Method for detecting viruses having hemagglutinin esterase activity

Abstract

Disclosed is a method of detecting a virus having hemagglutinin esterase activity in a sample, the method comprising: contacting the sample with a substrate for an enzyme of a hemagglutinin esterase (HE) of coronavirus or a hemagglutinin esterase fusion protein (HEF) of influenza C virus; and detecting the activity of the enzyme, the activity detection indicating that the sample contains coronavirus or influenza C virus.

Description

[0001] This application is a divisional application of the original application with the filing date of March 16, 2021, the application number of 202180022252.2, and the invention title of “Method of detecting viruses with hemagglutinin esterase activity”. This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 991,066, filed March 17, 2020, the disclosure of which is hereby incorporated by reference in its entirety herein for all purposes as if fully set forth herein. TECHNICAL FIELD

[0002] The present disclosure relates to a method of detecting viruses with hemagglutinin esterase activity.

[0003] Cross Reference to Related Applications This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 991,066, filed March 17, 2020, the disclosure of which is hereby incorporated by reference in its entirety herein for all purposes as if fully set forth herein. BACKGROUND

[0004] Coronaviruses are a group of related viruses that cause diseases in mammals and birds. In humans, coronaviruses can cause mild respiratory tract infections, as well as other potentially lethal infections such as SARS, MERS, and COVID-19.

[0005] Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The World Health Organization (WHO) declared COVID-19 a pandemic on March 11, 2020, and there is an urgent need for broad and rapid testing capabilities. Testing is critical because it allows infected individuals to avoid infecting others and to quickly get the care they need, and slows the spread of the disease.

[0006] There are several methods available for detecting coronaviruses, including reverse transcription-polymerase chain reaction (RT-PCR) and coronavirus-specific antibody (e.g., IgM / IgG) tests. However, these methods are labor-intensive and time-intensive. Moreover, test methods using coronavirus-specific antibodies cannot distinguish actively infected patients from recovered patients who still have antibodies after clearing the infection.

[0007] A method of detecting coronaviruses, including COVID-19, from human samples that is instant, inexpensive, and accurate has not been developed, but is urgently needed. Such a test could save many lives from a rapidly spreading and potentially deadly disease. SUMMARY

[0008] The present inventors recognized that hemagglutinin esterase (HE) present on the surface of the virus is one of the unique features of coronaviruses (including COVID-19 and influenza C) when compared to other viruses with similar symptoms (e.g., common cold rhinoviruses and influenza A and B), and completed the claimed invention that provides a rapid, inexpensive, and accurate method of detecting viruses with HE activity.

[0009] According to one aspect of the present invention, there is provided a method of detecting a virus with hemagglutinin esterase activity in a sample, comprising: contacting the sample with a substrate for the enzyme of a hemagglutinin esterase (HE) of a coronavirus or a hemagglutinin esterase fusion protein (HEF) of an influenza C virus; and detecting activity of the enzyme, wherein detection of activity indicates that the sample contains a coronavirus or an influenza C virus.

[0010] The virus can be a coronavirus and the enzyme can be HE, more specifically, the virus can be a coronavirus named COVID-19. The enzyme can catalyze hydrolysis or deacylation of a substrate. In the method, a product of hydrolysis or deacylation can be detected. The product formed by the deacylation or hydrolysis reaction between the substrate and the enzyme can emit a detectable signal. The detectable signal can include fluorescence or chemiluminescence. The signal can be fluorescence, 1,1´-oxydiacyl diimidazole (ODI) chemiluminescence, or peroxymalonic acid ester chemiluminescence (PO-CL). The substrate can be selected from 4-methylumbelliferyl acetate, fluorescein diacetate, and resorufin acetate. The sample can be a body fluid or tissue sample from a human subject, the sample being a nasopharynx or nasopharynx sputum sample. The sample and the substrate can be incubated at room temperature, and the result is shown from almost immediately to 60 minutes, for example, depending on the sample amount of each substrate. Incubation can be performed for about 3 minutes to 60 minutes. In the method, multiple readings of the activity of the enzyme can be taken as a function of reaction time. The pH of the mixture of the sample and the substrate can be 4 to 10.

