Patent Grant
12366581
The present disclosure relates to the detection of virus-neutralizing antibodies.
There is a need in the art for an assay that accurately assesses a subject's immune response to a virus such as SARS CoV-2. The 2019/2020 SARS CoV-2 outbreak in Wuhan China has provided sobering evidence that regional outbreaks of zoonotic virus infection have the potential to spread rapidly across much larger geographic regions. Given the widespread distribution of infections, SARS CoV-2 may now be established as an endemic virus in the world population.
Although the development of effective antiviral agents that rapidly suppress SARS CoV-2 virus replication will be invaluable in the therapeutic control of ongoing and/or future outbreaks, a preventative vaccine is currently viewed as the most effective approach to reducing the risk of future SARS CoV-2 outbreaks, epidemics, and pandemics. Literally hundreds of immunization strategies are being explored to develop safe and effective SARS CoV-2 vaccines. Many of the most promising approaches are designed to elicit broadly protective humoral immune responses resulting in neutralizing antibody (nAb) activity directed at the coronavirus spike protein.
Until such time that SARS CoV-2 infection can be effectively treated with existing and novel antiviral medications and/or immunotherapies, clinical treatment options for the severely ill will likely include treatment with convalescent sera/plasma donated by individuals that recently recovered from SARS CoV-2 infection.
However, the correlates of SARS CoV-2 protective immunity and therapeutic efficacy are unknown. Protection from virus infection, or suppression of virus replication may involve various components of the innate and adaptive immune systems. Evidence is emerging to indicate that B-cell mediated humoral immune responses (i.e., neutralizing antibodies) may be critical for protection and viral clearance. Currently there is an intense and comprehensive effort to develop, validate, and implement diagnostic assays to reliably characterize antibody responses to SARS CoV-2 infection and immunization.
The pathogenicity of SARS CoV-2 necessitates that all cell-based in vitro studies involving live (i.e. replication competent) virus are performed under BSL3 containment, including assessments of nAb activity. The assay as disclosed herein provides an accurate, reproducible, and high-throughput alternative that can be conducted under BSL2 containment resulting in reduced risk to laboratory personnel and lower costs to vaccine sponsors and providers of convalescent sera.
Such an assay allows for studies to establish protective immunity levels to SARS CoV-2 and would contribute to predicting individual prognoses and monitoring recovery following an infection with the pathogen. Additionally, the assay can be used to study vaccine and treatment responses and to assist in the selection of donor plasma for convalescent plasma therapy. Presently available assays are insufficient for these applications.
It is an object of the present disclosure to provide methods for detecting coronavirus neutralizing antibodies. The methods may be embodied in a variety of ways.
In some embodiments, the present disclosure provides a method for detecting whether a subject exposed to a coronavirus has developed a neutralizing antibody response, comprising (a) transfecting into a plurality of first cells: i) a nucleic acid encoding a coronavirus spike protein, and ii) a viral expression vector which comprises an indicator nucleic acid which produces a detectable signal; (b) incubating the first cells under conditions such that the first cells produce viral particles comprising the coronavirus spike protein; (c) contacting the viral particles of step (b) with a second cell in the presence or absence of a sample comprising an antibody from the subject, wherein the second cell expresses a cell surface receptor to which the coronavirus binds; (d) measuring the amount of the detectable signal produced by the second cell in order to determine the infectivity of the viral particles in the presence or absence of the sample; and (e) comparing the amount of signal measured in step (d) in the presence of the sample with the amount of signal produced in step (d) in the absence of the sample, wherein a reduced amount of signal measured in the presence of the sample indicates that the subject has developed a neutralizing antibody response capable of reducing infection.
In other embodiments, the present disclosure provides a method for determining whether a first subject infected by a virus is likely to respond to treatment with an antibody preparation, comprising: (a) transfecting into a plurality of first cells: i) a nucleic acid encoding a coronavirus spike protein from the subject, and ii) a viral expression vector which comprises an indicator nucleic acid which produces a detectable signal; (b) incubating the first cells under conditions such that the first cells produce viral particles comprising the coronavirus spike protein; (c) contacting the viral particles of step (b) with a second cell in the presence or absence of an antibody preparation, wherein the second cell expresses a cell surface receptor to which the virus binds; (d) measuring the amount of the detectable signal produced by the second cell in order to determine the infectivity of the viral particles in the presence or absence of the antibody preparation; and (e) comparing the amount of signal measured in step (d) in the presence of the antibody preparation with the amount of signal produced in step (d) in the absence of the antibody preparation, wherein a reduced amount of signal measured in the presence of the antibody preparation indicates that the subject is likely to be responsive to treatment with the antibody preparation.
In other embodiments, the present disclosure provides a method for detecting the level (i.e., titer) of neutralizing antibody response in a sample from a subject exposed to a coronavirus or to a coronavirus vaccine, comprising: (a) transfecting into a plurality of first cells: i) a nucleic acid encoding a coronavirus spike protein, and ii) an viral expression vector which comprises an indicator nucleic acid which produces a detectable signal; (b) incubating the first cells under conditions such that the first cells produce viral particles comprising the coronavirus spike protein; (c) contacting the viral particles of step (b) with a second cell in the presence or absence of a sample from the subject that was exposed to a coronavirus or to a coronavirus vaccine, wherein the second cell expresses a cell surface receptor to which the coronavirus binds; (d) measuring the amount of the detectable signal produced by the second cell in order to determine the infectivity of the viral particles in the presence or absence of the sample; and (e) comparing the amount of signal measured in step (d) in the presence of the sample with the amount of signal produced in step (d) in the absence of the sample, and; (f) determining the level of a neutralizing antibody response in the sample based on the extent of the reduction in infectivity of the viral particles exposed to the sample as compared to the infectivity of the viral particles not exposed to the sample.
