of PkSERA3 Ag2 and variants as species-specific indicators of acute P. knowlesi infection across epidemiological zones. 

Objective 3: Assessment of Technical Feasibility in the lateral flow system. The best performing mAbs will be assayed by ELISA, and further down-selection will lead to selected mAbs being printed onto test strips. Antibody reagents will be provided to a diagnostic test developer Contract Research Organization (CRO) to validate the technical feasibility of integrating the developed mAbs into a lateral-flow RDT.

Objective 4: Stakeholder consultation to understand the preferred test design, and to inform Product Design and generate evidence for a business case for this novel malaria RDT.

Target results: Validated P. knowlesi-specific mAb reagent for use in LFA development.

How can your partnership (project) address global health challenges?

Short-term impact: (i) progression towards the development of a P. knowlesi-specific PoC RDT; (ii) advance the characterisation of P. knowlesi biomarkers; (iii) communicate the diagnostic gap to incentivise 3rd party investment through PoC development, articulated use-case, and market size estimation. Long-term impact: (i) providing the tools to support improved patient diagnosis and outcomes, (ii) understanding spatial patterns of infection risk across endemic areas; and (iii) generate evidence to inform national elimination strategies.

What sort of innovation are you bringing in your project?

This project will involve the development and production of novel and innovative diagnostics to address the emerging problem of zoonotic malaria. The project activities are expected to deliver a range of new insights into P. knowlesi malaria, which will broadly support research and innovation alongside the primary intended outcome of reagent development and validation for diagnostic development.

Role and Responsibility of Each Partner

Ehime University will lead the laboratory activities to optimise and express at scale, then validate the new biomarker and associated monoclonal antibody (mAb) which will enable downstream diagnostic development. The Ehime team will use their in-house parasite cultures (P. knowlesi culture lines as well as P. falciparum) to assess the specificity of the generated mAb. They will also lead Objs 3 and 4 in collaboration with UMS.

Universiti Malaysia Sabah (UMS) will prospectively collect whole-blood clinical samples. They will validate the mAbs using banked serum and perform validation of clinical sample reactivity with recombinant proteins by ELISA, as well as IFA validation using candidate mAbs with patient slides. UMS will also contribute to the PPC development and lead the stakeholder discussions in Obj 4.

Both partners will contribute to assessing milestones and stage-gates through membership of the Steering Committee