The repeat region of the circumsporozoite protein is critical for sporozoite formation and maturation in Plasmodium.
ABSTRACT: The circumsporozoite protein (CSP) is the major surface protein of the sporozoite stage of malaria parasites and has multiple functions as the parasite develops and then migrates from the mosquito midgut to the mammalian liver. The overall structure of CSP is conserved among Plasmodium species, consisting of a species-specific central tandem repeat region flanked by two conserved domains: the NH2-terminus and the thrombospondin repeat (TSR) at the COOH-terminus. Although the central repeat region is an immunodominant B-cell epitope and the basis of the only candidate malaria vaccine in Phase III clinical trials, little is known about its functional role(s). We used the rodent malaria model Plasmodium berghei to investigate the role of the CSP tandem repeat region during sporozoite development. Here we describe two mutant parasite lines, one lacking the tandem repeat region (?Rep) and the other lacking the NH2-terminus as well as the repeat region (?N?Rep). We show that in both mutant lines oocyst formation is unaffected but sporozoite development is defective.
Project description:The Plasmodium falciparum circumsporozoite protein (CSP) is critical for sporozoite function and invasion of hepatocytes. Given its critical nature, a phase III human CSP malaria vaccine trial is ongoing. The CSP is composed of three regions as follows: an N terminus that binds heparin sulfate proteoglycans, a four amino acid repeat region (NANP), and a C terminus that contains a thrombospondin-like type I repeat (TSR) domain. Despite the importance of CSP, little is known about its structure. Therefore, recombinant forms of CSP were produced by expression in both Escherichia coli (Ec) and then refolded (EcCSP) or in the methylotrophic yeast Pichia pastoris (PpCSP) for structural analyses. To analyze the TSR domain of recombinant CSP, conformation-dependent monoclonal antibodies that recognized unfixed P. falciparum sporozoites and inhibited sporozoite invasion of HepG2 cells in vitro were identified. These monoclonal antibodies recognized all recombinant CSPs, indicating the recombinant CSPs contain a properly folded TSR domain structure. Characterization of both EcCSP and PpCSP by dynamic light scattering and velocity sedimentation demonstrated that both forms of CSP appeared as highly extended proteins (R(h) 4.2 and 4.58 nm, respectively). Furthermore, high resolution atomic force microscopy revealed flexible, rod-like structures with a ribbon-like appearance. Using this information, we modeled the NANP repeat and TSR domain of CSP. Consistent with the biochemical and biophysical results, the repeat region formed a rod-like structure about 21-25 nm in length and 1.5 nm in width. Thus native CSP appears as a glycosylphosphatidylinositol-anchored, flexible rod-like protein on the sporozoite surface.
Project description:The circumsporozoite protein (CSP) plays a key role in malaria sporozoite infection of both mosquito salivary glands and the vertebrate host. The conserved Regions I and II have been well studied but little is known about the immunogenic central repeat region and the N-terminal region of the protein. Rodent malaria Plasmodium berghei parasites, in which the endogenous CS gene has been replaced with the avian Plasmodium gallinaceum CS (PgCS) sequence, develop normally in the A. stephensi mosquito midgut but the sporozoites are not infectious. We therefore generated P. berghei transgenic parasites carrying the PgCS gene, in which the repeat region was replaced with the homologous region of P. berghei CS (PbCS). A further line, in which both the N-terminal region and repeat region were replaced with the homologous regions of PbCS, was also generated. Introduction of the PbCS repeat region alone, into the PgCS gene, did not rescue sporozoite species-specific infectivity. However, the introduction of both the PbCS repeat region and the N-terminal region into the PgCS gene completely rescued infectivity, in both the mosquito vector and the mammalian host. Immunofluorescence experiments and western blot analysis revealed correct localization and proteolytic processing of CSP in the chimeric parasites. The results demonstrate, in vivo, that the repeat region of P. berghei CSP, alone, is unable to mediate sporozoite infectivity in either the mosquito or the mammalian host, but suggest an important role for the N-terminal region in sporozoite host cell invasion.
