HOST CELL COMPRISING A NUCLEIC ACID CONSTRUCTION EXPRESSION VECTOR

Abstract

The present invention relates to a host cell comprising a vector integrating a nucleic acid construct encoding a protein comprising an N-terminal signal peptide, a first polypeptide of interest, a second polypeptide of interest, and an internal signal peptide sequence positioned between the first and second polypeptides. This signal peptide sequence can be cleaved by a protease to produce independent first and second polypeptides. The invention provides a novel method for utilizing integrated protease signal sites within a host cell to simultaneously express two or more antigens from a single polynucleotide construct.

Description

Title of the invention: HOST CELL COMPRISING A NUCLEIC ACID CONSTRUCTION EXPRESSION VECTOR Field of the invention

[0001] The present invention relates to the fields of molecular biology, biotechnology, and immunology. More particularly, it relates to a host cell comprising a vector integrating a nucleic acid construct encoding multiple polypeptide antigens or immunogens, or other polypeptides of interest. The invention provides a novel way of using integrated protease signal sites to simultaneously express two or more antigens from a single polynucleotide construct. Context of the invention

[0002] Various systems have been proposed for the expression of several recombinant proteins from the same vector or nucleic acid construct. Constructs that attempt to express several proteins from a single construct such as a plasmid, a viral vector, a polycistronic construct, or a self-replicating RNA construct suffer from a number of problems, including genetic interference, where the expression of one gene on the construct interferes with the expression of another gene; variable gene expression, where different genes are expressed to varying degrees; positional effects, where the relative positioning of genes upstream or downstream on a construct affects gene expression; and epigenetic modifications that differ between genes on the same construct and can lead to differential expression.Epigenetic modifications that differ between genes within the same construct and can lead to differential gene expression; transcriptional interference, where the transcription of one gene interferes with that of another, leading to altered expression levels; size limitations that restrict the number of non-coding segments in a construct; genetic incompatibility, where the co-expression of two or more genes leads to undesirable outcomes; promoter competition, which leads to different expression of different genes; or regulatory interactions between genes within the same construct leading to undesirable outcomes.

[0003] In view of the above, the inventors sought to develop a single polynucleotide construct capable of efficiently and reliably expressing several recombinant polypeptides of interest. This construct codes for a long A protein that, once expressed, is cleaved into individual proteins. For example, it can encode two or more structural viral antigens from an animal virus, which are then post-translated into individual viral antigens or immunogens. Because each independent antigen is derived from the same long protein, this method can produce stoichiometric amounts of each antigen. Since protein processing typically occurs in the endoplasmic reticulum (ER), the polypeptides of interest, once released from the protein, can be further processed or associate with each other, for example, to form tertiary or quaternary epitopes or structures absent from conventionally expressed recombinant proteins.

[0004] Maintaining the stoichiometry of the amounts of viral proteins or other types of proteins is beneficial because it allows the correct assembly, stability and efficient production of multiprotein complexes, such as viral capsids; it can allow correct post-translational trafficking and processing of recombinant proteins once expressed and can contribute to the formation of immunogenic complexes of recombinant proteins comprising conformational epitopes comprising chains or residues of different proteins in a quaternary complex. Summary of the invention

[0005] One aspect of the invention relates to a host cell comprising a vector that transfects or transforms cells, such as host cells, in a non-human animal to be vaccinated, as well as a method for vaccinating or immunizing a non-human animal. The vectors comprise nucleic acid constructs comprising polynucleotides encoding a protein or a recombinant protein comprising an N-terminal signal peptide, a first polypeptide of interest, a second polypeptide of interest, and an internal signal sequence, such as a 6K or 6K-like sequence, positioned between the first and second polypeptides of interest; wherein the internal signal sequence can be cleaved by a protease to produce independent first and second polypeptides. The N-terminal signal peptide can be used to translocate the protein from the cytoplasm to the endoplasmic reticulum.

[0006] The description also relates to methods of manufacturing and using polypeptides expressed by nucleic acid construction and transformed into individual polypeptides of interest by cellular proteases.

[0007] The preceding paragraphs have been provided by way of general introduction and are not intended to limit the scope of the following claims. The methods of The achievements described, as well as other advantages, will be better understood if one refers to the detailed description that follows, in conjunction with the accompanying drawings. Brief description of the figures

[0008] [FIG1 FIG. 1 represents replicons 1 and 3-6 which orient the SIV HA gene (swine influenza virus hemagglutinin) to the 5' end of an open reading frame (ORF), a linker, and then an NA gene (neuraminidase) to the 3' end of the ORF. Replicon 2 reverses the order of the SIV HA and NA genes in the ORF. As shown, the internal linker sequences for each replicon are derived from V6K (Venezuelan equine encephalitis virus), 6K, S6K (Semliki Forest virus), BSP (bunyavirus), HSP (hantavirus), and p7 (hepatitis C virus).

[0009] [Fig. 2] [Fig. 2] (from Owji et al., 2018, below) illustrates a general signal peptide structure and cleavage site comprising a positively charged N region, an H region which includes a hydrophobic core, and a C region involved in cleavage. A 6K element may comprise two signal peptides separated by other residues of a 6K or viral-type protein.

[0010] [Fig.3] The [Fig.3] (from Lober, et al., 2001) represents the precursor of the Hantaan virus glycoprotein and the cleavage site of the putative signal peptide comprising the N, H and C regions.

[0011] [Fig. 4] [Fig. 4] (from Lober et al., 2001) represents the cleavage sites of the putative signal peptide comprising the N, H, and C regions of different Bunyavirus glycoprotein precursors. The arrows indicate the potential cleavage sites between the two glycoproteins G1 and G2 (genera Hantavirus and Phlebovirus) or NSm and G1 (genus Bunyavirus).

[0012] [Fig.5] [Fig.5]. Analysis of the cleavage site for the H p7 strain of HCV, see Lin, et al, Processing in the hepatitis C virus E2-NS2 region: identification of p7 and two distinct E2-specific products with different C termini, J. VIROL. 1994, 68(8) 5063-5073. See also the sequences of [Fig.5] of Lin et al.

[0013] [Fig.6] [Fig.6]. Analysis of cleavage sites for Bunyamwera.

[0014] The signal peptide predictions in FIGS 4A and 4B were obtained from an online signal peptide predictor.

[0015] [Fig.7] Fig.7 describes the 6K and 6K-type sequences and their domain structures.

[0016] [Fig.8] Fig.8 describes the 6K and 6K-type sequences as well as the different types of amino acid residues.

[0017] [Fig.9] The [Fig.9] relates to the construction of the SIV-H3-H1 link. Detailed description of the invention

[0018] As stated in this document, the invention relates to a vector comprising a nucleic acid construct designed to include a coding sequence that expresses a protein comprising two, three, four, or more polypeptides of interest, separated by 6K, 6K-like, or internal signal peptide cleavage sites. Once expressed, the protein encoded by the coding sequence is considered to be transported to the endoplasmic reticulum by means of the N-terminal signal peptide. Proteases, such as signal peptidases, present in the endoplasmic reticulum then cleave the protein at the cleavage sites of the N-terminal or internal signal peptide, thereby releasing the two or more polypeptides of interest, which are then free to be processed separately or to associate.The invention also relates to vectors and host cells comprising the nucleic acid construct and expressing the protein comprising the first, second or subsequent polypeptides of interest.

[0019] In this document, the term "polynucleotide" or "nucleic acid" refers to a linear polymer whose molecule is composed of numerous nucleotide units. It may be a subsection of a nucleic acid molecule integrated into a longer polynucleotide or, conversely, an autonomous nucleic acid with discrete terminations such as a 5' terminal phosphate group and a 3' free hydroxyl group.

[0020] In this document, the term "polypeptide" or "protein" refers to a chain of amino acids linked by peptide bonds, whose N and C ends are defined, or a chain of amino acids forming a domain, region, or fragment of a larger polypeptide, such as part of a fusion protein, chimeric protein, or polyprotein. Thus, a polypeptide may be an independent, self-contained polypeptide or may be a subunit of a larger polypeptide adjacent to or flanked by other sequences.

