ES2660615T3 - Vectores de CMVH y CMVRh recombinantes y usos de los mismos - Google Patents
Vectores de CMVH y CMVRh recombinantes y usos de los mismos Download PDFInfo
- Publication number
- ES2660615T3 ES2660615T3 ES11781407.9T ES11781407T ES2660615T3 ES 2660615 T3 ES2660615 T3 ES 2660615T3 ES 11781407 T ES11781407 T ES 11781407T ES 2660615 T3 ES2660615 T3 ES 2660615T3
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- Prior art keywords
- cmvrh
- cmvh
- sequence
- response
- recombinant
- Prior art date
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Abstract
Un vector de CMVRh o CMVH recombinante que comprende una secuencia de ácido nucleico que codifica un antígeno heterólogo, en el que el antígeno heterólogo es un antígeno específico de patógeno humano aislado de virus de la inmunodeficiencia humana (VIH), en el que el vector incluye una supresión de uno o más genes de CMVRh o CMVH que son esenciales para o aumentan la replicación.
Description
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promotor.
Un promotor es una secuencia mínima suficiente para dirigir la transcripción. También se incluyen los elementos promotores que son suficientes para hacer la expresión génica dependiente de promotor controlable para especificidad de tipo celular, especificidad tisular o inducible por señales o agentes externos; dichos elementos pueden localizarse en regiones 5’ o 3’ del gen. Se incluyen promotores tanto constitutivos como inducibles (véase por ejemplo, Bitter et al., Methods in Enzymology 153: 516-544, 1987). Por ejemplo, cuando se clonen en sistemas bacterianos, pueden usarse promotores inducibles tales como pL de bacteriófago lambda, plac, ptrp, ptac (promotor híbrido de ptrp-lac) y similares. En una realización, cuando se clonan en sistemas celulares de mamífero, pueden usarse promotores derivados del genoma de células de mamífero (tales como promotor de metalotioneína) o de virus de mamífero (tales como la repetición terminal larga de retrovirus; el promotor tardío de adenovirus; el promotor de 7,5 K de virus vaccinia). También pueden usarse promotores producidos por ADN recombinante o técnicas sintéticas para proporcionar transcripción de las secuencias de ácido nucleico.
Un polinucleótido puede insertarse en un vector de expresión, incluyendo un vector vírico, que contiene una secuencia promotora, que facilita la transcripción eficaz de la secuencia genética insertada del hospedador. El vector de expresión contiene típicamente un origen de replicación, un promotor, así como secuencias de ácido nucleico específicas que permiten la selección fenotípica de las células transformadas.
Heterólogo: un polipéptido heterólogo (tal como un antígeno heterólogo) o polinucleótido heterólogo se refiere a una polipéptido o polinucleótido derivado de una fuente o especie diferente. En algunas realizaciones en el presente documento, la secuencia heteróloga es de una fuente genética diferente, tal como un virus u otro organismo, que la segunda secuencia. En ejemplos particulares, la secuencia heteróloga es una secuencia de ácido nucleico que codifica un antígeno tumoral o un antígeno específico de patógeno.
Péptido inmunogénico (o antigénico): un péptido que comprende un motivo específico de alelo u otra secuencia, tal como una repetición N terminal, de modo que el péptido se una con una molécula del MCH e induzca una respuesta de linfocitos T citotóxicos (“CTL”) o una respuesta de linfocitos B (por ejemplo producción de anticuerpos) contra el antígeno del que deriva el péptido inmunogénico. En una realización, se identifican péptidos inmunogénicos usando motivos de secuencia u otros métodos, tales como red neuronal o determinaciones polinominales conocidas en la técnica. Típicamente, se usan algoritmos para determinar el “umbral de unión” de péptidos para seleccionar los que tienen puntuaciones que les proporcionan una alta probabilidad de unión con una cierta afinidad y serán inmunogénicos. Los algoritmos se basan en los efectos sobre la unión del MHC de un aminoácido particular en una posición particular, los efectos en la unión de anticuerpo de un aminoácido particular en una posición particular, o los efectos en la unión de una sustitución particular en un péptido que contiene motivo. Dentro del contexto de un péptido inmunogénico, un “resto conservado” es uno que aparece en una frecuencia significativamente mayor de lo que se esperaría por distribución aleatoria en una posición particular en un péptido. En una realización, un resto conservado es uno en el que la estructura del MHC puede proporcionar un punto de contacto con el péptido inmunogénico.
