Exploring gold glyconanoparticles as multivalent carrier for specific molecules involved in hiv-1 infection

Resumo

ABSTRACTThe infection of the human immunodeficiency virus (HIV) is the cause of AIDS and isone of the greatest infectious diseases ever seen. HIV infects cells of the humanimmune system such as helper T cells (specifically CD4+ T cells), macrophages, anddendritic cells. The HIV membrane is decorated with proteins spikes. The envelopeglycoprotein (Env) spikes are formed by three transmembrane gp41 non-covalentlybound to three gp120, a highly glycosilated protein. The Env spikes initiate infection ofhost cells and are targets for vaccine development. The molecular mechanism of theentry process, where CD4 receptor, CCR5/CXCR4 co-receptors and the viral envelopglycoprotein gp120 are involved, is still not well understood, however, it wasdemonstrated that HIV entry is characterized by binding of multiple copies of Env, CD4and CCR5 or CXCR4. In this thesis we use gold nanoparticles, biofunctionalized withcarbohydrates (glyconanoparticles, GNPs) to mutimerize selected molecules that mayinterfere with concrete steps of HIV infection. Three different families of GNPs wereprepared. Sulphated gold glyconanoparticles (SO4-GNPs), able to bind positivecharged amino acids of gp120, were prepared and characterized. We demonstrate thatdepending on the sulphate ligand density, these nanoparticles can bind gp120 withhigh affinity as shown in SPR-based experiments and neutralize the in vitro HIVinfection of T-lymphocytes in the nanomolar range. A miniprotein that mimics the CD4receptor (miniCD4) and inhibits the HIV entry process was multimerized on GNPs(miniCD4-GNPs). The conformation of miniCD4 did not change once linked to theGNPs as showed by circular dichroism (CD) experiments. However, the multivalentpresentation of miniCD4 on GNP did not increase the activity of miniCD4. Indeed, HIV-1 neutralization assays did not show improved IC50 values for the miniCD4-GNPsrespect to the free miniCD4, probably because the distance between the miniCD4 onthe GNPs do not perfectly match the distance between CD4 binding site on HIV Env.An important peptide of gp120, the V3 variable loop, was also multimerized on GNPs.We found that GNPs bearing only glucose and carboxylic ending linkers are able tomodulate the conformation of V3 peptide (random coil) to obtain V3-GNP constructswith well defined conformations (¿-helix or ß-strand) as showed by CD. Studies by SPRshowe that only the V3ß-GNP are able to bind mAb the specific anti-V3 antibody 447-52D. HIV neutralization experiments show that V3-GNP were active only at highconcentration (100 ug/ml). However, a preliminary immunization study with V3ß-GNPon mice shows that mAb contained in mice sera are able to recognize V3ß-GNP. Amore extensive immunization study will be performed immunizing rabbits with differentkind of V3-GNP.