MULTISPECIFIC ANTIBODIES SPECIFICALLY BINDING TO ZIKA VIRUS EPITOPES AND USES THEREOF
20200317755 ยท 2020-10-08
Inventors
Cpc classification
G01N2333/185
PHYSICS
C07K16/1081
CHEMISTRY; METALLURGY
A61K48/00
HUMAN NECESSITIES
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
C07K2317/33
CHEMISTRY; METALLURGY
G01N2800/52
PHYSICS
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
A61K2039/545
HUMAN NECESSITIES
International classification
Abstract
The invention relates to multispecific antibodies, and antigen binding fragments thereof, that specifically bind to distinct Zika virus epitopes and potently neutralize infection of ZIKV. The invention also relates to nucleic acids that encode such antibodies and antibody fragments. In addition, the invention relates to the use of the antibodies and antibody fragments of the invention in prophylaxis and treatment of ZIKV infection.
Claims
1. An isolated multispecific antibody, or an antigen binding fragment thereof, specifically binding to distinct Zika virus epitopes.
2. The antibody, or the antigen binding fragment thereof, according to claim 1, wherein the antibody, or the antigen binding fragment thereof, neutralizes Zika virus infection.
3.-6. (canceled)
7. The antibody, or the antigen binding fragment thereof, according to claim 1, characterized in that the antibody, or antigen binding fragment thereof, is bispecific, trispecific, tetraspecific or pentaspecific, preferably the antibody, or the antigen binding fragment thereof, is bispecific, trispecific or tetraspecific, more preferably the antibody, or the antigen binding fragment thereof, is bispecific or trispecific, even more preferably the antibody, or the antigen binding fragment thereof, is bispecific.
8. The antibody, or the antigen binding fragment thereof, according to claim 1, characterized in that the antibody, or antigen binding fragment thereof, is bivalent, trivalent, tetravalent, hexavalent or octavalent, more preferably, the antibody, or the antigen binding fragment thereof, is bivalent or tetravalent, most preferably, the antibody, or the antigen binding fragment thereof, is tetravalent.
9.-11. (canceled)
12. The antibody, or the antigen binding fragment thereof, according to claim 1, characterized in that the antibody, or antigen binding fragment thereof, comprises an Fc moiety.
13.-14. (canceled)
15. The antibody, or the antigen binding fragment thereof, according to claim 1, characterized in that the antibody, or antigen binding fragment thereof, is of the Fabs-in-tandem-Ig antibody format, or is of the of the DVD-Ig antibody format.
16.-34. (canceled)
35. The antibody, or the antigen binding fragment thereof, according to claim 1, characterized in that the antibody or antigen binding fragment thereof, comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein at least one CDR, preferably the at least one heavy chain CDRH3, comprises or consists of an amino acid sequence according to any of SEQ ID NOs: 3, 75, 39, 21, 57, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, and 261, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
36.-39. (canceled)
40. The antibody, or the antigen binding fragment thereof, according to claim 1, characterized in that the antibody or antigen binding fragment thereof, comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein (i) the at least one CDRH1 comprises an amino acid sequence according to any of SEQ ID NOs: 1, 19, 37, 55, 73, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, and 259, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (ii) the at least one CDRH2 comprises an amino acid sequence according to any of SEQ ID NOs: 2, 20, 38, 56, 74, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, and 260, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (iii) the at least one CDRH3 comprises an amino acid sequence according to any of SEQ ID NOs: 3, 21, 39, 57, 75, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, and 261, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (iv) the at least one CDRL1 comprises an amino acid sequence according to any of SEQ ID NOs: 4, 22, and 40, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (v) the at least one CDRL2 comprises an amino acid sequence according to any of SEQ ID NOs: 5, 6, 23, 24, 41, and 42, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and/or (vi) the at least one CDRL3 comprises an amino acid sequence according to any of SEQ ID NOs: 7, 25, and 43, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
41.-48. (canceled)
49. The antibody, or the antigen binding fragment thereof, according to claim 1, characterized in that the antibody, or the antigen binding fragment thereof, comprises (a) a first epitope binding site comprising CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences (i) according to SEQ ID NOs: 1-5 and 7; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; or (ii) according to SEQ ID NOs: 1-4 and 6-7; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and (b) a second epitope binding site comprising CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences (i) according to SEQ ID NOs: 19-23 and 25; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; or (ii) according to SEQ ID NOs: 19-22 and 24-25; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
50. (canceled)
51. The antibody, or the antigen binding fragment thereof, according to claim 1, characterized in that the antibody, or the antigen binding fragment thereof, comprises (a) a first epitope binding site comprising CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences (i) according to SEQ ID NOs: 1-5 and 7; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; or (ii) according to SEQ ID NOs: 1-4 and 6-7; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and (b) a second epitope binding site comprising CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences (i) according to SEQ ID NOs: 37-41 and 43; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; or (ii) according to SEQ ID NOs: 37-40 and 42-43; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
52. The antibody, or the antigen binding fragment thereof, according to claim 1, characterized in that the antibody, or the antigen binding fragment thereof, comprises a heavy chain variable region (VH) and, optionally, a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises or consists of an amino acid sequence according to any of SEQ ID NOs: 8, 26, 44, 62, 80, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242, 246, 250, 254, 258, and 262; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
53. The antibody, or the antigen binding fragment thereof, according to claim 1, characterized in that the antibody, or the antigen binding fragment thereof, comprises (i) a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 8 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and/or a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 9 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (ii) a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 26 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and/or a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 27 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (iii) a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 44 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and/or a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 45 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; (iv) a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 62 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and/or a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 63 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; or (v) a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 80 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and/or a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 81 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
54.-55. (canceled)
56. The antibody, or the antigen binding fragment thereof, according to claim 1, characterized in that the antibody, or the antigen binding fragment thereof, comprises (a) a first epitope binding site comprising a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 8 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and/or a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 9 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and (b) a second epitope binding site comprising a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 26 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and/or a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 27 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
57. (canceled)
58. The antibody, or the antigen binding fragment thereof, according to claim 1, characterized in that the antibody, or the antigen binding fragment thereof, comprises (a) a first epitope binding site comprising a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 8 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and/or a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 9 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and (b) a second epitope binding site comprising a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 44 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and/or a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 45 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
59. The antibody, or the antigen binding fragment thereof, according to claim 1, characterized in that the antibody, or the antigen binding fragment thereof, is in the Fabs-in-tandem-Ig (FIT-Ig) format and an outer Fab of the FIT-Ig format comprises an epitope binding site comprising CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences (i) according to SEQ ID NOs: 1-5 and 7; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; or (ii) according to SEQ ID NOs: 1-4 and 6-7; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
60. The antibody, or the antigen binding fragment thereof, according to claim 59, characterized in that the outer Fab of the FIT-Ig format comprises an epitope binding site comprising a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 8 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and/or a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 9 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
61. The antibody, or the antigen binding fragment thereof, according to claim 59, characterized in that the inner Fab of the FIT-Ig format comprises an epitope binding site comprising CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences (i) according to SEQ ID NOs: 19-23 and 25; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; or (ii) according to SEQ ID NOs: 19-22 and 24-25; or functional sequence variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
62. The antibody, or the antigen binding fragment thereof, according to claim 61, characterized in that the inner Fab of the FIT-Ig format comprises an epitope binding site comprising a heavy chain variable region (VH) amino acid sequence according to SEQ ID NO: 26 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and/or a light chain variable region (VL) amino acid sequence according to SEQ ID NO: 27 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
63.-65. (canceled)
66. A nucleic acid molecule comprising at least one polynucleotide encoding the antibody, or the antigen binding fragment thereof, according to claim 1, or a fragment thereof, wherein the fragment comprises at least one CDR of the antibody, or the antigen binding fragment thereof.
67.-70. (canceled)
71. The nucleic acid molecule according claim 66, wherein the polynucleotide sequence comprises or consists of a nucleic acid sequence according to any one of SEQ ID NOs: 10-18, 28-36, 46-54, 64-72, and 82-90; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
72. A vector comprising the nucleic acid molecule according to claim 66.
73.-75. (canceled)
76. A cell expressing the antibody, or the antigen binding fragment thereof, according to claim 1.
77. A pharmaceutical composition comprising the antibody, or the antigen binding fragment thereof, according to claim 1.
78.-87. (canceled)
88. A method for monitoring the quality of an anti-Zika vaccine, the method comprising using the antibody, or the antigen binding fragment thereof, according to claim 1 to check that an antigen of said vaccine contains one or more distinct Zika virus epitope in a correct conformation.
89. A method for diagnosis of a Zika virus infection in a subject, the method comprising contacting the antibody, or the antigen binding fragment thereof, according to claim 1 with a sample from the subject and detecting an antigen/antibody complex.
90. A kit of parts comprising at least one antibody, or the antigen binding fragment thereof, according to claim 1, and at least one means for administration of the antibody or the antigen binding fragment thereof
91. A method of preventing and/or treating a Zika virus infection in a subject, wherein the method comprises administering to a subject in need thereof the antibody, or the antigen binding fragment thereof, according to claim 1.
92.-93. (canceled)
94. A method of preventing and/or treating a Zika virus infection in a subject, wherein the method comprises administering to a subject in need thereof the nucleic acid molecule according to claim 66.
Description
DESCRIPTION OF FIGURES
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EXAMPLES
[0363] Exemplary embodiments of the present invention are provided in the following examples. The following examples are presented only by way of illustration and to assist one of ordinary skill in using the invention. The examples are not intended in any way to otherwise limit the scope of the invention.
Example 1
Isolation of ZIKV-Specific Antibodies and Production of Monoclonal Antibodies
[0364] IgG+ memory B cells were isolated from cryopreserved peripheral blood mononuclear cells (PBMCs) of four ZIKV-infected donors (ZKA, ZKB, ZKC and ZKD) using CD22 microbeads (Miltenyi Biotec), followed by depletion of cells carrying IgM, IgD and IgA by cell sorting. Memory B cells from the ZIKV-infected donors were then immortalized with EBV (Epstein Barr Virus) and CpG (CpG oligodeoxynucleotide 2006) in multiple replicate wells as previously described (Traggiai, E. et al., Nat. Med. 10, 871-875, 2004) and culture supernatants were then tested in a primary screening using in parallel a 384-well based micro-neutralization assay and a binding assay (ELISA) to test their binding to ZIKV NS1 protein or to ZIKV E protein. Results of the binding assay (binding to ZIKV E protein) are shown in
[0365] Neutralization assays were undertaken on Vero cells. In a 384-well plate, ZIKV H/PF/2013 that resulted in an infection rate (m.o.i, multiplicity of infection) of 0.35 was incubated with superntanants for 1 h at 37% (5% CO2) before the addition to pre-seeded 5,000 Vero cells. These were incubated for a further 5 days, after which supernatant was removed and WST-1 reagent (Roche) was added. Positive cultures were collected and expanded. From positive cultures the VH and VL sequences were retrieved by RT-PCR. Antibodies were cloned into human IgG1 and Ig kappa or Ig lambda expression vectors (kindly provided by Michel Nussenzweig, Rockefeller University, N.Y., US) essentially as described (Tiller T, Meffre E, Yurasov S, Tsuiji M, Nussenzweig M C, Wardemann H (2008) Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J Immunol Methods 329: 112-124). Monoclonal antibodies were produced from EBV-immortalized B cells or by transient transfection of 293 Freestyle cells (Invitrogen). Supernatants from B cells or transfected cells were collected and IgG were affinity purified by Protein A or Protein G chromatography (GE Healthcare) and desalted against PBS.
[0366]
Example 2
Characterization of Antibodies ZKA190, ZKA185, ZKA230, ZKA64 and ZKA78
[0367] In Example 1, a large number of ZIKV-neutralizing antibodies were identified and characterized for their specificity to ZIKV, in particular ZIKV E protein and ZIKV EDIII as well as for their cross-reactivity towards DENY. Antibodies ZKA190 (SEQ ID NOs: 1-18), ZKA185 (SEQ ID NOs: 19-36), ZKA230 (SEQ ID NOs: 37-54), ZKA64 (SEQ ID NOs: 73-90) and ZKA 78 (SEQ ID NOs: 55-72) described in Example 1 were then selected and further tested against ZIKV E protein (ZIKV), ZIKV EDIII (DIIIZI) and also tested against the E protein of dengue virus (DENY, serotype number 1) by ELISA. To this end, a standard ELISA was used. Briefly, ELISA plates were coated with ZIKV E protein at 1 or 3 g/ml, blocked with 10% FCS in PBS, incubated with sera or human antibodies and washed. Bound antibodies were detected by incubation with AP-conjugated goat anti-human IgG (Southern Biotech). Plates were then washed, substrate (p-NPP, Sigma) was added and plates were read at 405 nm. The relative affinities of monoclonal antibody binding were determined by measuring the concentration of antibody (EC5o) required to achieve 50% maximal binding at saturation.