[0011] According to another aspect of the present invention, there is provided a method of detecting a coronavirus in a sample, comprising: contacting the sample with an antibody to a coronavirus; mixing the contacted sample with a substrate that is a substrate for an enzyme with hemagglutinin esterase (HE) activity; and detecting activity of the enzyme, wherein detection of activity indicates that the sample contains a coronavirus. The antibody to a coronavirus can be an antibody to SARS-CoV-2 or a specific variant thereof.

[0012] According to yet another aspect of the present application, there is provided a kit for detecting a virus having hemagglutinin esterase from a sample, the kit comprising a container holding a substrate that reacts with an enzyme having hemagglutinin esterase (HE) activity, wherein the enzyme catalyzes hydrolysis or deacylation of the substrate, and a product of the hydrolysis or deacylation emits a detectable signal.

[0013] These and other aspects will become apparent to those of ordinary skill in the art upon reading and understanding the following specification. BRIEF DESCRIPTION OF DRAWINGS

[0014] The above and other objects, features and advantages of the present application will become more apparent to those of ordinary skill in the art by Figure 1 SARS-CoV-2 (COVID-19) structure is shown (showing a cutaway view model of a coronavirus, e.g., in Wikipedia at https: / / en.wikipedia.org / wiki / Coronavirus).

[0015] Figure 2 Reaction mechanisms of ODI-CL and PO-CL using four different peroxyoxalate compounds are shown. X: high-energy intermediate, chemiluminescent probe in the ground state, and chemiluminescent probe* in the excited state.

[0016] Figure 3 Hydrolysis reactions between substrates and HE of coronaviruses or HEF of influenza C are shown. (1: 4-methylumbelliferyl acetate, 2: 3-(2-benzoxazolyl)umbelliferyl acetate, 3: fluorescein diacetate, 4: resorcinol acetate, 5: 4-methylumbelliferone, 6: 3-(2-benzoxazolyl)umbelliferone, 7: fluorescein, 8: resorufin).

[0017] Figure 4 Fluorescence emission of 5: 4-methylumbelliferone (4-MU), 6: 3-(2-benzoxazolyl)umbelliferone, 7: fluorescein, 8: resorufin formed from the hydrolysis reactions shown by Figure 3

[0018] Chemiluminescence of the luminophores (5 to 8) emitted in peroxyoxalate (PO) or 1,1´-oxaloyldiimidazole (ODI) chemiluminescence (CL) reactions is shown. Figure 5

[0019] Figure 6 ​is a calibration curve that enables quantification of HE in a sample using hydrolysis reaction of fluorescein diacetate and HE of coronavirus and chemiluminescence detection. (Relative CL intensity was measured with LUMAT 9507 luminometer).

[0020] Figure 7 shows hydrolysis reaction of HE and resorcylic acid diacetate over time at different concentrations of HE.

[0021] Figure 8 is a macroscopic observation photograph showing early diagnosis of coronavirus infection using hydrolysis reaction of resorcylic acid diacetate with HE of coronavirus.

[0022] Figure 9 is a calibration curve that enables rapid detection of trace level of HE of coronavirus.

[0023] Figure 10 shows one coronavirus captured by one spike antibody. DETAILED DESCRIPTION

[0024] Based on the hydrolysis reaction between a substrate and hemagglutinin esterase (HE) of coronavirus or hemagglutinin esterase fusion protein (HEF) of influenza C, a detection method using fluorescence and chemiluminescence was developed for rapid detection of specific viruses in human samples. It should be understood that alternative signaling methods are contemplated in addition to the specific examples provided herein.

[0025] As shown in Figure 1 , coronavirus has many HEs and RNAs with three different proteins (e.g., spike glycoprotein, nucleocapsid (N) protein, envelope). In addition, two HEs (dimer) can be found on each spot of coronavirus. HEF of influenza C is composed of three HEs (trimer).

