RESUMEN

Varicella-zoster virus (VZV) is the etiologic agent of varicella (chickenpox) and herpes zoster (shingles) infections commonly involving skin, mucous membranes, and less frequently the central nervous system. Traditional methods for the laboratory diagnosis of these infections are time-consuming, labor-intensive, and often insensitive. As such, these tests are being replaced by more sensitive and rapid molecular methods. This study evaluated the performance of two different molecular assays, the Simplexa VZV Direct and Simplexa VZV Swab Direct, to detect VZV DNA in cerebrospinal fluid (CSF) and lesion-swab specimens, respectively. The Simplexa VZV Direct and Simplexa VZV Swab Direct assays were compared against individual composite reference methods that varied depending on the sample cohort examined. A total of 883 CSF and 452 cutaneous and mucocutaneous prospective, retrospective, and contrived specimens were evaluated in this multicenter study. The results of this study showed that the Simplexa assays demonstrated near perfect agreement (k = 0.98) compared to the composite reference methods for the detection of VZV in CSF and lesion swab specimens. A further comparison between the standard of care molecular assays employed at the site of specimen collection and the Simplexa assays demonstrated excellent agreement (k = 1.0). The Simplexa assays offer rapid and reliable alternatives for the detection of VZV in certain clinical specimens without the need for nucleic acid extraction.

Asunto(s)

Varicela , Herpes Zóster , Varicela/diagnóstico , Herpes Zóster/diagnóstico , Herpesvirus Humano 3/genética , Humanos , Estudios Prospectivos , Estudios Retrospectivos , Manejo de Especímenes

RESUMEN

We genotyped a Somali population (n = 85; age < or =30 years) for 617 cytokine and cytokine receptor single nucleotide polymorphisms (SNPs) using Illumina GoldenGate genotyping to determine associations with measles, mumps and rubella immunity. Overall, 61 significant associations (P < or = 0.01) were found between SNPs belonging to cytokine receptor genes regulating T helper (Th)1 (IL12RB2, IL2RA and B) and Th2 (IL4R and IL10RB) immunity, and cytokine (IL1B, TNFA, IL6 and IFNB1) and cytokine receptor (IL1RA, IFNAR2, IL18R1, TNFRSF1A and B) genes regulating innate immunity and variations in antibody levels to measles, mumps and/or rubella. SNPs within two major inflammatory cytokine genes, TNFA and interleukin (IL) 6, showed associations with measles-specific antibodies. Specifically, the minor allele variant of rs1799964 (TNFA -1211 C>T) was associated with primarily seronegative values (median enzyme immunoassay index values < or =0.87; P = 0.002; q = 0.23) in response to measles disease and/or vaccination. A heterozygous variant CT for rs2069849 (IL6 +4272C>T; Phe201Phe) was also associated with seronegative values and a lower median level of antibody response to measles disease and/or vaccination (P = 0.004; q = 0.36) or measles vaccination alone (P = 0.008). Several SNPs within the coding and regulatory regions of cytokine and cytokine receptor genes showed associations with mumps and rubella antibody levels but were less informative as strong linkage disequilibrium patterns and lower frequencies for minor alleles were observed among these SNPs. Our study identifies specific SNPs in innate immune response genes that may play a role in modulating antibody responses to measles vaccination and/or infection in Somali subjects.

Asunto(s)

Citocinas/genética , Sarampión/inmunología , Paperas/inmunología , Receptores de Citocinas/genética , Rubéola (Sarampión Alemán)/inmunología , Adolescente , Anticuerpos Antivirales/sangre , Niño , Estudios de Cohortes , Citocinas/inmunología , Femenino , Genotipo , Humanos , Desequilibrio de Ligamiento , Masculino , Sarampión/genética , Vacuna contra el Sarampión-Parotiditis-Rubéola/genética , Vacuna contra el Sarampión-Parotiditis-Rubéola/inmunología , Paperas/genética , Polimorfismo de Nucleótido Simple , Grupos de Población , Receptores de Citocinas/inmunología , Rubéola (Sarampión Alemán)/genética , Somalia

RESUMEN

The ELISPOT assay is a highly sensitive technique used for the detection of individual cytokine releasing cells. We have developed an IFN-gamma ELISPOT assay utilizing unfractionated frozen peripheral blood mononuclear cells (PBMC) to quantify the frequency of measles virus (MV)-specific IFN-gamma-secreting T cells in 117 healthy children who had been previously immunized with two doses of the measles-mumps-rubella vaccine. We have also estimated the variability associated with the quantification of ELISPOT plates and compared the number of MV-specific IFN-gamma-secreting T cells for each subject as determined by two different operators of an ELISPOT reader. The median frequency of MV-specific IFN-gamma-producing memory T cells detected by this assay was 0.005 % and 0.01 % as determined by an in-house and commercial operator, respectively. Although we found a significant correlation (r = 0.83, p<0.0001) between the number of spots counted by the commercial and in-house operators of an ELISPOT reader, the median number of spots counted by the commercial operator was twice the number of spots counted by an in-house operator (p<0.001). This demonstrates the importance of using a common ELISPOT reader and operator, among other parameters, to quantify the number of spots when a large volume of plates are being scanned and analyzed.

Asunto(s)

Inmunoensayo/normas , Interferón gamma/análisis , Interferón gamma/metabolismo , Virus del Sarampión/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Adolescente , Niño , Femenino , Humanos , Interferón gamma/inmunología , Masculino , Vacuna contra el Sarampión-Parotiditis-Rubéola/inmunología , Reproducibilidad de los Resultados , Especificidad por Sustrato

RESUMEN

Immunity to measles is conferred by the interplay of humoral and cellular immune responses, the latter being critical in maintaining long-term recall response. Therefore, it is important to evaluate measles-specific humoral and cellular immunity in populations several years after vaccination and understand the correlations among these measures of immunity. We examined measles-specific antibodies, lymphoproliferation and the Th1/Th2 signature cytokines, interferon (IFN)-gamma and interleukin (IL)-4, in a population-based cohort of healthy children from Olmsted County, Minnesota after two doses of measles-mumps-rubella-II (MMR-II) vaccine. We detected positive measures of measles-specific cellular and humoral immunity in the majority of our study population. However, a small proportion of subjects demonstrated an immune response skewed towards the Th2 type, characterized by the presence of either IL-4 and/or measles-specific antibodies and a lack of IFN-gamma production. Further, we observed a significant positive correlation between lymphoproliferation and secretion of IFN-gamma (r = 0.20, P = 0.0002) and IL-4 (r = 0.15, P = 0.005). Measles antibody levels were correlated with lymphoproliferation (r = 0.12, P = 0.03), but lacked correlation to either cytokine type. In conclusion, we demonstrated the presence of both long-term cellular and humoral responses after MMR-II vaccination in a significant proportion of study subjects. Further, a positive correlation between lymphoproliferation and IL-4 and IFN-gamma suggests that immunity to measles may be maintained by both Th1 and Th2 cells. We speculate that the Th2 biased response observed in a subset of our subjects may be insufficient to provide long-term immunity against measles. Further examination of the determinants of Th1 versus Th2 skewing of the immune response and long-term follow-up is needed.

Asunto(s)

Anticuerpos Antivirales/inmunología , Citocinas/inmunología , Virus del Sarampión/inmunología , Vacuna contra el Sarampión-Parotiditis-Rubéola/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Adolescente , Factores de Edad , Anticuerpos Antivirales/biosíntesis , Especificidad de Anticuerpos/inmunología , Niño , Femenino , Humanos , Inmunidad Celular/inmunología , Inmunoglobulina G/análisis , Interferón gamma/inmunología , Interleucina-4/inmunología , Masculino , Vacuna contra el Sarampión-Parotiditis-Rubéola/administración & dosificación , Células TH1/inmunología , Células Th2/inmunología

