NS2B AS MARKER FOR ZIKA VIRUS INFECTIONS

Abstract

The present invention relates to protein NS2b or fragment(s) thereof as biomarker or diagnostic marker for the diagnosis and/or prognosis of Zika virus infections. The present invention further relates to peptides and cyclic peptides, compositions and arrays and multimer compounds comprising them. The present invention further relates to a method for the diagnosis and/or prognosis of Zika virus infections, comprising the use of protein NS2b or fragment(s) thereof, or of the peptides, cyclic peptides, compositions and/or arrays in immunoassays. The present invention further relates to peptide-based compounds comprising at least one fragment of protein NS2b and at least one further component and to methods for the diagnosis and/or prognosis of Zika virus infections.

Claims

1-26. (canceled)

27. A protein NS2b or at least one fragment thereof as a biomarker or diagnostic marker for use in a method for the diagnosis and/or prognosis of Zika virus infections.

28. The NS2b protein or at least one fragment thereof according to claim 27, wherein the fragment comprises an amino acid sequence of the ZIKV polyproteome of positions 1429-1449 of Zika Uganda Strain MR766_NIID (SEQ ID NO: 1).

29. The NS2b fragment according to claim 27, wherein the fragment comprises or is a peptide having an amino acid sequence selected from SEQ ID NOs: 1 to 10, or an amino acid sequence selected from SEQ ID NOs: 1 to 10 having one, two, three or four amino acid substitution(s) in position 9, 11, 15 and/or 17 in reference to the amino acid sequence of SEQ ID NO: 1.

30. The NS2b fragment according to claim 29, wherein the amino acid substitution(s) are A9P, A9E, A9M, A9S, A9T, A9K; V11D, V11E, V11T, V11A, V11N, V11S, V11M, V11L or I11D, I11E, I11T, I11A, I11N, I11 S, I11M, I11L; S15D, S15K, S15M, S15A, S15R, S15N; and/or R17D, R17E, R17T.

31. The NS2b fragment according to claim 27, wherein the fragment comprises or is a cyclic peptide having an amino acid sequence selected from SEQ ID NOs: 1 to 10, or an amino acid sequence selected from SEQ ID NOs: 1 to 10 having one, two, three or four amino acid substitution(s) in position 9, 11, 15 and/or 17 in reference to the amino acid sequence of SEQ ID NO: 1, wherein the cyclization is via thioether formation, wherein the peptide comprises an additional cysteine residue at or near the C-terminus, or head-to-tail cyclization.

32. A peptide or a cyclic peptide that is either: A) a peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10 having one, two, three or four amino acid substitution(s) in position 9, 11, 15 and/or 17 in reference to the amino acid sequence of SEQ ID NO: 1 or B) a cyclic peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10, or an amino acid sequence selected from SEQ ID NOs: 1 to 10 having one, two, three or four amino acid substitution(s) in position 9, 11, 15 and/or 17 in reference to the amino acid sequence of SEQ ID NO: 1, wherein the cyclization is via thioether formation, wherein the peptide comprises an additional cysteine residue at or near the C-terminus, or head-to-tail cyclization.

33. The peptide of claim 32 having a length of 5 to 130 amino acids.

34. The peptide or the cyclic peptide of claim 32, comprising at least one further component(s), which is/arc selected from label(s), tag(s), linker or anchoring group(s) or combinations thereof, wherein said at least one further component(s) is/are covalently coupled to said peptide via a linker, an amino acid side chain, and/or to the N- and/or C-terminus.

