Bruna Daniella de Souza Silva

Prime–Boost with Mycobacterium smegmatis Recombinant Vaccine Improves Protection in Mice Infected with Mycobacterium tuberculosis

The development of a new vaccine as a substitute for Bacillus Calmette–Guerin or to improve its efficacy is one of the many World Health Organization goals to control tuberculosis. Mycobacterial vectors have been used successfully in the development of vaccines against tuberculosis. To enhance the potential utility of Mycobacterium smegmatis as a vaccine, it was transformed with a recombinant plasmid containing the partial sequences of the genes Ag85c, MPT51, and HspX (CMX) from M. tuberculosis. The newly generated recombinant strain mc2-CMX was tested in a murine model of infection. The recombinant vaccine induced specific IgG1 or IgG2a responses to CMX. CD4+ and CD8+ T cells from the lungs and spleen responded ex vivo to CMX, producing IFN-γ, IL17, TNF-α, and IL2. The vaccine thus induced a significant immune response in mice. Mice vaccinated with mc2-CMX and challenged with M. tuberculosis showed better protection than mice immunized with wild-type M. smegmatis or BCG. To increase the safety and immunogenicity of the CMX antigens, we used a recombinant strain of M. smegmatis, IKE (immune killing evasion), to express CMX. The recombinant vaccine IKE-CMX induced a better protective response than mc2-CMX. The data presented here suggest that the expression of CMX antigens improves the immune response and the protection induced in mice when M. smegmatis is used as vaccine against tuberculosis.

MPT-51/CpG DNA vaccine protects mice against Mycobacterium tuberculosis

Tuberculosis (TB) is a severe infectious disease that kills approximately two million people worldwide every year. Because BCG protection is variable and does not protects adults, there is a great need for a new vaccine against TB that does not represent a risk for immunocompromised patients and that is also capable of protecting adult individuals. MPT-51 is a protein found in the genome of mycobacteria and binds to the fibronectin of the extracellular matrix, which may have a role in host tissue attachment and virulence. In order to test the usefulness of MPT-51 as a subunit vaccine, BALB/c were vaccinated and challenged with Mycobacterium tuberculosis. The infection of BALB/c with M. tuberculosis increased the number of IFN-gamma(+) T lymphocytes specific to MPT-51 in the spleen and lungs. Inoculation with rMPT-51/FIA and with rMPT-51/CpG DNA in non-infected BALB/c increased the amounts of IFN-gamma(+) T lymphocytes. Inoculation with rMPT-51/FIA also induced a humoral response specific to MPT-51. CFU counts of lung tissues done 60 days after infection showed a reduction of about 2 log in the bacteria load in the group of animals inoculated with rMPT-51/CpG DNA. These results make MPT-51 a valuable component to be further evaluated in the development of other subunit vaccines.

Using BCG, MPT-51 and Ag85 as antigens in an indirect ELISA for the diagnosis of bovine tuberculosis.

This study evaluated the Mycobacterium tuberculosis protein antigen MPT-51, the trimeric antigen 85 (Ag85) complex, and Bacillus Calmette-Guérin (BCG) in an indirect ELISA to diagnose bovine tuberculosis (TB) from serum samples. Serum was collected from 208 intra-dermal tuberculin test (ITT)-positive and 54 ITT-negative animals from a region where bovine TB is endemic. Using the Ag85 and BCG antigens, the indirect ELISA was able to discriminate ITT-positive from ITT-negative animals. This level of discrimination was not achieved when using the MPT-51 antigen. The highest sensitivity (Se) and specificity (Sp) of the test was found when BCG was used (Se, 82%; Sp, 91%). Further work in different epidemiological settings and with larger numbers of animals will be required to validate these findings.

Different phenotypes of CD8+ T cells associated with bacterial load in active tuberculosis.

Tuberculosis is an infectious disease that affects millions of people worldwide with an annual mortality rate of 1.3 million. The mechanisms contributing to the loss of balance of immune responses and progression to active tuberculosis disease are unknown. Although CD4+ and CD8+ T cells and the cytokines they produce are crucial for protection against tuberculosis they have different roles in tuberculosis immunology. The function of CD4+ T cells has been extensively studied; however, less is known about the phenotype and function of CD8+ T cells. This study evaluated the specific expression of IFN-γ, IL-17, IL-10, and TGF-β and ex vivo expression of perforin and granzyme-B by CD8+ T cells from active tuberculosis individuals compared with latent infected individuals and non-latent infected individuals. Tuberculosis responses were correlated with the baciloscopy score. We observed that the presence of IL-10 and TGF-β expression and down-expression of granzyme-B in CD8+ T cells correlated with increased sputum bacillary load in active tuberculosis individuals. These findings provide new insights into the role of CD8+ T cells in Mycobacterium tuberculosis disease.

Role of antibodies reactive to HspX in discriminating pulmonary tuberculosis contacts with high risk of developing active disease.

