WO2023235955A1 - Neutralization assay to rapidly assess neutralization activity of anti-sars-cov-2 antibodies - Google Patents

Abstract

A method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the method comprising: (a) forming a mixture comprising (i) the sample, (ii) an ACE2 receptor connected to a first peptide fragment of a split enzyme, (iii) a SARS-CoV-2 spike (S) protein connected to a second peptide fragment of the split enzyme, (vi) a third peptide fragment of the split enzyme and (v) a suitable substrate of the split enzyme, such that an enzymatic activity of the split enzyme when reconstituted on the substrate results in the emission of an optical signal from the mixture, and (b) detecting a level of the optical signal emitted from the mixture, wherein a decrease in the level detected optical signal relative to a standard optical signal indicates that the sample contains a neutralizing anti-SARS-CoV-2 antibody.

Description

Neutralization Assay to Rapidly Assess Neutralization Activity of Anti-SARS-CoV-2

Antibodies

FIELD OF TECHNOLOGY

The present disclosure relates to neutralization assay to rapidly assess neutralization activity of anti-SARS-CoV-2 antibodies.

BACKGROUND INFORMATION

SARS-CoV-2 continues to threaten the world’s health as emerging variants of concern have the potential to circumvent deployed vaccines. Simple and rapid SARS-CoV-2 serological tests are needed to accurately measure the level and duration of neutralization activity of antibodies that arise from natural infection or vaccination. Currently, there are several FDA-approved serological tests under Emergency Use Authorizations (EUA), many of which can detect IgM or IgG against SARS-CoV-2, but do not measure their neutralization efficacy specifically^1-3. Functional neutralizing antibody titers are often measured with pseudotyped viruses, however, long assay time and discrepancies in published assay protocols have limited their use^4-6. Furthermore, pseudotype viral assays require biosafety level-3 (BSL-3) laboratory conditions. Alternatively, surrogate virus neutralization assays have been developed to circumvent the use of pseudovirions^7-10. Although some of these assays have shown successful measurement for serosurveillance of clinical samples, they often resemble ELISA, requiring multiple time-consuming binding and washing steps, while others have not yet reported successful measurement of clinical samples, likely due to the instability of recombinant proteins in serum and plasma.

PCT/CA2021/051733 describes a homogeneous serological assay platform named “SATiN” that utilizes a tri-part NanoLuc®, which is split into two small peptide tags, β9 and 10 (each about 1 kDa), and one large fragment, Δ11S (18 kDa). The SATiN assay utilizes spike protein and Protein G tagged with either β9 or β10. Upon simultaneous binding of the tagged spike protein and Protein G to anti-SARS-CoV-2 antibody, β9 and β10 re brought into proximity which induces refolding of Δ11S into active luciferase, producing glow-type luminescence¹¹.

The SATiN assay, however, mainly focuses on capturing and detecting general antibodies binding to virus proteins (e.g., spike or nucleocapsid)². Since it is well known that mere binding does not necessarily imply neutralization³⁷, a true neutralization assay for the better understanding of protective immunity against SARS- CoV-2 is needed.

SUMMARY OF DISCLOSURE

Presented herein is a homogeneous surrogate neutralization assay called “Neu- SATiN” to quantify the degree of neutralization from antibodies directly from plasma or serum.

In one embodiment provided is a method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the method comprising: (a) forming a mixture comprising (i) the sample, (ii) an ACE2 receptor connected to a first peptide fragment of a split enzyme, (iii) a SARS-CoV-2 spike (S) protein connected to a second peptide fragment of the split enzyme, (vi) a third peptide fragment of the split enzyme and (v) a suitable substrate of the split enzyme, such that an enzymatic activity of the split enzyme when reconstituted on the substrate results in the emission of an optical signal from the mixture, and (b) detecting a level of the optical signal emitted from the mixture, wherein a decrease in the level detected optical signal relative to a standard optical signal indicates that the sample contains a neutralizing anti-SARS-CoV-2 antibody.

In one embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the standard optical signal is obtained from a reference mixture comprising (i) a negative control sample (i.e. , a sample known to be free of neutralizing anti-SARS-CoV-2 antibodies), (ii) the ACE2 receptor connected to a first peptide fragment of a split enzyme, (iii) the SARS-CoV-2 spike (S) protein connected to a second peptide of the split enzyme, (vi) the third peptide fragment of the split enzyme and (v) the suitable substrate of the split enzyme.

In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the standard optical signal is obtained from a reference mixture comprising (i) a test control sample that contains a known amount of neutralizing anti-SARS-CoV-2 antibody, (ii) the ACE2 receptor connected to a first peptide fragment of a split enzyme, (iii) the SARS-CoV-2 spike (S) protein connected to a second peptide of the split enzyme, (vi) the third peptide fragment of the split enzyme and (v) the suitable substrate of the split enzyme, thereby quantifying the level of neutralization in the sample.

In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the standard optical signal comprises a plurality of optical signals obtained from a plurality of reference mixtures, each reference mixture comprising (i) a test control sample that contains a known amount of neutralizing anti-SARS-CoV-2 antibody, (ii) the ACE2 receptor connected to a first peptide fragment of a split enzyme, (iii) the SARS-CoV-2 spike (S) protein connected to a second peptide of the split enzyme, (vi) the third peptide fragment of the split enzyme and (v) the suitable substrate of the split enzyme, thereby quantifying the level of neutralization in the sample.

In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the standard optical signal is obtained from a mixture comprising (i) the ACE2 receptor connected to a first peptide fragment of a split enzyme, (ii) the SARS-CoV-2 spike (S) protein connected to a second peptide of the split enzyme, (iii) the third peptide fragment of the split enzyme and (iv) the suitable substrate of the split enzyme.

In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the SARS-CoV-2 S protein is provided as a full-length S protein (SF). In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the first peptide fragment is connected to the N-terminus of the ACE2, and the second peptide fragment is connected to the N- terminus of the SF.

In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the ACE2 is provided as a dimerized ACE2 binder comprising a human IgG Fc fragment linked to the C-terminus of a N-terminal domain (amino acids at positions 16-614) of the ACE2 (ACE2-Fc).

In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the first peptide fragment of the split enzyme is connected to the N-terminus of the dimerized ACE2 binder and the second peptide fragment is connected to the N-terminus of the SF.

In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the second peptide fragment is connected to the N-terminus of the SF through a G/S linker.

In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the SARS-CoV-2 S protein is a partial length S protein of the SARS-CoV-2, and wherein the partial length S protein is one or more of a S1 subunit of the SF protein, a S2 subunit of the SF protein, or a receptor binding domain (RBD) of the SF protein.

In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the SARS-CoV-2 S protein is the RBD, and wherein the first peptide fragment is connected to the N-terminus of the ACE2, and the second peptide fragment is connected to the N-terminus of the RBD or to the C- terminus of the RBD or to both the N-Terminus and the C-terminus of the RBD.

In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the split enzyme is a split luciferase and the first peptide fragment, the second peptide fragment and the third peptide fragment are split fragments of the split luciferase.

