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

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium vivax </it>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 <it>Pf</it>RONs, <it>Pf</it>92, <it>Pf</it>38, <it>Pf</it>12 and <it>Pf</it>34, 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 <it>P. vivax </it>rhoptry neck protein 1 (<it>Pv</it>RON1) and examine its antigenicity in natural <it>P. vivax </it>infections.</p> <p>Methods</p> <p>The <it>Pv</it>RON1 encoding gene, which is homologous to that encoding the <it>P. falciparum </it>apical sushi protein (ASP) according to the plasmoDB database, was selected as our study target. The <it>pvron1 </it>gene transcription was evaluated by RT-PCR using RNA obtained from the <it>P. vivax </it>VCG-1 strain. Two peptides derived from the deduced <it>P. vivax </it>Sal-I <it>Pv</it>RON1 sequence were synthesized and inoculated in rabbits for obtaining anti-<it>Pv</it>RON1 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 <it>Pv</it>RON1 protein was assessed using human sera from individuals previously exposed to <it>P. vivax </it>malaria by ELISA.</p> <p>Results</p> <p>In the <it>P. vivax </it>VCG-1 strain, RON1 is a 764 amino acid-long protein. <it>In silico </it>analysis has revealed that <it>Pv</it>RON1 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 <it>Pv</it>RON1 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 <it>P. vivax</it>.</p> <p>Conclusions</p> <p>This study shows the identification and characterization of the <it>P. vivax </it>rhoptry neck protein 1 in the VCG-1 strain. Taking into account that <it>Pv</it>RON1 shares several important characteristics with other <it>Plasmodium </it>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 <it>Aotus </it>monkey model are thus recommended.</p

The in vitro antigenicity of Plasmodium vivax rhoptry neck protein 2 (PvRON2) B- and T-epitopes selected by HLA-DRB1 binding profile

Malaria caused by Plasmodium vivax is a neglected disease which is responsible for the highest morbidity in both Americas and Asia. Despite continuous public health efforts to prevent malarial infection, an effective antimalarial vaccine is still urgently needed. P. vivax vaccine development involves analyzing naturally-infected patients' immune response to the specific proteins involved in red blood cell invasion. The P. vivax rhoptry neck protein 2 (PvRON2) is a highly conserved protein which is expressed in late schizont rhoptries; it interacts directly with AMA-1 and might be involved in moving-junction formation. Bioinformatics approaches were used here to select B- and T-cell epitopes. Eleven high-affinity binding peptides were selected using the NetMHCIIpan-3.0 in silico prediction tool; their in vitro binding to HLA-DRB1*0401, HLA-DRB1*0701, HLA-DRB1*1101 or HLA-DRB1*1302 was experimentally assessed. Four peptides (39152 (HLA-DRB1*04 and 11), 39047 (HLA-DRB1*07), 39154 (HLADRB1*13) and universal peptide 39153) evoked a naturally-acquired T-cell immune response in P. vivax-exposed individuals from two endemic areas in Colombia. All four peptides had an SI greater than 2 in proliferation assays; however, only peptides 39154 and 39153 had significant differences compared to the control group. Peptide 39047 was able to significantly stimulate TNF and IL-10 production while 39154 stimulated TNF production. Allele-specific peptides (but not the universal one) were able to stimulate IL-6 production; however, none induced IFN-? production. The Bepipred 1.0 tool was used for selecting four B-cell epitopes in silico regarding humoral response. Peptide 39041 was the only one recognized by P. vivax-exposed individuals' sera and had significant differences concerning IgG subclasses; an IgG2 > IgG4 profile was observed for this peptide, agreeing with a protection-inducing role against P. falciparum and P. vivax as previously described for antigens such as RESA and MSP2. The bioinformatics results and in vitro evaluation reported here highlighted two T-cell epitopes (39047 and 39154) being recognized by memory cells and a B-cell epitope (39041) identified by P. vivax-exposed individuals' sera which could be used as potential candidates when designing a subunit-based vaccine. © 2018 López, Yepes-Pérez, Díaz-Arévalo, Patarroyo and Patarroyo

NClassG+: A classifier for non-classically secreted Gram-positive bacterial proteins

<p>Abstract</p> <p>Background</p> <p>Most predictive methods currently available for the identification of protein secretion mechanisms have focused on classically secreted proteins. In fact, only two methods have been reported for predicting non-classically secreted proteins of Gram-positive bacteria. This study describes the implementation of a sequence-based classifier, denoted as NClassG+, for identifying non-classically secreted Gram-positive bacterial proteins.</p> <p>Results</p> <p>Several feature-based classifiers were trained using different sequence transformation vectors (frequencies, dipeptides, physicochemical factors and PSSM) and Support Vector Machines (SVMs) with Linear, Polynomial and Gaussian kernel functions. Nested <it>k</it>-fold cross-validation (CV) was applied to select the best models, using the inner CV loop to tune the model parameters and the outer CV group to compute the error. The parameters and Kernel functions and the combinations between all possible feature vectors were optimized using grid search.</p> <p>Conclusions</p> <p>The final model was tested against an independent set not previously seen by the model, obtaining better predictive performance compared to SecretomeP V2.0 and SecretPV2.0 for the identification of non-classically secreted proteins. NClassG+ is freely available on the web at <url>http://www.biolisi.unal.edu.co/web-servers/nclassgpositive/</url></p

