We thank Sergio G&#243;mez-Medina for excellent technical support with mouse experiments. This work was supported by funds from the Leibniz Association and the Leibniz Center of Infection. A.L. is a recipient of a pre-doctoral fellowship from the Leibniz Graduate School.

Animal experiments were conducted according to approved protocols and following the guidelines of the German animal protection law. All staff carrying out animal experiments passed training programs according to category B or C of the Federation of European Laboratory Animal Science Associations.
1. Generation of Recombinant Live Attenuated Influenza Vaccines by Reverse Genetics
NOTE: The detailed protocol for the generation of recombinant influenza viruses by reverse genetics has been described by previous studies5 and is out of the scope of this report. Briefly, rescue of cold-adapted influenza vaccines (CAV) is done in a biosafety class II cabinet under biosafety level 2 (BSL2) containment, and involves steps detailed below. The transfection and infection protocol follows that of Martinez-Sobrido et al.5.
<ol>
	<li>Generate a reassortant cold-adapted influenza vaccine using as background the cold-adapted strain A/Ann Arbor/6/60 (H2N2), as well as the hemagglutinin (HA) and a modified neuraminidase (NA) of the A/PR/8/34 (H1N1) strain (Figure 1A). The modification in the NA gene consists of a PCR-based replacement of a conserved sequence in the protein stalk (residues 65 to 72) by a short cDNA sequence encoding the chicken ovalbumin (OVA)-derived peptide SIINFEKL (Figure 1B). The SIINFEKL peptide is a highly immunodominant peptide in the context of the H-2b MHC class I-restricted response of C57BL/6 mice12.</li>
	<li>Plate 106 293T cells per well in 6-well plates using DMEM 10% fetal bovine serum (FBS) supplemented with 1% penicillin/streptomycin (P/E).</li>
	<li>Prepare plasmid transfection mixture: Add 1 &#181;g of each one of the plasmids encoding cDNAs for PB2, PB1, PA, NP, M, NS from the influenza A/Ann Arbor/6/60 strain and 1 &#181;g of the plasmids encoding for the NA-SIINFEKL and HA from influenza A/PR/8/34. Add pre-warmed OptiMEM (15 &#181;l for a final volume of 100 &#181;l/well).</li>
	<li>Incubate transfection mix for 30 min at RT.</li>
	<li>Add 100 &#181;l of transfection mix/well and incubate for 16 hr&#160;at 37 &#176;C and 5% CO2.</li>
	<li>Change cell media by DMEM 0.3% BSA 1% P/E at 33 &#176;C and 5% CO2 for 5 hr.</li>
	<li>Add 106 influenza permissive MDCK cells in DMSO containing 1 &#181;g/ml of trypsin from bovine pancreas treated with L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK-trypsin). Maintain the 293T-MDCK co-cultures for 2 - 3 days at 33 &#176;C and 5% CO2.</li>
	<li>Collect supernatants of MDCK-293T cell co-cultures and clear by centrifugation at 260 x g for 5 min.</li>
	<li>Inoculate 9 - 10 day-old chicken embryonated eggs in sterile conditions. To do so, make a hole on the top of the eggshell as indicated by Martinez-Sobrido et al.5.</li>
	<li>Cover the eggshell with melted wax using a cotton swab and incubate the inoculated chicken eggs for 3 days at 33 &#176;C.</li>
	<li>Harvest the allantoic fluid from the infected embryonated eggs: Wash the eggshells with 70% ethanol. Open the egg with a very gently crack in the upper part and remove the allantoic membrane using sterile forceps. Use a 10 ml pipette to collect as much fluid as possible (typically 6 - 10 ml). Centrifuge at 260 x g for 10 min at 4 &#176;C and transfer the cleared allantoic fluid to new tubes.</li>
</ol>
2. Tracking Vaccine-specific Immunity
<ol>
	<li>Tracing of peptide-bearing dendritic cells (DCs)
	<ol>
		<li>For mouse vaccination, anesthetize mice with 5% isoflurane in oxygen using a precision vaporizer. Use a 200 &#181;l pipette to administer 50 &#181;l of PBS containing 103 plaque-forming units (pfu) of the vaccine expressing the SIINFEKL peptide via droplets directly to the mouse nostrils.</li>
		<li>Three days post-vaccination, euthanize mice via isoflurane anesthesia followed by cervical dislocation.</li>