In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the split enzyme is a split luciferase and wherein the first peptide fragment has at least 80% sequence similarity with SEQ ID NO: 4, the second peptide fragment has at least 80% sequence similarity with SEQ ID NO: 6 and the third peptide fragment has at least 80% sequence similarity with SEQ ID NO: 2.

In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the split enzyme is a split luciferase and wherein the first peptide fragment is identical to SEQ ID NO: 4, the second peptide fragment is identical to SEQ ID NO: 6 and the third peptide fragment is identical to SEQ ID NO: 2.

In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the sample is blood or a blood product.

In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the blood or blood product is obtained from a subject that has received at least one vaccine against SARS-CoV-2.

In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the blood or blood product is obtained from a subject that has not received a vaccine against SARS-CoV-2.

In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the S protein is the S protein of wild-type SARS-CoV-2.

In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the S protein is the S protein of a variant of the wild-type SARS-CoV-2 or the S protein of a subvariant of the variant of the wild- type SARS CoV-2. In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the variant of the wild-type SARS-CoV-2 includes variants alpha, beta, gamma, delta, and omicron.

In another embodiment of the method for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the subvariant of the variant of the wild-type SARS CoV-2 include omicron subvariants BA.1 , BA.2, BA.3, BA.4, and BA.5.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures illustrate various aspects and preferred and alternative embodiments.

Figs. 1A-1B - General schematic of the COVID-19 neutralization assay and molecular modeling of spike (S) protein and ACE2 interaction (“Neu-SATiN”).

IA) Tri-part split luciferase (NanoLuc®) peptide fragments β1011 and β9 12 are individually fused to recombinant S protein (13, purple) and ACE2 (14, tan). Interaction of S protein 13 and ACE2 14 in the presence of luciferase fragment Δ11S 16 induces complementation of the split-luciferase 17 and 'turns on’ luminescence (left). In the presence of neutralizing antibodies 15, the interaction between S 13 protein and ACE2 14 is blocked, preventing luminescence (right). Figure generated using BioRender.

1 B) Molecular model of the predicted refolding of the tri-part split luciferase (PDB ID: 5IBO) is shown (27, green) after complementation of fragments Δ11S 26, β9 22and β1021 is driven by the interaction between full spike protein 23 (trimer) and ACE2 24 (PBD ID: 7A97).

Figs. 2A-2E. Binder pair screening in human serum and patient samples (PS 1 - 18). 2A) Molecular modeling of the distances between N-terminus of ACE2 to the N- terminus of RBD is ~60 Å and to the C-terminus is ~53 Å (PBD ID: 6M0J). As such, luciferase fragments can be fused at either terminus of RBD and N-terminus of ACE2. 2B) (S)RBD-β9 and β10-ACE2 binder pair was screened with increasing concentrations of neutralizing Ab (NAb, Sino Biological 40592-MM57) in human serum. 2C) Using (S)RBD-β9 and β10-ACE2 binder pair, the signal between 0 μg/mL of NAb (darker color) vs. 100 μg/mL of NAb (lighter color adjacent bar) shows a 10- fold decrease upon neutralization. 2D) Testing 18 patient plasma samples: (S)RBD- β9 and β10-ACE2 were mixed directly with plasma samples, followed by the addition of the detection solution (Δ11S and substrate). Samples 16, 17, and 18 (indicated by above bar) are known to be convalescent whereas other samples are from ICU patients with unknown antibody presence/levels.

Figs. 3A-3G. Validation of full spike proteins (wild type and variants) and ACE2 binders in the serosurveillance of clinical samples. Validation of full spike proteins (wild type and variants) and ACE2 binders in the serosurveillance of clinical samples. 3A) Molecular modeling of the distances between N-terminus of ACE2 to the N-termini of nearest S protein is ~48 Å and ~88 Å, respectively (PBD ID: 7A97). 3B) Comparison of signal and background for the full spike (wild type and variants) and ACE2 pairs. 3C) Serial dilution of a commercially available NAb (Sino Biological, 40592-R001 ) in the presence of β10-(S)WT and β9-ACE2-Fc pair. 3D) Serum samples that have been tested previously on two different COVID-19 detection assays were also tested using Neu-SATiN. Wild type full spike protein with β10 tag (β10-(S)WT) and ACE2 with β9 tag (β9-ACE2-Fc) were used as the binders. Signals from patient samples were normalized to the signal from normal human serum with the binders alone (no neutralizing antibodies). The scaled data for individual sample were plotted where the red dotted line at 10% activity (i.e., 90% neutralization) indicates the cutoff line to distinguish neutralizing samples from the non-neutralizing samples; '+’ and signs below the sample number indicate the result from two prior tests detecting anti-SARS- CoV-2 antibodies (COV2G Siemens™ 1st Gen and EUROIMMUN™ EIA). 3E) Activity measured in protein-based surrogate neutralization assay (pbSNA) versus neu-SATiN for the wild-type (WT) variant shows high correlation with a Pearson’s r value of 0.88. N=66. 3F) Neutralization efficacy of patient samples against WT and variant S proteins. Samples used in Fig. 3d (n = 43) were tested with an additional n = 35 patient samples that were positive for anti-SARS-CoV-2 antibodies. Luminescence signals from each patient serum were normalized to the signal from corresponding binder pair in commercial human serum (no NAb). Then, the highest value in each set was used to compute normalized activity. Known post-vaccination, post-infection, and negative (no known infection or vaccination) samples were plotted separately. Red dotted line at 10% indicates the cutoff between the negative samples and the positive samples for distinction of neutralization. 3G) Comparison of serum data from patients that were vaccinated to patients that were vaccinated after a documented infection (n = 40).

Fig. 4A-4C. Assessing the potency of neutralizing antibodies. 4A) Neutralizing capabilities of FDA EUA approved therapeutic antibodies Regn 10933 (left, casirivimab), Regn10987 (center, imdevimab), and JS016 (right, etesevimab) were evaluated using Neu-SATiN against different S variants. 4B) Evaluation of neutralizing antibodies in individuals with unvaccinated sera (n = 2), with one vaccination dose (n = 4), with two vaccination doses and collected within 50 days after the second shot (n = 13, intervals between shots were 21-37 days), and with two vaccination doses collected more than 50 days after the second shot (n = 5, intervals between shots were 21-36 days). 4C) Aggregated data showing titers at 50% neutralization (NT50) for each group.

