WO2021262728A1 - Compositions and methods for viral detection - Google Patents

Abstract

Hypertonic solutions including combinations of at least 5-30% of at least one monosaccharide or disaccharide, at least 5-20% of polyethylene glycol, 0.01-0.2% of at least one bisbiguanide, and/or 0.01-0.2% quaternary ammonium are provided. Kits including the solutions are also provided. Methods of detecting presence of one or more of a viral nucleic acid, antigen, or immunoglobulin utilizing the hypertonic solutions are further provided.

Description

COMPOSITIONS AND METHODS FOR VIRAL DETECTION

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/042,793, filed June 23, 2020, which is incorporated herein by reference in its entirety.

FIELD

This disclosure relates to compositions and methods for sample collection and detection of virus, antibodies against the virus, or viral infection, particularly coronavirus detection.

BACKGROUND

The COVID-19 pandemic has created an urgent need for rapid diagnostics that can be used to screen large numbers of samples to monitor for outbreaks. The SARS-CoV-2 virus that causes COVID-19 is spreading very easily and sustainably between people. The primary test is a PCR- based molecular genetic assay for the SARS-CoV-2 genome that is not rapid and has a high false negative rate.

There are over 200 COVID diagnostic tests worldwide, the vast majority of which are the PCR-based genetic tests. There are at least 38 serological tests for IgG, IgM or both manufactured worldwide currently. About half of these tests are rapid lateral flow immunoassays. Most of the remaining being ELISA-based assays that must be run in a specialized lab.

SUMMARY

There is a need for improved detection of viral nucleic acids, antigens, and/or antibodies. In addition, samples for diagnostic and serological tests requires special handling and processing conditions because of their potential to contain live virus, and venous blood collection requires a phlebotomist.

Provided herein are compositions and methods for sample collection and testing for presence of viral antibodies, viral antigens, or nucleic acids in a sample (such as a sample from a subject or an environmental sample). The disclosed compositions and methods include at least one agent that can inhibit, inactivate, or decrease viability or amount of virus in the sample.

Hypertonic solutions including 5-20% polyethylene glycol, 5-30% of at least one monosaccharide or disaccharide, or a combination thereof and 0.01-0.2% cetylpyridinium chloride, 0.01-0.2% chlorhexidine, or a combination thereof are provided. In some embodiments, the solution includes 5-20% polyethylene glycol and 0.01-0.2% cetylpyridinium chloride. In other embodiments the solution includes 5-30% of at least one monosaccharide or disaccharide and 0.01- 0.2% chlorhexidine. In some embodiments, the disclosed solutions are non-ionic hypertonic solutions. In other embodiments, the disclosed solutions are substantially non-ionic. In some examples, the solution has osmolality of about 280-2500 mOsm/kg.

In some examples, the hypertonic solution includes a monosaccharide, such as D-glucose.

In one example, the solution includes 5-20% D-glucose and 0.01-0.2% chlorhexidine gluconate.

The solution may further include 0.01-0.2% cetylpyridinium chloride and/or 5-30% polyethylene glycol (such as polyethylene glycol 3350). In one example, the solution includes 20% D-glucose and 0.1% chlorhexidine gluconate. In another example, the solution includes 20% D-glucose, 0.1% chlorhexidine gluconate, and 0.05% cetylpyridinium chloride.

In other examples, the solution includes 5-20% polyethylene glycol (such as polyethylene glycol 3350) and 0.01-0.2% cetylpyridinium chloride. The solution may further include 0.01-0.2% chlorhexidine gluconate and/or 5-30% of at least one monosaccharide or disaccharide (such as D- glucose).

In some embodiments, the hypertonic solution includes one or more additional components, such as one or more of alcohol (e.g. , ethanol), a surfactant (e.g. , a quaternary ammonium compound such as benzalkonium chloride or benzethonium chloride), and soluble protein (e.g., casein, albumin, or gelatin).

Also provided are kits including the disclosed hypertonic solutions, in a container or an absorbent pad or sponge.

