CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Patent Application No. 63/290,728 filed December 17, 2021, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is filed in electronic format via EFS-Web and is hereby incorporated by reference into the specification in its entirety. The name of the XML file containing the Sequence Listing is 2207620. xml. The size of the XML file is 48,089 bytes, and the XML filed was created on December 15, 2022.

[0003] A challenge in confronting a viral pandemic is the lack of real-time, readily and universally accessible testing for individuals carrying a virus. This technical bottleneck, which is a consequence of the technical complexity of current assays, has limited surveillance, population monitoring, and contact tracing and quarantining of infectious individuals. As more organizations return to active and in-person functions, it is essential that there is a testing platform that can meet the needs of testing and containment everywhere. The challenges of scalable manufacture, instrument placement, and complex reagents and handling need to be solved to provide a detection platform with performance equivalent to lab-based QPCR, that can provide a robust ability to develop testing that can be performed immediately, on-site, by virtually anyone, with limited technical equipment and training.

[0004] There is a need for reagents, kits, and methods that can address limitations inherent in previous testing platforms and provide a robust and fast-operating testing platform that does not require resource-intensive equipment, processing, or technical expertise.

SUMMARY OF THE INVENTION

[0005] Provided herein is a technology for rapid point-of-need (PON) detection of viruses, such as infectious coronavirus, e.g. SARS CoV-2, virions. The described detection reagents are inexpensive and highly and rapidly scalable to manufacture. PON application will not require resource-intensive equipment, processing or expertise. The platform technology can serve as a rapid-prototyping platform for rapid and scalable development of new pathogen assays that are ready to meet the needs for urgent testing of a fast-moving novel viral infection. [0006] In one aspect or embodiment, a method of detecting a microbe in a bodily fluid of a patient is provided, comprising: preparing an agglutination reaction mixture by mixing a sample of a bodily fluid of a patient with a yeast expressing on its surface: a fusion protein comprising an anchor peptide amino acid sequence at an N-terminal or C-terminal portion of the fusion protein for surface display of the fusion protein on the yeast, a VHH amino acid sequence, and a fluorogen activating protein (FAP) amino acid sequence, and a fluorescent dye, wherein the VHH amino acid sequence is a natural or synthetic nanobody amino acid sequence that binds to a surface protein of the microbe, and the FAP is an scFv amino acid sequence that binds the fluorescent dye, such that fluorescence emission intensity from the fluorescent dye increases when bound by the FAP, and the anchor is a yeast cell wall-anchoring peptide selected for surface display of the fusion protein, such as an Aga2, Flol-derived anchors, SED1, SAG1, 649-stalk, CCW12, PIR3, PIR4, Agal peptide; and detecting agglutination of the yeast by either: stratifying, e.g., by gravity settling or centrifugation at from 50 to 500 X g, the reaction mixture in a container holding the reaction mixture and exposing the stratified reaction mixture with light at an excitation wavelength for the FAP-bound fluorescent dye, wherein an agglutinated fluorescent mass indicates presence of the virus in the sample of the bodily fluid of the patient; or filtering the reaction mixture through a filter substrate configured to capture agglutinated yeast and pass non-agglutinated yeast in a filtrate, e.g. ranging from 5 to 30 microns (p) or from 10 to 20 p, such as 15 p; and exposing the filter with light at an excitation wavelength for the FAP-bound fluorescent dye, wherein an agglutinated fluorescent retentate indicates presence of the virus in the sample of the bodily fluid of the patient.

[0007] In another aspect or embodiment, a yeast strain is provided, comprising, an integrated nucleic acid comprising a gene for expressing on its surface a fusion protein comprising an anchor peptide amino acid sequence at an N-terminal or C-terminal portion of the fusion protein for surface display of the fusion protein on the yeast, a VHH amino acid sequence, and a fluorogen activating protein (FAP) amino acid sequence, wherein the VHH amino acid sequence is a natural or synthetic nanobody amino acid sequence that binds to a surface protein of the microbe, and the FAP is an scFv amino acid sequence that binds the fluorescent dye, such that fluorescence emission intensity from the fluorescent dye increases when bound by the FAP, and the anchor is a yeast cell wall-anchoring peptide selected for surface display of the fusion protein, such as an Aga2, Flol-derived anchors, SED1, SAG1, 649-stalk, CCW12, PIR3, PIR4, Agal peptide.

[0008] Non-limiting aspects of the present invention will now be described in the following numbered clauses:

[0009] Clause 1. A method of detecting a microbe in a bodily fluid of a patient, comprising: preparing an agglutination reaction mixture by mixing a sample of a bodily fluid of a patient with a yeast expressing on its surface: a fusion protein comprising an anchor peptide amino acid sequence at an N-terminal or C-terminal portion of the fusion protein for surface display of the fusion protein on the yeast, a VHH amino acid sequence, and a fluorogen activating protein (FAP) amino acid sequence, and a fluorescent dye, wherein the VHH amino acid sequence is a natural or synthetic nanobody amino acid sequence that binds to a surface protein of the microbe, and the FAP is an scFv amino acid sequence that binds the fluorescent dye, such that fluorescence emission intensity from the fluorescent dye increases when bound by the FAP, and the anchor is a yeast cell wall-anchoring peptide selected for surface display of the fusion protein, such as an Aga2, Flol-derived anchors, SED1, SAG1, 649-stalk, CCW12, PIR3, PIR4, Agal peptide; and detecting agglutination of the yeast by either: stratifying, e.g., by gravity settling or centrifugation at from 50 to 500 X g, the reaction mixture in a container holding the reaction mixture and exposing the stratified reaction mixture with light at an excitation wavelength for the FAP-bound fluorescent dye, wherein an agglutinated fluorescent mass indicates presence of the microbe in the sample of the bodily fluid of the patient; or filtering the reaction mixture through a filter substrate configured to capture agglutinated yeast and pass non-agglutinated yeast in a filtrate, e.g. ranging from 5 to 30 microns (p) or from 10 to 20 p, such as 15 p; and exposing the filter with light at an excitation wavelength for the FAP-bound fluorescent dye, wherein an agglutinated fluorescent retentate indicates presence of the microbe in the sample of the bodily fluid of the patient.

[0010] Clause 2. The method of clause 1, the fusion protein having the structure: N-anchor- VHH-FAP-C, N-anchor-FAP-VHH-C, N-Vnu-FAP-anchor-C, or N-FAP-Vnu-anchor-C, wherein N and C refer to the N- and C-terminals of the protein, anchor is the anchor peptide amino acid sequence, VHH is the natural or synthetic nanobody amino acid sequence that binds to a surface protein of the microbe, and FAP is the scFv amino acid sequence that binds the fluorescent dye, such that fluorescence emission intensity from the fluorescent dye increases when bound by the FAP.

[0011] Clause 3. The method of clause 1 or 2, wherein the microbe is a virus.

[0012] Clause 4. The method of clause 3, wherein the virus is a coronavirus, such as

SARS-CoV-2.

[0013] Clause 5. The method of clause 1 or 2, wherein the microbe is a bacteria.

[0014] Clause 6. The method of clause 5, wherein the bacteria is a member of the

Mycobacterium tuberculosis complex, such as Mycobacterium tuberculosis.

[0015] Clause 7. The method of any one of clauses 1-6, wherein the yeast is Saccharomyces cerevisiae.

[0016] Clause 8. The method of any one of clauses 1-7, wherein the dye is a triaryl methine or a cyanine dye.

[0017] Clause 9. The method of clause 8, wherein the dye is malachite green and the FAP is dL5, or the dye is thiazole orange and the FAP is AM2-2.

[0018] Clause 10. The method of clause 1, wherein the VHH binds a surface antigen of a virus.

[0019] Clause 11. The method of any one of clauses 1-9, wherein the VHH is selected from a VHH of SEQ ID NOS: 26-34.

[0020] Clause 12. The method of any one of clauses 1-10, wherein the VHH- binds a SARS- CoV-2 spike protein receptor binding domain (RBD).

[0021] Clause 13. The method of any one of clauses 1-12, wherein detecting agglutination of the yeast is determined by filtering the reaction mixture through a filter substrate configured to capture agglutinated yeast and pass non- agglutinated yeast in a filtrate, e.g. ranging from 5 to 30 microns (p) or from 10 to 20 p, such as 15 p; and exposing the filter with light at an excitation wavelength for the FAP-bound fluorescent dye.

[0022] Clause 14. The method of clause 13, wherein the filter substrate is contained in a syringe filter unit, optionally having a transparent housing, and the reaction is passed through the syringe filter unit by a syringe, and optionally the syringe filter unit and syringe are connected by a luer connection.

[0023] Clause 15. The method of clause 13, wherein the filter substrate is retained within a housing having an opening exposing the filter substrate. [0024] Clause 16. The method of clause 15, wherein the filter substrate is arranged over an absorbent material configured to draw or retain liquid from the filter substrate.

[0025] Clause 17. The method of any one of clauses 1-12, wherein detecting agglutination of the yeast is determined by stratifying, e.g., by gravity settling or centrifugation, the reaction mixture in a container holding the reaction mixture and exposing the stratified reaction mixture with light at an excitation wavelength for the FAP-bound fluorescent dye.

[0026] Clause 18. The method of any one of clauses 1-12, further comprising, while exposing the stratified reaction mixture or filter with light at an excitation wavelength for the FAP-bound fluorescent dye imaging the container holding the reaction mixture or filter to image any agglutinated yeast in the stratified reaction mixture or on the filter.

[0027] Clause 19. The method of any one of clauses 1-18, wherein the sample of a bodily fluid of a patient is saliva or nasopharyngeal mucus (e.g. as obtained from a nasal swab).

[0028] Clause 20. A yeast strain comprising, an integrated nucleic acid comprising a gene for expressing on its surface a fusion protein comprising an anchor peptide amino acid sequence at an N-terminal or C-terminal portion of the fusion protein for surface display of the fusion protein on the yeast, a VHH amino acid sequence, and a fluorogen activating protein (FAP) amino acid sequence, wherein the VHH amino acid sequence is a natural or synthetic nanobody amino acid sequence that binds to a surface protein of the microbe, and the FAP is an scFv amino acid sequence that binds the fluorescent dye, such that fluorescence emission intensity from the fluorescent dye increases when bound by the FAP, and the anchor is a yeast cell wall-anchoring peptide selected for surface display of the fusion protein, such as an Aga2, Flol-derived anchors, SED1, SAG1, 649-stalk, CCW12, PIR3, PIR4, Agal peptide.

[0029] Clause 21. The yeast strain of clause 20, the fusion protein having the structure: N- anchor-VHH-FAP-C, N-anchor-FAP-Vnu-C, N-Vnu-FAP-anchor-C, or N-FAP-Vnu-anchor-C, wherein N and C refer to the N- and C-terminals of the protein, anchor is the anchor peptide amino acid sequence, VHH is the natural or synthetic nanobody amino acid sequence that binds to a surface protein of the microbe, and FAP is the scFv amino acid sequence that binds the fluorescent dye, such that fluorescence emission intensity from the fluorescent dye increases when bound by the FAP.