Figs. 5A-5F. Specificity of RBD binder pair in simulated human serum and IC50 values of each binder pair. 5A) Comparison of signals between 0 μg/mL of NAb (darker color) vs. 100 μg/mL of NAb (lighter color adjacent bar) from six different pairs. Although all pairs show good response, some pairs show more distinct fold-difference between without vs. with NAb as indicated by numbers above bars. 5B) Pairs of ACE2 binders and (S)RBD binders were screened with increasing concentrations of neutralizing Ab (NAb, Sino Biological 40592-MM57) spiked in human serum. All pairs show substantial decrease in signal with increasing concentrations of NAb, demonstrating a quantifiable dose-response. 5C) When each binder pair was incubated with control isotype IgG-spiked human serum (additional concentrations of IgG spiked into human serum), less than 20% inhibition from high IgG (100 μg/mL) was observed in all of the pairs. 5D) Representative samples from Fig. 2D were serially diluted and tested by Neu-SATiN (left). As this in-house PSV assay is sensitive to protein content, performing the assay using direct plasma samples was not feasible. Instead, we selected ten samples (non-black bars on Fig. 2D) and extracted plasma IgG using protein G magnetic beads. The purified samples were serially diluted and tested with PSV assay (right). Each assay was repeated four individual times (n = 4) and the samples were run in triplicate (technical repeats). Average activity is shown (symbols) and CV is ≤0.15 for each sample (not shown). 5E) Average measurements were plotted and highest concentrations (left) show high correlation between the two assays with an r value of 0.94 and good correlation across all dilutions (right) with an r value of 0.81. NAb was used as an internal positive control (gray). One potential source of discrepancy seen between the results is lack of IgMs in PSV assay. Protein G is effective only in purifying IgGs, therefore purified clinical plasma mainly contained IgGs to be tested on PSV assay. As Neu-SATiN is performed directly using whole plasma, the antiviral activities of both IgGs and IgMs may contribute to the level of inhibition.⁵⁰

Fig. 6. Neutralization of full S protein variant pairs. The efficacy of 100 μg/mL NAb (Sino Biological, 40592-R001 ) was tested with variant pairs. The Alpha variant showed the most decrease in signal followed by the Delta strain. Almost no neutralization was observed with the Beta or Gamma strain.

Fig. 7. 5-point curves of percentage of inhibition of all sample provided (n=66) against WT and Omicron trimeric spike protein. The data was fitted using a three-parameter logistic non-linear regression.

DETAILED DISCLOSURE

Definitions

In this specification and in the claims that follow, reference will be made to several terms that shall be defined to have the meanings below. All numerical designations, e.g., dimensions and weight, including ranges, are approximations that typically may be varied ( + ) or ( - ) by increments of 0.1 , 1.0, or 10.0, as appropriate. All numerical designations may be understood as preceded by the term “about”.

The term “about,” particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent. “COVID-19+ subjects” (or patients) or “COVID-19 subjects” are subjects who are confirmed SARS-COVID-19 positive.

The term “patient” as used herein refers to a subject that is COVID-19+ or suspected of being COVID-19+.

The term “sample” includes body fluids. The term "body fluid", as used herein, includes blood, serum, plasma, urine, cerebrospinal fluid, saliva and any other body fluid that includes IgGs.

The term “subject” as used herein refers all members of the animal kingdom including mammals, preferably humans.

The methods of the invention may include steps of comparing sequences to each other, including wild-type sequence to one or more mutants (sequence variants). Such comparisons typically comprise alignments of polymer sequences, e.g., using sequence alignment programs and/or algorithms that are well known in the art (for example, BLAST, FASTA and MEGALIGN, to name a few). The skilled artisan can readily appreciate that, in such alignments, where a mutation contains a residue insertion or deletion, the sequence alignment will introduce a “gap” (typically represented by a dash, or “A”) in the polymer sequence not containing the inserted or deleted residue.

The term “sequence similarity,” in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.

Throughout this specification and the claims, the terms “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the terms “include”, “has” and their grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items. “Consisting essentially of’ when used to define systems, compositions and methods, shall mean excluding other elements of any essential significance to the combination for the intended use. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for using the systems of the present disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

The term “NT” as used herein refers to the N-terminal portion of a protein.

The term “CT” is used to refer to the C-terminal portion of a protein.

By “isolated” is meant, when referring to a polypeptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro molecules of the same type. The term “isolated” with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.

“SF” is a recombinant full length S protein of SARS-CoV-2.

“RBD” is used to refer to the receptor binding domain of the SF.

“S1” is a recombinant S1 region of SF of SARS-CoV-2.

“S2” is a recombinant S2 region of SF of SARS-CoV-2.

“Partial length of the S protein” is used to refer to one or more of S1 , S2 and RBD.

“S protein” is used to refer to the SF or partial length forms of the spike protein of SARS-CoV-2.

An original or wild-type virus that has one or more mutations is referred to as a “variant.” A variant known to spread more easily, cause more serious illness, or impact treatment or vaccine effectiveness, it is designated as a Variant of Concern (VoC). Examples of VoC include CoV-2 variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1 .617.2), and Omicron (B.1 .1 .529). Each variant may include lineages or subvariants. The Omicron variants includes, for example, subvariants or lineages BA.1 and BA, 2, BA.3, BA.4 and BA.5, and sublineages (BA.2.12, BA.1.1* etc.). Delta includes many AY.* sublineages. Gamma includes sublineages P.1.1 and P.1.2. Alpha also includes the Q.* sublineages. In this document, the term “variant” is meant to include the variant itself as well as its subvariants and sublineages.

Overview

The present disclosure relates to a novel homogeneous surrogate virus neutralization assay (hsVNA) called “Neutralization SATiN” or “Neu-SATiN” (Serological Assay based on split Tri-part Nanoluciferase; SATiN)¹¹ to detect and/or quantify the degree of neutralization from antibodies directly from a biological sample, plasma or serum. In this approach, a tri-part split of a reporter protein is used as a sensor for detecting and/or quantifying the degree of neutralization from anti-SARS-CoV-2 antibodies.

In Neu-SATiN, fragment peptides identical to or having at least 80% sequence similarity to peptide β9 (SEQ ID NO: 4) and to peptide β10 (SEQ ID NO: 6) are fused to the ACE2 receptor, the target of infection, and to the SARS-CoV-2 spike (S) protein (full length S protein (SF) or partial length of the S protein (for example the RBD of the S protein)). When ACE2 and the S protein (SF or a partial length of the S protein) interact in the presence of a peptide identical to or having at least 80% sequence similarity to peptide Δ11S (SEQ ID NO: 2), the fused split- β9 and β10 fragments are driven to within -100 Å of each other, allowing Δ11S to reconstitute into fully functional nanoluciferase reporter complex. Importantly, when the interaction between the ACE2 and S proteins are blocked by neutralizing antibodies, the fragment peptides of the split nanoluciferase reporter complex are prevented from interacting and subsequent complementation of the nanoluciferase reporter complex is blocked (Fig. 1A). In Neu- SATiN, the level of neutralization correlates with the decrease of luminescence emitted by the reporter complex. As the assay is intentionally modular, full-length ectoderm of the S protein of SARS-CoV-2 variants of concern can be quickly produced and swapped with wild-type S protein to assess immunological protection against variants. Moreover, as Neu-SATiN is designed to be a “mix-and-read” assay that is performed on the conventional lab bench, the actual hands-on time is less than about 30 minutes, significantly improving turnaround time.

In one embodiment, the present disclosure is a method or assay for detecting in a sample the presence of a neutralizing anti-SARS-CoV-2 antibody, the method comprising:

(a) forming a mixture comprising (i) the sample, (ii) an ACE2 receptor connected to a first peptide fragment of a split enzyme, (iii) a SARS-CoV-2 spike (S) protein connected to a second peptide of the split enzyme, (vi) a third peptide fragment of the split enzyme and (v) a suitable substrate of the split enzyme, such that an enzymatic activity of the split enzyme when reconstituted on the substrate results in the emission of an optical signal from the mixture, and

(b) detecting a level of the optical signal emitted from the mixture, wherein a decrease in the level detected optical signal relative to a standard optical signal indicates that the sample contains a neutralizing anti-SARS-CoV-2 antibody.