Respuesta inmunitaria: un cambio en la inmunidad, por ejemplo una respuesta de una célula del sistema inmunitario, tal como un linfocito B, linfocito T, macrófago, monocito o polimorfonucleocito, a un agente inmunogénico en un sujeto. La respuesta puede ser específica para un antígeno particular (una “respuesta específica de antígeno”). En un ejemplo particular, una respuesta inmunitaria es una respuesta de linfocitos T, tal como una respuesta CD4+ o una respuesta CD8+. En otro ejemplo, la respuesta es una respuesta de linfocitos B y da como resultado la producción de anticuerpos específicos para el agente inmunogénico. En algunos ejemplos, dicha respuesta inmunitaria proporciona protección para el sujeto del agente inmunogénico o la fuente del agente inmunogénico. Por ejemplo, la respuesta puede tratar a un sujeto que tiene un tumor, por ejemplo, interfiriendo con la metástasis del tumor o reduciendo el número o tamaño de un tumor. En otro ejemplo, la respuesta inmunitaria puede tratar a un sujeto con una enfermedad infecciosa. Una respuesta inmunitaria puede ser activa e implicar la estimulación del sistema inmunitario del sujeto, o ser una respuesta que resulta de inmunidad adquirida de forma pasiva. Una respuesta inmunitaria “estimulada de forma repetida” es una respuesta inmunitaria a largo plazo que resulta de la estimulación periódica y repetitiva del sistema inmunitario por la producción repetida de un antígeno dentro de un hospedador. En algunos ejemplos, una respuesta inmunitaria aumentada o potenciada es un aumento en la capacidad de un sujeto para combatir una enfermedad, tal como un tumor o una enfermedad infecciosa.
Inmunidad: el estado de poder montar una respuesta protectora tras exposición a un agente inmunogénico. Las respuestas protectoras pueden ser mediadas por anticuerpos o mediadas por células inmunitarias, y pueden dirigirse hacia un patógeno o antígeno tumoral particular. La inmunidad puede adquirirse de forma activa (tal como por exposición a un agente inmunogénico, bien de forma natural o bien en una composición farmacéutica) o de forma pasiva (tal como mediante administración de anticuerpos o linfocitos T estimulados y expandidos in vitro). En algunas realizaciones desveladas en el presente documento, la inmunidad se adquiere por administración (tal como por administración intraperitoneal o intravenosa) de un vector de CMV recombinante que expresa un antígeno particular, tal como un antígeno específico de patógeno o un antígeno tumoral.
Enfermedad infecciosa: una enfermedad provocada por un patógeno, tal como un hongo, parásito, protozoo,
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266: 131-141, 1996; Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997; y Zhang y Madden, Genome Res. 7: 649-656, 1997.
Los ortólogos de proteínas se caracterizan típicamente por posesión de más de 75 % de identidad de secuencia contada sobre el alineamiento de longitud completa con la secuencia de aminoácidos de proteína específica usando ALIGN ajustado a parámetros por defecto. Las proteínas con aún mayor similitud con una secuencia de referencia mostrarán aumento de las identidades de porcentaje cuando se evalúen por este método, tal como al menos 80 %, al menos 85 %, al menos 90 %, al menos 92 %, al menos 95 % o al menos 98 % de identidad de secuencia. Además, la identidad de secuencias puede compararse sobre la longitud completa de dominios particulares de los péptidos desvelados.
