Field of Invention

The invention relates to a serological test for Zika virus infection. In particular, the invention relates to polypeptides that are distinguished from a wild-type Zika Nonstructual Protein 1 (NS1), which are suitable for use in a serological test for detecting a Zika virus infection in a subject.

Background

The Zika virus (ZIKV), a single-stranded RNA virus, belongs to the family Flaviviridae. It is transmitted by infected Aedes mosquitoes, the same vector that transmits the dengue virus (DENV) in tropical and subtropical areas. Patients infected by ZIKV are often asymptomatic or present mild symptoms similar to dengue infections, such as fever, rash, joint pain, and etc. However, the ZIKV outbreak in Brazil has drawn much attention due to its association with a marked increase in the number of newborns with microcephaly from infected mothers. Following that, other neurological diseases such as Guillain-Barre syndrome (GBS) has also been associated with ZIKV infections.

A number of molecular- or serological-based assays has since been approved by FDA for emergency use to diagnose Zika infections. Nucleic acid testing (NAT) in general showed good specificity, but high variation in assay sensitivity were reported. This variability can be due to (i) complicated experimental setup, (ii) genetic variability in different virus strains and (iii) narrow detection window due to low viremia load in ZIKV-infected patients. Thus, in NAT negative cases, complementary assays based on serology testing i.e. Zika IgM antibody capture enzyme-linked immunosorbent assay (Zika MAC-EFISA) and plaque-reduction neutralization test (PRNT) are required to validate the results. These secondary tests are not specific due to high cross-reactivity with other flaviviruses, further complicating the interpretation of test results. There is a need to develop a more reliable Zika diagnostic to facilitate outbreak control and improve patient care. Accordingly, it is generally desirable to overcome or ameliorate one or more of the above mentioned difficulties.

Summary

Disclosed herein is a polypeptide that is distinguished from a wild-type Zika Nonstructual Protein 1 (NS1) by at least one amino acid substitution in the wild-type NS1 amino acid sequence, wherein the at least one amino acid substitution is at a position corresponding to positions 341, 344, 348, 350, 351 and 352 as set forth in SEQ ID NO: 1.

Disclosed herein is an isolated polynucleotide comprising a nucleic acid that encodes a polypeptide as defined herein.

Disclosed herein is an expression construct comprising a nucleic acid that encodes a polypeptide as defined herein.

Disclosed herein is a composition comprising a polypeptide as defined herein.

Disclosed herein is a method of producing a polypeptide as defined herein, the method comprising the steps of a) transforming an expression construct as defined herein into a host cell and b) expressing the polypeptide in the host cell.

Disclosed herein is a method for detecting antibodies specific for Zika virus in a sample obtained from a subject, wherein a polypeptide as defined herein is used as a capture reagent and/or as a binding partner for antibodies specific against Zika virus.

Disclosed herein is a method for detecting antibodies specific for Zika virus in a sample obtained from a subject, the method comprising: (a) contacting the sample with a polypeptide as defined herein under conditions and for a sufficient time for antibodies present in the sample to bind to the polypeptide; and (b) detecting the presence and/or the concentration of antibody that is bound to the polypeptide.

Disclosed herein is a method for detecting a subject suffering from a Zika infection, the method comprising: (a) contacting a sample from the subject with a polypeptide as defined herein under conditions and for a sufficient time for antibodies present in the sample to bind to the polypeptide; and (b) detecting the presence and/or the concentration of antibody that is bound to the polypeptide to determine whether the subject suffers from a Zika infection.

Disclosed herein is a method for distinguishing a Zika infection from a non-Zika flaviviral infection in a subject, the method comprising: (a) contacting a sample from the subject with a polypeptide as defined herein under conditions and for a sufficient time for antibodies present in the sample to bind to the polypeptide; and (b) detecting the presence and/or the concentration of antibody that is bound to the polypeptide to determine whether the subject suffers from a Zika infection and not a non-Zika flaviviral infection.

Disclosed herein is the use of an engineered polypeptide as defined herein in an in vitro diagnostic test for the detection of anti-Zika virus antibodies.

Disclosed herein is a reagent kit for the detection of anti-Zika virus antibodies, wherein the reagent kit comprises a polypeptide as defined herein.

Brief Description of Drawings

Embodiments of the present invention are hereafter described, by way of non-limiting example only, with reference to the accompanying drawings in which:

Figure 1: Purified fraction of engineered Zika antigen and typical expression yield in CHO cells for engineered antigen variants.

Figure 2. Reactivity of NS1 antigens to ZIKV and DENV plasma. (A, i) Reactivity of H-zWT and H-zDl to commercial ZIKV IgG in ELISA format. (A, ii) Reactivity of H-zWT, H-zDl, ZIKV WT and DENV WT to samples from TTSH. (A, iii) Comparison of zDl-Trx and zD2-Trx activity to DSC-7, and (A, iv) comparison of H-zWT, H- zMutl, and H-zMut2 activity to DSC-7. The graphs showed mean OD measurements from 2 replicates. (B) H-zMut2 ELISA was tested with a training set for binding to IgM and IgG. Results are representative of replicates for each sample. Normalized O.D > 1.5 for plasma or serum sample was determined as positive for ZIKV infection.

Figure 3. H-zMut2 ELISA for validation set. (A, D) H-zMut2 reactivity to IgM and IgG present in plasma collected during acute and recent convalescent phase (ZIKV- A: n=70 (1-6 d.p.o) and ZIKV-C: n=48 (7-14 d.p.o); DENV-A: n= 81(1-6 d.p.o) and DENV-B: n=70 (7-21 d.p.o); d.p.o: days post onset of symptoms). Plasma samples were blinded and tested with H-zMut2 as the capture antigen. Normalized OD > 1.5 for plasma sample was determined as positive for ZIKV infection. Results are representative of 2 replicates for each plasma sample. See Table 4 for details of evaluation on 3 antigens — H-zMut2, H-zWT and WT-NS1. (B-C, E-F) The plots show distribution of number of plasma cases (x-axis) over number of days post infection (y- axis, d.p.o) for H-zMut2 ELISA tested with validation set; the number of positive plasma samples (black bar) was shown against the total (grey bar) for each d.p.o.

Figure 4. Immunochromatographic assay (IA): H-zMut2 FI IA for IgM and IgG detection. (A) H-zMut2 as capture antigen in the FI IA format was tested with training set for detecting IgM (top) and IgG (bottom). Representative strips show a comparison of performance for WT-NS1, H-zWT and H-zMut2. Overall, H-Mut2 showed higher specificity than H-zWT (against DENV plasma, right panel), though both H-Mut2 and H-zWT showed greater sensitivity compared to WT-NS1 (against ZIKV plasma, left panel). The arrows indicate positive signals at the test line (T), upstream of the control line (C). See Table 5 for performance with the training set for 3 antigens — H-zMut2, H-zWT and WT-NS1. (B-F) The plots show distribution of number of plasma cases (x- axis) over number of days post onset of symptoms (y-axis, d.p.i) for FI IA format tested with validation set in a blinded manner (TTSH plasma); positive plasma (black) and total plasma cases (grey) over d.p.i were also shown.

