この研究は、米国の国立衛生研究所（NIH）（RO1 AI68002）によってサポートされていました。

1。組換えSARS - CoVのRBDタンパク質の調製<ol><li>リン酸カルシウムトランスフェクション試薬を準備する<ol><li> 2X HBSバッファーの準備：一緒に塩化ナトリウム16gの、のNa 2 HPO 4 · 7H 2 O 0.4gの、およびHEPESの13.0グラムを混ぜる。 7.00 pHを調整し、蒸留水で1000mLとする総量を持ち出す。滅菌用溶液を濾過した後、分注して-20℃で保存する ヒント：pH値の任意の変化は、トランスフェクションの結果に影響を与えるだろう。従って、それは7.00（たとえば、6.99、7.00、または7.01）の周りにいくつかのpH値をテストし、下記の導入方法を用いてトランスフェクションに最適なものを見つけることをお勧めします。 </li><li> 2.5 M CaCl 2の準備：200mLの最終容量に蒸留水に塩化カルシウム2 * 2H 2 Oの73.5グラムを追加。 -20℃で溶液とストアをフィルタリング</li></ol></li><li>組換えプラスミドトランスフェクションとタンパク質精製<ol><li>トランスフェクションの前に50〜70パーセントコンフルエント24時間で293T細胞を分割する。 40mLのDMEMにFBSおよび1％ペニシリン/ストレプトマイシン（P / S）で37 10％熱不活性化（HI）を含む° Cを5％CO 2でのT - 175cm 2の組織培養フラスコで細胞を成長させる。 </li><li>すべてのトランスフェクション試薬、トランスフェクションの前に室温に立ち上げることが必要。これらの試薬 ​​は、2X HBS、2.5MのCaCl 2、DIH 2 O、及びrRBDプラスミド6を含む。 ヒント：タンパク質発現に使用される最終的な組換えプラスミド構築物は、培養上清中に発現した組換えタンパク質の分泌を確保するためのシグナルペプチドが含まれている必要があります。 6X Hisタグを簡単に精製するための発現タンパク質のC末端に追加されることがあります。 </li><li> 1つの50 mLの（A）と1つの15 mLの（B）BDファルコンチューブを準備し、表1に示されている試験管に試薬を加える。チューブBでチューブAに2X HBSバッファーを追加し、2.5M CaCl 2を追加し、プラスミドrRBD、およびDIH 2 Oの要件にボリュームをもたらす <table border="1"><tbody><tr><td colspan="4"> A. One T - 175cm 2の組織培養フラスコ（4000μL/フラスコ）：rRBD表現するための準備 </td></tr><tr><td colspan="2"> チューブ </td><td colspan="2"> チューブB </td></tr><tr><td> 2X HBS </td><td> 2000μL </td><td> 2.5MのCaCl 2 </td><td> 200μL </td></tr><tr><td></td><td></td><td> rRBDプラスミドDNA </td><td> 40μgの</td></tr><tr><td></td><td></td><td> DIH 2 Oへ </td><td> 2000μL </td></tr><tr><td colspan="4"> B. One 100 mmのペトリ皿（1000μL/皿）：SARSの偽の生産準備 </td></tr><tr><td colspan="2"> チューブ </td><td colspan="2"> チューブB </td></tr><tr><td> 2X HBS </td><td> 500μL </td><td> 2.5MのCaCl 2 </td><td> 50μL </td></tr><tr><td></td><td></td><td> SARS - CoVのSプラスミドDNA </td><td> 5μgの</td></tr><tr><td></td><td></td><td>プラスミドHIV - 1（pNL4 - 3.luc.RE） </td><td> 5μgの</td></tr><tr><td></td><td></td><td> DIH 2 Oへ </td><td> 500μL </td></tr></tbody></table> 表1。