[0368] Results are shown in
[0369] Moreover, none of ZKA190, ZKA185, ZKA230, and ZKA64 showed any detectable binding to DENV E proteins (
[0370] To further confirm those results, the ZIKV E protein binding antibodies ZKA190, ZKA64 and ZKA78 were additionally tested against E protein of dengue virus (DENV, serotypes number 1-4). ZKA64 and ZKA190 did not bind to DENV1-4 E protein, thereby confirming that ZKA64 and ZKA190 are specific for ZIKV. ZKA78, in contrast, bound to DENV1-4 E, confirming that ZKA78 is a cross-reactive antibody binding to the E protein of both ZIKV and DENV (cf.
Example 3
The Isolated Antibodies Potently Neutralize ZIKV Infection
[0371] The isolated antibodies ZKA190, ZKA185, ZKA230, ZKA64 and ZKA78 were tested for their ability to neutralize ZIKV and DENV1 infection in vitro.
[0372] Neutralization of DENV and ZIKV infection by antibodies was measured using a micro-neutralization flow cytometry-based assay. Different dilutions of antibodies were mixed with ZIKV (MOI of 0.35) or attenuated DENV1 (all at MOI of 0.04) for 1 hour at 37 C. and added to 5000 Vero cells/well in 96-well flat-bottom plates. After four days for ZIKV and five days for DENV, the cells were fixed with 2% formaldehyde, permeabilized in PBS 1% FCS 0.5% saponin, and stained with the mouse mAb 4G2. The cells were incubated with a goat anti-mouse IgG conjugated to Alexa Fluor488 (Jackson Immuno-Research, 115485164) and analyzed by flow cytometry. In other cases the ZIKV neutralization data are also determined measuring cell viability using the WST-1 reagent (Roche). The neutralization titer (50% inhibitory concentration [IC50]) was expressed as the antibody concentration that reduced the infection by 50% compared to cell-only control wells.
[0373] Results are shown in
[0374] It is important to note that the ultra-potent ZKA64 and ZKA190 antibodies in addition to their ability to neutralize the ZIKV H/PH/2013 strain (present example), also bound to the E protein and EDIII derived from the ZIKV strains MR766 and SPH2015, respectively (
Example 4
The LALA Mutation Inhibits Antibody-Dependent Enhancement of ZIKV Infection by Serum Antibodies
[0375] Neutralizing antibodies were also tested for their ability to enhance the infection of ZIKV in the non-permissive K562 cells (antibody-dependent enhancement assay, ADE assay). ADE was measured by a flow based assay using K562 cells. Antibodies and ZIKV H/PF/2013 (MOI 0.175) were mixed for 1 hour at 37 C. and added to 5000 K562 cells/well. After four days, cells were fixed, permeabilized, and stained with m4G2. The number of infected cells was determined by flow cytometry.
[0376] Results are shown in
[0377] In view thereof it was investigated whether ADE could be also induced by immune sera and whether this could be blocked by neutralizing antibodies delivered as a LALA variant. To obtain the LALA variant, each of the heavy chains was mutated at amino acids 4 and 5 of CH2 domain by substituting an alanine in place of the natural leucine using site-directed mutagenesis. As described above, LALA variants (of human IgG1 antibodies) do not bind to Fc-gamma-receptors and complement.
[0378] To investigate the effect of ZKA64-LALA antibody in ZIKV ADE, an inhibition of ADE assay was used. Since ADE of ZIKV is observed using ZIKV- or DENV-immune plasma, ZIKV (MOI 0.175) was mixed with plasma from primary ZIKV- or DENV-infected donors for 30 minutes at 37 C. ZKA64-LALA antibody was added at 50 g/ml, mixed with 5000 K562 cells/well and incubated for three days. Cells were then stained with 4G2 and analyzed by flow cytometry.
[0379] Results are shown in
[0380] Of note, the ADE effect of ZIKV- and DENV-immune plasma was completely blocked by the EDIII-specific ZKA64-LALA antibody. The ADE blocking ability of a single EDIII-specific LALA antibody could be related not only to its capacity to out-compete serum enhancing antibodies but also to neutralize virus once internalized into endosomes.
[0381] These results indicate that a potently neutralizing antibody, such as ZKA190, ZKA230, ZKA185 or ZKA64, developed in the LALA form, have a strong potential to be used in prophylactic or therapeutic settings to prevent congenital ZIKV infection, e.g. in pregnant women and/or in people living in high risk areas. The use of the LALA form avoids the risk of ZIKV ADE and, as shown above, could also block ADE of pre-existing cross-reactive antibodies, such as in the case of patients already immune to DENY.
Example 5
ZKA190 Neutralizes ZIKV More Potently than Prior Art Antibody EDE1 mAb C8
[0382] To compare the isolated neutralizing antibodies with highly neutralizing anti-ZIKV antibodies of the prior art, neutralization performance of ZKA190 was compared to that of prior art highly neutralizing mAb EDE1 CS (Barba-Spaeth G, Dejnirattisai W, Rouvinski A, Vaney M C, Medits I, Sharma A, Simon-Loriere E, Sakuntabhai A, Cao-Lormeau V M, Haouz A, England P, Stiasny K, Mongkolsapaya J, Heinz F X, Screaton G R, Rey F A. Structural basis of potent Zika-dengue virus antibody cross-neutralization. Nature. 2016 Aug. 4; 536(7614):48-53). Neutralization of both antibodies was tested against a panel of four distinct ZIKV strains (H/PF/2013; MR766, MRS-OPY and PV 10552).
[0383] Briefly, neutralization of ZIKV infection by mAbs was measured using a micro-neutralization flow cytometry-based assay. Different dilutions of mAbs were mixed with ZIKV (MOI of 0.35) for 1 hour at 37 C. and added to 5000 Vero cells/well in 96-well flat-bottom plates. After four days for ZIKV, the cells were fixed with 2% formaldehyde, permeabilized in PBS containing 1% fetal calf serum (Hyclone) and 0.5% saponin, and stained with the mouse mAb 4G2. The cells were incubated with a goat anti-mouse IgG conjugated to Alexa Fluor488 (Jackson Immuno- Research, 115485164) and analyzed by flow cytometry. The neutralization titer (50% inhibitory concentration [IC50]) is expressed as the antibody concentration that reduced the infection by 50% compared to virus-only control wells.
[0384] Results are shown in
Example 6
Further Characterization of Antibody ZKA190
[0385] The potency of antibody ZKA190 was further investigated in vitro and in vivo. To this end, the mAb was synthesized in IgG1 wild-type (wt) format and in an IgG1 Fc-LALA format. Briefly, the VH and VL sequences were cloned into human Ig1, Ig and Ig expression vectors (kindly provided by Michel Nussenzweig, Rockefeller University, New York, N.Y., USA), essentially as described (Tiller T, Meffre E, Yurasov S, Tsuiji M, Nussenzweig M C, Wardemann H: Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J Immunol Methods 2008, 329:112-124). Recombinant mAbs were produced by transient transfection of EXPI293 cells (Invitrogen), purified by Protein A chromatography (GE Healthcare) and desalted against PBS. To obtain the LALA variant, each of the heavy chains was mutated at amino acids 4 and 5 of CH2 domain by substituting an alanine in place of the natural leucine using site-directed mutagenesis. As described above, LALA variants (of human IgG1 antibodies) do not bind to Fc-gamma-receptors and complement.
[0386] As shown in
[0387] Since ZIKV has been shown to infect human neural progenitor cells (hNPC) leading to heightened cell toxicity, dysregulation of cell-cycle and reduced cell growth, ZKA190 and ZKA190-LALA were tested in hNPCs. To this end, adult male fibroblasts obtained from the Movement Disorders Bio-Bank (Neurogenetics Unit of the Neurological Institute Carlo Besta, Milan) were reprogrammed using the CytoTune-iPS 2.0 Sendai kit (Life Technologies). hiPSCs were maintained in feeder-free conditions in mTeSR1 (Stem Cell Technologies). To generate embryoid bodies (EBs), dissociated hiPSCs were plated into low adhesion plates in mTeSR1 supplemented with N2 (0.5) (ThermoFisher Scientific), human Noggin (0.5 mg/ml, R&D System), SB431542 (5 M, Sigma), Y27632 (10 M, Miltenyi Biotec) and penicillin/streptomycin (1%, Sigma) (as described in Marchetto M C N, Carromeu C, Acab A, Yu D, Yeo G W, Mu Y, Chen G, Gage F H, Muotri A R: A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell 2010, 143:527-539). To obtain rosettes, EBs were plated after 10 days onto matrigel-coated plates (1:100, matrigel growth factor reduced, Corning) in DMEM/F12 (Sigma) with N2 (1:100), non-essential amino acids (1%, ThermoFisher Scientific) and penicillin/streptomycin. After 10 days, cells were passaged with Accutase (Sigma) and seeded onto matrigel coated-flasks in NPC media containing DMEM/F12, N2 (0.25%), B27 (0.5%, ThermoFisher Scientific), penicillin/streptomycin and FGF2 (20 ng/ml, ThermoFisher Scientific). hNPCs (3104) were plated on coverslips in 24-well plates 3 days prior to infection with PRVABC59 strain. Virus stock was incubated with the mAbs 1 h prior to addition to hNPCs to obtain an MOI of 0.5. After 4 h of virus adsorption, culture supernatant was removed and fresh medium containing the mAbs was re-added. Supernatant was collected 96 h post-infection to measure virus titers by plaque assay on Vero cells. Cells were fixed in 4% paraformaldehyde (PFA, Sigma) solution in phosphate-buffered saline (PBS, Euroclone) for 30 min for indirect immunofluorescence. Fixed cells were permeabilized for 30 minutes (min) in blocking solution, containing 0.2% Triton X-100 (Sigma) and 10% donkey serum (Sigma), and incubated overnight at 4 C. with the primary antibodies in blocking solution. The following antibody was used for detection: anti-envelope (1:200, Millipore, MAB10216). Then, cells were washed with PBS and incubated for 1 h with Hoechst and anti-mouse Alexa Fluor-488 secondary antibodies (1:1,000 in blocking solution, ThermoFisher Scientific). After PBS washes, cells were washed again and mounted. Results are shown in
[0388] Next, the ability of ZKA190 and ZKA190-LALA to cause ADE was tested in the K562 cell line as described in Example 4. Briefly, ADE was measured by a flow based assay using K562 cells. Briefly, for ZKA190, ZKA190 and ZIKV H/PF/2013 (MOI 0.175) were mixed for 1 hour at 37 C. and added to 5000 K562 cells/well. After four days, cells were fixed, permeabilized, and stained with mAb m4G2. The number of infected cells was determined by flow cytometry. For ZKA190-LALA, ZIKV (MOI 0.175) was mixed with plasma from primary ZIKV-infected donors for 30 minutes at 37 C. ZKA190-LALA was added at 50 E g/ml, mixed with 5000 K562 cells/well and incubated for three days. Cells were then stained with 4G2 and analyzed by flow cytometry. Results are shown in
[0389] Anti-prM antibodies form part of the predominant antibodies elicited during the human immune response against flaviviruses and have been shown to enhance virus infection in vitro (Dejnirattisai, W., Jumnainsong, A., Onsirisakul, N., Fitton, P., Vasanawathana, S., Limpitikul, W., Puttikhunt, C., Edwards, C., Duangchinda, T., Supasa, S., et al. (2010). Cross-reacting antibodies enhance dengue virus infection in humans. Science 328, 745-748). K562 cells were pre-incubated with serial dilutions of prM cross-reactive antibody DV62 (Beltramello, M., Williams, K. L., Simmons, C. P., Macagno, A., Simonelli, L., Quyen, N. T. H., Sukupolvi-Petty, S., Navarro-Sanchez, E., Young, P. R., de Silva, A. M., et al. (2010). The human immune response to Dengue virus is dominated by highly cross-reactive antibodies endowed with neutralizing and enhancing activity. Cell Host Microbe 8, 271-283) derived from a DENV immune donor. Results are shown in
[0390] Finally, the ability of different concentrations of ZKA190, ZKA190-LALA and ZKA190 Fab to cause or block ADE of ZIKV in the presence of enhancing concentrations of human anti-DENV2 plasma or DV62 was tested. Results are shown in
Example 7
ZKA190 Binds to a Conserved and Highly Accessible Region of EDIII
[0391] To determine the ZKA190 epitope at the residue level, solution NMR spectroscopy was used as described in Bardelli, M., Livoti, E., Simonelli, L., Pedotti, M., Moraes, A., Valente, A. P., and Varani, L. (2015). Epitope mapping by solution NMR spectroscopy. J. Mol. Recognit. 28, 393-400; Simonelli, L., Beltramello, M., Yudina, Z., Macagno, A., Calzolai, L., and Varani, L. (2010). Rapid structural characterization of human antibody-antigen complexes through experimentally validated computational docking. J Mol Biol 396, 1491-1507; and Simonelli, L., Pedotti, M., Beltramello, M., Livoti, E., Calzolai, L., Sallusto, F., Lanzavecchia, A., and Varani, L. (2013). Rational Engineering of a Human Anti-Dengue Antibody through Experimentally Validated Computational Docking. PLoS ONE 8, e55561.
