Characterising PvRBSA: an exclusive protein from Plasmodium species infecting reticulocytes

Background: Plasmodium vivax uses multiple ligand-receptor interactions for preferential invasion of human reticulocytes. Several of these ligands have been identified by in silico approaches based on the role displayed by their orthologs in other Plasmodium species during initial adhesion or invasion. However, the cell adhesion role of proteins that are exclusive to species that specifically invade reticulocytes (as P. vivax and P. cynomolgi) has not been evaluated to date. This study aimed to characterise an antigen shared between Plasmodium species that preferentially infect reticulocytes with a focus on assessing its binding activity to target cells. Results: An in silico analysis was performed using P. vivax proteome data to identify and characterise one antigen shared between P. vivax and P. cynomolgi. This led to identification of the pvrbsa gene present in the P. vivax VCG-I strain genome. This gene is transcribed in mature schizonts and encodes a protein located on the parasite surface. rPvRBSA was antigenic and capable of binding to a population of reticulocytes with a different Duffy phenotype. Interestingly, the molecule showed a higher percentage of binding to immature human reticulocytes (CD71hi). Conclusions: This study describes for the first time, a molecule involved in host cell binding that is exclusive in reticulocyte-infecting Plasmodium species. This suggest that PvRBSA is an antigenic adhesin that plays a role in parasite binding to target cells. © 2017 The Author(s)

Identification, characterization and antigenicity of the Plasmodium vivax rhoptry neck protein 1 (PvRON1)

Background: Plasmodium vivax malaria remains a major health problem in tropical and sub-tropical regions worldwide. Several rhoptry proteins which are important for interaction with and/or invasion of red blood cells, such as PfRONs, Pf92, Pf38, Pf12 and Pf34, have been described during the last few years and are being considered as potential anti-malarial vaccine candidates. This study describes the identification and characterization of the P. vivax rhoptry neck protein 1 (PvRON1) and examine its antigenicity in natural P. vivax infections. Methods: The PvRON1 encoding gene, which is homologous to that encoding the P. falciparum apical sushi protein (ASP) according to the plasmoDB database, was selected as our study target. The pvron1 gene transcription was evaluated by RT-PCR using RNA obtained from the P. vivax VCG-1 strain. Two peptides derived from the deduced P. vivax Sal-I PvRON1 sequence were synthesized and inoculated in rabbits for obtaining anti-PvRON1 antibodies which were used to confirm the protein expression in VCG-1 strain schizonts along with its association with detergent-resistant microdomains (DRMs) by Western blot, and its localization by immunofluorescence assays. The antigenicity of the PvRON1 protein was assessed using human sera from individuals previously exposed to P. vivax malaria by ELISA. Results: In the P. vivax VCG-1 strain, RON1 is a 764 amino acid-long protein. In silico analysis has revealed that PvRON1 shares essential characteristics with different antigens involved in invasion, such as the presence of a secretory signal, a GPI-anchor sequence and a putative sushi domain. The PvRON1 protein is expressed in parasite's schizont stage, localized in rhoptry necks and it is associated with DRMs. Recombinant protein recognition by human sera indicates that this antigen can trigger an immune response during a natural infection with P. vivax. Conclusions: This study shows the identification and characterization of the P. vivax rhoptry neck protein 1 in the VCG-1 strain. Taking into account that PvRON1 shares several important characteristics with other Plasmodium antigens that play a functional role during RBC invasion and, as shown here, it is antigenic, it could be considered as a good vaccine candidate. Further studies aimed at assessing its immunogenicity and protection-inducing ability in the Aotus monkey model are thus recommended

Persistence, clearance and reinfection regarding six high risk human papillomavirus types in Colombian women : A follow-up study

Background: The design of new healthcare schemes which involve using molecular HPV screening means that both persistence and clearance data regarding the most prevalent types of HR-HPV occurring in cities in Colombia must be ascertained.Methods: This study involved 219 HPV positive women in all of whom 6 types of HR-HPV had been molecularly identified and quantified; they were followed-up for 2 years. The Kaplan-Meier survival function was used for calculating the time taken for the clearance of each type of HPV. The role of a group of independent variables concerning the time taken until clearance was evaluated using a Cox proportional-hazards regression model or parametric (log-logistic) methods when necessary. Regarding viral load, the Wilcoxon rank-sum test was used for measuring the difference of medians for viral load for each type, according to the state of infection (cleared or persistent). The Kruskal-Wallis test was used for evaluating the change in the women's colposcopy findings at the start of follow-up and at the end of it (whether due to clearance or the end of the follow-up period).Results: It was found that HPV-18 and HPV-31 types had the lowest probability of becoming cleared (1.76 and 2.75 per 100 patients/month rate, respectively). Women from Colombian cities other than Bogotá had a greater probability of being cleared if they had HPV-16 (HR 2.58: 1.51-4.4 95% CI) or HPV-58 (1.79 time ratio: 1.33-2.39 95% CI) infection. Regarding viral load, HPV-45-infected women having 1 × 106 to 9.99 × 109 viral copies had better clearance compared to those having greater viral loads (1.61 time ratio: 1.01-2.57 95% CI). Lower HPV-31 viral load values were associated with this type's persistence and changes in colposcopy findings for HPV-16 gave the worst prognosis in women having low absolute load values.Conclusions: HPV infection clearance in this study was related to factors such as infection type, viral load and the characteristics of the cities from which the women came. Low viral load values would indicate viral persistence and a worse prognosis regarding a change in colposcopy findings. © 2014 Soto-De León et al.; licensee BioMed Central Ltd