One or more calibration curves of known concentrations of neutralizing antibodies can be established and the detected signal emitted from the sample is compared to the one or more calibration curves to determine the concentrations of the neutralizing antibodies in the sample.

In embodiments, the SARS-CoV-2 S is provided as a S_F and the ACE2 is provided as a dimerized ACE2 binder created by fusing the ACE2 N-terminal domain (amino acids at positions 16-614) with the human IgG Fc fragment or portion, optionally including an interposed linker between the ACE2 and the Fc portion. The dimerized ACE2 binder is denoted “ACE2-Fc” (Table 1 ). In one embodiment, β10 (SEQ ID NO: 6) is connected to the N-terminus of SF protein (binder 010-S) and β9 (SEQ ID NO: 4) to the N-terminus of ACE2-Fc (binder β9-ACE2-Fc).

In embodiments, the detected optical signal emitted by the sample is compared to a plurality (more than one) reference optical signals having known amounts of neutralizing anti-SARS-CoV-2 antibodies, thereby quantifying the level of neutralization in the sample.

In embodiments, the split reporter complex is a split luciferase and the first, second and third peptide fragments are split fragments of the split luciferase.

In embodiments, the first peptide fragment has at least 80% (i.e., anywhere between 80% and 100%) sequence similarity with SEQ ID NO: 4, the second peptide fragment has at least 80% sequence similarity with SEQ ID NO: 6 and the third peptide fragment has at least 80% sequence similarity with SEQ ID NO: 2.

The assays or methods of the present disclosure can also be used to follow the efficacy of a treatment of infectious disorders or other diseases. For example, in the case of COVID-19, a patient can be tested for quantifiably amount of neutralizing a- SARS CoV-2 antibodies at different stages of the treatment using the systems, tests and methods of this disclosure. A reduction in the amount of a-SARS CoV-2 antibody being indicative of the efficacy of the COVID-19 treatment.

The assays or methods of the present disclosure can also be used to determine the efficacy of a COVID-19 vaccine in producing neutralizing antibodies.

The assays or methods of the present disclosure can be used to detect neutralizing antibodies against wild-type SARS CoV-2 as well as neutralizing antibodies against variants of the wild-type SARS CoV-2, including, without limitation, variants alpha, beta, gamma, delta and omicron, and including any subvariants of each of alpha, beta, gamma, delta and omicron, such as omicron subvariants BA.1 , BA.2, BA.3, BA.4, BA.5, and so forth).

In order to aid in the understanding and preparation of the present disclosure, the following illustrative, non-limiting examples are provided.

EXAMPLES

Example 1

Materials and Methods Cell culture media, antibodies, and cloning reagents

HEK293 cell culture reagents were purchased from Thermo Fisher Scientifics. Two neutralizing antibodies: 40592-MM57 (used for RBD pair screening) and 40592-R001 (used for WT pair screening) were both purchased from Sino Biological. Regn10933 (CPC511A), Regn10987 (CPC512A) and JS016 (CPC516A) were purchased from Cell Sciences.

Clinical samples

Samples were obtained from either the University of Utah School of Medicine, ARUP Laboratories, or from Unity Health. University of Utah School of Medicine (total n = 16) were obtained from infected patients within 48 hr of admission to ICU (n = 13) or within 3-5 weeks of positive PCR test for convalescent patients (n = 3). Samples for determining neutralization (total n = 63) in uninfected, infected and/or vaccinated were generously provided by ARUP Laboratories (IRB approved protocol 0007740). Samples for antibody titer and serosurveillance studies (total n = 24) were collected from Unity Health employees, enrolled through (REB approved protocol REB 20-107, Toronto). All samples were deidentified.

Vector Construction and Transient Transfection in HEK293 Cells

Vector cloning for the binder expression was performed as previously described¹¹. Briefly, all cDNA were cloned into an in-house mammalian expression vector derived from pCMV5 with a signal peptide sequence appended at the N-termini and an octa- histidine stretch at the C-termini. For the constructs that had the tag at the N-termini, either β9 or β10 sequence was placed after the signal peptide, followed by the binder sequence. Likewise, the constructs with C-terminus tag had β9 or β10 sequence right before octa-histidine (Table 1 ). The final products were transfected into HEK293 cells using polyethylenimine Max (Polysciences).

Production and Purification of Binders

HEK293 cells transfected with binder constructs were cultured in DMEM supplied with 10% FBS and 1X antimycotic-antibiotic mixture. Typically, cells were seeded at 50% confluency, and the media was collected every day until the cells became fully confluent. The collected media was filtered through 0.22 pm PES filter before purification. Purification was done on AKTA FPLC using HisPur™ Cobalt Resin. Tween-20 (0.01 %), trehalose (0.1 %), and glycerol (10%) was added to the final product and kept at -80 °C before use.

Reconstitution of Split-NanoLuc® Driven by (S)RBD and ACE2 interaction

For the screening of (S)RBD binders with ACE2 binders, 10 μL of commercial human serum (Sigma-Aldrich®, S1-100ML) spiked in with various concentrations of the neutralizing antibody (Sino Biological, 40592-MM57) was combined with 5 μL of (S)RBD binders (10 pmol) and 5 μL of ACE2 binders (10 pmol), and incubated for 30 min. The incubation was done using white, round bottom 96-well plates at room temperature with vigorous shaking. Then, the “detection solution” which consists of coelentrazine (substrate) and Δ11S was premixed, and 80 μL of the detection solution was added to each well. The final concentrations of the substrate, coelentrazine, was 10 μM and the large enzyme fragment, Δ11S, was 500 nM per well in a total volume of 100 μL. Luminescent signal was measured using TECAN Infinite M1000Pro in a kinetic cycle. The results reported here are from the 30-min timepoint.

Split NanoLuc®-based Virus Neutralization Assay: Testing Spiked Samples and Convalescent Samples

Measuring neutralization activity of clinical samples was done in a similar fashion as described above: 10 μL of clinical samples were mixed with 10 pmol of spike protein (5 μL; full S or (S)variant) and 10 pmol of ACE2 fusion (5 μL). All three components were incubated together for 30 min with vigorous shaking. Then, 80 μL of the detection solution (defined above) was added (final total volume per well was 100 μL) and the kinetic cycle of luminescent was measured. For serial dilutions to quantify activity of low titer samples, the assays were performed in a buffer containing 20 mM Tris (pH 7.5), 0.1% Tween 20, 2 mM TCEP, 2mM EDTA, 25 mM NaCI and 0.05% BSA. Serum samples (12.5 μL) were first diluted in a volume of 50 μL of buffer, followed by additional one-half serial dilutions up to 6 times. An aliquot (5 μL) of the diluted sample was mixed with 5 μL of β10 modified S binder (20 nM; full S or (S)Variant) and incubated for 30 minutes. An aliquot (5 μL) of the reaction mixture was further mixed with 45 μL of substrate mixture containing 555.6 nM β9-ACE2-Fc, 100 nM Δ11 S, 22.2 μM furimazine (substrate). After 1 hr incubation, luminescence signals were measured using a microplate reader. The inhibition curve of a sample against S protein variant was obtained by fitting the readings at different dilutions into the normalized response model with variable slope in GraphPad. The titer of 50% neutralization was calculated according to each model.

