Systems, methods and kits for detection of binding substances - Patents.com

Abstract

Described herein are cell-free in vitro systems, methods, and kits for detection of binding substances in a fluid sample that disrupt host-microbe interactions, fusion, and / or penetration. The systems, methods, and kits described herein include host nanoparticles expressing a surface receptor recognized by an exogenous ligand, and exogenous nanoparticles expressing the exogenous ligand, and pre-incubation of either the host nanoparticles or the exogenous nanoparticles with the fluid sample reduces binding / interaction between the host nanoparticles and the exogenous nanoparticles if the binding substance is present in the fluid sample.

Description

[Technical Field]

[0001] This specification relates to cell-free and pathogen-free systems, methods, and kits for the detection of binding agents. This specification further relates to the use of host and foreign nanoparticles that express surface receptors and ligands that mimic host-pathogen interactions to detect binding agents present in biological samples. [Background technology]

[0002] The detection of neutralizing substances (e.g., neutralizing antibodies) is a critical component of infectious disease research and development applications for human and veterinary medicine, particularly in vaccine and therapeutic drug development, epidemiological surveillance, herd immunity surveillance, contact tracing (duration of immunity), and monitoring commercial vaccines against emerging variants. Particularly in the context of rapidly evolving infectious diseases such as the SARS-CoV-2 pandemic and the emergence of new variants, methods for detecting neutralizing substances in human samples are critical for controlling the spread of disease. Existing methods and kits for detecting neutralizing substances, particularly for detecting pathogen-neutralizing or blocking molecules, do not closely mimic the physiological conditions of host-pathogen interactions in vitro. Furthermore, these methods are often time-consuming and rely on the production of recombinant proteins and monoclonal and / or polyclonal antibodies, which can reduce the sensitivity or specificity of the assay. Therefore, improved methods for detecting neutralizing substances that closely recapitulate host-pathogen interactions while being rapid, fully customizable, and safe for use outside of biosafety level 2 environments, would be highly desirable. Summary of the Invention

[0003] In a first aspect, an in vitro system for detecting binding substances (e.g., neutralizing or blocking substances that disrupt host-microbe interactions and / or fusion / penetration) in a fluid sample (e.g., a biological fluid sample) is described herein. The system generally comprises host nanoparticles comprising fragments of host cells that express or are designed to express a surface receptor recognized by the exogenous ligand, and foreign nanoparticles that express or are designed to express the exogenous ligand on their surface such that the exogenous ligand is recognizable by the surface receptor on the host nanoparticles, wherein the host nanoparticles and / or foreign nanoparticles further comprise a detectable label, and wherein the binding substance is present in the sample if pre-incubation of either the host nanoparticles or the foreign nanoparticles with the sample reduces binding / interaction between the host nanoparticles and the foreign nanoparticles compared to a corresponding fluid sample lacking the binding substance.

[0004] In a further aspect, described herein is a kit for screening a sample for the presence of a binding substance (e.g., that disrupts host-microbe interaction and / or fusion / penetration). The kit generally includes a first container containing host nanoparticles that comprise or consist of fragments of mammalian host cells expressing a surface receptor recognized by the exogenous ligand, and a second container containing exogenous nanoparticles that express the exogenous ligand on their surface such that the exogenous ligand is recognizable by the surface receptor of the host nanoparticles, wherein the host nanoparticles and / or the exogenous nanoparticles further comprise a detectable label, and wherein the binding substance is present in the sample if pre-incubation of the sample with either the host nanoparticles or the exogenous nanoparticles reduces binding / interaction between the host nanoparticles and the exogenous nanoparticles compared to a corresponding sample lacking the binding substance.

[0005] In a further aspect, described herein is an in vitro method for detecting a binding substance (e.g., a neutralizing or blocking substance that disrupts host-microbe interaction and / or fusion / penetration) in a fluid sample (e.g., a biological fluid sample). The method generally includes providing host nanoparticles and foreign nanoparticles as defined herein, pre-incubating the host nanoparticles or foreign nanoparticles with the fluid sample to allow binding / interaction of the binding substance, incubating the host nanoparticles with the foreign nanoparticles, and measuring the binding / interaction therebetween, wherein a decreased level of binding / interaction between the host nanoparticles and the foreign nanoparticles after pre-incubation with the fluid sample compared to pre-incubation with a corresponding fluid sample lacking the binding substance indicates the presence of the binding substance in the fluid sample.

[0006] This specification references a number of documents, the contents of which are incorporated herein by reference in their entireties.

[0007] General definition Headings and other identifiers, e.g., (a), (b), (i), (ii), etc., are provided merely to facilitate the reading of the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require that the steps or elements be performed in alphabetical or numerical order or in the order in which they are presented.

[0008] The use of the words "a" or "an" when used in the claims and / or this specification in conjunction with the term "comprising" may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0009] As used in this specification and claims, the words "comprising" (and any form of comprising, e.g., "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of including, e.g., "includes" and "include") or "containing" (and any form of containing, e.g., "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0010] The term "about" is used to indicate that a value includes the standard deviation of error for the device or method being used to determine the value. Generally, the term "about" is meant to indicate a possible variation of up to 10%. Thus, variations of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10% of a value are encompassed by the term "about." Unless otherwise specified, the use of the term "about" before a range applies to both ends of the range.

[0011] Other objects, advantages and features of the present specification will become more apparent from a reading of the following non-limiting description of specific embodiments thereof, given by way of example only, with reference to the accompanying drawings.

[0012] In the accompanying drawings: [Brief explanation of the drawings]

[0013] [Figure 1A]Figure 1A shows a schematic model of a binding agent detection assay according to an exemplary embodiment, comprising host nanoparticles (H) coated onto the wells of a microplate that express a surface host receptor, fluorescent foreign nanoparticles (F) that express a surface ligand specific for the host receptor, and a neutralizing agent. Figure 1A shows a neutralizing agent (e.g., an antibody) that is specific for the foreign ligand of the foreign nanoparticle, thereby preventing recognition of the corresponding surface receptor on the host nanoparticle. [Figure 1B] Figure 1B shows a schematic model of a binding agent detection assay according to an exemplary embodiment, comprising host nanoparticles (H) coated onto the wells of a microplate that express a surface host receptor, fluorescent foreign nanoparticles (F) that express a surface ligand specific for the host receptor, and a neutralizing agent. Figure 1B shows the blocking agent being specific for (i.e., blocking) the surface receptor of the host nanoparticle, thereby preventing recognition of the corresponding foreign ligand on the foreign nanoparticle. [Figure 2A] A schematic model of the vesicular morphology of host nanoparticle H with various proteins expressed on the plasma membrane and intracellularly expressed proteins is shown. [Figure 2B] Figure 1 shows a schematic diagram of the membrane morphology of host nanoparticle H with various proteins expressed on the plasma membrane. [Figure 3A] 1 shows a schematic model of the vesicular morphology of foreign nanoparticle F with various proteins expressed on the plasma membrane and intracellularly expressed proteins. [Figure 3B] FIG. 1 shows a schematic diagram of the membrane morphology of exogenous nanoparticle F with various proteins expressed on the plasma membrane. [Figure 3C] 1 shows a schematic model of the vesicular morphology of foreign nanoparticle F, which is a capsid and has various proteins expressed on the plasma membrane and intracellularly expressed proteins. [Figure 4A] Schematic models of various types of host-foreign nanoparticle interactions in the assay are shown: Figure 4A shows the binding of host nanoparticles and foreign nanoparticles in vesicular form. [Figure 4B]A schematic model of various types of host-foreign nanoparticle interactions in the assay is shown in Figure 4B. Figure 4B shows the binding of host nanoparticles and foreign nanoparticles in vesicular form, the subsequent fusion of membranes, and the import of intracellular contents and signal proteins (e.g., import of foreign intracellular signal proteins into the host). [Figure 4C] A schematic model of various types of host-foreign nanoparticle interactions in the assay is shown in Figure 4C. Figure 4C shows the binding of host nanoparticles and foreign nanoparticles in the form of vesicles, the subsequent fusion of membranes, the import of intracellular contents and proteins, and the binding of host intracellular proteins and foreign intracellular proteins to form functional signals. [Figure 4D] 4A and 4B show schematic models of various types of host-foreign nanoparticle interactions in the assay. Figure 4D shows the binding of membrane forms of host nanoparticles and foreign nanoparticles. [Figure 4E] Schematic models of various types of host-foreign nanoparticle interactions in the assay are shown: Figure 4E shows the binding of the membrane form of the foreign nanoparticle to the vesicular form of the host nanoparticle. [Figure 4F] Schematic models of various types of host-foreign nanoparticle interactions in the assay are shown: Figure 4F shows the binding of the vesicular form of the foreign nanoparticle to the membrane form of the host nanoparticle. [Figure 5A] Representative fluorescence microscopy of HEK293T cells incubated with control fluorescent viral nanoparticles lacking a ligand for binding to HEK293T cells is shown. With increasing amounts of viral nanoparticles, no binding was observed. [Figure 5B] Representative fluorescence microscopy observations of the binding of fluorescent nanoparticles to a vesicular stomatitis virus (VSV) model with increasing amounts of viral nanoparticles to HEK293T cells are shown. [Figure 5C] 5A and 5B show quantification of binding by flow cytometry with increasing amounts of viral nanoparticles. [Figure 6A]Representative fluorescence microscopy of HEK293T cells incubated with control fluorescent VSV viral nanoparticles expressing a plasmid containing a transactivatable GFP expression cassette is shown. No binding, and therefore no internalization, was observed with increasing amounts of viral nanoparticles (i.e., no fluorescence). [Figure 6B] Representative fluorescence microscopy observations of the binding and internalization of a vesicular stomatitis virus (VSV) model fluorescent nanoparticle expressing a plasmid containing a transactivatable GFP expression cassette with increasing amounts of viral nanoparticles into HEK293T cells are shown. [Figure 6C] 6A and 6B show quantification of binding by flow cytometry with increasing amounts of viral nanoparticles. [Figure 7] Representative flow cytometry analysis of HEK293T cells incubated with a mixture of fluorescent nanoparticle vesicular stomatitis virus (VSV) model and various concentrations of anti-VSV neutralizing antibody (Ab01402-2.0) is shown. [Figure 8] Representative flow cytometry analysis of HEK293T cells incubated with a mixture of VSV-G pseudotyped lentivirus encapsulating a transactivatable GFP expression cassette and various concentrations of anti-VSV neutralizing antibody (Ab01402-2.0) is shown. [Figure 9] 1 shows a representative flow cytometry analysis of binding of VSV-like viral nanoparticles to host nanoparticles expressing surface receptors specific for VSV glycoproteins. [Figure 10A] Representative flow cytometry analysis of a SARS-CoV-2 model of fluorescent viral nanoparticles and host nanoparticles expressing angiotensin-converting enzyme 2 (ACE2) receptors incubated with a mixture of various concentrations of either an irrelevant antibody (anti-VSV antibody; Ab01402-2.0) or a neutralizing anti-Spike RBD (receptor binding domain) antibody (Ab02019-12.1) is shown. [Figure 10B]Representative flow cytometry analysis of fluorescent viral nanoparticle SARS-CoV-2 models and host nanoparticles expressing angiotensin-converting enzyme 2 (ACE2) receptors incubated with a mixture of various concentrations of either FBS or pooled anti-SARS-CoV-2 antibody-positive human serum is shown. [Figure 10C] Representative flow cytometry analysis of a SARS-CoV-2 model of fluorescent viral nanoparticles and a host nanoparticle expressing the angiotensin-converting enzyme 2 (ACE2) receptor incubated with a mixture of purified neutralizing chicken IgY polyclonal antibody against the S1 region of the SARS-CoV-2 spike protein at various concentrations. [Figure 10D] Representative flow cytometry analysis of a SARS-CoV-2 model of fluorescent viral nanoparticles and a host nanoparticle expressing the angiotensin-converting enzyme 2 (ACE2) receptor incubated with a mixture of purified neutralizing chicken IgY polyclonal antibody against the S2 region of the spike protein of SARS-CoV-2 at various concentrations. [Figure 11A] Figure 1 shows the results of a fusion assay between R18-labeled SARS-CoV-2 foreign envelope nanoparticles and host ACE2 nanoparticles in suspension in either PBS or DMEM. [Figure 11B] Figure 1 shows the results of a fusion assay between R18-labeled SARS-CoV-2 foreign envelope nanoparticles and host ACE2 / TMPRSS2 nanoparticles in suspension in either PBS or DMEM. Fluorescence signals are expressed as relative fluorescence units (RFU) normalized to the control. [Figure 12] Figure 1 shows the results of a fusion assay of R18-labeled SARS-CoV-2 foreign envelope nanoparticles with host ACE2 nanoparticles or host ACE2 / TMPRSS2 nanoparticles in suspension at either 37°C or 4°C. Fluorescence signals are expressed as relative fluorescence units (RFU) normalized to the control. [Figure 13]Neutralization assay results of R18-labeled SARS-CoV-2 foreign envelope nanoparticles fusion with host ACE2 nanoparticles in suspension, either untreated or in the presence of either the MSA5 aptamer (10 μM) or anti-Spike RBD antibody (20 μg / ml). Fluorescence signals are expressed as relative fluorescence units (RFU) normalized to the control. [Figure 14] Figure 1 shows the results of a fusion assay between SARS-CoV-2 foreign envelope nanoparticles and coated host ACE2 nanoparticles, and subsequent neutralization in the presence of either anti-Spike RBD antibody (10 μg / ml) or anti-VSVg antibody (10 μg / ml). [Figure 15A] Neutralization assay results between SARS-CoV-2 foreign envelope nanoparticles and coated host ACE2 nanoparticles in the presence of either anti-Spike RBD antibody (20 μg / ml), soluble ACE2 receptor (10 μg / ml), Ouabain (120 nM), MSA5 aptamer (10 μM), soluble Spike RBD (10 μg / ml) or anti-VSVg antibody (10 μg / ml) are shown. [Figure 15B] Neutralization assay results between coated SARS-CoV-2 foreign envelope nanoparticles and host ACE2 nanoparticles in the presence of either anti-Spike RBD antibody (20 μg / ml), soluble ACE2 receptor (10 μg / ml), or soluble Spike RBD (10 μg / ml) are shown. Results are expressed as % neutralization in relative fluorescence units (RFU) normalized to the control. [Figure 16] Neutralization assay results between SARS-CoV-2 foreign envelope nanoparticles and coated host ACE2 nanoparticles in the presence of either anti-Spike RBD (10 μg / ml) or anti-VSVg antibodies (10 μg / ml) diluted in either urine (1 / 50), saliva (1 / 20), serum (1 / 20), or plasma (1 / 50) containing solutions are shown. Results are expressed as % neutralization in relative fluorescence units (RFU) normalized to the control. [Figure 17A]Neutralization assay results between SARS-CoV-2 foreign envelope nanoparticles and coated host ACE2 nanoparticles in the presence of anti-Spike RBD (10 μg / ml) or anti-VSVg antibodies (10 μg / ml) are shown, and the assay was performed at 37°C or room temperature using incubation times as previously described (1-hour preincubation of antibody and foreign nanoparticles; 3-hour incubation of host nanoparticles and foreign nanoparticle / antibody mixture). [Figure 17B] We present the results of a neutralization assay between SARS-CoV-2 foreign envelope nanoparticles and coated host ACE2 nanoparticles in the presence of anti-Spike RBD (10 μg / ml) or anti-VSVg antibodies (10 μg / ml). The assay was performed at 37°C or room temperature, and the pre-incubation period of SARS-CoV-2 nanoparticles and antibodies was reduced from 1 hour to 30 minutes. [Figure 17C] We present the results of a neutralization assay between SARS-CoV-2 foreign envelope nanoparticles and coated host ACE2 nanoparticles in the presence of anti-Spike RBD (10 μg / ml) or anti-VSVg antibodies (10 μg / ml). The assay was performed at 37°C or room temperature, and the host ACE2 nanoparticle / SARS-CoV-2 nanoparticle / antibody incubation period was reduced from 3 hours to 1 hour. [Figure 17D] Neutralization assays between SARS-CoV-2 foreign envelope nanoparticles and coated host ACE2 nanoparticles in the presence of anti-Spike RBD (10 μg / ml) or anti-VSVg antibodies (10 μg / ml) are shown. Assays were performed at 37°C or room temperature. The pre-incubation period for SARS-CoV-2 nanoparticles and antibodies was shortened from 1 hour to 30 minutes, and the host ACE2 nanoparticle / SARS-CoV-2 nanoparticle / antibody incubation period was shortened from 3 hours to 1 hour. Results are expressed as % neutralization in relative fluorescence units (RFU) normalized to the control. [Figure 18]Neutralization assay results between Bacillus anthracis foreign nanoparticles and coated host ANTXR2 nanoparticles in the presence of either anti-PA63 antibody (10 μg / ml) or anti-VSVg antibody (10 μg / ml) are shown. Results are expressed as % neutralization in relative fluorescence units (RFU) normalized to the control. [Figure 19A] 1 shows the results of a neutralization assay between foreign non-enveloped norovirus nanoparticles expressing surface VP1 protein and coated host FUT2 nanoparticles in the presence of anti-VP1 antibodies (10 μg / ml or 100 μg / ml). [Figure 19B] Figure 1 shows the results of a neutralization assay between foreign non-enveloped norovirus nanoparticles expressing the surface VP1 protein and coated host FUT2 nanoparticles in the presence of anti-VSVg antibodies (10 μg / ml). Results are expressed as % neutralization in relative fluorescence units (RFU) normalized to the control. [Figure 20] Figure 1 shows the results of a neutralization assay between SARS-CoV-2 foreign envelope nanoparticles and coated host ACE2 nanoparticles produced from HeLa cells in the presence of either anti-Spike RBD antibody (10 μg / ml or 100 μg / ml). Results are expressed as % neutralization in relative fluorescence units (RFU) normalized to the control. [Figure 21] Representative transmission electron microscopy images of Spike-SARS-CoV-2 foreign envelope nanoparticles (Figure 21A), ACE2 host envelope nanoparticles (Figure 21B), Norovirus VP1 foreign non-enveloped nanoparticles (Figure 21C), and linear spike-SARS-CoV-2 foreign envelope nanoparticles (Figure 21D) are shown. DETAILED DESCRIPTION OF THE INVENTION

[0014] Described herein are cell-free and / or pathogen-free in vitro systems, methods, and kits for detecting binding agents in biological samples. In some embodiments, the invention stems from the production of host and foreign nanoparticles that mimic host-pathogen interactions in vitro and can be used to detect neutralizing agents that can block host-foreign nanoparticle interactions.

