Biology subjects

Chmielewski, J.

Publications and source records attributed to Chmielewski, J..

6 recordsLinked to original sources

bioRxiv2026

Nanobodies against Plasmodium adhesins that block receptor engagement and malaria parasite invasion

Malaria is caused by Plasmodium parasites, and its clinical symptoms are a result of parasite invasion of red blood cells and the subsequent cycles of replication and proliferation. In human populations, Plasmodium vivax is responsible for the most widely distributed recurring malaria infections whereas Plasmodium falciparum inflicts the most mortality and morbidity. One well-characterized family of adhesins involved in red blood cell invasion is the reticulocyte-binding-like protein homolog family, known as the RBL superfamily which includes the PfRh family in P. falciparum and PvRBP family in P. vivax. Here we report a collection of nanobodies against three members of this adhesin family, PfRh5, PfRh4 and PvRBP2b. Nanobodies against these Plasmodium adhesins bind with high affinity across several epitopes and can block receptor engagement and inhibit parasite invasion of red blood cells. Using computational design, we generated stabilized PfRh4 variants that encompass the conserved scaffold present in the PfRh and PvRBP families of adhesins and show that several variants with improved expression retained binding to mouse monoclonal antibodies, nanobodies and Complement Receptor 1, the human receptor for PfRh4. We also observed that most of the inhibitory nanobodies against the three antigens recognized the conserved structural scaffold that define this family of adhesins. These results demonstrate the potential of nanobodies to block malaria parasite invasion into red blood cells.

microbiology↗

bioRxiv2025

Crystal structure and nanobodies against domain 3 of the malaria parasite fusogen Plasmodium falciparum HAP2

Malaria parasites are transmitted to humans through a bite from an infected female Anopheles mosquito. Within the mosquito midgut, malaria parasite gametes are activated and undergo fertilisation. If parasite fertilisation is perturbed, this stops the transmission of malaria parasites from mosquito to human. One proposed target of transmission-blocking interventions is Plasmodium falciparum fusogen PfHAP2, which is essential for gamete fusion during parasite fertilisation. However, to date, no monoclonal antibodies or structures of PfHAP2 have been generated. We have identified nanobodies that bind specifically to domain 3 of PfHAP2 with nanomolar affinities, two of which show some cross-species reactivity with HAP2 of other Plasmodium species. The crystal structure of one nanobody in complex with domain 3 of PfHAP2 provides the first structural insights into this transmission-blocking target in P. falciparum.

microbiology↗

bioRxiv2025

Differential importance of MSP4 and MSP5 for infection of red blood cells between human infecting malaria parasites

Plasmodium species malaria parasites require invasion and replication within red blood cells to cause disease. Merozoite surface proteins (MSPs) are proposed to play a role in attachment of merozoites to RBCs and have long been considered as potential vaccine targets, but their functions during invasion are largely unknown. We applied targeted gene editing to investigate MSP4 and 5 function in P. falciparum, which causes most malaria mortality, and P. knowlesi, an in vitro culturable zoonotic species closely related to the widespread P. vivax. CRISPR-Cas9 gene-editing revealed that P. knowlesi MSP4 was not required for parasite growth in vitro. While P. knowlesi MSP5 could be functionally replaced by P. vivax MSP5, it was refractory to gene deletion. We confirmed the opposite for two different P. falciparum laboratory isolates where MSP4 is essential but MSP5 is dispensable. Attempts to select for reliance on the non-essential MSP (e.g. P. knowlesi MSP4 or P. falciparum MSP5) through long-term growth of inducible knock-out parasites, or via chimeric complementation of the essential MSP4 or 5 with the essential MSP from the other species, were unsuccessful. Live cell filming revealed a severe cell-entry defect with conditional knock-down of MSP5 protein expression in P. knowlesi. This study demonstrates differential importance of MSP4 and MSP5 during merozoite RBC invasion across human infecting malaria species, emphasises that vaccine candidates must be considered individually for the two most prominent human malarias and promotes MSP5 as a potential vaccine candidate for P. knowlesi and P. vivax. SignificanceFor a malaria parasite to cause disease, the merozoite form of the lifecycle has to infect and replicate within human red blood cells. Proteins on the surface of the merozoite are considered as promising vaccine candidates, but the functions of these proteins are poorly understood. Here we demonstrate that two structurally similar merozoite surface proteins (MSP), MSP4 and MSP5, have differential importance between one human infecting malaria species compared to a second. The finding that MSP4 is essential for growth in one species, and MSP5 in the other, has implications for understanding invasion biology of malaria parasites and highlights that even structurally similar vaccine targets may need to be chosen specifically for each human infecting malaria species.

microbiology↗

bioRxiv2025

An abundant merozoite surface protein of Plasmodium falciparum modulates susceptibility to inhibitory antibodies

