AMA1 function is critical for both merozoites and sporozoites, and in in a mouse model16 and in a non-human primate malaria model17. in solution between the two vaccine components. To advance vaccine development, here we engineered chimeric antigens by replacing the AMA1 DII loop, displaced upon ligand binding, with RON2L. Structural analysis confirmed that the fusion chimera (Fusion-FD12) closely mimics the binary AMA1-RON2L complex. Immunization studies in female rats demonstrated that Fusion-FD12 immune sera, but not purified IgG, neutralized vaccine-type parasites more efficiently compared to apoAMA1, despite lower overall anti-AMA1 titers. Interestingly, Fusion-FD12 immunization enhanced antibodies targeting conserved epitopes on AMA1, leading to increased UNC 926 hydrochloride UNC 926 hydrochloride neutralization of non-vaccine type parasites. Identifying these cross-neutralizing antibody epitopes holds promise for developing an effective, strain-transcending malaria vaccine. Subject terms: Parasite host response, Malaria, Protein vaccines The AMA1-RON2 complex is important for invasion and a potential vaccine target. Here the authors engineer a fusion protein of AMA1 and RON2 loop that mimics the receptor-ligand complex and show that it induces antibodies that neutralize non-vaccine type parasites. Introduction Malaria caused by remains an immense global health and economic concern and is responsible for the majority of the 627,000 malaria-related deaths in 20211. Merozoite invasion of RBCs can be considered the gateway to disease as it is the parasites growing within the safety of the host cell that causes clinical symptoms in susceptible individuals. RTS,S, the first WHO authorized malaria vaccine targeting the clinically silent forms of the parasite has limited efficacy and there is a growing concern due to the development of resistance to frontline antimalarials2,3. There is an urgent need for a vaccine that can reduce the parasite burden in the blood and prevent disease. People living in endemic countries, Rabbit polyclonal to ZNF22 who are exposed to repeated malaria infections, can develop clinical immunity4. AMA1 is among the most immunogenic parasite targets, and anti-AMA1 antibodies inhibit merozoite invasion5,6. AMA1 function is critical for both merozoites and sporozoites, and in in a mouse model16 and in a non-human primate malaria model17. Vaccine efficacy was strongly correlated with the ability of the binary complex vaccine to increase the proportion of neutralizing antibodies targeting AMA1-RON2 interaction16,17. This enhancement in neutralizing antibodies was not only limited to vaccine-type parasites but also against some heterologous parasites16,17. Despite these encouraging results, manufacturing and deploying a vaccine that relies on producing and mixing two proteins that need to spontaneously assemble in solution, presents technical challenges. To facilitate vaccine development, we engineered a single chimeric antigen that would recapitulate the receptor-ligand complex and promote the effective development of neutralizing antibodies against RON2L to its AMA1 partner led to allosteric structural changes in domain 3 of TgAMA119. Such conformational changes in PfAMA1D123 may result in protein instability that is not tolerated in the Sf9 heterologous expression system. Open in a separate window Fig. 1 AMA1 domains 1 and 2 are sufficient for complex-mediated enhancement in antibody quality.A Schematic of AMA1 showing domains 1, 2 (purple) and 3 (gray). Domains 1 and 2 (purple) of AMA1 together form a hydrophobic groove, the binding site for RONL2. Domain 3 (gray) is modeled on PvAMA1 domain 3 (PDB: 1W8K). B AMA1-specific antibody titer in the purified IgG from apoAMA1 (blue) and AMA1?+?RON2L binary complex (green) immunized rats. Open circle and squares indicate the groups that used AMA1D123 (all three domains) and AMA1D12 (domains 1 and 2), respectively. Data are presented for individual animals (receptor-ligand binary complex.A Schematic showing the region of AMA1 DII loop that was replaced with RON2L. B Surface view of PfAMA1-RON2L fusion with the same color code as shown in A. C 2Fo-Fc electron density map for parasites in vivo remains to be determined. Open in a separate window Fig. 5 Antibody specificity and neutralizing activity differences in serum and affinity purified IgG.A, B (left panels) Serum IgG titer in animals immunized with the Fusion-FD12 (purple triangle) or apoAMA1D12 (blue square) antigens in AddaVax (A) and Freunds (B). The malaria-related deaths1 highlight the urgent need for an effective malaria vaccine. Age-related clinical protection is observed in malaria endemic population28 and infection-induced antibodies can clear parasites and UNC 926 hydrochloride resolve clinical symptoms in children29. Identifying the target(s) of such protective antibodies can help develop vaccines to prevent disease. Candidate antigens have shown promise in preclinical studies, however clinical trials have so far not proven successful30. AMA1 is a leading vaccine candidate as antibodies neutralize parasites in vitro and afford partial protection in NHP models of human malaria17,31,32. Numerous polymorphisms in AMA1 surrounding the binding site of its ligand RON2 suggest it is an important target of human neutralizing antibodies18. However.
