Sequence Conservation in Plasmodium falciparum α-Helical Coiled Coil Domains Proposed for Vaccine Development

AbstractGenetic diversity of surface exposed and stage specific Plasmodium falciparum immunogenic proteins pose a major roadblock to developing an effective malaria vaccine with broad and long-lasting immunity. We conducted a prospective genetic analysis of candidate antigens (msp1, ama1, rh5, eba175, glurp, celtos, csp, lsa3, Pfsea, trap, conserved chrom3, hyp9, hyp10, phistb, surfin8.2, and surfin14.1) for malaria vaccine development on 2375 P. falciparum sequences from 16 African countries. We described signatures of balancing selection inferred from positive values of Tajima’s D for all antigens across all populations except for glurp. This could be as a result of immune selection on these antigens as positive Tajima’s D values mapped to regions with putative immune epitopes. A less diverse phistb antigen was characterised with a transmembrane domain, glycophosphatidyl anchors between the N and C- terminals, and surface epitopes that could be targets of immune recognition. This study demonstrates the value of population genetic and immunoinformatic analysis for identifying and characterising new putative vaccine candidates towards improving strain transcending immunity, and vaccine efficacy across all endemic populations.

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Geographically restricted heterogeneity of the Plasmodium falciparum circumsporozoite protein: relevance for vaccine development.

Infection and Immunity ◽

10.1128/iai.60.2.675-682.1992 ◽

1992 ◽

Vol 60 (2) ◽

pp. 675-682 ◽

Cited By ~ 43

Author(s):

D L Doolan ◽

A J Saul ◽

M F Good

Keyword(s):

Plasmodium Falciparum ◽

Vaccine Development ◽

Circumsporozoite Protein

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The Plasmodium falciparum circumsporozoite protein produced in Lactococcus lactis is pure and stable

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011268 ◽

2019 ◽

Vol 295 (2) ◽

pp. 403-414 ◽

Cited By ~ 1

Author(s):

Susheel K. Singh ◽

Jordan Plieskatt ◽

Bishwanath Kumar Chourasia ◽

Vandana Singh ◽

Judith M. Bolscher ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Lactococcus Lactis ◽

Malaria Vaccine ◽

Vaccine Development ◽

Expression System ◽

Surface Protein ◽

Circumsporozoite Protein ◽

Full Length

The Plasmodium falciparum circumsporozoite protein (PfCSP) is a sporozoite surface protein whose role in sporozoite motility and cell invasion has made it the leading candidate for a pre-erythrocytic malaria vaccine. However, production of high yields of soluble recombinant PfCSP, including its extensive NANP and NVDP repeats, has proven problematic. Here, we report on the development and characterization of a secreted, soluble, and stable full-length PfCSP (containing 4 NVDP and 38 NANP repeats) produced in the Lactococcus lactis expression system. The recombinant full-length PfCSP, denoted PfCSP4/38, was produced initially with a histidine tag and purified by a simple two-step procedure. Importantly, the recombinant PfCSP4/38 retained a conformational epitope for antibodies as confirmed by both in vivo and in vitro characterizations. We characterized this complex protein by HPLC, light scattering, MS analysis, differential scanning fluorimetry, CD, SDS-PAGE, and immunoblotting with conformation-dependent and -independent mAbs, which confirmed it to be both pure and soluble. Moreover, we found that the recombinant protein is stable at both frozen and elevated-temperature storage conditions. When we used L. lactis–derived PfCSP4/38 to immunize mice, it elicited high levels of functional antibodies that had the capacity to modify sporozoite motility in vitro. We concluded that the reported yield, purity, results of biophysical analyses, and stability of PfCSP4/38 warrant further consideration of using the L. lactis system for the production of circumsporozoite proteins for preclinical and clinical applications in malaria vaccine development.

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Genetic diversity and antibody responses against Plasmodium falciparum vaccine candidate genes from Chhattisgarh, Central India: Implication for vaccine development

PLoS ONE ◽

10.1371/journal.pone.0182674 ◽

2017 ◽

Vol 12 (8) ◽

pp. e0182674 ◽

Cited By ~ 11

Author(s):

Priyanka Patel ◽

Praveen K. Bharti ◽

Devendra Bansal ◽

Rajive K. Raman ◽

Pradyumna K. Mohapatra ◽

...

