Les auteurs sont reconnaissants pour l&#39;aide au développement expérimental de la méthode de la vaccination chez les souris de intralymphatique Iris Erdmann, Barbara von Beust, Maria et Julia Martínez-Gómez. Merci aussi à Maggy Arras et Nikola Cesarovic pour nous laisser utiliser leur salle d&#39;opération pour cette production vidéo.

Une. Anesthésie de souris <ol><li> Préparer les anesthésiques par mélange de kétamine (anesthésique dissociatif) et de xylazine (sédatif et analgésique) dans une solution saline tamponnée. Les concentrations de kétamine et de xylazine dans la solution finale est de 12,5 et 2 mg / ml, respectivement. </li><li> Injecter l&#39;anesthésique à la souris par voie intrapéritonéale à l&#39;aide d&#39;une seringue avec une aiguille de 25 à 30 G. Utiliser 0,1 ml/10 g de poids corporel. </li><li> Appliquer une pommade ophtalmique aux yeux des souris afin d&#39;éviter le dessèchement de la cornée. </li><li> Assurez-vous que la souris est suffisamment anesthésiée par pincement son pied ou un orteil avec la pince. Si la souris ne réagit pas avec les réflexes au pincement, de procéder à une intervention chirurgicale. Si la souris réagit à la rigueur avec des réflexes ou des contractions musculaires, attendre 1-2 minutes et répéter le test de pincement. Si la souris réagit toujours, remplacez la souris avec un nouvel animal et répéter les procédures de l&#39;étape 1.2 partir. </li></ol> 2. Je chirurgicalencision de la région inguinale de la souris <ol><li> Placez la souris sur son dos. </li><li> Mouiller une région inguinale avec de l&#39;éthanol désinfection (70%). Pour assurer une meilleure désinfection, la région inguinale peut être rasé pour enlever la fourrure désinfection préalable avec de l&#39;éthanol. </li><li> Prenez la patte arrière et plier la hanche pour produire un angle d&#39;environ 90 ° de l&#39;articulation de la hanche. </li><li> L&#39;utilisation d&#39;un forceps courbes de microdissecting, prendre la peau de la souris dans la région inguinale où l&#39;articulation de la hanche est plié et tirez la peau mouillée légèrement. </li><li> Tout en maintenant la peau avec des pinces, couper une petite incision (&lt;5 mm) à travers la peau avec des ciseaux chirurgicaux. </li><li> Placez la pointe des ciseaux fermés dans l&#39;incision et ouvrir davantage l&#39;incision en ouvrant les ciseaux tout en maintenant à l&#39;intérieur de l&#39;incision. Cela entraînera la peau à se déchirer et devrait produire un diamètre inférieur à 10 mm. Remarque: Une source de lumière froide avec guides de lumière flexibles peut être utilisé pour améliorer la visibilité. En plusles souris mâles, en particulier, l&#39;identification d&#39;un ganglion lymphatique peut être difficile en raison de la quantité de tissu de matière grasse dans la région inguinale. </li></ol> 3. Injection intralymphatique <ol><li> Préparer une seringue (0,5 ml ou moins) avec une aiguille hypodermique G 28-30; biseau court peut être préférable à un long biseau. Aspirer 10 pi de vaccin à injecter et assurent que la seringue est exempte d&#39;air. </li><li> Localiser le ganglion lymphatique inguinal avec l&#39;aide de la pince courbes et la pointe de ciseaux fermé. Le ganglion