MERS vaccine antigen

Abstract

The field of the specification relates broadly to Middle East respiratory syndrome coronavirus vaccine (MERS-CoV) antigens and methods of using and manufacturing MERS-CoV antigens. The invention also relates to vaccines, kits, devices and strips comprising the MERS-CoV antigen. The invention also relates to ribonucleic acids encoding a S protein monomer of a coronavirus vaccine (MERS-CoV) antigen and methods of using and manufacturing the ribonucleic acid. The invention also relates to vectors, lipid nanoparticles, RNA vaccines, kits, devices and strips comprising the ribonucleic acid.

Description

[0001] MERS VACCINE ANTIGEN

[0002] FIELD

[0003] The field of the specification relates broadly to Middle East respiratory syndrome coronavirus vaccine (MERS-CoV) antigens and methods of using and manufacturing MERS-CoV antigens. The invention also relates to vaccines, kits, devices and strips comprising the MERS-CoV antigen. The invention also relates to ribonucleic acids encoding a S protein monomer of a coronavirus vaccine (MERS-CoV) antigen and methods of using and manufacturing the ribonucleic acid. The invention also relates to vectors, lipid nanoparticles, RNA vaccines, kits, devices and strips comprising the ribonucleic acid.

[0004] BACKGROUND

[0005] The Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a lethal zoonotic pathogen that emerged in Saudi Arabia in 2012. MERS CoV causes severe respiratory symptoms with a case fatality rate of ~36%. As of June 2024, 2,625 cases of MERS, including 951 deaths, have been reported by health authorities worldwide. The most recent deaths due to MERS CoV were in July 2024. Human transmission of MERS-CoV can occur via direct or indirect contact with infected dromedary camels, the host reservoir species, or with infected patients. High MERS CoV seroprevalence rates have been found in Saudi and Sudanese dromedary camels (87-93% seropositivity) (Tolah et al., 2020) as well as in camel workers with well-documented camel exposure (~50% seropositivity) (Alshukari et al., 2018). Intermittent sporadic cases continue to occur in the Middle East with considerable risk of spreading globally. MERS-CoV is thus identified by the WHO and CEPI as a priority pathogen.

[0006] MERS-CoV is a member of the betacoronavirus (pCoV) genus of the Coronaviridae family of viruses. MERS CoV-related viruses are predominantly found in bats suggesting that the virus originated and evolved in bats prior to crossing over into dromedary camels and then into humans in 2012 (Wong et al., 2019). MERS CoV-like viruses have also been found in game species that come into regular contact with humans such as hedgehogs and pangolins (Corman et al., 2014; Chen et al., 2023) providing further avenues fora spill-over event. Livestock animals such as alpacas, llamas and pigs can be experimentally infected with MERS CoV as can laboratory animals including macaques (Yao et al., 2014) and human dipeptidyl peptidase 4 (DPP4)-transgenic mice (van Doremalen et al., 2015). MERS CoV and its relatives comprise the merbecovirus subgenus of the pCoVs which has been further subdivided into 4 clades based on clustering of receptor binding domain (RBD) sequences (Catanzaro et al., 2024). The RBD is the domain of the viral spike glycoprotein (S) that is responsible for initiating infection by binding to cellular receptors. Clade 1 members, which includes MERS CoV use DPP4 as the entry receptor whereas clades 2, 3 and 4 merbecoviruses use ACE2.

[0007] An effective MERS CoV vaccine is not yet available for human use. DNA (Modjarrad et al., 2019) and viral vector (ChAdOxI [Bosaeed et al., 2022], and modified vaccinia Ankara [Koch et al., 2020]) vaccines based on the spike glycoprotein have reached human trials, while other vaccine modalities have shown promise in animal models. These experimental modalities include recombinant subunit vaccines: clamp-stabilised soluble trimeric spike (Pallesen et al., 2017; Ahuja et al., 2024; Wang et al., 2024; Chang et al., 2023) and various forms of recombinant RBD (Dai et al., 2020; Martinez et al., 2023), virus-like particles (Park et al., 2022), S nanoparticles (Coleman et al., 2017; Hutchison et al., 2023), mRNA encoding RBD (Tai et al., 2023), and rhabdovirus vectored vaccines (Kato et al., 2019; Liu et al., 2018; Chi et al., 2022).

[0008] Human-to-human transmission of MERS-CoV appears to be an inefficient process, generally occurring in healthcare settings and among family members (Memish et al., 2020). MERS CoV is thus genetically stable when compared to SARS CoV-2 (Lau et al., 2017; Markov et al., 2023). The high transmissibility of SARS CoV-2 and its associated rapid sequence diversification in humans enables immune escape. SARS CoV-2 spike based vaccines therefore require periodic updating to retain their effectiveness against newly emerged variants. In the case of SARS CoV-2, protective neutralizing antibody responses are largely directed to the region around the receptor binding motif which is a hot spot of amino acid variation due to immune selection (Dadoinate et al., 2024). This would likely be an issue if MERS CoV were to acquire the mutations required for efficient human-to-human transmission leading to an epidemic or pandemic.

[0009] The trimeric CoV spike glycoprotein (S) is the sole target of protective neutralizing antibodies (NAbs) and forms the basis of successful SARS CoV-2 vaccines: full-length S delivered either as mRNA (Pfizer-BioNTech, Moderna) or as nanoparticles (Novavax). While successful in ameliorating the effects of the COVID-19 pandemic, vaccinal immunity wanes over 2-6 months, requiring periodic boosting with updated vaccines, and their high retail cost (over USD100 / dose) limits access. While these vaccine modalities could theoretically be used in a MERS epidemic or pandemic, the availability of simpler alternative vaccine modalities would be beneficial. Soluble trimeric S subunit vaccines are one such modality as they include conserved and variable neutralization epitopes in the context of a native-like spike. However, their development can be problematic due to their low stability, necessitating the use of trimerization clamps derived from foreign proteins, such as T4 phage foldon to maintain quaternary structure. These clamps can elicit off-target antibody responses that hamper the passage of clamped trimer vaccine candidates to licensure for use in humans (Sliepen et al., 2015; Chappell et al., 2021). Thus, there is a need for improved antigens for eliciting immune responses to MERS coronaviruses. In particular, vaccine antigens with one or more of improved stability, melting temperature, immunogenicity, antigenicity and production yield.

[0010] SUMMARY OF THE DISCLOSURE

[0011] In an aspect, the present invention provides a Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen comprising a MERS-CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond.

[0012] In an embodiment, the non-endogenous inter-protomer disulfide bond is formed between cysteines selected from: i) cysteines at a position corresponding to amino acid numbers 429 and 1059 of SEQ ID NO: 1 (F14); and / or ii) cysteines at a position corresponding to amino acid numbers 580 and 60 of SEQ ID NO: 1 (A1).

[0013] In an aspect, the present invention provides a Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen comprising a MERS-CoV S protein trimer with a C-terminal truncation in the stem region.

[0014] In an aspect, the present invention provides a protein nanoparticle comprising the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen as described herein.

[0015] In an aspect, the present invention provides a virus-like particle comprising the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen as described herein.

[0016] In an aspect, the present invention provides a deoxyribonucleic acid encoding the Middle East respiratory syndrome coronavirus (MERS-CoV) antigen as described herein.

[0017] In an aspect, the present invention provides a ribonucleic acid encoding the Middle East respiratory syndrome coronavirus (MERS-CoV) antigen as described herein.

[0018] In an aspect, the present invention provides a ribonucleic acid encoding a S protein monomer of a Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen wherein the vaccine antigen comprises a MERS-CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond.

[0019] In an aspect, the present invention provides a vector comprising the deoxyribonucleic acid as described herein.

[0020] In an aspect, the present invention provides a host cell comprising the deoxyribonucleic acid as described herein or the vector as described herein.

[0021] In an aspect, the present invention provides a method of producing the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen as described herein comprising culturing the host cell as described herein in culture medium to produce the vaccine antigen.

[0022] In an aspect, the present invention provides a method of producing a Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine comprising culturing the host cell as described herein in culture medium to produce the ribonucleic acid as described herein or the deoxyribonucleic acid as described herein.

[0023] In an aspect, the present invention provides a vaccine comprising the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen as described herein, or the protein nanoparticle as described herein, or the virus-like particle as described herein, or the deoxyribonucleic acid as described herein, or the ribonucleic acid as described herein or the vector as described herein.

[0024] In an aspect, the present invention provides a vaccine comprising the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen as described herein, or the protein nanoparticle as described herein, or the virus-like particle as described herein.

[0025] In an embodiment, the vaccine is a tetravalent vaccine.

[0026] In an aspect, the present invention provides a vaccine comprising a merbecovirus vaccine antigen, a SARS-CoV2 vaccine antigen, a clade 1a sarbecovirus vaccine antigen, and a clade 3 sarbecovirus vaccine antigen.

[0027] In an embodiment, the merbecovirus vaccine antigen, SAR-CoV2 vaccine antigen, clade 1a sarbecovirus vaccine antigen and the clade 3 sarbecovirus vaccine antigen comprise at least one non-endogenous inter-protomer disulfide bond and / or a C-terminal truncation in the stem region.

[0028] In an embodiment, the merbecovirus vaccine antigen, SAR-CoV2 vaccine antigen, clade 1a sarbecovirus vaccine antigen and the clade 3 sarbecovirus vaccine antigen comprise at least one non-endogenous inter-protomer disulfide bond and a C-terminal truncation in the stem region.

[0029] In an aspect, the present invention provides a method of inducing an immune response to a Middle East respiratory syndrome coronavirus (MERS-CoV) in a subject, the method comprising delivering the vaccine antigen as described herein, the ribonucleic acid as described herein, the deoxyribonucleic acid as described herein or the vaccine as described herein to a subject.

[0030] In an aspect, the present invention provides a method of enhancing the immune response to a Middle East respiratory syndrome coronavirus (MERS-CoV) in a subject, the method comprising delivering the vaccine antigen as described herein, the ribonucleic acid as described herein, the deoxyribonucleic acid as described herein or the vaccine as described herein to a subject.

[0031] In an aspect, the present invention provides a method of preventing or reducing the likelihood of a Middle East respiratory syndrome coronavirus (MERS-CoV) infection in a subject, the method comprising delivering the vaccine antigen as described herein, the ribonucleic acid as described herein, the deoxyribonucleic acid as described herein or the vaccine as described herein to a subject.

[0032] In an aspect, the present invention provides a method of preventing, or reducing the likelihood or severity of a symptom of a Middle East respiratory syndrome coronavirus (MERS-CoV) infection in a subject, the method comprising delivering the vaccine antigen as described herein, the ribonucleic acid as described herein, the deoxyribonucleic acid as described herein or the vaccine as described herein to a subject.

[0033] In an aspect, the present invention provides a method of reducing the severity and / or duration of a Middle East respiratory syndrome coronavirus (MERS-CoV) infection in a subject, the method comprising delivering the vaccine antigen as described herein, the ribonucleic acid as described herein, the deoxyribonucleic acid as described herein or the vaccine as described herein to a subject.

[0034] In an aspect, the present invention provides a method of preventing or reducing viral shedding in a human individual infected with a Middle East respiratory syndrome coronavirus (MERS-CoV), the method comprising delivering the vaccine antigen as described herein, the ribonucleic acid as described herein, the deoxyribonucleic acid as described herein or the vaccine as described herein to a subject.

[0035] In an aspect, the present invention provides a method of treating a Middle East respiratory syndrome coronavirus (MERS-CoV) infection in a subject, the method comprising delivering the vaccine antigen as described herein, the ribonucleic acid as described herein, the deoxyribonucleic acid as described herein or the vaccine as described herein to a subject.ln an aspect, the present invention provides the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen as described herein, the ribonucleic acid as described herein, the deoxyribonucleic acid as described herein or the vaccine as described herein for use in one or more of: i) inducing an immune response to a MERS-CoV in a subject; ii) enhancing the immune response to a MERS-CoV in a subject; iii) preventing or reducing the likelihood of a MERS-CoV infection in a subject; iv) preventing or reducing the likelihood of severity of a MERS-CoV symptom in a subject; v) reducing the severity and / or duration of a MERS-CoV infection in a subject; vi) preventing or reducing viral shedding in a subject; and vii) treating a MERS-CoV infection in a subject.

[0036] In an aspect, the present invention provides kit, device, surface or strip comprising the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen as described herein or the ribonucleic acid as described herein.

