This study was supported by No. 31670938, 32070924, 32000651 of the National Natural Science Foundation Program of China, No. 2014jcyjA0107 and No. 2019jcyjA-msxmx0159 of the Natural Science Foundation Project Program of Chongqing, No. 2020XBK24 and No. 2020XBK26 of the Army Medical University Special projects, and No. 202090031021 and No. 202090031035 of National Innovation and Entrepreneurship Program for college students.

The animal experiments were conducted in accordance with the Laboratory Animal&#8212;Guideline for ethical review of animal welfare (GB/T 35892-2018) and were approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University.&#160;The mice were euthanized by an intraperitoneal injection of 100 mg/kg of 1% sodium pentobarbital.
1. Preparation of the I-OVA NE
<ol>
	<li>Admix 1 mg of monophosphoryl lipid A (MPLA) with 100 &#181;L of DMSO, vortex for 5 min, and allow it to stand for 4 h at room temperature (RT) to dissolve completely.</li>
	<li>Add Tween 80 and I-OVA quantitatively and mix. 
	NOTE: Tween 80 and I-OVA were mixed at a mass ratio of 25:119.</li>
	<li>Add squalene into the mixture prepared in step 1.2 (7:3, Smix: squalene).</li>
	<li>Add 100 &#181;L of MPLA solution (10 mg/mL) to the mixture prepared in step 1.3.</li>
	<li>Prepare the nanoemulsion vaccine using low-energy emulsification methods19: add the mixed solution to water droplets at approximately 70% of the total volume and stir gently to obtain a transparent and easily flowing mixture. 
	NOTE: The BNE control (blank emulsion) was prepared by the same method, replacing water with I-OVA.</li>
</ol>
2. Physicochemical characterization and stability
NOTE: Assess the droplet size distribution, zeta potential, and other physicochemical data of the I-OVA NE vaccine following steps 2.1-2.3; perform morphological characterization of the I-OVA NE vaccine following steps 2.4-2.7; and examine the 3D structure of the I-OVA NE vaccine following steps 2.8-2.9.
<ol>
	<li>Blend 50 mg of mucin protein with 100 mL of water for injection to prepare a 0.05% mucin solution.</li>
	<li>Dilute 4 mg/mL I-OVA NE 200-fold with 0.05 mg/mL mucin protein or deionized water.</li>
	<li>Observe the particle sizes, zeta potential, polydispersity index (PDI), and electrophoretic mobility at 25 &#176;C using a nanoanalyzer19.</li>
	<li>Dilute 10 &#181;L of I-OVA NE 200-fold with 2 mL of deionized water.</li>
	<li>Place 5 &#181;L of prediluted I-OVA NE vaccine (step 2.4) on a carbon-coated copper grid, and cover it with 10 &#181;L of 1% phosphotungstic acid for 3 min.</li>
	<li>Remove the excess phosphotungstic acid with filter paper.</li>
	<li>Obtain images using TEM. Place a 10 &#181;L sample diluted 50 times on a 100-mesh carbon copper grid and allow it to stand at room temperature (RT) for 5 min before adding 10 &#181;L of phosphotungstic acid (1%, pH 7.4). Examine all the samples by TEM at a voltage of 120 kV.</li>
	<li>Characterize the molecular morphology of the I-OVA NEusing a high-resolution atomic force microscope. Obtain the color images of I-OVA NE under the following conditions: for tungsten probes (force constant: 0.06 N&#183;m-1); scan range: 450 nm x 450 nm; tapping mode: imaging mode; and scanning method: point-by-point scanning at RT.</li>
</ol>
3. In vitro and in vivo toxicity assays
NOTE: The in vitro toxicity of the I-OVA NE vaccine was assessed following steps 3.1-3.9, and the in vivo toxicity of the I-OVA NE vaccine was assessed following steps 3.10-3.13.
<ol>
	<li>Revive human BEAS-2B epithelial cells following steps 3.1.1-3.1.4 and culture them in a complete growth medium at 37 &#176;C in a 5% CO2 incubator. 
	NOTE: To prepare the complete growth medium, add fetal bovine serum (FBS) and penicillin/streptomycin to the RPMI-1640 medium at final concentrations of 10% and 1%, respectively.
	<ol>
		<li>Turn on the water bath and adjust the temperature to 37 &#176;C. Remove the cell vials frozen in liquid nitrogen and thaw quickly in a 37 &#176;C water bath.</li>
