METHOD FOR DETERMINING THE POTENCY OF ANTIGENS
20230204567 · 2023-06-29
Assignee
Inventors
- Jill LIVENGOOD (Cambridge, MA, US)
- Michael JOHNSON (Cambridge, MA, US)
- James Gifford (Cambridge, MA, US)
- Tawnya NEAD (Cambridge, MA, US)
- Mark Lyons (Cambridge, MA, US)
- Jackie Marks (Cambridge, MA, US)
Cpc classification
C12N2770/24134
CHEMISTRY; METALLURGY
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
G01N33/542
PHYSICS
Abstract
The present disclosure relates to a method for determining the potency of an antigen sample such as a vaccine antigen sample. The present disclosure is also related to a method for monitoring the potency of a vaccine antigen during the production process including purifying, inactivating and formulating the vaccine antigen and to a method for producing a virus vaccine. Further, the present disclosure relates to vaccines obtainable by the methods disclosed. In certain embodiments of the present invention the antigen sample is a zika virus antigen sample.
Claims
1. A method for detecting a signal indicative for the potency of an antigen sample such as a vaccine antigen sample, wherein the antigen in the antigen sample provides at least two epitopes and the method comprises the steps of: Step 1: providing a kit comprising an acceptor kit and a donor kit, the acceptor kit comprising an amount of an acceptor microsphere and an amount of an acceptor antibody and the donor kit comprising an amount of a donor microsphere and an amount of a donor antibody, wherein the acceptor microsphere is capable to accept energy which is transferred in a proximity reaction to produce a signal and is capable of binding or is bound to the constant region of the acceptor antibody and is not capable of binding to the donor antibody, the acceptor antibody has a variable region which is capable of binding to one of the at least two epitopes of the antigen and a constant region which is capable of binding or is bound to said acceptor microsphere, wherein the acceptor antibody is not capable of binding to the donor microsphere, the donor microsphere is capable to donate energy which is transferred in a proximity reaction to produce a signal by the acceptor microsphere and is capable of binding or is bound to the constant region of the donor antibody and is not capable of binding to the acceptor antibody, and the donor antibody has a variable region which is capable of binding to the other of the at least two epitopes of the antigen and a constant region which is capable of binding to said donor microsphere, wherein the donor antibody is not capable of binding to the acceptor microsphere, Step 2: contacting the amount of said donor microsphere, the amount of said acceptor microsphere, the amount of said donor antibody and the amount of said acceptor antibody of step 1 with the sample to allow forming a complex of the antigen in the sample with the donor antibody bound to the donor microsphere and the acceptor antibody bound to the acceptor microsphere and the acceptor antibody bound to one of the at least two epitopes of the antigen and the donor antibody bound to the other of the at least two epitopes of the antigen, Step 3: conducting a proximity reaction to produce a signal indicative for the potency of the antigen sample, and Step 4: detecting the signal indicative for the potency of the antigen sample.
2. A method for determining the amount of the antigen in the antigen sample indicative for the potency of the antigen sample by detecting the signal in accordance with claim 1 and further comprising the step of: Step 5: determining the amount of the antigen in the antigen sample indicative for the potency of the antigen sample based on the detected signal.
3. A method for determining the potency of the antigen sample such as a vaccine antigen sample by detecting the amount of the antigen in accordance with claim 2 and further comprising the step of: Step 6: determining the potency of the antigen sample based on the amount of the antigen in the sample determined in step 5.
4. The method for determining the potency of an antigen sample in accordance with claim 3, wherein step 6 comprises the steps of Step 6.1: determining the potency of standardized samples of the antigen in human or non-human subjects by measuring the associated mean neutralizing antibody titers produced in said human or non-human subjects, Step 6.2: determining the amount of the antigen with at least two epitopes in said standardized samples according to the method of claim 2, Step 6.3: establishing a standard curve from the mean neutralizing antibody titers of step 6.1 and the amount of the antigen of step 6.2, and Step 6.4: determining the potency of the antigen sample by comparing the amount of antigen in the antigen sample determined in step 5 with the standard curve.
5. The method of any one of claims 1 to 4, wherein the at least two epitopes are the same epitopes and wherein the acceptor and donor antibody have the same variable region and/or are capable of binding to the same epitope.
6. The method of any one of claims 1 to 4, wherein the at least two epitopes are different epitopes and wherein the acceptor and donor antibody have different variable regions.
7. The method of claims 1 to 6, wherein in step 1 the acceptor microsphere is bound to the constant region of the acceptor antibody and/or the donor microsphere is bound to the constant region of the donor antibody.
8. The method of any one of claims 1 to 7 wherein the antigen sample is a vaccine antigen sample.
9. The method of claim 8, wherein the vaccine antigen in the vaccine antigen sample is a virus antigen.
10. The method of any one of claims 1 to 7 wherein the antigen sample is a virus antigen sample.
11. The method of claim 9 or 10, wherein the donor and acceptor antibody do not cross-react with other virus antigens than the virus antigen of claims 9 and 10.
12. The method of any one of claims 9 to 11, wherein at least one or both of the donor and acceptor antibodies neutralize the virus antigen to which they bind when tested in a plaque reduction neutralization test or reporter virus particle test or microneutralization test or focus forming assay.
13. The method of any one of claims 9 to 12, wherein the virus antigen is selected from the group consisting of zika virus antigen, dengue virus antigen, norovirus antigen, and poliovirus antigen.
14. The method of any one of claims 9 to 12, wherein the virus antigen is selected from the group consisting of a live virus, an inactivated virus, a live attenuated virus and a virus like particle.
15. The method of claim 14, wherein the virus antigen is an inactivated virus.
16. The method of claim 15, wherein the virus antigen is an inactivated zika virus.
17. The method of claim 16, wherein the antigen is an inactivated zika virus absorbed on alum.
18. The method of any one of claims 4 to 17, wherein the standardized samples in step 6.1 are provided by a forced degradation study or different doses of the antigen.
19. The method of any one of claims 4 to 18, wherein the subjects in step 6.1 are mice.
20. A method of monitoring the potency of a vaccine antigen during the production process including purifying, inactivating and formulating of said vaccine antigen to form a final vaccine by measuring the potency of the vaccine antigen in accordance with a method of any one of claims 1 to 19.
21. A method of producing a virus vaccine comprising the steps of: Step A: preparing various batches of vaccine antigen, Step B: determining the potency of the vaccine antigen of the various vaccine antigen batches produced in step A in accordance with the method of claims 1 to 20 and selecting the vaccine antigen batches in conformity with a predetermined potency requirement, Step C: preparing vaccine batches by formulating the vaccine antigen batches selected in step B into various batches of virus vaccine, and Step D: determining the potency of the vaccine antigen in the vaccine batches of the various batches produced in step C in accordance with the method of claims 1 to 20 and selecting the vaccine batches in conformity with the predetermined potency requirement.
22. The method of claim 21, wherein step A includes various sub-steps and step B is performed after each sub-step.
23. The method of claim 22, wherein the sub-steps comprise inactivation of a live virus to an inactivated virus.
24. The method of claim 23, wherein the live virus is a zika virus and the inactivation is accomplished with formaldehyde, or ultraviolet irradiation, or gamma irradiation, or beta-propiolactone.
25. Vaccine obtainable by the method of claims 21 to 24.
26. A kit comprising an acceptor kit and a donor kit, the acceptor kit comprising an amount of an acceptor microsphere and an amount of an acceptor antibody and the donor kit comprising an amount of a donor microsphere and an amount of a donor antibody, wherein the acceptor microsphere is capable to accept energy which is transferred in a proximity reaction to produce a signal and is capable of binding or is bound to the constant region of the acceptor antibody and is not capable of binding to the donor antibody, the acceptor antibody has a variable region which is capable of binding to one of the at least two epitopes of a zika virus antigen and a constant region which is capable of binding or is bound to said acceptor microsphere, wherein the acceptor antibody is not capable of binding to the donor microsphere, the donor microsphere is capable to donate energy which is transferred in a proximity reaction to produce a signal by the acceptor bead and is capable of binding or is bound to the constant region of the donor antibody and is not capable of binding to the acceptor antibody, and the donor antibody has a variable region which is capable of binding to the other of the at least two epitopes of the zika virus antigen and a constant region which is capable of binding to said donor microsphere, wherein the donor antibody is not capable of binding to the acceptor microsphere.
27. The kit of claim 26, wherein the donor and acceptor antibodies do not cross-react with dengue antigens.
28. The kit of any one of claims 26 to 27, wherein at least one or both of the donor and acceptor antibodies are zika virus neutralizing antibodies.
29. The kit of any one of claims 26 to 28, wherein the donor and acceptor antibodies provide an EC.sub.50 value towards the zika virus antigen of less than 100 ng/mL, or less than 80 ng/mL, or less than 60 ng/mL, or less than 40 ng/mL, or less than 30 ng/mL.
30. The kit of any one of claims 26 to 29, wherein the donor and acceptor antibodies bind to epitopes on the zika virus envelope glycoprotein domain III of the envelope glycoprotein encoded by SEQ ID NO: 1.
31. The kit of any one of claims 26 to 29, wherein the epitopes are two different epitopes and wherein the acceptor and donor antibody have different variable regions and wherein one of them is antibody 1 and the other is antibody 2.
32. The kit of claim 31, wherein antibody 1 binds to amino acid E370 of SEQ ID NO: 1 and antibody 2 binds to amino acids T397 and H398 of SEQ ID NO: 1.
33. The kit of claim 31, wherein antibody 1 and antibody 2 are each characterized by the heavy and light chain complementary determining regions, wherein the antibody 1 is characterized by a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 4, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 5, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 6, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 9, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 10, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 11, and the antibody 2 is characterized by a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 18, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 19, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 20, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 23, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 24, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 25.
