Structure of the Plasmodium falciparum Circumsporozoite Protein, a Leading Malaria Vaccine Candidate
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 (Rh 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. 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 (Rh 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. Malaria caused by Plasmodium falciparum is a serious global health issue, resulting in an estimated 1.5 million deaths annually, primarily among infants and young children. Ongoing multifaceted global intervention strategies to control malaria include drug treatment, insecticide usage, bed-net use, and vaccine development. However, parasite and mosquito control measures have met with limited success resulting from an increased drug and insecticide resistance within the Plasmodia and mosquito populations, respectively. Vaccine development represents an encouraging approach given that previous animal and human studies using irradiated sporozoites demonstrated the feasibility of producing an efficacious vaccine (1Clyde D.F. Most H. McCarthy V.C. Vanderberg J.P. Am. J. Med. Sci. 1973; 266: 169-177Crossref PubMed Scopus (427) Google Scholar, 2Nussenzweig R.S. Vanderberg J. Most H. Orton C. Nature. 1967; 216: 160-162Crossref PubMed Scopus (660) Google Scholar, 3Nussenzweig R.S. Vanderberg J.P. Most H. Orton C. Nature. 1969; 222: 488-489Crossref PubMed Scopus (114) Google Scholar). Although the exact immunologic correlates of protection remain elusive, an abundance of evidence indicates that protection against liver stage parasites is complex, involving multiple immune mechanisms (4Franke E.D. Sette A. 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To date, the majority of the pre-erythrocytic stage vaccine development has focused on the circumsporozoite protein (CSP), 2The abbreviations used are: CSPcircumsporozoite proteinTSRthrombospondin-like type I repeatmAbmonoclonal antibodyISIinhibition of sporozoite invasionECMEagle's essential minimum complete mediaDTTdithiothreitolBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolPBSphosphate-buffered salineSEC-MALS-QELS-HPLCsize exclusion chromatography-multiangle light scattering-quasi-elastic light scatter detection; high pressure liquid chromatographyqRTquantitative real timerrecombinantAFMatomic force microscopyEcCSPE. coli CSPPpCSPP. pastoris CSPESI-MSelectrospray ionization-mass spectrometry-MLmechanical microfluidization lysis-CLchemical detergent lysis. the predominant surface antigen on sporozoites. CSP can be segmented into three regions as follows: the N-terminal region containing region I; the central repeat region; and the C-terminal region containing the thrombospondin-like type I repeat (TSR). CSP vaccine development focused on the central repeat region that contains the D.F. G. M. J. S. 1987; PubMed Scopus Google Scholar). However, vaccine to both the central repeat region containing the and the C terminus containing the TSR and J.M. Nussenzweig R.S. B. C. S. R. J. Infect. 2000; PubMed Scopus Google Scholar, J.A. M. P. P. M. J. Med. PubMed Scopus Google Scholar). the and malaria is composed of a of the central repeat and the C-terminal regions to the surface antigen P. J. A. A. M. J. S. J. P. P. A. S. B. W.R. J. L. J. Med. PubMed Scopus Google Scholar). However, studies have the importance of the N-terminal region R. J. PubMed Scopus Google Scholar, S. M. P. R. Corradin G. PubMed Scopus Google Scholar, R. R. P. Kumar S. J. PubMed Scopus Google Scholar, Sacci P. J. PubMed Scopus Google Scholar). Sacci P. J. PubMed Scopus Google demonstrated the of the N-terminal region in liver with and R. J. PubMed Scopus Google an within the N-terminal region that with liver cells heparin sulfate R. R. P. Kumar S. J. PubMed Scopus Google Scholar). this to be the resulting antibodies were to be in a sporozoite invasion R. R. P. Kumar S. J. PubMed Scopus Google Scholar). to the N-terminal region and were recognized by from in regions S. M. P. R. Corradin G. PubMed Scopus Google Scholar). circumsporozoite protein thrombospondin-like type I repeat monoclonal of sporozoite invasion essential minimum complete exclusion chromatography-multiangle light scattering-quasi-elastic light scatter detection; high pressure liquid real recombinant atomic force microscopy coli CSP P. pastoris CSP ionization-mass microfluidization detergent lysis. To the structure of CSP and to recombinant protein for human we and recombinant CSP. expression Escherichia coli and Pichia to feasibility to CSP. To the of the we a of monoclonal antibodies that were to used to the of the and The have the of CSP as a flexible, rod-like To antibodies CSP, we a CSP by The to CSP amino to into the expression using the and The resulting a The of CSP to produced as R.L. PubMed Scopus Google and using a by exclusion to to a of were with a with an N-terminal amino acid were by against sporozoite and microscopy of sporozoites. to be were and using the the To or the the were using an in vitro to to sporozoite invasion of HepG2 of the invasion of HepG2 cells M.R. P. Am. J. Med. 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PubMed Scopus Google Scholar). the nm the to and by to a of for by and The using or lysis. the in of and a three The for to and the and EcCSP then from the using a and using a containing and the in of and for the of to the of of The for to and were with and then in EcCSP and using a EcCSP refolded on a of of a on a with and from the using EcCSP and on a and using a exclusion with the coli expression of CSP, the amino acid of CSP used to a Pichia for expression in the methylotrophic yeast P. pastoris CSP to amino to into the and of the Pichia expression as the The resulting a to with and into P. the of CSP protein produced in as V. J. R.L. L. C. R. Infect. Immun. PubMed Scopus Google Scholar, Jr., R.L. Miller L.H. J. PubMed Scopus Google Scholar). be However, in PpCSP to by on a by and with a exclusion The antigen to in and were using a with