		<li>After thawing, quickly pipette the cells into a 15 mL sterile centrifuge tube, add 2 mL of the complete growth medium, and centrifuge at 129 x g for 5 min.</li>
		<li>Remove the supernatant, add 2 mL of the complete growth medium to resuspend the cells, and centrifuge at 129 x g for 5 min.</li>
		<li>Remove the supernatant, add 6 mL of complete growth medium to resuspend the cells, and transfer the cells to a T25 culture flask to culture the cells at 37 &#176;C in a 5% CO2 incubator.</li>
	</ol>
	</li>
	<li>When the cell density reaches 80%-90%, discard the culture medium and wash the cells twice with 2 mL of PBS. Add 1 mL of 0.25% trypsin to digest the cells for 1-2 min. When rounding of the cells is observed, immediately add 4 mL of the complete growth medium to neutralize the trypsin.</li>
	<li>Mix and aspirate the samples into a 15 mL sterile centrifuge tube and centrifuge at 129 x g for 5 min.</li>
	<li>Remove the supernatant and resuspend the cells in 1 mL of RPMI-1640 complete medium. Use 20 &#181;L for cell counting, and dilute the cells to 1 x 105 cells/mL.</li>
	<li>Plate the BEAS-2B cells at a density of 1 x 104 cells/well in 96-well plates in 100 &#181;L of RPMI-1640 complete medium and preincubate the plates for 24 h at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Discard the supernatant, and add 100 &#181;L of I-OVA NE, 100 &#181;L of I-OVA+BNE (physically mixed), and 100 &#181;L of I-OVA prediluted with a complete growth medium at various final concentrations (0.5 mg/mL, 1 mg/mL, 2 mg/mL, 4 mg/mL, 8 mg/mL), with BNE as a control. Incubate for 24 h at 37 &#176;C. 
	NOTE: Add 100 &#181;L of complete growth medium to the negative control groups and 100 &#181;L of cell suspension (1 x 105/mL) to the positive control groups.</li>
	<li>Remove the medium and add 90 &#181;L of complete growth medium and 10 &#181;L of CCK-8 solution to each well of the plate.</li>
	<li>Incubate the plate for 2 h at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Measure the absorbance of each well at 450 nm using an enzyme-labeled plate reader.</li>
	<li>Calculate the survival ratio of BEAS-2B cells as indicated in the following equation: 
	(OD450 sample&#8722; OD450 negative control/OD450 positive control&#8722; OD450 negative control) &#215; 100%</li>
	<li>Randomly divide 6-week-old C57BL/6 mice into five groups (n = 5 in each group) and anesthetize them with 4% isoflurane for induction. Maintain anesthesia with 2% isoflurane. Use neomycin sulfate ointment on the eyes of the mice to prevent dryness.</li>
	<li>Use 10 &#181;L pipette tips to perform nasal immunization of the mice with 10 &#181;L/nostril of I-OVA, I-OVA+BNE, and IOVA NE at 4 mg/mL for all the&#160;3 days. Use BNE and PBS as the experimental control. 
	NOTE: Provide thermal support till the animal recovers from anesthesia.</li>
	<li>Euthanize all the mice by an intraperitoneal injection of 100 mg/kg 1% sodium pentobarbital on day 4.</li>
	<li>Cut approximately 3 mm thick samples of nasal tissues with scissors, and remove the lung tissues.</li>
	<li>Fix the nasal tissues and the whole lung tissues in 4% paraformaldehyde for 24 h. Dehydrate the tissues through a serial alcohol and xylene gradient and then embed in paraffin.Slice the finished wax blocks on a paraffin slicer at a thickness of 4 &#181;m.</li>
	<li>Stain the sections with hematoxylin and eosin (H&#38;E). Then, observe the mucosal toxicity, including hyperemia, edema, neutrophil infiltration, and structural damage in the nasal mucosa and lung tissue, under a microscope (100x and 200x)7.</li>
</ol>
4. In vitro cellular uptake
<ol>
	<li>Plate BEAS-2B cells at a density of 5 x 105 cells/well in 12-well plates with coverslips in 2 mL of the complete growth medium and preincubate the plates overnight at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Add 100 &#181;L of FITC-labeled I-OVA NE (purity: 98.3%, produced by the company, 4 mg/mL) or I-OVA (4 mg/mL) to 900 &#181;L of cell suspension and place at 37 &#176;C for 90 min. 
