KR20150044954A - 변형된 인간 로타바이러스 및 이의 용도 - Google Patents
변형된 인간 로타바이러스 및 이의 용도 Download PDFInfo
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- KR20150044954A KR20150044954A KR1020157007160A KR20157007160A KR20150044954A KR 20150044954 A KR20150044954 A KR 20150044954A KR 1020157007160 A KR1020157007160 A KR 1020157007160A KR 20157007160 A KR20157007160 A KR 20157007160A KR 20150044954 A KR20150044954 A KR 20150044954A
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Abstract
본 기술은 일반적으로 바이러스 백신 분야에 관한 것이다. 특히, 본 기술은 변형된 Vero-적응된 인간 로타바이러스 균주 및 고역가 바이러스를 생성하는 배양 방법, 로타바이러스 백신, 예방접종 프로토콜 및 진단 및 예후 검정법을 제공한다.
Description
본원은 발명의 명칭 "변형된 인간 로타바이러스 및 이의 용도"의 2012년 8월 27일에 출원된 호주 가특허 출원 번호 2012903702(이의 전문이 본원에 참조로 포함된다)와 관련되며 이를 우선권으로 주장한다.
본 개시는 일반적으로 바이러스 백신 분야에 관한 것이다. 특히, 본 개시는 변형된 인간 로타바이러스 균주(strain) 및 고역가 바이러스를 생성하는 배양 방법, 로타바이러스 백신, 예방접종 프로토콜 및 진단 및 예후 검정법을 제공한다.
본 명세서에서의 임의의 선행 기술에 대한 참조는, 이러한 선행 기술이 어떠한 나라에서든 공통된 일반적 지식의 일부를 형성한다는 것에 대한 인정 또는 어떠한 형태의 제안도 아니며, 그러한 것으로 간주되지 말아야 한다.
본 명세서에서 저자로 인용되는 간행문에 대한 서지정보는 본 명세서의 마지막에 알파벳순으로 총괄되어 있다.
세계적으로, 로타바이러스(RV)는 영아 및 유아에서 설사와 관련된 이환율 및 사망률의 주요 요인이다. RV 감염은, 주로 개발도상국에서, 매년 500,000명을 초과하는 죽음에 대해 책임이 있다. 세계보건기구(WHO)는 안전하고 효과적인 백신의 개발이 우선적이라고 진술하였다[Bern and Glass (1994) Impact of diarrheal diseases worldwide In Kapikian AZ (ed) Viral Infections of the grastrointestinal tract, 2nd edition, New York Marcel Dekker, Inc.:1-26; Jiang et al. (2002) Clin Infect Dis 34:1351-1361].
특히, RV는 영아 및 유아가 입원해야 하는 질환인 급성 위장염을 일으킨다. 미국, 호주 및 일본에서의 연구는 급성 설사 질환에 기인한 어린이 입원의 34 내지 63%가 RV 감염과 관련된다는 것을 입증하였다. 미국에 있는 건강 관리 기관에서의 RV 위장염에 기인한 입원은, 13 내지 24개월의 어린이에서는 100,000명당 222명이고 1년 미만의 어린이에서는 100,000명당 362명인 것으로 추정되었다. RV로의 감염은 이러한 소아과 집단에서 급성 설사에 기인한 총 입원 중 63%와 관련되었다. 1973년부터 1983년까지 미국에서의 사망률 자료 보고서는, 연당 500명의 죽음이 설사 질환에 기인하여 4세 미만의 어린이에서 발생하며, 미국에서 설사에 기인한 한겨울 죽음의 20 내지 80%가 RV 감염과 관련된다고 나타내었다. RV는 또한 제3세계 나라에서 설사 질환과 관련된 사망률의 상당한 부분에 책임이 있다. 따라서, 효과적인 RV 백신은 세계의 선진국 및 개발도상국 모두에서 어린이 건강에 대해 주요한 영향을 갖는다.
RV는, 캡시드가 바이러스 단백질(VP)의 3개의 동심층에 의해 형성되는, 외피를 갖지 않는 정20면체 바이러스이다. 맨 안쪽의 층은 11개 분절의 이중-가닥 RNA 및 각각 12개 카피의 VP1(RV 폴리머라제) 및 VP3(바이러스 코핑(copping) 효소)로 구성된 바이러스 게놈을 둘러싸는 VP2의 60개 이량체에 의해 형성된다. 단백질의 제2층은 VP2의 상단에 위치되어 이중층 입자(DLP)를 형성하는 VP6의 280개 삼량체에 의해 형성된다. 마지막으로, RV의 가장 바깥쪽 층을 구성하는 당단백질 VP7의 280개 삼량체 및 VP4 단백질의 60개 이량체 스파이크를 DLP에 첨가하는 것은 성숙한 감염성 RV를 나타내는 3중층 입자(TLP)를 형성하는 것을 돕는다.
VP7은 외부 캡시드 단백질이다. 이는 당단백질이며, VP4 및 VP6과 상호작용한다. 또 다른 단백질 그룹은 "비-구조 단백질"(NSP) 이다. 예를 들어, NSP5는 바이로플라즘(viroplasm)의 조립 시 및 바이러스 복제를 위해 NSP2와 함께 구조적 역할을 갖는 것으로 제안되었다.
문헌[Sato et al. (1981) Arch. Virol. 69:155-160]은 MA104 세포(리서스(Rhesus) 원숭이로부터의 태아 신장 세포)의 롤러 배양물을 사용하여 배설물(fecal) 시료로부터의 인간 RV를 성공적으로 배양하였다고 기재하였다. 그러나, MA104 세포는, 적어도 백신용의 바이러스를 생성하기 위한 비히클로서 사용하기에는, 상대적으로 비특성화되어 있다. RV 백신 개발에 사용하기 위해 잘 특성화된 세포 시스템에서 RV를 배양하기 위한 프로토콜을 개발할 필요가 있다. 또한, 피험자에서 방어적 면역 및 반응을 생성하기 하기에 충분한 역가를 갖는 바이러스가 필요하다.
뉴클레오타이드 및 아미노산 서열은 서열 식별자 번호(서열번호)로 언급된다. 서열번호는 숫자로 표기되는 서열 식별자 <400>1(서열번호 1), <400>2(서열번호 2) 등에 상응한다. 서열 식별자에 대한 요약이 표 1에 제공된다. 서열 목록은 특허청구범위 다음에 제공된다.
배양 방법은 Vero 세포에서 로타바이러스(RV)에 대해 제공된다. "Vero 세포”의 언급은 Vero 세포-유래된 세포주를 포함한다. RV는 "Vero-적응된(adapted)" RV 또는 "RV3-Vero" 또는 "RV3BB"로 지칭된다. 본 방법은 RV 접종물(inoculum)의 트립신처리 및 트립신처리된 RV의 Vero 세포에서의 배양을 수반한다. 이러한 트립신 방법은 RV3가 Vero 세포에서 성장할 수 있도록 바이러스를 활성화시키는데 있어서 다른 RV 생성에서 사용되는 것보다 훨씬 높은 농도의 트립신을 사용한다. "트립신처리"의 언급은 RV를 돼지, 반추 동물(예: 소) 중 하나 이상을 포함하는 임의의 공급원으로부터의 트립신 및 재조합 트립신에 대해 처리하는 것을 의미한다. 연속적인 세포주에서의 성장 적응시, RV 제제는 편리하게는VP1, VP3, VP4, NSP4, NSP5/6를 포함하는 구조 및 비-구조 단백질 중 하나 또는 둘 모두에서의 추론된 아미노산 변화 및/또는 VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6에서의 뉴클레오타이드 변화로 확인되는 게놈 변형을 겪는다. 다른 RV 단백질에서의 아미노산 변화 또는 RV 게놈에서의 뉴클레오타이드 변화를 이끄는 유전적 변형도 또한 본 개시에 의해 고려된다. Vero-적응된 RV는 백신 개발에 유용한 것으로 제안된다. 진단 및 예후 검정법이 또한 RV 감염, 백신 통합성 및/또는 치료 또는 예방 프로토콜의 효능을 모니터링하기 위해 제안된다.
따라서, MA104-적응된 RV에서의 상응하는 단백질과 비교하여, VP1, VP3, VP4, NSP4 및/또는 NSP5/6에 및/또는 VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6을 암호화하는 유전자에 돌연변이를 포함하는 Vero 세포-적응된("Vero-적응된") RV(또한 RV-Vero 또는 Vero-RV 또는 RV3BB 또는 Vero-적응된 RV3BB로 지칭됨)가 제공된다.
편의상, 뉴클레오타이드 및 아미노산 비교는 문헌[Rippinger et al. (2010) Virology 405(1):1201-1213]에 의해 공개된 MA104-적응된 RV3 서열을 참조하여 수행된다.
다른 RV 활성화 프로토콜과 비교하여 더욱 높은 농도의 트립신을 사용하여 RV 접종물 또는 분취물(aliquot)을 트립신처리하고 이어서 RV를 Vero 세포의 존재 하에서, 예컨대 MA104-적응된 RV와 비교하여, VP1, VP3, VP4, NSP4 및/또는 NSP5/6 중 하나 또는 둘 모두에서 적어도 아미노산 변화를 초래하는 유전적 변형 및/또는 VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6을 암호화하는 유전자에서의 유전적 변형에 충분한 시간 동안 및 조건 하에서 배양하는 것을 포함하는, Vero 세포에서 RV를 배양하는 방법이 또한 고려된다. 상기 기재되는 바와 같이, 트립신은 돼지 트립신, 반추동물 트립신 또는 재조합 트립신과 같은 임의의 공급원으로부터의 것일 수 있다. 반추 동물 트립신은 소[cattle(bovine)], 양[sheep(ovine)], 염소[goat(caprine)], 기린, 야크, 낙타[camel(camelus)], 라마, 영양 또는 마크로포드(macropod)를 포함한다. 일 실시형태에서, 트립신은 돼지, 소, 양, 염소, 낙타 또는 재조합 트립신이다.
일 실시형태에서, NSP4에서 53, 76, 85, 162 및 165 중에서 선택되는 코돈, NSP5/6에서 46 및 112 중에서 선택되는 코돈, VP1에서 886 중에서 선택되는 코돈, VP3에서 643 및 785 중에서 선택되는 코돈, 및 VP4에서 43, 374, 385, 388, 442 및 756 중에서 선택되는 코돈 및/또는 NSP2에서 433 중에서 선택되는 뉴클레오타이드, NSP4에서 197, 267, 294, 525 및 534 중에서 선택되는 뉴클레오타이드, NSP5/6에서 158, 165 및 355 중에서 선택되는 뉴클레오타이드, VP1에서 2692 중에서 선택되는 뉴클레오타이드, VP3에서 343, 1366, 1744, 1976 및 2402 중에서 선택되는 뉴클레오타이드 및/또는VP4에서 136, 369, 1130, 1162, 1171, 1334 및 2276 중에서 선택되는 뉴클레오타이드에서 아미노산 또는 뉴클레오타이드 변화가 있다.
또 다른 양태는 Vero-적응된 RV3BB 및 하나 이상의 약제학적으로 허용되는 담체, 희석제 및/또는 부형제를 포함하는 조성물에 관한 것이다. 이러한 조성물은 살아있는 약화된 RV 백신 또는 사멸된 RV 백신으로 여겨진다.
또 다른 양태는 피험자에서 RV 감염 또는 재감염 치료 약물의 제조시 Vero-RV, 예컨대 Vero-RV3 또는 이의 성분의 용도에 관한 것이다.
진단제, 예컨대 Vero-적응된 RV에 대한 항체가 또한 제공된다.
뉴클레오타이드 "변화"는 뉴클레오타이드 치환, 부가, 결실 또는 삽입을 포함한다. 상기 변화는 변화를 갖는 뉴클레오타이드 서열에 의해 암호화되는 단백질에서 상응하는 아미노산 변화를 초래할 수 있거나 초래하지 않을 수 있다. 이러한 뉴클레오타이드 또는 아미노산 변화는 그 중에서도 특히 바이러스 감염, 로드, 치료 효능 및 진화 또는 적응을 모니터링하기 위한 진단 목적에 유용하다. Vero-적응된 RV3과 MA104-적응된 RV3 사이의 아미노산 및 뉴클레오타이드 서열 차이에 대한 요약이 표 2 및 표 5에 제시된다.
서열번호 | 설명 |
1 | 프라이머 - JCVI_VP1_5'UTR |
2 | 프라이머 - GEN-VP1R |
3 | 프라이머 - GEN-VP2F |
4 | 프라이머 - GEN-VP2R |
5 | 프라이머 - GEN-VP3F |
6 | 프라이머 - GEN-VP3R |
7 | 프라이머 - JP_1_For_g4_RV3 |
8 | 프라이머 - JCVI_VP4_3'UTR |
9 | 프라이머 - GEN-VP6F |
10 | 프라이머 - GEN-VP6R |
11 | 프라이머 - Beg9 |
12 | 프라이머 - End9 |
13 | 프라이머 - JCVI_NSP1_5'UTR |
14 | 프라이머 - JCVI_NSP1_3'UTR |
15 | 프라이머 - GEN-NSP2F |
16 | 프라이머 - GEN-NSP2R |
17 | 프라이머 - GEN-NSP3F |
18 | 프라이머 - GEN-NSP3R |
19 | 프라이머 - 10.1 |
20 | 프라이머 - 10.2 |
21 | 프라이머 - GEN-NSP5F |
22 | 프라이머 - GEN-NSP5R |
23 | 뉴클레오타이드 서열 MA104-유래된 RV3 VP7 유전자 |
24 | MA104-적응된 RV3 VP7 단백질의 아미노산 서열 |
25 | 뉴클레오타이드 서열 RV3-Vero VP7 유전자 |
26 | RV3-Vero VP7 단백질의 아미노산 서열 |
27 | 뉴클레오타이드 서열 MA104-유래된 RV3 NSP5/6 유전자 |
28 | MA104-적응된 RV3 NSP5/6 단백질의 아미노산 서열 |
29 | 뉴클레오타이드 서열 RV3-Vero NSP5 유전자 |
30 | RV3-Vero NSP5 단백질의 아미노산 서열 |
31 | 뉴클레오타이드 서열 MA104-유래된 RV3 NSP2 유전자 |
32 | MA104-적응된 RV3 NSP2 단백질의 아미노산 서열 |
33 | 뉴클레오타이드 서열 RV3-Vero NSP2 유전자 |
34 | RV3-Vero NSP2 단백질의 암호화 영역의 아미노산 서열 |
35 | 뉴클레오타이드 서열 MA104-유래된 RV3 NSP4 유전자 |
36 | RV3 NSP4 단백질의 아미노산 서열 |
37 | 뉴클레오타이드 서열 RV3-Vero NSP4 유전자 |
38 | RV3-Vero NSP4 단백질의 아미노산 서열 |
39 | 뉴클레오타이드 서열 MA104-유래된 RV3 VP6 유전자 |
40 | MA104-유래된 RV3 VP6 단백질의 아미노산 서열 |
41 | 뉴클레오타이드 서열 RV3-Vero VP6 유전자 |
42 | RV3-Vero VP6 단백질의 아미노산 서열 |
43 | 뉴클레오타이드 서열 MA104-적응된 RV3 NSP3 유전자 |
44 | MA104-유래된 RV3 NSP3 단백질의 아미노산 서열 |
45 | 뉴클레오타이드 서열 RV3-Vero NSP3 유전자 |
46 | RV3 Vero NSP3 단백질의 아미노산 서열 |
47 | MA104-적응된 RV3 VP1의 뉴클레오타이드 서열 |
48 | MA104-적응된 RV3 VP1의 아미노산 서열 |
49 | Vero-적응된 RV3 VP1의 뉴클레오타이드 서열 |
50 | 아미노산 서열 Vero-적응된 RV3 VP1 |
51 | MA104-적응된 RV3 VP2 유전자의 뉴클레오타이드 서열 |
52 | MA104-적응된 RV3 VP2 단백질의 아미노산 서열 |
53 | Vero-적응된 RV3 VP2 유전자의 뉴클레오타이드 서열 |
54 | Vero-적응된 RV3 VP2 단백질의 아미노산 서열 |
55 | MA104-적응된 RV3 VP3 유전자의 뉴클레오타이드 서열 |
56 | MA104-적응된 RV3 VP7 단백질의 아미노산 서열 |
57 | Vero-적응된 RV3 VP3 유전자의 뉴클레오타이드 서열 |
58 | Vero-적응된 RV3 VP3 단백질의 아미노산 서열 |
59 | MA104-적응된 RV3 VP4 유전자의 뉴클레오타이드 서열 |
60 | MA104-적응된 RV3 VP4 유전자의 아미노산 서열 |
61 | Vero-적응된 RV3 VP4 유전자의 뉴클레오타이드 서열 |
62 | Vero-적응된 RV3 VP4 프로브의 아미노산 서열 |
63 | MA104-적응된 RV3 VP7 유전자의 뉴클레오타이드 서열 |
64 | MA104-적응된 RV3 VP7 단백질의 아미노산 서열 |
65 | Vero-적응된 RV3 VP7 유전자의 뉴클레오타이드 서열 |
66 | Vero-적응된 RV3 VP7 단백질의 아미노산 서열 |
유전자 또는 단백질 | MA104-적응된 RV3 | Vero-적응된 RV3 |
NSP1 유전자 | - | - |
NSP1 단백질 | - | - |
NSP2 유전자 | 387G | 433A |
NSP2 단백질 | - | - |
NSP3 유전자 | - | - |
NSP3 단백질 | - | - |
NSP4 유전자 | 157G 227T 254G 485T 494C |
197A 267C 294A 525G 534T |
NSP4 단백질 | 53A 76I 85C 162V 165P |
53T 76T 85Y 162G 165L |
NSP5/6 유전자 | 137G 144T 334T |
158A 165C 355A |
NSP5/6 단백질 | 46G 112L |
46E 112I |
VP1 유전자 | 2656G | 2692T |
VP1 단백질 | 886V | 886F |
VP2 유전자 | - | - |
VP2 단백질 | - | - |
VP3 유전자 | 294T 1317A 1695T 1927G 2353T |
343C 1366G 1744C 1976A 2402C |
VP3 단백질 | 643E 785Y |
643K 785H |
VP4 유전자 | 127C 360A 1121T 1153G 1162A 1325G 2267A |
136A 369G 1130C 1162A 1171C 1334A 2276T |
VP4 단백질 | 43P 374L 385D 388I 442R 756N |
43T 374S 385N 388L 442H 756I |
VP6 유전자 | - | - |
VP6 단백질 | - | - |
VP7 유전자 | - | - |
VP7 단백질 | - | - |
1 뉴클레오타이드 번호 또는 아미노산 자기 번호는 문헌[Rippinger et al, 2010 상기 참조]에 의해 채택된 번호매김에 기초한다.
도 1은 MA104-적응된 RV3 NSP1 유전자의 뉴클레오타이드 서열을 나타낸다.
도 2은 MA104-적응된 RV3 NSP1 단백질의 추론된 아미노산 서열을 나타낸다.
도 3은 Vero-적응된 RV3 NSP1 유전자의 뉴클레오타이드 서열을 나타낸다.
도 4은 Vero-적응된 NSP1 단백질의 추론된 아미노산 서열을 나타낸다.
도 5는 MA104-적응된 RV3 및 Vero-적응된 RV3 NSP1 유전자의 뉴클레오타이드 서열 비교를 나타낸다.
도 6는 MA104-적응된 RV3 및 Vero-적응된 RV3 NSP1 단백질의 추론된 아미노산 서열 비교를 나타낸다.
도 7은 MA104-적응된 RV3 NSP2 유전자의 뉴클레오타이드 서열을 나타낸다.
도 8은 MA104-적응된 RV3 NSP2 단백질의 추론된 아미노산 서열을 나타낸다.
도 9은 Vero-적응된 RV3 NSP2 유전자의 뉴클레오타이드 서열을 나타낸다.
도 10은 Vero-적응된 RV3 NSP2 단백질의 추론된 아미노산 서열을 나타낸다.
도 11는 MA104-적응된 RV3 및 Vero-적응된 RV3 NSP2 유전자의 뉴클레오타이드 서열 비교를 나타낸다.
도 12는 MA104-적응된 RV3 및 Vero-적응된 RV3 NSP2 단백질의 추론된 아미노산 서열 비교를 나타낸다.
도 13은 MA104-적응된 RV3 NSP3 유전자의 뉴클레오타이드 서열을 나타낸다.
도 14은 MA104-적응된 RV3 NSP3 단백질의 추론된 아미노산 서열을 나타낸다.
도 15은 Vero-적응된 RV3 NSP3 유전자의 뉴클레오타이드 서열을 나타낸다.
도 16은 Vero-적응된 RV3 NSP3 단백질의 추론된 아미노산 서열을 나타낸다.
도 17는 MA104-적응된 RV3 및 Vero-적응된 RV3 NSP3 유전자의 뉴클레오타이드 서열 비교를 나타낸다.
도 18는 MA104-적응된 RV3 및 Vero-적응된 RV3 NSP2 단백질의 추론된 아미노산 서열 비교를 나타낸다.
도 19은 MA104-적응된 RV3 NSP4 유전자의 뉴클레오타이드 서열을 나타낸다.
도 20은 MA104-적응된 RV3 NSP4 단백질의 추론된 아미노산 서열을 나타낸다.
도 21은 Vero-적응된 RV3 NSP4 유전자의 뉴클레오타이드 서열을 나타낸다.
도 22은 Vero-적응된 RV3 NSP4 단백질의 추론된 아미노산 서열을 나타낸다.
도 23는 MA104-적응된 RV3 및 Vero-유래된 RV3 NSP4 유전자의 뉴클레오타이드 서열 비교를 나타낸다.
도 24는 MA104-적응된 RV3 및 Vero-적응된 RV3 NSP4 단백질의 추론된 아미노산 서열 비교를 나타낸다.
도 25는 MA104-적응된 RV3 NSP5/6 유전자의 뉴클레오타이드 서열을 나타낸다.
도 25는 MA104-적응된 RV3 NSP5/6 단백질의 추론된 아미노산 서열을 나타낸다.
도 27은 Vero-적응된 RV3 NSP5/6 유전자의 뉴클레오타이드 서열을 나타낸다.
도 28은 Vero-적응된 RV3 NSP5/6 단백질의 추론된 아미노산 서열을 나타낸다.
도 29는 MA104-적응된 RV3 및 Vero-적응된 RV3 NSP5/6 유전자의 뉴클레오타이드 서열 비교를 나타낸다.
도 30는 MA104-적응된 RV3 및 Vero-적응된 RV3 NSP5/6 단백질의 추론된 아미노산 서열 비교를 나타낸다.
도 31은 MA104-적응된 RV3 VP1 유전자의 뉴클레오타이드 서열을 나타낸다.
도 32은 MA104-적응된 RV3 VP1 프로브의 추론된 아미노산 서열을 나타낸다.
도 33은 Vero-적응된 RV3 VP1 유전자의 뉴클레오타이드 서열을 나타낸다.
도 34은 Vero-적응된 RV3 VP1 단백질의 뉴클레오타이드 서열을 나타낸다.
도 35는 MA104-적응된 RV3 및 Vero-적응된 RV3 VP1 유전자의 비교를 나타낸다.
도 36는 MA104-적응된 RV3 및 Vero-적응된 RV3 VP1 단백질의 비교를 나타낸다.
도 37은 MA104-적응된 RV3 VP2 유전자의 뉴클레오타이드 서열을 나타낸다.
도 38은 MA104-적응된 RV3 VP2 프로브의 추론된 아미노산 서열을 나타낸다.
도 39은 Vero-적응된 RV3 VP2 유전자의 뉴클레오타이드 서열을 나타낸다.
도 40은 Vero-적응된 RV3 VP2 단백질의 뉴클레오타이드 서열을 나타낸다.
도 41는 MA104-적응된 RV3 및 Vero-적응된 RV3 VP2 유전자의 비교를 나타낸다.
도 42는 MA104-적응된 RV3 및 Vero-적응된 RV3 VP2 단백질의 비교를 나타낸다.
도 2은 MA104-적응된 RV3 NSP1 단백질의 추론된 아미노산 서열을 나타낸다.
도 3은 Vero-적응된 RV3 NSP1 유전자의 뉴클레오타이드 서열을 나타낸다.
도 4은 Vero-적응된 NSP1 단백질의 추론된 아미노산 서열을 나타낸다.
도 5는 MA104-적응된 RV3 및 Vero-적응된 RV3 NSP1 유전자의 뉴클레오타이드 서열 비교를 나타낸다.
도 6는 MA104-적응된 RV3 및 Vero-적응된 RV3 NSP1 단백질의 추론된 아미노산 서열 비교를 나타낸다.
도 7은 MA104-적응된 RV3 NSP2 유전자의 뉴클레오타이드 서열을 나타낸다.
도 8은 MA104-적응된 RV3 NSP2 단백질의 추론된 아미노산 서열을 나타낸다.
도 9은 Vero-적응된 RV3 NSP2 유전자의 뉴클레오타이드 서열을 나타낸다.
도 10은 Vero-적응된 RV3 NSP2 단백질의 추론된 아미노산 서열을 나타낸다.
도 11는 MA104-적응된 RV3 및 Vero-적응된 RV3 NSP2 유전자의 뉴클레오타이드 서열 비교를 나타낸다.
도 12는 MA104-적응된 RV3 및 Vero-적응된 RV3 NSP2 단백질의 추론된 아미노산 서열 비교를 나타낸다.
도 13은 MA104-적응된 RV3 NSP3 유전자의 뉴클레오타이드 서열을 나타낸다.
도 14은 MA104-적응된 RV3 NSP3 단백질의 추론된 아미노산 서열을 나타낸다.
도 15은 Vero-적응된 RV3 NSP3 유전자의 뉴클레오타이드 서열을 나타낸다.
도 16은 Vero-적응된 RV3 NSP3 단백질의 추론된 아미노산 서열을 나타낸다.
도 17는 MA104-적응된 RV3 및 Vero-적응된 RV3 NSP3 유전자의 뉴클레오타이드 서열 비교를 나타낸다.
도 18는 MA104-적응된 RV3 및 Vero-적응된 RV3 NSP2 단백질의 추론된 아미노산 서열 비교를 나타낸다.
도 19은 MA104-적응된 RV3 NSP4 유전자의 뉴클레오타이드 서열을 나타낸다.
도 20은 MA104-적응된 RV3 NSP4 단백질의 추론된 아미노산 서열을 나타낸다.
도 21은 Vero-적응된 RV3 NSP4 유전자의 뉴클레오타이드 서열을 나타낸다.
도 22은 Vero-적응된 RV3 NSP4 단백질의 추론된 아미노산 서열을 나타낸다.
도 23는 MA104-적응된 RV3 및 Vero-유래된 RV3 NSP4 유전자의 뉴클레오타이드 서열 비교를 나타낸다.
도 24는 MA104-적응된 RV3 및 Vero-적응된 RV3 NSP4 단백질의 추론된 아미노산 서열 비교를 나타낸다.
도 25는 MA104-적응된 RV3 NSP5/6 유전자의 뉴클레오타이드 서열을 나타낸다.
도 25는 MA104-적응된 RV3 NSP5/6 단백질의 추론된 아미노산 서열을 나타낸다.
도 27은 Vero-적응된 RV3 NSP5/6 유전자의 뉴클레오타이드 서열을 나타낸다.
도 28은 Vero-적응된 RV3 NSP5/6 단백질의 추론된 아미노산 서열을 나타낸다.
도 29는 MA104-적응된 RV3 및 Vero-적응된 RV3 NSP5/6 유전자의 뉴클레오타이드 서열 비교를 나타낸다.
도 30는 MA104-적응된 RV3 및 Vero-적응된 RV3 NSP5/6 단백질의 추론된 아미노산 서열 비교를 나타낸다.
도 31은 MA104-적응된 RV3 VP1 유전자의 뉴클레오타이드 서열을 나타낸다.
도 32은 MA104-적응된 RV3 VP1 프로브의 추론된 아미노산 서열을 나타낸다.
도 33은 Vero-적응된 RV3 VP1 유전자의 뉴클레오타이드 서열을 나타낸다.
도 34은 Vero-적응된 RV3 VP1 단백질의 뉴클레오타이드 서열을 나타낸다.
도 35는 MA104-적응된 RV3 및 Vero-적응된 RV3 VP1 유전자의 비교를 나타낸다.
도 36는 MA104-적응된 RV3 및 Vero-적응된 RV3 VP1 단백질의 비교를 나타낸다.
도 37은 MA104-적응된 RV3 VP2 유전자의 뉴클레오타이드 서열을 나타낸다.
도 38은 MA104-적응된 RV3 VP2 프로브의 추론된 아미노산 서열을 나타낸다.
도 39은 Vero-적응된 RV3 VP2 유전자의 뉴클레오타이드 서열을 나타낸다.
도 40은 Vero-적응된 RV3 VP2 단백질의 뉴클레오타이드 서열을 나타낸다.
도 41는 MA104-적응된 RV3 및 Vero-적응된 RV3 VP2 유전자의 비교를 나타낸다.
도 42는 MA104-적응된 RV3 및 Vero-적응된 RV3 VP2 단백질의 비교를 나타낸다.
본 명세서 전반에 걸쳐서, 문맥상 달리 요구되지 않는 한, 단어 "포함한다" 또는 "포함하는"과 같은 변형은 언급된 요소 또는 정수 또는 방법 단계 또는 요소 또는 정수 또는 방법 단계의 그룹을 포함하는 것을 의미하는 것으로, 임의의 요소 또는 정수 또는 방법 단계 또는 요소 또는 정수 또는 방법 단계의 그룹을 배제시키는 것을 의미하는 것이 아니라는 것이 이해될 것이다.
본 명세서에서 사용되는 단수 형태 "하나의" 및 "상기"는 문맥이 달리 명백히 지시하지 않는 한 복수의 양태를 포함한다는 것에 주목해야 한다. 따라서, 예를 들어, "로타바이러스"의 언급은 단독의 로타바이러스 및 2개 이상의 로타바이러스를 포함하고; "제제"의 언급은 단독 제제 및 2개 이상의 제제를 포함하며; "본 개시"의 언급은 "본 개시"에 대한 단독 언급 및 본 개시에 의해 교시되는 다수의 양태를 포함하고; 기타의 것도 동일하다. 본원에서 교시되고 제공되는 양태는 용어 "발명"에 의해 망라된다. 모든 이러한 양태가 본 발명의 범위 내에서 제공된다.
RV 분취물 또는 접종물을 다른 RV 활성화 프로토콜에 비해 고농도의 트립신을 사용하여 트립신처리하는 단계 및 상기 트립신처리된 RV를 Vero 세포 또는 Vero 세포-유래된 세포주에서 RV가 VP1, VP3, VP4, NSP4 및/또는 NSP5/6 중 하나 이상에서 아미노산 변화 및/또는 VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6 중 하나 이상에서 뉴클레오타이드 변화를 초래하는 유전적 변형을 겪기에 충분한 시간 동안 및 조건 하에서 배양하는 단계를 포함하는 RV 균주의 배양 방법이 고려된다.
"변화"의 언급은 아미노산 또는 뉴클레오타이드 치환, 결실, 부가 또는 삽입을 포함한다. "트립신처리"의 언급은 RV를 트립신으로 처리하는 것을 의미한다. 트립신은 임의의 공급원으로부터의 것일 수 있으며, 돼지 트립신, 반추 동물 트립신 및 재조합 트립신을 포함한다. 반추 동물 트립신은 소[cattle(bovine)], 양[sheep(ovine)], 염소[goat(caprine)], 기린, 야크, 낙타[camel(camelus)], 라마, 영양 또는 마크로포드(macropod)를 포함한다. 일 실시형태에서, 트립신은 돼지, 소, 양, 염소, 낙타 또는 재조합 트립신이다. 일 실시형태에서, 트립신은 돼지 트립신이다. 일 실시형태에서, 트립신은 소 트립신이다. 일 실시형태에서, 트립신은 돼지, 소 또는 재조합 트립신이다. 일 실시형태에서, 트립신은 재조합 트립신이다. "재조합 트립신"은 임의의 세포 유형(예: 세균, 곤충, 효모 또는 포유동물 세포)에서 조작된 유전 물질의 발현에 의해 생성되는 임의의 기원의 트립신이다.
일 실시형태에서, RV 균주는 RV3이다. 생성되는 RV는 편리하게는 "Vero 세포-적응된 RV" 또는 "Vero-적응된 RV" 또는 "Vero-RV" 또는 "RV-Vero" 또는 RV3BB로 지칭된다. 추가의 양태에서, 변형된 Vero-RV는, 예컨대 백신에서 사용하기 위해 또는 항체 생성을 위해 수거된다.
따라서, RV3 분취물 또는 접종물을 다른 RV3 활성화 프로토콜에 비해 고농도의 트립신을 사용하여 트립신처리하는 단계, 이어서 상기 트립신처리된 RV를 Vero 세포 또는 Vero 세포-유래된 세포주에서 RV가 VP1, VP3, VP4, NSP4 및/또는 NSP5/6에서 아미노산 변화 및/또는 VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6에서 뉴클레오타이드 변화를 초래하는 유전적 변형을 겪기에 충분한 시간 동안 및 조건 하에서 배양하는 단계, 및 이어서 변형된 RV3를 선택적으로 수거하는 단계를 포함하는, RV3 균주의 배양 방법이 고려된다.
RV3 분취물 또는 접종물을 다른 RV3 활성화 프로토콜에 비해 고농도의 돼지, 반추 동물 또는 재조합 트립신을 사용하여 트립신처리하는 단계, 이어서 상기 트립신처리된 RV를 Vero 세포 또는 Vero 세포-유래된 세포주에서 RV가 VP1, VP3, VP4, NSP4 및/또는 NSP5/6에서 아미노산 변화 및/또는 VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6에서 뉴클레오타이드 변화를 초래하는 유전적 변형을 겪기에 충분한 시간 동안 및 조건 하에서 배양하는 단계, 및 이어서 변형된 RV3를 선택적으로 수거하는 단계를 포함하는, RV3 균주의 배양 방법이 본원에서 고려될 수 있다.