[0015] As used herein, the term "host nanoparticle" refers to a nanoparticle produced or derived from a producer cell or host cell that naturally expresses or overexpresses, or is engineered to express or overexpress, one or more surface receptors or proteins that recognize an exogenous ligand. Host nanoparticles are preferably engineered to be replication-deficient by lacking the machinery necessary for replication (e.g., a nucleus). For example, host nanoparticles can be derived from or produced from eukaryotic or mammalian cells (e.g., cell lines or ex vivo cells). Host nanoparticles can include a plasma membrane, can be vesicles with an "intracellular" portion (Figure 2A), or can have a linear form (Figure 2B). In some embodiments, host nanoparticles consist of or comprise proteins derived from producer cells. Host nanoparticles can include other surface proteins (e.g., located on the plasma membrane) or intracellular proteins, such as ion channels, structural proteins / fatty acids / carbohydrates, or detectable molecules (e.g., fluorescent or luminescent proteins), or proteins necessary to generate functional detectable molecules. In some embodiments, the expressed surface or intracellular proteins include all physiological post-translational modifications (e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, or lipidation). In the SARS-CoV-2 model, for example, the surface receptors / proteins on the host nanoparticles can be angiotensin-converting enzyme 2 (ACE2) receptor and / or transmembrane serine protease 2 (TMPRSS2). The host nanoparticles can be any size ranging from 1 nm to 500 nm.

[0016] In some embodiments, the host-producer cells are eukaryotic (e.g., yeast or mammalian) cells. In some embodiments, the host-producer cells may be engineered to express or overexpress an exogenous ligand. In some embodiments, the host-producer cells may be engineered to express or overexpress a detectable label. In some embodiments, the host-producer cells may be engineered to express or overexpress an exogenous viral protein (e.g., from an enveloped or non-enveloped virus), an exogenous bacterial protein, or an exogenous fungal protein that induces the formation of extracellular vesicles or particles (e.g., biological nanoparticles or virus-like particles) within the host-producer cells.

[0017] In some embodiments, the host nanoparticle is a vesicular nanoparticle that contains an exogenous viral structural protein (e.g., a late assembly (L) domain protein or polyprotein, e.g., HIV Gag) that induces host cell budding. In some embodiments, the exogenous viral structural protein can be fused to a detectable label.

[0018] As used herein, the term "foreign nanoparticle" generally refers to a nanoparticle designed to mimic a microorganism or pathogen that interacts with, fuses with, penetrates, or infects a host cell. In some embodiments, the foreign nanoparticle naturally expresses or overexpresses, or is designed to express or overexpress, a foreign ligand specific for a receptor present on the surface of the host nanoparticle. Preferably, the foreign nanoparticle is designed to be replication-deficient by lacking the machinery necessary for replication. For example, the foreign nanoparticle can be derived from or produced by a microorganism, such as, but not limited to, any pathogen, including bacteria, fungi, viruses, or parasites. In some embodiments, the foreign nanoparticle can be produced from or derived from a eukaryotic (e.g., yeast or mammalian) producer cell. In some embodiments, the foreign nanoparticle can be formed by self-assembly of viral structural proteins (e.g., capsid proteins).

[0019] In some embodiments, the microbial or eukaryotic producer cells in which the exogenous nanoparticles are produced or derived can be engineered to express or overexpress a surface receptor; can be engineered to express or overexpress a detectable label; can be engineered to express or overexpress an exogenous viral protein (e.g., from an enveloped or non-enveloped virus), an exogenous bacterial protein, or an exogenous fungal protein that induces the formation of extracellular vesicles or particles (e.g., biological nanoparticles or virus-like particles) within the host producer cell; can be nanoparticles that fuse with and / or penetrate host nanoparticles upon contact; or any combination thereof.

[0020] In some embodiments, the foreign nanoparticle can be a vesicular foreign nanoparticle that includes an exogenous viral structural protein (e.g., a late assembly (L) domain protein or polyprotein, e.g., HIV Gag) that induces host cell budding.

[0021] In some embodiments, the foreign nanoparticles are designed to express or overexpress any surface antigen, autoantigen, or allergen for recognition by the corresponding receptor on the host nanoparticle. The foreign nanoparticles may contain a membrane (e.g., plasma membrane), may be vesicles with an "intracellular" portion (Figure 3A) (e.g., capsid form; Figure 3C), or may be linear (Figure 3B). The foreign nanoparticles may contain other surface proteins (e.g., located on the plasma membrane) or intracellular proteins, such as ion channels, structural proteins / fatty acids / carbohydrates, or detectable molecules (e.g., fluorescent or luminescent proteins), or proteins necessary to generate functional detectable molecules. In some aspects, the expressed surface or intracellular proteins include all physiological post-translational modifications (e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, or lipidation). In a SARS-CoV-2 model, for example, the foreign ligand may be the full-length Spike protein, S1, S2, or the receptor-binding domain (RBD) of the Spike protein. The exogenous nanoparticles can be any size ranging from 1 nm to 500 nm.

[0022] As used herein, the term "detectable molecule" or "detectable label" refers to a molecule that is easily detectable or can be converted or modified to be detectable. For example, a detectable molecule can be any fluorescent (e.g., GFP) or luminescent (e.g., chemiluminescent) molecule (i.e., directly detectable). In some aspects, a detectable molecule can be a precursor molecule that is first converted or modified by any modification (e.g., chemical) to render it functionally detectable. For example, a detectable molecule can be a gene encoding a fluorescent molecule that is translated into a functional fluorescent protein (e.g., GFP, luciferase, secreted alkaline phosphatase [SEAP]). In some aspects, conversion or modification of the detectable molecule by a protein or mechanism present on or expressed within the host nanoparticle (e.g., either on the surface or intracellularly) occurs upon binding of the foreign nanoparticle to the host nanoparticle and / or subsequent internalization of the foreign nanoparticle. In some embodiments, the detectable label is detectable upon contact with a substrate, protein, or enzyme (i.e., indirectly detectable).

[0023] As used herein, the term "binding agent" refers to a molecule that can prevent the recognition of a foreign ligand of a foreign nanoparticle by its corresponding surface receptor on a host nanoparticle (FIG. 1A) (e.g., a neutralizing agent). In some aspects, the binding agent is a blocking agent. In some aspects, the binding agent is specific for a surface receptor on a host nanoparticle or for a foreign ligand of a foreign nanoparticle (FIG. 1B) (e.g., a blocking agent). In some aspects, the binding agent is an antibody or any antigen-binding fragment thereof. In some aspects, the antibody is any monoclonal, polyclonal, chimeric, natural, non-natural, recombinant, isotype, or species of antibody. In some aspects, the neutralizing antibody is a natural or synthetic peptide, protein, nucleic acid (e.g., an aptamer or ribozyme), or chemical molecule (e.g., a small molecule). In some embodiments, the binding agent can inhibit fusion of the host nanoparticle membrane with the foreign nanoparticle membrane. In some aspects, the binding agent can inhibit internalization or penetration of the foreign nanoparticle into the host nanoparticle.

[0024] As used herein, the term "sample" refers to any sample, particularly a fluid sample, suspected of containing a binding substance. The sample may be any laboratory sample or biological sample. Samples may include, but are not limited to, cell extracts, extracellular fluids, body fluids collected from the body of a subject (e.g., animal or human), culture medium, blood, bone marrow, plasma, serum, tears, feces, saliva, nasal secretions, bronchoalveolar lavage fluid (BALF), spinal fluid, biopsy, or any organ. In some embodiments, the sample may be any solution containing one or more candidate binding substances to be screened by the methods, systems, or kits defined herein.

[0025] In some aspects, the in vitro systems described herein for detecting binding substances (e.g., neutralizing or blocking substances that disrupt host-microbe interactions) in a fluid sample (e.g., a biological fluid sample) generally include host nanoparticles comprising plasma membrane fragments of host cells that express or are designed to express a surface receptor recognized by the exogenous ligand, and exogenous nanoparticles that express or are designed to express the exogenous ligand on their surface such that the exogenous ligand is recognizable by the surface receptor of the host nanoparticles, wherein the host nanoparticles and / or the exogenous nanoparticles further comprise a detectable label, and wherein the binding substance is present in the sample if pre-incubation of either the host nanoparticles or the exogenous nanoparticles with the sample reduces binding / interaction between the host nanoparticles and the exogenous nanoparticles compared to a corresponding fluid sample lacking the binding substance. Figures 1A and 1B show exemplary embodiments of the systems described herein.

[0026] In a further aspect, kits described herein for screening a sample for the presence of a binding substance (e.g., that disrupts host-microbe interactions) generally include a first container containing host nanoparticles that comprise or consist of plasma membrane fragments of mammalian host cells expressing a surface receptor recognized by the exogenous ligand, and a second container containing exogenous nanoparticles that express the exogenous ligand on their surface such that the exogenous ligand is recognizable by the surface receptor of the host nanoparticles, wherein the host nanoparticles and / or the exogenous nanoparticles further comprise a detectable label, and wherein said binding substance is present in the sample if pre-incubation of either the host nanoparticles or the exogenous nanoparticles with the sample reduces binding / interaction between the host nanoparticles and the exogenous nanoparticles compared to a corresponding sample lacking the binding substance.