[0021] The term "polyprotein," as defined in this document, refers to a protein that, after being synthesized, is cleaved to produce two or more distinct polypeptides. A polyprotein is generally encoded by a single coding sequence of mRNA that is translated into a polyprotein. The single mRNA may be found in nature or be an artificial construct, such as a construct comprising coding sequences obtained from different viral species or strains, or a coding sequence that encodes a limited number of antigens from the same virus or microorganism, not necessarily in the same order as they appear in the chromosome or mRNA of the virus or microorganism. Polyproteins exist in nature. For the purposes of this document, they may also be synthetic or recombinant polypeptides, such as chimeric or cleavable fusion proteins comprising immunogenic parts. or antigenic amino acid residues from two, three or more different polypeptide antigens.

[0022] The term "coding sequence" refers to a sequence of polynucleotides, which may be RNA or DNA, that directly codes, or in the case of DNA as a last resort, a sequence of amino acids forming a polypeptide. A coding sequence may be found in nature or be artificially constructed.

[0023] The term "region" refers to different functional sub-parts of a polyprotein or chimeric protein encoded by a nucleic acid construct, such as amino acid portions that will be subsequently released as independent polypeptides or other functional parts, for example, "an N-terminal signal peptide region." The term "region," as well as the terms "fragment," "segment," or "domain," can be used to refer to sub-parts of a longer polyprotein, chimeric protein, or fusion protein.

[0024] An "open reading frame" or ORF, for example, in viral polynucleotides generally begins with a start codon and ends with a stop codon. It can code for one or more viral polypeptides.

[0025] The embodiments of the invention include, but are not limited to, the following elements.

[0026] Nucleic acid constructs. One aspect of the invention relates to a polynucleotide or nucleic acid construct that encodes a protein comprising an N-terminal signal peptide, a first polypeptide of interest, an internal or 6K or 6K-type signal sequence cleavable by a protease, and a second polypeptide of interest. Upon contact of the protein with a protease recognizing the signal peptide, such as those present in 6K or 6K-type sequences, the first and second polypeptides are cleaved and then released as independent polypeptides. In some cases, a single signal peptide cleavage sequence will be placed between an upstream and a downstream polypeptide of interest. In other cases, several signal peptide sequences will be placed between the polypeptides of interest encoded by the construct.These multiple signal peptide sequences include, but are not limited to, signal peptide sequences within a 6K or 6K-type polypeptide sequence.

[0027] The nucleic acid sequence encoding the protein can be considered as part of a single open reading frame (ORF), since this entire portion of polynucleotides can be translated into protein, i.e. into proteins or polypeptides as described herein.

[0028] Advantageously, the invention can express two, three, or more proteins that are never associated with each other in nature. For example, the coding sequence can code for antigens or epitopes of the influenza HA polypeptide as well as antigens or epitopes of porcine reproductive and respiratory viruses. It can also encode a subset of antigens from a particular virus (or microorganism), but not others. Thus, it could omit antigens that inhibit or dominate immune responses. It could also encode the antigens of a virus in a different order than that found in nature or in the same order. For example, if the viral antigens A, B, C, and D are expressed by the mRNA of a virus in the order ABCD in a viral ORF, a construct of the invention can express the same antigens in a different order such as BADC, DCBA, etc., based on their order of appearance in the coding sequence of the polynucleotide construct.

[0029] Three or more polypeptides of interest. In some cases, the nucleic acid construct described herein encodes additional polypeptides of interest. For example, in these embodiments, the protein may include an additional 6K or 6K-type region or an internal signal peptide and a third polypeptide of interest positioned downstream of the second polypeptide of interest. Similarly, the construct may encode a protein comprising a fourth, fifth, or other polypeptide of interest preceded by a 6K or 6K-type region or an internal signal sequence.

[0030] As stated in this document, a polyprotein is a large protein molecule that is a single protein chain containing multiple functional regions or subunits, each of which can be cleaved or transformed into a distinct functional protein. These functional proteins may include structural proteins, antigens, immunogens, enzymes, or other proteins of interest derived from a longer polyprotein or polypeptide construct. In some embodiments of the invention, a polyprotein comprises two, three, or more polypeptides of interest derived from an immunogen or an antigen, such as a viral immunogen or antigen. In another embodiment, the two, three, or more polypeptides of interest may be derived from the same gene of interest or from different genes of interest.In another embodiment, the two, three, or more polypeptides of interest are different from each other; for example, they are portions of different viral antigens.

[0031] The coding sequence of the construct of the invention codes for a protein or polypeptide comprising two, three, or more antigenic or functional segments separated by cleavable signal peptides, such as 6K or 6K-type polypeptides. Cleavage of the signal peptides releases these separated antigenic regions as independent polypeptides that can be transported, complexed, or folded independently. Often, the independent polypeptides produced by cleavage are present in stoichiometric amounts depending on the number of polypeptide regions of interest encoded by the construct. This helps to avoid problems related to the expression of known quantities of multiple antigens from different vectors or different RNA transcripts.

[0032] Without being bound by any particular theory or explanation, the inventors consider that when a protein encoded by the construct described herein is transported into the endoplasmic reticulum, it undergoes a series of events that lead to the cleavage and / or activation of its individual components, such as the first, second, or subsequent polypeptides of interest derived from different polypeptide antigens. The protein is then considered to be transported across the cytoplasmic membrane of the host cell and into the lumen of the ER, a process facilitated by the presence of a signal peptide at the N-terminus of the protein. This N-terminal signal peptide acts as a molecular tag that helps direct the polyprotein into the ER. The signal peptide is then cleaved by a signal peptidase, which is a specialized protease present in the lumen of the ER. This cleavage releases the mature protein from the N-terminal signal peptide.The mature protein is then cleaved into its individual components by proteases present in the lumen of the ER that recognize specific cleavage sites in the protein, such as 6K, 6K-like or signal peptidase cleavage sites incorporated into the nucleic acid construct.

[0033] Nucleic acid construct regulation elements. In some cases, the nucleic acid construct described herein further includes a promoter, enhancer, silencer or other regulatory region, such as a promoter or ribosome binding site upstream of the protein coding sequences or, for RNA constructs or transcripts, a 3' UTR sequence, a polyA tail or a terminator sequence.

[0034] Other peptide sequences in the polyprotein. In some embodiments, the protein may include other peptide sequences, such as a rigid or flexible linker or an affinity tag between the domains described above. A linker can facilitate folding and proper processing of the protein when transposed into the endoplasmic reticulum (ER). For applications involving the recovery of individual polypeptides of interest, an affinity tag (e.g., a His tag comprising 6 to 10 His residues or a FLAG:DYKDDDK tag) can facilitate the isolation or purification of a polypeptide of interest produced by processing the polyprotein. These additional peptide elements may or may not form parts of the protein regions comprising the two, three, or more polypeptides of interest.

[0035] The protein encoded by the nucleic acid construct described herein generally comprises an N-terminal signal peptide region. Preferably, in terms of the transformation of the protein into individual polypeptides, the peptide region The N-terminal signal region is the one that efficiently translocates a translated protein into the ER. In some embodiments, the N-terminal signal peptide region comprises 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 to 150 residues, or any intermediate length within this range. By comparison, the internal signal sequence peptides mentioned below often comprise 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 to 75 residues, or, in other embodiments, fewer than 15 or 20 residues.

[0036] Cleavage regions of the 6K peptide and the internal signal peptide. The protein encoded by the nucleic acid constructs shown herein comprises cleavable sequences, such as those of a 6K or 6K-like protein from alphaviruses or a cleavage site of the internal signal peptidase from other viruses. Cleavage of these sequences, which are interposed between the polypeptides of interest, releases two, three, or more of the polypeptides of interest from the polyprotein or polypeptide construct.

[0037] Internal signal peptide sequences. The constructs presented herein comprise sequences encoding one or more internal signal peptides, 6K or 6K-like proteins, which are placed alongside and between sequences encoding polypeptides of interest, such as sequences encoding different viral antigens. One or more internal signal peptide sequences may be incorporated into a construct as described herein.

[0038] The signal peptides and their cleavage sites can be identified for use in the constructs disclosed herein using a computer program such as Signal IP-6.0, which is described in more detail at<https: / / services.healthtech.dtu.dk / services / SignalP-6.0 / > (Last accessed May 10, 2023). The SignalP 6.0 server predicts the presence of signaling peptides and the location of their cleavage sites in the proteins of Archaea, Gram-positive bacteria, Gram-negative bacteria, and Eukarya. In bacteria and archaea, SignalP 6.0 can distinguish five types of signal peptides:

[0039] Sec / SPI: "standard" secretory signal peptides transported by the Sec translocon and cleaved by Signal Peptidase I (Lep),

[0040] Sec / SPII: lipoprotein signaling peptides transported by the Sec translocon and cleaved by signaling peptidase II (Lsp),

[0041] Tat / SPI: Tat signal peptides transported by the Tat translocon and cleaved by Signal Peptidase I (Lep), Tat / SPII: Tat lipoprotein signal peptides transported by the Tat translocon and cleaved by signal peptidase II (Lsp),

[0043] Sec / SPIII: Pilin and pilin-like signal peptides transported by the Sec translocon and cleaved by Signal Peptidase III (PilD / PibD).

[0044] In addition, SignalP 6.0 predicts signal peptide regions. Depending on the type, the positions of the n, h and c regions as well as other distinctive features are predicted.

[0045] SignalP 6.0 is based on a transforming protein language model with a conditional random field for structured prediction. The preferred parameters of this program are: organism: "Eukarya" (predicts Sec / SP1) or "other"; "long output" or "short output" (no figures); and model mode: "fast" or "slow". The slow model mode can be used to accurately describe the boundaries of the region.

[0046] The 6K protein or 6K-type proteins. The term "6K" or "6K-type protein" refers to a small membrane protein encoded by alphaviruses and certain other viruses and is named after its approximate molecular weight of 6 kDa.

[0047] The term 6K protein refers to a small alphavirus protein that separates the membrane glycoproteins E2 and EL. During translation, signal peptide (SP) sequences located at the N and C ends of the 6K protein are cleaved to release the membrane protein E2 upstream and the membrane protein El downstream of the small 6K protein. Alphavirus 6K proteins are small (58-61 amino acids), hydrophobic, and associate with membranes. Alphaviruses, such as Venezuelan equine encephalitis virus (VEEV), typically encode the 6K protein between their E2 and EL glycoproteins. Other viruses of the Alphavirus genus that encode a 6K protein include, but are not limited to, Sindbis virus (SINV), Chickungunya virus (CHIKV), Semliki forest virus (SFV), Ross River Virus (RRV), Sagiyama virus (SAGV), Getah virus (GETV), Middleburg virus (MIDV), Bebaru virus (BEBV), O'nyong nyong virus (ONNV), Ndumu (NDUV) and Barmah Forest virus (BFV).New World alphaviruses such as Venezuelan equine encephalitis virus (VEEV), Western equine encephalitis virus (WEEV), and Eastern equine encephalitis virus (EEEV) also encode a 6K protein. The 6K protein includes an internal signal peptide that facilitates the expression of two membrane-bound viral antigens. When a 6K protein is analyzed, an internal signal peptide region is found that allows cleavage of the 6K protein away from E1 and E2.

[0048] The cleavage of a 6K protein to generate individual structural proteins (e.g., the first and second polypeptides of interest encoded by the construct) occurs via cellular proteases such as signal peptidases. In some cases, a nucleic acid construct such as described herein may encode an exogenous protease that recognizes and cleaves the 6K domains of the polyprotein it encodes.

[0049] The term "type 6K protein" refers to a non-alphavirus protein that separates an upstream protein from a downstream protein. In other words, it has a Similar in structure to the alphavirus 6K protein described above, but not originating from an alphavirus. The HCV p7 protein is an example of a 6K-type protein. It separates the HCV E2 and NS2 proteins and also has SPs at its N and C ends that are used to cleave the E2 protein upstream and the NS2 protein downstream of the small p7 protein.

[0050] A 6K or 6K-type viral protein sequence need not be present between the upstream and downstream polypeptide sequences encoded by a construct when an alternative internal signal peptide separating them is sufficient to provide a cleavage site. As mentioned above, an example of a 6K-type protein is the HCV p7 protein, which is a viral protein found between and within the same open reading frame as E2 and NS2. Other examples of 6K or 6K-type proteins encoded by polynucleotide sequences that are incorporated into the constructs disclosed herein include: [0051 ] >p7: ALENLVILNAASLAGTHGLVSFLVFFCFAWYLKGRWVPGAVYAFYGM WPLLLLALPQRAYA.

[0052] >S6k:ASVAETMAYLWDQNQALFWLEFAAPVACILIITYCLRNVLCCCKSL SFLVLLSLGATARA.

[0053] >V6k:GRPRAETTWESLDHLWNNNQQMFWIQLLIPLAALIVVTRLLRCVCC VVPFLVM AGAAGAGA.

[0054] Signal peptide sequences distinct from those of 6K and 6K-like proteins include a Hantavirus signal peptide that is used to enable the viral maturation of two envelope proteins from the same open reading frame. Other examples include the Bunyavirus and Hantavirus sequences:

[0055] >BSP: SLIISILLSVLILSFVTPIEGT.

[0056] >HSP: RTNLLFRYKSRCYIFTMWIFLVLESILWAASA.

[0057] Signal sequence cleavage sites, including 6K or 6K type cleavage sites. N-terminal and internal signal peptide sequences and signal peptide cleavage sites can be determined using a program such as SignalP 6.0 or can be recognized by the presence of a structural motif such as those described herein.

[0058] An N-terminal signal peptide generally directs an immature protein to the endoplasmic reticulum (ER) where the immature protein is processed, for example, by cleavage of the N-terminal signal peptide and internal signal peptides separating the regions of the immature protein. For example, an immature protein or a polyprotein comprising regions of two, three, or more antigens separated by internal signal peptides can be transformed into distinct antigens of interest. Cleavage of the N-terminal signal peptide is generally the first step in the transformation of the protein into its mature form (e.g., without an N-terminal signal peptide). Then, The partially transformed protein is cleaved into two or more antigenic or immunogenic polypeptides of interest via internal signal peptidase sequence sites to produce independent monomeric polypeptides of interest.

[0059] The independent monomeric polypeptides of interest, processed from the larger immature protein or polyprotein, can then be transported for secretion from a cell or for insertion into the cell membrane. Independent monomeric polypeptides lacking a transmembrane domain and unable to bind to the membrane are often secreted by the cell.

[0060] In some cases, the internal signal peptide may remain fused to an upstream or downstream polypeptide of interest. When this signal peptide also includes a transmembrane domain, this may result in the insertion of the polypeptide of interest into the membrane.

[0061] In other embodiments, the proteases may be derived from viruses or be an exogenous protease encoded by the nucleic acid construct itself.

[0062] Cleavage motifs. Many signal sequences include common motifs; see Owji, H. et al, A comprehensive review of signal peptides: Structure, roles, and applications, EUR. J. CELL BIOL. 2018, 97:422-441 or Lober et al. The Hantaan virus glycoprotein precursor is cleaved at the conserved pentapeptide WAASA, VIROLOGY (2001), 289, 224-229.

[0063] The nucleic acid construct described herein may comprise an N-terminal or internal signal sequence that includes a cleavage motif comprising:

[0064] a region N comprising 1 to 5 charged residues (including, but not limited to, Arg and Lys),

[0065] a region H covering 7 to 15 contiguous hydrophobic residues, of which 3 to 5 are leucine residues (including, but not limited to, Leu and Gly or other residues forming an alpha helix),

[0066] a region C comprising 3 to 7 uncharged amino acid residues (including, but not limited to, Ala and Val, or other beta-sheet-forming residues), and / or

[0067] a region N comprising 1 to 6 charged amino acid residues, (including, but not limited to, Glu and Ala). See [Fig.2] of Owji, H. et al, supra.

[0068] In some cases, the nucleic acid construct described herein comprises N-terminal and / or internal signal peptide sequences including an AXA or VXA cleavage motif between a C region and a Pro region corresponding to an N region. [Fig.2] of Owji, H. et al, supra.

[0069] In other cases, the nucleic acid constructs presented here include one or more internal cleavage sites derived from a virus. These may be all or part of a 6K sequence from a virus of the genus Alphavirus, other 6K-type viral sequences, an E2 / p7 or p7 / NS2 cleavage site from the hepatitis C virus, or a cleavage site of the internal signal peptidase of a virus of the order Bunyavirales or of a cleavage site comprising a WAASA motif; see Lin et al, Processing in the E2-NS2 Region of Hepatitis C Viruses: Identification of p7 and Two Distinct E2-Speciufic Products with Different C Termini, J. VIROLOGY, 1994, 68(8), 5063-5076; Shi, X, et al, Visualizing the Replication Cycle of Bunyamwere Oprthobunyavirus Expressing Fluorescent Protein-Tagged Gc Glycoprotein, J. VIROLOGY, 84(7), 8460-8469; Shi, et al, Bunyamwera orthobunyavirus glycoprotein precursor is processed by cellular signal peptidase and signal peptide peptidase, PNAS, 2016, 2016-2016.<https: / / doi.org / 10.1073 / pnas. 1603364113> ; Loeber, C. et al., The Hantaan Virus Glycoprotein Precursor Is Cleaved at the Conserved Pentate Peptide WAASA. Virology, 2001, 289, 224-229.