RV3 분취물 또는 접종물을 다른 RV3 활성화 프로토콜에 비해 고농도의 돼지, 소 또는 재조합 트립신을 사용하여 트립신처리하는 단계, 이어서 상기 트립신처리된 RV를 Vero 세포 또는 Vero 세포-유래된 세포주에서 RV가 VP1, VP3, VP4, NSP4 및/또는 NSP5/6에서 아미노산 변화 및/또는 VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6에서 뉴클레오타이드 변화를 초래하는 유전적 변형을 겪기에 충분한 시간 동안 및 조건 하에서 배양하는 단계, 및 이어서 변형된 RV3를 선택적으로 수거하는 단계를 포함하는, RV3 균주의 배양 방법이 본원에서 고려될 수 있다.
일 실시형태에서, RV는 R 일렉트로페로타입(electropherotype), 예컨대 RV1, RV2 또는 RV3이다. 더욱 특히, 균주는 RV3이다. 또 다른 실시형태에서, RV는 M-일렉트로페로타입, 예컨대 RV5 또는 PA-일렉트로페로타입, 예컨대 RV6이다[참조: Rodger et al. (1981) J. Clin. Microbiol 13:272-278; Albert et al. (1983) J. Clin. Microbiol 17:162-164].
따라서, 본원에는 RV 분취물 또는 접종물을 다른 RV 활성화 프로토콜에 비해 고농도의 트립신을 사용하여 트립신처리하는 단계 및 이어서 상기 트립신처리된 RV를 Vero 세포 또는 Vero 세포-유래된 세포주에서 RV가 VP1, VP3, VP4, NSP4 및/또는 NSP5/6에서 아미노산 변화 및/또는 VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6에서 뉴클레오타이드 변화를 초래하는 유전적 변형을 겪기에 충분한 시간 동안 및 조건 하에서 배양하는 단계 및 이어서 변형된 RV를 선택적으로 수거하는 단계를 포함하는, Vero 세포에서 고역가로 RV 균주, 특히 RV3BB를 배양하는 방법이 기재된다.
본원에 기재되는 배양 프로토콜은 백신으로 사용하기 위해 또는 이에 대한 항체 또는 다른 진단제의 개발을 위해 살아있는 약독화된 RV의 생성을 가능하게 하거나, 사멸되거나 달리 불활성화될 수 있는 RV의 제조를 가능하게 한다.
따라서, RV의 약독화된 균주의 분취물 또는 접종물을 다른 RV3 활성화 프로토콜에 비해 고농도의 트립신을 사용하여 트립신처리하는 단계 및 이어서 상기 트립신처리된 RV를 Vero 세포 또는 Vero 세포-유래된 세포주에서 RV가 VP1, VP3, VP4, NSP4 및/또는 NSP5/6에서 아미노산 변화 및/또는 VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6에서 뉴클레오타이드 변화를 초래하는 유전적 변형을 겪기에 충분한 시간 동안 및 조건 하에서 배양하는 단계를 포함하는, 살아있는 약독화된 RV를 생성하는 방법이 제공된다.
대체적인 실시형태에서, 상기 지시되는 바와 같이, RV는 배양되고 수거된 후 사멸됨으로써 사멸된 RV 백신에 사용하기 위한 사멸된 RV 제제를 생성한다.
"유전적 변형"의 언급은 단일 또는 다수의 뉴클레오타이드의 치환, 부가, 결실 및/또는 삽입을 포함한다. 특정 실시형태에서, 유전적 변형은 NSP4, NSP5 또는 VP7와 같은 암호화된 단백질에 아미노산 변경(즉, 치환, 부가, 결실 또는 삽입)을 유도한다. 또 다른 실시형태에서, 변형은 VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6을 암호화하는 유전자에 뉴클레오타이드 치환, 부가, 결실 및/또는 삽입을 유도한다. 또 다른 실시형태에서, 변형은 VP1, VP3, VP4, NSP4 및/또는 NSP5/6 중 하나 이상에서 아미노산 변화를 유도한다.
RV에 대한 유전적 변형은 편리하게는 MA104 세포에서 배양되는 RV(즉, MA104-적응된 RV)와 비교된다. MA104 세포에서 배양된 이러한 RV는, MA104 세포가 충분한 자격을 갖추지 않았으며 이들의 잠재적 종양원성 프로파일이 현재 확인되지 않았으므로, 백신 생산에 적합하지 않다.
RV 분취물 또는 접종물을 다른 RV 활성화 프로토콜에 비해 고농도의 트립신을 사용하여 트립신처리하는 단계 및 이어서 상기 트립신처리된 RV를 Vero 세포 또는 Vero 세포-유래된 세포주에서 RV가 VP1, VP3, VP4, NSP4 및/또는 NSP5/6 중 하나 이상에서 아미노산 변형 및/또는 VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6에서 뉴클레오타이드 변화를 겪기에 충분한 시간 동안 및 조건 하에서 배양하는 단계를 포함하는 RV 균주의 배양 방법이 고려된다.
또한, RV 분취물 또는 접종물을 다른 RV 활성화 프로토콜에 비해 고농도의 트립신을 사용하여 트립신처리하는 단계 및 이어서 상기 트립신처리된 RV를 Vero 세포 또는 Vero 세포-유래된 세포주에서 RV가 VP1, VP3, VP4, NSP4 및/또는 NSP5/6 중 하나 이상에서 아미노산 변형 및/또는 VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6에서 뉴클레오타이드 변화를 겪기에 충분한 시간 동안 및 조건 하에서 배양하는 단계 및 이어서 변형된 RV를 선택적으로 수거하는 단계를 포함하는, RV1, RV2, RV3, RV5 및 RV6으로 이루어진 리스트 중에서 선택되는 RV 균주의 배양 방법이 기재된다.
일 실시형태와 관련하여, RV3 분취물 또는 접종물을 다른 RV3 활성화 프로토콜에 비해 고농도의 트립신을 사용하여 트립신처리하는 단계 및 이어서 상기 트립신처리된 RV3를 Vero 세포 또는 Vero 세포-유래된 세포주에서 RV3가 VP1, VP3, VP4, NSP4 및/또는 NSP5/6 에서 아미노산 변화 및/또는 VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6 에서 뉴클레오타이드 변화를 겪기에 충분한 시간 동안 및 조건 하에서 배양하는 단계를 포함하는, RV3 균주의 배양 방법이 고려된다.
수거된 RV는 백신 조성물을 포함하는 살아있는 약독화된 RV 조성물의 제조에 유용하다. 대안적으로, 수거된 RV는 사멸되며 사멸된 RV 조성물에 사용된다.
따라서, RV의 약독화된 균주의 분취물 또는 접종물을 다른 RV 활성화 프로토콜에 비해 고농도의 트립신을 사용하여 트립신처리하는 단계 및 이어서 상기 트립신처리된 RV를 Vero 세포 또는 Vero 세포-유래된 세포주에서 RV가 VP1, VP3, VP4, NSP4 및/또는 NSP5/6 중 하나 이상에서 아미노산 변화 및/또는 VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6 중 하나 이상에서 뉴클레오타이드 변화를 겪기에 충분한 시간 동안 및 조건 하에서 배양하는 단계를 포함하는, 살아있는 약독화된 RV를 생성하는 방법이 제공된다.
일 실시형태에서, RV는 RV3BB로도 지칭되는 RV3이며, 유전적 변형은 표 2 또는 표 5에 열거되는 것들이다. 본 개시는 표 2 또는 표 5에 열거되는 바와 같은 하나 또는 모든 변이를 고려한다.
따라서, RV를 다른 RV 활성화 프로토콜에 비해 고농도의 트립신을 사용하여 트립신처리하는 단계 및 이어서 상기 트립신처리된 RV를 Vero 세포 또는 Vero 세포-유래된 세포주에서 유전적 변형이 표 2 또는 표 5에 열거된 돌연변이의 리스트 중에서 선택되는 부위에서 발생하기에 충분한 시간 동안 및 조건 하에서 배양하는 단계를 포함하는, Vero 세포에서 RV를 배양하는 방법이 제공된다.
"등가물"의 언급은 RV3의 또 다른 균주 또는 또 다른 RV에서의 상응하는 뉴클레오타이드 또는 아미노산을 포함한다.
또한, VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6로 이루어진 리스트 중에서 선택되는 유전자 또는 단백질에서의 유전적 변형인 표 2 또는 표 5의 돌연변이 중에서 선택되는 돌연변이를 포함하는 분리된 Vero-적응된 RV가 기재된다.
약독화된 RV3의 분취물 또는 접종물을 트립신처리하는 단계 및 상기 트립신처리된 RV3를 Vero 세포 또는 Vero 세포-유래된 세포주에서 유전적 변형이 MA104-적응된 RV3과 비교하여 표 2 또는 표 5에 제시된 리스트 중에서 선택되는 유전자에서 발생하기에 충분한 시간 동안 및 조건 하에서 배양하는 단계를 포함하는, Vero 세포에서 약독화된 RV3를 생성하는 방법이 제공된다
상술된 돌연변이는 표 3에서 정의되는 바와 같은 단일 아미노산 코드를 사용하여 기재된다.
아미노산 | 3-문자 약어 | 1-문자 기호 |
알라닌 | Ala | A |
아르기닌 | Arg | R |
아스파라긴 | Asn | N |
아스파트산 | Asp | D |
시스테인 | Cys | C |
글루타민 | Gln | Q |
글루탐산 | Glu | E |
글리신 | Gly | G |
히스티딘 | His | H |
이소루이신 | Ile | I |
루이신 | Leu | L |
리신 | Lys | K |
메티오닌 | Met | M |
페닐알라닌 | Phe | F |
프롤린 | Pro | P |
세린 | Ser | S |
트레오닌 | Thr | T |
트립토판 | Trp | W |
타이로신 | Tyr | Y |
발린 | Val | V |
본 개시는 약독화된 RV3의 분취물 또는 접종물을 트립신처리하는 단계 및 상기 트립신처리된 RV3를 Vero 세포 또는 Vero 세포-유래된 세포주에서 유전적 변형이 NSP4에서 53, 76, 85, 162 및 165 중에서 선택되는 코돈, NSP5/6에서 46 및 112 중에서 선택되는 코돈, VP1에서 886 중에서 선택되는 코돈, VP3에서 643 및 785 중에서 선택되는 코돈, 및 VP4에서 43, 374, 385, 388, 442 및 756 중에서 선택되는 코돈 및/또는 NSP2에서 433 중에서 선택되는 뉴클레오타이드, NSP4에서 197, 267, 294, 525 및 534 중에서 선택되는 뉴클레오타이드, NSP5/6에서 158, 165 및 355 중에서 선택되는 뉴클레오타이드, VP1에서 2692 중에서 선택되는 뉴클레오타이드, VP3에서 343, 1366, 1744, 1976 및 2402 중에서 선택되는 뉴클레오타이드 및/또는VP4에서 136, 369, 1130, 1162, 1171, 1334 및 2276 중에서 선택되는 뉴클레오타이드에서 뉴클레오타이드 치환, 결실 및/또는 부가를 포함하는 리스트 중에서 선택적으로 일어나기에 충분한 시간 동안 및 조건 하에서 배양하는 단계를 포함하는, Vero 세포에서 약독화된 RV3를 생성하는 방법이 제공된다.
Vero-적응된 RV 및 특히 Vero-적응된 RV3의 생성은 약독화된 살아있는 RV 백신으로의 사용을 가능하게 한다. 대안적으로, Vero 세포에서 생성되는 RV는 사멸될 수 있으며 사멸된 RV 백신으로 사용될 수 있다.
따라서, 본원에는 VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6에서 하나 이상의 돌연변이를 갖고, 하나 이상의 약제학적으로 허용되는 담체, 희석제 및/또는 부형제를 추가로 포함하는, 약독화된 살아있는 또는 사멸된 RV 조성물이 교시된다.
일 실시형태에서, MA104-적응된 RV3와 비교하여, NSP4에서 53, 76, 85, 162 및 165 중에서 선택되는 코돈, NSP5/6에서 46 및 112 중에서 선택되는 코돈, VP1에서 886 중에서 선택되는 코돈, VP3에서 643 및 785 중에서 선택되는 코돈, 및 VP4에서 43, 374, 385, 388, 442 및 756 중에서 선택되는 코돈 및/또는 NSP2에서 433 중에서 선택되는 뉴클레오타이드, NSP4에서 197, 267, 294, 525 및 534 중에서 선택되는 뉴클레오타이드, NSP5/6에서 158, 165 및 355 중에서 선택되는 뉴클레오타이드, VP1에서 2692 중에서 선택되는 뉴클레오타이드, VP3에서 343, 1366, 1744, 1976 및 2402 중에서 선택되는 뉴클레오타이드 및/또는VP4에서 136, 369, 1130, 1162, 1171, 1334 및 2276 중에서 선택되는 뉴클레오타이드에서의 돌연변이 중 하나 이상으로부터 선택되는 유전적 변형을 갖는 약독화되거나 사멸된 Vero-적응된 RV를 포함하고, 하나 이상의 약제학적으로 허용되는 담체, 희석제 및/또는 부형제를 추가로 포함하는, 조성물이 제공된다.
MA104-적응된 RV3와 비교하여 표 2 또는 표5의 리스트 중에서 선택되는 하나 이상의 돌연변이를 포함하는 살아있는 약독화된 또는 사멸된 RV3를 포함하는 조성물이 제공된다.
일 실시형태에서, RV는 1 내지 38개의 돌연변이, 예컨대 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 또는 38개의 돌연변이를 포함한 표 2 또는 표 5에 열거되는 하나 이상의 돌연변이를 포함한다.
조성물의 제조는 편리하게는 세계보건기구(WHO) 기준에 따른다. 일반적으로, 조성물은 3개월 이전 및 출생 후 실행가능한 한 빨리 경구 투여된다. 또한, 조성물은 백신, 제형, 약물 및 기타 용어로도 지칭될 수 있다.
백신의 목적하는 면역학적 또는 생리학적 효과는 RV 감염의 억제, RV에 의한 재감염의 방지, 표적 피험자로부터 RV의 정화 또는 RV 감염 증상, 예컨대 급성 설사 및/또는 체액 소실(탈수 포함)의 개선이다. 구체적으로, 효과는 RV 또는 RV 성분, 예컨대 단백질, 탄수화물 또는 지질에 대한 면역 반응 및 특히 체액성 면역 반응의 생성을 포함한다.
또한, 백신은 RV의 2개 이상의 상이한 균주 또는 2개 이상의 이의 항원성 또는 면역원성 성분을 포함할 수 있다. 또한, "배합"은, 바이러스 제제가 별개로 제공되고 별개로 투여 또는 적용되거나 적용 전에 함께 혼합되는, 다수-부분의 조성물, 예컨대2개 부분의 조성물을 포함한다. 예를 들어, 다수-부분 약제학적 팩은 별개로 유지되는 2개 이상의 살아있는 약독화된 또는 사멸된 RV 제제 또는 이의 성분을 가질 수 있다.
본원에 기재되는 바이러스 제제는 백신, 제형, 약물 또는 조성물로 지칭되는 상태로 유지될 수 있거나 사용 전에 제형으로 재구성되는 건조, 동결건조, 냉동 또는 탈수된 형태로 존재할 수 있다. 용어 "백신"은, 제형이 RV-특이적 항체를 포함할 수 있기 때문에, 제형을 면역 반응을 유도하는 제제로 제한하는 것으로 간주되지 않는다.
본원에는 인간 피험자에게 RV 감염에 대한 면역 반응을 생성하기에 충분한 시간 동안 및 조건 하에서 유효량의 살아있는 약독화된 Vero-적응된 RV3 또는 사멸된 Vero-적응된 RV3를 투여하는 단계를 포함하는, RV 감염에 대해 인간 피험자를 예방접종하는 방법이 본원에서 제공된다.
특히, 면역 반응은 방어적 면역 반응이다. 면역 반응은 RV의 비-약독화된(병독성) 균주에 의한 감염 또는 재감염 전, 동안 또는 이후 유용할 수 있다.
인간 피험자에게 RV 감염에 대한 면역 반응을 생성하기에 충분한 시간 동안 및 조건 하에서 유효량의 살아있는 약독화된 Vero-적응된 RV3 또는 사멸된 Vero-적응된 RV3를 투여하는 단계를 포함하는, RV 감염을 갖거나 RV 감염 또는 재감염 발병 위험이 있는 인간 피험자를 치료하는 방법이 본 개시에 의해 교시된다. 이러한 방법은 RV에 의해 유발되는 설사를 갖는 인간 피험자를 치료하는 것을 포함한다.
"유효량"은 "치료학적 유효량"을 포함한다.
본원에서 사용되는 바와 같은 백신 또는 본원에서의 제제의 "유효량" 및 "치료학적 유효량"이란 용어는 RV 감염의 감소 또는 RV-특이적 항체의 생성인 목적하는 치료학적 또는 생리학적 효과 또는 결과를 제공하기에 충분한 양의 제제(예: 살아있는 약독화된 RV 또는 사멸된 RV)를 의미한다. 바람직하지 못한 효과, 예를 들어, 부작용은 때때로 목적하는 치료 효과와 함께 나타나며; 따라서, 의사는 적정한 "유효량"을 결정함에 있어 잠재적 이점과 잠재적 위험 간에 균형을 맞춘다. 정확한 필요량은 피험자의 나이 및 일반적 상태, 투여 방식 등에 좌우되며, 피험자마다 상이할 것이다. 따라서, 정확한 "유효량"을 구체화하는 것은 가능하지 않을 수 있다. 그러나, 임의의 개별 경우에서 적합한 "유료향"은 단지 통상의 실험으로 당업자에 의해 결정될 수 있다.
따라서, 본원에서 사용되는 "유효량"은 적절한 투여 요법에 따라 투여되는 경우 RV 감염 감소, RV 감염 증상의 개선 및/또는 RV-특이적 항체의 생성이라는 목적하는 효과를 제공하는 바이러스 제제 또는 이의 성분의 양을 지칭한다. 투여는 수분, 수시간, 수일, 수주일 또는 수개월의 간격으로 일어날 수 있다. 적합한 투여 용량 및 요법은 담당의 또는 또는 다른 의료 제공자에 의해 결정될 수 있다. 예를 들어, 살아있는 약독화된 RV의 투여는 105 내지 108 ffu/ml, 예컨대 105, 106, 107 및 108 ffu/ml를 포함한다.
"약제학적으로 허용되는" 담체, 부형제 또는 희석제는 생물학적이지 않은 물질 또는 달리 목적하지 않는 물질로 구성된 약제학적 비히클을 의미하며, 상기 물질은 피험자에게 임의의 또는 실질적인 부작용을 일으키지 않으면서 선택된 바이러스 제제 또는 이의 성분과 함께 피험자에게 투여될 수 있다. 담체는 부형제 및 다른 첨가제, 예컨대 희석제, 세제, 착색제, 습윤 또는 유화제, pH 완충제, 보존제 등을 포함할 수 있다.
본원에서 사용되는 용어 "치료하는" 및 "치료"는 RV 감염 증상의 중증도 및/또는 횟수 감소, RV 감염 증상의 제거, RV 감염 증상의 발생 방지 및 RV 감염 후 증상의 호전, 치유 또는 개선을 지칭한다. 증상은 급성 또는 만성 설사, 체액 손실 및/또는 발열을 포함한다.
피험자를 "치료하는"은 RV 감염의 예방 및 병태 증상 개선에 의한 임상적으로 증상을 갖는 개체의 치료를 포함할 수 있다. 또한, 무증상 피험자의 치료도 고려된다. 또한, 용어 "치료하는"은 RV의 예방 및 RV에 대한 면역학적 기억의 유도에도 적용된다. 인간 피험자는 직접적으로 치료될 수 있거나, 임산부는 태아나지 않은 유아로 전달되는 목적하는 면역 반응으로 치료될 수 있다.
본원에서 사용되는 "피험자"는 일반적으로 본원에서 기재되는 약제학적 제형 및 방법으로부터 혜택을 받을 수 있는 인간을 지칭한다. 피험자는 특히 개체, 환자, 호스트 또는 수령자로 지칭될 수 있다.
인간 피험자는 임의의 연령일 수 있다. 특정한 감염되기 쉬운 인간의 예는 0 내지 5세 및 50세 이상, 예컨대 51세 내지 60세, 61세 내지 70세, 71세 내지 80세, 81세 내지 90세 및 91세 이상을 포함한다. 또한, 6세 내지 10세, 11세 내지 15세 및 16세 내지 20세의 어린이도 21세 내지 50세의 인간과 같이 고려될 수 있다.
또한, 실험실 시험 동물에서 실험적 예방접종 또는 항체 생성이 실시될 수 있다. 실험실 시험 동물의 예는 마우스, 래트, 토끼, 원숭이 동물, 기니아 피그, 햄스터 및 영장류, 예컨대 오랑우탄, 고릴라, 마모셋 및 리서스 원숭이를 포함한다. 토끼, 설치류 및 원숭이 동물은 특별히 편리한 시험 시스템 또는 동물 모델을 제공한다.
본원에서 제공되는 조성물은 경구 투여에 적합한 조성물을 포함하며, 각각 예정된 양의 RV 및/또는 이의 성분을 포함하는, 개별 단위, 예컨대 액제, 캡슐제 또는 사세이 또는 정제로서; 산제 또는 과립제로서; 수상 또는 비-수성 액체 중의 용액제 또는 현탁제로서; 또는 수중유 액체 에멀젼 또는 유중수 에멀젼으로서 제시될 수 있다. 또한, 활성 성분은 볼루스, 연약 또는 페이스트로 제시될 수 있다.
또한, 조성물은 항원성 조성물로서 지칭될 수 있다.
백신으로서 유용할 항원성 조성물을 위해, 상기 조성물은 바이러스 또는 이의 성분에 대해 면역 반응을 유도해야 한다. 본원에서 사용되는 "항원성 조성물"은 항원(예: VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6), 항원을 암호화하는 핵산(예: 항원 발현 벡터) 또는 항원 또는 살아있는 약독화된 또는 사멸된 바이러스를 발현 또는 제시하는 세포를 포함할 수 있다. 또 다른 실시형태에서, 조성물은 RV 및 추가의 면역자극제 또는 이러한 제제를 암호화하는 핵산을 포함하는 혼합물로 존재한다. 면역자극제는, 이로 제한됨이 없이, 추가의 항원 또는 바이러스 균주, 면역조절제, 항원 제시 세포 또는 애주번트를 포함한다.
일 실시형태에서, 조성물 또는 면역학적 작용 등가물은 인간 피험자에서 RV에 대한 체액성 및/또는 세포-매개된 면역 반응 유도 시 효과적인 백신으로 사용된다. 능동 및 수동 면역화 실시형태 모두에서 사용하기 위한 하나 이상의 항원성 조성물 또는 백신이 본원에서 고려된다.
백신의 조성은 다양할 수 있다. 물론, 본원에서 기재되는 다양한 조성물이 다수 성분들을 추가로 포함할 수 있다는 것을 알 수 있을 것이다. 예를 들어, 백신은 하나 이상의 애주번트애주번트를 포함할 수 있다. 백신은 본 개시에 비추어 볼 때 당업자에게 공지된 임의의 방법으로 제조 및/또는 투여될 수 있다.
면역조절제가 세포 또는 환자의 반응을 증강시키기 위해 백신에 포함될 수 있다는 것이 고려된다. 면역조절제는 정제된 단백질, 면역조절제를 암호화하는 핵산 및/또는 백신 조성물 중에서 면역조절제를 발현하는 세포로서 포함될 수 있다. 본원에서 고려되는 면역조절제의 비제한적 예는 인터류킨(Ls), 사이토킨, 인터류킨 또는 사이토킨을 암호화하는 핵산 및/또는 이러한 화합물을 발현하는 세포를 포함한다. 인터류킨 및 사이토킨은, 비제한적으로, iIL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-18, β-인터페론, α-인터페론, γ-인터페론, 안지오스타틴, 트롬보스폰딘, 엔도스타틴, GM-CSF, G-CSF, M-CSF, METH-1, METH-2, 종양괴사인자, TGFβ, LT 및 이의 배합을 포함한다.
또한, 케모킨, 케모킨을 암호화하는 핵산 및/또는 이들을 발현하는 세포가 백신 성분으로 사용될 수 있다. 케모킨은 일반적으로 면역 이펙터 세포를 케모킨 발현 부위로 소집하기 위한 화학유인제로서 작용한다. 다른 면역계 성분의 치료 부위로의 소집을 증진시키기 위해 특정 케모킨 암호화 서열을, 예를 들어, 사이토킨 암호화 서열과 함께 발현시키는 것이 유리할 수 있다. 이러한 케모킨은, 예를 들어, RANTES, MCAF, MIP1-알파, MIP1-β, IP-10 및 이의 배합을 포함한다. 당업자는 특정한 사이토킨이 또한 화학유인제 효과를 갖는 것으로 알려져 있고 또한 케모킨 용어 하에 분류될 수 있었다는 것을 인식할 것이다.
T 세포 면역을 상향조절하거나 억제제 세포 활성을 하향조절하는 것으로 알려진 생물학적 반응 조절제(BRM)을 공동-투여하는 것이 바람직할 수 있다. 이러한 BRM은, 비제한적으로, 시메티딘(CIM; 1200 mg/d) (Smith/Kline, PA); 저용량 사이클로포스파미드(CYP; 300 mg/m2) (Johnson/Mead, NJ), 또는 하나 이상의 면역 헬퍼 기능에 수반되는 단백질, 예컨대 B-7을 암호화하는 유전자를 포함한다.
반응을 자극하기 위해 애주번트애주번트를 사용하는 것을 포함하는 면역화 프로토콜이 본원에서 고려된다. 일부 애주번트애주번트는 항원이 제시되는 방식에 영향을 끼친다.
특정 실시형태에서, 애주번트 효과는 인산염 완충된 식염수 중 약 0.05 내지 약 0.1% w/v의 용액으로 사용되는 백반과 같은 제제의 사용에 의해 달성된다. 대안적으로, 항원은 약 0.25% w/v 용액으로 사용되는 슈가의 합성 중합체(카보폴[Registered Trade Mark])와의 혼합물로서 제조된다.
일부 애주번트는, 예를 들어, 세균으로부터 수득되는 특정 유기 분자이며, 항원보다는 호스트에 작용한다. 일례는 세균성 펩티도글리칸인 무라밀 디펩타이드(N-아세틸무라밀-L-알라닐-D-이소글루타민[MDP])이다. 대부분의 애주번트와 같이 MDP의 효과는 완전히 이해되지 않는다. MDP는 대식세포를 자극하나 또한 B 세포도 직접적으로 자극하는 것으로 보인다. 따라서, 애주번트의 효과는 항원-특이적이 아니다. 그러나, 이들은 정제된 항원과 함께 투여되는 경우, 항원에 대한 반응을 선택적으로 증진시키는데 사용될 수 있다.
또한, 다양한 폴리사카라이드 애주번트가 사용될 수 있다. 예를 들어, 폐렴구균의 폴리사카라이드 애주번트가 사용될 수 있다. 최적 반응을 생성하거나 달리 억제를 생성하지 않는 용량이 지시되는 바와 같이 사용되어야 한다. 디아세틸화된 키틴을 포함한 키틴 및 키토산과 같은 폴리사카라이드의 폴리아민 종류가 특히 바람직하다.
애주번트의 또 다른 그룹은 세균성 펩티도글리칸의 무라밀 디펩타이드(MDP, N-아세틸무라밀-L-알라닐-D-이소글루타민) 그룹이다. 또한, 무라밀 디펩타이드의 유도체, 예컨대 아미노산 유도체 트레오닐-MDP 및 지방산 유도체 MTPPE가 고려된다.
본 개시에서 사용하기 위해 고려되는 또 다른 애주번트는 BCG이다. 또한, BCG(바실러스 갈메테-구에린, 미코박테리움의 약독화된 균주) 및 BCG-세포벽 골격(CWS)가 트레할로즈 디미콜레이트의 존재 또는 부재 하에 애주번트로서 사용될 수 있다. 트레할로즈 디미콜레이트는 그 자체로 사용될 수 있다. 트레할로즈 디미콜레이트 투여는 마우스에서 인플루엔자 바이러스 감염에 대한 증강된 내성과 관련되는 것으로 밝혀졌다. 트레할로즈 디미콜레이트는 미국 특허 번호 4,579,945에 기재되는 바와 같이 제조될 수 있다.
BCG는 이의 면역자극 특성 때문에 중요한 임상적 수단이다. BCG는 망상내피계를 자극하도록 작용하고, 천연 킬러 세포를 활성화시키며, 조혈모세포의 증식을 증가시킨다. BCG의 세포벽 추출물은 뛰어난 면역 애주번트 활성을 갖는 것으로 입증되었다. 미코박테리아에 대한 분자 유전학적 도구 및 방법은 BCG로 외래 유전자를 도입하기 위한 수단을 제공하였다.
또 다른 양태는 피험자에서 RV 감염/재감염 치료 약물의 제조시 Vero-RV, 예컨대 Vero-RV3 또는 이의 성분의 용도에 관한 것이다.
또한, Vero-적응된 RV 제제에 대한 항체가 본원에서 고려된다.
"항체"란 항원(즉, RV 제제 또는 이의 성분)과 조합, 상호작용 또는 달리 결합할 수 있는 면역글로불린 패밀리의 단백질을 의미한다. 따라서, 항체는 항원-결합 분자이다. "항체"는 면역상호작용 분자의 일례이며, 폴리클로날 또는 모노클로날 항체를 포함한다. 특정 면역상호작용 분자는 진단 또는 정제 절차에 사용하기 위한 모노클로날 항체 또는 인간 피험자에서 유도되는 경우 폴리클로날 항체이다. 또한, 용어 "항체"는 2개의 상이한 RV 항원, 예컨대 VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6에 대한 이중특이적 항체와 같은 조작된 항체를 포함한다.
용어 "항원"은 본원에서 면역 반응에서 반응할 수 있고/있거나 면역 반응을 유도할 수 있는 물질을 지칭하기 위해 광의로 사용된다. "항원"의 언급은 전체 바이러스 또는 이의 성분을 포함한다.
면역화 및 후속의 모노클로날 항체의 생성은, 예를 들어, 문헌[Kohler and Milstein (Kohler et al. (1975) Nature 256:495-499 and Kohler et al. (1976) Eur. J. Immunol. 6(7):511-519; Coligan et al (1991-1997) Current Protocols in Immunology or Toyama et al. (1987) Monoclonal Antibody, Experiment Manual, published by Kodansha Scientific]에 기재되는 바와 같은 표준 프로토콜을 이용하여 수행될 수 있다. 근본적으로, 동물은 항체-생성 세포, 특히 항체-생성 체세포(예: B 림프구)를 생성하기 위해 표준 방법에 의해 Vero-적응된 RV 또는 이의 면역원성 단편으로 면역화된다. 이어서, 이들 세포는 불멸화를 위해 면역화된 동물로부터 적출될 수 있다. 항원은 먼저 캐리어와 결합되어야 할 수 있다.
"캐리어"란 비-면역원성 물질 또는 저조한 면역원성 물질(에: 합텐)이 이의 면역원성을 증진시키기 위해 천연적으로 또는 인위적으로 결합되는 전형적으로 고분자량인 임의의 물질을 의미한다.
항체-생성 세포의 불멸화는 당해 분야에 잘 알려진 방법을 이용하여 수행될 수 있다. 예를 들어, 불멸화는 엡스타인-바르 바이러스(EBV)를 사용하여 형질전환 방법에 의해 달성될 수 있다[Kozbor et al. (1986) Methods in Enzymology 121:140]. 일 실시형태에서, 항체-생성 세포는 모노클로날 항체의 생성을 위해 널리 이용되는 세포 융합 방법[Coligan et al. (1991-1997) 상기 참조]을 이용하여 불멸화된다. 이러한 방법에서, 항체, 특히 B 세포를 생성할 잠재력을 갖는 항체-생성 체세포는 골수종 세포주와 융합된다. 이들 체세포는 프라이밍된 동물, 바람직하게는 설치류 동물, 예컨대 마우스 및 래트의 림프절, 비장 및 말초혈로부터 유도될 수 있다. 예시적 실시형태에서, 마우스 비장 세포가 사용된다. 그러나, 래트, 토끼, 양 또는 염소 세포 또는 다른 동물종으로부터의 세포가 대신 사용될 수 있을 것이다.
특화된 골수종 세포주는 하이브리도마-생성 융합 절차에 사용하기 위해 림프구성 종양으로부터 개발되었다[Kohler et al. (1976) 상기 참조; Kozbor et al. (1986) 상기 참조; 및 Volk et al. (1982) J. Virol. 42(1):220-227]. 이들 세포주는 적어도 3가지 이유로 개발되었다. 첫 번째는 비융합되고 유사하게 무한히 자가-증식하는 골수종 세포로부터 융합된 하이브리도마의 선별을 용이하게 하는 것이다. 보통, 이것은 하이브리도마의 성장을 지지하는 특정한 선별 배지에서 성장할 수 없게 만드는 효소 결핍을 갖는 골수종을 사용하여 달성된다. 두 번째는 자신의 항체를 생성하는 림프구성 종양 세포의 내재적 능력으로부터 기인한다. 하이브리도마에 의한 종양 세포 항체 생성을 제거하기 위해서, 내인성 면역글로불린 경쇄 또는 중쇄를 생성할 수 없는 골수종 세포주가 사용된다. 이들 세포주 선택에 대한 세 번째 이유는 융합 적합성 및 효율 때문이다.