Project description:The circumsporozoite protein (CSP) is the major surface protein of Plasmodium sporozoites, the infective stage of malaria. Although CSP has been extensively studied as a malaria vaccine candidate, little is known about its structure. Here, we show that CSP is proteolytically cleaved by a papain family cysteine protease of parasite origin. Our data suggest that the highly conserved region I, found just before the repeat region, contains the cleavage site. Cleavage occurs on the sporozoite surface when parasites contact target cells. Inhibitors of CSP processing inhibit cell invasion in vitro, and treatment of mice with E-64, a highly specific cysteine protease inhibitor, completely inhibits sporozoite infectivity in vivo.
Project description:The extended rod-like Plasmodium falciparum circumsporozoite protein (CSP) is comprised of three primary domains: a charged N terminus that binds heparan sulfate proteoglycans, a central NANP repeat domain, and a C terminus containing a thrombospondin-like type I repeat (TSR) domain. Only the last two domains are incorporated in RTS,S, the leading malaria vaccine in phase 3 trials that, to date, protects about 50% of vaccinated children against clinical disease. A seroepidemiological study indicated that the N-terminal domain might improve the efficacy of a new CSP vaccine. Using a panel of CSP-specific monoclonal antibodies, well-characterized recombinant CSPs, label-free quantitative proteomics, and in vitro inhibition of sporozoite invasion, we show that native CSP is N-terminally processed in the mosquito host and undergoes a reversible conformational change to mask some epitopes in the N- and C-terminal domains until the sporozoite interacts with the liver hepatocyte. Our findings show the importance of understanding processing and the biophysical change in conformation, possibly due to a mechanical or molecular signal, and may aid in the development of a new CSP vaccine.
Project description:Malaria is a global health concern, and research efforts are ongoing to develop a superior vaccine to RTS,S/AS01. To guide immunogen design, we seek a comprehensive understanding of the protective humoral response against Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP). In contrast to the well-studied responses to the repeat region and the C-terminus, the antibody response against the N-terminal domain of PfCSP (N-CSP) remains obscure. Here, we characterized the molecular recognition and functional efficacy of the N-CSP-specific monoclonal antibody 5D5. The crystal structure at 1.85-Å resolution revealed that 5D5 binds an ?-helical epitope in N-CSP with high affinity through extensive shape and charge complementarity and the unusual utilization of an antibody N-linked glycan. Nevertheless, functional studies indicated low 5D5 binding to live Pf sporozoites and lack of sporozoite inhibition in vitro and in vivo. Overall, our data do not support the inclusion of the 5D5 N-CSP epitope into the next generation of CSP-based vaccines.
Project description:Studies in animals and human volunteers demonstrate that antibodies against the repeat-region of the Plasmodium circumsporozoite protein (CSP) abrogate sporozoite infection. However, the realization that the N- and C- terminal regions flanking the repeats play essential roles in parasite infectivity raised the possibility that they could be targeted by protective antibodies. We characterized a monoclonal antibody (mAb5D5) specific for the N-terminus of the P. falciparum CSP, which inhibits the proteolytic cleavage of the CSP, a key requirement for parasite infection of hepatocytes. Adoptive transfer of mAb5D5 strongly inhibits the in vivo infection of sporozoites expressing the N-terminus of P. falciparum CSP, and this protection is greatly enhanced when combined with antirepeat antibodies. Our results show that antibodies interfering with molecular processes required for parasite infectivity can exert a strong in vivo protective activity and indicate that pre-erythrocytic vaccines against Plasmodium should include the CSP N-terminal region.
