Structural and Immunological Characterization of Recombinant 6-Cysteine Domains of the Plasmodium falciparum Sexual Stage Protein Pfs230

Abstract

Development of a Plasmodium falciparum (Pf) transmission blocking vaccine (TBV) has the potential to significantly impact malaria control. Antibodies elicited against sexual stage proteins in the human bloodstream are taken up with the blood meal of the mosquitoes and inactivate parasite development in the mosquito. In a phase 1 trial, a leading TBV identified as Pfs25-EPA/Alhydrogel® appeared safe and immunogenic, however, the level of Pfs25-specific antibodies were likely too low for an effective vaccine. Pfs230, a 230-kDa sexual stage protein expressed in gametocytes is an alternative vaccine candidate. A unique 6-cysteine-rich domain structure within Pfs230 have thwarted its recombinant expression and characterization for clinical evaluation for nearly a quarter of a century. Here, we report on the identification, biochemical, biophysical, and immunological characterization of recombinant Pfs230 domains. Rabbit antibodies generated against recombinant Pfs230 domains blocked mosquito transmission of a laboratory strain and two field isolates using an ex vivo assay. A planned clinical trial of the Pfs230 vaccine is a significant step toward the potential development of a transmission blocking vaccine to eliminate malaria. Development of a Plasmodium falciparum (Pf) transmission blocking vaccine (TBV) has the potential to significantly impact malaria control. Antibodies elicited against sexual stage proteins in the human bloodstream are taken up with the blood meal of the mosquitoes and inactivate parasite development in the mosquito. In a phase 1 trial, a leading TBV identified as Pfs25-EPA/Alhydrogel® appeared safe and immunogenic, however, the level of Pfs25-specific antibodies were likely too low for an effective vaccine. Pfs230, a 230-kDa sexual stage protein expressed in gametocytes is an alternative vaccine candidate. A unique 6-cysteine-rich domain structure within Pfs230 have thwarted its recombinant expression and characterization for clinical evaluation for nearly a quarter of a century. Here, we report on the identification, biochemical, biophysical, and immunological characterization of recombinant Pfs230 domains. Rabbit antibodies generated against recombinant Pfs230 domains blocked mosquito transmission of a laboratory strain and two field isolates using an ex vivo assay. A planned clinical trial of the Pfs230 vaccine is a significant step toward the potential development of a transmission blocking vaccine to eliminate malaria. Development of a malaria vaccine that effectively protects against parasite infection of both the natural host, Anopheles mosquitoes, and its secondary host, man, would effectively disrupt transmission and clinical disease. The most well known investigational vaccine against Plasmodium falciparum malaria that recently received a positive scientific opinion from the Committee for Medicinal Products for Human Use of the European Medicines Agency in July 2015, is identified as RTS,S (MosquirixTM). This vaccine targets the circumsporozoite protein that is present on the surface of the sporozoite, the parasite stage that infects man (1Agnandji S.T. Lell B. Soulanoudjingar S.S. Fernandes J.F. Abossolo B.P. Conzelmann C. Methogo B.G. Doucka Y. Flamen A. Mordmuller B. Issifou S. Kremsner P.G. Sacarlal J. Aide P. Lanaspa M. et al.First results of phase 3 trial of RTS,S/AS01 malaria vaccine in African children.N. Engl. J. Med. 2011; 365: 1863-1875Crossref PubMed Scopus (653) Google Scholar). RTS,S, a virus like-particle-based vaccine, is protective against clinical disease in about 30% of the young children who participated in a phase 3 trial (1Agnandji S.T. Lell B. Soulanoudjingar S.S. Fernandes J.F. Abossolo B.P. Conzelmann C. Methogo B.G. Doucka Y. Flamen A. Mordmuller B. Issifou S. Kremsner P.G. Sacarlal J. Aide P. Lanaspa M. et al.First results of phase 3 trial of RTS,S/AS01 malaria vaccine in African children.N. Engl. J. Med. 2011; 365: 1863-1875Crossref PubMed Scopus (653) Google Scholar, 2RTS,S. Clinical Trials Partnership Efficacy and safety of RTS,S/AS01 malaria vaccine with or without a booster dose in infants and children in Africa: final results of a phase 3, individually randomised, controlled trial.Lancet. 