We acknowledge at SciLifeLab, Sweden, the Affinity Proteomics-Stockholm Scilifelab Unit team for developing and applying the method described here, the Human Antibody Therapeutics Unit for providing scFvs and Fab reagents, and Jonas Klingstr&#246;m for the VeroE6 cells infected with SARS-CoV-2 isolates stemming from clinical samples. The authors thank Sherry Dunbar, PhD, MBA of Luminex Corporation (Austin, TX), for research support, and Matt Silverman MSci, PhD of Biomedical Publishing Solutions (Panama City, FL; mattsilver@yahoo.com) for scientific and writing assistance. This work was supported by funds from the Knut and Alice Wallenberg Foundation and Science for Life Laboratory (SciLifeLab) (VC2020-0015 to Claudia Fredolini and Francesca Chiodi and VC-2022-0028 to Claudia Fredolini).

Analysis of SARS-CoV-2 Using Dual-Reporter and Multiplex Technology

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The authors declare no conflict of interest.

Bead-based multiplex technology has been shown to be a valuable platform for high-throughput pathogen detection in a number of clinical applications. The high flexibility of the platform, based on flow-cytometry principles, allows targeting antibodies, proteins, and nucleic acids18,19,20,21,22, multiplexing hundreds of analytes simultaneously. However, to our knowledge, this technology has not previously been applied to detect intact viral particles. In this report, the technology was applied for the detection of intact viral particles by targeting three independent surface epitopes of SARS-CoV-2.
Enveloped RNA viruses show high structural similarity to extracellular vesicles (EVs), small phospholipid membranes carrying viral RNA and proteins along with host proteins23. Sandwich immunoassays have previously been applied to the detection of EVs, using an antibody pair targeting two distinct surface proteins24,25. The limitation of sandwich assays to simultaneously detect only two proteins is removed with multiplex approaches that allow the simultaneous detection of more than two proteins per reaction.
The three-laser dual-reporter detection system described here is the most advanced bead-based flow analysis instrument to date. With respect to single-reporter readout systems, the dual-reporter (RP1 and RP2 channels) allows the detection of three surface proteins/epitopes in parallel. Targeting multiple viral surface proteins and epitopes provides a more accurate representation of the viral protein load, which, beyond confirming that the virus is in fact, intact, also opens up the opportunity to further investigate viral surface antigens and the mechanisms of the viral and host protein interactions.
During the COVID-19 pandemic, the importance of promptly identifying individuals carrying active viral particles was important in efforts to contain virus spread. Genomic RNA is detected by quantitative RT-PCR regardless of its origin (intact virus particles or free). However, only an intact envelope with accessible S protein can mediate cell entry and subsequent virus replication. Previous studies with microfluidic chips in patient samples have shown how the detection of intact viral particles combined with point-of-care testing would enable frequent testing and enhanced surveillance of disease spread, including a more informed choice of individuals to be quarantined26. The application of a multiplexed microsphere-based assay would allow for the design of assays aimed at the screening of multiple viruses and their surface antigen variants, obtaining a more accurate picture of virus spread in the population.
Flow virometry is a recent development of flow cytometry aiming at the analysis of viral particles. Despite being capable of detecting discrete viral particles, the analysis of small viruses poses a current issue for flow virometry27,28. Similarly to the method described here, flow virometry involves the capture of intact virions by gold nanoparticles coupled to antibodies. Limitations for both methods include (i) the dependence on high-affinity capture and detection reagents for surface-expressed antigen targeted by the microspheres or nanoparticles, (ii) limited capability to discriminate between virus particles and extracellular vesicles, and (iii) lack of standards for proper particle quantification.