예를 들어, P3X63-Ag8, P3X63-AG8.653, P3/NS1-Ag4-1 (NS-1), Sp2/0-Ag14 및 S194/5.XXO.Bu.1을 포함한 많은 골수종 세포주가 융합된 세포 하이브리드의 생성에 사용될 수 있다. P3X63-Ag8 및 NS-1 세포주는 Kohler 및 Milstein에 의해 기재되었다[Kohler et al. (1976) 상기 참조]. Shulam 등[Shulman et al. (1978) Nature 276:269-270]은 Sp2/0-Ag14 골수종 주를 개발하였다. S194/5.XXO.Bu.1 주는 문헌[Trowbridge (1982) J. Exp. Med. 148(1):220-227]에 보고되었다.
항체-생성 비장 또는 림프절 세포와 골수종 세포의 하이브리드를 생성하는 방법은 세포막 융합을 촉진시키는 (화학적, 바이러스 또는 전기적) 제제 또는 제제들의 존재 하에서 체세포와 골수종 세포를 (약 20:1 내지 약 1:1로 다양할 수는 있으나) 10:1의 비율로 혼합하는 것을 포함한다. 융합 방법은 문헌[Kohler et al. (1975) 상기 참조; Kohler et al. (1976) 상기 참조; Gefter et al. (1977) Somatic Cell Genet. 3:231-236; 및 Volk et al. (1982) 상기 참조]에 기재되어 있다. 연구자들에 의해 사용되는 융합-촉진제는 센다이 바이러스 및 폴리에틸렌 글리콜(PEG)이었다.
융합 절차는 매우 낮은 빈도로 가시적 하이브리드를 생성하기 때문에(예를 들어, 비장이 체세포 공급원으로 사용되는 경우, 대략 1x105개의 비장 세포에 대해 단지 하나의 하이브리드가 수득된다), 남아 있는 비융합된 세포, 특히 비융합된 골수종 세포로부터 융합된 세포 하이브리드를 선별하는 수단을 갖는 것이 유용하다. 또한, 다른 생성된 융합된 세포 하이브리드 중에서 목적하는 항체-생성 하이브리도마를 검출하는 수단이 필요하다. 일반적으로, 융합된 세포 하이브리드의 선별은 하이브리도마의 성장은 지지하지만, 통상 무한정 분열하는 비융합된 골수종 세포의 성장은 방지하는 배지에서 세포를 배양함으로써 달성된다. 융합에 사용되는 체세포는 시험관내 배양에서 장기간 생존을 유지하지 못하므로 문제가 되지는 않는다.
융합된 세포 하이브리드를 선별적으로 배양하는데 수주가 걸린다. 이러한 시기 중 초기에 목적하는 항체를 생성하는 하이브리드를 확인해야 하며, 이로써 이들은 후속적으로 클로닝되고 증식될 수 있다. 비록 약 1 내지 30%가 드물지는 않으나, 일반적으로, 수득된 하이브리드의 약 10%가 목적하는 항체를 생성한다. 예를 들어, 미국 특허 번호 6,056,957에 기재되어 있는 바와 같이, 항체-생성 하이브리드의 검출은 효소-결합된 면역검정 및 방사선면역검정 기술을 포함한 수개의 표준 검정 방법 중 임의의 것에 의해 달성될 수 있다.
일단 목적하는 융합된 세포 하이브리드가 선별되고 각각의 항체-생성 세포주로 클로닝되면, 각각의 세포주는 2개의 표준 방식 중 임의의 방식으로 증식될 수 있다. 하이브리도마 세포의 현탁물은 조직적합 동물로 주사될 수 있다. 이어서, 주사된 동물은 융합된 세포 하이브리드에 의해 생성되는 특이적 모노클로날 항체를 분비하는 종양을 전개시킬 것이다. 동물의 체액, 예컨대 혈청 또는 복수액이 모노클로날 항체를 고농도로 제공하도록 태핑(tapping)될 수 있다. 대안적으로, 개별 세포주는 시험관내 실험실 배양 용기에서 증식될 수 있다. 단일의 특이적 모노클로날 항체를 포함하는 고농도의 배양 배지는 경사분리, 여과 또는 원심분리에 의해 수거된 후 정제될 수 있다.
세포주는 임의의 적합한 면역검출 수단에 의해 목적하는 바이러스 또는 바이러스 항원을 검출하기 위한 특이성에 대해 시험된다. 예를 들어, 세포주는 다수의 웰에 나누어 담기고, 항온처리되며, 각각의 웰로부터의 상등액이 효소-결합된 면역흡착 검정(ELISA), 간접적 형광 항체 기술 등에 의해 분석된다. 표적 항원은 인식할 수 있으나 비-표적 에피토프는 인식하지 않는 모노클로날 항체를 생성하는 세포주(들)가 확인되며, 이어서 직접적으로 시험관내에서 배양되거나 조직적합 동물에 주사되어 종양을 형성하고 필요한 항체를 생성하고 수집하며 정제한다.
따라서, 진단 또는 예후에 사용하기 위해 또는 RV 감염, 유지 또는 어셈블리를 감소시키는데 치료학적으로 사용하기 위해 Vero-적응된 RV, 예컨대 Vero-적응된 RV3 또는 이의 성분과 특이적으로 상호작용하는 폴리클로날 및 모노클로날 항체가 제공된다.
상기에서 언급되는 제1 기재된 항체에 대한 임의의 제2 항체(모노클로날, 폴리클로날 또는 항체 또는 합성 항체의 단편)를 포함하는 것이 본 개시의 범위에 속한다. 제1 항체와 제2 항체 모두가 검출 검정에 사용될 수 있거나, 제1 항체가 시판되는 항-면역글로불린 항체와 함께 사용될 수 있다. 따라서, RV 감염, 재감염, 감염 병력, 백신의 통합성을 모니터링하고 치료적 개입을 모니터링하기 위한 진단 및 예후 검정이 본원에 제공된다.
면역검정에서 모노클로날 항체의 사용은, 모노클로날 항체를 다량으로 생성하는 능력 및 생성물의 균일성 때문에, 특히 바람직하다. 불멸 세포주와 면역원성 제제에 대해 감작화된 림프구의 융합에 의해 유도되는 모노클로날 항체 생성을 위한 하이브리도마 세포주의 제조는 당업자에게 잘 알려진 기술로 수행될 수 있다.
따라서, 또 다른 양태는 생물학적 샘플을 항체-폴리펩타이드 복합체가 생성되기에 충분한 시간 동안 및 조건 하에서 RV 또는 이의 성분 또는 이의 유도체 또는 상동체에 대해 특이적인 항체와 접촉시키는 단계 및 이어서 복합체를 검출하는 단계를 포함하는, 피험자로부터의 생물학적 샘플 중에서 Vero-적응된 RV, 예컨대 RV3-Vero 또는 이의 성분을 검출하는 방법을 제공한다.
생물학적 샘플은 대변 샘플을 포함한다.
따라서, Vero-적응된 RV 또는 항체의 존재는 다수의 방식, 예컨대 웨스턴 블롯팅, ELISA 절차 및 RIP에 의해 검출될 수 있다. 미국 특허 번호 4,016,043, 4,424,279 및 4,018,653을 참조하여 알 수 있는 바와 같이 다양한 면역검정 기술이 이용될 수 있다.
샌드위치 검정이 특히 가장 유용하고 일반적으로 이용되는 검정이며, 본 개시에서 사용하기에 유리하다. 샌드위치 검정 기술에 대한 다수의 변형이 존재하며, 모두 본 개시에 의해 포함시키고자 한다. 간단히 설명하면, 전형의 전향 검정에서, 비표지된 항체가 고체 기체 상에 고정되고, RV 또는 RV 항원("항원")에 대해 시험될 샘플이 결합된 분자와 접촉된다. 적합한 항온처리 기간 후, 이어서 항체-항원 복합체를 형성하기에 충분한 시간 동안, 항원에 대해 특이적이고 검출가능한 시그널을 생성할 수 있는 리포터 분자로 표지된 제2 항체를 첨가하고, 항체-항원-표지된 항체의 또 다른 복합체를 형성하기 위한 충분한 시간이 가능하게 항온처리한다. 임의의 비반응된 물질을 세척해내고, 항원의 존재를 리포터 분자에 의해 생성되는 시그널 관측에 의해 측정한다. 그 결과는 가시적 시그널의 단순 관측에 의한 정성적인 것일 수 있거나, 공지된 양의 합텐을 포함하는 대조용 샘플과의 비교에 의해 정량될 수 있다. 전향 검정에 대한 변형은 샘플과 표지된 항체 모두가 결합된 항체에 동시에 첨가되는 동시적 검정을 포함한다. 쉽게 분명해질 수 있는 임의의 사소한 변형을 포함하는 이들 기술들은 당업자에게 잘 알려져 있다. 본 개시에 따라, 샘플은 조직 생검, 대변 샘플, 혈액, 활액 및/또는 림프를 포함하는 Vero-적응된 RV 또는 이에 대한 항체를 포함할 수 있는 것이다. 따라서, 샘플은 일반적으로 생물학적 유체를 포함하는 생물학적 샘플이다.
전형의 전향 샌드위치 검정에서, RV 또는 이의 항원성 부분에 대해 특이성을 갖는 제1 항체는 고체 표면에 공유적으로 또는 수동적으로 결합된다. 고체 표면은 전형적으로 유리 또는 중합체이며, 가장 일반적으로 사용되는 중합체는 셀룰로오스, 폴리아크릴아미드, 나일론, 폴리스티렌, 폴리비닐 클로라이드 또는 폴리프로필렌이다. 고체 지지체는 튜브, 비드, 마이크로플레이트의 디스크 또는 면역검정을 수행하기에 적합한 임의의 다른 표면의 형태일 수 있다. 결합 과정은 당해 분야에 잘 알려져 있으며, 일반적으로 가교-결합, 공유 결합 또는 물리적 흡수로 이루어지며, 중합체-항체 복합체는 시험 샘플의 제조시 세척된다. 이어서, 시험될 샘플의 분취물을 고체상 복합체에 첨가하고, 항체에 존재하는 임의의 아단위가 결합하기에 충분한 시간 동안(예: 2 내지 40분 또는 더욱 편리한 경우 밤새) 및 적합한 조건 하에(예: 약 20℃ 내지 약 40℃ 동안) 항온처리한다. 항온처리 기간 후, 항체 아단위 고체상을 세척하고, 건조시키며, 합텐의 일부에 특이적인 제2 항체와 함께 항온처리한다. 제2 항체는 합텐에 대한 제2 항체의 결합을 나타내는데 사용되는 리포터 분자에 결합된다.
대안적인 방법은 RV 항원을 고정시킨 후 고정된 표적물을 혈청 또는 혈장에 노출시키는 것을 포함한다. 표적물의 양 및 리포터 분자 시그널의 세기에 따라, 결합된 표적물은 항체를 사용한 직접적 표지화에 의해 검출될 수 있다. 대안적으로, 제1 항체에 대해 특이적인 제2의 표지된 항체는 표적물-제1 항체 복합체에 노출되어 표적물-제1항체-제2 항체의 3차 복합체를 형성한다. 복합체는 리포터 분자에 의해 방출되는 시그널에 의해 검출된다.
본 명세서에서 사용되는 "리포터 분자"란 이의 화학적 특성에 의해 항원-결합된 항체의 검출을 가능하게 하는 분석적으로 확인가능한 시그널을 제공하는 분자를 의미한다. 검출은 정성적 또는 정량적일 수 있다. 이러한 유형의 검정에서 가장 일반적으로 사용되는 리포터 분자는 효소, 형광단 또는 방사성핵종 포함 분자(즉, 방사성동위원소) 및 화학발광 분자이다. 효소 면역검정의 경우, 효소는 일반적으로 글루타르알데하이드 또는 페리오데이트에 의해 제2 항체에 접합된다. 그러나, 쉽게 인식될 수 있는 바와 같이, 당업자에 의해 쉽게 이용가능한 매우 다양한 상이한 접합 기술이 존재한다. 일반적으로 사용되는 효소는 특히 서양고추냉이 퍼옥시다제, 글루코즈 옥시다제, 베타-갈락토시다제 및 알칼리 포스파타제를 포함한다. 특정 효소와 함께 사용되는 기질은 일반적으로 상응하는 효소에 의한 가수분해시 검출가능한 색상 변화를 생성하기 위해 선택된다. 적합한 효소의 예는 알칼리 포스파타제 및 퍼옥시다제를 포함한다. 또한, 상기 언급되는 색소원 기질보다는 형광 생성물을 생성하는 형광원 기질을 사용할 수 있다. 모든 경우에, 효소-표지된 항체를 제1 항체-항원 복합체에 첨가하고, 결합시킨 후, 과량의 시약을 세척하여 제거시킨다. 이어서, 적합한 기질을 포함하는 용액을 항체-항원-항체의 복합체에 첨가한다. 기질은 제2 항체에 결합된 효소와 반응하여 정성적인 시각적 시그널을 생성하고, 이는 통상적으로 분광광도법으로 더욱 정량되어 샘플 중에 존재하였던 항원의 양을 나타낼 수 있다. 또한, "리포터 분자"는 세포 응집 또는 응집 억제, 예컨대 라텍스 비드 상의 적혈구 등의 사용까지 미친다.
대안적으로, 형광 화합물, 예컨대 플루오레세인 및 로다민은 이들의 결합 능력을 변화시키지 않으면서 항체에 화학적으로 커플링될 수 있다. 특정 파장의 광을 사용한 조사로 활성화되는 경우, 형광색소-표지된 항체는 광 에너지를 흡수하여 분자 내에 흥분 상태를 유도하고, 이어서 광학 현미경으로 가시적으로 검출가능한 특징적인 색상의 광을 방출한다. 형광 표지된 항체는 제1 항체-합텐 복합체에 결합되게 된다. 비결합된 시약을 세척하여 제거한 후, 남아있는 3차 복합체를 적합한 파장의 광에 노출시키고, 관측되는 형광으로 목적하는 합텐의 존재를 나타낸다. 면역형광 및 EIA 기술은 모두 당해 분야에 매우 잘 확립되어 있으며, 본 방법에 특히 바람직하다. 그러나, 다른 리포터 분자, 예컨대 방사성동위원소, 화학발광 또는 생물발광 분자도 또한 사용될 수 있다.
또한, 유전적 검정, 예컨대 Vero-적응된 RV, 예컨대 Vero-RV3을 검출하기 위해 PCR 분석을 수반하는 유전적 검정이 수행될 수 있으며, 본원에 포함된다. 이러한 유전적 검정은 본원에서 고려되는 뉴클레오타이드 변화를 검출하는데 유용하다.
또한, 키트, 예컨대 구획이 항체를 포함하는 시약, PCR 성분, 리포터 분자로부터의 기질 및/또는 고정화된 항체 또는 항원을 포함하는 고체 지지체를 포함하는 구획이 있는 형태의 키트가 제공된다. 또한, 일 세트의 설명서가 포함될 수 있다.
실시예
본 기술에 의해 교시되는 양태는 하기 비제한적 실시예에 의해 더욱 설명된다.
실시예 1
Vero 세포에서 로타바이러스 백신의 배양 절차
하기는 로타바이러스(RV3)를 Vero 세포에 적응시키는 방법을 기재한다.
1-1 재료
1. 바이러스 생성 혈청 비함유 배지(VP-SFM) GIBCO #11681.
2. L-글루타민(0.2M) ICN Biomedicals Inc. #16-801-49 use @ 9ml/litre
3. 페니실린/스트렙토마이신(100X)CSL use @100 IU/ml/
4. 트립신 1X Sigma Chemicals #T-7418(활성화에는 50?/ml를 사용하고 유지 배지에는 5?/ml를 사용).
5. RV3 로타바이러스: 로타바이러스의 RV3 균주는 혈청형 G3P2A[6], 아그룹 II, 긴 일렉트로페로타입(무증상 감염을 갖는 신생아로부터 분리됨)이다. 적합한 RV3 균주는 Hu/Australia/10-25-10/77/L(ATCC 확인 번호 VR2104, 기탁일: 1985년 2월 1일) 및 Hu/Australia/1-9-12/77/S(ATCC 확인 번호 R2105, 기탁일: 1985년 2월 1일)를 포함한다.
6. Vero 세포
1-2 초기 바이러스 접종물의 제조
RV3를 Vero 세포 감염 전에 트립신처리하였다. 간단히 설명하면, 튜브당 200?의 RV3 백신 스톡을 30분 동안 37℃에서 20?의 0.5mg/ml 트립신과 함께 항온처리한 후, 세포 배양 제조가 끝날 때까지 4℃에 두었다. 이는 세포로의 감염을 위해 RV를 활성화시키는데 일반적으로 사용되는 것보다 높은 농도이다. 표준 트립신 활성화는 5 내지 15μg/m의 돼지 트립신을 사용하여 로타바이러스 VP4 단백질을 절단함으로써 바이러스를 활성화되게 하고 종국적으로는 세포에 결합하고 들어가서 복제하게 한다. 이러한 과정에서, RV3BB를 활성화시키기 위한 표준량의 3배 초과량이 사용된다.
1-3 Vero 세포의 제조 및 RV3를 갖는 롤러 튜브의 항온처리
플라스크 중의 Vero 세포를 PBS로 2회 세척하고, 표준 세포 배양 프로토콜을 이용하여 트립신처리한 후 현탁물로 두었다. 트립신처리된 Vero 세포를 50 ml의PBS에 재현탁시키고, 1700rpm에서 10분 동안 원심분리하였다. 상등액을 제거하고, 펠렛화된 Vero 세포를 세척하고, 상기한 바와 같이 PBS 중에서 재원심분리하였다. 세포수를 세고, Vero 세포를 VP-SFM-T.(즉, VP-SFM + 5ug/ml 트립신) 중에 7x105개 세포/ml로 희석하였다. 총 약 2x106개의 세포(2.5 ml의 세포 현탁물)를 200ul의 트립신처리된 바이러스 현탁물 또는 대조군으로서 배지 및 트립신을 사용한 "모의-감염물"(VP-SFM-T)을 갖는 각각의 멸균 롤러 조직 배양 튜브에 두었다. 바이러스 및 세포를 37℃/5% v/v CO2/95% v/v 공기에서 롤러 기구 상에서 항온처리하였다. 24시간 후, 트립신처리되고 세척된 Vero 세포의 추가의 분취물을 가하였다(100ul 총 용적 VP-SFM 중 2x106개 세포). 감염을 진행시키고, 감염 40시간 후 바이러스를 수거하였다. RV3 바이러스를 수거하기 위해서, 세포를 2라운드의 냉동(-70℃) 및 해동으로 용해시켰다. 바이러스를 저속으로 원심분리시켜 세포 파편을 제거시키고, 정제된 바이러스 현탁물을 -70℃에서 저장하였다.
바이러스를 MA104 세포에서의 표준 절차로 적정하여 감염 병소의 수를 측정할 수 있다.
실시예 2
RNA 추출 및 RT-PCR 증폭 및 서열분석 방법
RNA 추출. 바이러스 RNA를 QIAamp Viral RNA 미니키트(QIAGEN, Westburg, Leusden, The Netherlands)를 사용하여 제조자의 프로토콜에 따라 Vero-적응된 RV-3 균주로부터 추출하였다.
RT-PCR. 역전사효소 PCR(RT-PCR)을 Invitrogen 원-스톱 RT-PCR 키트(Invitrogen, Carlsbad, California, USA)를 사용하여 수행하였다. 로타바이러스 유전자의 증폭에 사용된 프라이머가 표 1에 열거되어 있다. RT-PCR을 45oC에서 30분의 초기 역전사 후 95oC에서 15분 동안 PCR 활성화로 수행하였다. 증폭은 VP6, VP7, NSP1-5에 대해서는 94oC에서 45초, 45oC에서 45초 및 70oC에서 75초 및 VP1-4에 대해서는 94oC에서 45초, 45oC에서 45초 및 70oC에서 3분의 40주기로 구성되었다. 최종 연장은 70oC에서 7분으로 수행되었다. 앰플리콘을 전기영동에 의해 분리하고, 적합한 DNA 겔을 절단하여 하기 변형과 함께 제조자의 기재에 따라 QIAquick 겔 추출 키트(QIAGEN)를 사용하여 정제하였다. 단계 5에서 이소프로판올을 유화된 겔 DNA 혼합물에 가하는 것을 생략하였으며, 미량의 아가로즈를 제거하기 위한 추가의 세척 단계인 단계 9에서 제조자에 의한 선택사양으로서의 표지화를 포함시켰다.
서열분석. 서열분석은 Big Dye 버젼 3.1(ABI)을 사용하여 제조자의 방법에 따라 수행하였다. 또한, 표 4에 열거된 프라이머를 서열분석에 사용하였다. 추가의 내부 프라이머가 필요한 경우 사용되었다.
유전자 | 프라이머명 | 프라이머 서열 (5'→ 3') | 서열번호 |
VP1 | JCVI_VP1_5'UTR | TGTAAAACGACGGCCAGTGGCTATTAAAGCTGTAC | 서열번호 1 |
GEN-VP1R | GGTCACATCCTAAGCCGYTC | 서열번호 2 | |
VP2 | GEN-VP2F | GGCTATTRAAGGYTCAATGG | 서열번호 3 |
GEN-VP2R | GGTCATATCTCCACARTGG | 서열번호 4 | |
VP3 | GEN-VP3F | GGCTWTTAAAGCAGTACCA | 서열번호 5 |
GEN-VP3R | GGYCACATCATGACTAGTG | 서열번호 6 | |
VP4 | JP_1_For_g4_RV3 | GGCTATAAAATGGCTTCACTC | 서열번호 7 |
JCVI_VP4_3'UTR | CAGGAAACAGCTATGACCGGTCACATYCCTCRATR | 서열번호 8 | |
VP6 | GEN-VP6F | GGCTTTWAAACGAAGAAGTCTT | 서열번호 9 |
GEN-VP6R | GGT CAC ATC CTC TCA CT | 서열번호 10 | |
VP7 | Beg9 | GGCTTTAAAAGAGAGAATTTCCGTCTGG | 서열번호 11 |
End9 | GGTCACATCATACAATTCTAATCTAAG | 서열번호 12 | |
NSP1 | JCVI_NSP1_5'UTR | TGTAAAACGACGGCCAGTTATGAAAAGTCTTGTGGAAGC | 서열번호 13 |
JCVI_ NSP1_3'UTR | CAGGAAACAGCTATGACCCATTTTATGCTGCCTAGGCG | 서열번호 14 | |
NSP2 | GEN-NSP2F | GGCTTTTAAAGCGTCTCAG | 서열번호 15 |
GEN-NSP2R | GGTCACATAAGCGCTTTC | 서열번호 16 | |
NSP3 | GEN-NSP3F | GGCTTTTAATGCTTTTCAGTG | 서열번호 17 |
GEN-NSP3R | ACATAACGCCCCTATAGC | 서열번호 18 | |
NSP4 | 10.1 | GGCTTTTAAAAGTTCTGTCC | 서열번호 19 |
10.2 | GGTCACACTAAGACCATTCC | 서열번호 20 | |
NSP5 | GEN-NSP5F | GGCTTTTAAAGCGCTACAG | 서열번호 21 |
GEN-NSP5R | GGTCACAAAACGGGAGT | 서열번호 22 |
실시예 3
MA104 적응된 RV3와 비교하여 RV3-Vero에서 뉴클레오타이드 및 아미노산 변화 확인
독특한 뉴클레오타이드 변화가 MA104 세포에서 유래하는 RV3와 비교하여 RV3-Vero에서 확인되었다(도 1 내지 42 및 표 2 및 5).
독특한 뉴클레오타이드 변화가 NSP4에서 53, 76, 85, 162 및 165 중에서 선택되는 코돈, NSP5/6에서 46 및 112 중에서 선택되는 코돈, VP1에서 886 중에서 선택되는 코돈, VP3에서 643 및 785 중에서 선택되는 코돈, 및 VP4에서 43, 374, 385, 388, 442 및 756 중에서 선택되는 코돈 및/또는 NSP2에서 433 중에서 선택되는 뉴클레오타이드, NSP4에서 197, 267, 294, 525 및 534 중에서 선택되는 뉴클레오타이드, NSP5/6에서 158, 165 및 355 중에서 선택되는 뉴클레오타이드, VP1에서 2692 중에서 선택되는 뉴클레오타이드, VP3에서 343, 1366, 1744, 1976 및 2402 중에서 선택되는 뉴클레오타이드 및/또는VP4에서 136, 369, 1130, 1162, 1171, 1334 및 2276 중에서 선택되는 뉴클레오타이드에 위치되었다. MA104-RV3와 비교하여 Vero-RV3에서의 특이적인 뉴클레오타이드 돌연변이가 표 2 및 5에 나타나 있다.
단백질 (bp-크기) | 서열분석된 nt 영역* | 뉴클레오타이드 치환 1 | 아미노산 치환 |
VP1 (3268bp) | 37-3303 | 백신 2692 T 대 MA104 2656 G | 백신 886 F 대 MA104 886 V |
VP2 (2717bp) | 17-2686 | 없음 | 없음 |
VP3 (2591bp) | 50-2554 | 백신 343 C 대 MA104 294T 백신 1366 G 대 MA104 1317 A 백신 1744 C 대 MA104 1695 T 백신 1976 A 대 MA104 1927 G 백신 2402 C 대 MA104 2353 T |
백신 643 K 대 MA104 643 E 백신 785 H 대 MA104 785 Y |
VP4 (2359bp) | 10-2334 | 백신 136 A 대 MA104 127C 백신 369 G 대 MA104 360A 백신 1130 C 대 MA104 1121T 백신 1162 A 대 MA104 1153 G 백신 1171 C 대 MA104 1162 A 백신 1334 A 대 MA104 1325 G 백신 2276 T 대 MA104 2267 A |
백신 43 T 대 MA104 43 P 백신 374 S 대 MA104 374 L 백신 385 N 대 MA104 385 D 백신 388 L 대 MA104 388I 백신 442 H 대 MA104 442 R 백신 756I 대 MA104 756N |
VP6 (1359bp) | 27-1217 | 없음 | 없음 |
VP7 ((1062bp) | 49-1026 | 없음 | 없음 |
NSP1 (1461bp) | 47-1498 | 없음 | 없음 |
NSP2 (1059bp) | 47-997 | 백신 433 A 대 MA104 387 G | 없음 |
NPS3 (1070bp) | 35-964 | 없음 | 없음 |
NSP4 (750bp) | 41-565 | 백신 197 A 대 MA104 157 G 백신 267 C 대 MA104 227 T 백신 294 A 대 MA104 254 G 백신 525 G 대 MA104 485 T 백신 534 T 대 MA104 494 C |
백신 53 T 대 MA104 53 A 백신 76 T 대 MA104 761 백신 85 Y 대 MA104 85 C 백신 162 G 대 MA104 162 V 백신 165 L 대 MA104 165P |
NSP5/6 (664bp) | 22-612 | 백신 158 A 대 MA104 137G 백신 165 C 대 MA104 144 T 백신 355 A 대 MA104 334 T |
백신 46 E 대 MA104 46 G 백신 112 I 대 MA104 112 L |
1 각각의 유전자의 암호화 영역에 대한 뉴클레오타이드 서열은 문헌[Rippinger et al, 2010 상기 참조]에 의해 공개된 MA104-적응된 RV3 바이러스의 서열과 비교되었다.
당업자는 본원에 기재된 본 발명의 양태가 구체적으로 기재된 양태와 다르게 변화 및 변형될 수 있다는 것을 알 수 있을 것이다. 이러한 양태가 모든 이러한 변화 및 변형을 포함한다는 것을 알아야 한다. 본 기술은 본 명세서에서 개별적으로 또는 총체적으로 언급되거나 지시되는 단계, 특징, 조성물 및 화합물, 및 상기 단계 또는 특징 중 임의의2개 이상의 것의 임의의 모든 조합을 교시한다.
