Name: conformational evaluation of hiv-1 trimeric envelope glycoproteins using a cell-based elisa assay
Uploaded: 2014-09-14T15:00:00.000+00:00
Duration: 7 min 10 s
Description: Watch this Scientific Journal Video about Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay at JoVE.com

우리는 그의 관대 한 A32, 17B, 48D의 선물 및 C11 모노클로 날 항체 박사 제임스 로빈슨 감사합니다. PGT (121)는 NIH AIDS 시약 프로그램, AIDS, NIAID, NIH (고양이 # 12343) 부문을 통해 얻었다. 이 작품은 과학자 AF와 CRCHUM 연속체 보조금에 의해뿐만 아니라 CIHR 촉매 부여 # 126630로 # 24639을 부여 영 FRQS 설립하여, CIHR 운영 # 257792로, 혁신 프로그램 리더 # 29866에 대한 캐나다 재단에 의해 지원되었다 AF 및 MR합니다. AF는 FRSQ Chercheur Boursier 주니어 1 교제 번호에 24639를받는 사람이다. MV는 CIHR 박사 연구 상 번호 291485에 의해 지원되었다.

1 일 한 - 세포 배양 <ol><li> 플레이트 2 × 10 4 인간 골육종 (HOS) 발광 읽기에 적합한 불투명, 96 웰 세포 배양 접시에 잘 당 세포. 10 % 소 태아 혈청 (FBS) 및 100 U / ㎖ 페니실린 - 스트렙토 마이신이 보충 된 둘 베코 변형 이글 중간 (DMEM)를 사용합니다. 37 ° C, 5 % CO 2에서 다음 날까지 품어. </li></ol> 2 2 일 - 폴리에틸렌 이민 (PEI) 형질 <ol><li> 다음 단계에 따라 형질 믹스를 준비합니다. 동일한 봉투 형질하여야 웰의 수에 따른 시약 및 DNA의 양을 조정한다. </li><li> 튜브는 25 mM의 HEPES와 보충 5 μL의 DMEM에 봉투 인코딩 플라스미드 NG 10 NG 문신 인코딩 (예 : PTAT-III 27)를 플라스미드와 150를 추가합니다. </li><li> 같은 pSVIII으로 문신에 의존 봉투를 코딩하는 플라스미드를 사용하는 경우 문신 인코딩 플라스미드에만 필요합니다. </li><li> 튜브 B : 1에서 450 ng의 PEI를 (추가5 ㎕의 DMEM에 μg / μL 용액). </li><li> 철저하게 10 초 동안 볼 텍싱에 의해 튜브 A. 믹스에 관 B의 내용을 추가하고 형질 믹스에서 객실 온도 (22 ° C에서)에서 10 분을 품어. </li><li> 96 - 웰 플레이트 잘 당 형질 믹스 10 μl를 추가합니다. 37 ° C, 5 % CO 2에서 48 시간 동안 배양한다. </li></ol> 3 일 4 - ELISA <ol><li> 봉투 / 항체 복합체의 가능한 엔도 사이토 시스를 최소화하기 위해 실온에서 모든 실험을 수행합니다. </li><li> 동시에 사용되는 접시 당 세척 완충액 250 ㎖를 준비한다. 세척 버퍼 1X 트리스 - 완충 생리 식염수 (TBS)의 pH 7.5 (50 mM 트리스 - 염소, pH가 7.5, 150 밀리미터의 NaCl) 1 mM의 MgCl2를 1.8 mM의 염화칼슘이 보충. </li><li> 1 %의 탈지 분유 및 세척 버퍼에 5 mM 트리스 산도 8.0을 추가하여 접시 당 버퍼를 차단 125 ML을 준비합니다. </li><li> 96 웰 플레이트에서 세포 배양 매체와 형질 믹스 (뜨는)를 제거합니다. </li><li> 100 μ 추가; 잘 당 버퍼를 차단하고 실온에서 20 분을 품어의 리터. </li><li> 상층 액을 제거하고 버퍼를 차단에 적절한 농도로 희석 아니라 당 항체 (혈청)의 50 μl를 추가합니다. 통상적으로, 1 μg / ml의 농도를 사용한다. 실온에서 1 시간을 품어. </li><li> 100 μL가 버퍼를 차단 한 후 100 ㎕의 세척 버퍼로 세척 공정의 3 배를 반복 씻을 배. </li><li> , 상층 액을 제거 버퍼를 차단 100 μl를 추가하고, 실온에서 5 분을 품어. </li><li> 상층 액을 제거하고 버퍼를 차단에서 1 / 3000로 희석 차 항체의 50 μl를 추가합니다. 제조 업체의 차이에 따라 최적의 항체 희석 다릅니다. 실온에서 40 분을 품어. </li><li> 100 μL가 버퍼를 차단 한 후 100 ㎕의 세척 버퍼로 세척 공정의 3 배를 반복 씻을 배. </li></ol> (4) 데이터 수집 <ol><li> 판으로부터 상층 액을 제거하고 잘 당 30 ㎕의 1 배 강화 된 화학 발광 (ECL) 기판을 추가합니다. </li><li> 화학 획득1 초 동안 iluminescence 신호 / 웰에 대한 제조사의 지시에 따라 적절한 플레이트 리더. 시간을 읽는 것은 하드웨어의 차이에 따라 다를 수 있습니다. </li></ol>