Cells secrete EVs into their surroundings, and when infected by a virus, they may also secrete virions that are similarly sized as the EVs and may eventually express the same antigens29. Because the EVs will have similar membrane compositions as the virus, it could be hard to distinguish them from each other using only affinity-based methods such as the dual laser single-reporter approach. However, strategies described here feature a higher multiplex capacity, enabling a broader and deeper investigation of particles&#39; protein composition. Flow-based methods allow tracking of discrete particles, providing opportunities for digital quantification. One strategy to address the quantification issue in our method would be to use well-characterized synthetic vesicles expressing antigens of interest as virus-like particles (VLPs) for preparing standard curves.
A common path of entry and exit of SARS-CoV-2 from the host cells is through the interaction of the virus and host cell membrane2,15. In this process, the probability of host membrane proteins being incorporated into the virus surface is high. By screening incorporated host proteins, one can track the pathway of the infection and potentially predict disease&#160;course for different risk patients, allowing earlier treatment decisions. It also allows for the characterization of the viruses across different sample batches in research laboratories. This can be explored further by testing if different characteristics are related to different levels of viral infectivity and for screening of antibodies and drug molecules that target viral surface proteins.
An important aspect concerning the method described is that it relies on the affinity of the capture and detection reagents against their target proteins on the virus. The choice of affinity reagents is, therefore, a determining factor in assay performance. Possibly, multiple affinity reagents should be screened and tested for capture and detection to select those with the highest affinity. Here, the performance of ten scFvs and twelve Fab fragments was preliminarily assessed using recombinant RBD and on viral particles from the supernatant of SARS-Cov-2 infected Calu-3 lung epithelial cells (VeroE6 cells were used to culture/assess cytotoxicity in all subsequent studies). Anti-FLAG PE was used to detect the FLAG-tagged scFvs (Supplementary Table 1 and Supplementary Table 2). The five best-performing scFvs were then selected to be applied in the dual-reporter assay, together with commercial Hu-anti-S1 (Table 1), on supernatants from infected VeroE6 African green monkey kidney epithelial cells.
Another critical factor for the protocol&#39;s success is the procedure selected for microsphere coupling. The coupling method should be efficient and, at the same time, keep conformational epitopes or amino acid residues involved in the protein binding intact and unmodified. Here, the EDC-NHS reaction was applied to couple neutravidin directly to microspheres, adapting a protocol previously described30 and a neutravidin + biotin system to bind recombinant ACE2 to the coupled microspheres. Alternative coupling methods and their efficiency can be tested and compared. Finally, it was observed that different fluorescently labeled detection reagents (e.g., anti-FLAG PE (phycoerythrin) and anti-FLAG Brilliant Violet 421) may result in different MFI levels that may affect assay sensitivity.
In conclusion, the method described allows the detection of intact viral particles in solution, applying a dual-reporter strategy. The analysis of three surface determinants in parallel provides a more specific tool to characterize viral particles and eventually discriminate them from other EVs (e.g., not containing viral antigens). This strategy is an alternative to flow virometry. Although the current approach does not discriminate particle sizes, magnetic bead strategies using color-coded microspheres offer a broader capability in surface antigen profiling and experimental design by high-multiplex and high-throughput analysis. The assay shows high precision and robustness and can be extended to the analysis of any type of extracellular vesicle and any other type of bioparticle exposing surface antigens in body fluids or other liquid matrices. This was a proof-of-concept study that demonstrated the utility of using scFvs as one detection reagent in a multiplex analysis of multiple protein epitopes on viral particles. Future studies are necessary to determine the specific characteristics of scFvs (e.g., binding affinities, cross-reactivity with other reagents and targets) if they are to be used for quantitative or clinical purposes.