Methods of detecting one or more viral nucleic acids, antigens, or immunoglobulins in a sample are also provided. In some embodiments, the methods include collecting an oral rinse or oral swab from a subject using any one of the hypertonic solutions disclosed herein and measuring presence of the one or more one or more viral nucleic acids, antigens, or immunoglobulins. In other embodiments, the methods include collecting an oral rinse or oral swab from a subject using a non-ionic hypertonic solution (e.g., a solution including 5-20% D-glucose), mixing the oral rinse or oral swab with a solution including 0.01-0.2% chlorhexidine, and measuring presence of the one or more one or more viral nucleic acids, antigens, or immunoglobulins. In some examples, the one or more viral antigens or immunoglobulins are detected using an immunoassay, such as an ELISA or lateral flow immunoassay. In some examples, the oral rinse or swab is not processed or is minimally processed prior to measuring presence of the one or more one or more viral nucleic acids, antigens, or immunoglobulins. The virus in some non-limiting examples is a coronavirus, for example, SARS-CoV-2. The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of an exemplary absorbent sponge for use in sample collection methods described herein. While certain dimensions are shown, these are exemplary only and are non-limiting.

FIGS. 2A-2D show a schematic of an exemplary non-limiting method of use of a sample collection solution provided herein. FIG. 2A: A specimen is collected from a subject following oral rinse or a swab including oral rinse solution. FIG. 2B : An amount of the collected solution is deposited into assay well. FIG. 2C: the specimen is optionally diluted. FIG. 2D: test result is obtained.

FIGS. 3A-3C are a series of graphs showing concentration (pg/ml) of IgG (FIG. 3A), IgM (FIG. 3B), and IgA (FIG. 3C) in oral rinse solutions for two subjects. Paired t-tests within each subject showed a significant difference in concentration between water (open bars) and 20% dextrose (closed bars) rinse solutions. *p<0.05, ***p<.0001.

FIG. 4 is a graph showing that rinse samples containing CPC or CHG show less IgG degradation than untreated rinse samples. IgG concentrations after one week at room temperature. 20% dextrose solutions were used to collect samples from subjects 910 and 918. Samples were spiked to 0.05% CPC or CPC and 0.1% CHG.

FIG. 5 is a graph showing 0.2% CHG (biocide) in a 20% glucose solution (hypertonic solution) inhibited SARS-CoV-2 plaque formation. *** P < 0.001 compared to virus only control.

FIG. 6 is a diagram showing an exemplary lateral flow immunoassay for testing oral rinse or swabs disclosed herein. The solution is collected from a sample following oral rinse or swab. A membrane including anti-IgM, anti-IgG, anti-IgA, and a control is contacted with the rinse or swab solution and presence of antibodies in the sample is detected, for example, utilizing a horseradish peroxidase-conjugated secondary antibody followed by a substrate such as TMB or ABTS.

DETAILED DESCRIPTION

Humans have four major antibody types, with three of them responding to infections. IgA and IgM typically develop first, followed by IgG. One approach for detecting antibodies that is well developed, able to be mass produced, and low cost is lateral flow immunoassay (LFI) technology. LFI tests do not require equipment to be run, the results are easily read and typically produce results in fifteen minutes or less, and in some examples, can be utilized with oral samples, such as saliva or a mouth rinse or swab.

Oral sample collection is typically achieved by spitting saliva into a tube or using an absorbent pad. Oral IgG and IgM antibodies come from oral mucosal transudate (OMT), secreted at the gingival crevice where the gum tissue contacts teeth. IgA is the most abundant immunoglobulin in saliva and early anti-coronavirus, including anti-SARS-CoV-2 IgA antibodies have been reported (e.g., Woo et al., Clin. Diagn. Lab. Immunol. 4:665-668, 2004; Padoana et al, Clinica Chimica Acta 507:164-166, 2020; Guo et al., Clin. Infect. Dis. doi: 10.1093/cid/ciaa310, 2020).

Thus, provided herein are hypertonic solutions (such as non-ionic or substantially non- ionic solutions) for oral sample collection, which can be used in methods for detecting viruses, including coronaviruses in a sample. Hypertonic solutions increase oral antibody concentrations, and in particular examples, the hypertonic solutions include an anti-microbial agent, such as a bisbiguanide.