2015; 386: 31-45Abstract Full Text Full Text PDF PubMed Scopus (810) Google Scholar). Efforts toward development of a vaccine to disrupt parasite infection of the mosquito host, also identified as a transmission blocking vaccine have to date only been able to evaluate a sexual stage-specific protein, Pfs25, 2The abbreviations used are: Pfs25, P. falciparum 25-kDa zygote and ookinete protein; Pfs230, P. falciparum 230-kDa sexual stage protein; TBV, transmission blocking vaccine; EPA, ExoProtein A; TBA, transmission blocking activity; SMFA, standard membrane feeding assay; SEC-MALS, size exclusion chromatography-multiangle light scattering. 2The abbreviations used are: Pfs25, P. falciparum 25-kDa zygote and ookinete protein; Pfs230, P. falciparum 230-kDa sexual stage protein; TBV, transmission blocking vaccine; EPA, ExoProtein A; TBA, transmission blocking activity; SMFA, standard membrane feeding assay; SEC-MALS, size exclusion chromatography-multiangle light scattering. which is a 25-kDa protein expressed on the zygote and ookinete surfaces. A phase 1 study demonstrated that human antibodies raised against a recombinant Pfs25 (Pfs25H) protein formulated in Montanide ISA 51, a water-in-oil adjuvant formulation, were biologically active in an ex vivo feeding assay (3Wu Y. Ellis R.D. Shaffer D. Fontes E. Malkin E.M. Mahanty S. Fay M.P. Narum D. Rausch K. Miles A.P. Aebig J. Orcutt A. Muratova O. Song G. Lambert L. et al.Phase 1 trial of malaria transmission blocking vaccine candidates Pfs25 and Pvs25 formulated with montanide ISA 51.PLoS ONE. 2008; 3: e2636Crossref PubMed Scopus (310) Google Scholar), however, this formulation was not deemed suitable for a public health vaccine. More recently, in preclinical studies, Pfs25H has been shown to have enhanced immunogenic properties when chemically conjugated to a carrier molecule such as Neisseria meningitis outer membrane protein complex (4Wu Y. Przysiecki C. Flanagan E. Bello-Irizarry S.N. Ionescu R. Muratova O. Dobrescu G. Lambert L. Keister D. Rippeon Y. Long C.A. Shi L. Caulfield M. Shaw A. Saul A. Shiver J. Miller L.H. Sustained high-titer antibody responses induced by conjugating a malarial vaccine candidate to outer-membrane protein complex.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 18243-18248Crossref PubMed Scopus (104) Google Scholar), or Pseudomonas aeruginosa ExoProtein A (EPA) (5Qian F. Wu Y. Muratova O. Zhou H. Dobrescu G. Duggan P. Lynn L. Song G. Zhang Y. Reiter K. MacDonald N. Narum D.L. Long C.A. Miller L.H. Saul A. Mullen G.E. recombinant proteins to Pseudomonas aeruginosa ExoProtein a for of malaria vaccine PubMed Scopus Google Scholar, F. Rausch Muratova O. Zhou H. Song G. A. Lambert L. Narum D.L. Wu Y. Saul A. Miller L.H. Long C.A. Mullen G.E. of to recombinant Pseudomonas aeruginosa ExoProtein A of and Pfs25 the of 2008; PubMed Scopus Google Scholar). In the chemically conjugated has the of a with a of about in in size to that of the RTS,S C. Reiter K. B. Aebig J. Rausch K. Wu Y. Narum D.L. Development of a malaria transmission blocking vaccine as a chemically conjugated PubMed Scopus Google Scholar). In a phase 1 human trial, the Pfs25-EPA/Alhydrogel® formulation was shown to safe and immunogenic, and generated human antibodies that transmission by using an ex vivo standard membrane feeding assay R. R. D. H. A. E. N. A. K. D. O. C. D. J. S. N. J. M. P. S. A. A. P. D. L. Y. and P. E. R. R. D. H. A. E. N. A. K. D. O. C. D. J. S. N. J. M. P. S. A. A. P. D. L. Y. and P. E. The of Pfs25-specific however, appeared for of the mosquito a the a adjuvant formulation, the of sexual stage vaccine candidates or vaccine candidates have been identified S. of malaria vaccine in the of PubMed Scopus Google A. J. A. D. M. protein of Plasmodium falciparum malaria in Natl. Acad. Sci. U.S.A. 2008; PubMed Scopus Google Scholar, E. N. A malaria transmission blocking vaccine on expressed in ONE. PubMed Scopus Google Scholar, A. K. M. S. M. Muratova Wu Y. A Pfs230 vaccine candidate transmission of Plasmodium 2011; PubMed Scopus Google M. Wu Y. H. Muratova O. MacDonald J. S. H. M. of Pfs230 using a is to malaria 2011; PubMed Scopus Google Scholar). a quarter of a et R. J. N. Miller L.H. The 230-kDa surface protein of Plasmodium falciparum is also a for PubMed Google the potential of the P. falciparum sexual stage 230-kDa protein as a candidate for a transmission blocking vaccine. Pfs230 a complex with a protein of a