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1.
Front Immunol ; 15: 1360220, 2024.
Article in English | MEDLINE | ID: mdl-38650925

ABSTRACT

Background: Malaria remains a major global health priority, and monoclonal antibodies (mAbs) are emerging as potential new tools to support efforts to control the disease. Recent data suggest that Fc-dependent mechanisms of immunity are important mediators of protection against the blood stages of the infection, but few studies have investigated this in the context of mAbs. We aimed to isolate mAbs agnostic to cognate antigens that target whole merozoites and simultaneously induce potent neutrophil activity measured by the level of reactive oxygen species (ROS) production using an antibody-dependent respiratory burst (ADRB) assay. Methods: We used samples from semi-immune adults living in coastal Kenya to isolate mAbs that induce merozoite-specific ADRB activity. We then tested whether modifying the expressed IgG1 isotype to an IgG-IgA Fc region chimera would enhance the level of ADRB activity. Results: We isolated a panel of nine mAbs with specificity to whole merozoites. mAb J31 induced ADRB activity in a dose-dependent fashion. Compared to IgG1, our modified antibody IgG-IgA bi-isotype induced higher ADRB activity across all concentrations tested. Further, we observed a negative hook effect at high IgG1 mAb concentrations (i.e., >200 µg/mL), but this was reversed by Fc modification. We identified MSP3.5 as the potential cognate target of mAb J31. Conclusions: We demonstrate an approach to engineer mAbs with enhanced ADRB potency against blood-stage parasites.


Subject(s)
Antibodies, Monoclonal , Antibodies, Protozoan , Malaria, Falciparum , Merozoites , Neutrophils , Plasmodium falciparum , Plasmodium falciparum/immunology , Humans , Antibodies, Protozoan/immunology , Neutrophils/immunology , Neutrophils/metabolism , Malaria, Falciparum/immunology , Malaria, Falciparum/parasitology , Antibodies, Monoclonal/immunology , Merozoites/immunology , Respiratory Burst/immunology , Immunoglobulin G/immunology , Adult , Reactive Oxygen Species/metabolism , Kenya , Immunoglobulin Isotypes/immunology , Neutrophil Activation/immunology , Female , Antigens, Protozoan/immunology
2.
Sci Rep ; 12(1): 3040, 2022 02 23.
Article in English | MEDLINE | ID: mdl-35197516

ABSTRACT

The pathogenesis of malaria is associated with blood-stage infection and there is strong evidence that antibodies specific to parasite blood-stage antigens can control parasitemia. This provides a strong rational for applying blood-stage antigen components in a multivalent vaccine, as the induced antibodies in combination can enhance protection. The Plasmodium falciparum rhoptry-associated membrane antigen (PfRAMA) is a promising vaccine target, due to its fundamental role in merozoite invasion and low level of polymorphism. Polyclonal antibodies against PfRAMA are able to inhibit P. falciparum growth and interact synergistically when combined with antibodies against P. falciparum reticulocyte-binding protein 5 (PfRh5) or cysteine-rich protective antigen (PfCyRPA). In this study, we identified a novel PfRAMA-specific mAb with neutralizing activity, which in combination with PfRh5- or PfCyRPA-specific mAbs potentiated the neutralizing effect. By applying phage display technology, we mapped the protective epitope to be in the C-terminal region of PfRAMA. Our results confirmed previous finding of synergy between PfRAMA-, PfRh5- and PfCyRPA-specific antibodies, thereby paving the way of testing these antigens (or fragments of these antigens) in combination to improve the efficacy of blood-stage malaria vaccines. The results emphasize the importance of directing antibody responses towards protective epitopes, as the majority of anti-PfRAMA mAbs were unable to inhibit merozoite invasion of erythrocytes.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Protozoan/immunology , Malaria Vaccines/immunology , Plasmodium falciparum/immunology , Protozoan Proteins/immunology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Neutralizing/biosynthesis , Antibodies, Neutralizing/isolation & purification , Antibodies, Protozoan/chemistry , Antigens, Protozoan/immunology , Carrier Proteins/immunology , Cell Line , Drug Synergism , Epitopes/chemistry , Epitopes/immunology , Humans , Malaria Vaccines/chemistry , Malaria, Falciparum/prevention & control , Merozoites/immunology , Mice , Protein Binding , Protozoan Proteins/biosynthesis , Protozoan Proteins/chemistry , Protozoan Proteins/isolation & purification
3.
Sci Rep ; 11(1): 19183, 2021 09 28.
Article in English | MEDLINE | ID: mdl-34584166

ABSTRACT

Plasmodium falciparum, the parasite responsible for severe malaria, develops within erythrocytes. Merozoite invasion and subsequent egress of intraerythrocytic parasites are essential for this erythrocytic cycle, parasite survival and pathogenesis. In the present study, we report the essential role of a novel protein, P. falciparum Merozoite Surface Antigen 180 (PfMSA180), which is conserved across Plasmodium species and recently shown to be associated with the P. vivax merozoite surface. Here, we studied MSA180 expression, processing, localization and function in P. falciparum blood stages. Initially we examined its role in invasion, a process mediated by multiple ligand-receptor interactions and an attractive step for targeting with inhibitory antibodies through the development of a malaria vaccine. Using antibodies specific for different regions of PfMSA180, together with a parasite containing a conditional pfmsa180-gene knockout generated using CRISPR/Cas9 and DiCre recombinase technology, we demonstrate that this protein is unlikely to play a crucial role in erythrocyte invasion. However, deletion of the pfmsa180 gene resulted in a severe egress defect, preventing schizont rupture and blocking the erythrocytic cycle. Our study highlights an essential role of PfMSA180 in parasite egress, which could be targeted through the development of a novel malaria intervention strategy.


