ABSTRACT
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.
Subject(s)
Alphapapillomavirus/genetics , Coinfection/epidemiology , Genotype , Papillomavirus Infections/epidemiology , Adult , Coinfection/virology , Colombia/epidemiology , Cross-Sectional Studies , Female , Humans , Middle Aged , Papillomavirus Infections/virology , Prevalence , Risk Factors , Young AdultABSTRACT
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.
Subject(s)
Antigens, Bacterial/chemistry , Bacterial Proteins/chemistry , Epithelial Cells/drug effects , Macrophages/drug effects , Mycobacterium tuberculosis/chemistry , Peptides/metabolism , Amino Acid Sequence , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Binding Sites , Blotting, Western , Cell Line, Tumor , Cytochalasins/pharmacology , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Humans , Kinetics , Macrophages/metabolism , Macrophages/microbiology , Microscopy, Immunoelectron , Molecular Sequence Data , Mycobacterium tuberculosis/pathogenicity , Mycobacterium tuberculosis/physiology , Organ Specificity , Peptides/chemical synthesis , Peptides/pharmacology , Protein Binding , Tuberculosis, Pulmonary/microbiologyABSTRACT
Our ongoing search for a fully-effective vaccine against the Plasmodium falciparum parasite (causing the most lethal form of human malaria) has been focused on identifying and characterising proteins' amino acid sequences (high activity binding peptides or HABPs) involved in parasite invasion of red blood cells (RBC) by the merozoite and hepatocytes by the sporozoite. Many such merozoite HABPs have been recognised and molecularly and structurally characterised; however, native HABPs are immunologically silent since they do not induce any immune response or protection against P. falciparum malaria infection and they have to be structurally modified to allow them to fit perfectly into immune system molecules. A deeply structural analysis of these conserved merozoite HABPs and their modified analogues has led to rules or principles becoming recognised for constructing a logical and rational methodology for a minimal subunit-based, multi-epitope, multi-stage, chemically-synthesised vaccine. The same in-depth analysis of the most relevant sporozoite proteins involved in sporozoite cell-traversal and hepatocyte invasion as well as the hepatic stage is shown here. Specifically modifying these HABPs has resulted in a new set of potential pre-erythrocyte targets which are able to induce high, longlasting antibody titres in Aotus monkeys, against their corresponding recombinant proteins and the complete parasite native molecules. This review shows how these rules may be applied against the first stage of parasite invasion (i.e. the sporozoite) to mount the first line of defence against the malarial parasite, which may indeed be the most effective one. Our results strongly support including some of these modified sporozoite HABPs in combination with the previously-described modified merozoite HABPs for obtaining the aforementioned fully-protective, multiepitope, multi-stage, minimal subunit-based, chemically-synthesized, antimalarial vaccine.
Subject(s)
Malaria Vaccines/chemistry , Malaria Vaccines/immunology , Malaria/prevention & control , Peptides/chemistry , Peptides/immunology , Plasmodium/growth & development , Amino Acid Sequence , Animals , Antimalarials , Humans , Malaria/immunology , Malaria Vaccines/therapeutic use , Models, Molecular , Molecular Sequence Data , Peptides/therapeutic use , Plasmodium/immunology , Protozoan Proteins/chemistry , Protozoan Proteins/immunologyABSTRACT
The development of an adequate immune response against pathogens is mediated by molecular interactions between different cell types. Among them, binding of antigenic peptides to the Major Histocompatibility Complex (MHC) molecule expressed on the membrane of antigen presenting cells (APCs), and their subsequent recognition by the T cell receptor have been demonstrated to be crucial for developing an adequate immune response. The present review compiles computational quantum chemistry studies about the electrostatic potential variations induced on the MHC binding region by peptide's amino acids, carried out with the aim of describing MHC-peptide binding interactions. The global idea is that the electrostatic potential can be represented in terms of a series expansion (charge, dipole, quadrupole, hexadecapole, etc.) whose three first terms provide a good local approximation to the molecular electrostatic 'landscape' and to the variations induced on such landscape by targeted modifications on the residues of the antigenic peptide. Studies carried out in four MHC class II human allele molecules, which are the most representative alleles of their corresponding haplotypes, showed that each of these molecules have conserved as well as specific electrostatic characteristics, which can be correlated at a good extent with the peptide binding profiles reported experimentally for these molecules. The information provided by such characteristics would help increase our knowledge about antigen binding and presentation, and could ultimately contribute to developing a logical and rational methodology for designing chemically synthesized, multi-antigenic, subunit-based vaccines, through the application of quantum chemistry methods.
Subject(s)
Histocompatibility Antigens/chemistry , Peptides/chemistry , Vaccines/chemistry , HLA Antigens/chemistry , Humans , Peptides/immunology , Protein Binding , Quantum Theory , Static Electricity , Vaccines/immunologyABSTRACT
The prevalence of human papillomavirus (HPV) infections in 2109 females inhabiting five cities of Colombia was determined. Of the 49.2% with an HPV infection, 59.8% were infected with more than one viral type. Species 7 (of the the genus Alphapapillomavirus) was associated with multiple infections. Analysis of the socio-demographic data revealed a statistically significant protective effect associated with the status of civil union (civil recognition of cohabitation without marriage), and indigenous ethnicity proved to be a risk factor for HPV infection. This is the first study comparing HPV infection among women from geographical regions of Colombia with different socio-cultural structures.
Subject(s)
Alphapapillomavirus/genetics , Papillomavirus Infections/epidemiology , Adult , Alphapapillomavirus/isolation & purification , Colombia , Female , Humans , Logistic Models , Papillomavirus Infections/virology , Prevalence , Risk Factors , Surveys and Questionnaires , Uterine Cervical Neoplasms/prevention & controlABSTRACT
An anti-malarial vaccine against the extremely lethal Plasmodium falciparum is desperately needed. Peptides from this parasite's proteins involved in invasion and having high red blood cell-binding ability were identified; these conserved peptides were not immunogenic or protection-inducing when used for immunizing Aotus monkeys. Modifying some critical binding residues in these high-activiy binding peptides' (HABPs') attachment to red blood cells (RBC) allowed them to induce immunogenicity and protection against experimental challenge and acquire the ability to bind to specific HLA-DRp1* alleles. These modified HABPs adopted certain characteristic structural configurations as determined by circular dichroism (CD) and 1H nuclear magnetic resonance (NMR) associated with certain HLA-DRbeta1* haplotype binding activities and characteristics, such as a 2-angstroms-distance difference between amino acids fitting into HLA-DRp1 Pockets 1 to 9, residues participating in binding to HLA-DR pockets and residues making contact with the TCR, suggesting haplotype and allele-conscious TCR. This has been demonstrated in HLA-DR-like genotyped monkeys and provides the basis for designing high effective, subunit-based, multi-antigen, multi-stage, synthetic vaccines, for immediate human use, malaria being one of them.
Subject(s)
Epitopes/immunology , Malaria Vaccines/chemical synthesis , Plasmodium falciparum/immunology , Vaccines, Subunit/chemical synthesis , Vaccines, Synthetic/immunology , Animals , Epitopes/genetics , Epitopes/metabolism , HLA-DR Antigens/chemistry , HLA-DR Antigens/genetics , HLA-DR Antigens/immunology , Humans , Malaria/genetics , Malaria/immunology , Malaria Vaccines/genetics , Malaria Vaccines/immunology , Models, Biological , Plasmodium falciparum/pathogenicity , Vaccines, Subunit/chemistry , Vaccines, Subunit/immunology , Vaccines, Synthetic/genetics , Vaccines, Synthetic/metabolismABSTRACT
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.
Subject(s)
HLA-DR1 Antigen/chemistry , Peptides/chemistry , Amino Acid Motifs , Amino Acid Sequence , Animals , Aotidae , Conserved Sequence , Malaria Vaccines/immunology , Malaria, Falciparum/immunology , Malaria, Falciparum/prevention & control , Models, Molecular , Molecular Sequence Data , Peptides/immunology , Plasmodium falciparum/immunology , Protein Binding , Receptors, Antigen, T-Cell/chemistry , Structure-Activity Relationship , Vaccines, Subunit/immunologyABSTRACT
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.