In other embodiments, the present disclosure provides methods to evaluate the effect of a mutation in a SARS CoV-2 spike protein on susceptibility to an anti-SARS CoV-2 neutralizing antibody. For example, the method may comprise (a) transfecting into a first portion of a plurality of first cells: i) a nucleic acid encoding a control coronavirus spike protein, and ii) a viral expression vector which comprises an indicator nucleic acid which produces a detectable signal; and (b) transfecting into a second portion of a plurality of first cells: i) a nucleic acid encoding a coronavirus spike protein that comprises a mutation and ii) a viral expression vector which comprises an indicator nucleic acid which produces a detectable signal. The method may then further comprise (c) incubating the first and second portion of first cells separately under conditions such that the first portion cells produce first viral particles comprising the control coronavirus spike protein, and the second portion cells produce second viral particles comprising the coronavirus spike protein having the mutation. The method may further comprise (d) contacting the first viral particles of step (c) with a first portion of a plurality of second cells in the presence of a sample comprising an antibody that binds SARS CoV-2 (i.e., an anti-SARS CoV-2 antibody), wherein the second cell expresses a cell surface receptor to which the coronavirus binds. The method may further comprise (e) contacting the second viral particles of step (c) with a second portion of a plurality of second cells in the presence of the sample comprising an antibody that binds SARS CoV-2, wherein the second cell expresses a cell surface receptor to which the coronavirus binds. The method may further comprise (f) measuring the amount of the detectable signal produced by the second cell in steps (d) and (e), wherein a reduced amount of signal produced by the second cell in step (d) as compared to the signal produced by the second cell in step (e) indicates that the mutation in the spike protein confers a reduced susceptibility of the viral particles to the anti-SARS CoV-2 antibody.
Also disclosed are compositions for the detection of a compound such as a neutralizing antibody that can modulate SARS infectivity. In certain embodiments, the composition may comprise cells that have been engineered to be either producer cells or target cells as disclosed herein. For example, the system may comprise a first cell (or cells) that has been genetically modified to be a target cell that expresses an angiotensin-converting enzyme 2 receptor (ACE-2). In some embodiments, the target cell may be genetically modified to also express human airway transmembrane trypsin-like serine protease (TMPRSS2). In some embodiments, and as discussed herein, the ACE2 and/or TMPRSS2 may be introduced into the target cell in a manner that allows for transient expression. Or, the ACE2 and/or TMPRSS2 may be introduced into the target cell in a manner that allows for stable expression. Also, disclosed are genetic constructs for making producer and/or target cells.
Also disclosed are kits, systems, and computer-program products tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to perform any of the steps of the methods or run any part of the systems.
The following description recites various aspects and embodiments of the present compositions and methods. No particular embodiment is intended to define the scope of the compositions and methods. Rather, the embodiments merely provide non-limiting examples of various methods and systems that are at least included within the scope of the compositions and methods. The description is to be read from the perspective of one of ordinary skill in the art; therefore, information well known to the skilled artisan is not necessarily included.
The present disclosure now will be described more fully hereinafter. The disclosure may be embodied in many different forms and should not be construed as limited to the aspects set forth herein; rather, these aspects are provided so that this disclosure will satisfy applicable legal requirements. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entireties. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.
When introducing elements of the present disclosure or the embodiment(s) thereof, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. It is understood that aspects and embodiments of the disclosure described herein include “consisting” and/or “consisting essentially of” aspects and embodiments.
The term “and/or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or in combination with any one or more of the listed items. For example, the expression “A and/or B” is intended to mean either or both of A and B, i.e. A alone, B alone or A and B in combination. The expression “A, B, and/or C” is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.
Various aspects of this disclosure are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
“Sample” or “patient sample” or “biological sample” or “specimen” are used interchangeably herein. The source of the sample may be solid tissue as from a fresh tissue, frozen and/or preserved organ or tissue or biopsy or aspirate. The source of the sample may be a liquid sample. Non-limiting examples of liquid samples include cell-free nucleic acid, blood or a blood product (e.g., serum, plasma, or the like), urine, nasal swabs, biopsy sample (e.g., liquid biopsy for the detection of cancer) or combinations thereof. The term “blood” encompasses whole blood, blood product, or any fraction of blood, such as serum, plasma, buffy coat, or the like as conventionally defined. Suitable samples include those which are capable of being deposited onto a substrate for collection and drying including, but not limited to: blood, plasma, serum, urine, saliva, tear, cerebrospinal fluid, organ, hair, muscle, or other tissue sampler other liquid aspirate. In an embodiment, the sample body fluid may be separated on the substrate prior to drying. For example, blood may be deposited onto a sampling paper substrate which limits migration of red blood cells allowing for separation of the blood plasma fraction prior to drying in order to produce a dried plasma sample for analysis. For example, in certain embodiments (e.g., COVID-19) the biological sample comprises a specimen from either the upper or lower respiratory system. In an embodiment, the sample may comprise e.g., at least one of a nasopharyngeal swab, a mid-turbinate swab, anterior nares swab, an oropharyngeal swab, sputum, a lower respiratory tract aspirate, a bronchoalveolar lavage, a nasopharyngeal wash and/or aspirate, or a nasal aspirate. The sample may contain compounds that are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.
As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, the terms “subject” and “subjects” refer to an animal, preferably a mammal including a non-primate (e.g., a cow, pig, horse, donkey, goat, camel, cat, dog, guinea pig, rat, mouse, or sheep) and a primate (e.g., a monkey, such as a cynomolgus monkey, gorilla, chimpanzee, or a human).
As used herein, the terms “pseudovirus,” “pseudovirion,” and “viral particles” may be used interchangeably.
“Treatment,” and other forms of this word refer to the administration of an agent to impede a disease. Treatment may also refer to any course which one skilled, for example, a treating physician, deems expedient.
As used herein, the term “titer” refers to the concentration of an analyte of interest. As used herein, “titer” may refer to the concentration of an antibody. In some embodiments, “titer” may refer to the concentration of a neutralizing antibody, such as a neutralizing antibody that recognized the Sars-CoV-2 spike protein.
Methods
In some embodiments, the present disclosure provides a method for detecting whether a subject exposed to a coronavirus has developed a neutralizing antibody response, comprising (a) transfecting into a plurality of first cells: i) a nucleic acid encoding a coronavirus spike protein, and ii) a viral expression vector which comprises an indicator nucleic acid which produces a detectable signal; (b) incubating the first cells under conditions such that the first cells produce viral particles comprising the coronavirus spike protein; (c) contacting the viral particles of step (b) with a second cell in the presence or absence of a sample comprising an antibody from the subject, wherein the second cell expresses a cell surface receptor to which the coronavirus binds; (d) measuring the amount of the detectable signal produced by the second cell in order to determine the infectivity of the viral particles in the presence or absence of the sample; and (e) comparing the amount of signal measured in step (d) in the presence of the sample with the amount of signal produced in step (d) in the absence of the sample, wherein a reduced amount of signal measured in the presence of the sample indicates that the subject has developed a neutralizing antibody response capable of reducing infection.