I. Compositions

Provided herein are compositions, such as hypertonic solutions, that are useful for collection and/or analysis of a sample for presence of or immune response to a vims. In some non-limiting examples, the virus is a coronavirus (such as SARS-CoV-2). In particular embodiments, the solution includes an antimicrobial compound, which may inactivate or inhibit pathogens (including bacteria and/or viruses) in the sample.

In some embodiments, the disclosed hypertonic solutions are non-ionic or substantially non ionic. As used herein, “non-ionic” solutions have little or no ions present, for example, no added ionic compounds in the solution. A “substantially non-ionic solution” refers to a solution including a minimal amount of ionic compounds added. In some examples, a substantially non-ionic solution is one in which a bisbiguanide (such as CHG) is soluble. In some examples, a substantially non ionic solution is one in which at least 0.02% w/v CHG is soluble, such as at least 0.02% w/v, at least 0.05% w/v, at least 0.1% w/v, or at least 0.2% w/v CHG. In other examples, a substantially non-ionic solution includes less than 0.2% ions, such as less than 0.2%, less than 0.15%, less than 0.1%, or less than 0.05% ions.

Hypertonic solutions including at least 5% of a monosaccharide or disaccharide, at least 5% polyethylene glycol (PEG), or both, and an antimicrobial agent are provided. In particular embodiments, the solution includes about 5-30% w/v of at least one monosaccharide or disaccharide, at least 5-20% of a polymeric version of polyethylene glycol, or a combination thereof, and about 0.01-0.2% v/v of a bisbiguanide compound, about 0.01-0.2% of a quaternary ammonium compound, or a combination thereof. In some examples, the bisbiguanide is chlorhexidine (e.g., chlorhexidine digluconate (CHG) or chlorhexidine acetate). In other examples, the quaternary ammonium compound is cetylpyridinium chloride (CPC).

In some embodiments, the solution includes 5-30% of at least one monosaccharide or disaccharide, such as about 5-10%, about 10-15%, about 15-20%, about 20-25%, or about 25-30% of at least one monosaccharide or disaccharide, for example about 5%,

6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% of at least one monosaccharide or disaccharide. In some examples, the monosaccharide or disaccharide is at least one of D-glucose, D-galactose, D- mannose, sucrose, or trehalose.

In other embodiments, the solution includes 5-20% of at least one PEG, such as about 5- 10%, about 10-15%, or about 15-20% of at least one PEG, for example about 5%, 6%, 7%, 8%,

9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of at least one polymeric PEG. In some examples, the PEG is PEG 3350; however, other polymeric PEGs can be utilized, such as PEG 400, PEG 600, or PEG 1450.

In further embodiments, the solution includes about 5-30% of at least one monosaccharide or disaccharide (such as about 5-10%, about 10-15%, about 15-20%, about 20-25%, or about 25- 30% of at least one monosaccharide or disaccharide) and about 5-20% of at least one PEG (such as about 5-10%, about 10-15%, or about 15-20% of at least one PEG). In some examples, the solution includes about 5%, about 10%, about 15%, about 20%, about 25%, or about 30% of at least one monosaccharide or disaccharide (for example, D-glucose or sucrose) and about 5%, 10%, about 15%, or about 20% of at least one PEG (for example, PEG 3350).

In some embodiments, the solution also includes about 0.01-0.2% of a bisbiguanide compound, such as about 0.01-0.05%, about 0.05-0.075%, about 0.075-0.1%, about 0.1-0.15%, or about 0.15-0.2%, for example about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, or about 0.2%.

In some embodiments, the solution also includes about 0.01-0.2% of a quaternary ammonium compound, such as about 0.01-0.05%, about 0.05-0.075%, about 0.075-0.1%, about 0.1-0.15%, or about 0.15-0.2%, for example about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, or about 0.2%.

In particular examples, the solution includes 5-30% glucose (e.g., dextrose or sucrose) and 0.01-0.2% chlorhexidine, such as CHG. In other examples, the solution includes 5-30% PEG and 0.01-0.2% quaternary ammonium, such as CPC. In further examples, the solution includes 5-30% glucose (e.g., dextrose or sucrose), 0.01-0.2% chlorhexidine, and 0.01-0.2% CPC. In still further examples, the solution includes 5-30% glucose (e.g., dextrose or sucrose), 5-20% PEG, and 0.01- 0.2% chlorhexidine and/or CPC. In additional examples, the solution includes 10-20% glucose (e.g., dextrose or sucrose), 10-20% PEG (e.g., PEG 3350), 0.02% CHG and 0.01%-0.05% CPC. Exemplary solutions are provided in Table 1.

