KR20190015712A - 면역자극 및 항원 폴리펩티드를 포함하는 효모 백신 벡터, 및 그를 이용하는 방법 - Google Patents
면역자극 및 항원 폴리펩티드를 포함하는 효모 백신 벡터, 및 그를 이용하는 방법 Download PDFInfo
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- KR20190015712A KR20190015712A KR1020187034980A KR20187034980A KR20190015712A KR 20190015712 A KR20190015712 A KR 20190015712A KR 1020187034980 A KR1020187034980 A KR 1020187034980A KR 20187034980 A KR20187034980 A KR 20187034980A KR 20190015712 A KR20190015712 A KR 20190015712A
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- lys
- glu
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Abstract
본원에서는 면역자극성 폴리펩티드 및 임의적으로 항원성 폴리펩티드를 포함하는 효모를 포함하는 백신 조성물을 제공한다. 면역자극성 폴리펩티드 및 항원성 폴리펩티드는 효모 백신 조성물의 표면 상에서 발현되거나, 또는 디스플레이된다. 대상체에 백신접종하기 위해 백신 조성물을 사용하는 방법을 또한 제공한다.
Description
관련 특허 출원에 대한 상호 참조
본 특허 출원은 2016년 5월 3일 출원된 미국 가특허 출원 번호 62/331,044에 대한 우선권의 이익을 주장하고, 상기 특허 출원의 내용은 그 전문이 본원에서 참조로 포함된다.
서열 목록
본 출원은 EFS-Web을 통해 전자 방식으로 출원 중에 있고, .txt 포맷으로 전자 방식으로 제출된 서열 목록을 포함한다. .txt 파일은 2017년 5월 2일 작성된 파일명 "2017-05-01_5658-00379_ST25.txt"의 서열 목록을 포함하고, 그 크기는 118,371 바이트이다. 상기 .txt 파일에 포함된 서열 목록은 본 명세서의 일부이며, 그 전문이 본원에서 참조로 포함된다.
백신은 질환을 호전시키거나, 또는 예방하기 위해 항원, 특히, 병원체로부터의 항원에 대한 후천성 면역 반응을 개시시키는 데 사용된다. 불활성화된 또는 약독화된 미생물 백신이 대개는 완전한 방어성을 나타내는 강건한 면역 반응을 자극시키는 데 효과적이지만, 일부 경우에서는, 상기 백신들이 방어성을 나타내지 않거나, 또는 단지 부분적으로만 방어성을 나타내는 바, 다른 전략법을 사용하여 방어 백신을 개발하여야 한다. 미생물 기반 백신은 큰 항원성 단백질, 예컨대, 바이러스 단백질을 적절하게 발현하기 위해 글리코실화에 의한 번역 후 단백질 변형을 수행할 수 없는 바; 이에, 글리코실화할 수 있고, 이로써, 적절하게 폴딩된 큰 항원성 단백질을 생성할 수 있는, 지속적인 방어 면역 반응을 자극시키는 데 안전하고 효과적인, 효모 백신 벡터 개발이 요구된다.
본원에서는 효모 백신 벡터를 제공한다. 백신 벡터는 경구 투여에 적합하고, 항원에 대하여 신속하고, 장기간 지속된 면역을 일으키고, 표적화된 미생물에 의한 후속되는 감염으로부터 방어한다. 특히, 발생된 면역 반응은 점막 감염으로부터의 방어에 적합한 IgA 반응이다.
한 측면에서, 효모 백신 조성물을 제공한다. 조성물은 HMGB1 폴리펩티드 또는 CD40 리간드로부터 선택되는 면역자극성 폴리펩티드를 코딩하는 면역자극성 폴리뉴클레오티드를 포함하는 효모를 포함한다. 효모는 효모의 표면 상에 HMGB1 폴리펩티드 또는 CD40 리간드를 발현하도록 조작된다. 조성물은 또한 이 또한 효모의 표면 상에서 적합하게 발현되는 항원성 폴리펩티드를 포함할 수 있다. 조성물은 제약상 허용되는 담체 및/또는 애주번트와 조합하여 제약 조성물을 생성할 수 있다. 조성물은 1개 초과의 항원성 폴리펩티드를 포함할 수 있고, 상기 1개 초과의 항원성 폴리펩티드는 동일하거나, 또는 상이한 유기체 또는 종으로부터 유래된 것일 수 있다.
또 다른 측면에서, 대상체에게 본원에서 제공하는 백신 조성물 및 제약 조성물을, 백신 조성물 및 항원성 폴리펩티드와 관련된 감염체에 대한 대상체의 면역 반응을 증강시키는 데 효과적인 양으로 투여함으로써 대상체에서 면역 반응을 증강시키는 방법을 제공한다.
도 1은 살모넬라 엔테리디티스(Salmonella Enteriditis) aroA/htrA, 살모넬라 엔테리디티스 aroA/htrA-AgA-CD154, 및 살모넬라 엔테리디티스 aroA/htrA-AgA-AgB-HMGB1로 표지된 형광과 공동 배양 후 RAW 264 대식세포의 형광 강도를 비교한 그래프이다.
도 2는 글리코실포스파티딜이노시톨 고정 사카로마이세스 세레비시아에(Saccharomyces cerevisiae) 알파 응집소 세포 표면 발현 방법을 사용하여 피키아 파스토리스(Pichia pastoris) 상의 갈루스 갈루스(Gallus gallus) 특이적인 고 이동성 그룹 박스 1 (HMGB1) 단백질 발현을 포함하도록 조작된 pPICZ 플라스미드 지도의 개략도이다. pPICZ는 AOX1 프로모터를 사용하는 메탄올 유도성이다.
도 3은 피키아 파스토리스 (X33) 상의 HMGB1 세포 표면 발현을 보여주는 사진 세트이다. 피키아 파스토리스-HMGB1 구축물 #4 (도 3D: DIC 단독, 도 3E: 형광 단독, 도 3F: DIC/형광 오버레이) 및 피키아 파스토리스 (X33; 도 3A: DIC 단독, 도 3B: 형광 단독, 도 3C: DIC/형광 오버레이) 백본을 포스페이트 완충 염수 (PBS) 중의 1% 염소 혈청 중에서 1:1,000으로 알렉사(Alexa) 488과 접합된 염소 항-토끼 IgG의 F(ab)2 부위를 갖는 PBS 중에서 1:5로 희석된 토끼 폴리클로날 HMGB1 156-177을 사용하여 염색시켰다. 형질전환된 9개의 피키아 파스토리스-HMGB1 구축물 중 3개의 구축물 상에서 HMGB1 단백질 발현이 최적으로 발현되었다.
도 4는 피키아 파스토리스 특이적 혈청 항체 샘플/음성 대조군 혈청 비를 보여주는 그래프이다. 본 발명자들은 ELISA를 사용하여 측정된 피키아 파스토리스 특이적 항체의 전형적인 용량 반응 곡선을 관찰하였다. 107/브로일러로 SQ로 투여된 피키아 파스토리스는 너무 적어서 면역 반응을 증가시키지 못했고, 109/브로일러는 너무 많아서 심지어 X33 구축물 백본으로의 혈청전환을 일으켰다. 108/브로일러로 SQ로 투여된 피키아 파스토리스는 모든 HMGB1+ 피키아 파스토리스 구축물이 피키아 파스토리스 특이적 혈청 항체를 유의하게 상승시킨 (P<0.001) 최상의 용량이었다.
도 5는 백신접종을 받지 않은 대조군 브로일러 (1군) 혈청에 대해 정규화된 피키아 파스토리스 특이적 혈청 항체의 비를 보여주는 그래프이다. 본 발명자들이 예측한 용량 반응이 반복되었다. 본 발명자들은 전형적인 용량 반응 곡선을 관찰하였다. 107/브로일러로 SQ로 투여된 피키아 파스토리스는 너무 적어서 면역 반응을 증가시키지 못했고, 109/브로일러는 너무 많아서 심지어 X33 구축물 백본으로의 혈청전환을 일으켰다. 108/브로일러로 SQ로 투여된 피키아 파스토리스는 모든 HMGB1+피키아 파스토리스 구축물이 피키아 파스토리스 특이적 혈청 항체를 유의하게 상승시킨 (P=0.049) 최상의 용량이었다.
도 6은 시험감염 후 6일째의 병변 점수가 4인 동물의 백분율(%)을 보여주는 그래프이고, 각 막대 내에 표시된 백분율(%)은 시험감염 후 6일째 사망률(%)을 보여주는 것이다.
도 7은 모든 병변 점수의 분포가 또한 백신접종받은 동물에서 감소되었다는 것을 보여주는 그래프이다. 백신접종받은 동물은 더 낮은 병변 점수를 보였다.
도 2는 글리코실포스파티딜이노시톨 고정 사카로마이세스 세레비시아에(Saccharomyces cerevisiae) 알파 응집소 세포 표면 발현 방법을 사용하여 피키아 파스토리스(Pichia pastoris) 상의 갈루스 갈루스(Gallus gallus) 특이적인 고 이동성 그룹 박스 1 (HMGB1) 단백질 발현을 포함하도록 조작된 pPICZ 플라스미드 지도의 개략도이다. pPICZ는 AOX1 프로모터를 사용하는 메탄올 유도성이다.
도 3은 피키아 파스토리스 (X33) 상의 HMGB1 세포 표면 발현을 보여주는 사진 세트이다. 피키아 파스토리스-HMGB1 구축물 #4 (도 3D: DIC 단독, 도 3E: 형광 단독, 도 3F: DIC/형광 오버레이) 및 피키아 파스토리스 (X33; 도 3A: DIC 단독, 도 3B: 형광 단독, 도 3C: DIC/형광 오버레이) 백본을 포스페이트 완충 염수 (PBS) 중의 1% 염소 혈청 중에서 1:1,000으로 알렉사(Alexa) 488과 접합된 염소 항-토끼 IgG의 F(ab)2 부위를 갖는 PBS 중에서 1:5로 희석된 토끼 폴리클로날 HMGB1 156-177을 사용하여 염색시켰다. 형질전환된 9개의 피키아 파스토리스-HMGB1 구축물 중 3개의 구축물 상에서 HMGB1 단백질 발현이 최적으로 발현되었다.
도 4는 피키아 파스토리스 특이적 혈청 항체 샘플/음성 대조군 혈청 비를 보여주는 그래프이다. 본 발명자들은 ELISA를 사용하여 측정된 피키아 파스토리스 특이적 항체의 전형적인 용량 반응 곡선을 관찰하였다. 107/브로일러로 SQ로 투여된 피키아 파스토리스는 너무 적어서 면역 반응을 증가시키지 못했고, 109/브로일러는 너무 많아서 심지어 X33 구축물 백본으로의 혈청전환을 일으켰다. 108/브로일러로 SQ로 투여된 피키아 파스토리스는 모든 HMGB1+ 피키아 파스토리스 구축물이 피키아 파스토리스 특이적 혈청 항체를 유의하게 상승시킨 (P<0.001) 최상의 용량이었다.
도 5는 백신접종을 받지 않은 대조군 브로일러 (1군) 혈청에 대해 정규화된 피키아 파스토리스 특이적 혈청 항체의 비를 보여주는 그래프이다. 본 발명자들이 예측한 용량 반응이 반복되었다. 본 발명자들은 전형적인 용량 반응 곡선을 관찰하였다. 107/브로일러로 SQ로 투여된 피키아 파스토리스는 너무 적어서 면역 반응을 증가시키지 못했고, 109/브로일러는 너무 많아서 심지어 X33 구축물 백본으로의 혈청전환을 일으켰다. 108/브로일러로 SQ로 투여된 피키아 파스토리스는 모든 HMGB1+피키아 파스토리스 구축물이 피키아 파스토리스 특이적 혈청 항체를 유의하게 상승시킨 (P=0.049) 최상의 용량이었다.
도 6은 시험감염 후 6일째의 병변 점수가 4인 동물의 백분율(%)을 보여주는 그래프이고, 각 막대 내에 표시된 백분율(%)은 시험감염 후 6일째 사망률(%)을 보여주는 것이다.
도 7은 모든 병변 점수의 분포가 또한 백신접종받은 동물에서 감소되었다는 것을 보여주는 그래프이다. 백신접종받은 동물은 더 낮은 병변 점수를 보였다.
본원에서는 백신 조성물에 대해, 또는 백신 조성물에 의해 발현된 항원성 폴리펩티드에 대해 면역 반응을 유발할 수 있는 백신 조성물을 제공한다. 특히, 백신 조성물은 효모의 표면 상에서 면역자극성 폴리펩티드를 발현하도록 조작된 효모를 포함한다. 효모는 또한 효모의 표면 상에서 추가의 항원성 폴리펩티드를 발현하도록 조작될 수 있다. 특정 실시양태에서, 피키아 파스토리스 백신 벡터를 제공한다. 백신 벡터는 효모의 표면 상에서 디스플레이되거나, 또는 발현되는 면역자극성 폴리펩티드를 코딩하는 면역자극성 폴리뉴클레오티드 서열을 포함한다. 면역자극성 폴리펩티드는 고 이동성 그룹 박스 1 (HMGB1) 면역자극성 폴리펩티드 또는 CD40 리간드, 예컨대, CD154 폴리펩티드 또는 그의 단편 또는 다른 CD40 효능제, 예컨대, CD40 효능작용 항체일 수 있다. 면역자극성 폴리펩티드는 통상의 기술자에게 이용가능한 임의의 수단을 사용하여 효모, 예컨대, 피키아 파스토리스의 표면 상에서 발현될 수 있다. 예에서, 면역자극성 폴리펩티드는 사카로마이세스 세레비시아에 α-응집소의 3' 단부에 의해 코딩되는 글리코실포스파티딜이노시톨 (GPI)-고정 기전을 통해 효모의 표면에 부착된다. 통상의 기술자는 백신 조성물을 생성하기 위하여 다른 발현 시스템을 사용하여 효모에 포함된 면역자극성 및/또는 항원성 폴리펩티드를 표면 발현시킬 수 있다는 것을 쉽게 이해할 것이다.
HMGB1 단백질은 DNA 구조 및 안정성에 중요한 DNA 결합 단백질로서 최초로 확인되었다. 이는 서열 특이성 없이 DNA에 결합하는 편재성으로 발현되는 핵 단백질이다. 상기 단백질은 고도로 보존되고, 식물에서부터 포유동물까지에 이르는 범위의 유기체에서 발견된다. 닭, 지브라피시, 인간, 마우스, 래트, 게잡이 원숭이, 소, 말, 개, 돼지, 토끼, 레드 드럼(red drum), 메기, 험프헤드 도미(humphead snapper), 금붕어, 킹코브라, 브라인 슈림프 및 다른 HMGB1 아미노산 서열을 제공한다. 서열식별번호(SEQ ID NO): 2-30 및 94-105를 참조한다. 전 포유동물에 걸쳐 서열은 95% 아미노산 동일성으로 고도로 보존되고, 아미노산 변이는 보존적이다. 따라서, 가능하게는 한 종으로부터의 HMGB1 단백질이 또 다른 종으로부터의 것을 기능적으로 대체할 수 있을 것이다. 전장의 HMGB1 단백질 또는 그의 일부는 본원에 기술된 백신 벡터에서 HMGB1 폴리펩티드로서 사용될 수 있다. HMGB1은 A1 및 A2, 및 B1 및 B2로 명명되는 2개의 DNA 결합 영역을 가진다. 문헌 [Andersson and Tracey, Annu. Rev. Immunol. 2011, 29:139-162]를 참조한다.
HMGB1은 염증 매개인자이고, 예컨대, 괴사성 세포로부터의 핵 손상의 신호로서의 역할을 한다. HMGB1은 또한 단백질의 아세틸화, 핵을 가로지르는 전위 및 분비를 필요로 하는 프로세스에서 단핵구/대식세포 계통의 세포에 의해 활발하게 분비될 수 있다. 세포외 HMGB1은 최종 당화 생성물에 대한 수용체 (RAGE: Receptor for Advanced Glycated End-products), 및 톨(Toll)-유사 수용체 패밀리 (TLR)의 구성원, 특히, TLR4를 통한 신호전달에 의해 강력한 염증 매개인자로서 작용한다. RAGE 결합 활성이 확인되었고, 이는 HMGB1 RAGE 결합 도메인의 폴리펩티드를 필요로 한다. TLR4 결합은 닭 HMGB1 서열 (서열식별번호: 2)의 106번 위치의 시스테인을 필요로 하고, 이는 HMGB1의 B 박스 영역에서 발견된다.
적합하게, 백신 벡터는 닭 HMGB1 폴리펩티드 또는 그의 상동체의 아미노산 150-183 및 89-109를 포함하는 폴리펩티드를 코딩하는 폴리뉴클레오티드를 함유한다. 서열식별번호: 2를 참조한다. 실시예에서, HMGB1의 190 아미노산 폴리펩티드가 사용되었다. 적합하게, 폴리뉴클레오티드는 백신 조성물이 사용되는 대상체와 동일한 종으로부터의 HMGB1 폴리펩티드를 코딩한다. HMGB1은 상기 논의된 바와 같은 수많은 종에 걸쳐 고도로 보존되기 때문에, HMGB1 폴리펩티드 및 대상체의 이종성 조합 (즉, 닭 백신에서 사용하기 위한 인간 HMGB1 폴리펩티드)이 본 발명의 방법에서 유용할 수 있다. HMGB1 폴리펩티드는 효모 백신 조성물 중에 또는 그 상에 존재하는 임의의 외래 항원 또는 항원성 폴리펩티드에 대한 대상체에서의 면역 반응을 증강시키는 데 사용될 수 있다. 통상의 기술자는 HMGB1 폴리펩티드를 사용하여 효모 백신 조성물 중에 존재하는 1개 초과의 항원성 폴리펩티드에 대한 면역 반응을 증강시킬 수 있다는 것을 이해할 것이다. HMGB1로부터의 폴리펩티드는, 적어도 부분적으로, 수지상 세포 및 대식세포를 활성화시켜 시토카인, 예컨대, IL-1, IL-6, IFN-γ 및 TNF-α의 생산을 자극함으로써 면역 반응을 자극시킨다. 실시예에서, HMGB1의 폴리펩티드는 백신 조성물의 표면 상에서 발현되었다.