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저자는 관심 없음 충돌 선언하지 않습니다.

이 분석은 HIV-1 삼량 봉투는 세포 표면에 발현하여 특정 모노클로 날 항체의 상호 작용을 검출하기 위해 최적화된다. 프로토콜이 확립 한 후에는 전체적으로 낮은 재료 비용 및 항체의 적은 양의 높은 처리량으로 매체에서 사용될 수있다. 이 분석은 형질 기반이기 때문에, 용이하게 봉투의 형태에 미치는 영향을 연구하기 위해 같은 CD4와 같은 세포 단백질의 동시 발현을 위해 적응 될 수있다. 그러나,이 프로토콜의 형질베이스는 또한 가장 중요한 함정 한 것을 의미한다. 첫째, 항원이 기술로 연구 할 독립적 인 발현 벡터에 사용할 수 있도록해야합니다. 이와 같이, 다양한 임상 소스 또는 프로 바이러스 구조 유전자 봉투로부터 포유 동물 발현 벡터로 서브 클로닝 될 필요가있을 것이다. 전장 프로 바이러스 구조가 또한 이러한 방법에 사용할 수 있지만 (Veillette 외 참조. 18)이 또한 법을 암시필자 바이러스 입자 생산 따라서 적절한 biocontainment 시설에서 작업을 필요로. 또한,이 기술의 성공은 밀접하게 형질 전환 효율이 연결되어 있습니다. 얻어진 낮은 신호는 종종 형질 항원의 가난한 발현에 기인. 문제의 일반적인 소스는 플라스미드 DNA 품질, 형질 전환 시약입니다. 및 세포 생존 능력. 필요한 경우, 형질 전환 조건의 최적화는 또한 유동 세포 계측법 또는 웨스턴 블 롯팅과 같은 다른 기술을 사용하여 수행 될 수있다. 참고로, 그것은 몇 가지 봉투 구조의 발현이 최적이 될 수 있으므로 기술의 결과에 영향을 미칠 수 있다는 것을 인식하는 것이 중요하다. 여기에서 우리는 이전에 설명 CD4i 모노클로 날 항체를 사용하여 HIV-1 봉투의 형태를 프로빙에 초점을 맞추었다. 이 설정은 봉투의 점 돌연변이 또는 동시 발현 단백질의 구조적 결과에 영향을 프로빙 분석 등의 폭 넓은 범위를 허용한다. 더욱이, 그 기술은 설명다시 다양한 봉투의 입체 형태에 대해 서로 다른 단클론 항체의 특이성을 조사하기 위해 서로 다른 형태 적 성향과 잘 특성화 된 봉투 돌연변이와 함께 사용할 수 있습니다. 이것은 고도의 전문 장비 또는 노하우를 필요로하지 않는 동안 새로 고립 된 모노클로 날 항체의 쉽고 빠른 특성을 수 있습니다. 우리는 다양한 HIV-1 clades 다른 가까운 친척 계통 (HIV-2, SIV / Mac 용) 18 봉투에 대해이 방법을 사용하지만, 우리는이 분석은 다른 바이러스 가정들과 같은 추가 표면 항원에 대해 적용 할 수 있으리라 생각합니다.