Figure 5. Immunochromatographic assay (IA): H-zMutl as detector antigen in the F2 IA for detecting IgM and IgG. Representative strips showing F2 IA format tested with validation set in blinded manner. The arrows indicate the test line for IgM and IgG respectively, along with the control line (C). Figure 6. ELISA for ZIKV NS1 detection. (A) ROC curve analysis showing the performance of C12-C11 sandwich ELISA when tested against ZIKV infected or non ZIKA infected samples. (B) ZIKV NS1 quantification in patient samples using in-house antibody pair. Each dot represents an individual patient sample. C) The plot shows distribution of number of plasma cases (x-axis) over the number of days post onset of symptoms (y-axis) for ZIKV NS1 ELISA tested with the validation set; positive plasma (black) and the total plasma cases (grey) at each d.p.i are also shown.

Figure 7. ZIKV NS1 “sandwich” ELISA using in-house antibody pair. C12-C11 was tested by ELISA using recombinant antigens of different flaviviruses spiked into human control sera (A) and viral culture supernatant. PR, Puerto Rico strains (Y2015); TH, Thailand strain (KF993678) and SG, Singapore strain (NPHL). The data represent the average (± S.D.) of the replicates for each sample from one of the three independent experiments. (B) Standard curve for ZIKV NS1 ELISA established with recombinant protein spiked into human serum. The assay values represent the average of 4 experiments, and the error bars indicate the standard deviation (S.D.). (C) The cut-off for ELISA was established using 45 patient samples infected by DENV. Horizontal line indicates cut-off at 0.25 ng/ml.

Figure 8. shows an alignment of the Zika NS1 with other related viruses (at position 320 to 352).

Detailed Description

Disclosed herein is a polypeptide that is distinguished from a wild-type Zika Nonstructual Protein 1 (NS1) by at least one amino acid substitution in the wild-type NS1 amino acid sequence, wherein the at least one amino acid substitution is at a position corresponding to positions 341, 344, 348, 350, 351 and 352 as set forth in SEQ ID NO: 1.

Without being bound by theory, the inventors have developed specific serology tests that could differentiate ZIKV from DENV infections by engineering the ZIKV non- structural protein 1 (NS1). Both enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assays (IA) were established for specific and sensitive binding to anti-ZIKV IgM and IgG. In particular, the inventors have developed two IA assays, where the engineered antigens were used either as capture (FI format) or detector (F2 format), resulting in slight difference in sensitivity and specificity. Assay performance was assessed by testing plasma samples collected from Tan Tock Seng Hospital. The samples were collected from patients during acute (days 1-6 post onset of symptoms) and convalescent (days 7-21 post onset of symptoms) phases of infection. Collectively, the findings showed that the IgM/IgG tests performed better on convalescent samples, but the performance on the acute samples could be improved by combining with an NS1 detection test.

The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally-occurring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers. These terms do not exclude modifications, for example, glycosylations, acetylations, phosphorylations and the like. Soluble forms of the subject proteinaceous molecules are particularly useful. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids or polypeptides with substituted linkages.

The polypeptide as defined herein may be distinguished from a wild-type Zika Nonstructual Protein 1 (NS1) by at least one, two, three, four, five or six amino acid substitutions in the wild-type NS1 amino acid sequence, wherein the at least one, two, three, four, five or six amino acid substitutions is at a position (or are positions) corresponding to positions 341, 344, 348, 350, 351 and 352 as set forth in SEQ ID NO: 1.

The term "substitution" as used herein refers to the presence of an amino acid residue at a certain position of the derivative sequence which is different from the amino acid residue which is present or absent at the corresponding position in the reference sequence. The polypeptide as defined herein may be distinguished from wild- type Zika NS1 by additional amino acid substitutions that do not correspond to positions 341, 344, 348, 350, 351 and 352 as set forth in SEQ ID NO: 1.

The polypeptide as defined herein may, for example, have one or more “conservative amino acid substitution”. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows:

AMINO ACID SUB-CLASSIFICATION

The term “sequence identity” as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G and I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

The term “at least 70%” as used herein includes at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. Disclosed herein is a polypeptide that is distinguished from a wild- type Zika NS 1 by at least one amino acid substitution in the wild-type NS1 amino acid sequence, wherein the at least one amino acid substitution is at a position corresponding to positions 341, 344, 348, 351 and 352 as set forth in SEQ ID NO: 1.

In one embodiment, the at least one amino acid substitution is at a position corresponding to positions 341, 344, 348, 351 and 352 as set forth in SEQ ID NO: 1.

In one embodiment, the amino acid substitution corresponding to position 341 is a substitution to a polar neutral residue (e.g. H or Q). In one embodiment, the amino acid substitution corresponding to position 344 is an amino acid substitution to an acidic or basic residue (e.g. K, R, D or E). In one embodiment, the amino acid substitution corresponding to position 348 is an amino acid substitution to an acidic residue (e.g. D or E). In one embodiment, the amino acid substitution corresponding to position 351 is an amino acid substitution to a basic residue (e.g. H, K or R). In one embodiment, the amino acid substitution corresponding to position 352 is an amino acid substitution to an acidic residue (e.g. D or E).

In one embodiment, the polypeptide comprises an amino acid substitution selected from the group consisting of P341H, N344K, S348D, T351H and A352D. In one embodiment, the polypeptide comprises the amino acid substitutions of P341H, N344K, S348D, T351H and A352D. In one embodiment, the polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2.

Disclosed herein is a polypeptide that is distinguished from a wild- type Zika NS 1 by at least one amino acid substitution in the wild-type NS1 amino acid sequence, wherein the at least one amino acid substitution is at a position corresponding to positions 341, 344 and 350 as set forth in SEQ ID NO: 1.

In one embodiment, the at least one amino acid substitution is at a position corresponding to positions 341, 344 and 350 as set forth in SEQ ID NO: 1. In one embodiment, the amino acid substitution corresponding to position 341 is a substitution to a polar neutral residue (e.g. Q or H). In one embodiment, the amino acid substitution corresponding to position 344 is a substitution to an acidic residue (e.g. K, R, D or E). In one embodiment, the amino acid substitution corresponding to position 350 is a substitution to a polar neutral residue (e.g. T).

In one embodiment, the polypeptide comprises an amino acid substitution selected from the group consisting of P341Q, N344D and V350T. In one embodiment, the polypeptide comprises the amino acid substitutions of P341Q, N344D and V350T. In one embodiment, the polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 3.

In one embodiment, the polypeptide is a recombinant polypeptide.

The polypeptide as defined herein may be folded in native conformation or denatured with linear epitopes. It may be a fusion protein from one or more Zika virus protein or non-Zika virus protein or peptide.

In one embodiment, the polypeptide comprises a solubility tag. The solubility tag may be positioned at the N or C terminus of the polypeptide to improve the solubility of the polypeptide.

In one embodiment, the solubility tag is human albumin, thioredoxin or a fragment thereof. In one embodiment, the solubility tag comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 4.

In one embodiment, the polypeptide comprises an affinity tag (such as a polyhistidine tag).

In one embodiment, the polypeptide comprises a solubility and affinity tag having an amino acid sequence of at least 70% sequence identity to SEQ ID NO: 4.

In one embodiment, the polypeptide is suitable for specifically detecting antibodies against Zika virus in a biological sample.