Keyword(s):

Genetic Diversity ◽

Plasmodium Falciparum ◽

Candidate Genes ◽

Vaccine Development ◽

Vaccine Candidate ◽

Central India ◽

Antibody Responses

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Identification of a Potent Combination of Key Plasmodium falciparum Merozoite Antigens That Elicit Strain-Transcending Parasite-Neutralizing Antibodies

Infection and Immunity ◽

10.1128/iai.01107-12 ◽

2012 ◽

Vol 81 (2) ◽

pp. 441-451 ◽

Cited By ~ 40

Author(s):

Alok K. Pandey ◽

K. Sony Reddy ◽

Tajali Sahar ◽

Sonal Gupta ◽

Hina Singh ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Neutralizing Antibodies ◽

Malaria Vaccine ◽

Vaccine Development ◽

Blood Stage ◽

Content Type ◽

Receptor Blocking ◽

Erythrocyte Invasion ◽

Invasion Inhibition ◽

Blood Stage Malaria

ABSTRACTBlood-stage malaria vaccines that target singlePlasmodium falciparumantigens involved in erythrocyte invasion have not induced optimal protection in field trials. Blood-stage malaria vaccine development has faced two major hurdles, antigenic polymorphisms and molecular redundancy, which have led to an inability to demonstrate potent, strain-transcending, invasion-inhibitory antibodies. Vaccines that target multiple invasion-related parasite proteins may inhibit erythrocyte invasion more efficiently. Our approach is to develop a receptor-blocking blood-stage vaccine againstP. falciparumthat targets the erythrocyte binding domains of multiple parasite adhesins, blocking their interaction with their receptors and thus inhibiting erythrocyte invasion. However, with numerous invasion ligands, the challenge is to identify combinations that elicit potent strain-transcending invasion inhibition. We evaluated the invasion-inhibitory activities of 20 different triple combinations of antibodies mixedin vitroagainst a diverse set of six key merozoite ligands, including the novel ligandsP. falciparumapical asparagine-rich protein (PfAARP), EBA-175 (PfF2),P. falciparumreticulocyte binding-like homologous protein 1 (PfRH1), PfRH2, PfRH4, andPlasmodiumthrombospondin apical merozoite protein (PTRAMP), which are localized in different apical organelles and are translocated to the merozoite surface at different time points during invasion. They bind erythrocytes with different specificities and are thus involved in distinct invasion pathways. The antibody combination of EBA-175 (PfF2), PfRH2, and PfAARP produced the most efficacious strain-transcending inhibition of erythrocyte invasion against diverseP. falciparumclones. This potent antigen combination was selected for coimmunization as a mixture that induced balanced antibody responses against each antigen and inhibited erythrocyte invasion efficiently. We have thus demonstrated a novel two-step screening approach to identify a potent antigen combination that elicits strong strain-transcending invasion inhibition, supporting its development as a receptor-blocking malaria vaccine.

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Immunoglobulin G Subclass-Specific Responses against Plasmodium falciparum Merozoite Antigens Are Associated with Control of Parasitemia and Protection from Symptomatic Illness

Infection and Immunity ◽

10.1128/iai.01129-08 ◽

2009 ◽

Vol 77 (3) ◽

pp. 1165-1174 ◽

Cited By ~ 165

Author(s):

Danielle I. Stanisic ◽

Jack S. Richards ◽

Fiona J. McCallum ◽

Pascal Michon ◽

Christopher L. King ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Immunoglobulin G ◽

Vaccine Development ◽

Surface Protein ◽

Merozoite Surface Protein ◽

Igg Subclass ◽

Apical Membrane Antigen 1 ◽

Falciparum Merozoite ◽

Merozoite Antigens

ABSTRACT Substantial evidence indicates that antibodies to Plasmodium falciparum merozoite antigens play a role in protection from malaria, although the precise targets and mechanisms mediating immunity remain unclear. Different malaria antigens induce distinct immunoglobulin G (IgG) subclass responses, but the importance of different responses in protective immunity from malaria is not known and the factors determining subclass responses in vivo are poorly understood. We examined IgG and IgG subclass responses to the merozoite antigens MSP1-19 (the 19-kDa C-terminal region of merozoite surface protein 1), MSP2 (merozoite surface protein 2), and AMA-1 (apical membrane antigen 1), including different polymorphic variants of these antigens, in a longitudinal cohort of children in Papua New Guinea. IgG1 and IgG3 were the predominant subclasses of antibodies to each antigen, and all antibody responses increased in association with age and exposure without evidence of increasing polarization toward one subclass. The profiles of IgG subclasses differed somewhat for different alleles of MSP2 but not for different variants of AMA-1. Individuals did not appear to have a propensity to make a specific subclass response irrespective of the antigen. Instead, data suggest that subclass responses to each antigen are generated independently among individuals and that antigen properties, rather than host factors, are the major determinants of IgG subclass responses. High levels of AMA-1-specific IgG3 and MSP1-19-specific IgG1 were strongly predictive of a reduced risk of symptomatic malaria and high-density P. falciparum infections. However, no antibody response was significantly associated with protection from parasitization per se. Our findings have major implications for understanding human immunity and for malaria vaccine development and evaluation.

