Measles Virus Glycoprotein-Pseudotyped Lentiviral Vector-Mediated Gene Transfer into Quiescent Lymphocytes Requires Binding to both SLAM and CD46 Entry Receptors

Abstract

Gene transfer into quiescent T and B cells is of importance for gene therapy and immunotherapy approaches to correct hematopoietic disorders. Previously, we generated lentiviral vectors (LVs) pseudotyped with the Edmonston measles virus (MV) hemagglutinin and fusion glycoproteins (Hgps and Fgps) (H/F-LVs), which, for the first time, allowed efficient transduction of quiescent human B and T cells. These target cells express both MV entry receptors used by the vaccinal Edmonston strain, CD46 and signaling lymphocyte activation molecule (SLAM). Interestingly, LVs pseudotyped with an MV Hgp, blind for the CD46 binding site, were completely inefficient for resting-lymphocyte transduction. Similarly, SLAM-blind H mutants that recognize only CD46 as the entry receptor did not allow stable LV transduction of resting T cells. The CD46-tropic LVs accomplished vector-cell binding, fusion, entry, and reverse transcription at levels similar to those achieved by the H/F-LVs, but efficient proviral integration did not occur. Our results indicate that both CD46 and SLAM binding sites need to be present in cis in the Hgp to allow successful stable transduction of quiescent lymphocytes. Moreover, the entry mechanism utilized appears to be crucial: efficient transduction was observed only when CD46 and SLAM were correctly engaged and an entry mechanism that strongly resembles macropinocytosis was triggered. Taken together, our results suggest that although vector entry can occur through the CD46 receptor, SLAM binding and subsequent signaling are also required for efficient LV transduction of quiescent lymphocytes to occur.Measles virus (MV) belongs to the paramyxoviridae family and is the causative agent of measles disease. It has two envelope glycoproteins (gp's), the hemagglutinin (H) and fusion (F) glycoproteins (Hgp and Fgp, respectively), which mediate receptor binding and fusion, respectively (28, 29). Signaling lymphocyte activation molecule (SLAM) (CD150) is the receptor for both clinical isolates and vaccine strains (49, 55). However, vaccine strains like Edmonston (Edm) have gained, in addition to entry through the SLAM receptor, entry through the CD46 receptor after their adaptation in SLAM-negative cells (25,54). Moreover, recent findings suggest the existence of a third MV receptor in epithelial cells (54). CD46 is a complementregulatory molecule expressed on all human nucleated cells (27), whereas SLAM is constitutively expressed at the surfaces of some T and B cell subsets and upregulated upon proliferation of T and B lymphocytes and mature dendritic cells (DCs) (3,8). The cellular distribution of SLAM determines lymphoid tropism and explains in part the immunosuppressive character of measles virus.Importantly, even though wild-type and vaccine MV strains have been extensively studied at the levels of virulence (55), immunosuppression, and immune response (4, 21, 36) and the crystal structures of CD46 and SLAM receptor binding to MV hemagglutinin have recently been elucidated (7,17,41), there are still few data abo...

Measles virus envelope pseudotyped lentiviral vectors transduce quiescent human HSCs at an efficiency without precedent

“…The mechanism that explains this high-level H/F-LV entry and productive transduction needs to be investigated, but for T and B cells, we might speculate that CD46 binding induces an alternative cell entry (eg, macropinocytosis). 21 During engraftment, transplanted HSCs home to the BM niche, where they initiate both differentiation and self-renewal. There, they reside in a hypoxic niche, where they acquire dormancy and maintain stemness.…”

“…19 The SLAM-restricted vectors (H-D4/F-LVs), SLAM-blind Hgp (Ha533), and H-D4-YG were described previously 21,34 LV production Self-inactivating HIV-1-derived vectors were generated by transient transfection of 293T cells as described previously. 35 Viral vectors were produced either pseudotyped with VSV-G or with MV gp's.…”

“…[18][19][20] These vector particles (H/F-LVs) are able to mediate cell entry via the native MV receptors CD46 and signaling lymphocyte activation molecule (SLAM). 19,21 CD46 is a complement regulatory molecule expressed on all human nucleated cells, 22 whereas SLAM (CD150) is constitutively expressed at the surface of some T and B subsets and upregulated upon proliferation of T and B lymphocytes and mature dendritic cells. 23,24 The third MV receptor, nectin-4, is not expressed on lymphocytes.…”

Efficient transduction of healthy and malignant plasma cells by lentiviral vectors pseudotyped with measles virus glycoproteins

“…24 Therefore, we evaluated the presence of both receptors on malignant and healthy PCs. As illustrated for one representative patient in Figure 1a, primary malignant PCs expressed CD46 and SLAMF1/CD150 and this was confirmed for 10 other patients with MM (Figure 1b).…”

“…Both healthy and malignant PCs express the two receptors for measles glycoproteins, SLAMF1/CD150 and CD46, and both receptors were shown to be mandatory to obtain an efficient H/F-LVs transduction in primary lymphocytes. 24 The vast majority of primary malignant PCs (495%) is out of the cell cycle 26 in-vitro-generated healthy PCs harvested at day 7 of culture were transduced with H/F-LVs and cultured for 3 days on 10 4 bone marrow stromal cells in 1 ml of RPMI1640 culture medium, 10% FCS, IL-6 (10 ng/ ml) and IFNa (500 U/ml), washed and cultured for additional 11 days on 10 4 bone marrow stromal cells in 1 ml of RPMI1640 culture medium, 10% FCS and IL-6 (10 ng/ml). Data are GFP expression in CD138 þ PCs generated in one experiment representative of three at day 3 and day 14 after H/F-LV transduction assayed by FACS analysis.…”