Assessing the response of T cells to "Mycobacterium tuberculosis" lipids

Most vaccines used nowadays are created using inactivated or attenuated compounds
from micro-organisms. Advances in basic immunology and molecular biology opened the
gate for consequent improvement of vaccination strategies. The vast majority of successful
vaccines developed so far functions through production of specific antibodies, thus allowing
rapid eradication of the disease’s causative agent. However, intracellular bacterial pathogens
like Mycobacterium tuberculosis hide from immune attack by antibodies within cells, and
their virulence is related to their capacity to survive for prolonged times within macrophage
phagosomes by blocking lysosomal delivery and subsequent degradation. Therefore, design
of more effective vaccination strategies is needed to ensure efficient killing of this type of
pathogens. The discovery of CD1 molecules, that present lipidic antigens from the bacterial
cell wall to T cells, might be an additional step in this direction. In this dissertation, we used
chemical and biochemical approaches to identify and synthesize lipid antigens, as well as T
cell activation assays, molecular biology tools, and transgenic mice to evaluate the potential
of lipid antigens to be included in subunit vaccines.
In a first series of studies, we identified glycerol monomycolate (GroMM) as a mycobacterial
lipid antigen that activates CD1b-restricted T cells, and confirmed its immunogenicity during
the course of infection by M. tuberculosis. GroMM efficiently stimulates T cells from PPDpositive
healthy donors, but not from non-infected donors nor patients with active
tuberculosis. These data suggest that GroMM-reactive T cells are primed during infection and
may contribute to protection against pathogenic mycobacteria, rendering GroMM an
interesting candidate for further evaluation to be used in vaccination strategies.
A second series of studies dealt with a lipid antigen from M. tuberculosis previously identified in our laboratory, i.e. diacylated sulfoglycolipids (Ac2SGL). Sulfoglycolipid (SGL)
analogs were synthesized in order to study the structural constraints governing binding to
CD1b and generation of immunogenic CD1b-SGL complexes. Comparison of these analogs
sharing the same trehalose-sulfate polar head but differing in the structure of their acyl tails
showed that the number of C-methyl substituents, the configuration of the chiral centers, and
the respective localization of the two different acyl chains on the polar head are important structural elements that must be considered for the design of sulfoglycolipid analogs with
potential use as vaccine subunits.
In a third series of experiments, we began pre-clinical in vivo studies in CD1b-transgenic
mice that we have generated, using the most immunogenic synthetic SGL analog. CD1b:SGL
dimers allowed us to follow successful priming and expansion of CD1b-restricted SGLreactive
T cells after immunization. Finally, we have investigated different ways to increase
immunogenicity by facilitating lipid solubilisation and transport into antigen presenting cells
(APCs).
Altogether, the data obtained and discussed in the present dissertation go through the first
stages of a vaccine’s development, from identification of candidate antigens to pre-clinical in
vivo studies in mice. Confirmation of the protective effect of the lipid antigens described
herein will determine whether they can be considered as candidate compounds of a subunit
vaccine.