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1.
Vaccine ; 31(42): 4834-40, 2013 Oct 01.
Article in English | MEDLINE | ID: mdl-23933335

ABSTRACT

Accruing data strongly support the possible role of CD8+ T cells in immunity against tuberculosis (TB). Multivalent vaccines against Mycobacterium tuberculosis (Mtb) that incorporate CD8+ T cell antigens with those that elicit CD4+ T cells are therefore highly desirable. To screen for potential CD8+ T cell antigens that are produced by Mtb during infection, we isolated pathogen-derived peptides that bound to MHC Class I molecules expressed in adherent splenocytes obtained from Mtb-infected mice. Mass spectroscopy analysis revealed the following four nonamer peptides that had 100% homology with Mtb proteins: DGYVGAPAH (MT_0401), TTMPLFAD (MT_1164), RSGAATPVR (MT_2160.1) and LAAVVGVVL (MT_0078). The gene MT_0401 codes the protein 5'-phosphoribosylglycinamide transformylase 2 and the other three genes code for hypothetical proteins with unknown function. The NCBI/Blast analysis showed that among the four peptides DGYVGAPAH had the highest maximum alignment score and lowest E value (number of alignments expected by chance). Therefore, we assessed whether MT_0401 expressed in two genetic vaccine formulations was capable of stimulating CD8+ T cell response that is specific to DGYVGAPAH peptide. When mice were immunized with a recombinant plasmid DNA and an E1/E3-deleted Adenovirus 5 expressing MT0401 protein, using both homologous and heterologous prime-boost protocols, they developed strong DGYVGAPAH-specific CD8+ T cell response as well as antibody and CD4+ specific T cell response to the full length MT0401 protein. Equally important was the observation that mice infected with Mtb developed DGYVGAPAH-specific CD8+ T cell responses in both spleen and lungs. These results demonstrate that Mtb antigens that are processed and presented via MHC Class I machinery can be readily identified by the described approach and may be useful candidate antigens to stimulate specific CD8+ T cell responses in vaccine development programs.


Subject(s)
Antigens, Bacterial/immunology , CD8-Positive T-Lymphocytes/immunology , Peptides/immunology , Tuberculosis Vaccines/immunology , Tuberculosis/immunology , Tuberculosis/microbiology , Animals , Antibodies, Bacterial/blood , Antigens, Bacterial/chemistry , Antigens, Bacterial/isolation & purification , CD4-Positive T-Lymphocytes/immunology , Female , Lung/immunology , Mass Spectrometry , Mice , Mice, Inbred C57BL , Peptides/chemistry , Peptides/isolation & purification , Spleen/immunology , Tuberculosis Vaccines/administration & dosage , Tuberculosis Vaccines/isolation & purification , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunology , Vaccines, Synthetic/isolation & purification
2.
J Clin Microbiol ; 51(5): 1367-73, 2013 May.
Article in English | MEDLINE | ID: mdl-23390284

ABSTRACT

The development of an accurate antigen detection assay for the diagnosis of active tuberculosis (TB) would represent a major clinical advance. Here, we demonstrate that the Mycobacterium tuberculosis Rv1681 protein is a biomarker for active TB with potential diagnostic utility. We initially identified, by mass spectroscopy, peptides from the Rv1681 protein in urine specimens from 4 patients with untreated active TB. Rabbit IgG anti-recombinant Rv1681 detected Rv1681 protein in lysates and culture filtrates of M. tuberculosis and immunoprecipitated it from pooled urine specimens from two TB patients. An enzyme-linked immunosorbent assay formatted with these antibodies detected Rv1681 protein in unconcentrated urine specimens from 11/25 (44%) TB patients and 1/21 (4.8%) subjects in whom TB was initially clinically suspected but then ruled out by conventional methods. Rv1681 protein was not detected in urine specimens from 10 subjects with Escherichia coli-positive urine cultures, 26 subjects with confirmed non-TB tropical diseases (11 with schistosomiasis, 5 with Chagas' disease, and 10 with cutaneous leishmaniasis), and 14 healthy subjects. These results provide strong validation of Rv1681 protein as a promising biomarker for TB diagnosis.