Disclosed herein is a molecular complex between a polypeptide as defined herein and an antibody against Zika virus. The antibody against Zika virus may be present in a biological sample that has been obtained from a subject. The formation of a molecular complex between the polypeptide and the antibody may indicate the presence of a Zika virus infection in a subject.

Disclosed herein is an isolated polynucleotide comprising a nucleic acid that encodes a polypeptide as defined herein.

Disclosed herein is a cDNA comprising a nucleic acid that encodes a polypeptide as defined herein.

The term “polynucleotide” or “nucleic acid” are used interchangeably herein to refer to a polymer of nucleotides, which can be mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

An "isolated nucleic acid" refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

In one embodiment, the isolated nucleic acid is operably linked to one or more expression control sequences. Disclosed herein is an expression construct comprising a nucleic acid that encodes a polypeptide as defined herein.

The term “construct” refers to a recombinant genetic molecule including one or more isolated nucleic acid sequences from different sources. Thus, constructs are chimeric molecules in which two or more nucleic acid sequences of different origin are assembled into a single nucleic acid molecule and include any construct that contains (1) nucleic acid sequences, including regulatory and coding sequences that are not found together in nature (i.e., at least one of the nucleotide sequences is heterologous with respect to at least one of its other nucleotide sequences), or (2) sequences encoding parts of functional RNA molecules or proteins not naturally adjoined, or (3) parts of promoters that are not naturally adjoined. Representative constructs include any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single stranded or double stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecules have been operably linked. Constructs of the present invention will generally include the necessary elements to direct expression of a nucleic acid sequence of interest that is also contained in the construct, such as, for example, a target nucleic acid sequence or a modulator nucleic acid sequence. Such elements may include control elements such as a promoter that is operably linked to (so as to direct transcription of) the nucleic acid sequence of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the invention, the construct may be contained within a vector. In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and/or elements to facilitate stable integration of the construct into the genome of a host cell. Two or more constructs can be contained within a single nucleic acid molecule, such as a single vector, or can be containing within two or more separate nucleic acid molecules, such as two or more separate vectors.

An “expression construct” generally includes at least a control sequence operably linked to a nucleotide sequence of interest. In this manner, for example, promoters in operable connection with the nucleotide sequences to be expressed are provided in expression constructs for expression in an organism or part thereof including a host cell. For the practice of the present invention, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art, see for example, Molecular Cloning: A Laboratory Manual, 3rd edition Volumes 1, 2, and 3. J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.

By “control element” or “control sequence” is meant nucleic acid sequences (e.g. , DNA) necessary for expression of an operably linked coding sequence in a particular host cell. The control sequences that are suitable for prokaryotic cells for example, include a promoter, and optionally a r/ ^' s-acting sequence such as an operator sequence and a ribosome binding site. Control sequences that are suitable for eukaryotic cells include transcriptional control sequences such as promoters, polyadenylation signals, transcriptional enhancers, translational control sequences such as translational enhancers and internal ribosome binding sites (IRES), nucleic acid sequences that modulate mRNA stability, as well as targeting sequences that target a product encoded by a transcribed polynucleotide to an intracellular compartment within a cell or to the extracellular environment.

The term "encoding" or “encodes” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e. rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription of a gene and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

Disclosed herein is a vector comprising a nucleic acid that encodes a polypeptide as defined herein.

By the term "vector", as used herein, is meant any plasmid or virus encoding an exogenous nucleic acid. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into virions or cells, such as, for example, polylysine compounds and the like. The vector may be a viral vector which is suitable as a delivery vehicle for delivery of a nucleic acid that encodes a polypeptide of the present invention to the patient, or the vector may be a non-viral vector which is suitable for the same purpose. Examples of viral and non-viral vectors for delivery of DNA to cells and tissues are well known in the art and are described, for example, in Ma et al. (1997, Proc. Natl. Acad. Sci. U.S.A. 94: 12744-12746). Examples of viral vectors include, but are not limited to, a lentiviral vector, a recombinant adenovirus, a recombinant retrovirus, a recombinant adeno-associated virus, a recombinant avian pox virus, and the like (Cranage et al., 1986, EMBO J. 5:3057-3063; International Patent Application No. WO94/17810, published August 18, 1994; International Patent Application No. W094/23744, published October 27, 1994). Examples of non-viral vectors include, but are not limited to, liposomes, polyamine derivatives of DNA, and the like.

Disclosed herein is a composition comprising a polypeptide as defined herein.

The composition may be a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

By “pharmaceutically acceptable carrier” is meant a solid or liquid filler, diluent or encapsulating substance that can be safely used in topical or systemic administration to an animal, preferably a mammal, including humans. Representative pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington’s Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient(s), its use in the pharmaceutical compositions is contemplated. Disclosed herein is a method of producing a polypeptide as defined herein, the method comprising the steps of a) transforming an expression construct as defined herein into a host cell and b) expressing the polypeptide in the host cell.

In one embodiment, the method comprising purifying the polypeptide.

Disclosed herein is a method for detecting antibodies specific for Zika virus in a sample obtained from a subject, wherein a polypeptide as defined herein is used as a capture reagent and/or as a binding partner for antibodies specific against Zika virus.

As used herein, the terms “subject” and “patient ’ are used interchangeably. A subject may include any animal, including mammals. Mammals include, without limitation, farm animals (e.g., horse, cow, pig), companion animals (e.g„, dog, cat) laboratory animals (e.g., mouse, rat, rabbits), and non-human primates. In some embodiments, the subject is a human being.

The methods as defined herein may refer to an antibody capture assay that detects Zika antibodies that recognises a polypeptide as defined herein.

The term “immunoassay” as used herein refers to an analytical method which uses the ability of an antibody to bind a particular antigen as the means for determining the presence of the antibody or antigen. An antibody-capture immunoassay is an assay that provides an antigen which is used to detect antibodies against a particular pathogen in a sample from a subject.

It is contemplated that a range of immunoassay formats be encompassed by this definition, including but not limited to direct immunoassays, indirect immunoassays, and “sandwich” immunoassays (e.g. a sandwich enzyme-linked immunosorbent assay (ELISA)). In one embodiment, the antigen is immobilized on a support and is capable of binding an antibody in a sample. In a variation of the antibody-capture assay the antigen is mixed with the antibody in the biological sample and the antigen-antibody complex thus formed is captured by a second antibody against the antigen or antibody or both in the antigen- antibody complex which is immobilized on a support. Detection of an antibody-antigen complex can be performed by several methods. The antigen may be prepared with a label such as biotin, an enzyme, a fluorescent marker, or radioactivity, and may be detected directly using this label. Alternatively, a labeled “secondary antibody” or “reporter antibody” which recognizes the primary antibody may be added, forming a complex comprised of antigen-antibody-antibody. Again, appropriate reporter reagents are then added to detect the labeled antibody. Any number of additional antibodies may be added as desired. These antibodies may also be labeled with a marker, including, but not limited to an enzyme, fluorescent marker, or radioactivity. Either the antigen or the antibody (primary or secondary) may be immobilized on a solid support, but the labeled component cannot be immobilized because the detectable signal is precluded from being a measure of binding.