	NOTE: Prepare FITC-labeled I-OVA NE as described in step 4.1. Add 1 mL of complete growth medium to the control groups.</li>
	<li>Wash three times with 0.1 M PBS (1 mL/well) for 30 min at 37 &#176;C after the treatment.</li>
	<li>Fix these samples with 4% paraformaldehyde for 20 min in the dark. After fixation, remove the paraformaldehyde and wash 3 times with 0.1 M PBS (1 mL/well) for 30 min at 37 &#176;C.</li>
	<li>Preincubate the samples with DAPI (4&#39;,6-diamidino-2-phenylindole) at a final concentration of 10 &#181;g/mL for 10 min in the dark, and then wash 5 times with 0.1 M PBS (1 mL/well) for 30 min at 37 &#176;C after treatment.</li>
	<li>Obtain the cellular uptake by CLSM withthe following parameter settings: Frame size: 512 px x 512 px, Scan speed: 8; Line step: 1; Averaging: 2.</li>
</ol>
5. In vivo release
<ol>
	<li>Anesthetize nude mice with 4% isoflurane gas and maintain anesthesia at 2% isoflurane. Immunize nude mice intranasally with 10 &#181;L of PE-labeled I-OVA at 4 mg/mL or PE-labeled I-OVA NE at 4 mg/mL in each nostril.</li>
	<li>At 0 h, 0.5 h, 1.5 h, 3, 6 h, 9 h, 12 h, and 24 h, capture all the mice under anesthesia by the IVIS system.</li>
	<li>Perform a background scan immediately before intranasal administration to provide a threshold value (Min = 1.06 x 106) to adjust the images gathered at all time points.</li>
	<li>Click on the Living Image software to initiate the sequence and click Initialize to initialize the IVIS system</li>
	<li>When it is initialized, the temperature status light in the IVIS acquisition control panel is red. When the temperature status light changes to green, imaging can be performed.</li>
	<li>Click Imaging Wizard and then choose Fluorescence in the dialog box that appears.</li>
	<li>Adjust the following parameters: Exposure Time: auto sec, Binning: 8, F/Stop: 2, Field of View: D.</li>
	<li>Select the 620 nm excitation filter and the 670 nm emission filter. Click on Acquire Sequence to acquire the image.</li>
	<li>Process the live images of all the mice and the radiance data with Living Image software.
	<ol>
		<li>After acquiring the picture, click on ROI Tools in the Tool Palette, select Circle, and make an ROI circle.</li>
		<li>Click on Measure ROIs to obtain a quantitative value for the ROI area.</li>
	</ol>
	</li>
</ol>
6. In vivo antitumor efficacy
<ol>
	<li>Refresh and culture the mouse lymphoma E.G7-OVA cells from EL4 with a complete growth medium following steps 2.1.1-2.1.4. 
	NOTE: To prepare E.G7-OVA cell complete growth medium, mix RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate and supplemented with 0.05 mM 2-mercaptoethanol and 0.4 mg/mL G418, 90%, and fetal bovine serum, 10%.</li>
	<li>Subculture the cells at a ratio of 1:2 when the cells reach a density between 1 x 106 cells/mL and 1 x 107 cells/mL.</li>
	<li>Evaluate the preventive protective effect on mice as described in steps 6.3.1-6.3.4..
	<ol>
		<li>Randomly divide 6-week-old C57BL/6 mice into five groups (n = 8 in each group). Anesthetize nude mice with 4% isoflurane gas and maintain anesthesia with 2% isoflurane. Partially shave the back hair and neomycin sulfate ointment on the eyes of the mice to prevent dryness.</li>
		<li>Intranasally immunize the mice with 10 &#181;L of 1 mg/mL I-OVA, BNE+I-OVA, or I-OVA NE in each nostril, BNE (diluted four times with PBS), or a PBS control 3 times with a 7-day interval between each immunization.</li>
		<li>Inoculate all the mice hypodermically into the right back with 5 x 105 E. G7-OVA cells on the 7th day after the final immunization.</li>
		<li>At 0, 6, 9, 12, 15, and 18 days after the final immunization, monitor the tumor volumes by measuring two axes of the tumor using digital calipers, and record the 30-day (after the final immunization) survival of the mice.</li>
	</ol>