Table 1. Exemplary hypertonic solutions

In some examples, the osmolality of the solution is hypertonic, for example, greater than about 278 mOsm/kg. In particular examples, the osmolality of the solution is about 280-2500 mOsm/kg, such as about 280-600 mOsm/kg, about 500-850 mOsm/kg, about 800-1100 mOsm/kg, about 1000-1250 mOsm/kg, about 1200-1500 mOsm/kg, about 1300-1800 mOsm/kg, about 1750- 2000 mOsm/kg, or about 2000-2500 mOsm/kg. In other examples, the osmolality of the solution is about 280 mOsm/kg, about 300 mOsm/kg, about 400 mOsm/kg, about 500 mOsm/kg, about 600 mOsm/kg, about 700 mOsm/kg, about 800 mOsm/kg, about 900 mOsm/kg, about 1000 mOsm/kg, about 1100 mOsm/kg, about 1200 mOsm/kg, about 1500 mOsm/kg, about 1750 mOsm/kg, about 2000 mOsm/kg, about 2250 mOsm/kg, or about 2500 mOsm/kg. In particular examples, the osmolality of the solution is about 280 mOsm/kg, about 555 mOsm/kg, about 834 mOsm/kg, or about 1110 mOsm/kg.

In some examples, the osmolality of the solution is provided by non-ionic ingredients. In particular examples, the solution includes less than 270-280 mOsm/kg ionic components, e.g., less about than 4% w/v ionic components (such as less than about 3.5% w/v ionic components, less than about 3% w/v ionic components, less than about 2.5% w/v ionic components, less than about 2% w/v ionic components, less than about 1.5% w/v ionic components, less than about 1% w/v ionic components, or less than about 0.5% w/v ionic components). In some examples, the solution includes less than about 0.9% w/v NaCl (such as less than about 0.85% w/v NaCl, less than about 0.8% w/v NaCl, less than about 0.75% w/v NaCl, less than about 0.7% w/v NaCl, less than about 0.65% w/v NaCl, less than about 0.6% w/v NaCl, less than about 0.55% w/v NaCl, less than about 0.5% w/v NaCl, less than about 0.45% w/v NaCl, less than about 0.4% w/v NaCl, less than about 0.35% w/v NaCl, less than about 0.3% w/v NaCl, less than about 0.25% w/v NaCl, less than about 0.2% w/v NaCl, less than about 0.15% w/v NaCl, or less than about 0.1% w/v NaCl). In particular examples, the solution does not include NaCl.

In some embodiments, the solution includes one or more additional components. For example, the solution may include one or more additional ingredients including one or more stabilizers, surfactants or viscosity reducing agents, soluble proteins (e.g., to increase immunoassay analyte stability), and/or buffers. In some examples, the stability is an alcohol, such as ethanol, for example at about 7-15% (v/v) (e.g., about 7-10%, 8-12%, 10-12%, or 10-15%). In additional examples, the viscosity reducing agent includes a quaternary ammonium compound (e.g., benzalkonium chloride or benzethonium chloride), for example at about 0.01-0.1% (v/v). In further examples, the soluble protein is included at about 0.05-3%, and may include one or more of casein, albumin, and gelatin. The solution may also include one or more buffers. In some examples, the solution has a pH of about 7.0-7.5. An exemplary non-limiting buffer is phosphate buffered saline. In another example, the solution includes Hanks balanced salt solution and/or antibiotics (e.g., gentamicin and/or amphotericin B).

In any of the disclosed embodiments, the solution further includes water. In some examples, water makes up all or a portion of the volume of the solution remaining after inclusion of the other components.

Exemplary compositions are provided in Table 2. The composition includes at least monosaccharide or disaccharide, PEG, or both and a bisbiguanide, a quaternary ammonium, or both (and water). Additional components are optional. The impact of the components is provided; however, this is non-limiting, and other impacts/uses are contemplated as well.