RESUMEN

Mechanisms that lead to induction of life-long immunity to measles virus (MV) are poorly understood. In the present study, we have assessed the activation, proliferation and cytokine secreting function of peripheral blood T cells from MV immune individuals. Expression of cell blastogenesis markers, such as increased forward light scatter and CD38 expression, peaked 5-7 days after infection of peripheral blood mononuclear cells (PBMC) with the live attenuated Edmonston strain of MV. Subset analysis revealed that both CD3- and CD3+ cells expressed activation markers but that the CD3+ T cells predominated late in the culture period corresponding to maximal proliferation and cell recovery. The majority of CD3+ T cells consisted of CD4+CD8- cells. IFN-gamma and IL-4 production similarly showed optimal production late in culture. Depletion of CD4 cells prior to culture and MV stimulation completely abrogated both IFN-gamma and IL-4 production, whereas depletion of CD8 cells did not diminish production, suggesting that CD4+CD8- T cells were principally involved in production of these cytokines. Finally, optimal IFN-gamma production was elicited at high MV doses and IL-4 at much lower doses. These results suggest that among MV immune individuals, in vitro responses to measles are dominated by CD4+ T cells that, depending on antigen dose, primarily produce a Th1-like and, to a lesser extent, a Th1/Th2-mixed pattern of cytokine release.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Citocinas/biosíntesis , Activación de Linfocitos/inmunología , Virus del Sarampión/inmunología , Adulto , Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Células Cultivadas , Medio de Cultivo Libre de Suero , Relación Dosis-Respuesta Inmunológica , Humanos , Interferón gamma/biosíntesis , Interleucina-4/biosíntesis , Sarampión/inmunología , Subgrupos de Linfocitos T/inmunología , Células TH1/inmunología

RESUMEN

The transporter associated with antigen processing (TAP) and human leukocyte antigen-DM (HLA-DM) genes are involved in the antigen-processing pathway of both HLA class I and class II-restricted antigen presentation. We hypothesized that polymorphisms within the TAP and DM genes may influence antibody levels following measles vaccination. We examined TAP and DM polymorphisms in 242 school children from Olmsted County, Minnesota, USA who received one dose of measles-mumps-rubella-II (MMR-II) vaccine at the age of 15 months. Based on the level of serum measles-specific immunoglobulin G (IgG) antibodies, subjects were classified as seronegatives (n = 72) or seropositives (n = 170). We determined TAP1 and TAP2 allele types by polymerase chain reaction (PCR) amplification of specific alleles (PASA) and determined DM allele type by PCR amplification followed by direct sequencing of the polymorphic sites. We analysed the data for any TAP or DM allelic association with antibody levels post measles vaccination using the chi-square test and univariate linear regression analysis. We found no trend in the overall distribution of TAP and DM genotype frequencies between seronegative and seropositive subjects, suggesting that TAP and DM polymorphism and antibody levels following measles vaccination are not directly associated. In addition, we did not find an association between TAP (TAP1, P = 0.71; TAP2, P = 0.87) or DM (DMA, P = 0.42; DMB, P = 0.71) homozygosity and seronegativity to measles vaccine in this study group. Our study suggests that TAP and DM gene polymorphisms do not influence antibody levels post measles vaccination.

Asunto(s)

Antígenos HLA-D/genética , Antígenos de Histocompatibilidad Clase I/genética , Inmunoglobulina G/sangre , Vacuna Antisarampión/inmunología , Polimorfismo Genético , Transportadoras de Casetes de Unión a ATP , Formación de Anticuerpos/genética , Formación de Anticuerpos/inmunología , Femenino , Antígenos HLA-D/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Lactante , Masculino , Sarampión/prevención & control , Minnesota

RESUMEN

DNA microarray technology is a new and powerful tool that allows the simultaneous analysis of a large number of nucleic acid hybridization experiments in a rapid and efficient fashion. The development of the DNA microarray chip has been driven by modern techniques of microelectronic fabrication, miniaturization and integration to produce what is referred to as "laboratory-on-chip" devices. The application of DNA chip technology includes the comprehensive analysis of multiple gene mutations and expressed sequences with regard to newer drug designs, host-pathogen interactions and the design of new vaccines. An advantage of microarray technology is that it can assist researchers to better define and understand the expression profile of a given genotype associated with disease, adverse effects from exposure to certain stimuli, or the ability to understand or predict immune responses to specific antigens. This paper briefly reviews DNA microarray technology and its implications with special reference to vaccine design. The technical aspects comprising array manufacturing and design, array hybridization, formatting, scanning and data handling are also briefly discussed.

