WO2020208434A1 - Vaccin sous-unitaire contre le virus zika - Google Patents

Vaccin sous-unitaire contre le virus zika Download PDF

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WO2020208434A1
WO2020208434A1 PCT/IB2020/051462 IB2020051462W WO2020208434A1 WO 2020208434 A1 WO2020208434 A1 WO 2020208434A1 IB 2020051462 W IB2020051462 W IB 2020051462W WO 2020208434 A1 WO2020208434 A1 WO 2020208434A1
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zikv
vaccine
protein
rec
subunit
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PCT/IB2020/051462
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Rajgokul K. SHANMUGAM
Viswanathan RAMASAMY
Rahul Shukla
Upasana Arora
Sathyamangalam Swaminathan
Navin Khanna
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International Centre For Genetic Engineering And Biotechnology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to vaccine production. More specifically the present invention relates to the production of genetically engineered membrane-associated particulate subunit vaccine for Zika virus, a mosquito-borne flavivirus.
  • Zika virus Zika virus (ZIKV) is a flavivirus, phylogenetically related to West Nile virus (WNV), Yellow fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV) and the four antigenically distinct serotypes of dengue viruses (DENV-1, DENV-2, DENV-3 and DENV-4) (Pierson TC, Diamond MS. Flaviviruses. In: Knipe DM, Howley PM, editors-in-chief. Fields Virology, 6e. Philadelphia: Wolters Kluwer and Lippincott Williams & Wilkins; 2013. p. 747-794).
  • Zika virus a previously slow pandemic spreads rapidly through the Americas. J Gen Virol. 2016; 97: 269-273; Lazear HM, Diamond MS. Zika virus: New clinical syndromes and its emergence in the Western hemisphere. J Virol. 2016; 90: 4864-4875).
  • ZIKV may also be transmitted between humans sexually (Musso D, et. al. Potential sexual transmission of Zika virus. Emerg Infect Dis. 2015; 21: 359-361 ) or vertically (Besnard M, et al, Evidence of perinatal transmission of Zika virus, French Polynesia, December 2013 and February 2014. Euro Surveill.
  • ZIKV infections are beginning to be reported in India. It is currently estimated that more than 2 billion people live in areas considered suitable for ZIKV transmission (Messina E et al, Mapping global environmental suitability for Zika virus. eLife. 2016; 5: el 5272).
  • ZIKV vaccine development is complicated by the existence of antibody-dependent enhancement (ADE) phenomenon, stemming from the interaction between ZIKV on the one hand and DENVs on the other. Not only do these viruses share the same mosquito vector (WeavenSV et al, Zika virus: History, emergence, biology, and prospects for control. Antiviral Res. 2016; 130: 69-80), but also are genetically and antigenically similar.
  • ADE antibody-dependent enhancement
  • the ZIKV E protein is the major component involved in receptor binding, membrane fusion and in recognition by the host immune system and contains epitopes recognized by potent murine (Zhao H et al, Structural basis of Zika virus-specific antibody protection. Cell. 2016; 166: 1016-1027) and human (Stettler K et al, Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection. Science. 2016; 353: 823-826; Wang Q et al, Molecular determinants of human neutralizing antibodies isolated from a patient infected with Zika virus. SciTransl Med. 2016; 8: 369ral79) neutralizing antibodies (nAbs).
  • the ZIKV E protein is also organized into three distinct domains, referred to as envelope domain I (EDI), EDII and EDIII ⁇ Dai L et al, Structures of the Zika virus envelope protein and its complex with a flavivirus broadly protective antibody. Cell Host Microb. 2016; 19: 696-704).
  • the flaviviralprM protein appears to function as a chaperone of the E protein, preventing premature fusion of the immature virion as it transits the trans-Golgi network within the infected cell.
  • nucleic acid-based and most protein-based vaccine candidates are designed to encode the two ZIKV structural proteins prM and E (Barrett, Current status of Zika vaccine development: Zika vaccines advance into clinical evaluation npj Vaccines. 2018; 3: 24; Richner& Diamond, Zika virus vaccines: immune response, current status, and future challenges. Current Opinlmmunol. 2018; 53: 130-136; WHO Vaccine Pipeline Tracker)
  • compositions including a virus-like particle (VLP)-based vaccine displaying a portion of ZIKV envelope protein (E) domain III (Dill) and a portion of ZIKV envelope protein (E) and related methods are disclosed herein. Further, compositions including vaccines comprising a portion of ZIKA virus E protein, wherein the portion of ZIKA virus E protein is either a full-length version of ZIKA virus E protein or a functionally equivalent version of the full-length ZIKA virus E protein, are disclosed.
  • US’ 181 further mentions“Since ZIKV and DENV are closely related and co-circulate geographically, ZIKV vaccines that are based on common epitopes of the two viruses may elicit cross-reactive antibodies that augment infection of DENV in vaccinated subjects when they are exposed to DENV secondarily.