34. The kit of claim 31, wherein antibody 1 and antibody 2 are characterized by the heavy and light chain complementary determining regions, wherein the antibody 1 is characterized by a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 32, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 33, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 34, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 37, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 38, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 39, and the antibody 2 is characterized by a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (VH-CDR1) amino acid sequence of SEQ ID NO: 18, a heavy chain complementary determining region 2 (VH-CDR2) amino acid sequence of SEQ ID NO: 19, and a heavy chain complementary determining region 3 (VH-CDR3) amino acid sequence of SEQ ID NO: 20, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (VL-CDR1) amino acid sequence of SEQ ID NO: 23, a light chain complementary determining region 2 (VL-CDR2) amino acid sequence of SEQ ID NO: 24, and a light chain complementary determining region 3 (VL-CDR3) amino acid sequence of SEQ ID NO: 25.
35. The kit of claim 31, wherein antibody 1 and antibody 2 are characterized by the heavy and light chain variable regions, wherein the antibody 1 is characterized by a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 3 and a light chain variable region (VL) amino acid sequence of SEQ ID NO: 8, and the antibody 2 is characterized by a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 17 and a light chain variable region (VL) amino acid sequence of SEQ ID NO: 22.
36. The kit of claim 31, wherein antibody 1 and antibody 2 are characterized by the heavy and light chain variable regions, wherein the antibody 1 is characterized by a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 31 and a light chain variable region (VL) amino acid sequence of SEQ ID NO: 36, and the antibody 2 is characterized by a heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 17 and a light chain variable region (VL) amino acid sequence of SEQ ID NO: 22.
37. The kit of claim 31, wherein antibody 1 and antibody 2 are characterized by the heavy and light chain, wherein the antibody 1 is characterized by a heavy chain (H) amino acid sequence of SEQ ID NO: 2 and a light chain (L) amino acid sequence of SEQ ID NO: 7, and the antibody 2 is characterized by a heavy chain (H) amino acid sequence of SEQ ID NO: 16 and a light chain (L) amino acid sequence of SEQ ID NO: 21.
38. The kit of claim 31, wherein antibody 1 and antibody 2 are characterized by the heavy and light chain, wherein the antibody 1 is characterized by a heavy chain (H) amino acid sequence of SEQ ID NO: 30 and a light chain (L) amino acid sequence of SEQ ID NO: 35, and the antibody 2 is characterized by a heavy chain (H) amino acid sequence of SEQ ID NO: 16 and a light chain (L) amino acid sequence of SEQ ID NO: 21.
39. The kit of any one of claims 31 to 38, wherein antibody 1 is the donor antibody and antibody 2 is the acceptor antibody and wherein the donor antibody is biotinylated and the donor microsphere is coated with streptavidin and wherein the acceptor antibody is covalently bound to the acceptor microsphere.
40. Method of any one of claims 1 to 24, wherein the antigen is a zika antigen and the kit is defined by anyone of claims 26 to 39.
41. Antigen obtainable by a method of claim 40.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
Kit of Acceptor Antibody, Donor Antibody, Acceptor Microsphere, and Donor Microsphere
[0136] The present invention is directed to a kit comprising an acceptor kit and a donor kit, the acceptor kit comprising an amount of an acceptor microsphere and an amount of an acceptor antibody and the donor kit comprising an amount of a donor microsphere and an amount of a donor antibody, wherein [0137] the acceptor microsphere is capable to accept energy which is transferred in a proximity reaction to produce a signal and is capable of binding or is bound to the constant region of the acceptor antibody and is not capable of binding to the donor antibody, [0138] the acceptor antibody has a variable region which is capable of binding to one of the at least two epitopes of an antigen and a constant region which is capable of binding or is bound to said acceptor microsphere, wherein the acceptor antibody is not capable of binding to the donor microsphere, [0139] the donor microsphere is capable to donate energy which is transferred in a proximity reaction to produce a signal by the acceptor microsphere and is capable of binding or is bound to the constant region of the donor antibody and is not capable of binding to the acceptor antibody, and [0140] the donor antibody has a variable region which is capable of binding to the other of the at least two epitopes of the antigen and a constant region which is capable of binding to said donor microsphere, wherein the donor antibody is not capable of binding to the acceptor microsphere.
[0141] Other settings applying two Abs binding to two epitopes of an antigen include for instance the sandwich ELISA setting. Thereby one Ab is immobilized onto a plate, the antigen is applied to that plate and can be bound by the immobilized Ab. Afterwards, the second Ab is added and an enzyme-based detection is carried out. Although ELISA is a common method applied in the art, it has several 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, as well as time-consuming operation (multiple steps required including washing procedures). 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.
[0142] The microsphere useful for the invention ranges in the size from about 10 to about 500 nm in diameter, more preferably from about 50 to about 400 nm, even more preferably from about 200 to about 300 nm, and most preferably the microsphere has a diameter of about 200 to about 250 nm. The microsphere may be magnetic.
[0143] The microsphere may be constructed of any material to which molecules like Abs may be attached. 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.
[0144] The microsphere may comprise functional groups useful for attachment of molecules, such as the Abs of the present invention. Said functional groups may be, but are not limited to, carboxylates, esters, alcohols, carbamides, aldehydes, amines, sulfur oxides, nitrogen oxides, or halides. Molecules can be covalently coupled to the microspheres using chemical techniques described herein or in the prior art (see e.g. Bruckner, Springer Verlag 2010, Organic Mechanisms). For example, Abs can be coupled to the microsphere by a reductive amination. Therefore, an aldehyde on the surface of the microsphere reacts with an amine group within the molecule to result in an unstable imine which is further reduced to a stable amine using suitable reducing agents such as sodium cyanoborohydride (NaBH.sub.3CN) or sodium borohydride (NaBH.sub.4).
[0145] As amine-containing compounds other than those provided by the Ab that should be coupled to the microsphere may interfere with the reductive amination, amine containing compounds should be removed from the Ab solution with a suitable buffer exchange method. For instance, buffers containing amines (e.g. Tris(hydroxymethyl)aminomethan (Tris), glycine, bicine, tricine) should be avoided. For instance, suitable buffer include phosphate buffer saline (PBS), carbonate buffer, or sodium phosphate buffer. The pH for reductive amination may be about 8. As coupling efficiency can be reduced, Abs should be free of any protein or peptide-based stabilizer such as bovine serum albumin (BSA) or gelatin and the buffer should be free of glycerol.
[0146] The microsphere may comprise affinity groups for attachment of molecules, such as Abs of the present invention. Said affinity groups may be, but are not limited to, Ni.sup.2+(for immobilization of His-tagged molecules like His-tagged Abs), Protein A, Protein G, Protein L, anti-human IgG Ab, anti-rabbit IgG Ab, anti-mouse IgG Ab, anti-mouse IgM Ab, anti-rat IgG Ab, anti-sheep IgG Ab, anti-chicken IgY Ab, anti-goat IgG Ab, anti-FLAG Ab, streptavidin, avidin, and glutathione.
[0147] Microspheres may be one out of the list consisting of AlphaLISA® acceptor microspheres, AlphaScreen® acceptor microspheres, AlphaDonor microspheres as produced by Perkin Elmer (Waltham, US). In certain embodiments the acceptor microsphere is an AlphaLISA® acceptor microsphere.
[0148] The acceptor microsphere is capable to accept energy which is transferred in a proximity reaction and comprises one or more molecules that are able to accept energy which is transferred in a proximity reaction. In certain embodiments, the one or more molecules are fluorophores. Fluorophores include but are not limited to thioxene, anthracene, rubrene, and lanthanides like europium, europium chelates, or any derivatives thereof. The fluorophores are able to produce a detectable signal upon excitation, wherein the excitation is caused by accepting energy which is transferred in a proximity reaction.
[0149] The donor microsphere is capable to donate energy which is transferred in a proximity reaction and contains one or more molecules that are able to donate energy which is transferred in a proximity reaction. In certain embodiments, the one or more molecules are photosensitizers. Photosensitizers are molecules that produce a chemical change in another molecule in a photochemical process. In certain embodiments the photosensitizer is phthalocyanine. The donation of energy, which is transferred, can be induced by irradiation of the photosensitizer with a certain wavelength as part of a proximity reaction.
[0150] In one embodiment the donor microsphere is not capable of directly interacting with an acceptor microsphere and the acceptor microsphere is not capable of directly interacting with a donor microsphere. “Directly” within that context means, that the acceptor and donor microsphere do react with each other in another way than occurring during a proximity reaction. For instance, the functional groups of the donor and acceptor microspheres do chemically react with each other or the affinity groups of the donor and acceptor microsphere do non-covalently interact with each other. For instance, a protein A-coated donor microsphere is able to interact directly with an anti-human IgG Ab-coated acceptor microsphere. This interaction is resulting a false-positive signal and should therefore be avoided.
[0151] A proximity reaction is a reaction capable of producing a detectable signal. The proximity reaction is characterized by a donating step, wherein one of the two reaction partners (“the donor”, e.g. a donor microsphere) donates energy, which is transferred and by an accepting step, wherein the other of the two reaction partners (“the acceptor”, e.g. an acceptor microsphere) accepts the energy which is transferred and thereby produces a detectable signal.
