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N-Terminal Pfs230 Domain Produced in Baculovirus as a Biological Active Transmission-Blocking Vaccine Candidate.

ABSTRACT: 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 6-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 (amino acids 443 to 731) with a polyhistidine affinity tag was expressed in Super Sf9 cells. 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 Da (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 an 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 the generation of complex Plasmodium proteins.

SUBMITTER: Lee SM

PROVIDER: S-EPMC5629673 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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N-Terminal Pfs230 Domain Produced in Baculovirus as a Biological Active Transmission-Blocking Vaccine Candidate.

Lee Shwu-Maan SM Wu Chia-Kuei CK Plieskatt Jordan L JL Miura Kazutoyo K Hickey John M JM King C Richter CR

Clinical and vaccine immunology : CVI 20171005 10

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 6-cysteine (6-Cys) motifs with a multitude of disulfide bonds, the feasibility of expression of a full-length protein has been difficult. A priorit ...[more]

PMID: 28747311

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Project description:BACKGROUND:Control and elimination of malaria can be accelerated by transmission-blocking interventions such as vaccines. A surface antigen of Plasmodium falciparum gametocytes, Pfs230, is a leading vaccine target antigen, and has recently progressed to experimental clinical trials. To support vaccine product development, an N-terminal Pfs230 antigen was designed to increase yield, as well as to improve antigen quality, integrity, and homogeneity. METHODS:A scalable baculovirus expression system was used to express the Pfs230D1+ construct (aa 552-731), which was subsequently purified and analysed. Pfs230D1+ was designed to avoid glycosylation and protease digestion, thereby potentially increasing homogeneity and stability. The resulting Pfs230D1+ protein was compared to a previous iteration of the Pfs230 N-terminal domain, Pfs230C1 (aa 443-731), through physiochemical characterization and in vivo analysis. The induction of functional antibody responses was confirmed via the standard membrane feeding assay (SMFA). RESULTS:Pfs230D1+ was produced and purified to an overall yield of 23 mg/L culture supernatant, a twofold yield increase over Pfs230C1. The Pfs230D1+ protein migrated as a single band via SDS-PAGE and was detected by anti-Pfs230C1 monoclonal antibodies. Evaluation by SDS-PAGE, chromatography (size-exclusion and reversed phase) and capillary isoelectric focusing demonstrated the molecule had improved homogeneity in terms of size, conformation, and charge. Intact mass spectrometry confirmed its molecular weight and that it was free of glycosylation, a key difference to the prior Pfs230C1 protein. The correct formation of the two intramolecular disulfide bonds was initially inferred by binding of a conformation specific monoclonal antibody and directly confirmed by LC/MS and peptide mapping. When injected into mice the Pfs230D1+ protein elicited antibodies that demonstrated transmission-reducing activity, via SMFA, comparable to Pfs230C1. CONCLUSION:By elimination of an O-glycosylation site, a potential N-glycosylation site, and two proteolytic cleavage sites, an improved N-terminal Pfs230 fragment was produced, termed D1+, which is non-glycosylated, homogeneous, and biologically active. An intact protein at higher yield than that previously observed for the Pfs230C1 fragment was achieved. The results indicate that Pfs230D1+ protein produced in the baculovirus expression system is an attractive antigen for transmission-blocking vaccine development.

Dataset Information

N-Terminal Pfs230 Domain Produced in Baculovirus as a Biological Active Transmission-Blocking Vaccine Candidate.

Publications

N-Terminal Pfs230 Domain Produced in Baculovirus as a Biological Active Transmission-Blocking Vaccine Candidate.

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