ES2565846T3 - Vacuna combinada contra el sarampión-papiloma humano - Google Patents
Vacuna combinada contra el sarampión-papiloma humano Download PDFInfo
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- ES2565846T3 ES2565846T3 ES09828417.7T ES09828417T ES2565846T3 ES 2565846 T3 ES2565846 T3 ES 2565846T3 ES 09828417 T ES09828417 T ES 09828417T ES 2565846 T3 ES2565846 T3 ES 2565846T3
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- 201000005505 Measles Diseases 0.000 title abstract 3
- 208000003154 papilloma Diseases 0.000 title 1
- 229960005486 vaccine Drugs 0.000 title 1
- 241000700605 Viruses Species 0.000 abstract description 16
- 108090000623 proteins and genes Proteins 0.000 abstract description 5
- 239000000427 antigen Substances 0.000 abstract description 4
- 108091007433 antigens Proteins 0.000 abstract description 4
- 102000036639 antigens Human genes 0.000 abstract description 4
- 102000004169 proteins and genes Human genes 0.000 abstract description 4
- 241000712079 Measles morbillivirus Species 0.000 abstract 1
- 230000028993 immune response Effects 0.000 abstract 1
- 229940041323 measles vaccine Drugs 0.000 abstract 1
- 229960002566 papillomavirus vaccine Drugs 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 16
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- 238000001890 transfection Methods 0.000 description 6
- 210000003501 vero cell Anatomy 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
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- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
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- 101710127721 Membrane protein Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
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- 229910019142 PO4 Inorganic materials 0.000 description 1
- 101710177166 Phosphoprotein Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
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- 238000002826 magnetic-activated cell sorting Methods 0.000 description 1
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- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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Abstract
Una vacuna combinada contra el sarampión-HPV que comprende un virus de vacuna recombinante contra el sarampión que expresa antígenos derivados del HPV y virus del sarampión, en la que los antígenos pueden producir una respuesta inmunitaria y protección tanto contra el sarampión como el HPV, y en la que el antígeno derivado del HPV comprende: (a) proteína L1; y/o (b) proteína L2; y/o (c) proteína E6 y/o E7.
Description
-4-294 ORF de HPV-E7 -295-297 codón de parada
Figura 6. Rescate del virus recombinante MV2EZ-L1 en células auxiliares 293-3-46. Las células se cotransfectaron 5 con 25 ng de pEMCLa y 5 µg del plásmido recombinante p(+)MV2EZ-L1.
Figura 7. Formación de sincitio en células Vero transfectadas con MV2EZ-L1. A las 24 h p.i., las células se fijaron con etanol y se tiñeron con cristal violeta.
10 Figura 8. Curvas de crecimiento de virus asociados a células (CA) y sin células (CF) en células MRC5. Se infectaron monocapas con un MDI de 0,05 con MV2EZ-L1 de virus MVEZ.
Figura 9. Expresión de HPV-L1 a partir de MV recombinantes por análisis de inmunofluorescencia. Se infectaron células Vero con MV2EZ-L1 (A) o MVEZ (B) con una MDI de 0,05 durante 48 h y se procesaron según la
15 inmunofluorescencia indirecta. En los paneles superiores, se detectaron señales de HPV-L1 específicas con anticuerpo monoclonal de ratón dirigido contra HPV-L1, seguido de conjugado de anticuerpo de cabra dirigido contra IgG de ratón-FITC, en los paneles inferiores los mismos portaobjetos se tiñeron con DAPI (clorhidrato del 4’,6diamidina-2-fenilindol) y se observaron respectivamente con luz fluorescente y natural.
20 Figura 10. Expresión de HPV-L1 a partir de MV recombinantes por análisis de transferencia Western. Se infectaron células Vero con MVEZ (1) o MV2EZ-L1 (2) con una MDI de 0,05 durante 48 h. Expresión de HPV-L1 en el líquido sobrenadante (A) y el lisado celular (B) después de infección con MVEZ (banda 1) o MV2EZ-L1 (banda 2).
Figura 11. Análisis de transferencia Western de HPV-L1 en diferentes fracciones de gradiente de CsCl. Los ejemplos 25 describen la invención.
[0025] Todos los procedimientos de clonación eran básicamente como se describe en Sambrook y col. 30 (1989).
[0026] Todas las enzimas de restricción eran de New England BioLabs; los oligonucleótidos cebadores de la PCR eran de Invitrogen.
35 [0027] La secuencia de L1 se amplificó por PCR, y se clonó directamente en los vectores de MV definitivos, obteniendo dos plásmidos recombinantes de MV-HPV16-L1: p(+)MV2EZ-HPV-L1 y p(+)MV3EZ-HPV-L1.
[0028] La amplificación por PCR se llevó a cabo usando la ADN polimerasa Pfu de corrección de errores (Stratagene). Las secuencias de ADN de los cebadores oligonucleótidos sintéticos se dan con mayúsculas para los
40 nucleótidos del MV y en minúsculas para los nucleótidos no MV; están subrayadas las secuencias de sitios de reconocimiento de endonucleasas de restricción relevantes. Se han usado los siguientes cebadores: FOR-L1 5’ ttggcgcgccATGAGCCTGTGGCTGCCC -3’; REV-L1 5’ -atgacgtcTCACAGCTTCCTCTTCTTCCTC -3’.
[0029] For-L1 contiene un extremo saliente (en minúsculas) con sitio de restricción de BssHII (gcgcgc), 45 después del sitio de protección largo de 3 pb (ttg).
[0030] Rev-L1 contiene un extremo saliente (en minúsculas) con sitio de restricción de AatII (gacgtc).
[0031] El PCR-HPV16-L1 (1536 pb) obtenido se clonó en el vector p(+)MVEZ (figura 1) entre los genes para
50 la proteína P y M (posición 2, figura 2) o entre H y L (posición 3, figura 3), después de digestión con BssHII + AatII. Los ligados se llevaron a cabo durante la noche a 16ºC en una relación equimolar con respecto a los vectores MeV2EZ y MeV3EZ previamente digeridos con BssHII + AatII (19 Kb de longitud), usando una unidad de ADN ligasa de T4, obteniendo, respectivamente, p(+)MV2EZ-L1 (20561 pb) y p(+)MV3EZ-L1 (20561 pb). En las figuras 4 y 5 se dan respectivamente, las secuencias de los vectores p(+)MV2EZ-GFP y p(+)MV3EZ-GFP.
55 [0032] Después, células químicamente competentes XL10-Gold se transformaron con todo el volumen de ligado, siguiendo un protocolo de transformación estándar (Sambrook y col. 1989), se sembraron y seleccionaron en placas de LB-Agar según la resistencia a la ampicilina. Las colonias se cribaron por preparación de plásmido de ADN (QIAGEN, kit mini, midi y maxi) y digestión con enzimas de restricción. Los clones correctos se enviaron a
8
transfección.
[0039] Células 293-3-46 se sembraron en un pocillo de 35 mm para alcanzar ~50-70% de confluencia cuando eran transfectadas. 4 h antes de la transfección, el medio se sustituyó por 3 ml de DMEM que contenía FCS al 10%. 5 Todos los plásmidos recombinantes se prepararon de acuerdo con el kit de preparación de plásmidos de QIAGEN. El kit para la coprecipitación con fosfato de Ca2+ del ADN era de Invitrogen.
[0040] Las células se cotransfectaron con los plásmidos en las siguiente concentración final: pEMCLa 25 ng y el plásmido recombinante p(+)MV2EZ-L1 5 mg. Todos los plásmidos, diluidos en H2O se añadieron a un tubo 10 Eppendorf que contenía CaCl2 2 M, la mezcla se añadió a otro tubo Eppendorf que contenía tampón HEPES en condiciones de agitación, y se incubó 30 min a temperatura ambiente (t.a.). Después, los coprecipitados se añadieron gota a gota al cultivo y la transfección se llevó a cabo a 37ºC y 5% de CO2 durante aproximadamente 18
h. Después, el medio de transfección se sustituyó por 3 ml de DMEM que contenía FCS al 10%.
15 [0041] Los primeros sincitios aparecieron 3-4 días después de la transfección, cuando las células todavía eran subconfluentes como se muestra en la figura 6. Para dejar que la formación de sincitios progresara más fácilmente, las monocapas de células casi confluentes de cada pocillo de 35 mm después se transfirieron a una placa de 10 cm. Cada sincitio se recogió en 300 µl de medio de transfección y se puso en un tubo Eppendorf estéril que contenía 700 µl de OPTIMEM, se congeló y descongeló durante tres ciclos, y se almacenó a -80ºC.
20
[0042] Se llevaron a cabo diluciones seriadas de 10 veces de las preparaciones de virus usando OPTIMEM hasta un volumen final de 0,5 ml. Cada dilución se añadió a cultivos de células Vero de 35 mm. Después de 1 h de
25 adsorción de virus, el inóculo se separó y las células infectadas se cubrieron con 2 ml de DMEM que contenía FCS al 5% y agarosa de bajo punto de fusión al 1% (agarosa LMP). Después de 5 días de incubación a 37ºC y 5% de CO2, los cultivos se fijaron con 1 ml de TCA al 10% durante 1 h, después se reticularon con UV durante 30 min. Después de separar la capa superior de agarosa, las monocapas de células se tiñeron con cristal violeta disuelto en etanol al 4%, se lavaron con agua y se hizo el recuento en las placas con el microscopio invertido (figura 7).
30
[0043] Los virus rescatados se sometieron a pases sucesivos 10 veces en células MRC5, se sembraron en placas de 10 cm de diámetro, que se habían infectado con los virus MV de referencia y recombinante con una MDI
35 de 0,01 ufp/células. Después de que la monocapa estuviera totalmente infectada, se usó 1% del líquido sobrenadante de cada cultivo para infectar la subsiguiente monocapa de células MACS. Para el ensayo de la expresión del transgén y la estabilidad, se usaron los virus de los pases 1, 5 y 10 para la caracterización adicional de la expresión por transferencia Western e inmunofluorescencia.
[0044] Células MRC5 sembradas en placa de 35 mm (1-5x105) se vigilaron para el 90% de confluencia, y se infectaron con suspensión de virus clarificada de la fracción de virus asociados a células con una MDI de 0,05, incluyendo MVEZ como control. Las muestras, que correspondían a la llamada “fracción de virus sin células” y la 45 llamada “fracción de virus asociados a células”, se recogieron diariamente durante una semana y se valoraron. A partir de la comparación de las curvas de crecimiento, es interesante que la replicación de MV2EZ-L1 estaba solo ligeramente deteriorada: el virus recombinante alcanzó valoraciones máximas de 6,12 x 106 DICT50/ml 48 hpi (horas post-infección), mientras que MVEZ dio valoraciones finales de 6,8 x 106 DICT50/ml 36 hpi (fig. 8). El progreso de la replicación ligeramente más lento también se reflejaba en tamaños de placas algo reducidas. MV2EZ-L1 produjo
50 placas con un diámetro medio de 0,83 mm, mientras que MVEZ produjo placas con un diámetro medio de 0,91 mm.
[0045] Para analizar la expresión de antígeno de MV y HPV, se llevaron a cabo análisis de 55 inmunofluorescencia, transferencia Western y aislamiento de VLP.
Inmunofluorescencia
[0046] Se sembraron células Vero en cubreobjetos de vidrio de 24 mm x 24 mm en pocillos de 35 mm, se
10
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| EP1375670B1 (en) | 2002-06-20 | 2013-06-12 | Institut Pasteur | Recombinant measles viruses expressing epitopes of antigens of RNA viruses and use of the recombinant viruses for the preparation of vaccine compositions |
| DK1375512T3 (da) * | 2002-06-20 | 2009-10-26 | Pasteur Institut | Infektiös cDNA af en godkendt vaccinestamme af mæslingevirus. Anvendelse til immunogene sammensætninger |
| CA2432738A1 (en) | 2003-02-26 | 2004-08-26 | Philippe Despres | New dengue and west nile viruses proteins and genes coding the foregoing, and their use in vaccinal, therapeutic and diagnostic applications |
| US9119815B2 (en) * | 2009-05-05 | 2015-09-01 | Cadila Healthcare Limited | Combined measles-malaria vaccine |
| MX360449B (es) * | 2011-10-12 | 2018-11-01 | Univ Pennsylvania | Vacunas para el virus del papiloma humano y metodos para su uso. |
| US20130122038A1 (en) * | 2011-11-14 | 2013-05-16 | The United States Of America As Represented By The Secretary Of The Department | Heterologous prime-boost immunization using measles virus-based vaccines |
| WO2014043518A1 (en) | 2012-09-14 | 2014-03-20 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Brachyury protein, non-poxvirus non-yeast vectors encoding brachyury protein, and their use |
| AU2014248535B2 (en) | 2013-03-12 | 2017-03-30 | Inovio Pharmaceuticals, Inc. | Improved vaccines for human papilloma virus and methods for using the same |
| CN105801704B (zh) | 2014-12-31 | 2020-06-02 | 艾托金生物医药(苏州)有限公司 | 一种具有抗宫颈癌细胞活性的重组疫苗构建方法及应用 |
| CN104830869B (zh) * | 2015-04-30 | 2018-06-01 | 武汉凯德维斯生物技术有限公司 | 与宫颈癌发生相关的hpv整合的基因位点及其应用 |
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| RU2681174C1 (ru) * | 2018-07-12 | 2019-03-04 | Закрытое акционерное общество научно-производственная компания "Комбиотех" | Способ получения рекомбинантной вакцины для профилактики папилломавирусной инфекции человека, рекомбинантная вакцина |
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| ES2194839T3 (es) | 1991-07-19 | 2003-12-01 | Univ Queensland | Vacuna de papilomavirus. |
| EP1835029A1 (en) | 1992-06-25 | 2007-09-19 | Georgetown University | Papillomavirus vaccines |
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| ATE492289T1 (de) | 1993-03-09 | 2011-01-15 | Univ Rochester | Herstellung von menschlichen papillomavirus hbv- 11 kapsidprotein l1 und virus-ähnliche partikeln |
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| CA2722847A1 (en) | 2010-07-15 |
| BRPI0909547A2 (pt) | 2019-03-06 |
| TWI561244B (en) | 2016-12-11 |
| CN102099053A (zh) | 2011-06-15 |
| US9623098B2 (en) | 2017-04-18 |
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