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ROCKVILLE, MD, United States

Disrupting erythrocyte invasion by Plasmodium falciparum is an attractive approach to combat malaria. P. falciparum EBA-175 (PfEBA-175) engages the host receptor Glycophorin A (GpA) during invasion and is a leading vaccine candidate. Antibodies that recognize PfEBA-175 can prevent parasite growth, although not all antibodies are inhibitory. Here, using x-ray crystallography, small-angle x-ray scattering and functional studies, we report the structural basis and mechanism for inhibition by two PfEBA-175 antibodies. Structures of each antibody in complex with the PfEBA-175 receptor binding domain reveal that the most potent inhibitory antibody, R217, engages critical GpA binding residues and the proposed dimer interface of PfEBA-175. A second weakly inhibitory antibody, R218, binds to an asparagine-rich surface loop. We show that the epitopes identified by structural studies are critical for antibody binding. Together, the structural and mapping studies reveal distinct mechanisms of action, with R217 directly preventing receptor binding while R218 allows for receptor binding. Using a direct receptor binding assay we show R217 directly blocks GpA engagement while R218 does not. Our studies elaborate on the complex interaction between PfEBA-175 and GpA and highlight new approaches to targeting the molecular mechanism of P. falciparum invasion of erythrocytes. The results suggest studies aiming to improve the efficacy of blood-stage vaccines, either by selecting single or combining multiple parasite antigens, should assess the antibody response to defined inhibitory epitopes as well as the response to the whole protein antigen. Finally, this work demonstrates the importance of identifying inhibitory-epitopes and avoiding decoy-epitopes in antibody-based therapies, vaccines and diagnostics. © 2013 Chen et al.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 600.00K | Year: 2012

DESCRIPTION (provided by applicant): Malaria causes at least 250 million cases and nearly 1 million deaths per year. GSK's malaria vaccine, RTS, S/AS01 is being tested in a Phase 3 clinical trial, and is likely to be licensed for use in children in the developing world, if safe ad effective. This vaccine is based entirely on the repeat region and carboxy terminus of the Plasmodium falciparum (Pf) circumsporozoite protein (CSP). It is administered with an adjuvant AS01, which includes liposomes, monophosphoryl lipid A, and a purified plant extract, QS21, but was initially developed with an oil in water-based adjuvant. Downselection of adjuvants for clinical trials was done through a series of iterative studies in mice and non-human primates (NHPs). In its final formulation this vacccine protects 50% of volunteers against experimental challenge with Pf for 2 weeks after last dose, and 22% of volunteers for 6 months. Protection is thought to be primarily mediated by antibodies against the repeat region of PfCSPand possibly CD4+ T cell responses against the C' terminus of the PfCSP. The vaccine does not induce meaningful CD8+ T cell responses. However, many malariologists believe that long- term protection will be dependent on induction of Pf-specific CD8+ T cellimmunity, as has been obseved in mice and NHPs immunized with irradiated sporozoites. RTS, S/AS01 is not being considered for non-immune travelers and military personnel, because its protective efficacy is too low. A vaccine for this population needs to provide gt80% protective immunity for at least 6 months to have a substantial market. We hypothesize that by adding highly functional, protective CD8+ T cell responses to antibody responses against the PfCSP, such protective immunity can be achieved. Recombinant adenovirus (Ad) expressing proteins like the PfCSP is currently a popular method for inducing CD8+ T cell responses in humans. However, despite the induction of antigen-specific CD8+ T cell responses of very high magnitude such Ad-based vaccines havenot been highly protective in humans, especially against malaria. Recently, it was shown in mice that recombinant attenuated Listeria monocytogenes (Lm) induced much higher quality (functional) CD8+ T cell responses than did recombinant Ad5. We will use aheterologous prime-boost regimen combining an adjuvanted recombinant PfCSP protein (rPfCSP) and Lm expressing PfCSP (Lm-PfCSP). The goal of this strategy is to induce PfCSP- specific protective antibodies and protective CD8+ and CD4+ T cell responses thatprovide gt80% protection that is sustained for at least 6 months. In Phase I we will identify combinations of rPfCSP, adjuvant and Lm- PfCSP that induce high level antibodies, and CD8+ and CD4+ T cell responses in mice. In Phase II we will take the approach used by GSK, and use immunogenicity in NHPs to downselect combinations for clinical trials of a vaccine that is intended to have efficacy adequate to prevent gt80% of vaccinees from developing Pf parasitemia; a vaccine suitable for the potential multi-billion dollar non-immune traveler, business, and military markets, and for eliminating Pf in geographically focused campaigns in the developing world. PUBLIC HEALTH RELEVANCE: Malaria causes 400-500 million clinical cases and gt1 million deaths annually, is responsible for gt1% loss of GDP in Africa annually and is a serious concern for travelers and military personnel. Protein Potential's goal is to develop and commercialize a gt90% protective malaria vaccine for primary markets with a potentialfor gt 1 billion annual revenues; 1) travelers from the developed world, and 2) all populations in the developing world. Success in this project will significantly decrease the cost of development and time to market for this malaria vaccine.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 609.20K | Year: 2015