As used herein, the term “reporter reagent” is used in reference to compounds which are capable of detecting the presence of antibody bound to antigen. For example, a reporter reagent may be a calorimetric substance which is attached to an enzymatic substrate. Upon binding of antibody and antigen, the enzyme acts on its substrate and causes the production of a color. Other reporter reagents include, but are not limited to fluorogenic and radioactive compounds or molecules.

As used herein, the term “solid support” is used in reference to any solid material to which reagents such as antibodies, antigens, and other compounds may be attached. For example, in the EFISA method, the wells of microtiter plates often provide solid supports. Other examples of solid supports include nitrocellulose membrane, microscope slides, coverslips, beads, particles, cell culture flasks, as well as many other items.

As used herein, the terms “label” and means for detecting the antibody-antigen complex refer to molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex. Any label or indicating means can be linked to or incorporated in an expressed protein, peptide, or antibody molecule that is part of the present invention, or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well known in clinical diagnostic chemistry. The labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer. Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyante (FITC), 5- dimethylamine-l-natpthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like.

In preferred embodiments, the indicating group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, or the like. In such cases where the principle indicating group is an enzyme such as HRP or glucose oxidase, additional reagents are required to indicate that a receptor-ligand complex (immunoreactant) has formed. Such additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine. An additional reagent useful with glucose oxidase is 2,2,- azino-di-(3-ethyl-benzthiazoline-G-sulfonic acid) (ABTS).

Radioactive elements are also useful labeling agents and are used illustratively herein. An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as ¹²⁴ I, ¹²⁵ I, ¹²⁸ I, ¹³² I and ⁵¹ Cr represent one class of gamma ray emission-producing radioactive element indicating groups. Another group of useful labeling means are those elements such as ⁿ C, ¹⁸ F, ¹⁵ 0 and ¹³ N which themselves emit positrons. Also useful is a beta emitter, such as ^m indium or ³ H.

The linking of labels, i.e. labeling of peptides and proteins is well known in the art. For instance, monoclonal antibodies produced by a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture medium. The techniques of protein conjugation or coupling through activated functional groups are particularly applicable.

Disclosed herein is a method for detecting antibodies specific for Zika virus in a sample obtained from a subject, the method comprising: a) contacting the sample with a polypeptide as defined herein under conditions and for a sufficient time for antibodies present in the sample to bind to the polypeptide; and b) detecting the presence and/or the concentration of antibody that is bound to the polypeptide.

In one embodiment, the polypeptide as defined herein is immobilized on a solid support prior to step a). In an alternative embodiment, the polypeptide as defined herein is immobilized or conjugated to the surface of nanoparticles (e.g. gold nanoparticles).

In one embodiment, the detected antibody is an IgG or IgM antibody. The IgG or IgM antibody may, for example, be detected or measured using an anti-IgG or anti-IgM secondary antibody that is conjugated to a label, such as a HRP enzyme label for detection of the IgG or IgM antibody. Alternatively, nanoparticles that are bound to IgG or IgM antibodies may be captured by anti-IgG or anti-IgM antibodies that are immobilized on a solid support.

By “antibody” is meant a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity. Representative antigen-binding molecules that are useful in the practice of the present invention include polyclonal and monoclonal antibodies as well as their fragments (such as Fab, Fab’, F(ab’)2, Fv), single chain (scFv) and domain antibodies (including, for example, shark and camelid antibodies), and fusion proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding/recognition site. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof).

In one embodiment, the sample is a convalescent sample. The term “convalescent sample” may refers to a sample taken from a subject who has recovered from a disease, such as an infectious disease.

The sample as referred to herein may be one that is obtained a subject. In one embodiment, the sample is a serological sample. The sample may, for example, be blood (e.g. whole blood), plasma or serum. The sample may be one that has been further processed. For example, the sample may be further processed so that the antibodies to be detected are concentrated in comparison to the original sample, or present in higher purity. Methods for further processing the biological sample are known to those skilled in the art.

Disclosed herein is a method for detecting a subject suffering from a Zika infection, the method comprising: a) contacting a sample from the subject with a polypeptide as defined herein under conditions and for a sufficient time for antibodies present in the sample to bind to the polypeptide; and b) detecting the presence and/or the concentration of antibody that is bound to the polypeptide to determine whether the subject suffers from a Zika infection.

Methods for treating a subject found to be suffering from a Zika infection are also provided herein. In one embodiment, there is provided a method for detecting a subject suffering from a Zika infection, the method comprising: a) contacting a sample from the subject with a polypeptide as defined herein under conditions and for a sufficient time for antibodies present in the sample to bind to the polypeptide; b) detecting the presence and/or the concentration of antibody that is bound to the polypeptide to determine whether the subject suffers from a Zika infection and c) treating the subject found to be suffering from a Zika infection.

The term “treating" as used herein may refer to (1) preventing or delaying the appearance of one or more symptoms of the disorder; (2) inhibiting the development of the disorder or one or more symptoms of the disorder; (3) relieving the disorder, i.e., causing regression of the disorder or at least one or more symptoms of the disorder; and/or (4) causing a decrease in the severity of one or more symptoms of the disorder.

In one embodiment, there is provided a method for treating a subject suffering from a Zika infection, the method comprising: a) contacting a sample from the subject with a polypeptide as defined herein under conditions and for a sufficient time for antibodies present in the sample to bind to the polypeptide; b) detecting the presence and/or the concentration of antibody that is bound to the polypeptide to determine whether the subject suffers from a Zika infection and c) treating the subject found to be suffering from a Zika infection.

Disclosed herein is a method for distinguishing a Zika infection from a non-Zika flaviviral infection in a subject, the method comprising: a) contacting a sample from the subject with a polypeptide as defined herein under conditions and for a sufficient time for antibodies present in the sample to bind to the polypeptide; and b) detecting the presence and/or the concentration of antibody that is bound to the polypeptide to determine whether the subject suffers from a Zika infection and not a non-Zika flaviviral infection.

In one embodiment, the non-Zika flaviviral infection is Dengue.

Disclosed herein is the use of an engineered polypeptide as defined herein in an in vitro diagnostic test for the detection of anti-Zika virus antibodies.

Disclosed herein is a reagent kit for the detection of anti-Zika virus antibodies, wherein the reagent kit comprises a polypeptide as defined herein.

In one embodiment, the polypeptide is a capture reagent and/or a binding partner for antibodies specific against Zika virus.

In one embodiment, the kit is in an ELISA or lateral flow format.