[0392] Briefly, spectra were recorded on a Bruker Avance 700 MHz NMR spectrometer at 300 K. For assignments of backbone resonances standard triple resonance experiments (HNCO, HN(CA)CO, HN(CO)CACB, HNCACB were used, while sidechains were annotated using HCCH-TOCSY and HBHA(CO)NH experiments. All NMR experiments were processed using Topspin 2.1 (Bruker Biospin) and analysed with CARA. NOESY cross peaks were automatically assigned using the CYANA noeassign macro based on the manually assigned chemical shifts. Upper-distance restraints used for the structure calculations in CYANA using the standard simulated annealing protocol were derived from 70 ms .sup.15N- and .sup.13C-resolved NOESY spectra. Backbone dynamics of ZIKV EDIII were derived from .sup.15N relaxation measurements recorded on 600 and 700 MHz spectrometers. Proton-detected versions of the CPMG (R2), inversion-recovery (R1) and .sup.15N{.sup.1H}-steady-state NOE were utilized. Delay settings for the T2 series were in the range of 0 to 0.25 sec and for the T1 series between 0.02 to 2 sec. The .sup.15N{.sup.1H}-NOE experiment used a relaxation delay of 5 s. The R1 and R2 relaxation rates were derived from least-squares fits of corresponding exponential functions to the measured data using home-written scripts. The relaxation data were analyzed in a model-free approach using the software package DYNAMICS. The program ROTDIF was used to calculate the overall correlation time from the relaxation data (8.5 ns). NMR epitope mapping was performed as previously described (Bardelli et al., 2015; Simonelli et al., 2010; 2013). Briefly, overlay of .sup.15NHSQC spectra of labelled EDIII free or bound to ZKA190 Fab allowed identification of EDIII residues whose NMR signal changed upon complex formation, indicating that they were affected by ZKA190 binding. Changes were identified by manual inspection and by the Chemical Shift Perturbation (CSP), CSP=((.sub.H).sup.2+(.sub.N/10).sup.2).sup.1/2. NMR samples were typically 800 M of [.sup.15N, .sup.13C]-labeled EDIII in 20 mM sodium phosphate, 50 mM NaCl, pH 6.0. Perdeuterated (nominally 70%) .sup.2H,.sup.15N EDIII samples were used for NMR epitope mapping with a EDIII:ZKA190 Fab ratio of 1:1.1; EDIII concentration was typically 0.4 mM.
[0393] Since the NMR signal is strongly dependent on the local chemical environment, changes upon complex formation identify antigen residues that are affected by antibody binding, either directly or through allosteric effects. By comparing the NMR spectra of free and bound EDIII (
[0394] Computational docking followed by molecular dynamics simulation, guided and validated by NMR-derived epitope information as well as EDIII mutagenesis, showed that ZKA190 binds through an interface characterized by shape and charge complementarity (
Example 8
Mechanisms of ZKA190 Neutralization
[0395] The ability of ZKA190 to efficiently neutralize the virus may involve inhibition of either cell attachment or membrane fusion. A further mechanism might involve virus inactivation through cross-linking of viral particles.
[0396] ZKA190 Fab can neutralize ZIKV, albeit less efficiently than the corresponding IgG. By binding to the EDI-EDIII linker, ZKA190 (both Fab and IgG) might inhibit the 70 degree rotation of DIII required for viral fusion to the host cell membrane (Bressanelli, S., Stiasny, K., Allison, S. L., Stura, E. A., Duquerroy, S., Lescar, J., Heinz, F. X., and Rey, F. A. (2004). Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation. Embo J 23, 728-738; Modis, Y., Ogata, S., Clements, D., and Harrison, S. C. (2004). Structure of the dengue virus envelope protein after membrane fusion. Nature 427, 313-319). Alternatively, ZKA190 might prevent the attachment of ZIKV to target cells.
[0397] The ability of ZKA190 to inhibit membrane fusion is supported by confocal microscopy analysis. To this end, Vero cells were plated at 7,500 cells/well on 12 mm-diameter coverslips in 24-well plates and incubated overnight. Cells were infected with ZIKV H/PF/2013 (MOI of 100) in the presence or absence of neutralizing concentrations of Alexa-488 conjugated mAbs (0.7 M) at 37 C. for 3 h, washed with PBS, and fixed with 2% paraformaldehyde in PBS for 30 min at room temperature. Acidified endosome were identified with Lysotracker red (Invitrogen) by adding the dye (50 nM) to the cells for the last 30 min of the incubation prior to fixation. Fixation was followed by extensive washes in PBS and 50 mM glycine and finally the coverslips were prepared for microscopy analysis using Vectashield mounting medium for fluorescence with DAPI (Vector Laboratories). Samples were analyzed by confocal microscopy using a Leica TCS SP5 microscope with a 63/1.4 N.A. objective. Image analysis and processing was performed with FIJI software.
[0398] Results are shown in
Example 9
In Vivo Characterization of the EDIII-Specific mAb ZKA190
[0399] To evaluate their prophylactic and therapeutic properties, ZKA190 and ZKA190-LALA were tested in A129 mice challenged with a lethal dose of ZIKV strain MP1751 (African lineage). To test their prophylactic potencies, ZKA190 and ZKA190-LALA were administered one day before virus challenge.
[0400] Female A129 mice (IFN-alpha/beta receptor /) and wild-type 129Sv/Ev mice aged 5-8 weeks were administered mAbs (ZKA190, ZKA190-LALA and control antibody MPEG (Corti, D., et al. Cross-neutralization of four paramyxoviruses by a human monoclonal antibody. Nature 501, 439-443 (2013)) diluted in PBS at different doses via the intraperitoneal (i.p.) route in a volume of 500 l. MAbs were administered either 1 day before or 1, 2, 3 or 4 days after virus challenge. Animals were challenged subcutaneously with 102 pfu ZIKV (strain MP1751) and followed for 14 days. Weights and temperatures were monitored daily and clinical observations were recorded at least twice per day. On day 5 post-challenge, 50 l of blood was collected from each animal into a RNAprotect tube (Qiagen, UK) and frozen at 80 C. At the end of the study (14 days post-challenge) or when animals met human endpoints, necropsies were undertaken, and blood and sections of brain, spleen, liver, kidney and ovary were collected for virological analysis.
[0401] Tissue samples from A129 mice were weighed and homogenized into PBS using ceramic beads and an automated homogenizer (Precellys, UK) using six 5 second cycles of 6500 rpm with a 30 second gap. Two hundred l of tissue homogenate or blood solution was transferred into 600 L RLT buffer (Qiagen, UK) for RNA extraction using the RNeasy Mini extraction kit (Qiagen, UK); samples were passed through a QIAshredder (Qiagen, UK) as an initial step. A ZIKV specific realtime RT-PCR assay was utilized for the detection of viral RNA from subject animals. The primer and probe sequences were adopted from Quick et al., 2017 (Quick, J, Grubaugh N D, Pullan S T, Claro I M, Smith A D, Gangavarapu K, Oliveira G, Robles-Sikisaka R, Rogers T F, Beutler N A, et al.: Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nat Protoc 2017, 12:1261-1276) with in-house optimization and validation performed to provide optimal mastermix and cycling conditions. Real-time RT-PCR was performed using the SuperScript III Platinum One-step qRT-PCR kit (Life Technologies, UK). The final mastermix (15 l) was comprised of 10 l of 2 Reaction Mix, 1.2 l of PCR-grade water, 0.2 l of 50 mM MgSO4, 1 l of each primer (ZIKV 1086 and ZIKV 1162c both at 18 M working concentration), 0.8 l of probe (ZIKV 1107-FAM at 25 M working concentration) and 0.8 l of SSIII enzyme mix. Five l of template RNA was added to the mastermix, yielding a final reaction volume of 20 l. The cycling conditions used were 50 C. for 10 minutes, 95 C. for 2 minutes, followed by 45 cycles of 95 C. for 10 seconds and 60 C. for 40 seconds, plus a final cooling step of 40 C. for 30 seconds. Quantification analysis using fluorescence was performed at the end of each 60 C. step. Reactions were run and analyzed on the 7500 Fast platform (Life Technologies, UK) using 7500 software version 2.0.6. Quantification of viral load in samples was performed using a dilution series of quantified RNA oligonucleotide (Integrated DNA Technologies). The oligonucleotide comprised the 77 bases of ZIKV RNA targeted by the assay, based on GenBank accession AY632535.2 and was synthesized to a scale of 250 nmol with HPLC purification.
[0402] Results are shown in
[0403] To evaluate the therapeutic potential of ZKA190, we administered ZKA190 and ZKA190-LALA at different time-points following ZIKV infection. At a dose of 15 mg/kg, survival rates of 80%-100% were achieved, and the morbidity was greatly reduced even when treatment was given four days post-infection (
Example 10
In Vitro Selection of ZIKV Escape Mutants
[0404] Use of antibody therapeutics may result in the selection of escape mutants. To assess the ability of ZKA190 to select for resistant mutants (MARMs) in vitro, ZIKV (H/PF/2013) was passaged in the presence of sub-neutralizing concentrations of ZKA190.
[0405] Briefly, two-thousands TCID50 of H/PF/2013 ZIKV in 500 l were incubated with 250 l containing varying concentrations of mAb (8 different concentrations, starting with a final concentration of 200 g/ml and performing serial 1:4 dilutions). The mixture was incubated for 45 minutes at 37 C., followed by the addition of 250 l of a Vero cells suspension (3.2106 cells) and an incubation in a 24 well plate for three-four days at 37 C. to allow virus propagation to occur. After each step of selection, 500 l of supernatants from three conditions were selected: the lowest concentration of mAb at which full protection of the monolayer was observed, one concentration at which a partial CPE effect on the cell monolayer was observed and one concentration at which 100% of the cell monolayer was destroyed by the ZIKV CPE. The tube was spun down for 5 minutes at 1000g, aliquoted and stored at 80 C. Half of the volume was again pre-mixed with varying concentrations of mAb to repeat the selection and propagation process. The remaining supernatant was used for micro-neutralization assays and subsequent sequencing of the virus.
[0406] To identify the escape mutations of the selected MARMs virus, a genomic RNA extraction was done followed by a one-step-PCR to amplify and sequence the ZIKV E protein amplicon. Cell supernatant (140 l) from the MARMs selection was used for RNA extraction with the QIAamp Viral RNA mini kit (Qiagen). cDNA synthesis and PCR amplification were performed together using the SuperScript III One-Step RT-PCR with Platinium Taq (Invitrogen). For one reaction 25 l reaction mix, 8 l sterile water, 2 M of each primer, 1 l RNAse out (Life Technologies), 2 l Superscript III RT/Platinum TaqMix and 12 l RNA were used giving a final reaction volume of 50 l. For the E protein N-terminal part, the primer pair Zika-E-F1 5-TGCAAACGCGGTCGCAAACCTGGTTG-3 (SEQ ID NO: 266) and ZIKV-E-R1 5-CGTGCCAAGGTAATGGAATGTCGTG-3 (SEQ ID NO: 267) and for the C-terminal part the primer pair ZIKV-Ef1530 5-AGCCTAGGACTTGATTGTGAACCGA-3 (SEQ ID NO: 268) and ZIKV-E-R2769 5-TTACAGATCCCACAACGACCGTCAG-3 (SEQ ID NO: 269) were used. The cycling conditions were 54 C. for 40 minutes, 94 C. for 2 min followed by 45 cycles of 94 C. for 45 seconds, 50 C. for 45 seconds and 68 C. for 1.5 minutes with a final elongation step at 68 C. for 5 minutes and a final cooling step at 4 C. The PCR products were analyzed and extracted from a 1.5% agarose gel and further purified with the GFX PCR DNA and Gel Band Purification kit (GE Healthcare). For the sequencing reaction 8 l of purified PCR product was mixed with 2 M primer in a final volume of 10 l and sent for sequencing (Microsynth). E protein N-terminal products were sequenced with ZIKV-E-F2 5-ACTTGGTCATGATACTGCTGATTGC-3 (SEQ ID NO: 270) and ZIKV-E-R2 5-TCGGTTCACAATCAAGTCCTAGGCT-3 (SEQ ID NO: 271), C-terminal PCR products with ZIKV-E-f2058 5-GCTAACCCCGTAATCACTGAAAGCA-3 (SEQ ID NO: 272) and ZIKV-E-r2248 5-AAGACTGCCATTCTCTTGGCACCTC-3 (SEQ ID NO: 273). Sequences were assembled and analyzed using CLC Main Workbench software (CLC Bio, version 5).
