RESUMO
To eliminate the problems associated with the use of extraneous adjuvants we have designed a Self-Assembling Protein Nanoparticle (SAPN) containing epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) (designated FMP014) and portions of the TLR5 agonist flagellin (designated FMP014D0D1) as an intrinsic adjuvant. By combining different molar ratios of FMP014 to FMP014D0D1 monomers before self-assembly, we generated multiple nanoparticles and investigated their biophysical characteristics, immunogenicity and protective efficacy. Immunization with the construct formulated with the ratio 58:2 of FMP014 to FMP014D0D1 had the highest protective efficacy against a challenge with a transgenic P. berghei sporozoite expressing PfCSP. Increasing the proportion of flagellin per particle resulted in an inverse relationship with levels of both antibody titers and protection. The cytokine profiles of the various immunization groups were evaluated and quantitative amounts of the cytokines IL-2, IFN-γ, IL-12/p70 (Th1); IL4, IL5 (Th2); TNF-α, IL1ß, IL-6, KC/GRO (pro-inflammatory), and IL-10 (immunomodulatory) were measured. The relationship of the cytokines to each other revealed a strong immunomodulatory effect depending on the proportion of flagellin in the construct. Our results demonstrate that SAPNs with flagellin may be a promising strategy for the development and delivery of a safe vaccine for infectious diseases.
Assuntos
Flagelina/imunologia , Imunogenicidade da Vacina , Malária Falciparum/prevenção & controle , Nanopartículas , Plasmodium falciparum/imunologia , Domínios Proteicos/imunologia , Proteínas de Protozoários/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Antiprotozoários/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Flagelina/química , Flagelina/genética , Imunização , Malária Falciparum/imunologia , Malária Falciparum/metabolismo , Camundongos , Modelos Biológicos , Plasmodium falciparum/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos/genética , Dobramento de Proteína , Multimerização Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes , Receptor 5 Toll-Like/agonistasRESUMO
We have developed FMP014, a vaccine candidate against Plasmodium falciparum malaria, which is comprised of 60 identical monomer protein chains that form an icosahedral shaped self-assembling protein nanoparticle (SAPN). Each monomer contains selected P. falciparum Circumsporozoite Protein (PfCSP) CD4+ and CD8+ epitopes, universal TH epitopes, portions of the α-TSR domain, and 6 repeats of the NANP motifs of the PfCSP. Here we describe the conditions that are required for successful scale-up and cGMP manufacturing of FMP014 with a yield of ≈1.5g of drug substance per 100g of wet bacterial paste. When adjuvanted with an Army Liposomal Formulation (ALF) based adjuvant, the nanoparticle vaccine is highly immunogenic and prevents infection of mice by an otherwise lethal dose of transgenic P. berghei sporozoites expressing the full-length PfCSP.