[0037] In an aspect, the present invention provides use of the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen as described herein in the manufacture of a medicament for one or more of: i) inducing an immune response to a MERS-CoV in a subject; ii) enhancing the immune response to a MERS-CoV in a subject; iii) preventing or reducing the likelihood of a MERS-CoV infection in a subject; iv) preventing or reducing the likelihood of severity of a MERS-CoV symptom in a subject; v) reducing the severity and / or duration of a MERS-CoV infection in a subject; vi) preventing or reducing viral shedding in a subject; and vii) treating a MERS-CoV infection in a subject.

[0038] In an aspect, the present invention provides a method of increasing S protein trimer yield comprising modifying the MERS-CoV S protein trimer to comprise a stem region C-terminal truncation.

[0039] In an aspect, the present invention provides a method of stabilizing a MERS-CoV S protein trimer in a prefusion conformation comprising modifying the MERS-CoV S protein trimer to comprise at least one non-endogenous inter-protomer disulfide bond.

[0040] In an aspect, the present invention provides a method of increasing the melting temperature of a MERS-CoV S protein trimer comprising modifying the MERS-CoV S protein trimer to comprise a stem region C-terminal truncation.

[0041] In an aspect, the present invention provides a method of increasing the melting temperature of a MERS-CoV S protein trimer stabilised in the prefusion conformation comprising modifying the MERS-CoV S protein trimer to comprise a stem region C-terminal truncation.

[0042] In an aspect, the present invention provides a method of enhancing neutralising antibody responses comprising modifying the MERS-CoV S protein trimer to comprise at least one interprotomer disulfide bond and / or modifying the MERS-CoV S protein trimer to comprise a stem region C-terminal truncation.

[0043] In an aspect, the present invention provides a ribonucleic acid encoding a S protein monomer of a Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen wherein the vaccine antigen comprises a MERS-CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond.

[0044] In an embodiment, the non-endogenous inter-protomer disulfide bond is formed between cysteines selected from: i) cysteines at a position corresponding to amino acid numbers 429 and 1059 of SEQ ID NO: 1 (F14); and / or ii) cysteines at a position corresponding to amino acid numbers 580 and 60 of SEQ ID NO: 1 (A1).

[0045] In an aspect, the present invention provides a ribonucleic acid encoding a S protein monomer of a Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen wherein the vaccine antigen is a MERS-CoV S protein trimer and wherein the S protein monomer of the MERS-CoV S protein trimer has a C-terminal truncation in the stem region.

[0046] In an aspect, the present invention provides a vector comprising the ribonucleic acid as described herein. In an aspect, the present invention provides a lipid nanoparticle comprising the ribonucleic acid as described herein.

[0047] In an aspect, the present invention provides a host cell comprising the ribonucleic acid as described herein or the vector as described herein.

[0048] In an aspect, the present invention provides a method of producing a coronavirus vaccine comprising culturing the host cell as described herein in culture medium to produce the ribonucleic acid described herein.

[0049] In an aspect, the present invention provides a RNA vaccine comprising the ribonucleic acid as described herein, orthe vector as described herein, orthe lipid nanoparticle as described herein.

[0050] In an aspect, the present invention provides a method of inducing an immune response to a MERS-CoV in a subject, the method comprising delivering the ribonucleic acid as described herein or RNA vaccine as described herein to a subject.

[0051] In an aspect, the present invention provides a method of enhancing the immune response to MERS-CoV in a subject, the method comprising delivering the ribonucleic acid as described herein or RNA vaccine as described herein to a subject.

[0052] In an aspect, the present invention provides a method of preventing or reducing the likelihood of a MERS-CoV infection in a subject, the method comprising delivering ribonucleic acid as described herein or RNA vaccine as described herein to a subject.

[0053] In an aspect, the present invention provides a method of preventing, or reducing the likelihood or severity of a symptom of a MERS-CoV infection in a subject, the method comprising delivering the ribonucleic acid as described herein or RNA vaccine as described herein to a subject.

[0054] In an aspect, the present invention provides a method of reducing the severity and / or duration of a MERS-CoV infection in a subject, the method comprising delivering the RNA vaccine ribonucleic acid as described herein or RNA vaccine as described herein to a subject.

[0055] In an aspect, the present invention provides a method of preventing or reducing viral shedding in a human individual infected with a MERS-CoV, the method comprising delivering the ribonucleic acid as described herein or RNA vaccine as described herein to a subject.

[0056] In an aspect, the present invention provides a ribonucleic acid as described herein orthe RNA vaccine as described herein in one or more of: i) inducing an immune response to a MERS-CoV in a subject; ii) enhancing the immune response to a MERS-CoV in a subject; iii) preventing or reducing the likelihood of a MERS-CoV infection in a subject; iv) preventing or reducing the likelihood of severity of a MERS-CoV symptom in a subject; v) reducing the severity and / or duration of a MERS-CoV infection in a subject; vi) preventing or reducing viral shedding in a subject; and vii) treating a MERS-CoV infection in a subject. In an aspect, the present invention provides a kit, device, surface or strip comprising the Middle East respiratory syndrome coronavirus (MERS-CoV) ribonucleic acid as described herein.

[0057] In an aspect, the present invention provides use of the MERS-CoV ribonucleic acid as described herein in the manufacture of a medicament for one or more of: i) inducing an immune response to a MERS-CoV in a subject; ii) enhancing the immune response to a MERS-CoV in a subject; iii) preventing or reducing the likelihood of a MERS-CoV infection in a subject; iv) preventing or reducing the likelihood of severity of a MERS-CoV symptom in a subject; v) reducing the severity and / or duration of a MERS-CoV infection in a subject; vi) preventing or reducing viral shedding in a subject; and vii) treating a MERS-CoV infection in a subject.

[0058] In an aspect, the present invention provides a method of producing a CoV vaccine, the method comprising: (a) providing (i) a sugar; (ii) at least one buffering agent selected from one or more of: acetate, succinate, citrate, prolamine, arginine, glycine, histidine, borate, carbonate and phosphate and (iii) at least one CoV vaccine antigen; (b) combining (a) together to create a liquid formulation; (c) cooling the liquid formulation to below a freezing state in (b) to create a frozen formulation; and (d) lyophilizing the frozen formulation in (c) to create the CoV vaccine.

[0059] In an aspect, the present invention provides a method of producing a MERS-CoV vaccine, the method comprising: (a) providing (i) a sugar; (ii) at least one buffering agent selected from one or more of: acetate, succinate, citrate, prolamine, arginine, glycine, histidine, borate, carbonate and phosphate and (iii) at least one MERS-CoV vaccine antigen; (b) combining (a) together to create a liquid formulation; (c) cooling the liquid formulation to below a freezing state in (b) to create a frozen formulation; and (d) lyophilizing the frozen formulation in (c) to create the MERS-CoV vaccine.

[0060] Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise. For instance, as the skilled person would understand examples of non-endogenous inter-protomer disulfide bonds outlined above for the vaccine antigen of the invention equally apply to the ribonucleic acid encoding a S protein monomer of a Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen.

[0061] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

[0062] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter. The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

[0063] BRIEF DESCRIPTION OF THE FIGURES

[0064] Figure 1 Shows the linear schematic of the MERS spike showing key structural and functional elements. A) the full-length MERS CoV spike. B) the spike ectodomain comprising the leader peptide (amino acids 1-17), the head (amino acids 18-1219) and stem (amino acids 1220-1296). L: leader peptide; NTD: N-terminal domain; RBD: receptor binding domain; RBM: DPP4 receptor-binding motif; FP: fusion peptide; HR1: heptad repeat 1; 2P: diPro substitution; CH: central helix; MSS: membrane spanning sequence. C) Alignment of betacoronavirus stem sequences. MERS CoV spike amino acid numbers are at the top of the alignment and (except for 1221) indicate the C-terminal amino acid of the Spike truncation mutants described in Figures 2-4. SARS CoV-2 stem truncation points shown to improve soluble trimer yield in PCT / AU2024 / 050878 Vaccine Antigen are indicated at the bottom of the alignment. Conserved amino acids are highlighted in black, partially conserved amino acids in grey. A dot (.) indicates an amino acid deletion.

[0065] Figure 2 Shows the amino acid sequence of the MERS1 (Betacoronavirus England 1) spike (Genbank accession number AFY13307).

[0066] Figure 3 Shows the amino acid sequence of the MERS1 spike containing the ‘2P’ (V1060P / L1061P) mutation.

[0067] Figure 4 Shows the amino acid sequences of M2P glycoprotein stem truncation mutants. The C-terminal Gly-Ser-Gly-Ser-His6 sequence added to the MERS spike has not been included.

[0068] Figure 5 Shows DNA sequences encoding the M2P glycoprotein stem truncation mutants listed in Figure 2. The Gly-Ser-His6 sequence added to the C-terminal amino acid of the spike truncation mutants has not been included.

[0069] Figure 6 Shows the biochemical characteristics of secreted prefusion-stabilized MERS1 glycoproteins (M2P) terminating at K1240, N1248, D1258, E1265, Q1270, A1275. A) Superose 6 size exclusion chromatography (SEC) of total secreted spike protein obtained from 50 ml of Expi293F culture by affinity purification on TALON resin. The molecular weight markers are thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa) and ovalbumin (43 kDa). B) SEC of purified trimeric Spikes following a freeze (-80°C)-thaw cycle. C) Melting temperature of purified Spike trimers determined by differential scanning fluorimetry (DSF). D) SDS-PAGE under reducing conditions of purified Spike trimers. The protein bands were visualised following Coomassie blue staining, m, molecular weight markers. E) Yields of purified M2P trimers.

[0070] Figure 7 Shows the antigenic characteristic of purified spike trimers determined in biolayer interferometry with neutralizing ligands directed to the RBM: DPP4-Fc, CDC2-C2, KNIH90-F1; RBDup / non-RBM: MERS-4; NTD: 7D10, G2; and Stem / Pan-beta: CC95-108, CC99-103. The neutralizing ligands were immobilized on anti-human IgG Fc capture biosensors and the M2P trimers (30 nM) were in the analyte phase. Association was for the first 300 sec and dissociation was for the second 300 sec. The location of the epitope of each ligand is indicated in brackets. ‘Pan-beta’ indicates that a particular antibody has pan-betacoronavirus neutralizing activity.

[0071] Figure 8 Shows the location of paired Cys substitutions in the MERS1 spike trimer.

[0072] The model was drawn with PYMOL using the coordinates PDB ID 5X5F. The amino acid pairs that were replaced with Cys are shown in CPK and identified by the code used in Table 1. The amino acids shown in CPK and coloured black are in chain B while those coloured in grey are in chain C. The model at bottom was rotated ~90° to the right in relation to the model at top.

[0073] Figure 9 Shows the amino acid sequences of M2P-1258 glycoproteins containing diCys mutations. The mutants are coded according to Table 1 and Figure 8. MERS CoV MERS1 sequences are shown. The non-native C-terminal Gly-Ser-His6 sequence has not been included.

[0074] Figure 10 Shows DNA sequences encoding the M2P-1258 glycoproteins containing diCys mutations listed in Figure 8. The mutants are coded according to Table 1 and Figure 8. MERS CoV MERS1 sequences are shown. The DNA sequence encoding the non-native C-terminal Gly-Ser-His6 sequence has not been included.

[0075] Figure 11 Shows the biochemical characteristics of secreted Cys-substituted M2P-1258 glycoproteins. Superose 6 size exclusion chromatography (SEC) of total secreted M2P-1258 glycoproteins containing di-Cys mutations following affinity purification using TALON resin. The mutant code is shown in each chromatogram. The vertical dotted lines indicate fractions that were collected, pooled and concentrated for further analysis. The elution positions of thyroglobulin (669 kDa) and ferritin (440 kDa) are indicated by arrows at the top of the chromatogram.

[0076] Figure 12 Shows the SDS-PAGE of purified M2P-1258 spikes trimers in the presence (reducing) and absence (nonreducing) of reducing agent (betamercaptoethanol). The arrow shows the expected position of a disulfide-stabilised spike trimer. m, molecular weight markers. Figure 13 Shows the Biochemical characteristics of secreted M2P-1258 proteins carrying the A1 and F14 mutations. A) Superose 6 elution profiles of purified M2P-1258. A1 and M2P-1258. F14 after a freeze-thaw cycle. The vertical dotted lines indicate the elution positions of thyroglobulin (669 kDa) and ferritin (440 kDa). B) Thermal unfolding and melting temperature of purified M2P-1258. A1 and M2P-1258. F14 trimers in DSF. C) SDS-PAGE of purified M2P-1258. A1 and M2P-1258. F14 spikes trimers in the presence (reducing) and absence (nonreducing) of reducing agent (betamercaptoethanol). dslS and monS shows the expected position of disulfide-stabilised spike trimer, and unlinked monomer respectively, m, molecular weight markers.

[0077] Figure 14 Shows the antigenic characteristics of purified M2P-1258. A1 and M2P-1258. F14 spike trimers determined in biolayer interferometry with neutralizing ligands.

[0078] The neutralizing ligands were immobilized on anti-human IgG Fc capture biosensors and the spike trimers (30 nM) were in the analyte phase. Association was for the first 300 sec and dissociation was for the second 300 sec.

[0079] Figure 15 Shows the structural context of the A1 and F14 disulfides in the MERS spike (PDB ID 5X5F) structure. A) A1: Cys-60 of the NTD is bonded to Cys-580 of the RBD of an adjacent monomer; F14: Cys-429 of the RBD is bonded to Cys-1059 at the top of the S2 central helix of an adjacent monomer next to the 2P mutation (V1060P / L1061P). B) The F14 disulfide is close to the ‘hinge’ between the RBD and subdomain 1 (SD1) of S1.

[0080] Figure 16 Shows the A1 and F14 disulfides in the context of the 3-fold symmetry axis of the spike trimer, when viewed from the top of a trimer in the 3RBD-down conformation (PDB ID 7YN0).

[0081] Figure 17 Shows theoretical mRNA sequences encoding M2P-1258 glycoprotein variants. The RNA sequence encoding the non-native N-terminal leader peptide and C-terminal Gly-Ser-His6 sequence has not been included.

[0082] Figure 18 Shows the amino acid sequences of S6P. BA286-1192 glycoproteins. The non-native N-terminal leader peptide and C-terminal Gly-Ser-His6 sequence has not been included.

[0083] Figure 19 Shows DNA sequences encoding the S6P. BA286-1192 glycoproteins.

[0084] The DNA sequence encoding the non-native N-terminal leader peptide and C-terminal Gly-Ser-His6 sequence has not been included.

[0085] Figure 20 Shows the biochemical and antigenic analysis of purified trimeric parental, D17 (D571C / S967C) and 11+VI (V570C / S967C / A1016V / A1020l)-mutated S6P. BA286-1192 glycoproteins treated at 37°C for up to 28 days. A) Superose 6 SEC. B) DSF. The arrows indicate a novel glycoprotein species that appears after 7 days of treatment of the parental trimer. C) Reactivity with ACE2-Fc and NAbs in BLI; D) SDS-PAGE under nonreducing and reducing conditions. The gels were stained with Coomassie brilliant blue dye.

[0086] Figure 21 Shows the amino acid sequences of expected omicron BA.2.86 mature spike glycoproteins carrying the 6P mutation and truncated at N1192 without and with the D17 mutation. The non-native N-terminal tPAL and linking amino acids (AlaSer), and C-terminal Gly-Ser-His6 sequence have not been included.

[0087] Figure 22 Shows the DNA sequences of expected omicron BA.2.86 mature spike glycoproteins carrying the 6P mutation and truncated at N1192 without and with the D17 mutation. The non-native N-terminal tPAL and linking amino acids (AlaSer), and C-terminal Gly-Ser-His6 sequence have not been included.

[0088] Figure 23 Shows the amino acid sequences of expected PRD-0038 mature spike glycoproteins carrying the 6P mutation and truncated at the SARS CoV-2 S N1192-equivalent amino acid without and with the D17 mutation. The non-native N-terminal tPAL and linking amino acids (AlaSer), and C-terminal Gly-Ser-His6 sequence have not been included.

[0089] Figure 24 Shows the DNA sequences of expected PRD-0038 mature spike glycoproteins carrying the 6P mutation and truncated at the SARS CoV-2 S N1192-equivalent amino acid without and with the D17 mutation. The non-native N-terminal tPAL and linking amino acids (AlaSer), and C-terminal Gly-Ser-His6 sequence have not been included.

[0090] Figure 25 Shows the amino acid sequences of expected WIV1 mature spike glycoproteins carrying the 6P mutation and truncated at the SARS CoV-2 S N1192-equivalent amino acid without and with the D17 mutation. The non-native N-terminal tPAL and linking amino acids (AlaSer), and C-terminal Gly-Ser-His6 sequence have not been included.

[0091] Figure 26 Shows the DNA sequences of expected WIV1 mature spike glycoproteins carrying the 6P mutation and truncated at the SARS CoV-2 S N1192-equivalent amino acid without and with the D17 mutation. The non-native N-terminal tPAL and linking amino acids (AlaSer), and C-terminal Gly-Ser-His6 sequence have not been included.

[0092] Figure 27 Shows the amino acid sequence of the full-length MERS CoV Englandl spike. Figure 28 Shows the DNA sequence of the full-length MERS CoV Englandl spike.

[0093] Figure 29 Shows the C57BL / 6 immunization protocol. A) Animal groups; B) Immunization schedule.

[0094] Figure 30 Shows the neutralization activity of sera obtained after 3 immunizations with experimental vaccines against MERS CoV S-HIV pseudoviruses. Neutralization ID90s (reciprocal of the serum dilution required for 90% inhibition of infection) obtained with sera from individual animals are indicated with various symbols. The bars are geometric mean ID90s. The geometric mean pseudovirus neutralization ID90 of the control group receiving 3 doses of 50% Addavax-PBS was <20. ns, not significant; **, P < 0.01: ***, P < 0.001; ****, P < 0.0001, Kruskal-Wallis test.

[0095] Figure 31 Shows ELISA binding titres of sera obtained after 3 immunizations with experimental vaccines to purified spike trimers derived from SARS CoV-2 Omicron BA.2.86, MERS CoV Englandl, and bat sarbecoviruses WIV1 and PRD-0038. The variant RBDs are indicated above the graphs and the vehicle control and vaccine groups are indicated below the x axis. The endpoint was determined as 50-times background luciferase activity obtained in the absence of primary antibody. The horizontal bars are the geometric means. A Kruskal-Wallis test was used to determine whether the differences in binding titres are significant, ns, not significant; *, P < 0.05; **, P < 0.01: ***, P < 0.001; ****, P < 0.0001.

[0096] Figure 32 Shows ELISA binding titres of sera obtained after 3 immunizations with experimental vaccines to RBDs derived from SARS CoV-2 Omicron JN.1, MERS CoV Englandl, and bat sarbecoviruses WIV1 and PRD-0038. The variant RBDs are indicated above the graphs and the vehicle control and vaccine groups are indicated below the x axis. The endpoint was determined as 50-times background luciferase activity obtained in the absence of primary antibody. The horizontal bars are the geometric means. A Kruskal-Wallis test was used to determine whether the differences in binding titres are significant, ns, not significant; *, P < 0.05; **, P < 0.01: ***, P < 0.001; ****, P < 0.0001.

[0097] Figure 33 Shows ELISA binding titres of sera obtained after 3 immunizations with experimental vaccines to the stem region of the ancestral SARS CoV-2 Spike. Left, maltose-binding protein (MBP)-(S amino acids 1138-1208) chimeric protein; Right, Stem synthetic peptide (S amino acids 1138-1165); The vehicle control and vaccine groups are indicated below the x axis. The endpoint was determined as 50-times background luciferase activity obtained in the absence of primary antibody. The horizontal bars are the geometric means. A Kruskal-Wallis test was used to determine whetherthe differences in binding titres are significant, ns, not significant; *, P < 0.05; ****, P < 0.0001.

[0098] Figure 34 Shows Superose 6 SEC analysis of purified parental, and F14 -mutated M2P-1258 oligomers treated at 37°C for up to 28 days.

[0099] Figure 35 Shows the neutralization activity of sera obtained after 3 immunizations with experimental vaccines against authentic SARS CoV-2 omicron JN.1 virus.

[0100] Neutralization ID50s (reciprocal of the serum dilution required for 50% inhibition of infection) obtained with sera from individual animals are indicated with various symbols. The bars are geometric mean ID50s. The geometric mean virus neutralization ID50 of the control group receiving 3 doses of 50% Addavax-PBS was <20. ns, not significant; **, P < 0.01; ****, P < 0.0001, Kruskal-Wallis test.

[0101] Figure 36 Shows the C57BL / 6 immunization protocol in which 2 doses or 1 dose of monovalent or tetravalent spike vaccine formulations were administered.

[0102] A) Animal groups; B) Immunization schedule.

[0103] Figure 37 Shows the neutralization activity of sera obtained after 1, 2 or 3 immunizations with experimental vaccines against HIV pseudoviruses containing betacoronavirus spike glycoproteins or authentic SARS CoV-2. Data from the experiment outlined in Figure 36 are shown to the left of the vertical dotted line, while data from the experiment summarised in Figure 24 are shown to the right of the vertical dotted line. MERS CoV, NeoCoV, SARS CoV (Tor2), WIV1, PRD-0038 and BtKY72. KW at the top of graphs indicates that S-HIV pseudoparticles were used in the assay. SARS CoV-2 (JN.1) and SARS CoV-2 (XEC) at the top of graphs indicates that authentic viruses were used in the assay. The immunogens administered to mice are indicated under the x axis. Tet_2d, Tet_1d and Tet_3d indicate sera from mouse groups receiving 2, 1 and 3 doses, respectively of the tetravalent formulation described in Figures 36 and 24. Neutralization ID50s (reciprocal of the serum dilution required for 50% inhibition of infection) obtained with sera from individual animals are indicated with various symbols. The bars are geometric mean ID50s. The geometric mean neutralization ID50 of the control groups receiving 50% Addavax-PBS was <20. ns, not significant; *, P < 0.05; **, P < 0.01: ***, P < 0.001; ****, P < 0.0001, Kruskal-Wallis test, nd, not determined.

[0104] Figure 38 Shows the thermostability at 37°C of M2P-1258 oligomers without and with the A1 and F14 disulfide mutations. A) Superose 6 SEC analysis of purified parental, and A1- and F14-mutated S trimers treated at 37°C for up to 84 days. B) SDS-PAGE of 37°C-treated parental and F14-mutated M2P-1258 oligomers sampled at different time points. -pME, nonreducing, +pME, reducing conditions. dslS: disulfide-linked spike; monS monomeric spike.

[0105] Figure 39 Shows an alignment of the MERS CoV Englandl and MjHKU4r CoV-1 spikes. The alignment was generated with clustal Q. The RBD is underlined. S429 and D1059 of MERS CoV S and the equivalent amino acids of MjHKU4r CoV-1 S which were substituted with Cys to give the F14 mutant are highlighted with black boxes and white type. T1258 of MERS CoV S and the equivalent amino acid of MjHKU4r CoV-1 S which are the amino acids at which the stem was truncated are highlighted in grey. *, identical amino acids; highly similar amino acids;., similar amino acids; deletion. The Genbank accession numbers of the spike sequences are: AFY13307 for MERS CoV, and OQ786861.1 for MjHKU4r CoV-1.

[0106] Figure 40 Shows the amino acid sequences of MjH2P-1258 (parental) and F14 glycoproteins. The C-terminal Gly-Ser-Gly-Ser-His6 sequence added to the MjH2P-1258 spikes has not been included.

[0107] Figure 41 Shows DNA sequences encoding the MjH2P-1258 (parental) and MjH2P-1258. F14 glycoproteins. The Gly-Ser-His6 sequence added to the C-terminal amino acid of the spike truncation mutants has not been included.

[0108] Figure 42 Shows the biochemical characterisation of MjH2P-1258 parental and F14 glycoproteins expressed in Expi293F cells. Superose 6 size exclusion chromatography (SEC) of total secreted spike protein obtained from 50 ml of Expi293F culture by affinity purification on TALON resin. The molecular weight markers are thyroglobulin (669 kDa) and ferritin (440 kDa).

[0109] B) SEC of purified trimeric Spikes following a freeze (-80°C)-thaw cycle. C) Melting temperature of purified Spike trimers determined by differential scanning fluorimetry (DSF). D) SDS-PAGE under nonreducing (-pME) and reducing (+pME) conditions of purified Spike trimers. The protein bands were visualised following Coomassie blue staining, m, molecular weight markers. dslS: disulfide linked S; monS: monomeric S.

