[Show abstract][Hide abstract] ABSTRACT: Analysing human papillomavirus (HPV) viral load is important in determining the risk of developing cervical cancer (CC); most knowledge to date regarding HPV viral load and cervical lesions has been related to HPV-16. This study evaluated the association between the viral load of the six most prevalent high-risk viral types in Colombia and cervical intraepithelial neoplasia (CIN) frequency.
114 women without CIN and 59 women having CIN confirmed by colposcopy, all of them positive by conventional PCR for HPV infection in the initial screening, were included in the study. Samples were tested for six high-risk HPV types to determine viral copy number by real-time PCR. Crude and adjusted odds ratios (ORa) were estimated for evaluating the association between each viral type's DNA load and the risk of cervical lesions occurring.
The highest viral loads were identified for HPV-33 in CIN patients and for HPV-31 in patients without lesions (9.33 HPV copies, 2.95 interquartile range (IQR); 9.41 HPV copies, 2.58 IQR). Lesions were more frequent in HPV-16 patients having a low viral load (3.53 ORa, 1.16-10.74 95%CI) compared to those having high HPV-16 load (2.62 ORa, 1.08-6.35 95%CI). High viral load in HPV-31 patients was associated with lower CIN frequency (0.34 ORa, 0.15-0.78 95%CI).
An association between HPV DNA load and CIN frequency was seen to be type-specific and may have depended on the duration of infection. This analysis has provided information for understanding the effect of HPV DNA load on cervical lesion development.
BMC Cancer 12/2015; 15(1):1126. DOI:10.1186/s12885-015-1126-z · 3.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The review covers the functional and structural approach followed by our group for more than 34 years in the search for a methodology that allows the rational design of chemically synthesized vaccines. An analogy between Odysseus, the cunning hero of the epic poem Odyssey by Homer, and the elusive Plasmodium parasite has been made, to review our research group's main considerations when developing a rational methodology for designing second generation, modified peptide-based, minimal subunit, multi-antigen, multi-stage, chemically synthesised vaccines against Plasmodium falciparum malaria.
[Show abstract][Hide abstract] ABSTRACT: Studying proteins from the M. tuberculosis H37Rv envelop is important for understanding host-pathogen interaction regarding bacterial infection and survival within a host; such knowledge is indispensable regarding studies aimed at developing drugs or vaccines against tuberculosis, a disease which continues to cause more than one million deaths worldwide every year. The present work presents a study of the Rv3705c protein which has been described as being an outer protein. Several servers and bioinformatics' tools were used for predicting its location on mycobacterial surface and a 3D model of the protein was obtained which was then compared to experimental circular dichroism results for its peptides. PCR assays were used for corroborating rv3705c gene presence and transcription in a laboratory strain and immunoblotting and electron microscopy were used for confirming protein localisation on cell envelop.Receptor-ligand assays revealed two peptides having high specific binding (HABPs); peptide 38485 (121DRAFHRVVDRTVGTSGQTTA140) bound to both cell lines used as infection target (U937 and A549 epithelial cell line-derived macrophages) and 38488 (181RLRENVLLQAKVTQSGNAGP200) bound to U937 cells. It was found that peptide 38485 provided significant inhibition regarding mycobacterial entry to both cell lines in in vitro assays. These results led to proposing peptide 38485 as one of the epitopes to be used in future studies aimed at characterising the immune response of functionally important synthetic peptides which could be included in developing a synthetic anti-tuberculosis vaccine. This article is protected by copyright. All rights reserved
Journal of Cellular Biochemistry 09/2015; DOI:10.1002/jcb.25379 · 3.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Biochemical, structural and single amino acid level analysis of 49 Plasmodium falciparum protein regions (13 sporozoite and 36 merozoite proteins) has highlighted the functional role of each conserved high activity binding peptide (cHABP) in cell host-microbe interaction, involving biological functions such as gliding motility, traversal activity, binding invasion, reproduction, nutrient ion transport and the development of severe malaria. Each protein's key function in the malaria parasite's asexual lifecycle (pre-erythrocyte and erythro-cyte) is described in terms of cHABPs; their sequences were located in elegant work published by other groups regarding critical binding regions implicated in malarial parasite invasion. Such cHABPs represent the starting point for developing a logical and rational methodology for selecting an appropriate mixture of modified cHABPs to be used in a completely effective, synthetic antimalarial vaccine. Such methodology could be used for developing vaccines against diseases scourging humanity.