[0070] The signal peptidase cleavage sites of Hantaan and other viruses, including those of [Fig.3] and 4, are described by Lober et al, VIROLOGY (2001), 289, 224-229.

[0071] The specific cleavage motifs of mammalian signal peptides can vary from protein to protein and from cell type to cell type, but they involve short, conserved amino acid sequences that are recognized by host proteases. Cleavage of signal peptides is a regulated and controlled event that allows the protein to be transformed into its mature form and properly transported to its final destination in the cell. Several publications describe the signal sequence cleavage motifs, which are the specific amino acid sequences found in signal peptides that are recognized and cleaved by proteases. Translocation of proteins across the eukaryotic endoplasmic reticulum and bacterial plasma membranes. NATURE, 2007, 450(7170), 663-9; Walter, P.et al, Signal Sequence Recognition and Protein Targeting to the Endoplasmic Reticulum Membrane, ANNUAL REVIEW OF CELL BIOLOGY, 1994, 10.87; and Kapp, K. et al, Post-targeting functions of signal peptides, PROTEIN TRANSPORT INTO THE ENDOPLASMIC RETICULUM, 2009. .

[0072] These publications provide detailed information on the different types of signal sequences, their mechanisms of action, and the proteases involved in their cleavage. They also provide information on the different cleavage motifs that have been described for various signal sequences and their specificity for different proteases.

[0073] Signal peptide: This is a membrane protease that specifically cleaves signal peptides from newly synthesized proteins, for example by releasing them into the lumen of the endoplasmic reticulum (ER). These include:

[0074] Furin: This is a cysteine ​​protease that cleaves signaling peptides at specific locations, thus enabling the transport of the mature protein to its final destination.

[0075] Site-1 Protease (S1P): Member of the subtilisin-type protease family, it is involved in the maturation and sorting of newly synthesized proteins.

[0076] Site 2 (S2P) protease: This is another member of the subtilisin-type protease family which is involved in the maturation of newly synthesized proteins.

[0077] PACE4 (Protease-Activated Cysteine ​​Endopeptidase 4): This is a protease involved in the processing of growth factors and cytokines.

[0078] In some cases, cells expressing a protease described above are used as host cells to express and process the polyprotein encoded by the construct of the invention.

[0079] Signal peptide cleavage regions or sites. In some embodiments, there may be at least three different cleavage sites. The first may be the 6K alphavirus, which includes all viruses of the genus Alphavirus. The second site may be of the 6K type, for example, the p7 of HCV. Finally, the third site may consist of internal signal peptides, or ISPs for short. These often originate from viruses of the order Bunyavirales, which includes Hantaan, Bunyamwera, and others.

[0080] Another characteristic common to most of these cleavage sites is the presence of at least one transmembrane domain. The 6k and ISPs have one domain, and the p7 has two. A transmembrane domain can comprise 16 to 30, for example 20 to 25, amino acid residues, which are generally predominantly nonpolar residues.

[0081] The polypeptide regions of interest. The polynucleotide or nucleic acid construct described herein can be used to express two, three, or more polypeptides of interest. There is no particular limitation as to the type of polypeptide encoded by the construct. For applications involving immunization or vaccination, the polypeptide sequences of interest are generally derived from polypeptides comprising microbial T- or B-cell epitopes, such as polypeptides comprising one or more bacterial or viral antigen epitopes. These polypeptides may correspond to an entire microbial antigen or to an immunogenic or antigenic fragment, such as a fragment comprising one or more B- or T-cell epitopes.

[0082] In some cases, these microbial polypeptides are antigenic or immunogenic polypeptides of a virus that infects domestic animals. For example, the polypeptides of interest may be those of pathogens causing a specific disease, including the following:

[0083] poultry diseases: Newcastle disease (MN), avian influenza (AI), infectious bursitis (IBD), infectious bronchitis (IB), Marek's disease, avian cholera, infectious laryngotracheitis (ILT) and coccidiosis;

[0084] feline diseases: feline leukemia, feline infectious peritonitis;

[0085] Canine diseases: distemper, adenovirus, parvovirus and leptospirosis; and

[0086] swine diseases: Porcine circovirus, swine influenza virus, rotavirus, porcine reproductive and respiratory syndrome virus (PRRSV), Lawsonia, African swine fever (ASF), classical swine fever (CSF) and porcine epidemic diarrhea (PED).

[0087] A non-exhaustive list of antigens that can be encoded by the constructs includes the gB, gD, gE and gl antigens of ILTV; H5, H9, H7, H1N1 or H1N2 of AIV and SIV; the F protein for NDV; or VP1 or VP2 for IBV.

[0088] Vectors. A suitable vector for incorporating the polynucleotide or nucleic acid construct described herein may be a plasmid vector, a viral vector, an artificial chromosome, or a transposon. Some vectors are attenuated or live virus vectors. Typically, a vector is chosen that will efficiently transform or transfect a host cell with the nucleic acid construct described herein under conditions permitting expression of the polyprotein encoded by the construct.

[0089] In some cases, the vector is a modified live virus vector, such as a modified live pox, herpesvirus, adenovirus, lentivirus or retrovirus vector.

[0090] In some embodiments, the vector comprises DNA or modified DNA which may optionally replicate in a host cell. The vector may be a DNA launching platform which, when introduced into a host cell, transcribes and translates the nucleic acid construct into RNA, then into a polyprotein, and which may optionally include an origin of replication and / or a selectable marker.

[0091] In other cases, the vector may be composed of RNA or modified RNA that optionally replicates in a host cell. Such a vector may further include a 5' cap, a 3' UTR, and / or a poly A tail. A vector may include an RNA launching platform that, when introduced into a host cell, translates the nucleic acid construct into a polyprotein and optionally replicates upon introduction into the host cell. In some embodiments, the vector comprises a self-replicating RNA which includes a 5' cap, a nucleic acid sequence encoding the non-structural proteins nsPl, nsP2, nsP3 and nsP4 of the alphavirus, a 26S promoter, the nucleic acid sequence encoding the first polypeptide of interest, the nucleic acid encoding the signal sequence, the nucleic acid encoding the second polypeptide of interest, and a 3'UTR and polyA tail.

[0092] Other specific examples of vectors include a Togavirus vector, including, but not limited to, an SFV, SINV or VEEV vector; a vector Flaviviridae, including, but not limited to, a Flavivirus vector or a Pestivivus vector; an Orthomyxoviridae vector, including, but not limited to, an Influenza vector; a Rhabdoviridae vector, including a Lyssavirus vector, a Vesiculovirus vector or a Novirhabdovirus vector; a Paramyxoviridae vector, including a Pneumomyxovirinae vector, a Pneumovirus vector, a Paramyxovirinae vector, a Respirovirus vector, an Avulavirus vector, a Rubulavirus vector or a Morbilivirus vector; see Mogeler, MA & Kamrud, KI, RNA-based viral vectors, EXPERT REV. VACCINES Early online 1-30 (2014).

[0093] Host cells. Another aspect of the invention relates to a host cell transformed or transfected with the nucleic acid constructs or corresponding vectors described herein. The host cells may be those of a non-human animal such as a mammal or a bird. In some cases, the host cells will be those of a domestic animal or derived therefrom, such as canines, felines, cattle, equines, goats, sheep, pigs, or poultry, such as chickens or turkeys. In some cases, the host cells may be taken from or derived from amphibians, reptiles, or fish. In some cases, the host cell may be yeast, a fungus, a plant cell, or a prokaryotic cell, provided that these cells are capable of cleaving the signal peptide cleavage sites in the polyprotein.

[0094] In some cases, when the polypeptides of interest are derived from a microbial or viral pathogen, the host cell is chosen as the one for which the microorganism or virus has a tropism. Similarly, the cleavage sites of the N-terminal peptide and / or the internal signal peptide can be chosen from those found in the host cell or in a microbial or viral pathogen.

[0095] Method for the simultaneous expression of two, three, or more polypeptides of interest. Another aspect of the invention relates to a method for producing two, three, four, five, or more polypeptides, comprising the transformation or transfection of a vector comprising the nucleic acid construct described herein into a host cell capable of cleaving the domains of the N-terminal peptide and the internal signal peptide. The host cell may be an in vivo cell, for example, in a tissue or fluid from a subject such as a vaccinated subject, an ex vivo cell taken from a living organism, or an in vitro cultured cell, for example, a culture cell line such as Chinese hamster ovary (CHO) cells, HeLa cells, or cultured Sporodoptera frugiperda insect cells.

[0096] This method can be used to produce two or more polypeptides of interest in approximate stoichiometric ratios, such as a molar ratio of about 1:1 (or 1:1:1, 1:1:1, etc.) which may vary from a predicted stoichiometric ratio such as 1:1 or 1:1:1 of ±1.2, 5, 10, 20 mol%. Other stoichiometric ratios Polypeptides of interest can be produced, for example, by encoding the same polypeptide of interest in two positions of a construct expressing three polypeptides of interest. In some cases, co-expressed polypeptides of interest can associate with each other in the ER and form new epitopes or tertiary or quaternary structures.

[0097] Purification of the polypeptides of interest. Once the relevant polypeptides are released from the polyprotein, they can be isolated or purified by conventional biochemical techniques such as size exclusion, ion exchange, affinity chromatography, hydrophobic interaction or reversed-phase chromatography.

[0098] Compositions / vaccines. A related aspect of this technology is a composition comprising the vectors disclosed herein and a pharmaceutically acceptable carrier. In one instance, the composition comprises RNA or DNA including the constructs disclosed herein and inorganic lipid nanoparticles (LION). In some instances, a composition will comprise the nucleic acid construct disclosed herein that encodes a polyprotein encoding two, three, or more microbial or viral antigens or immunogenic fragments, and a carrier, adjuvant, or excipient in a form suitable for administration to produce a prophylactic or therapeutic response against infection. Such a composition can produce or enhance a humoral or cellular response in an immunized subject against the microbial or viral pathogen from which the polypeptides of interest were derived.

[0099] Therapeutic compositions. Nucleic acid constructs, vectors, host cells, and polypeptides of interest can be administered to a subject in need, depending on the subject, the desired immune response, and the type of vaccine or vector. An RNA or DNA construct can be administered in saline solution, in combination with lipids or polyethylene glycol, aluminum salts, or other adjuvants. In one instance, the construct or vector is administered in combination with an inorganic lipid nanoparticle.