The most common pathogenic viruses, such as influenza, HIV, human cytomegalovirus, and SARS-CoV strains, are enveloped viruses. Cell infection by enveloped viruses requires the fusion of viral and host cell membranes, resulting in the release of the viral genome into the cytoplasm. Viral RNA will then replicate before being packed into a new viral particle1,2. During these processes, not only viral proteins but also host membrane proteins may be incorporated into the envelope, becoming an integral part of the new viral particle. Host cell membrane proteins incorporated into the virus envelope may facilitate virus entry into a new host cell, exploiting the mechanisms of cell-cell interactions, homing, and immune system escape3,4.
Despite the importance of investigating virus-associated proteins, most of the currently available techniques for virus analysis5 do not support high-throughput and high-multiplex characterization of virus surface antigen. Neither are they capable of detecting individual viral particles or of discriminating between infectious intact virus particles, non-infectious RNA, viral proteins, and virus subpopulations expressing different antigens. Recently, flow cytometry has been modified and adapted into a novel method for the analysis of viral particles, namely, flow virometry. Flow virometry allows the investigation of single viral particles and their surface antigens. However, limitations including low throughput, low multiplex capability, complicated experimental setup and data analysis, and limited detectability of small-sized viral particles remain6,7.
Microsphere-based multiplexed quantification of proteins and nucleic acid is a well-established technology with numerous applications ranging from protein quantification in body fluids, protein-protein interaction studies, and diagnosis of viral infections8,9,10,11,12,13. A recently introduced flow analysis instrument features a dual-reporter channel, allowing the measurement of two fluorescent reporter molecules in the same reaction well. This new capability has shown to be particularly useful for the parallel profiling of different immunoglobulin isotypes14. Here, it is described how the dual reporter system can be used to detect intact viral particles, targeting multiple surface antigens in parallel.
As a proof of concept, this report details the development of a triple-detection system for SARS-CoV-2 virus particles. SARS-CoV-2 consists of four main proteins, one is the spike protein (S), which consists of two subunits. The first subunit, S1, makes the primary binding to the ACE2 expressed in human cell membranes. The second subunit, S2, facilitates entry into the target cell by a fusion peptide, creating a pore in the target cell membrane that the virion can enter through15. The three remaining building blocks of SARS-CoV-2 are the nucleocapsid (N), the membrane protein (M), and the envelope protein (E). The nucleocapsid is responsible for the packaging of the viral genome by forming ribonucleoprotein structures with RNA, while the membrane and envelope proteins play central roles in the virus assembly.
The assay described here targets three independent epitopes of the S1 subunit expressed on the envelope surface of SARS-CoV-2. Serial dilutions of both SARS-CoV-2 infected and uninfected cell supernatants are used. Viral particles are captured via ACE2-conjugated microspheres binding the S1 subunit on the virus. Surface virus S protein is then detected in parallel with a commercialy-available tagged immunoglobulin single-chain variable fragment (scFv) and a human monoclonal anti-S1 antibody (Hu-anti-S1) together with an in-house developed FLAG-tagged scFv. The Hu-anti-S1 is detected by the first channel (RP1) in the dual-reporter system with orange R-phycoerythrin (PE)-conjugated anti-human IgG-Fc secondary antibody, and the scFv is detected by the second channel (RP2) with a blue Brilliant Violet 421 (BV421)-conjugated secondary anti-FLAG antibody. The virus particle assay is represented in Figure 1.