[0152] In one embodiment, the proximity reaction is characterized by a donating step, wherein the donor microsphere donates energy which is transferred and by an accepting step, wherein the acceptor microsphere accepts the energy which is transferred and thereby produces a detectable signal. In one embodiment the first step of a proximity reaction comprises irradiation of the donor microsphere with a certain wavelength, thereby inducing a chemical change in another molecule by the photosensitizer. In one embodiment the donor microsphere contains phthalocyanine and is irradiated with a wavelength of about 680 nm. Excited phthalocyanine induces the production of singlet oxygen out of ambient oxygen near and/or at the surface of the donor microsphere. Further, the proximity reaction is characterized by the diffusion of singlet oxygen to the acceptor microsphere. In the next step of the proximity reaction energy is transferred from singlet oxygen to a fluorophore (as for instance rubrene, anthracene, a europium chelate, or thioxene) within the acceptor microsphere. The energy may be further transferred from the fluorophore to one or more other fluorophores until the energy is transferred to a final fluorophore which emits light (the signal). In certain embodiments, light at a wavelength from about 520 to 680 nm is emitted and can be detected between about 520 to 630 nm.
[0153] In one embodiment of the invention fluorophores within the acceptor microsphere include thioxene, anthracene, and rubrene. Thioxene is converted to a di-ketone derivative following its reaction with singlet oxygen. Energy is transferred from the di-ketone derivative of thioxene to anthracene by emission of light with a wavelength of about 340 to about 350 nm, which results in an excitation of anthracene. Excited anthracene transfers energy to rubrene as the final fluorophore by emission of light with a wavelength of about 450 to about 500 nm. Excited rubrene produces a signal in the form of emission of light with a wavelength of about 540 to about 680 nm (the signal) which can be detected between about 520 and about 620 nm. An example of acceptor microspheres comprising thioxene, anthracene, and rubrene are the AlphaScreen® acceptor microspheres as produced by Perkin Elmer (Waltham, US).
[0154] In another embodiment of the present invention fluorophores within the acceptor microsphere include thioxene and a europium chelate. Thioxene is converted to a di-ketone derivative following its reaction with singlet oxygen. Energy is transferred from the di-ketone derivative of thioxene by its emission of light with a wavelength from about 340 to about 350 nm to europium as the final fluorophore. Excited europium produces a signal in the form of emission of light with a wavelength from about 605 to about 625 nm (the signal) which can be detected between about 607 and about 623 nm. An example of acceptor microspheres comprising thioxene and europium chelate are the Alpha LISA® acceptor microspheres as produced by Perkin Elmer (Waltham, US).
[0155] A long excitation wavelength of about 680 nm combined with a shorter emission wavelength of about 520 to about 620 nm reduces interference from biological or other assay components and thereby ensures a low background signal.
[0156] The proximity reaction is dependent on the proximity of the two reaction partners and is thereby indicative for the proximity of two reaction partners.
[0157] In one embodiment the requirement of sufficient proximity can be realized by the requirement of the diffusion of singlet oxygen from donor to acceptor microsphere. Singlet oxygen has a lifetime of about 4 ps prior to falling back to ground state. Within that time, singlet oxygen is able to diffuse about 200 nm in solution. The diffusion of singlet oxygen as basis of a proximity reaction is well suitable for analyzing antigens which result in complexes where the distance between donor and acceptor microsphere is 200 nm or less, e.g. virus particles with a diameter not exceeding 150 nm such as zika virus particles with a diameter of about 50 nm.
[0158] In one embodiment of the invention the at least two epitopes of the antigen are the same epitopes, wherein acceptor and donor Ab are capable of binding to the same epitope and/or have the same variable region. An antigen with at least two same epitopes may be a virus carrying multiple copies of structural protein on its surface or a dimeric virus antigen (e.g. the dimeric E protein of ZIKV).
[0159] In another embodiment of the present invention, the at least two epitopes are different epitopes and the acceptor and donor antibody have different variable regions.
[0160] In another embodiment the donor and acceptor Ab do not cross-react with other antigens. For instance, if the antigen is a ZIKV antigen, the donor and acceptor Ab do not cross-react with DENV antigens.
[0161] In one embodiment of the present invention at least one of the donor and acceptor Abs neutralizes the virus antigen to which it binds when tested in a plaque reduction neutralization test or reporter virus particle test or microneutralization test or focus forming assay.
[0162] In one embodiment of the invention the donor and acceptor antibody each neutralize the virus antigen to which they bind when tested in a plaque reduction neutralization test or reporter virus particle test or microneutralization test or focus forming assay.
[0163] In one specific embodiment of the invention the antigen is a virus antigen, including virus antigens selected from the group consisting of zika virus antigen, dengue virus antigen, norovirus antigen, and poliovirus antigen. The virus antigen may be one or more of the structural proteins or one or more the non-structural proteins of the virus. The virus antigen may also be the whole virus.
[0164] In another specific embodiment the antigen is a virus antigen, wherein the virus antigen is selected from the group consisting of a live virus, an inactivated virus, a live attenuated virus, and a virus like particle. In certain embodiments the antigen is selected from the group of a live zika virus, an inactivated zika virus, a live attenuated zika virus, and a zika virus like particle. In certain embodiments the antigen is selected from the group of a live dengue virus, an inactivated dengue virus, a live attenuated dengue virus, and a dengue virus like particle. In another embodiment the antigen is selected from the group of a live poliovirus, an inactivated poliovirus, a live attenuated poliovirus, and a poliovirus like particle. In another embodiment the antigen is selected from the group of a live norovirus, an inactivated norovirus, a live attenuated norovirus, and a norovirus like particle.
[0165] In one embodiment the antigen is a vaccine antigen. In a specific embodiment the vaccine antigen is a virus antigen, wherein the virus antigen is selected from the group consisting of a live virus, an inactivated virus, a live attenuated virus, and a virus like particle. In certain embodiments the antigen is selected from the group of a live zika virus, an inactivated zika virus, a live attenuated zika virus, and a zika virus like particle. In one embodiment the antigen is selected from the group of a live dengue virus, an inactivated dengue virus, a live attenuated dengue virus, and a dengue virus like particle. In another embodiment the antigen is selected from the group of a live poliovirus, an inactivated poliovirus, a live attenuated poliovirus, and a poliovirus like particle. In another embodiment the antigen is selected from the group of a live norovirus, an inactivated norovirus, a live attenuated norovirus, and a norovirus like particle. Within that context a virus antigen further includes virus antigens selected from the group consisting of zika virus antigen, dengue virus antigen, norovirus antigen, and poliovirus antigen. The virus antigen may be one or more of the structural proteins or one or more of the non-structural proteins of the virus. The virus antigen may also be the whole virus.
[0166] In one embodiment the virus antigen is adsorbed to an adjuvant. In certain embodiments the adjuvant is alum. Alum within this context may refer to aluminum hydroxide or aluminum phosphate.
[0167] In a specific embodiment the virus antigen is an inactivated virus.
[0168] In one embodiment the virus antigen is an inactivated virus adsorbed to an adjuvant. In certain embodiments the adjuvant is alum. Alum within this context may refer to aluminum hydroxide or aluminum phosphate.
[0169] In one specific embodiment the virus antigen is an inactivated zika virus.
[0170] In a more specific embodiment the virus antigen is an inactivated zika virus adsorbed to an adjuvant. In certain embodiments the adjuvant is alum. Alum within this context may refer to aluminum hydroxide or aluminum phosphate.
[0171] According to one embodiment each of the acceptor antibody, the donor antibody, the acceptor microsphere, and the donor microsphere is in an unbound state.
[0172] According to one embodiment the acceptor microsphere is bound to the constant region of the acceptor antibody and/or the donor microsphere is bound to the constant region of the donor antibody.
[0173] According to one embodiment of the present invention the donor antibody is biotinylated and the donor microsphere is coated with streptavidin.
[0174] According to one embodiment of the present invention the acceptor antibody is covalently bound to the acceptor microsphere. In a specific embodiment the acceptor Ab is covalently bound to the acceptor microsphere by a reductive amination.
[0175] According to one specific embodiment the donor antibody is biotinylated and the donor microsphere is coated with streptavidin and the acceptor antibody is covalently bound to the acceptor microsphere.
Kit of Zika Binding Acceptor Antibody, Zika Binding Donor Antibody, Acceptor Microsphere, and Donor Microsphere
[0176] The present invention is directed to a kit, comprising an acceptor kit and a donor kit, the acceptor kit comprising an amount of an acceptor microsphere and an amount of an acceptor antibody and the donor kit comprising an amount of a donor microsphere and an amount of a donor antibody, wherein [0177] the acceptor microsphere is capable to accept energy which is transferred in a proximity reaction to produce a signal and is capable of binding or is bound to the constant region of the acceptor antibody and is not capable of binding to the donor antibody, [0178] the acceptor antibody has a variable region which is capable of binding to one of the at least two epitopes of a zika virus antigen and a constant region which is capable of binding or is bound to said acceptor microsphere, wherein the acceptor antibody is not capable of binding to the donor microsphere, [0179] the donor microsphere is capable to donate energy which is transferred in a proximity reaction to produce a signal by the acceptor bead and is capable of binding or is bound to the constant region of the donor antibody and is not capable of binding to the acceptor antibody, and [0180] the donor antibody has a variable region which is capable of binding to the other of the at least two epitopes of the zika virus antigen and a constant region which is capable of binding to said donor microsphere, wherein the donor antibody is not capable of binding to the acceptor microsphere.
[0181] Concerning the kit, reference is made to the chapter above entitled “Kit of acceptor antibody, donor antibody, acceptor microsphere, and donor microsphere”.
[0182] In one embodiment the donor and the acceptor Abs do not cross-react with dengue antigens.
[0183] In another embodiment at least one of the donor and the acceptor Abs is a ZIKV neutralizing Ab as for instance determined in a plaque reduction neutralization test or reporter virus particle test or microneutralization test or focus forming assay.
[0184] In another embodiment the donor and the acceptor Abs both are ZIKV neutralizing Abs as for instance determined in a plaque reduction neutralization test or reporter virus particle test or microneutralization test or focus forming assay.
[0185] In one embodiment the donor and acceptor Abs provide an EC.sub.50 value towards the zika virus antigen of less than 100 ng/mL, or less than 80 ng/mL, or less than 60 ng/mL, or less than 40 ng/mL, or less than 30 ng/mL. In a specific embodiment within that context the zika virus antigen is a PIZV.
[0186] In another embodiment of the invention the donor and acceptor Abs provide an EC.sub.50 value towards the zika virus antigen of less than 5e7 TCID.sub.50 titer, or less than 4e7 TCID.sub.50 titer, or less than 3e7 TCID.sub.50 titer. In a specific embodiment within that context the zika virus antigen is a zika live virus.
[0187] The EC.sub.50 value can be determined by detecting the signal indicative for the potency of the ZIKV antigen as described by the methods in the chapter below (“Method for determining the potency of an antigen sample”) for a serial dilution of the ZIKV antigen. By plotting the detected signal against the ZIKV antigen amount (which can be for instance either a concentration in ng/mL or a titer) and fitting the data with a non-linear regression according to a dose-response curve, the EC.sub.50 value can be calculated.
[0188] According to one embodiment of the present invention the donor and acceptor Abs bind to epitopes on ZIKV EDIII of the E protein encoded by SEQ ID NO: 1.
[0189] According to one embodiment the acceptor and donor antibody are antibody 1 and antibody 2 and have different variable regions. Antibody 1 and antibody 2 can be anti-ZIKV #1 and anti-ZIKV #2. Further, antibody 1 and antibody 2 can be anti-ZIKV #2 and anti-ZIKV #3. For further details and characterization of Abs reference is made to Example 1. Antibody 1 and antibody 2 may each 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. Antibody 1 and antibody 2 may each 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 antibody 1 and antibody 2. Antibody 1 and antibody 2 are presented together with their species origin, amino acid sequences, as well as corresponding nucleotide sequences of heavy chain (H), heavy chain variable region (VH), heavy chain complementary determining regions 1 to 3 (VH-CDR-1-3), light chain (L), light chain variable region (VL), as well as light chain complementary determining regions 1 to 3 (VL-CDR-1-3). Amino acid Nucleic acid Species sequence sequence Origin mAb mAb part (SEQ ID No) (SEQ ID No) Rabbit Anti-ZIKV #1 H 2 12 (IgG, Clone 242-3) VH 3 13 VH-CDR1 4 N/A VH-CDR2 5 N/A VH-CDR3 6 N/A L 7 14 VL 8 15 VL-CDR1 9 N/A VL-CDR2 10 N/A VL-CDR3 11 N/A Rabbit Anti-ZIKV #2 H 16 26 (IgG, Clone 306-2) VH 17 27 VH-CDR1 18 N/A VH-CDR2 19 N/A VH-CDR3 20 N/A L 21 28 VL 22 29 VL-CDR1 23 N/A VL-CDR2 24 N/A VL-CDR3 25 N/A Mouse Anti-ZIKV #3 H 30 40 (IgG2a kappa Clone VH 31 N/A D1-4G2-4-15) VH-CDR1 32 N/A VH-CDR2 33 N/A VH-CDR3 34 N/A L 35 41 VL 36 N/A VL-CDR1 37 N/A VL-CDR2 38 N/A VL-CDR3 39 N/A
TABLE-US-00002 TABLE 2 Critical amino acid residues in one-code letter code from the ZIKV E protein (SEQ ID NO: 1) important for binding of Anti-ZIKV #1 and 2 mAbs as evaluated by alanine scanning mutagenesis. T = Thr, E = Glu, H = His. mAb Critical Residues E Protein Domain Anti-ZIKV #1 (Clone 242-3) E370 III Anti-ZIKV #2 (Clone 306-2) T397, H398 III
[0190] According to one embodiment of the present invention, the antibody 1 is the donor antibody and the antibody 2 is the acceptor antibody.
[0191] According to another embodiment of the present invention, the donor antibody is biotinylated and the donor microsphere is coated with streptavidin.
[0192] According to another embodiment of the present invention, the acceptor antibody is covalently bound to the acceptor microsphere.
[0193] According to a specific embodiment of the present invention the antibody 1 is the donor antibody and antibody 2 is the acceptor antibody, the donor antibody is biotinylated and the donor microsphere is coated with streptavidin, and the acceptor antibody is covalently bound to the acceptor microsphere.
Method for Determining the Potency of an Antigen Sample
[0194] The invention is directed to a method for detecting a signal indicative for the potency of an antigen sample such as a vaccine antigen sample, wherein the antigen in the antigen sample provides at least two epitopes and the method comprises the steps of: [0195] Step 1: providing a kit comprising an acceptor kit and a donor kit, the acceptor kit comprising an amount of an acceptor microsphere and an amount of an acceptor antibody and the donor kit comprising an amount of a donor microsphere and an amount of a donor antibody, wherein [0196] the acceptor microsphere is capable to accept energy which is transferred in a proximity reaction to produce a signal and is capable of binding or is bound to the constant region of the acceptor antibody and is not capable of binding to the donor antibody, [0197] the acceptor antibody has a variable region which is capable of binding to one of the at least two epitopes of the antigen and a constant region which is capable of binding or is bound to said acceptor microsphere, wherein the acceptor antibody is not capable of binding to the donor microsphere, [0198] the donor microsphere is capable to donate energy which is transferred in a proximity reaction to produce a signal by the acceptor microsphere and is capable of binding or is bound to the constant region of the donor antibody and is not capable of binding to the acceptor antibody, and [0199] the donor antibody has a variable region which is capable of binding to the other of the at least two epitopes of the antigen and a constant region which is capable of binding to said donor microsphere, wherein the donor antibody is not capable of binding to the acceptor microsphere, [0200] Step 2: contacting the amount of said donor microsphere, the amount of said acceptor microsphere, the amount of said donor antibody and the amount of said acceptor antibody of step 1 with the sample to allow forming a complex of the antigen in the sample with the donor antibody bound to the donor microsphere and the acceptor antibody bound to the acceptor microsphere and the acceptor antibody bound to one of the at least two epitopes of the antigen and the donor antibody bound to the other of the at least two epitopes of the antigen, [0201] Step 3: conducting a proximity reaction to produce a signal indicative for the potency of the antigen sample, and [0202] Step 4: detecting the signal indicative for the potency of the antigen sample.
[0203] The invention is further directed to such a method for determining the amount of the antigen in the antigen sample indicative for the potency of the antigen sample by detecting the signal in accordance with the method as described above and further comprising the step of: [0204] Step 5: determining the amount of the antigen in the antigen sample indicative for the potency of the antigen sample based on the detected signal.
[0205] The invention is further directed to such a method for determining the potency of the antigen sample such as a vaccine antigen sample by detecting the amount of the antigen in accordance with the method as described above and further comprising the step of: [0206] Step 6: determining the potency of the antigen sample based on the amount of the antigen in the sample determined in step 5.
[0207] Concerning the kit, reference is made to the previous chapters entitled “Kit of acceptor antibody, donor antibody, acceptor microsphere, and donor microsphere” and “Kit of zika binding acceptor antibody, zika binding donor antibody, acceptor microsphere, and donor microsphere”.
[0208] According to one embodiment the antigen sample is a vaccine antigen sample.
[0209] According to one embodiment the vaccine antigen in the vaccine antigen sample is a virus antigen.
[0210] According to one embodiment the antigen sample is a virus antigen sample.
[0211] Concerning the virus antigen, reference is made to the previous chapters entitled “Kit of acceptor antibody, donor antibody, acceptor microsphere, and donor microsphere” and “Kit of zika binding acceptor antibody, zika binding donor antibody, acceptor microsphere, and donor microsphere”.
[0212] In one embodiment contacting the amount of said donor microsphere, the amount of said acceptor microsphere, the amount of said donor antibody and the amount of said acceptor antibody of step 1 with the sample to allow forming a complex of the antigen in the sample with the donor antibody bound to the donor microsphere and the acceptor antibody bound to the acceptor microsphere and the acceptor antibody bound to one of the at least two epitopes of the antigen and the donor antibody bound to the other of the at least two epitopes of the antigen in step 2 is carried out for about 14 to 28 hours.
[0213] The order of contacting the amount of said donor microsphere, the amount of said acceptor microsphere, the amount of said donor antibody and the amount of said acceptor antibody of step 1 with the sample to allow forming a complex of the antigen in the sample with the donor antibody bound to the donor microsphere and the acceptor antibody bound to the acceptor microsphere and the acceptor antibody bound to one of the at least two epitopes of the antigen and the donor antibody bound to the other of the at least two epitopes of the antigen in step 2 may vary.
[0214] In one embodiment the amount of donor Ab and the amount of acceptor Ab are contacted with the sample for a certain contacting time in a first step followed by contacting the amount of donor microsphere and the amount of acceptor microsphere with the amount of donor Ab, the amount of acceptor Ab, and the sample for a certain contacting time in a second step.
[0215] In another embodiment the acceptor microsphere is bound to the constant region of the acceptor Ab and the donor microsphere is bound to the constant region of the donor Ab and the amount of acceptor microsphere bound to the constant region of the acceptor Ab and the amount of donor microsphere bound to the constant region of the donor Ab are concomitantly contacted with the sample for a certain contacting time.
[0216] In another embodiment, the donor Ab is biotinylated, the donor microsphere is coated with streptavidin and the acceptor microsphere is bound to the constant region of the acceptor Ab and the amount of donor Ab, as well as the amount of acceptor microsphere bound to the constant region of the acceptor Ab are contacted with the sample for a certain contacting time in a first step followed by contacting the sample, the amount of donor Ab, and the amount of acceptor microsphere bound to the acceptor Ab with the amount of donor microsphere for a second contacting time in a second step. Contacting in the first step may be carried out for about 16 to about 24 hours and contacting in the second step may be carried out for about 2 hours.
