GLYCAN LOOP OF FLAVIVIRUSES ENVELOPE PROTEIN

FIELD OF THE INVENTION

The present invention relates to the field of vaccination against flavivimses, and in particular against Zika virus. The invention further relates to methods for producing or selecting antibodies directed against said flavivimses. The invention further relates to isolated polypeptides, recombinant cells, and attenuated viruses, suitable for eliciting an immune response.

BACKGROUND OF THE INVENTION

Flavivimses (family Flaviviridae) are enveloped vimses whose genome is a positive polarity RNA molecule. The flavivirion is composed of three structural proteins designated C (core protein), M (membrane protein) and E (envelope protein). The E protein that is exposed on the surface of the vims is responsible for the major biological functions of the virion including vims attachment and specific membrane fusion. The translation of the genomic RNA produces a precursor polyprotein which is cleaved simultaneously by cellular and viral proteases to generate the individual viral proteins: three structural proteins C, prM (the glycosylated precursor of the protein M), E, and seven nonprotein proteins. - structural NS1 to NS5. Flavivims replication occurs in the cytoplasm of infected cells. Flavivimses have complex natural cycles of transmission that involve several natural hosts (mostly mammals and birds) and vectors, the latter being hematophagous arthropods such as ticks and mosquitoes.

Mosquito- and tick-borne flavivimses are the main cause of severe human diseases such as haemorrhagic manifestations or meningo-encephalitic syndromes. Presently, the yellow fever vims (YFV), the Dengue vims (DENV), the West Nile Vims (WNV), the tick-borne encephalitis vims (TBEV), the Japanese encephalitis vims (JEV), the Tembusu vims (TMUV) (TMUV has no impact in public health but only in farmed birds); in contrario, Usutu vims is a new emerging WNV-related vims of medical concern and the Zika vims (ZIKV) are among the most medically-important flavivimses. For instance, the Dengue vims (DEN) is recognized as the cause of severe haemorrhagic fevers (diseases) around the world. Also, the mosquito-bome Zika vims (ZIKV) belonging to the flavivims genus of Flaviviridae family is the etiologic agent of Zika congenital syndrome and neurological disorders in humans.

The E protein has been proposed as a potential candidate for flavivirus prevention and treatment. It is established that elicitation of a protective antibody response is a critical step in the development of safe and efficient flavivirus vaccines. In particular, monoclonal antibodies isolated from Dengue Patients have been considered as protective against Zika Virus, as shown in Swanstrom (American Society of Microbiology 2016; Vol. 7; Issue 4; eOl 123-16).

The general structure and function of the Envelope glycoprotein in Flavivirus Infections is known. The envelope (E) protein of Zika virus (504 amino-acids long, by reference to the E protein of ZIKV MR766, as reference in SEQ ID N°ll) is responsible for vims entry into the host-cell. Illustratively, the ZIKV E ectodomain (residues 1-406) is divided in three structural envelope domains: Domain I (EDI), Domain II (ED II) and Domain III (EDIII). ZIKV EDI contains 132 residues distributed in three spaced segments: the N-terminal residues 1-52 and the central residues 132-193 followed by residues 280-296. ZIKV EDI comprises a flexible glycan-loop (EDI-GL, residues E-145 to E-164) which may be post-translationally N-glycosylated at Asnl54 for some viral strains.

WO2017/220748 reports an attenuated Zika virus with a protein E of an epidemic strain, wherein at least one residue at position 152, 156 or 158 is mutated.

W02018/007575 also reports an attenuated Zika vims comprising a mutation leading to the abrogation of a N-glycosylation site on the Envelope protein, for preventing the generation of auto-antibodies responsible for Guillain-Barre syndrome. According to some embodiments, an amino acid residue at position 152, 156 or 158 is mutated.

Besides, inoculation of the viral clone ZIKBeHMR-2 (a chimeric ZIKV derived from the MR766 strain in which the structural protein region was replaced with the one of a BeH819015 strain) in immunocompetent adult mice also results in production of neutralizing anti-ZIKV antibodies. Frumence (Vaccines 2019, 7, 66; doi:10.3390/vaccines7030066) further reports a neutralization test based on a recombinant GFP reporter Zika vims MR766 strain for detecting neutralizing antibodies.

Those strategies rely on the reconstitution of an envelope protein conformation and stabilization to conserve an epitope targeted by neutralizing antibodies. Those approaches thus all rely on the conservation of the whole structure of the Envelope proteins of those flaviviruses.

Others have developed strategies based on peptide-carrier vaccines, with a similar goal in the HIV-1 field, as taught in Combadiere (Vaccines 2019, 7, 105; doi: 10.3390/vaccines7030105).

However, there remains a need to develop new effective vaccine candidates against flaviviruses. In particular, there remains a need to develop vaccine candidates which remain easy to produce and formulate for the prevention or treatment of an infection of a family of Flaviviridae viruses such as Dengue virus, or Yellow Fever virus (YFV), or Japanese Encephalitis virus (JEV), or West Nile virus (WNV), or Zika virus.

There also remains a need to identify and/or select neutralizing antibodies from other non-neutralizing antibodies. There also remains a need to detect new epitopes which can bind to such neutralizing antibodies. In particular, there remains a need to develop or select novel neutralizing antibodies against a plurality of flaviviruses.

The invention has for purpose to meet the above-mentioned needs.

SUMMARY OF THE INVENTION

According to a first embodiment, the invention relates to an isolated polypeptide suitable for binding to an antibody directed against a Zika virus envelope (E) protein, said polypeptide having at most 500 amino acids and comprising sequence SEQ ID N°l:

Valine* -Asparagine* -Aspartic Acid*-Xaa2-Glycine*; wherein Xaa2 is any amino acid, in particular selected from threonine (T), isoleucine (I), leucine (L), glycine (G), alanine (A), valine (V) or a modified form thereof; wherein Valine*, Asparagine*, Aspartic Acid*, and Glycine* respectively refer to Valine, Asparagine, Aspartic Acid, and Glycine, or a modified form thereof.

The isolated peptide may be provided in the form of a modified peptide.

The isolated polypeptide may also be conjugated to an immunogenic moiety, in particular an immunogenic Keyhole limpet hemocyanin (KLH) polypeptide moiety.

According to a second embodiment, the invention relates to an attenuated flavivirus comprising a polypeptide as defined above. According to said second embodiment, the invention relates to an attenuated flavivims comprising a polypeptide suitable for binding to an antibody directed against a Zika virus envelope (E) protein, said peptide having at most 500 amino acids and comprising sequence SEQ ID N°l:

Valine* -Asparagine* -Aspartic Acid*-Xaa2-Glycine*; wherein Xaa2 is any amino acid, in particular selected from threonine (T), isoleucine (I), leucine (L), glycine (G), alanine (A), valine (V) or a modified form thereof; wherein Valine*, Asparagine*, Aspartic Acid*, and Glycine* respectively refer to Valine, Asparagine, Aspartic Acid, and Glycine, or a modified form thereof.

According to a third embodiment, the invention relates to a pharmaceutical composition, comprising a polypeptide as defined above, and/or an attenuated flavivims as defined above.

According to a fourth embodiment, the invention relates to a polynucleotide comprising or consisting of a nucleic acid encoding a polypeptide as defined above.

According to a fifth embodiment, the invention relates to a recombinant cell comprising a polynucleotide as defined above.

According to a sixth embodiment, the invention relates to a method for producing a polypeptide as defined above, wherein said method comprises the steps of: a) culturing a recombinant cell in conditions allowing the expression of the polypeptide, thereby producing the polypeptide; b) optionally, purifying the polypeptide obtained at step a).

According to a seventh embodiment, the invention relates to an isolated monoclonal antibody directed against a polypeptide or an attenuated flavivims as defined above.

According to a eighth embodiment, the invention relates to an in vitro method for recovering an antibody directed against a flavivims, wherein said method comprises the steps of: a) providing a sample susceptible to contain the said antibody; b) bringing into contact the sample at step a) with a polypeptide or an attenuated flavivims as defined above; c) recovering the said polypeptide or the said flavivirus, thereby recovering the said antibody.

According to a ninth embodiment, the invention relates to a nanovesicle or a nanoparticle, in particular an exosome, comprising at least one polypeptide as defined above.

According to a tenth embodiment, the invention relates to a recombinant cell suitable for producing a nanovesicle or a nanoparticle as defined as defined above.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Schematic diagram of EDI sequences from ZIKV. In (A), graph of recombinant EDI construct showing the fusion of the three segments of E protein from ZIKV strain BeH819015. The amino acids of EDI are numbered in E protein. The glycan loop (GL) sequence in EDI is shown as black hadched segment. In (B), sequence alignments of the rEDI constructs inserted into vector plasmid pcDNA-3.1. The three amino-acid substitutions between rEDI-BeH819015 and rEDI-MR766 are noted. The amino-acid change N154Q between rEDI-BeH819015 and mutant rEDI-(Q154) is noted. The N-terminal signal peptide and the two C-terminal FLAG and 6X(His) tags (underlined) separated by two glycine-serine spacers are shown in italics.

Figure 2. Immunoblot assay on HEK-293 cells expressing rEDI. Lysates obtained from HEK-293 cells transfected with plasmids expressing either rEDI-BeH819095, rEDI-MR766, or rEDI-(Q154) were tested with a pool of anti-ZIKBeHMR-2 immune sera at dilution 1:200 (ZIKBeHMR-2 serum) or anti-FLAG antibody (a-FLAG antibody) by immunoblot assay. Anti-inactivated ZIKBeHMR-2 immune serum was used as a negative serum (control serum). The GAPDH protein serves as a protein loading control.

Figure 3. Sequences of synthetic peptides representing E-GL sequences from ZIKV strains BeH819015 (peptGL-BeH819015) and MR766 (peptGL-MR766).

The mutant peptide peptGL-(T8, 112) derived from peptGL-BeH819015 bears the amino- acid substitutions I8T and T12I. A single amino-acid substitution H14Y has been introduced into peptGL-BeH819015 to generate the mutant peptGL-(Y14). The black points and underlined amino acids are positions that differ from peptGLBeH819015. An Alanin-rich (Ala-rich) peptide (also reported herein as ‘peptcontrol’) was used as a negative control. Figure 4. Dose-response curve of peptGL-MR766 to anti-ZIKBeHMR-2 immune serum measured by peptide-based ELISA. Plates were coated with increasing concentrations of peptGL-MR766 and incubated with a pooled anti-ZIKBeHMR-2 immune serum (ZIKBeHMR2) at dilution 1:50 (closed circles). Anti-inactivated ZIKBeHMR-2 immune serum at dilution 1:50 served as a control (closed boxes).

