Multispecific antibodies specifically binding to Zika virus epitopes

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 at least two distinct Zika virus epitopes, that comprises a first heavy chain comprising CDRH1, CDRH2, and CDRH3 amino acid sequences and a first light chain comprising CDRL1, CDRL2, and CDRL3 amino acid sequences, and a second heavy chain comprising CDRH1, CDRH2, and CDRH3 amino acid sequences and a second light chain comprising CDRL1, CDRL2, and CDRL3 amino acid sequences, each according to one of: (a) SEQ ID NOs: 1-5 and 7, respectively; (b) SEQ ID NOs: 1-4 and 6-7, respectively; (c) SEQ ID NOs: 19-23 and 25, respectively; (d) SEQ ID NOs: 19-22 and 24-25, respectively; (e) SEQ ID NOs: 37-41 and 34, respectively; (f) SEQ ID NOs: 37-40 and 42-43, respectively; (g) SEQ ID NOs: 55-59 and 61, respectively; (h) SEQ ID NOs: 55-58 and 60-61, respectively; (i) SEQ ID NOs: 73-77 and 79, respectively; or (j) SEQ ID NOs: 73-76 and 78-79, respectively, wherein the first heavy chain and first light chain and the second heavy chain and second light chain are different.

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. 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.

4. 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.

5. 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.

6. 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.

7. 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 (ii) according to SEQ ID NOs: 1-4 and 6-7; 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 (ii) according to SEQ ID NOs: 19-22 and 24-25.

8. 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 (ii) according to SEQ ID NOs: 1-4 and 6-7; 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 (ii) according to SEQ ID NOs: 37-40 and 42-43.

9. 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% sequence identity and 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% 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% sequence identity and 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% 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% sequence identity and 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% 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% sequence identity and 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% 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% sequence identity and 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% sequence identity.

10. 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% sequence identity and 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% 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% sequence identity and 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% sequence identity.

11. 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% sequence identity and 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% 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% sequence identity and 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% sequence identity.

12. 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 (ii) according to SEQ ID NOs: 1-4 and 6-7.

13. The antibody, or the antigen binding fragment thereof, according to claim 12, 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% sequence identity and 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% sequence identity.

14. The antibody, or the antigen binding fragment thereof, according to claim 12, 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 (ii) according to SEQ ID NOs: 19-22 and 24-25.

15. The antibody, or the antigen binding fragment thereof, according to claim 14, 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% sequence identity and 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% sequence identity.

16. The antibody, or the antigen binding fragment thereof, according to claim 3, characterized in that the antibody, or the antigen binding fragment thereof, is bispecific.

17. The antibody, or the antigen binding fragment thereof, according to claim 4, characterized in that the antibody, or the antigen binding fragment thereof, is tetravalent.

18. The antibody, or the antigen binding fragment thereof, according to claim 1, characterized in that the antibody, or the antigen binding fragment thereof, is bispecific, trispecific or tetraspecific.

19. A pharmaceutical composition comprising the antibody, or the antigen binding fragment thereof, according to claim 1.

20. 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.

21. An isolated multispecific antibody, or an antigen binding fragment thereof, specifically binding to at least two distinct Zika virus epitopes, that comprises a heavy chain comprising CDRH1, CDRH2, and CDRH3 amino acid sequences and a light chain comprising CDRL1, CDRL2, and CDRL3 amino acid sequences according to: (a) SEQ ID NOs: 1-5 and 7, respectively; (b) SEQ ID NOs: 1-4 and 6-7, respectively; (c) SEQ ID NOs: 19-23 and 25, respectively; (d) SEQ ID NOs: 19-22 and 24-25, respectively; (e) SEQ ID NOs: 37-41 and 34, respectively; (f) SEQ ID NOs: 37-40 and 42-43, respectively; (g) SEQ ID NOs: 55-59 and 61, respectively; (h) SEQ ID NOs: 55-58 and 60-61, respectively; (i) SEQ ID NOs: 73-77 and 79, respectively; or (j) SEQ ID NOs: 73-76 and 78-79, respectively.

Description

DESCRIPTION OF FIGURES

(1) FIG. 1 shows the reactivity (ELISA) and ZIKV and DENV1 neutralizing activity of antibodies derived from four ZIKV immune donors (ZKA, ZKB, ZKC and ZKD) to E protein of ZIKV and DENV1-4 and to EDIII-domain of ZIKV E protein; NNB-neutralizing, non-E-protein binding antibodies.

(2) FIG. 2 shows the binding of ZKA190, ZKA78 and ZKA64 antibodies to ZIKV and DENV1 E and to ZIKV EDIII proteins as measured by ELISA.

(3) FIG. 3 shows the binding of ZKA185 and ZKA190 antibodies to ZIKV E, DENV1 VLP and to ZIKV EDIII proteins as measured by ELISA.

(4) FIG. 4 shows for Example 3 the neutralizing activity of ZKA190, ZKA64, ZKA64-LALA, ZKA230 and ZKA78 antibodies against ZIKV (H/PF/2013 strain) and DENV1 on Vero cells as measured by flow-cytometry (% of infected cells).

(5) FIG. 5 shows for Example 3 the neutralizing activity of ZKA190, ZKA64, ZKA185, ZKA230 and ZKA78 antibodies against ZIKV (H/PF/2013 strain) on Vero cells as measured with a cell viability readout (wst-1, Roche).

(6) FIG. 6 shows for Example 4 the infection enhancing activity (ADE, antibody-dependent enhancement) of ZKA190, ZKA64, ZKA64-LALA, ZKA185, ZKA230 and ZKA78 antibodies for ZIKV (H/PF/2013 strain) on non-permissive K562 cells as measured by flow-cytometry (% of infected cells).

(7) FIG. 7 shows for Example 4 that four ZIKV-immune plasma and one DENV-immune plasma showed similar capacity to enhance ZIKV infection of K562 cells (upper panel). This ADE effect was completely blocked in all five immune plasma by the EDIII-specific ZKA64-LALA antibody (lower panel).

(8) FIG. 8 shows the amino acid alignment of the EDIII region of 39 ZIKV strains from the Asian lineage since 2013 (including the prototypic strain MR766 of the African lineage isolated in 1947).

(9) FIG. 9 shows for Example 3 the neutralizing activity of ZKA190 and ZKA190-LALA antibody against three strains of ZIKV (H/PF/2013, MR766 and MRS_OPY_Martinique_PaRi_2015) on Vero cells as measured by flow-cytometry (% of infected cells).

(10) FIG. 10 shows for Example 5 neutralization of ZKA190 and CS mAbs tested against a panel of four strains of ZIKV, as determined by the percentage of infected Vero cells in the presence of increasing amounts of the mAbs (A). Shown are also the IC50 values (B) and statistics (C). Data are representative of at least two independent experiments.

(11) FIG. 11 shows for Example 6 the neutralization and enhancement of ZIKV infection by antibody ZKA190. (A) Neutralization of ZIKV PRVABC59 strain infection of hNPCs by ZKA190, ZKA190-LALA and a control mAb as determined by plaque assay on Vero cells (left panel) and indirect immunofluorescence of infected hNPCs using fluorophore-labelled anti-E antibody (right panel). (B) ADE of ZIKV infection of non-permissive K562 cells by ZKA190 and ZKA190-LALA. (C) ADE induced in K562 cells when ZIKV is pre-incubated with serial dilutions of plasma serum from different ZIKV-positive patients (left panel). When ZKA190 LALA is added to the ZIKV-serum complexes, ADE is inhibited (right panel). (D) ADE induced in K562 cells when ZIKV is pre-incubated with serial dilutions of a prM cross-reactive mAb (DV62) derived from a DENV-immune donor. ZKA190-LALA inhibits ADE of ZIKV when complexed with prM-reactive antibody DV62. (E) Effect on ADE induced by peak enhancing dilution of a DENV2 plasma (left panel) or anti-prM DV62 mAb (right panel) by serial dilutions of indicated mAbs.