[0217] The complex allowed to form in step 2 brings the donor microsphere and acceptor microsphere in sufficient proximity that a proximity reaction can occur. Consequently, if no complex is formed, the donor microsphere and acceptor microsphere do not react in a proximity reaction. Therefore, the signal produced in the proximity reaction in step 3 is proportional to the amount of formed complex and therefore to the amount of antigen in the antigen sample.
[0218] In one embodiment of the invention the signal produced in the proximity reaction in step 3 is generated by the final fluorophore within the acceptor microsphere. In this context the final fluorophores may be a europium chelate or rubrene. The signal is emission of light with a wavelength in the range of about 520 to about 680 nm, in particular of about 615 nm. The signal can be detected by any suitable detection instrument.
[0219] In one embodiment the detection instrument is capable of excitation at about 680 nm and reading the emission at about 520 to about 630 nm, in particular at about 615 nm. A laser or a light emitting diode (LED) may be used as the excitation source. Preferred detection instruments may include but are not limited to EnVision®, EnSpire™, EnSight™, or VICTOR® Nivo™ Multilabel Plate Readers from Perkin Elmer.
[0220] The signal produced in the proximity reaction in step 3 is proportional to the amount of formed complex and therefore proportional to the amount of antigen in the antigen sample. Therefore, determining the amount of the antigen in the antigen sample in step 5 can be carried out by comparing the signal indicative for the potency of the antigen sample with a standard curve. The standard curve may be a sigmoidal-shaped dose-response curve or a linear curve plotting different amounts of the type of antigen to be analyzed within the sample against the corresponding signal. The amount of antigen can be for instance expressed as a concentration or a titer.
[0221] As the potency i.e. the capability of an antigen to induce an immune response in a subject depends on the amount of antigen within an antigen sample, the amount of antigen is indicative for the potency of the antigen sample, and therefore the signal indicative for the amount of antigen is also indicative for the potency of an antigen sample.
[0222] The invention is further directed to such a method for determining the potency of an antigen sample in accordance with the method as described above, wherein step 6 comprises the steps of [0223] Step 6.1: determining the potency of standardized samples of the antigen in human or non-human subjects by measuring the associated mean neutralizing antibody titers produced in said human or non-human subjects, [0224] Step 6.2: determining the amount of the antigen with at least two epitopes in said standardized samples according to the method as described above, [0225] Step 6.3: establishing a standard curve from the mean neutralizing antibody titers of step 6.1 and the amount of the antigen of step 6.2, and [0226] Step 6.4: determining the potency of the antigen sample by comparing the amount of antigen in the antigen sample determined in step 5 with the standard curve.
[0227] According to one embodiment the standardized antigen samples are provided by a forced degradation study or different doses of the antigen.
[0228] According to one embodiment the non-human subjects in step 6.1 include mice, rats, cats, rabbits, primates, and non-human primates.
[0229] According to one embodiment the subjects in step 6.1 are mice.
[0230] Mean neutralizing Ab titers can be determined by methods well known in the art including a MNT, a PRNT, a RVP assay, or a FFA. According to one embodiment of the invention mean neutralizing Ab titers are determined by a RVP assay.
[0231] According to one embodiment the standard curve is generated by plotting the potency of the standardized antigen samples expressed as the mean neutralizing Ab titers against the determined amount of the antigen in the standardized samples.
[0232] The present invention is further directed to the method as described above, wherein the antigen sample is a zika antigen sample. The zika antigen may be an inactivated virus. In certain embodiments of the present invention the method for monitoring the potency of a ZIKV antigen sample is referred to as Zika Antigen Potency Assay (ZAPA).
Method for Monitoring the Potency of a Vaccine Antigen During the Production Process
[0233] The present invention is further directed to a method of monitoring the potency of a vaccine antigen during the production process including purifying, inactivating and formulating of said vaccine antigen to form a final vaccine by measuring the potency of the vaccine antigen in accordance with the method as described above.
[0234] In one embodiment the vaccine antigen is a ZIKV antigen and the potency of the ZIKV antigen is monitored during the production process including purifying, inactivating and formulating of said ZIKV antigen to form a final ZIKV vaccine by measuring the potency of the ZIKV antigen in accordance with the ZAPA method as described above.
[0235] Purifying can be carried out by filtration and/ or chromatography.
Method of Producing a Virus Vaccine
[0236] The present invention is directed to a method of producing a virus vaccine comprising the steps of: [0237] Step A: preparing various batches of vaccine antigen, [0238] Step B: determining the potency of the vaccine antigen of the various vaccine antigen batches produced in step A in accordance with the method as described above and selecting the vaccine antigen batches in conformity with a predetermined potency requirement, [0239] Step C: preparing vaccine batches by formulating the vaccine antigen batches selected in step B into various batches of virus vaccine, and [0240] Step D: determining the potency of the vaccine antigen in the vaccine batches of the various batches produced in step C in accordance with the method as described above and selecting the vaccine batches in conformity with the predetermined potency requirement.
[0241] Concerning the method for determining the potency of the vaccine antigen reference is made to the previous chapters entitled “Method for determining the potency of an antigen sample” and “Monitoring the potency of a vaccine antigen during the production process”.
[0242] In one embodiment of the present invention step A includes various sub-steps and step B is performed after each sub-step.
[0243] In certain embodiments of the present invention the sub-steps include purification (as for instance by chromatography or filtration) and inactivation (as for instance with formaldehyde, or ultraviolet irradiation, or gamma irradiation, or beta-propiolactone).
[0244] In a specific embodiment the sub-steps comprise inactivation of a live virus to an inactivated virus.
[0245] In a specific embodiment of the present invention the live virus is a zika virus and the inactivation is accomplished with formaldehyde, or ultraviolet irradiation, or gamma irradiation, or beta-propiolactone.
[0246] The invention is further directed to a method as described above wherein the vaccine antigen is a zika antigen.
Vaccines Obtainable by the Method for Producing a Virus Vaccine
[0247] The invention is further directed to a vaccine obtainable by the method described above.
[0248] The invention is further directed to a zika antigen obtainable by the method described above.
Alternative Donor and Acceptor Structures
[0249] Microspheres have been described above as one suitable structure for an acceptor and a donor capable of reacting in a proximity reaction. However, the invention also encompasses other embodiments wherein alternative donor and acceptor structures are applied.
[0250] One example for alternative acceptor and donor structures is a pair which is able to react by a Förster Resonance Energy Transfer (FRET). For instance, in one specific embodiment a pair of two light-sensitive molecules (chromophores) are reacting in the proximity reaction as donor and acceptor. The donor chromophore is excited and can transfer the energy from the excitation to an acceptor chromophore through non-radiative dipole-dipole coupling. The excited acceptor chromophore is then capable to produce a detectable signal (e.g. by the emission of light). The efficiency of the energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor. For instance, one common FRET pair of chromophores is cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), both are color variants of green fluorescent protein (GFP).
[0251] Another example for alternative acceptor and donor structures is a pair which is able to react in a Bioluminescence Resonance Energy Transfer (BRET). This technique uses a bioluminescent enzyme (e.g. Renilla luciferase) as a donor to produce an initial photon emission compatible with a fluorophore as YFP as an acceptor. BRET does not require external illumination to initiate the energy transfer, which decreases possible background noise.
[0252] In these embodiments the donor antibody may be covalently bound to a donor structure (such as the donor chromophore or the bioluminescent enzyme) and the acceptor antibody may be covalently bound to an acceptor structure (such as the acceptor chromophore), wherein the donor structure is capable of transferring energy (such as excitation energy in the case of a donor chromophore) to the acceptor structure if both structures are sufficiently close to each other.
[0253] The present invention is therefore further directed to kits and methods as described above, wherein the microspheres are exchanged by alternative donor and acceptor structures.
EXAMPLES
[0254] 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: Biotinylation of Donor Ab and Coupling of Acceptor Ab to Acceptor Microspheres
[0255] mAbs applied in the Zika antigen potency assay (ZAPA) set-up are listed in Table 1 and 3.