Subject(s)
Bacterial Proteins/urine , Biomarkers/urine , Mycobacterium tuberculosis/metabolism , Tuberculosis, Pulmonary/diagnosis , Amino Acid Sequence , Antibodies, Bacterial/immunology , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Chagas Disease/urine , Escherichia coli/isolation & purification , Escherichia coli Infections/urine , Humans , Immunoglobulin G/immunology , Leishmaniasis, Cutaneous/urine , Molecular Sequence Data , Schistosomiasis/urine
3.
Sci Transl Med ; 5(170): 170ra13, 2013 Jan 30.
Article in English | MEDLINE | ID: mdl-23363977

ABSTRACT

Mycobacterium tuberculosis (Mtb) can persist in hostile intracellular microenvironments evading immune cells and drug treatment. However, the protective cellular niches where Mtb persists remain unclear. We report that Mtb may maintain long-term intracellular viability in a human bone marrow (BM)-derived CD271(+)/CD45(-) mesenchymal stem cell (BM-MSC) population in vitro. We also report that Mtb resides in an equivalent population of BM-MSCs in a mouse model of dormant tuberculosis infection. Viable Mtb was detected in CD271(+)/CD45(-) BM-MSCs isolated from individuals who had successfully completed months of anti-Mtb drug treatment. These results suggest that CD271(+) BM-MSCs may provide a long-term protective intracellular niche in the host in which dormant Mtb can reside.


Subject(s)
Bone Marrow Cells/cytology , Mesenchymal Stem Cells/cytology , Mycobacterium tuberculosis/physiology , Nerve Tissue Proteins/metabolism , Receptors, Nerve Growth Factor/metabolism , Stem Cell Niche , AC133 Antigen , Aerosols , Animals , Antigens, CD/metabolism , Bone Marrow/microbiology , Bone Marrow/pathology , Bone Marrow Cells/metabolism , Cell Differentiation , Disease Models, Animal , Female , Glycoproteins/metabolism , Humans , Lung/microbiology , Lung/pathology , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Microbial Viability , Peptides/metabolism , Tuberculosis/drug therapy , Tuberculosis/microbiology , Tuberculosis/pathology
4.
Clin Vaccine Immunol ; 19(6): 935-43, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22518013

ABSTRACT

Despite the clear need to control visceral leishmaniasis (VL), the existing diagnostic tests have serious shortcomings. Here, we introduce an innovative approach to directly identify Leishmania infantum antigens produced in vivo in humans with VL. We combined reverse-phase high-performance liquid chromatography (RP-HPLC) with mass spectrometry and categorized three distinct L. infantum proteins presumably produced in bone marrow/spleen/liver and excreted in the urine of patients with VL. The genes coding for these proteins (L. infantum iron superoxide dismutase, NCBI accession number XP_001467866.1; L. infantum tryparedoxin, NCBI accession number XP_001466642.1; and L. infantum nuclear transport factor 2, NCBI accession number XP_001463738.1) were cloned, and the recombinant molecules were produced in Escherichia coli. Antibodies to these proteins were produced in rabbits and chickens and were used to develop a capture enzyme-linked immunosorbent assay (ELISA) designed to detect these L. infantum antigens in the urine of VL patients. Specificity of the antibodies was confirmed by a Western blot analysis using both recombinant proteins and whole parasite extract. Importantly, a urinary antigen detection assay assembled with pairs of antibodies specific for each of these antigens identified 17 of 19 patients with VL. These results indicate that an improved antigen detection assay based on L. infantum proteins present in the urine of patients with VL may represent an important new strategy for the development of a specific and accurate diagnostic test that has the potential to both distinguish active VL from asymptomatic infection and serve as an important tool to monitor therapy efficacy.


