This study was supported by the National Institutes of Health (NIH) of the United States (RO1 AI68002).

1. Recombinant SARS-CoV RBD Protein Preparation
<ol>
<li>Prepare calcium phosphate transfection reagent
<ol>
<li>2X HBS buffer preparation: Mix together 16 g of NaCl, 0.4 g of Na2HPO4*7H2O, and 13.0 g of HEPES. Adjust pH to 7.00 and bring up total volume to 1000 mL in distilled water. After filtering the solution for sterilization, aliquot and store it at -20&deg;C. 
Hint: Any variation of the pH value would affect the transfection results. Thus, it is advisable to test several pH values around 7.00 (for example, 6.99, 7.00, or 7.01) and find the best one for the transfection using the method introduced below.</li>
<li>2.5 M CaCl2 preparation: Add 73.5 g of CaCl2*2H2O to distilled water in a final volume of 200 mL. Filter the solution and store at -20&deg;C.</li>
</ol>
</li>
<li>Recombinant plasmid transfection and protein purification
<ol>
<li>Split 293T cells at 50-70% confluency 24 h before transfection. Grow cells in T-175 cm2 tissue culture flasks in 40 mL DMEM containing 10% heat-inactivated (HI) FBS and 1% Penicillin/Streptomycin (P/S) at 37&deg;C in 5% CO2.</li>
<li>All transfection reagents should be brought up to room temperature before transfection. These reagents include 2X HBS, 2.5M CaCl2, DiH2O, and rRBD plasmid 6. 
Hint: The final recombinant plasmid construct used for protein expression should contain a signal peptide to ensure secretion of expressed recombinant proteins to the culture supernatant. A 6x His tag may be added to the C-terminal of the expressed protein for easy purification.</li>
<li>Prepare one 50 mL (A) and one 15 mL (B) BD Falcon tube, and add reagents to the tubes as indicated in Table 1. Add 2X HBS buffer to tube A. In tube B, add 2.5M CaCl2 and rRBD plasmid, and bring the volume to the requirement in DiH2O. 
 <table border="1">
<tbody>
 <tr>
 <td colspan="4">A. One T-175 cm2 tissue culture flask (4000 &mu;L/flask): prepare for rRBD expression</td>
 </tr>
 <tr>
 <td colspan="2">Tube A </td>
 <td colspan="2">Tube B</td>
 </tr>
 <tr>
 <td>2X HBS</td>
 <td>2000 &mu;L</td>
 <td>2.5M CaCl2</td>
 <td>200 &mu;L</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>rRBD plasmid DNA</td>
 <td>40 &mu;g</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>DiH2O to</td>
 <td>2000 &mu;L</td>
 </tr>
 <tr>
 <td colspan="4">B. One 100-mm petri dish (1000 &mu;L/dish): prepare for SARS pseudovirus production</td>
 </tr>
 <tr>
 <td colspan="2">Tube A </td>
 <td colspan="2">Tube B</td>
 </tr>
 <tr>
 <td>2X HBS</td>
 <td>500 &mu;L</td>
 <td>2.5M CaCl2</td>
 <td>50 &mu;L</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>SARS-CoV S plasmid DNA</td>
 <td>5 &mu;g</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>HIV-1 plasmid (pNL4-3.luc.RE)</td>
 <td>5 &mu;g</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>DiH2O to</td>
 <td>500 &mu;L</td>
 </tr>
 </tbody>
</table>
Table 1. Transfection mixture preparation and volumes 
The volumes listed in Table 1 are for one transfection unit. If more flasks or dishes are used for the transfection, adjust volumes accordingly.</li>
<li>Add the DNA-calcium solution in tube B into tube A in a dropwise manner, while maintaining constant and gentle mixing in a vortex. Let the mixture sit at room temperature for 20-30 min. The key for successful calcium phosphate transfection depends on the size and shape of the precipitate formed. Thus, the mixture should be vortexed constantly and slowly to meet this requirement and, as a result, improve transfection efficacy.</li>
<li>Add mixture in a dropwise and even manner into 293T cells (4000 &mu;L/flask). Culture cells in incubator at 37&deg;C in 5% CO2.</li>
<li>Replace the culture medium with fresh serum-free OPTI-MEM I Reduced-Serum Medium (50 mL/flask) 8-10 h post-transfection. Continue to culture cells for two more days in the same condition.</li>
<li>Collect supernatant containing expressed rRBD protein 72 h post-transfection. Centrifuge at 6000 rpm for 15 min to remove cell debris. Add protease inhibitor cocktail to the collected supernatant and store at 4&deg;C overnight.</li>
<li>On the next day, purify rRBD recombinant protein from the culture supernatant using Ni-NTA Superflow following manufacturer's instructions.</li>