[0026] The chemical and physical properties of the esterases of HE and HEF are the same as those of acetyl (acetyl xylan) esterase that catalyzes the deacetylation of xylan and xylo-oligosaccharide. Therefore, due to the same chemical and physical properties, the present inventors used acetyl (acetyl xylan) esterase, which can be easily obtained, to show the results of the present invention. In addition, the present inventors fully expect to obtain the same or similar results if HE or HEF is used.

[0027] Taking advantage of the properties of acetyl esterase, four different substrates, 4-methylumbelliferyl acetate, 3-(2-benzoxazolyl)umbelliferyl acetate, fluorescein diacetate, and resorcylic acid diacetate, were synthesized and commercialized to monitor the activity of acetyl esterase in a sample. The products formed by the deacetylation (or hydrolysis) reaction of acetyl esterase with the substrates emit fluorescence, 1,1´-oxydiacyl diimidazole (ODI) chemiluminescence, and peroxymalonic acid ester chemiluminescence (PO-CL).

[0028] Based on the mechanism of deacetylation (or hydrolysis) reaction, new analytical methods and kits have been designed and developed for early diagnosis of infection of coronavirus or influenza C. The detection of fluorescence, 1,1'-oxydiacyl diimidazole chemiluminescence (ODI-CL) and peroxymonosuccinate chemiluminescence (PO-CL) is applied to the present invention because the chemiluminescent probe formed by the rapid hydrolysis reaction of non-fluorescent and non-chemiluminescent substrates with HE (or HEF) of coronavirus or influenza C can emit light. As shown in Figure 2 , the high-energy intermediate X formed by ODI-CL and PO-CL reactions produces fluorescence as a light source (laser, LED, xenon).

[0029] As shown in Figure 3 , three different fluorescent compounds (5 to 8) are formed by the hydrolysis reaction of substrates (1, 2, 3 or 4) and acetyl (acetyl xylan) esterase (HE or HEF of coronavirus or influenza C present in sputum, nasopharynx, nasopharynx or saliva samples). The yield of the hydrolysis reaction depends on the pH (4 to 10), the composition of the buffer solution, the chemical and physical properties of the substrate and the reaction (incubation) time of the substrate with HE (or HEF).

[0030] The 4-MU, 3-(2-benzoxazolyl) xanthenyl, fluorescein and resorcinol formed by the hydrolysis reaction shown in Figure 3 emit blue, green and red fluorescence (see Figure 4 ). As the concentration of HE (or HEF) in the hydrolysis reaction increases, the relative fluorescence intensity of the product increases proportionally. Using a simple method of fluorescence detection, it is confirmed that the new analytical method can quickly diagnose the disease of patients infected with coronavirus or influenza C. The diagnostic method of fluorescence detection is operated with a fluorescence detection well plate reader or a fluorometer operated with a cuvette or borosilicate glass tube. A diagnostic kit is designed and developed based on the fluorescence detection method. The optimal incubation (reaction) time of the substrate with HE (or HEF) can be 3 minutes to 30 minutes at room temperature to detect low concentrations of coronavirus or influenza C in the sample. The sensitivity of the analytical method depends on the quantum efficiency of the fluorescent compound formed by the hydrolysis reaction. In addition, the sensitivity of the analytical method using fluorescence detection depends on the concentration of the substrate. As the concentration of the substrate increases, the sensitivity of the analytical method is enhanced. The range of the substrate used in the present invention is 0.01 mM to 0.5 mM. The composition of the diagnostic kit is the substrate, the reaction buffer and the microplate (such as 96, 384 microplate) or glass test tube.