[0110] Figure 43 Shows an amino acid alignment of the spike sequences that were combined in the tetravalent vaccine formulation used in the experiments summarised in Figures 29 and 36. Prepared with Clustal Q. *, identical amino acids,: highly similar amino acids,. similar amino acids, - deletion

[0111] Figure 44 Shows the spike amino acid sequences used to test neutralizing activity in Figures 30, 35, and 37. The SARS CoV-2 omicron BA.2.86 is included because this isolate was used to make S6P. BA286-1192. D17 in the tetravalent vaccine formulation described in Figure 36. Figure 45 Shows an alignment of spike amino acid sequences used to test neutralizing activity in Figures 30, 35, and 37. Prepared with Clustal Q. *, identical amino acids,: highly similar amino acids,. similar amino acids, - deletion. The SARS CoV-2 omicron BA.2.86 is included because this was used in the tetravalent vaccine formulation described in Figure 36.

[0112] Figure 46 shows a percent amino acid identity matrix determined from the alignment of spike amino acid sequences used to test neutralizing activity in Figures 30, 35, and 37. Prepared with Clustal Q. The SARS CoV-2 omicron BA.2.86 is included because this was used in the tetravalent vaccine formulation described in Figure 36.

[0113] Figure 47 Shows ELISA binding titres of sera obtained from the experiments described in Figures 29 and 36 to the stem region of the MERS CoV and ancestral SARS CoV-2 Spike. Top, maltose-binding protein (MBP)-(MERS CoV S amino acids 1221-1275) chimeric protein; Bottom, (MBP)-(SARS CoV-2 Hu1 S amino acids 1138-1208) The vehicle control and vaccine groups are indicated below the x axis. The data from the experiments described in Figures 36 and 29, respectively, are to the left and right of the vertical dotted line. The endpoint was determined as 50-times background luciferase activity obtained in the absence of primary antibody. The horizontal bars are the geometric means. A Kruskal-Wallis test was used to determine whether the differences in binding titres are significant, ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0114] Figure 48 Shows MRCoV S-HIV pseudovirus neutralization ID50s elicited in mice receiving 3 and 2 doses of monovalent parental, F14- and A1 -mutated M2P-1258 trimers.

[0115] Neutralization ID50s (reciprocal of the serum dilution required for 50% inhibition of infection) obtained with sera from individual animals are indicated with various symbols, ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, Kruskal-Wallis test.

[0116] Figure 49 Shows MRCoV S-HIV pseudovirus neutralization ID50s elicited in mice receiving 3 and 2 doses of monovalent M2P-1258. F14 or the tetravalent (Tet) formulation of covalently stabilized MERS CoV and Sarbecovirus S trimers. Neutralization ID50s (reciprocal of the serum dilution required for 50% inhibition of infection) obtained with sera from individual animals are indicated with various symbols, ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, Kruskal-Wallis test.

[0117] Figure 50 Shows phylogenetic tree of selected Merbecovirus and Sarbecovirus spike amino acid sequences. Isolates from which Spike sequences were derived for use in neutralization assays are in bold type. The virus clade is shown to the right in bold italic type. Tree constructed using MEGAX.

[0118] Figure 51 Shows the spike amino acid sequences for MERS-CoV, SARS-CoV2, SARS-CoV and OC43, along with predicted membrane spanning sequences.

[0119] KEY TO SEQUENCE LISTING SEQ ID NO: 1 Middle East respiratory syndrome Amino acid sequence coronavirus 1 (MERS1) spike residues 1 to

[0120] 1353 (full length spike comprising the

[0121] transmembrane domain and cytoplasmic tail)

[0122] SEQ ID NO: 2 MERS1 spike with 2P mutation (M2P), Amino acid sequence residues 1 to 1353

[0123] SEQ ID NO 3 M2P-1275 Amino acid sequence SEQ ID NO 4 M2P-1275 Nucleotide sequence SEQ ID NO 5 M2P-1240 Amino acid sequence SEQ ID NO 6 M2P-1248 Amino acid sequence SEQ ID NO 7 M2P-1258 Amino acid sequence SEQ ID NO 8 M2P-1265 Amino acid sequence SEQ ID NO 9 M2P-1270 Amino acid sequence SEQ ID NO 10 M2P-1240 Nucleotide sequence SEQ ID NO 11 M2P-1248 Nucleotide sequence SEQ ID NO 12 M2P-1258 Nucleotide sequence SEQ ID NO 13 M2P-1265 Nucleotide sequence SEQ ID NO 14 M2P-1270 Nucleotide sequence SEQ ID NO 15 A1: M2P-1258 D580C / Q60C Amino acid sequence SEQ ID NO 16 B7: M2P-1258 S362C / T803C Amino acid sequence SEQ ID NO 17 C44: M2P-1258 Q364C / Q808C Amino acid sequence SEQ ID NO 18 D17: M2P-1258 N637C / N1042C Amino acid sequence SEQ ID NO 19 E9: M2P-1258 S365C / A930C Amino acid sequence SEQ ID NO 20 F14: M2P-1258 S429C / D1059C Amino acid sequence SEQ ID NO 21 G44: M2P-1258 D509C / S435C Amino acid sequence SEQ ID NO 22 H2: M2P-1258 N765C / S950C Amino acid sequence SEQ ID NO 23 I1: M2P-1258 N636C / N1042C Amino acid sequence SEQ ID NO 24 J23: M2P-1258 S781C / Q857C Amino acid sequence SEQ ID NO 25 K35: M2P-1258 S1114C / N1104C Amino acid sequence SEQ ID NO 26 L21: M2P-1258 S1199C / Q988C Amino acid sequence SEQ ID NO 27 M2: M2P-1258 D1182C / S966C Amino acid sequence SEQ ID NO 28 A1: M2P-1258 D580C / Q60C Nucleotide sequence SEQ ID NO 29 B7: M2P-1258 S362C / T803C Nucleotide sequence SEQ ID NO 30 C44: M2P-1258 Q364C / Q808C Nucleotide sequence SEQ ID NO 31 D17: M2P-1258 N637C / N1042C Nucleotide sequence SEQ ID NO 32 E9: M2P-1258 S365C / A930C Nucleotide sequence SEQ ID NO 33 F14: M2P-1258 S429C / D1059C Nucleotide sequence SEQ ID NO 34 G44: M2P-1258 D509C / S435C Nucleotide sequence SEQ ID NO 35 H2: M2P-1258 N765C / S950C Nucleotide sequence SEQ ID NO 36 I1: M2P-1258 N636C / N1042C Nucleotide sequence SEQ ID NO 37 J23: M2P-1258 S781C / Q857C Nucleotide sequence SEQ ID NO 38 K35: M2P-1258 S1114C / N1104C Nucleotide sequence SEQ ID NO 39 L21: M2P-1258 S1199C / Q988C Nucleotide sequence SEQ ID NO 40 M2: M2P-1258 D1182C / S966C Nucleotide sequence

[0124]

[0125] SEQ ID NO 41 Forward primer for M2P-1275 Nucleotide sequence SEQ ID NO: 42 Reverse primer (K1240) Nucleotide sequence SEQ ID NO: 43 Reverse primer (N1248) Nucleotide sequence SEQ ID NO: 44 Reverse primer (T1258) Nucleotide sequence SEQ ID NO: 45 Reverse primer (E1265) Nucleotide sequence SEQ ID NO: 46 Reverse primer (Q1270) Nucleotide sequence SEQ ID NO: 47 Avitag Amino acid sequence SEQ ID NO: 48 MERS COV spike glycoprotein stem, with Amino acid sequence predicted membrane spanning sequence

[0126] SEQ ID NO: 49 SARS COV-2 spike glycoprotein stem, with Amino acid sequence predicted membrane spanning sequence

[0127] SEQ ID NO: 50 SARS COV spike glycoprotein stem, with Amino acid sequence membrane spanning sequence

[0128] SEQ ID NO: 51 OC43 spike glycoprotein stem, with Amino acid sequence membrane spanning sequence

[0129] SEQ ID NO: 52 MERS COV predicted membrane spanning Amino acid sequence sequence

[0130] SEQ ID NO: 53 SARS COV-2 membrane spanning Amino acid sequence sequence

[0131] SEQ ID NO: 54 SARS COV predicted membrane spanning Amino acid sequence sequence

[0132] SEQ ID NO: 55 OC43 predicted membrane spanning Amino acid sequence sequence

[0133] SEQ ID NO: 56 M2P-1258 RNA construct Nucleotide sequence SEQ ID NO: 57 A1: M2P-1258 D580C / Q60C RNA construct Nucleotide sequence SEQ ID NO: 58 F14: M2P-1258 S429C / D1059C RNA Nucleotide sequence construct

[0134] SEQ ID NO 59 S6P. BA286-1192 Amino acid sequence SEQ ID NO 60 S6P. BA286-1192. D17 Amino acid sequence SEQ ID NO 61 S6P. BA286-1192.11 +VI Amino acid sequence SEQ ID NO 62 S6P. BA286-1192 Nucleotide sequence SEQ ID NO 63 S6P. BA286-1192. D17 Nucleotide sequence SEQ ID NO 64 S6P. BA286-1192.11 +VI Nucleotide sequence SEQ ID NO 65 S6P. BA286-1192 Amino acid sequence SEQ ID NO 66 S6P. BA286-1192. D17 Amino acid sequence SEQ ID NO 67 S6P. BA286-1192 Nucleotide sequence SEQ ID NO 68 S6P. BA286-1192. D17 Nucleotide sequence SEQ ID NO 69 S6P. PRD-1192 Amino acid sequence SEQ ID NO 70 S6P. PRD-1192. D17 Amino acid sequence SEQ ID NO 71 S6P. PRD-1192 Nucleotide sequence SEQ ID NO 72 S6P. PRD-1192. D17 Nucleotide sequence SEQ ID NO 73 S6P. WIV1-1192 Amino acid sequence SEQ ID NO 74 S6P. WIV1-1192. D17 Amino acid sequence SEQ ID NO 75 S6P. WIV1-1192 Nucleotide sequence SEQ ID NO 76 S6P. WIV1-1192. D17 Nucleotide sequence SEQ ID NO 77 MERS CoV Englandl spike Amino acid sequence SEQ ID NO 78 MERS CoV Englandl spike Nucleotide sequence SEQ ID NO 79 MRCoV spike Amino acid sequence SEQ ID NO 80 MjH2P-1258 Amino acid sequence SEQ ID NO 81 MjH2P-1258. F14 Amino acid sequence SEQ ID NO 82 MjH2P-1258 Nucleotide sequence SEQ ID NO 83 MjH2P-1258. F14 Nucleotide sequence SEQ ID NO 84 NeoCoV spike Amino acid sequence SEQ ID NO 85 WIV1 spike Amino acid sequence

[0135]

[0136] SEQ ID NO 86 SARS CoV Tor2 spike Amino acid sequence SEQ ID NO: 87 PRD-0038 spike Amino acid sequence SEQ ID NO: 88 BtKY72. YW spike Amino acid sequence SEQ ID NO: 89 SARS CoV-2 omicron BA.2.86 spike Amino acid sequence SEQ ID NO: 90 SARS CoV-2 omicron JN.1 spike Amino acid sequence

[0137]

[0138] SEQ ID NO: 91 SARS CoV-2 omicron XEC spike Amino acid sequence

[0139] DISCUSSION OF EMBODIMENTS

[0140] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any materials and methods similar or equivalent to those described herein can be used to practice or test the present disclosure. Practitioners are particularly directed to Ausubel et al., Current Protocols in Molecular Biology, Supplement 47, John Wiley & Sons, New York, 1999; CoIowick and Kaplan, eds., Methods In Enzymology, Academic Press, Inc.; Weir and Blackwell, eds., Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications, 1986; Kontermann and Dubel (Ed), Antibody Engineering, Vol 1-2, Ed., Springer Press, 2010) for definitions and terms of the art and other methods known to the person skilled in the art.

[0141] Reference to any prior art in this specification is not, and should not be taken as, acknowledgement or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[0142] The term "and / or", e.g., " X and / or Y" shall be understood to mean either " X and Y" or " X or Y" and shall be taken to provide explicit support for both meanings or for either meaning. As used herein, the term “about”, unless stated to the contrary, refers to + / - 10%, more preferably + / - 5%, even more preferably + / - 1%, of the designated value.

[0143] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0144] As used herein, the singular form "a", "an" and "the" include singular and plural references unless the context indicates otherwise. Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

[0145] Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). A sequence listing is submitted electronically herewith and is incorporated by reference in its entirety.