[0030] Clause 22. The yeast strain of clause 20 or 21, wherein the microbe is a virus.

[0031] Clause 23. The yeast strain of clause 22, wherein the virus is a coronavirus, such as

SARS-CoV-2.

[0032] Clause 24. The yeast strain of clause 20 or 21, wherein the microbe is a bacteria. [0033] Clause 25. The yeast strain of clause 24, wherein the bacteria is a member of the Mycobacterium tuberculosis complex, such as Mycobacterium tuberculosis.

[0034] Clause 26. The yeast strain of clause 20, wherein the yeast is Saccharomyces cerevisiae.

[0035] Clause 27. The yeast strain any one of clauses 20-26, wherein the dye is a triaryl methine or a cyanine dye.

[0036] Clause 28. The yeast strain of clause 27, wherein the dye is malachite green and the FAP is dL5, or the dye is thiazole orange and the FAP is AM2-2.

[0037] Clause 29. The yeast strain of clause 20, wherein the VHH binds a surface antigen of a virus.

[0038] Clause 30. The yeast strain of any one of clause 20-22, wherein the VHH is selected from a VHH of SEQ ID NOS: 26-34.

[0039] Clause 31. The yeast strain of clause 20, wherein the VHH- binds a SARS-CoV-2 spike protein receptor binding domain (RBD).

[0040] Clause 32. A kit comprising a vessel containing a yeast cell as claimed in any one of clauses 20-31.

[0041] Clause 33. The kit of clause 32, wherein the yeast is S. cerevisiae.

[0042] Clause 34. The kit of clause 32 or 33, wherein the yeast is dried.

[0043] Clause 35. The kit of clause 32 or 33, wherein the yeast comprises live stationary phase yeast cells in growth media for the yeast.

[0044] Clause 36. The kit of clause 32 or 33, wherein the yeast is frozen in growth media for the yeast with glycerol, e.g. from 30% to 50% v/v glycerol.

[0045] Clause 37. The kit of any one of clauses 32-36, further comprising a fermentation vessel and growth media for the yeast.

[0046] Clause 38. The kit of any one of clauses 32-37, further comprising a fluorescent dye that is bound by the FAP and which increases fluorescence when bound by the FAP.

[0047] Clause 39. The kit of any one of clauses 32-38, further comprising a filter unit containing a filter substrate with a mesh size ranging from 5 p to 30 p, e.g., 15 p.

[0048] Clause 40. The kit of clause 39, wherein the filter unit is a syringe filter and the kit further comprises a syringe that couples with the syringe filter, such as by a luer lock.

[0049] Clause 41. The kit of clause 39, wherein the filter unit comprises a housing having an opening, a filter substrate retained within the opening of the housing and optionally arranged over an absorbent material configured to draw or receive liquid from the filter substrate. [0050] Clause 42. The kit of clause 39, wherein the filter unit comprises a housing having an opening, a removable filter substrate retained within the opening of the housing, and arranged over a waste vessel configured to allow passage of liquid through the filter substrate under gravity (1 X g).

BRIEF DESCRIPTION OF THE DRAWING(S)

[0051] FIG. 1 provides exemplary amino acid sequences of FAP proteins (Prior Art) (SEQ ID NOS: 1-5), from Ozhalici-Unal, H., et al. (2008) A rainbow of fluoromodules: a promiscuous scFv protein binds to and activates a diverse set of Anorogenic cyanine dyes. J. Am. Chem. Soc. 130, 12620-12621.

[0052] FIG. 2 provides exemplary FAP sequences, identifying the cognate Auorophore (MG or TO1; SEQ ID NOS: 6-15) (Prior Art, International Patent Publication No. WO 2008/092041). Of note, HE1.0.1-TO1 is also referred to as AM2-2 and HE1-TO1 also is referred to as scFvl.

[0053] FIG. 3 provides further examples of FAP sequences that bind cyanine dyes (Prior Art, Zanotti KJ, et al. Blue Auorescent dye -protein complexes based on Anorogenic cyanine dyes and single chain antibody fragments. Org Biomol Chem. 2011 Feb 21;9(4):1012-20, SEQ ID NOS: 16-21).

[0054] FIG. 4 provides further examples of dE5 FAP sequences that bind cyanine dyes (Prior Art, see, e.g., International Patent Publication No. WO 2011/150079 Al, the disclosure of which is incorporated herein by reference in its entirety, SEQ ID NOS: 22-25).

[0055] FIG. 5 depicts schematically the structure of VHH reagents, as compared to conventional antibody structure.

[0056] FIGS. 6A-6E show schematically versions of the pathogen-detection method described herein. FIG. 6A shows a method by which a pathogen-containing sample may be detected by settling, e.g. at 1g in a tube, or > 1g by centrifugation. Depicted is use of a Auorescent indicator that Auoresces upon illumination with light at an excitation wavelength for the Auorescent indicator. FIG. 6B shows a method by which a pathogen-containing sample may be detected by filtration. FIG. 6C is a schematic side view of filters, depicting the filtering process, as shown in FIG. 6B. FIG. 6D is a schematic view of the method depicted in FIGS. 6B and 6C using a syringe and an in-line filter. FIG. 6E is a schematic side view of filters, depicting the filtering process, as shown in FIG. 6B, using an absorbent pad to draw filtrate through the filter. [0057] FIGS. 7 A and 7B show schematically an exemplary device for implementing the methods described herein.

[0058] FIGS. 8A and 8B. Schematic of yeast agglutination-based virion detection. FIG. 8A. Brewers/Baker’s yeast cells expressing a virion-binding molecule and a sensitive reporter agglutinate into larger aggregates when virions are present in a specimen. FIG. 8B. Simple physical separation methods (e.g. filtration or settling) allow isolation of yeast agglutinates and measurement of reporter signal proportional to the quantity of virion in the specimen.

[0059] FIG. 9. Yeast agglutination schemes and pilot experiment with model system. (A) Yeast/virion agglutinate. Agglutination requires a modest excess of virions (estimated -10- fold) to provide sufficient bridging contacts between yeast cells. (B) Binding of SARS Cov-2 virion to yeast cell. A single 100 nm diameter virion bears 25-50 flexible spike proteins that potentially can form multivalent contacts with NBs displayed on a 3000-5000 nm diameter yeast cell. The large number of fluorescent dL5 FAPs on each yeast cell (10 ⁴ -10 ⁵ ) provides efficient signal amplification. C) A yeast/yeast model system demonstrates NB -mediated agglutination.

[0060] FIG. 10 is a graph depicting cell population proportions of the filtrate and retentate after a cell mixture of yeast cells displaying GFP and dL5-NB was filtered through a 5 pm, 10 pm, 15 pm, or 20 pm filter and washed with buffer.

[0061] FIG.ll is a graph depicting cell population proportions of the filtrate and retentate after a cell mixture of yeast cells displaying GFP and dL5-NB was filtered through a 5 pm, 10 pm, 15 pm, or 20 pm filter and washed with buffer.

[0062] FIG. 12 is a graph depicting the percentage of cells that agglutinated as a function of the initial concentration of dL5 to the initial concentration of EGFP.

[0063] FIG. 13 is a graph depicting the percentage of cells that agglutinated as a function of the EGFP yeast cell concentration.

[0064] FIG. 14 is a graph (top) depicting the agglutinate ratio of dL5 cells to EGFP cells as a function of the initial concentration of dL5 to the initial concentration of EGFP with theoretical depictions (bottom) of the agglutinates at a ratio of 100:1 and at a ratio of 1:1.

[0065] FIGS. 15A and 15B show schematically a plasmid vector (FIG. 15A) for introducing an NB-dL5 fusion protein into a yeast genome. Genomic insert portion, excised at BsmBI sites, is shown in FIG. 15B, broken in half at dotted line (pTDH3 AGA2 H11-H4 NB dL5 tADHl KanMX) integrated into yeast Chr III ARS416d locus).

[0066] FIG. 16 provides amino acid sequences of exemplary SARS-CoV-2 anti-spike RBD sybodies (SEQ ID NOS: 26-34). [0067] FIGS. 17A and 17B provide a plasmid map (FIG. 17A) and amino acid sequences (FIG. 17B) of exemplary fusion proteins: (top) yeast- secreted SARS-CoV-2 RBD SAG1 AM2-2 fusion protein (SEQ ID NO: 35), used to construct model virions by attachment to 100 nm beads, and (botom) yeast-displayed H11-H4 NB dL5 fusion protein (SEQ ID NO: 36), comprising an anti-SARS-CoV-2 NB sequence and dL5, an scFv that activates MG. The yeast- displayed Hl 1-H4 NB dL5 fusion protein comprises, in order: AGA2p anchor protein, a linker, H11-H4 NB, a linker, dL5 NP138 scFv (anti-malachite green), and a His tag.

DESCRIPTION OF THE INVENTION

[0068] Other than in the operating examples, or where otherwise indicated, the use of numerical values in the various ranges specified in this application are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word “about”. In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values. Further, as used herein, all numbers expressing dimensions, physical characteristics, processing parameters, quantities of ingredients, reaction conditions, and the like, used in the specification and claims are to be understood as being modified in all instances by the term “about”. Moreover, unless otherwise specified, all ranges disclosed herein are to be understood to encompass the beginning and ending range values and any and all subranges subsumed therein. For example, a stated range of “1 to 10” should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more and ending with a maximum value of 10 or less, e.g., 1 to 3.3, 4.7 to 7.5, 5.5 to 10, and the like.

[0069] As used herein “a” and “an” refer to one or more. The term “comprising” is open- ended and may be synonymous with “including”, “containing”, or “characterized by”. The term “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0070] As used herein, spatial or directional terms, such as “left”, “right”, “inner”, “outer”, “above”, “below”, “over”, “under”, and the like, relate to the invention as it is shown in the drawing figures are provided solely for ease of description and illustration, and do not imply directionality, unless specifically required for operation of the described aspect of the invention. It is to be understood that the invention can assume various alternative orientations and, accordingly, such terms are not to be considered as limiting.

[0071] As used herein, a "patient" or “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a nonprimate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose). As used herein, the terms "treating”, or "treatment" refer to a beneficial or desired result, such as improving one of more functions, or symptoms of a disease.

[0072] Unless stated otherwise, nucleotide sequences are recited herein in a 5’ to 3’ direction, and amino acid sequences are recited herein in an N-terminal to C-terminal direction according to convention.

[0073] The methods, compositions, and kits described herein rely on agglutination of yeast cells by virus particles. Agglutination of S. cerevisiae and other yeasts is a dynamic straindependent natural behavior (Soares, E. (2011), Flocculation in Saccharomyces cerevisiae: a review. Journal of Applied Microbiology, 110: 1-18). Multicellular agglutination (aka flocculation) mediated by yeast surface lectin/yeast surface sugar binding has long been relevant to the brewing of lagers and ales. Many bacterial strains can induce yeast multicelluar agglutination via yeast surface mannose. Highly specific protein/protein interactions can also mediate pairwise two-cell yeast agglutination, as in the case of yeast mating between a and a haploid cells.

