Introduction and Background of the Project

Plasmodium vivax, the predominant malaria parasite in the Asia-Pacific and the Americas, has the ability to form long-lasting dormant stages, called hypnozoites, which can remain in the patient’s liver for many months. When these hypnozoites reactivate, they can initiate a new infection in a patient’s blood that can cause an acute disease episode and contribute to onwards transmission to mosquitoes and new human hosts. Such relapsing infections are the main reason why it is more difficult to control and eventually eliminate P. vivax from its tropical heartland.

Hypnozoites are difficult to target, as there is no diagnostic test that can directly detect their presence in a patient’s liver and patients can have liver infection in the absence of a blood-stage infection. As a consequence, if only people with blood-stage infections are treated, a substantial part of the parasite reservoir will be missed and these medical interventions will not be efficient. Typical P. vivax strains found in tropical settings cause a primary infection followed by a primary relapse. A diagnostic test that detects exposure to P. vivax infections that can identify most, if not all, individuals who potentially harbor hypnozoites is crucial. In order to develop such a test, a well-validated panel of carefully selected, highly immunogenic antigens is required.

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

Asia-Pacific countries aim to eliminate malaria from their region by 2030. The tendency towards fragmentation of transmission into ‘hot-spots’ of high transmission within large areas of otherwise little or no transmission and the challenges in eliminating P. vivax represent major obstacles to achieving this goal. The development of relevant serological markers of P. vivax transmission will contribute to addressing these challenges in two ways:

By strategically identifying relevant serological markers this will address a major gap in our surveillance tool kit for malaria control and elimination. Firstly, such an assay could be used for monitoring the impact of intensified control on malaria transmission at a population level, even when overall transmission levels are low, including remote areas with poor surveillance systems that are most resilient to malaria elimination. Serological markers will thus give program managers the ability to stratify their control approaches according to transmission risk and specifically target areas with high residual transmission, thereby greatly improve their programs’ effectiveness.

Secondly, current diagnostic tools cannot directly identify dormant liver stage infections. Therefore, mass-drug administration (MDA) is the only current approach effective at eliminating the hypnozoites in asymptomatic individuals. While technically feasible the logistical challenges of MDA mean that it has only been successfully applied either on small islands or in countries with relatively strong health systems. Serological screening and restricting treatment to antibody-positive individuals will reduce the number of people targeted by mass treatment by 80-90%, making the intervention logistically simpler, safer and socially more acceptable. This will make drug-based interruption of P. vivax transmission equally feasible in higher transmission areas with weak(er) health systems. Serological markers could thus play an essential role in achieving a P. vivax malaria free Asia-Pacific.

What sort of innovation are you bringing in your project?

The concept of diagnosing not only concurrent but recent infections and the application of these tests to identifying people who are at high risk of carrying dormant liver stage parasites represents a paradigm shift in malaria diagnostics. If successful, this diagnostic test would be a first of its kind.

Several innovative approaches are employed in both the discovery and validation of these candidate marker antigens. The validation will be done in cohort studies in 4 different populations and evaluates the performance of each antigen individual and in combination for the prediction of exposure. By using multiple endpoints and completely separate clinical studies, we are thus conducting completely independent discovery and validation studies, thereby strengthening our ability to identify markers that work both globally and in different age groups. 

Role and Responsibility of Each Partner

Ehime University will be responsible for expressing 40 preliminary candidate antigens in the superior Wheat Germ Cell-Free System (WGCFS) selected from a list of antigens with decaying antibody titers discovered using a very large E. coli cell-free protein microarray. Ehime University will screen the decay of antibody titers against these antigens with plasma samples from Thai and Brazilian P. vivax patients. Together with results from 308 previously screened WGCFS expressed antigens short-list of 24 candidate antigens will be obtained. In order to obtain good quality protein for validation, Ehime University will optimize candidate protein constructs and develop improved expression vectors for the larger-scale expression.

CellFree Sciences will be responsible for the expression of 24 candidate proteins at 1-2 mg scale, and delivery quality controlled protein batches to be used for validation at the WEHI.

WEHI will be responsible for the overall coordination and management of the project. In addition, WEHI will conduct the statistical analyses of high-throughput screening data and in collaboration with Ehime University be responsible to for the downselection of candidate antigens. WEHI will then conduct the validation of candidate proteins (expressed by CFS). For this, we will measure antibodies to candidate proteins in people from longitudinal cohort studies in Thailand, Brazil, Papua New Guinea (PNG) and the Solomon Islands using a newly developed multiplex quantitative bead array assay and determine the performance of these antibodies, individual or in combinations, as biomarkers of recent and asymptomatic infections of P. vivax.

FIND’s role is to leverage its expertise in the development of new diagnostic solutions to ensure that the serological markers that are being developed in the frame of this project can ultimately be deployed on an appropriate technology platform and result in a diagnostic test that meet the specific needs of malaria endemic countries. FIND is managing consultations with P. vivax experts to develop target product profiles for serology tests and identify the specific requirements associated with the detection of recent past P. vivax infections. FIND is also coordinating a technology landscape assessment and a market analysis for this type of test to pave the way for the development of viable new diagnostic solutions for P. vivax malaria.

Final Report

1. Project objective 

Plasmodium vivax, the predominant malaria parasite in the Asia-Pacific and the Americas, has the ability to form long-lasting dormant stages, called hypnozoites, which can remain in the patient’s liver for many months. These stages, which cannot be detected with any currently available diagnostic test, are the key hurdle to malaria elimination in the Asia-Pacific region. This project thus aimed to validate a panel of novel serological biomarkers as indicators of recent exposure to P. vivax infections. By detecting recent-past infections these can serve as an indirect marker for people at the higher risk of recurrent P. vivax infections and thus possibly having hypnozoites.  

2. Project design

The project objective was obtained in 3 steps: First, the project validated candidates obtained in an earlier high-throughput screen using high quality wheat-germ cell-free proteins and an ultra-sensitive magnetic bead assay. The sensitivity and specificity of these markers for predicting the presence of asymptomatic and symptomatic infections in previous 9 months was then determined in cohort studies from low-transmission settings in Thailand, Brazil and Solomon Islands. Lastly, the feasibility of different technological approaches for the simple, rapid and robust detection of serological biomarkers in blood was determined through an evaluation of existing and near market technologies.

3. Results, lessons learned

Out of 55 candidates identified in our earlier discovery studies, 41 were successfully produced at sufficiently high yield and purity. All but one of these were successfully transferred to a high-throughput assay format that allowed accurate measurement of antibodies in clinical samples. Antibodies to these 40 candidate antigens as well as 13 vaccine and 12 highly immunogenic control antigens were then measured in 3 cohorts from Thailand, Brazil and Solomon Islands with > 2,500 participants. A majority of antigens showed strong associations of antibody titers with time since last infection. Titers decreased significantly from participants with concurrent and recent infections (

Two specific target product profiles, one for the identification and treatment of likely hypnozoite carriers (TPP1) and one for general surveillance application (TPP2) were developed with the help of expert panels. The best 5-antigen combination achieved highly promising performance in line with the requirements identified for both TPPs. The validation of candidate markers was thus able to confirm the technical feasibility of developing serological biomarkers of recent exposure to P. vivax infections and identified a validated short-list of 12 antigens to be considered for further development.

A detailed assessment of both commercially available as well as platforms currently under development was conducted. The outcome of multiple rounds of assessment identified a number of promising platforms that can even meet the challenging requirements associated TPP1 calling for a point-of-care assay. These lead-platforms are now being considered for further in-depth evaluation.

All in all the data and the technical feasibility information generated by this project support a GO decision to commencing the development feasibility phase for a novel sero-diagnostic test of recent exposure to P. vivax infections.