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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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+SARS-CoV-2+spike+pseudotyped+virus+for+viral+entry+and+neutralization+assays:+a+1-week+protocol.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxycycline+inhibition+of+a+pseudotyped+virus+transduction+does+not+translate+to+inhibition+of+SARS-CoV-2+infectivity.">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.
<p class="jov...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.45 &#956;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&#39;s Modified Eagle Medium&#160;</td>
			<td>SIGMA-ALDRICH</td>
			<td>D6546 - 500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;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 &#39;data analysis program&#39;</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&#160;</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&#160; &#39;luminometer&#39;&#160;</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 &#39;reduced serum medium&#39;&#160;</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 &#39;luciferase reading reagent&#39;</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 ...

pseudotyped-viruses-as-molecular-tool-to-monitor-humoral-immune

Research

JoVE Journal

Immunology and Infection

1.5K Views.  University of Verona. 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.

Video: Author Spotlight: Studying Host-Virus Interactions with Pseudotyped Viruses 

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 ...

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at &#8805;21 days from symptom onset but had higher sensitivity with samples collected at &#8804;5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The increasing number of SARS-CoV-2 infections raises the need for better antibody detection assays. Current antibody detection methods compromise sensitivity for speed or are sensitive but time-consuming. A large proportion of SARS-CoV-2-neutralizing antibodies target the receptor-binding domain (RBD), one of the primary immunogenic compartments of SARS-CoV-2. We have recently designed and developed a highly sensitive, bioluminescent-tagged RBD (NanoLuc HiBiT-RBD) to detect SARS-CoV-2 antibodies. The following text describes the procedure to produce the HiBiT-RBD complex and a fast assay to evaluate the presence of RBD-targeting antibodies using this tool. Due to the durability of the HiBiT-RBD protein product over a wide range of temperatures and the shorter experimental procedure that can be completed within 1 h, the protocol can be considered as a more efficient alternative to detect SARS-CoV-2 antibodies in patient serum samples.

The outlined protocol describes the procedure for producing the HiBiT-receptor-binding domain protein complex and its application for fast and sensitive detection of SARS-CoV-2 antibodies.

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe ...

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses.&#160;Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has been responsible for over 242 million infections and more than 4.9 million deaths worldwide. Similar to other viruses, studying SARS-CoV-2 requires the use of experimental methods to detect the presence of virus in infected cells and/or in animal models. To overcome this limitation, we generated replication-competent recombinant (r)SARS-CoV-2 that expresses bioluminescent (nanoluciferase, Nluc) or fluorescent (Venus) proteins. These reporter-expressing rSARS-CoV-2 allow tracking viral infections in vitro and in vivo based on the expression of Nluc and Venus reporter genes. Here the study describes the use of rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus to detect and track SARS-CoV-2 infection in the previously described K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of infection using in vivo imaging systems (IVIS). This rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus show rSARS-CoV-2/WT-like pathogenicity and viral replication in vivo. Importantly, Nluc and Venus expression allow us to directly track viral infections in vivo and ex vivo, in infected mice. These rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus represent an excellent option to study the biology of SARS-CoV-2 in vivo, to understand viral infection and associated COVID-19 disease, and to identify effective prophylactic and/or therapeutic treatments to combat SARS-CoV-2 infection.

This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has ...

Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by a high rate of transmission and low mortality, the COVID-19 pandemic necessitated accurate and efficient diagnostic testing, particularly in closed populations such as residential universities. Initial availability of nucleic acid testing, like nasopharyngeal swabs, was limited due to supply chain pressure which also delayed reporting of test results. Saliva-based reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) testing has shown to be comparable in sensitivity and specificity to other testing methods, and saliva collection is less physically invasive to participants. Consequently, we developed a multiplex RT-qPCR diagnostic assay for population surveillance of Clemson University and the surrounding community. The assay utilized open-source liquid handling robots and thermocyclers instead of complex clinical automation systems to optimize workflow and system flexibility. Automation of saliva-based RT-qPCR enables rapid and accurate detection of a wide range of viral RNA concentrations for both large- and small-scale testing demands. The average turnaround for the automated system was &#60; 9 h for 95% of samples and &#60; 24 h for 99% of samples. The cost for a single test was $2.80 when all reagents were purchased in bulk quantities.

The protocol describes a SARS-CoV-2 diagnostic method that utilizes open-source automation to perform RT-qPCR molecular testing of saliva samples. This scalable approach can be applied to clinical public health surveillance as well as to increase the capacity of smaller university laboratories.

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by ...