  • This hypothesis is supported by the finding that cross-reactive antibodies targeting the highly conserved fusion loop in EDII (EDII-FL) of zE generated during natural ZIKV infection can enhanced DENV infection both in cell culture and in mice. Therefore, vaccine strategies based on antigens that can avoid induction of cross-reactive antibodies should minimize the risk of ADE of DENV infections.”
  • US2018340181 describes the use of the plant expression system (N.
  • CN108503696 teaches a subunit Zika virus vaccine expressed by yeast cells.
  • the subunit Zika virus vaccine developed by using yeast cells in the invention has the advantages of high yield, high purity, good stability and easy purification; meanwhile, because the subunit Zika virus vaccine contains no viral nucleic acid component, the subunit Zika virus vaccine is free of the possibility of mutation restoration and has high safety.
  • CN108503697 discloses similar invention where the expression host is Drosophila. These two inventions concern the production of ZIKV EDIII and ZIKV E80 proteins as vaccine antigens using the yeast expression system (CN ‘696) or the Drosophila insect cell expression system (CN‘697).
  • the two vaccine antigens are designed to be secreted into the culture supernatant, using either the a-factor secretion signal (CN‘696) or the Drosophila BiP secretion signal (CN’697).
  • the CN’696 vaccine is a monomer and the CN’697 vaccine is a dimer and these vaccines do not form higher ordered structures.
  • neutralizing antibody titers elicited the yeast- expressed ZIKV EDIII and insect cell-expressed ZIKV E80 appear to be the preferred vaccine candidates, however the two vaccines have not been tested for ADE.
  • MAP membrane-associated particle
  • a subunit Zika virus (ZIKV) vaccine comprising: a membrane-associated particulate form of immunogen rec ZIKV envelope (E) protein, wherein said rec ZIKV E protein comprises of domains I, II and III and said domain III is placed on the membrane surface and is freely accessible to ZIKV EDIII-specific antibodies.
  • ZIKV Zika virus
  • kits comprising a membrane-associated particulate form of immunogen rec ZIKV envelope (E) protein, said rec ZIKV E protein comprises of domains I, II and III and said domain III is freely accessible to ZIKV EDIII-specific antibodies, wherein said kit is capable of detecting ZIKV-specific antibodies in biofluids such as blood, plasma, serum, urine and saliva.
  • E immunogen rec ZIKV envelope
  • Figure 1 illustrates the recombinantZIKV E antigen in accordance with the present invention.
  • Figure 2 illustrates the comparison of ZIKV and DENV E proteins.
  • FIG. 3 illustrates the expression screening of transformants in accordance with the present invention.
  • FIG. 4 illustrates the localization and purification of rec ZIKV E protein.
  • Figure 5 illustrates thephysical characterization of purified rec ZIKV E protein.
  • Figure 6 illustrates the alum formulation of recombinant ZIKV E MAPs and the immunization schedules.
  • Figure 7 illustrates the immunological evaluation of rec ZIKV E MAP -induced antibodies.
  • Figure 8 illustrates the evaluation of DENV enhancement by BALB/c anti-rec ZIKV E MAP antiserum/// vitro.
  • Figure 9 illustrates the evaluation of the enhancement potential of anti-rec ZIKV E MAP antibodies in vivo.
  • Figure 10 illustrates the in vivo ADE model utilized to assess the ZIKV-enhancement potential of anti-rec ZIKV E MAP antibodies.
  • the present invention provides a highly immunogenic ZIKV prM-lacking recombinant membrane-associated particulate subunit vaccine comprising the recombinant ZIKV E protein, comprising envelope domains I, II and III, and surface-displaying ZIKV EDIIF
  • This vaccine by virtue of displaying ZIKV EDIII is endowed with the capacity to elicit an immune response in mammals, which is capable of specifically neutralizing ZIKV infection of susceptible mammalian cells.
  • this membrane-associated particulate vaccine by virtue of not containing prM and not exposing cross-reactive epitopes, does not enhance infection of susceptible cells byflaviviruses, including but not limited to, ZIKV, DENV-1, DENV-2, DENV-3 and DENV-4.
  • the present invention discloses a method to produce a membrane-associated Zika virus subunit vaccine using a suitable host expression system, such as the yeast Pichia pastoris.
  • the subunit vaccine is in particulate form displaying the Zika virus envelope domain III so that it may elicit Zika virus-specific neutralizing antibodies without enhancement potential against the closely related dengue viruses.
  • a nucleic acid molecule comprising a nucleotide sequence encoding a signal peptide, ZIKV E protein ectodomainand an affinity tag for easy purification.
  • This nucleic acid molecule is hereinafter referred to as ZIKV E gene (SEQ ID 1).
  • SEQ ID 1 nucleotide 1-6, containing the initiator codon (ATG), were introduced to facilitate cloning and expression.
  • Nucleotides 7-108 encode the C-terminal 34 aa residues of ZIKV prM (of ZikaSPH2015).
