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Principal Investigator: Niraj Tolia
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2023
Award: $723,116
Funding agency: National Institute of Allergy and Infectious Diseases
Malaria is caused by eukaryotic parasites that display distinct surface antigens during three independent stages of the life cycle: 1) initial infection caused by the pre-erythrocytic stage, 2) clinical symptoms as a result of the blood stage, and 3) transmission by the mosquito stage. While both T-cell and B-cell responses play a role in naturally acquired immunity to malaria, focusing the B-cell responses on conserved broadly-neutralizing functional epitopes significantly improves protection and may lead to sterile immunity. Four aspects of parasite biology confound malaria vaccine development: 1) antigenic variability across strains and species, 2) the presence of immunodominant but non-neutralizing epitopes in antigens, 3) the diverse, numerous, and often redundant parasite antigens required for each stage of the life cycle, and 4) poor immune response upon vaccination with designed parasite antigens. Rapid major advances in the structural definition of neutralizing epitopes on key malaria antigens, as well as in nanoparticle technology, motivate structure-guided design of immunogens for malaria vaccines targeting all stages of the Plasmodium life cycle.
We propose to leverage existing structural information of malaria antigen and neutralizing antibody complexes to design improved immunogens and nanoparticles that will elicit protective immune responses. Immunogens will be improved through protein design to retain neutralizing epitopes, eliminate non-neutralizing epitopes, and present optimized immunogens on nanoparticles for efficient delivery and increased immunogenicity. Proteins will be designed using computational approaches to stabilize protein conformations, reduce large proteins to immunogenic subdomains, and scaffold epitopes for efficient presentation. Diverse established nanoparticle platforms will be evaluated for their ability to effectively present antigens, and novel nanoparticles will be developed for delivery. All designed immunogens and nanoparticles will be structurally characterized through x-ray crystallography and cryo-electron microscopy to ensure the correct conformational 3D structure of the antigen is retained. We have developed a computational design procedure using the NIAID Locus and Biowulf high performance computer clusters, and an in vitro screening platform to validate computationally designed antigens.
In FY23, we published the first report in Science Advances of our novel computational design and screening platform termed SPEEDesign that was first developed to improve malaria antigens. We showcased this method using a SARS-CoV-2 vaccine antigen. Designed immunogens elicited 10-fold higher neutralizing antibody titers than the unmodified antigen in mice, and the antibody response was focused towards neutralizing epitopes. The designed immunogens also had enhanced yields and stability and retained the proper 3D structure. We have now successfully applied this pipeline to improve several malaria antigens.
We used SPEEDesign to design enhanced immunogens for the malaria transmission blocking vaccine antigen Pfs48/45 as published in NPJ Vaccines. The designed Pfs48/45 immunogen retained a desired neutralizing epitope, eliminated an undesired glycan, and had vastly improved biophysical characteristics compared to the native antigen with higher production yields and higher thermostability. The designed immunogens elicited a potent transmission blocking response, while the native antigen had no protective response in preclinical animal studies. Further development of this designed antigen as a nanoparticle resulted in a potent malaria transmission blocking vaccine.
We further extended the ability of SPEEDesign to enhance immunogens by redesigning the SARS-CoV-2 RBD to enable nanoparticle display as published in Cell Reports. The RBD contains a single glycan that precludes high density display. Effective elimination of this glycan while retaining the neutralizing epitopes in the RBD and stabilizing the domain resulted in an immunogen (noNAG-RBD) that could be fused to three distinct nanoparticle platforms with no adverse effects. We established a 60-copy nonNAG-RBD nanoparticle produced similar neutralizing activity in rodents and non-human primates as full-length Spike comparator and the nanoparticle could serve as an effective booster to vaccinees immunized with full-length Spike. We are now applying these approaches to malaria antigens to create optimized immunogens for nanoparticle display.
We continue to develop novel nanoparticle-platforms using Plasmodium proteins, which can assemble into heptamers, dodecamers, tetradecamers, 24-mers and 60-mers. In addition to the Plasmodium based platforms, we have also utilized three established bacterial protein platforms that can assemble into particles containing up to 60 subunits to display designed immunogens. As published in NPJ Vaccines, we designed a single-component 60-copy Pfs230D1 transmission blocking vaccine that elicits potent and durable protection in rabbits. This study demonstrated that nanoparticle display successfully increased functional antibody titers. All nanoparticle platforms have been designed in a modular fashion to allow plug-and-play screening of designed immunogens in various expression systems expediting the identification of optimized vaccines and several different antigen-nanoparticle combinations are now in pre-clinical testing in rodents.
Finally, we published a comprehensive review on the structural basis of the Plasmodium vivax Duffy Binding Protein (PvDBP) interaction with the host-receptor Duffy Antigen/Receptor for Chemokines (DARC) and the role of structure-based design in developing vaccine for P. vivax malaria. We also contributed to a collaborative study published in Protein Science that designed a novel inhibitor for the PvDBP:DARC interaction essential for P. vivax parasite invasion of host red cells.
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