lymphatique apparaît grisâtre dans le tissu adipeux plus blanchâtre et peut encore être identifié par capillaires afférents et efférents qui entrent et sortent du ganglion lymphatique. </li><li> Immobiliser le ganglion lymphatique en le maintenant entre les branches de la pince incurvés. </li><li> Prenez la seringue et insérer l&#39;aiguille dans le ganglion lymphatique avec les coniques orientés vers le haut; s&#39;assurer que l&#39;ensemble du biseau pénètre dans le ganglion lymphatique. </li><li> Injecter le vaccin (10 pi). Si le feu vert de la lymphee est un gonflement (sauter), l&#39;injection peut supposer succès. Si l&#39;aiguille n&#39;est pas placé assez profondément dans le ganglion lymphatique, une partie ou la totalité du vaccin peut s&#39;échapper et ne pas entrer dans le ganglion lymphatique. Si l&#39;aiguille est insérée trop profondément, le vaccin injecté sera publié voie sous-cutanée en dessous du noeud lymphatique. Dans ce dernier cas, répéter l&#39;injection ou de remplacer la souris avec une nouvelle souris non traitées. </li></ol> 4. Fermeture de l&#39;incision par suture <ol><li> Ouvrez un paquet de suture chirurgicale stérile. </li><li> Saisir l&#39;aiguille de suture et d&#39;un support d&#39;aiguille à la portion distale du corps d&#39;aiguille. Serrer le porte-aiguille en le serrant jusqu&#39;à ce que les premières prises de cliquet. </li><li> Saisir la peau d&#39;un côté de l&#39;incision avec une pince (en fonction de la préférence du chirurgien, pinces ou crochets de la peau dentée ou non dentée peut être utilisé). Insérer l&#39;aiguille ca. 2-3 mm du bord de l&#39;incision, l&#39;entrée de l&#39;extérieur de la peau. </li><li> Loosen attente de la peau avec la pince et à saisir le côté opposé de l&#39;incision avec la pince. Insérer l&#39;aiguille ca. 2-3 mm du bord de l&#39;incision, l&#39;entrée de l&#39;intérieur de la peau. </li><li> Desserrer le porte-aiguille et de saisir l&#39;extrémité de l&#39;aiguille avec la pince. Retirez l&#39;aiguille de la bande de roulement à travers la peau, laissant une longueur convenable de la bande de roulement à l&#39;extérieur du premier point insertion de l&#39;aiguille (si plusieurs souris sont à traiter, laisser un extrémité la plus courte; 5-10 souris peut être utilisée sur une seule aiguille à suture / bande de roulement). </li><li> Faire le noeud préféré selon les protocoles de chirurgien standard. Un point est suffisante si l&#39;incision n&#39;est pas supérieure à 10 mm. Pour les grandes incisions, faire un deuxième point. </li></ol> 5. Traitement post-opératoire <ol><li> Placez la souris dans la cage et couvrir avec les tissus pour garder au chaud. Lors du traitement de plusieurs souris, placez-les près de l&#39;autre pour se réchauffer. Sinon, placez la souris sur un tapis chaud jusqu&#39;à ce qu&#39;ils se sont réveillésup. </li><li> Observez la souris jusqu&#39;à ce qu&#39;ils se réveillent. </li><li> Respecter les souris quotidiennement en ce qui concerne la cicatrisation ainsi que d&#39;autres symptômes cliniques tels que des infections de plaies. La blessure est généralement suffisamment fermée dans les deux jours et guérit dans les sept jours de la procédure. </li></ol>