		<li>Lift the mouse skin at the midline just below the ribcage with forceps and cut a small incision through the skin with scissors. From the point of incision, make a 3 - 5 cm long cut through the skin towards both head and tail, then two 2 cm incisions from the midline laterally from each end. Peel back the skin to reveal the peritoneal and thoracic cavities. Carefully cut through the membranes surrounding the peritoneum to expose the thorax.</li>
		<li>To access the mediastinum cut the diafragma and the bottom of the cage rib. Find and remove 2 mediastinal lymph nodes (mLNs), slightly dorsal and laterally adjacent to the thymus, medially adjacent to the brachiocephalic artery, near the ventral side of the trachea.</li>
		<li>To harvest the mLNs use curved forceps. Place forceps underneath the lymph nodes and pull them up gently. Place lymph nodes in a 6-well plate containing 1 ml of DMEM and remove any connective or fat tissue surrounding the node.
		<ol>
			<li>Tease each sample thoroughly with two 26 G needles attached to 1 ml syringes. To tease the lymph node, hold it down with one needle while breaking open the lymph node with the other needle. When this is done correctly, concentrated cells bursting from the lymph node are easily observed in the media. Pass all tissue material through a 70 &#181;m nylon filter to obtain single cell suspensions.</li>
		</ol>
		</li>
		<li>Centrifuge single cell suspensions for 10 min at 260 x g in a refrigerated centrifuge. Resuspend the cell pellet in 2 ml of Red Blood Cell Lysing (RBCL) Buffer to lyse red blood cells. Allow lysis to proceed for 3 min at RT.</li>
		<li>Neutralize RBCL buffer via centrifugation and cell resuspension in 2 ml of ice-cold PBS.</li>
		<li>Plate cells in U-shaped 96-well plates at a concentration of 107 cells/ml in a final volume of 100 &#181;l.</li>
		<li>For flow cytometry analysis, block cell Fc-receptors using 1 &#181;g of anti-mouse CD16/CD32 antibodies per 106 cells for 15 min on ice.</li>
		<li>Centrifuge plates and discard supernatants by plate flicking. Incubate individual wells with the surface antibody combination of choice. Typically for detection of dendritic cells in lymph nodes, the antibody cocktail should include: 0.25 &#181;g of anti-CD11c, 0.25 &#181;g of CD11b, 0.5 &#181;g of MHC class II, 0.25 &#181;g of CD103 and 0.15 &#181;g of CD8&#945; in a final volume of 100 &#181;l.</li>
		<li>For negative gating, add 0.25 &#181;g of anti-CD3, CD19, NK1.1 or a lineage cocktail (Figure 2A). Alternative gating strategies as well as protocols for proper compensation of multiple fluorescent dyes can be found at the website of the Immunological Genome Project. In addition to the surface markers of choice, add 1 &#181;g of an antibody against H-2Kb bound to SIINFEKL to the cocktail. This commercially available antibody will allow visualization of DCs in which the SIINFEKL peptide is bound to surface MHC class I.</li>
		<li>Incubate cells with surface antibodies for 30 min on ice. Protect plate from light. Centrifuge plates at 260 x g for 5 min and wash with 2 ml of PBS two times. Transfer cells to flow cytometry tubes for analysis.</li>
	</ol>
	</li>
	<li>Assessment of vaccine-specific T cell proliferation
	<ol>
		<li>Obtain donor T cells from commercially available TCR-transgenic mice (OT-I mice) (C57BL/6-Tg(Tcr aTcrb)1100Mjb/J) in which all the generated CD8 T cells are specific for the SIINFEKL peptide. Euthanize mice by isoflurane anesthesia followed by cervical dislocation. Harvest spleen by performing an incision in the abdominal cavity. Place spleen/s in 2 ml of DMEM/spleen and remove connective and fat tissue.</li>
		<li>Generate single cell suspensions from spleen following the procedure described in 2.1.2 and 2.1.3.</li>
		<li>To further enrich the single cell suspension on T cells, apply magnetic bead separation protocols using either positive or negative selection procedures12. To differentiate donor from recipient cells, use congenic mice so that for example recipient mice express the leukocyte marker CD45.1 while donor T cells express CD45.212.</li>