HMGB1의 염증성 활성은 전장의 단백질을 필요로 하지 않고, 기능적 단편이 확인되었다. B 박스가 HMGB1의 염증유발성 효과를 매개하는 데 충분한 것으로 나타났고, 따라서, HMGB1 박스 b1 및 HMGB1 박스 b2가 본 발명의 맥락에서 HMGB1 폴리펩티드 또는 그의 기능적 단편이다. 예컨대, 서열식별번호: 35 및 36을 참조한다. 추가로, RAGE 결합 부위 및 염증유발성 시토카인 활성을 맵핑하였다. 각각 서열식별번호: 37 및 38을 참조한다. 따라서, 이들 폴리펩티드가 본 발명의 맥락에서 HMGB1 폴리펩티드의 기능적 단편이다. 서열식별번호: 31-38을 참조한다.
통상의 기술자는 예컨대, 국제 공개 번호 WO2002 092004 (이는 그 전문이 본원에서 참조로 포함된다)에서의 것과 같은 방법을 사용하여 염증유발성 시토카인 활성을 자극시킬 수 있는 HMGB1 폴리펩티드 및 그의 단편을 확인할 수 있다. 적합하게, HMGB1 폴리펩티드는 닭 HMGB1 서열의 아미노산 150-183의 RAGE 결합 도메인 (HMGB1 RAGE 결합 도메인 또는 그의 상동체) 및 닭 HMGB1 서열의 아미노산 89-109의 염증유발성 시토카인 활성 도메인 (서열식별번호: 2; HMGB1 염증유발성 시토카인 활성 또는 그의 상동체)을 포함한다. 특히, HMGB1 폴리펩티드 및 그의 기능적 단편 또는 상동체는 닭 HMGB1 서열 또는 HMGB1 박스 a1, HMGB1 박스 a2, HMGB1 박스 b1, HMGB1 박스 b2, HMGB1 RAGE 결합 도메인, 또는 HMGB1 염증유발성 시토카인 활성의 HMGB1 폴리펩티드와 각각 동일하거나, 또는 그와 적어도 99% 동일한, 적어도 98% 동일한, 적어도 95% 동일한, 적어도 90% 동일한, 적어도 85% 동일한, 또는 적어도 80% 동일한 폴리펩티드를 포함한다.
면역자극성 폴리펩티드는 또한 CD40 리간드 또는 CD40 효능제일 수 있다. 대상체에서 CD40에 결합할 수 있고, 대상체가 백신 조성물 및 그의 회합된 외래 항원성 폴리펩티드에 반응하도록 자극시킬 수 있는 CD154 폴리펩티드는 면역자극성 폴리펩티드로서 사용될 수 있다. CD154 폴리펩티드는 전장의 CD154일 수 있거나, 또는 그 길이는 50개 이하의 아미노산, 더욱 적합하게, 40개 이하, 30개 이하, 또는 20개 이하의 아미노산 길이일 수 있다. 폴리펩티드 길이는 10 내지 15개의 아미노산, 10 내지 20개의 아미노산, 10 내지 25개의 아미노산 길이일 수 있다. CD154 서열 및 CD40 결합 영역은 다양한 종들 사이에 고도로 보존되지 않는다. 닭 및 인간의 CD154 서열은 각각 서열식별번호: 106 및 서열식별번호: 107에 제공되어 있다.
CD154의 CD40 결합 영역이 인간, 닭, 오리, 마우스 및 소를 비롯한, 다수의 종에 대해 결정되었고, 서열식별번호: 108, 서열식별번호: 109, 서열식별번호: 110, 서열식별번호: 111, 및 서열식별번호: 112에 각각 제시되어 있다. 서열식별번호: 106-112에 제공된 CD154 서열과 동일하거나, 또는 적어도 99% 동일한, 적어도 98% 동일한, 적어도 95% 동일한, 적어도 90% 동일한, 적어도 85% 동일한, 또는 적어도 80% 동일한 폴리펩티드를 또한 포함한다. 종들 사이에서 CD40 결합 영역에서의 서열이 가변적이지만, CD154의 CD40에의 종간 교차 결합이 보고되었다. 예를 들어, 인간 CD154 폴리펩티드는 닭에서 면역 반응을 증강시킬 수 있다. 그러므로, 종 특이적 CD154 폴리펩티드 또는 이종성 CD154 폴리펩티드를 사용하여 본 발명을 실시할 수 있다.
또 다른 대안으로, CD40 리간드는 CD40 효능작용 항체 또는 그의 일부일 수 있다. 상기 CD40 효능작용 항체는 적어도 국제 출원 번호 WO2015/187969에 개시되어 있다. CD40 항체 및 효능작용 CD40 항체는 또한 여러 종, 특히, 마우스 및 인간에 대해 상업적으로 이용가능하다. 항체는 항체가 CD40을 발현하는 표적 세포 내에서 신호전달을 유도할 수 있다면, 항체는 CD40에 대해 효능작용성을 나타내는 것이다. CD40을 통해 신호전달이 이루어지면, 항원 제시 세포의 표면 상에서의 CD40 및 TNF 수용체의 발현은 증가되고, 반응성 산소 종 및 산화질소 생산, 및 B 세포 활성화가 유도되고, 이로써, 이소형 전환이 일어나게 된다.
적합한 닭 CD40 효능작용 항체는 2C5로서 지칭된 서열식별번호: 113 (중쇄) 및 서열식별번호: 114 (경쇄)로서, 또는 DAG-1로서 지칭된 서열식별번호: 115 (단일 쇄 가변 단편 (scFv))로서 본원에서 제공되는 항체를 포함한다. 이들 항체는 Fc 부분 및 비-CDR 영역을 상동성 숙주-화합성 항체 백본 서열로 대체시켜 이러한 항체 백본 그 자체에 대한 면역 반응을 최소화시킬 수 있도록 "닭화(chickenized)" 형태로 제조될 수 있다. 추가로, 항체는 재조합적으로 또는 효소 소화 (즉, 파파인 또는 펩신)를 통하여 더 작은 항체 부분으로 제조될 수 있고, 이는 항체의 F(ab) 부분, 예컨대, F(ab)2 단편만을 포함할 수 있다. 닭 CD40 항체 둘 모두에 대한 CDR 영역이 확인되었다. 2C5로서 명시되고, 서열식별번호: 113 및 서열식별번호: 114에 제공된 항체의 경우, 중쇄 가변 영역은 서열식별번호: 116에 기재된 아미노산 서열을 포함하는 CDR1, 서열식별번호: 117에 기재된 아미노산 서열을 포함하는 CDR2, 및 서열식별번호: 118에 기재된 아미노산 서열을 포함하는 CDR3을 포함하고, 경쇄 가변 영역은 서열식별번호: 119에 기재된 아미노산 서열을 포함하는 CDR1, 서열식별번호: 120에 기재된 아미노산 서열을 포함하는 CDR2, 및 서열식별번호: 121에 기재된 아미노산 서열을 포함하는 CDR3을 포함한다. DAG-1로서 명시되고, 서열식별번호: 115에 제공된 항체의 경우, 중쇄 가변 영역은 서열식별번호: 122에 기재된 아미노산 서열을 포함하는 CDR1, 서열식별번호: 123에 기재된 아미노산 서열을 포함하는 CDR2, 및 서열식별번호: 124에 기재된 아미노산 서열을 포함하는 CDR3을 포함하고, 경쇄 가변 영역은 서열식별번호: 125에 기재된 아미노산 서열을 포함하는 CDR1, 서열식별번호: 126에 기재된 아미노산 서열을 포함하는 CDR2, 및 서열식별번호: 127에 기재된 아미노산 서열을 포함하는 CDR3을 포함한다. 또 한, 서열식별번호: 113-127 중 적어도 하나와 동일하거나, 또는 적어도 99% 동일한, 적어도 98% 동일한, 적어도 95% 동일한, 적어도 90% 동일한, 적어도 85% 동일한, 또는 적어도 80% 동일한 폴리펩티드를 또한 포함한다.
본원에서 제공되는 백신 조성물은 효모를 포함한다. 효모는 하기 효모 속: 사카로마이세스, 칸디다(Candida) 속, 크립토코쿠스(Cryptococcus) 속, 한세눌라(Hansenula) 속, 클루이베로마이세스(Kluyveromyces) 속, 피키아, 로도토룰라(Rhodotorula) 속, 시조사카로마이세스(Schizosaccharomyces) 속 및 야로위아(Yarrowia) 속 중 임의의 것으로부터 선택될 수 있다. 효모는 사카로마이세스 세레비시아에, 칸디다 알비칸스(Candida albicans), 한세눌라 폴리모르파(Hansenula polymorpha), 피키아 파스토리스 및 시조사카로마이세스 폼베(Schizosaccharomyces pombe)로 이루어진 군으로부터 선택되는 종의 효모일 수 있다. 실시예에서, 피키아 파스토리스가 사용되었다. 효모는 면역자극성 폴리펩티드를 코딩하는 면역자극성 폴리뉴클레오티드를 포함하고, 항원성 폴리펩티드를 코딩하는 항원성 폴리뉴클레오티드를 추가로 포함할 수 있다. 적합하게, 면역자극성 폴리펩티드 및 항원성 폴리펩티드는 효모에 의해 자연적으로 발현되지는 않는다. 효모는 면역자극성 폴리펩티드 및 항원성 폴리펩티드를 발현하도록, 및 효모의 표면 상에서 상기 폴리펩티드들을 디스플레이하거나, 또는 발현하도록 조작된다.
항원성 폴리펩티드의 적어도 일부, 및 면역자극성 폴리펩티드의 적어도 일부는 피키아 파스토리스 또는 다른 효모 기반 백신 조성물의 표면 상에 존재한다. 백신 백신 조성물의 표면 상에 존재하는 것은 막횡단 단백질 내에 포함되거나, 막횡단 단백질, 막 지질 또는 막-GPI-고정 탄수화물 또는 단백질과 상호작용하거나, 그에 공유결합으로 또는 화학적으로 가교 결합된 폴리펩티드를 포함한다. 폴리펩티드를 이루는 아미노산들을 펩티드 결합을 통해 N-말단, C-말단 또는 막횡단 단백질 내의 임의의 위치 (즉, 막횡단 단백질의 2개의 아미노산 사이에 삽입됨)에 또는 막횡단 단백질의 하나 이상의 아미노산 대신 (즉, 결실-삽입)에 연결시킴으로써 폴리펩티드가 막횡단 단백질 내에 포함될 수 있다. 면역자극성 또는 항원성 폴리펩티드의 표면 발현을 수득하기 위해 비-천연 면역자극성 폴리뉴클레오티드 또는 항원성 폴리뉴클레오티드를 막횡단 단백질 또는 세포 벽 단백질의 세포외 루프 내의 프레임 내에 삽입될 수 있다는 것을 통상의 기술자는 이해할 것이다. 실시예에서, 효모의 공유적으로 연결된 GPI-고정 시스템을 이용하는 α-응집소 C-말단 고정 방법이 사용되지만, 다른 유사한 고정 방법이 통상의 기술자에게 이용가능하다.
대안적으로, 폴리펩티드는 통상의 기술자가 이용가능한 방법들을 통해 막 내의 단백질, 지질 또는 탄수화물에 공유적으로 또는 화학적으로 연결될 수 있다. 예를 들어, 디-술피드 결합 또는 비오틴-아비딘 가교 결합을 사용하여 항원성 및 면역자극성 폴리펩티드를 백신 조성물 중의 효모의 표면 상에 제시시킬 수 있다. 적합하게, 항원성 폴리펩티드 및 면역자극성 폴리펩티드는 융합 단백질의 일부분이다. 두 폴리펩티드는 펩티드 결합을 통해 직접적으로 연결될 수 있거나, 또는 폴리뉴클레오티드에 의해 코딩되는 링커 펩티드, 또는 이들이 내부로 삽입되는 제3 단백질의 절편에 의해 이격될 수 있다. 일부 실시양태에서, 면역자극성 폴리펩티드 및/또는 항원성 폴리펩티드 각각의 카피를 1개 초과로 포함하는 융합 단백질이 효모에 포함된다. 일부 실시양태에서, 면역자극성 폴리펩티드의 다중 카피는 동일한 면역자극성 폴리펩티드 또는 상이한 폴리펩티드의 카피를 1개 초과로 포함한다. 동일하거나, 또는 유사한 항원성 폴리펩티드의 다중 카피가 효모에 포함될 수 있고, 예컨대, 가능하게는 단일의 또는 단지 몇 안 되는 아미노산 서열 차이를 갖는 조류 인플루엔자 M2e 항원의 다중 카피가 효모에 포함될 수 있다는 점에서, 항원성 폴리펩티드의 경우에도 동일하게 적용된다. 대안적으로, 효모는 백신 조성물의 단일 투여가 상이한 종으로부터의 상이한 항원에 대한 면역 반응을 유도하거나, 또는 증강시킬 수 있도록 하기 위해 다중의 상이하고, 독특한 항원성 폴리펩티드를 발현하도록 조작될 수 있다. 예를 들어, 백신 조성물은 동일한 백신 조성물에 포함될 수 있는 서열식별번호: 55 및 서열식별번호: 61의 항원성 폴리펩티드를 포함시킴으로써 캄필로박터(Campylobacter) 및 에이메리아(Eimeria)에 의한 후속되는 감염에의 면역 반응을 증강시키기 위해 제조될 수 있다.
항원성 폴리펩티드 또는 면역자극성 폴리펩티드를 코딩하는 폴리뉴클레오티드가 백신 조성물의 효모 내로 삽입되고 발현되어 항원성 폴리펩티드 및 면역자극성 폴리펩티드가 생성될 수 있다. 폴리뉴클레오티드는 백신 조성물의 염색체 내로 삽입될 수 있거나, 또는 플라스미드 또는 다른 염색체외 DNA 상에, 예컨대, YAC (효모 인공 염색체) 상에서 코딩될 수 있다. 적합하게, 항원성 폴리펩티드 및/또는 면역자극성 폴리펩티드를 코딩하는 폴리뉴클레오티드는 독립적으로 발현될 수 있거나, 또는 발현되는 효모 백신 폴리뉴클레오티드 내로 삽입된다. 효모 백신 폴리뉴클레오티드는 효모 백신의 표면 상에서 발현되는 폴리펩티드, 예컨대, 막횡단 단백질을 코딩할 수 있다. 항원성 폴리펩티드 및/또는 면역자극성 폴리펩티드를 코딩하는 폴리뉴클레오티드는 항원성 폴리펩티드 및/또는 면역자극성 폴리펩티드가 효모의 표면 상에서 발현될 수 있도록 효모 백신 폴리뉴클레오티드 서열 내로 삽입될 수 있다.
대안적으로, 항원성 폴리펩티드 및/또는 면역자극성 폴리펩티드를 코딩하는 폴리뉴클레오티드는 효모 백신의 표면 상의 단백질, 지질 또는 탄수화물과의 회합을 통해 효모 백신의 표면 상에 디스플레이되거나, 또는 제시되는 분비형 폴리펩티드 내로 삽입될 수 있다. 통상의 기술자는 항원성 폴리펩티드 및/또는 면역자극성 폴리펩티드를 코딩하는 폴리뉴클레오티드가, 항원성 폴리펩티드 및/또는 면역자극성 폴리펩티드를 발현시키고, 그를 효모로 처치되는 대상체의 면역 세포에 제시하도록 광범위한 효모 폴리뉴클레오티드 내로 삽입될 수 있음을 이해할 것이다.
상기 논의에서 언급된 바와 같이, 본원에 기술된 백신은 또한 항원성 폴리펩티드를 코딩하는 항원성 폴리뉴클레오티드를 포함할 수 있다. 항원성 폴리펩티드는 후천성 면역계에 의해 특이적으로 인식될 수 있는 폴리펩티드이다. 항원성 폴리펩티드는 백신 벡터로서 작용하는 효모에 대해 백신접종하기 위해 벡터로서 선택되는 효모에 의해 자연적으로 발현될 수 있다. 대안적으로, 효모 백신 벡터는 항원성 폴리펩티드로서 백신 벡터와 자연적으로 회합되어 있지 않은 이종성 폴리펩티드를 코딩하는 이종성 폴리뉴클레오티드를 보유할 수 있다. 항원성 폴리펩티드로는 면역원성인 임의의 폴리펩티드를 포함한다. 항원성 폴리펩티드로는 병원체-관련, 알레르겐-관련, 종양-관련 또는 질환-관련 항원을 포함하나, 이에 제한되지 않는다. 병원체로는 바이러스, 기생충, 진균 및 박테리아 병원체 뿐만 아니라, 단백질 병원체, 예컨대, 프리온을 포함한다. 항원성 폴리펩티드는 전장의 단백질 또는 그의 일부일 수 있다.
다수의 단백질의 면역계 인식은 대개는 에피토프로 지칭되는 비교적 적은 개수의 아미노산을 기초로 한다는 것이 잘 확립되어 있다. 에피토프는 단지 8-10개의 아미노산일 수 있다. 항원성 폴리펩티드라는 용어는 대상체에서 항체 또는 T 세포 면역 반응의 생성 대상이 되는 에피토프를 포함할 수 있다. 에피토프 및 항원 또는 항원성 폴리펩티드라는 용어는 상호교환적으로 사용될 수 있다. 따라서, 본원에 기술된 항원성 폴리펩티드는 전장의 단백질, 길이가 8개의 아미노산 길이인 에피토프 또는 상기 극값 사이의 임의의 일부분일 수 있다. 실제로, 항원성 폴리펩티드는 단일 병원체 또는 단백질로부터의 1개 초과의 에피토프를 포함할 수 있다. 항원성 폴리펩티드는 예컨대, 유선염 감염과 연관된 것, 살모넬라, 클로스트리디움(Clostridium), 캄필로박터, 에스쉐리키아(Escherichia), 시겔라(Shigella), 헬리코박터(Helicobacter), 비브리오(Vibrio), 플레시오모나스(Plesiomonas), 에드와르디아(Edwardia), 클렙시엘라(Klebsiella), 스타필로코쿠스(Staphylococcus), 스트렙토코쿠스(Streptococcus), 아에로모나스(Aeromonas)와 같은, 박테리아의 큰 세그먼트 또는 박테리아의 작은 폴리펩티드; 인플루엔자, 구제역 바이러스, 돼지 유행성 설사 바이러스 (PEDv), 및 돼지 생식기 호흡 증후군 바이러스 (PRRSV)를 포함하나, 이에 제한되지 않는 바이러스 단백질; 에이메리아 아종, 톡소플라스마(Toxoplasma), 말라리아, 또는 다른 기생충을 포함하나, 이에 제한되지 않는 기생충 감염; 및 종양 항원을 포함할 수 있지만, 이에 제한되지는 않을 것이다. 예를 들어, 미국 특허 번호 8,604,198, 국제 공개 번호 WO2009/059018, WO2009/059298, WO2011/091255, WO2011/156619, WO2014070709, WO 2014/127185 또는 WO 2014/152508에서 확인된 항원 또는 에피토프가 사용될 수 있다. 항원성 폴리펩티드는 서열식별번호: 39-93에 제공된 것 중 임의의 하나 이상의 것을 포함할 수 있고, 서열식별번호: 39-93에 제공된 것과 동일하거나, 또는 적어도 99% 동일한, 적어도 98% 동일한, 적어도 95% 동일한, 적어도 90% 동일한, 적어도 85% 동일한, 또는 적어도 80% 동일한 폴리펩티드를 포함한다. 통상의 기술자는 서열식별번호: 39-93에 포함된 펩티드 중 일부가 항원성 에피토프로서 작용하는 데 요구될 가능성이 있는 것보다 더 길고, 따라서, 이들 항원성 폴리펩티드의 단편 또한 그에 포함된다는 것을 알고 있다. 통상의 기술자는 항원성 폴리펩티드는 추가의 아미노산 또한 포함할 수 있거나, 또는 링커 아미노산을 통해 서로에게 또는 면역자극성 폴리펩티드에 연결되어 융합 단백질 종류를 형성할 수 있다는 것 또한 이해할 것이다. 링커 아미노산은 임의의 아미노산일 수 있지만, 세린 및 글리신이 가장 보편적으로 사용된다. 링커는 1 또는 2개의 아미노산인 정도로 짧을 수도 있지만, 그 길이는 4, 5, 6, 8, 10, 12, 14, 15개 또는 그 초과의 아미노산 길이일 수 있다.
