scholarly journals Antigenic Characterization of an Intrinsically Unstructured Protein, Plasmodium falciparum Merozoite Surface Protein 2

2012 ◽  
Vol 80 (12) ◽  
pp. 4177-4185 ◽  
Author(s):  
Christopher G. Adda ◽  
Christopher A. MacRaild ◽  
Linda Reiling ◽  
Kaye Wycherley ◽  
Michelle J. Boyle ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Surface Protein ◽  
Variable Region ◽  
Intrinsic Property ◽  
Merozoite Surface Protein ◽  
Surface Proteins ◽  
Structural Basis ◽  
Content Type ◽  
Intrinsically Unstructured Protein ◽  
Unstructured Protein

ABSTRACTMerozoite surface protein 2 (MSP2) is an abundant glycosylphosphatidylinositol (GPI)-anchored protein ofPlasmodium falciparum, which is a potential component of a malaria vaccine. As all forms of MSP2 can be categorized into two allelic families, a vaccine containing two representative forms of MSP2 may overcome the problem of diversity in this highly polymorphic protein. Monomeric recombinant MSP2 is an intrinsically unstructured protein, but its conformational properties on the merozoite surface are unknown. This question is addressed here by analyzing the 3D7 and FC27 forms of recombinant and parasite MSP2 using a panel of monoclonal antibodies raised against recombinant MSP2. The epitopes of all antibodies, mapped using both a peptide array and by nuclear magnetic resonance (NMR) spectroscopy on full-length recombinant MSP2, were shown to be linear. The antibodies revealed antigenic differences, which indicate that the conserved N- and C-terminal regions, but not the central variable region, are less accessible in the parasite antigen. This appears to be an intrinsic property of parasite MSP2 and is not dependent on interactions with other merozoite surface proteins as the loss of some conserved-region epitopes seen using the immunofluorescence assay (IFA) on parasite smears was also seen on Western blot analyses of parasite lysates. Further studies of the structural basis of these antigenic differences are required in order to optimize recombinant MSP2 constructs being evaluated as potential vaccine components.

scholarly journals Production and Preclinical Evaluation of Plasmodium falciparum MSP-119and MSP-311Chimeric Protein, PfMSP-Fu24

2014 ◽  
Vol 21 (6) ◽  
pp. 886-897 ◽  
Author(s):  
Puneet K. Gupta ◽  
Paushali Mukherjee ◽  
Shikha Dhawan ◽  
Alok K. Pandey ◽  
Suman Mazumdar ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Malaria Vaccine ◽  
Chimeric Protein ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Surface Proteins ◽  
Content Type ◽  
Freund’S Adjuvant ◽  
Freund's Adjuvant

ABSTRACTAPlasmodium falciparumchimeric protein, PfMSP-Fu24, was constructed by genetically coupling immunodominant, conserved regions of two merozoite surface proteins, the 19-kDa region C-terminal region of merozoite surface protein 1 (PfMSP-119) and an 11-kDa conserved region of merozoite surface protein 3 (PfMSP-311), to augment the immunogenicity potential of these blood-stage malaria vaccine candidates. Here we describe an improved, efficient, and scalable process to produce high-quality PfMSP-Fu24. The chimeric protein was produced inEscherichia coliSHuffle T7 ExpresslysYcells that express disulfide isomerase DsbC. A two-step purification process comprising metal affinity followed by cation exchange chromatography was developed, and we were able to obtain PfMSP-Fu24with purity above 99% and with a considerable yield of 23 mg/liter. Immunogenicity of PfMSP-Fu24formulated with several adjuvants, including Adjuplex, Alhydrogel, Adjuphos, Alhydrogel plus glucopyranosyl lipid adjuvant, aqueous (GLA-AF), Adjuphos+GLA-AF, glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE), and Freund's adjuvant, was evaluated. PfMSP-Fu24formulated with GLA-SE and Freund's adjuvant in mice and with Alhydrogel and Freund's adjuvant in rabbits produced high titers of PfMSP-119and PfMSP-311-specific functional antibodies. Some of the adjuvant formulations induced inhibitory antibody responses and inhibitedin vitrogrowth ofP. falciparumparasites in the presence as well as in the absence of human monocytes. These results suggest that PfMSP-Fu24can form a constituent of a multistage malaria vaccine.

