When Dr. Stephen Smith of Seattle Children’s Research Institute came down with muscle aches, gastrointestinal distress and a sudden loss of smell in late February, he suspected he had COVID-19.
The testing criteria had yet to be expanded to include individuals with Smith’s symptoms and so he did what many scientists with his expertise would do: he developed a way to test himself.
The fruits of his curiosity, now published in The Journal of Infectious Diseases, offer a reliable way to quantify whether an individual has neutralizing antibodies that could prevent the novel coronavirus from infecting cells using a method that is more broadly applicable than those currently available.
“If you think you’ve had COVID-19 and go to the doctor, they can test your blood and tell you whether or not you have antibodies to COVID-19, but that doesn’t tell you whether your antibodies are any good at functionally blocking the virus from binding to cells,” Smith said. “
There are tests on the market now that can tell you that, but they are expensive and take a long time to get results. We wanted to develop a way to give you additional information about your immune status without all the barriers that make it difficult to use in a community setting.”
The newly developed diagnostic could have a range of potential commercial applications from broad community testing to assessing vaccine responses and screening for convalescent plasmas that have particularly high levels of neutralizing antibodies as a potential treatment.
Cell-free test looks at protein interactions
The novel coronavirus enters cells when the viral spike protein binds to the ACE2 protein on the surface of human cells. Neutralizing antibodies that block this binding are thought to contribute to immunity to the virus in people who recover from COVID-19.
Smith applied a technique called immunoprecipitation detected by flow cytometry (IP-FCM) to study the interactions between the proteins and to look for evidence that antibodies were inhibiting the interaction and blocking the virus from binding to cells.
Instead of relying on live cells and viruses like other available blood tests, IP-FCM uses recombinant—or lab-made—proteins and instruments commonly available in commercial serological labs.
“Other tests that provide insight into immunity work by taking antibodies from your blood and mixing them together with a virus and then exposing that mixture to live cells.
Three days later they can determine immunity based on whether your blood prevented the viruses from infecting the cells or not,” Smith said. “Our cell-free test can provide that same information overnight.”
Collaborative science launches innovative study
Smith is among a small group of scientists in the U.S. who have pioneered IP-FCM to study the interactions between proteins. His lab in Seattle Children’s Center for Integrative Brain Research uses IP-FCM to uncover new treatments for autism by studying the more than 100 genetic variations known to contribute to the condition.
To apply his expertise to the current pandemic, Smith collaborated with Drs. Lisa Frenkel and Whitney Harrington from the research institute’s Center for Global Infectious Global Disease Research who are following a community cohort of Seattle Children’s employees who were never hospitalized and had recovered from mild to moderate COVID-19.
The researchers hope by tracking their recovery and taking blood samples over time as part of the Seattle Children’s Recovered SARS2 Cohort study they can shed light on the immune responses to the novel coronavirus.
Funding in part by Seattle Children’s COVID-19 Research Fund helped Smith design and launch the study.
Using IP-FCM, Smith tested the blood samples from 24 cohort participants.
The test showed that 92% of the participants had antibodies to the novel coronavirus at an average of a little over a month post-infection. Results were validated with 30 control samples.
“Not only did the participants have antibodies, but our test also showed that their antibodies were pretty effective at neutralizing the binding between the spike protein and the cell’s receptor,” he said.
“It’s consistent with other studies from cell-based tests showing that people who get COVID do make neutralizing antibodies.”
Interestingly, when researchers looked at the test results against other data gathered from the cohort, they found that those who mounted a fever had higher levels of antibodies. The research team also plans to retest the samples to see how antibody levels change over time.
“It’s going to be very important to look at people over a longer time period to track their antibody levels and whether or not they get re-infected,” Smith said. “Until we do those studies, we really don’t know how these clinical measures of antibody neutralization relate to susceptibility in the real world.”
Identifying new drug candidates for COVID-19
In addition to exploring opportunities to commercialize the diagnostic, Smith and his team are now using the test to rapidly screen thousands of approved drugs that could potentially interfere with the binding between ACE2 and the spike protein.
Lab manager, Edward Gniffke, and Stanford University undergraduate and summer intern, Kaleb Tsegay, helped run the initial screen that could potentially identify drugs capable of preventing or treating COVID-19.
“We already have some compounds that look like they are inhibiting, which is pretty exciting,” Smith said. “This first line screen will help us pinpoint the most promising agents for further tests.”
Flow cytometry is a laser-based cell biology technique that is used to analyze, count and sort cells of interest from a mixed population. Fluorescence-activated cell sorting (FACS) is a method of choice for analysis and purification of isolated single cells (viz., bacteria, algae, plant and animal cells).
FACS can detect and discriminate cells as well as suspended particles by its properties of light scattering and fluorescence (excitation/emission mode). The fluorescence of cells may be obtained using specific fluorochrome reagents, or by using antibodies tagged with a fluorochrome targeted against a cell surface antigen and/or internal constituents in permeabilized cells.
Flow cytometry has been used for monitoring cells expressing fluorescent proteins (e.g., GFP) [1] and undergoing DNA replication and cell cycle as well as apoptosis. The tool has also been used for immunophenotyping.
FACS has been successfully used for generating qualitative and quantitative data in a broad range of biomedical, clinical and therapeutic research, thereby widening its applications from research to clinical studies.
The analysis of viruses by flow cytometry was termed as ‘flow virometry’ [2,3].
The recent coronavirus pandemic COVID-2019 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The virus belongs to the Sarbecovirus subgenus (genus Betacoronavirus, family Coronaviridae). This virus has unique clinical characteristics and is highly contagious with unclear pathological mechanisms.
The virus can cause a life-threatening respiratory illness in humans, especially to the people in their late 50s and above, with or without co-morbidities such as diabetes, kidney diseases, heart diseases, etc.
Early symptoms of SARS-CoV-2 infection are very similar to that of mild-to-moderate flu, which makes it extremely difficult to classify and shortlisting infected individuals. In addition, inefficient contact tracing of infected individuals is becoming increasingly hard.
Until a successful treatment strategy is appropriately identified, the key to managing this pandemic is greatly dependent on quick and faster detection of infected individuals, followed by isolation of patients from the healthy population.
Presently, the detection of the COVID-19 is done by quantitative real-time PCR (qRT-PCR) using unique set of PCR primers.
The test detects the viral RNA in the samples, and the first step of the process is making cDNA from viral RNA using the reverse transcriptase enzyme.
Immunological detection of SARS-CoV-2 antibodies in the blood of infected humans is another method being used for development of rapid detection kits using lateral flow immunochemistry.
Reverse transcriptase – LAMP (loop-mediated isothermal amplification) is another potential detection system for SARS-CoV-2, which uses isothermal amplification of the viral nucleic acid using specially designed oligo-nucleotide primers. Several PCR-based high-throughput diagnostics kits for detection of SARS-CoV-2 are also under development [4].
qRT-PCR, despite being a gold standard and a widely used tool, is time consuming and the throughput of the system is low. The antibody-based detection is not accurate and is prone to false or nonspecific results. Therefore, to cover the larger population, we need an accurate, high-throughput and faster sample testing tool for screening.
India is among the top three countries with respect to the number of COVID-19 cases and over the last 2 months the rate of infection has grown exponentially.
At present, India is testing close to nine thousand people per million of population, which is clearly insufficient, and therefore a large-scale and robust screening process needs to be developed.
Recently, a human monoclonal antibody that binds to a conserved receptor-binding domain on the spike of the SARS-CoV-2 has been reported [5].
Such monoclonal antibodies against SARS-CoV-2 will be useful for development of antigen detection tests and serological assays. Döhla et al. reported 88.9% specificity in qRT-PCR, whereas 36.4% specificity was reported in antibody-based rapid detection kits for diagnosis of COVID-19 cases [5].
