이 연구는 미국 국립 보건원 (NIH) (RO1 AI68002)에 의해 지원되었다.

1. 재조합 SARS - CoV RBD 단백질 준비 <ol><li> 칼슘 인산염 transfection 시약 준비 <ol><li> 2X HBS 버퍼 준비 : 함께 NaCl의 16g, 나 2 HPO 4 * 7H 2 O의 0.4 g, 그리고 HEPES의 13.0 g를 섞습니다. 7.00로 산도를 조절하고 증류수 1000 ML에 총 볼륨을 가져 오십시오. 살균, 나누어지는에 대한 솔루션을 필터링 및 -20 ° C.에서 저장 후 힌트 : 산도 가치의 모든 변동은 transfection 결과에 영향을 미치지합니다. 따라서 그것은 7.00 (예를 들어, 6.99, 7.00, 또는 7.01) 주위 여러 ​​산도 값을 테스트하고 아래에 소개된 방법을 사용하여 transfection에 가장 적합한 하나를 찾을 것을 권장합니다. </li><li> 2.5 M CaCl 2 준비 : 200 ML의 최종 볼륨 증류수에 CaCl 2 * 2 시간 2 O의 73.5 g을 추가합니다. -20 ° C.에 솔루션 및 상점 필터 </li></ol></li><li> 재조합 플라스미드 transfection하여 단백질 정화 <ol><li> transfection 전에 confluency 24 H는 50~70%에서 293T 세포 분할. 40 ML DMEM는 FBS, 1 % 페니실린 / 스트렙토 마이신 (P / S) 37 10 % 열 inactivated (HI)를 포함 ° C를 5% CO 2의 T - 175cm 두 조직 문화 flasks에 세포를 성장. </li><li> 모든 transfection의 시약은 transfection 전에 상온까지 가지고 있어야합니다. 이 시약은 2X HBS, 2.5M CaCl 2, DiH 2 O, 그리고 rRBD 플라스미드 6 포함되어 있습니다. 힌트 : 단백질 표정은 문화 뜨는로 표현 재조합 단백질의 분비를 보장하기 위해 신호 펩티드를 포함해야합니다에 대한 최종 재조합 플라스미드를 사용 제작합니다. 배의 그의 태그는 쉽게 정화를위한 표현 단백질의 C - 터미널에 추가할 수 있습니다. </li><li> 한 50 ML (A) 한 15 ML (B) BD 팰컨 튜브를 준비하고, 같은 표 1에 표시된 튜브에 시약을 추가합니다. 튜브 B에 관 A.에 2X HBS 버퍼를 추가 2.5M CaCl 2를 추가하고 플라스미드 rRBD, 그리고 DiH 2 O.의 요구 사항에 볼륨을 가지고 <table border="1"><tbody><tr><td colspan="4"> A. 하나의 T - 175cm 두 조직 문화 플라스크 (4000 μL / 술병) : rRBD 표현을위한 준비 </td></tr><tr><td colspan="2"> 튜브 </td><td colspan="2"> 튜브 B </td></tr><tr><td> 2X HBS </td><td> 2000 μL </td><td> 2.5M CaCl 2 </td><td> 200 μL </td></tr><tr><td></td><td></td><td> rRBD 플라스미드 DNA </td><td> 40 μg </td></tr><tr><td></td><td></td><td> DiH 2 O로 </td><td> 2000 μL </td></tr><tr><td colspan="4"> B. 하나의 100 - mm 배양 접시 (1000 μL / 접시) : 사스 pseudovirus 생산을위한 준비 </td></tr><tr><td colspan="2"> 튜브 </td><td colspan="2"> 튜브 B </td></tr><tr><td> 2X HBS </td><td> 500 μL </td><td> 2.5M CaCl 2 </td><td> 50 μL </td></tr><tr><td></td><td></td><td> SARS - CoV S 플라스미드 DNA </td><td> 5 μg </td></tr><tr><td></td><td></td><td> (pNL4 - 3.luc.RE) HIV - 1 플라스미드 </td><td> 5 μg </td></tr><tr><td></td><td></td><td> DiH 2 O로 </td><td> 500 μL </td></tr></tbody></table> 표 1. Transfection 혼합물의 준비와 볼륨 표 1에 나열된 볼륨 하나 transfection 유닛위한 것입니다. 