Subject(s)
Antigens, Protozoan/metabolism , Antigens, Surface/metabolism , Malaria, Falciparum/parasitology , Plasmodium falciparum/pathogenicity , Protozoan Proteins/metabolism , Animals , Antigens, Protozoan/genetics , Antigens, Surface/genetics , Disease Models, Animal , Erythrocytes/parasitology , Gene Knockout Techniques , Humans , Malaria Vaccines/therapeutic use , Malaria, Falciparum/blood , Malaria, Falciparum/immunology , Malaria, Falciparum/prevention & control , Merozoites/genetics , Merozoites/immunology , Merozoites/metabolism , Mice , Plasmodium falciparum/immunology , Plasmodium falciparum/metabolism , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/genetics , Rabbits , Vaccine Development
4.
J Exp Med ; 218(9)2021 09 06.
Article in English | MEDLINE | ID: mdl-34342640

ABSTRACT

We previously identified a Plasmodium falciparum (Pf) protein of unknown function encoded by a single-copy gene, PF3D7_1134300, as a target of antibodies in plasma of Tanzanian children in a whole-proteome differential screen. Here we characterize this protein as a blood-stage antigen that localizes to the surface membranes of both parasitized erythrocytes and merozoites, hence its designation as Pf erythrocyte membrane and merozoite antigen 1 (PfEMMA1). Mouse anti-PfEMMA1 antisera and affinity-purified human anti-PfEMMA1 antibodies inhibited growth of P. falciparum strains by up to 68% in growth inhibition assays. Following challenge with uniformly fatal Plasmodium berghei (Pb) ANKA, up to 40% of mice immunized with recombinant PbEMMA1 self-cured, and median survival of lethally infected mice was up to 2.6-fold longer than controls (21 vs. 8 d, P = 0.005). Furthermore, high levels of naturally acquired human anti-PfEMMA1 antibodies were associated with a 46% decrease in parasitemia over 2.5 yr of follow-up of Tanzanian children. Together, these findings suggest that antibodies to PfEMMA1 mediate protection against malaria.


Subject(s)
Antigens, Protozoan/metabolism , Erythrocyte Membrane/parasitology , Malaria, Falciparum/parasitology , Merozoites/metabolism , Plasmodium falciparum/physiology , Protozoan Proteins/genetics , Animals , Antibodies, Protozoan/immunology , Antigens, Protozoan/genetics , Antigens, Protozoan/immunology , Child, Preschool , Female , Host-Parasite Interactions/physiology , Humans , Infant , Malaria Vaccines/genetics , Malaria Vaccines/immunology , Malaria, Falciparum/immunology , Malaria, Falciparum/mortality , Merozoites/immunology , Mice, Inbred BALB C , Plasmodium falciparum/immunology , Plasmodium falciparum/pathogenicity , Polymorphism, Single Nucleotide , Protozoan Proteins/chemistry , Protozoan Proteins/immunology , Protozoan Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Tanzania
5.
J Immunol ; 206(8): 1817-1831, 2021 04 15.
Article in English | MEDLINE | ID: mdl-33789984

ABSTRACT

Plasmodium falciparum merozoite surface protein (PfMSP)2 is a target of parasite-neutralizing Abs. Inclusion of recombinant PfMSP2 (rPfMSP2) as a component of a multivalent malaria vaccine is of interest, but presents challenges. Previously, we used the highly immunogenic PfMSP8 as a carrier to enhance production and/or immunogenicity of malaria vaccine targets. In this study, we exploited the benefits of rPfMSP8 as a carrier to optimize a rPfMSP2-based subunit vaccine. rPfMSP2 and chimeric rPfMSP2/8 vaccines produced in Escherichia coli were evaluated in comparative immunogenicity studies in inbred (CB6F1/J) and outbred (CD1) mice, varying the dose and adjuvant. Immunization of mice with both rPfMSP2-based vaccines elicited high-titer anti-PfMSP2 Abs that recognized the major allelic variants of PfMSP2. Vaccine-induced T cells recognized epitopes present in both PfMSP2 and the PfMSP8 carrier. Competition assays revealed differences in Ab specificities induced by the two rPfMSP2-based vaccines, with evidence of epitope masking by rPfMSP2-associated fibrils. In contrast to aluminum hydroxide (Alum) as adjuvant, formulation of rPfMSP2 vaccines with glucopyranosyl lipid adjuvant-stable emulsion, a synthetic TLR4 agonist, elicited Th1-associated cytokines, shifting production of Abs to cytophilic IgG subclasses. The rPfMSP2/8 + glucopyranosyl lipid adjuvant-stable emulsion formulation induced significantly higher Ab titers with superior durability and capacity to opsonize P. falciparum merozoites for phagocytosis. Immunization with a trivalent vaccine including PfMSP2/8, PfMSP1/8, and the P. falciparum 25 kDa sexual stage antigen fused to PfMSP8 (Pfs25/8) induced high levels of Abs specific for epitopes in each targeted domain, with no evidence of antigenic competition. These results are highly encouraging for the addition of rPfMSP2/8 as a component of an efficacious, multivalent, multistage malaria vaccine.


