Bu çalışmada, Amerika Birleşik Devletleri Ulusal Sağlık Enstitüleri (NIH) (RO1 AI68002) tarafından desteklenmiştir.

1. Rekombinant SARS-CoV RBD Protein Hazırlık <ol><li> Kalsiyum fosfat transfeksiyon reaktif hazırlayın <ol><li> 2X HBS tampon hazırlanması: 16 g NaCl, 0,4 g Na 2 HPO 4 * 7H 2 O ve Hepes 13,0 g birlikte karıştırın. PH 7.00 ayarlayın ve toplam hacmi 1000 ml distile su getirmek. Sterilizasyon, kısım için çözüm filtreleme ve -20 ° C&#39;de saklayın sonra İpucu: pH değerinin herhangi bir değişiklik transfeksiyon sonuçlarını etkileyebilir. Böylece, etrafında birkaç pH değerleri 7.00 (örneğin, 6.99, 7.00, veya 7.01) test etmek ve aşağıda tanıtılan yöntem kullanarak transfeksiyon için en uygun olanı bulmak için tavsiye edilir. </li><li> 2.5 M CaCl 2 hazırlık: 73.5 g CaCl 2 * 2H 2 O son hacim 200 mL distile su ekleyin. Filtre -20 ° C&#39;de çözüm ve mağaza </li></ol></li><li> Rekombinant plazmid transfeksiyon ve protein saflaştırılması <ol><li> Transfeksiyon 24 saat önce confluency 293T hücrelerin% 50-70 Böl. 40 ml DMEM FBS ve% 1 penisilin / Streptomisin (P / S) 37% 10 ısı ile inaktive edilmiş (HI) içeren ° C,% 5 CO 2 T-175 cm 2 doku kültürü şişeler hücrelerin büyütün . </li><li> Tüm transfeksiyon ayıraçları, önce oda sıcaklığına kadar transfeksiyon getirilmelidir. Bu reaktifler 2X HBS, 2.5M CaCl 2 dih 2 O, ve rRBD plazmid 6 içerir. İpucu: protein ekspresyonu kültür süpernatant ifade rekombinant proteinlerin salgılanmasını sağlamak için bir sinyal peptid içermelidir son rekombinant plazmid inşa. 6x Onun etiketi için kolay arıtma ifade protein C-terminal eklenebilir. </li><li> 50 ml (A) ve 15 ml (B) BD Falcon tüp hazırlanması ve Tablo 1&#39;de gösterildiği gibi tüplere reaktifler eklemek. Tüp B tüp A. 2X HBS tampon, 2.5M CaCl 2 eklemek ve plazmid rRBD ve dih 2 O. gereksinimi ses getirmek <table border="1"><tbody><tr><td colspan="4"> A. Bir T-175 cm 2 doku kültürü balonuna (4000 mcL / şişesi): rRBD ifade hazırlanmak </td></tr><tr><td colspan="2"> Tüp A </td><td colspan="2"> Tüp B </td></tr><tr><td> 2X HBS </td><td> 2000 mcL </td><td> 2.5M CaCl 2 </td><td> 200 mcL </td></tr><tr><td></td><td></td><td> rRBD plazmid DNA </td><td> 40 mikrogram </td></tr><tr><td></td><td></td><td> Dih 2 O </td><td> 2000 mcL </td></tr><tr><td colspan="4"> B. Bir 100-mm petri (1000 mcL / yemek): SARS pseudovirus üretim için hazırlamak </td></tr><tr><td colspan="2"> Tüp A </td><td colspan="2"> Tüp B </td></tr><tr><td> 2X HBS </td><td> 500 mcL </td><td> 2.5M CaCl 2 </td><td> 50 mcL </td></tr><tr><td></td><td></td><td> SARS-CoV S plazmid DNA </td><td> 5 mikrogram </td></tr><tr><td></td><td></td><td> (PNL4 3.luc.RE) HIV-1 plazmid </td><td> 5 mikrogram </td></tr><tr><td></td><td></td><td> Dih 2 O </td><td> 500 mcL </td></tr></tbody></table> Tablo 1: Transfeksiyon karışım hazırlama ve birimler Tablo 1&#39;de belirtilen miktarlar bir transfeksiyon ünitesi için. Daha fazla matara veya yemekler transfeksiyon için kullanılırsa, buna göre hacimleri ayarlayabilirsiniz. </li><li> Bir girdap sabit ve yumuşak bir karıştırma korurken, tüp damla damla bir şekilde bir tüp B DNA kalsiyum çözüm ekleyin. Karışımı 20-30 dakika oda sıcaklığında bekletin. Başarılı kalsiyum fosfat transfeksiyon için anahtar boyutu ve şekli, oluşan çökelti bağlıdır. Böylece, karışımın bir sonucu olarak, transfeksiyon etkinliğini arttırmak, bu gereksinimi karşılamak için sürekli ve yavaş yavaş vortekslenmiş ve olmalıdır. </li><li> 293T hücreleri (4000 mcL / balon) içine damla damla ve hatta bir şekilde karışımı ekleyin. 37 ° inkübatör Kültür hücreleri ° C,% 5 CO 2 . </li><li> Taze serum OPTI-MEM İndirgenmiş-Serum Orta (50 ml / balon) 8-10 saat sonrası transfeksiyon ile kültür ortamı değiştirin. Kültür hücreleri aynı durumun iki gün daha devam edin. </li><li> Süpernatant içeren ifade rRBD protein 72 saat sonrası transfeksiyon toplayın. Hücre enkaz kaldırmak için 6000 rpm&#39;de 15 dakika santrifüjleyin. 