Testing Spiked Samples and Convalescent Samples with Pseudovirus Neutralization (PSV) Assay

The active sera (from patients in ICU) and convalescent sera were purified using protein G magnetic beads (Promega Corporation, G7471) as per manufacturer’s instruction. The concentrations of purified IgG were measured using NanoDrop 2000. Pseudovirions were produced by co-transfecting 293T human embryonic kidney cells using PEI transfection reagent (Polysciences, Inc., Warrington, PA) with NL4-3 HIV-1 genome (pNL4-3.Luc.R-E-, including the firefly luciferase gene inserted into the nef coding sequence and frameshift mutations in Env and Vpr) and a plasmid encoding the desired virus fusion protein (pCAGGS-SARS2-S-cFlag D614G, kind gift of M. Farzan⁴⁸ for SARS-CoV-2 S or μMDG VSV-G for VSV as a specificity control). Forty hours post-transfection, pseudovirus-containing supernatant was filtered (0.45 μM) and concentrated by ultracentrifugation (26,000 RPM, 2 hours) through a 20% sucrose/TNE (10 mM Tris pH 7.6, 100 mM NaCI, 1 mM EDTA) cushion, and the pellet resuspended in TNE, aliquoted and stored at -80 °C. To measure inhibition of infectivity, 50 μL of 2× IgG (purified from patient sera) diluted in media was added to CaLu-3 cells (ATCC HTB-55) in a 96-well format, each concentration in triplicate. 50 μL of pseudovirus diluted in media+ 16 μg/mL DEAE-dextran was added and plates were spinoculated at 2100 x g, 30 min, 10 °C. At 20 hours, virus and inhibitor were removed via aspiration, and fresh media was replenished. At 40 hours, the cells were lysed, and the luciferase activity was measured (Bright-Glo luciferase assay system, Promega, Madison, Wl). To determine the normalized luciferase value, average luciferase activity for no virus wells were first subtracted and then the luciferase signals were normalized to the average luciferase activity for no inhibitor wells.

Protein-based surrogate neutralization assay (pbSNA)

The complete methodology and performance data of the assay is described previously⁴⁹. Briefly, 384-well high-binding polystyrene Nunc plates (Thermo Fisher Scientific, #460372) were coated with 100 ng/well of full-length trimeric spike protein provided by the National Research Council Canada (NRC). The plates were centrifuge at 216×g for 2 min to ensure even coating and incubated overnight with rocking at4°C. The serum samples were diluted using a Hamilton MicroLab STAR robotic liquid handler. All plate washing steps included four washes with 100 μL of PBST and were carried using a 405 TS/LS LHC2 plate washer (Biotek Instruments). After the coating step, the plates were washed and blocked for 1 hour at room temperature with 80 μL of 3% w/v skim milk powder in PBST. The plates were washed once more and the serum, diluted in 1 % w/v skim milk powder in PBST, was added at a final volume of 20uL per well and incubated with shaking for 2 hours. A standard curve of purified neutralizing monoclonal antibody (NRCoV2-20-Fc, NRC) was added alongside a set of pooled negative/positive serum. The plates were then washed and 20μL of recombinant biotinylated ACE2 (NRC) was added (6.5 ng/well) and incubated with shaking for 1 hour. The plates were then washed to remove unbound ACE2 and 20μL of Streptavidin-Peroxidase Polymer (Sigma #S2438) was added to each well (25 ng/well) and incubated for 1 hour with shaking. Plates were washed one last time and developed by adding 20 μL of ELISA Pico Chemiluminescent Substrate (diluted 1 :2 in MilliQ H₂O) was dispensed into each well. After a 5-min incubation, plates were read on an Neo2 plate reader (BioTek Instruments) at 20 ms/well and a read height of 1.0 mm. Luminescence signal were blank adjusted and percentage of ACE2-Spike interaction determined by dividing the luminescence values by the maximal signal (No serum control; maximal ACE2-spike binding; 0% inhibition).

Results We considered three options for fusing the split-luciferase fragments to the SARS- CoV-2 spike (S) protein and its target, the human ACE2 receptor: 1 ) N-terminus, 2) C- terminus, or 3) both termini. Although surface loops are possible fusion points, modifying these domains may interfere with neutralizing antibody binding. Based on our previous experience, we know that reconstitution of split-NanoLuc® is most efficient when the fusion locations are within -50-100 Å^13-15. We used molecular modeling to determine possible fusion points on S protein and ACE2 that place fragments within this proximity (Fig. 2A and Fig. 3A).

To test feasibility, we initially built the system using only the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and ACE2. Based on molecular modeling of RBD and ACE2 (Fig. 2A)¹⁶, the distance from the N-terminus of ACE2 to the N- terminus of RBD is -60 Å and to the C-terminus is -53 Å (PBD ID: 6M0J). In contrast, the C-terminus of ACE2 is more than 100 Å away from either terminus of RBD and thus was excluded as a potential tag fusion site. The complete list of binders used in this study is listed in Table 1. Using purified recombinant proteins, we confirmed that the engineered RBD and ACE2 binders with complementary tags produced detectable luminescent signal when combined with the large fragment of the split luciferase, Δ11S, in human serum. Each binder combination shows substantial luminescence; however, there are differences in signal-to-background ratios (Fig. 5A). We also confirmed substantial decrease in the signal when the binders were incubated with neutralizing antibody (NAb) (Sino Biological, 40592-MM57), indicating inhibition of RBD binding to ACE2 to prevent complementation of the split-luciferase fragments. For any given pair, the fractional decrease in luminescent signal as a function of increasing concentration of NAb displays a typical dose-response curve for inhibition that is specific to the NAb (Fig. 5B). This suggests that, although the maximum signal intensity produced by each binder pair is different, the proportional decrease in the signal in the presence of NAb is similar. Therefore, the ability to measure NAbB antiviral activity is feasible with several different pairs. We characterized binder pairs further by calculating IC50 of the NAb using each pair and stability of the complex in high concentrations of non-binding IgG (Figs. 5C and Table 4). We selected the pair (S)RBD-β9 and β10-ACE2 based on the lowest discrepancy in IC50 value compared to the manufacturer’s report and in resolving concentration to response.

After validating Neu-SATiN’s ability to detect NAb activity, we validated the assay with clinical plasma samples from actively infected (from ICU) and convalescent patients (n = 18). The plasma samples were tested and binned into 5 different groups in respect to their relative luminescence compared to the control NAb (Fig. 2D). Of the convalescent patient samples (PS 16, 17, 18), patient sample 17 (PS 17) showed inhibition similar to 100 μg/mL of the control NAb. PS 16 and PS 18 were not as effective as PS 17, but still showed significant decrease in luminescence intensity compared to 0 μg/mL of NAb. As the neutralization activity depends on the epitope, affinity and concentration of the antibodies, the comparison between the control antibody and the antibodies in patient plasma is only relative. Nevertheless, our data confirm that Neu-SATiN is able to distinguish the presence or absence of antiviral antibodies and quantify the relative level of neutralization directly in clinical samples.