トランスフェクション混合物の準備とボリューム 表1に記載されているボリュームは一トランスフェクションユニット用です。より多くのフラスコや皿をトランスフェクションに使用されている場合は、それに応じてボリュームを調整します。 </li><li>渦の定数と、穏やかに混合維持しながら、チューブ滴下方法で中にチューブのBのDNA -カルシウム溶液を加える。混合物は20〜30分間室温で放置します。成功したリン酸カルシウムトランスフェクションのためのキーが形成された沈殿物の大きさと形状に依存する。このように、混合物は、結果として、トランスフェクション効率を向上させる、この要件を満たすために絶えずゆっくりとボルテックスとすべきである。 </li><li> 293T細胞（4000μL/フラスコ）にさえ滴下し、方法で混合物を追加。 37℃インキュベーターで培養細胞を℃、5％CO 2でC。 </li><li>新鮮な無血清OPTI - MEM I削減血清培地（50mLの/フラスコ）80から10時間後にトランスフェクション培地を交換してください。同じ条件でさらに2日間、培養細胞に進みます。 </li><li>を含む上清に発現rRBDタンパク質の72時間のトランスフェクション後を収集する。細胞破片を除去するために15分間6000rpmで遠心する。 4℃で一晩上清を回収し、ストアにプロテアーゼ阻害剤カクテルを添加。 </li><li>次の日に、製造元の指示に従ってのNi - NTA Superflowを使用して培養上清よりrRBD組換えタンパク質を精製する。 </li><li>アミコンウルトラ-15濃度のチューブを使用してタンパク質を精製した集中。タンパク質濃度の後、濃度のチューブにPBSを追加し、溶出バッファーにイミダゾールを削除するには、もう一度遠心してください。 ° C使用時まで-80タンパク質を精製タンパク質の濃度とストアを計算する。 </li></ol></li></ol> 2。マウスの免疫化およびサンプル採集<ol><li>プレ暖かいシグマアジュバントシステム（SAS）40から45℃までは、製造元の指示に従って。バイアル当たり1mLのPBSを加えてよく混ぜる。 </li><li>表2のプロトコルに従って、タンパク質-アジュバントエマルジョンを準備します。ピペットで1.5 mlチューブにrRBDタンパク質を算出。チューブとエマルジョンを形成するために2〜3分間激しくボルテックスするSASアジュバントの等量を追加。 ヒント：表2にリストされているボリュームは、つのマウス用です。実際に使用されるマウスの数に応じてボリュームを調整します。各グループについて、それぞれのワクチンに必要なタンパク質とアジュバントを一緒に混ぜる。常に正確さを確保するためにワクチン接種が1回余分のサンプルを準備する。 <table border="1"><tbody><tr><td> グループ </td><td> 第1回ワクチン （200μL/マウス） </td><td> 第2回のワクチン （200μL/マウス） </td><td> 第3 回ワクチン （200μL/マウス） </td></tr><tr><td> rRBDタンパク質</td><td> PBSで20μgのタンパク質 （100μL）+ 100μLSAS </td><td> PBS中の10μgのタンパク質（100μL）+ 100μLSAS </td><td> PBS中の10μgのタンパク質（100μL）+ 100μLSAS </td></tr><tr><td> PBS制御</td><td> 100μLのPBS + 100μLSAS </td><td> 100μLのPBS + 100μLSAS </td><td> 100μLのPBS + 100μLSAS </td></tr></tbody></table> 表2。