mRNA VACCINE

FIELD

The present specification relates to vaccines. In particular, the specification relates to vaccines comprising an mRNA polynucleotide encoding an antigen from an infectious microorganism, particularly an HPV antigen, and an amphipathic cell penetrating peptide from the RALA family of peptides. The specification further relates to methods of preparing such vaccines and to their use in therapy.

BACKGROUND

Vaccines involve introducing antigens—a substance that the immune system will attack—either directly or indirectly into the body, typically via injection. Vaccines fall into two broad categories: prophylactic and therapeutic. Prophylactic vaccines are designed to build immunity—the antigens provoke the body's immune system to create antibodies and a cellular response to protect against future infection and/or disease. Therapeutic vaccines are designed to help the body do a better job of fighting an infection and/or disease it already has.

To produce a therapeutic vaccine, a particular type of immune response, known as a CD8+ response, is required. CD8+ T cells are cytotoxic T cells of the immune system. They are provoked on encountering a pathogenic stimulus, and kill cells presenting foreign immunogenic antigen fragments. CD8+ T-cells are a critical component of the cellular immune response and play an important role in the control of viral infection. CD8+ T-cells are able to recognize and destroy infected cells, as well as suppress viral binding and transcription. A CD8+ mediated response is considered essential to treat active, latent, and persistent viral pathogens (for example Cosma et al., 2018 Apr. 27; 7: F1000 Faculty Rev-508, Beura et al., Immunity, 2018 Feb. 20; 48 (2): 327-338.e5, and McMichael, Cold Spring Harb. Perspect. Biol., 2018 Sep. 4; 10 (9): a029124), and requires specific delivery of the antigen into the antigen presenting cells (Gessani et al., Toxins (Basel), 2014 May 26; 6 (6): 1696-723). Current antigenic protein vaccines delivered via intramuscular injection utilise a CD4+ mediated approach with adjuvants, to produce either a cytolytic CD4+ immune responses or antigen cross-presentation to elicit a CD8+ response. CD4+ T cells are “helper” cells, they regulate the immune response to a particular antigen and operate more indirectly than CD8+ T-cells. A vaccine provoking a CD8+ dominated response has remained elusive due to the triggering of innate, cellular immunity, and an inability to deliver the antigen to the right location within the right immune cells.

There are several types of vaccines, including inactivated vaccines; live-attenuated vaccines; messenger ribonucleic acid (mRNA) vaccines; subunit, recombinant, polysaccharide, and conjugate vaccines; toxoid vaccines; and viral vector vaccines. These different vaccines introduce the antigen into the body in different ways.

mRNA vaccines convey instructions to cells to make antigens, which in turn provoke an immune response. The mRNA selected encodes for an antigen of the infection and/or disease to be treated. mRNA vaccines have several benefits compared to other types of vaccines including shorter manufacturing times, and since mRNA cannot be incorporated into the cellular genome, they carry no risk of causing the disease in the person getting vaccinated. However, unmodified synthetic mRNA is not stable, and the mRNA itself can stimulate the activation of an unwanted (e.g. CD4+) immune response. Chemical modifications on the ribose, the RNA termini, and nucleobases may therefore be required to improve stability and reduce immunogenicity (see for example Gao et al., Acta. Biomater. 2021 Sep. 1; 131:1-15).

Human papilloma virus (HPV) is the most common sexually transmitted disease, with an estimated 200 million people infected worldwide (WHO Global HPV Market Study 2018). Infection with HPV can lead to genital warts, precancerous lesions and cancer. Most HPV infections are subclinical and more than 90 percent of all new HPV infections are undetectable within two years, even without treatment. Unfortunately, some infections persist and lead to complications, and as an etiological agent, HPV is responsible for 5% of all cancers worldwide, including 99% of all cervical cancers and 60% of all cutaneous non-melanoma cancers such as basal cell and squamous cell (de Martel et al., New Microbiol. 2017 April; 40 (2): 80-85 and Brianti et al., Int J Cancer. 2017 Aug. 15; 141 (4): 664-670). HPV induced cervical cancer is the second most common cause of death in females after breast cancer.

Of the more than 150 strains of HPV, 40 affect the genital area, but most don't pose a serious risk to health. A person can be infected with more than one HPV strain at a time, and strains are identified by number. HPV strains are categorised as low-risk HPV or high-risk HPV. Infections with most low-risk HPV strains are asymptomatic, and these strains are not associated with cancer. Infections with high-risk HPV are more problematic, and can lead to cervical dysplasia and certain types of cancer. There are at least 12 high-risk strains of HPV, including HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 52, HPV 56, HPV 58, and HPV 59, of which two-HPV 16 and HPV 18-cause the majority of HPV-related cancers.

Current HPV vaccines are prophylactic vaccines, designed to be administered to 11-14-year-olds to prevent infection (Cook et al., Pediatrics. 2018 September; 142 (3)). These vaccines have no effect on those already infected with HPV, and even if prophylactic coverage was universal, it would take at least 20 more years for it to translate into significant reduction of HPV-related morbidity (Kumar et al., Med J Armed Forces India, 2015 April; 71 (2): 171-7). Meanwhile, incidence rates for cervical cancer are predicted to rise by 43% in the UK alone, with 17 new cases per 100,000 females by 2035 (British Gynaecological Cancer Society (BGCS) Cervical Cancer Guidelines: Recommendations for Practice). There is clearly a need for a therapeutic vaccine, or vaccine capable of acting both therapeutically and prophylactically, to address existing HPV, particularly HPV 16 and HPV 18, infection.

There are several known HPV proteins (L1, L2, E1, E2, E4, E5, E6 and E7) that can function as antigens in a vaccination approach for the treatment of HPV infection. Prophylactic vaccines on the market utilize the major capsid (viral surface) protein L1 (Kirnbauer et al., Proc. Natl. Acad. Sci. USA, 1992 Dec. 15; 89 (24), and Schiller et al., Vaccine, 2018 Aug. 6; 36 (32 Pt A): 4768-4773). However, L1 (and L2) proteins get inactivated or deleted during the integration of the HPV genome into the cellular genome, and as a consequence, vaccines employing L1 and L2 antigens are ineffective against an HPV infection once it is established. The early proteins E1, E2, E4, E5, E6 and E7 are expressed for a longer period in the life cycle of HPV and represent a better target for therapeutic vaccines, even for advanced HPV infection (Pal et al., Front Microbiol. 2019; 10:3116).

The antigen delivery system employed in mRNA vaccination, plays a key role in determining the type of immune response. The ratio of carrier:mRNA also plays a role-mRNA molecules are negatively charged polynucleotides and the carrier needs both to protect the cargo from degradation as well as deliver it to the correct target cells. Optimisation of the carrier:mRNA ratio facilitates production of nanoparticles with suitable size and charge characteristics to ensure uptake by antigen-presenting cells.

The RALA family of peptides are amphipathic peptides composed of repeating RALA units that are capable of overcoming biological barriers to gene delivery, both in vitro and in vivo. The term “RALA” has been used inconsistently in the literature, but typically refers to an amphipathic peptide or group of peptides composed of repeating RALA units generally of less than approximately 50 amino acid residues. Cohen-Avrahami et al. (J. Phys. Chem. B 2011, 115:10 189-1 097 and Colloids and Surfaces B: Biointerfaces 77 (2010) 131-138) disclose an amphipathic 16-mer peptide referred to as “RALA”. Faranack et al., (Biomacromolecules 2013, 14, 2033-2040) use the term “RALA” to describe a 30-mer RALA peptide, as does Mccarthy et al. (Journal of Controlled Release 189 (2014) 141-149) but for a different 30-mer peptide. WO 2014/087023 and WO 2015/189205 defined the term “RALA” as a generic term for a group of peptides falling within the scope of the invention as described therein.

The RALA family of peptides have been used to deliver genetic material such as plasmid DNA (McCarthy H O et al. J Control Release, 2014 Sep. 10; 189:141-9; and Ali A A et al., Nanomedicine, 2017 April; 13 (3): 921-932), mRNA (Udhayakumar et al., Adv. Healthc. Mater. 2017 July; 6 (13)), siRNA (Mulholland E. J. et al., J Control Release, 2019 Dec. 28; 316:53-65), and small molecules such as bisphosphonates (Jena L N et al., J. Nanobiotechnology. 2021 May 4; 19 (1): 127), and calcium phosphates (Sathy B. N. et al., J. Mater. Chem. B. 2017 Mar. 7; 5 (9): 1753-1764).

The specific RALA peptide WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) is a 30 amino acid, non-toxic, peptide with a +6 electric charge at a physiological pH that converts to a +8 helical cell penetrating conformation at acidic conditions found inside the endosome of a cell. When complexed with certain payloads, e.g. DNA and mRNA, in water it has been shown to be capable of spontaneous self-assembly into nanoparticles (McCarthy et al. 2014 and Udhayakumar et al. 2017). This pH dependent change allows for escape of the peptide and the cargo within a cell, resulting in highly efficiency cellular entry and cargo delivery, without any associated toxicity at the physiological pH of 7.4 outside the cell. The nanoparticles have been shown to be extremely stable at a range of temperatures and over time, and RALA peptides do not themselves provoke an immunological response.