3D Analysis of the TCR/pMHCII Complex Formation in Monkeys Vaccinated with the First Peptide Inducing Sterilizing Immunity against Human Malaria

T-cell receptor gene rearrangements were studied in Aotus monkeys developing high antibody titers and sterilizing immunity against the Plasmodium falciparum malaria parasite upon vaccination with the modified synthetic peptide 24112, which was identified in the Merozoite Surface Protein 2 (MSP-2) and is known to bind to HLA-DRβ1*0403 molecules with high capacity. Spectratyping analysis showed a preferential usage of Vβ12 and Vβ6 TCR gene families in 67% of HLA-DRβ1*0403-like genotyped monkeys. Docking of peptide 24112 into the HLA-DRβ1*0401–HA peptide–HA1.7TCR complex containing the VDJ rearrangements identified in fully protected monkeys showed a different structural signature compared to nonprotected monkeys. These striking results show the exquisite specificity of the TCR/pMHCII complex formation needed for inducing sterilizing immunity and provide important hints for a logical and rational methodology to develop multiepitopic, minimal subunit-based synthetic vaccines against infectious diseases, among them malaria

Using the PfEMP1 Head Structure Binding Motif to Deal a Blow at Severe Malaria

Plasmodium falciparum (Pf) malaria causes 200 million cases worldwide, 8 million being severe and complicated leading to similar to 1 million deaths and similar to 100,000 abortions annually. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) has been implicated in cytoadherence and infected erythrocyte rosette formation, associated with cerebral malaria; chondroitin sulphate-A attachment and infected erythrocyte sequestration related to pregnancy-associated malaria and other severe forms of disease. An endothelial cell high activity binding peptide is described in several of this similar to 300 kDa hypervariable protein's domains displaying a conserved motif (GACxPxRRxxLC); it established H-bonds with other binding peptides to mediate red blood cell group A and chondroitin sulphate attachment. This motif (when properly modified) induced PfEMP1-specific strain-transcending, fully-protective immunity for the first time in experimental challenge in Aotus monkeys, opening the way forward for a long sought-after vaccine against severe malaria

Validating subcellular localization prediction tools with mycobacterial proteins

<p>Abstract</p> <p>Background</p> <p>The computational prediction of mycobacterial proteins' subcellular localization is of key importance for proteome annotation and for the identification of new drug targets and vaccine candidates. Several subcellular localization classifiers have been developed over the past few years, which have comprised both general localization and feature-based classifiers. Here, we have validated the ability of different bioinformatics approaches, through the use of SignalP 2.0, TatP 1.0, LipoP 1.0, Phobius, PA-SUB 2.5, PSORTb v.2.0.4 and Gpos-PLoc, to predict secreted bacterial proteins. These computational tools were compared in terms of sensitivity, specificity and Matthew's correlation coefficient (MCC) using a set of mycobacterial proteins having less than 40% identity, none of which are included in the training data sets of the validated tools and whose subcellular localization have been experimentally confirmed. These proteins belong to the TBpred training data set, a computational tool specifically designed to predict mycobacterial proteins.</p> <p>Results</p> <p>A final validation set of 272 mycobacterial proteins was obtained from the initial set of 852 mycobacterial proteins. According to the results of the validation metrics, all tools presented specificity above 0.90, while dispersion sensitivity and MCC values were above 0.22. PA-SUB 2.5 presented the highest values; however, these results might be biased due to the methodology used by this tool. PSORTb v.2.0.4 left 56 proteins out of the classification, while Gpos-PLoc left just one protein out.</p> <p>Conclusion</p> <p>Both subcellular localization approaches had high predictive specificity and high recognition of true negatives for the tested data set. Among those tools whose predictions are not based on homology searches against SWISS-PROT, Gpos-PLoc was the general localization tool with the best predictive performance, while SignalP 2.0 was the best tool among the ones using a feature-based approach. Even though PA-SUB 2.5 presented the highest metrics, it should be taken into account that this tool was trained using all proteins reported in SWISS-PROT, which includes the protein set tested in this study, either as a BLAST search or as a training model.</p

