METHOD FOR DETECTING VIRUS PARTICLES AND KITS THEREFOR

Abstract

Disclosed is a method for detecting virus particles in a sample, comprising the steps of: (a) incubating the sample with at least one virus-binding molecule bound to a solid phase; and (b) detecting binding of virus particles to the at least one virus-binding molecule bound to the solid phase. Also disclosed is a kit for use in this method.

Claims

1. A method for detecting virus particles in a sample, comprising the steps of: (a) incubating the sample with at least one virus-binding molecule bound to a solid phase; and (b) detecting binding of virus particles to the at least one virus-binding molecule bound to the solid phase.

2. The method of claim 1, wherein the virus-binding molecules comprise a virus entry receptor.

3. The method of claim 1, wherein the at least one virus-binding molecule comprises angiotensin-converting enzyme 2 (ACE2), selected from the group consisting of native human ACE2, recombinant human ACE2, and modified recombinant human ACE2; wherein the virus particles are severe acute respiratory syndrome coronavirus (SARS-CoV)-1 particles, SARS-CoV-2 particles or HCoV-NL63 particles.

4. The method of claim 1, wherein the sample is a clinical sample, comprising bronchoalveolar lavage (BAL) fluid, sputum, tracheal aspirate, epithelial cells obtained by an epithelial swab, or a body fluid such as blood, serum, plasma or urine.

5. The method of claim 1, wherein the binding is detected directly or indirectly.

6. The method of claim 1, wherein the binding is detected by one or more labelled antibodies.

7. The method of claim 1, wherein the binding is detected by one or more soluble recombinant virus entry receptors.

8. The method of claim 1, wherein the binding is detected by PCR.

9. The method of claim 1, wherein the sample is an aerosol.

10. The method of claim 1, wherein the sample is a native or inactivated virus or pseudo virus preparation.

11. The method of claim 1, wherein the sample is incubated in the presence of body fluids.

12. The method of claim 1, wherein the sample is incubated in the presence of neutralizing antibodies.

13. The method of claim 1, wherein the virus particles are infectious virus particles.

14. The method of claim 1, wherein the at least one virus-binding molecule bound to the solid phase comprises a virus entry receptor bound to the solid phase, wherein the virus entry receptor bound to the solid phase is enzymatically inactive.

15. The method of claim 1, further comprising the steps of: incubating with a washing solution; incubating with a soluble virus entry receptor for the virus particles; and incubating with a washing solution.

16. The method of claim 1, wherein said detecting step (b) comprises detecting soluble virus entry receptor bound to the virus particles bound to at least one virus-binding molecule bound to the solid phase.

17. The method of claim 1, wherein the virus entry receptor bound to the solid phase is enzymatically inactive ACE2, wherein the soluble virus entry receptor is soluble enzymatically active ACE2, wherein the virus particles are SARS-CoV-1 particles, SARS-CoV-2 particles or HCoV-NL63 particles.

18. The method of claim 1, wherein the solid phase is a plate such as a 96-well plate, beads such as agarose beads or magnetic beads, a solid phase having a graphene surface or a solid phase having a semiconducting surface.

19. A kit for performing the method of claim 1, comprising a manual, one or more solvents, one or more buffers, and/or one or more solid phases and/or one or more enzymes and/or one or more antibodies and/or one or more primers and/or one or more enzyme substrates and/or one or more inactivated virus or pseudo virus preparations.

20. A kit for detecting virus particles in a sample, preferably for use in the method of claim 14, comprising: a solid phase being one of a plate, beads such as agarose beads, a solid phase having a graphene surface, and a solid phase having a semiconducting surface; a virus entry receptor for the virus particles bound to the solid phase; a soluble virus entry receptor for the virus particles; at least one substrate for the enzymatic activity of the soluble virus entry receptor; at least one washing solution; and at least one inactivated virus or pseudo virus preparations; wherein: the virus entry receptor bound to the solid phase is enzymatically inactive ACE2, the soluble virus entry receptor is soluble enzymatically active ACE2, wherein the virus particles are SARS-CoV particles, SARS-CoV-2 particles or HCoV-NL63 particles.

Description

[0079] The present invention is further explained by the following figures and examples, without being restricted thereto.

[0080] In FIG. 1, the assay principle based on capturing virus particles with a molecule resembling the virus's receptor for entry into human cells (e.g., inactivated recombinant ACE2) and detecting virus particles using the enzymatic activity of recombinant ACE2 as reporter is schematically visualized (virus entry receptor binding test, VERB test). In the figure, the principle of a VERB assay using a microtiter plate coated with a virus-binding molecule (red) is shown. The scheme describes detection using the activity of soluble virus entry receptor molecules (green) inducing a fluorescence-, color- or chemiluminescence-generating substrate/product conversion.