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TMK est désigné comme l&#39;inventeur de brevets portant sur l&#39;immunothérapie intralymphatique chez l&#39;homme. TMK a été conseiller scientifique et a reçu les frais de voyage de ImVisioN GmbH, Cytos Biotechnology, MannKind Corporation, et XBiotech USA Inc. PJ n&#39;a aucun intérêt à divulguer des conflits.

Intralymphatique immunisation et l&#39;immunothérapie se sont révélés être appropriés pour la stimulation des deux réponses d&#39;anticorps et de réponses des cellules T. Comme démontré dans cette vidéo-article, la procédure intralymphatique de la vaccination est une méthode rapide et facile pour stimuler une forte réponse immunitaire chez les souris. Un chirurgien qualifié peut effectuer la procédure pendant 3-4 min. La session peut également être partagé entre deux chirurgiens où l&#39;on est gén?...

Les vaccins sont typiquement injectés par voie intramusculaire ou sous-cutanée pour la stimulation des réponses immunitaires. La réussite de ce procédé nécessite un drainage efficace de vaccin vers les ganglions lymphatiques où les cellules présentant l&#39;antigène peuvent interagir avec les lymphocytes pour la production de T-et de réponses des cellules B. En outre, la force et le type de réponses immunitaires induites dépendent aussi de la densité ou de la fréquence de ces interactions ainsi que le micro-environnement lui-même, en particulier la teneur en cytokines. Comme seule une petite fraction d&#39;un vaccin injecté dans un tissu périphérique atteint un ganglion lymphatique, la vaccination de souris et l&#39;homme par injection directe du vaccin dans les ganglions lymphatiques, le site ont été les réponses immunitaires sont produits, ont été réalisées. Chez l&#39;homme, la procédure est guidée par ultrasons, un procédé également utilisé pour l&#39;administration d&#39;agents d&#39;imagerie pour la visualisation et de diagnostic dans le système lymphatique. Chez la souris, la procédure est invasive. Ici, une petite (5-10 mm) incision est fabriqué dans la région inguinale des animaux anesthésiés 1, le ganglion lymphatique est localisé et immobilisé avec une pince, et un volume de 10-20 ul du vaccin est injecté sous contrôle visuel, 10 pi est utilisé pour les premières injections et chez de jeunes souris avec de petits ganglions lymphatiques, tandis que 20 pl peuvent être injectés dans les ganglions lymphatiques de souris âgée ou déjà amorcés, qui ont de plus grands ganglions lymphatiques. L&#39;incision peut être fermé avec un seul point à l&#39;aide de sutures chirurgicales. Par cette méthode, les souris ont vaccinés avec l&#39;ADN plasmidique de 2,3, l&#39;ARN messager 4, 1,3,5,6 peptide, protéine 7-10, les particules 11, 12 des bactéries, ainsi que des adjuvants 7,13, et une forte amélioration de l&#39;abri réponses contre tous les types de vaccins ont été observés. La méthode intralymphatique de la vaccination est particulièrement approprié dans les situations où la vaccination classique produit une immunité insuffisante ou lorsque la quantité de vaccin disponible est limité ou very coûteux. Chez l&#39;homme, le procédé intralymphatique de vaccination a été appliquée à des patients allergiques à 14,15 ou 16 à 21 patients atteints d&#39;un cancer. Bien que la notion de courant est que la méthode est intralymphatique plus invasif que d&#39;autres méthodes injectables tels que des injections intramusculaires et sous-cutanées, la perception de la douleur n&#39;est pas plus élevée qu&#39;après une ponction veineuse 15. Il est prévu que la vaccination intralymphatique deviendra une alternative ou un complément à d&#39;autres méthodes de vaccins prophylactiques et thérapeutiques en particulier. Cet article décrit en détail la façon dont la procédure de vaccination intralymphatique est réalisée chez la souris. Toutes les procédures décrites, ont été approuvés par l&#39;Agence vétérinaire cantonal de Zurich et réalisés selon des lignes directrices et des directives sur la protection des animaux utilisés à des fins scientifiques fédéraux suisses.

<table border="0" cellpadding="0" cellspacing="0" width="653">
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		<col />
		<col />
		<col />
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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Ketamine (Ketasol-100)</td>
			<td style="width:135px;">Graeub AG, Switzerland</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Anesthetics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Xylazine (Rompun)</td>
			<td style="width:135px;">Bayer, Germany</td>
			<td style="width:119px;"></td>
			<td>Anesthetics</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Viscotears Eye-Gel</td>
			<td style="width:135px;">Novartis, Switzerland</td>
			<td style="width:119px;"></td>
			<td>To keep eyes from drying out during anesthesia.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">BD Micro-Fine 0.5 ml</td>
			<td style="width:135px;">BD Medical, France</td>
			<td style="width:119px;"></td>
			<td>29 G Insulin syringes with permanently attached needles</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">6-0 Dermalon Monofilament nylon</td>
			<td style="width:135px;">Covidien, MA, USA</td>
			<td style="width:119px;"></td>
			<td>For sutures (0.7 metric, 18G, 45 cm, Blue)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Curved forceps, 4.5 inch</td>
			<td style="width:135px;">Polymed, Switzerland</td>
			<td style="width:119px;"></td>
			<td>For incision and holding of lymph node</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Straight surgical scissors, 4.5 inch</td>
			<td style="width:135px;">Polymed, Switzerland</td>
			<td style="width:119px;"></td>
			<td>For incision</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Needle holder, 5.5 inch</td>
			<td style="width:135px;">Polymed, Switzerland</td>
			<td style="width:119px;"></td>
			<td>To close incision with suture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
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intralymphatic immunotherapy and vaccination in mice