Subject(s)
Antibodies, Protozoan , Antigens, Protozoan/analysis , Clinical Laboratory Techniques/methods , Leishmania infantum/chemistry , Leishmaniasis, Visceral/diagnosis , Protozoan Proteins/analysis , Urine/chemistry , Adolescent , Adult , Animals , Antibodies, Protozoan/isolation & purification , Antigens, Protozoan/genetics , Antigens, Protozoan/isolation & purification , Blotting, Western , Chickens , Child , Child, Preschool , Chromatography, High Pressure Liquid , Cloning, Molecular , Escherichia coli/genetics , Female , Humans , Leishmania infantum/genetics , Male , Mass Spectrometry , Parasitology/methods , Protozoan Proteins/genetics , Protozoan Proteins/isolation & purification , Rabbits , Urine/parasitology , Young Adult
5.
Immunology ; 135(3): 216-25, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22043824

ABSTRACT

An efficacious tuberculosis (TB) vaccine will probably need to induce both CD4 and CD8 T-cell responses specific to a protective Mycobacterium tuberculosis antigen(s). To achieve this broad cellular immune response we tested a heterologous DNA/protein combination vaccine strategy. We used a purified recombinant protein preparation of a unique M. tuberculosis antigen (rMT1721) found in the urine of TB patients, an optimized plasmid DNA expressing this protein (DNA-MT1721), and a Toll-like receptor 4 agonist adjuvant. We found that priming mice with DNA-MT1721 and subsequently boosting with rMT1721 elicited high titres of specific IgG1 and IgG2a antibodies as well as high magnitude and polyfunctional CD4(+) T-cell responses. However, no detectable CD8(+) T-cell response was observed using this regimen of immunization. In contrast, both CD4(+) and CD8(+) T-cell responses were detected after a prime/boost vaccination regimen using rMT1721 as the priming antigen and DNA-MT1721 as the boosting immunogen. These findings support the exploration of heterologous DNA/protein immunization strategies in vaccine development against TB and possibly other infectious diseases.


Subject(s)
Bacterial Proteins/administration & dosage , Bacterial Proteins/immunology , Mycobacterium tuberculosis/immunology , Tuberculosis Vaccines/administration & dosage , Adjuvants, Immunologic/administration & dosage , Animals , Antibodies, Bacterial/biosynthesis , Antigens, Bacterial/administration & dosage , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , DNA, Bacterial/administration & dosage , DNA, Bacterial/genetics , Female , Immunization, Secondary , Immunoglobulin G/biosynthesis , Interferon-gamma/biosynthesis , Mice , Mice, Inbred C57BL , Toll-Like Receptor 4/agonists , Tuberculosis Vaccines/genetics , Vaccines, DNA/administration & dosage , Vaccines, DNA/genetics , Vaccines, Synthetic/administration & dosage
6.
Open J Immunol ; 1(1): 1-7, 2011 Jun 01.
Article in English | MEDLINE | ID: mdl-23264930

ABSTRACT

DPPD (Rv0061) is a difficult to express protein of Mycobacterium tuberculosis that elicits strong and specific delayed type hypersensitivity reactions in humans infected with M. tuberculosis. Therefore e DPPD is a molecule that can improve the specificity of the tuberculin skin test, which is widely used as an aid for the diagnosis of tuberculosis. However, a pitfall of our initial studies was that the DPPD molecule used to perform the skin tests was engineered as fusion molecule with another Mycobacterium protein. This approach was used because no expression of DPPD could be achieved either as a single molecule or as a fusion protein using a variety of commercially available expression systems. Here, we report the production and purification of rDPPD using a synthetic gene engineered to contain E. coli codon bias. The gene was cloned into pET14b expression vector, which was subsequently used to transform Rosetta 2(DE3) pLysS or BL-21(DE3)pLysS host cells. The recombinant protein was over-expressed after induction with IPTG and its purification was easily achieved at levels of 5 - 10 mg/l of bacterial broth cultures. The purified protein was confirmed to be DPPD by Mass Spectroscopy sequencing analysis. Moreover, purified rDPPD stimulated peripheral blood mononuclear cells of PPD positive blood donors to produce high levels of IFN-γ, thus confirming that this molecule is biologically active. Because of the DPPD gene is restricted to the tuberculosis-complex organisms of Mycobacterium genus, this highly purified molecule should be useful for the identification of individuals sensitized with tubercle bacilli.