Udhayakumar et al. (Udhayakumar et al. 2017) evaluated the capacity of this RALA peptide in mRNA nanocomplexes to prime CD8+ T cells by immunising mice with RALA complexed mRNA encoding the model antigen ovalbumin (the main protein found in egg white). Udhayakumar et al. concluded that multiple different mRNA nucleotide modifications and a very high N/P ratio (i.e. ratio of positively charged nitrogen atoms in the peptide to negatively charged phosphates in the mRNA backbone) were needed for optimum complexation of Ova mRNA and to enable efficient CD8+ T cell priming.

Ali et al. (Ali et al. 2017) investigated a DNA vaccination for cervical cancer; delivering RALA condensed E6/E7 plasmid DNA nanoparticles via polymeric microneedles. However, in order for DNA vaccines to work, the cargo must reach the nucleus, be transcribed to mRNA, and the protein translated. mRNA vaccines remove the transcription step, and the need for the nucleic acid to reach the target cell nucleus.

Coolen et al (Biomaterials, vol. 195, 2019 al., p. 23-37) investigated the ability of poly(lactic acid) nanoparticles coupled with cell-penetrating peptides to potentiate mRNA-based vaccine expression in dendritic cells triggering their activation. However, a RALA peptide complexed with eGFP (Green Fluorescent Protein) failed to efficiently transfect DC2.4 dendritic cells and was not selected for further evaluation.

The present specification describes vaccines comprising an mRNA polynucleotide encoding an antigen from an infectious microorganism, particularly an HPV antigen, and a specific RALA peptide. These vaccines result in intracellular delivery of the mRNA cargo, protecting it from degradation, and do not provoke an innate (CD4+) immune response. Furthermore, the RALA complexed mRNA nanoparticles can be readily lyophilized (retaining functionality), are stable and readily reconstituted, do not require cold chain storage, and are relatively inexpensive to manufacture.

SUMMARY

This specification describes, in part, a vaccine, comprising:

- i) an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof; and
- ii) an amphipathic cell penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) or a sequence with at least 80% sequence identity or homology.

This specification also describes, in part, vaccines as described herein for use in therapy, particularly for use in the treatment of viral infections and in the treatment of cancer.

DETAILED DESCRIPTION OF THE INVENTION

Many embodiments of the invention are detailed throughout the specification and will be apparent to a reader skilled in the art. The invention is not to be interpreted as being limited to any of the recited embodiments.

“A” (or “an”) means “at least one”. In any embodiment where “a” is used to denote a given material or element, “a” may mean one.

“Comprising” means that a given material or element may contain other materials or elements. In any embodiment where “comprising” is mentioned the given material or element may be formed of at least 10% w/w, at least 20% w/w, at least 30% w/w, or at least 40% w/w of the material or element. In any embodiment where “comprising” is mentioned, “comprising” may also mean “consisting of” (or “consists of”) or “consisting essentially of” (or “consists essentially of”) a given material or element.

“Consisting of” or “consists of” means that a given material or element is formed entirely of the material or element. In any embodiment where “consisting of” or “consists of” is mentioned, the given material or element may be formed of 100% w/w of the material or element.

“Consisting essentially of” or “consists essentially of” means that a given material or element consists almost entirely of that material or element. In any embodiment where “consisting essentially of” or “consists essentially of” is mentioned the given material or element may be formed of at least 50% w/w, at least 60% w/w, at least 70% w/w, at least 80% w/w, at least 90% w/w, at least 95% w/w or at least 99% w/w of the material or element.

In any embodiment where “is” or “may be” is used to define a material or element, “is” or “may be” may mean the material or element “consists of” or “consists essentially of” the material or element.

Embodiments may be combined.

Claims are embodiments.

mRNA polynucleotide

A polynucleotide is a compound and/or substance that comprises a polymer of nucleotides (nucleotide monomers). The polynucleotides of the present disclosure function as messenger RNA (mRNA). “Messenger RNA” refers to any polynucleotide that encodes a polypeptide (both naturally-occurring (wild-type) and non-naturally-occurring) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo.

The basic components of an mRNA molecule typically include at least one open reading frame, a 5′ untranslated region (UTR), a 3′ UTR, a 5′ cap and a poly-A tail. The mRNA polynucleotides described herein function as mRNA but may differ from wild-type mRNA in both functional and/or structural design features.

A “5′ untranslated region” (5′UTR) refers to a region of an mRNA that is directly upstream (i.e., 5′) from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide.

A “3′ untranslated region” (3′UTR) refers to a region of an mRNA that is directly downstream (i.e., 3′) from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide.

A “poly-A tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3′), from the 3′ UTR that contains multiple, consecutive adenosine monophosphates. A poly-A tail may contain 10 to 150 adenosine monophosphates. In one embodiment, a poly-A tail contains 50 to 150 adenosine monophosphates. In one embodiment, a poly-A tail contains 75 to 125 adenosine monophosphates. In one embodiment, a poly-A tail contains about 120 adenosine monophosphates. In one embodiment, a poly-A tail contains 120 adenosine monophosphates. In one embodiment, a poly-A tail contains about 80 adenosine monophosphates. In one embodiment, a poly-A tail contains 80 adenosine monophosphates. In a relevant biological setting (e.g., in cells, in vivo) the poly-A tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, export of the mRNA from the nucleus, and translation.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the mRNA polynucleotide comprises 200 to 5,000 nucleotides.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame, a 5′ UTR, a 3′ UTR, a 5′ cap and a poly-A tail encoding an antigen from an infectious microorganism or an immunogenic fragment thereof.

An Open Reading Frame

An “open reading frame” (ORF) is a continuous stretch of polynucleotide beginning with a start codon (e.g., methionine (AUG)), and ending with a stop codon (e.g., UAA, UAG or UGA). It encodes, and has the potential to be translated into, a polypeptide (in this case the antigen or the immunogenic fragment thereof). It is also known as a coding region.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the open reading frame comprises about 1950 nucleotides.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the open reading frame comprises about 477 nucleotides.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the open reading frame comprises about 297 nucleotides.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the open reading frame comprises 45-90% of the total nucleotides of the mRNA.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the open reading frame comprises 45-70% of the total nucleotides of the mRNA.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the open reading frame comprises 50-65% of the total nucleotides of the mRNA.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the open reading frame comprises 90% of the total nucleotides of the mRNA.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the open reading frame comprises about 90% of the total nucleotides of the mRNA.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the open reading frame comprises 63% of the total nucleotides of the mRNA.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the open reading frame comprises about 63% of the total nucleotides of the mRNA.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the open reading frame comprises 52% of the total nucleotides of the mRNA.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the open reading frame comprises about 52% of the total nucleotides of the mRNA.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an E1, E2, E4, E5, E6, E7, L1, or L2 HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an E1, E2, E4, E5, E6 or E7 HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an E6 or E7 HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an E1 or E6 or E7 HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an E1 HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an E6 HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an E7 HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine does not comprise an mRNA polynucleotide comprising an open reading frame encoding an E5 HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an L1 or L2 HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an L1 HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an L2 HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding a high-risk HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and/or 68 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an HPV 6, 8, 11, 16 and/or 18 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an HPV 16 and/or 18 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding a low-risk HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an HPV 6 and/or 11 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an E1 HPV 16 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an E1 HPV 18 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an E6 HPV 16 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an E6 HPV 18 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an E7 HPV 16 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an E7 HPV 18 antigen or an immunogenic fragment thereof.

Table 1 describes certain mRNA open reading frame sequences that encode antigens from infectious microorganisms that may be incorporated into the mRNA polynucleotides in the vaccines 10 described herein.

TABLE 1

mRNA polynucleotides encoding antigens

mRNA Open Reading Frame Sequence

(all uridines “U” are N1-methylpseudouridine

Antigen
nucleotides)
SEQ ID_No

HPV 16 E6
AUGCACCAGAAGCGGACCGCCAUGUUCCAGGACCCCCAGGAGAGACCUA
SEQ ID_No 2

GAAAGCUGCCUCAGCUGUGCACCGAGCUGCAGACCACCAUCCACGACAU

CAUCCUGGAAUGCGUGUAUUGUAAACAGCAACUGCUGAGACGGGAAG

UGUACGACUUCGCCUUCAGAGAUCUGUGCAUCGUGUACAGAGACGGC

AACCCCUACGCCGUGUGCGAUAAGUGCCUGAAGUUCUACAGCAAGAUC

AGCGAGUACCGGCACUACUGCUACAGCCUGUACGGCACCACACUGGAAC

AGCAGUACAACAAGCCCCUGUGCGACCUGCUGAUCCGGUGCAUCAACU

GCCAGAAACCUCUGUGUCCUGAGGAAAAGCAGAGACACCUGGACAAGA

AGCAGCGAUUUCACAACAUCAGAGGAAGAUGGACAGGCAGAUGCAUGA

GCUGCUGCAGAUCUAGCAGAACCAGAAGAGAGACACAGCUGUGA

HPV 16 E7
AUGCACGGCGACACCCCUACACUGCACGAGUACAUGCUGGACCUGCAGC
SEQ ID_No 3

CUGAAACCACCGACCUGUACUGCUACGAGCAGCUGAACGACUCUAGCG

AGGAAGAGGACGAGAUCGACGGCCCUGCCGGCCAGGCCGAGCCUGAUA

GAGCCCACUACAACAUCGUGACCUUCUGCUGCAAGUGCGACAGCACCCU

UAGACUGUGCGUGCAGAGCACACACGUGGACAUCAGAACCCUGGAAGA

UCUGCUGAUGGGCACCUUGGGCAUCGUGUGCCCCAUCUGCAGCCAGAA

GCCUUGA

HPV16 E1
AUGGCGGACCCCGCUGGCACCAACGGAGAAGAAGGCACCGGCUGCAAC
SEQ ID_No 4

GGCUGGUUCUACGUGGAAGCCGUGGUGGAAAAGAAGACCGGCGACGC

CAUCAGCGACGACGAGAAUGAGAACGAUAGCGACACCGGCGAGGAUCU

CGUCGAUUUCAUAGUCAAUGAUAACGAUUACCUGACGCAGGCCGAAAC

AGAAACGGCCCAUGCGCUGUUCACCGCACAAGAGGCGAAGCAGCAUAG

AGAUGCAGUGCAAGUAUUGAAAAGGAAGUAUUUGGUGAGCCCUCUGU

CUGACAUAAGCGGAUGUGUGGACAAUAAUAUAAGCCCACGCCUUAAGG

CAAUCUGUAUCGAGAAGCAGAGCAGGGCCGCGAAGAGGCGGCUGUUCG

AGAGCGAAGACAGCGGAUAUGGCAAUACCGAAGUGGAGACACAGCAAA

UGUUGCAAGUAGAAGGCCGCCACGAGACAGAAACCCCCUGUAGCCAGU

AUAGCGGUGGUAGCGGAGGUGGAUGUAGCCAGUACAGCAGCGGAAGC

GGCGGCGAAGGGGUUAGCGAGAGACAUACCAUCUGCCAAACUCCGCUG

ACGAACAUUUUGAAUGUGCUCAAGACGAGCAACGCCAAAGCUGCAAUG

CUUGCGAAGUUUAAAGAAUUGUACGGCGUUAGCUUUAGCGAAUUGGU

GAGACCCUUCAAGAGCAAUAAGAGCACUUGUUGUGAUUGGUGCAUCG

CUGCAUUUGGCCUUACACCGAGCAUUGCGGACAGCAUAAAGACUUUGC

UGCAACAGUACUGUCUGUACCUGCACAUUCAGAGCCUCGCAUGUAGCU

GGGGAAUGGUCGUGCUGCUCCUGGUGCGAUACAAGUGCGGAAAAAAU

AGAGAAACUAUAGAGAAGCUCCUCAGCAAGCUGCUCUGCGUUAGCCCA

AUGUGCAUGAUGAUUGAGCCCCCAAAGUUGCGAAGCACCGCCGCCGCA

CUUUACUGGUACAAGACUGGUAUAAGCAACAUCAGCGAAGUGUACGG

AGAUACACCCGAGUGGAUCCAGCGCCAAACCGUUCUCCAGCAUAGCUU

UAACGACUGUACGUUUGAAUUGAGCCAGAUGGUCCAGUGGGCGUAUG

ACAACGAUAUCGUUGACGACUCAGAAAUAGCUUACAAAUACGCACAGC

UCGCGGACACCAACAGCAACGCAAGCGCGUUUUUGAAGAGUAACAGCC

AAGCCAAGAUCGUUAAGGAUUGUGCGACUAUGUGUAGACACUAUAAG

CGCGCUGAGAAGAAACAAAUGUCAAUGAGCCAAUGGAUUAAAUACCGA

UGUGAUCGGGUUGAUGACGGUGGCGAUUGGAAGCAAAUUGUCAUGU

UUCUUAGAUAUCAGGGGGUUGAGUUCAUGAGCUUCUUGACAGCGCUC

AAGAGAUUUCUGCAAGGGAUCCCAAAGAAGAAUUGUAUACUCCUUUAC

GGAGCUGCCAAUACAGGGAAGAGUCUGUUCGGAAUGAGCCUGAUGAA

AUUCCUCCAAGGGAGCGUCAUCUGCUUUGUCAACAGCAAAAGCCACUU

UUGGCUUCAACCUCUCGCCGAUGCGAAAAUUGGUAUGCUCGAUGAUG

CUACCGUCCCAUGCUGGAACUACAUCGACGAUAACCUUAGAAAUGCAC

UUGACGGGAAUCUGGUGAGCAUGGAUGUUAAGCACAGACCUCUUGUG

CAGUUGAAAUGCCCUCCACUUCUCAUCACAUCAAAUAUUAACGCGGGG

ACGGACAGCCGAUGGCCCUAUCUGCACAAUCGGCUUGUGGUGUUCACG

UUCCCAAAUGAGUUCCCUUUCGACGAGAACGGAAAUCCAGUAUAUGAA

CUGAAUGACAAAAACUGGAAGAGUUUCUUCUCCCGGACCUGGAGCAGA

CUGAGCCUGCAUGAGGACGAGGAUAAAGAAAAUGACGGGGACAGCCUC

CCCACUUUCAAAUGCGUCUCUGGACAGAACACUAACACCUUGUAA

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 2 or a sequence with at least 90% sequence identity or homology.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 2 or a sequence with at least 95% sequence identity or homology.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 2 or a sequence with at least 98% sequence identity or homology.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 2.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame consisting essentially of a sequence selected from SEQ ID_No 2.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame consisting of a sequence selected from SEQ ID_No 2.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 3 or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 3 or a sequence with at least 90% sequence identity or homology.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 3 or a sequence with at least 95% sequence identity or homology.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 3 or a sequence with at least 98% sequence identity or homology.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 3.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame consisting essentially of a sequence selected from SEQ ID_No 3.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame consisting of a sequence selected from SEQ ID_No 3.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 4 or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 4 or a sequence with at least 90% sequence identity or homology.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 4 or a sequence with at least 95% sequence identity or homology.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 4 or a sequence with at least 98% sequence identity or homology.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 4.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame consisting essentially of a sequence selected from SEQ ID_No 4.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame consisting of a sequence selected from SEQ ID_No 4.

Encoding an Antigen

An antigen is a protein or a polypeptide from an infectious microorganism that binds to a specific antibody or T-cell receptor, triggering an immune response.

The antigens from an infectious microorganism encoded by the mRNA polynucleotide in the vaccines described herein can be “wild-type” i.e. naturally occurring antigens, or modified naturally occurring antigens. Non naturally occurring antigens that may be encoded by the mRNA polynucleotides in the vaccines herein are similar enough (e.g. 80% sequence identity or homology) to provoke the same immunogenic reaction as the equivalent wild-type antigen.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, or an immunogenic fragment thereof, wherein the antigen, or an immunogenic fragment thereof, has at least 80% sequence identity or homology with the wild-type antigen or a fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, or an immunogenic fragment thereof, wherein the antigen, or an immunogenic fragment thereof, has at least 90% sequence identity or homology with the wild-type antigen or a fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, or an immunogenic fragment thereof, wherein the antigen, or an immunogenic fragment thereof, has at least 95% sequence identity or homology with the wild-type antigen or a fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, or an immunogenic fragment thereof, wherein the antigen, or an immunogenic fragment thereof, has at least 98% sequence identity or homology with the wild-type antigen or a fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, or an immunogenic fragment thereof, wherein the antigen, or an immunogenic fragment thereof, is identical the wild-type antigen or a fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the antigen or immunogenic fragment thereof is a polypeptide.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the antigen or immunogenic fragment thereof is a polypeptide consisting of 80-700 amino acid residues.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the antigen or immunogenic fragment thereof is a polypeptide consisting of 80-180 amino acid residues.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the antigen or immunogenic fragment thereof is a polypeptide consisting of 600-700 amino acid residues.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the antigen or immunogenic fragment thereof is a polypeptide consisting of about 649 amino acid residues.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the antigen or immunogenic fragment thereof is a polypeptide consisting of 649 amino acid residues.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the antigen or immunogenic fragment thereof is a polypeptide consisting of 140-170 amino acid residues.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the antigen or immunogenic fragment thereof is a polypeptide consisting of about 158 amino acid residues.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the antigen or immunogenic fragment thereof is a polypeptide consisting of 158 amino acid residues.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the antigen or immunogenic fragment thereof is a polypeptide consisting of 90-110 amino acid residues.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the antigen or immunogenic fragment thereof is a polypeptide consisting of about 98 amino acid residues.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the antigen or immunogenic fragment thereof is a polypeptide consisting of 98 amino acid residues.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an HPV antigen or an immunogenic fragment thereof forming part or all of the HPV viral capsid.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an HPV antigen or an immunogenic fragment thereof responsible for binding the HPV to a cell being infected.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism that interacts with retinoblastoma protein (pRb), or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism that interacts with p53, or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism that attaches to cell receptors, or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism that causes fusion of viral and cellular membranes, or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism that is responsible for binding of the virus to a cell being infected, or an immunogenic fragment thereof.

Table 2 describes sequences of antigens from an infectious microorganism that the mRNA polynucleotides of the vaccines described herein may encode for:

TABLE 2

Antigens

Antigen
Sequence
SEQ ID_No

HPV 16 E6
MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAF
SEQ ID_No 5

RDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIR

CINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL

HPV 16 E7
MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHY
SEQ ID_No 6

NIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP

HPV 16 E1
MADPAGTNGEEGTGCNGWFYVEAVVEKKTGDAISDDENENDSDTGEDLVD
SEQ ID_No 7

FIVNDNDYLTQAETETAHALFTAQEAKQHRDAVQVLKRKYLVSPLSDISGCVD

NNISPRLKAICIEKQSRAAKRRLFESEDSGYGNTEVETQQMLQVEGRHETETPC

SQYSGGSGGGCSQYSSGSGGEGVSERHTICQTPLTNILNVLKTSNAKAAMLAKFK

ELYGVSFSELVRPFKSNKSTCCDWCIAAFGLTPSIADSIKTLLQQYCLYLHIQSL

ACSWGMVVLLLVRYKCGKNRETIEKLLSKLLCVSPMCMMIEPPKLRSTAAALY

WYKTGISNISEVYGDTPEWIQRQTVLQHSFNDCTFELSQMVQWAYDNDIVD

DSEIAYKYAQLADTNSNASAFLKSNSQAKIVKDCATMCRHYKRAEKKQMSMS

QWIKYRCDRVDDGGDWKQIVMFLRYQGVEFMSFLTALKRFLQGIPKKNCILL

YGAANTGKSLFGMSLMKFLQGSVICFVNSKSHFWLQPLADAKIGMLDDATVP

CWNYIDDNLRNALDGNLVSMDVKHRPLVQLKCPPLLITSNINAGTDSRWPYL

HNRLVVFTFPNEFPFDENGNPVYELNDKNWKSFFSRTWSRLSLHEDEDKEND

GDSLPTFKCVSGQNTNTL

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID_No 5 or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID_No 5.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen consisting essentially of a sequence selected from SEQ ID_No 5.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen consisting of SEQ ID_No 5.

In one embodiment there is provided an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID_No 5.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID_No 6 or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID_No 6.

In one embodiment there is provided an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID_No 6.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen consisting essentially of a sequence selected from SEQ ID_No 6.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen consisting of SEQ ID_No 6.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID_No 7 or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID_No 7.

In one embodiment there is provided an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from SEQ ID_No 7.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen consisting essentially of a sequence selected from SEQ ID_No 7.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen consisting of SEQ ID_No 7.

Table 3 shows gene bank accession numbers for the sequences of certain HPV antigens from an infectious microorganism that the mRNA polynucleotides of the vaccines described herein may encode for:

TABLE 3

Gene bank accession numbers for certain HPV antigens

HPV
GenBank/UniProtKB/Swiss-Prot

HPV strain
protein
Accession

Human papilloma
L1
QEE83786.1

virus type 6
L2
QEE83785.1

E1
QEE83780.1

E2
QEE83781.1

E4
QEE83782.1

E5A
QEE83783.1

E5B
QEE83784.1

E6
QEE83778.1

E7
QEE83779.1

Human papilloma
L1
P06417.1

virus type 8
L2
P06419.1

E1
P06420.1

E2
P06422.1

E4
P06425.1

E6
P06428.1

E7
P06430.1

Human papilloma
L1
QEE83895.1

virus type 11
L2
QEE83894.1

E1
QEE83889.1

E2
QEE83890.1

E4
QEE83891.1

E5A
QEE83892.1

E5B
QEE83893.1

E6
QEE83887.1

E7
QEE83888.1

Human papilloma
L1
AYV61481.1

virus type 16
L2
AYV61480.1

E1
AYV61476.1

E2
AYV61477.1

E4
AYV61478.1

E5
AYV61479.1

E6
AAL01351.1

E7
NP_041326.1

Human papilloma
L1
ATL15246.1

virus type 18
L2
ATL15245.1

E1
AGU90874.1

E2
ATL15234.1

E4
ATL15243.1

E5
ATL15244.1

E6
ATL15239.1

E7
ATL15240.1

Human papilloma
L1
ASL24648.1

virus type 31
L2
CAD1807847.1

E1
CAD1807839.1

E2
CAD1807842.1

E4
CAD1807843.1

E5
CAD1814371.1

E6
CAD1807836.1

E7
CAD1807837.1

Human papilloma
L1
ASL24649.1

virus type 33
L2
AEI61868.1

E1
AEI61432.1

E2
AEI61865.1

E4
CAD1814538.1

E5
CAD1814539.1

E6
AFO63342.1

E7
AFO63343.1

Human papilloma
L1
QJD33099.1

virus type 35
L2
QJD38258.1

E1
QJD33301.1

E2
QJD36478.1

E4
QJD38256.1

E5
QJD38249.1

E6
QJD38252.1

E7
QJD38253.1

Human papilloma
L1
CAD1814503.1

virus type 39
L2
CAD1814501.1

E1
AGU90577.1

E2
CAD1814494.1

E4
CAD1814496.1

E5
AGU90579.1

E6
CAD1814490.1

E7
CAD1814491.1

Human papilloma
L1
CAD1814430.1

virus type 45
L2
CAD1814429.1

E1
AGU90627.1

E2
CAD1814428.1

E4
CAD1807061.1

E5
CAD1807062.1

E6
CAD1814425.1

E7
CAD1814426.1

Human papilloma
L1
AMK51256.1

virus type 51
L2
AMK51255.1

E1
AMK51253.1

E2
AMK51252.1

E4
AMK51254.1

E5
P26553.1

E6
P26554.1

E7
P26558.1

Human papilloma
L1
CAD1814438.1

virus type 52
L2
CAD1814437.1

E1
CAD1814433.1

E2
CAD1814434.1

E4
CAD1814435.1

E5
CAD1814436.1

E6
CAD1814431.1

E7
CAD1814432.1

Human papilloma
L1
CAD1814189.1

virus type 56
L2
CAD1814186.1

E1
CAD1814181.1

E2
CAD1814183.1

E4
CAD1814185.1

E6
CAD1814586.1

E7
CAD1814179.1

Human papilloma
L1
CAD1813847.1

virus type 58
L2
CAD1813846.1

E1
CAD1813842.1

E2
CAD1813843.1

E4
CAD1813844.1

E5
CAD1813845.1

E6
CAD1813840.1

E7
CAD1813841.1

Human papilloma
L1
CAD1814219.1

virus type 59
L2
CAD1814217.1

E1
CAD1814209.1

E2
CAD1814211.1

E4
CAD1814213.1

E5
CAD1814215.1

E6
CAD1814206.1

E7
CAD1814207.1

Human papilloma
L1
CAD1814518.1

virus type 66
L2
CAD1814517.1

E1
CAD1814510.1

E2
CAD1814512.1

E4
CAD1814515.1

E6
CAD1814506.1

E7
CAD1814507.1

Human papilloma
L1
CAD1807650.1

virus type 68
L2
CAD1807649.1

E1
CAD1807646.1

E2
CAD1807647.1

E4
CAD1807371.1

E5
CAD1807648.1

E6
CAD1807644.1

E7
CAD1807645.1

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from Table 3 or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen comprising a sequence selected from Table 3.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen consisting essentially of a sequence selected from Table 3.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen consisting of a sequence selected from Table 3.

Immunogenic Fragment Thereof

An immunogenic fragment of an antigen (also known as an epitope, or antigenic determinant) is the fragment of the antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells, and induces an immune response. Immunogenic fragments that provoke a CD8+ response are typically 8-10 amino acids long.

An Infectious Microorganism

Infectious microorganisms fall into 5 main categories-bacteria, parasites (for example protozoa), fungi, viruses and prions. These pathogenic microorganisms can be transmitted by animals, humans, insects or other agents causing diseases in the host organism.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from a bacterium, parasite, fungus, virus or prion or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from a bacterium or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from a parasite or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from a fungus or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from a virus or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from a prion or an immunogenic fragment thereof.

Multiple Component Vaccines

In one embodiment the vaccine comprises multiple, e.g. 2-10, mRNA polynucleotides comprising open reading frames each encoding a single antigen from an infectious microorganism or an immunogenic fragment thereof. The two or more mRNA polynucleotides may encode the same or different antigens, or sequences from different parts of the same antigen, additionally or alternatively from different strains of infectious microorganisms.

In one embodiment the vaccine comprises one mRNA polynucleotide comprising open reading frames encoding multiple, e.g. 2-10, antigens from an infectious microorganism or an immunogenic fragment thereof (e.g. as a fusion polypeptide). The open reading frames encoding multiple antigens from an infectious microorganism or an immunogenic fragment thereof may encode the same or different antigens, or sequences from different parts of the same antigen, additionally or alternatively from different strains of infectious microorganisms.

In one embodiment, the vaccines described herein are multivalent. A multivalent vaccine combines antigens from different strains of one pathogen to immunize against one disease.

In one embodiment the vaccine comprises a first mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, and a second mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, and a third mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof.

In one embodiment the vaccine comprises one mRNA polynucleotide comprising open reading frames encoding two antigens from an infectious microorganism or an immunogenic fragment thereof.

In one embodiment the vaccine comprises one mRNA polynucleotide comprising open reading frames encoding three antigens from an infectious microorganism or an immunogenic fragment thereof.

In one embodiment the vaccine comprises:

- i) a first mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof;
- ii) a second mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof; and
- iii) an amphipathic cell penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine comprises:

- i) a first mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof;
- ii) a second mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof;
- ii) a third mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof; and
- iv) an amphipathic cell penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine comprises two mRNA polynucleotides comprising an open reading frame each encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the two mRNA polynucleotides are present in about a 50:50% w/w ratio.

In one embodiment the vaccine comprises three mRNA polynucleotides comprising an open reading frame each encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the three mRNA polynucleotides are present in about equal % w/w ratio.

“Copy number” refers to the number of copies of the mRNA present. To determine copy numbers of mRNA, the molecular weight (MW) of one copy of mRNA is calculated by addition of the molecular weights of all constituent nucleotides in the entire nucleotide sequence, where:

- A=329.2 g/mol
- C=305.2 g/mol
- G=345.2 g/mol
- T=306.2 g/mol

Any proprietary sequences (non-ORF), for which the precise nucleotides are not known, are accounted for by using the average molecular weight of A/G/C/T=321.45 g/mol.

The copy numbers present in a known mass of mRNA (m) are then calculated as follows:

Copy Number=(m/MW)*NA

- (where NA=Avogadro's Number=6.0221409×10{circumflex over ( )}23)

In one embodiment the vaccine comprises an mRNA polynucleotide comprising two or more open reading frames each encoding an antigen from an infectious microorganism or an immunogenic fragment thereof wherein the open reading frames are present in an equal copy number ratio.

In one embodiment the vaccine comprises three mRNA polynucleotides comprising open reading frames each encoding an HPV antigen selected from E1, E6 and E7 or an immunogenic fragment thereof wherein the three mRNA polynucleotides are present in 1 (E1):3 (E6):4 (E7) copy number ratio. In one embodiment the vaccine comprises three mRNA polynucleotides comprising open reading frames each encoding an HPV antigen selected from E1, E6 and E7 or an immunogenic fragment thereof wherein the three mRNA polynucleotides are present in about 1 (E1):3.2 (E6):4.2 (E7) copy number ratio.

In one embodiment the vaccine comprises two mRNA polynucleotides comprising open reading frames each encoding an HPV antigen selected from E1, E2, E4, E5, E6, E7, L1, or L2 or an immunogenic fragment thereof.

In one embodiment the vaccine comprises three mRNA polynucleotides comprising open reading frames each encoding an HPV antigen selected from E1, E2, E4, E5, E6, E7, L1, or L2 or an immunogenic fragment thereof.

In one embodiment the vaccine comprises two mRNA polynucleotides comprising open reading frames each encoding an HPV antigen selected from E1, E2, E4, E5, E6 or E7 or an immunogenic fragment thereof.

In one embodiment the vaccine comprises three mRNA polynucleotides comprising open reading frames each encoding an HPV antigen selected from E1, E2, E4, E5, E6 or E7 or an immunogenic fragment thereof.

In one embodiment the vaccine comprises two mRNA polynucleotides comprising open reading frames one encoding an E6 HPV antigen or an immunogenic fragment thereof, and the other encoding an E7 HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises three mRNA polynucleotides comprising open reading frames one encoding an E6 HPV antigen or an immunogenic fragment thereof, one encoding an E7 HPV antigen or an immunogenic fragment thereof, and one encoding an E1 HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises two mRNA polynucleotides comprising open reading frames one encoding an L1 HPV antigen or an immunogenic fragment thereof, and the other encoding an L2 HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises two mRNA polynucleotides comprising an open reading frame each encoding a high-risk HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises three mRNA polynucleotides comprising an open reading frame each encoding a high-risk HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises two mRNA polynucleotides comprising an open reading frame each encoding an HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and/or 68 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises three mRNA polynucleotides comprising an open reading frame each encoding an HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and/or 68 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises two mRNA polynucleotides comprising an open reading frame each encoding an HPV 6, 8, 11, 16 and/or 18 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises three mRNA polynucleotides comprising an open reading frame each encoding an HPV 6, 8, 11, 16 and/or 18 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises two mRNA polynucleotides comprising an open reading frame each encoding an HPV 16 and/or 18 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises three mRNA polynucleotides comprising an open reading frame each encoding an HPV 16 and/or 18 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises two mRNA polynucleotides comprising an open reading frame each encoding a low-risk HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises three mRNA polynucleotides comprising an open reading frame each encoding a low-risk HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises two mRNA polynucleotides comprising an open reading frame each encoding an HPV 6 and/or 11 antigen or an immunogenic fragment thereof. In one embodiment the vaccine comprises three mRNA polynucleotides comprising an open reading frame each encoding an HPV 6 and/or 11 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises two mRNA polynucleotides comprising an open reading frame one encoding a high-risk HPV antigen or an immunogenic fragment thereof, and the other encoding a low-risk HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises three mRNA polynucleotides comprising an open reading frame at least one encoding a high-risk HPV antigen or an immunogenic fragment thereof, and at least one encoding a low-risk HPV antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises two mRNA polynucleotides comprising open reading frames one encoding an E6 HPV 16 antigen or an immunogenic fragment thereof, and the other encoding an E7 HPV 16 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises three mRNA polynucleotides comprising open reading frames one encoding an E6 HPV 16 antigen or an immunogenic fragment thereof, one encoding an E7 HPV 16 antigen or an immunogenic fragment thereof, and one encoding an E1 HPV 16 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises two mRNA polynucleotides comprising open reading frames one encoding an E6 HPV 18 antigen or an immunogenic fragment thereof, and the other encoding an E7 HPV 18 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises three mRNA polynucleotides comprising open reading frames one encoding an E6 HPV 18 antigen or an immunogenic fragment thereof, one encoding an E7 HPV 18 antigen or an immunogenic fragment thereof, and one an E1 HPV 18 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises three mRNA polynucleotides comprising open reading frames one encoding an E6 HPV 16 antigen or an immunogenic fragment thereof, one encoding an E7 HPV 18 antigen or an immunogenic fragment thereof, and one encoding an E1 HPV 18 antigen or an immunogenic fragment thereof.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 2 or a sequence with at least 80% sequence identity or homology, and an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 3 or a sequence with at least 80% sequence identity or homology, and an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 4 or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 2 or a sequence with at least 80% sequence identity or homology and an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 3 or a sequence with at least 80% sequence identity or homology wherein the two mRNA polynucleotides are present in about a 50:50% w/w ratio.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 2 or a sequence with at least 80% sequence identity or homology and an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 3 or a sequence with at least 80% sequence identity or homology wherein the two mRNA polynucleotides are present in a 50:50% w/w ratio.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 2 and an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 3 wherein the two mRNA polynucleotides are present in a about a 50:50% w/w ratio.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 2, and an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 3, and an mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 4 wherein the three mRNA polynucleotides are present in a about a equal % w/w ratio.

In one embodiment the vaccine comprises two mRNA polynucleotides comprising open reading frames one encoding an E6 HPV antigen selected from SEQ ID_No 5 or a sequence with at least 80% sequence identity or homology, and the other encoding an E7 HPV antigen selected from SEQ ID_No 6 or a sequence with at least 80% sequence identity or homology wherein the two mRNA polynucleotides are present in a 50:50% w/w ratio.

In one embodiment the vaccine comprises three mRNA polynucleotides comprising open reading frames one encoding an E6 HPV antigen selected from SEQ ID_No 5 or a sequence with at least 80% sequence identity or homology, one encoding an E7 HPV antigen selected from SEQ ID_No 6 or a sequence with at least 80% sequence identity or homology, and one encoding an E1 HPV antigen selected from SEQ ID_No 7 or a sequence with at least 80% sequence identity or homology wherein the three mRNA polynucleotides are present in a equal % w/w ratio.

In one embodiment the vaccine comprises two mRNA polynucleotides comprising open reading frames one encoding an E6 HPV antigen selected from SEQ ID_No 5, and the other encoding an E7 HPV antigen selected from SEQ ID_No 6 wherein the two mRNA polynucleotides are present in a 50:50% w/w ratio.

In one embodiment the vaccine comprises three mRNA polynucleotides comprising open reading frames one encoding an E6 HPV antigen selected from SEQ ID_No 5, one encoding an E7 HPV antigen selected from SEQ ID_No 6, and one encoding an E1 HPV antigen selected from SEQ ID_No 7 wherein the three mRNA polynucleotides are present in equal % w/w ratio.

Immunoadjuvants

In one embodiment the vaccine further comprises an immunoadjuvant. An immunoadjuvant is a substance that acts to accelerate, prolong, or enhance an antigen-specific immune response when used in combination with specific vaccine antigens.

Suitable immunoadjuvants include inorganic compounds, for example potassium alum KAI(SO₄)₂, aluminium hydroxide, aluminium phosphate, and calcium phosphate hydroxide; oils, for example paraffin oil, propolis, and peanut oil; and cytokines, for example IL-1 (interleukin-1), IL-2, IL-7, IL-12 and IL-15. Other suitable immunoadjuvants include granulocyte-macrophage colony-stimulating factor (GM-CSF).

In one embodiment the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant. In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, and an open reading frame encoding an immunoadjuvant.

In one embodiment the vaccine comprises:

- i) a first mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 2;
- ii) a second mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 3;
- iii) a third mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 8; and
- iv) an amphipathic cell penetrating peptide comprising the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1).

In one embodiment the vaccine comprises:

- i) a first mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 2;
- ii) a second mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 3;
- iii) a third mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 4;
- iv) a fourth mRNA polynucleotide comprising an open reading frame comprising a sequence selected from SEQ ID_No 8; and
- v) an amphipathic cell penetrating peptide comprising the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1).