[0074] Yeast may also be engineered to express and display surface proteins that specifically-mediate yeast agglutination. A pairwise agglutination screen has been described based on yeast display of heterologous proteins in opposite mating types that mediate mating at a frequency that reflects the strength of the protein/protein interaction (Younger D, et al. High-throughput characterization of protein-protein interactions by reprogramming yeast mating. Proc Natl Acad Sci U S A. 2017 Nov 14; 114(46): 12166-12171. Another screen has been described for the detection of malarial parasite-induced circulating antibodies that mediate multicellular yeast agglutination (Cruz CJG, et al. Malarial Antibody Detection with an Engineered Yeast Agglutination Assay. ACS Synth Biol. 2022 Sep 16; 11 (9):2938-2946). These screens are not designed for whole cell-mediated, or virion-mediated yeast multicellular agglutination and visualization, and differ radically from the methods described herein.

[0075] The methods, reagents and kits described herein may be generally be applied to detecting microbes, such as virus particles (virions), bacteria, and single-cell fungi. The methods may exclude microbes that self-agglutinate or flocculate, e.g. without an agglutinating agent, such as the modified yeast cells described herein. In one example, the microbe is a virus, such as a coronavirus. In another example, the virus is a SARS or MERS virus, such as SARS- CoV2. In another example the microbe is a bacteria cell. In a further example, the microbe is a member of the Mycobacterium tuberculosis complex.

[0076] An antibody is an immunoglobulin molecule produced by B lymphoid cells with a specific amino acid sequence. Antibodies are evoked in humans or other animals, such as camelids (llamas, alpacas, camels, etc.), by a specific antigen (immunogen). Antibodies are characterized by reacting specifically with the antigen in some demonstrable way, antibody and antigen each being defined in terms of the other. “Eliciting an antibody response” refers to the ability of an antigen or other molecule to induce the production of antibodies.

[0077] An immunogen is a compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen traditionally reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens, and in the context of the present disclosure, reacts with naturally-generated VHH peptides (generally, nanobodies), or synthetically modified or mutated VHH peptides (also nanobodies, but also referred to as sybodies when synthetically modified). In the context of the present disclosure an antigen is a surface protein or other surface-expressed antigen by which a microbe can be agglutinated. For example the antigen may be a viral surface protein, and more specifically the antigen may be a receptor-binding ectodomain of a virus, such as a fragment of a coronavirus spike protein, e.g., an ectodomain or fragment thereof or a protein comprising an epitope of a coronavirus protein, such as a spike protein. A microbe is a single-cell organism, such as, for example and without limitation, a virus, bacteria, fungus, or protozoa, that may be pathogenic (disease-causing). A parasite may be, for example and without limitation, single-cell as in the exemplary cases of Trypanosome or Plasmodium, multicellular as in the exemplary cases of hookworm and lice, of fungal as in the exemplary case of ringworm. In one embodiment, the microbe is a virus, such as a coronavirus, for example a SARS-CoV-2 virus particle and the antigen is a coronavirus spike protein ectodomain.

[0078] In one example, the microbe is a member of the Mycobacterium tuberculosis complex. The Mycobacterium tuberculosis complex (MTC or MTBC) is a genetically related group of Mycobacterium species that can cause tuberculosis in humans or other animals. It includes: Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium orygis, Mycobacterium bovis and the Bacillus Calmette-Guerin strain, Mycobacterium microti, Mycobacterium canetti, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium suricattae. and Mycobacterium mungi.

[0079] Many nanobodies (NBs) bind to surface protein epitopes of viruses, for example and without limitation, respiratory syncytial virus F glycoprotein (Moliner-Morro et al., Nanobodies in the limelight: Multifunctional tools in the fight against viruses. J Gen Virol. 2022 May; 103(5) and influenza A H7N9 hemagglutinin (Gaiotto et al., Nanobodies mapped to cross-reactive and divergent epitopes on A(H7N9) influenza hemagglutinin using yeast display. Sci Rep. 2021 Feb 4; 11(1):3126), and surface protein epitopes of bacteria, for example and without limitation, Pseudomonas aeruginosa 7G flagellum proteins, P. aeruginosa 9D flagellum proteins, enterotoxigenic E. coli (ETEC) CfaE adhesion protein (Qin et al., Single Domain Antibody application in bacterial infection diagnosis and neutralization. Front Immunol. 2022 Sep 29;13:1014377)). NB reagents that bind the surface protein epitopes of these classes of pathogens may be candidate effectors for yeast-mediated agglutination.

[0080] As a non-limiting example in relation to NB -binding antigens or targets, coronaviruses (Coronoviridae) are members of the Nidovirales order, and are enveloped, nonsegmented positive-sense RNA viruses. The most prominent feature of coronaviruses is the club-shape spike projections emanating from the surface of the virion. These spikes are a defining feature of the virion and give them the appearance of a solar corona, prompting the name, coronaviruses. Homotrimers of the virus encoded S protein make up the distinctive spike structure on the surface of the virus. The trimeric S glycoprotein is a class I fusion protein and mediates attachment to the host receptor. In most, but not all, coronaviruses, S is cleaved by a host cell furin-like protease into two separate polypeptides noted SI and S2. SI makes up the large receptor-binding domain of the S protein while S2 forms the stalk of the spike. Coronavirus proteins include, for example and without limitation, and among others: spike (SI and S2) proteins, M (membrane) protein, N (nucleocapsid) protein, ORFlab (ORFla and ORFlb) proteins, ORF3 proteins, the sequences of which are broadly published and are freely available (See, e.g., NCBI Reference Sequence: NC_045512.2).

[0081] A “codon-optimized” nucleic acid refers to a nucleic acid sequence that has been altered such that the codons are optimal for expression in a particular system (such as a particular species of group of species). For example, a nucleic acid sequence can be optimized for expression in yeast cells. Codon optimization does not alter the amino acid sequence of the encoded protein.

[0082] A conservative substitution is a substitution of one amino acid residue in a protein sequence for a different amino acid residue having similar biochemical properties. Typically, conservative substitutions have little to no impact on the activity of a resulting polypeptide. For example, a VHH polypeptide sequence may include one or more conservative substitutions (for example 1-10, 2-5, or 10-20, or no more than 2, 5, 10, 20, 30, 40, or 50 substitutions) yet retain its antigen-binding function, namely, in the context of the present disclosure, the ability to bind to a target agglutinating antigen. A polypeptide can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence that encodes that polypeptide using, for example, standard procedures such as site-directed mutagenesis or PCR. Methods are provided herein to ascertain proper expression of any TPD sequence.

[0083] The term “contacting” refers to placement in direct physical association; includes both in solid and liquid form. “Contacting” is often used interchangeably with “exposed.” In some cases, “contacting” includes transfecting, such as transfecting a nucleic acid molecule into a cell. In other examples, “contacting” refers to incubating a molecule (such as an antibody) with a biological sample.

[0084] A fusion protein or fusion polypeptide refers to a protein or polypeptide generated, for example, by expression of a nucleic acid sequence engineered from nucleic acid sequences encoding at least a portion of two different (heterologous) proteins. To create a fusion protein, the nucleic acid sequences are in the same reading frame and contain no internal stop codons. For example, as described in further detail herein, a fusion protein includes a nanobody fused to a FAP.

[0085] An epitope is an antigenic determinant capable of inducing humoral and/or cell- mediated immune response or immunity, that is the portion of an antigen that is recognized by the immune system, and in the case of a protein antigen, comprises a specific amino acid sequence. Epitopes may be identified by any useful epitope mapping method, e.g., as are broadly-known in the arts.

[0086] An “isolated” or “purified” biological component (such as a nucleic acid, peptide, protein, protein complex, or particle) refers to a component that has been substantially separated, produced apart from, or purified away from other components in a preparation or other biological components in the cell of the organism in which the component occurs, that is, other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins that have been “isolated” or “purified”, thus, include, for example and without limitation, nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins. The term “isolated” or “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, an isolated biological component is one in which the biological component is more enriched than the biological component is in its natural environment within a cell, or other production vessel. A preparation may be purified such that the biological component represents at least 50%, such as at least 70%, at least 90%, at least 95%, or greater, of the total biological component content of the preparation.

[0087] A linker refers to a molecule or group of atoms positioned between two moieties (portions of a molecule). Linkers may be bifunctional, e.g., the linker includes a functional group at each end, wherein the functional groups are used to couple the linker to the two moieties. The two functional groups may be the same, e.g., a homobifunctional linker, or different, e.g., a heterobifunctional linker. A peptide linker or spacer may be used to link the C-terminus of a first polypeptide to the N-terminus of a second polypeptide in a fusion protein or peptide. Non-limiting examples of peptide linkers or spacers include glycine- serine peptide linkers. Typically, such linkage is accomplished using molecular biology techniques to genetically manipulate DNA encoding the first polypeptide linked to the second polypeptide by the peptide linker in an open reading frame. Spacers or linkers are common to fusion proteins, and may be inserted between subunits of the fusion protein as described herein. Use and characterization of spacer amino acid sequences is routine, and choice of which is well within the abilities of a person of ordinary skill in the art. For example and without limitation, spacer amino acids may include polar uncharged or charged amino acids. Peptide linkers or spacers may include amino acids, e.g. of from 1 to 50 amino acids in length, corresponding to flanking polypeptides naturally present in a polypeptide included in the described fusion proteins, such as sequences flanking a VHH peptide. Spacers may be of sufficient length and rigidity, e.g. forming an alpha helix, such that functional elements of the fusion protein are sufficiently separated to maintain their desired functionality, such as the FAP, NB, and target antigen portions of the fusion proteins (See, e.g., Chen X, el al. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct;65(10): 1357-69). Spacers may have a length ranging from 1-100 A (Angstroms), or from 1 to 50 amino acids. As above, codon degeneracy and choice of amino acid for spacers may be unique to each clone, such that the DNA sequence acts as a barcode.

[0088] A nucleic acid molecule (a nucleic acid) refers to a polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. The term “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.” The term includes single- and double- stranded forms of DNA. A polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.

[0089] A first nucleic acid is said to be operably linked to a second nucleic acid when the first nucleic acid is placed in a functional relationship with the second nucleic acid. Generally, operably linked DNA sequences are contiguous (e.g., in cis) and, where the sequences act to join two protein coding regions, in the same reading frame (e.g., open reading frame or ORF), for example to produce a fusion protein. Operably linked nucleic acids include a first nucleic acid contiguous with the 5' or 3' end of a second nucleic acid. In other examples, a second nucleic acid is operably linked to a first nucleic acid when it is embedded within the first nucleic acid, for example, where the nucleic acid construct includes (in order) a portion of the first nucleic acid, the second nucleic acid, and the remainder of the first nucleic acid.

[0090] A polypeptide is a polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used. The terms “polypeptide”, “peptide”, or “protein” as used herein are intended to encompass any amino acid sequence and include proteins and modified sequences such as glycoproteins. The term “polypeptide” is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced. The term “residue” or “amino acid residue” includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide.

[0091] Conservative amino acid substitutions are those substitutions that, when made, least or minimally interfere with the properties of the original protein, that is, in the context of the end-use, the structure and function of the protein is conserved and not significantly changed by such substitutions, and may be identified by use of matrices, such as the BLOSUM series of matrices, and other matrices. Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, for example, seryl or threonyl, is substituted for (or by) a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine.