4 ° C gecede toplanan süpernatant ve mağaza proteaz inhibitörü kokteyl ekleyin. </li><li> Ertesi gün, Ni-NTA Superflow aşağıdaki üreticinin talimatlarına kullanarak kültür süpernatant rRBD rekombinant protein arındırmak. </li><li> Amicon Ultra -15 konsantrasyon tüpler kullanılarak proteinin saflaştırılmış konsantre ol. Protein konsantrasyonuPBS toplama tüplerine ekleyin ve imidazol elüsyon tampon kaldırmak için tekrar santrifüj. -80 ° C kullanana kadar az protein saflaştırılmış protein konsantrasyonu ve mağaza hesaplayın. </li></ol></li></ol> 2. Fare Bağışıklama ve Örnek Toplama <ol><li> 40-45 Pre-sıcak Sigma adjuvan sistemi (SAS) ° C ile üreticinin talimatlarına göre. Flakon başına 1 ml PBS ekleyin ve iyice karıştırın. </li><li> Tablo 2&#39;de protokole göre protein-adjuvan emülsiyon hazırlayın. Pipet, 1.5 mL tüp içine rRBD protein hesaplanır. SAS adjuvan eşit hacim ve emülsiyon oluşturmak için 2-3 dakika boyunca kuvvetlice vorteks tüp ekleyin. İpucu: Tablo 2&#39;de belirtilen miktarlar bir fare. Hacimleri kullanılan gerçek fare numaralarına göre ayarlayın. Her grup için, her aşı için gerekli protein ve adjuvan karıştırın. Doğruluğunu sağlamak için aşılama için her zaman ekstra bir örnek hazırlamak. <table border="1"><tbody><tr><td> Grup </td><td> 1 st aşı (200 mcL / fare) </td><td> 2 nci aşı (200 mcL / fare) </td><td> 3 üncü aşı (200 mcL / fare) </td></tr><tr><td> rRBD protein </td><td> PBS içinde 20 mg protein (100 mcL) + 100 mcL SAS </td><td> PBS içinde 10 mg protein (100 mcL) + 100 mcL SAS </td><td> PBS içinde 10 mg protein (100 mcL) + 100 mcL SAS </td></tr><tr><td> PBS kontrolü </td><td> 100 mcL PBS + 100 mcL SAS </td><td> 100 mcL PBS + 100 mcL SAS </td><td> 100 mcL PBS + 100 mcL SAS </td></tr></tbody></table> Tablo 2: Fare bağışıklama protokolü </li><li> Subkutan prime-bağışıklık kadın BALB / c farelerde (eski 4-6 hafta, 5 fare / grup) ve rRBD ve SAS ile iki kat artırmak gibi Tablo 2&#39;de gösterilmiştir. PBS grup kontrol olarak kullanın. Genellikle seçilen subkutan enjeksiyonlar için site, kürek kemikleri arasındaki gevşek deri. Alternatif olarak, ventral karın enjekte etmek kolaydır, çünkü yaygın olarak kullanılır, ve herhangi bir sızıntı enjeksiyon siteleri gözlemlemek olabilir. Tekrarlanan doz aşı kullanıldığında, potansiyel yerel deri reaksiyonları önlemek, çeşitli enjeksiyon siteleri seçmek kolaydır. </li><li> Her aşıdan sonra aşılama ve 10 gün önce 56 ° C&#39;de 30 dakika için tamamlayıcı inaktive anestezi ile retro-orbital, ısı ve sera ile fareler Bleed. Kullanana kadar -20 ° C&#39;de saklayın fare sera. </li></ol> 3. Nötralizasyon Algılama kullanarak Pseudovirus Nötralizasyon Testi <ol><li> SARS pseudovirus hazırlayın <ol><li> Transfeksiyon önce 100 mm doku kültürü petri kaplarına (2x10 6 hücre / yemek) 16 saat 293T hücreler Split, ve yukarıdaki gibi hücreleri büyümek. </li><li> Tablo 1&#39;de gösterildiği gibi transfeksiyon ayıraçları hazırlayın. Co-transfect plazmid kodlama SARS-CoV S protein ve plazmid kodlama Env kusurlu, lusiferaz-HIV-1 genom ifade (pNL4 3.luc.RE) kalsiyum fosfat transfeksiyon reaktifi kullanılarak. </li><li> 10 ml taze DMEM% 10 FBS ve% 1 P / S 8-10 saat sonrası transfeksiyon içeren orta değiştirin. Süpernatant içeren SARS pseudovirus 72 saat sonrası transfeksiyon toplayın. </li><li> 0.45 mikron filtre ile filtre pseudovirus. Kısım ve mağaza -80 ° C kullanana kadar. </li></ol></li><li> SARS pseudovirus nötralizasyon tayini <ol><li> 16 saat önce 96-iyi doku kültürü plakaları 4 cells/100 mcL / 10 SARS-CoV reseptörü ACE2 (ACE2/293T) ifade 293T hücreleri enfeksiyon Bölünmüş. </li><li> ACE2/293T hücrelerde virüs titresi tespit etmek için 2 kat SARS pseudovirus sulandırınız. </li><li> Seri 96-iyi doku kültürü plakaları fare sera sulandırmak ve titre SARS pseudovirus eşit miktarda ekleyin. 1 saat 37 ° C&#39;de plakaları Preincubate </li><li> ACE2/293T hücreleri inkübasyondan sonra, sera pseudovirus karışımı 100 mcL ekleyin ve 37 ° hücreler büyümeye devam ° C,% 5 CO 2 . Taze DMEM 24 saat sonra ekleyin. </li><li> Tamamen kültür süpernatantlar plakaları 72 saat enfeksiyon sonrası çıkarın. 1X lusiferaz hücre kültürü reaktifi (60 mcL / iyi) ekleyin ve oda sıcaklığında 1-2 saat süreyle sabit plaka titreme ile hücre parçalama teşvik. </li><li> Luminometre tabak içine transfer hücre ilginçtir (50 mcL / iyi) (Microfluor 96-kuyucuğu). Lusiferaz tahlil sistemi dahil lusiferaz substrat (50 mcL / kuyu) ekleyin ve Ultra 384 luminometre göreli lusiferaz aktivitesini algılar. </li><li> % 50 nötralizan antikor titresi (NT 50) 6 olarak SARS pseudovirus nötralizasyon titresi ve mevcut hesaplayın. </li></ol></li></ol>