[0407] Resistant mutant MARM2 was isolated after three rounds of selection, and its E protein showed a E370K mutation in DIII. The mutation abolished neutralization by ZKA190, although the antibody can bind to the mutated DIII (
Example 11
Development of Bispecific Antibodies According to the Present Invention
[0408] Viral escape mutants can greatly hinder the efficacy of therapeutic antibodies. To overcome this problem, the present inventors hypothesized that the possibility of virus escaping would be greatly reduced when combining two highly neutralizing antibodies. In view thereof, a series of bispecific antibodies combining ZKA190 with other potently neutralizing mAbs directed towards distinct sites on the E protein was generated. Thereby, it was focused on two mAbs, ZKA185 and ZKA230, that are highly neutralizing and do not compete with ZKA190.
[0409] Firstly, their ability to cross-neutralize four ZIKV strains was analyzed as described above. ZKA185, and to a lesser extent ZKA230, potently neutralized African, Asian and American strains with an IC50 ranging from 0.02 to 0.62 nM (
[0410] To identify the ZKA185 and ZKA230 epitopes and also their propensity to generate escape mutants, MARMs against ZKA185 (MARM3) and ZKA230 (MARM4) were isolated by passaging virus in the presence of sub-neutralizing antibody concentrations as described above. MARM3 contained substitutions at K84E and D67H, which are both located on DII (
[0411] To gain insight into the development of MARMs capable of escaping from the pressure of multiple antibodies, ZKA190 MARM2 (E370K) were serially passaged in the presence of ZKA185 or ZK230. Thereby, it was found that double MARMs emerged after 3 to 4 passages. ZKA230 introduced an extra K84E mutation while ZKA185, selected for a D76G mutation. These findings indicate that ZIKV can escape the neutralization by multiple antibodies targeting distinct sites when the selection is performed in a stepwise fashion, and confirmed a high plasticity of the ZIKV E protein.
[0412] In conclusion, ZKA185 was selected to be used together with ZKA190 for the development of a bispecific antibody since it potently cross-neutralizes ZIKV strains, binds to an alternative site, and does not compete with ZKA190. The bispecific antibody was produced in a tetravalent symmetric format called Fabs-in-tandem-Ig (FIT-Ig). FIT-Igs are described in detail, for example, in WO 2015/103072 A1 and in Gong S, Ren F, Wu D, Wu X, Wu C: Fabs-in-tandem immunoglobulin is a novel and versatile bispecific design for engaging multiple therapeutic targets. MAbs 2017.
[0413] FIT-Ig may be produced using three polypeptides. Polypeptide 1 usually comprises the light chain of the outer Fab fused, preferably without linkers, to the N-terminal region of the inner Fab heavy chain. Polypeptide 2 usually comprises the heavy chain variable and CH1 regions of the outer Fab, and polypeptide 3 usually comprises by the light chain of the inner Fab. Accordingly, an antibody of the FIT-Ig format usually comprises an inner Fab and an outer Fab. Two types of FIT-Igs were generated with ZKA190 Fab either in the outer or inner position. Briefly, the three genes encoding for FIT-Ig were codon optimized, synthesized by Genscript and cloned as follows: i) the VL of the outer Fab, followed by the full constant region (lambda or kappa), is fused with the VH of the inner and cloned into the Ig1 expression vector (modified to encode for the LALA mutation). The resulting polypeptide 1 is formed by VL and CL of the outer Fab, VH of the inner Fab fused to IgG1 CH1-hinge-CH2-CH3 domains; ii) the VH gene of the outer Fab (encoding for polypeptide 2 formed by VH and CH1 of the outer Fab) was cloned into the Fab expression vector (Ig1 expression vector in which a stop codon is introduced after the codon encoding for the CH1 cysteine residue 220); iii) the VL gene of the inner Fab is cloned into the Ig or Ig expression vectors (encoding for polypeptide 3 formed by VL and CL of the inner Fab). Recombinant FIT-Ig mAbs were produced by transient transfection of EXPI293 cells (Invitrogen) using a molar ratio of 1:3:3 of the three constructs described above (as described in WO 2015/103072 A1), purified by Protein A chromatography (GE Healthcare) and desalted against PBS. The proteins were analyzed by SDS-PAGE in both reduced and non-reduced conditions and their concentrations determined by BCA (Pierce, Rockford, Ill.). In non-reduced conditions, FIT-Ig migrated as a major single band of approximately 250 KDa. In reducing conditions, each of the FIT-Ig proteins yielded two bands, one higher MW band is polypeptide 1 of approximately 75 KDa, and one lower MW band corresponds to both polypeptide 2 and 3 overlapped at approximately 25 KDa. To further study the physical properties of FIT-Ig in solution, size exclusion chromatography (SEC) was used to analyze each protein. Purified FIT-Ig, in PBS, was applied on a Superdex 200 Increase 5/150 GL. All proteins were determined using UV detection at 280 nm and 214 nm. FIT-Ig proteins exhibited a single major peak, demonstrating physical homogeneity as monomeric proteins.
Example 12
In Vitro Characterization of an Antibody According to the Present Invention (FIT-1)
[0414] The FIT-Ig bispecific antibody (here designated FIT-1) with ZKA190 in the outer and ZKA185 in the inner Fab positions (
[0415] Next, the ability of FIT-1 to select MARMs was tested (as described above). However, despite eight rounds of serial passages, no MARMs could be isolated. By contrast, MARMs appeared after 3 to 4 passages when individual mAbs were used. These results suggest that the use of FIT-1 as a therapeutic is safer, since simultaneous mutations in both DIII and DII are less likely to occur.
[0416] Confocal microscopy studies using Vero cells also showed that FIT-1, as ZKA190, likely inhibits virus infection at a post-attachment step, likely fusion (
[0417] Finally, FIT-1, as well as its Fab fragment, blocked ADE of human anti-DENV2 plasma or DV62 at concentrations above 0.1 nM and 10 nM, respectively (
Example 13
In Vivo Therapeutic Potential of FIT-1
[0418] To evaluate the therapeutic potential of FIT-1, three different doses (15, 5 and 1 mg/kg) were administered at three different time-points to female A129 mice following ZIKV infection. Briefly, female A129 mice were assigned to 10 distinct groups (control and three different doses with three different administration time points for each dose). All animals were challenged subcutaneously with 102 pfu ZIKV (strain MP17.51) and followed for 14 days. Mice of the nine treatment groups were administered with bispecific antibody FIT-1 diluted in PBS at 15, 5 or 1 mg/kg via the intraperitoneal (i.p.) route in a volume of 500 l on day 1, day 2, or day 3 after ZIKV virus challenge. Weights and temperatures of all animals were monitored daily and clinical observations were recorded at least twice per day. On day 5 post-challenge, 50 l of blood was collected from each animal into a RNAprotect tube (Qiagen,UK) and frozen at 80 C. At the end of the study (14 days post-challenge) or when animals met human endpoints, necropsies were undertaken, and blood and sections of brain and ovary were collected for virological analysis.
[0419] Results are shown in
Example 14
Effect of FIT-1 on Disease in a Congenital Infection Model in AG129 Mice
[0420] The effect of treatment of infected dams with FIT-1 on the pups born to the infected and treated dams was assessed in a mouse model of congenital infection with ZIKV, which results in live offspring after congenital exposure with ZIKV. In this model, AG-129 mice, deficient in IFN receptors, are used, which results in viral replication in the placenta, transmission of virus to the fetus, and potential long-term implications such as intrauterine growth restriction and hearing deficits.
[0421] The effect of FIT-1 treatment on various parameters in the context of intrauterine infection was tested. To this end, mice were infected with ZIKV 7 days post-coitus (dpc) and treated with FIT-1 1 3 days after virus challenge (cf Example 13). Various outcomes of disease, such as virus titer in the fetus and placenta and intrauterine growth restriction, were assessed to determine the effect of FIT-1 treatment.
[0422] Materials and Methods:
[0423] Animals: 42 female AG-129 mice were used. Groups of animals were randomly assigned to experimental groups and individually marked with ear tags. Hormonal treatment was used to induce estrous in females. Females were individually placed with males and examined for a vaginal plug 0.5 days post-coitus (dpc).
[0424] Virus: Zika virus Malaysia (P6-740) was used. A suitable challenge dose was administered via s.c. injection in a volume of 0.1 ml.
[0425] Test agent: FIT-1 was administered at a dose of 45 mg/kg. The non-specific control antibody MPEG-LALA Ctr IgG1 was used as an isotype control placebo treatment.
[0426] Quantification of virus: The virus titer of various tissues was quantified using a quantitative RT-PCR assay. Total RNA was extracted from tissue samples using TRIzol (ThermoFisher Scientific, Cat#15596018). A volume of 2 l of the RNA preparation was used for amplification. Serial dilutions of synthetic RNA spanning the amplification region were used to make a positive control; undiluted synthetic ZIKV RNA had 10.sup.8.0 copies/l. Samples were subjected to 40 cycles of 15 seconds at 95 C. and 60 seconds at 60 C. following an initial single cycle of 30 min at 50 C. and 10 min at 95 C. Samples of unknown quantity were quantified by extrapolation of C(t) values using a curve generated from serial dilutions of synthetic ZIKV RNA.
[0427] Experiment Design: Dams were challenged 7 days-post coitus (dpc), and were treated with FIT-1 24 or 72 hours post virus challenge. 2 dams per group were necropsied 11 days after virus challenge. The placenta, fetal tissues, brain tissue, and spleen tissue were collected for determination of virus titer by QRT-PCR. 2 females per group were followed through parturition. Occipito-frontal diameter (OF) of the head and crown rump length (CRL) of pups was recorded with a digital caliper and intrauterine growth restriction was determined by determining the pup size by the formula: CRL X OF. Weights of pups and dams were measured at various times.
[0428] Statistical analysis: Survival data were analyzed using the Wilcoxon log-rank survival analysis (Prism 5, GraphPad Software, Inc).
[0429] Results and Discussion:
[0430] Treatment with FIT-1 was evaluated in a mouse model of congenital infection and disease associated with ZIKV infection. Treatment with FIT-1 was protective to pregnant females with the large majority of treated females being protected from mortality, regardless of when treatment was administered (
[0431] A trend towards improved measurements in average pup size. The average size of pups from dams treated with FIT-1 were higher than those treated the control MPEG and were similar to sham-infected animals (
[0432] The virus titer of various tissues is shown in
[0433] Conclusions:
[0434] Overall, these data support a protective role of FIT-1 in preventing or treating disease in fetuses congenitally exposed to ZIKV. A trend towards improvement in fetal and placental size parameters was observed, with a highly significant reduction in virus titer in various maternal and fetal tissues.
Example 15
Effect of FIT-1 on Disease in a Testis Infection Model in AG129 Mice
[0435] Sexual transmission and persistent infection of the male reproductive tract has been documented in men infected with ZIKV (D'Ortenzio E, Matheron S, Yazdanpanah Y, et al. Evidence of Sexual Transmission of Zika Virus. N Engl J Med 2016; 374:2195-8). In the AG-129 mouse strain, severe disease is usually observed around 2 weeks after virus challenge, including significant replication of the virus in the testes of mice (Julander J G, Siddharthan V, Evans J, et al. Efficacy of the broad-spectrum antiviral compound BCX4430 against Zika virus in cell culture and in a mouse model. Antiviral Res 2016; 137:14-22). Key sites of virus replication in the reproductive tract of male AG-129 mice include the epididymis and testicle, as well as various accessory sex glands.
[0436] In the present study, the effect of FIT-1 on ZIKV infected male mice is assessed in the testis infection model. To this end, male AG129 mice were infected with ZIKV and the pathology in the male reproductive tract after ZIKV infection and treatment with FIT-1 is assessed.
[0437] Materials and Methods:
[0438] Animals: Male AG129 mice were used. Groups of animals were randomly assigned to experimental groups and individually marked with ear tags.