[0146] The sequence of the Middle East respiratory syndrome Coronavirus (MERS-CoV) Betacoronavirus England 1 strain is described in GenBank Reference Sequence: KC164505.2. The spike (S) protein from the MERS-CoV Betacoronavirus England 1 strain is described in Genbank accession number AFY13307, and is as defined in SEQ ID NO: 1. This strain may also be referred to as "wild-type", “ancestral” and "parental" strain herein. However, distinct from SARS-CoV-2 strains, there are multiple “ancestral” or “parental” strains since MERS-CoV strains are divided into various clades and there are no dominant MERS-CoV strains presently circulating. It is anticipated that in the event of an outbreak, the rapid replication of viruses while the infection spreads, leading to subsequent mutated strains emerging, would result in a key “ancestral” or “parental” strain (from which the outbreak initially began) being identified. The various MERS-CoV clades or different “ancestral” or “parental” strains are discussed further below.

[0147] As used herein “antigen” refers to a substance capable of stimulating an immune response.

[0148] As used herein, “protomer” refers to the basic structural unit of an oligomeric protein (e.g. the S protein monomer of an S protein trimer). In some embodiments, protomers can be chemically linked (e.g. via disulfide bond) to form or stabilise part of an oligomeric structure (e.g. three S protein monomers can be linked to form S protein trimer oligomeric structure). It would be clear to a person skilled in the art that an S protein trimer comprises three protomers.

[0149] As used herein, “endogenous” refers to developing or originating within the native virus. As used herein, “non-endogenous” refers to something that has not developed or originated within the native virus (e.g. something that a virus has been altered to comprise e.g. something that is man-made).

[0150] As used herein, “melting temperature” refers to the temperature at which 50% of a protein is unfolded. Temperature can break the chemical interactions that hold a protein structure in shape. Accordingly, melting temperature can be used to reflect or indicate the stability of a protein. In an embodiment, melting temperature can be determined using differential scanning fluorimetry (DSF).

[0151] As used herein, “signal sequence” refers to the residues corresponding to 1 to 17 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0152] As used herein, “N-terminal domain” or “NTD” refers to the residues corresponding to 18 to 350 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0153] As used herein, the “receptor binding domain” or “RBD” refers to the residues corresponding to 381-588 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0154] As used herein, “receptor binding motif" or “RBM” refers to the residues corresponding to Y499, N501, K502, D510, R511, E513, P515, W535, E536, D539, Y540, R542, W553 and V555 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0155] As used herein, “stem region” refers to the residues corresponding to 1220 to 1296 of SEQ ID: NO: 1. In an embodiment, the stem region comprises the residues corresponding to 1220 to 1296 of SEQ ID NO: 1 or SEQ ID NO: 2. As used herein, “trimerization sequence” refers to a sequence found at the C terminal region of the S-protein monomers which facilitates trimerization of the S protein trimer. In some embodiments, the trimerization domain is a heterologous sequence, not found within coronaviruses. In an embodiment, the trimerization sequence is a heterologous sequence, not found within MERS-CoV. In some embodiments, the trimerization sequence is the trimeric foldon domain of bacteriophage T4 fibritin or a modified version thereof. In some embodiments, the trimerization sequence is a coiled-coil, an artificial coiled-coil or a modified coiled-coil. In some embodiments, the trimerization sequences was designed de novo. In some embodiments, the trimerization sequence is a trimeric foldon domain of bacteriophage T4 fibritin or a modified version thereof.

[0156] As used herein, “truncation” refers to shortening a molecule by removing a portion of it. In an embodiment, the molecule is a protein and the truncation is at the C-terminal end of the protein sequence.

[0157] As used herein, the term “increase” or “increases” or “increased” or “increasing” refers to having a higher or greater level of a given parameter compared to the level of a given parameter at baseline or compared to a control.

[0158] As used herein, “a control” refers to a standard of comparison for checking the results of a survey or experiment. In an embodiment, the control is a vaccine antigen lacking one or more of: i) the non-endogenous inter-protomer disulfide bond and ii) the C-terminal truncation in the stem region. In an embodiment, the control is a vaccine antigen lacking the non-endogenous inter-protomer disulfide bond. In an embodiment, the control is a vaccine antigen lacking the C-terminal truncation in the stem region.

[0159] As used herein, the term “reduce” or “reduces” or “reduced” or “reducing” refers to having a lower or lesser level of a given parameter compared to the level of a given parameter at baseline or compared to a control.

[0160] When used in the context of a polypeptide, such as an antigen, the polypeptide may be exposed to “reducing conditions”, such as by exposure to a reducing agent such as betamercaptoethanol. A polypeptide may be exposed to “reducing conditions” in order to assess the stability of the polypeptide.

[0161] As used herein, the term “epitope” refers to particular peptide sequences on a molecule that are antigenic, such that they elicit a specific immune response. An epitope is the region of an antigen to which B and / or T cells respond. An antibody can bind to a particular antigenic epitope which may be formed both from contiguous amino acids or non-contiguous amino acids.

[0162] As used herein, the “yield of the trimer” or the “trimer yield” refers to micrograms of pure trimeric spike protein obtained from 50 ml of cell culture. As used herein, the term “multivalent vaccine” refers to a vaccine that comprises more than one antigen, with each antigen being different from one another. For example, the vaccine may comprise two antigens encoding different proteins from the same virus. In another example, the vaccine may comprise two antigens encoding the same protein from different viruses. In another example, the vaccine may comprise two antigens encoding different proteins from different viruses. As used herein, the term “bivalent vaccine” refers to a vaccine that comprises two antigens that are different from one another. As used herein, the term “trivalent vaccine” refers to a vaccine that comprises three antigens that are different from one another. As used herein, the term “tetravalent vaccine” refers to a vaccine that comprises four antigens that are different from one another.

[0163] Coronavirus

[0164] " Coronavirus" or " CoV" are enveloped, positive sense, single-stranded RNA viruses. There are two subfamilies of Coronaviridae, Letovirinae and Orthocoronavirinae. In an embodiment, the CoV is selected from the genera alphacoronavirus (alphaCoV), betacoronavirus (betaCoV), gammacoronavirus (gammaCoV) and deltacoronavirus (deltaCoV). In an embodiment, the alphaCoV is selected from coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), feline infectious peritonitis virus (FIPV) and canine coronavirus (CCoV). In an embodiment, the CoV is a betacoronavirus. In an embodiment, the betaCoV is selected from: a bat sarbecovirus (such as PRD-0038 and WIV1), Middle-East respiratory syndrome-related coronavirus (MERS-CoV), human coronavirus HKU1 (HCoV-HKU1), Human coronavirus OC43 (HCoV-OC43), Severe acute respiratory syndrome-related coronavirus (SARS-CoV), Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2), murine hepatitis virus (MHV) and / or bovine coronavirus (BCoV). In an embodiment, the CoV is mink respiratory coronavirus (MRCoV). “Merbecovirus” is a subgenus of viruses in the genus betacoronavirus, which includes MERS-CoV. In an embodiment, the CoV is a merbecovirus. In an embodiment, the CoV is a bat merbecovirus. In an embodiment, the CoV is a divergent merbecovirus. In an embodiment, the CoV is a bat divergent merbecovirus. In an embodiment, the CoV is MjHKU4r. In an embodiment, the CoV is a sarbecovirus. In an embodiment, the CoV is a bat sarbecovirus. In an embodiment, the CoV is a divergent Sarbecovirus. In an embodiment, the CoV is a divergent bat sarbecovirus. In an embodiment, the CoV is PRD-0038. In an embodiment, the CoV is WIV1. In an embodiment, the CoV is capable of infecting a human. In an embodiment, the CoV capable of infecting a human is selected from: MERS-CoV, SARS-CoV-2, HCoV-OC43, HCoV-HKLH, HCoV-229E, HCoV-NL63 and SARS-CoV or a subtype or variant thereof. Reference to “MERS-CoV” should be considered to also include subtypes or variants thereof. “MERS-CoV” may also be referred to as “betacoronavirus England 1”, “human betacoronavirus 2c England-Qatar” or “human betacoronavirus 2C Jordan-N3”.

[0165] Examples of MERS-CoV variants are described, for example, in Azhar et al. 2023, Farrag et al. 2021, Kim et al., 2016, Schroeder et al. (2021) and Hassan et al. (2025).

[0166] In an embodiment, the MERS-CoV is MERS-CoV Hu / England-1 / 2012 or a variant thereof. In an embodiment, the MERS-CoV is HCoV-EMC / 2012 or a variant thereof. In an embodiment, the MERS-CoV comprises the sequence as described in GenBank accession number KC164505.2 or a variant thereof. In an embodiment, the MERS-CoV is MERS-CoV Hu / Jordan-N3 / 2012 or a variant thereof. In an embodiment, the MERS-CoV comprises the sequence as described in GenBank accession number KC776174.1 or a variant thereof. In an embodiment, the MERS-CoV is MERS-CoV Hu / Bisha-1 / 2012 or a variant thereof. In an embodiment, the MERS-CoV is MERS-CoV Hu / Qatar-3 / 2013 or a variant thereof. In an embodiment, the MERS-CoV is MERS-CoV Hu / Munich-UAE / 2013 or a variant thereof.

[0167] In an embodiment, the CoV is a merbecovirus HKU4 clade virus. In an embodiment, the CoV is a merbecovirus HKU5 clade virus. In an embodiment, the CoV is a sarbecovirus clade 1a virus. In an embodiment, the CoV is a sarbecovirus clade 1b virus. In an embodiment, the CoV is a sarbecovirus clade 3 virus.

[0168] In an embodiment, the MERS-CoV is a MERS-CoV clade A virus. In an embodiment, the MERS-CoV is a MERS-CoV clade B virus. In an embodiment, the MERS-CoV is a MERS-CoV clade B lineage 2 virus. In an embodiment, the MERS-CoV is a MERS-CoV clade B lineage 3 virus. In an embodiment, the MERS-CoV is a MERS-CoV clade B lineage 4 virus. In an embodiment, the MERS-CoV is a MERS-CoV clade B lineage 5 virus. In an embodiment, the MERS-CoV is a MERS-CoV clade B lineage 6 virus. In an embodiment, the MERS-CoV is a MERS-CoV clade B lineage 7 virus. In an embodiment, the MERS-CoV is a MERS-CoV clade C virus. In an embodiment, the MERS-CoV is an Al-Hasa clade virus. In an embodiment, the MERS-CoV is a Buraidah-1 clade virus. In an embodiment, the MERS-CoV is a Hafr-AI-Batin-1 clade virus. In an embodiment, the MERS-CoV is a Jeddah-Riyadh clade virus. In an embodiment, the MERS-CoV is a Riyadh-3 clade virus.

[0169] In an embodiment, the MERS-CoV is a B5-2023 clade virus. In an embodiment, the MERS-CoV is a B5-2023.1 clade virus (B5-2023 subclade 1). In an embodiment, the MERS-CoV is a B5-2023.2 clade virus (B5-2023 subclade 2). In an embodiment, the MERS-CoV is a B5-2023.3 clade virus (B5-2023 subclade 3). In an embodiment, the MERS-CoV is a B5-2023.4 clade virus (B5-2023 subclade 4). In an embodiment, the MERS-CoV is a B5-2023.5 clade virus (B5-2023 subclade 5). In an embodiment, the MERS-CoV is MERS-CoV Camel / AI-Quwayiyah or a variant thereof. In an embodiment, the MERS-CoV is MERS-CoV Camel / AI-Riyadh or a variant thereof. In an embodiment, the MERS-CoV is MERS-CoV Camel / Sajr or a variant thereof. In an embodiment, the MERS-CoV is MERS-CoV Camel / Shaqra or a variant thereof. In an embodiment, the MERS-CoV is MERS-CoV Camel / Jeddah or a variant thereof. In an embodiment, the MERS-CoV is MERS-CoV Camel / AI-Duwadimi or a variant thereof.