(12) FIG. 12 shows for Example 7 the identification of ZKA190 epitope and analysis of its conservation in ZIKV strains. (A) Overlay of [.sup.15N,.sup.1H]-HSQC spectra of .sup.15N-labeled ZIKV EDIII in absence (black) or presence (red) of unlabelled ZKA190 Fab. Differences identify EDIII residues affected by antibody binding. (B) NMR epitope mapping of ZKA190 Fab in complex with ZKV EDIII. The chemical shift perturbation (CSP, y-axis) is plotted against the EDIII residue number. Residues affected by antibody binding are in red. (C) Residues in FG loop identified by NMR epitope mapping is partially hidden in E protein mol A but largely exposed in mols B and C. EDIII of E protein was coloured in blue. Residues identified by NMR epitope mapping are coloured in magenta except those in the FG loop are coloured in green. Adjacent E proteins are shown as grey surface. (D) Level of amino acid residue conservation in ZKA190 epitope as calculated by the analysis of sequences from 217 ZIKV strains found in ZIKV Resources (NCBI) databases as of Nov. 24, 2016. (E) Open-book representation showing charge complementarity between the epitope and paratope of the docking result. Boundaries of the epitope and paratope are circled in green. The borders between heavy and light chains of Fab and its corresponding footprint on EDIII are shown as yellow dashed lines.

(13) FIG. 13 shows for Example 7 the ZKA190 epitope identified by NMR and Docking. (A) Cartoon representation of the 12 lowest energy NMR structures of ZIKV EDIII, with residues affected by ZKA190 binding in red. Flexibility in the N-terminus of the construct is apparent. (B) Model of the ZKA190:EDIII complex derived by computational docking and molecular simulation validated by NMR results. The NMR identified epitope on EDIII (grey) is in red. The ZKA190 heavy and light chain are colored in dark and light green, respectively. EDIII residues that affect or not antibody binding when mutated are shown as orange and blue sticks, respectively. (C) NMR identified ZKA190 epitope (red) is accessible on the virus surface (white).

(14) FIG. 14 shows for Examples 7 and 10 the binding of wt or mutated EDIII to ZKA190 IgG. SPR data and binding kinetics are shown. EDIII mutants that affect (red highlights) or do not affect binding are shown as indicated in the figure.

(15) FIG. 15 shows for Example 8 the results of the confocal microscopy experiments. ZIKV incubated with a concentration exceeding 10,000-fold the 1050 value of either ZKA190 Fab or full IgG were added to Vero cells. The ZIKV:antibody complex is detected inside the cells (green) and co-localizes with endosomes (red, yellow overlay). Endosomes and acidic organelles are marked by Lysotracker red; Alexa-488 conjugated ZKA190 is in green. Nuclei are stained with DAPI (blue).

(16) FIG. 16 shows for Example 9 prophylactic and therapeutic efficacy of ZKA190. (A) ZKA190 is strongly protective against ZIKV infection when administered prophylactically to mice (A129 in (A) and AG129 in (B)) challenged with a lethal dose of ZIKV strain MP17451. Experiments used N=4-8 mice per group. Kaplan-Meier survival curves are shown (A). Significance was determined by using the Mantel-Cox log-rank test. Panel A, top left: ZKA190 at 5, 1 and 0.2 mg/kg versus Ctr mAb, P=0.0031; ZKA190 at 0.04 mg/kg versus Ctr mAb, P=0.0116; ZKA190-LALA at 5, 1, 0.2 and 0.04 mg/kg versus Ctr mAb, P=0.0031. Panel A, top right: Morbidity score of mice monitored over a 14-15 day period (two different scoring methods were used; see (Dowall, S. D., Graham, V. A., Rayner, E., Atkinson, B., Hall, G., Watson, R. J., Bosworth, A., Bonney, L. C., Kitchen, S., and Hewson, R. (2016). A Susceptible Mouse Model for Zika Virus Infection. PLoS Negl Trop Dis 10, e0004658-13). Panel A, lower panels: body weight of mice. Panels B: ZKA190 or ZKA190-LALA were administered at 15 mg/kg at different time-points after ZIKV infection. Panel B, top left: A Kaplan-Meier survival curve is shown. Experiments used N=5 mice per group. Significance was determined by using the Mantel-Cox log-rank test. ZKA190 and ZKA190-LALA given either on day 1, 2, 3 or 4 versus Ctr., P=0.0016. Panel B, top right: Morbidity score of mice monitored over a 14-day according to (Dowall et al., 2016). Mice were monitored over a 14 day period for body weight loss (Panel B, lower panels). Control antibody is MPEG specific for RSV F protein (Corti, D., et al. Cross-neutralization of four paramyxoviruses by a human monoclonal antibody. Nature 501, 439-443 (2013)).

(17) FIG. 17 shows for Example 9 the prophylactic efficacy of the anti-ZIKV EDIII-specific mAb ZKA190 against ZIKV strains MP1741. (A) Shown is the viremia measured as PFU/ml on day 5 in blood of all animals. (B) Viral load was measured as genomic copies/ml by qPCR on day 5 in blood of all animals and in blood and indicated tissues when animals were culled at the end of the study or when the humane end points were met. (C) Mice were monitored over a 14 day period for body weight loss (D) Human serum IgG concentration in day 5 blood samples. Significance was determined compared to control antibody treatment by nonparametric unpaired Mann-Whitney U test. *p<0.05; **p<0.01; ***p<0.001.

(18) FIG. 18 shows for Example 9 the therapeutic efficacy of the anti-ZIKV EDIII-specific mAb ZKA190. (A) Viral loads were measured as PFUs on day 5 in blood of all animals. (B) Viral loads were measured as genomic copies by qPCR on day 5 in blood of all animals and in blood and indicated tissues when animals were culled at the end of the study or when the human end points were met. Significance was determined compared to control antibody treatment by nonparametric unpaired Mann-Whitney U test. *p<0.05; **p<0.01. (C) Human serum IgG concentration in day 5 blood samples.

(19) FIG. 19 shows for Example 11 and 12 the engineering of ZKA190 into the bispecific FIT-1 mAb and its in vitro characterization. (A) ZKA185 and ZKA230 mAbs were tested for neutralization of four strains of ZIKV, as determined by the percentage of infected Vero cells in the presence of increasing amounts of mAbs. Data are representative of at least two independent experiments. (B) Binding of ZKA185 and ZKA230 IgG and Fab to recombinant ZIKV VLP, E and DIII antigens as assessed by ELISA. (C) ZKA190, ZKA185 and ZKA230 were tested for neutralization of H/PF/2013 (wt) and MARMs 1-4. (D) Surface representation of two E protein dimers bound by ZKA190 (green); the ZKA190 NMR derived epitope is in red; positions mutated in MARMs are indicated in yellow (E370), blue (T335), orange (D67) and magenta (K84). (E) Model of FIT-1. The natural linkers between inner and outer Fabs allow a flexible movement of Fabs in the FIT-1 antibody. The variable regions of ZKA185 and ZKA190 are highlighted in blue and green, respectively. (F) Binding of FIT-1 IgG and Fab to recombinant ZIKV VLP, E and DIII antigens as assessed by ELISA. (G) ZKA190, ZKA185 and FIT-1 mAbs were tested for neutralization of four strains (IC50 values, G) and four MARMs (H) of ZIKV. (I) Neutralization of ZIKV H/PF/2013 strain by ZKA185, ZKA230 and FIT-1 IgG and Fab determined as in (A). (J) Confocal microscopy experiments as shown in FIG. 3G. (K) Effect on ADE induced by peak enhancing dilution of anti-prM DV62 mAb or DENV2 plasma by serial dilutions of FIT-1 IgG and Fab.

