METHOD FOR DETECTION OF ZIKA VIRUS SPECIFIC ANTIBODIES
20230324404 · 2023-10-12
Assignee
Inventors
- Erick PEREZ-GUZMAN (Cambridge, MA, US)
- Eduardo NASCIMENTO (Cambridge, MA, US)
- Hansi DEAN (Cambridge, MA, US)
- Ralph BRAUN (Cambridge, MA, US)
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
C12N2770/24134
CHEMISTRY; METALLURGY
G01N2469/20
PHYSICS
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
Abstract
The present invention is directed to a method, i.e. an immunoassay, for determining the presence and/or the amount of anti-zika Anti-ZIKV #1 virus antibodies, i.e. zika virus-specific antibodies in a sample. Therefore, the present invention is directed to a microsphere complex comprising a microsphere coupled to a zika virus like particle, as well as to a kit comprising said microsphere complex and an amount of reporter antibody that binds to the zika virus like particle. The present invention further relates to a method for determining an antibody correlate of protection against zika virus infection for a zika virus vaccine. Moreover, the present invention is directed to a method for diagnosing the protection of a human or non-human subject against a zika virus infection.
Claims
1. A microsphere complex comprising a microsphere coupled to a zika virus like particle.
2. The microsphere complex of claim 1, wherein the zika virus like particle comprises the envelope glycoprotein, membrane protein, and/or pre-membrane protein which are at least 70%, or at least 75%, or least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to corresponding parts of SEQ ID NO: 2.
3. A kit comprising: an amount of the microsphere complex of claim 1, and an amount of a reporter antibody that binds to the zika virus like particle of the microsphere complex, preferably wherein the reporter antibody is a zika virus neutralizing antibody, and/or does not cross-react with antigens from other flaviviruses, such as dengue virus, West Nile virus, Japanese encephalitis virus, Yellow Fever Virus, St. Louis Encephalitis virus, and Tick Borne Encephalitis virus.
4.-5. (canceled)
6. The kit of claim 3, wherein the reporter antibody is attached to at least one detectable label, optionally by the heavy chain constant region of the reporter antibody.
7. The kit of claim 6, wherein the at least one detectable label is a fluorescence label, such as xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin, cyanine, coumarin, and any derivative thereof.
8. The kit of claim 3, wherein the reporter antibody provides an EC.sub.50 value towards the zika virus like particle coupled to the microsphere within the microsphere complex of less than 0.5 μg/mL, or less than 0.4 μg/mL or less than 0.3 μg/mL or less than 0.2 μg/mL or less than 0.15 μg/mL or less than 0.1 μg/mL or less than 0.09 μg/mL or less than 0.08 μg/mL or less than 0.07 μg/mL or less than 0.05 μg/mL or less than 0.04 μg/mL or less than 0.03 μg/mL or less than 0.01 μg/mL.
9. The kit of claim 3, wherein the reporter antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 7, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 8, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 9, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 12, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 13, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 14, or a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 21, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 22, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 23, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 26, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 27, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 28; or a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 35, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 36, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 37, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 40, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 41, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 42, or a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 49, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 50, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 51, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 54, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 55, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 56, or a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 63, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 64, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 65, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 68, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 69, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 70, or a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 76, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 77, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 78, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 80, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 81, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 82; or a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 84, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 85, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 86, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 88, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 89, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 90, or a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 95, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 96, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 97, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 100, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 101, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 102, or a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 109, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 110, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 111, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 114, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 115, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 116, or a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 123, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 124, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 125, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 128, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 129, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 130.
10.-18. (canceled)
19. A method for detecting a signal from a reporter antibody indicative for the presence and/or amount of anti-zika virus antibodies in a sample from a subject comprising the steps of: Step 1: providing a kit according to claim 3, including an amount of said microsphere complex and an amount of said reporter antibody, Step 2: contacting the amount of said microsphere complex and the amount of said reporter antibody of step 1 with the sample to allow binding of the anti-zika virus antibodies in the sample to the zika virus like particles coupled to the microspheres in the microsphere complex while competing with the reporter antibody, and Step 3: detecting a signal from the reporter antibody bound to the zika virus like particles coupled to the microspheres in the microsphere complex in step 2
20. The method of claim 19, comprising the steps of: Step 1: providing a kit according to claim 3, including an amount of said microsphere complex and an amount of said reporter antibody, Step 2.1: contacting the amount of said microsphere complex of step 1 with the sample to allow binding of the anti-zika virus antibodies in the sample to the zika virus like particles coupled to the microspheres in the microsphere complex, Step 2.2: contacting said amount of reporter antibody with said microsphere complex and the sample of step 2.1 to allow binding of the reporter antibody to the zika virus like particles coupled to the microspheres in the microsphere complex, and Step 3: detecting a signal from the reporter antibody bound to the zika virus like particles coupled to the microspheres in the microsphere complex in step 2.2.
21. The method of claim 19, comprising the steps of: Step 1: providing a kit according to claim 3, including an amount of said microsphere complex and an amount of said reporter antibody, Step 2.1: contacting the amount of said microsphere complex of step 1 with the sample to allow binding of anti-zika virus antibodies in the sample to the zika virus like particles coupled to the microspheres in the microsphere complex, Step 2.2: contacting said amount of reporter antibody with said microsphere complex and the sample of step 2.1 to allow binding of the reporter antibody to the zika virus like particles coupled to the microspheres, Step 2.3: contacting said amount of reporter antibody, said amount of microsphere complex, and the sample of step 2.2 with an amount of a secondary reporter antibody to allow binding of the secondary reporter antibody to the constant region of the reporter antibody, and Step 3: detecting a signal from the secondary reporter antibody bound to the reporter antibody in step 2.3, wherein the reporter antibody is bound to the zika virus like particles coupled to the microspheres in the microsphere complex in step 2.2.
22. The method of claim 19 for detecting the presence and/or amount of anti-zika virus antibodies in a sample from a subject comprising the further steps of: Step 4: determining the presence and/or the amount of the reporter antibody bound to the zika virus like particles coupled to the microspheres in the microsphere complex from the signal of step 3, and Step 5: determining the presence and/or the amount of anti-zika virus antibodies in the sample based on the presence and/or the amount of the reporter antibody determined in step 4.
23. The method of claim 19, wherein the sample is a sample from the group consisting of blood, urine, serum, blood plasma, cerebrospinal fluid, and lymph fluid.
24. The method of claim 19, wherein the subject is a subject from the group consisting of mouse, primate, non-human primate, human, rabbit, cat, rat, horse, and sheep.
25. A method for determining an antibody correlate of protection against zika virus infection for a zika virus vaccine in a type of non-human subjects comprising the steps of: Step 1: selecting a group of said subjects which are zika virus naive, Step 2: dividing the group of subjects into at least two subgroups, wherein one subgroup functions as control group and at least one subgroup functions as inoculation group, Step 3: inoculating said at least one inoculation group with a dose of the zika virus vaccine, Step 4: challenging all subjects with an infectious amount of the zika virus, Step 5: determining the amount of anti-zika virus antibodies for each subject according to claim 19 at least after inoculation with the zika virus vaccine and before challenging with the infectious amount of the zika virus, Step 6: determining presence or absence of viremia in all subjects after challenging with the infectious amount of the zika virus, Step 7: repeating steps 3 to 6 with further inoculation groups with increasing vaccine doses until absence of viremia is determined in all subjects of one inoculation group in step 6, and Step 8: determining the amount of anti-zika virus antibodies after inoculation with the zika virus vaccine and before challenging with the infectious amount of the zika virus associated with absence of viremia after challenging with the infectious amount of zika virus as antibody correlate of protection.
26. A method for determining an antibody correlate of protection against zika virus infection in human subjects by mathematically modeling the correlate of protection of a non-human subject as determined according to claim 25 to fit human subjects.
27. A method for diagnosing the protection of a human subject against a zika virus infection comprising the steps of: Step 1: providing a sample from the human subject outside the human body, Step 2: determining the amount of anti-zika virus antibodies in the sample from the human subject according to claim 19, and Step 3: determining protection by comparing the amount of anti-zika virus antibodies determined in step 2 to the antibody correlate of protection against zika virus infection in human subjects.
28. A method for diagnosing the protection of a non-human subject against a zika virus infection comprising the steps of: Step 1: providing a sample from the non-human subject outside the non-human body, Step 2: determining the amount of anti-zika virus antibodies in the sample from the non-human subject according to claim 19, and Step 3: determining protection by comparing the amount of anti-zika virus antibodies determined in step 2 to the antibody correlate of protection.
29. A method for diagnosing a zika virus infection in a subject comprising the steps of: Step 1: providing a sample from the subject outside the subject body, Step 2: determining the amount of anti-zika virus antibodies in the sample according to claim 19, and Step 3: determining infection by comparing said amount of anti-zika virus antibodies to established amounts of anti-zika virus antibodies in zika virus infected subjects.
30. The method for diagnosing a zika virus infection according to claim 29, wherein the subject is a human.
31. The method of claim 29, wherein the zika virus infection is acute.
32. The method of claim 29, wherein the zika virus infection is convalescent.
33. A method for detecting a signal from a detection antibody indicative for the presence and/or amount of anti-zika virus antibodies in a sample from a subject comprising the steps of: Step 1: contacting an amount of a microsphere complex according to claim 1 with the sample to allow binding of anti-zika virus antibodies in the sample to the zika virus like particles coupled to the microspheres in the microsphere complex, Step 2: contacting an amount of a detection antibody with the microsphere complex and the sample of step 1 to allow binding of the detection antibody to the heavy chain constant region of the anti-zika virus antibodies bound to the zika virus like particles coupled to the microspheres in the microsphere complex, wherein the detection antibody binds to the anti-zika virus antibodies with the variable region of the detection antibody and wherein the detection antibody is attached to at least one detectable label, and Step 3: detecting a signal from the detection antibody bound to the anti-zika virus antibodies in step 2.
34. The method according to claim 33 for determining the presence and/or amount of anti-zika virus antibodies in a sample from a subject, wherein the method comprises the further steps of: Step 4: determining the presence and/or amount of the detection antibody bound to the anti-zika virus antibodies from the signal of step 3, and Step 5: determining the presence and/or amount of anti-zika virus antibodies in the sample from the presence and/or amount of the detection antibody determined in step 4.
35. A method for preventing zika disease in a human subject comprising the steps of: Step 1: obtaining a sample from the human subject, Step 2: determining the amount of anti-zika virus antibodies in the sample from the human subject according to claim 19, Step 3: determining whether the human subject has an amount of anti-zika virus antibodies to confer protection by comparing the amount of anti-zika virus antibodies determined in step 2 to the antibody correlate of protection against zika virus infection in human subjects, and Step 4: administering to the human subject a zika virus vaccine if the human subject has an amount of anti-zika antibodies that is lower than the antibody correlate of protection against zika virus infection in human subjects.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
Microsphere Complex
[0201] It is an object of the present invention to provide an immobilizable binding partner for anti-ZIKV Abs. Such binding partners are required to bind and thereby to detect anti-ZIKV Abs. These binding partners are immobilized to allow detection of the binding in immunoassays. The usual immobilization is carried out on a plate as is e.g. known from the enzyme linked immunosorbent assay (ELISA) setting. Such a set-up including a plate and an enzyme-based detection, however, has certain disadvantages. These disadvantages include the risk of false results due to insufficient blocking, the risk that the activity of the enzyme used for detection (e.g. horseradish peroxidase) may be hampered by sample constituents, time-consuming operation (multiple steps required including washing procedures), as well as a separate assay requirement for each antigen to be analyzed, thereby requirement of larger amounts of reagents. Moreover, the colorimetric readout of the ELISA often lacks sensitivity as enzyme amplification is required and therefore is prone to variability and errors in the amount of amplification. Therefore, assays based on antigen immobilization on microspheres as in Microsphere Immune Assays (MIAs), such as the Luminex set-up, have been developed, which have the advantages of high specificity and reactivity, flexibility to single- or multiplex antigens from different viruses in one single experiment, the possibility for high-throughput, cost-effectiveness, low sample volumes and short turnaround times. Moreover, the data are more accurate and reliable compared to conventional methods such as ELISA, as the data are calculated from the mean of hundreds of microspheres, each of which functions as an individual replicate. The immobilization of an appropriate antigen on such a microsphere is, however, challenging and the precondition to use the advantages of the MIA setting.
[0202] The present invention therefore provides a microsphere complex comprising a microsphere coupled to a zika antigen, in particular in the form of a microsphere coupled to a zika virus like particle (ZIKV VLP).
Microsphere
[0203] The microsphere useful for the invention ranges in the size from about 0.01 to about 100 μm in diameter, more preferably from about 1 to about 20 μm, and most preferably a microsphere has a diameter from about 5 to about 7 μm. In a preferred embodiment the microsphere has a diameter of about 6.5 μm. The size of a microsphere can be determined in practically any flow cytometry apparatus by so-called forward or small-angle scatter light.
[0204] The microsphere may be constructed of any material to which molecules like VLPs or EDIII may be attached to. For example, acceptable materials for the construction of microspheres include but are not limited to: polystyrene, polyacrylic acid, polyacrylonitrile, polyacrylamide, polyacrolein, polybutadiene, polydimethylsiloxane, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene, polymethylmethacrylate, or combinations thereof. In a preferred embodiment of the present invention, microspheres are constructed of polystyrene.
[0205] The microsphere may comprise affinity groups for attachment of molecules, such as antigens of the present invention. Said affinity groups may be, but are not limited to, Ni.sup.2+ (for immobilization of His-tagged molecules like EDIII), Protein A, Protein G, Protein L, anti-human IgG Ab, anti-rabbit IgG Ab, anti-mouse IgG Ab, anti-goat IgG Ab, anti-FLAG Ab, streptavidin, avidin, and glutathione.
[0206] The microsphere may comprise functional groups on the surface useful for attachment of molecules, such as the antigens of the present invention. Said functional groups may be, but are not limited to, carboxylates, esters, alcohols, carbamides, aldehydes, amines, sulfur oxides, nitrogen oxides, maleimides, or halides. In a preferred embodiment the microsphere comprises carboxylates on the surface. Molecules like antigens can be covalently coupled to the microspheres using chemical techniques described herein or in the prior art (e.g. Bruckner, Springer Verlag 2010, Organic Mechanisms; Angeloni et al., xMAP Cookbook, Luminex, 4.sup.th edition). In a preferred embodiment molecules like antigens (i.e. VLPs or EDIII) can be coupled to the microsphere by carbodiimide coupling using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS). Thereby, the EDC is reacting to an unstable o-acylisourea ester with a carboxylate on the surface of the microspheres. The unstable o-acylisourea ester readily reacts with Sulfo-NHS to form a semi-stable amine reactive NHS-ester. The NHS-ester finally reacts with an amine group provided by an antigen, thereby forming a stable amide bond.
[0207] As amine-containing compounds other than those provided by the antigen, glycerol, urea, imidazole, azide, and some detergents may interfere with the carbodiimide coupling, they should be removed from the antigen preparation with a suitable buffer exchange method. For instance, a suitable buffer for carbodiimide coupling is 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer. The pH value for coupling may be between about 5 and about 9. Coupling of the antigen to the microsphere may be carried out by incubation for about 2 hours.
[0208] The microsphere may be magnetic. In a preferred embodiment, the microsphere may be superparamagnetic. Magnetic microspheres can be easily captured by a magnetic plate separator for instance to wash the microspheres. A magnetic plate separator can be used for separating the microspheres within the 96-well plate from the solution within the wells of the 96-well plate by magnetic capture and refers to a construction for holding a 96-well plate. A magnetic plate separator enables the user to quickly decant the supernatant within the wells and washing of the wells, while fixing the microspheres at the bottom of the 96-well plate by magnetic capture. Application of a magnetic plate separator as is possible in the MIA set-up reduces the risk that microspheres are getting lost during washing procedures.
[0209] The microsphere may be identified by a specific feature. This specific feature refers to a specific property of the microsphere, which allows it to be identified by a detection instrument. Identification of a microsphere likewise allows identification of the antigen, which is coupled to the microsphere. The specific feature may be that the microsphere comprises one or more fluorescent dyes having a specific emission spectrum and/or that the microsphere may comprise one or more fluorescent dyes at a specific concentration and/or that the microsphere is of a certain size.
[0210] The microsphere may be part of a microsphere set. A microsphere set refers to a plurality of microspheres that share the same specific feature. Microspheres of other sets are characterized by different specific features as one or more fluorescent dyes having another specific emission spectrum and/or one or more fluorescent dyes at another specific concentration and/or another certain size. By detection of the specific feature, a microsphere can be identified as part of a set and distinguished from microspheres of other sets. If the specific feature is that the microsphere may be of a certain size, all microspheres within the same set should be relatively the same size and all microspheres not part of this specific microsphere set should be of a different size. Microspheres of different microsphere sets can be coupled to different antigens. These antigens may comprise VLPs or EDIII of different flaviviruses. The unique specific features of the microspheres being part of different microsphere sets allow the different microspheres to be distinguished from each other. Moreover, when coupling different antigens to microspheres of different microsphere sets, the microspheres of the sets can be mixed, retaining the ability to be distinguished by their specific feature and thereby to identify the coupled antigen. This allows for multiplexing e.g. the simultaneous determination of the coupled antigens and/or the presence and/or amount of different Abs directed to the different antigens in one single experiment.
[0211] In one embodiment, the specific feature is that the microsphere is of a certain size ranging from about 0.01 to about 100 μm in diameter, more preferably from about 1 to about 10 μm in diameter. For instance, microspheres of one microsphere set may be about 6 μm in diameter, microspheres of another microsphere set may be about 6.5 μm in diameter.
[0212] In other embodiments, the specific feature is that the microsphere comprises one or more fluorescent dyes having a specific emission spectrum and/or comprises one or more fluorescent dyes at a specific concentration.
[0213] For instance, microspheres of one microsphere set may comprise two fluorescent dyes having an emission spectrum with an emission maximum at 675 nm, microspheres of another microsphere set may comprise two fluorescent dyes having an emission spectrum with an emission maximum at 700 nm. In another example, microspheres of one microsphere set may comprise a fluorescent dye at one specific concentration, microspheres of another microsphere set may comprise a fluorescent dye at another specific concentration, therefore providing two emission spectra with different emission intensities.
[0214] In other embodiments the specific feature is that the microsphere comprises one or more fluorescent dyes having a specific emission spectrum.
[0215] In one embodiment, the fluorescent dyes of the microspheres being part of different microsphere sets allow for excitation of the fluorescent dyes by the same light source (e.g. the fluorescent dyes of microspheres being part of different microsphere sets are excitable by the same wavelength). In specific embodiments, the microspheres being part of different microsphere sets are excitable with a wavelength within the range from about 600 nm to about 650 nm, more preferably with a wavelength of about 615 nm to about 640 nm, and even more preferably with a wavelength of about 620 nm to about 635 nm. In one embodiment, the different microspheres are excitable with a wavelength of about 621 nm. An advantage of such a set-up is, that only one light source is needed for distinguishing all microspheres present within a microsphere mixture and thereby further simplifying a set-up in which multiple virus like particles can be applied in one single experiment (multiplexing set-up).
[0216] Microspheres may be one out of the list consisting of MagPlex® microspheres, MicroPlex® microspheres, LumAvidin® microspheres, MagPlex®-Avidin microspheres, and SeroMAP® microspheres produced by the Luminex Corporation (Austin, Texas). In a preferred embodiment, the microspheres are the MagPlex® microspheres, which are superparamagnetic polystyrene microspheres with surface carboxyl groups produced by Luminex Corporation (Austin, Texas). MagPlex® microspheres comprise one or more fluorescent dyes having a specific emission spectrum as the specific feature allowing each microsphere to be identified by a detection instrument as for instance a MAGPIX® instrument as produced by the Luminex Corporation (Austin, Texas). Different MagPlex® microsphere catalog numbers (Luminex Corporation, Austin, Texas) refer to different microsphere sets, wherein the microspheres of the different sets comprise one or more fluorescent dyes having different specific emission spectra. The emission spectra of MagPlex® microspheres allow for excitation of the different fluorescent dyes by the same light source and therefore for simultaneous detection of the microspheres by one excitation wavelength. In specific embodiments, the excitation wavelength is from about 600 nm to about 650 nm, more preferably from about 620 nm to about 640 nm, such as about 621 nm.
ZIKV VLP
[0217] The antigen coupled to the microsphere according to the invention is a ZIKV VLP. The ZIKV VLP is derived from a ZIKV strain. ZIKV strains include but are not limited to ZikaSPH (Brazil 2015, GenBank accession No. KU321639.1), Brazil-ZKV (Brazil 2015, GenBank accession No. KU497555.1), PRVABC59 (Puerto Rico 2015, GenBank accession No. KU501215.1), Haiti1225 (Haiti 2014, GenBank accession No. KU509998.1), Natal RGN (Brazil, GenBank accession No. KU527068.1), SV0127-14 (Thailand 2014, GenBank accession No. KU681081.3), SPH2015 (GenBank accession No. KU321639.1), CPC-0740 (Philippine 2012, GenBank accession No. KU681082.3), SSABR1 (Brazil, GenBank accession No. KU707826.1), VE_Ganxian (China, GenBank accession No. KU744693.1), MR766-NIID (Uganda, GenBank accession No. LC002520.1), MR 766 (Uganda 1947, GenBank accession No. AY632535.2), and H/PF (French Polynesia 2013, GenBank accession No KJ776791.1) (WO 2017/109225). Further, ZIKV strains include Cambodia 2010 (GenBank accession No JN860885) or Micronesia 2007 (GenBank accession No EU545988) (Mlakar et al., N Engl J Med. 2016 Mar. 10; 374(10):951-8). Further, ZIKV strains include FLR (Colombia 2015) strain (WO 2018/017497), Z1106031 isolated in Suriname (Asian genotype; GenBank accession No KU312314), Z1106027 isolated in Suriname (Asian genotype; GenBank accession No KU312315); Z1106032 isolated in Suriname (Asian genotype; GenBank accession No KU312313), and Z1106033 isolated in Suriname (Asian genotype; Enfissi et al., Lancet 2016, 387(10015):227-228; GenBank Accession No. KU312312.1, SEQ ID NO: 1 and SEQ ID NO: 2).
[0218] According to one embodiment of the invention the ZIKV VLP is derived from ZIKV strain Z1106033 characterized by SEQ ID NO: 1 and/or SEQ ID NO: 2.
[0219] According to one embodiment of the invention the ZIKV VLP comprises structural proteins of ZIKV strain Z1106033 characterized by SEQ ID NO: 1 and/or SEQ ID NO: 2.
[0220] According to one embodiment of the invention the ZIKV VLP comprises the envelope glycoprotein, membrane protein, and/or pre-membrane protein of ZIKV strain Z1106033 characterized by SEQ ID NO: 1 and/or SEQ ID NO: 2.
[0221] According to one embodiment of the invention the ZIKV VLP comprises the envelope glycoprotein, membrane protein, and pre-membrane protein of ZIKV strain Z1106033 characterized by SEQ ID NO: 1 and/or SEQ ID NO: 2.
[0222] According to one embodiment, the ZIKV VLP is produced in human embryonic kidney (HEK293) cells.
[0223] In one embodiment, the ZIKV strain is Z1106033 isolated in Suriname (Asian genotype; Enfissi et al., Lancet 2016, 387(10015):227-228; GenBank Accession No. KU312312.1, SEQ ID NO: 1 and SEQ ID NO: 2) and the ZIKV VLP is produced in HEK293 cells.
[0224] Within the meaning of this invention, a ZIKV VLP comprising the envelope glycoprotein (E protein), membrane (M) protein, and/or pre-membrane (prM) protein of a ZIKV strain such as the Z1106033 strain refers to a ZIKV VLP comprising an envelope glycoprotein, membrane protein, and/or pre-membrane protein which are at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to corresponding parts of the protein sequence of the ZIKV strain such as the Z1106033 strain (SEQ ID NO: 2). Corresponding parts within that context mean parts of the protein sequence (such as SEQ ID NO: 2 for Z1106033 strain) that encode for E protein, M protein, or prM protein, respectively.
Kit of Microsphere Complex and Reporter Antibody
[0225] The present invention is directed to a kit comprising an amount of a microsphere complex as described in the previous chapter with the heading “Microsphere complex” and an amount of a reporter antibody that binds to the zika virus like particle (ZIKV VLP) of the microsphere complex.
[0226] Regarding the microsphere complex reference is made to the previous chapter with the heading “Microsphere complex”.
[0227] According to one embodiment of the invention, the reporter antibody is an immunoglobulin (Ig) molecule, comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds
[0228] According to one embodiment of the invention, the reporter Ab is a zika virus neutralizing antibody.
[0229] According to one embodiment of the invention, the reporter Ab is a recombinant Ab.
[0230] According to one embodiment the reporter Ab does not cross-react with dengue virus antigens.
[0231] According to one embodiment the reporter Ab does not cross-react with flavivirus antigens, such as DENV antigens, WNV antigens, JEV antigens, YFV antigens, SLEV antigens, and/or TBEV antigens.
[0232] According to one embodiment of the invention, the reporter antibody is a monoclonal antibody.
[0233] According to one embodiment of the invention, the reporter antibody is derived from a non-human origin.
[0234] According to one embodiment of the invention, the reporter Ab is attached to at least one detectable label. In preferred embodiments, the reporter Ab is attached to at least one detectable label by the heavy chain constant region of the reporter Ab.
[0235] According to one specific embodiment of the invention, the reporter Ab is directly attached to at least one detectable label. In preferred embodiments, the reporter Ab is directly attached to at least one detectable label by the heavy chain constant region of the reporter Ab. In embodiments, wherein the reporter Ab is directly attached to at least one detectable label, no secondary report Ab is necessary for detection of the reporter Ab.
[0236] According to another specific embodiment of the invention, the reporter Ab is indirectly attached to at least one detectable label, wherein the reporter Ab reacts with a secondary reporter Ab directly attached to at least one detectable label. In even more specific embodiments, the reporter Ab is indirectly attached to at least one detectable label by the heavy chain constant region of the reporter Ab, wherein the reporter Ab reacts with a secondary reporter Ab directly attached to at least one detectable label.
[0237] According to one embodiment of the invention, the detectable label is a compound or moiety that comprises one or more appropriate chemical substances or enzymes, which directly or indirectly generate a detectable compound or signal in a chemical, physical or enzymatic reaction. Labeling can be achieved by methods well known in the art (see, for example, Lottspeich, F., and Zorbas H., Springer Spektrum 2012, Bioanalytik).
[0238] According to one embodiment of the invention, the detectable label is selected from the group of fluorescent labels, magnetic labels, enzyme labels, colored labels, chromogenic labels, luminescent labels, radioactive labels, haptens, biotin, metal complexes, metals, and colloidal gold. All these types of labels are well established in the art.
[0239] According to one embodiment of the invention, the label is selected from such which provide the emission of fluorescence or phosphorescence upon irradiation or excitation or the emission of X-rays when using a radioactive label.
[0240] According to one embodiment of the invention, the label is an enzyme label, which include but are not limited to alkaline phosphatase, horseradish peroxidase (HRP), β-galactosidase, and p-lactamase. Enzyme labels catalyze the formation of chromogenic reaction products.
[0241] In specific embodiments, the detectable labels are fluorescent labels. Numerous fluorescent labels are well established in the art and commercially available from different suppliers (see, for example, The Handbook—A Guide to Fluorescent Probes and Labeling Technologies, 10th ed. (2006), Molecular Probes, Invitrogen Corporation, Carlsbad, CA, USA). Examples of fluorescent labels include but are not limited to fluorescein isothiocyanate (FITC), rhodamine, phycoerythrin (PE), cyanine, or coumarin. For detecting such labels any suitable detection system may be used.
[0242] According to one embodiment of the invention, the label is a fluorescent label such as PE.
[0243] Concerning the detection of such labels with suitable detection systems, reference is made to the following chapter with the heading “Method of detecting anti-zika virus antibodies”.
[0244] In specific embodiments, the reporter Ab provides an EC.sub.50 value towards the ZIKV VLP coupled to the microsphere within the microsphere complex of less than 0.5 μg/mL, or less than 0.4 μg/mL or less than 0.3 μg/mL or less than 0.2 μg/mL or less than 0.15 μg/mL or less than 0.1 μg/mL or less than 0.09 μg/mL or less than 0.08 μg/mL or less than 0.07 μg/mL or less than 0.05 μg/mL or less than 0.04 μg/mL or less than 0.03 μg/mL or less than 0.01 μg/mL. In other specific embodiments, the reporter Ab provides an EC.sub.25 value towards the ZIKV VLP coupled to the microsphere within the microsphere complex of less than 0.05 μg/mL, or less than 0.04 μg/mL or less than 0.03 μg/mL or less than 0.02 μg/mL or less than 0.01 μg/mL or less than 0.008 μg/mL or less than 0.006 μg/mL or less than 0.005 μg/mL or less than 0.004 μg/mL or less than 0.003 μg/mL. Examples of EC.sub.50 (and EC.sub.25 values) determined for the anti-ZIKV Abs as used herein can be found in Table 5. EC.sub.50 (and EC.sub.25 values) may be determined after incubation of the reporter Ab with the ZIKV VLP for about 10 min, or for about 60 min, or for about 120 min. In preferred embodiments, EC.sub.25 values are determined after incubation of the reporter Ab with the ZIKV VLP for about 120 min.
[0245] In other embodiments, the reporter antibody is a ZIKV specific reporter Ab, providing an EC.sub.50 value towards the zika VLP coupled to the microsphere within the microsphere complex, which is lower than each EC.sub.50 value, which said reporter antibody provides when tested in binding towards other microsphere complexes comprising a microsphere coupled to a DENV VLP, such as a DENV1 VLP, and/or DENV2 VLP, and/or DENV3 VLP, and/or DENV4 VLP. According to such embodiments, the difference between the EC.sub.50 values may be at least 2-fold, or 3-fold, or 4-fold, or 5-fold, or 8-fold, or 10-fold, or 12-fold, or 15-fold, or 20-fold.
[0246] In specific embodiments, the reporter antibody is a ZIKV specific reporter Ab, providing an EC.sub.50 value towards the ZIKV VLP coupled to the microsphere within the microsphere complex which is lower than each EC.sub.50 value which said reporter antibody provides when tested in binding towards other microsphere complexes comprising a microsphere coupled to DENV1 VLP, and/or DENV2 VLP, and/or DENV3 VLP, and/or DENV4 VLP. According to such embodiments, the difference between the EC.sub.50 values may be at least 2-fold, or 3-fold, or 4-fold, or 5-fold, or 8-fold, or 10-fold, or 12-fold, or 15-fold, or 20-fold.
[0247] In more specific embodiments, the reporter antibody is a ZIKV specific reporter Ab, providing an EC.sub.50 value towards the ZIKV VLP coupled to the microsphere within the microsphere complex of less than 0.5 μg/mL or less than 0.4 μg/mL or less than 0.3 μg/mL or less than 0.2 μg/mL or less than 0.15 μg/mL or less than 0.1 μg/mL or less than 0.09 μg/mL or less than 0.08 μg/mL or less than 0.07 μg/mL or less than 0.05 μg/mL or less than 0.04 μg/mL or less than 0.03 μg/mL or less than 0.01 μg/mL and an EC.sub.50 value towards other microsphere complexes comprising a microsphere coupled to DENV VLP, such as a DENV1 VLP, and/or DENV2 VLP, and/or DENV3 VLP, and/or DENV4 VLP of at least 1 μg/mL or of at least 1.1 μg/mL or of at least 1.2 μg/mL or of at least 1.3 μg/mL or of at least 1.4 μg/mL.
[0248] Regarding the ZIKV VLP reference is made to the previous chapter entitled “Microsphere complex”.
[0249] In specific embodiments of the invention, the DENV1 VLP is derived from DENV1 strain Puerto Rico/US/BID-V853/1998 (GenBank accession No. EU482592.1; SEQ ID NO: 179 and 180). DENV1 VLP may be produced in HEK293 cells. In more specific embodiments the DENV1 VLP comprises structural proteins from DENV1 strain Puerto Rico/US/BID-V853/1998 (GenBank accession No. EU482592.1; SEQ ID NO: 179 and 180). In even more specific embodiments, the DENV1 VLP comprises the E protein, M protein, and/or prM protein which are at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to those encoded by DENV1 strain Puerto Rico/US/BID-V853/1998 (GenBank accession No. EU482592.1; SEQ ID NO: 179 and 180).
[0250] In specific embodiments of the invention, the DENV2 VLP is derived from DENV2 strain Thailand/16681/84 (EMBL-EBI accession No: U87411.1; SEQ ID NO: 181 and 182). DENV2 VLP may be produced in HEK293 cells. In more specific embodiments the DENV2 VLP comprises structural proteins from DENV2 strain Thailand/16681/84 (EMBL-EBI accession No: U87411.1; SEQ ID NO: 181 and 182). In even more specific embodiments, the DENV2 VLP comprises the E protein, M protein, and/or prM protein which are at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to those encoded by DENV2 strain Thailand/16681/84 (EMBL-EBI accession No: U87411.1; SEQ ID NO: 181 and 182).
[0251] In specific embodiments of the invention, the DENV3 VLP is derived from DENV3 strain Sri Lanka D3/H/IMTSSA-SRI/2000/1266 (GenBank accession No. AY099336.1; SEQ ID NO: 183 and 184). DENV3 VLP may be produced in HEK293 cells. In more specific embodiments the DENV3 VLP comprises structural proteins from DENV3 strain Sri Lanka D3/H/IMTSSA-SRI/2000/1266 (GenBank accession No. AY099336.1; SEQ ID NO: 183 and 184). In even more specific embodiments, the DENV3 VLP comprises the E protein, M protein, and/or prM protein which are at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to those encoded by DENV3 strain Sri Lanka D3/H/IMTSSA-SRI/2000/1266 (GenBank accession No. AY099336.1; SEQ ID NO: 183 and 184).
[0252] In specific embodiments of the invention, the DENV4 VLP is derived from DENV4 strain Dominica/814669/1981 (EMBL-EBI accession No: AF326825.1; SEQ ID NO: 185 and 186). DENV4 VLP may be produced in HEK293 cells. In more specific embodiments the DENV4 VLP comprises structural proteins from DENV4 strain Dominica/814669/1981 (EMBL-EBI accession No: AF326825.1; SEQ ID NO: 185 and 186). In even more specific embodiments, the DENV4 VLP comprises the E protein, M protein, and/or prM protein which are at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to those encoded by DENV4 strain Dominica/814669/1981 (EMBL-EBI accession No: AF326825.1; SEQ ID NO: 185 and 186).
[0253] In specific embodiments of the present invention for production of DENV1-4 VLPs, the C-terminal 20% of DENV E protein were replaced by the corresponding Japanese encephalitis virus (JEV) SA-14 sequence (EMBL-EBI accession No: M55506.1, SEQ ID NO: 177 and 178; E protein amino acids 399-497 (DENV1 VLP), 397-495 (DENV2 VLP), 399-492 (DENV3 VLP), 400-495 (DENV4 VLP)). The replaced sequence corresponds to the transmembrane and intraparticle portion of the protein.
[0254] In a preferred embodiment, the EC.sub.50 and EC.sub.25 values are determined by incubation of the ZIKV VLP and/or DENV VLPs coupled to the microspheres in the microsphere complex with a serial dilution of the reporter Ab and determining the binding of the reporter Ab towards the ZIKV VLP and/or DENV VLP coupled to the microspheres by detecting the signal from the reporter Ab bound to the ZIKV VLP and/or DENV VLP coupled to the microspheres in the microsphere complex.
[0255] Concerning the detection of the signal from the reporter Ab bound to the ZIKV VLP and/or DENV VLP coupled to the microspheres in the microsphere complex reference is made to the following chapter with the heading “Method of detecting anti-zika virus antibodies”.
[0256] In certain embodiments, for determining the EC.sub.50 and/or EC.sub.25 values, the serial dilution of the reporter Ab is incubated with the ZIKV VLP and/or DENV VLPs coupled to the microspheres in the microsphere complex for about 10 min previous to determining the binding of the reporter Ab towards the ZIKV VLP and/or DENV VLP coupled to the microspheres by detecting the signal from the reporter Ab bound to the ZIKV VLP and/or DENV VLP coupled to the microspheres in the microsphere complex.
[0257] In certain embodiments, for determining the EC.sub.50 and/or EC.sub.25 values, the serial dilution of the reporter Ab is incubated with the ZIKV VLP and/or DENV VLPs coupled to the microspheres in the microsphere complex for about 120 min previous to determining the binding of the reporter Ab towards the ZIKV VLP and/or DENV VLP coupled to the microspheres by detecting the signal from the reporter Ab bound to the ZIKV VLP and/or DENV VLP coupled to the microspheres in the microsphere complex.
[0258] In certain embodiment, for determining the EC.sub.50 and/or EC.sub.25 values, the serial dilution of the reporter Ab is incubated with the ZIKV VLP and/or DENV VLPs coupled to the microspheres in the microsphere complex for about 60 min previous to determining the binding of the reporter Ab towards the ZIKV VLP and/or DENV VLP coupled to the microspheres by detecting the signal from the reporter Ab bound to the ZIKV VLP and/or DENV VLP coupled to the microspheres in the microsphere complex.
[0259] In one embodiment, the serial dilution of the reporter Ab ranges from about 0.0001 to about 100 μg/mL, more preferable from about 0.001 to about 35 μg/mL, and even more preferable from about 0.001 to about 20 μg/mL, wherein the concentrations of the reporter Ab refer to the concentrations previous to 2-fold dilution with the microspheres (see also Example 2).
[0260] In one embodiment, the reporter antibody is incubated with a secondary reporter Ab directly attached to at least one detectable label for detecting the signal from the reporter Ab bound to the ZIKV VLP and/or DENV VLP coupled to the microspheres in the microsphere complex.
[0261] In another embodiment, the reporter antibody is incubated with a secondary reporter Ab directly attached to at least one detectable label for about 30 min. In a preferred embodiment the at least one detectable label is a fluorescence label. In an even more preferred embodiment, the fluorescence label is PE.
[0262] In other embodiments, the reporter Ab is directly attached to at least one detectable label, preferably to at least one fluorescence label, more preferably to PE. In these embodiments, a signal from the reporter Ab bound to the ZIKV VLP and/or DENV VLP coupled to the microspheres in the microsphere complex can be directly detected without the need of further incubation with a secondary reporter Ab.
[0263] Concerning the detection of detectable labels, reference is made to the following chapter with the heading “Method of detecting anti-zika virus antibodies”.
[0264] In specific embodiments the detected signal for each serial dilution of the reporter Ab is analyzed using a suitable data analysis software e.g. Prism (GraphPad). The detected signals for each reporter antibody are sigmoidal fitted according to a dose-response curve and the EC.sub.50 value is calculated for the reporter Ab. The EC.sub.50 value is calculated using mAb concentrations previous to 2-fold dilution by incubation with the microspheres.
[0265] In certain embodiments, the EC.sub.50 value can be reliably determined when a sufficient number of dilutions of reporter Ab is examined per reporter Ab. A sufficient number of dilutions within that context may mean that the number of dilutions is sufficient to reach a plateau for the minimum signal and the maximum signal in the dose-response curve. A sufficient number of dilutions may be about 10 or more.
[0266] It may be possible that the reporter Ab binds to the other microsphere complexes such as microsphere complexes comprising microspheres coupled to DENV1 VLP, DENV2 VLP, DENV3 VLP, or DENV4 VLP to such a less extent that the EC.sub.50 value cannot be calculated in a statistically reliable way. In such cases, the EC.sub.50 value can be assumed to be at least 1 μg/mL.
[0267] According to one embodiment, the reporter antibody is one of the Abs selected from the group consisting of anti-ZIKV #1, anti-ZIKV #2, anti-ZIKV #3, anti-ZIKV #4, anti-ZIKV #5, anti-ZIKV #6, anti-ZIKV #7, anti-ZIKV #8, anti-ZIKV #9, anti-ZIKV #10, or anti-ZIKV #11. For further details and characterization of Abs reference is made to Example 2. The reporter antibody may be characterized by the sequence of the VH-CDR1 and/or VH-CDR2 and/or VH-CDR3 and/or VL-CDR1 and/or VL-CDR2 and/or VL-CDR3. The reporter antibody may alternatively or additionally be characterized by the sequence of the VH and/or VL and/or H and/or L. The sequence referred to may be an amino acid sequence or a nucleic acid sequence encoding the amino acid sequence. The sequences and critical amino acid residues for binding are provided in Table 1 and 2, respectively. Critical residues are those amino acids whose side chains make the highest energetic contribution to the Ab-epitope interaction and whose mutation gave the lowest binding reactivities (<10% of wild-type) by alanine scanning mutagenesis (Bogan and Thorn, J. Mol. Biol. 1998, 280, 1-9; Lo Conte et al., J. Mol. Biol. 1999, 285, 2177-2198).
TABLE-US-00001 TABLE 1 Sequence information for reporter Abs. Reporter Abs are presented together with their amino acid sequences, as well as corresponding nucleotide sequences (where available) of heavy chain (H), heavy chain variable region (VH), heavy chain complementary determining regions 1 to 3 (VH-CDR1-3), light chain (L), light chain variable region (VL), as well as light chain complementary determining regions 1 to 3 (VL-CDR1-3). H and L sequences were not available from the vendor, publications, or databases for commercial reporter Abs (Anti-ZIKV #6 and 7). Amino acid Nucleic acid mAb sequence sequence mAb part (SEQ ID No) (SEQ ID No) Examples Anti-ZIKV #1 H 5 15 VH 6 16 VH-CDR1 7 N/A VH-CDR2 8 N/A VH-CDR3 9 N/A L 10 17 VL 11 18 VL-CDR1 12 N/A VL-CDR2 13 N/A VL-CDR3 14 N/A Anti-ZIKV #2 H 19 29 VH 20 30 VH-CDR1 21 N/A VH-CDR2 22 N/A VH-CDR3 23 N/A L 24 31 VL 25 32 VL-CDR1 26 N/A VL-CDR2 27 N/A VL-CDR3 28 N/A Anti-ZIKV #3 H 33 43 VH 34 44 VH-CDR1 35 N/A VH-CDR2 36 N/A VH-CDR3 37 N/A L 38 45 VL 39 46 VL-CDR1 40 N/A VL-CDR2 41 N/A VL-CDR3 42 N/A Anti-ZIKV #4 H 47 57 VH 48 58 VH-CDR1 49 N/A VH-CDR2 50 N/A VH-CDR3 51 N/A L 52 59 VI 53 60 VL-CDR1 54 N/A VL-CDR2 55 N/A VL-CDR3 56 N/A Anti-ZIKV #5 H 61 71 VH 62 72 VH-CDR1 63 N/A VH-CDR2 64 N/A VH-CDR3 65 N/A L 66 73 VL 67 74 VL-CDR1 68 N/A VL-CDR2 69 N/A VL-CDR3 70 N/A Anti-ZIKV #6 VH 75 N/A VH-CDR1 76 N/A VH-CDR2 77 N/A VH-CDR3 78 N/A VL 79 N/A VL-CDR1 80 N/A VL-CDR2 81 N/A VL-CDR3 82 N/A Anti-ZIKV #7 VH 33 91 VH-CDR1 84 N/A VH-CDR2 85 N/A VH-CDR3 86 N/A VL 87 92 VL-CDR1 88 N/A VL-CDR2 89 N/A VL-CDR3 90 N/A Anti-ZIKV #8 H 93 103 VH 94 104 VH-CDR1 95 N/A VH-CDR2 96 N/A VH-CDR3 97 N/A L 98 105 VL 99 106 VL-CDR1 100 N/A VL-CDR2 101 N/A VL-CDR3 102 N/A Anti-ZIKV #9 H 107 117 and #10 VH 108 118 VH-CDR1 109 N/A VH-CDR2 110 N/A VH-CDR3 111 N/A L 112 119 VL 113 120 VL-CDR1 114 N/A VL-CDR2 115 N/A VL-CDR3 116 N/A Anti-ZIKV #11 H 121 131 VH 122 132 VH-CDR1 123 N/A VH-CDR2 124 N/A VH-CDR3 125 N/A L 126 133 VL 127 134 VL-CDR1 128 N/A VL-CDR2 129 N/A VL-CDR3 130 N/A
TABLE-US-00002 TABLE 2 Critical amino acid residues in one-letter code from the ZIKV E protein (SEQ ID NO: 3) important for binding of Anti-ZIKV #1 to 5 mAbs as evaluated by alanine scanning mutagenesis. T = Thr, G = Gly, E = Glu, K = Lys, H = His. Critical E Protein mAb Residues Domain Anti-ZIKV #1 (Clone 102-1) T309, G337 III Anti-ZIKV #2 (Clone 242-3) E370 III Anti-ZIKV #3 (Clone 270-12) E370 III Anti-ZIKV #4 (Clone 289-3) E162, G181, G182, K301 I, III Anti-ZIKV #5 (Clone 306-2) T397, H398 III
Method of Detecting Anti-Zika Virus Antibodies
[0268] Regarding the microsphere complex and the kit reference is made to the respective chapters above.
[0269] The present invention is further directed to a method for detecting a signal from a reporter antibody indicative for the presence and/or amount of anti-zika virus antibodies in a sample from a subject comprising the steps of: [0270] Step 1: providing a kit comprising an amount of a microsphere complex comprising a microsphere coupled to a zika virus like particle and an amount of a reporter antibody that binds to the zika virus like particle of the microsphere complex, [0271] Step 2: contacting the amount of said microsphere complex and the amount of said reporter antibody of step 1 with the sample to allow binding of anti-zika virus antibodies in the sample to the zika virus like particles coupled to the microspheres in the microsphere complex while competing with the reporter antibody, and [0272] Step 3: detecting a signal from the reporter antibody bound to the zika virus like particles coupled to the microspheres in the microsphere complex in step 2.
[0273] According to one specific embodiment, contacting in step 2 is carried out for about 10 to 100 min.
[0274] According to one specific embodiment of the invention, the amount of microsphere complex and the amount of reporter antibody are concomitantly contacted with the sample in step 2.
[0275] According to one specific embodiment of the invention the method comprises the steps of: [0276] Step 1: providing a kit comprising an amount of a microsphere complex comprising a microsphere coupled to a zika virus like particle and an amount of a reporter antibody that binds to the zika virus like particle of the microsphere complex, [0277] Step 2.1: contacting the amount of said microsphere complex of step 1 with the sample to allow binding of anti-zika virus antibodies in the sample to the zika virus like particles coupled to the microspheres in the microsphere complex, [0278] Step 2.2: contacting said amount of reporter antibody with said microsphere complex and the sample of step 2.1 to allow binding of the reporter antibody to the zika virus like particles coupled to the microspheres in the microsphere complex, and [0279] Step 3: detecting a signal from the reporter antibody bound to the zika virus like particles coupled to the microspheres in the microsphere complex in step 2.2.
[0280] According to another specific embodiment of the invention the method comprises the steps of: [0281] Step 1: providing a kit comprising an amount of a microsphere complex comprising a microsphere coupled to a zika virus like particle and an amount of a reporter antibody that binds to the zika virus like particle of the microsphere complex, [0282] Step 2.1: contacting the amount of said microsphere complex of step 1 with the amount of said reporter antibody to allow binding of the reporter antibody to the zika virus like particles coupled to the microspheres in the microsphere complex, [0283] Step 2.2: contacting the sample with said microsphere complex and said reporter antibody of step 2.1 to allow binding of anti-zika virus antibodies in the sample to the zika virus like particles coupled to the microspheres in the microsphere complex, and [0284] Step 3: detecting a signal from the reporter antibody bound to the zika virus like particles coupled to the microspheres in the microsphere complex in step 2.1.