삼량 바이러스 엔벨로프 당 단백질 (봉투)에 의해 매개되는 인간 면역 결핍 바이러스 1 형 (HIV-1) 항목은 전염성주기의 첫 번째 단계입니다. 비리의 표면에서 발표 만 노출 바이러스 항원이기 때문에, 봉투 삼량은 중화 및 nonneutralizing 항체를 이끌어 낸다. 따라서, 그것은 백신의 면역원 디자인에 대한 흥미있는 후보를 나타냅니다. 그러나, 수용성 또는 재조합 형태로 봉투와 함께 예방 접종 시험 차 HIV-1가 1-3 분리 대부분에 대한 최소한의 효과와 반응을 이끌어 냈다. 그럼에도 불구하고, 부분적인 효능 면역원 후보로 HIV-1 봉투의 RV144 백신 재판 4 새로운 관심에서 관찰. 이는 백신 유도 항 봉투 항체는 SIV에 대한 보호의 어느 정도를 생성하기에 충분했고, HIV 5 도전 것을 나타내는 최근 연구에 의해 확증되었다. 소포체에서 합성되고, 봉투의 glycoprote 후전구체, gp160에, 바이러스 성 융합 프로세스 연료하는 능력에 중요한 다양한 번역 후 변형을 겪는다. 봉투 전구체는 gp120의-gp41의 연락을 유지 비공유 상호 작용으로, 그 여분의 세포질 gp120의 및 횡단 gp41 서브 유닛 6-10로 분해되기 전에 삼량에 적절하고 동료 접어해야합니다. 감염된 세포는 주로 기계의 전체 질량 (11, 12)의 50 % 정도를 포함하는, 봉투 glycosylating을 담당한다. 더 나은 다른 입체 형태를 이해하는 중요성을 강조 봉투는 적응하고, 그렇지 않으면 15 ~ 19 노출 될 수있는 특정 높은 면역 원성 항원을 숨길 수 있도록 생각하는 준 안정성을 제공하면서 그 결과 복잡한 구조는, 봉투는 13, 14 구조적으로 유연하게 할 수 있습니다 기본 봉투 삼량 체에 의해 샘플링. 지금까지 여러 가지 기술들이 개발되고 성공적 봉투 구조적 CH를 연구하는데 사용되어왔다Anges의. 그러나, 그들은 종종 특정 봉투 컨텍스트로 제한되고, 자신의 한계에 따라 다릅니다. 예를 들어, 표면 플라즈몬 공명 형태 또는 특정 모노클로 날 항체를 사용하여 면역 침전 분석법 (모노클로 날 항체)은, 하나는 자신의 삼량 체 형태 20,21 immunogenetically에서 상이한 것으로 알려진 모노머 용해 또는 가용화 된 봉투 분자에 의존한다. 최근의 연구는 분열은 주로 항체 14,22,23를 nonneutralizing 인식 항원의 노출이 봉투의 입체 형태에 영향을 미치는 것이 좋습니다. 여기에서 우리는 세부 사항에서 cellularly 발현 봉투의 삼량 18,24-26의 형태를 빠르고 쉽게 결정을 허용하는 방법을 설명합니다. 인간 점착성 세포주에서 봉투의 일시적인 형질 감염에 이어 봉투 특정 항체의 결합은 단순한 화학 발광 반응을 이용하여 검출된다. 이 기술은 또한 환경의 구조적 특성을 사용할 수있는 형태를-의존적이다덴트 항체. 따라서, 이러한 분석은 강력하고 매우 유연한 검출 방법을 제공한다.

<table><tbody><tr><td>HOS cells</td><td>ATCC</td><td>CRL-1543</td><td></td></tr><tr><td>White Opaque Tissue Culture Plate, 96-well, Flat Bottom</td><td>BD</td><td>353296</td><td></td></tr><tr><td>Polyethylenimine, linear, 25,000 MW</td><td>Polysciences</td><td>23966</td><td>Prepared in 1 mg/ml solution</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Invitrogen</td><td>11995</td><td></td></tr><tr><td>Goat Anti-human IgG, Peroxidase Conjugated</td><td>Pierce</td><td>31413</td><td></td></tr><tr><td>Enhanced Chemiluminescence Substrate</td><td>PerkinElmer</td><td>NEL105001EA</td><td></td></tr><tr><td>TriStar LB 941, Plate Reader</td><td>Berthold Technologies</td><td></td><td></td></tr></tbody></table>

conformational evaluation of hiv-1 trimeric envelope glycoproteins using a cell-based elisa assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

상술 한 일반적인 절차를 사용하여, 우리는 야생형 (중량) 또는 돌연변이 된 봉투 하나에 CD4i 에피토프의 노출에 가용성 CD4 (sCD4)과 동시 발현 세포 CD4의 영향을 검정 프로토콜을 적응 18,24 전술 한 바와 같이, 25, 28. 한 개략적 일반적 절차와 sCD4 또는 CD4 세포 (18)의 동시 발현에 의해 치료 다음 CD4i 에피토프의 노출을 나타내는 도표. 18,24 예상대로 단클론 항체 2G12을 인식하는 외부 도메인이 처리에 의해 영향을받지 않는 반면, 그림 2에서, 우리는 CD4i 모노클로 날 항체의 17 (b)와 공 수용체 결합 부위 24,29 중복 48D 항원에 노출 봉투 구조 변화를 유도하기 위해 sCD4을 사용했다. 도 3에 제시된 바와 같이 입체 봉투에서 점 돌연변이의 영향은 또한 여기.,이 분석을 이용하여 평가 될 수는 a 감소하는 것으로 알려져 층 돌연변이 H66A 1 봉투를 사용D 성향은 자발적으로 CD4 결합 된 형태 18,24,30,31 또는 CD4 바인딩 상태 32에 봉투를 걸리기과 조화 된 결과 (그림 3A)를 얻은 돌연변이 (S375W)을 샘플링합니다. 다른 봉투의 익스가 사용되는 경우, 이는 발현 수준에 따른 상대적인 광 유니트 (RLU)로 표현 된 원시 데이터를 정규화하는 것이 필요하다. 이 경우, 우리는 정규화 된 항체 (도 3b)로서, PGT121, 봉투 실드 33-35 글리 칸의 부분을 인식하는 항체를 사용. 우리가 최근 18과 같이, 동일한 셀에있는 봉투와의 상호 작용이 CD4 CD4i 에피토프를 노출 구조 변화를 ENV하도록 이끈다. 도 4에서는, 셀 기반 ELISA 분석에서 봉투와 함께 CD4의 익스 양의 증가와 동시 형질 감염된 gp12의 내부 영역에서 불연속 에피토프를 인식 CD4i A32 모노클로 날 항체 및 C11 18,36-38위한 신호를 증가 수득영, 구조적 독립적 2G12 항체에 의해 봉투 인식 (그림 4A)를 영향을받지 않았다 반면. 조건 사이의 형질 전환 효율을 제어하기 위해, 원시 데이터는 2G12 (도 4b)로 정상화시켰다. 증가 된 신호 A32에 대해 획득 및 C11 항체는 봉투가 봉투 (39)와 상호 작용할 수 감소 능력 CD4 돌연변이 (F43H)로 동시 형질 감염된 때 A32과 C11 변조의 부재로 나타내는 바와 같이 봉투-CD4 상호 작용에 의존. <img alt="그림 1" fo:content-width="5in" src="/files/ftp_upload/51995/51995fig1highres.jpg" width="500" /> HOS 세포가 세포 표면에서 삼량 봉투 표현 형질되는 프로 시저의 방지 봉투 셀 기반 ELISA. (A) 일반 구조도 1의 도식 표현. 