[0092] A promoter is an array of nucleic acid control sequences which direct transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements. A “constitutive promoter” is a promoter that is continuously active and is not subject to regulation by external signals or molecules. In contrast, the activity of an “inducible promoter” is regulated by an external signal or molecule (for example, a transcription factor). Non-limiting examples of yeast promoters, useful in the production of the fusion protein described herein, include: GALI, GAL1/10 (bicistronic), TDH3, CCW12, ENO2, TEF1, TEF2, RPL3, PGK1, HSP26, SSA1, HSP82, as are broadly-known.

[0093] A recombinant nucleic acid refers to a nucleic acid molecule (or protein or virus) that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques which are broadly-known. The term recombinant includes nucleic acids and proteins that have been altered solely by addition, substitution, or deletion of a portion of a natural nucleic acid molecule or protein.

10094] “Sequence identity” refers to the similarity between nucleic acid or amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity may be measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs, orthologs, or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in the art, for example, see: Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237- 44, 1988; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0095] Once aligned, the number of matches may be determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166^-1554*100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer.

[0096] Homologs and variants of a polypeptide are typically characterized by possession of at least about 75%, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity counted over the full length alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.

[0097] For sequence comparison of nucleic acid sequences, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are used. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example and without limitation, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection. One example of a useful algorithm is PILEUP. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360, 1987. Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., Nucl. Acids Res. 12:387-395, 1984).

[0098] Another example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and the BLAST 2.0 algorithm, which are described in Altschul et al., J. Mol. Biol. 215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLASTP program (for amino acid sequences) uses as defaults a word length (W) of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see. Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989). An oligonucleotide is a linear polynucleotide sequence of up to about 100 nucleotide bases in length.

[0099J As used herein, reference to “at least 80% identity” (or similar language) refers to “at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence. As used herein, reference to “at least 90% identity” (or similar language) refers to “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence.

[00100] Complementary refers to the ability of polynucleotides (nucleic acids) to hybridize to one another, forming inter-strand base pairs. Base pairs are formed by hydrogen bonding between nucleotide units in polynucleotide strands that are typically in antiparallel orientation. Complementary polynucleotide strands can base pair (hybridize) in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. In RNA as opposed to DNA, uracil rather than thymine is the base that is complementary to adenosine. Two sequences comprising complementary sequences can hybridize if they form duplexes under specified conditions, such as in water, saline (e.g., normal saline, or 0.9% w/v saline) or phosphate-buffered saline), or under other stringency conditions, such as, for example and without limitation, 0.1X SSC (saline sodium citrate) to 10X SSC, where IX SSC is 0.15M NaCl and 0.015M sodium citrate in water. Hybridization of complementary sequences is dictated, e.g., by the nucleobase content of the strands, the presence of mismatches, the length of complementary sequences, salt concentration, temperature, with the melting temperature (Tm) lowering with shorter complementary sequences, increased mismatches, and increased stringency. Perfectly matched sequences are said to be “fully complementary”, though one sequence (e.g., a target sequence in an mRNA) may be longer than the other.

[00101] A “transformed” cell is a cell into which has been introduced a nucleic acid molecule (such as a heterologous nucleic acid) by any useful molecular biology technique. The term encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including, for example and without limitation, transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, or particle gun acceleration.

[00102] A vector is a nucleic acid molecule allowing insertion of foreign nucleic acid without disrupting the ability of the vector to replicate and/or integrate in a host cell. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. An insertional vector is capable of inserting itself into a host nucleic acid. A vector can also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of inserted gene or genes. Many vectors are available for constructing the nucleic acids described herein for expression of the described fusion protein in yeast cells, such as in S. cerevisiae cells.

[00103] By "expression" or “gene expression,” it is meant the overall flow of information from a gene or functional/structural RNA, and a polyadenylation sequence), to produce a gene product (typically a protein, optionally post-translationally modified or a functional/structural RNA). A “gene” refers to a functional genetic unit for producing a gene product, such as RNA or a protein in a cell, or other expression system encoded on a nucleic acid and comprising: a transcriptional control sequence, such as a promoter and other cis-acting elements, such as transcriptional response elements (TREs) and/or enhancers; an expressed sequence that may encode a protein (referred to as an open-reading frame or ORF), and a poly adenylation sequence. By "expression of genes under transcriptional control of," or alternately "subject to control by," a designated sequence such as TRE or transcription control element, it is meant gene expression from a gene containing the designated sequence operably linked (functionally attached, typically in cis) to the gene. A "gene for expression of" a stated gene product is a gene capable of expressing that stated gene product when placed in a suitable environment— that is, for example, when transformed, transfected, transduced, etc. into a cell, and subjected to suitable conditions for expression. In the case of a constitutive promoter "suitable conditions" means that the gene typically need only be introduced into a host cell. In the case of an inducible promoter, "suitable conditions" means when factors that regulate transcription, such as DNA-binding proteins, are present or absent - for example an amount of the respective inducer is available to the expression system (e.g., cell), or factors causing suppression of a gene are unavailable or displaced - effective to cause expression of the gene.

[00104J A coronavirus polypeptide is a polypeptide encoded by a coronavirus or a portion of a polypeptide encoded by a coronavirus, as in epitopes and immunogenic fragments thereof, and polypeptides with significant sequence identity (e.g., at least 85%, 90%, 95%, 98%, or 99%) with a polypeptide encoded by a natural coronavirus, and retaining or improving upon native function as an immunogen, epitope, or TPD in the case of the present disclosure.

[00105] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It is to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Unless otherwise indicated, polymer molecular weight is expressed as number- average molecular weight (Mri). Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[00106] Nucleic acids and vectors encoding the described fusion proteins may be provided. In some non-limiting examples, disclosed is a recombinant vector, such as a yeast plasmid, that expresses the disclosed fusion proteins. One of skill in the art can readily use the genetic code to construct a variety of functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same protein sequence due to codon degeneracy. In some embodiments, the polynucleotide is codon-optimized for expression in yeast cells.

[00107] Exemplary nucleic acids may be prepared by cloning techniques, e.g., as are broadly-known and implemented either commercially, or in the art. Multiple textbooks and reference manuals describe and provide examples of useful and appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through such techniques are known. Commercial and public product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, Calif.), Addgene, and Applied Biosystems (Foster City, Calif.), as well as many other commercial sources.

[00108 Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill.

[00109] The nucleic acids for expressing the disclosed fusion proteins can include a recombinant DNA which is incorporated into an autonomously replicating plasmid or into the genomic DNA of a yeast cell. The term includes single- and double- stranded forms of DNA.

[00110] Nucleic acid sequences encoding a disclosed fusion protein sequence can be operatively linked to expression control sequences. An expression control sequence operatively linked to a coding sequence is placed in the sequence such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, a start codon (e.g., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.

[00111] Hosts can include yeast cells, such as, for example and without limitation, Pichia pastoris, Saccharomyces cerevisiae, Kluyveromyces lactis, or Hansenula polymorpha cells. Methods for growth, storage, transformation, and propagation of yeast cells are broadly-known. Transformation of a host cell with recombinant DNA can be carried out by conventional techniques as are well known to those skilled in the art. Such methods of transfection of DNA include calcium phosphate co-precipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or viral vectors can be used. One of skill in the art can readily use an expression systems such as plasmids and vectors for use in producing proteins in yeast cells.

[00112] As described herein, “yeast” is a single-celled, eukaryotic microbe that can grow quickly in complex or defined media with doubling times typically around 2.5 h in glucose- containing media. Yeast is easier and less expensive to use for recombinant protein production than insect or mammalian cells. The most commonly employed yeasts in the laboratory are Saccharomyces cerevisiae and certain members of the Pichia genus, such as Pichia pastoris (Komagataella phaffii). While S. cerevisiae may be commonly used, and used in the examples herein, other yeasts may be effectively employed in the systems and methods described herein. Other exemplary yeasts include Kluyveromyces lactis and Hansenula polymorpha.

[00113] Additional amino acid sequences for inclusion in the described fusion proteins, include, for example and without limitation, anchor peptides, linkers, spacers, carriers (see, e.g., US 2020/0031874, incorporated herein by reference), signal peptides, self-cleaving sequences, and affinity tags, may be included in the fusion protein, so long as they do not interfere to any significance with the operation of the fusion proteins as described herein. The fusion peptides described herein are anchored in the cell wall of the yeast. Yeast display technologies are broadly-known, and are useful in expression of the fusion proteins described herein. The fusion proteins comprise a VHH nanobody peptide and an scFv FAP peptide operably linked in frame to a surface display peptide (anchor). Anchoring peptides on yeast cells, such as S. cerevisiae is readily achieved using suitable anchor sequences, and any ancillary sequences specific to a selected anchor sequence. Non-limiting examples of anchor peptides include: a- agglutinin or a-agglutinin, e.g., Aga2, Flo 1 -derived anchors, SED1, SAG1, 649-stalk, CCW 12, PIR3, PIR4, AGA1 (see, e.g., Uchanski T, et al. An improved yeast surface display platform for the screening of nanobody immune libraries. Sci Rep. 2019 Jan 23;9(1):382. doi: 10.1038/s41598-018-37212-3; Teymennet-Ramfrez KV, et al. Yeast Surface Display System: Strategies for Improvement and Biotechnological Applications. Frontiers in Bioengineering and Biotechnology. 2021;9:794742; Zhao H, et al. Interaction of alphaagglutinin and a-agglutinin, Saccharomyces cerevisiae sexual cell adhesion molecules. J Bacterial. 2001 May;183(9):2874-80; and Kajiwara, K., Aoki, W. & Ueda, M. Evaluation of the yeast surface display system for screening of functional nanobodies. AMB Expr 10, 51 (2020). https://doi.org/10.1186/sl3568-020-00983-y).

[00114] The VHH peptide may be located at the N-terminal end of the fusion peptide, thus the fusion peptide may have the overall structure: N- Anchor- VHH-FAP-C, where the dashes can represent a spacer, such as a Serinyl-glycinyl-rich spacer. FIG. 17B provides an exemplary amino acid sequence of a fusion protein useful in the methods described herein for SARS-CoV- 2 detection. The gene for expressing the fusion protein may be synthesized/assembled (cloned) by any useful recombinant DNA methodology, for example and without limitation, in yeast or E. coli. As above, useful cloning methods are broadly-known, though Golden Gate cloning methods may find particular use in preparing and aligning the subunits of the fusion protein. While not every fusion protein, e.g., combination of FAP and NB, is expected to function optimally in the described display and agglutination methods, NBs are readily obtained and cloned, as are FAPs and suitable linker/spacer sequences, and have been proven to function well in fusion proteins, including yeast display systems. The ability of any given fusion protein to agglutinate a microbe is easily tested and optimized. For example, prior to cloning, an FAP/NB fusion protein can be attached to micro-beads, and agglutination can be assessed.

