P. J. Cardona

Construction, characterization and preclinical evaluation of MTBVAC, the first live-attenuated M. tuberculosis-based vaccine to enter clinical trials

The development of a new tuberculosis vaccine is an urgent need due to the failure of the current vaccine, BCG, to protect against the respiratory form of the disease. MTBVAC is an attenuated Mycobacterium tuberculosis vaccine candidate genetically engineered to fulfil the Geneva consensus requirements to enter human clinical trials. We selected a M. tuberculosis clinical isolate to generate two independent deletions without antibiotic-resistance markers in the genes phoP, coding for a transcription factor key for the regulation of M. tuberculosis virulence, and fadD26, essential for the synthesis of the complex lipids phthiocerol dimycocerosates (DIM), one of the major mycobacterial virulence factors. The resultant strain MTBVAC exhibits safety and biodistribution profiles similar to BCG and confers superior protection in preclinical studies. These features have enabled MTBVAC to be the first live attenuated M. tuberculosis vaccine to enter clinical evaluation.

Double-blind, randomized, placebo-controlled Phase I Clinical Trial of the therapeutical antituberculous vaccine RUTI®

A Phase I interventional Clinical Trial was performed with a potential tuberculosis vaccine, based on detoxified cellular fragments of M. tuberculosis, named RUTI. The objective was to evaluate the safety profile and T-cell immune responses over a 6-month period following subcutaneous inoculation. The double-blind, randomized and placebo-controlled trial was conducted in healthy volunteers, all recruited at one site. RUTI, at each of the four tested doses, starting from 5microg and going up to 200microg, and placebo were inoculated to groups of 4 and 2 volunteers respectively, consecutively. RUTI appeared to be well tolerated as judged by local and systemic clinical evaluation, though vaccine dose dependent local adverse reactions were recorded. T-cell responses of blood lymphocytes to PPD and a number of antigen subunits were elevated, when compared with controls subjects. These results support the feasibility of future evaluation, to be targeted at subjects with latent tuberculosis infection (LTBI).

Extended safety studies of the attenuated live tuberculosis vaccine SO2 based on phoP mutant.

Safety is one of the main concerns for attenuated live vaccine candidates. Here we extend the stability and attenuation studies of the promising tuberculosis vaccine candidate based on Mycobacterium tuberculosis phoP mutant strain, SO2. Stability of the phoP mutation was tested after sub-culturing SO2 strain for 6 months in laboratory media and also after 3 months of infection in SCID mice. Results showed no reversion of the phoP mutation either in vitro or in vivo. In addition, SO2 was fully sensitive to four major first-line antituberculous drugs against tuberculosis. Safety and toxicity studies were performed in guinea pigs. Animals were infected with a quantity of SO2 equivalent to 50 vaccination doses (2.5x10(6) CFUs) and weight was monitored for 6 months. All animals survived and no histological lesions were found, showing full attenuation of SO2. Studies in a post-exposure model of guinea pigs and mice, previously infected with M. tuberculosis, were performed and no toxicity effects were found after inoculation of SO2. All these results together confirm that SO2 has a secure safety profile that encourages its use in clinical trials.

The tuberculin skin test increases the responses measured by T cell interferon-gamma release assays.

RUTI is a vaccine consisting of Mycobacterium tuberculosis bacilli grown in stress conditions that is fragmented, detoxified and liposomed. RUTI was designed to shorten the treatment of latent tuberculosis infection (LTBI) with isoniazid from 9 months to just 1 month, by additional treatment with two inoculations of RUTI 4 weeks apart. During the validation process for monitoring the immunogenicity of administration of RUTI in a Phase I clinical trial, the question arose whether to introduce the tuberculin skin test (TST) in the screening of non‐LTBI volunteers. This study was designed to evaluate the effect of TST on subsequent different T‐cell interferon‐gamma release assay (TIGRA) responses, using a spectrum of M. tuberculosis‐related antigens (ESAT‐6, CFP‐10, 16 kDa, 19 kDa, MPT64, Ag 85B, 38 kDa, hsp65, PPD and BCG). The results showed an increase in post‐TST response even in non‐LTBI subjects for most antigens tested, as measured both by whole blood assay (WBA) and ELISPOT. Increased ELISPOT response decreased toward pre‐TST levels within 1 month whereas the WBA response did not. Taking into account that there is no definitive correlation between TST and TIGRA tests to diagnose LTBI and the feasibility that TST might alter the immune monitoring included in clinical trials, these data suggest that TST determination should be carefully planned to avoid any interference with TIGRA.

