Immunogenic zika virus compositions and uses thereof

Abstract

The present invention relates to a vaccine for the prevention or treatment of a Zika virus infection and / or a Zika virus associated clinical illness of any severity. Further provided are methods for producing and inactivating Zika virus as well as processes for manufacturing compositions comprising Zika virus.

Description

[0001] Immunogenic Zika virus compositions and uses thereof

[0002] FIELD OF THE INVENTION

[0003] The disclosure relates to methods for the inactivation of Zika viruses for use in vaccines. Further disclosed are Zika virus pharmaceutical compositions and vaccines for the treatment or prevention of a Zika virus infection and / or a Zika virus associated clinical illness of any severity, as well as a method for treating a subject at risk of Zika virus infection. Further provided are methods for producing Zika virus particles in a bioreactor.

[0004] BACKGROUND OF THE INVENTION

[0005] Zika virus was detected in 2007 for the first time outside of the endemic regions of Asia and Africa since its discovery in a Rhesus monkey in Uganda in 1947. Since then, the vims has caused a large epidemic in French Polynesia, spreading through islands in the Pacific and into South and Central America by 2015 (WHO “Zika Situation Report” February 5, 2016). Evidence suggests that in addition to being transmitted by Aedes species mosquitos, other vectors may exist, and the vims may be transmitted by blood transfusion, transplacentally, and through sexual transmission (WHO Zika Vims Fact Sheet, Feb. 2016). Though the symptoms of Zika vims infection include mild fever, rash, and conjunctivitis, there is a likely correlation between infection and neurological disorders, including Guillain-Barre syndrome and microcephaly in fetuses / neonates subsequent to infection during pregnancy. There is currently no specific treatment or vaccine for Zika vims and the only preventative measures involve control of the mosquito vector. Zika vims presents a substantial public health threat due to the wide circulation of the Aedes mosquito, multiple routes of transmission, and potentially severe neurological effects of infection.

[0006] A preventative vaccine against Zika vims is a pressing medical need in endemic areas and in geographical areas where the vector is spreading. Furthermore, as Zika infection has grave consequences on embryonic and fetal development, a safe and effective vaccine for women of childbearing potential or pregnant women is needed. Vaccines administered during pregnancy must be very safe for both the mother and the developing fetus. While live attenuated viral vaccines are highly effective, they are often not considered safe enough for administration to pregnant women. In this regard, inactivated viral vaccines, which lack the ability to propagate in the vaccinated subject, are considered much safer. Development of an inactivated Zika vims vaccine for administration to at-risk patients would fill this need.

[0007] Several methods have been developed to inactivate viral stocks, that are used for vaccine development. In addition to formaldehyde, beta-propiolactone (BPL) has been a very popular chemical agent in various vaccine approaches due to its high inactivation potency and relatively low damage to protein antigens.

[0008] BPL is interacting primarily with nucleic acids where it modifies the Nitrogen-7 atom of guanosine, and to a lesser extent, the N 1 position of adenosine resulting in misread by the polymerase and a point mutation at each modified nucleotide position. These multiple point mutations render the virus replication incompetent, resulting in its complete inactivation. This reaction occurs very fast at low temperatures resulting in total inactivation within hours. Although BPL inactivation is mainly accomplished through modification of genetic material, certain amino acids may also display nucleophilic moieties with which BPL can interact. As with formaldehyde, the interaction of BPL with viral proteins could induce conformational changes on the viral surface, potentially resulting in the alteration of epitopes necessary for the induction of neutralizing antibodies against the pathogen. In studies using synthesized peptides, it has been observed that BPL treatment can result in alkylation or acylation modifications of up to 9 different amino acids (C, H, M, D, E, Y, K, E, S) depending on actual pH (Uittenbogaard et al., 2011. Reactions of P-Propiolactone with Nucleobase Analogues, Nucleosides, and Peptides: Implications for the inactivation of viruses. JBC 286(42)36198-36214). Within a pH range of 7 to 9, for example, an especially high percentage of modifications were observed for cysteine (>95 %), histidine (15 - 25 %) and methionine (36 %) residues. Of the Zika virus structural proteins, immunogenicity is mainly derived from the envelope (E) protein with minor contributions of the membrane (M) and Capsid (C) proteins (Culshaw, et al., 2018. The immunology of Zika Virus. FlOOOResearch 7(F1000 Faculty Rev):203). It was shown that BPL concentrations of 750 ppm and higher resulted in reduced immunogenicity of Zika virus, possibly due to increased modification of immunogenic epitopes on the virus surface (Chida et al., 2021. Comparison of Zika virus inactivation methods for reagent production and disinfection methods. J. of Virol. Methods 287: 114004). Therefore, the maximal concentration of BPL used for Zika virus inactivation should not exceed safe concentrations (500 - 600 ppm).

[0009] While BPL is highly toxic, its half-life in aqueous solution is low and depending on temperature and buffer composition (e.g. <30 min at 37°C) and the final degradation product propionic acid is harmless to humans. Therefore, inactivation by BPL is routinely followed by a hydrolyzation step at elevated temperatures to facilitate this chemical reaction.

[0010] The Thl / Th2 dichotomy has been considered the cornerstone of immune responses since the late 1980s (Mosmann et al. 1989. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Ann Rev Immunol 7: 145-173). In response to viral infection, the host mounts both Thl and Th2 responses. Stimulation of the adaptive immune response via IFN and Antigen Presenting Cells (APCs) drives the expansion and differentiation of cytotoxic T-cells (CD8+) and mediates the transition of naive T helper cells (ThO) into mature Th cells (CD4+). Generally, Thl and Th2 responses are the most important responses to viral infections; Thl being primarily responsible for the pro-inflammatory response to intracellular parasites, and Th2, being mainly characterized by antibody production (Howard, et al. 2022. Understanding Immune Responses to Viruses — Do Underlying Thl / Th2 Cell Biases Predict Outcome? Viruses 14: 1493. https: / / doi.org / 10.3390 / vl4071493). While the use of alum adjuvants in inactivated virus vaccines has a track record of safety and efficacy, alum mainly induces Th2 type immune responses. There is therefore a need to develop adjuvant systems better able to provoke both arms of immunity to provide a more balanced and effective immune response. As described herein, the use of alum in combination with an adjuvant stimulating a more Thl -skewed response in an inactivated Zika virus vaccine preparation both reduces the amount of antigen required (dose sparing) as well as providing a strong Thl response.

[0011] SUMMARY OF THE INVENTION

[0012] Previously, a first generation inactivated Zika virus vaccine was prepared by formaldehyde inactivation and formulated with alum, as described in WO2017 / 109225A1, which is incorporated herein by reference in its entirety. In some cases, formaldehyde can alter the surface structure of viruses and thus result in a poor immune response to the inactivated virus (Delrue, et al. 2012. Inactivated vims vaccines from chemistry to prophylaxis: merits, risks and challenges. Expert Rev. Vaccines 11(6):695-719). Therefore, with the aim of minimizing surface protein modifications, an inactivation process targeting mainly viral nucleic acids using beta-propiolactone (BPL) was developed for the second generation Zika virus vaccine as described herein. Further, to provide a superior immune response compared with alum adjuvantation alone, the inactivated Zika virus was adjuvanted with both alum and a Toll-like receptor (TLR) agonist. Also provided are methods of administering the disclosed Zika virus vaccines for the treatment or prevention of a Zika virus infection and / or a Zika virus associated clinical illness of any severity.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings are not intended to be drawn to scale. The Figures are illustrative only and are not required for enablement of the disclosure. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

[0015] Figure 1. Schematic overview of the plaque assay procedure for determining Zika virus yield and inactivation. Figure 2. Western blot analysis of BPL inactivated vims compared to Zika vims inactivated with increasing concentrations of formaldehyde. The Zika vims E protein was detected by use of the commercially available Zika vims specific monoclonal antibody mAb 40543-MM09.

[0016] Figure 3. SE-HPLC (UV214nm) overlay of an exemplary BPL inactivated Zika vims and Zika vims inactivated with three different concentrations of formaldehyde.

[0017] Figure 4. Zika vims BPL inactivation method summary.

[0018] Figure 5. Immunogenicity of BPL inactivated Zika vims three weeks after a single immunization. Neutralization capacity of individual mouse sera per group is depicted. Mice showing a titer above 50% (dotted line) are taken as seroconverted. A. Formulation based on Zika vims antigen content as assessed by mass (BSA units=ng). B. Formulation based on Zika vims antigen content as assessed by ELISA units (Antigen units=AU). All doses were formulated with 1 mg / mL A1(OH)3 (100 pg / dose).

[0019] Figure 6. Immunogenicity of BPL inactivated Zika vims formulation delivered in low, medium and high antigen doses, each formulated with alum alone (1 mg / mL) or alum (1 mg / mL) + 3M-052-AF (10 pg / mL) and delivered on dO and d21. A. Low antigen dose (200 ng / 10 AU) d21 and d42, B. Medium antigen dose (600 ng / 30 AU) d21 and d42, and C. High antigen dose (1200 ng / 60 AU) d21 and d42. An unadjuvanted medium antigen dose (400 ng / 20 AU) was included as a control and results shown in Fig. 6B.

[0020] Figure 7. Interferon-gamma (IFN-y) production by splenocytes recovered from mice vaccinated once or twice. A. IFN-y production in splenocytes after in vitro restimulation with peptide libraries as assessed by Multiplex. CD-I mice were immunized twice with 1200 ng inactivated Zika vims formulated with alum alone (100 pg) or alum (100 pg) + 3M-052-AF (1 pg). 7 days after the 2ndimmunization, mice were euthanized, spleens removed and splenocytes isolated. IxlO6cells were stimulated with either a specific peptide library encoding the E protein of Zika vims or with an unspecific library encoding Hu-Actin at 2pg / mL. After 18-24h, supernatants were collected and analyzed for cytokine production according to the supplier’s protocol. Results are presented as pg / mL. B. IFN-y production in splenocytes after in vitro restimulation with peptide libraries as assessed by ELISpot. Mice were immunized as in part A. above and splenocytes harvested. 3xl05cells were stimulated with specific or unspecific peptide libraries as in part A. above. After 18-24h, cells were discarded and anti-IFN-y coated plate was analyzed for cytokine production according to the supplier’s protocol. Results are presented as Spot Forming Units (SFU) per 3xl05cells. C. Comparison of ELISpot IFN-y production in splenocytes after in vitro restimulation with peptide libraries after one and two immunizations. CD-I mice were i.p. vaccinated twice as in part A. above and splenocytes were harvested 7 days after the 1stand 7 days after the 2ndimmunization. 3xl05cells were stimulated with specific or unspecific peptide libraries as in part A. above. After 18-24h, cells were discarded and anti-IFN-y coated plate was analyzed for cytokine production according to the supplier’s protocol. Results are presented as Spot Forming Units (SFU) per 3xl05cells. In all cases, the geometric mean (GM) from each of three individual mice was calculated. Analysis was done in GraphPad Prism 10.

[0021] Figure 8. Zika virus vaccine candidate immunogenicity assessed in rats during the course of toxicology studies. A. Immunizations were done on days 0, 15 and 29; Blood draws were done on days 15, 29 and 59. B. Neutralizing antibody titers (pNTso) in individual rat sera collected after one dose (d 15), two doses (d29) and three doses (d59) of inactivated Zika virus at a high dose (320 AU) adjuvanted with alum (1 mg / mU) and a low dose (80 AU) adjuvanted with alum (1 mg / mU) and 3M- 052-AF (6.25 pg / mU). The dosages were delivered in a volume of 0.4 mU.

[0022] Figure 9. Staggered dose escalation scheme for clinical Phase 1 study — open-label sentinels.

[0023] Figure 10. Overall clinical Phase 1 study design — including an open-label sentinel phase and a double-blind randomized phase.

[0024] Figure 11. Examples of bioreactors using a falling film component for circulation / oxygenation of cell culture medium.

[0025] A) A perspective view of a bioreactor for culturing cells, wherein the bioreactor includes an external casing or housing 112 forming an interior compartment and a removable cover 114 for covering the interior compartment, which may include various openings or ports P with removable covers or caps C for allowing for the selective introduction or removal of fluid, gas (including by way of a sparger), probes, sensors, samplers, or the like. The bioreactor may be used in connection with an external reservoir and conduits (e.g., forward and return) to form a continuous loop for circulating fluid to the bioreactor (adapted from WO2019122239A1, Fig. 1).

[0026] B) The bioreactor according to A) above, showing the medium recirculation loop (adapted from WO2019122239A1, Fig. 2C).

[0027] C) A bioreactor arranged such that an upper plate 330 is provided with peripheral openings 330c to allow fluid to flow directly along the inner wall formed by tube 334. In this manner, a thin layer or film of fluid may be created, which flows downwardly while passing through the fifth chamber 328. This may serve to increase the volume of the fluid exposed to gas (air) within the fifth chamber 328, prior to it being returned to the first chamber 316. This implementation can allow for more oxygen transfer which may be needed for larger sizes or otherwise to increase cell growth rates or adjust process parameters based on the biologic being produced (adapted from WO2019122239A1, Figs. 11 and 12).

[0028] D) A structured bag bioreactor 100, which is placed in fluid communication with a circulation tube 410. A first end of the circulation tube 410 is fluidly connected to a dip tube 110 in the structured bag 100 and a second end of the circulation tube 410 is fluidly connected to a circulation port 420 disposed in the fitment of the structured bag 100. The circulation port 420 fluidly connects the circulation tube 410 to the interior compartment of the structured bag 100. The circulation system also includes a pump configured to move fluid and / or other components out of the structured bag 100, into the circulation system and back into the structured bag 100. Fluid and / or other components are circulated using the pump 400 to increase oxygenation. Media in the interior compartment of the structured bag 100 is drawn into the circulation tube 410 via the dip tube 110. Media is then returned to the structured bag 100 through the circulation port 420. As the media flows into the bag though the circulation port 420, oxygen diffuses into the media while the media cascades down the bag, for example, down the interior of the sidewall to minimize splashing or disruption of cells in the structured bag 100. The cascading of the media functions like a "falling film oxygenator" (adapted from WO2017197154A1, Fig. 4).

[0029] E) An exemplary fixed-bed bioreactor (iCELLis 500), showing the falling film component (side-cuts). The cell culture medium flows through the fixed bed from the bottom to the top. At the top, the medium falls as a thin film down the outer wall where it takes up O2 to maintain high a Volumetric Oxygen Transfer Coefficient (kLa) in the bioreactor. The arrows represent the direction of the liquid circulation (adapted from PALL Biotech USD3262d “Productivity and Reduced Footprint for Industrialization of Animal Cell Culture Processes in a Fixed-Bed Bioreactor”, 2021).

[0030] Figure 12. The process flow of the optimized aeration strategy to minimize the falling film during virus infection and propagation in a fixed bed bioreactor such as e.g. an iCELLis 500 A) Virus infection phase (-2-3.5 hr); B) Virus propagation phase day 1 (up to approximately 26 hours PI); C) Virus propagation phase day 2 (from approximately 26 hours PI) to harvest (approximately 48-52 hr PI).

[0031] DETAILED DESCRIPTION OF THE INVENTION

[0032] Disclosed herein are Zika virus pharmaceutical compositions and vaccines. Also disclosed are related methods of inactivation of a Zika virus with beta-propiolactone (BPL). Further, methods of administering said Zika virus vaccines for the treatment or prevention of a Zika virus infection and / or a Zika virus associated clinical illness of any severity are provided. An advantage of the invention is that the herein disclosed method of inactivation with beta-propiolactone provides a minimally modified E-protein compared with Zika virus inactivated with formalin. Furthermore, the pharmaceutical compositions provided herein are formulated with at least two adjuvants to provide a superior immune response.

[0033] Therefore, in a first aspect, provided is a pharmaceutical composition comprising i) an inactivated Zika virus, ii) an aluminium salt adjuvant, and iii) at least one additional adjuvant, wherein said at least one additional adjuvant is a toll-like receptor (TLR) agonist. In a preferred embodiment, the inactivated Zika virus comprises an RNA genome corresponding to the DNA sequence provided by SEQ ID NO: 2 or 3, or a variant sequence that is at least 80% identical to SEQ ID NO: 2 or 3 and able to pack a virulent Zika virus. In one embodiment, the variant sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.99% identical to SEQ ID NO: 2 or 3 and able to pack a virulent Zika virus. In a preferred embodiment, the pharmaceutical composition comprises an aluminium adjuvant as the at least one adjuvant. In a preferred aspect, the inactivated Zika virus is a beta-propiolactone (BPL) inactivated Zika virus.

[0034] The examples provided herein use the H / PF / 2013 strain of Zika virus; however, any strain of Zika virus may be used in the methods and compositions described herein. In some embodiments, the Zika virus is an isolate from an infected subject during a Zika virus outbreak. In some embodiments, the Zika virus is a strain isolated from Africa or from the African virus lineage. In some embodiments, the Zika virus is a strain isolated from Asia or from the Asian lineage (includes also strains from French Polynesia). In some embodiments, the Zika virus is a strain isolated from the Americas (South America, Central America, or North America), such as a Suriname Zika virus strain.

[0035] In one embodiment, the inactivated Zika virus of the invention as provided herein is the publicly available sequence of the French Polynesian Zika virus strain H / PF / 2013 polyprotein gene (SEQ ID NO: 1; accession no: KJ776791.1), which is a Zika virus from the Asian lineage. The genomic sequence provided by SEQ ID NO: 2 was sequenced from the H / PF / 2013 strain after passaging in the lab, whereas SEQ ID NO: 3 is the same genomic sequence which has been adapted or further adapted to passaging on Vero cells and comprises two mutations compared with SEQ ID Nos: 1 and 2.

[0036] In one embodiment, the inactivated Zika virus comprises an RNA genome corresponding to the DNA sequence provided by SEQ ID NO: 2 or 3. In one embodiment, the inactivated Zika virus comprises an RNA genome corresponding to a variant sequence that is at least 80% identical to SEQ ID NO: 2 or 3 and able to pack a virulent Zika virus. In one embodiment, the variant sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.99% identical to SEQ ID NO: 2 or 3 and able to pack a virulent Zika virus. In one embodiment, the inactivated Zika vims comprises an E protein as defined by SEQ ID NO: 4. In some embodiments, the inactivated Zika vims comprises a variant amino acid sequence of the E protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to SEQ ID NO: 4 and able to pack a vimlent Zika vims. In one embodiment, the inactivated Zika vims comprises a variant amino acid sequence of the E protein that is at least 95% identical, especially at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 4 and able to pack a vimlent Zika vims. In one embodiment, the inactivated Zika vims comprises an E protein comprising one or more modified amino acids. In one embodiment, one or more modified amino acids result from BPL inactivation. In one embodiment, the one or more modified amino acids are selected from the group consisting of M125, H210, M295, H398, H399, H401 and H446. In a preferred aspect, the inactivated Zika vims comprises an E protein wherein each of the said modified amino acids, i.e., M125, H210, M295, H398, H399, H401 and H446, is present at a frequency of greater than 15%. In a preferred aspect, the inactivated Zika vims has an E protein with the amino acid sequence provided by SEQ ID NO: 4.

[0037] The terms "identical" or percent "identity" in the context of two or more nucleic acids or amino acid sequences refer to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) over a specified region or over the entire sequence, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length. In some embodiments, the identity exists over the length of a protein, such as the E protein.

[0038] The similarity between amino acid sequences and / or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity; the higher the percentage, the more similar the two sequences are. Homologs, orthologs, or variants of a polynucleotide or polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.

[0039] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5: 151-3, 1989; Corpet et al., Nuc. Acids Res. 16: 10881-90, 1988; Huang et al. Computer Appls. In the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al, J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0040] Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. Preferably, the percentage sequence identity is determined over the full length of the sequence. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166=1554* 100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer.

[0041] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs BLASTP, BLASTN, BLASTX, TBLASTN and TBLASTX. A description of how to determine sequence identity using this program is available on the NCBI website on the internet. The BLAST and the BLAST 2.0 algorithms are also described in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, alignments (B) of 50, expectation I of 10, M=5, N=-4, and a comparison of both strands. The BLASTP program (for amino acid sequences) uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915, 1989).

[0042] Homologs and variants of a polynucleotide or polypeptide are typically characterized by possession of at least about 75%, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over at least 50, 100, 150, 250, 500, 1000, 2000, 5000 or 10,000 nucleotide or amino acid residues of the reference sequence, over the full length of the reference sequence or over the full length alignment with the reference amino acid sequence of interest. Polynucleotides or proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. For sequence comparison of amino acid or nucleic acid sequences, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are used.

[0043] One example of a useful algorithm is PILEUP. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, Mol. Evol. 35:351-360, 1987. The method used is similar to the method described by Higgins & Sharp, supra. Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-395, 1984).

[0044] As used herein, reference to "at least 80% identity" refers to at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity to a specified reference sequence, e.g. to at least 50, 100, 150, 250, 500, 1000, 5000 or 10,000 nucleotide or amino acid residues of the reference sequence or to the full length of the sequence. As used herein, reference to "at least 90% identity" refers to at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity to a specified reference sequence, e.g. to at least 50, 100, 150, 250, 500, 1000, 5000 or 10,000 nucleotide or amino acid residues of the reference sequence or to the full length of the sequence.

[0045] In one embodiment, the inactivated Zika virus comprises a heterologous mixture of Zika virus with 1 to 10 genomic mutations compared with SEQ ID Nos: 2 or 3 and able to pack a virulent Zika virus. In one embodiment, the inactivated Zika virus comprises an RNA genome corresponding to the DNA sequence provided by SEQ ID NO: 2 or 3. In one embodiment, the inactivated Zika virus comprises a heterologous mixture of RNA genomes corresponding to the DNA sequences provided by SEQ ID NO: 2 and 3.

[0046] In one aspect, the inactivated Zika virus is provided at 1 to 10 pg / dose, preferably at 1.5 to 6 pg / dosc. As described herein, the mass per unit volume (e.g., pg / mL) of inactivated Zika virus may be determined by the use of size exclusion chromatography (SEC) with a reference protein, e.g., BSA, as a standard for comparison, such as, e.g., the SEC assay as described in Example 2 in the current disclosure. In such case, the mass may also be referred to as “BSA units”. In further aspects, other protein assays known in the art (e.g., Bradford assay) may be used to determine inactivated Zika virus mass per unit volume. In one embodiment, the inactivated Zika virus is provided at 1 to 20 pg / dose. In a preferred embodiment, the inactivated Zika virus is provided at 1 to 10 pg / dose, such as 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 pg / dose, especially about 2, 4 or 8 pg / dose.

[0047] In one aspect, the inactivated Zika virus is provided at a dose of between 100 to 800 AU / dose, preferably 100 to 600 AU / dose, preferably 100 to 400 AU, especially 100, 200 or 400 AU / dose. As described herein, the antigen units per unit volume (AU / mU) may be determined by the use of an EUISA-based method. In one aspect, the EUISA is a standard plate-based “sandwich” ELISA for quantifying the amount of a particular epitope which binds to the specific antibodies used in the assay, such as, e.g., the “second generation” ELISA as described herein. In such an ELISA, the antigen content in the inactivated Zika virus preparation may be determined relative to a standard virus preparation. Vaccine formulation using the antigen content of an inactivated virus may be more accurate batch to batch than using a simple protein mass determination, as it measures the amount of relative antigen units (AU) present. In this regard, an antigen unit (AU) as measured by the “second generation” ELISA as described herein is approximately equal to 20 ng of virus protein as measured in the SEC assay above; however, this will vary depending on the amount / quality of antigen in any given batch of inactivated Zika virus. Assessment of antigen concentration in terms of ELISA antigen units is thought to be a more accurate way of formulating doses across batches. In general, the total viral protein content as estimated by SEC-HPLC (BSA units) of 2 pg- 100 AU, 4 pg~200 AU and 8 pg~400 AU, when AU units are determined by the ELISA assays as described herein, particularly when the AU units are determined by the competitive liquid phase ELISA as described herein. In a preferred aspect, the AU / mL of the inactivated Zika virus is determined by a “liquid” ELISA assay as described herein in the Examples. A liquid ELISA, also referred to herein as “competitive liquid phase ELISA”, is an ELISA-like assay wherein a step of antibody-antigen binding is done in a liquid format (instead of on an antibody-coated plate), which step is followed by quantification of unbound antibody in an antigen coated plate. Thus, in a liquid ELISA, the antigen content is inversely proportional to the remaining (unbound) Zika specific antibody. In such an assay, a particularly relevant antibody is used for antigen content determination, i.e., one that binds to one or more epitopes known to be important for virus neutralization. In one aspect, a polyclonal antibody may be used. In a preferred aspect, a monoclonal antibody or a mix of monoclonal antibodies are used. In a most preferred embodiment, a single monoclonal antibody is used, particularly a monoclonal antibody such as, e.g., Anti-Zika-Envelope Protein Antibody ZKA64 [Human IgGl] or a similarly relevant monoclonal antibody. Therefore, in one aspect, the antigen concentration determination may be assessed by an ELISA assay, such as any known in the art. In a preferred aspect, the ELISA assay is the “second generation” ELISA assay as described in Example 2 of the current disclosure. In a most preferred aspect, the AU determination is done by a competitive liquid phase ELISA assay, particularly as described herein in Example 2 below.

[0048] In one aspect, the aluminium salt adjuvant provided in the pharmaceutical composition is aluminium hydroxide (Al(0U)3) or aluminium phosphate. A preferred aluminium salt is aluminium hydroxide with reduced Cu content, e.g. lower than 1.25 ppb Cu based on the weight of the inactivated Zika virus composition, an adjuvant described in detail in WO2013 / 083726A1 and Schlegl et al., Vaccine 33 (2015) 5989-5996. As referred to herein, the weight of the alum component refers to the weight of the Al3+in the solution, regardless of what type of aluminium salt is used. For example, 0.5 mg of Al3+corresponds to 1.5 mg alum. In one embodiment, the amount of alum (esp. A1(OH)3) present in the pharmaceutical composition is between about 0.1 and 2 mg / mL, between about 0.2 and 1.5 mg / mL, between about 0.5 and 1.3 mg / mL, especially between about 0.8 to 1.2 mg / mL, most preferably about 1 mg / mL, i.e., 0.5 mg / 0.5 mL dose for human subjects. In a preferred embodiment, the pharmaceutical composition is administered in a volume (dose) of between 0.1 and 1 mL, preferably about 0.25 to 0.75 mL, preferably 0.45 to 0.55 mL, most preferably about 0.45 mL or 0.5 mL, e.g., ± 10% or ±5%, or exactly 0.45 mL or 0.5 mL.

[0049] In one aspect, the pharmaceutical composition further comprises a second adjuvant, wherein such adjuvant is an adjuvant stimulating a Thl-skewed immune response. Thl response-directing adjuvants promote the induction of a T helper type 1 (i.e. Thl) immune response in an immunized subject (rather than a solely Th2 type response), i.e. a “Thl-biased response”. It has surprisingly been found that using an adjuvant that promotes a Thl response can improve immunogenicity of the vaccine and thus antiviral responses, as well as reduce the risk of disadvantageous effects such as immunopathology, which may arise from a predominantly Th2 type response (possibly due to hypersensitivity against viral components). In a particularly preferred embodiment, the adjuvant stimulating a Thl-skewed immune response is a compound or molecule which is a toll -like receptor (TLR) agonist. The Thl- or Th2 -directing properties of commonly used adjuvants are known in the art. Neutralizing antibodies, the production of which is critical for anti-viral immunity, are strongly stimulated by Th2-stimulating adjuvants, such as e.g. alum. Also important for anti-viral immunity are cellular immune responses, which are only weakly stimulated by alum. Delivered together, alum and Thl -inducing adjuvants, such as TLR agonists, can provide a potent anti-viral response. In short, the use of TLR agonist(s) and alum together in a vaccine formulation can provide a more balanced immune response to antigens. Th2 responses may be assessed based on levels of IgGl (murine) as well as IgG4 (human) antibodies and / or cytokines such as, e.g., IL-4, IL-5 and / or IL-12. Thl responses may be assess based on levels of IgG2a (mouse) and IgGl / IgG3 (human) antibodies and / or cytokines such as, e.g., IL-2 and / or IFN-y. Patern recognition receptors (PRR) are present mostly on immune cells and are activated by distinct structural motifs of microbes and / or damaged cells or tissues. These molecular structures are also known as pathogen- or damage-associated molecular paterns (PAMPs and DAMPs) and are recognized by the immune system as “non-self As such, PRRs are important to innate (cellular) immunity and include several types such as toll-like receptors (TLR), nucleotide-binding oligomerization domain-like receptors (NLR), C-type lectin receptors (CLR) and RIG- 1 like receptors (RLR) (Zaru, Patern recognition receptor (PRRs) ligands; www.immunology.org / public- information / bitesized-immunology / receptors-molecules / patem-recognition-receptor-prrs; downloaded 24-Jan-2024).

[0050] TLR agonists for use herein may include particularly TLR2, TLR4, TLR7, TLR8 and / or TLR9 agonists, particularly TLR9 agonists or mixed TLR7 / 8 agonists. In some embodiments, the TLR agonists are natural substances, are derived from natural substances or are fully synthetic substances. In some embodiments, the TLR agonist is comprised of two components. In some embodiments, the 2 -component adjuvant comprises an antibacterial peptide and a TLR9 agonist. In some embodiments, the antibacterial peptide is provided by the amino acid sequence KLKL5KLK (SEQ ID NO: 5). In some embodiments, the TLR9 agonist is a deoxyinosine-containing immunostimulatory oligodeoxynucleic acid molecule (I-ODN). In some embodiments, the I-ODN comprises the nucleic acid sequence (dldC)is (SEQ ID NO: 6). In some embodiments, the adjuvant is IC31®, i.e. KLKL5KLK (SEQ ID N0:5) and the nucleic acid sequence (dldC)is (SEQ ID NO: 6). In some embodiments, the IC31 adjuvant is in nanoparticle form (See, e.g., US Patent No. 8,765,148 B2, incorporated by reference in its entirety). In some embodiments, the second adjuvant is a TLR7 agonist, such as e.g. imiquimod. In some embodiments, the second adjuvant is a TLR8 agonist or a mixed TLR7 / 8 agonist. Such agonists are generally synthetic compounds (small molecules) designed to mimic the natural viral single-stranded RNA ligands. Examples include the imidazoquinolines (such as, e.g., resiquimod; aka imidazoquinoline R848) and oxoadenines (such as, e.g., SM360320) and derivatives thereof (see e.g., Bazin, et al., Optimization of 8-oxoadenines with toll-like -receptor 7 and 8 activity. 2021. Bioorg Med Chem Let. 30(6) : 126984. doi: 10.1016 / j.bmcl.2020.126984).

[0051] In a preferred embodiment, the TLR agonist is a TLR7, TLR8 and / or mixed TLR7 / 8 agonist, such as, e.g., an imidazoquinoline or derivative thereof. In a preferred embodiment, the agonist is a 3M imidazoquinoline immune response modifier (IRM), particularly wherein the IRM is selected from a lipid-modified IRM such as those described in US 7,799,800, EP3222621 and / or US9,795,669. In particular, the lipid-modified IRM is selected from N-(2-{2-[4-amino-2-(2-methoxyethyl)-lH- imidazo[4,5-c]quinolin-l-yl]ethoxy}ethyl)octadecanamide, N-(2-{2-[4-amino-2-(2-methoxyethyl)- lH-imidazo[4,5-c]quinolin-l-yl]ethoxy}ethyl)dodecanamide, N-(2-{2-[4-amino-2-(2-methoxyethyl)- lH-imidazo[4,5-c]quinolin-l-yl]ethoxy}ethyl)tetradecanamide, N-(2-{2-[4-amino-2-(2- methoxyethyl)-lH-imidazo[4,5-c]quinolin-l-yl]ethoxy}ethyl)hexadecanamide, N-(4-((4-Amino-2- butyl-lH-imidazo[4,5-c]quinolin-l-yl)oxy)butyl)stearamide, N-(2-{2-[4-amino-2-(2-methoxyethyl)- lH-imidazo[4,5-c]quinolin-l-yl]ethoxy}ethyl)hexadecanamide and / or N-(4-{[4-amino-2 -butyl- 1H- imidazo[4,5-c]quinolin-l-yl]oxy}butyl)octadecanamide (a.k.a. 3M-052), preferably N-(4-{[4-amino- 2-butyl-lH-imidazo[4,5-c]quinolin-l-yl]oxy}butyl)octadecanamide (3M-052). 3M-052 is a synthetic derivative of a natural molecule which is an IRM bearing a Cl 8 lipid moiety. It was designed for slow dissemination from the site of application to prevent the activation of a cytokine storm (Smirnov, et al., Vaccine adjuvant activity of 3M-052: An imidazoquinoline designed for local activity without systemic cytokine induction. 2011. Vaccine 29:5434-5442).