Project description:Combinations of monoclonal antibodies (mAbs) against different epitopes on the same antigen synergistically neutralize many viruses. However, there are limited studies assessing whether combining human mAbs against distinct regions of the Plasmodium falciparum (Pf) circumsporozoite protein (CSP) enhances in vivo protection against malaria compared to each mAb alone or whether passive transfer of PfCSP mAbs would improve protection following vaccination against PfCSP. Here, we isolated a panel of human mAbs against the subdominant C-terminal domain of PfCSP (C-CSP) from a volunteer immunized with radiation-attenuated Pf sporozoites. These C-CSP-specific mAbs had limited binding to sporozoites in vitro that was increased by combination with neutralizing human "repeat" mAbs against the NPDP/NVDP/NANP tetrapeptides in the central repeat region of PfCSP. Nevertheless, passive transfer of repeat- and C-CSP-specific mAb combinations did not provide enhanced protection against in vivo sporozoite challenge compared to repeat mAbs alone. Furthermore, combining potent repeat-specific mAbs (CIS43, L9, and 317) that respectively target the three tetrapeptides (NPDP/NVDP/NANP) did not provide additional protection against in vivo sporozoite challenge. However, administration of either CIS43, L9, or 317 (but not C-CSP-specific mAbs) to mice that had been immunized with R21, a PfCSP-based virus-like particle vaccine that induces polyclonal antibodies against the repeat region and C-CSP, provided enhanced protection against sporozoite challenge when compared to vaccine or mAbs alone. Collectively, this study shows that while combining mAbs against the repeat and C-terminal regions of PfCSP provide no additional protection in vivo, repeat mAbs do provide increased protection when combined with vaccine-induced polyclonal antibodies. These data should inform the implementation of PfCSP human mAbs alone or following vaccination to prevent malaria infection.
Project description:Circumsporozoite protein (CSP) coats the Plasmodium falciparum sporozoite surface and is a major malaria subunit vaccine target. We measured epitope-specific reactivity to field-derived CSP haplotypes in serum samples from Malian adults and children on a custom peptide microarray. Compared to children, adults showed greater antibody responses and responses to more variants in regions proximal to and within the central repeat region. Children acquired short-lived immunity to an epitope proximal to the central repeat region but not to the central repeat region itself. This approach has the potential to differentiate immunodominant from protective epitope-specific responses when combined with longitudinal infection data.
Project description:Antibodies against the central repeat of the Plasmodium falciparum (Pf) circumsporozoite protein (CSP) inhibit parasite activity and correlate with protection from malaria. However, the humoral response to the PfCSP C terminus (C-PfCSP) is less well characterized. Here, we describe B cell responses to C-PfCSP from European donors who underwent immunization with live Pf sporozoites (PfSPZ Challenge) under chloroquine prophylaxis (PfSPZ-CVac), and were protected against controlled human malaria infection. Out of 215 PfCSP-reactive monoclonal antibodies, only two unique antibodies were specific for C-PfCSP, highlighting the rare occurrence of C-PfCSP-reactive B cells in PfSPZ-CVac-induced protective immunity. These two antibodies showed poor sporozoite binding and weak inhibition of parasite traversal and development, and did not protect mice from infection with PfCSP transgenic Plasmodium berghei sporozoites. Structural analyses demonstrated that one antibody interacts with a polymorphic region overlapping two T cell epitopes, suggesting that variability in C-PfCSP may benefit parasite escape from humoral and cellular immunity. Our data identify important features underlying C-PfCSP shortcomings as a vaccine target.
Project description:Circumsporozoite protein (CSP) of Plasmodium falciparum is a protective human malaria vaccine candidate. There is an urgent need for models that can rapidly down-select novel CSP-based vaccine candidates. In the present study, the mouse-mosquito transmission cycle of a transgenic Plasmodium berghei malaria parasite stably expressing a functional full-length P. falciparum CSP was optimized to consistently produce infective sporozoites for protection studies. A minimal sporozoite challenge dose was established, and protection was defined as the absence of blood-stage parasites 14 days after intravenous challenge. The specificity of protection was confirmed by vaccinating mice with multiple CSP constructs of differing lengths and compositions. Constructs that induced high NANP repeat-specific antibody titers in enzyme-linked immunosorbent assays were protective, and the degree of protection was dependent on the antigen dose. There was a positive correlation between antibody avidity and protection. The antibodies in the protected mice recognized the native CSP on the parasites and showed sporozoite invasion inhibitory activity. Passive transfer of anti-CSP antibodies into naive mice also induced protection. Thus, we have demonstrated the utility of a mouse efficacy model to down-select human CSP-based vaccine formulations.