7.
Am J Pathol ; 176(1): 198-204, 2010 Jan.
Article in English | MEDLINE | ID: mdl-20008132

ABSTRACT

Recent evidence points to lung draining lymph nodes as the site that initiates the immune response in mice infected with aerosolized Mycobacterium tuberculosis. Here we expanded these studies and showed that infection of mice that lack lymph nodes with aerosolized M. tuberculosis results in a massive mononuclear cell infiltrate in the lungs within 14 days postinfection. This infiltration clearly resembles an expansion of the bronchus-associated lymphoid tissue. As expected, no bronchus-associated lymphoid tissue was observed in M. tuberculosis-infected wild-type control mice. Importantly, acquired specific immune response to M. tuberculosis antigens could be detected in lung lymphocytes harvested from mice lacking lymph nodes as early as 14 days postinfection. In addition, the bacterial burden in these mice was indistinguishable from that observed in wild-type C57BL/6 control mice. These results indicate that in the absence of lymph nodes, priming of the immune response occurs in the lung tissues after infection of mice with aerosolized M. tuberculosis and clearly illustrate the enormous plasticity of the immune system to develop resistance to foreign pathogens.


Subject(s)
Adaptive Immunity/immunology , Lung/immunology , Lung/microbiology , Lymph Nodes/microbiology , Lymph Nodes/pathology , Mycobacterium tuberculosis/immunology , Aerosols , Animals , Immunohistochemistry , Lung/pathology , Lymph Nodes/immunology , Lymphotoxin-alpha/deficiency , Lymphotoxin-alpha/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Splenectomy , Tuberculosis, Pulmonary/immunology , Tuberculosis, Pulmonary/microbiology
8.
Infect Control Hosp Epidemiol ; 30(4): 392-5, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19236281

ABSTRACT

To evaluate the utility of the QuantiFERON-TB Gold assay for monitoring latent tuberculosis treatment efficacy, the assay was performed serially for healthcare workers receiving isoniazid therapy. After 9 months of isoniazid therapy, all of these healthcare workers remained QuantiFERON-TB Gold positive, and cellular proliferation assays revealed persistently strong purified protein derivative responses. These results do not support the use of the QuantiFERON-TB Gold assay to monitor therapy.


Subject(s)
Antitubercular Agents/therapeutic use , Interferon-gamma/blood , Isoniazid/therapeutic use , Mycobacterium tuberculosis/immunology , Reagent Kits, Diagnostic , Tuberculosis/drug therapy , Adult , Female , Humans , Lymphocyte Activation , Male , Middle Aged , Skin Tests , Treatment Outcome , Tuberculin/immunology , Tuberculosis/immunology , Tuberculosis/microbiology
9.
Microbes Infect ; 10(14-15): 1469-76, 2008.
Article in English | MEDLINE | ID: mdl-18817888

ABSTRACT

Although guinea pigs are considered one of the best animal models of tuberculosis, little data exist describing latent or dormant tuberculosis infection in these animals. Here we address this issue using a streptomycin auxotrophic mutant of Mycobacterium tuberculosis. This mutant grows unimpaired in the presence of streptomycin but in its absence shifts to latency/dormancy (lack growth and over-expression of alpha-crystallin). To establish infection animals are inoculated with the mutant followed by daily administration of streptomycin (three weeks), which allows initial microbial multiplication in the animal's tissues. Withdrawal of streptomycin establishes latency/dormancy and few viable organisms are recovered from the animals' lungs and spleen six months later. During the infectious process guinea pigs steadily gained weight and presented no clinical signs (scuff fur and lethargy) of disease. Histopathology of organs mimicked tuberculous lesions in humans and PBMC from infected animals strongly responded to stimulation with PPD. Finally, tuberculin skin test (a hallmark of latent infection diagnosis) performed in infected animals was strongly positive (>or=15 mm induration). These results point to an interesting and reliable model of latent/dormant tuberculosis infection in guinea pigs.


