eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of Quantified Glycerol Stocks of Bacillus Calmette-Guerin (BCG) Danish and Mycobacterium tuberculosis H37Rv
NOTE: All the protocols described were performed under bio-safety level-3 (BSL3) conditions.
<ol>
	<li>Thaw frozen glycerol stocks of BCG Danish or H37Rv strains and inoculate 100 &#956;l in 10 ml of 7H9 medium supplemented with Tween-80 0.05% (v/v) and ADC (Albumin, Dextrose, Catalase) 10% (v/v).</li>
	<li>Incubate under static conditions for one week at 37 &#176;C until the culture is in log-phase growth.</li>
	<li>Scale up bacterial culture by transferring the 10-ml culture to 200 ml of fresh medium. Incubate for 20 additional days under static conditions.</li>
	<li>Transfer to 50-ml centrifuge tubes and centrifuge for 15 min at 2500 x g.</li>
	<li>Discard supernatants and resuspend the bacterial pellet in the residual volume. Add sterile 3-mm-diameter glass beads to each tube (10 - 15 per tube).</li>
	<li>Vortex vigorously for 1 min in order to dissociate bacterial clumps. Resuspend each pellet in 10 ml of phosphate-buffered saline (PBS) with Tween-80 0.05% (v/v).</li>
	<li>Leave tubes for 10 min allowing the greatest bacterial aggregates and glass beads to settle down.</li>
	<li>Recover 9.5 ml of supernatant from each of the 4 tubes containing small clumps and single bacteria and transfer volumes to a single fresh 50-ml centrifuge tube (final recovered volume 38 ml). Centrifuge for 10 min at 400 x g.</li>
	<li>Recover 35 ml of supernatant (containing mainly single bacteria) in a single fresh 50-ml centrifuge tube and add 15 ml of PBS with glycerol 50% (v/v), obtaining a final glycerol concentration of 15% (v/v).</li>
	<li>Mix gently and distribute in 1 ml aliquots in appropriate tubes for freezing. Store at -80 &#176;C (one lot provides approximately 50 aliquots of glycerol stock).</li>
	<li>To quantify glycerol stocks, thaw glycerol the day after freezing and perform seven ten-fold serial dilutions in separate 1.5-ml tubes containing 900 &#956;l of PBS.</li>
	<li>Plate 100 &#956;l of each dilution in 7H10 solid-agar medium supplemented with ADC 10% (v/v) prepared in 55-mm diameter Petri dishes.</li>
	<li>Carefully add 3 mm-diameter glass beads (approx. 10 per plate) and gently shake the plate to equally distribute volume over the agar plate. Discard beads and seal the agar plate with parafilm.</li>
	<li>Incubate for 21 days at 37 &#176;C and determine bacterial concentration by counting colony-forming units (CFU) in the dilutions where single colonies can be accurately distinguished.</li>
</ol>
2. Mouse Vaccination
<ol>
	<li>Thaw a pre-quantified BCG glycerol and dilute it in PBS to prepare two suspensions. Calculate final volume considering the dose per animal is 100 &#956;l for subcutaneous administration (107 CFU per ml for subcutaneous vaccination) and 40 &#956;l for intranasal administration (2.5 x 107 CFU for intranasal vaccination).</li>
	<li>Use a specialized anesthesia workstation for rodents to anesthetize mice with a mixture of isoflurane and oxygen. Use isoflurane 5% to induce anesthesia and 2% to maintain the mice in the anesthesia chamber. (Other accepted anesthesia method can be used).</li>
	<li>For subcutaneous vaccine administration fill a 1-ml syringe with a 26 G needle with 1 ml of bacterial suspension (107 CFU/ml). Remove air bubbles.</li>
	<li>Place an anesthetized mouse on a flat surface in prone position inside a laminar flow hood and inoculate subcutaneously 100 &#956;l (106 CFU/dose) of vaccine in a flank of the mouse back. Return the mouse to the cage and monitor it to ensure that it properly recovers from anesthesia.</li>
	<li>Change the syringe needle between each mouse administration. Removed air bubbles as described above.</li>
	<li>For intranasal administration, place an anesthetized mouse in supine position inside the hood.</li>
	<li>With a micropipette take 20 &#956;l of the suspension with 2.5 x 107 CFU/ml. Place the inoculum drop-by-drop between the two nostrils until the whole volume has been deposited, leaving time between drops to allow the mouse to breathe the volume in. 
	NOTE: mice can suffer from respiratory distress during this procedure so they should be left to assimilate each drop prior to administrate the next one to prevent it as much as possible.</li>
	<li>Re-fill the micropipette with another 20 &#956;l and repeat the operation. If the mouse starts to wake up from the anesthesia after the first inoculation, place it in the anesthesia chamber prior to the second one. Return the mouse to the cage and ensure that it properly recovers from anesthesia.</li>
</ol>
3. Mouse Intranasal Challenge with H37Rv Strain
<ol>
	<li>Thaw a pre-quantified H37Rv glycerol and dilute it in PBS to prepare a bacterial suspension of 2,500 CFU per ml. Calculate final volume considering the dose per animal is 40 &#956;l.</li>
	<li>H37Rv is inoculated intranasally as described in 2.5 - 2.7. Final challenge dose per mouse is 102 CFU/40 &#181;l.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Middlebrook 7H9 broth</td>
			<td>BD</td>
			<td>271310</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook ADC Enrichment</td>
			<td>BD</td>
			<td>211887</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Scharlau</td>
			<td>TW00800250</td>
			<td></td>
		</tr>
		<tr>
			<td>3-mm diameter Glass Beads</td>
			<td>Scharlau</td>
			<td>038-138003</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook 7H10 Agar</td>
			<td>BD</td>
			<td>262710</td>
			<td></td>
		</tr>
		<tr>
			<td>1-ml syringe 26GA 0.45x10 mm</td>
			<td>BD</td>
			<td>301358</td>
			<td></td>
		</tr>
		<tr>
			<td>C tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>M tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-236</td>
			<td></td>
		</tr>
		<tr>
			<td>LACHRYMAL OLIVE LUER LOCK 0.60 x 30 mm. 23G x 1 1/4&#34;</td>
			<td>UNIMED</td>
			<td>27.134</td>
			<td>Used as trachea cannula for BAL</td>
		</tr>
		<tr>
			<td>Albumin, from bovine serum</td>
			<td>Sigma</td>
			<td>A4503</td>
			<td></td>
		</tr>
	</tbody>
</table>