<table><tbody><tr><td>ACE2-Biotin</td><td>Acro Biosystems (Newark, DE)</td><td>AC2-H82E6-25 ug</td><td>Conc: 340 &amp;micro;g/mL, LOT#BV35376-203HFI-2128</td></tr><tr><td>Anti-Goat IgG, PE-conjugated</td><td>Jackson ImmunoResearch (West Grove, PA)&amp;nbsp;</td><td>705-116-147</td><td>Host species: Donkey&amp;nbsp;</td></tr><tr><td>Anti-Human IgG R-PE&amp;nbsp;</td><td>Life Technologies/Thermo Fisher (Waltham, MA)</td><td>H10104</td><td>Conc: 0.15 mg/mL, LOT#2079224, Host species: Goat</td></tr><tr><td>Anti-Mouse IgG, PE-conjugated</td><td>Jackson ImmunoResearch (West Grove, PA)&amp;nbsp;</td><td>115-116-146</td><td>Host species: Goat&amp;nbsp;</td></tr><tr><td>Anti-Rabbit IgG, PE-conjugated&amp;nbsp;</td><td>Jackson ImmunoResearch (West Grove, PA)&amp;nbsp;</td><td>111-116-144</td><td>Host species: Goat&amp;nbsp;</td></tr><tr><td>Biotin</td><td>Thermo-Fisher Scientific (Waltham, MA)</td><td>20RUO</td><td>100 mM, pH 10 Conc. 1 mg/mL</td></tr><tr><td>Blocker Casein in PBS</td><td>Thermo-Fisher Scientific (Waltham, MA)</td><td>37528</td><td>LOT#VD301372</td></tr><tr><td>Blocker reagent for ELISA (BRE)</td><td>Roche (Basel, Switzerland)</td><td>11112589001</td><td></td></tr><tr><td>Brilliant Violet 421 anti-DYKDDDDK Tag Antibody (Anti-FLAG) 0.2 mg/ml, rat IgG2a, &amp;lambda;</td><td>BioLegend (Amsterdam, The Netherlands)</td><td>637321</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Saveen &amp;amp; Werner (Limhamn, Sweden)</td><td>B2000-500</td><td>LOT#04D5865</td></tr><tr><td>EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride)</td><td>Proteochem (Hurricane, UT)</td><td>C1100-custom (65 mg)</td><td>LOT# MK3857</td></tr><tr><td>Fetal calf serum (FCS)</td><td>Gibco/Thermo Fisher (Waltham, MA)</td><td>10270-106</td><td></td></tr><tr><td>Goat anti-ACE2 polyclonal antibody</td><td>R&amp;amp;D Systems/Bio-Techne (Minneapolis, MN)</td><td>AF933</td><td>Host species: Goat&amp;nbsp;</td></tr><tr><td>Goat IgG</td><td>Bethyl Labs (Montgomery, TX)</td><td>P50-200</td><td>LOT#P50-200-6</td></tr><tr><td>L-glutamine</td><td>Thermo-Fisher Scientific (Waltham, MA)</td><td>25030024</td><td></td></tr><tr><td>Low-bind 1.5 mL microfuge tubes&amp;nbsp;</td><td>VWR (Radnor, PA)</td><td>525-0133</td><td></td></tr><tr><td>MagPlex-C Microspheres</td><td>Luminex Corporation (Austin, TX)</td><td>MC10XXX-01</td><td></td></tr><tr><td>MEM tissue cuture media</td><td>Gibco/Thermo Fisher (Waltham, MA)</td><td>21430-020</td><td></td></tr><tr><td>Microplate, 96-Well, Polystyrene, Half-area, Clear</td><td>Greiner Bio-One (Kremsm&amp;uuml;nster, Austria)</td><td>675101</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Gibco/Thermo Fisher (Waltham, MA)</td><td>25080-060</td><td></td></tr><tr><td>Neutravidin&amp;nbsp;</td><td>Thermo-Fisher