Name: in vitro cellular activity evaluation of the nanoemulsion vaccine adjuvant ophiopogonin d
Uploaded: 2022-12-09T13:35:00
Description: Watch this Scientific Journal Video about In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D at JoVE.com

This study was supported by grant No. 2021YFC2302603 of the National Key Research and Development Program of China, grants No. 31670938, 32070924, 82041045, and 32000651 of the National Natural Science Foundation Program of China, grants No. 2014jcyjA0107 and No. 2019jcyjA-msxmx0159 of the Natural Science Foundation Project Program of Chongqing, grant No. CYS21519 of the Postgraduate Research and Innovation Project of Chongqing, grant No. 2020XBK24 of the Army Medical University Special projects, and grant No. 202090031021 of the National Innovation and Entrepreneurship Program for college students.

All cell experiments were performed in a cell engineering laboratory equipped with basic operating rooms, buffer rooms, sterile culture rooms, and identification and analysis rooms. The working environment and conditions were free from microbial contamination and other harmful factors. The animal experiments were conducted based on the Guidelines for the Care and Use of Laboratory Animals and were approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University.
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<ol>
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Subunit vaccines provide excellent safety but poor immunogenicity. The main strategy to enhance the immunogenicity is to physically adsorb or couple antigens with adjuvants and incorporate them into the drug delivery systems to promote the uptake and presentation by DCs. Natural plant saponins such as quillaia saponin and its derivatives are highly toxic and are not suitable for the development of human vaccines17. Therefore, the study of the toxic effects of vaccines or adjuvants on cells is a ne...