Table 2. Exemplary hypertonic solution components

II. Kits

Also provided are kits including one or more of the solutions provided herein. In some embodiments, the kit includes a disclosed solution in a container (for example, a tube, syringe, or vial), such as a single-use container. In some examples, the disclosed solution is provided in an amount suitable for sample collection from a subject, for example by oral rinsing (for example, about 2-15 ml of solution). In other examples, the kit includes an absorbent sponge or pad that contains a disclosed solution (e.g., is wet with or soaked with the solution), for example as shown in FIG. 1. In some examples, the sponge or pad is attached to a rod (such as a wooden or plastic rod), which can be used to place the sponge or pad in a subject’s mouth and/or for handling the sponge or pad. In one example, the kit includes a sponge or pad including the solution in a container (such as a tube).

In additional examples, the kit includes a second collection tube that optionally contains additional components, into which the oral rinse or sponge is placed following use. In some examples, the additional components in the tube include RNase inhibitors, protein denaturing agents, preservatives (e.g., ProClin™ preservatives), and/or non-ionic surfactants.

The kits may contain additional components or reagents, for example, for detection of virus nucleic acids, proteins, and/or antibodies in a sample, such as a lateral flow assay (e.g., a “dip stick”). Additional components in the kit may include reagents for detection of nucleic acids, such as buffers, enzymes, detection reagents, viral proteins, viral proteins conjugated to enzymes, and/or enzyme-labeled antibodies. Components that serve as calibrators, positive or negative controls may also be included in the kits. In some examples, the kit further includes instructions for use.

III. Methods of Use

Provided here are methods of using the disclosed compositions, for example in detection of viral nucleic acids, antigens, or immunoglobulins in a subject. The solution is non-toxic to eukaryotic cells, non-ionic or substantially non-ionic, and microbicidal, e.g., reducing the potential infectivity of the sample.

In some embodiments, the methods include rinsing the mouth of a subject with a disclosed solution and collecting the rinse from the subject and measuring the presence of one or more of viral nucleic acids, antigens, or immunoglobulins. In some examples, the solution is rinsed in the subject’s mouth (for example, by swishing in the mouth) for about 10-60 seconds (for example, about 15-30 seconds) prior to collection, for example by collecting in a tube or vial. In some examples, the subject expresses the rinse into a tube or vial, while in other examples, the rinse is absorbed onto an absorbent pad or sponge. An exemplary method is shown in FIG. 2. In other embodiments, the mouth of the subject is rinsed with a non-ionic hypertonic solution (such as a solution including about 5-20% monosaccharide and/or disaccharide) and the solution is then combined with a solution including a bisbiguanide (such as about 0.01-0.2% chlorhexidine) upon collection (e.g., in a collection tube or vial). The collection tube or vial containing the bisbiguanide may include additional components, such as one or more of RNase inhibitors, protein denaturing agents, preservatives (e.g. , ProClin™ preservatives), and/or non-ionic surfactants.

In additional embodiments, the methods include providing the rinse to the subject on an absorbent pad or sponge including a disclosed solution (e.g., about 2-5 ml of the solution). The pad or sponge may be used to deliver the rinse to the subject’s mouth, followed by swishing and collection of the solution, either by reabsorption in the sponge or pad or by collection in a tube or vial (for example, by the subject expressing the solution into a tube or vial). In other examples, the pad or sponge is placed in the subject’s mouth and rubbed on the outer gingival mucosa for about 10-60 seconds (for example, about 15-30 seconds). The pad or sponge is then placed in a tube or vial for further processing. In some embodiments, the sponge or pad includes a non-ionic or substantially non-ionic hypertonic solution (such as a solution including about 5-20% monosaccharide and/or disaccharide) and the collected solution is then combined with a solution including a bisbiguanide (such as about 0.01-0.2% chlorhexidine). The collection tube or vial containing the bisbiguanide may include additional components, such as one or more of RNase inhibitors, protein denaturing agents, preservatives (e.g., ProClin™ preservatives), and/or non-ionic surfactants.