Scientific (Waltham, MA)</td><td>31000</td><td>LOT#UK292857</td></tr><tr><td>PBS tablets</td><td>Medicago AB (Uppsala, Sweden)</td><td>09-9400-100</td><td>LOT#272320-01</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco/Thermo Fisher (Waltham, MA)</td><td>15140122</td><td></td></tr><tr><td>Poly(vinyl alcohol)</td><td>Sigma-Aldrich (St. Louis, MO)&amp;nbsp;</td><td>360627</td><td></td></tr><tr><td>Polyvinylpyrrolidone</td><td>Sigma-Aldrich (St. Louis, MO)&amp;nbsp;</td><td>437190</td><td></td></tr><tr><td>ProClin 300</td><td>Sigma-Aldrich (St. Louis, MO)&amp;nbsp;</td><td>48915-U</td><td></td></tr><tr><td>Rabbit IgG</td><td>Bethyl Labs (Montgomery, TX)</td><td>P120-301</td><td>LOT#12</td></tr><tr><td>scFv-FAb1</td><td>In-house production</td><td></td><td>Human Antibody Therapeutics Unit, Scilifelab, Sweden. Monoclonal scFv. Conc: 0.12 mg/mL.</td></tr><tr><td>scFv-FAb2</td><td>In-house production</td><td></td><td>Human Antibody Therapeutics Unit, Scilifelab, Sweden. Monoclonal scFv. Conc batch1: 0.38 mg/mL. Conc batch2: 0.45 mg/mL</td></tr><tr><td>scFv-FAb3</td><td>In-house production</td><td></td><td>Human Antibody Therapeutics Unit, Scilifelab, Sweden. Monoclonal scFv. Conc: 0.34 mg/mL.</td></tr><tr><td>scFv-FAb4</td><td>In-house production</td><td></td><td>Human Antibody Therapeutics Unit, Scilifelab, Sweden. Monoclonal scFv. Conc: 2.85 mg/mL.</td></tr><tr><td>scFv-FAb5</td><td>In-house production</td><td></td><td>Human Antibody Therapeutics Unit, Scilifelab, Sweden. Monoclonal scFv. Conc:2.7mg/mL.</td></tr><tr><td>SARS-CoV-2 infectious particles, Swedish isolate</td><td>In-house production&amp;nbsp;</td><td></td><td>The Public Health Agency of Sweden</td></tr><tr><td>SARS-CoV-2 Spike Antibody (Hu-anti-S1)</td><td>Novus Biologicals (Centennial, CO)</td><td>NBP2-90980&amp;nbsp;</td><td>Monoclonal antibody. Conc: 1 mg/mL. Host: Human. Clone: CR3022. Isotype: IgG1 Kappa. LOT#T201B06</td></tr><tr><td>Sodium phosphate monobasic, anhydrous</td><td>Sigma-Aldrich (St. Louis, MO)&amp;nbsp;</td><td>S3139</td><td></td></tr><tr><td>Sulfo-NHS (N-hydroxysulfosuccinimide)</td><td>Thermo-Fisher Scientific (Waltham, MA)</td><td>24510</td><td>LOT# XH321563</td></tr><tr><td>Tween</td><td>Thermo-Fisher Scientific (Waltham, MA)</td><td>BP337-50</td><td>LOT#194435</td></tr><tr><td>Ultraviolet lamp</td><td>Vilber Lourmat GmbH (Eberhardzell, Germany)</td><td>VL-215.G</td><td>Wavelength = 254 nm; 2 &amp;times; 15-watt bulbs</td></tr><tr><td>Vero E6 cells</td><td>ATCC (Manassus, VA)</td><td>CRL-1586</td><td></td></tr><tr><td>xMAP INTELLIFLEX DR-SE (dual-reporter flow instrument)</td><td>Luminex Corporation (Austin, TX)</td><td>INTELLIFLEX-DRSE-RUO</td><td></td></tr></tbody></table>