(20) FIG. 20 shows the therapeutic efficacy of FIT-1. FIT-1 is strongly effective against ZIKV infection when administered therapeutically at different time-points to mice (A129) challenged with a lethal dose of ZIKV strain MP17451. Experiments used N=5-6 mice per group. (A) Kaplan-Meier survival curves are shown. Significance was determined by using the Mantel-Cox log-rank test. FIT-1 at 15, 5 and 1 mg/kg given either on day 1, 2 versus Ctr mAb, P=0.0012; ZKA190 at 15 and 5 mg/kg given on day 3 versus Ctr mAb, P=0.0012; ZKA190 at 1 mg/kg given on day 3 versus Ctr mAb, P=0.0170. (B) Morbidity score of mice monitored over a 21 day period (Dowall et al., 2016). (C) Viral loads were measured as PFUs on day 5 in blood of all animals. (D) Mice were monitored over a 21 day period for body weight loss. Control mAb in panel A is MPEG mAb (specific for RSV F protein (Corti, D., et al. Cross-neutralization of four paramyxoviruses by a human monoclonal antibody. Nature 501, 439-443 (2013)). (E) Viral loads were measured as genomic copies by qPCR on day 5 in blood of all animals and in blood and indicated tissues when animals were culled at the end of the study or when the human end points were met. Significance was determined compared to control antibody treatment by nonparametric unpaired Mann-Whitney U test. *p<0.05; **p<0.01.

(21) FIG. 21 shows for Example 14 that female AG129 mice treated with FIT-1 after challenge with Malaysian ZIKV were protected from mortality as compared with placebo-treated mice.

(22) FIG. 22 shows for Example 14 intrauterine growth restriction (IUGR) in pups born to females treated with FIT-1 and challenged with ZIKV.

(23) FIG. 23 shows for Example 14 average weight of pups on the date of birth born to females treated with FIT-1 and infected with ZIKV.

(24) FIG. 24 shows for Example 14 the weight of placenta collected 11 dpi from females treated with FIT-1.

(25) FIG. 25 shows for Example 14 quantification of viral RNA in (A) fetus, (B) placenta, (C) maternal spleen and (D) maternal brain (***P<0.001, **P<0.01, as compared with MPE8 treatment).

(26) FIG. 26 shows for Example 15 the survival of male AG129 mice infected with ZIKV and treated with FIT-1 24 or 72 h after virus challenge (*P<0.05, as compared with MPE8 negative control treatment).

(27) FIG. 27 shows for Example 15 mean percent weight change of AG129 mice treated with FIT-1 at various times after challenge with ZIKV.

(28) FIG. 28 shows for Example 15 the disease score of the A) testicle or B) epididymis of male AG129 mice treated with FIT-1 24 or 72 h after challenge with ZIKV. Treatment with reactive Ab results in reduction in disease of the testicle and the epididymis.

(29) FIG. 29 shows for Example 16 the viral load in serum of the 3 groups tested. The horizontal line indicates the LLOQ of 860 GC/mL.

EXAMPLES

(30) 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

(31) 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 FIG. 1.

(32) 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, New York, 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.

(33) FIG. 1 provides an overview over selected ZIKV neutralizing antibodies (cf Tables 1 and 2 for the amino acid sequences of their CDRs and heavy/light chain variable regions). The last two columns of FIG. 1 provide the neutralization activities (IC.sub.50) of ZIKV and DENV1 (if tested). The other columns provide binding activities (EC.sub.50) of the antibodies to ZIKV E protein (ZIKV E), DENV1 E protein (DENV1 E), DENV2 E protein (DENV2 E), DENV3 E protein (DENV3 E), DENV4 E protein (DENV4 E), DENV1 virus-like particle (DENV1 VLP), DENV2 virus-like particle (DENV2 VLP), DENV3 virus-like particle (DENV3 VLP), DENV4 virus-like particle (DENV4 VLP), and to EDIII-domain of ZIKV E protein (DIII ZKA).

Example 2: Characterization of Antibodies ZKA190, ZKA185, ZKA230, ZKA64 and ZKA78

(34) 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 (EC50) required to achieve 50% maximal binding at saturation.

(35) Results are shown in FIGS. 3 and 4. Of note, ZKA64 and ZKA190 bound to ZIKV E and ZIKV EDIII (DIII ZI) with low EC50 values, thereby indicating that ZKA64 and ZKA190 are binding to domain III of ZIKV E protein (EDIII). ZKA78 bound to ZIKV E, but not to ZIKV EDIII, indicating that ZKA78 is binding to ZIKV E, but not targeting the EDIII region. Despite their considerable ZIKV neutralizing activity (cf FIG. 1), antibodies ZKA185 and ZKA230 did not show any detectable binding to ZIKV E and ZIKV EDIII (FIG. 3). Accordingly, ZKA185 and ZKA230 were referred to as neutralizing-non-E-binding (NNB) antibodies. Those NNB antibodies are assumed to recognize quaternary epitopes that are displayed on the ZIKV infectious virions but not on soluble proteins.

(36) Moreover, none of ZKA190, ZKA185, ZKA230, and ZKA64 showed any detectable binding to DENV E proteins (FIG. 1, DENV1-4 serotypes, and FIGS. 3 and 4), indicating that ZKA190, ZKA185, ZKA230, and ZKA64 are specific for ZIKV and not cross-reactive to dengue virus. ZKA78, in contrast, which is assumed to bind to ZIKV EDI/II, but not to ZIKV EDIII (cf FIG. 2), bound to DENV E proteins (FIGS. 1 and 3), indicating that ZKA78 is a cross-reactive antibody binding to both, ZIKV and DENV.

(37) 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. FIG. 1).

Example 3: The Isolated Antibodies Potently Neutralize ZIKV Infection

(38) The isolated antibodies ZKA190, ZKA185, ZKA230, ZKA64 and ZKA78 were tested for their ability to neutralize ZIKV and DENV1 infection in vitro.

(39) 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.

(40) Results are shown in FIGS. 4, 5 and 9. The EDIII-specific mAbs ZKA64 and ZKA190 and the NNB mAb ZKA230 were highly potent in ZIKV neutralization (strain H/PF/2013), with IC50 values of 93, 9 and 10 ng/ml, respectively (FIG. 4, upper panel). In contrast, the cross-reactive antibody ZKA78 only partially neutralized ZIKV infectivity and cross-neutralized DENV1 infectivity (FIG. 4, lower panels). Similar data were obtained by measuring the ZIKV-induced cytopathic effect as measured with the WST-1 reagent (FIG. 5). In this second assay, NNB antibody ZKA185 was also included in the panel of tested antibodies and showed an IC50 similar to the most potent antibodies ZKA190 (EDIII-specific) and ZKA230 (NNB).

(41) 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 (FIG. 1 and FIG. 2). ZKA190 and ZKA190-LALA was also confirmed to effectively neutralize two additional ZIKV strains (MR766 and MRS_OPY_Martinique_PaRi_2015) (FIG. 9). Taken together the results indicate that the ultra-potent ZKA64 and ZKA190 antibodies cross-react with multiple strains of ZIKV belonging to different genotypes and origins (East African and Asian from Uganda, French Polynesia, Martinique and Brazil).

Example 4: The LALA Mutation Inhibits Antibody-Dependent Enhancement of ZIKV Infection by Serum Antibodies

(42) 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.

(43) Results are shown in FIG. 6. All antibodies enhanced infection of ZIKV in the non-permissive K562 cells at a broad range of concentrations, including those that fully neutralized ZIKV infection on Vero cells (FIG. 6). Of note, while EDIII-specific antibodies ZKA64 and ZKA190 fully neutralized ZIKV infections of K562 cells above 1 g/ml, the NNB antibody ZKA230 failed to do so, a result that might be due to the different mechanisms of neutralization of free viruses versus Fc-gamma-receptor-internalized viruses. In contrast, the cross-reactive ZKA78 that only partially neutralized ZIKV infectivity, effectively enhanced ZIKV infection of K562 cells. These results show that cross-reactive antibodies elicited by either ZIKV or DENV infection can mediate heterologous ADE.

(44) 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.

(45) 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.