In other embodiments, the present disclosure provides for a method for determining whether a subject infected by a virus is likely to respond to treatment with an antibody preparation, comprising: (a) transfecting into a plurality of first cells: i) a nucleic acid encoding a coronavirus spike protein from the subject, and ii) a viral expression vector which comprises an indicator nucleic acid which produces a detectable signal; (b) incubating the first cells under conditions such that the first cells produce viral particles comprising the coronavirus spike protein from the subject; (c) contacting the viral particles of step (b) with a second cell in the presence or absence of the antibody preparation, wherein the second cell expresses a cell surface receptor to which the virus binds; (d) measuring the amount of the detectable signal produced by the second cell in the presence or absence of the antibody preparation; and (e) comparing the amount of signal measured in step (d) in the presence of the antibody preparation with the amount of signal produced in step (d) in the absence of the antibody preparation, wherein a reduced amount of signal measured in the presence of the antibody preparation from the second subject indicates that the subject is likely to be responsive to treatment with the antibody preparation. In certain embodiments, the antibody preparation is convalescent sera from a second subject.
In other embodiments, the present disclosure provides a method for detecting the level (e.g., titer) of neutralizing antibody response in a sample from subject exposed to a coronavirus comprising: (a) transfecting into a plurality of first cells: i) a nucleic acid encoding a coronavirus spike protein, and ii) a viral expression vector which comprises an indicator nucleic acid which produces a detectable signal; (b) incubating the first cells under conditions such that the first cells produce viral particles comprising the coronavirus spike protein; (c) contacting the viral particles of step (b) with a second cell in the presence or absence of the sample from the subject that was exposed to a coronavirus, wherein the second cell expresses a cell surface receptor to which the coronavirus binds; (d) measuring the amount of the detectable signal produced by the second cell in order to determine the infectivity of the viral particles in the presence or absence of the sample; and (e) comparing the amount of signal measured in step (d) in the presence of the sample with the amount of signal produced in step (d) in the absence of the sample, and; (f) determining the level of a neutralizing antibody response in the sample based on the extent of the reduction in infectivity of the viral particles exposed to the sample as compared to the infectivity of the viral particles not exposed to the sample. In some embodiments, the sample is a serum sample. In an embodiment, the measured level of neutralizing antibody is correlated to the World Health Organization International Standard having a defined level of International Units (IU) per milliliter (mL).
In other embodiments, the present disclosure provides a method for detecting the level of neutralizing antibody response (e.g., titer) in a sample from subject exposed to a coronavirus vaccine comprising: (a) transfecting into a plurality of first cells: i) a nucleic acid encoding a coronavirus spike protein, and ii) a viral expression vector which comprises an indicator nucleic acid which produces a detectable signal; (b) incubating the first cells under conditions such that the first cells produce viral particles comprising the coronavirus spike protein; (c) contacting the viral particles of step (b) with a second cell in the presence of a sample obtained from the subject before the subject was exposed to a coronavirus vaccine or a sample from the subject obtained after the sample was exposed to the coronavirus vaccine, wherein the second cell expresses a cell surface receptor to which the coronavirus binds; (d) measuring the amount of the detectable signal produced by the second cell in order to determine the infectivity of the viral particles in the presence of the sample obtained before the subject was exposed to the coronavirus vaccine; and (e) measuring the amount of the detectable signal produced by the second cell in order to determine the infectivity of the viral particles in the presence of the sample obtained after the subject was exposed to the coronavirus vaccine; (f) comparing the amount of signal measured in step (d) with the amount of signal produced in step (e), and determining the level of a neutralizing antibody response in the sample based on the difference in the amount of signal measured in step (d) with the amount of signal produced in step (e).
In some embodiments of the methods disclosed herein, the antibody response may be a neutralizing antibody response that completely inhibits infection. In some embodiments of the methods disclosed herein, the neutralizing antibody response may partially inhibit infection, but to a level that is therapeutically effective.
In some embodiments of the methods disclosed herein, the coronavirus spike protein may be a coronavirus S protein. SARS CoV-2 Spike (S) protein is a trimeric protein that mediates the binding and entry of the virus into host cells. S protein is a major target of neutralizing antibodies. Each S protein monomer consists of an N-terminal S1 domain that mediates receptor binding and a membrane-proximal S2 domain that mediates membrane fusion. SARS CoV-2 can use the angiotensin converting enzyme-2 (ACE-2) as its cellular receptor.
The SARS CoV-2 S protein is known to mutate, leading to variants. SARS-CoV-2 variants are defied by specific point mutations along the length of the spike gene causing amino acid deletions or substitutions. These changers may confer enhanced spike protein binding efficiency to the ACE 2 receptor, allowing the virus to infect cells more readily. Additionally and/or alternatively, these changes may affect the capability of the virus to escape from antibodies that are raised after natural infection, after vaccine, or from antibody therapies. In some embodiments, the S protein may be from a subject infected with a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant. In some embodiments the variant may be the B.1.1.7 (UK) variant, B.1.351 (South Africa) variant, the B.1.1.28.1 (Brazil) variant, or the B.1.427/B/1/429 (California) variant.
In some embodiments of the methods disclosed herein, the indicator nucleic acid may comprise an indicator gene. In some embodiments, the indicator gene may be a luciferase gene. Or other gene sequences that encode detectable indicators may be used.