동일한 에피토프 또는 항원성 폴리펩티드의 다중 카피, 또는 상이한 단백질들로부터의 다중 에피토프가 백신 벡터 내에 포함될 수 있다. 동일하거나, 또는 상이한 병원체 또는 질환으로부터의 여러 에피토프 또는 항원이 다중 항원에 대하여 증강된 면역 반응을 발생시키기 위해 효모 백신 벡터로 조합되어 투여될 수 있다는 것이 고려된다. 효모 백신 벡터는 다중 병원성 미생물, 바이러스 또는 종양 연관 항원으로부터의 항원을 코딩할 수 있다. 다중 항원을 발현할 수 있는 백신 벡터의 투여는 2가지 이상의 질환에 대한 면역을 동시에 유도한다는 장점을 가지고 있다.
항원성 폴리펩티드에 대한 면역 반응을 증강시킬 수 있는 면역자극성 폴리펩티드를 코딩하는 폴리뉴클레오티드는 항원성 폴리펩티드 또한 코딩할 수 있다. 면역자극성 폴리펩티드를 코딩하는 폴리뉴클레오티드는 항원성 폴리펩티드를 코딩하는 폴리뉴클레오티드에 연결될 수 있고, 이로써, 백신 벡터 중 면역자극성 폴리펩티드 및 항원성 폴리펩티드는 동일한 폴리뉴클레오티드에 의해 코딩된다. 항원성 폴리펩티드 및 면역자극성 폴리펩티드 중 적어도 일부는 효모 백신 벡터의 표면 상에 존재한다. 백신 조성물은 항원성 폴리펩티드를 코딩하는 항원성 폴리뉴클레오티드, 및 면역자극성 폴리펩티드를 코딩하는 면역자극성 폴리뉴클레오티드를 포함할 수 있다. 면역자극성 폴리펩티드 및 항원성 폴리펩티드는 예컨대, 융합 단백질로 연결될 수 있다. 면역자극성 폴리펩티드 및 항원성 폴리펩티드는 둘 모두 막횡단 단백질의 외부 루프 내에 삽입될 수 있거나, 또는 GPI-고정 기전을 통해 표면에 부착될 수 있다.
이종성 폴리뉴클레오티드로는 효모 백신 벡터 이외의 다른 병원성 미생물 또는 바이러스로부터 선택되는 항원을 코딩하는 폴리뉴클레오티드를 포함하나, 이에 제한되지 않는다. 상기 이종성 또는 항원성 폴리뉴클레오티드는 병원성 바이러스, 예컨대, 인플루엔자 (예컨대, M2e, 혈구응집소, 또는 뉴라미니다제), 헤르페스바이러스 (예컨대, 헤르페스바이러스의 구조 단백질을 코딩하는 유전자), 레트로바이러스 (예컨대, gp160 외피 단백질), 아데노바이러스, 파라믹소바이러스, 코로나바이러스 등으로부터 유래되는 것일 수 있다. 이종성 폴리뉴클레오티드는 또한, 예컨대, 박테리아 단백질, 예컨대, 독소 및 외막 단백질을 코딩하는 유전자와 같이, 병원성 박테리아로부터 수득될 수 있다. 추가로, 기생충, 예컨대 에이메리아로부터의 이종성 폴리뉴클레오티드가 효모 벡터 백신 조성물에서 사용하기 위한 것으로 관심의 대상이 되는 후보물질이다.
면역계를 일으키는 데 관여하는 추가의 면역자극성 폴리펩티드 또한 본원에 기술된 백신 조성물에 포함될 수 있다. 상기 폴리뉴클레오티드는 자신의 자극 효과에 대해 공지된 면역계 분자, 예컨대, 인터루킨, 종양 괴사 인자 또는 인터페론, 또는 면역 조절에서 관여하는 또 다른 폴리펩티드, 예컨대, CD40 리간드 또는 CD40 효능제를 코딩할 수 있다. 따라서, 효모 백신 벡터는 1개 초과의 면역자극성 폴리펩티드 또는 1개 초과의 항원성 폴리펩티드를 함유할 수 있다. 효모 백신 벡터는 폴리펩티드의 발현 수준을 증가시키기 위해 동일한 폴리펩티드의 1개 초과의 카피를 포함한다. 대안적으로, 다중의 상이한 면역자극성 폴리펩티드, 또는 그를 코딩하는 뉴클레오티드, 또는 다중 항원성 폴리펩티드, 또는 그를 코딩하는 뉴클레오티드가 단일 재조합 효모에 포함될 수 있다. 다중 항원성 폴리펩티드는 유사 항원의 다중 카피, 예컨대, M2e 항원 (서열식별번호: 41 및 42)의 2개의 상이한 에피토프일 수 있다. 항원성 폴리펩티드는 완전히 다른 항원에 대한 것이지만, 동일한 감염체와 관련이 있는 항원, 예컨대, M2e 및 HA5일 수 있다. 서열식별번호: 41-44를 참조한다. 항원성 폴리펩티드는 또한 단일의 단위 백신을 이용하여 1개 초과의 병원체에 대하여 백신접종하기 위해 상이한 종에 대한 항원일 수 있다. 예컨대, E. 콜라이(E. coli)로부터의 PAL에 대한 서열식별번호: 54, 또는 에이메리아로부터의 MPP에 대한 서열식별번호: 61과 함께 조합된 인플루엔자 M2e에 대한 서열식별번호: 41이 있다. 실시예에서, MPP-TRAP-HMGB-1 (서열식별번호: 61 연결 서열식별번호: 65 연결 서열식별번호: 2)을 발현하는 효모를 포함하는 백신 조성물을 생성하였고, 이는 에이메리아 맥시마(Eimeria maxima)에 의한 시험감염과 연관된 이환율 및 사망률, 둘 모두를 감소시키는 것으로 나타났다.
백신 조성물 및 제약상 허용되는 담체를 포함하는 조성물을 또한 제공한다. 제약상 허용되는 담체는 생체내 투여에 적합한 임의의 담체이다. 적합하게, 제약상 허용되는 담체는 경구, 비강, 또는 점막 전달용으로 허용되는 것이다. 제약상 허용되는 담체는 물, 완충 용액, 글루코스 용액 또는 박테리아 배양액을 포함할 수 있다. 조성물의 추가 성분은 부형제, 예컨대, 안정제, 보존제, 희석제, 유화제 및 윤활제를 적합하게 포함할 수 있다. 제약상 허용되는 담체 또는 희석제의 예는 안정제, 예컨대, 탄수화물 (예컨대, 소르비톨, 만닛톨, 전분, 수크로스, 글루코스, 덱스트란), 단백질, 예컨대, 알부민 또는 카세인, 단백질 함유 작용제, 예컨대, 소 혈청 또는 탈지유, 및 완충제 (예컨대, 포스페이트 완충제)를 포함한다. 특히 상기 안정제가 조성물에 첨가되는 경우, 조성물은 동결 건조 또는 분무 건조에 적합하다.
백신 조성물은 대상체에서 복제불가능할 수 있다. 효모는 예컨대, 약독화된 형태의 효모와 같이, 실험실 환경 밖에서는 성장이 불가능할 수 있다. 적합하게, 효모는 백신 조성물에 첨가되기 이전에 불활성화되거나 또는 사멸된다. 백신 조성물은 또한 애주번트를 포함할 수 있다. 애주번트는 관련 기술분야에 공지되어 있고, 실시예에서는 만노실화된 키토산 애주번트가 사용되었다. WO 2014/070709를 참조한다.
본원에 기술된 조성물은 면역 반응, 예컨대, 항원성 폴리펩티드에 대한, 또는 백신 벡터 그 자체에 대한 항체 반응을 증강시키는 데 사용될 수 있다. 본원에 기술된 조성물 및 백신 벡터는 질환 기간을 단축시킴으로써, 질환과 연관된 이환율 또는 사망률을 감소시킴으로써, 또는 질환에 걸릴 가능성을 감소시킴으로써 추후 질환의 중증도를 감소시킬 수 있다. 본원에 기술된 백신 벡터 투여 이후의 질환과 연관된 이환율 또는 사망률은 백신 벡터를 제공받지 않은 유사 대상체와 비교하여 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 또는 심지어 100%만큼 감소될 수 있다.
백신 조성물을 투여함으로써 대상체에서 면역 반응을 증강시키는 방법을 또한 제공한다. 백신 조성물은 백신 조성물 및 그의 회합된 항원성 폴리펩티드에 대한 면역 반응을 자극시킬 수 있는 전장의 면역자극성 폴리펩티드 또는 그의 일부를 함유할 수있다. 면역자극성 폴리펩티드를 함유하는 백신 조성물은 백신, 및 특히, 항원성 폴리펩티드에 대한 대상체의 면역 반응을 증강시키는 데 효과적인 양으로 대상체에게 투여된다. 면역 반응을 증강시키는 것은 대상체의 면역계에 의해 매개되는 치료 또는 예방 효과를 유도하는 것을 포함하나, 이에 제한되지 않는다. 효과는 항원성 폴리펩티드에 대한, 또는 항원성 폴리펩티드를 발현하는 감염원 또는 암성 물질에 대한 반응을 시험함으로써 측정될 수 있다. 구체적으로, 면역 반응을 증강시키는 것은 항체 생산 증강, 항체 중쇄 부류 전환 증강, 항원 제시 세포의 성숙, 헬퍼 T 세포의 자극, 세포용해성 T 세포의 자극, 또는 T 및/또는 B 세포 메모리 유도를 포함할 수 있지만, 이에 제한되지 않는다.
조성물은 피하로, 경구로, 비내로, 및 점막으로 이루어진 것을 포함하나, 이에 제한되지 않는, 다양한 수단에 의해 투여될 수 있다. 예를 들어, 백신 조성물 또는 백신 벡터는 에어로졸에 의해, 분무에 의해, 식품 또는 물에의 첨가에 의해, 경구 위관영양에 의해, 또는 점안제를 통해 전달될 수 있다. 일부 실시양태에서, 조성물은 주사에 의해, 예컨대, 진피내로, 비경구로, 피하로, 복강내로, 정맥내로, 두개내로 또는 근육내로 이루어지는 주사에 의해 투여된다. 닭 또는 다른 가금류의 경우, 조성물은 알에 투여될 수 있다. 투여 수단의 조합이 또한 사용될 수 있다. 실시예에서, 피하로 백신접종을 수행한 후, 백신 조성물의 부스트를 경구로 제공할 수 있다. 예컨대, 비내로 또는 에어로졸 또는 분무를 통해 전달한 후, 이어서, 경구 위관영양, 또는 음식물 또는 음용수에 포함시키는 것과 같은 다른 조합이 또한 사용될 수 있다.
대상체는 척추동물, 적합하게, 포유동물, 적합하게, 인간, 소, 고양이, 개, 돼지, 수산 양식 어류, 적합한 메기, 도미, 금붕어, 또는 조류, 적합하게, 가금류, 예컨대, 닭 또는 칠면조를 포함하나, 이에 제한되지 않는다. 다른 감염 동물 모델이 또한 사용될 수 있다. 면역 반응을 증강시키는 것은 대상체의 면역계에 의해 매개되는 치료 또는 예방 효과를 유도하는 것을 포함하나, 이에 제한되지 않는다. 구체적으로, 면역 반응을 증강시키는 것은 예컨대, 도 4 및 5에서 입증된, 항체 생산 증강을 포함할 수 있다. 일부 실시양태에서, IgA 반응이 일어난다.
투여될 유용한 투여량은 대상체의 연령, 체중 및 종, 투여 모드 및 경로, 및 면역 반응의 추구 대상이 되는 병원체 또는 질환의 유형에 따라 달라질 것이다. 조성물은 면역 반응을 유발하는 데 충분한 효모 백신 벡터의 임의의 용량으로 투여될 수 있다. 105 내지 1010개 효모 벡터 카피 범위인 용량이 충분한 것으로 고려된다. 구체적으로, 백신 벡터 특이적인 면역 반응을 유도함으로써, 궁극적으로는 효모 세포 표면 상에서 HMGB1에 인접한 항원성 카르고에 대한 면역 반응의 자극을 나타내도록 하는 데에는, 400X의 확대 배율하에서 혈구계를 사용하여 1 ㎣당 효모의 개수를 계수함으로써 측정된 108개 피키아 파스토리스-HMGB1 백신 벡터 카피인 용량이 최적이었다.
조성물은 단 1회만 투여될 수 있거나, 또는 면역 반응을 증가시키기 위해 2회 이상 투여될 수 있다. 조성물이 1회 초과로 투여되는 경우, 조성물은 상이한 투여 경로를 통해 투여될 수 있으며, 매회 백신은 상기 논의된 바와 같이 투여된다. 예를 들어, 조성물이 1주, 2주만큼, 또는 3주만큼, 1개월, 2개월, 3개월, 6개월 또는 그 초과인 기간만큼 간격을 두고 2회 이상 투여될 수 있다. 효모는 투여 전에 생존가능할 수 있지만, 일부 실시양태에서, 효모는 투여 이전에 사멸되거나, 또는 불활성화될 수 있다. 일부 실시양태에서, 효모는 대상체 내에서 복제가능할 수 있는 한편, 다른 실시양태에서, 효모는 대상체 내에서 복제불가능할 수 있다. 실시예에 제시된 바와 같이, 효모 백신 벡터는 투여 이전에 포르말린, 글루타르알데히드, 에탄올, 산성화, 열 또는 항생제를 사용하여 불활성화될 수 있다. 통상의 기술자는 효모 백신 벡터를 불활성화시키는 다른 수단이 또한 사용될 수 있다는 것을 이해할 것이다.
본 개시내용은 본원에 기술된 구성, 성분 배열, 또는 방법 단계의 구체적 세부 사항에 제한되지 않는다. 본원에 개시된 조성물 및 방법은 하기의 개시내용을 고려하여 통상의 기술자에게 명백할 각종 방식으로 제조되고/거나, 실시되고/거나, 사용되고/거나, 수행되고/거나 형성될 수 있다. 본원에 사용된 어법 및 전문 용어는 단지 기술하기 위한 것이고, 본 특허청구범위의 범주를 제한하는 것으로 간주되지 않아야 한다. 다양한 구조 또는 방법 단계를 지칭하기 위해 본 설명 및 특허청구범위에 사용된 바와 같은, 서수 표시, 예컨대 제1, 제2 및 제3은 임의의 특정 구조 또는 단계, 또는 상기 구조 또는 단계에 대한 임의의 특정 순서 또는 입체 배치를 표시하는 것으로 간주되지 않는다. 본원에 기술된 모든 방법은 본원에서 달리 명시되지 않는 한, 또는 문맥상 달리 명백하게 모순되지 않는 한, 임의의 적합한 순서로 수행될 수 있다. 본원에 제공된 임의의 및 모든 예, 또는 예시적 표현 (예컨대, "예컨대")의 사용은 단지 본 개시내용을 용이하게 하기 위한 것이고, 달리 청구되지 않는 한, 본 개시내용의 범주에 대한 어떤 제한도 의미하지 않는다. 본 명세서에서의 어떤 표현도, 및 도면에 제시된 어떤 구조도 임의의 비-청구된 요소가 개시된 주제를 실시하는 데 본질적이라는 것을 의미하는 것으로 해석되지 않아야 한다. 본원에서 "포함하는(including)," "포함하는(comprising)" 또는 "갖는"이라는 용어 및 그의 파생어 사용은 그 다음에 열거되는 요소 및 그의 등가물 뿐만 아니라, 추가 요소를 포함하는 것을 의미한다. 특정 요소를 "포함하는," "포함하는" 또는 "갖는" 것으로 언급되는 실시양태는 또한 상기 특정 요소로 "본질적으로 이루어지는 것" 및 그로 "이루어지는 것"으로서 고려된다.
본원에서 값을 범위로 언급하는 것은 본원에서 달리 명시되지 않는 한, 단지 그 범위 내에 속하는 각각의 별도의 값을 개별적으로 지칭하는 약칭 방법으로서 의도되는 것이고, 각각의 별도의 값은 마치 그가 본원에 개별적으로 언급된 것처럼 본 명세서 내 포함된다. 예를 들어, 농도 범위가 1% 내지 50%인 것으로 언급되는 경우, 예컨대, 2% 내지 40%, 10% 내지 30%, 또는 1% 내지 3% 등과 같은 값이 본 명세서 내에 명확하게 열거되는 것으로 의도된다. 이들은 구체적으로 의도된 것의 예일 뿐이고, 열거된 최저 값과 최고 값 사이 및 이들 값을 포함한 수치의 가능한 모든 조합이 본 개시내용에서 명확하게 언급되는 것으로 간주되어야 한다. 특별히 열거된 양 또는 양의 범위를 기술하기 위하여 "약"이라는 단어를 사용하는 것은 바로 그 언급된 양에 매우 인접한 값이 상기 양, 예컨대, 측정치를 형성하는 데 있어서의 제작 허용 오차, 기구 및 인간 오차 등에 기인하여 고려될 수 있거나, 또는 자연적으로 고려되는 값에 포함된다는 것을 나타내는 것으로 한다. 양을 지칭하는 모든 백분율(%)은 달리 명시되지 않는 한, 중량%이다.