[0100] LION. Inorganic lipid nanoparticles (LION). In some embodiments, the platform is administered in the form of a nanoemulsion particle having a hydrophobic core and comprising a mixture of liquid oil and one or more inorganic solid nanoparticles. The nanoemulsion particle may also be referred to herein as inorganic lipid nanoparticles (LION). The liquid oil is mixed with one or more inorganic nanoparticles to form a hydrophobic core. The liquid oil is generally metabolizable. The suitable liquid oil may be a vegetable oil, an animal oil, or a synthetically prepared oil. In some cases, the liquid oil is fish oil. In some cases, the liquid oil is a natural or synthetic terpenoid. In some cases, the liquid oil is squalene, a triglyceride (such as caprylic / capric triglyceride or myristic acid triglyceride), vitamin E, lauryl polyoxyglyceride, monoacylglycerol, soy lecithin, sunflower oil, soybean oil, olive oil, grapeseed oil, or a combination of these oils. In one case, the liquid oil is squalene, a triglyceride (such as caprylic / capric triglyceride or myristic acid triglyceride), vitamin E, lauryl polyoxyglyceride, monoacylglycerol, soy lecithin, or a combination of these.In one case, the liquid oil is squalene, a triglyceride (such as caprylic / capric triglyceride or myristic acid triglyceride), sunflower oil, soybean oil, olive oil, grapeseed oil, or a combination thereof. In some cases, the liquid oil is squalene (of natural or synthetic origin, possibly in combination with one of the aforementioned liquid oils). The inorganic nanoparticles can be formed from one or more of the same or different metals (all metals, including transition metals), such as metal salts, metal oxides, metal hydroxides, and metal phosphates.Examples include silicon dioxide (SiO2), iron oxides (FeO34, FeO23, FeO, or combinations thereof), aluminum oxide (AlO123), aluminum oxyhydroxide (Al10(OH)), aluminum hydroxyphosphate (Al(OH)x(PO4)4y), calcium phosphate (Ca3(PO4)42), calcium hydroxyapatite (CalO(PO4)6(OH)2), iron gluconate, or iron sulfate. In some cases, the inorganic solid nanoparticle is a metal oxide, such as a transition metal oxide. In one case, the inorganic solid nanoparticle is an iron oxide, for example magnetite (Fe O34), maghemite (γ-Fe O23), wustite (FeO), hematite (α-Fe O23), or combinations thereof. In some cases, the inorganic solid nanoparticle is a metal hydroxide, such as aluminum hydroxide or aluminum oxyhydroxide.The inorganic solid nanoparticle may contain a reporter element detectable by imaging methods to enable the imaging and tracking of the resulting nanoemulsion particles within the body. For example, the inorganic solid nanoparticle may contain a reporter element detectable by magnetic resonance imaging (MRI), such as a paramagnetic, superparamagnetic, ferrimagnetic, or ferromagnetic compound. Iron oxides, iron gluconates, and iron sulfates are examples of inorganic solid nanoparticles detectable by MRI. Inorganic solid nanoparticles typically have a mean diameter (number-weighted average diameter) ranging from approximately 3 nm to approximately 50 nm. For example, the inorganic solid nanoparticle may have a mean diameter of approximately 5 nm. of approximately 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, or 50 nm. The inorganic solid nanoparticle can be surface-modified before being mixed with the liquid oil. For example, if the surface of the inorganic solid nanoparticle is hydrophilic, it can be coated with hydrophobic molecules (or surfactants) to facilitate its miscibility with the liquid oil in the "oil" phase of the nanoemulsion particle. Phosphate-terminated lipids (such as phosphatidylated lipids), phosphorus-terminated surfactants, carboxylate-terminated surfactants, sulfate-terminated surfactants, or amine-terminated surfactants can be used for surface modification of inorganic solid nanoparticles.Typical phosphate-terminated lipids or phosphorus-terminated surfactants include trioctylphosphine oxide (TOPO) and distearylphosphatidic acid (DSPA). Typical sulfate-terminated surfactants include, but are not limited to, sodium dodecyl sulfate (SDS). Typical carboxylate-terminated surfactants include oleic acid. Amine-terminated surfactants include oleylamine. In one case, the inorganic solid nanoparticle is a metal oxide such as an iron oxide, and a surfactant, such as oleic acid, oleylamine, SDS, DSPA, or TOPO, is used to coat the inorganic solid nanoparticle before it is mixed with liquid oil to form the hydrophobic core.In one case, the inorganic solid nanoparticle is a metal hydroxide, such as aluminum hydroxide or aluminum oxyhydroxide, and a phosphate-terminated lipid or surfactant, such as oleic acid, oleylamine, SDS, TOPO, or DSPA, is used to coat the inorganic solid nanoparticle before it is mixed with liquid oil to form the hydrophobic core. The lipids used to form the nanoemulsion particles can be cationic lipids, anionic lipids, neutral lipids, or mixtures thereof. In some cases, the lipids used are cationic lipids. For example, positively charged lipids that can interact favorably with negatively charged bioactive agents (such as DNA or RNA) can be used in the nanoemulsion composition. Suitable cationic lipids include 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP); 3.beta.[N(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC Cholesterol); dimethyldioctadecylammonium (DDA); l,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP); dipalmitoyl(C16:0)trimethylammonium propane (DPTAP); distearoyltrimethylammonium propane (DSTAP); N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); chloride. of N,N-dioleoyl-N,N-dimethylammonium (DODAC); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); and 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA); 1,1'-((2-(4-(bis(2-hydroxydodecyl)amino)ethyl) (2-hydroxydodecyl)amino)ethyl)piperazin-l-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200); and their combinations. A typical cationic lipid is DOTAP. Other examples of suitable lipids include, but are not limited to, phosphatidylcholines (PCs), such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), l-palmitoyl-2-oleoylphosphatidylcholine (POPC), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylcholine (DMPC), etc.; phosphatidylethanolamines (PEs), such as l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), etc.; and phosphatidylglycerol (PG). and PEGylated lipids, including the PEGylated version of any of the above lipids (e.g., DSPE-PEG). The nanoemulsion particle may also contain one or more surfactants, which may be hydrophobic or hydrophilic. In some cases, the nanoemulsion particle further comprises a hydrophobic surfactant. In some cases, the nanoemulsion particle further comprises a hydrophilic surfactant. In one case, the nanoemulsion particle further comprises both a hydrophobic and a hydrophilic surfactant. Suitable hydrophobic surfactants include those with a hydrophilic-lipophilic balance (HLB) value less than or equal to 10, for example, less than or equal to 5, between 1 and 5, or between 4 and 5. An example of a hydrophobic surfactant is a sorbitan ester (such as sorbitan monoester or sorbitan trimester). For example, the hydrophobic surfactant may be a sorbitan ester with an HLB value of 1 to 5, or 4 to 5.In some embodiments, the hydrophobic surfactant is a sorbitan monoester or a sorbitan triester. Exemplary sorbitan monoesters include sorbitan monostearate and sorbitan monooleate. Examples of sorbitan triesters include sorbitan tristearate and sorbitan trioleate. Suitable hydrophilic surfactants include polyethylene oxide-based surfactants, for example, polyoxyethylene sorbitan ester (polysorbate). In some cases, the hydrophilic surfactant is a polysorbate. Examples of polysorbates include polysorbate 80 (polyoxyethylene sorbitan monooleate or Tween 80), polysorbate 60 (polyoxyethylene sorbitan monostearate or Tween 60), polysorbate 40 (polyoxyethylene sorbitan monopalmitate or Tween 40), and polysorbate 20 (polyoxyethylene sorbitan monolaurate or Tween 20). In one instance, the hydrophilic surfactant is polysorbate 80.The nanoemulsion particle. may have an oil / surfactant molar ratio ranging from about 0.1:1 to about 20:1, from about 0.5:1 to about 12:1, from about 0.5:1 to about 9:1, from about 0.5:1 to about 5:1, from about 0.5:1 to about 3:1, or from about 0.5:1 to about 1:1. The nanoemulsion particle may have a hydrophilic surfactant / lipid (e.g., cationic lipid) ratio ranging from about 0.1:1 to about 2:1, from about 0.2:1 to about 1.5:1, from about 0.3:1 to about 1:1, from about 0.5:1 to about 1:1, or from about 0.6:1 to about 1:1. The nanoemulsion particle can have a hydrophobic surfactant / lipid (e.g., cationic lipid) ratio ranging from about 0.1:1 to about 5:1, from about 0.2:1 to about 3:1, from about 0.3:1 to about 2:1, from about 0.5:1 to about 2:1, or from about 1:1 to about 2:1.The nanoemulsion particle may comprise from about 0.2% to about 40% w / v of liquid oil, from about 0.001% to about 10% w / v of inorganic solid nanoparticle, from about 0.2% to about 10% w / v of lipid (e.g., cationic lipid), from about 0.25% to about 5% w / v of hydrophobic surfactant (e.g., sorbitan ester), and from about 0.5% to about 10% w / v of hydrophilic surfactant.In some embodiments, the nanoemulsion particle comprises: a hydrophobic core comprising a mixture of one or more inorganic nanoparticles containing at least one metal oxide nanoparticle optionally coated with a phosphate-terminated lipid, a phosphorus-terminated surfactant, a carboxylate-terminated surfactant, a sulfate-terminated surfactant or an amine-terminated surfactant, and a liquid oil containing squalene of natural or synthetic origin; a cationic lipid comprising DOTAP; a hydrophobic surfactant comprising a sorbitan ester selected from the group consisting of sorbitan monostearate, sorbitan monooleate and sorbitan trioleate; and a hydrophilic surfactant comprising a polysorbate.In one embodiment, the nanoemulsion particle comprises: a hydrophobic core comprising a mixture of: one or more inorganic nanoparticles containing iron oxide nanoparticles, and a liquid oil containing natural or synthetic squalene; the cationic lipid DOTAP; a hydrophobic surfactant comprising sorbitan monostearate; and a hydrophilic surfactant comprising polysorbate 80. In this LION composition, the LION particle may comprise from about 0.2% to about 40% w / v of squalene, from about 0.001% to about 10% w / v of iron oxide nanoparticles, from about 0.2% to about 10% w / v of DOTAP, from about 0.25% to about 5% w / v of sorbitan monostearate and from about 0.5% to about 10% w / v of polysorbate 80.In one embodiment, the LION particle comprises about 2% to about 6% w / v of squalene, about 0.01% to about 1% w / v of iron oxide nanoparticles, about 0.2% to about 1% w / v of DOTAP, about 0.25% to about 1% w / v of sorbitan monostearate, and about 0.5% to about 5% w / v of polysorbate. 80. In certain embodiments, the nanoemulsion particle comprises: a hydrophobic core comprising a mixture of: one or more inorganic nanoparticles containing at least one metal hydroxide or oxyhydroxide nanoparticle optionally coated with a phosphate-terminated lipid, a phosphorus-terminated surfactant, a carboxylate-terminated surfactant, a sulfate-terminated surfactant or an amine-terminated surfactant, and a liquid oil containing squalene of natural or synthetic origin; a cationic lipid comprising DOTAP; a hydrophobic surfactant comprising a sorbitan ester selected from the group consisting of sorbitan monostearate, sorbitan monooleate and sorbitan trioleate; and a hydrophilic surfactant comprising a polysorbate.In one embodiment, the nanoemulsion particle comprises: a hydrophobic core comprising a mixture of one or more inorganic nanoparticles containing aluminum hydroxide or aluminum oxyhydroxide nanoparticles optionally coated with TOPO, and a liquid oil containing natural or synthetic squalene; the cationic lipid DOTAP; a hydrophobic surfactant comprising sorbitan monostearate; and a hydrophilic surfactant comprising polysorbate 80. In this LION composition, the LION particle may comprise from about 0.2% to about 40% w / v of squalene, from about 0.001% to about 10% w / v of aluminium hydroxide or aluminium oxyhydroxide nanoparticles, from about 0.2% to about 10% w / v of DOTAP, from about 0.25% to about 5% w / v of sorbitan monostearate and from about 0.5% to about 10% w / v of polysorbate 80.In one case, the LION particle comprises approximately 2% to 6% w / v of squalene, approximately 0.01% to 1% w / v of aluminum hydroxide or aluminum oxyhydroxide nanoparticles, approximately 0.2% to 1% w / v of DOTAP, approximately 0.25% to 1% w / v of sorbitan monostearate, and approximately 0.5% to 5% w / v of polysorbate 80. Nanoparticles and nanoemulsions have been described in the literature, and these terms are used here to refer to particles smaller than 1000 nanometers. The nanoemulsion particle (LION) typically has a mean diameter (hydrodynamic mean diameter z, measured by dynamic light scattering) ranging from approximately 20 nm to approximately 200 nm. In some cases, the average diameter z of the LION particle is between about 20 nm and about 150 nm, between about 20 nm and about 100 nm, between about 20 nm and about 80 nm, between about 20 nm and about 60 nm.In some cases, the mean z-diameter of the LION particle is between approximately 40 nm and approximately 200 nm, between approximately 40 nm and approximately 150 nm, between approximately 40 nm and approximately 100 nm, between approximately 40 nm and approximately 90 nm, between approximately 40 nm and approximately 80 nm, or between approximately 40 nm and approximately 60 nm. In one case, the mean z-diameter of the LION particle is between approximately . 40 nm and approximately 80 nm. In one case, the mean z-diameter of the LION particle is between approximately 40 nm and approximately 60 nm. The mean polydispersity index (PDI) of the nanoemulsion (LION) particles can be between approximately 0.1 and approximately 0.5. For example, the mean polydispersity index of the LION particles can range from approximately 0.2 to approximately 0.5, from approximately 0.1 to approximately 0.4, from approximately 0.2 to approximately 0.4, from approximately 0.2 to approximately 0.3, or from approximately 0.1 to approximately 0.3.