DESCRIPTION provided by applicant Vaccines are a rational and cost effective means for protecting against infectious diseases in travelers military personnel and in endemic developing country populations Our goal in this proposal is to address several significant vaccine needs the need for an easy to administer needle free and safe oral vaccine vector platform for stable expression and delivery of multiple foreign antigens that generates long term efficacy following a rapid immunization regimen and which can be distributed without the need for refrigeration the lack of a licensed vaccine for prevention of morbidity and mortality due o shigellosis and development of a multivalent oral vaccine that will simultaneously protect against multiple disease agents i e enteric fever plus the major causes of shigellosis To address these challenges we exploit the extensive safety record of the existing live oral attenuated Salmonella Typhi Ty a typhoid vaccine by utilizing it as our lead candidate vector to develop a combination oral vaccine that will simultaneously protect against both typhoid fever with cross protection against some paratyphoid fevers and shigellosis Further we hypothesize that this vaccine can be formulated to be safe stable and highly immunogenic and can be easily administered orally The current proposal is aimed at creating multivalent vaccine strains Ty a expressing S sonnei form O polysaccharide Ty a Ss and Ty a expressing S flexneri a form O polysaccharide Ty a Sf a We will also create acid resistant Ty a Ss and Ty a Sf a by adding Shigella glutamate decarboxylase GAD genes into vaccine candidates and fully characterize each strain genetically and biochemically Immunogenicity and protective efficacy against S Typhi Ty S sonnei G and S flexneri a T in mice of Ty a Ss Ty a Sf a Ty a Ss GAD and Ty a Sf a GAD will be assessed by immunization via intranasal installation of doses followed by mucosal challenge In Phase II we will conduct similar work for S flexneri a and finalize a temperature stable dried product as rapidly dissolvable wafers or tablets conduct all pre clinical IND enabling studies finalize a clinical protocol and submit an IND This Phase I project will provide the foundation for the Phase II to complete construction of a quadrivalent anti shigellosis vaccine that will protect against more than of shigellosis worldwide PUBLIC HEALTH RELEVANCE Our goal is to develop a multivalent oral vaccine that will simultaneously protect against multiple disease agents is easy to administer needle free is a safe oral vaccine vector platform for stable expression and delivery of multiple foreign antigens that generates long term efficacy following a rapid immunization regimen and which can be distributed without the need for refrigeration To address these challenges we exploit the extensive safety record of the existing live oral attenuated Salmonella Typhi Ty a typhoid vaccine by utilizing it as our lead candidate vector to develop a combination oral vaccine that will simultaneously protect against both typhoid fever with cross protection against some paratyphoid fevers and shigellosis We hypothesize that this vaccine can be formulated to be safe stable highly immunogenic and can be easily administered orally


Grant
Agency: Department of Defense | Branch: Navy | Program: SBIR | Phase: Phase II | Award Amount: 749.99K | Year: 2005