In some embodiments of the methods disclosed herein, the cell surface receptor may be ACE-2. As is depicted in
In other aspects, disclosed are methods to evaluate mutations in SARS CoV-2 for their ability to confer increased or decreased susceptibility to anti-SARS CoV-2 neutralizing antibodies. For example, the method may comprise (a) transfecting into a first portion of a plurality of first cells: i) a nucleic acid encoding a control coronavirus spike protein, and ii) a viral expression vector which comprises an indicator nucleic acid which produces a detectable signal; and (b) transfecting into a second portion of a plurality of first cells: i) a nucleic acid encoding a coronavirus spike protein that comprises a mutation. The method may then further comprise (c) incubating the first and second portion of first cells separately under conditions such that the first portion cells produce first viral particles comprising the control coronavirus spike protein, and the second portion cells produce second viral particles comprising the coronavirus spike protein having the mutation. The method may further comprise (d) contacting the first viral particles of step (c) with a second cell in the presence of a sample comprising an antibody that recognizes SARS CoV-2 (i.e., an anti-SARS CoV-2 antibody), wherein the second cell expresses a cell surface receptor to which the coronavirus binds. The method may further comprise (e) contacting the second viral particles of step (c) with a second cell in the presence of a sample comprising an antibody that recognizes SARS CoV-2, wherein the second cell expresses a cell surface receptor to which the coronavirus binds. The method may further comprise (f) measuring the amount of the detectable signal produced by the second cells in (d) and (e), wherein a reduced amount of signal produced by the second cell in step (d) as compared to the signal produced by the second cell in step (e) indicates that the mutation in the spike protein confers a reduced susceptibility of the viral particles to the anti-SARS CoV-2 antibody. In certain embodiments, the control coronavirus spike protein is a spike protein from a wild type coronavirus. In certain other embodiments, the control coronavirus spike protein is a spike protein from a different subject or from the same subject at a different time point.
In other embodiments, the present disclosure provides a method for detecting the level of neutralizing antibody response (e.g., titer) to a mutated or variant SARS-CoV-2 in a subject previously exposed to a coronavirus vaccine developed for a non-mutated SARS-CoV-2 comprising: (a) transfecting into a plurality of first cells: i) a nucleic acid encoding a coronavirus spike protein (i.e., the mutated or variant spike protein), and ii) a viral expression vector which comprises an indicator nucleic acid which produces a detectable signal; (b) incubating the first cells under conditions such that the first cells produce viral particles comprising the mutant/variant coronavirus spike protein; (c) contacting the viral particles of step (b) with a second cell in the presence of a sample obtained from the subject before the subject was exposed to a coronavirus vaccine or a sample from the subject obtained after the sample was exposed to the coronavirus vaccine, wherein the second cell expresses a cell surface receptor to which the coronavirus binds; (d) measuring the amount of the detectable signal produced by the second cell in order to determine the infectivity of the viral particles in the presence of the sample obtained before the subject was exposed to the coronavirus vaccine; and (e) measuring the amount of the detectable signal produced by the second cell in order to determine the infectivity of the viral particles in the presence of the sample obtained after the subject was exposed to the coronavirus vaccine; (f) comparing the amount of signal measured in step (d) with the amount of signal produced in step (e), and determining the level of a neutralizing antibody response in the sample based on the difference in the amount of signal measured in step (d) with the amount of signal produced in step (e). In an embodiment, the spike protein of step (a) is isolated from the subject. In other embodiments, the spike protein is genetically modified in vitro. In some embodiments, the results with the mutated spike protein are compared to a normal (i.e., non-mutated) spike protein control.
In some embodiments, the coronavirus spike protein may comprise a mutation associated with increased viral infectivity. In come embodiments, the coronavirus spike protein may contain a D614G mutation. The mutation D614G in the carboxy terminal region of the SARS CoV-2 S1 domain is associated with increased viral loads in COVID-19 patients.
In some embodiments of the methods disclosed herein, the subject was infected with a coronavirus. In some embodiments of the methods disclosed herein, the subject was infected with severe acute respiratory syndrome coronavirus 2 (SARS CoV-2).
In some embodiments of the methods disclosed herein, the first cells may be mammalian cells. In some embodiments of the methods disclosed herein, the first cells may be human cells. In some embodiments of the methods disclosed herein, the first cells may be human embryonic kidney cells. In some embodiments of the methods disclosed herein, the human embryonic kidney cells may be 293 cells.
In some embodiments of the methods disclosed herein, the second cell may be a mammalian cell. In some embodiments of the methods disclosed herein, the second cell may be a human cell. In some embodiments of the methods disclosed herein, the second cell may be a human embryonic kidney cell. In some embodiments, the human embryonic kidney cell is a 293 cell. In certain embodiments, the second cell (also referred to herein as a target cell) is engineered to stably express ACE2 and/or TMPRSS2. In certain embodiments, the second cell is the 5A10 cell line, In certain embodiments, the second cell is the 5G7 cell line.
In some embodiments of the methods disclosed herein, the sample may be serum or plasma.
In some embodiments of the methods disclosed herein, the viral expression vector may be a human immunodeficiency virus (HIV) expression vector.
In some embodiments of the methods disclosed herein, the level of neutralizing antibody response of the sample is correlated to the subject's level of post-infection protective immunity.
In some embodiments of the methods disclosed herein, the level of neutralizing antibody response in the sample is correlated to a titer of total anti-coronavirus antibody as measured in the subject.
Compositions
Also disclosed are compositions for the detection of a compound such as a neutralizing antibody that can modulate SARS infectivity. In certain embodiments, the composition may comprise cells that have been engineered to be either producer cells or target cells as disclosed herein.
For example, the composition may comprise a cell (or cells) genetically modified to be a target cell that expresses an angiotensin-converting enzyme 2 receptor (ACE-2). In some embodiments, the target cell may be genetically modified to also express human airway transmembrane trypsin-like serine protease (TMPRSS2). In some embodiments, and as discussed herein, the ACE2 and/or TMPRSS2 may be introduced into the target cell in a manner that allows for transient expression. Or, the ACE2 and/or TMPRSS2 may be introduced into the target cell in a manner that allows for stable expression. In certain embodiments, the target cell is from the 5A10 cell line, In certain embodiments, the target cell is from the 5G7 cell line.