Vaccines are an important means of preventing and treating infectious and noncommunicable diseases. The appropriate addition of adjuvants and delivery vehicles to vaccine formulations is beneficial for enhancing the immunogenicity of antigens and generating long-lasting immune responses1. In addition to the classical adjuvant alum (aluminum salt), there are six kinds of adjuvants for vaccines that are currently marketed: MF592,3, AS043, AS033, AS013, CpG10184, and Matrix-M5. Generally, when the human body encounters a viral attack, the first and second lines of defense (skin, mucosa, and macrophages) take the lead in clearing the virus, and finally, the third line of defense, involving the immune organs and immune cells, is activated. Aluminum and aluminum salts have been the most widely used adjuvants for human vaccines since the early 1920s, eliciting an effective innate immune response6. However, it has been proposed that the activation of antigen-presenting cells (APCs) by classical adjuvants, which stimulates the immune cells to generate specific sets of cytokines and chemokines, is the mechanism by which adjuvants work and may be one of the reasons why adjuvants exert only transient effects on specific immune responses7. The presence of limited licensed adjuvants for human use is a restrictive factor for developing vaccines that elicit effective immune responses8.
Currently, an increasing number of adjuvant studies are demonstrating the ability to induce a strong cellular immune response in mice. QS-21 has been shown to induce a balanced T-helper 1 (Th1) and T-helper 2 (Th2) immune response, produce higher levels of antibody titers, and prolong the protection as an adjuvant, but its strong toxicity and hemolytic properties limit its development as a standalone clinical adjuvant9,10. OP-D (ruscogenin-O-&#945;-L-rhamnopyranosy1-(1&#8594;2)-&#946;-D-xylopyranosyl-(1&#8594;3)-&#946;-D-fucopyranoside) is one of the steroidal saponins isolated from the root of the Chinese medicinal plant Ophiopogon japonicas4. Additionally, it is the chief pharmacologically active component (Shen Mai San) found in Radix Ophiopogonis and is known to have certain pharmacological properties11. Moreover, it is a member of the Liliaceae family and is widely utilized for its inhibitory and protective effects in cellular inflammation and myocardial injury. For example, OP-D ameliorates DNCB-induced atopic dermatitis-like lesions and tumor necrosis factor alpha (TNF-&#945;) inflammatory HaCaT cells in BALB/c mice12. Importantly, OP-D promotes the antioxidative protection of the cardiovascular system and protects the heart against doxorubicin-induced autophagic injury by reducing both reactive oxygen species generation and disrupting mitochondrial membrane damage. Experiments have shown that taking OP-D with mono-desmoside helps to boost immune health, increase white blood cell counts and DNA synthesis, and make antibodies last longer13. It has previously been found that OP-D has an adjuvant effect14.
Nanoemulsions are oil-in-water nanoformulations composed of a combination of surfactants, oil, cosurfactants, and water12,15. These nanovaccine designs allow antigens and adjuvants to be encapsulated together to enhance immune stimulation, protect the antigens, and promote dendritic cell (DC) maturation16. For development of these novel adjuvants obtained from screening, it is important to find appropriate methods to evaluate their cellular response abilities.
The purpose of this protocol is to systematically evaluate whether adjuvants can enhance phagocytosis and the expression of immune cells in in vitro cell culture and to elaborate on the main experimental methods. The experiment is divided into four subsections: (1) the toxicity of OP-D and NOD to L929 cells is determined by the cell counting kit-8 (CCK-8) assay; (2) the cytokine levels of endocrine IFN-&#947; and IL-17A and the corresponding cell numbers in immunized mice are detected by splenocyte stimulation and ELISpot assays; (3) the antigen presentation ability of DCs after adjuvant stimulation is observed using confocal microscopy; and (4) the three kinds of cytokines, IL-1&#946;, IL-6, and TNF-&#945;, in the supernatants obtained from peritoneal macrophages (PMs) in normal mice cocultured with adjuvants are detected.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin-EDTA (1x)</td>
			<td>GIBCO, USA</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well filter plates</td>
			<td>Millipore. Billerica, MA</td>
			<td>CLS3922</td>
			<td></td>
		</tr>
		<tr>
			<td>AlPO4</td>
			<td>General Chemical Company, USA</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td>Countstar, China</td>
			<td>IC1000</td>
			<td></td>
		</tr>
		<tr>
			<td>BALB/c mice and C57BL/6 mice</td>
			<td>Beijing HFK Bioscience Co. Ltd</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>caprylic/capric triglyceride (GTCC)</td>
			<td>Beijing Fengli Pharmaceutical Co. Ltd., Beijing, China</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>CCK-8 kits</td>
			<td>Dojindo, Japan</td>
			<td>CK04</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counting Plate</td>
			<td>Costar, Corning, USA</td>
			<td>CO010101</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Sieve</td>
			<td>biosharp, China</td>
			<td>BS-70-CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810 R</td>
			<td>Eppendorf, Germany&#160;</td>
			<td>5811000398</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM basic(1x) medium</td>
			<td>GIBCO, USA</td>
			<td>C11885500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>DSZ5000X Inverted Microscope</td>
			<td>Nikon,Japan</td>
			<td>DSZ5000X</td>
			<td></td>
		</tr>
		<tr>
			<td>EL-35 (Cremophor-35)</td>
			<td>Mumbai, India</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISpot classic</td>
			<td>AID, Germany</td>
			<td>ELR06</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>GIBCO, USA</td>
			<td>10099141C</td>
			<td></td>
		</tr>
		<tr>
			<td>Full-function Microplate Reader</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>VL0000D2</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>P42212</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Invitrogen, USA</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Granulocyte Macrophage Colony-Stimulating Factor</td>
			<td>GM-CSF, R&#38;D Systems, USA</td>
			<td>315-03</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Invitrogen, USA</td>
			<td>15630106</td>
			<td></td>
		</tr>
		<tr>
			<td>HF 90/240 Incubator</td>
			<td>Heal Force, Switzerland</td>
			<td>null</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-4</td>
			<td>PeproTech, USA</td>
			<td>042149</td>
			<td></td>
		</tr>
		<tr>
			<td>L929 cell line</td>
			<td>FENGHUISHENGWU, China</td>
			<td>&#160;NCTC clone 929 (RRID:CVCL_0462)</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal Microscopy</td>
			<td>Zeiss, Germany</td>
			<td>LSM 980</td>
			<td></td>
		</tr>
		<tr>
			<td>MONTANE 85 PPI</td>
			<td>SEPPIC, France</td>
			<td>L12910</td>
			<td></td>
		</tr>
		<tr>
			<td>MONTANOX 80 PPI</td>
			<td>SEPPIC, France</td>
			<td>36372K</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IFN-&#947; ELISA kit</td>
			<td>Dakewe, China</td>
			<td>1210002</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IFN-&#947; precoated ELISPOT kit</td>
			<td>Dakewe, China</td>
			<td>DKW22-2000-096</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-17A ELISA kit</td>
			<td>Dakewe, China</td>
			<td>1211702</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-17A ELISpotPLUS Kit</td>
			<td>ebiosciences, USA</td>
			<td>3521-4HPW-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-1&#946; ELISA kit</td>
			<td>Dakewe, China</td>
			<td>1210122</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-6 ELISA kit</td>
			<td>Dakewe, China</td>
			<td>1210602</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse TNF-&#945; ELISA kit</td>
			<td>Dakewe, China</td>
			<td>1217202</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids(100x)</td>
			<td>Invitrogen, USA</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophiopogonin-D</td>
			<td>Chengdu Purui Technology Co. Ltd</td>
			<td>945619-74-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution</td>
			<td>Invitrogen, USA</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin</td>
			<td>Solarbio, China</td>
			<td>CA1620</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>ZSGB-BIO, China</td>
			<td>ZLI-9062</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis Buffer</td>
			<td>Solarbio, China</td>
			<td>R1010</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Hyclone (Life Technology), USA</td>
			<td>SH30809.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate(100 mM)</td>
			<td>Invitrogen, USA</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Squalene</td>
			<td>Sigma, USA</td>
			<td>S3626</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;- Mercaptoethanol</td>
			<td>Invitrogen, USA</td>
			<td>21985023</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro cellular activity evaluation of the nanoemulsion vaccine adjuvant ophiopogonin d