Asunto(s)

Diseño de Fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Vacunas , Animales , Antígenos/química , Antígenos/genética , Antígenos/inmunología , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Análisis por Conglomerados , Biología Computacional , Análisis Mutacional de ADN/instrumentación , Análisis Mutacional de ADN/métodos , Sondas de ADN , ADN Complementario/genética , ADN de Neoplasias/genética , Bases de Datos Factuales , Predicción , Perfilación de la Expresión Génica/instrumentación , Perfilación de la Expresión Génica/métodos , Genoma Bacteriano , Genoma Viral , Antígenos HLA/inmunología , Humanos , Ligandos , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Técnica de Sustracción , Moldes Genéticos , Vacunas/química , Vacunas de ADN/química , Vacunas de ADN/genética , Vacunas Sintéticas/química , Vacunas Sintéticas/genética

RESUMEN

Burkholderia cepacia is an emerging opportunistic pathogen that causes fatal infections in patients suffering from cystic fibrosis (CF) and chronic granulomatous disease. Various environmental isolates of B. cepacia are, however, capable of degrading environmental pollutants, such as trichloroethylene, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), etc., and are also highly effective in controlling plant diseases caused by nematodes and fungi. Such strains have therefore been proposed for environmental release to clean up toxic dump sites or as biopesticides. Various efforts to distinguish between clinical and environmental isolates of B. cepacia with regard to their virulence characteristics have produced ambiguous results, suggesting that newer methods are needed to test for the presence or absence of pathogenic potential in B. cepacia strains proposed for environmental release. We now report that several clinical strains of B. cepacia secrete cytotoxic factors that allow macrophage and mast cell death in the presence of external ATP. Several environmental strains had reduced activity in this regard. We also demonstrate that, while all the strains secrete enzymes that have nucleoside diphosphate kinase (Ndk), adenylate kinase (Ak) and 5'-nucleotidase activity, the level of secretion of the 5'-nucleotidase (and/or ATPase/phosphatase) appears to be lower in the environmental strains than in the clinical strains. The secretion of these enzymes is specifically activated in the presence of eukaryotic proteins such as alpha2-macroglobulin. As macrophage-or mast cell surface-associated P2Z receptors promote their cell death in the presence of mM concentrations of ATP, and as the secreted ATP-using enzymes generate various phosphorylated or non-phosphorylated adenine nucleotides that may even be better agonists than ATP in activating the P2Z receptors or may act through the activation of additional purinergic receptors, such enzymes may play an important role in allowing B. cepacia to evade host defence.

Asunto(s)

Infecciones por Burkholderia/microbiología , Burkholderia cepacia/patogenicidad , Macrófagos/microbiología , Mastocitos/microbiología , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Adenilato Quinasa/metabolismo , Animales , Burkholderia cepacia/aislamiento & purificación , Burkholderia cepacia/metabolismo , Muerte Celular , Línea Celular , Células Cultivadas , Cromatografía Líquida de Alta Presión , Pruebas Inmunológicas de Citotoxicidad , Ambiente , Femenino , Macrófagos/citología , Masculino , Mastocitos/citología , Ratones , Ratones Endogámicos BALB C , Nucleósido-Difosfato Quinasa/metabolismo , Virulencia