		<li>To label T cells, incubate them in 1 ml of PBS containing 5 &#181;M of carboxyfluorescein succinimidyl ester (CFSE) in an optimal cell density of 5 x 106 cells/ml. Incubate cells for 20 min at 37 &#176;C protected from light exposure. After incubation, centrifuge cells at 260 x g for 5 min, discard supernatant, and resuspend cells in 3 ml fetal bovine serum (FBS) to neutralize CFSE.</li>
		<li>Centrifuge cells at 260 x g during 10 min and resuspend in the adequate volume of PBS so that 100 &#181;l of solution contains 2 x 106 cells. Infuse recipient congenic mice with 2 x 106 cells/mouse via retro-orbital sinus injection13.</li>
		<li>Euthanize recipient mice at days 3, 4 and 5 post-vaccination using isoflurane anesthesia followed by cervical dislocation. Harvest mediastinal lymph nodes and prepare single cell suspensions for flow cytometry as indicated in 2.1.2-2.1.5.</li>
		<li>Identify donor cells by staining the single cell suspensions in 100 &#181;l of an antibody cocktail containing: 0.25 &#181;g of anti-mouse CD3, CD4 and CD8 antibodies; 0.5 &#181;g of anti-mouse CD45.1 or CD45.2 antibody (depending on the phenotype of donor cells). Leave the FL-1 channel of the flow cytometer open (FITC) to determine the dilution profile of CFSE12 (Figure 2B).</li>
	</ol>
	</li>
</ol>
3. Chimeric Mice to Dissect Gene-specific Vaccine Responses
NOTE: The generation of competitive bone marrow chimeras using DTR-based mouse models allows the discrimination of cell-specific functions during immune response to vaccines. Here we show an additional strategy by which combination of DTR-expressing donor cells with cells from knockout mice permits to study gene specific functions during immunity in specific cell compartments. In the example we generate mice with specific deletion of toll-like receptor 3 (TLR3) in Langerin+ CD103+ DCs.
<ol>
	<li>Preparation of bone marrow donor cells
	<ol>
		<li>Work under a biosafety level II cabinet. Avoid contamination by using exclusively autoclaved instruments.</li>
		<li>To generate bone marrow chimeric mice, use congenic CD45.1+ C57BL/6 female mice as recipients, and use TLR3-/-, Langerin-DTR/EGFP, or wt mice expressing CD45.2+ as donors. Using congenic donor and recipient mice allows the differentiation of donor and recipient cells via flow cytometry and this allows engraftment to be evaluated.</li>
		<li>To obtain donor bone marrow cells, euthanize donor mice with isoflurane anesthesia followed by cervical dislocation. With sharp scissors perform incisions around the ankles so that the mouse musculature is exposed.</li>
		<li>Using blunt forceps pull mouse skin and fur gently from the ankle towards the hips so that leg muscles are visible.</li>
		<li>With sharp scissors cut mouse legs at the height of the hips and above the head of the femur.</li>
		<li>Place legs on a petri dish and on ice. Work fast and remove all muscles to expose the bones.</li>
		<li>Separate femurs and tibiae. Place bones in a solution of 70% ethanol for 5 min to remove possible bacteria and place them immediately on another petri dish containing 5 ml of serum-free DMEM. Cut bone ends using sharp scissors and flush the bone marrow into the DMEM. To do so, flush 1 ml of DMEM into the bone by inserting a 26 G needle attached to a 5 ml syringe. Make sure the bones look white (empty) after flushing.</li>
		<li>Collect all bone marrow cells from donor mice in 10 ml of DMEM. Generate single cell suspensions from bone marrow cells by passing the harvest through a 70 &#181;m cell strainer.</li>
		<li>Clear single cell suspensions of bone marrow cells by centrifugation at 260 x g for 10 min at 4 &#176;C in a refrigerated centrifuge. Resuspend cell pellet in 10 ml of PBS. Deplete red blood cells using RBCL solution as described in 2.1.3 and 2.1.4.</li>
		<li>Count bone marrow cells using either a hemocytometer or an automated cell counter. Assess cell viability by determination of dead cells via trypan blue staining13. Optimally, one single animal yields around 3 x 107 white blood cells/mouse with a viability of at least 80%.</li>