  • Nucleotides 109-1317 encode the first 403 aa residues of ZIKV E protein, comprising domains I, II and III (of ZikaSPH2015).
  • Nucleotides 1318-1332 encode a pentaglycl peptide linker.
  • Nucleotides 1333-1350 encode the 6x His tag.
  • Nucleotides 1351-1353 (TAG) denote the stop codon.
  • the corresponding protein encoded is referred to hereinafter as recombinant (rec) ZIKV E protein.
  • the ectodomain of the ZIKV E protein representing the N-terminal 80% of the full-length ZIKV E molecule, is comprised of three domains, I, II and IP (EDI, EDII and EDIII, respectively).
  • the rec ZIKV E protein may comprise the C- terminal 34 amino acid residues of ZIKV prM( signal peptide), the N-terminal 403 amino acid residues of the full-length ZIKV Eprotein (ZIKV E ectodomain) and a run of 6 histidine residues (6x His tag).
  • the ZIKV E ectodomain may be derived from the ZIKV strain, ZikaSPH2015.
  • the ZIKV E gene sequence may be varied to match the codon bias of the expression host.
  • the ZIKV E ectodomain-encoding polynucleotide sequences of ZIKV E gene may be replaced with corresponding ectodomain-encoding polynucleotide sequences derived from any one of prevalent/available and known ZIKV strains or isolates.
  • ZIKV strains/isolates include, but are not limited to, ZIKA YAP 2007, H/PF/2013, BeH818995, PRVABC59, ZKV-16-097, MR766, ARB7701, ArD 128000 and IbH30656.
  • the prM signal encoding sequences of SEQ ID 1 may be replaced by equivalent sequences from any of the other flaviviruses or by any other signal peptide known to be functional in the chosen host expression system.
  • flaviviruses from which the signal peptide may be derived include, but are not limited to,WNV, YFV, JEV, TBEV, DENV-1, DENV-2, DENV-3 and DENV-4.
  • other signal peptides include, but not limited to, a mating factor signal peptide of Saccharomyces cerevisiase and IgG leader peptide.
  • the 6x His tag affinity may be replaced by any other affinity tag for ease of purification of the rec ZIKV E protein.
  • affinity purification tags include, but are not limited to, glutathione S transferase, maltose-binding protein and intein.
  • the rec ZIKV E protein may be expressed without any additional affinity purification tag.
  • the host in which the rec ZIKV E protein-encoding nucleic acid molecule is introduced may be any suitable prokaryotic or eukaryotic host.
  • the ZIKV E gene may exist as an independent plasmid-borne entity in the host. In another embodiment, one or more copies of the ZIKV E gene may be integrated into the host DNA.
  • the host used may be either prokaryotic or eukaryotic, compatible with the expression of rec ZIKV E protein in a form conducive to its self- assembly into 30-50 nm particulate structures displaying ZIKV EDIII on the surface.
  • the host for rec ZIKV E expression may be a yeast host.
  • yeast hosts include, but are not limited to, Pichia pastoris and Hansenulapolymorpha .
  • rec ZIKV E protein expression may be achieved under the control of any suitablesynthetic or natural, constitutive promoter.
  • rec ZIKV E protein expression may be achieved under the control of a synthetic or natural, regulated promoter.
  • the expressed rec ZIKV E protein may be localized to the soluble fraction of host cell lysate.
  • the expressed rec ZIKV E protein may be localized to the insoluble (membrane-associated) fraction of the host cell lysate.
  • the expressed ZIKV E protein may be purified by conventional or affinity chromatographic methods under native conditions provided the purified protein can self-assemble into ZIKV EDIII-displaying particulate form of 30-50 nm size.
  • conventional or affinity chromatographic purification may be carried out under denaturing conditions.
  • the purification may exploit any inherent affinity binding of the rec ZIKV E protein to a specific liganded matrix.
  • the rec ZIKV E protein purified under denaturing conditions may be refolded in such a way as to promote self-assembly into highly immunogenic ZIKV ED Ill-displaying particulate form of 30-50 nm size.
  • a ZIKV vaccine formulation comprising the 30-50 nm particulate rec ZIKV E protein preparation, a human use-compatible adjuvant, such as alum, and any pharmacologically acceptable ingredient may be inoculated into a mammal.
  • the inoculation may be performed in one of multiple accepted ways, such as intradermal, intramuscular, sub-cutaneous etc.
  • the inoculation may be performed using a range of vaccine dosages.
  • the inoculation may be administered more than once and at varied intervals.
  • the inoculation of the ZIKV vaccine formulation may be shown to elicit an appropriate ZIKV-specific immune response.
  • the antibodies elicited by inoculation into the mammal may be shown to be specific for unique ZIKV conformational or quaternary epitopes.
  • these antibodies may be shown to potently neutralize ZIKV infection of susceptible mammalian cells.
  • these antibodies may be shown to lack any ADE potential towards various ZIKV isolates as well as other flaviviruses, including but not restricted to, DENV-1, DENV-2, DENV-3 and DENV-4 in a mammal.