본 명세서에 인용된 임의의 비-특허 또는 특허 문서를 비롯한, 임의의 참고문헌이 선행 기술을 구성하는 것으로 인정되지 않는다. 특히, 달리 언급되지 않는 한, 본원에서의 임의의 문헌에 대한 언급은 이들 문헌 중 어느 것도 미국 또는 임의의 다른 국가에서 관련 기술분야의 통상적 일반 지식의 일부를 형성한다는 인정을 구성하지 않는다는 것을 이해할 것이다. 참고문헌에 대한 임의의 논의는 그들 저자가 주장하는 내용을 기술하고 있고, 본 출원인은 본원에 인용된 문서 중 임의의 것의 정확성과 적합성에 이의를 제기할 수 있는 권리를 보유하고 있다. 본원에 인용된 모든 참고문헌은 달리 분명하게 명시되지 않는 한, 완전히 참조로 포함된다. 인용된 참고문헌에 기재된 임의의 정의 및/또는 설명 사이에 임의의 불일치 사항이 있는 경우, 본 개시내용이 우선되어야 한다.
하기 실시예는 단지 예시적인 것이며, 본 발명의 범주 또는 첨부된 특허청구범위의 범주를 제한하는 것으로 의도되지 않는다.
실시예
본 발명자들은 항원성 카르고에 대한 면역 반응을 유의하게 증가시키기 위해 전장의 고 이동성 그룹 박스 1 (HMGB1)을 발현하는 피키아 파스토리스 백신 벡터를 개발하였다. 앞서, 본 발명자들은 말단절단된 CD154, 또는 CD40 리간드, 폴리펩티드 및/또는 전장의 HMGB1을 이중 약독화된 살모넬라 엔테리디티스 (SE) 내로 삽입하고, Raw 264 뮤린 대식세포인 세포에 의한 SE의 포식세포성 흡수를 비교하였다 (도 1). 형광 활성화된 세포 분류 (FACS) 분석법을 사용하여 Raw 264 내에서의 상대적인 형광 강도를 측정하였다. S. 아우레우스(S. aureus) pHrodo 입자 상에서의 형광 신호는 더욱 산성을 나타내는 pH에서 증가되기 때문에, 형광 강도를 측정하였다. 일단 박테리아가 포식세포 작용에 의해 대식세포 내로 흡수되고 나면, 대식세포 내에 파고리소좀이 생성된다. 대식세포는 파고리소좀 내부의 pH를 산성화시킴으로써 박테리아를 분해시킨다. 그러므로, 박테리아를 활발하게 분해시킨 대식세포는 더욱 큰 형광 강도를 가지게 될 것이다. 도 1의 데이터는, 뮤린 대식세포는 약독화된 SE 백신 벡터 단독인 것과 비교하였을 때, HMGB1을 함유하는 SE 백신 벡터를 우선적으로 포식하였고 (P=0.011), CD154는 뮤린 대식세포에 의한 포식세포성 흡수를 유의하게 변경시키지 않았다는 것 (P=0.057)을 입증한다.
본 발명자들은 백신 카르고에 대한 면역 반응을 증가시키면서, HMGB1 단백질을 염색체로 피키아 파스토리스 내로 삽입시키기 위해 플라스미드 통합 시스템을 사용하여 HMGB1의 세포 표면 발현을 위해 피키아 파스토리스를 조작하였다. 피키아 파스토리스는 사카로마이세스 세레비시아에보다 10 내지 100배 더 높은 단백질 발현을 일으킨다. HMGB1은 면역계에 위험 신호를 보내고, 이로써, RAGE 반응을 일으킨다. 상기 기술된 포식세포 작용 검정법은, HMGB1이 캐리어 시스템, 예를 들어, 살모넬라 엔테리디티스의 뮤린 대식세포 내로의 흡수를 증가시킬 수 있는 강력한 면역 자극성 분자라는 것을 보여준다. 효모의 세포 표면 상에서의 HMGB1 발현은 살모넬라 엔테리디티스 구축물에서 관찰되는 바와 같은, 포식세포 작용에 의한 효모 벡터 백신의 대식세포 내로의 흡수를 증강시켜야 한다. 본 발명자들은 피키아 파스토리스에서의 발현을 위한 pPICZ 발현 벡터를 포함하는, 이지셀렉트 피키아 익스프레션 키트(EasySelect Pichia Expression Kit)를 인비트로겐(Invitrogen)®으로부터 입수하였다. 피키아 파스토리스용 세포 표면 발현 키트는 현재 이용불가능하지만, 여러 연구원들은 GPI-고정 단백질을 사용하여 효모 시스템에서의 세포 표면 발현을 개시하였다 (Wasilenko et al., 2009). 사용된 GPI-고정 단백질은 사카로마이세스 세레비시아에로부터의 α-응집소의 C-말단부이다. 세린 스페이서 영역에 의해 사카로마이세스 세레비시아에 α-응집소의 C-말단부에 연결된 HMGB1을 pPICZ 세포내 발현 플라스미드 (도 2 및 서열식별번호: 1)로 스플라이싱하였다. pPICZ는 신속한 고수준의 단백질 발현을 위한 메탄올 유도성 플라스미드이다. 오직 형질전환된 효모 세포에서만 HMGB-1의 존재를 나타내는 면역형광을 보여주는 (도 3E 및 도 3F) 도 3에서 HMGB1의 세포 표면 발현이 확인되었다.
피키아 파스토리스의 세포 표면 상에서 HMGB1 단백질 발현을 확인한 후, 본 발명자들은 브로일러 닭 (n=15마리 닭/군)에서 시험하기 위해 3개의 HMGB1 양성 피키아 파스토리스 클론을 선택하였다. 멸균수 중 0.3% 글루타르알데히드를 사용하여 피키아 파스토리스-HMGB1 양성 클론 #3, 4, 및 6을 불활성화시키고, 만노실화된 키토산 애주번트 중에서 1:2로 혼합하였다. WO 2014/070709 (이는 그 전문이 본원에서 참조로 포함된다)를 참조한다. 본 발명자들은 0.25 mL의 백신/닭을 피하 주사하여 각 피키아 파스토리스-HMGB1 양성 구축물을 3개 용량 107-109로 투여하였다 (하기 표 1). 부화당일 및 14일째에 브로일러 닭에 백신접종하였다. 21일째 IgG 항체 역가 측정을 위해 혈청을 수집하였다. 피키아 파스토리스에 특이적인 IgG를 측정하는 직접적인 ELISA를 본 발명자들의 실험실시에서 최적화시켰다. ELISA 검정법 내에서의 플레이트 대 플레이트의 변동성을 설명하기 위해 샘플/음성 대조군 혈청 비를 기록하였다. 각 브로일러 닭으로부터 피키아 파스토리스에 특이적인 항체 역가를 측정하였다. 피키아 파스토리스-HMGB1 백신접종받은 닭의 IgG를 비변형된 피키아 파스토리스 (X33) 백신접종받은 닭의 IgG와 비교하였다. 본 결과는 도 4에 제시되어 있다.
<표 1>
브로일러 닭에서의 피키아 파스토리스 백신접종 용량 전략법
이어서, 본 발명자들은 유사한 IgG 항체 반응이 관찰되는지 여부를 측정하기 위해 피키아 파스토리스-HMGB1 구축물 # 3, 4, 또는 6을 3주령된 브로일러 닭에 백신접종하였다. 본 발명자들은 0.25 mL의 각 피키아 파스토리스 백신을 3주령된 브로일러 닭에 백신접종하였다 (표 1: n=10마리 닭/군). 멸균수 중 0.3% 글루타르알데히드를 사용하여 피키아 파스토리스-HMGB1 양성 구축물 #3, 4, 또는 6을 불활성화시키고, 만노실화된 키토산 애주번트 중에서 1:2로 혼합하였다. 21일째 및 35일째 브로일러 닭에 백신접종하였다. 21일째 IgG 항체 역가 측정을 위해 혈청을 수집하였다. 피키아 파스토리스에 특이적인 IgG를 측정하는 직접적인 ELISA를 본 발명자들의 실험실시에서 최적화시켰다. ELISA 검정법 내에서의 플레이트 대 플레이트의 변동성을 설명하기 위해 백신접종을 받지 않은 닭 (1군)에 대하여 정규화된 450 nm에서의 흡광도를 기록하였다. 각 브로일러 닭으로부터 피키아 파스토리스에 특이적인 항체 역가를 측정하였다. 피키아 파스토리스-HMGB1 백신접종받은 닭의 IgG를 비변형된 피키아 파스토리스 (X33) 백신접종받은 닭의 IgG와 비교하였다. 본 결과는 도 5에 도시되어 있다.
HMGB1은 비변형된 피키아 파스토리스를 주사맞은 브로일러와 비교하여, 불활성화된 HMGB1+피키아 파스토리스를 SQ 주사맞은 브로일러에서 피키아 파스토리스에 특이적인 IgG 항체 역가를 유의하게 상승시켰다 (도 4 및 도 5). HMGB1은 피키아 파스토리스 백신 벡터에 대한 면역 반응을 증가시켰고, 이는 동일한 피키아 파스토리스에 의해 발현된 임의의 항원성 카르고가, HMGB1 부재하에서 항원성 카르고가 피키아 파스토리스에서 발현된다면, 그 경우에서보다 더 높은 면역 반응을 유도할 것이라는 것을 제안하는 것이다.
물질 및 방법
피키아 파스토리스 X-33 (야생형)을 이지셀렉트™ 피키아 익스프레션 키트의 일부로서 인비트로겐 (미국 캘리포니아주 칼즈배드)으로부터 입수하였다. 피키아 파스토리스에서의 발현을 위해 최적화된 HMGB1 코딩 서열은 진스크립트 (Genscript: 미국 뉴저지주 피츠카타웨이)에 의해 합성되었고, pUC57 클로닝 벡터로 본 발명자들의 실험실로 공급되었다. 백신 구축 동안 모든 필요한 플라스미드 증식을 위해 TOP10 전기수용성 E. 콜라이 (인비트로겐)를 사용하였다. 플라스미드 DNA 형질전환 후, 100 ㎍/mL 암피실린(Ampicillin)으로 보충된 LB 배지, 또는 50 ㎍/mL 제오신(Zeocin)을 함유하는 저염 LB 배지를 사용하여 E. 콜라이를 37℃에서 증식시켰다. 적절할 경우, 50 ㎍ 제오신으로 보충된 YPD 배지를 사용하여 30℃에서 피키아 파스토리스를 통상적으로 증식시켰다. 후속되는, 재조합 백신 균주 스크리닝을 위해, 히스티딘을 함유하는 효모용 최소 배지 (MMH) 및 덱스트로스를 함유하는 효모용 최소 배지 (MDH)를 사용하였다. 글리세롤을 함유하는 최소 배지 (MGY) 및 메탄올을 함유하는 최소 배지 (MM)를 사용하여 배양물 중에서의 피키아 파스토리스로부터의 HMGB1 발현을 유도시켰다.
백신 벡터 구축
피키아 파스토리스-HMGB1발현 벡터를 제조하기 위해, pPICZ를 KpnI 및 PmeI로 소화시켜 클로닝을 위한 벡터 백본을 제조하였다. pUC57/HMGB1-알파 응집소를 KpnI 및 EcoRV로 소화시키고, 이어서, 1.6 kb HMGB1-알파 응집소 삽입체를 겔 정제하였다. 라이게이션 및 TOP10 E. 콜라이로의 형질전환 후, 콜로니 PCR을 수행하여 적절한 pPICZ/HMGB1-알파 응집소 라이게이션된 플라스미드를 보유하는 콜로니를 확인하였다. 상기 PCR을 위한 프라이머 - AOXSeqF (5' GACTGGTTCCAATTGACAAGC 3'; 서열식별번호: 128); AOXSeqR (5' GCAAATGGCATTCTGACATCC 3'; 서열식별번호: 129)은 이지셀렉트™ 키트로 제공받았다. KOD DNA 폴리머라제 (밀리포어(Millipore: 독일 다름슈타트))를 사용하여 앰플리콘을 제조하였다. 본 반응을 위한 사이클링 파라미터는 하기와 같다: 98℃에서 10분; 이어서, 98℃에서 15초; 55℃에서 5초로 진행된 25회 사이클. 아칸소 대학교 DNA 코어 연구 시설(University of Arkansas DNA core laboratory facility: 미국 아칸소주 페이엣빌)에서 서열분석하여 라이게이션된 플라스미드 pPICZ/HMGB1-알파 응집소를 추가로 확인하였다. 이어서, pPICZ/HMGB1-알파 응집소를 PmeI 소화를 통해 선형화시키고, 정제하여 전기천공을 위해 피키아 파스토리스 X-33으로 제조하였다.
전기천공을 위해, 5 mL의 피키아 파스토리스 X-33을 YPD 브로쓰 중에서 30℃에서 밤새도록 성장시켰다. 다음날, 500 ml의 신선한 YPD 브로쓰에 5 mL 배양물을 접종하고, OD가 1.5가 될 때까지 성장시켰다. 이어서, 세포를 빙냉 멸균수로 2회, 및 빙냉 멸균 소르비톨 (1 M)로 1회 세척하였다. 최종적으로는 세포를 1 mL 빙냉 소르비톨 중에 재현탁시켰다. 80 ㎕의 컴피턴트 피키아 파스토리스를 10 ㎍의 선형화된 pPICZ/HMGB1-알파 응집소와 혼합하고, 2.0 KV에서 1회 펄싱하여 효모 세포를 전기천공시켰다. 100 ㎍/mL 제오신을 함유하는 YPD 플레이트 상에서 제오신 내성 유전자를 함유하는 선형 백신 구축물이 성공적으로 염색체에 통합된 형질전환체를 선택하였다. 다시 콜로니 PCR을 사용하여 형질전환체를 분석하였다. 상기 언급된 프라이머 및 사이클링 파라미터를 사용하였다.
피키아 파스토리스 pPICZ / HMGB1 AOX1 유전자의 스크리닝.
HMGB1-알파 응집소의 발현을 구동시키는 데 필요한 AOX1 유전자의 존재 및 안정성을 확인하기 위하여 생성된 백신 균주를 시험하였다. GS115 Mut- (이지셀렉트™ 피키아 익스프레션 키트로 제공받은 비기능성 AOX1 유전자를 포함하는 음성 대조군 균주) 이외에도 9개의 제오신 내성 균주를 MDH 및 MMH 아가 상에 플레이팅시켜 시험하였다. AOX1 결핍 균주는 MDH에서보다 MMH 배지 상에서 훨씬 더 느린 성장을 보인다. 상기 두 배지 상에서의 성장 시간을 사용하여 AOX1 결핍 균주를 확인한다. 음성 대조군 GS115 Mut-과 함께 9개의 균주를 각각 MMH 및 MDH 배지 상에서 복제시켰고, 3일 동안 인큐베이션시켰다. 배양물을 매 24시간마다 체크하고, 성장 수준을 기록하였다.
HMGB1
-알파 응집소 단백질 유도.
배양물 중에서 HMGB1의 발현을 유도하기 위해, 50 mL 피키아 파스토리스 pPICZ/HMGB1-알파 응집소 배양물을 단일 콜로니로부터 MGY 브로쓰 중에서 30℃에서 밤새도록 성장시켰다. 밤새도록 배양된 상기 배양물 25 ml를 250 mL 또는 미리 가온된 MM 브로쓰로 옮겨 놓고, 적절한 통기를 위해 멸균된 치즈 클로스(cheese-cloth)로 커버하였다. 이어서, 상기 배양물을 96시간 동안 활발하게 진탕 (250 rpm)시키면서 성장시켰고; 유도를 유지시키기 위해 매 24시간마다 최종 농도가 0.5%가 되도록 100% 메탄올을 첨가하였다. HMGB1 -알파 응집소 유전자 생성물의 유도는 96시간 경과 후에 최대화되었다.
유도 후, 면역형광 검정법을 사용하여 각각의 9개의 피키아 파스토리스-HMGB1 양성 구축물을 피키아 파스토리스 세포 표면 상에서의 HMGB1 단백질 발현에 대하여 시험하였다. 피키아 파스토리스-HMGB1 구축물 #1, 3, 4, 5, 6, 7, 8, 9, 및 10, 및 X33 백본을 PBS 중의 1% 염소 혈청 중에서 1:1,000으로 알렉사 488과 접합된 염소 항-토끼 IgG의 F(ab)2 부위를 갖는 포스페이트 완충 염수 (PBS) 중에서 1:5로 희석된 토끼 폴리클로날 HMGB1 156-177을 사용하여 염색시켰다. 형질전환된 9개의 피키아 파스토리스-HMGB1 구축물 중 3개의 구축물 (#3, 4, 및 6) 상에서 HMGB1 단백질 발현이 최적으로 발현되었다 (도 3).
효모 벡터 백신 후보물질로의 백신접종 이후의 S. 엔테리티디스 (S. Enteritidis) 회수 평가.
이어서, 본 발명자들은 시험감염 이후, S. 엔테리티디스 (SE), 혈청군 D 살모넬라의 회수를 감소시키는 효모 벡터 PAL-HMGB1 (서열식별번호: 54 연결 서열식별번호: 2) 및 항체 가이드된 PAL 백신 후보물질의 효능을 시험하였다. 60마리의 SPF 레그혼종 닭을 양계장에서 부화시켰다. 부화당일, 닭을 2개 군, 대조군 또는 백신접종 군으로 나누었다. 백신접종받는 닭에 피하로 및 경구 위관영양에 의해 4.25 x 109 cfu/새 효모-PAL-HMGB1을 백신접종하였고, 이어서, 상기 2개 군 모두를 철조망 바닥이 있는 양계장에 놓아 두었고, 먹이 및 물을 임의 제공하였다. 백신접종 전, 샘플링을 위해 1일된 닭 10마리를 CO2 흡입에 의해 안락사시켜 닭이 살모넬라 음성임을 확인하였다. 7일째, 백신접종받은 닭을 피하로 및 경구 위관영양에 의해 5 x 109 cfu/새로 부스팅하였다. 14일째, 경구 위관영양에 의해 1.2 x 108 cfu/새 SE로 모든 닭을 시험감염시켰다. 21 및 28일째, 군당 10개의 배설강 스왑을 수득하여 시험감염 균주의 쉐딩(shedding)을 평가하였다. 32일째, 20마리의 닭 (대조군) 또는 10마리의 닭 (백신접종받은 닭)으로부터 간 및 비장 (LS), 및 맹장 (CT)을 수집하고, 직접 플레이팅 (CFU/g) 및 SE 출현 강화에 의해 배양하였다.