[0439] Virus: Zika virus (Puerto Rican strain, PRVABC-59). A challenge dose of 10.sup.2 CCID.sub.50 was administered via s.c. injection in the inguinal fold in a volume of 0.1 ml. This challenge dose is typically lethal in untreated AG129 mice with mortality occurring around 2 weeks after challenge.
[0440] Test agent: FIT-1 was administered at a dose of 15 mg/kg. The non-specific control antibody MPEG-LALA Ctr IgG1 was used as an isotype control placebo treatment.
[0441] Histopathology: Tissues were collected and incubated for 24 hours in neutral buffered formalin. Following appropriate fixation, all collected tissues were trimmed and held in 70% ethanol until routine processing, paraffin embedding and sectioning were performed. All tissues were blindly analyzed independently by a veterinary anatomic pathology resident and a board certified veterinary anatomic pathologist. A scoring system was developed to grade the severity of inflammation in the reproductive tract.
[0442] Experiment Design: Mice were infected with ZIKV and were monitored for 28 days post-virus challenge for survival and weight change. Treatment with 15 mg/kg of FIT-1 was performed 24 or 72 h after virus challenge. A single treatment was administered i.p. in a volume of 0.1 ml. An isotype matched control Ab, MPEG-LALA Crt IgG1, was administered as described above with treatment occurring 24 h after virus challenge. A group of mock-infected, FIT-1-treated mice were included as toxicity controls and a group of normal controls was included. Individual weights were taken on 0, and every other day from 7-21 dpi. Mice were observed daily for signs of disease including conjunctivitis, hunching, and limb weakness or paralysis and initial onset of disease signs was recorded. A cohort of 3 animals was necropsied on 6 dpi and tissue samples were collected for histopathologic analysis.
[0443] Statistical analysis: Survival data were analyzed using the Wilcoxon log-rank survival analysis (Prism 5, GraphPad Software, Inc).
[0444] Results and Discussion:
[0445] Zika virus (ZIKV) can persist in male reproductive tissues for extended periods up to six months, representing a clear target for antiviral treatment. To determine the efficacy of a bispecific anti-ZIKV Ab in preventing or reducing pathology to the male reproductive tract, groups of mice were treated 24 or 72 h after virus challenge with a Puerto Rican isolate of ZIKV. Survival, weight change, and histopathology were used to determine the efficacy of mAb treatment. An isotype control mAb, MPEG-LALA Ctr IgG1, was used as a placebo treatment.
[0446] A significant (P<0.0.5) improvement in survival was observed as compared with placebo mAb treatment (
[0447] Mean weight change of mice treated with FIT-1 was similar to that of sham-infected treatment controls, while mean weight of mice treated with MPEG declined rapidly after 7 dpi, which further demonstrates potent protection of the mice from disease (
[0448] Since the primary purpose of this study was to evaluate the effects of treatment on the male reproductive tract, and in previous studies the testes and epididymides were the most severely impacted after infection, specific attention was paid to the histopathology of these tissues. No disease was observed in the testicle or epididymis of mice treated with FIT-1, aside from a single animal in the 24 h treatment group (
[0449] Conclusions:
[0450] Treatment with FIT-1 was effective in reducing all evaluated disease parameters. Therapeutic treatment up to 72 h after virus challenge was highly efficacious.
Example 16
Prophylactic and Therapeutic Efficacy of FIT-1 in Rhesus Macaques Challenged with ZIKV
[0451] This study evaluated the prophylactic and therapeutic efficacy of a bi-specific antibody targeting Zika virus (ZIKV) in Indian Rhesus Macaques (IRM) challenged with ZIKV strain PRVABC59. Sixteen (16) IRMs were randomized into three treatment groups. One group received FIT-1 one day prior to challenge (5 mg/kg) and a second received the treatment one day following challenge (15 mg/kg). A third group was treated with an isotype control (FIT-3, 5 mg/kg) one day prior to challenge. On Day 0, all IRMs were challenged with 110.sup.5 PFU of ZIKV strain PRVABC59 delivered by subcutaneous (SC) injection. Sera, urine, and saliva were collected at predetermined time points and tested for ZIKV load as measured by quantitative RT-PCR.
[0452] Methods:
[0453] Prior to Study Day 0, sixteen (16) IRMs were randomized into respective groups according to gender/weight using Provantis Software. On Days 1 or 1, animals received either FIT-1 or isotype control FIT-3 delivered intravenously at the dose listed in Table 4 below. On Day 0, all macaques were anesthetized and challenged with 0.5 mL of wild type ZIKV strain PRVABC59 with a target challenge dose of 1010.sup.5 PFU per animal by subcutaneous injection. Blood, urine, and saliva samples were collected at predetermined time points as indicated in Table 5 for an assessment of viral load by RT-qPCR.
TABLE-US-00007 Animal Groupings Treatment Route, Group Treatment Day Dosage Challenge.sup.1 1 FIT-1 Day 1 i.v., PRVABC59 (3M/3F) 5 mg/kg (1 10.sup.5 PFU) 2 FIT-1 Day +1 i.v., PRVABC59 (3M/3F) 15 mg/kg (1 10.sup.5 PFU) 3 FIT-3 Day 1 i.v., PRVABC59 (2M/2F) 5 mg/kg (1 10.sup.5 PFU) .sup.1Subcutaneously challenged on Day 0 with 0.5 mL of wild type ZIKV.
TABLE-US-00008 TABLE 5 Key Activities Study Day 1 0 1 5 10 15 20 25 30 Treatment ZIKV challenge (s.c.) Daily Observations All animals were observed two times daily Body Weight Daily Body Temperature Daily Blood Collections.sup.1 Daily Urine/Saliva Collections Viral Load: RT- Daily qPCR Blood Volume (mL) 2 2 2 2 20 Euthanasia .sup.1Day 0 and 1 blood was collected prior to challenge and treatment
[0454] On Study Days 1 (Groups 1 and 3) and 1 (Group 2) anesthetized animals were administered either FIT-1 (stock concentration: 3.67 mg/mL) or FIT-3 (stock concentration: 3.79 mg/mL) via intravenous injection into the right saphenous or cephalic veins.
[0455] ZIKV strain PRVABC59; Human/2015/Puerto Rico (American Isolate) was the challenge material used in this study. Preparation of the virus inoculum was performed in a Class II Biological Safety Cabinet under BSL-2 conditions. The virus stock was thawed in a 371 C. water bath, vortexed, and diluted with VP-SFM to yield an inoculum of the appropriate concentration of 210.sup.5 PFU/mL. Each syringe was filled with 0.5 mL of virus inoculum and kept on ice until transferred to the animal facility for dosing. On Study Day 0, all animals were anesthetized, the injection site clipped, wiped with alcohol and marked with an indelible marker. Animals were inoculated SC on the anterior surface of the left forearm with 0.5 mL of the ZIKV isolate and dose indicated in Table 4.
[0456] All macaques were observed twice daily throughout the quarantine and study periods for signs of morbidity and mortality. Animals were observed twice daily (at no less than 8 hour intervals) for responsiveness and clinical signs including rash, erythema, conjunctivitis, ocular discharge and swelling. For all animals, blood was collected at the time points indicated in Table 5 for in vitro testing via RT-qPCR viral load. During the terminal bleed on Day 30, a volume not exceeding 20 mL per animal was collected. For all animals, urine was collected at the time points indicated in Table 5 for in vitro testing via RT-qPCR for monitoring of virus shedding. Animals were individually housed during the urine collection periods of the study. Urine was collected directly from the cage pans, placed on wet ice following collection, separated into aliquots and stored at 70 C. or below until ready for assay testing. Saliva (drool) was collected from anesthetized animals directly into tubes (up to approximately 0.5 mL). Samples were kept on wet ice following collection and stored at 70 C. or below until ready for assay testing.
[0457] Viral loads were measured using a RT-qPCR method for detection of ZIKV genomes in the serum, urine, and saliva samples collected at the time points indicated in Table 5. Samples recovered from virus inoculated animals were tested using primers and probes designed for the detection of ZIKV strain PRVABC59. A description of the PCR methods, including primer and probe sequences, has been published (Goebel et. al., 2016, A Sensitive virus yield assay for evaluation of antivirals against Zika virus. J. Virol. Methods). Viral RNA was isolated from biological fluids using the QIAmp Viral RNA mini kit (Qiagen, 52906). The viral RNA was eluted with sterile RNase and DNase free H.sub.2O and stored at 70 C. or below. The lower limit of quantitation (LLOQ) of this assay was determined to be 10 copies per reaction.
[0458] An aliquot of the challenge virus inocula was back-titrated by standard plaque assay on Vero cells to confirm the actual delivered dose. Ten-fold serial dilutions of the challenge inocula were used to infect confluent monolayers of Vero cells in 6-well plates that were plated the day before. Plates were incubated at 37 C. and 5% CO2 for 1 hour before the addition of overlay media containing 0.5% agarose. Plates were incubated for 3 days until discernable plaques form after which they were fixed, stained with crystal violet and counted.
[0459] Results:
[0460] Macaques were monitored twice daily for signs of mortality and morbidity for the duration of the study. All animals survived to the scheduled termination. Body weights and temperatures of all animals were measured at the time points indicated in Table 5. There was no significant body weight loss during the course of the study. All animals maintained a normal range of body temperatures throughout the study. Clinical observations were limited to mild redness at the challenge site in a few animals.
[0461] Viral loads were measured using a RT-qPCR method for detection of ZIKV genomes in serum, urine, and saliva samples collected at the time points indicated in Table 5. Viral load in the serum is presented in
[0462] Animals in Group 3, treated with the isotype control, had detectable viral load in the serum the day following challenge. Viral load in three of the four animals peaked above 110.sup.5 genome copies/mL (GC/mL) on either Day 2 or 3. The average serum viral load peaked in this group on Day 3 at 1.1510.sup.5 GC/mL. Viral loads decreased below the LLOQ of the assay (860 GC/mL) by Day 4 in one animal and by Day 5 in the remaining three.
[0463] Group 2 animals, treated with 15 mg/kg of FIT-1 the day after challenge, had an average viral load of 3.9910.sup.3 GC/mL prior to treatment. On Day 2, average serum viral load decreased to only 96.5 GC/mL. While low levels of viral RNA were sporadically detected after Day 1, at no point following treatment were viral loads detected in Group 2 animals at or above the LLOQ of the RT-qPCR assay.
[0464] Pretreatment with 5 mg/kg of FIT-1 decreased Day 1 average serum viral loads by greater than 50-fold compared to Group 3. At no time point after challenge were viral loads detected at or above the LLOQ of the assay, and by Day 2 no viral RNA was detected in the serum of any of the Group 1 animals.
[0465] Only sporadic, low levels of ZIKV RNA were detected in the urine or saliva from any of the animals. At no point were viral loads detected at or above the LLOQ of the assay in urine or saliva from any animal.
[0466] An aliquot of the challenge virus inocula was back-titrated by standard plaque assay on Vero cells to confirm the actual delivered dose. The plaque assay yielded a titer of 1.710.sup.5 PFU/mL.
SUMMARY AND CONCLUSIONS
[0467] This study evaluated the prophylactic and therapeutic efficacy of FIT-1 in IRMs challenged with ZIKV. There was no mortality throughout the course of the study and clinical observations were limited to mild redness at the challenge site in a few animals. Viral load as detected by RT-qPCR in serum collected following challenge was the primary end point of the study. Viral RNA was readily detected in the serum of all animals treated with the isotype control, with viral loads peaking on Days 2 or 3 and sustaining at levels above the LLOQ of 860 GC/mL until Days 4 or 5. Average peak load was 1.1.510.sup.5 GC/mL on Day 3. In contrast, prophylactic treatment with 5 mg/kg of FIT-1 effectively reduced peak viral loads and time to viral clearance in ZIKV challenged IRMs. Low levels of viral RNA were detected in all six animals from this group on Day 1 but by Day 2, no viral RNA was detected in any animal. At no point was ZIKV RNA detected above the LLOQ of 860 GC/mL in the serum of any animal from this group. Similarly, therapeutic treatment with 15 mg/kg of FIT-1 the day following challenge reduced both peak viral loads and time to viral clearance from the serum compared to animals treated with the isotype control. Group 2 macaques had a mean peak viral load of 3.9910.sup.3 GC/mL on Day 1 but after treatment on Day 1, average viral loads in the serum of this group decreased to only 96.5 GC/mL by Day 2. No animals in this group had viral loads above the LLOQ of 860 GC/mL for the remainder of the observation period.