RESUMEN

Vibrio cholerae strain VB1 secretes a number of enzymes into the outside medium that utilize ATP as a substrate. Such enzymes are found in the outside medium during the mid-log phase of growth, when the optical density at 650 nm is about 0.4, and they demonstrate nucleoside diphosphate kinase (Ndk), 5' nucleotidase, and adenylate kinase (Ak) activities. We report that the filtered growth medium of V. cholerae, as well as the flowthrough fraction of a green Sepharose column during fractionation of the growth medium, had very little cytotoxicity by itself towards macrophages and mast cells but exhibited significant cytotoxicity in the presence of exogenous ATP. Such fractions, harboring 5' nucleotidase, Ndk, and presumably other ATP-utilizing enzymes, demonstrated enhanced macrophage and mast cell death; periodate-oxidized-ATP (oATP)-treated macrophage and mast cells or such cells exposed to 0.1 mM Mg(2+), where surface-associated P2Z receptors could not be activated, were not susceptible to subsequent ATP addition. Microscopic visualization of mast cells clearly demonstrated cell morphological changes such as swelling, vacuolization, and nuclear fragmentation following treatment with ATP and the growth medium of V. cholerae; however, these effects were suppressed if the mast cells were pretreated with oATP. These results strongly imply that the secreted ATP-utilizing enzymes of V. cholerae modulate the external ATP levels of the macrophage and mast cells, leading to their accelerated death, presumably through activation of P2Z receptors. Thus, development of inhibitors for such enzymes may reduce the level of V. cholerae infection; alternatively, mutations in such genes may eliminate V. cholerae survival in the gut and contribute to a safer live vaccine.

Asunto(s)

Adenosina Trifosfato/metabolismo , Fagocitos/patología , Vibrio cholerae/patogenicidad , 5'-Nucleotidasa/metabolismo , Adenilato Quinasa/metabolismo , Animales , Muerte Celular , Cromatografía Líquida de Alta Presión , Macrófagos/patología , Mastocitos/patología , Ratones , Ratones Endogámicos BALB C , Receptores Purinérgicos P2/fisiología , Virulencia

RESUMEN

Peptide mapping by limited proteolysis of a highly protective 71-kDa cell wall-associated protein of Mycobacterium tuberculosis H37Ra was carried out in order to identify key protective determinants within the native protein. The 71-kDa protein, which had an isoelectric point of 4.25, was digested into eight major bands at 48 h using trypsin and pepsin at equal enzyme to protein ratios (pH 5.5). The in vitro lymphocyte reactivity of individual peptides suggested P1, P2 and P5 to be significantly immunoreactive in mice immunized with native 71-kDa-polylactide-coglyeolide (PLG); however, the reactivity was significantly lower than that of the native 71-kDa protein. Immunization of mice with a pooled fraction (upper fraction-71 kDa) of more immunoreactive peptides (consisting of P1 and P2) did not further boost their immunoreactivity. However, P1 and P2 exhibited comparable or even higher lymphocyte proliferation in human tuberculous and control subjects. These data suggest distinct antigenic specificities in humans and mice and further substantiate the use of the 71-kDa protein or its peptides P1 and P2 as potential vaccine candidates for tuberculosis.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/inmunología , Mycobacterium tuberculosis/metabolismo , Animales , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/aislamiento & purificación , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Humanos , Inmunización , Focalización Isoeléctrica , Activación de Linfocitos , Ratones , Mycobacterium tuberculosis/genética , Pepsina A , Mapeo Peptídico , Linfocitos T/inmunología , Tripsina

RESUMEN

Current vaccination and drug delivery strategies emphasize on the development of controlled release techniques for persistent and sustained effects. In the recent years, polymer based systems for the delivery of bioactive agents have gained considerable attention due to their marked adjuvanticity, established biodegradability and biocompatibility, excellent mechanical strength and controlled release profiles. This review deals with the potential applications of synthetic polymers mainly PLG polymers in delivery of vaccines and drugs.

Asunto(s)

Portadores de Fármacos , Ácido Láctico/administración & dosificación , Ácido Poliglicólico/administración & dosificación , Polímeros/administración & dosificación , Vacunas/administración & dosificación , Copolímero de Ácido Poliláctico-Ácido Poliglicólico

RESUMEN

Cellular and humoral immunity induced by Mycobacterium tuberculosis has led to identification of newer vaccine candidates, but despite this, many questions concerning the protection against tuberculosis remain unanswered. Recent progress in this field has centered on T cell subset responses and cytokines that these cells secrete. There has been a steady progress in identification and characterization of several classes of major mycobacterial proteins which includes secretory/export proteins, cell wall associated proteins, heat shock proteins and cytoplasmic proteins. The protein antigens are now believed to represent the key protective immunity inducing antigens in the bacillus. In this review, various mycobacterial protein antigens of vaccination potential are compared for their efficacy in light of current immunological knowledge.