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C., Sun, S., Chen, M., Liu, Z., Guo, H., He, Y., Zhou, Y., Zheng, B. J., Jiang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+receptor-binding+domain+of+SARS-CoV+spike+protein+expressed+in+mammalian,+insect+and+E.+coli+cells+elicits+potent+neutralizing+antibody+and+protective+immunity.">Recombinant receptor-binding domain of SARS-CoV spike protein expressed in mammalian, insect and E. coli cells elicits potent neutralizing antibody and protective immunity.</a> Virology. 393, 144-150 (2009).</li><li>Ambrosino, D. 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Hücre yoğunluğu kalsiyum fosfat tabanlı transfeksiyon etkinliğini etkileyen önemli bir faktördür. Deneyimlerimize göre,% 70 daha az hücrelerin confluency en iyi sonuçları getiriyor. Böylece, transfeksiyon verimliliğini artırmak için, hücre yoğunluğu confluency, yaklaşık% 50-70 tutulması tavsiye edilir. Kalsiyum fosfat transfeksiyon yöntem genellikle lipid transfeksiyon 16 gibi diğer transfeksiyon yöntemleri ile karşılaştırıldığında daha az etkili olduğu düşünülmektedir. Ancak, bu protokolde, böylece Transfeksiyonu verimliliği artırmak, sürekli ve yavaş bir girdap transfeksiyon çözüm karıştırma uygun boyutu ve şekli ile bir çökelti oluşumunu sağlar böylece değiştirilmiş bir kalsiyum fosfat transfeksiyon yöntemi kullandı.