profiling of surface protein epitopes on viral particles by multiplex dual-reporter strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

Conjugation test 
The conjugation test showed that goat-IgG and neutravidin-biotinylated ACE2 were successfully conjugated to the microspheres. The assay detection specificity was confirmed by probing ACE2-conjugated microspheres with PE-labeled secondary antibodies generated in different animal species (Figure 2). No cross-reactivity between the different detection antibodies was observed. When the bead mixtures were probed with goat anti-ACE2 + anti-goat IgG PE, a median fluorescence intensity (MFI; arbitrary units) value above the background was detected for both ACE2 and goat IgG-conjugated microspheres but not for the unconjugated microsphere (bare) or for the biotin-coated microspheres. Anti-mouse IgG PE and anti-rabbit IgG PE were used as negative controls to check for false-positive signals. A negligible fluorescence signal was generated upon incubation with the microspheres, indicating that the positive signals for the ACE2 and the goat IgG were specific.
Viral particle detectability in cell supernatants 
Magnetic beads coupled to recombinant human ACE2 were used to capture SARS-CoV-2 viral particles from infected and control (no virus) VeroE6 cell culture supernatants and were then simultaneously probed for two distinct viral spike regions using a monoclonal antibody and one of five distinct scFvs. A concentration-dependent signal in the dilutions of SARS-Cov-2 infected cell supernatants was observed in both reporter channels (RP1 and RP2) (Figure 3), indicating that both the commercial Hu-anti-S1 antibody and the different scFvs detected the viral particle bound to the ACE2-conjugated microsphere. With three out of five scFvs, the virus is detectable in dilutions down to 1:18 (scFv2, scFv3, scFv5); for the remaining two scFvs (scFv7 and scFv9), it is detectable down to 1:6 dilutions. This could be attributable to a different affinity for the target. As shown in Figure 3 and Table 1, scFv3 provides the highest MFI intensity, followed by scFv5, scFv2, scFv7, and scFv9, respectively.
Globally, scFvs detection results in lower MFI in comparison to the Hu-anti-S1. This could indicate lower affinity, but it could also be an artifact due to the labeling with different fluorescent dyes (PE and BV421). Another trend that can be seen for scFv7 and scFv9 is that the MFI values are slightly lower for the RP1 channel (anti-spike) as well compared to the other three configurations. This could indicate that the scFvs are either cross-reacting or interfering in another way with the ACE2-Hu-anti-S1 interaction, which could also explain the lower signal in the RP2 channel. No viral particles were detected in the supernatant of the non-infected Vero E6 cells in either the RP1 or the RP2 channel.
The neutravidin-biotin conjugated microsphere, the goat-IgG microsphere, and the unconjugated microspheres are used as negative control beads. The viral particles were captured with magnetic microspheres coupled to ACE2 and tested with commercial human anti-spike in the RP1 reporter channel and with different scFvs in the RP2 reporter channel (scFv is indicated in the top left of each panel). No virus particles were detected in any of the infected and non-infected samples.
Assay precision and robustness 
To evaluate assay precision, all the conditions were run in triplicate. A coefficient of variance (CV) for the ACE2 microsphere was calculated for each dilution point. All of the calculated CVs for the assay were below 15%, where the highest measured CV was 13%, and the lowest CV was 1% (Table 2). As can be seen in the density plot (Figure 4) of the RP1 channel, PE detection of the commercial Hu-anti-S1 shows higher precision, mainly concentrated around a CV of 3%. The RP2 channel, BV detection of scFVs, shows higher CVs. However, as can be seen in Table 2, the higher range of CVs is driven by the samples with low concentrations of viral particles, such as the blank. To test the robustness of the protocol, the assay was repeated twice by different operators, using bead mixtures generated on different days and a lower sample volume (72% lower). A very good Pearson correlation, ranging between 0.98 and 1, was observed for both the RP1 and the RP2 channels (p-value &#60; 0.01), confirming assay robustness and the possibility of applying the assay when less sample is available (Figure 5). This flow analysis technology follows the &#34;ambient analyte theory&#34;17, making the assay sensitive to concentration but not volume.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66230/66230fig01.jpg" /> 
Figure 1: The virus particle assay. (A) Cell supernatant from both infected and un-infected Vero E6 cells are added in a serial dilution to either a 96-well or 384-well plate, together with magnetic microspheres conjugated with neutravidin, and then coupled to either biotinylated human ACE2 or Biotin. Unconjugated microspheres coupled with goat-IgG and bare microspheres are used as negative controls together with the neutravidin-biotin conjugated microsphere. (B) Microsphere-virus particle complexes that have formed are detected with a detection cocktail consisting of Hu-anti-S1 and one of the different scFvs with FLAG-tag. A fluorescent mix with anti-human IgG PE targeting the Hu-anti-S1 and anti-FLAG Brilliant Violet 421 targeting the scFvs is then added. (C) The three-laser, dual-detection system emits a red, green, and violet laser to detect the microparticle complex. The red laser detects the microsphere dye label, while the green and violet lasers detect the anti-S1 and the scFvs, respectively. The generated data are then analyzed. <a href="https://www.jove.com/files/ftp_upload/66230/66230fig01large.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/66230/66230fig02.jpg" /> 