봉투의 형태이어서 다른 구체적인 인식 항체를 사용하여 샘​​플링 될 수있다(예 : CD4i 단클론 항체 등) 입체 형태. 신호는 HRP-결합 된 항 - 인간 단클론 항체로 염색 한 후 화학 발광에 의해 검출된다. sCD4 (B) 또는 CD4 세포 (C)의 동시 발현은 CD4i 에피토프의 노출을 초래할 봉투 형태 적 변화를 유도하기 위해 사용될 수있다. <img alt="그림이" fo:content-width="5in" src="/files/ftp_upload/51995/51995fig2highres.jpg" width="500" /> 그림 2 sCD4는 CD4i 항원 결정기와 sCD4의. 상호 작용의 노출로 이어지는 봉투 구조 변화를 유도 HIV-1 CO-JRFL ΔCT 봉투는 CD4i의 항원 결정기 (17B, 48D)을 대상으로 항체에 의해 인식,하지만 gp120의 외부 도메인 인식하는 항체에 의해 강화 2G12. HIV-1 CO-JRFLΔCT 봉투를 코딩하는 플라스미드로 형질 전환 된 각각의 웰 이상 세포가 계속되기 전에 RT에서 30 분 동안 존재 또는 4 μg / ㎖ sCD4의 부재 하에서 세정 및 배양 48 시간표준 프로토콜 inuing (4 일 - ELISA). 봉투의 형태는 다음 실온에서 1 시간 동안 0.25 μg / ml의 2G12, 1 μg / ml의 17B 또는 48D 방지 봉투 모노클로 날 항체와 함께 배양하여 프로빙했다. 신호 RT에서 45 분 동안 HRP-공액 항 - 인간 항체와 인큐베이션 후, 화학 발광에 의해 검출 하였다. 관련이없는 플라스미드로 형질 우물에서 얻은 신호 여섯 반복 실험의 SD ± 평균 RLU 값을 보이고있는 바와 같다 (더 봉투는) 차감하지 않습니다. 중요성이 일원 분산 분석에 의해 테스트와 데이터, 3 개의 독립적 인 실험에서 얻은 결과를 나타낸다 (NS, 중요하지, ****, p &lt;0.0001). <img alt="그림 3" fo:content-width="5in" src="/files/ftp_upload/51995/51995fig3highres.jpg" width="500" /> 봉투의 형태의 그림 3 변조. HIV-1 YU2 ΔCT 층 한 봉투 돌연변이 (H66A)는 S375W 변형 전시 반면 CD4i의 17B 인식을 감소S 17B는 신호 증가 및 층 (1)의 돌연변이 표현형을 회복하기에 충분하다. (A) 항 봉투 PGT121 및 17B 모노클로 날 항체를 사용하여 얻은 신호의 값을 RLU. (B) 또는 sCD4 치료없이 다음 CD4i 단클론 항체 (17B)의 PGT121 정규화 신호. 관련이없는 플라스미드로 형질 우물에서 얻은 신호에서 삼중의 SD ± 평균 값을 보이고있는 바와 같다 (더 봉투)를 차감하지 않습니다. 중요성이 일원 분산 분석에 의해 테스트와 데이터, 3 개의 독립적 인 실험에서 얻은 결과를 나타낸다 (** p &lt;0.01, *** p &lt;0.001, ****, p &lt;0.0001). <img alt="그림 4" fo:content-width="5in" src="/files/ftp_upload/51995/51995fig4highres.jpg" width="500" /> 세포 CD4의 그림 4와 동시 발현은 CD4i 항체에 의해 인식을 향상시킵니다. CD4 인코딩 플라스미드를 선호하기 위해 HIV1 YU2 ΔCT 봉투와 동시 형질 감염된했다CD4 결합 된 형태 18. (A) 항 봉투 2G12, 또는 C11 A32 모노클로 날 항체 및 항-CD4 OkT4 단클론 항체를 사용하여 얻은 신호의 값 RLU. (B) CD4i 모노클로 날 항체 A32과 C11의 2G12 정규화 신호. 관련이없는 플라스미드로 형질 우물에서 얻은 신호에서 삼중의 SD ± 평균 값을 보이고있는 바와 같다 (더 봉투)를 차감하지 않습니다. 데이터는 3 개의 독립적 인 실험에서 얻어진 결과의 대표입니다. 증가 파란색 막대는 CD4의 양을 단계적 증가를 나타냅니다 익스 (1.7 NG, 3.5 NG, 7 NG)를 형질 전환되는과 빨간색 막대 (F43H을 CD4 돌연변이의 형질을 나타내는 반면, 회색 바, CD4의 부재에 표시 gp120의 상호 작용으로 감소 용량, 7 NG).