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		<div>
		<table border="1">
		<thead>
		<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>
		</table>
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protocol for recombinant rbd-based sars vaccines: protein preparation, animal vaccination and neutralization detection

Güvenlik profili ve enfeksiyonlara karşı güçlü bir bağışıklık yanıtları uyardığı yeteneği dayanarak, alt ünite aşıları 1-3 patojenleri geniş bir yelpazede aday olarak kullanılmaktadır. Memeli hücresi sistemi, böylece düzgün katlanmış ve glikozile proteinleri oluşturan, post-translasyonel değiştirme yeteneğine sahip olduğu için, memeli hücrelerinde ifade rekombinant proteinlerin yüksek antijenik ve immünojenite 4-6 korumak için en büyük potansiyel göstermiştir. SARS, 2004 yılından bu yana hiçbir yeni vaka bildirilmiş olmasına karşın, gelecekte salgınları sürekli bir tehdit ve bu nedenle, SARS-CoV karşı aşıların geliştirilmesi, ihtiyatlı bir önleyici adımdır ve yapılmalıdır. SARS-CoV S protein RBD reseptör bağlayıcı ve virüs enfeksiyonu 7-9 karşı spesifik nötralizan antikorlar indüksiyon önemli rol oynar. Bu nedenle bu protokol, RBD tabanlı altbirim SARS karşı aşı geliştirmek için yeni yöntemler açıklanmaktadır. Kısaca, rekombinant RBD protein (rRBD), uygun konformasyon ve yüksek immünojenite 6 ile doğru katlanmış bir protein elde etmek için, memeli 293T hücrelerinin kültür süpernatant ifade edildi. Hücrelere rekombinant plazmid kodlama RBD transfeksiyon sonra, bazı değişikliklerle birlikte kalsiyum fosfat transfeksiyon yöntemi 6,10 kullanılarak gerçekleştirildi . Lipid transfeksiyon yöntemi 11,12 ile karşılaştırıldığında, bu değiştirilmiş kalsiyum fosfat transfeksiyon yöntemi işlemek için ucuz, kolay ve uygun boyutu ve şekli ile transfeksiyon kompleksi 13,14 oluşur kez yüksek etkinliğini ulaşmak için bir potansiyele sahiptir. Son olarak, SARS pseudovirus nötralizasyon tayini protokol tanıttı ve sera rRBD protein ile aşılanan farelerin nötralize aktivite tespit etmek için kullanılır oldu. Bu testte, nispeten güvenli, bulaşıcı bir SARS-CoV dahil değildir ve bir biyogüvenlik-3 laboratuvar 15 gereksinimi olmadan yapılabilir . Burada açıklanan protokol aynı zamanda örneğin, HIV, respiratuar sinsisyal virüs (RSV), Ebola virüsü, grip virüsü gibi, Nipah ve Handra virüsler, tasarım ve diğer virüslere karşı rekombinant altbirim aşıları sınıf I füzyon proteinleri ile çalışma kullanılabilir. Buna ek olarak tüm bu virüslerin pseudovirus üreten ve daha sonra pseudovirus nötralizasyon tayini kurulması için yöntemler uygulanabilir.