Current issues in molecular biology 08/2015; 18(1):57-78. · 5.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction:
Obtaining an effective antimalarial vaccine has represented one of the biggest public health challenges over the last 50 years. Despite efforts by many laboratories around the world using whole-organism, recombinant proteins and genome-based approaches, the results have been disappointing. One of the main problems when designing an antimalarial vaccine is the poor immunogenicity induced by the functionally relevant and conserved protein regions of the parasite. Areas covered: This review focuses on the logical and rational methodology followed to identify Plasmodium falciparum conserved functional regions with the ability to bind to target cells conserved high activity binding peptides (cHABPs) and the physicochemical and immunological characteristics that should be taken into account for modifying them into highly immunogenic and protection-inducing peptides (mHABPs) into highly immunogenic and protection-inducing in Aotus monkeys. Expert opinion: The functional approach taken to develop a fully protective, minimal subunit-based, multiantigenic, multistage and synthetic peptide-based antimalarial vaccine has shown promising results. The clear relationship observed between mHABPs structure and their immunological properties highlights the challenges and opportunities arising from this methodology, as well as the universal principles and rules derived therefrom.
[Show abstract][Hide abstract] ABSTRACT: Determining immune protection-inducing protein structures (IMPIPS) involves defining the stereo-electron and topochemical characteristics which are essential in MHC-p-TCR complex formation. Modified high activity binding peptides (mHABP) were thus synthesised to produce a large panel of IMPIPS measuring 26.5 ±3.5Å between the farthest atoms fitting into Pockets 1 to 9 of HLA-DRβ1∗structures. They displayed a polyproline II-like (PPIIL) structure with their backbone O and N atoms orientated to establish H-bonds with specific residues from HLA-DRβ∗-peptide binding regions (PBR). Residues having specific charge and gauche+ orientation regarding p3χ1, p5χ2, and p7χ1 angles determined appropriate rotamer orientation for perfectly fitting into the TCR to induce an appropriate immune response. Immunological assays in Aotus monkeys involving IMPIPS mixtures led to promising results; taken together with the aforementioned physicochemical principles, noninterfering, long-lasting, protection-inducing, multi-epitope, multistage, minimal subunit-based chemically-synthesised peptides can be designed against diseases scourging humankind.
PLoS ONE 04/2015; 10(4):e0123249. DOI:10.1371/journal.pone.0123249 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Malaria parasites have their Achilles' heel; they are vulnerable in small parts of their relevant molecules where they can be wounded and killed. These are sporozoite and merozoite protein conserved high activity binding peptides (cHABPs), playing a critical role in binding to and invasion of host cells (hepatocytes and erythrocytes, respectively). cHABPs can be modified by specific amino acid replacement, according to previously published physicochemical rules, to produce analogues (mHABPs) having left-handed polyproline II (PPIIL)-like structures which can modulate an immune response due to fitting perfectly into the HLA-DRβ1* peptide binding region (PBR) and having an appropriate presentation to the T-cell receptor (TCR).
Current issues in molecular biology 04/2015; 18(1):11-20. · 5.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Developing novel generations of subunit-based antimalarial vaccines in the form of chemically-defined macromolecule systems for multiple antigen presentation represents a classical problem in the field of vaccine development. Many efforts involving synthesis strategies leading to macromolecule constructs have been based on dendrimer-like systems, the condensation of large building blocks and conventional asymmetric double dimer constructs, all based on lysine cores. This work describes novel symmetric double dimer and condensed linear constructs for presenting selected peptide multi-copies from the apical sushi protein expressed in Plasmodium falciparum. These molecules have been proved to be safe and innocuous, highly antigenic and have shown strong protective efficacy in rodents challenged with two Plasmodium species. Insights into systematic design, synthesis and characterisation have led to such novel antigen systems being used as potential platforms for developing new anti-malarial vaccine candidates.