In one embodiment the vaccine comprises:

- i) a first mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof;
- ii) a second mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof;
- iii) a third mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant; and
- iv) an amphipathic cell penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine comprises:

- i) a first mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof;
- ii) a second mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof;
- iii) a third mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof;
- iv) a fourth mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant; and
- v) an amphipathic cell penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding a cytokine.

In one embodiment the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding IL-1, IL-2, IL-7, IL-12 or IL-15.

In one embodiment the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding IL-15.

In one embodiment the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding human IL-15.

In one embodiment the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding granulocyte-macrophage colony-stimulating factor.

In one embodiment the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding human granulocyte-macrophage colony-stimulating factor.

In one embodiment the vaccine comprises:

- i) a first mRNA polynucleotide comprising an open reading frame encoding an HPV E6 antigen or an immunogenic fragment thereof;
- ii) a second mRNA polynucleotide comprising an open reading frame encoding an HPV E7 antigen or an immunogenic fragment thereof;
- iii) a third mRNA polynucleotide comprising an open reading frame encoding human IL-15; and
- iv) an amphipathic cell penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine comprises:

- i) a first mRNA polynucleotide comprising an open reading frame encoding an HPV E6 antigen or an immunogenic fragment thereof;
- ii) a second mRNA polynucleotide comprising an open reading frame encoding an HPV E7 antigen or an immunogenic fragment thereof;
- iii) a third mRNA polynucleotide comprising an open reading frame encoding an HPV E1 antigen or an immunogenic fragment thereof;
- iv) a fourth mRNA polynucleotide comprising an open reading frame encoding human IL-15; and
- v) an amphipathic cell penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine comprises:

- i) a first mRNA polynucleotide comprising an open reading frame encoding an HPV 16 E6 antigen or an immunogenic fragment thereof;
- ii) a second mRNA polynucleotide comprising an open reading frame encoding an HPV 16 E7 antigen or an immunogenic fragment thereof;
- iii) a third mRNA polynucleotide comprising an open reading frame encoding human IL-15; and
- iv) an amphipathic cell penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine comprises:

- i) a first mRNA polynucleotide comprising an open reading frame encoding an HPV 16 E6 antigen or an immunogenic fragment thereof;
- ii) a second mRNA polynucleotide comprising an open reading frame encoding an HPV 16 E7 antigen or an immunogenic fragment thereof;
- iii) a third mRNA polynucleotide comprising an open reading frame encoding an HPV 16 E1 antigen or an immunogenic fragment thereof;
- iv) a fourth mRNA polynucleotide comprising an open reading frame encoding human IL-15; and
- v) an amphipathic cell penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine comprises:

- i) a first mRNA polynucleotide comprising an open reading frame encoding an antigen selected from SEQ ID_No 5;
- ii) a second mRNA polynucleotide comprising an open reading frame encoding an antigen selected from SEQ ID_No 6;
- iii) a third mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant selected from SEQ ID_No 10; and
- iv) an amphipathic cell penetrating peptide comprising the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1).

In one embodiment the vaccine comprises:

- i) a first mRNA polynucleotide comprising an open reading frame encoding an antigen selected from SEQ ID_No 5;
- ii) a second mRNA polynucleotide comprising an open reading frame encoding an antigen selected from SEQ ID_No 6;
- iii) a third mRNA polynucleotide comprising an open reading frame encoding an antigen selected from SEQ ID_No 7;
- iv) a fourth mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant selected from SEQ ID_No 10; and
- v) an amphipathic cell penetrating peptide comprising the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1).

In one embodiment the vaccine further comprises up to 20% w/w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant. 20% w/w (total mRNA) means that the mRNA encoding the immunoadjuvant makes up 20% by weight of the total amount of mRNA in the vaccine.

In one embodiment the vaccine further comprises up to 20% w/w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding human IL-15.

In one embodiment the vaccine further comprises 5-20% w/w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant.

In one embodiment the vaccine further comprises 5-20% w/w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding human IL-15.

In one embodiment the vaccine further comprises 5% w/w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant.

In one embodiment the vaccine further comprises about 5% w/w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding human IL-15.

In one embodiment the vaccine further comprises 10% w/w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant.

In one embodiment the vaccine further comprises about 10% w/w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding human IL-15.

In one embodiment the vaccine further comprises 15% w/w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant.

In one embodiment the vaccine further comprises about 15% w/w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding human IL-15.

In one embodiment the vaccine further comprises 20% w/w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant.

In one embodiment the vaccine further comprises about 20% w/w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding human IL-15.

In one embodiment the vaccine further comprises up to 20% w/w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding GM-CSF.

In one embodiment the vaccine further comprises 5-20% w/w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding GM-CSF.

In one embodiment the vaccine further comprises about 5% w/w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding GM-CSF.

In one embodiment the vaccine further comprises about 10% w/w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding GM-CSF.

In one embodiment the vaccine further comprises about 15% w/w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding GM-CSF.

In one embodiment the vaccine further comprises about 20% w/w (total mRNA) of an mRNA polynucleotide comprising an open reading frame encoding GM-CSF.

Table 4 describes certain mRNA open reading frame sequences encoding immunoadjuvants that may be incorporated mRNA polynucleotides and added to the vaccines described herein.

TABLE 4

mRNA polynucleotides encoding immunoadjuvants

mRNA Open Reading Frame Sequence

Immunoadjuvant
(all uridines “U” are N1-methylpseudouridine nucleotides)
SEQ ID_No

hIL-15 (human
AUGAGAAUCAGCAAGCCGCACCUGAGAAGCAUCAGCAUUCAGU
SEQ ID_No 8

interleukin 15)
GCUACCUCUGCCUGCUGCUCAACAGCCACUUCCUGACCGAAGC

CGGAAUCCACGUAUUCAUCCUGGGAUGCUUCAGCGCCGGCCUC

CCCAAAACAGAGGCAAACUGGGUAAACGUAAUAAGCGACCUCA

AAAAGAUCGAAGACCUGAUCCAGAGCAUGCACAUCGACGCAAC

CCUGUACACCGAAAGCGACGUACACCCAAGCUGCAAAGUAACC

GCGAUGAAAUGCUUCCUGCUGGAGCUGCAAGUAAUCAGCCUG

GAGAGCGGCGACGCCAGCAUCCACGACACCGUGGAAAACCUGA

UUAUCUUGGCAAACAACAGCCUGAGCAGCAACGGAAACGUGAC

AGAGAGCGGAUGCAAAGAGUGCGAGGAGCUGGAGGAAAAGAA

CAUCAAAGAAUUCCUCCAAAGCUUCGUGCACAUAGUGCAGAUG

UUCAUAAACACAAGCUGA

hGM-CSF
AUGUGGCUGCAAAGCCUUCUGCUCUUGGGGACUGUGGCCUGC
SEQ ID_No 9

UCAAUUUCUGCACCUGCCCGAUCGCCCAGUCCAAGCACGCAGC

CCUGGGAGCAUGUGAAUGCCAUCCAGGAGGCGCGGAGGUUAC

UGAACCUGAGUAGAGACACUGCUGCUGAGAUGAAUGAAACAG

UAGAAGUGAUAUCAGAAAUGUUUGAUCUCCAGGAGCCUACCU

GCCUACAGACACGCCUGGAGCUGUAUAAACAGGGCCUGAGGG

GCUCCCUCACCAAGCUCAAAGGACCCUUGACAAUGAUGGCCAG

CCACUACAAGCAACACUGUCCUCCAACCCCGGAAACUUCCUGU

GCAACCCAGAUUAUCACAUUUGAAUCCUUCAAAGAGAACCUGA

AGGAUUUCCUACUUGUUAUCCCCUUCGACUGCUGGGAGCCAG

UCCAGGAGUGA

In one embodiment the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant comprising a sequence selected from SEQ ID_No 8 or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant comprising a sequence selected from SEQ ID_No 8.

In one embodiment there is provided an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant comprising a sequence selected from SEQ ID_No 8.

In one embodiment the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant comprising a sequence selected from SEQ ID_No 9 or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant comprising a sequence selected from SEQ ID_No 9 or a sequence with at least 90% sequence identity or homology.

In one embodiment the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant comprising a sequence selected from SEQ ID_No 9 or a sequence with at least 95% sequence identity or homology.

In one embodiment the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant comprising a sequence selected from SEQ ID_No 9 or a sequence with at least 98% sequence identity or homology.

In one embodiment the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant comprising a sequence selected from SEQ ID_No 9.

In one embodiment there is provided an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant comprising a sequence selected from SEQ ID_No 9.

Table 5 describes sequences of immunoadjuvant that the mRNA polynucleotides of the vaccines described herein may encode for:

TABLE 5

Immunoadjuvant

Immunoadjuvant
Sequence
SEQ ID_No

Human IL-15
MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANW
SEQ ID_

VNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS
No 10

LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQ

SFVHIVQMFINTS

Human GM-CSF
MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSR
SEQ ID_

DTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLT
No 11

MMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWE

PVQE

In one embodiment the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant selected from SEQ ID_No 10 or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant selected from SEQ ID_No 10.

