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

Direct measurement of neutrons induced in lead by cosmic muons at a shallow underground site

Neutron production in lead by cosmic muons has been studied with a Gadolinium doped liquid scintillator detector. The detector was installed next to the Muon-Induced Neutron Indirect Detection EXperiment (MINIDEX), permanently located in the T\"ubingen shallow underground laboratory where the mean muon energy is approximately 7 GeV. The MINIDEX plastic scintillators were used to tag muons; the neutrons were detected through neutron capture and neutron-induced nuclear recoil signals in the liquid scintillator detector. Results on the rates of observed neutron captures and nuclear recoils are presented and compared to predictions from GEANT4-9.6 and GEANT4-10.3. The predicted rates are significantly too low for both versions of GEANT4. For neutron capture events, the observation exceeds the predictions by factors of $1.65\,\pm\,0.02\,\textrm{(stat.)}\,\pm\,0.07\,\textrm{(syst.)}$ and $2.58\,\pm\,0.03\,\textrm{(stat.)}\,\pm\,0.11\,\textrm{(syst.)}$ for GEANT4-9.6 and GEANT4-10.3, respectively. For neutron nuclear recoil events, which require neutron energies above approximately 5 MeV, the factors are even larger, $2.22\,\pm\,0.05\,\textrm{(stat.)}\,\pm\,0.25\,\textrm{(syst.)}$ and $3.76\,\pm\,0.09\,\textrm{(stat.)}\,\pm\,0.41\,\textrm{(syst.)}$, respectively. Also presented is the first statistically significant measurement of the spectrum of neutrons induced by cosmic muons in lead between 5 and 40 MeV. It was obtained by unfolding the nuclear recoil spectrum. The observed neutron spectrum is harder than predicted by GEANT4. An investigation of the distribution of the time difference between muon tags and nuclear recoil signals confirms the validity of the unfolding procedure and shows that GEANT4 cannot properly describe the time distribution of nuclear recoil events. In general, the description of the data is worse for GEANT4-10.3 than for GEANT4-9.6.Comment: 29 pages, 22 figures, 4 table

Associating Approximate Paths and Temporal Sequences of Noisy Detections: Application to the Recovery of Spatio-temporal Cancer Cell Trajectories

In this paper we address the problem of recovering spatio-temporal trajectories of cancer cells in phase contrast video-microscopy where the user provides the paths on which the cells are moving. The paths are purely spatial, without temporal information. To recover the temporal information associated to a given path we propose an approach based on automatic cell detection and on a graph-based shortest path search. The nodes in the graph consist of the projections of the cell detections onto the geometrical cell path. The edges relate nodes which correspond to different frames of the sequence and potentially to the same cell and trajectory. In this directed graph we search for the shortest path and use it to define a temporal parametrization of the corresponding geometrical cell path. An evaluation based on 286 paths of 7 phase contrast microscopy videos shows that our algorithm allows to recover 92% of trajectory points with respect to the associated ground truth. We compare our method with a state-of-the-art algorithm for semi-automated cell tracking in phase contrast microscopy which requires interactively placed starting points for the cells to track. The comparison shows that supporting geometrical paths in combination with our algorithm allow us to obtain more reliable cell trajectories.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

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

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

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

Influence of particle amount on neutralization.

<p>Equal amounts of vector particles as determined by p24 ELISA were incubated in serial plasma dilutions of two different α-MV antibody-positive donors. The dilutions were added to (<b>a</b>) CD133-positive HuH7 cells that were seeded 24 h before transduction at a density of 1.0×10⁴ (CD133-specific vectors) and 5.0×10⁴ (MV_NSe-LV) cells per 96 well, respectively, to apply similar MOIs. Alternatively, the dilutions were added to (<b>b</b>) 1.0×10⁴ (CD20-LV) and 5.0×10⁴ (MV_NSe-LV) CD20-positive Raji cells, respectively. Seventy-two h later, the percentage of EGFP-positive cells was determined by FACS analysis. The relative transduction efficiency compared to transduction in absence of plasma of one representative donor is shown.</p

Targeting vectors are protected against the neutralizing antibody K71.

<p>Equal amounts of physical particles of the indicated vector types were incubated in presence of increasing amounts of antibody K71 (K71-Ab; putative epitope near the mutation sites in H_mut-scFv constructs) and L77 (L77-Ab; putative epitope distant to the mutation sites in H_mut-scFv constructs), respectively, in a final volume of 100 µl. After incubation at room temperature for 1 h, (<b>a</b>) 3×10⁴ (CD20-LV) or 4.5×10⁵ (MV_NSe-LV) CD20-positive Raji cells were added, or the vectors were added to (<b>b</b>) 9.3×10⁴ (CD105-LV) or 3.4×10⁴ (MV_NSe-LV) CD105-positive HT1080 cells or (<b>c</b>) 3.4×10⁴ (CD133-LV) or 2.7×10⁴ (MV_NSe-LV) CD133-positive HuH7 cells, to apply an MOI of 0.25 for each vector type. Forty-eight hours later, the fraction of EGFP-positive cells was quantified by FACS analysis. Mean values (n = 3) and s.d. of the relative transduction efficiency compared to transduction in absence of antibody is shown for each cell line. (<b>d</b>) The indicated H proteins were expressed on the surface of HEK-293T cells. The control antibody K83 and the antibodies K71 and L77 were incubated with the cells, respectively, and a FITC-labeled secondary antibody was used to detect antibody binding to the different H proteins by FACS analysis. The percentage of FITC-positive cells subtracted by the staining of secondary antibody alone is shown. Arrows indicate 0% cell staining.</p