더 flasks이나 요리가 transfection에 사용하는 경우 적절하게 볼륨을 조절할 수 있습니다. </li><li> 와동에 지속적인 부드러운가 혼합 유지하면서, 튜브 dropwise 방식에 관 B의 DNA - 칼슘 솔루션을 추가합니다. 혼합물은 20-30 분 실온에서 앉아 보자. 성공적인 칼슘 인산염 transfection에 대한 열쇠는 형성 촉진의 크기와 모양에 따라 달라집니다. 따라서 혼합물은 그 결과로 transfection의 효능을 향상시키고,이 요건을 충족하기 위해 지속적으로 천천히 vortexed하고 있어야합니다. </li><li> 293T 세포 (4000 μL / 플라스크)에도 dropwise와 방식으로 혼합을 추가합니다. 37 인큐베이터 문화 전지 ° 5% CO 2에서 C. </li><li> 신선한 혈청이없는 OPTI - 멤 I 감소 - 세럼 중간 (50 ML / 술병) 80-10 H 게시할 - transfection있는 문화 매체를 교체합니다. 같은 조건으로 이틀 더 문화 세포로 진행합니다. </li><li> 뜨는 포함하는 표현 rRBD 단백질 72 H 포스트 - transfection를 수집합니다. 세포 파편을 제거 15 분 6,000 rpm으로 원심 분리기. 4 ° C 하룻밤에 수집된 뜨는 및 저장 프로 테아제 억제제 칵테일을 추가합니다. </li><li> 다음날, 니켈 - NTA의 Superflow 다음과 같은 제조 업체의 지침을 사용하여 문화 뜨는에서 rRBD 재조합 단백질 정화. </li><li> Amicon 울트라 -15 농도 튜브를 사용하여 단백질을 정화 집중. 단백질 농도 후,농도 튜브에 PBS를 추가하고 용출 버퍼에 이미다졸을 제거하는 다시 원심 분리기. ° C를 사용하기 전까지 -80에서 단백질을 정제 단백질 농도 및 저장을 계산합니다. </li></ol></li></ol> 2. 마우스 예방 접종 및 샘플 수집 <ol><li> 40-45 사전 따뜻한 시그마 도움이되는 시스템 (SAS) ° C 제조 업체의 지시에 따라. 유리병 당 1 ML PBS를 추가하고 철저히 섞는다. </li><li> 표 2의 프로토콜에 따라 도움이되는 단백질의 감광 유제를 준비합니다. 피펫은 1.5 ML 튜브에 rRBD 단백질을 풀었다. 튜브 및 유제를 형성하기 위해 2-3 분 적극적으로 와동에 SAS 도움이되는 동등한 볼륨을 추가합니다. 힌트 : 표 2에 나열된 볼륨 한 마우스입니다. 사용되는 실제 마우스 번호에 따라 볼륨을 조정합니다. 각 그룹에 대한 각 백신에 필요한 단백질과 도움이되는 함께 섞는다. 항상 정확성을 보장하기 위해 예방 접종을 한 여분의 샘플을 준비합니다. <table border="1"><tbody><tr><td> 그룹 </td><td> 1 일 백신 (200 μL / 마우스) </td><td> 2 차 예방 접종 (200 μL / 마우스) </td><td> 제 3 백신 (200 μL / 마우스) </td></tr><tr><td> rRBD 단백질 </td><td> PBS 20 μg 단백질 (100 μL) + 100 μL SAS </td><td> PBS 10 μg의 단백질 (100 μL) + 100 μL SAS </td><td> PBS 10 μg의 단백질 (100 μL) + 100 μL SAS </td></tr><tr><td> PBS 제어 </td><td> 100 μL PBS + 100 μL SAS </td><td> 100 μL PBS + 100 μL SAS </td><td> 100 μL PBS + 100 μL SAS </td></tr></tbody></table> 표 2. 