Zika WT NS1

DVGCSVDFSKKETRCGTGVFVYNDVEAWRDRYKYHPDSPRRLAAAVKQAWEDGIC

GIS S V SRMENIMWRS VEGELNAILEENG V QLTV V V GS VKNPM WRGPQRLP VPVNELP

HGWKAWGKSYFVRAAKTNNSFVVDGDTLKECPLKHRAWNSFLVEDHGFGVFHTSV

WLKVREDYSLECDPAVIGTAVKGKEAVHSDLGYWIESEKNDTWRLKRAHLIEMKTC

EWPKSHTLWTDGIEESDLIIPKSLAGPLSHHNTREGYRTQMKGPWHSEELEIRFEEC PG

TKVHVEETCGTRGPSLRSTTASGRVIEEWCCRECTMPPLSFRAKDGCWYGMEIRPRK

EPESNLVRSMVTA (SEQ ID NO: 1) zMut2

DVGCSVDFSKKETRCGTGVFVYNDVEAWRDRYKYHPDSPRRLAAAVKQAWEDGIC GIS S V SRMENIMWRS VEGELNAILEENG V QLTV V V GS VKNPM WRGPQRLP VPVNELP HGWKAWGKSYFVRAAKTNNSFVVDGDTLKECPLKHRAWNSFLVEDHGFGVFHTSV WLKVREDYSLECDPAVIGTAVKGKEAVHSDLGYWIESEKNDTWRLKRAHLIEMKTC EWPKSHTLWTDGIEESDLIIPKSLAGPLSHHNTREGYRTQMKGPWHSEELEIRFEECPG TKVHVEETCGTRGPSLRSTTASGRVIEEWCCRECTMPPLSFRAKDGCWYGMEIRPRK EHESKLVRDMVHD (SEQ ID NO: 2) zMutl

DVGCSVDFSKKETRCGTGVFVYNDVEAWRDRYKYHPDSPRRLAAAVKQAWEDGIC

GIS S V SRMENIMWRS VEGELNAILEENG V QLTV V V GS VKNPM WRGPQRLP VPVNELP

HGWKAWGKSYFVRAAKTNNSFVVDGDTLKECPLKHRAWNSFLVEDHGFGVFHTSV

WFKVREDYSFECDPAVIGTAVKGKEAVHSDFGYWIESEKNDTWRFKRAHFIEMKTC

EWPKSHTLWTDGIEESDLIIPKSLAGPLSHHNTREGYRTQMKGPWHSEELEIRFEEC PG

TKVHVEETCGTRGPSLRSTTASGRVIEEWCCRECTMPPLSFRAKDGCWYGMEIRPRK

QPESDLVRSMTT A (SEQ ID NO: 3)

N-terminal soluble tag (H)*

HHHHHHAAADAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVT EFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQ HKDDNPNLPRLVRPEVD VMCT AFHDNEETFLKKYLYEI ARRHP YFY APELLFFAKRY KAAFTECCQAADKAACLLPKLDELRDEGKASSAKQGAASAPSTGSKPTAP (SEQ ID NO: 4)

*H-zMut2 would be the N-terminal tag sequence followed by zMut2, etc.

As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

As used in this application, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof.

Throughout this specification and the statements which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Those skilled in the art will appreciate that the invention described herein in susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

EXAMPLES

Example 1

Material and Methods

Study Approval

Whole-blood samples were collected with ethylenediaminetetraacetic acid-lined Vacutainer tubes (Becton Dickinson) from patients referred to the Communicable Disease Centre, Tan Tock Seng Hospital, Singapore. Blood specimens were obtained from patients consenting to the study. All patients gave separate written informed consent. The study protocols were approved by the SingHealth Centralized Institutional Review Board (reference 2016/2219) and by the National Healthcare Group Domain Specific Review Board (reference 2015/00528).

Patients Samples This study included plasma samples obtained from 94 ZIKV patients admitted to the Communicable Disease Centre at Tan Tock Seng Hospital (TTSH) from 27 August 2016 through 14 August 2017 and 70 DENV patients from 29 April 2016 through 28 March 2017. Samples were collected at 2 phases: acute (1-6 days post onset of symptoms) and early convalescent (7-21 days post onset of symptoms). Patients may donate blood samples multiple times during each phase. Only 11/94 (12%) of patients from the Zika cohort and 12/70 (17%) of patients from the Dengue cohort had travelled within 2 weeks upon recruitment. Therefore, it could be concluded that the majority of the patients were infected due to local transmission.

Among the ZIKV patients, 41 (43.62%) were females and 53 (56.38%) were males. The recruited patients were mostly Chinese (77 or 81.91%), with 7 Malays (7.45%) and 5 (5.32%) Indians and other ethnic groups each. The median age of the patient was 39 years (range, 14-72 years). These patients were confirmed to be infected with ZIKV according to RT-PCR using an adapted protocol (Lanciotti, R.S., Kosoy, O.L., Laven, J.J., Velez, J.O., Lambert, A.J., Johnson, A.J. et al. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis. 2008; 14: 1232-1239) performed on plasma and urine samples obtained during their first visit to the Communicable Disease Centre. In addition, all ZIKV patients were tested for dengue NS1 using the SD BIOLINE Dengue Duo rapid test, and 3/94 patients were further confirmed DENV NSl-positive by DENV RT-PCR, indicating a concurent DENV infection (22).

Among the DENV patients, 19 (27.14%) were females, and 51 (72.86%) were males. The recruited patients were mostly Chinese (41 or 58.57%), with 7 Indians (10%), 5 Malays (7.14%), and 17 (24.29%) from other ethnic groups making up the cohort. The median age of the patients was 35 years (range: 22-60 years). The DENV patients recruited were tested NS1 positive from hospital routine diagnostics using the SD BIOLINE Dengue Duo rapid test. All NS1 positive samples were confirmed to be dengue positive using the FTD Zika / dengue/ chikungunya RT-PCR (Fast Track Diagnostics). Dengue serotypes were further determined by FTD dengue differentiation RT-PCR test (Fast Track diagnostics) according to manufacturer’s instructions (see Supplementary Materials). For the validation tests, there were 70 ZIKV samples with 62 unique patients (as 9 patients had more than one sample collected during the time period), and 81 DENV samples with 68 unique patients (13 patients having more than 1 sample collected) in the acute phase (1-6 days post onset of symptoms). From the early convalescent phase (7-21 days post onset of symptoms) there were 48 ZIKV samples with 44 unique patients and 70 DENV samples with 53 unique patients with some patients having more than one sample in each phase. Samples were randomized and blinded during testing.

During assay optimization, a subset of samples from TTSFi and a commercial vendor (Seracare) were used, and this combined sample pool was designated as the training set (ZIKV = 37, DENV = 67). TTSFi samples have records of the day of collection post onset of symptoms (ZIKV = 27, DENV = 46 ), whereas this information was not available for the commercial samples (ZIKV = 10, DENV = 21). Seracare panel 0845- 0142 (ZIKV) and 0845-0074 (DENV) were respectively used for training, while samples DSC-7, 12 and 20 from Seracare panel 0845-0051 (DENV) and ZPC-1, 2, 4 and 8 (ZIKV, country of origin Columbia) acquired via Singapore vendor (Precision Technologies) were used for characterization of engineered ZIKV NS1.

Development of the Zika serological tests

Cloning, expression, and purification of Zika NS1 antigens

Flaviviruses NS1 and ZIKV WT (WT-NS1, Uganda strain) were purchased from Native Antigen (Oxford, UK). Zika NS1 construct (French Polynesia/10087PF/2013; accession KX447521.1) was used for in house protein engineering and expression. ZIKV NS1 mutants, peptide fragments, and domain fragments were generated from either gene synthesis (Integrated DNA Technologies (IDT) or Bio Basic) or primers containing the designed residue mutations (IDT). Plasmids containing related ZIKV NS1 antibodies and ZIKV NS1 proteins were expressed in mammalian cells with ExpiCFIO system (Thermo Fisher). Culture supernatants were harvested and purified using FiisPur Ni- NTA resin (Thermo Fisher) for Fiis-tagged proteins and protein A beads (Amintra) for antibodies, respectively. Proteins were buffer exchanged with PBS buffer (Thermo Fisher) using Amicon Ultra 15 (Millipore), and quantified by using NanoDrop spectrophotometer.