실험 방법 요약:
하기 표 2에 제시된 바와 같이, 시험감염 후 18일째까지 효모-PAL-HMGB-1 백신으로 백신접종받은 닭으로부터 유의하게 더 적은 양의 살모넬라가 회수되었다. 하기 표 3에 제시된 바와 같이, 배설강 스왑으로부터도 또한 유의하게 더 적은 양의 살모넬라가 회수되었고, 빠르면 시험감염 후 2주째 완전히 제거되었다.
<표 2>
<표 3>
효모 벡터 백신 후보물질로의 백신접종 이후의
콕시디아
(
Coccidia
) 감염 평가.
이어서, 본 발명자들은 시험감염 후, 에이메리아 맥시마, M6 균주에 의한 감염 이후의 이환율 및 사망률을 감소시키는 효모 (피키아 파스토리스) 벡터 MPP-TRAP-HMGB1 (서열식별번호: 61 연결 서열식별번호: 65 연결 서열식별번호: 2)의 효능을 시험하였다. 면역형광에 의해 MPP-TRAP-HMGB1의 발현이 표면에서 발현된 것을 확인하였다. 80마리의 닭을 부화장으로부터 부화당일 입수하였다. 부화당일, 닭을 무작위로 4개 군, 음성 대조군, 양성 대조군, 부화당일 및 14일째 경구 위관영양에 의해 MCA 백신 중 피키아-MPP-TRAP-HMGB-1로 백신접종받는 닭, 또는 4일째 및 14일째 음용수 중의 MCA 백신 중 피키아-MPP-TRAP-HMGB-1로 백신접종받는 닭으로 나누었다. 백신접종받는 닭에 경구 위관영양에 의해, 또는 음용수 중에서 경구 위관영양에 의해 5 x 107 cfu새 효모-MPP-TRAP-HMGB1을 백신접종하였다. 만노실화된 키토산 애주번트 (MCA) 스톡 용액 (1.5% 키토산 w:v)을 피키아 현탁액 (0.5% 최종 농도)을 이용하여 1:2로 희석시켰다. 경구 위관영양을 위해, MCA (0.5%) + 피키아 구축물 (1x107개의 세포)을 프라임 및 부스트, 둘 모두를 위해 경구 위관영양에 의해 0.25 mL로 전달하였다. 음용수 중에서 투여하는 경우, 음용수 중의 MCA의 최종 농도는 0.004%였고, 피키아의 최종 농도는 음용수 1 mL당 2.3 x 106개의 세포였고, 이는 프라임 및 부스트 투여, 둘 모두를 위해 사용되었다. 모든 닭에 개별적으로 태그부착하고, 음용수 백신접종 동안 및 먹이 및 물을 임의 제공하는 동안은 예외로 하여, 모든 닭 (군당 N=20)을 뒤섞이게 하였다.
20일째 모든 군에 시험감염하였고, 접종 후 6일째의 병변 점수를 측정하였다. 일부 낭포체는 초기 시험감염으로부터 탈출하고 (변함없이 통과하고), 이로써, 상기 뒤섞인 닭 중 시험감염되지 않은 대조군에서는 매우 적은 시험감염이 예상된다. 20일째, 양성 대조군 새, 및 백신접종받은 새 세트 둘 모두를 100,000개의 에이메리아 맥시마 (균주 M6) 난모세포로 시험감염시켰다. 26일째, 각 새를 존슨 및 레이드 병변 점수 인덱스(Johnson and Reid Lesion Score Index)를 사용하여 병변에 대하여 점수화하였다 (문헌 [Johnson, J. and W. M. Reid 1970. Experimental Parasitology 28: 30-36]). 상기 병변 점수 방법에서, 수치 점수는 하기를 의미한다: 0: 육안상 병변 없음; 1: 중장의 장막측 상에 작은 붉은색 점상 출혈이 출현할 수 있고, 비록 소량의 오렌지색 점액이 존재할 수는 있지만, 장의 풍선화 또는 비후화는 없음; 2: 장막 표면에 다수의 붉은색 점상 출혈과 함께 작은 반점들이 있을 수 있고; 장은 오렌지색 점액으로 채워질 수 있고; 장의 풍선화는 거의 없거나, 또는 전혀 없고; 벽의 비후화가 있음; 3: 장벽은 풍선화 또는 비후화되고, 점막 표면은 울퉁불퉁한 모양을 나타내고; 장 내용물은 극소 혈전 및 점액으로 채워짐; 4: 장벽은 그 길이 대부분에서 풍선화될 수 있고; 다수의 혈전 및 소화된 적혈구를 함유하여 특징적인 색상 및 아주 불쾌한 악취를 내고; 벽은 크게 비후화되고; 이 점수에서는 사망한 닭이 보고됨.
도 6은 시험감염 후 6일째의 병변 점수가 4인 동물의 비율(%)을 보여주는 그래프이고, 각 막대 내에 표시된 백분율(%)은 시험감염 후 6일째 사망률(%)을 보여주는 것이다. 특히, 6일째까지 양성 대조군 동물의 경우 15%인 것에 반해, 백신접종받은 새 중에서는 사망한 새는 없었다. 도 6에 나타낸 p 값 계산치로 제시된 바와 같이 (음용수 백신접종인 경우, p= 0.037, 경구 위관영양인 경우, p= 0.067), 병변 점수 또한 감소되었다. 면역자극성 폴리펩티드로서 HMGB1과 함께 MPP 및 TRAP 항원, 둘 모두를 발현하는 피키아가 음용수에 포함되었을 때, 병변 점수는 유의하게 감수되었다. 하기와 같이 통계 분석을 수행하였다. 점수 0과 1 사이의 중증도 차이는 점수 1과 2 사이의 중증도 차이 유사하고, 그 다음의 점수 차이도 그러하다고 가정하고, SAS에서 PROC MIXED ANOVA 모델을 사용하여 병변 데이터를 분석하였다. 상기 가정하에서, PROC MIXED ANOVA 분석을 위해 점수 평균을 분석할 수 있다. 문헌 [Johnson and Reid (1970)]에 의해 기술된 바와 같이, 병변 점수는 0 내지 4 범위이다. 랜덤 및 고정 효과 검정을 수행하였다. 평균차를 계산하여 처리군 간의 병변 점수 사이의 임의의 유의한 차이를 측정하였다. 데이터를 측정하여 푸아송(Poisson) 분포를 갖도록 하고, 터키-크레이머(Tukey Kramer) 검정을 사용하여 처리군 사이에 임의의 통계학상 유의한 차이가 있는지 여부를 측정하였다.
도 7에 제시된 바와 같이, 백신접종받은 동물에서는 모든 병변 점수 분포 또한 감소되었다. 백신접종받은 동물은 더 낮은 병변 점수를 보였다. 따라서, 음용수 중의 또는 경구 위관영양을 통한 백신접종이 에이메리아에 의한 시험감염 이후, 사망률을 감소시켰고, 이환율을 감소시켰다.
SEQUENCE LISTING
<110> BOARD OF TRUSTEES OF THE UNIVERSITY OF ARKANSAS
THE TEXAS A&M UNIVERSITY SYSTEM
FAULKNER, Olivia B.
BIELKE, Lisa
CALHOUN, Nicole
BERGHMAN, Luc
HARGIS, Billy
<120> YEAST VACCINE VECTOR INCLUDING IMMUNOSTIMULATORY AND ANTIGENIC
POLYPEPTIDES AND METHODS OF USING THE SAME
<130> 5658-00379
<150> US 62/331,044
<151> 2016-05-03
<160> 129
<170> PatentIn version 3.5
<210> 1
<211> 1578
<212> DNA
<213> Saccharomyces cerevisiae
<220>
<221> misc_feature
<222> (1)..(1578)
<223> HMGB1 + 3 portion of Saccharomyces cerevisiae alpha-agglutinin
<400> 1
cacgtgatgg gtaaaggtga tccaaaaaaa ccaagaggta aaatgtcttc ttatgctttt 60
tttgttcaaa cttgtcgtga agaacataaa aaaaaacatc cagatgcttc tgttaatttt 120
tctgaatttt ctaaaaaatg ttctgaaaga tggaaaacta tgtcttctaa agaaaaaggt 180
aaatttgaag atatggctaa agctgataaa ctacgttatg aaaaagaaat gaaaaattat 240
gttccaccaa aaggtgaaac taaaaaaaaa tttaaagatc caaatgctcc aaaaagacca 300
ccatctgctt tttttctatt ttgttctgaa tttagaccaa aaattaaagg tgaacatcca 360
ggtttatcta ttggtgatgt tgctaaaaaa ttaggtgaaa tgtggaataa tactgctgct 420
gatgataaac aaccatatga aaaaaaagct gctaaattaa aagaaaaata tgaaaaagat 480
attgctgctt atagagctaa aggtaaagtt gatgctggta aaaaagttgt tgctaaagct 540
gaaaaatcta aaaaaaaaaa agaagaagaa gaagattcct cctccggtcg gaacctcggt 600
acagctagcg ccaaaagctc ttttatctca accactacta ctgatttaac aagtataaac 660
actagtgcgt attccactgg atccatttcc acagtagaaa caggcaatcg aactacatca 720
gaagtgatca gccatgtggt gactaccagc acaaaactgt ctccaactgc tactaccagc 780
ctgacaattg cacaaaccag tatctattct actgactcaa atatcacagt aggaacagat 840
attcacacca catcagaagt gattagtgat gtggaaacca ttagcagaga aacagcttcg 900
accgttgtag ccgctccaac ctcaacaact ggatggacag gcgctatgaa tacttacatc 960
tcgcaattta catcctcttc tttcgcaaca atcaacagca caccaataat ctcttcatca 1020
gcagtatttg aaacctcaga tgcttcaatt gtcaatgtgc acactgaaaa tatcacgaat 1080
actgctgctg ttccatctga agagcccact tttgtaaatg ccacgagaaa ctccttaaat 1140
tccttctgca gcagcaaaca gccatccagt ccctcatctt atacgtcttc cccactcgta 1200
tcgtccctct ccgtaagcaa aacattacta agcaccagtt ttacgccttc tgtgccaaca 1260
tctaatacat atatcaaaac gaaaaatacg ggttactttg agcacacggc tttgacaaca 1320
tcttcagttg gccttaattc ttttagtgaa acagcagtct catctcaggg aacgaaaatt 1380
gacacctttt tagtgtcatc cttgatcgca tatccttctt ctgcatcagg aagccaattg 1440
tccggtatcc aacagaattt cacatcaact tctctcatga tttcaaccta tgaaggtaaa 1500
gcgtctatat ttttctcagc tgagctcggt tcgatcattt ttctgctttt gtcgtacctg 1560
ctattctaat aaggtacc 1578
<210> 2
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<210> 3
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Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu
180 185 190
Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Asp Glu
195 200 205
Glu Glu Asp Asp Asp Asp Glu
210 215
<210> 5
<211> 215
<212> PRT
<213> Mus musculus
<220>
<221> misc_feature
<222> (1)..(215)
<223> HMGB1
<400> 5
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Asp Asp Glu Glu
180 185 190
Asp Glu Glu Asp Glu Glu Glu Glu Glu Glu Glu Glu Asp Glu Asp Glu
195 200 205
Glu Glu Asp Asp Asp Asp Glu
210 215
<210> 6
<211> 215
<212> PRT
<213> Rattus norvegicus
<220>
<221> misc_feature
<222> (1)..(215)
<223> HMGB1
<400> 6
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Asp Asp Glu Glu
180 185 190
Asp Glu Glu Asp Glu Glu Glu Glu Glu Glu Glu Glu Asp Glu Asp Glu
195 200 205
Glu Glu Asp Asp Asp Asp Glu
210 215
<210> 7
<211> 180
<212> PRT
<213> Cricetulus griseus
<220>
<221> misc_feature
<222> (1)..(180)
<223> HMGB1
<400> 7
Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg Trp Lys Thr
1 5 10 15
Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala Lys Ala Asp
20 25 30
Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro Pro Lys Gly
35 40 45
Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro
50 55 60
Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys Ile Lys Gly
65 70 75 80
Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys Leu Gly Glu
85 90 95
Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr Glu Lys Lys
100 105 110
Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala Ala Tyr Arg
115 120 125
Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val Lys Ala Glu
130 135 140
Lys Ser Lys Lys Lys Lys Glu Glu Glu Asp Asp Glu Glu Asp Glu Glu
145 150 155 160
Asp Glu Glu Glu Glu Glu Glu Glu Glu Asp Glu Asp Glu Glu Glu Asp
165 170 175
Asp Asp Asp Glu
180
<210> 8
<211> 215
<212> PRT
<213> Macaca mulatta
<220>
<221> misc_feature
<222> (1)..(215)
<223> HMGB1
<400> 8
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu
180 185 190
Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Asp Glu
195 200 205
Glu Glu Asp Asp Asp Asp Glu
210 215
<210> 9
<211> 215
<212> PRT
<213> Macaca fascicularis
<220>
<221> misc_feature
<222> (1)..(215)
<223> HMGB1
<400> 9
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu
180 185 190
Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Asp Glu
195 200 205
Glu Glu Asp Asp Asp Asp Glu
210 215
<210> 10
<211> 227
<212> PRT
<213> Otolemur garnettii
<220>
<221> misc_feature
<222> (1)..(227)
<223> HMGB1
<400> 10
Met Asp Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Cys
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Gly Glu Glu Arg Glu Lys Lys His
20 25 30
Pro Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu
35 40 45
Arg Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met
50 55 60
Ala Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile
65 70 75 80
Pro Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro
85 90 95
Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro
100 105 110
Lys Ile Lys Gly Glu His His Leu Ser Thr Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Ser Asn Pro Ala Ala Gly Asp Lys Gln Pro Glu
130 135 140
Glu Lys Lys Ala Ala Lys Leu Glu Glu Lys Asp Lys Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Val Gly Ser Ser Arg Leu Lys Lys
165 170 175
Ala Arg Lys Arg Arg Lys Arg Arg Lys Met Arg Lys Met Lys Arg Lys
180 185 190
Lys Met Met Asn Lys Leu Val Leu Ala Gln Phe Leu Phe Leu Val Tyr
195 200 205
Lys Ala Phe Asn Pro Leu Val Tyr Asn Ser Leu Leu Leu Lys Lys Lys
210 215 220
Ile Glu Met
225
<210> 11
<211> 211
<212> PRT
<213> Xenopus laevis
<220>
<221> misc_feature
<222> (1)..(211)
<223> HMGB1
<400> 11
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Tyr Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ala Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Ser Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Val Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Phe Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Ser Thr Ile Gly Asp Ile Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Thr Asp Asp Lys Leu Pro Phe
130 135 140
Glu Arg Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Val Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Glu Pro Ala Lys Lys Ala Pro Ala
165 170 175
Lys Pro Glu Lys Ala Lys Lys Lys Glu Glu Asp Asp Glu Asp Asp Asp
180 185 190
Glu Glu Asp Glu Asp Glu Glu Asp Glu Glu Glu Glu Glu Glu Glu Asp
195 200 205
Asp Asp Glu
210
<210> 12
<211> 211
<212> PRT
<213> Xenopus (Silurana) tropicalis
<220>
<221> misc_feature
<222> (1)..(211)
<223> HMGB1
<400> 12
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Tyr Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ala Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Ser Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Val Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Phe Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Ser Thr Ile Gly Asp Ile Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Thr Asp Asp Lys Leu Pro Tyr
130 135 140
Glu Arg Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Val Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Glu Pro Ala Lys Lys Ala Pro Ala
165 170 175
Lys Phe Glu Lys Ala Lys Lys Lys Glu Asp Asp Asp Asp Asp Glu Asp
180 185 190
Asp Asp Asp Glu Glu Glu Glu Asp Glu Glu Glu Glu Asp Glu Glu Asp
195 200 205
Asp Asp Glu
210
<210> 13
<211> 215
<212> PRT
<213> Papio Anubis
<220>
<221> misc_feature
<222> (1)..(215)
<223> HMGB1
<400> 13
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu
180 185 190
Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Asp Glu
195 200 205
Glu Glu Asp Asp Asp Asp Glu
210 215
<210> 14
<211> 215
<212> PRT
<213> Callicebus moloch
<220>
<221> misc_feature
<222> (1)..(215)
<223> HMGB1
<400> 14
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu
180 185 190
Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Asp Glu
195 200 205
Glu Glu Asp Asp Asp Asp Glu
210 215
<210> 15
<211> 215
<212> PRT
<213> Callithrix jacchus
<220>
<221> misc_feature
<222> (1)..(215)
<223> HMGB1
<400> 15
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu
180 185 190
Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Asp Glu
195 200 205
Glu Glu Asp Asp Asp Asp Glu
210 215
<210> 16
<211> 215
<212> PRT
<213> Bos taurus
<220>
<221> misc_feature
<222> (1)..(215)
<223> HMGB1
<400> 16
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu
180 185 190
Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Glu Glu
195 200 205
Glu Glu Asp Asp Asp Asp Glu
210 215
<210> 17
<211> 214
<212> PRT
<213> Bos indicus
<220>
<221> misc_feature
<222> (1)..(214)
<223> HMGB1
<400> 17
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Lys
165 170 175
Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu Asp
180 185 190
Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Glu Glu Glu
195 200 205
Glu Asp Asp Asp Asp Glu
210
<210> 18
<211> 215
<212> PRT
<213> Equus caballus
<220>
<221> misc_feature
<222> (1)..(215)
<223> HMGB1
<400> 18
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu
180 185 190
Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Asp Glu
195 200 205
Glu Glu Asp Asp Asp Asp Glu
210 215
<210> 19
<211> 215
<212> PRT
<213> Canis lupus familiaris
<220>
<221> misc_feature
<222> (1)..(215)
<223> HMGB1
<400> 19
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu
180 185 190
Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Asp Glu
195 200 205
Glu Glu Asp Asp Asp Asp Glu
210 215
<210> 20
<211> 215
<212> PRT
<213> Sus scrofa
<220>
<221> misc_feature
<222> (1)..(215)
<223> HMGB1
<400> 20
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys His Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu
180 185 190
Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Glu Glu
195 200 205
Glu Glu Asp Asp Asp Asp Glu
210 215
<210> 21
<211> 215
<212> PRT
<213> Oryctolagus cuniculus
<220>
<221> misc_feature
<222> (1)..(215)
<223> HMGB1
<400> 21
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu
180 185 190
Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Asp Glu
195 200 205
Glu Glu Asp Asp Asp Asp Glu
210 215
<210> 22
<211> 206
<212> PRT
<213> Sciaenops ocellatus
<220>
<221> misc_feature
<222> (1)..(206)
<223> HMGB1
<400> 22
Met Val Lys Glu Gln Gly Lys Pro Arg Gly Lys Met Ser Ser Tyr Ala
1 5 10 15
Tyr Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro Asp
20 25 30
Ala Ser Val Asn Phe Ala Glu Phe Ser Lys Lys Cys Ser Gly Arg Trp
35 40 45
Lys Thr Met Ser Ser Lys Glu Lys Gly Lys Phe Glu Asp Leu Ala Arg
50 55 60
Gln Asp Lys Ala Arg Tyr Glu Arg Glu Met Met Ser Tyr Val Pro Ala
65 70 75 80
Arg Gly Gly Lys Lys Lys Lys Tyr Lys Asp Pro Asn Ala Pro Lys Arg
85 90 95
Pro Pro Ser Ala Phe Phe Ile Phe Cys Ser Glu Phe Arg Pro Lys Val
100 105 110
Lys Gly Glu Ala Pro Gly Leu Thr Ile Gly Glu Val Ala Lys Arg Leu
115 120 125
Gly Glu Met Trp Asn Gly Thr Ala Ser Glu Asp Lys Gln Pro Phe Glu
130 135 140
Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Glu Val Ala Ala
145 150 155 160
Tyr Arg Gln Lys Thr Lys Ala Gly Ala Gly Pro Ala Ala Lys Ala Pro
165 170 175
Ala Lys Val Glu Lys Lys Val Glu Asp Asp Asp Asp Asp Asp Asp Asp
180 185 190
Asp Glu Glu Glu Glu Glu Asp Asp Tyr Asp Asp Asp Asp Glu
195 200 205
<210> 23
<211> 182