Malaria merozoite surface proteins (MSPs), are thought to have important roles in red blood cell (RBC) invasion and their exposure on the parasite surface makes them attractive vaccine candidates. However, their role in invasion has not been directly demonstrated and their biological functions are unknown. One of the most abundant proteins is PfMSP2, which is likely an ancestral protein that has been maintained in the Plasmodium falciparum lineage and is a focus of vaccine development, whose function remains unknown. Using CRISPR-Cas9 gene-editing, we removed PfMSP2 from two different P. falciparum lines with no impact on parasite replication or phenotype in vitro, demonstrating that it is not essential for RBC invasion. However, loss of PfMSP2 led to increased inhibitory potency of antibodies targeting other merozoite proteins involved in invasion, particularly PfAMA1. In a solid-phase model, increasing concentrations of PfMSP2 protein reduced binding of different antibodies against PfAMA1 in a dose dependent manner. These data suggest that PfMSP2 can modulate the susceptibility of merozoites to protective inhibitory antibodies. The results of this study change our understanding of the potential functions of PfMSP2 and establishes a new concept in malaria where a surface protein can reduce the protective efficacy of antibodies targeting a different antigen. These findings have important implications for understanding malaria immunity and informing vaccine development.

microbiology↗

bioRxiv2025

Pfs48/45 nanobodies block Plasmodium falciparum transmission

Malaria parasite fertilisation occurs within the Anopheles mosquito midgut. Interventions that inhibit parasite fertilisation prevent ongoing transmission and are important for malaria elimination efforts. Pfs48/45 and Pfs230 are two leading transmission-blocking vaccine candidates. Both proteins form a complex on the surface of sexual stage parasites and are essential for male fertility. Here we have identified nanobodies against Pfs48/45 that recognise gametocytes and have strong transmission-reducing activity. The crystal structure of our most potent nanobody in complex with Pfs48/45 reveals it binds a distinct epitope to TB31F, a leading transmission-blocking monoclonal antibody. In addition, we generated bispecific nanobodies that can target both Pfs48/45 and Pfs230 simultaneously and are fused to a human Fc domain. Our results show that these bispecific nanobodies recognise both Pfs48/45 and Pfs230 and reduce malaria parasite fertilisation in Anopheles stephensi. These results demonstrate the potential of nanobodies as a versatile antibody format that can reduce malaria transmission. Author SummaryMalaria is spread when an infected Anopheles mosquito bites a human. Within the female Anopheles mosquito, malaria parasite fertilisation occurs in the mosquito midgut. If you inhibit parasite fertilisation in the mosquito, you can prevent onward transmission of the malaria parasite from mosquito to humans. Transmission blocking vaccines work by stopping parasite fertilisation and development in the mosquito and are key for malaria elimination by preventing community spread. Pfs48/45 and Pfs230 are two leading transmission-blocking vaccine candidates, and both are critical for male fertility. Here we describe the generation of nanobodies that target Pfs48/45. We show that when nanobodies against Pfs48/45 were added to infected blood meals for Anopheles mosquitoes, the nanobodies significantly reduced parasite transmission. In addition, we generated bispecific nanobodies that target both Pfs48/45 and Pfs230 and these bispecific nanobodies also significantly reduced oocyst development. Our work demonstrates the potential of nanobodies as a versatile antibody format that can reduce malaria transmission.

microbiology↗

bioRxiv2025

Cryo-EM structure of endogenous Plasmodium falciparum Pfs230 and Pfs48/45 fertilization complex

Plasmodium falciparum Pfs230 and Pfs48/45, part of a core fertilization complex, are leading malaria transmission-blocking vaccine candidates. However, how the two proteins interact is unknown. Here we report a 3.36 [A] resolution cryo-electron microscopy structure of the endogenous Pfs230-Pfs48/45 complex. We show that Pfs48/45 interacts with Pfs230 domains 13 and 14, domains that are not included in current Pfs230 vaccine immunogens. Using a transgenic parasite line with a domain 13 to 14 deletion, we show that these domains are essential for Pfs230 localization on the gamete surface. Furthermore, this line significantly reduced oocyst formation in the mosquito midgut, showing that the presence of Pfs230 domains 13 and 14 is critical for successful fertilization. Nanobodies against domains 13 and 14 inhibit Pfs230-Pfs48/45 complex formation, reduce transmission and structural analyses reveal their binding epitopes. Furthermore, domains 13 and 14 are targets of naturally acquired immunity and when delivered as mRNA-LNP immunizations induce potent immune responses and blocked transmission of malaria parasites. Our comprehensive structural insights on a core P. falciparum fertilization complex will guide the design of novel transmission-blocking vaccine candidates against malaria.

microbiology↗