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

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.

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Potent Malaria Transmission-Blocking Antibody Responses Elicited by Plasmodium falciparum Pfs25 Expressed in Escherichia coli after Successful Protein Refolding

Infection and Immunity ◽

10.1128/iai.01438-13 ◽

2014 ◽

Vol 82 (4) ◽

pp. 1453-1459 ◽

Cited By ~ 32

Author(s):

Rajesh Kumar ◽

Evelina Angov ◽

Nirbhay Kumar

Keyword(s):

Escherichia Coli ◽

Plasmodium Falciparum ◽

Malaria Transmission ◽

Strong Support ◽

Target Antigen ◽

Transmission Blocking ◽

Content Type ◽

E Coli ◽

Blocking Activity ◽

Transmission Blocking Vaccine

ABSTRACTProduction of Pfs25, aPlasmodium falciparumtransmission-blocking vaccine target antigen, in functional conformation with the potential to elicit effective immunogenicity still remains a major challenge. In the current study, codon-harmonized recombinant Pfs25 (CHrPfs25) was expressed inEscherichia coli, and purified protein after simple oxidative refolding steps retained reduction-sensitive conformational epitopes of transmission-blocking monoclonal antibodies. CHrPfs25 formulated in several adjuvants elicited strong immunogenicity in preclinical studies in mice. Antibodies elicited after immunization recognized native Pfs25 on the surface of live gametes ofP. falciparumand demonstrated complete malaria transmission-blocking activity. The transmission-blocking efficacy was 100% even after a 1:128 dilution of sera from immunized mice in the complete Freund's adjuvant and Montanide ISA51 groups and after a 1:16 dilution of sera from mice in the alum group. The blocking was mediated by antibodies; purified IgG at concentrations as low as 31.25 μg/ml exhibited 100% transmission blocking in membrane feeding assays employing two different species of mosquitoes,Anopheles gambiaeandAnopheles stephensi. This study provides the first evidence for successful expression of biologically functional rPfs25 inE. coli. The extremely potent malaria transmission-blocking activity of antibodies elicited by immunization with purified protein provides strong support for further evaluation ofE. coli-derived CHrPfs25 as a malaria transmission-blocking vaccine in human clinical trials.

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

npj Vaccines ◽

10.1038/s41541-021-00423-3 ◽

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.

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

Clinical and Vaccine Immunology ◽

10.1128/cvi.00140-17 ◽

2017 ◽

Vol 24 (10) ◽

Cited By ~ 17

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.

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Predicting the public health impact of a malaria transmission-blocking vaccine

Nature Communications ◽

10.1038/s41467-021-21775-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Joseph D. Challenger ◽

Daniela Olivera Mesa ◽

Dari F. Da ◽

R. Serge Yerbanga ◽

Thierry Lefèvre ◽

...

Keyword(s):

Public Health ◽

Malaria Transmission ◽

Health Impact ◽

Public Health Impact ◽

Substantial Impact ◽

Transmission Blocking ◽

Block Transmission ◽

Membrane Feeding ◽

Field Efficacy ◽

Transmission Blocking Vaccine

AbstractTransmission-blocking vaccines that interrupt malaria transmission from humans to mosquitoes are being tested in early clinical trials. The activity of such a vaccine is commonly evaluated using membrane-feeding assays. Understanding the field efficacy of such a vaccine requires knowledge of how heavily infected wild, naturally blood-fed mosquitoes are, as this indicates how difficult it will be to block transmission. Here we use data on naturally infected mosquitoes collected in Burkina Faso to translate the laboratory-estimated activity into an estimated activity in the field. A transmission dynamics model is then utilised to predict a transmission-blocking vaccine’s public health impact alongside existing interventions. The model suggests that school-aged children are an attractive population to target for vaccination. Benefits of vaccination are distributed across the population, averting the greatest number of cases in younger children. Utilising a transmission-blocking vaccine alongside existing interventions could have a substantial impact against malaria.