(46) Results are shown in FIG. 7. In a homologous setting, four ZIKV-immune plasma collected from convalescent patients and one DENV-immune plasma showed similar capacity to enhance ZIKV infection of K562 cells (FIG. 7, upper panel), and this ADE effect was completely blocked by the EDIII-specific ZKA64-LALA antibody (FIG. 7, lower panel).

(47) 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.

(48) 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

(49) 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).

(50) 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.

(51) Results are shown in FIG. 10. ZKA190 mAb potently neutralized African, Asian and American strains with an IC50 ranging from 0.6 to 8 ng/ml. In comparison, prior art antibody C8 was about 24-fold less potent.

Example 6: Further Characterization of Antibody ZKA190

(52) 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, NY, 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.

(53) As shown in FIG. 10A and described in Example 5, ZKA190 was tested against a panel of four ZIKV strains. ZKA190 mAb potently neutralized African, Asian and American strains with an IC50 ranging from 0.004 to 0.05 nM (FIG. 10A; 0.6 to 8 ng/ml).

(54) 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 FIG. 11A. Both, ZKA190 and ZKA190-LALA, fully abolished infection and replication of ZIKV in hNPCs.

(55) 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 FIG. 11B. ZKA190 supports ADE from 0.0001 to 1 nM; as expected, ZKA190-LALA did not show any ADE activity. The ability of ZKA190-LALA to inhibit ADE induced by plasma from four ZIKV-immune donors in K562 cells was also tested. Results are shown in FIG. 11C. It was found that ZKA190-LALA completely inhibited the ADE induced by plasma antibodies (FIG. 11C).

(56) 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 FIG. 11D. DV62 cross-reacted with ZIKV prM protein and caused ADE at a broad range of concentrations (FIG. 11D). ZKA190-LALA can fully block anti-prM DV62 mAb-induced ADE of immature or partially immature ZIKV particles (FIG. 11D).

(57) 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 FIG. 11E. ZKA190 at low concentrations increased the prM DV62-mediated ADE of ZIKV infection, consistent with its ability to promote the entry of both immature and mature virions, while at concentrations above 1.3 nM (i.e., 200 ng/ml) ZKA190 blocked ADE induced by both DENV plasma and mAb DV62. ZKA190-LALA, as well as its Fab fragment, reduced ADE at concentrations above 0.06 nM, indicating that both inhibited virus infection at a post-attachment step, such as fusion.

Example 7: ZKA190 Binds to a Conserved and Highly Accessible Region of EDIII

(58) 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.

(59) 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.

(60) 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 (FIG. 12A), residues affected by ZKA190 were mapped to the LR of EDIII, in particular to the BC, DE and FG loops, as well as to part of the EDI-EDIII hinge (FIG. 13A). These residues are nearly identical among 217 known ZIKV strains, with the exception of substitutions at V3411 and E393D in the Uganda 1947 isolate (FIG. 12D). These mutations are also present in the MR766 strain that was efficiently neutralized by ZKA190 (FIG. 10A). Analysis of the ZKA190 epitope on the uncomplexed ZIKV structure showed that the epitope is highly accessible, except for the FG loop in the 5-fold vertex (FIGS. 13B and 12C, molecule A).

(61) 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 (FIGS. 13B and 12E). Docking indicates that there are no direct contacts between ZKA190 and the FG loop on EDIII, suggesting that changes in its NMR signals upon antibody binding derive from allosteric effects. This notion is supported by the fact that mutations of FG loop residues in recombinant EDIII, but not in other epitope regions, did not affect the binding affinity of ZKA190 for EDIII (FIGS. 13B and 14).

Example 8: Mechanisms of ZKA190 Neutralization

(62) 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.

(63) 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.

(64) 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.

(65) Results are shown in FIG. 15. Confocal microscopy analysis shows that ZKA190 (Fab or IgG) can enter Vero cells only when complexed with ZIKV, at neutralizing concentrations exceeding the IC50 by 10,000-fold (FIG. 15).

Example 9: In Vivo Characterization of the EDIII-Specific mAb ZKA190

(66) 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.

(67) 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.

(68) 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.

(69) Results are shown in FIGS. 16, 17 and 18. ZKA190 and ZKA190-LALA were shown to protect mice from mortality and morbidity at concentrations of 5, 1 or 0.2 mg/kg (FIG. 16A-B). ZKA190-LALA, and to a lesser extent ZKA190, delayed morbidity and mortality as compared to the control group at 0.04 mg/kg. Viral titers in blood and organs were reduced significantly compared to control antibody-treated animals, even in the presence of serum antibody levels below 1 g/ml (FIG. 17A-D).

(70) 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 (FIG. 16E-G). ZKA190 and ZKA190-LALA treatment at all post-infection time-points resulted in significantly reduced viral titers, compared to animals treated with control antibody, with a clear trend for greater reduction with earlier treatment (FIG. 18A-16C). Of note, ZKA190-LALA showed a significantly reduced antiviral activity in the blood day 5 sample as compared to ZKA190 when mAbs were given four days post-infection, a result that might be related to the impaired ability of the LALA variant to facilitate rapid clearance of coated virions. Of the 16 treated mice, one in vivo escape mutant (Monoclonal Antibody Resistant Mutant 1, MARM1), containing an amino acid substitution in DIII (T335R, in the center of the epitope) was isolated, while viruses from the other treated mice did not contain any E mutations. Introduction of the T335R mutation into recombinant DIII showed that it abrogated ZKA190 binding, as determined by SPR (FIG. 14; cf Example 7 for experimental methods).

Example 10: In Vitro Selection of ZIKV Escape Mutants

(71) 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.

(72) 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.

(73) 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).

(74) 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 (FIG. 14). The mutations in the in vivo (T335R) and in vitro (E370K) MARMS are located on the BC and DE loops of DIII, respectively, and are consistent with the epitope identified by NMR.

Example 11: Development of Bispecific Antibodies According to the Present Invention

(75) 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.

(76) 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 (FIG. 19A). ZKA185 binds with high affinity to recombinant ZIKV E protein and to Zika virus-like particles (VLP) but not to the isolated DIII (FIG. 19B). Conversely, ZKA230 bound to ZIKV VLPs, but not to recombinant E or DIII, suggesting that it recognizes a quaternary epitope displayed only on the viral surface (FIG. 19B). ZKA185 IgG and Fab were shown to bind to E and VLP antigens with similar high affinity by ELISA.

(77) 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 (FIG. 19D). MARM4 showed a mixture of different amino acid substitutions at position 84 (from K to G, E or R), confirmed in multiple sequencing experiments. Finally, MARMs 1 to 4 were tested against ZKA190, ZKA185 and ZKA230. ZKA190 neutralized ZKA185 and ZKA230 MARMs as well as the parental virus (FIG. 19C). ZKA185 neutralized both ZKA190 and ZKA230 MARMs. ZKA230 neutralized only ZKA190 MARM2 and did not neutralize either ZKA190 MARM1 or ZKA185 MARM3.

(78) 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.

(79) 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.

(80) 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, IL). 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)

(81) The FIT-Ig bispecific antibody (here designated FIT-1) with ZKA190 in the outer and ZKA185 in the inner Fab positions (FIG. 19E) was selected and further characterized. ELISA showed FIT-1 to bind DIII, E and VLP (FIG. 19F). FIT-1 retained high neutralizing potency against ZIKV strains, with IC50 values largely similar to those of the parental ZKA190 and ZKA185 antibodies (FIG. 19G). FIT-1 was produced using the backbone IgG1 antibody in the LALA format, thereby eliminating any possibility of causing ADE. Several lines of evidence suggest that the ZKA190 and ZKA185 moieties in the FIT-1 format are both active. Firstly, FIT-1 bound to E protein with higher affinity than either the parental ZKA190 and ZKA185 antibodies (KD values: ZKA185 1.8 nM, ZKA190 9.3 nM and FIT-1 KD<1 M due to slower dissociation rate, presumably through avidity effects). Secondly, FIT-1 effectively neutralized all the ZKA190, ZKA185 and ZKA230 MARMs (FIG. 19H) in contrast to the individual mAbs. The neutralizing activity of the Fab fragment of FIT-1 (each comprising one ZKA190 and one ZKA185) was reduced only by about 6-fold (FIG. 19I, right panel).

