scholarly journals Preclinical development of a Pfs230-Pfs48/45 chimeric malaria transmission-blocking vaccine

npj Vaccines ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Susheel K. Singh ◽  
Jordan Plieskatt ◽  
Bishwanath K. Chourasia ◽  
Vandana Singh ◽  
Karin Lövgren Bengtsson ◽  
...  
Keyword(s):  
Malaria Transmission ◽  
Clinical Evaluation ◽  
Disulfide Bonds ◽  
Expression System ◽  
Mass Spectrometry Analysis ◽  
Preclinical Development ◽  
Transmission Blocking ◽  
Spectrometry Analysis ◽  
Functional Antibodies ◽  
Transmission Blocking Vaccine

AbstractThe Plasmodium falciparum Pfs230 and Pfs48/45 proteins are leading candidates for a malaria transmission-blocking vaccine (TBV). Previously, we showed that a Pfs230–Pfs48/45 fusion protein elicits higher levels of functional antibodies than the individual antigens, but low yields hampered progression to clinical evaluation. Here we identified a modified construct (ProC6C) with a circumsporozoite protein (CSP) repeat-linker sequence that enhances expression. A scalable and reproducible process in the Lactococcus lactis expression system was developed and ProC6C was successfully transferred for manufacturing under current Good Manufacturing Practices (cGMP). In addition, a panel of analytical assays for release and stability were developed. Intact mass spectrometry analysis and multiangle light scattering showed that the protein contained correct disulfide bonds and was monomeric. Immunogenicity studies in mice showed that the ProC6C adsorbed to Alhydrogel®, with or without Matrix-MTM, elicited functional antibodies that reduced transmission to mosquitoes and sporozoite invasion of human hepatocytes. Altogether, our data support manufacture and clinical evaluation of ProC6C as a multistage malaria-vaccine candidate.

scholarly journals N-Terminal Prodomain of Pfs230 Synthesized Using a Cell-Free System Is Sufficient To Induce Complement-Dependent Malaria Transmission-Blocking Activity

2011 ◽  
Vol 18 (8) ◽  
pp. 1343-1350 ◽  
Author(s):  
Mayumi Tachibana ◽  
Yimin Wu ◽  
Hideyuki Iriko ◽  
Olga Muratova ◽  
Nicholas J. MacDonald ◽  
...  
Keyword(s):  
Malaria Transmission ◽  
Expression System ◽  
Surface Protein ◽  
Western Blot Assay ◽  
Free System ◽  
Transmission Blocking ◽  
Content Type ◽  
Membrane Feeding ◽  
Blocking Activity ◽  
Transmission Blocking Vaccine

ABSTRACTThe aim of a malaria transmission-blocking vaccine is to block the development of malaria parasites in the mosquito and thus prevent subsequent infection of the human host. Previous studies have demonstrated that the gametocyte/gamete surface protein Pfs230 can induce transmission-blocking immunity and have evaluatedEscherichia coli-produced Pfs230 as a transmission-blocking vaccine candidate. In this study, we used the wheat germ cell-free expression system to produce N-terminal fragments of Pfs230 and evaluated the transmission-blocking activity of antisera raised against the recombinant Pfs230 protein. The rabbit antisera reacted to the surface of cultured gametocytes and gametes of thePlasmodium falciparumNF54 line, recognized the 360-kDa form of parasite-produced Pfs230 by Western blot assay, and reduced the infectivity of NF54 parasites toAnopheles stefensimosquitoes in the presence of complement in a standard membrane feeding assay. Thus, our data demonstrate that the N-terminal pro domain of Pfs230 is sufficient to induce complement-dependent transmission-blocking activity againstP. falciparum.

scholarly journals Elucidating functional epitopes within the N-terminal region of malaria transmission blocking vaccine antigen Pfs230

npj Vaccines ◽  
2022 ◽  
Vol 7 (1) ◽  
Author(s):  
Kazutoyo Miura ◽  
Eizo Takashima ◽  
Thao P. Pham ◽  
Bingbing Deng ◽  
Luwen Zhou ◽  
...  
Keyword(s):  
Malaria Transmission ◽  
Synthetic Peptide ◽  
Vaccine Antigen ◽  
Parasite Development ◽  
Transmission Blocking ◽  
Peptide Antigens ◽  
Membrane Feeding ◽  
Functional Antibodies ◽  
Enrichment Techniques ◽  
Transmission Blocking Vaccine

AbstractPfs230 is a leading malaria transmission blocking vaccine (TBV) candidate. Comprising 3135 amino acids (aa), the large size of Pfs230 necessitates the use of sub-fragments as vaccine immunogens. Therefore, determination of which regions induce functional antibody responses is essential. We previously reported that of 27 sub-fragments spanning the entire molecule, only five induced functional antibodies. A “functional” antibody is defined herein as one that inhibits Plasmodium falciparum parasite development in mosquitoes in a standard membrane-feeding assay (SMFA). These five sub-fragments were found within the aa 443–1274 range, and all contained aa 543–730. Here, we further pinpoint the location of epitopes within Pfs230 that are recognized by functional antibodies using antibody depletion and enrichment techniques. Functional epitopes were not found within the aa 918–1274 region. Within aa 443–917, further analysis showed the existence of functional epitopes not only within the aa 543–730 region but also outside of it. Affinity-purified antibodies using a synthetic peptide matching aa 543–588 showed activity in the SMFA. Immunization with a synthetic peptide comprising this segment, formulated either as a carrier-protein conjugate vaccine or with a liposomal vaccine adjuvant system, induced antibodies in mice that were functional in the SMFA. These findings provide key insights for Pfs230-based vaccine design and establish the feasibility for the use of synthetic peptide antigens for a malaria TBV.