Evaluation of a Commercial Probe Assay for Detection of Rifampin Resistance in Mycobacterium tuberculosis Directly from Respiratory and Nonrespiratory Clinical Samples

A commercial assay (Inno-Line Probe Assay; Innogenetics, Belgium) was evaluated to determine its ability to detect rifampin resistance inMycobacterium tuberculosis directly from clinical specimens. Fifty-nine selected specimens (42 respiratory and 17 nonrespiratory) culture positive forMycobacterium tuberculosis were tested along with their corresponding isolates in culture. The results were compared with those obtained by in vitro susceptibility testing. The results of the line probe assay to detect rifampin resistance inMycobacterium tuberculosis present in clinical specimens and in cultured isolates were concordant for 58 of 59 (98.3%) isolates (95% confidence limits = 90.9–99.9%). The line probe assay failed only once, when a fecal specimen was tested; no amplification was observed due to the presence of inhibitory compounds. The most frequently observed mutation was His526→Asp (58.7%), followed by the His526→Tyr (23.9%); together, they represented 82.6% of rifampin-resistant samples. In conclusion, the Inno-Line Probe Assay is a rapid, useful method for detecting the presence ofMycobacterium tuberculosis complex and its resistance to rifampin directly from clinical specimens and culture. Moreover, since rifampin resistance is a potential marker for multidrug resistance inMycobacterium tuberculosis, this assay may constitute an important tool for the control of tuberculosis.

Towards a ‘Human‐like’ Model of Tuberculosis: Intranasal Inoculation of LPS Induces Intragranulomatous Lung Necrosis in Mice Infected Aerogenically with Mycobacterium tuberculosis

It is well known that one of the differences between murine and human tuberculosis is the lack of intragranulomatous necrosis in the former. The aim of this study was to create a feasible and reproducible model of an experimental model of murine tuberculosis in which this necrosis should be present. Considering the Shwartzman reaction as a possible explanation for intragranulomatous necrosis in human tuberculosis, C57Bl/6 mice, infected aerogenically with a virulent strain of Mycobacterium tuberculosis, were intranasally inoculated with lipopolysaccharide (LPS) on day 19 postinfection (p.i.). Twenty‐four hours later, neutrophils infiltrated the lung parenchyma in a significant level, and 10 days after necrosis could be detected in the centres of primary granulomas, that showed scanty macrophages and large amounts of collagen on an eosinophilic background. On the other hand, a significant decrease in the concentration of colony forming units (CFU) could be appreciated 24 h after the LPS inoculation. Afterwards, nonbronchogenic spreading of granulomas increased and higher levels of interferon (IFN)‐γ mRNA were detected. These results lend support to the Shwartzman reaction as the origin of the intragranulomatous necrosis in the M. tuberculosis infection, and provides a useful tool to improve experimental murine models in tuberculosis.

Comparative evaluation of two commercial assays for direct detection of Mycobacterium tuberculosis in respiratory specimens.

Two commercial systems for the amplification and detection ofMycobacterium tuberculosis directly from respiratory samples were compared. The Roche Cobas Amplicor MTB Test and the Roche manual Amplicor MTB Test (Roche Diagnostic Systems, USA) were applied to 755 decontaminated respiratory specimens collected from 470 patients. Results were compared with those of acid-fast staining and culture. A total of 251 specimens were collected from 156 patients diagnosed with pulmonary tuberculosis, including 28 specimens corresponding to 13 patients that were receiving antituberculous treatment. Given the overall positivity rate of 33.2% (251/755), the sensitivity, specificity, and positive and negative predictive values were 92.4, 100, 100, and 96.5%, respectively, for the Cobas Amplicor MTB Test and 90.8, 100, 100, and 95.8%, respectively, for the Amplicor MTB Test. For 204 (81.3%) smear positive specimens and 47 (19.7%) smear negative specimens, the sensitivity values were 100 and 59.6%, respectively, for the Cobas Amplicor MTB Test and 100 and 51%, respectively, for the Amplicor MTB Test. There were no statistically significant differences in sensitivity or specificity between the two assays and culture (p>0.05). The overall results of both assays were concordant for 99.5% of the samples. It is concluded that although both nucleic acid amplification methods are rapid and specific for the detection ofMycobacterium tuberculosis complex in respiratory specimens, the Cobas Amplicor MTB Test appears to be slightly more sensitive than the Amplicor MTB Test when smear negative specimens are investigated.