(82) 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.

(83) Confocal microscopy studies using Vero cells also showed that FIT-1, as ZKA190, likely inhibits virus infection at a post-attachment step, likely fusion (FIG. 19J).

(84) 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 (FIG. 19K), demonstrating that FIT-1 did not cause ADE and could block ADE of monocytic cells by poorly neutralizing cross-reactive antibodies.

Example 13: In Vivo Therapeutic Potential of FIT-1

(85) 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.

(86) Results are shown in FIG. 20. The data show that at a dose of 15 mg/kg, survival rates were 100% without signs of morbidity even when treatment was given three days post-infection (FIG. 20A); viral titers were abrogated and no escape mutants were detected, indicating high in vivo efficacy. Administration of 5 mg/kg resulted in survival rates of 70-100%, also no escape mutants were detected on day 5 after infection. The lowest dose tested (i.e., 1 mg/kg) protected when administered on day 1 or 2, but not day 3, after infection.

Example 14: Effect of FIT-1 on Disease in a Congenital Infection Model in AG129 Mice

(87) 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.

(88) 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.

(89) Materials and Methods:

(90) 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).

(91) 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.

(92) 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.

(93) 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.

(94) 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.

(95) Statistical analysis: Survival data were analyzed using the Wilcoxon log-rank survival analysis (Prism 5, GraphPad Software, Inc).

(96) Results and Discussion:

(97) 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 (FIG. 21). Females treated with a non-specific negative control antibody had a mortality rate that was similar to that observed in previous studies.

(98) 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 (FIG. 22). This difference was less pronounced in regard to fetal weight, where all groups had similar averages (FIG. 23). There was a trend towards higher placenta weight in dams treated with FIT-1 (FIG. 24).

(99) The virus titer of various tissues is shown in FIG. 25. Significant reduction in viral RNA in the fetus and placenta of dams treated with FIT-1 were observed (FIGS. 25A and 25B, respectively). The reduction was especially evident in placental tissues with an approximate 5-log.sub.10 reduction in ZIKV RNA levels. Maternal spleen and brain also had significantly reduced viral RNA in FIT-1-treated animals as compared with MPEG, with several log.sub.10 reductions in levels (FIGS. 25C and 25D, respectively).

(100) Conclusions:

(101) 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

(102) 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.

(103) 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.

(104) Materials and Methods:

(105) Animals: Male AG129 mice were used. Groups of animals were randomly assigned to experimental groups and individually marked with ear tags.

(106) 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.

(107) 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.

(108) 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.

(109) 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.

(110) Statistical analysis: Survival data were analyzed using the Wilcoxon log-rank survival analysis (Prism 5, GraphPad Software, Inc).

(111) Results and Discussion:

(112) 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.

(113) A significant (P<0.0.5) improvement in survival was observed as compared with placebo mAb treatment (FIG. 26). Complete survival was observed in mice treated 24 h post-challenge with FIT-1, while mice treated at 72 h post-virus inoculation resulted in one animal that succumbed to virus infection. This single animal was euthanized 24 days after virus challenge, which was much later than animals treated with placebo (FIG. 26). Since infection of AG-129 mice with ZIKV results in lethality, which is much more severe than a typical natural infection with this virus, high levels of protection that are seen with FIT-1 are very promising.

(114) 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 (FIG. 27).

(115) 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 (FIG. 28), further demonstrating the efficacy of FIT-1 treatment. FIT-1 also protected mice from disease when treatment was initiated 72 h after virus challenge and none of the mice treated at this time had observable disease in the testicle or epididymis (FIG. 28). The majority of mice in the placebo treatment group had inflammation in the testicle (2/3) and epididymis (3/3) (FIG. 28), although disease severity in this study was fairly mild.

(116) Conclusions:

(117) 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

(118) 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.

(119) Methods:

(120) 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.

(121) TABLE-US-00007 TABLE 4 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.

(122) 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

(123) 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.

(124) 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.

(125) 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.

(126) 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.

(127) 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.

(128) Results:

(129) 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.

(130) 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 FIG. 29.

(131) 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.

(132) 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.

(133) 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.

(134) 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.

(135) 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

(136) 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.