참고문헌
Albert et al. (1983) J. Clin. Microbiol 17:162-164
Bern and Glass (1994) Impact of diarrheal diseases worldwide In Kapikian AZ (ed) Viral Infections of the grastrointestinal tract, 2nd edition, New York Marcel Dekker, Inc.:1-26
Coligan et al. (1991-1997) Current Protocols in Immunology
Gefter et al. (1977) Somatic Cell Genet. 3:231-236
Jiang et al. (2002) Clin Infect Dis 34:1351-1361
Kohler et al. (1975) Nature 256:495-499
Kohler et al (1976) Eur. J. Immunol. 6(7):511-519
Kozbor et al. 1986) Methods in Enzymology 121:140
Rippinger et al. (2010) Virology 405(1):1201-1213
Rodger et al. (1981) J. Clin. Microbiol 13:272-278
Sato et al. (1981) Arch. Virol. 69:155-160
Toyama et al. (1987) Monoclonal Antibody, Experiment Manual, published by Kodansha Scientific
Trowbridge (1982) J. Exp. Med. 148(1):220-227
Volk et al. (1982) J. Virol. 42(1):220-227
<110> MURDOCH CHILDRENS RESEARCH INSTITUTE
<120> MODIFIED HUMAN ROTAVIRUSES AND USES THEREFOR
<130> IPA150140-AU
<150> AU 2012903702
<151> 2012-02-27
<160> 66
<170> KopatentIn 2.0
<210> 1
<211> 35
<212> DNA
<213> artificial
<220>
<223> Primer - JCV1 VP1 5'UTR
<400> 1
tgtaaaacga cggccagtgg ctattaaagc tgtac 35
<210> 2
<211> 18
<212> DNA
<213> artificial
<220>
<223> Primer - GEN-VP1R
<400> 2
ggtcacatct aagcgytc 18
<210> 3
<211> 20
<212> DNA
<213> artificial
<220>
<223> Primer - GEN-VP2F
<400> 3
ggctattraa ggytcaatgg 20
<210> 4
<211> 19
<212> DNA
<213> artificial
<220>
<223> Primer - GEN-VP2R
<400> 4
ggtcatatct ccacartgg 19
<210> 5
<211> 19
<212> DNA
<213> artificial
<220>
<223> Primer - GEN-VP3F
<400> 5
ggctwttaaa gcagtacca 19
<210> 6
<211> 19
<212> DNA
<213> artificial
<220>
<223> Primer - GEN-VP3R
<400> 6
ggycacatca tgactagtg 19
<210> 7
<211> 21
<212> DNA
<213> artificial
<220>
<223> Primer - JP 1 for g4 RV3
<400> 7
ggctataaaa tggcttcact c 21
<210> 8
<211> 35
<212> DNA
<213> artificial
<220>
<223> Primer - JCV1 VP3 3'UTR
<400> 8
caggaaacag ctatgaccgg tcacatycct cratr 35
<210> 9
<211> 22
<212> DNA
<213> artificial
<220>
<223> Primer - GEN VP6F
<400> 9
ggctttwaaa cgaagaagtc tt 22
<210> 10
<211> 17
<212> DNA
<213> artificial
<220>
<223> Primer - GEN VP6R
<400> 10
ggtcacatcc tctcact 17
<210> 11
<211> 28
<212> DNA
<213> artificial
<220>
<223> Primer - Beg9
<400> 11
ggctttaaaa gagagaattt ccgtctgg 28
<210> 12
<211> 27
<212> DNA
<213> artificial
<220>
<223> Primer - End9
<400> 12
ggtcacatca tacaattcta atctaag 27
<210> 13
<211> 39
<212> DNA
<213> artificial
<220>
<223> Primer - JCV1 NSP1 5'UTR
<400> 13
tgtaaaacga cggccagtta tgaaaagtct tgtggaagc 39
<210> 14
<211> 38
<212> DNA
<213> artificial
<220>
<223> Primer - JCV1 NSP1 3'UTR
<400> 14
caggaaacag ctatgaccca ttttatgctg cctaggcg 38
<210> 15
<211> 19
<212> DNA
<213> artificial
<220>
<223> Primer - GeN-NSP2F
<400> 15
ggcttttaaa gcgtctcag 19
<210> 16
<211> 18
<212> DNA
<213> artificial
<220>
<223> Primer - GEN-NSP2R
<400> 16
ggtcacataa gcgctttc 18
<210> 17
<211> 21
<212> DNA
<213> artificial
<220>
<223> Prinmer - GEN-NSP3F
<400> 17
ggcttttaat gcttttcagt g 21
<210> 18
<211> 18
<212> DNA
<213> artificial
<220>
<223> Primer - GEN-NSP3R
<400> 18
acataacgcc cctatagc 18
<210> 19
<211> 21
<212> DNA
<213> artificial
<220>
<223> Primer - 10.1
<400> 19
ggcttttaaa agttctgttc c 21
<210> 20
<211> 20
<212> DNA
<213> artificial
<220>
<223> Primer - 10.2
<400> 20
ggtcacacta agaccattcc 20
<210> 21
<211> 18
<212> DNA
<213> artificial
<220>
<223> Primer - GeN-NSP5F
<400> 21
ggctttaaag cgctacag 18
<210> 22
<211> 17
<212> DNA
<213> artificial
<220>
<223> Primer - GeN-NSP5R
<400> 22
ggtcacaaaa cgggagt 17
<210> 23
<211> 1461
<212> DNA
<213> artificial
<220>
<223> MA104-RV3 VP7
<400> 23
atggctactt ttaaagatgc ttgttattat tataagagaa ttaacaagtt gaatcacgca 60
gtcttgaagt taggagttaa tgatacatgg agaccatcac ctccaactaa gtataaagga 120
tggtgtctgg attgttgcca acacactgat ttaacttact gtcgaggttg taccatgtat 180
catgtatgtc aatggtgtag tcaatatggt agatgctttc ttgatagtga accacatcta 240
ttaagaatga gaactttcaa gaatgaagtg acgaaaaatg atttaatgaa tttaattgac 300
atgtacaata cattatttcc tataaatcaa agaatagtag ataaattcat taatagtaca 360
agacaacata aatgtagaaa tgaatgtatg acacagtggt acaatcactt agtactacca 420
ataacattac agtctctatc aatcgaatta gatggtgata tttattacgt gtttggatat 480
tatgatagta tgaatgacat taatcaaact ccattctcat ttacaaattt aatagatatg 540
tatgataagt tgctacttga taatataaat tttaacagaa tgtcattctt accagtagca 600
ttacaacaag aatatgcact cagatatttt tcaaaatcaa ggtttattag tgaaaagagg 660
aaatgtatca gtgatgtaca tttttccgct agcgtaatag aaaatttaca caatccaagt 720
tttaagatac agattacacg caattgtagt gaattatcct ctgattggaa cggagcatgc 780
aaacttgtta aagatatgag cgcttatttt gatgtcctga aaacatcaca tattgaattt 840
tatagtattt caactagatg tagagtgttt acgcagtata aacttaaagt agcatctaag 900
catataaaac caaattatgt gacatcaaat catagaacat ctgcgactga ggtacataat 960
tgtaaatggt gctcaattaa tagtagttat actgtatgga atgattttag aattaagaag 1020
atatatgata acattttcaa ttttctacga gctttagtca aatcaaatgc taatgttgga 1080
cattgttcgt cacaggaaaa gatgtatgag catattgaag atgttctgga tgtatgtgat 1140
gatgaaaaat ggaaaatggc ggtaacagaa atattcaatt ggttagaacc agtagaactt 1200
aatactgtta aatatgttct gttcaatcat gaggtaaatt gggatgtcat taatttatta 1260
gttcagagta ttggtaaagt accacaaata ctgactttga atgatattgt cataattatg 1320
aaatctatca tatatgagtg gtttgatatc agatatatga ggaacacacc aatgactaca 1380
tttacagttg acaaattaag acggttatgc acaggagtga aggctgttga ttatgattcc 1440
ggcatatctg acgttgaata a 1461
<210> 24
<211> 486
<212> PRT
<213> artificial
<220>
<223> MA104-adapted RV3 VP7
<400> 24
Met Ala Thr Phe Lys Asp Ala Cys Tyr Tyr Tyr Lys Arg Ile Asn Lys
1 5 10 15
Leu Asn His Ala Val Leu Lys Leu Gly Val Asn Asp Thr Trp Arg Pro
20 25 30
Ser Pro Pro Thr Lys Tyr Lys Gly Trp Cys Leu Asp Cys Cys Gln His
35 40 45
Thr Asp Leu Thr Tyr Cys Arg Gly Cys Thr Met Tyr His Val Cys Gln
50 55 60
Trp Cys Ser Gln Tyr Gly Arg Cys Phe Leu Asp Ser Glu Pro His Leu
65 70 75 80
Leu Arg Met Arg Thr Phe Lys Asn Glu Val Thr Lys Asn Asp Leu Met
85 90 95
Asn Leu Ile Asp Met Tyr Asn Thr Leu Phe Pro Ile Asn Gln Arg Ile
100 105 110
Val Asp Lys Phe Ile Asn Ser Thr Arg Gln His Lys Cys Arg Asn Glu
115 120 125
Cys Met Thr Gln Trp Tyr Asn His Leu Val Leu Pro Ile Thr Leu Gln
130 135 140
Ser Leu Ser Ile Glu Leu Asp Gly Asp Ile Tyr Tyr Val Phe Gly Tyr
145 150 155 160
Tyr Asp Ser Met Asn Asp Ile Asn Gln Thr Pro Phe Ser Phe Thr Asn
165 170 175
Leu Ile Asp Met Tyr Asp Lys Leu Leu Leu Asp Asn Ile Asn Phe Asn
180 185 190
Arg Met Ser Phe Leu Pro Val Ala Leu Gln Gln Glu Tyr Ala Leu Arg
195 200 205
Tyr Phe Ser Lys Ser Arg Phe Ile Ser Glu Lys Arg Lys Cys Ile Ser
210 215 220
Asp Val His Phe Ser Ala Ser Val Ile Glu Asn Leu His Asn Pro Ser
225 230 235 240
Phe Lys Ile Gln Ile Thr Arg Asn Cys Ser Glu Leu Ser Ser Asp Trp
245 250 255
Asn Gly Ala Cys Lys Leu Val Lys Asp Met Ser Ala Tyr Phe Asp Val
260 265 270
Leu Lys Thr Ser His Ile Glu Phe Tyr Ser Ile Ser Thr Arg Cys Arg
275 280 285
Val Phe Thr Gln Tyr Lys Leu Lys Val Ala Ser Lys His Ile Lys Pro
290 295 300
Asn Tyr Val Thr Ser Asn His Arg Thr Ser Ala Thr Glu Val His Asn
305 310 315 320
Cys Lys Trp Cys Ser Ile Asn Ser Ser Tyr Thr Val Trp Asn Asp Phe
325 330 335
Arg Ile Lys Lys Ile Tyr Asp Asn Ile Phe Asn Phe Leu Arg Ala Leu
340 345 350
Val Lys Ser Asn Ala Asn Val Gly His Cys Ser Ser Gln Glu Lys Met
355 360 365
Tyr Glu His Ile Glu Asp Val Leu Asp Val Cys Asp Asp Glu Lys Trp
370 375 380
Lys Met Ala Val Thr Glu Ile Phe Asn Trp Leu Glu Pro Val Glu Leu
385 390 395 400
Asn Thr Val Lys Tyr Val Leu Phe Asn His Glu Val Asn Trp Asp Val
405 410 415
Ile Asn Leu Leu Val Gln Ser Ile Gly Lys Val Pro Gln Ile Leu Thr
420 425 430
Leu Asn Asp Ile Val Ile Ile Met Lys Ser Ile Ile Tyr Glu Trp Phe
435 440 445
Asp Ile Arg Tyr Met Arg Asn Thr Pro Met Thr Thr Phe Thr Val Asp
450 455 460
Lys Leu Arg Arg Leu Cys Thr Gly Val Lys Ala Val Asp Tyr Asp Ser
465 470 475 480
Gly Ile Ser Asp Val Glu
485
<210> 25
<211> 1588
<212> DNA
<213> artificial
<220>
<223> RV3-Vero VP7
<400> 25
tgtaaaacga cggccagtta tgaaaagtct tgtggaagcc atggctactt ttaaagatgc 60
ttgttattat tataagagaa ttaacaagtt gaatcacgca gtcttgaagt taggagttaa 120
tgatacatgg agaccatcac ctccaactaa gtataaagga tggtgtctgg attgttgcca 180
acacactgat ttaacttact gtcgaggttg taccatgtat catgtatgtc aatggtgtag 240
tcaatatggt agatgctttc ttgatagtga accacatcta ttaagaatga gaactttcaa 300
gaatgaagtg acgaaaaatg atttaatgaa tttaattgac atgtacaata cattatttcc 360
tataaatcaa agaatagtag ataaattcat taatagtaca agacaacata aatgtagaaa 420
tgaatgtatg acacagtggt acaatcactt agtactacca ataacattac agtctctatc 480
aatcgaatta gatggtgata tttattacgt gtttggatat tatgatagta tgaatgacat 540
taatcaaact ccattctcat ttacaaattt aatagatatg tatgataagt tgctacttga 600
taatataaat tttaacagaa tgtcattctt accagtagca ttacaacaag aatatgcact 660
cagatatttt tcaaaatcaa ggtttattag tgaaaagagg aaatgtatca gtgatgtaca 720
tttttccgct agcgtaatag aaaatttaca caatccaagt tttaagatac agattacacg 780
caattgtagt gaattatcct ctgattggaa cggagcatgc aaacttgtta aagatatgag 840
cgcttatttt gatgtcctga aaacatcaca tattgaattt tatagtattt caactagatg 900
tagagtgttt acgcagtata aacttaaagt agcatctaag catataaaac caaattatgt 960
gacatcaaat catagaacat ctgcgactga ggtacataat tgtaaatggt gctcaattaa 1020
tagtagttat actgtatgga atgattttag aattaagaag atatatgata acattttcaa 1080
ttttctacga gctttagtca aatcaaatgc taatgttgga cattgttcgt cacaggaaaa 1140
gatgtatgag catattgaag atgttctgga tgtatgtgat gatgaaaaat ggaaaatggc 1200
ggtaacagaa atattcaatt ggttagaacc agtagaactt aatactgtta aatatgttct 1260
gttcaatcat gaggtaaatt gggatgtcat taatttatta gttcagagta ttggtaaagt 1320
accacaaata ctgactttga atgatattgt cataattatg aaatctatca tatatgagtg 1380
gtttgatatc agatatatga ggaacacacc aatgactaca tttacagttg acaaattaag 1440
acggttatgc acaggagtga aggctgttga ttatgattcc ggcatatctg acgttgaata 1500
ctgaaataga ggtcacattt gccaccgcaa gactccctgc actagagtag cgcctaggca 1560
gcataaaatg ggtcatagct gtttcctg 1588
<210> 26
<211> 486
<212> PRT
<213> artificial
<220>
<223> RV3-Vero VP7
<400> 26
Met Ala Thr Phe Lys Asp Ala Cys Tyr Tyr Tyr Lys Arg Ile Asn Lys
1 5 10 15
Leu Asn His Ala Val Leu Lys Leu Gly Val Asn Asp Thr Trp Arg Pro
20 25 30
Ser Pro Pro Thr Lys Tyr Lys Gly Trp Cys Leu Asp Cys Cys Gln His
35 40 45
Thr Asp Leu Thr Tyr Cys Arg Gly Cys Thr Met Tyr His Val Cys Gln
50 55 60
Trp Cys Ser Gln Tyr Gly Arg Cys Phe Leu Asp Ser Glu Pro His Leu
65 70 75 80
Leu Arg Met Arg Thr Phe Lys Asn Glu Val Thr Lys Asn Asp Leu Met
85 90 95
Asn Leu Ile Asp Met Tyr Asn Thr Leu Phe Pro Ile Asn Gln Arg Ile
100 105 110
Val Asp Lys Phe Ile Asn Ser Thr Arg Gln His Lys Cys Arg Asn Glu
115 120 125
Cys Met Thr Gln Trp Tyr Asn His Leu Val Leu Pro Ile Thr Leu Gln
130 135 140
Ser Leu Ser Ile Glu Leu Asp Gly Asp Ile Tyr Tyr Val Phe Gly Tyr
145 150 155 160
Tyr Asp Ser Met Asn Asp Ile Asn Gln Thr Pro Phe Ser Phe Thr Asn
165 170 175
Leu Ile Asp Met Tyr Asp Lys Leu Leu Leu Asp Asn Ile Asn Phe Asn
180 185 190
Arg Met Ser Phe Leu Pro Val Ala Leu Gln Gln Glu Tyr Ala Leu Arg
195 200 205
Tyr Phe Ser Lys Ser Arg Phe Ile Ser Glu Lys Arg Lys Cys Ile Ser
210 215 220
Asp Val His Phe Ser Ala Ser Val Ile Glu Asn Leu His Asn Pro Ser
225 230 235 240
Phe Lys Ile Gln Ile Thr Arg Asn Cys Ser Glu Leu Ser Ser Asp Trp
245 250 255
Asn Gly Ala Cys Lys Leu Val Lys Asp Met Ser Ala Tyr Phe Asp Val
260 265 270
Leu Lys Thr Ser His Ile Glu Phe Tyr Ser Ile Ser Thr Arg Cys Arg
275 280 285
Val Phe Thr Gln Tyr Lys Leu Lys Val Ala Ser Lys His Ile Lys Pro
290 295 300
Asn Tyr Val Thr Ser Asn His Arg Thr Ser Ala Thr Glu Val His Asn
305 310 315 320
Cys Lys Trp Cys Ser Ile Asn Ser Ser Tyr Thr Val Trp Asn Asp Phe
325 330 335
Arg Ile Lys Lys Ile Tyr Asp Asn Ile Phe Asn Phe Leu Arg Ala Leu
340 345 350
Val Lys Ser Asn Ala Asn Val Gly His Cys Ser Ser Gln Glu Lys Met
355 360 365
Tyr Glu His Ile Glu Asp Val Leu Asp Val Cys Asp Asp Glu Lys Trp
370 375 380
Lys Met Ala Val Thr Glu Ile Phe Asn Trp Leu Glu Pro Val Glu Leu
385 390 395 400
Asn Thr Val Lys Tyr Val Leu Phe Asn His Glu Val Asn Trp Asp Val
405 410 415
Ile Asn Leu Leu Val Gln Ser Ile Gly Lys Val Pro Gln Ile Leu Thr
420 425 430
Leu Asn Asp Ile Val Ile Ile Met Lys Ser Ile Ile Tyr Glu Trp Phe
435 440 445
Asp Ile Arg Tyr Met Arg Asn Thr Pro Met Thr Thr Phe Thr Val Asp
450 455 460
Lys Leu Arg Arg Leu Cys Thr Gly Val Lys Ala Val Asp Tyr Asp Ser
465 470 475 480
Gly Ile Ser Asp Val Glu
485
<210> 27
<211> 594
<212> DNA
<213> artificial
<220>
<223> MA104 RV3 NSP5/6
<400> 27
atgtctctca gcattgacgt agcaagtctt ccctcaattt cttctagtat ctttaaaaat 60
gaatcgtctt ctacaacgtc aactctttct ggaaaatcta ttggtaggag tgaacagtac 120
atttcaccag atgcaggagc atttaataaa tacatgttgt cgaagtctcc agaggatatt 180
ggaccgtctg attctgcttc aaacgatcca ctcaccagct tttcgattag atcgaatgca 240
gttaaaacaa atgcagacgc tggcgtgtct atggattcat cgacgcaatc acgaccttca 300
agcaacgttg ggtgcgatca attggatttc tccttaacta aaggtattaa tgttagtgct 360
aatcttgatt catgtatatc aatttcaact gatcataaga aggagaaatc caagaaagat 420
aaaagtagga aacactaccc gagaattgaa gcagattctg attctgaaga ttatgttttg 480
gatgattcag atagtgatga cggtaaatgt aagaattgta aatataagaa aaaatatttc 540
gcactaagaa tgaggatgaa gcgagtcgca atgcaattga tcgaggattt gtaa 594
<210> 28
<211> 197
<212> PRT
<213> artificial
<220>
<223> MA104-adapted RV3 NSP5/6
<400> 28
Met Ser Leu Ser Ile Asp Val Ala Ser Leu Pro Ser Ile Ser Ser Ser
1 5 10 15
Ile Phe Lys Asn Glu Ser Ser Ser Thr Thr Ser Thr Leu Ser Gly Lys
20 25 30
Ser Ile Gly Arg Ser Glu Gln Tyr Ile Ser Pro Asp Ala Gly Ala Phe
35 40 45
Asn Lys Tyr Met Leu Ser Lys Ser Pro Glu Asp Ile Gly Pro Ser Asp
50 55 60
Ser Ala Ser Asn Asp Pro Leu Thr Ser Phe Ser Ile Arg Ser Asn Ala
65 70 75 80
Val Lys Thr Asn Ala Asp Ala Gly Val Ser Met Asp Ser Ser Thr Gln
85 90 95
Ser Arg Pro Ser Ser Asn Val Gly Cys Asp Gln Leu Asp Phe Ser Leu
100 105 110
Thr Lys Gly Ile Asn Val Ser Ala Asn Leu Asp Ser Cys Ile Ser Ile
115 120 125
Ser Thr Asp His Lys Lys Glu Lys Ser Lys Lys Asp Lys Ser Arg Lys
130 135 140
His Tyr Pro Arg Ile Glu Ala Asp Ser Asp Ser Glu Asp Tyr Val Leu
145 150 155 160
Asp Asp Ser Asp Ser Asp Asp Gly Lys Cys Lys Asn Cys Lys Tyr Lys
165 170 175
Lys Lys Tyr Phe Ala Leu Arg Met Arg Met Lys Arg Val Ala Met Gln
180 185 190
Leu Ile Glu Asp Leu
195
<210> 29
<211> 664
<212> DNA
<213> artificial
<220>
<223> RV3-Vero NSP5
<400> 29
ggcttttaaa gcgctacagt gatgtctctc agcattgacg tagcaagtct tccctcaatt 60
tcttctagta tctttaaaaa tgaatcgtct tctacaacgt caactctttc tggaaaatct 120
attggtagga gtgaacagta catttcacca gatgcagaag cattcaataa atacatgttg 180
tcgaagtctc cagaggatat tggaccgtct gattctgctt caaacgatcc actcaccagc 240
ttttcgatta gatcgaatgc agttaaaaca aatgcagacg ctggcgtgtc tatggattca 300
tcgacgcaat cacgaccttc aagcaacgtt gggtgcgatc aattggattt ctccataact 360
aaaggtatta atgttagtgc taatcttgat tcatgtatat caatttcaac tgatcataag 420
aaggagaaat ccaagaaaga taaaagtagg aaacactacc cgagaattga agcagattct 480
gattctgaag attatgtttt ggatgattca gatagtgatg acggtaaatg taagaattgt 540
aaatataaga aaaaatattt cgcactaaga atgaggatga agcgagtcgc aatgcaattg 600
atcgaggatt tgtaatgtca acctgagagc acactaggga gctccccact cccgttttgt 660
gacc 664
<210> 30
<211> 197
<212> PRT
<213> artificial
<220>
<223> RV3-Vero NSP5
<400> 30
Met Ser Leu Ser Ile Asp Val Ala Ser Leu Pro Ser Ile Ser Ser Ser
1 5 10 15
Ile Phe Lys Asn Glu Ser Ser Ser Thr Thr Ser Thr Leu Ser Gly Lys
20 25 30
Ser Ile Gly Arg Ser Glu Gln Tyr Ile Ser Pro Asp Ala Glu Ala Phe
35 40 45
Asn Lys Tyr Met Leu Ser Lys Ser Pro Glu Asp Ile Gly Pro Ser Asp
50 55 60
Ser Ala Ser Asn Asp Pro Leu Thr Ser Phe Ser Ile Arg Ser Asn Ala
65 70 75 80
Val Lys Thr Asn Ala Asp Ala Gly Val Ser Met Asp Ser Ser Thr Gln
85 90 95
Ser Arg Pro Ser Ser Asn Val Gly Cys Asp Gln Leu Asp Phe Ser Ile
100 105 110
Thr Lys Gly Ile Asn Val Ser Ala Asn Leu Asp Ser Cys Ile Ser Ile
115 120 125
Ser Thr Asp His Lys Lys Glu Lys Ser Lys Lys Asp Lys Ser Arg Lys
130 135 140
His Tyr Pro Arg Ile Glu Ala Asp Ser Asp Ser Glu Asp Tyr Val Leu
145 150 155 160
Asp Asp Ser Asp Ser Asp Asp Gly Lys Cys Lys Asn Cys Lys Tyr Lys
165 170 175
Lys Lys Tyr Phe Ala Leu Arg Met Arg Met Lys Arg Val Ala Met Gln
180 185 190
Leu Ile Glu Asp Leu
195
<210> 31
<211> 954
<212> DNA
<213> artificial
<220>
<223> MA104 RV3 NSP2
<400> 31
atggctgagc tagcttgctt ttgttatcct catttagaga acgatagcta taaatttatt 60
cctttcaata gtttggcaat aaaatgtatg ttgacagcta aagtggataa gaaagatcaa 120
gataaatttt ataattctat tgtatacgga attgcgccac caccacaatt caaaaaacgt 180
tataatacta gtgataattc aagaggcatg aattatgaaa caactatgtt taataaggtg 240
gctattttaa tttgtgaagc acttaattca attaaagtca ctcaatctga cattgctaat 300
gtcctctcaa gagtagtttc tattagacat ttggaaaatt tggtgttaag aaaagaaaat 360
catcaagatg tgctgtttca ttcgaaggag ctactcttaa aatccgtttt gatagctatt 420
ggtcaatcaa aagaaattga aactactgct actgccgagg gaggagaaat agtgttccaa 480
aatgcagctt ttactatgtg gaaattgact tatttagatc ataaactaac gcctattttg 540
gaccaaaatt ttatcgaata taagattaca ttaaatgaag ataaaccaat ttcagatgta 600
tgtgttaaag aacttgttgc tgaattgaga tggcagtata acagatttgc tgtaataacg 660
catggtaaag gtcactatag agtcgttaaa tattcatcag ttgctaatca cgcagataga 720
gtatttgtcg catacaaaaa taatgctaag agtggtaatg ttattaattt taatttactg 780
gatcaaagaa taatttggca aaattggtac gcatttacat cttcaatgaa acaaggtaat 840
acacttgaag tatgtaagaa actgctcttt caaaagatga agcaggagaa gaatccattt 900
aaaggactgt caactgatag aaaaatggat gaagtctcac atgttggagt ttag 954
<210> 32
<211> 317
<212> PRT
<213> artificial
<220>
<223> MA104-adapted RV3 NSP2
<400> 32
Met Ala Glu Leu Ala Cys Phe Cys Tyr Pro His Leu Glu Asn Asp Ser
1 5 10 15
Tyr Lys Phe Ile Pro Phe Asn Ser Leu Ala Ile Lys Cys Met Leu Thr
20 25 30
Ala Lys Val Asp Lys Lys Asp Gln Asp Lys Phe Tyr Asn Ser Ile Val
35 40 45
Tyr Gly Ile Ala Pro Pro Pro Gln Phe Lys Lys Arg Tyr Asn Thr Ser
50 55 60
Asp Asn Ser Arg Gly Met Asn Tyr Glu Thr Thr Met Phe Asn Lys Val
65 70 75 80
Ala Ile Leu Ile Cys Glu Ala Leu Asn Ser Ile Lys Val Thr Gln Ser
85 90 95
Asp Ile Ala Asn Val Leu Ser Arg Val Val Ser Ile Arg His Leu Glu
100 105 110
Asn Leu Val Leu Arg Lys Glu Asn His Gln Asp Val Leu Phe His Ser
115 120 125
Lys Glu Leu Leu Leu Lys Ser Val Leu Ile Ala Ile Gly Gln Ser Lys
130 135 140
Glu Ile Glu Thr Thr Ala Thr Ala Glu Gly Gly Glu Ile Val Phe Gln
145 150 155 160
Asn Ala Ala Phe Thr Met Trp Lys Leu Thr Tyr Leu Asp His Lys Leu
165 170 175
Thr Pro Ile Leu Asp Gln Asn Phe Ile Glu Tyr Lys Ile Thr Leu Asn
180 185 190
Glu Asp Lys Pro Ile Ser Asp Val Cys Val Lys Glu Leu Val Ala Glu
195 200 205
Leu Arg Trp Gln Tyr Asn Arg Phe Ala Val Ile Thr His Gly Lys Gly
210 215 220
His Tyr Arg Val Val Lys Tyr Ser Ser Val Ala Asn His Ala Asp Arg
225 230 235 240
Val Phe Val Ala Tyr Lys Asn Asn Ala Lys Ser Gly Asn Val Ile Asn
245 250 255
Phe Asn Leu Leu Asp Gln Arg Ile Ile Trp Gln Asn Trp Tyr Ala Phe
260 265 270
Thr Ser Ser Met Lys Gln Gly Asn Thr Leu Glu Val Cys Lys Lys Leu
275 280 285
Leu Phe Gln Lys Met Lys Gln Glu Lys Asn Pro Phe Lys Gly Leu Ser
290 295 300
Thr Asp Arg Lys Met Asp Glu Val Ser His Val Gly Val
305 310 315
<210> 33
<211> 1059
<212> DNA
<213> artificial
<220>
<223> RV3-VEro NSP2
<400> 33
ggcttttaaa gcgtctcagt cgccgtttga gccttgcggt gtagccatgg ctgagctagc 60
ttgcttttgt tatcctcatt tagagaacga tagctataaa tttattcctt tcaatagttt 120
ggcaataaaa tgtatgttga cagctaaagt ggataagaaa gatcaagata aattttataa 180
ttctattgta tacggaattg cgccaccacc acaattcaaa aaacgttata atactagtga 240
taattcaaga ggcatgaatt atgaaacaac tatgtttaat aaggtggcta ttttaatttg 300
tgaagcactt aattcaatta aagtcactca atctgacatt gctaatgtcc tctcaagagt 360
agtttctatt agacatttgg aaaatttggt gttaagaaaa gaaaatcatc aagatgtgct 420
gtttcattcg aaagagctac tcttaaaatc cgttttgata gctattggtc aatcaaaaga 480
aattgaaact actgctactg ccgagggagg agaaatagtg ttccaaaatg cagcttttac 540
tatgtggaaa ttgacttatt tagatcataa actaacgcct attttggacc aaaattttat 600
cgaatataag attacattaa atgaagataa accaatttca gatgtatgtg ttaaagaact 660
tgttgctgaa ttgagatggc agtataacag atttgctgta ataacgcatg gtaaaggtca 720
ctatagagtc gttaaatatt catcagttgc taatcacgca gatagagtat ttgtcgcata 780
caaaaataat gctaagagtg gtaatgttat taattttaat ttactggatc aaagaataat 840
ttggcaaaat tggtacgcat ttacatcttc aatgaaacaa ggtaatacac ttgaagtatg 900
taagaaactg ctctttcaaa agatgaagca ggagaagaat ccatttaaag gactgtcaac 960
tgatagaaaa atggatgaag tctcacatgt tggagtttag ttcgctttcg atttgaaaat 1020
gatgatgacg aagcaggaat agaaagcgct tatgtgacc 1059
<210> 34
<211> 317
<212> PRT
<213> artificial
<220>
<223> RV3-Vero NSP2
<400> 34
Met Ala Glu Leu Ala Cys Phe Cys Tyr Pro His Leu Glu Asn Asp Ser
1 5 10 15
Tyr Lys Phe Ile Pro Phe Asn Ser Leu Ala Ile Lys Cys Met Leu Thr
20 25 30
Ala Lys Val Asp Lys Lys Asp Gln Asp Lys Phe Tyr Asn Ser Ile Val
35 40 45
Tyr Gly Ile Ala Pro Pro Pro Gln Phe Lys Lys Arg Tyr Asn Thr Ser
50 55 60
Asp Asn Ser Arg Gly Met Asn Tyr Glu Thr Thr Met Phe Asn Lys Val
65 70 75 80
Ala Ile Leu Ile Cys Glu Ala Leu Asn Ser Ile Lys Val Thr Gln Ser
85 90 95
Asp Ile Ala Asn Val Leu Ser Arg Val Val Ser Ile Arg His Leu Glu
100 105 110
Asn Leu Val Leu Arg Lys Glu Asn His Gln Asp Val Leu Phe His Ser
115 120 125
Lys Glu Leu Leu Leu Lys Ser Val Leu Ile Ala Ile Gly Gln Ser Lys
130 135 140
Glu Ile Glu Thr Thr Ala Thr Ala Glu Gly Gly Glu Ile Val Phe Gln
145 150 155 160
Asn Ala Ala Phe Thr Met Trp Lys Leu Thr Tyr Leu Asp His Lys Leu
165 170 175
Thr Pro Ile Leu Asp Gln Asn Phe Ile Glu Tyr Lys Ile Thr Leu Asn
180 185 190
Glu Asp Lys Pro Ile Ser Asp Val Cys Val Lys Glu Leu Val Ala Glu
195 200 205
Leu Arg Trp Gln Tyr Asn Arg Phe Ala Val Ile Thr His Gly Lys Gly
210 215 220
His Tyr Arg Val Val Lys Tyr Ser Ser Val Ala Asn His Ala Asp Arg
225 230 235 240
Val Phe Val Ala Tyr Lys Asn Asn Ala Lys Ser Gly Asn Val Ile Asn
245 250 255
Phe Asn Leu Leu Asp Gln Arg Ile Ile Trp Gln Asn Trp Tyr Ala Phe
260 265 270
Thr Ser Ser Met Lys Gln Gly Asn Thr Leu Glu Val Cys Lys Lys Leu
275 280 285
Leu Phe Gln Lys Met Lys Gln Glu Lys Asn Pro Phe Lys Gly Leu Ser
290 295 300
Thr Asp Arg Lys Met Asp Glu Val Ser His Val Gly Val
305 310 315
<210> 35
<211> 528
<212> DNA
<213> artificial
<220>
<223> MA104-derived RV3 NSP4
<400> 35
atggataagc ttgccgacct caactacaca ttgagtgtaa tcactttaat gaatgacaca 60
ttgcattcta taattcaaga tcctggaatg gcgtatttta catatattgc atctgttcta 120