In some embodiments, the collected solution is utilized in one or more assays for detection of viral nucleic acids (e.g., PCR or RT-PCR assays) or viral antigens or antibodies (e.g., immunoassays, such as ELISA or lateral flow immunoassays). Exemplary immunoassays include, but are not limited to, Vazyme 2019-nCoV IgG/IgM detection kit, or modifications thereof, for example, to include IgA detection. Additional exemplary commercially available detection assays include Architect SARS-CoV-2 IgG ELISA (Abbott), Elecsys anti-SARS-CoV-2 (IgG) (Roche), Platelia SARS-CoV-2 Total Ab assay (Bio-Rad), SARS-CoV-2 ELISA (IgG) (Euroimmun AG), SCoV-2 Detect IgG ELISA (InBios), SCoV-2 Detect IgM ELISA (InBios), Anti-SARS-CoV-2 Rapid Test (AutoBio Diagnostics), qSARS-CoV-2 IgG/IgM Rapid Test (Cellex, Inc.), RightSign COVID-19 IgG/IgM Rapid Test Cassette (Hangzhou Biotech), COVID-19 IgG/IgM Rapid Test Cassette (Healgen), Alinity SARS-CoV-2 IgG (Abbott), LIAISON SARS-CoV-2 S1/S2 IgG (DiaSorin), and VITROS Anti-SARS-CoV-2 IgG test (Ortho-Clinical Diagnostics). In some examples, a commercially available detection assay is modified to include IgA detection. In particular examples, the collected solution can be used directly or can be processed, such as by adding solvents, preservatives, buffers, or other compounds or substances. In some examples, the collected sample is used directly or with minimal pre-processing.

In some embodiments, the subject is a human or other mammal that has or is suspected to have a viral infection. In some examples, the subject has symptoms of a viral infection. In other examples, the subject is asymptomatic. In further examples, the subject had contact with or suspected contact with another individual known or suspected to have a viral infection. In particular examples, the disclosed methods are used to detect coronavirus (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) nucleic acids, antigens, or immunoglobulins. However, the methods can be utilized with other viruses. In some examples, the virus is an enveloped vims, for example, herpesviruses, poxviruses, hepadnaviruses, asfarviridae, flaviviruses, alphaviruses, togaviruses, coronaviruses, hepatitis D vims, orthomyxoviruses, paramyxovimses, rhabdoviruses, bunyavimses, filovimses, and retroviruses. In particular non-limiting examples, the vims is a respiratory syncytial vims, herpes simplex virus type 1/type 2, equine infectious vims, variola virus, equine influenza virus, hog cholera vims, bovine viral diarrhea, parainfluenza virus, rabies virus, canine distemper virus, Newcastle virus, pseudorabies virus, cytomegalovirus, or human immunodeficiency virus type 1.

EXAMPLES

The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.

- it - Example 1

Solubility of Chlorhexidine in Non-Ionic Hypertonic Solutions

Chlorhexidine is used as an antimicrobial compound/preservative in topical agents and in solutions such as mouthwashes and eyedrops. Chlorhexidine gluconate (CHG) has been demonstrated to have viricidal properties, including against coronaviruses (Lim et al., Anaesth. Intensive Care 36:502-512, 2008). However, CHG is not compatible with ionic hypertonic solutions, such as sodium chloride, at concentrations greater than 0.01%. We have found that 0.02% CHG precipitated in 3% sodium chloride (NaCl). However, when added to 10% glucose, 0.02% CHG, 0.05% CHG and 0.1% CHG remained soluble and did not precipitate.

Example 2

Testing Solutions for Virus Neutralization

Oral rinse solutions are prepared with different antimicrobial concentrations and then evaluated for SARS-CoV-2 inactivation by adding the solution to SARS-CoV-2 prior to the virus being used to infect a susceptible cell culture. Solutions include 10% glucose with 0%, 0.01%, 0.02%, 0.05%, or 0.10% CHG. Samples including SARS-CoV-2 in the oral rinse without the antimicrobial agent and SARS-CoV-2 in PBS are positive controls and uninfected cell culture is a negative control. If effects of the rinse solution on the cell culture are observed, viral solutions will either be diluted by at least two orders of magnitude (1:100), or the vims in the solutions will be separated from the oral rinse by centrifugal ultrafiltration prior to being used to infect the cell culture.