Transcriptome profiling of gene expression during immunisation trial against Fasciola hepatica : Identification of genes and pathways involved in conferring immunoprotection in a murine model

Background: Fasciolosis remains a significant food-borne trematode disease causing high morbidity around the world and affecting grazing animals and humans. A deeper understanding concerning the molecular mechanisms by which Fasciola hepatica infection occurs, as well as the molecular basis involved in acquiring protection is extremely important when designing and selecting new vaccine candidates. The present study provides a first report of microarray-based technology for describing changes in the splenic gene expression profile for mice immunised with a highly effective, protection-inducing, multi-epitope, subunit-based, chemically-synthesised vaccine candidate against F. hepatica. Methods: The mice were immunised with synthetic peptides containing B- and T-cell epitopes, which are derived from F. hepatica cathepsin B and amoebapore proteins, as novel vaccine candidates against F. hepatica formulated in an adjuvant adaptation vaccination system; they were experimentally challenged with F. hepatica metacercariae. Spleen RNA from mice immunised with the highest protection-inducing synthetic peptides was isolated, amplified and labelled using Affymetrix standardised protocols. Data was then background corrected, normalised and the expression signal was calculated. The Ingenuity Pathway Analysis tool was then used for analysing differentially expressed gene identifiers for annotating bio-functions and constructing and visualising molecular interaction networks. Results: Mice immunised with a combination of three peptides containing T-cell epitopes induced high protection against experimental challenge according to survival rates and hepatic damage scores. It also induced differential expression of 820 genes, 168 genes being up-regulated and 652 genes being down-regulated, p value <0.05, fold change ranging from -2.944 to 7.632. A functional study of these genes revealed changes in the pathways related to nitric oxide and reactive oxygen species production, Interleukin-12 signalling and production in macrophages and Interleukin-8 signalling with up-regulation of S100 calcium-binding protein A8, Matrix metallopeptidase 9 and CXC chemokine receptor 2 genes. Conclusion: The data obtained in the present study provided us with a more comprehensive overview concerning the possible molecular pathways implied in inducing protection against F. hepatica in a murine model, which could be useful for evaluating future vaccine candidates. © 2017 The Author(s)

On the Evolution and Function of Plasmodium vivax Reticulocyte Binding Surface Antigen (pvrbsa)

The RBSA protein is encoded by a gene described in Plasmodium species having tropism for reticulocytes. Since this protein is antigenic in natural infections and can bind to target cells, it has been proposed as a potential candidate for an anti-Plasmodium vivax vaccine. However, genetic diversity (a challenge which must be overcome for ensuring fully effective vaccine design) has not been described at this locus. Likewise, the minimum regions mediating specific parasite-host interaction have not been determined. This is why the rbsa gene’s evolutionary history is being here described, as well as the P. vivax rbsa (pvrbsa) genetic diversity and the specific regions mediating parasite adhesion to reticulocytes. Unlike what has previously been reported, rbsa was also present in several parasite species belonging to the monkey-malaria clade; paralogs were also found in Plasmodium parasites invading reticulocytes. The pvrbsa locus had less diversity than other merozoite surface proteins where natural selection and recombination were the main evolutionary forces involved in causing the observed polymorphism. The N-terminal end (PvRBSA-A) was conserved and under functional constraint; consequently, it was expressed as recombinant protein for binding assays. This protein fragment bound to reticulocytes whilst the C-terminus, included in recombinant PvRBSA-B (which was not under functional constraint), did not. Interestingly, two PvRBSA-A-derived peptides were able to inhibit protein binding to reticulocytes. Specific conserved and functionally important peptides within PvRBSA-A could thus be considered when designing a fully-effective vaccine against P. vivax