[0285] According to one specific embodiment of the invention the method comprises the steps of: [0286] Step 1: providing a kit comprising an amount of a microsphere complex comprising a microsphere coupled to a zika virus like particle and an amount of a reporter antibody that binds to the zika virus like particle of the microsphere complex, [0287] Step 2.1: contacting the amount of said microsphere complex with the sample to allow binding of anti-zika virus antibodies in the sample to the zika virus like particles coupled to the microspheres in the microsphere complex, [0288] Step 2.2: contacting said amount of reporter antibody with said microsphere complex and the sample of step 2.1 to allow binding of the reporter antibody to the zika virus like particles coupled to the microspheres, [0289] Step 2.3: contacting said amount of reporter antibody, said amount of microsphere complex, and the sample of step 2.2 with a secondary reporter antibody to allow binding of the secondary reporter antibody to the constant region of the reporter antibody, and [0290] Step 3: detecting a signal from the secondary reporter antibody bound to the reporter antibody in step 2.3, wherein the reporter antibody is bound to the zika virus like particles coupled to the microspheres in the microsphere complex in step 2.2.
[0291] According to another specific embodiment of the invention the method comprises the steps of: [0292] Step 1: providing a kit comprising an amount of a microsphere complex comprising a microsphere coupled to a zika virus like particle and an amount of a reporter antibody that binds to the zika virus like particle of the microsphere complex, [0293] Step 2.1: contacting the amount of said microsphere complex with the amount of said reporter antibody to allow binding of the reporter antibody to the zika virus like particles coupled to the microspheres in the microsphere complex, [0294] Step 2.2: contacting the sample with said microsphere complex and said reporter antibody of step 2.1 to allow binding of anti-zika virus antibodies in the sample to the zika virus like particles coupled to the microspheres in the microsphere complex, [0295] Step 2.3: contacting said amount of reporter antibody, said amount of microsphere complex, and the sample of step 2.2 with a secondary reporter antibody to allow binding of the secondary reporter antibody to the constant region of the reporter antibody, and [0296] Step 3: detecting a signal from the secondary reporter antibody bound to the reporter antibody in step 2.3, wherein the reporter antibody is bound to the zika virus like particles coupled to the microspheres in the microsphere complex in step 2.1.
[0297] According to one embodiment, contacting in step 2.1 is carried out for about 40 min to about 80 min and contacting in step 2.2 is carried out for about 5 min to about 60 min.
[0298] According to one embodiment, contacting in step 2.1 is carried out for about 60 min and contacting in step 2.2 is carried out for about 10 min.
[0299] According to one embodiment contacting in step 2.1 is carried out for about 40 min to about 80 min and contacting in step 2.2 is carried out for about 5 min to about 60 min and contacting in step 2.3 is carried out for about 10 min to about 50 min.
[0300] According to one embodiment contacting in step 2.1 is carried out for about 60 min and contacting in step 2.2 is carried out for about 10 min and contacting in step 2.3 is carried out for about 30 min.
[0301] According to one embodiment of the invention, the sample is a sample from the group consisting of blood, urine, serum, plasma, cerebrospinal fluid, and lymph fluid. According to one embodiment, the sample is a blood plasma sample. According to another embodiment, the sample is a serum sample. According to the invention, the sample is provided outside the human or animal body. Within this invention, the term “plasma” refers to blood plasma.
[0302] According to a specific embodiment of the invention, the plasma sample is heat-inactivated. Heat-inactivation may occur by incubating the sample for about 30 min at about 56° C.
[0303] According to the invention, the sample is from a subject from the group consisting of mouse, primate, non-human primate, human, rabbit, cat, rat, horse, and sheep. According to one embodiment, the subject is human. According to another embodiment, the subject is a non-human primate.
[0304] According to one embodiment of the present invention, the signal in step 3 is resulting from the at least one detectable label. According to another embodiment, the signal in step 3 is a fluorescence signal. In a specific embodiment, the fluorescence signal results from phycoerythrin. The signal can be detected by any suitable detection instrument.
[0305] According to the invention, the detection system refers to any system, which is suitable for determining values indicative for the presence and/or amount of reporter antibody captured on the microsphere.
[0306] According to the invention, the detection system may also be able to determine values indicative for the presence and/or amount of a microsphere by identifying the specific feature of the microsphere.
[0307] The selection of a suitable detection system depends on several parameters such as the type of detectable labels used for detection or the kind of analysis performed. Various optical and non-optical detection systems are well established in the art. A general description of detection systems that can be used with the method can be found, e.g., in Lottspeich, F., and Zorbas H., Springer Spektrum 2012, Bioanalytik.
[0308] According to one embodiment of the invention, the detection system is an optical detection system. In some embodiments, performing the method involves simple detection systems, which may be based on the measurement of parameters such as fluorescence, optical absorption, resonance transfer, and the like.
[0309] According to one embodiment of the invention, the detection system measures fluorescence. Such systems measure the capacity of particular molecules to emit their own light when excited by light of a particular wavelength resulting in a characteristic absorption and emission behavior. In particular, quantitative detection of fluorescence signals is performed by means of modified methods of fluorescence microscopy (for review see, e.g., Lichtman, J. W., and Conchello, J. A. (2005) Nature Methods 2, 910-919; Zimmermann, T. (2005) Adv. Biochem. Eng. Biotechnol. 95, 245-265). Thereby, the signals resulting from light absorption and light emission, respectively, are separated by one or more filters and/or dichroites and imaged on suitable detectors. Data analysis is performed by means of digital image processing. Image processing may be achieved with several software packages well known in the art (such as Mathematica Digital Image Processing, EIKONA, or Image-PRO). Another suitable software for such purposes is the Iconoclust software (Clondiag Chip Technologies GmbH, Jena, Germany). Suitable detection systems may be based on “classical” methods for measuring a fluorescent signal such as epifluorescence or darkfield fluorescence microscopy (reviewed, e.g., in: Lakowicz, J. R. (1999) Principles of Fluorescence Spectroscopy, 2nd ed., Plenum Publishing Corp., NY). Another optical detection system that may be used is confocal fluorescence microscopy, wherein the object is illuminated in the focal plane of the lens by a point light source. Importantly, the point light source, object and point light detector are located on optically conjugated planes. Examples of such confocal systems are described in detail, for example, in Diaspro, A. (2002) Confocal and 2-photon-microscopy: Foundations, Applications and Advances, Wiley-Liss, Hobroken, NJ. The fluorescence-optical system is usually a fluorescence microscope without an autofocus, for example a fluorescence microscope having a fixed focus. Further fluorescence detection methods that may also be used include inter alia total internal fluorescence microscopy (see, e.g., Axelrod, D. (1999) Surface fluorescence microscopy with evanescent illumination, in: Lacey, A. (ed.) Light Microscopy in Biology, Oxford University Press, New York, 399-423), fluorescence lifetime imaging microscopy (see, for example, Dowling, K. et al. (1999) J. Mod. Optics 46, 199-209), fluorescence resonance energy transfer (FRET; see, for example, Periasamy, A. (2001) J. Biomed. Optics 6, 287-291), bioluminescence resonance energy transfer (BRET; see, e.g., Wilson, T., and Hastings, J. W. (1998) Annu. Rev. Cell Dev. Biol. 14, 197-230), and fluorescence correlation spectroscopy (see, e.g., Hess, S. T. et al. (2002) Biochemistry 41, 697-705). In specific embodiments, detection is performed using FRET or BRET, which are based on the respective formation of fluorescence or bioluminescence quencher pairs. The use of FRET is also described, e.g., in Liu, B. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 589-593; and Szollosi, J. et al. (2002) J. Biotechnol. 82, 251-266. The use of BRET is detailed, for example, in Prinz, A. et al. (2006) Chembiochem. 7, 1007-1012; and Xu, Y. et al. (1999) Proc. Natl. Acad. Sci. USA 96, 151-156.
[0310] In one embodiment the detection system comprises a first light source, e.g. an argon laser or a light emitting diode (LED), which has an excitation wavelength in the range of 300 to 700 nm and a second light source, e.g. an argon laser or a LED, which has an excitation wavelength in the range of 400 to 700 nm and a suitable detection component as for instance a photodiode such as an avalanche photodiode (APD) in combination with a photomultiplier or a charge-coupled device (CCD) sensor. The first light source may be used for excitation of a detectable label. The second light source may be used for the identification of the specific feature of a microsphere, wherein the specific feature may be that the microsphere comprises one or more fluorescent dyes having a specific emission spectrum, and/or one or more fluorescent dyes at a specific concentration.
[0311] In a preferred embodiment, the detection system comprises a first light source, e.g. an argon laser or a LED, which has an excitation wavelength in the range of 500 to 600 nm and a second light source, e.g. an argon laser or a LED, which has an excitation wavelength in the range of 600 to 700 nm. In a more preferred embodiment, the detection system comprises a first light source, e.g. an argon laser or a LED, which has an excitation wavelength in the range of 510 to 540 nm and a second light source, e.g. an argon laser or a LED, which has an excitation wavelength in the range of 615 to 645 nm. In an even more preferred embodiment the first light source, e.g. the argon laser or LED has an excitation wavelength in the range of about 510 to about 535 nm and the second light source, e.g. the argon laser or LED has an excitation wavelength in the range of about 620 to about 635 nm. For instance, the detection system comprises a first light source, e.g. an argon laser or a LED, which has an excitation wavelength of about 525 nm and a second light source, e.g. an argon laser or a LED, which has an excitation wavelength of about 635 nm.
[0312] The detection system may be also capable of distinguishing the individual size of a microsphere from one microsphere set from the individual size of a microsphere of another microsphere set, thereby allowing individual identification of the microsphere.
[0313] The detection system may be one of the group consisting of MAGPIX®, Luminex 200©, and FLEXMAP 3D® (Luminex Corp. Austin, Tex.). In a preferred embodiment, the detection system is the MAGPIX® (Luminex Corp. Austin, Tex.). These detection systems may be operated by a specific software, including the xPONENT® software (Luminex Corp. Austin, Tex.). These detection systems are capable of detecting both, the signal from the at least one detectable label of the reporter or the detection Ab, as well as the specific feature of the microsphere present in the microsphere complex, wherein the specific feature is that the microsphere comprises one or more fluorescent dyes having a specific emission spectrum, and/or one or more fluorescent dyes at a specific concentration.
[0314] The detection system may be capable of analyzing one microsphere after the other thereby identifying the microsphere by detecting the specific feature of the microsphere and detecting the signal from the at least one detectable label of the reporter or detection antibody such as flow cytometry based detection systems (e.g. Luminex 200® and FLEXMAP 3D®). The flow cytometry based detection systems Luminex 200® and FLEXMAP 3D® include two lasers each one for irradiation of the one or more fluorescent dyes of the microsphere (the specific feature of microspheres that can be identified by these specific detection systems) and the at least one detectable label of the reporter or detection Ab. As flow cytometry based detection systems are not capturing the microspheres with a magnet, the Luminex 200® and FLEXMAP 3D® systems are compatible with both, magnetic microspheres such as the MagPlex® microspheres and non-magnetic microspheres such as the Microplex® microspheres. The Luminex 200® and FLEXMAP 3D® systems detect signals from the microspheres and reporter or detection Ab by avalanche photodiodes (APD) in combination with photomultipliers (PMT).
[0315] Alternatively, the detection system may be capable of analyzing multiple microspheres at once. Therefore, a monolayer of magnetic microspheres is captured by a magnet and the microspheres are excited with two LEDs, one LED for excitement of the one or more fluorescent dyes of the microspheres (the specific feature of microspheres that can be identified by these specific detection systems) and the other LED for excitement of the at least one detectable label of the reporter or detection Ab. The signals from the microspheres and reporter or detection Ab are recorded by a CCD imager, which allows identification of each microsphere and the corresponding antigen to which the microsphere is coupled to. An example for a LED-based detection system is the MAGPIX® instrument. As analyses with the MAGPIX® instrument involves capture of the microspheres with a magnet, the MAGPIX® instrument is solely compatible with magnetic microspheres such as MagPlex® microspheres.
[0316] The present invention is further directed to such a method for detecting the presence and/or amount of anti-zika virus antibodies in a sample from a subject comprising the further steps of: [0317] Step 4: determining the presence and/or the amount of the reporter antibody bound to the zika virus like particles coupled to the microspheres in the microsphere complex from the signal of step 3, and [0318] Step 5: determining the presence and/or the amount of anti-zika virus antibodies in the sample based on the presence and/or the amount of the reporter antibody determined in step 4.
[0319] In specific embodiments of the invention, the amount of reporter Ab bound to the ZIKV VLPs coupled to the microspheres in the microsphere complex is determined by comparing the signal of step 3 to a standard curve, wherein the standard curve comprises signals resulting from known amounts of reporter Ab bound to ZIKV VLPs coupled to microspheres in a microsphere complex.
[0320] In specific embodiments of the invention, the amount of anti-ZIKV Abs in the sample is determined based on the amount of the reporter Ab determined in step 4 by comparing the amount of reporter Ab to a standard curve, wherein the standard curve comprises amounts of reporter Ab resulting from known amounts of anti-ZIKV Abs present within a sample.
[0321] According to another embodiment of the present invention, the anti-ZIKV Abs from the sample of the subject are ZIKV specific Abs.
[0322] In further embodiments, the anti-ZIKV Abs from the sample of the subject are ZIKV neutralizing Abs.
[0323] Further, the invention refers to a multiplexing method for detecting signals from multiple reporter Abs indicative for the presence and/or amount of Abs directed to different viruses within one sample. Therefore, different microsphere complexes are mixed, wherein the different microsphere complexes comprise microspheres of different microsphere sets coupled to a specific virus antigen (e.g. flavivirus antigens including DENV, ZIKV, WNV, JEV, YFV). The microspheres of a specific microsphere set can be identified by a specific feature, which may be that the microspheres comprise one or more fluorescent dyes having a specific emission spectrum and/or one or more fluorescent dyes at a specific concentration and/or are of a certain size. By identification of the specific feature, the specific virus antigen coupled to the microsphere can be identified simultaneously. For instance, a first microsphere complex comprising a microsphere comprising one or more fluorescent dyes having a specific emission spectrum is coupled to a zika virus like particle and a second microsphere complex comprising a microsphere comprising one or more fluorescent dyes having another specific emission spectrum is coupled to a dengue virus like particle and both microsphere complexes are mixed. The fluorescent dyes of the microspheres may be excited by the same light source i.e. the same wavelength. By detecting the specific feature of the microspheres, the immobilized antigen is concomitantly detected. Further, when the microsphere complex mixture is incubated with a sample comprising anti-ZIKV and anti-DENV Abs, both anti-ZIKV and anti-DENV Abs can be detected within one single experiment by the application of two reporter Abs, wherein one reporter Ab binds to the ZIKV VLP and the other reporter Ab binds to the DENV VLP. Both reporter Abs may be attached to phycoerythrin as the detectable label. By exciting, for instance, one microsphere after the other with two different wavelengths, wherein one wavelength is suitable for exciting the one or more fluorescent dyes of the microsphere and the other wavelength is suitable for exciting the detectable label, a signal indicative for the specific feature of the microsphere (and therefore for the antigen immobilized) can be detected and a signal indicative for the amount of bound reporter Ab can be detected. Both, anti-ZIKV and anti-DENV Abs can be determined in one single experiment using the multiplexing approach.
[0324] Method for Determining an Antibody Correlate of Protection Against Zika Virus Infection
[0325] The present invention is further directed to a method for determining an antibody correlate of protection against zika virus infection for a zika virus vaccine in a type of non-human subjects, the method comprising the steps of: [0326] Step 1: selecting a group of said subjects which are zika virus naive, [0327] Step 2: dividing the group of subjects into at least two subgroups, wherein one subgroup functions as control group and at least one subgroup functions as inoculation group, [0328] Step 3: inoculating said at least one inoculation group with a dose of the zika virus vaccine, [0329] Step 4: challenging all subjects with an infectious amount of the zika virus, [0330] Step 5: determining the amount of anti-zika virus antibodies for each subject according to the methods as described above at least after inoculation with the zika virus vaccine and before challenging with the infectious amount of the zika virus, [0331] Step 6: determining presence or absence of viremia in all subjects after challenging with the infectious amount of the zika virus, [0332] Step 7: repeating steps 3 to 6 with further inoculation groups with increasing vaccine doses until absence of viremia is determined in all subjects of one inoculation group in step 6, and [0333] Step 8: determining the amount of anti-zika virus antibodies after inoculation with the zika virus vaccine and before challenging with the infectious amount of the zika virus associated with absence of viremia after challenging with the infectious amount of zika virus as antibody correlate of protection.
[0334] In specific embodiments of the invention the type of non-human subjects is selected from the group consisting of mice, primates, non-human primates, rabbits, cats, rats, horses, or sheep.
[0335] In a more specific embodiment of the invention, the type of non-human subjects is non-human primates.
[0336] In further embodiments of the invention, the zika virus vaccine in step 3 is a purified inactivated zika virus vaccine (PIZV).
[0337] In more specific embodiments, the zika virus vaccine in step 3 is a PIZV and the dose of the PIZV in step 3 is between about 0.3 μg and about 20 μg. In embodiments, where more than one subgroup is selected in step 2, each subgroup receives a different dose of the zika virus vaccine in step 3. For instance, a first subgroup receives a dose of the PIZV of about 0.4 μg, a second subgroup receives a dose of the PIZV of about 2 μg, and a third subgroup receives a dose of the PIZV of about 10 μg.
[0338] In specific embodiments, the zika virus in step 4 is ZIKV strain PRVABC59.
[0339] In more specific embodiments, the zika virus in step 4 is ZIKV strain PRVABC59 and the infectious amount in step 4 is a nominal dose of about 104 focus forming units (FFU) present in about 0.5 mL.
[0340] In a specific embodiment of the invention, challenging in step 4 is performed between about 60 and about 80 days, more preferable about 71 days, after inoculating the at least one inoculation group with a dose of a ZIKV vaccine.
[0341] In another embodiment, the presence of viremia is determined in step 6 by monitoring the typical symptoms caused by an infection with the ZIKV of step 4 and/or determining the amount of ZIKV of step 4 and/or determining the amount of neutralizing Abs directed against ZIKV of step 4. In more specific embodiments, the amount of ZIKV is determined by RT-PCR and/or the amount of neutralizing Abs directed against ZIKV is determined by a RVP assay, a FFA, a MNT, or a PRNT.
[0342] In another embodiment, the presence of viremia is determined in step 6 after 0, and/or 1, and/or 2, and/or 3, and/or 4, and/or 5, and/or 6, and/or 7, and/or 8, and/or 9, and/or 10, and/or 11, and/or 12, and/or 13, and/or 14, and/or 15, and/or 20, and/or 30 days after challenging with the ZIKV in step 4.
[0343] This invention is further directed to a method for determining an antibody correlate of protection against ZIKV infection in human subjects by mathematically modelling the correlate of protection of a non-human subject like a non-human primate to fit human subjects.
Method for Diagnosing Protection Against Zika Virus Infection
[0344] The present invention is further directed to a method for diagnosing the protection of a human subject against a zika virus infection comprising the steps of: [0345] Step 1: providing a sample from the human subject outside the human body, [0346] Step 2: determining the amount of anti-ZIKV antibodies in the sample from the human subject as described above, and [0347] Step 3: determining protection by comparing the amount of anti-ZIKV antibodies determined in step 2 to the antibody correlate of protection against zika virus infection in human subjects, optionally determined according to the method described above.
[0348] In preferred embodiments, the method for diagnosing the protection of a human subject against a zika virus infection comprising the steps of: [0349] Step 1: providing a sample from the human subject outside the human body, [0350] Step 2: determining the amount of anti-ZIKV antibodies in the sample from the human subject as described above, and [0351] Step 3: determining protection by comparing the amount of anti-ZIKV antibodies determined in step 2 to the antibody correlate of protection against zika virus infection in human subjects determined according to the method described above.
[0352] The present invention is further directed to a method for diagnosing the protection of a non-human subject against a zika virus infection comprising the steps of: [0353] Step 1: providing a sample from the non-human subject outside the non-human body, [0354] Step 2: determining the amount of anti-ZIKV antibodies in the sample from the non-human subject as described above, and [0355] Step 3: determining protection by comparing the amount of anti-ZIKV antibodies determined in step 2 to the antibody correlate of protection determined in this type of non-human subjects according to the method described above.
[0356] In a particular embodiment the sample of step 1 is a blood sample, in particular a blood plasma sample. Method for diagnosing zika virus infection
[0357] The present invention is further directed to a method for diagnosing a ZIKV infection in a human subject comprising the steps of: [0358] Step 1: providing a sample from the human subject outside the human body [0359] Step 2: determining the amount of anti-ZIKV antibodies in the sample as described above, [0360] Step 3: determining infection by comparing said amount of anti-ZIKV antibodies to established amounts of anti-ZIKV antibodies in ZIKV infected human subjects.
[0361] The present invention is further directed to a method for diagnosing a ZIKV infection in a non-human subject comprising the steps of: [0362] Step 1: providing a sample from the non-human subject outside the non-human body [0363] Step 2: determining the amount of anti-ZIKV antibodies in the sample as described above, [0364] Step 3: determining infection by comparing said amount of anti-ZIKV antibodies to established amounts of anti-ZIKV antibodies in ZIKV infected non-human subjects.
[0365] In certain embodiments, the sample is a blood sample, in particular a blood plasma sample.
[0366] In certain embodiments, the ZIKV infection is convalescent.
[0367] In certain embodiments, the ZIKV infection is acute.
Method for Assaying the Presence of a Zika Virus Infection
[0368] The present invention is further directed to a method for assaying the presence of a zika virus infection in a subject comprising the steps of: [0369] Step 1: obtaining a sample from the subject, [0370] Step 2: determining the amount of anti-zika virus antibodies in the sample as described above under the section “Method of detecting anti-zika virus antibodies”, and [0371] Step 3: determining the presence of a zika virus infection by comparing said amount of anti-zika virus antibodies to established amounts of anti-zika virus antibodies in zika virus infected subjects.
[0372] In certain embodiments, the subject is a human.
[0373] In certain embodiments, the zika virus infection is acute.
[0374] In certain embodiments, the zika virus infection is convalescent.
[0375] In certain embodiments, the sample is a blood sample, in particular a blood plasma sample.
Method for Preventing Zika Disease
[0376] The present invention is further directed to a method for preventing zika disease in a human subject, the method comprising the steps of: [0377] Step 1: obtaining a sample from the human subject, [0378] Step 2: determining the amount of anti-zika virus antibodies in the sample from the human subject as described above under the section “Method of detecting anti-zika virus antibodies”, [0379] Step 3: determining whether the human subject has an amount of anti-zika virus antibodies to confer protection by comparing the amount of anti-zika virus antibodies determined in step 2 to the antibody correlate of protection against zika virus infection in human subjects, optionally determined as described above under the section “Method for determining an antibody correlate of protection”, and [0380] Step 4: administering to the human subject a zika virus vaccine if the human subject has an amount of anti-zika antibodies that is lower than the antibody correlate of protection against zika virus infection in human subjects, optionally determined as described above under the section “Method for determining an antibody correlate of protection”.
[0381] “Confer protection” within that context means that the amount of anti-zika virus antibodies present in the human subject is sufficient to prevent the human subject from developing zika disease after infection with a zika virus.
[0382] In preferred embodiments, the method for preventing zika disease in a human subject comprises the steps of: [0383] Step 1: obtaining a sample from the human subject, [0384] Step 2: determining the amount of anti-zika virus antibodies in the sample from the human subject as described above under the section “Method of detecting anti-zika virus antibodies”, [0385] Step 3: determining whether the human subject has an amount of anti-zika virus antibodies to confer protection by comparing the amount of anti-zika virus antibodies determined in step 2 to the antibody correlate of protection against zika virus infection in human subjects determined as described above under the section “Method for determining an antibody correlate of protection”, and [0386] Step 4: administering to the human subject a zika virus vaccine if the human subject has an amount of anti-zika antibodies that is lower than the antibody correlate of protection against zika virus infection in human subjects determined as described above under the section “Method for determining an antibody correlate of protection”.