마우스 예방 접종 프로토콜 </li><li> Subcutaneously 프라임 - 면역 여성 BALB / C 마우스 (오래된 4-6주, 5 마우스 / 그룹) 및 rRBD 및 SAS와 배 향상은 표 2에서 지적했다. 컨트롤로 PBS 그룹을 사용합니다. 일반적으로 선택한 피하 주사에 대한 사이트는 양어깨 사이의 느슨한 피부이다. 또는 복부 복부는 그것이 주사 쉽게되기 때문에 일반적으로 사용되며, 사출 사이트에서 어떠한 누수를 관찰 수 있습니다. 백신의 반복 복용을 사용하는 경우, 그것은 잠재적인 지역의 피부 반응을 방지, 각종 사출 사이트를 선택 쉽습니다. </li><li> ° C 30 분 보완을 inactivate하는 56 각 예방 접종 후 예방 접종을 10 일 전에 마취와 함께 복고풍 - 궤도 및 열을 세라를 통해 쥐를 출혈. 사용하기 전까지 -20 ° C에서 보관 마우스 세라. </li></ol> 3. Pseudovirus의 중화 분석을 사용하여 중화 감지 <ol><li> 사스 pseudovirus 준비 <ol><li> transfection 전에 (2x10 6 셀 / 요리) 100mm 조직 문화를 배양 접시에서 16 H를 293T 세포를 분리하고, 상기 세포를 성장. </li><li> 로 표 1에 표시된 transfection의 시약을 준비합니다. 공동 transfect 플라스미드 인코딩 SARS - CoV S 단백질과 플라스미드 인코딩 환경을 - 결함, 루시페라제 - 표현 HIV - 1 유전자 (pNL4 - 3.luc.RE) 칼슘 인산염 transfection 시약을 사용합니다. </li><li> 10 ML 신선한 DMEM 10 % FBS, 1 % P / S 80-10 H 포스트 transfection을 포함와 매체를 대체합니다. 뜨는 포함 사스 pseudovirus 72 H 포스트 - transfection를 수집합니다. </li><li> 0.45 μm의 필터를 통해 필터 pseudovirus. -80에서 나누어지는하고 저장 ° C까지 사용. </li></ol></li><li> 사스 pseudovirus의 중화 분석 <ol><li> 16 H 감염 전에 96 잘 조직 배양 플레이트 4 cells/100 μL / 음 10 SARS - CoV 수용체 ACE2 (ACE2/293T)를 표현 293T 세포를 분할. </li><li> ACE2/293T 세포에서 바이러스 titer를 감지 2 배로 사스 pseudovirus을 희석. </li><li> 순차적으로 96 - 웰 조직 문화 접시에 마우스 세라를 희석하고, titrated 사스 pseudovirus 동등한 볼륨을 추가합니다. 1 H.위한 37 ° C에서 접시를 Preinc​​ubate </li><li> 부화 후, ACE2/293T 세포에 세라 - pseudovirus 혼합물 100 μL를 추가하고, 37 세포의 성장을 계속 ° C를 5% CO 2인치 나중에 신선한 DMEM 24 H를 추가합니다. </li><li> 완전히 문화 supernatants는 접시에서 72 H 게시 - 감염을 제거합니다. 1X 루시페라제 세포 배양 용해 시약 (물론 60 μL /)를 추가하고 지속은 실온에서 1-2 H에 대한 번호판을 잡고 세포 용해를 촉진. </li><li> luminometer 플레이트로 전송 세포 lysates (물론 50 μL /) (Microfluor 96 - 웰 플레이트). 루시페라제 분석 시스템에 포함된 루시페라제 기판 (50 μL / 음) 추가하고 울트라 384 luminometer에서 상대 루시페라제 활동을 감지합니다. </li><li> 50% 중화 항체 titer (NT 50) 6 등 사스 pseudovirus의 중화 titer와 선물을 계산합니다. </li></ol></li></ol>

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R., Prieto, Y., Oramas, N., Sanchez, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylenimine-based+transfection+method+as+a+simple+and+effective+way+to+produce+recombinant+lentiviral+vectors.">Polyethylenimine-based transfection method as a simple and effective way to produce recombinant lentiviral vectors.</a> Appl. Biochem. Biotechnol. 157, 538-544 (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

세포 밀도는 칼슘 인산염 기반 transfection의 효능에 영향을 미치는 중요한 요소이다. 우리의 경험에서 세포의 confluency 미만 70 % 최상의 결과를 제공합니다. 따라서, transfection 효율을 개선하기 위해, 그것은 세포 밀도가 confluency의 50-70 % 정도에서 보관하는 것이 좋습니다. 칼슘 인산염 transfection 방법은 일반적으로 지질 transfection 16 같은 다른 transfection 방식에 비해 덜 효율적인 것으로 생각됩니다. 그러나,이 프로토콜에서 우리는 따라서 transfection 효율을 향상, 소용돌이에 transfection 솔루션의 혼합 지속과 느린 해당 크기와 모양과 침전물의 형성을 보장 의하여 수정된 칼슘 인산염 transfection 방법을 사용합니다.

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
<tr>
<td>Na2HPO4*7H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S2429</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2*2H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C5080</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12430</td>
<td>&nbsp;</td>
<tr>
<td>HI FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10438</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>OPTI-MEM I Medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31985</td>
<td>&nbsp;</td>
<tr>
<td>Protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11836170001</td>
<td>&nbsp;</td>
<tr>
<td>Ni-NTA Superflow</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>30450</td>
<td>&nbsp;</td>
<tr>
<td>Sigma adjuvant system</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S6322</td>
<td>&nbsp;</td>
<tr>
<td>Luciferase assay system</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>E1501</td>
<td>&nbsp;</td>
<tr>
<td>Luciferase cell culture lysis reagent (5X)</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>E1531</td>
<td>&nbsp;</td>
<tr>
<td>Amicon Ultra - 15</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC901024</td>
<td>&nbsp;</td>
<tr>
<td>Microfluor 96-well plates</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>7905</td>
<td>&nbsp;</td>
<tr>
<td>Ultra 384 luminometer</td>
<td>&nbsp;</td>
<td>Tecan Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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protocol for recombinant rbd-based sars vaccines: protein preparation, animal vaccination and neutralization detection

그들의 안전을 프로필과 감염에 대한 강력한 면역 반응을 유도하는 능력을 기반으로, subunit 백신은 1-3 병원균의 다양한 후보로 사용되었습니다. 포유 동물 세포 시스템이 있으므로 제대로 접힌과 glycosylated 단백질을 형성, 사후 translational 수정이 가능하기 때문에, 포유 동물 세포에 표현 재조합 단백질은 높은 항원성 및 immunogenicity 4-6을 유지하기 위해 가장 큰 잠재력을 보여주었다. SARS에 대한 새로운 사건이 2004 년 이후보고되지 않고있다지만, 미래 발생은 지속적인 위협하므로, SARS - CoV에 대한 백신의 개발은 신중하게 예방 단계와 진행해야합니다. SARS - CoV S 단백질의 RBD는 수용체 바인딩 및 바이러스 감염에 대한 구체적인 7-9 중화 항체의 유도에 중요한 역할을 담당하고 있습니다. 따라서,이 프로토콜에서 우리는 사스에 대한 RBD 기반 subunit 백신을 개발하기위한 새로운 방법을 설명합니다. 간단히, 재조합 단백질 RBD (rRBD)는 적절한 형태와 높은 immunogenicity 6 제대로 접힌 단백질을 얻기 위해 포유 동물 293T 세포의 문화 뜨는으로 표현되었다. 세포에 재조합 플라스미드 인코딩 RBD의 transfection 그 다음 수정과 칼슘 인산염 transfection 방법 6,10를 사용하여 수행되었습니다. 지질 transfection 방법 11,12 비해,이 수정된 칼슘 인산염 transfection 방법은 처리 저렴, 쉽게하고, 적당한 크기와 모양과 transfection 복합은 13,14을 형성되면 높은 효능을 도달하는 가능성이있다. 마지막으로, 사스 pseudovirus의 중화 분석은 프로토콜의 도입 rRBD 단백질과 예방 접종을 생쥐의 세라의 중화 활동을 탐지하는 데 사용되었다. 이 분석은 상대적으로 안전, 전염병 사스 - CoV를 포함하지 않으며, biosafety - 3 실험실 15 요구하지 않고 수행할 수 있습니다. 프로토콜은 또한 예를 들어, HIV, 호흡 syncytial 바이러스 (RSV), 에볼라 바이러스, 인플루엔자 바이러스뿐만 아니라 Nipah과 Handra 바이러스를 설계하고 클래스 I 융합 단백질로 다른 바이러스에 대한 재조합 subunit 백신을 연구하는 데 사용할 수 있습니다 여기에 설명되어 있습니다. 또한, pseudovirus을 생성하고 이후 pseudovirus의 중화 분석 수립을위한 방법은 모든 바이러스에 적용할 수 있습니다.

Watch this Scientific Journal Video about Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection at JoVE.com

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

이 프로토콜은 사스에 대한 재조합 수용체 결합 도메인 (RBD) 기반 subunit 백신을 연구를위한 일반적인 절차를 설명합니다. 그것은 293T 세포에 RBD 단백질, RBD와 설립 사스 pseudovirus의 중화 분석을 사용하여 마우스 세라의 중화 활동의 검출과 생쥐의 예방 접종의 transfection과 표현을위한 방법을 포함하고 있습니다.