  • the rec ZIKV E protein may be incorporated in a diagnostic test of kit to detect the presence of anti -ZIKV antibodies in patient samples as a means of identifying ZIKV infection cases.
  • the present inventors have surprisingly found that the immunogenic ZIKV prM-lacking recombinant membrane-associated particulate subunit has higher order structuresas evident in Fig 5, panels D and E, even in the absence of prM. To achieve this, the present inventors have added the C-terminal 34 amino acid residues of prM as a signal peptide to the N-terminus of the ZIKV E ectodomain. During expression in P.
  • this prMsignal (which helps the recombinant protein traverse the endoplasmic reticulum and in doing so causes membrane association) is cleaved off and the resulting E ectodomain self-assembles into MAPs.
  • VLP virus-like particle
  • prM and E Normally ZIKV virus-like particle
  • the present invention shows that higher order structures similar to VLP can be formed in the absence of prM. These have been named as‘membrane-associated particles (MAPs)’ as they have been purified from the membrane-fraction (of induced P. pastoris cells) and this also helps to distinguish them from classic VLPs, which also by definition contain prM and E.
  • the MAPs contain only one protein.
  • this ZIKV- specificity is a result of the uniqueness of the rec ZIKV E MAPs which, by display of ZIKV EDIII, elicit a ZIKV ED Ill-directed Ab response.
  • Figure 1 illustrates the recombinant ZIKV E antigen in accordance with the present invention.
  • A Schematic representation of the ZIKV polyprotein. ⁇ 2 N’ and‘COOFF indicate the amino terminus and carboxy-terminus, respectively of the polyprotein. Proteins prM and E are indicated by black and purple boxes, respectively. The region of the polyprotein included in the antigen is bounded by the two white lines in the C- terminal regions of prM and E.
  • B Schematic representation of the design of the rec ZIKV E antigen consisting of the last 34 amino acid residues of prM and the first 403 amino acid residues of E.
  • the C-terminally located grey and red boxes denote the pentaglycyl peptide linker and the hexa-histidine (H6) tag, respectively.
  • C Predicted amino acid sequence of the recombinant polypeptide is shown in‘B’. The color scheme corresponds to that shown in‘B’. PrMamino acid residues are underlined. The N-terminal dipeptide‘MS’ was introduced during cloning. The downward arrow in‘B’ and‘C’ denotes the signal peptide cleavage site.
  • D Map of the rec ZIKV E expression plasmid, pPIC-ZIKV E.
  • the synthetic ZIKV E gene (ZIKV-Envelope) is inserted between the AOX1 promoter (5’ AOX1) on the 5’ side and the transcriptional terminator (TT) on the 3’ side. It carries the selection marker (Zeo R ), which is functional in both E. coli and P. pastoris , and the E. coli origin of replication (Ori), for bacterial propagation.
  • Figure 2 illustrates the comparison of ZIKV and DENV E proteins. Shown is the amino acididentity between the E protein of ZikaSPH2015 (GenBank ID: ALU33341.1) strain, on which the recombinant ZIKV E protein is based, on the one hand, and the E proteins encoded by typical Asian and African ZIKV strains/isolates as well as each typical DENV serotype, on the other. Genbank accession numbers of the polyproteins of each virus is shown in parenthesis below the isolate/strain name. The values in the bottom row represent percent amino acididentity between ZikaSPH2015 E protein and the E proteins of the virus indicated in the cell above. Identity was determined by comparing the amino acid sequences between pairs using NCBI’s protein-protein blastp algorithm.
  • Figure 3 illustrates the expression screening of transformants.
  • Test-tube cultures of zeocin-positive/f /3 ⁇ 4/.s/ /7.stran sfor ants (1-11) were induced with methanol (1.5%) for 3 days and the urea-solubilized M fractions of the total lysates were separated by SDS- PAGE, transferred onto nitrocellulose membrane and probed with mAb 24A12.
  • Aliquots of un-induced P. pastoris cultures were analyzed in lanes‘IT as negative controls.
  • two positive controls were also analyzed.
  • Figure 4 illustrates the localization and purification of rec ZIKV E protein.
  • A Analysis of localization of rec ZIKV E protein in P. pastoris. An aliquot of methanol-induced (Ind) culture of P. pastoris was lysed with glass beads and separated into supernatant ( S) and membrane-enriched pellet ( M) fractions. Total (7) extract prepared from an equivalent aliquot of the induced culture, the ⁇ fraction, and the urea-solubilized M fraction were run on SDS-polyacrylamide gel and subjected to Western blot analysis using mAb 24A12. Total (7) extract prepared from an equivalent aliquot of the un-induced (U) culture was analyzed in parallel. Pre-stained protein ladder was analyzed in lane‘L’.
  • Figure 5 illustrates thephysical characterization of purified rec ZIKV E protein.
  • A Coomassie-stained SDS-polyacrylamide gel analysis of pooled peaks 1 and 2 (zE) after dialysis.