Comparison of a Nonradiometric System with Bactec 12B and Culture on Egg-Based Media for Recovery of Mycobacteria from Clinical Specimens

Abstract The MB/BacT (Organon-Teknika, USA) is a fully automated, rapid, nonradiometric system for the culture of mycobacteria from clinical samples. The rate of recovery of mycobacteria and the time to detection obtained with the MB/BacT were compared with those obtained with Löwenstein-Jensen and Coletsos solid media and Bactec 7H12 (12B) (Becton-Dickinson, USA) broth when 600 processed specimens were inoculated into all media in parallel. Specimens included 383 respiratory samples, 20 urine samples, 23 purulent exudates, 13 stool samples, 103 blood samples, 14 bone marrow aspirates, and 44 body fluid samples or aspirates. Overall, 106 mycobacterial isolates comprising six species were recovered, of which 100 (94.3%) were detected with MB/BacT, 98 (92.5%) with egg-based media, and 96 (90.2%) with Bactec 12B. The recovery rates of Mycobacteriumtuberculosis complex with MB/BacT, egg-based media, and Bactec 12B were 98.7%, 93.7, and 89.9%, respectively. The average number of days to detection of single mycobacterial isolates was 14.2 days for MB/BacT, 26.1 days for egg-based media, and 11.7 days for Bactec 12B. The contamination rates were higher in MB/BacT (5%) than in Bactec 12B (1.8%) or egg-based media (1.5%). MB/BacT is a reliable, nonradiometric, less labor-intensive alternative to Bactec 12B for recovery of mycobacteria, but, as with other liquid culture methods, MB/BacT should be used in combination with a solid medium, not on its own.

Production of Antibodies against Glycolipids from the Mycobacterium tuberculosis Cell Wall in Aerosol Murine Models of Tuberculosis

Evolution of antibodies against glycolipids from the Mycobacterium tuberculosis cell wall has been studied for the first time in experimental murine models of tuberculosis induced by aerosol, in which infection, reinfection, reactivation, prophylaxis and treatment with antibiotics have been assayed. Results show a significant humoral response against these antigens, where diacyltrehaloses (DAT) and sulpholipid I (SL‐I) elicited higher antibody levels than protein antigens like antigen 85 protein complex (Ag85), culture filtrate proteins (CFP) and purified protein derivative (PPD). Only immunoglobulin M (IgM) antibodies have been detected against DAT and SL‐I. Their evolution has a positive correlation with bacillary concentration in tissues.

Usefulness of acr Expression for Monitoring Latent Mycobacterium tuberculosis Bacilli in ‘In Vitro’ and ‘In Vivo’ Experimental Models

Real‐time RT‐PCR was used to quantify the expression of genes possibly involved in Mycobacterium tuberculosis latency in in vitro and murine models. Exponential and stationary phase (EP and SP) bacilli were exposed to decreasing pH levels (from 6.5 to 4.5) in an unstirred culture, and mRNA levels for 16S rRNA, sigma factors sigA,B,E,F,G,H and M, Rv0834c, icl, nirA, narG, fpbB, acr, rpoA, recA and cysH were quantified. The expression of acr was the one that best correlated with the CFU decrease observed in SP bacilli. In the murine model, the expressions of icl, acr and sigF tended to decrease when bacillary counts increased and vice versa. Values from immunodepressed mice (e.g. α/β T cells, TNF, IFN‐γ and iNOs knock out strains), with accelerated bacillary growth rate, confirmed this fact. Finally, the expression of acr was maintained in mice following long‐term treatment with antibiotics. The quantification of acr expression could be useful for monitoring the presence of latent bacilli in some murine models of tuberculosis.