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated with antigens. Ophiopogonin D (OP-D), a purified component extracted from the plant Ophiopogon japonicus, has been found to be useful as a vaccine adjuvant. The problems of the low solubility and toxicity of OP-D can be effectively overcome by using a low-energy emulsification method to prepare nanoemulsion ophiopogonin D (NOD). In this article, a series of in vitro protocols for cellular activity evaluation are examined. The cytotoxic effects of L929 were determined using a cell counting kit-8 assay. Then, the secreted cytokine levels and corresponding immune cell numbers after the stimulation and culture of splenocytes from immunized mice were detected by ELISA and ELISpot methods. In addition, the antigen uptake ability in bone marrow-derived dendritic cells (BMDCs), which were isolated from C57BL/6 mice and matured after incubation with GM-CSF plus IL-4, was observed by laser scanning confocal microscopy (CLSM). Importantly, macrophage activation was confirmed by measuring the levels of IL-1&#946;, IL-6, and tumor necrosis factor alpha (TNF-&#945;) cytokines by ELISA kits after coculturing peritoneal macrophages (PMs) from blank mice with the adjuvant for 24 h. It is hoped that this protocol will provide other researchers with direct and effective experimental approaches to evaluate the cellular response efficacies of novel vaccine adjuvants.

The cellular activity evaluation of the adjuvants OP-D and NOD was completed in vitro according to the protocol. L929 fibroblasts are a useful screening model for the in vitro toxicity testing of NOD (Figure 1). The quantification of inflammatory cytokine levels in the spleen can help researchers better understand the immune response (Figure 2). Monitoring CTLs with ELISpot is the gold standard for assessing antigen-specific T-cell immunity in ...

Watch this Scientific Journal Video about In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D at JoVE.com