[00115] The gene for expressing the fusion protein may be integrated into the yeast genome. Yeast genomic integration is well-studied and is readily achievable by a person of ordinary skill with respect to yeast display systems. Examples of yeast display technologies include, without limitation, those disclosed in: Amen T, Kaganovich D. Integrative modules for efficient genome engineering in yeast. Microb Cell. 2017 Jun 5;4(6):182-190; Ronda C, et al. CrEdit: CRISPR mediated multi-loci gene integration in Saccharomyces cerevisiae. Microb Cell Fact. 2015 Jul 7; 14:97; Reider Apel A, et al. A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. Nucleic Acids Res. 2017 Jan 9;45(l):496-508; Lee ME, et al. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth Biol. 2015 Sep 18;4(9):975-86; Siddiqui MS, et al. A system for multilocus chromosomal integration and transformation-free selection marker rescue. FEMS Yeast Res. 2014 Dec;14(8):1171-85; Jensen NB, et al. EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae. FEMS Yeast Res. 2014 Mar;14(2):238-48.

[00116] A polymer “comprises” or is “derived from” a stated monomer if that monomer is incorporated into the polymer. Thus, the incorporated monomer (monomer residue) that the polymer comprises is not the same as the monomer prior to incorporation into a polymer, in that at the very least, certain groups/moieties are missing and/or modified when incorporated into the polymer backbone. A polymer is said to comprise a specific type of linkage if that linkage is present in the polymer, such as, without limitation: ester, amide, carbonyl, ether, thioester, thioether, disulfide, sulfonyl, amine, carbonyl, phosphodiester, or carbamate bonds. As such, peptides comprise amino acid residues, and nucleic acids comprise nucleotide residues. [00117] Nanobodies (NBs) have become the predominant small immunoreagent in research, biotechnology, and medicine. Although classical antibodies have been mainline reagents and therapeutics for over 30 years, NBs are (1) smaller and simpler which makes them easy to recombinantly engineer; (2) generally free from autoimmune reactions that compromise natural antibodies; and (3) can be inexpensively and rapidly synthesized without host animals.

[00118] A fluorogen activating protein (FAP) is an activator polypeptide that selectively binds a fluorogen and increases fluorescent emission from the fluorogen. As such, a FAP is a binding partner of its cognate fluorogen (fluorophore), such as a triaryl methine or a monomethine dye, such as a cyanine dye. In the examples below, the fluorophores malachite green (MG, an example of a triaryl methine dye, see, e.g., US Patent No. 9,249,306, incorporated herein by reference) and thiazole orange (e.g., sulfonated TO1, as shown below). The fluorogen may have an excitation wavelength such that when bound by the FAP activator and exposed to light at the excitation wavelength the fluorogen produces a detectably different, and in one instance increased, fluorescent emission as compared to unbound fluorogen exposed to light at the same excitation wavelength. The excitation light can be produced by any lightemitting device, such as a lamp, a light-emitting diode, or a laser, as are broadly known by those of skill in the art. International Patent Publication No. WO 2008/092041, incorporated herein by reference in its entirety, describes and provides amino acid sequences of suitable FAPs for binding MG moieties and TO1 or other monomethine cyanine dye moieties. Also described in WO 2008/092041 are PEG-modified versions of MG and TO1.

[00119] In further detail, Fluorogen activating proteins (FAPs) are a class of fluorescencebased molecular tags that have been used in a variety of trafficking assays due to the rapid noncovalent association and activation of a Anorogenic dye by the expressed protein tag. One FAP can exhibit distinct properties when combined with various dye derivatives. FAPs may be derived from single chain variable fragment antibodies (scFv) that specifically recognize and activate Anorogenic dyes with high binding affinity and provide modularity in targeted labeling without the need for direct conjugation of dyes. Fluorogens useful in combination with FAPs are organic dyes that have low Auorescence signal when free in solution and show significantly enhanced Auorescence output upon binding to the FAP. These dye-FAP Auoromodules show several advantages as Auorescent tags in cell biological studies. FAPs are small expressible protein modules that have typically been cloned as a fusion to proteins of interest. Work has demonstrated the use as recombinant affinity tags for secondary detection of Auorescein (Saunders, M. J., et al. (2014) A bifunctional converter: Auorescein quenching scFv/Auorogen activating protein for photostability and improved signal to noise in Auorescence experiments. Bioconjugate Chem. 25 (8), 1556-1564) or biotin modified proteins (Gallo, E., and Jarvik, J. (2014) Fluorogen-activating scFv biosensors target surface markers on live cells via streptavidin or single-chain avidin. Mol. Biotechnol. 56, 585-590). The fast association and activation of fluorogen/FAP complexes shortens the time for labeling protocols. Since the fluorescence is dependent on the association of flu orogen to the FAP, the fluoromodules allow order-of-addition and compartment selectivity to achieve subpopulation labeling for internalized receptors. A number of modifications to fluoro gens enrich the functional properties of a single FAP for optical labeling, such as membrane permeability/exclusion (Szent-Gyorgyi, C., et al. (2008) Fluorogen-activating single-chain antibodies for imaging cell surface proteins. Nat. Biotechnol. 26, 235-240), fluorescence brightness (Szent-Gyorgyi, C., et al. (2010) Fluorogenic dendrons with multiple donor chromophores as bright genetically targeted and activated probes. J. Am. Chem. Soc. 132, 11103-11109), and environmental sensitivity (Grover, A., et al. (2012) Genetically encoded pH sensor for tracking surface proteins through endocytosis. Angew. Chem., Int. Ed. Engl. 51, 4838-4842 and Szent-Gyorgyi, C., et al. (2010) J. Am. Chem. Soc. 132, 11103-11109). Thus, fusion of a nanobody or target antigen to a FAP should provide a compact probe and tethering mechanism to instantaneously label and bind nanobody paratopes with their cognate epitopes in the screening process described herein.

[00120] Several FAPs have been reported to activate fluorescence of malachite green (MG) and thiazole orange (TO) derivatives, as well as dimethylindole red (DIR), oxazolethiazole- blue (OTB), and various derivatives of these dyes, resulting in a range of fluoromodules with excitation/emission properties at any desired laser wavelength and emission range, typically with affinities for fluorogens in the low nanomolar to picomolar range (Szent-Gyorgyi, C., et al. (2008) Nat. Biotechnol. 26, 235-240; Ozhalici-Unal, H., et al. (2008) A rainbow of fluoromodules: a promiscuous scFv protein binds to and activates a diverse set of fluorogenic cyanine dyes. J. Am. Chem. Soc. 130, 12620-12621; Senutovitch, N., et al. (2012) A variable light domain fluorogen activating protein homodimerizes to activate dimethylindole red. Biochemistry 51, 2471-2485; ZaEbtti, K. J., et al. (2011) Blue fluorescent dye -protein complexes based on fluorogenic cyanine dyes and single chain antibody fragments. Org. Biomol. Chem. 9, 1012-1020; Wu Y, et al. Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair. J Biomol Screen. 2016 Jan;21(l):74-87). [00121] The dL5 FAP (examples provided in FIGS. 2A and 4, all of which are considered to be examples of a dL5 FAP) is relatively small (24.2 kDa), and binds to MG with a low picomolar equilibrium dissociation constant (Szent-Gyorgyi, C., et al. (2013) Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces. J. Mol. Biol. 425, 4595-4613). The extremely low unbound fluorescence background and the high fluorescent signal upon binding allow high contrast and no-wash signal detection (See, e.g., Wang Y, et al. Fluorogen activating protein- affibody probes: modular, no-wash measurement of epidermal growth factor receptors. Bioconjug Chem. 2015 Jan 21;26(l):137-44). FIGS. 1-4 provide non-limiting examples of FAP amino acid sequences that bind triaryl methine dyes, such as malachite green dyes, and monomethine cyanine dyes, such as thiazole orange dyes.

[00122] Heavy-chain only antibodies (HcAbs) are found in camelids, such as llamas and alpacas. Compared to conventional mAbs, HcAbs consist of just two heavy chains, with a single variable domain (VHH, ~15kDa) as the antigen-binding region. These nanoscale VHHS also may be referred to as “nanobodies” (NBs), retaining full antigen-binding potential upon isolation, establishing them as the smallest, naturally-derived antigen-binding fragment. Nanobodies have spurred the development of commercial companies and have been used in applications such as biosensing, affinity-capture, and protein crystallization; however, their most significant potential lies in therapeutics, e.g., for cancer (See, e.g., Mitchell, LS, et al. Proteins. 2018; 86: 697- 706 and Yang EY, Shah K. Nanobodies: Next Generation of Cancer Diagnostics and Therapeutics. Front Oncol. 2020 Jul 23;10:1182. doi: 10.3389/fonc.2020.01182).

[00123] Unlike other antibody fragments, nanobodies do not require extensive assembly or molecular optimization to create complex constructs and can be readily incorporated as functional subunits of a wide array of nanobody-fusion molecules, including, without limitation: bivalent, biparatopic, bispecific, NB-scFv, NB-cytokine, NB-fluorophore, and NB- drug fusion proteins, as well as NB -nanoparticle and NB-virus complexes. In general, NB antigen specificity is determined at the exposed ends of each variable domain through three peptide loops, or complementarity determining regions (CDRs) (See, FIG. 5). The CDR3 loop provides a significant contribution to an antibody's specificity and diversity, and on average, nanobodies have a much greater CDR3 length compared to that of human VH domains, which strengthens their interactions with target antigens (Desmyter A, et al. Antigen specificity and high affinity binding provided by one single loop of a camel single-domain antibody. J Biol Chem. 2001 Jul 13;276(28):26285-90. doi: 10.1074/jbc.M 102107200). The NB CDR3 regions can form finger-like projections that enable high-affinity binding to traditionally inaccessible cavity-like epitopes (De Genst E, et al. Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. Proc Natl Acad Sci USA. (2006) 103:4586-91). The CDR1 and CDR2 regions also aid in antigen binding, which enables greater paratope diversity than that of mAbs (Mitchell LS, et al. Protein Eng Des Sei. 2018 Jul 1 ;3 l(7-8):267-275).

[00124] Development and production of nanobodies may be performed commercially, such as by Abcore, Inc. of Ramona, CA and the VIB Nanobody Core at Vrije Universiteit Brussel, Belgium, among others, and numerous patents are directed to nanobody technology, or immunoglobulin single variable domain proteins, for example as illustrated in International Patent Application Publication No. WO 2012/175741 Al, the disclosure of which is incorporated herein by reference. Commercial providers provide a range of products ranging from camelid (e.g., llama) blood, PBMCs, RNA or cDNA, a library of nanobody clones, as well as screened clones and large-scale production of positive clones.