Here, we recommend an approach for diagnosis of SARS-CoV-2 by screening of test samples (swabs) using flow cytometry. In this approach, we have proposed a process of indirect immunofluorescence where the virus particles are first bound to primary antibodies followed by the complex being labeled by fluorescent secondary antibodies for detection in a flow cell.
Process & method of COVID-19 detection
In this communication, we have discussed a process of advanced flow virometry to enhance testing scale of COVID-19 cases. SARS-CoV-2 belongs to the family Coronaviridae, consisting of a 29–30 kb chain of positive single-stranded RNA. The virus particle size ranges from 70 to 90 nm [23].
Extensive studies on the dengue virion (40–60 nm) was done using combination of fluorescently labeled antibodies and MNPs [9]. Here, we propose the approach of labeling the surface of the viral particles with antigen-specific primary antibodies and secondary antibody conjugated to a fluorescent dye (e.g., fluorescein isothiocyanate, PE, Cy5®, etc.) to detect the SARS-CoV-2.
Proposed steps for sample preparation and assay are discussed in following points (a–h). However, there are potential risks of handling the live SARS-CoV-2 particles in the flow cytometer.
Therefore, the process requires a biosafety level 3 (BSL-3) facility and a negative pressure enclosed chamber or a specially designed biohood for reduction of chances of infection caused by potential microdroplets. The steps are:
(a) – Collection of oral/nasal swabs in tubes containing viral transport media, and filtering samples with a 0.45-μm cut-off membrane, which is required to minimize aggregation and artifacts in the sample.
(b) – Suspension of the samples in filter-sterilized ice-cold phosphate-buffered saline (PBS) solution is the first step of sample preparation. Reports suggest that 1% w/v sodium azide in ice-cold PBS helps to prevent the modulation and internalization of surface antigens, which can help in detection process by improving fluorescence intensity of virion particles.
(c) – In 1 ml of the PBS suspension, 0.1–10 μg of the primary antibodies is added. The suspension is mixed, and the tube is incubated in dark for 30–60 min at room temperature. The tube could also be incubated for longer time at 4°C in the dark. Dilutions, if necessary, should be made in solution containing 3% (w/v) bovine serum albumin (BSA) in ice-cold PBS.
(d) – After incubation, the washing step is done for three-times by centrifugation at 400 × g for 5 min at 4°C. The pellet is resuspended in ice-cold PBS by gentle tapping (vigorous vortexing may reduce efficiency in detection step).
(e) – Dilution of the fluorochrome-labeled secondary antibodies could be done in 3% w/v BSA in ice-cold PBS (or according to the manufacturer’s instructions). In 1 ml of the suspended virion-antibody mix from the previous step, 0.2–10 μg of secondary antibodies is added, and the tubes are incubated in dark for at least 30 min at room temperature.
(f) – The cells are to be washed three-times by centrifugation at 400 × g for 5 min using 1 ml of ice-cold PBS containing 3% (w/v) BSA, 1% (w/v) sodium azide. The supernatant is removed using micropipette and the pellet is suspended in 100–200 μl of ice-cold PBS.
(g) – Analysis of the cells on the flow cytometer should be done as soon as possible. We recommend that for virus studies, filtration of the sheath with 0.1-μm filter instead of 0.22-μm filter paper. Viruses are small, therefore, proper thresholds needs to be set for forward side scatter (FSC) and side scatter (SSC). For example, for T4/lambda particle (70 × 200 nm) FSC photomultiplier tubes (PMT) was set at 1000 and SSC at 200 to maximize signal-to-noise ratios. We propose to optimize FSC and SSC (1000 and 400) for enumeration of SARS-CoV-2.
(h) – Controls: prior to sample analysis, a blank, in other words, filtered PBS, needs to be analyzed for background event recognition. The analysis needs to be done at low flow rate and readings should be captured on biexponential plots for fluorescence signals (linear scale for FSC and SCC).
Surface labeling with primary antibodies and the antigen–primary antibodies–secondary antibodies interaction may not be strong enough if sample processing is not done carefully. Poor sample processing may result in shedding of labeled antibodies from viral surface, which could give false-negative results. Therefore, a viral positive control with known fluorescent intensities should be used as internal control for large-scale analysis.
