Expression vector comprising a DNA sequence encoding a Zika virus premembrane and an envelope protein, its use, and vaccines.

Abstract

The present invention relates to the expression and secretion of the Zika virus envelope protein. Elements of the pre-membrane and envelope sequence have been modified to enhance the expression of the envelope protein as a secreted product in the culture medium of transformed insect cell lines. The expressed and purified product is suitable as a vaccine antigen.

Description

1 / 51 "EXPRESSION VECTOR COMPRISING A DNA SEQUENCE ENCODING A ZIKA VIRUS PRE-MEMBRANE AND AN ENVELOPE PROTEIN, ITS USE, AND VACCINES"

[0001] This application claims the benefit of priority under section 35 U.S.C. §119(e) of U.S. Application Serial No. 62 / 367,310 filed July 26, 2016, U.S. Application Serial No. 62 / 410,572 filed October 20, 2016, and U.S. Application Serial No. 62 / 442,346 filed January 4, 2017, the full texts of which are incorporated herein by reference. Incorporation of Sequence Listing

[0002] The material in the accompanying sequence listing is incorporated herein by reference. The accompanying sequence listing text file, named HBI1130_3WO_Sequence_Listing.txt, was created on July 25, 2017, and is 58 KB in size. The file can be viewed using Microsoft Word on a computer running Windows OS. Field of Invention

[0003] The invention relates generally to the design of optimized Zika virus envelope genes and their expression, and more specifically to vaccines against the Zika virus. Background of the Invention

[0004] Zika virus (ZIKV) is a mosquito-borne flavivirus whose recent spread throughout the Americas has led the WHO to declare Zika a public health emergency of international concern (WHO 12 Feb 2016; PAHO 14 Feb 2016). Transmitted primarily by Aedes mosquitoes, infection with positive-sense strand RNA can lead to mild symptoms similar to dengue: fever and skin rashes sometimes associated with conjunctivitis, arthralgia, or myalgia. Originally isolated in 1947 from a rhesus monkey during a yellow fever surveillance study in Uganda, ZIKV has already caused Petition 870260052369, dated 01 / 06 / 2026, p. 8 / 131 2 / 51 sporadic outbreaks of acute, but mild, disease have been reported in Africa, Asia, and the Pacific Islands (Dick et al., 1952). During a large ZIKV outbreak in 2007 on Yap Island, 18% of people presented with clinical disease while 82% of those infected with ZIKV were asymptomatic (Duffy et al., 2009). ZIKV reached the Western Hemisphere, Easter Island, and Chile in 2014 (Tognarelli et al., 2014), then appeared in Brazil in early 2015 and has since spread to 33 countries and territories in the Caribbean, Central America, South America, and Mexico (PAHO 02Apr2016). In addition to mosquito transmission, there are reports of ZIKV being sexually transmitted. The first indication that ZIKV can cause more severe neurological symptoms was the report of a 20-fold increase in the incidence of Guillain-Barré syndrome (GBS) during an outbreak in 2013 / 2014 in French Polynesia (Oehler et al., 2014).A similar increase in GBS was also observed in the Americas coinciding with the spread of ZIKV (WHO 02Apr2016). In Bahia, Brazil, 26 of 42 patients with GBS had a history of symptoms consistent with ZIKV infection (PAHO 14Feb2016; Schuller-Faccini et al., 2015). El Salvador and Colombia reported a 3-fold increase in GBS incidence; 12 of 22 patients presented with fever-related skin rashes 7 to 15 days prior to the onset (Schuller-Faccini et al., 2015; WHO 14 Feb2016). Honduras, Venezuela, and Suriname also reported an increase in GBS incidence; GBS cases were also reported during the ZIKV outbreak in Puerto Rico, Martinique, Panama, French Guiana, and Haiti (WHO 02Apr2016). During the long and ongoing ZIKV pandemic in Brazil, there has been an increase in the number of reports of newborns with microcephaly; up to 6480 suspected cases of microcephaly have been reported in Brazil (PAHO 14 Feb 2016; WHO 02 Apr 2016).Given the association of GBS, microcephaly, and other birth defects with ZIKV infection, the development of... Petition 870260052369, dated 01 / 06 / 2026, p. 9 / 131 3 / 51 A safe and effective vaccine against ZIKV is of paramount importance.

[0005] Currently there are no approved vaccines against ZIKV. Multiple vaccine candidates have been discussed; all are in preclinical development (WHO 02Apr2016). The WHO has identified at least 18 efforts in the search for an active vaccine using a range of vaccine technologies, including inactivated Zika virus, live attenuated viruses, viral vectors expressing ZIKV antigens, DNA-based vaccines, RNA-based vaccines, peptide-based vaccines, VLP-based vaccines, and recombinant protein-based vaccines. It has been suggested that DNA-based vaccine candidates being developed by the Vaccine Research Center at NIH and Innovio Pharmaceuticals, Inc. might be ready to begin Phase 1 clinical trials later this year. The target population for a ZIKV vaccine may include women of childbearing age and pregnant women. The risks of any replicating or genetic vaccine vector in this population would be significant.Therefore, a non-replicating recombinant protein subunit is an attractive approach for a ZIKV vaccine since vaccines based on purified recombinant proteins provide an improved safety profile. The key to success with the recombinant subunit approach for vaccines is the ability to efficiently produce high-quality proteins that result in immune responses equivalent to or better than those of traditional approaches with live or inactivated viruses.

[0006] The choice of recombinant protein expression system to be employed depends on the desired application. The chosen system must satisfy key criteria such as appropriate folding and processing, consistency and productivity (reduced costs) of the desired protein product (Schmidt, Appl. Microbiol. Biotechnol. (2004) 65:363-372). Systems of Petition 870260052369, dated 01 / 06 / 2026, page 10 / 131 4 / 51 Insect cell-based expression systems have the potential to meet capacity requirements based on ease of culture, greater tolerance to osmolality and byproduct concentrations during large-scale culture, and generally higher expression levels (Ikonomou et al., Appl. Microbiol Biotechnol. (2003) 62:1-20). Recently, the use of insect cell-based expression systems has become more common. These systems provide most of the desired characteristics of eukaryotic cells and offer additional advantages such as lower input costs. Insect cell-based systems either rely on infecting host cells with insect virus vectors (e.g., baculovirus) or generating stable cell lines by integrating expression plasmids into the host cell genome.

[0007] The baculovirus expression system (BES) has emerged as the main insect cell culture system used for recombinant protein expression. This system is based on the use of vectors derived from insect viruses known as baculoviruses. These vectors are used to generate recombinant viruses that encode the desired protein product. The recombinant viruses are used to infect host insect cells, which then express the desired recombinant proteins. Although there are advantages to this system in terms of ease of cloning and "time to product," there are also several disadvantages. The main challenge in using BES is that it relies on viral infection of host cells. This results in cell lysis and cell death 72-96 hours after infection (Farrell et al., Biotech. Biogen. (1998) 60:656-663; Deo and Park, Biotechnol. Appl. Biochem. (2006) 43:129-135).As a result, during the later stages of infection, the insect cell processing equipment becomes compromised. Petition 870260052369, dated 01 / 06 / 2026, page 11 / 131 5 / 51 as the processing of the desired product is also compromised. This limits the time that cells can produce product and, possibly even more importantly, leads to altered forms of the product being produced. In addition, cell lysis releases cellular enzymes that can also affect the quality of the desired product.

[0008] The use of stably transformed insect cells for the expression of recombinant proteins is an alternative to the use of BES. Expression systems based on stably transformed insect cells are non-lytic and allow for the long-term stable production of secreted products that require appropriate folding and post-translational modifications. Secretion of the product into the culture medium provides a cleaner starting material for the purification process and allows the final protein product to be purified using basic methods. This leads to higher quality products (Kirkpatrick and Shatzman in Gene Expression Systems: Using Nature for the Art of Expression (1999) pp. 289-330).

[0009] The Drosophila melanogaster cell expression system (“Drosophila expression system”) is a consolidated heterologous protein expression system based on the use of expression vectors containing Drosophila promoters and Drosophila S2 cells (“S2 cells”) (Schneider, Embryol. Exp. Morph. (1972) 27:353-365). S2 cells are transformed with these vectors in order to establish stable cell lines expressing proteins corresponding to the heterologous sequences introduced into the vector (Johansen, H. et al., Genes Dev. (1989) 3:882-889; Ivey-Hoyle, M., Curr. Opin. Biotechnol. (1991) 2:704-707; Culp, JS, et al., Biotechnology (NY) (1991) 9:173-177; US Patents 5,550,043; 5,681,713; 5,705,359; 6,046,025). This insect cell expression system has been shown to successfully produce numerous proteins from different sources. Examples of proteins that were Petition 870260052369, dated 01 / 06 / 2026, page 12 / 131 Six out of five proteins successfully expressed in the Drosophila S2 cell system include HIV gp120 (Culp, JS, et al., Biotechnology (NY) (1991) 9:173-177; Ivey-Hoyle, M., Curr. Opin. Biotechnol. (1991) 2:704-707), human dopamine β-hydrolase (Bin et al., Biochem. J. (1996) 313:5764), and human vascular cell adhesion protein (Bernard et al., Cytotechnol. (1994) 15:139-144). In each of these examples, expression levels were higher than in other previously used expression systems.

[0010] In addition to high levels of expression, the Drosophila expression system has been shown to be capable of expressing heterologous proteins that maintain biological function similar to the native proteins (Bin et al., Biochem. J. (1996) 313:57-64), (Incardona and Rosenberry, Mol. Biol. Cell. (1996) 7:595-611). More recent examples have shown through X-ray crystallography studies that this expression system is capable of producing molecules with a structure similar to the native proteins (Modis et al., Proc. Natl. Acad. Sci. USA (2003) 100:6986-6991), (Modis et al., Nature (2004) 427:313-319), (Xu et al., Acta. Crystallogr. D Biol. Crystallogr (2005) 61: 942-950). Two other recent publications have also demonstrated the ability of the Drosophila expression system to produce high-quality products. In the first work, Schmetzer et al. (J. Immun.(2005) 174: 942-952) compares the EpCAM protein expressed in baculovirus with the EpCAM protein expressed in Drosophila in terms of folding and native protein conformation. Specifically, the EpCAM expressed in BES and the EpCAM expressed in Drosophila were compared to the denatured EpCAM expressed in Drosophila. It was determined that the EpCAM expressed in BES was in a partially folded state compared to the undenatured and denatured EpCAM protein expressed in Drosophila. This indicates that the protein expressed in BES is in an incompletely folded state. The EpCAM protein. Petition 870260052369, dated 01 / 06 / 2026, page 13 / 131 7 / 51 expressed in Drosophila, on the other hand, adopted a more completely folded state. The authors of this article considered that the protein expressed in Drosophila was in the “natural” state, whereas the protein expressed in baculovirus was not. In the second study, Gardsvoll et al. (Prot. Exp. Purif. (2004) 34:284-295) demonstrate that the expression of the urokinase-type plasminogen activator receptor (uPAR) in S2 cells results in a more homogeneous product with respect to glycosylation (5 N-linked sites) than the uPAR expressed in CHO cells.

[0011] The development of a vaccine containing a recombinant subunit for ZIKV requires the selection of appropriate gene sequences from the ZIKV genome that encode proteins that are the target of neutralizing antibodies. Like other members of the flavivirus family, the ZIKV envelope glycoprotein is the primary target of neutralizing antibodies. In addition to selecting an appropriate ZIKV gene sequence, efforts to optimize the expression of the selected gene sequences are also desirable to increase the ability to effectively express the selected sequences so that the resulting products are soluble, stable, and conformationally relevant.

[0012] Although there are examples of flavivirus envelope gene sequences being expressed, there are no clear examples of optimizing the expression of these gene sequences, as most examples involve the use of naturally occurring sequences from the parental viruses. There are several ways to optimize expression. However, each selected sequence requires multiple rounds of experimentation to determine which methods or combination of methods will result in the most effective expression in a given expression system while maintaining appropriate native or biologically relevant characteristics that have the potential to... Petition 870260052369, dated 01 / 06 / 2026, page 14 / 131 8 / 51 immunogenic to induce a neutralizing antibody response in the case of the ZIKV envelope gene sequence.

[0013] Problems are frequently encountered with viral envelope sequences that require the maintenance of the appropriate native or biologically relevant characteristic, which can hinder optimal expression. These problems include non-optimal truncations that define the amino-terminal and carboxy-terminal of the expressed product, poor or ineffective post-translational processing, and poor matching of native codon usage with codon usage from the host cells of the expression system.

[0014] The efficiency of heterologous protein expression in eukaryotic systems depends on many factors, such as the promoter and associated regulatory elements, transcription start sequences, and polyadenylation signals. Since the expression vectors used in a typical system are optimized for the given host cell, optimization of the gene sequence of interest is generally of great importance to ensure optimal expression of the desired protein product. This is typically done by adapting the use of codons from the gene sequence to the use of codons typical of the host cells. Although the gene sequence is altered through codon optimization, the amino acid sequence of the encoded protein is not modified through the optimization process (Gustafsson et al., 2004).Optimizing the use of basic codons involves replacing random codons in the target gene sequence with those most frequently used by host cells. Alternatively, the entire gene sequence can be altered to align with the codon usage of host cells used to express the desired product. With the current efficiency of de novo gene synthesis, the latter approach has become the preferred method for codon optimization. As a... Petition 870260052369, dated 01 / 06 / 2026, page 15 / 131 9 / 51 Heterologous protein expression is an important part of the biotechnology industry; methods such as codon optimization are often useful for increasing expression levels.

[0015] Most proteins secreted from cells contain an N-terminal signal sequence that directs the protein into the cell's secretion pathway. Optimizing the signal sequence or signal peptide sequence that interacts with the endoplasmic membrane to initiate the secretion process has the potential to increase processing efficiency and thus increase protein expression. The eukaryotic signal sequence has been divided into three structural regions: basic, hydrophobic, and polar, starting from the N-terminus and continuing to the C-terminus, respectively (von Heijne, 1986 and Bendtsen et al. 2004). Over the years, numerous secretion signals have been identified and used to direct the secretion of recombinant proteins. Although many signal sequences have been used and have proven functional, few studies have defined the optimal sequences for a given cell type.The general characteristics and norms related to the three structural regions are well established, as presented in detail by von Heijne (1986) and Bendtsen et al. (2004). However, there is little comparative experimental data on what constitutes an optimal secretion signal in a given expression system or in a given heterologous protein being expressed. Most published work deals with the characterization and optimization of secretion signals from Gram-positive bacteria and yeast (Le Loir et al., 2005 and Hofmann and Schultz, 1991). One study describing the optimization of the IL-2 secretion signal clearly demonstrates the advantages of optimization (Zhang et al., 2005).

[0016] Many eukaryotic proteins are modified by N-linked (asparagine-linked) glycosylation. The number of sites and the Petition 870260052369, dated 01 / 06 / 2026, p. 16 / 131 10 / 51 Glycosylation efficiency by the oligosaccharyltransferase enzyme can vary, as each expressed protein can vary based on numerous factors. This can influence its expression and function. N-linked glycosylation normally occurs at Asn residues in the Asn-X-Ser / Thr motif, where X is any amino acid acceptor for Pro. However, many Asn-X-Ser / Thr sequences are not glycosylated or are glycosylated inefficiently (Mellquist et al., 1998). Inefficient glycosylation at one or more Asn-X-Ser / Thr sequences in a protein results in the production of heterogeneous glycoprotein products. The work of Mellquist et al. revealed that the amino acid at the Y position (amino acid residue immediately following the Ser or Thr residue) is an important determinant of core glycosylation efficiency. This represents an example of a means to optimize the glycosylation efficiency of heterologously expressed proteins.

[0017] The methods described above depict methods for altering structural aspects of gene sequences to increase the expression level of the desired protein product. Alternatively, modification of internal protein sequences to enhance selected epitopes or to remove selected epitopes can be employed to create a more desirable product, such as a protein product that has altered immunogenic potential. As an example, epitopes in the E protein of the dengue virus believed to generate cross-reactive antibody responses with flaviviruses were altered to reduce the potential for immunological enhancement (Hughes et al., 2012). These included the immunodominant B cell fusion peptide epitope and domain III epitopes.

[0018] Although the structure of flavivirus envelope proteins has been extensively studied and the structures established by cryoEM and X-ray crystallography, it constitutes an optimal gene sequence for the expression of heterologous proteins in systems of Petition 870260052369, dated 01 / 06 / 2026, page 17 / 131 11 / 51 The expression of proteins in eukaryotic host cells is not yet well defined. Current technology and methods provide the potential to assemble gene sequences and make modifications to internal sequences, defining new points of interest that could lead to improvements in structure and function. While there is potential to make such modifications, it is common knowledge that not all attempts to do so are successful. Modifications or optimization attempts that work with a protein in a given expression system do not always work with other proteins or in other expression systems. For example, removing the first 58 amino acids from the N-terminus of the ectodomain of the West Nile virus envelope protein abolishes expression.In another example, the expression of the C-terminal domain (domain III) of the West Nile virus or tick-borne encephalitis virus envelope protein is readily expressed; however, these protein subunits are suboptimal in terms of their ability to stimulate functional immune responses, although it is not obvious why this occurs. Therefore, a systematic evaluation is needed to determine the potential of various efforts to modify and optimize a given gene sequence so that high levels of high-quality heterologous protein are expressed and so that such alterations do not negatively impact the desired functional attributes. In the field of biotechnology, the ability to efficiently produce recombinant proteins at a convenient cost of inputs is key to success.To achieve this goal for a particular protein, in this case the Zika virus envelope protein using the Drosophila S2 cell expression system, further experimentation is needed to define the parameters that result in the optimal expression of a high-quality protein product. Petition 870260052369, dated 01 / 06 / 2026, page 18 / 131 12 / 51

[0019] The combination of multiple optimizations appropriately targeted to different aspects of the gene sequence and / or structure of a protein in order to obtain an additional advantage can further increase the utility of the optimized protein product. In the case of flavivirus envelope proteins, there are several examples of the expression of various truncated products as soluble recombinant proteins; however, all these examples depend on sequences derived from native viral sequences or synthetic versions of the native sequence. No significant effort has been made to optimize expression and secretion beyond the work to define the carboxy-terminal of the envelope protein that is most suitable for the expression of a soluble product. There are no examples of combinations of multiple optimizations to further improve the expression and secretion of soluble flavivirus envelope proteins.Therefore, the technical problems to be solved are: (1) identification of the translational, post-translational, or structural components of the envelope protein or associated components that, when optimized, result in increased expression levels and potentially improve the structural quality of the protein so that it is a more potent immunogen; (2) design of the synthetic component where possible to aid in optimization; and (3) determination of the optimal combination of multiple components so that the combination results in an additive increase in productivity and quality of protein expression. Further improvements in the expression of the Zika envelope protein could potentially offer effective immunogens at an improved cost of inputs, increasing the production capacity of recombinant proteins suitable for use in vaccines to combat the spread of the Zika virus.The use of such improvements could also be applied to other members of the flavivirus family, including, but not limited to, other similar viruses. Petition 870260052369, dated 01 / 06 / 2026, page 19 / 131 13 / 51 limitation, West Nile virus, dengue virus, tick-borne encephalitis virus, yellow fever virus, Zika virus and several other viruses that can cause encephalitis, https: / / en.wikipedia.org / wiki / Flavivirus - cite note-ShiP-Y-2 as well as insect-specific flaviviruses (ISFs) such as cell fusing agent virus (CFAV), Palm Creek virus (PCV), and Parramatta River virus (PaRV).

[0020] Summary of the Invention

[0021] The invention features the optimized expression of a soluble ZIKV envelope protein subunit that results in high levels of expression of a native-like protein or a biologically relevant protein; and is therefore an effective immunogen for the production of neutralizing antibodies. Specifically, the invention is directed at the expression of optimized ZIKA gene sequences when Drosophila melanogaster S2 cells are used as the host cell.

[0022] The optimized ZIKV gene sequence for expression of a soluble and stable envelope protein is composed of a contiguous prM-E sequence with optimized codons, containing an optimized secretion signal for the E protein segment, and having an optimized C-terminus that enhances the expression and stability of the expressed E product. The optimized gene sequence is contained in an expression vector for use in Drosophila S2 cells. The codon optimization of the gene sequence is designed for optimal expression in Drosophila S2 cells. The synthetic optimized secretion signal used is designed to result in effective post-translational processing at the pM-E junction by the host cell signal peptide protease. The newly defined C-terminus for the envelope protein, Glycine436, provides the interaction between the domain III region and the domain I region that helps to Petition 870260052369, dated 01 / 06 / 2026, page 20 / 131 14 / 51 stabilize the expressed and secreted Zika envelope protein. The combination of optimization methods resulted in a unique gene sequence that provides expression of a soluble Zika envelope protein with improved stability and at high levels. This improved Zika envelope protein is suitable for use as a vaccine to protect against diseases caused by Zika virus infection.

[0023] The optimized ZIKV gene sequences of the present invention are capable of high-level expression and secretion of the encoded envelope protein in the culture medium of transformed S2 cells. Specifically, the described ZIKV envelope gene sequence has been optimized for 1) codon use in Drosophila S2 cells, 2) an optimized synthetic secretion signal sequence and processing site, and 3) a C-terminal truncation point that adds stability to the expressed product.

[0024] The invention also provides methods for using products encoded by optimized ZIKV gene sequences in vaccine formulations to protect against diseases caused by ZIKV infection.

[0025] One aspect of the present invention is to provide an expression vector that includes a DNA sequence of optimized codons encoding a Zika virus pre-membrane and an envelope protein. Expression of the DNA sequence results in the secretion of a soluble envelope protein into the culture medium.

[0026] In one embodiment, the optimized codon DNA sequence includes SEQ ID NO:1.

[0027] In another embodiment, the optimized codon DNA sequence is further optimized to increase the secretion of the soluble envelope protein by optimizing the secretion signal sequence of protein E. This optimized secretion signal codon sequence Petition 870260052369, dated 01 / 06 / 2026, p. 21 / 131 15 / 51 improvement includes SEQ ID NO:2.

[0028] In another embodiment, the optimized codon DNA sequence is further optimized for soluble envelope protein expression, the carboxy-terminus having been extended to stabilize the expressed envelope protein. This extended optimized codon sequence includes SEQ ID NO:3.

[0029] In another embodiment, the optimized codon DNA sequence is further optimized to combine the enhanced E secretion signal with the extended carboxy-terminal to increase the expression and secretion of the soluble envelope protein. This optimized codon sequence with combined enhancements includes SEQ ID NO:4.

[0030] In another embodiment, the expression vector pHH202 of Drosophila, which includes SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, is used to express and secrete the ZIKV-encoded heterologous protein E from cultured insect cells. This pHH202 vector expression cassette includes SEQ ID NO:7.

[0031] In one embodiment, expression vectors are used in Drosophila cells.

[0032] In another embodiment, expression vectors are used in Drosophila S2 cells.

[0033] In one embodiment, the invention provides a vaccine comprising an effective amount of purified envelope protein (E) from the Zika virus, wherein the protein is secreted into the growth medium when recombinantly expressed in a host cell; and an effective amount of an aluminum-based adjuvant, wherein the vaccine induces the production of neutralizing antibodies in human subjects. In one aspect, the E protein is produced and recombinantly expressed in insect host cells. In one aspect, the E protein is produced and recombinantly expressed in insect host cells. Petition 870260052369, dated 01 / 06 / 2026, page 22 / 131 16 / 51 recombinant in Drosophila melanogaster Schneider 2 (S2) host cells. Preferably, the vaccine is in a pharmaceutically acceptable excipient. In one aspect, protein E is encoded by a nucleic acid sequence that begins at nucleotide 505 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 which is correlated with amino acid residue 169 of SEQ ID NO:6.

[0034] Another aspect of the present invention is to provide a method for eliciting an immune response that offers protection against diseases caused by the Zika virus. The method includes administering to an individual in need thereof a composition that includes a soluble envelope protein expressed and secreted by an expression vector that includes an optimized codon DNA sequence that includes SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0035] In one embodiment, the composition includes an adjuvant to enhance the immune response. The vaccine may also include an effective amount of a saponin-based adjuvant, for example, QS21, an aluminum-based adjuvant (collectively referred to as Alum), for example Alhydrogel®, or an adjuvant based on a stable oil-in-water emulsion (SE) that may include squalene. In the embodiments, a TLR-4 agonist is a fully synthetic lipid A derivative (SLA). In one embodiment, the vaccine includes a mixture of SLA and a saponin-based adjuvant, such as QS21, where the mixture is a liposomal mixture with these adjuvant components (SLA-LSQ).

[0036] Other aspects and advantages of the invention will become apparent from the following description and the appended claims. Brief Description of the Drawings

[0037] Figure 1 shows the sequence of the pre-membrane and the Petition 870260052369, dated 01 / 06 / 2026, p. 23 / 131 17 / 51 envelope of the Zika virus strain French Polynesia H / PF / 2013 (SEQ ID NO:5) with translation (SEQ ID NO:6)

[0038] Figure 2 shows the optimized codon sequence of the Zika virus (SEQ ID NO:1) pre-membrane and envelope (80E)

[0039] Figure 3 shows the expression of ZIPFP-80E-CoOp of optimized codons in Drosophila S2 cells. Samples were examined on 10% SDS PAGE gel under non-reducing conditions and stained with Coomassie blue.

[0040] Figure 4 shows a comparison of flavivirus secretion signals at the prM-E junction along with a synthetic secretion signal optimized for Zika virus envelope protein expression (SEQ ID NOS: 8 - 21).

[0041] Figure 5 is an alignment of flavivirus envelope proteins at junction 80E (SEQ ID NOS: 22 - 37).

[0042] Figures 6A-6B show results of the titration by Mouse serum ELISA after two or three doses of ZIKFP-80E-CoOp formulated with multiple adjuvants.

[0043] Figures 7A-7B show results of the titration by PRNT of mouse serum after two or three doses of ZIKFP-80E-CoOp formulated with multiple adjuvants.

[0044] Figure 8 shows the expression of ZIPFP-80E-WT, ZIKFP80E-CoOp and ZIKFP OpE-436-CoOp in Drosophila S2 cells. Samples were examined on 10% SDS gel under non-reducing conditions and stained with Coomassie blue. Detailed Description of the Invention

[0045] The invention presents an optimized gene sequence of ZIKV is used for the expression of a soluble and stable envelope protein composed of a contiguous prM-E sequence with optimized codons, containing an optimized secretion signal for a segment of the E protein, and having an optimized C-terminus that enhances expression. Petition 870260052369, dated 01 / 06 / 2026, page 24 / 131 18 / 51 and the stability of the expressed E product. The optimized gene sequence is inserted into a Drosophila S2 cell expression vector that induces the expression of high levels of high-quality Zika envelope proteins in S cells that have been stably transformed with expression vectors carrying the optimized gene sequence. The use of the optimized gene sequence results in increased productivity and quality of the expressed Zika envelope protein. Increased expression of the Zika envelope protein provides an effective immunogen at an improved cost of inputs that can increase the production capacity of recombinant proteins suitable for use in vaccines to combat the spread of the Zika virus.

[0046] The term “gene sequence” refers to a sequence of DNA is transcribed into an RNA molecule that can function directly or be translated into a chain of amino acids.

[0047] The term “optimized” refers to sequences that have been derived from naturally occurring sequences and have been altered to improve their functions.

[0048] The term “optimized codon” refers to a nucleic acid coding region that has been adapted for expression in the cells of a given host by replacing at least, or more than one, or a significant number of, codons with one or more codons that are used more frequently in the genes of that host.

[0049] The term “synthetic” refers to sequences that are not seen to occur naturally. More specifically, the synthetic elements described in this report are not found in the gene sequences of the Zika virus or related flaviviruses.

[0050] The term “operationally linked” refers to a juxtaposition where the components thus described are in a relationship that allows them to function in the manner Petition 870260052369, dated 01 / 06 / 2026, p. 25 / 131 19 / 51 intended.

[0051] “Expression cassette” means the combination of promoter elements with other transcriptional and translational regulatory control elements that are operationally linked to a gene sequence to be expressed. A gene sequence can be inserted into the expression cassette for expression of said gene sequence. The expression cassette is capable of directing transcription that results in the production of an mRNA for the desired gene product which is then translated into protein by host cell translation systems. The expression cassette is integral to the expression vector (plasmid). Such an expression vector directs the expression of the protein encoded by the gene sequence after being introduced into host cells.

[0052] The term “transformed” refers to DNA-mediated cell transformation. This term refers to the introduction of plasmid DNA into insect cells in the process of generating stable cell lines subsequent to the integration of the introduced DNA into the cell genome. This term is used in place of the term “transfection,” which is generally used in the same context. The term transformation is used for the introduction of plasmid DNA into cultured cells to distinguish it from the introduction of viral DNA into cultured cells, which was originally called transfection. Since there are no viral DNA sequences in the present invention being introduced into the host that result in the production of virus-like particles or cell lysis, the term transformed is preferred.

[0053] “Expression” or “expressed” means the production of proteins using expression vectors and a host cell, for example, Drosophila S2 cells, to produce a recombinant protein product that is readily detectable as a cell-associated product or as a product secreted into the culture medium. Petition 870260052369, dated 01 / 06 / 2026, page 26 / 131 20 / 51

[0054] “Secretion” means secretion of a recombinant protein expressed from host cells grown in culture medium. The expressed and secreted protein is the result of a given gene sequence being operationally linked to an expression cassette such that the sequence codes for the given protein.

[0055] The term “product” refers to any recombinant protein, full-length or subunit thereof, that is expressed by a host cell into which an expression vector carrying the gene sequence encoding the product has been introduced.

[0056] Insect cells are an alternative eukaryotic expression system that provides the ability to adequately express folded and post-translationally modified proteins while offering simple and relatively inexpensive growth conditions. The use of stably transformed insect cell expression systems offers advantages over those based on baculovirus infection of insect host cells. Based on this, S2 cells were selected as the preferred insect host cells.As a result, efforts to optimize expression vectors for stably transformed insect cells were based on data obtained from the analysis of specific Drosophila genes as well as the complete Drosophila genome.

[0057] In a preferred embodiment of the invention, the E protein secretion signal located at the carboxy terminus of the prM sequence and immediately preceding the N-terminus of the sequence is a synthetic sequence designed to improve the processing of these sequences. This synthetic secretion sequence has an enhanced hydrophobic core region, and amino acid residues -2 and -1 have been optimized to increase recognition of the signal protease cleavage site at the prM-E junction. The amino acid residues at +1 and Petition 870260052369, dated 01 / 06 / 2026, page 27 / 131 21 / 51 +4 of sequence E were also optimized to aid in the recognition of the signal protease cleavage site. The amino acid sequence of the synthetic secretion signal including the optimized residues in sequence E is shown in Figure 4, and the nucleotide sequence encoding these elements in the optimized prM-80E codon sequence (Op80E-CoOp) is shown in detail in SEQ ID NO:2.

[0058] In a preferred embodiment of the invention, the C-terminal of the E protein has been extended beyond the 80E terminus to stabilize the soluble envelope protein that is expressed. Specifically, the C-terminal of the soluble E protein has been extended from Gly-404 to Ile-436 as shown in Figure 5. The optimized nucleotide sequence of prM-E codons encoding the extended C-terminal E protein (E-436-CoOp) is shown in detail in SEQ ID NO:3.

[0059] In a more preferred embodiment of the invention, the combination of optimized and synthetic elements has been combined into a single gene sequence for the expression of the Zika envelope protein, resulting in increased efficiency and yield of the product. The product is also improved in terms of stability as a soluble product and as an immunogen for use as a vaccine. The nucleotide sequence of prM-E containing the assembled optimized Zika virus envelope protein components, codon optimization, synthetic secretion signal, and the C-terminal E-436 extension (OpE-436-CoOp) is presented in SEQ ID NO:4.

[0060] Thus, the present invention presents the combination of multiple optimizations targeting different aspects of the Zika virus prM-E gene sequence in such a way that an additive advantage is obtained, resulting in high levels of envelope protein expression. The optimized Zika prM-E sequence when Petition 870260052369, dated 01 / 06 / 2026, page 28 / 131 The 22 / 51 sequence used to express the envelope protein in Drosophila S2 cells results in the economical production of large quantities of high-quality proteins. The examples below show that the use of individual optimized elements in the Zika gene sequence results in improved or increased expression of the envelope protein. The highest levels of envelope protein expression are achieved in the gene sequence where all identified optimized elements are combined.

[0061] Although the descriptions presented above and the accompanying examples are primarily directed toward the use of expression vectors optimized with Drosophila S2 cells, the vectors and methods can be applied to other insect cell lines that result in stable cell lines after transformation of host cells with plasmid DNA.

[0062] The following examples are offered for illustrative purposes only and not as a limitation. EXAMPLES

[0063] The following examples describe the development of optimized ZIKV gene sequences for envelope protein expression in insect cells. The examples demonstrate the ability to effectively express the proteins in Drosophila S2 cells at levels that are commercially suitable for product development.

[0064] The examples demonstrate the ability of individual regulatory elements to enhance the ability to express proteins in S2 cells and the efforts made to determine which alterations contributed to the enhanced function of these elements. The results presented below demonstrate that different elements and modifications of these elements can result in high levels of expression or in very low or undetectable expression. Thus, the Petition 870260052369, dated 01 / 06 / 2026, page 29 / 131 23 / 51 The selection of functional and effective regulatory elements must be determined through experimentation. Therefore, the invention described in this application is unique in that the expression cassette described is mostly of synthetic composition and targets high levels of protein expression. EXAMPLE 1 Expression of wild-type and optimized ZIKV 80E proteins in Drosophila S2 cells.

[0065] In an effort to identify gene sequences optimized to induce high levels of high-quality ZIKV envelope protein in S2 cells, the wild-type prM-E gene sequence was compared with a codon-optimized prM-E gene sequence. Both WT and codon-optimized sequences were synthetically produced (DNA2.0, Menlo Park, CA). For codon optimization, the standard Drosophila melanogaster codon table (Kazusa DNA Research Institute, http: / / www.kazusa.or.jp / codon / ) was used. As the goal is to improve expression efficiency, which is partly controlled by the translation process, a 10% usage threshold was used in codon assignment (any codon used < 10% is excluded). Additionally, based on our analysis of highly expressed proteins in Drosophila, we added the exclusion of the following codons: CGA for Arg, ATA for Ile, and GTA for Val.The synthesized gene sequences included appropriate restriction enzyme sites at the ends, and a stop codon was included at the end of the envelope protein coding region.

[0066] For the expression of the ZIKV envelope protein, the genomic sequence representing the ZIKV premembrane protein and the envelope protein truncated at the carboxy-terminal (prM-80E) was used. The sequence used for expression Petition 870260052369, dated 01 / 06 / 2026, page 30 / 131 24 / 51 is based on the French Polynesian strain 2013 H / PF / 2013 (GenBank Accession # KJ776791). The WT sequence of prM-80E from the French Polynesian strain 2013 H / PF / 2013 along with the translation is shown in Figure 1. The optimized codon sequence is detailed in SEQ ID NO:1 and in Figure 2. Although the sequence of the optimized codon sequence is different, it codes for the same protein sequence shown in Figure 1 for the WT sequence. Viral strains in the Asian lineage such as the French Polynesian strain 2013 H / PF / 2013 are responsible for the outbreak occurring in the Americas (22). The first amino acid codon for the prM sequence is fused “in frame” with the secretion signal of the expression vector. When the prM-80E sequence is expressed in S2 cells, the prM-E junction is processed by an S2-encoded signal protease. This results in the secretion of an 80E product with a native N-terminus in the culture medium.

[0067] Synthetic DNA fragments were digested with appropriate restriction enzymes and inserted into the expression cassette (SEQ ID NO:7) of the pHH202 expression vector, which had been digested with Nhe I and Xho I. The pHH202 expression cassette contains the following elements: metallothionein promoter, optimized Kozak sequence, influenza HA secretion signal, and the early 3'UTR of SV40. The hygromycin-encoding gene is also incorporated into the pHH202 expression plasmid downstream of the expression cassette. The pHH202 expression plasmid is designed to allow directional cloning of the gene of interest at unique Nhe I and Xho I sites. Complete splices and inserts of all constructs were sequenced to verify that the various introduced components are correct and that the appropriate reading frame has been maintained.

[0068] For this work, traditional S2 cell culture and transformation methods were used (Van der Straten, Methods in Petição 870260052369, de 01 / 06 / 2026, pág. 31 / 131 25 / 51 Mol. and Cell Biol. (1989) 1:1-8; Culp et al., Biotechnology (1991) 9:173177; Kirkpatrick and Shatzman, In Gene Expression Systems: Using Nature for the Art of Expression, Eds. Fernandez and Hoeffler, Academic Press, (1999) 289-330). Células S2 de Drosophila (Schneider, J. Samples from Embryol. Exp. Morph. (1972) 27:353-365) acquired from ATCC were used. S2 cells were adapted to grow in Excell 420 medium (SAFC, St. Louis, MO), and all procedures and cultivation described in this application were performed in Excell 420 medium. Cultures are typically seeded at a density of 1x10⁶ cells / ml and incubated for days 5 to 7. All cultures were incubated at a temperature of 26° to 27°C. Expression plasmids into which genes of interest were inserted were transformed into S2 cells using the ExpreS2 TR reagent (Expres2ion Bio, Horsholm, Denmark). After transformation, cells resistant to hygromycin B, 0.3 mg / ml, were selected. After stable cell lines were selected, they were analyzed for the expression of appropriate products.For expression assessment, 5 ml cultures of selected cell lines were seeded at 2x10⁶ cells / ml and cultured in the presence of 0.2 mM copper sulfate at 26°C for 7 days. The cultures were analyzed for recombinant protein expression in both cell-associated fractions and the culture medium. Proteins were separated by SDS-PAGE and stained with Coomassie blue or transferred to nitrocellulose for western blot analysis. Expression levels > 1 pg / ml (1 mg / L) are easily detected in S2 cultures by staining SD-PAGE gels with Coomassie blue.

[0069] The parental S2 cell lines expressing ZIKFP 80E-WT and ZIKFP-80E-CoOp were established using traditional methods developed at HBI (23). The expression of the ZIKFP-80E product was identified using the monoclonal antibody Petition 870260052369, dated 01 / 06 / 2026, page 32 / 131 26 / 51 conformationally sensitive (mAb) 4G2 that recognizes most flavivirus envelope proteins (24). Expression data for two parental S2 cell lines expressing the optimized codon ZIKFP-80ECoOp are shown in Figure 3. West Nile virus 80E (WN-80E) is included for comparison. The 4G2 mAb is also used for purification using immunoaffinity chromatography (IAC) methods. IAC-based 4G2 mAb purification is analogous to the process currently used for the WN-80E vaccine program and has been successfully transferred to cGMP production.

[0070] The use of the prM-E gene sequence of optimized codons in transformed S2 cells resulted in the expression of ZIKFP-80ECoOp at approximately 30 pg / ml. An SDS-PAGE gel stained with Coomassie blue is shown in Figure 3 with both non-concentrated culture media samples containing ZIKFP-80E-CoOp (lanes 25) and purified ZIKFP-80E-CoOp (lanes 9-10). Samples of WN-80E are also included in the gel for comparison.

[0071] Another adjuvant that can be used in the vaccine formulation described herein is a stable oil-based emulsion. In one embodiment, the emulsion is a stable oil-in-water (SE) emulsion that may optionally include squalene.

[0072] Another adjuvant that can be used in the vaccine formulation described herein is a saponin-based adjuvant, such as QS21. QS21 is a purified plant extract that enhances the immune system's ability to respond to vaccine antigens. It is derived from the soap tree (Quillaja saponaria) and contains water-soluble triterpenoid glycosidic compounds, which are members of a family of phytocompounds called saponins. In one embodiment, a saponin-based adjuvant is combined with SLA to form a liposomal formulation. In one embodiment, SLA is Petition 870260052369, dated 01 / 06 / 2026, page 33 / 131 27 / 51 combined with QS21 to form a liposomal formulation (SLALSQ).

[0073] The vaccine formulation of the present invention may also include one or more diluents, carriers, solubilizers, emulsifiers, preservatives and / or adjuvants. EXAMPLE 2 Synthetic sequence design of the secretion signal for enhanced expression in Drosophila S2 cells.

[0074] The secretion signal peptide plays an important role in the expression of proteins that are targeted for secretion from the cell. Therefore, the use of optimal sequences that target the desired recombinant protein for secretion in the culture medium during production is important for protein processing efficiency and potential increases in product yield. The example of secretion signal optimization presented by Zhang et al. (J. Gene Med. (2005) 7:354-365) clearly demonstrates the benefits of secretion signal optimization. However, this specific example applies to plants, and it is unclear whether the described changes in the secretion signal apply to other types of eukaryotic cells. Furthermore, since Zika envelope expression is achieved through prM-E polypeptide expression, secretion is directed by the integral prM protein sequence that serves as a transmembrane anchor component for the prM protein.The second membrane-spanning sequence of the prM protein also serves as a secretion signal peptide for the E protein. There is no clear guidance on how best to optimize this transmembrane anchor peptide / secretion signal peptide sequence to increase secretion and envelope protein yield. Therefore, research was conducted on the secretion signals of prM. 28 / 51 And from flavivirus. Putative secretion signals from prM and the N-terminus of the E protein were analyzed using the SignalP program described by Petersen et al. (Nat. Methods, (2011) 8(10):785-6), which predicts the strength of the secretion signal based on an established algorithm and also predicts the cleavage site of the analyzed sequence.

[0075] Surprisingly, as shown in Figure 4, the prediction scores for all flaviviruses analyzed were poor. A score equal to > 0.500 is defined as a satisfactory secretion signal. Therefore, an effort was made to establish the necessary changes to improve the predicted secretion signal peptide score. It is believed that such changes will result in increased expression of the desired protein product in the culture medium of stably transformed S2 cells. The designs of the synthetic secretion signal followed the matrix table first described by von Heijne (Nuc. Acids Res. (1986) 14:4683-4690), and further improved by Bendtsen et al. (J. Mol. Biol. (2004) 340:783-795). The design aimed to maintain the secretion signal length across the 17 amino acids, include a single charged residue in the basic region, enhance the hydrophobic region, and improve positions -1 and -2.Initially, only the secretion signal (SyntheticZ) was designed, leaving the N-terminus of protein E unchanged. However, the score obtained for this sequence was only 0.374 despite optimization. Amino acids +1 to +4 (in this case, the N-terminus of protein E) can also impact the efficiency of the cleavage site; therefore, these were also altered at positions +1 and +4. The combination of the synthetic secretion sequence and the alterations at positions +1 and +4 (Synthetic+) resulted in a score of 0.649. Thus, the combination of both alterations was necessary to achieve a score > 0.500. To confirm that both alterations are necessary, only the... Petition 870260052369, dated 01 / 06 / 2026, p. 35 / 131 29 / 51 +1 and +4 changes were made following Zika WT (Zika +) and analyzed. This resulted in a small increase in the score from 0.231 to 0.310, but still below a score of 0.500. The amino acid sequences of the described signal peptides are listed below along with their Signal P scores. -1+1 | | Score for QKVIYLVMILLIAPAYS IRCIGV “Zika”: 0.231 Score for MRTIIALLLLLVSGAHG IRCIGV “SyntheticZ”: 0.374 Score for MRTIIALLLLLVSGAHA SRCVGV “Synthetic+”: 0.703 Score for QKVIYLVMILLIAPAYS SRCVGV “Zika+”: 0.310

[0076] The Synthetic+ secretion signal is operationally linked to the optimized Zika codon prM-80E sequence (SEQ ID NO:1) to create the combination of codon optimization and secretion signal optimization as detailed in SEQ ID NO:2. SEQ ID NO:2 is then inserted into the pHH202 vector expression cassette (SEQ ID NO: 7). The protein E that is expressed and secreted is called ZIKFP-Op80E-CoOp. EXAMPLE 3 ALTERATION OF THE CARBOX TERMINAL OF THE E PROTEIN SEQUENCE TO IMPROVE SECRETION AND STABILITY

[0077] The C-terminal of the E ectodomain of flavivirus E is typically defined by the Gly residue in the WXK / RXG motif of the sequence. In the case of Zika, this is Gly404. Although this truncation results in the secretion of the ZIKFP-80E product as shown in Example 1, efforts to increase the stability of the expressed E protein are still desirable, both in terms of expression levels and structural integrity. An analysis of Zika virus cyroEM data (Sirohi et al., 2016) suggests that an extension of Petition 870260052369, dated 01 / 06 / 2026, page 36 / 131 The 30 / 51 ectodomain E from Gly404 to Gly436 may provide a stabilizing effect. The C-terminal extension to Gly436 has the potential to provide interaction between the domain III region and the domain I region and help stabilize the expressed and secreted ZIKV envelope protein. In the extended E protein, the Phe431 residue has the potential to interact with a hydrophobic pocket in domain I composed of Val12, Val23, and Val24. An extension of the E protein in this way could lead to improved proper folding (native-like structure) and protein stability.

[0078] The optimized Zika codon prM-E sequence that extends the E protein to amino acid residue 436 is named prM-E-436 and is detailed in SEQ ID NO:3. SEQ ID NO:3 is then inserted into the expression cassette (SEQ ID NO: 7) of the pHH202 vector. The expressed and secreted E protein is named ZIKFP-E-436-CoOp.

[0079] A fully optimal Zika rM-E prM sequence combining the three attributes, codon optimization, optimized synthetic secretion signal, and extended E sequence, was also generated as shown in detail in SEQ ID NO:4. SEQ ID NO:4 is then inserted into the expression cassette (SEQ ID NO: 7) of the pHH202 vector. The expressed and secreted E protein is named ZIKFP-OpE-436-CoOp. The expression and secretion of ZIKFP-80E-WT, ZIKFP-80E-CoOp, and ZIKFP-OpE-436-CoOp from S2 cells was evaluated by SDS-PAGE. A Coomassie-stained gel is shown in Figure 8. All samples represent non-concentrated culture media to allow for a direct comparison of expression levels between the different recombinant Zika E proteins. Although expression is detected for each of the expressed sequences, ZIKFP-80E-WT, ZIKFP-80E-CoOp, and ZIKFP-OpE-436-CoOp, ZIKFP-80E-CoOp results in the highest level of expression. EXAMPLE 4 Petition 870260052369, dated 01 / 06 / 2026, page 37 / 131 31 / 51 Immunogenic evaluation of the ZIKFP-80E protein from optimized codons in mice.

[0080] The immunogenicity of the ZIKFP-80ECoOp protein subunit expressed in Drosophila S2 was analyzed in inbred and outbred mice with several different adjuvants to evaluate the immunogenic potential. Mice were immunized intramuscularly with two or three doses of ZIKFP-80E separated by 3-week intervals. Two quantities of ZIKFP80E-CoOp were analyzed, 5.0 pg and 2.5 pg. The adjuvants tested were Alhydrogel, GPI-0100, and GLA-SE. Mice were exudated 2 weeks after the second or third dose to prepare serum samples for antibody analysis. The immunogenicity study design is presented in Table 1. Petition 870260052369, dated 01 / 06 / 2026, page 38 / 131 32 / 51 Table 1. Immunogenicity study design for ZIKFP-80E-CoOp Group Article of Test Antigen Dose Elem Al GPI-0100 GLA SE Mouse # SW Mouse # 129 # of Mouse dose 2 # of Mouse dose 3 1 ZIKFP-80E-CoOp with Adjuvant GPI-0100 in SW 5 pg — 100 pg — 10 — 5 5 2 ZIKFP-80E-CoOp with Adjuvant Alum in SW 5 pg 120 pg — — 10 — 5 5 3 ZIKFP-80E-CoOp with Adjuvant GLA-SE in SW 5 pg — — 5 pg 10 — 5 5 4 ZIKFP-80E-CoOp with Adjuvant GLA-SE in SW 2.5 pg — — 5 pg 10 — 5 5 5 No antigen (Negative Control) Adjuvant GLA-SE — — — 5 pg 5 — 2 3 Petition 870260052369, dated 01 / 06 / 2026, page 39 / 131 33 / 51 in SW 6 ZIKFP-80E-CoOp with Adjuvant GPI-0100 on 129S6 / SvEvTac 5 pg — 100 pg — — 10 5 5 7 ZIKFP-80E-CoOp with Adjuvant GLA-SE on 129S6 / SvEvTac 5 pg — — 5 pg — 10 5 5 8 ZIKFP-80E-CoOp with Adjuvant GLA-SE on 129S6 / SvEvTac 2.5 pg — — 5 pg — 10 5 5 9 No antigen (Negative Control) Adjuvant GLA-SE on 129S6 / SvEvTac — — — 5 pg — 5 2 3 Petition 870260052369, dated 01 / 06 / 2026, page 40 / 131 34 / 51

[0081] Mice were immunized intramuscularly two or three times at 3-week intervals with the purified protein subunit at a dose of 5.0 pg or 2.5 pg. Five mice were exudated two weeks after dose two and 5 mice were exudated two weeks after dose three. The sera were then analyzed for anti-80E antibody titers by ELISA. The sera were also evaluated for the presence of virus-neutralizing antibodies using the plaque reduction neutralization test (PRNT).

[0082] The ELISA results for sera collected after two or three doses of ZIKFP-80E-CoOp formulated with multiple adjuvants are presented in Figure 6. The ELISA results indicate that ZIKFP-80E-CoOp is immunogenic and the responses in 129S6 / Sv mice are more robust and consistent than in Swiss Webster mice. References

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Zika virus, Microcephaly and Guillain-Barré Syndrome Situation Report: WHO; 2016 [cited 2016 April 2]. Available from: http: / / apps.who.int / iris / bitstream / 10665 / 204718 / 1 / zikasitrep_31Mar2016_eng.pdf?ua=1. WHO. Zika Product Landscape 2016 [cited 2016 April 2]. Available from: http: / / www.who.int / csr / research-and-development / zika-rdpipeline.pdf. Xu T, Logsdon NJ, Água MR. Structure of IL-22 derived from insect cells. Acta Crystallogr D Biol Crystallogr. 2005; 61(pt 7): 942-50. Zhang L, Leng Q, Mixson AJ. Alteration in the IL2 signal peptide affects Petition 870260052369, dated 01 / 06 / 2026, p. 45 / 131 39 / 51 secretion of proteins in vitro and in vivo. J. Gene Med. 2005; 7:354365. SEQ ID NO:1 - Sequência de nucleotídeos de códon otimizado para o prM-80E do Zika (Produto = ZIKFP-80E-CoOp) GCAGAAGTGACCCGCCGCGGCAGCGCATACTATATGTACCTCGA TCGTAACGACGCGGGC GAAGCTATCTCCTTCCCGACCACGCTGGGCATGAACAAGTGCTAT ATTCAGATTATGGAC CTGGGCCATATGTGCGACGCGACCATGTCCTACGAATGTCCGATG CTGGACGAAGGAGTT GAGCCTGATGACGTCGATTGCTGGTGCAATACCACTTCCACCTGG GTGGTGTACGGTACT TGCCATCACAAAAAGGGCGAAGCCCGCCGTTCCCGTCGCGCTGT CACTCTGCCAAGCCAC AGCACACGCAAATTGCAGACGAGGAGTCAGACGTGGTTGGAGTC GCGCGAGTACACAAAG CACCTGATTCGGGTGGAAAATTGGATCTTCCGGAATCCGGGCTTT GCTTTGGCGGCAGCC GCTATTGCGTGGCTGCTCGGCAGTAGCACGTCGCAGAAAGTGATT TACCTGGTCATGATC CTCCTCATCGCCCCCGCCTATTCGATCCGTTGCATTGGCGTCAGC AACCGCGATTTCGTG GAGGGCATGAGCGGTGGAACCTGGGTCGACGTTGTGCTGGAACA TGGCGGCTGCGTCACA GTGATGGCTCAGGACAAGCCGACCGTGGACATCGAGTTGGTTAC CACGACGGTTTCCAAC ATGGCGGAGGTTCGCAGCTACTGCTACGAAGCCAGCATCAGCGA TATGGCATCGGACAGC CGGTGCCCGACCCAGGGAGAAGCATATCTCGACAAGCAGTCCGA CACGCAATATGTCTGT Petição 870260052369, de 01 / 06 / 2026, pág. 46 / 131 40 / 51 AAAAGGACGCTCGTTGACCGCGGCTGGGGCAACGGCTGCGGCCT GTTTGGAAAAGGCTCC CTGGTCACATGCGCGAAGTTTGCATGGTCGAAGAAGATGACGGGC AAAAGCATCCAACCA GAGAATCTGGAAATACCGGATCATGTTGTCCGTGCACGGCAGCCA GCATAGTGGCATGATT GTGAACGACACCGGTCACGAAACCGACGAGAACCGCGCTAAAGT TGAGATCACCCGAAC AGTCCCCGGGCCGAGGCCACGCTGGGAGGCTTCGGATCGCTGG GTCTGGATTGCGAACCC CGCACCGGACTGGATTTCTCGGATCTCTACTACCTGACGATGAAC AATAAGCACTGGCTG GTGCACAAAGAGTGGTTCCATGATATCCCATTGCCCTGGCATGCC GGTGCCGATACCGGA ACACCCCACTGGAACAATAAGGAGGCCCTGGTCGAGTTTAAGGAC GCGCAGCCTAAGCGT CAAACGGTGGTGGTGCTGGGATCCCCAAGAGGGCGCCGTGCACAC GGCCCTGGCCGGCGCG CTGGAGGCCGAGATGGACGGTGCCAAGGGACGCTTGAGCTCCG GACACCTGAAATGCCGC CTCAAGATGGACAAGCTGCGTCTGAAAGGAGTGTCTCTACTCCTC TGCACCGCCGCGTTC ACCTTCACTAAGATTCCCGCCGAGACTTTGCACGGTACAGTGACC GTTGAGGTGCAGTAT GCCGGAACCGATGGCCTTGCAAAGTCCCGGCCCAAATGGCGGT GGATATGCAGACGCTG ACGCCTGTGGGCCGGCTCATTACCGCAAACCCAGTCATCACGGA GAGTACCGAGAACTCG AAGATGATGCTGGAGTTGGACCCCCCGTTTGGCGACAGTTACATC GTGATCGGAGTGGGC Petition 870260052369, dated 01 / 06 / 2026, page 47 / 131 41 / 51 GAAAAGAAGATTACGCACCATTGGCACCGTAGCGGC SEQ ID NO:2 - Codon nucleotide sequence optimized for Zika prM-80E with synthetic E secretion signal (Product = ZIKFPOp80E-CoOp) GCAGAAGTGACCCGCCGCGGCAGCGCATACTATATGTACCTCGA TCGTAACGACGCGGGC GAAGCTATCTCCTTCCCGACCACGCTGGGCATGAACAAGTGCTAT ATTCAGATTATGGAC CTGGGCCATATGTGCGACGCGACCATGTCCTACGAATGTCCGATG CTGGACGAAGGAGTT GAGCCTGATGACGTCGATTGCTGGTGCAATACCACTTCCACCTGG GTGGTGTACGGTACT TGCCATCACAAAAAGGGCGAAGCCCGCCGTTCCCGTCGCGCTGT CACTCTGCCAAGCCAC AGCACACGCAAATTGCAGACGAGGAGTCAGACGTGGTTGGAGTC GCGCGAGTACACAAAG CACCTGATTCGGGTGGAAAATTGGATCTTCCGGAATCCGGGCTTT GCTTTGGCGGCAGCC GCTATTGCGTGGCTGCTCGGCAGTAGCACGTCGATGCGCACCAT CATTGCCCTGCTCTTG CTGCTCGTGAGCGGTGCCCACGCCAGCCGTTGCGTGGGCGTCA GCAACCGCGATTTCGTG GAGGGCATGAGCGGTGGAACCTGGGTCGACGTTGTGCTGGAACA TGGCGGCTGCGTCACA GTGATGGCTCAGGACAAGCCGACCGTGGACATCGAGTTGGTTAC CACGACGGTTTCCAAC ATGGCGGAGGTTCGCAGCTACTGCTACGAAGCCAGCATCAGCGA TATGGCATCGGACAGC CGGTGCCCGACCCAGGGAGAAGCATATCTCGACAAGCAGTCCGA Petição 870260052369, de 01 / 06 / 2026, pág. 48 / 131 42 / 51 CACGCAATATGTCTGT AAAAGGACGCTCGTTGACCGCGGCTGGGGCAACGGCTGCGGCCT GTTTGGAAAAGGCTCC CTGGTCACATGCGCGAAGTTTGCATGTTCGAAGAAGATGACGGGC AAAAGCATCCAACCA GAGAATCTGGAATACCGGATCATGTTGTCCGTGCACGGCAGCCA GCATAGTGGCATGATT GTGAACGACACCGGTCACGAAACCGACGAGAACCGCGCTAAAGT TGAGATCACCCCGAAC AGTCCCCGGGCCGAGGCCACGCTGGGAGGCTTCGGATCGCTGG GTCTGGATTGCGAACCC CGCACCGGACTGGATTTCTCGGATCTCTACTACCTGACGATGAAC AATAAGCACTGGCTG GTGCACAAAGAGTGGTTCCATGATATCCCATTGCCCTGGCATGCC GGTGCCGATACCGGA ACACCCCACTGGAACAATAAGGAGGCCCTGGTCGAGTTTAAGGAC GCGCACGCTAAGCGT CAAACGGTGGTGGTGCTGGGATCCCAAGAGGGCGCCGTGCACAC GGCCCTGGCCGGCGCG CTGGAGGCCGAGATGGACGGTGCCAAGGGACGCTTGAGCTCCG GACACCTGAAATGCCGC CTCAAGATGGACAAGCTGCGTCTGAAAGGAGTGTCCTACTCCCTC TGCACCGCCGCGTTC ACCTTCACTAAGATTCCCGCCGAGACTTTGCACGGTACAGTGACC GTTGAGGTGCAGTAT GCCGGAACCGATGGCCCTTGCAAAGTCCCGGCCCAAATGGCGGT GGATATGCAGACGCTG ACGCCTGTGGGCCGGCTCATTACCGCAAACCCAGTCATCACGGA GAGTACCGAGAACTCG AAGATGATGCTGGAGTTGGACCCCCCGTTTGGCGACAGTTACATC Petição 870260052369, de 01 / 06 / 2026, pág. 49 / 131 43 / 51 GTGATCGGAGTGGGC GAAAAGAAGATTACGCACCATTGGCACCGTAGCGGC SEQ ID NO:3 - Sequência de nucleotídeos de códon otimizado para o prM-E-436 do Zika (Produto = ZIKFP-E-436-CoOp) GCAGAAGTGACCCGCCGCGGCAGCGCATACTATATGTACCTCGA TCGTAACGACGCGGGC GAAGCTATCTCCTTCCCGACCACGCTGGGCATGAACAAGTGCTAT ATTCAGATTATGGAC CTGGGCCATATGTGCGACGCGACCATGTCCTACGAATGTCCGATG CTGGACGAAGGAGTT GAGCCTGATGACGTCGATTGCTGGTGCAATACCACTTCCACCTGG GTGGTGTACGGTACT TGCCATCACAAAAAGGGCGAAGCCCGCCGTTCCCGTCGCGCTGT CACTCTGCCAAGCCAC AGCACACGCAAATTGCAGACGAGGAGTCAGACGTGGTTGGAGTC GCGCGAGTACACAAAG CACCTGATTCGGGTGGAAAATTGGATCTTCCGGAATCCGGGCTTT GCTTTGGCGGCAGCC GCTATTGCGTGGCTGCTCGGCAGTAGCACGTCGCAGAAAGTGATT TACCTGGTCATGATC CTCCTCATCGCCCCCGCCTATTCGATCCGTTGCATTGGCGTCAGC AACCGCGATTTCGTG GAGGGCATGAGCGGTGGAACCTGGGTCGACGTTGTGCTGGAACA TGGCGGCTGCGTCACA GTGATGGCTCAGGACAAGCCGACCGTGGACATCGAGTTGGTTAC CACGACGGTTTCCAAC ATGGCGGAGGTTCGCAGCTACTGCTACGAAGCCAGCATCAGCGA TATGGCATCGGACAGC CGGTGCCCGACCCAGGGAGAAGCATATCTCGACAAGCAGTCCGA Petição 870260052369, de 01 / 06 / 2026, pág. 50 / 131 44 / 51 CACGCAATATGTCTGT AAAAGGACGCTCGTTGACCGCGGCTGGGGCAACGGCTGCGGCCT GTTTGGAAAAGGCTCC CTGGTCACATGCGCGAAGTTTGCATGTTCGAAGAAGATGACGGGC AAAAGCATCCAACCA GAGAATCTGGAATACCGGATCATGTTGTCCGTGCACGGCAGCCA GCATAGTGGCATGATT GTGAACGACACCGGTCACGAAACCGACGAGAACCGCGCTAAAGT TGAGATCACCCCGAAC AGTCCCCGGGCCGAGGCCACGCTGGGAGGCTTCGGATCGCTGG GTCTGGATTGCGAACCC CGCACCGGACTGGATTTCTCGGATCTCTACTACCTGACGATGAAC AATAAGCACTGGCTG GTGCACAAAGAGTGGTTCCATGATATCCCATTGCCCTGGCATGCC GGTGCCGATACCGGA ACACCCCACTGGAACAATAAGGAGGCCCTGGTCGAGTTTAAGGAC GCGCACGCTAAGCGT CAAACGGTGGTGGTGCTGGGATCCCAAGAGGGCGCCGTGCACAC GGCCCTGGCCGGCGCG CTGGAGGCCGAGATGGACGGTGCCAAGGGACGCTTGAGCTCCG GACACCTGAAATGCCGC CTCAAGATGGACAAGCTGCGTCTGAAAGGAGTGTCCTACTCCCTC TGCACCGCCGCGTTC ACCTTCACTAAGATTCCCGCCGAGACTTTGCACGGTACAGTGACC GTTGAGGTGCAGTAT GCCGGAACCGATGGCCCTTGCAAAGTCCCGGCCCAAATGGCGGT GGATATGCAGACGCTG ACGCCTGTGGGCCGGCTCATTACCGCAAACCCAGTCATCACGGA GAGTACCGAGAACTCG AAGATGATGCTGGAGTTGGACCCCCCGTTTGGCGACAGTTACATC Petição 870260052369, de 01 / 06 / 2026, pág. 51 / 