[00125] Cloning of nanobodies, and modification of nanobodies to improve affinity are broadly-described (See, for example and without limitation: Itoh K, Sokol SY. Expression cloning of camelid nanobodies specific for Xenopus embryonic antigens. PLoS One. 2014 Oct 6;9(10):el07521; Pardon E, et al., A general protocol for the generation of Nanobodies for structural biology. Nat Protoc. 2014 Mar;9(3):674-93; Muyldermans S. A guide to: generation and design of nanobodies. FEBS J. 2021 Apr;288(7):2084-2102; Vincke C, et al. Generation of single domain antibody fragments derived from camelids and generation of manifold constructs. Methods Mol Biol. 2012;907:145-76; Uchanski, T., et al. An improved yeast surface display platform for the screening of nanobody immune libraries. Sci Rep 9, 382 (2019); Guttler T, et al. Neutralization of SARS-CoV-2 by highly potent, hyperthermostable, and mutation- tolerant nanobodies. EMBO J. 2021 Oct l;40(19):el07985; and Sulea T, et al., Application of Assisted Design of Antibody and Protein Therapeutics (ADAPT) improves efficacy of a Clostridium difficile toxin A single-domain antibody. Sci Rep. 2018 Feb 2;8( 1 ):2260).

[00126] In the context of the present disclosure, the respective sequences of the VHH nanobodies is secondary to the point that, as would be recognized by a person of ordinary skill in the art, VHH sequences and structures are very well-characterized and can be readily inserted into a yeast expression cassette, and in-frame with an FAP sequence in the fusion proteins described herein to achieve the desired antigen recognition for purposes herein. Further, many VHH antibodies to SARS-CoV-2 stalk RBD have been produced and characterized, with their amino acid sequences being made publicly-available. As described herein, such that a person of ordinary skill can readily obtain and insert a suitable Vnu-encoding sequence into a suitable yeast expression vector, for example as described herein. Though the framework region sequences, e.g., FR1, FR2, FR3, and FR4, are conserved, there still remains a degree of variability in those regions not only inter- species, but within species. As an example, the following is an example of a VHH framework consensus sequence obtained from 21 alpaca and llama VHH germline peptides, which show at least -90% sequence identity with exemplary consensus sequence:

QVQLVESGGGLVQAGGSLRLSCAAS(X) _m RQAPGKERE(X)„YADSVKGRFTISRDNAK NTVYLQMNSLKPEDTAVYYCA(X)pYDYWGQGTQVTVS (SEQ ID NOS: 37-40), where n is 12, m is 12 or 13, and p ranges from 6 to 26, inclusive, and (X) _m , (X) _n , and (X) correspond to CRD1, CDR2, and CDR3, respectively of the camelid VHH.

[00127] NB framework regions may be synthetically-modified to change one or more amino acid residues to improve any quality of the NB, and efficacy of those modified NBs are readily screened in the assay described herein for binding to a virus particle and for the ability to agglutinate yeast cells. Reference to Mitchell, LS, et al. Proteins. 2018; 86: 697- 706 and Mitchell LS, et al. Protein Eng Des Sei. 2018 Jul l;31(7-8):267-275, as well as the multitude of additional references and sequences of NBs/camelid VHH regions/sybodies that are publicly- available, can guide a person of ordinary skill in the selection and cloning of NB -encoding nucleic acids.

[00128] A full description of molecular cloning methods useful for production of proteins, including nanobodies, in yeast, including plasmids, clone selection, evaluation and propagation, and mutagenesis is not necessary as those methods are routine and well within the abilities of a person of ordinary skill in the art. Further, cloning services are broadly-available both through research institutions and businesses. As an example, Golden Gate cloning: Golden Gate cloning technology relies on Type IIS restriction enzymes, first discovered in 1996. Type IIS restriction enzymes are unique from "traditional" restriction enzymes in that they cleave outside of their recognition sequence, creating four base flanking overhangs. Since these overhangs are not part of the recognition sequence, they can be customized to direct assembly of DNA fragments. When designed correctly, the recognition sites do not appear in the final construct, allowing for precise, scarless cloning. The cloning scheme is as follows: the gene of interest is designed with Type IIS sites (such as Bsal or BbsI), that are located on the outside of the cleavage site. As a result, these sites are eliminated by digestion/ligation and do not appear in the final construct. The destination vector contains sites with complementary overhangs that direct assembly of the final ligation product (See, e.g., Marillonnet, S., & Griitzner, R. (2020). Synthetic DNA assembly using golden gate cloning and the hierarchical modular cloning pipeline. Current Protocols in Molecular Biology, 13Q,el l5, also, see, Otto M, et al. Expansion of the Yeast Modular Cloning Toolkit for CRISPR-Based Applications, Genomic Integrations and Combinatorial Libraries. ACS Synth Biol. 2021 Dec 17; 10(12):3461- 3474).

[00129] Provided herein is a nucleic acid, and a yeast cell comprising the nucleic acid. The yeast cell may be an S. cerevisiae cell. The nucleic acid comprises a gene for expressing a fusion protein. The gene comprises a promoter, such as a constitutive or inducible yeast promoter for controlling production of an mRNA encoding the fusion protein, and ORF encoding the fusion protein, and a suitable termination sequence. The fusion protein comprises, linked together: a VHH (NB) peptide; an FAP peptide, and an anchor peptide. The VHH peptide is a nanobody peptide that binds a viral antigen, such as a viral surface protein. The viral surface protein may be a surface protein, and typically an ectodomain thereof. The antigen may be a coronavirus, e.g., SARS or MERS, spike protein ectodomain. The antigen may be a SARS-CoV-2 spike protein ectodomain, a number of which have been developed by various research facilities and commercial entities (see, e.g., Koenig PA, et al. Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape. Science. 2021 Feb 12;371(6530):eabe6230. doi: 10.1126/science.abe6230; Justin D. Walter, et al. bioRxiv 2020.04.16.045419; and Sun X, et al. Nanobody-Functionalized Cellulose for Capturing SARS-CoV-2. Appl Environ Microbiol. 2022 Mar 8;88(5):e0230321, also see International Patent Application No. PCT/IB2022/062388 describing a screening method for screening, identifying, and quantifying the affinity of antigen-binding VHH peptides).

[00130] Nanobodies targeting the SARS-CoV-2 spike protein RBD (receptor binding domain) are expected to be useful in the agglutination methods described herein. Plasmids encoding useful nanobodies are commercially available, e.g. from Addgene (Watertown, Massachusetts), including plasmids encoding sybody (SB) Nos. 14, 15, 16, 42, 45, and 68 (SEQ ID NOS: 29-34), the amino acid sequences of which are provided in Walter et al. (Justin D. Walter, et al. bioRxiv 2020.04.16.045419, providing amino acid sequences of 63 unique SARS-CoV-2 spike RBD-binding sybodys), and which can be subcloned into a fusion protein with an FAP by any useful method, such as, for example and without limitation, by PCR using primers containing a Type IIS restriction endonuclease recognition site, for seamless cloning using Golden Gate cloning methods.

[00131] Non-limiting embodiments of the agglutination assay described herein are depicted in FIGS. 6A-6C. In one example, depicted in FIG. 6A, yeast comprising a gene encoding a surface-display NB-FAP fusion protein are either deposited into, or provided in a suitable vessel, such as a tube, well, or any other suitable container. A dye binding partner with the FAP also is present in, or added into the reaction mixture. A liquid sample is also deposited into the same vessel (FIG. 6A, left) and the reaction mixture is mixed in any suitable manner, such as by inversion, shaking, vortexing, etc. (FIG. 6A, middle). The liquid sample may be a bodily fluid, such as, without limitation: saliva, urine, mucus, blood, serum, plasma. The liquid sample may be diluted as needed. Once mixed, the sample (FIG. 6A, right) is allowed to settle. The settling may be either achieved by holding the tube motionless for a time adequate for any agglutinated material to settle by gravity (1 X g, or 1g, where g is the earth’s gravitational force) at the bottom of the vessel, or by centrifugation to hasten settling at >lg. If centrifuged the centrifugation should be at a g-force able to pellet or layer the agglutinated material separate from the non-agglutinated yeast cells. Rheology modifiers, such as glycerol, polysaccharides, mono- or disaccharides, non-ionic detergents, block polymer detergents, mono- or divalent cations, chelating agents, and buffering agents, may be included in the reaction mixture to modify pelleting or to create a layering effect.

[00132] FIG 6B - 6D depict a second screening method in which, referring to FIG. 6B, yeast comprising a gene encoding a surface-display NB-FAP fusion protein are either deposited into, or provided in a suitable vessel, such as a tube, well, or any other suitable container. A dye binding partner of the FAP, such as malachite green if the FAP is dL5 or TO1 if the FAP is AM2-2, also is present in, or added into the reaction mixture. A liquid sample is also deposited into the same vessel (FIG. 6B, left) and the reaction mixture is mixed in any suitable manner, such as by inversion, shaking, vortexing, etc. (FIG. 6B, middle) for a period of time sufficient to promote agglutination, such as by vortexing at low to moderate speed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, including any increment therebetween, for example for 30 minutes. The liquid sample may be a bodily fluid, such as, without limitation: saliva, urine, mucus, blood, serum, plasma. The liquid sample may be diluted as needed. The mix 10 is then transferred to, and passed through a filter (FIG. 6B, right), with filtrate 12 passing through the filter 14, and retentate 16, if present, retained on the filter (Positive). In one example, the mix 10 is placed on the filter 14 and is allowed to percolate through the filter 14 at IXg, to be followed by washes that also percolate through the filter at 1 X g. The filter then may be examined, e.g. imaged or scanned, visualization and quantification. Alternatively, the mix 10 may be forced through the filter, e.g., using a syringe as described below. The filter 14 may be a 10 micron (p, mesh size) filter, or a filter ranging from 3 p to 30 p, including any increment therebetween. For FIGS. 6B-6D, filter size is selected to retain yeast cells agglutinated in the presence of virus, yet to pass in the filtrate 12 non-agglutinated yeast cells. The retentate 16 is an agglutinated mass of yeast cells and virus, as described herein. The filtering of the mix 10 shown in FIG. 6B is further depicted in FIG. 6C, depicting the filter 14, filtrate 12, and retentate 16, with exposure of the filter 14 to light 17 at an excitatory wavelength for the dye binding partner of the FAP bound to the FAP, and depicting fluorescence 18 of the retentate 16 in the positive sample, and no retentate or fluorescence on the negative sample when exposed to light at an excitatory wavelength for the dye binding partner of the FAP bound to the FAP.

[00133] FIG. 6D depicts an example of the method shown in FIGS. 6B and 6C. A sample 10 of a patient’s bodily fluid is provided, such as saliva or a nasal swab mixed in water, saline, PBS, or a suitable aqueous solution. The sample 11 is drawn into a (medical) syringe 20 by pulling the plunger 22 of the syringe. The syringe 22 can contain the yeast and dye as in FIGS. 6A-6C, such that drawing the sample 11 into the syringe mixes the sample, yeast, and dye, or those ingredients may be mixed with the sample 11 prior to being drawn into the syringe. The syringe 20 containing mix 10 is attached to a disposable filter unit 24 containing the filter 14. The filter unit 24 is optionally transparent, allowing excitation of any dye in retentate retained by the filter 14 in retentate, and viewing of any fluorescence retained on the filter 14. The filter unit 24 may be opened to reveal the filter after use, to visualize the retentate, or the filter 24 may be otherwise removable from the filter unit 24. Once the syringe 20 is attached to the filter unit 24, the plunger 22 is pressed to force the mix 10 through the filter, trapping any agglutinated material on the filter.