  • B Western blot analysis of the pooled peaks (zE) using mAb 24A12.
  • protein ladder was run in lanes‘L’. The sizes (in kDa) of the markers in the protein ladder are shown to the left of each panel. The arrow on the right, of both these panels, indicates the position of the rec ZIKV E protein.
  • Figure 6 illustrates thealum formulation of recombinant ZIKV E MAPs and the immunization schedules.
  • a suspension of recombinant ZIKV E MAPs coated onto alum (20 pg in 100 m ⁇ ) was spun down and 10 m ⁇ of the resultant supernatant (lane 3) was analyzed by SDS-PAGE.
  • aliquots of purified rec ZIKV E MAPs (un-adsorbed on alum) adjusted to 20 pg/100 pi, equivalent to 1 pg (5 pi, lane 2) and 2 pg (10 pi, lane 1) protein, were analyzed for comparison.
  • Figure 7 illustrates theimmunological evaluation of rec ZIKV E MAP-induced antibodies.
  • Figure 8 illustrates theevaluation of DENV enhancement by BALB/c anti-rec ZIKV E MAP antiserum in vitro.
  • DENV-1 magenta
  • DENV-2 green
  • DENV-3 blue
  • DENV-4 black
  • PBS+alum mock-immunized mice
  • K562 alum-formulated rec ZIKV E MAPs
  • Percent DENV-infected cells were determined by flow cytometry.
  • C Experiment similar to those described in panels‘A’ and‘B’, except that DENVs were pre-incubated with serial dilutions of mAb 4G2 instead of murine immune serum before K562 infection.
  • Figure 9 illustrates theevaluation of the enhancement potential of anti-rec ZIKV E MAP antibodies in vivo.
  • (B) Kaplan-Meier survival curves for groups (z/ 6) of AG129 mice which were intravenously administrated with a sub-lethal dose of DENV-2 S221 (pre-incubated with NMS, green curve) or with ICs generated in vitro by mixing the sub-lethal dose of DENV-2 S221 either with mAb 4G2 (grey curve), anti -DENV-2 antiserum (black, dashed curve), or serum from BALB/c immunized (on days 0, 14, 28) with rec ZIKV E MAPs (purple curve). Survival was observed for 12 days post-challenge.
  • Figure 10 illustrates them vivo ADE model utilized to assess the ZIKV-enhancement potential of anti-rec ZIKV E MAP antibodies.
  • A Immune sera were injected into riVa/ mice (derived from C57BL/6 strain) two hours before ZIKV challenge. Mice were monitored over 12 days post sub-lethal ZIKV infection for survival, clinical symptoms and weigh loss.
  • B-D Stall 1 mice were inoculated IP with NMS (dotted curve), anti- DENV-3 immune serum (dashed curve, black) or anti-rec ZIKV E MAP antiserum (solid curve) and then challenged with ZIKV (PRVABC59) two hours later.
  • the synthetic ZIKV E gene which was codon-optimized for expression in P. pastoris , encoded the last 34 amino acid ( aa ) residues of the ZIKV prM protein, to serve as the signal peptide, followed by the first 403 aa residues of the full-length ZIKV E protein, the vaccine antigen.
  • Nucleotide sequences encoding a penta-glycine peptide linker followed by a hexa-histidine tag were appended at the 3’ end of the ZIKV E gene. This synthetic gene which was placed under the P.
  • the ZIKV E ectodomain-encoding sequences of the synthetic ZIKV E gene were derived from the ZikaSPH2015 Brazilian strain.
  • ZikaSPH2015 E protein displays at least 99% sequence identity with E proteins of Asian ZIKV strains, at least 96% sequence identity with E proteins of African ZIKV strains and 55-57% sequence identity with respect to E proteins of the four DENVs ( Figure 2).
  • Plasmid pPIC-ZIKV E (pPICZ A vector into which the synthetic ZIKV E gene was inserted in the polylinker) was integrated into the genome of P. pastoris strain KM71H by electroporation according to the vendor’s manual (Invitrogen Life Technologies, Thermo Fisher Scientific). The resultant transformants, obtained through zeocin selection, were subjected to an expression screening step to identify clones capable of expressing the rec ZIKV E protein. Rec ZIKV E protein was identified with an‘ in-house’flavivirus E- specific mAb 24A12 in Western blots ( Figure 3).
  • This Western blot may also be probed with the flavivirus-specificmAb 4G2 (Henchal EA et al, Dengue virus-specific and flavivirus group determinants identified with monoclonal antibodies by indirect immunofluorescence. Am J Trop Med Hyg. 1982; 3: 830-836) or any of the ZIKV E- specific mAbs described in the literature (Zhao H et al, Structural basis of Zika virus- specific antibody protection. Cell. 2016; 166: 1016-1027;Stett ⁇ er et al, Specificity, cross reactivity, and function of antibodies elicited by Zika virus infection. Science.
