Multiple high-risk HPV genotypes are grouped by type and are associated with viral load and risk factors.

Investigating whether high-risk human papillomavirus (HR-HPV) types tend to become grouped in a particular way and whether factors are associated with such grouping is important for measuring the real impact of vaccination. In total, 219 women proving positive for HPV as detected by real-time PCR were included in the study. Each sample was analysed for detecting and quantifying six viral types and the hydroxymethylbilane synthase gene. Multiple correspondence analysis led to determining grouping patterns for six HR-HPV types and simultaneous association with multiple variables and whether viral load was related to the coexistence of other viral types. Two grouping profiles were identified: the first included HPV-16 and HPV-45 and the second profile was represented by HPV-31, HPV-33 and HPV-58. Variables such as origin, contraceptive method, births and pregnancies, educational level, healthcare affiliation regime, atypical squamous cells of undetermined significance and viral load were associated with these grouping profiles. Different socio-demographic characteristics were found when coinfection occurred by phylogenetically related HPV types and when coinfection was due to non-related types. Biological characteristics, the number of viral copies, temporality regarding acquiring infection and competition between viral types could influence the configuration of grouping patterns. Characteristics related to women and HPV, influence such interactions between coexisting HPV types reflecting the importance of their evaluation.

Bovine leukaemia virus DNA in fresh milk and raw beef for human consumption.

Bovine leukaemia virus (BLV) is the causative agent of enzootic bovine leucosis, which has been reported worldwide. BLV has been found recently in human tissue and it could have a significant impact on human health. A possible hypothesis regarding viral entry to humans is through the consumption of infected foodstuffs. This study was aimed at detecting the presence of BLV DNA in raw beef and fresh milk for human consumption. Nested PCR directed at the BLV gag gene (272 bp) was used as a diagnostic test. PCR products were confirmed by Sanger sequencing. Forty-nine per cent of the samples proved positive for the presence of proviral DNA. This is the first study highlighting the presence of the BLV gag gene in meat products for human consumption and confirms the presence of the viral DNA in raw milk, as in previous reports. The presence of viral DNA in food products could suggest that viral particles may also be found. Further studies are needed to confirm the presence of infected viral particles, even though the present findings could represent a first approach to BLV transmission to humans through foodstuff consumption.

Peptides derived from Mycobacterium tuberculosis Rv2301 protein are involved in invasion to human epithelial cells and macrophages.

The specific function of putative cut2 protein (or CFP25), encoded by the Rv2301 gene from Mycobacterium tuberculosis H37Rv, has not been identified yet. The aim of this study was to assess some of CFP25 characteristics and its possible biological role in Mycobacterium tuberculosis H37Rv invasion process to target cells. Molecular assays indicated that the gene encoding Rv2301 is present and transcribed in M. tuberculosis complex strains. The presence of Rv2301 protein over the bacilli surface was confirmed by Western blot and immunoelectron microscopy analyses, using goats sera inoculated with synthetic peptides derived from Rv2301 protein. Receptor-ligand binding assays with carcinomic human alveolar basal epithelial cells (A549) and macrophages derived from human histolytic lymphoma monocytes (U937) allowed us to identify five high activity binding peptides (HABPs) in both cell lines, and two additional HABPs only in A549 cells. U937 HABPs binding interactions were characterized by saturation assays, finding dissociation constants (Kd) within the nanomolar range and positive cooperativity (nH>1). Inhibition assays were performed to assess the possible biological role of Rv2301 identified HABPs, finding that some of them were able to inhibit invasion at a 5 µM concentration, compared with the cytochalasin control. On the other hand, HABPs, and especially HABP 36507 located at the N-terminus of the protein, facilitated the internalization of fluorescent latex beads into A549 cells. These findings are of vital importance for the rational selection of Rv2301 HABPs, to be included as components of an antituberculosis vaccine.

Increased sensitivity of human lymphoid lines to natural killer cells after induction of the Epstein-Barr viral cycle by superinfection or sodium butyrate.