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A Plant-Produced Pfs230 Vaccine Candidate Blocks Transmission of Plasmodium falciparum

Clinical and Vaccine Immunology ◽

10.1128/cvi.05105-11 ◽

2011 ◽

Vol 18 (8) ◽

pp. 1351-1357 ◽

Cited By ~ 58

Author(s):

Christine E. Farrance ◽

Amy Rhee ◽

R. Mark Jones ◽

Konstantin Musiychuk ◽

Moneim Shamloul ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Vaccine Candidate ◽

Expression System ◽

Leaf Tissue ◽

Sexual Cycle ◽

New Host ◽

Transmission Blocking ◽

Content Type ◽

Block Transmission ◽

Membrane Feeding

ABSTRACTPlasmodium falciparumis transmitted to a new host after completing its sexual cycle within a mosquito. Developing vaccines against the parasite sexual stages is a critical component in the fight against malaria. We are targeting multiple proteins ofP. falciparumwhich are found only on the surfaces of the sexual forms of the parasite and where antibodies against these proteins have been shown to block the progression of the parasite's life cycle in the mosquito and thus block transmission to the next human host. We have successfully produced a region of the Pfs230 antigen in our plant-based transient-expression system and evaluated this vaccine candidate in an animal model. This plant-produced protein, 230CMB, is expressed at approximately 800 mg/kg in fresh whole leaf tissue and is 100% soluble. Administration of 230CMB with >90% purity induces strong immune responses in rabbits with high titers of transmission-blocking antibodies, resulting in a greater than 99% reduction in oocyst counts in the presence of complement, as determined by a standard membrane feeding assay. Our data provide a clear perspective on the clinical development of a Pfs230-based transmission-blocking malaria vaccine.

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Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant Pfs25, a Leading Transmission-Blocking Vaccine Candidate

Infection and Immunity ◽

10.1128/iai.00486-17 ◽

2017 ◽

Vol 86 (1) ◽

Cited By ~ 5

Author(s):

Elizabeth M. Parzych ◽

Kazutoyo Miura ◽

Aarti Ramanathan ◽

Carole A. Long ◽

James M. Burns

Keyword(s):

Neutralizing Antibodies ◽

Malaria Vaccine ◽

Vaccine Candidate ◽

Surface Protein ◽

Merozoite Surface Protein ◽

Carrier Protein ◽

Transmission Blocking ◽

Content Type ◽

Vaccine Carrier ◽

Transmission Blocking Vaccine

ABSTRACTChallenges with the production and suboptimal immunogenicity of malaria vaccine candidates have slowed the development of aPlasmodium falciparummultiantigen vaccine. Attempting to resolve these issues, we focused on the use of highly immunogenic merozoite surface protein 8 (MSP8) as a vaccine carrier protein. Previously, we showed that a genetic fusion of the C-terminal 19-kDa fragment of merozoite surface protein 1 (MSP119) toP. falciparumMSP8 (PfMSP8) facilitated antigen production and folding and the induction of neutralizing antibodies to conformational B cell epitopes of MSP119. Here, using thePfMSP1/8 construct, we further optimized the recombinantPfMSP8 (rPfMSP8) carrier by the introduction of two cysteine-to-serine substitutions (CΔS) to improve the yield of the monomeric product. We then sought to test the broad applicability of this approach using the transmission-blocking vaccine candidatePfs25. The production of rPfs25-based vaccines has presented challenges. Antibodies directed against the four highly constrained epidermal growth factor (EGF)-like domains ofPfs25 block sexual-stage development in mosquitoes. The sequence encoding maturePfs25 was codon harmonized for expression inEscherichia coli. We produced a rPfs25-PfMSP8 fusion protein [rPfs25/8(CΔS)] as well as unfused, mature rPfs25. rPfs25 was purified with a modest yield but required the incorporation of refolding protocols to obtain a proper conformation. In comparison, chimeric rPfs25/8(CΔS) was expressed and easily purified, with thePfs25 domain bearing the proper conformation without renaturation. Both antigens were immunogenic in rabbits, inducing IgG that bound nativePfs25 and exhibited potent transmission-reducing activity. These data further demonstrate the utility ofPfMSP8 as a parasite-specific carrier protein to enhance the production of complex malaria vaccine targets.

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A C-terminal Pfs48/45 malaria transmission-blocking vaccine candidate produced in the baculovirus expression system

Scientific Reports ◽

10.1038/s41598-019-57384-w ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Shwu-Maan Lee ◽

John M. Hickey ◽

Kazutoyo Miura ◽

Sangeeta B. Joshi ◽

David B. Volkin ◽

...

Keyword(s):

Malaria Transmission ◽

Vaccine Candidate ◽

Expression System ◽

Baculovirus Expression System ◽

Transmission Blocking ◽

Baculovirus Expression ◽

Transmission Blocking Vaccine

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

npj Vaccines ◽

10.1038/s41541-021-00383-8 ◽

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.

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