[00134] FIG. 6E depicts an example of the method shown in FIGS. 6B and 6C. Referring to FIG. 6B, in one example, the mix 10 is placed on the filter 14 and is allowed to percolate through the filter 14 at 1 X g, to be followed by washes that also percolate through the filter at 1 X g. In such a case, the filter 14 may be of sufficient size, e.g. thickness, to absorb the filtrate 12 and any wash solution. Referring to FIG. 6E, where the filter is of insufficient size to retain all liquid, a mix 110 essentially as described in relation to FIGS. 6A-6C is deposited on filter 114. Rather than being forced through the filter 114 as depicted in the embodiment shown in FIG. 6D, the filtrate is drawn through the filter by absorbent pad 112. The absorbent pad can be any suitable absorbent material selected to, and configured to draw or retain liquid from the filter 114, such as a cellulosic material, cotton, gauze, a sponge, dried silica, dried acrylamide material, or any other suitable absorbing or wicking material, including any suitable combination of materials. Alternatively, a vessel or container may be configured under the filter 114 to receive waste filtrate. Exposure of the filter 114 to light 117 at an excitatory wavelength for the dye binding partner of the FAP bound to the FAP, and depicting fluorescence 118 of the retentate 116 in the positive sample, and no retentate or fluorescence on the negative sample when exposed to light at an excitatory wavelength for the dye binding partner of the FAP bound to the FAP. In this example, and in other examples shown in FIGS. 6A-6E, the filter may be washed with a suitable aqueous wash solution such as water, saline, PBS or any other solution that does not interfere with the agglutination. The wash step may reduce background fluorescence by washing away remaining non-agglutinated yeast and dye from any retentate.

[00135] Referring to FIGS. 7A and 7B, the filter 214 and absorbent pad 212 essentially as described in reference to FIG. 6E is retained in device 205. If the filter is of sufficient size to retain any filtrate and wash, the absorbent pad may be omitted from the device 205. Alternatively, a vessel or container may be configured under the filter 214 to receive waste filtrate. As shown, device 205 comprises a housing 230 that facilitates handling and use of the filter 214 and absorbent pad 212. In use, mix (e.g. 10 and 110, above) is deposited on the filter 214 through the hole 232 in the housing 230, and filtrate is absorbed into the absorbent pad 212. Any agglutinated retentate remains on the filter 214. The sample may be washed with as suitable wash solution to further reduce background fluorescence. An image of the filter may be taken with a smartphone or other suitable imaging device (see, Banik, S., et al. Recent trends in smartphone-based detection for biomedical applications: a review. Anal Bioanal Chem 413, 2389-2406 (2021)) with detection limits ranging down to as little as 10 fluorescent molecules (Vietz C, et al. Benchmarking Smartphone Fluorescence-Based Microscopy with DNA Origami Nanobeads: Reducing the Gap toward Single-Molecule Sensitivity. ACS Omega. 2019 Jan 31;4(l):637-642). As such, an illumination source, such as a small LED light with appropriate filters, can be provided to illuminate any retentate at an excitation frequency for the fluorogen, and the filter 214 and any retentate can be imaged using a simple camera, such as a cellphone camera.

[00136] For all reactions, the yeast cells may be live so that the fusion protein is expressed and prevalent on the surfaces of the yeast cells. Alternatively, the yeast cells may be preserved, or cryopreserved in a state in which the fusion protein is prevalent on the surface of the cells. Because the methods and cells are intended to be used in a simple and user-friendly manner, it may be preferable to provide the live cells grown immediately before use.

[00137] FIGS. 7A-7D show schematically an exemplary device 205 for implementing the methods described herein. The combination of the filter 214 and absorbent pad 212, as show in FIGS. 6D and 6E is retained in housing 230 with an opening 232 exposing the filter 214. Housing 230 can be a box, solid structure, or any structure configured to house the filter 214 and absorbent pad 212. Filter 214 is optionally covered by a foil, paper, and/or plastic cover (not shown) to preserve the filter 214 and absorbent pad 212 during storage and handling prior to use. The housing 230 may be two cardboard members glued or otherwise affixed at the edges, or may be a box. The absorbent pad 212 and/or filter 214 may be affixed to the housing using any suitable adhesive. Although housing 230 is depicted as a rectangular box, it may have any size, shape, or topology so long as the filter 214 is exposed for observation.

[00138] A kit also is provided comprising at a minimum, the yeast encoding the fusion protein as described herein, in suitable packaging for storage, transport, and handling. The yeast may be provided in a vessel, and the yeast may be in a dried form. Methods of drying yeast are broadly-known in the baking and brewing arts. For example yeast may be dried by pressing the yeast, extruding the pressed yeast, and then by drying the extruded yeast under low humidity, e.g., in a fluidized bed, e.g. to about 8% water content. The kit may include a container including dried or liquid medium for growth of the yeast prior to use. Alternatively, the yeast may be suspended in a liquid reaction mixture and preserved in a suitable preservative such as glycerol, and may be stored refrigerated or frozen prior to use. A suitable dye that binds to the FAP of the fusion protein may be included in the kit, e.g. in a reaction mix or separately. The kit also may include a syringe and syringe filter for example as described in reference to FIG. 6D, or a device with a filter and absorbent pad, for example as described in reference to FIGS. 7A and 7B. The kit may also include an LED light, such as a small, battery- powered penlight or flashlight, providing a light source for illuminating the filter at an excitatory wavelength for the dye. The elements of a kit may be included in one or more cartridges and automated systems for handling the sample, growing the yeast, preparing the reaction mixture, filtering or settling the reaction mixture, illuminating the filter or any settled materials, and/or imaging or otherwise scanning the filter or vessel in which any agglutinated material may be settled.

Examples

[00139] Rather than assay for viral RNA using qPCR or test for viral protein using antibodies, COVID-19 infectiousness can be tested by detecting intact, but inactivated virions in saliva. The technology is based on virion-mediated agglutination of specially created yeast cells that can surface display 10 ⁴ -10 ⁵ copies of a virus-binding protein and carry a readily detectable reporter protein that facilitates low-technology demand quantitation of virion- mediated signals in patient-derived specimens. By separating and quantifying the agglutinated yeast clusters from unicellular yeast based on settling or filtration, the degree of agglutination and the size of individual aggultinates can be assessed to provide quantitative properties correlated to the abundance of virion in the specimen. The reporter in the yeast provides a sensitive signal proportional to the number of agglutinated cells retained on a filter or settled to the bottom of a tube (FIGS. 8A and 8B).

Example 1 - Yeast Agglutination

[00140] A 1:1 cell mixture respectively displaying enhanced green fluorescent protein (EGFP) or a cognate dL5-anti-GFP nanobody (dL5-NB) (red channel) formed agglutinates that were quantitatively assayed on a flow cytometer. EGFP is expressed on centromere plasmids such that approximately 40% of cells suffer plasmid loss and do not surface display; dL5-NB is expressed from a chromosomal integrant such that all cells surface display. About 85% of the cells that display are incorporated into agglutinates of widely varying size (mean cell number 12-13) with roughly a 6:7 EGFP:dL5-NB stoichiometry (see, FIG. 9). Control experiments using EGFP-displaying cells and dL5-displaying cells show only a low background of red/green dimers. Thus, agglutination is largely mediated by NB/EGFP binding.

Example 2 - Filtration Optimization and Agglutination Enrichment

[00141] Cell mixtures from Example 1 were poured over a 5 pm, 10 pm, 15 pm, or 20 pm filter (Nylon mesh pluriStrainer) and vacuum filtered. A cell mixture was also prepared and was not filtered. The filters were washed IX with buffer (PBS, 0.1% Triton X-100), where the buffer was applied onto the surface of the filters using a syringe. The filtrate, i.e. the yeast cells, growth media, and buffer that passed through the filter, and the retentate, i.e. the yeast cells that were retained on the filter, were quantitatively assayed on a flow cytometer. The resulting cell population proportions of the filtrate and retentate can be found in FIG. 10. There was an increased proportion of red fluorescent agglutinated cells, as compared to unicellular non-fluorescent yeast cells, in the retentate. The agglutinates were effectively isolated using a 15 pm and a 20 pm filter.

[00142] Cell mixtures from Example 1 were poured over a 5 pm, 10 pm, 15 pm, or 20 pm filter (nylon mesh) and were vacuum filtered. A cell mixture was poured over a 15 pm filter and was gravity filtered. A cell mixture was also prepared and was not filtered. The filters were washed IX with buffer (PBS, 0.1% Triton X-100). The filtrate, i.e. the yeast cells, growth media, and buffer that passed through the filter, and the retentate, i.e. the yeast cells that were retained on the filter, were quantitatively assayed on a flow cytometer. The resulting cell population proportions of the filtrate and retentate can be found in FIG. 11. There was an increased proportion of red fluorescent agglutinated cells, as compared to unicellular non- fluorescent yeast cells, in the retentate. The agglutinates were effectively isolated using a 15 pm and a 20 |im filter. A higher agglutinate population was obtained for agglutinates that were washed with buffer without the use of a syringe, as the agglutinates were broken apart by the pressure of the syringe.

Example 3 - Dose Response

[00143] The following cell mixtures were prepared, where the ratio is the initial concentration of dL5 to the initial concentration of EGFP: (1) a 100:1 cell mixture; (2) a 50:1 cell mixture; (3) a 20:1 cell mixture; (4) a 10:1 cell mixture; a 5:1 cell mixture; (5) a 2:1 cell mixture; and (6) a 1:1 cell mixture. The control was a cell mixture having no EGFP. The formed agglutinates were quantitatively assayed on a flow cytometer.

[00144] The resulting percentage of cells that agglutinated for each cell mixture can be found in FIG. 12. The percentage of cells that agglutinated as a function of EGFP yeast concentration (10 ⁶ cells per milliliter (cells/mL)) can be found in FIG. 13. The agglutination ratio of dL5 cells to EGFP cells as a function of the initial concentration of dL5 cells to the initial concentration of EGFP cells can be found in FIG. 14 (top). The agglutinate composition changed with concentration, as depicted in FIG.14 (bottom).

Example 4 - Development of Rapid Point-of-Need Detection of SARS-CoV-2 Virions

[00145] Coronavirus Disease 2019 (COVID-19) infectiousness will be detected using intact, but inactivated SARS-CoV-2 virions in saliva. The detection is based on virion-mediated agglutination of specially created yeast cells. The yeast cells will surface display 10 ⁴ -10 ⁵ copies of a virion-binding protein and will carry a readily detectable reporter protein that can facilitate the quantitation of virion-mediated signals in patient-derived specimens. The detectable reporter protein in the yeast cells will provide a sensitive signal that is proportional to the number of agglutinated cells retained on a filter or settled to the bottom of a tube. By separating the agglutinated yeast clusters from unicellular yeast (by settling or filtration) and quantifying, the degree of agglutination and the size of individual agglutinates can be assessed in order to provide quantitative properties that correlate to the abundance of virion in the patient-derived specimen.