Subject(s)
Disease Models, Animal , Mycobacterium tuberculosis/physiology , Tuberculosis/microbiology , Animals , Body Weight , Colony Count, Microbial , Female , Guinea Pigs , Liver/pathology , Lung/microbiology , Lung/pathology , Mutation , Mycobacterium tuberculosis/genetics , Spleen/microbiology , Spleen/pathology , Streptomycin/administration & dosage , Streptomycin/metabolism , Tuberculin Test , Tuberculosis/pathology , Tuberculosis/physiopathology
10.
Microbes Infect ; 10(6): 664-72, 2008 May.
Article in English | MEDLINE | ID: mdl-18457976

ABSTRACT

In previous studies we showed that biasing the immune response to Porphyromonas gingivalis antigens to the Th1 phenotype increases inflammatory bone resorption caused by this organism. Using a T cell screening strategy we identified eight P. gingivalis genes coding for proteins that appear to be involved in T-helper cell responses. In the present study, we characterized the protein encoded by the PG_1841 gene and evaluated its relevance in the bone resorption caused by P. gingivalis because subcutaneous infection of mice with this organism resulted in the induction of Th1 biased response to the recombinant PG1841 antigen molecule. Using an immunization regime that strongly biases toward the Th1 phenotype followed by challenge with P. gingivalis in dental pulp tissue, we demonstrate that mice pre-immunized with rPG1841 developed severe bone loss compared with control immunized mice. Pre-immunization of mice with the antigen using a Th2 biasing regime resulted in no exacerbation of the disease. These results support the notion that selected antigens of P. gingivalis are involved in a biased Th1 host response that leads to the severe bone loss caused by this oral pathogen.


Subject(s)
Bacterial Proteins/toxicity , Bone Resorption/chemically induced , Gram-Negative Bacterial Infections/immunology , Porphyromonas gingivalis/chemistry , Th1 Cells/drug effects , Animals , Antigens, Bacterial/immunology , Bone Resorption/immunology , Gram-Negative Bacterial Infections/physiopathology , Mice , Porphyromonas gingivalis/pathogenicity , Th1 Cells/immunology , Th2 Cells/immunology
11.
Clin Vaccine Immunol ; 15(4): 638-43, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18305107

ABSTRACT

Although the antigen detection assay has the potential to discriminate active tuberculosis from latent infection, development of such a test for the accurate diagnosis of this serious disease has only recently become a matter of interest. Here we present evidence that a Mycobacterium tuberculosis protein (ornithine carboamyltransferase, coded for by MT_1694; Rv1656 [argF]) is an interesting candidate molecule for this test development. The protein was initially discovered by mass spectroscopy in urine of patients with pulmonary tuberculosis and shown by Western blot analysis to be present in M. tuberculosis crude cell extract as well as in the culture supernatant ("secreted" protein). In addition, a recombinant ornithine carboamyltransferase (rMT1694) produced in Escherichia coli was recognized by immunoglobulin G (IgG) antibodies from patients with active tuberculosis but not by IgG from uninfected healthy subjects. Moreover, rMT1694 was strongly recognized by peripheral blood mononuclear cells from both healthy tuberculin purified protein derivative (PPD)-positive individuals and patients with pulmonary tuberculosis. More importantly, a capture enzyme-linked immunosorbent assay formatted with rabbit IgG antibodies specific to rMT1694 was able to identify the presence of this antigen in urine samples from 6 of 16 patients with pulmonary tuberculosis and in none of 16 urine samples collected from healthy PPD(+) controls. These results indicate that an improved antigen detection assay based on M. tuberculosis ornithine carboamyltransferase may represent an important new strategy for the development of a specific and accurate diagnostic test for tuberculosis.