Inferring Plasmodium vivax protein biology by using omics data

Deciphering Plasmodium vivax biology has long been a challenge for groups working on this parasite, mainly due to the complications involved in propagating it in vitro. However, adapting P. vivax strains in non-human primates and the arrival of high-performance analysis methods has led to increased knowledge regarding parasite protein composition and the ability of some molecules to trigger an immune response or participate in protein-protein interactions. This review describes the state of the art concerning proteomics-, immunomics- and interatomics-related P. vivax omic studies, discussing their potential use in developing disease control methods

Vacunas contra Plasmodium vivax : un desafío de investigación

La malaria causada por Plasmodium vivax sigue siendo un problema de salud pública en áreas tropicales y subtropicales en todo el mundo. A pesar de la importancia epidemiológica de esta especie, su complejidad biológica ha obstaculizado los avances en el campo del desarrollo de vacunas. Pocos antígenos se han descrito y analizado hasta la fecha en estudios preclínicos y clínicos, destacando así el gran desafío que enfrentan los grupos que actualmente trabajan en esta especie de parásito. Esta revisión resume el trabajo más representativo realizado durante los últimos años y analiza los enfoques adoptados para avanzar hacia una vacuna contra Plasmodium vivax .Malaria caused by Plasmodium vivax continues being a public health problem in tropical and subtropical areas throughout the whole world. In spite of this species’ epidemiological importance, its biological complexity has hampered advances being made in the field of vaccine development. Few antigens have been described and analyzed to date in preclinical and clinical studies, thereby highlighting the great challenge facing groups currently working on this parasite species. This review summarizes the most representative work done during the last few years and discusses the approaches adopted in making progress towards an anti-Plasmodium vivax vaccine

Characterizing PvARP, a novel Plasmodium vivax antigen

Background Plasmodium vivax continues to be the most widely distributed malarial parasite species in tropical and sub-tropical areas, causing high morbidity indices around the world. Better understanding of the proteins used by the parasite during the invasion of red blood cells is required to obtain an effective vaccine against this disease. This study describes characterizing the P. vivax asparagine-rich protein (PvARP) and examines its antigenicity in natural infection. Methods The target gene in the study was selected according to a previous in silico analysis using profile hidden Markov models which identified P. vivax proteins that play a possible role in invasion. Transcription of the arp gene in the P. vivax VCG-1 strain was here evaluated by RT-PCR. Specific human antibodies against PvARP were used to confirm protein expression by Western blot as well as its subcellular localization by immunofluorescence. Recognition of recombinant PvARP by sera from P. vivax-infected individuals was evaluated by ELISA. Results VCG-1 strain PvARP is a 281-residue-long molecule, which is encoded by a single exon and has an N-terminal secretion signal, as well as a tandem repeat region. This protein is expressed in mature schizonts and is located on the surface of merozoites, having an apparent accumulation towards their apical pole. Sera from P. vivax-infected patients recognized the recombinant, thereby suggesting that this protein is targeted by the immune response during infection

Reticulocytes: Plasmodium vivax target cells

Reticulocytes represent the main invasion target for Plasmodium vivax, the second most prevalent parasite species around the world causing malaria in humans. In spite of these cells' importance in research into malaria, biological knowledge related to the nature of the host has been limited, given the technical difficulties present in working with them in the laboratory. Poor reticulocyte recovery from total blood, by different techniques, has hampered continuous in vitro P. vivax cultures being developed, thereby delaying basic investigation in this parasite species. Intense research during the last few years has led to advances being made in developing methodologies orientated towards obtaining enriched reticulocytes from differing sources, thereby providing invaluable information for developing new strategies aimed at preventing infection caused by malaria. This review describes the most recent studies related to obtaining reticulocytes and discusses approaches which could contribute towards knowledge regarding molecular interactions between target cell proteins and their main infective agent, P. vivax. © 2013 Société Française des Microscopies and Société de Biologie Cellulaire de France

Reticulocytes: Plasmodium vivax target cells

Reticulocytes represent the main invasion target for Plasmodium vivax, the second most prevalent parasite species around the world causing malaria in humans. In spite of these cells' importance in research into malaria, biological knowledge related to the nature of the host has been limited, given the technical difficulties present in working with them in the laboratory. Poor reticulocyte recovery from total blood, by different techniques, has hampered continuous in vitro P. vivax cultures being developed, thereby delaying basic investigation in this parasite species. Intense research during the last few years has led to advances being made in developing methodologies orientated towards obtaining enriched reticulocytes from differing sources, thereby providing invaluable information for developing new strategies aimed at preventing infection caused by malaria. This review describes the most recent studies related to obtaining reticulocytes and discusses approaches which could contribute towards knowledge regarding molecular interactions between target cell proteins and their main infective agent, P. vivax. © 2013 Société Française des Microscopies and Société de Biologie Cellulaire de France