Figure 5. Dose-response curves of peptGL-MR766 and peptGL-BeH819015 to anti-ZIKBeHMR-2 immune sera measured by peptide-based ELISA. Plates were coated with increasing concentrations of peptGL-MR766 or peptGL-BeH819015 incubated with a pooled anti-ZIKBeHMR-2 immune serum (ZIKBeHMR2 serum) at dilution 1:200. Anti-inactivated ZIKBeHMR-2 immune serum (control serum) at dilution 1:200 served as a control. The Ala-rich peptide was used as a negative control (peptcontrol).

Figure 6. Reactivity of individual anti-ZIKBeHMR-2 immune sera to peptGL-MR766 and peptGL-BeH819015. Plates were coated with 300 ng of peptide per well and then incubated with individual anti-ZIKBeHMR-2 immune sera (n=14) (ZIKBeHMR-2) at dilution 1:200. Anti-inactivated ZIKBeHMR-2 immune serum (control) served as a control. The Ala-rich peptide was used as a negative control (peptcontrol).

Figure 7. Reactivity of anti-ZIKBeHMR-2 immune sera to peptGL-(T8, 112) mutant. Plates were coated with 300 ng of peptide (peptGL-MR766 or peptGL-(T8, 112) mutant of peptGL-BeH819015) per well and then incubated with individual anti- ZIKBeHMR-2 immune sera (n=8) (ZIKBeHMR-2) at dilution 1:200. Anti-inactivated ZIKBeHMR-2 immune serum (control) served as a negative control.

Figure 8. Reactivity of anti-ZIKBeHMR-2 immune sera to peptGL-(T8, 112) and peptGL-(Y14) mutant. Plates were coated with 300 ng of peptide (peptGL- MR766 or peptGL-(Y14) mutant of peptGL-BeH819015) per well and then incubated with individual anti-ZIKBeHMR-2 immune sera (n=8) (ZIKBeHMR-2) at dilution 1:200. Anti inactivated ZIKBeHMR-2 immune serum (control) served as a negative control. Figure 9. Antibody reactivity of KLH-peptGL ^MR766 immune serum with peptGL peptides. The O.D.450 or signal intensity of mouse pre-immune serum SI-RE and KLH-peptGL-MR766 immune serum SI -RE at dilution 1:200 in relation to peptGL- MR766 (SEQ ID N°4) or peptGL-BeH819015 (SEQ ID N°5) through peptide-based ELISA. Peptcontrol was used as a negative peptide control. The results are the mean (+S.D.) of three repeats. Statistical values correspond to differences between the two peptides. Pairwise comparison was performed and statistically significant comparison is shown as * p < 10 ^-4 .

Figure 10. Recognition of rEDI by peptGL ^MR766 reactive antibody. HEK- 293T cells were transfected 24 h with plasmids expressing rEDI-MR766 or rEDI- BeH819015 or mock-transfected (mock). FACS analysis was performed to detect expression of rEDI in transfected cells. For FACS analysis, cells were incubated with KLH-peptGL- MR766 immune serum SI-RE as primary antibody and then Alexa 488-conjugated anti mouse IgG antibody as secondary antibody. The MFI values of FITC were determined. The error bars represent standard errors of two independent experiments. MFI values generated by rEDI-MR766 or rEDI-BeH819015 in comparison with mock. Pairwise comparisons were performed and statistically significant comparisons are shown as ** p < 10 ^-2 , * p < 0.05.

DETAILED DESCRIPTION OF THE INVENTION

The inventors now provide experimental evidence that the glycan-loop (E-GL) of the envelope E protein influences the accessibility of neutralizing antibody epitopes recognized by an anti- ZIKBeHMR-2 immune serum.

In particular, it is shown that residues E-152, E-156, and E-158 from the E-GL from Domain I (E-DI) of the Zika vims E protein play a key role in the difference of neutralization between BeH819015 and MR766 strains.

Hence, the inventors provide evidence of the antigenic reactivity of recombinant EDI (rEDI) from ZIKV strains MR766 and BeH819015 as well as synthetic short peptide representing their E-GL (peptGL).

Immunoblot assay show that anti-ZIKBeHMR-2 immune serum is reactive only with rEDI of MR766. Thus, evidence is provided herein that a 20-mer peptide representing the E-GL of the Envelope protein of ZIKV virus, especially the strain MR766, enables a new approach for the detection of specific antibodies, especially neutralizing antibodies, as well as the development of candidate vaccines against ZIKV.

This is surprising, because the immunogenic properties and antigenic reactivity of such isolated fragments had not previously been reported experimentally.

Without wishing to be bound by the theory, the inventors now propose that the three EDI-GL residues E-152, E-156, and E-158 play a key role in the accessibility of neutralizing antibody epitopes on ZIKV.

To the knowledge of the inventors, this is the first report demonstrating that the short peptide GSQHSGMTVNDIGYETDENR (SEQ ID N°4) representing the glycan-loop (GL) of the Zika virus (ZIKV) strain MR766 is antigenic in a peptide-based ELISA format.

To the knowledge of the inventors, this is also the first report demonstrating the antigenicity of a fragment of the Envelope protein of a flavivirus, within Domain I (E-DI), which explicitly includes the glycan-loop itself.

Thus, an isolated polypeptide bearing this motif is now found suitable for the development of a serologic test based on the detection of anti-flavivirus antibodies, in particular anti-ZIKV E-GL antibodies, in serum specimens. Conversely, the live ZIKBeHMR-2 virus which contains the E-GL sequence GSQHSGMTVNDIGYETDENR (SEQ ID N°4) is also able to elicit production of neutralizing antibodies against different ZIKV strains. Thus, the isolated Zika virus Envelope polypeptide fragment can now be included in the formulation of a candidate vaccine against ZIKV, but also against other flavivimses due to cross -reactivity and cross-protection.

Other variants of the polypeptide are further provided, as controls for the differentiation between neutralizing and non-neutralizing antibodies.

Definitions

As used herein, the singular form " a ", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a pharmaceutically acceptable carrier " encompasses a plurality of pharmaceutically acceptable carriers, including mixtures thereof.

As used herein, « a plurality of» may thus include « two » or « two or more ».

As used herein, « comprising » may include « consisting of». As used herein, « an amino acid sequence having from 0 to 200 amino acids in length » generally encompasses any peptide having from 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,

36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,

60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,

84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,

106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,

124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,

142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,

160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,

178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,

196, 197, 198, 199, 200 amino acids. It will be readily understood that when the amino acid sequence is composed of 0 amino acids, i.e. when referring to a N-ter or C-ter part of a polypeptide, this refers to the absence of additional amino acids.

As used herein, a “ peptide having at most 500 amino acids” generally encompasses any peptide having from 5 to 500 amino acids in length; which may thus encompass any peptide having from 5 to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,

21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,

45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,

69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,

93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,

131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,

149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,

167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,

185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,

203, 204, 205, 206, 207, 208, 209, 210, 212, 212, 213, 214, 215, 216, 217, 218, 219, 220,

221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,

239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256,

257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274,

275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292,

293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,

329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,

347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,

365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382,

383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400,

401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418,

419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436,

437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,

455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,

473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490,

491, 492, 493, 494, 495, 496, 497, 498, 499, 500 amino acids.

As used herein, the term “ amino acid ” refers to natural or unnatural amino acids in their D and L stereoisomers for chiral amino acids. It is understood to refer to both amino acids and the corresponding amino acid residues, such as are present, for example, in peptidyl structure. Natural and unnatural amino acids are well known in the art. Common natural amino acids include, without limitation, alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (lie), leucine (Leu), Lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Uncommon and unnatural amino acids include, without limitation, allyl glycine (AllylGly), norleucine, norvaline, biphenylalanine (Bip), citrulline (Cit), 4- guanidinophenylalanine (Phe(Gu)), homoarginine (hArg), homolysine (hLys), 2- naphtylalanine (2-Nal), ornithine (Om) and pentafluorophenylalanine.

As used herein, a “ modified form” of a given amino acid encompasses any equivalent, translationally or post-translationally modified amino acid or non-naturally occurring amino acid, including non standard amino acids, that result from a reaction at an amino group, carboxy group, side-chain functional group, or from the replacement of any hydrogen by a heteroatom. Hence, a modified amino acid may encompass an amino acid derivative which results from either one of modifications selected from: N-linked glycosylation, O-linked glycosylation, phosphorylation, methylation, acetylation, amidation, formation of pyrrolidone carboxylic acid, isomerization, hydroxylation, sulfation, flavin-binding, cysteine oxidation, nitrosylation and ubiquity lation. As used herein and above, an "equivalent amino acid" means an amino acid which may be substituted for another amino acid in the peptide compounds according to the invention without any appreciable loss of function. Equivalent amino acids will be recognized by those of ordinary skill in the art. Substitution of like amino acids is made on the basis of relative similarity of side chain substituents, for example regarding size, charge, hydrophilicity and hydrophobicity as described herein. The phrase "or an equivalent amino acid thereof' when used following a list of individual amino acids means an equivalent of one or more of the individual amino acids included in the list.

As used herein, the term "antibody" or "immunoglobulin" have the same meaning. The term "antibody" refers to folded immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immuno- specifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments.

The terms " monoclonal antibody", " monoclonal Ab", " monoclonal antibody composition" , "mAb" , or the like, as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

As used herein, the term “ specificity ” refers to the ability of an antibody to detectably bind an epitope presented on an antigen while having relatively little detectable reactivity with non-antigen proteins or structures. Specificity can be relatively determined by binding or competitive binding assays, using, e.g., Biacore instruments, a described elsewhere herein. Specificity can be exhibited by, e.g., an about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or greater ratio of affinity /avidity in binding to the specific antigen versus nonspecific binding to other irrelevant molecules.

As used herein, the term “ affinity ”, as used herein, means the strength of the binding of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined by 1/Kd. Preferred methods for determining the affinity of mAbs can be found in Harlow, et ah, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. One method well known in the art for determining the affinity of mAbs is the use of Biacore instruments.

As used herein, an " immune response " or " immunity " or “ immunogenicity ” as the terms are interchangeably used herein, is meant the induction of a humoral (i.e., B cell) and/or cellular (i.e., T cell) response. Suitably, a humoral immune response may be assessed by measuring the antigen- specific antibodies present in serum of immunized animals in response to introduction of the antigen into the host . The immune response may be assessed by the enzyme linked immunosorbant assay of sera of immunized mammals, or by microneutralization assay of immunized animal sera. A CTL assay can be employed to measure the T cell response from lymphocytes isolated from the spleen or other organs of immunized animals.

As used herein, a “ diagnosis ” may also encompass the “ follow-up ” of a given patient or population of patients over time.

As used herein, an “ immunologically active fragment ” generally refers to a fragment of a given antigen having at least five (5) consecutive amino acids from the said antigen. Thus, this definition may encompass fragments having at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 26, 27, 28, 29, or 30 consecutive aminoacids from the said antigen.

As used herein, the term “ biocompatible ” is meant to refer to compounds which do not cause a significant adverse reaction in a living animal when used in pharmaceutically relevant amounts.

As used herein, a “ pharmaceutically acceptable carrier” is intended to include any and ah carrier (such as any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like) which is compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances are known. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. As used herein, a “ pharmaceutically acceptable carrier ” is intended to include any and all carrier (such as any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like) which is compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances are known. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention.

As used herein, the term “ nanoparticles ” is meant to refer to particles having an average size (such as a diameter, for spherical or nearly spherical nanoparticles) of 1000 nanometers (nm) in size or less, in particular of 200 nm in size or less. The “ diameter ” is typically defined as the “ crystalline diameter ” or as the “ hydrodynamic diameter”. The crystalline size (or “ diameter ” if applicable) of a population of nanoparticles can be determined herein by transmission electron microscopy whereas the hydrodynamic size related to surface functionalization is measured by dynamic laser light scattering (DLS), in a physiological medium, for example NaCl 0.9% , NaCl 0.9%/Glucose 5%, or other buffer media at a physiological pH, used for biological evaluation as well as in vitro and in vivo experiments, as described in the Material & Methods section.

As used herein, the term “ nanovesicles ” is meant to refer to an extracellular vesicle that is a nanoparticle having generally an average size of 200 nm or less. Nanovesicles are lipid membrane bound particles that carry biologically active signaling molecules (e.g. microRNAs, proteins) among other molecules. Generally, the nanovesicle is limited by a lipid bilayer, and the biological molecules are enclosed and/or can be embedded in the bilayer. Thus, a nanovesicle includes a lumen surrounded by plasma membrane. The different types of vesicles can be distinguished based on diameter, subcellular origin, density, shape, sedimentation rate, lipid composition, protein markers, nucleic acid content and origin, such as from the extracellular matrix or secreted.

As used herein, the term " exosome " refers to a membranous nanovesicle which is secreted by a cell, and ranges generally in diameter from 10 to 200 nm, in particular from 10 to 100 nm. Exosomes generally are produced from late endosomes or multivesicular bodies, as intralumenal vesicles which are formed by the inward budding and scission of vesicles from the limited endosomal membrane into these enclosed nanovesicles. These intralumenal vesicles are then released from the multivesicular body lumen into the extracellular environment, typically into a body fluid such as blood, cerebrospinal fluid or saliva, during exocytosis upon fusion with the plasma membrane. An exosome is created intracellularly when a segment of membrane invaginates and is endocytosed.

As used herein, the term “ conjugated to”, or “ linked to”, such as in “a ligand linked to the nanoparticles” may refer either to a covalent link or to a non-covalent link. In a non-limitative manner, such non-covalent interactions may occur due to electrostatic interactions, Van der Walls forces, p-effects, and hydrophobic effects. Alternatively, covalent-interactions occur as a consequence of the formation of a covalent bond, such as the coupling of a ligand which can bind to an aryl hydrocarbon receptor (AHR) transcription factor, and a functional (reactive) chemical group at the surface of the nanoparticle.

As used herein, “ treating ” means any manner in which one or more of the symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. As used herein, amelioration of the symptoms of a particular disorder refers to any lessening of the symptoms, whether permanent or temporary, lasting or transient, that can be attributed to or associated with treatment by the compositions and methods of the present invention. Accordingly, the expression “ treating ” may include “ reversing partially or totally the effect” of a given condition, or even “ curing ” when permanent reversal is considered.

As used herein, “ preventing ” encompasses “ reducing the likelihood of occurrence” and “ reducing the likelihood of re-occurrence” .

As used herein, the term “ isolated ” or “purified" refers to those molecules that have been altered by humans in their native state, that is, if such molecules exist in nature, that they have been changed and/or removed from their original environment.

As used herein, a "vaccine composition " is a composition suitable for administration to a human is capable of eliciting a specific immune response against a pathogen, in particular a Flavivirus, such as Zika virus.

As used herein, the “ percentage identity” between two sequences of nucleic acids or proteins means the percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after optimal alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly along their length. The comparison of sequences is traditionally carried out by comparing the sequences after having optimally aligned them, said comparison being able to be conducted by segment or by using an “alignment window”. Optimal alignment of the sequences for comparison can be carried out, in addition to comparison by hand, by means of the local homology algorithm of Smith and Waterman (1981), by means of the local homology algorithm of Neddleman and Wunsch (1970), by means of the similarity search method of Pearson and Lipman (1988) or by means of computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group,

575 Science Dr., Madison, WI, or by the comparison software BEAST NR or BEAST P).

The percentage identity between two sequences is determined by comparing the two optimally-aligned sequences in which the sequence to compare can have additions or deletions compared to the reference sequence for optimal alignment between the two sequences. Percentage identity is calculated by determining the number of positions at which the nucleotide or amino acid residue is identical between the two sequences, preferably between the two complete sequences, dividing the number of identical positions by the total number of positions in the alignment window and multiplying the result by 100 to obtain the percentage identity between the two sequences.

As used herein, the term ‘‘ flaviviru ” refers to members of the genus Flavivirus, which is classified within the family Flaviviridae. The flaviviruses are largely pathogenic to humans and other mammals. Flaviviruses that inflict disease upon humans and animals include Alfuy, Apoi, Aroa, Bagaza, Banzi, Batu Cave, Bouboui, Bukalasa bat, Bussuquara, Cacipacore, Carey Island, Cowbone Ridge, Dakar bat, Dengue (serotypes 1, 2, 3 and 4), Edge Hill, Entebbe bat, Gadgets Gully, Iguape, Ilheus, Israel turkey meningoencephalitis, Japanese encephalitis, Jugra, Jutiapa, Kadam, Karshi, Kedougou, Kokobera, Koutango, Kunjin, Kyasanur Forest disease, Langat, Meaban, Modoc, Montana myotis leukoencephalitis, Murray Valley encephalitis, Naranjal, Negishi, Ntaya, Omsk hemorrhagic fever, Phnom Penh bat, Potiskum, Powassan, Rio Bravo, Rocio, Royal Farm, Russian spring summer encephalitis, Saboya, Sal Vieja, San Perlita, Saumarez Reef, Sepik, Sokuluk, Spondweni, St. Louis encephalitis, Stratford, Tick-bome encephalitis — central European subtype, Tick-borne encephalititis — far eastern subtype, Tembusu, THCAr, Tyuleniy, Uganda S, Usutu, West Nile, Yaounde, Yellow fever, Yokose, Zika, Cell fusing agent and other related flaviviruses, as listed in Kuno et al. (J. Virol. 72: 73-83 (1998)).

Isolated peptides According to a first embodiment, the invention relates to an isolated polypeptide suitable for binding to an antibody directed against a Zika virus envelope (E) protein, said peptide having at most 500 amino acids and comprising sequence SEQ ID N°l:

Valine* -Asparagine* -Aspartic Acid*-Xaa2-Glycine*; wherein Xaa2 is any amino acid, in particular selected from threonine (T), isoleucine (I), leucine (L), glycine (G), alanine (A), valine (V) or a modified form thereof; wherein Valine*, Asparagine*, Aspartic Acid*, and Glycine* respectively refer to Valine, Asparagine, Aspartic Acid, and Glycine, or a modified form thereof.

In particular, the isolated polypeptide may comprise amino acid sequence SEQ

ID N°2:

V aline* - Asparagine* - Aspartic Acid* -Xaa2-Glycine* -Xaa3 ; wherein Xaa3 is tyrosine (Y), histidine (H), or a modified form thereof, and preferably is a tyrosine (Y).

It will be readily understood that the provided isolated polypeptides are meant to include an amino acid sequence which is prone to mimic the glycan-loop of the Zika vims Envelope protein. Fragments and extended constructs including more than the amino acid sequence SEQ ID N°1 are also considered, with the proviso that the isolated polypeptide does not consist of a whole, full-length, naturally-occuring Zika vims Envelope protein.

According to some embodiments, the isolated polypeptide may still include a polypeptide sequence corresponding to parts of a naturally-occuring Zika vims Envelope protein, such as all or part of the Domain I, Domain II, and/or Domain III of a naturally- occuring Zika vims Envelope protein.

For reference, a full-length, naturally-occuring Zika vims Envelope protein is referenced in the Genbank database under the n° LC002520 for ZIKV strain MR766 (SEQ ID N°13); and under n°KU365778 for ZIKV strain BeH819015 (SEQ ID N°14).

By reference to SEQ ID N°13, the region corresponding to Domain I (EDI) corresponds to polypeptides sequences 1 to 52, 132 to 193, and 280 to 296.

By reference to SEQ ID N°13, the region corresponding to Domain II (EDII) corresponds to polypeptides sequences 52 to 132, and 193 to 280.

By reference to SEQ ID N°13, the region corresponding to Domain IIII (EDIII) corresponds to polypeptides sequences 296 to 406. According to a particular embodiment, the isolated peptide as defined above is an isolated polypeptide suitable for binding to an antibody directed against a Zika vims envelope (E) protein, said polypeptide comprising sequence SEQ ID N°l:

[N-ter] -V aline* - Asparagine* - Aspartic Acid* -Xaa2-Glycine* - [C-ter] ; wherein Xaa2 is any amino acid, in particular selected from threonine (T), isoleucine (I), leucine (L), glycine (G), alanine (A), valine (V) or a modified form thereof; wherein Valine*, Asparagine*, Aspartic Acid*, and Glycine* respectively refer to Valine, Asparagine, Aspartic Acid, and Glycine, or a modified form thereof; and with [N-ter] being an amino acid sequence having from 0 to 200 amino acids in length; with [C-ter] being an amino acid sequence having from 0 to 200 amino acids in length.

