Olga Gil

Evolution of foamy macrophages in the pulmonary granulomas of experimental tuberculosis models

The chronic phase of Mycobacterium tuberculosis infection in mouse experimental models is characterized by the accumulation of foamy macrophages (FM)--which shape the outer ring of the granuloma - in the alveolar spaces, as detected in paraffin-embedded tissues stained with hematoxylin-eosin. In this study, the use of semi- and ultra-thin sections offers more detailed information about the origin of FM both in mouse and guinea-pig experimental models. Lipid bodies (LB) are present in macrophages from the beginning of infection and accumulate in the chronic phase. LB progress from an early (ELB) to a late (LLB) stage, defined according to their progressive capacity to generate cholesterol crystals, resembling atherosclerotic lesions. FM arise from massive accumulation of LLB. Electronic microscopy reveals intracellular lipophilic inclusions (ILIs) in those M. tuberculosis bacilli inside FM. It is our hypothesis that the accumulation of lipids in M. tuberculosis concomitant to the establishment of the non-replicating state prepares the bacilli for future reactivation and for facing future stressful environments.

Granuloma Encapsulation Is a Key Factor for Containing Tuberculosis Infection in Minipigs

A transthoracic infection involving a low dose of Mycobacterium tuberculosis has been used to establish a new model of infection in minipigs. The 20-week monitoring period showed a marked Th1 response and poor humoral response for the whole infection. A detailed histopathological analysis was performed after slicing the formalin-fixed whole lungs of each animal. All lesions were recorded and classified according to their microscopic aspect, their relationship with the intralobular connective network and their degree of maturity in order to obtain a dissemination ratio (DR) between recent and old lesions. CFU counts and evolution of the DR with time showed that the proposed model correlated with a contained infection, decreasing from week 9 onwards. These findings suggest that the infection induces an initial Th1 response, which is followed by local fibrosis and encapsulation of the granulomas, thereby decreasing the onset of new lesions. Two therapeutic strategies were applied in order to understand how they could influence the model. Thus, chemotherapy with isoniazid alone helped to decrease the total number of lesions, despite the increase in DR after week 9, with similar kinetics to those of the control group, whereas addition of a therapeutic M. tuberculosis fragment-based vaccine after chemotherapy increased the Th1 and humoral responses, as well as the number of lesions, but decreased the DR. By providing a local pulmonary structure similar to that in humans, the mini-pig model highlights new aspects that could be key to a better understanding tuberculosis infection control in humans.

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.

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.

Intragranulomatous necrosis in pulmonary granulomas is not related to resistance against Mycobacterium tuberculosis infection in experimental murine models induced by aerosol

Intragranulomatous necrosis is a primary feature in the natural history of human tuberculosis (TB). Unfortunately, this phenomenon is not usually seen in the experimental TB murine model. Artificial induction of this necrosis in pulmonary granulomas (INPG) may be achieved through aerosol inoculation of lipopolysaccharide (LPS) 3 weeks after Mycobacterium tuberculosis infection. At week 9 post‐infection, the centre of primary granulomas became larger, showing eosinophilic necrosis. Interestingly, INPG induction was related to mice strains C57BL/6 and 129/Sv, but not to BALB/c and DBA/2. Furthermore, the same pattern was obtained with the induction of infection using a clinical M. tuberculosis strain (UTE 0335R) that naturally induces INPG. In all the mice strains tested, the study of pulmonary mRNA expression revealed a tendency to increase or to maintain the expression of RANTES, interferon‐γ, tumour necrosis factor and iNOS, in both LPS‐ and UTE 0335R‐induced INPG, thus suggesting that this response must be necessary but not sufficient for inducing INPG. Our work supports that INPG induction is a local phenomenon unrelated to the resistant (C57BL/6 and BALB/c) or susceptible (129/Sv and DBA/2) background of mice strains against M. tuberculosis infection.

Newborn Mice Vaccination with BCG.HIVA222 + MVA.HIVA Enhances HIV-1-Specific Immune Responses: Influence of Age and Immunization Routes

We have evaluated the influence of age and immunization routes for induction of HIV-1- and M. tuberculosis-specific immune responses after neonatal (7 days old) and adult (7 weeks old) BALB/c mice immunization with BCG.HIVA222 prime and MVA.HIVA boost. The specific HIV-1 cellular immune responses were analyzed in spleen cells. The body weight of the newborn mice was weekly recorded. The frequencies of HIV-specific CD8+ T cells producing IFN-γ were higher in adult mice vaccinated intradermally and lower in adult and newborn mice vaccinated subcutaneously. In all cases the IFN-γ production was significantly higher when mice were primed with BCG.HIVA222 compared with BCGwt. When the HIV-specific CTL activity was assessed, the frequencies of specific killing were higher in newborn mice than in adults. The prime-boost vaccination regimen which includes BCG.HIVA222 and MVA.HIVA was safe when inoculated to newborn mice. The administration of BCG.HIVA222 to newborn mice is safe and immunogenic and increased the HIV-specific responses induced by MVA.HIVA vaccine. It might be a good model for infant HIV and Tuberculosis bivalent vaccine.

Enhanced gamma interferon responses of mouse spleen cells following immunotherapy for tuberculosis relapse.

ABSTRACT Gamma interferon responses of spleen cells in mice were examined during postchemotherapy relapse of intraperitoneally induced latent tuberculous infection. The mycobacterial extract RUTI, which prevented the relapse, significantly enhanced the immune responses to secreted and structural recombinant mycobacterial antigens, suggesting that RUTI-mediated protection was mediated by activated T cells.