Additionally and/or alternatively, the composition may comprise a producer cell (or cells). In certain embodiments, the producer cell may comprise: i) a nucleic acid encoding a SARS CoV-2 spike protein or a portion thereof, and ii) a genomic viral expression vector comprising sequences from a second virus that is not a coronavirus and that comprises an indicator nucleic acid that produces a detectable signal. For example, in certain embodiments, the producer cell may be a genetically modified cell made by the steps of: transfecting into the cell: i) a nucleic acid encoding a SARS CoV-2 spike protein or a portion thereof, and ii) a genomic viral expression vector comprising sequences from a second virus that is not a coronavirus and that comprises an indicator nucleic acid that produces a detectable signal. In some embodiments, the coronavirus spike protein is a coronavirus S protein. In some embodiments, the viral expression vector comprising sequences from a second virus that is not a coronavirus and that comprises an indicator nucleic acid that produces a detectable signal is introduced into the cell in a manner that allows for transient expression. In some embodiments, the viral expression vector comprising sequences from a second virus that is not a coronavirus and that comprises an indicator nucleic acid that produces a detectable signal is introduced into the cell in a manner that allows for stable expression.
In certain embodiments, the second virus is HIV or a genetically modified HIV. Thus, in certain embodiments, disclosed is a composition comprising an HIV nucleic acid modified to remove genomic sequences that encode the HIV envelope protein and genetically modified to encode and express SARS CoV-2 spike protein or a portion thereof and/or cells transfected with an HIV nucleic acid modified encode and express SARS CoV-2 spike protein or a portion thereof.
A variety of constructs may be used as an indicator nucleic acid. In certain embodiments, the indicator nucleic acid comprises an indicator gene. For example, the indicator gene may be a luciferase gene. Or, other indicator genes may be used.
In certain embodiments, the S protein is from a subject infected with a coronavirus. In certain embodiments, the subject is infected with severe acute respiratory syndrome coronavirus 2 (SARS CoV-2). The SARS CoV-2 S protein is known to mutate, leading to variants. SARS-CoV-2 variants are defied by specific point mutations along the length of the spike gene causing amino acid deletions or substitutions. These changers may confer enhanced spike protein binding efficiency to the ACE 2 receptor, allowing the virus to infect cells more readily. Additionally, these changes may affect the capability of the virus to escape from antibodies that are raised after natural infection, after vaccine, or from antibody therapies. In some embodiments, the S protein may be from a subject infected with a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant. In some embodiments the variant may be the B.1.1.7 (UK) variant, B.1.351 (South Africa) variant, the B.1.1.28.1 (Brazil) variant, or the B.1.427/B/1/429 (California) variant.
A variety of cells may be used to generate the target cell or the producer cell. In certain embodiments, the producer cells and/or the target cells are mammalian cells. Also, in some embodiments, the producer cells and/or the target cells are human cells. In some cases the producer cells and/or the target cells are human embryonic kidney (HEK) cells, as for example 293 HEK cells.
Kits
Also disclosed are kits for performing any of the steps of the disclosed methods and/or using any of the disclosed compositions and a computer-program product tangibly embodied in a non-transitory machine-readable storage medium (e.g., software) for performing any of the steps of the disclosed methods, and/or using any of the disclosed compositions, or running any of the parts of the disclosed systems.
Thus, the kit may comprise cells that have been engineered to be either producer cells or target cells as disclosed herein. In certain embodiments, the producer cell may comprise: i) a nucleic acid encoding a SARS CoV-2 spike protein or a portion thereof, and ii) a genomic viral expression vector comprising sequences from a second virus that is not a coronavirus and that comprises an indicator nucleic acid that produces a detectable signal. In certain embodiments, the producer cell may be made by the steps of: transfecting into a producer cell: i) a nucleic acid encoding a SARS CoV-2 spike protein or a portion thereof, and ii) a genomic viral expression vector comprising sequences from a second virus that is not a coronavirus and that comprises an indicator nucleic acid that produces a detectable signal.
Additionally and/or alternatively, the kit may comprise a target cell (or cells) that has been engineered to express an angiotensin-converting enzyme 2 receptor (ACE-2) and/or TMPRSS2. In some embodiments, and as discussed herein, the ACE2 and/or TMPRSS2 may be introduced into the target cell in a manner that allows for transient expression. Or, the ACE2 and/or TMPRSS2 may be introduced into the target cell in a manner that allows for stable expression. In an embodiment, the cells may comprise one of the 5A10 and/or 5G7 cell lines disclosed herein.
The kit may further comprise a reagent and/or equipment for contacting the target cell with a viral particle comprising at least a portion of the SARS CoV-2 spike protein in the absence and the presence of a compound to be tested as a potential modulator of SARS CoV-2 infectivity. In one embodiment, the compound may be a subject or a patient sample believed to contain neutralizing antibodies. Additionally, the kit may comprise a reagent and/or equipment and/or software for measuring levels of infection of the target cell in order to determine infectivity of the viral particle in the presence or absence of the compound. In one embodiment, infected cells may comprise a detectable marker. For example, in one embodiment, the target cells may comprise luciferase. Or, the viral particle may comprise the detectable marker (e.g., luciferase). Thus, in certain embodiments, the kit may comprise a reagent and/or equipment and/or software for measuring signal from the detectable marker (e.g., luciferase) due to infection of the target cells.
As disclosed herein, in certain embodiments, the viral particle is an HIV pseudovirion. For example, in one embodiment, the method may utilize the PhenoSense SARS CoV-2 neutralizing antibody (nAb) Assay format as disclosed herein.
The kit may further comprise reagents and/or equipment for incubating the producer cell under conditions such that pseudovirions that comprise the SARS CoV-2 spike protein or a portion thereof are generated. Also, the kit may comprise reagents and/or equipment for contacting the pseudovirions made by the producer cell with a target cell under conditions such that the pseudovirions infect the target cell and in the presence or absence of a compound to be tested as a potential modulator of SARS CoV-2 infectivity. In one embodiment, the compound may be a subject or patient sample believed to contain neutralizing antibodies.
Additionally, the kit may comprise a reagent and/or equipment and/or software for measuring levels of infection of the target cell in order to determine infectivity of the viral particle in the presence or absence of the compound. In one embodiment, infected cells may comprise a detectable marker. For example, in one embodiment, the target cells may comprise luciferase. Or, the viral particle may comprise luciferase. Thus, in certain embodiments, the kit may comprise a reagent and/or equipment and/or software for measuring signal from luciferase due to infection of the target cells.