Stocks of SARS-CoV-2 are quantified by plaque assay on Vero cells (or other SARS-CoV- 2 susceptible cells). A defined amount of vims (e.g., 10,000 plaque forming units) is mixed with the oral rinse solution and incubated at room temperature for various times (e.g., 5 min, 15 min, 30 min, 1 h). Following incubation, samples are diluted in phosphate-buffered saline and quantified on Vero cells.

Example 3

Testing Solutions for Immunoglobulin Degradation and Virus Inactivation

Subjects swished with Oral rinse for 20 seconds. Samples were placed on ice, aliquoted and frozen. For analysis by ELISA samples were thawed on ice, spun for 5 minutes and used in ELISA assays. Immunoglobulins were measured using Salimetrics Salivary Human Total ELISA Kits.

IgG and IgM were detected by indirect sandwich ELISA Kits in which a “sandwich” is formed when the pre-coated capture Anti-Human IgG or Ig M antibodies present on the plate binds IgG or IgM in standards & samples. The total amount of IgG or IgM Antibody Enzyme Conjugate detected is directly proportional to the amount of Total Human IgG or IgM present in the sample. The Salimetrics® SIgA Indirect Enzyme Immunoassay Kit is a competitive immunoassay specifically designed and validated for the quantitative measurement of salivary SIgA. This is an indirect competitive immunoassay kit. The amount of SIgA Antibody Enzyme Conjugate detected is inversely proportional to the amount of SIgA present in the sample

The effect of oral rinse solutions on presence of immunoglobulins were tested. Oral rinse with 20% dextrose contained significantly more immunoglobulins (IgG, IgM, and IgA) than water rinse (FIGS. 3A-3C) from two subjects.

The effect of including CPC and CHG in the rinse was also tested. Solution containing 20% dextrose was used to collect samples from two subjects, the samples were spiked to contain 0.05% CPC or 0.05% CPC and 0.1 CHG, then stored at room temperature for one week. Rinse samples containing CPC or CPC and CHG showed less IgG degradation than untreated rinse samples (20% dextrose alone) (FIG. 4).

The ability of 0.2% CHG in 20% dextrose to inhibit SARS-CoV-2 plaque formation was assessed. SARS-CoV-2 infectious viral titer was measured via plaque reduction assays with neutral red staining 48 hours post treatment with CHG and infection with SARS-CoV-2 in a confluent 12 well plate of confluent Vero E6 cells. There was a 10-fold reduction in virus titer in the presence of 0.2% CHG (FIG. 5). Fold-change was calculated compared to the vims only control using a one way ANOVA multiple comparisons test.

Example 4

Testing Solutions for Compatibility with Lateral Flow Immunoassay

Oral rinse solutions are tested for use in LFI. Solutions include 5% glucose with 0.05% or 0.10% CHG and 10% glucose with 0.05% or 0.10% CHG. The oral sample may have lower viscosity than a serum, plasma or whole blood sample, which could impact the performance characteristics of the LFI. Membranes from LFI development kits are obtained and used to evaluate the performance of the oral rinse solutions for lateral flow transport and antibody binding and are set up to capture IgG, IgM and IgA antibodies.

Nitrocellulose strips are dot blotted with anti-IgA, anti-IgG and anti-IgM antibodies (FIG. 6). Oral rinse solutions with known concentrations of anti-SARS-CoV-2 IgG, IgM and IgA antibodies are tested with these strips by allowing the rinse solution to wick up the membrane. Once the membrane has been saturated it is incubated in a buffer that contains HRP-conjugated anti-human immunoglobulin secondary antibody and then developed with a solution containing a substrate/chromogen for color-based detection. Purified anti-SARS-CoV-2 IgG, IgM and IgA antibodies from COVID patients are used to spike oral rinse solutions for testing.

Example 5 Testing Lateral Flow Immunoassay with Saliva Samples

Matched oral rinsed-saliva samples and blood samples from COVID- 19 positive patients and COVID- 19 negative control subjects are obtained and both blood and saliva are tested on rapid LFI tests (e.g., Vazyme LFI Detection kit). Prior to testing, the saliva samples are divided, with half the sample being applied to the LFI with the kit diluent. The other half of the sample is mixed with the oral rinse solution prior to being applied to the LFI. Test results are interpreted in accordance with the manufacturer’s instructions.