Immunochromatographic Assays

Format 1: Nitrocellulose membrane strips (Millipore) were spotted with 0.33 pi of 0.5 mg/ml H-zMut2 (test line) and 0.33 mΐ of 0.2 mg/ml polyclonal goat anti-human IgG or 0.33 mΐ of 0.2 mg/ml mouse anti- human IgM antibody (control line). The strips were dried at 37 °C for 5 min and blocked with blocker casein in TBS for 30 min. The strips were then washed with borate buffer supplemented with 1% sucrose and 0.01% SDS and dried for an hour at 37 °C. The strips were assembled with the glass fiber filter and absorption pad. For the IgM test, 5 mΐ of diluted sample (1:220) was passed through the membrane, followed by 15 mΐ of PBS buffer containing 0.05% Tween and 1% BSA. The strips were then added with polyclonal goat anti- human IgM-AP (Fitzgerald), followed by 10 mΐ of washing buffer. Subsequently the strip was dipped in 200 mΐ of BCIP/NBT (MOSS) for 7.5 min. The reaction was stopped by dipping the membrane strip in 0.3 M NaOH. For the IgG test, 5 mΐ of diluted sample (1 :400) was passed through the membrane, followed by 15 mΐ of the washing buffer. The strips were then added with goat anti-human IgG Fc HRP (Thermo Fisher), followed by 10 mΐ washing buffer. The membrane strip was dipped in 200 mΐ of metal-enhanced DAB solution (Thermo Scientific) for 5 min. The strips were dipped in water before imaging. All the strips were imaged using Bio-Rad ChemiDoc™ MP Imaging System under autoscale setting.

Format 2: To prepare the test strips, 1 mΐ of anti-human IgG and IgM capture antibodies (1 mg/ml) were immobilized on nitrocellulose membrane strips (3 mm width x 25 mm length) at the downstream and upstream portion, respectively, via vacuum drying. The test strips were then blocked with casein (1% w/v), washed with borate buffer, and vacuum dried before use. To prepare the conjugate pad, the H-zMutl antigen was first conjugated to 40-nm gold nanoparticles (Au NPs) via covalent binding. The conjugated Au NPs were then diluted to 0.5 OD using casein buffer, and then dried on glass fiber strips (3 mm width x 30 mm length). The nitrocellulose test strips were assembled with the glass fiber and an absorbent pad (10 mm width x 20 mm length). Patient plasma (neat, 5 mΐ) were applied to the upstream portion of the nitrocellulose test strip. 60 mΐ of chasing buffer (lx PBS) was then applied to the glass fiber conjugate pad. As the patient plasma and Au NPs flowed past the IgG/IgM test spots, a visible red signal could be observed by the naked eye within 15 minutes.

ZIKV IgM and IgG ELISA assay

Polystyrene plates were coated overnight with 1 pg/ml of ZIKV NS 1 -related antigen in PBS buffer, and blocked with blocking buffer (PBS with 10% non-fat dry milk (Bio- Rad)). To perform the ZIKV IgM ELISA assay, patient serum diluted in blocking buffer was mixed with IgG/Rf stripper (Bio-Rad) (e.g. 0.5 pi of sample with 2 pi of IgG stripper in a total of 60 mΐ to make a 1:120 sample dilution) and incubated for 30-45 min. After incubation, the sample was transferred to the ZIKV NS 1 -coated plates and incubated for 25 min at 37°C. Plates were washed, and anti-human IgM-HRP (Abeam) (1:4300 dilution) was added for additional 10 min incubation at 37°C. Plates were washed again, and the TMB substrate was added for 10 min before stopping with stop solution (KPL). For ZIKV IgG ELISA assay, blocking buffer containing patient serum (1:250 dilution) was transferred to ZIKV NS1 antigens-coated plates and incubated for 12 min at 37°C. Upon washing, the plates were added with anti-human IgG-HRP (Thermo Fisher) (1:5500 dilution) for 10 min incubation at room temperature, followed by substrate incubation for 7 min, and stopped by adding stop solution and measuring absorbance at 450nm. Sera samples mean OD were measured from 2 replicates. When the ELISA assays was using sera samples from training set and validation set, mean optical density (OD) of the sera samples (P) divided by the mean OD of internal standard (I). P/I ratio of >1.5 were considered as Zika positive for both IgM and IgG assays. The internal standard was built based on a commercial dengue sample that consistently showed minimal cross reactivity in the assays.

Monoclonal antibody generation and production

The anti-ZIKV NS1 antibodies Cll and C12 were generated in New Zealand White rabbits according to an approved IACUC protocol. Briefly, animal was immunized with recombinant ZIKV WT NS1 (The Native Antigen) and the titer was checked after every round of immunization. Upon achieving a high titer, B cells were isolated and sorted for culture. Supernatants from the B cells were tested using ZIKV NS1 antigen ELISA and variable regions were then recovered from positive clones for sub-cloning into expression vector containing rabbit constant regions. Monoclonal antibodies were expressed in CHO cells for large-scale production according to manufacturer’s protocol (Gibco). Antibodies were purified with Protein A resins (Amintra), buffer-exchanged into PBS (Gibco), and concentrated using Amicon Ultra centrifugal filters (Merck Millipore).

ZIKV NS 1 ELISA

Polystyrene plates were coated with 1 pg/ml of C12 in PBS and incubated overnight at 4°C. The plates were blocked with 2% bovine serum albumin, fraction V (BSA, Capricorn) in PBS before use. After washing with PBS, 45 pi of serially diluted recombinant ZIKV NS1 antigen (ranging from 0 to 6.4 ng/ml) in normal human serum control or patient sample were co-incubated with 5 mΐ of 10% BSA and 1% PBST for 1 hr at 37°C. 10 mg/ml of recombinant DENV1 NS1 in normal human serum control was included as negative control. Plates were then washed 5 times with 0.2% PBST and once with PBS. Next, an optimized amount of biotinylated Cll in 1% BSA 0.1% PBST was added for 1 hour incubation at room temperature. The plates were washed and incubated at room temperature with streptavidin-poly-HRP (SDT) diluted in 1% BSA 0.1% PBST for 30min. The plates were then developed with 100 mΐ of TMB for 15 min and terminated with 50 mΐ of stop solution. The absorbance at 450 nm was taken using Tecan M200 plate reader. The limit of detection was set at 2x the OD450 of the background (normal human serum). The ZIKV NS1 level in the samples was estimated through interpolation from a standard curve. The cut-off was established by 3 standard deviations (S.D) from the mean values of 45 DENV patient samples interpolated from the standard curve.

Example 1

Engineering the full-length NS1 protein for serological assays

It was first hypothesized that the ZIKV NS1 can be used to develop a specific and sensitive serological test, since it was possible to generate monoclonal antibodies specific for this antigen without cross reactivity to NS1 from other flaviviruses. This suggested that NS1 contained epitopes that are specific to ZIKV immune sera and low reactivity to DENV immune sera. This idea was tested by generating a series of NS1 peptides predicted to be immunogenic, but these ZIKV NS1 peptides showed no reactivity to commercial ZIKV human sera (ZPC-1, ZPC-2, ZPC-4, ZPC-8) (data not shown). Furthermore, as the full-length wildtype Zika NS1 was poorly expressed in the mammalian system, the inventors turned to derive various ZIKV NS1 domains and full- length proteins fused to different carriers at the N or C terminus. The protein solubility of engineered ZIKV NS 1 , and also reactivity to the ZIKV and DENV sera, respectively, were considered.