acagttttgt tcacattaca caaagcttca attccagcca tgaaaatagc attgaaaaca 180
tcaaaatgtt catataaagt aattaaatat tgtatagtca cgataattaa tactctttta 240
aaattggctg gatgtaaaga gcaggttact acaaaagacg aaattgagca acagatggac 300
agaattgtta aagagatgag acgtcagctg gagatgattg ataaactaac tactcgtgaa 360
attgaacagg ttgaattgct taaatgtata tatgacaacc tgataactag accagttaac 420
gttatagata tgtcgaagga attcaatcaa aaaaacatca aaacgctaga tgaatgggag 480
agtgtaaaaa atccatatga accgtcagaa gtgactgcat ccatgtaa 528
<210> 36
<211> 175
<212> PRT
<213> artificial
<220>
<223> RV3 NSP4
<400> 36
Met Asp Lys Leu Ala Asp Leu Asn Tyr Thr Leu Ser Val Ile Thr Leu
1 5 10 15
Met Asn Asp Thr Leu His Ser Ile Ile Gln Asp Pro Gly Met Ala Tyr
20 25 30
Phe Thr Tyr Ile Ala Ser Val Leu Thr Val Leu Phe Thr Leu His Lys
35 40 45
Ala Ser Ile Pro Ala Met Lys Ile Ala Leu Lys Thr Ser Lys Cys Ser
50 55 60
Tyr Lys Val Ile Lys Tyr Cys Ile Val Thr Ile Ile Asn Thr Leu Leu
65 70 75 80
Lys Leu Ala Gly Cys Lys Glu Gln Val Thr Thr Lys Asp Glu Ile Glu
85 90 95
Gln Gln Met Asp Arg Ile Val Lys Glu Met Arg Arg Gln Leu Glu Met
100 105 110
Ile Asp Lys Leu Thr Thr Arg Glu Ile Glu Gln Val Glu Leu Leu Lys
115 120 125
Cys Ile Tyr Asp Asn Leu Ile Thr Arg Pro Val Asn Val Ile Asp Met
130 135 140
Ser Lys Glu Phe Asn Gln Lys Asn Ile Lys Thr Leu Asp Glu Trp Glu
145 150 155 160
Ser Val Lys Asn Pro Tyr Glu Pro Ser Glu Val Thr Ala Ser Met
165 170 175
<210> 37
<211> 749
<212> DNA
<213> artificial
<220>
<223> RV3-Vero NSP4
<400> 37
ggcttttaaa agttctgtcc gagagagcgc gtgcggaaag atggataagc ttgccgacct 60
caactacaca ttgagtgtaa tcactttaat gaatgacaca ttgcattcta taattcaaga 120
tcctggaatg gcgtatttta catatattgc atctgttcta acagttttgt tcacattaca 180
caaagcttca attccaacca tgaaaatagc attgaaaaca tcaaaatgtt catataaagt 240
aattaaatat tgtatagtca cgataactaa tactctttta aaattggctg gatataaaga 300
gcaggttact acaaaagacg aaattgagca acagatggac agaattgtta aagagatgag 360
acgtcagctg gagatgattg ataaactaac tactcgtgaa attgaacagg ttgaattgct 420
taaatgtata tatgacaacc tgataactag accagttaac gttatagata tgtcgaagga 480
attcaatcaa aaaaacatca aaacgctaga tgaatgggag agtggaaaaa atctatatga 540
accgtcagaa gtgactgcat ccatgtaaga ggttgagttg ccgtcgtctg tcttcggaag 600
cggcggaact cttcaccgca agccccatta gacctgatga ttgactgaga agccacagtc 660
aatcatatcg cgtgtggctc agccttaatc ccgtttaacc aatccagcga gtgttggacg 720
ttaatggaag gaatggtctt agtgtgacc 749
<210> 38
<211> 175
<212> PRT
<213> artificial
<220>
<223> RV3-Vero NSP4
<400> 38
Met Asp Lys Leu Ala Asp Leu Asn Tyr Thr Leu Ser Val Ile Thr Leu
1 5 10 15
Met Asn Asp Thr Leu His Ser Ile Ile Gln Asp Pro Gly Met Ala Tyr
20 25 30
Phe Thr Tyr Ile Ala Ser Val Leu Thr Val Leu Phe Thr Leu His Lys
35 40 45
Ala Ser Ile Pro Thr Met Lys Ile Ala Leu Lys Thr Ser Lys Cys Ser
50 55 60
Tyr Lys Val Ile Lys Tyr Cys Ile Val Thr Ile Thr Asn Thr Leu Leu
65 70 75 80
Lys Leu Ala Gly Tyr Lys Glu Gln Val Thr Thr Lys Asp Glu Ile Glu
85 90 95
Gln Gln Met Asp Arg Ile Val Lys Glu Met Arg Arg Gln Leu Glu Met
100 105 110
Ile Asp Lys Leu Thr Thr Arg Glu Ile Glu Gln Val Glu Leu Leu Lys
115 120 125
Cys Ile Tyr Asp Asn Leu Ile Thr Arg Pro Val Asn Val Ile Asp Met
130 135 140
Ser Lys Glu Phe Asn Gln Lys Asn Ile Lys Thr Leu Asp Glu Trp Glu
145 150 155 160
Ser Gly Lys Asn Leu Tyr Glu Pro Ser Glu Val Thr Ala Ser Met
165 170 175
<210> 39
<211> 1194
<212> DNA
<213> artificial
<220>
<223> MA104-derived RV3 VP6
<400> 39
atggaggttc tgtattcatt gtcaaaaact cttaaagatg ctagggataa gattgttgaa 60
ggtacattat attctaatgt tagtgatctc attcagcaat ttaatcaaat gatagtaacc 120
atgaatggaa atgactttca aactggagga attggcaatt tacctattag aaattggaca 180
tttgactttg gtctactagg tactacgctg ttaaaccttg atgctaatta cgttgagacc 240
gcaagaacta caattaagta ttttattgac tttattgata atgtatgtat ggatgaaatg 300
gcaagagagt ctcaaagaaa tggagtagct ccacaatctg aggcattgag gaagctagcc 360
ggtattaaat ttaaaagaat aaattttaat aattcatcag aatatataga aaattggaat 420
ttacaaaata gaagacagcg taccggattt gttttccata aacctaatat atttccatac 480
tcagcatcat ttactttaaa taggtctcaa ccaatgcatg acaatttaat gggaaccatg 540
tggcttaacg ctggatcaga aattcaagtg gctggatttg actactcgtg tgccctaaat 600
gctccagcaa atattcagca gtttgaacat attgtccagc ttaggcgtgc gctaactaca 660
gctactataa ctttgctacc tgatgcagaa agatttagtt ttccaagagt tattaattca 720
gcagatggcg caaccacatg gttctttaat ccaattatcc taagaccaaa caatgtagag 780
gtagaatttt tactgaatgg acaaattatt aatacatatc aagctagatt tggcactatt 840
atcgcaagaa attttgatac aattcgtcta tcattccaat taatgcgtcc accaaacatg 900
acgccagccg taaatgcatt atttccgcaa gcacaacctt ttcaacatca tgcaacagtt 960
ggacttacgt tacgtattga gtctgcagtt tgtgaatcag tgcttgcgga tgcaaatgaa 1020
actttattgg cgaatgttac tgcagtacgt caagagtatg ctataccggt tggaccagta 1080
tttccaccag gcatgaattg gactgagctg attactaact attcaccatc cagggaagat 1140
aatttgcaac gtgtctttac agtagcctct atcagaagca tgttaattaa gtga 1194
<210> 40
<211> 397
<212> PRT
<213> artificial
<220>
<223> MA104-derived RV3 VP6
<400> 40
Met Glu Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp
1 5 10 15
Lys Ile Val Glu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu Ile Gln
20 25 30
Gln Phe Asn Gln Met Ile Val Thr Met Asn Gly Asn Asp Phe Gln Thr
35 40 45
Gly Gly Ile Gly Asn Leu Pro Ile Arg Asn Trp Thr Phe Asp Phe Gly
50 55 60
Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val Glu Thr
65 70 75 80
Ala Arg Thr Thr Ile Lys Tyr Phe Ile Asp Phe Ile Asp Asn Val Cys
85 90 95
Met Asp Glu Met Ala Arg Glu Ser Gln Arg Asn Gly Val Ala Pro Gln
100 105 110
Ser Glu Ala Leu Arg Lys Leu Ala Gly Ile Lys Phe Lys Arg Ile Asn
115 120 125
Phe Asn Asn Ser Ser Glu Tyr Ile Glu Asn Trp Asn Leu Gln Asn Arg
130 135 140
Arg Gln Arg Thr Gly Phe Val Phe His Lys Pro Asn Ile Phe Pro Tyr
145 150 155 160
Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Met His Asp Asn Leu
165 170 175
Met Gly Thr Met Trp Leu Asn Ala Gly Ser Glu Ile Gln Val Ala Gly
180 185 190
Phe Asp Tyr Ser Cys Ala Leu Asn Ala Pro Ala Asn Ile Gln Gln Phe
195 200 205
Glu His Ile Val Gln Leu Arg Arg Ala Leu Thr Thr Ala Thr Ile Thr
210 215 220
Leu Leu Pro Asp Ala Glu Arg Phe Ser Phe Pro Arg Val Ile Asn Ser
225 230 235 240
Ala Asp Gly Ala Thr Thr Trp Phe Phe Asn Pro Ile Ile Leu Arg Pro
245 250 255
Asn Asn Val Glu Val Glu Phe Leu Leu Asn Gly Gln Ile Ile Asn Thr
260 265 270
Tyr Gln Ala Arg Phe Gly Thr Ile Ile Ala Arg Asn Phe Asp Thr Ile
275 280 285
Arg Leu Ser Phe Gln Leu Met Arg Pro Pro Asn Met Thr Pro Ala Val
290 295 300
Asn Ala Leu Phe Pro Gln Ala Gln Pro Phe Gln His His Ala Thr Val
305 310 315 320
Gly Leu Thr Leu Arg Ile Glu Ser Ala Val Cys Glu Ser Val Leu Ala
325 330 335
Asp Ala Asn Glu Thr Leu Leu Ala Asn Val Thr Ala Val Arg Gln Glu
340 345 350
Tyr Ala Ile Pro Val Gly Pro Val Phe Pro Pro Gly Met Asn Trp Thr
355 360 365
Glu Leu Ile Thr Asn Tyr Ser Pro Ser Arg Glu Asp Asn Leu Gln Arg
370 375 380
Val Phe Thr Val Ala Ser Ile Arg Ser Met Leu Ile Lys
385 390 395
<210> 41
<211> 1359
<212> DNA
<213> artificial
<220>
<223> RV3-Vero VP6
<400> 41
ggcttttaaa cgaagaagtc ttcgacatgg aggttctgta ttcattgtca aaaactctta 60
aagatgctag ggataagatt gttgaaggta cattatattc taatgttagt gatctcattc 120
agcaatttaa tcaaatgata gtaaccatga atggaaatga ctttcaaact ggaggaattg 180
gcaatttacc tattagaaat tggacatttg actttggtct actaggtact acgctgttaa 240
accttgatgc taattacgtt gagaccgcaa gaactacaat taagtatttt attgacttta 300
ttgataatgt atgtatggat gaaatggcaa gagagtctca aagaaatgga gtagctccac 360
aatctgaggc attgaggaag ctagccggta ttaaatttaa aagaataaat tttaataatt 420
catcagaata tatagaaaat tggaatttac aaaatagaag acagcgtacc ggatttgttt 480
tccataaacc taatatattt ccatactcag catcatttac tttaaatagg tctcaaccaa 540
tgcatgacaa tttaatggga accatgtggc ttaacgctgg atcagaaatt caagtggctg 600
gatttgacta ctcgtgtgcc ctaaatgctc cagcaaatat tcagcagttt gaacatattg 660
tccagcttag gcgtgcgcta actacagcta ctataacttt gctacctgat gcagaaagat 720
ttagttttcc aagagttatt aattcagcag atggcgcaac cacatggttc tttaatccaa 780
ttatcctaag accaaacaat gtagaggtag aatttttact gaatggacaa attattaata 840
catatcaagc tagatttggc actattatcg caagaaattt tgatacaatt cgtctatcat 900
tccaattaat gcgtccacca aacatgacgc cagccgtaaa tgcattattt ccgcaagcac 960
aaccttttca acatcatgca acagttggac ttacgttacg tattgagtct gcagtttgtg 1020
aatcagtgct tgcggatgca aatgaaactt tattggcgaa tgttactgca gtacgtcaag 1080
agtatgctat accggttgga ccagtatttc caccaggcat gaattggact gagctgatta 1140
ctaactattc accatccagg gaagataatt tgcaacgtgt ctttacagta gcctctatca 1200
gaagcatgtt aattaagtga ggaccagact aaccatctgg tatccaatct taattagcat 1260
gtagctatgt caagtcattc agactctaca agtaaggaca tgatttcatg ttcgctacgt 1320
agagtaactg catgaatgat gtagtgagag gatgtgacc 1359
<210> 42
<211> 397
<212> PRT
<213> artificial
<220>
<223> RV3-Vero VP6
<400> 42
Met Glu Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp
1 5 10 15
Lys Ile Val Glu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu Ile Gln
20 25 30
Gln Phe Asn Gln Met Ile Val Thr Met Asn Gly Asn Asp Phe Gln Thr
35 40 45
Gly Gly Ile Gly Asn Leu Pro Ile Arg Asn Trp Thr Phe Asp Phe Gly
50 55 60
Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val Glu Thr
65 70 75 80
Ala Arg Thr Thr Ile Lys Tyr Phe Ile Asp Phe Ile Asp Asn Val Cys
85 90 95
Met Asp Glu Met Ala Arg Glu Ser Gln Arg Asn Gly Val Ala Pro Gln
100 105 110
Ser Glu Ala Leu Arg Lys Leu Ala Gly Ile Lys Phe Lys Arg Ile Asn
115 120 125
Phe Asn Asn Ser Ser Glu Tyr Ile Glu Asn Trp Asn Leu Gln Asn Arg
130 135 140
Arg Gln Arg Thr Gly Phe Val Phe His Lys Pro Asn Ile Phe Pro Tyr
145 150 155 160
Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Met His Asp Asn Leu
165 170 175
Met Gly Thr Met Trp Leu Asn Ala Gly Ser Glu Ile Gln Val Ala Gly
180 185 190
Phe Asp Tyr Ser Cys Ala Leu Asn Ala Pro Ala Asn Ile Gln Gln Phe
195 200 205
Glu His Ile Val Gln Leu Arg Arg Ala Leu Thr Thr Ala Thr Ile Thr
210 215 220
Leu Leu Pro Asp Ala Glu Arg Phe Ser Phe Pro Arg Val Ile Asn Ser
225 230 235 240
Ala Asp Gly Ala Thr Thr Trp Phe Phe Asn Pro Ile Ile Leu Arg Pro
245 250 255
Asn Asn Val Glu Val Glu Phe Leu Leu Asn Gly Gln Ile Ile Asn Thr
260 265 270
Tyr Gln Ala Arg Phe Gly Thr Ile Ile Ala Arg Asn Phe Asp Thr Ile
275 280 285
Arg Leu Ser Phe Gln Leu Met Arg Pro Pro Asn Met Thr Pro Ala Val
290 295 300
Asn Ala Leu Phe Pro Gln Ala Gln Pro Phe Gln His His Ala Thr Val
305 310 315 320
Gly Leu Thr Leu Arg Ile Glu Ser Ala Val Cys Glu Ser Val Leu Ala
325 330 335
Asp Ala Asn Glu Thr Leu Leu Ala Asn Val Thr Ala Val Arg Gln Glu
340 345 350
Tyr Ala Ile Pro Val Gly Pro Val Phe Pro Pro Gly Met Asn Trp Thr
355 360 365
Glu Leu Ile Thr Asn Tyr Ser Pro Ser Arg Glu Asp Asn Leu Gln Arg
370 375 380
Val Phe Thr Val Ala Ser Ile Arg Ser Met Leu Ile Lys
385 390 395
<210> 43
<211> 964
<212> DNA
<213> artificial
<220>
<223> MA104-adapted RV3 NSP3
<400> 43
ttttaatgct tttcagtggt tgctgctcaa gatggagtct actcagcaga tggtaagctc 60
tattattaac acttcttttg aagctgcagt tgttgctgcc acttcaacat tagaattaat 120
gggtattcaa tatgattaca atgaagtatt tactagagtt aaaagtaaat ttgattatgt 180
gatggatgac tctggtgtta aaaacaatct tttgggtaaa gctataacta ttgatcaggc 240
gttaaatgga aagtttggct cagctattag aaatagaaat tggatgactg attctaaaac 300
ggttgctaaa ttagatgaag acgtgaataa acttagaatg acattatctt ctaaaggaat 360
cgaccaaaag atgagagtac ttaatgcttg ttttagtgta aaaagaatac caggaaaatc 420
atcatcaata attaaatgct ctagacttat gaaggataaa atagaacgtg gagaagttga 480
ggttgatgat tcatatgttg atgagaaaat ggaaattgat actattgatt ggaaatctcg 540
ttatgatcag ttagaaaaaa gatttgagtc actaaaacaa agagttaatg agaaatacaa 600
tacttgggta caaaaagcga agaaagtaaa tgaaaatatg tactctcttc agaatgtcat 660
ttcacaacag caaaaccaaa tagcagatct tcaacaatat tgtaataaat tggaagctga 720
tttgcaaggc aaatttagtt cattagtgtc atcagttgag tggtatctaa ggtctatgga 780
attgccaaat gatgtaaaga atgatattga acagcagtta aattcaattg atttaattaa 840
tcccattaat gctatagatg atatcgaatc attgattaga aatttaattc aagattatga 900
cagaacattt ttaatgttaa aaggactgtt gaagcaatgc aactatgaat atacatatga 960
gtag 964
<210> 44
<211> 310
<212> PRT
<213> artificial
<220>
<223> MA104-derived RV3 NSP3
<400> 44
Met Glu Ser Thr Gln Gln Met Val Ser Ser Ile Ile Asn Thr Ser Phe
1 5 10 15
Glu Ala Ala Val Val Ala Ala Thr Ser Thr Leu Glu Leu Met Gly Ile
20 25 30
Gln Tyr Asp Tyr Asn Glu Val Phe Thr Arg Val Lys Ser Lys Phe Asp
35 40 45
Tyr Val Met Asp Asp Ser Gly Val Lys Asn Asn Leu Leu Gly Lys Ala
50 55 60
Ile Thr Ile Asp Gln Ala Leu Asn Gly Lys Phe Gly Ser Ala Ile Arg
65 70 75 80
Asn Arg Asn Trp Met Thr Asp Ser Lys Thr Val Ala Lys Leu Asp Glu
85 90 95
Asp Val Asn Lys Leu Arg Met Thr Leu Ser Ser Lys Gly Ile Asp Gln
100 105 110
Lys Met Arg Val Leu Asn Ala Cys Phe Ser Val Lys Arg Ile Pro Gly
115 120 125
Lys Ser Ser Ser Ile Ile Lys Cys Ser Arg Leu Met Lys Asp Lys Ile
130 135 140
Glu Arg Gly Glu Val Glu Val Asp Asp Ser Tyr Val Asp Glu Lys Met
145 150 155 160
Glu Ile Asp Thr Ile Asp Trp Lys Ser Arg Tyr Asp Gln Leu Glu Lys
165 170 175
Arg Phe Glu Ser Leu Lys Gln Arg Val Asn Glu Lys Tyr Asn Thr Trp
180 185 190
Val Gln Lys Ala Lys Lys Val Asn Glu Asn Met Tyr Ser Leu Gln Asn
195 200 205
Val Ile Ser Gln Gln Gln Asn Gln Ile Ala Asp Leu Gln Gln Tyr Cys
210 215 220
Asn Lys Leu Glu Ala Asp Leu Gln Gly Lys Phe Ser Ser Leu Val Ser
225 230 235 240
Ser Val Glu Trp Tyr Leu Arg Ser Met Glu Leu Pro Asn Asp Val Lys
245 250 255
Asn Asp Ile Glu Gln Gln Leu Asn Ser Ile Asp Leu Ile Asn Pro Ile
260 265 270
Asn Ala Ile Asp Asp Ile Glu Ser Leu Ile Arg Asn Leu Ile Gln Asp
275 280 285
Tyr Asp Arg Thr Phe Leu Met Leu Lys Gly Leu Leu Lys Gln Cys Asn
290 295 300
Tyr Glu Tyr Thr Tyr Glu
305 310
<210> 45
<211> 1070
<212> DNA
<213> artificial
<220>
<223> RV3-Vero NSP3
<400> 45
ggcttttaat gcttttcagt ggttgctgct caagatggag tctactcagc agatggtaag 60
ctctattatt aacacttctt ttgaagctgc agttgttgct gccacttcaa cattagaatt 120
aatgggtatt caatatgatt acaatgaagt atttactaga gttaaaagta aatttgatta 180
tgtgatggat gactctggtg ttaaaaacaa tcttttgggt aaagctataa ctattgatca 240
ggcgttaaat ggaaagtttg gctcagctat tagaaataga aattggatga ctgattctaa 300
aacggttgct aaattagatg aagacgtgaa taaacttaga atgacattat cttctaaagg 360
aatcgaccaa aagatgagag tacttaatgc ttgttttagt gtaaaaagaa taccaggaaa 420
atcatcatca ataattaaat gctctagact tatgaaggat aaaatagaac gtggagaagt 480
tgaggttgat gattcatatg ttgatgagaa aatggaaatt gatactattg attggaaatc 540
tcgttatgat cagttagaaa aaagatttga gtcactaaaa caaagagtta atgagaaata 600
caatacttgg gtacaaaaag cgaagaaagt aaatgaaaat atgtactctc ttcagaatgt 660
catttcacaa cagcaaaacc aaatagcaga tcttcaacaa tattgtaata aattggaagc 720
tgatttgcaa ggcaaattta gttcattagt gtcatcagtt gagtggtatc taaggtctat 780
ggaattgcca aatgatgtaa agaatgatat tgaacagcag ttaaattcaa ttgatttaat 840
taatcccatt aatgctatag atgatatcga atcattgatt agaaatttaa ttcaagatta 900
tgacagaaca tttttaatgt taaaaggact gttgaagcaa tgcaactatg aatatacata 960
tgagtagtca cataattaaa agtattaacc atctacacat gaccctctat gagcacaata 1020
gttaaaagct aacactgtca aaaacctaaa tggctatagg ggcgttatgt 1070
<210> 46
<211> 310
<212> PRT
<213> artificial
<220>
<223> RV3 Vero NSP3
<400> 46
Met Glu Ser Thr Gln Gln Met Val Ser Ser Ile Ile Asn Thr Ser Phe
1 5 10 15
Glu Ala Ala Val Val Ala Ala Thr Ser Thr Leu Glu Leu Met Gly Ile
20 25 30
Gln Tyr Asp Tyr Asn Glu Val Phe Thr Arg Val Lys Ser Lys Phe Asp
35 40 45
Tyr Val Met Asp Asp Ser Gly Val Lys Asn Asn Leu Leu Gly Lys Ala
50 55 60
Ile Thr Ile Asp Gln Ala Leu Asn Gly Lys Phe Gly Ser Ala Ile Arg
65 70 75 80
Asn Arg Asn Trp Met Thr Asp Ser Lys Thr Val Ala Lys Leu Asp Glu
85 90 95
Asp Val Asn Lys Leu Arg Met Thr Leu Ser Ser Lys Gly Ile Asp Gln
100 105 110
Lys Met Arg Val Leu Asn Ala Cys Phe Ser Val Lys Arg Ile Pro Gly
115 120 125
Lys Ser Ser Ser Ile Ile Lys Cys Ser Arg Leu Met Lys Asp Lys Ile
130 135 140
Glu Arg Gly Glu Val Glu Val Asp Asp Ser Tyr Val Asp Glu Lys Met
145 150 155 160
Glu Ile Asp Thr Ile Asp Trp Lys Ser Arg Tyr Asp Gln Leu Glu Lys
165 170 175
Arg Phe Glu Ser Leu Lys Gln Arg Val Asn Glu Lys Tyr Asn Thr Trp
180 185 190
Val Gln Lys Ala Lys Lys Val Asn Glu Asn Met Tyr Ser Leu Gln Asn
195 200 205
Val Ile Ser Gln Gln Gln Asn Gln Ile Ala Asp Leu Gln Gln Tyr Cys
210 215 220
Asn Lys Leu Glu Ala Asp Leu Gln Gly Lys Phe Ser Ser Leu Val Ser
225 230 235 240
Ser Val Glu Trp Tyr Leu Arg Ser Met Glu Leu Pro Asn Asp Val Lys
245 250 255
Asn Asp Ile Glu Gln Gln Leu Asn Ser Ile Asp Leu Ile Asn Pro Ile
260 265 270
Asn Ala Ile Asp Asp Ile Glu Ser Leu Ile Arg Asn Leu Ile Gln Asp
275 280 285
Tyr Asp Arg Thr Phe Leu Met Leu Lys Gly Leu Leu Lys Gln Cys Asn
290 295 300
Tyr Glu Tyr Thr Tyr Glu
305 310
<210> 47
<211> 3267
<212> DNA
<213> artificial
<220>
<223> MA104-adapted RV3 VP1
<400> 47
atggggaagt acaatctaat cttgtcagaa tatttatcat ttgtttataa ttcacaatct 60
gcagttcaaa taccaattta ttattcttcc aattcagaat tagaaaaaag atgtattgaa 120
tttcatgcta aatgtgttga cagttcaaaa aaaggtctat cattaaaacc tttattcgaa 180
aaatataaag atgtaataga taatgcaact ttactatctc tattatcata ttcttatgat 240
aaatacaacg ctgtagaaag gaaactagtc aattatgcta aaggtaaacc attagaggct 300
gatttaacgg caaacgagct tgattatgaa aataataaaa taacttctga attgtttcaa 360
tcagctgaag aatatactga ttcattaatg gatcctgcta tattaacttc gttatcttca 420
aatctaaatg cagtcatgtt ttggttagaa cgacactcaa atgacgttgc tgatgcaaac 480
aaaatttata aacgtagact ggatctattt accatagtag catctacaat aaataaatat 540
ggagtaccta gacataatga aaaatataga tatgaatacg aggtgatgaa ggataaaccg 600
tactacttag taacctgggc caactcatct atagaaatgc ttatgtcagt gttttcccat 660
gaagattatt taatagcaaa agagttaata atcttatcat attcaaatag atcaacgtta 720
gcaaaactag tttcatctcc aatgtcgata ttggttgcat taatagatat caatggtacg 780
tttattacaa atgaagagtt agaactcgag ttttcagata aatatgttaa ggcaattgtg 840
cctgatcaaa ttttcgatga attacaggaa atgatcgaca atatgaggaa agttggttta 900
gtagatatac caaaaatgat tcaagaatgg ttaattgatt gttcattaga gaaatttaca 960
ttgatgtcaa aaatttattc ttggtcattt catgttggtt ttagaaaaca aaagatgatt 1020
gatgcagcat tagaccaatt gaaaacagag tacactgaag atgtagataa tgagatgtat 1080
aatgagtata cgatgttaat tagagatgaa atagttaaaa tgctagaagt accagttaaa 1140
catgatgatc atctacttcg cgattcagaa ttagccggat tgttatcaat gtcatcagct 1200
tcaaatggtg aatcaaggca acttaaattt ggtcgtaaaa caatattttc aactaagaaa 1260
aatatgcatg tcatggatga tatcgcacat ggaaaatata ctccgggtgt cattcctcca 1320
gtaaatgtag atagaccaat tccactaggt cgtagagatg ttcctggacg aagaacaaga 1380
attatattca tattaccata tgaatacttt attgcgcagc acgctgtcgt agaaaaaatg 1440
ttatcatacg caaagcatac tagagagtac gcagaatttt attcgcagtc aaatcaattg 1500
ctatcatatg gtgatgttac aagattctta tccaataatt ctatggtatt atacacagac 1560
gtttcacaat gggattcgtc acaacataac acacaaccat ttagaaaagg aataattatg 1620
ggtttagata tgctatccaa tatgactaat gatccaaaag tagtacaaac gctaaattta 1680
tataaacaaa cacaaattaa tctcatggat tcatatgttc aaatacctga cggtaatgta 1740
ataaaaaaga ttcagtatgg tgctgttgct tcaggagaaa aacaaaccaa ggcagctaat 1800
tctatagcta atttggcact cattaaaacg gtattgtcaa gaattgcaaa taaatattct 1860
tttataacca aaataatcag agtcgatggt gatgataatt atgcagtact acaatttaat 1920
accgatgtca ctaaacaaat ggtccaagat gtgtcaaacg atgtgaggta tatatattct 1980
agaatgaatg ctaaagttaa agcactggta tctacagtcg gtatcgaaat agcaaaaaga 2040
tatatagcag gaggaaaaat atttttcaga gctggtataa acttattaaa taatgagaaa 2100
cgtggacaaa gtacacaatg ggatcaagca gctattttat actcaaacta cattgttaac 2160
aaattacgag gatttgagac tgatagagaa tttgtactaa ctaaaattat acaaatgaca 2220
tctatagcta ttactggatc actaaggcta tttccgtcag aacgagtgtt aactactaat 2280
tctacattca aagtatttga ctcagaagat ttcattatag agtatggaac aactgacgat 2340
gaagtatata tacaaagagc atttatgtca ttatctagtc aaaagtcagg aatagctgat 2400
gaaattgctt cttcacaaac atttaaaaat tatgtcaata aattatctga ccaactatta 2460
gtatcaaaaa acgtaattgt atctaaaggt atagctgtaa cagaaaaggc gaaattgaat 2520
tcatatgcac cagtttattt agaaaaacgt cgtgcacaaa tatcagcgct attaactatg 2580
ttgcagaaac cagtgtcatt taaatcaaat aaaattacta ttaatgacat tttgcgtgac 2640
ataaaaccat tttttgtcac ttctgaagct aatttgtcaa ttcaatacag aaaatttatg 2700
ccaacactac ctgataatgt ccaatatgtt atacaatgta taggatcgag gacgtaccag 2760
atagaagata gtgggtcaaa atcatcgatt tcgaagttaa tatcaaaata ttcagtttac 2820
aaaccatcga ttgaagaact atataaagtt atatctttaa gagaacaaga aatacagttg 2880
tatttagttt cattaggagt tccgctagtt gatgcaagca cgtacgtcgg gtcaagaata 2940
tattcgcaag ataaatataa aatattagaa tcttacgtat ataatttatt atcaattaat 3000
tatggatgtt atcaattatt cgattttaat tctccagatt tagagagact tatacgaatt 3060
ccttttaaag gtaagatacc agctgtaaca tttatattac atctttacgc taaacttgaa 3120
ataataaact atgctattaa gaacggagct tggatcagct tgttttgtaa ctatccaaaa 3180
tctgagatga ttaaattatg gaagaaaatg tggaatataa cagcattacg gtctccctat 3240
actagtgcga atttctttca agattag 3267
<210> 48
<211> 1088
<212> PRT
<213> artificial
<220>
<223> MA104-adapted RV3 VP1
<400> 48
Met Gly Lys Tyr Asn Leu Ile Leu Ser Glu Tyr Leu Ser Phe Val Tyr
1 5 10 15
Asn Ser Gln Ser Ala Val Gln Ile Pro Ile Tyr Tyr Ser Ser Asn Ser
20 25 30
Glu Leu Glu Lys Arg Cys Ile Glu Phe His Ala Lys Cys Val Asp Ser
35 40 45
Ser Lys Lys Gly Leu Ser Leu Lys Pro Leu Phe Glu Lys Tyr Lys Asp
50 55 60
Val Ile Asp Asn Ala Thr Leu Leu Ser Leu Leu Ser Tyr Ser Tyr Asp
65 70 75 80
Lys Tyr Asn Ala Val Glu Arg Lys Leu Val Asn Tyr Ala Lys Gly Lys
85 90 95
Pro Leu Glu Ala Asp Leu Thr Ala Asn Glu Leu Asp Tyr Glu Asn Asn
100 105 110
Lys Ile Thr Ser Glu Leu Phe Gln Ser Ala Glu Glu Tyr Thr Asp Ser
115 120 125
Leu Met Asp Pro Ala Ile Leu Thr Ser Leu Ser Ser Asn Leu Asn Ala
130 135 140
Val Met Phe Trp Leu Glu Arg His Ser Asn Asp Val Ala Asp Ala Asn
145 150 155 160
Lys Ile Tyr Lys Arg Arg Leu Asp Leu Phe Thr Ile Val Ala Ser Thr
165 170 175
Ile Asn Lys Tyr Gly Val Pro Arg His Asn Glu Lys Tyr Arg Tyr Glu
180 185 190
Tyr Glu Val Met Lys Asp Lys Pro Tyr Tyr Leu Val Thr Trp Ala Asn
195 200 205
Ser Ser Ile Glu Met Leu Met Ser Val Phe Ser His Glu Asp Tyr Leu
210 215 220
Ile Ala Lys Glu Leu Ile Ile Leu Ser Tyr Ser Asn Arg Ser Thr Leu
225 230 235 240
Ala Lys Leu Val Ser Ser Pro Met Ser Ile Leu Val Ala Leu Ile Asp
245 250 255
Ile Asn Gly Thr Phe Ile Thr Asn Glu Glu Leu Glu Leu Glu Phe Ser
260 265 270
Asp Lys Tyr Val Lys Ala Ile Val Pro Asp Gln Ile Phe Asp Glu Leu
275 280 285
Gln Glu Met Ile Asp Asn Met Arg Lys Val Gly Leu Val Asp Ile Pro
290 295 300
Lys Met Ile Gln Glu Trp Leu Ile Asp Cys Ser Leu Glu Lys Phe Thr
305 310 315 320