	</li>
	<li>Evaluate the therapeutic protective effect on mice after inoculating E.G7-OVA cells as described in steps 6.4.1-6.4.3. For the grouping and the handling of mice refer to step 6.3.1.
	<ol>
		<li>On day 0, inject the C57BL/6 mice subcutaneously into the right back with E.G7-OVA cells (5 x 105 cells/mouse).</li>
		<li>At 0, 7, and 14 days after injection, immunize all the mice intranasally immunized with 10 &#181;L of I-OVA, BNE+I-OVA, I-OVA NE (all at concentrations of 1 mg/mL), BNE, or PBS three times in each nostril.</li>
		<li>At 0, 6, 9, 12, 15, and 18 days after injection, monitor the tumor volume and record the 30-day survival of the mice. 
		NOTE: If the tumor volume exceeds 3,000 mm3,&#160;the mice should be euthanized for humane reasons, and these mice will be considered dead in the survival curve. Tumor volume is calculated by a modified ellipsoidal formula as indicated in the following equation: 
		Volume = &#960;/6 x L x W2 
		where L represents the tumor length, and W represents the tumor width (length unit: mm, volume unit: mm3).</li>
	</ol>
	</li>
</ol>
7. Statistical analysis
<ol>
	<li>Analyze the differences in data between the different groups using appropriate statistical software with one-way ANOVA, Tukey&#39;s multiple comparisons, or a Student&#39;s t-test. Use the Kaplan-Meier method to estimate the survival outcomes and compare the groups with log-rank statistics. Express all the results as mean &#177; SD. The significance of P values P &#60; 0.05, P &#60; 0.01, and P &#60; 0.001 is represented using *, **, and ***, respectively, on the plots.</li>
</ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispersity+effects+in+polymer+self-assemblies:+A+matter+of+hierarchical+control.">Dispersity effects in polymer self-assemblies: A matter of hierarchical control.</a> Chemical Society Reviews. 46 (14), 4119-4134 (2017).</li><li>Pei, M., Li, H., Zhu, Y., Lu, J., Zhang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+evidence+of+oncofetal+antigen+and+TLR-9+agonist+co-delivery+by+alginate+nanovaccines+for+liver+cancer+immunotherapy.">In vitro evidence of oncofetal antigen and TLR-9 agonist co-delivery by alginate nanovaccines for liver cancer immunotherapy.</a> Biomaterials Science. 10 (11), 2865-2876 (2022).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Titanium+dioxide+nanoparticles+induced+reactive+oxygen+species+(ROS)+related+changes+of+metabolomics+signatures+in+human+normal+bronchial+epithelial+(BEAS-2B)+cells.">Titanium dioxide nanoparticles induced reactive oxygen species (ROS) related changes of metabolomics signatures in human normal bronchial epithelial (BEAS-2B) cells.</a> Toxicology and Applied Pharmacology. 444, 116020 (2022).</li><li>Kumar, V., Sharma, N., Maitra, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+toxicity+assessment+of+nanoparticles.">In vitro and in vivo toxicity assessment of nanoparticles.</a> International Nano Letters. 7 (4), 243-256 (2017).</li><li>Tong, Y. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+immunopotentiator,+ophiopogonin+D,+encapsulated+in+a+nanoemulsion+as+a+robust+adjuvant+to+improve+vaccine+efficacy.">An immunopotentiator, ophiopogonin D, encapsulated in a nanoemulsion as a robust adjuvant to improve vaccine efficacy.</a> Acta Biomaterialia. 77, 255-267 (2018).</li><li>Elliott, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy:+Principles+and+modern+practices.">Confocal microscopy: Principles and modern practices.</a> Current Protocols in Cytometry. 92 (1), 68 (2020).</li><li>Huang, Y., Zou, Y., Lin, L., Zheng, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ginsenoside+Rg1+activates+dendritic+cells+and+acts+as+a+vaccine+adjuvant+inducing+protective+cellular+responses+against+lymphomas.">Ginsenoside Rg1 activates dendritic cells and acts as a vaccine adjuvant inducing protective cellular responses against lymphomas.</a> DNA and Cell Biology. 36 (12), 1168-1177 (2017).</li></ol>

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Nanovaccines functionalized with immunocyte membranes have great advantages in disease-targeted therapy, and the side effects are minimized by properties such as unique tumor tropism, the identification of specific targets, prolonged circulation, enhanced intercellular interactions, and low systemic toxicity. They can also be easily integrated with other treatment modules to treat cancers cooperatively16,20. Desirable attributes can be obtained by controllingphysical and chemical properties such as the measurement, shape, and electric charge. Hence, nanovaccines have become important in a wide range of applications21. These properties are major decisive factors regarding uptake and toxicity, and nanovaccines can be rendered nontoxic only by manipulation22. Therefore, this protocol for the study of physicochemical characteristics, including shape, size, and charge, is vital. TEM is a highly precise instrument that has been widely used in the scientific community for many years23. It has become an essential tool to understand the properties of nanostructured materials and to manipulate their behaviors. In addition, atomic force microscopy (AFM) has emerged as a powerful technique for the nanomechanical characterization of biological samples24. It can provide high-resolution 3D and nanoscale information while also analyzing surface details at the atomic level. In addition to determining changes in particle size distribution, DLS can measure both size and charge to provide information about the aggregation state of nanoparticles in solution25.
It has been reported that high zeta potential values are essential for the good stability of colloidal suspensions26. In this study, we used these protocols to assess the physicochemical characteristics of nanovaccines with TEM, AFM, and DLS. Moreover, the surface of the nasal mucosa contains a large amount of mucus, which provides lubrication, moisture, and a chemical protective barrier. This maybe due to the interaction between the mucus andsome antigens or delivery systems, resulting in the accumulation and &#34;capture&#34; of antigens or delivery systems and their subsequent removal, enormously reducing the delivery efficiency7. It is well known that the stability of nanovaccines is vital for nasal administration. Therefore, we used Nano ZS to determine a series of stability parameters, including the particle size, polydispersity indexes, zeta potentials, and electrophoresis mobility, after treatment with 0.5% mucin protein.
The in vivo histocompatibility and in vitro cell viability assays showed that this novel nanovaccine was nontoxic in the range of the tested concentrations27. Human normal bronchial epithelial (BEAS-2B) cells are standard cell lines used to study the human respiratory tract28. Due to its lower cost, rapidity, and minimal ethical concerns, in vitro toxicity assessment is an important method. In this study, the in vitro cell viability assay was determined by a CCK8 assay. In addition, in vivo toxicity assessment is usually performed in animal models such as mice and rats. Histopathological examination is often performed on tissues exposed to nanoparticles, such as the heart, eye, brain, liver, kidney, lung, and spleen29. Therefore, we used these methods to assess&#160;the toxicity of this novel nanovaccine in vitro and in vivo.
Antigen uptake and prolongation are prerequisites for antigen submission to trigger a subsequent immune response30. It is necessary that the cellular uptake of BEAS-2B and the release profiles of the novel nanovaccine in vivo and in vitro&#160;be determined. CLSM is the most common commercial implementation of the relevant technology, which can be found in the great majority ofimaging laboratories and has a wide range of applications. These instruments are widely used and relatively easy to use; however, they are usually not optimal for quantitative data collection.
In our protocol, cellular uptake was detected by CLSM because of the clear resolution, optical sectioning capability, and versatility with 3D imaging31. In addition, IVIS was used to obtain the in vivo release profiles of the novel nanomaterial because it can provide an imaging chamber with exterior light excluded for quantitative bioluminescence and fluorescence imaging in vivo and in vitro. Therefore, in this protocol, we used these methods to determine the cellular uptake and release profiles of novel nanovaccines in vivo and in vitro.
It is also important to assess the tumor efficacy of the novel nanovaccine. In our study, the E.G7 tumor-bearing model was used to determine the therapeutic and protective effects of the vaccine. EL4 cells are derived from the T lymphocytes of C57BL/6 mice with high-grade malignancy. E.G7 cells are derived from EL4 lymphoma cells transfected by electroporation32. In our study, this nanovaccine induced protective immunity in E.G7-OVA tumor-bearing mice. In summary, it is necessary to establish a series of protocols to study the physicochemical characteristics, stability, toxicity, release profiles, cellular uptake, and antitumor effects of nanovaccines in vitro and in vivo. These protocols will provide useful results for the novel nasal nanovaccine.

As one of the most critical public health innovations, vaccines play a key role in fighting the global burden of human disease1. For example, at present, more than 120 candidate vaccines for COVID-19 diseases are being tested, some of which have been approved in many countries2. Recent reports state that cancer vaccines have effectively improved the progress of clinical cancer treatments because they direct the immune system of cancer patients to recognize antigens as foreign to the body3. Moreover, multiple T cell epitopes located inside or outside tumor cells can be used to design peptide vaccines, which have shown advantages in the treatment of metastatic cancers because of the lack of the significant toxicity associated with radiotherapy and chemotherapy4,5. Since the mid-1990s, preclinical and clinical trials for tumor treatment have been conducted mainly using antigen peptide vaccines, but few vaccines exhibit an adequate therapeutic effect on cancer patients6. Furthermore, cancer vaccines with peptide epitopes have poor immunogenicity and insufficient delivery efficiency, which may be due to the rapid degradation of extracellular peptides that diffuse rapidly from the site of administration, which leads to insufficient antigen uptake by immune cells7. Therefore, it is necessary to overcome these obstacles with vaccine delivery technology.
OVA257-264, the MHC class I-binding 257-264 epitope expressed as a fusion protein, is a frequently used model epitope8. In addition, OVA257-264 is crucial to the adaptive immune response against tumors, which depends on the cytotoxic T lymphocyte (CTL) response. It is mediated by antigen-specific CD8+ T cells in the tumor, which are induced by the OVA257-264 peptide. It is characterized by insufficient granzyme B, which is released by cytotoxic T cells, leading to the apoptosis of target cells8. However, free OVA257-264 peptide administration may induce little CTL activity because the uptake of these antigens occurs in nonspecific cells rather than antigen-presenting cells (APCs). The deficiency of appropriate immune stimulation results in CTL activity5. Therefore, the induction of efficacious CTL activity demands considerable advancement.
Owing to the barrier provided by epithelial cells and the continuous secretion of mucus, vaccine antigens are rapidly removed from the nasal mucous9,10. Developing an efficient vaccine vector that can pass through the mucosal tissue is crucial because the antigen-presenting cells are situated under the mucosal epithelium9. Intranasal injection of vaccines theoretically induces mucosal immunity to fight mucosal infection11. In addition, nasal delivery is an effective and safe administration method for vaccines due to its convenience, the avoidance of intestinal administration, and improved patient compliancy7. Therefore, nasal delivery is a good means of administration for the novel peptide epitope nanovaccine.
Several synthetic biomaterials have been devised to combine epitopes of cell-tissue and cell-cell interactions. Certain bioactive proteins, such as Ile-Lys-Val-Ala-Val (IKVAV), have been introduced as part of the structure of the hydrogel to confer bioactivity12. This peptide likely contributes to cell attachment, migration, and outgrowth13 and binds integrins &#945;3&#946;1 and &#945;6&#946;1 to interact with different cancer cell types. IKVAV is a cell adhesion peptide derived from the laminin basement membrane protein &#945;1 chain that was originally used to model the neural microenvironment and cause the neuronal differentiation14. Therefore, finding an efficient delivery vehicle for this novel vaccine is important for disease control.
Recently reported emulsion systems, such as W805EC and MF59, have also been compounded for the nasal cavity delivery of inactivated influenza vaccine or recombinant hepatitis B surface antigen and illustrated to trigger both mucosal and systemic immunity15. Nanoemulsions (NEs) have the advantages of easy administration and convenient co-formation with effective adjuvants compared with particulate mucosal delivery systems16. Nanoemulsion vaccines have been reported to alter the allergic phenotype in a sustained manner different from traditional desensitization, which results in long-term suppressive effects17. Others reported that nanoemulsions combined with Mtb-specific immunodominant antigens could induce potent mucosal cell responses and confer significant protection18. Therefore, a novel intranasal self-assembled nanovaccine with the synthetic peptide IKVAV-OVA257-264 (I-OVA, the peptide consisting of IKVAV bound to OVA257-264) was designed. It is important to assess this novel nanovaccine systematically.
The purpose of this protocol is to systematically evaluate the physicochemical characteristics, toxicity, and stability of the nanovaccine, detect whether antigen uptake and protective and therapeutic effects are enhanced using technical means, and elaborate on the main experimental contents. In this study, we established a series of protocols to study the physicochemical characteristics and stability, determine the magnitude of toxicity of the I-OVA NE to BEAS-2B cells by CCK-8, and observe the antigen-presenting ability of BEAS-2B cells to the vaccine using confocal microscopy, evaluate the release profiles of this novel nanovaccine in vivo and in vitro, and detect the protective and therapeutic effect of this vaccine by using an E.G7-OVA tumor-bearing mouse model.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well plates</td>
			<td>Corning Incorporated, USA</td>
			<td>CLS3922</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad 6.0 microplate reader</td>
			<td>Bio-Rad Laboratories Incorporated Limited Co., CA, USA</td>
			<td>&#160;Bio-Rad 6.0</td>
			<td></td>
		</tr>
		<tr>
			<td>CCK-8 kits</td>
			<td>Dojindo, Japan</td>
			<td>CK04</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>fetal bovine serum (FBS)</td>
			<td>Hyclone (Life Technology, USA)</td>
			<td>SH30088.03</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-labeled I-OVA</td>
			<td>Shanghai Botai 
			Biotechnology Co., Ltd.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>HF 90/240 Incubator</td>
			<td>Heal Force, Switzerland</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC&#160;</td>
			<td>Shanghai Botai Biotechology Co., Ltd.</td>
			<td>E2695</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Nikon,Japan</td>
			<td>DSZ5000X</td>
			<td></td>
		</tr>
		<tr>
			<td>IPC-208</td>
			<td>Chong Qing University, China</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS system&#160;</td>
			<td>Caliper Life Science Limited Company</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>JEM-1230 TEM</td>
			<td>JEOL Limited Company of Japan</td>
			<td>1230 TEM</td>
			<td></td>
		</tr>
		<tr>
			<td>Malvern NANO ZS</td>
			<td>Malvern Instruments Ltd., UK</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>MPLA</td>
			<td>&#160;Invivogen 
			Lit. Co.</td>
			<td>tlrl-mpla</td>
			<td></td>
		</tr>
		<tr>
			<td>Neomycin Sulfate Ointment</td>
			<td>Shanghai CP General Pharmaceutical Co. , Ltd.</td>
			<td>H31022262</td>
			<td></td>
		</tr>
		<tr>
			<td>OVA257&#8211;264</td>
			<td>Shanghai Botai 
			Biotechnology Co., Ltd.</td>
			<td>NA</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>Synthetic peptide (I-OVA) conjugation of IKVAV-PA</td>
			<td>Shanghai Botai 
			Biotechnology Co., Ltd.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM800 laser scanning confocal fluorescence microscope</td>
			<td>Zeiss, Germany</td>
			<td>Zeiss LSM800</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

According to the protocol, we completed the preparation and in vitro and in vivo experimental evaluation of the nasal tumor nanovaccine delivery. TEM, AFM, and DLS are effective means for the assessment of the basic characteristics of the surface zeta potential and the particle size of the nanovaccine (Figure 1). BEAS-2B epithelial cells are a useful screening model for the in vitro toxicity testing of nasal vaccines (Figure 2A). The microphotographs stained with H&#38;E illustrate that I-OVA NE had no obvious mucosal toxicity, including tissue damage, bleeding, or inflammatory cell infiltration (Figure 2B). Efficient uptake of the antigen by BEAS-2B cells in the nasal cavity is a prerequisite for antigen presentation to elicit subsequent immune responses (Figure 3). The IVIS system helps to elucidate the sustained-release effect of I-OVA NE in vitro and suggests that this nanovaccine can delay rapid release, prolong the time in the nasal area, and improve the uptake of peptides in cells (Figure 4). The preventive protective models and the therapeutic protective models directly reflect the protective effect of the I-OVA NE vaccine and the ability of the I-OVA NE vaccine to inhibit tumor growth and prolong the median survival time of mice (Figure 5). The above experimental results have been published by Yang et al.7.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64299/64299fig01.jpg" /> 
Figure 1: Physical characteristics and stability of I-OVA NE. (A) Transmission electron micrograph (TEM), scale bar = 100 nm. (B) Atomic force microscopy (AFM) micrograph. The X and Y axes both have a total length of 450 nm. (C) Size diameter and distribution. (D) Zeta potential and distribution of I-OVA NE analyses performed using Nano ZS. (E) Particle sizes, (F) polydispersity indexes, (G) zeta potentials, and (H) electrophoresis mobility of I-OVA NE in mucin stability analyses performed using Nano ZS. This figure has been adapted with permission from Yang et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64299/64299fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64299/64299fig02.jpg" /> 
Figure 2: In vitro and in vivo toxicity of I-OVA NE. (A) Relative viability of BEAS-2B cells in culture exposed to different peptide concentrations of I-OVA, BNE+I-OVA, and I-OVA NE for 24 h. BNE was used as a control. The data are expressed as the mean &#177; SD (n = 3). (B) Microscopic examination of pathological sections of the nasal mucosa and lung tissues fixed 5 h after the challenge. Images were captured at 100x and 200x magnification. This figure has been adapted with permission from Yang et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64299/64299fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64299/64299fig03.jpg" /> 
Figure 3: Cellular uptake of the I-OVA NE. In vitro confocal fluorescence imaging of BEAS-2B cells treated for 1 h with I-OVA or I-OVA NE. PBS was used as a control, I-OVA was labeled with FITC (green fluorescence), and the nuclei were stained with DAPI (blue fluorescence) (scale bar = 50 &#181;m). This figure has been adapted with permission from Yang et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64299/64299fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64299/64299fig04.jpg" /> 
Figure 4: In vitro release of I-OVA NE. (A) In vivo fluorescence imaging of PE-labeled I-OVA in the mouse nasal cavity. Relative fluorescence intensity recorded at 0 h, 0.5 h, 1.5 h, 3 h, 6 h, 9 h, 12 h, and 24 h after nasal administration of I-OVA or I-OVA NE. (B) Quantitation of fluorescence intensity. The data are expressed as the mean &#177; SD (n = 5). *: P &#60; 0.05; **: P &#60;0.01; and ***: P &#60; 0.001. This figure has been adapted with permission from Yang et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64299/64299fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64299/64299fig05.jpg" /> 
Figure 5: In vivo anti-tumor efficacy of I-OVA NE. (A) Average tumor growth curves of the vaccinated mice in the preventive protective models. (B) Percent survival rate of the vaccinated mice in the preventive protective models. (C) Average tumor growth curves of the vaccinated mice in the therapeutic protective models. (D) Percent survival rate of the vaccinated mice in the therapeutic protective models. *: P &#60; 0.05; **: P &#60; 0.01; and ***: P &#60; 0.001. This figure has been adapted with permission from Yang et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64299/64299fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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.

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; ...

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Nanyang Technological University

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1.6K Views.  Third Military Medical University of Chinese PLA. Here, we present detailed methods for the preparation and evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo.

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

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 ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

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 the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

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 ...

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

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.

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.

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

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

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Chapters in this video

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

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

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

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

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

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