[0031] As shown in Figure 5As shown, 1,1'-oxydiacyl diimidazole (ODI) and several peroxyoxalic acid ester (PO) compounds have been used in the invented analytical method using chemiluminescence detection that can enable early and rapid sensing of trace levels of coronavirus and influenza C. The peroxyoxalic acid ester compounds in the invention are bis(2,4-dinitrophenyl) oxalate (DNPO), bis(2,4,6-trichlorophenyl) oxalate (TCPO), bis[2-(3,6,9-trioxa decyloxy carbonyl)-4-nitrophenyl] oxalate (TDPO), and bis-(2,4,5-trichloro-6-(pentyloxy carbonyl) phenyl) oxalate (CPPO). To obtain a fast PO-CL reaction, sodium salicylate, pyridine, and imidazole derivatives such as imidazole, 2-methylimidazole, 4-methylimidazole, 2,4-dimethylimidazole, or 2-ethylimidazole can be added as a base or nucleophilic catalyst.

[0032] The time required to complete the test can depend on Figure 3 the chemical and physical properties of the substrate as shown. Thus, the time range for the quantitative analysis of coronavirus (or influenza C) in the sample at room temperature can be 1 minute to 30 minutes. In addition, the time required to generate the chemiluminescent probe from the hydrolysis reaction as shown depends on the properties of the buffer solution used to dissolve the substrate. Furthermore, the analysis time will depend on the human sample collection method, such as nasopharyngeal or oropharyngeal swab, nasopharyngeal or nasal aspirate, and saliva collection. The reactivity of the hydrolysis reaction depends on the temperature. For example, the hydrolysis reaction at 37 °C is faster than the hydrolysis reaction at room temperature. Figure 3

[0033] The intensity of 4-MU, 3-(2-benzoxazolyl)umbelliferone, fluorescein, and resorufin depends on the concentration of coronavirus or influenza C in the sample. As the viral concentration increases, the relative CL intensity increases proportionally. The sensitivity of the method depends on the properties of the chemiluminescent probe formed by the hydrolysis of the substrate and HE (or HEF). The sensitivity of the method using resorufin diacetate and HE (or HEF) reaction to form resorufin is superior to 4-MU and fluorescein. The method can be operated with a luminometer or a well plate reader with CL detection.

[0034] As shown, 4-MU, 5, emits blue or green light upon the addition of PO or ODI with H2O2. The color of the emitted light of 4-MU depends on Figure 5 Figure 3 ​​The pH used in the hydrolysis reaction shown. The optimal buffer for the 4-methylumbelliferyl acetate and HE (or HEF) hydrolysis reaction is 1 x PBS (pH 7.4). The 4-MU formed under acidic conditions (<pH 7) emits green light, while the 4-MU formed under neutral and basic conditions (pH 7 and above) emits bright blue light. In addition, 4MU under basic conditions is brighter than under acidic conditions. The time required to complete the analysis in PBS at room temperature can be 30 minutes. The concentration of 4-methylumbelliferyl acetate used in the hydrolysis reaction of the present invention can range from 0.05 mM to 0.5 mM.

[0035] As shown in Figure 5 3-(2-benzoxazolyl)umbelliferone, 6, emits blue light upon the addition of PO or ODI with H2O2. The assay method using fluorescein is more sensitive than the assay method using 4-MU. Therefore, the analysis time using fluorescein diacetate (5 minutes to 10 minutes) is shorter than the analysis time using 4-methylumbelliferyl acetate. In addition, this method uses 10 mM Tris-hydrochloric acid buffer (pH 7 to pH 8.5). As shown in Figure 6 the brightness of fluorescein increases with the increase of HE in the sample. In the present invention, the concentration of fluorescein diacetate that produces fluorescein from the hydrolysis reaction ranges from 0.01 mM to 0.1 mM.

[0036] As shown in Figure 5 fluorescein 7 emits green light upon the addition of PO or ODI with H2O2. The assay method using fluorescein is more sensitive than the assay method using 4-MU. Therefore, the analysis time using fluorescein diacetate (10 minutes to 20 minutes) is shorter than the analysis time using 4-methylumbelliferyl acetate. In addition, this method uses 10 mM phosphate buffer (pH 8.5). As shown in Figure 6 the brightness of fluorescein increases with the increase of HE in the sample. In the present invention, the concentration of fluorescein diacetate that produces fluorescein from the hydrolysis reaction ranges from 0.01 mM to 0.2 mM.