Superinfection of latently Epstein-Barr virus (EBV)-carrying Raji cells with the P3HR-1 substrain EBV, known to induce the entry of a substantial fraction of cells into an abortively lytic cycle, increased the susceptibility of the cells to natural killer (NK) effect of human blood lymphocytes. Reciprocal cold-target competition tests with known NK-cell sensitive and -resistant lymphoid cell ines showed that the increased susceptibility is a result of the appearance of an NK-sensitive target, rather than to a general increase in membrane fragility. Lymphocytes of EBV-seropositive and -negative donors were equally effective killers against P3HR-1 virus-superinfected targets. EBV-induced NK sensitivity increased with time. It was a result of some event associated with the intracellular viral cycle, and not to the adherence of viral particles to the cell surface. Induction of EBV-carrying P3HR-1 cells to entry into the viral cycle with n-butyrate also increased their NK sensitivity. A transforming, noncytopathic prototype strain of EBV, B95-8, failed to increase the susceptibility of theRaji cells to NK-lysis, although it had some effect on the Daudi line. Because NK cells can kill virus-producing cells at an early stage of the cycle, before the virus particles are assembled, they may restrict, in vivo, the spread of the virus from latently infected cells.

Disease associations of the Ia-like human alloantigens. Contrasting patterns in rheumatoid arthritis and systemic lupus erythematosus.

Increasing evidence has been obtained of the special value of Ia-like B-cell alloantisera for demonstrating disease associations with histocompatibility antigens. This was particularly evident for the study of the immunogenetics of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), two conditions frequently considered related. The profiles of antigens recognized by the alloantisera in patients from each disease group was distinctive. Two types of alloantisera were obtained that illustrated the divergence between the twod iseases. One type showed a higher than normal incidence in RA but lower than normal in SLE; the other showed a higher incidence in SLE. While these sera were not totally defined, evidence was obtained that the SLE-reactive alloantiserum related to two alleles of the major histocompatibility complex DRw2 and DRw3, while the RA-reactive alloantiserum related to a common specificity shared by cells positive for either DRw4, DRw7, or DRw10. The data indicate that immunogenetic factors are relevant to the development of both RA and SLE, but that these are distinct for each disease.

Antibodies in malarial sera to parasite antigens in the membrane of erythrocytes infected with early asexual stages of Plasmodium falciparum.

Monolayers of human erythrocytes (E) infected with Plasmodium falciparum were briefly fixed with 1% glutaraldehyde and air dried. They were then exposed to sera from patients with P. falciparum malaria or from donors immune to this parasite and tested in an indirect immunofluorescence assay (IFA). Parasites in infected E were made visible by counterstaining with ethidium bromide. Immunofluorescence (IF) was restricted to the surface of infected E. No antibody binding was detected unless the E were dried, suggesting that the relevant antigens were not available on the outer layers of the E surface. Staining over large parts of the E surface was seen already when the merozoite penetrated noninfected cells and was strong in E containing early stages of the parasite (rings, trophozoites). It was weak or absent from E containing schizonts. Antibodies in sera from different parts of Africa, Colombia, or Sweden reacted similarly with E infected with a Tanzanian P. falciparum strain kept in culture for many years and with parasitized E freshly drawn from African, Swedish, or Colombian patients. All sera from residents of a holoendemic area (Liberia) were IFA positive. In contrast, some sera from Colombian or Swedish patients with primary infection gave negative results. The results of the IFA and of an enzyme-linked immunosorbent assay in which fixed and dried E were the targets were well-correlated, suggesting that the same antibodies were detected by these assays. The antigens involved in the IFA were susceptible to pronase but not to trypsin or neuraminidase. E surface IF was inhibited by lysates of infected E, merozoite extracts, or soluble antigens present in P. falciparum culture supernatants but not by lysates of normal E or ghost extracts. The inhibitory antigens were heat stable (100 degrees C, 5 min). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting of either antigen-enriched preparations from culture supernatants or merozoite extracts showed that antibodies eluted from monolayers of infected E reacted consistently with a predominant polypeptide of Mr 155,000 and two to four minor polypeptides of lower molecular weights. Metabolic labeling of the parasites with 75Se-methionine indicated that these antigens were parasite derived. We conclude that the antigens involved in these reactions are released from bursting schizonts or merozoites and are deposited in the E membrane in the course of invasion.(ABSTRACT TRUNCATED AT 400 WORDS)

An analysis of in vitro T cell responsiveness in lepromatous leprosy.