Subject(s)
Antigens, Protozoan/chemistry , Protozoan Proteins/chemistry , Protozoan Proteins/immunology , Trypanosoma cruzi/immunology , Amino Acid Sequence , Animals , Antibodies, Protozoan/blood , Antigens, Protozoan/genetics , Chagas Disease/immunology , Epitopes/chemistry , Epitopes/genetics , Humans , Immunoglobulin G/blood , Immunoglobulin Isotypes/blood , Models, Molecular , Protein Structure, Secondary , Protozoan Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Trypanosoma cruzi/geneticsABSTRACT
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.
Subject(s)
Antigens, Protozoan/chemistry , Antigens, Protozoan/metabolism , Hepatocytes/parasitology , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Peptide Fragments/metabolism , Plasmodium falciparum/pathogenicity , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , Amino Acid Sequence , Animals , Binding Sites , Circular Dichroism , Humans , Models, Molecular , Molecular Sequence Data , Neuraminidase/pharmacology , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Protein ConformationABSTRACT
A vaccine against malaria is desperately needed, and Aotus monkeys are highly susceptible to experimental infection with malarial parasites. A thorough analysis of this monkey's immune system molecules was thus undertaken in our institute. Cloning and sequencing, followed by three-dimensional analysis, has revealed high homology with some HLA-DRB1 molecules in terms of their peptide binding region pockets. Molecules such as HLA-DRB1*03, 11, 08, and HLA-DRB1*04 are so similar to Aotus MHC-DRB molecules that peptides identified as binding to these molecules and inducing protective immunity in these monkeys could be used in humans without further refinement, while small modifications seem to be needed for those binding to HLA-DRB1*07, HLA-DRB1*15, 16, and HLA-DRB1*10-like molecules, making this New World monkey an excellent model for tailor-made vaccine development, especially against malaria.
Subject(s)
Drug Design , HLA-DR Antigens/chemistry , Histocompatibility Antigens Class II/chemistry , Malaria Vaccines/chemistry , Platyrrhini/immunology , Amino Acid Sequence , Binding Sites , HLA-DR Antigens/genetics , HLA-DR Antigens/immunology , HLA-DRB1 Chains , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/immunology , Humans , Malaria Vaccines/genetics , Molecular Sequence Data , Peptides/chemistry , Peptides/genetics , Protein Conformation , Sequence AlignmentABSTRACT
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.
Subject(s)
Alleles , HLA-DR Antigens/immunology , Receptors, Antigen, T-Cell/immunology , Vaccines, Synthetic/immunology , Amino Acid Sequence , Animals , Binding Sites/genetics , Binding Sites/immunology , HLA-DR Antigens/chemistry , HLA-DR Antigens/genetics , Humans , Malaria Vaccines/genetics , Malaria Vaccines/immunology , Molecular Sequence Data , Plasmodium falciparum/genetics , Plasmodium falciparum/immunology , Protozoan Proteins/genetics , Protozoan Proteins/immunology , Receptors, Antigen, T-Cell/genetics , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Vaccines, Synthetic/geneticsABSTRACT
In order to characterize immunoglobulin heavy-chain variable (IGHV) genes in Aotus nancymaae monkeys, different mRNAs encoded by five IGHV families in this non-human primate were molecularly analysed considering their paramount importance in antibody production in an immune response. This study reports gene products exhibiting 91% amino acid similarity with IGHV1, IGHV2, IGHV3, IGHV4 and IGHV7 human IGHV families. Our analyses suggest that the IGHV gene has several conserved characteristics in humans and A. nancymaae. Several amino acid residues that are highly conserved in all family members described in humans were also present in these families in A. nancymaae. Antibody diversity in these families has remained the same since divergence of both species. Our study continues to provide evidence supporting the use of A. nancymaae monkey as an animal model for studying antibody response.