Figure 2: Conjugation confirmation plot. The bead mixtures consisted of four different microsphere IDs, each conjugated with a different protein: neutravidin-biotin-ACE2 (ACE2), unconjugated microsphere (Bare Bead), neutravidin-biotin (Biotin), and goat-IgG (Goat IgG). In the conjugation test three different configurations of detection fluorophores were used. Namely, goat anti-ACE2 + anti-goat IgG PE, anti-mouse IgG PE, and anti-rabbit IgG PE. The Y-axis shows the average measured MFI (median fluorescence intensity; arbitrary units) signal from each microsphere with the three different conditions. The X-axis shows the different capture antibodies applied. <a href="https://www.jove.com/files/ftp_upload/66230/66230fig02large.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/66230/66230fig03.jpg" /> 
Figure 3: Multiplexed detection of surface proteins. Y-axis: Mean MFI (median fluorescence intensity; arbitrary units &#177; standard deviation) for each sample, analyzed in triplicate wells per condition. X-axis: Serial dilution points of cell supernatant. Orange: Virus particles in supernatant from Vero E6 infected with SARS-CoV-2 WT detected with human anti-spike + anti-human PE (phycoerythrin). Blue: Supernatant from Vero E6 infected with SARS-CoV-2 WT detected with the different scFvs + anti-FLAG Brilliant Violet 421. Grey: Non-infected cell supernatant detected with human anti-spike + anti-human PE. Black: Non-infected cell supernatant detected with the five different scFvs + anti-FLAG Brilliant Violet 421. The viral particles were captured with magnetic microspheres coupled to ACE2 and tested with commercial human anti-spike antibodies in the RP1 reporter channel and with different scFvs in the RP2 reporter channel (scFv is indicated in the top left of each panel). No virus particles were detected in any of the non-infected samples. The epitope targeted by scFv3 had the highest affinity. <a href="https://www.jove.com/files/ftp_upload/66230/66230fig03large.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/66230/66230fig04.jpg" /> 
Figure 4: Variation dispersion plot. The Y-axis is the frequency of events, and the X-axis shows the coefficient of variance (CV) in percentage for each replicate of the different samples. RP1 and RP2 are the first and second reporter channels that detect fluorescence associated with phycoerythrin and Brilliant Violet 421, respectively. <a href="https://www.jove.com/files/ftp_upload/66230/66230fig04large.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/66230/66230fig05.jpg" /> 
Figure 5: Run correlation matrix. (A,B) Y-axis: Pearson correlation matrix in log10-scale between three separate runs, run by three different operators and with different bead mixtures. A lower sample volume was applied in the third run. The histograms show the distribution of the different variable clusters based on measured MFI. (A) Correlation for the RP1 reporter channel between the different runs. (B) Correlation for the RP2 reporter channel between the different runs. MFI=median fluorescence intensity in arbitrary units. ***p &#60; 0.001. <a href="https://www.jove.com/files/ftp_upload/66230/66230fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Detection</td>
			<td>Reactivity</td>
		</tr>
		<tr>
			<td>scFv2</td>
			<td>++</td>
		</tr>
		<tr>
			<td>scFv3</td>
			<td>+++</td>
		</tr>
		<tr>
			<td>scFv5</td>
			<td>++</td>
		</tr>
		<tr>
			<td>scFv7</td>
			<td>+</td>
		</tr>
		<tr>
			<td>scFv9</td>
			<td>+</td>
		</tr>
		<tr>
			<td>Human anti-Spike IgG</td>
			<td>++++</td>
		</tr>
	</tbody>
</table>
Table 1: Ranking of scFvs in detection based on the MFI intensity obtained in the standard curves. 
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>RP1 (PE)</td>
			<td>RP2 (BV421)</td>
		</tr>
		<tr>
			<td>Sample Dilution</td>
			<td>CV range [%]</td>
			<td>CV range [%]</td>
		</tr>
		<tr>
			<td>Blank</td>
			<td>3&#8211;11</td>
			<td>2&#8211;13</td>
		</tr>
		<tr>
			<td>1:1458</td>
			<td>1&#8211;7</td>
			<td>2&#8211;7</td>
		</tr>
		<tr>
			<td>1:456</td>
			<td>4&#8211;6</td>
			<td>3&#8211;8</td>
		</tr>
		<tr>
			<td>1:162</td>
			<td>3&#8211;6</td>
			<td>3&#8211;7</td>
		</tr>
		<tr>
			<td>1:54</td>
			<td>2&#8211;4</td>
			<td>2&#8211;4</td>
		</tr>
		<tr>
			<td>1:18</td>
			<td>2&#8211;4</td>
			<td>1&#8211;4</td>
		</tr>
		<tr>
			<td>1:6</td>
			<td>2&#8211;6</td>
			<td>1&#8211;6</td>
		</tr>
		<tr>
			<td>1:2</td>
			<td>1&#8211;5</td>
			<td>1&#8211;3</td>
		</tr>
	</tbody>
</table>
Table 2: CV% (mean/standard deviation &#215; 100) range of each dilution point of the SARS-CoV-2 infected supernatant for both the RP1 and the RP2 reporter channels.
Supplementary File 1: Immunoglobulin single-chain variable fragment (scFv) generation. <a href="https://www.jove.com/files/ftp_upload/66230/Supplementary File 1.docx" target="_blank">Please click here to download this File.</a>
Supplementary Table 1: Screening scFvs in pairs with Fabs against serial dilution of recombinant Spike (RBD). To evaluate the performance of different detection peptides, 12 combinations of spike protein, Fab, were used as capture in buffer spiked with recombinant RBD. Ten (10) scFvs targeting different epitopes of the spike protein were applied as detection. Depending on the performance of the capture-detection pair, they were either marked as failed (-) or successful (+). <a href="https://www.jove.com/files/ftp_upload/66230/Supplementary Table 1.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Table 2: Screening scFvs in pairs with Fabs against serial dilution of SARS-Cov-2 infected Calu-3 cell supernatant. For evaluation of different detection peptides performance, 12 combinations of spike protein, Fab, were used as capture in SARS-Cov-2 infected Calu-3 cell supernatant. Ten (10) scFvs targeting different epitopes of the spike protein were applied as detection. Depending on the performance of the capture-detection pair, they were either marked as failed (-) or successful (+). <a href="https://www.jove.com/files/ftp_upload/66230/Supplementary Table 2.xlsx" target="_blank">Please click here to download this File.</a>