the were were to the of the recombinant and were to the using an to the The surface with an of by of in in or in were with of the in or cells and were and then to as the recombinant proteins were in from nm to were a of for The using of for and with a were using and a the on a as protein in to a of protein as and were in and nm, and to the The using of for and with a a to nm, the and as To in were in were the nm in a length using a of nm, a of nm, and a of of on structure in from to structure using the L. PubMed Scopus Google Scholar, L. PubMed Scopus Google Scholar). To the and of EcCSP and exclusion with light scattering and light scatter on an to a light scattering and a light scattering were and as by S. J. Jr., R.L. L. Miller L.H. A. PubMed Scopus Google Scholar). sedimentation velocity were in velocity a of The with of protein a of PpCSP or were to the velocity into as P. J. 2000; PubMed Scopus Google using the domain by The of this is a of the of in the of a this treatment, the sedimentation velocity were to for the of P. J. 2000; PubMed Scopus Google that the is from the and this for the of the to a the of The by and used to the of the PpCSP and the and for the used in the sedimentation and for the to be for were and as by S. J. Jr., R.L. L. Miller L.H. A. PubMed Scopus Google with as of the protein a of both in and using with a of a a type and a or S. J. Jr., R.L. L. Miller L.H. A. PubMed Scopus Google Scholar, R. J. PubMed Scopus Google Scholar). protein by of of of of in on by with of and complete an The then into the by S. J. Jr., R.L. L. Miller L.H. A. PubMed Scopus Google Scholar). were within the and to of and for and structure of the N-terminal domain structure using R.L. PubMed Scopus Google and J. 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To the of recombinant CSP in coli (EcCSP) and P. pastoris (PpCSP) a of by against sporozoite to for three of the in and the CSP Nussenzweig R.S. Infect. Immun. PubMed Google Scholar, P. Nussenzweig V. Nussenzweig R.S. J. Exp. Med. 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The TSR domain contains and and and and forms an extended structure about nm in length and nm in width. be by a from about to the C terminus The repeat region forms a about nm in length and 1.5 nm in composed of with a of about or repeat of in the of the a to the structure and a to the N terminus of the repeat region multiple vaccine the of a CSP have against from to and against malaria in J. A. J. B. C. M. P. S. M. F. C. W.R. R. J. PubMed Scopus Google Scholar, P. M. J. S. A. A. M. J. B. M. B. W.R. J. PubMed Scopus Google Scholar, M. L. A. W.R. P. L. M. G. A. PubMed Scopus Google Scholar). However, the the N-terminal region of CSP, has to be a critical domain R. J. PubMed Scopus Google Scholar, S. M. P. R. Corradin G. PubMed Scopus Google Scholar, R. R. P. Kumar S. J. PubMed Scopus Google Scholar, Sacci P. J. PubMed Scopus Google Scholar). to and recombinant CSP the N repeat and C with the of a of the structure of CSP. we the and the and biophysical of CSP from expression coli and P. 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However, for vaccine development is of the or on the structure. of both proteins a the recombinant proteins an of structure Furthermore, we to the a from or or the structure The for both EcCSP and PpCSP structure is from to or is a the resulting in is to have a on structure for recombinant biophysical studies of the EcCSP and PpCSP by and a predominant for both and in as The of for is the of is of an the and Furthermore, EcCSP and PpCSP extended on the by sedimentation and sedimentation velocity the that CSP a highly extended structure. To and to the structural of CSP, we all three recombinant proteins by high resolution that of studies the of using an to both and sedimentation velocity in the of CSP. The and the on a that CSP is an extended and flexible with These the evidence CSP has an structure and with previous studies that the structure of the NANP repeat region of CSP. studies NANP by and C. C. J. J.A. J. Am. Scopus Google by C. L. F. C. H. G. J.A. J. Am. Scopus Google and by A. A. J.A. Google all of demonstrated the a H. M. Sci. PubMed Scopus Google Scholar). by C. C. J.A. J. PubMed Scopus Google that by multiple a structure be sedimentation and the that the NANP repeat region of CSP a an and flexible the of the repeat region to the repeat the TSR domain is an structure. the N-terminal domain and the repeat the repeat region and the TSR domain. These to the of and multiple with a of 1.5 1.5 The the the and is to by the Although can S. J. Jr., R.L. L. Miller L.H. A. PubMed Scopus Google Scholar, R. J. PubMed Scopus Google is for the CSP protein with its and high of structure to be in the ribbon-like forms the and the is for the in both the length and the of CSP the of cells has in vitro PubMed Scopus Google Scholar, C. PubMed Scopus Google and to include both of cells in is formed and of cells by of a A. Med. PubMed Scopus Google Scholar, G. Vanderberg J.P. Nussenzweig R.S. Nussenzweig V. A. PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar, R. S. P. B. M. R. PubMed Scopus Google Scholar). However, the for the protein within this and is abundance of evidence both the N-terminal domain and C-terminal domain in the invasion of from to liver heparin sulfate on the surface of R. J. PubMed Scopus Google Scholar, S. M. P. R. Corradin G. PubMed Scopus Google Scholar, R. R. P. Kumar S. J. PubMed Scopus Google Scholar, Sacci P. J. PubMed Scopus Google Scholar, A. R. R. P. PubMed Scopus Google Scholar, C. P. B. P. Nussenzweig V. PubMed Scopus Google Scholar). the structure of the protein as an flexible rod-like structure on the of or is of we have from expression PpCSP to and to as by the of an and in These evidence of a highly the evidence CSP as an flexible rod-like protein on the surface of sporozoites. to for the and of for and on B. for with the for sporozoites for and for and protein respectively. with