		<li>In the case of total bone marrow chimeras, prepare TLR-3-/- or wt donor cells. The optimal cell concentration is 2 x 107 cells/ml since 2 x 106 cells in 100 &#181;l of PBS will be used for transplantation. If not used immediately, preserve donor cells at -80 &#176;C using slow freezing techniques and FBS with 10% DMSO. For the mixed chimeric model, mix Langerin-DTR-donor cells and TLR3-/- donor cells in a 75:25 ratio. Prepare a mix of 75 : 25 Langerin-DTR : wt bone marrow cells to generate control mice.</li>
	</ol>
	</li>
	<li>Mouse irradiation and transplantation
	<ol>
		<li>Irradiate female mice between 6 - 8 weeks of age in a Cesium-137 source irradiator. Give mice two doses of 550 rad 4 hr&#160;apart. Split irradiation minimizes acute hepatic toxicity and therefore reduces mouse lethality due to the irradiation protocol.</li>
		<li>After irradiation, keep mice under treatment with antibiotics for two weeks. Give antibiotics with the drinking water at 2.5 - 5 mg of Baytril/kg.</li>
		<li>2 - 4 hr&#160;after the second irradiation, transplant mice with donor bone marrow cells intravenously. Perform the injection of donor cells via retro-orbital sinus injection13 and under isoflurane anesthesia using maximum volume of 100 &#181;l of PBS containing 2 x 106 cells.</li>
		<li>Four weeks after transplantation, evaluate engraftment in a sample of 100 &#181;l of peripheral blood via flow cytometry using a machine able to read at least two colors. To distinguish between donor and radio-resistant recipient cells, use commercially available fluorescently labeled CD45.1 and CD45.2 anti-mouse antibodies using dilutions between 1/100 and 1/200. Optimal engraftment of donor cells should be at least 80% of total hematopoietic cells in peripheral blood.</li>
	</ol>
	</li>
	<li>Vaccination and targeted depletion
	<ol>
		<li>Vaccinate mice with recombinant live-attenuated influenza vaccine via intranasal. For mouse vaccination, anesthetize mice with 5% isoflurane in oxygen using a precision vaporizer. Use a 200 &#181;l pipette to administer 50 &#181;l of PBS containing 103 plaque-forming units (pfu) of the vaccine.</li>
		<li>To generate the final mouse model, treat mice with 50 ng of DT diluted in 50 &#181;l of PBS intranasally. Administer DT drop by drop directly to the nostrils of anesthetized mice as described in 3.3.1. Treat mice with daily doses starting treatment at day -2 before vaccination until day 2 post-vaccination (Figure 3B).</li>
	</ol>
	</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+the+control+of+immunity.">Dendritic cells and the control of immunity.</a> Nature. 392, 245-252 (1998).</li><li>Mart&#237;nez-Sobrido, L., Garc&#237;a-Sastre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+recombinant+influenza+virus+from+plasmid+DNA.">Generation of recombinant influenza virus from plasmid DNA.</a> J Vis Exp. 3, (2010).</li><li>Haniffa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Tissues+Contain+CD141hi+Cross-Presenting+Dendritic+Cells+with+Functional+Homology+to+Mouse+CD103++Nonlymphoid+Dendritic+Cells.">Human Tissues Contain CD141hi Cross-Presenting Dendritic Cells with Functional Homology to Mouse CD103+ Nonlymphoid Dendritic Cells.</a> Immunity. 37, 60-73 (2012).</li><li>Haniffa, M., Collin, M., Ginhoux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+and+functional+specialization+of+dendritic+cells+in+human+and+mouse.">Ontogeny and functional specialization of dendritic cells in human and mouse.</a> Adv. Immunol. 120, 1-49 (2013).</li><li>Jung, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+depletion+of+CD11c++dendritic+cells+abrogates+priming+of+CD8++T+cells+by+exogenous+cell-associated+antigens.">In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens.</a> Immunity. 17, 211-220 (2011).</li><li>Lahl, K., Sparwasser, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+depletion+of+FoxP3++Tregs+using+the+DEREG+mouse+model.">In vivo depletion of FoxP3+ Tregs using the DEREG mouse model.</a> Methods Mol. Biol. 17, 211-220 (2011).</li><li>Duffield, J. 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The authors have nothing to disclose.