TABLE-US-00003 TABLE 3 Anti-ZIKV #4 mAb applied in the ZAPA. Anti-ZIKV #4 is presented together with its species origin and amino acid sequences of heavy chain (H), heavy chain variable region (VH), heavy chain complementary determining regions 1 to 3 (VH-CDR-1-3), light chain (L), light chain variable region (VL), as well as light chain complementary determining regions 1 to 3 (VL-CDR-1-3). Amino acid Species sequence Origin mAb mAb part (SEQ ID No) Human Anti-ZIKV #4 H 42 (Clone EDE1-C10) VH 43 VH-CDR1 44 VH-CDR2 45 VH-CDR3 46 L 47 VL 48 VL-CDR1 49 VL-CDR2 50 VL-CDR3 51
[0256] Anti-ZIKV #1 and 2 mAbs were generated and characterized as described in co-pending application PCT/US2019/052189 (Takeda Ig Application). In brief, rabbits were immunized with purified inactivated Zika vaccine (PIZV) and ZIKV virus like particles (VLPs). Afterwards, the spleen was isolated for generation of hybridoma cells. Hybridoma supernatants were examined for reactivity towards ZIKV VLPs and E protein, as well as cross-reactivity towards inactivated DENV 1-4 by enzyme linked immunosorbent assay (ELISA). Therefore, hybridoma supernatants were screened against inactivated DENV1 (West Pacific 74, Microbix), DENV2 (16681; Microbix), DENV3 (CH53489, Microbix), DENV4 (TVP-360, Microbix), ZIKV E protein (Native Antigen), and ZIKV VLP (Native Antigen). DENV1, 3, and 4 were inactivated with gamma-irradiation and DENV2 with formalin by the manufacturer as a part of the production process. Both ZIKV E protein and ZIKV VLP were used as positive control antigens. In brief, antigens were coated onto Nunc Polysorp ELISA plates at 1 μg/mL in carbonate coating buffer (pH 9.4) at 4° C. overnight prior to use. Then, plates were washed with PBS containing 0.05% Tween-20 (PBS-T). A 5% non-fat dry milk blocking solution was added to the plates for a minimum of 1 hour at room temperature to reduce non-specific binding. Plates were washed and hybridoma supernatants were added to the plates. Plates were then incubated at 37° C. for 1 to 2 hours. Plates were again washed with PBS-T. Goat-derived anti-rabbit IgG (H+L) horseradish peroxidase conjugated secondary Ab (Jackson ImmunoResearch, Lot. No. L2416-X326F) was diluted 1:5,000 in 5% milk blocking solution and added to the plates. Plates were incubated 37° C. for 1.5 hours and then washed again with PBS-T. 3,3′, 5,5′-Tetramethylbenzidine substrate was added and incubation was carried out for 10 min at room temperature. The reaction was stopped with 1 N HCI and the plates were scanned for absorbance at 450 nm and 630 nm using an EnSpire reader (Perkin Elmer). Positive binding cut-off was set at 0.5 optical density reading. Both, anti-ZIKV #1 and 2 did not show binding to any of DENV1 to 4 verifying that both Abs are ZIKV-selective (Table 4). Moreover, hybridoma supernatants were screened for their neutralizing activity in a microneutralization test (MNT) as well as a reporter virus particle (RVP) assay. Anti ZIKV #1 showed strong neutralization activity, whereas anti-ZIKV #2 showed weak neutralization activity. Affinity of hybridoma supernatants towards ZIKV VLPs was determined by a Bio-layer interferometry (BLI) assay. In addition, epitope binning was examined using a competitive BLI assay, binding a primary mAb to the VLP, followed by cross-binding a secondary mAb. Binning experiments showed that Anti-ZIKV #1 and 2 bind to different regions within the antigen. Further, mAbs were sequenced (comp. Table 1). Finally, amino acid residues within the antigen critical for binding of 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 reactivity (<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). Both mAbs were shown to bind to ZIKV EDIII (comp. Table 2). Anti-ZIKV #1 and 2 were stored in PBS, pH 7.4 at a final concentration in the range of 1.3 to 1.4 mg/mL.
TABLE-US-00004 TABLE 4 Reactivity of Anti-ZIKV #1 and 2 hybridoma supernatants against ZIKV VLP, ZIKV E protein, and DENV1 to 4 examined by ELISA. Presented are optical density (OD) values for each antigen and Ab. ZIKV ZIKV E mAb VLP protein DENV1 DENV2 DENV3 DENV4 Anti-ZIKV #1 0.97 0.33 0.04 0.034 0.04 0.038 Anti-ZIKV #2 1.15 0.61 0.037 0.034 0.034 0.048
[0257] Anti-ZIKV #3 was originally generated as described previously using DENV-2 whole virus for immunization of mice (Gentry et al., Am J Trop Med Hyg 1982, 31(3): 548-555). The mAb binds the fusion loop at EDII and shows cross-reactivity with other flaviviruses like ZIKV (Aubry et al., Transfusion 2016, 56:33-40). Anti-ZIKV #4 was originally generated as described previously using DENV-2 whole protein for immunization. The mAb binds to E protein dimer epitope and shows cross-reactivity with ZIKV (Barba-Spaeth et al., Nature 2016; 536:48-53).
[0258] Anti-ZIKV #3 and #4 are commercially available from Wuxi AppTec. Anti-ZIKV #3 is additionally available from Absolute Antigen (Protein A purified, supplied in PBS, pH 7.4 with 0.02% Proclin-300 at 1 mg/mL, Cat. No. Ab00230.2.0). Wuxi expressed both Abs in Chinese hamster ovary (CHO) cells. The supernatant of the transfected cells was affinity purified using Protein G sepharose column (GE Healthcare) and analyzed with SDS-PAGE. Heavy chain (H) sequence of anti-ZIKV #3 (SEQ ID NO: 30 and 40) is deposited in GenBank under the accession codes AHX42424.1 (amino acid sequence) and KJ438785.1 (coding sequence and amino acid sequence), light chain (L) sequence of anti-ZIKV #3 (SEQ ID NO: 35 and 41) is deposited in GenBank under the accession codes AHX42423.1 (amino acid sequence) and KJ438784.1 (coding sequence and amino acid sequence). Anti-ZIKV #4 has been crystalized complexed with DENV2 E protein (PDB: 4UT9; Rouvinski et al., Nature 2015, 520(7545): 109-113) and ZIKV (PDB: 5H37; Zhang et al., Nat Commun 2016, 7, 13679).
[0259] mAbs serving as acceptor Abs were coupled to acceptor microspheres as described in the following. For conjugation, 25 mg of acceptor microspheres (0.25 mL of a 100 mg/mL stock, unconjugated AlphaLISA® acceptor microspheres, Perkin Elmer, Cat. No. 6772001-3) were mixed with 0.5 mg of acceptor Ab to result in a coupling ratio of 1:50 (mg protein : mg microspheres). Next, corresponding volumes of 10% Tween-20 to result in a 160-fold dilution, corresponding volumes of a 25 mg/mL solution of NaBH.sub.3CN (prepared freshly in water; Sigma Aldrich, Cat. No. 152159) to result in a 20-fold dilution, and corresponding volumes of 0.13 M phosphate buffer pH 8.0 were added to obtain a final reaction volume of 1 mL. For example, 0.374 mL of mAb concentrated at 1.34 mg/mL were added to 0.25 mL of the 100 mg/mL microsphere stock. Afterwards, 0.445 mL of 0.13 M phosphate buffer pH 8.0 were added, followed by 6 μL of 10% Tween-20 and 50 μL of a 25 mg/mL solution of NaBH.sub.3CN. The mixture was incubated for 18-19 hours at 37° C. under mild agitation (6-10 rpm). For blocking, 50 μL of a 65 mg/mL solution of carboxy-methoxylamine (CMO; Sigma Aldrich, Cat. No. C13408) prepared freshly in 0.8 M NaOH were added to the reaction resulting in a final concentration of 3.25 mg/mL CMO and incubation was carried out for 1 hour at 37° C. For purification, the tube was centrifuged for 40 min at 16,000×g and 4° C., supernatant was removed, and the microsphere pellet was resuspended in 5 mL 0.1 M Tris-HCl, pH 8.0. After centrifugation for 40 min at 16,000×g and 4° C., supernatant was removed. The washing step was repeated once. After centrifugation for 40 min at 16,000×g and 4° C., supernatant was removed and the microspheres were resuspended to 5 mg/mL in storage buffer (PBS, pH 7.4 with 0.05% Proclin-300). The conjugated acceptor microspheres were stored at 4° C. until further use.
[0260] mAbs serving as donor Abs were biotinylated as described in the following. N-hydroxysuccinimido-ChromaLink™ Biotin (NHS-ChromaLink™ Biotin 354S, 10 mg/mL stock concentration in dimethylformamide (DMF); SoluLink Inc., Cat. No. B1001-105, Lot. No. WOTL26127) was prepared freshly at 2 mg/mL in PBS, pH 7.4. NHS-ChromaLink™ Biotin 354S contains a chromophore (aryl hydrazine) with an absorbance maximum at 354 nm linked by a triethylenglycol (PEG3) linker to biotin. The succinimidyl ester functional group enables modification of lysines in aqueous buffers. Diluted NHS-ChromaLink™ Biotin was mixed with the donor Ab to result in a 30-fold molar excess of biotin over Ab, wherein the reaction concentration of the Ab was kept at 0.5 mg/mL. The reaction volume was adjusted with PBS, pH 7.4 previous to addition of NHS-ChromaLink™ Biotin. For instance, 937 μL of 1.26 mg/mL mAb were mixed with 1333.3 μL PBS, pH 7.4. Next, 89.7 μL of 2 mg/mL NHS-ChromaLink™ Biotin were added, resulting in a total volume of 2360 μL and 1.18 mg mAb (7.375 nmoles) and 0.179 mg NHS-ChromaLink™ Biotin (221.250 nmoles) in the reaction. Incubation was carried out for 2 hours at 21-23° C. Afterwards, free biotin was removed using a desalting column equilibrated with PBS, pH 7.4 (Zeba desalting columns, 5 mL, Pierce (Thermo Fisher Scientific), Cat. No. 89882). This step was repeated once with a second desalting column. For characterization, absorbance at 280 nm (A.sub.280nm, referring to the protein amount) and at 354 nm (A.sub.354nm, referring to the biotin amount) were determined, wherein the 280 nm value was corrected for the absorbance of the label at 280 nm as determined as 0.23×A.sub.354nm. With the extinction coefficient at 280 nm (214,400 M.sup.−1) and the molecular weight (160,000 g/mol) of the mAb the protein concentration was determined from the A.sub.280nm value. Likewise, the concentration of biotin was determined from the A.sub.354nm value using an extinction coefficient of biotin at 354 nm of 29,000 M.sup.−1 and a molecular weight of 810.92 g/mol. The ratio of biotin per Ab was then determined by dividing the concentration of biotin by the concentration of Ab. A final biotinylated Ab concentration of 0.5 μM (80 μg/mL) was adjusted by dilution with stabilization buffer (PBS, pH 7.4, 0.1% Tween-20, 0.05% sodium azide). Biotinylated mAbs were stored at 4° C. until further use.