Read this Scientific Article Protocol about Author Spotlight: Advancing Antiviral Strategies through Novel Immunocapture and Mass Spectrometry Techniques at JoVE.com

Author Spotlight: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

profiling-surface-protein-epitopes-on-viral-particles-multiplex-dual

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Immunology and Infection

637 Views.  KTH Royal Institute of Technology. Proteomische analyse van het envelopvirus onthulde dat gastheereiwitten worden opgenomen in nieuw gevormd virus, wat de replicatie, infectiviteit en pathogenese beïnvloedt. Ons onderzoek richt zich op het profileren van oppervlakte-eiwitten in virale deeltjes om sleutelelementen in de interactie tussen virus en gastheer te vinden, wat leidt tot nieuwe doelen voor antivirale middelen en vaccins. Immuno-elektronenmicroscopie is de enige beeldvormingstechniek d...

Video: Auteur in de schijnwerpers: het bevorderen van antivirale strategieën door middel van nieuwe immunocapture- en massaspectrometrietechnieken

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent 1, 2. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene 3, 4. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction 5, 6.
There are three main components necessary for efficient lentiviral packaging 1, 5, 6, 7. 
1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 
2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 
3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance.
An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at:
<a href="http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf" target="_blank">http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf</a> 8.
This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification1.
A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs).
The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types2,3. However, no single &#x2018;marker&#x2019; has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. 
A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. 
In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response5, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody6. 
For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells7. The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.

We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray). Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes.

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase ...

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens.
This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera.
Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes.

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge ...

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 Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate antigen-displaying VLPs. Surface antigen-specific antibodies are coupled to, and thus immobilized on a solid and insoluble matrix such as beads. Due to their high affinity to the target antigen, these antibodies can capture VLPs decorated with the cognate antigen anchored in the membrane envelope of the VLPs. This protocol describes the binding of antigen-specific antibodies to protein A- or G-conjugated magnetic beads. In our study, human immunodeficiency virus (HIV)-derived VLPs formed by the group-specific antigen (Gag) viral core precursor protein p55 Gag and displaying the envelope glycoproteins (Env) of HIV are examined. The VLPs are captured utilizing broadly neutralizing antibodies (bNAbs) directed against neutralization-sensitive epitopes in Env. The VLP capture assay outlined here represents a sensitive and easy-to-perform method to demonstrate that (i) the VLPs are decorated with the respective target antigen, (ii) the surface antigen retained its structural integrity as demonstrated by the epitope-specific binding of bNAbs used in the assay and (iii) the structural integrity of the VLPs revealed by the detection of Gag proteins in a subsequent Western blot-analysis. Consequently, the utilization of bNAbs for immunoprecipitation facilitates a prediction of whether VLP vaccines will be able to elicit a neutralizing B cell response in vaccinated humans. We anticipate that this protocol will furnish other researchers with a valuable and straightforward experimental approach to examine potential VLP-based vaccines.

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle VLP-Based Vaccines Using a Capture Assay

Here, we present a protocol to detect neutralization epitopes on antigen-displaying virus-like particles (VLPs). Immunoprecipitation of the human immunodeficiency virus (HIV)-derived VLPs is performed using envelope glycoproteins-specific monoclonal antibodies coupled to protein G-conjugated magnetic beads. Captured VLPs are subsequently subjected to SDS-PAGE and Western blot-analysis employing viral core protein Gag-specific antibodies.

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate ...

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

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.

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

Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes on the same analyte remain limited. This report describes the development and optimization of a multiplexed immunobead assay for serological testing of common immunoglobulin isotypes (e.g., IgA, IgM, and IgG) associated with an immune response to SARS-CoV-2 infection or vaccination. Assays were accomplished using a flow-based, multiplex fluorescent reader with dual-channel capability. Optimizations focused on analyte capture time, detection antibody concentration, and detection antibody incubation time. Analytical assay performance characteristics (e.g., assay range (including lower and upper limits of quantitation); and intra- and inter-assay precision) were established for either IgG/IgM or IgA/IgM serotype combination in tandem using the 'dual channel' mode. Analyte capture times of 30 min for IgG, 60 min for IgM, and 120 min for IgA were suitable for most applications, providing a balance of assay performance and throughput. Optimal detection antibody incubations at 4 &#181;g/mL for 30 min was observed and are recommended for general applications, given the overall excellent precision (percent coefficient of variance (%CV) &#8804; 20%) and sensitivity values observed. The dynamic range for the IgG isotype spanned several orders of magnitude for each assay (Spike S1, Nucleocapsid, and Membrane glycoproteins), which supports robust titer evaluations at a 1:500 dilution factor for clinical applications. Finally, the optimized protocol was applied to monitoring Spike S1 seroconversion for subjects (n = 4) that completed a SARS-CoV-2 vaccine regimen. Within this cohort, Spike S1 IgG levels were observed to reach maximum titers at 14 days following second dose administration, at a much higher (~40-fold) signal intensity than either IgM or IgA isotypes. Interestingly, we observed highly variable Spike S1 IgG titer decay rates that were largely subject-dependent were observed, which will be the topic of future studies.

This article describes a convenient method for monitoring dynamic changes in antibody titers for two immunoglobulin isotypes concurrently (either IgA, IgM, or IgG) resulting from an immune response to SARS-CoV-2 infection or vaccination. This 'Multianalyte Covid-19 Immune Response Panel' employs three indirect immunoassays built on coded microspheres that are read using a flow-based multiplex reader with 'dual-channel' capability.

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes ...

Medicine

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Samenvatting

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