[0052] In one embodiment, the TLR agonist, especially the mixed TLR7 / 8 agonist, is provided in an emulsion formulation, such as, e.g., 3M-052-SE. In a preferred embodiment, the mixed TLR7 / 8 agonist is provided as an aqueous formulation, such as e.g., 3M-052-AF. Insoluble or poorly soluble pharmaceutical excipients, including active ingredients, may be solubilized in a variety of ways as described in the art (e.g., as described by Strickley, Solubilizing Excipients in Oral and Injectable Formulations. 2004. Pharmaceutical Research, Vol. 21(2): 201-230). The TLR agonist may be solubilized by formulating with various solubilizing excipients such as water-soluble organic solvents, non-ionic surfactants, water-insoluble lipids, organic liquids / semi-solids, cyclodextrins and phospholipids. The chemical techniques to solubilize water-insoluble drugs for oral and injection administration include pH adjustment, cosolvents, complexation, microemulsions, self-emulsifying drug delivery systems, micelles, liposomes, and emulsions. In a preferred embodiment, the TLR 7 / 8 agonist, especially any 3M immune response modifier, is solubilized by formulating with a phospholipid solubilizing compound such as e.g., distearoyl phosphatidylglycerol (DSPG). Most preferably, the TLR7 / 8 agonist is 3M-052 solubilized with DSPG, i.e., AP60-702 or “3M-052-AF” (aqueous formulation). In a preferred embodiment, the 3M-052 adjuvant, especially the 3M-052-AF adjuvant, is provided in the pharmaceutical composition at a concentration of about 0.1 to 100 pg / mL, especially about 1 to 10 pg / mL, preferably about 2 to 8 pg / mL, preferably about 5 to 7 pg / mL, especially about 6.25 pg / mL; i.e., about 3.125 pg / 0.5 mL human dose, or about 3.125 pg / 0.45 mL human dose. The concentration specified refers to the adjuvant molecule itself, disregarding other possible compounds contained in the 3M-052 adjuvant preparation.

[0053] In a preferred embodiment, the TLR agonist is a TLR9 agonist such as CpG, preferably CpG 1018. As used herein, “CpG” refers to a cytosine-phospho-guanosine (CpG) motif-containing oligodeoxynucleotide (or CpG-ODN), e.g. which is capable of acting as a toll-like receptor 9 (TLR9) agonist. The CpG motif refers to an unmethylated cytidine-phospho-guanosine dinucleotide sequence, e.g. which is capable of binding to TLR9. In one embodiment, the CpG adjuvant comprised in the vaccine of the invention is a class A, class B or class C CpG, preferably a class B CpG. Class B CpG molecules include CpG 1018, CpG 1826 and CpG 7909 (SEQ ID Nos: 7, 8 and 9, respectively). Most preferred is CpG 1018. CpG 1018 may be adsorbed onto alum and thus used as a combination adjuvant that induces both Thl and Th2 responses (as described in e.g. Tian et al. The novel complex combination of alum, CpG ODN and HH2 as adjuvant in cancer vaccine effectively suppresses tumor growth in vivo. 2017. Oncotarget 8(28):45951-45964). The presence of A13+may reduce the required dose of CpG, i.e., have a “dose-sparing” effect. In one embodiment, the two adjuvants are both comprised in the formulation of the vaccine of the invention; i.e. a “single-vial formulation”. In a preferred embodiment, the inactivated Zika virus vaccine is formulated with A13+, and combined with a separate TLR agonist-containing solution directly before vaccination of a subject; i.e. “bed-side mixing”.

[0054] In one embodiment, the A13+:CpG weight / weight (w / w) ratio, preferably the A13+provided in the form of Al(OH)3:CpG 1018 (w / w) ratio, in the vaccine composition is about 1: 10, about 1:5, about 1:4, about 1:3, about 1:2, about 1: 1, about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 10: 1, preferably between about 1:3 and 3: 1, more preferably between about 1:2 and 1: 1, most preferably about 1:2, even more preferably 1:2 in humans. In this regard, the effects of CpG in mice and humans are known to be very different, in part due to different TLR9 receptors which respond differently to CpG-DNA sequences as well as unique cellular distribution patterns of TLR9 expression in mice and humans (Chuang TS et al., Toll-like receptor 9 mediates CpG-DNA signaling. 2002. J Leukocyte Biol. (71)538-544). Therefore, the alunrCpG ratio giving an optimal response to any given antigen is likely to differ, perhaps substantially, in humans and mice. In a preferred embodiment, a relatively high amount of alum (compared with currently licensed alum-adjuvanted vaccines) is used to ensure complete binding of antigen, as well as binding of at least a portion of the total CpG in the formulation. In this regard, the ratio of alunrCpG affects the amount of “free” desorbed CpG, i.e., the CpG which is not bound to alum and / or antigen in the vaccine composition. In a preferred embodiment, the amount of free (unbound) CpG in the vaccine composition is greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, preferably about 70% to 95%, most preferably about 80% to 90%, e.g. by weight (based on the total weight of CpG in the vaccine composition). In particular, the alunrCpG ratio should facilitate a majority of the CpG content as “free” (desorbed) CpG; i.e., the CpG is not bound to components of the vaccine such that it remains in a depot. In a preferred embodiment, the amount of free CpG versus bound CpG is greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 85%, greater than 90%, preferably greater than between 70 to 90%, especially between about 80 and 90%, e.g. by weight (i.e. the amount of free CpG by weight based on the total weight of CpG in the vaccine composition). In one embodiment, the amount of CpG in the composition of the current invention is between about 0.25 and 6 mg / mL, between about 0.5 and 3 mg / mL, between about 1 and 3 mg / mL, especially between about 1.5 to 2.5 mg / mL, most preferably about 2 mg / mL, i.e., 1 mg / dose. In an especially preferred embodiment, the vaccine composition of the current invention is adjuvanted with 1 mg / mL A13+and 2 mg / mL CpG 1018; i.e., 0.5 mg A13+and 1 mg / mL CpG 1018 per dose.

[0055] In one embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients. Such excipients may include sugars, such as e.g. glucose, sorbitol or sucrose; buffer components, such as e.g., potassium and / or sodium salts; buffer systems such as e.g. HEPES, phosphate buffered saline (PBS) or Dulbecco’s PBS (DPBS) with or without calcium and magnesium; amino acids, such as e.g., L-methionine; and / or stabilizers, such as proteins, e.g., human serum albumin. In a preferred embodiment, the HSA is a recombinant HSA (rHSA). In one embodiment, the pharmaceutically acceptable excipients essentially consist of sucrose, potassium phosphate and sodium citrate and, optionally, magnesium chloride, D-sorbitol, L-methionine and recombinant human serum albumin (rHSA). In a further embodiment, said pharmaceutically acceptable excipients essentially consist of about 5% (w / v) sucrose, about 10 mM potassium phosphate, about 25 mM sodium citrate and about 0.01% (w / v) recombinant human serum albumin (rHSA). In a preferred embodiment, the pharmaceutically acceptable excipients essentially consist of about 5% (w / v) sucrose; about 5 mM potassium phosphate; about 25 mM sodium citrate; about 5 mM MgC12; about 0.5 % (w / v) D-sorbitol; about 10 mM L-methionine; and about 0.01% (w / v) recombinant human serum albumin (rHSA). As used herein “recombinant human serum albumin” is used interchangeably with “recombinant serum albumin” (rHA). Neither of these products is derived from human sera, but is instead produced recombinantly.

[0056] In some embodiments, the purification of the Zika virus is carried out using protamine sulphate (PS) precipitation followed by sucrose gradient centrifugation as previously set forth in WO2017 / 109225, WO2017 / 109228, WO2017 / 109227 (included herein by reference in their entirety). Therefore, in a preferred embodiment, the pharmaceutical composition of the current disclosure contains protamine sulphate at amounts too low to detect by HPLC, i.e., below 1 pg / mL, below 100 ng / mL, below 10 ng / mL, or below 1 ng / mL. Because only very low levels of protamine sulphate remain after purification by this method, in a preferred embodiment, protamine sulphate may be detected in the pharmaceutical composition by mass spectroscopy or another sensitive method. In some embodiments, the sucrose gradient pool is subjected to a further purification step which results in a final formulation virtually devoid of residual protamine sulphate; i.e., below detectable levels in any standard assay. In some embodiments, the further purification step is an ultra / diafiltration step following collection of the sucrose gradient pool (SGP). In a preferred embodiment, the ultra / diafiltration step is performed using a hollow fiber membrane having a molecular weight cut-off of equal to or less than 1000 kDa. In a preferred embodiment, the hollow fiber membrane has a molecular weight cut-off of about 300 kDa, or about 100 kDa. In one embodiment, a process for producing the pharmaceutical compositions of the current invention is provided. In one embodiment, the process comprises the steps of a) propagating a Zika virus in a bioreactor; b) harvesting the Zika virus to obtain a Zika virus composition (a); c) inactivating Zika virus composition (a) to obtain a Zika virus composition (b); d) subjecting Zika virus composition (b) to one or more further purification steps to obtain a Zika virus composition (c); and e) combining Zika virus composition (c) with i) one or more pharmaceutically acceptable carriers or excipients, ii) an aluminium salt adjuvant, and iii) at least one additional adjuvant, wherein said at least one additional adjuvant is a toll-like receptor (TLR) agonist. In a preferred embodiment, the specific Zika virus, the mode of propagation of Zika virus in a bioreactor, the harvest and / or inactivation steps, the further purification steps and the carriers, excipients and / or adjuvants to be combined with the inactivated Zika virus are selected from those provided herein.

[0057] In further embodiments, the pharmaceutical composition according to the invention is delivered to a subject in a priming series followed by one or more boosters. As used herein, the priming series consists of at least one dose of the inactivated Zika virus vaccine. In one embodiment, the priming series consists of at least two doses of the inactivated Zika virus vaccine. In one embodiment, the priming series consists of at least three doses of the inactivated Zika virus vaccine. In one embodiment, the priming series consists of a first dose followed by a second dose administered at least 14 days, at least 28 days, at least 60 days, at least 70 days, at least 80 days or 90 days, at least 6 months or at least 9 months after the first dose, especially at least 28 days, at least 60 days, at least 70 days, at least 80 days or 90 days after the first dose. Thus, in some embodiments, the second dose of the inactivated Zika virus vaccine is administered 30 to 120 days or 1 to 4 months (preferably about 3 months, especially about 1 or 2 months) after the first dose, especially wherein the first two doses of the priming series are administered 1 to 4 weeks apart. In some embodiments, the priming series optionally also comprises a third dose. In one embodiment, the optional third dose of the priming series is administered between 6 and 12 months after the first dose.

[0058] In a preferred embodiment, the priming series is followed by one or more booster shots. In one embodiment, the one or more boosters are provided at regular intervals, such as every one to twenty years, especially every 2, every 3, every 4, every 5 years or every 10 years following the priming series. In a preferred embodiment, all doses are administered in a volume of 0.45 mb or 0.5 mb. The pharmaceutical composition as disclosed herein may be administered to the subject orally or by a parenteral route selected from the group consisting of subcutaneous, intracutaneous, intradermal, intravenous, intramuscular, intraarticular, intraperitoneal, intrathecal or by infusion. In some embodiments, the BPL inactivated Zika vims pharmaceutical composition is administered to a subject once as a priming series; i.e., the first dose or series of doses which primes the subject for an anamnestic response to a later delivered booster. In a preferred embodiment, the priming series of the BPL inactivated Zika vims pharmaceutical composition comprises administration to the subject more than once, preferably two times, e.g., at an interval of between one and four weeks. For example, in one embodiment, the priming series comprises administration of the pharmaceutical composition once on day 0 and once again at about day 7. In some embodiments, the priming series comprises administration of the pharmaceutical composition once on day 0 and once again at about day 14. In some embodiments, the priming series comprises administration of the pharmaceutical composition once on day 0 and once at about day 21. In some embodiments, the priming series comprises administration of the pharmaceutical composition once on day 0 and once again at about day 28. In a preferred embodiment, the priming series comprises administration of the pharmaceutical composition on day 0 and day 28. In another preferred embodiment, the priming series comprises administration of the pharmaceutical composition on day 0 and day 14. In some embodiments, the priming series comprises administration of the pharmaceutical composition three times, e.g., once on day 0, once again on about day 7 to 28, and once again at about month 3 to 24, preferably at month 6 to 12.

[0059] In further embodiments, a further dose or doses of the BPL inactivated Zika virus pharmaceutical composition is administered after the priming series as described above, referred to herein as a “booster” dose or doses. In one embodiment, the booster dose of the BPL inactivated Zika virus pharmaceutical composition is administered at least 6 months, at least one year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, at least 15 years, at least 20 years after the first dose of the priming series. In some embodiments, the booster dose of the BPL inactivated Zika virus pharmaceutical composition is administered 6 months to 2 years (preferably about 1 year) after the priming series.

[0060] In one embodiment, a booster dose of the BPL inactivated Zika virus pharmaceutical composition is administered at regular intervals (“regular booster”) following the priming series and a first booster dose. In one embodiment, the regular booster is administered every year, every 2 years, every 3 years, every 4 years, every 5 years, every 6 years, every 7 years, every 8 years, every 9 years, every 10 years, throughout the life of the subject or as long as the subject requires protection from Zika virus infection or disease. In a preferred embodiment, the regular booster is administered every 3 to 10 years, especially every 5 years. As used herein, the administration of the pharmaceutical composition, whether as a single dose or part of a series, is referred to as “vaccination” and / or “immunization”, and a subjecting receiving one or more doses may be referred to as “vaccinated” and / or “immunized”. In a further preferred embodiment, the pharmaceutical composition according to the disclosure is a vaccine. Also within the scope of the present disclosure are kits for use in prophylactic administration to a subject, for example to prevent or reduce the severity of Zika virus infection. Such kits can include one or more containers comprising a composition containing inactivated Zika virus, such as a BPL inactivated Zika virus pharmaceutical composition according to the current disclosure. In some embodiments, the kit may further include one or more additional components comprising a second composition, such as a second adjuvant for addition to the Zika virus composition and / or a second vaccine. In some embodiments, the second vaccine is a Japanese encephalitis virus vaccine, a tick borne encephalitis (TBE) vaccine, a Human Papilloma virus (HPV) vaccine, a Dengue virus vaccine, a Yellow fever virus vaccine and / or a Chikungunya virus vaccine.

[0061] In some embodiments, the kit comprises instructions for use in accordance with any of the methods described herein. The included instructions may comprise a description of administration of the composition containing inactivated Zika virus vaccine to prevent, delay the onset of, or reduce the severity of Zika vims infection. The kit may further comprise a description of selecting a subject suitable for administration based on identifying whether that subject is at risk for exposure to Zika vims or contracting a Zika vims infection. In still other embodiments, the instructions comprise a description of administering a composition containing inactivated Zika vims to a subject at risk of exposure to Zika vims or contracting Zika vims infection.

[0062] The instructions relating to the use of the composition containing inactivated Zika vims generally include information as to the dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instmctions are also acceptable.

[0063] The kits of the present disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for use in combination with a specific device, such as a syringe or an infusion device. The container may have a sterile access port, for example the container may be a vial having a stopper pierceable by a hypodermic injection needle. At least one active agent in the composition is an inactivated Zika vims, as described herein.

[0064] In one embodiment, the pharmaceutical composition according to the current disclosure is administered to the subject orally or by a parenteral route, including subcutaneous, intracutaneous, intradermal, intravenous, intramuscular, intraarticular, intraperitoneal, intrathecal or by infusion. In a preferred embodiment, the pharmaceutical composition is administered subcutaneously or intramuscularly, most preferably intramuscularly.

[0065] In one embodiment the subject receiving the pharmaceutical composition of the disclosure is a human subject. In a preferred embodiment, the subject is a resident of or is travelling to an epidemic or endemic region. A subject living in or traveling to an epidemic or endemic region, or a region that is susceptible to outbreaks, has a higher risk of becoming infected by Zika virus and / or for suffering a Zika virus associated clinical illness of any severity. In a further preferred embodiment, the subject is 15 to 49 years old. In another embodiment, the subject is a child, i.e. from 5 to 18 years old. In another embodiment, the subject is an infant or young child, i.e., from 0 to 5 years old. In yet another preferred embodiment, the subject is a woman of childbearing potential. In certain embodiments, the woman of childbearing potential may be pregnant or planning to become pregnant at any time during the course of the priming series, such as at the administration of the first dose, the second dose or any eventual third dose. The woman of childbearing potential who is pregnant or planning to become pregnant may also, in one embodiment, receive one or more booster doses of the pharmaceutical composition.

[0066] In one embodiment, the pharmaceutical composition of the disclosure is able to increase serum antibody titers to Zika virus in a human by at least 1 log relative to a control within about 7 days from booster immunization. In a further embodiment, the pharmaceutical composition of the disclosure is able to increase serum antibody titers to Zika virus in a human by at least 1 log relative to a control within about 14 days from booster immunization. In one embodiment, the serum antibody titers are measured in an ELISA assay. In a preferred embodiment, the serum antibody titers are neutralizing antibody titers as measured by a Plaque Reduction Neutralizing test (PRNT) or a micro-neutralizing test (pNT), such as the assays described herein. As used herein, a booster immunization can mean the second dose in a two-dose priming series and / or any further dose administered after the two-dose priming series. Alternatively, a booster immunization can mean the third dose in a three-dose priming series and / or any further dose administered after the three-dose priming series.

[0067] In one embodiment, the pharmaceutical composition of the disclosure is able to stimulate seroconversion in at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to 100% of vaccinated subjects within 7 days of booster vaccination, wherein seroconversion is defined as reaching a neutralizing Zika virus antibody titer of at least 10, preferably at least 20. In some embodiments, seroconversion can be defined as reaching a 2-fold to 10-fold or higher neutralizing Zika virus antibody titer when compared with baseline levels, such as a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold or higher neutralizing Zika virus antibody titer when compared with baseline levels. In a preferred embodiment, seroconversion is defined as reaching a 4-fold or higher neutralizing Zika virus antibody titer when compared with baseline levels. In one embodiment, the pharmaceutical composition of the disclosure is able to stimulate seroconversion in at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to 100% of vaccinated subjects within 14 days of booster vaccination, wherein seroconversion is defined as reaching a neutralizing Zika virus antibody titer of at least 10, preferably at least 20. In a preferred embodiment, seroconversion is defined as reaching a 4-fold or higher neutralizing Zika virus antibody titer when compared with baseline levels. Baseline levels, as used herein, refers to the levels of antibodies present directly before administration of the booster vaccination. In one embodiment, the neutralizing Zika virus antibody titers are measured by a Plaque Reduction Neutralizing test (PRNT), such as e.g. the PRNT assay as described herein. In a preferred embodiment, the neutralizing Zika virus antibody titers are measured by a micro-neutralizing test (pNT), such as e.g. the pNT assay as described herein.

[0068] In one embodiment, the pharmaceutical composition of the disclosure is able to stimulate seroconversion in at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to 100% of vaccinated subjects within 7 days of booster vaccination, wherein seroconversion is defined as reaching a neutralizing Zika virus antibody titer of at least 10, preferably at least 20. In one embodiment, the pharmaceutical composition of the disclosure is able to stimulate seroconversion in at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to 100% of vaccinated subjects within 14 days of booster vaccination, wherein seroconversion is defined as reaching a neutralizing Zika virus antibody titer of at least 10, preferably at least 20. In a preferred embodiment, the neutralizing Zika virus antibody titers are measured by a Plaque Reduction Neutralizing test (PRNT) or a micro-neutralizing test (pNT), such as the assays described herein.

[0069] An immune response to a second or further exposure to an antigen or vaccine is referred to as an “anamnestic response”. Seroconversion is defined as the state of having sufficient specific circulating antibodies to block infection or to greatly reduce the severity of a Zika virus associated clinical illness. An immune response which stimulates seroconversion as used herein is also referred to as a “protective” immune response. In one embodiment, the seroconversion or protective immune response lasts for at least 6 months. In one embodiment, the seroconversion or protective immune response lasts for at least 12 months. In a preferred embodiment, the seroconversion or protective immune response lasts for at least 24 months. In a preferred embodiment, the seroconversion or protective immune response lasts for at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, at least 20 years. In one embodiment, the seroconversion or protective immune response lasts for at least one pregnancy duration; such as at least about 12 months, i.e., a period sufficient for conception and pregnancy.

[0070] In one aspect, the pharmaceutical compositions as disclosed herein are suitable for use in a method of treating or preventing a Zika virus infection and / or a Zika virus associated clinical illness of any severity.

[0071] In one aspect is provided a method of treating or preventing a Zika virus infection in a subject in need thereof, wherein the method comprising administering an effective amount of any one of the pharmaceutical compositions according to the current disclosure. In a preferred aspect, the pharmaceutical composition as disclosed herein is a vaccine or vaccine composition.

[0072] In one embodiment, the current disclosure provides a method for Zika virus inactivation with betapropiolactone (BPL). In one embodiment, the method comprises contacting the Zika virus with BPL for longer than is required to completely inactivate the Zika virus as measured by plaque assay. In one embodiment, the Zika virus is contacted with BPL for a time period of between 6 and 48 hours, such as 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours, preferably for about 30±2 hours, especially about 30 hours. In a preferred embodiment, the BPL inactivation is performed at a temperature of between 4°C and 10°C; i.e. a temperature of 7°C ± 3°C. In one embodiment, a buffer solution is added to stabilize the Zika virus during inactivation. In a preferred embodiment, the buffer solution comprises HEPES.

[0073] In one embodiment, BPL is added only once during the inactivation period, i.e., at the beginning of the inactivation period. In a preferred embodiment, BPL is added twice during the inactivation period. In particular, it is preferred that BPL is added at the beginning of the inactivation period (first addition) and again after about 8 to 10 hours of incubation (second addition), preferably after 9 hours of incubation. In one embodiment, the first addition of BPL is added at a concentration of about 400 to 600 ppm, preferably a concentration of 500 ppm. In one embodiment, the second addition of BPL is added at a concentration of about 100 to 300 ppm, preferably at a concentration of 200 ppm. In an additional embodiment, the Zika virus is optionally concurrently transferred to another vessel directly after one or both additions of BPL to avoid any areas of low BPL concentration. In particular, the Zika virus is transferred to a second vessel following the addition of BPL at the beginning of the inactivation period and / or to a third vessel wherein the composition is optionally concurrently transferred to a second vessel. Preferably the transfer to another vessel is performed at both BPL addition timepoints. Concurrent transfer as used herein can be construed as anywhere from simultaneously with the addition of BPL to within about 5, 10 or 15 minutes of the addition of BPL. In one embodiment, the optional transfer to a third vessel is performed via a fdter, preferably a 0.45 / 0.2 pm filter.

[0074] In a preferred embodiment, the BPL concentration does not exceed 600 ppm at any time during the course of the inactivation period. In one embodiment, the BPL is hydrolyzed following the inactivation period. In one embodiment, the hydrolyzation is achieved by elevating the temperature of the solution. In a preferred embodiment, the temperature of the solution is elevated to 35°C ± 2°C until the BPL is hydrolyzed. In a preferred embodiment, the temperature is elevated for a period of 2.5 ± 0.5 hours. In one embodiment, the solution is cooled to 5 ± 3°C for storage following BPL hydrolysis. In one embodiment, the inactivated Zika virus may be stored at 5 ± 3 °C for up to 25 days before further processing, e.g., packaging.

[0075] In a preferred embodiment, BPL inactivation of Zika virus is performed as follows:

[0076] Phase I: Virus inactivation with BPL Prior to inactivation concentrated harvest is supplemented with 25 mM HEPES (e.g. 1 M stock solution, pH 7.2 - 7.5, ThermoFisher Scientific) to stabilize the pH during hydrolysis of BPL to propionic acid. BPL is added to the 7 ± 3°C pre-cooled virus concentrate inside the 1st vessel (e.g. PETG bottles) at a final concentration of 500 ppm BPL (0.05%) and mixed for 5 min at ~80 rpm. The suspension is then transferred to the 2nd vessel and static incubated at 7 ± 3°C. After 8 - 9 h, 200 ppm BPL (0.02%) is added to the 2ndvessel and, after initial mixing, the solution is transferred to the 3rd vessel. To remove aggregates that could have formed during the initial inactivation, a 0.45 / 0.2 pm filter is included in this 2nd transfer step. The inactivation is considered to start after the initial transfer is completed. After 30 h the inactivation Phase I is completed and Phase II is started immediately.

[0077] Phase II: Hydrolysis of remaining BPL The inactivated viral solution (IVS) is warmed by transferring the vessel containing the IVS to a temperature controlled waterbath or cryostat set to 35°C. Phase II begins when the suspension has reached a temperature of 32°C and continues for 2.5 ± 0.5 h. After 2.5 h the inactivation vessel is transferred to a temperature-controlled fridge set to 5 ± 3°C and Phase III begins.

[0078] Phase III: Cooling and storage of IVS The IVS is cooled to a temperature of 5 ± 3 °C and stored until proof of successful inactivation is completed, such as up to 10 days or up to 25 days.

[0079] Also provided in the current disclosure is a pharmaceutical composition comprising a BPL inactivated Zika virus, wherein said Zika virus is inactivated by the methods disclosed herein. In one embodiment, the pharmaceutical composition is suitable for use in a method of treating or preventing a Zika virus infection and / or a Zika virus associated clinical illness of any severity.

[0080] In a further aspect is provided a method of treating or preventing a Zika virus infection in a subject in need thereof, wherein such method comprises administering an effective amount of pharmaceutical composition comprising i) an inactivated Zika virus, ii) an aluminium salt adjuvant and iii) at least one additional adjuvant, wherein the at least one additional adjuvant is a toll-like receptor (TLR) agonist. In a preferred embodiment, the method treats or prevents a Zika virus infection and / or a Zika virus associated clinical illness of any severity.

[0081] As used herein, the term “bioreactor” refers to a device that supports a biologically active environment in which a biological process such as propagation of virus and vectors under controlled conditions may be carried out. Bioreactors may be designed for small-scale cultures such as those used in research laboratories, as well as large-scale bioreactors comprising vessels or vats to produce and harvest biological macromolecules such as vaccine virus, antigens, and vectors on a pilot plant or commercial scale. A bioreactor may be used to propagate both suspended and adherent cells. The bioreactor is a controlled environment wherein the dissolved oxygen (DO), nitrogen, carbon dioxide, and pH levels may be adjusted. Parameters such as DO, pH, temperature, and biomass are measured at periodic intervals.

[0082] A “fixed-bed bioreactor” (FBBR) means a type of bioreactor which includes a fixed-bed of packing material that promotes cell adhesion and growth. Fixed-bed bioreactors have been used to produce viral vaccine products at both small and large scale due to the ability to perfuse high cell densities with low shear force. The fixed-bed bioreactor may be a single-use bioreactor such as e.g. the commercially available iCELLis system (Pall Corporation). The iCELLis system platform offers a novel fixed-bed technology comprising carriers composed of woven medical-grade polyethylene terephthalate (PET) fibers in a robust, single, closed system that does not require any aseptic handling. Additionally, this system incorporates high rates of gas exchange using “waterfall” or “falling film” technology through the control of temperature, O2, pH, carbon dioxide (CO2), and nitrogen (N2), in addition, the use of a magnetic impeller that produces low cell shear stress and evenly distributed media circulation. For most viruses, production titers from the iCELLis system are significantly increased when compared to classical adherent cell flat-stock flasks. The iCELLis technology may be used at small-scale such as in the iCELLis Nano, where the growing area is between 0.5 to 4 m2and manufacturing scale, such as in iCELLis 500 where the growing area ranges from 66 to 500 m2. Processes developed in the small-scale system may be scaled up to that of the manufacturing scale. A fixed-bed bioreactor may have sensors that measure and monitor the pH, temperature, dissolved oxygen, and the biomass, which indicates adherent cell density. A fixed-bed bioreactor may also have different ports that enable the addition of oxygen or nitrogen, a media exchange port, ports for the addition of sodium hydroxide (NaOH) and / or CO2 to adjust the pH.

[0083] The host cells of the disclosed method may be cultivated on microcarriers, which may be in suspension in bioreactors or on microcarrier strips. Preferably, the host cells are cultivated on microcarrier strips or other carriers in a fixed bed of a fixed-bed bioreactor. Preferably, the fixed-bed bioreactor is a commercially available iCELLis Nano (Pall Corporation), iCELLis 500 bioreactor (Pall Corporation), or a Univercells fixed-bed bioreactor (Univercells SA). The fixed-bed may provide a maximum of 40,000 cm2in an 800mL fixed-bed bioreactor such as the iCELLis Nano, and up to 5,000,000 cm2in a 25L or more fixed-bed bioreactor such as iCELLis 500 (Fig. IE). The fixed- bed height may range from 10 to 20 mm, providing a growth area of 5300 cm2to 40,000 cm2in an 800 mL fixed-bed bioreactor and 660,000 cm2to 5,000,000 cm2in a 25L or more fixed-bed bioreactor.

[0084] Host cells may be cultivated by using a seeding density ranging from 2000 to 20,000 cells per cm2. The seeding density may be adjusted based on the type of host cell, the volume of the bioreactor, the height of fixed-bed in a fixed-bed bioreactor, etc. It is within the knowledge of one skilled in the art to select the optimum seeding density for the process.

[0085] As used herein, “culture media”, “media” or “cell culture medium” refers to a liquid used to culture the host cells in the bioreactor. The media used in the procedure of the disclosure may include various ingredients that support the growth of the host cells, including but not limited to amino acids, vitamins, organic and inorganic salts, carbohydrates. The media may be serum-free media, which is media formulated without any serum, such as e.g. animal serum. A serum -free medium when used may be selected from, but not limited to, DMEM, DMEM / F12, Medium 199, MEM, RPMI, OptiPRO-SFM, VP-SFM, VP-SFM AGT, HyQ PF-Vero, MP-Vero, most preferably OptiPRO-SFM. The culture media may also be animal-free media; that is, not containing any product of animal origin. The culture media may also be protein-free media; that is, media is formulated without added proteins. The serum -free or protein-free media may be formulated without serum or protein but may contain cellular protein derived from the host cells, and optionally proteins specifically added to the serum-free or the protein-free media.

[0086] The pH for cultivation can be, for example, between 6.5-7.5, depending on the pH stability of the host cells. Preferably the cells are cultivated at a pH of 7.4. The host cells may be cultivated at a temperature of between 20 and 40°C, specifically between 30 and 40°C, and preferably between 35 and 37°C for mammalian cells, especially at 35°C or 37°C. Preferably, the host cells are expanded, i.e., increased in number in preparation for infection, at 37°C. In a preferred embodiment, the temperature is lowered to and maintained at 35°C for virus infection and propagation phases. As used herein, (virus) “production” and “propagation” are used interchangeably.

[0087] The host cell or host cell line or cells used for the cultivation of virus in the method of the disclosure may be any eukaryotic cell that is suitable for the production of virus antigen, viral vector, or virus production. Preferably the host cell may be an “adherent cell” or an “anchorage-dependent cell”. Adherent cells are cells that adhere to a surface in culture condition, anchorage may be required for their growth, and they may also be called anchorage -dependent cells. Adherent cells suitable for the process of the disclosure include but are not limited to Vero cells, MBCK cells, MDBK cells, MRC-5 cells, BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, murine cells, human cells, avian cells, insect cells, HeLa cells, HEK-293 cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK 15 cells, Wl-38 cells, T-FLY cells, BHK cells, SP2 / 0 cells, NSO cells, NTCT cells, and PerC6 cells, 3T3 cells, or a combination, derivative or modification thereof. The preferred adherent cell is an anchorage -dependent cell that may be grown on a carrier such as e.g. a PET strip, but suspension cells that may be adapted to grow as adherent cells may also be used. More preferably, the anchorage-dependent cells of the disclosure are Vero cells or a derivative or modification thereof. It is within the knowledge of one skilled in the art to select an adherent host cell suitable for use in the process of the disclosure.

[0088] Aspects of the invention provide processes for producing Zika viral particles in host cells in a bioreactor system that uses a falling film for medium oxygenation, characterized in that shear stress and / or medium foaming are minimized by maintaining the Falling Film Height (FFH) at the lowest possible level while still maintaining required dissolved oxygen (DO) saturation levels at the respective stages of the process, thereby minimizing damage to virus particles compared with more turbulent incubation conditions. As used herein, the Falling Film Height is defined as the vertical height difference between the top of the falling film and the bottom of the falling film, i.e., the surface of the cell culture medium. The falling film is a result of the continuous circulation of the cell culture medium through the bioreactor.

[0089] In bioprocesses, the dissolved oxygen (DO) concentration of the cell culture medium in the bioreactor (also referred to herein as “DO”, “(oxygen) saturation” or “percent (oxygen) saturation”) is a key parameter to obtain optimal cell growth and product yield. To maintain dissolved oxygen (DO) saturation in a fixed bed bioreactor utilizing an adjustable falling film height (FFH), an interaction of falling film height and stirrer speed (regulating the linear speed of media through the fixed-bed) is important. This interplay is important in different types of fixed bed bioreactors, such as e.g., wherein the bioreactor comprises a fixed bed provided in a spiral shape wherein the medium recirculates through the bed, such as the bioreactor provided in Figs 11A and 11B, wherein the bioreactor provides a fixed bed embedded in a chamber through which the medium circulates such as the bioreactor shown in Fig. 11C; a bioreactor comprising a flask, wherein the medium is pumped out through a dip tube and returned to the chamber in a waterfall such as shown in Fig. 11D; and a bioreactor wherein the FFH is adjusted by varying the weight of the medium introduced into the system such as shown in Fig. HE. This also applies to the iCELLis Nano development system for which however the option to change the falling film height is not given, since it is fixed by design to 3 cm. The goal is to find a balance between maintaining DO levels at a set point and to allow sufficient media recirculation through the fixed-bed to ensure adequate nutrient supply and oxygen to the cells but at the same time introducing as little energy as possible into the system to avoid shear stress and extensive media foaming, both of which should be avoided due to potential negative impact on cell growth and product loss. It is well known in the field that bioreactor products such as proteins and virus particles can start to unfold at gas-liquid interfaces, resulting in aggregation and ultimately product loss. Protein denaturation in foam has been extensively studied and shown to result in unfavorable conformational changes of biomolecules (Protein Denaturation in Foam: Surface Activity and Conformational Change. Clarkson et al. (1999) Journal of Colloid and Interface Science 215:333- 338; Protein Denaturation by Combined Effect of Shear and Air-Liquid Interface. Yuh-Fun Maa et al. (1997) Biotechnology and Bioengineering 54(6):503-512). It is therefore important to control gasliquid interfaces and foaming in bioreactors.

[0090] The level of efficiency of oxygen transfer in the bioreactor medium to regulate DO is dependent on the oxygen transfer rate (OTR) from the gaseous phase to the liquid phase, which amongst others is affected by the volumetric mass transfer coefficient kLa. The kLa describes the general aeration capacity of a bioreactor system and can be influenced by several process parameters. This value reflects how efficiently a cultivation system can introduce oxygen to the liquid phase which is in direct contact with the cultivated cells. kLa values are system specific and can change rapidly, depending upon the process parameters applied. In general, the greatest impact on kLa values is achieved by adjusting gas flow rates and / or stirrer speeds. The special design of bioreactors utilizing a falling film or waterfall type of oxygenation, such as e.g. the iCELLis 500 bioreactor as shown in Fig. 11E, however, allows for an additional modification by modulating the falling film height (FFH). The FFH is generally determined by the media level in the bioreactor which in turn can be adjusted by controlling its weight in recirculation mode. The falling film is created by the media circulation inside the bioreactor, which is achieved by a built-in magnetic drive impeller located in the center of the bioreactor. The impeller creates a flow of cell culture media through the fixed-bed from the bottom to the top, where the medium falls as a thin film down the outer wall (falling film) and oxygen transfer from gaseous phase to the media takes place, maintaining high kLa in the bioreactor (see e.g., Fig. HE).

[0091] In a stirred-tank bioreactor (i.e., a fixed-bed bioreactor with a magnetic stir bar or impeller, such as e.g., the iCELLis 500, scale-X and other bioreactors), both the stirrer and the application of a thin film of falling medium (falling film) at the top of the fixed bed are important components for gas exchange (Evaluation of the Single-Use Fixed-Bed Bioreactors in Scalable Virus Production. Lesch et al. (2021) Biotechnology Journal 16, 2000020). These two variables may be adjusted to vary gas exchange in the system; i.e., increase or reduce stirrer speed and adjust the magnitude of the falling film. In addition, the speed of air flow to the headspace of the bioreactor influences gas exchange. In the iCELLis 500, for example, it has been shown that a 6 cm falling film and a linear speed of 0.6 cm / sec corresponds to a kLa value of ~2.4 h1which can be significantly enhanced (nearly doubled) by increasing the linear speed to 1.0 cm / sec (kLa value of ~4.2 h1). Although increasing the falling film height and / or stirrer speed can greatly increase the kLa in a bioreactor, the increase can also lead to the formation of foam and turbulent flow which not only reduces the efficiency of gas transfer, but may also damage nascent viral particles in the system.

[0092] “Gas flow” and “oxygen (O2) flow” are used interchangeably herein; however, it should be noted that some fixed bed bioreactors enable the adjustment (increase or decrease) of the percentage of oxygen comprised in the gas flow, depending on the DO of the medium. This adjustment may be automatic, as part of the bioreactor’s controls, or may be performed manually. This dynamic oxygen percentage parameter is part of the iCELLis 500 bioreactor which is used in the Examples herein. Therefore, a particular gas flow setting does not indicate a constant O2 percentage flow rate, but a dynamic O2 percentage flow rate based on the DO of the cell culture medium.

[0093] In some embodiments, the process of the invention comprises at least the steps of (i) a virus infection phase with a dissolved oxygen (DO) level; i.e. percent oxygen saturation, of no less than 10%, preferably no less than 15%, and (ii) a virus production phase wherein the DO saturation level is maintained at 30% or higher, preferably at 40% or higher, most preferably at 50%, or around 50%, i.e. at between 45% to 55%. In some embodiments the Falling Film Height (FFH) is maintained at the lowest possible level required for adequate dissolved oxygen (DO) levels at the respective stages of the process. By “adequate dissolved oxygen (DO) levels” it is typically meant the DO levels mentioned in steps (i) and (ii) above. By “the lowest possible level” it is typically meant that the FFH is maintained at a minimum level (e.g. zero in step (i), or 2 cm in step (ii)), and only increased above the minimum level if the DO saturation level approaches the minimum DO level, e.g. if the DO level falls to below 15% in step (i) or below 50% in step (ii). In some embodiments, the DO saturation level in the vims infection phase is maintained at 10% or higher, e.g. at least 11%, at least 12%, at least 13%, at least 14%, or at least 15%. Preferably the DO saturation level in the vims infection phase is 10 to 30%, 10 to 20%, or 10 to 15%.

[0094] In some embodiments, the DO saturation level in the vims production phase is maintained at 40% or higher, e.g. at least 45% or at least 50%. Preferably the DO saturation level in the vims production phase is 30 to 70%, 40 to 60%, 45 to 55%, 48 to 52%, or about 50%.

[0095] Broadly, the process of producing viral particles in a bioreactor includes a host cell propagation phase, lasting for several days, such as 3 to 10 days, especially 5 to 9 days or 6 to 8 days, approximately 7 days. Following propagation of the host cells, when a favorable cell density (confluency) is reached, vims can be introduced in a vims infection phase. “Favorable cell density” as used herein means a range of cell numbers per unit area (e.g. cells / cm2), as applied to adherent cells, which range is suitable for vims infection of the cells for the purpose of vims propagation. As known by the person skilled in the art, a favorable cell density may depend on several factors, including the cell type, the medium used, the vessel for growth and the vims to be propagated, among others. Cell density may also be measured by percent confluency, meaning what percentage of the cell growth surface is covered by adherent cells. Favorable cell density may be a lesser or greater cell confluency depending on the cell type and purpose, but generally comprises confluency of about 40%, 50%, 60%, 70%, 80%, 90% or 100% confluency. In general, a favorable cell density for Vero cells as used herein means about 0.5 to 2.5 x 105cells / cm2, especially about 1.0 to 2.0 x 105cells / cm2, or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9 x 105 / cm2. The optimal cell density is generally determined empirically for any given vims and / or host cell. During the vims infection phase (i), the vims particles adhere to the host cells and are taken up. The vims infection phase generally lasts a period of a few hours, particularly about 1 to 5 hours, especially about 2 to 4 hours, or 2.5 to 3.5 hours, particularly about 2 to 3.5 hours.

[0096] Following infection of the host cells, the vims propagation phase (ii) begins. In some embodiments, the vims propagation phase (ii) has a duration of 20 to 52 hours, preferably 26 to 52 hours, more preferably 30 to 52 hours, more preferably 34 to 52 hours, more preferably 38 to 52 hours, most preferably 48 to 52 hours. The vims propagation phase is also referred to herein as vims production phase. This phase can be considered in two parts: the early and the late parts; i.e., “day 1” and “day 2” post-infection, respectively. The early part of vims propagation, lasting essentially the duration of the first day after infection or post infection (also referred to herein as “PI”), is characterized by a more active host cell metabolism than the later part, requiring relatively more nutrients and oxygen. The earlier part of the vims propagation phase is defined generally as that part of the process directly following infection of the cells up to about 26 hours post infection (PI), especially to about 18 to 30 hours PI, to about 20 to 28 hours PI, to about 22 to 26 hours PI, especially to about 24 or 26 hours PI. The later part of the virus propagation phase, lasting essentially the duration of the second day PI, is characterized by a slowing down of cell metabolism and increasing cytopathic effect (CPE) as virus production and cell density are both higher. The later part of the virus propagation phase is broadly defined as greater than 26 hours post infection (PI) until virus harvest, particularly in the time period of between about 20 to 56 hours PI, especially between about 24 to 54 hours PI, between about 25 to 52 hours PI, particularly between about 26 to 48 hours PI or 26 to 50 hours PI.

[0097] “Harvesting” or “virus harvesting” as used herein refers to the collection of the virus by collecting unclarified culture media from the host cells in the bioreactor. The harvesting of the virus may generally be performed 24 to 72 hours post-infection, preferably 35 to 65 hours post-infection, preferably 40 to 60 hours post-infection, more preferably 45 to 55 hours post-infection, even more preferably 48 to 52 hours post-infection, most preferably about 48, 49, 50, 51 or 52 hours postinfection. Some viruses may require an addition step of host cell lysis before harvest.

[0098] Following harvest, viruses of the disclosure may be quantified by methods including but not limited to plaque assays, end-point dilution assays, hemagglutination assays, bicinchoninic acid assay, or electron microscopy. Preferably, the virus may be quantified by a plaque assay method. As used herein, a plaque assay method is a method to measure the number of infectious virus particles, based on its measurement of plaque -forming units (pfu). In the plaque assay, cell monolayers are infected with a serial dilution of the virus stock solution. The infected cells release progeny virus, which in turn infect neighboring cells. The cells are lysed to produce clear regions surrounded by uninfected cells, called plaques, which are visualized using a dye. A higher sample virus titer leads to a higher number of plaques.

[0099] In some embodiments, the FFH is maintained at 0 cm (i.e., no falling film or “zero falling film”) for at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably for 100% of the duration of the virus infection phase (i). In some embodiments, the FFH is maintained at 0 cm for as long as dissolved oxygen (DO) levels are not less than 10%. In other embodiments, the FFH is maintained at 0 cm for as long as dissolved oxygen (DO) levels are not less than 15%. The DO levels in the medium during the virus infection phase according to the current invention should generally not fall below 10%, preferably not below 15%. Increased turbulence during the virus infection phase has been observed during the course of the current invention to reduce the final yield of virus antigen obtained by the process. In a preferred embodiment, therefore, the FFH is maintained at zero for the duration of the virus infection phase, in order to minimize any turbulence in the system. An example of adjustments to be made in the system to keep turbulence low and DO saturation levels adequate in the virus infection as well as virus propagation phases are summarized in Table la. Table la. Technical setting / adjustments for infection and propagation phases for virus production in a fixed-bed bioreactor.

[0100] In some embodiments, the stirrer speed of the bioreactor remains constant for the entire duration of the virus infection phase (i) and the virus propagation phase (ii). Thus, the stirrer speed is maintained at between approximately 200 and 300 rpm, more preferably between 220 and 280 rpm, especially between 233 and 263 rpm, for the duration of the virus infection phase (i) and the virus propagation phase (ii).

[0101] In some embodiments, if the DO falls below 15%, especially 14%, 13%, 12%, 11% or 10%, during the virus infection phase (i), an initial FFH of 1 cm is set until the DO levels increase to 15%. If the 1 cm FFH is not sufficient, the FFH is increased by 0.5 cm increments until a DO of at least 15%, especially about 15%, can be maintained. The FFH can therefore be increased to 1.5 cm, 2.0 cm, 2.5 cm, 3.0 cm or 3.5 cm; however, in a preferred embodiment, the FFH is not increased to more than 4 cm during the virus infection phase (i), preferably not more than 3 cm, especially not more than 2.5 cm or 2 cm.

[0102] Refer to Figure 12A for a flowchart summarizing the control strategy for the virus infection phase (i).

[0103] In some embodiments, the FFH at the beginning of the virus production phase (ii) is set at a minimum level necessary to obtain a target DO saturation level during this phase, e.g. at least 30%, at least 40% or at least 50% DO saturation. In one embodiment, the FFH at the beginning of the virus production phase (ii) is set at 2 cm and is increased by 0.5 cm increments to a maximum of 4 cm, only as a way to mitigate DO levels falling below about 50%. Having a falling fdm during the virus production phase seems not to be as critical to antigen yield optimization as during the virus infection phase; however, keeping it low is still advantageous. Therefore, in a preferred embodiment, the FFH during the virus production phase (ii) is maintained at less than 4 cm, preferably less than 3.5 cm, preferably less than 3.0 cm, preferably less than 2.5 cm, preferably less than 2 cm, most preferably about 2 to 3 cm. When the DO level starts to trend down from the 50% set point with the O2 flow (gas flow) at ~3 L / min (maximum flow) then an adjustment of the target falling film height and agitation speed set point are required. See Table 2a for details.

[0104] Table 2a. DO Control Strategy - Increase of Falling Film Height or Agitation Speed Adjustment The adjustment of the falling film height target will follow the sequence shown in the table below (from 1 to 6). The starting point depends on the current falling film height target and agitation speed e.g. if the process is currently running at sequence no. 1, adjustment will be made corresponding to sequence no. 2.

[0105] On Day 2 post-infection, the cell biomass starts to decline due to virus infecting an increasing number of cells, resulting in lower overall oxygen demand indicated by lower O2 flow rate into the bioreactor headspace. As a consequence, agitation and falling film height can slowly be decreased in 0.5 cm increments if possible, as proposed in Table 3a.

[0106] Table 3a. Day 2 DO Control Strategy - Reduction of Falling Film Height or Agitation Speed

[0107] Where the O2 flow (gas flow) has been reduced to < 2.5 L / min then an adjustment to reduce the agitation speed set point or falling film height target will be performed. The adjustment of the agitation speed or falling film height will follow the sequence shown in the table below (from 1 to 6). The starting point for the adjustment depends on the current falling film height target and agitation speed, e.g. if the process is currently running at sequence no. 2, adjustment will be made corre spending to sequence no, 3, Refer to Figure 12B (Day 1 post infection) and Figure 12C (Day 2 post infection) for flowcharts summarizing the control strategy for the virus propagation phase (ii).

[0108] In one embodiment, the bioreactor is a fixed-bed bioreactor. In one embodiment, the bioreactor comprises a fixed bed provided in a spiral shape wherein the medium recirculates through the bed, such as the bioreactor provided in Figs 11A and 11B, wherein the bioreactor provides a fixed bed embedded in a chamber through which the medium circulates such as the bioreactor shown in Fig. 11C; a bioreactor comprising a flask, wherein the medium is pumped out through a dip tube and returned to the chamber in a waterfall such as shown in Fig. 11D; a bioreactor wherein the FFH is adjusted by the weight of the medium introduced into the system such as shown in Fig. HE. In the system shown in Fig. HE, i.e., the iCELLis 500 bioreactor, the volume / weight of the medium may be varied, e.g., the addition of approximately one liter of cell culture medium results in an approximately 1 cm decrease in the FFH; conversely, the removal of approximately one liter of cell culture medium results in an approximately 1 cm increase in FFH.

[0109] In a preferred embodiment, the bioreactor is selected from a Scale-X™ bioreactor (described in Univercells Technologies Application Note entitled “scale-X™ bioreactor for viral vector production”, 2020), an iCELLis Nano bioreactor system, and an iCELLis 500 bioreactor system (described in USTR 3473 “iCELLis® 500+ Generation R Bioreactors”, published by Pall, May 2022), preferably an iCELLis 500 bioreactor system.

[0110] As used herein, “infection” or “virus infection” refers to the entry of a virus into the host cell and the subsequent replication of the virus in the cell. The infection of a host cell in the method of the disclosure may be carried out when the cells reach a specific biomass. Preferably, the cells may be infected when they reach a high growth rate, indicated by high biomass, and high conductivity as measured by the biomass sensor or confluency of the cells. The host cells are infected by at least one virus particle. As used herein, multiplicity of infection (MOI) is the average number of virus particles infecting each cell. In one embodiment, the multiplicity of infection (MOI) used to infect the host cells is about 0.001 to 1, about 0.01 to 0.1, preferably about 0.05 to about 0.5; i.e., 0.001 to 1, 0.01 to 0.1, preferably 0.05 to 0.5. In a preferred embodiment, the MOI is 0.03. The number of virus particles necessary for sufficient infection is within the knowledge of one skilled in the art.

[0111] In some embodiments, the virus is inactivated, such as by e.g., treatment with ultraviolet (UV) light and / or treatment with chemical agents. In one embodiment, the chemical agent may be formaldehyde. In another embodiment, the chemical agent may be beta-propiolactone. Preferably, the chemical inactivating step(s) are performed under mild conditions, comprising e.g., mixing the chemical viral inactivating agent and the liquid composition comprising the viral particles under conditions of laminar flow but not turbulent flow, and incubating for a time sufficient to inactivate the viral particles. The mild inactivation step is optionally performed in a flexible bioreactor bag. The mild inactivation step preferably comprises five or less container inversions during the period of inactivation. Preferably, the mixing of the chemical viral inactivating agent and the composition comprising native virus particles comprises subjecting the container to rocking, rotation, orbital shaking, or oscillation for not more than 10 minutes at not more than 10 rpm during the period of incubation.

[0112] The virus of the disclosure may be a whole or split Zika virus, a Zika virus antigen, a Zika viral vector or combination, derivative or modification thereof. In a preferred embodiment, the Zika virus is a whole virus. In a preferred embodiment, the Zika virus particle has a RNA genome corresponding to the DNA sequence provided by the nucleic acid sequence of SEQ ID NO: 2, or a variant nucleic acid sequence that is at least 88% identical the nucleic acid sequence of SEQ ID NO: 2 and able to pack a virulent Zika virus. In one embodiment, the variant sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.99% identical to SEQ ID NO: 2 and able to pack a virulent Zika virus.

[0113] The French Polynesian Zika virus strain H / PF / 2013 polyprotein gene (accession no: KJ776791.1), is a Zika virus from the Asian lineage. The genomic sequence provided by SEQ ID NO: 2 was sequenced from the H / PF / 2013 strain after passaging in the lab, whereas SEQ ID NO: 3 is the same genomic sequence which has been adapted or further adapted to passaging on vero cells and comprises two mutations compared with SEQ ID NO: 2. This sequence comprises the mutations T2923C and G4319A, corresponding to an M32I mutation in the NS2B protein and an L145P mutation in the NS1 protein, respectively. It was additionally noted that three heterogeneities were sometimes present; namely, an F135L mutation in the preM protein and two silent mutations (III in the E protein and L636L in the NS5 protein).

[0114] Therefore, in a preferred embodiment, the Zika virus particle has an RNA genome corresponding to the DNA sequence provided by the nucleic acid sequence of SEQ ID NO: 3, or a variant nucleic acid sequence that is at least 88% identical the nucleic acid sequence of SEQ ID NO: 3 and able to pack a virulent Zika virus. In one embodiment, the variant sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.99% identical to SEQ ID NO: 3 and able to pack a virulent Zika virus. In a further preferred embodiment, the Zika virus of the invention comprises a heterogeneous mixture of Zika virus particles comprising particles with a genomic sequence as defined in SEQ ID NO: 3 or a variant thereof as defined above, optionally comprising one or more mutations selected from a mutation resulting in an F135L mutation in the preM protein, a mutation resulting in the silent mutation III in the E protein and a mutation resulting in the L636L in the NS5 protein.

[0115] In one embodiment, provided is a pharmaceutical composition obtained or obtainable by the process according to the current disclosure. In one embodiment, the pharmaceutical composition provided by the current invention is a vaccine. In one embodiment, the pharmaceutical composition or vaccine provided in the current invention is provided in lyophilized form. Lyophilized (i.e., freeze-dried) pharmaceutical compositions or vaccines are typically more stable than liquid formulations and can be stored at 2-8° C for a predefined length of time.

[0116] Any of the pharmaceutical compositions or vaccines provided in the current invention may further comprise an adjuvant and / or a pharmaceutically acceptable excipient. Excipients include substances such as buffers, stabilizers or further active ingredients, especially ingredients known in connection with pharmaceutical compositions and / or vaccine production. In particular, the compositions or vaccines described herein may be administered to a subject with, prior to, or after administration of one or more adjuvants. An adjuvant is a molecule or formulation that enhances a response in a subject, such as an immune response, to an antigen or other molecule. In some embodiments, an adjuvant may stabilize an antigen or other molecule. Determining whether a virus vaccine or pharmaceutical composition is administered with an adjuvant depends on various factors (e.g., type and extent of response desired) and will be evident to one of skill in the art.

[0117] The term “preventing” can also be interpreted herein as “protecting from”. Any of the methods or uses described herein may be used for the prevention or treatment of a Zika virus infection in a subject. As used herein, the terms “prevent,” “preventing” and “protecting from” include the administration of a virus vaccine or composition to a subject to reduce or delay the onset of the manifestation of clinical or subclinical symptoms, complications, pathologies or biochemical indicia of a disease or infection, or to reduce or inhibit the spread / transmission of a virus; i.e., to treat or prevent a Zika virus infection and / or a Zika vims associated clinical illness of any severity. As used herein, antigen(s), such as an inactivated, live attenuated or vector vims provided by the processes described herein, that is administered to a subject prophylactically (e.g., prior to infection) may be referred to as a vaccine.

[0118] The current invention is not limited in its application to the details of construction and the arrangement of components set forth in the description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including”, “comprising”, or “having”, “containing”, “involving”, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0119] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, virology, cell or tissue culture, genetics and protein and nucleic acid chemistry described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0120] The present invention is further illustrated by the following examples, which in no way should be construed as further limiting. The entire contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference, in particular for the teaching that is referenced hereinabove. However, the citation of any reference is not intended to be an admission that the reference is prior art.

[0121] EXAMPLES

[0122] Example la: A process for propagating infectious Zika virus

[0123] Influence of duration and amplitude of falling film during virus infection phase (i) and virus propagation phase (ii) on viral antigen yield across two iCELLis 500 Zika virus production batches

[0124] During the course of the invention, two batches of Zika virus (ZIKV) were produced in an iCELLis 500 bioreactor on Vero cells under varying conditions (see summary in Table 4a). It has been observed that the duration and / or amplitude of the Falling Film has an influence on antigen yield. The relative productivity of Zika virus batches was measured as number of viral particles by size exclusion high performance liquid chromatography (SE-HPLC) and the area under the curve was used to calculate productivity normalized to cell counts at infection [reported as mAU / 109cells] (see Table 4a).

[0125] Table 4a. Exemplary ZIKV production runs

[0126] A long zero Falling Film duration during infection phase (i) and a moderate Falling Film height during production phase (ii) for the ZVPIV00001 batch resulted in higher productivity compared to the TECH-ZVPIV01 batch. This result show that applied Falling Film conditions could be an explanation for the difference in productivity.

[0127] In summary, it was shown for ZIKV in the iCELLis 500 system that a better virus yield at harvest is expected when applying a longer time of “zero falling film” during infection phase and a lower FFH during the virus production phase. Application of both of these conditions to the greatest extent possible while still maintaining acceptable DO levels reduces mechanical stress to the small amount of active virus inoculum added into the bioreactor to infect the cells and also helps to maintain the structural integrity of the virus during the virus production phase.

[0128] Example 1: Zika virus inactivation with beta-propiolactone

[0129] General considerations Two major goals should be addressed for inactivating a virus for vaccine purposes: 1) the infectious virus must be completely inactivated in order to be safe, and 2) epitopes important for the induction of protective immunity should be conserved / unmodified after the inactivation procedure. Zika virus particles, like all flaviviruses, have an outer surface composed of an envelope (E) protein, which binds to host cell receptors and mediates virus entry. This protein has three domains EDI, EDII and EDIII, whereas the other structural proteins are generally not accessible. The Zika E protein is highly similar to those of other flaviviruses, leading to production of antibodies which can be cross-reactive with other flavivirus. Generation of these cross-reactive antibodies can provoke a phenomenon known as antibody-dependent enhancement (ADE), wherein weakly- neutralizing antibodies against one viral strain can “improve” the infectivity of another strain.

[0130] Development of a “second generation ” Zika virus production process A first generation Zika virus vaccine, developed earlier, was characterized in part by formaldehyde inactivation. One of the major differences in the second generation Zika virus vaccine process as described herein is the use of beta- propiolactone, a nucleic acid based inactivator, instead of formaldehyde, a protein based inactivator. This change was expected to result in virus particles with less modified structural surface epitopes and a concomitant increase in immunogenicity. Another change in the second generation process is that the virus particles are inactivated directly after harvesting using the PS-treated, clarified cell culture supernatant, not after purification as in the first generation process. This was adapted from a similar process developed for BPL inactivation of SARS-CoV-2 (WO2021 / 204825, incorporated by reference herein in its entirety). For comparison of the effects of formaldehyde versus BPL inactivated on Zika virus qualities, the formaldehyde inactivation process used herein was also performed on clarified Zika virus harvest.

[0131] Two drawbacks of the first generation Zika virus production included propagation of the virus in roller bottles (RB), i.e., with limited scale-up capabilities, and the usage of fetal bovine serum (FBS) in the cell culture medium. In the second-generation process disclosed herein, Vero cells were expanded under serum-free conditions using OptiPro® media in the iCELLis fixed-bed bioreactor system. This animal-component-free bioreactor process can feasibly deliver up to twice the specific productivity in terms number of virus particles produced per area growth surface compared to the RB based process with FBS-supplemented media. Furthermore, the iCellis-nano platform may be used for lab-scale experiments to optimize virus propagation conditions and the high capacity iCellis-500+ can then be used for commercial production.

[0132] Study Material The Zika virus strain H / PF / 2013 was originally isolated from a 51 -year-old woman infected in French Polynesia in 2013. This isolate was propagated on Vero cells for three passages by the European Virus Archive (EVAg). A sample of this P3 (passage 3) Zika virus material was obtained and used to generate a research master virus seed bank (rMVSB) using Vero cells as cell substrate. The active Zika virus harvest materials were produced in a lab scale fixed-bed bioreactor (iCellis-nano). All inactivation kinetic samples were stabilized 1+1 with FBS and stored immediately at <-65°C until analysis by plaque assay.

[0133] Plaque Assay A plaque assay for determining Zika virus yield and inactivation was performed according the schematic overview of the provided in Figure 1. In brief, on Day 0, a confluent monolayer of Vero cells is inoculated with positive control Zika virus (WVSB) or a negative control diluent as well as test samples at specified dilutions. Plates are incubated for 75 + 5 min at 35 ± 3°C and 5 + 1% CO2, before a first overlay of 1.8% agarose supplemented with 2x EMEM, FBS, L- Glutamine and Amphotericin B is added. Subsequently, plates are incubated for 72 h at 35 ± 3°C and 5 + 1% CO2. A second overlay of 1.0% Agarose with Neutral Red solution is added and plates are incubated for 18 - 24 h at 35 ± 3°C and 5 + 1% CO2. Plaques, which appear as clear spots on a red background, are then counted using a light box. The virus titer is calculated and expressed as pfu / mL and logic pfu / mL. If no plaques are counted in any of the kinetic samples, the titer is indicated as <4 pfu / mL (0.6 logic pfii / mL); if neat, the titer can be evaluated directly. If only the 1: 10 dilution can be assessed (e.g. early inactivation kinetic samples) and no plaques are detected, then a titer of <40 (1.6 log 10 pfu / mL) is given to the sample.

[0134] Inactivation by BPL treatment Overall the inactivation by BPL for Flaviviruses is less time consuming than for formaldehyde, as it requires in many cases <24-48 h incubation for complete inactivation (Chida et al., 2021, supra,' Chowdhury et al., Comparison of P-Propiolactone and Formalin Inactivation on Antigenicity and Immune Response of West Nile Virus. 2015. Advances in Virology; Volume 2015, Article ID 616898; https: / / doi.org / 10.1155 / 2015 / 616898). Longer incubations require multiple additions to maintain the effective BPL concentration over the whole process. BPL concentration and hydrolysis in OptiPro production medium (the sample matrix) was analyzed at different temperatures using gas-chromatography coupled with mass spectrometry (GC- MS) based on a method described by Lei et al, 2018 (https: / / doi.org / 10.1016 / j jpha.2018.06.003). The values were compared to theoretical kinetics based on published rate constants for BPL in physiological buffers. For these experiments BPL was added at a starting concentration of 500 ppm to OptiPro production media equilibrated to the different temperatures. Results are summarized in Table 1 and compared to calculated half-life based on literature data.

[0135] Table 1. Comparison of the theoretical (calculated) half-life of BPL versus the measured half-life of BPL in OptiPro Serum Free Medium (SFM) at increasing temperatures. The starting BPL concentration was 500 ppm.

[0136] The determined BPL hydrolyzation rates in the Zika vaccine product matrix were comparable to or even lower than the theoretical values (Uittenbogaard et al, 2011, J. Biol. Chem. 286:36198) indicating a suitable stability of BPL in the OptiPro media. At the end of inactivation Phase I prior to warming, about 50% of the initial BPL concentration remained. Taken together the remaining ~50% of BPL after 30 h at 2 - 8°C and the inactivation kinetic of Zika virus described before (no infectious particles detectable after ~12 h; data not shown), the amount of BPL used in the Zika inactivation process includes a large safety margin to ensure complete inactivation of the Zika virus in the product.

[0137] Analysis of Zika virus in-process samples using size exclusion chromatography A Superose 6 Increase 10 / 300 column (GE Healthcare) equilibrated with PBS +250 mM NaCl, pH 7.4 at a flow rate of 1 mL / min and operated at 25°C, was used to detect Zika virus at 214 nm detection wavelength in harvest samples and throughout the downstream process. The HPLC system used was a Thermo Vanquish HPLC System running with Chromeleon software. It is a semi -quantitative (relative yield) and qualitative (purity) method that separates intact viral particles from virus aggregates and host cell proteins (HCPs). The method cannot distinguish between infectious and non-infectious viral particles due to their same retention time. Since the analysis time is quite fast (45 min per sample), it can be used as in-process control during process transfer and production.

[0138] Analysis ofbeta-propiolactone modifications Identification of BPL modification sites in the Envelope (E) protein covering the viral surface was carried out by LC-MS / MS. Briefly, samples were concentrated by filtration and digested with PNGase F to remove N-linked glycans. Viral proteins were separated by SDS-PAGE and the deglycosylated E protein band was cut out, reduced, alkylated, and finally digested in-gel with trypsin. Following clean-up and de-salting, the samples were analyzed by LC-MS / MS. LC-MS / MS raw spectra were processed with MaxQuant (v2. 1.4.0) software using a protein database combing the Vero cell proteome and the translated Zika virus genome for peptide matching. The data processing step applied standard search parameters including trypsin digest, methionine oxidation, N-terminal acetylation, carbamido-methylation of cysteine residues and BPL modification as variable modifications. All data were filtered with a false discovery rate (FDR) of 1% and BPL modifications were manually validated.

[0139] The identified BPL modification sites were matched against known and characterized epitopes deposited in the Immune Epitope Database (IEDB) using a custom R script. Visualizations of the modification sites were prepared by the software program VMD (vl.9.4.a48, Humphrey et al., VMD: Visual Molecular Dynamics. 1996. J. Molec. Graphics 14:33-38) using the Envelope-Membrane protein heterodimer Cryo EM structure as template as described in AD-ME-0022 (pdb accession number: 7jyi.2, DiNunno et al., Identification of a pocket factor that is critical to Zika virus assembly. 2020. Nat. Commun. 11:4953, doi: 10.1038 / s41467-020-18747-4).

[0140] Formaldehyde inactivation of Zika virus In the Zika virus first generation process the virus was inactivated with formaldehyde using the routine JEV commercial process. Briefly, concentrated sucrose gradient pool is diluted 1:4 with 5% PBS-sucrose pH 7.3 solution to reduce the sucrose concentration to a final concentration of 10 - 12% (w / w), then inactivated by incubation with 0.02% formaldehyde over a period of ten days at 22 ± 2°C. To remove any larger particles that could contain only partially inactivated virus the formaldehyde treated virus solution is filtered through a 0.2 pm filter on day 3 post formaldehyde addition. On day 10, the formaldehyde is neutralized by addition of sodium metabisulphite (2 mM end concentration). Neutralized, inactivated virus (NIV) solution is aliquoted and frozen at <70°C until final dilution to drug substance.

[0141] Comparison of formaldehyde and BPL inactivated Zika virus Two concentrated lab-scale Zika virus harvests were combined and inactivated at 5°C with an initial concentration of 500 ppm BPL plus an addition of 200 ppm BPL after 10 h with a total inactivation time of 32 h. After hydrolyzation of BPL the inactivated material was subjected to downstream processing. Aliquots of the purified Zika virus material were also treated with increasing concentrations of formaldehyde (100, 200 and 400 ppm) in order to assess the cross-linking effects at higher concentrations. After neutralization of formaldehyde, the samples were analyzed by Western Blot targeting the viral E protein and for total virus particles by SE-HPLC.

[0142] Western Blot analysis using the commercially available Zika virus specific monoclonal antibody mAb 40543-MM09 is shown in Figure 2. This antibody recognizes epitopes on domain III of surface protein E specific to Zika virus. A strong band at ~55 kDa corresponding to protein E was detected for the BPL inactivated Zika virus. For the formaldehyde inactivated samples, additional high molecular weight signals representing aggregated viral particles were detected. The signal intensity for E protein decreased significantly at higher formaldehyde concentration, possibly partially due to a reduction in Zika virus monomers; however, the lower signal strength may also be a result of masking of antibody binding sites due to cross-linking.

[0143] The Zika virus particle content of BPL and formaldehyde inactivated samples were also analyzed by SE-HPLC. The overlay of the inactivated Zika virus preparations revealed that they have a similar peak shape and area (Figure 3), in contrast to the pronounced cross-linking effect observed for E protein by formaldehyde as shown in Figure 2. This indicates that formaldehyde as expected cross links the proteins on the surface of the virus particle but does not lead to virus aggregation and / or precipitation.

[0144] Basis for the BPL inactivation procedure Developmental studies for establishing the BPL inactivation method for Zika virus as disclosed herein included side by side comparisons of formaldehyde and BPL inactivated Zika virus, several different concentrations of BPL (from 50 to 700 BPL) as well as conditions of inactivation (temperature, pH, one or two BPL additions), hydrolysis parameters and storage duration. The applied inactivation procedure was based on observations collected during these studies.