  • Rec ZIKV E protein was purified using Ni 2+ -NTA affinity chromatography, taking advantage of the C-terminally engineered 6x-His tag. As it was associated with the insoluble M fraction of induced lysates, purification was performed under denaturing conditions. 6M guanidine hydrochloride (Gu-HCl) was preferred for extraction compared to 8M urea as the former was twice as efficient as the latter. However, as Gu-HCl- containing column fractions are not compatible with SDS-PAGE analysis, the denaturant was switched from Gu-HCl to urea after column binding as described below.
  • Gu-HCl 6M guanidine hydrochloride
  • a 2 L culture of P. pastoris harboring the ZIKV E gene grown to logarithmic phase was induced with 1.5 % methanol for 3 days at 30°C.
  • This culture was centrifuged and the resultant induced cell pellet ( ⁇ 50 g wet weight) was washed in sterile l x phosphate buffered saline (PBS) and re-suspended in 300 ml cell suspension buffer, CSB (50mM Tris-HCl (pH 8.5)/500mM NaCl/1 mM phenyl methyl sulfonyl fluoride).
  • PBS sterile l x phosphate buffered saline
  • CSB 50mM Tris-HCl (pH 8.5)/500mM NaCl/1 mM phenyl methyl sulfonyl fluoride
  • This suspension was subjected to lysis in a Dynomill (WAB, Muttenz, Switzerland) and clarified by centrifugation at 16,000 xg to separate out the membrane-enriched M fraction.
  • the M fraction was stirred in 200 ml membrane extraction buffer MEB-1 [CSB supplemented with 6M guanidine hydrochloride (Gu-HCl) and 20 mM imidazole] for ⁇ 4 hours at room temperature (RT).
  • the resultant extract was centrifuged (13,000 rpm, 1 hour, RT) and filtered (0.45 m).
  • the resultant clarified extract was bound to Ni 2+ -NTA resin (25 ml of a 50% slurry, pre-equilibrated in MEB-1) in batch mode (at RT overnight).
  • Ni 2+ -NTA resin plus bound sample slurry was packed into a chromatographic column which was connected to an AKTA purifier.
  • the column was washed extensively with MEB-1 followed by MEB-2 (CSB supplemented with 8M urea and 20 mM imidazole) to replace 6M Gu-HCl with 8M urea. Elution was performed using a step imidazole gradient in MEB-2. Column fractions were analyzed by SDS-PAGE.
  • mAb 24A12 which recognizes ZIKV EDIII, in a Western blot ( Figure 5B).
  • mAb 24A12 in this step may be replaced with others antibodies including, but not limited to, flavivirus-specific mAb 4G2, ZIKV specific mAbs ZV-48, ZV- 64, ZV-67, polyclonal animal sera or ZIKV-infected human sera.
  • a prM signal peptide 34 ad was included at the N-terminus of the recombinant protein to ensure proper processing.
  • N-terminal sequence analysis revealed that the prM signal peptide had been cleaved off from the P. pastoris-ex pressed rec ZIKV E protein.
  • N-linked glycan analysis of the purified rec ZIKV E protein was carried out using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
  • MALDI-TOF MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
  • the MS glycan profile, as well as the mass composition and relative abundance of the N-glycan moieties of rec ZIKV E protein are summarized in Figure 5C. Oligomannose structures up to Manl3 were observed with Man9 being the most abundant species.
  • VLPs virus-like particles
  • insect cells Dai S et al, Zika virus baculovirus-expressed virus-like particles induce neutralizing antibodies in mic e.VirolSinica 2018; 33: 213-226
  • mammalian cells Boigard H et al, Zika virus-like particle (VLP) based vaccin e.PLoSNegl Trop Dis. 2017; 11: e0005608; Garg H et al, Development of virus-like-particle vaccine and reporter assay for Zika virus. J Virol.
  • ELISA microtiter wells were coated with purified rec ZIKV E MAPs in 0.1 M sodium bicarbonate buffer (pH 9.6), overnight at 4°C (0.2 pg/0.1 ml/well).
  • wells were also coated with P. pastoris produced purified rec DENV-2 E protein, for comparison.
  • Coated wells were washed three times with lx PBS containing 0.1% Tween-20 (PBST) and blocked with 5% skim milk (200pl/well), prepared in lx PBS, for 2 hours at 37°C.
  • PBST 0.1% Tween-20
  • the rec ZIKV E protein-containing MAPs were recognized efficiently by the murine mAbs ZV-48 and ZV-67, but not by ZV-2. All three are ZIKV-specific murine mAbs. None of them recognized recombinant DENV-2 E protein. The binding sites of these mAbson the ZIKV E protein have been mapped using recombinant protein-binding studies in conjunction with X-ray crystallography (Zhao H et al, Structural basis of Zika virus-specific antibody protection. Cell. 2016; 166: 1016-1027).
  • ZV-2, ZV-48 and ZV-67 bind to the ABDE sheet, the C-C’ loop, and the lateral ridge (LR), respectively of ZIKV EDIII.
