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Awarded Amount$595,650DiseaseMalariaInterventionVaccineDevelopment StageAntigen IdentificationCollaboration PartnersEhime University , PATH Malaria Vaccine InitiativePublication
Tachibana M, Miura K, Takashima E, Morita M, Nagaoka H, Zhou L, Long CA, Richter King C, Torii M, Tsuboi T, Ishino T. Identification of domains within Pfs230 that elicit transmission blocking antibody responses. Vaccine. 2019 Mar 22;37(13):1799-1806. doi: 10.1016/j.vaccine.2019.02.021. Epub 2019 Feb 26. PMID: 30824357; PMCID: PMC6708081.
Introduction and Background of the Project
There is an unprecedented opportunity to eliminate malaria from the developing world through the discovery and development of new tools designed to interrupt multiple stages of the parasite life cycle. Such efforts include the development of new curative drugs, vector control methods, and vaccines that prevent the spread of disease. This project seeks to complement these efforts through the discovery of novel vaccines that block parasite transmission from human to mosquito, thereby reducing spread of infection and contributing to elimination within a population. While such “transmission-blocking” vaccines (TBVs) have been an objective of malaria vaccine researchers for many years, new approaches to the discovery and optimization of antigen targets, as well as novel ways to focus the immune system, are now applicable to vaccine design. Our project is based on a solid foundation of existing information, leverages new technology, and will be implemented by a proven collaboration of scientists. We will dissect a protein found on the sexual-stage parasite, Pfs230, to identify antigenic regions that elicit the most potent vaccine responses, generate these using scalable systems suitable for their production, and present these antigens using the latest technology to augment immune responses using nanoparticle delivery.
Objective 1: Generate fragments of Pfs230 that mimic the intact native protein and identify those that, as antigens, elicit the most potent transmission-blocking antibodies.
The Pfs230 protein is a well established transmission-blocking target antigen; however, its utility has been limited by the inability to produce the protein due to its large size and complex structure. This project will dissect the Pfs230 protein and employ Ehime University's cell-free technology that adapts well to complex malaria proteins.
Objective 2: Efficiently produce Pfs230 antigens retaining potent immunogenicity.
We will build on recent published results (including from the applicant) that have identified systems suitable for both early vaccine discovery and product development to produce novel potent Pfs230 antigens using scalable expression systems.
Objective 3: Generate vaccine candidates suitable for advancement to testing in humans.
We will build on recent discoveries (including by the applicant and its partners) and leverage novel nanoparticle delivery platforms for the presentation of native configuration Pfs230 on particles which lead to augmented immunogenicity.
This proposal will employ cutting-edge technology to achieve its goals and objectives and deploy successful partnerships, including with the world-renowned malaria vaccine discovery investigator, Dr. Takafui Tsuboi, of Ehime University.
Objective 1: The production of Pfs230 protein as a vaccine antigen has proved challenging, due to its complex structure that includes more than 60 cysteines. Our strategy is to divide the Pfs230 protein into folding regions for expression in the wheat germ cell-free system (WGCFS).
Objective 2: While the WGCFS is preferred for the initial characterization of vaccine antigens, it is not applicable to the production of vaccines for clinical testing and development. In keeping with MVI’s goal of rapid translation of innovative vaccines to the clinic, we will employ production of the most active Pfs230 antigens in insect cell scalable production, which has major advantages for malaria antigens. MVI has also focused on identification of analytical testing protocols to assure native configuration of antigens.
Objective 3: Recent results indicate that display of antigens on a virus-like nanoparticle augments immunogenicity. We will employ two recently discovered nanoparticle platforms for display of the selected Pfs230 antigens. These platforms are designed to maintain the native configuration of the antigens consistent with our overall design objectives. The nanoparticles will be used for the presentation of the most potent Pfs230 antigens.
How can your partnership (project) address global health challenges?
Despite the successful scale-up of multiple interventions, there were an estimated 212 million cases of malaria and an estimated 429,000 malaria deaths worldwide in 2015, according to the most recent WHO World Malaria Report. Vaccines are urgently needed to help control malaria and to support future elimination and eventual eradication efforts.
While drugs and insecticides have had a significant impact on the burden of disease, resistance poses an ongoing challenge. A TBV that prevents human-to-mosquito transmission could break the cycle of parasite transmission, and thus play an important role in malaria elimination and eradication. The availability of a TBV vaccine would represent a critical additional tool for the fight against malaria that would work in synergy with other interventions, since blocking transmission of the parasite would reduce the pressure on other measures, by slowing the development of resistance and thus extending their effectiveness.
This proposal aims at fast-tracking the discovery and validation of the most potent target Pfs230 domain for full vaccine development.