In this study we describe how reverse genetics and chimeric mouse models can be utilized to elucidate the physiologic and molecular mechanisms of vaccine-induced immunity. Influenza reverse genetics is established in many laboratories and has played a chief role in understanding influenza pathogenesis, replication, and transmission17. A key point in our protocol is rescue of cold-adapted influenza vaccines expressing foreign epitopes. While the strategy of introducing short cDNAs into the stalk of the neuramin...

The generation of immunological memory in the absence of disease is the physiological basis of efficient vaccination1. Recently, systems biology-based approaches have revealed that successful vaccines such as the yellow fever vaccine, induce a strong induction of innate immune responses and activation of several subsets of dendritic cells (DCs), which in turn, lead to multilineage activation of antigen-specific T cells2,3. Since DCs are the only immune cell population with the ability to activate antigen-specific na&#239;ve T cells4, the study of their function during vaccination is critical to understand immune responses to vaccines and to design future strategies against challenging pathogens.
A system allowing tracing of different DCs subsets during immune responses to vaccines would be desirable in order to establish an accurate kinetics of DC migration to lymphoid tissues, and therefore to provide insight into the physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. Reverse genetics-based approaches offer the possibility to generate modified, live-attenuated vaccines that can be used experimentally with this purpose. Since its implementation on influenza research, plasmid-based reverse genetics has been widely employed to generate recombinant influenza strains including LAIVs. Standard protocols to rescue recombinant influenza viruses require multi-transfection of highly transfectable cell lines with ambisense plasmids (producing both positive and negative sense RNA) containing the eight influenza viral segments as well as amplification in a permissive system such as Madin-Darby canine kidney (MDCK) cells and/or chicken embryonated eggs5. However, the application of reverse genetics to generate molecular tools in order to study the immune mechanisms of vaccination remains unexplored.
The generation of new mouse models allowing specific depletion of immune cell subsets, including DCs, has opened new possibilities to understand the basic immune mechanisms underlying vaccine-elicited protection. The comparison between DC subset functions in mice and humans has revealed that, to a great extent, mouse and human DCs are functionally homologous6,7, these findings, strongly suggest that the development of mouse models allowing specific depletion of DCs in the steady state and during inflammatory conditions, may serve to understand the physiology of DC responses in humans. In recent years a number of mouse models have been generated carrying transgenes expressing the simian diphtheria toxin (DT) receptor (DTR) under the control of the promoter region of a gene of interest8,9. Since mouse tissues do not naturally express DTR, these models allow conditional depletion of cell subsets carrying the targeted gene of interest upon mouse inoculation with DT. Thus, our ability to deplete specific DCs and other leukocytes in vivo during physiological processes, has been greatly enhanced by the development of DTR-based ro. However, while these transgenic mouse models have been used extensively to understand the ontogeny of the immune system, their application to vaccine development has been scarcely tested. Here, by combining influenza reverse genetics and DTR-based competitive bone marrow chimeras, we propose a method to study the kinetics of vaccine immunity as well as individual gene function during immune responses to vaccines in vivo. The application of this technology for preclinical evaluation of new vaccines against challenging infectious diseases could help to rationalize vaccine design and to test vaccine candidates in vivo.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#180;s Modified Eagle Medium (DMEM 1X)</td>
			<td>Gibco RL-Life Technologies</td>
			<td>41965-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti MEM</td>
			<td>Gibco RL-Life Technologies</td>