scholarly journals Comparative Immunogenicities of Full-Length Plasmodium falciparum Merozoite Surface Protein 3 and a 24-Kilodalton N-Terminal Fragment

2011 ◽  
Vol 18 (8) ◽  
pp. 1221-1228 ◽  
Author(s):  
Maryam Imam ◽  
Yengkhom Sangeeta Devi ◽  
Akhilesh K. Verma ◽  
Virander Singh Chauhan
Keyword(s):  
Plasmodium Falciparum ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Terminal Region ◽  
Content Type ◽  
Conserved Domain ◽  
Terminal Fragment ◽  
Falciparum Merozoite ◽  
Form Part ◽  
Parasite Lysate

ABSTRACTRecombinantPlasmodium falciparummerozoite surface protein 3 (PfMSP3F) and a 24-kDa fragment from its N terminus (MSP3N) that includes the essential conserved domain, which elicits the maximum antibody (Ab)-dependent cellular inhibition (ADCI), were expressed as soluble proteins inEscherichia coli. Both proteins were found to be stable in both soluble and lyophilized forms. Immunization with MSP3F and MSP3N formulated separately with two human-compatible adjuvants, aluminum hydroxide (Alhydrogel) and Montanide ISA 720, produced significant antibody responses in mice and rabbits. Polyclonal Abs against both antigens recognized native MSP3 in the parasite lysate. These two Abs also recognized two synthetic peptides, previously characterized to possess B cell epitopes from the N-terminal region. Antibody depletion assay showed that most of the IgG response is directed toward the N-terminal region of the full protein. Anti-MSP3F and anti-MSP3N rabbit antibodies did not inhibit merozoite invasion or intraerythrocytic development but significantly reduced parasitemia in the presence of human monocytes. The ADCI demonstrated by anti-MSP3N antibodies was comparable to that exhibited by anti-MSP3F antibodies (both generated in rabbit). These results suggest that the N-terminal fragment of MSP3 can be considered a vaccine candidate that can form part of a multigenic vaccine against malaria.

scholarly journals Systematic Genetic Analysis of the Plasmodium falciparum MSP7-Like Family Reveals Differences in Protein Expression, Location, and Importance in Asexual Growth of the Blood-Stage Parasite

2010 ◽  
Vol 9 (7) ◽  
pp. 1064-1074 ◽  
Author(s):  
Madhusudan Kadekoppala ◽  
Solabomi A. Ogun ◽  
Steven Howell ◽  
Ruwani S. Gunaratne ◽  
Anthony A. Holder
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Expression ◽  
Surface Protein ◽  
Gene Families ◽  
Merozoite Surface Protein ◽  
Blood Stage ◽  
Asexual Blood Stage ◽  
Content Type ◽  
Specific Proteins ◽  
Proteolytic Cleavages

ABSTRACT Proteins located on Plasmodium falciparum merozoites, the invasive form of the parasite's asexual blood stage, are of considerable interest in vaccine research. Merozoite surface protein 7 (MSP7) forms a complex with MSP1 and is encoded by a member of a multigene family located on chromosome 13. The family codes for MSP7 and five MSP7-related proteins (MSRPs). In the present study, we have investigated the expression and the effect of msrp gene deletion at the asexual blood stage. In addition to msp7, msrp2, msrp3, and msrp5 are transcribed, and mRNA was easily detected by hybridization analysis, whereas mRNA for msrp1 and msrp4 could be detected only by reverse transcription (RT)-PCR. Notwithstanding evidence of transcription, antibodies to recombinant MSRPs failed to detect specific proteins, except for antibodies to MSRP2. Sequential proteolytic cleavages of MSRP2 resulted in 28- and 25-kDa forms. However, MSRP2 was absent from merozoites; the 25-kDa MSRP2 protein (MSRP225) was soluble and secreted upon merozoite egress. The msrp genes were deleted by targeted disruption in the 3D7 line, leading to ablation of full-length transcripts. MSRP deletion mutants had no detectable phenotype, with growth and invasion characteristics comparable to those of the parental parasite; only the deletion of MSP7 led to a detectable growth phenotype. Thus, within this family some of the genes are transcribed at a significant level in asexual blood stages, but the corresponding proteins may or may not be detectable. Interactions of the expressed proteins with the merozoite also differ. These results highlight the potential for unexpected differences of protein expression levels within gene families.

scholarly journals Factors Associated with Immunoglobulin G Subclass Polarization in Naturally Acquired Antibodies to Plasmodium falciparum Merozoite Surface Proteins: a Cross-Sectional Survey in Brazilian Amazonia

2006 ◽  
Vol 13 (7) ◽  
pp. 810-813 ◽  
Author(s):  
Kézia K. G. Scopel ◽  
Cor J. F. Fontes ◽  
Marcelo U. Ferreira ◽  
Érika M. Braga
Keyword(s):  
Plasmodium Falciparum ◽  
Immunoglobulin G ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Surface Proteins ◽  
Cross Sectional Survey ◽  
Cross Sectional ◽  
Falciparum Merozoite ◽  
Immunoglobulin G Subclass ◽  
Current Exposure

ABSTRACT We investigated immunoglobulin G (IgG) subclass antibody responses to Plasmodium falciparum merozoite surface protein 1 (MSP-1) and MSP-2 in 112 malaria-exposed subjects in Brazil. IgG3 polarization was primarily epitope driven, being little affected by cumulative or current exposure to malaria and not affected by a subject's age and Fcγ receptor IIA genotype.

scholarly journals Plasmodium falciparum Malaria in the Peruvian Amazon, a Region of Low Transmission, Is Associated with Immunologic Memory

2012 ◽  
Vol 80 (4) ◽  
pp. 1583-1592 ◽  
Author(s):  
Eva H. Clark ◽  
Claudia J. Silva ◽  
Greta E. Weiss ◽  
Shanping Li ◽  
Carlos Padilla ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Surface Protein ◽  
Plasmodium Falciparum Malaria ◽  
Merozoite Surface Protein ◽  
Content Type ◽  
Low Transmission ◽  
Apical Membrane Antigen 1 ◽  
Specific Memory ◽  
Erythrocyte Binding ◽  
Transmission Region

ABSTRACTThe development of clinical immunity toPlasmodium falciparummalaria is thought to require years of parasite exposure, a delay often attributed to difficulties in developing protective antibody levels. In this study, we evaluated severalP. falciparumvaccine candidate antigens, including apical membrane antigen 1 (AMA-1), circumsporozoite protein (CSP), erythrocyte binding antigen 175 (EBA-175), and the 19-kDa region of merozoite surface protein 1 (MSP119). After observing a more robust antibody response to MSP119, we evaluated the magnitude and longevity of IgG responses specific to this antigen in Peruvian adults and children before, during, and afterP. falciparuminfection. In this low-transmission region, even one reported prior infection was sufficient to produce a positive anti-MSP119IgG response for >5 months in the absence of reinfection. We also observed an expansion of the total plasmablast (CD19+CD27+CD38high) population in the majority of individuals shortly after infection and detected MSP1-specific memory B cells in a subset of individuals at various postinfection time points. This evidence supports our hypothesis that effective antimalaria humoral immunity can develop in low-transmission regions.

scholarly journals Deletion of the Plasmodium falciparum Merozoite Surface Protein 7 Gene Impairs Parasite Invasion of Erythrocytes

2008 ◽  
Vol 7 (12) ◽  
pp. 2123-2132 ◽  
Author(s):  
Madhusudan Kadekoppala ◽  
Rebecca A. O'Donnell ◽  
Munira Grainger ◽  
Brendan S. Crabb ◽  
Anthony A. Holder
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Interactions ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Significant Degree ◽  
Surface Proteins ◽  
Transcriptional Level ◽  
Parental Line ◽  
Parasite Line ◽  
Asexual Blood Stage

