Effects of galectins and antibodies in HIV infection; Novel Assays

Sammanfattning: The high variability of the HIV envelope glycoproteins (Env), and their heavy glycan coating, contributes to the limited host immune control. Still, broadly neutralizing antibodies (bnAbs) are found in some chronically infected HIV-infected individuals, which has spurred the research on antibody-based vaccines. An important tool in detecting and studying bnAbs, are neutralization assays. Here we developed an image-based, high-content automated version of a plaque reduction (PR) assay, which uses green fluorescent protein expression as a reporter of HIV infection. This permitted simultaneous detection of antibodies mediating neutralization and inhibition of virus induced cell-cell fusion. In a multicenter study, Neutnet II, the assay compared well with other neutralization assays and was suggested to be an alternative to the traditional peripheral blood monocyte (PBMC)-based assay and the TZMbl assay. The glycans of Env can also take part in HIV host cell adhesion and infection, via host glycan-binding protein, such as galectins. To explore this, we examined the interaction of gp120 (the surface Env protein) with a panel of galectins, by adapting the fluorescent anisotropy (FA) assay to microscale. Galectin-8, a galectin with two carbohydrate recognition domains (CRDs), had high affinity for gp120 as well as the HIV receptor CD4. The N-terminal CRD mediated the strongest interaction with gp120. The results of the FAassay correlated well with binding of whole virions screened in another assay against the same panel of galectins, now immobilized on beads. In the PR assay described above, here used as an infectivity assay, added intact galectin-8 enhanced infectivity of some HIV-1- strains, while this was not seen with the N-CRD, demonstrating that both CRDs of galectin-8 are required for the effect on infectivity. The enhancement effect mediated by galectin-8 was most pronounced with HIV-1 isolates obtained during the relative immune competent chronic phase, as compared to viruses isolated after AIDS onset. Hence, galectin-8 binding carbohydrate motifs on Env appear to be altered at severe immunodeficiency, adding to the knowledge on the evolution of Env glycosylation patterns related to HIV pathogenesis. These results add to the basic knowledge of virus-host interactions, which hopefully could be used for identification of antibodies and galectin-inhibitors effective in HIV prophylactic interventions.