Flow cytometry could detect DENV after 24 h postinfection in Vero 76 (African Green monkey kidney) cell line [24]. The detection was made possible using fluorescein isothiocyanate-labeled 4G2 monoclonal antibody [25]. Therefore, we propose that early detection of SARS-CoV-2 in suspected patients is possible in flow virometry.
Samples with lower viral load may still work better for qRT-PCR-based detection, whereas for the samples with moderate to high viral load, the flow virometry method discussed here will work at an acceptable range of reliability and reproducibility. However, the antigen–antibody-based fluorometric detection, we describe here requires further validation and comparison with other test methods at a larger-scale across various countries.
Results & discussion
Our hypothesis of screening COVID-19 samples using flow virometry could be tested in all hospitals, institutes and diagnostic centers equipped with BSL-3 facilities. We also strongly recommend the researchers to follow the personal protective equipment guidelines to test this method in a dedicated instrument.
The indirect immunofluorescence protocol discussed in this article where sequential binding of virus particles with primary and fluorescent-tagged secondary antibodies (specific binding to primary antibodies) would give sensitive and faster detection of SARS-CoV-2 in test samples.
Outline of process-flow is depicted in Figure 1, where the test sample is incubated with the primary antibodies against SARS-CoV-2, followed by secondary antibodies tagged with a fluorochrome. Similar methods were applied for detection of other pathogenic viruses such as DENV at 3 × 105 particles per ml of culture [25].
Using 18-color SORP sorter (BD FACSAria II) with 355–640 nm lasers, 1.0 FSC ND filter and PMT – SSC detector, flow cytometry could detect 80 virion/ml [26].
The primary antibody needs to be specific and can be one from human anti-SARS-CoV-2 S1 or human anti-SARS-CoV-2 spike receptor-binding domain. To study the limit of detection of an assay setup, titration of the antibodies is required against known concentration of virion. In BD FACSAria II, SSC detector could be positioned at the right angle to the stream and FSC around 10−5° angle will work best for labeled virus particles.
The flow diagram showing steps in sample preparation and detection of the severe acute respiratory syndrome coronavirus-2 using flow cytometer.
Test samples in viral transport media mixed with primary antibodies and then with secondary antibodies labeled with fluorescent tag. Samples were analyzed in flow cytometer using laser. (A) Binding of viral surface proteins with primary antibodies. (B) Fluorescent tagging of virus–primary antibody complex with secondary antibodies.
Antigen selection is the most crucial step in order to distinguish SARS-CoV-2 from other coronaviruses. Immune-informatics studies [27] identified an epitope, ITLCFTLKR, which has not only higher binding scores but also 99.8% structural favorability as per Ramachandran-plot analysis root-mean-square deviation (RMSD; values: 0–1 Å).
More than 20 epitopes were proposed, and biological stability of the peptides were scored after detailed molecular dynamics simulation and in silico codon adaptation experiments [28]. Process optimization and comparative studies with a bunch of epitopes will help in identifying the top three epitopes, which will work best for the method described here.
Overall process of labeling the virion with the antibodies is expected to take around 90–120 min, and sample analysis in flow cytometer takes around 30 s. In a 96-well plate processing of 85–90 samples (including controls) could be done in 180 min in manual operations.
However, the whole process should be done in lesser time if a robotic liquid handler is added in the processing step. Therefore, in a robot-assisted screening platform, 1500–1800 samples could be screened in a day using single FACS instrument in a cost-effective manner. Pooled sample analysis for scoring community spread studies will help to scale-up the analysis by ten- to 20-times per day based on design of the experiment. Overall objective to add robotic automated and liquid handler in the process is to minimize human interaction and achieve throughput.
reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434223/
More information: Edward P Gniffke et al, Plasma From Recovered COVID-19 Patients Inhibits Spike Protein Binding to ACE2 in a Microsphere-Based Inhibition Assay, The Journal of Infectious Diseases (2020). DOI: 10.1093/infdis/jiaa508