[0027] In further aspects, in vitro methods described herein for detecting binding substances (e.g., neutralizing or blocking substances that disrupt host-microbe interactions) in a fluid sample (e.g., a biological fluid sample) generally include providing host nanoparticles and foreign nanoparticles as defined herein, pre-incubating the host nanoparticles or foreign nanoparticles with the fluid sample to allow binding / interaction of the binding substances, incubating the host nanoparticles with the foreign nanoparticles, and measuring the binding / interaction therebetween, wherein a decreased level of binding / interaction between the host nanoparticles and the foreign nanoparticles after pre-incubation with the fluid sample compared to pre-incubation with a corresponding fluid sample lacking the binding substance indicates the presence of the binding substance in the fluid sample.

[0028] In some embodiments, the binding / interaction between the host nanoparticle and the foreign nanoparticle refers to the ability of the binding substance to neutralize the foreign nanoparticle (neutralizing activity), block the host nanoparticle receptor (blocking activity), inhibit fusion of the host nanoparticle membrane with the foreign nanoparticle membrane, and / or inhibit internalization / penetration of the foreign nanoparticle into the host nanoparticle. In some embodiments, the systems, methods, or kits described herein include a blocking step using any suitable blocking solution (e.g., bovine serum albumin, milk, or FBS) to reduce non-specific binding. In some embodiments, the systems and methods described herein include one or more washing steps after incubating the foreign nanoparticle with the host nanoparticle, particularly to remove foreign nanoparticles that are not bound / internalized (e.g., neutralized via the binding substance). In some embodiments, detection of the detectable molecule can be performed by any common method / instrument for detecting luminescence or fluorescence.

[0029] In some embodiments, the host nanoparticles and / or the foreign nanoparticles can be immobilized on a solid support or are intended for immobilization on a solid support. In some embodiments, the solid support can be on any suitable surface for coating or hybridization, such as a microplate, tube, or bead. The host nanoparticles can be pre-coated on the support or coated by known methods for coatings used in immunoassays such as ELISA. In some embodiments, the addition of the foreign nanoparticles to the host nanoparticles can be performed in the presence of a binding substance (e.g., a pre-incubation step) or after incubation of the host nanoparticles with a binding substance (e.g., a blocking step). The steps of the methods and systems described herein can generally be performed at 4°C to 37°C (e.g., room temperature) in the presence or absence of CO2 (e.g., 5%). For example, the coating step can be performed at any temperature between 4°C and 37°C for a period ranging from 1 to 48 hours. Incubation of the host nanoparticles with the foreign nanoparticles (or the host nanoparticles and foreign nanoparticles pre-incubated with the binding agent) may be carried out at any temperature between 4°C and 37°C for a period ranging from 0.5 to 8 hours.

[0030] In some embodiments, the host nanoparticles and / or the foreign nanoparticles can be in a suspension.

[0031] In some embodiments, the host nanoparticles and / or foreign nanoparticles, and subsequent incubation to detect the binding agent, can be in any solution or gel (e.g., hydrogel), such as an aqueous solution, buffer (e.g., PBS), or culture medium (e.g., DMEM).

[0032] In some embodiments, the absence or reduction in the detection of the detectable molecule indicates the presence of the binding substance. In some embodiments, the amount or concentration of the binding substance negatively correlates with the amount of the detected detectable molecule. For example, increasing concentrations of the binding substance may bind to the host nanoparticles or the foreign nanoparticles, preventing the binding of the foreign nanoparticles bearing the detectable molecule. In a subsequent washing step, unbound / uninternalized foreign nanoparticles are washed away from the system, thereby reducing the detection of the detectable molecule. In some embodiments, the binding substance present in a sample can be determined quantitatively (e.g., by using a standard curve), semi-quantitatively, or qualitatively (e.g., presence or absence).

[0033] In some embodiments, the foreign nanoparticle binds to the host nanoparticle in either vesicular or membrane form through recognition of the foreign ligand and surface receptor (Figure 4A, Figure 4D, Figure 4E, or Figure 4F). In some cases, binding of the foreign nanoparticle to the host nanoparticle induces fusion of the two respective membranes (Figure 4B or Figure 4C). In some cases, membrane fusion is followed by one or two rounds of import of intracellular contents (e.g., proteins). In some cases, binding of the foreign nanoparticle to the host nanoparticle induces internalization of the foreign nanoparticle. In some cases, internalization is followed by disruption of the foreign nanoparticle membrane and release of its intracellular contents. For example, a detectable molecule may be imported into the host nanoparticle or may be converted into a functional detectable molecule upon import.

[0034] The principles of the methods and systems described herein offer several advantages over existing methods and assays for detecting binding agents. First, the systems and methods described herein do not utilize living cells or pathogens (e.g., bacteria or viruses), making them safe for use outside of Biosafety Level 2 environments. Second, physiological host-pathogen interactions are closely mimicked, which facilitates neutralizing / blocking agent activity, including preservation of surface receptors / proteins and post-translational modifications of foreign ligands (e.g., glycoproteins). Third, the systems and methods described herein provide a rapid response (e.g., 0.5-8 hours) to the presence of binding agents. Fourth, the systems and methods described herein may be partially or fully automated.

[0035] In some embodiments, the kit may include a microplate (e.g., a 96-well plate) or tube pre-coated with host nanoparticles. The kit may further include coating, blocking, washing, dilution, and / or detection solutions. The host and / or foreign nanoparticles may be in solution (ready to use) or dehydrated / lyophilized (to be reconstituted). The kit may further include one or more positive controls (e.g., commercially available or purified neutralizing antibodies) and / or standard curves, either in solution or lyophilized. The kit may further include a bottle of ready-to-use or reconstituted washing solution. The kit may also include instructions for use.

[0036] item In various embodiments, one or more of the following items are described herein:

[0037] 1. An in vitro system for detecting a binding substance (e.g., a neutralizing or blocking substance that disrupts host-microbe interaction and / or fusion / penetration) in a fluid sample (e.g., a biological fluid sample), the system comprising: host nanoparticles comprising fragments of host cells that express or are designed to express a surface receptor recognized by an exogenous ligand; and exogenous nanoparticles that express or are designed to express an exogenous ligand on their surface such that the exogenous ligand is recognizable by the surface receptor on the host nanoparticles; the host nanoparticles and / or the exogenous nanoparticles further comprise a detectable label; and the binding substance is present in the sample if pre-incubation of either the host nanoparticles or the exogenous nanoparticles with the sample reduces binding / interaction between the host nanoparticles and the exogenous nanoparticles compared to a corresponding fluid sample lacking the binding substance.

[0038] 2. A kit for screening a sample for the presence of a binding substance (e.g., that disrupts host-microbe interaction and / or fusion / penetration), the kit comprising: a first container containing host nanoparticles that comprise or consist of fragments of mammalian host cells expressing a surface receptor recognized by the exogenous ligand; and a second container containing exogenous nanoparticles that express the exogenous ligand on their surface such that the exogenous ligand is recognizable by the surface receptor of the host nanoparticles, wherein the host nanoparticles and / or the exogenous nanoparticles further comprise a detectable label; and wherein the binding substance is present in the sample if pre-incubation of either the host nanoparticles or the exogenous nanoparticles with the sample reduces binding / interaction between the host nanoparticles and the exogenous nanoparticles compared to a corresponding sample lacking the binding substance.

[0039] 3. An in vitro method for detecting a binding substance (e.g., a neutralizing or blocking substance that disrupts host-microbe interaction and / or fusion / penetration) in a fluid sample (e.g., a biological fluid sample), the method comprising providing host nanoparticles and foreign nanoparticles according to items 1 or 2; pre-incubating the host nanoparticles or foreign nanoparticles with the fluid sample to allow binding / interaction of the binding substance; incubating the host nanoparticles with the foreign nanoparticles and measuring the binding / interaction therebetween, wherein a decreased level of binding / interaction between the host nanoparticles and the foreign nanoparticles after pre-incubation with the fluid sample compared to pre-incubation with a corresponding fluid sample lacking the binding substance indicates the presence of the binding substance in the fluid sample.

[0040] 4. The system, kit or method of any one of items 1 to 3, wherein the host and / or foreign nanoparticles are in a vesicular or linear shape.

[0041] 5. The system, kit or method of any one of items 1 to 4, wherein the host nanoparticles are produced from or derived from host producer cells.

[0042] 6. The system, kit or method of item 5, wherein the host producer cell is a eukaryotic (e.g., yeast or mammalian) cell.

[0043] 7. The system, kit, or method of item 5 or 6, wherein the host producer cells are (a) engineered to express or overexpress an exogenous ligand, (b) engineered to express or overexpress a detectable label, (c) engineered to express or overexpress an exogenous viral protein (e.g., from an enveloped or non-enveloped virus), an exogenous bacterial protein, or an exogenous fungal protein that induces the formation of extracellular vesicles or particles (e.g., biological nanoparticles or virus-like particles) in the host producer cells, or (d) any combination of (a)-(c).

[0044] 8. The system, kit, or method of any one of items 1 to 7, wherein the host nanoparticle is a vesicular nanoparticle comprising an exogenous viral structural protein (e.g., a late assembly (L) domain protein or polyprotein, e.g., HIV Gag) that induces host cell budding.

[0045] 9. The system, kit or method of item 8, wherein the exogenous viral structural protein is fused to a detectable label.

[0046] 10. The system, kit, or method of any one of items 1-9, wherein the exogenous nanoparticles are produced from or derived from a microorganism (e.g., a bacterium, fungus, virus, or parasite), produced from or derived from a eukaryotic (e.g., yeast or mammalian) producer cell, or formed by self-assembly of a viral structural protein (e.g., capsid protein).