TABLE-US-00009 TablesofSequencesandSEQIDNumbers ZKA190 SEQIDNO. Aminoacidsequence CDRH.sub.1 1 GFTFSKYG CDRH.sub.2 2 ISYEGSNK CDRH.sub.3 3 AKSGTQYYDTTGYEYRGLEYFGY CDRL.sub.1 4 QSVSSSY CDRL.sub.2 5 DAS CDRL.sub.2 6 LIYDASSRA long CDRL.sub.3 7 QQYGRSRWT VH 8 QVQLVESGGGVVQPGRSLRLSCAASGFTFSKYGMHWVRQAPGKGLE WVAVISYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCAKSGTQYYDTTGYEYRGLEYFGYWGQGTLVTVSS VL 9 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKRGQAPR LLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY GRSRWTFGQGTKVEIK ZKA190 SEQIDNO. Nucleicacidsequence CDRH.sub.1 10 ggattcaccttcagtaaatatggc CDRH.sub.2 11 atatcatatgagggaagtaataaa CDRH.sub.3 12 gcgaaatcggggacccaatactatgatactactggttatg agtataggggtttggaatactttggctac CDRL.sub.1 13 cagagtgttagtagcagttac CDRL.sub.2 14 gatgcatcc CDRL.sub.2 15 ctcatctatgatgcatccagcagggcc long CDRL.sub.3 16 cagcagtatggtaggtcaaggtggaca VH 17 caggtgcagctggtggagtctgggggaggcgtggtccagc ctgggaggtccctgagactctcctgtgcagcctctggatt caccttcagtaaatatggcatgcactgggtccgccaggct ccaggcaaggggctggagtgggtggcagttatatcatatg agggaagtaataaatattatgcagactccgtgaagggccg attcaccatctccagagacaattccaagaacacgctgtat ctgcaaatgaacagcctgagagctgaggacacggcagtgt attactgtgcgaaatcggggacccaatactatgatactac tggttatgagtataggggtttggaatactttggctactgg ggccagggaaccctggtcaccgtctcctcag VL 18 gaaattgtgttgacgcagtctccaggcaccctgtctttgt ctccaggggaaagagccaccctctcctgcagggccagtca gagtgttagtagcagttacttagcctggtaccagcagaaa cgtggccaggctcccaggctcctcatctatgatgcatcca gcagggccactggcatcccagacaggttcagtggcagtgg gtctgggacagacttcactctcaccatcagcagactggag cctgaagattttgcagtgtattactgtcagcagtatggta ggtcaaggtggacattcggccaagggaccaaggtggaaat caaac ZKA185 SEQIDNO. Aminoacidsequence CDRH.sub.1 19 GYSFTSYW CDRH.sub.2 20 FDPSDSQT CDRH.sub.3 21 ARRYCSSSSCYVDN CDRL.sub.1 22 ALPNKF CDRL.sub.2 23 EDN CDRL.sub.2 24 VIYEDNKRP long CDRL.sub.3 25 YSTDSSSNPLGV VH 26 EVQLVQSGAEVKKPGESLRISCKGSGYSFTSYWITWVRQMPGKGLE WMAKFDPSDSQTNYSPSFQGHVTISVDKSISTAYLQWSSLKASDTA MYYCARRYCSSSSCYVDNWGQGTLVTIFS VL 27 SYELTQPPSVSVSPGQTARITCSGDALPNKFAYWYRQKSGQAPVLV IYEDNKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYHCYSTDS SSNPLGVFGGGTKLTVL ZKA185 SEQIDNO. Nucleicacidsequence CDRH.sub.1 28 ggatatagttttaccagttactgg CDRH.sub.2 29 tttgatcctagtgactctcaaacc CDRH.sub.3 30 gcgagaagatattgtagtagtagtagttgttatgtggacaa t CDRL.sub.1 31 gcattgccaaataaattt CDRL.sub.2 32 gaggacaac CDRL.sub.2 33 gtcatctatgaggacaacaaacgaccc long CDRL.sub.3 34 tactcaacagacagcagttctaatcccctgggagta VH 35 gaagtgcagctggtgcagtccggagcagaggtgaaaaagcc cggggagtctctgaggatctcctgtaagggttctggatata gttttaccagttactggatcacctgggtgcgccagatgccc gggaaaggcctggagtggatggcgaagtttgatcctagtga ctctcaaaccaactacagcccgtccttccaaggccacgtca ccatctcagttgacaagtccatcagcactgcctacttgcag tggagcagcctgaaggcctcggacaccgccatgtattactg tgcgagaagatattgtagtagtagtagttgttatgtggaca attggggccagggaaccctggtcaccatcttctcag VL 36 tcctatgagctgacacagccaccctcggtgtcagtgtcccc aggacaaacggccaggatcacctgctctggagatgcattgc caaataaatttgcttattggtaccggcagaagtcaggccag gcccctgttctggtcatctatgaggacaacaaacgaccctc cgggatccctgagagattctctggctccagctcagggacaa tggccaccttgactatcagtggggcccaggtggaggatgaa gctgactaccactgttactcaacagacagcagttctaatcc cctgggagtattcggcggagggaccaagctgaccgtcctag ZKA230 SEQIDNO. Aminoacidsequence CDRH.sub.1 37 GGSISSDY CDRH.sub.2 38 IYYSGST CDRH.sub.3 39 ARRRKYDSLWGSFAFDI CDRL.sub.1 40 SSNIGGNY CDRL.sub.2 41 IND CDRL.sub.2 42 LICINDHRP long CDRL.sub.3 43 ATWDDSLGGLV VH 44 QVQLQESGPGLVKPSETLSLTCAVSGGSISSDYWSWIRQPPGKGLE WIGYIYYSGSTNYNPSLKSRVTISVDTSKNHFSLKLNSVTAADTAV YYCARRRKYDSLWGSFAFDIWGQGTMVTVSS VL 45 QSVLTQPPSASGTPGQRVTISCSGSSSNIGGNYVYWYQQLPGTAPK LLICINDHRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCATW DDSLGGLVFGGGTKLTVL ZKA230 SEQIDNO. Nucleicacidsequence CDRH.sub.1 46 ggtggctccatcagtagtgactac CDRH.sub.2 47 atctattacagtgggagcacc CDRH.sub.3 48 gcgaggaggaggaagtatgattccctttgggggagttttgc ttttgatatc CDRL.sub.1 49 agctccaacatcggaggtaattat CDRL.sub.2 50 attaatgat CDRL.sub.2 51 ctcatctgtattaatgatcaccggccc long CDRL.sub.3 52 gcaacatgggatgacagcctgggtggccttgta VH 53 caggtgcagctgcaggagtcgggcccaggcctggtgaagcc ttcggagaccctgtccctcacctgcgcagtctctggtggct ccatcagtagtgactactggagctggatccggcagccccca gggaagggactggagtggattgggtatatctattacagtgg gagcaccaactacaacccctccctcaagagtcgagtcacca tatcagtagacacgtccaagaaccacttctccctgaagctg aactctgtgaccgctgcggacacggccgtgtattactgtgc gaggaggaggaagtatgattccctttgggggagttttgctt ttgatatctggggccaagggacaatggtcaccgtctcttca g VL 54 cagtctgtgctgactcagccaccctcagcgtctgggacccc cgggcagagggtcaccatctcttgttctggaagcagctcca acatcggaggtaattatgtatactggtaccagcagctccca ggaacggcccccaaactcctcatctgtattaatgatcaccg gccctcaggggtccctgaccgattctctggctccaagtctg gcacctcagcctccctggccatcagtgggctccagtccgag gatgaggctgattattactgtgcaacatgggatgacagcct gggtggccttgtattcggcggagggaccaagctgaccgtcc tag ZKA78 SEQIDNO. Aminoacidsequence CDRH.sub.1 55 GFTFSNYA CDRH.sub.2 56 IGRNGDSI CDRH.sub.3 57 VKDLAIPESYRIEADY CDRL.sub.1 58 QSVLYRSNNKNY CDRL.sub.2 59 WAS CDRL.sub.2 60 LIYWASTRE long CDRL.sub.3 61 QQYYSSPRT VH 62 EVQLAESGGGLVQPGGSLTLSCSGSGFTFSNYAMVWARQAPGKGLE YVSGIGRNGDSIYYTDSVKGRFTISRDNSKSMVYLQMSSLRTEDTA VYYCVKDLAIPESYRIEADYWGQGTLVIVSA VL 63 DIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNKNYLSWYQQKP GQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISPLQAEDVAVY YCQQYYSSPRTFGQGTKVEIK ZKA78 SEQIDNO. Nucleicacidsequence CDRH.sub.1 64 ggcttcacttttagtaactatgca CDRH.sub.2 65 atcgggcgcaacggggactctatc CDRH.sub.3 66 gtgaaagatctggccatccccgagtcctacagaattgaag ctgattat CDRL.sub.1 67 cagtccgtgctgtaccgctctaacaacaagaattac CDRL.sub.2 68 tgggcttca CDRL.sub.2 69 ctgatctattgggcttcaacccgggaa long CDRL.sub.3 70 cagcagtactattctagtcctcgaact VH 71 gaggtgcagctggcagaatcaggcgggggactggtccagc ctggcggcagcctgacactgtcttgcagtggatcaggctt cacttttagtaactatgcaatggtgtgggcaaggcaggct cctgggaagggactggagtatgtctctggcatcgggcgca acggggactctatctactatactgatagtgtgaagggccg gttcaccatcagcagagacaatagcaaatccatggtgtac ctgcagatgagctccctgcgaaccgaagacacagcagtgt actattgcgtgaaagatctggccatccccgagtcctacag aattgaagctgattattggggacagggcaccctggtcatc gtgagcgccg VL 72 gacatcgtgatgacacagtctccagatagtctggcagtca gtctgggggagagggccactattaactgcaagagctccca gtccgtgctgtaccgctctaacaacaagaattacctgtct tggtatcagcagaagcccggacagccccctaaactgctga tctattgggcttcaacccgggaaagcggcgtcccagacag attctcaggcagcgggtccggaacagacttcaccctgaca attagccccctgcaggcagaggacgtggctgtctactatt gtcagcagtactattctagtcctcgaactttcggccaggg gaccaaggtggaaatcaaac ZKA64 SEQIDNO. Aminoacidsequence CDRH.sub.1 73 GYTFTGYH CDRH.sub.2 74 INPNSGGT CDRH.sub.3 75 ARMSSSIWGFDH CDRL.sub.1 76 QSVLIN CDRL.sub.2 77 GAS CDRL.sub.2 78 LIYGASSRA long CDRL.sub.3 79 QQYNDWPPIT VH 80 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYHIDWVRQARGQGLE WMGRINPNSGGTNYAQKFQGRVTMTRDTSISTAYMQLSRLRSDDSA VYYCARMSSSIWGFDHWGQGTLVTVSS VL 81 EIVMTQSPATLSVSPGERATLSCRASQSVLINLAWYQQKPGQAPRL LIYGASSRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYN DWPPITFGQGTRLEIK ZKA64 SEQIDNO. Nucleicacidsequence CDRH.sub.1 82 ggctacaccttcacagggtatcac CDRH.sub.2 83 attaaccctaattctggcgggacc CDRH.sub.3 84 gctcggatgagctcctctatttggggcttcgatcat CDRL.sub.1 85 cagtctgtgctgattaac CDRL.sub.2 86 ggagcatcc CDRL.sub.2 87 ctgatctatggagcatcctccagggct long CDRL.sub.3 88 cagcagtacaatgattggccccctatcaca VH 89 caggtgcagctggtccagagcggagcagaggtgaagaaacc cggcgcctcagtgaaggtcagctgcaaagcttccggctaca ccttcacagggtatcacatcgactgggtgaggcaggcaaga ggacagggactggaatggatgggacggattaaccctaattc tggcgggaccaactacgcccagaagtttcagggccgagtga ctatgaccagagacaccagcatctccacagcttatatgcag ctgtcccggctgagatctgacgatagtgccgtctactattg tgctcggatgagctcctctatttggggcttcgatcattggg ggcagggaacactggtgactgtcagttcag VL 90 gagatcgtgatgactcagtctccagccaccctgtcagtcag cccaggagaacgggcaaccctgtcttgcagagcctcccagt ctgtgctgattaacctggcttggtaccagcagaagccaggc caggcaccccgactgctgatctatggagcatcctccagggc taccggcattcctgcacgcttcagtggatcaggaagcggaa cagagtttaccctgacaatctctagtctgcagtccgaagac ttcgctgtctactattgtcagcagtacaatgattggccccc tatcacatttggccaggggactagactggagatcaagc SEQID Constantregions NO. Sequence IgG.sub.1CH.sub.1-CH.sub.2- 91 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV CH.sub.3aa TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K IgG.sub.1CH.sub.1-CH.sub.2- 92 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV CH.sub.3LALAaa TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgGCKaa 93 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC IgGCLaa 94 GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLT PEQWKSHRSYSCQVTHEGSTVEKTVAPTECS IgG.sub.1CH.sub.1-CH.sub.2- 95 gcgtcgaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctct CH.sub.3nucl gggggcacagcggccctgggctgcctggtcaaggactacttccccgaacctgtg acggtctcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctg tcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagca gcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaag gtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtg cccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaa ggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtg agccacgaAgaCcctgaggtcaagttcaactggtacgtggacggcgtggaggt gcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgt ggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaag tgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagcc aaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggag atgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcga catcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccac gcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtgga caagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggct ctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaa IgG.sub.1CH.sub.1-CH.sub.2- 96 gcgtcgaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctct CH3LALAnucl gggggcacagcggccctgggctgcctggtcaaggactacttccccgaacctgtg acggtctcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctg tcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagca gcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaag gtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtg cccagcacctgaaGCCGCGgggggaccgtcagtcttcctcttccccccaaaac ccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtgga cgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtgga ggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccg tgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtac aagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaa gccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggag gagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccag cgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaaga ccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgt ggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatga ggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaa IgGCKnucl 97 cgTacGgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaa atctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaa agtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtc acagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctga gcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcaggg cctgagctcgcccgtcacaaagagcttcaacaggggagagtgt IgGCLnucl 98 ggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagctt caagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgt gacagtggcttggaaagcagatagcagccccgtcaaggcgggagtggagacca ccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctg acgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaag ggagcaccgtggagaagacagtggcccctacagaatgttca ZKA3 SEQIDNO. Aminoacidsequence CDRH.sub.1 99 GFIFSNYA CDRH.sub.2 100 IGGKGDSI CDRH.sub.3 101 VKDLAVLESDRLEVDQ VH 102 EVQLAESGGGLVQPGGSLRLSCSGSGFIFSNYAMVWARQAP GKGLEYVSGIGGKGDSIYHIDSVKGRFTISRDNSKRTVYLQ MSRLRTEDTAVYYCVKDLAVLESDRLEVDQWGQGTLVIVSA ZKA4 SEQIDNO. Aminoacidsequence CDRH.sub.1 103 GFTFSSYV CDRH.sub.2 104 TSYDGSNK CDRH.sub.3 105 ARGPVPYWSGESYSGAYFDF VH 106 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYVMHWVRQAP GKGLEWVTVTSYDGSNKYYADSVKGRFTISRDNAKNTLYLQ MNSLRGEDTAIYYCARGPVPYWSGESYSGAYFDFWGQGILV TVSS ZKA5 SEQIDNO. Aminoacidsequence CDRH.sub.1 107 GFTFSNYY CDRH.sub.2 108 MSSSETIK CDRH.sub.3 109 ARSGIETVAGSIDYYGMDV VH 110 QVQLVESGGGLVKPGGSLRLSCAGSGFTFSNYYMTWIRQAP GKGLELVSYMSSSETIKYYADSVKGRFTISRDNAKNSLYLQ MNSLRADDTARYYCARSGIETVAGSIDYYGMDVWGHGTPVT VSS ZKA6 SEQIDNO. Aminoacidsequence CDRH.sub.1 111 DFTVSNYA CDRH.sub.2 112 VSYDGSNK CDRH.sub.3 113 ATGVTMFQGAQTNAEYLHY VH 114 QVHLVESGGGVVQPGRSLRLSCEASDFTVSNYAMHWVRQAP GKGLEWVAVVSYDGSNKYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTALYYCATGVTMFQGAQTNAEYLHYWGQGSLVT ISS ZKA7 SEQIDNO. Aminoacidsequence CDRH.sub.1 115 GFTFSRYG CDRH.sub.2 116 VSGDGSST CDRH.sub.3 117 VKDFWSGDQSLESDF VH 118 EVQLVESGGGLVQPGGSLRLSCSASGFTFSRYGMVWARQAP GKGLEYLSGVSGDGSSTYYANSVKGRFTISRDNSKNTLYLH MSRLRDEDTAMYYCVKDFWSGDQSLESDFWGQGALVTVSS ZKA8 SEQIDNO. Aminoacidsequence CDRH.sub.1 119 GFTFSAHA CDRH.sub.2 120 ISRNEDYT CDRH.sub.3 121 VKDFGTSPQTDF VH 122 DERLVESGGGLVQPGGSLRLVCSASGFTFSAHAMHWVRQPP GKGLEYVSTISRNEDYTYYADSVKGRFTISRDNSKNSLYLQ MRRLRPEDTAIYYCVKDFGTSPQTDFWGQGTLVAVSS ZKA76 SEQIDNO. Aminoacidsequence CDRH.sub.1 123 GFTFSTYF CDRH.sub.2 124 ISSTGSYK CDRH.sub.3 125 ARPFHSEYTYGLDAFDI VH 126 EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYFMHWVRQAP GKGLEWVASISSTGSYKFYADSVKGRFTISRDNTKNSLFLQ MNSLRAEDTAVFYCARPFHSEYTYGLDAFDIWGQGTMLTVS S ZKA117 SEQIDNO. Aminoacidsequence CDRH.sub.1 127 GGSIRRTNSY CDRH.sub.2 128 ISYSGST CDRH.sub.3 129 ARLNDGSTVTTSSYFDY VH 130 QLQLQESGPGLVKPSETLSLTCTVSGGSIRRTNSYWGWIRQ TTGKGLQWIGSISYSGSTFYNPSLKSRVTISLDTSKDHFSL ELSSVTAADTAIYYCARLNDGSTVTTSSYFDYWGQGTLVTV SS ZKB27 SEQIDNO. Aminoacidsequence CDRH.sub.1 131 GYSFTSSW CDRH.sub.2 132 IDPSDSYT CDRH.sub.3 133 ARHDYSVSENGMDV VH 134 EVQLVQSGAEVKKPGESLRISCKASGYSFTSSWINWVRQMP GKGLEWMGRIDPSDSYTTYNPSFQGHVTISVDKSIGTAYLQ WNSLRASDTAMYYCARHDYSVSENGMDVWGQGTTVIVSS ZKB29 SEQIDNO. Aminoacidsequence CDRH.sub.1 135 GFTFSSYT CDRH.sub.2 136 ISYDGSHK CDRH.sub.3 137 ARRSYSISCFDY VH 138 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAP GKGLEWVAVISYDGSHKFYADSVKGRFTISRDNSKDTLYLQ MNSLRAEDTALYYCARRSYSISCFDYWGQGTLVTISS ZKB34 SEQIDNO. Aminoacidsequence CDRH.sub.1 139 GFTFSRSG CDRH.sub.2 140 VSYDGSNK CDRH.sub.3 141 AKDLTMVRGVHYYYYVMDV VH 142 QVQLVESGGGVVQPGRSLRLSCAASGFTFSRSGMHWVRQAP GKGLEWVAVVSYDGSNKYYSDSVKGRFTISRDNSKNTLYLQ MNSLRVEDTAVYYCAKDLTMVRGVHYYYYVMDVWGQGTIVT VSS ZKB39 SEQIDNO. Aminoacidsequence CDRH.sub.1 143 GYTFDDYY CDRH.sub.2 144 INPHRGGT CDRH.sub.3 145 VRDQYCDGGNCYGIHQPHYGMDV VH 146 QVQLVQSGAEVKKPGASLKVSCKASGYTFDDYYIHWVRQAP GQGLEWLGRINPHRGGTNYAQKFQGRVIMTLDMSISTTYME LRRITSDDAAVYYCVRDQYCDGGNCYGIHQPHYGMDVWGQG TTVTVSS ZKB46 SEQIDNO. Aminoacidsequence CDRH.sub.1 147 GYSFTSYW CDRH.sub.2 148 IDPSDSYT CDRH.sub.3 149 ARREYSSSSGQEDWFDP VH 150 EVQLVQSGAEVKKPGESLRISCKGSGYSFTSYWISWVRQMP GKGLEWMGRIDPSDSYTNYSPSFQGHVTISADKSISTAYLQ WSSLKASDTAMYYCARREYSSSSGQEDWFDPWGQGTLVTVS S ZKB53 SEQIDNO. Aminoacidsequence CDRH.sub.1 151 GFTFSSYA CDRH.sub.2 152 ISYDGSNR CDRH.sub.3 153 ARHVEQLPSSGYFQH VH 154 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQTP GKGLEWVTVISYDGSNRYYADSVKGRFTISRDNSKNTLYLQ MNSLRSEDTAVYYCARHVEQLPSSGYFQHWGQGTLVTVSS ZKC26 SEQIDNO. Aminoacidsequence CDRH.sub.1 155 GFIFSDFY CDRH.sub.2 156 IGHDGSYI CDRH.sub.3 157 ARAHGGFRH VH 158 QVQVVESGGGLVKPGGSLRLSCAASGFIFSDFYMSWMRQAP GKGLEWVAYIGHDGSYILYADSVKGRFTISRDNAKNSLFLR MNSLRVEDTAVYYCARAHGGFRHWGQGTVVAVSP ZKD5 SEQIDNO. Aminoacidsequence CDRH.sub.1 159 GFTFTSYG CDRH.sub.2 160 ISYDGSNK CDRH.sub.3 161 ARDRDHYDLWNAYTFDY VH 162 QVQLVESGGGVVQPGRSLRLSCAASGFTFTSYGMHWVRQTP GKGLDWVAVISYDGSNKYYADSVKGRFTISRDNSKDTLYLQ MNSLRAADTALYYCARDRDHYDLWNAYTFDYWGQGTLVTVS S ZKD7 SEQIDNO. Aminoacidsequence CDRH.sub.1 163 GFTFSNYA CDRH.sub.2 164 ISYDVSDK CDRH.sub.3 165 AGGPLGVVVIKPSNAEHFHH VH 166 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYAMHWVRQAP GKGLEWVAVISYDVSDKYYADSVKGRFTISRDNSKNTLFLQ MNSLRAEDTAAYYCAGGPLGVVVIKPSNAEHFHHWGQGTLV TVSS ZKD8 SEQIDNO. Aminoacidsequence CDRH.sub.1 167 GFTFINYA CDRH.sub.2 168 ISYDGSNK CDRH.sub.3 169 ATDADAYGDSGANFHY VH 170 QVQLVESGGGVVQPGKSLRLSCAASGFTFINYAIHWVRQAP GKGLEWVAVISYDGSNKFYTDSVKGRFTISRDNSKNTLYLQ MNSLRADDTAVYYCATDADAYGDSGANFHYWGQGTLVTVSS ZKD15 SEQIDNO. Aminoacidsequence CDRH.sub.1 171 DASISSGGFS CDRH.sub.2 172 IYSSGDT CDRH.sub.3 173 