In lepromatous leprosy, there is extensive replication of Mycobacterium leprae (M. leprae) within dermal macrophages. This lack of microbial resistance has been attributed to a defective cell-mediated immune response to M. leprae antigens. We have examined the in vitro response of T cells to M. leprae to determine if hyporesponsiveness could be reversed. The study included 40 unselected patients from New York and from Colombia, most with the severe lepromatous form of the disease. We first noted that lepromatous leprosy patients were of two types: those unable to respond, as assessed by T cell proliferation and immune (gamma) interferon (IFN-gamma) release, and a second group, exhibiting low but detectable responses relative to tuberculoid controls. When the effect of exogenous recombinant interleukin-2 (IL-2) on the response to M. leprae antigens was compared in the two groups, many of the low responders, but not the nonresponders, showed enhanced proliferation and IFN-gamma release. To evaluate a possible suppressive effect of monocytes, these cells were eliminated with a cell-specific monoclonal antibody and complement. Depletion of monocytes often expanded preexisting weak responses but did not reverse the anergy of the M. leprae nonresponders. The enhancement was not M. leprae-specific, since it was also observed when bacillus Calmette-Guerin was the antigenic stimulus for proliferation and IFN-gamma production. Removal of the suppressor T cell subset, with OKT8 antibody and complement, also did not restore responses in nonresponder patients. We conclude that a sizable number of lepromatous leprosy patients exhibit a low degree of responsiveness to M. leprae and that the responses can be enhanced in vitro with IL-2 or with monocyte depletion. Nonresponsiveness, however, cannot be reversed. Since currently available assays measure the function of previously sensitized T cells, suppressor mechanisms may yet contribute to defective cell-mediated immunity by impairing the initial sensitization to M. leprae antigens.

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.

Correction to: Comparison of healing of full-thickness skin wounds grafted with multidirectional or unidirectional autologous artificial dermis: differential delivery of healing biomarkers.

In the original article text presenting and discussing results shown in Fig. 6 omitted to mention that quantification of TGF-ß2 and TGF-ß3 was not included in Fig. 6a, c, e.

Shifting the polarity of some critical residues in malarial peptides' binding to host cells is a key factor in breaking conserved antigens' code of silence.

As microbes use many mechanisms for avoiding immunological pressure, new strategies must be developed to bypass the immunological code of silence of conserved, functionally-important amino acid sequences, such as those involved in high activity binding peptides' (HABPs) attaching to their host cells. Hundreds of experiments in large numbers of Aotus monkeys revealed that this immunological code of silence could be broken by shifting the polarity of some critical host cell binding residues in these HABPs by substituting F for R and vice versa, Y<-->W, L<-->H, I<-->N, P<-->D, M<-->K or E, C<-->T, V<-->N or S; there are special rules for A, G and S. (1)H-nuclear magnetic resonance of these modified, immunogenic, protection-inducing HABPs and molecular modelling revealed that such modifications induced appropriate fitting into specific HLA-DRbeta1 Pockets, suggesting the presence of new pockets and a haplotype- and allele-specific conscious TCR. A highly immunogenic and protection-inducing anti-malarial vaccine can thus be produced.

Comparison of healing of full-thickness skin wounds grafted with multidirectional or unidirectional autologous artificial dermis: differential delivery of healing biomarkers.