[0047] 11. The system, kit, or method of item 10, wherein the microbial or eukaryotic producer cells from which the foreign nanoparticles are produced or derived are (a) engineered to express or overexpress a surface receptor, (b) engineered to express or overexpress a detectable label, (c) engineered to express or overexpress an exogenous viral protein (e.g., from an enveloped or non-enveloped virus), exogenous bacterial protein, or exogenous fungal protein that induces the formation of extracellular vesicles or particles (e.g., biological nanoparticles or virus-like particles) within the host producer cells, (d) nanoparticles that fuse with and / or penetrate host nanoparticles upon contact, or (e) any combination of (a)-(d).

[0048] 12. The system, kit, or method of any one of items 1 to 11, wherein the foreign nanoparticle is a vesicular foreign nanoparticle comprising an exogenous viral structural protein (e.g., a late assembly (L) domain protein or polyprotein, e.g., HIV Gag) that induces host cell budding.

[0049] 13. The system, kit, or method of any one of items 1 to 12, wherein the foreign nanoparticles are derived from the SARS-CoV-2 virus and / or express or are engineered to express the SARS-CoV-2 Spike protein or a portion thereof on their surface.

[0050] 14. The system, kit, or method of any one of items 1 to 13, wherein the host nanoparticles express or are engineered to express angiotensin-converting enzyme 2 (ACE2) and / or transmembrane serine protease 2 (TMPRSS2) on their surface.

[0051] 15. The system, kit, or method of any one of items 1 to 14, wherein the exogenous ligand is a surface ligand expressed on a microbial cell, an autoantigen, or an allergen.

[0052] 16. The system, kit, or method of any one of items 1 to 15, wherein the detectable label is expressed internally or on the surface of the foreign nanoparticle or the host nanoparticle.

[0053] 17. The system, kit, or method of any one of items 1 to 16, wherein the detectable label is a fluorescent or luminescent molecule (e.g., a protein).

[0054] 18. The system, kit or method of any one of items 1 to 17, wherein the detectable label is detectable upon contact with a substrate, protein or enzyme.

[0055] 19. The system, kit, or method of any one of items 1 to 18, wherein the sample is blood, serum, tears, saliva, plasma, urine, nasal secretion, bronchoalveolar lavage fluid (BALF), feces, or cerebrospinal fluid.

[0056] 20. The system, kit, or method of any one of items 1 to 19, wherein the binding agent is an antibody or antigen-binding fragment thereof, a peptide, a protein, a nucleic acid (e.g., an aptamer or ribozyme), or a small molecule.

[0057] 21. The system, kit or method of any one of items 1 to 20, wherein the binding agent recognizes a surface receptor on a host nanoparticle or a foreign ligand on a foreign nanoparticle.

[0058] 22. The system, kit or method of any one of items 1 to 21, wherein the binding agent prevents or attenuates fusion of the membrane of the host nanoparticle with the membrane of the foreign nanoparticle.

[0059] 23. The system, kit or method of any one of items 1 to 22, wherein the binding agent prevents or attenuates internalization / penetration of the foreign nanoparticle into the host nanoparticle.

[0060] 24. The system, kit, or method of any one of items 1 to 23, wherein the host nanoparticle comprises a plasma membrane fragment of a host cell that expresses or is engineered to express a surface receptor that is recognized by the exogenous ligand.

[0061] 25. The system, kit, or method of any one of items 1 to 24, wherein the host nanoparticles and / or the foreign nanoparticles are immobilized on a solid support or are intended for immobilization on a solid support.

[0062] 26. The system, kit or method of any one of items 1 to 24, wherein the host nanoparticles and / or the foreign nanoparticles are in suspension.

[0063] 27. The kit of any one of items 2 and 4-26, further comprising instructions for use.

[0064] example Example 1: Production of host (H) and foreign (F) nanoparticles For this neutralizing and blocking molecule detection assay, we produced two types of nanoparticles: host and foreign nanoparticles. They are complex biologically mixed structures based on the assembly of biological molecules of various natures to create a membrane composed of lipids, proteins, and / or polysaccharides (glycans). We fabricated nanoparticles in either a vesicular form (Figures 2A, 3A, and 3C) with an internal compartment and an extracellular surface, or in a linear form (Figures 2B and 3B). The nanoparticles were derived from cultured eukaryotic or prokaryotic producer cells. They can be composed of all the elements conventionally found in native plasma membranes, such as various types of lipids, polysaccharides, proteins, and the natural post-translational modifications associated with these various elements (e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation).

[0065] To functionalize these complex biological membrane structures, specifically selected proteins (i.e., antigens, ligands, receptors) are overexpressed in producer cells (e.g., adherents or cells in suspension) and incorporated into these structures using various techniques, such as expression vectors (e.g., plasmids). In addition, other overexpressed proteins contribute to the production process of these membrane-type complex biological structures. Some of these associated proteins generate fluorescent or optical signals. All of the proteins that make up complex biological structures have three-dimensional conformations and natural post-translational modifications. By incorporating antigens or ligands into these structures, it is possible to simulate the outer surface of pathogens and form foreign nanoparticles that can be used to design neutralizing molecule detection assays. Based on the same principle, by incorporating receptors and specific proteins into these complex structures, it is possible to mimic the outer surface of cells and form host nanoparticles that can be used to design neutralizing molecule detection assays.

[0066] Upon expression / overexpression of the protein of interest, producer cells were cultured for a period of time to induce nanoparticle formation / budding, and the nanoparticles were further harvested by centrifugation, filtration (0.45 μm), and / or chromatography techniques. Ultracentrifugation on a sucrose cushion was typically performed to concentrate the nanoparticles. The nanoparticles were then resuspended, quantified for total protein, and stored at -80°C. Expression of specific proteins, such as HIV Gag protein, along with the protein of interest, was found to aid in the development of the nanoparticle's internal structure and trigger the nanoparticle budding mechanism from the producer cells.

[0067] We have produced another type of vesicular foreign nanoparticle that relies solely on protein assembly. These proteins have the inherent ability to self-assemble into capsids in cells and / or in vitro in the absence of cells (Figure 3C). Other proteins, such as fluorescent proteins or luminescent enzymes, may be contained within the structures. These proteins, which make up these complex capsid-type biological structures, possess three-dimensional conformations and native post-translational modifications. This type of complex biological structure is used to simulate the surface of non-enveloped viruses in neutralizing molecule detection assays. We have produced norovirus (a non-enveloped virus) foreign nanoparticles expressing VP1 on their surface (as further described in Example 9). Transmission electron microscopy images of these nanoparticles are shown in Figure 21C.

[0068] These nanoparticles were produced by engineered proteins and culturing eukaryotic or prokaryotic cells. Briefly, eukaryotic or prokaryotic cells were modified to overexpress native, exogenous, and / or chimeric proteins. To fabricate complex membrane-type biological structures, specific overexpressed proteins were assembled on the inner surface and / or through the membrane envelope. Furthermore, some of the overexpressed proteins contributed to the membrane budding process, leading to the formation and excretion of these vesicular nanoparticles in the extracellular medium. These vesicular forms were linearized by mechanical force induced during their production and extraction. Linear forms of SARS-CoV-2 foreign nanoparticles expressing the Spike protein were produced. Transmission electron microscopy images of these nanoparticles are shown in Figure 21D.

[0069] To produce complex capsid-type biological structures, proteins were auto-assembled within producer cells to form these structures, which were then excreted into the extracellular medium. Host nanoparticles were engineered with plasma membranes from host producer cells to express surface receptors and proteins that aid in the recognition of surface ligands / proteins on the surface of foreign nanoparticles. These nanoparticles lacked any genetic material (DNA or RNA) or machinery that would enable functional replication. For example, for the SARS-CoV-2 model, host nanoparticles were engineered to express the angiotensin-converting enzyme 2 (ACE2) receptor and transmembrane serine protease 2 (TMPRSS2) for recognition of the SARS-CoV-2 Spike glycoprotein (mutant D614G) expressed on the surface of foreign nanoparticles. Because the nanoparticles were derived directly from producer cells expressing these proteins, the surface proteins possessed all necessary post-translational modifications. In another model, foreign nanoparticles were engineered to express the vesicular stomatitis virus (VSV)-G surface glycoprotein, and host nanoparticles were engineered to express the VSV-G surface receptor. To generate fluorescent signals within the host or foreign nanoparticles, GFP was fused to Gag or the expressed surface receptor / exogenous ligand. For example, for the SARS-CoV-2 model, producer cells were transfected with a plasmid expressing Gag fused to the fluorescent protein GFP and a plasmid expressing the SARS-CoV-2 Spike mutant D614G. Luciferase or other known fluorescent proteins, such as secreted alkaline phosphatase (SEAP), were used to generate fluorescent signals.

[0070] Example 2: Characterization of foreign (F) nanoparticles To demonstrate the ability of foreign nanoparticles to interact with native / living cells (i.e., binding to the plasma membrane), increasing amounts of fluorescent VSV viral nanoparticles produced as described in Example 1 were incubated with HEK293T cells (10,000 cells / well in a 96-well plate). Fluorescent viral nanoparticles lacking specific antigen or ligand were used as a negative control to assess any nonspecific interactions. HEK293T cells are naturally susceptible to VSV infection because they possess receptors that recognize VSV glycoproteins. After incubation of the cells with VSV nanoparticles (18 hours at 37°C and 5% CO), the cultures were washed to remove nonspecific binding and subsequently analyzed by flow cytometry. The incubation was performed according to the following protocol:

[0071] Day 1: HEK293T cells were seeded at 10,000 cells / well in a 96-well plate and incubated at 37°C, 5% CO for 24 hours.