Example 2: Evaluation of mAb Pairs for ZAPA
[0261] Next, different combinations of mAbs were evaluated for their performance in the ZAPA (Table 5, mAb pairs #1 to 3). Donor Abs were biotinylated and acceptor Abs were coupled to acceptor microspheres according to Example 1.
TABLE-US-00005 TABLE 5 Different donor and acceptor Ab combinations tested in the ZAPA. mAb pair Acceptor Ab Donor Ab #1 Anti-ZIKV #2 Anti-ZIKV #1 #2 Anti-ZIKV #2 Anti-ZIKV #3 #3 Anti-ZIKV #2 Anti-ZIKV #4
[0262] For testing of the mAb pairs, purified inactivated zika vaccine (PIZV) and ZIKV strain PRVABC59 were evaluated in ZAPA. PIZV was provided at a stock concentration of 10 μg/mL drug substance (DS; purified, liquid, formalin-inactivated ZIKV) as determined by a Bradford assay. PIZV is formulated by the absorbance of DS on aluminum hydroxide (Al(OH).sub.3; alum; Alhydrogel® 2%, Brenntag, Lot. No. 5414).
[0263] ZIKV TCID.sub.50 (50% Tissue Culture Infectious Dose) was determined by an endpoint dilution assay including the observation of cytopathic effects (CPE) after inoculating Vero cells with the virus. Therefore, Vero cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Corning, Cat. No. 15-017-CV) supplemented with 10% (v/v) Fetal Bovine Serum (FBS; Sigma, Cat. No. 12007C), 2% (v/v) L-glutamine (from a 200 mM stock; Hyclone, Cat. No. SH30034.01), and 1% (v/v) Penicillin/Streptomycin (Pen/Strep, from a 10-fold stock; Hyclone, Cat. No. SV30010) at 36±2° C. and 5% CO.sub.2. Cells were seeded at 1.4×10.sup.4 cells per well in 100 μL medium in a 96-well plate (Costar, Cat. No. 3596) and allowed to settle down and grow for 2 days to achieve a confluency of >90% at the time of virus addition. Then, cells were incubated with a serial dilution of ZIKV (prepared in dilution medium: DMEM supplemented with 2% (v/v) FBS, 2% (v/v) L-glutamine, and 1% (v/v) Pen/Strep) for 5 days±4 hours by decanting the supernatant from the cells and addition of 100 μL per well of corresponding virus dilution. The serial dilution was examined in duplicates. Each of the two equivalent dilution series was plated in quadruplicates. Negative controls were included by addition of 100 μL dilution medium lacking ZIKV. After the incubation time, the absorbance at 560 nm and 420 nm was recorded after incubating the plate for 15 min at room temperature to account for color changes in heavily infected wells in which the cells have died. Absorbance at 420 nm was subtracted from the absorbance at 560 nm. A value >0 accounted for CPE negative, a value <0 for CPE positive. From the CPE results for the dilutions and replicates, the mean TCID.sub.50 titer per mL was calculated according to the method of Reed and Muench. The results were confirmed by scoring the plate visually with a light microscope and corrected if needed.
[0264] PIZV and ZIKV were serially diluted in assay buffer (25 mM HEPES pH 7.4, 0.5% Triton X-100, 0.1% Casein, 1 mg/mL Dextran-500, and 0.5% Proclin-300; prepared from a 10-fold stock from Perkin Elmer, Cat. No. AL000F) to result in a 5-fold amount of the final assay concentrations or titers, respectively, for each dilution (for final concentrations or titers see
[0265] Biotinylated anti-ZIKV mAbs were prepared as described under Example 1 to result in a final concentration of 80 μg/mL biotinylated mAb. In a first step, the stock was vortexed for 5 to 20 sec and diluted 1:600 in assay buffer (e.g. 5 μL of biotinylated anti-ZIKV mAb to 2985 μL assay buffer). The dilution was again vortexed for 5 to 20 sec. Anti-ZIKV #2 was conjugated to acceptor microspheres as described under Example 1. In a second step, the 5 mg/mL stock of conjugated acceptor microspheres was vortexed (5 to 20 sec) and diluted 1:300 in assay buffer in the same tube as biotinylated anti-ZIKV mAbs were diluted (e.g. 10 μL of conjugated acceptor microspheres were added to 2990 μL diluted biotinylated mAb from the step before). The dilution was again vortexed for 5 to 20 sec. 30 μL of the dilution of biotinylated anti-ZIKV mAb and conjugated acceptor microspheres were added per well into the 96-well plate. The sides of the plate were tapped to collect contents to the bottom of the wells. The plate was sealed with a foil sealer (Adhesive PCR Sealing Foil Sheets, Thermo Fischer, Cat. No. AB-0626) to block light and incubation was carried out at 37° C. for 16 to 24 hours.
[0266] Streptavidin-coated donor microspheres at a 5 mg/mL stock concentration (PerkinElmer, Cat. No. 6760002) were vortexed (5 to 20 sec) and diluted 1:100 in assay buffer. 10 μL of the dilution were added per well. The sides of the plate were tapped to collect contents to the bottom of the wells. The plate was sealed with a foil sealer to block light and incubation was carried out at 37° C. for 2 hours ±10 min.
[0267] The plate was removed from the incubator and read within 10 min. Therefore, the foil sealer was removed from the plate immediately before reading to minimize light exposure. The plate was analyzed in an EnSpire multimode plate reader (PerkinElmer) with the “96-well AlphaLISA protocol”. ZAPA signal counts in relative fluorescence units (RFU) from the PIZV and ZIKV dilutions were normalized to the medium background signal resulting from the blank wells, and plotted against the corresponding PIZV concentrations and the ZIKV titers, respectively. The data were independently fitted for each mAb pair with a four parameter logistic (4PL) regression model (
[0268] mAb pairs #1 and 2 resulted in high signals for both, the ZIKV and PIZV samples. Contrarily, only weak signal for high PIZV concentrations and almost no signal even at the highest ZIKV titer was observed using mAb pair #3. The data show that the ZAPA set-up using mAb pairs #1 and 2 is able to efficiently determine PIZV and ZIKV in a concentration dependent manner, resulting in a good signal-to-noise ratio.
[0269] In summary, mAb pairs #1 and 2 resulted in high signals compared to mAb pair #3. ZAPA analysis was shown to fit for purpose of analyzing both, ZIKV strain PRVABC59 and PIZV. In conclusion, ZAPA mAb pairs #1 and 2 are able to efficiently measure the epitope availability from the live virus as well as from PIZV.
Example 3: Evaluation of Stability Indication by ZAPA
[0270] In a next step, ZAPA was applied to analyze samples including different amounts of heat-inactivated DS, as well as PIZV samples formulated with the heat-inactivated DS samples by adsorbing DS on alum. The aim of this forced-degradation study was to evaluate if ZAPA is capable of reliably indicating the amount of intact DS, either present alone, or adsorbed on alum within the PIZV samples and therefore is a read out for antigen stability, i.e. intact epitopes.
[0271] Therefore, a portion of DS was heat treated for 1 h at 85° C. in order to degrade the material. DS samples were prepared by mixing untreated and heat-treated DS to result in 0, 25, 50, 75, and 100% of total heat-treated DS amount within the samples. Previous to preparing PIZV samples, DS samples were analyzed with a Bradford assay, using ZIKV recombinant E protein (Meridian Life Sciences, Inc.; Lot. No. 1J29317) as a standard. Of note, the total protein amount detected remained stable even after heat-treatment (Table 6).
TABLE-US-00006 TABLE 6 Analysis of DS samples with Bradford assay. Presented is the total protein amount in μg/mL. DS Sample (% heat-inactivated DS) Total protein amount (μg/mL) 100 53.9 75 53.5 50 53.1 25 54.8 0 53.4
[0272] Next, DS samples were diluted to result in a DS amount of 1 μg per 100 μL volume (10 μg/mL). For formulation of PIZV samples, 40 μg of alum (Alhydrogel® 2%, Brenntag, Lot. No. 5414) were added per 1 μg DS sample and samples were stirred for 2 hours at room temperature. The DS and PIZV samples were stored at 5±3° C. until analysis.
[0273] DS and PIZV samples were analyzed with ZAPA using mAb pair #1 (see Table 5). In addition to DS and PIZV samples, a PIZV reference (stock concentration: 20 μg/mL) was included. The reference was serially diluted in assay buffer (25 mM HEPES pH 7.4, 0.5% Triton X-100, 0.1% Casein, 1 mg/mL Dextran-500, and 0.5% Proclin-300; prepared from a 10-fold stock from Perkin Elmer, Cat. No. AL000F) as described under Example 2.
[0274] 10 μL per reference dilution or DS or PIZV sample were added per well into a white 96-well plate (1/2 area plate-96, Perkin Elmer, Cat. No. 6002299). In addition, blank wells were included by addition of 10 μL of assay buffer per well to account for background signal. Each reference dilution, as well as the samples and blank controls were evaluated in duplicates. ZAPA was further carried out as described under Example 2.
[0275] ZAPA signal counts in relative fluorescence units (RFU) from the reference dilutions, as well as from the DS and PIZV samples were normalized to the medium background signal resulting from the blank wells. The data from the reference material were independently fitted with a four parameter logistic (4PL) regression model. Corresponding ZAPA signal counts for a certain amount of intact DS within the PIZV reference dilutions were interpolated to the ZAPA signals from DS and PIZV samples and the thereby resulting ZAPA values were reported as antigen units per mL (AU/mL) for corresponding DS and PIZV samples (
[0276] DS and PIZV samples solely containing heat-treated DS resulted in the lowest ZAPA values compared to the other samples, whereas the DS and PIZV samples that contained 100% untreated DS resulted in the highest ZAPA values. The values of all examined DS and PIZV samples fit a linear response with an R.sup.2 value of 0.986 (DS samples; linear regression: y=439.44x+1557.2) and 0.954 (PIZV samples; linear regression: y=122.14x+718.53), indicating that this method accurately and selectively detects changing antigen availability within the DS and PIZV samples after heat-degradation independent of the presence of additional ingredients such as alum.