(137) TABLE-US-00009 TablesofSequencesandSEQIDNumbers ZKA190 SEQIDNO. Aminoacidsequence CDRH1 1 GFTFSKYG CDRH2 2 ISYEGSNK CDRH3 3 AKSGTQYYDTTGYEYRGLEYFGY CDRL1 4 QSVSSSY CDRL2 5 DAS CDRL2 6 LIYDASSRA long CDRL3 7 QQYGRSRWT VH 8 QVQLVESGGGVVQPGRSLRLSCAASGFTFSKYGMHWVRQAPGKGLE WVAVISYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCAKSGTQYYDTTGYEYRGLEYFGYWGQGTLVTVSS VL 9 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKRGQAPR LLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY GRSRWTFGQGTKVEIK ZKA190 SEQIDNO. Nucleicacidsequence CDRH1 10 ggattcaccttcagtaaatatggc CDRH2 11 atatcatatgagggaagtaataaa CDRH3 12 gcgaaatcggggacccaatactatgatactactggttatg agtataggggtttggaatactttggctac CDRL1 13 cagagtgttagtagcagttac CDRL2 14 gatgcatcc CDRL2 15 ctcatctatgatgcatccagcagggcc long CDRL3 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 CDRH1 19 GYSFTSYW CDRH2 20 FDPSDSQT CDRH3 21 ARRYCSSSSCYVDN CDRL1 22 ALPNKF CDRL2 23 EDN CDRL2 24 VIYEDNKRP long CDRL3 25 YSTDSSSNPLGV VH 26 EVQLVQSGAEVKKPGESLRISCKGSGYSFTSYWITWVRQMPGKGLE WMAKFDPSDSQTNYSPSFQGHVTISVDKSISTAYLQWSSLKASDTA MYYCARRYCSSSSCYVDNWGQGTLVTIFS VL 27 SYELTQPPSVSVSPGQTARITCSGDALPNKFAYWYRQKSGQAPVLV IYEDNKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYHCYSTDS SSNPLGVFGGGTKLTVL ZKA185 SEQIDNO. Nucleicacidsequence CDRH1 28 ggatatagttttaccagttactgg CDRH2 29 tttgatcctagtgactctcaaacc CDRH3 30 gcgagaagatattgtagtagtagtagttgttatgtggacaa t CDRL1 31 gcattgccaaataaattt CDRL2 32 gaggacaac CDRL2 33 gtcatctatgaggacaacaaacgaccc long CDRL3 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 CDRH1 37 GGSISSDY CDRH2 38 IYYSGST CDRH3 39 ARRRKYDSLWGSFAFDI CDRL1 40 SSNIGGNY CDRL2 41 IND CDRL2 42 LICINDHRP long CDRL3 43 ATWDDSLGGLV VH 44 QVQLQESGPGLVKPSETLSLTCAVSGGSISSDYWSWIRQPPGKGLE WIGYIYYSGSTNYNPSLKSRVTISVDTSKNHFSLKLNSVTAADTAV YYCARRRKYDSLWGSFAFDIWGQGTMVTVSS VL 45 QSVLTQPPSASGTPGQRVTISCSGSSSNIGGNYVYWYQQLPGTAPK LLICINDHRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCATW DDSLGGLVFGGGTKLTVL ZKA230 SEQIDNO. Nucleicacidsequence CDRH1 46 ggtggctccatcagtagtgactac CDRH2 47 atctattacagtgggagcacc CDRH3 48 gcgaggaggaggaagtatgattccctttgggggagttttgc ttttgatatc CDRL1 49 agctccaacatcggaggtaattat CDRL2 50 attaatgat CDRL2 51 ctcatctgtattaatgatcaccggccc long CDRL3 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 CDRH1 55 GFTFSNYA CDRH2 56 IGRNGDSI CDRH3 57 VKDLAIPESYRIEADY CDRL1 58 QSVLYRSNNKNY CDRL2 59 WAS CDRL2 60 LIYWASTRE long CDRL3 61 QQYYSSPRT VH 62 EVQLAESGGGLVQPGGSLTLSCSGSGFTFSNYAMVWARQAPGKGLE YVSGIGRNGDSIYYTDSVKGRFTISRDNSKSMVYLQMSSLRTEDTA VYYCVKDLAIPESYRIEADYWGQGTLVIVSA VL 63 DIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNKNYLSWYQQKP GQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISPLQAEDVAVY YCQQYYSSPRTFGQGTKVEIK ZKA78 SEQIDNO. Nucleicacidsequence CDRH1 64 ggcttcacttttagtaactatgca CDRH2 65 atcgggcgcaacggggactctatc CDRH3 66 gtgaaagatctggccatccccgagtcctacagaattgaag ctgattat CDRL1 67 cagtccgtgctgtaccgctctaacaacaagaattac CDRL2 68 tgggcttca CDRL2 69 ctgatctattgggcttcaacccgggaa long CDRL3 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 CDRH1 73 GYTFTGYH CDRH2 74 INPNSGGT CDRH3 75 ARMSSSIWGFDH CDRL1 76 QSVLIN CDRL2 77 GAS CDRL2 78 LIYGASSRA long CDRL3 79 QQYNDWPPIT VH 80 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYHIDWVRQARGQGLE WMGRINPNSGGTNYAQKFQGRVTMTRDTSISTAYMQLSRLRSDDSA VYYCARMSSSIWGFDHWGQGTLVTVSS VL 81 EIVMTQSPATLSVSPGERATLSCRASQSVLINLAWYQQKPGQAPRL LIYGASSRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYN DWPPITFGQGTRLEIK ZKA64 SEQIDNO. Nucleicacidsequence CDRH1 82 ggctacaccttcacagggtatcac CDRH2 83 attaaccctaattctggcgggacc CDRH3 84 gctcggatgagctcctctatttggggcttcgatcat CDRL1 85 cagtctgtgctgattaac CDRL2 86 ggagcatcc CDRL2 87 ctgatctatggagcatcctccagggct long CDRL3 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 IgG1CH1-CH2- 91 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV CH3aa TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K IgG1CH1-CH2- 92 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV CH3LALAaa TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgGCKaa 93 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC IgGCLaa 94 GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLT PEQWKSHRSYSCQVTHEGSTVEKTVAPTECS IgG1CH1-CH2- 95 gcgtcgaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctct CH3nucl gggggcacagcggccctgggctgcctggtcaaggactacttccccgaacctgtg acggtctcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctg tcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagca gcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaag gtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtg cccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaa ggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtg agccacgaAgaCcctgaggtcaagttcaactggtacgtggacggcgtggaggt gcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgt ggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaag tgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagcc aaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggag atgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcga catcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccac gcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtgga caagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggct ctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaa IgG1CH1-CH2- 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 CDRH1 99 GFIFSNYA CDRH2 100 IGGKGDSI CDRH3 101 VKDLAVLESDRLEVDQ VH 102 EVQLAESGGGLVQPGGSLRLSCSGSGFIFSNYAMVWARQAP GKGLEYVSGIGGKGDSIYHIDSVKGRFTISRDNSKRTVYLQ MSRLRTEDTAVYYCVKDLAVLESDRLEVDQWGQGTLVIVSA ZKA4 SEQIDNO. Aminoacidsequence CDRH1 103 GFTFSSYV CDRH2 104 TSYDGSNK CDRH3 105 ARGPVPYWSGESYSGAYFDF VH 106 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYVMHWVRQAP GKGLEWVTVTSYDGSNKYYADSVKGRFTISRDNAKNTLYLQ MNSLRGEDTAIYYCARGPVPYWSGESYSGAYFDFWGQGILV TVSS ZKA5 SEQIDNO. Aminoacidsequence CDRH1 107 GFTFSNYY CDRH2 108 MSSSETIK CDRH3 109 ARSGIETVAGSIDYYGMDV VH 110 QVQLVESGGGLVKPGGSLRLSCAGSGFTFSNYYMTWIRQAP GKGLELVSYMSSSETIKYYADSVKGRFTISRDNAKNSLYLQ MNSLRADDTARYYCARSGIETVAGSIDYYGMDVWGHGTPVT VSS ZKA6 SEQIDNO. Aminoacidsequence CDRH1 111 DFTVSNYA CDRH2 112 VSYDGSNK CDRH3 113 ATGVTMFQGAQTNAEYLHY VH 114 QVHLVESGGGVVQPGRSLRLSCEASDFTVSNYAMHWVRQAP GKGLEWVAVVSYDGSNKYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTALYYCATGVTMFQGAQTNAEYLHYWGQGSLVT ISS ZKA7 SEQIDNO. Aminoacidsequence CDRH1 115 GFTFSRYG CDRH2 116 VSGDGSST CDRH3 117 VKDFWSGDQSLESDF VH 118 EVQLVESGGGLVQPGGSLRLSCSASGFTFSRYGMVWARQAP GKGLEYLSGVSGDGSSTYYANSVKGRFTISRDNSKNTLYLH MSRLRDEDTAMYYCVKDFWSGDQSLESDFWGQGALVTVSS ZKA8 SEQIDNO. Aminoacidsequence CDRH1 119 GFTFSAHA CDRH2 120 ISRNEDYT CDRH3 121 VKDFGTSPQTDF VH 122 DERLVESGGGLVQPGGSLRLVCSASGFTFSAHAMHWVRQPP GKGLEYVSTISRNEDYTYYADSVKGRFTISRDNSKNSLYLQ MRRLRPEDTAIYYCVKDFGTSPQTDFWGQGTLVAVSS ZKA76 SEQIDNO. Aminoacidsequence CDRH1 123 GFTFSTYF CDRH2 124 ISSTGSYK CDRH3 125 ARPFHSEYTYGLDAFDI VH 126 EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYFMHWVRQAP GKGLEWVASISSTGSYKFYADSVKGRFTISRDNTKNSLFLQ MNSLRAEDTAVFYCARPFHSEYTYGLDAFDIWGQGTMLTVS S ZKA117 SEQIDNO. Aminoacidsequence CDRH1 127 GGSIRRTNSY CDRH2 128 ISYSGST CDRH3 129 ARLNDGSTVTTSSYFDY VH 130 QLQLQESGPGLVKPSETLSLTCTVSGGSIRRTNSYWGWIRQ TTGKGLQWIGSISYSGSTFYNPSLKSRVTISLDTSKDHFSL ELSSVTAADTAIYYCARLNDGSTVTTSSYFDYWGQGTLVTV SS ZKB27 SEQIDNO. Aminoacidsequence CDRH1 131 GYSFTSSW CDRH2 132 IDPSDSYT CDRH3 133 ARHDYSVSENGMDV VH 134 EVQLVQSGAEVKKPGESLRISCKASGYSFTSSWINWVRQMP GKGLEWMGRIDPSDSYTTYNPSFQGHVTISVDKSIGTAYLQ WNSLRASDTAMYYCARHDYSVSENGMDVWGQGTTVIVSS ZKB29 SEQIDNO. Aminoacidsequence CDRH1 135 GFTFSSYT CDRH2 136 ISYDGSHK CDRH3 137 ARRSYSISCFDY VH 138 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAP GKGLEWVAVISYDGSHKFYADSVKGRFTISRDNSKDTLYLQ MNSLRAEDTALYYCARRSYSISCFDYWGQGTLVTISS ZKB34 SEQIDNO. Aminoacidsequence CDRH1 139 GFTFSRSG CDRH2 140 VSYDGSNK CDRH3 141 AKDLTMVRGVHYYYYVMDV VH 142 QVQLVESGGGVVQPGRSLRLSCAASGFTFSRSGMHWVRQAP GKGLEWVAVVSYDGSNKYYSDSVKGRFTISRDNSKNTLYLQ MNSLRVEDTAVYYCAKDLTMVRGVHYYYYVMDVWGQGTIVT VSS ZKB39 SEQIDNO. Aminoacidsequence CDRH1 143 GYTFDDYY CDRH2 144 INPHRGGT CDRH3 145 VRDQYCDGGNCYGIHQPHYGMDV VH 146 QVQLVQSGAEVKKPGASLKVSCKASGYTFDDYYIHWVRQAP GQGLEWLGRINPHRGGTNYAQKFQGRVIMTLDMSISTTYME LRRITSDDAAVYYCVRDQYCDGGNCYGIHQPHYGMDVWGQG TTVTVSS ZKB46 SEQIDNO. Aminoacidsequence CDRH1 147 GYSFTSYW CDRH2 148 IDPSDSYT CDRH3 149 ARREYSSSSGQEDWFDP VH 150 EVQLVQSGAEVKKPGESLRISCKGSGYSFTSYWISWVRQMP GKGLEWMGRIDPSDSYTNYSPSFQGHVTISADKSISTAYLQ WSSLKASDTAMYYCARREYSSSSGQEDWFDPWGQGTLVTVS S ZKB53 SEQIDNO. Aminoacidsequence CDRH1 151 GFTFSSYA CDRH2 152 ISYDGSNR CDRH3 153 ARHVEQLPSSGYFQH VH 154 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQTP GKGLEWVTVISYDGSNRYYADSVKGRFTISRDNSKNTLYLQ MNSLRSEDTAVYYCARHVEQLPSSGYFQHWGQGTLVTVSS ZKC26 SEQIDNO. Aminoacidsequence CDRH1 155 GFIFSDFY CDRH2 156 IGHDGSYI CDRH3 157 ARAHGGFRH VH 158 QVQVVESGGGLVKPGGSLRLSCAASGFIFSDFYMSWMRQAP GKGLEWVAYIGHDGSYILYADSVKGRFTISRDNAKNSLFLR MNSLRVEDTAVYYCARAHGGFRHWGQGTVVAVSP ZKD5 SEQIDNO. Aminoacidsequence CDRH1 159 GFTFTSYG CDRH2 160 ISYDGSNK CDRH3 161 ARDRDHYDLWNAYTFDY VH 162 QVQLVESGGGVVQPGRSLRLSCAASGFTFTSYGMHWVRQTP GKGLDWVAVISYDGSNKYYADSVKGRFTISRDNSKDTLYLQ MNSLRAADTALYYCARDRDHYDLWNAYTFDYWGQGTLVTVS S ZKD7 SEQIDNO. Aminoacidsequence CDRH1 163 GFTFSNYA CDRH2 164 ISYDVSDK CDRH3 165 AGGPLGVVVIKPSNAEHFHH VH 166 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYAMHWVRQAP GKGLEWVAVISYDVSDKYYADSVKGRFTISRDNSKNTLFLQ MNSLRAEDTAAYYCAGGPLGVVVIKPSNAEHFHHWGQGTLV TVSS ZKD8 SEQIDNO. Aminoacidsequence CDRH1 167 GFTFINYA CDRH2 168 ISYDGSNK CDRH3 169 ATDADAYGDSGANFHY VH 170 QVQLVESGGGVVQPGKSLRLSCAASGFTFINYAIHWVRQAP GKGLEWVAVISYDGSNKFYTDSVKGRFTISRDNSKNTLYLQ MNSLRADDTAVYYCATDADAYGDSGANFHYWGQGTLVTVSS ZKD15 SEQIDNO. Aminoacidsequence CDRH1 171 DASISSGGFS CDRH2 172 IYSSGDT CDRH3 173 ARAHTPTSKFYYYYAMDV VH 174 QLQLQESGSGLVKPSQTLSLTCTVSDASISSGGFSWSWIRQ PLGKGLEWLGYIYSSGDTFYNPSLQGRVTMSVDIFRSQFSL KLTSVTAADTAMYYCARAHTPTSKFYYYYAMDVWGQGTTVT VSS ZKD16 SEQIDNO. Aminoacidsequence CDRH1 175 GFTFSDHF CDRH2 176 SRNKPNSYTT CDRH3 177 AKVGGCYGGDCHVENDY VH 178 EVQLVESGGDLVQPGGSLRLSCVASGFTFSDHFMDWVRQAP GKGLEWVGRSRNKPNSYTTEYAASVKGRFSISRDDSKKALY LQMNSLQTEDTAVYYCAKVGGCYGGDCHVENDYWGQGTLVT VSS ZKD17 SEQIDNO. Aminoacidsequence CDRH1 179 GFIFSDYA CDRH2 180 ISYDGSSR CDRH3 181 ARGYCSSGTCFSTNAEYFHP VH 182 QVQMVESGGGVVQPGTSLRLSCATSGFIFSDYAMHWVRQAP GKGLEWVAVISYDGSSRLYADSVKGRFTVSRDNSKNTLYLQ MHSLRAGDTAVYYCARGYCSSGTCFSTNAEYFHPWGQGTLA TISS ZKD20 SEQIDNO. Aminoacidsequence CDRH1 183 GFTFSDHF CDRH2 184 SRNKPNSYTT CDRH3 185 ARVGGCNGGDCHVENDY VH 186 EVQLVESGGGLVQPGGSLRLSCVASGFTFSDHFMDWVRQAP GKGLEWVGRSRNKPNSYTTEYAASVKGRFTISRDDSKNSLY LQMNSLQTEDTAVYYCARVGGCNGGDCHVENDYWGQGTLVT VSS ZKA134 SEQIDNO. Aminoacidsequence CDRH1 187 GGTFSAYA CDRH2 188 IIPFFGTA CDRH3 189 ARSDIVSTTRGYHHYGMDV VH 190 QVHLVQSGAEVKKPGSSVNVSCKASGGTFSAYAISWVRQAP GQGLEWMGGIIPFFGTAYYAQKFKGRVTVTADKSISTVYME MISLRSEDTAVYYCARSDIVSTTRGYHHYGMDVWGQGTTVT VSS ZKA246 SEQIDNO. Aminoacidsequence CDRH1 191 GYTFSDYY CDRH2 192 INPYSGGT CDRH3 193 ARGFTMISDREFDP VH 194 QVQLVQSGAEVKRPGASVKVSCKASGYTFSDYYMHWVRQAP GQGLEWMGRINPYSGGTNYAQKFHGRVTVTRDTSISTVYME LRGLRSDDTAVYYCARGFTMISDREFDPWGQGTLVTVSS ZKA256 SEQIDNO. Aminoacidsequence