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Force Spectroscopy of the Plasmodium falciparum Vaccine Candidate Circumsporozoite Protein Suggests a Mechanically Pliable Repeat Region.

ABSTRACT: The most effective vaccine candidate of malaria is based on the Plasmodium falciparum circumsporozoite protein (CSP), a major surface protein implicated in the structural strength, motility, and immune evasion properties of the infective sporozoites. It is suspected that reversible conformational changes of CSP are required for infection of the mammalian host, but the detailed structure and dynamic properties of CSP remain incompletely understood, limiting our understanding of its function in the infection. Here, we report the structural and mechanical properties of the CSP studied using single-molecule force spectroscopy on several constructs, one including the central region of CSP, which is rich in NANP amino acid repeats (CSPrep), and a second consisting of a near full-length sequence without the signal and anchor hydrophobic domains (CSP?HP). Our results show that the CSPrep is heterogeneous, with 40% of molecules requiring virtually no mechanical force to unfold (<10 piconewtons (pN)), suggesting that these molecules are mechanically compliant and perhaps act as entropic springs, whereas the remaining 60% are partially structured with low mechanical resistance (?70 pN). CSP?HP having multiple force peaks suggests specifically folded domains, with two major populations possibly indicating the open and collapsed forms. Our findings suggest that the overall low mechanical resistance of the repeat region, exposed on the outer surface of the sporozoites, combined with the flexible full-length conformations of CSP, may provide the sporozoites not only with immune evasion properties, but also with lubricating capacity required during its navigation through the mosquito and vertebrate host tissues. We anticipate that these findings would further assist in the design and development of future malarial vaccines.

SUBMITTER: Patra AP

PROVIDER: S-EPMC5313086 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Force Spectroscopy of the <i>Plasmodium falciparum</i> Vaccine Candidate Circumsporozoite Protein Suggests a Mechanically Pliable Repeat Region.

Patra Aditya Prasad AP Sharma Shobhona S Ainavarapu Sri Rama Koti SR

The Journal of biological chemistry 20161227 6

The most effective vaccine candidate of malaria is based on the <i>Plasmodium falciparum</i> circumsporozoite protein (CSP), a major surface protein implicated in the structural strength, motility, and immune evasion properties of the infective sporozoites. It is suspected that reversible conformational changes of CSP are required for infection of the mammalian host, but the detailed structure and dynamic properties of CSP remain incompletely understood, limiting our understanding of its functio ...[more]

PMID: 28031457

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Project description:BackgroundFully efficient vaccines against malaria pre-erythrocytic stage are still lacking. The objective of this dose/adjuvant-finding study was to evaluate the safety, reactogenicity and immunogenicity of a vaccine candidate based on a peptide spanning the C-terminal region of Plasmodium falciparum circumsporozoite protein (PfCS102) in malaria naive adults.Methodology and principal findingsThirty-six healthy malaria-naive adults were randomly distributed into three dose blocks (10, 30 and 100 microg) and vaccinated with PfCS102 in combination with either Montanide ISA 720 or GSK proprietary Adjuvant System AS02A at days 0, 60, and 180. Primary end-point (safety and reactogenicity) was based on the frequency of adverse events (AE) and of abnormal biological safety tests; secondary-end point (immunogenicity) on P. falciparum specific cell-mediated immunity and antibody response before and after immunization. The two adjuvant formulations were well tolerated and their safety profile was good. Most AEs were local and, when systemic, involved mainly fatigue and headache. Half the volunteers in AS02A groups experienced severe AEs (mainly erythema). After the third injection, 34 of 35 volunteers developed anti-PfCS102 and anti-sporozoite antibodies, and 28 of 35 demonstrated T-cell proliferative responses and IFN-gamma production. Five of 22 HLA-A2 and HLA-A3 volunteers displayed PfCS102 specific IFN-gamma secreting CD8(+) T cell responses. Responses were only marginally boosted after the 3(rd) vaccination and remained stable for 6 months. For both adjuvants, the dose of 10 microg was less immunogenic in comparison to 30 and 100 microg that induced similar responses. AS02A formulations with 30 microg or 100 microg PfCS102 induced about 10-folds higher antibody and IFN-gamma responses than Montanide formulations.Conclusions/significancePfCS102 peptide was safe and highly immunogenic, allowing the design of more advanced trials to test its potential for protection. Two or three immunizations with a dose of 30 microg formulated with AS02A appeared the most appropriate choice for such studies.Trial registrationSwissmedic.ch 2002 DR 1227.