Cytokines, chemokines, and growth and remodeling factors orchestrate wound healing when skin damage occurs. During early stages, when the wound is still open, detection and quantification of these compounds might provide biomarkers of skin wound healing, which could aid to complete the scenario provided by clinical follow-up data and histological and histomorphometric analyses. This work assessed and compared the healing of full-thickness skin wounds grafted with artificial dermis made with autologous skin fibroblasts and unidirectional or multidirectional type I collagen scaffolds to test this hypothesis. Biomarkers of healing were detected and quantified in the culture medium of artificial dermis and exudates from the grafted wounds. Clinical follow-up of animals and histological and histomorphometric analysis showed differences in graft integration, wound closure, and histological and histomorphometric parameters. Surface plasmon resonance quantification of 13 healing biomarkers indicated differential secretion of most of the quantified factors in culture medium by the multidirectional and unidirectional artificial dermis. Also, there were significant differences between the concentration of some of the factors analyzed in the exudates of wounds grafted with the evaluated artificial dermis. These findings suggest that differential delivery of healing biomarkers induced by the directionality of the scaffold used to produce the multidirectional and unidirectional dermis was sufficient to create two skin wound microenvironments that determined a different outcome of healing. Overall, data indicate that healing of wounds grafted with multidirectional autologous artificial dermis is better than that of the wounds grafted with the unidirectional one.

Immunological and structural characterization of an epitope from the Trypanosoma cruzi KMP-11 protein.

The K1 peptide is an HLA-A*0201-restricted cytotoxic epitope derived from the Trypanosoma cruzi KMP-11 protein, this being the etiological agent of Chagas' disease. This work describes the K1 peptide's secondary structure and its recognition by sera from chagasic patients. Circular dichroism and NMR spectroscopy analysis revealed that the K1 peptide adopts an alpha-helical conformation. Fifty-six percent of individuals had anti-K1 and 86% anti-KMP-11 antibodies by ELISA in the chronic Chagas' group and 28 and 68% in the indeterminate Chagas' group, respectively. By contrast, no reactivity was observed in sera from healthy individuals and tuberculosis patients. Antibody response subclass specificity to the K1 peptide was IgG1 and IgG3. Taken together these results support the idea that the K1 peptide acts as a B-cell-inducer epitope during Chagas' disease.

The Aotus nancymaae erythrocyte proteome and its importance for biomedical research.

The Aotus nancymaae species has been of great importance in researching the biology and pathogenesis of malaria, particularly for studying Plasmodium molecules for including them in effective vaccines against such microorganism. In spite of the forgoing, there has been no report to date describing the biology of parasite target cells in primates or their biomedical importance. This study was thus designed to analyse A. nancymaae erythrocyte protein composition using MS data collected during a previous study aimed at characterising the Plasmodium vivax proteome and published in the pertinent literature. Most peptides identified were similar to those belonging to 1189 Homo sapiens molecules; >95% of them had orthologues in New World primates. GO terms revealed a correlation between categories having the greatest amount of proteins and vital cell function. Integral membrane molecules were also identified which could be possible receptors facilitating interaction with Plasmodium species. The A. nancymaae erythrocyte proteome is described here for the first time, as a starting point for more in-depth/extensive studies. The data reported represents a source of invaluable information for laboratories interested in carrying out basic and applied biomedical investigation studies which involve using this primate. SIGNIFICANCE: An understanding of the proteomics characteristics of A. nancymaae erythrocytes represents a fascinating area for research regarding the study of the pathogenesis of malaria since these are the main target for Plasmodium invasion. However, and even though Aotus is one of the non-human primate models considered most appropriate for biomedical research, knowledge of its proteome, particularly its erythrocytes, remains unknown. According to the above and bearing in mind the lack of information about the A. nancymaae species genome and transcriptome, this study involved a search for primate proteins for comparing their MS/MS spectra with the available information for Homo sapiens. The great similarity found between the primate's molecules and those for humans supported the use of the monkeys or their cells for continuing assays involved in studying malaria. Integral membrane receptors used by Plasmodium for invading cells were also found; this required timely characterisation for evaluating their therapeutic role. The list of erythrocyte protein composition reported here represents a useful source of basic knowledge for advancing biomedical investigation in this field.