intranasal immunization with bcg in a mouse model

Source: Uranga, S. et al., <a href="https://app.jove.com/v/54440">Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice.</a>&#160;J. Vis. Exp. (2016)
This video demonstrates intranasal BCG immunization in mice, activating both innate and adaptive immune responses, leading to the development of lung-resident memory T cells and effector B cells, thus enhancing protection against Mycobacterium infections.

Intranasal Immunization with BCG in a Mouse Model

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Start with an anesthetized mouse and administer a live-attenuated BCG strain of Mycobacterium bovis, which has reduced disease-causing ability, through the mouse&#39;s nose.
The injected bacteria stimulate an immune response in the lung tissue and nearby lymph node.
This leads to the generation of effector T cells and B cells that enter the mouse&#39;s circulation.
The effector B cells produce antibodies against mycobacteria.
Meanwhile, the effector T cells settle in the lung tissue as resident memory T cells or TRM cells, providing long-term immunity.
To assess the effectiveness of immunization, administer live, virulent mycobacteria with disease-causing ability through the mouse&#39;s nose.
This triggers the activation of TRM cells in the lung tissue.
Activated TRM cells release cytokines, attracting other immune cells, including effector B cells.
These B cells secrete antibodies that bind to mycobacteria, enhancing their clearance by neutrophils, indicating successful immunization.

intranasal-immunization-with-bcg-in-a-mouse-model

Research

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Immunogenics and Vaccine Development

117 Views. Source: Uranga, S. et al., Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice. J. Vis. Exp. (2016) This video demonstrates intranasal BCG immunization in mice, activating both innate and adaptive immune responses, leading to the development of lung-resident memory T cells and effector B cells, thus enhancing protection against Mycobacterium infections. 

Video: Intranasal Immunization with BCG in a Mouse Model - Concept

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for protection against children&#39;s severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

JoVE Journal

Immunology and Infection

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

Medicine

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-&#947; and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-&#947; secretion assessed using ELISA showed a significantly higher titer (**p &#60; 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...

Intranasal Immunization with BCG in a Mouse Model

Transcript

Intranasal Immunization with BCG in a Mouse Model

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells