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Recent work demonstrating that asymptomatic carriers of P. falciparum parasites make up a large part of the infectious reservoir highlights the need for an effective malaria vaccine. Given the historical challenges of vaccine development, multiple parasite stages have been targeted, including the sexual stages required for transmission. Using flow cytometry to efficiently screen for P. falciparum gamete/zygote surface reactivity, we identified 82 antibodies that bound live P. falciparum gametes/zygotes. Ten antibodies had significant transmission-reducing activity (TRA) in a standard membrane feeding assay and were subcloned along with 9 nonTRA antibodies as comparators. After subcloning, only eight of the monoclonals obtained have significant TRA. These eight TRA mAbs do not recognize epitopes present in any of the current recombinant transmission-blocking vaccine candidates, Pfs230D1M, Pfs48/45.6C, Pf47 D2 and rPfs25. One TRA mAb immunoprecipitates two surface antigens, Pfs47 and Pfs230, that are expressed by both gametocytes and gametes/zygotes. These two proteins have not previously been reported to associate and the recognition of both by a single TRA mAb suggests the Pfs47/Pfs230 complex is a new vaccine target. In total, Pfs230 was the dominant target antigen, with five of the eight TRA mAbs and 8 of 11 nonTRA gamete/zygote surface reactive mAbs interacting with Pfs230. Of the three remaining TRA mAbs, two recognized non-reduced, parasite-produced Pfs25 and one bound non-reduced, parasite-produced Pfs48/45. None of the TRA mAbs bound protein on an immunoblot of reduced gamete/zygote extract and two TRA mAbs were immunoblot negative, indicating none of the new TRA epitopes are linear. The identification of eight new TRA mAbs that bind epitopes not included in any of the constructs currently under advancement as transmission-blocking vaccine candidates may provide new targets worthy of further study., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Pfs230 is essential for Plasmodium falciparum transmission to mosquitoes and is the protein targeted by the most advanced malaria-transmission-blocking vaccine candidate. Prior understanding of functional epitopes on Pfs230 is based on two monoclonal antibodies (mAbs) with moderate transmission-reducing activity (TRA), elicited from subunit immunization. Here, we screened the B cell repertoire of two naturally exposed individuals possessing serum TRA and identified five potent mAbs from sixteen Pfs230 domain-1-specific mAbs. Structures of three potent and three low-activity antibodies bound to Pfs230 domain 1 revealed four distinct epitopes. Highly potent mAbs from natural infection recognized a common conformational epitope that is highly conserved across P. falciparum field isolates, while antibodies with negligible TRA derived from natural infection or immunization recognized three distinct sites. Our study provides molecular blueprints describing P. falciparum TRA, informed by contrasting potent and non-functional epitopes elicited by natural exposure and vaccination., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Malaria kills around 409,000 people a year, mostly children under the age of five. Malaria transmission-blocking vaccines work to reduce malaria prevalence in a community and have the potential to be part of a multifaceted approach required to eliminate the parasites causing the disease. Pfs25 is a leading malaria transmission-blocking antigen and has been successfully produced in a plant expression system as both a subunit vaccine and as a virus-like particle. This study demonstrates an improved version of the virus-like particle antigen display molecule by eliminating known protease sites from the prior A85 variant. This re-engineered molecule, termed B29, displays three times the number of Pfs25 antigens per virus-like particle compared to the original Pfs25 virus-like particle. An improved purification scheme was also developed, resulting in a substantially higher yield and improved purity. The molecule was evaluated in a mouse model and found to induce improved transmission-blocking activity at lower doses and longer durations than the original molecule., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Malaria elimination requires interruption of the highly efficient transmission of Plasmodium parasites by mosquitoes. TB31F is a humanised monoclonal antibody that binds the gamete surface protein Pfs48/45 and inhibits fertilisation, thereby preventing further parasite development in the mosquito midgut and onward transmission. We aimed to evaluate the safety and efficacy of TB31F in malaria-naive participants., Methods: In this open-label, first-in-human, dose-escalation, phase 1 clinical trial, healthy, malaria-naive, adult participants were administered a single intravenous dose of 0·1, 1, 3, or 10 mg/kg TB31F or a subcutaneous dose of 100 mg TB31F, and monitored until day 84 after administration at a single centre in the Netherlands. The primary outcome was the frequency and magnitude of adverse events. Additionally, TB31F serum concentrations were measured by ELISA. Transmission-reducing activity (TRA) of participant sera was assessed by standard membrane feeding assays with Anopheles stephensi mosquitoes and cultured Plasmodium falciparum gametocytes. The trial is registered with Clinicaltrials.gov, NCT04238689., Findings: Between Feb 17 and Dec 10, 2020, 25 participants were enrolled and sequentially assigned to each dose (n=5 per group). No serious or severe adverse events occurred. In total, 33 grade 1 and six grade 2 related adverse events occurred in 20 (80%) of 25 participants across all groups. Serum of all participants administered 1 mg/kg, 3 mg/kg, or 10 mg/kg TB31F intravenously had more than 80% TRA for 28 days or more, 56 days or more, and 84 days or more, respectively. The TB31F serum concentration reaching 80% TRA was 2·1 μg/mL (95% CI 1·9-2·3). Extrapolating the duration of TRA from antibody kinetics suggests more than 80% TRA is maintained for 160 days (95% CI 136-193) following a single intravenous 10 mg/kg dose., Interpretation: TB31F is a well tolerated and highly potent monoclonal antibody capable of completely blocking transmission of P falciparum parasites from humans to mosquitoes. In areas of seasonal transmission, a single dose might cover an entire malaria season., Funding: PATH's Malaria Vaccine Initiative., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

The Plasmodium falciparum gametocyte surface protein, Pfs48/45, is a potential target for malaria transmission-blocking vaccines. However, due to its size and complexity, expression of the full-length protein has been difficult, leading to focus on the C-terminal six cysteine domain (6C) with the use of fusion proteins to facilitate expression and folding. In this study, we utilized the baculovirus system to evaluate the expression of three Pfs48/45 proteins including the full-length protein, the 6C domain fragment and the 6C domain mutant to prevent glycosylation. Expression of the recombinant Pfs48/45 proteins was conducted in super Sf9 cells combined with the use of tunicamycin to prevent N-glycosylation. The proteins were then evaluated as immunogens in mice to demonstrate the induction of functionally active polyclonal antibody responses as measured in the standard membrane feeding assay (SMFA). Only the 6C protein was found to exhibit significant transmission-reducing activity. Further characterization of the biologically active 6C protein demonstrated it was homogeneous in terms of size, charge, conformation, absence of glycosylation, and containing proper disulfide bond pairings. This study presents an alternative expression system, without the need of a fusion protein partner, for the Pfs48/45 6C protein fragment including further evaluation as a potential transmission-blocking vaccine candidate.

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.

Pfs25 is a malaria transmission-blocking vaccine antigen candidate, but its apparently limited immunogenicity in humans has hindered clinical development. Here, we show that recombinant, polyhistidine-tagged (his-tagged) Pfs25 can be mixed at the time of immunization with pre-formed liposomes containing cobalt porphyrin-phospholipid, resulting in spontaneous nanoliposome antigen particleization (SNAP). Antigens are stably presented in uniformly orientated display via his-tag insertion in the cobalt porphyrin-phospholipid bilayer, without covalent modification or disruption of antigen conformation. SNAP immunization of mice and rabbits is well tolerated with minimal local reactogenicity, and results in orders-of-magnitude higher functional antibody generation compared with other 'mix-and-inject' adjuvants. Serum-stable antigen binding during transit to draining lymph nodes leads to enhanced antigen uptake by phagocytic antigen-presenting cells, with subsequent generation of long-lived, antigen-specific plasma cells. Seamless multiplexing with four additional his-tagged Plasmodium falciparum polypeptides induces strong and balanced antibody production, illustrating the simplicity of developing multistage particulate vaccines with SNAP immunization.

A liquid chromatography tandem-mass spectrometry method was developed to map the eleven disulfide bonds in Pfs25, a malaria transmission-blocking vaccine candidate. The compact and complex nature of Pfs25 has led to difficulties in prior peptide mapping efforts. Here, we report confirmation of proper disulfide pairing of a recombinant Pfs25, by optimizing denaturation and digestion with trypsin/Lys-C. The digested peptides were separated by reversed phase HPLC to obtain the peptide map and elucidate the disulfide linkages. MS E fragmentation confirmed the digested peptides and disulfide bonds. The eleven disulfide bonds and locations matched the predicted Pvs25 crystal structure, a Pfs25 homologue., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Transmission-blocking vaccines (TBVs) have become a focus of strategies to control and eventually eliminate malaria as they target the entry of sexual stage into the Anopheles stephensi mosquito thereby preventing transmission, an essential component of the parasite life cycle. Such vaccines are envisioned as complements to vaccines that target human infection, such as RTS,S as well as drug treatment, and vector control strategies. A number of conserved proteins, including Pfs25, have been identified as promising TBV targets in research or early stage development. Pfs25 is a 25 kDa protein of Plasmodium falciparum expressed on the surface of zygotes and ookinetes. Its complex tertiary structure, including numerous cysteines, has led to difficulties in the expression of a recombinant protein that is homogeneous, with proper conformation, and free of glycosylation, a phenomenon not found in native parasite machinery., Methods: While the expression and purification of Pfs25 in various systems, has been previously independently reported, here a parallel analysis of Pfs25 is presented to inform on the biochemical features of Pfs25 and their impact on functionality. Three scalable expression systems were used to express, purify, and evaluate Pfs25 both in vitro and in vivo, including the ability of each protein to produce functional antibodies through the standard membrane feeding assay., Results: Through numerous attempts, soluble, monomeric Pfs25 derived from Escherichia coli was not achieved, while Pichia pastoris presented Pfs25 as an inhomogeneous product with glycosylation. In comparison, baculovirus produced a pure, monomeric protein free of glycosylation. The glycosylation present for Pichia produced Pfs25, showed no notable decrease in the ability to elicit transmission reducing antibodies in functional evaluation, while a reduced and alkylated Pfs25 (derived from plant and used as a control) was found to have significantly decreased transmission reducing activity, emphasizing the importance of ensuring correct disulfide stabilized conformation during vaccine design and production., Conclusions: In this study, the biochemical features of Pfs25, produced from different expression systems, are described along with their impact on the ability of the protein to elicit functional antibodies. Pfs25 expressed using baculovirus and Pichia showed promise as candidates for vaccine development.