<212> PRT
<213> Ictalurus punctatus
<220>
<221> misc_feature
<222> (1)..(182)
<223> HMGB1
<400> 23
Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro Asp Thr Ser Val
1 5 10 15
Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg Trp Lys Thr Met
20 25 30
Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala Arg Leu Asp Lys
35 40 45
Ala Arg Tyr Glu Arg Glu Met Lys Asn Tyr Val Pro Pro Arg Gly Glu
50 55 60
Lys Lys Lys Arg Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser
65 70 75 80
Ala Phe Phe Ile Phe Cys Ala Glu Tyr Arg Pro Lys Val Lys Glu Glu
85 90 95
Thr Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys Leu Gly Glu Met
100 105 110
Trp Asn Lys Thr Ser Ala Glu Glu Lys Gln Pro Tyr Glu Lys Lys Ala
115 120 125
Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala Ala Tyr Arg Lys
130 135 140
Gly Lys Val Val Gly Gly Ala Ala Lys Ala Pro Thr Lys Pro Asp Lys
145 150 155 160
Ala Asp Asp Asp Glu Asp Asp Asp Asp Asp Glu Asp Asp Asp Asp Asp
165 170 175
Asp Asp Glu Asp Asp Glu
180
<210> 24
<211> 209
<212> PRT
<213> Lutjanus sanguineus
<220>
<221> misc_feature
<222> (1)..(209)
<223> HMGB1
<400> 24
Met Gly Arg Glu Pro Arg Glu Pro Gly Lys Pro Arg Gly Lys Met Ser
1 5 10 15
Ser Tyr Ala Tyr Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys
20 25 30
His Pro Asp Ala Ser Val Asn Phe Ala Glu Phe Ser Lys Lys Cys Ser
35 40 45
Glu Arg Trp Lys Thr Met Ser Pro Lys Glu Lys Ser Lys Phe Glu Asp
50 55 60
Leu Ala Arg Gln Asp Lys Ala Arg Tyr Glu Arg Glu Met Leu Thr Tyr
65 70 75 80
Val Pro Ala Arg Gly Gly Lys Lys Lys Lys Phe Lys Asp Pro Asn Ala
85 90 95
Pro Lys Arg Pro Pro Ser Ala Phe Phe Ile Phe Cys Ser Glu Phe Arg
100 105 110
Pro Lys Val Lys Gly Glu Ser Pro Gly Leu Ser Ile Gly Glu Val Ala
115 120 125
Lys Arg Leu Gly Glu Met Trp Asn Gly Thr Ser Ser Glu Asp Lys Gln
130 135 140
Pro Phe Glu Lys Lys Ala Ala Lys Leu Lys Glu Arg Tyr Glu Lys Glu
145 150 155 160
Val Ala Ala Tyr Arg Gln Lys Thr Lys Gly Gly Ser Ala Pro Ala Gly
165 170 175
Lys Ala Pro Ala Lys Ala Glu Lys Lys Val Glu Glu Asp Asp Asp Asp
180 185 190
Glu Glu Asp Asp Asp Asp Glu Glu Glu Glu Asp Tyr Asp Asp Asp Asp
195 200 205
Glu
<210> 25
<211> 204
<212> PRT
<213> Carassius auratus
<220>
<221> misc_feature
<222> (1)..(204)
<223> HMGB1
<400> 25
Met Gly Lys Asp Pro Thr Lys Pro Arg Gly Lys Met Ser Ser Tyr Ala
1 5 10 15
Tyr Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro Glu
20 25 30
Ala Thr Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg Trp
35 40 45
Lys Thr Met Ser Gly Lys Glu Lys Gly Lys Phe Glu Asp Met Ala Lys
50 55 60
Gln Asp Lys Val Arg Tyr Glu Arg Glu Met Lys Asn Tyr Ile Pro Pro
65 70 75 80
Lys Gly Glu Lys Lys Lys Arg Phe Lys Asp Pro Asn Ala Pro Lys Arg
85 90 95
Pro Pro Ser Ala Phe Phe Ile Phe Cys Ser Glu Phe Arg Ser Lys Val
100 105 110
Lys Glu Glu Thr Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Arg Leu
115 120 125
Gly Glu Met Trp Asn Lys Thr Ser Ala Glu Asp Lys Gln Pro Phe Glu
130 135 140
Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala Ala
145 150 155 160
Tyr Arg Ser Lys Gly Lys Val Val Gly Gly Ala Ala Lys Ala Pro Ser
165 170 175
Lys Pro Val Lys Val Asn Asp Asp Asp Asp Asp Asp Asp Glu Asp Glu
180 185 190
Asp Glu Asp Asp Asp Asp Glu Glu Glu Asp Asp Glu
195 200
<210> 26
<211> 198
<212> PRT
<213> Salmo salar
<220>
<221> misc_feature
<222> (1)..(198)
<223> HMGB1
<400> 26
Met Gly Lys Asp Pro Arg Lys Pro Arg Gly Lys Met Ser Ser Tyr Ala
1 5 10 15
Tyr Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro Glu
20 25 30
Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg Trp
35 40 45
Arg Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Leu Ala Lys
50 55 60
Leu Asp Lys Val Arg Tyr Glu Arg Glu Met Arg Ser Tyr Ile Pro Pro
65 70 75 80
Lys Gly Glu Lys Lys Lys Arg Phe Lys Asp Pro Asn Ala Pro Lys Arg
85 90 95
Pro Ser Ser Ala Phe Phe Ile Phe Cys Ala Asp Phe Arg Pro Gln Val
100 105 110
Lys Gly Glu Thr Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys Leu
115 120 125
Gly Glu Lys Trp Asn Asn Leu Thr Ala Glu Asp Lys Val Pro Tyr Glu
130 135 140
Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Thr Ala
145 150 155 160
Tyr Arg Asn Lys Gly Lys Val Pro Val Ser Val Pro Ala Lys Ala Ala
165 170 175
Ala Pro Thr Lys Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp
180 185 190
Glu Asp Asp Asp Asp Asp
195
<210> 27
<211> 206
<212> PRT
<213> Anoplopoma fimbria
<220>
<221> misc_feature
<222> (1)..(206)
<223> HMGB1
<400> 27
Met Val Lys Glu Leu Gly Lys Pro Lys Gly Lys Met Ser Ser Tyr Ala
1 5 10 15
Tyr Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro Glu
20 25 30
Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg Trp
35 40 45
Lys Thr Met Ser Leu Lys Glu Lys Gly Lys Phe Glu Asp Leu Ala Arg
50 55 60
Gln Asp Lys Ala Arg Tyr Glu Arg Glu Met Met Ser Tyr Ile Pro Pro
65 70 75 80
Arg Gly Ile Lys Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg
85 90 95
Pro Pro Ser Ala Phe Phe Ile Phe Cys Ala Glu Tyr Arg Pro Lys Val
100 105 110
Lys Gly Glu Thr Pro Gly Ala Thr Ile Gly Asp Val Ala Lys Arg Leu
115 120 125
Gly Glu Met Trp Asn Gly Thr Ala Ser Glu Asp Arg Gln Pro Phe Glu
130 135 140
Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Glu Val Ala Ala
145 150 155 160
Tyr Arg Ala Lys Thr Lys Pro Gly Ala Cys Ala Ala Ala Ala Pro Ser
165 170 175
Lys Ala Pro Ala Lys Val Glu Lys Lys Val Glu Asp Asp Asp Asp Asp
180 185 190
Asp Asp Asp Glu Glu Glu Asp Asp Phe Asp Asp Asp Asp Asp
195 200 205
<210> 28
<211> 194
<212> PRT
<213> Oncorhynchus mykiss
<220>
<221> misc_feature
<222> (1)..(194)
<223> HMGB1
<400> 28
Met Gly Lys Asp Pro Arg Lys Pro Arg Gly Lys Met Ser Ser Tyr Ala
1 5 10 15
Tyr Phe Val Gln Thr Cys Arg Ala Glu His Lys Lys Lys His Pro Glu
20 25 30
Ala Ser Val Asn Phe Ala Glu Phe Ser Lys Lys Cys Ser Glu Arg Trp
35 40 45
Lys Pro Met Ser Pro Lys Glu Lys Gly Lys Phe Glu Asp Met Ala Lys
50 55 60
Gln Asp Lys Val Arg Tyr Glu Gly Glu Met Lys Asn Tyr Ile Pro Pro
65 70 75 80
Asn Gly Gln Lys Lys Lys Arg Phe Lys Asp Pro Asn Ala Pro Lys Arg
85 90 95
Pro Pro Ser Ala Phe Phe Ile Phe Cys Ala Asp Phe Arg Ala Lys Ile
100 105 110
Lys Ser Glu His Pro Gly Leu Ser Ile Gly Asp Thr Ala Lys Lys Leu
115 120 125
Gly Val Met Trp Asn Ser Ser Ala Ala Glu Glu Lys Lys Pro Tyr Glu
130 135 140
Lys Lys Ala Ala Thr Leu Lys Glu Lys Tyr Asp Lys Asp Ile Ala Ser
145 150 155 160
Tyr Arg Thr Asn Gly Arg Val Asp Thr Ala Ser Ser Ala Ala Ala Asp
165 170 175
Asp Glu Glu Glu Asp Asp Glu Glu Asp Asp Asp Glu Asp Glu Asp Asp
180 185 190
Asp Glu
<210> 29
<211> 208
<212> PRT
<213> Lethenteron camtschaticum
<220>
<221> misc_feature
<222> (1)..(208)
<223> HMGB1
<400> 29
Met Gly Lys Gly Asp Pro Lys Lys Pro Lys Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Tyr Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys Asn Pro
20 25 30
Glu Ala Ser Val Asn Phe Ala Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Glu Lys Glu Lys Thr Arg Phe Glu Asp Met Ala
50 55 60
Lys Val Asp Lys Val Arg Tyr Asp Arg Glu Met Lys Thr Tyr Val Pro
65 70 75 80
Pro Lys Gly Glu Arg Gly Ser Arg Lys Lys Lys Asp Pro Asn Ala Pro
85 90 95
Lys Arg Pro Pro Ser Ala Phe Phe Ile Tyr Cys Ala Glu Tyr Arg Ser
100 105 110
Lys Val Arg Ala Glu Asn Pro Gly Leu Thr Ile Gly Ser Ile Ala Lys
115 120 125
Lys Leu Gly Glu Met Trp Asn Asn Ala Pro Ala Asp Glu Lys Ser Ile
130 135 140
Tyr Glu Arg Lys Thr Ala Lys Leu Lys Glu Lys Tyr Asp Lys Asp Met
145 150 155 160
Ala Ser Tyr Arg Ser Lys Gly Lys Val Glu Thr Ser Lys Val Ala Ser
165 170 175
Lys Pro Ala Ser Lys Gln Arg Asp Asp Asp Asp Asp Glu Asp Asp Asp
180 185 190
Glu Asp Glu Asp Glu Asp Glu Asp Glu Asp Asp Asp Asp Asp Asp Glu
195 200 205
<210> 30
<211> 172
<212> PRT
<213> Ctenopharyngodon idella
<220>
<221> misc_feature
<222> (1)..(172)
<223> HMGB1
<400> 30
Met Gly Lys Asp Pro Arg Lys Pro Lys Gly Lys Met Ser Ser Tyr Ala
1 5 10 15
Tyr Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro Glu
20 25 30
Ala Thr Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg Trp
35 40 45
Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala Lys
50 55 60
Gln Asp Lys Val Arg Phe Glu Arg Glu Met Lys Asn Tyr Ile Pro Pro
65 70 75 80
Lys Gly Glu Lys Lys Arg Arg Phe Lys Asp Pro Asn Ala Pro Lys Arg
85 90 95
Pro Pro Ser Ala Phe Phe Ile Phe Cys Gly Asp Tyr Arg Pro Lys Ile
100 105 110
Arg Gly Glu Asn Pro Gly Leu Ser Ile Gly Asp Ile Ala Lys Lys Leu
115 120 125
Gly Glu Met Trp Asn Ser Ser Ser Ala Glu Val Lys Gln Pro Tyr Glu
130 135 140
Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Asp Lys Asp Ile Ala Leu
145 150 155 160
Tyr Arg Thr Lys Gly Ile Ala Gly Leu Ser Lys Lys
165 170
<210> 31
<211> 106
<212> PRT
<213> Ophiophagus hannah
<220>
<221> misc_feature
<222> (1)..(106)
<223> HMGB1
<400> 31
Met Ala Lys Ala Asp Lys Val Arg Tyr Asp Arg Glu Met Lys Asp Tyr
1 5 10 15
Gly Pro Ala Lys Gly Gly Lys Lys Lys Lys Asp Pro Asn Ala Pro Lys
20 25 30
Arg Pro Pro Ser Gly Phe Phe Leu Phe Cys Ser Glu Phe Cys Ser Glu
35 40 45
Phe Arg Pro Lys Ile Lys Ser Thr Asn Pro Gly Ile Ser Ile Gly Asp
50 55 60
Val Ala Lys Lys Leu Gly Glu Met Trp Asn Asn Leu Ser Asp Ser Glu
65 70 75 80
Lys Gly Pro Tyr Asn Asn Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu
85 90 95
Lys Val Arg Leu Gly Cys Trp Cys Trp Cys
100 105
<210> 32
<211> 51
<212> PRT
<213> Artemia franciscana
<220>
<221> misc_feature
<222> (1)..(51)
<223> HMGB1
<400> 32
Met Pro Arg Ser Lys Asp Glu Ser Lys Pro Arg Gly Lys Leu Thr Ala
1 5 10 15
Tyr Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Arg Lys His
20 25 30
Pro Asp Glu Asn Val Val Phe Ala Glu Phe Ser Lys Lys Cys Ala Glu
35 40 45
Arg Trp Lys
50
<210> 33
<211> 85
<212> PRT
<213> Gallus gallus
<220>
<221> misc_feature
<222> (1)..(85)
<223> HMGB1 box a1
<400> 33
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ser Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Leu Arg Tyr Glu Lys Glu Met Lys Asn Tyr Val Pro
65 70 75 80
Pro Lys Gly Glu Thr
85
<210> 34
<211> 54
<212> PRT
<213> Gallus gallus
<220>
<221> misc_feature
<222> (1)..(54)
<223> HMGB1 box a2
<400> 34
Pro Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu
1 5 10 15
Arg Trp Lys Thr Met Ser Ser Lys Glu Lys Gly Lys Phe Glu Asp Met
20 25 30
Ala Lys Ala Asp Lys Leu Arg Tyr Glu Lys Glu Met Lys Asn Tyr Val
35 40 45
Pro Pro Lys Gly Glu Thr
50
<210> 35
<211> 73
<212> PRT
<213> Gallus gallus
<220>
<221> misc_feature
<222> (1)..(73)
<223> HMGB1 box b1
<400> 35
Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe
1 5 10 15
Cys Ser Glu Phe Arg Pro Lys Ile Lys Gly Glu His Pro Gly Leu Ser
20 25 30
Ile Gly Asp Val Ala Lys Lys Leu Gly Glu Met Trp Asn Asn Thr Ala
35 40 45
Ala Asp Asp Lys Gln Pro Tyr Glu Lys Lys Ala Ala Lys Leu Lys Glu
50 55 60
Lys Tyr Glu Lys Asp Ile Ala Ala Tyr
65 70
<210> 36
<211> 69
<212> PRT
<213> Gallus gallus
<220>
<221> misc_feature
<222> (1)..(69)
<223> HMGB1 box b2
<400> 36
Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu
1 5 10 15
Phe Arg Pro Lys Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp
20 25 30
Val Ala Lys Lys Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp
35 40 45
Lys Gln Pro Tyr Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu
50 55 60
Lys Asp Ile Ala Ala
65
<210> 37
<211> 21
<212> PRT
<213> Gallus gallus
<220>
<221> misc_feature
<222> (1)..(21)
<223> HMGB1 RAGE binding domain
<400> 37
Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala Phe Phe Leu Phe
1 5 10 15
Cys Ser Glu Phe Arg
20
<210> 38
<211> 33
<212> PRT
<213> Gallus gallus
<220>
<221> misc_feature
<222> (1)..(33)
<223> HMGB1 proinflammatory cytokine activity
<400> 38
Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala Ala Tyr Arg Ala Lys Gly
1 5 10 15
Lys Val Asp Ala Gly Lys Lys Val Val Ala Lys Ala Glu Lys Ser Lys
20 25 30
Lys
<210> 39
<211> 182
<212> PRT
<213> Gallus gallus
<220>
<221> misc_feature
<222> (1)..(182)
<223> Clostridium perfringens antigen
<400> 39
Ser Lys Glu Tyr Ala Arg Gly Phe Ala Lys Thr Gly Lys Ser Ile Tyr
1 5 10 15
Tyr Ser His Ala Ser Met Ser His Ser Trp Asp Asp Trp Asp Tyr Ala
20 25 30
Ala Lys Val Thr Leu Ala Asn Ser Gln Lys Gly Thr Ala Gly Tyr Ile
35 40 45
Tyr Arg Phe Leu His Asp Val Ser Glu Gly Asn Asp Pro Ser Val Gly
50 55 60
Lys Asn Val Lys Glu Leu Val Ala Tyr Ile Ser Thr Ser Gly Glu Lys
65 70 75 80
Asp Ala Gly Thr Asp Asp Tyr Met Tyr Phe Gly Ile Lys Thr Lys Asp
85 90 95
Gly Lys Thr Gln Glu Trp Glu Met Asp Asn Pro Gly Asn Asp Phe Met
100 105 110
Thr Gly Ser Lys Asp Thr Tyr Thr Phe Lys Leu Lys Asp Glu Asn Leu
115 120 125
Lys Ile Asp Asp Ile Gln Asn Met Trp Ile Arg Lys Arg Lys Tyr Thr
130 135 140
Ala Phe Pro Asp Ala Tyr Lys Pro Glu Asn Ile Lys Val Ile Ala Asn
145 150 155 160
Gly Lys Val Val Val Asp Lys Asp Ile Asn Glu Trp Ile Ser Gly Asn
165 170 175
Ser Thr Tyr Asn Ile Lys
180
<210> 40
<211> 6
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(6)
<223> protective epitope of CPa
<400> 40
Ala Arg Gly Phe Ala Lys
1 5
<210> 41
<211> 8
<212> PRT
<213> Avian Influenza virus
<220>
<221> misc_feature
<222> (1)..(8)
<223> Avian Influenza virus m2e
<400> 41
Glu Val Glu Thr Pro Ile Arg Asn
1 5
<210> 42
<211> 8
<212> PRT
<213> Avian Influenza virus
<220>
<221> misc_feature
<222> (1)..(8)
<223> Avian Influenza virus m2e
<400> 42
Glu Val Glu Thr Pro Thr Arg Asn
1 5
<210> 43
<211> 12
<212> PRT
<213> Avian Influenza virus
<220>
<221> misc_feature
<222> (1)..(12)
<223> Avian Influenza virus (HA5 UA)
<400> 43
Leu Leu Ser Arg Ile Asn His Phe Glu Lys Ile Gln
1 5 10
<210> 44
<211> 19
<212> PRT
<213> Avian Influenza virus
<220>
<221> misc_feature
<222> (1)..(19)
<223> Avian Influenza virus (HA5 LB)
<400> 44
Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asp Phe Asn Asp Tyr
1 5 10 15
Glu Glu Leu
<210> 45
<211> 16
<212> PRT
<213> Avian Influenza virus
<220>
<221> misc_feature
<222> (1)..(16)
<223> Avian Influenza virus (NP 54-69)
<400> 45
Gly Arg Leu Ile Gln Asn Ser Ile Thr Ile Glu Arg Met Val Leu Ser
1 5 10 15
<210> 46
<211> 14
<212> PRT
<213> Avian Influenza virus
<220>
<221> misc_feature
<222> (1)..(14)
<223> Avian Influenza virus (NP 147-160)
<400> 46
Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp
1 5 10
<210> 47
<211> 19
<212> PRT
<213> E. coli
<220>
<221> misc_feature
<222> (1)..(19)
<223> PAL bis from E. coli
<400> 47
Glu Gly His Ala Asp Glu Arg Gly Thr Pro Glu Tyr Asn Ile Ser Leu
1 5 10 15
Gly Glu Arg
<210> 48
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: peptide
<400> 48
Gly His Ala Asp Glu Arg
1 5
<210> 49
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: peptide
<400> 49
Asp Glu Arg Gly Thr Pro
1 5
<210> 50
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: peptide
<400> 50
Glu Tyr Asn Ile Ser Leu
1 5
<210> 51
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: peptide
<400> 51
Ile Ser Leu Gly Glu Arg
1 5
<210> 52
<211> 19
<212> PRT
<213> Vibrio spp.