Subject(s)
Mycobacterium tuberculosis/enzymology , Ornithine Carbamoyltransferase/urine , Tuberculosis, Pulmonary/urine , Animals , Antibody Specificity , Antigens, Bacterial/urine , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay/methods , Humans , Immunoglobulin G/immunology , Mycobacterium tuberculosis/immunology , Neutrophils/immunology , Ornithine Carbamoyltransferase/genetics , Ornithine Carbamoyltransferase/immunology , Rabbits , Recombinant Proteins/immunology , Tuberculosis, Pulmonary/blood , Tuberculosis, Pulmonary/immunology , Tuberculosis, Pulmonary/microbiology
12.
Microbes Infect ; 9(3): 308-16, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17306589

ABSTRACT

In human and experimental paracoccidioidomycosis the severe disease is characterized by depressed cellular immunity whereas the mild disease is associated with persistent T cell immunity. Since the subcutaneous route of antigen inoculation is an efficient inducer of cellular immunity, we decided to study this route of infection and verify its effect on a lethal secondary infection of susceptible hosts. It was observed that the s.c. infection induces positive delayed type hypersensitivity (DTH) responses in 9 different mouse strains, is a self healing process and susceptible mice develop more intense DTH reactions than resistant mice to Paracoccidioides brasiliensis infection. Unexpectedly, the previous s.c. infection of susceptible mice led to immunoprotection or disease exacerbation depending on the route of fungal challenge. Immunoprotection was achieved after intraperitoneal challenge and was associated with persistent cell-mediated immunity and a mixed type-1/type-2 immunity. Exacerbated disease was found after intravenous challenge, was associated with cellular immunity anergy and prevalent type-2 immune response. As a whole, our work demonstrates that susceptibility to P. brasiliensis infection cannot be ascribed to intrinsic inability to mount cellular immune responses, that a single immunization procedure can result in opposite disease outcomes and immunoprotection can be achieved by a balanced Th1/Th2 immunity.


Subject(s)
Antigens, Fungal/administration & dosage , Paracoccidioides/pathogenicity , Paracoccidioidomycosis/immunology , Paracoccidioidomycosis/physiopathology , Animals , Antibodies, Fungal/blood , Antigens, Fungal/immunology , Cytokines/metabolism , Female , Hypersensitivity, Delayed , Injections, Intraperitoneal , Injections, Intravenous , Injections, Subcutaneous , Male , Mice , Mice, Inbred Strains , Paracoccidioides/immunology , Paracoccidioidomycosis/microbiology , Paracoccidioidomycosis/mortality , Th1 Cells/immunology , Th2 Cells/immunology , Time Factors , Virulence
13.
Clin Vaccine Immunol ; 13(9): 1014-21, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16960113

ABSTRACT

Most individuals exposed to Mycobacterium tuberculosis become infected but hinder the infectious process in dormant foci, known as latent tuberculosis. This limited infection usually stimulates strong T-cell responses, which provide lifelong resistance to tuberculosis. However, latent tuberculosis is still poorly understood, particularly because of the lack of a reliable animal model of dormant infection. Here we show that inoculation of mice with a unique streptomycin-auxotrophic mutant of Mycobacterium tuberculosis recapitulates dormant infection. The mutant grows unimpaired in the presence of streptomycin and no longer grows but remains viable for long periods of time after substrate removal, shifting from the log growth phase to the latent stage, as indicated by augmented production of alpha-crystallin. Mice challenged with the mutant and inoculated with streptomycin for approximately 3 weeks developed a limited infection characterized by a low bacteriological burden and the presence of typical granulomas. After substrate withdrawal, the infection was hindered but few microorganisms remained viable (dormant) in the animals' tissues for at least 6 months. In addition, the animals developed both potent T-cell responses to M. tuberculosis antigens, such as early culture filtrate, Ag85B, and ESAT-6, and resistance to reinfection with virulent M. tuberculosis. Therefore, infection of mice or other animals (e.g., guinea pigs) with M. tuberculosis strain 18b constitutes a simple and attractive animal model for evaluation of antituberculosis vaccines in the context of an M. tuberculosis-presensitized host, a prevailing condition among humans in need of a vaccine.