Asunto(s)

Vacuna BCG/inmunología , Proteínas Bacterianas/inmunología , Mycobacterium/inmunología , Animales , Antígenos Bacterianos , Humanos , Tuberculosis/inmunología , Tuberculosis/prevención & control , Vacunas Sintéticas/inmunología

RESUMEN

Mechanism of immunoprotection and specificity of two highly immunoprotective mycobacterial proteins, viz. 71 and 30 kDa were investigated. The adoptive transfer studies indicated that immunoprotection was mainly mediated by cooperative effect of CD4+ and CD8+ (66.7-73.3% on the basis of percent survival) which was further enhanced marginally by supplementation of B cells, natural killer cells, dendritic cells, macrophages and other immune cells. The specificity studies indicated that both the proteins did not cross react with the unrelated intracellular pathogens i.e. Aspergillus fumigatus, Salmonella typhi and Leishmania donovani as seen by T cell proliferation assay. The protection imparted by these mycobacterial proteins was also specific as the 71 and 30 kDa primed mice did not exhibit any cross protection against sublethal challenge of S typhi. The results indicate 71 and 30 kDa mycobacterial proteins to contain T cell specific epitopes responsible for specific immunoprotection, thus indicating their potential role as antituberculous vaccine candidates.

Asunto(s)

Proteínas Bacterianas/inmunología , Inmunoterapia , Mycobacterium tuberculosis , Tuberculosis/prevención & control , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos , Tuberculosis/inmunología

RESUMEN

Microparticles composed of poly (DL-lactide-co-glycolide) (DL-PLG) were used as delivery vehicles for evaluating the immunoreactive and immunoprotective properties of 71-kDa cell wall associated protein of Mycobacterium tuberculosis H37Ra. Mice immunized with 71-kDa microparticles entrapped in DL-PLG (PLG-MPs) exhibited significantly higher T-cell stimulation and cytokine release in comparison to 71-kDa emulsified in Freund's incomplete adjuvant (FIA) as well as a BCG vaccinated group throughout the post-immunization (p.im.) period. Further, the protective efficacy of 71-kDa was evaluated on the basis of survival rates and viable bacilli load in different organs at 30 days post challenge (p.c.), with the median lethal dose (LD50) of M. tuberculosis H37Rv at weeks 8 and 16 p.im. Both 71-kDa-PLG and 71-kDa-FIA immunized groups exhibited a comparable protection (90%) which was significantly higher (P < 0.5) than in the BCG group (70%) at week 8 p.im. and it was consistent with the decreased bacterial load in the target organs. However, on increasing the interval of challenge to 16 weeks p.im., the protective efficacy of 71-kDa-PLG was sustained (85%) while that of 71-kDa-FIA began to wane (70%). Further. the 71-kDa-PLG immunized group exhibited a significantly higher (P < 0.001) clearance of bacterial load from the lungs and livers in comparison to the 71-kDa-FIA immunized group. The results suggest the long-term protective potential of a PLG-microparticle based antigen delivery system for tuberculosis.