Project description:BackgroundThe 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.MethodsA 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.ResultsIn 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.ConclusionsIn 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.

Project description:The circumsporozoite protein (CSP) of Plasmodium falciparum is a major surface protein, which forms a dense coat on the sporozoite's surface. Preclinical research on CSP and clinical evaluation of a CSP fragment-based RTS, S/AS01 vaccine have demonstrated a modest degree of protection against P. falciparum, mediated in part by humoral immunity and in part by cell-mediated immunity. Given the partial protective efficacy of the RTS, S/AS01 vaccine in a recent Phase 3 trial, further improvement of CSP-based vaccines is crucial. In this report, we describe the preclinical development of a full-length, recombinant CSP (rCSP)-based vaccine candidate against P. falciparum malaria suitable for current Good Manufacturing Practice (cGMP) production. Utilizing a novel high-throughput Pseudomonas fluorescens expression platform, we demonstrated greater efficacy of full-length rCSP as compared to N-terminally truncated versions, rapidly down-selected a promising lead vaccine candidate, and developed a high-yield purification process to express immunologically active, intact antigen for clinical trial material production. The rCSP, when formulated with various adjuvants, induced antigen-specific antibody responses as measured by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), as well as CD4+ T-cell responses as determined by ELISpot. The adjuvanted rCSP vaccine conferred protection in mice when challenged with transgenic P. berghei sporozoites containing the P. falciparum repeat region of CSP. Furthermore, heterologous prime/boost regimens with adjuvanted rCSP and an adenovirus type 35-vectored CSP (Ad35CS) showed modest improvements in eliciting CSP-specific T-cell responses and anti-malarial protection, depending on the order of vaccine delivery. Collectively, these data support the importance of further clinical development of adjuvanted rCSP, either as a stand-alone product or as one of the components in a heterologous prime/boost strategy, ultimately acting as an effective vaccine candidate for the mitigation of P. falciparum-induced malaria.

Project description:BackgroundThe malaria vaccine candidate RTS,S/AS01 (GSK Vaccines) induces high IgG concentration against the circumsporozoite protein (CSP) of Plasmodium falciparum. In human vaccine recipients circulating anti-CSP antibody concentrations are associated with protection against infection but appear not to be the correlate of protection. However, in a humanized mouse model of malaria infection prophylactic administration of a human monoclonal antibody (MAL1C), derived from a RTS,S/AS01-immunized volunteer, directed against the CSP repeat region, conveyed full protection in a dose-dependent manner suggesting that antibodies alone are able to prevent P. falciparum infection when present in sufficiently high concentrations. A competition ELISA was developed to measure the presence of MAL1C-like antibodies in polyclonal sera from RTS,S/AS01 vaccine recipients and study their possible contribution to protection against infection.ResultsMAL1C-like antibodies present in polyclonal vaccine-induced sera were evaluated for their ability to compete with biotinylated monoclonal antibody MAL1C for binding sites on the capture antigen consisting of the recombinant protein encompassing 32 NANP repeats of CSP (R32LR). Serum samples were taken at different time points from participants in two RTS,S/AS01 vaccine studies (NCT01366534 and NCT01857869). Vaccine-induced protection status of the study participants was determined based on the outcome of experimental challenge with infected mosquito bites after vaccination. Optimal conditions were established to reliably detect MAL1C-like antibodies in polyclonal sera. Polyclonal anti-CSP antibodies and MAL1C-like antibody content were measured in 276 serum samples from RTS,S/AS01 vaccine recipients using the standard ELISA and MAL-1C competition ELISA, respectively. A strong correlation was observed between the results from these assays. However, no correlation was found between the results of either assay and protection against infection.ConclusionsThe competition ELISA to measure MAL1C-like antibodies in polyclonal sera from RTS,S/AS01 vaccine recipients was robust and reliable but did not reveal the elusive correlate of protection.

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Force Spectroscopy of the Plasmodium falciparum Vaccine Candidate Circumsporozoite Protein Suggests a Mechanically Pliable Repeat Region.

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Force Spectroscopy of the <i>Plasmodium falciparum</i> Vaccine Candidate Circumsporozoite Protein Suggests a Mechanically Pliable Repeat Region.

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