Synthetic peptides from Plasmodium falciparum apical membrane antigen 1 (AMA-1) specifically interacting with human hepatocytes.

Plasmodium falciparum apical membrane antigen 1 (AMA-1) is expressed during both the sporozoite and merozoite stage of the parasite's life cycle. The role placed by AMA-1 during sporozoite invasion of hepatocytes has not been made sufficiently clear to date. Identifying the sequences involved in binding to hepatocytes is an important step towards understanding the structural basis for sporozoite-hepatocyte interaction. Binding assays between P. falciparum AMA-1 peptides and HepG2 cell were performed in this study to identify possible AMA-1 functional regions. Four AMA-1 high activity binding peptides (HABPs) bound specifically to hepatocytes: 4310 ((74)QHAYPIDHEGAEPAPQEQNL(93)), 4316 ((194)TLDEMRHFYKDNKYVKNLDE(213)), 4321 ((294)VVDNWEKVCPRKNLQNAKFGY(313)) and 4332 ((514)AEVTSNNEVVVKEEYKDEYA(533)). Their binding to these cells became saturable and resistant to treatment with neuraminidase. Most of these peptides were located in AMA-1 domains I and III, these being target regions for protective antibody responses. These peptides interacted with 36 and 58 kDa proteins on the erythrocyte surface. Some of the peptides were found in exposed regions of the AMA-1 protein, thereby facilitating their interaction with host cells. It is thus probable that AMA-1 regions defined by the four peptides mentioned above are involved in sporozoite-hepatocyte interaction.

Evidence for separate control by phorbol esters of CD18-dependent adhesion and translocation of protein kinase C in U-937 cells.

Treatment of U-937 GTB cells with tumor-promoting phorbol esters induced adherence of the cells to plastic, with a t1/2 of 20 min. The ED50 was determined to 3.3 nM for phorbol-12,13-dibutyrate and 0.3 nM for 12-O-tetradecanoyl-4 beta-phorbol-13-acetate, whereas the non-tumor-promoting analogue 12-O-tetradecanoyl-4 alpha-phorbol-13-acetate was ineffective at concentrations up to 100 nM. The adherence process showed characteristics typical of leucocyte adhesion and was inhibited by a monoclonal antibody to the leucocyte adhesion molecule CD18. The sublines of U-937, RES and RESREV made resistant to the action of low doses of phorbol ester regarding inhibition of DNA synthesis and containing lower levels of protein kinase C compared to U-937 GTB, were desensitized with respect to the adhesion response. Translocation of protein kinase C from cytosol to the particulate fraction occurred at about 10-fold higher concentrations of phorbol ester than the adhesion response in U-937 GTB cells, under otherwise similar conditions, whereas no difference in sensitivity was observed between the sublines. Also phorbol ester stimulation of choline incorporation into lipids exhibited lower sensitivity compared to the adhesion response with no difference observed between the various cell lines. The results indicate that CD18-dependent adhesion, like DNA synthesis, is controlled by phorbol esters in a manner unrelated to the translocation of protein kinase C and that the control mechanism might involve forms of protein kinase C which are subject to stable down-modulation following TPA adaption of the cells.

Tetraspanin CD151 and integrin α6ß1 mediate platelet-enhanced endothelial colony forming cell angiogenesis.