マウスの免疫化プロトコール</li><li>皮下プライム接種雌のBALB / cマウス（4-6週齢、5匹/群）、およびrRBDとSASで二回ブーストは、表2に示した。コントロールとしてPBS群を使用してください。通常、選択された皮下注射のためのサイトは、肩甲骨の間のたるんだ皮膚になります。また、腹側腹部は、それは注入することが簡単なので、一般的に、使用されており、注射部位からの漏れが観察される場合があります。ワクチンの反復投与が使用されている場合、それは潜在的な地元の皮膚反応を防止するため、様々な注射部位を選択することは簡単です。 </li><li> ° Cで30分間補数を失活させる56の各ワクチン接種後にワクチン接種し、10日の前に麻酔薬による眼窩、そして熱血清を経由してマウスをブリード。使用するまで-20℃で保存するマウス血清。 </li></ol> 3。偽ウイルス中和アッセイを使用して中和検出<ol><li> SARSの偽を準備<ol><li> 16時間は、トランスフェクションの前に100mmの組織培養ペトリ皿（2 × 10 6細胞/ディッシュ）で293T細胞を分割し、上記のように細胞を成長させる。 </li><li>表1に示されるトランスフェクション試薬を準備します。同時トランスフェクションをコードするプラスミドSARS - CoVのSタンパク質とをコードするプラスミドEnvの欠損、ルシフェラーゼを発現するHIV - 1ゲノム（pNL4 - 3.luc.RE）リン酸カルシウムトランスフェクション試薬を使用する。 </li><li> 10mLの新鮮なDMEMを含む10％FBSと1％P / S 80から10時間後にトランスフェクション培地を交換してください。偽72時間トランスフェクション後上清を含むSARSを収集する。 </li><li> 0.45μmのフィルターを通してフィルター偽。 -80℃で分注し、店舗℃で使用するまで。 </li></ol></li><li> SARSの偽ウイルス中和アッセイ<ol><li> 16時間は感染する前に、96ウェル組織培養プレートに4 cells/100μL/ウェル10でSARS - CoVの受容体ACE2（ACE2/293T）を発現する293T細胞を分割する。 </li><li> ACE2/293T細胞でウイルス力価を検出するために、2倍でSARSの偽ウイルスを希釈する。 </li><li>シリアル96ウェル組織培養プレートにマウス血清を希釈し、滴定SARSの偽の等量を追加します。 1時間37℃でプレートをPreinc​​ubate </li><li>インキュベーション後、ACE2/293T細胞に血清-偽の混合物の100μLを追加し、37℃で細胞を成長し続ける° Cを5％CO 2で。後で新鮮なDMEM 24時間を追加。 </li><li>完全にプレート72時間後の感染から培養上清を取り除く。 1Xルシフェラーゼの細胞培養溶解試薬（60μL/ウェル）を追加し、一定の室温で1-2時間プレートを振盪しながら細胞溶解を促進する。 </li><li>ルミノメーターのプレート（Microfluor 96ウェルプレート）に、細胞溶解物（50μL/ウェル）を転送する。ルシフェラーゼアッセイ系に含まれるルシフェラーゼ基質（50μL/ウェル）を追加し、ウルトラ384ルミノメーターでの相対的なルシフェラーゼ活性を検出する。 </li><li> 50パーセント中和抗体価（NT 50）6としてSARSの偽の中和力価と現在を計算する。 </li></ol></li></ol>