35. A composition or an array comprising: (a) at least one peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10, or an amino acid sequence having at least about 50% sequence identity to an amino acid sequence of SEQ ID NOs: 1 to 10, or (b) at least one peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10 having one, two, three or four amino acid substitution(s) in position 9, 11, 15 and/or 17 in reference to the amino acid sequence of SEQ ID NO: 1, and/or (c) at least one cyclic peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10, or an amino acid sequence selected from SEQ ID NOs: 1 to 10 having one, two, three or four amino acid substitution(s) in position 9, 11, 15 and/or 17 in reference to the amino acid sequence of SEQ ID NO: 1, wherein the cyclization is via thioether formation, wherein the peptide comprises an additional cysteine residue at or near the C-terminus, or head-to-tail cyclization, (d) a protein or at least one fragment thereof of claim 1.

36. A multimer compound comprising at least two of component(s) (a) to (d): (a) a peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10, or an amino acid sequence having at least about 50% sequence identity to an amino acid sequence of SEQ ID NOs: 1 to 10, or (b) a peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10 having one, two, three or four amino acid substitution(s) in position 9, 11, 15 and/or 17 in reference to the amino acid sequence of SEQ ID NO: 1, (c) a cyclic peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10, or an amino acid sequence selected from SEQ ID NOs: 1 to 10 having one, two, three or four amino acid substitution(s) in position 9, 11, 15 and/or 17 in reference to the amino acid sequence of SEQ ID NO: 1, wherein the cyclization is via thioether formation, wherein the peptide comprises an additional cysteine residue at or near the C-terminus, or head-to-tail cyclization, and/or (d) a protein NS2b or at least one fragment thereof of claim 1, wherein at least two of component(s) (a) to (d) are covalently coupled to each other or are connected via a linear or cyclic scaffold.

37. A peptide-based compound comprising: (i) at least one peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10, or an amino acid sequence having at least about 50% sequence identity to an amino acid sequence of SEQ ID NOs: 1 to 10, or an amino acid sequence selected from SEQ ID NOs: 1 to 10 having one, two, three or four amino acid substitution(s) in position 9, 11, 15 and/or 17 in reference to the amino acid sequence of SEQ ID NO: 1, and/or at least one cyclic peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10, or an amino acid sequence selected from SEQ ID NOs: 1 to 10 having one, two, three or four amino acid substitution(s) in position 9, 11, 15 and/or 17 in reference to the amino acid sequence of SEQ ID NO: 1 wherein the cyclization is via thioether formation, wherein the peptide comprises an additional cysteine residue at or near the C-terminus, or head-to-tail cyclization and (ii) at least one further component coupled to the peptide and/or cyclic peptide (i).

38. The peptide-based compound of claim 37, wherein the peptide and/or cyclic peptide has a length of at least 5 amino acids.

39. The peptide-based compound of claim 37, wherein the at least one further component is selected from label(s), tag(s), linker or anchoring group(s), or combinations thereof, wherein the at least one further component(s) is/are preferably covalently coupled to said peptide and/or cyclic peptide via a linker, an amino acid side chain, and/or to the N- and/or C-terminus.