Subject(s)
Antigens, Protozoan/immunology , Malaria Vaccines/immunology , Malaria/immunology , Merozoites/metabolism , Plasmodium falciparum/immunology , Protozoan Proteins/immunology , Th1 Cells/immunology , Animals , Antibodies, Neutralizing/metabolism , Antibodies, Protozoan/metabolism , Antigens, Protozoan/genetics , Epitope Mapping , Female , Glucosides , Immunodominant Epitopes , Immunoglobulin G/metabolism , Lipid A , Malaria Vaccines/genetics , Male , Merozoites/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Phagocytosis , Protozoan Proteins/genetics
6.
Front Immunol ; 12: 565625, 2021.
Article in English | MEDLINE | ID: mdl-33679730

ABSTRACT

Sub-Saharan Africa has generally experienced few cases and deaths of coronavirus disease 2019 (COVID-19). In addition to other potential explanations for the few cases and deaths of COVID-19 such as the population socio-demographics, early lockdown measures and the possibility of under reporting, we hypothesize in this mini review that individuals with a recent history of malaria infection may be protected against infection or severe form of COVID-19. Given that both the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Plasmodium falciparum (P. falciparum) merozoites bind to the cluster of differentiation 147 (CD147) immunoglobulin, we hypothesize that the immunological memory against P. falciparum merozoites primes SARS-CoV-2 infected cells for early phagocytosis, hence protecting individuals with a recent P. falciparum infection against COVID-19 infection or severity. This mini review therefore discusses the potential biological link between P. falciparum infection and COVID-19 infection or severity and further highlights the importance of CD147 immunoglobulin as an entry point for both SARS-CoV-2 and P. falciparum into host cells.


Subject(s)
Basigin/immunology , COVID-19 , Immunologic Memory , Malaria, Falciparum , Plasmodium falciparum/immunology , SARS-CoV-2/immunology , Africa South of the Sahara/epidemiology , COVID-19/epidemiology , COVID-19/immunology , Humans , Malaria, Falciparum/epidemiology , Malaria, Falciparum/immunology , Merozoites/immunology , Severity of Illness Index
7.
Infect Immun ; 89(6)2021 05 17.
Article in English | MEDLINE | ID: mdl-33722929

ABSTRACT

High-avidity antibodies (Abs) are acquired after a few Plasmodium falciparum infections in low transmission areas, but it remains unclear if Ab avidity to different merozoite antigens increases with age in individuals with persistent antigenemia and, if so, when a fully mature Ab response occurs. The study used plasma samples collected between 1996 and 1998 from 566 individuals aged 4 to 84 years in Simbok, Cameroon, where residents received an estimated 1.6 infectious mosquito bites/person/night. Plasma samples were examined for Ab levels (median fluorescence intensity [MFI]) and Ab avidity index (AI) (where AI = [MFI after treatment with 2 M NH4SCN/MFI without salt] × 100) using a bead-based multiplex immunoassay for recombinant AMA1, EBA-175, MSP1-42 (3D7, FVO), MSP2 (3D7, Fc27), and MSP3. Blood-smear positivity for P. falciparum declined with age from 54.3% at 4 to 5 years to 18% at 16 to 40 years and <11% at >40 years of age, although most individuals had submicroscopic parasitemia. Ab affinity maturation, based on age-related patterns of median AI, percentage of individuals with AI of ≥50, and strength of association between MFI and AI, occurred at different rates among the antigens; they developed rapidly before age 4 years for AMA1, increased gradually with age for EBA-175 and MSP1 until ∼16 to 25 years, but occurred negligibly for MSP2 and MSP3. In a hyperendemic area with perennial transmission, affinity maturation resulting in an increase in the proportion of high-avidity Abs occurred for some merozoite antigens, in parallel with a decline in malaria slide passivity, but not for others.


Subject(s)
Antibodies, Protozoan/immunology , Antibody Affinity/immunology , Antigens, Protozoan/immunology , Malaria, Falciparum/epidemiology , Malaria, Falciparum/immunology , Merozoites/immunology , Plasmodium falciparum/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Cameroon , Child , Child, Preschool , Female , Humans , Malaria, Falciparum/parasitology , Male , Middle Aged , Young Adult
8.
PLoS One ; 15(12): e0243943, 2020.
Article in English | MEDLINE | ID: mdl-33332459

ABSTRACT

Developing a vaccine against Plasmodium falciparum malaria has been challenging, primarily due to high levels of antigen polymorphism and a complex parasite lifecycle. Immunization with the P. falciparum merozoite antigens PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5 has been shown to give rise to growth inhibitory and synergistic antisera. Therefore, these five merozoite proteins are considered to be promising candidates for a second-generation multivalent malaria vaccine. Nevertheless, little is known about IgG and IgM responses to these antigens in populations that are naturally exposed to P. falciparum. In this study, serum samples from clinically immune adults and malaria exposed children from Ghana were studied to compare levels of IgG and IgM specific for PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5. All five antigens were found to be specifically recognized by both IgM and IgG in serum from clinically immune adults and from children with malaria. Longitudinal analysis of the latter group showed an early, transient IgM response that was followed by IgG, which peaked 14 days after the initial diagnosis. IgG levels and parasitemia did not correlate, whereas parasitemia was weakly positively correlated with IgM levels. These findings show that IgG and IgM specific for merozoite antigens PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5 are high in children during P. falciparum malaria, but that the IgM induction and decline occurs earlier in infection than that of IgG.


