Peter Lawætz Andersen

Proteomic Profiling of Mycobacterium tuberculosis Identifies Nutrient-starvation-responsive Toxin–antitoxin Systems

In order to successfully enter the latent stage, Mycobacterium tuberculosis must adapt to conditions such as nutrient limitation and hypoxia. In vitro models that mimic latent infection are valuable tools for describing the changes in metabolism that occur when the bacterium exists in a non-growing form. We used two complementary proteomic approaches, label-free LC-MS/MS analysis and two-dimensional difference gel electrophoresis, to determine the proteome profile of extracellular proteins from M. tuberculosis cultured under nutrient starvation. Through the label-free LC-MS/MS analysis of fractionated samples, 1176 proteins were identified from culture filtrates of log phase and nutrient-starved cultures, and the protein levels of 230 proteins were increased in nutrient-starved culture filtrates, whereas those of 208 proteins were decreased. By means of Gene Ontology clustering analysis, significant differences in the overall metabolism during nutrient starvation were detected. Notably, members of the toxin–antitoxin systems were present in larger quantities in nutrient-starved cultures, supporting a role for these global modules as M. tuberculosis switches its metabolism into dormancy. Decreased abundance of proteins involved in amino acid and protein synthesis was apparent, as well as changes in the lipid metabolism. Further analysis of the dataset identified increased abundance of lipoproteins and decreased abundance of ESAT-6 family proteins. Results from the two-dimensional difference gel electrophoresis proteomics demonstrated overall agreement with the LC-MS/MS data and added complementary insights about protein degradation and modification.

The HyVac4 subunit vaccine efficiently boosts BCG-primed anti-mycobacterial protective immunity.

Background The current vaccine against tuberculosis (TB), BCG, has failed to control TB worldwide and the protective efficacy is moreover limited to 10–15 years. A vaccine that could efficiently boost a BCG-induced immune response and thus prolong protective immunity would therefore have a significant impact on the global TB-burden. Methods/Findings In the present study we show that the fusion protein HyVac4 (H4), consisting of the mycobacterial antigens Ag85B and TB10.4, given in the adjuvant IC31® or DDA/MPL effectively boosted and prolonged immunity induced by BCG, leading to improved protection against infection with virulent M. tuberculosis (M.tb). Increased protection correlated with an increased percentage of TB10.4 specific IFNγ/TNFα/IL-2 or TNFα/IL-2 producing CD4 T cells at the site of infection. Moreover, this vaccine strategy did not compromise the use of ESAT-6 as an accurate correlate of disease development/vaccine efficacy. Indeed both CD4 and CD8 ESAT-6 specific T cells showed significant correlation with bacterial levels. Conclusions/Significance H4-IC31® can efficiently boost BCG-primed immunity leading to an increased protective anti-M.tb immune response dominated by IFNγ/TNFα/IL-2 or TNFα/IL2 producing CD4 T cells. H4 in the CD4 T cell inducing adjuvant IC31® is presently in clinical trials.

Cationic liposomal vaccine adjuvants in animal challenge models: overview and current clinical status

Cationic liposome formulations can function as efficient vaccine adjuvants. However, due to the highly diverse nature of lipids, cationic liposomes have different physical–chemical characteristics that influence their adjuvant mechanisms and their relevance for use in different vaccines. These characteristics can be further manipulated by incorporation of additional lipids or stabilizers, and inclusion of carefully selected immunostimulators is a feasible strategy when tailoring cationic liposomal adjuvants for specific disease targets. Thus, cationic liposomes present a plasticity, which makes them promising adjuvants for future vaccines. This versatility has also led to a vast amount of literature on different experimental liposomal formulations in combination with a wide range of immunostimulators. Here, we have compiled information about the animal challenge models and administration routes that have been used to study vaccine adjuvants based on cationic liposomes and provide an overview of the applicability, progress and clinical status of cationic liposomal vaccine adjuvants.

A New Experimental Infection Model in Ferrets Based on Aerosolised Mycobacterium bovis

There is significant interest in developing vaccines to control bovine tuberculosis, especially in wildlife species where this disease continues to persist in reservoir species such as the European Badger (Meles meles). However, gaining access to populations of badgers (protected under UK law) is problematic and not always possible. In this study, a new infection model has been developed in ferrets (Mustela furo), a species which is closely related to the badger. Groups of ferrets were infected using a Madison infection chamber and were examined postmortem for the presence of tuberculous lesions and to provide tissue samples for confirmation of Mycobacterium bovis by culture. An infectious dose was defined, that establishes infection within the lungs and associated lymph nodes with subsequent spread to the mesentery lymph nodes. This model, which emphasises respiratory tract infection, will be used to evaluate vaccines for the control of bovine tuberculosis in wildlife species.

An attenuated Mycobacterium tuberculosis clinical strain with a defect in ESX-1 secretion induces minimal host immune responses and pathology

Although Mycobacterium tuberculosis (M.tb) DK9897 is an attenuated strain, it was isolated from a patient with extrapulmonary tuberculosis and vaccination with a subunit vaccine (H56) induced poor protection against it. Both attenuation and lack of protection are because M.tb DK9897 cannot secrete the EsxA virulence factor nor induce a host response against it. Genome sequencing identified a frameshift mutation in the eccCa1 gene. Since the encoded EccCa1 protein provides energy for ESX-1 secretion, it suggested a defect in the ESX-1 type VII secretion system. Genetic complementation with a plasmid carrying the M.tb H37Rv sequence of eccCa1-eccCb1-pe35 re-established EsxA secretion, host specific EsxA T-cell responses, and increased strain virulence. The ESX-1 secretion defect prevents several virulence factors from being functional during infection and therefore attenuates M.tb. It precludes specific T-cell responses against strong antigens and we found very little in vivo cytokine production, gross pathology or granuloma formation in lungs from M.tb DK9897 infected animals. This coincides with M.tb DK9897 being unable to disrupt the phagosome membrane and make contact to the cytosol.