Subject(s)
Aotidae/genetics , Genes, Immunoglobulin , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Amino Acid Sequence , Animals , Antibody Diversity/genetics , Aotidae/immunology , Cloning, Molecular , Humans , Molecular Sequence Data , Multigene Family , Phylogeny , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Homology, Amino AcidABSTRACT
Transmembrane protein tyrosine phosphatase (PTPase) CD45 has been implicated in activating, differentiating and the development of different immune system cells. It regulates T-or B-cell activation during receptor-specific recognition by dephosphorylating tyrosine residues in protein kinase substrates. Aotus nancymaae, Aotus nigriceps, and Aotus vociferans CD45 nucleotide and deduced amino acid sequences are presented here, where we found 90-92% identity with the human counterpart in the nucleotide sequence and 83-86% in the amino acid sequence. Aotus CD45 alternative splicing isoforms include the same exons used in human CD45, producing several identical molecular weight nucleotide fragments. Most of the non-synonymous substitutions were found in the extracellular domain. The more conserved CD45 cytoplasmic portion has two intracellular phosphatase domains (D1 and D2) separated by a short spacer and some residues and motifs involved in signaling or molecular docking, intra- and intermolecular interactions and CD45 activity and activity regulation. All invariant residues and structural/functional motifs found in PTPases were totally conserved, suggesting that Aotus CD45 is a functional enzyme. Phylogenetic analysis has shown that the Aotus CD45 molecules are more related to the human homologs than to other reported vertebrate sequences and that the ancestral group of Aotus clade is A. vociferans. When Aotus species were compared, A. nigriceps and A. vociferans were the two most distant species, while A. nancymaae and A. nigriceps appeared to be a sister group. This could be relevant in deciding which Aotus species is to be used for studying particular immunological processes during lymphocyte activation or development.
Subject(s)
Aotidae/genetics , Leukocyte Common Antigens/genetics , Alternative Splicing , Amino Acid Sequence , Animals , Aotidae/immunology , Female , Humans , Male , Molecular Sequence Data , Primates/genetics , Sequence AlignmentABSTRACT
Stability studies on the SPf66 antimalarial peptide with different pH and temperature conditions were carried out. The degradation mechanism was elucidated by the size-exclusion chromatography (SEC) technique and the experimental data obtained at 37 degrees C and different pH were fitted to a kinetic degradation model that could explain the loss of its immunogenic capacity. At 5, 25, 37, and 70 degrees C and pH 2, changes were detected in the areas of the different species, although the values obtained could not be fitted to any known degradation kinetics.
Subject(s)
Malaria Vaccines/chemistry , Protozoan Proteins/chemistry , Recombinant Proteins/chemistry , Chromatography, Gel , Drug Stability , Hydrogen-Ion Concentration , Temperature , Vaccines, Synthetic/chemistryABSTRACT
The immunogenicity of the synthetic malaria vaccine SPf66 has been recently improved by the application of new adjuvants as QS-21 saponin or poly-D,L-lactide-co-glycolide (PLGA) polymers. The search for less invasive administration routes made us test the immunogenicity of SPf66-loaded microparticles by the nasal route in Balb/c mice. We report here that the intranasal administration of the adequate PLGA vaccine formulations greatly improves and maintains higher antibody levels compared to the conventional alum adjuvant and to the administration of the particles by other routes (subcutaneous, oral). Systemic immune responses were characterized as mixed Th1/Th2-type: IFN-gamma and IgG2a isotype were found as signs of Th1 activation, whilst IgE and IgG1 secretions indicate Th2 response. Since both types of response have been associated to protective immunity in malaria, we postulate that this new approach supposes an advantage over the traditional adjuvants and routes.