Watch this Scientific Journal Video about Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay at JoVE.com

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

셀 기반의 ELISA 분석을 사용하여 HIV-1 삼량 봉투 당 단백질의 구조적 평가

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

conformational-evaluation-hiv-1-trimeric-envelope-glycoproteins-using

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

14.3K 조회수.  Université de Montréal. Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

비디오: Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' surface envelope glycoprotein. We previously proposed that the crown of the V3 loop, although dynamic and sequence variable, is constrained throughout the population of HIV-1 viruses to an immunologically relevant &beta;-hairpin tertiary structure. Importantly, there are thousands of different V3 loop crown sequences in circulating HIV-1 viruses, making 3D structural characterization of trends across the diversity of viruses difficult or impossible by crystallography or NMR. Our previous successful studies with folding of the V3 crown1, 2 used the ab initio algorithm 3 accessible in the ICM-Pro molecular modeling software package (Molsoft LLC, La Jolla, CA) and suggested that the crown of the V3 loop, specifically from positions 10 to 22, benefits sufficiently from the flexibility and length of its flanking stems to behave to a large degree as if it were an unconstrained peptide freely folding in solution. As such, rapid ab initio folding of just this portion of the V3 loop of any individual strain of the 60,000+ circulating HIV-1 strains can be informative. Here, we folded the V3 loop of the R2 strain to gain insight into the structural basis of its unique properties. R2 bears a rare V3 loop sequence thought to be responsible for the exquisite sensitivity of this strain to neutralization by patient sera and monoclonal antibodies4, 5. The strain mediates CD4-independent infection and appears to elicit broadly neutralizing antibodies. We demonstrate how evaluation of the results of the folding can be informative for associating observed structures in the folding with the immunological activities observed for R2.

Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

The crown region of different V3 loop sequences of the surface envelope glycoprotein (gp120) of HIV-1 can be structurally characterized in many cases by in silico folding of positions 10 to 22 of the loop using a state-of-the-art ab initio folding algorithm. Here we demonstrate the folding and evaluation of this region of the V3 loop from the R2 strain of HIV-1, a uniquely neutralization sensitive strain with puzzling functional properties.

하여 HIV - 1 V3 루프 크라운 R2 시퀀스 Immunologically 관련 동적 차 구조 기능의 평가 순이 론적 접는

The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' ...