[0170] In an embodiment, the MERS-CoV variant is at least 90% identical to the MERS-CoV parental strain. In an embodiment, the MERS-CoV variant is at least 92% identical to the MERS-CoV parental strain. In an embodiment, the MERS-CoV variant is at least 93% identical to the MERS-CoV parental strain. In an embodiment, the MERS-CoV variant is at least 94% identical to the MERS-CoV parental strain. In an embodiment, the MERS-CoV variant is at least 95% identical to the MERS-CoV parental strain. In an embodiment, the MERS-CoV variant is at least 96% identical to the MERS-CoV parental strain. In an embodiment, the MERS-CoV variant is at least 97% identical to the MERS-CoV parental strain. In an embodiment, the MERS-CoV variant is at least 98% identical to the MERS-CoV parental strain. In an embodiment, the MERS-CoV variant is at least 99% identical to the MERS-CoV parental strain. In an embodiment, the MERS-CoV parent strain (also referred to as the ancestral strain) is MERS-CoV Hu / England-1 / 2012. In an embodiment, the MERS-CoV parent strain (also referred to as the ancestral strain) comprises the sequence as described in GenBank accession number KC164505.2. In an embodiment, the MERS-CoV parent strain (also referred to as the ancestral strain) is HCoV-EMC / 2012. In an embodiment, the MERS-CoV parent strain (also referred to as the ancestral strain) is MERS-CoV Hu / Jordan-N3 / 2012. In an embodiment, the MERS-CoV parent strain (also referred to as the ancestral strain) comprises the sequence as described in GenBank accession number KC776174.1. In an embodiment, the MERS-CoV parent strain (also referred to as the ancestral strain) is MERS-CoV Hu / Hafr-AI-batin-1 / 2013. In an embodiment, the MERS-CoV parent strain (also referred to as the ancestral strain) is MERS-CoV Hu / Bisha-1 / 2012. In an embodiment, the MERS-CoV parent strain (also referred to as the ancestral strain) is MERS-CoV Hu / Qatar-3 / 2013. In an embodiment, the MERS-CoV parent strain (also referred to as the ancestral strain) is MERS-CoV Hu / Munich-UAE / 2013.

[0171] In an embodiment, the CoV is a “Variant of Interest” also referred to as a “VOI”. As used herein a VOI is a variant of a CoV that is associated with genetic changes that are predicted or known to affect virus characteristics such as transmissibility, disease severity, immune escape, diagnostic or therapeutic escape; and identified to cause significant community transmission or multiple disease clusters (which could be MERS-CoV disease clusters, or in the case of SARS-CoV-2 COVID19 clusters), in multiple countries with increasing relative prevalence alongside increasing number of cases over time, or other apparent epidemiological impacts to suggest an emerging risk to global public health.

[0172] In an embodiment, the CoV is a “Variant of Concern” also referred to as a “VOC”. As used herein a VOC is a variant of a CoV that is associated with one or more of the following changes at a degree of global public health significance: increase in transmissibility ordetrimental change in epidemiology (which could be a detrimental change in MERS-CoV epidemiology or in the case of SARS-CoV-2 the detrimental change is in COVID-19 epidemiology); increase in virulence or change in clinical disease presentation; or a decrease in effectiveness of public health and social measures or available diagnostics, vaccines, therapeutics. In an embodiment, the CoV is classified as a VOC, VOI or VHC by a health regulatory body e.g. the World Health Organisation (WHO), the United States Center of Disease Control (CDC), the European Centre for Disease Prevention and Control (ECDC) or an equivalent local government health regulatory body in a specific jurisdiction. In an embodiment, the CoV is classified as a VOC or VOI by WHO. In an embodiment, the CoV is classified as a VOC, VOI or VHC by the CDC. In an embodiment, the CoV is classified as a VOC or VOI by ECDC.

[0173] In an embodiment, the CoV is a “Variant of High Consequence” also referred to as a “VHC”. In an embodiment the VHC as clear evidence that prevention measures or medical countermeasures have significantly reduced effectiveness relative to previously circulating variants. In addition to the characteristics of a VOC, a VHC can have one or more of the following impacts on medical countermeasures: demonstrated failure of diagnostic test targets; evidence to suggest a significantly reduction in vaccine effectiveness, a disproportionately high number of vaccine breakthrough cases, or very low vaccine-induced protection against severe disease; significantly reduced susceptibility to multiple emergency use authorization or approved therapeutics and more severe clinical disease and increased hospitalizations.

[0174] In an embodiment, the MERS-CoV comprises one or more mutations selected from the group: V27A, V27L, G159Y, H194Y, K369I, S390F, L411F, L450F, S457G, S460F, A434S, Y447X, D509G, I529T, D510G, A597V, R626P, E666K, M696T, L745F and A756Q

[0175] In an embodiment, the MERS-CoV comprises one or more mutations selected from the group: V27A, V27L, G159Y, H194Y, S390F, L411F, L450F, I529T, D510G, A597V, R626P, E666K, M696T, L745F and A756Q.

[0176] In an embodiment, the MERS-CoV comprises one or more mutations in the receptor binding domain (RBD) selected from the group: K369I, S390F, S457G, S460F, A434S, Y447X and D509G.

[0177] In an embodiment, the CoV is SARS-CoV-2 or a subtype or variant thereof. In an embodiment, SARS-CoV-2 is SARS-CoV-2 hCoV-19 / Australia / VIC01 / 2020. In an embodiment, SARS-COV-2 comprises the sequences as described in NCBI Reference Sequence: NC_045512.2. In an embodiment, SARS-CoV-2 comprises the sequence as described in GenBank: MN908947.3 or a variant thereof. Examples of SARS-CoV-2 variants are described, for example, in Shen et al., 2020, Tang et al., 2020, Phan et al., 2020, Khan et al., 2020, Forster et al., 2020, Vasireddy et al., 2021, Winger et al., 2021, Sanyaolu et al., 2021, Ou et al., 2022 and Fernandes et al., 2022.

[0178] In an embodiment, the SARS-CoV-2 is an omicron variant or a subtype or variant thereof. In an embodiment, the SARS-CoV-2 is a non-omicron variant. In an embodiment, the SARS-CoV-2 is a delta variant or a subtype or variant thereof.

[0179] In an embodiment, the CoV is SARS-CoV or a subtype or variant thereof. In an embodiment, the SARS-CoV is SARS-CoV Tor2 strain. In an embodiment, the SARS-CoV is SARS-CoV Urbani strain.

[0180] In an embodiment, the CoV variant is at least 90% identical to the parental strain. In an embodiment, the variant is at least 92% identical to the parental strain. In an embodiment, the variant is at least 93% identical to the parental strain. In an embodiment, the variant is at least 94% identical to the parental strain. In an embodiment, the variant is at least 95% identical to the parental strain. In an embodiment, the variant is at least 96% identical to the parental strain. In an embodiment, the variant is at least 97% identical to the parental strain. In an embodiment, the variant is at least 98% identical to the parental strain. In an embodiment, the variant is at least 99% identical to the parental strain. In an embodiment, the parent strain (also referred to as the ancestral strain) is Hu-1 strain is described by Wu et al., 2020. In some embodiments, the parental strain is SARS-CoV-2 hCoV-19 / Australia / VIC01 / 2020. In some embodiments, the parental strain is BetaCoV / Ancestral / WIV04 / 2019.

[0181] In an embodiment, the CoV is a VOC or VOI as described in Vasireddy et al., 2021, Winger et al., 2021 or Sanyaolu et al., 2021, Chavda et al., 2022.

[0182] In an embodiment, where the CoV is SARS-CoV-2 VOC, the VOC comprises one or more of the following mutations: H69del, V70del, G142del / D, Y144del, V213G, S371 F / L, D405N, R408S, E484K / Q / A, S494P, N501Y, A570D, D614G, P681 H / R, T716I, S982A, D1118H, V1176F, K1191N, D80A, D215G, 241del, 242del, 243del, K417N, N501Y, D614G, A701 V, T19R, V70F, T95I, E156-, F157-, R158G, A222V*, W258L*, K417N / T*, L452R, T478K, D614G, D950N, L18F, T20N, P26S, D138Y, R190S, K417T, N501Y, D614G, H655Y, T1027I, H655Y A67V, del69-70, T95I, del142-144, Y145D, N211del, L212I, ins214EPE, G339D, S373P, S375F, K417N, N440K, G446S, S477N, T478K, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, N764K, D796Y, N856K, Q954H, N969K and L981F.

[0183] In an embodiment, where the CoV is SARS-CoV-2 VOC, the VOC comprises one or more of the following RBD mutations: G339D / H, R346T, L368I, S371F / L, S373P, S375F, T376A, D405N, R408S, K417N / T, N440K, K444T, V445P, G446S, L452R, F456L, N460K, S477N, T478K / R, E484K / Q / A, F486V / S / P, F490S, Q493R, G496S, Q498R, S494P, N501Y, Y505H. In an embodiment, the VOC comprises one or more ofthe following NTD mutations: L18F, T19R / I, T20N, del24-26, P26S, A27S, Q52H, A67V, del69-70, H69del, V70del, V70F, D80A, V83A, T95I, D138Y, del142-144, G142del, G142D, Y144del, Y145D, K147Q, deE156, delF157, dell 57-158, R158G, E180V, Q183E, R190S, N211del, L212I, V213G / E, ins214EPE, D215G, 241del, 242del, 243del, G252V, A222V*, and W258L*

[0184] In an embodiment, the VOC is B.1.1.7 or a variant thereof. In an embodiment, the VOC is B.1.351 or a variant thereof. In an embodiment, the VOC is B.1.351.2 or a variant thereof. In an embodiment, the VOC is B.1.351.2 or a variant thereof. In an embodiment, the VOC is B.1.351.3 or a variant thereof. In an embodiment, the VOC is P1 or a variant thereof. In an embodiment, the VOC is P1.1 or a variant thereof. In an embodiment, the VOC is P1.2 or a variant thereof. In an embodiment, the VOC is B.1.617.2 or a variant thereof. In an embodiment, the VOC is AY.1 or a variant thereof. In an embodiment, the VOC is AY.2 or a variant thereof. In an embodiment, the VOC is AY.3 or a variant thereof. In an embodiment, the VOC is B.1.1.529 or a variant thereof. In an embodiment, the VOC is BA.1 or a variant thereof. In an embodiment, the VOC is BA.1.1 or a variant thereof. In an embodiment, the VOC is BA.2 or a variant thereof. In an embodiment, the VOC is BA.2.74 or a variant thereof. In an embodiment, the VOC is BA.2.86 or a variant thereof. In an embodiment, the VOC is BA.3 or a variant thereof. In an embodiment, the VOC is BA.4 or a variant thereof. In an embodiment, the VOC is BA.5 or a variant thereof. In an embodiment, the VOC is BA.4-5 (also known as BA.4 / 5) ora variant thereof. In an embodiment, the VOC is XBB 1.5 or a variant thereof. In an embodiment, the VOC is XBB 1.16 or a variant thereof. In an embodiment, the VOC is XBB 2.3 or a variant thereof. In an embodiment, the VOC is XBB 1.9.2 ora variant thereof. In an embodiment, the VOC is XBB 1.9.1 or a variant thereof. In an embodiment, the VOC is BA.2.86 or a variant thereof. In an embodiment, the VOC is JN.1 or a variant thereof. In an embodiment, the VOC is XEC or a variant thereof. In an embodiment, the VOC is CH.1.1 or a variant thereof.

[0185] In an embodiment, where the CoV is SARS-CoV-2 VOI, the VOI comprises one or more of the following mutations: L452R, D614G, S13I, W152C, A67V, 69del, 70del, 144del, E484K, Q677H, F888L, L5F, D80G, T95I, Y144, F157S, D253G, L452R, S477N, E484K, A701 V, T859N, D950H and Q957R, N501Y, P681R, P681H, E484Q, P681R, S477N, L452Q and F490S. In an embodiment, the VOI is B.1.525 or a variant thereof. In an embodiment, the VOI is B.1.526 or a variant thereof. In an embodiment, the VOI is B.1.617.1 or a variant thereof. In an embodiment, the VOI is C37 or a variant thereof. In an embodiment, the VOI is B.1.427 or a variant thereof. In an embodiment, the VOI is B.1.429 or a variant thereof. In an embodiment, the VOI is P2 or a variant thereof. In an embodiment, the VOI is B.1.525 or a variant thereof. In an embodiment, the VOI is P3 or a variant thereof. In an embodiment, the VOI is B.1.620 or a variant thereof. In an embodiment, the VOI is B.1.621 or a variant thereof. In an embodiment, the VOI is C.37 or a variant thereof. In an embodiment, the VOI is BA2.75 or a variant thereof. In an embodiment, the VOI is BQ.1 or a variant thereof.

[0186] CoV infections, including MERS-CoV, can cause respiratory, enteric, hepatic, and neurological diseases in different animal species, including camels, cattle, cats, and bats. CoV can be transmitted from one individual to another through contact of viral droplets with mucosa. Typically, viral droplets are airborne and inhaled via the respiratory tract including the nasal airway. Typically, the individual is a human individual. In some embodiments, the individual is a selected from a non-human primate, a mammal, a livestock animal, a laboratory test animal, a captive wild animal, a zoo animal, and a reservoir animal. Typically, during an infection, CoV can be found in the upper respiratory tract, for example the nasal passages. In some examples, CoV can be found in the lower respiratory tract, for example the bronchi and / or alveoli.