UNLABELLED: ESSENTIALS: Platelet releasates (PRs) enhance endothelial colony forming cell (ECFC) angiogenesis. The impact of platelet membrane components on ECFC angiogenesis was studied by a tube formation assay. Platelets enhanced ECFC angiogenesis more potently than PR, via tetraspanin CD151 and integrin α6ß1. Optimal enhancement of ECFC angiogenesis by platelets requires both membrane proteins and PR. BACKGROUND: Platelets promote angiogenesis of endothelial colony forming cells (ECFCs), with the underlying mechanisms not being fully understood. OBJECTIVE: To investigate if platelets regulate the angiogenic property of ECFCs via mechanisms beyond platelet-released angiogenic regulators. METHODS AND RESULTS: Endothelial colony forming cells were generated by ECFC-directed cell culture of peripheral blood mononuclear cells. Capillary-like tube formation of ECFCs was assessed using a Matrigel assay. Platelets promoted ECFC tube formation in both basic and complete ECFC medium. Importantly, the ECFC angiogenic responses induced by platelets were stronger than those induced by platelet releasates. Thus, the branching points of ECFC tube formation (30.5 ± 9.0/field, ECFC alone) were increased by platelet releasates (58.2 ± 8.3/field) and even more profoundly by platelets (95.5 ± 17.6/field), indicating that platelet membrane components also promoted ECFC tube formation. The latter was further supported by evidence that fixed platelets did enhance ECFC tube formation. Subsequent experiments revealed that the promotion was dependent on platelet-surface glycoproteins, as removal of sialic acid from platelet glycoproteins by neuraminidase abolished the enhancement. Furthermore, platelet-expressed, but not ECFC-expressed, CD151 was important for the enhancement, as pretreatment of platelets, but not ECFCs, with a CD151-blocking antibody attenuated the effect. Integrin α6ß1 on both ECFCs and platelets also participated in platelet-promoted tube formation, as integrin α6 or ß1 blockade of either cell type markedly or totally inhibited the phenomenon. Moreover, platelets exerted the enhancement via the Src-PI3K signaling pathway of ECFCs. CONCLUSION: Platelet-enhanced ECFC angiogenesis requires platelet tetraspanin CD151 and α6ß1 integrin, as well as ECFC α6ß1 integrin and Src-PI3K signaling.

Based on HLA-DR beta1* allele binding specificities, striking differences in distance and TCR Contacting Residue Orientation can be observed in modified protection-inducing malarial synthetic peptides.

An anti-malarial vaccine is urgently needed, especially against P. falciparum which causes 2 to 3 million deaths each year, mostly in Sub-Saharan African children. This vaccine should contain molecules from the parasite's different developmental stages due to the parasite's remarkable complexity and genetic variability. The first approach using synthetic peptides from different parasite stage molecules (the SPf66 malaria vaccine) conferred limited protective efficacy in Aotus monkeys and in large field-trials carried out in different parts of the world SPf66 contains red blood cell (RBC) binding merozoite peptides for which immune responses against them are genetically controlled by HLA-DR region. Therefore, a systematic search of conserved high activity binding peptides (HABP) was undertaken aimed at using them as immunogens. However, these peptides were poorly immunogenic and had poor protection-inducing capacity against experimental challenge with a P. falciparum strain highly infective for Aotus monkeys an experimental model with an immune system quite similar to humans. Modifications were thus made to key residues to render them immunogenic and protection-inducing. These native and modified HABPs' three-dimensional structure was determined by (1)H-NMR studies and their ability in forming stable Major Histocompatibility Class II - peptide (MHCII-peptide) complexes was correlated with their ability to bind in vitro to purified HLA-DR beta1* molecules. Our experimental data suggests a correlation between modified HABPs' three-dimensional structure, HLA-DR beta1* binding preferences and their protection-inducing capacity in monkeys. Furthermore, the data presented here indicates that a synthetic peptide vaccine's three-dimensional structural features dictate both HLA-DR beta1* allele binding preference (imposing genetic restriction on the immune response) and on these vaccines' protection-inducing value. Basic knowledge of a parasite's functionally active peptides, their 3D structure and their interaction for forming the MHC II- peptide-TCR complex will thus contribute towards designing fully effective multi-component, multi-stage subunit-based malarial vaccines.

Mapping of antigenic determinants of the T. cruzi hsp70 in chagasic and healthy individuals.