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Immunization with VAR2CSA-DBL5 Recombinant Protein Elicits Broadly Cross-Reactive Antibodies to Placental Plasmodium falciparum-Infected Erythrocytes

Infection and Immunity ◽

10.1128/iai.00410-09 ◽

2010 ◽

Vol 78 (5) ◽

pp. 2248-2256 ◽

Cited By ~ 29

Author(s):

Marion Avril ◽

Megan M. Cartwright ◽

Marianne J. Hathaway ◽

Mirja Hommel ◽

Salenna R. Elliott ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Amino Acid ◽

Recombinant Protein ◽

Recombinant Proteins ◽

Vaccine Development ◽

Amino Acid Identity ◽

Clinical Syndrome ◽

Acid Identity ◽

Geographic Origins ◽

Pregnancy Malaria

ABSTRACTPregnancy-associated malaria is a severe clinical syndrome associated with the sequestration ofPlasmodium falciparum-infected erythrocytes in the placenta. Placental binding is mediated by VAR2CSA, a member of the large and diverseP. falciparumerythrocyte membrane 1 (PfEMP1) protein family. To better understand if conserved regions in VAR2CSA can be targeted by antibodies, we immunized rabbits with VAR2CSA-DBL1 and -DBL5 recombinant proteins produced inPichia pastorisand developed a panel of seven chondroitin sulfate A (CSA)-binding parasites from diverse geographic origins. Overall, no two parasites in the panel expressed the same VAR2CSA sequence. The DBL1 domains averaged 80% amino acid identity (range, 72 to 89%), and the DBL5 domains averaged 86% amino acid identity (range, 83 to 99%), similar to a broader sampling of VAR2CSA sequences from around the world. Whereas antibodies generated against the VAR2CSA-DBL1 recombinant protein had only limited breadth and reacted with three or four parasites in the panel, immunization with DBL5 recombinant proteins elicited broadly cross-reactive antibodies against all or most parasites in the panel, as well as to fresh clinical isolates from pregnant women. These findings demonstrate that the major PfEMP1 variant expressed by placental isolates exposes strain-transcendent epitopes that can be targeted by vaccination and may have application for pregnancy malaria vaccine development.

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Plasmodium falciparum schizont stage transcriptome variation among clinical isolates and laboratory-adapted clones

10.1101/329532 ◽

2018 ◽

Cited By ~ 1

Author(s):

Sarah J Tarr ◽

Ofelia Díaz-Ingelmo ◽

Lindsay B Stewart ◽

Suzanne E Hocking ◽

Lee Murray ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Vaccine Development ◽

Ex Vivo ◽

Experimental Studies ◽

Differentially Expressed ◽

Clinical Isolates ◽

Schizont Stage ◽

Variable Expression ◽

The Impact

AbstractMalaria parasite genes exhibit variation in both sequence and expression level. There is much information on sequence polymorphism, but less resolution on natural variation in transcriptomes of parasites at specific developmental stages. This is largely because it is challenging to obtain highly replicated sampling of transcriptomes to overcome potentially confounding technical and biological variation. We address the issue in the major human parasite Plasmodium falciparum by obtaining RNA-seq profiles of multiple independent replicate preparations of mature schizont-stage parasites from a panel of clinical isolates recently established in culture and from long-term laboratory-adapted clones. With a goal of robustly identifying variably expressed genes, we show that increasing the numbers of biological sample replicates greatly improves the discovery rate. Generally, six independent replicates of each parasite culture is recommendable as being significantly to lower numbers, although for highly expressed genes variable expression can be detected when fewer replicates are available. A broad comparison identifies genes differing in relative expression between cultured clinical isolates and laboratory-adapted clones. Genes more highly expressed in the laboratory-adapted clones include an AP2 transcription factor gene Pf3D7_0420300 and putative methyl transferase genes. The variable expression of several known merozoite invasion ligands is confirmed, and previously uncharacterised genes are shown to be differentially expressed among clinical isolates. New RT-qPCR assays validate the variation in transcript levels of these genes, and allow quantitation of expression to be extended to a wider panel of clinical isolate samples. These variably expressed genes are new candidates for investigation as potential determinants of alternative parasite developmental pathways or targets of immunity.Author summaryUnderstanding parasite diversity and adaptation may require characterisation of gene expression variation, and is vital if chemotherapeutic or vaccine development is to consider new candidate targets, but it is technically challenging to generate precise data on clinical isolates. Here, we analyse the transcriptomes of mature Plasmodium falciparum schizonts using RNA-sequencing, using large numbers of biological replicate samples to minimise the impact of inter-replicate variation on observed patterns of differential expression. This identifies genes that are differentially expressed in long term laboratory-adapted parasites and recently cultured clinical isolates, as well as among different clinical isolates. In additional samples of schizonts grown in the first cycle ex vivo prior to any erythrocyte invasion, expression levels of a selected panel of these genes vary among isolates, but mean levels are similar to those in the continuously cultured clinical isolates, indicating that the latter are useful for experimental studies requiring biological replication.

