High-Throughput Generation of P. falciparum Functional Molecules by Recombinational Cloning

Abstract

Large-scale functional genomics studies for malaria vaccine and drug development will depend on the generation of molecular tools to study protein expression. We examined the feasibility of a high-throughput cloning approach using the Gateway system to create a large set of expression clones encoding Plasmodium falciparum single-exon genes. Master clones and their ORFs were transferred en masse to multiple expression vectors. Target genes (n = 303) were selected using specific sets of criteria, including stage expression and secondary structure. Upon screening four colonies per capture reaction, we achieved 84% cloning efficiency. The genes were subcloned in parallel into three expression vectors: a DNA vaccine vector and two protein expression vectors. These transfers yielded a 100% success rate without any observed recombination based on single colony screening. The functional expression of 95 genes was evaluated in mice with DNA vaccine constructs to generate antibody against various stages of the parasite. From these, 19 induced antibody titers against the erythrocytic stages and three against sporozoite stages. We have overcome the potential limitation of producing large P. falciparum clone sets in multiple expression vectors. This approach represents a powerful technique for the production of molecular reagents for genome-wide functional analysis of the P. falciparum genome and will provide for a resource for the malaria resource community distributed through public repositories.

Protective immunity to pre-erythrocytic stage malaria

“…Outstanding issues in malaria vaccine development Ab-mediated immunity Refs Identify mechanisms that maintain high titers of CS-protein-specific Abs for a prolonged period of time [33][34][35][36]66,67,69] Determine the role of Abs specific for non-CS-protein sporozoite Ags [16,73,74] Establish the contribution to protection of Ab responses targeting malaria proteins expressed on the surface of infected hepatocytes [75] Identify mechanisms to induce production of high-avidity Abs [38] T cell-mediated immunity Identify the key sporozoite and liver-stage Ags that stimulate protective CD4 and CD8 T cell-mediated immunity [23][24][25]64] Determine if there is a requirement for persisting CD4/CD8 T E cells or CD4/CD8 T CM cells (or both) for long-term protection [50,51,54,58,59,61,76] Establish whether there is a need for chronic stimulation from a persisting Ag depot to maintain long-lived intra-hepatic CD4/CD8 T E cells [36,49,60,61] Characterize the relative contribution of multi-functional CD4/CD8 T cells versus unifunctional CD4/CD8 T cells in conferring protection [27,41,42] Identify the primary site (liver or lymph node draining the liver) of pre-erythrocytic stage Ag-specific CD4/CD8 T cell activation [15,51,54,69] Determine whether CD4/CD8 T cells induced by subcutaneous or intramuscular immunization with subunit vaccines migrate to the liver [51] Identify a cross-presentation pathway that can be targeted by exogenous Ag to activate CD8 T cells ...…”

“…Several CD4 and CD8 T cell epitopes have been mapped to the C terminus of the CS protein, and some of these are universal epitopes [19][20][21][22]. Currently, there are also extensive efforts to identify additional sporozoite proteins [23][24][25].…”

“…A principal hindrance to answering these questions in the RAS model is our lack of knowledge of the Ags expressed by the liver stage of the parasite. Consequently, extensive efforts employing both genomics and proteomics are being directed to discovering relevant liver stage Ags [23][24][25]. For example, 16 novel Pf proteins have been recently identified which peripheral blood mononuclear cells (PBMC) from RAS-immunized volunteers recognized more frequently than 'traditional' Pf Ags such as the CS protein and exported protein-1 (EXP-1) [64].…”

Malaria vaccines 1985–2005: a full circle?

“…At the molecular level, the advances have been remarkable during this time and are set to continue with the genomic and proteomic data that are accumulating [23,24]. That said, the asexual-bloodstage target antigen that is undergoing the most intensive investigation is still, as in 1985, the merozoite surface protein MSP-1.…”

“…A fair conclusion that is drawn frequently from studies of natural immunity is that, for vaccine development, it would be better to focus on cryptic epitopes [24] rather than epitopes of the highly immunogenic polymorphic or clonally variant domains [29]. However, some promising liver-and blood-stage candidate vaccine molecules have been selected after analysis of naturally acquired immune responses [30,31].…”