An N-terminal Pfs230 domain produced in baculovirus as a biological active transmission-blocking vaccine candidate.

Transmission-blocking vaccines have the potential to accelerate malaria parasite elimination by inducing antibodies that block parasite transmission from humans to mosquitoes. Pfs230, a gametocyte surface protein involved in gamete function, has long been a promising candidate. Due to the large size (3,135 amino acids), complex domains, and repeating six-cysteine (6-Cys) motifs with a multitude of disulfide bonds, the feasibility of expression of a full-length protein has been difficult. A priority focus, therefore, has been on the generation of single domains, including N-terminal fragments. Here we utilized a heterologous expression system, baculovirus, to produce an N-terminal domain of Pfs230 (Pfs230C1).Pfs230C1 (aa 443-731) was expressed in Super Sf9 cells with a poly-histidine affinity tag. Since the native host lacks glycosylation machinery, a single N585Q mutation was made to eliminate potential N-linked glycosylation. The expressed protein, purified by nickel affinity, ion exchange and size exclusion chromatography to >90% purity, was present in monomeric form, with an observed mass of 33,510 daltons (matching oxidized form). Peptide mapping and disulfide analysis confirmed the proper formation of predicted disulfide bonds.Antibodies, generated against Pfs230C1 in mice, bound to the gametocyte in immunofluorescence assay (IFA) and demonstrated functional activity in both the standard membrane feeding assay (SMFA) and the exflagellation assay (EXA).The biochemical, biophysical, and immunological results reported herein support the continued advancement of an N-terminal Pfs230 antigen (Pfs230C1) as a component of a transmission-blocking vaccine. Our results also support the continued use of the scalable baculovirus expression system for generation of complex Plasmodium proteins.