Biochemical and Biophysical Research Communications 07/2014; 451(1). DOI:10.1016/j.bbrc.2014.06.143 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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).
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.
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.
[Show abstract][Hide abstract] ABSTRACT: Tuberculosis (TB) continues being one of the diseases having the greatest mortality rates around the world, 8.7 million cases having been reported in 2011. An efficient vaccine against TB having a great impact on public health is an urgent need. Usually, selecting antigens for vaccines has been based on proteins having immunogenic properties for patients suffering TB and having had promising results in mice and non-human primates. Our approach has been based on a functional approach involving the pathogen–host interaction in the search for antigens to be included in designing an efficient, minimal, subunit-based anti-TB vaccine. This means that Mycobacterium tuberculosis has mainly been involved in studies and that lipoproteins represent an important kind of protein on the cell envelope which can also contribute towards this pathogen's virulence. This study has assessed the expression of four lipoproteins from M. tuberculosis H37Rv, that is, Rv1411c (LprG), Rv1911c (LppC), Rv2270 (LppN) and Rv3763 (LpqH), and the possible biological activity of peptides derived from these. Five peptides were found for these proteins which had high specific binding to both alveolar A549 epithelial cells and U937 monocyte-derived macrophages which were able to significantly inhibit mycobacterial entry to these cells in vitro.
Chemical Biology & Drug Design 07/2014; 84(6). DOI:10.1111/cbdd.12365 · 2.49 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
HIV infection leads to a decreasing immune response, thereby facilitating the appearance of other infections, one of the most important ones being HPV. However, studies are needed for determining associations between immunodeficiency caused by HIV and/or the presence of HPV during the course of cervical lesions and their degree of malignancy. This study describes the cytological findings revealed by the Papanicolaou test, laboratory characteristics and HPV molecular profile in women with and without HIV infection.
A total of 216 HIV-positive and 1,159 HIV-negative women were invited to participate in the study; PCR was used for the molecular detection of HPV in cervical samples. Statistical analysis (such as percentages, Chi-square test and Fisher’s exact test when applicable) determined human papillomavirus (HPV) infection frequency (single and multiple) and the distribution of six types of high-risk-HPV in women with and without HIV infection. Likewise, a logistic regression model was run to evaluate the relationship between HIV-HPV infection and different risk factors.
An association was found between the frequency of HPV infection and infection involving 2 or more HPV types (also known as multiple HPV infection) in HIV-positive women (69.0% and 54.2%, respectively); such frequency was greater than that found in HIV-negative women (44.3% and 22.7%, respectively). Statistically significant differences were observed between both groups (p = 0.001) regarding HPV presence (both in infection and multiple HPV infection). HPV-16 was the most prevalent type in the population being studied (p = 0.001); other viral types had variable distribution in both groups (HIV-positive and HIV-negative). HPV detection was associated with <500 cell/mm3 CD4-count (p = 0.004) and higher HIV-viral-load (p = 0.001). HPV-DNA detection, <200 cell/mm3 CD4-count (p = 0.001), and higher HIV-viral-load (p = 0.001) were associated with abnormal cytological findings.
The HIV-1 positive population in this study had high multiple HPV infection prevalence. The results for this population group also suggested a greater association between HPV-DNA presence and cytological findings. HPV detection, together with low CD4 count, could represent useful tools for identifying HIV-positive women at risk of developing cervical lesions.
BMC Cancer 06/2014; 14(1):451. DOI:10.1186/1471-2407-14-451 · 3.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Non-human primates belonging to the Aotus genus have been shown to be excellent experimental models for evaluating drugs and vaccine candidates against malaria and other human diseases. The immune system of this animal model must be characterised to assess whether the results obtained here can be extrapolated to humans. Class I and II major histocompatibility complex (MHC) proteins are amongst the most important molecules involved in response to pathogens; in spite of this, the techniques available for genotyping these molecules are usually expensive and/or time-consuming. Previous studies have reported MHC-DRB class II gene typing by microsatellite in Old World primates and humans, showing that such technique provides a fast, reliable and effective alternative to the commonly used ones. Based on this information, a microsatellite present in MHC-DRB intron 2 and its evolutionary patterns were identified in two Aotus species (A. vociferans and A. nancymaae), as well as its potential for genotyping class II MHC-DRB in these primates.