Subject(s)
Mycobacterium tuberculosis/immunology , Tuberculosis Vaccines/immunology , Tuberculosis/prevention & control , Animals , Antigens, Bacterial/immunology , BCG Vaccine/immunology , CD11b Antigen/immunology , CD4 Antigens/immunology , CD8 Antigens/immunology , Colony Count, Microbial , Disease Models, Animal , Immunohistochemistry , In Vitro Techniques , Lung/immunology , Lung/microbiology , Lung/pathology , Mice , Mice, Inbred BALB C , Mutation , Mycobacterium tuberculosis/genetics , T-Lymphocytes/immunology , Tuberculosis/immunology , alpha-Crystallins/metabolism
14.
J Immunol ; 175(8): 5298-305, 2005 Oct 15.
Article in English | MEDLINE | ID: mdl-16210635

ABSTRACT

The existence of therapeutic agents and the bacille Calmette-Guérin (BCG) vaccine have not significantly affected the current tuberculosis pandemic. BCG vaccine protects against serious pediatric forms of tuberculosis but not against adult pulmonary tuberculosis, the most common and contagious form of the disease. Several vaccine candidates, including Mycobacterium tuberculosis recombinant proteins formulated in newer adjuvants or delivered in bacterial plasmid DNA have recently been described. An attractive source of vaccine candidates has been M. tuberculosis Ags present in culture supernatants of the initial phases of the bacterial growth in vitro. In this study we describe an Ag discovery approach to select for such Ags produced in vivo during the initial phases of the infection. We combined RP-HPLC and mass spectrometry to identify secreted or shed M. tuberculosis proteins eliminated in animal urine within 14 days after the infection. A peptide containing sequence homology with a hypothetical M. tuberculosis protein was identified and the recombinant protein produced in Escherichia coli. The protein was recognized by Ab (IgG2a and IgG1) and T cells (Th1) of mice infected with M. tuberculosis and by lymphoid cells from healthy donors who had a positive purified protein derivative skin test but not from tuberculosis patients. Moreover, this Ag induced protection in mice against M. tuberculosis at levels comparable to protection induced by BCG vaccine. These results validate the Ag discovery approach of M. tuberculosis proteins secreted or shed in vivo during the early phases of the infection and open new possibilities for the development of potential vaccine candidates or of markers of active mycobacterial multiplication and therefore active disease.


Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Mycobacterium tuberculosis/genetics , Tuberculosis, Pulmonary/microbiology , Animals , Antigens, Bacterial/chemistry , Antigens, Bacterial/genetics , Antigens, Bacterial/immunology , Antigens, Bacterial/urine , Bacterial Proteins/immunology , Bacterial Proteins/urine , Cells, Cultured , Chromatography, High Pressure Liquid , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Humans , Mass Spectrometry , Mice , Mice, Inbred C57BL , Mycobacterium tuberculosis/immunology , Mycobacterium tuberculosis/metabolism , Spleen/cytology , Spleen/immunology , Tuberculosis Vaccines/administration & dosage , Tuberculosis Vaccines/immunology , Tuberculosis, Pulmonary/urine
15.
Exp Parasitol ; 110(1): 12-21, 2005 May.
Article in English | MEDLINE | ID: mdl-15804374

ABSTRACT

Cattle present variable levels of resistance to ticks and the immune correlates of these heritable phenotypes must be known in order to develop effective vaccines. The antibody responses to tick salivary antigens were examined in cattle of tick-susceptible (Holstein) and tick-resistant (Nelore) breeds. After heavy infestations, levels of IgG1 and IgG2 antibodies decreased in Holsteins and remained the same in Nelores. Conversely, levels of IgE antibodies increased in Holsteins. Different sizes of tick burdens modulated the IgG1 antibody response in a susceptible breed (Aberdeen): levels were higher than in controls in heavily infested animals, but not in those undergoing intermediary or minimal infestations. The three experimental groups presented similar levels of IgG2 antibodies. Levels of IgE antibodies were higher only in animals undergoing intermediate infestations. These results indicate that tick infestations suppress the IgG antibody response in susceptible breeds, that IgE antibodies are not protective, and that the dose of tick saliva modulates the isotype of host antibody responses.