In one embodiment the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant selected from SEQ ID_No 11 or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine further comprises an mRNA polynucleotide comprising an open reading frame encoding an immunoadjuvant selected from SEQ ID_No 11.

mRNA Modifications

In one embodiment the mRNA polynucleotides described herein comprise one or more modifications relative to the naturally occurring “wild-type” mRNA. Modification refers to modifications of the adenosine (A), guanosine (G), uridine (U), or cytidine (C) nucleotides in at least one of their position, pattern, percent and/or structure. Modifications may comprise naturally-occurring, non-naturally-occurring or a combination of naturally-occurring and non-naturally-occurring modifications. Modifications may be of a sugar, a nucleobase, or an internucleotide linkage (e.g., to a phosphate group). Modifications may be introduced during synthesis or post-synthesis, and may be introduced with chemical synthesis or with a polymerase enzyme. Any of the regions of the mRNA polynucleotide may be modified.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising one or more modifications resulting in reduced degradation of the mRNA after administration relative to an unmodified (i.e. wild-type) polynucleotide.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising one or more modifications resulting in reduced immunogenicity (e.g., a reduced innate response) after administration relative to an unmodified polynucleotide. Modifications of the mRNA polynucleotides described herein may include codon optimisation, capping, for example a five-prime 5′ cap, and uridine modification.

i) Codon Optimisation

In one embodiment the mRNA polynucleotide may be codon optimized. Codon optimisation ensures the most efficient translation in humans. Codon optimization, in one embodiment, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias G/C 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 one embodiment, the open reading frame (ORF) sequence is optimized using optimization algorithms.

In one embodiment codon-optimized mRNA may, for instance, be one in which the levels of guanosine (G) and/or cytidine (C) residues are enhanced. The G/C-content of nucleic acid molecules may influence the stability of the mRNA. mRNA having an increased amount of guanosine (G) and/or cytidine (C) residues may be functionally more stable than nucleic acids containing a large amount of adenosine (A) and uridine (T) nucleotides. 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 the open reading frame(s) of the mRNA.

In one embodiment codon-optimized mRNA may include uridine depletion relative to an unmodified polynucleotide. Uridine depletion reduces the likelihood of an innate immune response to the mRNA.

ii) Capping

In one embodiment, the mRNA polynucleotide comprises a five-prime cap (5′ cap). The 5′ cap is a specially altered nucleotide on the 5′ end of the mRNA. mRNA capping may assist with the production of the most biologically active enhancing translation to protein, and least immunogenic mRNA (as in less likely to provoke an innate immune response).

In one embodiment, the mRNA polynucleotide comprises a five-prime cap (5′ cap) selected from CleanCap® available from TriLink Biotechnologies® (e.g. those described in WO 2017/053297). Suitable caps include:

- CleanCap® Reagent AG for co-transcriptional capping of mRNA: m7G(5′)ppp(5′)(2′OMeA)pG, or
- ARCA (Anti Reverse Cap Analog): 3′-O-Me-m⁷G(5′)ppp(5′)G.

5′ capping prevents degradation of the mRNA in the cytoplasm and promotes ribosome recruitment and protein translation.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism wherein the mRNA polynucleotide comprises a 5′ cap.

iii) Uridine Modification

In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, wherein the mRNA polynucleotide comprises one or more modified uridine nucleotides selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine nucleotides.

In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof, wherein the mRNA polynucleotide comprises one or more modified uridine nucleotides selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine nucleotides.

iv) Cytidine Modification

In one embodiment, the vaccine comprises an mRNA polynucleotide comprising modified cytidine nucleotides. Cytidine modification may be employed to impart desirable characteristics such as increased nuclease stability, increased translation or reduced interaction of innate immune receptors with in vitro transcribed RNA.

In one embodiment, the vaccine comprises an mRNA polynucleotide comprising one or more modified cytidine nucleotides selected from N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), 5-methylcytidine-5′-triphosphate (5-methyl-CTP), and 2-thio-5-methyl-cytidine.

In one embodiment the mRNA polynucleotide comprises no cytidine modifications.

In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides wherein at least 80% of the uridine nucleotides are N1-methylpseudouridine nucleotides and the cytidine nucleotides are unmodified.

In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides wherein at least 90% of the uridine nucleotides are N1-methylpseudouridine nucleotides and the cytidine nucleotides are unmodified.

In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides wherein at least 95% of the uridine nucleotides are N1-methylpseudouridine nucleotides and the cytidine nucleotides are unmodified.

In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides wherein at least 97% of the uridine nucleotides are N1-methylpseudouridine nucleotides and the cytidine nucleotides are unmodified.

In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides wherein at least 99% of the uridine nucleotides are N1-methylpseudouridine nucleotides and the cytidine nucleotides are unmodified.

In one embodiment, the vaccine comprises an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism, wherein the mRNA polynucleotide comprises modified uridine nucleotides wherein all uridine nucleotides are N1-methylpseudouridine nucleotides and the cytidine nucleotides are unmodified.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising one or more modifications selected from a 5′ cap and uridine modification.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising two modifications selected from a 5′ cap and uridine modification.

In one embodiment the vaccine comprises an mRNA polynucleotide uniformly modified (e.g., fully modified throughout the entire sequence) for a particular modification.

In one embodiment the mRNA polynucleotides described herein comprise one or more modifications selected from a 5′ cap, and uridine modification fully modified throughout the entire sequence.

In one embodiment the vaccine comprises an mRNA polynucleotide comprising one or more modifications selected from a 5′ cap, and uridine modification in one region of the polynucleotide.

Amphipathic Cell Penetrating Peptide

In accordance with convention, the peptides described herein are drawn “N-terminus” first, i.e. on the left hand side.

In one embodiment the vaccine comprises an amphipathic cell penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine comprises an amphipathic cell penetrating peptide comprising the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine comprises an amphipathic cell penetrating peptide consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) or a sequence with at least 80% sequence identity or homology.

In one embodiment the vaccine comprises an amphipathic cell penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1).

In one embodiment the vaccine comprises an amphipathic cell penetrating peptide comprising or the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1).

In one embodiment the vaccine comprises an amphipathic cell penetrating peptide consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1).

In one embodiment a sequence with at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) consists of less than or equal to 35 amino acid residues.

In one embodiment a sequence with at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) consists of less than or equal to 30 amino acid residues.

In one embodiment a sequence with at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) consists of 26-30 amino acid residues.

In one embodiment a sequence with at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) comprises at least 5 arginine residues (R).

In one embodiment a sequence with at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) comprises at least 6 arginine residues (R).

In one embodiment a sequence with at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) comprises at least 10 alanine residues (A).

In one embodiment a sequence with at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) comprises at least 12 alanine residues (A).

In one embodiment a sequence with at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) comprises at least 5 leucine residues (L).

In one embodiment a sequence with at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) comprises at least 6 leucine residues (L).

In one embodiment a sequence with at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) comprises at least one cysteine residue (C).

In one embodiment a sequence with at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) comprises at least two but no more than three glutamic acid (E) residues.

In one embodiment a sequence with at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) comprises at least 6 arginine residues (R), at least 12 Alanine Residues (A), at least 6 leucine residues (L), optionally at least one cysteine residue (C) and at least two but no more than three glutamic acids residues (E).

In one embodiment a sequence with at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) comprises the consensus sequences EARLARALARALAR and/or LARALARALRA.

In one embodiment a sequence with at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) comprises the consensus sequence EARLARALARALAR.

In one embodiment a sequence with at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) comprises the consensus sequence LARALARALRA.

In one embodiment a sequence with at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) comprises the consensus sequences EARLARALARALAR and LARALARALRA.

In one embodiment a sequence with at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) does not comprise glycine (G).

A Sequence with at Least 80% Sequence Identity or Homology

Any of a variety of sequence alignment methods can be used to determine percent sequence identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Conventional methods include Altschul et al., Bull. Math. Bio. 48:603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992 where two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the “blosum 62” scoring matrix of Henikoff and Henikoff.

“Sequence identity” between two or more sequences is expressed as a percentage and is a function of the number of identical positions shared by the sequences. Thus, sequence identity may be calculated as the number of identical amino acids or nucleotides divided by the total number of amino acids or nucleotides, multiplied by 100. Calculations of % sequence identity may also take into account the number of gaps, and the length of each gap that needs to be introduced to optimize alignment of two or more sequences. Sequence comparisons and the determination of percent identity between two or more sequences can be carried out using specific mathematical algorithms, such as BLAST, which will be familiar to a skilled person.

Homologous sequences may be characterized as having one or more amino acid or nucleotide substitutions, deletions or additions/insertions. These changes are of a minor nature that do not significantly affect the folding or activity of the peptide. These may be small amino acid or nucleotide substitutions; small deletions; and small terminal extensions or other small additions/insertions. An algorithm (e.g. BLAST) looks for the minimal number of edit operations (inserts, deletes, and substitutions) in order to transform the one sequence into an exact copy of the other sequence and assigns a % similarity (homology).

In one embodiment the term “sequence identity or homology” refers to sequence identity.

In one embodiment the term “sequence identity or homology” refers to sequence homology.

Nanoparticles

In one embodiment the vaccines described herein comprise or consist of nanoparticles. Nanoparticles may be formed by self-assembly by adding the mRNA polynucleotide, and the amphipathic cell penetrating peptide together in ultrapure water. The formulation may be lyophilised and then reconstituted for administration.