<li>Concentrate purified protein using Amicon Ultra -15 concentration tubes. After protein concentration, add PBS to the concentration tubes and centrifuge again to remove imidazole in elution buffer. Calculate protein concentration and store purified protein at -80&deg;C until use.</li>
</ol>
</li>
</ol>
2. Mouse Immunization and Sample Collection
<ol>
<li>Pre-warm Sigma adjuvant system (SAS) to 40-45&deg;C according to manufacturer's instructions. Add 1 mL PBS per vial and mix thoroughly.</li>
<li>Prepare protein-adjuvant emulsion according to the protocol in Table 2. Pipette calculated rRBD protein into a 1.5 mL tube. Add equal volume of SAS adjuvant to the tube and vortex vigorously for 2-3 min to form emulsion. 
Hint: The volumes listed in Table 2 are for one mouse. Adjust volumes according to the actual mouse numbers used. For each group, mix together the proteins and adjuvant required for each vaccine. Always prepare one extra sample for the vaccination to ensure accuracy. 
<table border="1">
<tbody>
 <tr>
 <td>Group</td>
 <td>1st vaccine 
 (200 &mu;L/mouse)</td>
 <td>2nd vaccine 
 (200 &mu;L/mouse)</td>
 <td>3rd vaccine 
 (200 &mu;L/mouse)</td>
 </tr>
 <tr>
 <td>rRBD protein</td>
 <td>20 &mu;g protein in PBS 
 (100 &mu;L) + 100 &mu;L SAS</td>
 <td>10 &mu;g protein in PBS (100 &mu;L) + 100 &mu;L SAS</td>
 <td>10 &mu;g protein in PBS (100 &mu;L) + 100 &mu;L SAS</td>
 </tr>
 <tr>
 <td>PBS control</td>
 <td>100 &mu;L PBS + 
 100 &mu;L SAS</td>
 <td>100 &mu;L PBS + 
 100 &mu;L SAS</td>
 <td>100 &mu;L PBS + 
 100 &mu;L SAS</td>
 </tr>
</tbody>
</table>
Table 2. Mouse immunization protocol</li>
<li>Subcutaneously prime-immunize female BALB/c mice (4-6 weeks old, 5 mice/group), and boost twice with rRBD and SAS as indicated in Table 2. Use PBS group as the control. The site for subcutaneous injections usually chosen is the loose skin between the shoulder blades. Alternatively, the ventral abdomen is commonly used, because it is easier to inject, and may observe any leakage from the injection sites. When repeated doses of vaccines are used, it is easy to select various injection sites, preventing potential local skin reactions.</li>
<li>Bleed mice via retro-orbital with anesthetic before immunization and 10 days after each vaccination, and heat sera at 56&deg;C for 30 min to inactivate complement. Store mouse sera at -20&deg;C until use.</li>
</ol>
3. Neutralization Detection using Pseudovirus Neutralization Assay
<ol>
<li>Prepare SARS pseudovirus
<ol>
<li>Split 293T cells in 100 mm tissue culture petri dishes (2x106 cells/dish) 16 h before transfection, and grow cells as above.</li>
<li>Prepare transfection reagents as indicated in Table 1. Co-transfect a plasmid encoding SARS-CoV S protein and a plasmid encoding Env-defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.RE) using calcium phosphate transfection reagent.</li>
<li>Replace medium with 10 mL fresh DMEM containing 10% FBS and 1% P/S 8-10 h post-transfection. Collect supernatant containing SARS pseudovirus 72 h post-transfection.</li>
<li>Filter pseudovirus through a 0.45 &mu;m filter. Aliquot and store at -80&deg;C until use.</li>
</ol>
</li>
<li>SARS pseudovirus neutralization assay
<ol>
<li>Split 293T cells expressing SARS-CoV receptor ACE2 (ACE2/293T) at 104 cells/100 &mu;L/well in 96-well tissue culture plates 16 h before infection.</li>
<li>Dilute SARS pseudovirus by 2-fold to detect the virus titer in ACE2/293T cells.</li>
<li>Serially dilute mouse sera in 96-well tissue culture plates, and add equal volume of titrated SARS pseudovirus. Preincubate the plates at 37&deg;C for 1 h. </li>
<li>After incubation, add 100 &mu;L of the sera-pseudovirus mixture to ACE2/293T cells, and continue to grow cells at 37&deg;C in 5% CO2. Add fresh DMEM 24 h later.</li>
<li>Completely remove culture supernatants from the plates 72 h post-infection. Add 1X luciferase cell culture lysis reagent (60 &mu;L/well), and promote cell lysis with constant shaking of plates for 1-2 h at room temperature.</li>
<li>Transfer cell lysates (50 &mu;L/well) into luminometer plates (Microfluor 96-well plates). Add luciferase substrate (50 &mu;L/well) included in luciferase assay system, and detect relative luciferase activity in Ultra 384 luminometer.</li>
<li>Calculate SARS pseudovirus neutralization titer and present as 50% neutralizing antibody titer (NT50) 6.</li>
</ol>
</li>
</ol>