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

Bu protokol, SARS karşı rekombinant reseptör-bağlayıcı etki alanı (RBD) tabanlı altbirim aşılar eğitim için genel bir yordam açıklanır. RBD 293T hücrelerinin protein, RBD ve fare sera kurulmuş bir SARS pseudovirus nötralizasyon tayini ile nötralizasyonu aktivitesi tespit ile farelerin bağışıklık transfeksiyon ve anlatım yöntemleri içerir.

Rekombinant RBD tabanlı SARS Aşılar için Protokol: Protein hazırlanması, Hayvan Aşılama ve Nötralizasyon Algılama

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

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Research

JoVE Journal

Immunology and Infection

42.4K Görüntüleme.  New York Blood Center.  中文 

Video: 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).

Yapışkan bant tabanlı Örnekleme ve Floresans kombinasyonu In situ</emHızlı Algılama> Hibridizasyon Salmonella Taze üretin

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

İmmünoloji ve Enfeksiyon

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.

Nanopartikül Aşılama sonrası analizi antijene özgü CD8 + T hücresi tepkileri için tüm hayvan Görüntüleme ve Akış Sitometrisi Teknikleri

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.

Kimerik fare modellerinde Rekombinant Grip aşıların burun içinde yoluyla mukozal bağışıklık Okumak

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.

Vivo Tahlil antijen spesifik T-hücre Cytolytic işlevi bir aşı modeli kullanılarak tespiti için

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.

Rekombinant kuş Herpesvirus vektörleri ile CRISPR/Cas9 üreten gen düzenleme

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.

Küçük Hint firavunları (Herpestes Auropunctatus) sera bir oral kuduz aşılama biyolojik Marker olarak kullanım Için Iophenoxic asit analogları Analizi

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.

Çok Katlı Boncuk Tabanlı Akış Analiz Sistemi için Hızlı, Çok Katlı Çift Muhabir IgG ve IgM SARS-CoV-2 Nötralizasyon Tahlil

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 Tabanlı Bir Biyoreporter Kullanılarak SARS-CoV-2 Reseptör Bağlayıcı Etki Alanı Antikorunun Tespiti

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.

K18 hACE2 Transgenik Farelerde Viral Enfeksiyonun Canlı Görüntülenmesi ve Raporlayıcı İfade Eden Rekombinant SARS-CoV-2 Kullanılarak Ölçülmesi

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.

Virüs Benzeri Parçacık (VLP) Tabanlı Aşılarda Bir Yakalama Tahlili Kullanılarak Görüntülenen Antijenlerde Nötralizasyona Duyarlı Epitopların Tespiti

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 ...