<ol>
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利害の衝突は宣言されません。

細胞密度は、リン酸カルシウムベースのトランスフェクションの有効性に影響を与える重要な要因です。我々の経験では、細胞のコンフルエンシーが70％未満では、最良の結果をもたらします。このように、トランスフェクション効率を向上させるためには、それは細胞密度がコンフルエントの50〜70％前後に維持することをお勧めします。リン酸カルシウムトランスフェクション法は、一般的にこのような脂質トランスフェクション16のような他のトランスフェクション法に比べ非効率的であると考えられている。しかし、このプロトコルでは、我々はこのようにトランスフェクション効率を向上させる、渦のトランスフェクション溶液の混合定数、遅いが適切なサイズと形状の沈殿物の形成を確保することにより修正されたリン酸カルシウムトランスフェクション法を使用。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<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>
		</table>
		</div>

protocol for recombinant rbd-based sars vaccines: protein preparation, animal vaccination and neutralization detection

それらの安全性プロファイルと感染症に対して強力な免疫応答を誘導する能力に基づいて、サブユニットワクチンは1-3病原体の様々な候補として使用されている。哺乳類細胞系がこのように適切に折り畳まとグリコシル化タンパク質を形成し、翻訳後修飾が可能なので、哺乳動物細胞で発現した組換えタンパク質は、高い抗原性および免疫原性4-6を維持するために最大の可能性を示している。 SARSの新たな症例は2004年以来報告されていないものの、将来の発生が一定の脅威であるため、SARS - CoVのワクチンの開発には慎重な予防手段であり、実施されるべきである。 SARS - CoVのSタンパク質のRBDは、ウイルス感染7から9に対する特異的中和抗体の受容体結合と誘導に重要な役割を果たしている。したがって、このプロトコルでは、我々はSARSに対するRBDベースのサブユニットワクチンを開発するための新規の方法を説明します。簡単に言えば、組換えRBDタンパク質（rRBDは）適切なコンフォメーションと高い免疫原性6で正しく折り畳まれたタンパク質を得るために哺乳類293T細胞の培養上清中に発現した。細胞への組換えプラスミドのエンコーディングRBDのトランスフェクションは、いくつかの修正とリン酸カルシウムトランスフェクション法6,10を用いて行った。脂質トランスフェクション法11,12と比較して、この変更されたリン酸カルシウムトランスフェクション法は、処理する方が簡単、安価であり、そして適切なサイズと形状を持つトランスフェクション複合体は13,14を形成されると、高い有効性に到達する可能性を秘めています。最後に、SARSの偽ウイルス中和アッセイは、プロトコルで導入され、rRBDタンパク質をワクチン接種したマウスの血清の中和活性を検出するために使用されていました。このアッセイは比較的安全である、伝染性SARS - CoVのが関与していない、およびバイオ- 3実験室15の要件なしで行うことができます。 プロトコルはここで説明するも、クラスIの融合タンパク質、例えば、HIV、呼吸器合胞体ウイルス（RSV）、エボラウイルス、インフルエンザウイルスだけでなく、ニパウイルスとHandraウイルスと他のウイルスに対する組換えサブユニットワクチンを設計し、研究するために使用することができます。さらに、偽を生成し、その後、偽ウイルス中和アッセイを確立するための方法はすべて、これらのウイルスに適用することができます。

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

このプロトコルは、組換え受容体結合ドメイン（RBD）ベースのSARSに対するサブユニットワクチンを研究するための一般的な手順を説明します。それは、293T細胞におけるRBDタンパク質のトランスフェクションと発現、RBDおよび確立されたSARSの偽ウイルス中和アッセイを用いたマウス血清の中和活性の検出によるマウスの免疫化のためのメソッドが含まれています。

タンパク質調製物、動物の予防接種と中和の検出：遺伝子組換えRBDベースのSARSのワクチンのためのプロトコル

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

Research

JoVE Journal

Immunology and Infection

42.4K ビュー.  New York Blood Center.  中文 

ビデオ: 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).

粘着テープベースのサンプリングと蛍光の組み合わせその場で</emの迅速検出のための>ハイブリダイゼーションサルモネラ生鮮食品について

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

免疫・感染学

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.

ナノ粒子ワクチン接種後の抗原特異的CD8 + T細胞応答の分析のための全動物イメージングとフローサイトメトリー技術

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.

キメラマウスモデルにおける組換えインフルエンザワクチンの鼻腔内投与は、粘膜免疫を研究するために、

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.

生体内で予防接種モデルを用いた抗原特異的 T 細胞細胞傷害性関数の検出のための試金

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/Cas9 組換え鳥ヘルペス ウイルスのベクトルを生成する遺伝子の編集

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.

小さなインドのマングースにおけるイオフェノキ酸類似体の分析 (ヘルペステス・オーロプンクタトゥス)経口狂犬病ワクチン接種生物学的マーカーとしての使用のためのセラ

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.

多重化ビードベースの流れ分析システムのための高速多重デュアルレポーターIgGおよびIgM SARS-CoV-2中和アッセイ

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ベースバイオレポーターを用いたSARS-CoV-2受容体結合ドメイン抗体の検出

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.

レポーター発現組換えSARS-CoV-2を用いたK18 hACE2トランスジェニックマウスにおけるウイルス感染のライブイメージングと定量

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.

捕捉アッセイを用いたウイルス様粒子(VLP)ベースワクチンに対する抗原中中和感受性エピトープの検出

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 ...