Subject(s)
Immunoglobulin G/immunology , Immunoglobulin M/immunology , Malaria, Falciparum/immunology , Plasmodium falciparum/immunology , Adolescent , Adult , Aged , Antibodies, Protozoan/immunology , Antigens, Protozoan/immunology , Child , Child, Preschool , Female , Humans , Infant , Malaria Vaccines/immunology , Malaria, Falciparum/parasitology , Male , Merozoites/immunology , Merozoites/pathogenicity , Middle Aged , Plasmodium falciparum/pathogenicity , Young Adult
9.
Parasit Vectors ; 13(1): 602, 2020 Dec 01.
Article in English | MEDLINE | ID: mdl-33261638

ABSTRACT

BACKGROUND: Thrombospondin-related anonymous protein (TRAP) has been described as a potential vaccine candidate for several diseases caused by apicomplexan parasites. However, this protein and members of this family have not yet been characterized in Babesia bigemina, one of the most prevalent species causing bovine babesiosis. METHODS: The 3186-bp Babesia bigemina TRAP-1 (BbiTRAP-1) gene was identified by a bioinformatics search using the B. bovis TRAP-1 sequence. Members of the TRAP and TRAP-related protein families (TRP) were identified in Babesia and Theileria through a search of the TSP-1 adhesive domain, which is the hallmark motif in both proteins. Structural modeling and phylogenetic analysis were performed with the identified TRAP proteins. A truncated recombinant BbiTRAP-1 that migrates at approximately 107 kDa and specific antisera were produced and used in Western blot analysis and indirect fluorescent antibody tests (IFAT). B-cell epitopes with neutralizing activity in BbiTRAP-1 were defined by enzyme-linked immunosorbent assays (ELISA) and invasion assays. RESULTS: Three members of the TRAP family of proteins were identified in B. bigemina (BbiTRAP-1 to -3). All are type 1 transmembrane proteins containing the von Willebrand factor A (vWFA), thrombospondin type 1 (TSP-1), and cytoplasmic C-terminus domains, as well as transmembrane regions. The BbiTRAP-1 predicted structure also contains a metal ion-dependent adhesion site for interaction with the host cell. The TRP family in Babesia and Theileria species contains the canonical TSP-1 domain but lacks the vWFA domain and together with TRAP define a novel gene superfamily. A variable number of tandem repeat units are present in BbiTRAP-1 and could be used for strain genotyping. Western blot and IFAT analysis confirmed the expression of BbiTRAP-1 by blood-stage parasites. Partial recognition by a panel of sera from B. bigemina-infected cattle in ELISAs using truncated BbiTRAP-1 suggests that this protein is not an immunodominant antigen. Additionally, bovine anti-recombinant BbiTRAP-1 antibodies were found to be capable of neutralizing merozoite invasion in vitro. CONCLUSIONS: We have identified the TRAP and TRP gene families in several Babesia and Theileria species and characterized BbiTRAP-1 as a novel antigen of B. bigemina. The functional relevance and presence of neutralization-sensitive B-cell epitopes suggest that BbiTRAP-1 could be included in tests for future vaccine candidates against B. bigemina.


Subject(s)
Babesia/immunology , Babesiosis/parasitology , Cattle Diseases/parasitology , Merozoites/immunology , Protozoan Proteins/chemistry , Protozoan Proteins/immunology , Thrombospondin 1/chemistry , Thrombospondin 1/immunology , Amino Acid Motifs , Amino Acid Sequence , Animals , Babesia/classification , Babesia/genetics , Babesia/growth & development , Cattle , Female , Male , Merozoites/chemistry , Merozoites/genetics , Merozoites/growth & development , Mice , Mice, Inbred BALB C , Multigene Family , Phylogeny , Protozoan Proteins/genetics , Sequence Alignment , Thrombospondin 1/genetics
10.
PLoS One ; 15(11): e0242012, 2020.
Article in English | MEDLINE | ID: mdl-33170876

ABSTRACT

BACKGROUND: Co-infection with malaria and intestinal parasites is common in children in Africa and may affect their immune response to a malaria parasite infection. Prior studies suggest that co-infections may lead to increased susceptibility to malaria infection and disease severity; however, other studies have shown the reverse. Knowledge on how co-morbidities specifically affect the immune response to malaria antigens is limited. Therefore, this study sought to determine the prevalence of co-infection of malaria and intestinal parasites and its association with antibody levels to malaria merozoite antigens. METHODS: A cross sectional study was carried out in two villages with high transmission of malaria in Cameroon (Ngali II and Mfou) where mass drug administration (MDA) had been administered at ~6-month intervals (generally with albendazole or mebendazole). Children aged 1-15 years were enrolled after obtaining parental consent. A malaria rapid diagnostic test was used on site. Four (4) ml of peripheral blood was collected from each participant to determine Plasmodium falciparum infections by microscopy, haemoglobin levels and serology. Fresh stool samples were collected and examined by wet mount, Kato-Katz method and modified Ritchie concentration techniques. A Multiplex Analyte Platform assay was used to measure antibody levels. RESULTS: A total of 320 children were enrolled. The prevalence of malaria by blood smear was 76.3% (244/320) and prevalence of malaria and intestinal parasites was 16.9% (54/320). Malaria prevalence was highest in young children; whereas, intestinal parasites (IP+) were not present until after 3 years of age. All children positive for malaria had antibodies to MSP142, MSP2, MSP3 and EBA175. No difference in antibody levels in children with malaria-co infections compared to malaria alone were found, except for antibody levels to EBA-175 were higher in children co-infected with intestinal protozoa (p = 0.018), especially those with Entamoeba histolytica infections (p = 0.0026). CONCLUSION: Antibody levels to EBA175 were significantly higher in children co-infected with malaria and E. histolytica compared to children infected with malaria alone. It is important to further investigate why and how the presence of these protozoans might modulate the immune response to malaria antigens.