scholarly journals A Reproducible and Scalable Process for Manufacturing a Pfs48/45 Based Plasmodium falciparum Transmission-Blocking Vaccine

2021 ◽  
Vol 11 ◽  
Author(s):  
Susheel K. Singh ◽  
Jordan Plieskatt ◽  
Bishwanath K. Chourasia ◽  
Amanda Fabra-García ◽  
Asier Garcia-Senosiain ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Clinical Evaluation ◽  
Disulfide Bonds ◽  
Process Development ◽  
Drug Product ◽  
Protein A ◽  
Surface Protein ◽  
Size Exclusion ◽  
Transmission Blocking ◽  
Transmission Blocking Vaccine

The cysteine-rich Pfs48/45 protein, a Plasmodium falciparum sexual stage surface protein, has been advancing as a candidate antigen for a transmission-blocking vaccine (TBV) for malaria. However, Pfs48/45 contains multiple disulfide bonds, that are critical for proper folding and induction of transmission-blocking (TB) antibodies. We have previously shown that R0.6C, a fusion of the 6C domain of Pfs48/45 and a fragment of PfGLURP (R0), expressed in Lactococcus lactis, was properly folded and induced transmission-blocking antibodies. Here we describe the process development and technology transfer of a scalable and reproducible process suitable for R0.6C manufacturing under current Good Manufacturing Practices (cGMP). This process resulted in a final purified yield of 25 mg/L, sufficient for clinical evaluation. A panel of analytical assays for release and stability assessment of R0.6C were developed including HPLC, SDS-PAGE, and immunoblotting with the conformation-dependent TB mAb45.1. Intact mass analysis of R0.6C confirmed the identity of the product including the three disulfide bonds and the absence of post-translational modifications. Multi-Angle Light Scattering (MALS) coupled to size exclusion chromatography (SEC-MALS), further confirmed that R0.6C was monomeric (~70 kDa) in solution. Lastly, preclinical studies demonstrated that the R0.6C Drug Product (adsorbed to Alhydrogel®) elicited functional antibodies in small rodents and that adding Matrix-M™ adjuvant further increased the functional response. Here, building upon our past work, we filled the gap between laboratory and manufacturing to ready R0.6C for production under cGMP and eventual clinical evaluation as a malaria TB vaccine.

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

2017 ◽  
Vol 24 (10) ◽  
Author(s):  
Shwu-Maan Lee ◽  
Chia-Kuei Wu ◽  
Jordan L. Plieskatt ◽  
Kazutoyo Miura ◽  
John M. Hickey ◽  
...  
Keyword(s):  
Amino Acids ◽  
Disulfide Bonds ◽  
Peptide Mapping ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Size Exclusion ◽  
Affinity Tag ◽  
Transmission Blocking ◽  
Content Type ◽  
Transmission Blocking Vaccine

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.

scholarly journals Evaluation of two lead malaria transmission blocking vaccine candidate antibodies in natural parasite-vector combinations

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Anais Bompard ◽  
Dari F. Da ◽  
Rakiswendé S. Yerbanga ◽  
Sumi Biswas ◽  
Melissa Kapulu ◽  
...  
Keyword(s):  
Malaria Transmission ◽  
Vaccine Candidate ◽  
Transmission Blocking ◽  
Transmission Blocking Vaccine

Pinpointing cysteine oxidation sites by high-resolution proteomics reveals a mechanism of redox-dependent inhibition of human STING

2021 ◽  
Vol 14 (680) ◽  
pp. eaaw4673
Author(s):  
Natalia Zamorano Cuervo ◽  
Audray Fortin ◽  
Elise Caron ◽  
Stéfany Chartier ◽  
Nathalie Grandvaux
Keyword(s):  
Posttranslational Modifications ◽  
Protein Function ◽  
Disulfide Bonds ◽  
Redox Regulation ◽  
Mass Spectrometry Analysis ◽  
Type I ◽  
Cysteine Oxidation ◽  
Spectrometry Analysis ◽  
Host Interactions ◽  
In Cells

Protein function is regulated by posttranslational modifications (PTMs), among which reversible oxidation of cysteine residues has emerged as a key regulatory mechanism of cellular responses. Given the redox regulation of virus-host interactions, the identification of oxidized cysteine sites in cells is essential to understand the underlying mechanisms involved. Here, we present a proteome-wide identification of reversibly oxidized cysteine sites in oxidant-treated cells using a maleimide-based bioswitch method coupled to mass spectrometry analysis. We identified 2720 unique oxidized cysteine sites within 1473 proteins with distinct abundances, locations, and functions. Oxidized cysteine sites were found in numerous signaling pathways, many relevant to virus-host interactions. We focused on the oxidation of STING, the central adaptor of the innate immune type I interferon pathway, which is stimulated in response to the detection of cytosolic DNA by cGAS. We demonstrated the reversible oxidation of Cys148 and Cys206 of STING in cells. Molecular analyses led us to establish a model in which Cys148 oxidation is constitutive, whereas Cys206 oxidation is inducible by oxidative stress or by the natural ligand of STING, 2′3′-cGAMP. Our data suggest that the oxidation of Cys206 prevented hyperactivation of STING by causing a conformational change associated with the formation of inactive polymers containing intermolecular disulfide bonds. This finding should aid the design of therapies targeting STING that are relevant to autoinflammatory disorders, immunotherapies, and vaccines.

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