Leu Met Ser Lys Ile Tyr Ser Trp Ser Phe His Val Gly Phe Arg Lys
325 330 335
Gln Lys Met Ile Asp Ala Ala Leu Asp Gln Leu Lys Thr Glu Tyr Thr
340 345 350
Glu Asp Val Asp Asn Glu Met Tyr Asn Glu Tyr Thr Met Leu Ile Arg
355 360 365
Asp Glu Ile Val Lys Met Leu Glu Val Pro Val Lys His Asp Asp His
370 375 380
Leu Leu Arg Asp Ser Glu Leu Ala Gly Leu Leu Ser Met Ser Ser Ala
385 390 395 400
Ser Asn Gly Glu Ser Arg Gln Leu Lys Phe Gly Arg Lys Thr Ile Phe
405 410 415
Ser Thr Lys Lys Asn Met His Val Met Asp Asp Ile Ala His Gly Lys
420 425 430
Tyr Thr Pro Gly Val Ile Pro Pro Val Asn Val Asp Arg Pro Ile Pro
435 440 445
Leu Gly Arg Arg Asp Val Pro Gly Arg Arg Thr Arg Ile Ile Phe Ile
450 455 460
Leu Pro Tyr Glu Tyr Phe Ile Ala Gln His Ala Val Val Glu Lys Met
465 470 475 480
Leu Ser Tyr Ala Lys His Thr Arg Glu Tyr Ala Glu Phe Tyr Ser Gln
485 490 495
Ser Asn Gln Leu Leu Ser Tyr Gly Asp Val Thr Arg Phe Leu Ser Asn
500 505 510
Asn Ser Met Val Leu Tyr Thr Asp Val Ser Gln Trp Asp Ser Ser Gln
515 520 525
His Asn Thr Gln Pro Phe Arg Lys Gly Ile Ile Met Gly Leu Asp Met
530 535 540
Leu Ser Asn Met Thr Asn Asp Pro Lys Val Val Gln Thr Leu Asn Leu
545 550 555 560
Tyr Lys Gln Thr Gln Ile Asn Leu Met Asp Ser Tyr Val Gln Ile Pro
565 570 575
Asp Gly Asn Val Ile Lys Lys Ile Gln Tyr Gly Ala Val Ala Ser Gly
580 585 590
Glu Lys Gln Thr Lys Ala Ala Asn Ser Ile Ala Asn Leu Ala Leu Ile
595 600 605
Lys Thr Val Leu Ser Arg Ile Ala Asn Lys Tyr Ser Phe Ile Thr Lys
610 615 620
Ile Ile Arg Val Asp Gly Asp Asp Asn Tyr Ala Val Leu Gln Phe Asn
625 630 635 640
Thr Asp Val Thr Lys Gln Met Val Gln Asp Val Ser Asn Asp Val Arg
645 650 655
Tyr Ile Tyr Ser Arg Met Asn Ala Lys Val Lys Ala Leu Val Ser Thr
660 665 670
Val Gly Ile Glu Ile Ala Lys Arg Tyr Ile Ala Gly Gly Lys Ile Phe
675 680 685
Phe Arg Ala Gly Ile Asn Leu Leu Asn Asn Glu Lys Arg Gly Gln Ser
690 695 700
Thr Gln Trp Asp Gln Ala Ala Ile Leu Tyr Ser Asn Tyr Ile Val Asn
705 710 715 720
Lys Leu Arg Gly Phe Glu Thr Asp Arg Glu Phe Val Leu Thr Lys Ile
725 730 735
Ile Gln Met Thr Ser Ile Ala Ile Thr Gly Ser Leu Arg Leu Phe Pro
740 745 750
Ser Glu Arg Val Leu Thr Thr Asn Ser Thr Phe Lys Val Phe Asp Ser
755 760 765
Glu Asp Phe Ile Ile Glu Tyr Gly Thr Thr Asp Asp Glu Val Tyr Ile
770 775 780
Gln Arg Ala Phe Met Ser Leu Ser Ser Gln Lys Ser Gly Ile Ala Asp
785 790 795 800
Glu Ile Ala Ser Ser Gln Thr Phe Lys Asn Tyr Val Asn Lys Leu Ser
805 810 815
Asp Gln Leu Leu Val Ser Lys Asn Val Ile Val Ser Lys Gly Ile Ala
820 825 830
Val Thr Glu Lys Ala Lys Leu Asn Ser Tyr Ala Pro Val Tyr Leu Glu
835 840 845
Lys Arg Arg Ala Gln Ile Ser Ala Leu Leu Thr Met Leu Gln Lys Pro
850 855 860
Val Ser Phe Lys Ser Asn Lys Ile Thr Ile Asn Asp Ile Leu Arg Asp
865 870 875 880
Ile Lys Pro Phe Phe Val Thr Ser Glu Ala Asn Leu Ser Ile Gln Tyr
885 890 895
Arg Lys Phe Met Pro Thr Leu Pro Asp Asn Val Gln Tyr Val Ile Gln
900 905 910
Cys Ile Gly Ser Arg Thr Tyr Gln Ile Glu Asp Ser Gly Ser Lys Ser
915 920 925
Ser Ile Ser Lys Leu Ile Ser Lys Tyr Ser Val Tyr Lys Pro Ser Ile
930 935 940
Glu Glu Leu Tyr Lys Val Ile Ser Leu Arg Glu Gln Glu Ile Gln Leu
945 950 955 960
Tyr Leu Val Ser Leu Gly Val Pro Leu Val Asp Ala Ser Thr Tyr Val
965 970 975
Gly Ser Arg Ile Tyr Ser Gln Asp Lys Tyr Lys Ile Leu Glu Ser Tyr
980 985 990
Val Tyr Asn Leu Leu Ser Ile Asn Tyr Gly Cys Tyr Gln Leu Phe Asp
995 1000 1005
Phe Asn Ser Pro Asp Leu Glu Arg Leu Ile Arg Ile Pro Phe Lys
1010 1015 1020
Gly Lys Ile Pro Ala Val Thr Phe Ile Leu His Leu Tyr Ala Lys
1025 1030 1035
Leu Glu Ile Ile Asn Tyr Ala Ile Lys Asn Gly Ala Trp Ile Ser
1040 1045 1050
Leu Phe Cys Asn Tyr Pro Lys Ser Glu Met Ile Lys Leu Trp Lys
1055 1060 1065
Lys Met Trp Asn Ile Thr Ala Leu Arg Ser Pro Tyr Thr Ser Ala
1070 1075 1080
Asn Phe Phe Gln Asp
1085
<210> 49
<211> 3322
<212> DNA
<213> artificial
<220>
<223> Vero-adatped RV3 VP1
<400> 49
tgtaaaacga cggccagtgg ctattaaagc tgtacaatgg ggaagtacaa tctaatcttg 60
tcagaatatt tatcatttgt ttataattca caatctgcag ttcaaatacc aatttattat 120
tcttccaatt cagaattaga aaaaagatgt attgaatttc atgctaaatg tgttgacagt 180
tcaaaaaaag gtctatcatt aaaaccttta ttcgaaaaat ataaagatgt aatagataat 240
gcaactttac tatctctatt atcatattct tatgataaat acaacgctgt agaaaggaaa 300
ctagtcaatt atgctaaagg taaaccatta gaggctgatt taacggcaaa cgagcttgat 360
tatgaaaata ataaaataac ttctgaattg tttcaatcag ctgaagaata tactgattca 420
ttaatggatc ctgctatatt aacttcgtta tcttcaaatc taaatgcagt catgttttgg 480
ttagaacgac actcaaatga cgttgctgat gcaaacaaaa tttataaacg tagactggat 540
ctatttacca tagtagcatc tacaataaat aaatatggag tacctagaca taatgaaaaa 600
tatagatatg aatacgaggt gatgaaggat aaaccgtact acttagtaac ctgggccaac 660
tcatctatag aaatgcttat gtcagtgttt tcccatgaag attatttaat agcaaaagag 720
ttaataatct tatcatattc aaatagatca acgttagcaa aactagtttc atctccaatg 780
tcgatattgg ttgcattaat agatatcaat ggtacgttta ttacaaatga agagttagaa 840
ctcgagtttt cagataaata tgttaaggca attgtgcctg atcaaatttt cgatgaatta 900
caggaaatga tcgacaatat gaggaaagtt ggtttagtag atataccaaa aatgattcaa 960
gaatggttaa ttgattgttc attagagaaa tttacattga tgtcaaaaat ttattcttgg 1020
tcatttcatg ttggttttag aaaacaaaag atgattgatg cagcattaga ccaattgaaa 1080
acagagtaca ctgaagatgt agataatgag atgtataatg agtatacgat gttaattaga 1140
gatgaaatag ttaaaatgct agaagtacca gttaaacatg atgatcatct acttcgcgat 1200
tcagaattag ccggattgtt atcaatgtca tcagcttcaa atggtgaatc aaggcaactt 1260
aaatttggtc gtaaaacaat attttcaact aagaaaaata tgcatgtcat ggatgatatc 1320
gcacatggaa aatatactcc gggtgtcatt cctccagtaa atgtagatag accaattcca 1380
ctaggtcgta gagatgttcc tggacgaaga acaagaatta tattcatatt accatatgaa 1440
tactttattg cgcagcacgc tgtcgtagaa aaaatgttat catacgcaaa gcatactaga 1500
gagtacgcag aattttattc gcagtcaaat caattgctat catatggtga tgttacaaga 1560
ttcttatcca ataattctat ggtattatac acagacgttt cacaatggga ttcgtcacaa 1620
cataacacac aaccatttag aaaaggaata attatgggtt tagatatgct atccaatatg 1680
actaatgatc caaaagtagt acaaacgcta aatttatata aacaaacaca aattaatctc 1740
atggattcat atgttcaaat acctgacggt aatgtaataa aaaagattca gtatggtgct 1800
gttgcttcag gagaaaaaca aaccaaggca gctaattcta tagctaattt ggcactcatt 1860
aaaacggtat tgtcaagaat tgcaaataaa tattctttta taaccaaaat aatcagagtc 1920
gatggtgatg ataattatgc agtactacaa tttaataccg atgtcactaa acaaatggtc 1980
caagatgtgt caaacgatgt gaggtatata tattctagaa tgaatgctaa agttaaagca 2040
ctggtatcta cagtcggtat cgaaatagca aaaagatata tagcaggagg aaaaatattt 2100
ttcagagctg gtataaactt attaaataat gagaaacgtg gacaaagtac acaatgggat 2160
caagcagcta ttttatactc aaactacatt gttaacaaat tacgaggatt tgagactgat 2220
agagaatttg tactaactaa aattatacaa atgacatcta tagctattac tggatcacta 2280
aggctatttc cgtcagaacg agtgttaact actaattcta cattcaaagt atttgactca 2340
gaagatttca ttatagagta tggaacaact gacgatgaag tatatataca aagagcattt 2400
atgtcattat ctagtcaaaa gtcaggaata gctgatgaaa ttgcttcttc acaaacattt 2460
aaaaattatg tcaataaatt atctgaccaa ctattagtat caaaaaacgt aattgtatct 2520
aaaggtatag ctgtaacaga aaaggcgaaa ttgaattcat atgcaccagt ttatttagaa 2580
aaacgtcgtg cacaaatatc agcgctatta actatgttgc agaaaccagt gtcatttaaa 2640
tcaaataaaa ttactattaa tgacattttg cgtgacataa aaccattttt tttcacttct 2700
gaagctaatt tgtcaattca atacagaaaa tttatgccaa cactacctga taatgtccaa 2760
tatgttatac aatgtatagg atcgaggacg taccagatag aagatagtgg gtcaaaatca 2820
tcgatttcga agttaatatc aaaatattca gtttacaaac catcgattga agaactatat 2880
aaagttatat ctttaagaga acaagaaata cagttgtatt tagtttcatt aggagttccg 2940
ctagttgatg caagcacgta cgtcgggtca agaatatatt cgcaagataa atataaaata 3000
ttagaatctt acgtatataa tttattatca attaattatg gatgttatca attattcgat 3060
tttaattctc cagatttaga gagacttata cgaattcctt ttaaaggtaa gataccagct 3120
gtaacattta tattacatct ttacgctaaa cttgaaataa taaactatgc tattaagaac 3180
ggagcttgga tcagcttgtt ttgtaactat ccaaaatctg agatgattaa attatggaag 3240
aaaatgtgga atataacagc attacggtct ccctatacta gtgcgaattt ctttcaagat 3300
tagagcggct taggatgtga cc 3322
<210> 50
<211> 1088
<212> PRT
<213> artificial
<220>
<223> Vero-adapted RV3 VP1
<400> 50
Met Gly Lys Tyr Asn Leu Ile Leu Ser Glu Tyr Leu Ser Phe Val Tyr
1 5 10 15
Asn Ser Gln Ser Ala Val Gln Ile Pro Ile Tyr Tyr Ser Ser Asn Ser
20 25 30
Glu Leu Glu Lys Arg Cys Ile Glu Phe His Ala Lys Cys Val Asp Ser
35 40 45
Ser Lys Lys Gly Leu Ser Leu Lys Pro Leu Phe Glu Lys Tyr Lys Asp
50 55 60
Val Ile Asp Asn Ala Thr Leu Leu Ser Leu Leu Ser Tyr Ser Tyr Asp
65 70 75 80
Lys Tyr Asn Ala Val Glu Arg Lys Leu Val Asn Tyr Ala Lys Gly Lys
85 90 95
Pro Leu Glu Ala Asp Leu Thr Ala Asn Glu Leu Asp Tyr Glu Asn Asn
100 105 110
Lys Ile Thr Ser Glu Leu Phe Gln Ser Ala Glu Glu Tyr Thr Asp Ser
115 120 125
Leu Met Asp Pro Ala Ile Leu Thr Ser Leu Ser Ser Asn Leu Asn Ala
130 135 140
Val Met Phe Trp Leu Glu Arg His Ser Asn Asp Val Ala Asp Ala Asn
145 150 155 160
Lys Ile Tyr Lys Arg Arg Leu Asp Leu Phe Thr Ile Val Ala Ser Thr
165 170 175
Ile Asn Lys Tyr Gly Val Pro Arg His Asn Glu Lys Tyr Arg Tyr Glu
180 185 190
Tyr Glu Val Met Lys Asp Lys Pro Tyr Tyr Leu Val Thr Trp Ala Asn
195 200 205
Ser Ser Ile Glu Met Leu Met Ser Val Phe Ser His Glu Asp Tyr Leu
210 215 220
Ile Ala Lys Glu Leu Ile Ile Leu Ser Tyr Ser Asn Arg Ser Thr Leu
225 230 235 240
Ala Lys Leu Val Ser Ser Pro Met Ser Ile Leu Val Ala Leu Ile Asp
245 250 255
Ile Asn Gly Thr Phe Ile Thr Asn Glu Glu Leu Glu Leu Glu Phe Ser
260 265 270
Asp Lys Tyr Val Lys Ala Ile Val Pro Asp Gln Ile Phe Asp Glu Leu
275 280 285
Gln Glu Met Ile Asp Asn Met Arg Lys Val Gly Leu Val Asp Ile Pro
290 295 300
Lys Met Ile Gln Glu Trp Leu Ile Asp Cys Ser Leu Glu Lys Phe Thr
305 310 315 320
Leu Met Ser Lys Ile Tyr Ser Trp Ser Phe His Val Gly Phe Arg Lys
325 330 335
Gln Lys Met Ile Asp Ala Ala Leu Asp Gln Leu Lys Thr Glu Tyr Thr
340 345 350
Glu Asp Val Asp Asn Glu Met Tyr Asn Glu Tyr Thr Met Leu Ile Arg
355 360 365
Asp Glu Ile Val Lys Met Leu Glu Val Pro Val Lys His Asp Asp His
370 375 380
Leu Leu Arg Asp Ser Glu Leu Ala Gly Leu Leu Ser Met Ser Ser Ala
385 390 395 400
Ser Asn Gly Glu Ser Arg Gln Leu Lys Phe Gly Arg Lys Thr Ile Phe
405 410 415
Ser Thr Lys Lys Asn Met His Val Met Asp Asp Ile Ala His Gly Lys
420 425 430
Tyr Thr Pro Gly Val Ile Pro Pro Val Asn Val Asp Arg Pro Ile Pro
435 440 445
Leu Gly Arg Arg Asp Val Pro Gly Arg Arg Thr Arg Ile Ile Phe Ile
450 455 460
Leu Pro Tyr Glu Tyr Phe Ile Ala Gln His Ala Val Val Glu Lys Met
465 470 475 480
Leu Ser Tyr Ala Lys His Thr Arg Glu Tyr Ala Glu Phe Tyr Ser Gln
485 490 495
Ser Asn Gln Leu Leu Ser Tyr Gly Asp Val Thr Arg Phe Leu Ser Asn
500 505 510
Asn Ser Met Val Leu Tyr Thr Asp Val Ser Gln Trp Asp Ser Ser Gln
515 520 525
His Asn Thr Gln Pro Phe Arg Lys Gly Ile Ile Met Gly Leu Asp Met
530 535 540
Leu Ser Asn Met Thr Asn Asp Pro Lys Val Val Gln Thr Leu Asn Leu
545 550 555 560
Tyr Lys Gln Thr Gln Ile Asn Leu Met Asp Ser Tyr Val Gln Ile Pro
565 570 575
Asp Gly Asn Val Ile Lys Lys Ile Gln Tyr Gly Ala Val Ala Ser Gly
580 585 590
Glu Lys Gln Thr Lys Ala Ala Asn Ser Ile Ala Asn Leu Ala Leu Ile
595 600 605
Lys Thr Val Leu Ser Arg Ile Ala Asn Lys Tyr Ser Phe Ile Thr Lys
610 615 620
Ile Ile Arg Val Asp Gly Asp Asp Asn Tyr Ala Val Leu Gln Phe Asn
625 630 635 640
Thr Asp Val Thr Lys Gln Met Val Gln Asp Val Ser Asn Asp Val Arg
645 650 655
Tyr Ile Tyr Ser Arg Met Asn Ala Lys Val Lys Ala Leu Val Ser Thr
660 665 670
Val Gly Ile Glu Ile Ala Lys Arg Tyr Ile Ala Gly Gly Lys Ile Phe
675 680 685
Phe Arg Ala Gly Ile Asn Leu Leu Asn Asn Glu Lys Arg Gly Gln Ser
690 695 700
Thr Gln Trp Asp Gln Ala Ala Ile Leu Tyr Ser Asn Tyr Ile Val Asn
705 710 715 720
Lys Leu Arg Gly Phe Glu Thr Asp Arg Glu Phe Val Leu Thr Lys Ile
725 730 735
Ile Gln Met Thr Ser Ile Ala Ile Thr Gly Ser Leu Arg Leu Phe Pro
740 745 750
Ser Glu Arg Val Leu Thr Thr Asn Ser Thr Phe Lys Val Phe Asp Ser
755 760 765
Glu Asp Phe Ile Ile Glu Tyr Gly Thr Thr Asp Asp Glu Val Tyr Ile
770 775 780
Gln Arg Ala Phe Met Ser Leu Ser Ser Gln Lys Ser Gly Ile Ala Asp
785 790 795 800
Glu Ile Ala Ser Ser Gln Thr Phe Lys Asn Tyr Val Asn Lys Leu Ser
805 810 815
Asp Gln Leu Leu Val Ser Lys Asn Val Ile Val Ser Lys Gly Ile Ala
820 825 830
Val Thr Glu Lys Ala Lys Leu Asn Ser Tyr Ala Pro Val Tyr Leu Glu
835 840 845
Lys Arg Arg Ala Gln Ile Ser Ala Leu Leu Thr Met Leu Gln Lys Pro
850 855 860
Val Ser Phe Lys Ser Asn Lys Ile Thr Ile Asn Asp Ile Leu Arg Asp
865 870 875 880
Ile Lys Pro Phe Phe Phe Thr Ser Glu Ala Asn Leu Ser Ile Gln Tyr
885 890 895
Arg Lys Phe Met Pro Thr Leu Pro Asp Asn Val Gln Tyr Val Ile Gln
900 905 910
Cys Ile Gly Ser Arg Thr Tyr Gln Ile Glu Asp Ser Gly Ser Lys Ser
915 920 925
Ser Ile Ser Lys Leu Ile Ser Lys Tyr Ser Val Tyr Lys Pro Ser Ile
930 935 940
Glu Glu Leu Tyr Lys Val Ile Ser Leu Arg Glu Gln Glu Ile Gln Leu
945 950 955 960
Tyr Leu Val Ser Leu Gly Val Pro Leu Val Asp Ala Ser Thr Tyr Val
965 970 975
Gly Ser Arg Ile Tyr Ser Gln Asp Lys Tyr Lys Ile Leu Glu Ser Tyr
980 985 990
Val Tyr Asn Leu Leu Ser Ile Asn Tyr Gly Cys Tyr Gln Leu Phe Asp
995 1000 1005
Phe Asn Ser Pro Asp Leu Glu Arg Leu Ile Arg Ile Pro Phe Lys
1010 1015 1020
Gly Lys Ile Pro Ala Val Thr Phe Ile Leu His Leu Tyr Ala Lys
1025 1030 1035
Leu Glu Ile Ile Asn Tyr Ala Ile Lys Asn Gly Ala Trp Ile Ser
1040 1045 1050
Leu Phe Cys Asn Tyr Pro Lys Ser Glu Met Ile Lys Leu Trp Lys
1055 1060 1065
Lys Met Trp Asn Ile Thr Ala Leu Arg Ser Pro Tyr Thr Ser Ala
1070 1075 1080
Asn Phe Phe Gln Asp
1085
<210> 51
<211> 2673
<212> DNA
<213> artificial
<220>
<223> MA104-adapted RV3 VP2
<400> 51
atggcgtaca ggaagcgcgg agttaaacgt gaaaacttac cacaacaaaa tgaacgtctg 60
caagaaaaag aaactgaaaa taatacagac gtaaccatgg aaaataaaaa taacaataga 120
aggcagcaat tatctgacaa agtgttatca caaaaagagg aaataataac tgatgtacaa 180
gatgacatta aaatagctga tgaggttaaa aaatcatcaa aagaagagtc aaaacagtta 240
cttgaaatat taaaaacgaa agaagatcat cagaaagaag tacagtacga aattctacaa 300
aaaacaatac cgacttttga accaaaagaa tcaattttga aaaaattaga agatataaga 360
ccagaacaag ctaagaagca aatgaaattg tttagaatat ttgaaccaag acaattacca 420
atctatcgag caaatggtga gaaagagtta aggaatagat ggtattggaa attgaaaaag 480
gatacactgc cggacggaga ttatgacgta cgagagtatt ttttaaattt atatgatcaa 540
atactgatag aaatgccaga ttatttatta ctaaaagata tggctgtaga aaataaaaac 600
tctagagatg ctggtaaagt tgtagattct gagactgcaa gtatttgtga tgctatattt 660
caagatgagg agacagaggg agttattaga agattcatcg cagatatgag acaacaagtt 720
caagctgata gaaatattgt caattatcca tcaattttgc atccgattga tcacgcattt 780
aatgaatact ttttgaatca tcaattagtt gaaccattga ataacgaaat tatttttaat 840
tatataccag aaagaataag gaatgatgtt aactatattt tgaatatgga tatgaatttg 900
ccatcaacag cgcgatatat tagaccaaat ctattgcaag atagactaaa tttacatgat 960
aattttgaat cattatggga cacgataact acatcaaatt acatattagc aagatcagtc 1020
gtgcctgatt tgaaggaaaa agaattagtt tcaactgaag ctcagatcca gaaaatgtct 1080
caagatttgc agcttgaagc tttaacaata caatctgaaa cgcagtttct ggccggtata 1140
aattcacaag cagcgaatga ttgttttaaa acactgatag cagctatgtt aagccaacgt 1200
acaatgtcat tagattttgt aactacgaat tatatgtcac ttatatctgg catgtggcta 1260
ttgactgtta taccaaatga tatgtttctt cgtgagtcgc tagtcgcatg cgaattggct 1320
ataataaata ctatagttta tccagcattt ggaatgcaaa gaatgcatta tagaaatggc 1380
gatcctcaga ctccatttca aatagcagaa caacagatac aaaattttca agtagctaat 1440
tggttacatt ttattaacaa taatagattt aggcaagttg ttattgatgg agtgttaaat 1500
caaacactta atgacaacat taggaatgga caagttatta atcaattaat ggaagcatta 1560
atgcagttat ctagacaaca atttccgacc atgccagttg attataaaag atcaatccag 1620
agaggaatat tgttattatc taacagatta ggtcagttgg ttgatttaac aagattatta 1680
tcatacaatt atgaaactct aatggcttgt ataactatga acatgcaaca tgttcaaact 1740
ctcactaccg aaaaattaca attaacttct gtcacatctt tatgtatgtt aattggaaat 1800
accacagtaa ttccaggtcc acaaacacta tttcactatt ataacgtaaa tgtaaatttt 1860
cattcaaatt ataacgaacg aattaatgat gctgtggcta ttattacggc tgctaataga 1920
ttaaacttat atcagaaaaa aatgaaatca atagttgagg attttttgaa aagattgcaa 1980
atttttgatg taccacgagt gccagatgac caaacgtaca ggttgagaga cagacttaga 2040
ttattgccag ttgaaagacg aagacttgat atatttaact taatattaat gaatatggag 2100
cagatcgaac gagcttcaga caaaattgct caaggagtaa taattgctta cagagatatg 2160
cagctagaaa gagatgagat gtatgggttt gttaacatcg ctaggaacct cgatggatat 2220
caacaaatca atttggaaga gttgatgaga actggggact atggtcaaat tactaatatg 2280
ctattaaaca accagcctgt agctttagta ggagcattac catttgtgac agattcatca 2340
gttatatcgc tcattgcaaa attggatgcc acagtttttg ctcaaatagt taaacttaga 2400
aaagtggaca ctttaaaacc aatattgtat aaaataaatt ctgattctaa tgatttctat 2460
ttagttgcaa attatgattg gataccaact tcaaccacaa aagtttataa gcaagtacca 2520
caaccttttg atttcagagc gtcaatgcat atgttaacgt ctaatttaac ttttactgtt 2580
tattccgatt tgctatcttt cgtttctgca gacacggttg aacctattaa cgcaattgct 2640
tttgacaata tgcgcattat gaacgaactg taa 2673
<210> 52
<211> 890
<212> PRT
<213> artificial
<220>
<223> MA104-adapted RV3 VP2
<400> 52
Met Ala Tyr Arg Lys Arg Gly Val Lys Arg Glu Asn Leu Pro Gln Gln
1 5 10 15
Asn Glu Arg Leu Gln Glu Lys Glu Thr Glu Asn Asn Thr Asp Val Thr
20 25 30
Met Glu Asn Lys Asn Asn Asn Arg Arg Gln Gln Leu Ser Asp Lys Val
35 40 45
Leu Ser Gln Lys Glu Glu Ile Ile Thr Asp Val Gln Asp Asp Ile Lys
50 55 60
Ile Ala Asp Glu Val Lys Lys Ser Ser Lys Glu Glu Ser Lys Gln Leu
65 70 75 80
Leu Glu Ile Leu Lys Thr Lys Glu Asp His Gln Lys Glu Val Gln Tyr
85 90 95
Glu Ile Leu Gln Lys Thr Ile Pro Thr Phe Glu Pro Lys Glu Ser Ile
100 105 110
Leu Lys Lys Leu Glu Asp Ile Arg Pro Glu Gln Ala Lys Lys Gln Met
115 120 125
Lys Leu Phe Arg Ile Phe Glu Pro Arg Gln Leu Pro Ile Tyr Arg Ala
130 135 140
Asn Gly Glu Lys Glu Leu Arg Asn Arg Trp Tyr Trp Lys Leu Lys Lys
145 150 155 160
Asp Thr Leu Pro Asp Gly Asp Tyr Asp Val Arg Glu Tyr Phe Leu Asn
165 170 175
Leu Tyr Asp Gln Ile Leu Ile Glu Met Pro Asp Tyr Leu Leu Leu Lys
180 185 190
Asp Met Ala Val Glu Asn Lys Asn Ser Arg Asp Ala Gly Lys Val Val
195 200 205
Asp Ser Glu Thr Ala Ser Ile Cys Asp Ala Ile Phe Gln Asp Glu Glu
210 215 220
Thr Glu Gly Val Ile Arg Arg Phe Ile Ala Asp Met Arg Gln Gln Val
225 230 235 240
Gln Ala Asp Arg Asn Ile Val Asn Tyr Pro Ser Ile Leu His Pro Ile
245 250 255
Asp His Ala Phe Asn Glu Tyr Phe Leu Asn His Gln Leu Val Glu Pro
260 265 270
Leu Asn Asn Glu Ile Ile Phe Asn Tyr Ile Pro Glu Arg Ile Arg Asn
275 280 285
Asp Val Asn Tyr Ile Leu Asn Met Asp Met Asn Leu Pro Ser Thr Ala
290 295 300
Arg Tyr Ile Arg Pro Asn Leu Leu Gln Asp Arg Leu Asn Leu His Asp
305 310 315 320
Asn Phe Glu Ser Leu Trp Asp Thr Ile Thr Thr Ser Asn Tyr Ile Leu
325 330 335
Ala Arg Ser Val Val Pro Asp Leu Lys Glu Lys Glu Leu Val Ser Thr
340 345 350
Glu Ala Gln Ile Gln Lys Met Ser Gln Asp Leu Gln Leu Glu Ala Leu
355 360 365
Thr Ile Gln Ser Glu Thr Gln Phe Leu Ala Gly Ile Asn Ser Gln Ala
370 375 380
Ala Asn Asp Cys Phe Lys Thr Leu Ile Ala Ala Met Leu Ser Gln Arg
385 390 395 400
Thr Met Ser Leu Asp Phe Val Thr Thr Asn Tyr Met Ser Leu Ile Ser
405 410 415
Gly Met Trp Leu Leu Thr Val Ile Pro Asn Asp Met Phe Leu Arg Glu
420 425 430
Ser Leu Val Ala Cys Glu Leu Ala Ile Ile Asn Thr Ile Val Tyr Pro
435 440 445
Ala Phe Gly Met Gln Arg Met His Tyr Arg Asn Gly Asp Pro Gln Thr
450 455 460
Pro Phe Gln Ile Ala Glu Gln Gln Ile Gln Asn Phe Gln Val Ala Asn
465 470 475 480
Trp Leu His Phe Ile Asn Asn Asn Arg Phe Arg Gln Val Val Ile Asp
485 490 495
Gly Val Leu Asn Gln Thr Leu Asn Asp Asn Ile Arg Asn Gly Gln Val
500 505 510
Ile Asn Gln Leu Met Glu Ala Leu Met Gln Leu Ser Arg Gln Gln Phe
515 520 525
Pro Thr Met Pro Val Asp Tyr Lys Arg Ser Ile Gln Arg Gly Ile Leu
530 535 540
Leu Leu Ser Asn Arg Leu Gly Gln Leu Val Asp Leu Thr Arg Leu Leu
545 550 555 560
Ser Tyr Asn Tyr Glu Thr Leu Met Ala Cys Ile Thr Met Asn Met Gln
565 570 575
His Val Gln Thr Leu Thr Thr Glu Lys Leu Gln Leu Thr Ser Val Thr
580 585 590
Ser Leu Cys Met Leu Ile Gly Asn Thr Thr Val Ile Pro Gly Pro Gln
595 600 605
Thr Leu Phe His Tyr Tyr Asn Val Asn Val Asn Phe His Ser Asn Tyr
610 615 620
Asn Glu Arg Ile Asn Asp Ala Val Ala Ile Ile Thr Ala Ala Asn Arg
625 630 635 640
Leu Asn Leu Tyr Gln Lys Lys Met Lys Ser Ile Val Glu Asp Phe Leu
645 650 655
Lys Arg Leu Gln Ile Phe Asp Val Pro Arg Val Pro Asp Asp Gln Thr
660 665 670
Tyr Arg Leu Arg Asp Arg Leu Arg Leu Leu Pro Val Glu Arg Arg Arg
675 680 685
Leu Asp Ile Phe Asn Leu Ile Leu Met Asn Met Glu Gln Ile Glu Arg
690 695 700
Ala Ser Asp Lys Ile Ala Gln Gly Val Ile Ile Ala Tyr Arg Asp Met
705 710 715 720
Gln Leu Glu Arg Asp Glu Met Tyr Gly Phe Val Asn Ile Ala Arg Asn
725 730 735
Leu Asp Gly Tyr Gln Gln Ile Asn Leu Glu Glu Leu Met Arg Thr Gly
740 745 750
Asp Tyr Gly Gln Ile Thr Asn Met Leu Leu Asn Asn Gln Pro Val Ala
755 760 765
Leu Val Gly Ala Leu Pro Phe Val Thr Asp Ser Ser Val Ile Ser Leu
770 775 780
Ile Ala Lys Leu Asp Ala Thr Val Phe Ala Gln Ile Val Lys Leu Arg
785 790 795 800
Lys Val Asp Thr Leu Lys Pro Ile Leu Tyr Lys Ile Asn Ser Asp Ser
805 810 815
Asn Asp Phe Tyr Leu Val Ala Asn Tyr Asp Trp Ile Pro Thr Ser Thr
820 825 830
Thr Lys Val Tyr Lys Gln Val Pro Gln Pro Phe Asp Phe Arg Ala Ser
835 840 845
Met His Met Leu Thr Ser Asn Leu Thr Phe Thr Val Tyr Ser Asp Leu
850 855 860
Leu Ser Phe Val Ser Ala Asp Thr Val Glu Pro Ile Asn Ala Ile Ala
865 870 875 880
Phe Asp Asn Met Arg Ile Met Asn Glu Leu
885 890
<210> 53
<211> 2717
<212> DNA
<213> artificial
<220>
<223> Vero-adapted RV3 VP2
<400> 53
ggctattaaa ggttcaatgg cgtacaggaa gcgcggagtt aaacgtgaaa acttaccaca 60
acaaaatgaa cgtctgcaag aaaaagaaac tgaaaataat acagacgtaa ccatggaaaa 120
taaaaataac aatagaaggc agcaattatc tgacaaagtg ttatcacaaa aagaggaaat 180
aataactgat gtacaagatg acattaaaat agctgatgag gttaaaaaat catcaaaaga 240
agagtcaaaa cagttacttg aaatattaaa aacgaaagaa gatcatcaga aagaagtaca 300
gtacgaaatt ctacaaaaaa caataccgac ttttgaacca aaagaatcaa ttttgaaaaa 360
attagaagat ataagaccag aacaagctaa gaagcaaatg aaattgttta gaatatttga 420
accaagacaa ttaccaatct atcgagcaaa tggtgagaaa gagttaagga atagatggta 480
ttggaaattg aaaaaggata cactgccgga cggagattat gacgtacgag agtatttttt 540
aaatttatat gatcaaatac tgatagaaat gccagattat ttattactaa aagatatggc 600
tgtagaaaat aaaaactcta gagatgctgg taaagttgta gattctgaga ctgcaagtat 660
ttgtgatgct atatttcaag atgaggagac agagggagtt attagaagat tcatcgcaga 720
tatgagacaa caagttcaag ctgatagaaa tattgtcaat tatccatcaa ttttgcatcc 780
gattgatcac gcatttaatg aatacttttt gaatcatcaa ttagttgaac cattgaataa 840
cgaaattatt tttaattata taccagaaag aataaggaat gatgttaact atattttgaa 900
tatggatatg aatttgccat caacagcgcg atatattaga ccaaatctat tgcaagatag 960
actaaattta catgataatt ttgaatcatt atgggacacg ataactacat caaattacat 1020
attagcaaga tcagtcgtgc ctgatttgaa ggaaaaagaa ttagtttcaa ctgaagctca 1080
gatccagaaa atgtctcaag atttgcagct tgaagcttta acaatacaat ctgaaacgca 1140
gtttctggcc ggtataaatt cacaagcagc gaatgattgt tttaaaacac tgatagcagc 1200
tatgttaagc caacgtacaa tgtcattaga ttttgtaact acgaattata tgtcacttat 1260
atctggcatg tggctattga ctgttatacc aaatgatatg tttcttcgtg agtcgctagt 1320
cgcatgcgaa ttggctataa taaatactat agtttatcca gcatttggaa tgcaaagaat 1380
gcattataga aatggcgatc ctcagactcc atttcaaata gcagaacaac agatacaaaa 1440
ttttcaagta gctaattggt tacattttat taacaataat agatttaggc aagttgttat 1500
tgatggagtg ttaaatcaaa cacttaatga caacattagg aatggacaag ttattaatca 1560
attaatggaa gcattaatgc agttatctag acaacaattt ccgaccatgc cagttgatta 1620
taaaagatca atccagagag gaatattgtt attatctaac agattaggtc agttggttga 1680
tttaacaaga ttattatcat acaattatga aactctaatg gcttgtataa ctatgaacat 1740
gcaacatgtt caaactctca ctaccgaaaa attacaatta acttctgtca catctttatg 1800
tatgttaatt ggaaatacca cagtaattcc aggtccacaa acactatttc actattataa 1860
cgtaaatgta aattttcatt caaattataa cgaacgaatt aatgatgctg tggctattat 1920
tacggctgct aatagattaa acttatatca gaaaaaaatg aaatcaatag ttgaggattt 1980
tttgaaaaga ttgcaaattt ttgatgtacc acgagtgcca gatgaccaaa cgtacaggtt 2040
gagagacaga cttagattat tgccagttga aagacgaaga cttgatatat ttaacttaat 2100
attaatgaat atggagcaga tcgaacgagc ttcagacaaa attgctcaag gagtaataat 2160
tgcttacaga gatatgcagc tagaaagaga tgagatgtat gggtttgtta acatcgctag 2220
gaacctcgat ggatatcaac aaatcaattt ggaagagttg atgagaactg gggactatgg 2280
tcaaattact aatatgctat taaacaacca gcctgtagct ttagtaggag cattaccatt 2340
tgtgacagat tcatcagtta tatcgctcat tgcaaaattg gatgccacag tttttgctca 2400
aatagttaaa cttagaaaag tggacacttt aaaaccaata ttgtataaaa taaattctga 2460
ttctaatgat ttctatttag ttgcaaatta tgattggata ccaacttcaa ccacaaaagt 2520
ttataagcaa gtaccacaac cttttgattt cagagcgtca atgcatatgt taacgtctaa 2580
tttaactttt actgtttatt ccgatttgct atctttcgtt tctgcagaca cggttgaacc 2640
tattaacgca attgcttttg acaatatgcg cattatgaac gaactgtaaa cgccaacccc 2700
actgtggaga tatgacc 2717
<210> 54
<211> 890
<212> PRT
<213> artificial
<220>
<223> Vero-adapated RV3 VP2
<400> 54
Met Ala Tyr Arg Lys Arg Gly Val Lys Arg Glu Asn Leu Pro Gln Gln
1 5 10 15
Asn Glu Arg Leu Gln Glu Lys Glu Thr Glu Asn Asn Thr Asp Val Thr
20 25 30
Met Glu Asn Lys Asn Asn Asn Arg Arg Gln Gln Leu Ser Asp Lys Val
35 40 45
Leu Ser Gln Lys Glu Glu Ile Ile Thr Asp Val Gln Asp Asp Ile Lys
50 55 60
Ile Ala Asp Glu Val Lys Lys Ser Ser Lys Glu Glu Ser Lys Gln Leu
65 70 75 80
Leu Glu Ile Leu Lys Thr Lys Glu Asp His Gln Lys Glu Val Gln Tyr
85 90 95
Glu Ile Leu Gln Lys Thr Ile Pro Thr Phe Glu Pro Lys Glu Ser Ile
100 105 110
Leu Lys Lys Leu Glu Asp Ile Arg Pro Glu Gln Ala Lys Lys Gln Met
115 120 125
Lys Leu Phe Arg Ile Phe Glu Pro Arg Gln Leu Pro Ile Tyr Arg Ala
130 135 140
Asn Gly Glu Lys Glu Leu Arg Asn Arg Trp Tyr Trp Lys Leu Lys Lys
145 150 155 160
Asp Thr Leu Pro Asp Gly Asp Tyr Asp Val Arg Glu Tyr Phe Leu Asn
165 170 175
Leu Tyr Asp Gln Ile Leu Ile Glu Met Pro Asp Tyr Leu Leu Leu Lys
180 185 190
Asp Met Ala Val Glu Asn Lys Asn Ser Arg Asp Ala Gly Lys Val Val
195 200 205
Asp Ser Glu Thr Ala Ser Ile Cys Asp Ala Ile Phe Gln Asp Glu Glu
210 215 220
Thr Glu Gly Val Ile Arg Arg Phe Ile Ala Asp Met Arg Gln Gln Val
225 230 235 240
Gln Ala Asp Arg Asn Ile Val Asn Tyr Pro Ser Ile Leu His Pro Ile
245 250 255
Asp His Ala Phe Asn Glu Tyr Phe Leu Asn His Gln Leu Val Glu Pro
260 265 270
Leu Asn Asn Glu Ile Ile Phe Asn Tyr Ile Pro Glu Arg Ile Arg Asn
275 280 285
Asp Val Asn Tyr Ile Leu Asn Met Asp Met Asn Leu Pro Ser Thr Ala
290 295 300
Arg Tyr Ile Arg Pro Asn Leu Leu Gln Asp Arg Leu Asn Leu His Asp
305 310 315 320
Asn Phe Glu Ser Leu Trp Asp Thr Ile Thr Thr Ser Asn Tyr Ile Leu
325 330 335
Ala Arg Ser Val Val Pro Asp Leu Lys Glu Lys Glu Leu Val Ser Thr
340 345 350
Glu Ala Gln Ile Gln Lys Met Ser Gln Asp Leu Gln Leu Glu Ala Leu
355 360 365
Thr Ile Gln Ser Glu Thr Gln Phe Leu Ala Gly Ile Asn Ser Gln Ala
370 375 380
Ala Asn Asp Cys Phe Lys Thr Leu Ile Ala Ala Met Leu Ser Gln Arg
385 390 395 400
Thr Met Ser Leu Asp Phe Val Thr Thr Asn Tyr Met Ser Leu Ile Ser
405 410 415
Gly Met Trp Leu Leu Thr Val Ile Pro Asn Asp Met Phe Leu Arg Glu
420 425 430
Ser Leu Val Ala Cys Glu Leu Ala Ile Ile Asn Thr Ile Val Tyr Pro
435 440 445
Ala Phe Gly Met Gln Arg Met His Tyr Arg Asn Gly Asp Pro Gln Thr
450 455 460
Pro Phe Gln Ile Ala Glu Gln Gln Ile Gln Asn Phe Gln Val Ala Asn
465 470 475 480
Trp Leu His Phe Ile Asn Asn Asn Arg Phe Arg Gln Val Val Ile Asp
485 490 495
Gly Val Leu Asn Gln Thr Leu Asn Asp Asn Ile Arg Asn Gly Gln Val
500 505 510
Ile Asn Gln Leu Met Glu Ala Leu Met Gln Leu Ser Arg Gln Gln Phe
515 520 525
Pro Thr Met Pro Val Asp Tyr Lys Arg Ser Ile Gln Arg Gly Ile Leu
530 535 540
Leu Leu Ser Asn Arg Leu Gly Gln Leu Val Asp Leu Thr Arg Leu Leu
545 550 555 560
Ser Tyr Asn Tyr Glu Thr Leu Met Ala Cys Ile Thr Met Asn Met Gln
565 570 575
His Val Gln Thr Leu Thr Thr Glu Lys Leu Gln Leu Thr Ser Val Thr
580 585 590
Ser Leu Cys Met Leu Ile Gly Asn Thr Thr Val Ile Pro Gly Pro Gln
595 600 605
Thr Leu Phe His Tyr Tyr Asn Val Asn Val Asn Phe His Ser Asn Tyr
610 615 620
Asn Glu Arg Ile Asn Asp Ala Val Ala Ile Ile Thr Ala Ala Asn Arg
625 630 635 640
Leu Asn Leu Tyr Gln Lys Lys Met Lys Ser Ile Val Glu Asp Phe Leu
645 650 655
Lys Arg Leu Gln Ile Phe Asp Val Pro Arg Val Pro Asp Asp Gln Thr
660 665 670
Tyr Arg Leu Arg Asp Arg Leu Arg Leu Leu Pro Val Glu Arg Arg Arg
675 680 685
Leu Asp Ile Phe Asn Leu Ile Leu Met Asn Met Glu Gln Ile Glu Arg
690 695 700
Ala Ser Asp Lys Ile Ala Gln Gly Val Ile Ile Ala Tyr Arg Asp Met
705 710 715 720
Gln Leu Glu Arg Asp Glu Met Tyr Gly Phe Val Asn Ile Ala Arg Asn
725 730 735
Leu Asp Gly Tyr Gln Gln Ile Asn Leu Glu Glu Leu Met Arg Thr Gly
740 745 750
Asp Tyr Gly Gln Ile Thr Asn Met Leu Leu Asn Asn Gln Pro Val Ala
755 760 765
Leu Val Gly Ala Leu Pro Phe Val Thr Asp Ser Ser Val Ile Ser Leu
770 775 780
Ile Ala Lys Leu Asp Ala Thr Val Phe Ala Gln Ile Val Lys Leu Arg
785 790 795 800
Lys Val Asp Thr Leu Lys Pro Ile Leu Tyr Lys Ile Asn Ser Asp Ser
805 810 815
Asn Asp Phe Tyr Leu Val Ala Asn Tyr Asp Trp Ile Pro Thr Ser Thr
820 825 830
Thr Lys Val Tyr Lys Gln Val Pro Gln Pro Phe Asp Phe Arg Ala Ser
835 840 845
Met His Met Leu Thr Ser Asn Leu Thr Phe Thr Val Tyr Ser Asp Leu
850 855 860
Leu Ser Phe Val Ser Ala Asp Thr Val Glu Pro Ile Asn Ala Ile Ala
865 870 875 880
Phe Asp Asn Met Arg Ile Met Asn Glu Leu
885 890
<210> 55
<211> 2508
<212> DNA
<213> artificial
<220>
<223> MA104-adapated RV3 VP3
<400> 55
atgaaagtat tagctttaag acacagtgtg gctcaggtgt atgcagatac tcaaacctat 60
ttgcatgatg attcaaaaga tgagtatgaa aatgcatttc tcatttctaa tctaactaca 120
cataatatat tatacctgaa ttacagcctt aaaacattaa aaatattaaa tgaatcaggt 180
atagcagcag ttgaagtaca gtctccagat gaattatttg cgttaataag gtgtaatttt 240
acgtacgatt atgagaataa catagtttat ttacatgatt attcatatta tactaacaat 300
gaaataagaa cagatcaaca ttggataact aaaactgata taattgacta tctattacct 360
ggatggaaat taacatatgt aggctataat ggaaagaaca cacgaggtca ctatgatttt 420
tcatttatat gtcaaaatgc agctacagat gatgatataa taattgaata tatatactcc 480
aatgagttag actttcaaaa ctttctgtta agaaaaatta aagagagaat gaccacatct 540
cttccaattg ctagattatc aaatcgtgtg tttagagata aattatttcc atctattgta 600
aacatacata aaaaagtgat aaacgttggg ccaaggaatg aatctatgtt cacattctta 660
aattttccaa ctattaagca attttcaaac ggtgcgtata ttgtgaagca tactattaaa 720
ttgaagcagg agaaatggtt gggtaaaaga gtatcacaat ttgacatcgg acaatataaa 780
aacatgttaa atgtggttac tactatttac tattactata atttatacta ttcgaaacct 840
ataatataca tgcttggttc agctccatct tattggattt atgatattaa acagtattct 900
gattttacat ttgaaacatg ggatccatta gatactccat attctacaac acatcataaa 960
gaattatttt ttgataagga tgtcaataag cttaaagata attcagtttt atatatagac 1020
ataagaactg atagaggtaa tatggattgg aaagagtgga gaaaaatagt agaacaacaa 1080
actgttagca atctaaatat tgcgtataaa tatctatcaa caggaaaagc aaaggtgtgt 1140
tgtgttaaat taactgctat ggacctagaa ttaccaataa cagcaaaatt acttcatcat 1200
ccaactactg aagttagatc agaattttat gcaatattgg atgtatggga tattattacc 1260
attaaaagat ttataccaaa aggtgtattt tatgctttta taaataatgt aaccacagag 1320
aatgtgttta tacaacctcc tttcaaatta aaaacgtcac ctactgatta catagtggca 1380
ttgtatgctt tatccaatga cttcaactcg aggcaagatg ttattaattt gattaataaa 1440
caaaaacaat cactcattac tgttcgtata aataatactt ttaaagacga accaaaagta 1500
aattttaaaa atatttatga ttggacgttt ctgccaacag attttgaact taaagattcg 1560
ataattacat catatgatgg ttgtttaggc atattcggat tatcgatatc tttatcgtct 1620
aaacccactg gaaataatca tttatttata ataaatggaa ctgacaaata tgataaacta 1680
gaccaatatg caaatcatat gggtatttct agaagatcgc atcaaattag attctcagag 1740
tctgcgacat catattcggg atacattttt agagatcttt caaataataa tttcaatcta 1800
ataggtacta atgtggagaa ttcagtttca ggacatgtct ataatgcatt aatttactat 1860
agatataatt atgcgtttga tcttaaaaga tggatttatc tacattcaat tgggaaagtc 1920
gctgtagagg gtggaagata ttatgaacat gcaccaatcg aactgatata tgcatgccga 1980
tcagctaaag aatttgctat attacaagat gacttaacgg tattgagata cgctaatgaa 2040
attgaagggt atataagcaa agtgtacagt ataacctatg ctgacgatcc caattacttt 2100
ataggaatta aatttaatag tataccttat gaatacgatg ttaaaattcc acatttgacg 2160
ctaggagtat tatttatatc tgataatatg atacatgatg taatcacagt gttgaagaaa 2220
atgaagacag agctatttaa aatggaaatt agtactagtt atacttatat gctatctgat 2280
aatacgtatg tagcaaatgc tagtggtgtg ttatcaactt attttaaatt atataatatg 2340
ttctatagaa attatatcac gtttggacaa tcacgaatgt ttatcccaca cattacgctt 2400
agttttagca acaaacagac ggttagaata gaaagtacaa aactcagaat caattcaatt 2460
tacttaagaa agattaaagg tgaaaccgta tttgatatgt ctgagtga 2508
<210> 56
<211> 835
<212> PRT
<213> artificial
<220>
<223> MA104-adapated RV3 VP3
<400> 56
Met Lys Val Leu Ala Leu Arg His Ser Val Ala Gln Val Tyr Ala Asp
1 5 10 15
Thr Gln Thr Tyr Leu His Asp Asp Ser Lys Asp Glu Tyr Glu Asn Ala
20 25 30
Phe Leu Ile Ser Asn Leu Thr Thr His Asn Ile Leu Tyr Leu Asn Tyr
35 40 45
Ser Leu Lys Thr Leu Lys Ile Leu Asn Glu Ser Gly Ile Ala Ala Val
50 55 60
Glu Val Gln Ser Pro Asp Glu Leu Phe Ala Leu Ile Arg Cys Asn Phe
65 70 75 80
Thr Tyr Asp Tyr Glu Asn Asn Ile Val Tyr Leu His Asp Tyr Ser Tyr
85 90 95
Tyr Thr Asn Asn Glu Ile Arg Thr Asp Gln His Trp Ile Thr Lys Thr
100 105 110
Asp Ile Ile Asp Tyr Leu Leu Pro Gly Trp Lys Leu Thr Tyr Val Gly
115 120 125
Tyr Asn Gly Lys Asn Thr Arg Gly His Tyr Asp Phe Ser Phe Ile Cys
130 135 140
Gln Asn Ala Ala Thr Asp Asp Asp Ile Ile Ile Glu Tyr Ile Tyr Ser
145 150 155 160
Asn Glu Leu Asp Phe Gln Asn Phe Leu Leu Arg Lys Ile Lys Glu Arg
165 170 175
Met Thr Thr Ser Leu Pro Ile Ala Arg Leu Ser Asn Arg Val Phe Arg
180 185 190
Asp Lys Leu Phe Pro Ser Ile Val Asn Ile His Lys Lys Val Ile Asn
195 200 205
Val Gly Pro Arg Asn Glu Ser Met Phe Thr Phe Leu Asn Phe Pro Thr
210 215 220
Ile Lys Gln Phe Ser Asn Gly Ala Tyr Ile Val Lys His Thr Ile Lys
225 230 235 240
Leu Lys Gln Glu Lys Trp Leu Gly Lys Arg Val Ser Gln Phe Asp Ile
245 250 255
Gly Gln Tyr Lys Asn Met Leu Asn Val Val Thr Thr Ile Tyr Tyr Tyr
260 265 270
Tyr Asn Leu Tyr Tyr Ser Lys Pro Ile Ile Tyr Met Leu Gly Ser Ala
275 280 285
Pro Ser Tyr Trp Ile Tyr Asp Ile Lys Gln Tyr Ser Asp Phe Thr Phe
290 295 300
Glu Thr Trp Asp Pro Leu Asp Thr Pro Tyr Ser Thr Thr His His Lys
305 310 315 320
Glu Leu Phe Phe Asp Lys Asp Val Asn Lys Leu Lys Asp Asn Ser Val
325 330 335
Leu Tyr Ile Asp Ile Arg Thr Asp Arg Gly Asn Met Asp Trp Lys Glu
340 345 350
Trp Arg Lys Ile Val Glu Gln Gln Thr Val Ser Asn Leu Asn Ile Ala
355 360 365
Tyr Lys Tyr Leu Ser Thr Gly Lys Ala Lys Val Cys Cys Val Lys Leu
370 375 380
Thr Ala Met Asp Leu Glu Leu Pro Ile Thr Ala Lys Leu Leu His His
385 390 395 400
Pro Thr Thr Glu Val Arg Ser Glu Phe Tyr Ala Ile Leu Asp Val Trp
405 410 415
Asp Ile Ile Thr Ile Lys Arg Phe Ile Pro Lys Gly Val Phe Tyr Ala
420 425 430
Phe Ile Asn Asn Val Thr Thr Glu Asn Val Phe Ile Gln Pro Pro Phe
435 440 445
Lys Leu Lys Thr Ser Pro Thr Asp Tyr Ile Val Ala Leu Tyr Ala Leu
450 455 460
Ser Asn Asp Phe Asn Ser Arg Gln Asp Val Ile Asn Leu Ile Asn Lys
465 470 475 480
Gln Lys Gln Ser Leu Ile Thr Val Arg Ile Asn Asn Thr Phe Lys Asp
485 490 495
Glu Pro Lys Val Asn Phe Lys Asn Ile Tyr Asp Trp Thr Phe Leu Pro
500 505 510
Thr Asp Phe Glu Leu Lys Asp Ser Ile Ile Thr Ser Tyr Asp Gly Cys
515 520 525
Leu Gly Ile Phe Gly Leu Ser Ile Ser Leu Ser Ser Lys Pro Thr Gly
530 535 540
Asn Asn His Leu Phe Ile Ile Asn Gly Thr Asp Lys Tyr Asp Lys Leu
545 550 555 560
Asp Gln Tyr Ala Asn His Met Gly Ile Ser Arg Arg Ser His Gln Ile
565 570 575
Arg Phe Ser Glu Ser Ala Thr Ser Tyr Ser Gly Tyr Ile Phe Arg Asp
580 585 590
Leu Ser Asn Asn Asn Phe Asn Leu Ile Gly Thr Asn Val Glu Asn Ser
595 600 605
Val Ser Gly His Val Tyr Asn Ala Leu Ile Tyr Tyr Arg Tyr Asn Tyr
610 615 620
Ala Phe Asp Leu Lys Arg Trp Ile Tyr Leu His Ser Ile Gly Lys Val
625 630 635 640
Ala Val Glu Gly Gly Arg Tyr Tyr Glu His Ala Pro Ile Glu Leu Ile
645 650 655
Tyr Ala Cys Arg Ser Ala Lys Glu Phe Ala Ile Leu Gln Asp Asp Leu
660 665 670
Thr Val Leu Arg Tyr Ala Asn Glu Ile Glu Gly Tyr Ile Ser Lys Val
675 680 685
Tyr Ser Ile Thr Tyr Ala Asp Asp Pro Asn Tyr Phe Ile Gly Ile Lys
690 695 700
Phe Asn Ser Ile Pro Tyr Glu Tyr Asp Val Lys Ile Pro His Leu Thr
705 710 715 720
Leu Gly Val Leu Phe Ile Ser Asp Asn Met Ile His Asp Val Ile Thr
725 730 735
Val Leu Lys Lys Met Lys Thr Glu Leu Phe Lys Met Glu Ile Ser Thr
740 745 750
Ser Tyr Thr Tyr Met Leu Ser Asp Asn Thr Tyr Val Ala Asn Ala Ser
755 760 765
Gly Val Leu Ser Thr Tyr Phe Lys Leu Tyr Asn Met Phe Tyr Arg Asn
770 775 780
Tyr Ile Thr Phe Gly Gln Ser Arg Met Phe Ile Pro His Ile Thr Leu
785 790 795 800
Ser Phe Ser Asn Lys Gln Thr Val Arg Ile Glu Ser Thr Lys Leu Arg
805 810 815
Ile Asn Ser Ile Tyr Leu Arg Lys Ile Lys Gly Glu Thr Val Phe Asp
820 825 830
Met Ser Glu
835
<210> 57
<211> 2591
<212> DNA
<213> artificial
<220>
<223> Vero-adapated RV3 VP3
<400> 57
ggctattaaa gcagtaccag tagtgtgttt tacctctgat ggtgtaagca tgaaagtatt 60
agctttaaga cacagtgtgg ctcaggtgta tgcagatact caaacctatt tgcatgatga 120
ttcaaaagat gagtatgaaa atgcatttct catttctaat ctaactacac ataatatatt 180
atacctgaat tacagcctta aaacattaaa aatattaaat gaatcaggta tagcagcagt 240
tgaagtacag tctccagatg aattatttgc gttaataagg tgtaatttta cgtacgatta 300
tgagaataac atagtttatt tacatgatta ttcatattat accaacaatg aaataagaac 360
agatcaacat tggataacta aaactgatat aattgactat ctattacctg gatggaaatt 420
aacatatgta ggctataatg gaaagaacac acgaggtcac tatgattttt catttatatg 480
tcaaaatgca gctacagatg atgatataat aattgaatat atatactcca atgagttaga 540
ctttcaaaac tttctgttaa gaaaaattaa agagagaatg accacatctc ttccaattgc 600
tagattatca aatcgtgtgt ttagagataa attatttcca tctattgtaa acatacataa 660
aaaagtgata aacgttgggc caaggaatga atctatgttc acattcttaa attttccaac 720
tattaagcaa ttttcaaacg gtgcgtatat tgtgaagcat actattaaat tgaagcagga 780
gaaatggttg ggtaaaagag tatcacaatt tgacatcgga caatataaaa acatgttaaa 840
tgtggttact actatttact attactataa tttatactat tcgaaaccta taatatacat 900
gcttggttca gctccatctt attggattta tgatattaaa cagtattctg attttacatt 960
tgaaacatgg gatccattag atactccata ttctacaaca catcataaag aattattttt 1020
tgataaggat gtcaataagc ttaaagataa ttcagtttta tatatagaca taagaactga 1080
tagaggtaat atggattgga aagagtggag aaaaatagta gaacaacaaa ctgttagcaa 1140
tctaaatatt gcgtataaat atctatcaac aggaaaagca aaggtgtgtt gtgttaaatt 1200
aactgctatg gacctagaat taccaataac agcaaaatta cttcatcatc caactactga 1260
agttagatca gaattttatg caatattgga tgtatgggat attattacca ttaaaagatt 1320
tataccaaaa ggtgtatttt atgcttttat aaataatgta accacggaga atgtgtttat 1380
acaacctcct ttcaaattaa aaacgtcacc tactgattac atagtggcat tgtatgcttt 1440
atccaatgac ttcaactcga ggcaagatgt tattaatttg attaataaac aaaaacaatc 1500
actcattact gttcgtataa ataatacttt taaagacgaa ccaaaagtaa attttaaaaa 1560
tatttatgat tggacgtttc tgccaacaga ttttgaactt aaagattcga taattacatc 1620
atatgatggt tgtttaggca tattcggatt atcgatatct ttatcgtcta aacccactgg 1680
aaataatcat ttatttataa taaatggaac tgacaaatat gataaactag accaatatgc 1740
aaaccatatg ggtatttcta gaagatcgca tcaaattaga ttctcagagt ctgcgacatc 1800
atattcggga tacattttta gagatctttc aaataataat ttcaatctaa taggtactaa 1860
tgtggagaat tcagtttcag gacatgtcta taatgcatta atttactata gatataatta 1920
tgcgtttgat cttaaaagat ggatttatct acattcaatt gggaaagtcg ctgtaaaggg 1980
tggaagatat tatgaacatg caccaatcga actgatatat gcatgccgat cagctaaaga 2040
atttgctata ttacaagatg acttaacggt attgagatac gctaatgaaa ttgaagggta 2100
tataagcaaa gtgtacagta taacctatgc tgacgatccc aattacttta taggaattaa 2160
atttaatagt ataccttatg aatacgatgt taaaattcca catttgacgc taggagtatt 2220
atttatatct gataatatga tacatgatgt aatcacagtg ttgaagaaaa tgaagacaga 2280
gctatttaaa atggaaatta gtactagtta tacttatatg ctatctgata atacgtatgt 2340
agcaaatgct agtggtgtgt tatcaactta ttttaaatta tataatatgt tctatagaaa 2400
tcatatcacg tttggacaat cacgaatgtt tatcccacac attacgctta gttttagcaa 2460
caaacagacg gttagaatag aaagtacaaa actcagaatc aattcaattt acttaagaaa 2520
gattaaaggt gaaaccgtat ttgatatgtc tgagtgagct agaaacttaa cacactagtc 2580
atgatgtgac c 2591
<210> 58
<211> 835
<212> PRT
<213> artificial
<220>
<223> Vero-adapated RV3 VP3
<400> 58
Met Lys Val Leu Ala Leu Arg His Ser Val Ala Gln Val Tyr Ala Asp
1 5 10 15
Thr Gln Thr Tyr Leu His Asp Asp Ser Lys Asp Glu Tyr Glu Asn Ala
20 25 30
Phe Leu Ile Ser Asn Leu Thr Thr His Asn Ile Leu Tyr Leu Asn Tyr
35 40 45
Ser Leu Lys Thr Leu Lys Ile Leu Asn Glu Ser Gly Ile Ala Ala Val
50 55 60
Glu Val Gln Ser Pro Asp Glu Leu Phe Ala Leu Ile Arg Cys Asn Phe
65 70 75 80
Thr Tyr Asp Tyr Glu Asn Asn Ile Val Tyr Leu His Asp Tyr Ser Tyr
85 90 95
Tyr Thr Asn Asn Glu Ile Arg Thr Asp Gln His Trp Ile Thr Lys Thr
100 105 110
Asp Ile Ile Asp Tyr Leu Leu Pro Gly Trp Lys Leu Thr Tyr Val Gly
115 120 125
Tyr Asn Gly Lys Asn Thr Arg Gly His Tyr Asp Phe Ser Phe Ile Cys
130 135 140
Gln Asn Ala Ala Thr Asp Asp Asp Ile Ile Ile Glu Tyr Ile Tyr Ser
145 150 155 160
Asn Glu Leu Asp Phe Gln Asn Phe Leu Leu Arg Lys Ile Lys Glu Arg
165 170 175
Met Thr Thr Ser Leu Pro Ile Ala Arg Leu Ser Asn Arg Val Phe Arg
180 185 190
Asp Lys Leu Phe Pro Ser Ile Val Asn Ile His Lys Lys Val Ile Asn
195 200 205
Val Gly Pro Arg Asn Glu Ser Met Phe Thr Phe Leu Asn Phe Pro Thr
210 215 220
Ile Lys Gln Phe Ser Asn Gly Ala Tyr Ile Val Lys His Thr Ile Lys
225 230 235 240
Leu Lys Gln Glu Lys Trp Leu Gly Lys Arg Val Ser Gln Phe Asp Ile
245 250 255
Gly Gln Tyr Lys Asn Met Leu Asn Val Val Thr Thr Ile Tyr Tyr Tyr
260 265 270
Tyr Asn Leu Tyr Tyr Ser Lys Pro Ile Ile Tyr Met Leu Gly Ser Ala
275 280 285
Pro Ser Tyr Trp Ile Tyr Asp Ile Lys Gln Tyr Ser Asp Phe Thr Phe
290 295 300
Glu Thr Trp Asp Pro Leu Asp Thr Pro Tyr Ser Thr Thr His His Lys
305 310 315 320
Glu Leu Phe Phe Asp Lys Asp Val Asn Lys Leu Lys Asp Asn Ser Val
325 330 335
Leu Tyr Ile Asp Ile Arg Thr Asp Arg Gly Asn Met Asp Trp Lys Glu
340 345 350
Trp Arg Lys Ile Val Glu Gln Gln Thr Val Ser Asn Leu Asn Ile Ala
355 360 365
Tyr Lys Tyr Leu Ser Thr Gly Lys Ala Lys Val Cys Cys Val Lys Leu
370 375 380
Thr Ala Met Asp Leu Glu Leu Pro Ile Thr Ala Lys Leu Leu His His
385 390 395 400
Pro Thr Thr Glu Val Arg Ser Glu Phe Tyr Ala Ile Leu Asp Val Trp
405 410 415
Asp Ile Ile Thr Ile Lys Arg Phe Ile Pro Lys Gly Val Phe Tyr Ala
420 425 430
Phe Ile Asn Asn Val Thr Thr Glu Asn Val Phe Ile Gln Pro Pro Phe
435 440 445
Lys Leu Lys Thr Ser Pro Thr Asp Tyr Ile Val Ala Leu Tyr Ala Leu
450 455 460
Ser Asn Asp Phe Asn Ser Arg Gln Asp Val Ile Asn Leu Ile Asn Lys
465 470 475 480
Gln Lys Gln Ser Leu Ile Thr Val Arg Ile Asn Asn Thr Phe Lys Asp
485 490 495
Glu Pro Lys Val Asn Phe Lys Asn Ile Tyr Asp Trp Thr Phe Leu Pro
500 505 510
Thr Asp Phe Glu Leu Lys Asp Ser Ile Ile Thr Ser Tyr Asp Gly Cys
515 520 525
Leu Gly Ile Phe Gly Leu Ser Ile Ser Leu Ser Ser Lys Pro Thr Gly
530 535 540
Asn Asn His Leu Phe Ile Ile Asn Gly Thr Asp Lys Tyr Asp Lys Leu
545 550 555 560
Asp Gln Tyr Ala Asn His Met Gly Ile Ser Arg Arg Ser His Gln Ile
565 570 575
Arg Phe Ser Glu Ser Ala Thr Ser Tyr Ser Gly Tyr Ile Phe Arg Asp
580 585 590
Leu Ser Asn Asn Asn Phe Asn Leu Ile Gly Thr Asn Val Glu Asn Ser
595 600 605
Val Ser Gly His Val Tyr Asn Ala Leu Ile Tyr Tyr Arg Tyr Asn Tyr
610 615 620
Ala Phe Asp Leu Lys Arg Trp Ile Tyr Leu His Ser Ile Gly Lys Val
625 630 635 640
Ala Val Lys Gly Gly Arg Tyr Tyr Glu His Ala Pro Ile Glu Leu Ile
645 650 655
Tyr Ala Cys Arg Ser Ala Lys Glu Phe Ala Ile Leu Gln Asp Asp Leu
660 665 670
Thr Val Leu Arg Tyr Ala Asn Glu Ile Glu Gly Tyr Ile Ser Lys Val
675 680 685
Tyr Ser Ile Thr Tyr Ala Asp Asp Pro Asn Tyr Phe Ile Gly Ile Lys
690 695 700
Phe Asn Ser Ile Pro Tyr Glu Tyr Asp Val Lys Ile Pro His Leu Thr
705 710 715 720
Leu Gly Val Leu Phe Ile Ser Asp Asn Met Ile His Asp Val Ile Thr
725 730 735
Val Leu Lys Lys Met Lys Thr Glu Leu Phe Lys Met Glu Ile Ser Thr
740 745 750
Ser Tyr Thr Tyr Met Leu Ser Asp Asn Thr Tyr Val Ala Asn Ala Ser
755 760 765
Gly Val Leu Ser Thr Tyr Phe Lys Leu Tyr Asn Met Phe Tyr Arg Asn
770 775 780
His Ile Thr Phe Gly Gln Ser Arg Met Phe Ile Pro His Ile Thr Leu
785 790 795 800
Ser Phe Ser Asn Lys Gln Thr Val Arg Ile Glu Ser Thr Lys Leu Arg
805 810 815
Ile Asn Ser Ile Tyr Leu Arg Lys Ile Lys Gly Glu Thr Val Phe Asp
820 825 830
Met Ser Glu
835
<210> 59
<211> 2328
<212> DNA
<213> artificial
<220>
<223> MA104-adapted RV3 VP4
<400> 59
atggcttcac tcatttatag acagctactc actaattcat acacagttga attatcagat 60
gagattaata cgattggatc agaaaaaagt caaaatgtaa cgattaatcc cggaccgttt 120
gctcaaccaa attatgcacg agtaacttgg agtcatgggg aagtgaatga ttcgacaaca 180
atagagccag tacttgatgg cccttatcaa tcaacaagtt ttaagccacc aagcgattac 240
tggatattat tgaatccaac taatcaacaa gttgtattag agggtaccaa taaaactgat 300
atttgggttg ccttattact tgttgaacca aacgtaacca atcaaagtag acaatacaca 360
ttatttggag aaacgaaaca aattactgta gaaaataaca caaacaaatg gaaattcttc 420
gaaatgttca gaagcagtgt tagtgccgaa tttcaacata agcgcacttt aacatcagac 480
actaaattag ctgggttttt aaaacattat aacagtgttt ggactttcca cggtgaaacg 540
ccacatgcta caactgatta ctcatcaact tcaaatttat ctgaagtaga aactacaata 600
catgttgagt tttatataat accaagatcg caggaatcta agtgtgttga atatataaat 660
actggattgc caccaatgca gaatacaagg aatatagttc cagttgcact atcatctaga 720
gcagtgactt atcaacgtgc tcaggttaat gaagatatca tcatatcaaa gacatcgttg 780
tggaaggaaa tgcaatataa cagagacatc gtaataaggt ttaaatttaa taatagtata 840
gtaaaacttg gtgggctagg ttataaatgg tcagaaattt cgtttaaagc tgctaattat 900
cagtacaatt acttgcgaga tggagaacaa gttacggcac atactacttg ttcagtcaat 960
ggtgtgaata acttcagtta taatggagga tcactgccaa ctgattttag tgtatcaaga 1020
tatgaagtga ttaaggagaa ttcttatgtc tatgttgatt attgggatga ttcacaagca 1080
tttaggaaca tggtatacgt caggtcattg gcagcaaatt taaattcagt aaagtgtagt 1140
ggaggaaatt atgattttca aataccagtt ggtgcatggc cagtgatgag tggaggtgcg 1200
gtatctttgc attttgcggg agtcacctta tccactcaat ttactgactt tgtatcactt 1260
aattcgttaa gatttagatt cagtttgacc gttgaagatc cacctttttc aatttcacgc 1320
acacgtgtgt caggattata cgggctacca gcatttaatc cgaatagcgg acatgaatat 1380
tatgaaatag ctgggggatt ttctcttatt tcattagtac cgtctaatga cgattatcaa 1440
actccaatca tgaattcagt tacagtgcga caagatcttg aacgtcaact aggtgattta 1500
agggaggaat tcaattctct atcacaagaa atagcgatga cgcaattgat agatttagca 1560
ttattaccgt tagatatgtt ttctatgttc tcaggtatta aaagcacaat tgacgcagct 1620
aaatcaatgg ccacaaaggt gatgaaaaag tttaagagat cgggattagc tacatcaatc 1680
tctgaattaa ctggatcatt atcaaacgct gcttcatcag tttccagaag ttcatctatt 1740
agatctaaca tatcatccat ttcagtgtgg acggatgttt ccgaacagat agcgggttcg 1800
tcagactctg tcaggaatat ttccacgcaa acgtcagcta ttagtagaag attgcgacta 1860
cgcgaaatta ctacacaaac tgaaggtatg aattttgatg atatttcagc ggcagttctt 1920
aaaactaaga tagataaatc aactcatata agcccagata cattaccaga cataataact 1980
gagtcatctg aaaaattcat accaaaacga gcttatagag ttctaaaaga tgatgaagtg 2040
atggaagctg acgtggatgg gaagttcttt gcatataagg ttgacacttt tgaagaagtg 2100
ccatttgacg tagataaatt tgtcgatttg gtaactgatt ctcctgtaat ttcagctata 2160
atcgatttta agacgttgaa gaatttaaac gacaattatg gtataacgcg atctcaagcg 2220
ttagacttaa tcagatctga tcccagagtt ttacgcgatt ttatcaatca gaataatcca 2280
attattaaaa atagaattga acagctaata ctgcaatgta gactgtga 2328
<210> 60
<211> 775
<212> PRT
<213> artificial
<220>
<223> MA104-adapated RV3 VP4
<400> 60
Met Ala Ser Leu Ile Tyr Arg Gln Leu Leu Thr Asn Ser Tyr Thr Val
1 5 10 15
Glu Leu Ser Asp Glu Ile Asn Thr Ile Gly Ser Glu Lys Ser Gln Asn
20 25 30
Val Thr Ile Asn Pro Gly Pro Phe Ala Gln Pro Asn Tyr Ala Arg Val
35 40 45
Thr Trp Ser His Gly Glu Val Asn Asp Ser Thr Thr Ile Glu Pro Val
50 55 60
Leu Asp Gly Pro Tyr Gln Ser Thr Ser Phe Lys Pro Pro Ser Asp Tyr
65 70 75 80
Trp Ile Leu Leu Asn Pro Thr Asn Gln Gln Val Val Leu Glu Gly Thr
85 90 95
Asn Lys Thr Asp Ile Trp Val Ala Leu Leu Leu Val Glu Pro Asn Val
100 105 110
Thr Asn Gln Ser Arg Gln Tyr Thr Leu Phe Gly Glu Thr Lys Gln Ile
115 120 125
Thr Val Glu Asn Asn Thr Asn Lys Trp Lys Phe Phe Glu Met Phe Arg
130 135 140
Ser Ser Val Ser Ala Glu Phe Gln His Lys Arg Thr Leu Thr Ser Asp
145 150 155 160
Thr Lys Leu Ala Gly Phe Leu Lys His Tyr Asn Ser Val Trp Thr Phe
165 170 175
His Gly Glu Thr Pro His Ala Thr Thr Asp Tyr Ser Ser Thr Ser Asn
180 185 190
Leu Ser Glu Val Glu Thr Thr Ile His Val Glu Phe Tyr Ile Ile Pro
195 200 205
Arg Ser Gln Glu Ser Lys Cys Val Glu Tyr Ile Asn Thr Gly Leu Pro
210 215 220
Pro Met Gln Asn Thr Arg Asn Ile Val Pro Val Ala Leu Ser Ser Arg
225 230 235 240
Ala Val Thr Tyr Gln Arg Ala Gln Val Asn Glu Asp Ile Ile Ile Ser
245 250 255
Lys Thr Ser Leu Trp Lys Glu Met Gln Tyr Asn Arg Asp Ile Val Ile
260 265 270
Arg Phe Lys Phe Asn Asn Ser Ile Val Lys Leu Gly Gly Leu Gly Tyr
275 280 285
Lys Trp Ser Glu Ile Ser Phe Lys Ala Ala Asn Tyr Gln Tyr Asn Tyr
290 295 300
Leu Arg Asp Gly Glu Gln Val Thr Ala His Thr Thr Cys Ser Val Asn
305 310 315 320
Gly Val Asn Asn Phe Ser Tyr Asn Gly Gly Ser Leu Pro Thr Asp Phe
325 330 335
Ser Val Ser Arg Tyr Glu Val Ile Lys Glu Asn Ser Tyr Val Tyr Val
340 345 350
Asp Tyr Trp Asp Asp Ser Gln Ala Phe Arg Asn Met Val Tyr Val Arg
355 360 365
Ser Leu Ala Ala Asn Leu Asn Ser Val Lys Cys Ser Gly Gly Asn Tyr
370 375 380
Asp Phe Gln Ile Pro Val Gly Ala Trp Pro Val Met Ser Gly Gly Ala
385 390 395 400
Val Ser Leu His Phe Ala Gly Val Thr Leu Ser Thr Gln Phe Thr Asp
405 410 415
Phe Val Ser Leu Asn Ser Leu Arg Phe Arg Phe Ser Leu Thr Val Glu
420 425 430
Asp Pro Pro Phe Ser Ile Ser Arg Thr Arg Val Ser Gly Leu Tyr Gly
435 440 445
Leu Pro Ala Phe Asn Pro Asn Ser Gly His Glu Tyr Tyr Glu Ile Ala
450 455 460
Gly Gly Phe Ser Leu Ile Ser Leu Val Pro Ser Asn Asp Asp Tyr Gln
465 470 475 480
Thr Pro Ile Met Asn Ser Val Thr Val Arg Gln Asp Leu Glu Arg Gln
485 490 495
Leu Gly Asp Leu Arg Glu Glu Phe Asn Ser Leu Ser Gln Glu Ile Ala
500 505 510
Met Thr Gln Leu Ile Asp Leu Ala Leu Leu Pro Leu Asp Met Phe Ser
515 520 525
Met Phe Ser Gly Ile Lys Ser Thr Ile Asp Ala Ala Lys Ser Met Ala
530 535 540
Thr Lys Val Met Lys Lys Phe Lys Arg Ser Gly Leu Ala Thr Ser Ile
545 550 555 560
Ser Glu Leu Thr Gly Ser Leu Ser Asn Ala Ala Ser Ser Val Ser Arg
565 570 575
Ser Ser Ser Ile Arg Ser Asn Ile Ser Ser Ile Ser Val Trp Thr Asp
580 585 590
Val Ser Glu Gln Ile Ala Gly Ser Ser Asp Ser Val Arg Asn Ile Ser
595 600 605
Thr Gln Thr Ser Ala Ile Ser Arg Arg Leu Arg Leu Arg Glu Ile Thr
610 615 620
Thr Gln Thr Glu Gly Met Asn Phe Asp Asp Ile Ser Ala Ala Val Leu
625 630 635 640
Lys Thr Lys Ile Asp Lys Ser Thr His Ile Ser Pro Asp Thr Leu Pro
645 650 655
Asp Ile Ile Thr Glu Ser Ser Glu Lys Phe Ile Pro Lys Arg Ala Tyr
660 665 670
Arg Val Leu Lys Asp Asp Glu Val Met Glu Ala Asp Val Asp Gly Lys
675 680 685
Phe Phe Ala Tyr Lys Val Asp Thr Phe Glu Glu Val Pro Phe Asp Val
690 695 700
Asp Lys Phe Val Asp Leu Val Thr Asp Ser Pro Val Ile Ser Ala Ile
705 710 715 720
Ile Asp Phe Lys Thr Leu Lys Asn Leu Asn Asp Asn Tyr Gly Ile Thr
725 730 735
Arg Ser Gln Ala Leu Asp Leu Ile Arg Ser Asp Pro Arg Val Leu Arg
740 745 750
Asp Phe Ile Asn Gln Asn Asn Pro Ile Ile Lys Asn Arg Ile Glu Gln
755 760 765
Leu Ile Leu Gln Cys Arg Leu
770 775
<210> 61
<211> 2378
<212> DNA
<213> artificial
<220>
<223> Vero-adapated RV3 VP4
<400> 61
ggctataaaa tggcttcact catttataga cagctactca ctaattcata cacagttgaa 60
ttatcagatg agattaatac gattggatca gaaaaaagtc aaaatgtaac gattaatccc 120
ggaccgtttg ctcaaacaaa ttatgcacga gtaacttgga gtcatgggga agtgaatgat 180
tcgacaacaa tagagccagt acttgatggc ccttatcaat caacaagttt taagccacca 240
agcgattact ggatattatt gaatccaact aatcaacaag ttgtattaga gggtaccaat 300
aaaactgata tttgggttgc cttattactt gttgaaccaa acgtaaccaa tcaaagtaga 360
caatacacgt tatttggaga aacgaaacaa attactgtag aaaataacac aaacaaatgg 420
aaattcttcg aaatgttcag aagcagtgtt agtgccgaat ttcaacataa gcgcacttta 480
acatcagaca ctaaattagc tgggttttta aaacattata acagtgtttg gactttccac 540
ggtgaaacgc cacatgctac aactgattac tcatcaactt caaatttatc tgaagtagaa 600
actacaatac atgttgagtt ttatataata ccaagatcgc aggaatctaa gtgtgttgaa 660
tatataaata ctggattgcc accaatgcag aatacaagga atatagttcc agttgcacta 720
tcatctagag cagtgactta tcaacgtgct caggttaatg aagatatcat catatcaaag 780
acatcgttgt ggaaggaaat gcaatataac agagacatcg taataaggtt taaatttaat 840
aatagtatag taaaacttgg tgggctaggt tataaatggt cagaaatttc gtttaaagct 900
gctaattatc agtacaatta cttgcgagat ggagaacaag ttacggcaca tactacttgt 960
tcagtcaatg gtgtgaataa cttcagttat aatggaggat cactgccaac tgattttagt 1020
gtatcaagat atgaagtgat taaggagaat tcttatgtct atgttgatta ttgggatgat 1080
tcacaagcat ttaggaacat ggtatacgtc aggtcattgg cagcaaattc aaattcagta 1140
aagtgtagtg gaggaaatta taattttcaa ctaccagttg gtgcatggcc agtgatgagt 1200
ggaggtgcgg tatctttgca ttttgcggga gtcaccttat ccactcaatt tactgacttt 1260
gtatcactta attcgttaag atttagattc agtttgaccg ttgaagatcc acctttttca 1320
atttcacgca cacatgtgtc aggattatac gggctaccag catttaatcc gaatagcgga 1380
catgaatatt atgaaatagc tgggggattt tctcttattt cattagtacc gtctaatgac 1440
gattatcaaa ctccaatcat gaattcagtt acagtgcgac aagatcttga acgtcaacta 1500
ggtgatttaa gggaggaatt caattctcta tcacaagaaa tagcgatgac gcaattgata 1560
gatttagcat tattaccgtt agatatgttt tctatgttct caggtattaa aagcacaatt 1620
gacgcagcta aatcaatggc cacaaaggtg atgaaaaagt ttaagagatc gggattagct 1680
acatcaatct ctgaattaac tggatcatta tcaaacgctg cttcatcagt ttccagaagt 1740
tcatctatta gatctaacat atcatccatt tcagtgtgga cggatgtttc cgaacagata 1800
gcgggttcgt cagactctgt caggaatatt tccacgcaaa cgtcagctat tagtagaaga 1860
ttgcgactac gcgaaattac tacacaaact gaaggtatga attttgatga tatttcagcg 1920
gcagttctta aaactaagat agataaatca actcatataa gcccagatac attaccagac 1980
ataataactg agtcatctga aaaattcata ccaaaacgag cttatagagt tctaaaagat 2040
gatgaagtga tggaagctga cgtggatggg aagttctttg catataaggt tgacactttt 2100
gaagaagtgc catttgacgt agataaattt gtcgatttgg taactgattc tcctgtaatt 2160
tcagctataa tcgattttaa gacgttgaag aatttaaacg acaattatgg tataacgcga 2220
tctcaagcgt tagacttaat cagatctgat cccagagttt tacgcgattt tatcattcag 2280
aataatccaa ttattaaaaa tagaattgaa cagctaatac tgcaatgtag actgtgagag 2340
ctccatcgag gaatgtgacc ggtcatagct gtttcctg 2378
<210> 62
<211> 775
<212> PRT
<213> artificial
<220>
<223> Vero-adapted RV3 VP4
<400> 62
Met Ala Ser Leu Ile Tyr Arg Gln Leu Leu Thr Asn Ser Tyr Thr Val
1 5 10 15
Glu Leu Ser Asp Glu Ile Asn Thr Ile Gly Ser Glu Lys Ser Gln Asn
20 25 30
Val Thr Ile Asn Pro Gly Pro Phe Ala Gln Thr Asn Tyr Ala Arg Val
35 40 45
Thr Trp Ser His Gly Glu Val Asn Asp Ser Thr Thr Ile Glu Pro Val
50 55 60
Leu Asp Gly Pro Tyr Gln Ser Thr Ser Phe Lys Pro Pro Ser Asp Tyr
65 70 75 80
Trp Ile Leu Leu Asn Pro Thr Asn Gln Gln Val Val Leu Glu Gly Thr
85 90 95
Asn Lys Thr Asp Ile Trp Val Ala Leu Leu Leu Val Glu Pro Asn Val
100 105 110
Thr Asn Gln Ser Arg Gln Tyr Thr Leu Phe Gly Glu Thr Lys Gln Ile
115 120 125
Thr Val Glu Asn Asn Thr Asn Lys Trp Lys Phe Phe Glu Met Phe Arg
130 135 140
Ser Ser Val Ser Ala Glu Phe Gln His Lys Arg Thr Leu Thr Ser Asp
145 150 155 160
Thr Lys Leu Ala Gly Phe Leu Lys His Tyr Asn Ser Val Trp Thr Phe
165 170 175
His Gly Glu Thr Pro His Ala Thr Thr Asp Tyr Ser Ser Thr Ser Asn
180 185 190
Leu Ser Glu Val Glu Thr Thr Ile His Val Glu Phe Tyr Ile Ile Pro
195 200 205
Arg Ser Gln Glu Ser Lys Cys Val Glu Tyr Ile Asn Thr Gly Leu Pro
210 215 220
Pro Met Gln Asn Thr Arg Asn Ile Val Pro Val Ala Leu Ser Ser Arg
225 230 235 240
Ala Val Thr Tyr Gln Arg Ala Gln Val Asn Glu Asp Ile Ile Ile Ser
245 250 255
Lys Thr Ser Leu Trp Lys Glu Met Gln Tyr Asn Arg Asp Ile Val Ile
260 265 270
Arg Phe Lys Phe Asn Asn Ser Ile Val Lys Leu Gly Gly Leu Gly Tyr
275 280 285
Lys Trp Ser Glu Ile Ser Phe Lys Ala Ala Asn Tyr Gln Tyr Asn Tyr
290 295 300
Leu Arg Asp Gly Glu Gln Val Thr Ala His Thr Thr Cys Ser Val Asn
305 310 315 320
Gly Val Asn Asn Phe Ser Tyr Asn Gly Gly Ser Leu Pro Thr Asp Phe
325 330 335
Ser Val Ser Arg Tyr Glu Val Ile Lys Glu Asn Ser Tyr Val Tyr Val
340 345 350
Asp Tyr Trp Asp Asp Ser Gln Ala Phe Arg Asn Met Val Tyr Val Arg
355 360 365
Ser Leu Ala Ala Asn Ser Asn Ser Val Lys Cys Ser Gly Gly Asn Tyr
370 375 380
Asn Phe Gln Leu Pro Val Gly Ala Trp Pro Val Met Ser Gly Gly Ala
385 390 395 400
Val Ser Leu His Phe Ala Gly Val Thr Leu Ser Thr Gln Phe Thr Asp
405 410 415
Phe Val Ser Leu Asn Ser Leu Arg Phe Arg Phe Ser Leu Thr Val Glu
420 425 430
Asp Pro Pro Phe Ser Ile Ser Arg Thr His Val Ser Gly Leu Tyr Gly
435 440 445
Leu Pro Ala Phe Asn Pro Asn Ser Gly His Glu Tyr Tyr Glu Ile Ala
450 455 460
Gly Gly Phe Ser Leu Ile Ser Leu Val Pro Ser Asn Asp Asp Tyr Gln
465 470 475 480
Thr Pro Ile Met Asn Ser Val Thr Val Arg Gln Asp Leu Glu Arg Gln
485 490 495
Leu Gly Asp Leu Arg Glu Glu Phe Asn Ser Leu Ser Gln Glu Ile Ala
500 505 510
Met Thr Gln Leu Ile Asp Leu Ala Leu Leu Pro Leu Asp Met Phe Ser
515 520 525
Met Phe Ser Gly Ile Lys Ser Thr Ile Asp Ala Ala Lys Ser Met Ala
530 535 540
Thr Lys Val Met Lys Lys Phe Lys Arg Ser Gly Leu Ala Thr Ser Ile
545 550 555 560
Ser Glu Leu Thr Gly Ser Leu Ser Asn Ala Ala Ser Ser Val Ser Arg
565 570 575
Ser Ser Ser Ile Arg Ser Asn Ile Ser Ser Ile Ser Val Trp Thr Asp
580 585 590
Val Ser Glu Gln Ile Ala Gly Ser Ser Asp Ser Val Arg Asn Ile Ser
595 600 605
Thr Gln Thr Ser Ala Ile Ser Arg Arg Leu Arg Leu Arg Glu Ile Thr
610 615 620
Thr Gln Thr Glu Gly Met Asn Phe Asp Asp Ile Ser Ala Ala Val Leu
625 630 635 640
Lys Thr Lys Ile Asp Lys Ser Thr His Ile Ser Pro Asp Thr Leu Pro
645 650 655
Asp Ile Ile Thr Glu Ser Ser Glu Lys Phe Ile Pro Lys Arg Ala Tyr
660 665 670
Arg Val Leu Lys Asp Asp Glu Val Met Glu Ala Asp Val Asp Gly Lys
675 680 685
Phe Phe Ala Tyr Lys Val Asp Thr Phe Glu Glu Val Pro Phe Asp Val
690 695 700
Asp Lys Phe Val Asp Leu Val Thr Asp Ser Pro Val Ile Ser Ala Ile
705 710 715 720
Ile Asp Phe Lys Thr Leu Lys Asn Leu Asn Asp Asn Tyr Gly Ile Thr
725 730 735
Arg Ser Gln Ala Leu Asp Leu Ile Arg Ser Asp Pro Arg Val Leu Arg
740 745 750
Asp Phe Ile Ile Gln Asn Asn Pro Ile Ile Lys Asn Arg Ile Glu Gln
755 760 765
Leu Ile Leu Gln Cys Arg Leu
770 775
<210> 63
<211> 981
<212> DNA
<213> artificial
<220>
<223> MA104-adapated RV3 VP7
<400> 63
atgtatggta ttgaatatac cacagttttg acctttttga tatcaattat attgttgaat 60
tacgtactca aatcattaac tagaataatg gactttatta tctacagatt tcttttgatt 120
atagttatac tgtcaccact ccttaatgca caaaattatg gaataaatct tccgattact 180
ggatcaatgg acacaccata tacgaactca acgcgagagg aagtattcct aacttcgact 240
ttatgtttgt attacccaac cgaagcagca acagaaataa atgataatgc atggaaggat 300
acactttctc aactattttt aactaaagga tggccgacag gatctattta ttttaaagat 360
tatactgata ttgcctcgtt ttcagttgat ccacaactgt actgtgatta taatttggta 420
ttaatgaaat acgacgctac actacaactg gacatgtctg aactagcaga tttgttactt 480
aatgagtggt tatgtaatcc tatggatatt actttgtatt attatcaaca aactgatgag 540
gcaaataaat ggatttcaat gggatcatct tgtaccataa aggtatgtcc actaaatacg 600
caaacattag gaattgggtg tctaactact gatacaaaca cgttcgaaga agttgcaaca 660
gctgaaaaat tagtgattac tgacgttgta gatggggtca atcataaatt gaacgtgacg 720
acaaacactt gtacgattag aaattgtaaa aaattaggac caagggaaaa cgtagcagtt 780
atacaggttg gtggctcaga tgtacttgac ataacagctg atcctacgac agcgccacga 840
acagaaagaa tgatgcgagt gaattggaag aaatggtggc aagtatttta tacaatagtt 900
gactacgtga atcaaattgt gcaagcgatg tccaaaagat cgagatcgtt aaattctgct 960
gcattttact acagggtata g 981
<210> 64
<211> 326
<212> PRT
<213> artificial
<220>
<223> MA104-adapated RV3 VP7
<400> 64
Met Tyr Gly Ile Glu Tyr Thr Thr Val Leu Thr Phe Leu Ile Ser Ile
1 5 10 15
Ile Leu Leu Asn Tyr Val Leu Lys Ser Leu Thr Arg Ile Met Asp Phe
20 25 30
Ile Ile Tyr Arg Phe Leu Leu Ile Ile Val Ile Leu Ser Pro Leu Leu
35 40 45
Asn Ala Gln Asn Tyr Gly Ile Asn Leu Pro Ile Thr Gly Ser Met Asp
50 55 60
Thr Pro Tyr Thr Asn Ser Thr Arg Glu Glu Val Phe Leu Thr Ser Thr
65 70 75 80
Leu Cys Leu Tyr Tyr Pro Thr Glu Ala Ala Thr Glu Ile Asn Asp Asn
85 90 95
Ala Trp Lys Asp Thr Leu Ser Gln Leu Phe Leu Thr Lys Gly Trp Pro
100 105 110
Thr Gly Ser Ile Tyr Phe Lys Asp Tyr Thr Asp Ile Ala Ser Phe Ser
115 120 125
Val Asp Pro Gln Leu Tyr Cys Asp Tyr Asn Leu Val Leu Met Lys Tyr
130 135 140
Asp Ala Thr Leu Gln Leu Asp Met Ser Glu Leu Ala Asp Leu Leu Leu
145 150 155 160
Asn Glu Trp Leu Cys Asn Pro Met Asp Ile Thr Leu Tyr Tyr Tyr Gln
165 170 175
Gln Thr Asp Glu Ala Asn Lys Trp Ile Ser Met Gly Ser Ser Cys Thr
180 185 190
Ile Lys Val Cys Pro Leu Asn Thr Gln Thr Leu Gly Ile Gly Cys Leu
195 200 205
Thr Thr Asp Thr Asn Thr Phe Glu Glu Val Ala Thr Ala Glu Lys Leu
210 215 220
Val Ile Thr Asp Val Val Asp Gly Val Asn His Lys Leu Asn Val Thr
225 230 235 240
Thr Asn Thr Cys Thr Ile Arg Asn Cys Lys Lys Leu Gly Pro Arg Glu
245 250 255
Asn Val Ala Val Ile Gln Val Gly Gly Ser Asp Val Leu Asp Ile Thr
260 265 270
Ala Asp Pro Thr Thr Ala Pro Arg Thr Glu Arg Met Met Arg Val Asn
275 280 285
Trp Lys Lys Trp Trp Gln Val Phe Tyr Thr Ile Val Asp Tyr Val Asn
290 295 300
Gln Ile Val Gln Ala Met Ser Lys Arg Ser Arg Ser Leu Asn Ser Ala
305 310 315 320
Ala Phe Tyr Tyr Arg Val
325
<210> 65
<211> 1062
<212> DNA
<213> artificial
<220>
<223> Vero-adapated RV3 VP7
<400> 65
ggctttaaaa gagagaattt ccgtctggct agcggttagc tccttttaat gtatggtatt 60
gaatatacca cagttttgac ctttttgata tcaattatat tgttgaatta cgtactcaaa 120
tcattaacta gaataatgga ctttattatc tacagatttc ttttgattat agttatactg 180
tcaccactcc ttaatgcaca aaattatgga ataaatcttc cgattactgg atcaatggac 240
acaccatata cgaactcaac gcgagaggaa gtattcctaa cttcgacttt atgtttgtat 300
tacccaaccg aagcagcaac agaaataaat gataatgcat ggaaggatac actttctcaa 360
ctatttttaa ctaaaggatg gccgacagga tctatttatt ttaaagatta tactgatatt 420
gcctcgtttt cagttgatcc acaactgtac tgtgattata atttggtatt aatgaaatac 480
gacgctacac tacaactgga catgtctgaa ctagcagatt tgttacttaa tgagtggtta 540
tgtaatccta tggatattac tttgtattat tatcaacaaa ctgatgaggc aaataaatgg 600
atttcaatgg gatcatcttg taccataaag gtatgtccac taaatacgca aacattagga 660
attgggtgtc taactactga tacaaacacg ttcgaagaag ttgcaacagc tgaaaaatta 720
gtgattactg acgttgtaga tggggtcaat cataaattga acgtgacgac aaacacttgt 780
acgattagaa attgtaaaaa attaggacca agggaaaacg tagcagttat acaggttggt 840
ggctcagatg tacttgacat aacagctgat cctacgacag cgccacgaac agaaagaatg 900
atgcgagtga attggaagaa atggtggcaa gtattttata caatagttga ctacgtgaat 960
caaattgtgc aagcgatgtc caaaagatcg agatcgttaa attctgctgc attttactac 1020
agggtatagg tatagcttag attagaattg tatgatgtga cc 1062
<210> 66
<211> 326
<212> PRT
<213> artificial
<220>
<223> Vero-adapated RV3 VP7
<400> 66
Met Tyr Gly Ile Glu Tyr Thr Thr Val Leu Thr Phe Leu Ile Ser Ile
1 5 10 15
Ile Leu Leu Asn Tyr Val Leu Lys Ser Leu Thr Arg Ile Met Asp Phe
20 25 30
Ile Ile Tyr Arg Phe Leu Leu Ile Ile Val Ile Leu Ser Pro Leu Leu
35 40 45
Asn Ala Gln Asn Tyr Gly Ile Asn Leu Pro Ile Thr Gly Ser Met Asp
50 55 60
Thr Pro Tyr Thr Asn Ser Thr Arg Glu Glu Val Phe Leu Thr Ser Thr
65 70 75 80
Leu Cys Leu Tyr Tyr Pro Thr Glu Ala Ala Thr Glu Ile Asn Asp Asn
85 90 95
Ala Trp Lys Asp Thr Leu Ser Gln Leu Phe Leu Thr Lys Gly Trp Pro
100 105 110
Thr Gly Ser Ile Tyr Phe Lys Asp Tyr Thr Asp Ile Ala Ser Phe Ser
115 120 125
Val Asp Pro Gln Leu Tyr Cys Asp Tyr Asn Leu Val Leu Met Lys Tyr
130 135 140
Asp Ala Thr Leu Gln Leu Asp Met Ser Glu Leu Ala Asp Leu Leu Leu
145 150 155 160
Asn Glu Trp Leu Cys Asn Pro Met Asp Ile Thr Leu Tyr Tyr Tyr Gln
165 170 175
Gln Thr Asp Glu Ala Asn Lys Trp Ile Ser Met Gly Ser Ser Cys Thr
180 185 190
Ile Lys Val Cys Pro Leu Asn Thr Gln Thr Leu Gly Ile Gly Cys Leu
195 200 205
Thr Thr Asp Thr Asn Thr Phe Glu Glu Val Ala Thr Ala Glu Lys Leu
210 215 220
Val Ile Thr Asp Val Val Asp Gly Val Asn His Lys Leu Asn Val Thr
225 230 235 240
Thr Asn Thr Cys Thr Ile Arg Asn Cys Lys Lys Leu Gly Pro Arg Glu
245 250 255
Asn Val Ala Val Ile Gln Val Gly Gly Ser Asp Val Leu Asp Ile Thr
260 265 270
Ala Asp Pro Thr Thr Ala Pro Arg Thr Glu Arg Met Met Arg Val Asn
275 280 285
Trp Lys Lys Trp Trp Gln Val Phe Tyr Thr Ile Val Asp Tyr Val Asn
290 295 300
Gln Ile Val Gln Ala Met Ser Lys Arg Ser Arg Ser Leu Asn Ser Ala
305 310 315 320
Ala Phe Tyr Tyr Arg Val
325
Claims (22)
- 로타바이러스(RV) 균주(strain)를 배양하는 방법으로서,
상기 방법은 RV 분취물(aliquot) 또는 접종물(inoculum)을 트립신처리하는 단계 및 상기 트립신처리된 RV를 Vero 세포 또는 Vero 세포-유래된 세포주에서 상기 RV가 VP1, VP3, VP4, NSP4 및/또는 NSP5/6 중 하나 이상에서 아미노산 변화 및/또는 VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6 중 하나 이상에서 뉴클레오타이드 변화를 초래하는 유전적 변형을 겪기에 충분한 시간 동안 및 조건 하에서 배양하는 단계를 포함하는 방법. - 제1항에 있어서,
상기 RV 균주가 인간 RV3인 방법. - 제1항 또는 제2항에 있어서,
상기 RV를 수거하는 추가의 단계를 포함하는 방법. - 제1항에 있어서,
상기 트립신이 돼지, 반추 동물 또는 재조합 트립신인 방법. - 제4항에 있어서,
상기 반추 동물 트립신이 소, 양, 염소 또는 낙타 트립신인 방법. - 제1항 내지 제5항 중 어느 한 항에 있어서,
상기 아미노산 또는 뉴클레오타이드 변화가 MA104-적응된 RV와 비교되는 방법. - 제1항 내지 제6항 중 어느 한 항에 있어서,
상기 아미노산 또는 뉴클레오타이드 변화가 NSP4에서 53, 76, 85, 162 및 165 중에서 선택되는 코돈, NSP5/6에서 46 및 112 중에서 선택되는 코돈, VP1에서 886 중에서 선택되는 코돈, VP3에서 643 및 785 중에서 선택되는 코돈, 및 VP4에서 43, 374, 385, 388, 442 및 756 중에서 선택되는 코돈 및/또는 NSP2에서 433 중에서 선택되는 뉴클레오타이드, NSP4에서 197, 267, 294, 525 및 534 중에서 선택되는 뉴클레오타이드, NSP5/6에서 158, 165 및 355 중에서 선택되는 뉴클레오타이드, VP1에서 2692 중에서 선택되는 뉴클레오타이드, VP3에서 343, 1366, 1744, 1976 및 2402 중에서 선택되는 뉴클레오타이드 및/또는 VP4에서 136, 369, 1130, 1162, 1171, 1334 및 2276 중에서 선택되는 뉴클레오타이드에 존재하는 방법. - 제1항 내지 제7항 중 어느 한 항에 있어서,
상기 아미노산 또는 뉴클레오타이드 변화가 표 2 또는 표 5에 제공되는 리스트 중에서 선택되는 방법. - Vero 세포에서 약독화된 RV3를 생성하는 방법으로서,
상기 방법은 약독화된 RV3의 분취물 또는 접종물을 트립신처리하는 단계 및 상기 트립신처리된 RV3를 Vero 세포 또는 Vero 세포-유래된 세포주와 함께 MA104-적응된 RV3와 비교하여 표 2 또는 표 5에서 제공되는 리스트 중에서 선택되는, 유전적 변형이 발생하기에 충분한 시간 동안 및 조건 하에서 배양하는 단계를 포함하는 방법. - 제9항에 있어서,
상기 트립신이 돼지, 반추 동물 또는 재조합 트립신인 방법. - 제10항에 있어서,
상기 반추 동물 트립신이 소, 양, 염소 또는 낙타 트립신인 방법. - NSP4에서 53, 76, 85, 162 및 165 중에서 선택되는 코돈, NSP5/6에서 46 및 112 중에서 선택되는 코돈, VP1에서 886 중에서 선택되는 코돈, VP3에서 643 및 785 중에서 선택되는 코돈, 및 VP4에서 43, 374, 385, 388, 442 및 756 중에서 선택되는 코돈 및/또는 NSP2에서 433 중에서 선택되는 뉴클레오타이드, NSP4에서 197, 267, 294, 525 및 534 중에서 선택되는 뉴클레오타이드, NSP5/6에서 158, 165 및 355 중에서 선택되는 뉴클레오타이드, VP1에서 2692 중에서 선택되는 뉴클레오타이드, VP3에서 343, 1366, 1744, 1976 및 2402 중에서 선택되는 뉴클레오타이드 및/또는 VP4에서 136, 369, 1130, 1162, 1171, 1334 및 2276 중에서 선택되는 뉴클레오타이드로 이루어진 리스트 중에서 선택되는 하나 이상의 돌연변이를 포함하는, 분리된 Vero-적응된 RV3 (Vero-adapted RV3).
- 제12항에 있어서,
표 2 또는 5의 리스트 중에서 선택되는 돌연변이를 포함하는 분리된 Vero-적응된 RV3. - VP1, VP3, VP4, NSP4 및/또는 NSP5/6에서의 하나 이상의 돌연변이 중에서 선택되는 유전적 변형을 갖는 RV를 포함하고, 하나 이상의 약제학적으로 허용되는 담체, 희석제 및/또는 부형제를 추가로 포함하는, 약독화된 살아있는 또는 사멸된 RV 조성물.
- 제14항에 있어서,
상기 약독화된 또는 사멸된 Vero-적응된 RV가 표 2 또는 표 5에 열거된 하나 이상의 돌연변이 중에서 선택되는 유전적 변형을 갖고, 상기 조성물이 하나 이상의 약제학적으로 허용되는 담체, 희석제 및/또는 부형제를 추가로 포함하는 조성물. - RV 감염에 대해 인간 피험자를 예방접종하는 방법으로서,
상기 방법은 상기 피험자에게 유효량의 살아있는 약독화된 Vero-적응된 RV3 또는 사멸된 Vero-적응된 RV3를 RV 감염에 대한 면역 반응을 발생시키기에 충분한 시간 동안 및 조건 하에 투여하는 단계를 포함하는 방법. - 제16항에 있어서,
상기 면역 반응이 방어적 면역 반응인 방법. - RV 감염을 갖거나 RV 감염 또는 재감염 발병 위험이 있는 인간 피험자를 치료하는 방법으로서,
상기 방법은 상기 피험자에게 유효량의 살아있는 약독화된 Vero-적응된 RV3 또는 사멸된 Vero-적응된 RV3를 RV 감염에 대한 면역 반응을 발생시키기에 충분한 시간 동안 및 조건 하에 투여하는 단계를 포함하는 방법. - 피험자에서 RV 감염을 치료하는 약물의 제조시 Vero-RV의 용도.
- Vero-RV에 대한 분리된 항체.
- Vero-RV를 검출 또는 모니터링하는 방법으로서,
상기 방법은 VP1, VP3, VP4, NSP4 및/또는 NSP5/6 중 하나 이상에서의 아미노산 변화 및/또는 VP1, VP3, VP4, NSP2, NSP4 및/또는 NSP5/6 중 하나 이상에서 뉴클레오타이드 변화를 초래하는 유전적 변형을 스크리닝하는 단계를 포함하는 방법. - Vero-RV의 감염, 치료, 역학(epidemiology) 또는 적응(adaptation)을 모니터링하기 위한 진단제 제조시 Vero-RV의 용도.
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