[0037] As shown in Figure 5As shown, resorufm, 8, emits red light upon addition of PO or ODI with H202. The sensitivity of the analytical method using resorufm is superior to that using 4-MU, 3-(2-benzoxazolyl) umbelliferone, and fluorescein. Thus, the analytical time (1 to 5 minutes) using resorufm acetate is faster than that using 4-methylumbelliferone acetate and fluorescein diacetate. The optimal buffer solution in this method is 1 x TBS (pH 7.5). The concentration range (0.001 to 0.1 mM) of resorufm acetate used in the hydrolysis reaction is much wider than that of other substrates, such as 4-methylumbelliferone acetate, 3-(2-benzoxazolyl) umbelliferone acetate, and fluorescein diacetate. The time required for quantification of HE (or HEF) in samples collected using nasopharyngeal swabs or oropharyngeal swabs can be very short. For example, the time required for quantification of acetyl(xylan) esterase in samples is as short as 3 minutes. In addition, the time required for quantification of samples collected using nasopharyngeal swabs can be less than 10 minutes. Furthermore, the time required for quantification of saliva samples diluted 20-fold with 1 x TBS (pH 7.5) is as short as 5 minutes. The limit of detection (LOD = 3S) determined using the analytical method is as low as 25 pg / ml. S is the standard deviation determined after 20 replicates of the background. The LOD of the analytical method for chemiluminescent detection is at least 10-fold lower than that for fluorescent detection. See Figure 6 In addition, the analytical method for chemiluminescent detection is at least 30 minutes faster than that for fluorescent detection because the former is much more sensitive than the latter.

[0038] As the viral concentration increases, the relative CL intensity increases proportionally. The method can be operated with a luminometer or a well-plate reader with CL detection.

[0039] As shown in Figure 7 resorufm is formed from the rapid hydrolysis of resorufm acetate with HE (or HEF). Resorufm (pink color) formed in the presence of relatively high concentrations (>0.015 mU / ml or >0.56 ng / ml) of HE can be observed by the naked eye within 60 seconds. This result indicates that it is possible to rapidly observe by the naked eye whether a patient is infected with a coronavirus (or influenza C) or not. As evidence, Figure 4 shows the color difference between 0.004 mU of HE and a negative control that does not contain HE. Figure 8 indicates that it is possible to perform early diagnosis of coronavirus by the naked eye.

[0040] As a tool for observing coronavirus (or influenza C) infection with the naked eye, lateral flow assay (LFA) can be applied. The LFA applied in the present invention is simpler than conventional LFA such as pregnancy, cancer, and infectious disease test kits because the former does not require any antibody such as a capture or detection antibody. For example, a sample (5 µl to 100 µl) diluted in 1x TBS is loaded on a sample pad. The sample flows to a test line containing resorcinol acetate. When HE (or HEF) is present in the sample, the color of the test line changes from white (or light yellow) to deep pink with a rapid hydrolysis reaction.

[0041] As shown in Figure 9 , chemiluminescence detection can quantify trace levels of HE in a sample. The results show that, using the hydrolysis reaction of resorcinol acetate and HE, an analysis method for chemiluminescence detection in 3 minutes makes it possible to diagnose coronavirus infection at an early stage.

[0042] The composition of the diagnostic kit based on the analysis method using chemiluminescence detection is a substrate (e.g., 4-methylumbelliferone (4-MU) 5, 3-(2-benzoxazolyl)umbelliferone 6, fluorescein 7, resorcinol 8), a reaction buffer, a chemiluminescence reagent (e.g., ODI or PO, H2O2), and a 96 (or 384) microwell plate or a borosilicate glass tube. For example, using a diagnostic kit prepared using resorcinol acetate, it is possible to conveniently and quickly diagnose coronavirus or influenza C infection on-site, for example, at public transportation hubs such as airports, train stations, ports, and bus terminals.