[0072] Day 2: Diluting fluorescent VSV nanoparticles (antigenic / ligand and negative models) in complete medium Removing medium from 96-well plates Droplet injection of diluted fluorescent VSV nanoparticles onto HEK293T cells Incubate at 37°C, 5% CO2 for 18 hours.

[0073] Day 3: Removing medium from 96-well plates Adherent HEK293T cells are washed with PBS containing 0.01% SDS and 10 mM EDTA to remove non-specific binding, then washed with PBS alone, and finally added with complete medium. Capture images by fluorescence microscopy Washing adherent HEK293T cells with PBS Add trypsin and incubate at 37°C to detach the cells. Inhibit trypsin using complete medium Harvest the cells by centrifugation (remove the supernatant) and resuspend the cells in PBS. Analyze cells by flow cytometry for fluorescent signal (GFP)

[0074] Cells incubated with fluorescent viral nanoparticles lacking the VSV G glycoprotein exhibited low fluorescence signals under microscopic observation (Figure 5A), thereby indicating the absence of binding. Cells incubated with fluorescent VSV nanoparticles expressing the VSV G glycoprotein exhibited fluorescent signals that increased with increasing amounts of nanoparticles (a dose-response effect) (Figure 5B). Furthermore, images revealed that the fluorescent signal was specifically located on the cells (as seen by phase contrast) via either surface binding or internalization, rather than on the plastic support. Although flow cytometry analysis did not identify the cellular location (membrane and / or interior of the cell) of the fluorescent viral nanoparticles, these results demonstrated that VSV nanoparticles can specifically interact with live cells (Figure 5C).

[0075] To determine whether viral nanoparticles can penetrate or become internalized in live cells, fluorescent VSV nanoparticles expressing a plasmid containing a transactivatable GFP expression cassette, as described above, were incubated with HEK293T cells. Because HEK293T cells possess the machinery for translation of functional GFP protein, successful internalization of viral nanoparticles is indicated by GFP expression. As seen in Figures 6B and 6C, GFP expression was observed by fluorescence microscopy and flow cytometry upon incubation of HEK293T cells with VSV nanoparticles expressing the VSV G glycoprotein, but not with negative control viral nanoparticles lacking the VSV G glycoprotein (Figure 6A). These data confirm that viral nanoparticles can be internalized upon surface recognition and that the intracellular contents of viral nanoparticles can be shared with host cells.

[0076] Finally, to determine whether viral nanoparticle binding to cells could be neutralized, fluorescent VSV nanoparticles expressing the VSV G glycoprotein were preincubated with various concentrations of an anti-VSV neutralizing antibody (Ab01402-2.0; clone IE9F9) specific for the VSV G protein for 1 hour before incubation with host HEK293T cells. As shown in Figure 7, after washing the cells, increasing concentrations of the neutralizing antibody inhibited VSV nanoparticle binding to HEK293T cells, as demonstrated by a decrease in fluorescence by flow cytometry. To confirm these results, we generated VSV-G pseudotyped lentivirus encapsulating a transactivatable GFP expression cassette, incubated it with the anti-VSV neutralizing antibody, and then incubated it with HEK293T cells. This data demonstrates that the assay can be used to detect binding agents that prevent the internalization of foreign nanoparticles. Similar results were observed, with a decrease in fluorescence with increasing concentrations of the neutralizing antibody (Figure 8).

[0077] Example 3: Detection of neutralization of interactions between host nanoparticles and foreign nanoparticles Binding of foreign nanoparticles to host nanoparticles was first assessed by incubating fluorescent VSV nanoparticles with host nanoparticles produced as described in Example 1. The host nanoparticles were first coated onto the bottom of a 96-well plate and then incubated with VSV nanoparticles. Incubation was performed using the following protocol:

[0078] Day 1: Coat the host nanoparticles in a 96-well plate with coating buffer overnight at 4°C.

[0079] Day 2: Removing liquid from the 96-well plate Various concentrations of fluorescent VSV nanoparticles are added to the wells. Incubate overnight at 4°C. Remove liquid from the 96-well plate Wash with PBS Read fluorescence using a plate reader instrument

[0080] As shown in Figure 9, an increase in fluorescence was observed with increasing amounts of VSV nanoparticles incubated with the host nanoparticles, indicating binding of the two nanoparticles.

[0081] Next, we used a nanoparticle SARS-CoV-2 model, as described in Example 1, to determine whether neutralization could be detected. Briefly, host nanoparticles exposing the ACE2 receptor and TMPRSS2 on their surface, which bind to the Spike glycoprotein of SARS-CoV-2, were coated onto a 96-well plate. Defined amounts of SARS-CoV-2 fluorescent nanoparticles were then mixed and incubated with various concentrations of various biological samples containing or lacking neutralizing antibodies. After incubation, the wells were washed to remove nonspecific binding and analyzed for fluorescence detection using a plate reader instrument. If inhibition of binding between the viral nanoparticles and the host nanoparticles occurred (i.e., via neutralizing antibodies), an absence or decrease in fluorescence would be observed. The assay was performed using the following protocol.

[0082] Day 1: Coat the host nanoparticles in a 96-well plate with coating buffer overnight at 4°C.

[0083] Day 2: Incubate fluorescent SARS-CoV-2 viral nanoparticles with increasing concentrations of biological samples for 4 hours at 37 °C. Remove liquid from the 96-well plate Wash with PBS Add the pre-incubated mixture of viral nanoparticles / neutralizing antibodies to the host nanoparticles Incubate for 4 hours at 37°C. Wash with PBS Read fluorescence using a plate reader instrument

[0084] Neutralization of the binding between host and viral nanoparticles was observed upon addition of increasing concentrations of anti-Spike RBD antibody (human IgG monoclonal; Ab02019-12.1), as indicated by a decrease in fluorescence levels (Figure 10A). However, neutralization was not observed in the presence of other antibodies, such as anti-VSV IgG antibody (Ab01402-2.0). These data demonstrate that antibody-mediated neutralization can be detected by this assay.

[0085] To determine whether neutralizing agents could be detected within complex biological samples such as serum, we performed the same experiment by incubating fluorescent SARS-CoV-2 nanoparticles with either pooled human serum from SARS-CoV-2-positive patients or FBS. These samples were pre-tested for the presence of anti-SARS-CoV-2 antibodies. As shown in Figure 10B, a decrease in fluorescence was observed with increasing concentrations of pooled human serum compared to FBS.

[0086] Finally, to determine whether this assay would be useful for detecting antibody-mediated neutralization of various isotypes, species of origin, or targets on the Spike protein, we performed the same experiment using purified chicken polyclonal IgY antibodies targeting the S1 or S2 region of the SARS-CoV-2 Spike protein. As shown in Figures 10C and 10D, anti-S1- or anti-S2-mediated neutralization was readily detected.

[0087] These data demonstrate that this assay can be used safely and efficiently to detect a variety of neutralizing substances present in complex biological assays.

[0088] Example 4: Detection of neutralization of interactions between host nanoparticles and foreign nanoparticles As used herein, the term "envelope" in the phrase "foreign enveloped nanoparticles" refers to nanoparticles intended to mimic enveloped viruses. The produced foreign enveloped nanoparticles were composed of a lipid membrane with biological molecules derived from the lipid membrane of producer cells. In these experiments, the foreign enveloped nanoparticles presented the SARS-CoV-2 spike glycoprotein anchored to the lipid membrane on their surface and were produced using ectopic coexpression of Gag protein and viral glycoproteins in mammalian cells. However, instead of GFP, the SARS-CoV-2 foreign nanoparticles were modified with the fluorescent dye octadecylrhodamine B chloride (R18). This fluorescent dye was inserted into the membrane of the foreign nanoparticles at a surface density that caused self-quenching of the fluorescent dye. Fusion of the membrane of the labeled foreign nanoparticle with the unlabeled target membrane (host nanoparticle) resulted in relief of self-quenching and a proportional increase in relative fluorescence intensity. Therefore, this assay confirmed that the fusion process between the foreign nanoparticles and the host nanoparticles occurred directly. Additionally, a transmission electron microscopy image of enveloped foreign SARS-CoV-2 nanoparticles expressing Spike protein is shown in Figure 21A.

[0089] Host ACE2 nanoparticles and host ACE2 / TMPRSS2 nanoparticles were produced as described in Example 1. A transmission electron microscope image of the enveloped host ACE2 nanoparticles is shown in Figure 21B.

[0090] The fusion assay consisted of incubating the host nanoparticles with the foreign nanoparticles (i.e., without a coating step) in suspension in PBS or culture medium (DMEM) for 3 hours at 37°C. Mixing was performed in a microwell plate. After substrate addition, the luminescence signal was detected over time using a plate reader (emission = 400 nm). As shown in Figure 11A, fusion of the foreign Spike SARS-CoV-2 nanoparticles with the host ACE2 nanoparticles was confirmed in both PBS and DMEM. The results shown represent normalized fluorescence levels from the control (background fluorescence signal from nanoparticles in a monoculture). Fusion of the foreign Spike SARS-CoV-2 nanoparticles with the host ACE2 / TMPRSS2 nanoparticles was also successfully confirmed in both PBS and DMEM (Figure 11B).