[0081] In another embodiment of this invention, an alternative technical setup is described, which enables higher sample volumes. FIG. 2 shows an assay principle based on capture with inactivated recombinant ACE2 bound to beads. This leads to a significant improvement in the surface/volume ratio and thus to a more efficient binding of virus particles from the sample. Furthermore, this principle can be carried out as liquid extraction column or even aerosol test to analyze respiratory secretions, saliva, breathing air, but also plasma or urine or other body fluids. Based on this assay principle, a chromogenic point-of-care test is feasible, that reports the presence of infectious virus particles as a color change within a few minutes. In the figure, the principle of a VERB assay using agarose beads coated with a virus-binding molecule (red) is shown. The scheme describes detection using the activity of soluble virus entry receptor molecules (green) inducing a fluorescence-, color- or chemiluminescence-generating substrate/product conversion. Different types of beads could be used as a capture matrix, including but not limited to agarose beads, magnetic beads or luminex beads.

[0082] FIG. 3: Comparative analysis of COVID-19 VERB results for tracheal aspirate (TA) and serum from two COVID-19 (CoV) patients, also investigating matrix (Serum+TA-1 CoV) using detection of viral entry receptor as biochemical readout (HRP substrate).

[0083] FIG. 4: Comparative analysis of COVID-19 VERB results for tracheal aspirate (TA) and serum from two COVID-19 (CoV) patients, also investigating matrix (Serum+TA-1 CoV) using RT-PCR based detection.

[0084] FIG. 5: Comparing the results obtained from RT-PCR analysis of the VERB input with the VERB result following RT-PCR detection.

[0085] FIG. 6: Effects of 1% serum from COVID-19 negative (CNS) and COVID-19 positive convalescent serum (CPS) on VERB signals obtained from tracheal aspirate of a COVID-19 patient.

[0086] FIG. 7: VERB capture of a dilution series of retrovirus pseudotyped with SARS-CoV-2 spike protein using RT-PCR based detection.

[0087] FIG. 8: Fraction of SARS-CoV-2 RNA (% of Input) obtained after VERB capture from nasopharyngeal swab samples from COVID-19 patients.

[0088] FIG. 9: Correlation of infectivity in classical plaque assay to RT-qPCR analysis from Input (Total RNA) and VERB Capture in nasopharyngeal swab samples from COVID-19 patients. Boxes represent the interquartile range; horizontal lines indicate median values; “+” indicate mean values.

Example 1: Binding of SARS-CoV-2 Particles to Solid Phase Capture Matrix Coated with rhACE2

[0089] The capture of SARS-CoV-2 was performed with His-tagged recombinant human ACE2. Samples used included tracheal aspirate (TA) and serum from SARS-CoV-2 infected patients. We used Ni-NTA plates (96-well) as a solid phase to immobilize His-tagged ACE2 for virus capture. ACE2 coated plates were incubated with pretreated tracheal aspirate or serum samples as indicated, followed by multiple washing steps. Following virus-binding, wells were incubated with soluble biotin labeled recombinant human ACE2 to saturate free ACE2 binding sites on virus particles, followed by multiple washing steps. For detection, we used streptavidin labeled HRP in combination with a high sensitivity chemiluminescence substrate (FIG. 3). Luminescence signals (RLU) were detected in all samples containing tracheal aspirate. Tracheal aspirate diluted 1:1 in serum from a healthy volunteer resulted in a reduced signal corresponding to the dilution factor employed. Following biochemical detection, sample were subjected to RT-PCR for determination of virus titers in VERB wells (FIG. 4). Signals for RT-PCR based detection and biochemical detection of analyzed samples were highly correlating. Given that biochemical detection requires the presence of multivalent binding sites on captured particles, we conclude that intact virus can be efficiently captured on solid phase capture matrix. To our surprise, comparing the VERB input with the VERB output using RT-PCR revealed that the ratio between output and input was significantly different between the two individual patients analyzed, suggesting the presence of large amounts of viral RNA in patient 1 (TA-1), that is not associated with intact virus particles and could therefore not be detected in the VERB assay (FIG. 5). Importantly, the ratio between TA-2 and TA-1 for results obtained in a classical cellular plaque assay for COVID-19 was comparable to the ratios obtained from VERB assays using either RT-PCR or biochemical detection of the viral entry receptor (Table 1, below), while the RT-PCR based determination of titers in input samples revealed a clearly different ratio between TA-2 and TA-1, suggesting that the outcome of the VERB assay reflects classical cellular plaque assays. We conclude, that the VERB assay can be used to discriminate between intact and infectious virus particles and disintegrated virus fragments, still leading to (false positive) signals in RT-PCR based analysis of clinical samples, as shown for tracheal aspirates.