Cuando se compare significativamente menos que la secuencia completa para identidad de secuencia, las secuencias homólogas poseerán típicamente al menos 80 % de identidad de secuencia sobre ventanas cortas de 10-20 aminoácidos, y pueden poseer identidades de secuencias de al menos 85 %, al menos 90 %, al menos 95 %
o al menos 99 % dependiendo de su similitud con la secuencia de referencia. La identidad de secuencia sobre dichas ventanas cortas puede determinarse usando LFASTA; se describen métodos en el sitio web de NCSA. Un experto en la materia apreciará que estos intervalos de identidad de secuencias se proporcionan solamente como guía; es completamente posible que puedan obtenerse homólogos fuertemente significativos que quedan fuera de los intervalos proporcionados.
Una indicación alternativa de que dos moléculas de ácido nucleico están estrechamente relacionadas es que las dos moléculas hibridan entre sí en condiciones rigurosas. Las condiciones rigurosas son dependientes de secuencia y son diferentes en diferentes parámetros ambientales. En general, las condiciones rigurosas se seleccionan para que sean aproximadamente 5 ºC a 20 ºC menores que el punto de fusión térmico I para la secuencia específica a una fuerza iónica y pH definidos. La Tm es la temperatura (a fuerza iónica y pH definidos) a la que el 50 % de la secuencia diana hibrida con una sonda perfectamente coincidente. Las condiciones para hibridación de ácidos nucleicos y cálculo de rigurosidades pueden encontrarse en Sambrook et al. (En Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, Nueva York, 1989) y Tijssen (Laboratory Techniques in Biochemistry and Molecular Biology Parte I, C. 2, Elsevier, Nueva York, 1993).
Las condiciones de hibridación que dan como resultado grados de rigurosidad particulares variarán dependiendo de la naturaleza del método de hibridación elegido y la composición y longitud de las secuencias de ácido nucleico hibridantes. En general, la temperatura de hibridación y la fuerza iónica (especialmente la concentración de Na+) del tampón de hibridación determinarán la rigurosidad de la hibridación, aunque los tiempos residuales también influyen en la rigurosidad. Se analizan cálculos con respecto a condiciones de hibridación requeridos para conseguir grados particulares de rigurosidad en Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2ª ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, capítulos 9 y 11. A continuación hay un conjunto ejemplar de condiciones de hibridación:
Rigurosidad muy alta (detecta secuencias que comparten 90 % de identidad)
Hibridación: SSC 5x a 65 ºC durante 16 horas Lavar dos veces: SSC 2x a temperatura ambiente (TA) durante 15 minutos cada uno Lavar dos veces: SSC 0,5x a 65 ºC durante 20 minutos cada uno
Alta rigurosidad (detecta secuencias que comparten 80 % de identidad o más)
Hibridación: SSC 5x-6x a 65 ºC-70 ºC durante 16-20 horas
Lavar dos veces: SSC 2x a TA durante 5-20 minutos cada uno
Lavar dos veces: SSC 1x a 55 ºC-70 ºC durante 30 minutos cada uno
Baja rigurosidad (detecta secuencias que comparten más del 50 % de identidad)
Hibridación: SSC 6x a TA hasta 55 ºC durante 16-20 horas Lavar al menos dos veces: SSC 2x-3x a TA hasta 55 ºC durante 20-30 minutos cada uno.
Las secuencias de ácido nucleico que no muestran un alto grado de identidad pueden no obstante codificar secuencias de aminoácidos similares, debido a la degeneración del código genético. Se entiende que pueden realizarse cambios en la secuencia de ácido nucleico usando esta degeneración para producir múltiples secuencias de ácido nucleico que codifican cada una sustancialmente la misma proteína.
Sujeto: organismos vertebrados multicelulares vivos, una categoría que incluye tanto seres humanos como mamíferos no humanos. Este término abarca individuos tanto conocidos como desconocidos, de modo que no hay ningún requisito de que una persona que trabaje con una muestra de un sujeto sepa quién es el sujeto, o incluso de dónde se adquirió la muestra.