[0387] In certain embodiments, the human subject is a woman. In certain embodiments, the woman is a woman of childbearing potential.
[0388] In certain embodiments, the human subject is living in a zika endemic region. In certain embodiments, the human subject is living in a zika non-endemic region traveling to a zika endemic region.
[0389] In certain embodiments, the zika virus vaccine is a purified inactivated zika virus vaccine. A suitable zika virus vaccine is described, for instance, in WO 2019/090228.
Method for Detecting Total Anti-Zika Virus Antibodies in a Sample
[0390] The present invention is further directed to a method for detecting essentially the complete panel of anti-zika virus antibodies (which may be of a certain antibody class, such as IgG) in a sample. When compared to the method for detecting anti-zika virus antibodies as described above, the method for detecting total anti-zika virus antibodies does not include the application of a reporter Ab, which is competing with the anti-zika virus antibodies in a sample. Whereas the method for detecting anti-zika virus antibodies as described above provides a read-out for antibodies in a sample, which are capable of competing with the reporter Ab (ZIKV-specific Abs), the method for detecting total anti-zika virus antibodies as described under this section provides a read-out of essentially the complete panel of anti-zika virus antibodies (which may be of a certain antibody class, such as IgG) in a sample.
[0391] Thus, the present invention in one embodiment is further directed to a method for detecting a signal from a detection antibody indicative for the presence and/or amount of anti-zika virus antibodies in a sample from a subject comprising the steps of: [0392] Step 1: contacting an amount of a microsphere complex as described above with the sample to allow binding of anti-zika virus antibodies in the sample to the zika virus like particles coupled to the microspheres in the microsphere complex, [0393] Step 2: contacting an amount of a detection antibody with the microsphere complex and the sample of step 1 to allow binding of the detection antibody to the heavy chain constant region of the anti-zika virus antibodies bound to the zika virus like particles coupled to the microspheres in the microsphere complex, wherein the detection antibody binds to the anti-zika virus antibodies with the variable region of the detection antibody and wherein the detection antibody is attached to at least one detectable label, and [0394] Step 3: detecting a signal from the detection antibody bound to the anti-zika virus antibodies in step 2.
[0395] The present invention in one embodiment is further directed to a method for determining the presence and/or amount of anti-zika virus antibodies in a sample from a subject, wherein the method comprises the further steps of: [0396] Step 4: determining the presence and/or amount of the detection antibody bound to the anti-zika virus antibodies from the signal of step 3, and [0397] Step 5: determining the presence and/or amount of anti-zika virus antibodies in the sample from the presence and/or amount of the detection antibody determined in step 4.
[0398] The amount of anti-zika virus antibodies in the sample may be determined from the amount of the detection antibody by comparison to a standard curve. For recording the standard curve, samples with known anti-zika antibody amounts may be analyzed in the method. Corresponding signals derived from the detection antibody upon analysis of the samples can then be matched to the corresponding anti-zika antibody amounts in the samples.
[0399] In one embodiment, contacting in step 1 is carried out for about 60 min and contacting in step 2 is carried out for about 30 min.
[0400] In one embodiment, the detection antibody is attached to the at least one detectable label by the heavy chain constant region. In certain embodiments, the at least one detectable label the detection antibody is attached to is a fluorescence label, such as phycoerythrin.
[0401] In certain embodiments, the signal in step 3 is resulting from the at least one detectable label, preferably the signal is a fluorescence signal.
[0402] Concerning the detection of the signal from the detection antibody, reference is made to the chapter “Method for detecting anti-zika virus antibodies”.
[0403] In certain embodiments, the sample is a sample from the group consisting of blood, urine, serum, blood plasma, cerebrospinal fluid, and lymph fluid, in particular the sample is a serum or blood plasma sample.
[0404] In certain embodiments, the subject is from the group consisting of mouse, primate, non-human primate, human, rabbit, cat, rat, horse, and sheep, in particular the subject is a human.
[0405] The present invention is further directed to a method for determining an antibody correlate of protection against zika virus infection as described under the chapter “Method for determining an antibody correlate of protection against zika virus infection”, wherein the amount of anti-zika virus antibodies for each subject in step 5 is determined according to the method for detecting total anti-zika virus antibodies in a sample as described under this chapter.
[0406] The present invention is further directed to a method for diagnosing the protection of a human subject against a zika virus infection as described under the chapter “Method for diagnosing zika virus protection”, [0407] wherein the amount of anti-zika virus antibodies in the sample in step 2 is determined according to the method for detecting total anti-zika virus antibodies in a sample as described under this chapter, and [0408] wherein protection in step 3 is determined by comparing the amount of anti-zika virus antibodies to the antibody correlate of protection determined in human subjects as described above using the method for detecting total anti-zika virus antibodies in a sample as described under this chapter.
[0409] The present invention is further directed to a method for diagnosing the protection of a non-human subject against a zika virus infection as described under the chapter “Method for diagnosing zika virus protection”, [0410] wherein the amount of anti-zika virus antibodies in the sample in step 2 is determined according to the method for detecting total anti-zika virus antibodies in a sample as described under this chapter, and [0411] wherein protection in step 3 is determined by comparing the amount of anti-zika virus antibodies to the antibody correlate of protection determined in this type of non-human subjects as described above using the method for detecting total anti-zika virus antibodies in a sample as described under this chapter.
[0412] In certain embodiments, the detection antibody is capable of binding to a certain antibody class or subclass (isotype). In preferred embodiments, the detection antibody is capable of binding to antibodies of class IgG.
Alternative Zika Antigens Useful for Coupling to the Microspheres
[0413] The present invention is alternatively directed to a kit comprising an amount of a microsphere complex coupled to a zika antigen and an amount of a reporter antibody that binds to the zika antigen of the microsphere complex and corresponding methods.
[0414] The ZIKV antigen may be a ZIKV structural protein or a ZIKV non-structural protein.
Microsphere Complex Comprising a Microsphere Coupled to a Dengue Virus Like Particle
[0415] The present invention further provides a microsphere complex comprising a microsphere coupled to a dengue virus like particle (DENV VLP). Concerning the properties of the microsphere, reference is made to the chapter “microsphere complex” above.
[0416] In one embodiment, the microsphere is coupled to a dengue-1 virus like particle.
[0417] In one embodiment, the dengue-1 virus like particle is derived from dengue-1 virus strain Puerto Rico/US/BID-V853/1998 characterized by SEQ ID NO: 179 and/or SEQ ID NO: 180.
[0418] In one embodiment, the dengue-1 virus like particle comprises structural proteins of dengue-1 virus strain Puerto Rico/US/BID-V853/1998 characterized by SEQ ID NO: 179 and/or SEQ ID NO: 180.
[0419] In one embodiment, the dengue-1 virus like particle comprises the envelope protein, the membrane protein, and/or the pre-membrane protein, which are at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to corresponding parts of SEQ ID NO: 180. In another embodiment, the dengue-1 virus like particle comprises the envelope protein, the membrane protein, and the pre-membrane protein, which are at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to corresponding parts of SEQ ID NO: 180.
[0420] In one embodiment, the microsphere is coupled to a dengue-2 virus like particle.
[0421] In one embodiment, the dengue-2 virus like particle is derived from dengue-2 virus strain Thailand/16681/84 characterized by SEQ ID NO: 181 and/or SEQ ID NO: 182.
[0422] In one embodiment, the dengue-2 virus like particle comprises structural proteins of dengue-2 virus strain Thailand/16681/84 characterized by SEQ ID NO: 181 and/or SEQ ID NO: 182.
[0423] In one embodiment, the dengue-2 virus like particle comprises the envelope protein, the membrane protein, and/or the pre-membrane protein, which are at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to corresponding parts of SEQ ID NO: 182. In another embodiment, the dengue-2 virus like particle comprises the envelope protein, the membrane protein, and the pre-membrane protein, which are at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to corresponding parts of SEQ ID NO: 182.
[0424] In one embodiment, wherein the microsphere is coupled to a dengue-3 virus like particle.
[0425] In one embodiment, the dengue-3 virus like particle is derived from dengue-3 virus strain Sri Lanka D3/H/IMTSSA-SRI/2000/1266 characterized by SEQ ID NO: 183 and/or SEQ ID NO: 184.
[0426] In one embodiment, the dengue-3 virus like particle comprises structural proteins of dengue-3 virus strain Sri Lanka D3/H/IMTSSA-SRI/2000/1266 characterized by SEQ ID NO: 183 and/or SEQ ID NO: 184.
[0427] In one embodiment, the dengue-3 virus like particle comprises the envelope protein, the membrane protein, and/or the pre-membrane protein, which are at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to corresponding parts of SEQ ID NO: 184. In another embodiment, the dengue-3 virus like particle comprises the envelope protein, the membrane protein, and the pre-membrane protein, which are at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to corresponding parts of SEQ ID NO: 184.
[0428] In one embodiment, the microsphere is coupled to a dengue-4 virus like particle.
[0429] In one embodiment, the dengue-4 virus like particle is derived from dengue-4 virus strain Dominica/814669/1981 characterized by SEQ ID NO: 185 and/or SEQ ID NO: 186.
[0430] In one embodiment, the dengue-4 virus like particle comprises structural proteins of dengue-4 virus strain Dominica/814669/1981 characterized by SEQ ID NO: 185 and/or SEQ ID NO: 186.
[0431] In one embodiment, the dengue-4 virus like particle comprises the envelope protein, the membrane protein, and/or the pre-membrane protein, which are at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to corresponding parts of SEQ ID NO: 186. In another embodiment, the dengue-4 virus like particle comprises the envelope protein, the membrane protein, and the pre-membrane protein, which are at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to corresponding parts of SEQ ID NO: 186.
[0432] In one embodiment, the dengue virus like particle is produced in human embryonic kidney (HEK293) cells.
[0433] Within the meaning of this invention, a DENV VLP comprising the envelope glycoprotein (E protein), membrane (M) protein, and/or pre-membrane (prM) protein of a DENV strain refers to a DENV VLP comprising an envelope glycoprotein, membrane protein, and/or pre-membrane protein which are at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to corresponding parts of the protein sequence of the DENV virus strain. Corresponding parts within that context mean parts of the protein sequence that encode for E protein, M protein, or prM protein, respectively.
EXAMPLES
[0434] The following Examples are included to demonstrate certain aspects and embodiments of the invention as described in the claims. It should be appreciated by those of skill in the art, however, that the following description is illustrative only and should not be taken in any way as a restriction of the invention.
Example 1: Coupling of ZIKV and DENV Antigens to Microspheres
[0435] Microspheres used for coupling were MagPlex® microspheres (Luminex Corporation, Austin, Texas). MagPlex® microspheres are superparamagnetic polystyrene microspheres with surface carboxyl groups. The microspheres were delivered in a volume of 4 to 4.1 mL with an average concentration of 1.2 to 1.3×10.sup.7 microspheres per mL (microspheres/mL). Microspheres used in this example were catalog numbers MC10014-04 (Product Lot. B75253), MC10025-04 (Product Lot. B76003), MC10033-04 (Product Lot. B71929), MC10045-04 (Product Lot. B76946), MC10047-04 (Product Lot. B75165), and MC10076-04 (Product Lot. B76373). The different microsphere catalog numbers refer to different microsphere sets wherein the microspheres of the sets comprise one or more fluorescent dyes having a specific emission spectrum (the specific feature) for distinguishing the microspheres upon detection. As the coupling mechanism involving the surface carboxyl groups is independent of the specific feature of the microspheres, catalog numbers of the MagPlex® microspheres may be exchanged according to variations in experimental set-ups.
[0436] DENV antigens for coupling to microspheres were DENV1 VLP (0.46 mg/mL liquid stock in 10 mM sodium phosphate, 20 mM sodium citrate, 154 mM sodium chloride pH 7.4; The Native Antigen Company, Product Code: DENV1-VLP-500, Batch No. 19040109), DENV2 VLP (0.52 mg/mL liquid stock in 10 mM sodium phosphate, 20 mM sodium citrate, 154 mM sodium chloride pH 7.4; The Native Antigen Company, Product Code: DENV2-VLP-500, Batch No. 19040816), DENV3 VLP (0.72 mg/mL liquid stock in 10 mM sodium phosphate, 20 mM sodium citrate, 154 mM sodium chloride pH 7.4; The Native Antigen Company, Product Code: DENV3-VLP-500, Batch No. 18111415), and DENV4 VLP (0.14 mg/mL liquid stock in Dulbecco's phosphate-buffered saline (DPBS) pH 7.4, 30% sucrose; The Native Antigen Company, Product Code: DENV4-VLP-500, Batch No. 18110614). DENV1-4 VLPs are consisting of DENV prM, M, and E protein produced in human embryonic kidney (HEK 293) cells. For production of DENV1-4 VLPs, the C-terminal 20% of DENV E protein were replaced by the corresponding Japanese encephalitis virus (JEV) SA-14 sequence (EMBL-EBI accession No: M55506.1, SEQ ID NO: 177 and 178; residues replaced: E protein amino acids 399-497 (DENV1 VLP), 397-495 (DENV2 VLP), 399-492 (DENV3 VLP), 400-495 (DENV4 VLP)). The replaced sequence corresponds to the transmembrane and intraparticle portion of the protein. DENV1 VLP was produced using the sequence from strain Puerto Rico/US/BID-V853/1998 (GenBank accession No. EU482592.1; SEQ ID NO: 179 and 180). DENV2 VLP was produced using the sequence from strain Thailand/16681/84 (EMBL-EBI accession No: U87411.1; SEQ ID NO: 181 and 182). DENV3 VLP was produced using the sequence from strain Sri Lanka D3/H/IMTSSA-SRI/2000/1266 (GenBank accession No. AY099336.1; SEQ ID NO: 183 and 184). DENV4 VLP was produced using the sequence from strain Dominica/814669/1981 (EMBL-EBI accession No: AF326825.1; SEQ ID NO: 185 and 186).
[0437] ZIKV antigens for coupling to microspheres were ZIKV VLP, and comparative examples, including recombinant 6×His-SUMO-tagged ZIKV EDIII (rZIKV-EDIII-1), recombinant 6×His-tagged ZIKV EDIII (rZIKV-EDIII-2), and recombinant human IgG1 Fc-tagged ZIKV EDIII (rZIKV-EDIII-3).
[0438] ZIKV VLPs (0.15 mg/mL liquid stock in DPBS pH 7.4, 20% sucrose; The Native Antigen Company, Product Code: ZIKV-VLP-250, Batch No. 19051017) comprise prM, M, and E protein of ZIKV isolate Z1106033 isolated in Suriname (Asian genotype; Enfissi et al., Lancet 2016, 387(10015):227-228; GenBank Accession No. KU312312.1, SEQ ID NO: 1 and SEQ ID NO: 2) and are produced in HEK 293 cells. [00341] r-ZIKV-EDIII-1 derived from the EDIII (SEQ ID NO: 4) of ZIKV strain H/PF/2013 (GenBank Accession No. KJ776791) and was cloned into a pETite vector (Lucigen, Cat. No. 49003-1). The pETite vector is designed for expression of the target protein as a fusion protein with an amino (N)-terminal 6×His-SUMO tag. r-ZIKV-EDIII-1 was expressed in Escherichia coli HI-Control BL21 (DE3) cells (Lucigen, Cat. No. 60435-1) and purified with immobilized metal affinity chromatography (IMAC) using a His-column. To further evaluate purity and integrity of rZIKV-EDIII-1 after the His-column run, elution fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analytical size exclusion chromatography (SEC) revealing a monomeric state of the recombinant protein. Finally, antigenicity of rZIKV-EDIII-1 was validated in a Bio-layer interferometry (BLI) assay using anti-ZIKV #1 to 6 and anti-PanDENV1-4 EDIII. The Octet results revealed that rZIKV-EDIII-1 showed good affinity for Anti-ZIKV #1-3 and 5, but solely weak association with anti-ZIKV #4 and it did not associate with Anti-ZIKV #6 and Anti-PanDENV1-4 EDIII mAb. rZIKV-EDIII-2 (Asian strain; The Native Antigen Company, Product Code: REC31775-100, Lot. No.: 20012409) was manufactured in E. coli and provided with an amount of 100 μg in 20 mM carbonate buffer pH 10.0. rZIKV-EDIII-3 (Creative Biolabs, Product ID: VAng-Wyb7346) is derived from ZIKV strain SPH2015 (GenBank accession No. ALU33341.1) and comprises ZIKV EDIII (amino acids V593-L699) with the Fc-region of human IgG1 at the carboxyl (C)-terminus expressed in HEK293 cells. The lyophilized protein (50 μg) was reconstituted to achieve a 0.25 mg/mL stock concentration in phosphate buffered saline (PBS) pH 7.4, 5% trehalose, 5% mannitol, 0.01% Tween-80 according to the Manufacture's protocol.
[0439] Antigens were coupled to the microspheres as described in Angeloni S., Das S. Dunbar S., Stone V, Swift S., xMAP Cookbook 4.sup.th edition, “A collection of methods and protocols for developing multiplexing assays with xMAP Technology” (Luminex Corporation, Austin, Texas). Different microspheres comprising one or more fluorescent dyes having a specific emission spectrum were applied for coupling of multiple antigens (Luminex Corporation, Austin, Texas; Cat. No. MC10014-04, MC10025-04, MC10033-04, MC10045-04, MC10047-04, MC10076-04) to provide the possibility to distinguish the microspheres according to their coupled antigens when analyzed within one sample. For example, ZIKV VLP was coupled to MagPlex® Cat. No. MC10047-04 and DENV3 VLP to MagPlex® Cat. No. MC10025-04.
[0440] The uncoupled stocks of MagPlex® microsphere suspensions (1.2 to 1.3×10.sup.7 microspheres/mL, Luminex Corporation, Austin, Texas) were resuspended by vortexing (30 sec) and 1 up to 12.5×10.sup.6 microspheres of each stock were transferred to 1.5 mL microcentrifuge tubes and placed into a 1.5 mL tubes magnetic separator (Life Technologies, Cat. No. 44578578). Separation of the microspheres from the suspension occurred for 30-60 sec. Supernatant was carefully removed without disrupting the microsphere pellet while the tubes were still positioned in the magnetic separator. Afterwards, the tubes were removed from the magnetic separator and the microspheres were resuspended in 100 μL distilled H.sub.2O (dH.sub.2O) by vortexing and sonication for approximately 20 sec. The tubes were again placed into the magnetic separator and separation of the microspheres from the suspension occurred for 30-60 sec. Supernatant was removed without disrupting the microsphere pellet while the tubes were still positioned in the magnetic separator. The microspheres were resuspended in 80 μl of activation buffer (0.1 M sodium phosphate (monobasic) pH 6.2) and mixed by vortexing and sonication for 20 sec. Then, 10 μL of 50 mg/mL M hydroxysulfosuccinimide (Sulfo-NHS; 2 mg of Sulfo-NHS in 40 μL of dH.sub.2O; Thermo Fisher Scientific, Cat. No. A39269, Lot. No. UI284573) were added to each microsphere tube and gentle mixing was carried out by vortexing (5 sec). Further, 10 μL of 50 mg/mL 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; 1 mg EDC in 20 μL of dH.sub.2O; Thermo Fisher Scientific, Cat. No. A35391, Lot. No. UD277513) were added to each microsphere tube and gentle mixing was carried out by vortexing (5 sec). Samples were incubated for 20 min at room temperature with gentle mixing by vortexing after 10 min. The tubes were placed into the magnetic separator and separation of the microspheres from the suspension occurred for 30-60 sec. Supernatant was removed without disrupting the microsphere pellet while the tubes were still positioned in the magnetic separator. 50 mM 2-(A morpholino)ethanesulfonic acid (MES) buffer was prepared by dilution of a stock solution (1 M MES buffer) in dH.sub.2O. The buffer pH varied depending on the optimum pH value required for coupling of each viral antigen. Optimum pH values for each antigen were evaluated by the microsphere immunoassays (MIAs) performed under Example 2. MES buffer at pH 6 (Boston Bioproducts, Cat. No. BBMS-60, Lot. No. F03K118) was used for coupling of DENV1-4 VLP. MES buffer pH of 6 was the optimum for coupling of rZIKV-EDIII-1 (coupling also evaluated in MES buffer at pH 7; Boston Bioproducts, Cat. No. BBMS-70, Lot. No. E02K118) and rZIKV-EDIII-3 (coupling also evaluated in MES buffer at pH 5; Boston Bioproducts, Cat. No. BBMS-50, Lot. No. F10K112 and pH 7). MES buffer at pH 7 was used for coupling of ZIKV VLP. rZIKV-EDIII-2 was coupled using MES buffer at pH 8 (Boston Bioproducts, Cat. No. BBMS-80, Lot. No. A24M126; coupling also evaluated in MES buffer at pH 9; Alfa Aesar, Cat. No. AAJ61646AK, Lot. No. X11E562). The tubes were removed from the magnetic separator and the microspheres were resuspended in 250 μL of corresponding 50 mM MES buffer by vortexing and sonication for 20 sec. The tubes were placed into the magnetic separator and separation of the microspheres from the suspension occurred for 30-60 sec. Supernatant was removed without disrupting the microsphere pellet while the tubes were still positioned in the magnetic separator. The tubes were removed from the magnetic separator and the microspheres were resuspended in 250 μL of corresponding 50 mM MES buffer by vortexing and sonication for 20 sec. The tubes were placed into the magnetic separator and separation of the microspheres from the suspension occurred for 30-60 sec. Supernatant was removed without disrupting the microsphere pellet while the tubes were still positioned in the magnetic separator. The tubes were removed from the magnetic separator and 400 μl of corresponding 50 mM MES buffer were added. Afterwards, 100 μL of a corresponding solution of each antigen (diluted in corresponding 50 mM MES buffer) were transferred to the respective 1.5 mL tube containing the activated microspheres to result in a ratio of 5 μg antigen per 10.sup.6 microspheres in a total volume of 500 μL. The mixture was vortex for 20 sec. For coupling, samples were incubated for 2 hours under rotation at room temperature. The tubes were placed into the magnetic separator and separation of the microspheres from the suspension occurred for 30-60 sec. Supernatant was removed without disrupting the microsphere pellet while the tubes were still positioned in the magnetic separator. The tubes were removed from the magnetic separator and the microspheres were resuspended in 1 mL of 1% BSA in 1-fold PBS pH 7.4 by vortexing for approximately 20 sec. The tubes were placed into the magnetic separator and separation of the microspheres from the suspension occurred for 30-60 sec. Supernatant was removed without disrupting the microsphere pellet while the tubes were still positioned in the magnetic separator. The tubes were removed from the magnetic separator and the microspheres were resuspended in 1 mL of 1% BSA in 1-fold PBS pH 7.4 by vortexing for approximately 20 sec. The tubes were placed into the magnetic separator and separation of the microspheres from the suspension occurred for 30-60 sec. Supernatant was removed without disrupting the microsphere pellet while the tubes were still positioned in the magnetic separator. The tubes were removed from the magnetic separator and the microspheres were resuspended in 250 μL of 1% BSA in 1-fold PBS pH 7.4 by vortexing for approximately 20 sec. The microspheres were kept in the 1.5 mL tubes or alternatively transferred to larger tubes (i.e. 2 mL tubes) when higher amounts of microspheres were coupled such as 12.5×10.sup.6 microspheres. In order to count the microspheres recovered after the coupling reaction, the microsphere suspension was diluted 2-fold in 1% BSA in 1-fold PBS pH 7.4 (e.g. 15 μL microsphere suspension diluted with 15 μL of 1% BSA in 1-fold PBS pH 7.4). The number of microspheres recovered after the coupling reaction was determined using an automated cell counter (Countes II, Thermo Fisher Scientific, Cat. No. AMQAX1000) by correlating the determined “dead cells” concentration provided by the cell counter to the microspheres. The coupled microspheres were stored at 2-8° C. in the dark (blocking step). Previous to use, the coupled microspheres were allowed to pre-warm for at least 30 min at room temperature.