Submitted on: 05/21/2011 Approved on: 05/25/2011Correspondence to: Ana Paula Junqueira-KipnisDepartamento de Microbiologia, Imunologia, Patologia e ParasitologiaInstituto de Patologia Tropical e Saude PublicaUniversidade Federal de GoiasRua 235 S/N 74605-050Goiania, GO, BrazilFax: +55 62 3209-6363anapaula@iptsp.ufg.brFinancial Support: CNPq (project no. 575907/08-8), FUNAPE and FAPEG (project no. 001/2007). We declare no conflict of interest.

The use of Mycobacterium tuberculosis HspX and GlcB proteins to identify latent tuberculosis in rheumatoid arthritis patients

Rheumatoid arthritis (RA) is an autoimmune disease characterised by the destruction of articular cartilage and bone damage. The chronic treatment of RA patients causes a higher susceptibility to infectious diseases such as tuberculosis (TB); one-third of the world’s population is latently infected (LTBI) with Mycobacterium tuberculosis (Mtb). The tuberculin skin test is used to identify individuals LTBI, but many studies have shown that this test is not suitable for RA patients. The goal of this work was to test the specific cellular immune responses to the Mtb malate synthase (GlcB) and heat shock protein X (HspX) antigens of RA patients and to correlate those responses with LTBI status. The T-helper (Th)1, Th17 and Treg-specific immune responses to the GlcB and HspX Mtb antigens were analysed in RA patients candidates for tumour necrosis factor-α blocker treatment. Our results demonstrated that LTBI RA patients had Th1-specific immune responses to GlcB and HspX. Patients were followed up over two years and 14.3% developed active TB. After the development of active TB, RA patients had increased numbers of Th17 and Treg cells, similar to TB patients. These results demonstrate that a GlcB and HspX antigen assay can be used as a diagnostic test to identify LTBI RA patients.

Tuberculosis in rheumatoid arthritis patients: the difficulty in making the diagnosis of latent infection.

Since the beginning of the use of anti-TNF in the treatment of rheumatoid arthritis and other inflammatory diseases, cases of pulmonary tuberculosis and extrapulmonary tuberculosis have been reported in patients receiving such treatment. In most cases, the disease develops by the time the patient has received the sixth infusion. Every patient should be evaluated for latent tuberculosis infection prior to the use of a TNF inhibitor. However, the diagnosis of latent tuberculosis infection is a challenge. The tuberculin test, which was the only test available to detect latent tuberculosis infection for nearly a century, presents a number of limitations. Tests based on the detection of the in-vitro production of IFN-gamma by mononuclear cells activated by specific antigens appear to be more accurate and have been studied in patients with rheumatoid arthritis.

Tuberculosis latente en la artritis reumatoide. Evaluación de la respuesta celular y tomografía computarizada de alta resolución

INTRODUCTION The diagnosis of latent tuberculosis (LTB) in patients with rheumatoid arthritis (RA) has become important with the introduction of anti-tumor necrosis factor (anti-TNF-α) agents and the appearance of active tuberculosis cases in these patients. The tuberculin skin test (TST) has limited value in patients with RA. Tests based on the release of interferon-gamma (IFN-γ) are being studied, but their role has not been well established for this group of patients. OBJECTIVES To compare the diagnosis of LTB in patients with RA by using cellular immune response to the TST and T.SPOT-TB. Additionally, findings of tomography studies compatible with LTB were used. METHODS Clinical evaluation, TST, T.SPOT-TB and high-resolution computed tomography (HRCT) in a group of patients with RA at the University Hospital of the Federal University of Goiás. RESULTS Response to the TST was lower in patients with RA (13.5%) compared to the predicted values of the general population. T.SPOT-TB identified a higher number of patients with LTB than the TST (36.8%). HRCT showed changes compatible with LTB in 52.9% of the patients, including 8 of the 11 patients with negative TST and T.SPOT-TB. CONCLUSIONS The TST by itself is insufficient to diagnose LTB. A higher number of positive results were obtained with T.SPOT-TB when compared to the TST. Nevertheless, it was negative in a large percentage of patients with tomography findings consistent with LTB. HRCT is readily available in most large health-care centers and it could be incorporated into the diagnostic strategy for LTB in patients with RA.

Sensitization with atypical mycobacteria is a potent risk factor for cross-reaction with the delayed type hypersensitivity assay in mice

Tuberculosis is a disease that infects approximately two billion people worldwide. The current diagnostic test utilizes purified protein derivative (PPD) obtained from Mycobacterium tuberculosis cultures to elicit a host delayed type hypersensitivity reaction to identify infected individuals. This reaction is manifested as an induration at 48 hours following intradermal i njection. These experiments demonstrate that in a mouse model, repeated administrations of PPD do not elicit a positive tuberculin skin test (TST). However, prior sensitization to environmental mycobacteria does induce a positive TST when administered via the intraperitoneal or oral route. This work has important implications regarding the specificity of the PPD reagent, disease diagnosis, and environmental mycobacteria.