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

This protocol can provide other researchers with direct and effective experimental approaches and the detail steps to evaluate the cellular response efficacies of novel vaccine adjuvants. It cannot only provide researchers with specific cellular evaluation methods for adjuvants, but can also break down extraction methods such as BM disease. This protocols not only demonstrate no toxicity and high cellular delivery, but also provide detailed procedures for adjuvant evaluation field.To begin, turn on the water bath and adjust the temperature to 37 degrees Celsius. Then after collecting a tube of frozen L929 cells from liquid nitrogen, thaw quickly in the water bath. After pipetting the cells into a 15 milliliters sterile centrifuge tube, add two milliliters of DMEM and mix well.Centrifuge the samples at 129 x g for five minutes. And after discarding the supernatant, add two milliliters of DMEM to resuspend the cells. Centrifuge the samples again.And after discarding the supernatant, add six milliliters of DMEM complete medium for resuspension, and transferred to a T25 centimeter squared culture flask in a 37 degree Celsius incubator with 5%carbon dioxide to culture for 48 hours. On day 24 after the primary immunization, remove the mice from the animal room, place it in a glass dish and soak in 75%alcohol for five minutes. Place the centrifuge tubes on a centrifuge tube rack, numbered the disposable Petri dishes, and add five milliliters of PBS to each Petri dish with a 10 milliliter pipette.Make a six to eight centimeter incision with scissors in the middle of the left ventral side of the mouse. Tear open the skin to expose the abdominal wall and locate the long red strip of the spleen. Next, lift the peritoneum on the inferior side of the spleen with forceps.Cut it open and turn it upward to expose the spleen. Lift the spleen with forceps. Separate the connective tissue beneath the spleen with ophthalmic scissors and remove the spleen.Place the spleen in a Petri dish containing five milliliters of PBS and mill with a sieve and syringe plunger. After grinding, transfer the liquid into a 15 milliliter centrifuge tube with a pipette in accordance with the numbering. Next, centrifuge the liquid at 453 x g for five minutes.And after discarding the supernatant, add three milliliters of red blood cell lysis buffer to each tube. Resuspend the cells and lyse at room temperature for 10 minutes. Then add 10 to 12 milliliters of PBS to each tube.And after mixing and centrifuging at 453 x g for five minutes, discard the supernatant and add 10 milliliters of PBS to each centrifuge tube and resuspend the cells. Take 20 microliters of each sample in a well of the cell counting plate and record the number of live cells using an automated cell counter. Next, centrifuge the samples at 453 x g for five minutes.And after discarding the supernatant, dilute to 2.5 x 10 to the sixth cells per milliliter with RF10 medium and add to a 96-well plate at 100 microliters per well. Cut a six to eight centimeters incision below the abdomen of the mouse with scissors and clamp the two ends of the opening to separate it in different directions and expose the legs of the mouse. Separate the mouse femur from the mouse body and the tibia from the joint.And keep the bones intact at both ends. Next, remove the residual tissue and cartilage from the articular joints at both ends of the femur with scissors and forceps. Soak the femurs in 75%alcohol for five minutes and then soak in a sterile PBS solution to wash off the surface alcohol.Then cut off the ends of the femurs with scissors and rinse the bone marrow in a sterile Petri dish with sterile PBS solution, followed by aspiration with a one milliliter syringe. Repeat the washing three to five times. Next, filter by a cell sieve and collect the bone marrow-derived dendritic cells into a 15 milliliter centrifuge tube.After centrifuging the samples at 290 x g for five minutes, discard the supernatant and add four milliliters of red blood cell lysis buffer, resuspend and lyse at room temperature for five minutes. Next, add 10 milliliters of sterile PBS solution to neutralize the lysate, and after centrifuging at 290 x g for five minutes, discard the supernatant. Resuspend the cells in one milliliter of DMEM containing 1%penicillin-streptomycin solution and 10%fetal bovine serum and count.Then add granulocyte macrophage colony stimulating factor plus interleukin-4 to the medium. Adjust the cell concentration to 5 x 10 to the fifth per milliliter and inoculate the cells on cover slips. After adding the cell suspension to a six-well plate at two milliliters per well, place the plate in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide for 48 hours.Change the medium completely after two days and change half of the medium after four days. Immerse the mice in 75%alcohol after euthanasia and place them face up in a glass Petri dish in numbered order. Pass the mice through the transfer window into the sterile operation room and place them on the operation table for five minutes.Using the syringe, aspirate 10 milliliters of saline. Tilt the mice downward at approximately 45 degrees and inject into the middle of the abdominal cavity. Draw about five milliliters of cell suspension into a 15 milliliter centrifuge tube for each 10 milliliters and repeat the injection three times.Centrifuge the cell suspension at 129 x g for five minutes to obtain mouse peritoneal primary macrophages. L929 fibroblasts are a useful screening model for the in vitro toxicity testing of NOD. The quantification of inflammatory cytokine levels in the spleen can help researchers better understand the immune response.Monitoring cytotoxic T lymphocytes with enzyme-linked immunospot is the gold standard for assessing antigen specific T-cell immunity in clinical trials and for screening vaccine candidates. The increased uptake of an antigen by dendritic cells can elicit enhanced adaptive immune responses. Macrophages play an important role in presenting antigens to T-cells, as well as inducing other antigen presenting cells to express co-stimulatory molecules, thereby initiating adaptive immune responses.The isolation of cells under the ratio of drug to cell action are of utmost importance.