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Tech. 9, 387-403 (1990).</li><li>Qi, Z., Zhou, L., Zhang, Q., Ren, L., Dai, R., Wu, B., Wang, T., Zhu, Z., Yang, Y., Cui, B., Wang, Z., Wang, H., Qiu, Y., Guo, Z., Yang, R., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+mouse,+guinea+pig+and+rabbit+models+for+evaluation+of+plague+subunit+vaccine+F1+rV270.">Comparison of mouse, guinea pig and rabbit models for evaluation of plague subunit vaccine F1+rV270.</a> Vaccine. 28, 1655-1660 (2010).</li><li>Rosser, M. P., Xia, W., Hartsell, S., McCaman, M., Zhu, Y., Wang, S., Harvey, S., Bringmann, P., Cobb, R. 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C., Sun, S., Chen, M., Liu, Z., Guo, H., He, Y., Zhou, Y., Zheng, B. J., Jiang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+receptor-binding+domain+of+SARS-CoV+spike+protein+expressed+in+mammalian,+insect+and+E.+coli+cells+elicits+potent+neutralizing+antibody+and+protective+immunity.">Recombinant receptor-binding domain of SARS-CoV spike protein expressed in mammalian, insect and E. coli cells elicits potent neutralizing antibody and protective immunity.</a> Virology. 393, 144-150 (2009).</li><li>Ambrosino, D. 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Protoc. 1, 695-700 (2006).</li><li>Jiang, M., Deng, L., Chen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Ca(2+)-phosphate+transfection+efficiency+enables+single+neuron+gene+analysis.">High Ca(2+)-phosphate transfection efficiency enables single neuron gene analysis.</a> Gene Ther. 11, 1303-1311 (2004).</li><li>Han, D. P., Kim, H. G., Kim, Y. B., Poon, L. L., Cho, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+safe+neutralization+assay+for+SARS-CoV+and+characterization+of+S-glycoprotein.">Development of a safe neutralization assay for SARS-CoV and characterization of S-glycoprotein.</a> Virology. 326, 140-149 (2004).</li><li>Toledo, J. 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No conflicts of interest declared.

Cell density is an important factor affecting the efficacy of calcium phosphate-based transfection. In our experience, less than 70% confluency of the cells brings the best results. Thus, in order to improve transfection efficiency, it is recommended that cell density be kept at around 50-70% of confluency. The calcium phosphate transfection method is generally thought to be less efficient compared with other transfection methods, such as lipid transfection 16. However, in this protocol, we used a modified calcium phosphate transfection method whereby constant and slow mixing of the transfection solution in a vortex ensures the formation of a precipitate with the appropriate size and shape, thus improving transfection efficiency.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
<tr>
<td>Na2HPO4*7H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S2429</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2*2H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C5080</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12430</td>
<td>&nbsp;</td>
<tr>
<td>HI FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10438</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>OPTI-MEM I Medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31985</td>
<td>&nbsp;</td>
<tr>
<td>Protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11836170001</td>
<td>&nbsp;</td>
<tr>
<td>Ni-NTA Superflow</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>30450</td>
<td>&nbsp;</td>
<tr>
<td>Sigma adjuvant system</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S6322</td>
<td>&nbsp;</td>
<tr>
<td>Luciferase assay system</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>E1501</td>
<td>&nbsp;</td>
<tr>
<td>Luciferase cell culture lysis reagent (5X)</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>E1531</td>
<td>&nbsp;</td>
<tr>
<td>Amicon Ultra - 15</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC901024</td>
<td>&nbsp;</td>
<tr>
<td>Microfluor 96-well plates</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>7905</td>
<td>&nbsp;</td>
<tr>
<td>Ultra 384 luminometer</td>
<td>&nbsp;</td>
<td>Tecan Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

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

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 ...

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

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

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

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.&#160;intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

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

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

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

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

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

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

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

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 ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

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.

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

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

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.

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