  • the LR epitope is quite complex in that it is comprised of several secondary structure elements that include the A-strand, the B-C loop, D-E loop and F-G loop involving 21 contact residues (Zhao H et al, Structural basis of Zika virus-specific antibody protection. Cell. 2016; 166: 1016-1027).
  • the efficient recognition by mAb ZV- 67, a potent neutralizer of ZIKV infectivity suggests that the antigenic integrity of the LR epitope is largely preserved in the rec ZIKV E MAPs.
  • ABDE sheet recognized by mAb ZV-2, a weak neutralizer, and also not predicted to be accessible on the ZIKV particle was not detectable on the rec ZIKV E MAPs as well.
  • the rec ZIKV E MAPs were also recognized by a human mAb, ZKA-64 (highly potent nAb), specific to ZIKV EDIII (StettlerKe/ al, Specificity, cross reactivity, and function of antibodies elicited by Zika virus infection. Science. 2016; 353: 823-826). Taken collectively, the mAb probing data suggest that the rec ZIKV EMAPs preserve the overall antigenic integrity of EDIII, but with subtle differences.
  • Example 5 Investigation of the capacity of P. yastoris- produced rec ZIKV E MAPs to induce ZIKV-specific, ZIKV EDIII-directed antibody response
  • Sera were collected 10-15 days after the 3 rd dose for determination of total Ab titers by indirect ELISA, using 5 different recombinant flaviviralE proteins (corresponding to ZIKV and DENV serotypes 1-4) as the capture antigens.
  • ELISA microtiter wells were treated overnight at 4°C with purified recombinant protein in 0.1 M sodium bicarbonate buffer, pH 9.6 (0.2 pg/0.1 ml/well). Coated wells were washed with lx PBS containing 0.1% Tween-20 (PBST) and blocked for 2 hours at 37°C, with 5% skim milk prepared in lx PBS.
  • PBST Tween-20
  • the blocked wells were washed (3x with PBST) and treated (0.1 ml/well, 1 hour, 37°C) with serial 2-fold dilutions of different immune sera.
  • Wells were washed (6x with PBST) and incubated with 0.1 ml anti -mouse IgG HRPO conjugate (0.1 pg/ml in 2.5% skim milk, prepared in IX PBS) for 1 hour at 37°C.
  • wells were washed (6x with PBST), treated with TMB substrate (0.1 ml/well) for 30 minutes at 37°C, followed by 1 N H 2 S0 (0.1 ml/well).
  • the absorbance was read at 450 nm, with 650 nm as reference.
  • ELISA absorbance values were ⁇ 0.05.
  • ELISA end-point titers defined as the highest reciprocal serum dilution that yielded an absorbance 4-fold over background (that is, a cut-off absorbance value of 0.2), were determined from extrapolation of non-linear regression curves using GraphPad Prism (v7.0) software.
  • ZIKV reporter virus particles were used instead of wild-type ZIKV.
  • RVPs ZIKV reporter virus particles
  • These ZIKV RVPs are identical to wild- type ZIKV on the outside and infect susceptible cells once but do not produce infectious progeny as they lack the structural genes. Instead they encode Renilla luciferase reporter whose activity provides a read-out of ZIKV RVP entry into cells (Shan C et al, Evaluation of a novel reporter virus neutralization test for serological diagnosis of zika and dengue virus infection. J ClinMicrobiol.
  • the ZIKV RVP inhibition assay was performed as follows. Vero cells were seeded in 96- well plates at 1.5xl0 4 /well/200 m ⁇ a day in advance (day 0). Replicates of ZIKV RVPs (10 m ⁇ each) were pre-incubated with serial two-fold dilutions of heat-inactivated (D, 56°C, 30 min) immune sera, in a total volume of 100 m ⁇ , for 1 hour. On day 1, Vero cells were exposed to this pre-incubation mixture (100 m ⁇ ZIKV-RVP + immune serum/well).
  • luciferase activity was measured as follows. Media was aspirated off from the wells which were rinsed once with lx PBS and followed by the addition of freshly diluted (in lx PBS containing 10% AFBS, final concentration 60 mM) ViviRen Live Cell substrate (100 m ⁇ /well).
  • Luminiscence was measured 2 minutes after substrate addition using SpecrtaMax M3 microplate reader (Molecular Devices, USA) set to‘luminiscence’ read mode.
  • ZIKV neutralizing activity was determined in terms of RNT 50 titer, defined as the serum dilution capable of causing a 50% reduction in the ZIKV RVP-expressed luciferase activity with reference to the activity expressed by ZIKV-RVP, in the absence of immune serum, taken asl00%
  • mice were allowed to rest for >4 months after the 3 rd immunization, and then given a 4 th dose (day 160). Mice were bled one day before (day 159) and 10 days after (day 170) the 4 th dose for nAb estimation using the ZIKV RVP luciferase assay (Figure 6C).