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de los CMVRh que son homólogos de ORF de CMVH que se sabe que no son esenciales para la replicación en fibroblastos se enumeran en la Tabla 1 a continuación. En algunas realizaciones, estos ORF son sitios para inserción de antígenos patógenos para la expresión en los vectores de vacuna basados en CMV desvelados.
5 En comparación, el CMV murino codifica 170 ORF, de los que 78 (45,9 %) son homólogos de proteínas de CMVH conocidas. Resulta importante que a diferencia de CMV murino, CMVRh codifica un complemento completo de genes de evasión inmunitaria de tipo CMVH, y proteínas del tegumento con suficiente homología con CMVH para formar cuerpos densos.
10 Tabla 1. Ejemplos representativos de ORF de CMVRh con homólogos en el genoma de CMVH
- ORF de CMVRh
- ORF de CMVH Mutante de CMVRh
- Rh01
- RL1
- Rh05
- UL153
- Rh17
- UL4
- Rh19
- UL7
- Rh20
- UL6
- Rh31
- UL13
- Rh33
- UL14 25698, 25739
- Rh35a
- UL19
- Rh36
- UL20
- Rh40
- UL23 31242
- Rh42
- UL24 31782, 32619
- Rh43
- UL25 33323, 33711
- Rh54
- UL31
- Rh56
- UL33
- Rh59
- UL35
- Rh68
- UL42
- Rh69
- UL43 54274
- Rh72
- UL45 57859, 58210
- Rh107
- UL78
- Rh123
- UL88 122332, 122472
- Rh143
- UL111A
- Rh148
- UL116
- Rh151/2
- UL118/9
- Rh155
- UL121 155860
- Rh158
- UL147
- Rh159
- UL148 165110
- Rh160
- UL132
- Rh160a
- UL130
- Rh162
- UL145
- Rh163
- UL144
- Rh164
- UL141
- Rh181
- US1
- Rh182
- US2
- Rh184
- US3
- Rh189
- US11
- Rh190
- US12
- Rh192
- US13
- Rh198
- US17
- Rh199
- US18
- Rh200
- US19
- Rh201
- US20
- Rh202
- US21
- Rh203
- US22
- Rh221
- US29
- Rh223
- US30
- Rh225
- US31
Cada ORF de CMVRh anotado, así como varios ORF no reconocidos previamente (Rh35a y Rh160a), se comparó con el conjunto completo de ORF de CMVH usando el algoritmo BlastP. Las puntuaciones con una significación de 15 ≤10-5 se consideraron aciertos. Si más de un ORF de CMVRh correspondía a un ORF de CMVH (por ejemplo, Rh111 y 112 son homólogos de UL83) la ORF de CMVRh se excluyó de la lista. Nueve de las ORF de CMVRh se
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Claims (1)
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Application Number | Priority Date | Filing Date | Title |
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US29930P | 1996-11-01 | ||
US33497610P | 2010-05-14 | 2010-05-14 | |
US334976P | 2010-05-14 | ||
US37691110P | 2010-08-25 | 2010-08-25 | |
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PCT/US2011/029930 WO2011119920A2 (en) | 2010-03-25 | 2011-03-25 | Cmv glycoproteins and recombinant vectors |
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- 2018-05-09 AU AU2018203259A patent/AU2018203259A1/en not_active Abandoned
- 2018-12-10 HK HK18115806.2A patent/HK1256651A1/zh unknown
- 2018-12-18 HK HK18116180.6A patent/HK1257045A1/zh unknown
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2019
- 2019-07-11 AU AU2019204982A patent/AU2019204982B2/en active Active
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2020
- 2020-04-30 HR HRP20200698TT patent/HRP20200698T1/hr unknown
- 2020-05-05 CY CY20201100410T patent/CY1124015T1/el unknown
- 2020-09-01 CY CY20201100816T patent/CY1123362T1/el unknown
- 2020-09-03 HR HRP20201406TT patent/HRP20201406T1/hr unknown
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2022
- 2022-02-22 AU AU2022201186A patent/AU2022201186A1/en active Pending
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