40. A method for the diagnosis and/or prognosis of Zika virus infections, comprising the steps of (a) providing a sample of a patient to be tested, (b) providing (1) protein NS2b or at least one fragment thereof; (2) a peptide-based compound comprising (i) at least one peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10, or an amino acid sequence having at least about 50% sequence identity to an amino acid sequence of SEQ ID NOs: 1 to 10, or an amino acid sequence selected from SEQ ID NOs: 1 to 10 having one, two, three or four amino acid substitution(s) in position 9, 11, 15 and/or 17 in reference to the amino acid sequence of SEQ ID NO: 1, and/or at least one cyclic peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10, or an amino acid sequence selected from SEQ ID NOs: 1 to 10 having one, two, three or four amino acid substitution(s) in position 9, 11, 15 and/or 17 in reference to the amino acid sequence of SEQ ID NO: 1 wherein the cyclization is via thioether formation, wherein the peptide comprises an additional cysteine residue at or near the C-terminus, or head-to-tail cyclization and (ii) at least one further component coupled to the peptide and/or cyclic peptide (i); (3) a peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10 having one, two, three or four amino acid substitution(s) in position 9, 11, 15 and/or 17 in reference to the amino acid sequence of SEQ ID NO: 1; (4) at least one cyclic peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10, or an amino acid sequence selected from SEQ ID NOs: 1 to 10 having one, two, three or four amino acid substitution(s) in position 9, 11, 15 and/or 17 in reference to the amino acid sequence of SEQ ID NO: 1, wherein the cyclization is via thioether formation, wherein the peptide comprises an additional cysteine residue at or near the C-terminus, or head-to-tail cyclization (5) a composition or an array comprising (a) at least one peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10, or an amino acid sequence having at least about 50% sequence identity to an amino acid sequence of SEQ ID NOs: 1 to 10, or (b) at least one peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10 having one, two, three or four amino acid substitution(s) in position 9, 11, 15 and/or 17 in reference to the amino acid sequence of SEQ ID NO: 1, and/or (c) at least one cyclic peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10, or an amino acid sequence selected from SEQ ID NOs: 1 to 10 having one, two, three or four amino acid substitution(s) in position 9, 11, 15 and/or 17 in reference to the amino acid sequence of SEQ ID NO: 1, wherein the cyclization is via thioether formation, wherein the peptide comprises an additional cysteine residue at or near the C-terminus, or head-to-tail cyclization, (d) a protein NS2b or at least one fragment thereof of claim 1; and/or (6) a multimer compound comprising at least two of component(s) (a) to (d): (a) a peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10, or an amino acid sequence having at least about 50% sequence identity to an amino acid sequence of SEQ ID NOs: 1 to 10, or (b) a peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10 having one, two, three or four amino acid substitution(s) in position 9, 11, 15 and/or 17 in reference to the amino acid sequence of SEQ ID NO: 1, (c) a cyclic peptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 10, or an amino acid sequence selected from SEQ ID NOs: 1 to 10 having one, two, three or four amino acid substitution(s) in position 9, 11, 15 and/or 17 in reference to the amino acid sequence of SEQ ID NO: 1, wherein the cyclization is via thioether formation, wherein the peptide comprises an additional cysteine residue at or near the C-terminus, or head-to-tail cyclization, and/or (d) a protein NS2b or at least one fragment thereof, wherein at least two of component(s) (a) to (d) are coupled to each other or are connected via a linear or cyclic scaffold; (c) performing an immunoassay, comprising detecting an anti-Zika antibody response in said patient sample.

41. The method of claim 40, comprising the use of a negative control, wherein said negative control is a NS2b peptide, cyclic peptide or a peptide-based compound comprising an amino acid substitution in position K7 in reference to the amino acid sequence of SEQ ID NO: 1.

42. The method according to claim 40, wherein the NS2b fragment comprises an amino acid sequence of the ZIKV polyproteome of positions 1429-1449 of Zika Uganda Strain MR766 NIID (SEQ ID NO: 1)

43. The method according to claim 40, wherein the NS2b fragment has the following substitutions: A9P, A9E, A9M, A9S, A9T, A9K; V11D, V11E, V11T, V11A, V11N, V11S, V11M, V111, or I11D, I11E, MT, I11A, I11N, I11S, I11M, I11L; S15D, S15K, S15M, S15A, S15R, S15N; and/or R17D, R17E, R17T.

44. The method according to claim 40, wherein one fragment is used that comprises more than one of said peptides, or more than one fragment each comprising or consisting of one peptide are used.

45. The NS2b fragment according to claim 29, wherein the amino acid substitution(s) are A9P, A9E, V11D, MD, V11E, I11E, S15D, S15K, and/or R17D, R17E.

46. The NS2b fragment according to claim 29, comprising an amino acid sequence selected from SEQ ID NOs: 11 to 16.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0272] FIG. 1: Course of disease and diagnostics (detection of RNA and antibodies).