According to another particular embodiment, the isolated peptide as defined above is an isolated polypeptide suitable for binding to an antibody directed against a Zika virus envelope (E) protein, said polypeptide comprising sequence SEQ ID N°2:

[N-ter]-Valine*-Asparagine*-Aspartic Acid*-Xaa2-Glycine*-Xaa3*-[C-ter]; wherein Xaa2 is any amino acid, in particular selected from threonine (T), isoleucine (I), leucine (L), glycine (G), alanine (A), valine (V) or a modified form thereof; wherein Xaa3 is tyrosine (Y), histidine (H), or a modified form thereof, and preferably is a tyrosine (Y); wherein Valine*, Asparagine*, Aspartic Acid*, and Glycine* respectively refer to Valine, Asparagine, Aspartic Acid, and Glycine, or a modified form thereof; and with [N-ter] being an amino acid sequence having from 0 to 200 amino acids in length; with [C-ter] being an amino acid sequence having from 0 to 200 amino acids in length.

In particular, the isolated polypeptide may comprise amino acid sequence SEQ

ID N°3:

[N-ter] -Xaal- V aline* - Asparagine* - Aspartic Acid* -Xaa2-Glycine* -Xaa3- [C-ter] ; wherein Xaal is any amino acid, in particular selected from threonine (T), isoleucine (I), leucine (L), glycine (G), alanine (A), valine (V) or a modified form thereof; wherein Xaa2 is any amino acid, in particular selected from threonine (T), isoleucine (I), leucine (L), glycine (G), alanine (A), valine (V) or a modified form thereof; wherein Xaa3 is tyrosine (Y), histidine (H), or a modified form thereof, and preferably is a tyrosine (Y);

[N-ter] is an amino acid sequence having from 0 to 200 amino acids in length;

[C-ter] is an amino acid sequence having from 0 to 200 amino acids in length.

The isolated polypeptide as defined above may thus comprise amino acid sequence SEQ ID N°l, in particular amino acid sequence SEQ ID N°2, more particularly amino acid sequence SEQ ID N°3, wherein Xaal and Xaa2 are selected from threonine (T) or isoleucine (I), or a modified form thereof, and Xaa3 is tyrosine (Y), histidine (H), or a modified form thereof.

Preferably, the isolated polypeptide as defined above may thus comprise amino acid sequence SEQ ID N°l, in particular amino acid sequence SEQ ID N°2, more particularly amino acid sequence SEQ ID N°3, wherein Xaal and Xaa2 are selected from threonine (T) or isoleucine (I), and Xaa3 is tyrosine (Y), histidine (H).

According to one preferred embodiment, Xaal and Xaa2 are selected from threonine (T) or isoleucine (I), and Xaa3 is tyrosine (Y).

The isolated polypeptide as defined above may comprise or even consist of, a polypeptide sequence selected from:

- TVNDIGY (SEQ ID N°16);

- IVNDTGH (SEQ ID N°17);

- TVNDIGH (SEQ ID N°18);

- IVNDIGY (SEQ ID N°19);

- TVNDTGY (SEQ ID N°20) ;

- IVNDIGH (SEQ ID N°21) ;

- TVNDTGH (SEQ ID N°22)

- IVNDTGY (SEQ ID N°23).

The isolated polypeptide as defined above may comprise or even consist of, a polypeptide sequence selected from:

- GS QHS GMT VNDIG YETDENR (SEQ ID N°4);

- GS QHS GMI VNDTGHETDENR (SEQ ID N°5); - GS QHS GMT VNDIGHETDENR (SEQ ID N°6);

- GS QHS GMI VNDIG YETDENR (SEQ ID N°7);

- GS QHS GMT VNDTGYETDENR (SEQ ID N°8);

- GS QHS GMI VNDIGHETDENR (SEQ ID N°9);

- GS QHS GMT VND TGHETDENR (SEQ ID N°10)

- GS QHS GMI VNDTGYETDENR (SEQ ID N°ll).

According to a most preferred embodiment, the invention relates to such isolated polypeptides comprising (or even consisting of) amino acid sequences SEQ ID N°2 or 3; wherein Xaa3 is a Tyrosine.

Hence, according to a most preferred embodiment, the invention relates to such isolated polypeptides comprising (or even consisting of) amino acid sequences derived from the E protein wherein residue corresponding to position 158 is a Tyrosine.

The isolated polypeptides as defined above may also, or alternatively, comprise a sequence having at least 80% of sequence identity with either one of SEQ ID N°4 to 11; in particular which may comprise a sequence having at least 85% of sequence identity with either one of SEQ ID N°4 to 11; more particularly, which may comprise a sequence having at least 90% of sequence identity with either one of SEQ ID N°4 to 11; preferably, which may comprise a sequence having at least 95% of sequence identity with either one of SEQ ID N°4 to 11.

For instance, the isolated polypeptide as defined above may comprise a sequence having at least 80% of sequence identity with SEQ ID N°4.

Alternatively, the isolated polypeptide as defined above may comprise a sequence having at least 80% of sequence identity with SEQ ID N°4.

Alternatively, the isolated polypeptide as defined above may comprise a sequence having at least 80% of sequence identity with SEQ ID N°5.

Alternatively, the isolated polypeptide as defined above may comprise a sequence having at least 80% of sequence identity with SEQ ID N°6.

Alternatively, the isolated polypeptide as defined above may comprise a sequence having at least 80% of sequence identity with SEQ ID N°7. Alternatively, the isolated polypeptide as defined above may comprise a sequence having at least 80% of sequence identity with SEQ ID N°8.

Alternatively, the isolated polypeptide as defined above may comprise a sequence having at least 80% of sequence identity with SEQ ID N°9.

Alternatively, the isolated polypeptide as defined above may comprise a sequence having at least 80% of sequence identity with SEQ ID N°10.

Alternatively, the isolated polypeptide as defined above may comprise a sequence having at least 80% of sequence identity with SEQ ID N°ll.

Alternatively, the isolated polypeptide as defined above may comprise a sequence having at least 90% of sequence identity with SEQ ID N°4.

Alternatively, the isolated polypeptide as defined above may comprise a sequence having at least 90% of sequence identity with SEQ ID N°5.

Alternatively, the isolated polypeptide as defined above may comprise a sequence having at least 90% of sequence identity with SEQ ID N°6.

Alternatively, the isolated polypeptide as defined above may comprise a sequence having at least 90% of sequence identity with SEQ ID N°7.

Alternatively, the isolated polypeptide as defined above may comprise a sequence having at least 90% of sequence identity with SEQ ID N°8.

Alternatively, the isolated polypeptide as defined above may comprise a sequence having at least 90% of sequence identity with SEQ ID N°9.

Alternatively, the isolated polypeptide as defined above may comprise a sequence having at least 90% of sequence identity with SEQ ID N°10.

Alternatively, the isolated polypeptide as defined above may comprise a sequence having at least 90% of sequence identity with SEQ ID N°ll.

The isolated polypeptide as defined above may comprise a sequence having at least 80% of sequence identity with SEQ ID N°12:

IRCIGVSNRDFVEGMS GGTWVD VVLEHGGC VT VMAQDKPT VDIELVTTT V S NPENLE YRIMLS VHGS QHS GMT VNDIG YETDENR AKVEITPN S PR AE ATLGG FGSLGLDCEPRAKGRLS S GHLCRLKMD (SEQ ID N°12).

In particular, the isolated polypeptide as defined above may comprise a sequence having at least 90% of sequence identity with SEQ ID N°12: IRCIGVSNRDFVEGMS GGTWVD VVLEHGGC VT VMAQDKPT VDIELVTTT V S NPENLEYRIMLSVHGSQHSGMTVNDIGYETDENRAKVEITPNSPRAEATLGG FGSLGLDCEPRAKGRLS S GHLCRLKMD (SEQ ID N°12).

In particular, the isolated polypeptide as defined above may comprise a sequence having at least 95% of sequence identity with SEQ ID N°12:

IRCIGVSNRDFVEGMS GGTWVD VVLEHGGC VT VMAQDKPT VDIELVTTT V S NPENLEYRIMLSVHGSQHSGMTVNDIGYETDENRAKVEITPNSPRAEATLGG FGS LGLDCEPR AKGRLS S GHLCRLKMD (SEQ ID N°12).

Due to the presence of a motif mimicking the glycan-loop of Zika vims Envelope protein, the GL motif corresponding to SEQ ID N°1 may be either glycosylated on Asparagine (Asn or “N”), or alternatively not glycosylated.

According to one embodiment, the isolated peptide is glycosylated on Asparagine*, by reference to SEQ ID N°l.

According to one embodiment, the isolated peptide is glycosylated on Asparagine*, by reference to SEQ ID N°l.

The isolated polypeptide of SEQ ID N°1 may further comprise additional amino acids beyond the GL motif corresponding to SEQ ID N°l.

According to one embodiment, the isolated peptide is of at most 400 amino acids in length. According to one embodiment, the isolated peptide is of at most 300 amino acids in length. According to one embodiment, the isolated peptide is of at most 200 amino acids in length. According to one embodiment, the isolated peptide is of at most 100 amino acids in length. According to one embodiment, the isolated peptide is of at most 50 amino acids in length. According to one embodiment, the isolated peptide is of at most 40 amino acids in length. According to one embodiment, the isolated peptide is of at most 30 amino acids in length. According to one embodiment, the isolated peptide is of at most 25 amino acids in length.

According to a particular embodiment, the isolated peptide as defined above is an isolated polypeptide suitable for binding to an antibody directed against a Zika vims envelope (E) protein, said polypeptide having at most 400 amino acids and comprising sequence SEQ ID N°l:

[N-ter] -V aline* - Asparagine* - Aspartic Acid* -Xaa2-Glycine* - [C-ter] ; wherein Xaa2 is any amino acid, in particular selected from threonine (T), isoleucine (I), leucine (L), glycine (G), alanine (A), valine (V) or a modified form thereof; wherein Valine*, Asparagine*, Aspartic Acid*, and Glycine* respectively refer to Valine, Asparagine, Aspartic Acid, and Glycine, or a modified form thereof; and [N-ter] is an amino acid sequence having from 0 to 200 amino acids in length;

[C-ter] is an amino acid sequence having from 0 to 200 amino acids in length.

According to another particular embodiment, the isolated peptide as defined above is an isolated polypeptide suitable for binding to an antibody directed against a Zika vims envelope (E) protein, said polypeptide having at most 400 amino acids and comprising sequence SEQ ID N°2:

[N-ter]-Valine*-Asparagine*-Aspartic Acid*-Xaa2-Glycine*-Xaa3*-[C-ter]; wherein Xaa2 is any amino acid, in particular selected from threonine (T), isoleucine (I), leucine (L), glycine (G), alanine (A), valine (V) or a modified form thereof; wherein Xaa3 is tyrosine (Y), histidine (H), or a modified form thereof wherein Valine*, Asparagine*, Aspartic Acid*, and Glycine* respectively refer to Valine, Asparagine, Aspartic Acid, and Glycine, or a modified form thereof; and [N-ter] is an amino acid sequence having from 0 to 200 amino acids in length;

[C-ter] is an amino acid sequence having from 0 to 200 amino acids in length.

The isolated polypeptides according to the invention, may be modified polypeptides. Hence, according to one embodiment, the isolated polypeptides may comprise one or more modified amino acids. According to one of such embodiments, the isolated polypeptides may comprise one or more chemically modified amino acids.

The N- and C-termini of the peptides described herein may be protected against proteolysis. For instance, the N-terminus or or the C-terminus may be in the form of an acetyl group, and the N-terminus or the C-terminus may be in the form of an amide group. Internal modifications of the peptides to be resistant to proteolysis are also envisioned, e.g. wherein at least a -CONH peptide bond is modified and replaced by a (CH2NH) reduced bond, a (NHCO) retro-inverso bond, a (CH2-0) methylene-oxy bond, a (CH2-S) thiomethylene bond, a (CH2CH2) carba bond, a (CO-CH2) cetomethylene bond, a (CHOH-CH2) hydroxyethylene bond), a (N-N) bound, a E-alcene bond or also a -CH=CH-bond. The peptides described herein may also be protected against proteolysis by the technique of stapled peptides, such as the technique described by Walensky et al. Science. 2004, 305,

1466-70.

In certain embodiments, the isolated polypeptide according to the invention is covalently or non-covalently linked (conjugated) to a carrier.

The types of carrier molecules used for enhancing the immunogenicity of a given construct (i.e. the isolated polypeptide) linked to a carrier are well within the general knowledge of the one skilled in the art. The carrier to which the polypeptide is optionally conjugated can be selected from a wide variety of known carriers (or immunogenic moieties). Hence, according to one embodiment, the polypeptide of the invention may be further conjugated to an immunogenic moiety.

In a non-exhaustive manner, the immunogenic moiety may be an antigen as described herein is any substance that under appropriate conditions results in an immune response in a subject, including, but not limited to, polypeptides, peptides, proteins, glycoproteins, and polysaccharides, nanovesicles and nanoparticles.

Antigens that may be conjugated to the isolated polypeptide include antigens from an animal, a plant, a vims, a protozoan, a parasite, a bacterium, or an antigen associated with a disease state, such as cancer, for example a tumor antigen, or a combination of antigens from the same or different sources.

The isolated polypeptide of the invention may be conjugated to one or more more antigens. The antigen may be any viral peptide, protein, polypeptide, or a fragment thereof derived from a vims including, but not limited to, influenza viral proteins, e. g. , influenza vims neuraminidase, influenza vims hemagglutinin, respiratory syncytial vims (RSV) -viral proteins, e. g. , RSV F glycoprotein, RSV G glycoprotein, herpes simplex vims (HSV) viral protein, e. g., herpes simplex vims glycoprotein including for example, gB, gC, gD, and gE. Examples of bacterial antigens include the chlamydia MOMP and PorB antigens . Antigen of a pathogenic vims that may be used in the immunogenic compositions of the invention include adenovirdiae (e. g . , mastadenovims and aviadenovims), herpesviridae (e. g., herpes simplex virus 1, herpes simplex virus 2, herpes simplex virus 5, and herpes simplex virus 6), leviviridae (e. g., levivirus, enterobacteria phase MS2, allolevirus), poxviridae (e. g. , chordopoxvirinae , parapoxvirus , avipoxvirus, capripoxvirus , leporiipoxvirus, suipoxvirus, molluscipoxvirus, and entomopoxvirinae), papovaviridae (e. g., polyomavirusand papillomavirus), paramyxoviridae (e. g paramyxovirus , parainfluenza virusl, mobillivirus (e. g., measles virus), rubulavirus (e. g., mumps virus), pneumonovirinae (e. g., pneumovirus, human respiratory syncytial virus), and metapneumo virus (e. g. , avian pneumovirus and human metapneumovirus) ), picomaviridae (e.g., enterovirus, rhinovirus, hepatovirus (e. g., human hepatits A virus) , cardiovirus, andapthovirus), reoviridae (e. g. , orthoreo virus , orbivirus, rotavirus, cypovirus, fijivirus, phytoreovirus, and oryzavirus) , retroviridae (e. g. , mammalian type B retroviruses, mammalian type C retroviruses, avian type C retroviruses, type D retrovirus group, BLV- HTLV retroviruses, lentivirus (e. g. human immunodeficiency virus 1 and human immunodeficiency virus 2), spumavirus), flaviviridae (e. g., hepatitis C virus), hepadnaviridae (e. g. , hepatitis B virus), togaviridae (e. g . , alphavirus (e. g., sindbis virus) and rubivirus (e. g., rubella virus) ) , rhabdoviridae (e. g. vesiculovirus, lyssavirus, ephemerovirus, cytorhabdovirus, and necleorhabdovirus), arenaviridae (e. g., arenavirus, lymphocytic choriomeningitis virus, Ippy virus, and lassa virus), and coronaviridae (e. g . , coronavirus and torovirus).

The antigen may be an infectious disease agent including, but not limited to, influenza virus hemagglutinin, human respiratory syncytial virus G glycoprotein, core protein, matrix protein or other protein of Dengue virus, measles virus hemagglutinin, herpes simplex virus type 2 glycoprotein gB, poliovirus I VP1, envelope glycoproteins of HIV I, hepatitis B surface antigen, diptheria toxin, streptococcus 24M epitope, gonococcal pilin, pseudorabies virus g50 (gpD) , pseudorabies virusll (gpB) , pseudorabies virusglll (gpC) , pseudorabies virus glycoprotein H, pseudorabies virus glycoprotein E, transmissible gastroenteritis glycoprotein 195, transmissible gastroenteritis matrix protein, swine rotavirus glycoprotein 38, swine parvovirus capsid protein, Serpulinahydodysenteriae protective antigen, bovine viral diarrhea glycoprotein 55, Newcastle disease virus hemagglutinin- neuraminidase, swine flu hemagglutinin, swine flu neuraminidase, foot and mouth disease virus, hog colera virus, swine influenza virus, African swine fever virus, Mycoplasmaliyopneutiioniae, infectious bovine rhinotracheitis virus (e. g. , infectious bovine rhinotracheitis virus glycoprotein E or glycoprotein G) , or infectious laryngotracheitis virus (e. g., infectious laryngotracheitis virus glycoprotein G or glycoprotein I) , a glycoprotein of La Crosse virus, neonatal calf diarrhoea virus, Venezuelan equineencephalomyelitis virus, punta toro virus, murine leukemia virus, mouse mammary tumor virus, hepatitis B virus core protein and/or hepatitis B virus surface antigen or a fragment or derivative thereof, antigen of equine influenza virus or equine herpesvirus (e.g., equine influenza virus type A/ Alaska 91 neuraminidase, equine influenza virus typeA/Miami 63 neuraminidase, equine influenza virus type A/Kentucky81 neuraminidase equine herpesvirus type 1 glycoprotein B, and equine herpesvirus type 1 glycoprotein D, antigen of bovine respiratory syncytial virus or bovine parainfluenza virus (e.g., bovine respiratory syncytial virus attachment protein (BRSV G) , bovine respiratory syncytial virus fusion protein (BRSV F) , bovine respiratory syncytial virus nucleocapsid protein (BRSVN) , bovine parainfluenza virus type 3 fusion protein, and the bovine parainfluenza virus type 3 hemagglutinin neuraminidase) , bovine viral diarrhea virus glycoproteins or glycoprotein 53. The antigen may also be a cancer antigen or a tumor antigen. Any cancer or tumor antigen known to one skilled in the art may be used in the present invention including, but not limited to, KS 1/4 pan-carcinoma antigen, ovarian carcinoma antigen (CA125) , prostatic acid phosphate, prostate specific antigen, melanoma-associated antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen (HMW- MAA) , prostate specific membrane antigen, carcinoembryonic antigen (CEA) , polymorphic epithelial mucin antigen, human milk fat globule antigen, colorectal tumor- associated antigens such as: CEA, TAG- 72, LEA, Burkitt ' s lymphoma antigen-38.13 , CD19, human B-lymphoma antigen-CD20, CD33, melanoma specific antigens such as ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside GM3 , tumor- specific transplantation type of cell-surface antigen (TSTA) such as virally- induced tumor antigens including T-antigen DNA tumor viruses and Envelope antigens of RNA tumor viruses, oncofetal antigen- alpha-fetoprotein such as CEA of colon, bladder tumor oncofetal antigen, differentiation antigen such as human lung carcinoma antigen L6, L20, antigens of fibrosarcoma, human leukemia T cell antigen-Gp37, neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR (Epidermal growth factor receptor) , HER2 antigen (pl85HER2 ), polymorphic epithelial mucin (PEM), malignant human lymphocyte antigen-APO-1, differentiation antigen, such as I antigen found in fetal erythrocytes, primary endoderm, I antigen found in adult erythrocytes, preimplantation embryos, I (Ma) found in gastricadenocarcinomas,M18, M39 found in breast epithelium, SSEA-I found in myeloid cells, VEP8, VEP9, Myl,VIM-D5,Du56-22 found in colorectal cancer, TRA-1-85 (blood group H) , C14 found in colonic adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastric cancer, Y hapten, LeY found in embryonal carcinoma cells, TL5 (blood group A) , EGF receptor found inA431 cells, El series (blood group B) found in pancreatic cancer, FCIO. 2 found in embryonal carcinoma cells, gastric adenocarcinoma antigen, CO-514 (blood group Lea) found in Adenocarcinoma, NS-IO found in adenocarcinomas, CO-43 (blood groupLeb) , G49 found in EGF receptor of A431 cells, MH2 (blood groupALeb/Ley) found in colonic adenocarcinoma, 19.9 found in colon cancer, gastric cancer mucins, TsA7 found in myeloid cells, R24 found in melanoma, 4.2,GD3,D1.1, OFA-I, GM2, OFA-2, GD2, and Ml : 22: 25: 8 found in embryonal carcinoma cells, and SSEA-3 and SSEA-4 found in 4 to 8- cell stage embryos. The antigen may comprise a virus, against which an immune response is desired. The virus may be a recombinant or chimeric viruses. The virus may be attenuated. Production of recombinant, chimeric and attenuated viruses may be performed using standard methods known to one skilled in the art. The invention encompasses a live recombinant viral antigens or inactivated recombinant viral antigens.

In particular, the immunogenic moiety may be selected from an immunogenic Keyhole limpet hemocyanin (KLH) polypeptide moiety.

The isolated polypeptide according to invention is suitable for use as a medicament. In particular, the isolated polypeptide is suitable for use in a method for preventing and/or eliciting an immune response against a flavivirus infection, or for the in vivo diagnosis of a flavivirus infection. The isolated polypeptide can thus be formulated as an immunogenic or vaccine composition.

Thus, according to one particular embodiment, the invention relates to an attenuated flavivirus comprising the said isolated polypeptide. In particular, the recombinant cell may express, intracellularly or extracellularly, or secrete, the said isolated polypeptide. Preferably, the attenuated flavivirus is an attenuated Zika virus comprising the said isolated polypeptide.

According to one embodiment, the invention relates to a nanovesicle or a nanoparticle, in particular an exosome, comprising at least one polypeptide as defined above. In particular, the nanovesicle or nanoparticle may be suitable for use as a medicament. According to one embodiment, the invention relates to a recombinant cell suitable for producing a nanovesicle or a nanoparticle as defined above.

According to an exemplary embodiment, such nanoparticles may be metal nanoparticles (i.e. gold, platinum, silver, titanium, zinc, cerium, iron, and/or thallium nanoparticles), such as gold (Au) nanoparticles; and in particular functionalized gold nanoparticles comprising the said isolated polypeptides.

Such nanoparticles may be covalently linked to a polypeptide according to the protocol set forth in Ma (« Modular assembly of proteins on nanoparticles » ; Nature Communications 2018 ; 9 : 1489).

Such nanovesicles, in particular such exosomes, may be produced as described in W02009115561 or WO2011036416. According to some embodiments, the polypeptide may thus comprise at least one polypeptide sequence selected from : a membrane domain having the ability to anchor in the lipid bilayer of a cell membrane ; and/or a cytoplasmic domain (CD) of a membrane protein, for addressing said chimeric polypeptide to the membrane vesicles, in particular to the vesicles forming exosomes, and / or to the cell compartment (s) ( s) involved in the formation of these membrane vesicles, or a mutated derivative of this CD domain, this mutated derivative being defined by the substitution, the deletion and / or the insertion of one or more residue (s) in the sequence of the reference CD domain and this mutated derivative retaining the aforementioned addressability of the CD domain.

The polypeptides and/or the attenuated flaviviruses and/or the nanovesicle and/or the nanoparticle according to the invention can be formulated in a pharmaceutical composition, optionally in combination with other reagents. According to one embodiment, the invention thus relates to a pharmaceutical composition, comprising a polypeptide and/or an attenuated flavivirus and/or a nanovesicle and/or a nanoparticle as defined above.

A pharmaceutical composition can be generally defined as a composition comprising the polypeptide and/or the attenuated flavivirus according to the invention; and preferably a pharmaceutically acceptable carrier. A vaccine composition according to the invention is aimed at generating antibodies, and/or eliciting an immune response, directed against a polypeptide as defined above in the mammal organism to which the said vaccine composition is administered.

A vaccine composition can thus be generally defined as a pharmaceutical composition comprising the polypeptide and/or the attenuated flavivirus according to the invention; and preferably an immune-adjuvant agent.

Immuno-adjuvant agents encompass, but are not limited to, Stimulon™, QS-21 (Aquila Biopharmaceuticals, Inc., Framingham, Mass.); MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Mont.), 529 (an amino alkyl glucosamine phosphate compound, Corixa, Hamilton, Mont.), IL-12 (Genetics Institute, Cambridge, Mass.); GM-CSF (Immunex Corp., Seattle, Wash.); N-acetyl-muramyl-L-theronyl-D- isoglutamine (thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(r-2'- dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy-ethylamine) (CGP 19835A, referred to as MTP-PE); and cholera toxin. Other immuno-adjuvant agents or compounds which may be used encompass non-toxic derivatives of cholera toxin, including its A subunit, and/or conjugates or genetically engineered fusions of the N. meningitidis polypeptide with cholera toxin or its B subunit ("CTB"), procholeragenoid, fungal polysaccharides, including schizophyllan, muramyl dipeptide, muramyl dipeptide ("MDP") derivatives, phorbol esters, the heat labile toxin of E. coli, block polymers or saponins.

Illustratively, the examples herein illustrate a pharmaceutical composition, in particular a vaccine composition, comprising (i) an isolated polypeptide according to the invention, and preferably (ii) a pharmaceutically acceptable carrier.

A vaccine composition preferably includes such immune-adjuvants, such as Freund’s adjuvant.

The formulation of such immunogenic compositions is well known to persons skilled in the art. Immunogenic compositions of the invention preferably include a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody. The preparation and use of pharmaceutically acceptable carriers is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the immunogenic compositions of the present invention is contemplated.

Such immunogenic compositions can be administered parenterally, e.g., by injection, either subcutaneously or intramuscularly, as well as orally or intranasally. Other modes of administration employ oral formulations, pulmonary formulations, suppositories, and transdermal applications, for example, without limitation. Oral formulations, for example, include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like, without limitation.

The invention further relates to a polynucleotide comprising or consisting of a nucleic acid encoding a polypeptide as defined above.

Hence, the polynucleotide defined herein may comprise regulatory sequences (such as a suitable promoter(s), enhancer(s), terminator(s), etc.) allowing the expression (e.g. transcription and translation) of a polypeptide according to the invention in a host cell. The genetic constructs of the invention may be DNA or RNA, and are preferably double- stranded DNA. The genetic constructs of the invention may also be in a form suitable for transformation of the intended host cell or host organism, in a form suitable for integration into the genomic DNA of the intended host cell or in a form suitable for independent replication, maintenance and/or inheritance in the intended host organism. For instance, the genetic constructs of the invention may be in the form of a vector, such as for example a plasmid, cosmid, YAC, a viral vector or transposon. In particular, the vector may be an expression vector, i.e. a vector that can provide for expression in vitro and/or in vivo (e.g. in a suitable host cell, host organism and/or expression system).

In a preferred but non-limiting aspect, a genetic construct of the invention comprises i) at least one nucleic acid of the invention; operably connected to ii) one or more regulatory elements, such as a promoter and optionally a suitable terminator; and optionally also iii) one or more further elements of genetic constructs known per se; in which the terms "regulatory element", "promoter", "terminator" and "operably connected" have their usual meaning in the art (as further described herein); and in which said "further elements" present in the genetic constructs may for example be 3'- or 5'-UTR sequences, leader sequences, selection markers, expression markers/reporter genes, and/or elements that may facilitate or increase (the efficiency of) transformation or integration. These and other suitable elements for such genetic constructs will be clear to the skilled person, and may for instance depend upon the type of construct used, the intended host cell or host organism; the manner in which the nucleotide sequences of the invention of interest are to be expressed (e.g. via constitutive, transient or inducible expression); and/or the transformation technique to be used. For example, regulatory requences, promoters and terminators known per se for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments) may be used in an essentially analogous manner.

Preferably, in the genetic constructs of the invention, said at least one nucleic acid of the invention and said regulatory elements, and optionally said one or more further elements, are "operably linked" to each other, by which is generally meant that they are in a functional relationship with each other. For instance, a promoter is considered "operably linked" to a coding sequence if said promoter is able to initiate or otherwise control/regulate the transcription and/or the expression of a coding sequence (in which said coding sequence should be understood as being "under the control of" said promotor). Generally, when two nucleotide sequences are operably linked, they will be in the same orientation and usually also in the same reading frame. They will usually also be essentially contiguous, although this may also not be required.

Preferably, the regulatory and further elements of the genetic constructs of the invention are such that they are capable of providing their intended biological function in the intended host cell or host organism.

For instance, a promoter, enhancer or terminator should be "operable" in the intended host cell or host organism, by which is meant that (for example) said promoter should be capable of initiating or otherwise controlling/regulating the transcription and/or the expression of a nucleotide sequence as defined herein, e.g. a coding sequence, to which it is operably linked. Some particularly preferred promoters include, but are not limited to, promoters known per se for the expression in the host cells mentioned herein; and in particular promoters for the expression in the bacterial cells.

A selection marker should be such that it allows, i.e. under appropriate selection conditions, host cells and/or host organisms that have been (successfully) transformed with the nucleotide sequence of the invention to be distinguished from host cells/organisms that have not been (successfully) transformed. Some preferred, but non-limiting examples of such markers are genes that provide resistance against antibiotics (such as kanamycin or ampicillin), genes that provide for temperature resistance, or genes that allow the host cell or host organism to be maintained in the absence of certain factors, compounds and/or (food) components in the medium that are essential for survival of the non-transformed cells or organisms.

A leader sequence should be such that in the intended host cell or host organism- -it allows for the desired post-translational modifications and/or such that it directs the transcribed mRNA to a desired part or organelle of a cell. A leader sequence may also allow for secretion of the expression product from said cell. As such, the leader sequence may be any pro-, pre-, or prepro- sequence operable in the host cell or host organism.

An expression marker or reporter gene should be such that— in the host cell or host organism— it allows for detection of the expression of a gene or nucleotide sequence present on the genetic construct. An expression marker may optionally also allow for the localisation of the expressed product, e.g. in a specific part or organelle of a cell and/or in (a) specific cell(s), tissue(s), organ(s) or part(s) of a multicellular organism. Such reporter genes may also be expressed as a protein fusion with the amino acid sequence of the invention. Some preferred, but non-limiting examples include fluorescent proteins such as GFP.

Some preferred, but non-limiting examples of suitable promoters, terminator and further elements include those that can be used for the expression in the host cells mentioned herein; and in particular those that are suitable for expression in bacterial cells, such as those mentioned herein. For some (further) non-limiting examples of the promoters, selection markers, leader sequences, expression markers and further elements that may be present/used in the genetic constructs of the invention, such as terminators, transcriptional and/or translational enhancers and/or integration factors, reference is made to the general handbooks such as Sambrook et al. Other examples will be clear to the skilled person.

The genetic constructs of the invention may generally be provided by suitably linking the nucleotide sequence(s) of the invention to the one or more further elements described above, for example using the techniques described in the general handbooks such as Sambrook et al.

Often, the genetic constructs of the invention will be obtained by inserting a nucleotide sequence of the invention in a suitable (expression) vector known per se.

The nucleic acids of the invention and/or the genetic constructs of the invention may be used to transform a host cell or host organism, i.e. for expression and/or production of the peptides of the invention.

Hence, the invention further relates to a recombinant cell comprising a polynucleotide as defined above.

According to one further embodiment, the invention relates to an isolated monoclonal antibody directed against a polypeptide as defined above.

Such antibody may be selected from a group comprising or consisting of: non human antibodies, human antibodies, humanized antibodies, synthetic antibodies, and chimeric antibodies.

Methods & uses

The provided polypeptides are endowed with advantageous properties for binding to neutralizing antibodies directed against a pluraliy of flaviviruses, and more particularly against Zika virus. Hence, they can be advantageously used to discriminate neutralizing antibodies from non-neutralizing antibodies.

Alternatively, they can be administered to a host to elicite an immune response, in particular toward the production of antibodies.

According to one embodiment, the invention thus also relates to a method for producing a polypeptide according to the invention, wherein said method comprises the steps of: a) culturing a recombinant cell in conditions allowing the expression of the polypeptide, thereby producing the polypeptide; b) optionally, purifying the polypeptide obtained at step a).

According to one further embodiment, the invention relates to a method for producing a flavivirus according to the invention, in particular an attenuated flavivirus, wherein said method comprises the steps of: a) culturing a recombinant cell in conditions allowing the expression of the flavivirus, thereby producing the flavivirus; b) optionally, purifying the flavivirus obtained at step a).

According to one further embodiment, the invention relates to an in vitro method for recovering an antibody directed against a polypeptide or a flavivirus, wherein said method comprises the steps of: d) providing a sample susceptible to contain the said antibody; e) bringing into contact the sample at step a) with at least one polypeptide or an attenuated flavivirus as defined above; f) recovering the said polypeptide or the said flavivirus, thereby recovering the said antibody.

EXAMPLES

A. MATERIALS & METHODS

A.l. Cells and Reagents

HEK-293 cells (ATCC, CRL-1573) were culture at 37°C under 5%C0 ₂ in MEM medium supplemented with 10% of heat-inactivated fetal bovine serum (FBS, Dutscher, Brumath, France) and antibiotics. Mouse anti-DDDK tag (FLAG epitope) and donkey anti mouse IgG, HRP conjugate, horseradish peroxidase (HRP) were purchased from Abeam (Birmingham, UK). DAPI was purchased from Euromedex (Souffelweyersheim, France).

A.2. Mouse immunization with live ZIKV or KLH-peptide conjugates and serum specimens A total of 29 individualized mouse immune serum specimens was selected for this study (Frumence et al., Vaccines 7,55 and 7,66, 2019) A first group of adult BALB/c mice (n=5) was inoculated with heat-inactivated ZIKBeHMR-2, and a second group (n=14) received two doses of 5 log PFU of ZIKBeHMR-2 with a 6-week interval. All the serum specimens were heat-inactivated at 56°C for 30 min.

Also, a group of adult BALB/c mice were inoculated with 20-30 pg of KLH- peptGL-MR766 conjugates in complete Freund’s adjuvant by intradermal administration. Two weeks after the prime, the immunized mice were twice boosted with the same dose of KLH-peptGL-MR766 conjugates in incomplete Freund’s adjuvant with 2-3 weeks lapsing between immunizations. Mice were bled two weeks after the last immunization.

A.3. Synthetic peptides: free or conjugated to KLH

The synthetic peptides peptGL representing the 20 amino-acid residues of the glycan-loop from EDI of ZIKV strains BeH819015 (Genbank access KU365778) and MR766 (Genbank access LC002520) were chemically synthesized by Genecust (Boynes, France). The two peptides were free or conjugated to the carrier protein keyhole limpet hemocyanin (KLH) by Genecust (Boynes, France). Peptides peptGL mutants and peptide control peptcontrol were also produced by Genecust (Boynes, France).

A.4. Recombinant ZIKV EDI constructs

The mammalian-codon-optimized sequence coding for a signal peptide, followed by the amino-acid residues [1-52], [132-193], and [280-295] which compose the Domain I from the E protein (EDI) of ZIKV strain BeH819015 (Genbank access KU365778) or MR766 (Genbank access LC002520) and ended by FLAG and 6X(His) tags in tandem with glycine- serine spacers were synthetized by Genecust (Boynes, France). The synthetic genes were cloned into Nhe I and Not I restriction site of the pcDNA3.1-Hygro plasmid to generate pcDNA3/ZIKV-rEDI-BeH819015 and pcDNA3/ZIKV-rEDI-MR766. The single amino-acid substitution N154Q was introduced into pcDNA3/ZIKV-rEDI-BeH819015 to generate a plasmid mutant entitled pcDNA3/ZIKV-rEDI(Glnl54). By directed mutagenesis, the amino-acid substitutions I152T, T156I, and H158Y were introduced into pcDNA3/ZIKV-rEDI-BeH819015 to generate a plasmid mutant intitled pcDNA3/ZIKV- rEDI-MR766. All the plasmid sequences were verified by Sanger method (GeneCust, Boynes, France). HEK-293 cells were transient transfected with pcDNA3/ZIKV-rEDI using Lipofectamine 3000 (Thermo Fisher Scientific, Les Ulis, France ).

A.5. Immunoblot assay

Cells were lysed with RIPA lysis buffer (Sigma, Fyon, France) containing protease inhibitors. Equal quantity of proteins was loaded on a NuPAGE Novex 4 to 12% bis-Tris protein gel and transferred to nitrocellulose membrane. After blocking of the membrane with 90% FBS in PBS supplemented with Tween-20, the blot was incubated with appropriate dilution of the primary antibody in the same buffer. Anti-ZIKBeHMR-2 immune sera were used at dilution 1:200. Anti-FFAG mAb was used at dilution 1 :2,000. Anti-mouse IgG HRP-conjugated secondary antibodies were used at dilution 1:2,000. The membranes were developed with Pierce ECF Western blotting substrate (Thermo Fisher Scientific, Fes Ulis, France) and exposed on an Amersham imager 600 (GE Healthcare).

A.6. Peptide-based ELISA

A 96-well plates were coated with 300 ng of peptide diluted in 0.1 ml of PBS at 4°C overnight. Plates were incubated at 37°C with mouse sera diluted in PBS-Tween plus 3% milk. The plates were washed in PBS Tween and then incubated in the presence of HRP- conjugated anti-mouse IgG antibody (ImmunoReagents, Raleigh, USA). After washes in PBS-Tween, plates were incubated with TMB substrate ( Thermo Fisher Scientific, Fes Ulis, France) and absorbance was measured at 450 nm.

A.7. Statistical Analysis

Data obtained with different treatments were compared by one-way or two-way ANOVA tests as appropriate. Values of p< 0.05 were considered statistically significant for a post-hoc Tukey's test. All statistical tests were done using the software Graph-Pad Prism version 9.0.

B. RESULTS

B.l. Antigenic reactivity of ZIKV rEDI

In order to evaluate the ability of anti-ZIKBeHMR-2 immune serum to recognize EDI-GL, a recombinant rEDI protein (132 amino-acid residues) was generated by joining the three ZIKV E segments [1-52], [132-193], and [280-295] which compose the EDI of epidemic Brazilian ZIKV strain BeH819015 (Fig. 1A). The middle EDI segment of rEDI- BeH819015 contains the 20 residues E-145 to E-163 which comprise the EDI-GL sequence (Fig. 1A). The mammalian codon-optimized rEDI sequence was preceded by a mammalian signal peptide for targeting the protein into the secretory pathway and ended with the C- terminal FLAG and 6x(His) antigen sequences which are separated by two short Gly-Ser spacers (Fig. IB). By directed mutagenesis, the three substitutions I152T, T156I, and H158Y were introduced into rEDI-BeH819015 to generate a rEDI mutant intitled rEDI-MR766 (Fig. 1A). The N154Q mutation was introduced into rEDI-BeH819015 to generate the rEDI- (Q154) mutant (mutant plasmid pcDNA3/rEDI-(Glnl54)) (Fig. IB). The substitution from Asn to Gin (N154Q) is expected to cause the loss of the N-glycosylation site of ZIKV E protein from BeH819015, and thus a lack of glycan.

We analyzed the expression of rEDI by indirect immunofluorescence (IF) assay. Human epithelial HEK-293T cells were transfected 24 hours with pcDNA3/rEDI- BeH819015, pcDNA3/rEDI-MR766 or pcDNA3rEDI-(Glnl54). Immunofluorescence (IF) assay with anti-tag antibody readily detected intracellular expression of rEDI-BeH819015, rEDI-MR766 and mutant rEDI-(Glnl54) in transfected HEK-293 cells. Thus, we validate that the plasmids expressing different ZIKV rEDI are suitable for further experiments.

Immunoblot assay on RIPA cell lysates was performed for assessing rEDI protein expression in transfected HEK-293T cells (Fig. 2). Anti-FLAG antibody detected the expression of rEDI-BeH819015 and its rEDI-(Q154) mutant as well as rEDI-MR766 in HEK-293T cells. We observed that both rEDI-(Q154) mutant and rEDI-MR766 migrated faster than rEDI-BeH819015 (Fig. 2). The fact that mutants rEDI-(Q154) and rEDI-MR766 have comparable migration profile is consistent with a lack of glycan linked to rEDI-MR766. Thus, the slower migration of rEDI-BeH819015 is presumably due to a glycan linked to N154.

We assessed the reactivity of rEDI-MR766, rEDI-BeH819015, and its mutant rEDI-(Q154) with a pooled serum from B ALB/c mice that received two high doses of live ZIKBeHMR-2 virus with an interval of one month (Fig. 2). Pooled sera from mice inoculated with heat- inactivated ZIKBeHMR-2 virus served as negative control (Ref). As a control, no reactivity was observed with anti-inactivated ZIKBeHMR-2 immune serum regardless the rEDI constructs tested. There was no reactivity of anti-ZIKBeHMR-2 immune serum with rEDI-BeH819015. The very weak recognition of mutant rEDI-(Q154) suggests that the removal of a glycan linked to rEDI-BeH819015 has a minor influence on its antigenic reactivity in relation with anti-ZIKBeHMR-2 immune serum (Fig. 2).

Taken together, these results show that the amino-acid substitutions I152T, T156I, and H158T which differentiate rEDI-MR766 from rEDI-BeH819015 yield a stronger recognition of rEDI by anti-ZIKBeHMR-2 immune serum.

We conclude that the residues E-152, E-156, and E-158 play a key role in the antigenic reactivity of ZIKV EDI in relation with anti-ZIKBeHMR-2 immune serum regardless of the presence or not of a glycan linked to glycan loop region

B.2. Antigenic reactivity of peptides representing E-GL from ZIKV

Our results suggest a pivotal role for the three residues at positions E-152, E- 156, and E-158 in the reactivity of anti-ZIKBeHMR-2 immune serum to EDI. To better understand the role of GL in the antigenic reactivity of EDI, we decided to generate two chemically synthetized peptides peptGL-BeH819015 and peptGLMR766 representing the residues E-145 to E-164 of ZIKV strains BeH819015 and MR766, respectively (Fig. 3). An Ala-rich peptide of 16 amino-acid residues (hereafter intitled peptcontrol) served as a negative control.

In order to evaluate the sensitivity of an ELISA based on E-GL peptides, a dose- response curve was performed using peptGL-MR766 and ZIKBeHMR-2 immune serum at dilution of 1:50 (Fig. 4). Peptide-based ELISA showed that ZIKBeHMR-2 immune serum strongly reacted with peptGLMR766. In contrast, heat-inactivated ZIKBeHMR-2 immune serum showed no reactivity with the peptGL-MR766 peptide regardless the peptide concentration. Thus, a peptide-based ELISA using peptGL-MR766 is valid for the detection of anti-GL antibodies in mice inoculated with live ZIKBeHMR-2 virus. The minimal detectable concentration of peptGL-MR766 was approximately 0.1 pg.mL-l.

A peptide-based ELISA was then performed to determine whether anti- ZIKBeHMR-2 immune serum was also reactive with peptGL-BeH819015 (Fig. 5). Heat- inactivated ZIKBeHMR-2 immune serum was used as a negative serum control. The peptides peptGL-MR766 and peptcontrol served as positive or negative peptide control, respectively. A dose-response curve showed that anti-ZIKBeHMR-2 immune serum has no reactivity with peptGL-BeH819015 regardless the peptide concentration tested.

Thus, anti-ZIKBeHMR-2 immune serum has high level of reactivity with peptGL-MR766 but not peptGL-BeH819015 by peptide-based ELISA.

We can conclude that immunization of with live ZIKBeHMR-2 serum induces the production of GL-reactive antibodies able to recognize a peptide representing the GL region of ZIKV strain MR766 but not BeH819015.

We then examined whether BALB/c mice twice inoculated with ZIKBeHMR-2 virus elicited different antibody levels against peptGL-MR766. Individual anti-ZIKBeHMR- 2 immune sera served as negative serum control. Both peptGL-BeH819015 and peptcontrol were used as negative controls. A group of fifteen individual anti-ZIKBeHMR-2 immune sera at dilution 1:200 was tested for their reactivity to peptGL-MR766 by peptide-based ELISA (Fig. 6). Among them, four anti-ZIKBeHMR-2 immune serum samples showed a strong reactivity with peptGL-MR766. A weaker reactivity was also observed with three other serum samples. Thus, individual variation in GL-related antibody response can take place in mice inoculated with ZIKBeHMR-2 virus.

Comparative sequence analysis of peptGL-MR766 and peptGL-BeH819015 identified a permutation of the two amino-acid residues lie and Thr occurring at positions 8 and 12 of the peptide (Fig. 3). Such interchange could contribute to the lack of antigenic reactivity of peptGL-BeH819015 in relation with anti-ZIKBeHMR2 immune serum. Consequently, peptGL-BeH819015 was modified at positions 8 and 12 to generate a mutant peptGL-(T8, 112) (Fig. 3). Peptide-based ELISA was performed with individual anti- ZIKBeHMR-2 immune sera showing a strong positive reactivity to peptGL-MR766 at serum dilution 1:200 (Fig. 7). Anti-inactivated ZIKBeHMR-2 immune sera were used as negative controls. Anti-ZIKBeHMR-2 immune sera showed no reactivity with mutant peptGL-(T8, 112) (Fig. 7). Thus, the two amino-acid substitutions I8T and T12I resulted in no change in the antigenic reactivity of peptGL-BeH819015 in relation with anti-ZIKBeHMR-2 immune serum. We next investigated whether the polar amino-acid residue at position 14 (H14) might play a role in the weak reactivity of peptGL-BeH819015. Consequently, the mutation H14Y was introduced into peptGL-BeH819015 generating peptGL-(Y14) mutant (Fig. 3). A similar antigenic reactivity was observed between peptGL-(Y 14) and peptGL-MR766 in relation with anti-ZIKBeHMR-2 immune serum (Fig. 8).

This suggests that residue at position 14 influences antigenic reactivity of GL region in relation with anti-ZIKBeHMR-2 immune serum.

Thus, we identify the polar residue at position E-158, in particular Y158, as key antigenic determinant in the reactivity of GL region of ZIKV in relation with anti- ZIKBeHMR-2 immune serum

B.3. A ZIKV GL-derived peptide (SEQ ID N°4) coupled to KLH is immunogenic in mice and the obtained serum reacts with a recombinant antigenic domain I (rEDI) of the ZIKV E protein.

A group of adult BALB/c mice were twice inoculated with KLH-peptGL- MR766 conjugates (which correspond to the peptide of SEQ ID N°4 coupled to an immunogenic compound) in presence of adjuvant. The mouse immune sera were tested individually using peptGL-MR766 as coating antigen through peptide-based ELISA. The KLH-peptGL-MR766 immune serum SI -RE has been identified as a positive serum for GL peptide-reactive antibodies at dilution 1:200 (Figure 9). As a control, a such immune serum showed no reactivity with peptcontrol peptide. Also, the pre-immune serum SI -RE has no reactivity with peptGL peptide. The peptGL-BeH819015 peptide corresponding to SEQ ID N°5 was assayed with KLH-peptGL-MR766 immune serum SI-RE (Figure 9). We found that the positive mouse serum for GL-MR766 peptide-reactive antibodies has ability to recognize pepGL-BeH819015 peptide but at a lower extent as compared to peptGL-MR766.

We can conclude that immunization with KLH-peptGL-MR766 conjugates induces production of GL region-reactive antibodies able to recognize peptides representing the GL regions of ZIKV of Africa and Asia genotypes including contemporary epidemic strains. The reactivity of KLH-peptGL-MR766 immune serum SI -RE was assayed on HEK-293 cells expressing ZIKV rEDI by flow-cytometry analysis (Fig. 10). We found that peptGL-MR766-reactive antibodies can recognize rEDI-MR766 but also rEDI-BeH819015 although a much lower extent.

We can conclude that immunization with KLH-peptGL-MR766 conjugates induces production of GL region-reactive antibodies able to recognize EDI domain of ZIKV of Africa and Asia genotypes including contemporary epidemic strains. The weaker antigenic reactivity of rEDI-BeH819015 compared to rEDI-MR766 highlights a critical role for residues E-152, E-156, and E-158 in the ability of ZIKV EDI to be recognized by GL-reactive antibodies.

Taken together, these results show that the KLH-peptGL ^MR766 immune serum has ability to recognize the GL region.

FACS analysis was then performed to evaluate the reactivity of KLH- peptGL ^MR766 immune serum with rEDI ^MR766 and its mutants expressed in HEK-293T cells. Anti-FLAG antibody showed that their expression levels were similar at 24 h post transfection. As shown in Figure 10, KLH-peptGL ^MR766 immune serum has ability to react with rEDI ^MR766 expressed in HEK-293T cells. The mean fluorescence intensity (MFI) values of GL peptide-reactive antibody and anti-FLAG antibody were similar, indicating a high level of rEDI ^MR766 antigenic reactivity in relation with GL peptide-reactive antibodies. Introduction of amino-acid substitutions E-T152I, E-I156T, and E-Y158H reduced by at least 70% the antigenic reactivity of rEDI ^MR766 (Figure 10). Analysis of mutant rEDI ^MR766 (I152, T156) revealed that the two amino-acid substitutions E-T152I and E-I156T decreased recognition of rEDI ^MR766 . An amino-acid substitution N154Q was also introduced in glycosylated mutant rEDI ^MR766 (1152, T156, H158). The loss of the N-glycosylation site showed no effect on immunoreactivity of mutant rEDI ^MR766 . Thus, it seems unlikely that the glycan linked to N154 may play a role in the weaker reactivity of the GL peptide-reactive antibody in relation with rEDI ^MR766 mutants bearing the mutations E-T152I and E-I156T.

Taken together, these results show that the GL peptide-reactive antibody has ability to recognize the GL region into a reconstituted EDI regardless of the 5 presence of a glycan linked to N154. Those results suggest that the two residues E-152 and E-156 have a key role in the antigenic reactivity of GL region in relation with a change in GL structural conformation.

SEQUENCE LISTING