We compared Neu-SATiN to a pseudovirus neutralization (PSV) assay based on a human immunodeficiency virus (HIV) system with luciferase gene reporter. As PSV assays are sensitive to protein content and often require dilution of clinical samples (typically one-half dilutions or more) with specific matrix (e.g., cell culture compatible media and buffer), performing the assay using direct plasma samples was not feasible. Instead, we selected ten samples (non-black bars on Fig. 2D) and extracted plasma IgG using protein G magnetic beads. The purified samples were serially diluted and tested with PSV assay and compared to the Neu-SATiN (Table 4). The results obtained from Neu-SATiN and PSV assay show good correlation at highest antibody concentration (Fig. 5E-5F) with a Pearson’s rvalue of 0.86. Correlation is slightly lower across all IgG concentrations (Fig. 5E) with a Pearson’s r value of 0.81. Both PSV assay and Neu-SATiN showed that PS 17 had the highest inhibition activity and this was similar to the positive control (IgG purified from commercial human serum spiked with 100 μg/mL NAb). One potential source of discrepancy seen between the results is lack of IgMs in PSV assay. Protein G is effective only in purifying IgGs, therefore purified clinical plasma mainly contained IgGs to be tested on PSV assay. As Neu- SATiN is performed directly using whole plasma, the antiviral activities of both IgGs and IgMs may contribute to the level of inhibition.

Even though many of the antibodies produced by vaccines target the RBD of S protein^{17 18}, the more pressing concern is protection against variants of concern which often have mutations outside of the RBD.^{19 20} In fact, all currently approved vaccines in US and Canada are based on full-length S protein sequences and have been generated before the onset of variants in 2021. As such, we expanded Neu-SATiN using ACE2 and the full ectodomain of S protein, trimerized through the foldon domain, including known amino acid substitutions to stabilize conformation²². We hypothesized that using the full-length S protein (SF) should provide a more comprehensive measurement of the neutralization effect. Molecular modeling was used again to examine the distances between the termini of two proteins (Fig. 3A) using PBD ID: 7A9721. Based on this analysis, we decided to fuse β10 to the N-terminus of SF protein and β9 to the N-terminus of ACE2. The length of linker connecting the proteins to the tags was also doubled from nine to eighteen amino acids to increase the flexibility of the tags and to encompass a broader range of potential binding confirmations. Consistent with previous studies²⁴, we found the interaction between S ectodomain and ACE2 monomer is relatively low, likely due to ACE2 instability, and decided to use a dimerized ACE2 binder created by fusing the ACE2 N-terminal domain (a. a. 16-614) with the human IgG Fc fragment (Table 1 ). These two binders, denoted as “β10-(S)- WT” and “β9-ACE2-Fc”, were validated for binding-induced luminescence (Fig. 3B). The full wild-type spike protein (WT) version of the binder pair shows average luminescence signal of 1.6 x 106 RLU indicating successful complementation of the split-NanoLuc® fragments and shows a robust 3,000-fold signal-to-background ratio. We produced mutated S proteins for each of the major SARS-CoV-2 variants of concern: Alpha, Beta, Gamma, Delta, and Omicron¹⁹²⁰ (mutation sequences in Table 2). The mutations were made throughout the ectodomain of S protein and not just limited to the RBD. Additional mutations were made to the furin cleavage site to prevent unwanted degradation of the binders by cell culture proteases and to enable purification of recombinant proteins²⁵²⁶. The orientation of the tags on each protein were kept the same: β10-(S)Variant and β9-ACE2-Fc. All four variants (Alpha, Beta, Gamma, and Delta) produced distinct luminescence signals with high signal-to- background (Fig. 3B). The neutralization of the WT pair (WT S protein with ACE2) corresponds well with concentrations of a NAb (Sino Biological, 40592-R001) (Fig. 30); however, there was limited neutralization of most of the variants using this NAb (Fig. 6).

We obtained another set of serum samples (n = 43) that have been tested with two different Emergency Use Authorized COVID-19 S protein binding antibody assays: 1) C0V2G Siemens™ 1st Gen (target antigen: RBD, cutoff: ≥ 1.0), and 2) EUROIMMUN™ (target antigen: S1 , cutoff: ≥ 1.1). The samples can be further categorized into three different groups: 1) SARS-CoV-2 exposed with no vaccination history (post-infection, Samples 1 - 18), 2) SARS-CoV-2 exposed with vaccination history (post-infection and post-vaccination, Samples 19 - 28), and 3) neither exposed nor vaccinated (no infection and no vaccination, Samples 29 - 43). All of the samples were screened with our full-length-WT S protein Neu-SATiN for the presence of neutralizing antibodies. Commercial human serum spiked with NAb (Sino Biological, 40592-R001) was used as a positive control and to determine relative neutralization activity of the patient samples.

Samples with previous infection history (Samples 1 - 28, n = 28) showed normalized activity score < 10% (Fig. 3D - indicated by red dotted line) in Neu-SATiN, with the exception of Sample 2 (frequency = 27/28, 96%). These were also the samples that scored > 20.00 in Siemens™ 1st Gen and > 8 in EUROIMMUN™ EIA, both indicating the presence of anti-SARS-CoV-2 antibodies against the WT S protein (Table 3). Intriguingly, when the patients were previously infected and then vaccinated (Samples 19-28, n = 10), their sera showed normalized activity scores of 5% or below (frequency = 10/10, 100%). The various levels of neutralization shown in Samples 1 - 18 are possibly due to difference in sample collection day post-infection, as it is known that antibodies levels are highest 4-5 weeks after symptom onset²⁷’ ²⁸. Conversely, all SARS-CoV-2 negative patients (Samples 29-43, n = 15), based on standard antibody test, showed signals above 10% (frequency = 15/15, 100%), indicating no neutralization activity. Further, the activity of Neu-SATiN was benchmarked against a protein-based neutralization assay (Fig. 7) of the wild-type variant spike protein, showing good correlation with a Pearson’s r value of 0.88 (N=66) (Fig. 3E).