In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A hypertonic solution comprising:

5-20% polyethylene glycol, 5-30% of at least one monosaccharide or disaccharide, or a combination thereof and

0.01-0.2% cetylpyridinium chloride, 0.01-0.2% chlorhexidine, or a combination thereof.

2. The hypertonic solution of claim 1, comprising:

5-20% polyethylene glycol; and

0.01-0.2% cetylpyridinium chloride.

3. The hypertonic solution of claim 1, comprising:

5-30% of at least one monosaccharide or disaccharide; and 0.01-0.2% chlorhexidine.

4. The hypertonic solution of any one of claims 1 to 3, wherein the solution is a non-ionic or substantially non-ionic hypertonic solution.

5. The hypertonic solution of any one of claims 1 to 4, wherein the solution has osmolality of about 280-2500 mOsm/kg.

6. The hypertonic solution of any one of claims 1 or 3 to 5, wherein the monosaccharide comprises D-glucose.

7. The hypertonic solution of claim 6, comprising 5-20% D-glucose and 0.01-0.2% chlorhexidine gluconate.

8. The hypertonic solution of claim 7, further comprising 0.01-0.2% cetylpyridinium chloride.

9. The hypertonic solution of any one of claims 6 to 8, further comprising 5-30% polyethylene glycol.

10. The hypertonic solution of any one of claims 1, 2, 4, 5, or 9 wherein the polyethylene glycol comprises polyethylene glycol 3350.

11. The hypertonic solution of claim 10, comprising 5-20% polyethylene glycol 3350 and 0.01- 0.2% cetylpyridinium chloride.

12. The hypertonic solution of claim 11, further comprising 0.01-0.2% chlorhexidine gluconate.

13. The hypertonic solution of any one of claims 10 to 12, further comprising 5-30% of at least one monosaccharide or disaccharide.

14. The hypertonic solution of any one of claims 1 to 13, further comprising one or more of an alcohol, a surfactant, and a soluble protein.

15. The hypertonic solution of claim 14, wherein the alcohol comprises ethanol.

16. The hypertonic solution of claim 14, wherein the surfactant comprises a quaternary ammonium compound.

17. The hypertonic solution of claim 16, wherein the quaternary ammonium compound is benzalkonium chloride or benzethonium chloride.

18. The hypertonic solution of claim 14, wherein the soluble protein comprises casein, albumin, or gelatin.

19. A kit comprising the hypertonic solution of any one of claim 1 to 18 in a container or an absorbent pad or sponge.

20. A method of detecting one or more viral nucleic acids, antigens, or immunoglobulins in a sample, comprising: collecting from a subject an oral rinse or swab comprising the hypertonic solution of any one of claims 1 to 18; and measuring presence of the one or more one or more viral nucleic acids, antigens, or immunoglobulins .

21. The method of claim 20, further comprising: providing the hypertonic solution to the subject; and obtaining the oral rinse or swab.

22. A method of detecting one or more viral nucleic acids, antigens, or immunoglobulins in a sample, comprising: collecting from a subject an oral rinse or swab comprising a non-ionic or substantially non ionic hypertonic solution; mixing the oral rinse or swab with a solution comprising 0.01-0.2% chlorhexidine and/or 0.01-0.2% cetylpyridinium chloride; and measuring presence of the one or more one or more viral nucleic acids, antigens, or immunoglobulins .

23. The method of any one of claims 20 to 22, wherein measuring presence of the one or more one or more viral antigens or immunoglobulins comprises immunoassay.

24. The method of claim 23, wherein the immunoassay is ELISA or lateral flow immunoassay.

25. The method of any one of claims 20 to 24, wherein the oral rinse or swab is not processed prior to measuring presence of the one or more one or more viral nucleic acids, antigens, or immunoglobulins .

26. The method of any one of claims 20 to 25, wherein the virus comprises a coronavirus.

27. The method of claim 26, wherein the coronavirus is SARS-CoV-2.