  • EDIII-specific Abs were specifically depleted from the anti-rec ZIKV E MAP antiserum on immobilized ZIKV EDIII as follows. Amylose resin was washed 3x with sterile water and 3x with column buffer (20 mMTris-HCl/200 mMNaCl/1 mM EDTA, pH 7.4). For each washing step, the resin was spun at 3,000 rpm for 5 min at RT.
  • Indirect ELISA revealed that the total ZIKV EDIII-specific Ab titers of both sera, which were not significantly affected following mock depletion using immobilized MBP, underwent considerable decrease following depletion on immobilized MBP ZIKV EDIII fusion protein. For example at a 1000-fold dilution of serum, ELISA absorbance values decreased >50% following depletion on immobilized MBP ZIKV EDIII fusion protein.
  • nAb titers in the un-depleted and MBP ZIKV EDIII-depleted immune sera were determined.
  • ZIKV-specific nAb titers can be abrogated to as high as 80% by additional rounds of depletion of the immune sera on immobilized ZIKV EDIII. It is evident that depletion of ZIKV EDIII-specific Abs in the immune sera strongly correlates with a decrease in nAb titer.
  • Example 7 Evaluation of flavivirus infection-enhancing capacity of rec ZIKV E MAP-induced Abs
  • DENV ADE assays were performed using the FcyllR-expressing K562 cell line.
  • Virus and serum dilutions were prepared in D-MEM+2% AFBS.
  • K562 cells (5X10 4 cells/20 m ⁇ /well) were mixed with the immune complex (IC) and allowed to incubate for 1 hour at 37°C in a humidified 10% CO2 incubator (final volume of 100 m ⁇ /well). Next, the cells were washed once with D- MEM+2% AFBS and suspended in fresh D-MEM+2% AFBS (200 m ⁇ /well) and incubated for 24 hours at 37°C in a humidified 10% C0 2 incubator.
  • ICs were first generated in vitro by pre-incubating (1 hour on ice) a sub-lethal dose of DENV-2 S221 (2xl0 4 FACS Infectious Units) separately with 5 m ⁇ (neat) of either NMS or anti-rec ZIKV E MAP-antiserum in a total volume of 50 m ⁇ .
  • Control IC was generated in parallel by using either 10 pg mAh 4G2, or 5 m ⁇ of anti -DENV-2 antiserum, during pre-incubation with DENV-2 S221.
  • VC virus control
  • NMS normal mouse serum
  • rec-ZIKV E MAPs by virtue of their lack of DENV-2 enhancing potential, are unique in that they offer an inherent safety aspect.
  • the finding that rec ZIKV E MAP -induced Abs do not escalate a sub-lethal DENV-2 infection is an unexpected and novel finding.
  • ZIKVs of the Asian lineage cause delayed onset of morbidity and clinical signs and rarely cause death. These mice usually recover by ⁇ 2 weeks post-challenge (Tripathi et al, A novel Zika virus mouse model reveals strain specific differences in virus pathogenesis and host inflammatory immune responses. PLoSPathog. 2017; 13: el 006258).
  • mice were injected with NMS, anti-DENV-3 antiserum or anti-rec ZIKV E MAP antiserum (20 m ⁇ /mouse, i.p.) and challenged 2 hours later with ZIKV strain PRVABC59 (5xl0 3 PFU/mouse, i.d.). These were monitored over 12 days post-ZIKV infection, for survival, clinical symptoms (fore- and hind-limb paralysis, hunched back etc) and weight loss(Figure 10, panels B-D).
  • Flavivirus-nAbs are postulated to facilitate enhancement of the homologous flavivirus when they are at sub -neutralizing concentrations (Pierson TC et al, The stoichiometry of antibody-mediated neutralization and enhancement of West Nile virus infection. Cell Host Microbe. 2007; 1: 135-145).
  • the circulating ZIKV nAb titers would have undergone -75 -fold dilution (with RNT50 dropping to sub -neutralizing levels of -3).

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Abstract

La présente invention se rapporte au domaine du développement de vaccins contre le virus Zika (ZIKV). Le développement du vaccin ZIKV est compliqué par l'existence d'un phénomène de facilitation de l'infection par des anticorps (ADE), issu de l'interaction entre ZIKV d'une part et les virus de la dengue d'autre part. La présente invention concerne un vaccin sous-unitaire ZIKV qui assure une protection contre le virus Zika sans causer d'ADE. Selon la présente invention, le vaccin sous-unitaire ZIKV comprend une forme particulaire associée à une membrane d'une protéine d'enveloppe ZIKV recombinante immunogène (E), ladite protéine E ZIKV recombinante comprenant des domaines I, II et III. Le domaine III est placé sur la surface de la membrane et peut être librement accédé par les anticorps spécifiques de ZIKV EDIII. L'invention concerne également des procédés de préparation du vaccin sous-unitaire ZIKV et de kits de détection d'anticorps spécifiques de ZIKV dans des biofluides tels que le sang, le plasma, le sérum, l'urine et la salive.
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