Measles virus glycoprotein-based lentiviral targeting vectors that avoid neutralizing antibodies

Lentiviral vectors (LVs) are potent gene transfer vehicles frequently applied in research and recently also in clinical trials. Retargeting LV entry to cell types of interest is a key issue to improve gene transfer safety and efficacy. Recently, we have developed a targeting method for LVs by incorporating engineered measles virus (MV) glycoproteins, the hemagglutinin (H), responsible for receptor recognition, and the fusion protein into their envelope. The H protein displays a single-chain antibody (scFv) specific for the target receptor and is ablated for recognition of the MV receptors CD46 and SLAM by point mutations in its ectodomain. A potential hindrance to systemic administration in humans is pre-existing MV-specific immunity due to vaccination or natural infection. We compared transduction of targeting vectors and non-targeting vectors pseudotyped with MV glycoproteins unmodified in their ectodomains (MV-LV) in presence of α-MV antibody-positive human plasma. At plasma dilution 1:160 MV-LV was almost completely neutralized, whereas targeting vectors showed relative transduction efficiencies from 60% to 90%. Furthermore, at plasma dilution 1:80 an at least 4-times higher multiplicity of infection (MOI) of MV-LV had to be applied to obtain similar transduction efficiencies as with targeting vectors. Also when the vectors were normalized to their p24 values, targeting vectors showed partial protection against α-MV antibodies in human plasma. Furthermore, the monoclonal neutralizing antibody K71 with a putative epitope close to the receptor binding sites of H, did not neutralize the targeting vectors, but did neutralize MV-LV. The observed escape from neutralization may be due to the point mutations in the H ectodomain that might have destroyed antibody binding sites. Furthermore, scFv mediated cell entry via the target receptor may proceed in presence of α-MV antibodies interfering with entry via the natural MV receptors. These results are promising for in vivo applications of targeting vectors in humans

CD20 and CD19 targeted vectors induce minimal activation of resting B lymphocytes

B lymphocytes are an important cell population of the immune system. However, until recently it was not possible to transduce resting B lymphocytes with retro- or lentiviral vectors, making them unsusceptible for genetic manipulations by these vectors. Lately, we demonstrated that lentiviral vectors pseudotyped with modified measles virus (MV) glycoproteins hemagglutinin, responsible for receptor recognition, and fusion protein were able to overcome this transduction block. They use either the natural MV receptors, CD46 and signaling lymphocyte activation molecule (SLAM), for cell entry (MV-LV) or the vector particles were further modified to selectively enter via the CD20 molecule, which is exclusively expressed on B lymphocytes (CD20-LV). It has been shown previously that transduction by MV-LV does not induce B lymphocyte activation. However, if this is also true for CD20-LV is still unknown. Here, we generated a vector specific for another B lymphocyte marker, CD19, and compared its ability to transduce resting B lymphocytes with CD20-LV. The vector (CD19ds-LV) was able to stably transduce unstimulated B lymphocytes, albeit with a reduced efficiency of about 10% compared to CD20-LV, which transduced about 30% of the cells. Since CD20 as well as CD19 are closely linked to the B lymphocyte activation pathway, we investigated if engagement of CD20 or CD19 molecules by the vector particles induces activating stimuli in resting B lymphocytes. Although, activation of B lymphocytes often involves calcium influx, we did not detect elevated calcium levels. However, the activation marker CD71 was substantially up-regulated upon CD20-LV transduction and most importantly, B lymphocytes transduced with CD20-LV or CD19ds-LV entered the G1b phase of cell cycle, whereas untransduced or MV-LV transduced B lymphocytes remained in G0. Hence, CD20 and CD19 targeting vectors induce activating stimuli in resting B lymphocytes, which most likely renders them susceptible for lentiviral vector transduction

Resting lymphocyte transduction with measles virus glycoprotein pseudotyped lentiviral vectors relies on CD46 and SLAM

AbstractThe measles virus (MV) glycoproteins hemagglutinin (H) and fusion (F) were recently shown to mediate transduction of resting lymphocytes by lentiviral vectors. MV vaccine strains use CD46 or signaling lymphocyte activation molecule (SLAM) as receptor for cell entry. A panel of H protein mutants derived from vaccine strain or wild-type MVs that lost or gained CD46 or SLAM receptor usage were investigated for their ability to mediate gene transfer into unstimulated T lymphocytes. The results demonstrate that CD46 is sufficient for efficient vector particle association with unstimulated lymphocytes. For stable gene transfer into these cells, however, both MV receptors were found to be essential

α-MV antibody-negative serum does not neutralize MV_NSe-LV or targeting vectors.

<p>Equal amounts of physical particles of the indicated vector types were incubated in serial serum dilutions of an α-MV antibody-negative donor. Then, (<b>a</b>) 1×10⁴ (CD20-LV) and 2×10⁵ (MV_NSe-LV) CD20-positive Raji cells were added, respectively, or the dilutions were added to (<b>b</b>) CD133-positive HuH7 cells. These were seeded at a density of 1.0×10⁴ (targeting vectors) and 5.0×10⁴ (MV_NSe-LV) cells per 96 or 48 well, respectively, 24 h before transduction, to apply similar MOIs of vector particles. Forty-eight to 72 h later, the percentage of EGFP-positive cells was determined by FACS analysis. As control, medium without serum was used.</p

CD20-VLP and CD19ds-VLP do not induce calcium influx into unstimulated primary human B lymphocytes.

<p>Freshly isolated primary human B lymphocytes were labeled with Calcium Sensor Dye eFluor® 514 and labeled cells were assessed by flow cytometry to establish a baseline level of fluorescence. Then cells were removed, incubated with the indicated stimulants, and replaced immediately for further flow cytometric analysis. The transient increase in intracellular calcium concentration was recorded by monitoring the change in MFI of the cells. To determine the background auto-fluorescence of the cells, one sample was recorded without the addition of any stimulant after baseline monitoring (mock). The gap in the histogram reflects the time-period when the tube containing the cells was removed from the instrument to add the stimulants. The arrow indicates the time-point of addition of the stimulant. VLP: virus like particles; MFI: mean fluorescence intensity.</p

Targeting vectors are protected against MV neutralizing antibodies.

<p>The indicated vector particles were incubated in serial plasma dilutions of two different α-MV antibody-positive donors. (<b>a</b>) 3×10⁴ CD20-positive Raji cells (MOI 0.4) were added, or the dilutions were added to (<b>b</b>) CD105/CD20-positive HT1080-CD20 (MOI 0.3) or (<b>c</b>) HT1080-CD133 cells (MOI 0.3) that were seeded at a density of 1.7×10⁴ and 0.75×10⁴ cells per 96 well, respectively, 24 h before transduction. Forty-eight to 72 h later, the fraction of EGFP-positive cells was quantified by FACS analysis. The relative transduction efficiency compared to transduction in absence of plasma (medium control) of one representative donor is shown for each cell line.</p

Stable transduction of unstimulated primary human B lymphocytes by CD20-LV and CD19ds-LV.

<p>(<b>A</b>) Freshly isolated primary human B lymphocytes from six different donors were transduced with CD20-LV, CD19ds-LV or MV-LV at an MOI of 2, or VSVG-LV at an MOI of 100. Forty-eight hours later, B lymphocytes were identified by their CD20 expression and the percentage of EGFP⁺ cells in the CD20-gated cells was determined by FACS analysis. Results are expressed as mean ± SEM. *, ** and *** indicate <i>P</i><0.05, <i>P</i><0.01 and <i>P</i><0.001 versus transduction with CD20-LV, respectively. Data were analyzed by one-way ANOVA. (<b>B–C</b>) To verify stable integration of the <i>egfp</i> gene into the B lymphocyte genome, freshly isolated primary human B lymphocytes (purity was ∼99.8%) from three different donors were transduced with the indicated vectors. Transduced cells were cultivated in presence of 10 ng/ml rhIL-15 and 10 ng/ml rhIL-2 for 10 days on MS-5 feeder cells. The percentage of CD19⁺/EGFP⁺ cells in the CD20-gated cells was determined by FACS analysis at the indicated time points. (<b>B</b>) FACS blots of one representative experiment are shown (at least 2000 B lymphocytes were gated for analysis). (<b>C</b>) Diagram showing a summary of all three independent experiments. Dark gray column: CD20-LV; light gray column: CD19ds-LV; white column: VSVG-LV. Results are expressed as mean ± SEM. * and ** indicate <i>P</i><0.05 and <i>P</i><0.01 for CD20-LV and CD19ds-LV versus VSVG-LV, respectively. Data were analyzed by Student’s t-test without adjustment of multiple comparisons.</p

Schematic drawing of cytoplasmic tail-truncated hemagglutinin envelope proteins used for pseudotyping of lentiviral vectors.

<p>In the mutated hemagglutinin protein (H_mut) that is derived from the NSe variant of the measles virus (MV) vaccine strain Edmonston B, mutations in the MV receptor recognition regions Y481A, R533A, S548L and F549S (ectodomain) are indicated by asterisks. Glycine-serine linker ((G₄S)₃) or the factor Xa cleavage site (IEGR) were used as linker region between H_mut and single-chain antibody (scFv). A histidine tag (H6) is present at the scFv C-terminus. The hemagglutinin protein derived from the NSe variant of the MV vaccine strain Edmonston B that is not mutated and does not display a scFv is labeled H_NSe. The hemagglutinin protein derived from the wild-type measles virus strain IC-B is labeled H_wt. All hemagglutinin proteins are truncated by 18 amino acids in their cytoplasmic tail (Δ18) to allow incorporation into the lentiviral envelope. The names of the respective vector particles pseudotyped with the depicted H variants are indicated on the left site. w/o: without.</p

Cell cycle progression of resting primary human B lymphocytes after transduction with targeted vectors.^1.

1<p>Cells were analyzed 48 h after transduction.</p>2<p>Unstimulated B lymphocytes were analyzed.</p>3<p>B lymphocytes were stimulated for 48 h with a cytokine cocktail consisting of 50 ng/ml IL-2, 300 ng/ml CD40Ligand, 10 ng/ml IL-4 and 10 ng/ml IL-10.</p>4<p>not applicable.</p>5<p>not done.</p