131 45 / 51 GTGATCGGAGTGGGC GAAAAGAAGATTACGCACCATTGGCACCGTAGCGGC AGCACCAT CGGCAAGGCCTTCGAG GCCACCGTGCGCGGCGCCAAGCGCATGGCCGTGCTGGGCGACA CCGCCTGGGACTTCGGC TCCGTGGGCGGC SEQ ID NO:4 - Zika prM-E-436 optimized codon nucleotide sequence with synthetic E secretion signal (Product = ZIKFP-OpE-436-CoOp) GCAGAAGTGACCCGCCGCGGCAGCGCATACTATATGTACCTCGA TCGTAACGACGCGGGC GAAGCTATCTCCTTCCCGACCACGCTGGGCATGAACAAGTGCTAT ATTCAGATTATGGAC CTGGGCCATATGTGCGACGCGACCATGTCCTACGAATGTCCGATG CTGGACGAAGGAGTT GAGCCTGATGACGTCGATTGCTGGTGCAATACCACTTCCACCTGG GTGGTGTACGGTACT TGCCATCACAAAAAGGGCGAAGCCCGCCGTTCCCGTCGCGCTGT CACTCTGCCAAGCCAC AGCACACGCAAATTGCAGACGAGGAGTCAGACGTGGTTGGAGTC GCGCGAGTACACAAAG CACCTGATTCGGGTGGAAAATTGGATCTTCCGGAATCCGGGCTTT GCTTTGGCGGCAGCC GCTATTGCGTGGCTGCTCGGCAGTAGCACGTCGATGCGCACCAT CATTGCCCTGCTCTTG CTGCTCGTGAGCGGTGCCCACGCCAGCCGTTGCGTGGGCGTCA GCAACCGCGATTTCGTG GAGGGCATGAGCGGTGGAACCTGGGTCGACGTTGTGCTGGAACA TGGCGGCTGCGTCACA Petition 870260052369, dated 01 / 06 / 2026, p. 52 / 131 46 / 51 GTGATGGCTCAGGACAAGCCGACCGTGGACATCGAGTTGGTTAC CACGACGGTTTCCAAC ATGGCGGAGGTTCGCAGCTACTGCTACGAAGCCAGCATCAGCGA TATGGCATCGGACAGC CGGTGCCGACCCAGGGAGAAGCATATCTCGACAAGCAGTCCGA CACGCAATATGTCTGT AAAAGGACGCTCGTTGACCGCGGCTGGGGCAACGGCTGCGGCCT GTTTGGAAAAGGCTCC CTGGTCACATGCGCGAAGTTTGCATGGTCGAAGAAGATGACGGGC AAAAGCATCCAACCA GAGAATCTGGAAATACCGGATCATGTTGTCCGTGCACGGCAGCCA GCATAGTGGCATGATT GTGAACGACACCGGTCACGAAACCGACGAGAACCGCGCTAAAGT TGAGATCACCCGAAC AGTCCCCGGGCCGAGGCCACGCTGGGAGGCTTCGGATCGCTGG GTCTGGATTGCGAACCC CGCACCGGACTGGATTTCTCGGATCTCTACTACCTGACGATGAAC AATAAGCACTGGCTG GTGCACAAAGAGTGGTTCCATGATATCCCATTGCCCTGGCATGCC GGTGCCGATACCGGA ACACCCCACTGGAACAATAAGGAGGCCCTGGTCGAGTTTAAGGAC GCGCAGCCTAAGCGT CAAACGGTGGTGGTGCTGGGATCCCCAAGAGGGCGCCGTGCACAC GGCCCTGGCCGGCGCG CTGGAGGCCGAGATGGACGGTGCCAAGGGACGCTTGAGCTCCG GACACCTGAAATGCCGC CTCAAGATGGACAAGCTGCGTCTGAAAGGAGTGTCCTACTCCCTC TGCACCGCCGCGTTC ACCTTCACTAAGATTCCCGCCGAGACTTTGCACGGTACAGTGACC GTTGAGGTGCAGTAT Petition 870260052369, dated 01 / 06 / 2026, page 53 / 131 47 / 51 GCCGGAACCGATGGCCCTTGCAAAGTCCCGGCCCAAATGGCGGT GGATATGCAGACGCTG ACGCCTGTGGGCCGGCTCATTACCGCAAACCCAGTCATCACGGA GAGTACCGAGAACTCG AAGATGATGCTGGAGTTGGACCCCCCGTTTGGCGACAGTTACATC GTGATCGGAGTGGGC GAAAAGAAGATTACGCACCATTGGCACCGTAGCGGC AGCACCAT CGGCAAGGCCTTCGAG GCCACCGTGCGCGGCGCCAAGCGCATGGCCGTGCTGGGCGACA CCGCCTGGGACTTCGGC TCCGTGGGCGGC SEQ ID NO:5 - Polynesian prM-E nucleotide sequence French (Product = ZIKFP-80E-WT) GCGGAGGUCACUAGACGUGGGAGUGCAUACUAUAUGUACUUGG ACAGAAACGACGCUGGG 60 GAGGCCAUAUCUUUUCCAACCACAUUGGGGAUGAAUAAGUGUUA UAUACAGAUCAUGGAU 120 CUUGGACACAUGUGUGAUGCCACCAUGAGCUAUGAAUGCCCUA UGCUGGAUGAGGGGGUG 180 GAACCAGAUGACGUCGAUUGUUGGUGCAACACGACGUCAACUU GGGUUGUGUACGGAACC 240 UGCCAUCACAAAAAGGUGAAGCACGGAGAUCUAGAAGAGCUGU GACGCUCCCCUCCCAU 300 UCCACUAGGAAGCUGCAAACGCGGUCGCAAACCUGGUUGGAAU CAAGAGAAUACACAAAG 360 CACUUGAUUAGAGUCGAAAAUUGGAUAUUCAGGAACCCUGGCUU CGCGUUAGCAGCAGCU 420 GCCAUCGCUUGGCUUUUGGGAAGCUCAACGAGCCAAAAAGUCA UAUACUUGGUCAAUGAUA 480 Petition 870260052369, de 01 / 06 / 2026, pág. 54 / 131 48 / 51 CUGCUGAUUGCCCCGGCAUACAGCAUCAGGUGCAUAGGAGUCA GCAAUAGGGACUUUGUG 540 GAAGGUAUGUCAGGUGGACUUGGGGUUGAUGUUGUGCUUGGAAC AUGGAGGUGUGUGUCACC 600 GUAAUGGCACAGGACAAACCGACUGUCGACAUAGAGCUGGUUA CACAACAGCAGCAAC 660 AUGGCGGAGGUAAGAUCCUAUCUGCUAUGAGGCAUCAAUAUCGG ACAUGGCUUCGGACAGC 720 CGCUGCCCAACAACGGUGAAGCCUACCUUGACAAGCAAUCAGA CACUCAAUAUGUCUGC 780 AAAAGAACGUUAGUGGACAGAGGCUGGGGAAAUGGAUGUGGAC UUUUUGGCAAAGGGAGC 840 CUGGUGCAUCCGCUAGUUUGCAUGCCAAAAAAUGACCG GGAAGAGCAUCCAGCCA 900 GAGAAUCUGGAGUACCGGAAUAAUGCUGUCAGUGGCUCCC AGCAGUGGGAUGAUC 960 GUUAAUGACACAGGAAGGU UGAGAUAACGCCCAAU1020 UCACCAAGAGCCGAAGCCACCCUGGGGGUUUUGGAAGCCUAG STORAGEACCG 1080 AGGACAGGCHOUSEHOUSE UAACA SUMMARY 1140 REPLACEMENTACCAUCAUACCUUGGCCACG CUGGGGCAGACACCGGA 1200 ACCUCAQUACACAAAGAAGACQUACUTAQUE CGCACAUGCCAAAAGG 1260 WHAT TO BECOME CGGCCUGCUGGAGCU 1320 STUDYCUGCAUGGUGCAAAGGGAAGGCUCUCUG GCCACUUGAAAAUGUCGC 1380 Petition 870260052369, dated 01 / 06 / 2026, p. 55 / 131 49 / 51 CUGAAAAUGGAUAAACUUAGAUUGAAGGGCGUGUCAUACUCCUU GUGUACCGCAGCGUUC 1440 ACAUUCACCAAGAUCCCGGCUGAAACACUGCACGGGACAGUCAC AGUGGAGGUACAGUAC 1500 GCAGGGACAGAUGGACCUUGCAAGGUUCCAGCUCAGAUGGCGG UGGACAUGCAAACUCUG 1560 ACCCCAGUUGGGAGGUUGAUAACCGCUAACCCCGUAAUCACUG AAAGCACUGAGAACUCU 1620 AAGAUGAUGCUGGAACUUGAUCCACAUUUGGGGACUCUUACA UUGUCAUAGGAGUCGGG 1680 GAGAAGAAGAUCACCCACCACUGGCACAGGAGUGGCAGCACCAU UGGAAAAGCAUUUGAA 1740 GCCACUGUGAGAGGUGCCAAGAGAAUGGCAGUCUUGGGAGACA CAGCCUGGGACUUUGGA 1800 UCAGUUGGAGGCGCUCUCAACUCAUUGGGCAAGGGCAUCCAUC AAAUUUUUGGAGCAGCU 1860 UUCAAAUCAUUGUUUGGAGGAAUGUCCUGGUUCUCACAAAUUCU CAUUGGAACGUUGCUG 1940 AUGUGGUUGGGUCUGAACACAAAGAAUGGAUCUAUUUCCCUUA UGUGCUUGGCCUUAGGG 2000 GGAGUGUUGAUCUUCUUAUCCACAGCUGUCUCUGCUG 2017 SEQ ID NO:6 - Amino acid sequence of prM-E from French Polynesia A E V T R R G S A Y Y M Y L D R N D A G 20 E A I S F P T T L G M N K C Y I Q I M D 40 L G H M C D A T M S Y E C P M L D E G V 60 E P D D V D C W C N T T S T W V V Y G T 80 C H H K K G E A R R S R R A V T L P S H 100 Petição 870260052369, de 01 / 06 / 2026, pág. 56 / 131 50 / 51 S T R K L Q T R S Q T W L E S R E Y T K 120 H L I R V E N W I F R N P G F A L A A A 140 A I A W L L G S S T S Q K V I Y L V M I160 L L I A P A Y S I R C I G V S N R D F V180 E G M S G G T W V D V V L E H G G C V T200 V M A Q D K P T V D I E L V T T T V S N 220 M A E V R S Y C Y E A S I S D M A S D S 240 R C P T Q G E A Y L D K Q S D T Q Y V C 260 K R T L V D R G W G N G C G L F G K G S 280 L V T C A K F A C S K K M T G K S I Q P 300 E N L E Y R I M L S V H G S Q H S G M I 320 V N D T G H E T D E N R A K V E I T P N 340 S P R A E A T L G G F G S L G L D C E P 360 R T G L D F S D L Y Y L T M N N K H W L 380 V H K E W F H D I P L P W H A G A D T G400 T P H W N N K E A L V E F K D A H A K R420 Q T V V V L G S Q E G A V H T A L A G A440 L E A E M D G A K G R L S S G H L K C R460 L K M D K L R L K G V S Y S L C T A A F 480 T F T K I P A E T L H G T V T V E V Q Y 500 A G T D G P C K V P A Q M A V D M Q T L 520 T P V G R L I T A N P V I T E S T E N S 540 K M M L E L D P P F G D S Y I V I G V G560 E K K I T H H W H R S G S T I G K A F E580 A T V R G A K R M A V L G D T A W D F G 600 S V G G A L N S L G K G I H Q I F G A A620 F K S L F G G M S W F S Q I L I G T LL 640 M W L G L N T K N G S I S L M C L A L G 660 G V L I F L S T A V S A672 Petition 870260052369, of 01 / 06 / 2026, p. 57 / 131 51 / 51 SEQ ID NO:7 - pHH202 expression vector cassette sequence GGTACCGTTGCAGGACAGGATGTGGTGCCGATGTGACTAGCTC TTTGCTGCAGGCCGTCCTATCCTCTGGTTCCGATAAGAGACCCAG AACTCCGGCCCCCCACCGCCCACCGCCACCCCCATACATATGTG GTACGCAAGTAAGAGTGCCTGCGCATGCCCCATGTGCCCCACCA AGAGTTTTGCATCCCCATACAAGTCCCCAAAGTGGAGAACCGAACC AATTCTTCGCGGGCAGAACAAGCTTCTGCACACGTCTCCACTC GAATTTGGAGCCGGCCGGCGTGTGCAAAAGAGGTGAATCGAACG AAGACCCGTGTGTAAAGCCGCGTTTCCAAATGTATAAAACGA GAGCATCTGGCCAATGTGCATCAGTTGTGGTCAGCAGCAAAATCA AGTGAATCATCTCAGTGCAACTAAAGGGGGaATCTAGAaacaacATG AAGACCATTATCGCCCTGTCGTACATCTTTTGCCTGGTGTTCgctag cTCTAGctagAggctcgagGCCTTCGAAGAGACCAGGATACCATACCAT ATTGATGAGTTTGGACAAACCAACTAGAATGCAGTGAAAAAAAT GCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATT ATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTAT GTTTCAGGTTCAGGGGGAGGTGGGAGTTTTTAAG

[0084] Although the present invention has been described in terms of exemplary embodiments and specific examples, it will be appreciated that the embodiments described in this application are only illustrative and various modifications and alterations may be made by those skilled in the art without departing from the spirit and scope of the invention set forth in the claims that follow. Petition 870260052369, dated 01 / 06 / 2026, p. 58 / 131

Claims

1 / 2 CLAIMS 1. Expression vector, characterized in that it comprises a DNA sequence encoding a Zika virus pre-membrane and an envelope protein, wherein the expression of the DNA sequence results in the secretion of a soluble envelope protein in a culture medium, wherein the DNA sequence comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:

4.

2. Expression vector, according to claim 1, characterized in that it further comprises an expression cassette comprising SEQ ID NO:

7.

3. Use of expression vectors, as defined in claim 1, characterized in that they are used in Drosophila cells.

4. Use of expression vectors, as defined in claim 1, characterized in that they are used in Drosophila S2 cells.

5. Vaccine, characterized in that it comprises: (a) a purified envelope protein (E) from Zika virus encoded by SEQ ID NO:1, or SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or a nucleic acid sequence beginning at nucleotide 505 of SEQ ID NO:4, or beginning at amino acid residue 169 of SEQ ID NO:6, wherein the protein is secreted into growth medium when recombinantly expressed in a host cell; and (b) an adjuvant, wherein the vaccine induces the production of neutralizing antibodies in human subjects.

6. Vaccine, according to claim 5, characterized in that protein E is produced and expressed recombinantly in insect host cells. Petition 870260052369, dated 01 / 06 / 2026, pp. 119 / 131 2 / 2 7. Vaccine, according to claim 5, characterized in that the E protein is produced and expressed in recombinant form in Drosophila melanogaster Schneider 2 (S2) host cells.

8. Vaccine, according to claim 5, characterized in that it further comprises a pharmaceutically acceptable excipient.

9. A vaccine, according to claim 5, characterized in that human individuals are immunodeficient.

10. A vaccine according to claim 8, characterized in that human individuals are immunodeficient.

11. Vaccine, characterized in that it comprises any of the soluble envelope proteins expressed and secreted by the expression vectors, as defined in claim 1 or 2.

12. Vaccine, according to any one of claims 6 to 11, characterized in that it induces immunogenicity against the Zika virus in humans.

13. Vaccine, according to any one of claims 6 to 12, characterized in that the adjuvant is a saponin-based adjuvant, an aluminum (Alum)-based adjuvant, a stable oil-in-water emulsion (SE)-based adjuvant, SLA, or combinations thereof.

14. Vaccine, according to claim 13, characterized in that the adjuvant is a saponin-based adjuvant.

15. Vaccine, according to claim 14, characterized in that the saponin adjuvant is QS21.

16. Vaccine, according to any one of claims 13 to 15, characterized in that SLA and QS21 are combined in a liposomal formulation (SLA-LSQ). Petition 870260052369, dated 01 / 06 / 2026, pp. 120 / 131

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