Subject(s)
Antibodies, Protozoan/immunology , Antigens, Protozoan/immunology , Immunity, Cellular/immunology , Malaria Vaccines/immunology , Th1 Cells/immunology , Th2 Cells/immunology , Administration, Intranasal , Animals , Antibodies, Protozoan/analysis , Antigens, Protozoan/administration & dosage , Enzyme-Linked Immunosorbent Assay , Immunoglobulin E/analysis , Immunoglobulin E/biosynthesis , Immunoglobulin G/analysis , Immunoglobulin G/biosynthesis , Interferon-gamma/metabolism , Lactic Acid , Malaria Vaccines/administration & dosage , Mice , Mice, Inbred BALB C , Microspheres , Particle Size , Polyglycolic Acid , Polylactic Acid-Polyglycolic Acid Copolymer , PolymersABSTRACT
The presence of several Mycobacterium species was determined in 68 New World monkeys kept captive in the Cali Zoo. One hundred and thirty-three gastric lavage and blood samples were evaluated for mycobacterial presence by Ziehl-Neelsen (ZN) staining, culture and PCR amplification of the Mycobacterium tuberculosis Mtp40 species-specific gene. Mycobacteria other than tuberculosis (MOTT) were identified by PCR restriction fragment length polymorphism (RFLP). Different species of mycobacteria were detected in 65% of the primate population studied by Alpha Antigen PCR. Eleven percent were positive for Mtp40 PCR amplification, being diagnosed as having M. tuberculosis, and acid-fast bacilli were observed in 23% by ZN staining. MOTT were isolated from samples taken from 37 primates by culturing; according to the RFLP analysis, three strains were classified as belonging to the MAISS complex (Mycobacterium avium-intracellulare-scrofulaceum-simiae) and eight more, isolated from soil inside the cages, were categorized as environmental contaminants. Mycobacterium spp. were detected in 13 different New World primate species showing that PCR amplification of the Mtp40 gene is a better tool than culture for M. tuberculosis detection in captive animals and that RFLP is a useful technique for MOTT identification.
Subject(s)
Cebidae , Monkey Diseases/microbiology , Mycobacterium Infections/veterinary , Mycobacterium/isolation & purification , Animals , Animals, Zoo , Antigens, Bacterial/chemistry , Antigens, Bacterial/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Monkey Diseases/epidemiology , Mycobacterium/classification , Mycobacterium/genetics , Mycobacterium Infections/blood , Mycobacterium Infections/epidemiology , Mycobacterium Infections/microbiology , Nucleic Acid Hybridization , Phylogeny , Polymerase Chain Reaction/veterinary , Polymorphism, Restriction Fragment Length , Type C Phospholipases/chemistry , Type C Phospholipases/geneticsABSTRACT
Gammadelta T lymphocytes have a heterodimeric complex formed by the association of gamma and delta chains as receptor. Proliferation of this lymphocyte population has been observed, when infection by several pathogens such as Mycobacterium tuberculosis and Plasmodium spp. occurs. The New World Monkey Aotus nancymaae has become a very good experimental model for the immunological and physiopathological study of these infectious agents. The A. nancymaae gamma-variable region was characterized from peripheral blood samples by using cDNA and genomic DNA polymerase chain reaction amplification, DNA sequencing, and dot-blot hybridization techniques. Seventeen different T-cell receptor gamma-variable (TCRGV) sequences were obtained. These sequences were distributed among TCRGV subsets 1, 2, or 3, according to human subset classification. Although no subset 4 amplification was obtained, this subset was detected by dot-blot hybridization. The presence of these 4 subsets resembles the behavior displayed by 'gammadelta-low species' (humans and mice), where high diversity among these lymphocytes can be observed. Homologies greater than 70% were found with respect to humans. Sequence convergence between human and A. nancymaae subsets 1 and 3 highlights Aotus as a promising model for studying these lymphocyte functions.
Subject(s)
Aotidae/blood , Aotidae/genetics , Genes, T-Cell Receptor gamma/physiology , Amino Acid Sequence , Animals , Evolution, Molecular , Hybridization, Genetic , Molecular Sequence Data , Phylogeny , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
Parasitic diseases remain as a major public health problem worldwide, not only based on their historically high morbidity and mortality rates, but also because risk factors associated with their transmission are increasing. Laboratory diagnosis and particularly immunodiagnosis is a basic tool for the demonstration, clinical management and control of these infections. Classically, the serological tests for the detection of antibodies or antigens are based on the use of crude and purified antigens. Synthetic peptides have opened a new field and perspectives, as the source of pure epitopes and molecules for diagnosis of malaria, Chagas' disease, leishmaniasis, schistosomiasis, hidatidosis, cysticercosis and fasciolosis based on the detection of antibodies and circulating antigens. Herein, are critically reviewed the relevant advances and applications of the synthetic peptides on immunodiagnosis of parasitic diseases. A variety of sequences, constructs (monomers, polymers, MAPs), immunological methods and samples have been used, demonstrating their diagnostic potential. However, in most parasitic infections it is necessary to use more than a single peptide in order to avoid the genetic restriction against certain epitopes, as well as to test them in well characteized groups of patients, in order to confirm their sensitivity and specificity. The concept of multidiagnosis with synthetic peptides, using a novel multi-dot blot assay is introduced. Finally, the chemical imitation of antigens, offers a tremendous posibilities in the diagnosis of parasitic infections in developing countries since this strategy is cheaper, simpler, reproducible, useful for large scale testing and in most cases, specific and sensitive.