[0273] A typical time course of virus and antibody kinetics in (flavi-)viral infections after virus infection: After infection, the virus replicates, until the immune system successfully overcomes the infection, generating specific IgM and eventually (after isotype switch) IgG antibodies against the viral proteins.

[0274] FIG. 2: First screening round (Arrays with whole proteome content):

[0275] (a) The IgG antibody reactivity of Zika patients of different groups was screened against the whole ZIKV proteome (only protein NS2b shown). The most prominent interaction was observed with the protein NS2b. A representative selection of data for relevant peptides (bracket with black asterisk) is shown in (b). Framed peptides represent Seq-IDs NO. 2-7. Fluorescence values are shown in gradual 3-color scale (green: >0; black: >100; red: >1000)

[0276] FIG. 3: Second screening round (Specific arrays with NS1, NS2a, NS2b content).

[0277] The IgG antibody reactivity showed clear signals in the NS2b protein. A major epitope was identified.

[0278] FIG. 4: 3D structure of the NS2b-NS3 serine protease complex.

[0279] The identified major epitope is only at the beginning (13 amino acids) part of an ordered 3D crystal structure (GDITWEKDAEVTG) [SEQ ID NO. 2], the missing 8 amino acids of this epitope (highlighted in the circle) are part of a probably unordered linear protrusion of 54 amino acids length (orange line), connected to the amino acid (highlighted with a star).

[0280] FIG. 5: Comparison of specific Zika NS2b and dengue NS2b reactivity in patients with and without dengue/Zika infection:

[0281] The identified major epitope in the Zika NS2b protein is only prevalent in patients after infection with Zika (convalescent samples, highlighted in circles). In the dengue virus NS2b protein, there is almost no reactivity.

[0282] FIG. 6: Comparison of specific Zika NS2b IgG reactivity in the second study in Zika and Dengue patients.

[0283] The second more comprehensive study analyzed the IgG and IgM antibody responses to the proteomes of 119 Zika virus strains using a total of 375 disease and control human plasma samples. While we did not observe any specific IgM reactivity against framed peptides representing SEQ ID NOs. 1-10, the major epitope in the Zika NS2b protein shows a clear and differential IgG reactivity in patients after infection with Zika in nearly all convalescent and a high number of acute samples. In contrast, the Zika negative/Dengue positive and Zika negative/Dengue negative samples hardly showed any IgG reactivity. Fluorescence values are shown in gradual 3-color scale (light gray: >0; gray: >100; dark gray: >1000)

[0284] FIG. 7: Full substitution scan of SEQ ID 9.

[0285] 7A and B, top: Fluorescence pattern of four arrays stained with Zika acute and convalescent plasma samples. Arrays are framed with polio and hemagglutinin peptides as positive controls.

[0286] 7A and B, bottom: Amino acid plots calculated from the IgG fluorescence intensities. The IgG reactivity of each amino acid exchange and each peptide variant was referenced to the IgG fluorescence intensity of wild type peptide WEKDAEVTGNSPRLD (SEQ ID NO. 9) set to 100%. The wild type peptide sequence is plotted from the N- to the C-terminus on the x-axis, the relative IgG reactivity of each amino acid substitution as percentage of the wild type peptide at 100% on the y-axis.

[0287] FIG. 8: IgG reactivity in different patient sera towards the WT peptide and cyclic Zika NS2b peptide variants.

[0288] The cyclic peptides and WT peptide were tested by a chemiluminescence ELISA using sera of Zika acute and Zika convalescent patients. A healthy control (HC) sera was included. The ELISA assays were performed under stringent conditions, that is at high serum dilutions (1:1000) to better evaluate the diagnostic potential of the peptide marker. The ELISA results are given in chemiluminescence units (CLUs). Samples were considered as positive, if the CLU of a respective sera is >=2 times over HC value. Each Zika serum exhibited a positive response towards at least one of the cyclic peptide variants.

[0289] A) IgG reactivity towards test peptides in Zika acute patient sera; the numbers at the y-axis indicate the chemiluminescence intensity, serum identifier are plotted at the x-axis.

[0290] B) IgG reactivity towards test peptides in Zika convalescent patient sera; the numbers at the y-axis indicate the chemiluminescence intensity, serum identifier are plotted at the x-axis.

EXAMPLES

[0291] 1. NS2b ZIKV Peptide Microarray

[0292] The whole proteome sequences of all at that time point publicly available 15 zika virus strains were retrieved from the NCBI database and translated into 15-mer peptides with a peptide-peptide overlap of 12 amino acids. Peptide arrays were produced by the company PEPperPRINT GmbH (Heidelberg, Germany) in a laser printing process on glass slides, coated with a PEGMA/PMMA graft copolymer, which were functionalized with a Ala-Ala-linker. In brief, a layer of amino acid particles, containing Fmoc-amino acid pentafluorophenyl esters, was printed layer after layer onto the functionalized glass slides, with intermittent melting (i.e. coupling) steps at 90 C. and chemical washing and capping steps (Stadler et al., 2008). Peptides were generated in duplicates on the arrays, which were screened for IgG responses in human sera.

[0293] 2. Proteome-Wide Zika Virus Peptide Microarrays

[0294] The proteome sequences of 119 Zika virus strains from Nicaragua, Colombia, Guatemala, Honduras, Mexico, Panama and the USA were obtained from sequencing data. A homology analysis and sequence alignment were applied to remove redundant sequences, and unique Zika virus sequences translated into microarrays with 4356 different 15 amino acid peptides, which were printed in duplicate. Peptide microarrays were produced by PEPperPRINT GmbH (Heidelberg, Germany) in a laser printing process on glass slides, coated with a PEGMA/PMMA graft copolymer, which were functionalized with a Ala-Ala-linker. In brief, a layer of amino acid particles, containing Fmoc-amino acid pentafluorophenyl esters, was printed layer after layer onto the functionalized glass slides, with intermittent melting (i.e. coupling) steps at 90 C. and chemical washing and capping steps (Stadler et al., 2008).

[0295] 3. Immunostaining of Peptide Microarrays

[0296] Peptide microarrays were placed in incubation trays (PEPperPRINT GmbH, Heidelberg, Geimany) and blocked for 30 min at room temperature at 120 RPM orbital shaking with western blot blocking buffer MB-070 (Rockland, USA).

[0297] In the first study, sera were diluted 1:1000 in PBS buffer with 0.05% Tween 20 pH 7.4 (PBST) and 10% blocking buffer, incubating the sera for 16 h at 4 C. and 50 RPM orbital shaking. Peptide microarrays were washed three times shortly with PBST, followed by an incubation with a 1:2500 dilution of the secondary antibody (goat anti-human IgG Fc specific DyLight 680, Rockland, USA), together with the control antibody, diluted 1:500 (anti-c-Myc antibody, PEPperPRINT, Germany), for 30 min at room temperature and 120 RPM orbital shaking. The peptide microarrays were washed 310 s with PBST and rinsed with deionized water. After drying in a stream of air, fluorescent images were acquired using an Odyssey Imaging System (LI-COR, USA) at 700 nm with a resolution of 21 m and a scanning sensitivity of 7. Image analysis and quantification was performed with the PepSlide Analyzer software (Sicasys Software GmbH, Heidelberg, Germany).

[0298] In the second study, human plasma samples were diluted 1:250 in PBS buffer with 0.05% Tween 20 pH 7.4 (PBST) and 10% blocking buffer, incubating the sera for 16 h at 4 C. and 140 RPM orbital shaking. Peptide microarrays were washed three times shortly with PBST, followed by an incubation with a 1:5000 dilution of the secondary antibodies (goat anti-human IgG Fc specific DyLight680 and goat anti-human IgM chain specific DyLight800, Rockland, USA) for 30 min at room temperature and 140 RPM orbital shaking.

[0299] Samples used for the peptide array experiments of the first study were collected from individuals with acute febrile illnesses enrolled in a prospective cohort study from May 2015 to April 2016. The cohort was established in an urgent health care clinic in the Recife Metropolitan Region as part of the International Research Consortium on Dengue Risk Assessment, Management and Surveillance-IDAMS (www.idams.eu; Jaenisch et al., 2013; Jaenisch et al., 2016). The age of patients from whom sera were used for the assays varied from 9 to 57 years old, where 7 were females and 5 were males. Sample collection was performed in the first day of recruitment (Day 1acute sample), which following the IDAMS protocol corresponds to the period within the first 72h of the febrile period, and on the convalescent phase (Day 10-30 after recruitmentconvalescent sample). For molecular viral diagnosis, quantitative real-time PCR (qRT-PCR) for Dengue (DENV) and Zika (ZIKV) viruses was performed. Protocols were slightly modified from previously reported assays (Warrilow et al., 2002; Lances et al., 2012; Lanciotti et al., 2007). Positive controls were viruses extracted from cell culture, and the negative control was water. As for serology, samples were assayed for anti-DENV IgM and IgG and anti-ZIKV IgM, through ELISA. The Panbio Dengue Capture ELISA was used for the anti-DENV IgM and IgG assays, following the manufacturer's protocol. The anti-ZIKV IgM ELISA protocol was that of the Centers for Diseases Control and Prevention (CDC) (MMWR 2016; Granger et al., 2017).

[0300] According to the assays results, each sample was classified as: [0301] 1) ZIKV Positive/DENV Nave, if they were positive for ZIKV qRT-PCR and negative for DENV qRT-PCR in the acute phase and/or positive for anti-ZIKV IgM with titers >2 times those for anti-DENV IgM in the convalescent phase, and negative for anti-DENV IgG in the acute phase; [0302] 2) ZIKA Positive/DENV Exposed, if they were positive for ZIKV qRT-PCR and negative for DENV qRT-PCR in the acute phase and/or positive for anti-ZIKV IgM with titers >2 times those for anti-DENV IgM in the convalescent phase, and positive for anti-DENV IgG in the acute phase; [0303] 3) Nave, if they were negative for DENV and ZIKV qRT-PCRs in the acute phase, and negative for anti-DENV and anti-ZIKV IgM and IgG in both acute and convalescent phases.

[0304] In the second study, human plasma samples were pre-collected and purchased from the Nicaraguan Biorepository at the Sustainable Sciences Institute and tested by PCR for Dengue (DENV) and Zika (ZIKV) viremia by polymerase chain reaction. The day of enrollment after onset of symptoms was in the range of 4-6 days for acute plasma samples. Collection day of convalescent sample after onset of symptoms was in the range of 14-21 days. The 375 samples were classified as: [0305] 1) ZIKV Positive/DENV Nave, if they were positive for ZIKV PCR and negative for DENV PCR in the acute or convalescent phase; [0306] 2) ZIKA Positive/DENV Positive, if they were positive for ZIKV PCR and positive for DENV PCR in the acute or convalescent phase; [0307] 3) ZIKA Negative/DENV Positive, if they were positive for DENV PCR in the convalescent phase and collected before the Zika virus outbreak in 2015. [0308] 2) ZIKA Negative/DENV Negative, if they were negative for ZIKV PCR and negative for DENV PCR.

[0309] 4. Full Substitution Scan

[0310] A substitution analysis was performed with the WT peptide WEKDAEVTGNSPRLD (SEQ ID No. 9) and cyclic peptide KDAEVTGNS (SEQ ID NO. 10) to determine essential, conserved and less-conserved/variable amino acid positions and to identify stronger reacting peptide variants.