[0145] These observations identified a BPL concentration dependent inactivation kinetic of at ~1 logic PFU per hour at 500 ppm BPL. A total inactivation time of 30h was found to be sufficient and also to include a significant safety margin. The BPL inactivated Zika virus was found to be stable at 2-8°C up to 25 days, allowing time for assessment of inactivation. It is important to ensure that enough BPL is present throughout the whole inactivation time; for this purpose, a second addition of 200 ppm (0.02%) after 8 - 9 h was found to be effective. When 200 ppm BPL is added at the second timepoint, the total BPL concentration remained below 600 ppm, but was high enough to prolong the virus inactivation kinetic until the end of the inactivation period. To stabilize the pH of the inactivated viral solution, PS-treated concentrated harvest pre-cooled to 7 ± 3°C is supplemented with 25 mM HEPES pH 7.2 - 7.5.

[0146] Initial studies at lab scale from 15 mL up to 400 mL indicated an acceptable inactivation kinetic for Zika virus by BPL. A starting viral titer of 9 logic pfu / mL was reduced below detectable levels within 10 h after addition of 500 ppm BPL with an inactivation rate of -Ilog per hour. At lower BPL concentrations, the inactivation was significantly slower, highlighting the need to maintain high BPL concentrations throughout the incubation time. To reduce remaining BPL (hydrolysis) after the inactivation the solution is warmed to temperatures above 32°C for a total time of 2.5 h ± 0.5 h in a temperature-controlled incubator set to 35 ± 2°C. The total time of the hydrolyzation step for the current process volume of about 1 L was between 5 and 6 hours including the warming to and the incubation above 32°C. After completion of the hydrolysis, the inactivated viral solution (IVS) was immediately cooled down to 5 ± 3°C in a temperature-controlled fridge and stored there until inactivation was confirmed by neat large volume plaque assay.

[0147] Kinetic samples taken during inactivation during developmental studies were analyzed by SE-HPLC to ensure virus integrity. An overlay of samples taken throughout the BPL inactivation up to 30 h and after hydrolysis showed no change in peak shape (data not shown). This finding indicated the applied inactivation method does not lead to physical modifications of the viral particles (e.g. compared to aggregation via cross-linking by formaldehyde). As mechanical stress is minimized in the applied procedure there was also no significant loss of viral particles during the inactivation step (recovery >95 %).

[0148] The sum of Zika virus inactivation development data as summarized above indicates that the applied BPL treatment can be considered efficient and includes a significant safety margin for inactivation of Zika virus concentrated harvest material. Critical parameters for the inactivation step were identified as the concentration of BPL at time of addition and throughout the inactivation time, a constant temperature of viral solution during incubation, sufficient inactivation time, pH of the solution during incubation and the initial virus titer.

[0149] Pollowing initial development at lab scale, the viral inactivation process was transferred to GMP production. The final inactivation process for commercial production is shown in Figure 4. Briefly, the scale for the inactivation is 30-35 L of protamine sulphate treated and filtered / concentrated harvest using a 30 h total incubation time at a setpoint of 7°C. BPL is added in two steps, 500 ppm (0.05%) at the beginning and 200 ppm (0.02%) after 9 h. In total 3 vessels are used. After the initial addition of BPL in vessel 1 the solution is mixed and immediately transferred to inactivation vessel 2 where the early inactivation kinetic samples are collected (Ih, 3h, 5h, 8h). The second addition of BPL to vessel 2 is followed by mixing and a transfer to the final vessel 3 via a 0.45 / 0.2 gm filter (Satorius Sartopore). Further kinetic samples are taken from vessel 3. After hydrolysis the final sample is collected and the IVS is stored at 5 ± 3 °C until further processing. Kinetic samples were analyzed by plaque assay and SE-HPLC. The plaque assay data from an example large scale study are summarized in Table 2. Briefly, the starting titer was lower than observed for small-scale bioreactors with about 8 log pfu / mL (data not shown). Already at the 3h timepoint, only 6 plaques could be detected in the 1: 100 dilution which would correspond to ~3 logic pfii / mL. The titer at 5h was calculated at <2.4 and this was the latest timepoint at which any plaques were detected. The large scale inactivation kinetic can therefore be considered equivalent to the data generated during development.

[0150] Table 2. Inactivation kinetic of large scale Techrun.

[0151] Characterization of BPL modification of the Zika virus E protein by LC-MS / MS BPL modifications were analyzed in the E-protein only, as it is the major antigen for immunity. The focus of the study was identification of amino acid residue modification in the E protein and whether the final inactivation method of two-step addition of BPL showed a different modification pattern compared with a single dose addition of 500 ppm BPL. In total, seven tryptic peptides were found to be modified with a frequency higher than 15%, with no differences between the two tested inactivation protocols (500 ppm BPL, or 500 ppm BPL followed by 200 ppm BPL)(see Table 3). These seven peptides contained a total of 13 modification sites (cysteine, histidine, and methionine), out of which seven were modified at frequencies higher than 15%, thus <1.4% of the total E-protein amino acids are modified.

[0152] Table 3. Overview over the modified amino acid residues in two analyzed PIV lots (PIV #1 and PIV #2). Percentage modifications are based on automatic calculations by MaxQuant and may differ from manual calculations. M: Methionine, H: Histidine. PIV #2 was inactivated according to the two-step BPL addition method as disclosed herein.

[0153] Next, these modified amino acid residues were matched against the Immune Epitope Database (IEDB). This showed that twelve out of the 68 characterized partly overlapping Zika virus E-protein epitope in the IEDB were affected by BPL modifications (Table 4). Two of these twelve epitopes namely l l l-GSLVTCAKFACSKKMTGKSIQ-131 and 391-VGEKKITHHWHRSGSTIGKAF-411 were shown to elicit dominant CD4+ T-cell responses (Koblischke et al., Structural Influence on the Dominance of Virus-Specific CD4 T Cell Epitopes in Zika Virus Infection. 2018. Front. Immunol. 9: 1196; doi: 10.3389 / fimmu.2018.01196; Smith et al., Rapid microsphere-assisted peptide screening (MAPS) of promiscuous MHCII-binding peptides in Zika virus envelope protein. 2019. AIChE Journal 66(3); https: / / doi.org / 10.1002 / aic.16697). Overall, only eight amino acids in twelve out of 68 overlapping epitopes were found to be modified. Thus, the overall impact of BPL on antigenicity can be considered low.

[0154] Table 4. Characterized Zika virus E-protein epitopes with modified amino acids. The epitopes are partly overlapping due to experimental reasons and different study designs. The two CD4+ T-cell response inducing epitopes - l l l-GSLVTCAKFACSKKMTGKSIQ-131 and 391-VGEKKITHHWHRSGSTIGKAF-411 are highlighted in bold. Modified amino acid residues are underscored. All epitopes analyzed are linear.

[0155] Like all flavivirus E-proteins, the Zika virus E-protein possesses four domains: the ectodomains (ED) I, II, and III, as well as the stem-transmembrane domain (Sirohi and Kuhn, 2017). The ectodomains are very similar among Flaviviridae and often give rise to cross-neutralizing antibodies. However, antibodies against the EDIII seem to be less cross-reactive than antibodies against domains I and II while still being potently neutralizing (Smith et al., 2019, supra). Of the two BPL modified epitopes eliciting a dominant CD4+ response, one (G111-Q131) is located in the EDII of the E-protein, while the other (V391-F411) is located in EDIII. This latter epitope (V391-F411) was not only shown to elicit a CD4+T-cell response, but also to contain an amino acid (K394) crucial for neutralization. The importance of this residue was demonstrated by the introduction of a K394A mutation in the Zika virus E-protein, leading to abolished neutralizing antibody binding (Robbiani et al., Recurrent Potent Human Neutralizing Antibodies to Zika Virus in Brazil and Mexico. 2017. Cell 169(4):597-609.el 1. doi: 10.1016 / j .cell.2017.04.024). Importantly, BPL at the concentration used for Zika virus inactivation as disclosed herein did not modify the crucial position K394.

[0156] Analysis of amino acid modifications of the viral E-protein by LC-MS / MS showed that less than 1.4% of the amino acids were modified by BPL (using a threshold of 15%). However, the mean modification rate of the modified amino acid residues was 66% indicating that the majority of the viral particles carried the same modifications. This was in contrast to findings for SARS-CoV-2, which showed much lower modification frequencies per amino acid in the Spike protein (< 15%).

[0157] Example 2: Immunogenicity of a BPL inactivated Zika virus vaccine

[0158] Neutralizing antibody studies with alum adjuvanted inactivated Zika virus

[0159] Methods for quantification of inactivated Zika virus antigen

[0160] Size Exclusion Chromatography Size exclusion chromatography (SEC) was used for detection and characterization of viral particles in Purified Inactivated Zika virus (PIV) and Drug Substance (DS) samples using a Superose 6 Increase 10 / 300 SEC column coupled to a Thermo Vanquish Flex HPLC system. The column was equilibrated with PBS + 250 mM NaCl, pH 7.4 with a flow rate of 1 mL / min at 25°C. The eluate was monitored at 214 nm. The relative virus content was calculated by external calibration using the standard protein Bovine Serum Albumin (BSA) and expressed in BSA units (1 BSA unit = 1 pg BSA / mb).

[0161] Zika virus second Generation Ag ELISA The second generation Ag ELISA is used for antigen content determination of Zika vims Drug Product (DP) Supernatant (SN), which is used to assess and calculate the degree of adsorption (DoA) to Aluminum Hydroxide (Al(OH)s) for release and stability testing of the Zika virus vaccine candidate. The method is based on the principle of a sandwich ELISA, which requires a plate coating with antibodies (anti-Flavivirus group antigen immunoglobulins [4G2, Mouse IgG2a]) and a detection antibody (Zika Virus Anti-Envelope Protein antibody [ZKA64], HRP conjugated). The detection is performed by the addition of a TMB substrate (chromogenic substrate); the hydrolyzed TMB forms a stable-colored conjugate, quantified by spectrophotometric detection at X450nm (Z630nm reference), that is directly proportional to the concentration of antigen content in the sample. Hence, the more antigen is contained in sample (i.e. DP SN) the higher the signal detected. An internal Zika virus reference substance (diluted PIV or DS) is used to determine the relative potency (REP) by a parallel line analysis with a linear regression fitting using Gen5 Secure software as well as the antigen concentration of the samples in Antigen Units (AU) / mL. Zika virus Competitive Liquid Phase ELISA The Zika virus Competitive Liquid Phase ELISA is used for antigen content determination in Inactivated Virus Solution (IVS), IPC samples (e.g. Captocore load, Captocore Flowthrough, TFF2 Pool and PIV prior to stabilization), Purified Inactivated Virus Solution (PIV), Drug Substance (DS), Drug Product (DP; Final Bulk Vaccine [FBV] and Final Vaccine Lot [FVL]) and DP bed-side mixed with 3M-052-AF. The method is based on the principle of an indirect competitive ELISA, which is comprised by two different antigen-antibody incubation phases (i.e. a liquid phase followed by an immobilized phase). In brief, a test sample is incubated in solution (= liquid phase) with a monoclonal antibody against Zika virus Envelope protein (i.e. Anti- Zika-Envelope Protein Antibody ZKA64 [Human IgGl]), which is able to bind to the antigen in Zika virus in-process samples. The antibody forms complexes with the target antigen during this liquid incubation phase while unbound antibody (if applicable) remains in solution. Subsequently, a part of this liquid phase is transferred to a second plate, which is coated with the same target antigen (e.g. PIV), (= immobilized phase). Unbound antibody (from the pre -incubation plate) will bind to the coated antigen, which can subsequently be detected through a secondary antibody conjugated with HRP (i.e., Goat anti-Human IgG HRP conjugate). The detection of the secondary antibody is performed by the addition of a TMB substrate (chromogenic substrate). The hydrolyzed TMB forms a stable-colored conjugate that is inversely proportional to the concentration of antigen content in the sample. Hence, the more antigen contained in the analyte (i.e. PIV / DS or DP sample), the less antibody is available to bind to the coated antigen, and the lower the signal detected.

[0162] Antigen quantification is carried out by spectrophotometric detection at X450nm (Z630nm reference) using a standard curve generated in an automated plate reader using Gen5 Software. A four-parameter logistic fit is applied to all samples and controls (reference substance (RS) - with a known concentration; assay control (AC) and samples) and samples’ concentration estimated by a parallelism analysis and relative potency determined.

[0163] Neutralizing antibody quantification

[0164] Plaque Reduction Neutralization Test (PRNT) - single dilution This assay allows determination of the 50% Effective Dose (ED50) in sera of mice injected with different doses of the Zika virus vaccine candidate or placebo by assessing the titer of Zika virus-neutralizing antibodies. In brief, for a final 1 :20 dilution, animal sera are diluted in assay diluent 1: 10, mixed with the same volume of Zika Virus solution with fixed number of plaque forming units per mL and incubated for 30 minutes at 35°C. Thereafter, sera-virus mixtures are transferred to the 6-well plates seeded with Vero cells and incubated for 60 minutes at 35°C, 5±1%CC>2 before adding the first agarose overlay (0.9 % agarose). Plates are further incubated for 3 days at 35°C, 5±1%CC>2, then second agarose overlay is added (5% Neutral Rd Solution in 1% agarose) and plates are incubated for another 18 to 24 hours at 35°C, 5±1%CC>2, after which plaques are counted. Each serum sample is tested in triplicate (on 3 wells) and % neutralization is calculated by normalizing the median plaque number over triplicates to the assay Negative Control (100 % infection) - a serum sample obtained from animals injected with placebo (i.e., without Zika virus-specific antibodies). Animals with >50% neutralization are considered to be seroconverted. The 50% Effective Dose (ED50) is calculated by Probit analyses, that assesses the vaccine dose against the number of animals that seroconverted in each dose group.

[0165] Plaque Reduction Neutralization Test (PRNT) - serial dilution This assay allows detection and quantification of neutralizing antibodies in sera of mice treated with the Zika vims vaccine candidate or placebo. In brief, all animal sera in a dose group are pooled and diluted in assay diluent in 4-fold series of dilutions starting at 1: 10. Same volume of Zika Vims solution with fixed number of plaque forming units per m is added to diluted sera pools and incubated for 30 minutes at 35°C. Thereafter, sera-virus mixtures are transferred to the 6-well plates seeded with Vero cells and incubated for 60 minutes at 35°C, 5±1%CC>2 before adding the first agarose overlay (0.9 % agarose). Plates are further incubated for 3 days at 35°C, 5±1%CC>2, then second agarose overlay is added (5% Neutral Rd Solution in 1% agarose) and plates are incubated for another 18 to 24 hours at 35 °C, 5±1%CC>2, after which plaques are counted. Each sera pool dilution is tested in triplicate (on 3 wells) and % neutralization is calculated by normalizing the median plaque number over triplicates to the assay Negative Control (100 % infection) - a serum sample obtained from animals injected with placebo (i.e., without Zika vims-specific antibodies). The 50% neutralization titer for each dose group is extrapolated from the 3 -parameter-fitted sigmoid curve as the reciprocal value of the sera pool dilution that corresponds to 50% neutralization.

[0166] Neutralizing antibody titers of individual mice following single immunizations

[0167] Immunogenicity of a Zika virus lot produced on roller bottle platform and formulated based on ELISA antigen units (A U)

[0168] Swiss (CD-I) mice 10 mice / group were immunized intraperitoneally (i.p.) with 6 different doses of BPL inactivated Zika vims (100 pL / dose), ranging from 0.005 to 1.2 AU / dose. Zika vims doses were based on ELISA units (AU) as determined by second generation Ag ELISA as described herein. All doses were adjuvanted with 1 mg / mL Al(0H)3. Sera was taken 21 days after a single immunization and tested for neutralizing capacity in a single dilution PRNT assay (1:20 semm dilution). Neutralizing capacity of individual mouse sera per group is depicted. Neutralization capacity of individual mouse sera per group is depicted in Fig. 5A Mice showing a titer above 50% (dotted line) are taken as seroconverted. Formulation based on Ag ELISA values (undiluted vims preparation from roller bottles: 221 AU / mL (2.1 pg / mL BSA units)).

[0169] Immunogenicity of a Zika virus lot produced using an iCellis platform and formulated based on antigen content (BSA units (ng)) Swiss (CD-I) mice 10 mice / group were immunized intraperitoneally (i.p.) with different doses of the BPL inactivated Zika virus ranging from 0.37 to 90 ng / dose. Formulation was done based on BSA units (ng), determined by the SEC assay as described herein. All doses were adjuvanted with 1 mg / mL Al(OH)s and delivered in a volume of 100 pL. Sera was taken 21 days after single immunization and tested for neutralization capacity in a single dilution PRNT assay (1:20 serum dilution). Neutralization capacity of individual mouse sera per group is depicted in Fig. 5B. Mice showing a titer above 50% (dotted line) are taken as seroconverted. Formulation was based on BSA units (undiluted virus preparation from iCellis: 20.5 pg / mL BSA units (1835 AU / mL)).

[0170] Neutralizing antibody titers of pooled sera following one or two immunizations

[0171] Neutralizing studies with increased antigen dose and double adjuvantation CD-I mice were i.p. immunized on dO and d21 and sera were harvested on d21 and d42 and assessed for Zika neutralizing activity. Material used for immunization, as well as EC50 values are provided in Table 5. Serum serial dilution curves for d21 and d42 are shown in Fig. 6.

[0172] Table 5. Summary of dose response results shown in Figure 6. Dosages are provided as both AU and ng. A1(OH)3provided at 1 mg / mL; 3M-052-AF provided at 10 pg / mL (mouse dose=0. 1 ml).

[0173] No clear dose response was observed. The medium dose (600 ng) dose benefit of second adjuvant seen compared to Alum only. Other observations include that the immediate (d21) response was higher with the double adjuvant combination and the no adjuvant formulation showed an impaired immune response

[0174] Cytokine production studies with alum and alum / 3M-052-AF adjuvanted inactivated Zika virus

[0175] The Zika virus specific cellular immune response to the BPL inactivated Zika virus present in the Zika virus vaccine candidate was investigated in CD-I mice by measuring IFN-y production after in vitro restimulation of splenocytes isolated after one or two immunizations. Analysis was done using Cytokine Multiplex ELISA and ELISpot measurements as readout parameters. Experimental design CD-I mice were immunized twice intraperitoneally (i.p.) with 100 pL P- propiolactone (BPL) inactivated Zika virus (referred as Drug Substance, DS) formulated with a) alum (1 mg / mL) or b) alum (1 mg / mL) in combination with 3M-052-AF (10 pg / mL) at an interval of 21 days. A control group with alum alone was also included (see Table 6). On days 7 and 28, mice were euthanized, and splenocytes collected for testing. Zika virus specific IFN-y production was assessed by multiplex and ELISpot as described below and shown in Fig. 7. For all IFN-y experiments, the geometric mean (GM) from three single mice was calculated and analysis was done in GraphPad Prism 10.

[0176] Table 6. Overview of the CD-I mice immunization Scheme on Day 0 and Day 21. Each group contains three mice. Splenocytes were isolated on days 7 and 28. DS=BPL inactivated Zika virus. A1(OH)3 provided at 1 mg / mL; 3M-052-AF provided at 10 pg / mL (mouse dose=0. 1 m ).

[0177] Multiplex assessment of antigen-specific IFN-y production by d28 splenocytes In a 96-well round bottom culture plate, IxlO6splenocytes per well were stimulated for 18-24h with 2 pg / mL of either a specific peptide library encoding the E protein of Zika virus or an unspecific library encoding Hu- Actin as a background control. Each peptide covers 15 amino acids with an 11 amino acid overlap between adjacent peptides. After 18-24h, supernatants were collected and analyzed for cytokine production according to the supplier’s protocol. Briefly, the collected supernatants containing secreted cytokines and cytokine standards were mixed with magnetic beads coated with the cytokine antibodies to be studied. The Ab bead-cytokine complexes were incubated with a biotinylated detection antibody and stained with streptavidin-R-phycoerythrin (SAPE). Secreted cytokines were measured in a Luminex 200 machine. Results are presented as pg / mL (Fig. 7A).

[0178] Zika virus-specific production of IFN-y was detected in all groups ranging from 526 pg / mL geometric mean (GM) in the Alum test group to over 6650 pg / mL GM in the Alum / 3M-052-AF test group. The production of IFN-y in the placebo group (Alum only) was low (56 pg / mL GM) as well as the unspecifically stimulated cells (55 pg / mL GM in the Alum group only and 583 pg / mL GM in the Alum / 3M-052-AF group) (Fig. 7A).

[0179] ELISpot assessment of antigen-specific IFN-y production by d28 splenocytes Splenocytes were isolated 7 days after the 2nd immunization. In a 96-well anti-IFN-y membrane coated plate, 3xl05splenocytes per well were stimulated with the peptide libraries mentioned above. After 18-24h, cells were discarded, plate washed and incubated with a biotinylated detection antibody. After incubation with Streptavidin-HRP, spots were developed with a TMB substrate. Spot forming units were measured in a Bioreader 6000 (BioSys, US). Results are presented as Spot Forming Units (SFU) per 3xl05cells (Fig. 7B).

[0180] The EUISpot analysis showed a similar pattern to that of the multiplex analysis. The detected IFN-y ranged from 15 Spot Forming Units (SFU) per 3x105stimulated lymphocytes in the Alum group (GM) to 412 SFU in the Alum / 3M-052-AF group (GM). Background production of IFN-y was low in the Placebo group and in cells stimulated with unspecific peptides.

[0181] Comparison of IFN-y production by splenocytes after one or two immunizations Harvested splenocytes were also assessed for a potential boosting effect of the adjuvant / antigen combinations 7 days after the first immunization (d7) and 7 days after the second immunization (d28). After preparation, 3xl05splenocytes per well were plated in a 96-well anti -IFN-y membrane coated plate and stimulated with peptide libraries as described above. After 18-24h, the spots were developed as described above. Results are presented as Spot Forming Units (SFU) per 3xl05cells (Fig. 7C).

[0182] The results demonstrated a strong boosting effect after the second immunization with double- adjuvanted Zika virus in EUISpot as the Zika-specific IFN-y production rose from 85 SFU after the first vaccination to 412 SFU after the second vaccination.

[0183] In summary, it was found that the synthetic TLR7 / 8 agonist 3M-052-AF formulated together with Alum elicited an antigen-specific Thl cellular immune response against the Zika virus Envelope protein as shown by the production of IFN-y via two different methodologies. The results also show a strong boosting effect after two immunizations with Alum / 3M-052-AF, suggesting the accumulation of memory T cells over time, which is beneficial for the development of a vaccine against Zika virus for human use.

[0184] Example 3: Immunogenicity testing in rat toxicology study

[0185] A 29-day toxicology study of the inactivated Zika virus candidate with adjuvant(s) was carried out in Wistar Han IGS rats (Charles River). In the study, 30 animals were used: 20 for the main study (immunization at dl and dl 5) and 10 in the post-treatment study (immunization at dl, dl 5 and d29). Immunizations were administered i.m. at two different sites in two 0.2 mb volumes (0.4 mb total / animal).

[0186] Table 7. Experimental design of the rat toxicology study. Each group consisted of 30 rats (15 male and 15 female); 10 of which were terminated on each of days 15, 29 and 59. A1(OH)3 provided at 1 mg / mL; 3M-052- AF provided at 6 25 ug / ml, (rat dose=0.4 mL; for comparison, the human dose is approximately 0.45-0.5 mL)

[0187] Table 8. Immunogenicity of the Zika virus vaccines administered during the rat toxicology study. Neutralizing antibody titers were measured in the jiNT assay. Shown here are the GMTs of each timepoint as measured. Data from individual rat sera are shown in Figure 8. No neutralizing activity was observed in any of the placebo control animals or in the pre -vaccination pool per treatment group.

[0188] Microneutralization assay ( NT) The microneutralization assay is a cell -based assay for the quantification of Zika virus specific neutralizing antibodies in serum samples. Briefly, the Zika virus pNT assay typically includes up to twenty 96-well microtiter plates, each plate containing Virus Control (VC), Medium Control (MC) and 10 replicates per dilution of the respective serum sample (SPL). Vero cells are seeded on day 0 at a cell density of 2xl05cells / mL in 96-well plates and are grown overnight at 37°C, 5% CO2. On the following day (day 1) eight 2-fold serial dilutions of serum are prepared in the pre -incubation plate (deep well plate). The serum samples are mixed with an equal volume of Zika virus clarified (filtered) harvest material, protamine sulfate-treated and Tris / Sucrose stabilized. Additionally, one pre -incubation plate contains virus sample used as virus titration sample. Pre -incubation plates are incubated for 60-100 min at room temperature (RT), preferably, on a shaker set to 800 rpm. After this time the samples are transferred in 10 replicates to a monolayer of Vero cells seeded in 96-well plates. The plates are incubated at 35°C, 5% CO2 for 6 days (assay day 7) until staining. For staining, Resazurin solution is added to each well and plates are incubated for 3-4 hours at 35°C, 5% CO2. The staining reaction is stopped by adding SDS solution. Viable, metabolically active cells continuously convert Resazurin (blue color) to Resorufin (pink color) thereby generating a measure of viability. Absorbance is quantified at 570 nm and 600 nm reference wavelength using a SpectraMax Multi Mode iD3 microplate reader equipped with SoftMax Pro 7.0.3 GxP software. Each well (10 wells per sample dilution) is evaluated against an OD cutoff value, wells with OD lower than the cutoff is counted as CPE positive.

[0189] The virus neutralization titer (pNTso) is defined as the reciprocal value of serum dilution that corresponds to 50% virus neutralization (i.e., protection from cytopathic effect), extrapolated from the 4-parameter curve fit parallel to the assay positive control (PC). As shown in Fig. 8 and Table 8, vaccination of rats with alum adjuvanted Zika vims at the high dose (320 AU+alum) was immunogenic in all animals as assessed by the induction of neutralizing antibodies. Further, addition of a TLR adjuvant (3M-052-AF) to the alum adjuvanted Zika vims at the low dose (80 AU) generated a faster induction of neutralizing antibodies as seen after the first vaccination on day 15. The maximum neutralizing antibody response was greater in the double adjuvanted formulation group compared to the alum only group. It should be noted that the dosing schedule of two-week intervals was chosen not for optimal immunogenicity, but only for purposes of toxicology testing of the vaccine formulations. The observed results, therefore, may not reflect the level of antibodies that could be reached by use of a broader vaccination scheme, such as e.g., the third shot (such as e.g., a booster) administered at 4-12 weeks or later following the first two shots.

[0190] Example 4: A clinical phase 1 study of a Zika virus vaccine

[0191] Background The Zika vims vaccine candidate, as described in the previous Examples, is a highly purified, inactivated, whole Zika vims vaccine designed for active immunization for the prevention of disease caused by the Flaviviridae Zika vims. The candidate vaccine is intended to prevent Zika vims disease in (1) the population living in endemic regions, or in areas of an ongoing Zika vims epidemic or imminent outbreak, as well as (2) to serve as a prophylactic measure for travelers to endemic or epidemic areas or areas at risk for a potential outbreak. The Zika vims vaccine candidate is adsorbed on aluminum hydroxide (alum) and administered with or without the addition of a TLR adjuvant (e.g. bedside mixing with 3M-052-AF [AAHI]). The Zika vims vaccine candidate has been developed using the same manufacturing platform as the one used for Valneva’s licensed vaccines: IXIARO®, a vaccine to protect from Japanese Encephalitis vims, another member of the same Flaviviridae family, and COVID- 19 Vaccine (inactivated, adjuvanted).

[0192] This is a phase 1 study, composed of an initial unblinded (open-label) safety mn-in (N=20 sentinels; see Fig. 9) and a randomized, double-blind, dose-finding, multicenter study (N=100; see Fig. 10) in Flavivims naive adults aged 18 to 49 years. The sentinel safety data was closely monitored by the sponsor’s Safety Review Committee (SRC). Three dose levels of the Zika vims vaccine candidate (measured by Liquid ELISA as described herein) will be evaluated: a low dose of 100 antigen units (AU) of inactivated Zika vims, a medium dose of 200 AU, and a high dose of 400 AU, each dose formulated with alum adjuvant (aluminum hydroxide 1 mg / mL). In addition, to potentially improve the immunogenicity profile, the 100 AU (low) dose formulation is further adjuvanted during bedside mixing with the toll-like receptor agonist 3M-052-AF (TLR7 / 8) prior to administration. The study contains 4 study arms each enrolling 30 participants (5 sentinel / mn-in and 25 randomized) per arm. Each vaccination is administered intramuscularly (i.m.; 0.45 mL) in the deltoid muscle in a two-dose regimen (Days 1 and 29). The screening period before the first vaccination can last up to 21 days and the study is then conducted in three parts: Part A (Day 1 to Day 57), Part B (Day 58 to Day 208) and Part C (Day 209 to Day 395). Part A analysis will be performed after all participants have received the second vaccination and completed Visit 7 (see Fig. 10). All participants are followed up for safety and immunogenicity up to 6 months after the second immunization (i.e. Day 208 / Month 7). Part B analysis is performed after all participants have completed their Visit 8. Following Sponsor’s review of available safety and immunogenicity data from Part A and Part B, all sentinels and randomized participants from selected treatment arms with the most favorable safety and immunogenicity profile will be asked to come back for an additional blood draw and on-site study visit at their Visit 9 (i.e. Day 395 / Month 13). The randomized participants from the remaining treatment arms will be followed by phone-call for assessment of long-term safety. Part C analysis will be performed after all participants have completed their Visit 9.

[0193] A total of approximately 90 participants will be enrolled in the study to receive either a low dose of 100 AU, medium dose of 200 AU or a high dose of 400 AU of the Zika virus vaccine candidate. Moreover, a further treatment group (n=30) will receive the low dose of the Zika virus vaccine candidate with an additional adjuvant (3M-052-AF) added by bedside mixing prior to vaccination. The participants will be randomized into these 4 treatment groups of 30 participants each. Randomization will be done 1 to 1 in all study arms. The participants in the treatment groups receive Zika vaccine (Uow dose w / Alum), Zika vaccine (Medium dose w / Alum), Zika vaccine (High dose w / Alum) or Zika vaccine (Uow dose w / Alum+3M-052) (see Table 9 for Treatment Groups and Figure 10 for overall study design).

[0194] As a safety precaution, the study began with enrolment of 20 sentinel subjects (5 participants per treatment group / 4 treatment groups) in a sequential open-label, run-in following staggered doseescalation (Fig. 9). The sentinel safety data was closely monitored by the sponsor’s Safety Review Committee (SRC) as defined in the SRC charter. The SRC confirmed the start of the vaccinations in the next treatment arm(s) during this part of the trial.

[0195] An independent Data and Safety Monitoring Board (DSMB) reviewed the accrued safety data of all vaccinated sentinels until Day 57 of the last sentinel participant. The decision to either halt or continue with all or specific treatment arms into the randomized phase was determined by a recommendation from the DSMB, based on accrued Day 57 safety data and a documented sponsor decision taking into account DSMB recommendation and available immunogenicity data.

[0196] Up to approximately 100 participants will thereafter be randomized into the 4 treatment arms. The 100 AU, 200 AU and 400 AU vaccine doses (all in combination with Alum) will be administered in a final injected volume of approximately 0.45 mL (or 0.5 mL), including the doses prepared by bedside mixing with the TLR agonist 3M-052-AF.

[0197] Table 9. Study treatment groups. Each group comprises 25 subjects to be vaccinated on day 1 and day 29 with a vaccine injection volume of approximately 0.45 mL. Each treatment group comprises five sentinel participants which are open-label with staggered dose escalation and receive the same 2-dose regimen of other group participants.

[0198] Participant vaccination As a safety precaution measure, the first 20 sentinel participants (5 participants per treatment group in each of the 4 treatment groups) were vaccinated in the study in an open-label fashion according to the dose-escalation scheme outlined in Figure 9. As more than one trial site was involved in the recruitment of sentinel participants during the staggered dose-escalation phase, a process was implemented by the sponsor to ensure the timely exchange of relevant safety information amongst the involved sites.

[0199] The dose-escalation phase started with the vaccination of the first five sentinel participants with the Zika vaccine Low dose w / Alum (i.e. Zika vaccine 100 AU w / Alum). On Day 1, the first sentinel participant was vaccinated and observed at the study site for 3 hours after vaccination for safety and reactogenicity. Prior to discharge from the study site, vital signs were measured, and the participant was instructed to use the eDiary. This first participant will be followed up for safety with a safety phone call on the next day (Day 2, Visit 1-S) to assess the participant’s well-being / condition: the provided information to the investigator was to be compared with the entries in the participant’s eDiary and discrepancies were to be discussed and documented by the investigator. The minimum time before the next participant could be vaccinated was therefore 24 hours. Subsequently, the next 4 sentinel participants will be vaccinated with at least one-hour observation period between the participants of the Zika vaccine Low dose w / Alum group. Safety telephone calls were performed by the study site approximately 24 hours after the first vaccination. All 5 sentinel participants had to return to the study site for their next visit on Day 8 (Visit 2-S) for safety follow-up. The Visit 2-S safety data of these five sentinels were reviewed by an SRC. The review identified no safety concerns nor any stopping criteria, therefore the trial continued with the next treatment arm.

[0200] Upon SRC Review and approval to continue, five sentinel participants in the next dose level, Medium Zika vaccine dose w / Alum (i.e. Zika vaccine 200 AU w / Alum) were vaccinated. In parallel, five participants were vaccinated in the arm with Zika vaccine Low dose w / Alum plus addition of 3M- 052-AF (i.e. Zika vaccine 100 AU w / Alum + 3M-052-AF). The vaccination procedure as described for the first treatment arm were repeated.