CDRH1 195 GFTFSTYW CDRH2 196 IKQDGSEK CDRH3 197 ARDPGYDDFWSGSYSGSFDI VH 198 EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYWMTWVRQAP GKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNTKNSLYLQ VNSLRAEDTAIYYCARDPGYDDFWSGSYSGSFDIWGQGTMV TVSS ZKB42 SEQIDNO. Aminoacidsequence CDRH1 199 GFTFNNYG CDRH2 200 ISYDGNKK CDRH3 201 VKYGERINGYSDPFDH VH 202 QVQVVESGGGVVQPGRSLRLFCAASGFTFNNYGMHWVRQAP GKGLEWVALISYDGNKKYYADSVKGRFSISRDNSKNTLYLQ MNRLRSGDTAVYHCVKYGERINGYSDPFDHWGQGTLVTVSS ZKB85 SEQIDNO. Aminoacidsequence CDRH1 203 GYTFTTYA CDRH2 204 INTNTGNP CDRH3 205 ARVIVPYAFDI VH 206 QVQLVQSGSELKKPGASVKVSCKASGYTFTTYAMNWVRQAP GQGPEWVGWINTNTGNPTYAQGFTGRFVLSLDTSVSTAFLQ ISSLKAEDTAVYYCARVIVPYAFDIWGQGTMVTVSS ZKB47 SEQIDNO. Aminoacidsequence CDRH1 207 GYTFTNYY CDRH2 208 INPSGGPT CDRH3 209 ARDQYGGYARYGMDV VH 210 QVQLVQSGAEVKKPGASVKVSCQASGYTFTNYYMHWVRQAP GQGLEWMGIINPSGGPTSYAQKFQGRVTMTTDTSTSTVYME LSSLRSEDTAVYYCARDQYGGYARYGMDVWGQGTTVIVSS ZKC6 SEQIDNO. Aminoacidsequence CDRH1 211 GYTFTGYY CDRH2 212 INPNSGGT CDRH3 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 CDRH1 219 GYIFTRYW CDRH2 220 IDPSDSYT CDRH3 221 ARQETAREDGMAV VH 222 EVQLVQSGAEVKKPGKSLRISCKGSGYIFTRYWISWVRQMP GKGLEWMGRIDPSDSYTNYSPSFQGHVTISADKSISTAYLQ WSSLKASDTAMYYCARQETAREDGMAVWGQGTTVTVSS ZKA174 SEQIDNO. Aminoacidsequence CDRH1 223 GGSMSNSYYH CDRH2 224 IYYSGST CDRH3 225 ARNPVFNPLTLTHDAFDI VH 226 QLQLQESGPGLVKPSETLSLTCTVSGGSMSNSYYHWGWIRQ PPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSL KLNSVTAADTAVYYCARNPVFNPLTLTHDAFDIWGQGTMVT VSS ZKA189 SEQIDNO. Aminoacidsequence CDRH1 227 GFTFSSYA CDRH2 228 ISGSGDNT CDRH3 229 AKWPYYDFWSGSESYFDP VH 230 GVQLLESGGALVQPGKSLRLSCAASGFTFSSYALTWVRQAP GKGLQWVSAISGSGDNTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKWPYYDFWSGSESYFDPWGQGTLVTV SS ZKA195 SEQIDNO. Aminoacidsequence CDRH1 231 GYNFPSYW CDRH2 232 IDPSDSYT CDRH3 233 ARADCRSTSCYLVFE VH 234 EVQLVQSGAEVKKPGESLRISCKDSGYNFPSYWIHWVRQMP GKGLEWMGTIDPSDSYTNYSPSFQGHVTISADKSISTAYLQ WSSLKASDTAMYYCARADCRSTSCYLVFEGQGTLVTVSS ZKA215 SEQIDNO. Aminoacidsequence CDRH1 235 GYTFTSYW CDRH2 236 IDPSDSHT CDRH3 237 ARHALPNYFDS VH 238 EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWISWVRQMP GKGLEWMGRIDPSDSHTDYSPSFQGHVTISADKSISAAYLQ WSSLKASDTAMYYCARHALPNYFDSWGQGTLVTVSS ZKA218 SEQIDNO. Aminoacidsequence CDRH1 239 GFPFSSYW CDRH2 240 INSDGRNT CDRH3 241 ARGGYDYDSSGCFDY VH 242 EVQLVESGGGLVQPGGSLRLSCAASGFPFSSYWMHWVRQAP GKGLVWVSRINSDGRNTNYADSVKGRFTISRDNAENTVYLQ MNSLRAEDTAVYYCARGGYDYDSSGCFDYWGQGTLVTVSS ZKB75 SEQIDNO. Aminoacidsequence CDRH1 243 GFTFSNYA CDRH2 244 ISGTGGST CDRH3 245 AKDSASRGGYCSGGVCYLNPGHHDY VH 246 EVQVLESGGGLLQPGGSLRLSCAASGFTFSNYAMSWVRQAP GKGLEWVSTISGTGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKDSASRGGYCSGGVCYLNPGHHDYWG QGTLVTVSS ZKB83 SEQIDNO. Aminoacidsequence CDRH1 247 GYSFTNYW CDRH2 248 IDPSDSYT CDRH3 249 ARLRGSLYCSGGRCYSVPGETPNWFDP VH 250 EVQLVQSGAEVKKPGESLRISCKGSGYSFTNYWITWVRQMP GKGLEWMGSIDPSDSYTNYSPSFQGHVTISADWSINTAYLQ WSSLKASDTAKYYCARLRGSLYCSGGRCYSVPGETPNWFDP WGQGTLVTVSS ZKC3 SEQIDNO. Aminoacidsequence CDRH1 251 GGSITSYY CDRH2 252 IYYSGST CDRH3 253 ARVGGAPYYYYGMDV VH 254 QVQLQESGPGLVKPSETLSLTCTVSGGSITSYYWSWIRQPP GKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKL SSVTAADTAVYYCARVGGAPYYYYGMDVWGQGTTVTVSS ZKC18 SEQIDNO. Aminoacidsequence CDRH1 255 GFTFGDYA CDRH2 256 IRSKAYGGTT CDRH3 257 SRDHTGTTYAFDI VH 258 EVQLVESGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAP GKGLEWVGFIRSKAYGGTTEYAASVKGRFTISRDDSKSIAY LQMNSLKTEDTAVYYCSRDHTGTTYAFDIWGQGTMVTVSQ ZKD1 SEQIDNO. Aminoacidsequence CDRH1 259 GFTFSSYG CDRH2 260 IWYDGSNK CDRH3 261 ARDRRGYGDYVGYYYGMDV VH 262 QVQLVESGGGVVQPGRSLRLSCASGFTFSSYGMHWVRQAP GKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCARDRRGYGDYVGYYYGMDVWGQGTTVT VSS Name SEQIDNO. Aminoacidsequence ZIKVEDIII 263 TAAFTFTKXPAEXXHGTVTVEXQYXGXDGPCKXPXQ generic MAVDXQTLTPVGRLITANPVITEXTENSKMMLELDPP FGDSYIVIGXGXKKITHHWHRS ZIKV 264 TAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQM H/PF/2013 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 X1maybeany(naturallyoccurring)aminoacid,preferably K,A,orE; X2maybeany(naturallyoccurring)aminoacid,preferably V,F,orL; X3maybeany(naturallyoccurring)aminoacid,preferably SorF; X4maybeany(naturallyoccurring)aminoacid,preferably IorV; X5maybeany(naturallyoccurring)aminoacid,preferably TorV; X6maybeany(naturallyoccurring)aminoacid,preferably LorD; X7maybeany(naturallyoccurring)aminoacid,preferably VorG; X8maybeany(naturallyoccurring)aminoacid,preferably AorG; X9maybeany(naturallyoccurring)aminoacidexceptR, preferablyTorA; X10maybeany(naturallyoccurring)aminoacid,preferably VorI; X11maybeany(naturallyoccurring)aminoacid,preferably AorV; X12maybeany(naturallyoccurring)aminoacid,preferably MorT; X13maybeany(naturallyoccurring)aminoacid,preferably SorG; X14maybeany(naturallyoccurring)aminoacid,preferably EorK; X15maybeany(naturallyoccurring)aminoacid,preferably VorI; X16maybeany(naturallyoccurring)aminoacid,preferably E,A,K,orD;and X17maybeany(naturallyoccurring)aminoacid,preferably E,A,orK,morepreferablyKorA Zika-E-F1 266 TGCAAACGCGGTCGCAAACCTGGTTG primer ZIKV-E-R1 267 CGTGCCAAGGTAATGGAATGTCGTG primer ZIKV-Ef1530 268 AGCCTAGGACTTGATTGTGAACCGA primer ZIKV-E- 269 TTACAGATCCCACAACGACCGTCAG R2769primer ZIKV-E-F2 270 ACTTGGTCATGATACTGCTGATTGC ZIKV-E-R2 271 TCGGTTCACAATCAAGTCCTAGGCT ZIKV-E-f2058 272 GCTAACCCCGTAATCACTGAAAGCA ZIKV-E- 273 AAGACTGCCATTCTCTTGGCACCTC r2248 *the sequences highlighted in bold are CDR regions (nucleotide or aa) and the underlined residues are mutated residues as compared to the germlinesequence.