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

FACTORY OF PROCESSES – NEW FORMAT OF ORGANIZATION OF EDUCATIONAL PROCESS IN HIGHER EDUCATIONAL INSTITUTION

The article deals with the technology of organization of the educational process in a higher educational institution with the application of the interactive method of training – factorу of processes. The comparative characteristics of the methodology of factorу of processes are given, general content-technological determinants with the format of the business game are shown, as well as their distinctive features. A sufficiently large variational scenario potential of factorу of processes with the obligatory inclusion of a number of stages is shown. The experience of implementing the factorу of processes in the educational process of the Tyumen State Medical University in the framework of the program "Lean production in health care" is demonstrated. The article shows the high potential for incorporating the format of the factorу of processes into the educational process with obtaining the necessary competencies and aiming at using the acquired skills in further practical activitie

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

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

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

Historical experience of the rise of a regional quality system of medical care

The variety of professional and ethical features of clinicians’ activities requires improving the capabilities of monitoring technologies. This paper reviews the regional experience of the formation of the quality and safety management system of medical care (based on the 10-year-period materials of the Tyumen region). The key area of this study is to analyze the development of trends and features of regional experience in the formation of a quality and safety management system for medical care, the formation of technologies, and to develop insurance mechanisms for professional liability of medical workers and insurance risks (historical perspective based on the materials of the Tyumen region, Russian Federation). A regional, departmental medical care quality control technology has been created to reduce morbidity, mortality and prevent patient health risks. A technological base for the management of medical care has been formed as a factor in reducing morbidity and as the basis for the formation of a system for reducing health risks, which includes issues of continuous, systematic training of medical personnel, indicators of medical care quality, the level of quality of forensic examinations. To improve the efficiency of departmental control of medical care quality, during the study a medical care and clinical and expert work control department was established in the Department of Health of the Tyumen Region (DH TR). The key link in the departmental control of the medical care quality is the "expert commissions" of medical institutions (MI), the key link in the interdepartmental control of the medical care quality is the "health, social and medical insurance services". The studies were initiated during the transition to single-channel funding and assessments of medical institutions on a completed case basis.La variedad de características profesionales y éticas de las actividades de los médicos requiere mejorar las capacidades de las tecnologías de monitoreo. Este documento revisa la experiencia regional de la formación del sistema de gestión de calidad y seguridad de la atención médica (basado en los materiales del período de 10 años de la región de Tyumen, Federación Rusa. Se creó una tecnología regional departamental de control de calidad de la atención médica como medio para reducir la morbilidad, mortalidad y prevenir riesgos para la salud de los pacientes. Se conformó una base tecnológica para la gestión de la atención médica, como factor de reducción de la morbilidad y como base para la conformación de un sistema de reducción de riesgos a la salud, que incluye temas de capacitación sistemática continua del personal médico, indicadores de calidad de la atención médica, el nivel de calidad de los exámenes forenses. Para mejorar la eficiencia del control departamental de la calidad de la atención médica, durante el estudio se estableció un departamento de control de atención médica y trabajo clínico y experto en el Departamento de Salud de la Región de Tyumen (DH TR). El eslabón clave en el control departamental de la calidad de la atención médica son las "comisiones de expertos" de las instituciones médicas (MI), el eslabón clave en el control interdepartamental de la calidad de la atención médica son los "servicios de seguro médico, social y de salud". Los estudios se iniciaron durante la transición a la financiación de un solo canal y las evaluaciones de las instituciones médicas sobre la base de un caso completo