Subject(s)
Coinfection/epidemiology , Intestinal Diseases, Parasitic/epidemiology , Malaria, Falciparum/epidemiology , Adolescent , Animals , Antibodies, Protozoan/blood , Antibody Formation , Antigens, Protozoan/immunology , Cameroon/epidemiology , Child , Child, Preschool , Cross-Sectional Studies , Female , Humans , Immunoglobulin G/blood , Immunologic Tests , Infant , Malaria/epidemiology , Malaria/immunology , Malaria/parasitology , Malaria, Falciparum/immunology , Malaria, Falciparum/parasitology , Male , Merozoites/immunology , Parasites/immunology , Plasmodium falciparum/immunology , Prevalence , Protozoan Proteins/immunology
11.
Parasitol Res ; 119(11): 3639-3648, 2020 Nov.
Article in English | MEDLINE | ID: mdl-32930858

ABSTRACT

Due to its wide presence in apicomplexan parasites as well as high polymorphism and antigenic diversity, the variable merozoite surface antigen (VMSA) family in Babesia sp. has attracted increasing attention of researchers. Here, all the reported VMSA genes of Babesia spp. were obtained from GenBank, and multiple alignments were performed by using conserved regions to blast the Babesia orientalis genome database (unpublished data). Five MSA genes (named MSA-2a1, MSA-2a2, MSA-2c1, MSA-1, and MSA-2c2, respectively) were identified, sequenced, and cloned from B. orientalis, which were shown to encode proteins with open reading frames ranging in size from 266 (MSA-2c1) to 317 (MSA-1) amino acids. All the five proteins contain an MSA-2c superfamily conserved domain, with an identical signal peptide and glycosyl phosphatidyl inositol (GPI)-anchor for each of them. The five proteins were also predicted to contain B cell epitopes, with only three for BoMSA-2c1, the smallest protein in the BoVMSA family, while at least six for each of the others. Notably, BoMSA-2a1 has 2 identical copies, a specific phenomenon only present in B. orientalis. This research has determined the MSA genes of B. orientalis and provides a genetic basis for further research of functional genes in B. orientalis.


Subject(s)
Antigens, Protozoan/genetics , Babesia/genetics , Protozoan Proteins/genetics , Animals , Antigens, Protozoan/immunology , Antigens, Surface/genetics , Babesia/immunology , Epitopes, B-Lymphocyte , Glycosylphosphatidylinositols/analysis , Merozoite Surface Protein 1/genetics , Merozoites/chemistry , Merozoites/immunology , Open Reading Frames , Polymorphism, Genetic , Protozoan Proteins/immunology
12.
PLoS One ; 15(8): e0237786, 2020.
Article in English | MEDLINE | ID: mdl-32822376

ABSTRACT

Plasmodium falciparum malaria killed 451,000 people in 2017. Merozoites, the stage of the parasite that invades RBCs, are a logical target for vaccine development. Treatment with the protease inhibitor E64 followed by filtration through a 1.2 µm filter is being used to purify merozoites for immunologic assays. However, there have been no studies to determine the effect of these treatments on the susceptibility of merozoites to complement or antibodies. To address this gap, we purified merozoites with or without E64 followed by filtration through either a 1.2 or 2.7 µm filter, or no filtration. Merozoites were then incubated in either 10% fresh or heat-inactivated serum followed by surface staining and flow cytometry with monoclonal antibodies against the complement effector molecules C3b or C5b9. To determine the effect of anti-merozoite antibodies, we incubated merozoites with MAb5.2, a mouse monoclonal antibody that targets the merozoite surface protein 1. We used an amine-reactive fluorescent dye to measure membrane integrity. Treatment with E64 resulted in an insignificant increase in the proportion of merozoites that were C3b positive but in a significant increase in the proportion that were C5b9 positive. Filtration increased the proportion of merozoites that were either C3b or C5b9-positive. The combination of filtration and E64 treatment resulted in marked deposition of C3b and C5b9. MAb5.2 induced greater complement deposition than serum alone or an IgG2b isotype control. The combination of E64 treatment, filtration, and MAb5.2 resulted in very rapid and significant deposition of C5b9. Filtration through the 1.2 µm filter selected a population of merozoites with greater membrane integrity, but their integrity deteriorated rapidly upon exposure to serum. We conclude that E64 treatment and filtration increase the susceptibility of merozoites to complement and antibody. Filtered or E64-treated merozoites are not suitable for immunologic studies that address the efficacy of antibodies in vitro.


Subject(s)
Merozoites/drug effects , Merozoites/isolation & purification , Plasmodium falciparum/drug effects , Plasmodium falciparum/isolation & purification , Protease Inhibitors/pharmacology , Animals , Antibodies, Protozoan/immunology , Complement Activation/drug effects , Filtration , Flow Cytometry , Humans , Malaria, Falciparum/immunology , Malaria, Falciparum/parasitology , Merozoites/immunology , Mice , Plasmodium falciparum/immunology
13.
Malar J ; 19(1): 171, 2020 May 01.
Article in English | MEDLINE | ID: mdl-32357882