Also disclosed is a computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to use the kit and/or perform a step or steps of the kit of any of the disclosed embodiments. In one embodiment, the kit comprises a computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to determine changes in SARS CoV-2 infectivity caused by a compound of interest.
Systems
Also disclosed are systems for performing any of the steps of the disclosed methods and/or using any of the disclosed compositions and computer-program product tangibly embodied in a non-transitory machine-readable storage medium (i.e., software) for performing any of the steps of the disclosed methods, and/or using any of the disclosed compositions, or running any of the parts of the disclosed systems.
Thus, the system may comprise cells that have been engineered to be either producer cells or target cells as disclosed herein. In certain embodiments, the producer cell may comprise: i) a nucleic acid encoding a SARS CoV-2 spike protein or a portion thereof, and ii) a genomic viral expression vector comprising sequences from a second virus that is not a coronavirus and that comprises an indicator nucleic acid that produces a detectable signal. In certain embodiments, the producer cell may be made by the steps of: transfecting into a producer cell: i) a nucleic acid encoding a SARS CoV-2 spike protein or a portion thereof, and ii) a genomic viral expression vector comprising sequences from a second virus that is not a coronavirus and that comprises an indicator nucleic acid that produces a detectable signal.
The target cell (or cells) may be engineered to express an angiotensin-converting enzyme 2 receptor (ACE-2) and/or TMPRSS2. In some embodiments, and as discussed herein, the ACE2 and/or TMPRSS2 may be introduced into the target cell in a manner that allows for transient expression. Or, the ACE2 and/or TMPRSS2 may be introduced into the target cell in a manner that allows for stable expression. In certain embodiments, the target cell is from the 5A10 cell line, In certain embodiments, the target cell is from the 5G7 cell line.
The system may further comprise a station and/or equipment for contacting the target cell with a viral particle comprising at least a portion of the SARS CoV-2 spike protein in the absence and the presence of a compound to be tested as a potential modulator of SARS CoV-2 infectivity. In one embodiment, the compound may be a subject or a patient sample believed to contain neutralizing antibodies. Additionally, the system may comprise a station and/or equipment for measuring levels of infection of the target cell in order to determine infectivity of the viral particle in the presence or absence of the compound. In one embodiment, infected cells may comprise a detectable marker. For example, in one embodiment, the target cells may comprise luciferase. Or, the viral particle may comprise the detectable marker (e.g., luciferase). Thus, in certain embodiments, the system may comprise a station for measuring signal from the detectable marker (e.g. luciferase) due to infection of the target cells.
As disclosed herein, in certain embodiments, the viral particle is an HIV pseudovirion. For example, in one embodiment, the method may utilize the PhenoSense SARS CoV-2 neutralizing antibody (nAb) Assay format as disclosed herein.
The system may further comprise a station and/or equipment for incubating the producer cell under conditions such that pseudovirions that comprise the SARS CoV-2 spike protein or a portion thereof are generated. Also, the system may comprise a station and/or equipment for contacting the pseudovirions made by the producer cell with a target cell under conditions such that the pseudovirions infect the target cell and in the presence or absence of a compound to be tested as a potential modulator of SARS CoV-2 infectivity. In one embodiment, the compound may be a subject or patient sample believed to contain neutralizing antibodies.
Additionally, the system may comprise a station and/or equipment for measuring levels of infection of the target cell in order to determine infectivity of the viral particle in the presence or absence of the compound. In one embodiment, infected cells may comprise a detectable marker. For example, in one embodiment, the target cells may comprise luciferase. Or, the viral particle may comprise luciferase. Thus, in certain embodiments, the system may comprise a station for measuring signal from luciferase due to infection of the target cells.
In some embodiments, the system or any of the stations of the system further comprises a computer and/or a data processor. In certain embodiments, the system may comprise one or more computers, and/or a computer product tangibly embodied in a non-transitory computer readable storage medium containing instructions which, when executed on the one or more data processors, cause the one or more data processors to perform actions for performing the methods or implementing the systems of any of embodiments disclosed herein. One or more embodiments described herein can be implemented using programmatic modules, engines, or components. A programmatic module, engine, or component can include a program, a sub-routine, a portion of a program, or a software component or a hardware component capable of performing one or more stated tasks or functions. As used herein, a module or component can exist on a hardware component independently of other modules or components. Alternatively, a module or component can be a shared element or process of other modules, programs or machines. For example, as disclosed below, the system may comprise a computer and/or computer-program product tangibly embodied in a non-transitory machine-readable storage medium for relating changes in infectivity of the viral particles or pseudovirions to an autoimmune response. Thus, in certain embodiments, the system may comprise components to quantify the measurement. Also, the system may comprise components to perform statistical analysis of the data.
Also disclosed is a computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to run the systems and/or perform a step or steps of the methods of any of the disclosed embodiments. In one embodiment, the system comprises a computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to determine changes in SARS CoV-2 infectivity caused by a compound of interest.
The SARS CoV-2 nAb Assay has been developed by leveraging the proprietary PhenoSense Assay platform that was developed to evaluate antiretroviral drug susceptibility (Petropoulos et al., Antimicrob. Agents Chemother. 44:920-28, 2000) and later adapted to evaluate entry inhibitors, nAb activity (Richman et al., PNAS 100:4144-49, 2003) and co-receptor tropism (Whitcomb et al., Antimicrob. Agents Chemother. 51:566-75, 2007). The production of luciferase is dependent on virus entry and the completion of a single round of virus replication. Agents that inhibit pseudovirus entry or replication reduce luciferase activity in a dose-dependent manner, providing a quantitative measure of drug and antibody susceptibility. Over time, the PhenoSense assay platform has been successfully adapted to evaluate vaccines and entry inhibitors that target a variety of enveloped viruses, including Orthomyxovirus (Influenza A & B), Paramyxovirus (RSV A & B), Filovirus (Ebola), and most recently Coronavirus (SARS CoV-2, SARS CoV).