Malaria caused by Plasmodium falciparum affects 300-500 million and kills 1-3 million individuals annually. For more than 150 years during every military campaign conducted where malaria was transmitted, U.S. forces have had more casualties from malaria than from hostile fire. For these reasons malaria vaccine development is one of the highest ranked objectives for DoD biomedical R&D. Five P. falciparum proteins, PfCSP, PfSSP2, PfLSA1, PfAMA1 and PfMSP1 are considered prime targets for inclusion in a malaria vaccine. In a Phase I SBIR we demonstrated the feasibility of producing one of these proteins, PfCSP, in a highly immunogenic form. In Phase II we will produce 20-50 mg of purified, well characterized, recombinant proteins based on these antigens, demonstrate that the proteins are immunogenic in mice, and provide the proteins to the Naval Medical Research Center for use as reagents in their vaccine development program. In addition we will produce protocols and reagents that are transferable to Investigational New Drug (IND) applications and FDA directed studies. Thus, by the end of Phase II, we will have the data, material, and protocols required to take these proteins forward, not only as reagents, but also as vaccines on their own


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 592.66K | Year: 2008

DESCRIPTION (provided by applicant): Malaria causes an estimated 500 million clinical cases, up to 2.7 million deaths, and a loss of gt1% of GDP in Africa annually, and poses a serious concern for travelers and military. Protein Potential's goal is to deve lop alone or contribute to the development and commercialization of a gt90% protective subunit recombinant vaccine for 2 primary markets with a potential for gt 1 billion in annual revenues: 1) Travelers from the developed world and 2) Infants and young ch ildren in the developing world. The gold standard for such a vaccine is immunization with whole attenuated Plasmodium falciparum (Pf) sporozoites (spz) by the bite of irradiated infected mosquitoes, which protects greater than 90% of experimentally challen ged volunteers for at least 10 months. This protection is thought to be primarily mediated by CD8+ T cells, and to a lesser extent CD4+ T cells, against multiple parasite proteins expressed in spz and infected hepatocytes, and secondarily by antibodies aga inst multiple spz proteins. Thus far only immune responses directed against the Pf circumsporozoite protein (PfCSP) have been shown to reproducibly prevent Pf infection in volunteers, but this protection is only about 40% for 2-3 weeks. Thus, there have be en intense efforts to identify other spz or liver (pre-erythrocytic) stage proteins that are targets of the highly protective immunity engendered by the irradiated spz vaccine. PfCelTOS, also know as PfAg2, was discovered through analysis of the Pf genome. P. berghei CelTOS has been shown to be involved in spz invasion of the liver, P. falciparum CelTOS was recognized by T cells from 8/8 human volunteers immunized with irradiated P. falciparum spz, and P. yoelii CelTOS protected 64% of mice against infectio n in the P. yoelii rodent malaria model system when administered as a vaccine. These findings have established PfCelTOS as a prime candidate for being a stand-alone pre-erythrocytic stage Pf vaccine, or part of a multi-protein Pf vaccine designed to elicit protective T cell and antibody responses. Accordingly, we have produced a full length, recombinant PfCelTOS protein in Pichia pastoris, and shown that outbred mice immunized with this protein produce antibodies that recognize Pf spz (titer in indirect flu orescent antibody test, 12,800) and block invasion and development of Pf spz in hepatocytes in vitro (68% inhibition at serum dilution of 1:20). Herein, we propose to optimize production and purification of this protein. Many vaccinologists consider sequen tial immunization with recombinant virus and recombinant (rec) proteins as the best current approach to optimize antibody and T cell responses in the same recipients. Therefore, we will assess the immunogenicity of the Pf CelTOS rec protein alone with adju vant, and in a prime boost (sequential immunization) strategy with rec virus expressing PfCelTOS to provide data to support design of Phase II studies in rhesus monkeys and the first clinical trials of these immunogens. In addition, to strengthen the argum ent for moving the PfCelTOS rec protein to Phase II, we will produce a PyCelTOS rec protein, and conduct studies to achieve significant protective immunity in mice immunized with PyCelTOS immunogens. PUBLIC HEALTH RELEVANCE: Malaria causes 500 million clin ical cases and 1-3 million deaths annually, is responsible for gt1% loss of GDP in Africa annually and is a serious concern for travelers and military personnel. Protein Potential's goal is to develop and commercialize a gt90% protective malaria vaccine fo r primary markets with a potential for gt 1 billion annual revenues; 1) travelers from the developed world, and 2) infants, young children, and adolescent girls in the developing world. Success in this project will significantly decrease the cost of develo pment and time to market for this malaria vaccine.

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