This study was supported by the National Institute of Health grant 1K22AI097291-01 and by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR000433. We also acknowledge Prof. Darrell Irvine at MIT and Prof. Matthias Stephan at Fred Hutchinson Cancer Center for their contribution on the initial work on the vaccine nanoparticles and OT-I/Luc transgenic mice.

All experiments described in this protocol were approved by the University Committee on Use and Care of Animals (UCUCA) at University of Michigan and performed according to the established policies and guidelines.
1. Synthesis and Characterization of ICMVs Co-loaded with Protein Antigen and Adjuvant Molecules
<ol>
	<li>Mix 1:1 molar ratio of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidop...

The protocol provided in this article describes the synthesis and characterization of a new lipid-based nanoparticle system, termed ICMVs, and provides the process of validating effectiveness of nanoparticle-based vaccine formulations to induce antigen-specific CD8+ T cell responses. ICMV synthesis is completed in all aqueous condition, which is a major advantage compared with other commonly used polymeric nanoparticle systems (e.g., poly(lactide-co-glycolide) acid particles), which typically require organic sol...

Traditional vaccine development has mainly employed the empirical approach of trial-and-error. However, with the recent development of a wide array of biomaterials and discovery of molecular determinants of immune activation, it is now possible to rationally design vaccine formulations with biophysical and biochemical cues derived from pathogens1,2. In particular, various particulate drug delivery platforms have been examined as vaccine carriers as they can be co-loaded with subunit antigens and immunostimulatory agents, protect vaccine components from degradation, and enhance their co-delivery to antigen presenting cells (APCs) residing in lymph nodes (LNs), thus maximizing immune stimulation and activation3-5. In this report, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs), which have been previously demonstrated as a potent vaccine platform for elicitation of robust cytotoxic T lymphocyte (CTL) and humoral immune responses in both systemic and mucosal tissue compartments6-9. In particular, vaccination with ICMVs achieved substantially enhanced serum IgG levels against a malaria antigen, compared with vaccination with conventional adjuvants (e.g., alum and Montanide)7 and also elicited potent CTL responses against tumor cells and viral challenge models in mice9. Here, using ICMVs as a model vaccine nanoparticle system, we describe methods for characterization of vaccine nano-formulations, including particle size and zeta potential measurements and tracking of particle trafficking to draining LNs (dLNs) utilizing confocal imaging of cryosectioned tissues7. In addition, we present a whole-animal imaging-based method of analyzing expansion of CTL responses in mice after adoptive transfer of luciferase-expressing antigen-specific CD8+ T cells9,10. Finally, we describe tetramer staining of peripheral blood mononuclear cells (PBMCs) for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles6,9.
ICMVs are a lipid-based nanoparticle formulation synthesized by controlled fusion of simple liposomes into multilamellar structures, which are then chemically stabilized by cross-linking maleimide-functionalized phospholipid head groups within lipid layers with dithiol crosslinkers6. Once ICMVs are synthesized, a small fraction of nanoparticles can be used to determine particle size and zeta potential (i.e., surface charge of particles) with a dynamic light scattering (DLS) system and a zeta potential analyzer. DLS measures changes in light scattering in particle suspensions, allowing determination of the diffusion coefficient and the hydrodynamic size of particles11. Achieving consistent particle size from batch to batch synthesis is critical since particle size is one of the major factors influencing lymphatic draining of vaccine particles to dLNs and subsequent cellular uptake by APCs12,13. In addition, zeta potential can be obtained by measuring the particle velocity when an electric current is applied, which allows determination of the electrophoretic mobility of particles and particle surface charge11. Ensuring consistent zeta potential values of particles is important since surface charge of particles determines colloidal stability, which has direct impact on particle dispersion during storage and after in vivo administration14,15. In order to track the particle localization to dLNs, ICMVs can be labeled with desired fluorophores including lipophilic dyes and covalently-tagged antigens. Following immunization, mice can be euthanized at various time points, dLNs resected, cryosectioned, and analyzed with confocal microscopy. This technique allows visualization of lymphatic draining of both the nanoparticle vaccine carriers and antigen to dLNs. The tissue sections can additionally be stained with fluorescently labeled antibodies and utilized to obtain more information, such as types of cells associated with the antigen and formation of germinal centers as we have shown previously7.
Once the particle synthesis is optimized and trafficking to the dLNs is confirmed, it is important to validate elicitation of in vivo CTL expansion. In order to analyze elicitation of antigen-specific CD8+ T cells in response to nanoparticle vaccination, we have utilized a model antigen, ovalbumin (OVA), with OVA257-264 peptide (SIINFEKL) immunodominant CD8+ T cell epitope, which allows detailed immunological analyses of antigen-specific T cell responses for initial vaccine development16,17. In particular, to interrogate the dynamics of expansion and migration of antigen-specific CD8+ T cells, we have generated a double-transgenic mouse model by crossing firefly luciferase-expressing transgenic mice (Luc) with OT-I transgenic mice that possess CD8+ T cells with T-cell receptor (TCR) specific for SIINFEKL (in association with H-2Kb). From these OT-I/Luc mice, luciferase-expressing, OT-I CD8+ T cells can be isolated and prepared for adoptive transfer into na&#239;ve C57BL/6 mice. Once seeded, successful immunization with OVA-containing nanoparticles will result in expansion of the transferred T cells, which can be tracked by monitoring the bioluminescence signal with a whole animal imaging system9,10. This non-invasive whole-body imaging technique has been used with other viral or tumor antigens in the past18-20, revealing processes involved in T cell expansion in lymphoid tissues and dissemination to peripheral tissues in a longitudinal manner.