Subject(s)
Parasitic Diseases/diagnosis , Peptides , Animals , Cysticercosis/diagnosis , Cysticercosis/immunology , Echinococcosis/diagnosis , Echinococcosis/immunology , Enzyme-Linked Immunosorbent Assay , Fascioliasis/diagnosis , Fascioliasis/immunology , Humans , Immunologic Tests/methods , Leishmaniasis/diagnosis , Leishmaniasis/immunology , Malaria/diagnosis , Malaria/immunology , Parasitic Diseases/immunology , Peptides/chemical synthesis , Schistosomiasis/diagnosis , Schistosomiasis/immunology , Sensitivity and Specificity , Trypanosomiasis/diagnosis , Trypanosomiasis/immunologyABSTRACT
The erythrocyte-binding antigen 140 (EBA140) sequence was chemically synthesized in 61 20-mer sequential peptides covering the entire 3D7 protein strain, each of which was tested in erythrocyte-binding assays. Peptides 26135, 26144, 26147, 26160, 26170 and 26177 presented high erythrocyte-binding activity, with affinity constants ranging from 350 to 750 nM. Critical erythrocyte-binding residues were determined by competition-binding assays with glycine analogous peptides. Cross-linking assays with SDS-PAGE from high erythrocyte membrane protein binding peptides showed that all these peptides bound specifically to 25, 52 and 75 kDa erythrocyte membrane proteins. The nature of these receptor sites was studied in peptide-binding assays using enzyme-treated erythrocytes, showing that these protein receptors are susceptible to structural changes provoked by enzyme treatment (neuraminidase, trypsin or chymotrypsin). Inhibition invasion assays in 'in vitro' cultures showed that all specific high binding sequences were able to inhibit invasion by 11-69% at 200 microM concentration.
Subject(s)
Carrier Proteins/metabolism , Erythrocytes/metabolism , Plasmodium falciparum/metabolism , Protozoan Proteins/metabolism , Amino Acid Sequence , Animals , Antigens, Protozoan/immunology , Antigens, Protozoan/metabolism , Binding Sites, Antibody , Binding, Competitive , Biological Assay/methods , Carrier Proteins/chemistry , Carrier Proteins/immunology , Chymotrypsin/metabolism , Cross-Linking Reagents/chemistry , Erythrocytes/parasitology , Humans , Kinetics , Membrane Proteins , Molecular Sequence Data , Neuraminidase/metabolism , Plasmodium falciparum/immunology , Protozoan Proteins/chemistry , Protozoan Proteins/immunology , Radioligand Assay , Trypsin/metabolismABSTRACT
The synthetic peptide SPf66 adsorbed on alum is one of the few Plasmodium falciparum vaccines which have been tested in field trials. We previously reported that subcutaneous administration of SPf66 loaded PLGA microparticles (MP) enhances the antibody response to this antigen compared to the conventional alum formulation. We now evaluate the suitability of polymeric formulations to obtain systemic immune responses by gastric intubation of Balb/c mice. Formulations composed of 1:1 mixtures of PLGA 50:50 and 75:25 (lactic:glycolic) microparticles were administered by the oral route, and when animals were boosted 3 weeks later significant systemic IgG antibody responses were elicited, comparable to alum triple shot and superior to the aqueous vaccine given by the oral route. The finding of IgG2a isotype for PLGA-vaccinated mice compared to the absent levels of this isotype for the alum-vaccinated group could be interpreted as a sign of Th1-like immune response and cellular immune response activation. Our results confirm that using the appropriate schedule the oral administration of PLGA particles is suitable to obtain systemic immune responses to the carried antigen.