The measurement of nAb activity using the PhenoSense SARS CoV-2 nAb Assay is performed by generating HIV-1 pseudovirions that express the SARS CoV-2 spike protein (See, e.g.,
% Inhibition=100%−(((RLU(Vector+Sample+Diluent)−RLU(Background))/(RLU(Vector+Diluent)−RLU(Background)))×100%) Equation 1:
The results of the PhenoSense SARS CoV-2 nAb can be reported as an ID50 titer (1/Dilution) or qualitatively (positive, negative) based on a pre-defined dilution cutoff (e.g >50% inhibition at 1:40 dilution). Examples of Anti-SARS CoV-2 nAb titration profiles can be found in
To ensure that the measured nAb activity is SARS CoV-2 nAb specific, each test specimen is also assessed using a non-specific pseudovirus (specificity control) that expresses a non-reactive envelope protein of one or more unrelated viruses (e.g. avian influenza virus).
Critical Reagents
A critical reagent is defined as an assay component essential to obtaining the anti-SARS CoV-2 nAb titer. Critical reagents for the anti-SARS CoV-2 nAb assay include the following:
Assay Accuracy and Inclusivity was determined by assessing the nAb titers exhibited in serology confirmed SARS CoV-2 Positive/Reactive and Negative/Non-Reactive sera, or samples collected prior to 2019.
Serology Positive specimens are defined as IgG or total Ig positive as determined using antibody binding assays. Serology Negative samples are defined as IgG or Total Ig negative as determined using antibody binding assays. Historical Negative samples are defined as samples with collection dates that pre-date the SARS CoV-2 pandemic. All serology positive SARS CoV-2 Positive/Reactive sera were confirmed SARS CoV-2 PCR Positive and all SARS CoV-2 Negative/Non-Reactive sera were confirmed SARS CoV-2 PCR Negative. Examples of correlations between the results of the antibody binding assays and nAb titers generated using the assay disclosed herein are in
Assay Accuracy and Inclusivity test results are recorded in
Based on the combined results of qualitative serology and PCR assays, the sensitivity of the PhenoSense anti-SARS CoV-2 Assay is 100% (no false negatives among 49 samples), and the specificity is 98% (one false positive among 50 samples). The single false positive result occurred at the 1:40 minimum required dilution.
Based on the combined results of qualitative serology assays and historic negative samples, the specificity of the PhenoSense anti-SARS CoV-2 nAb assay is 98.8% (one false positive among 82 samples).
The acceptance criteria for quantitative determinations of nAb titer were not pre-defined. The qualitative assessment of SARS CoV-2 nAb positive and negative samples satisfied the pre-defined acceptance criteria:
Intra-Assay Precision was determined by assessing the variation in ID50 observed across six replicates of four serum samples consisting of a nAb High-Titer Positive, Intermediate-Titer Positive, Low-Titer Positive and Negative sample.
Intra-Assay Precision was evaluated within each of four separate assay runs.
Intra-Assay Precision test results are recorded in
The assessment for each arm of Intra-Assay Precision satisfied the pre-defined acceptance criteria:
Inter-Assay Precision was determined by assessing the variation in ID50 mean observed across six replicates of four serum samples consisting of a High-Titer Positive, Intermediate-Titer Positive, Low-Titer Positive and Negative sample (Precision and Reproducibility Panel).
Inter-Assay Precision was evaluated across all four independent runs performed by two operators using two independent lots of critical reagents (pseudovirus, cells, assay controls).
Inter-Assay Precision test results are recorded in
The assessment across all arms of Inter-Assay Precision satisfied the pre-defined acceptance criteria:
The contribution of manual versus automated serum dilution and transient versus stable ACE2 expression to Inter-Assay Precision were interrogated using appropriate statistical methods.
Manual vs. Automated Serum Dilution
The inter-assay precision of assays conducted using manual and automated serum dilutions were also compared to assess the equivalence of the two dilution methods. Comparative results of the manual and automated dilution processes are recorded in
Transient vs. Stable ACE2 Expression
The inter-assay precision of assays conducted using HEK293 cells that transiently or stably express ACE2 was compared to assess the equivalence of the two target cell types. Comparative results of the ACE2 transient and stable cell types are recorded in
Inter-Assay Run Comparison
Across all possible paired comparisons, differences in mean ID50 for the HTS, ITS and LTS samples did not exceed 3-fold, satisfying the pre-defined acceptance criteria
An ANOVA analysis comparing Runs A, B, C, D indicated differences in Runs A, B, C, D. Based on a Tukey Honest Significant Differences test, the difference was entirely driven by the results of Run A compared to Runs B, C, D. Additional studies are planned to investigate the potential sources of the observed variation. Currently, the preferred assay modality corresponds to Run C (automated sample dilution, transient ACE2 expression), which does not differ significantly modalities of Run B (manual sample dilution, stable ACE2 expression) and Run D (automated sample dilution, stable ACE2 expression).
Assay Linearity was determined by creating and testing five three-fold dilutions (approximately one half log 10) of a High-Titer Positive serum in negative serum. Each dilution was tested independently in the assay. A serum with an ID50 titer of 22,224 was used to generate six contrived samples with nominal (expected) ID50 titers of 7408, 2469, 823, 274, 123, 91. The observed titers of each of the contrived samples was compared to the nominal titers based on the undiluted sample ID50 (
Assay linearity satisfied the pre-defined acceptance criteria: The variation in true (observed) titer (ID50) extrapolated from each neighboring start dilution of the linearity sample must exhibit ≤3-fold variation (observed titer÷predicted titer ≤3-fold).
Specificity was evaluated by assessing assay reactivity (true positives), assay cross-reactivity (false positives) and assay interference (false negatives). Specificity testing was performed using a High-Titer Positive, Intermediate-Titer Positive, Low-Titer Positive and Negative sample (Assay Specificity Panel).
Assay reactivity was evaluated by depleting the immunoglobulin of two low-titer anti-SARS CoV-2 nAb samples (LTS-1, LTS-2) and one negative titer sample (NTS) and assessing the impact on anti-SARS CoV-2 nAb activity. Antibody depletion was performed using protein G sepharose affinity chromatography.
The depletion on anti-SARS CoV-2 nAb activity is recorded in
The results of assay reactivity testing satisfied the pre-defined assay acceptance criteria:
Assay cross reactivity/selectivity was evaluated based on two approaches:
The summary results of non-specific nAb activity in pre-pandemic HN sera are recorded in
nAb activity observed in the presence of high concentrations (200 μg/mL) of antiviral antibodies was compared to nAb activity in the presence of antiviral antibody vehicle alone (
The results of cross reactivity testing satisfied the pre-defined assay acceptance criteria.