			<td>31985-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen-Life Technologies</td>
			<td>11668-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10.000 U/ml)</td>
			<td>PAA</td>
			<td>p11-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Embryonated eggs</td>
			<td>Valo biomedia Gmbh</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (1X)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;M Nylon Filters</td>
			<td>Greiner-Biorad</td>
			<td>542-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysing buffer (RBCL) 10X</td>
			<td>BD Bioscience</td>
			<td>555899</td>
			<td></td>
		</tr>
		<tr>
			<td>CD16/CD32 Mouse BD Fc Block (2.4G2)</td>
			<td>BD Pharmigen</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-Anti-mouse SIINFEKL-H2kb (25 D1.16)</td>
			<td>Biolegend</td>
			<td>141605</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-Anti-mouse CD11c (HLA3)</td>
			<td>BD Biosciences</td>
			<td>553802</td>
			<td></td>
		</tr>
		<tr>
			<td>eFluor 450-Anti-mouse MHCII (Md/114.15.2)</td>
			<td>eBioscience</td>
			<td>48-5321-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Pe-Cy7-Anti-mouse CD11b (M1/70)</td>
			<td>Biolegend</td>
			<td>101216</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCp/Cy5.5-Anti-mouse CD103 (2E7)</td>
			<td>Biolegend</td>
			<td>121416</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-Anti-mouse CD45.1 (A20)</td>
			<td>eBioscience</td>
			<td>12-0453-82</td>
			<td></td>
		</tr>
		<tr>
			<td>V500-Anti-mouse CD45.2 (1O4)</td>
			<td>BD Bioscience</td>
			<td>562130</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCp-eFluor710 -Anti-mouse CD8a (53-6.7)</td>
			<td>eBioscience</td>
			<td>46-0081-80</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-Cy7-Anti-mouse CD3&#949; (145-2611)</td>
			<td>Biolegend</td>
			<td>100325</td>
			<td></td>
		</tr>
		<tr>
			<td>eFluor450-Anti-mouse CD4 (GK 1.5)</td>
			<td>eBioscience</td>
			<td>48-0041-80</td>
			<td></td>
		</tr>
		<tr>
			<td>CFSE Proliferation dye</td>
			<td>eBioscience</td>
			<td>65-0850-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Baytril 2.5%</td>
			<td>Bayer</td>
			<td>65-0850-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Dymethil-Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovalbumin&#160;</td>
			<td>Molecular probes&#160;</td>
			<td>O23020</td>
			<td></td>
		</tr>
		<tr>
			<td>Diphteria Toxin (DT)</td>
			<td>Sigma-Aldrich</td>
			<td>D0564</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-TPCK</td>
			<td>Sigma-Aldrich</td>
			<td>T1426</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACsCanto II Flow cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo cell analysis software 9.5</td>
			<td>Flowjo inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)&#160;</td>
			<td>Life technologies</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Automatic Cell Counter</td>
			<td>Invitrogen-Life Technologies</td>
			<td>C10227</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Generation of recombinant live-attenuated influenza vaccines can be achieved by transfection of plasmids encoding the eight segments of influenza virus under the control of bidirectional promoters5. A cold-adapted influenza vaccine usually contains six segments of a cold-adapted strain as well as the HA and NA of the influenza strain of choice (e.g., H1N1) (Figure 1A). The principle of cold-adaptation is based on virus-restricted replication at 33 &#176;C, the temperature of the upper...

Watch this Scientific Journal Video about Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity at JoVE.com

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

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.

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 ...

intranasal-administration-recombinant-influenza-vaccines-chimeric

Research

JoVE Journal

Immunology and Infection

12.4K Views.  Heinrich Pette Institute, Leibniz Institute for Experimental Virology. 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.

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

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

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.

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

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-&alpha; knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...