Example 2: Selection of mAbs (Reporter Abs) for Microsphere Immunoassay (MIA)
[0441] mAbs tested for binding to antigen-coupled microspheres prepared as described in Example 1 are listed in Table 3 with available sequence information provided in Table 1 and 4.
TABLE-US-00003 TABLE 3 mAbs tested for binding to antigen-coupled microspheres. mAbs are presented together with their species origin. Anti-ZIKV #9 (Clone 181-4) and anti-ZIKV #10 (Clone 329-2) have the same protein and nucleic acid sequences. Species Origin mAb Examples Rabbit Anti-ZIKV #1 (Clone 102-1) Rabbit Anti-ZIKV #2 (Clone 242-3) Rabbit Anti-ZIKV #3 (Clone 270-12) Rabbit Anti-ZIKV #4 (Clone 289-3) Rabbit Anti-ZIKV #5 (Clone 306-2) Mouse Anti-ZIKV #6 (IgG2c kappa, Clone ZV-67) Mouse Anti-ZIKV #7 (IgG2a kappa, Clone ZKA-64) Rabbit Anti-ZIKV #8 (Clone 260-2) Rabbit Anti-ZIKV #9 (Clone 181-4) Rabbit Anti-ZIKV #10 (Clone 329-2) Rabbit Anti-ZIKV #11 (Clone 11-3) Comparative Examples Rabbit Anti-Flavivirus #1 (IgG kappa, Clone D1-4G2-4-15) Mouse Anti-Flavivirus #2 (IgG2a kappa, Clone D1-4G2-4-15) Human Anti-ZIKV E Protein (IgG1 kappa, Clone ZKA-78) Mouse Anti-PanDENV1-4 EDIII (IgG1 kappa, Clone 2D73) Rabbit Antibody Clone 78-2 Rabbit Antibody Clone 278-11 Controls Rabbit IgG Isotype Control Human Purified Human IgG1 Mouse IgG1 Isotype Control
TABLE-US-00004 TABLE 4 Sequence information for comparative example mAbs anti-ZIKV E protein, anti-Flavivirus #1 and 2, Antibody Clone 278-11, and Antibody Clone 78-2 tested for binding to antigen-coupled microspheres. For comparative example mAb Anti-PanDENV1-4 EDIII, which is a commercial mAb, sequence information was neither available from the vendor, nor publications, nor databases. (H = heavy chain; L = light chain; VH = heavy chain variable region; VL = light chain variable region; VH-CDR 1-3 = complementary determining regions 1-3 of heavy chain variable region; VL-CDR 1-3 = complementary determining regions 1-3 of light chain variable region). Amino acid Nucleic acid mAb sequence sequence mAb part (SEQ ID No) (SEQ ID No) Anti-ZIKV E Protein VH 135 143 VH-CDR 1 136 N/A VH-CDR 2 137 N/A VH-CDR 3 138 N/A VL 139 144 VL-CDR 1 140 N/A VL-CDR 2 141 N/A VL-CDR 3 142 N/A Anti-Flavivirus # 1 H 145 147 and 2 L 146 148 Antibody Clone 78-2 H 149 159 VH 150 160 VH-CDR 1 151 N/A VH-CDR 2 152 N/A VH-CDR 3 153 N/A L 154 161 VL 155 162 VL-CDR 1 156 N/A VL-CDR 2 157 N/A VL-CDR 3 158 N/A Antibody Clone 278-11 H 163 173 VH 164 174 VH-CDR 1 165 N/A VH-CDR 2 166 N/A VH-CDR 3 167 N/A L 168 175 VL 169 176 VL-CDR 1 170 N/A VL-CDR 2 171 N/A VL-CDR 3 172 N/A
[0442] Anti-ZIKV #1 to 5 mAbs (stock concentrations: 2.2, 1.77, 1.84, 2.4, 2.2 mg/mL, respectively, in phosphate buffer saline (PBS), pH 7.4), anti-ZIKV #8 to 11 mAbs, as well as Antibodies Clone 278-11 and 78-2 were generated and characterized as described in co-pending application PCT/US2019/052189 (WO 2020/106358; Takeda Ig Application). In brief, rabbits were immunized with purified inactivated ZIKV vaccine (PIZV) and ZIKV VLPs. Afterwards, the spleen was isolated for generation of hybridoma cells. Hybridoma supernatants were examined for reactivity towards ZIKV VLPs and ZIKV E protein, as well as cross-reactivity towards inactivated DENV1-4 by enzyme linked immunosorbent assay (ELISA). Moreover, hybridoma supernatants were screened for their neutralizing activity in a microneutralization titer (MNT) as well as a reporter virus particle (RVP) assay. Anti ZIKV #1 to 4, and #8 to 10 showed strong neutralization activity, whereas anti-ZIKV #5 showed weak neutralization activity. Anti-ZIKV #11 and Antibody Clone 78-2 showed no neutralization activity over the tested concentration range. Affinity of hybridoma supernatants towards ZIKV VLPs was determined by a Bio-layer interferometry (BLI) assay. In addition, epitope binning was examined in one experiment for anti-ZIKV #1-5 using a competitive BLI assay, binding a primary mAb to the ZIKV VLP, followed by cross-binding a secondary mAb. Binning experiments showed that anti-ZIKV #1 and 2, anti-ZIKV #3 and 4, and anti-ZIKV #5 built up three different clusters, indicating that mAbs belonging to different clusters bind to different regions of ZIKV VLP. Epitope binning was also examined in another experiment for anti-ZIKV #1-5, #8-11 and Antibody Clone 78-2. Binning experiments showed that anti-ZIKV #1-4 and #8-10 built up one cluster, whereas anti-ZIKV #5, #11, and antibody clone 78-2 each were highly diverse from one another and from the other mAbs (anti-ZIKV #1-4 and #8-10). Further, mAbs were sequenced (comp. Table 1 and Table 4). Finally, amino acid residues within the antigen critical for binding of anti-ZIKV #1-5 mAbs were evaluated using an alanine scanning mutagenesis library. Critical residues are those amino acids whose side chains make the highest energetic contribution to the Ab-epitope interaction and whose mutation gave the lowest binding reactivities (<10% of wild-type; Bogan and Thorn, J. Mol. Biol. 1998, 280, 1-9; Lo Conte et al., J. Mol. Biol. 1999, 285, 2177-2198). All mAbs were shown to bind to ZIKV EDIII. Notably, anti-ZIKV #4 also binds to EDI, an epitope associated with EDIII (comp. Table 2).
[0443] Anti-ZIKV #6 (Protein A affinity purified, 1 mg/mL stock concentration in PBS with 0.02% Proclin-300, Absolute Antibody, Cat. No. Ab00812-4.0, Lot. No. T1650A08) and 7 (Protein A purified, 1 mg/mL stock concentration in PBS with 0.02% Proclin-300, Absolute Antibody, Cat. No. Ab00779-2.0, Lot. No. T1839B41), as well as comparative example mAbs anti-Flavivirus #1 (Protein A purified, 1 mg/mL stock concentration in PBS with 0.02% Proclin-300, Absolute Antibody, Cat. No. Ab00230-23.0, Lot. No. T1812A57) and 2 (Protein A purified, 1 mg/mL stock concentration in PBS with 0.02% Proclin-300, Absolute Antibody, Cat. No. Ab00230-2.0, Lot. No. T1650A04), anti-ZIKV E Protein (Absolute Antibody, 1 mg/mL stock concentration, Cat. No. Ab00780-10.0, Lot. No. T1644B07) and anti-PanDENV1-4 EDIII (Absolute Antibody, 1 mg/mL stock concentration, Cat. No. Ab00948-1.1, Lot. No. T1749B16) were commercially available. According to the manufacturer, anti-ZIKV #6 was generated by infecting mice with 1000 focus forming units (FFU) of ZIKV MR-766 (GenBank accession No. AY632535.2) followed by a booster 30 days post infection of ZIKV H/PF/2013 (GenBank Accession No. KJ776791) and a final intravenous boost with live ZIKV EDIII (amino acids 299 to 407 of the ZIKV E protein). According to the manufacturer, anti-ZIKV #7 was selected from Epstein-Barr-Virus-immortalized memory B cells derived from ZIKV-infected, DENV-naïve human donors. According to the manufacturer, anti-Flavivirus #1 and #2 have been produced with DENV2 as the immunogen. According to the manufacturer, anti-ZIKV E Protein was selected from Epstein-Barr-Virus-immortalized memory B cells derived from ZIKV-infected, DENV-naïve human donors. According to the manufacturer, anti-PanDENV1-4 EDIII was generated from BALB/c mice immunized with recombinant DENV3 EDIII. Splenocytes were obtained from immunized mice and fused with the NS-1 myeloma cell line to generate hybridomas. Anti-PanDENV1-4 EDIII reacts with residues 309-319 (DKEMAETQHGT) within DENV EDIII.
[0444] Anti-ZIKV #6 (Zhao et al., Cell 2016, 166(4), 1016-1027) and Anti-ZIKV #7 (GenBank accession No. KX496860; Stettler et al., Science 2016, 353(6301), 823-6) were generated and characterized previously. Both anti-ZIKV #6 and 7 were demonstrated to bind to ZIKV EDIII. Anti-Flavivirus #1 and 2 were generated and characterized previously (Gentry et al., Am J Trop Med Hyg 1982, 31(3): 548-555; Nawa et al., J. Virol. Meth. 2001, 92, 65-70). Anti-Flavivirus #1 and 2 recognize the fusion loop within EDII that is conserved among different flaviviruses i.e. DENV, WNV, JEV, ZIKV, and YFV (Aubry et al., Transfusion 2016, 56:33-40). Anti-ZIKV E Protein was generated and characterized previously (Stettler et al., Science 2016, 353(6301), 823-6). Anti-ZIKV E Protein is directed against ZIKV EDI/II and cross-reacts with DENV1-4. Anti-PanDENV1-4 EDIII was generated and characterized previously (Li et al., J. Gen. Virol. 2013, 94, 2191-2201) and shows binding to all four DENV serotypes.
[0445] In addition to example and comparative example mAbs, control mAbs were tested for binding to antigen-coupled microspheres. As control mAbs served a rabbit IgG isotype control (Invitrogen, 1 mg/mL stock concentration, Cat. No.: 02-6106, Lot. No.: SJ257848), purified human IgG1 (UniProt ID: P01857; Molecular Innovations, 2.49 mg/mL stock concentration in 20 mM sodium phosphate, 150 mM NaCl, 0.05% sodium azide, pH 7.4, Cat. No.: HU-IGG1-1.0MG, Lot. No.: HU-IGG1M-5284), and mouse IgG1 isotype control (Invitrogen, 1 mg/mL stock concentration in 10 mM PBS, pH 7.4, 0.1& sodium azide, Cat. No.: 02-6100, Lot. No.: UD283794).
Binding Specificity of mAbs Towards Antigen-Coupled Microspheres
Determination of EC.sub.50 Values after 10 or 60 Min of Incubation of the mAb with the Antigen-Coupled Microspheres
[0446] For evaluation of binding specificity of mAbs towards antigen-coupled microspheres, EC.sub.50 values were determined after 10 or 60 min of incubation of the mAb with the antigen-coupled microspheres, respectively.
[0447] Therefore, microspheres were vortexed for 20 sec. A working microsphere mixture was prepared by diluting the coupled microsphere stock to a final concentration of 25 microspheres/μL in assay buffer (1% (w/v) BSA in 1-fold PBS, pH 7.4) and vortexing for 5 sec. The working mixture was kept at room temperature until 50 μL of the prepared working microsphere mixture were added per well in a black flat bottom 96-well assay plate (Corning Inc., Cat. No. 3915). mAbs were diluted from the corresponding stock concentrations in assay buffer to result in concentrations ranging from 0.001 to 20 μg/mL. mAb concentrations applied in the different experimental set-ups are indicated in the figures. 50 μL of each serially diluted mAb, or 50 μL assay buffer (2 blank wells per plate) were added to the microspheres and the suspension was pipetted up and down three times. Each mAb dilution was examined in duplicates. The plates were covered with a foil sealing sheet and incubated for 10 or 60 min (t 5 min), respectively, at room temperature on a plate shaker at 600 rpm. After incubation, the assay plate was washed two times with PBS-T (0.05% (v/v) Tween-20 in PBS pH 7.4) in a magnetic plate washer (BioTek Instruments, Product Id. 400072). Afterwards, the plate was placed in a 96-well plate magnet (Life Technologies, Product Id. 32513) and incubated for 30 sec while covering the plate. Following the incubation step, the supernatant was removed. For detection, F(ab′)2-goat anti-rabbit IgG (heavy and light chain) cross-adsorbed phycoerythrin (PE)-conjugated secondary Ab (Invitrogen, Cat. No. 31846, Lot. No. TL2684941, 0.5 mg/mL), R-PE AffiniPure F(ab′).sub.2 fragment goat anti-mouse IgG secondary Ab (heavy and light chain; Jackson ImmunoResearch, Cat. No. 115-116-146, Lot. No. 143867, 0.5 mg/mL), or PE-conjugated goat anti-human IgG secondary antibody (Southern Biotech, Cat. No. 2040-09, Lot. No. B3919-X449B, 0.5 mg/mL) were diluted 1:50 in assay buffer to achieve a final working concentration of 10 μg/mL by vortexing for 5 sec. 50 μL of the corresponding diluted detection Abs were added to each well. The plate was covered with a foil sealing sheet and incubation carried out for 30 min (t 2 min) at room temperature on a plate shaker at 600 rpm. The assay plate was washed two times with PBS-T in the magnetic plate washer. After the washing steps, the plate was placed in the 96-well plate magnet and incubated for 30 sec while covering the plate. Following the incubation step, the supernatant was removed. The microspheres were resuspended in 100 μL assay buffer per well. At this point, storage of the plate sealed with foil sealing sheet overnight at 4° C. is possible. Previous to sample read-out, the plate is allowed to re-equilibrate to room temperature for 20 min (5 min) if stored overnight at 4° C. The plate was put on an orbital shaker at 600 rpm for at least 5 min in order to allow for complete resuspension of immunocomplexed microspheres. Finally, the plate was placed in the multiplexing MAGPIX® plate reader (Luminex Corporation, Austin, Texas). The program used was xPONENT® (Build 4.2.1705.0) and is set-up with sample volume: 50 μL per well; plate protocol: 96-well plate format; and microsphere protocol: Map, BP 50 regions, Type MagPlex.
Determination of EC.sub.50 and EC.sub.25 Values after 2 Hours of Incubation of the mAb with the Antigen-Coupled Microspheres
[0448] In addition, binding specificity of mAbs towards antigen-coupled microspheres was evaluated by determining EC.sub.50 and EC.sub.25 values after 2 hours of incubation of the mAb with the antigen-coupled microspheres.
[0449] Therefore, microspheres were vortexed for 20 sec. A working microsphere mixture was prepared by diluting the coupled microsphere stock to a final concentration of 25 microspheres/μL in assay buffer (1% (w/v) BSA in 1-fold PBS, pH 7.4) and vortexing for 5 sec. The working mixture was kept at room temperature until 50 μL of the prepared working microsphere mixture were added per well in a black flat bottom 96-well assay plate (Corning Inc., Cat. No. 3915). After working microsphere mixture addition, the assay plate was washed two times with PBS-T (0.05% (v/v) Tween-20 in PBS pH 7.4) in a magnetic plate washer (BioTek Instruments, Product Id. 400072). Afterwards, the plate was placed in a 96-well plate magnet (Life Technologies, Product Id. 32513) and incubated for 30 sec while covering the plate. Following the incubation step, the supernatant was removed. mAbs were diluted from the corresponding stock concentrations in assay buffer to result in concentrations ranging from 0.001 to 20 μg/mL. After microsphere wash, 50 μL of each serially diluted mAb, or 50 μL assay buffer (2 blank wells per plate) were added to the microspheres. Each mAb dilution was examined in duplicates. The plates were covered with a foil sealing sheet and incubated for 2 hours at room temperature on a plate shaker at 600 rpm. After incubation, the assay plate was washed two times with PBS-T (0.05% (v/v) Tween-20 in PBS pH 7.4) in the magnetic plate washer. Afterwards, the plate was placed in the 96-well plate magnet and incubated for 30 sec while covering the plate. Following the incubation step, the supernatant was removed. For detection, F(ab′)2-goat anti-rabbit IgG (heavy and light chain) cross-adsorbed phycoerythrin (PE)-conjugated secondary Ab (Invitrogen, Cat. No. 31846, Lot. No. TL2684941, 0.5 mg/mL), R-PE AffiniPure F(ab′).sub.2 fragment goat anti-mouse IgG secondary Ab (heavy and light chain; Jackson ImmunoResearch, Cat. No. 115-116-146, Lot. No. 143867, 0.5 mg/mL), or PE-conjugated goat anti-human IgG secondary antibody (Southern Biotech, Cat. No. 2040-09, Lot. No. B3919-X449B, 0.5 mg/mL) were diluted 1:50 in assay buffer to achieve a final working concentration of 10 μg/mL by vortexing for 5 sec. 50 μL of the corresponding diluted detection Abs were added to each well. The plate was covered with a foil sealing sheet and incubation carried out for 30 min (t 2 min) at room temperature on a plate shaker at 600 rpm. The assay plate was washed two times with PBS-T in the magnetic plate washer. After the washing steps, the plate was placed in the 96-well plate magnet and incubated for 30 sec while covering the plate. Following the incubation step, the supernatant was removed. The microspheres were resuspended in 100 μL assay buffer per well. At this point, storage of the plate sealed with foil sealing sheet overnight at 4° C. is possible. Previous to sample read-out, the plate is allowed to re-equilibrate to room temperature for 20 min (t 5 min) if stored overnight at 4° C. The plate was put on an orbital shaker at 600 rpm for at least 5 min in order to allow for complete resuspension of immunocomplexed microspheres. Finally, the plate was placed in the multiplexing MAGPIX® plate reader (Luminex Corporation, Austin, Texas). The program used was xPONENT® (Build 4.2.1705.0) and is set-up with sample volume: 50 μL per well; plate protocol: 96-well plate format; and microsphere protocol: Map, BP 50 regions, Type MagPlex.
Determination of EC.sub.50 and EC.sub.25 Values
[0450] Data were analyzed and plotted using GraphPad Prism 8 version 8.1.0 (GraphPad Software, Inc.;
TABLE-US-00005 TABLE 5 EC.sub.50 and EC.sub.25 values for example mAbs incubated with ZIKV VLP for 120, 60 and/or 10 min. EC.sub.25 (μg/ml) EC.sub.50 (μg/mL) Incubation time: Incubation time: Incubation time: Incubation time: mAb 120 min 120 min 60 min 10 min Anti-ZIKV #1 (Clone 102-1) 0.0021 0.0066 0.029 0.1095 Anti-ZIKV #2 (Clone 242-3) 0.0024 0.0080 0.044 0.1397 Anti-ZIKV #3 (Clone 270-12) 0.0045 0.0144 0.061 0.1813 Anti-ZIKV #4 (Clone 289-3) 0.0025 0.0076 0.030 0.1607 Anti-ZIKV #5 (Clone 306-2) 0.0047 0.0158 0.066 0.2563 Anti-ZIKV #6 (IgG2c Clone ZV-67) 0.0081 0.0273 0.134 0.6769 Anti-ZIKV #7 (IgG2a Clone ZKA-64) 0.0296 0.1010 0.423 1.1760 Anti-ZIKV #8 (Clone 260-2) 0.0075 0.0252 N/A N/A Anti-ZIKV #9 (Clone 181-4) 0.0059 0.0156 N/A N/A Anti-ZIKV #10 (Clone 329-2) 0.0043 0.0145 N/A N/A Anti-ZIKV #11 (Clone 11-3) 0.0032 0.0105 N/A N/A
[0451] The data show that ZIKV VLP as the antigen coupled to the microspheres resulted in strong binding of all example mAbs anti-ZIKV #1 to 7 after 1 hour incubation of the mAbs with the microspheres (
[0452] rZIKV-EDIII-1 as the antigen coupled to the microspheres resulted in overall strong binding of anti-ZIKV #1-4 and 6-7 (
[0453] Anti-ZIKV #1 showed overall strong binding towards rZIKV-EDIII-3 as the antigen coupled to the microspheres (
[0454] In contrast, binding of mAbs to rZIKV-EDIII-2 was comparatively low (
[0455] Notably, the comparative example mAb Anti-PanDENV1-4 EDIII did not bind to any of the ZIKV antigens evaluated (
[0456] In summary, the ZIKV VLPs coupled to the microspheres showed consistent binding specificity results with all example mAbs. rZIKV-EDIII-2 seemed not suitable for use in the MIA.
Example 3: Selection and Pre-Characterization of Human Plasma Samples
[0457] Human plasma samples #1 to 4 for testing in the MIA were obtained from a commercially available panel (ABO Pharmaceuticals; Table 6). Samples were collected in Colombia from single blood donations.
TABLE-US-00006 TABLE 6 Human plasma samples for testing in the microsphere immunoassay (MIA). Presented are the date of symptoms onset, as well as the date of specimen collection, ethnicity, gender, and age of the blood donors at the time point of donation. Symptoms Specimen Sample ID onset collection Ethnicity Gender Age Plasma 11 May 2015 6 Jun. 2015 Hispanic Male 27 sample #1 Plasma 29 Apr. 2015 22 May 2015 Hispanic Female 41 sample #2 Plasma 11 Apr. 2016 14 Apr. 2016 Hispanic Male 30 sample #3 Plasma 2 May 2016 3 Jun. 2016 Hispanic Female 31 sample #4
[0458] Plasma sample #1 (Specimen ID: PLA_116, Internal Specimen ID: PLA_116, Source ID: ABOFCOL01, Sample No. 0262), plasma sample #2 (Specimen ID: RBB2560, Internal Specimen ID: 757{circumflex over ( )}2, Source ID: ABOFCOL01, Sample No. 0221), plasma sample #3 (Specimen ID: Z_0087_B, Internal Specimen ID: PARS_71, Source ID: ABOFCOL01, Sample No. 1038) and plasma sample #4 (Specimen ID: Z_0096_B, Internal Specimen ID: PARS_81, Source ID: ABOFCOL01, Sample No. 1043) were further characterized with a ZIKV reporter virus particle (RVP) assay as described in Young et al. Sci Rep 2020, 16(3488). In brief, samples were heat-inactivated in a water bath for 30 min at 56° C. (t 0.2° C.) and incubated with ZIKV RVP (Integral Molecular, Cat. No. ZIKV SPH2015 RVP-Renilla, Lot. No. P-229A) for 1 hour at 37° C. The mixture was incubated with Vero cells for 72 hours at 37° C. Renilla-Glo substrate (Promega, Cat. No. E2710) was then added and incubation carried out for 15 min at room temperature. Finally, plates were analyzed in a luminometer. The effective concentration at 50% (EC.sub.50 RVP) was determined by a non-linear regression curve fit with the lower asymptote constrained to 0 (GraphPad Prism). The human lower limit of quantification (LLOQ) is log.sub.10 EC.sub.50 RVP titer <2.0 Relative Luciferase Units (RLU; EC.sub.50 RVP titer <100 RLU), below which the matrix interfered with the measurement. Plasma samples #1 and 2 showed lower EC.sub.50 RVP titers (1,227 and 519.5 RLU, respectively) when compared to the titers of plasma samples #3 and 4 (15,316 and 3,269 RLU, respectively). Consequently, plasma samples #1-2 and #3-4 were categorized as ZIKV low- or high-reactive samples, respectively. ZIKV low-reactive samples were not expected to contain ZIKV specific Abs, whereas ZIKV high-reactive samples were expected to contain ZIKV specific Abs. ZIKV low-reactive samples were considered to contain anti-DENV Abs due to RVP titers above the human LLOQ.