<220>
<221> misc_feature
<222> (1)..(19)
<223> PALbis from vibrio spp.
<400> 52
Glu Gly His Ala Asp Glu Arg Gly Thr Pro Glu Tyr Asn Ile Ala Leu
1 5 10 15
Gly Glu Arg
<210> 53
<211> 17
<212> PRT
<213> Campylobacter spp.
<220>
<221> misc_feature
<222> (1)..(17)
<223> corresponding peptide from Campylobacter spp.
<400> 53
Glu Gly Asn Cys Asp Glu Trp Gly Thr Asp Glu Tyr Asn Gln Ala Leu
1 5 10 15
Gly
<210> 54
<211> 19
<212> PRT
<213> E. coli
<220>
<221> misc_feature
<222> (1)..(19)
<223> PAL from E. coli
<400> 54
Thr Val Glu Gly His Ala Asp Glu Arg Gly Thr Pro Glu Tyr Asn Ile
1 5 10 15
Ser Leu Gly
<210> 55
<211> 21
<212> PRT
<213> Campylobacter jejuni
<220>
<221> misc_feature
<222> (1)..(21)
<223> Campylobacter jejuni Cj0113
<400> 55
Gly Val Ser Ile Thr Val Glu Gly Asn Cys Asp Glu Trp Gly Thr Asp
1 5 10 15
Glu Tyr Asn Gln Ala
20
<210> 56
<211> 19
<212> PRT
<213> Vibrio spp.
<220>
<221> misc_feature
<222> (1)..(19)
<223> Vibrio spp. alternative PAL epitope
<400> 56
Thr Val Glu Gly His Ala Asp Glu Arg Gly Thr Pro Glu Tyr Asn Ile
1 5 10 15
Ala Leu Gly
<210> 57
<211> 57
<212> DNA
<213> E. coli
<220>
<221> misc_feature
<222> (1)..(57)
<223> E. coli nucleotide sequence for PAL epitope
<400> 57
gaaggtcacg cggacgaacg tggtaccccg gaatacaaca tctctctggg tgaacgt 57
<210> 58
<211> 9
<212> PRT
<213> E. coli
<220>
<221> misc_feature
<222> (1)..(9)
<223> Epitope of PAL from E. coli
<400> 58
Glu Tyr Asn Ile Ser Leu Gly Glu Arg
1 5
<210> 59
<211> 9
<212> PRT
<213> Vibrio spp.
<220>
<221> misc_feature
<222> (1)..(9)
<223> Epitope of PAL from Vibrio spp.
<400> 59
Glu Tyr Asn Ile Ala Leu Gly Glu Arg
1 5
<210> 60
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: composite minimal epitope
<220>
<221> misc_feature
<222> (2)..(6)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (13)..(13)
<223> Xaa can be any naturally occurring amino acid
<400> 60
Pro Xaa Xaa Xaa Xaa Xaa Gly Tyr Gly Ala Cys Glu Xaa Asn Leu Gly
1 5 10 15
<210> 61
<211> 43
<212> PRT
<213> Eimeria maxima
<220>
<221> misc_feature
<222> (1)..(43)
<223> MPP; Eimeria maxima
<400> 61
Pro Ser His Asp Ala Pro Glu Ser Glu Arg Thr Pro Arg Val Ile Ser
1 5 10 15
Phe Gly Tyr Gly Ala Cys Glu His Asn Leu Gly Val Ser Leu Phe Arg
20 25 30
Arg Glu Glu Thr Lys Lys Asp Pro Arg Gly Arg
35 40
<210> 62
<211> 28
<212> PRT
<213> Neospora canium
<220>
<221> misc_feature
<222> (1)..(28)
<223> Neospora canium
<400> 62
Pro Arg Ile Val Ser Phe Gly Tyr Gly Ala Cys Glu His Asn Leu Gly
1 5 10 15
Met Ser Leu Tyr Asp Arg Gln Gly Leu Gln Arg Gln
20 25
<210> 63
<211> 21
<212> PRT
<213> Eimeria tenella
<220>
<221> misc_feature
<222> (1)..(21)
<223> Eimeria tenella
<400> 63
Glu Ser Gln Arg Ala Pro Met Val Ile Arg Tyr Gly Tyr Gly Ala Cys
1 5 10 15
Glu Tyr Asn Leu Gly
20
<210> 64
<211> 10
<212> PRT
<213> Eimeria maxima
<220>
<221> misc_feature
<222> (1)..(10)
<223> Eimeria maxima TRAP-1
<400> 64
Gly Gly Gly Phe Pro Thr Ala Ala Val Ala
1 5 10
<210> 65
<211> 40
<212> PRT
<213> Eimeria maxima
<220>
<221> misc_feature
<222> (1)..(40)
<223> Eimeria maxima TRAP-02
<400> 65
Ala Ala Pro Glu Thr Pro Ala Val Gln Pro Lys Pro Glu Glu Gly His
1 5 10 15
Glu Arg Pro Glu Pro Glu Glu Glu Glu Glu Lys Lys Glu Glu Gly Gly
20 25 30
Gly Phe Pro Thr Ala Ala Val Ala
35 40
<210> 66
<211> 40
<212> PRT
<213> Eimeria maxima
<220>
<221> misc_feature
<222> (1)..(40)
<223> Eimeria maxima TRAP-03
<400> 66
Gly Gly Gly Phe Pro Thr Ala Ala Val Ala Gly Gly Val Gly Gly Val
1 5 10 15
Leu Leu Ile Ala Ala Val Gly Gly Gly Val Ala Ala Phe Thr Ser Gly
20 25 30
Gly Gly Gly Ala Gly Ala Gln Glu
35 40
<210> 67
<211> 21
<212> PRT
<213> Campylobacter jejuni
<220>
<221> misc_feature
<222> (1)..(21)
<223> Campylobacter jejuni Cj0982
<400> 67
Lys Asp Ile Val Leu Asp Ala Glu Ile Gly Gly Val Ala Lys Gly Lys
1 5 10 15
Asp Gly Lys Glu Lys
20
<210> 68
<211> 35
<212> PRT
<213> Campylobacter jejuni
<220>
<221> misc_feature
<222> (1)..(35)
<223> Campylobacter jejuni Cj0420
<400> 68
Lys Val Ala Leu Gly Val Ala Val Pro Lys Asp Ser Asn Ile Thr Ser
1 5 10 15
Val Glu Asp Leu Lys Asp Lys Thr Leu Leu Leu Asn Lys Gly Thr Thr
20 25 30
Ala Asp Ala
35
<210> 69
<211> 16
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(16)
<223> Clostridium perfringens Alpha toxin
<400> 69
Asn Ala Trp Ser Lys Glu Tyr Ala Arg Gly Phe Ala Lys Thr Gly Lys
1 5 10 15
<210> 70
<211> 9
<212> PRT
<213> Avian influenza
<220>
<221> misc_feature
<222> (1)..(9)
<223> Avian influenza M2e peptide
<400> 70
Cys Glu Val Glu Thr Pro Thr Arg Asn
1 5
<210> 71
<211> 9
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(9)
<223> 1-Alpha-31
<400> 71
Gly Lys Ile Asp Gly Thr Gly Thr His
1 5
<210> 72
<211> 15
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(15)
<223> 2-Alpha-51
<400> 72
Glu Asn Asp Met Ser Lys Asn Glu Pro Glu Ser Val Arg Lys Asn
1 5 10 15
<210> 73
<211> 20
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(20)
<223> 3-Alpha-71
<400> 73
Glu Asn Met His Glu Leu Gln Leu Gly Ser Thr Tyr Pro Asp Tyr Asp
1 5 10 15
Lys Asn Ala Tyr
20
<210> 74
<211> 20
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(20)
<223> 4-Alpha-81
<400> 74
Thr Tyr Pro Asp Tyr Asp Lys Asn Ala Tyr Asp Leu Tyr Gln Asp His
1 5 10 15
Phe Trp Asp Pro
20
<210> 75
<211> 20
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(20)
<223> 5-Alpha-91
<400> 75
Asp Leu Tyr Gln Asp His Phe Trp Asp Pro Asp Thr Asp Asn Asn Phe
1 5 10 15
Ser Lys Asp Asn
20
<210> 76
<211> 10
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(10)
<223> 6-Alpha-117
<400> 76
Ile Pro Asp Thr Gly Glu Ser Gln Ile Arg
1 5 10
<210> 77
<211> 10
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(10)
<223> 7-Alpha-136
<400> 77
Glu Trp Gln Arg Gly Asn Tyr Lys Gln Ala
1 5 10
<210> 78
<211> 23
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(23)
<223> 8-Alpha-158
<400> 78
Asp Ile Asp Thr Pro Tyr His Pro Ala Asn Val Thr Ala Val Asp Ser
1 5 10 15
Ala Gly His Val Lys Phe Glu
20
<210> 79
<211> 20
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(20)
<223> 9-Alpha-170
<400> 79
Val Asp Ser Ala Gly His Val Lys Phe Glu Thr Phe Ala Glu Glu Arg
1 5 10 15
Lys Glu Gln Tyr
20
<210> 80
<211> 20
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(20)
<223> 10-Alpha-181
<400> 80
Thr Phe Ala Glu Glu Arg Lys Glu Gln Tyr Lys Ile Asn Thr Ala Gly
1 5 10 15
Cys Lys Thr Asn
20
<210> 81
<211> 21
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(21)
<223> 11-Alpha-191
<400> 81
Lys Ile Asn Thr Val Gly Cys Lys Thr Asn Glu Asp Phe Tyr Ala Asp
1 5 10 15
Ile Leu Lys Asn Lys
20
<210> 82
<211> 20
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(20)
<223> 12-Alpha-200
<400> 82
Glu Asp Phe Tyr Ala Asp Ile Leu Lys Asn Lys Asp Phe Asn Ala Trp
1 5 10 15
Ser Lys Glu Tyr
20
<210> 83
<211> 20
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(20)
<223> 13-Alpha-210
<400> 83
Lys Asp Phe Asn Ala Trp Ser Lys Glu Tyr Ala Arg Gly Phe Ala Lys
1 5 10 15
Thr Gly Lys Ser
20
<210> 84
<211> 17
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(17)
<223> 14-Alpha-220
<400> 84
Ala Arg Gly Phe Ala Lys Thr Gly Lys Ser Ile Tyr Tyr Ser His Ala
1 5 10 15
Ser
<210> 85
<211> 17
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(17)
<223> 15-Alpha-233
<400> 85
Ser His Ala Ser Met Ser His Ser Trp Asp Asp Trp Asp Tyr Ala Ala
1 5 10 15
Lys
<210> 86
<211> 20
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(20)
<223> 16-Alpha-240
<400> 86
Ser Trp Asp Asp Trp Asp Tyr Ala Ala Lys Val Thr Leu Ala Asn Ser
1 5 10 15
Gln Lys Gly Thr
20
<210> 87
<211> 16
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(16)
<223> 17-Alpha-270
<400> 87
Asp Val Ser Glu Gly Asn Asp Pro Ser Val Gly Asn Asn Val Lys Glu
1 5 10 15
<210> 88
<211> 12
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(12)
<223> 18-Alpha-291
<400> 88
Ser Thr Ser Gly Glu Lys Asp Ala Gly Thr Asp Asp
1 5 10
<210> 89
<211> 13
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(13)
<223> 19-Alpha-309
<400> 89
Lys Thr Lys Asp Gly Lys Thr Gln Glu Trp Glu Met Asp
1 5 10
<210> 90
<211> 21
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(21)
<223> 20-Alpha-320
<400> 90
Asp Asn Pro Gly Asn Asp Phe Met Ala Gly Ser Lys Asp Thr Tyr Thr
1 5 10 15
Phe Lys Leu Lys Asp
20
<210> 91
<211> 20
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(20)
<223> 21-Alpha-330
<400> 91
Ser Lys Asp Thr Tyr Thr Phe Lys Leu Lys Asp Glu Asn Leu Lys Ile
1 5 10 15
Asp Asp Ile Gln
20
<210> 92
<211> 16
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(16)
<223> 22-Alpha-354
<400> 92
Arg Lys Arg Lys Tyr Thr Ala Phe Pro Asp Ala Tyr Lys Pro Glu Asn
1 5 10 15
<210> 93
<211> 19
<212> PRT
<213> Clostridium perfringens
<220>
<221> misc_feature
<222> (1)..(19)
<223> 23-Alpha-379
<400> 93
Val Val Asp Lys Asp Ile Asn Glu Trp Ile Ser Gly Asn Ser Thr Tyr
1 5 10 15
Asn Ile Lys
<210> 94
<211> 215
<212> PRT
<213> Amazona aestiva
<220>
<221> misc_feature
<222> (1)..(215)
<223> High mobility group protein B1
<400> 94
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ser Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Leu Arg Tyr Glu Lys Glu Met Lys Asn Tyr Val Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Phe Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Val Asp Ala Gly Lys Lys Val Val Ala
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Asp
180 185 190
Glu Asp Glu Glu Asp Glu Asp Asp Glu Glu Glu Glu Glu Glu Glu Glu
195 200 205
Glu Asp Asp Asp Asp Asp Glu
210 215
<210> 95
<211> 213
<212> PRT
<213> Chlamydotis macqueenii
<220>
<221> misc_feature
<222> (1)..(213)
<223> High mobility group protein B1, partial
<400> 95
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ser Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Leu Arg Tyr Glu Lys Glu Met Lys Asn Tyr Val Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Phe Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Val Asp Ala Gly Lys Lys Val Val Ala
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Asp
180 185 190
Glu Asp Glu Glu Asp Glu Glu Asp Glu Glu Glu Glu Glu Glu Glu Asp
195 200 205
Glu Asp Asp Asp Asp
210
<210> 96
<211> 209
<212> PRT
<213> Tyto alba
<220>
<221> misc_feature
<222> (1)..(209)
<223> High mobility group protein B1, partial
<400> 96
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ser Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Leu Arg Tyr Glu Lys Glu Met Lys Asn Tyr Val Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Phe Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Val Asp Ala Gly Lys Lys Val Val Ala
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Asp
180 185 190
Glu Asp Glu Glu Asp Glu Asp Asp Glu Glu Glu Glu Glu Glu Glu Asp
195 200 205
Glu
<210> 97
<211> 209
<212> PRT
<213> Podiceps cristatus
<220>
<221> misc_feature
<222> (1)..(209)
<223> High mobility group protein B1, partial
<400> 97
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ser Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Leu Arg Tyr Glu Lys Glu Met Lys Asn Tyr Val Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Phe Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Val Asp Ala Gly Lys Lys Val Val Ala
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Asp
180 185 190
Glu Asp Glu Glu Asp Glu Asp Glu Glu Glu Glu Glu Glu Glu Asp Glu
195 200 205
Asp
<210> 98
<211> 206
<212> PRT
<213> Chaetura pelagic
<220>
<221> misc_feature
<222> (1)..(206)
<223> High mobility group protein B1, partial
<400> 98
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ser Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Leu Arg Tyr Glu Lys Glu Met Lys Asn Tyr Val Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Phe Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Val Asp Ala Gly Lys Lys Val Val Ala
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Asp
180 185 190
Glu Asp Glu Glu Asp Glu Asp Asp Glu Glu Glu Glu Glu Glu
195 200 205
<210> 99
<211> 206
<212> PRT
<213> Tauraco erythrolphus
<220>
<221> misc_feature
<222> (1)..(206)
<223> High mobility group protein B1, partial
<400> 99
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ser Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Leu Arg Tyr Glu Lys Glu Met Lys Asn Tyr Val Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Phe Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Val Asp Ala Gly Lys Lys Val Val Ala
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Asp
180 185 190
Glu Glu Glu Glu Asp Glu Asp Asp Glu Glu Glu Glu Glu Glu
195 200 205
<210> 100
<211> 203
<212> PRT
<213> Phaethon lepturus
<220>
<221> misc_feature
<222> (1)..(203)
<223> High mobility group protein B1, partial
<400> 100
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ser Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Leu Arg Tyr Glu Lys Glu Met Lys Asn Tyr Val Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Phe Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Val Asp Ala Gly Lys Lys Val Val Ala
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Asp
180 185 190
Glu Asp Glu Glu Asp Glu Asp Asp Glu Glu Glu
195 200
<210> 101
<211> 201
<212> PRT
<213> Pterocles gutturalis
<220>
<221> misc_feature
<222> (1)..(201)
<223> High mobility group protein B1, partial
<400> 101