[0091] Fusion between host and foreign envelope nanoparticles was also demonstrated using a split luciferase complementation assay based on NanoLuc™ luciferase (Sasaki, M et al., 2018). This method is a well-characterized technique for testing protein-protein interactions. NanoLuc luciferase (Nluc) is a relatively small protein (approximately 19 kDa) capable of producing bright luminescence. Nluc can be split into two non-functional subunits: a large (18 kDa) subunit called LgBiT and a small (11 amino acid) subunit called HiBiT, creating a complementation reporter system for testing protein-protein interactions. This Nluc split complementation system was successfully used to monitor the cellular entry and release of virus-like particles and viral particles.

[0092] In the following experiments, LgBiT and HiBiT fragments were generated upon fusion with Gag protein during nanoparticle production. Thus, ACE2 host envelope nanoparticles encapsulate LgBiT protein fragments, while Spike SARS-CoV-2 foreign envelope nanoparticles encapsulate HiBiT protein fragments. Upon fusion between the host and foreign nanoparticles, following interaction between the ACE2 receptor and the Spike SARS-CoV-2 antigen, the LgBiT and HiBiT fragments combine to form an active enzyme, which can generate a bright luminescent signal in the presence of substrate. To ensure specific detection of fused nanoparticles, we measured luminescence generation over time using a non-lytic detection reagent (Nano-Glo® Live Cell Assay System - Promega) that can penetrate the lipid membrane without any disruption. As shown in Figure 14, this method confirmed fusion between the foreign Spike SARS-CoV-2 nanoparticles and the coated host ACE2 nanoparticles.

[0093] Figure 12 further confirms fusion between enveloped host nanoparticles and enveloped foreign nanoparticles. Fusion of native viruses with live cells is known to be temperature-dependent, as low temperatures limit the mobility of viral glycoproteins. Furthermore, it is well documented that low temperatures inhibit some active biological pathways (energy-dependent), such as cell infection by enveloped viruses.

[0094] To determine whether fusion between foreign envelope nanoparticles and host nanoparticles involves protein motility and other active biological pathways, such as those that occur during the natural infection process, fusion assays were performed at 4°C and 37°C. Briefly, R18-labeled foreign envelope nanoparticles were mixed with host nanoparticles and incubated at 37°C or 4°C for 3 hours. After the incubation period, formaldehyde was added to halt the biological process. Fluorescence signals were then detected using a plate reader (excitation = 544 nm, emission = 590 nm). As shown in Figure 12, fusion was shown to occur with both ACE2- and ACE2 / TMPRSS-host nanoparticles at 37°C but dramatically decreased at 4°C.

[0095] Next, we investigated the neutralization of fusion between foreign nanoparticles and host nanoparticles induced by both methods. As shown in Figure 13, fusion between Spike SARS-CoV-2 foreign nanoparticles in suspension and coated host ACE2 nanoparticles was neutralized in the presence of anti-Spike RBD antibodies (Seydoux et al., 2020) or a SARS-CoV-2 Spike protein-specific aptamer (MSA5; (Li et al., 2021)), compared to untreated controls. As shown in Figure 14, fusion between treated coated ACE2 host nanoparticles and SARS-CoV-2 foreign nanoparticles (mediated by luciferase complementation) was significantly inhibited in the presence of anti-Spike RBD antibodies, but not anti-VSVg antibodies.

[0096] These data suggest that foreign and host nanoparticles, either in suspension or coated on a surface, exhibit a fusion process similar to that observed in natural viral infection. Furthermore, this system can be successfully used to detect neutralizing substances.

[0097] Example 5: Detection of various types of neutralizing and blocking substances The system described herein can be used to detect neutralizing substances such as antibodies, but can also be used to detect various types of neutralizing substances. As shown in Figure 15A, we demonstrated neutralization of fusion between Spike SARS-CoV-2 foreign nanoparticles and coated host ACE2 nanoparticles using an anti-Spike RBD antibody, a soluble ACE2 protein (Chaouat et al., 2021), or a SARS-CoV-2 Spike protein-specific aptamer (MSA5; (Li et al., 2021)), compared with an anti-VSVg negative control antibody. Neutralization was further blocked in the presence of the small molecule ouabain, which was shown to bind to the SARS-CoV-2 Spike protein (Caohuy et al., 2021). Furthermore, fusion between Spike SARS-CoV-2 foreign nanoparticles and coated host ACE2 nanoparticles was successfully blocked via soluble RBD, a blocker that binds to the ACE2 receptor on the host nanoparticle (Figure 15A).

[0098] Next, we coated the foreign nanoparticles and performed neutralization in a system where host nanoparticles were added in solution. As shown in Figure 15B, the addition of neutralizing or blocking substances such as anti-RBD or soluble ACE2, soluble RBD, successfully neutralized / blocked the fusion of the coated Spike SARS-CoV-2 foreign nanoparticles with host ACE2 nanoparticles.

[0099] These data suggest that the system described herein can be used to detect various types of neutralizing or blocking agents, such as antibodies, proteins, nucleic acids, and small molecules. Furthermore, the system can be adapted in various ways, for example, by coating either the foreign or host nanoparticles, or by having both nanoparticles in suspension.

[0100] Example 6: Compatibility with various types of biological samples Neutralizing substances can be present in various bodily fluids. Therefore, we next determined the effectiveness of this system in various biological fluid samples. As shown in Figure 16, we evaluated the neutralization of Spike SARS-CoV-2 foreign nanoparticles and coated host ACE2 nanoparticles in the presence of anti-Spike RBD Ab or anti-VSVg Ab (negative control) diluted in either urine, saliva, or serum. Anti-Spike RBD-mediated neutralization was successfully demonstrated in all fluids. Similar results were observed in plasma, although the effect was reduced, likely due to the presence of heparin (not present in serum), which has been shown to inhibit SARS-CoV-2 entry into cells (Bewley et al., 2021). This effect was even more evident when the amount of plasma was increased (not shown).

[0101] These data suggest that the system can be used to detect neutralizing or blocking substances in a variety of biological fluid samples.

[0102] Example 7: Effect of temperature and incubation timing To further optimize the system, various temperatures and incubation times were investigated. As described in the previous example, foreign nanoparticles were generally incubated with a solution containing a neutralizing agent at 37°C for 1 hour, and then the mixture with the host nanoparticles was incubated at 37°C for 3 hours (Figure 17A). Neutralization was also demonstrated under these conditions at room temperature instead of 37°C, but the effect was slightly reduced.

[0103] Neutralization was maintained at both temperatures when the incubation time of the foreign nanoparticles and neutralizing agent was shortened to 30 minutes (FIG. 17B) or when the incubation time of the host nanoparticles and foreign nanoparticle / neutralizing agent was shortened to 1 hour (FIG. 17C). With both incubation times shortened, the neutralization effect was slightly reduced at 37°C and significantly reduced at room temperature.

[0104] These data suggest that the system is highly flexible in that it can be used with various incubation times for the various components and at either 37°C (e.g., in an incubator) or room temperature (e.g., on the bench or in the field). Furthermore, incubation times can be increased or decreased to increase the sensitivity of the system depending on the conditions (e.g., temperature).

[0105] Example 8: Detection of neutralizing substances against non-viral foreign nanoparticles To determine whether this system could be adapted to detect neutralizing substances against microorganisms other than viruses, bacterial exogenous nanoparticles were developed.

[0106] Here, we used this system to detect neutralizing antibodies against the PA63 fragment of anthrax toxin from Bacillus anthracis (UniProt accession number: P13423). PA63 is a non-toxic fragment of anthrax toxin that binds to a host cell receptor (ANTXR2 - UniProt accession number: P58335) and is the antigen targeted by neutralizing antibodies.

[0107] In these experiments, the foreign nanoparticles were fluorescent envelope nanoparticles whose surfaces were modified with PA63 fragments. The foreign envelope nanoparticles were produced from HEK293 cells expressing a GAG protein fused to GFP and a chimeric protein, thereby enabling linkage between the target antigen (PA63 fragment) and the nanoparticle surface, as described in Example 1. The chimeric protein consists of a membrane localization signal and a peptide tag capable of forming a spontaneous amide bond based on the harnessing reaction of an adhesion protein derived from the bacterium Streptococcus pyogenes. The resulting unmodified nanoparticles consist of a lipid membrane derived from producer cells encapsulating GFP (or another protein, such as luciferase), containing the chimeric protein across the membrane, and displaying the linking tag on the outer surface. The target antigen was produced separately, and the sequence of the protein was fused to another peptide tag derived from an adhesion protein of the bacterium Streptococcus pyogenes. Nanoparticle functionalization can be achieved after incubation with a target antigen. The two partner tags (located on the nanoparticle surface and fused to the target antigen) form a spontaneous and irreversible isopeptide bond together. This functionalization method can be used regardless of antigen origin and can be applied to produce a series of nanoparticles modified with proteins from various sources, such as bacteria, parasites, yeast, mammals, or fungi.

[0108] Co-expression of GAG proteins and the cellular receptor targeted by PA63 (ANTXR2) was used to produce host nanoparticles as previously described in Example 1.

[0109] As shown in Figure 18, neutralization of the interaction between coated host ANTXR2 nanoparticles and foreign B. anthracis PA63 nanoparticles was demonstrated in the presence of anti-PA63 antibody, but not anti-VSVg antibody (negative control).