Amongst the different construct designs, it was determined that the His-tagged albumin domain (H, residue 1-200 aa) N-terminally fused to the NS1 variants, resulting in H- zWT (NS1: 1-352 aa) and H-zDl (NS1: 172-352 aa), showed reasonable solubility (at least 1 mg per 40-80ml of culture). Using IgG ELISA, it was shown that the 2 constructs had good reactivity to the commercial ZIKV samples (ZPC-1, ZPC-2, ZPC-4, and ZPC- 8, with OD >1.0; Fig 2A, i), except that H-zDl only showed reactivity with OD > 1.0 in 1 TTSH ZIKV sera , compared to H-zWT (Fig 2A, ii). Interestingly, it was observed that ZIKV WT and DENV WT (obtained from Native Antigen) both showed similar binding activity as H-zDl for these TTSH sera (Fig 2A, ii).

While the full-length ZIKV NS1 was not expressed in soluble form with the thioredoxin (Trx) at the C-terminus, the inventors were able to produce 2 soluble forms of ZIKV C- terminal constructs: zDl-Trx (residue 172-352 aa) and zD2-Trx (172-339 aa). It was asked if truncation at the C-terminus would differentiate zD2-Trx from zDl-Trx in DENV IgG cross reactivity. Amongst the DENV samples from the Seracare commercial panel 0845_0051 that were available at the time (DSC-7, DSC-12 and DSC-20), it was found that DSC-7 showed cross reactivity to the ZIKV WT. It was then shown that the zD2-Trx has drastic reduced IgG ELISA activity to DSC-7, compared to zDl-Trx (Fig 2A, iii). Though this was observed only with 1 DENV sera, it was hypothesized that perturbing residues conserved between DENV and ZIKV in region of 339-352 could reduce DENV IgG cross reactivity.

To validate this hypothesis, a series of mutants spanning the 339-352 amino acids region of H-zWT construct was generated, since this format was the most reactive to ZIKV sera IgG. Of all the mutants, H-zMutl (V350T, N344D P341Q) and H-zMut2 (A352D, T351H, S348D, N344K, P341H) were selected for their reasonable soluble expression, and also their ability to reduce DENV cross reactivity without greatly compromising ZIKV signal, in both the ELISA and IA formats. It was first shown that H-zMut2 had a greater reduction to DSC-7 in IgG activity, compared to H-zWT and H-zMutl in IgG ELISA (Fig 2A, iv). H-zMut2 was then used as the capture antigen for optimizing the ELISA for specific binding to IgM and IgG with a collection of plasma samples designated the “training set”, which consisted of samples from TTSH and commercial sources. Indeed, under the optimized ELISA conditions and presented as normali ed OD, H-zMut2 resulted in IgM/G detection with sensitivity and specificity greater than 80% (Fig 2B, i and ii). The cross reactivity to DENV sera was further validated and it was found to be indeed lower for that of H-zMut2 compared to H-zWT and ZIKV WT, in the blinded test evaluation.

H-zMut2 ELISA for blinded test evaluation

Upon achieving the desired performance with the training set, the assay was evaluated on a larger group of samples in a blinded manner. This “validation set” consisted of 269 ZIKV and DENV patient samples collected by Tan Tock Seng Hospital. Among the three engineered antigens, H-zMut2 showed greater detection sensitivity and specificity than ZIKV WT, but only slightly lower in sensitivity though with higher in specificity, compared to H-zWT (Fig.3, Table 4). In the ELISA test, H-zMut2 showed low sensitivity with acute samples (IgM/IgG: 41%/23%) albeit high specificity (IgM/IgG: 100% 191%) (Fig. 3, Table 1). The result reflected the low IgG titer during the acute phase of Zika infection, consistent with other studies (Fig 3D, 3E, Table 1, Table 4). Comparing to H-zMut2, ZIKV WT showed much lower sensitivity (IgM/IgG: 3%/14%) (Table 4). In contrast to the acute samples, H-zMut2 capture antigen showed relatively high sensitivity when tested on convalescent samples (sensitivity IgM/IgG: 79%/83%, specificity IgM/IgG: 95%/84%), and continued to outperform ZIKV WT (sensitivity IgM/IgG: 33%/56%, specificity IgM/G: 98%/73%) (Fig. 3, Table 1, Table 4).

Given that the IgM or IgG ELISA with H-zMut2 each detected a different subset of ZIKV samples, combining the IgM/IgG test results could achieve a greater sensitivity for both acute (17% [WT] < 52% [mut2]) and convalescent samples (83% [WT] < 89% [mut2] ) (Table 4). Although H-zWT was more sensitive than ZIKV WT in individual IgM/IgG tests, both antigens showed comparable combined sensitivity (Table 4). The WT sequence, however, was more cross-reactive to DENV IgG (specificity: 54%[H- zWT] < 71%[ZIKV WT] < 80%[H-zMut2]).

Table 1. Summary of sensitivity and specificity results for the validation set in blinded evalution. ELISA and IA assays were evaluated for the detection of NS1, IgM and IgG with TTSH plasma samples (ZIKV: n = 70 with 1-6 d.p.o, and n = 48 with 7- 16 d.p.o; DENV: n = 81 with 1-6 d.p.o, and n = 70 with 7-21 d.p.o; d.p.o: days post onset of symptoms). Sensitivity and specificity were determined with positive plasmas over the total number of respective ZIKV and DENV plasma samples.

Table 2. Summary for sensitivity and specificity comparison between GenBody and in-house IA assays. All IA assays were evaluated with TTSH plasma for IgM and IgG test (ZIKV n = 42, DENV n = 39, 7-16 d.p.o, subset of blinded test samples). GenBody strips were tested in a non-blinded approach, and compared to that of FI and F2 results that were obtained from the blinded test of the validation set.

Table 3. H-zMut2 ELISA for training set. Both IgM and IgG ELISA were tested with plasma as indicated by using H-zMut2 as the capture antigen. Detail of the training set was described in the Material and Methods section (main text). For ZIKV IgM test, 27 ZIKV plasma and 31 DENV plasma samples were obtained from TTSH, and 10 ZIKV sera and 21 DENV sera were obtained from commercially available source. For ZIKV IgG test, 27 ZIKV plasma and 46 DENV plasma samples were obtained from TTSH, and 10 ZIKV sera and 21 DENV sera were obtained from commercially available source. Table 4. Capture antigens-ELISA for validation set. The capture antigens — H- zMut2, WT-NS1 and H-zWT, were tested respectively for detecting IgM and IgG in a blinded manner; sensitivity and specificity were determined against ZIKV and DENV plasma, respectively. In the combined IgM/IgG tests, normalized OD > 1.5 either in IgM or IgG test for respective plasma sample was determined as positive. Plasma sample details and information were described in the Material and Methods section.