In the present paper we describe the analysis of the immunological recognition by sera of healthy individuals and chagasic patients of the Trypanosoma cruzi heat shock 70 kDa protein. By a Falcon Assay Screening Test, using as antigen an ATP-agarose purified T. cruzi hsp70, it has been found that the sera of infected patients as well as of that of healthy individuals show reactivity against the hsp70 protein but that the reactivity of the sera of patients is in general significantly higher than that of healthy individuals. The analysis of the reactivity of the chagasic sera against a collection of peptides covering 92% of the protein has shown that more than 50% of the peptides gave a positive response but only against a few peptides did we observe high reactivity in a wide spectrum of sera. Only four peptides (numbers 9, 12, 14 and 47) were recognized by all sera tested with high reactivity values. The sera of healthy individuals also showed reactivity against a large percentage of peptides but with lower values. It was observed that particular peptides showing high reactivity against the sera of healthy donors also show high reactivity against patients' sera. However, the general pattern of reactivity against the peptides is different in chagasic and healthy sera. The immunodominant peptides map in the highly conserved as well as in the less conserved part of the hsp70 molecule. The 1/3 C-terminal, being the least conserved part of the molecule, seems to be the least immunogenic. Mapping of the epitopes led to the identification of particular immunogenic motifs within individual peptides.

Determining the Plasmodium vivax VCG-1 strain blood stage proteome.

Plasmodium vivax is the second most prevalent parasite species causing malaria in humans living in tropical and subtropical areas throughout the world. There have been few P. vivax proteomic studies to date and they have focused on using clinical isolates, given the technical difficulties concerning how to maintain an in vitro culture of this species. This study was thus focused on identifying the P. vivax VCG-1 strain proteome during its blood lifecycle through LC-MS/MS; this led to identifying 734 proteins, thus increasing the overall number reported for P. vivax to date. Some of them have previously been related to reticulocyte invasion, parasite virulence and growth and others are new molecules possibly playing a functional role during metabolic processes, as predicted by Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis. This is the first large-scale proteomic analysis of a P. vivax strain adapted to a non-human primate model showing the parasite protein repertoire during the blood lifecycle. Database searches facilitated the in silico prediction of proteins proposed for evaluation in further experimental assays regarding their potential as pharmacologic targets or as component of a totally efficient vaccine against malaria caused by P. vivax. BIOLOGICAL SIGNIFICANCE: P. vivax malaria continues being a public health problem around world. Although considerable progress has been made in understanding genome- and transcriptome-related P. vivax biology, there are few proteome studies, currently representing only 8.5% of the predicted in silico proteome reported in public databases. A high-throughput proteomic assay was used for discovering new P. vivax intra-reticulocyte asexual stage molecules taken from parasites maintained in vivo in a primate model. The methodology avoided the main problem related to standardising an in vitro culture system to obtain enough samples for protein identification and annotation. This study provides a source of potential information contributing towards a basic understanding of P. vivax biology related to parasite proteins which are of significant importance for the malaria research community.

Alpha helix shortening in 1522 MSP-1 conserved peptide analogs is associated with immunogenicity and protection against P. falciparum malaria.

1522 is a nonimmunogenic conserved high-activity binding peptide (HABP) belonging to Plasmodium falciparum MSP-1 protein N-terminal fragment. The key amino acids in binding to red blood cells (RBC) were identified and replaced by others having similar mass but different charge. Because conserved HABPs are not antigenic nor immunogenic, immunogenicity and protectivity studies were then conducted on them in the Aotus monkey. 1H-NMR studies included the lead peptide 1522 as well as the analogs 9782, 13446, 13448, and 13442 to relate their structure to biological function. All the peptides presented alpha-helical structure, with differences observed in helix location and extension. The nonprotective 1522 peptide was totally helical from the N- to the C-terminus, very similar to nonprotective 13442 and 13448 peptides whose extension was almost totally helical. The 9782 and 13446 protective peptides, however, possessed a shorter helical region where modified critical binding residues were not included. A more flexible region was generated at the C-terminus in those peptides with a shorter helical region, leading to a greater number of conformers. These data suggest that peptide flexibility results in increased interaction with immune system molecules, generating protective immunity.