RU2021132097A - Белки rsv f в предшествующей слиянию конформации и их применение - Google Patents
Белки rsv f в предшествующей слиянию конформации и их применение Download PDFInfo
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Claims (44)
1. Выделенный иммуноген для индукции иммунного ответа на белок F RSV, содержащий:
рекомбинантный белок F RSV или его фрагмент, содержащий по меньшей мере одну аминокислотную замену по сравнению с нативным белком F RSV, которая стабилизирует рекомбинантный белок F RSV в предшествующей слиянию конформации, где по меньшей мере одна аминокислотная замена включает замены:
(A) S155C и S290C замены, которые формируют неприродную дисульфидную связь; и/или
(B) первую цистеиновую замену в одном из положений 137-216 белка F RSV и вторую цистеиновую замену в одном положений 26-61 белка F RSV, которые формируют неприродную дисульфидную связь;
рекомбинантный белок RSV F или его фрагмент содержит белок F2 и эктодомен F1, содержащие аминокислотные последовательности, имеющие по меньшей мере 80% идентичность остаткам 26-109 и 137-513 соответственно или 26-103 и 145-513 соответственно последовательности SEQ ID NO: 371.
2. Иммуноген по п. 1, где положения белка F RSV первого и второго цистеина имеют Cβ-Cβ расстояние от 2,0-5,5 Ангстрем в трехмерной структуре, представленной структурными координатами, приведенными в Таблице 1.
3. Иммуноген по п. 1, где иммуноген специфически связывается с предшествующим слиянию специфическим антителом D25 или AM22 после инкубации при 20°C в фосфатно-солевом буфере при физиологическом pH в течение по меньшей мере 24 часов в отсутствие антитела.
4. Иммуноген по п. 1, где нативный белок F RSV представляет собой белок или F RSV A, B или белок F бычьего RSV.
5. Иммуноген по п. 1, где рекомбинантный белок F RSV представляет собой одноцепочечный белок F RSV, и полипептиды F2 и F1 связаны через гетерологичный пептидный линкер или связаны прямо.
6. Иммуноген по п. 5, где положение 105 полипептида F2 связано с положением 145 полипептида F1 через линкер Gly-Ser или положение 103 полипептида F2 прямо связано с положением 145 полипептида F1.
7. Иммуноген по п. 5, где положение 105 полипептида F2 связано с положением 145 полипептида F1 через линкер Gly-Ser или положение 103 полипептида F2 прямо связано с положением 145 полипептида F1.
8. Иммуноген по п. 1, где рекомбинантный белок F RSV дополнительно содержит одну или более дополнительных аминокислотных замен, которые стабилизируют рекомбинантный белок F RSV в предшествующей слиянию конформации.
9. Иммуноген по п. 1, где рекомбинантный белок F RSV дополнительно стабилизирован в предшествующей слиянию конформации с помощью заполняющей полость замены, включающей F, L, W, Y, H или M замену в положении 190, положении 207 или положениях 190 и 207.
10. Иммуноген по п.1, где рекомбинантный белок RSV F дополнительно включает дисульфидную связь между промоторами между A149C и Y458C заменами; дисульфидную связь между промоторами между N183C и N428C заменами, и белок RSV F дополнительно содержит вставку G между положениями 182/183 или дисульфидную связь между промоторами между T369C и N455C заменами.
11. Иммуноген по п. 1, где рекомбинантный белок F RSV содержит остатки 26-109 и 137-513 соответственно или 26-103 и 145-513 соответственно, или 26-105 и 145-513 соответственно SEQ ID NO: 185.
12. Иммуноген по п. 1, где полипептид F1 содержит спираль α10 RSV, содержащую от положения 492 до одного из положений 510-529 RSV, и где полипептид F1 содержит по меньшей мере две замены на цистеин, которые образуют неприродную дисульфидную связь между протомерами.
13. Иммуноген по п. 1, содержащий мультимер рекомбинантного белка F RSV или его фрагмента.
14. Иммуноген по п. 1, где рекомбинантный белок F RSV или его фрагмент связаны с доменом тримеризации.
15. Иммуноген по п. 14, где C-конец полипептида F1 в рекомбинантном белке F RSV связан с доменом тримеризации.
16. Иммуноген по п. 14, где домен тримеризации представляет собой домен Foldon.
17. Иммуноген по п. 14, где рекомбинантный белок F RSV, связанный с доменом тримеризации, содержит остатки 26-109 и 137-544 или 26-103 и 145-544 SEQ ID NO: 185.
18. Иммуноген по п. 1, где рекомбинантный белок F RSV или его фрагмент связаны с субъединицей белковой наночастицы.
19. Иммуноген по п. 18, где C-конец рекомбинантного белка F RSV или его фрагмента связан с субъединицей белковой наночастицы.
20. Иммуноген по п. 1, где рекомбинантный белок F RSV образует тример в фосфатно-солевом буфере при физиологических значениях pH.
21. Иммуноген по п. 1, где: (a) рекомбинантный белок F RSV или его фрагмент не включают дисульфидную связь между положениями 481 и 489 F RSV, или между положениями 509 и 510 F RSV; (b) рекомбинантный белок F RSV или его фрагмент не включают остаток цистеина в положениях 481, 489, 509, 510 F RSV или их комбинации; (c) комбинация (a) и (b).
22. Иммуноген по п. 1, где Fab моноклонального антитела D25 или AM22 специфически связывается с иммуногеном, вирусоподобной частицей или белковой наночастицей с Kd 1 мкМ или менее.
23. Иммуноген по п. 1, где C-концевой остаток F1 полипептида на рекомбинантном белке RSV F соответствует одному из положений 513-529 RSV F и связан с доменом тримеризации Foldon гетерологичным пептидным линкером.
24. Иммуноген по п. 1, где рекомбинантный белок F RSV содержит трансмембранный домен.
25. Вирусоподобная частица, содержащая иммуноген по п. 1.
26. Белковая наночастица, содержащая иммуноген по п. 1.
27. Молекула нуклеиновой кислоты для получения иммуногена по п. 1, где указанная молекула кодирует иммуноген по п. 1.
28. Молекула нуклеиновой кислоты по п. 27, кодон-оптимизированная для экспрессии в клетке человека или животного семейства бычьих.
29. Молекула нуклеиновой кислоты по п. 27, где указанная молекула представляет собой РНК молекулу.
30. Вектор для экспрессии иммуногена по п. 1, содержащий молекулу нуклеиновой кислоты по пп. 27-29.
31. Вектор по п. 30, где вектор представляет собой вирусный вектор.
32. Вектор по п. 30, где вирусный вектор представляет собой вектор на основе парагриппа крупного рогатого скота, вектор на основе вируса парагриппа человека, вектор на основе вируса болезни Ньюкасла, вектор на основе вируса Сендай, вектор на основе вируса кори, ослабленный вектор RSV, вектор на основе парамиксовируса, вектор на основе аденовируса, вектор на основе альфавируса, вектор на основе вируса венесуэльского энцифалита лошадей, вектор на основе вируса леса Семлики, вектор на основе вируса Синдбис, вектор на основе аденоассоциированного вируса, вектор на основе поксвируса, вектор на основе рабдовируса, вектор на основе вируса везикулярного стоматита, вектор на основе пикорнавируса или вектор на основе вируса герпеса.
33. Иммуногенная композиция для индукции иммунного ответа на белок F RSV, содержащая эффективное количество иммуногена по п. 1, и фармацевтически приемлемый носитель.
34. Иммуногенная композиция по п. 33, дополнительно содержащая адъювант.
35. Способ индукции иммунного ответа на F RSV у индивидуума, включающий введение индивидууму эффективного количества иммуногенной композиции по п. 33 для индукции иммунного ответа.
36. Способ предупреждения инфекции RSV у индивидуума, включающий введение индивидууму терапевтически эффективного количества иммуногенной композиции по п. 33, тем самым осуществляя предупреждение инфекции RSV у индивидуума.
37. Способ обнаружения антитела, связывающего F RSV, у индивидуума, включающий предоставление эффективного количества иммуногена, по п. 1; контактирование биологического образца от индивидуума с рекомбинантным белком F RSV или белковой наночастицей в условиях, достаточных для образования иммунного комплекса между рекомбинантным белком F RSV или белковой наночастицей и антителом, связывающим F RSV; и обнаружение иммунного комплекса, тем самым обнаруживая антитело, связывающее F RSV, у индивидуума.
38. Набор для индукции иммунного ответа на белок F RSV у индивидуума, содержащий иммуноген по п. 1, и инструкции по применению набора.
39. Применение иммуногена по п. 1 для предупреждения инфекции RSV у индивидуума.
40. Применение иммуногена по п. 1 для индукции иммунного ответа на белок F RSV у индивидуума.
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- 2014-03-13 DK DK14775224.0T patent/DK2970398T3/da active
- 2014-03-13 RU RU2015142981A patent/RU2761631C2/ru active
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