TABLE-US-00001 TABLE 1 Direct comparison of plaque assay with RT-PCR and VERB detection in tracheal aspirates of two COVID-19 patients. INPUT VERB RT-PCR Plaque Assay RT-PCR ACE2 [copies/ml] [PFU/ml] [copies/ml] [RLU] TA-1 CoV 332699 1.4E+05 144815 4448.4 TA-2 CoV 77605 4.9E+04 58813 1530.1 TA-2/TA-1 23% 36% 41% 34%

Example 2: Monitoring Neutralizing Antibodies

[0090] In another experiment, we performed the binding step for tracheal aspirates in the presence of serum. Serum from healthy volunteers (CNS, COVID-19 negative serum) and serum from convalescent COVID-19 patients (CPS, COVID-19 positive serum) was used at a dilution of 1:100 during virus capture. Serum was mixed with the virus containing samples (TA-1 CoV and TA-2 CoV) and incubated for 60 min before incubating with His-tag ACE2 coated Ni-NTA plates. Following binding and washing as described before, chemiluminescence signals were analyzed. The presence of serum from convalescent COVID-19 patients selectively resulted in a profound suppression of obtained VERB signals, suggesting the inhibition of the interaction between plate immobilized virus entry receptor and virus particles. Therefore we conclude that a modified setup of the VERB assay is suitable to detect neutralizing antibodies against COVID-19 in serum samples.

Example 3: Binding of Spike-Pseudotyped Retroviral Particles to Magnetic Beads Coated with ACE2 or Enzymatically Inactive ACE2

[0091] The capture of retroviruses pseudotyped with SARS-CoV-2 Spike protein was performed with magnetic beads coated either with enzymatically active ACE2 or an enzymatically inactive mutant of ACE2. Specifically, streptavidin magnetic beads were used as a solid phase to immobilize biotinylated ACE2 for virus capture. In addition, non-coated beads (“MOCK”) were included as a control for unspecific binding of particles to the solid phase. ACE2-coated beads were incubated with a dilution series of pseudotyped retrovirus in phosphate buffered saline, followed by multiple washing steps. For detection, the beads were incubated in RNA lysis buffer and subsequently, RNA was extracted using a commercially available kit. VERB captured RNA in comparison to total RNA extracted from input samples was quantified by RT-qPCR (see FIG. 7). Captured RNA (VERB) clearly correlated with total RNA (Input) in a dose-dependent manner, when using ACE2-coated beads. In contrast, significantly less RNA was detected after incubation with uncoated beads (Mock), thus suggesting that the capture of RNA from a Spike-pseudotype virus preparation is specific for ACE2. Surprisingly, a linear relationship of input to captured RNA was observed over the entire dilution series, covering five orders of magnitude. In addition, equal capture efficiencies were obtained by using an enzymatically inactive mutant of ACE2. We conclude that VERB capture is highly efficient, works in a quantitative and dose-dependent manner over a wide range of virus concentrations and is independent of the enzymatic activity of ACE2.

Example 4: A Semi-Automated VERB Capture Platform for the Analysis of Clinical Samples

[0092] The capture of SARS-CoV-2 from nasopharyngeal swab (NPS) samples was performed using biotinylated recombinant human ACE2 immobilized on streptavidin magnetic beads. To compare VERB captured SARS-CoV-2 to total RNA (input) from unprocessed samples, samples from different patients were each diluted 1:2 in saline and each of the samples was divided into two equal parts. The first part of each sample was used for direct RNA extraction (called “Input” in FIG. 8), whereas the second part of each sample was subjected to VERB capture, followed by RNA extraction. Briefly, samples were incubated with VERB beads for 30 minutes at room temperature with constant agitation, followed by two wash steps in phosphate buffered saline using an automated magnetic handler (TANbead Maelstrom 8). Subsequently, beads were incubated in RNA lysis buffer and RNA from Input samples and VERB capture samples, were extracted in parallel. We analysed the RNA content of the samples by RT-qPCR using a primer-probe set targeting the nucleocapsid gene of SARS-CoV-2.

[0093] Surprisingly, the fraction of captured virus varied dramatically between different patients (see FIG. 8), which suggests that classical RT-qPCR analysis does not allow a proper estimate of the amount of infectious virus present in clinical samples.

[0094] To directly establish the link to infectivity, a subset of samples was examined in a classical cellular plaque assay (see also FIG. 9). Already in input samples (total RNA), a difference RNA copies/ml was apparent in samples with a positive signal in plaque assay compared to samples not yielding any plaques (negative). Although the ratio of median RNA copies/ml for positive samples (median=5.8e7) to the median RNA copies/ml of negative samples (median=1.6e3) was already more than 10,000-fold, this difference became even more pronounced after VERB capture. Plaque assay positive samples still showed median RNA copies/ml of 5.1e6, whereas negative samples were mostly also negative in RT-qPCR, resulting in a median RNA copies/ml of 2.5. In summary, we conclude that results obtained after VERB capture are a good predictor of infectivity and correlate well with a classical plaque assay.