PLoS ONE 05/2014; 9(5):e96973. DOI:10.1371/journal.pone.0096973 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Topological and stereo-electron characteristics are essential in major histocompability class II-peptide-T-cell receptor (MHC-p-TCR) complex formation for inducing an appropriate immune response. Modified high activity binding peptides (mHABPs) were synthesised for complete full protection antimalarial vaccine development producing a large panel of individually fully protection-inducing protein structures (FPIPS) and very high long-lasting antibody-inducing (VHLLAI) mHABPs. Most of those which did not interfere, compete, inhibit or suppress their individual VHLLAI or FPIPS activity contained or displayed a polyproline II-like (PPIIL) structure when mixed. Here we show that amino acid side-chains located in peptide binding region (PBR) positions p3 and p7 displayed specific electron charges and side-chain gauche(+) orientation for interacting with the TCR. Based on the above, and previously described physicochemical principles, non-interfering, long-lasting, full protection-inducing, multi-epitope, multistage, minimal subunit-based chemically synthesised mHABP mixtures can be designed for developing vaccines against diseases scourging humankind, malaria being one of them.
[Show abstract][Hide abstract] ABSTRACT: Plasmodium falciparum (Pf) malaria causes 200 million cases worldwide, 8 million being severe and complicated leading to ∼1 million deaths and ∼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 ∼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.
PLoS ONE 02/2014; 9(2):e88420. DOI:10.1371/journal.pone.0088420 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous BAC clone analysis of the Platyrrhini owl monkey KIRs have shown an unusual genetic structure in some loci. Therefore, cDNAs encoding KIR molecules from eleven Aotus vociferans monkeys were characterized here; ten putative KIR loci were found, some of which encoded atypical proteins such as KIR4DL and transcripts predicted to encode a D0+D1 configuration (AOTVOKIR2DL1*01v1) which appear to be unique in the Aotus genus. Furthermore, alternative splicing was found as a likely mechanism for producing activator receptors in A. vociferans species. KIR proteins from New World monkeys may be split into three new lineages according to domain by domain phylogenetic analysis. Although the A. vociferans KIR family displayed a high divergence among paralogous genes, individual loci were limited in their genetic polymorphism. Selection analysis showed that both constrained and rapid evolution may operate within the AvKIR family. The frequent alternative splicing (as a likely mechanism generating activator receptors), the presence of KIR4DL and KIR2DL1 (D0+D1) molecules and other data reported here suggest that the KIR family in Aotus has had a rapid evolution, independent from its Catarrhini counterparts.
PLoS ONE 11/2013; 8(11):e79731. DOI:10.1371/journal.pone.0079731 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human papillomavirus (HPV) is the main risk factor associated with the development of cervical cancer (CC); however, there are other factors that favour the progression of the illness such as immunosuppression caused by the human immunodeficiency virus (HIV). This study was thus aimed at evaluating the functionality of classical polymerase chain reaction (PCR)-based molecular tests for the generic identification of HPV-DNA (GP5+/6+, MY09/11, pU1M/2R individually or in combination) using cervical and urine samples from 194 HIV-positive women. Infected samples were tested with type-specific primers for six high-risk types (HPV-16, -18, -31, -33, -45 and -58) and two low risk ones (HPV-6/11). HPV infection prevalence in cervical samples was 70.1% and 63.9% in urine samples. HPV-16 was the most prevalent viral type in both cervical and urine samples, having high multiple infection frequency compared to single infections detected in such samples. HPV-DNA detection by PCR (mainly pU1M/2R primer set) in urine samples was positively associated with abnormal cytological findings (ASCUS/SIL). It was determined that the operative characteristics for detecting cytological abnormalities were similar for cervical and urine samples. This suggested using PCR for detecting HPV-DNA in urine samples as a potential screening strategy for CC prevention in future prevention and control programmes along with currently implemented strategies for reducing the impact of the disease, i.e. they are economic, easy to collect, have wide acceptability amongst women and have similar operative characteristics to those of the cervical samples.