[0311] Each amino acid position in the WT sequence was systematically exchanged by all 20 physiologic amino acids, and the resulting peptides and peptide variants were printed in triplicate on peptide microarrays. The immunoreactivity of the peptide variants was tested by incubation with Zika acute and convalescent plasma samples. The assay was carried out in a multiplexed format with fluorescently labeled secondary anti-human IgG and anti-human IgM antibodies to simultaneously analyze the IgG and IgM profiles of each sample.

[0312] Results are shown in FIGS. 7A and 7B. The IgG specific epitopes are strongly conserved in positions K7, E10, T12, G13, N14 (numbering in reference to SEQ ID NO. 1). In these positions, no amino acid exchange is tolerated. Other amino acid positions are less conserved or even variable. Strongest IgG reactivities were observed for peptide variants of A9P,E; V11D,E; S15D,K and R17D,E. Interestingly, amino acid position K7 turned out to be essential for binding of anti-Zika virus antibodies in all tested human samples, and was shown to be differential for Zika virus in the corresponding regions of NS2b of other flaviviruses.

[0313] 5. Cyclic Peptides

[0314] For ELISA tests, four cyclic peptides with a Biotin tag preceding the C-terminal cysteine residue were synthesized. The non-cyclic WT peptide (WT peptide) was synthesized with a C-terminal Biotin tag at the C-terminus, the amino acid sequence was identical to SEQ ID NO. 9.

[0315] The cyclic peptides were cyclized via a thioether bond. Therefore, a C-terminal cysteine residue was added to the amino acid sequence of the peptide and the N-terminus carried a bromoacetyl group. The cyclization was via the thiol group, i.e. the cysteine side chain, and the bromacetylated N-terminus.

TABLE-US-00012 SEQ ID Peptide NO. WT WEKDAEVTGNSPRLD 9 Peptide 1c (cyclized WT) [00002] 9 Peptide 2c (cyclic, di-substituted) [00003] 11 Peptide 3c (cyclic, mono- substituted) [00004] 12 Peptide 4c (cyclic, tri-substituted) [00005] 13 [0316] 6. ELISA Tests

[0317] 6.1 Protocol

[0318] Peptide ELISA-Tests were performed on Streptavidin functionalized 96 well microtiterplates (Lumitrac, Greiner BioOne, Germany). The plates were coated with the respective biotinylated peptides with a biotin tag positioned at the C-terminus, preceding the C-terminal cysteine. The plates were incubated overnight at 4 C. with test sera at a dilution of 1:1000 (v/v) in PBST/10% Rockland (PBST=Phosphate buffered saline pH 7.2, 0.05% Tween 20). Thereafter, plates were washed 3 times with PBST and incubated for 1 h at 20 C. with the detection antibody (goat anti-human Fc peroxidase conjugate) at a dilution of 1: 10000 in PBST/10% Rockland. The plates were washed again 3 times with PBST, followed by addition of the peroxidase substrate solution (BM Chemiluminescence ELISA substrate, Sigma Aldrich). The chemiluminescence was measured after 10 minutes at 425 nm using an automated plate reader (CLARIOstar. BMG LABTECH GmbH, Germany). The chemiluminescence intensity is expressed in chemiluminescence units (CLU), the intensity expressing the immunoreactivity of tested sera.

[0319] 6.2 Samples/Tested Sera

[0320] Samples used for the ELISA tests were pre-collected and purchased from the Nicaraguan Biorepository at the Sustainable Sciences Institute and tested for Dengue (DENV) and Zika (ZIKV) viremia by polymerase chain reaction.

[0321] 6.3 ELISA Results:

[0322] The ELISA results are given in CLUs and shown in FIGS. 8A and 8B. Samples were considered as positive, if the CLU of a respective sera is >=2 times over HC value. All tested cyclic peptides and the WT peptide detected Zika specific antibody responses in acute patient sera as well as in convalescent patient sera. A positive IgG response was observed for each Zika serum to at least one of the tested peptide variants.

[0323] The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

REFERENCES

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