ARAHTPTSKFYYYYAMDV VH 174 QLQLQESGSGLVKPSQTLSLTCTVSDASISSGGFSWSWIRQ PLGKGLEWLGYIYSSGDTFYNPSLQGRVTMSVDIFRSQFSL KLTSVTAADTAMYYCARAHTPTSKFYYYYAMDVWGQGTTVT VSS ZKD16 SEQIDNO. Aminoacidsequence CDRH.sub.1 175 GFTFSDHF CDRH.sub.2 176 SRNKPNSYTT CDRH.sub.3 177 AKVGGCYGGDCHVENDY VH 178 EVQLVESGGDLVQPGGSLRLSCVASGFTFSDHFMDWVRQAP GKGLEWVGRSRNKPNSYTTEYAASVKGRFSISRDDSKKALY LQMNSLQTEDTAVYYCAKVGGCYGGDCHVENDYWGQGTLVT VSS ZKD17 SEQIDNO. Aminoacidsequence CDRH.sub.1 179 GFIFSDYA CDRH.sub.2 180 ISYDGSSR CDRH.sub.3 181 ARGYCSSGTCFSTNAEYFHP VH 182 QVQMVESGGGVVQPGTSLRLSCATSGFIFSDYAMHWVRQAP GKGLEWVAVISYDGSSRLYADSVKGRFTVSRDNSKNTLYLQ MHSLRAGDTAVYYCARGYCSSGTCFSTNAEYFHPWGQGTLA TISS ZKD20 SEQIDNO. Aminoacidsequence CDRH.sub.1 183 GFTFSDHF CDRH.sub.2 184 SRNKPNSYTT CDRH.sub.3 185 ARVGGCNGGDCHVENDY VH 186 EVQLVESGGGLVQPGGSLRLSCVASGFTFSDHFMDWVRQAP GKGLEWVGRSRNKPNSYTTEYAASVKGRFTISRDDSKNSLY LQMNSLQTEDTAVYYCARVGGCNGGDCHVENDYWGQGTLVT VSS ZKA134 SEQIDNO. Aminoacidsequence CDRH.sub.1 187 GGTFSAYA CDRH.sub.2 188 IIPFFGTA CDRH.sub.3 189 ARSDIVSTTRGYHHYGMDV VH 190 QVHLVQSGAEVKKPGSSVNVSCKASGGTFSAYAISWVRQAP GQGLEWMGGIIPFFGTAYYAQKFKGRVTVTADKSISTVYME MISLRSEDTAVYYCARSDIVSTTRGYHHYGMDVWGQGTTVT VSS ZKA246 SEQIDNO. Aminoacidsequence CDRH.sub.1 191 GYTFSDYY CDRH.sub.2 192 INPYSGGT CDRH.sub.3 193 ARGFTMISDREFDP VH 194 QVQLVQSGAEVKRPGASVKVSCKASGYTFSDYYMHWVRQAP GQGLEWMGRINPYSGGTNYAQKFHGRVTVTRDTSISTVYME LRGLRSDDTAVYYCARGFTMISDREFDPWGQGTLVTVSS ZKA256 SEQIDNO. Aminoacidsequence CDRH.sub.1 195 GFTFSTYW CDRH.sub.2 196 IKQDGSEK CDRH.sub.3 197 ARDPGYDDFWSGSYSGSFDI VH 198 EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYWMTWVRQAP GKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNTKNSLYLQ VNSLRAEDTAIYYCARDPGYDDFWSGSYSGSFDIWGQGTMV TVSS ZKB42 SEQIDNO. Aminoacidsequence CDRH.sub.1 199 GFTFNNYG CDRH.sub.2 200 ISYDGNKK CDRH.sub.3 201 VKYGERINGYSDPFDH VH 202 QVQVVESGGGVVQPGRSLRLFCAASGFTFNNYGMHWVRQAP GKGLEWVALISYDGNKKYYADSVKGRFSISRDNSKNTLYLQ MNRLRSGDTAVYHCVKYGERINGYSDPFDHWGQGTLVTVSS ZKB85 SEQIDNO. Aminoacidsequence CDRH.sub.1 203 GYTFTTYA CDRH.sub.2 204 INTNTGNP CDRH.sub.3 205 ARVIVPYAFDI VH 206 QVQLVQSGSELKKPGASVKVSCKASGYTFTTYAMNWVRQAP GQGPEWVGWINTNTGNPTYAQGFTGRFVLSLDTSVSTAFLQ ISSLKAEDTAVYYCARVIVPYAFDIWGQGTMVTVSS ZKB47 SEQIDNO. Aminoacidsequence CDRH.sub.1 207 GYTFTNYY CDRH.sub.2 208 INPSGGPT CDRH.sub.3 209 ARDQYGGYARYGMDV VH 210 QVQLVQSGAEVKKPGASVKVSCQASGYTFTNYYMHWVRQAP GQGLEWMGIINPSGGPTSYAQKFQGRVTMTTDTSTSTVYME LSSLRSEDTAVYYCARDQYGGYARYGMDVWGQGTTVIVSS ZKC6 SEQIDNO. Aminoacidsequence CDRH.sub.1 211 GYTFTGYY CDRH.sub.2 212 INPNSGGT CDRH.sub.3 213 ARVSDWGFAFDI VH 214 QVQLVQSGTEVKKPGASVKVSCKASGYTFTGYYMHWVRQAP GQGLEWMGRINPNSGGTNYAQKFQGRVTMTRDTSISTAYME LSGLRSDDTAVYYCARVSDWGFAFDIWGQGTMVTVSQ ZKA160 SEQIDNO. Aminoacidsequence CDRH1 215 GGSITSYS CDRH2 216 IFYSGST CDRH3 217 ARDQTMPVWVGGMDV VH 218 QVQLQESGPGLVKPSETLSLTCTVSGGSITSYSWSWIRQPP GKGLEWIGYIFYSGSTDYNPSLKSRVTISVDTSKDQFSLRL RSVTAADTAVYYCARDQTMPVWVGGMDVWGQGTTVTVSS ZKA172 SEQIDNO. Aminoacidsequence CDRH.sub.1 219 GYIFTRYW CDRH.sub.2 220 IDPSDSYT CDRH.sub.3 221 ARQETAREDGMAV VH 222 EVQLVQSGAEVKKPGKSLRISCKGSGYIFTRYWISWVRQMP GKGLEWMGRIDPSDSYTNYSPSFQGHVTISADKSISTAYLQ WSSLKASDTAMYYCARQETAREDGMAVWGQGTTVTVSS ZKA174 SEQIDNO. Aminoacidsequence CDRH.sub.1 223 GGSMSNSYYH CDRH.sub.2 224 IYYSGST CDRH.sub.3 225 ARNPVFNPLTLTHDAFDI VH 226 QLQLQESGPGLVKPSETLSLTCTVSGGSMSNSYYHWGWIRQ PPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSL KLNSVTAADTAVYYCARNPVFNPLTLTHDAFDIWGQGTMVT VSS ZKA189 SEQIDNO. Aminoacidsequence CDRH.sub.1 227 GFTFSSYA CDRH.sub.2 228 ISGSGDNT CDRH.sub.3 229 AKWPYYDFWSGSESYFDP VH 230 GVQLLESGGALVQPGKSLRLSCAASGFTFSSYALTWVRQAP GKGLQWVSAISGSGDNTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKWPYYDFWSGSESYFDPWGQGTLVTV SS ZKA195 SEQIDNO. Aminoacidsequence CDRH.sub.1 231 GYNFPSYW CDRH.sub.2 232 IDPSDSYT CDRH.sub.3 233 ARADCRSTSCYLVFE VH 234 EVQLVQSGAEVKKPGESLRISCKDSGYNFPSYWIHWVRQMP GKGLEWMGTIDPSDSYTNYSPSFQGHVTISADKSISTAYLQ WSSLKASDTAMYYCARADCRSTSCYLVFEGQGTLVTVSS ZKA215 SEQIDNO. Aminoacidsequence CDRH.sub.1 235 GYTFTSYW CDRH.sub.2 236 IDPSDSHT CDRH.sub.3 237 ARHALPNYFDS VH 238 EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWISWVRQMP GKGLEWMGRIDPSDSHTDYSPSFQGHVTISADKSISAAYLQ WSSLKASDTAMYYCARHALPNYFDSWGQGTLVTVSS ZKA218 SEQIDNO. Aminoacidsequence CDRH.sub.1 239 GFPFSSYW CDRH.sub.2 240 INSDGRNT CDRH.sub.3 241 ARGGYDYDSSGCFDY VH 242 EVQLVESGGGLVQPGGSLRLSCAASGFPFSSYWMHWVRQAP GKGLVWVSRINSDGRNTNYADSVKGRFTISRDNAENTVYLQ MNSLRAEDTAVYYCARGGYDYDSSGCFDYWGQGTLVTVSS ZKB75 SEQIDNO. Aminoacidsequence CDRH.sub.1 243 GFTFSNYA CDRH.sub.2 244 ISGTGGST CDRH.sub.3 245 AKDSASRGGYCSGGVCYLNPGHHDY VH 246 EVQVLESGGGLLQPGGSLRLSCAASGFTFSNYAMSWVRQAP GKGLEWVSTISGTGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKDSASRGGYCSGGVCYLNPGHHDYWG QGTLVTVSS ZKB83 SEQIDNO. Aminoacidsequence CDRH.sub.1 247 GYSFTNYW CDRH.sub.2 248 IDPSDSYT CDRH.sub.3 249 ARLRGSLYCSGGRCYSVPGETPNWFDP VH 250 EVQLVQSGAEVKKPGESLRISCKGSGYSFTNYWITWVRQMP GKGLEWMGSIDPSDSYTNYSPSFQGHVTISADWSINTAYLQ WSSLKASDTAKYYCARLRGSLYCSGGRCYSVPGETPNWFDP WGQGTLVTVSS ZKC3 SEQIDNO. Aminoacidsequence CDRH.sub.1 251 GGSITSYY CDRH.sub.2 252 IYYSGST CDRH.sub.3 253 ARVGGAPYYYYGMDV VH 254 QVQLQESGPGLVKPSETLSLTCTVSGGSITSYYWSWIRQPP GKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKL SSVTAADTAVYYCARVGGAPYYYYGMDVWGQGTTVTVSS ZKC18 SEQIDNO. Aminoacidsequence CDRH.sub.1 255 GFTFGDYA CDRH.sub.2 256 IRSKAYGGTT CDRH.sub.3 257 SRDHTGTTYAFDI VH 258 EVQLVESGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAP GKGLEWVGFIRSKAYGGTTEYAASVKGRFTISRDDSKSIAY LQMNSLKTEDTAVYYCSRDHTGTTYAFDIWGQGTMVTVSQ ZKD1 SEQIDNO. Aminoacidsequence CDRH.sub.1 259 GFTFSSYG CDRH.sub.2 260 IWYDGSNK CDRH.sub.3 261 ARDRRGYGDYVGYYYGMDV VH 262 QVQLVESGGGVVQPGRSLRLSCASGFTFSSYGMHWVRQAP GKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCARDRRGYGDYVGYYYGMDVWGQGTTVT VSS Name SEQIDNO. Aminoacidsequence ZIKVEDIII 263 TAAFTFTKXPAEXXHGTVTVEXQYXGXDGPCKXPXQ generic MAVDXQTLTPVGRLITANPVITEXTENSKMMLELDPP FGDSYIVIGXGXKKITHHWHRS ZIKV 264 TAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQM H/PF/2613 AVDMQTLTPVGRLITANPVITESTENSKMMLELDPPF EDIII GDSYIVIGVGEKKITHHWHRS ZIKVEDIII 265 X.sub.1GX.sub.2X.sub.3YSLCTAAFTFTKX.sub.4PAEX.sub.5X.sub.6HGTVTVEX.sub.7QYX.sub.8 generic GX.sub.9DGPCKX.sub.10PX.sub.11QMAVDX.sub.12QTLTPVGRLITANPVITE X.sub.13TX.sub.14NSKMMLELDPPFGDSYIVIGX.sub.15GX.sub.16X.sub.17KITHH WHRSG wherein X.sub.1maybeany(naturallyoccurring)aminoacid,preferably K,A,orE; X.sub.2maybeany(naturallyoccurring)aminoacid,preferably V,F,orL; X.sub.3maybeany(naturallyoccurring)aminoacid,preferably SorF; X.sub.4maybeany(naturallyoccurring)aminoacid,preferably IorV; X.sub.5maybeany(naturallyoccurring)aminoacid,preferably TorV; X.sub.6maybeany(naturallyoccurring)aminoacid,preferably LorD; X.sub.7maybeany(naturallyoccurring)aminoacid,preferably VorG; X.sub.8maybeany(naturallyoccurring)aminoacid,preferably AorG; X.sub.9maybeany(naturallyoccurring)aminoacidexceptR, preferablyTorA; X.sub.10maybeany(naturallyoccurring)aminoacid,preferably VorI; X.sub.11maybeany(naturallyoccurring)aminoacid,preferably AorV; X.sub.12maybeany(naturallyoccurring)aminoacid,preferably MorT; X.sub.13maybeany(naturallyoccurring)aminoacid,preferably SorG; X.sub.14maybeany(naturallyoccurring)aminoacid,preferably EorK; X.sub.15maybeany(naturallyoccurring)aminoacid,preferably VorI; X.sub.16maybeany(naturallyoccurring)aminoacid,preferably E,A,K,orD;and X.sub.17maybeany(naturallyoccurring)aminoacid,preferably E,A,orK,morepreferablyKorA Zika-E-F.sub.1 266 TGCAAACGCGGTCGCAAACCTGGTTG primer ZIKV-E-R.sub.1 267 CGTGCCAAGGTAATGGAATGTCGTG primer ZIKV-Ef.sub.1530 268 AGCCTAGGACTTGATTGTGAACCGA primer ZIKV-E- 269 TTACAGATCCCACAACGACCGTCAG R.sub.2769primer ZIKV-E-F.sub.2 270 ACTTGGTCATGATACTGCTGATTGC ZIKV-E-R.sub.2 271 TCGGTTCACAATCAAGTCCTAGGCT ZIKV-E-f.sub.2058 272 GCTAACCCCGTAATCACTGAAAGCA ZIKV-E- 273 AAGACTGCCATTCTCTTGGCACCTC r.sub.2248 *the sequences highlighted in bold are CDR regions (nucleotide or aa) and the underlined residues are mutated residues as compared to the germlinesequence.