[0187] In an embodiment, a CoV infection causes one or more symptoms selected from one or more of: fever, cough, sore throat, shortness of breath, viral shedding respiratory insufficiency, runny nose, nasal congestion, malaise, bronchitis, headache, muscle pain, dyspnoea, moderate pneumonia, severe pneumonia, acute respiratory distress syndrome (ARDS). In an embodiment, the ARDS is selected from mild ARDS (defined as 200 mmHg < PaO2 / FiO2 < 300 mmHg), moderate ARDS (defined as 100 mmHg < PaO2 / FiO2 < 200 mmHg) and severe ARDS (defined as PaO2 / FiO2 < 100 mmHg). In an embodiment, a CoV infection can cause one or more symptoms selected from one or more of: fever, cough, sore throat, shortness of breath, viral shedding, respiratory insufficiency, runny nose, nasal congestion, malaise, bronchitis, headache, muscle pain, dyspnoea, moderate pneumonia, severe pneumonia, acute respiratory distress syndrome (ARDS). In an embodiment, the CoV infection is asymptomatic.

[0188] MERS-CoV

[0189] MERS-CoV has four major structural proteins: spike (S), membrane (M) and envelope (E) proteins, and nucleocapsid (N) protein. S, M and E are embedded in the viral surface envelope and N is located in the ribonucleoprotein. The S protein recognizes the host cellular receptor to initiate virus entry via attachment to dipeptidyl peptidase 4 (DPP4).

[0190] MERS-CoV cell entry is mediated by the viral spike protein through several viral-host interactions: (1) the N-terminal domain of spike attaches virus to lectins present on the host cell surface, (2) the receptor binding domain (RBD) binds to the host cell receptor molecule, (3) and receptor binding reorganizes structural elements in the S2 subunit of the spike protein that mediate membrane fusion and subsequent cell entry. Each function is performed by a separate region of the spike protein, the RBD being responsible for the core interaction with the host receptor. MERS CoV S is synthesised as a highly glycosylated trimeric precursor that is cleaved by cellular furin during translocation through the secretory pathway to yield the mature S1 and S2 subunits that remain noncovalently associated. S1 contains the RBD while S2 contains the membrane fusion machinery, membrane anchor and cytoplasmic tail, which directs incorporation into virions. MERS-CoV S can adopt open and closed conformations in which the receptor binding site is exposed and occluded, respectively. Upon binding to the host receptor, DPP4, cleavage by the cellular protease TMPRSS2 activates S2-mediated fusion between viral and cytoplasmic membranes leading to entry. Alternatively, MERS-CoV is trafficked to the endosomes of target cells where cathepsin L or other proteases promote membrane fusion and entry. Membrane fusion involves large conformational changes in Sthat appose viral and cellular membranes such that they fuse to form a pore which expands and allows the viral core to enter the cytoplasm. These changes include N-terminal extension of the central helices of the S2 trimer to form a coiled coil that that translocate fusion peptides to the cellular membrane where they insert. The C-terminal portion of S2 then packs against hydrophobic grooves on the surface of the central coiled coil, apposing transmembrane domains and fusion peptides and enabling membrane fusion (Tortorici et al., 2019).

[0191] Vaccine antigen

[0192] S protein monomers of the MERS-CoV vaccine antigen

[0193] Suitable Spike proteins or fragments thereof are obtained or derived from MERS-CoV isolates, strains, clades, and / or sequences. Alternatively, they can be produced recombinantly or synthetically.

[0194] In an embodiment, the S protein monomer in the S-protein trimer is an ancestral (parental) MERS-CoV sequence as described herein (e.g. GenBank accession number AFY13307 or a sequence corresponding to SEQ ID NO: 1) or can be a more recent variant such as a variant having at least 90% identity to the MERS-CoV parental strain. In an embodiment, the S protein monomer in the S-protein trimer is from a VOC, VOI or a VHC as described herein. In an embodiment, the S protein monomer is SEQ ID NO: 1 or variant or modified version thereof. In an embodiment, the S protein monomer is an ancestral (parental) MERS-CoV sequence modified to comprise one or more mutations present in a variant as described herein. In an embodiment, the modification is selected from one or more of: V27A, V27L, G159Y, H194Y, K369I, S390F, L411F, L450F, S457G, S460F, A434S, Y447X, D509G, I529T, D510G, A597V, R626P, E666K, M696T, L745F and A756Q.

[0195] In an embodiment, the S protein monomer is of a MERS-CoV clade A virus. In an embodiment, the S protein monomer is of a MERS-CoV clade B virus. In an embodiment, the S protein monomer is of a MERS-CoV clade B lineage 2 virus. In an embodiment, the S protein monomer is of a MERS-CoV clade B lineage 3 virus. In an embodiment, the S protein monomer is of a MERS-CoV clade B lineage 4 virus. In an embodiment, the S protein monomer is of a MERS-CoV clade B lineage 5 virus. In an embodiment, the S protein monomer is of a MERS-CoV clade B lineage 6 virus. In an embodiment, the S protein monomer is of a MERS-CoV clade B lineage 7 virus. In an embodiment, the S protein monomer is of a MERS-CoV clade C virus. In an embodiment, the S protein monomer is of an Al-Hasa clade virus. In an embodiment, the S protein monomer is of a Buraidah-1 clade virus. In an embodiment, the S protein monomer is of a Hafr-AI-Batin-1 clade virus. In an embodiment, the S protein monomer is of a Jeddah-Riyadh clade virus. In an embodiment, the S protein monomer is of a Riyadh-3 clade virus.

[0196] In an embodiment, the S protein monomer comprises residues corresponding to 18 to 1296 of the amino acid sequence SEQ ID NO: 1 or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. In an embodiment, the S protein monomer comprises residues corresponding to 18 to 1296 of the amino acid sequence SEQ ID NO: 2 or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0197] In an embodiment, the S protein monomer comprises residues corresponding to 18 to 1318 of the amino acid sequence of SEQ ID NO: 1 or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. In an embodiment, the S protein monomer comprises residues corresponding to 18 to 1318 of the amino acid sequence of SEQ ID NO: 2 ora sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0198] In an embodiment, the S protein monomer comprises residues corresponding to 18 to 1353 of the amino acid sequence of SEQ ID NO: 1 or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. In an embodiment, the S protein monomer comprises residues corresponding to 18 to 1353 of the amino acid sequence of SEQ ID NO: 2 ora sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0199] In an embodiment, the S protein monomer comprises an endogenous or exogenous signal sequence. In an embodiment, the S protein monomer lacks an endogenous or exogenous signal sequence. In an embodiment, the endogenous signal sequence comprises residues 1 to 17 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0200] In an embodiment, the S protein monomer comprises a sequence encoding the transmembrane domain of a coronavirus. In an embodiment, the S protein monomer comprises a sequence encoding the transmembrane domain of MERS-CoV. In an embodiment, the transmembrane domain comprises residues corresponding to 1297 to 1318 of the amino acid sequence SEQ ID NO: 1 or a sequence at least 90% identical thereto. In an embodiment, the transmembrane domain comprises residues corresponding to 1297 to 1318 of the amino acid sequence SEQ ID NO: 2 or a sequence at least 90% identical thereto. In an embodiment, the S protein monomer lacks an endogenous or exogenous transmembrane domain. In an embodiment, the S protein monomer does not comprise a sequence encoding the transmembrane domain of a MERS-CoV.

[0201] In an embodiment, the S protein monomer comprises the 2P mutation as described herein. In an embodiment, the S protein monomer does not comprise the 2P mutation as described herein.

[0202] In an embodiment, the S protein monomer comprises the amino acid sequence of one or more of the variant mutations as described herein.

[0203] In an embodiment, the S protein monomer does not comprise a trimerization sequence. In an embodiment, the S protein monomer does not comprise a transmembrane domain sequence. In an embodiment, the S-protein monomer does not comprise a foldon sequence. In an embodiment, the S-protein monomer does not comprise a fibritin foldon sequence. In an embodiment, the S-protein monomer does not comprise FHA.

[0204] MERS-CoV vaccine antigen

[0205] In an aspect, the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen of the present invention comprises a MERS-CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond.

[0206] In an aspect, the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen of the present invention comprises a MERS-CoV S protein trimer with a C-terminal truncation in the stem region.

[0207] In an aspect, the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen of the present invention comprises a MERS-CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond and a C-terminal truncation in the stem region.

[0208] In an embodiment, the S protein monomer of the S protein trimer lacks a signal sequence. In an embodiment, the S protein monomer of the S protein trimer does not comprise a sequence that encodes a functional non-endogenous trimerization sequence.

[0209] In an embodiment, the S protein trimer is soluble.

[0210] In an embodiment, the MERS-CoV vaccine antigen additionally comprises the 2P modification.

[0211] In an embodiment, the MERS-CoV vaccine antigen is a pan-coronavirus vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen is a MERS-CoV clade A vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen is a MERS-CoV clade B vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen is a MERS-CoV clade B lineage 2 vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen is a MERS-CoV clade B lineage 3 vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen is a MERS-CoV clade B lineage 4 vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen is a MERS-CoV clade B lineage 5 vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen is a MERS-CoV clade B lineage 6 vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen is a MERS-CoV clade B lineage 7 vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen a MERS-CoV clade C vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen a MERS-CoV Al-Hasa clade vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen a MERS-CoV Buraidah-1 clade vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen a MERS-CoV Hafr-AI-Batin-1 clade vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen a MERS-CoV Jeddah-Riyadh clade vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen a MERS-CoV Riyadh-3 clade vaccine antigen.

[0212] In an embodiment, the S protein trimer is stabilised in the prefusion conformation. In an embodiment, the S protein trimer has modified antigenicity compared to an S protein trimer lacking an inter-protomer disulfide bond.

[0213] In an embodiment, the S protein trimer has modified immunogenicity compared to an S protein trimer lacking an inter-protomer disulfide bond.

[0214] In an embodiment, when administered to a subject the S protein trimer elicits a neutralising antibody response as described herein.

[0215] Ribonucleic acid encoding a S protein monomer of a Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen

[0216] In an aspect, the present invention provides a ribonucleic acid encoding the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen as described herein.

[0217] In an aspect, the present invention provides a ribonucleic acid encoding a S protein monomer of a Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen wherein the vaccine antigen comprises a MERS-CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond.

[0218] In an aspect, the present invention provides a ribonucleic acid encoding a S protein monomer of a coronavirus Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen wherein the vaccine antigen is a MERS-CoV S protein trimer and wherein the S protein monomer of the MERS-CoV S protein trimer has a C-terminal truncation in the stem region.

[0219] In an aspect, the present invention provides a ribonucleic acid encoding a S protein monomer of a coronavirus Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen wherein the vaccine antigen comprises a MERS-CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond and a C-terminal truncation in the stem region.

[0220] In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence selected from: SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 or a sequence at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% identical thereto.

[0221] In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence corresponding to SEQ ID NO: 4, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence corresponding to SEQ ID NO: 10, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence corresponding to SEQ ID NO: 11, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence corresponding to SEQ ID NO: 12, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence corresponding to SEQ ID NO: 13, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence corresponding to SEQ ID NO: 14, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence corresponding to SEQ ID NO: 28, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence corresponding to SEQ ID NO: 29, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence corresponding to SEQ ID NO: 30, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence corresponding to SEQ ID NO: 31, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence corresponding to SEQ ID NO: 32, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence corresponding to SEQ ID NO: 33, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence corresponding to SEQ ID NO: 34, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence corresponding to SEQ ID NO: 35, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence corresponding to SEQ ID NO: 36, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence corresponding to SEQ ID NO: 37, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence corresponding to SEQ ID NO: 38, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence corresponding to SEQ ID NO: 39, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises an RNA sequence encoded by a sequence corresponding to SEQ ID NO: 40, or a sequence at least 70% identical thereto.

[0222] In an embodiment, the ribonucleic acid comprises a sequence selected from: SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 or a sequence at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% identical thereto.

[0223] In an embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 56, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 57, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 58, or a sequence at least 70% identical thereto.

[0224] In an embodiment, the ribonucleic acid encodes an amino acid sequence selected from: SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27.

[0225] In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 3, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 5, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 6, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 7, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 8, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 9, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 15, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 16, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 17, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 18, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 19, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 20, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 21, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 22, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 23, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 24, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 25, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 26, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 27, or a sequence at least 70% identical thereto.

[0226] Coronavirus vaccine antigen

[0227] In an aspect, the present invention provides a coronavirus (CoV) vaccine antigen comprising a CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond.

[0228] In an aspect, the present invention provides a coronavirus (CoV) vaccine antigen comprising a CoV S protein trimer with a C-terminal truncation in the stem region.