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Research

JoVE Journal

Immunology and Infection

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

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 ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard manufacturing. To achieve an effective protein-based subunit vaccine, the protein antigen often needs to adopt a native-like conformation. This is particularly important for pathogen-surface antigens that are membrane-bound proteins. Cell-free methods have been successfully used to produce correctly folded functional membrane protein through the co-translation of nanolipoprotein particles (NLPs), commonly known as nanodiscs. 
This strategy can be used to produce subunit vaccines consisting of membrane proteins in a lipid-bound environment. However, cell-free protein production is often limited to small scale (&lt;1 mL). The amount of protein produced in small-scale production runs is usually sufficient for biochemical and biophysical studies. However, the cell-free process needs to be scaled up, optimized, and carefully tested to obtain enough protein for vaccine studies in animal models. Other processes involved in vaccine production, such as purification, adjuvant addition, and lyophilization, need to be optimized in parallel. This paper reports the development of a scaled-up protocol to express, purify, and formulate a membrane-bound protein subunit vaccine. 
Scaled-up cell-free reactions require optimization of plasmid concentrations and ratios when using multiple plasmid expression vectors, lipid selection, and adjuvant addition for high-level production of formulated nanolipoprotein particles. The method is demonstrated here with the expression of a chlamydial major outer membrane protein (MOMP) but may be widely applied to other membrane protein antigens. Antigen effectiveness can be evaluated in vivo through immunization studies to measure antibody production, as demonstrated here.

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

This protocol describes using commercial, cell-free protein expression kits to produce membrane proteins supported in nanodisc that can be used as antigens in subunit vaccines.

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard ...

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; in particular, some MHC I-restricted epitopes can induce effective cytotoxic T lymphocyte activity to clear tumor cells. Additionally, nasal administration is an effective and safe delivery technique for tumor vaccines due to its convenience and improved patient compliance. However, epitope peptides are unsuitable for nasal delivery because of their poor immunogenicity and lack of delivery efficiency. Nanoemulsions (NEs) are thermodynamically stable systems that can be loaded with antigens and delivered directly to the nasal mucosal surface. Ile-Lys-Val-Ala-Val (IKVAV) is the core pentapeptide of laminin, an integrin-binding peptide expressed by human respiratory epithelial cells. In this study, an intranasal self-assembled epitope peptide NE tumor vaccine containing the synthetic peptide IKVAV-OVA257-264 (I-OVA) was prepared by a low-energy emulsification method. The combination of IKVAV and OVA257-264 can enhance antigen uptake by nasal mucosal epithelial cells. Here, we establish a protocol to study the physicochemical characteristics by transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering (DLS); stability in the presence of mucin protein; toxicity by examining the cell viability of BEAS-2B cells and the nasal and lung tissues of C57BL/6 mice; cellular uptake by confocal laser scanning microscopy (CLSM); release profiles by imaging small animals in vivo; and the protective and therapeutic effect of the vaccine by using an E.G7 tumor-bearing model. We anticipate that the protocol will provide technical and theoretical clues for the future development of novel T cell epitope peptide mucosal vaccines.

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Here, we present detailed methods for the preparation and evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo.

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; ...

Immunogenicity Enhancement by Mycobacterium paratuberculosis in Autoimmune Encephalomyelitis

Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in complete Freund&#39;s adjuvant (CFA) containing inactivated Mycobacterium tuberculosis. The antigenic components of the mycobacterium activate dendritic cells to stimulate T-cells to produce cytokines that promote the Th1 response via toll-like receptors. Therefore, the amount and species of mycobacteria present during the antigenic challenge are directly related to the development of EAE. This methods paper presents an alternative protocol to induce EAE in C57BL/6 mice using a modified incomplete Freund&#39;s adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis strain K-10.
M. paratuberculosis, a member of the Mycobacterium avium complex, is the causative agent of Johne&#39;s disease in ruminants and has been identified as a risk factor for several human T-cell-mediated disorders, including multiple sclerosis. Overall, mice immunized with Mycobacterium paratuberculosis showed earlier onset and greater disease severity than mice immunized with CFA containing the strain of M. tuberculosis H37Ra at the same doses of 4 mg/mL. The antigenic determinants of Mycobacterium avium subspecies paratuberculosis (MAP)&#160;strain K-10 were able to induce a strong Th1 cellular response during the effector phase, characterized by significantly higher numbers of T-lymphocytes (CD4+ CD27+), dendritic cells (CD11c+ I-A/I-E+), and monocytes (CD11b+ CD115+) in the spleen compared to mice immunized with CFA. Furthermore, the proliferative T-cell response to the MOG peptide appeared to be highest in M. paratuberculosis-immunized mice. The use of an encephalitogen (e.g., MOG35-55) emulsified in an adjuvant containing M. paratuberculosis in the formulation may be an alternative and validated method to activate dendritic cells for priming myelin epitope-specific CD4+ T-cells during the induction phase of EAE.

Author Spotlight: Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis  

Here, we present an alternative protocol to actively induce experimental autoimmune encephalomyelitis in C57BL/6 mice, using the immunogenic epitope myelin oligodendrocyte glycoprotein (MOG)35-55 suspended in incomplete Freund's adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis.

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in ...