Introduction and Background of the Project

Introduction

The human complement system is a part of the innate immune response and consists of a cascade of enzymes that attack pathogens. It can act alone but it also enhances the efficacy of antibodies. Recent data suggest that Plasmodium falciparum, the causative agent of malignant malaria, can use the complement system for its own advantage to invade red blood cells and, in addition, it can recruit host complement regulators to protect itself from complement attack. We hypothesize that parasite proteins involved in this interaction are potential targets of the vaccine development.

Project objective

Identification of P. falciparum proteins that interact with host complement regulators and evaluation of their potentials as vaccine targets.

Project design

P. falciparum proteins that interact with host complement regulators will be identified by pulldown assays, mass spectrometry, and proteome microarray analysis. Recombinant proteins for the identified proteins will be produced and their binding capacity to the complement regulators will be evaluated. Specific antibodies against recombinant proteins will be produced and their effect on parasite viability and red blood cell (RBC) invasion capacity will be evaluated.

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

According to WHO, Plasmodium falciparum malaria is responsible for the deaths of around 438,000 worldwide in 2015. Commonly used anti malaria drugs are losing their effcitiveness due to anti malaria drug resistance in many places like in Africa. An highly effective vaccine against malaria will be an important tool towards achieving WHO goal to reduce mortality by ≥ 40% by 2020 and by ≥ 90% by 2030. Our study will fill a major gap in the vaccine development strategy which until now has ignored the parasite’s ability to evade complement and the identified proteins could serve as a single or a part of multicomponent highly effective malaria vaccine.

What sort of innovation are you bringing in your project?

The use of full P. falciparum proteome microarray with approximately 90% coverage of the entire protein coding genome to identify complex parasite protein ligands is a new and innovative approach to vaccine development. Antigen Discovery Inc. has developed 3D7 P. falciparum proteome microarray contains ~8,000 full length or fragmented proteins as a sole source.  

Role and Responsibility of Each Partner

José A. Stoute, MD, The Pennsylvania State University College of Medicine will serve as overall PI for the program. He will be in charge of identifying parasite protein ligands using pulldown assays and mass spectrometry. In addition, his laboratory will test the effect of antibodies against the protein ligands on parasite viability and RBC invasion capacity. Osamu Kaneko, MD, PhD, Institute of Tropical Medicine, Nagasaki University will lead the design of the protein expression plan and will produce recombinant proteins for testing and perform binding assay. Joseph Campo, PhD, Antigen Discovery, Inc. will direct the proteome microarray studies with samples provided by Dr. Stoute.

Others (including references if necessary)

https://www.ghvap.org/platforms/Pages/Antigen-Arrays.aspx

Final Report

1. Project objectives

Identification of P. falciparum (Pf) proteins that interact with host complement regulator X and evaluation of their potentials as vaccine targets.

2. Project design

P. falciparum proteins that interact with host complement regulators will be identified by pulldown assays, mass spectrometry, and proteome microarray analysis. Recombinant proteins for the identified proteins will be produced and their binding capacity to the complement regulators will be evaluated. Specific antibodies against recombinant proteins will be produced and their effect on parasite viability will be evaluated.

3. Results, lessons learned

A total of 12 experiments were performed with different versions of Pf partial proteome and full proteome (~91% Pf coverage) microarrays. Recombinant X was sourced from three vendors. However, we observed inconsistency in binding through replicated experiments and concluded that binding strength and stability were affected by, to date, unknown experimental factors. Therefore, this approach was abandoned. We conducted multiple pulldown experiments in which biotinylated X was incubated with intact merozoites with or without BS3 linker. In four experiments Pf formate nitrite transporter (PfFNT) was identified always in the presence of X. PfFNT is a transmembrane protein found on the surface of merozoites and is known to facilitate the movement of lactate across the membrane. A second protein, merozoite surface protein 9 (PfMSP9), is a part of a multiprotein complex on the merozoite surface and is shed during cell entry, which was found in 3 out of 4 screens in the presence of biotinylated X. Therefore, we decided to concentrate on these two proteins to determine whether either or both could interact with X. To this end, three transgenic Pf lines were generated; one expressed myc-tagged PfFNT, another expressed myc-tagged PfMSP9, and the third expressed myc-tagged Pf92, a protein known to interact with factor H and that we planned to use as a positive control. Co-immunoprecipitation experiments were inconclusive as to whether X interacted with PfFNT or PfMSP9. Part of the reason is due to lack of the appropriate negative controls and the failure to demonstrate whether Pf92 interacts with factor H as described. Both antibodies raised against recombinant PfFNT or PfMSP9 showed growth inhibitory activities, although involvement of X in the inhibition was not confirmed.