Complementary to analysis of adoptively transferred antigen-specific CD8+ T cells, endogenous T cell responses post vaccination can be examined with the peptide-major histocompatibility complex (MHC) tetramer assay21, in which a peptide-MHC tetramer complex, consisting of four fluorophore-tagged MHC-class I molecules loaded with peptide epitopes, is employed to bind TCR and label CD8+ T cells in an antigen-specific manner. The peptide-MHC tetramer assay can be performed either in terminal necropsy studies to identify antigen-specific CD8+ T cells in lymphoid and peripheral tissues or in longitudinal studies with peripheral blood mononuclear cells (PBMCs) obtained from serial blood draws. After staining lymphocytes with peptide-MHC tetramer, flow cytometry analysis is performed for detailed analyses on the phenotype of CTLs or quantification of their frequency among CD8+ T cells.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>1. Synthesis and characterization of ICMVs co-loaded with protein antigen and adjuvant molecules</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] (sodium salt) (MPB)</td>
			<td>Avanti Polar Lipids, INC.</td>
			<td>870012</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)</td>
			<td>Avanti Polar Lipids, INC.</td>
			<td>850375</td>
			<td></td>
		</tr>
		<tr>
			<td>Monophosphoryl Lipid A (Synthetic) (PHAD&#8482;) (MPLA)</td>
			<td>Avanti Polar Lipids, INC.</td>
			<td>699800</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL glass vials</td>
			<td>Wheaton</td>
			<td>0334125D</td>
			<td></td>
		</tr>
		<tr>
			<td>Symphny Vacuum Oven</td>
			<td>VWR</td>
			<td>414004-580</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovalbumin (OVA)</td>
			<td>Worthington</td>
			<td>3054</td>
			<td></td>
		</tr>
		<tr>
			<td>Bis-Tris Propane (BTP)</td>
			<td>Fisher</td>
			<td>BP2943</td>
			<td></td>
		</tr>
		<tr>
			<td>Q125 Sonicator (125W/20kHz)</td>
			<td>Qsonica</td>
			<td>Q125-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Fisher</td>
			<td>BP172</td>
			<td></td>
		</tr>
		<tr>
			<td>2 kDa Thiolated Polyethylene Glycol (PEG-SH)</td>
			<td>Laysan Bio</td>
			<td>MPEG-SH-2000-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>Malvern ZetaSizer Nano ZSP&#160;</td>
			<td>Malvern</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ZetaSizer Cuvettes</td>
			<td>Malvern</td>
			<td>DTS1070</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2. Examination of lymph node draining of fluorescence-tagged ICMVs with confocal microscopy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1,1&#39;-Dioctadecyl-3,3,3&#39;,3&#39;-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt (DID)</td>
			<td>Life Technologies</td>
			<td>D-7757</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 555-succinimidyl ester (AF555-NHS)</td>
			<td>Life Technologies</td>
			<td>A37571</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek OCT freezing medium&#160;</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Cryomolds</td>
			<td>VWR</td>
			<td>25608-922</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3. Monitoring expansion of antigen-specific, luciferase-expressing CD8+ T cells after nanoparticle vaccination with whole animal imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Jackson</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Albino C57BL/6 mice</td>
			<td>Jackson</td>
			<td>000058</td>
			<td></td>
		</tr>
		<tr>
			<td>OT-1 C57BL/6 mice</td>
			<td>Jackson</td>
			<td>003831</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m nylon strainer</td>
			<td>BD</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep&#8482; Mouse CD8+ T Cell Isolation Kit</td>
			<td>StemCell</td>
			<td>19853</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS&#174; whole animal imaging system</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4. Peptide-MHC tetramer staining of peripheral blood mononuclear cells (PBMCs) for flow cytometric analysis of antigen-specific CD8+ T cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>K2EDTA tubes</td>
			<td>BD</td>
			<td>365974</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK lysis buffer</td>
			<td>Life Technologies</td>
			<td>A10492-01&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD16/32 Fc Block</td>
			<td>Ebioscience</td>
			<td>14-0161-86</td>
			<td></td>
		</tr>
		<tr>
			<td>H-2Kb OVA Tetramer</td>
			<td>MBL</td>
			<td>TS-5001-1C</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD8-APC</td>
			<td>BD</td>
			<td>553031</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD44-FITC</td>
			<td>BD</td>
			<td>553133</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD62L-PECy7</td>
			<td>Ebioscience</td>
			<td>25-0621-82</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td>
			<td>SIGMA</td>
			<td>D8417-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>CyAn Flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo Software</td>
			<td>FlowJo</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

whole-animal imaging and flow cytometric techniques for analysis of antigen-specific cd8+ t cell responses after nanoparticle vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

The steps involved in the synthesis of ICMVs are illustrated in Figure 16. Briefly, a lipid film containing any lipophilic drugs or fluorescent dyes is hydrated in the presence of hydrophilic drugs. Divalent cations, such as Ca2+, are added to drive fusion of anionic liposomes into multilamellar vesicles. Dithiol crosslinker, such as DTT, is added to &#8220;staple&#8221; maleimide-functionalized lipids on apposing lipid layers, and finally remaining external maleimide groups are que...

Watch this Scientific Journal Video about Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination at JoVE.com

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

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Immunology and Infection

13.1K Views.  University of Michigan. We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.