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ser Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Leu Arg Tyr Glu Lys Glu Met Lys Asn Tyr Val Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Phe Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Val Asp Ala Thr Lys Lys Val Val Ala
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Asp
180 185 190
Glu Asp Glu Glu Asp Glu Asp Asp Glu
195 200
<210> 102
<211> 200
<212> PRT
<213> Gavia stellata
<220>
<221> misc_feature
<222> (1)..(200)
<223> High mobility group protein B1, partial
<400> 102
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ser Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Leu Arg Tyr Glu Lys Glu Met Lys Asn Tyr Val Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Phe Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Val Asp Ala Gly Lys Lys Val Val Ala
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Asp
180 185 190
Glu Asp Glu Glu Asp Glu Asp Glu
195 200
<210> 103
<211> 176
<212> PRT
<213> Nannospalax ehrenbergi
<220>
<221> misc_feature
<222> (1)..(176)
<223> High mobility group protein
<400> 103
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Glu Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
<210> 104
<211> 176
<212> PRT
<213> Gekko japonicas
<220>
<221> misc_feature
<222> (1)..(176)
<223> High mobility group protein B1
<400> 104
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Glu Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
<210> 105
<211> 175
<212> PRT
<213> Tupaia chinensis
<220>
<221> misc_feature
<222> (1)..(175)
<223> High mobility group protein B1
<400> 105
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala His Tyr Glu Arg Glu Met Lys Thr Phe Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ser Lys Lys Gly Val
165 170 175
<210> 106
<211> 272
<212> PRT
<213> Gallus gallus
<220>
<221> misc_feature
<222> (1)..(272)
<223> CD154 chicken (Gallus gallus)
<400> 106
Met Asn Glu Ala Tyr Ser Pro Ala Ala Pro Arg Pro Met Gly Ser Thr
1 5 10 15
Ser Pro Ser Thr Met Lys Met Phe Met Cys Phe Leu Ser Val Phe Met
20 25 30
Val Val Gln Thr Ile Gly Thr Val Leu Phe Cys Leu Tyr Leu His Met
35 40 45
Lys Met Asp Lys Met Glu Glu Val Leu Ser Leu Asn Glu Asp Tyr Ile
50 55 60
Phe Leu Arg Lys Val Gln Lys Cys Gln Thr Gly Glu Asp Gln Lys Ser
65 70 75 80
Thr Leu Leu Asp Cys Glu Lys Val Leu Lys Gly Phe Gln Asp Leu Gln
85 90 95
Cys Lys Asp Arg Thr Ala Ser Glu Glu Leu Pro Lys Phe Glu Met His
100 105 110
Arg Gly His Glu His Pro His Leu Lys Ser Arg Asn Glu Thr Ser Val
115 120 125
Ala Glu Glu Lys Arg Gln Pro Ile Ala Thr His Leu Ala Gly Val Lys
130 135 140
Ser Asn Thr Thr Val Arg Val Leu Lys Trp Met Thr Thr Ser Tyr Ala
145 150 155 160
Pro Thr Ser Ser Leu Ile Ser Tyr His Glu Gly Lys Leu Lys Val Glu
165 170 175
Lys Ala Gly Leu Tyr Tyr Ile Tyr Ser Gln Val Ser Phe Cys Thr Lys
180 185 190
Ala Ala Ala Ser Ala Pro Phe Thr Leu Tyr Ile Tyr Leu Tyr Leu Pro
195 200 205
Met Glu Glu Asp Arg Leu Leu Met Lys Gly Leu Asp Thr His Ser Thr
210 215 220
Ser Thr Ala Leu Cys Glu Leu Gln Ser Ile Arg Glu Gly Gly Val Phe
225 230 235 240
Glu Leu Arg Gln Gly Asp Met Val Phe Val Asn Val Thr Asp Ser Thr
245 250 255
Ala Val Asn Val Asn Pro Gly Asn Thr Tyr Phe Gly Met Phe Lys Leu
260 265 270
<210> 107
<211> 261
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<222> (1)..(261)
<223> CD154 human Homo sapiens
<400> 107
Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly
1 5 10 15
Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu
20 25 30
Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg
35 40 45
Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val
50 55 60
Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser
65 70 75 80
Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys
85 90 95
Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu
100 105 110
Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser
115 120 125
Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly
130 135 140
Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln
145 150 155 160
Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr
165 170 175
Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser
180 185 190
Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala
195 200 205
Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His
210 215 220
Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn
225 230 235 240
Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe
245 250 255
Gly Leu Leu Lys Leu
260
<210> 108
<211> 11
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<222> (1)..(11)
<223> CD154 partial human Homo sapiens
<400> 108
Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Cys
1 5 10
<210> 109
<211> 11
<212> PRT
<213> Gallus gallus
<220>
<221> misc_feature
<222> (1)..(11)
<223> Chicken CD154 peptide Gallus gallus
<400> 109
Trp Met Thr Thr Ser Tyr Ala Pro Thr Ser Ser
1 5 10
<210> 110
<211> 10
<212> PRT
<213> Anas sp.
<220>
<221> misc_feature
<222> (1)..(10)
<223> Duck CD154 peptide (Anas sp.)
<400> 110
Trp Asn Lys Thr Ser Tyr Ala Pro Met Asn
1 5 10
<210> 111
<211> 10
<212> PRT
<213> Mus sp.
<220>
<221> misc_feature
<222> (1)..(10)
<223> Mouse CD154 peptide (Mus p.)
<400> 111
Trp Ala Lys Lys Gly Tyr Tyr Thr Met Lys
1 5 10
<210> 112
<211> 10
<212> PRT
<213> Bos taurus
<220>
<221> misc_feature
<222> (1)..(10)
<223> Cow CD154 peptide (Bos taurus)
<400> 112
Trp Ala Pro Lys Gly Tyr Tyr Thr Leu Ser
1 5 10
<210> 113
<211> 139
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: Heavy chain, Leader
sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
<400> 113
Met Ala Val Leu Ala Leu Leu Leu Cys Leu Val Ala Phe Pro Ser Cys
1 5 10 15
Thr Leu Ser Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala
20 25 30
Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu
35 40 45
Thr Thr Tyr Asp Ile Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
50 55 60
Glu Trp Leu Gly Ile Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser
65 70 75 80
Ala Phe Met Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln
85 90 95
Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr
100 105 110
Tyr Cys Val Arg Asp Arg Gly Tyr Tyr Val Tyr Tyr Ser Met Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
130 135
<210> 114
<211> 127
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: Light chain, Leader
sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
<400> 114
Met Met Ser Ser Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe Gln
1 5 10 15
Gly Thr Arg Cys Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser
20 25 30
Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp
35 40 45
Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val
50 55 60
Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser
85 90 95
Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn
100 105 110
Met Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
115 120 125
<210> 115
<211> 264
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: amino acid
<400> 115
Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val
1 5 10 15
Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp
20 25 30
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile Tyr Ser Ala
35 40 45
Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser
50 55 60
Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu
65 70 75 80
Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Leu Thr Phe Gly
85 90 95
Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Ser Ser Arg Ser Ser Leu
100 105 110
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Thr
115 120 125
Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
130 135 140
Tyr Ile Tyr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
145 150 155 160
Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe
165 170 175
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
180 185 190
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
195 200 205
Ala Arg Arg Gly Thr Gly Thr Val Val Phe Asp Tyr Trp Gly His Gly
210 215 220
Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Thr
225 230 235 240
Ser Gly Gln Ala Gly Gln His His His His His His Gly Ala Tyr Pro
245 250 255
Tyr Asp Val Pro Asp Tyr Ala Ser
260
<210> 116
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: Heavy chain CDR1
<400> 116
Gly Phe Ser Leu Thr Thr Tyr Asp Ile Asn
1 5 10
<210> 117
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: Heavy chain CDR2
<400> 117
Ile Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met Ser
1 5 10 15
<210> 118
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: Heavy chain CDR3
<400> 118
Asp Arg Gly Tyr Tyr Val Tyr Tyr Ser Met Asp Tyr
1 5 10
<210> 119
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: Light chain CDR1
<400> 119
Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn
1 5 10
<210> 120
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: Light chain CDR2
<400> 120
Tyr Thr Ser Arg Leu His Ser
1 5
<210> 121
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: Light chain CDR3
<400> 121
Gln Gln Gly Asn Met Phe Pro Trp Thr
1 5
<210> 122
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: VH, CDR1
<400> 122
Asn Tyr Tyr Ile Tyr
1 5
<210> 123
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: VH, CDR2
<400> 123
Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe Lys
1 5 10 15
Gly
<210> 124
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: VH, CDR3
<400> 124
Arg Gly Thr Gly Thr Val Val Phe Asp Tyr Trp
1 5 10
<210> 125
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: VL, CDR1
<400> 125
Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala
1 5 10
<210> 126
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: VL, CDR2
<400> 126
Ser Ala Ser Tyr Arg Tyr Ser
1 5
<210> 127
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic: VL, CDR3
<400> 127
Gln Gln Tyr Ser Ser Tyr Pro Leu Thr
1 5
<210> 128
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic: AOXSeqF
<400> 128
gactggttcc aattgacaag c 21
<210> 129
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic: AOXSeqR
<400> 129
gcaaatggca ttctgacatc c 21
Claims (28)
- HMGB1 폴리펩티드 또는 CD40 리간드를 코딩하는 면역자극성 폴리뉴클레오티드를 포함하는 효모를 포함하는 효모 백신 조성물이며, 여기서 효모는 효모의 표면 상에 HMGB1 폴리펩티드 또는 CD40 리간드를 발현하는 것인 효모 백신 조성물.
- 제1항에 있어서, 항원성 폴리펩티드를 코딩하는 항원성 폴리뉴클레오티드를 추가로 포함하는 백신 조성물.
- 제2항에 있어서, 효모가 효모의 표면 상에 항원성 폴리펩티드를 발현하는 것인 백신 조성물.
- 제2항 또는 제3항에 있어서, 항원성 폴리펩티드가 인플루엔자(Influenza) 폴리펩티드, 캄필로박터(Campylobacter) 폴리펩티드, 클로스트리디움(Clostridium) 폴리펩티드, 살모넬라(Salmonella) 폴리펩티드, 에이메리아(Eimeria) 폴리펩티드, 및 종양 연관 폴리펩티드로 이루어진 군으로부터 선택되는 것인 백신 조성물.
- 제4항에 있어서, 항원성 폴리펩티드가 서열식별번호: 39-93 중 임의의 것으로부터 선택되는 것인 백신 조성물.
- 제2항 내지 제5항 중 어느 한 항에 있어서, 효모가 1개 초과의 항원성 폴리뉴클레오티드를 포함하는 것인 백신 조성물.
- 제6항에 있어서, 1개 초과의 항원성 폴리뉴클레오티드가 1개 초과의 종으로부터 유래된 것인 백신 조성물.
- 제1항 내지 제7항 중 어느 한 항에 있어서, 효모가 피키아(Pichia)인 백신 조성물.
- 제8항에 있어서, 피키아가 피키아 파스토리스(Pichia pastoris)인 백신 조성물.
- 제1항 내지 제9항 중 어느 한 항에 있어서, HMGB1 폴리펩티드가 전장의 HMGB1 폴리펩티드인 백신 조성물.
- 제10항에 있어서, HMGB1 폴리펩티드가 서열식별번호: 2-30 및 94-105에 제공된 HMGB1 서열, 및 상기 서열과 95%의 동일성을 갖는 서열로 이루어진 군으로부터 선택되는 것인 백신 조성물.
- 제1항 내지 제11항 중 어느 한 항에 있어서, CD40 리간드가 CD154 폴리펩티드 및 CD40 효능작용 항체 또는 그의 일부로부터 선택되는 것인 백신 조성물.
- 제1항 내지 제12항 중 어느 한 항에 있어서, CD40 리간드가 서열식별번호: 106-127 중 적어도 하나를 포함하는 것인 백신 조성물.
- 제2항 내지 제13항 중 어느 한 항에 있어서, 항원성 폴리펩티드 및 HMGB1 폴리펩티드 또는 CD40 리간드가 융합 단백질의 일부인 백신 조성물.
- 제1항 내지 제14항 중 어느 한 항에 있어서, HMGB1 폴리펩티드 또는 CD40 리간드가 GPI-고정된 것인 백신 조성물.
- 제15항에 있어서, 면역자극성 폴리뉴클레오티드가 사카로마이세스 세레비시아에(Saccharomyces cerevisiae)로부터의 α-응집소를 코딩하는 폴리뉴클레오티드 내로 삽입되는 것인 백신 조성물.
- 제1항 내지 제16항 중 어느 한 항에 있어서, 면역자극성 폴리뉴클레오티드가 적어도 2개의 링커 아미노산을 코딩하는 링커 올리고뉴클레오티드를 추가로 포함하는 것인 백신 조성물.
- 제1항 내지 제17항 중 어느 한 항의 백신 조성물 및 제약상 허용되는 담체를 포함하는 제약 조성물.
- 제18항에 있어서, 제약상 허용되는 담체가 경구 또는 비강 투여용으로 허용되는 것인 제약 조성물.
- 제18항 또는 제19항에 있어서, 효모가 복제불가능한 것인 제약 조성물.
- 제18항 내지 제20항 중 어느 한 항에 있어서, 효모가 불활성화되거나 또는 사멸되는 것인 제약 조성물.
- 대상체에게 제1항 내지 제17항 중 어느 한 항의 백신 조성물, 또는 제18항 내지 제21항 중 어느 한 항의 제약 조성물을, 백신 조성물에 대한 대상체의 면역 반응을 증강시키는 데 효과적인 양으로 투여하는 단계를 포함하는, 대상체에서 면역 반응을 증강시키는 방법.
- 제22항에 있어서, 백신 조성물이 경구로, 또는 비내로 투여되는 것인 방법.
- 제22항 또는 제23항에 있어서, 대상체가 인간, 소, 고양이, 개, 돼지, 어류, 메기, 도미, 금붕어, 조류, 가금류, 닭 및 칠면조로 이루어진 군으로부터 선택되는 것인 방법.
- 제22항 내지 제24항 중 어느 한 항에 있어서, 백신 조성물에 대한, 또는 백신 조성물 중의 폴리펩티드에 대한 항체 반응이 백신 조성물 투여 이후에 증강되는 것인 방법.
- 제25항에 있어서, 면역 반응이 IgA 항체 반응인 방법.
- 제22항 내지 제26항 중 어느 한 항에 있어서, 백신 조성물 중의 효모가 대상체에서 복제불가능한 것인 방법.
- 제22항 내지 제27항 중 어느 한 항에 있어서, 백신 조성물 중의 효모가 대상체에게로의 투여 이전에 불활성화되거나 또는 사멸되는 것인 방법.
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KR1020237015693A KR20230070521A (ko) | 2016-05-03 | 2017-05-03 | 면역자극 및 항원 폴리펩티드를 포함하는 효모 백신 벡터, 및 그를 이용하는 방법 |
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TW202224701A (zh) | 2022-07-01 |
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JP2022040389A (ja) | 2022-03-10 |
AR108688A1 (es) | 2018-09-19 |
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US20170319671A1 (en) | 2017-11-09 |
CA3023144A1 (en) | 2017-11-09 |
TWI758288B (zh) | 2022-03-21 |
WO2017192671A1 (en) | 2017-11-09 |
BR112018072592A2 (pt) | 2019-04-16 |
EP3452069A4 (en) | 2020-02-12 |
AU2017260323A1 (en) | 2018-12-20 |
JP7467027B2 (ja) | 2024-04-15 |
MX2018013331A (es) | 2019-06-10 |
EP3452069A1 (en) | 2019-03-13 |
AU2023255055A1 (en) | 2023-11-16 |
SG11201809686SA (en) | 2018-11-29 |
AU2017260323B2 (en) | 2023-11-16 |
US10682398B2 (en) | 2020-06-16 |
JP2024023648A (ja) | 2024-02-21 |
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