[0459] The results from RVP assay are also in line with the timing of sample collection. ZIKV low-reactive samples were collected between May and June 2015, whereas ZIKV high-reactive samples were collected between April and June 2016. The first cluster of PCR-confirmed locally-acquired ZIKV cases in Colombia were reported to Pan American Health Organization/World Health Organization (PAHO/WHO) in October 2015 (https://www.paho.org/en/documents/zika-epidemiological-report-colombia-2).
[0460] In addition to plasma samples #1 to 4, a human serum sample was included as negative control (Human Serum from a male, 52-aged donor; BioreclamationIVT, Cat. No. HMSRM, Lot. No. BRH1140253). This control did not react or solely reacted to a very low extent with DENV1-4 NS1 proteins in an ELISA. Moreover, in the ZIKV RVP assay (performed as described above) the negative control showed almost no neutralizing capacity compared to a RVP positive control, indicating that the sample does not contain anti-flavivirus Abs (
[0461] For analysis, a certain amount of each sample was heat-inactivated previous to testing in a water bath for 30 min at 56° C. (±0.2° C.).
Example 4: Selection of Antigen-Coupled Microspheres for MIA
[0462] In the next step, suitability of antigen-coupled microspheres for application in the MIA was evaluated using the human plasma samples from Example 3 and determining total anti-ZIKV IgG levels within the samples.
[0463] Antigen-coupled microspheres were prepared as described under Example 1 and vortexed for 20 sec. A working microsphere mixture was prepared by diluting the coupled microsphere stock to a final concentration of 25 microspheres/μL in assay buffer (1% (w/v) BSA in 1-fold PBS, pH 7.4) and vortexed for 5 sec. The working mixture was kept at room temperature until 50 μL of the prepared working microsphere mixture were added per well in a black flat bottom 96-well assay plate (Corning Inc., Cat. No. 3915). Heat-inactivated human plasma samples #1 to 4 as well as the negative control human serum sample were prepared as described under Example 3 and diluted 5-fold in assay buffer by pipetting up and down 20 times. From this 5-fold dilution, samples were 10-fold serially diluted in assay buffer in a deep-well 2 mL plate (ThermoFisher Scientific, Cat. No. 278752) by pipetting up and down 5 times between the dilutions to result in final dilutions of 1:5, 1:50, 1:500, and 1:5,000 when referred to the undiluted samples. For ZIKV VLP and rZIKV-EDIII-1 as antigens coupled to the microspheres, 50 μL of each serially diluted sample and assay buffer (2 blank wells per plate) were added per well to the microspheres to result in final sample dilutions of 1:10, 1:100, 1:1,000, and 1:10,000. For rZIKV-EDIII-2 and 3, 50 μL of the 5-fold dilution and assay buffer (2 blank wells per plate) were added per well to the microspheres to result in a final sample dilution of 1:10. Every dilution was examined in duplicates. The samples and the microspheres were mixed by pipetting up and down three times. The plates were covered with a foil sealing sheet and incubated for 60 min (5 min) at room temperature on a plate shaker at 600 rpm. After incubation, the assay plate was washed two times with PBS-T (0.05% (v/v) Tween-20 in PBS pH 7.4) in a magnetic plate washer (BioTek Instruments, Product Id. 400072). Afterwards, the plate was placed in a 96-well plate magnet (Life Technologies, Product Id. 32513) and incubated for 30 sec while covering the plate. Following the incubation step, the supernatant was removed. For detection, a goat anti-human IgG detection Ab conjugated to PE (SouthernBiotech, Cat. No. 2040-09, Lot. No. B3919-X449B) was diluted 1:50 in assay buffer to achieve a final working concentration of 10 μg/mL by vortexing for 5 sec. 50 μL of the diluted detection Ab were added to each well. The plate was covered with a foil sealing sheet and incubation carried out for 30 min (±2 min) at room temperature on a plate shaker at 600 rpm. The assay plate was washed two times with PBS-T in the magnetic plate washer. After the washing steps, the plate was placed in the 96-well plate magnet and incubated for 30 sec while covering the plate. Following the incubation step, the supernatant was removed. The microspheres were resuspended in 100 μL assay buffer per well. At this point, storage of the plate sealed with foil sealing sheet overnight at 4° C. is possible. Before the read-out, the plate is allowed to re-equilibrate to room temperature for 20 min (t 5 min) if stored overnight at 4° C. The plate was put on an orbital shaker at 600 rpm for at least 5 min in order to allow for complete resuspension of immunocomplexed-microspheres. Finally, the plate was placed in the multiplexing MAGPIX® plate reader (Luminex Corporation, Austin, Texas). The program used was xPONENT® (Build 4.2.1705.0) and is set-up with sample volume: 50 μL per well; plate protocol: 96-well plate format; and microsphere protocol: Map, BP 50 regions, Type MagPlex. For an assay to be considered as valid, the MFI values from the blank controls were required to be very low compared to the MFI values resulting from the sample wells.
[0464] The total anti-ZIKV IgG levels of samples from Example 3 were determined using ZIKV VLPs, as well as rZIKV-EDIII-1-3 as antigens coupled to the microspheres (cf. Example 1 for information on antigens and coupling procedure).
[0465] No binding of the negative control to ZIKV VLPs was observed (
[0466] Contrarily, strong background binding of the negative control to rZIKV-EDIII-1 was observed, which might be caused by the 6×His-SUMO-tag and/or a (partially) disturbed antigen structure after coupling (
[0467] In addition to analysis of heat-inactivated samples, the samples as described under Example 3 were tested in the total anti-ZIKV IgG MIA without previous heat-inactivation. The assay was performed as described above for the heat-inactivated samples using ZIKV VLPs as antigens coupled to the microspheres, with the difference that more sample dilutions were examined. Interestingly, the total IgG MIA resulted in the same results for all examined samples independent of sample heat-inactivation (
[0468] In summary, ZIKV VLPs as microsphere-coupled antigens provide a consistent data set. This is in conformity with the mAb-binding data from Example 2.
Example 5: Evaluation of ZIKV Specific Ab Levels by a Competitive MIA (cMIA)
[0469] In the next step, human samples and non-human primate samples were evaluated for the presence of ZIKV specific Abs within the samples by a competitive MIA (cMIA) using antigen-coupled microspheres and mAbs from Examples 1 and 2. In addition to a qualitative proof-of-concept set-up, the cMIA was also performed in a quantitative way.
Example 5.1: Qualitative Proof-of-Concept Set-Up
[0470] Microspheres as prepared under Example 1 were vortexed for 20 sec. A working microsphere mixture was prepared by diluting the coupled microsphere stock to a final concentration of 50 microspheres/μL in assay buffer (1% (w/v) BSA in 1-fold PBS, pH 7.4) and vortexing for 5 sec. The working mixture was kept at room temperature. Each 25 μL of the prepared working microsphere mixture were added per well of a black flat bottom 96-well assay plate (Corning Inc., Cat. No. 3915). Human plasma samples (plasma samples #1-4), as well as the negative control of Example 3 were heat-inactivated as described under Example 3 and were vortexed for 5 sec before transferring them into a deep-well 2 mL plate (ThermoFisher Scientific, Cat. No. 278752) to perform serial dilutions in assay buffer. Serial dilutions were prepared to result in a 5-, 10-, and 20-fold sample dilution for the cMIA using ZIKV VLP as microsphere-coupled antigens. Samples were mixed in between the dilutions by pipetting up and down 20 times (5-fold dilution) or 5 times (10- and 20-fold dilution), respectively. For rZIKV-EDIII-3 as antigen, samples were applied at a 5-fold dilution. Afterwards, 25 μL of each diluted sample were aliquoted into the assay plate per well in duplicates. Two controls were included by aliquoting 25 μL of assay buffer per well in duplicates to later account for 0% and 100% mAb binding. Afterwards, the mixture of microspheres and samples were pipetted up and down three times, which results in a 2-fold dilution of samples resulting in 10-, 20-, and 40-fold final dilutions. The plate was covered with a foil sealing sheet and incubated for 60 min (t 5 min) at room temperature on a plate shaker at 600 rpm. Meanwhile, an intermediary 50-fold (1:50) dilution of each example mAb (Anti-ZIKV #1 to 7) and Anti-PanDENV1-4 EDIII mAb was prepared by mixing 10 μL of the stock mAb solution with 490 μL of assay buffer. The 50-fold dilution was vortexed for 5 sec. Next, the 50-fold dilution was further diluted in assay buffer to reach the final mAb concentration, which corresponds to the EC.sub.50 concentration of the mAb for ZIKV VLP-binding calculated under Example 2 after 10 min incubation with the ZIKV VLP coupled-microspheres (Table 5). The final mAb concentration solution was vortexed for 10 sec. After incubation of the samples with the microspheres no additional washing steps were carried out. 50 μL of the final mAb concentration solution were added to the wells. 50 μL of assay buffer were added to the wells corresponding to 0% mAb-binding. No additional mixing was performed. The plate was covered with a foil sealing sheet and incubated for 10 min at room temperature on a plate shaker at 600 rpm. After incubation, the assay plate was washed two times with PBS-T (0.05% (v/v) Tween-20 in PBS pH 7.4) in a magnetic plate washer (BioTek Instruments, Product Id. 400072). Afterwards, the plate was placed in a 96-well plate magnet (Life Technologies, Product Id. 32513) and incubated for 30 sec while covering the plate. Following the incubation step, the supernatant was removed. For detection of rabbit or mouse mAbs, respectively, a F(ab′)2-Goat anti-rabbit IgG (heavy and light chain) cross-adsorbed PE-conjugated (Invitrogen, Cat. No. 31846, Lot. No. TL2684941, 0.5 mg/mL) or a R-PE AffiniPure F(ab′).sub.2 fragment goat anti-mouse IgG (heavy and light chain; Jackson ImmunoResearch, Cat. No. 115-116-146, Lot. No. 143867, 0.5 mg/mL) secondary reporter Ab was applied. Secondary Abs were diluted 1:50 in assay buffer to achieve a final working concentration of 10 μg/mL by vortexing for 5 sec. 50 μL of the corresponding diluted detection Abs were added to each well. The plate was covered with a foil sealing sheet and incubation carried out for 30 min (t 2 min) at room temperature on a plate shaker at 600 rpm. The assay plate was washed two times with PBS-T in a magnetic plate washer. After the washing steps, the plate was placed in a 96-well plate magnet and incubated for 30 sec while covering the plate. Following the incubation step, the supernatant was removed. The microspheres were resuspended in 100 μL assay buffer per well. At this point, storage of the plate sealed with foil sealing sheet overnight at 4° C. was possible. Before sample read-out, the plate was allowed to re-equilibrate to room temperature for 20 min (t 5 min) if stored at 4° C. overnight. The plate was put on an orbital shaker at 600 rpm for at least 5 min in order to allow for complete resuspension of immunocomplexed microspheres. Finally, the plate was placed in the multiplexing MAGPIX® plate reader (Luminex Corporation, Austin, Texas). The program used was xPONENT® (Build 4.2.1705.0) and is set-up with sample volume: 50 μL per well; plate protocol: 96-well plate format; and microsphere protocol: Map, BP 50 regions, Type MagPlex. For an assay to be considered as valid, the MFI values from the blank controls were required to be very low compared to the MFI values resulting from the sample wells.
[0471] Data generated were analyzed and plotted using GraphPad Prism 8 version 8.1.0 (GraphPad Software, Inc.). MFI values reported by the MAGPIX® reader resulting from binding of Anti-ZIKV #1 to 7 and Anti-PanDENV1-4 EDIII mAbs are presented for the analyzed plasma samples and the negative control dependent on the plasma dilution (
[0472] Satisfyingly, BoB values resulting from incubation of the negative control and human plasma samples #1 and 2, which are considered to be ZIKV low-reactive, with ZIKV VLPs as microsphere-coupled antigens were comparatively low for all anti-ZIKV #1 to 7 mAbs independent of the plasma dilution. On the other hand, higher BoB values were observed using the ZIKV high-reactive samples (human plasma samples #3 and 4;
[0473] Contrarily, low MFI values were observed for human plasma samples #1 and 2 considered as ZIKV low-reactive when compared to the 100% mAb binding control using the rZIKV-EDIII-3 as antigen coupled to the microspheres, indicating unspecific blocking of mAb binding, as ZIKV specific Abs should be absent from these samples (
Example 5.2: Quantitative cMIA
Example 5.2.1: Initial Quantitative cMIA Set-Up
[0474] To evaluate the suitability of the cMIA set-up for a quantitative analysis of samples, an initial quantitative set-up was evaluated. Therefore, the cMIA was performed as described above in Example 5.1 using ZIKV VLP-coupled microspheres and final sample dilutions in the range of 10-, 40-, 80-, 160-, and 320-fold in the assay (cf. also
[0475] For a quantitative analysis, MFI raw data obtained from the analysis in the MAGPIX Luminex reader were analyzed and plotted using GraphPad Prism 8 version 8.1.0 (GraphPad Software, Inc.). A mean value from the replicates of the 100% mAb binding control was calculated. From the mean value of the 100% mAb binding control signal, the value referring to 40% of the 100% binding control signal was calculated. The sample dilution values in normal scale (e.g. 10-, 20-, 40-fold dilution) were logarithmized (final sample dilution for
[0476] Plasma samples from Example 3 were analyzed with the initial quantitative set-up of the cMIA using anti-ZIKV #7. Human plasma samples #3 and 4, which were considered to be ZIKV high-reactive, showed a concentration dependent increase of blocking of mAb binding (indicated by the lower MFI values) to almost complete blockade of binding for the lowest sample dilution (
TABLE-US-00007 TABLE 7 ZIKV-specific blockade titers for samples examined in the initial quantitative cMIA using anti-ZIKV #7 calculated based on the initial sample dilution (cf. FIG. 37B). Presented are the titers together with the coefficient of determination (R.sup.2 value) for the titers. The titers refer to the dilution of the sample that inhibits 60% of the maximal binding of the anti-ZIKV #7 to the ZIKV VLPs. ZIKV-specific Sample Blockade Titer R.sup.2 Negative control N/A 0.77240 Human plasma sample #1 N/A 0.66370 Human plasma sample #2 4.10 (considered 0.61510 invalid) Human plasma sample #3 32.78 (considered 0.98620 valid) Human plasma sample #4 11.80 (considered 0.99730 valid)
[0477] In summary, the initial quantitative cMIA set-up was able to distinguish between presumptive ZIKV high-reactive (ZIKV specific Ab containing samples) and ZIKV low-reactive samples independent of the presence of cross-reactive Abs resulting from other flavivirus infections as DENV infection.
Example 5.2.2: Optimized Quantitative cMIA Set-Up
[0478] In a next step, the initial quantitiative cMIA set-up, which was shown to be capable to distinguish between ZIKV high- and low-reactive samples in Example 5.2.1, was further optimized as outlined below.
[0479] In the optimized quantitative cMIA set-up, samples were not heat-inactivated, as the MIA set-up was shown to work in a robust manner, independent of heat-inactivation (cf. Example 4). Samples were prepared by vortexing for 5 sec before transferring them into a deep-well 2 mL plate (ThermoFisher Scientific, Cat. No. 278752) for preparation of the serial sample dilutions. An initial 5-fold dilution per sample was performed using assay buffer (1% (w/v) BSA in 1-fold PBS, pH 7.4) in row A of the deep well plate. The initial dilution was mixed by pipetting up and down 10 times in row A. Then, 2-fold serial dilutions were performed by combining half volume of solution in row A with same volume of assay buffer in row B, and so on until row H. Mixing between dilution was performed by pipetting up and down 5 times. A total of eight 2-fold dilutions (5-fold to 640-fold) per sample (rows A-H; 5-, 10-, 20-, 40-, 80-, 160-, 320-, and 640-fold dilution, respectively) were performed. Deep well dilution plate was covered with a sealing sheet while a ZIKV VLP coupled-microspheres working mixture was prepared. ZIKV VLP coupled-microspheres prepared under Example 1 were vortexed for 20 sec. The coupled microsphere stock was diluted to a final concentration of 50 microspheres/μL in assay buffer and vortexed for 5 sec. The working mixture was kept at room temperature and 25 μL of the prepared working microsphere mixture were added per well of a black flat bottom 96-well assay plate (Corning Inc., Cat. No. 3915). Afterwards, 25 μL of each diluted sample were aliquoted into the assay plate per well in duplicates in a vertical plate layout (e.g. the 8 dilutions of the first sample were added in rows A-H and columns 1-2), resulting in a 2-fold dilution of each sample dilution (=final sample dilution; 10-, 20-, 40-, 80-, 160-, 320-, 640-, 1280-fold dilution, respectively) when combined with the 25 μL of the microsphere mixture per well. Two controls were included by aliquoting 25 μL of assay buffer per well in duplicates (columns 11-12) to later account for 0% and 100% mAb binding. The assay plate was covered with a foil sealing sheet and incubated for 60 min at room temperature on a plate shaker at 600 rpm. Meanwhile, an intermediary 50- or 1000-fold dilution of the mAb used (50-fold for Anti-ZIKV #6 to 7; 1000-fold for Anti-ZIKV #1 to 5) was prepared by mixing 5 μL of the stock mAb solution with 245 or 4995 μL of assay buffer, respectively. The intermediary dilution was vortexed for 5 sec. Next, the intermediary dilution was further diluted in assay buffer to reach the final mAb dilution, which corresponds to the EC.sub.25 or EC.sub.50 concentration (EC.sub.50 for Anti-ZIKV #6 to 7; EC.sub.25 for Anti-ZIKV #1 to 5) of the mAb for ZIKV VLP-binding calculated under Example 2 after 2 hr incubation with the ZIKV VLP coupled-microspheres (Table 5). The final mAb concentration solution was vortexed for 5 sec. After incubation of the samples with the microspheres, the assay plate was washed two times with PBS-T (0.05% (v/v) Tween-20 in PBS pH 7.4) in a magnetic plate washer (BioTek Instruments, Product Id. 400072). Afterwards, the plate was placed in a 96-well plate magnet (Life Technologies, Product Id. 32513) and incubated for 60 sec while covering the plate. Following the incubation step, the supernatant was removed. Then, 50 μL of the final mAb concentration solution were added to the sample-containing wells (columns 1-10) and 100% mAb binding (columns 11-12, rows E-H) wells and 50 μL of assay buffer were added to the wells corresponding to 0% mAb-binding (columns 11-12, rows A-D). The plate was covered with a foil sealing sheet and incubated for 2 hr at room temperature on a plate shaker at 600 rpm. After incubation with the mAb, the assay plate was washed two times with PBS-T (0.05% (v/v) Tween-20 in PBS pH 7.4) in a magnetic plate washer. Afterwards, the plate was placed in a 96-well plate magnet and incubated for 60 sec while covering the plate. Following the incubation step, the supernatant was removed. For detection of rabbit (Anti-ZIKV #1 to 5) or mouse (Anti-ZIKV #6 to 7) mAbs, respectively, a F(ab′)2-Goat anti-rabbit IgG (heavy and light chain) cross-adsorbed PE-conjugated (Invitrogen, Cat. No. 31846, Lot. No. TL2684941, 0.5 mg/mL) or a R-PE AffiniPure F(ab′).sub.2 fragment goat anti-mouse IgG (heavy and light chain; Jackson ImmunoResearch, Cat. No. 115-116-146, Lot. No. 143867, 0.5 mg/mL) secondary reporter Ab was applied. Secondary Abs were diluted 1:50 in assay buffer to achieve a final working concentration of 10 μg/mL by vortexing for 5 sec. 50 μL of the corresponding diluted secondary reporter Ab were added to all wells, including the 0% and 100% mAb binding control wells. The plate was covered with a foil sealing sheet and incubation carried out for 30 min at room temperature on a plate shaker at 600 rpm. After incubation of mAb, the assay plate was washed two times with PBS-T (0.05% (v/v) Tween-20 in PBS pH 7.4) in a magnetic plate washer. Afterwards, the plate was placed in a 96-well plate magnet and incubated for 60 sec while covering the plate. Following the incubation step, the supernatant was removed. After the washing steps, the microspheres were resuspended in 100 μL assay buffer per well. At this point, the plate can be stored with foil sealing sheet overnight at 4° C. Before sample read-out, the plate was allowed to re-equilibrate to room temperature for 20 min (t 5 min) if stored at 4° C. overnight. The plate was put on an orbital shaker at 600 rpm for at least 5 min in order to allow for complete resuspension of immunocomplexed microspheres. Finally, the plate was placed in the multiplexing MAGPIX® plate reader (Luminex Corporation, Austin, Texas). The program used was xPONENT® (Build 4.2.1705.0) and is set-up with sample volume: 50 μL per well; plate protocol: 96-well plate format; and microsphere protocol: Map, BP 50 regions, Type MagPlex.
[0480] The MFI data obtained from analysis in the MAGPIX Luminex reader were analyzed and plotted as described under Example 5.2.1 and ZIKV-specific blockade titers were calculated for each sample based on the final sample dilution.
Example 5.2.3 the Quantitative cMIA is Able to Distinguish Samples Containing ZIKV-Specific Abs from Samples Containing Other Anti-Flaviviruses Abs
[0481] In a next step, it was evaluated whether the quantitative cMIA set-up is able to distinguish samples comprising ZIKV-specific Abs from samples comprising other anti-flavivirus Abs (including ZIKV cross-reactive Abs) in a robust manner. Therefore, human samples containing antibodies directed against ZIKV, DENV, Yellow Fever Virus (YFV), St. Louis Encephalitis Virus (SLEV), or West Nile Virus (WNV) as well as samples containing antibodies directed against both, ZIKV and DENV or WNV and DENV, as determined in corresponding immunoassays, were selected for analysis. In addition to human samples, also non-human primate samples from animals infected with ZIKV or DENV, or vaccinated with either YFV vaccine (STAMARIL; Sanofi Pasteur), Japanese Encephalitis virus (JEV) vaccine (IXIARO; Valneva Scotland Ltd.), ZIKV vaccine (purified inactivated vaccine, PIZV; see, for instance, WO 2019/090228), WNV vaccine (INNOVATOR; Fort Dodge Animal Health), or Tick-borne encephalitis virus (TBEV) vaccine (ENCEPUR; GlaxoSmithKline) were analyzed. The quantitative cMIA was carried out using anti-ZIKV #7 as described above under Example 5.2.2.
Human Samples
[0482] Human samples evaluted were serum samples, except for the negative control (which did not comprise anti-flavivirus Abs; “FV-Naïve control”) and the sample that contained both, anti-ZIKV and anti-DENV Abs, which were plasma samples. mAb binding was almost completely blocked by the five samples containing anti-ZIKV antibodies at lower sample dilutions (designated as “+ZIKV #1-5 H”;
[0483] In contrast, mAb binding was not diminished over the sample dilution range similar to the negative control (“FV-Naïve control”) for the three samples containing anti-DENV Abs (designated as “+DENV #1-3 H”;
[0484] Consequently, the quantitative cMIA enables a robust differentiation between samples comprising ZIKV-specific antibodies, and samples comprising other flavivirus Abs (including ZIKV cross-reactive Abs) and thus enables characterization of the immune status of a human subject. No false-positives were detected.
Non-Human Primate Samples
[0485] The assay set-up was further applied for analyzing serum samples from non-human primates (NHP). Therefore, serum samples from rhesus macaques after ZIKV primary infection were evaluated in the quantitative cMIA (three of them are depicted in
[0486] Moreover, similar results than with the ZIKV natural infected animals were observed for samples from four rhesus macaques each vaccinated with two doses of the purified inactivated zika vaccine (PIZV) on days 1 and 29. MFI values resulting from analysis of samples from two of those animals are depicted in
[0487] Contrarily, samples from rhesus macaques vaccinated with YFV, JEV, WNV, or TBEV vaccines were not capable of blocking mAb binding. Samples from two animals each vaccinated with two doses of a JEV vaccine (first dose at day 1, second dose at day 29; designated as “JEV Vac. pre PIZV #1-2 NHP”;
[0488] However, mAb binding was efficiently blocked by samples from the same vaccinated rhesus macaques described above, which were taken after an additional subsequent vaccination of those animals with two doses of PIZV 168 days after the last YFV, JEV, WNV, or TBEV vaccine dose, respectively (designated as “JEV Vac. post PIZV #1-2 NHP”, “YFV Vac. post PIZV #1-2 NHP”, “WNV Vac. post PIZV #1-2 NHP”, and “TBEV Vac. post PIZV #1-2 NHP”;
Example 5.2.4 Quantitative cMIA Using mAbs #1-5
[0489] To demonstrate that the quantitative cMIA is suitable to be performed with sevaral different mAbs, anti-ZIKV #1-5 were applied in the quantitative cMIA as described under Example 5.2.2. As described under Example 5.2.2., anti-ZIKV #1-5 were applied at final mAb concentrations corresponding to the EC.sub.25 concentration of the mAb for ZIKV VLP-binding calculated under Example 2 after 2 hr incubation with the ZIKV VLP coupled-microspheres (Table 5). Anti-ZIKV #7 was applied at a final mAb concentration corresponding to the EC.sub.50 concentration (as described above under Example 5.2.2 and applied under Example 5.2.3) and in addition also at a final mAb concentration corresponding to the EC.sub.25 concentration of the mAb for ZIKV VLP-binding calculated under Example 2 after 2 hr incubation with the ZIKV VLP coupled-microspheres (Table 5).
[0490] Therefore, the human sample containing antibodies directed against both, ZIKV and DENV (designated as “+ZIKV/+DENV H”, as well as the negative control (which did not comprise anti-flavivirus Abs; designated as “FV-Naïve control”) described in Example 5.2.3 above were applied. In addition, the human samples containing antibodies directed against West Nile Virus (WNV) described in Example 5.2.3 above were pooled and analyzed (designated as “+WNV (human pool)”). Moreover, also the non-human primate samples from animals infected with DENV described in Example 5.2.3 were pooled for analysis (designated as “DENV nat NHP pool”). Finally, also the non-human primate samples from animals immunized with PIZV described in Example 5.2.3 were pooled for analysis (designated as “PIZV NHP pool”).
[0491] Each of anti-ZIKV #1-5 was able to differentiate the “PIZV NHP pool” and “+ZIKV/+DENV H” samples from the “DENV nat NHP pool” and the “+WNV (human pool)” samples, indicating a robust assay performance also with different mAbs (
TABLE-US-00008 TABLE 8 ZIKV-specific blockade titers for samples examined in the quantitative cMIA using anti- ZIKV #1-5 and #7 calculated based on the final sample dilution. Presented are the titers for the different samples together with the final mAb concentration (EC.sub.25 or EC.sub.50 concentrations, respectively). The titers refer to the dilution of the sample that inhibits 60% of the maximal binding of the anti-ZIKV mAbs to the ZIKV VLPs. mAb FV-Naïve DENV nat +WNV PIZV NHP Anti-ZIKV concentration control NHP pool (human pool) pool +ZIKV/+DENV H #1 EC.sub.25 0 0 0 8 35 #2 EC.sub.25 0 0 0 19 46 #3 EC.sub.25 0 0 0 19 52 #4 EC.sub.25 0 0 0 16 41 #5 EC.sub.25 0 0 0 25 46 #7 EC.sub.25 0 0 0 14 65 EC.sub.50 0 0 0 19 46
CONCLUSION
[0492] Although competitive ELISA set-ups have been used with ZIKV EDIII as plate-immobilized antigen (WO 2020/087038), our experimental data show that EDIII is not suitable for application as microsphere-coupled antigen. In particular, as used in WO 2020/087038, ZIKV EDIII C-terminally fused to a human IgG1 Fc tag (rZIKV-EDIII-3 in the present application) resulted in strong background signal in the total human IgG MIA when coupled to the microspheres (comp. Example 4). Moreover, rZIKV-EDIII-3 as microsphere coupled-antigen was not able to distinguish between ZIKV and other flavivirus infections in the cMIA. As rZIKV-EDIII-3, also other recombinant ZIKV EDIII tested herein were not suitable for the microsphere set-up. Solely ZIKV VLP showed satisfying and reliable results in both, the total IgG MIA, as well as the cMIA.
[0493] In summary, we could show that the cMIA set-up is able to reliably and efficiently detect and quantify anti-ZIKV Abs. With the use of different reporter Abs, we are able to detect both, strong-neutralizing, as well as weak-neutralizing Abs (i.e. by the use of Anti-ZIKV #5). With the application of a non-neutralizing reporter Ab, non-neutralizing anti-ZIKV Abs can be detected as well. Moreover, we could demonstrate that the reporter Abs used are highly selective for ZIKV VLPs, as the mAbs did not show binding to DENV1-4 VLPs within the same mAb concentration range applied for evaluation of binding to ZIKV VLPs. Therefore, our set-up is able to distinguish between ZIKV and other flavivirus infections by measuring ZIKV specific Abs instead of cross-reactive Abs, which underlines its suitability for use in diagnostics.
[0494] Compared to traditional assays such as competitive ELISA set-ups, a microsphere-based assay provides several advantages. This approach increases sensitivity and specificity, among other advantages such as the flexibility to single- or multi-plex antigens from different viruses in a single reaction (by the application of microspheres with different specific features), high-throughput (e.g. simplified washing procedures due to magnetic microspheres), cost-effectiveness (e.g. due to reduced sample volume, consumables, and labor), and short turnaround time.
[0495] Recently, it has been shown that results from competitive sample set-ups (i.e. set-ups using mAbs directed against ZIKV EDIII) measuring anti-ZIKV Ab titers correlate with protection in vivo (WO 2020/087038). This reinforces the fact that our cMIA is as well suitable for determining protection in subjects against ZIKV infection by measuring anti-ZIKV Abs. For instance, anti-ZIKV Abs may be induced by vaccination of the subjects or by natural ZIKV infections. Moreover, as the cMIA is selective for determination of anti-ZIKV Abs it is able to reliably distinguish between Abs induced by a ZIKV infection and cross-reactive Abs induced by any other flavivirus infection, such as a DENV, WNV, JEV, YFV, or SLEV infection.
[0496] In conclusion, ZIKV VLPs as antigens coupled to the microspheres in combination with all example mAbs (Anti-ZIKV #1-11) selected from Example 2 are well suitable for the cMIA. The results demonstrate the capability of the cMIA for reliable detection and quantification of ZIKV specific Abs in different samples from different origins (such as human or NHP) or in different sample types (such as serum or plasma). In addition, the assay is capable of distinguishing ZIKV specific Ab containing samples from ZIKV non- or low-reactive samples, independent of the presence of other (also ZIKV cross-reactive) anti-flavivirus Abs such as DENV, YFV, WNV, SLEV, or TBEV immunities, required by natural infection or vaccination.
[0497] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. As used herein the terms “about” and “approximately” means within 10 to 15% of the number, preferably within 5 to 10% of the number. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[0498] Numerous references have been made throughout this specification. Each of the above-cited references is individually incorporated herein by reference in its entirety. In case of conflict, the information in the present application prevail.
[0499] In the following, CDR sequences of anti-ZIKV Abs as listed in the sequence listing are additionally reproduced:
TABLE-US-00009 SEQ ID NO in the sequence mAb CDR listing CDR sequence Anti-ZIKV #1 VH-CDR1 7 GFSFNSNYW (Clone 102-1) VH-CDR2 8 FGGIHVT VH-CDR3 9 IISTGGSHRFNL VL-CDR1 12 ESIYTY VL-CDR2 13 RAS VL-CDR3 14 QATDVGGSGRGA Anti-ZIKV #2 VH-CDR1 21 GFSFTDRHY (Clone 242-3) VH-CDR2 22 YPGSSGST VH-CDR3 23 ARSSYPDSSGYSYGMDL VL-CDR1 26 QNINSN VL-CDR2 27 LTS VL-CDR3 28 QTYYDISNYGYA Anti-ZIKV #3 VH-CDR1 35 GFSFTDRHY (Clone 270- VH-CDR2 36 YPGSSGST 12) VH-CDR3 37 ARSSYPDSSGYSYGMDL VL-CDR1 40 QDINSN VL-CDR2 41 LTS VL-CDR3 42 QTYYDISNYGYA Anti-ZIKV #4 VH-CDR1 49 GFSFSSGAY (Clone 289-3) VH-CDR2 50 YTGDGRT VH-CDR3 51 ARAIAVGAGYGVGNYFTL VL-CDR1 54 ENIYGY VL-CDR2 55 KAS VL-CDR3 56 QSYYTSSSNADGSENA Anti-ZIKV #5 VH-CDR1 63 GFDFSDRYY (Clone 306-2) VH-CDR2 64 YVGSGDT VH-CDR3 65 ARHPGTYF VL-CDR1 68 QNIVNN VL-CDR2 69 DTS VL-CDR3 70 QTYYYYNKIING Anti-ZIKV #6 VH-CDR1 76 GYTFTSY (Clone ZV-67) VH-CDR2 77 YPRSGN VH-CDR3 78 ENYGSVY VL-CDR1 80 CKASQNVGTAVA VL-CDR2 81 SASNRYT VL-CDR3 82 QQFSSYPYT Anti-ZIKV #7 VH-CDR1 84 GYTFTGYH (Clone ZKA- VH-CDR2 85 INPNSGGT 64) VH-CDR3 86 ARMSSSIWGFDH VL-CDR1 88 QSVLIN VL-CDR2 89 LIYGASSRA VL-CDR3 90 QQYNDWPPIT Anti-ZIKV #8 VH-CDR1 95 GFSFTDRHY (Clone 260-2) VH-CDR2 96 YPGSSGST VH-CDR3 97 ARSSYPDSSGYSYGMDL VL-CDR1 100 QNINSN VL-CDR2 101 LTS VL-CDR3 102 QTYYDISNYGYA Anti-ZIKV #9 VH-CDR1 109 GFSFTDRHY and #10 VH-CDR2 110 YPGSSGST (Clone 181- VH-CDR3 111 ARSSYPDSSGYSYGMDL 4/329-2) VL-CDR1 114 QNINSN VL-CDR2 115 LTS VL-CDR3 116 QTYYDISNYGYA Anti-ZIKV #11 VH-CDR1 123 GFSFSSRFY (Clone 11-3) VH-CDR2 124 YGGSSGST VH-CDR3 125 ARGGSTAAAGFNL VL-CDR1 128 EDIYNL VL-CDR2 129 YAS VL-CDR3 130 QCNDYGGTYVPNA Anti-ZIKV E VH-CDR1 136 GFTFSNYA Protein VH-CDR2 137 IGRNGDSI (Clone ZKA- VH-CDR3 138 VKDLAIPESYRIEADY 78) VL-CDR1 140 QSVLYRSNNKNY VL-CDR2 141 LIYWASTRE VL-CDR3 142 QQYYSSPRT Antibody VH-CDR1 151 GLDFSTNSY Clone 78-2 VH-CDR2 152 IYVGDSSEI VH-CDR3 153 ARDLPSFTAPYAGYLRL VL-CDR1 156 TGYNVGDYP VL-CDR2 157 YHTEEFKH VL-CDR3 158 YTVHATESSLHYVF Antibody VH-CDR1 165 EFDSSSNA Clone 278-11 VH-CDR2 166 IYSGSGTI VH-CDR3 167 ARYNTGGFYYDL VL-CDR1 170 QRIGTN VL-CDR2 171 KAS VL-CDR3 172 QQGYSSNDADNT
SEQUENCE LISTING FREE TEXT
[0500] With respect to the requirements within WIPO Standard ST.25 concerning the presentation of nucleotide and amino acid sequence listings in patent applications, the free text as used in the sequence listing is repeated in the following:
TABLE-US-00010 SEQ SEQ SEQ ID ID ID NO: Free Text NO: Free Text NO: Free Text 3 ZIKV E-Protein 34 Anti-ZIKV #3 VH 64 Anti-ZIKV #5 VH-CDR2 4 EDIII domain of Zika virus 35 Anti-ZIKV #3 VH-CDR1 65 Anti-ZIKV #5 VH-CDR3 6 Anti-ZIKV #1 VH 36 Anti-ZIKV #3 VH-CDR2 67 Anti-ZIKV #5 VL 7 Anti-ZIKV #1 VH-CDR1 37 Anti-ZIKV #3 VH-CDR3 68 Anti-ZIKV #5 VL-CDR1 8 Anti-ZIKV #1 VH-CDR2 39 Anti-ZIKV #3 VL 69 Anti-ZIKV #5 VL-CDR2 9 Anti-ZIKV #1 VH-CDR3 40 Anti-ZIKV #3 VL-CDR1 70 Anti-ZIKV #5 VL-CDR3 11 Anti-ZIKV #1 VL 41 Anti-ZIKV #3 VL-CDR2 72 Anti-ZIKV #5 VH DNA 12 Anti-ZIKV #1 VL-CDR1 42 Anti-ZIKV #3 VL-CDR3 74 Anti-ZIKV #5 VL DNA 13 Anti-ZIKV #1 VL-CDR2 44 Anti-ZIKV #3 VH DNA 75 Anti-ZIKV #6 VH 14 Anti-ZIKV #1 VL-CDR3 46 Anti-ZIKV #3 VL DNA 76 Anti-ZIKV #6 VH-CDR1 16 Anti-ZIKV #1 VH DNA 48 Anti-ZIKV #4 VH 77 Anti-ZIKV #6 VH-CDR2 18 Anti-ZIKV #1 VL DNA 49 Anti-ZIKV #4 VH-CDR1 78 Anti-ZIKV #6 VH-CDR3 20 Anti-ZIKV #2 VH 50 Anti-ZIKV #4 VH-CDR2 79 Anti-ZIKV #6 VL 21 Anti-ZIKV #2 VH-CDR1 51 Anti-ZIKV #4 VH-CDR3 80 Anti-ZIKV #6 VL-CDR1 22 Anti-ZIKV #2 VH-CDR2 53 Anti-ZIKV #4 VL 81 Anti-ZIKV #6 VL-CDR2 23 Anti-ZIKV #2 VH-CDR3 54 Anti-ZIKV #4 VL-CDR1 82 Anti-ZIKV #6 VL-CDR3 25 Anti-ZIKV #2 VL 55 Anti-ZIKV #4 VL-CDR2 83 Anti-ZIKV #7 VH 26 Anti-ZIKV #2 VL-CDR1 56 Anti-ZIKV #4 VL-CDR3 84 Anti-ZIKV #7 VH-CDR1 27 Anti-ZIKV #2 VL-CDR2 58 Anti-ZIKV #4 VH DNA 85 Anti-ZIKV #7 VH-CDR2 28 Anti-ZIKV #2 VL-CDR3 60 Anti-ZIKV #4 VL DNA 86 Anti-ZIKV #7 VH-CDR3 30 Anti-ZIKV #2 VH DNA 62 Anti-ZIKV #5 VH 87 Anti-ZIKV #7 VL 32 Anti-ZIKV #2 VL DNA 63 Anti-ZIKV #5 VH-CDR1 88 Anti-ZIKV #7 VL-CDR1 89 Anti-ZIKV #7 VL-CDR2 102 Anti-ZIKV #8 VL-CDR3 118 Anti-ZIKV #9 and #10 VH DNA 90 Anti-ZIKV #7 VL-CDR3 104 Anti-ZIKV #8 VH DNA 120 Anti-ZIKV #9 and #10 VL DNA 91 Anti-ZIKV #7 VH DNA 106 Anti-ZIKV #8 VL DNA 122 Anti-ZIKV #11 VH 92 Anti-ZIKV #7 VL DNA 108 Anti-ZIKV #9 and #10 VH 123 Anti-ZIKV #11 VH-CDR1 94 Anti-ZIKV #8 VH 109 Anti-ZIKV #9 and #10 VH- 124 Anti-ZIKV #11 VH-CDR2 CDR1 95 Anti-ZIKV #8 VH-CDR1 110 Anti-ZIKV #9 and #10 VH- 125 Anti-ZIKV #11 VH-CDR3 CDR2 96 Anti-ZIKV #8 VH-CDR2 111 Anti-ZIKV #9 and #10 VH- 127 Anti-ZIKV #11 VL CDR3 97 Anti-ZIKV #8 VH-CDR3 113 Anti-ZIKV #9 and #10 VL 128 Anti-ZIKV #11 VL-CDR1 99 Anti-ZIKV #8 VL 114 Anti-ZIKV #9 and #10 VL- 129 Anti-ZIKV #11 VL-CDR2 CDR1 100 Anti-ZIKV #8 VL-CDR1 115 Anti-ZIKV #9 and #10 VL- 130 Anti-ZIKV #11 VL-CDR3 CDR2 101 Anti-ZIKV #8 VL-CDR2 116 Anti-ZIKV #9 and #10 VL- 132 Anti-ZIKV #11 VH DNA CDR3 134 Anti-ZIKV #11 VL DNA 139 Anti-ZIKV E Protein VL 144 Anti-ZIKV E Protein VL DNA 135 Anti-ZIKV E Protein VH 140 Anti-ZIKV E Protein VL-CDR1 150 Antibody Clone 78-2 VH 136 Anti-ZIKV E Protein VH- 141 Anti-ZIKV E Protein VL-CDR2 151 Antibody Clone 78-2 VH- CDR1 CDR1 137 Anti-ZIKV E Protein VH- 142 Anti-ZIKV E Protein VL-CDR3 152 Antibody Clone 78-2 VH- CDR2 CDR1 138 Anti-ZIKV E Protein VH- 143 Anti-ZIKV E Protein VH DNA 153 Antibody Clone 78-2 VH- CDR3 CDR3 155 Antibody Clone 78-2 VL 164 Antibody Clone 278-11 VH 171 Antibody Clone 278-11 VL- CDR2 156 Antibody Clone 78-2 VL- 165 Antibody Clone 278-11 VH- 172 Antibody Clone 278-11 VL- CDR1 CDR1 CDR3 157 Antibody Clone 78-2 VL- 166 Antibody Clone 278-11 VH- 174 Antibody Clone 278-11 VH CDR2 CDR2 DNA 158 Antibody Clone 78-2 VL- 167 Antibody Clone 278-11 VH- 176 Antibody Clone 278-11 VL CDR3 CDR3 DNA 160 Antibody Clone 78-2 VH 169 Antibody Clone 278-11 VL DNA 162 Antibody Clone 78-2 VL 170 Antibody Clone 278-11 VL- DNA CDR1
Items of the Invention
Microsphere Complex Comprising Microsphere Coupled to ZIKV VLP
[0501] 1. A microsphere complex comprising a microsphere coupled to a zika virus like particle. [0502] 2. The microsphere complex of item 1, wherein the zika virus like particle is derived from zika virus strain Z1106033 characterized by SEQ ID NO: 1 and/or SEQ ID NO: 2. [0503] 3. The microsphere complex of item 1, wherein the zika virus like particle comprises structural proteins of zika virus strain Z1106033 characterized by SEQ ID NO: 1 and/or SEQ ID NO: 2. [0504] 4. The microsphere complex of item 1, wherein the zika virus like particle comprises the envelope glycoprotein, membrane protein, and/or pre-membrane protein which are at least 70%, or at least 75%, or least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to corresponding parts of SEQ ID NO: 2. [0505] 5. The microsphere complex of any one of items 1 to 4, wherein the zika virus like particle is produced in human embryonic kidney (HEK293) cells. [0506] 6. The microsphere complex of any one of items 1 to 5, wherein the microsphere is a polystyrene microsphere. [0507] 7. The microsphere complex of any one of items 1 to 6, wherein the microsphere is magnetic. [0508] 8. The microsphere complex of any one of items 1 to 7, wherein the microsphere has a diameter in the range from about 0.01 to about 100 μm, preferably in the range from about 1 to 10 μm. [0509] 9. The microsphere complex of any one of items 1 to 8, wherein the microsphere contains carboxylate groups at the microsphere surface. [0510] 10. The microsphere complex of item 9, wherein coupling of the microsphere to the zika virus like particle occurs by formation of an amide bond between a carboxylate group of the microsphere and an amine group of the zika virus like particle. [0511] 11. The microsphere complex of any of items 1 to 10, wherein the microsphere can be identified by a specific feature. [0512] 12. The microsphere complex of item 11, wherein the specific feature is that the microsphere comprises one or more fluorescent dyes having a specific emission spectrum. [0513] 13. The microsphere complex of item 12, wherein the one or more fluorescent dyes are selected from the group consisting of squaraine, phthalocyanine, naphthalocyanine, and any derivative thereof.