[0101] Methods of administration. In certain treatment regimens, the nucleic acid constructs described herein, or the vectors comprising them, are formulated for in vivo administration. For administration to a non-human animal, the constructs or vectors according to this application may be administered by any enteral or parenteral route. In some cases, the construct or vector is administered subcutaneously, intravenously, intramuscularly, intra-articularly, intra-synovially, intrasternally, intrathecally, intrahepatically, intrathymically, into a sexual organ, intralesionally, intracranially, intraventricularly, orally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir.

[0102] Treatment. Another aspect of this technology is a method of preventing or treating a microbial or viral disease, its symptoms, comorbidities, or sequelae by administering a vector comprising the construct disclosed herein that encodes and expresses two, three, or more microbial or viral antigens, immunogens, or epitopes. Administration of the vaccine containing the nucleic acid construct or vector can induce innate, humoral, and / or cellular immune responses to the encoded microbial or viral antigens (the polypeptides or the target of interest), including the induction of neutralizing antibodies or antigen-specific T cells that recognize cells infected by the microbial or viral pathogens.

[0103] Applications for expressed polypeptides of interest. In some cases, polypeptides of interest, alone or in combination with other polypeptides of interest expressed by the same nucleic acid construct, can be formulated as diagnostic or therapeutic products. For example, virus-derived polypeptides of interest can be used to detect antibodies directed against the virus or to induce a humoral or cellular immune response against the virus. Examples

[0104] Example 1: Molecular construction, replicon generation and formulation of 6 SIV replicons with HA and NA genes linked by 6K or 6K-type elements. Materials and methods

[0105] Replicons comprising nucleic acid constructs encoding portions of the HA and NA genes from an H1N1 isolate of SIV (swine influenza virus) are produced. The portions of the constructs encoding the HA and NA genes are separated by sequences encoding a 6K or 6K-type linkage.

[0106] Small 6K or 6K-like linker elements from the following viruses are used: Venezuelan equine encephalitis virus (VEE), called 6K (V6K); Semliki forest virus (SFV), also called 6K (S6K); hepatitis C virus (HCV), called p7; bunyavirus (Bunya), called signal peptide (SP, BSP); and hantavirus (Hanta), also called SP (HSP). Similar elements from other viruses may also be used. Many viruses encode 6K or 6K-like sequences that are translated as part of a larger polyprotein, which is then processed into independent monomeric proteins.

[0107] Figure 1 graphically represents these constructs. Each of the six constructs comprises a 6K or 6K-like protein sequence separating the two coding sequences of the viral antigen, either neuraminidase (NA) or rhemagglutinin (HA). Five of the GOIs (replicons 1 and 3-6 of Figure 1) have the SIV HA gene upstream (5') relative to the linker element, followed by the NA gene downstream (3') (relative to the linker element).

[0108] The sixth model of GOI has the NA gene in position 5' and the HA gene in position 3', relative to the linker element.

[0109] The six constructs respectively incorporate V6K (from Venezuelan equine encephalitis virus), V6K (from Venezuelan equine encephalitis virus), S6K (from Semliki Forest Virus (SFV), BSP (from bunyavirus), HSP (from hantavirus), and p7 (from hepatitis C virus).

[0110] The 5' and 3' ends of a GOI are modified if necessary to contain additional nucleotides to facilitate cloning into the vector.

[0111] At the 5' end, the EcoRV restriction endonuclease site (GATATC) and the Kozak sequence (GCCACC) are added upstream of the 5' gene start codon.

[0112] At the 3' end, the GCTGC sequence and the Pacl restriction endonuclease site (TTAATTAA) are added downstream of the 3' gene stop codon.

[0113] IGOs ​​are fragments of double-stranded DNA.

[0114] Ligation of genes of interest in the VEE skeleton. The pSRT_463_HA_v2 (pSRT) vector, which contains the genes of the Venezuelan equine encephalitis virus replication machinery nsPl-4 and a 26S promoter that drives GOI expression, is used as the replicon expression vector. The pSRT-463_HA_v2 vector is based on a synthetically modified replicon alpha RNA technology (SMARRT). This vector and technology are described in Maine, CJ, et al, Self-Replicating RNAs Drive Protective Anti-tumor T Cell Responses to Neoantigen Vaccine Targets in a Combinatorial Approach and Maine, CJ, et al, Self-Replicating RNAs Drive Protective Anti-tumor T Cell Responses to Neoantigen Vaccine Targets in a Combinatorial Approach, MOL. THER. 2021, 3; 29(3):1186-1198. In some embodiments of the invention, a SMARRT-type vector or platform is used to deliver the coding sequence according to the invention.

[0115] A GOI and pSRT are both digested with the restriction endonucleases EcoRV and Pacl to prepare the ends for cloning. Each digestion reaction is purified to remove the endonucleases and any unwanted DNA fragments.

[0116] A GOI is ligated into the pSRT vector using ElectroLigase®. The products are electroporated into TransforMax™ EPI300™ Electropcometent E. coli and the resulting colonies are analyzed for the presence of the GOI by isolating the plasmid DNA and analyzing it by restriction fragment length polymorphism and next-generation sequencing.

[0117] Clones that have been correctly ligated without mutations are converted into glycerol stocks for long-term storage.

[0118] Replicon DNA amplification. Samples taken from each glycerol stock for each replicon are struck to isolate on animal-derived LB agar plates containing the selection antibiotic kanamycin, independently, and allowed to replicate overnight. The isolated colonies are harvested and used to inoculate cultures in animal-derived Terrifie broth containing the selection antibiotic kanamycin. The cultures are incubated overnight at 37°C with shaking to allow replication of the replicon plasmids containing the GOI. The bacterial paste is harvested by centrifugation, and the NucleoBond Xtra Maxi EF kit is used to isolate the purified, endotoxin-free plasmid DNA. This DNA is tested for quantity (OD260), quality (% supercoiled), and identity (Sanger or Lumina DNA Sequencing).

[0119] In vitro transcription of RNA. The purified DNA for each replicon is linearized by digesting the DNA with the restriction endonuclease Notl to facilitate transcription. The linearized DNA is transcribed into RNA overnight at 30°C using DNA-dependent RNA polymerase T7 and rNTPs of equal molarity. Subsequently, the transcription reaction products are treated with DNase I to digest the DNA template. The synthetic RNA product is purified by centrifugal filtration. The 5' end of the uncapped RNA product is enzymatically capped with guanyltransferase, rGTP, and SAM. The final capped synthetic RNA product is purified again by centrifugal filtration.

[0120] Formulation. The capped RNA transcripts of each replicon are encapsulated in lipid inorganic nanoparticles (LION) and formulated according to the study protocol.

[0121] Example 2: Summary of the experiment on linkers H3 and H1

[0122] Materials and methods for experiment H3 / H1 6K

[0123] Electroporation. Synthetic RNA of each construct - Hl, H3, H3H1, H1H3, H3-RVSP-H1, H3-V6K-H1 and H3-p7-Hl - as well as a cell control were electroporated into BHK-21 cells using the Lonza 4D-Nucleofector® X unit and the SF Cell Line Nucleofector® X Kit L (Lonza).

[0124] BHK-21 cells were harvested using 0.25% trypsin-EDTA and resuspended in SF buffer at a cell density of 1x106 cells / 100 pL of electroporation reaction.

[0125] Electroporated cells were recovered in Dulbecco's modified Eagle medium containing 2% fetal bovine serum and distributed into four wells of a 96-well tissue culture plate and into one well of a 6-well tissue culture plate.

[0126] The cell plates were then incubated at 37 ± 2°C and 5% CO2 for 21.5 hours.

[0127] After incubation, the cell plates were removed from the incubator and protein expression was assessed by indirect immunofluorescence (IFA) and by Western blot.

[0128] IFA. The cell culture medium from the 96-well tissue culture plate was decanted and the cells were washed twice with PBS IX, then fixed for 20 minutes at room temperature using Fix and Perm Medium A (Invitrogen).

[0129] After fixation, the cells were washed three times and blocked with PBS buffer containing 0.1% tween 20 and 3% FBS for 1 hour at 37 ± 2 °C. The cells were washed and incubated with hyperimmune antisera against swine influenza virus (SIV) H1 gamma and H3 IV-B for 1 hour.

[0130] After washing, the cells were incubated with an anti-goat IgG-FITC conjugate (Invitrogen) for 1 hour, then washed again.

[0131] Images of each of the wells were obtained using an EVOS™ FL inverted digital fluorescence microscope (Invitrogen).