Plasmodium falciparum which and PubMed Scopus Google on the surface of sexual stage J. and expression of the for the transmission blocking of Plasmodium PubMed Scopus Google Scholar, N. of malaria transmission blocking as a membrane PubMed Scopus Google Scholar). The complex is in of and Pfs230 is a surface protein that is development the of Plasmodium falciparum surface Pfs230, PubMed Scopus Google Scholar). J. Keister Muratova O. of of Plasmodium falciparum Pfs230, to malaria PubMed Scopus Google Scholar, J. D. P. S. R. E. of Plasmodium falciparum by human is with antibodies to the surface PubMed Google and R. J. N. Miller L.H. The 230-kDa surface protein of Plasmodium falciparum is also a for PubMed Google Scholar, K. Plasmodium a of the of antibodies in an assay of and PubMed Scopus Google antibodies have been shown to parasite transmission using the ex vivo in a Pfs230 has been to have a with a of Pfs230 were shown to to to mosquitoes was by S. B. Pfs230, human blood to and 2006; PubMed Scopus Google Scholar). or domains of Pfs230 have been expressed in recombinant expression and S. of malaria vaccine in the of PubMed Scopus Google Scholar, A. K. M. S. M. Muratova Wu Y. A Pfs230 vaccine candidate transmission of Plasmodium 2011; PubMed Scopus Google Scholar, M. Wu Y. H. Muratova O. MacDonald J. S. H. M. of Pfs230 using a is to malaria 2011; PubMed Scopus Google Scholar, Keister Muratova O. Pfs230, a Plasmodium falciparum protein, that the of Plasmodium falciparum to PubMed Scopus Google to date of the recombinant of Pfs230 have been suitable for and characterization to effectively a antibody A in recombinant protein has been the of an unique structure by et D.L. A. S. R. for the protein by surface protein Pfs230 of Plasmodium Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). More characterization of a E. recombinant P. falciparum parasite protein by to the that the within are to a protein identified as M. Y. MacDonald L. Miller L.H. of the Plasmodium Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). the to recombinant proteins with a in a suitable for and we to a and recombinant protein of suitable and for human clinical and were we report on the identification, and characterization of an E. expressed and Pfs230 protein and a Pfs230 protein of domains or domain of recombinant Pfs230 induced transmission blocking antibodies in however, on the P. protein was effective transmission blocking a a protein without identified as was and is a of a transmission blocking vaccine. by et Keister Muratova O. Pfs230, a Plasmodium falciparum protein, that the of Plasmodium falciparum to PubMed Scopus Google identified a natural of Pfs230 domain which was used as the for the The for the on the expression In P. the recombinant protein the domain domain 1 and in E. the the domain domain recombinant and proteins a to A recombinant of Pfs230, was to the protein for the of the The E. expressed protein was and as the with the as by demonstrated the of as by a by by size exclusion with light and demonstrated that the protein was in with an of and by was as a and the P. expression was to a recombinant protein that the domain domain 1 the shown in by was on and was with an with This a of a on a was such that the and protein were with the for the The recombinant protein, identified as the and a to the of the The was with the in the of was of with the of two The demonstrated that and secondary The secondary structure of using was to and that and were in with of and and not of by a for recombinant protein and not we the of a and a in the secondary structure using A in the was and and and to the secondary structure appeared to the the is suitable for a vaccine the of recombinant the and protein were also The were of protein and the of in for the P. expressed proteins using a assay were D. Saul Miles A.P. A assay for protein in recombinant proteins expressed in E. PubMed Scopus Google Scholar). the recombinant a of antibodies were against P. and for against and recombinant of identified as both and recombinant of Pfs230 by and The was a of with by and identified for Pfs230 not also Pfs230 on the surface of by A Pfs25-specific was in the study as a positive for the assay antibody were for parasite not mosquito in the ex vivo by to with a in parasite as the of mosquitoes with the of mosquitoes with or without human The Pfs25-specific which blocked mosquito