1. Volume 20, Issue 9, Pages 1699-1712 (September 2012)
Lentiviral Vectors Displaying Modified Measles Virus gp Overcome Pre-existing Immunity in In Vivo-like Transduction of Human T and B Cells 
Camille Lévy, Fouzia Amirache, Caroline Costa, Cecilia Frecha, Claude P Muller, Hasan Kweder, Robin Buckland, François-Loïc Cosset, Els Verhoeyen 
Molecular Therapy 
Volume 20, Issue 9, Pages (September 2012)
DOI: /mt
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Measles virus (MV)-derived lentiviral vectors (LVs) are neutralized by MV-specific antibodies present in human and macaque serum but resistant to macaque complement. (a) 293T (CD46+) cells were transduced with H/F-LVs at a multiplicity of infection (MOI) of 0.2 after preincubation of the lentivectors with serial dilutions of human serum from seven different donors. (b) 293T cells were transduced with H/F-LVs or VSV-G-LVs at a MOI of 0.2 upon preincubation with heat-treated complement-inactivated human sera or purified IgGs from the sera 3 and 7; sera were applied at 100-fold dilutions. (c) 293T cells were transduced with the indicated LVs upon incubation with heat-treated complement-inactivated sera from four preimmune (0 day) or vaccinated macaques (14 days and 230 days) at a dilution of 1/40 (means ± SD; n = 3); In (d) the indicated vectors were incubated with fresh and complement-inactivated sera from two naive macaques applied at a twofold dilution and subsequently their transduction efficiency was evaluated on 293T cells Neutralization was calculated as the transduction efficiency in presence of serum relative to the transduction efficiency in the absence of serum (means ± SD; n = 3).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Measles virus gp-pseudotyped lentiviral vectors (LVs) mutated in the major epitopes of the hemagglutinin escape from measles virus (MV)-specific monoclonal antibodies. (a) Schematic representation of the different MV hemagglutinin constructs. In Edm-Hgp (H) the binding sites to SLAM and CD46 are presented. In addition, the Noose and NE major epitopes that bind anti-MV antibodies in H are indicated; point mutations were introduced in Edm-H to knock out the Noose epitope55 and both the Noose and NE epitopes (HNoNE). (b) Surface staining of H gp on 293T cells transfected with hemagglutin encoding constructs (H, HNo, HNoNE) was performed with mouse monoclonal antibodies (mAbs) specific to the SLAM binding site (cl55), the Noose (BH216) and the NE (BH129) epitopes in H gp. The data are representative of three experiments. (c) 293T (CD46+), CHO-SLAM (SLAM+) and Raji (CD46+SLAM+) cells were transduced with H/F-, HNo/F-, HNoNE/F-, or VSV-G-LVs at a multiplicity of infection (MOI) of 0.2 after preincubation of these LVs with mAbs specific for the Noose (BH216, BH6) or NE (BH47, BH59, BH129) epitopes (means ± SD; n = 3). The specificity of the antibodies is shown in (a). Neutralization was calculated as the transduction efficiency in the presence of neutralizing antibodies relative to the transduction efficiency in the absence of antibodies.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Introduction of an extra glycosylation site in H-Edm allows the H/F-LVs to escape neutralization by anti-measles virus (MV) antibodies in human sera. (a) Schematic representation of the different MV hemagglutinin glycoproteins. In Edm-Hgp (H) the binding sites for SLAM and CD46 are presented. In addition, the Noose and NE major epitopes that bind specific anti-MV antibodies in H are indicated. The MV clinical strain H gp (HD4) contains only SLAM binding residues and the Noose and NE epitopes. As indicated, the HD4 contains an extra glycosylation site (D416N) compared to H; the D416N glycosylation site was introduced in HNoNE mutant (HNoNE-D416N). (b) Immunoblots of lentiviral particles displaying H, HD4, or HNoNE-D416N and F gps at their surface. Lentiviral vectors (LVs) were purified over a sucrose cushion by ultracentrifugation. The upper part of the membrane was stained with a monoclonal antibody (mAb) against the ectodomain of H, the middle part with a monoclonal antibody against the ectodomain of F and the lower part of the membrane was stained with anti-HIV p24 antibody directed against the HIV capsid. (c) Raji (CD46+SLAM+) cells were transduced with H/F-, HNoNE/F-, HNoNE-D416N/F, or VSV-G-LVs at a multiplicity of infection (MOI) of 0.2 after preincubation of these LVs with mAbs specific for the Noose (BH216, BH6) or NE (BH47, BH59, BH129) epitopes or an antibody (BH99) sensitive to the D416N glycosylation (means ± SD; n = 3); (d) Raji cells were transduced with H/F-LVs or HNoNE-D416N/F-LVs at a MOI of 0.2 after preincubation with serial dilutions of human serum from two different donors. Neutralization was calculated as the transduction efficiency in presence of neutralizing antibodies or serum relative to the transduction efficiency in absence of antibodies or serum. Both results obtained for each serum of a different donor are expressed on the same graph; the line represents the mean of both results while the points represent the actual data.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
The HD4-NoNE gp that gained CD46 receptor-binding resists neutralization by human serum. (a) Schematic representation of the different measles virus (MV) hemagglutinin gps. The indicated residues, 481Y and 492G (marked in white), confer CD46 binding in Edm-Hgp while the clinical strain based gps HD4 and HD4NoNE contain the 481N and 492E residues (marked in black). The N481Y and E492G mutations were introduced in HD4NoNE resulting in HD4-NoNE-YG. In parallel, a chimeric mutant HChim5 was engineered by swapping the domain corresponding domain in H D4 for the one encoding for the mutated Noose region and the CD46-binding sites in HNoNEgp (see Figure 2a). (b,d) Raji cells were transduced with H/F-LVs, H-D4/F-LVs and HD4-NoNE/F-LVs (b), HD4-NoNE-YG/F-LVs or HChim5-LVs (d) at a multiplicity of infection (MOI) of 0.2 upon preincubation of these vectors with serial dilutions of human sera from two different donors. Both results obtained for each serum of a different donor are expressed on the same graph; the line represents the mean of both results while the points represent the actual data. (c) Immunoblots of lentiviral particles displaying H, HD4, HD4-NoNE-YG, or HChim5 at their surface. Lentiviral vectors (LVs) were purified over a sucrose cushion by ultracentrifugation. The upper part of the membrane was stained with a monoclonal antibody (mAb) against the ectodomain of H, the central part with a mAb against the ectodomain of F and the lower part of the membrane with an anti-HIV p24 antibody directed against the capsid.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
HD4Escape-Mut/F-LVs efficiently transduce memory and naive T and B cells. T and B cells were purified from peripheral blood by negative selection. These unstimulated lymphocytes were immediately transduced with H/F-LVs, HNoNE-D416N/F-LVs, HD4-NoNE-YG/F-LVs, Hchim5/F-LVs (multiplicity of infection (MOI) of 1) and VSV-G-LVs (MOI of 50). Two days post-transduction surface expression of naive (CD27–) and memory B-cell (CD27+) subsets was detected by fluorescence-activated cell sorting (FACS) analysis. These data are representative of four experiments. (a) The GFP+ cells in these B-cell subsets are indicated in the upper right quadrant. Three days post-transduction surface staining for naive (CD45RA+) and memory (CD45RO+) subsets of T cells was performed by anti-CD45RA/anti-CD45RO double staining. (b) The GFP+ cells in these T-cell subsets are indicated in the upper right quadrant. These data are representative of four experiments. (c,d) represent long-term cultures of the LV transductions of unstimulated B and T cells. Transduced B cells were washed twice after transduction and cultured for 6 more days on MS5 feeder cells and were then analyzed for GFP-expressing naive (CD27–) and memory (CD27+) cells by (c) FACS, while transduced T cells were washed, and then cultured in RPMI medium supplemented with rIL-7 (10 ng/ml) for 6 days before FACS analysis of GFP-expressing naive (CD45RA+) and memory (CD45RO+) cells (d; means ± SD; n = 4). LVs, lentiviral vectors.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
HD4Escape-Mut/F-LVs transduce quiescent peripheral blood lymphocytes (PBLs) more efficiently than the original H/F-LVs in the presence of human serum. Freshly isolated PBLs were transduced with H/F-LVs, HNoNE-D416N/F-LVs, HD4NoNE-YG/F-LVs, Hchim5/F-LVs (MOI of 1) in the absence of or with increasing human serum concentrations from four different donors. Three days post-transduction surface staining for (a) T cells and (b) B cells was performed with anti-CD3 and anti-CD19 antibodies, respectively and transduction efficiencies in these subpopulations were analyzed by fluorescence-activated cell sorting (FACS). The transduction efficiencies in the presence of serum were expressed as the percentage of GFP+-cells relative to the percentage of transduced T or B cells in the absence of human serum was which was set to 100% (means ± SD; n = 3). LVs, lentiviral vectors.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
Novel engineered H/F-LVs allow efficient gene transfer into T cells within a blood sample. (a) Schematic representation of the transduction protocol. Four different H/F-LVs (H/F-LVs, HNoNE-D416N/F-LVs, HD4-NoNE-YG/F-LVs, and Hchim5/F-LVs) were used for transduction (multiplicity of infection (MOI) = 1) of total blood and control PBMC transduction. (b) Transduction efficiency of CD3+ T cells determined by fluorescence-activated cell sorting (FACS) analysis. The percentage of transduction obtained for the PBMCs present in the whole blood was expressed relative to the transduction efficiency of PBMCs in the absence of anti-measles virus (MV) antibodies which was set to 100%. The data for three different donors and three different sets of vector preparations are shown for day 5 as well as day 10 of culture in the presence of rIL-7. (c) Determination of transduction efficiency of CD3+ T cells by FACS analysis upon incubation of isolated PBMCs or whole blood with the different H/F-LV pseudotypes. In the dot blots the expression levels (MFI) of the GFP+ cells are indicated. LVs, lentiviral vectors.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions