We acknowledge the contribution of the health-care workers volunteers. This project was supported by the Department of Excellence 2023/2027, MUR, Italy. AR and DZ were supported by PRIN2022 (EU fundings; NextGenerationEU)

The present protocol has been approved by and follows the guidelines of the Ethical Committee of the University of Verona (approval protocol number 1538). Informed written consent was obtained from the human subjects participating in the study. Whole blood samples were collected from healthcare worker (HCW) volunteers who were in the process of receiving anti-SARS-CoV-2 vaccines. These samples were collected in plastic tubes containing anticoagulants for the subsequent isolation of serum15.
All the following processes must be performed in a Class-2 biological hood, working under sterile conditions. Virus handling must be performed with care, and all waste products must be neutralized in a diluted bleach solution. An overview of the protocol is displayed in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65658/65658fig01v2.jpg" /> 
Figure 1: Graphical representation of a neutralization assay. (A)&#160;PV production, (B)&#160;PV titration, and (C)&#160;neutralization assay. All the procedures are performed in a class-2 biological hood under sterile conditions. Titration step (B) needs to be performed to standardize the infectivity levels of PVs before use in the neutralization assay (C). This figure was created with BioRender. <a href="https://www.jove.com/files/ftp_upload/65658/65658fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. SARS-CoV-2 PVs production and infectivity test
<ol>
	<li>Seed 5 x 105 HEK293T cells in complete Dulbecco&#39;s Modified Eagle Medium (DMEM, high-glucose, 10% foetal bovine serum (FBS), 1% L-glutamine, 1% penicillin/streptomycin) in a 6-well plate (6WP) to reach a suitable cell density compatible with the transfection reagent used. In the case of performing transfection with polyehtylenimine (PEI) (prepare the reagent following the manufacturer instructions), ensure that the cells reach 40-60% density on the day of transfection (step 1.3). Keep the cells in a humidified incubator at 37 &#176;C and 5% CO2.</li>
	<li>Prior to transfection, replace the spent cell medium with fresh medium without antibiotics (DMEM, high-glucose, 10% FBS, 1% L-glutamine) to achieve higher transfection efficiency. 
	NOTE: The day after seeding, HEK293T cells are ready to be transfected.</li>
	<li>Transfect adherent HEK293T cells with a suitable transfection reagent according to the manufacturer&#39;s instructions. If using PEI, prepare two mixes and follow the steps below.
	<ol>
		<li>To prepare mix A, add 500 ng of pCMV-dR8.91 packaging plasmid16, 750 ng of pCSFLW reporter plasmid16, and 450 ng of SARS-CoV-2 Spike expressing plasmid in 100 &#181;L of reduced serum medium.</li>
		<li>To prepare mix B, add 17.5 &#181;L of PEI (concentration: 1 mg/mL) to 100 &#181;L of the reduced serum medium.</li>
		<li>Allow both mixes to incubate at room temperature (RT) for 5 min. Next, mix the contents of both tubes together by adding the PEI mix B to DNA mix A.</li>
		<li>Incubate the tube for 20-30 min at RT. Flick the tube gently every 3-4 min to enhance the mixing. Finally, add the mixture to the HEK293T cells.</li>
	</ol>
	</li>
	<li>16-20 h after the transfection, replace the culture medium with fresh, complete DMEM. Incubate at 37 &#176;C and 5% CO2, to allow for the production of PVs by transfected cells.</li>
	<li>72 h after the transfection, harvest the supernatant containing PVs. Then centrifuge at 1600 x g for 7 min at room temperature to remove cell debris and dead cells and filter it through a 0.45 &#181;m cellulose acetate filter.</li>
	<li>OPTIONAL STEP: To increase the final yield of PV titer, perform multiple transfections, pool the cell media containing PVs, and concentrate it using concentrating tubes.</li>
	<li>Proceed directly with the next steps (&#34;PVs titration&#34;, section 2) or aliquot the PV-containing medium in suitable tubes to store at -80 &#176;C until use. Prepare an additional aliquot (400-500 &#181;L) to be used for titration. 
	NOTE: Making multiple aliquots will guarantee reproducibility between experiments by avoiding excessive thaw-freeze cycles.</li>
</ol>
2. PVs titration
<ol>
	<li>Use the fresh PV-containing medium for the next steps or thaw the testing aliquot (step 1.7) to perform the titration of the new viral stock. Freezing aliquots of the same PV stock will guarantee reproducibility.</li>
	<li>Add 50 &#181;L of complete DMEM (or complete medium compatible with the target cell line in usage) in all the wells of a 96 well-plate (96WP) necessary to test in duplicate the PV stock, leaving row &#34;A&#34; empty. Add 100 &#181;L of PVs stock to row &#34;A&#34;. Based on the number of preparations to be tested, leave one column without the virus as a &#34;cell only&#34; control (Figure 2).</li>
	<li>Pipette 50 &#181;L from row A to row B and repeat this process up to row G to obtain serial dilutions of the initial stock. Discard the excess volume from the last row.</li>
	<li>Detach cellsusing trypsin/ethylenediaminetetraacetic acid 1x (EDTA) in Dulbecco&#39;s phosphate buffer saline 1x (DPBS 1x), after removing the spent medium and washing cells with DPBS 1x twice. Prepare cells to a density of 4 x 105 cells/mL. 
	NOTE: In this protocol, PVs infection was tested on the susceptible cell line HEK293T/ACE2; such cells were derived from HEK293T, transduced using a lentiviral vector to express ACE2 receptor.</li>
	<li>Add 50 &#181;L of the cell suspension into each well to ensure a cell count of 2 x 104 cells per well.</li>
	<li>Incubate at 37 &#176;C and 5% CO2, for 48 h.</li>
	<li>After the incubation, perform the Luciferase assay to obtain the reading as per the manufacturer&#39;s instructions. Add 100 &#181;L of the luciferase reagent to the wells and incubate in the dark at RT for 2 min. Move the content of each well to a black 96 well plate (compatible with the available plate reader) and read the plates in a 96 well plate reader. 
	NOTE: The luminometer used for the luciferase readout will produce a spreadsheet file with the raw, unprocessed data that will be used for downstream analysis (in this case, an Excel file). The virus&#39; infectivity will be expressed as relative luminescence units (RLU) (described in paragraph 4.1).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65658/65658fig02.jpg" /> 
Figure 2: Representative layout of a 96 well plate for PVs titration. A fixed volume of PV-containing supernatant is added to row A, columns 1-11, and serially diluted. The last column is left as the &#34;cell only&#34; control. This figure was created with BioRender. <a href="https://www.jove.com/files/ftp_upload/65658/65658fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Neutralization assay
<ol>
	<li>Thaw patients&#39; sera on ice. Inactivate serum samples by incubating them at 56 &#176;C for 30 min.</li>
	<li>In a 96 well plate, add 50 &#181;L of the fresh, complete DMEM (or complete medium compatible with the target cell line used)in each of the following wells: from row B (columns 1-10) to row H (columns 1-10). Put 95 &#181;L of the fresh, complete DMEM in row A (columns 1-10). Add 50 &#181;L and 100 &#181;L of complete DMEM into the wells of columns 11 and 12, respectively. These will be the infected (virus control, or VC) and uninfected (cell only, or CC) controls, respectively (Figure 3).</li>
	<li>Add 5 &#181;L of heat-inactivated serum/plasma samples in row A (columns 1-10). Each sample will be in duplicate. With a multichannel pipette, mix the samples in the first row and move 50 &#181;L of medium containing serum from row A to row B. Repeat this process up to the last row (Figure 3). Discard the remaining 50 &#181;L.</li>
	<li>Thaw the necessary number of PVs&#39; aliquots and dilute to &#8805; 104 RLU/mL. Add 50 &#181;L of the diluted PV-containing medium to each well (from column 1 to column 11) using a multichannel pipette to reach a 1:1 dilution of heat inactivated serum/plasma to virus. Incubate at 37 &#176;C and 5% CO2, for 1 h to allow the antibodies in the serum samples to bind to the SARS-CoV-2 spike protein on the PVs.</li>
	<li>Prepare at least 5 mL suspension of susceptible cells (HEK293T/ACE2) at a cell density of 4 x 105 cells/mL. Add 50 &#181;L of the cell suspension to each well and incubate at 37 &#176;C and 5% CO2, for 48 h.</li>
	<li>After the incubation, perform the luciferase assay reading according to the manufacturer&#39;s instructions, as described in step 2.7. 
	NOTE: The luminometer used for luciferase readout will produce a spreadsheet file (in this case, .xlsx) with the raw, unprocessed data that will be used for downstream analysis (the Luciferase assay file).</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65658/65658fig03.jpg" /> 
Figure 3: Plate representation based on serum dilution. Bright red corresponds to a higher quantity of serum, and bright blue lane (column 11) corresponds to infected cell control (VC, virus control). Light blue lane (column 12) corresponds to uninfected cells (CC, cell control). This figure was created with BioRender. <a href="https://www.jove.com/files/ftp_upload/65658/65658fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Titration analysis
<ol>
	<li>On the Luciferase assay file, assign the names/titles to the corresponding samples.</li>
	<li>Multiply the RLU measure by the dilution factors (from the top to the bottom of the grid: 20x, 40x, 80x, 160x, 320x, 640x, 1,280x, 2,560x) to obtain RLU/mL. If different dilution factors are used, change the multiplication factors accordingly.</li>
	<li>Calculate the average RLU/mL for each PV preparation.</li>
</ol>
5. PVs neutralization assay analysis
<ol>
	<li>On the Luciferase assay spreadsheet file (in this case, .xlsx), assign the corresponding titles to the tested samples. Enter the dilution factor of the sample (40s, 80x, 160x, 320x, 640x, 1,280x, 2,560x, 5,120x). Calculate the Log10 of the dilution factors.</li>
	<li>Calculate the average RLU of uninfected and infected control (Figure 3, columns 11 and 12, respectively). These values will be useful for the normalization in step 5.5.</li>
	<li>Open a new document for data analysis. Select X/Y analysis, input X as Numbers and Y as Enter 2 replicate values in side-by-side sub-columns.</li>
	<li>Enter Log10 (dilution) values as X numbers. Enter the duplicate RLU of the samples.</li>
	<li>Go to Analyze &#62; Normalize &#62; Flag all the samples on the same sheet. Input the average VC and CC values in How is 0% defined?, and How is 100% defined?, respectively. Click OK.</li>
	<li>On the normalized data sheet, go to Analyze &#62; XY analyses &#62; Nonlinear analyses (curve fit). Flag all the samples and click OK. For the Dose-response - Inhibition, select log(inhibitor) vs normalized response - variable slope.</li>
	<li>Under Constrain, change HillSlope to Must be less than 0.</li>
	<li>Under Output, flag Create summary table and graph. Click on OK to obtain the final analyses. A working sheet with a template for the analysis is provided in Supplementary File 1.</li>
</ol>

Assessing Serum Neutralization Against SARS-CoV-2 with Pseudotyped Viruses

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<li>Piubelli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subjects+who+developed+SARS-CoV-2+specific+IgM+after+vaccination+show+a+longer+humoral+immunity+and+a+lower+frequency+of+infection%5BTitle%5D)+AND+%22eBioMedicine%22%5BJournal%5D)">Subjects who developed SARS-CoV-2 specific IgM after vaccination show a longer humoral immunity and a lower frequency of infection.</a> eBioMedicine. 89, 104471(2023).</li>
<li>Zhang, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infectivity+of+pseudotyped+SARS-CoV-2+variants+of+concern+in+different+human+cell+types+and+inhibitory+effects+of+recombinant+spike+protein+and+entry-related+cellular+factors%5BTitle%5D)+AND+%22Journal+of+Medical+Virology%22%5BJournal%5D)">Infectivity of pseudotyped SARS-CoV-2 variants of concern in different human cell types and inhibitory effects of recombinant spike protein and entry-related cellular factors.</a> Journal of Medical Virology. 95 (1), e28437(2023).</li>
<li>da Costa, K. A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influenza+A+%28N1-N9%29+and+Influenza+B+%28B%2FVictoria+and+B%2FYamagata%29+neuraminidase+pseudotypes+as+tools+for+pandemic+preparedness+and+improved+influenza+vaccine+design%5BTitle%5D)+AND+%22Vaccines%22%5BJournal%5D)">Influenza A (N1-N9) and Influenza B (B/Victoria and B/Yamagata) neuraminidase pseudotypes as tools for pandemic preparedness and improved influenza vaccine design.</a> Vaccines. 10 (9), 1520(2022).</li>
<li>Condor Capcha, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+SARS-CoV-2+spike+pseudotyped+virus+for+viral+entry+and+neutralization+assays%3A+a+1-week+protocol%5BTitle%5D)+AND+%22Frontiers+in+Cardiovascular+Medicine%22%5BJournal%5D)">Generation of SARS-CoV-2 spike pseudotyped virus for viral entry and neutralization assays: a 1-week protocol.</a> Frontiers in Cardiovascular Medicine. 7, 618651(2021).</li>
<li>Diomede, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Doxycycline+inhibition+of+a+pseudotyped+virus+transduction+does+not+translate+to+inhibition+of+SARS-CoV-2+infectivity%5BTitle%5D)+AND+%22Viruses%22%5BJournal%5D)">Doxycycline inhibition of a pseudotyped virus transduction does not translate to inhibition of SARS-CoV-2 infectivity.</a> Viruses. 13 (9), 1745(2021).</li>
</ol>

The authors declare to have no conflict of interest.

Although using a wildtype virus simulates the actual infection, lentiviral PVs are a safer option to study the mechanisms associated with viral entry and infection without the strict safety requirements necessary to work with pathogenic viruses4,20,21. PVs are composed of a replication-defective viral core surrounded by the surface envelope glycoprotein of a pathogenic virus which is the objective of the study.
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Pseudotyped viruses (PVs) are molecular tools used in microbiology to study host-virus and pathogen-pathogen interactions1,2,3,4. PVs consist of an inner part, the viral core that protects the viral genome, and an outer part, the envelope glycoproteins on the surface of the virus that defines the tropism5. A pseudovirus is replication-incompetent in the target cell because it does not contain all the genetic information to generate new viral particles. This combination of peculiar features makes PVs a safe alternative to a wildtype virus. Wildtype viruses, on the other hand, are highly pathogenic and cannot be used in BSL 2 laboratories for analysis6.
The infectivity of PVs can be monitored by a reporter gene, usually coding for a fluorescent protein (GFP, RFP, YFP) or an enzyme that produces chemiluminescent products (luciferase). This is contained in one of the plasmids used for PV production and incorporated in the genome of the pseudovirus7.
Several types of PV cores currently exist, including lentiviral-derived particles based on the HIV-1 genome. The great advantage of HIV-1-based PVs over other platforms is their intrinsic integration process in the target cell genome8. Although HIV-1 is a highly contagious virus and is the causative agent of AIDS, these lentiviral vectors are safe to use because of the extensive optimization steps over the years. Optimal safety conditions were achieved with the introduction of 2nd-generation lentiviral vectors, in which viral genes were depleted without influencing transduction capabilities9. The 3rd and 4th generations contributed to the increased safety of lentiviral vector handling with the further splitting of the viral genome into separate plasmids10, 11. The latest generations of PVs are generally employed to produce lentiviral vectors for gene therapy.
PVs can be used to study interactions between viruses and host cells, during both the production and the infection phases. PVs are especially employed in pseudovirus neutralization assays (PVNA). PVNAs are widely validated to assess the neutralization potential of serum or plasma by targeting the viral glycoprotein on the PV&#39;s envelope12,13. Neutralization activity, expressed as the inhibitory concentration 50 (IC50), is defined as the dilution of serum/plasma that blocks 50% of viral particle entry14. In this protocol, we described the set-up of a PVNA to test the antibody activity against Severe Acute Respiratory Syndrome - Coronavirus 2 (SARS-CoV-2) in sera collected before and after receiving a booster vaccine dose.

<table><tbody><tr><td>0.45 &amp;mu;m filter</td><td>SARSTEDT</td><td>83 1826</td><td></td></tr><tr><td>6-well plate</td><td>SARSTEDT</td><td>83 3920</td><td></td></tr><tr><td>96-well plate</td><td>SARSTEDT</td><td>8,33,924</td><td></td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Units</td><td>Merck</td><td>10403892</td><td></td></tr><tr><td>Black Opaque 96-well Microplate</td><td>Perkin Elmer</td><td>60005270</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium&amp;nbsp;</td><td>SIGMA-ALDRICH</td><td>D6546 - 500ML</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (PBS 1x)</td><td>AUROGENE</td><td>AU-L0615-500</td><td></td></tr><tr><td>Foetal Bovine Serum</td><td>AUROGENE</td><td>AU-S1810-500</td><td></td></tr><tr><td>Graphpad Prism version 7</td><td>graphpad dotmatics</td><td>NA</td><td>In the manuscript, we replace the commercial name with &#039;data analysis program&#039;</td></tr><tr><td>HEK293T cells</td><td>ATCC</td><td>CRL-3216</td><td></td></tr><tr><td>HEK293T/ACE2 cells</td><td>ATCC</td><td>CRL-3216</td><td>HEK293T has been transduced to overexpress ACE2 with a lentiviral vector.</td></tr><tr><td>L-glutamine&amp;nbsp;</td><td>AUROGENE</td><td>AU-X0550-100</td><td></td></tr><tr><td>Luminometer - Victor3</td><td>Perkin Elmer</td><td>HH35000500</td><td>In the manuscript, we replace the commercial name with&amp;nbsp; &#039;luminometer&#039;&amp;nbsp;</td></tr><tr><td>Opti-MEM</td><td>Thermo Fisher Scientific</td><td>11058021</td><td>In the manuscript, we replace the commercial name with &#039;reduced serum medium&#039;&amp;nbsp;</td></tr><tr><td>p8.91 packaging plasmid</td><td>Di Genova et al., 2021</td><td></td><td>A kind gift from Prof. Nigel Temperton (ref 16.)</td></tr><tr><td>pCSFLW reporter plasmid</td><td>Di Genova et al., 2021</td><td></td><td>A kind gift from Prof. Nigel Temperton (ref 16.)</td></tr><tr><td>Penicillin/streptomycin</td><td>AUROGENE</td><td>AU-L0022-100</td><td></td></tr><tr><td>Polyethylenimine, branched (PEI) (25 kDa)</td><td>SIGMA-ALDRICH</td><td>408727</td><td></td></tr><tr><td>RRL.sin.cPPT.SFFV/Ace2.IRES-puro.WPRE (MT126)</td><td>Addgene</td><td>145839</td><td>This plasmid was used to generate HEK293Tcells/ACE2</td></tr><tr><td>SARS-CoV-2 Spike expressing plasmid</td><td>Addgene</td><td>pGBW-m4137382</td><td></td></tr><tr><td>steadylite plus Reporter Gene Assay System</td><td>Perkin Elmer</td><td>6066759</td><td>In the manuscript, we replaced the commercial name with &#039;luciferase reading reagent&#039;</td></tr><tr><td>Trypsin EDTA 1x</td><td>AUROGENE</td><td>AU-L0949-100</td><td></td></tr></tbody></table>

pseudotyped viruses as a molecular tool to monitor humoral immune responses against sars-cov-2 via neutralization assay

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in addition to their better-known use in gene therapy for the delivery of a gene of interest. PVs are replication defective because the viral genome is divided into different plasmids that are not incorporated into the PVs. This safe and versatile system allows the use of PVs in biosafety level 2 laboratories. Here, we present a general methodology to produce lentiviral PVs based on three plasmids as mentioned here: (1) the backbone plasmid carrying the reporter gene needed to monitor the infection; (2) the packaging plasmid carrying the genes for all the structural proteins needed to generate the PVs; (3) the envelope surface glycoprotein expression plasmid that determines virus tropism and mediates viral entry into the host cell. In this work, SARS-CoV-2 Spike is the envelope glycoprotein used for the production of non-replicative SARS-CoV-2 pseudotyped lentiviruses.
Briefly, packaging cells (HEK293T) were co-transfected with the three different plasmids using standard methods. After 48 h, the supernatant containing the PVs was harvested, filtered, and stored at -80 &#176;C. The infectivity of SARS-CoV-2 PVs was tested by studying the expression of the reporter gene (luciferase) in a target cell line 48 h after infection. The higher the value for relative luminescence units (RLUs), the higher the infection/transduction rate. Furthermore, the infectious PVs were added to the serially diluted serum samples to study the neutralization process of pseudoviruses' entry into target cells, measured as the reduction in RLU intensity: lower values corresponding to high neutralizing activity.

This protocol describes the production of SARS-CoV-2 PVs and a downstream application of these PVs to analyze the neutralization activity of serum/plasma of subjects receiving anti-COVID-19 vaccination17. Furthermore, this protocol can be applied to produce pseudotypes of each SARS-CoV-2 variant of concern (VOC) to test the evolution of the neutralizing response. Despite this protocol facilitating the study of humoral immune response after COVID-19 vaccination, it can be adapted to easily test the...

Watch this Scientific Journal Video about Studying Host-Virus Interactions with Pseudotyped Viruses at JoVE.com

Author Spotlight: Studying Host-Virus Interactions with Pseudotyped Viruses 

Pseudotyped viruses (PVs) are replication-defective virions that are used to study host-virus interactions under safer conditions than handling authentic viruses. Presented here is a detailed protocol that shows how SARS-CoV-2 PVs can be used to test the neutralizing ability of patients' serum after COVID-19 vaccination.

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in ...

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Immunology and Infection

1.6K Views.  University of Verona. Gepseudotypeerde virussen zijn moleculaire hulpmiddelen die kunnen worden gebruikt om interacties tussen gastheer en virus te bestuderen en om neutraliserende activiteit van serummonsters te testen. In het werk dat we vandaag presenteren, zal een algemene methodologie worden gepresenteerd om pseudogetypeerde virussen te produceren, gebaseerd op vrij plasmide. Voor neutralisatietests worden over het algemeen Y-type virussen gebruikt, maar ze zijn zeer pathogeen en kunnen niet worden behandeld ...

Video: Auteur in de schijnwerpers: Bestuderen van gastheer-virusinteracties met gepseudotypeerde virussen 

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...

Quantifying Vaccine-Induced Neutralizing Antibodies in SARS-CoV-2 Variants

Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 variants and the effectiveness of vaccine-induced neutralizing antibodies against viral variants. These assays are essential for pandemic surveillance and validating vaccines and variant-specific boosters. This manuscript demonstrates the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) for quick quantification of SARS-CoV-2 variant infectivity and vaccine-induced neutralizing antibodies to viral variants. Ha-CoV-2 is a SARS-CoV-2 virus-like particle consisting of viral structural proteins (S, M, N, and E) and a fast-expressing RNA genome derived from an alphavirus, Semliki Forest Virus (SFV). Ha-CoV-2 also contains both green fluorescent protein (GFP) and luciferase reporter genes that allow for quick quantification of viral infectivity. As an example, the infectivity of the SARS-CoV-2 Delta (B.1.617.2) and the Omicron (B.1.1.529) variants are quantified, and their sensitivities to a neutralizing antibody (27VB) are also measured. These examples demonstrate the great potential of Ha-CoV-2 as a robust platform for rapid quantification of SARS-CoV-2 variants and their susceptibility to neutralizing antibodies.

Author Spotlight: A Pseudotype Virus System for Assessing Omicron Subvariants and Neutralizing Antibodies in SARS-CoV-2 Research

This protocol describes the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) as a platform for rapid quantification of infectivity of SARS-CoV-2 variants and their sensitivity to neutralizing antibodies.

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 ...

Assessing Functional SARS-CoV-2 Neutralizing Antibodies

This video showcases a neutralization assay measuring serum efficacy in neutralizing SARS-CoV-2 viruses. It involves pseudotyped viruses, serum dilution, and luminescence-based quantification, revealing the neutralization titer.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Pseudotype-based neutralization ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

Assessing Functional Neutralizing Antibody Responses Against a Panel of SARS-CoV-2 Variants of Concern Using Standardized, Quantitative Pseudotype Neutralization Assays

To begin, add 50 microliters of DMEM with 10 percent FBS and 1 percent Penicillin-Streptomycin to rows B to H, columns 1 to 12 of a white 96-well plate.
Add 5 microliters of convalescent sera into wells of row A, columns 2 to 10, in a total volume of 100 microliters DMEM with 10 percent FBS and 1 percent Penicillin-Streptomycin. Add 5 microliters of positive and negative antisera into wells A11 and A12 as controls.
Remove 50 microliters of the solution from the wells of row A and perform two-fold serial dilutions of all the wells beneath it. For each dilution, use a multichannel pipette to mix the solutions 5-10 times by pipetting up and down without creating air bubbles.
After completing the serial dilution, discard the final 50 microliters from the last well of each column.
Centrifuge the plate for 1 minute at 500 rotations per minute to ensure no liquid is left on the walls of the well. Calculate the amount of DMEM required to dilute the SARS-CoV-2 PV as described in the text manuscript.
Mix the diluted PV solution in a pipette basin and aliquot 50 microliters into each well, except for wells A6 to A12 which comprise the cell-only control. The A1 to A6 wells serve as PV-only control.
Centrifuge plate for 1 minute at 500 rotations per minute to ensure no virus is left on the walls of the well. Then, incubate the plates at 37 degrees Celsius with 5 percent carbon dioxide for 1 hour to allow the serum to bind with the SARS-CoV-2 glycoprotein.
Next, prepare a plate of HEK 293T/17 target cells transfected 24 hours earlier with ACE2 and TMPRSS2 plasmids. Remove the culture media from the plate, and wash it twice with 2 milliliters of PBS, by adding it to one side of the dish to avoid cell detachment.
Discard the PBS and add 2 milliliters of trypsin to the plate. Place the plate in an incubator until the cells are detached.
After cells have detached, add 6 milliliters of DMEM with 10 percent FBS and 1 percent Penicillin-Streptomycin to the plate to quench trypsin activity, and resuspend cells gently.
Count the cells using an automated cell counter or by counting the chamber slide. Add 1 x 104 cells aliquoted in a total volume of 50 microliters to each well and incubate the plate at 37 degrees Celsius with 5 percent carbon dioxide for 48 to 72 hours.
After incubation, remove the culture media from all wells, and add 25 microliters of a 1:1 mix of PBS and a commercial luciferase assay reagent. Read the plate using a commercial luciferase assay system with a microplate luminometer.
Using the raw data generated by the luminometer, calculate the IC50 neutralizing antibody titers as described in the text manuscript.

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

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