ABSTRACT Merozoite surface proteins have been implicated in the initial attachment to the host red blood cell membrane that begins the process of invasion, an important step in the life cycle of the malaria parasite. In Plasmodium falciparum, merozoite surface proteins include several glycosylphosphatidyl inositol-anchored proteins and peripheral proteins attached to the membrane through protein-protein interactions. The most abundant of these proteins is the merozoite surface protein 1 (MSP1) complex, encoded by at least three genes: msp1, msp6, and msp7. The msp7 gene is part of a six-member multigene family in Plasmodium falciparum. We have disrupted msp7 in the Plasmodium falciparum D10 parasite, as confirmed by Southern hybridization. Immunoblot and indirect immunofluorescence analyses confirmed the MSP7 null phenotype of D10ΔMSP7 parasites. The synthesis, distribution, and processing of MSP1 were not affected in this parasite line. The level of expression and cellular distribution of the proteins MSP1, MSP3, MSP6, MSP9, and SERA5 remained comparable to those for the parental line. Furthermore, no significant change in the expression of MSP7-related proteins, except for that of MSRP5, was detected at the transcriptional level. The lack of MSP7 was not lethal at the asexual blood stage, but it did impair invasion of erythrocytes by merozoites to a significant degree. Despite this reduction in efficiency, D10ΔMSP7 parasites did not show any obvious preference for alternate pathways of invasion.

scholarly journals A Chimeric Plasmodium falciparum Merozoite Surface Protein Vaccine Induces High Titers of Parasite Growth Inhibitory Antibodies

2013 ◽  
Vol 81 (10) ◽  
pp. 3843-3854 ◽  
Author(s):  
James R. Alaro ◽  
Andrea Partridge ◽  
Kazutoyo Miura ◽  
Ababacar Diouf ◽  
Ana M. Lopez ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
T Cell ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Plasmodium Yoelii ◽  
T Cell Response ◽  
Blood Stage ◽  
Fusion Partner ◽  
Content Type ◽  
Cpg Oligodeoxynucleotide

ABSTRACTThe C-terminal 19-kDa domain ofPlasmodium falciparummerozoite surface protein 1 (PfMSP119) is an established target of protective antibodies. However, clinical trials ofPfMSP142, a leading blood-stage vaccine candidate which contains the protective epitopes ofPfMSP119, revealed suboptimal immunogenicity and efficacy. Based on proof-of-concept studies in thePlasmodium yoeliimurine model, we produced a chimeric vaccine antigen containing recombinantPfMSP119(rPfMSP119) fused to the N terminus ofP. falciparummerozoite surface protein 8 that lacked its low-complexity Asn/Asp-rich domain, rPfMSP8 (ΔAsn/Asp). Immunization of mice with the chimeric rPfMSP1/8 vaccine elicited strong T cell responses to conserved epitopes associated with the rPfMSP8 (ΔAsn/Asp) fusion partner. While specific forPfMSP8, this T cell response was adequate to provide help for the production of high titers of antibodies to bothPfMSP119and rPfMSP8 (ΔAsn/Asp) components. This occurred with formulations adjuvanted with either Quil A or with Montanide ISA 720 plus CpG oligodeoxynucleotide (ODN) and was observed in both inbred and outbred strains of mice.PfMSP1/8-induced antibodies were highly reactive with two major alleles ofPfMSP119(FVO and 3D7). Of particular interest, immunization withPfMSP1/8 elicited higher titers ofPfMSP119-specific antibodies than a combined formulation of rPfMSP142and rPfMSP8 (ΔAsn/Asp). As a measure of functionality,PfMSP1/8-specific rabbit IgG was shown to potently inhibit thein vitrogrowth of blood-stage parasites of the FVO and 3D7 strains ofP. falciparum. These data support the further testing and evaluation of this chimericPfMSP1/8 antigen as a component of a multivalent vaccine forP. falciparummalaria.

