A Reproducible and Scalable Process for Manufacturing a Pfs48/45 Based Plasmodium falciparum 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.

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Global diversity of the gene encoding the Pfs25 protein—a Plasmodium falciparum transmission-blocking vaccine candidate

Parasites & Vectors ◽

10.1186/s13071-021-05078-6 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Pornpawee Sookpongthai ◽

Korawich Utayopas ◽

Thassanai Sitthiyotha ◽

Theerakamol Pengsakul ◽

Morakot Kaewthamasorn ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Vaccine Development ◽

Protein A ◽

Distribution Patterns ◽

Haplotype Network ◽

Malarial Parasite ◽

Transmission Blocking ◽

Global Diversity ◽

Geographical Populations ◽

Transmission Blocking Vaccine

Abstract Background Vaccines against the sexual stages of the malarial parasite Plasmodium falciparum are indispensable for controlling malaria and abrogating the spread of drug-resistant parasites. Pfs25, a surface antigen of the sexual stage of P. falciparum, is a leading candidate for transmission-blocking vaccine development. While clinical trials have reported that Pfs25-based vaccines are safe and effective in inducing transmission-blocking antibodies, the extent of the genetic diversity of Pfs25 in malaria endemic populations has rarely been studied. Thus, this study aimed to investigate the global diversity of Pfs25 in P. falciparum populations. Methods A database of 307 Pfs25 sequences of P. falciparum was established. Population genetic analyses were performed to evaluate haplotype and nucleotide diversity, analyze haplotypic distribution patterns of Pfs25 in different geographical populations, and construct a haplotype network. Neutrality tests were conducted to determine evidence of natural selection. Homology models of the Pfs25 haplotypes were constructed, subjected to molecular dynamics (MD), and analyzed in terms of flexibility and percentages of secondary structures. Results The Pfs25 gene of P. falciparum was found to have 11 unique haplotypes. Of these, haplotype 1 (H1) and H2, the major haplotypes, represented 70% and 22% of the population, respectively, and were dominant in Asia, whereas only H1 was dominant in Africa, Central America, and South America. Other haplotypes were rare and region-specific, resulting in unique distribution patterns in different geographical populations. The diversity in Pfs25 originated from ten single-nucleotide polymorphism (SNP) loci located in the epidermal growth factor (EGF)-like domains and anchor domain. Of these, an SNP at position 392 (GGA/GCA), resulting in amino acid substitution 131 (Gly/Ala), defined the two major haplotypes. The MD results showed that the structures of H1 and H2 variants were relatively similar. Limited polymorphism in Pfs25 could likely be due to negative selection. Conclusions The study successfully established a Pfs25 sequence database that can become an essential tool for monitoring vaccine efficacy, designing assays for detecting malaria carriers, and conducting epidemiological studies of P. falciparum. The discovery of the two major haplotypes, H1 and H2, and their conserved structures suggests that the current Pfs25-based vaccines could be used globally for malaria control. Graphical Abstract

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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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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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Global Diversity of the Gene Encoding the Pfs25 Protein - A Plasmodium Falciparum Transmission-Blocking Vaccine Candidate

10.21203/rs.3.rs-779485/v1 ◽

2021 ◽

Author(s):

Pornpawee Sookpongthai ◽

Korawich Utayopas ◽

Thassanai Sitthiyotha ◽

Theerakamol Pengsakul ◽

Morakot Kaewthamasorn ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Vaccine Development ◽

Protein A ◽

Distribution Patterns ◽

Haplotype Network ◽

Malarial Parasite ◽

Transmission Blocking ◽

Global Diversity ◽

Geographical Populations ◽

Transmission Blocking Vaccine

Abstract Background: Vaccines against the sexual stages of the malarial parasite Plasmodium falciparum are indispensable for controlling malaria and abrogating the spread of drug-resistant parasites. Pfs25, a surface antigen of the sexual stage of P. falciparum, is a leading candidate for transmission-blocking vaccine development. While clinical trials have reported that Pfs25-based vaccines are safe and effective in inducing transmission-blocking antibodies, the extent of the genetic diversity of Pfs25 in malaria endemic populations has been rarely studied. Thus, this study aimed to investigate the global diversity of Pfs25 in P. falciparum populations.Methods: A database of 307 Pfs25 sequences of P. falciparum was established. Population genetic analyses were performed to evaluate haplotype and nucleotide diversity, analyze haplotypic distribution patterns of Pfs25 in different geographical populations, and construct a haplotype network. Neutrality tests were conducted to determine evidence of natural selection. Homology models of the Pfs25 haplotypes were constructed, subjected to molecular dynamics (MD), and analyzed in terms of flexibility and percentages of secondary structures.Results: Pfs25 of P. falciparum comprised 11 unique haplotypes. Of these, haplotype 1 (H1) and H2, the major haplotypes, represented 70% and 22% of the population, respectively. H1 and H2 were dominant in Asia, whereas H1 was dominant in Africa, Central America, and South America. Other haplotypes were rare and region-specific, resulting in unique distribution patterns in different geographical populations. The diversity in Pfs25 originated from 10 single nucleotide polymorphism (SNP) loci located in EGF-like domains and anchor domain. Of these, an SNP at position 392 (GGA/GCA), resulting in amino acid substitution 131 (Gly/Ala), defined the two major haplotypes. MD results showed that structures of H1 and H2 variants were relatively similar. Limited polymorphism in Pfs25 could be likely due to negative selection. Conclusions: The study successfully established a Pfs25 sequence database that can become an essential tool for monitoring vaccine efficacy, designing assays for detecting malaria carriers, and conducting epidemiological studies of P. falciparum. The discovery of the two major haplotypes, H1 and H2, and their conserved structures suggests that the current Pfs25-based vaccines could be globally used for malaria control.