In one embodiment there is provided a nanoparticle formulation comprising:

- i) an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof; and
- ii) an amphipathic cell penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) or a sequence with at least 80% sequence identity or homology.

In one embodiment there is provided a vaccine as described herein comprising a nanoparticle formulation of:

- i) an mRNA polynucleotide comprising an open reading frame encoding an antigen from an infectious microorganism or an immunogenic fragment thereof; and
- ii) an amphipathic cell penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID_No 1) or a sequence with at least 80% sequence identity or homology.

In one embodiment there is provided a vaccine as described herein formulated in a nanoparticle.

In one embodiment there is provided a nanoparticle comprising a vaccine as described herein.

In one embodiment there is provided a nanoparticle formulation comprising a vaccine as described herein.

In one embodiment there is provided a vaccine as described herein, comprising nanoparticles.

In one embodiment there is provided a vaccine as described herein, comprising nanoparticles with a Z-Average of 30-150 nm. The Z average is the intensity weighted mean hydrodynamic size of the ensemble collection of particles measured by dynamic light scattering (DLS).

In one embodiment there is provided a vaccine as described herein, comprising nanoparticles with a Z-Average of 60-100 nm.

In one embodiment there is provided a vaccine as described herein, comprising nanoparticles with a polydispersity index of =<0.3. The polydispersity index (PI) is a measure of the heterogeneity of a sample based on size.

In one embodiment there is provided a vaccine as described herein, comprising nanoparticles with a polydispersity index of =<0.5.

In one embodiment there is provided a vaccine as described herein, comprising nanoparticles with a net positive charge at a neutral pH.

N:P Ratio of mRNA: Amphipathic Cell Penetrating Peptide

The amount of the RALA peptide to mRNA is indicated by the N:P ratio, and represents the molar ratio of positively charged nitrogen atoms in the peptide to negatively charged phosphates in the mRNA. The mRNA quoted in the ratio may indicate one mRNA, or a mixture of mRNAs depending on the product.

N:P ratio is widely used to describe the contents of peptide or protein based nucleic acid nanoparticles due to its simplicity in comparison to masses. It is the molar ratio of positively charged nitrogen atoms in the amino acids to the negatively charged phosphates contributed by the nucleic acid backbone and can be described as the mass of protein or peptide required to neutralise the charge of 1 μg of nucleic acid. The N:P ratio can be calculated using the following equation:

$N : P = \frac{MRALA}{MmRNA . CNP}$

where M_RALAis the mass of protein in the nanoparticle, M_mRNAis the mass of mRNA in the nanoparticle and C_NPis the N:P constant. The N:P constant is the ratio of the positive charge density of the amino acid chain to the negative charge density of the mRNA backbone with charge density being defined as the charge divided by the molecular mass. This N:P constant can be calculated based on the knowledge that arginine is the positively charged amino acids and that the mass and charge of the bases in the mRNA backbone are constant leaving only molecular mass of the protein and charge of the protein as variables. As such the N:P constant calculation can be simplified to:

$NP 1 = 1 \times \frac{MWRALA}{3 4 0 * QRALA} = \frac{3 3 2 7.9 8}{3 4 0 * 7} = 1.4 0$

Where Q_RALAis the positive charge provided by the seven arginine residues in the sequence of RALA, and 340 is the average molecular weight of an mRNA nucleoside.

For example an N:P ratio (peptide to mRNA ratio) of 1:1 is 1.40 μg peptide (SEQ ID_No 1):1 μg mRNA. An N:P ratio of 7:1 is 9.79 μg peptide (SEQ ID_No 1):1 μg mRNA, and for an N:P ratio of 9:1 is 12.6 μg peptide (SEQ ID_No 1):1 μg mRNA

In one embodiment, the N:P ratio of the amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine may be varied. This may have a beneficial effect on the physiochemical characteristics (for example the Z-Average, zeta potential (particle charge), and/or polydispersity index), the cellular uptake, and/or the treatment efficacy.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is about 1-12:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide is 1-12:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is about 5-12:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is 5-12:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is about 7-10:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is 7-10:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is about 8.5-9.5:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is 8.5-9.5:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is 1:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is about 1:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is 2:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is about 2:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is 3:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is about 3:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is 4:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is about 4:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is 5:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is about 5:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is 6:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is about 6:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is 7:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is about 7:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is 8:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is about 8:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is 9:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is about 9:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is 10:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is about 10:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is 11:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is about 11:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is 12:1.

In one embodiment the N:P ratio of amphipathic cell penetrating peptide:mRNA polynucleotide in the vaccine is about 12:1.

Bulking Agents, Cryoprotectants and Agents Inferring Tonicity

In one embodiment, a bulking agent may be added prior to lyophilisation of nanoparticles for transport and storage. Bulking agents are additives that increase the bulk-volume of a product without affecting its properties.

In one embodiment, a cryoprotectant may be added prior to lyophilisation of nanoparticles. A cryoprotectant is a substance used to protect biological material from freezing damage.

In one embodiment, a solute may be added to infer tonicity, e.g. to produce an isotonic formulation once water is added to the formulation. An isotonic formulation possesses the same concentration of solutes as the blood, i.e. 290-310 mOsmol/kg.

In one embodiment, the osmolality of a solution of a vaccine as described herein in water is 10-1000 mOsmol/kg.

In one embodiment, the osmolality of a solution of a vaccine as described herein in water is 100-500 mOsmol/kg.

In one embodiment, the osmolality of a solution of a vaccine as described herein in water is 200-400 mOsmol/kg.

In one embodiment, the osmolality of a solution of a vaccine as described herein in water is 290-310 mOsmol/kg.

In one embodiment, the osmolality of a solution of a vaccine as described herein in water is about 300 mOsmol/kg.

In one embodiment, the osmolality of a solution of a vaccine as described herein in water is 300 mOsmol/kg.

Suitable bulking agents include trehalose, sucrose, mannose, dextrose or any mixture of such agents. These agents may also be employed as cryoprotectants and/or agents to infer tonicity.

In one embodiment, the vaccines described herein additionally comprise trehalose, sucrose, mannose, dextrose or any mixture of such agents.

In one embodiment, the vaccines described herein additionally comprise >85% w/w trehalose, sucrose, mannose, dextrose or any mixture of such agents.

In one embodiment, the vaccines described herein additionally comprise >90% w/w trehalose, sucrose, mannose, dextrose or any mixture of such agents.

In one embodiment, the vaccines described herein additionally comprise >95% w/w trehalose, sucrose, mannose, dextrose or any mixture of such agents.

In one embodiment the vaccine described herein comprises a bulking agent.

In one embodiment the vaccine described herein comprises a bulking agent selected from trehalose, sucrose, mannose and dextrose.

In one embodiment the vaccine described herein comprises trehalose.

In one embodiment the vaccines described herein additionally comprise trehalose.

In one embodiment the vaccines described herein additionally comprise >85% w/w trehalose.

In one embodiment the vaccines described herein additionally comprise >90% w/w trehalose.

In one embodiment the vaccines described herein additionally comprise >95% w/w trehalose.

In one embodiment the vaccines described herein additionally comprise >98% w/w trehalose.

In one embodiment the vaccines described herein additionally comprise about 99% w/w trehalose.

In one embodiment the vaccines described herein additionally comprise 99% w/w trehalose.

In one embodiment the vaccine described herein comprises sucrose.

In one embodiment the vaccines described herein additionally comprise sucrose.

In one embodiment the vaccines described herein additionally comprise >85% w/w sucrose.

In one embodiment the vaccines described herein additionally comprise >90% w/w sucrose.

In one embodiment the vaccines described herein additionally comprise >95% w/w sucrose.

In one embodiment the vaccine described herein comprises mannose.

In one embodiment the vaccines described herein additionally comprise mannose.

In one embodiment the vaccines described herein additionally comprise >85% w/w mannose.

In one embodiment the vaccines described herein additionally comprise >90% w/w mannose.

In one embodiment the vaccines described herein additionally comprise >95% w/w mannose.

In one embodiment the vaccine described herein comprises dextrose.

In one embodiment the vaccine described herein comprises mannose and trehalose.

In one embodiment the vaccine described herein comprises 5-20% w/v of mannose and trehalose. 5-20% w/v of mannose and trehalose means 5-20% w/v (mannose plus trehalose).

In one embodiment the vaccine described herein comprises about 10% w/v of mannose and trehalose.

In one embodiment the vaccine described herein comprises 10% w/v of mannose and trehalose.

In one embodiment the vaccine described herein comprises trehalose and mannose in an 85:15 w/v ratio.

In one embodiment the vaccine described herein comprises trehalose and mannose in about an 85:15 w/v ratio.

In one embodiment the vaccine described herein comprises trehalose and mannose in a 70:30 w/v ratio.

In one embodiment the vaccine described herein comprises trehalose and mannose in about a 70:30 w/v ratio.

In one embodiment the vaccine described herein comprises 5-20% w/v of trehalose and mannose in about an 85:15 w/v ratio.

In one embodiment the vaccine described herein comprises 5-20% w/v of trehalose and mannose in about a 70:30 w/v ratio.

In one embodiment the vaccine described herein comprises about a 10% w/v of trehalose and mannose in about an 85:15 w/v ratio.

In one embodiment the vaccine described herein comprises about a 10% w/v of trehalose and mannose in about a 70:30 w/v ratio.