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

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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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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In silico characterisation of putative Plasmodium falciparum vaccine candidates in African malaria populations

Scientific Reports ◽

10.1038/s41598-021-95442-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

O. Ajibola ◽

M. F. Diop ◽

A. Ghansah ◽

L. Amenga-Etego ◽

L. Golassa ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Malaria Vaccine ◽

Transmembrane Domain ◽

Vaccine Development ◽

Balancing Selection ◽

Immune Recognition ◽

African Countries ◽

Vaccine Candidates ◽

D Values ◽

Tajima’S D

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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Transgenic Parasites Stably Expressing Full-Length Plasmodium falciparum Circumsporozoite Protein as a Model for Vaccine Down-Selection in Mice Using Sterile Protection as an Endpoint

Clinical and Vaccine Immunology ◽

10.1128/cvi.00066-13 ◽

2013 ◽

Vol 20 (6) ◽

pp. 803-810 ◽

Cited By ~ 31

Author(s):

Michael D. Porter ◽

Jennifer Nicki ◽

Christopher D. Pool ◽

Margot DeBot ◽

Ratish M. Illam ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Malaria Vaccine ◽

Circumsporozoite Protein ◽

Full Length ◽

Content Type ◽

Transmission Cycle ◽

Degree Of Protection ◽

Antibody Titers ◽

Malaria Vaccine Candidate ◽

Transgenic Parasites

ABSTRACTCircumsporozoite protein (CSP) ofPlasmodium falciparumis a protective human malaria vaccine candidate. There is an urgent need for models that can rapidly down-select novel CSP-based vaccine candidates. In the present study, the mouse-mosquito transmission cycle of a transgenicPlasmodium bergheimalaria parasite stably expressing a functional full-lengthP. falciparumCSP was optimized to consistently produce infective sporozoites for protection studies. A minimal sporozoite challenge dose was established, and protection was defined as the absence of blood-stage parasites 14 days after intravenous challenge. The specificity of protection was confirmed by vaccinating mice with multiple CSP constructs of differing lengths and compositions. Constructs that induced high NANP repeat-specific antibody titers in enzyme-linked immunosorbent assays were protective, and the degree of protection was dependent on the antigen dose. There was a positive correlation between antibody avidity and protection. The antibodies in the protected mice recognized the native CSP on the parasites and showed sporozoite invasion inhibitory activity. Passive transfer of anti-CSP antibodies into naive mice also induced protection. Thus, we have demonstrated the utility of a mouse efficacy model to down-select human CSP-based vaccine formulations.

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A Full-Length Plasmodium falciparum Recombinant Circumsporozoite Protein Expressed by Pseudomonas fluorescens Platform as a Malaria Vaccine Candidate

PLoS ONE ◽

10.1371/journal.pone.0107764 ◽

2014 ◽

Vol 9 (9) ◽

pp. e107764 ◽

Cited By ~ 24

Author(s):

Amy R. Noe ◽

Diego Espinosa ◽

Xiangming Li ◽

Jordana G. A. Coelho-dos-Reis ◽

Ryota Funakoshi ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Pseudomonas Fluorescens ◽

Malaria Vaccine ◽

Vaccine Candidate ◽

Circumsporozoite Protein ◽

Full Length ◽

Malaria Vaccine Candidate

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Generation of a Genetically Modified Chimeric Plasmodium falciparum Parasite Expressing Plasmodium vivax Circumsporozoite Protein for Malaria Vaccine Development

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2020.591046 ◽

2020 ◽

Vol 10 ◽

Author(s):

Yukiko Miyazaki ◽

Catherin Marin-Mogollon ◽

Takashi Imai ◽

António M. Mendes ◽

Rianne van der Laak ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Malaria Vaccine ◽

Vaccine Development ◽

Human Hepatocytes ◽

Circumsporozoite Protein ◽

Wild Type ◽

Rodent Malaria ◽

Preclinical Evaluation ◽

Plasmodium Falciparum Parasite ◽

Falciparum Parasite

Chimeric rodent malaria parasites with the endogenous circumsporozoite protein (csp) gene replaced with csp from the human parasites Plasmodium falciparum (Pf) and P. vivax (Pv) are used in preclinical evaluation of CSP vaccines. Chimeric rodent parasites expressing PfCSP have also been assessed as whole sporozoite (WSP) vaccines. Comparable chimeric P. falciparum parasites expressing CSP of P. vivax could be used both for clinical evaluation of vaccines targeting PvCSP in controlled human P. falciparum infections and in WSP vaccines targeting P. vivax and P. falciparum. We generated chimeric P. falciparum parasites expressing both PfCSP and PvCSP. These Pf-PvCSP parasites produced sporozoite comparable to wild type P. falciparum parasites and expressed PfCSP and PvCSP on the sporozoite surface. Pf-PvCSP sporozoites infected human hepatocytes and induced antibodies to the repeats of both PfCSP and PvCSP after immunization of mice. These results support the use of Pf-PvCSP sporozoites in studies optimizing vaccines targeting PvCSP.

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Assessment of genetic diversity of Plasmodium falciparum circumsporozoite protein in Sudan: the RTS,S leading malaria vaccine candidate

Malaria Journal ◽

10.1186/s12936-021-03971-0 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Nouh Saad Mohamed ◽

Hanadi AbdElbagi ◽

Ahad R. Elsadig ◽

Abdalla Elssir Ahmed ◽

Yassir Osman Mohammed ◽

...

Keyword(s):

Genetic Diversity ◽

Plasmodium Falciparum ◽

Malaria Vaccine ◽

Cell Epitope ◽

Malaria Diagnosis ◽

Terminal Region ◽

Circumsporozoite Protein ◽

Nested Polymerase Chain Reaction ◽

Cross Sectional ◽

Haplotype Networks

Abstract Background The currently used malaria vaccine, RTS,S, is designed based on the Plasmodium falciparum circumsporozoite protein (PfCSP). The pfcsp gene, besides having different polymorphic patterns, can vary between P. falciparum isolates due to geographical origin and host immune response. Such aspects are essential when considering the deployment of the RTS,S vaccine in a certain region. Therefore, this study assessed the genetic diversity of P. falciparum in Sudan based on the pfcsp gene by investigating the diversity at the N-terminal, central repeat, and the C-terminal regions. Methods A cross-sectional molecular study was conducted; P. falciparum isolates were collected from different health centres in Khartoum State between January and December 2019. During the study period, a total of 261 febrile patients were recruited. Malaria diagnosis was made by expert microscopists using Giemsa-stained thick and thin blood films. DNA samples were examined by the semi-nested polymerase chain reaction (PCR). Single clonal infection of the confirmed P. falciparum cases, were used to amplify the pfcsp gene. The amplified amplicons of pfcsp have been sequenced using the Sanger dideoxy method. The obtained sequences of pfcsp nucleotide diversity parameters including the numbers of haplotypes (Hap), haplotypes diversity (Hapd), the average number of nucleotide differences between two sequences (p), and the numbers of segregating sites (S) were obtained. The haplotype networks were constructed using the online tcsBU software. Natural selection theory was also tested on pfcsp using Fuand Li’s D, Fuand Li’s F statistics, and Tajima’s D test using DnaSP. Results In comparison with the different pfcsp reference strains, the Sudanese isolates showed high similarity with other African isolates. The results of the N-terminal region showed the presence of 2 different haplotypes with a Hapd of 0.425 ± 0.00727. The presence of the unique insertion of NNNGDNGREGKDEDKRDGNN was reported. The KLKQP motif was conserved in all the studied isolates. At the central repeat region, 11 haplotypes were seen with a Hapd of 0.779 ± 0.00097. The analysis of the genetic diversity in the C-terminal region showed the presence of 10 haplotypes with a Hapd of 0.457 ± 0.073. Several non-synonymous amino acids changes were also seen at the Th2R and the Th3R T-cell epitope regions including T317K, E317K, Q318E, K321N, I322K, T322K, R322K, K324Q, I327L, G352N, S354P, R355K, N356D, Q357E, and E361A. Conclusions In this study, the results indicated a high conservation at the pfcsp gene. This may further contribute in understanding the genetic polymorphisms of P. falciparum prior to the deployment of the RTS,S vaccine in Sudan.

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Assessment of Genetic Diversity of Plasmodium Falciparum Circumsporozoite Protein in Sudan; The RTS,S Leading Malaria Vaccine Candidate

10.21203/rs.3.rs-619335/v1 ◽

2021 ◽

Author(s):

Nouh Saad Mohamed ◽

Hanadi AbdElbagi ◽

Ahad R. Elsadig ◽

Abdalla Elssir Ahmed ◽

Yassir Osman Mohammed ◽

...

Keyword(s):

Genetic Diversity ◽

Plasmodium Falciparum ◽

Malaria Vaccine ◽

Geographical Origin ◽

Vaccine Candidate ◽

Haplotype Diversity ◽

Terminal Region ◽

Circumsporozoite Protein ◽

High Conservation ◽

Malaria Vaccine Candidate

Abstract The currently used malaria vaccine; the RTS,S, is designed based on the Plasmodium falciparum circumsporozoite protein (PfCSP). The PfCSP gene, besides having different polymorphic patterns, can vary between P. falciparum isolates due to geographical origin and host immune response. Such aspects are essential when considering the deployment of the RTS,S vaccine in a certain region. Therefore, in this study we assessed the genetic diversity of P. falciparum in Sudan based on the PfCSP gene by investigating the diversity at the N-terminal, central repeat, and the C-terminal regions. The results of the N-terminal region showed the presence of 2 different haplotypes with a haplotype diversity (Hapd) of 0.425 ± 0.00727. The presence of the unique insertion of NNNGDNGREGKDEDKRDGNN was reported. The KLKQP motif was conserved in all the studied isolates. At the central repeat region, 11 haplotypes were seen with a Hapd of 0.779 ± 0.00097. The analysis of the genetic diversity in the C-terminal region showed the presence of 10 haplotypes with a Hapd of 0.457 ± 0.073. In this study, the results indicated a high conservation at the PfCSP gene. This may further contribute in understanding the genetic polymorphisms of P. falciparum prior to the deployment of the RTS,S vaccine in Sudan.

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