Altogether, the data supports that our newly developed Neu-SATiN neutralization assay is a reliable surrogate test that shows congruent results compared to already established tests. As we recognize that immunities generated by vaccination versus infection are known to produce antibodies targeting different parts of S protein²⁹ and that the protection against variants based on vaccination and/or previous infection is variable, we tested the same patient samples used to validate β10-(S)WT/ β9-ACE2- Fc, along with an additional set of infected patients (n = 35), for the samples’ neutralization efficacies towards different variants of concern. Since a universal NAb that can neutralize WT and all variants is not available, we were not able to determine relative degree of neutralization compared to a known concentration of NAb for each of the variants, but only for the WT. Instead, we compared fractional decrease in luminescence signal from the positive samples to negative samples for each variant pair; the highest signal observed from the negative group was considered 100% activity (i.e., no decrease in signal) and subsequent decrease in signal was determined for the positive groups. As shown in Fig. 3F, the NAb negative samples (darkest color violin plots) tend to display a wide range of signal compared to the NAb positive groups. Overall, the mean fractional signal observed from the positive samples (post- infection or post-vaccination) tested either with WT or variant pairs were 10% or lower. In other words, the signal measured from the positive samples were less than 10% of the signal from the negative group. This suggests that most of the samples within the positive groups have some level of neutralization ability towards each of the variant SARS-CoV-2s. In particular, both WT and Gamma variant were neutralized almost fully by immunity generated by vaccination; however, there appears to be a subset of samples within the Delta, Alpha, and Beta variant groups with minimal neutralization even after infection and/or vaccination (Fig. 3G). It was also interesting to note that natural immunity generated by infection with SARS-CoV-2 was not sufficient to neutralize the Gamma variant and that vaccination was required for stronger neutralization (Fig 3F). This observation is potentially due to antibodies produced by different immunities targeting different parts of S proteins and can cause differences in recognizing mutated sites^{29 30}. Another potential explanation is that the infective strains are also unknown and could induce different responses. Without further information on which vaccines were received by these patients or the sample collection dates post onset of symptoms, we are not able to determine the exact correlation between vaccination and difference in protection against variants.

We next sought to expand on our observations and tested serial dilutions of FDA EUA approved neutralizing antibodies Regn10933 (casirivimab), Regn10987 (imdevimab), and JS016 (etesevimab), as well as sera with known vaccination history (n = 24). Having such data is informative in detecting the lowest effective concentrations (titers) and therefore determining the potency of NAbs. Of the FDA EUA antibodies, imdevimab was the most effective against all variants of the S protein. The other two antibodies, casirivimab and etesevimab, were most potent against WT but showed variable potency against variant S proteins. There was no effective neutralization observed for the Beta strain with either antibody (Fig. 4A). For the serosurveillance of patient samples (Fig. 4B), no neutralizing activity was observed in patients that were not vaccinated, as expected. The patients with one dose showed negligible potency compared to the patients with two doses with samples collected < 50 days after the last dose. For samples collected > 50 days after receiving two doses, the potency of antibodies significantly reduced as indicated by shift in titration curve towards the right, consistent with recent reports of waning immunity for some vaccines³¹. In quantifying these patient samples in terms of the 50% neutralization titer (Fig. 4C), the majority show strong neutralization against WT and the least potency against Beta variant, with the other variants of concern intermediate.

Since the outbreak of the COVID-19 pandemic, the importance of a virus neutralization assay has risen more than ever. Virus neutralization assays are the main tools for developing vaccine and therapeutic strategies^32-34. Although the PSV assay is effective in measuring the degree of infection, maintaining cell cultures and making pseudovirus particles are labor intensive with potential safety concerns³⁵. Moreover, batch-to-batch variability in virus production and cell transfection efficiency limit standardization and robust assay results³⁶. Numerous immunoassays for rapid detection of anti-SARS-CoV-2 antibodies have been developed, however, these assays mainly focus on capturing and detecting antibodies binding to virus proteins (e.g., spike or nucleocapsid)². Since it is well known that mere binding does not necessarily imply neutralization³⁷, a true neutralization assay for the better understanding of protective immunity against SARS-CoV-2 is needed. To circumvent the use of virus particles and cells, surrogate versions of neutralization assays have been developed^7-9. These assays often use ELISA or similar platforms, with multiple time-consuming binding and washing steps, preventing high-throughput screening³⁸. In need of an accurate yet more rapid virus neutralization assay, we developed Neu- SATiN as a homogeneous surrogate virus neutralization assay (hsVNA) called, using a split-luciferase system.

There have been several reports describing efforts to develop surrogate virus neutralization (sVNA) assays for the detection of antibodies against SARS-CoV-2^39-42. These reports show capabilities as surrogate assays; however, many of these platforms require chemical modification and conjugation of proteins to beads or other solid surfaces, requiring intermediate purification and/or washing steps to remove unbound reagents and analytes. In contrast, Huang et al. reported the development of an hsVNA using the binary version of split-NanoLuc®. Although the approach is similar to the platform we report here, the authors used a monomeric or dimeric form of S1 domain of spike protein tagged with β10 (SmBiT), paired with ACE2 (LgBiT, NanoLuc® beta strands 1 through 9)³⁹, which was shown to have limited detection of non-RBD targeting antibodies. The importance of conformational changes in spike protein altering ACE2 binding has been emphasized in many studies^{23 43}. Also, with new variants of concern emerging, many of the deployed vaccines and second-wave vaccines in development are targeting full spike protein^44-46. We initially designed our assay by investigating the interactions between ACE2 and RBD¹⁰. The result is in agreement with reports that neutralization activities of NAb mostly come from anti- RBD immunoglobulins^{37 47}; however, immunologic response can also produce antibodies against epitopes outside of the RBD and may require the trimeric structure of the full spike protein for binding. This compelled us to develop stabilized, full-length- spike (both WT and major variants of concern) versions of the binders and stable, dimerized ACE2 receptor. The data presented in Fig. 3 demonstrates that β10-(S)WTZ β9-ACE2-Fc can differentiate the degree of neutralization directly from patient sera and that variant versions of the spike binders can distinguish anti-SARS-CoV-2 NAb positive serum samples from negative samples (Fig. 3E, 3F). In addition, Neu-SATiN can provide quantitative analysis of NT50 and thus enables the measurement of potency of anti-SARS-CoV-2 antibodies against different strains (Fig. 4). Combined, this provides a comprehensive screen of a patient’s level of protection against the current variants of concern. As the assay is modular, emerging variants of interest can be quickly produced and incorporated as we have demonstrated with the inclusion of the Omicron variant that become prominent during the initial review of this manuscript.

The results obtained with Neu-SATiN correlate with PSV assays and other antigen- based assays in detecting the neutralization potential of antibodies in clinical samples. It is important to note that natural immunity can produce antibodies that bind several antigens and function through alternate mechanisms, including antibody-dependent phagocytosis (ADP) or antibody-dependent cellular cytotoxicity (ADCC). Although the current format of Neu-SATiN cannot measure these types of antiviral activity, the majority of vaccines use Spike domains¹⁷’ ¹⁸; therefore, this assay can be used to assess protection developed from immunization. One advantage of Neu-SATiN is that it can be performed homogenously and directly using plasma or serum, which significantly reduces hands-on assay time to < 30 min and can be run under standard lab conditions. Importantly, we have demonstrated that the split-NanoLuc® based Neu-SATiN can be applied to full-length spike proteins of the original strain and variants to test neutralization levels of convalescent patient sera. Having a modular technology as a surrogate assay that can be easily adopted as a point-of-care tool is important in tracing and adapting to the evolution of the current pandemic.