scholarly journals Malaria Immunoepidemiology in Low Transmission: Correlation of Infecting Genotype and Immune Response to Domains of Plasmodium falciparum Merozoite Surface Protein 3

2011 ◽  
Vol 79 (5) ◽  
pp. 2070-2078 ◽  
Author(s):  
Stephen J. Jordan ◽  
Ana L. Oliveira ◽  
Jean N. Hernandez ◽  
Robert A. Oster ◽  
Debasish Chattopadhyay ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Effective Vaccine ◽  
Serum Samples ◽  
Content Type ◽  
Low Transmission ◽  
Rational Development ◽  
Time Of Infection ◽  
Potential Vaccine

ABSTRACTMalaria caused byPlasmodium falciparumis a major cause of global infant mortality, and no effective vaccine currently exists. Multiple potential vaccine targets have been identified, and immunoepidemiology studies have played a major part in assessing those candidates. When such studies are carried out in high-transmission settings, individuals are often superinfected with complex mixtures of genetically distinctP. falciparumtypes, making it impossible to directly correlate the genotype of the infecting antigen with the antibody response. In contrast, in regions of low transmissionP. falciparuminfections are often genetically simple, and direct comparison of infecting genotype and antigen-specific immune responses is possible. As a test of the utility of this approach, responses against several domains and allelic variants of the vaccine candidateP. falciparummerozoite surface protein 3 (PfMSP3) were tested in serum samples collected near Iquitos, Peru. Antibodies recognizing both the conserved C-terminal and the more variable N-terminal domain were identified, but anti-N-terminal responses were more prevalent, of higher titers, and primarily of cytophilic subclasses. Comparing antibody responses to different PfMSP3 variants with thePfMSP3genotype present at the time of infection showed that anti-N-terminal responses were largely allele class specific, but there was some evidence for responses that cross-reacted across allele classes. Evidence for cross-reactive responses was much stronger when variants within one allele class were tested, which has implications for the rational development of genotype-transcending PfMSP3-based vaccines.

Interactions with heparin-like molecules during erythrocyte invasion by Plasmodium falciparum merozoites

Blood ◽  
2010 ◽  
Vol 115 (22) ◽  
pp. 4559-4568 ◽  
Author(s):  
Michelle J. Boyle ◽  
Jack S. Richards ◽  
Paul R. Gilson ◽  
Wengang Chai ◽  
James G. Beeson
Keyword(s):  
Plasmodium Falciparum ◽  
Current Knowledge ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Surface Proteins ◽  
Invasion Pathways ◽  
Erythrocyte Invasion ◽  
Ligand Interactions ◽  
Processed Form ◽  
Multiple Receptor

AbstractDuring erythrocyte invasion, Plasmodium falciparum merozoites use multiple receptor-ligand interactions in a series of coordinated events, but current knowledge of these interactions is limited. Using real-time imaging of invasion, we established that heparin-like molecules block early, and essential, events in erythrocyte invasion by merozoites. All P falciparum isolates tested, and parasites using different invasion pathways were inhibited to comparable levels. Furthermore, it was not possible to select for heparin-resistant parasites. Heparin-like molecules occur naturally on the surface of human erythrocytes, where they may act as receptors for binding of merozoite surface proteins. Consistent with this, we demonstrated that MSP1-42, a processed form of merozoite surface protein 1 (MSP1) involved in invasion, bound heparin in a specific manner; furthermore, binding was observed with the secondary processing fragment MSP1-33, but not MSP1-19. We defined key structural requirements of heparin-like molecules for invasion inhibition and interactions with MSP1-42. Optimal activity required a degree of sulfation more than or equal to 2, disulfation of the N-acetylglucosamine or hexuronic acid residue, and a minimum chain length of 6 monosaccharides. These findings have significant implications for understanding P falciparum invasion of erythrocytes and the development of novel therapeutics and vaccines.

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