ABSTRACT

BACKGROUND: Antibodies (Ab) play a significant role in immunity to Plasmodium falciparum malaria. Usually, following repeated exposure to pathogens, affinity maturation and clonal selection take place, resulting in increased antibody avidity. However, some studies suggest affinity maturation may not occur to malaria antigens in endemic areas. Information on development of antibody avidity is confusing and conflicting, in part, because different techniques have been used to measure avidity. Today, bead-based multiplex immunoassays (MIA) are routinely used to simultaneously quantitate antibody levels to multiple antigens. This study evaluated the feasibility of developing an avidity MIA with 5 merozoite antigens (AMA1, EBA-175, MSP1-42, MSP2, MSP3) that uses a single chaotropic concentration. METHODS: The most common ELISA protocols that used the chaotropic reagents guanidine HCl (GdHCl), urea, and ammonium thiocyanate (NH4SCN) were adapted to a multiplex MIA format. Then, different concentrations of chaotropes and incubation times were compared and results were expressed as an Avidity Index (AI), i.e., percentage of antibody remaining bound in the presence of chaotrope. Experiments were conducted to (i) identify the assay with the widest range of AI (discriminatory power), (ii) determine the amount of chaotrope needed to release 50% of bound Ab using plasma from adults and infants, and (iii) evaluate assay repeatability. RESULTS: Overall, 4 M GdHCl and 8 M urea were weaker chaotropes than 3 M NH4SCN. For example, they failed to release significant amounts of Ab bound to MSP1-42 in adult plasma samples; whereas, a range of AI values was obtained with NH4SCN. Titration of NH4SCN revealed that 2 M NH4SCN gave the widest range of AI for the 5 antigens. Binding studies using plasma from 40 adults and 57 1-year old infants in Cameroon showed that 2.1 M ± 0.32 (mean ± SD) NH4SCN (adults) and 1.8 M ± 0.23 M (infants) released 50% of bound Ab from the merozoite antigens. CONCLUSIONS: An avidity MIA is feasible for the 5 merozoite antigens that uses a single concentration (2 M) of NH4SCN. The assay provides a simple method to quickly obtain information about Ab quantity and quality in the acquisition of immunity to malaria in endemic populations.


Subject(s)
Antibodies, Protozoan/immunology , Antibody Affinity/immunology , Antigens, Protozoan/immunology , Plasmodium falciparum/immunology , Adolescent , Adult , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoassay , Infant , Malaria, Falciparum/immunology , Male , Merozoites/immunology , Middle Aged , Young Adult
14.
Sci Rep ; 10(1): 1498, 2020 01 30.
Article in English | MEDLINE | ID: mdl-32001728

ABSTRACT

Despite significant progress in controlling malaria, the disease remains a global health burden. The intricate interactions the parasite Plasmodium falciparum has with its host allows it to grow and multiply in human erythrocytes. The mechanism by which P. falciparum merozoites invade human erythrocytes is complex, involving merozoite proteins as well as erythrocyte surface proteins. Members of the P. falciparum reticulocyte binding-like protein homolog (PfRh) family of proteins play a pivotal role in merozoite invasion and hence are important targets of immune responses. Domains within the PfRh2b protein have been implicated in its ability to stimulate natural protective antibodies in patients. More specifically, a 0.58 kbp deletion, at the C-terminus has been reported in high frequencies in Senegalese and Southeast Asian parasite populations, suggesting a possible role in immune evasion. We analysed 1218 P. falciparum clinical isolates, and the results show that this deletion is present in Ghanaian parasite populations (48.5% of all isolates), with Kintampo (hyper-endemic, 53.2%), followed by Accra (Hypo-endemic, 50.3%), Cape Coast (meso-endemic, 47.9%) and Sogakope (meso-endemic, 43.15%). Further analysis of parasite genomes stored in the MalariaGEN database revealed that the deletion variant was common across transmission areas globally, with an overall frequency of about 27.1%. Interestingly, some parasite isolates possessed mixed PfRh2b deletion and full-length alleles. We further showed that levels of antibodies to the domain of PfRh2 protein were similar to antibody levels of PfRh5, indicating it is less recognized by the immune system.


Subject(s)
Malaria, Falciparum/parasitology , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , Adolescent , Adult , Aged , Amino Acid Sequence , Animals , Antibodies, Protozoan/blood , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/immunology , Child , Child, Preschool , Cohort Studies , Cross-Sectional Studies , Erythrocytes/parasitology , Female , Gene Dosage , Gene Duplication , Genes, Protozoan , Ghana/epidemiology , Host-Parasite Interactions/genetics , Host-Parasite Interactions/immunology , Humans , Immune Evasion/genetics , Infant , Malaria, Falciparum/epidemiology , Malaria, Falciparum/transmission , Male , Merozoites/genetics , Merozoites/immunology , Middle Aged , Plasmodium falciparum/immunology , Protein Domains , Protozoan Proteins/chemistry , Protozoan Proteins/immunology , Sequence Deletion , Sequence Homology, Amino Acid , Young Adult
15.
Infect Immun ; 88(4)2020 03 23.
Article in English | MEDLINE | ID: mdl-31964745

ABSTRACT

Development of a successful blood-stage vaccine against Plasmodium falciparum malaria remains a high priority. Immune-epidemiological studies are effective tools for the identification of antigenic targets of naturally acquired immunity (NAI) against malaria. However, differences in study design and methodology may compromise interstudy comparisons. Here, we assessed antibody responses against intact merozoites and a panel of 24 recombinant merozoite antigens in longitudinal cohort studies of Ghanaian (n = 115) and Indian (n = 121) populations using the same reagents and statistical methods. Anti-merozoite antibodies were associated with NAI in both the Indian (hazard ratio [HR] = 0.41, P = 0.020) and the Ghanaian (HR = 0.17, P < 0.001) participants. Of the 24 antigen-specific antibodies quantified, 12 and 8 were found to be protective in India and Ghana, respectively. Using least absolute shrinkage and selection operator (LASSO) regression, a powerful variable subselection technique, we identified subsets of four (MSP6, MSP3.7, MSPDBL2, and Pf12) and five (cMSP33D7, MSP3.3, MSPDBL1, GLURP-R2, and RALP-1) antigens that explained NAI better than the individual antibodies in India (HR = 0.18, P < 0.001) and Ghana (HR = 0.31, P < 0.001), respectively. IgG1 and/or IgG3 subclasses against five antigens from these subsets were associated with protection. Through this comparative study, maintaining uniformity of reagents and methodology, we demonstrate that NAI across diverse geographic regions may result from antibodies to multiple antigenic targets that constitute the peripheral merozoite surface protein complexes.