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Antibody Targets and Properties for Complement-Fixation Against the Circumsporozoite Protein in Malaria Immunity

Frontiers in Immunology ◽

10.3389/fimmu.2021.775659 ◽

2021 ◽

Vol 12 ◽

Author(s):

Liriye Kurtovic ◽

Damien R. Drew ◽

Arlene E. Dent ◽

James W. Kazura ◽

James G. Beeson

Keyword(s):

Plasmodium Falciparum ◽

Malaria Vaccine ◽

Vaccine Development ◽

Complement Fixation ◽

Terminal Region ◽

Antibody Responses ◽

Circumsporozoite Protein ◽

Full Length ◽

Acquired Immunity ◽

Vaccine Candidates

The Plasmodium falciparum circumsporozoite protein (CSP) forms the basis of leading subunit malaria vaccine candidates. However, the mechanisms and specific targets of immunity are poorly defined. Recent findings suggest that antibody-mediated complement-fixation and activation play an important role in immunity. Here, we investigated the regions of CSP targeted by functional complement-fixing antibodies and the antibody properties associated with this activity. We quantified IgG, IgM, and functional complement-fixing antibody responses to different regions of CSP among Kenyan adults naturally exposed to malaria (n=102) and using a series of rabbit vaccination studies. Individuals who acquired functional complement-fixing antibodies had higher IgG, IgM and IgG1 and IgG3 to CSP. Acquired complement-fixing antibodies targeted the N-terminal, central-repeat, and C-terminal regions of CSP, and positive responders had greater antibody breadth compared to those who were negative for complement-fixing antibodies (p<0.05). Using rabbit vaccinations as a model, we confirmed that IgG specific to the central-repeat and non-repeat regions of CSP could effectively fix complement. However, vaccination with near full length CSP in rabbits poorly induced antibodies to the N-terminal region compared to naturally-acquired immunity in humans. Poor induction of N-terminal antibodies was also observed in a vaccination study performed in mice. IgG and IgM to all three regions of CSP play a role in mediating complement-fixation, which has important implications for malaria vaccine development.

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Analysis of the Role of TpUB05 Antigen from Theileria parva in Immune Responses to Malaria in Humans Compared to Its Homologue in Plasmodium falciparum the UB05 Antigen

Pathogens ◽

10.3390/pathogens9040271 ◽

2020 ◽

Vol 9 (4) ◽

pp. 271

Author(s):

Jerome Nyhalah Dinga ◽

Stephanie Numenyi Perimbie ◽

Stanley Dobgima Gamua ◽

Francis N. G. Chuma ◽

Dieudonné Lemuh Njimoh ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Immune Responses ◽

Protective Immunity ◽

Vaccine Development ◽

Theileria Parva ◽

Peripheral Blood Mononuclear ◽

Significant Difference ◽

First Time

Despite the amount of resources deployed and the technological advancements in molecular biology, vaccinology, immunology, genetics, and biotechnology, there are still no effective vaccines against malaria. Immunity to malaria is usually seen to be species- and/or strain-specific. However, there is a growing body of evidence suggesting the possibility of the existence of cross-strain, cross-species, and cross-genus immune responses in apicomplexans. The principle of gene conservation indicates that homologues play a similar role in closely related organisms. The homologue of UB05 in Theileria parva is TpUB05 (XP_763711.1), which has been tested and shown to be associated with protective immunity in East Coast fever. In a bid to identify potent markers of protective immunity to aid malaria vaccine development, TpUB05 was tested in malaria caused by Plasmodium falciparum. It was observed that TpUB05 was better at detecting antigen-specific antibodies in plasma compared to UB05 when tested by ELISA. The total IgG raised against TpUB05 was able to block parasitic growth in vitro more effectively than that raised against UB05. However, there was no significant difference between the two study antigens in recalling peripheral blood mononuclear cell (PBMC) memory through IFN-γ production. This study suggests, for the first time, that TpUB05 from T. parva cross-reacts with UB05 from P. falciparum and is a marker of protective immunity in malaria. Hence, TpUB05 should be considered for possible development as a potential subunit vaccine candidate against malaria.

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