Les vaccins sont typiquement injectés par voie intramusculaire ou sous-cutanée pour la stimulation des réponses immunitaires. Le succès de cette nécessite un drainage efficace de vaccin vers les ganglions lymphatiques où les cellules présentant l&#39;antigène peuvent interagir avec les lymphocytes pour la génération de réponses immunitaires recherchés. La force et le type de réponses immunitaires induites dépendent aussi de la densité ou la fréquence des interactions ainsi que le micro-environnement, en particulier la teneur en cytokines. Comme seule une infime partie de la périphérie vaccins injectés atteint les ganglions lymphatiques, la vaccination de souris et de l&#39;homme ont été réalisées par injection directe de vaccin dans les ganglions lymphatiques inguinaux, à savoir l&#39;injection intralymphatique. Chez l&#39;homme, la procédure est guidée par échographie. Chez la souris, une petite (5-10 mm) incision est pratiquée dans la région inguinale des animaux anesthésiés, le ganglion lymphatique est localisé et immobilisé avec une pince, et un volume de 10-20 ul du vaccin est injecté sous contrôle visuel.L&#39;incision est fermée avec un seul point à l&#39;aide de sutures chirurgicales. Les souris ont été vaccinées avec de l&#39;ADN plasmidique, l&#39;ARN, un peptide, une protéine, des particules, et les bactéries ainsi que des adjuvants, et une forte amélioration de la réponse immunitaire contre tous les types de vaccins ont été observées. La méthode intralymphatique de la vaccination est particulièrement approprié dans les situations où la vaccination classique produit une immunité insuffisante ou lorsque la quantité de vaccin disponible est limitée.

La procédure d&#39;injections intralymphatique chez la souris, en dépit de la nature chirurgicale, est simple et relativement rapide. Une personne formée peut effectuer la procédure en 3-4 min. L&#39;incision est fermée avec une couture guérit normalement dans les deux jours (figure 1) Intralymphatique vaccination ou l&#39;immunisation a été réalisée avec l&#39;ARNm, l&#39;ADN plasmidique, les peptides, les protéines, les virus et les bactéries. Figure 2<...

Watch this Scientific Journal Video about Intralymphatic Immunotherapy and Vaccination in Mice at JoVE.com

Intralymphatic Immunotherapy and Vaccination in Mice

La vaccination prophylactique et thérapeutique échoue souvent pour stimuler une forte réponse immunitaire due à la semaine drainage du vaccin vers les ganglions lymphatiques et par conséquent faible implication des cellules immunitaires. Par injection directe de vaccin vers les ganglions lymphatiques, dite injection intralymphatique, l&#39;efficacité du vaccin peut être fortement améliorée, et les doses de vaccin peut être réduite.

Intralymphatique Immunothérapie et vaccination chez la souris

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

intralymphatic-immunotherapy-and-vaccination-in-mice

Research

JoVE Journal

Immunology and Infection

22.9K vues.  University Hospital Zurich.  La vaccination prophylactique et thérapeutique échoue souvent pour stimuler une forte réponse immunitaire due à la semaine drainage du vaccin vers les ganglions lymphatiques et par conséquent faible implication des cellules immunitaires. Par injection directe de vaccin vers les ganglions lymphatiques, dite injection intralymphatique, l'efficacité du vaccin peut être fortement améliorée, et les doses de vaccin peut être réduite. 

Vidéo: Intralymphatic Immunotherapy and Vaccination in Mice

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Métastases expérimentales et CTL Modèle adoptifs souris Transfert immunothérapie

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

Recherche

Immunologie et infection

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

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

Protocole pour vaccins recombinants contre le SRAS RBD-base: préparation de protéines, vaccination des animaux et de détection de neutralisation

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

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies 2.
Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production.
DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 &deg;C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine 3. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4 - 20 x 106 cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum specifications before they are injected into patients.

The most commonly used method for generating large numbers of autologous dendritic cells (DCs) for use in tumor immunotherapy is described. The method uses IL-4 and GM-CSF to differentiate DCs from monocytes. The immature DCs are stimulated to mature and then loaded with antigens before they are injected back into the patient.

Préparation de la tumeur cellules dendritiques matures antigène chargées pour l&#39;immunothérapie

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

Criblage à haut débit pour à large spectre inhibiteurs chimiques de l&#39;ARN virus

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Génération de la RCA cellules T pour la thérapie adoptive dans le contexte de glioblastome norme de diligence

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

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.

In Vivo Test pour la détection de cellules T spécifiques de l’antigène fonction cytolytique en utilisant un modèle de Vaccination

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

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Génération de discriminatif anticorps monoclonaux humains de cellules rares spécifiques de l’antigène B circulant dans le sang

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Stéréotaxique transfert adoptif de cellules immunitaires cytotoxiques dans des modèles murins de xénogreffes de glioblastome Multiforme Orthotopic homme

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

Test ELISpot interféron-gamma optimisé à mesure les réponses des cellules T dans le modèle cobaye après Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

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.

Analyse des analogues de l'acide iophénoxique chez la petite mangouste indienne (Herpestes Auropunctatus) Sera pour utilisation comme marqueur biologique de vaccination contre la rage buccale

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