[Show abstract][Hide abstract] ABSTRACT: Tuberculosis (TB) remains one of the most worrying infectious diseases affecting public health around the world; 8.7 million new TB cases were reported in 2011. The search for an Mycobacterium tuberculosis H37Rv protein sequence which is functionally important in host-pathogen interaction has been proposed for developing a new vaccine which will allow efficient and safe control of the spread of this disease. The present study thus reports the results obtained for the Rv1268c protein described in the M. tuberculosis H37Rv genome as a hypothetical unknown, probably secreted, protein based on a highly robust, specific, sensitive and functional approach to the search for potential epitopes to be included in an anti-tuberculosis vaccine. Rv1268c presence was determined by immunoblotting after obtaining polyclonal sera against mycobacterial total sonicate or subcellular fractions. Such sera were used in electron immunomicroscopy (EIM) for confirming protein localisation on the M. tuberculosis envelop by recognising colloidal gold-labelled immunoglobulin. Screening assays revealed the presence of two sequences having high binding activity: one binding A549 alveolar epithelial cells ((141)TGMAALEQYLGSGHAVIVSI(160)) and other binding U937 monocyte-derived macrophages ((21)AVALGLASPADAAAGTMYGD(40)). Such sequences' ability to inhibit mycobacterial entry during in vitro assays was analysed. The structure of synthetic peptides binding to target cells was also determined, bearing in mind the structure-function relationship. These results, together with those obtained for other proteins, have been involved in selecting peptides which might be included in a subunit-based anti-tuberculosis vaccine.
[Show abstract][Hide abstract] ABSTRACT: Plasmodium falciparum malaria parasite invasion of erythrocytes is an essential step in host infection and the proteins involved in such invasion are the main target in developing an antimalarial vaccine. Secretory organelle-derived proteins (micronemal AMA1 protein and the RON2, 4, and 5 rhoptry neck proteins) have been recently described as components of moving junction complex formation allowing merozoites to move into a newly-created parasitophorous vacuole. This study led to identifying RON5 regions involved in binding to human erythrocytes by using a highly robust, sensitive and specific receptor-ligand interaction assay; it is further shown that the RON5 protein remains highly conserved throughout different parasite strains. It is shown that the binding peptide-erythrocyte interaction is saturable and sensitive to chymotrypsin and trypsin. Invasion inhibition assays using erythrocyte binding peptides showed that the RON5-erythrocyte interaction could be critical for merozoite invasion of erythrocytes. This work provides evidence (for the first time) suggesting a fundamental role for RON5 in erythrocyte invasion.
[Show abstract][Hide abstract] ABSTRACT: The aim of obtaining novel vaccine candidates against malaria and other transmissible diseases can be partly based on selecting non-polymorphic peptides from relevant antigens of pathogens, which have to be then precisely modified for inducing a protective immunity against the disease. Bearing in mind the high degree of the MSA-2(21-40) peptide primary structure's genetic conservation among malaria species, and its crucial role in the high RBC binding ability of Plasmodium falciparum (the main agent causing malaria), structurally defined probes based on non-natural peptide-bond isosteres were thus designed. Thus, two peptide mimetics were obtained (so-called reduced amide pseudopeptides), in which naturally made amide bonds of the (30)FIN(32)-binding motif of MSA-2 were replaced with ψ-[CH2-NH] methylene amide isostere bonds, one between the F-I and the second between I-N amino acid pairs, respectively, coded as ψ-128 ψ-130. These peptide mimetics were used to produce poly- and monoclonal antibodies in Aotus monkeys and BALB/c mice. Parent reactive mice-derived IgM isotype cell clones were induced to Ig isotype switching to IgG sub-classes by controlled in vitro immunization experiments. These mature isotype immunoglobulins revealed a novel epitope in the MSA-2(25-32) antigen and two polypeptides of rodent malaria species. Also, these antibodies' functional activity against malaria was tested by in vitro assays, demonstrating high efficacy in controlling infection and evidencing neutralizing capacity for the rodent in vivo malaria infection. The neutralizing effect of antibodies induced by site-directed designed peptide mimetics on Plasmodium's biological development make these pseudopeptides a valuable tool for future development of immunoprophylactic strategies for controlling malarial infection.