Kit
[0514] 14. A kit comprising: [0515] an amount of the microsphere complex of any one of items 1 to 13, and [0516] an amount of a reporter antibody that binds to the zika virus like particle of the microsphere complex. [0517] 15. The kit of item 14, wherein the reporter antibody is a zika virus neutralizing antibody. [0518] 16. The kit of item 14 or 15, wherein the reporter antibody does not cross-react with antigens from other flaviviruses, such as dengue virus, West Nile virus, Japanese encephalitis virus, Yellow Fever Virus, St. Louis Encephalitis virus, and Tick Borne Encephalitis virus. [0519] 17. The kit of item 16, wherein the reporter antibody does not cross-react with dengue virus antigens, such as dengue virus like particles. [0520] 18. The kit of any one of items 14 to 17, wherein the reporter antibody is a monoclonal antibody. [0521] 19. The kit of any one of items 14 to 18, wherein the reporter antibody is derived from a non-human origin. [0522] 20. The kit of any of items 14 to 19, wherein the reporter antibody is attached to at least one detectable label, preferably by the heavy chain constant region of the reporter antibody. [0523] 21. The kit of item 20, wherein the reporter antibody is directly attached to the at least one detectable label, preferably by the heavy chain constant region of the reporter antibody. [0524] 22. The kit of item 20, wherein the reporter antibody is indirectly attached to the at least one detectable label, preferably by the heavy chain constant region of the reporter antibody, wherein the reporter antibody reacts with a secondary reporter antibody directly attached to at least one detectable label. [0525] 23. The kit of item 22, wherein the secondary reporter antibody is directly attached to the at least one detectable label, preferably by the heavy chain constant region of the secondary reporter antibody. [0526] 24. The kit of any of items 20 to 23, wherein the at least one detectable label is a fluorescence label, such as xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin, cyanine, coumarin, and any derivative thereof. [0527] 25. The kit of item 24, wherein the at least one detectable label is phycoerythrin. [0528] 26. The kit of any of items 14 to 25, wherein the reporter antibody provides an EC.sub.50 value towards the zika virus like particle coupled to the microsphere within the microsphere complex of less than 0.5 μg/mL, or less than 0.4 μg/mL or less than 0.3 μg/mL or less than 0.2 μg/mL or less than 0.15 μg/mL or less than 0.1 μg/mL or less than 0.09 μg/mL or less than 0.08 μg/mL or less than 0.07 μg/mL or less than 0.05 μg/mL or less than 0.04 μg/mL or less than 0.03 μg/mL or less than 0.01 μg/mL. [0529] 27. The kit of any of items 14 to 25, wherein the reporter antibody is a zika virus specific reporter antibody, wherein the reporter antibody provides an EC.sub.50 value towards the zika virus like particle coupled to the microsphere within the microsphere complex which is lower than each EC.sub.50 value which said reporter antibody provides when tested in binding towards other microsphere complexes comprising a microsphere coupled to a dengue virus like particle. [0530] 28. The kit of the item 27, wherein the dengue virus like particle is a dengue serotype 1 virus like particle and/or a dengue serotype 2 virus like particle and/or a dengue serotype 3 virus like particle and/or a dengue serotype 4 virus like particle. [0531] 29. The kit of any of items 27 or 28, wherein the EC.sub.50 value towards the zika virus like particle is less than 0.5 μg/mL, or less than 0.4 μg/mL or less than 0.3 μg/mL or less than 0.2 μg/mL or less than 0.15 μg/mL or less than 0.1 μg/mL or less than 0.09 μg/mL or less than 0.08 μg/mL or less than 0.07 μg/mL or less than 0.05 μg/mL or less than 0.04 μg/mL or less than 0.03 μg/mL or less than 0.01 μg/mL and the EC.sub.50 towards the dengue virus like particle is at least 1 μg/mL or at least 1.1 μg/mL or at least 1.2 μg/mL or at least 1.3 μg/mL or at least 1.4 μg/mL. [0532] 30. The kit of any one of items 14 to 29, wherein the reporter antibody binds to the zika virus envelope glycoprotein domain III of the envelope glycoprotein encoded by SEQ ID NO: 3. [0533] 31. The kit of item 30, wherein the reporter antibody binds to amino acids T309 and G337 of SEQ ID NO: 3. [0534] 32. The kit of item 30, wherein the reporter antibody binds to amino acid E370 of SEQ ID NO: 3. [0535] 33. The kit of item 30, wherein the reporter antibody binds to amino acids T397 and H398 of SEQ ID NO: 3. [0536] 34. The kit of item 30, wherein the reporter antibody binds to amino acids E162, G181, G182, and K301 of SEQ ID NO: 3. [0537] 35. The kit of any one of items 14 to 30, wherein the reporter antibody comprises [0538] a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 7, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 8, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 9, and [0539] a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 12, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 13, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 14. [0540] 36. The kit of any one of items 14 to 30, wherein the reporter antibody comprises [0541] a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 21, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 22, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 23, and [0542] a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 26, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 27, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 28. [0543] 37. The kit of any one of items 14 to 30, wherein the reporter antibody comprises [0544] a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 35, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 36, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 37, and [0545] a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 40, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 41, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 42. [0546] 38. The kit of any one of items 14 to 30, wherein the reporter antibody comprises [0547] a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 49, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 50, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 51, and [0548] a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 54, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 55, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 56. [0549] 39. The kit of any one of items 14 to 30, wherein the reporter antibody comprises [0550] a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 63, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 64, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 65, and [0551] a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 68, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 69, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 70. [0552] 40. The kit of any one of items 14 to 30, wherein the reporter antibody comprises [0553] a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 76, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 77, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 78, and [0554] a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 80, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 81, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 82. [0555] 41. The kit of any one of items 14 to 30, wherein the reporter antibody comprises [0556] a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 84, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 85, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 86, and [0557] a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 88, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 89, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 90. [0558] 42. The kit of any one of items 14 to 30, wherein the reporter antibody comprises [0559] a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 95, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 96, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 97, and [0560] a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 100, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 101, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 102. [0561] 43. The kit of any one of items 14 to 30, wherein the reporter antibody comprises [0562] a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 109, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 110, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 111, and [0563] a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 114, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 115, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 116. [0564] 44. The kit of any one of items 14 to 30, wherein the reporter antibody comprises [0565] a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 123, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 124, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 125, and [0566] a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 128, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 129, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 130. [0567] 45. The kit of any one of items 14 to 30, wherein the reporter antibody comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 6, and a light chain variable region (VL) amino acid sequence of SEQ ID NO: 11. [0568] 46. The kit of any one of items 14 to 30, wherein the reporter antibody comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 20, and a light chain variable region (VL) amino acid sequence of SEQ ID NO: 25. [0569] 47. The kit of any one of items 14 to 30, wherein the reporter antibody comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 34, and a light chain variable region (VL) amino acid sequence of SEQ ID NO: 39. [0570] 48. The kit of any one of items 14 to 30, wherein the reporter antibody comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 48, and a light chain variable region (VL) amino acid sequence of SEQ ID NO: 53. [0571] 49. The kit of any one of items 14 to 30, wherein the reporter antibody comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 62, and a light chain variable region (VL) amino acid sequence of SEQ ID NO: 67. [0572] 50. The kit of any one of items 14 to 30, wherein the reporter antibody comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 75, and a light chain variable region (VL) amino acid sequence of SEQ ID NO: 79. [0573] 51. The kit of any one of items 14 to 30, wherein the reporter antibody comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 83, and a light chain variable region (VL) amino acid sequence of SEQ ID NO: 87. [0574] 52. The kit of any one of items 14 to 30, wherein the reporter antibody comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 94, and a light chain variable region (VL) amino acid sequence of SEQ ID NO: 99. [0575] 53. The kit of any one of items 14 to 30, wherein the reporter antibody comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 108, and a light chain variable region (VL) amino acid sequence of SEQ ID NO: 113. [0576] 54. The kit of any one of items 14 to 30, wherein the reporter antibody comprises a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 122, and a light chain variable region (VL) amino acid sequence of SEQ ID NO: 127. [0577] 55. The kit of any one of items 14 to 30, wherein the reporter antibody comprises a heavy chain (H) amino acid sequence of SEQ ID NO: 5, and a light chain (V) amino acid sequence of SEQ ID NO: 10. [0578] 56. The kit of any one of items 14 to 30, wherein the reporter antibody comprises a heavy chain (H) amino acid sequence of SEQ ID NO: 19, and a light chain (V) amino acid sequence of SEQ ID NO: 24. [0579] 57. The kit of any one of items 14 to 30, wherein the reporter antibody comprises a heavy chain (H) amino acid sequence of SEQ ID NO: 33, and a light chain (V) amino acid sequence of SEQ ID NO: 38. [0580] 58. The kit of any one of items 14 to 30, wherein the reporter antibody comprises a heavy chain (H) amino acid sequence of SEQ ID NO: 47, and a light chain (V) amino acid sequence of SEQ ID NO: 52. [0581] 59. The kit of any one of items 14 to 30, wherein the reporter antibody comprises a heavy chain (H) amino acid sequence of SEQ ID NO: 61, and a light chain (V) amino acid sequence of SEQ ID NO: 66. [0582] 60. The kit of any one of items 14 to 30, wherein the reporter antibody comprises a heavy chain (H) amino acid sequence of SEQ ID NO: 93, and a light chain (V) amino acid sequence of SEQ ID NO: 98. [0583] 61. The kit of any one of items 14 to 30, wherein the reporter antibody comprises a heavy chain (H) amino acid sequence of SEQ ID NO: 107, and a light chain (V) amino acid sequence of SEQ ID NO: 112. [0584] 62. The kit of any one of items 14 to 30, wherein the reporter antibody comprises a heavy chain (H) amino acid sequence of SEQ ID NO: 121, and a light chain (V) amino acid sequence of SEQ ID NO: 126.
Method for Detecting Anti-Zika Virus Antibodies
[0585] 63. A method for detecting a signal from a reporter antibody indicative for the presence and/or amount of anti-zika virus antibodies in a sample from a subject comprising the steps of: [0586] Step 1: providing a kit according to any one of items 14 to 62, including an amount of said microsphere complex and an amount of said reporter antibody, [0587] Step 2: contacting the amount of said microsphere complex and the amount of said reporter antibody of step 1 with the sample to allow binding of the anti-zika virus antibodies in the sample to the zika virus like particles coupled to the microspheres in the microsphere complex while competing with the reporter antibody, and [0588] Step 3: detecting a signal from the reporter antibody bound to the zika virus like particles coupled to the microspheres in the microsphere complex in step 2. [0589] 64. The method of item 63, wherein in step 2 the amount of said microsphere complex and the amount of said reporter antibody of step 1 are concomitantly contacted with the sample. [0590] 65. The method of item 63, comprising the steps of: [0591] Step 1: providing a kit according to any of items 14 to 62, including an amount of said microsphere complex and an amount of said reporter antibody, [0592] Step 2.1: contacting the amount of said microsphere complex of step 1 with the sample to allow binding of the anti-zika virus antibodies in the sample to the zika virus like particles coupled to the microspheres in the microsphere complex, [0593] Step 2.2: contacting said amount of reporter antibody with said microsphere complex and the sample of step 2.1 to allow binding of the reporter antibody to the zika virus like particles coupled to the microspheres in the microsphere complex, and [0594] Step 3: detecting a signal from the reporter antibody bound to the zika virus like particles coupled to the microspheres in the microsphere complex in step 2.2. [0595] 66. The method of item 63, comprising the steps of: [0596] Step 1: providing a kit according to any of items 14 to 62, including an amount of said microsphere complex and an amount of said reporter antibody, [0597] Step 2.1: contacting the amount of said microsphere complex of step 1 with the sample to allow binding of anti-zika virus antibodies in the sample to the zika virus like particles coupled to the microspheres in the microsphere complex, [0598] Step 2.2: contacting said amount of reporter antibody with said microsphere complex and the sample of step 2.1 to allow binding of the reporter antibody to the zika virus like particles coupled to the microspheres, [0599] Step 2.3: contacting said amount of reporter antibody, said amount of microsphere complex, and the sample of step 2.2 with an amount of a secondary reporter antibody to allow binding of the secondary reporter antibody to the constant region of the reporter antibody, and [0600] Step 3: detecting a signal from the secondary reporter antibody bound to the reporter antibody in step 2.3, wherein the reporter antibody is bound to the zika virus like particles coupled to the microspheres in the microsphere complex in step 2.2. [0601] 67. The method of item 63, wherein contacting in step 2 is carried out for about 10 to about 250 min. [0602] 68. The method of item 65, wherein contacting in step 2.1 is carried out for about 60 min and contacting in step 2.2 is carried out for about 10 min or for about 120 min. [0603] 69. The method of item 66, wherein contacting in step 2.1 is carried out for about 60 min and contacting in step 2.2 is carried out for about 10 min or for about 120 min and contacting in step 2.3 is carried out for about 30 min. [0604] 70. The method of any of items 63 to 69, wherein the signal in step 3 is resulting from the at least one detectable label. [0605] 71. The method of any of items 63 to 70, wherein the signal in step 3 is a fluorescence signal. [0606] 72. The method of item 71, wherein the signal in step 3 is a fluorescence signal resulting from phycoerythrin. [0607] 73. The method of any of items 63 to 72 for detecting the presence and/or amount of anti-zika virus antibodies in a sample from a subject comprising the further steps of: [0608] Step 4: determining the presence and/or the amount of the reporter antibody bound to the zika virus like particles coupled to the microspheres in the microsphere complex from the signal of step 3, and [0609] Step 5: determining the presence and/or the amount of anti-zika virus antibodies in the sample based on the presence and/or the amount of the reporter antibody determined in step 4. [0610] 74. The method of any of items 63 to 73, wherein the sample is a sample from the group consisting of blood, urine, serum, blood plasma, cerebrospinal fluid, and lymph fluid. [0611] 75. The method of item 74, wherein the sample is a blood plasma sample or serum sample. [0612] 76. The method of any of items 63 to 75, wherein the anti-zika virus antibodies from the sample of the subject are zika virus neutralizing antibodies. [0613] 77. The method of any of items 63 to 76, wherein the subject is a subject from the group consisting of mouse, primate, non-human primate, human, rabbit, cat, rat, horse, and sheep. [0614] 78. The method of item 77, wherein the subject is a non-human primate. [0615] 79. The method of item 77, wherein the subject is a human.
Method for Determining an Antibody Correlate of Protection Against Zika Virus Infection
[0616] 80. A method for determining an antibody correlate of protection against zika virus infection for a zika virus vaccine in a type of non-human subjects comprising the steps of: [0617] Step 1: selecting a group of said subjects which are zika virus naive, [0618] Step 2: dividing the group of subjects into at least two subgroups, wherein one subgroup functions as control group and at least one subgroup functions as inoculation group, [0619] Step 3: inoculating said at least one inoculation group with a dose of the zika virus vaccine, [0620] Step 4: challenging all subjects with an infectious amount of the zika virus, [0621] Step 5: determining the amount of anti-zika virus antibodies for each subject according to items 63 to [0622] 76 at least after inoculation with the zika virus vaccine and before challenging with the infectious amount of the zika virus, [0623] Step 6: determining presence or absence of viremia in all subjects after challenging with the infectious amount of the zika virus, [0624] Step 7: repeating steps 3 to 6 with further inoculation groups with increasing vaccine doses until absence of viremia is determined in all subjects of one inoculation group in step 6, and [0625] Step 8: determining the amount of anti-zika virus antibodies after inoculation with the zika virus vaccine and before challenging with the infectious amount of the zika virus associated with absence of viremia after challenging with the infectious amount of zika virus as antibody correlate of protection. [0626] 81. The method for determining an antibody correlate of protection according to item 80, wherein the zika virus vaccine in step 3 is a purified inactivated zika virus vaccine. [0627] 82. The method for determining an antibody correlate of protection according to any of items 80 or 81, wherein the zika virus in step 4 is zika virus strain PRVABC59. [0628] 83. The method for determining an antibody correlate of protection according to any of items 80 to 82, wherein the type of non-human subjects is selected from the group consisting of mice, primates, non-human primates, rabbits, cats, rats, horses, or sheep. [0629] 84. The method for determining an antibody correlate of protection according to item 83, wherein the type of non-human subjects is non-human primates. [0630] 85. A method for determining an antibody correlate of protection against zika virus infection in human subjects by mathematically modeling the correlate of protection of a non-human subject as determined according to any one of items 80 to 84 to fit human subjects.
Method for Diagnosing the Protection of a Subject Against a Zika Virus Infection
[0631] 86. A method for diagnosing the protection of a human subject against a zika virus infection comprising the steps of: [0632] Step 1: providing a sample from the human subject outside the human body, [0633] Step 2: determining the amount of anti-zika virus antibodies in the sample from the human subject according to items 63 to 76, and [0634] Step 3: determining protection by comparing the amount of anti-zika virus antibodies determined in step 2 to the antibody correlate of protection against zika virus infection in human subjects, which optionally has been determined according to item 85. [0635] 87. A method for diagnosing the protection of a non-human subject against a zika virus infection comprising the steps of: [0636] Step 1: providing a sample from the non-human subject outside the non-human body, [0637] Step 2: determining the amount of anti-zika virus antibodies in the sample from the non-human subject according to items 63 to 76, and [0638] Step 3: determining protection by comparing the amount of anti-zika virus antibodies determined in step 2 to the antibody correlate of protection determined according to items 80 to 84 in this type of non-human subjects.
Method for Diagnosing a Zika Virus Infection
[0639] 88. A method for diagnosing a zika virus infection in a subject comprising the steps of: [0640] Step 1: providing a sample from the subject outside the subject body, [0641] Step 2: determining the amount of anti-zika virus antibodies in the sample according to items 63 to 79, and [0642] Step 3: determining infection by comparing said amount of anti-zika virus antibodies to established amounts of anti-zika virus antibodies in zika virus infected subjects. [0643] 89. The method for diagnosing a zika virus infection according to item 88, wherein the subject is a human. [0644] 90. The method of any of items 88 or 89, wherein the zika virus infection is acute. [0645] 91. The method of any of items 88 or 89, wherein the zika virus infection is convalescent.
Method for Detecting Total Anti-Zika Antibodies
[0646] 92. A method for detecting a signal from a detection antibody indicative for the presence and/or amount of anti-zika virus antibodies in a sample from a subject comprising the steps of: [0647] Step 1: contacting an amount of a microsphere complex according to any one of items 1 to 13 with the sample to allow binding of anti-zika virus antibodies in the sample to the zika virus like particles coupled to the microspheres in the microsphere complex, [0648] Step 2: contacting an amount of a detection antibody with the microsphere complex and the sample of step 1 to allow binding of the detection antibody to the heavy chain constant region of the anti-zika virus antibodies bound to the zika virus like particles coupled to the microspheres in the microsphere complex, wherein the detection antibody binds to the anti-zika virus antibodies with the variable region of the detection antibody and wherein the detection antibody is attached to at least one detectable label, and [0649] Step 3: detecting a signal from the detection antibody bound to the anti-zika virus antibodies in step 2. [0650] 93. The method according to item 92 for determining the presence and/or amount of anti-zika virus antibodies in a sample from a subject, wherein the method comprises the further steps of: [0651] Step 4: determining the presence and/or amount of the detection antibody bound to the anti-zika virus antibodies from the signal of step 3, and [0652] Step 5: determining the presence and/or amount of anti-zika virus antibodies in the sample from the presence and/or amount of the detection antibody determined in step 4. [0653] 94. The method according to items 92 or 93, wherein contacting in step 1 is carried out for about 60 min and contacting in step 2 is carried out for about 30 min. [0654] 95. The method according to any one of items 92 to 94, wherein the detection antibody is attached to the at least one detectable label by the heavy chain constant region. [0655] 96. The method according to any one of items 92 to 95, wherein the at least one detectable label is a fluorescence label, such as phycoerythrin. [0656] 97. The method according to any one of items 92 to 96, wherein the signal in step 3 is resulting from the at least one detectable label, preferably the signal is a fluorescence signal. [0657] 98. The method according to any one of items 92 to 97, wherein the sample is a sample from the group consisting of blood, urine, serum, blood plasma, cerebrospinal fluid, and lymph fluid, in particular the sample is a serum or blood plasma sample. [0658] 99. The method according to any one of items 92 to 98, wherein the subject is from the group consisting of mouse, primate, non-human primate, human, rabbit, cat, rat, horse, and sheep, in particular the subject is a human. [0659] 100. The method for determining an antibody correlate of protection against zika virus infection according to any one of items 80 to 85, wherein the amount of anti-zika virus antibodies for each subject in step 5 is determined according to the method of any one of items 92 to 99. [0660] 101. The method for diagnosing the protection of a human subject against a zika virus infection according to item 86, [0661] wherein the amount of anti-zika virus antibodies in the sample in step 2 is determined according to the method of any one of items 92 to 98, and [0662] wherein protection in step 3 is determined by comparing the amount of anti-zika virus antibodies to the antibody correlate of protection determined in human subjects according to item 100. [0663] 102. The method for diagnosing the protection of a non-human subject against a zika virus infection according to item 87, [0664] wherein the amount of anti-zika virus antibodies in the sample in step 2 is determined according to the method of any one of items 92 to 98, and [0665] wherein protection in step 3 is determined by comparing the amount of anti-zika virus antibodies to the antibody correlate of protection determined in this type of non-human subjects according to item 100.
Microsphere Complex Comprising a Microsphere Coupled to a DENV VLP and Corresponding Kit
[0666] 103. A microsphere complex comprising a microsphere coupled to a dengue virus like particle. [0667] 104. The microsphere complex of item 103, wherein the microsphere is coupled to a dengue-1 virus like particle. [0668] 105. The microsphere complex of item 104, wherein the dengue-1 virus like particle is derived from dengue-1 virus strain Puerto Rico/US/BID-V853/1998 characterized by SEQ ID NO: 179 and/or SEQ ID NO: 180. [0669] 106. The microsphere complex of item 104, wherein the dengue-1 virus like particle comprises structural proteins of dengue-1 virus strain Puerto Rico/US/BID-V853/1998 characterized by SEQ ID NO: 179 and/or SEQ ID NO: 180. [0670] 107. The microsphere complex of item 104, wherein the dengue-1 virus like particle comprises the envelope protein, the membrane protein, and/or the pre-membrane protein which are at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to corresponding parts of SEQ ID NO: 180. [0671] 108. The microsphere complex of item 103, wherein the microsphere is coupled to a dengue-2 virus like particle. [0672] 109. The microsphere complex of item 108, wherein the dengue-2 virus like particle is derived from dengue-2 virus strain Thailand/16681/84 characterized by SEQ ID NO: 181 and/or SEQ ID NO: 182. [0673] 110. The microsphere complex of item 108, wherein the dengue-2 virus like particle comprises structural proteins of dengue-2 virus strain Thailand/16681/84 characterized by SEQ ID NO: 181 and/or SEQ ID NO: 182. [0674] 111. The microsphere complex of item 108, wherein the dengue-2 virus like particle comprises the envelope protein, the membrane protein, and/or the pre-membrane protein which are at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to corresponding parts of SEQ ID NO: 182. [0675] 112. The microsphere complex of item 103, wherein the microsphere is coupled to a dengue-3 virus like particle. [0676] 113. The microsphere complex of item 112, wherein the dengue-3 virus like particle is derived from dengue-3 virus strain Sri Lanka D3/H/IMTSSA-SRI/2000/1266 characterized by SEQ ID NO: 183 and/or SEQ ID NO: 184. [0677] 114. The microsphere complex of item 112, wherein the dengue-3 virus like particle comprises structural proteins of dengue-3 virus strain Sri Lanka D3/H/IMTSSA-SRI/2000/1266 characterized by SEQ ID NO: 183 and/or SEQ ID NO: 184. [0678] 115. The microsphere complex of item 112, wherein the dengue-3 virus like particle comprises the envelope protein, the membrane protein, and/or the pre-membrane protein which are at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to corresponding parts of SEQ ID NO: 184. [0679] 116. The microsphere complex of item 103, wherein the microsphere is coupled to a dengue-4 virus like particle. [0680] 117. The microsphere complex of item 116, wherein the dengue-4 virus like particle is derived from dengue-4 virus strain Dominica/814669/1981 characterized by SEQ ID NO: 185 and/or SEQ ID NO: 186. [0681] 118. The microsphere complex of item 116, wherein the dengue-4 virus like particle comprises structural proteins of dengue-4 virus strain Dominica/814669/1981 characterized by SEQ ID NO: 185 and/or SEQ ID NO: 186. [0682] 119. The microsphere complex of item 116, wherein the dengue-4 virus like particle comprises the envelope protein, the membrane protein, and/or the pre-membrane protein which are at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or 100% identical to corresponding parts of SEQ ID NO: 186. [0683] 120. The microsphere complex of any one of items 103 to 119, wherein the dengue virus like particle is produced in human embryonic kidney (HEK293) cells.
Method for Preventing Zika Disease
[0684] 121. A method for preventing zika disease in a human subject comprising the steps of: [0685] Step 1: obtaining a sample from the human subject, [0686] Step 2: determining the amount of anti-zika virus antibodies in the sample from the human subject according to items 63-76, [0687] Step 3: determining whether the human subject has an amount of anti-zika virus antibodies to confer protection by comparing the amount of anti-zika virus antibodies determined in step 2 to the antibody correlate of protection against zika virus infection in human subjects, which optionally has been determined according to item 85, and [0688] Step 4: administering to the human subject a zika virus vaccine if the human subject has an amount of anti-zika antibodies that is lower than the antibody correlate of protection against zika virus infection in human subjects, which optionally has been determined according to item 85. [0689] 122. A zika virus vaccine for use in a method for preventing zika disease in a human subject, the method comprising the steps of: [0690] Step 1: providing a sample from the human subject outside the human body, [0691] Step 2: determining the amount of anti-zika virus antibodies in the sample from the human subject according to items 63-76, [0692] Step 3: determining whether the human subject has an amount of anti-zika virus antibodies to confer protection by comparing the amount of anti-zika virus antibodies determined in step 2 to the antibody correlate of protection against zika virus infection in human subjects, which optionally has been determined according to item 85, and [0693] Step 4: administering to the human subject the zika virus vaccine if the human subject has an amount of anti-zika antibodies that is lower than the antibody correlate of protection against zika virus infection in human subjects, which optionally has been determined according to item 85. [0694] 123. The method according to item 121, wherein the zika virus vaccine is a purified inactivated zika virus vaccine. [0695] 124. The method according to item 121 or 123, wherein the human subject is a woman. [0696] 125. The zika virus vaccine for use according to item 122, wherein the zika virus vaccine is a purified inactivated zika virus vaccine. [0697] 126. The zika virus vaccine for use according to item 122 or 125, wherein the human subject is a woman.
Method for Assaying the Presence of a Zika Virus Infection
[0698] 127. A method for assaying the presence of a zika virus infection in a subject comprising the steps of: [0699] Step 1: obtaining a sample from the subject, [0700] Step 2: determining the amount of anti-zika virus antibodies in the sample according to items 63-79, and [0701] Step 3: determining the presence of a zika virus infection by comparing said amount of anti-zika virus antibodies to established amounts of anti-zika virus antibodies in zika virus infected subjects. [0702] 128. The method for assaying the presence of a zika virus infection according to item 127, wherein the subject is a human. [0703] 129. The method of any of items 127 or 128, wherein the zika virus infection is acute. [0704] 130. The method of any of items 127 or 128, wherein the zika virus infection is convalescent.