[0277] It can be seen from the data that one or both of the epitopes responsible for binding of the mAbs used in ZAPA has or have been disrupted by heat treatment. The data indicate that ZAPA efficiently detects presence of intact epitopes within the samples upon heat-inactivation. In conclusion, the assay is sensitive to changes in sample stability.
[0278] Taken together, other than the Bradford assay which provides the total amount of protein independent of heat-degradation, ZAPA provides information about the amount of intact antigen. ZAPA shows robust performance and reliable evaluation of the amount of intact epitopes in the DS and PIZV samples, verifying that the method is stability indicating and not affected by the presence of additional ingredients such as alum.
Example 4: Characterization of DS Batches Formulated to PIZV by ZAPA
[0279] In a next step, different DS batches formulated to PIZV were analyzed using ZAPA and compared to immune responses induced by the PIZV in CD-1 mice to evaluate if ZAPA is potency indicating.
[0280] Therefore, four different DS batches (#1 to 4) were analyzed. Total protein concentrations of DS batches were determined with Bradford as described under Example 3. Serial dilutions of the DS batches were prepared in Tris buffer (10 mM Hydroxymethyl aminomethane base (Fisher, Cat. No. T395-500) containing 150 mM sodium chloride (Fisher, Cat. No. S271-500), pH 7.6) to result in 0.001, 0.005, 0.01, 0.05, 0.1 μg of total antigen in 100 μL sample (reference is made to co-pending application U.S. 62/845,024). For formulation of PIZV samples, antigen was adsorbed on 50 μg alum (Alhydrogel® 2%, Brenntag, Lot. No. 5414) per 100 μL sample. The diluted samples were stored at 5±3° C. until analysis.
[0281] Next, PIZV samples from the different batches were analyzed with ZAPA using mAb pair #1 (see Table 5). In addition to PIZV samples, a PIZV reference (stock concentration: 20 μg/mL) was included. The reference was serially diluted in assay buffer (25 mM HEPES pH 7.4, 0.5% Triton X-100, 0.1% Casein, 1 mg/mL Dextran-500, and 0.5% Proclin-300; prepared from a 10-fold stock from Perkin Elmer, Cat. No. AL000F) as described under Example 2.
[0282] 10 μL per reference dilution or PIZV sample were added per well into a white 96-well plate (1/2 area plate-96, Perkin Elmer, Cat. No. 6002299). In addition, blank wells were included by addition of 10 μL of assay buffer per well to account for background signal. Each reference dilution, as well as the samples and blank controls were evaluated in duplicates. ZAPA was further carried out as described under Example 2.
[0283] ZAPA signal counts in relative fluorescence units (RFU) from the reference dilutions, as well as from the PIZV samples were normalized to the medium background signal resulting from the two blank wells. The data from the reference material were independently fitted with a four parameter logistic (4PL) regression model. Corresponding ZAPA signal counts for a certain amount of intact DS within the PIZV reference dilutions were interpolated to the ZAPA signals from PIZV samples and thereby resulting ZAPA values were reported as antigen units per 100 μL (AU/100 μL) for the corresponding PIZV samples (
[0284] ZAPA values for the dilution series of each DS batch followed a linear response, demonstrating robust performance of the assay as the ZAPA value linearly increases with epitope amount (
[0285] To link these in vitro ZAPA results indicative for antigenicity to immunogenicity and potency, CD-1 mice were vaccinated with corresponding PIZV samples resulting from the different DS batches. Therefore, for each of the PIZV samples eight mice including four male and four female mice were vaccinated by the intramuscular route with each one dose (volume of 100 μL) of PIZV sample. Neutralizing Ab titers after immunization were determined with a reporter virus particle (RVP) assay. Therefore, serum samples from mice, as well as a negative control lacking anti-ZIKV Abs (Innovative Research, Cat. No. IGRS-SER) and a positive control (Takeda) were heat-inactivated in a water bath at 56±2° C. for 30±2 min. After that, samples as well as negative and positive controls were serially diluted in assay media (1×Opti-MEM, Gibco, Cat. No. 11058-021, supplemented with 10% (v/v) FBS (Sigma, F4135) and 1% (v/v) Pen/Strep (100-fold stock, Gibco, 15140-122). 7.5 μL per dilution were added into one well of a white 384-well plate (Corning, Cat. No. 3570). ZIKV RVP particles (including C, E, prM, and M proteins; Integral Molecular) were diluted in assay media and 7.5 μL of the dilution were added per well into the 384-well plate. Incubation carried out for 60±2 min in a humidified incubator at 37±2° C. and 5% CO.sub.2. Vero cells were cultured as described for the TCID.sub.50 assay under Example 2. Cells were trypsinized, harvested, and resuspended in assay media prior to counting. 4625 cells in 15 μL assay media were added per well. Incubation carried out for 72±2 hours in a humidified incubator at 37±2° C. and 5% CO.sub.2. Next, Renilla-Glo substrate (Promega, Cat. No. E2750) was diluted 1:100 in buffer according to the manufactures protocol. 30 μL of substrate dilution were added per well and incubation carried out for 15±2 min in the dark. Finally, the plate was analyzed with an Enspire reader (Perkin Elmer) and the half maximal effective concentration (EC.sub.50) titer of neutralizing Abs is determined by regression of the recorded luminescence signal for the different dilutions.
[0286] Of note, as already indicated by the different ZAPA values, neutralizing Ab titers differed for equal antigen doses depending on the DS batch. For instance, for a dose of 0.01 μg DS according to the Bradford assay, neutralizing Ab titers differed with a log.sub.10 RVP value around 3 for DS batch #1 and log.sub.10 RVP values around 2.1 for DS batches #2 to 4 (
[0287] To examine whether ZAPA data correlate with the immunogenicity and potency results from the mouse model, data were analyzed using Prism (GraphPad, Version 8.2.0). Dose response curves (obtained by four parameter logistic (4PL) regression model) were compared using an F-test, examining two models. The first model (model 1) concludes that each agonist (meaning each PIZV dilution series) elicits the same dose response curve, whereas the second model (model 2) concludes that each agonist elicits a different dose response curve. The F ratio quantifies the relationship between the relative increase in the sum of squares from model 2 to model 1 and the relative increase in the degrees of freedom. If model 1 is correct, it is expected to measure an F ratio near 1.0. If the F ratio »1.0 there are two possibilities: model 2 is correct, or model 1 is correct, but random scatter led to a better fit using model 2. The p-value output qualifies how rare this ‘random scatter’ coincidence would be. In the case that the F ratio »1, and the p-value is low (less than α), it is concluded that model 2 is significantly better (more likely to be correct) than model 1. If the p-value is high, it is concluded that there is no compelling evidence supporting model 2 and model 1 is accepted.
[0288] When comparing log.sub.10-transformed AU-values (per 100 μL dose of sample) obtained by the ZAPA with the medium log.sub.10-transformed RVP values within each diluted sample, model 1 is correct according to the F-test (F ratio=1.024, p-value 0.4301), meaning the same dose-response curve can be applied for each agonist (each PIZV dilution series;
[0289] Taken together, these data underline the benefit of the ZAPA for analyzing and characterizing different PIZV batches. Corresponding potency can be reliably predicted using the ZAPA, as assay results correlate well with immunogenicity of analyzed samples. In comparison, even if total antigen amounts as determined by Bradford are equal, epitopes and therefore potency of a sample can vary. ZAPA is a useful tool to account for such variations, as epitopes are reliably determined and the ZAPA signal is a direct indicator for antigenicity and in vivo immunogenicity, and therefore potency of the PIZV samples.
Example 5: Determination of Relative Potency of PIZV Batches
[0290] ZAPA was shown to correlate well with antigenicity and in vivo immunogenicity and therefore potency of the PIZV samples under Example 4. Therefore, ZAPA can be applied to examine the relative potency of any PIZV batch compared to a PIZV reference of which ZAPA values have been correlated with induced neutralizing Ab titers (and therefore potency) as for example in a mouse as described under Example 4.
[0291] For this, two PIZV test samples (#1 and 2) and one PIZV reference (stock concentration: 20 μg/mL) were examined by ZAPA using mAb pair #1 (see Table 5). The samples and the reference were serially diluted in assay buffer (25 mM HEPES pH 7.4, 0.5% Triton X-100, 0.1% Casein, 1 mg/mL Dextran-500, and 0.5% Proclin-300; prepared from a 10-fold stock from Perkin Elmer, Cat. No. AL000F) as described under Example 2.
[0292] 10 μL per reference dilution or PIZV test sample dilution were added per well into a white 96-well plate (1/2 area plate-96, Perkin Elmer, Cat. No. 6002299). In addition, blank wells were included by addition of 10 μL of assay buffer per well to account for background signal. Each dilution or sample, as well as blank was evaluated in duplicates. ZAPA was further carried out as described under Example 2.
[0293] ZAPA signal was analyzed by a four parameter logistic (4PL) regression independently for each dilution series as described under Example 2 (
[0294] In summary, ZAPA can be routinely applied to monitor different PIZV batches by evaluating the relative potency compared to a characterized reference in a fast, efficient, and reliable way.
[0295] 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%, preferably within 5 to 10%. 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.
[0296] Numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.
[0297] 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: “synthetic peptide”, “synthetic nucleotide”.