What sort of innovation are you bringing in your project?
This project will employ Ehime University's cutting-edge WGCFS technology to tackle the challenges of producing a complex and large protein like Pfs230. We will employ state-of-the-art nanoparticle platforms that are designed to maintain the native configuration of the antigens and enable the formulation of vaccine nanoparticles more efficientlt than is possible using traditional conjugation. The nanoparticle platform has the advantage of effectively augmenting immune responses and thus can help us accelerate the development of a Pfs230 nanoparticle vaccine candidate for human testing.
Role and Responsibility of Each Partner
MVI is the coordinating institution and driving force for this proposed project. MVI has a long track record in malaria vaccine development and will ensure the fast-tracking of the identification and testing of the most potent Pfs230 candidate.
The Ehime University team will focus on the production of domains covering the entire Pf230 antigen in the WGCFS, which will then be used (1) to raise polyclonal antibodies and (2) as reference materials to benchmark testing of the scalable expression system. MVI's Reference Laboratory at the US National Institutes of Health will make available of its qualified standard membrane feeding assay (SMFA) to assess vaccine function. The SMFA is an ex vivo assay used to measure the transmission-blocking potential of antibodies elicited in response to vaccination with a TBV.
Others (including references if necessary)
1. World Health Organization (WHO). World Malaria Report 2016. Geneva: WHO; 2016.
1. Project objective
• Identify the component domains of Pfs230 responsible for eliciting potent transmission-blocking responses.
• Produce recombinant Pfs230 fragments using scalable expression systems.
• Leverage novel delivery platforms for development of Pfs230 transmission-blocking vaccine candidates.
2. Project design
• Overlapping domains/fragments encompassing entire Pfs230 were recombinantly produced in wheat-germ cell-free system and evaluated for functional immunogenicity in mice.
• The fragments capable of inducing transmission-reducing immunity were produced in a scalable baculovirus expression system. The products were evaluated in confirmatory in vivo studies. The production process was evaluated for product quality (identity, purity, integrity, and stability) and the production yield.
• Promising products were evaluated in in vivo study after formulating with available adjuvants and delivery platforms, including conjugation to CRM197, HBsAg-SpyTag mediated virus-like particles, CoPoP/PHAD, an adjuvanted liposome, and SA-1, a novel adjuvant being developed by Sumitomo Dainippon Pharma Co., Ltd.
3. Results, lessons learned
We achieved all three milestones on the project as described below.
MILESTONE 1: Select up to five domains that induce potent transmission-blocking activity for scalable expression systems.
A total of 30 fragments/subdomains of Pfs230 were expressed in WGCFS followed by evaluation of immunogenicity in mice and transmission-reducing activity (TRA) by standard membrane feeding assay (SMFA). Among these, five fragments induced strong TRA and all contain cysteine-motif 1 (CM1). The work was published (Tachibana M. et al., Vaccine, 2019, 37(13):1799-1806).
MILESTONE 2: Identify the Pfs230 antigen domain that elicits the most potent functional antibody when generated in a scalable expression system (with high yield and appropriate quality characteristics).
Three CM1-containing fragments, Pfs230C1, Pfs230D1 and Pfs230D1+, were expressed in a baculovirus expression system. TRA was confirmed in mice and rats immunized with the recombinant Pfs230C1 and Pfs230D1+. Subsequently, scalable downstream processes were developed in baculovirus system for both Pfs230C1 and Pfs230D1+. While both met a set of predetermined specifications on identity, purity, integrity, and stability, a higher yield was obtained with Pfs230D1+. We are preparing a manuscript to publish these findings.
MILESTONE 3: Select vaccine candidate(s) suitable for advancement to testing in humans (including testing of nanoparticle delivery).
Because of the larger-scale availability, we evaluated multiple delivery platforms and adjuvants using Pfs230C1, including conjugation to CRM197, HBsAg-SpyTag mediated virus-like-particles, and CoPoP/PHAD, adjuvanted liposomes capable of incorporating polyhistidine-tagged Pfs230C1 antigen through interaction between cobalt and his-tag. All three formulations induced stronger functional immunity compared to the alum formulation, among which the CoPoP/PHAD displayed the strongest durable immune enhancement.
Since the last progress report in March, we completed in vivo studies in mice and rats to evaluate Pfs230C1 and Pfs230D1+ formulated with the SA-1 adjuvant. The SA-1 adjuvant improved functional activity of anti-Pfs230 antibodies. While both Pfs230C1 and Pfs230D1+ with the SA-1 adjuvant induced antibodies with strong, comparable TRA, the higher production yield of Pfs230D1+ indicated this could be a better choice for advancement to testing in humans.