Subject(s)
Adaptive Immunity , Antibodies, Protozoan/blood , Malaria, Falciparum/immunology , Membrane Proteins/immunology , Merozoites/immunology , Plasmodium falciparum/immunology , Protozoan Proteins/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Ghana , Humans , India , Infant , Longitudinal Studies , Middle Aged , Young Adult
16.
Virulence ; 11(1): 88-103, 2020 12.
Article in English | MEDLINE | ID: mdl-31900030

ABSTRACT

Individuals growing up in malaria endemic areas gradually develop protection against clinical malaria and passive transfer experiments in humans have demonstrated that this protection is mediated in part by protective antibodies. However, neither the target antigens, specific effector mechanisms, nor the role of continual parasite exposure have been elucidated, which complicates vaccine development. Progress has been made in defining the innate signaling pathways activated by parasite components, including DNA, RNA, hemozoin, and phospholipids, which initiate the immune response and will be the focus of this review. The challenge that remains within the field is to understand the role of these early responses in the development of protective adaptive responses that clear iRBC and block merozoite invasion so that optimal vaccines and therapeutics may be produced.


Subject(s)
Erythrocytes/parasitology , Immunity/immunology , Life Cycle Stages/immunology , Merozoites/immunology , Parasites/immunology , Adaptive Immunity/immunology , Animals , Antigens, Protozoan/immunology , Antimalarials/immunology , Dendritic Cells , Hemeproteins , Humans , Killer Cells, Natural , Malaria/immunology , Malaria/prevention & control , Malaria, Falciparum/immunology , Malaria, Falciparum/prevention & control , Phospholipids/immunology , Pigments, Biological/immunology , Plasmodium falciparum/immunology , Protozoan Proteins/immunology , Protozoan Vaccines/immunology
17.
Malar J ; 19(1): 6, 2020 Jan 06.
Article in English | MEDLINE | ID: mdl-31906953

ABSTRACT

BACKGROUND: Vaccines are the most reliable alternative to elicit sterile immunity against malaria but their development has been hindered by polymorphisms and strain-specificity in previously studied antigens. New vaccine candidates are therefore urgently needed. Highly conserved Plasmodium falciparum reticulocyte-binding protein homologue-5 (PfRH5) has been identified as a potential candidate for anti-disease vaccine development. PfRH5 is essential for erythrocyte invasion by merozoites and crucial for parasite survival. However, there is paucity of data on the extent of genetic variations on PfRH5 in field isolates of Plasmodium falciparum. This study described genetic polymorphisms at the high affinity binding polypeptides (HABPs) 36718, 36727, 36728 of PfRH5 in Nigerian isolates of P. falciparum. This study tested the hypothesis that only specific conserved B and T cell epitopes on PfRH5 HABPs are crucial for vaccine development. METHODS: One hundred and ninety-five microscopically confirmed P. falciparum samples collected in a prospective cross-sectional study of three different populations in Lagos, Nigeria. Genetic diversity and haplotype construct of Pfrh5 gene were determined using bi-directional sequencing approach. Tajima's D and the ratio of nonsynonymous vs synonymous mutations were utilized to estimate the extent of balancing and directional selection in the pfrh5 gene. RESULTS: Sequence analysis revealed three haplotypes of PfRH5 with negative Tajima's D and dN/dS value of - 1.717 and 0.011 ± 0.020, respectively. A single nucleotide polymorphism, SNP (G → A) at position 608 was observed, which resulted in a change of the amino acid cysteine at position 203 to tyrosine. Haplotype and nucleotide diversities were 0.318 ± 0.016 and 0.0046 ± 0.0001 while inter-population genetic differentiation ranged from 0.007 to 0.037. Five polypeptide variants were identified, the most frequent being KTKYH with a frequency of 51.3%. One B-cell epitope, 151 major histocompatibility complex (MHC) class II T-cell epitopes, four intrinsically unstructured regions (IURs) and six MHC class I T-cell epitopes were observed in the study. Phylogenetic analysis of the sequences showed clustering and evidence of evolutionary relationship with 3D7, PAS-2 and FCB-2 RH5 sequences. CONCLUSIONS: This study has revealed low level of genetic polymorphisms in PfRH5 antigen with B- and T-cell epitopes in intrinsically unstructured regions along the PfRH5 gene in Lagos, Nigeria. A broader investigation is however required in other parts of the country to support the possible inclusion of PfRH5 in a cross-protective multi-component vaccine.


Subject(s)
Carrier Proteins/genetics , Carrier Proteins/immunology , Malaria Vaccines/genetics , Malaria Vaccines/immunology , Polymorphism, Single Nucleotide , Antibodies, Protozoan/immunology , Antigens, Protozoan/genetics , Antigens, Protozoan/immunology , Cross-Sectional Studies , Epitopes, B-Lymphocyte , Epitopes, T-Lymphocyte , Erythrocytes/parasitology , Gene Flow , Haplotypes , Histocompatibility , Malaria, Falciparum/immunology , Malaria, Falciparum/parasitology , Malaria, Falciparum/prevention & control , Merozoites/immunology , Nigeria , Phylogeny , Plasmodium falciparum/genetics , Plasmodium falciparum/isolation & purification , Prospective Studies , Sequence Analysis
18.
Infect Immun ; 88(2)2020 01 22.
Article in English | MEDLINE | ID: mdl-31712270