[0132] Protein extraction. The proteins from each of the constructs were extracted using the commercial Mem-PER™ Plus membrane protein extraction kit (Thermo Fisher Scientific) following the manufacturer's instructions.

[0133] The secreted, cytosolic and membrane protein fractions were collected and frozen at < -50°C.

[0134] Random selection (H1 and H3). Western blots were performed using the membrane protein fraction of each construct. Protein concentrations were obtained by measuring UV absorbance at 280 nm using the Nanodrop™ One (Thermo Fisher Scientific).

[0135] The proteins of each construct were denatured under reducing conditions at 70°C for 10 minutes using 10X Boit™ Sample Reducing Agent and 4X Boit™ LDS Sample Buffer (Thermo Fisher Scientific).

[0136] The denatured samples were loaded into the wells of a 4-12% Bis-Tris Plus gel with a Western MagicMark™ XP protein standard (Invitrogen). Two gels were loaded in the same orientation with 20 pg of protein loaded per well and run at 200 V for 22 minutes.

[0137] The transfer of proteins onto nitrocellulose membranes was carried out using the iBlot™ 2 gel transfer device (Thermo Fisher Scientific).

[0138] The membranes were then probed separately with porcine antisera, one blot receiving the antiserum against SIV H1 gamma and the other receiving the antiserum against SIV H3 IV-B, both detected using a horseradish peroxidase (HRP)-based goat anti-swine secondary antibody (EMD Millipore) using the iBind™ Flex Western Device (Thermo Fisher Scientific).

[0139] After an incubation of 2.5 hours at room temperature, the membranes were then washed with deionized water and incubated with the chemiluminescent substrate SuperSignal™ West Pico PLUS (Thermo Fisher Scientific) for five minutes.

[0140] Western blot images were obtained using the C-Digit® Blot Scanner (LL COR).

[0141] Summary of results. In the H1 and H3 Western blots, a band of approximately 80 kDa representing glycosylated hemagglutinin (HA0) was observed for the monogenic constructs H1 and H3 when probed with corresponding antisera, indicating correct expression of the protein; see arrows in [Fig.9].

[0142] Cellular control of [Fig.9], last column, showed no fluorescence in the immunofluorescence assay (IFA) nor band in the Western blot.

[0143] The linking constructions exhibited a variable expression of H1 and H3.

[0144] The H3-RVSP-H1 structure showed relatively high and equivalent expression of the H3 and Hl monomers, as observed in Western blot and IFA. The H3-p7-Hl and H3-V6K-H1 constructs also showed good expression of the H3 and Hl monomers.

[0145] The H3H1 construct showed expression of Hl and H3 proteins by both Western blot and IFA.

[0146] No HA0 band was observed for the H1H3 construct in the Hl blot, indicating that HA0 was not properly expressed by this construct.

[0147]

[0148] Terminology. The terminology used in this document is solely for the purpose of describing particular embodiments and is not intended to limit the invention.

[0149] Unless expressly stated otherwise, the terms used in this document have a simple and ordinary meaning, as understood by persons with ordinary knowledge of art.

[0150] The following definitions are intended to assist the reader in understanding this document, but are not intended to modify or limit the meaning of these terms unless otherwise indicated.

[0151] Although certain aspects of this disclosure have been described in connection with the specific embodiments offered as examples, alternatives, modifications, and variations may be made to the examples. The description and specific examples, while indicating embodiments of the technology, are for illustrative purposes only and are not intended to limit the scope of the technology. Furthermore, the enumeration of several embodiments exhibiting the stated features is not intended to exclude other embodiments with additional features or other embodiments incorporating different combinations of the stated features.Specific examples are provided to illustrate how to manufacture and use the compositions and methods of this technology and, unless explicitly stated otherwise, are not intended to represent any given realizations of this technology that have, or have not, been made or tested.

[0152] In this document, the terms "preferred" and "preferred" refer to embodiments of the technology that offer certain advantages under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, mentioning one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the technology.

[0153] In this document, the term "and / or" includes all combinations of one or more of the associated listed elements and may be abbreviated to A and / or B includes A, B and (A + B).

[0154] As used in the specification, but not in the claims, including as used in the examples, and unless expressly stated otherwise, all numbers may be read as if preceded by the word "substantially", "about" or "approximately", even if the term does not expressly appear. The expression "about" or "approximately", when used in the specification or the claims, may be used to describe a A magnitude and / or position should be used to indicate that the described value and / or position falls within a reasonable range of values ​​and / or positions. A reasonable range of values ​​based on disclosure and record history should be used when an explicit numerical range is not specified.

[0155] A numeric value can also be expressly defined by a numeric range, such as a value corresponding to + / - 0.1% of the stated value (or range of values), + / - 0.2% of the stated value (or range of values), + / - 0.5% of the stated value (or range of values), + / - 1% of the stated value (or range of values), + / - 2% of the stated value (or range of values), + / - 5% of the stated value (or range of values), + / - 10% of the stated value (or range of values), + / - 15% of the stated value (or range of values), + / - 20% of the stated value (or range of values), etc.

[0156] Any numeric range mentioned in this document is intended to include all subranges and values ​​contained therein. Where a range of values ​​is provided, it is understood that every intermediate value between an upper and lower limit of the range and any other declared or intermediate value within that declared range is included in the disclosure. Where the stated range includes upper and lower limits, ranges excluding either of those limits are also included.

[0157] In this document, the terms "may" and "can" and their variants are not limiting, so that the fact that an embodiment may include certain elements or features does not exclude other embodiments of the present invention which do not contain these elements or features.

[0158] Unless otherwise indicated, accession numbers, brand formulations or products, or commercial products are the latest versions available at the filing date of this application.

[0159] The citation of references in this document does not constitute an acknowledgment of the prior existence of such references or their relevance to the patentability of the technology disclosed herein. Any discussion of the content of the cited references is intended merely to provide a general summary of the claims made by the authors of the references and does not constitute an admission of the accuracy of the content of those references.

Claims

Demands

1. Host cell comprising a vector, characterized in that said vector comprises a nucleic acid construct comprising polynucleotides encoding a protein comprising an N-terminal signal peptide, a first polypeptide of interest, a second polypeptide of interest and an internal signal peptide sequence positioned between the first polypeptide and the second polypeptide; wherein the internal signal sequence can be cleaved by a protease to produce independent first and second polypeptides.

2. Host cell according to claim 1, characterized in that it is derived from a mammal.

3. Host cell according to claim 1, characterized in that it is an avian cell.

4. Host cell according to any one of claims 1 to 3, characterized in that the vector is a modified live virus vector.

5. Host cell according to any one of claims 1 to 4, characterized in that the vector is an RNA vector or a modified RNA vector which optionally replicates in a host cell.

6. Host cell according to any one of claims 1 to 5, characterized in that the vector is a self-replicating RNA vector comprising a 5' cap, a 5' UTR, a nucleic acid sequence encoding the non-structural proteins nsPl, nsP2, nsP3 and nsP4 of the alphavirus, a 26S promoter, the nucleic acid sequence encoding the first polypeptide of interest, the nucleic acid encoding the signal sequence, the nucleic acid encoding the second polypeptide of interest, and a 3'UTR tail and poly A.

7. Host cell according to any one of claims 1 to 6, characterized in that the vector is selected from vectors of Togavirus, Flaviviridae, Orthomyxoviridae, Rhabdoviridae, Pneumomyxovirinae, Picomaviridae, Astroviridae or Nidovirales.

8. Host cell according to any one of claims 1 to 7, characterized in that the N-terminal signal peptide of the nucleic acid construct directs the encoded protein into the endoplasmic reticulum, and preferably comprises 15 to 150 residues, preferably still 20 to 30 residues.

9. Host cell according to any one of claims 1 to 8, characterized in that the nucleic acid construct comprises N-terminal or internal signal peptide coded sequences comprising a motif comprising an N region comprising 1 to 5 charged residues, an H region covering 7 to 15 contiguous hydrophobic residues, of which 3 to 5 are leucine residues, a C region comprising 3 to 7 uncharged amino acid residues and / or a Pro region or a negative region comprising 1 to 6 charged amino acid residues.

10. Host cell according to any one of claims 1 to 9, characterized in that the nucleic acid construct comprises an N-terminal peptide-encoded sequence and / or internal signal comprising all or part of a 6K polypeptide from a virus of the genus Alphavirus or another 6K-type viral polypeptide.

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