Prevalencia de VPH-ADN y anticuerpos anti-VPH en mujeres de girardot, Colombia

Objective: To assess the frequency of HPV-DNA detection, human papillomavirus (HPV) seropositivity, presence of cervical lesions, and its relationship with certain socio-demographic factors in women from Girardot, Colombia from 2006 to 2007. Methods: Nine hundred fifty-three women attending their regular Pap smear control voluntarily provided cervical cells and blood samples for HPV-DNA analysis and ELISA detection of anti-L1 peptides and virus-like particles (VLPs) antibodies after answering a questionnaire regarding sexual behaviors, number of births, smoking habits, and socio-demographic background. Results: Twenty-six of the 953 women being examined (2.73%) presented cervical cell abnormalities. A frequency of 36.62% (95% CI: 33.52%–39.7%) HPV seropositivity was detected with peptide 18301, 35.36% (95% CI: 32.3%–38.4%) with 18283, and 32.95% (95% CI: 29.9%–36%) with 18294, whereas VLPs detected a 43% seropositivity (95% CI: 39.8%– 46.2%). Antibody frequency found with all peptides was significantly higher in women having cervical abnormalities (atypical squamous cells of undetermined significance and high-grade squamous intraephitelial lesions) compared with those having normal cytologies. Peptide 18283 reported a significantly higher seropositivity (35.71%) in women 44 years old, whereas peptides 18301 and 18294 evidenced a significantly lower seropositivity in those who had never given birth. HR-HPV-DNA was detected in 157 (20.50%) of 766 cervical samples amplifying positively for the -globin housekeeping gene. Conclusion: Peptides 18283, 18294, and 18301 were more specific and more sensitive than VLPs for detecting women with HR-HPV-DNA positive cervical lesions. Therefore, they could be useful in the design of a serological test for detecting HR-HPV-infected women having cervical lesions at a risk of progressing to cervical cancer

Molecular modeling and in silico characterization of Mycobacterium tuberculosis TlyA: Possible misannotation of this tubercle bacilli-hemolysin

<p>Abstract</p> <p>Background</p> <p>The TlyA protein has a controversial function as a virulence factor in <it>Mycobacterium tuberculosis </it>(<it>M. tuberculosis</it>). At present, its dual activity as hemolysin and RNA methyltransferase in <it>M. tuberculosis </it>has been indirectly proposed based on <it>in vitro </it>results. There is no evidence however for TlyA relevance in the survival of tubercle bacilli inside host cells or whether both activities are functionally linked. A thorough analysis of structure prediction for this mycobacterial protein in this study shows the need for reevaluating TlyA's function in virulence.</p> <p>Results</p> <p>Bioinformatics analysis of TlyA identified a ribosomal protein binding domain (S4 domain), located between residues 5 and 68 as well as an FtsJ-like methyltranferase domain encompassing residues 62 and 247, all of which have been previously described in translation machinery-associated proteins. Subcellular localization prediction showed that TlyA lacks a signal peptide and its hydrophobicity profile showed no evidence of transmembrane helices. These findings suggested that it may not be attached to the membrane, which is consistent with a cytoplasmic localization. Three-dimensional modeling of TlyA showed a consensus structure, having a common core formed by a six-stranded β-sheet between two α-helix layers, which is consistent with an RNA methyltransferase structure. Phylogenetic analyses showed high conservation of the <it>tlyA </it>gene among <it>Mycobacterium </it>species. Additionally, the nucleotide substitution rates suggested purifying selection during <it>tlyA </it>gene evolution and the absence of a common ancestor between TlyA proteins and bacterial pore-forming proteins.</p> <p>Conclusion</p> <p>Altogether, our manual <it>in silico </it>curation suggested that TlyA is involved in ribosomal biogenesis and that there is a functional annotation error regarding this protein family in several microbial and plant genomes, including the <it>M. tuberculosis </it>genome.</p

Mycobacterium tuberculosis Rv0679c protein sequences involved in host-cell infection: Potential TB vaccine candidate antigen

Hasta la fecha, la función de muchas proteínas de membrana hipotéticas de Mycobacterium tuberculosis aún se desconoce y su participación en las interacciones patógeno-huésped aún no se ha definido claramente. En este estudio, se evaluó la actividad biológica de los péptidos derivados de la proteína de membrana hipotética Rv0679c de M. tuberculosis y su participación en las interacciones patógeno-huésped. La transcripción del gen Rv0679c se estudió en 26 Mycobacteriumspp. Son. Los anticuerpos generados contra los supuestos epítopos de células B de Rv0679c se usaron en ensayos de inmunotransferencia y microscopía inmunoelectrónica. Los péptidos sintéticos que abarcan toda la longitud de la proteína fueron probados por su capacidad para unirse a las células A549 y U937. Los péptidos de unión de alta actividad (HABP) identificados en Rv0679c se probaron para determinar su capacidad para inhibir la invasión de micobacterias en las células.To date, the function of many hypothetical membrane proteins of Mycobacterium tuberculosis is still unknown and their involvement in pathogen-host interactions has not been yet clearly defined. In this study, the biological activity of peptides derived from the hypothetical membrane protein Rv0679c of M. tuberculosis and their involvement in pathogen-host interactions was assessed. Transcription of the Rv0679c gene was studied in 26 Mycobacterium spp. Strains. Antibodies raised against putative B-cell epitopes of Rv0679c were used in Western blot and immunoelectron microscopy assays. Synthetic peptides spanning the entire length of the protein were tested for their ability to bind to A549 and U937 cells. High-activity binding peptides (HABPs) identified in Rv0679c were tested for their ability to inhibit mycobacterial invasion into cells