ABSTRACT

Cytoadherence-linked asexual gene 9 (Clag9), a conserved Plasmodium protein expressed during the asexual blood stages, is involved in the cytoadherence of infected red blood cells (RBCs) to the endothelial lining of blood vessels. Here, we show that Plasmodium falciparum Clag9 (PfClag9) is a component of the PfClag9-RhopH complex that is involved in merozoite binding to human erythrocytes. To characterize PfClag9, we expressed four fragments of PfClag9, encompassing the entire protein. Immunostaining analysis using anti-PfClag9 antibodies showed expression and localization of PfClag9 at the apical end of the merozoites. Mass spectrometric analysis of merozoite extracts after immunoprecipitation using anti-PfClag9 antibody identified P. falciparum rhoptry-associated protein 1 (PfRAP1), PfRAP2, PfRAP3, PfRhopH2, and PfRhopH3 as associated proteins. The identified rhoptry proteins were expressed, and their association with PfClag9 domains was assessed by using protein-protein interaction tools. We further showed that PfClag9 binds human RBCs by interacting with the glycophorin A-band 3 receptor-coreceptor complex. In agreement with its cellular localization, PfClag9 was strongly recognized by antibodies generated during natural infection. Mice immunized with the C-terminal domain of PfClag9 were partially protected against a subsequent challenge infection with Plasmodium berghei, further supporting a biological role of PfClag9 during natural infection. Taken together, these results provide direct evidence for the existence of a PfRhopH-Clag9 complex on the Plasmodium merozoite surface that binds to human RBCs.


Subject(s)
Cell Adhesion Molecules/immunology , Erythrocytes/immunology , Merozoites/immunology , Plasmodium falciparum/immunology , Protozoan Proteins/immunology , Animals , Antibodies, Protozoan/immunology , Antigens, Protozoan/immunology , Humans , Malaria, Falciparum/immunology , Mice , Mice, Inbred BALB C , Plasmodium berghei/immunology , Protein Interaction Maps/immunology
19.
Mem Inst Oswaldo Cruz ; 114: e190158, 2019.
Article in English | MEDLINE | ID: mdl-31596312

ABSTRACT

As phagocytosis is the first line of defense against malaria, we developed a phagocytosis assay with Plasmodium vivax (P. vivax) merozoites that can be applied to evaluate vaccine candidates. Briefly, after leukocyte removal with loosely packed cellulose powder in a syringe, P. vivax trophozoites matured to the merozoite-rich schizont stages in the presence of the E64 protease inhibitor. The Percoll gradient-enriched schizonts were chemically disrupted to release merozoites that were submitted to merozoite opsonin-dependent phagocytosis in two phagocytic lines with human and mouse antibodies against the N- and C-terminus of P. vivax Merozoite Surface Protein-1 (Nterm-PvMSP1 and MSP119). The resulting assay is simple and efficient for use as a routine phagocytic assay for the evaluation of merozoite stage vaccine candidates.


Subject(s)
Antibodies, Protozoan/immunology , Merozoites/immunology , Phagocytosis/physiology , Plasmodium vivax/immunology , Protozoan Proteins/immunology , Animals , Female , Flow Cytometry , Merozoites/cytology , Mice , Mice, Inbred BALB C , Plasmodium vivax/physiology
20.
Exp Parasitol ; 206: 107757, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31494216

ABSTRACT

Equine theileriosis is a severe equine disease caused by the protozoan Theileria equi, which is prevalent in tropical and subtropical areas. In this study, a recombinant equi merozoite antigen-2 (rEMA-2) of T. equi was used as an immunogen. Two groups of 10 mice each were divided into control and vaccinated groups. Sixty mares seronegative for theileriosis were divided in two groups, one vaccinated and another group as a control animal. Mice and mares of the vaccinated groups were inoculated with 150 µL of the vaccine containing 50 µg of rEMA-2 and 2 mL of the vaccine containing 200 µg of rEMA-2, respectively, at days 0 and 21. The immunogenicity of rEMA-2 was evaluated by ELISA and fluorescent antibody test (IFAT) using serum from vaccinated mice, mares and antigenicity in naturally infected horse. At every point throughout the ELISA study, there were significant differences between the vaccinated and control groups (p < 0.05). The vaccine induced 3- and 4-fold IgG increases in mice at the 14th and 28th day, respectively, compared to the control group. The horses' IgG dynamics showed a significant (p < 0.05) increase in the total IgG titer as early as day 7, which increased until day 28 at which time a more significant (p < 0.001) IgG titer was observed. In evaluating the isotypes, we observed a trend similar to that of total IgG, where IgG(T) (IgG3-5) were significantly (p < 0.05) more elevated than the other isotypes analyzed, followed by IgGb (IgG4-7) and IgGa (IgG1). Positive fluorescence was detected by IFAT, suggesting that the protein is immunogenic and conserves some epitopes identical to the native T. equi antigens present in the equine blood smear. Thus, our results suggest that rEMA-2 can be a promising vaccinal antigen.


Subject(s)
Antigens, Protozoan/immunology , Pichia/immunology , Theileria/immunology , Analysis of Variance , Animals , Antibodies, Protozoan/blood , Antibodies, Protozoan/immunology , Antigens, Protozoan/biosynthesis , Antigens, Protozoan/genetics , Enzyme-Linked Immunosorbent Assay , Female , Fluorescent Antibody Technique , Horses , Immunoglobulin G/blood , Immunoglobulin G/immunology , Merozoites/immunology , Mice , Mice, Inbred BALB C , Polymerase Chain Reaction , Random Allocation , Recombinant Proteins/immunology
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