Table 1 - Amino acid sequences of the binders. Spike protein and ACE2 sequences were obtained from literature sources. Then, each binder was PCR- modified with appropriate tags and linkers to produce binders. Octa-histidine was used as the purification tag. M denotes the location of the START codon and * denotes STOP codon. PDGFRB signal peptide (RLPGAMPALALKGELLLLSLLLLLEPQISQG) (SEQ ID NO: 29) was used to promote protein secretion. The sequences for β9 and β10 are GSMLFRVTINS (SEQ ID NO: 4) and VSGWRFKKIS (SEQ ID NO: 6), respectively.

Table 2- Spike protein variant mutations. Mutation information was obtained from literature sources. Additional mutations were made (R682G, R683S, R685S, K986P, and V987P) to the furin cleavage site to prevent degradation of proteins by host cell protease. Del denotes for deletion.

Table 3. Results from two prior tests detecting anti-SARS-CoV-2 antibodies (C0V2G Siemens™ 1st Gen and EUROIMMUN™ EIA) for corresponding individual samples tested in Fig. 3d. Interpretation, indicated as either positive (+) or negative (-), denotes the presence of anti-SARS-CoV-2 antibodies.

Table 4 - IC50 (μg/mL) of the same NAb calculated using six different binder pairs with

AAT Bioquest IC50 calculator

Sequence Listing

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Although various embodiments of the disclosure have been described and illustrated, it will be apparent to those skilled in the art in light of the present description that numerous modifications and variations can be made. The scope of the invention is defined more particularly in the appended claims.

Claims

CLAIMS What is claimed is:

1 . A method for detecting in a sample the presence of a neutralizing anti-SARS-CoV- 2 antibody, the method comprising:

(a) forming a mixture comprising (i) the sample, (ii) an ACE2 receptor connected to a first peptide fragment of a split enzyme, (iii) a SARS-CoV-2 spike (S) protein connected to a second peptide fragment of the split enzyme, (vi) a third peptide fragment of the split enzyme and (v) a suitable substrate of the split enzyme, such that an enzymatic activity of the split enzyme when reconstituted on the substrate results in the emission of an optical signal from the mixture, and

(b) detecting a level of the optical signal emitted from the mixture, wherein a decrease in the level detected optical signal relative to a standard optical signal indicates that the sample contains a neutralizing anti-SARS-CoV-2 antibody.

2. The method of claim 1 , wherein the standard optical signal is obtained from a reference mixture comprising (i) a negative control sample (i.e. , a sample known to be free of neutralizing anti-SARS-CoV-2 antibodies), (ii) the ACE2 receptor connected to a first peptide fragment of a split enzyme, (iii) the SARS-CoV-2 spike (S) protein connected to a second peptide of the split enzyme, (vi) the third peptide fragment of the split enzyme and (v) the suitable substrate of the split enzyme.

3. The method of claim 1 , wherein the standard optical signal is obtained from a reference mixture comprising (i) a test control sample that contains a known amount of neutralizing anti-SARS-CoV-2 antibody, (ii) the ACE2 receptor connected to a first peptide fragment of a split enzyme, (iii) the SARS-CoV-2 spike (S) protein connected to a second peptide of the split enzyme, (vi) the third peptide fragment of the split enzyme and (v) the suitable substrate of the split enzyme, thereby quantifying the level of neutralization in the sample.

4. The method of claim 1 , wherein the standard optical signal comprises a plurality of optical signals obtained from a plurality of reference mixtures, each reference mixture comprising (i) a test control sample that contains a known amount of neutralizing anti- SARS-CoV-2 antibody, (ii) the ACE2 receptor connected to a first peptide fragment of a split enzyme, (iii) the SARS-CoV-2 spike (S) protein connected to a second peptide of the split enzyme, (vi) the third peptide fragment of the split enzyme and (v) the suitable substrate of the split enzyme, thereby quantifying the level of neutralization in the sample.

5. The method of claim 1 , wherein the standard optical signal is obtained from a mixture comprising (i) the ACE2 receptor connected to a first peptide fragment of a split enzyme, (ii) the SARS-CoV-2 spike (S) protein connected to a second peptide of the split enzyme, (iii) the third peptide fragment of the split enzyme and (iv) the suitable substrate of the split enzyme.

6. The method of any one of claims 1 to 5, wherein the SARS-CoV-2 S protein is provided as a full-length S protein (SF).

7. the method of claim 6, wherein the first peptide fragment is connected to the N- terminus of the ACE2, and the second peptide fragment is connected to the N-terminus of the SF.

8. The method of claim 6, wherein the ACE2 is provided as a dimerized ACE2 binder comprising a human IgG Fc fragment linked to the C-terminus of a N-terminal domain (amino acids at positions 16-614) of the ACE2 (ACE2-Fc).

9. The method of claim 8, wherein the first peptide fragment of the split enzyme is connected to the N-terminus of the dimerized ACE2 binder and the second peptide fragment is connected to the N-terminus of the SF.

10. The method of any one of claims 7 to 9, wherein the second peptide fragment is connected to the N-terminus of the SF through a G/S linker.

11. The method of any one of claims 1 to 5, wherein the SARS-CoV-2 S protein is a partial length S protein of the SARS-CoV-2, and wherein the partial length S protein is one or more of a S1 subunit of the SF protein, a S2 subunit of the SF protein, or a receptor binding domain (RBD) of the SF protein.

12. The method of claim 11 , wherein the SARS-CoV-2 S protein is the RBD, and wherein the first peptide fragment is connected to the N-terminus of the ACE2, and the second peptide fragment is connected to the N-terminus of the RBD or to the C- terminus of the RBD or to both the N-Terminus and the C-terminus of the RBD.

13. The method of any one of claims 1 to 12, wherein the split enzyme is a split luciferase and the first peptide fragment, the second peptide fragment and the third peptide fragment are split fragments of the split luciferase.

14. The method of any one of claims 1 to 13, wherein the split enzyme is a split luciferase and wherein the first peptide fragment has at least 80% sequence similarity with SEQ ID NO: 4, the second peptide fragment has at least 80% sequence similarity with SEQ ID NO: 6 and the third peptide fragment has at least 80% sequence similarity with SEQ ID NO: 2.

15. The method of any one of claims 1 to 13, wherein the split enzyme is a split luciferase and wherein the first peptide fragment is identical to SEQ ID NO: 4, the second peptide fragment is identical to SEQ ID NO: 6 and the third peptide fragment is identical to SEQ ID NO: 2.

16. The method of any one of claims 1 to 15, wherein the sample is blood or a blood product.

17. The method of claim 16, wherein the blood or blood product is obtained from a subject that has received at least one vaccine against SARS-CoV-2.

18. The method of claim 16, wherein the blood or blood product is obtained from a subject that has not received a vaccine against SARS-CoV-2.

19. The method according to any one of claims 1 to 18, wherein the S protein is the S protein of wild-type SARS-CoV-2.

20. The method according to any one of claims 1 to 18, wherein the S protein is the S protein of a variant of the wild-type SARS-CoV-2 or the S protein of a subvariant of the variant of the wild-type SARS CoV-2.

21. The method of claim 20, wherein the variant of the wild-type SARS-CoV-2 includes variants alpha, beta, gamma, delta, and omicron.

22. The method of claim 20, wherein the subvariant of the variant of the wild-type SARS CoV-2 include omicron subvariants BA.2, BA.4, and BA.5.