transmission by was as a positive L. Wu Y. A. Muratova Song G. Fay M.P. Miller L.H. Long C.A. K. The of antibody in assay PubMed Scopus Google membrane feeding assay results for and Pfs25-specific and is shown as an assay using a of which to of was of without human with or without human not with human with or without human with or without human Pfs25-specific is shown as an assay using a of which to of was of with or without human not in a were with the recombinant of Pfs230 formulated in a water-in-oil adjuvant The generated against of the Pfs230 proteins Pfs230 on the surface of by using a 3 or not or a standard as shown for the the was with the Pfs25-specific In this Pfs230 was shown to with Pfs25 on the surface of a sexual stage likely a young were for transmission blocking using the ex vivo The results with the antibody as by and as a an are in from of generated transmission blocking a to to the of and a that induced an enhanced level of blocking This was by in which antibodies against a of or to of not the and induced by were shown to for J. D. P. S. R. E. of Plasmodium falciparum by human is with antibodies to the surface PubMed Google Scholar), human was for blocking using or in the of human a blocking not the significant in generated by the recombinant the recombinant protein was for membrane feeding assay results for generated against and with human as an not of are from the as of in adjuvant and of are from the as of in adjuvant and of are from the as of in adjuvant and of are from the as of in adjuvant and of are from the as of in adjuvant and of are from the as of in adjuvant and of are from the as of in adjuvant and of are from the as of in adjuvant and of and are from a as the of in adjuvant and of and are from a as the of in adjuvant and of and are from a as the of in adjuvant 51, and of and are from a as the of in adjuvant 51, and of and are from a as the of in adjuvant 51, and of and are from a as the of in adjuvant 51, and as an not of are from the as of in adjuvant and of are from the as of in adjuvant and of and are from a as the of in adjuvant and of and are from a as the of in adjuvant 51, and in a evaluate the blocking of the was for blocking transmission of isolates of P. falciparum isolates of P. falciparum from were with or and to mosquitoes and the was using a membrane feeding assay. shown in 3, or nearly blocked a of from blood from the two the was and A which is was in both to a to of Pfs230 feeding assay results using human P. falciparum with in a a of the of the protein, the of were using and by The structure was identified as to and to was with not Pfs230 is expressed in gametocytes and is known to the of in for in the P. falciparum M. O. S. S. J. G. J. A. O. P. P. A. et of Plasmodium falciparum in natural by PubMed Scopus Google Scholar), which a of P. falciparum malaria. two with a were and with of and In the in in the for and were and with the the and structure of a was using the of and as The secondary structure of the and is with the secondary structure by The of the are shown on a surface of the two for and in to a of are to on the of the of domain 1 up of and The evaluation of Pfs230 as a transmission blocking vaccine candidate for investigational human has not been The was to an to a recombinant of Pfs230 with the and for human a of that the of proteins not we report on the expression and characterization of Pfs230 this two expression were on by et Keister Muratova O. Pfs230, a Plasmodium falciparum protein, that the of Plasmodium falciparum to PubMed Scopus Google and recently by et M. Wu Y. H. Muratova O. MacDonald J. S. H. M. of Pfs230 using a is to malaria 2011; PubMed Scopus Google Scholar). a E. Pfs230 protein of the domain domain and using the P. a recombinant protein was that the domain domain 1 proteins a to The expression the which was in P. as was by that the was of a of This was with the of by and by the of appeared and was used for immunological antibodies generated in were shown to P. falciparum transmission in a in a to SMFA, antibodies were to a blocking was a in the domain the and antibody responses by on a by was used to a P. expression such that the was in with the protein, and was to the of were in the expressed appeared and to with the of the In the two within were and as to and to which was in with the of recombinant malaria protein with a 6-cysteine-rich N. Miller L.H. and