Table 5. Capture antigens in immunochromatographic assay (FI IA) for IgM and IgG detection. The capture antigens — H-zMut2, WT-NS1 and H-zWT were tested respectively with the training set as defined in the Material and Methods section. Both IgM and IgG detection in FI IA format were evaluated with samples as indicated. For ZIKV IgM and IgG test, 27 ZIKV plasma and 46 DENV plasma were obtained from TTSH, and 10 ZIKV sera and 21 DENV sera were obtained from commercially available source.

Table 6. H-zMut2 ELISA for patient plasma samples collected over 2 time points.

Total of 35 patient specimens were assayed for detecting ZIKV IgM and IgG. Increase in the normalized OD for both IgM and IgG can be observed in most cases at the second time point, upon disease progression (30 of 35 cases). 28 out of the 35 patients were tracked from acute to convalescent phase (n = 28). Normalized OD > 1.5 was highlighted in grey color.

Example 2

Engineered NS1 antigens for rapid test assay

To develop immunochromatographic assay that would allow rapid diagnosis of ZIKV infections, both candidates - Fl-zMutl and Fl-zMut2 - were evaluated using two different assay formats. The first format (FI), similar to the ELISA approach, utilized the engineered proteins as capture antigens for ZIKV IgM and IgG on two independent strips, and a detector antibody conjugated to enzyme for signal amplification (Fig 4A). In the second format (F2), the antigens were conjugated to gold nanoparticles and served as a detector for binding patient IgM and IgG that were captured on two different spots on the same strip (Fig 5).

With the FI format, H-zMut2 showed greater sensitivity than H-zMutl for capturing ZIKV IgM/IgG (data not shown). In general, H-zMut2 showed greater detection sensitivity and specificity than ZIKV WT (except slight lower in IgM specificity, 89.6%[H-zMut2] vs 95.5%[ZIKV WT]), bwith greater IgG specicity than H-zWT though with comparable sensitivity (Fig. 4A, table 5). While H-zWT also showed improved sensitivity compared to ZIKV WT (IgM: 49% [WT] < 81% [H-zWT]; IgG: 70% [WT] < 97% [H-zWT]), this engineered WT had lower IgG specificity than H- zMut2 and ZIKV WT (Fig. 4A, table 5).

H-zMut2-Fl and H-zMutl-F2 for blinded test evaluation

When H-zMut2-Fl assay was evaluated with the validation set in a blinded manner, it showed 51%/95%(IgM) and 44%/93% (IgG) sensitivity/specificity for the acute phase samples (Table 1). Unlike the acute plasma samples, FI assay could achieve > 70% test performance for convalescent samples (sensitivity: IgM/IgG: 71%/90%; specificity: IgM/IgG: 87%/79%). Combining both IgM and IgG tests increased the sensitivity for acute phase samples (69%), without greatly lowering the specificity (89% vs 95%) (Table 1). Although the combined tests showed no major change in sensitivity with convalescent samples (90%), there was a slight decrease in the specificity (69% [IgM + IgG] < 79% [IgG] < 87%[IgM]) (Table 1).

Conversely, H-zMutl antigen showed greater sensitivity than H-zMut2 in the F2 format (data not shown). Table 1 showed that the F2-H-zMutl assay had lower sensitivity, noticeably in IgG detection, when evaluated in the blinded test. Flowever, when both IgM and IgG tests were combined, the result showed greater sensitivity — 60% for acute and 88% for convalescent samples, while maintaining excellent specificity of 96% and 84% for acute and convalescent samples, respectively (Table 1).

Performance comparison: F1/F2 I A format and a commercial kit

A commercially available ZIKV IgM/IgG rapid test kit (GenBody) was evaluated with TTSF1 samples, and the results were compared to the FI and F2 IA formats obtained from the blinded samples test. The Genbody kit utilized the anti E (envelope) and NS1 antibodies in complex with E/NS1 antigen for detecting ZIKV IgM/IgG. This commercial kit was previously reported to exhibit high sensitivity and specificity for both IgM and IgG (> 90%) (24). As shown in Table 2, the Genbody tests did not perform as well as FI and F2 IA when applied to the samples from the validation set. In particular, the Genbody test showed low sensitivity for IgM (29%) and low specificity for IgG (62%). The combined IgM/IgG test from Genbody showed low specificity (56%) but reasonable sensitivity (79%).

Addition ofZIKVNSl test improved the sensitivity for acute phase samples

The detection of DENV NS1 in serum has been reported to be a suitable method for diagnosing acute DENV infections. It was hypothesized that by detecting NS1 antigen in acute ZIKV -infected plasma, this assay could improve the sensitivity of the IgM/IgG test, since ZIKV belongs to the same flavivirus family as DENV. Monoclonal antibodies specific against ZIKV NS1 antigen were generated, and antibody pairing for quantitative ELISA (Fig. 7A) was optimized. Using normal human serum spiked with recombinant ZIKV NS1, 0.1 ng/ml was established as the detection limit in the assay (Fig. 7B). Based on testing 45 DENV samples, a cut-off above 0.25 ng/ml was set as being ZIKV NS1 positive (Fig. 7B).

The performance of The NS1 ELISA was next evaluated by testing the validation set in a blinded fashion. The area under the receiver operating characteristics (ROC) curve plotted with ZIKV infected and non ZIKV infected samples was 0.715 suggesting the assay was able to differentiate between these two group of patients with sensitivity/specificity of 41%/ 98% for acute phase samples (Table 1 and Fig. 6A). Importantly, it was found that the ZIKV NS1 concentration was extremely low or undetectable in most of the patient samples. Among ah the ZIKV infected acute samples, only 7% had NS1 > 1 ng/ml; 34% was in the range of 0.25-1 ng/ml, and 60% of the samples had NS1 level below the detection limit (Fig. 6B, C). However, when complementing NS1 antigen detection with either IgM or IgG ELISA, the sensitivity of detection can be improved for acute phase infections (53% [IgM+IgG] < 56% [IgM+NSl] < 61% [IgG+NSl]) (Table 1). Upon combining all three tests (NSl/IgM/IgG), the ELISA sensitivity was further improved to 67% while maintaining a high specificity (96%).

Analysis of acute phase patient samples

A total of 151 acute phase samples were tested with ELISA and IA methods (1-6 d.p.o, ZIKV n = 70, DENV n = 81). The data suggested that combination of three immuoassays - NS1, IgM and IgG were needed to achieve a reasonable detection sensitivity in the acute phase. Among the 70 acute serum samples, the ELISA tests were able to detect ZIKV infection as early as 2 days after fever onset, through detecting NS1 (7 cases), IgM (4 cases), or IgG (2 cases). The overall detection rate for the 70 acute samples was 41% for NS1 (29 cases), 41% for IgM (29 cases), and 22% for IgG (16 cases). Only 8 out of the 70 acute cases were positive for both IgM and IgG. Among the 29 samples positive for NS 1, 19 were positive for IgM and 2 were positive for IgG. Within the validation set (acute and convalescence, n = 118), 35 patients provided their blood samples at two different time points (Table 6). It was observed increased IgM and IgG levels in most of the samples by ELISA, upon disease progression over time (30 of 35 cases). For 28 of these patients, the first collection was in the acute phase and the second in the convalescent phase.

Table 7 shows the rationale of making the various mutations to the polypeptide (see also Fig. 8).