IL288634A - Improving the translation and protein secretion efficiency of mrna vaccines - Google Patents

Improving the translation and protein secretion efficiency of mrna vaccines

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Publication number
IL288634A
IL288634A IL288634A IL28863421A IL288634A IL 288634 A IL288634 A IL 288634A IL 288634 A IL288634 A IL 288634A IL 28863421 A IL28863421 A IL 28863421A IL 288634 A IL288634 A IL 288634A
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Israel
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sequence
vaccine
mrna
protein
virus
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IL288634A
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Hebrew (he)
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Eric GERST Jeffrey
HAIMOVICH Gal
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Yeda Res & Dev
Eric GERST Jeffrey
HAIMOVICH Gal
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Priority to IL288634A priority Critical patent/IL288634A/en
Publication of IL288634A publication Critical patent/IL288634A/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/04—Fusion polypeptide containing a localisation/targetting motif containing an ER retention signal such as a C-terminal HDEL motif
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source

Description

IMPROVING THE TRANSLATION AND PROTEIN SECRETION EFFICIENCY OF MRNA VACCINES FIELD AND BACKGROUND OF THE INVENTION The present invention, in some embodiments thereof, relates to mRNA vaccines and methods of optimizing the sequences thereof. Human infection with Coronaviridae (CoV) viruses can result in severe acute respiratory distress leading to lethality. The recent outbreak of the SARS-CoV-2 virus emphasizes the potent ability of human coronaviruses (hCoVs) to infect and rapidly spread throughout the human population, given the absence of immune prophylaxis (e.g. vaccination) or curative treatment. SARS-CoV-2 is a positive (+) single-strand RNA [(+)ssRNA] virus comprising a genome of ~30kb encoding at least 29 viral proteins (VPs) involved in viral infection, replication, and release. The genome is organized into a 5’untranslated region (UTR)-leader sequence, followed by a large open reading frame (ORF1ab) that encodes 16 non-structural VPs (nsp1-16), then by ORFs encoding the viral accessory proteins and structural proteins (e.g. spike (S), envelope (E), membrane (M), nucleocapsid (N)), and terminating with a 3’UTR-polyA tail. The non-structural VPs are involved in the cleavage of polypeptide1ab (NSP5), suppression of host antiviral response (nsp1), creation of the viral replication center from the endoplasmic reticulum (ER) (nsp2,3,4,6), and viral RNA replication (nsp7,8,9,10,12,13,14,15,16). Four structural VPs (S,E,M,N) form the coat of the virus and along with other small ORFs facilitate virion assembly, release, and infection. As with other (+)ssRNA viruses, upon infection the SARS-CoV-2 RNA acts as an mRNA for the direct translation of viral ORF1ab. As with other members of the hCoVs, infection of the lung epithelia with SARS- CoV-2 likely induces a reticulo-vesicular network of ER-derived double membrane vesicles (DMVs) that form a discrete viral replication organelle (or center; VRC). VPs are translated on the VRC surface, with many (i.e. soluble and membrane-anchored proteins) translocated into the membrane of the newly forming structure. VRC formation, therefore, represents an essential step for both vRNA replication and virion assembly, and hence, progressive infection upon virion release. Yet, little is known of the organellar dynamics and interactions with either the viral RNA or VPs to create the replication membrane, although morphological alteration of the ER (and, perhaps, other secretory pathway organelles, e.g. lipid droplets, Golgi, endosomes) is consistent with the secretory nature of viral replication, which first involves nsp translation and translocation. The ER is the primary site for the translation and translocation of soluble secreted and membrane (secretome) proteins. Thus, vRNA interactions with ER-associated RNA-binding proteins (RBPs) likely constitute a critical rate-limiting step in VP production. Recently, a cis RNA element has been identified which is present in nearly all secretome proteins from bacteria to humans. This motif, entitled "secretion-enhancing cis regulatory targeting element" (SECReTE), is based upon extended (>10 consecutive) triplet nucleotide repeats whereby a pyrimidine nucleotide is present every third base, whether in coding regions (as NYN or NNY; where N is any nucleotide and Y = U or C) or in the UTR regions. Mutational analyses performed using several yeast genes encoding secreted proteins (e.g. SUC2, CCW12, HSP150) revealed that the addition or removal of SECReTE motifs in yeast mRNAs could enhance or inhibit mRNA stability and association with the ER, respectively, thereby affecting protein secretion and cell physiology. Thus, SECReTE is important for mRNA-protein interactions at the level of the ER that facilitate protein production. Numerous SECReTE motifs encoded in many of SARS-CoV-2 VPs have also been identified, particularly in those encoding membrane-associated proteins (Haimovich et al. 2020 doi: https://doi.org/10.1101/2020.04.20.050088). Additional background art includes WO2019/150373; Cohen-Zontag, O. et al. PLoS Genet 15, e1008248 (2019) and Haimovich et al., bioRxiv, 2020.2004.2020.050088 (2020). SUMMARY OF THE INVENTION According to an aspect of the present invention there is provided a vaccine comprising an immunologically acceptable carrier and a mRNA encoding a protein, or a fragment thereof, of a pathogen, wherein the mRNA comprises at least one heterologous ER targeting sequence as set forth in SEQ ID NO: 8, wherein the mRNA does not comprise the sequence as set forth in SEQ ID NO: 6. According to an aspect of the present invention there is provided a method of generating the vaccine described herein, comprising: (a) selecting a sequence of a mRNA encoding a protein of a pathogen which comprises a heterologous ER targeting sequence as set forth in SEQ ID NO: 8; and (b) synthesizing mRNA comprising the sequence, thereby generating the mRNA based vaccine. According to embodiments of the invention, at least one heterologous ER targeting sequence as set forth in SEQ ID NO: 2. According to embodiments of the invention, the mRNA comprises a chemically modified nucleotide. According to embodiments of the invention, the mRNA comprises a non-natural cap. According to embodiments of the invention, the mRNA is encapsulated in a particle. According to embodiments of the invention, the particle comprises lipids. According to embodiments of the invention, the pathogen is a virus. According to embodiments of the invention, the mRNA is codon optimized for expression in human cells. According to embodiments of the invention, the ER targeting sequence does not comprise more than 5 consecutive repeats of the sequence TG. According to embodiments of the invention, the ER targeting sequence comprises at least 15 consecutive repeats of the sequence NNY, wherein N is any base and Y is a pyrimidine. According to embodiments of the invention, the ER targeting sequence does not comprise more than 10 consecutive thymines. According to embodiments of the invention, the mRNA further encodes a signal peptide sequence. According to embodiments of the invention, the signal peptide sequence is heterologous to the protein. According to embodiments of the invention, the signal peptide sequence comprises at least one heterologous ER targeting sequence as set forth in SEQ ID NO: 8.
According to embodiments of the invention, the protein is of a virus selected from the group consisting of a coronavirus, an influenza virus, cytomegalovirus (CMV), human immunodeficiency virus (HIV-1), rabies virus, measles virus, chickenpox virus, Respiratory syncytial virus (RSV), Epstein-Barr virus (EBV) and a Zika virus. According to embodiments of the invention, the protein is of a coronavirus. According to embodiments of the invention, the coronavirus is SARS-CoV2. According to embodiments of the invention, the protein is a spike protein. According to embodiments of the invention, the spike protein comprises a sequence as set forth in SEQ ID NOs: 10, 11, 12, 13, 14, 15 or 16. According to embodiments of the invention, the protein is a membrane protein. According to embodiments of the invention, the protein is a secreted protein. According to embodiments of the invention, the method further comprises encapsulating the mRNA with a carrier. According to embodiments of the invention, the carrier comprises a lipid particle. Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced. In the drawings: FIGs. 1A-C illustrate preliminary analysis of Spike mRNA variants. A ) Schematic of the five Spike variants (Wu, Wuhan (SEQ ID NO: 17); Max, Maximized SECReTE length and pyrimidine content (SEQ ID NO: 21); hS, humanized Spike (SEQ ID NO: 19); hS-Max, humanized Spike with maximized SECReTE length and pyrimidine content (SEQ ID NO: 23); No, no SECReTE Spike (SEQ ID NO: 25)). Arrow indicate the position of the prefusion (2P) mutations in SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 26. Below the five variants is a depiction of the conservation of SECReTE elements localization within Spike mRNAs of the 7 human-infecting coronaviruses. B) MCF7 cells were transiently transfected with 8 spike variants (four variants (SEQ ID NO: 17, 19, 21 and 25) and four variants containing the 2-proline pre-folding mutation (SEQ ID NO: 18, 20, 22 and 26), an irrelevant protein (cFos-YFP-C) or left untransfected as a control (Ctrl). hours later, the cells were fixed with 4% paraformaldehyde in PBS, blocked with 3% BSA and stained with a Rabbit anti-spike antibody (Abcam ab272504; 1:1000) and a secondary antibody goat anti-Rabbit IgG-alexa647. Samples were imaged on a Zeiss AxioObserver Z1 DuoLink dual camera imaging system with 63x objective. 0.5 μm step z-stack images were taken. Maximum projection images of the z-stacks were created by FIJI, and the resulting images were automatically analyzed for Alexa4fluorescence intensity using Cell Profiler. Each dot on the graph depicts a single cell. a.u. – arbitrary units of fluorescence. 2P indicates the prefusion 2-proline mutation. C) MCF7 cells were transfected with hS2P (SEQ ID NO: 20) or Wu2P (SEQ ID NO: 18) constructs. 30 hours later, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 3% BSA, and stained with a mouse anti-FLAG (M2) (Sigma F3165) and a secondary antibody goat anti-mouse IgG-alexa488. Samples were imaged on the Zeiss AxioObserver Z1 with a 100x objective. Representative maximum projections images of cells expressing hS2P, Wu2P, and untransfected cells are shown. DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION The present invention, in some embodiments thereof, relates to mRNA vaccines and methods of optimizing the sequences thereof. Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Messenger RNA (mRNA) vaccines transfects molecules of synthetic RNA into viable human cells. Once inside the cells, the vaccine's RNA functions as mRNA, causing the cells to build the foreign protein that would normally be produced by a pathogen (such as a virus) or by a cancer cell. These protein molecules stimulate an adaptive immune response which teaches the body how to identify and destroy the corresponding pathogen or cancer cells. The delivery of mRNA is achieved by a co-formulation of the molecule into lipid nanoparticles which protect the RNA strands and helps their absorption into the cells. The advantages of RNA vaccines over traditional protein vaccines are superior design and production speed, lower cost of production, and the induction of both cellular as well as humoral immunity. Vaccination of the human population against SARS-Cov2 using mRNA vaccines (the Moderna COVID-19 vaccine (mRNA-1273) and the Pfizer–BioNTech COVID-19 vaccine (BNT162b2)) encoding the SARS-Cov2 spike protein has yielded promising results in reducing infection, severe disease, mortality and transmission. Both vaccines encode for the full-length Spike protein consisting of a signal sequence, S1, and S2 domains (comprising the large extracellular domain including the RBD), followed by the short transmembrane and cytoplasmic domains. Upon ribosomal translation, the signal sequence targets the protein to the endoplasmic reticulum (ER), where it starts undergoing significant post-translational modifications. Aside from removal of the signal sequence, glycosylation (both N and O-type) as well as disulfide bond formation occur in this organelle. The end product in host cells expressing these mRNA vaccines is a surface-exposed, membrane-anchored, glycosylated, and trimerized Spike protein resembling the 3-D structure of the native viral Spike protein, to the extent that it interacts with its cognate receptor, hACE2. The wild-type SARS-CoV-2 Spike mRNA has 7 SECReTE elements, a repetitive motif consisting of >10 consecutive NNY repeats (see for example US Patent Application No. 20200354731, incorporated herein by reference). However, in the case of the SARS-CoV-2 Spike mRNA vaccine, the viral RNA sequence was codon-optimized to match the human codon bias, thus, destroying several SECReTE elements.
The present inventors set out to determine whether SECReTE content in the Spike RNA can affect Spike protein expression. The present inventors expressed different FLAG-tagged Spike proteins from transcripts bearing either modified or nullified vSECReTEs (i.e. the Wuhan, humanized, no-SECReTE, and maximized SECReTE motif; Figure 1A) having synonymous mutations in MCF7 cells. Immunofluorescence staining for Spike proteins on the cell surface shows that the higher the SECReTE content, the higher level of the secretion (Figure 1B). Furthermore, the humanized Spike was shown to be concentrated in the perinuclear region (probably ER) compared to the Wuhan transcript (Figure 1C). Thus, the present inventors conclude that humanized Spike might be retained on ER membranes (and the Golgi) and, therefore, less presented on the cell surface. Accordingly, the present inventors propose that mRNA sequences of vaccines can be further optimized to increase protein translation and secretion by taking into account the SECReTE content of the mRNA encoding the antigenic protein. Thus, according to a first aspect of the present invention, there is provided a vaccine comprising an immunologically acceptable carrier and a mRNA encoding a protein, or a fragment thereof, of a pathogen, wherein said mRNA comprises at least one heterologous ER targeting sequence as set forth in SEQ ID NO: 2, wherein said mRNA does not comprise the sequence as set forth in SEQ ID NO: 6. The term "vaccine" as used herein refers to a composition which comprises a mRNA which encodes for at least one antigenic protein, for administration to a subject (e.g. human), against which an immune response is elicited. In some embodiments a vaccine is a therapeutic. In some embodiments, a vaccine is prophylactic. The mRNA has an open reading frame encoding at least one antigenic protein or an immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response). As used herein, the term "open reading frame", abbreviated as "ORF", refers to a segment or region of an mRNA molecule that encodes a protein. The ORF comprises a continuous stretch of non-overlapping, in-frame codons, beginning with the initiation codon and ending with a stop codon, and is translated by the ribosome. As used herein, the term "protein of a pathogen" refers to a protein (or a fragment thereof that induces an immunogenic response to the protein) that is expressed by the pathogen. In one embodiment, the protein refers to the post-translationally modified protein (i.e. one that does not include its signal peptide). Accordingly, the term "protein of a pathogen" refers to a protein which is devoid of its signal peptide. In still another embodiment, the protein is at least 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % identical or homologous to the amino acid sequence of the wild-type protein. In one embodiment, the protein of the vaccine is derived from (i.e. naturally found in) an organism (e.g. virus, bacteria, parasite, fungus) which is pathogenic to a mammal. The pathogenic organism is one that brings about a disease (e.g. infectious disease) in a mammal. The term "infectious disease" refers to a transmissible disease caused by a pathogenic organism. According to a particular embodiment, the infectious disease is a respiratory disease – e.g. COVID, influenza. The infectious disease may be transmitted in any way including droplet contact, fecal-oral transmission, sexual transmission, oral transmission, direct contact and vehicle transmission. In another embodiment, the protein of the vaccine is a cancer-specific protein or peptide. The protein of the vaccine may originate from, but is not limited to any of the following families of virus: Adenovirus, arenaviridae, astroviridae, bunyaviridae, caliciviridae, coronaviridae, flaviviridae, herpesviridae, orthomyxoviridae, paramyxoviridae, picornaviridae, poxviridae, reoviridae, retroviridae, rhabdoviridae and togaviridae. More specifically at least one antigen or antigenic sequence may be derived from any of the following virus: Influenza A such as H1N1, H1N2, H3N2 and H5N1 (bird flu), Influenza B, Influenza C virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rotavirus, any virus of the Norwalk virus group, enteric adenoviruses, parvovirus, Dengue fever virus, Monkey pox, Mononegavirales, Lyssavirus such as rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, European bat virus 1 & 2 and Australian bat virus, Ephemerovirus, Vesiculovirus, Vesicular Stomatitis Virus (VSV), Herpesviruses such as Herpes simplex virus types 1 and 2, varicella zoster, cytomegalovirus, Epstein-Bar virus (EBV), human herpesvirusses (HHV), human herpesvirus type 6 and 8, Human immunodeficiency virus (HIV), papilloma virus, murine gammaherpesvirus, Arenaviruses such as Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, Lymphocytic choriomeningitis virus (LCMV), Bunyaviridiae such as Crimean-Congo hemorrhagic fever virus, Hantavirus, hemorrhagic fever with renal syndrome causing virus, Rift Valley fever virus, Filoviridae (filovirus) including Ebola hemorrhagic fever and Marburg hemorrhagic fever, Flaviviridae including Kaysanur Forest disease virus, Omsk hemorrhagic fever virus, Tick-borne encephalitis causing virus and Paramyxoviridae such as Hendra virus and Nipah virus, variola major and variola minor (smallpox), alphaviruses such as Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus, SARS-associated coronavirus (SARS-CoV), SARS-associated coronavirus (SARS-CoV-2), Middle East respiratory syndrome CoV (MERS-CoV), West Nile virus, any encephaliltis causing virus. According to a specific embodiment, the protein of the vaccine is derived from a coronavirus, such as human coronavirus 229E, human coronavirus OC43, SARS-CoV, HCoV NL63, HKU1, MERS-CoV and SARS-CoV-2. According to a particular embodiment, the coronavirus is SARS-CoV-2. SARS-CoV-2 contains four major structural proteins, namely spike (S), membrane (M) and envelope (E) proteins, all of which are embedded in the viral surface envelope, and nucleocapsid (N) protein, which is in the ribonucleoprotein core. S proteins are responsible for recognition of the host cellular receptor to initiate virus entry. M proteins are embedded in the envelope and shape the virion envelope. E proteins are small polypeptides that are crucial for CoV infectivity. N proteins make up the helical nucleocapsid and bind along the viral RNA genome. In addition to these structural proteins, SARS-CoV-2 encodes 16 non-structural proteins (nsp1–16) and 9 accessory proteins. Examples of SARS-CoV-2 antigenic proteins include the spike protein (having the amino acid sequence at least 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % identical or homologous to the sequence as set forth in SEQ ID NO: 1). According to a particular embodiment, the mRNA of the vaccine encodes a spike protein having an amino acid sequence as set forth in SEQ ID NO: 29. The spike protein consists of a membrane-distal S1 subunit and a membrane- proximal S2 subunit and exists in the virus envelope as a homotrimer. The S1 subunit determines receptor recognition via its receptor-binding domain (RBD), whereas the Ssubunit is responsible for membrane fusion, which is required for virus entry. In MERS-CoV, SARS-CoV or SARS-CoV-2, the RBD is located in the C-terminal domain of the S1 subunit. In some CoVs, the N-terminal domain (NTD) of the S1 subunit can be used for receptor binding (such as in mouse hepatitis virus) or might also be involved in virus attachment to host cells by recognizing specific sugar molecules (such as in TGEV, BCoV and IBV) or has an important role in the pre-fusion to post-fusion transition of the S protein. The S2 subunit contains the fusion peptide (FP), connecting region (CR), heptad repeat 1 (HR1) and HR2 around a central helix as a helix-turn-helix structure. According to a particular embodiment, the protein of the vaccine is the full-length Spike protein of SARS-CoV-2 with two proline substitutions (K986P and V987P). According to still another embodiment, the protein of the vaccine is the receptor binding domain (RBD) of SARS-CoV-2 (or fragments thereof). Additional viral targets that can be used in vaccines are disclosed in Lianpan Dai and George F. Gao, Nature Reviews, Immunology, Volume 21, February 2021, pages 73-82, the contents of which are incorporated herein by reference. An embodiment of the present invention includes at least one antigenic protein or peptide or fragment of an antigenic protein or peptide from a bacteria. More specifically, the protein of the vaccine may be derived from one of the following bacteria: Anthrax (Bacillus anthracis), Mycobacterium tuberculosis, Salmonella (Salmonella gallinarum, S. pullorum, S. typhi, S. enteridtidis, S. paratyphi, S. dublin, S. typhimurium), Clostridium botulinum, Clostridium perfringens, Corynebacterium diphtheriae, Bordetella pertussis, Campylobacter such as Campylobacter jejuni, Crytococcus neoformans, Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis, Listeria monocytogenes, Leptospira species, Legionella pneumophila, Borrelia burgdorferi, Streptococcus species such as Streptococcus pneumoniae, Neisseria meningitides, Haemophilus influenzae, Vibrio species such as Vibrio cholerae O1, V. cholerae non-O1, V. parahaemolyticus, V. parahaemolyticus, V. alginolyticus, V. fumissii, V. carchariae, V. hollisae, V. cincinnatiensis, V. metschnikovii, V. damsela, V. mimicus, V. fluvialis, V. vulnificus, Bacillus cereus, Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria & Aeromonas veronii, Plesiomonas shigelloides, Shigella species such as Shigella sonnei, S. boydii, S. flexneri, and S. dysenteriae, Enterovirulent Escherichia coli EEC (Escherichia coli-enterotoxigenic (ETEC), Escherichia coli-enteropathogenic (EPEC), Escherichia coli O157:H7 enterohemorrhagic (EHEC), Escherichia coli-enteroinvasive (EIEC)), Staphylococcus species, such as S. aureus and especially the vancomycin intermediate/resistant species (VISA/VRSA) or the multidrug resistant species (MRSA), Shigella species, such as S. flexneri, S. sonnei, S. dysenteriae, Cryptosporidium parvum, Brucella species such as B. abortus, B. melitensis, B. ovis, B. suis, and B. canis, Burkholderia mallei and Burkholderia pseudomallei, Chlamydia psittaci, Coxiella bumetii, Francisella tularensis, Rickettsia prowazekii, Histoplasma capsulatum, Coccidioides immitis. An embodiment of the present invention includes at least one antigenic protein or peptide or fragment of an antigenic protein or peptide from a parasite, examples of which include, but are not limited to: Plasmodium species such as Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Plasmodium falciparum, Endolimax nana, Giardia lamblia, Entamoeba histolytica, Cryptosporidium parvum, Blastocystis hominis, Trichomonas vaginalis, Toxoplasma gondii, Cyclospora cayetanensis, Cryptosporidium muris, Pneumocystis carinii, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Leishmania mexicana, Acanthamoeba species such as Acanthamoeba castellanii, and A. culbertsoni, Naegleria fowleri, Trypanosoma cruzi, Trypanosoma brucei rhodesiense, Trypanosoma brucei gambiense, Isospora belli, Balantidium coli, Roundworm (Ascaris lumbricoides), Hookworm (Necator Americanus, Ancylostoma duodenal), Pinworm (Enterobius vermicularis), Roundworm (Toxocara canis, Toxocara cati), Heart worm (Dirofilaria immitis), Strongyloides (Stronglyoides stercoralis), Trichinella (Trichinella spiralis), Filaria (Wuchereria bancrofti, Brugia malayi, Onchocerca volvulus, Loa loa, Mansonella streptocerca, Mansonella perstans, Mansonella ozzardi), and Anisakine larvae (Anisakis simplex (herring worm), Pseudoterranova (Phocanema, Terranova) decipiens (cod or seal worm), Contracaecum species, and Hysterothylacium (Thynnascaris species) Trichuris trichiura, Beef tapeworm (Taenia saginata), Pork tapeworm (Taenia solium), Fish tapeworm (Diphyllobothrium latum), and Dog tapeworm (Dipylidium caninum), Intestinal fluke (Fasciolopsis buski), Blood fluke (Schistosoma japonicum, Schistosoma mansoni) Schistosoma haematobium), Liver fluke (Clonorchis sinensis), Oriental lung fluke (Paragonimus westermani), and Sheep liver fluke (Fasciola hepatica), Nanophyetus salmincola and N. schikhobalowi. The protein of the vaccine may be derived from pathogens that infect and cause disease in domestic animals, especially commercially relevant animals such as pigs, cows, horses, sheep, goats, llamas, rabbits, mink, mice, rats, dogs, cats, ferrets, poultry such as chicken, turkeys, pheasants and others, fish such as trout, salmon, cod and other farmed species. Examples of diseases or agents here of from which at least one antigen or antigenic sequence may be derived include, but are not limited to: Multiple species diseases such as: Anthrax, Aujeszky's disease, Bluetongue, Brucellosis such as: Brucella abortus, Brucella melitensis or Brucella suis; Crimean Congo haemorrhagic fever, Echinococcosis/hydatidosis, virus of the family Picornaviridae, genus Aphthovirus causing Foot and Mouth disease especially any of the seven immunologically distinct serotypes: A, O, C, SAT1, SAT2, SAT3, Asial, or Heartwater, Japanese encephalitis, Leptospirosis, Newworld screwworm (Cochliomyia hominivorax), Old world screwworm (Chrysomya bezziana), Paratuberculosis, Q fever, Rabies, Rift Valley fever, Rinderpest, Trichinellosis, Tularemia, Vesicular stomatitis or West Nile fever; Cattle diseases such as: Bovine anaplasmosis, Bovine babesiosis, Bovine genital campylobacteriosis, Bovine spongiform encephalopathy, Bovine tuberculosis, Bovine viral diarrhoea, Contagious bovine pleuropneumonia, Enzootic bovine leukosis, Haemorrhagic septicaemia, Infectious bovine rhinotracheitis I infectious pustular vulvovaginitis, Lumpky skin disease, Malignant catarrhal fever, Theileriosis, Trichomonosis or Trypanosomosis (tsetse-transmitted); Sheep and goat diseases such as: Caprine arthritis I encephalitis, Contagious agalactia, Contagious caprine pleuropneumonia, Enzootic abortion of ewes (ovine chlamydiosis), Maedi-visna, Nairobi sheep disease, Ovine epididymitis (Brucella ovis), Peste des petits ruminants, Salmonellosis (S. abortusovis), Scrapie, Sheep pox and goat pox; Equine diseases such as: African horse sickness, Contagious equine metritis, Dourine, Equine encephalomyelitis (Eastem), Equine encephalomyelitis (Western), Equine infectious anaemia, Equine influenza, Equine piroplasmosis, Equine rhinopneumonitis, Equine viral arteritis, Glanders, Surra (Trypanosoma evansi) or Venezuelan equine encephalomyelitis; Swine diseases such as: African swine fever, Classical swine fever, Nipah virus encephalitis, Porcine cysticercosis, Porcine reproductive and respiratory syndrome, Swine vesicular disease or Transmissible gastroenteritis; Avian diseases such as: Avian chlamydiosis, Avian infectious bronchitis, Avian infectious laryngotracheitis, Avian mycoplasmosis (M. gallisepticum), Avian mycoplasmosis (M. synoviae), Duck virus hepatitis, Fowl cholera, Fowl typhoid, Highly pathogenic avian influenza this being any Influenzavirus A or B and especially H5N1, Infectious bursal disease (Gumboro disease), Marek's disease, Newcastle disease, Pullorum disease or Turkey rhinotracheitis; Lagomorph and rodent diseases such as: Virus enteritis, Myxomatosis or Rabbit haemorrhagic disease; Fish diseases such as: Epizootic haematopoietic necrosis, Infectious haematopoietic necrosis, Spring viraemia of carp, Viral haemorrhagic septicaemia, Infectious pancreatic necrosis, Infectious salmon anaemia, Epizootic ulcerative syndrome, Bacterial kidney disease (Renibacterium salmoninarum), Gyrodactylosis (Gyrodactylus salaris), Red sea bream iridoviral disease; or other diseases such as Camelpox or Leishmaniosis. According to a particular embodiment, the protein is derived from a cancer-causing pathogen including, but not limited to H. pylori, human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), and Epstein-Barr virus (EBV). The mRNA of the vaccine may further include 5′ and 3 ′ untranslated sequences to optimize mRNA stability and translation efficiency. The untranslated sequences may be heterologous to the native mRNA which encodes the wild-type protein in the pathogen or may be those sequences that are used by the pathogen to control expression thereof (i.e. the UTR is endogenous to the ORF encoding the antigen polypeptide). In some embodiments, the polynucleotide comprises two or more 5'UTRs or functional fragments thereof, each of which have the same or different nucleotide sequences. In some embodiments, the polynucleotide comprises two or more 3'UTRs or functional fragments thereof, each of which have the same or different nucleotide sequences.
In addition the mRNA of the vaccine may further include a sequence that encodes a signal peptide. As used herein, the phrase "signal peptide" refers to a peptide linked in frame to the amino terminus of a polypeptide and directs the encoded polypeptide into a cell's secretory pathway. The signal sequence is typically located N-terminal to the protein sequence. A signal sequence is normally absent from the mature protein. A signal sequence is typically cleaved from the protein by a signal peptidase after the protein is transported. In one embodiment, the signal peptide sequence is endogenous to the protein of the pathogen. In another embodiment, the signal peptide sequence is heterologous (i.e. is not native, or is exogenous, non-endogenous) to the protein of the pathogen. In one embodiment, the signal peptide has an amino acid sequence as set forth in SEQ ID NO: 7. Other exemplary signal sequences that can be used in the mRNA vaccines disclosed herein include MDSKGSSQKGSRLLLLLVVSNLLLPQGVVG (SEQ ID NO: 30), MDWTWILFLVAAATRVHS (SEQ ID NO: 31); METPAQLLFLLLLWLPDTTG (SEQ ID NO: 32); MLGSNSGQRVVFTILLLLVAPAYS (SEQ ID NO: 33); MKCLLYLAFLFIGVNCA (SEQ ID NO: 34); MWLVSLAIVTACAGA (SEQ ID NO: 35). As mentioned, the mRNA of the vaccine comprises at least one, at least two, at least three, at least four or more heterologous ER targeting sequence as set forth in SEQ ID NO: 8 and more preferably as set forth in SEQ ID NO: 2. As used herein, the term "heterologous" refers to a sequence of mRNA that is not present in the native mRNA that encodes (or regulates expression of) the wild-type protein of the pathogen (and further if the mRNA also comprises a signal peptide which is not native to the protein of the pathogen, the heterologous ER targeting sequence is not native to the signal peptide either). In some cases the mRNA sequence encoding the wild-type protein is the original sequence when first identified. In other cases, the sequence of the wild-type protein refers to the most prevalent sequence of the protein at the time of designing the vaccine. The ER targeting sequence of this aspect of the present invention may serve to aid in the localization of the mRNA (or the newly expressed protein encoded thereby) to the ER. In one embodiment, the ER targeting sequence promotes uptake of the mRNA or the newly expressed protein into the ER. In another embodiment, the ER targeting sequence promotes binding of the mRNA to ER-associated binding proteins. The NNY repeat of this sequence may be repeated in the mRNA at least 7 times (as in SEQ ID NO: 8), 8 times, 9 times, 10 times, 11 times, 12 times. 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times or more. According to a particular embodiment, the NNY repeat is repeated at least times (as in SEQ ID NO: 2). In a particular embodiment, the N of the NNY repeat is a pyrimidine. In another embodiment, the N of the NNY repeat is a purine. In all embodiments, the Y of the NNY repeat is a pyrimidine. According to a particular embodiment, the ER targeting sequence does not comprise nucleotides that encode for the expressed, post-translationally modified protein. According to still another embodiment, the ER targeting sequence does not comprise nucleotides that encode for a sequence as set forth in SEQ ID NO: 9. (KDEL). Preferably, the ER targeting sequence does not comprise more than 10, 15, 20, 25, 30 or more consecutive uridines (or modified uridines such as N1-Methylpseudouridine). Preferably, the ER targeting sequence does not comprise more than 10, 15, 20, 25, 30 or more consecutive cytosines. In one embodiment, the ER targeting sequence of this aspect of the present invention is positioned 3’ to the sequence that encodes the protein of the pathogen. In another embodiment the ER targeting sequence of this aspect of the present invention is positioned 5’ to the sequence that encodes the protein of the pathogen. In still another embodiment, at least one of the heterologous ER targeting sequences is not in the sequence which encodes the signal peptide. In still further embodiments, at least one of the ER targeting sequences is encoded in the sequence that encodes for the protein of the pathogen. Preferably, when the ER targeting sequence is comprised in the mRNA that encodes the protein of the pathogen, the codons of the nucleic acid sequence are selected to comprise the ER targeting sequence (such that a novel heterologous ER targeting sequence is created and the amino acid sequence of the expressed protein of the pathogen is identical to the native (or wild-type) amino acid sequence). The mRNA of the present disclosure, in some embodiments, are codon optimized for expression in human cells. Codon optimization methods are known in the art and may be used as provided herein. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g. glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art--non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and/or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms. In some embodiments, a codon optimized sequence shares less than 95% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally- occurring or wild-type mRNA sequence encoding a polypeptide or protein of the pathogen. In some embodiments, a codon optimized sequence shares less than 90% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of the pathogen. In some embodiments, a codon optimized sequence shares less than 85% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of the pathogen. In some embodiments, a codon optimized sequence shares less than 80% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of the pathogen. In some embodiments, a codon optimized sequence shares less than 75% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally- occurring or wild-type mRNA sequence encoding a polypeptide or protein of the pathogen. In some embodiments, a codon optimized sequence shares between 65% and 85% (e.g., between about 67% and about 85% or between about 67% and about 80%) sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally- occurring or wild-type mRNA sequence encoding a polypeptide or protein of the pathogen. In some embodiments, a codon optimized sequence shares between 65% and 75% or about 80% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding a polypeptide or protein of the pathogen. In some embodiments a codon optimized RNA may, for instance, be one in which the levels of G/C are enhanced. The G/C-content of nucleic acid molecules may influence the stability of the RNA. RNA having an increased amount of guanine (G) and/or cytosine (C) residues may be functionally more stable than nucleic acids containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. WO02/098443 discloses a pharmaceutical composition containing an mRNA stabilized by sequence modifications in the translated region. Due to the degeneracy of the genetic code, the modifications work by substituting existing codons for those that promote greater RNA stability without changing the resulting amino acid. The approach is limited to coding regions of the RNA. Care should be taken that the selected codon does not introduce unwanted secondary sequence functions that impede expression of the resulting open reading frames. Thus, for example in the case of a COVID vaccine which encodes for the SARS-CoV-2 spike protein (having the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 29), at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten of the following codons are suggested. (i) the sequence of the codon which encodes the amino acid at position 1 is AUG; (ii) the sequence of the codon which encodes the amino acid at position 2 is UUU; (iii) the sequence of the codon which encodes the amino acid at position 3 is GUU; (iv) the sequence of the codon which encodes the amino acid at position 4 is UUC; (v) the sequence of the codon which encodes the amino acid at position 5 is UUG; (vi) the sequence of the codon which encodes the amino acid at position 6 is GUU; (vii) the sequence of the codon which encodes the amino acid at position 7 is CUU; (viii) the sequence of the codon which encodes the amino acid at position 8 is CUC; (ix) the sequence of the codon which encodes the amino acid at position 9 is CCC; (x) the sequence of the codon which encodes the amino acid at position 10 is CUC; (xi) the sequence of the codon which encodes the amino acid at position is GUC; (xii) the sequence of the codon which encodes the amino acid at position is UCU; (xiii) the sequence of the codon which encodes the amino acid at position is UCU; (xiv) the sequence of the codon which encodes the amino acid at position is UUU; (xv) the sequence of the codon which encodes the amino acid at position 80 is GAU; (xvi) the sequence of the codon which encodes the amino acid at position is AAC; (xvii) the sequence of the codon which encodes the amino acid at position is CCU; (xviii) the sequence of the codon which encodes the amino acid at position is GUC; (xix) the sequence of the codon which encodes the amino acid at position is CUC; (xx) the sequence of the codon which encodes the amino acid at position is CCC; (xxi) the sequence of the codon which encodes the amino acid at position 86 is UUU; (xxii) the sequence of the codon which encodes the amino acid at position is AAC; (xxiii) the sequence of the codon which encodes the amino acid at position is GAC; (xxiv) the sequence of the codon which encodes the amino acid at position is GGC; (xxv) the sequence of the codon which encodes the amino acid at position is GUU; (xxvi) the sequence of the codon which encodes the amino acid at position 91 is UAC; (xxvii) the sequence of the codon which encodes the amino acid at position is UUU; (xxviii) the sequence of the codon which encodes the amino acid at position is GCC; (xxix) the sequence of the codon which encodes the amino acid at position is UCC; (xxx) the sequence of the codon which encodes the amino acid at position is ACC; (xxxi) the sequence of the codon which encodes the amino acid at position 116 is UCC; (xxxii) the sequence of the codon which encodes the amino acid at position 1is CUC; (xxxiii) the sequence of the codon which encodes the amino acid at position 1is CUC; (xxxiv) the sequence of the codon which encodes the amino acid at position 1is AUC; (xxxv) the sequence of the codon which encodes the amino acid at position 1is GUC; (xxxvi) the sequence of the codon which encodes the amino acid at position 1is AAC; (xxxvii) the sequence of the codon which encodes the amino acid at position 122 is AAC; (xxxviii) the sequence of the codon which encodes the amino acid at position 1is GCC; (xxxix) the sequence of the codon which encodes the amino acid at position 1is ACC; (xl) the sequence of the codon which encodes the amino acid at position 1is AAC; (xli) the sequence of the codon which encodes the amino acid at position 1is GUC; (xlii) the sequence of the codon which encodes the amino acid at position 127 is GUC; (xliii) the sequence of the codon which encodes the amino acid at position 1is AUC; (xliv) the sequence of the codon which encodes the amino acid at position 3is CGC; (xlv) the sequence of the codon which encodes the amino acid at position 3is AUC; (xlvi) the sequence of the codon which encodes the amino acid at position 3is UCC; (xlvii) the sequence of the codon which encodes the amino acid at position 360 is AAC; (xlviii) the sequence of the codon which encodes the amino acid at position 3is UGU; (xlix) the sequence of the codon which encodes the amino acid at position 3is GUC; (l) the sequence of the codon which encodes the amino acid at position 3is GCU; (li) the sequence of the codon which encodes the amino acid at position 3is GAU; (lii) the sequence of the codon which encodes the amino acid at position 3is UAU; (liii) the sequence of the codon which encodes the amino acid at position 366 is UCC; (liv) the sequence of the codon which encodes the amino acid at position 3is GUC; (lv) the sequence of the codon which encodes the amino acid at position 3is CUC; (lvi) the sequence of the codon which encodes the amino acid at position 3is UAU; (lvii) the sequence of the codon which encodes the amino acid at position 3is AAU; (lviii) the sequence of the codon which encodes the amino acid at position 371 is UCC; (lix) the sequence of the codon which encodes the amino acid at position 3is GCC; (lx) the sequence of the codon which encodes the amino acid at position 3is UCC; (lxi) the sequence of the codon which encodes the amino acid at position 3is UUU; (lxii) the sequence of the codon which encodes the amino acid at position 3is UCC; (lxiii) the sequence of the codon which encodes the amino acid at position 376 is ACC; (lxiv) the sequence of the codon which encodes the amino acid at position 3is UUU; (lxv) the sequence of the codon which encodes the amino acid at position 5is UUU; (lxvi) the sequence of the codon which encodes the amino acid at position 5is GGC; (lxvii) the sequence of the codon which encodes the amino acid at position 5is CGC; (lxviii) the sequence of the codon which encodes the amino acid at position 5is GAC (lxix) the sequence of the codon which encodes the amino acid at position 569 is AUC; (lxx) the sequence of the codon which encodes the amino acid at position 5is GCC; (lxxi) the sequence of the codon which encodes the amino acid at position 5is GAC; (lxxii) the sequence of the codon which encodes the amino acid at position 5is ACC; (lxxiii) the sequence of the codon which encodes the amino acid at position 5is ACC; (lxxiv) the sequence of the codon which encodes the amino acid at position 574 is GAC; (lxxv) the sequence of the codon which encodes the amino acid at position 5is GCC; (lxxvi) the sequence of the codon which encodes the amino acid at position 5is GUC; (lxxvii) the sequence of the codon which encodes the amino acid at position 5is CGC; (lxxviii) the sequence of the codon which encodes the amino acid at position 5is GAC; (lxxix) the sequence of the codon which encodes the amino acid at position 579 is UUU; (lxxx) the sequence of the codon which encodes the amino acid at position 8is UAC; (lxxxi) the sequence of the codon which encodes the amino acid at position 8is GGC; (lxxxii) the sequence of the codon which encodes the amino acid at position 8is GAC; (lxxxiii) the sequence of the codon which encodes the amino acid at position 8is UGC; (lxxxiv) the sequence of the codon which encodes the amino acid at position 8is CUC; (lxxxv) the sequence of the codon which encodes the amino acid at position 842 is GGC; (lxxxvi) the sequence of the codon which encodes the amino acid at position 8is GAC; (lxxxvii) the sequence of the codon which encodes the amino acid at position 8is AUC; (lxxxviii) the sequence of the codon which encodes the amino acid at position 8is GCC; (lxxxix) the sequence of the codon which encodes the amino acid at position 8is GCC; (xc) the sequence of the codon which encodes the amino acid at position 847 is CGC; (xci) the sequence of the codon which encodes the amino acid at position 8is GAC; (xcii) the sequence of the codon which encodes the amino acid at position 8is CUC; (xciii) the sequence of the codon which encodes the amino acid at position 8is AUC; (xciv) the sequence of the codon which encodes the amino acid at position 8is UGU; (xcv) the sequence of the codon which encodes the amino acid at position 852 is GCC; (xcvi) the sequence of the codon which encodes the amino acid at position 12is CUC; (xcvii) the sequence of the codon which encodes the amino acid at position 12is AUC; (xcviii) the sequence of the codon which encodes the amino acid at position 12is GCC; (xcix) the sequence of the codon which encodes the amino acid at position 12is AUC; (c) the sequence of the codon which encodes the amino acid at position 12is GUC; (ci) the sequence of the codon which encodes the amino acid at position 1229 is AUG; (cii) the sequence of the codon which encodes the amino acid at position 12is GUC; (ciii) the sequence of the codon which encodes the amino acid at position 12is ACC; (civ) the sequence of the codon which encodes the amino acid at position 12is AUC; (cv) the sequence of the codon which encodes the amino acid at position 12is AUG; (cvi) the sequence of the codon which encodes the amino acid at position 1234 is CUG; Particular sequences of heterologous SECReTE sequences which can be included in the mRNA encoding the spike protein of SARS-CoV2 vaccine include: AUGUUUGUUUUUCUUGUUCUCCUCCCCCUCGUCUCUUCU (SEQ ID NO: 10); UUUGAUAACCCUGUCCUCCCCUUUAACGACGGCGUUUACUUUGCCUCCACC (SEQ ID NO: 11); UCCCUCCUCAUCGUCAACAACGCCACCAACGUCGUCAUC (SEQ ID NO: 12); CGCAUCUCCAACUGUGUCGCUGAUUAUUCCGUCCUCUAUAAUUCC GCCUCCUUUUCCACCUUU (SEQ ID NO: 13); UUUGGCCGCGACAUCGCCGACACCACCGACGCCGUCCGCGACCCC (SEQ ID NO: 14); UACGGCGACUGCCUCGGCGACAUCGCCGCCCGCGACCUCAUCUGUGCC (SEQ ID NO: 15); and CCUCAUCGCCAUCGUCAUGGUCACCAUCAUGCUC (SEQ ID NO: 16).
In one embodiment, the nucleic acid sequence of the mRNA encoding the spike protein of SARS-CoV2 comprises the sequence as set forth in SEQ ID NO: 27 or SEQ ID NO: 28. Upon selection of an optimized nucleic acid sequence which incorporates a heterologous SECReTE sequence, the nucleic acid sequence may then be synthesized (in order to prepare the vaccine). Thus, according to another aspect of the present invention there is provided a method of generating a nucleic acid based vaccine for the treatment or prevention of a disease comprising: (a) selecting a sequence of a mRNA encoding a protein of a pathogen which comprises a heterologous ER targeting sequence as set forth in SEQ ID NO: 8; and (b) synthesizing mRNA comprising said sequence, thereby generating the mRNA based vaccine. The optimized nucleic acid sequence may be in vitro transcribed from an optimized DNA template to generate an RNA based vaccine or may be chemically synthesized. Defined chemical synthesis of an optimized mRNA as described herein, in the 3′->5′ direction is well established in prior art. The technology utilizes a ribonucleoside with suitable N-protecting group: generally 5T-Protecting group, the most popular being dimethoxytriphenyl, i.e. the DMT group; T-protecting group, out of which most popular is t-Butyldimethylsilyl ether; and, a 3 ′-phosphoramidite, the most popular of which is cyanoethyl diisopropyl (component 1). This component is then coupled with a nucleoside with a suitable N-protecting group, 2 ′ or 3′ succinate of a ribonucleoside attached to a solid support (component 2). The coupling of component 1 and 5 ′-OH-n-protected -2 ′,3′-protected-nucleoside (component 3) are also achieved in solution phase in presence of an activator leading to dimers and oligoribonucleotides, followed by oxidation (3 ′->5′ direction synthesis), also leads to a protected dinucleotide having a 3 ′-5′-internucleotide linkage (Ogilvie, K. K., Can. J. Chem., 58, 2686, 1980). Other technologies for chemical RNA synthesis, i.e. for the synthesis of the modified RNA according to the invention are known in prior art, such as, e.g. the method disclosed in US 20110275793 A1.
In another embodiment, the optimized mRNA of the present invention may be obtained by in vitro transcription, e.g. by bacteriophage-mediated in vitro transcription, preferably by Sp6 polymerase in vitro transcription and/or T3 polymerase-mediated in vitro transcription, more preferably by T7 polymerase-mediated in vitro transcription. Highly efficient in vitro transcription systems, particularly ones using phage, are known in the art and include polymerases such as T7, SP6, and T3. The DNA-dependent phage T7, T3, and SP6 RNA polymerases are widely used to synthesize a large quantity of RNAs. These enzymes are highly processive and are thus capable of generating long RNA molecules of up to thousands of nucleotides in length with low probability of falling off DNA templates during transcription and may thus be used for in vitro transcription in the present invention. Phage RNA polymerases specifically recognize their 18-bp promoter sequences (T7, 5 ′-TAATACGACTCACTATAG (SEQ ID NO: 5); T3, 5 ′-AATTAACCCTCACTAAAG (SEQ ID NO: 3); and SP6, 5 ′-ATTTAGGTGACACTATAG (SEQ ID NO: 4)) and initiate transcription precisely at the 18th nucleotide guanosine. With a T7, T3, or SP6 promoter fused to the 5 ′ end of a DNA template, the transcription reaction is expected to generate an RNA molecule with the predicted sequence. In this method, a DNA molecule corresponding to the optimized mRNA of the present invention is transcribed in vitro for the production of the mRNA. This DNA matrix has a suitable promoter, for example a T7 and/or SP6 and/or T3 promoter, for the in vitro transcription, followed by the desired nucleotide sequence for the mRNA to be produced and a termination signal for the in vitro transcription. According to the invention the DNA molecule that forms the matrix of the RNA construct to be produced, such as e.g. the optimized mRNA according to the present invention is prepared by fermentative replication and subsequent isolation as part of a plasmid replicable in bacteria. Suitable plasmids for in vitro transcription of the mRNA molecules according to the present invention are known in the art and are commercially available. For example the following plasmids may be mentioned as examples pT7Ts (GeneBank Accession No. U26404), the pGEM® series, for example pGEM®-1 (GeneBank Accession No. X65300) and pSP64 (GeneBank-Accession No. X65327); see also Mezei and Storts, Purification of PCR Products, in: Griffin and Griffin (Eds.), PCR Technology: Current Innovation, CRC Press, Boca Raton, Fla., 2001. The in vitro transcription of the mRNA according to the present invention may also include ribonucleoside triphosphates (rNTPs) analogues, such as those, e.g. required for 5 ′ capping of the in vitro transcribed mRNA according to the invention. According to a particular embodiment, the optimized mRNA that is obtainable by the method according to the present invention may be synthesized by in vitro transcription including naturally occurring rNTP analogues, for example 5-methyl-cytidine triphosphate and/or pseudouridine triphosphate. Thus, in this embodiment, the mRNAs of the vaccines comprise at least one N1-Methylpseudouridine. In another embodiment, all the uridines of the mRNA are replaced with N1-Methylpseudouridine. The term "ribonucleoside triphosphate analogues" as used herein refers to ribonucleoside triphosphate compounds comprising a chemical modification, wherein the chemical modification may comprise a backbone modification, a sugar modification, or a base modification. These ribonucleoside triphosphate analogues are also termed herein as modified nucleoside triphosphates, modified ribonucleosides or modified nucleosides. In this context, the modified nucleoside triphosphates as defined herein are nucleotide analogs/modifications, e.g. backbone modifications, sugar modifications or base modifications. A backbone modification in the context of the present invention is a modification, in which phosphates of the backbone of the nucleotides are chemically modified. A sugar modification in the context of the present invention is a chemical modification of the sugar of the nucleotides. Furthermore, a base modification in in the context of the present invention is a chemical modification of the base moiety of the nucleotides. In this context nucleotide analogs or modifications are preferably selected from nucleotide analogs, which are applicable for transcription and/or translation. 5′ Capping The 5 ′ cap structure of an mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5 ′ proximal introns removal during mRNA splicing. mRNA molecules which have been optimized according to embodiments described herein may be 5 ′-end capped generating a 5 ′-ppp-5′-triphosphate linkage between a terminal guanosine cap residue and the 5 ′-terminal transcribed sense nucleotide of the mRNA molecule. This 5 ′-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5 ′ end of the mRNA may optionally also be 2′-O-methylated. 5 ′-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation. Modifications to mRNAs of the present invention may generate a non- hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5 ′-ppp-5′ phosphorodiester linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, Mass.) may be used with α-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5 ′-ppp-5 ′ cap. Additional modified guanosine nucleotides may be used such as α-methyl-phosphonate and seleno-phosphate nucleotides. Additional modifications include, but are not limited to, 2 ′-O-methylation of the ribose sugars of 5 ′-terminal and/or 5 ′-anteterminal nucleotides of the mRNA (as mentioned above) on the 2 ′-hydroxyl group of the sugar ring. Multiple distinct 5 ′-cap structures can be used to generate the 5 ′-cap of a nucleic acid molecule, such as an mRNA molecule. Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e. endogenous, wild-type or physiological) 5 ′-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e. non-enzymatically) or enzymatically synthesized and/or linked to a nucleic acid molecule. According to the present invention, 5 ′ terminal caps may include endogenous caps or cap analogs. According to the present invention, a 5 ′ terminal cap may comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, N1- methyl-guanosine, 2 ′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. Thus, the mRNA which has been optimized according to embodiments of the present invention may also undergo capping and/or tailing reactions. A capping reaction may be performed by methods known in the art to add a 5 ′ cap to the 5 ′ end of the primary construct. Methods for capping include, but are not limited to, using a Vaccinia Capping enzyme (New England Biolabs, Ipswich, Mass.). Additional examples of ribonucleoside triphosphate analogues and other modifications that can be made to the RNA nucleic acid molecules are disclosed in 11,034,729, 10,064,959, 9868692, 10,577,403 and 10,703,789, the contents of which are incorporated herein by reference. IRES sequencesThe mRNA molecules may also be synthesized such that they contain an internal ribosome entry site (IRES). First identified as a feature Picorna virus RNA, IRES plays an important role in initiating protein synthesis in absence of the 5 ′ cap structure. An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. Polynucleotides, primary constructs or mmRNA containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes ("multicistronic nucleic acid molecules"). Examples of IRES sequences that can be used according to the invention include without limitation, those from picornaviruses (e.g. FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV). Poly-A TailsDuring RNA processing, a long chain of adenine nucleotides (poly-A tail) may be added to a polynucleotide such as an mRNA molecules in order to increase stability. Immediately after transcription, the 3 ′ end of the transcript may be cleaved to free a 3 ′ hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A tail that can be between, for example, approximately 100 and 250 residues long.
Unique poly-A tail lengths may provide certain advantages to the polynucleotides of the present invention. Generally, the length of a poly-A tail of mRNAs which have been optimized according to embodiments described herein is greater than 30 nucleotides in length. In another embodiment, the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the nucleic acid molecules include from about 30 to about 3,0nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 1to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000). In one embodiment, the poly-A tail is designed relative to the length of the overall mRNA. This design may be based on the length of the coding region, the length of a particular feature or region (such as the first or flanking regions), or based on the length of the ultimate product expressed from the mRNA. A poly-A tailing reaction may be performed by methods known in the art, such as, but not limited to, 2′ O-methyltransferase and by methods as described herein. If the primary construct generated from cDNA does not include a poly-T, it may be beneficial to perform the poly-A-tailing reaction before the primary construct is cleaned. Following synthesis of the optimized nucleic acid, the nucleic acid may be purified. mRNA purification may include, but is not limited to, mRNA clean-up, quality assurance and quality control. mRNA clean-up may be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, Mass.), poly-T beads, LNA™ oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term "purified" when used in relation to a nucleic acid such as a "purified mRNA" refers to one that is separated from at least one contaminant. As used herein, a "contaminant" is any substance which makes another unfit, impure or inferior. Thus, a purified polynucleotide (e.g., DNA and RNA) is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment or purification method. A quality assurance and/or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC. In another embodiment, the mRNA may be sequenced by methods including, but not limited to reverse-transcriptase-PCR. In one embodiment, the mRNA may be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis). A non-limiting example of a UV/Vis spectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, Mass.). The quantified mRNA may be analyzed in order to determine if the mRNA may be of proper size, check that no degradation of the mRNA has occurred. Degradation of the mRNA may be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE). Once synthesized the mRNA is typically formulated with an immunologically acceptable carrier. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the mRNA is combined to facilitate administration. According to a particular embodiment, the carrier comprises a particle - e.g. a lipid particle.
Thus, the mRNAs of the invention can be formulated using one or more lipidoids, liposomes, lipoplexes, or lipid nanoparticles. Further details on these formulations are provided in 11,034,729, 10,064,959, 9868692, 10,577,403 and 10,703,789, the contents of which are incorporated herein by reference. According to a particular embodiment, the vaccines of the present invention do not comprise adjuvants (e.g. aluminium, saponin derived substances etc.). The nucleic acid in the vaccine can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation of the nucleic acid; (4) alter the biodistribution to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein in vivo. In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients of the present invention can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the nucleic acid, hyaluronidase, nanoparticle mimics and combinations thereof. Accordingly, the formulations of the invention can include one or more excipients, each in an amount that together increases the stability of the nucleic acid, increases the expression of polynucleotide, and/or alters the release profile of the mRNA encoded proteins. Formulations of the mRNA vaccines described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of associating the mRNA with an excipient and/or one or more other accessory ingredients. The mRNA vaccine in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a "unit dose" refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the mRNA. The amount of the mRNA may generally be equal to the dosage of the mRNA which would be administered to a subject and/or a convenient fraction of such a dosage including, but not limited to, one-half or one-third of such a dosage.