characterization of Plasmodium falciparum a unique and the potential for an with Full Text Full Text PDF PubMed Scopus Google Scholar). is to that the is to the for and N. Miller L.H. and characterization of Plasmodium falciparum a unique and the potential for an with Full Text Full Text PDF PubMed Scopus Google Scholar, F. The of Plasmodium falciparum an for ONE. 2015; PubMed Scopus Google Scholar). preclinical the P. protein appeared to the E. expressed and the protein a of The for this is protein was and was by with which a within Pfs230 domain 1 demonstrated a for the of by against the that is a of protein this the for the shown alternative that the domain domain with to domain 1 such that the development of antibodies against domain demonstrated that blocking transmission by F. P. B. N. R. of Plasmodium falciparum malaria by antibodies is PubMed Google Scholar, D. S. S. A. R. antibodies against of the Plasmodium falciparum surface Pfs230 are PubMed Scopus Google and human antibodies Pfs230 J. D. P. S. R. E. of Plasmodium falciparum by human is with antibodies to the surface PubMed Google Scholar). This is the report that a with an which not by the A. N. J. M. K. D. M. protects against disease in a of 2015; PubMed Scopus Google transmission in the or of human This that the of Pfs230 by the of an complex or with the of Pfs230 protein N. A. G. protein of malaria gametocytes parasite transmission to the PubMed Scopus Google Scholar). human antibodies have been low in from malaria vaccine candidate toward PubMed Scopus Google Scholar, S. L. J. D. R. M. S. E. K. C. antibody responses to recombinant Pfs230 and transmission blocking vaccine 2015; Full Text Full Text PDF PubMed Scopus Google Scholar). Pfs230 Pfs230 is expressed This a for the of Pfs230 in a TBV to blocking Pfs230 has been to a of in Plasmodium falciparum J. Med. PubMed Scopus Google Scholar), TBV candidate and Pfs25, which is not expressed in to the in the which are by field for that the are to with phase 1 trial in a of isolates from and in with ex vivo by membrane feeding with a parasite clinical have been with recombinant Pfs25 to evaluate safety and and this the of Pfs230 clinical In the trial, recombinant Pfs25 Duggan Miller L.H. Narum D.L. of or Plasmodium falciparum protein and of a malaria vaccine candidate expressed in P. 2006; PubMed Scopus Google was formulated in Montanide (3Wu Y. Ellis R.D. Shaffer D. Fontes E. Malkin E.M. Mahanty S. Fay M.P. Narum D. Rausch K. Miles A.P. Aebig J. Orcutt A. Muratova O. Song G. Lambert L. et al.Phase 1 trial of malaria transmission blocking vaccine candidates Pfs25 and Pvs25 formulated with montanide ISA 51.PLoS ONE. 2008; 3: e2636Crossref PubMed Scopus (310) Google Scholar). Human Pfs25-specific transmission blocking antibodies were as shown by the ex vivo SMFA, however, the formulation was to for a public health vaccine to its A human study a that was shown to have the of a C. Reiter K. B. Aebig J. Rausch K. Wu Y. Narum D.L. Development of a malaria transmission blocking vaccine as a chemically conjugated PubMed Scopus Google Scholar). In the the formulated on was safe and of the vaccine, significant was of as by the ex vivo chemically conjugated to or in with a formulated on in is in a phase 1 study only two of in in the two with antibody responses also the in the this size was for not A. A. C. K. D. L. D. L. N. J. D. S. J. O. J. C. S. E. Y. and P. E. the development of the potential for the of a vaccine to malaria The study for blood from was by the from the of and the of used for this were by the of and of and Use the of which is and The and Use and for the and of in by The of the Pfs230 protein was used to the and to to and to to to the The were the of a of in which the been to an The a the in of protein that and protein that the The expression were with and P. The E. expressed protein to was by a for expression in E. The was the and of of the The expression was the E. expression for recombinant E. expressed protein the was and O. R. Reiter K. S. K. D. J. Long C. Miller L.H. Narum D.L. of the and of the Plasmodium falciparum proteins and PubMed Scopus Google Scholar). In was in 1 and by using and The and were as Duggan Miller L.H. Narum D.L. of or Plasmodium falciparum protein and of a malaria vaccine candidate expressed in P. 2006; PubMed Scopus Google with the that the used were and of for to was from and in with to using