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면역학 및 감염병학

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Cryo - 전자 Tomography 및 자동 하위 tomogram 평균을 사용하여 HIV의 봉투 Glycoproteins의 분자 구조 결정

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

siRNA 전달을위한 셀 타입의 특정 안티 HIV gp120 aptamers의 개발

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

의 이미징 HIV-1 봉투 유발 Virological 시냅스와 합성 지질 Bilayers에 신호

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an essential step for virus particle formation, since a defect in Gag membrane binding results in severe impairment of viral particle production. To gain mechanistic details of Gag-lipid membrane interactions, in vitro methods based on NMR, protein footprinting, surface plasmon resonance, liposome flotation centrifugation, or fluorescence lipid bead binding have been developed thus far. However, each of these in vitro methods has its limitations. To overcome some of these limitations and provide a complementary approach to the previously established methods, we developed an in vitro assay in which interactions between HIV-1 Gag and lipid membranes take place in a &#34;cell-like&#34; environment. In this assay, Gag binding to lipid membranes is visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique. Here we describe the background and the protocols to obtain myristoylated full-length Gag proteins and GUV membranes necessary for the assay and to detect Gag-GUV binding by microscopy.

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle GUV Membranes

We illustrate here an in vitro membrane binding assay in which interactions between HIV-1 Gag and lipid membranes are visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique.

거대한 단일 층 소포 (GUV) 막 바인딩 HIV-1 개그의 시각화

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

비리의 항원 스펙트럼과 세포 외 소포를 분석하는 Virometry 흐름

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

높은 처리량 Cre Lox 활성화 에이즈-1 바이러스 막 융해의 억제제를 식별 하기 위해 바이러스 막 융합 분석 결과

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

광견병 백신 효능을 평가하기 위한 시험관 내 ELISA 테스트

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate antigen-displaying VLPs. Surface antigen-specific antibodies are coupled to, and thus immobilized on a solid and insoluble matrix such as beads. Due to their high affinity to the target antigen, these antibodies can capture VLPs decorated with the cognate antigen anchored in the membrane envelope of the VLPs. This protocol describes the binding of antigen-specific antibodies to protein A- or G-conjugated magnetic beads. In our study, human immunodeficiency virus (HIV)-derived VLPs formed by the group-specific antigen (Gag) viral core precursor protein p55 Gag and displaying the envelope glycoproteins (Env) of HIV are examined. The VLPs are captured utilizing broadly neutralizing antibodies (bNAbs) directed against neutralization-sensitive epitopes in Env. The VLP capture assay outlined here represents a sensitive and easy-to-perform method to demonstrate that (i) the VLPs are decorated with the respective target antigen, (ii) the surface antigen retained its structural integrity as demonstrated by the epitope-specific binding of bNAbs used in the assay and (iii) the structural integrity of the VLPs revealed by the detection of Gag proteins in a subsequent Western blot-analysis. Consequently, the utilization of bNAbs for immunoprecipitation facilitates a prediction of whether VLP vaccines will be able to elicit a neutralizing B cell response in vaccinated humans. We anticipate that this protocol will furnish other researchers with a valuable and straightforward experimental approach to examine potential VLP-based vaccines.

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle VLP-Based Vaccines Using a Capture Assay

Here, we present a protocol to detect neutralization epitopes on antigen-displaying virus-like particles (VLPs). Immunoprecipitation of the human immunodeficiency virus (HIV)-derived VLPs is performed using envelope glycoproteins-specific monoclonal antibodies coupled to protein G-conjugated magnetic beads. Captured VLPs are subsequently subjected to SDS-PAGE and Western blot-analysis employing viral core protein Gag-specific antibodies.

포획 분석기를 사용하여 바이러스와 같은 입자(VLP) 기반 백신에 표시되는 항원에서 중화에 민감한 에피토프 검출

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate ...

<meta charset="utf8"></head><body>Dual-Reporter 및 Multiplex 기술을 사용한 SARS-CoV-2 분석

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

<meta charset="utf8"></head><body>저자 스포트라이트: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques (새로운 면역포획 및 질량 분석 기법을 통한 항바이러스 전략 발전)

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Multiplex Dual-Reporter Strategy를 통한 바이러스 입자의 표면 단백질 에피토프 프로파일링

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...