[0201] After at least the Zika vaccine Medium dose had been given, and safety follow up had been performed, the trial progressed to the highest dose (i.e., Zika vaccine 400 AU w / Alum) upon written confirmation by the SRC. The last five sentinels then received the highest dose (i.e., Zika vaccine 400 AU w / Alum). The SRC continued with close safety monitoring and decided that it was safe to proceed with the second vaccination in the sentinel treatment arm(s). As described above, written communication was provided to the trial site confirming that it was safe to continue with second vaccinations.

[0202] In line with their Charter, the DSMB reviewed all accrued safety data at the time point when the last sentinel participant had completed Day 57, including:

[0203] 1. Adverse events (AE), including solicited and unsolicited, SAEs and AESIs

[0204] 2. Results of symptom-driven physical examinations

[0205] 3. Relevant safety laboratory results

[0206] 4. Any other safety relevant issues

[0207] In the further conduct of the trial, the DSMB will convene for regular meetings at predefined time points to review accrued safety data (all AEs, SAEs, AESIs and Pregnancies) according to the DSMB Charter. Ad hoc DSMB meetings may be called at any time if an independent opinion or advice is needed.

[0208] Based on the specific safety and immunogenicity requirements having been met in the sentinel subjects as described in Figure 9 (data on file), the clinical trial study has been advanced to the double-blind randomized phase as outlined in Figure 10.

[0209] Study Objectives

[0210] Primary:

[0211] 1. To assess the safety and tolerability of the Zika vaccine up to 7 days after completion of each vaccination in a healthy adult population aged 18 to 49 years.

[0212] 2. To assess the immunogenicity of the Zika vaccine at Day 57, i.e. up to 28 days after the last vaccination in a healthy adult population aged 18 to 49 years.

[0213] Secondary:

[0214] 3. To identify the optimal dose level of the Zika vaccine in a healthy adult population aged 18 to 49 years. 4. To identify the optimal adjuvant combination with the Zika vaccine low dose in a healthy adult population aged 18 to 49 years.

[0215] 5. To assess the immunogenicity of the Zika vaccine up to 6 months after the second vaccination in a healthy adult population aged 18 to 49 years.

[0216] 6. To assess the long-term safety of the Zika vaccine up to 12 months after the second vaccination in a healthy adult population aged 18 to 49 years.

[0217] 7. To assess the immunogenicity of the Zika vaccine up to 12 months after the second vaccination in all sentinels and randomized participants in the selected treatment arm(s).

[0218] Exploratory:

[0219] 8. To evaluate the cellular immune response (e.g IFN-y / IL-4 ELISpot analysis) of the Zika vaccine up to 6 months after the second vaccination in a healthy adult population aged 18 to 49 years.

[0220] 9. To evaluate the cellular immune response (e.g IFN-y / IL-4 ELISpot analysis) of the Zika vaccine up to 12 months after the second vaccination in all sentinels and randomized participants in the selected treatment arm(s).

[0221] Inclusion Criteria

[0222] Participants who meet ALL of the following criteria are eligible for this study:

[0223] 1. Participant is 18 to 49 years of age on the day of ICF signing.

[0224] 2. Participant has a BMI of >18.5 and <30 kg / m2on the day of screening (Visit 0).

[0225] 3. Participant has an understanding of the study and its procedures, agrees to its provisions, and gives written informed consent prior to any trial -related procedures.

[0226] 4. Participant is generally healthy as determined by the Investigator’s clinical judgement based on medical history, physical examination, and screening laboratory tests.

[0227] 5. If female participant is of childbearing potential and is sexually active with a man: i. Female participant has a negative serum pregnancy or urine pregnancy test at screening (Visit 0) at screening (Visit 0) or Visit 1, respectively, and ii. Female participant agrees to employ adequate birth control measures up to Day 208 (Visit 8). This includes one of the following measures. a. Hormonal contraceptives (e.g. implants, birth control pills, patches). b. Intrauterine hormone-release systems and Intrauterine device. c. Barrier type of birth control measure (e.g. condoms, diaphragms, cervical caps). d. Vasectomy in the male sex partner >3 months prior to first vaccination.

[0228] 6. If female participant is of non-childbearing potential as: i. Women who have had surgical sterilization (hysterectomy or bilateral oophorectomy or tubal ligation or ii. Women >40 years of age who have had a cessation of menses for at least 12 months and a follicle-stimulating hormone (FSH) test confirming non-childbearing potential (FSH >40 mIU / mL)

[0229] 7. Male participant who is sexually active with a woman agrees to employ adequate birth control measures up to 90 days after last vaccination.

[0230] Exclusion Criteria

[0231] Participants who meet ANY of the following criteria are NOT eligible for this study:

[0232] 1. Participant has a known history of the following flavivirus infection; Zika Virus (ZIKV), Japanese Encephalitis Virus (JEV), Dengue Virus (DENV), Yellow Fever Virus (YFV), West- Nile Virus (WNV) or Tick-Borne Encephalitis Virus (TBEV) up until Visit 1.

[0233] 2. Participant has received or has plans to receive a licensed or investigational flavivirus vaccine during the course of the trial;

[0234] 3. Participant has travelled within 4 weeks prior to trial enrollment or has plans to travel to areas (including within the US) with Zika Virus (ZIKV), Japanese Encephalitis Virus (JEV), Dengue Virus (DENV) or Yellow Fever Virus (YFV) active transmission / circulation during the course of the trial.

[0235] 4. Participant has received an active or passive immunization within 4 weeks prior or planned to get such vaccination after any trial vaccination; except for influenza (seasonal or pandemic vaccines which may be administered outside a 7-day interval before or after any trial vaccination). Participants who planned to receive such vaccination within 4 weeks after the 1st vaccination will be excluded from the second vaccination.

[0236] 5. Participant has clinically significant abnormal laboratory values, as determined by the Investigator.

[0237] 6. Participant tests positive for human immunodeficiency virus (HIV), hepatitis B surface antigen (HBsAg) or hepatitis C virus (HCV) within 21 days before Visit 1.

[0238] 7. Participant had or has a history of significant cardiovascular, respiratory (including asthma), metabolic, psychiatric, neurological (including Guillain-Barre syndrome [GBS]), hepatic, rheumatic, autoimmune, hematological, gastrointestinal, or renal disorder.

[0239] 8. Participant has a known or suspected defect of the immune system that would prevent an immune response to the vaccine, such as participants with congenital or acquired immunodeficiency, including infection with human immunodeficiency vims (HIV), post organ transplantation status or other autoimmune diseases.

[0240] 9. Participant received immuno-suppressive therapy within 4 weeks prior to Visit 1. Radiation therapy or immunosuppressive cytotoxic drugs / monoclonal antibodies in the previous 3 years.

[0241] 10. Participant has a history of severe hypersensitivity reactions or anaphylaxis. 11. Participant has a history of any vaccine related contraindicating event (e.g., anaphylaxis, allergy to components of the candidate vaccine, other known contraindications).

[0242] 12. Participant had acute febrile infections within two weeks prior to vaccination in this study.

[0243] 13. Participant has donated blood within 4 weeks or received blood-derived products (e.g. plasma) within 12 weeks prior to vaccination in this study or plans to donate blood or use blood products during the course of the trial.

[0244] 14. Participant has a rash, dermatological condition or tattoos that would, in the opinion of the Investigator, interfere with injection site reaction rating.

[0245] 15. Participant presents with clinical conditions representing a contraindication to intramuscular vaccination and blood draws.

[0246] 16. Participant is currently enrolled (ICF signed) or has participated in another clinical trial involving an investigational medicinal product (IMP) or device within 4 weeks prior to study enrollment or is scheduled to participate in another clinical study involving an IMP or investigational device during the course of this study.

[0247] 17. Participant has plans to become pregnant up to Day 208 (Visit 8), or to father a child within 90 days after the last vaccination, or is pregnant (positive serum pregnancy test at screening) or lactating at the time of screening.

[0248] 18. Participant has a known or suspected problem with alcohol or drug abuse as determined by the Investigator.

[0249] 19. Participant is committed to an institution (by virtue of an order issued either by the judicial or the administrative authorities);

[0250] 20. Participant is a member of the team conducting this trial or is in a dependent relationship with one of the trial team members. Dependent relationships include close relatives (i.e., children, partner / spouse, siblings, parents) as well as employees of the Investigator or site personnel conducting the trial.

[0251] 21. Participant has any condition that, in the opinion of the Investigator, may compromise the participant’s well-being, might interfere with evaluation of study endpoints, or would limit the participant’s ability to complete the study.

[0252] Delay Criteria for Vaccination

[0253] Vaccination will be delayed if:

[0254] 1. Participant has an acute illness with or without elevated body temperature (>100.4 °F [38.0 °C]) within 2 days prior to the scheduled vaccination. Participants may be rescheduled for vaccination at a later date provided that the illness has resolved (body temperature <100.4 °F [38.0 °C]);

[0255] 2. Participant has received antipyretics within 6 hours prior to the scheduled time of vaccination. In this case the vaccination should be performed at a later date in adherence to the following criteria: 1) For a rescheduled first vaccination:

[0256] 1. All inclusion and none of the exclusion criteria are met;

[0257] 2. The rescheduled visit is within the specified time window for the first vaccination visit.

[0258] The participant will be excluded from the trial if these criteria are not all met.

[0259] 2) For a rescheduled second vaccination:

[0260] 1. All inclusion and none of the exclusion criteria are met;

[0261] 2. The rescheduled visit should be within the specified time window for the respective visit. In case the time window for the rescheduled visit cannot be met, the participant may still be vaccinated, but deviation from protocol specified procedures must be properly annotated.

[0262] Note: Participants not receiving the second vaccination due to an exclusion criterion met at Day 29 will be followed up until Visit 8 (Day 208) for safety and immunogenicity.

[0263] Study Endpoints

[0264] Primary Endpoints

[0265] Safety:

[0266] -Frequency and severity of solicited AEs (injection site and systemic reactions) within 7 days after each vaccination.

[0267] Immunogenicity :

[0268] -Geometric mean titer (GMT) for neutralizing antibodies against ZIKA determined by virus neutralizing assay at Day 57.

[0269] Secondary Endpoints

[0270] Safety:

[0271] -Frequency and severity of solicited adverse events (injection site and systemic reactions) within 7 days after any vaccination.

[0272] -Frequency and severity of any unsolicited AE until Day 395.

[0273] -Frequency and severity of any vaccine-related unsolicited AE until Day 395.

[0274] -Frequency and severity of any AE until Day 395.

[0275] -Frequency and severity of any vaccine-related AE until Day 395.

[0276] -Frequency and severity of any AESI until Day 395.

[0277] -Frequency and severity of any vaccine related AESI until Day 395.

[0278] -Frequency and severity of any SAE until Day 395.

[0279] -Frequency and severity of any vaccine-related SAE until Day 395.

[0280] Immunogenicity : -GMT for ZIKV-specific neutralizing antibodies at Day 1, Day 15, Day 29, Day 43, Day 208 and Day 395 (for all sentinels and randomized participants in the selected treatment arms) as determined by virus neutralizing assay.

[0281] -Rate of participants with seroconversion at Day 15, Day 29, Day 43, Day 57, Day 208 and Day 395 (for all sentinels and randomized participants in the selected treatment arms) as compared to baseline (Day 1) determined by virus neutralizing assay.

[0282] -Geometric Mean Fold increase (GMFI) of ZIKV-specific neutralizing antibodies at Day 15, Day 29, Day 43, Day 57, Day 208 and Day 395 (for all sentinels and randomized participants in the selected treatment arms) as compared to baseline (Day 1) determined by virus neutralizing assay.

[0283] Exploratory Endpoint:

[0284] -Cellular immune response on Day 1, Day 15, Day 43, Day 57, Day 208 and Day 395 (for all sentinels and randomized participants in the selected treatment arms).

[0285] Example 5. Influence of duration and amplitude of falling film during virus infection phase (i) and virus propagation phase (ii) on viral antigen yield across two iCELLis 500 Zika virus production batches

[0286] During the course of the invention, two batches of ZIKV were produced in an iCELLis 500 bioreactor on Vero cells under varying conditions (see summary in Table 10). It was observed that the duration and / or amplitude of the Falling Film could have an influence on the antigen yield, similar to observations with regard to SARS-CoV-2 in as described in WO2024 / 153686A1, which is incorporated herein by reference in its entirety. For Zika, the relative productivity of the batches was measured as number of viral particles by size exclusion high performance liquid chromatography (SE- HPLC) and the area under the curve was used to calculate productivity normalized to cell counts at infection [reported as mAU / 109cells].

[0287] Table 10. Exemplary ZIKV production runs

[0288] A long zero Falling Film duration during infection phase (i) and a moderate Falling Film height during production phase (ii) for the ZVPIV00001 batch resulted in higher productivity compared to the TECH-ZVPIV01 batch. This result is consistent with the idea that applied Falling Film conditions could be an explanation for differences in productivity. In summary, it was also shown for Zika virus in the iCELLis 500 system that a better virus yield at harvest is expected when applying a longer time of “zero falling film” during infection phase and a lower FFH during the virus production phase. Application of both of these conditions to the greatest extent possible while still maintaining acceptable DO levels reduces mechanical stress to the small amount of active virus inoculum added into the bioreactor to infect the cells and also helps to maintain the structural integrity of the virus during the virus production phase.

[0289] Further more detailed aspects of the invention:

[0290] Al. A pharmaceutical composition comprising i) an inactivated Zika virus, ii) an aluminium salt adjuvant and iii) at least one additional adjuvant, wherein said at least one additional adjuvant is a toll -like receptor (TLR) agonist.

[0291] A2. The pharmaceutical composition according to aspect Al, wherein the inactivated Zika virus is a beta-propiolactone (BPL)-inactivated Zika virus.

[0292] A3. The pharmaceutical composition according to aspect Al or A2, wherein the inactivated Zika virus comprises an RNA genome corresponding to the DNA sequence provided by SEQ ID NO: 2 or 3, or a variant sequence that is at least 80% identical to SEQ ID NO: 2 or 3 and able to pack a virulent Zika virus.

[0293] A4. The pharmaceutical composition according to aspect A3, wherein the variant sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.99% identical to SEQ ID NO: 2 or 3 and able to pack a virulent Zika vims.

[0294] A5. The pharmaceutical composition according to any one of aspects A2 to A4, wherein the Zika vims comprises an E protein as defined by SEQ ID NO: 4 or a variant amino acid sequence that is at least 95% identical to SEQ ID NO: 4 and able to pack a virulent Zika vims, and wherein the E protein comprises one or more modified amino acids selected from the group consisting of M125, H210, M295, H398, H399, H401 and H446.

[0295] A6. The pharmaceutical composition according to aspect A5, wherein each of the said modified amino acids is present at a percentage of greater than 15%. A7. The pharmaceutical composition according to any one of the preceding aspects, wherein the inactivated Zika virus comprises a heterologous mixture of Zika virus with 1 to 10 genomic mutations compared with SEQ ID Nos: 2 or 3 and able to pack a virulent Zika virus.

[0296] A8. The pharmaceutical composition according to any one of the preceding aspects, wherein the inactivated Zika virus comprises an RNA genome corresponding to the DNA sequence provided by SEQ ID NO: 2 or 3, or a heterologous mixture thereof.

[0297] A9. The pharmaceutical composition according to any one of the preceding aspects, wherein the Zika virus has an E protein with the amino acid sequence provided by SEQ ID NO: 4.

[0298] A 10. The pharmaceutical composition according to any one of the preceding aspects, wherein the inactivated Zika virus is provided at 1 to 10 pg / dose, preferably at 1.5 to 6 pg / dosc.

[0299] Al l. The pharmaceutical composition according to any one of the preceding aspects, wherein the inactivated Zika virus is provided at a dose of between 100 to 800 AU / dose, preferably 100 to 600 AU / dose, preferably 100 to 400 AU, especially 100, 200 or 400 AU / dose.

[0300] A 12. The pharmaceutical composition according to any one of the preceding aspects, wherein the aluminium salt adjuvant is aluminium hydroxide or aluminium phosphate.

[0301] A13. The pharmaceutical composition according to aspect A 12, wherein the aluminium hydroxide adjuvanted composition comprises less than 1.25 ppb Cu.

[0302] A 14. The pharmaceutical composition according to any one of the preceding aspects, wherein the aluminium adjuvant is present in the composition at a concentration of 1 mg / mL.

[0303] A15. The pharmaceutical composition according to any one of the preceding aspects, wherein the Toll-like receptor (TLR) agonist is a TLR4, a TLR7, a TLR8, a mixed TLR7 / 8 and / or a TLR9 agonist.

[0304] A 16. The pharmaceutical composition according to any one of the preceding aspects, wherein the TLR agonist comprises a mixture comprising a peptide and a deoxyinosine-containing immunostimulatory oligodeoxynucleic acid molecule (I-ODN). A 17. The pharmaceutical composition according to aspect A 16, wherein the peptide comprises the sequence KLKL5KLK (SEQ ID NO: 5) and the I-ODN comprises oligo-d(IC)i3 (SEQ ID NO: 6).

[0305] Al 8. The pharmaceutical composition according to any one of the preceding aspects, wherein the TLR agonist is a synthetic TLR7 / 8 ligand, particularly a synthetic TLR7 / 8 ligand provided in an aqueous formulation.

[0306] A 19. The pharmaceutical composition according to any one of the preceding aspects, further comprising one or more pharmaceutically acceptable excipients.

[0307] A20. The pharmaceutical composition according to aspect A 19, wherein the pharmaceutically acceptable excipients essentially consist of sucrose, potassium phosphate and sodium citrate and, optionally, magnesium chloride, D-sorbitol, L-methionine and recombinant human serum albumin (rHSA).

[0308] A21. The pharmaceutical composition according to aspect A19 or A20, wherein said pharmaceutically acceptable excipients essentially consist of about 5% (w / v) sucrose, about 10 mM potassium phosphate, about 25 mM sodium citrate and about 0.01% (w / v) recombinant human serum albumin (rHSA).

[0309] A22. The pharmaceutical composition according to any one of aspects A 19 to A21, wherein said pharmaceutically acceptable excipients essentially consist of about 5% (w / v) sucrose; about 5 mM potassium phosphate; about 25 mM sodium citrate; about 5 mM MgC12; about 0.5 % (w / v) D-sorbitol; about 10 mM L-methionine; and about 0.01% (w / v) recombinant human serum albumin (rHSA).

[0310] A23. The pharmaceutical composition according to any one of the preceding aspects, wherein the composition is delivered to a subject in a priming series followed by one or more boosters.

[0311] A24. The pharmaceutical composition according to aspect A23, wherein the priming series consists of 2 or 3 doses.

[0312] A25. The pharmaceutical composition according to aspect A23 or A24, wherein the first two doses of the priming series are administered 1 to 4 weeks apart. A26. The pharmaceutical composition according to any one of the aspects 23 to 25, wherein an optional third dose of the priming series is administered between 6 and 12 months after the first two doses.

[0313] A27. The pharmaceutical composition according to any one of aspects A23 to A26, wherein the one or more boosters are provided at regular intervals, such as every 2, every 3, every 4, every 5 years or every 10 years, following the priming series.

[0314] A28. The pharmaceutical composition according to any one of the preceding aspects, wherein the dose is administered in a volume of 0.5 mb.

[0315] A29. The pharmaceutical composition according to any one of aspects A23 to A28, wherein the composition is administered to the subject orally or by a parenteral route selected from the group consisting of subcutaneous, intracutaneous, intradermal, intravenous, intramuscular, intraarticular, intraperitoneal, intrathecal or by infusion.

[0316] A30. The pharmaceutical composition according to any one of the preceding aspects, wherein the subject is a human subject.

[0317] A31. The pharmaceutical composition according to any one of the preceding aspects, wherein the subject is a resident of or is travelling to an endemic region.

[0318] A32. The pharmaceutical composition according to any one of the preceding aspects, wherein the subject is 15 to 49 years old.

[0319] A33. The pharmaceutical composition according to any one of the preceding aspects, wherein the subject is a woman of childbearing potential.

[0320] A34. The pharmaceutical composition according to any one of the preceding aspects, wherein the composition is able to increase serum antibody titers to Zika virus in a human by at least 1 log relative to a control within about 7 days from booster immunization.

[0321] A35. The pharmaceutical composition according to any one of the preceding aspects, wherein the composition is able to increase serum antibody titers to Zika virus in a human by at least 1 log relative to a control within about 14 days from booster immunization. A36. The pharmaceutical composition according to any one of the preceding aspects, wherein the composition is able to stimulate seroconversion in at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to 100% of vaccinated subjects within 7 days of booster vaccination, wherein seroconversion is defined as reaching a neutralizing Zika virus antibody titer of at least 10, preferably at least 20.

[0322] A37. The pharmaceutical composition according to any one of the preceding aspects, wherein the composition is able to stimulate seroconversion in at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to 100% of vaccinated subjects within 14 days of booster vaccination, wherein seroconversion is defined as reaching a neutralizing Zika virus antibody titer of at least 10, preferably at least 20.

[0323] A38. The pharmaceutical composition according to aspects A36 or A37, wherein the protective immune response lasts for at least 6 month.

[0324] A39. The pharmaceutical composition according to any one of aspects 36 to 38, wherein the protective immune response lasts for at least 12 months.

[0325] A40. The pharmaceutical composition according to any one of aspects 36 to 39, wherein the protective immune response lasts for at least 24 months.

[0326] A41. The pharmaceutical composition according to any one of aspects 36 to 40, wherein the protective immune response lasts for at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, at least 20 years.

[0327] A42. The pharmaceutical composition according to any one of the preceding aspects, wherein the composition is a vaccine.

[0328] A43. The pharmaceutical composition according to any one of the preceding aspects, for use in a method of treating or preventing a Zika virus infection and / or a Zika vims associated clinical illness of any severity.

[0329] Bl. A method of treating or preventing a Zika virus infection in a subject in need thereof, comprising administering an effective amount of the pharmaceutical composition according to any one of the preceding aspects. Cl. A method for Zika virus inactivation with beta-propiolactone (BPL), comprising contacting the Zika virus with BPL for longer than is required to completely inactivate the Zika virus as measured by plaque assay.

[0330] C2. The method according to aspect Cl, wherein the Zika virus is contacted with BPL for a time period of between 6 and 48 hours, preferably for about 30 hours.

[0331] C3. The method according to aspect Cl or C2, wherein the BPL inactivation is performed at a temperature of between 4°C and 10°C; i.e. a temperature of 7°C ± 3°C.

[0332] C4. The method according to any one of aspects Cl to C3, wherein a buffer solution is added to stabilize the Zika virus during inactivation.

[0333] C5. The method according to any one of aspects Cl to C4, wherein BPL is added twice during the inactivation period.

[0334] C6. The method according to any one of aspects Cl to C5, wherein BPL is added at the beginning of the inactivation period at a concentration of 500 ppm, and wherein the composition is optionally concurrently transferred to a second vessel.

[0335] C7. The method according to any one of aspects Cl to C6, wherein additional BPL is added at about 8 to 9 hours from the beginning of the inactivation period at a concentration of 200 ppm, and wherein the composition is optionally concurrently transferred to a third vessel.

[0336] C8. The method according to aspect C7, wherein the optional transfer to a third vessel is performed via a filter, preferably a 0.45 / 0.2 pm filter.

[0337] C9. The method according to any one of aspects Cl to C8, wherein the BPL concentration does not exceed 600 ppm at any time during the course of the inactivation period.

[0338] CIO. The method according to any one of aspects Cl to C9, wherein the BPL is hydrolyzed following the inactivation period by elevating the temperature of the solution to 35°C ± 2°C for a period of 2.5 ± 0.5 hours.

[0339] Cl 1. The method according to aspect CIO, wherein the solution is cooled to 5 ± 3°C for storage following BPL hydrolysis. DI. A pharmaceutical composition comprising a BPL inactivated Zika virus, wherein said Zika virus is inactivated by the method according to any one of aspects C 1 to C 11.

[0340] D2. The pharmaceutical composition according to aspect DI, for use in a method of treating or preventing a Zika virus infection and / or a Zika virus associated clinical illness of any severity.

[0341] El. A method of treating or preventing a Zika virus infection in a subject in need thereof, comprising administering an effective amount of pharmaceutical composition comprising i) an inactivated Zika virus, ii) an aluminium salt adjuvant and iii) at least one additional adjuvant, wherein said at least one additional adjuvant is a toll-like receptor (TLR) agonist.

[0342] E2. The method according to aspect El, wherein the inactivated Zika virus is a beta-propiolactone (BPL)-inactivated Zika virus.

[0343] E3. The method according to aspect El or E2, wherein the BPL inactivated Zika virus comprises an RNA genome corresponding to the DNA sequence provided by SEQ ID NO: 2 or 3, or a variant sequence that is at least 80% identical to SEQ ID NO: 2 or 3 and able to pack a virulent Zika virus.

[0344] E4. The method according to aspect E3, wherein the variant sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.99% identical to SEQ ID NO: 2 or 3 and able to pack a virulent Zika virus.

[0345] E5. The method according to any one of aspects El to E4, wherein the BPL inactivated Zika virus comprises an E protein as defined by SEQ ID NO: 4 or a variant amino acid sequence that is at least 95% identical to SEQ ID NO: 4 and able to pack a virulent Zika virus, and wherein the E protein comprises one or more modified amino acids selected from the group consisting of M125, H210, M295, H398, H399, H401 and H446, wherein said modifications result from BPL inactivation, and optionally wherein each of the said modified amino acids is present at a frequency of greater than 15%.

[0346] E6. The pharmaceutical composition according to any one of aspects El to E5, wherein the BPL inactivated Zika virus comprises a heterologous mixture of Zika virus with 1 to 10 genomic mutations compared with SEQ ID Nos: 2 or 3 and able to pack a virulent Zika virus. E7. The method according to any one of aspects El to E4, wherein the inactivated Zika virus comprises an RNA genome corresponding to the DNA sequence provided by SEQ ID NO: 2 or 3, or a heterologous mixture thereof.

[0347] E8. The method according to any one of aspects El to E7, wherein the Zika virus has an E protein with the amino acid sequence provided by SEQ ID NO: 4.

[0348] E9. The method according to any one of aspects El to E8, wherein the inactivated Zika virus is provided at 1 to 20 pg / dose, preferably at 1 to 10 pg / dose, especially at about 2, 4 or 8 pg / dose.

[0349] E10. The method according to any one of aspects El to E8, wherein the inactivated Zika virus is provided at a dose of between 100 to 800 AU / dose, preferably 100 to 600 AU / dose, preferably 100 to 400 AU, especially 100, 200 or 400 AU / dose.

[0350] El l. The method according to aspect E10 wherein the dosage in AU is determined by use of a competitive liquid phase ELISA assay as disclosed herein.

[0351] E12. The method according to any one of aspects El to El l, wherein the aluminium salt adjuvant is aluminium hydroxide or aluminium phosphate.

[0352] E13. The method according to aspect El 2, wherein the aluminium hydroxide adjuvanted composition comprises less than 1.25 ppb Cu.

[0353] E14. The method according to any one of aspects El to El 3, wherein the aluminium adjuvant is present in the composition at a concentration of 1 mg / mL.

[0354] E15. The method according to any one of aspects El to El 4, wherein the Toll-like receptor (TLR) agonist is a TLR4, a TLR7, a TLR8, a mixed TLR7 / 8 and / or a TLR9 agonist.

[0355] E16. The method according to any one of aspects El to E15, wherein the TLR agonist comprises a mixture comprising a peptide and a deoxyinosine-containing immunostimulatory oligodeoxynucleic acid molecule (I-ODN).

[0356] El 7. The method according to aspect El 6, wherein the peptide comprises the sequence KLKL5KLK (SEQ ID NO: 5) and the I-ODN comprises oligo-d(IC)i3 (SEQ ID NO: 6). El 8. The method according to any one of aspects El to E 15, wherein the TLR agonist is a synthetic TLR7 / 8 ligand, particularly a synthetic TLR7 / 8 ligand provided in an aqueous formulation.

[0357] E19. The method according to any one of aspects El to E18, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0358] E20. The method according to aspect El 9, wherein the pharmaceutically acceptable excipients essentially consist of sucrose, potassium phosphate and sodium citrate and, optionally, magnesium chloride, D-sorbitol, L-methionine and recombinant human serum albumin (rHSA).

[0359] E21. The method according to aspect E19 or E20, wherein said pharmaceutically acceptable excipients essentially consist of about 5% (w / v) sucrose, about 10 mM potassium phosphate, about 25 mM sodium citrate and about 0.01% (w / v) recombinant human serum albumin (rHSA).

[0360] E22. The method according to any one of aspects El 9 to E21, wherein said pharmaceutically acceptable excipients essentially consist of about 5% (w / v) sucrose; about 5 mM potassium phosphate; about 25 mM sodium citrate; about 5 mM MgC12; about 0.5 % (w / v) D-sorbitol; about 10 mM L-methionine; and about 0.01% (w / v) recombinant human serum albumin (rHSA).

[0361] E23. The method according to any one of aspects E19 to E22, wherein the pharmaceutical composition is delivered to a subject in a priming series followed by one or more boosters.

[0362] E24. The method according to aspect E23, wherein the priming series consists of 2 or 3 doses.

[0363] E25. The method according to aspect E23 or E24, wherein the first two doses of the priming series are administered 1 to 4 weeks apart.

[0364] E26. The method according to any one of aspects E23 to E25, wherein an optional third dose of the priming series is administered between 6 and 12 months after the first two doses.

[0365] E27. The method according to any one of aspects E23 to E26, wherein the one or more boosters are provided at regular intervals, such as every 2, every 3, every 4, every 5 years or every 10 years, following the priming series. E28. The method according to any one of aspects E23 to E27, wherein the dose is administered in a volume of 0.5 mL.

[0366] E29. The method according to any one of aspects E23 to E28, wherein the pharmaceutical composition is administered to the subject orally or by a parenteral route selected from the group consisting of subcutaneous, intracutaneous, intradermal, intravenous, intramuscular, intraarticular, intraperitoneal, intrathecal or by infusion.

[0367] E30. The method according to any one of aspects El to E29, wherein the subject is a human subject.

[0368] E31. The method according to any one of aspects El to E30, wherein the subject is a resident of or is travelling to an endemic region.

[0369] E32. The method according to any one of aspects El to E31, wherein the subject is 15 to 49 years old.

[0370] E33. The method according to any one of aspects El to E32, wherein the subject is a woman of childbearing potential.

[0371] E34. The method according to any one of aspects El to E33, wherein the pharmaceutical composition is able to increase serum antibody titers to Zika virus in the subject by at least 1 log relative to a control within about 7 days from booster immunization.

[0372] E35. The method according to any one of aspects El to E34, wherein the pharmaceutical composition is able to increase serum antibody titers to Zika virus in the subject by at least 1 log relative to a control within about 14 days from booster immunization.

[0373] E36. The method according to any one of aspects El to E35, wherein the pharmaceutical composition is able to stimulate seroconversion in at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to 100% of vaccinated subjects within 7 days of booster vaccination, wherein seroconversion is defined as reaching a neutralizing Zika virus antibody titer of at least 10, preferably at least 20.

[0374] E37. The method according to any one of aspects El to E36, wherein the pharmaceutical composition is able to stimulate seroconversion in at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to 100% of vaccinated subjects within 14 days of booster vaccination, wherein seroconversion is defined as reaching a neutralizing Zika virus antibody titer of at least 10, preferably at least 20.

[0375] E38. The method according to aspect E36 or E37, wherein the protective immune response lasts for at least 6 months.

[0376] E39. The method according to aspect E36 or E37, wherein the protective immune response lasts for at least 12 months.

[0377] E40. The method according to aspect E36 or E37, wherein the protective immune response lasts for at least 24 months.

[0378] E41. The method according to aspect E36 or E37, wherein the protective immune response lasts for at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, at least 20 years.

[0379] E42. The method according to any one of aspects El to E41, wherein the pharmaceutical composition is a vaccine.

[0380] E43. The method according to any one of aspects El to E42, wherein the method treats or prevents a Zika virus infection and / or a Zika virus associated clinical illness of any severity.

[0381] Fl. A process for producing Zika virus particles in host cells in a bioreactor system that uses a falling film for medium oxygenation, characterized in that shear stress and / or medium foaming are minimized by maintaining the Falling Film Height (FFH) at the lowest possible level needed to maintain the required dissolved oxygen (DO) saturation levels at the respective stages of the process, thereby minimizing damage to virus particles compared with more turbulent incubation conditions.

[0382] F2. A process for producing Zika virus particles in host cells in a bioreactor system that uses a falling film for medium oxygenation, wherein said process comprises at least the following steps:

[0383] (i) a virus infection phase with a dissolved oxygen (DO) saturation level of no less than 10%, such as 10 to 30%, 10 to 20%, or 10 to 15%, especially at least 11%, at least 12%, at least 13%, at least 14%, or at least 15%, most preferably no less than 15%, and (ii) a virus production phase with a DO saturation level of around 50%; i.e. between 30 to 70%, preferably between 40 to 60%, more preferably between 45 to 55%, more preferably between 48 to 52%, most preferably 50%; characterized in that the Falling Film Height (FFH) is maintained at the lowest possible level needed to maintain the desired dissolved oxygen (DO) saturation levels at the respective stages of the process.

[0384] F3. The process according to aspect F2, wherein the virus infection phase (i) has a duration of 1 to 5 hours, more preferably 1.5 to 4 hours, most preferably 2.0 to 3.5 hours, especially 1.5 to 3.5 hours, and the virus production phase (ii) has a duration of 20 to 52 hours, preferably 26 to 52 hours, more preferably 30 to 52 hours, more preferably 34 to 52 hours, more preferably 38 to 52 hours, most preferably 48 to 52 hours

[0385] F4. The process according to aspect F2 or F3, wherein the FFH is maintained at 0 cm for at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably for 100% of the duration of the virus infection phase (i).

[0386] F5. The process according to any one of aspects Fl to F4, wherein stirrer speed of the bioreactor remains constant for the entire duration of the virus infection phase.

[0387] F6. The process according to any one of aspects Fl to F5, wherein, if the DO falls below 15% during the virus infection phase (i), an initial FFH of 1 cm is set and increased by 0.5 cm increments until a DO of at least 15% can be maintained.

[0388] F7. The process according to aspect F6, wherein the FFH is not increased to more than 4 cm.

[0389] F8. The process according to any one of aspects Fl to F7, wherein the FFH at the beginning of the virus production phase (ii) is set at 2 cm and is increased by 0.5 cm increments to a maximum of 4 cm.

[0390] F9. The process according to any one of aspects Fl to F8, wherein the FFH during the virus production phase (ii) is maintained at less than 4 cm, preferably less than 3.5 cm, preferably less than 3.0 cm, preferably less than 2.5 cm, preferably less than 2 cm, most preferably about 2 to 3 cm. F10. The process according to any one of aspects Fl to F9, wherein the bioreactor is a fixed-bed bioreactor.

[0391] Fl 1. The process according to any one of aspects Fl to F10, wherein the bioreactor comprises a fixed bed provided in a spiral shape wherein the medium recirculates through the bed, such as the bioreactor provided in Figs 13A and 13B; wherein the bioreactor provides a fixed bed embedded in a chamber through which the medium circulates, such as the bioreactor shown in Fig. 11C; wherein the bioreactor comprises a flask, wherein the medium is pumped out through a dip tube and returned to the chamber in a waterfall, such as shown in Fig. 1 ID; or wherein in the bioreactor FFH is adjusted by the weight of the medium introduced into the system, such as shown in Fig. 1 IE.

[0392] F12. The process according to any one of aspects Fl to Fl 1, wherein the bioreactor is selected from a Scale-XTM bioreactor, an iCELLis Nano bioreactor system and an iCELLis 500 bioreactor system.

[0393] F13. The process according to any one of aspects Fl to Fl 2, wherein the bioreactor is an iCellis 500 bioreactor system.

[0394] F14. The process according to any one of aspects Fl to Fl 3, wherein the host cells are at an optimized density at the time of infection.

[0395] F15. The process according to aspect F14, wherein optimized density means that the cells are at a level sufficient to produce virus, but low enough to enable a minimized FFH.

[0396] F16. The process according to aspect F14 or F15, wherein the optimized density is about 0.5 to 2.5 x 105cells / cm2, especially about 1.0 to 2.0 x 105cells / cm2, or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9 x 105 / cm2.

[0397] F17. The process according to any one of aspects Fl to F16, wherein the multiplicity of infection (MOI) used to infect the host cells is 0.001 to 1, 0.01 to 0.1, preferably 0.03 to 0.5, most preferably 0.03.

[0398] F18. The process according to any one of aspects Fl to F17, wherein the host cell is a mammalian cell, preferably a vero cell line. F19. The process according to any one of aspects Fl to F18, wherein the Zika virus particle comprises an RNA genome corresponding to the DNA sequence provided by the nucleic acid sequence of SEQ ID NO: 2, or a variant nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO: 2 and able to pack a virulent Zika virus.

[0399] F20. The process according to any one of aspects Fl to F18, wherein the Zika virus particle comprises an RNA genome corresponding to the DNA sequence provided by the nucleic acid sequence of SEQ ID NO: 3, or a variant nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO: 3 and able to pack a virulent Zika virus.

[0400] F21. The process according to aspect F19 or F20, wherein the Zika virus particle comprises an E protein comprising or consisting of (i) an amino acid sequence as defined by SEQ ID NO: 4, or (ii) an amino acid sequence having at least 95%, at least 97% or at least 99% identity to SEQ ID NO: 4, and able to pack a virulent Zika virus particle.

[0401] Gl. A process for producing a pharmaceutical composition according to any one of aspects Al to A43, wherein the process comprises the steps of a) propagating a Zika virus in a bioreactor; b) harvesting the Zika virus to obtain a Zika virus composition (a); c) inactivating Zika virus composition (a) to obtain a Zika virus composition (b); d) subjecting Zika virus composition (b) to one or more further purification steps to obtain a Zika virus composition (c); and e) combining Zika virus composition (c) with i) one or more pharmaceutically acceptable carriers or excipients, ii) an aluminium salt adjuvant, and iii) at least one additional adjuvant, wherein said at least one additional adjuvant is a toll-like receptor (TLR) agonist.

[0402] SEQUENCES

[0403] SEQ ID NO: 1

[0404] KJ776791.1, Zika virus strain H / PF / 2013 polyprotein gene, complete cds

[0405] AGTATCAACAGGTTTTATTTTGGATTTGGAAACGAGAGTTTCTGGTCATGAAAAACCCAAAAAAGAAATCCGGAGGATTCCGGATTGTC

[0406] AATATGCTAAAACGCGGAGTAGCCCGTGTGAGCCCCTTTGGGGGCTTGAAGAGGCTGCCAGCCGGACTTCTGCTGGGTCATGGGCCCA

[0407] TCAGGATGGTCTTGGCGATTCTAGCCTTTTTGAGATTCACGGCAATCAAGCCATCACTGGGTCTCATCAATAGATGGGGTTCAGTGGGG

[0408] AAAAAAGAGGCTATGGAAATAATAAAGAAGTTCAAGAAAGATCTGGCTGCCATGCTGAGAATAATCAATGCTAGGAAGGAGAAGAAG

[0409] AGACGAGGCGCAGATACTAGTGTCGGAATTGTTGGCCTCCTGCTGACCACAGCTATGGCAGCGGAGGTCACTAGACGTGGGAGTGCAT

[0410] ACTATATGTACTTGGACAGAAACGACGCTGGGGAGGCCATATCTTTTCCAACCACATTGGGGATGAATAAGTGTTATATACAGATCATG

[0411] GATCTTGGACACATGTGTGATGCCACCATGAGCTATGAATGCCCTATGCTGGATGAGGGGGTGGAACCAGATGACGTCGATTGTTGGT

[0412] GCAACACGACGTCAACTTGGGTTGTGTACGGAACCTGCCATCACAAAAAAGGTGAAGCACGGAGATCTAGAAGAGCTGTGACGCTCCC

[0413] CTCCCATTCCACTAGGAAGCTGCAAACGCGGTCGCAAACCTGGTTGGAATCAAGAGAATACACAAAGCACTTGATTAGAGTCGAAAATT

[0414] GGATATTCAGGAACCCTGGCTTCGCGTTAGCAGCAGCTGCCATCGCTTGGCTTTTGGGAAGCTCAACGAGCCAAAAAGTCATATACTTG

[0415] GTCATGATACTGCTGATTGCCCCGGCATACAGCATCAGGTGCATAGGAGTCAGCAATAGGGACTTTGTGGAAGGTATGTCAGGTGGGA

[0416] CTTGGGTTGATGTTGTCTTGGAACATGGAGGTTGTGTCACCGTAATGGCACAGGACAAACCGACTGTCGACATAGAGCTGGTTACAACA ACAGTCAGCAACATGGCGGAGGTAAGATCCTACTGCTATGAGGCATCAATATCGGACATGGCTTCGGACAGCCGCTGCCCAACACAAG GTGAAGCCTACCTTGACAAGCAATCAGACACTCAATATGTCTGCAAAAGAACGTTAGTGGACAGAGGCTGGGGAAATGGATGTGGACT TTTTGGCAAAGGGAGCCTGGTGACATGCGCTAAGTTTGCATGCTCCAAGAAAATGACCGGGAAGAGCATCCAGCCAGAGAATCTGGAG TACCGGATAATGCTGTCAGTTCATGGCTCCCAGCACAGTGGGATGATCGTTAATGACACAGGACATGAAACTGATGAGAATAGAGCGA AGGTTGAGATAACGCCCAATTCACCAAGAGCCGAAGCCACCCTGGGGGGTTTTGGAAGCCTAGGACTTGATTGTGAACCGAGGACAGG CCTTGACTTTTCAGATTTGTATTACTTGACTATGAATAACAAGCACTGGTTGGTTCACAAGGAGTGGTTCCACGACATTCCATTACCTTGG CACGCTGGGGCAGACACCGGAACTCCACACTGGAACAACAAAGAAGCACTGGTAGAGTTCAAGGACGCACATGCCAAAAGGCAAACT GTCGTGGTTCTAGGGAGTCAAGAAGGAGCAGTTCACACGGCCCTTGCTGGAGCTCTGGAGGCTGAGATGGATGGTGCAAAGGGAAGG CTGTCCTCTGGCCACTTGAAATGTCGCCTGAAAATGGATAAACTTAGATTGAAGGGCGTGTCATACTCCTTGTGTACCGCAGCGTTCACA TTCACCAAGATCCCGGCTGAAACACTGCACGGGACAGTCACAGTGGAGGTACAGTACGCAGGGACAGATGGACCTTGCAAGGTTCCAG CTCAGATGGCGGTGGACATGCAAACTCTGACCCCAGTTGGGAGGTTGATAACCGCTAACCCCGTAATCACTGAAAGCACTGAGAACTCT AAGATGATGCTGGAACTTGATCCACCATTTGGGGACTCTTACATTGTCATAGGAGTCGGGGAGAAGAAGATCACCCACCACTGGCACAG GAGTGGCAGCACCATTGGAAAAGCATTTGAAGCCACTGTGAGAGGTGCCAAGAGAATGGCAGTCTTGGGAGACACAGCCTGGGACTTT GGATCAGTTGGAGGCGCTCTCAACTCATTGGGCAAGGGCATCCATCAAATTTTTGGAGCAGCTTTCAAATCATTGTTTGGAGGAATGTCC TGGTTCTCACAAATTCTCATTGGAACGTTGCTGATGTGGTTGGGTCTGAACACAAAGAATGGATCTATTTCCCTTATGTGCTTGGCCTTAG GGGGAGTGTTGATCTTCTTATCCACAGCTGTCTCTGCTGATGTGGGGTGCTCGGTGGACTTCTCAAAGAAGGAGACGAGATGCGGTACA GGGGTGTTCGTCTATAACGACGTTGAAGCCTGGAGGGACAGGTACAAGTACCATCCTGACTCCCCCCGTAGATTGGCAGCAGCAGTCA AGCAAGCCTGGGAAGATGGTATCTGTGGGATCTCCTCTGTTTCAAGAATGGAAAACATCATGTGGAGATCAGTAGAAGGGGAGCTCAA CGCAATCCTGGAAGAGAATGGAGTTCAACTGACGGTCGTTGTGGGATCTGTAAAAAACCCCATGTGGAGAGGTCCACAGAGATTGCCC GTGCCTGTGAACGAGCTGCCCCACGGCTGGAAGGCTTGGGGGAAATCGTACTTCGTCAGAGCAGCAAAGACAAATAACAGCTTTGTCG TGGATGGTGACACACTGAAGGAATGCCCACTCAAACATAGAGCATGGAACAGCTTTCTTGTGGAGGATCATGGGTTCGGGGTATTTCAC ACTAGTGTCTGGCTCAAGGTTAGAGAAGATTATTCATTAGAGTGTGATCCAGCCGTTATTGGAACAGCTGTTAAGGGAAAGGAGGCTGT ACACAGTGATCTAGGCTACTGGATTGAGAGTGAGAAGAATGACACATGGAGGCTGAAGAGGGCCCATCTGATCGAGATGAAAACATGT GAATGGCCAAAGTCCCACACATTGTGGACAGATGGAATAGAAGAGAGTGATCTGATCATACCCAAGTCTTTAGCTGGGCCACTCAGCCA TCACAATACCAGAGAGGGCTACAGGACCCAAATGAAAGGGCCATGGCACAGTGAAGAGCTTGAAATTCGGTTTGAGGAATGCCCAGGC ACTAAGGTCCACGTGGAGGAAACATGTGGAACAAGAGGACCATCTCTGAGATCAACCACTGCAAGCGGAAGGGTGATCGAGGAATGG TGCTGCAGGGAGTGCACAATGCCCCCACTGTCGTTCCGGGCTAAAGATGGCTGTTGGTATGGAATGGAGATAAGGCCCAGGAAAGAAC CAGAAAGTAACTTAGTAAGGTCAATGGTGACTGCAGGATCAACTGATCACATGGATCACTTCTCCCTTGGAGTGCTTGTGATTCTGCTCA TGGTGCAGGAAGGGCTGAAGAAGAGAATGACCACAAAGATCATCATAAGCACATCGATGGCAGTGCTGGTAGCTATGATCCTGGGAG GATTTTCAATGAGTGACCTGGCTAAGCTTGCAATTTTGATGGGTGCCACCTTCGCGGAAATGAACACTGGAGGAGATGTAGCTCATCTG GCGCTGATAGCGGCATTCAAAGTCAGACCAGCGTTGCTGGTATCTTTCATCTTCAGAGCTAATTGGACACCCCGTGAAAGCATGCTGCTG GCCTTGGCCTCGTGTCTTTTGCAAACTGCGATCTCCGCCTTGGAAGGCGACCTGATGGTTCTCATCAATGGTTTTGCTTTGGCCTGGTTGG CAATACGAGCGATGGTTGTTCCACGCACTGATAACATCACCTTGGCAATCCTGGCTGCTCTGACACCACTGGCCCGGGGCACACTGCTTG TGGCGTGGAGAGCAGGCCTTGCTACTTGCGGGGGGTTTATGCTCCTCTCTCTGAAGGGAAAAGGCAGTGTGAAGAAGAACTTACCATTT GTCATGGCCCTGGGACTAACCGCTGTGAGGCTGGTCGACCCCATCAACGTGGTGGGACTGCTGTTGCTCACAAGGAGTGGGAAGCGGA GCTGGCCCCCTAGCGAAGTACTCACAGCTGTTGGCCTGATATGCGCATTGGCTGGAGGGTTCGCCAAGGCAGATATAGAGATGGCTGG GCCCATGGCCGCGGTCGGTCTGCTAATTGTCAGTTACGTGGTCTCAGGAAAGAGTGTGGACATGTACATTGAAAGAGCAGGTGACATC ACATGGGAAAAAGATGCGGAAGTCACTGGAAACAGTCCCCGGCTCGATGTGGCGCTAGATGAGAGTGGTGATTTCTCCCTGGTGGAGG ATGACGGTCCCCCCATGAGAGAGATCATACTCAAGGTGGTCCTGATGACCATCTGTGGCATGAACCCAATAGCCATACCCTTTGCAGCT GGAGCGTGGTACGTATACGTGAAGACTGGAAAAAGGAGTGGTGCTCTATGGGATGTGCCTGCTCCCAAGGAAGTAAAAAAGGGGGAG ACCACAGATGGAGTGTACAGAGTAATGACTCGTAGACTGCTAGGTTCAACACAAGTTGGAGTGGGAGTTATGCAAGAGGGGGTCTTTC ACACTATGTGGCACGTCACAAAAGGATCCGCGCTGAGAAGCGGTGAAGGGAGACTTGATCCATACTGGGGAGATGTCAAGCAGGATCT GGTGTCATACTGTGGTCCATGGAAGCTAGATGCCGCCTGGGACGGGCACAGCGAGGTGCAGCTCTTGGCCGTGCCCCCCGGAGAGAG AGCGAGGAACATCCAGACTCTGCCCGGAATATTTAAGACAAAGGATGGGGACATTGGAGCGGTTGCGCTGGATTACCCAGCAGGAACT TCAGGATCTCCAATCCTAGACAAGTGTGGGAGAGTGATAGGACTTTATGGCAATGGGGTCGTGATCAAAAATGGGAGTTATGTTAGTG CCATCACCCAAGGGAGGAGGGAGGAAGAGACTCCTGTTGAGTGCTTCGAGCCTTCGATGCTGAAGAAGAAGCAGCTAACTGTCTTAGA CTTGCATCCTGGAGCTGGGAAAACCAGGAGAGTTCTTCCTGAAATAGTCCGTGAAGCCATAAAAACAAGACTCCGTACTGTGATCTTAG CTCCAACCAGGGTTGTCGCTGCTGAAATGGAGGAAGCCCTTAGAGGGCTTCCAGTGCGTTATATGACAACAGCAGTCAATGTCACCCAC TCTGGAACAGAAATCGTCGACTTAATGTGCCATGCCACCTTCACTTCACGTCTACTACAGCCAATCAGAGTCCCCAACTATAATCTGTATA TTATGGATGAGGCCCACTTCACAGATCCCTCAAGTATAGCAGCAAGAGGATACATTTCAACAAGGGTTGAGATGGGCGAGGCGGCTGC CATCTTCATGACCGCCACGCCACCAGGAACCCGTGACGCATTTCCGGACTCCAACTCACCAATTATGGACACCGAAGTGGAAGTCCCAG AGAGAGCCTGGAGCTCAGGCTTTGATTGGGTGACGGATCATTCTGGAAAAACAGTTTGGTTTGTTCCAAGCGTGAGGAACGGCAATGA GATCGCAGCTTGTCTGACAAAGGCTGGAAAACGGGTCATACAGCTCAGCAGAAAGACTTTTGAGACAGAGTTCCAGAAAACAAAACAT

[0417] CAAGAGTGGGACTTTGTCGTGACAACTGACATTTCAGAGATGGGCGCCAACTTTAAAGCTGACCGTGTCATAGATTCCAGGAGATGCCT AAAGCCGGTCATACTTGATGGCGAGAGAGTCATTCTGGCTGGACCCATGCCTGTCACACATGCCAGCGCTGCCCAGAGGAGGGGGCGC ATAGGCAGGAATCCCAACAAACCTGGAGATGAGTATCTGTATGGAGGTGGGTGCGCAGAGACTGACGAAGACCATGCACACTGGCTTG AAGCAAGAATGCTCCTTGACAATATTTACCTCCAAGATGGCCTCATAGCCTCGCTCTATCGACCTGAGGCCGACAAAGTAGCAGCCATTG AGGGAGAGTTCAAGCTTAGGACGGAGCAAAGGAAGACCTTTGTGGAACTCATGAAAAGAGGAGATCTTCCTGTTTGGCTGGCCTATCA GGTTGCATCTGCCGGAATAACCTACACAGATAGAAGATGGTGCTTTGATGGCACGACCAACAACACCATAATGGAAGACAGTGTGCCG GCAGAGGTGTGGACCAGACACGGAGAGAAAAGAGTGCTCAAACCGAGGTGGATGGACGCCAGAGTTTGTTCAGATCATGCGGCCCTG AAGTCATTCAAGGAGTTTGCCGCTGGGAAAAGAGGAGCGGCTTTTGGAGTGATGGAAGCCCTGGGAACACTGCCAGGACACATGACA GAGAGATTCCAGGAAGCCATTGACAACCTCGCTGTGCTCATGCGGGCAGAGACTGGAAGCAGGCCTTACAAAGCCGCGGCGGCCCAAT TGCCGGAGACCCTAGAGACCATTATGCTTTTGGGGTTGCTGGGAACAGTCTCGCTGGGAATCTTTTTCGTCTTGATGAGGAACAAGGGC ATAGGGAAGATGGGCTTTGGAATGGTGACTCTTGGGGCCAGCGCATGGCTCATGTGGCTCTCGGAAATTGAGCCAGCCAGAATTGCAT GTGTCCTCATTGTTGTGTTCCTATTGCTGGTGGTGCTCATACCTGAGCCAGAAAAGCAAAGATCTCCCCAGGACAACCAAATGGCAATCA TCATCATGGTAGCAGTAGGTCTTCTGGGCTTGATTACCGCCAATGAACTCGGATGGTTGGAGAGAACAAAGAGTGACCTAAGCCATCTA ATGGGAAGGAGAGAGGAGGGGGCAACCATAGGATTCTCAATGGACATTGACCTGCGGCCAGCCTCAGCTTGGGCCATCTATGCTGCCT TGACAACTTTCATTACCCCAGCCGTCCAACATGCAGTGACCACTTCATACAACAACTACTCCTTAATGGCGATGGCCACGCAAGCTGGAG TGTTGTTTGGTATGGGCAAAGGGATGCCATTCTACGCATGGGACTTTGGAGTCCCGCTGCTAATGATAGGTTGCTACTCACAATTAACAC CCCTGACCCTAATAGTGGCCATCATTTTGCTCGTGGCGCACTACATGTACTTGATCCCAGGGCTGCAGGCAGCAGCTGCGCGTGCTGCCC AGAAGAGAACGGCAGCTGGCATCATGAAGAACCCTGTTGTGGATGGAATAGTGGTGACTGACATTGACACAATGACAATTGACCCCCA AGTGGAGAAAAAGATGGGACAGGTGCTACTCATAGCAGTAGCCGTCTCCAGCGCCATACTGTCGCGGACCGCCTGGGGGTGGGGGGA GGCTGGGGCCCTGATCACAGCGGCAACTTCCACTTTGTGGGAAGGCTCTCCGAACAAGTACTGGAACTCCTCTACAGCCACTTCACTGT GTAACATTTTTAGGGGAAGTTACTTGGCTGGAGCTTCTCTAATCTACACAGTAACAAGAAACGCTGGCTTGGTCAAGAGACGTGGGGGT GGAACAGGAGAGACCCTGGGAGAGAAATGGAAGGCCCGCTTGAACCAGATGTCGGCCCTGGAGTTCTACTCCTACAAAAAGTCAGGC ATCACCGAGGTGTGCAGAGAAGAGGCCCGCCGCGCCCTCAAGGACGGTGTGGCAACGGGAGGCCATGCTGTGTCCCGAGGAAGTGCA AAGCTGAGATGGTTGGTGGAGCGGGGATACCTGCAGCCCTATGGAAAGGTCATTGATCTTGGATGTGGCAGAGGGGGCTGGAGTTAC TACGCCGCCACCATCCGCAAAGTTCAAGAAGTGAAAGGATACACAAAAGGAGGCCCTGGTCATGAAGAACCCATGTTGGTGCAAAGCT ATGGGTGGAACATAGTCCGTCTTAAGAGTGGGGTGGACGTCTTTCATATGGCGGCTGAGCCGTGTGACACGTTGCTGTGTGACATAGG TGAGTCATCATCTAGTCCTGAAGTGGAAGAAGCACGGACGCTCAGAGTCCTCTCCATGGTGGGGGATTGGCTTGAAAAAAGACCAGGA GCCTTTTGTATAAAAGTGTTGTGCCCATACACCAGCACTATGATGGAAACCCTGGAGCGACTGCAGCGTAGGTATGGGGGAGGACTGG TCAGAGTGCCACTCTCCCGCAACTCTACACATGAGATGTACTGGGTCTCTGGAGCGAAAAGCAACACCATAAAAAGTGTGTCCACCACG AGCCAGCTCCTCTTGGGGCGCATGGACGGGCCCAGGAGGCCAGTGAAATATGAGGAGGATGTGAATCTCGGCTCTGGCACGCGGGCT GTGGTAAGCTGCGCTGAAGCTCCCAACATGAAGATCATTGGTAACCGCATTGAAAGGATCCGCAGTGAGCACGCGGAAACGTGGTTCT TTGACGAGAACCACCCATATAGGACATGGGCTTACCATGGAAGCTATGAGGCCCCCACACAAGGGTCAGCGTCCTCTCTAATAAACGGG GTTGTCAGGCTCCTGTCAAAACCCTGGGATGTGGTGACTGGAGTCACAGGAATAGCCATGACCGACACCACACCGTATGGTCAGCAAA GAGTTTTCAAGGAAAAAGTGGACACTAGGGTGCCAGACCCCCAAGAAGGCACTCGTCAGGTTATGAGCATGGTCTCTTCCTGGTTGTGG AAAGAGCTAGGCAAACACAAACGGCCACGAGTCTGTACCAAAGAAGAGTTCATCAACAAGGTTCGTAGCAATGCAGCATTAGGGGCAA TATTTGAAGAGGAAAAAGAGTGGAAGACTGCAGTGGAAGCTGTGAACGATCCAAGGTTCTGGGCTCTAGTGGACAAGGAAAGAGAGC ACCACCTGAGAGGAGAGTGCCAGAGTTGTGTGTACAACATGATGGGAAAAAGAGAAAAGAAACAAGGGGAATTTGGAAAGGCCAAG GGCAGCCGCGCCATCTGGTATATGTGGCTAGGGGCTAGATTTCTAGAGTTCGAAGCCCTTGGATTCTTGAACGAGGATCACTGGATGG GGAGAGAGAACTCAGGAGGTGGTGTTGAAGGGCTGGGATTACAAAGACTCGGATATGTCCTAGAAGAGATGAGTCGCATACCAGGAG GAAGGATGTATGCAGATGACACTGCTGGCTGGGACACCCGCATCAGCAGGTTTGATCTGGAGAATGAAGCTCTAATCACCAACCAAAT

[0418] GGAGAAAGGGCACAGGGCCTTGGCATTGGCCATAATCAAGTACACATACCAAAACAAAGTGGTAAAGGTCCTTAGACCAGCTGAAAAA GGGAAGACAGTTATGGACATTATTTCGAGACAAGACCAAAGGGGGAGCGGACAAGTTGTCACTTACGCTCTTAACACATTTACCAACCT AGTGGTGCAACTCATTCGGAATATGGAGGCTGAGGAAGTTCTAGAGATGCAAGACTTGTGGCTGCTGCGGAGGTCAGAGAAAGTGAC CAACTGGTTGCAGAGCAACGGATGGGATAGGCTCAAACGAATGGCAGTCAGTGGAGATGATTGCGTTGTGAAGCCAATTGATGATAG GTTTGCACATGCCCTCAGGTTCTTGAATGATATGGGAAAAGTTAGGAAGGACACACAAGAGTGGAAACCCTCAACTGGATGGGACAAC TGGGAAGAAGTTCCGTTTTGCTCCCACCACTTCAACAAGCTCCATCTCAAGGACGGGAGGTCCATTGTGGTTCCCTGCCGCCACCAAGAT GAACTGATTGGCCGGGCCCGCGTCTCTCCAGGGGCGGGATGGAGCATCCGGGAGACTGCTTGCCTAGCAAAATCATATGCGCAAATGT GGCAGCTCCTTTATTTCCACAGAAGGGACCTCCGACTGATGGCCAATGCCATTTGTTCATCTGTGCCAGTTGACTGGGTTCCAACTGGGA GAACTACCTGGTCAATCCATGGAAAGGGAGAATGGATGACCACTGAAGACATGCTTGTGGTGTGGAACAGAGTGTGGATTGAGGAGA ACGACCACATGGAAGACAAGACCCCAGTTACGAAATGGACAGACATTCCCTATTTGGGAAAAAGGGAAGACTTGTGGTGTGGATCTCT CATAGGGCACAGACCGCGCACCACCTGGGCTGAGAACATTAAAAACACAGTCAACATGGTGCGCAGGATCATAGGTGATGAAGAAAA GTACATGGACTACCTATCCACCCAAGTTCGCTACTTGGGTGAAGAAGGGTCTACACCTGGAGTGCTGTAAGCACCAATCTTAGTGTTGTC AGGCCTGCTAGTCAGCCACAGCTTGGGGAAAGCTGTGCAGCCTGTGACCCCCCCAGGAGAAGCTGGGAAACCAAGCCTATAGTCAGGC CGAGAACGCCATGGCACGGAAGAAGCCATGCTGCCTGTGAGCCCCTCAGAGGACACTGAGTCAAAAAACCCCACGCGCTTGGAGGCGC AGGATGGGAAAAGAAGGTGGCGACCTTCCCCACCCTTCAATCTGGGGCCTGAACTGGAGATCAGCTGTGGATCTCCAGAAGAGGGACT AGTGGTTAGAGGAG

[0419] SEQ ID N0: 2

[0420] Zika virus strain H / PF / 2013 as sequenced CAGACTGCGACAGTTCGAGTTTGAAGCGAAAGCTAGCAACAGTATCAACAGGTTTTATTTTGGATTTGGAAACGAGAGTTTCTGGTCAT GAAAAACCCAAAAAAGAAATCCGGAGGATTCCGGATTGTCAATATGCTAAAACGCGGAGTAGCCCGTGTGAGCCCCTTTGGGGGCTTG AAGAGGCTGCCAGCCGGACTTCTGCTGGGTCATGGGCCCATCAGGATGGTCTTGGCGATTCTAGCCTTTTTGAGATTCACGGCAATCAA GCCATCACTGGGTCTCATCAATAGATGGGGTTCAGTGGGGAAAAAAGAGGCTATGGAAATAATAAAGAAGTTCAAGAAAGATCTGGCT GCCATGCTGAGAATAATCAATGCTAGGAAGGAGAAGAAGAGACGAGGCGCAGATACTAGTGTCGGAATTGTTGGCCTCCTGCTGACCA CAGCTATGGCAGCGGAGGTCACTAGACGTGGGAGTGCATACTATATGTACTTGGACAGAAACGACGCTGGGGAGGCCATATCTTTTCC AACCACATTGGGGATGAATAAGTGTTATATACAGATCATGGATCTTGGACACATGTGTGATGCCACCATGAGCTATGAATGCCCTATGCT GGATGAGGGGGTGGAACCAGATGACGTCGATTGTTGGTGCAACACGACGTCAACTTGGGTTGTGTACGGAACCTGCCATCACAAAAAA GGTGAAGCACGGAGATCTAGAAGAGCTGTGACGCTCCCCTCCCATTCCACTAGGAAGCTGCAAACGCGGTCGCAAACCTGGTTGGAAT CAAGAGAATACACAAAGCACTTGATTAGAGTCGAAAATTGGATATTCAGGAACCCTGGCTTCGCGTTAGCAGCAGCTGCCATCGCTTGG CTTTTGGGAAGCTCAACGAGCCAAAAAGTCATATACTTGGTCATGATACTGCTGATTGCCCCGGCATACAGCATCAGGTGCATAGGAGT CAGCAATAGGGACTTTGTGGAAGGTATGTCAGGTGGGACTTGGGTTGATGTTGTCTTGGAACATGGAGGTTGTGTCACCGTAATGGCA CAGGACAAACCGACTGTCGACATAGAGCTGGTTACAACAACAGTCAGCAACATGGCGGAGGTAAGATCCTACTGCTATGAGGCATCAA TATCGGACATGGCTTCGGACAGCCGCTGCCCAACACAAGGTGAAGCCTACCTTGACAAGCAATCAGACACTCAATATGTCTGCAAAAGA ACGTTAGTGGACAGAGGCTGGGGAAATGGATGTGGACTTTTTGGCAAAGGGAGCCTGGTGACATGCGCTAAGTTTGCATGCTCCAAGA AAATGACCGGGAAGAGCATCCAGCCAGAGAATCTGGAGTACCGGATAATGCTGTCAGTTCATGGCTCCCAGCACAGTGGGATGATCGT TAATGACACAGGACATGAAACTGATGAGAATAGAGCGAAGGTTGAGATAACGCCCAATTCACCAAGAGCCGAAGCCACCCTGGGGGG TTTTGGAAGCCTAGGACTTGATTGTGAACCGAGGACAGGCCTTGACTTTTCAGATTTGTATTACTTGACTATGAATAACAAGCACTGGTT GGTTCACAAGGAGTGGTTCCACGACATTCCATTACCTTGGCACGCTGGGGCAGACACCGGAACTCCACACTGGAACAACAAAGAAGCA CTGGTAGAGTTCAAGGACGCACATGCCAAAAGGCAAACTGTCGTGGTTCTAGGGAGTCAAGAAGGAGCAGTTCACACGGCCCTTGCTG GAGCTCTGGAGGCTGAGATGGATGGTGCAAAGGGAAGGCTGTCCTCTGGCCACTTGAAATGTCGCCTGAAAATGGATAAACTTAGATT GAAGGGCGTGTCATACTCCTTGTGTACCGCAGCGTTCACATTCACCAAGATCCCGGCTGAAACACTGCACGGGACAGTCACAGTGGAGG TACAGTACGCAGGGACAGATGGACCTTGCAAGGTTCCAGCTCAGATGGCGGTGGACATGCAAACTCTGACCCCAGTTGGGAGGTTGAT AACCGCTAACCCCGTAATCACTGAAAGCACTGAGAACTCTAAGATGATGCTGGAACTTGATCCACCATTTGGGGACTCTTACATTGTCAT AGGAGTCGGGGAGAAGAAGATCACCCACCACTGGCACAGGAGTGGCAGCACCATTGGAAAAGCATTTGAAGCCACTGTGAGAGGTGC CAAGAGAATGGCAGTCTTGGGAGACACAGCCTGGGACTTTGGATCAGTTGGAGGCGCTCTCAACTCATTGGGCAAGGGCATCCATCAA ATTTTTGGAGCAGCTTTCAAATCATTGTTTGGAGGAATGTCCTGGTTCTCACAAATTCTCATTGGAACGTTGCTGATGTGGTTGGGTCTG AACACAAAGAATGGATCTATTTCCCTTATGTGCTTGGCCTTAGGGGGAGTGTTGATCTTCTTATCCACAGCTGTCTCTGCTGATGTGGGG TGCTCGGTGGACTTCTCAAAGAAGGAGACGAGATGCGGTACAGGGGTGTTCGTCTATAACGACGTTGAAGCCTGGAGGGACAGGTAC AAGTACCATCCTGACTCCCCCCGTAGATTGGCAGCAGCAGTCAAGCAAGCCTGGGAAGATGGTATCTGTGGGATCTCCTCTGTTTCAAG AATGGAAAACATCATGTGGAGATCAGTAGAAGGGGAGCTCAACGCAATCCTGGAAGAGAATGGAGTTCAACTGACGGTCGTTGTGGG ATCTGTAAAAAACCCCATGTGGAGAGGTCCACAGAGATTGCCCGTGCCTGTGAACGAGCTGCCCCACGGCTGGAAGGCTTGGGGGAAA TCGTACTTCGTCAGAGCAGCAAAGACAAATAACAGCTTTGTCGTGGATGGTGACACACTGAAGGAATGCCCACTCAAACATAGAGCATG GAACAGCTTTCTTGTGGAGGATCATGGGTTCGGGGTATTTCACACTAGTGTCTGGCTCAAGGTTAGAGAAGATTATTCATTAGAGTGTG ATCCAGCCGTTATTGGAACAGCTGTTAAGGGAAAGGAGGCTGTACACAGTGATCTAGGCTACTGGATTGAGAGTGAGAAGAATGACAC ATGGAGGCTGAAGAGGGCCCATCTGATCGAGATGAAAACATGTGAATGGCCAAAGTCCCACACATTGTGGACAGATGGAATAGAAGA GAGTGATCTGATCATACCCAAGTCTTTAGCTGGGCCACTCAGCCATCACAATACCAGAGAGGGCTACAGGACCCAAATGAAAGGGCCAT GGCACAGTGAAGAGCTTGAAATTCGGTTTGAGGAATGCCCAGGCACTAAGGTCCACGTGGAGGAAACATGTGGAACAAGAGGACCAT CTCTGAGATCAACCACTGCAAGCGGAAGGGTGATCGAGGAATGGTGCTGCAGGGAGTGCACAATGCCCCCACTGTCGTTCCGGGCTAA AGATGGCTGTTGGTATGGAATGGAGATAAGGCCCAGGAAAGAACCAGAAAGTAACTTAGTAAGGTCAATGGTGACTGCAGGATCAACT GATCACATGGATCACTTCTCCCTTGGAGTGCTTGTGATTCTGCTCATGGTGCAGGAAGGGCTGAAGAAGAGAATGACCACAAAGATCAT CATAAGCACATCGATGGCAGTGCTGGTAGCTATGATCCTGGGAGGATTTTCAATGAGTGACCTGGCTAAGCTTGCAATTTTGATGGGTG CCACCTTCGCGGAAATGAACACTGGAGGAGATGTAGCTCATCTGGCGCTGATAGCGGCATTCAAAGTCAGACCAGCGTTGCTGGTATCT TTCATCTTCAGAGCTAATTGGACACCCCGTGAAAGCATGCTGCTGGCCTTGGCCTCGTGTCTTTTGCAAACTGCGATCTCCGCCTTGGAA GGCGACCTGATGGTTCTCATCAATGGTTTTGCTTTGGCCTGGTTGGCAATACGAGCGATGGTTGTTCCACGCACTGATAACATCACCTTG GCAATCCTGGCTGCTCTGACACCACTGGCCCGGGGCACACTGCTTGTGGCGTGGAGAGCAGGCCTTGCTACTTGCGGGGGGTTTATGCT CCTCTCTCTGAAGGGAAAAGGCAGTGTGAAGAAGAACTTACCATTTGTCATGGCCCTGGGACTAACCGCTGTGAGGCTGGTCGACCCCA TCAACGTGGTGGGACTGCTGTTGCTCACAAGGAGTGGGAAGCGGAGCTGGCCCCCTAGCGAAGTACTCACAGCTGTTGGCCTGATATG CGCATTGGCTGGAGGGTTCGCCAAGGCAGATATAGAGATGGCTGGGCCCATGGCCGCGGTCGGTCTGCTAATTGTCAGTTACGTGGTC TCAGGAAAGAGTGTGGACATGTACATTGAAAGAGCAGGTGACATCACATGGGAAAAAGATGCGGAAGTCACTGGAAACAGTCCCCGG CTCGATGTGGCGCTAGATGAGAGTGGTGATTTCTCCCTGGTGGAGGATGACGGTCCCCCCATGAGAGAGATCATACTCAAGGTGGTCCT GATGACCATCTGTGGCATGAACCCAATAGCCATACCCTTTGCAGCTGGAGCGTGGTACGTATACGTGAAGACTGGAAAAAGGAGTGGT GCTCTATGGGATGTGCCTGCTCCCAAGGAAGTAAAAAAGGGGGAGACCACAGATGGAGTGTACAGAGTAATGACTCGTAGACTGCTAG GTTCAACACAAGTTGGAGTGGGAGTTATGCAAGAGGGGGTCTTTCACACTATGTGGCACGTCACAAAAGGATCCGCGCTGAGAAGCGG TGAAGGGAGACTTGATCCATACTGGGGAGATGTCAAGCAGGATCTGGTGTCATACTGTGGTCCATGGAAGCTAGATGCCGCCTGGGAC GGGCACAGCGAGGTGCAGCTCTTGGCCGTGCCCCCCGGAGAGAGAGCGAGGAACATCCAGACTCTGCCCGGAATATTTAAGACAAAG GATGGGGACATTGGAGCGGTTGCGCTGGATTACCCAGCAGGAACTTCAGGATCTCCAATCCTAGACAAGTGTGGGAGAGTGATAGGAC TTTATGGCAATGGGGTCGTGATCAAAAATGGGAGTTATGTTAGTGCCATCACCCAAGGGAGGAGGGAGGAAGAGACTCCTGTTGAGTG CTTCGAGCCTTCGATGCTGAAGAAGAAGCAGCTAACTGTCTTAGACTTGCATCCTGGAGCTGGGAAAACCAGGAGAGTTCTTCCTGAAA TAGTCCGTGAAGCCATAAAAACAAGACTCCGTACTGTGATCTTAGCTCCAACCAGGGTTGTCGCTGCTGAAATGGAGGAAGCCCTTAGA GGGCTTCCAGTGCGTTATATGACAACAGCAGTCAATGTCACCCACTCTGGAACAGAAATCGTCGACTTAATGTGCCATGCCACCTTCACT TCACGTCTACTACAGCCAATCAGAGTCCCCAACTATAATCTGTATATTATGGATGAGGCCCACTTCACAGATCCCTCAAGTATAGCAGCA AGAGGATACATTTCAACAAGGGTTGAGATGGGCGAGGCGGCTGCCATCTTCATGACCGCCACGCCACCAGGAACCCGTGACGCATTTC CGGACTCCAACTCACCAATTATGGACACCGAAGTGGAAGTCCCAGAGAGAGCCTGGAGCTCAGGCTTTGATTGGGTGACGGATCATTCT GGAAAAACAGTTTGGTTTGTTCCAAGCGTGAGGAACGGCAATGAGATCGCAGCTTGTCTGACAAAGGCTGGAAAACGGGTCATACAGC TCAGCAGAAAGACTTTTGAGACAGAGTTCCAGAAAACAAAACATCAAGAGTGGGACTTTGTCGTGACAACTGACATTTCAGAGATGGG CGCCAACTTTAAAGCTGACCGTGTCATAGATTCCAGGAGATGCCTAAAGCCGGTCATACTTGATGGCGAGAGAGTCATTCTGGCTGGAC CCATGCCTGTCACACATGCCAGCGCTGCCCAGAGGAGGGGGCGCATAGGCAGGAATCCCAACAAACCTGGAGATGAGTATCTGTATGG AGGTGGGTGCGCAGAGACTGACGAAGACCATGCACACTGGCTTGAAGCAAGAATGCTCCTTGACAATATTTACCTCCAAGATGGCCTCA TAGCCTCGCTCTATCGACCTGAGGCCGACAAAGTAGCAGCCATTGAGGGAGAGTTCAAGCTTAGGACGGAGCAAAGGAAGACCTTTGT GGAACTCATGAAAAGAGGAGATCTTCCTGTTTGGCTGGCCTATCAGGTTGCATCTGCCGGAATAACCTACACAGATAGAAGATGGTGCT TTGATGGCACGACCAACAACACCATAATGGAAGACAGTGTGCCGGCAGAGGTGTGGACCAGACACGGAGAGAAAAGAGTGCTCAAAC CGAGGTGGATGGACGCCAGAGTTTGTTCAGATCATGCGGCCCTGAAGTCATTCAAGGAGTTTGCCGCTGGGAAAAGAGGAGCGGCTTT TGGAGTGATGGAAGCCCTGGGAACACTGCCAGGACACATGACAGAGAGATTCCAGGAAGCCATTGACAACCTCGCTGTGCTCATGCGG GCAGAGACTGGAAGCAGGCCTTACAAAGCCGCGGCGGCCCAATTGCCGGAGACCCTAGAGACCATTATGCTTTTGGGGTTGCTGGGAA CAGTCTCGCTGGGAATCTTTTTCGTCTTGATGAGGAACAAGGGCATAGGGAAGATGGGCTTTGGAATGGTGACTCTTGGGGCCAGCGC ATGGCTCATGTGGCTCTCGGAAATTGAGCCAGCCAGAATTGCATGTGTCCTCATTGTTGTGTTCCTATTGCTGGTGGTGCTCATACCTGA GCCAGAAAAGCAAAGATCTCCCCAGGACAACCAAATGGCAATCATCATCATGGTAGCAGTAGGTCTTCTGGGCTTGATTACCGCCAATG AACTCGGATGGTTGGAGAGAACAAAGAGTGACCTAAGCCATCTAATGGGAAGGAGAGAGGAGGGGGCAACCATAGGATTCTCAATGG ACATTGACCTGCGGCCAGCCTCAGCTTGGGCCATCTATGCTGCCTTGACAACTTTCATTACCCCAGCCGTCCAACATGCAGTGACCACTTC ATACAACAACTACTCCTTAATGGCGATGGCCACGCAAGCTGGAGTGTTGTTTGGTATGGGCAAAGGGATGCCATTCTACGCATGGGACT TTGGAGTCCCGCTGCTAATGATAGGTTGCTACTCACAATTAACACCCCTGACCCTAATAGTGGCCATCATTTTGCTCGTGGCGCACTACAT GTACTTGATCCCAGGGCTGCAGGCAGCAGCTGCGCGTGCTGCCCAGAAGAGAACGGCAGCTGGCATCATGAAGAACCCTGTTGTGGAT GGAATAGTGGTGACTGACATTGACACAATGACAATTGACCCCCAAGTGGAGAAAAAGATGGGACAGGTGCTACTCATAGCAGTAGCCG TCTCCAGCGCCATACTGTCGCGGACCGCCTGGGGGTGGGGGGAGGCTGGGGCCCTGATCACAGCGGCAACTTCCACTTTGTGGGAAGG CTCTCCGAACAAGTACTGGAACTCCTCTACAGCCACTTCACTGTGTAACATTTTTAGGGGAAGTTACTTGGCTGGAGCTTCTCTAATCTAC ACAGTAACAAGAAACGCTGGCTTGGTCAAGAGACGTGGGGGTGGAACAGGAGAGACCCTGGGAGAGAAATGGAAGGCCCGCTTGAA CCAGATGTCGGCCCTGGAGTTCTACTCCTACAAAAAGTCAGGCATCACCGAGGTGTGCAGAGAAGAGGCCCGCCGCGCCCTCAAGGAC GGTGTGGCAACGGGAGGCCATGCTGTGTCCCGAGGAAGTGCAAAGCTGAGATGGTTGGTGGAGCGGGGATACCTGCAGCCCTATGGA AAGGTCATTGATCTTGGATGTGGCAGAGGGGGCTGGAGTTACTACGCCGCCACCATCCGCAAAGTTCAAGAAGTGAAAGGATACACAA AAGGAGGCCCTGGTCATGAAGAACCCATGTTGGTGCAAAGCTATGGGTGGAACATAGTCCGTCTTAAGAGTGGGGTGGACGTCTTTCA TATGGCGGCTGAGCCGTGTGACACGTTGCTGTGTGACATAGGTGAGTCATCATCTAGTCCTGAAGTGGAAGAAGCACGGACGCTCAGA GTCCTCTCCATGGTGGGGGATTGGCTTGAAAAAAGACCAGGAGCCTTTTGTATAAAAGTGTTGTGCCCATACACCAGCACTATGATGGA AACCCTGGAGCGACTGCAGCGTAGGTATGGGGGAGGACTGGTCAGAGTGCCACTCTCCCGCAACTCTACACATGAGATGTACTGGGTC TCTGGAGCGAAAAGCAACACCATAAAAAGTGTGTCCACCACGAGCCAGCTCCTCTTGGGGCGCATGGACGGGCCCAGGAGGCCAGTGA AATATGAGGAGGATGTGAATCTCGGCTCTGGCACGCGGGCTGTGGTAAGCTGCGCTGAAGCTCCCAACATGAAGATCATTGGTAACCG CATTGAAAGGATCCGCAGTGAGCACGCGGAAACGTGGTTCTTTGACGAGAACCACCCATATAGGACATGGGCTTACCATGGAAGCTAT GAGGCCCCCACACAAGGGTCAGCGTCCTCTCTAATAAACGGGGTTGTCAGGCTCCTGTCAAAACCCTGGGATGTGGTGACTGGAGTCAC AGGAATAGCCATGACCGACACCACACCGTATGGTCAGCAAAGAGTTTTCAAGGAAAAAGTGGACACTAGGGTGCCAGACCCCCAAGAA GGCACTCGTCAGGTTATGAGCATGGTCTCTTCCTGGTTGTGGAAAGAGCTAGGCAAACACAAACGGCCACGAGTCTGTACCAAAGAAG AGTTCATCAACAAGGTTCGTAGCAATGCAGCATTAGGGGCAATATTTGAAGAGGAAAAAGAGTGGAAGACTGCAGTGGAAGCTGTGA ACGATCCAAGGTTCTGGGCTCTAGTGGACAAGGAAAGAGAGCACCACCTGAGAGGAGAGTGCCAGAGTTGTGTGTACAACATGATGG GAAAAAGAGAAAAGAAACAAGGGGAATTTGGAAAGGCCAAGGGCAGCCGCGCCATCTGGTATATGTGGCTAGGGGCTAGATTTCTAG AGTTCGAAGCCCTTGGATTCTTGAACGAGGATCACTGGATGGGGAGAGAGAACTCAGGAGGTGGTGTTGAAGGGCTGGGATTACAAA GACTCGGATATGTCCTAGAAGAGATGAGTCGCATACCAGGAGGAAGGATGTATGCAGATGACACTGCTGGCTGGGACACCCGCATCAG CAGGTTTGATCTGGAGAATGAAGCTCTAATCACCAACCAAATGGAGAAAGGGCACAGGGCCTTGGCATTGGCCATAATCAAGTACACAT ACCAAAACAAAGTGGTAAAGGTCCTTAGACCAGCTGAAAAAGGGAAGACAGTTATGGACATTATTTCGAGACAAGACCAAAGGGGGA GCGGACAAGTTGTCACTTACGCTCTTAACACATTTACCAACCTAGTGGTGCAACTCATTCGGAATATGGAGGCTGAGGAAGTTCTAGAG ATGCAAGACTTGTGGCTGCTGCGGAGGTCAGAGAAAGTGACCAACTGGTTGCAGAGCAACGGATGGGATAGGCTCAAACGAATGGCA GTCAGTGGAGATGATTGCGTTGTGAAGCCAATTGATGATAGGTTTGCACATGCCCTCAGGTTCTTGAATGATATGGGAAAAGTTAGGAA GGACACACAAGAGTGGAAACCCTCAACTGGATGGGACAACTGGGAAGAAGTTCCGTTTTGCTCCCACCACTTCAACAAGCTCCATCTCA AGGACGGGAGGTCCATTGTGGTTCCCTGCCGCCACCAAGATGAACTGATTGGCCGGGCCCGCGTCTCTCCAGGGGCGGGATGGAGCAT CCGGGAGACTGCTTGCCTAGCAAAATCATATGCGCAAATGTGGCAGCTCCTTTATTTCCACAGAAGGGACCTCCGACTGATGGCCAATG CCATTTGTTCATCTGTGCCAGTTGACTGGGTTCCAACTGGGAGAACTACCTGGTCAATCCATGGAAAGGGAGAATGGATGACCACTGAA GACATGCTTGTGGTGTGGAACAGAGTGTGGATTGAGGAGAACGACCACATGGAAGACAAGACCCCAGTTACGAAATGGACAGACATTC CCTATTTGGGAAAAAGGGAAGACTTGTGGTGTGGATCTCTCATAGGGCACAGACCGCGCACCACCTGGGCTGAGAACATTAAAAACAC AGTCAACATGGTGCGCAGGATCATAGGTGATGAAGAAAAGTACATGGACTACCTATCCACCCAAGTTCGCTACTTGGGTGAAGAAGGG TCTACACCTGGAGTGCTGTAAGCACCAATCTTAGTGTTGTCAGGCCTGCTAGTCAGCCACAGCTTGGGGAAAGCTGTGCAGCCTGTGAC CCCCCCAGGAGAAGCTGGGAAACCAAGCCTATAGTCAGGCCGAGAACGCCATGGCACGGAAGAAGCCATGCTGCCTGTGAGCCCCTCA GAGGACACTGAGTCAAAAAACCCCACGCGCTTGGAGGCGCAGGATGGGAAAAGAAGGTGGCGACCTTCCCCACCCTTCAATCTGGGGC CTGAACTGGAGATCAGCTGTGGATCTCCAGAAGAGGGACTAGTGGTTAGAGGAGACCCCCCGGAAAACGCAAAACAGCATATTGACGC TGGGAAAGACCAGAGACTCCATGAGTTTCCACCACGCTGGCCGCCAGGCACAGATCGCCGAATAGCGGCGGCCGGTGTGGGG

[0421] SEQ ID N0: 3

[0422] Vero cell adapted Zika virus strain H / PF / 2O13 as sequenced

[0423] AGTTGTTGATCTGTGTGAATCAGACTGCGACAGTTCGAGTTTGAAGCGAAAGCTAGCAACAGTATCAACAGGTTTTATTTTGGATTTGGA AACGAGAGTTTCTGGTCATGAAAAACCCAAAAAAGAAATCCGGAGGATTCCGGATTGTCAATATGCTAAAACGCGGAGTAGCCCGTGT GAGCCCCTTTGGGGGCTTGAAGAGGCTGCCAGCCGGACTTCTGCTGGGTCATGGGCCCATCAGGATGGTCTTGGCGATTCTAGCCTTTT TGAGATTCACGGCAATCAAGCCATCACTGGGTCTCATCAATAGATGGGGTTCAGTGGGGAAAAAAGAGGCTATGGAAATAATAAAGAA GTTCAAGAAAGATCTGGCTGCCATGCTGAGAATAATCAATGCTAGGAAGGAGAAGAAGAGACGAGGCGCAGATACTAGTGTCGGAAT TGTTGGCCTCCTGCTGACCACAGCTATGGCAGCGGAGGTCACTAGACGTGGGAGTGCATACTATATGTACTTGGACAGAAACGACGCTG GGGAGGCCATATCTTTTCCAACCACATTGGGGATGAATAAGTGTTATATACAGATCATGGATCTTGGACACATGTGTGATGCCACCATG AGCTATGAATGCCCTATGCTGGATGAGGGGGTGGAACCAGATGACGTCGATTGTTGGTGCAACACGACGTCAACTTGGGTTGTGTACG GAACCTGCCATCACAAAAAAGGTGAAGCACGGAGATCTAGAAGAGCTGTGACGCTCCCCTCCCATTCCACTAGGAAGCTGCAAACGCG GTCGCAAACCTGGTTGGAATCAAGAGAATACACAAAGCACTTGATTAGAGTCGAAAATTGGATATTCAGGAACCCTGGCTTCGCGTTAG CAGCAGCTGCCATCGCTTGGCTTTTGGGAAGCTCAACGAGCCAAAAAGTCATATACTTGGTCATGATACTGCTGATTGCCCCGGCATACA GCATCAGGTGCATAGGAGTCAGCAATAGGGACTTTGTGGAAGGTATGTCAGGTGGGACTTGGGTTGATGTTGTCTTGGAACATGGAGG TTGTGTCACCGTAATGGCACAGGACAAACCGACTGTCGACATAGAGCTGGTTACAACAACAGTCAGCAACATGGCGGAGGTAAGATCC TACTGCTATGAGGCATCAATATCGGACATGGCTTCGGACAGCCGCTGCCCAACACAAGGTGAAGCCTACCTTGACAAGCAATCAGACAC TCAATATGTCTGCAAAAGAACGTTAGTGGACAGAGGCTGGGGAAATGGATGTGGACTTTTTGGCAAAGGGAGCCTGGTGACATGCGCT AAGTTTGCATGCTCCAAGAAAATGACCGGGAAGAGCATCCAGCCAGAGAATCTGGAGTACCGGATAATGCTGTCAGTTCATGGCTCCCA GCACAGTGGGATGATCGTTAATGACACAGGACATGAAACTGATGAGAATAGAGCGAAGGTTGAGATAACGCCCAATTCACCAAGAGCC GAAGCCACCCTGGGGGGTTTTGGAAGCCTAGGACTTGATTGTGAACCGAGGACAGGCCTTGACTTTTCAGATTTGTATTACTTGACTAT GAATAACAAGCACTGGTTGGTTCACAAGGAGTGGTTCCACGACATTCCATTACCTTGGCACGCTGGGGCAGACACCGGAACTCCACACT GGAACAACAAAGAAGCACTGGTAGAGTTCAAGGACGCACATGCCAAAAGGCAAACTGTCGTGGTTCTAGGGAGTCAAGAAGGAGCAG TTCACACGGCCCTTGCTGGAGCTCTGGAGGCTGAGATGGATGGTGCAAAGGGAAGGCTGTCCTCTGGCCACTTGAAATGTCGCCTGAA AATGGATAAACTTAGATTGAAGGGCGTGTCATACTCCTTGTGTACCGCAGCGTTCACATTCACCAAGATCCCGGCTGAAACACTGCACG GGACAGTCACAGTGGAGGTACAGTACGCAGGGACAGATGGACCTTGCAAGGTTCCAGCTCAGATGGCGGTGGACATGCAAACTCTGA CCCCAGTTGGGAGGTTGATAACCGCTAACCCCGTAATCACTGAAAGCACTGAGAACTCTAAGATGATGCTGGAACTTGATCCACCATTT GGGGACTCTTACATTGTCATAGGAGTCGGGGAGAAGAAGATCACCCACCACTGGCACAGGAGTGGCAGCACCATTGGAAAAGCATTTG AAGCCACTGTGAGAGGTGCCAAGAGAATGGCAGTCTTGGGAGACACAGCCTGGGACTTTGGATCAGTTGGAGGCGCTCTCAACTCATT GGGCAAGGGCATCCATCAAATTTTTGGAGCAGCTTTCAAATCATTGTTTGGAGGAATGTCCTGGTTCTCACAAATTCTCATTGGAACGTT GCTGATGTGGTTGGGTCTGAACACAAAGAATGGATCTATTTCCCTTATGTGCTTGGCCTTAGGGGGAGTGTTGATCTTCTTATCCACAGC TGTCTCTGCTGATGTGGGGTGCTCGGTGGACTTCTCAAAGAAGGAGACGAGATGCGGTACAGGGGTGTTCGTCTATAACGACGTTGAA GCCTGGAGGGACAGGTACAAGTACCATCCTGACTCCCCCCGTAGATTGGCAGCAGCAGTCAAGCAAGCCTGGGAAGATGGTATCTGTG GGATCTCCTCTGTTTCAAGAATGGAAAACATCATGTGGAGATCAGTAGAAGGGGAGCTCAACGCAATCCTGGAAGAGAATGGAGTTCA ACTGACGGTCGTTGTGGGATCTGTAAAAAACCCCATGTGGAGAGGTCCACAGAGATTGCCCGTGCCTGTGAACGAGCTGCCCCACGGC TGGAAGGCTTGGGGGAAATCGTACTTCGTCAGAGCAGCAAAGACAAATAACAGCTTTGTCGTGGATGGTGACACACTGAAGGAATGCC CACCCAAACATAGAGCATGGAACAGCTTTCTTGTGGAGGATCATGGGTTCGGGGTATTTCACACTAGTGTCTGGCTCAAGGTTAGAGAA GATTATTCATTAGAGTGTGATCCAGCCGTTATTGGAACAGCTGTTAAGGGAAAGGAGGCTGTACACAGTGATCTAGGCTACTGGATTGA GAGTGAGAAGAATGACACATGGAGGCTGAAGAGGGCCCATCTGATCGAGATGAAAACATGTGAATGGCCAAAGTCCCACACATTGTG GACAGATGGAATAGAAGAGAGTGATCTGATCATACCCAAGTCTTTAGCTGGGCCACTCAGCCATCACAATACCAGAGAGGGCTACAGG ACCCAAATGAAAGGGCCATGGCACAGTGAAGAGCTTGAAATTCGGTTTGAGGAATGCCCAGGCACTAAGGTCCACGTGGAGGAAACAT GTGGAACAAGAGGACCATCTCTGAGATCAACCACTGCAAGCGGAAGGGTGATCGAGGAATGGTGCTGCAGGGAGTGCACAATGCCCC CACTGTCGTTCCGGGCTAAAGATGGCTGTTGGTATGGAATGGAGATAAGGCCCAGGAAAGAACCAGAAAGTAACTTAGTAAGGTCAAT GGTGACTGCAGGATCAACTGATCACATGGATCACTTCTCCCTTGGAGTGCTTGTGATTCTGCTCATGGTGCAGGAAGGGCTGAAGAAGA GAATGACCACAAAGATCATCATAAGCACATCGATGGCAGTGCTGGTAGCTATGATCCTGGGAGGATTTTCAATGAGTGACCTGGCTAAG CTTGCAATTTTGATGGGTGCCACCTTCGCGGAAATGAACACTGGAGGAGATGTAGCTCATCTGGCGCTGATAGCGGCATTCAAAGTCAG ACCAGCGTTGCTGGTATCTTTCATCTTCAGAGCTAATTGGACACCCCGTGAAAGCATGCTGCTGGCCTTGGCCTCGTGTCTTTTGCAAACT GCGATCTCCGCCTTGGAAGGCGACCTGATGGTTCTCATCAATGGTTTTGCTTTGGCCTGGTTGGCAATACGAGCGATGGTTGTTCCACGC ACTGATAACATCACCTTGGCAATCCTGGCTGCTCTGACACCACTGGCCCGGGGCACACTGCTTGTGGCGTGGAGAGCAGGCCTTGCTAC TTGCGGGGGGTTTATGCTCCTCTCTCTGAAGGGAAAAGGCAGTGTGAAGAAGAACTTACCATTTGTCATGGCCCTGGGACTAACCGCTG TGAGGCTGGTCGACCCCATCAACGTGGTGGGACTGCTGTTGCTCACAAGGAGTGGGAAGCGGAGCTGGCCCCCTAGCGAAGTACTCAC AGCTGTTGGCCTGATATGCGCATTGGCTGGAGGGTTCGCCAAGGCAGATATAGAGATGGCTGGGCCCATAGCCGCGGTCGGTCTGCTA ATTGTCAGTTACGTGGTCTCAGGAAAGAGTGTGGACATGTACATTGAAAGAGCAGGTGACATCACATGGGAAAAAGATGCGGAAGTCA CTGGAAACAGTCCCCGGCTCGATGTGGCGCTAGATGAGAGTGGTGATTTCTCCCTGGTGGAGGATGACGGTCCCCCCATGAGAGAGAT CATACTCAAGGTGGTCCTGATGACCATCTGTGGCATGAACCCAATAGCCATACCCTTTGCAGCTGGAGCGTGGTACGTATACGTGAAGA

[0424] CTGGAAAAAGGAGTGGTGCTCTATGGGATGTGCCTGCTCCCAAGGAAGTAAAAAAGGGGGAGACCACAGATGGAGTGTACAGAGTAA TGACTCGTAGACTGCTAGGTTCAACACAAGTTGGAGTGGGAGTTATGCAAGAGGGGGTCTTTCACACTATGTGGCACGTCACAAAAGG ATCCGCGCTGAGAAGCGGTGAAGGGAGACTTGATCCATACTGGGGAGATGTCAAGCAGGATCTGGTGTCATACTGTGGTCCATGGAAG CTAGATGCCGCCTGGGACGGGCACAGCGAGGTGCAGCTCTTGGCCGTGCCCCCCGGAGAGAGAGCGAGGAACATCCAGACTCTGCCC GGAATATTTAAGACAAAGGATGGGGACATTGGAGCGGTTGCGCTGGATTACCCAGCAGGAACTTCAGGATCTCCAATCCTAGACAAGT GTGGGAGAGTGATAGGACTTTATGGCAATGGGGTCGTGATCAAAAATGGGAGTTATGTTAGTGCCATCACCCAAGGGAGGAGGGAGG AAGAGACTCCTGTTGAGTGCTTCGAGCCTTCGATGCTGAAGAAGAAGCAGCTAACTGTCTTAGACTTGCATCCTGGAGCTGGGAAAACC AGGAGAGTTCTTCCTGAAATAGTCCGTGAAGCCATAAAAACAAGACTCCGTACTGTGATCTTAGCTCCAACCAGGGTTGTCGCTGCTGA AATGGAGGAAGCCCTTAGAGGGCTTCCAGTGCGTTATATGACAACAGCAGTCAATGTCACCCACTCTGGAACAGAAATCGTCGACTTAA TGTGCCATGCCACCTTCACTTCACGTCTACTACAGCCAATCAGAGTCCCCAACTATAATCTGTATATTATGGATGAGGCCCACTTCACAGA TCCCTCAAGTATAGCAGCAAGAGGATACATTTCAACAAGGGTTGAGATGGGCGAGGCGGCTGCCATCTTCATGACCGCCACGCCACCA GGAACCCGTGACGCATTTCCGGACTCCAACTCACCAATTATGGACACCGAAGTGGAAGTCCCAGAGAGAGCCTGGAGCTCAGGCTTTG ATTGGGTGACGGATCATTCTGGAAAAACAGTTTGGTTTGTTCCAAGCGTGAGGAACGGCAATGAGATCGCAGCTTGTCTGACAAAGGCT GGAAAACGGGTCATACAGCTCAGCAGAAAGACTTTTGAGACAGAGTTCCAGAAAACAAAACATCAAGAGTGGGACTTTGTCGTGACAA CTGACATTTCAGAGATGGGCGCCAACTTTAAAGCTGACCGTGTCATAGATTCCAGGAGATGCCTAAAGCCGGTCATACTTGATGGCGAG AGAGTCATTCTGGCTGGACCCATGCCTGTCACACATGCCAGCGCTGCCCAGAGGAGGGGGCGCATAGGCAGGAATCCCAACAAACCTG GAGATGAGTATCTGTATGGAGGTGGGTGCGCAGAGACTGACGAAGACCATGCACACTGGCTTGAAGCAAGAATGCTCCTTGACAATAT TTACCTCCAAGATGGCCTCATAGCCTCGCTCTATCGACCTGAGGCCGACAAAGTAGCAGCCATTGAGGGAGAGTTCAAGCTTAGGACGG AGCAAAGGAAGACCTTTGTGGAACTCATGAAAAGAGGAGATCTTCCTGTTTGGCTGGCCTATCAGGTTGCATCTGCCGGAATAACCTAC ACAGATAGAAGATGGTGCTTTGATGGCACGACCAACAACACCATAATGGAAGACAGTGTGCCGGCAGAGGTGTGGACCAGACACGGA GAGAAAAGAGTGCTCAAACCGAGGTGGATGGACGCCAGAGTTTGTTCAGATCATGCGGCCCTGAAGTCATTCAAGGAGTTTGCCGCTG GGAAAAGAGGAGCGGCTTTTGGAGTGATGGAAGCCCTGGGAACACTGCCAGGACACATGACAGAGAGATTCCAGGAAGCCATTGACA ACCTCGCTGTGCTCATGCGGGCAGAGACTGGAAGCAGGCCTTACAAAGCCGCGGCGGCCCAATTGCCGGAGACCCTAGAGACCATTAT GCTTTTGGGGTTGCTGGGAACAGTCTCGCTGGGAATCTTTTTCGTCTTGATGAGGAACAAGGGCATAGGGAAGATGGGCTTTGGAATG GTGACTCTTGGGGCCAGCGCATGGCTCATGTGGCTCTCGGAAATTGAGCCAGCCAGAATTGCATGTGTCCTCATTGTTGTGTTCCTATTG CTGGTGGTGCTCATACCTGAGCCAGAAAAGCAAAGATCTCCCCAGGACAACCAAATGGCAATCATCATCATGGTAGCAGTAGGTCTTCT GGGCTTGATTACCGCCAATGAACTCGGATGGTTGGAGAGAACAAAGAGTGACCTAAGCCATCTAATGGGAAGGAGAGAGGAGGGGGC AACCATAGGATTCTCAATGGACATTGACCTGCGGCCAGCCTCAGCTTGGGCCATCTATGCTGCCTTGACAACTTTCATTACCCCAGCCGTC CAACATGCAGTGACCACTTCATACAACAACTACTCCTTAATGGCGATGGCCACGCAAGCTGGAGTGTTGTTTGGTATGGGCAAAGGGAT GCCATTCTACGCATGGGACTTTGGAGTCCCGCTGCTAATGATAGGTTGCTACTCACAATTAACACCCCTGACCCTAATAGTGGCCATCATT TTGCTCGTGGCGCACTACATGTACTTGATCCCAGGGCTGCAGGCAGCAGCTGCGCGTGCTGCCCAGAAGAGAACGGCAGCTGGCATCA TGAAGAACCCTGTTGTGGATGGAATAGTGGTGACTGACATTGACACAATGACAATTGACCCCCAAGTGGAGAAAAAGATGGGACAGGT GCTACTCATAGCAGTAGCCGTCTCCAGCGCCATACTGTCGCGGACCGCCTGGGGGTGGGGGGAGGCTGGGGCCCTGATCACAGCGGCA ACTTCCACTTTGTGG G AAG G CTCTCCG AACA AGTACTG G AACTCCTCTAC AG CCACTTCACTGTGTAACATTTTTAG GG G AAGTTACTTG GCTGGAGCTTCTCTAATCTACACAGTAACAAGAAACGCTGGCTTGGTCAAGAGACGTGGGGGTGGAACAGGAGAGACCCTGGGAGAG AAATGGAAGGCCCGCTTGAACCAGATGTCGGCCCTGGAGTTCTACTCCTACAAAAAGTCAGGCATCACCGAGGTGTGCAGAGAAGAGG CCCGCCGCGCCCTCAAGGACGGTGTGGCAACGGGAGGCCATGCTGTGTCCCGAGGAAGTGCAAAGCTGAGATGGTTGGTGGAGCGGG GATACCTGCAGCCCTATGGAAAGGTCATTGATCTTGGATGTGGCAGAGGGGGCTGGAGTTACTACGCCGCCACCATCCGCAAAGTTCAA GAAGTGAAAGGATACACAAAAGGAGGCCCTGGTCATGAAGAACCCATGTTGGTGCAAAGCTATGGGTGGAACATAGTCCGTCTTAAGA GTGGGGTGGACGTCTTTCATATGGCGGCTGAGCCGTGTGACACGTTGCTGTGTGACATAGGTGAGTCATCATCTAGTCCTGAAGTGGAA GAAGCACGGACGCTCAGAGTCCTCTCCATGGTGGGGGATTGGCTTGAAAAAAGACCAGGAGCCTTTTGTATAAAAGTGTTGTGCCCATA CACCAGCACTATGATGGAAACCCTGGAGCGACTGCAGCGTAGGTATGGGGGAGGACTGGTCAGAGTGCCACTCTCCCGCAACTCTACA CATGAGATGTACTGGGTCTCTGGAGCGAAAAGCAACACCATAAAAAGTGTGTCCACCACGAGCCAGCTCCTCTTGGGGCGCATGGACG GGCCCAGGAGGCCAGTGAAATATGAGGAGGATGTGAATCTCGGCTCTGGCACGCGGGCTGTGGTAAGCTGCGCTGAAGCTCCCAACAT GAAGATCATTGGTAACCGCATTGAAAGGATCCGCAGTGAGCACGCGGAAACGTGGTTCTTTGACGAGAACCACCCATATAGGACATGG GCTTACCATGGAAGCTATGAGGCCCCCACACAAGGGTCAGCGTCCTCTCTAATAAACGGGGTTGTCAGGCTCCTGTCAAAACCCTGGGA TGTGGTGACTGGAGTCACAGGAATAGCCATGACCGACACCACACCGTATGGTCAGCAAAGAGTTTTCAAGGAAAAAGTGGACACTAGG GTGCCAGACCCCCAAGAAGGCACTCGTCAGGTTATGAGCATGGTCTCTTCCTGGTTGTGGAAAGAGCTAGGCAAACACAAACGGCCAC GAGTCTGTACCAAAGAAGAGTTCATCAACAAGGTTCGTAGCAATGCAGCATTAGGGGCAATATTTGAAGAGGAAAAAGAGTGGAAGAC TGCAGTGGAAGCTGTGAACGATCCAAGGTTCTGGGCTCTAGTGGACAAGGAAAGAGAGCACCACCTGAGAGGAGAGTGCCAGAGTTG TGTGTACAACATGATGGGAAAAAGAGAAAAGAAACAAGGGGAATTTGGAAAGGCCAAGGGCAGCCGCGCCATCTGGTATATGTGGCT AGGGGCTAGATTTCTAGAGTTCGAAGCCCTTGGATTCTTGAACGAGGATCACTGGATGGGGAGAGAGAACTCAGGAGGTGGTGTTGAA GGGCTGGGATTACAAAGACTCGGATATGTCCTAGAAGAGATGAGTCGCATACCAGGAGGAAGGATGTATGCAGATGACACTGCTGGCT GGGACACCCGCATCAGCAGGTTTGATCTGGAGAATGAAGCTCTAATCACCAACCAAATGGAGAAAGGGCACAGGGCCTTGGCATTGGC CATAATCAAGTACACATACCAAAACAAAGTGGTAAAGGTCCTTAGACCAGCTGAAAAAGGGAAGACAGTTATGGACATTATTTCGAGAC AAGACCAAAGGGGGAGCGGACAAGTTGTCACTTACGCTCTTAACACATTTACCAACCTAGTGGTGCAACTCATTCGGAATATGGAGGCT GAGGAAGTTCTAGAGATGCAAGACTTGTGGCTGCTGCGGAGGTCAGAGAAAGTGACCAACTGGTTGCAGAGCAACGGATGGGATAGG CTCAAACGAATGGCAGTCAGTGGAGATGATTGCGTTGTGAAGCCAATTGATGATAGGTTTGCACATGCCCTCAGGTTCTTGAATGATAT GGGAAAAGTTAGGAAGGACACACAAGAGTGGAAACCCTCAACTGGATGGGACAACTGGGAAGAAGTTCCGTTTTGCTCCCACCACTTC AACAAGCTCCATCTCAAGGACGGGAGGTCCATTGTGGTTCCCTGCCGCCACCAAGATGAACTGATTGGCCGGGCCCGCGTCTCTCCAGG GGCGGGATGGAGCATCCGGGAGACTGCTTGCCTAGCAAAATCATATGCGCAAATGTGGCAGCTCCTTTATTTCCACAGAAGGGACCTCC GACTGATGGCCAATGCCATTTGTTCATCTGTGCCAGTTGACTGGGTTCCAACTGGGAGAACTACCTGGTCAATCCATGGAAAGGGAGAA TGGATGACCACTGAAGACATGCTTGTGGTGTGGAACAGAGTGTGGATTGAGGAGAACGACCACATGGAAGACAAGACCCCAGTTACG AAATGGACAGACATTCCCTATTTGGGAAAAAGGGAAGACTTGTGGTGTGGATCTCTCATAGGGCACAGACCGCGCACCACCTGGGCTG AGAACATTAAAAACACAGTCAACATGGTGCGCAGGATCATAGGTGATGAAGAAAAGTACATGGACTACCTATCCACCCAAGTTCGCTAC TTGGGTGAAGAAGGGTCTACACCTGGAGTGCTGTAAGCACCAATCTTAGTGTTGTCAGGCCTGCTAGTCAGCCACAGCTTGGGGAAAG

[0425] CTGTGCAGCCTGTGACCCCCCCAGGAGAAGCTGGGAAACCAAGCCTATAGTCAGGCCGAGAACGCCATGGCACGGAAGAAGCCATGCT GCCTGTGAGCCCCTCAGAGGACACTGAGTCAAAAAACCCCACGCGCTTGGAGGCGCAGGATGGGAAAAGAAGGTGGCGACCTTCCCCA CCCTTCAATCTGGGGCCTGAACTGGAGATCAGCTGTGGATCTCCAGAAGAGGGACTAGTGGTTAGAGGAGACCCCCCGGAAAACGCAA AACAGCATATTGACGCTGGGAAAGACCAGAGACTCCATGAGTTTCCACCACGCTGGCCGCCAGGCACAGATCGCCGAATAGCGGCGGC CGGTGTGGGGAAATCCATGGGTCT

[0426] SEQ I D N0: 4

[0427] H / PF / 2013.AHZ13508.1.French_Polynesia / 291-794 Flavivirus envelope glycoprotein E

[0428] IRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYV CKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGF GSLGLDCEPRTGLDFSDLYYLTMNNKHWLVH KEWFH DI PLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGA LEAEM DGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVI TESTENSKM MLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLF GGMSWFSQI LIGTLLMWLGLNTKNGSISLMCLALGGVLI FLSTAVSA

[0429] SEQ I D NO: 5

[0430] KLK peptide KLKLLLLLKLK

[0431] SEQ I D NO: 6

[0432] Oligo-(dldC)i3 dldC dldC dldC dldC dldC dldC dldC dldC dldC dldC dldC dldC dldC SEQIDNO:7

[0433] CpG 1018

[0434] TGACTGTGAACGTTCGAGATGA SEQIDN0:8

[0435] CpG 1826

[0436] TCCATGACGTTCCTGACGTT

[0437] SEQIDN0:9 CpG 7909

[0438] TCGTCGTTTTGTCGTTTTGTCGTT

Claims

CLAIMSWhat is claimed is:

1. A pharmaceutical composition comprising i) an inactivated Zika virus, ii) an aluminium salt adjuvant and iii) at least one additional adjuvant, wherein said at least one additional adjuvant is a toll -like receptor (TLR) agonist.

2. The pharmaceutical composition according to claim 1, wherein the inactivated Zika virus is a beta-propiolactone (BPL)-inactivated Zika virus.

3. The pharmaceutical composition according to claim 1 or 2, wherein the inactivated Zika virus comprises an RNA genome corresponding to the DNA sequence provided by SEQ ID NO: 2 or 3, or a variant sequence that is at least 80% identical to SEQ ID NO: 2 or 3 and able to pack a virulent Zika virus.

4. The pharmaceutical composition according to claim 3, wherein the variant sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.99% identical to SEQ ID NO: 2 or 3 and able to pack a virulent Zika vims.

5. The pharmaceutical composition according to any one of claims 2 to 4, wherein the Zika virus comprises an E protein as defined by SEQ ID NO: 4 or a variant amino acid sequence that is at least 95% identical to SEQ ID NO: 4 and able to pack a virulent Zika virus, and wherein the E protein comprises one or more modified amino acids selected from the group consisting of M125, H210, M295, H398, H399, H401 and H446.

6. The pharmaceutical composition according to claim 5, wherein each of the said modified amino acids is present at a percentage of greater than 15%.

7. The pharmaceutical composition according to any one of the preceding claims, wherein the inactivated Zika vims comprises a heterologous mixture of Zika vims with 1 to 10 genomic mutations compared with SEQ ID Nos: 2 or 3 and able to pack a virulent Zika vims.

8. The pharmaceutical composition according to any one of the preceding claims, wherein the inactivated Zika vims comprises an RNA genome corresponding to the DNA sequence provided by SEQ ID NO: 2 or 3, or a heterologous mixture thereof.

9. The pharmaceutical composition according to any one of the preceding claims, wherein the Zika virus has an E protein with the amino acid sequence provided by SEQ ID NO: 4.

10. The pharmaceutical composition according to any one of the preceding claims, wherein the inactivated Zika virus is provided at 1 to 10 pg / dose, preferably at 1.5 to 6 pg / dosc.

11. The pharmaceutical composition according to any one of the preceding claims, wherein the inactivated Zika virus is provided at a dose of between 100 to 800 AU / dose, preferably 100 to 600 AU / dose, preferably 100 to 400 AU, especially 100, 200 or 400 AU / dose.

12. The pharmaceutical composition according to any one of the preceding claims, wherein the aluminium salt adjuvant is aluminium hydroxide or aluminium phosphate.

13. The pharmaceutical composition according to claim 12, wherein the aluminium hydroxide adjuvanted composition comprises less than 1.25 ppb Cu.

14. The pharmaceutical composition according to any one of the preceding claims, wherein the aluminium adjuvant is present in the composition at a concentration of 1 mg / mL.

15. The pharmaceutical composition according to any one of the preceding claims, wherein the Toll-like receptor (TLR) agonist is a TLR4, a TLR7, a TLR8, a mixed TLR7 / 8 and / or a TLR9 agonist.

16. The pharmaceutical composition according to any one of the preceding claims, wherein the TLR agonist comprises a mixture comprising a peptide and a deoxyinosine-containing immunostimulatory oligodeoxynucleic acid molecule (I-ODN).

17. The pharmaceutical composition according to claim 16, wherein the peptide comprises the sequence KLKL5KLK (SEQ ID NO: 5) and the I-ODN comprises oligo-d(IC)i3 (SEQ ID NO: 6).

18. The pharmaceutical composition according to any one of the preceding claims, wherein the TLR agonist is a synthetic TLR7 / 8 ligand, particularly a synthetic TLR7 / 8 ligand provided in an aqueous formulation.

19. The pharmaceutical composition according to any one of the preceding claims, further comprising one or more pharmaceutically acceptable excipients.

20. The pharmaceutical composition according to claim 19, wherein the pharmaceutically acceptable excipients essentially consist of sucrose, potassium phosphate and sodium citrate and, optionally, magnesium chloride, D-sorbitol, L-methionine and recombinant human albumin (rHA).

21. The pharmaceutical composition according to claim 19 or 20, wherein said pharmaceutically acceptable excipients essentially consist of about 5% (w / v) sucrose, about 10 mM potassium phosphate, about 25 mM sodium citrate and about 0.01% (w / v) recombinant human albumin (rHA).

22. The pharmaceutical composition according to any one of claims 19 to 21, wherein said pharmaceutically acceptable excipients essentially consist of about 5% (w / v) sucrose; about 5 mM potassium phosphate; about 25 mM sodium citrate; about 5 mM MgC12; about 0.5 % (w / v) D-sorbitol; about 10 mM L-methionine; and about 0.01% (w / v) recombinant human albumin (rHA).

23. The pharmaceutical composition according to any one of the preceding claims, wherein the composition is delivered to a subject in a priming series followed by one or more boosters.

24. The pharmaceutical composition according to claim 23, wherein the priming series consists of 2 or 3 doses.

25. The pharmaceutical composition according to claim 23 or 24, wherein the first two doses of the priming series are administered 1 to 4 weeks apart.

26. The pharmaceutical composition according to any one of the claims 23 to 25, wherein an optional third dose of the priming series is administered between 6 and 12 months after the first two doses.

27. The pharmaceutical composition according to any one of claims 23 to 26, wherein the one or more boosters are provided at regular intervals, such as every 2, every 3, every 4, every 5 years or every 10 years, following the priming series.

28. The pharmaceutical composition according to any one of the preceding claims, wherein the dose is administered in a volume of 0.45 mL or 0.5 mL.

29. The pharmaceutical composition according to any one of claims 23 to 28, wherein the composition is administered to the subject orally or by a parenteral route selected from the group consisting of subcutaneous, intracutaneous, intradermal, intravenous, intramuscular, intraarticular, intraperitoneal, intrathecal or by infusion.

30. The pharmaceutical composition according to any one of the preceding claims, wherein the subject is a human subject.

31. The pharmaceutical composition according to any one of the preceding claims, wherein the subject is a resident of or is travelling to an endemic region.

32. The pharmaceutical composition according to any one of the preceding claims, wherein the subject is 15 to 49 years old.

33. The pharmaceutical composition according to any one of the preceding claims, wherein the subject is a woman of childbearing potential.

34. The pharmaceutical composition according to any one of the preceding claims, wherein the composition is able to increase serum antibody titers to Zika virus in a human by at least 1 log relative to a control within about 7 days from booster vaccination.

35. The pharmaceutical composition according to any one of the preceding claims, wherein the composition is able to increase serum antibody titers to Zika virus in a human by at least 1 log relative to a control within about 14 days from booster vaccination.

36. The pharmaceutical composition according to any one of the preceding claims, wherein the composition is able to stimulate seroconversion in at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to 100% of vaccinated subjects within 7 days of booster vaccination, wherein seroconversion is defined as reaching a 4-fold or higher neutralizing Zika virus antibody titer compared with baseline levels.

37. The pharmaceutical composition according to any one of the preceding claims, wherein the composition is able to stimulate seroconversion in at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to 100% of vaccinated subjects within 14 days of boostervaccination, wherein seroconversion is defined as reaching a 4-fold or higher neutralizing Zika virus antibody titer compared with baseline levels.

38. The pharmaceutical composition according to claim 36 or 37, wherein the 4-fold or higher neutralizing Zika virus antibody titer compared with baseline lasts for at least 6 months.

39. The pharmaceutical composition according to any one of claims 36 to 38, wherein the 4-fold or higher neutralizing Zika virus antibody titer compared with baseline lasts for at least 12 months.

40. The pharmaceutical composition according to any one of claims 36 to 39, wherein the 4-fold or higher neutralizing Zika virus antibody titer compared with baseline lasts for at least 24 months.

41. The pharmaceutical composition according to any one of claims 36 to 40, wherein the 4-fold or higher neutralizing Zika virus antibody titer compared with baseline lasts for at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, at least 20 years.

42. The pharmaceutical composition according to any one of claims 34 to 41, wherein the Zika virus antibody titers are measured by a microneutralization (pNT) assay.

43. The pharmaceutical composition according to any one of claims 34 to 42, wherein the booster vaccination is defined as a second or further vaccination with the pharmaceutical composition.

44. The pharmaceutical composition according to any one of the preceding claims, wherein the composition is a vaccine.

45. The pharmaceutical composition according to any one of the preceding claims, for use in a method of treating or preventing a Zika virus infection and / or a Zika vims associated clinical illness of any severity.

46. A method of treating or preventing a Zika virus infection in a subject in need thereof, comprising administering an effective amount of the pharmaceutical composition according to any one of the preceding claims.

47. A method for Zika virus inactivation with beta-propiolactone (BPL), comprising contacting the Zika virus with BPL for longer than is required to completely inactivate the Zika virus as measured by plaque assay.

48. The method according to claim 47, wherein the Zika virus is contacted with BPL for a time period of between 6 and 48 hours, preferably for about 30 hours.

49. The method according to claim 47 or 48, wherein the BPL inactivation is performed at a temperature of between 4°C and 10°C; i.e. a temperature of 7°C ± 3°C.

50. The method according to any one of claims 47 to 49, wherein a buffer solution is added to stabilize the Zika virus during inactivation.

51. The method according to any one of claims 47 to 50, wherein BPL is added twice during the inactivation period.

52. The method according to any one of claims 47 to 51, wherein BPL is added at the beginning of the inactivation period at a concentration of 500 ppm, and wherein the composition is optionally concurrently transferred to a second vessel.

53. The method according to any one of claims 47 to 52, wherein additional BPL is added at about 8 to 9 hours from the beginning of the inactivation period at a concentration of 200 ppm, and wherein the composition is optionally concurrently transferred to a third vessel.

54. The method according to claim 53, wherein the optional transfer to a third vessel is performed via a filter, preferably a 0.45 / 0.2 pm filter.

55. The method according to any one of claims 47 to 54, wherein the BPL concentration does not exceed 600 ppm at any time during the course of the inactivation period.

56. The method according to any one of claims 47 to 55, wherein the BPL is hydrolyzed following the inactivation period by elevating the temperature of the solution to 35 °C ± 2°C for a period of 2.5 ± 0.5 hours.

57. The method according to claim 56, wherein the solution is cooled to 5 ± 3 °C for storage following BPL hydrolysis.

58. A pharmaceutical composition comprising a BPL inactivated Zika virus, wherein said Zika virus is inactivated by the method according to any one of claims 47 to 57.

59. The pharmaceutical composition according to claim 58, for use in a method of treating or preventing a Zika virus infection and / or a Zika virus associated clinical illness of any severity.

60. A method of treating or preventing a Zika virus infection in a subject in need thereof, comprising administering an effective amount of pharmaceutical composition comprising i) an inactivated Zika virus, ii) an aluminium salt adjuvant and iii) at least one additional adjuvant, wherein said at least one additional adjuvant is a toll-like receptor (TLR) agonist.

61. The method according to claim 60, wherein the inactivated Zika virus is a beta-propiolactone (BPL)-inactivated Zika virus.

62. The method according to claim 60 or 61, wherein the BPL inactivated Zika virus comprises an RNA genome corresponding to the DNA sequence provided by SEQ ID NO: 2 or 3, or a variant sequence that is at least 80% identical to SEQ ID NO: 2 or 3 and able to pack a virulent Zika virus.

63. The method according to claim 62, wherein the variant sequence is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.99% identical to SEQ ID NO: 2 or 3 and able to pack a virulent Zika virus.

64. The method according to any one of claims 60 to 63, wherein the BPL inactivated Zika virus comprises an E protein as defined by SEQ ID NO: 4 or a variant amino acid sequence that is at least 95% identical to SEQ ID NO: 4 and able to pack a virulent Zika virus, and wherein the E protein comprises one or more modified amino acids selected from the group consisting of M125, H210, M295, H398, H399, H401 and H446, wherein said modifications result from BPL inactivation, and optionally wherein each of the said modified amino acids is present at a frequency of greater than 15%.

65. The pharmaceutical composition according to any one of claims 60 to 64, wherein the BPL inactivated Zika virus comprises a heterologous mixture of Zika virus with 1 to 10 genomic mutations compared with SEQ ID Nos: 2 or 3 and able to pack a virulent Zika virus.

66. The method according to any one of claims 60 to 63, wherein the inactivated Zika virus comprises an RNA genome corresponding to the DNA sequence provided by SEQ ID NO: 2 or 3, or a heterologous mixture thereof.

67. The method according to any one of claims 60 to 66, wherein the Zika virus has an E protein with the amino acid sequence provided by SEQ ID NO: 4.

68. The method according to any one of claims 60 to 67, wherein the inactivated Zika virus is provided at 1 to 20 pg / dose, preferably at 1 to 10 pg / dose, especially at about 2, 4 or 8 pg / dose.

69. The method according to any one of claims 60 to 67, wherein the inactivated Zika virus is provided at a dose of between 100 to 800 AU / dose, preferably 100 to 600 AU / dose, preferably 100 to 400 AU, especially 100, 200 or 400 AU / dose.

70. The method according to claim 69 wherein the dosage in AU is determined by use of a competitive liquid phase ELISA assay as disclosed herein.

71. The method according to any one of claims 60 to 70, wherein the aluminium salt adjuvant is aluminium hydroxide or aluminium phosphate.

72. The method according to claim 71, wherein the aluminium hydroxide adjuvanted composition comprises less than 1.25 ppb Cu.

73. The method according to any one of claims 60 to 72, wherein the aluminium adjuvant is present in the composition at a concentration of 1 mg / mL.

74. The method according to any one of claims 60 to 73, wherein the Toll-like receptor (TLR) agonist is a TLR4, a TLR7, a TLR8, a mixed TLR7 / 8 and / or a TLR9 agonist.

75. The method according to any one of claims 60 to 74, wherein the TLR agonist comprises a mixture comprising a peptide and a deoxyinosine-containing immunostimulatory oligodeoxynucleic acid molecule (I-ODN).

76. The method according to claim 75, wherein the peptide comprises the sequence KLKL5KLK (SEQ ID NO: 5) and the I-ODN comprises oligo-d(IC)i3 (SEQ ID NO: 6).

77. The method according to any one of claims 60 to 74, wherein the TLR agonist is a synthetic TLR7 / 8 ligand, particularly a synthetic TLR7 / 8 ligand provided in an aqueous formulation.

78. The method according to any one of claims 60 to 77, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

79. The method according to claim 78, wherein the pharmaceutically acceptable excipients essentially consist of sucrose, potassium phosphate and sodium citrate and, optionally, magnesium chloride, D-sorbitol, L-methionine and recombinant human albumin (rHA).

80. The method according to claim 78 or 79, wherein said pharmaceutically acceptable excipients essentially consist of about 5% (w / v) sucrose, about 10 mM potassium phosphate, about 25 mM sodium citrate and about 0.01% (w / v) recombinant human albumin (rHA).

81. The method according to any one of claims 78 to 80, wherein said pharmaceutically acceptable excipients essentially consist of about 5% (w / v) sucrose; about 5 mM potassium phosphate; about 25 mM sodium citrate; about 5 mM MgC12; about 0.5 % (w / v) D-sorbitol; about 10 mM L-methionine; and about 0.01% (w / v) recombinant human albumin (rHA).

82. The method according to any one of claims 60 to 81, wherein the pharmaceutical composition is delivered to a subject in a priming series followed by one or more boosters.

83. The method according to claim 82, wherein the priming series consists of 2 or 3 doses.

84. The method according to claim 82 or 83, wherein the first two doses of the priming series are administered 1 to 4 weeks apart.

85. The method according to any one of claims 82 to 84, wherein an optional third dose of the priming series is administered between 6 and 12 months after the first two doses.

86. The method according to any one of claims 82 to 85, wherein the one or more boosters are provided at regular intervals, such as every 2, every 3, every 4, every 5 years or every 10 years, following the priming series.

87. The method according to any one of claims 60 to 86, wherein the dose is administered in a volume of 0.45 mL or 0.5 mL.

88. The method according to any one of claims 60 to 87, wherein the pharmaceutical composition is administered to the subject orally or by a parenteral route selected from the group consisting of subcutaneous, intracutaneous, intradermal, intravenous, intramuscular, intraarticular, intraperitoneal, intrathecal or by infusion.

89. The method according to any one of claims 60 to 88, wherein the subject is a human subject.

90. The method according to any one of claims 60 to 89, wherein the subject is a resident of or is travelling to an epidemic or endemic region.

91. The method according to any one of claims 60 to 90, wherein the subject is 15 to 49 years old.

92. The method according to any one of claims 60 to 91, wherein the subject is a woman of childbearing potential.

93. The method according to any one of claims 60 to 92, wherein the pharmaceutical composition is able to increase serum antibody titers to Zika virus in the subject by at least 1 log relative to a control within about 7 days from booster immunization.

94. The method according to any one of claims 60 to 92, wherein the pharmaceutical composition is able to increase serum antibody titers to Zika virus in the subject by at least 1 log relative to a control within about 14 days from booster immunization.

95. The method according to any one of claims 60 to 92, wherein the pharmaceutical composition is able to stimulate seroconversion in at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to 100% of vaccinated subjects within 7 days of booster vaccination, wherein seroconversion is defined as reaching a 4-fold or higher neutralizing Zika virus antibody titer compared with baseline levels.

96. The method according to any one of claims 60 to 92, wherein the pharmaceutical composition is able to stimulate seroconversion in at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to 100% of vaccinated subjects within 14 days of booster vaccination, wherein seroconversion is defined as reaching a 4-fold or higher neutralizing Zika virus antibody titer compared with baseline levels.

97. The method according to claim 95 or 96, wherein the 4-fold or higher neutralizing Zika virus antibody titer compared with baseline lasts for at least 6 months.

98. The method according to any one of claims 95 to 97, wherein the 4-fold or higher neutralizing Zika virus antibody titer compared with baseline lasts for at least 12 months.

99. The method according to any one of claims 95 to 98, wherein the 4-fold or higher neutralizing Zika virus antibody titer compared with baseline lasts for at least 24 months.

100. The method according to any one of claims 95 to 99, wherein the 4-fold or higher neutralizing Zika virus antibody titer compared with baseline lasts for at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, at least 20 years.

101. The method according to any one of claims 95 to 100, wherein the Zika virus antibody titers are measured by a microneutralization (pNT) assay.

102. The pharmaceutical composition according to any one of claims 95 to 101, wherein the booster vaccination is defined as a second or further vaccination with the pharmaceutical composition.

103. The method according to any one of claims 60 to 102, wherein the pharmaceutical composition is a vaccine.

104. The method according to any one of claims 60 to 103, wherein the method treats or prevents a Zika virus infection and / or a Zika virus associated clinical illness of any severity.

105. A process for producing Zika virus particles in host cells in a bioreactor system that uses a falling film for medium oxygenation, characterized in that shear stress and / or medium foaming are minimized by maintaining the Falling Film Height (FFH) at the lowest possible level needed to maintain the required dissolved oxygen (DO) saturation levels at the respective stages of the process, thereby minimizing damage to virus particles compared with more turbulent incubation conditions.

106. A process for producing Zika virus particles in host cells in a bioreactor system that uses a falling film for medium oxygenation, wherein said process comprises at least the following steps:(i) a virus infection phase with a dissolved oxygen (DO) saturation level of no less than 10%, such as 10 to 30%, 10 to 20%, or 10 to 15%, especially at least 11%, at least 12%, at least 13%, at least 14%, or at least 15%, most preferably no less than 15%, and(ii) a virus production phase with a DO saturation level of around 50%; i.e. between 30 to 70%, preferably between 40 to 60%, more preferably between 45 to 55%, more preferably between 48 to 52%, most preferably 50%; characterized in that the Falling Film Height (FFH) is maintained at the lowest possible level needed to maintain the desired dissolved oxygen (DO) saturation levels at the respective stages of the process.

107. The process according to claim 106, wherein the virus infection phase (i) has a duration of 1 to 5 hours, more preferably 1.5 to 4 hours, most preferably 2.0 to 3.5 hours, especially 1.5 to 3.5 hours, and the virus production phase (ii) has a duration of 20 to 52 hours, preferably 26 to 52 hours, more preferably 30 to 52 hours, more preferably 34 to 52 hours, more preferably 38 to 52 hours, most preferably 48 to 52 hours108. The process according to claim 106 or 107, wherein the FFH is maintained at 0 cm for at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably for 100% of the duration of the virus infection phase (i).

109. The process according to any one of claims 105 to 108, wherein stirrer speed of the bioreactor remains constant for the entire duration of the virus infection phase.

110. The process according to any one of claims 105 to 109, wherein, if the DO falls below 15% during the virus infection phase (i), an initial FFH of 1 cm is set and increased by 0.5 cm increments until a DO of at least 15% can be maintained.

111. The process according to claim 110, wherein the FFH is not increased to more than 4 cm.

112. The process according to any one of claims 105 to 111, wherein the FFH at the beginning of the virus production phase (ii) is set at 2 cm and is increased by 0.5 cm increments to a maximum of 4 cm.

113. The process according to any one of claims 105 to 112, wherein the FFH during the virus production phase (ii) is maintained at less than 4 cm, preferably less than 3.5 cm, preferablyless than 3.0 cm, preferably less than 2.5 cm, preferably less than 2 cm, most preferably about 2 to 3 cm.

114. The process according to any one of claims 105 to 113, wherein the bioreactor is a fixed-bed bioreactor.

115. The process according to any one of claims 104 to 114, wherein the bioreactor comprises a fixed bed provided in a spiral shape wherein the medium recirculates through the bed, such as the bioreactor provided in Figs 13A and 13B; wherein the bioreactor provides a fixed bed embedded in a chamber through which the medium circulates, such as the bioreactor shown in Fig. 11C; wherein the bioreactor comprises a flask, wherein the medium is pumped out through a dip tube and returned to the chamber in a waterfall, such as shown in Fig. 1 ID; or wherein in the bioreactor FFH is adjusted by the weight of the medium introduced into the system, such as shown in Fig. 1 IE.

116. The process according to any one of claims 105 to 115, wherein the bioreactor is selected from a Scale-XTM bioreactor, an iCELLis Nano bioreactor system and an iCELLis 500 bioreactor system.

117. The process according to any one of claims 105 to 116, wherein the bioreactor is an iCellis 500 bioreactor system.

118. The process according to any one of claims 105 to 117, wherein the host cells are at an optimized density at the time of infection.

119. The process according to claim 118, wherein optimized density means that the cells are at a level sufficient to produce virus, but low enough to enable a minimized FFH.

120. The process according to claim 118 or 119, wherein the optimized density is about 0.5 to 2.5 x 105cells / cm2, especially about 1.0 to 2.0 x 105cells / cm2, or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9 x 105 / cm2.

121. The process according to any one of claims 105 to 120, wherein the multiplicity of infection (MOI) used to infect the host cells is 0.001 to 1, 0.01 to 0.1, preferably 0.03 to 0.5, most preferably 0.03.

122. The process according to any one of claims 105 to 121, wherein the host cell is a mammalian cell, preferably a vero cell line.

123. The process according to any one of claims 105 to 122, wherein the Zika virus particle comprises an RNA genome corresponding to the DNA sequence provided by the nucleic acid sequence of SEQ ID NO: 2, or a variant nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO: 2 and able to pack a virulent Zika virus.

124. The process according to any one of claims 105 to 122, wherein the Zika virus particle comprises an RNA genome corresponding to the DNA sequence provided by the nucleic acid sequence of SEQ ID NO: 3, or a variant nucleic acid sequence that is at least 88% identical to the nucleic acid sequence of SEQ ID NO: 3 and able to pack a virulent Zika virus.

125. The process according to claims 123 or 124, wherein the Zika virus particle comprises an E protein comprising or consisting of (i) an amino acid sequence as defined by SEQ ID NO: 4, or (ii) an amino acid sequence having at least 95%, at least 97% or at least 99% identity to SEQ ID NO: 4, and able to pack a virulent Zika virus particle.

126. A process for producing a pharmaceutical composition according to any one of claims 1 to 45, wherein the process comprises the steps of a) propagating a Zika virus in a bioreactor; b) harvesting the Zika virus to obtain a Zika virus composition (a); c) inactivating Zika virus composition (a) to obtain a Zika virus composition (b); d) subjecting Zika virus composition (b) to one or more further purification steps to obtain a Zika virus composition (c); and e) combining Zika virus composition (c) with i) one or more pharmaceutically acceptable carriers or excipients, ii) an aluminium salt adjuvant, and iii) at least one additional adjuvant, wherein said at least one additional adjuvant is a toll-like receptor (TLR) agonist.