a in and with a of or a step with was and using a with with and to a in the was with an of was The was a step with The was and in for a in the The from the in 1 The was to a to for a size exclusion The size exclusion was in The protein were and a The was recombinant protein was using to that O. R. Reiter K. S. K. D. J. Long C. Miller L.H. Narum D.L. of the and of the Plasmodium falciparum proteins and PubMed Scopus Google Scholar, R. C. K. A. M. Reiter K. R. P. D. Shi L. M. et in the Plasmodium falciparum circumsporozoite protein its 2015; PubMed Scopus Google for and with and light was on a and were by the was on a as O. R. Reiter K. S. K. D. J. Long C. Miller L.H. Narum D.L. of the and of the Plasmodium falciparum proteins and PubMed Scopus Google Scholar). was on an as O. R. Reiter K. S. K. D. J. Long C. Miller L.H. Narum D.L. of the and of the Plasmodium falciparum proteins and PubMed Scopus Google Scholar). was on a as Reiter K. S. B. Zhang Y. M. O. R. J. J. et of the Plasmodium falciparum circumsporozoite protein, a leading malaria vaccine Full Text Full Text PDF PubMed Scopus Google Scholar). structure was using the L. secondary structure from and 2008; PubMed Scopus Google Scholar). The of was identified using in with a was to of a final of and to the protein was for The was to the blocked by with for in the for the a without was with a in with were with was used to the and the The of the was to by of 1 for effective on the were with the for was on in positive using a controlled by were on the and to a with and for a of from the were and with in to the The were on the with a from 3 to phase in low and from to was used to and were a of 1 The was and of the was were from to were in were and against Pfs230 protein using identified the with The of identified was within were with were and with of or formulated in ISA were for on and for Rabbit used as by the were in The the blocking of parasite transmission of P. falciparum in and membrane feeding assay using isolates from were as L. Wu Y. A. Muratova Song G. Fay M.P. Miller L.H. Long C.A. K. The of antibody in assay PubMed Scopus Google Scholar). The used for Pfs230 the for Pfs25 K. Keister Muratova J. Long C.A. Saul A. induced by malaria vaccine candidates is a and of antibody J. PubMed Scopus Google Scholar). were by by with of P. falciparum for development were by against gametocytes and by using the recombinant Pfs230 assay was as S. B. Pfs230, human blood to and 2006; PubMed Scopus Google Scholar). In were in by with antibody or for for with secondary and in for the surface the has been using R. C. K. A. M. Reiter K. R. P. D. Shi L. M. et in the Plasmodium falciparum circumsporozoite protein its 2015; PubMed Scopus Google Scholar). In the gametocytes or were the antibody and on and were and with and secondary antibodies in blocking and secondary antibody were by with The were using and were by using an or as O. R. Reiter K. S. K. D. J. Long C. Miller L.H. Narum D.L. of the and of the Plasmodium falciparum proteins and PubMed Scopus Google Scholar, R. C. K. A. M. Reiter K. R. P. D. Shi L. M. et in the Plasmodium falciparum circumsporozoite protein its 2015; PubMed Scopus Google Scholar). The of was with Y. F. D. Miles C. J. D. with Full Text Full Text PDF PubMed Scopus Google using the of N. Miller L.H. and characterization of Plasmodium falciparum a unique and the potential for an with Full Text Full Text PDF PubMed Scopus Google Scholar), and F. The of Plasmodium falciparum an for ONE. 2015; PubMed Scopus Google Scholar), as structure was with D. P. protein secondary structure PubMed Scopus Google Scholar). The of the two P. falciparum isolates were blocked by was The was and from from blood using and and was by The of the transmission blocking was using a on of mosquitoes, which potential within of mosquitoes K. B. G. A. E. M. Fay M.P. Long C.A. of standard assay with Plasmodium falciparum malaria and potential for ONE. PubMed Scopus Google Scholar). N. J. M. K. R. R. K. O. J. L. K. N. J. Y. J. and D. and N. J. M. K. R. R. K. O. J. J. P. E. Y. J. and D. L. N. N. J. K. K. and D. L. N. the and for within the of and from for and from for from for with studies, and for the with of Pfs230 from the we Miller for and This from the as in M. O. S. S. J. G. J. A. O. P. P. A. et of Plasmodium falciparum in natural by PubMed Scopus Google was by the and the is by the with from the and