Relative amounts of the mRNA, the pharmaceutically acceptable excipient, and/or any additional ingredients in the vaccine in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1% and 99% (w/w) of the mRNA. In some embodiments, the formulations described herein may contain at least one mRNA. As a non-limiting example, the formulations may contain 1, 2, 3, 4 or mRNA. In one embodiment the formulation may contain modified mRNA encoding proteins selected from categories such as, but not limited to, human proteins, veterinary proteins, bacterial proteins, viral proteins, biological proteins, antibodies, immunogenic proteins, therapeutic peptides and proteins, secreted proteins, plasma membrane proteins, cytoplasmic and cytoskeletal proteins, intracellular membrane bound proteins, nuclear proteins, proteins associated with human disease and/or proteins associated with non-human diseases. In one embodiment, the formulation contains at least three modified mRNA encoding proteins. In one embodiment, the formulation contains at least five modified mRNA encoding proteins. In one embodiment, the vaccine comprises an excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia. Pharmaceutically acceptable excipients used in the manufacture of vaccines include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical compositions. Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof. Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof. Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEENn®60], polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ®45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [BRIJ®30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLUORINC® F 68, POLOXAMER®188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof. Exemplary binding agents include, but are not limited to, starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and combinations thereof. Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal. Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL®115, GERMABEN® II, NEOLONE™, KATHON™, and/or EUXYL®. Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and/or combinations thereof. Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof. Exemplary oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinations thereof. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator. The vaccine of the present invention may be administered by any route which results in a therapeutically effective outcome. These include, but are not limited to enteral, gastroenterol, epidural, oral, transdermal, epidural (peridural), intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection, (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), or in ear drops. In specific embodiments, compositions may be administered in a way which allows them cross the blood-brain barrier, vascular barrier, or other epithelial barrier. In a particular embodiment, the vaccine is administered intramuscularly. It is expected that during the life of a patent maturing from this application many relevant vaccines will be developed and the scope of the term vaccine is intended to include all such new technologies a priori. As used herein the term "about" refers to  10 % The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". The term "consisting of" means "including and limited to". The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure. As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof. Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween. As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. As used herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition. When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements. Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLESReference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion. Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques which are believed to be well known in the art. MATERIALS AND METHODS Plasmid construction Plasmid for humanized Spike (SEQ ID NO: 19) was obtained from Nevan Krogan (University of California San Francisco). Sequences for Wuhan-1, Max, and No Spike (SEQ ID No: 17, 21, 25) were ordered from Twist Bioscience and cloned by the company into pTwist-EF1alpha plasmid. For the prefolding mutation (2P), site directed mutagenesis was performed using PCR with primers 5’ ACCCACCTGAAGCCGAAGTCCAAATAG (SEQ ID NO: 36) 3’ and 5’ TCAGGTGGGTCGAGTCGGGAAAGAATATC 3’ (SEQ ID NO: 37) for the humanized Spike and 5’ ACCCACCTGAGGCTGAAGTGCAAATTG 3’ (SEQ ID NO: 38) and 5’ CAGGTGGGTCAAGACGTGAAAGG 3’ (SEQ ID NO: 39) for the Wuhan-1, maximized and No SECReTE variants. PCR program: 98 °C 3min, 18 cycles of 98 °C 20sec, 52 °C 20sec, 72 °C 10 min, 1 cycle of 72 °C 15min. PCR products were treated with DpnI (NEB) for 1 hour, 37 °C and 5 μl of the reaction was used for transformation to DH5 α E. coli cells. single colonies were picked, plasmid was purified using Wizard®Plus SV Minipreps DNA Purification System (Promega) according to manufacturer instructions and the correct Spike-2P gene was verified by Sanger sequencing at the Weizmann Institute of Science DNA sequencing unit.
Cell culture MCF7 cells were cultured in DMEM (high glucose), supplemented with 10% Fetal Bovine Serum (FBS), 1 mM sodium pyruvate and antibiotics (0.1 mg/mL streptomycin and 10 U/mL penicillin) at 37 °C with 5% CO2. The cells were routinely cultured in 10 cm dishes. For the experiments in Figure 1B-C, cells were plated in 12- well plated (approximately 100,000 cells per well) on fibronectin-coated glass coverslips. On the next day, cells in each well were transfected with 1 μg DNA of a different Spike variant containing plasmid. Transfection was performed with Jetprime (Polyplus) transfection reagent according to manufacturer’s instructions. Immunofluorescence For Figure 1B, 28 hours after transfection, the cells were fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature, washed twice in PBS, blocked for 30 minutes with 3% BSA in PBS. Coverslips were incubated with a Rabbit anti-spike antibody (Abcam ab272504; 1:1000) (4 °C over-night), washed 2 times with PBS, blocked again for 15 minutes with 3% BSA and then stained with a secondary antibody goat anti-Rabbit IgG-alexa647 1:1000 (1 hour, 37°C). Samples were washed twices with PBS, stained with DAPI, washed once and mounted on slides using Prolong glass anti-fade (Invitrogen). Samples were imaged on a Zeiss AxioObserver ZDuoLink dual camera imaging system with 63x objective. 0.5 μm step z-stack images were taken. Maximum projection images of the z-stacks were created by FIJI, and the resulting images were automatically analyzed for Alexa647 fluorescence intensity using Cell Profiler. For Figure 1C, 30 hours after transfection, the cells were fixed with 4% paraformaldehyde in PBS, washed twice in PBS, permeabilized with 0.1% Triton X-100 in PBS (10 minutes room temperature), blocked for 30 minutes with 3% BSA, and stained with a mouse anti-FLAG (M2) 1:200 (Sigma F3165) (2 hours, room temperature), washed and blocked as described for panel B, and stained with a secondary antibody goat anti-mouse IgG-alexa488 1:400 (1 hour, room temperature) followed by wash, DAPI stin and mounting as described for panel B. Samples were imaged on the Zeiss AxioObserver Z1 with a 100x objective.

Claims (24)

1.WHAT IS CLAIMED IS: 1. A vaccine comprising an immunologically acceptable carrier and a mRNA encoding a protein, or a fragment thereof, of a pathogen, wherein said mRNA comprises at least one heterologous ER targeting sequence as set forth in SEQ ID NO: 8, wherein said mRNA does not comprise the sequence as set forth in SEQ ID NO: 6.
2. The vaccine of claim 1, wherein said at least one heterologous ER targeting sequence as set forth in SEQ ID NO:
3. The vaccine of claims 1 or 2, wherein said mRNA comprises a chemically modified nucleotide.
4. The vaccine of claims 1 or 2, wherein said mRNA comprises a non-natural cap.
5. The vaccine of any one of claims 1-4, wherein said mRNA is encapsulated in a particle.
6. The vaccine of claim 5, wherein said particle comprises lipids.
7. The vaccine of any one of claims 1-6, wherein said pathogen is a virus.
8. The vaccine of any one of claims 1-7, wherein said mRNA is codon optimized for expression in human cells.
9. The vaccine of any one of claims 1-8, wherein said ER targeting sequence does not comprise more than 5 consecutive repeats of the sequence TG.
10. The vaccine of any one of claims 1-9, wherein said ER targeting sequence comprises at least 15 consecutive repeats of the sequence NNY, wherein N is any base and Y is a pyrimidine.
11. The vaccine of any one of claims 1-10, wherein said ER targeting sequence does not comprise more than 10 consecutive thymines.
12. The vaccine of any one of claims 1-11, wherein said mRNA further encodes a signal peptide sequence.
13. The vaccine of claim 12, wherein said signal peptide sequence is heterologous to said protein.
14. The vaccine of claim 12, wherein said signal peptide sequence comprises at least one heterologous ER targeting sequence as set forth in SEQ ID NO: 8.
15. The vaccine of any one of claims 1-14, wherein said protein is of a virus selected from the group consisting of a coronavirus, an influenza virus, cytomegalovirus (CMV), human immunodeficiency virus (HIV-1), rabies virus, measles virus, chickenpox virus, Respiratory syncytial virus (RSV), Epstein-Barr virus (EBV) and a Zika virus.
16. The vaccine of any one of claims 1-15, wherein said protein is of a coronavirus.
17. The vaccine of claim 16, wherein said coronavirus is SARS-CoV2.
18. The vaccine of claim 16, wherein said protein is a spike protein.
19. The vaccine of claim 18, wherein said spike protein comprises a sequence as set forth in SEQ ID NOs: 10, 11, 12, 13, 14, 15 or 16.
20. The vaccine of any one of claims 1-17, wherein said protein is a membrane protein.
21. The vaccine of any one of claims 1-17, wherein said protein is a secreted protein.
22. A method of generating the vaccine of claims 1-21, comprising: (a) selecting a sequence of a mRNA encoding a protein of a pathogen which comprises a heterologous ER targeting sequence as set forth in SEQ ID NO: 8; and (b) synthesizing mRNA comprising said sequence, thereby generating the mRNA based vaccine.
23. The method of claim 22, further comprising encapsulating said mRNA with a carrier.
24. The method of claim 23, wherein said carrier comprises a lipid particle. Dr. Hadassa Waterman Patent Attorney G.E. Ehrlich (1995) Ltd. 11 Menachem Begin Road 5268104 Ramat Gan
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US10577403B2 (en) * 2012-04-02 2020-03-03 Modernatx, Inc. Modified polynucleotides for the production of secreted proteins
WO2019150373A1 (en) * 2018-01-31 2019-08-08 Yeda Research And Development Co. Ltd. Endoplasmic reticulum targeting signal
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