Asunto(s)

Adyuvantes Inmunológicos , Proteínas Bacterianas/inmunología , Sistemas de Liberación de Medicamentos , Ácido Láctico , Mycobacterium tuberculosis/inmunología , Ácido Poliglicólico , Polímeros , Tuberculosis/prevención & control , Animales , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/inmunología , Vacuna BCG , Proteínas Bacterianas/administración & dosificación , Pared Celular/inmunología , Citocinas/análisis , Preparaciones de Acción Retardada , Portadores de Fármacos , Femenino , Hígado/microbiología , Pulmón/microbiología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos , Microesferas , Mycobacterium tuberculosis/aislamiento & purificación , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Bazo/citología , Bazo/microbiología , Linfocitos T/inmunología , Vacunación

RESUMEN

Proteins associated with the cell wall peptidoglycan (CW-Pr) of Mycobacterium tuberculosis H37Ra were isolated to evaluate their immunoreactivity and immunoprophylactic properties against experimental tuberculosis. Chemical treatment of the cell wall with trifluoromethanesulfonic acid: anisole (2:1) resulted in the release of three proteins of 71, 60 and 45 kDa as resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A comparative study of immune responses elicited to individual proteins in mice immunized with CW-Pr emulsified in incomplete Freund's adjuvant showed the 71-kDa protein to be the most immunoreactive antigen. This 71-kDa protein was found to cross-react with the 70-kDa heat shock protein from M. leprae and possessed ATPase activity. Mice immunized with the 71-kDa protein exhibited significantly higher immune responses, on the basis of T and B cell reactivity, as compared to a M. bovis Bacillus Calmette Guerin (BCG)-vaccinated group. The culture supernatants collected from 71-kDa stimulated lymphocytes stimulated exhibited increased interferon-gamma and interleukin-2 production. The protective efficacy of the 71-kDa protein in comparison to BCG was determined by challenging the mice with a virulent strain M. tuberculosis H37Rv. The 71-kDa protein was found to be more protective in animals challenged at 8 and 16 weeks post immunization, shown by increased survival rates and decreased viable bacilli counts in the target organs as compared to BCG-vaccinated animals.

Asunto(s)

Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Mycobacterium tuberculosis/inmunología , Animales , Pared Celular/química , Femenino , Masculino , Ratones , Peso Molecular

RESUMEN

We have isolated cell wall peptidoglycan associated proteins (CW-Pr) of Mycobacterium tuberculosis H37Ra by chemical treatment with trifluoromethanesulfonic acid:anisole (2:1), which further resolved into 71, 60 and 45 kDa proteins on SDS-PAGE. A study was carried out to investigate the immunoreactivity of these proteins with blood samples from 4 categories, including 15 tuberculous patients (TB), 5 tuberculous patients on ATT (TBT), 10 PPD non-reactive healthy controls (HPPD-) and 11 PPD reactive healthy controls (HPPD+). Comparing the proliferative responses to cell wall protein antigens, it was observed that the 71 kDa protein gave maximum stimulation with PBMCs from the TB and HPPD+ groups. The adherent PBMCs from the TB group also demonstrated enhanced phagocytosis, particularly in the presence of 71 and 45 kDa proteins, and the phagocytic index was significantly higher (P < 0.05) than the TBT group. However, PBMCs from of the groups recognized the 60 kDa cell wall antigen. Our results suggest that the 71 kDa protein from the cell wall of M. tuberculosis is highly immunogenic.

Asunto(s)

Antígenos Bacterianos/inmunología , Pared Celular/inmunología , Inmunidad Celular , Peptidoglicano/inmunología , Tuberculosis Pulmonar/inmunología , Antígenos Bacterianos/aislamiento & purificación , Adhesión Celular , Reacciones Cruzadas , Humanos , Activación de Linfocitos , Activación de Macrófagos , Fagocitosis

RESUMEN

A 71 kDa cell wall associated protein ofM.tuberculosis H(37)Ra, on encapsulation in biodegradable microparticles composed of poly-DL-lactide-co-glycolide (DL-PLG) exhibited higher level of T-cell stimulation and cytokine release as compared to 71 kDa-FIA in mice as evaluated till sixteenth week post immunization (p.im.). Further, the protection imparted by immunization with 71 kDa-PLG microparticles (71 kDa-PLG-MPs) was significantly higher (p<0.5) than the 71 kDa-FIA (70%) and BCG (65%) immunized group on the basis of survival rates, when challenged at sixteenth week p.im. The protective effect was consistent with the decreased bacterial load in the infected organs of 71 kDa-PLG primed group as compared to 71 kDa-FIA and BCG immunized groups.