[00146] Brewer’s (aka baker’s) yeast Saccharomyces cerevisiae can be used as an engineered agglutinating scaffold to surface display proteins that specifically bind to virion spike proteins, and also express a high level of reporter protein. The expression of a high level of reporter protein will improve signal amplification and sensitivity when coupled with a low technology-demand digital photographic analysis technique. The proteins that bind to virion spike proteins may be fragments of the native target. For example, the fragment of the native target may be angiotensin-converting enzyme 2 (ACE2), without the native enzyme activity, or peptide mimics. Alternatively, the protein may be an antibody-like molecule, such as a nanobody, that binds to the receptor binding domain (RBD).

[00147] Brewer’s Yeast is a Generally Regarded as Safe (GRAS) organism that can be grown at large scale in relatively simple fermenters (e.g., brewing equipment, yogurt equipment, or bread making equipment). The yeast cells will be developed as chromosomal integrants of the required constructs to ensure that the proteins for binding virion and detecting yeast cells are uniformly displayed on all cells that are grown from the starting culture. Approximately 1.0 milliliter (mL) of growth culture will suffice to produce enough yeast reporter cells (approximately 10 ⁸ ) for 10 to 100 tests and 1 liter (L) of growth culture will suffice to produce enough yeast reporter cells (approximately 10 ¹¹ ) for approximately 10,000- 100,000 test aliquots daily. The harvested reporter yeast cells may be stored and distributed as live cells in frozen glycerol slurries, in partially dried formats, or in frozen “single test” aliquots, to be combined with a patient-derived specimen, processed, and then read.

[00148] Provided herein are Saccharomyces cerevisiae yeast strains that are suitable for agglutination in the presence of target virions, and that carry reporters to facilitate direct quantification will be established. The influence of the affinity of the virion-binding protein, the dose-response of the agglutination of the yeast strains in the presence of multivalent particles, and the rate of agglutination after the addition of a specimen materials will be evaluated using flow cytometry, light scatter, and/or cell counting.

[00149] Fluorogenic dyes and activating binder proteins, such as Fluorogen Activating Proteins (FAPs will also be evaluated. These fluorogenic signal-generating systems will be evaluated for sensitivity and limit of linearity for the detection of yeast cells on filter devices that retain agglutinates, and for agglutinates that form with reference nanoparticle or microparticle agglutinators. Stains for yeast-contained nucleic acid content or other substrates that can be activated by native yeast enzymes will also be evaluated for detection.

[00150] Simple standardization materials will be developed, which can be distributed and used as reference specimens. These reference specimens may be based on nanoparticles or microparticles that mimic the size and/or valence of natural virions and that bind irreversibly to recombinant protein antigens via polyhistidine or biotin-modified fusion tags. For example, the reference nanoparticles or microparticles may have a density and size that is similar to intact SARS-CoV-2 virion (100 nanometers).

[00151] Methods to: (1) separate and quantify the agglutinated yeast from non- agglutinated yeast (unicellular yeast); and (2) use imaging analysis or particle analysis of the agglutinates to identify properties of agglutinates that correspond to viral load in the sample will be established. These agglutinate properties may include, but are not limited to agglutinate number, individual agglutinate size and/or density, agglutinate shape, or combinations thereof, as evaluated using flow cytometry, light scatter, and/or cell counting.

[00152] The developed and tested assay yeast will be used to evaluate a panel of previously collected and quantified patient-derived specimens. This validation will provide a benchmark for the bioanalytical performance of the test metrics and sample scoring metrics against current testing methods, such as quantitative PCR (QPCR), antigen, and ID-Now tests. Receiver- Operating-Characteristic curves will be evaluated for correspondence to QPCR results based on various simple quantitation approaches to determine the potential assay performance relative to current tests for SARS CoV-2.

[00153] Storage, shipping, and reviving of the live yeast strains for “on-site” generation and use will be evaluated. For example, kitchen equipment will be modified in order to determine if the growth of the yeast in home-scale fermentation equipment is both practical and contamination-free. In addition, approaches to kill the yeast, in a manner that does not disrupt the structure, abundance, or function of the genetically encoded, expressed binding protein and reporter proteins of the yeast or otherwise compromise assay quality, will be evaluated.

Example 5 - Yeast Agglutination Assay

[00154] A 2-color yeast agglutination assay may comprise the following steps:

1. Collect 5 mL of patient saliva in a 15 mL graduated disposable test tube.

2. Add 4 mL of an assay solution. For example, the assay solution may include 100 millimolar (mM) of pH 6 buffer; 1% Triton X-100 (to disperse mucus, lyse cells, and reduce non-specific binding); 2% Poly(ethylene glycol) (PEG) 8000 (to accelerate agglutination kinetics); and 100 nanomolar (nM) malachite green (MG-2p) to induce fluorescence. 0.3% of tri-(N-butyl)-phosphate (TNBP) may be used in combination with the Triton to inactivate the virus. The Triton/TNBP system is World Health Organization (WHO) validated for inactivation of enveloped virions but may need to be adjusted specifically for the yeast assay.

3. Cap the test tube and manually shake the tube. Wait 10 minutes before proceeding to the next step.

4. Add a pre-packaged yeast reporter slurry from a 1 mL cryotube (containing approximately 10 ⁷ cells) into the tube.

5. Cap the test tube and manually shake the tube. Wait 10 minutes before proceeding to the next step.

6. Screw a disposable 10 micron (pm) filter with a syringe adapter onto the test tube (e.g., pluriStrainer by pluriSelect Life Science, Leipzig, Germany).

7. Use syringe to manually draw the solution through filter until damp dry.

8. Place filter into a fluorescence imaging adapter for a cellular telephone. (Banik, S., el al. Recent trends in smartphone-based detection for biomedical applications: a review. Anal Bioanal Chem 413, 2389-2406 (2021)). The imagining adapter may include a small LED that excites at about 636 nm and a 650 nm bandpass emission filter at the cellular telephone.

9. Take photos at several exposures.

10. Compare the collected photos with the photo of the dummy filter having known fluorophore standards.

11. Use a phone application to estimate the virion number.

Example 6 - alternate validation methods using AM2.2-RBD fusion proteins on target yeast or virion-size beads.

[00155] The “probe” yeast cells may display an anti-spike dL5-NB fusion (red FAP) and the “target” yeast cells may display an AM2.2-RBD fusion protein (green FAP), which is analogous to the system described in Example 1. This system will enable the use of flow cytometry to evaluate protocols and reagent compositions, and to optimize the expression and accessibility of engineered variants of the “probe” dL5-NB fusion protein.

[00156] To better simulate the asymmetric nature of yeast/virion agglutination, the AM2.2- RBD fusion protein will be secreted and purified from yeast. The secreted AM2.2-RBD fusion protein may then be bound in varying stoichiometries to streptavidin-coated virus-size beads via a TOl-PEG-biotin “tie-dye” (a dye tethered to a biotin moiety via a PEG-containing linker/spacer). Flow cytometry can be then be used to assay, in 2 colors, agglutination that is driven by an excess of virion-size targets that display a realistic numbers of spike protein RBDs (such as, 25-50).

[00157] If a higher sensitivity or lower-technology for quantitation is needed, the yeast cells may be engineered with enzymatic reporters that can generate signal based on light absorption or colored spots that result from the action of the enzyme on invisible detection substrate molecules. The signal could then be measured with a camera or a document scanner after exposure to a suitable detection substrate. Enzyme-substrate pairs may be detected based on absorption, fluorescence, and/or luminescence.

Example 7 - assembly and genomic integration of gene encoding fusion protein

[00158] A scarless Golden Gate cassette system for efficient integration of NBs into a targeted yeast chromosomal locus is shown in FIG 15. The chosen yeast locus (chr III ARS 416d) has been demonstrated to support high levels of gene expression using the constitutive yeast TDH3 promoter paired with the yeast ADH1 terminator. In vitro synthesized NB DNA is inserted into a pUC9 (pTDH3 AGA2 dL5 tADHl KanMX ARS416d locus) host vector that has been gapped by Bsal digestion, and the ligated pUC construct is then produced in E. coli (FIG 15). For integration into the yeast chromosome, linearized pTDH3 Hl 1-H4 NB tADHl KanMX ARS416d locus DNA is excised by BsmBI digestion (an H11-H4 NB is described in Huo J, et al. (Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2. Nat Struct Mol Biol. 2020 Nov;27(l l):1094. doi: 10.1038/s41594-020-00527-9. Erratum for: Nat Struct Mol Biol. 2020 Sep;27(9):846-854). Efficient yeast chromosomal integration is driven by use of the well-characterized KanMX marker that enables G418 selection in rich medium. Confirmation of proper integration is accomplished by PCR of the engineered locus and Sanger sequencing of PCR-amplified NB inserts (see, FIG. 16). This system provides an inexpensive means to rapidly insert NBs into a characterized locus to create yeast agglutination reporter reagents that may be used in resource-poor third world environments. FIGS. 17A and 17B provide a plasmid map (FIG. 17A) and an amino acid sequence (FIG. 17B) of an exemplary fusion protein (Yeast-displayed Hl 1-H4 NB dL5 fusion protein) comprising an anti-SARS-CoV-2 NB sequence and dL5, an scFv that activates MG. [00159] FIG. 17A depicts a plasmid construct that when linearized within the TRP1 marker (e.g. using EcoRV) and integrated into the yeast chromosome at the trpl locus creates an NB- displaying strain that agglutinates in presence of the virions. It presents an alternate strategy as compared to constructs in FIG.15 because the FIG. 17A construct requires galactose medium to induce the GALI promoter as opposed to constitutive expression driven by the TDH3 promoter in FIG.15.

Example 8 - Smartphone detection of agglutination

[00160] Experiments were conducted using three fluorescence quantitation laboratory instruments (an IVIS animal imager, a Tecan SPARK plate reader, and a laboratory-built gel imager) to assess the potential for accurately quantitating fluorescence signal of dL5 reporter yeast captured on filters. On the plate reader, the response is linear down to about 10,000 yeast cells (-500,000,000 individual dL5 fluorophores). These experiments are carried out on instruments wholly uncustomized for the task at hand. Over the past 15 years, cell phones have been incorporated into relatively inexpensive fluorescence imaging hardware for point-of-care diagnostics and environmental sensors (see, Banik, S., et al. Recent trends in smartphone-based detection for biomedical applications: a review. Anal Bioanal Chem 413, 2389-2406 (2021)) with detection limits ranging down to as little as 10 fluorescent molecules (Vietz C, et al. Benchmarking Smartphone Fluorescence-Based Microscopy with DNA Origami Nanobeads: Reducing the Gap toward Single-Molecule Sensitivity. ACS Omega. 2019 Jan 31;4(1):637- 642). The dL5/MG-2p reporter FAP is exceptionally bright and stable to photobleaching (Saurabh S, el al. Super-resolution Imaging of Live Bacteria Cells Using a Genetically Directed, Highly Photostable Fluoromodule. J Am Chem Soc. 2016 Aug 24; 138(33): 10398- 401). Taken together, these observations suggest that one skilled-in-the-art can use smart phone cameras and phone data processing capabilities to devise point-of-care hardware and software to quantitate filter-immobilized virion-agglutinated yeast.

[00161 J Having described this invention, it will be understood to those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any embodiment thereof. References incorporated herein by reference are incorporated for their technical disclosure and only to the extent that they are consistent with the present disclosure.