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Safety, Immunogenicity and Efficacy of Pfs230D1M-EPA/AS01 Vaccine, a Transmission Blocking Vaccine Against Plasmodium Falciparum, in an Age De-Escalation Trial of Children and a Family Compound Trial in Mali

Case Medical Research ◽

10.31525/ct1-nct03917654 ◽

2019 ◽

Author(s):

Keyword(s):

Plasmodium Falciparum ◽

Transmission Blocking ◽

Compound Trial ◽

Transmission Blocking Vaccine

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Safety and immunogenicity of Pfs25H-EPA/Alhydrogel, a transmission-blocking vaccine against Plasmodium falciparum: a randomised, double-blind, comparator-controlled, dose-escalation study in healthy Malian adults

The Lancet Infectious Diseases ◽

10.1016/s1473-3099(18)30344-x ◽

2018 ◽

Vol 18 (9) ◽

pp. 969-982 ◽

Cited By ~ 34

Author(s):

Issaka Sagara ◽

Sara A Healy ◽

Mahamadoun H Assadou ◽

Erin E Gabriel ◽

Mamady Kone ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Dose Escalation ◽

Dose Escalation Study ◽

Double Blind ◽

Transmission Blocking ◽

Transmission Blocking Vaccine

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Engineering a Virus-Like Particle as an Antigenic Platform for a Pfs47-Targeted Malaria Transmission-Blocking Vaccine

Scientific Reports ◽

10.1038/s41598-019-53208-z ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Lampouguin Yenkoidiok-Douti ◽

Adeline E. Williams ◽

Gaspar E. Canepa ◽

Alvaro Molina-Cruz ◽

Carolina Barillas-Mury

Keyword(s):

Plasmodium Falciparum ◽

Amino Acid ◽

Malaria Transmission ◽

Transmission Blocking ◽

High Affinity ◽

Virus Like Particle ◽

Antibody Titers ◽

Reducing Activity ◽

Sexual Stages ◽

Transmission Blocking Vaccine

AbstractWe recently characterized Pfs47, a protein expressed on the surface of sexual stages and ookinetes of Plasmodium falciparum, as a malaria transmission-blocking vaccine (TBV) target. Mice immunization induced antibodies that conferred strong transmission-reducing activity (TRA) at a concentration of 200 μg/mL. Here, we sought to optimize the Pfs47 vaccine to elicit higher titers of high-affinity antibodies, capable of inducing strong TRA at a lower concentration. We report the development and evaluation of a Pfs47-based virus-like particle (VLP) vaccine generated by conjugating our 58 amino acid Pfs47 antigen to Acinetobacter phage AP205-VLP using the SpyCatcher:SpyTag adaptor system. AP205-Pfs47 complexes (VLP-P47) formed particles of ~22 nm diameter that reacted with polyclonal anti-Pfs47 antibodies, indicating that the antigen was accessible on the surface of the particle. Mice immunized with VLP-P47 followed by a boost with Pfs47 monomer induced significantly higher antibody titers, with higher binding affinity to Pfs47, than mice that received two immunizations with either VLP-P47 (VLP-P47/VLP-P47) or the Pfs47 monomer (P47/P47). Purified IgG from VLP-P47/P47 mice had strong TRA (83–98%) at concentrations as low as 5 μg/mL. These results indicate that conjugating the Pfs47 antigen to AP205-VLP significantly enhanced antigenicity and confirm the potential of Pfs47 as a TBV candidate.

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Does Host Complement Kill Borrelia burgdorferi within Ticks?

Infection and Immunity ◽

10.1128/iai.71.2.822-829.2003 ◽

2003 ◽

Vol 71 (2) ◽

pp. 822-829 ◽

Cited By ~ 17

Author(s):

Sivaprakash Rathinavelu ◽

Anne Broadwater ◽

Aravinda M. de Silva

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Outer Surface ◽

Protein A ◽

Surface Protein ◽

Block Transmission ◽

Outer Surface Protein A ◽

Tick Vector ◽

Host Blood ◽

Transmission Blocking Vaccine

ABSTRACT The Lyme disease spirochete, Borrelia burgdorferi, inhabits the gut lumen of the tick vector. At this location the spirochete is exposed to host blood when a tick feeds. We report here on studies that were done with normal and complement-deficient (C3-knockout) mice to determine if the host complement system killed spirochetes within the vector. We found that spirochete numbers within feeding nymphs were not influenced by complement, most likely because host complement was inactivated within the vector. The Lyme disease outer surface protein A (OspA) vaccine is a transmission-blocking vaccine that targets spirochetes in the vector. In experiments with mice hyperimmunized with OspA, complement was not required to kill spirochetes within nymphs and to block transmission from nymphs to the vaccinated host. However, host complement did enhance the ability of OspA antibody to block larvae from acquiring spirochetes. Thus, the effects of OspA antibody on nymphal transmission and larval acquisition appear to be based on different mechanisms.

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