재조합 RBD 기반 SARS 백신에 대한 프로토콜 : 단백질 준비, 동물 예방 접종 및 중화 감지

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

protocol-for-recombinant-rbd-based-sars-vaccines-protein-preparation

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JoVE Journal

Immunology and Infection

42.4K 조회수.  New York Blood Center.  中文 

비디오: Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

접착제 테이프 기반의 샘플링 및 형광의 조합 현장에서</em의 신속한 감지를 위해> 하이브 리다이 제이션 살모넬라균 신선한 농산물에 대한

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

연구

면역학 및 감염병학

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

나노 입자 백신 접종 후 분석 항원 특이 CD8 + T 세포 반응에 대한 전체 동물 이미징 및 유세포 기술

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

키메라 마우스 모델에서 재조합 인플루엔자 백신의 비강 관리는 점막 면역을 연구하기 위해

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Vivo에서 예방 접종 모델을 사용 하는 항 원 특정 T-셀 Cytolytic 함수의 검색에 대 한 분석 결과

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

CRISPR/Cas9와 재조합 조류 Herpesvirus 벡터 생성 편집 진

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

We offered captive mongooses placebo oral rabies vaccine baits with ethyl or methyl iophenoxic acid as a biomarker and verified bait uptake using a novel liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

작은 인도 몽구스(헤르페스 오로 punctatus)에서이오페녹산 유사체의 분석은 구강 광견병 예방 접종 생물학적 마커로 사용하기위한 세라

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at &#8805;21 days from symptom onset but had higher sensitivity with samples collected at &#8804;5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

멀티플렉스 비드 기반 유량 분석 시스템을 위한 신속한 멀티플렉스 듀얼 리포터 IgG 및 IgM SARS-CoV-2 중화 분석

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The increasing number of SARS-CoV-2 infections raises the need for better antibody detection assays. Current antibody detection methods compromise sensitivity for speed or are sensitive but time-consuming. A large proportion of SARS-CoV-2-neutralizing antibodies target the receptor-binding domain (RBD), one of the primary immunogenic compartments of SARS-CoV-2. We have recently designed and developed a highly sensitive, bioluminescent-tagged RBD (NanoLuc HiBiT-RBD) to detect SARS-CoV-2 antibodies. The following text describes the procedure to produce the HiBiT-RBD complex and a fast assay to evaluate the presence of RBD-targeting antibodies using this tool. Due to the durability of the HiBiT-RBD protein product over a wide range of temperatures and the shorter experimental procedure that can be completed within 1 h, the protocol can be considered as a more efficient alternative to detect SARS-CoV-2 antibodies in patient serum samples.

The outlined protocol describes the procedure for producing the HiBiT-receptor-binding domain protein complex and its application for fast and sensitive detection of SARS-CoV-2 antibodies.

HiBiT 기반 바이오 리포터를 사용하여 SARS-CoV-2 수용체 결합 도메인 항체 검출

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe ...

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has been responsible for over 242 million infections and more than 4.9 million deaths worldwide. Similar to other viruses, studying SARS-CoV-2 requires the use of experimental methods to detect the presence of virus in infected cells and/or in animal models. To overcome this limitation, we generated replication-competent recombinant (r)SARS-CoV-2 that expresses bioluminescent (nanoluciferase, Nluc) or fluorescent (Venus) proteins. These reporter-expressing rSARS-CoV-2 allow tracking viral infections in vitro and in vivo based on the expression of Nluc and Venus reporter genes. Here the study describes the use of rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus to detect and track SARS-CoV-2 infection in the previously described K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of infection using in vivo imaging systems (IVIS). This rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus show rSARS-CoV-2/WT-like pathogenicity and viral replication in vivo. Importantly, Nluc and Venus expression allow us to directly track viral infections in vivo and ex vivo, in infected mice. These rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus represent an excellent option to study the biology of SARS-CoV-2 in vivo, to understand viral infection and associated COVID-19 disease, and to identify effective prophylactic and/or therapeutic treatments to combat SARS-CoV-2 infection.

This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.

리포터 발현 재조합 SARS-CoV-2를 이용한 K18 hACE2 트랜스제닉 마우스에서의 바이러스 감염의 라이브 이미징 및 정량화

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has ...

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 ...