Subject(s)
Cattle Diseases/immunology , Immunoglobulin G/biosynthesis , Ixodidae/immunology , Tick Infestations/veterinary , Animals , Cattle , Cattle Diseases/parasitology , Disease Susceptibility/immunology , Disease Susceptibility/veterinary , Female , Immunity, Innate/immunology , Immunoglobulin A/biosynthesis , Immunoglobulin A/blood , Immunoglobulin E/biosynthesis , Immunoglobulin E/blood , Immunoglobulin G/blood , Male , Saliva/immunology , Tick Infestations/immunology , Tick Infestations/parasitology
16.
Infect Immun ; 72(7): 3932-40, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15213137

ABSTRACT

Resistance to paracoccidioidomycosis, the most important endemic mycosis in Latin America, is thought to be primarily mediated by cellular immunity and the production of gamma interferon. To assess the role of interleukin-4 (IL-4), a Th2 cytokine, pulmonary paracoccidioidomycosis in IL-4-depleted susceptible (B10.A) and intermediate (C57BL/6) mice was studied. Two different protocols were used to neutralize endogenous IL-4 in B10.A mice: 1 mg of anti-IL-4 monoclonal antibody (MAb)/week and 8 mg 1 day before intratracheal infection with 10(6) Paracoccidioides brasiliensis yeast cells. Unexpectedly, both protocols enhanced pulmonary infection but did not alter the levels of pulmonary cytokines and specific antibodies. Since in a previous work it was verified that C57BL/6 mice genetically deficient in IL-4 were more resistant to P. brasiliensis infection, we also investigated the effect of IL-4 depletion in this mouse strain. Treatment with the MAb at 1 mg/week led to less severe pulmonary disease associated with impaired synthesis of Th2 cytokines in the lungs and liver of control C57BL/6 mice. Conversely, in IL-4-depleted C57BL/6 mice, increased levels of tumor necrosis factor alpha and IL-12 were found in the lungs and liver, respectively. In addition, higher levels of immunoglobulin G2a (IgG2a) and lower levels of IgG1 antibodies were produced by IL-4-depleted mice than by control mice. Lung pathologic findings were equivalent in IL-4-depleted and untreated B10.A mice. In IL-4-depleted C57BL/6 mice, however, smaller and well-organized granulomas replaced the more extensive lesions that developed in untreated mice. These results clearly showed that IL-4 can have a protective or a disease-promoting effect in pulmonary paracoccidioidomycosis depending on the genetic background of the host.


Subject(s)
Interleukin-4/metabolism , Lung Diseases/microbiology , Paracoccidioidomycosis/metabolism , Animals , Antibody Formation/immunology , Immunoglobulin Isotypes/immunology , Lung/microbiology , Lung/pathology , Lung Diseases/immunology , Lung Diseases/metabolism , Male , Mice , Paracoccidioidomycosis/immunology , Th2 Cells/immunology
17.
Clin Immunol ; 103(2): 185-95, 2002 May.
Article in English | MEDLINE | ID: mdl-12027424

ABSTRACT

Paracoccidioides brasiliensis is a facultative, intracellular pathogen causing the most important deep mycosis in Latin America. As the production of IFN-gamma and induction of cell-mediated immunity to P. brasiliensis is of critical importance in host defense, the immunotherapeutic effect of exogenous IL-12 administration was studied in a murine model of susceptibility to pulmonary infection. rIL-12 treatment led to a less disseminated disease, as confirmed by decreased fungal loads in liver and spleen. Administration of rIL-12 did not affect fungal growth in the lungs, although it did induce an augmented pulmonary mononuclear cell inflammation. IL-12 treatment induced an early (week 1) increase in pulmonary IFN-gamma, but decreased cytokine and specific antibody (IgG1 and IgG3) production at week 8 after infection. These results show that IL-12 administration induces a less severe infection, but the high inflammatory response detected in the lungs precludes its possible use as a new therapeutic tool for severe paracoccidioidomycosis.


Subject(s)
Interleukin-12/pharmacology , Paracoccidioidomycosis/immunology , Animals , Antibodies, Fungal/biosynthesis , Cytokines/biosynthesis , Disease Models, Animal , Humans , Hypersensitivity, Delayed , Immunotherapy , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/pathology , Lung/immunology , Lung/microbiology , Lung/pathology , Male , Mice , Paracoccidioides/immunology , Paracoccidioides/isolation & purification , Paracoccidioidomycosis/etiology , Paracoccidioidomycosis/pathology , Paracoccidioidomycosis/prevention & control
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