Interference by endogenous and exogenous agents was evaluated.
The results of sample matrix interference testing are recorded in
Interference related to specimen type/collection is recorded in
The results of interference testing satisfied the pre-defined assay acceptance criteria:
The ability of the assay to reliably assess anti-SARS CoV-2 nAb activity at the 1:40 assay MRD was interrogated using two anti-SARS CoV-2 Low Titer Positive Sera that were diluted 1:2 and 1:3 and tested in the assay in triplicate in two separate assay runs. Thus, Anti-SARS CoV-2 nAb Positive Serum diluted in Negative Serum to achieve nominal titers of 1:60 and 1:40.
The results of Sensitivity testing are recorded in
The results of sensitivity testing satisfied the pre-defined assay acceptance criteria:
Sample and reagent stability was demonstrated by evaluating the consistency of anti-SARS CoV-2 nAb activity over time and under different storage conditions. Stability studies through 14 days were performed in the initial validation studies.
Two replicates of a low titer sample (LTS) and an intermediate titer sample (ITS) were tested following various handling and storage conditions. Two lots of critical reagents (CR) were used for replicate testing. Stability testing included a freshly thawed tube of sample for comparison (Time 0). Sample stability conditions were selected to reflect possible conditions associated with sample collection, shipment and storage prior to performing the assay.
The results of sample stability testing are recorded in
Critical Reagent Stability
The in-process stability of critical reagents was assessed by simulating routine sample testing conditions. Critical reagents consist of the SARS CoV-2 Pseudovirus, Specificity Control Pseudovirus and HEK293-ACE2 Target Cells. The performance of two lots of critical reagents was evaluated by comparing test results generated immediately after recovery from storage (Hour 0) versus six hours (Hour 6) at ambient temperature. In-process assessment of critical reagents was performed using low titer serum (LTS) and intermediate titer serum (ITS) specimens.
The results of Critical Reagent Stability testing are summarized in
The results of sample and critical reagent stability and handling testing satisfied the pre-defined assay acceptance criteria:
As described in Example 1 and in
Alternative to transiently expressing target cells, the target cells may be a cell line engineered to stably express ACE2 or both ACE2 and TMPRSS2. The 5A10 cell line that stably expresses ACE2 was produced by transfecting HEK293VL cells with 5 μg/mL ACE2 DNA. Hygromycin antibiotic selection was used (at 150 μg/mL) to select for ACE2 expressing cells. The stable ACE2 expressing cell pool was then screened for SARS CoV-2 pseudovirus infectivity and neutralizing antibody efficiency against SARS CoV-2 pseudovirus. A monoclonal cell line was generated from the stable cell pool using limiting dilution and was maintained under 150 μg/mL hygromycin selection. The stable monoclonal ACE2 expressing culture was then screened for SARS CoV-2 pseudovirus infectivity and neutralizing antibody efficiency against SARS CoV-2 pseudovirus (See
The 5G7 cell line that stably expresses both ACE2 and TMPRSS2 was produced by transfecting the 5A10 cell line (ACE2/HEK293VL) with 1 μg/mL TMPRSS2 DNA. Blasticidin antibiotic selection (at 10 μg/mL) was used to select for TMPRSS2 expressing cells, and the cells were maintained under hygromycin selection (at 150 μg/mL) to maintain ACE2 expression. The stable ACE2/TMPRSS2 expressing cell pool was then screened for SARS CoV-2 pseudovirus infectivity and neutralizing antibody efficiency against SARS CoV-2 pseudovirus. A monoclonal cell line was generated from the stable ACE2/TMPRSS2 expressing cell pool using limiting dilution and was maintained under the 10 μg/mL blasticidin and 150 μg/mL hygromycin antibiotic selection. The stable monoclonal ACE2/TMPRSS2 expressing culture was then screened for SARS CoV-2 pseudovirus infectivity and neutralizing antibody efficiency against SARS CoV-2 pseudovirus (See
The results with these three target cell types for the assays of pseudovirus infectivity and neutralizing antibody efficiency against SARS CoV-2 pseudovirus are shown in
The efficacy of neutralizing antibodies raised against the original SARS-CoV-2 strains (Wuhan) were tested against the latest SARS-CoV-2 variants circulating in the population. The variants studied included B.1.1.7 (UK) variant, B.1.351 (South Africa) variant, B.1.1.28.1 (Brazil) variant, and B.1.427/B.1.429 (California) variant, along with many of the single mutations that are included in each variant, shown in
As is shown in
Pseudoviruses were also tested against a panel of 12 sera pools known to neutralize SARS-CoV-2 (Wuhan or D614G) pseudoviruses. Sera pools were generated by mixing 8-16 samples of similar titer.
No single mutation was fully responsible for the nAB escape capability of B.1.1.7 or B.1.351. In this assay, the N501Y variant had little to no enhanced nAb escape capability. The mutations that contributed to B.1.1.7 nAb escape capability in the assay were T716I and/or D1118H. The mutations that contributed to B.1.351 nAb escape capability were DEL242-244, R426I, and E484K. However, each of these nAb escape contributing mutations showed lower infectivity compared to other pseudoviruses.
Listed hereafter are non-limiting examples of certain embodiments of the technology.
A1. A method for detecting whether a subject exposed to a coronavirus has developed a neutralizing antibody response, comprising:
This application claims priority to U.S. Provisional Application No. 63/044,070, filed Jun. 25, 2020, U.S. Provisional Application No. 63/126,164, filed Dec. 16, 2020 and U.S. Provisional Application No. 63/143,592, filed Jan. 29, 2021. The disclosures of U.S. Provisional Application Nos. 63/044,070, 63/126,164 and 63/143,592 are incorporated by reference herein in their entireties.
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| Number | Date | Country | |
|---|---|---|---|
| 20220065869 A1 | Mar 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| 63143592 | Jan 2021 | US | |
| 63126164 | Dec 2020 | US | |
| 63044070 | Jun 2020 | US |