[0043] As shown in Figure 10 , in order to confirm infection with a specific coronavirus, appropriate antibodies are applied in the present invention. The antibodies used in this method, which can capture the spike glycoprotein or nucleocapsid (N) protein of coronavirus, are likely to bind to the proteins of the coronavirus shown in Figure 1 . After capturing the coronavirus using the antibody, the substrate and the HE of the coronavirus bound to the antibody react for 10 minutes. Finally, using CL measurement with the addition of a chemiluminescence reagent, it is possible to confirm whether a patient is infected.

[0044] In summary, the fluorescent or chemiluminescent detection analysis method can be applied to rapidly detect coronavirus or influenza C in a sample. This is because the accuracy, precision, and reliability of the method are good within a statistically acceptable error range.

[0045] It should be understood that the above-described methods are merely illustrative embodiments of the principles of the present disclosure, and those of ordinary skill in the art can design other compositions and methods of using them without departing from the spirit and scope of the present invention. It should also be understood that the present disclosure relates to embodiments including and consisting of the disclosed parts.

Claims

1. A method for detecting an enzyme or fusion protein, the method comprising: Contact the enzyme or the fusion protein with the substrate; and The activity of the enzyme or the fusion protein was detected. The enzyme is hemagglutinin esterase (HE), and the fusion protein is hemagglutinin esterase fusion protein (HEF).

2. The method according to claim 1, wherein, The enzyme or the fusion protein catalyzes the hydrolysis or deacylation of the substrate.

3. The method according to claim 2, wherein, The products of the hydrolysis or deacylation are detected.

4. The method according to claim 3, wherein, The product formed by the deacylation or hydrolysis reaction between the substrate and the enzyme or the fusion protein emits a detectable signal.

5. The method according to claim 4, wherein, The signal is a change in solution color that can be observed with the naked eye, fluorescence, or chemiluminescence.

6. The method according to claim 5, wherein, The signal is fluorescence, 1,1'-oxalodiimidazole (ODI) chemiluminescence, or peroxyoxalate chemiluminescence (PO-CL).

7. The method according to claim 1, wherein, The substrates are selected from 4-methylumbelliferone acetate, 3-(2-benzoxazolyl)umbelliferone acetate, fluorescein diacetate, and halogen acetate.

8. The method according to claim 1, wherein, The enzyme or the fusion protein and substrate are incubated at room temperature for less than 1 second to 60 minutes.

9. The method according to claim 8, wherein, The culture is carried out for approximately 3 to 60 minutes for chemiluminescence detection.

10. The method according to claim 8, wherein, The culture is carried out for approximately 20 to 60 minutes for fluorescence detection.

11. The method according to claim 1, wherein, Multiple readings of the enzyme's activity were taken as a function of reaction time.

12. The method according to claim 1, wherein, The pH of the mixture of the enzyme or the fusion protein and the substrate is between 4 and 10.

13. A method for detecting the reaction product of an enzyme or fusion protein with a substrate, the method comprising: Contact the enzyme or the fusion protein with the substrate; and Detect the reaction product of the enzyme or the fusion protein with the substrate. The enzyme is hemagglutinin esterase (HE), and the fusion protein is hemagglutinin esterase fusion protein (HEF).

14. A kit for detecting viruses containing hemagglutinin esterase in a sample, comprising a container containing a substrate for reacting with an enzyme having hemagglutinin esterase (HE) activity. in, The enzyme catalyzes the hydrolysis or deacylation of the substrate, and The products of hydrolysis or deacylation emit detectable signals.

15. The kit according to claim 14, wherein, The sample is a fluid or tissue sample from a human subject.

16. The method according to claim 15, wherein, The samples were nasopharyngeal, nasopharyngeal sputum, and saliva samples.

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