[0110] These data suggest that this system can be used to detect neutralizing / blocking substances that neutralize / block the interactions between host cells and various microbial cells.

[0111] Example 9: Detection of neutralizing substances against non-enveloped virus foreign nanoparticles Non-enveloped viruses consist of a protein capsid without a lipid membrane. To evaluate whether our system is effective in detecting neutralizing / blocking substances for non-enveloped viruses, we used foreign non-enveloped nanoparticles mimicking norovirus and host enveloped nanoparticles displaying the FUT2 receptor on their surface. Foreign non-enveloped nanoparticles were produced by expressing the norovirus capsid protein (VP1, UniProt accession number Q83884) in HEK293 cells. Following expression, VP1 can spontaneously self-assemble to form norovirus capsids without the involvement of other viral proteins. These capsids were used as foreign non-enveloped nanoparticles in this experiment.

[0112] The corresponding host nanoparticles were enveloped nanoparticles produced as described in Example 1 using co-expression of the Gag protein and cellular receptor used by norovirus (FUT2 - UniProt accession number Q10981).

[0113] As shown in Figures 19A and 19B, neutralization of the interaction between coated host FUT2 nanoparticles and foreign norovirus VP1 nanoparticles was demonstrated in the presence of anti-VP1 antibody, but not anti-VSVg antibody (negative control).

[0114] These data suggest that this system can be used to detect neutralizing / blocking substances that neutralize / block the interaction of non-enveloped viruses with host cells.

[0115] Example 10: Production of host and foreign nanoparticles from various cell types As shown in the previous example, host and foreign nanoparticles were produced from HEK293T cells. To determine whether other cells could be used to produce nanoparticles, ACE2 host nanoparticles and SARS-CoV-2 Spike foreign nanoparticles were produced using HeLa cells under the same conditions as in Example 1. Neutralization of the interaction between the nanoparticles was then confirmed in the presence of anti-spike RBD antibodies (Figure 20).

[0116] References JPEG2025500402000002.jpg169168

Claims

1. 1. An in vitro system for detecting binding substances (e.g., neutralizing or blocking substances that disrupt host-microbe interactions and / or fusion / penetration) in a fluid sample (e.g., a biological fluid sample), the system comprising: a host nanoparticle comprising a fragment of a host cell that expresses or is engineered to express a surface receptor that is recognized by the exogenous ligand; a foreign nanoparticle that expresses or is designed to express the foreign ligand on its surface such that the foreign ligand is recognizable by the surface receptor of the host nanoparticle; Including, the host nanoparticle and / or the foreign nanoparticle further comprise a detectable label; The binding agent is present in the sample if pre-incubation of the sample with either the host nanoparticles or the foreign nanoparticles reduces the binding / interaction between the host nanoparticles and the foreign nanoparticles compared to a corresponding fluid sample lacking the binding agent. In vitro system.

2. 1. A kit for screening a sample for the presence of a binding agent (e.g., that disrupts host-microbe interactions and / or fusion / penetration), said kit comprising: a first container containing host nanoparticles comprising or consisting of fragments of mammalian host cells expressing a surface receptor recognized by the exogenous ligand; a second container containing exogenous nanoparticles expressing the exogenous ligand on their surface such that the exogenous ligand is recognizable by the surface receptor of the host nanoparticle; Including, the host nanoparticles and / or the foreign nanoparticles further comprise a detectable label, and if pre-incubation of the sample with either the host nanoparticles or the foreign nanoparticles reduces the binding / interaction between the host nanoparticles and the foreign nanoparticles compared to a corresponding sample lacking the binding agent, then the binding agent is present in the sample. kit.

3. 1. An in vitro method for detecting binding substances (e.g., neutralizing or blocking substances that disrupt host-microbe interactions and / or fusion / penetration) in a fluid sample (e.g., a biological fluid sample), the method comprising: Providing a host nanoparticle and a foreign nanoparticle according to claim 1; pre-incubating the host nanoparticles or the foreign nanoparticles with the fluid sample to allow binding / interaction of the binding agents; incubating the host nanoparticles with the foreign nanoparticles and measuring the binding / interaction therebetween, wherein a decrease in the level of binding / interaction between the host nanoparticles and the foreign nanoparticles after the pre-incubation with the fluid sample compared to pre-incubation with a corresponding fluid sample lacking the binding substance indicates the presence of a binding substance in the fluid sample. In vitro methods.

4. The system, kit or method of any one of claims 1 to 3, wherein the host and / or foreign nanoparticles are in vesicular or linear form.

5. The system, kit or method of any one of claims 1 to 3, wherein the host nanoparticles are produced or derived from host producer cells.

6. 6. The system, kit or method of claim 5, wherein the host producer cell is a eukaryotic (e.g., yeast or mammalian) cell.

7. the host producer cell (a) engineered to express or overexpress said exogenous ligand; (b) engineered to express or overexpress said detectable label; (c) engineered to express or overexpress an exogenous viral protein (e.g., from an enveloped or non-enveloped virus), exogenous bacterial protein, or exogenous fungal protein that induces the formation of extracellular vesicles or particles (e.g., biological nanoparticles or virus-like particles) in said host producer cells; or (d) Any combination of (a) to (c) The system, kit or method of claim 5, wherein

8. 4. The system, kit or method of any one of claims 1 to 3, wherein the host nanoparticle is a vesicular nanoparticle comprising an exogenous viral structural protein (e.g., a late assembly (L) domain protein or polyprotein, e.g., HIV Gag) that induces host cell budding.

9. 9. The system, kit or method of claim 8, wherein the exogenous viral structural protein is fused to the detectable label.

10. 4. The system, kit or method of any one of claims 1 to 3, wherein the exogenous nanoparticles are produced from or derived from a microorganism (e.g., a bacterium, fungus, virus or parasite), produced from or derived from a eukaryotic (e.g., yeast or mammalian) producer cell, or formed by self-assembly of viral structural proteins (e.g., capsid proteins).

11. the microbial or eukaryotic producer cell from which the exogenous nanoparticles are produced or derived, (a) engineered to express or overexpress said surface receptor; (b) engineered to express or overexpress said detectable label; (c) engineered to express or overexpress an exogenous viral protein (e.g., from an enveloped or non-enveloped virus), exogenous bacterial protein, or exogenous fungal protein that induces the formation of extracellular vesicles or particles (e.g., biological nanoparticles or virus-like particles) in said host producer cells; (d) a nanoparticle that fuses with and / or penetrates said host nanoparticle upon contact; or (e) Any combination of (a) to (d) The system, kit or method of claim 10, wherein

12. 4. The system, kit or method of any one of claims 1 to 3, wherein the foreign nanoparticle is a vesicular foreign nanoparticle comprising an exogenous viral structural protein (e.g., a late assembly (L) domain protein or polyprotein, e.g., HIV Gag) that induces host cell budding.

13. 4. The system, kit or method of any one of claims 1 to 3, wherein said foreign nanoparticles are derived from the SARS-CoV-2 virus and / or express or are engineered to express the SARS-CoV-2 Spike protein or a portion thereof on their surface.

14. 4. The system, kit or method of any one of claims 1 to 3, wherein the host nanoparticles express or are engineered to express angiotensin-converting enzyme 2 (ACE2) and / or transmembrane serine protease 2 (TMPRSS2) on their surface.

15. The system, kit or method of any one of claims 1 to 3, wherein the exogenous ligand is a surface ligand expressed on a microbial cell, an autoantigen or an allergen.

16. The system, kit or method of any one of claims 1 to 3, wherein the detectable label is expressed internally or on the surface of the foreign nanoparticle or the host nanoparticle.

17. The system, kit or method according to any one of claims 1 to 3, wherein the detectable label is a fluorescent or luminescent molecule (eg a protein).

18. The system, kit or method of any one of claims 1 to 3, wherein the detectable label is detectable upon contact with a substrate, protein or enzyme.

19. The system, kit or method of any one of claims 1 to 3, wherein the sample is blood, serum, tears, saliva, plasma, urine, nasal secretion, bronchoalveolar lavage fluid (BALF), feces or cerebrospinal fluid.

20. The system, kit or method of any one of claims 1 to 3, wherein the binding agent is an antibody or antigen-binding fragment thereof, a peptide, a protein, a nucleic acid (e.g., an aptamer or ribozyme), or a small molecule.

21. The system, kit or method of any one of claims 1 to 3, wherein the binding agent recognizes the surface receptor of the host nanoparticle or the foreign ligand of the foreign nanoparticle.

22. The system, kit or method of any one of claims 1 to 3, wherein the binding agent prevents or attenuates fusion of the membrane of the host nanoparticle with the membrane of the foreign nanoparticle.

23. The system, kit or method of any one of claims 1 to 3, wherein the binding agent prevents or attenuates internalization / penetration of the foreign nanoparticle into the host nanoparticle.

24. 4. The system, kit or method of any one of claims 1 to 3, wherein the host nanoparticle comprises a plasma membrane fragment of the host cell that expresses or is engineered to express the surface receptor recognized by the exogenous ligand.

25. 4. The system, kit or method of any one of claims 1 to 3, wherein the host nanoparticles and / or the foreign nanoparticles are immobilized on a solid support or are for immobilization on a solid support.

26. The system, kit or method of any one of claims 1 to 3, wherein the host nanoparticles and / or the foreign nanoparticles are in suspension.

27. 3. The kit of claim 2, further comprising instructions for use.

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