[0229] In an aspect, the coronavirus (CoV) vaccine antigen of the present invention comprises a CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond.

[0230] In an aspect, the coronavirus (CoV) vaccine antigen of the present invention comprises a CoV S protein trimer with a C-terminal truncation in the stem region.

[0231] In an aspect, the coronavirus (CoV) vaccine antigen of the present invention comprises a CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond and a C-terminal truncation in the stem region.

[0232] In an aspect, the coronavirus (CoV) vaccine antigen of the present invention comprises a CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond and a structural modification reduces the size of the alanine cavity.

[0233] In an aspect, the coronavirus (CoV) vaccine antigen of the present invention comprises a CoV S protein trimer with a C-terminal truncation in the stem region and a structural modification reduces the size of the alanine cavity. In an aspect, the coronavirus (CoV) vaccine antigen of the present invention comprises a CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond, a C-terminal truncation in the stem region and a structural modification reduces the size of the alanine cavity.

[0234] In an embodiment, the S protein monomer of the S protein trimer lacks a signal sequence. In an embodiment, the S protein monomer of the S protein trimer lacks at least part of the trimerization sequence.

[0235] In an embodiment, the S protein monomer of the S protein trimer does not comprise a sequence that encodes a functional endogenous trimerization sequence.

[0236] In an embodiment, the S protein monomer of the S protein trimer does not comprise a sequence that encodes a functional non-endogenous trimerization sequence.

[0237] In an embodiment, the S protein trimer is soluble.

[0238] In an embodiment, the CoV vaccine antigen additionally comprises the 2P modification. In an embodiment, the CoV vaccine antigen additionally comprises the 6P modification. In an embodiment, the CoV vaccine antigen is a pan-coronavirus vaccine antigen. In an embodiment, the CoV vaccine antigen is a SARS-CoV-2 vaccine antigen. In an embodiment, the CoV vaccine antigen is a SARS-CoV-2 omicron vaccine antigen. In an embodiment, the CoV vaccine antigen a SARS-CoV-2 non-omicron vaccine antigen.

[0239] In an embodiment, the S protein trimer is stabilised in the prefusion conformation.

[0240] In an embodiment, the S protein trimer has modified antigenicity compared to an S protein trimer lacking an inter-protomer disulfide bond.

[0241] In an embodiment, the S protein trimer has modified immunogenicity compared to an S protein trimer lacking an inter-protomer disulfide bond.

[0242] In an embodiment, when administered to a subject the S protein trimer elicits a neutralising antibody response as described herein.

[0243] In an embodiment, the S protein trimer further comprises a structural modification which reduces the size of the alanine cavity in the coiled-coil region of the S protein trimer and wherein the S protein trimer.

[0244] In an embodiment, the CoV vaccine antigen comprises a sequence selected from: SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 80, SEQ ID NO: 81 or a sequence at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% identical thereto.

[0245] In an embodiment, the CoV vaccine antigen comprises a sequence corresponding to SEQ ID NO: 59, or a sequence at least 70% identical thereto. In an embodiment, the CoV vaccine antigen comprises a sequence corresponding to SEQ ID NO: 60, or a sequence at least 70% identical thereto. In an embodiment, the CoV vaccine antigen comprises a sequence corresponding to SEQ ID NO: 61, or a sequence at least 70% identical thereto. In an embodiment, the CoV vaccine antigen comprises a sequence corresponding to SEQ ID NO: 65, or a sequence at least 70% identical thereto. In an embodiment, the CoV vaccine antigen comprises a sequence corresponding to SEQ ID NO: 66, or a sequence at least 70% identical thereto. In an embodiment, the CoV vaccine antigen comprises a sequence corresponding to SEQ ID NO: 69, or a sequence at least 70% identical thereto. In an embodiment, the CoV vaccine antigen comprises a sequence corresponding to SEQ ID NO: 70, or a sequence at least 70% identical thereto. In an embodiment, the CoV vaccine antigen comprises a sequence corresponding to SEQ ID NO: 73, or a sequence at least 70% identical thereto. In an embodiment, the CoV vaccine antigen comprises a sequence corresponding to SEQ ID NO: 74, or a sequence at least 70% identical thereto. In an embodiment, the CoV vaccine antigen comprises a sequence corresponding to SEQ ID NO: 80, or a sequence at least 70% identical thereto. In an embodiment, the CoV vaccine antigen comprises a sequence corresponding to SEQ ID NO: 81, or a sequence at least 70% identical thereto.

[0246] Ribonucleic acid encoding a S protein monomer of a coronavirus (CoV) vaccine antigen

[0247] In an aspect, the present invention provides a ribonucleic acid encoding the coronavirus vaccine antigen as described herein.

[0248] In an aspect, the present invention provides a ribonucleic acid encoding a S protein monomer of a coronavirus (CoV) vaccine antigen wherein the vaccine antigen comprises a CoV S protein trimerwith at least one non-endogenous inter-protomer disulfide bond.

[0249] In an aspect, the present invention provides a ribonucleic acid encoding a S protein monomer of a coronavirus (CoV) vaccine antigen wherein the vaccine antigen is a CoV S protein trimer and wherein the S protein monomer of the CoV S protein trimer has a C-terminal truncation in the stem region.

[0250] In an aspect, the present invention provides a ribonucleic acid encoding a S protein monomer of a coronavirus (CoV) vaccine antigen wherein the vaccine antigen comprises a CoV S protein trimerwith at least one non-endogenous inter-protomer disulfide bond and a C-terminal truncation in the stem region.

[0251] In an aspect, the present invention provides a ribonucleic acid encoding a S protein monomer of a coronavirus (CoV) vaccine antigen wherein the vaccine antigen comprises a CoV S protein trimerwith at least one non-endogenous inter-protomer disulfide bond and a structural modification reduces the size of the alanine cavity.

[0252] In an aspect, the present invention provides a ribonucleic acid encoding a S protein monomer of a coronavirus (CoV) vaccine antigen wherein the vaccine antigen is a CoV S protein trimer and wherein the S protein monomer of the CoV S protein trimer has a C-terminal truncation in the stem region and a structural modification reduces the size of the alanine cavity.

[0253] In an embodiment, the ribonucleic acid comprises an RNA sequence corresponding to a sequence selected from: SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 82, SEQ ID NO: 83, or a sequence at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% identical thereto.

[0254] In an embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 62, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 63, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 64, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 67, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 68, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 71, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 72, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 75, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 76, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 82, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid comprises a sequence corresponding to SEQ ID NO: 83, or a sequence at least 70% identical thereto.

[0255] In an embodiment, the ribonucleic acid encodes an amino acid sequence selected from: SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 80, SEQ ID NO: 81, or a sequence at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% identical thereto.

[0256] In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 59, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 60, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 61, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 65, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 66, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 69, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 70, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 73, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 74, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 80, or a sequence at least 70% identical thereto. In an embodiment, the ribonucleic acid encodes an amino acid sequence corresponding to SEQ ID NO: 81, or a sequence at least 70% identical thereto.

[0257] Cysteine modification

[0258] As used herein, “disulfide bond” refers to a covalent bond formed between the thiol or sulfhydryl (R-SH) side chain of two cysteine residues in one or more proteins. In some embodiments, the disulfide bond is formed between two cysteine residues within a protein.

[0259] As used herein, an “inter-protomer disulfide bond” refers to a disulfide bond between two protomers of an oligomeric structure. In some embodiments, the inter-protomer disulfide bond is formed between two or more protomers (S protein monomers) of the S protein trimer (the oligomeric structure). In some embodiments, each protomer of the S protein trimer are bonded to another protomer of the S protein trimer by a non-endogenous inter-protein disulfide bond.

[0260] As used herein, the “down” conformation of the prefusion S trimer, also known as the “three RBD-down” (“3RBD-down”) conformation, refers to a structure similar to the structure shown in Protein Data Bank ID (PDB ID) 7yn0 (Tsai et al., 2024). This configuration partially occludes the RBM, making it inaccessible to the receptor ACE2. When the RBD flips to the “up” conformation, either in the one RBD-up (1RBD-up) or two RBD-up (2RBD-up) conformations, and exposes the RBM, the adjacent CTD1 and NTD also shift away to accommodate the RBD movement. Thus, the transition of the RBD from the down conformation to the up conformation is a critical step before the receptor can fully engage.

[0261] In an embodiment, the S protein trimer comprises an endogenous or a non-endogenous membrane spanning sequence.

[0262] In an embodiment, the endogenous membrane spanning sequence comprises a sequence selected from: SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54 and SEQ ID NO: 55. In an embodiment, the endogenous membrane spanning sequence comprises a sequence corresponding to SEQ ID NO: 52. In an embodiment, the endogenous membrane spanning sequence comprises a sequence corresponding to SEQ ID NO: 53. In an embodiment, the endogenous membrane spanning sequence comprises a sequence corresponding to SEQ ID NO: 54. In an embodiment, the endogenous membrane spanning sequence comprises a sequence corresponding to SEQ ID NO: 55.

[0263] In an embodiment, the S protein trimer is stabilised in the prefusion conformation. In an embodiment, the non-endogenous inter-protomer disulfide bond is formed between cysteines selected from: i) cysteines at a position corresponding to amino acid numbers 429 and 1059 of SEQ ID NO: 1 or SEQ ID NO: 2 (F14); and / or ii) cysteines at a position corresponding to amino acid numbers 580 and 60 of SEQ ID NO: 1 or SEQ ID NO: 2 (A1).

[0264] In an embodiment, the non-endogenous inter-protomer disulfide bond is formed between cysteines at a position corresponding to amino acid numbers 429 and 1059 of SEQ ID NO: 1 or SEQ ID NO: 2 (F14).

[0265] In an embodiment, the non-endogenous inter-protomer disulfide bond is formed between cysteines at a position corresponding to amino acid numbers 437 and 1064 of SEQ ID NO: 80 (F14).

[0266] In an embodiment, the non-endogenous inter-protomer disulfide bond is formed between cysteines at a position corresponding to amino acid numbers 580 and 60 of SEQ ID NO: 1 or SEQ ID NO: 2 (A1). In an embodiment, the non-endogenous inter-protomer disulfide bond stabilizes the RBD in the down conformation.

[0267] In some embodiments, the non-endogenous inter-protomer disulfide bond is formed between cysteines at a position corresponding to amino acid numbers 429 and 1059 of SEQ ID NO: 1 (F14). In some embodiments, the non-endogenous inter-protomer disulfide bond is formed between cysteines at a position corresponding to amino acid numbers 580 and 60 of SEQ ID NO: 1 (A1). In some embodiments, the non-endogenous inter-protomer disulfide bond is formed between cysteines at a position corresponding to amino acid numbers 580 and 60 of SEQ ID NO: 2 (A1). In some embodiments, the non-endogenous inter-protomer disulfide bond is formed between cysteines at a position corresponding to amino acid numbers 580 and 60 of SEQ ID NO: 2 (A1). In some embodiments, the MERS-CoV vaccine antigen comprises a non-endogenous inter-protomer disulfide bond formed between cysteines at a position corresponding to amino acid numbers 580 and 60 of SEQ ID NO: 1, and a non-endogenous inter-protomer disulfide bond formed between cysteines at a position corresponding to amino acid numbers 429 and 1059 of SEQ ID NO: 1 (AF). In some embodiments, the MERS-CoV vaccine antigen comprises a non-endogenous inter-protomer disulfide bond formed between cysteines at a position corresponding to amino acid numbers 580 and 60 of SEQ ID NO: 1, and a non-endogenous inter-protomer disulfide bond formed between cysteines at a position corresponding to amino acid numbers 429 and 1059 of SEQ ID NO: 2 (AF). In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 429 and 1059 of SEQ ID NO: 1 with cysteine (F14). In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 580 and 60 of SEQ ID NO: 1 with cysteine (A1). In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 580 and 60 of SEQ ID NO: 2 with cysteine (A1). In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 580 and 60 of SEQ ID NO: 2 with cysteine (A1). In some embodiments, the MERS-CoV vaccine antigen comprises a non-endogenous inter-protomer disulfide bond formed as a result of the substitution of the residues corresponding to amino acid numbers 580 and 60 of SEQ ID NO: 1 with cysteine, and a non-endogenous inter-protomer disulfide bond formed as a result of the substitution of the residues corresponding to amino acid numbers 429 and 1059 of SEQ ID NO: 1 with cysteine (AF). In some embodiments, the MERS-CoV vaccine antigen comprises a non-endogenous inter-protomer disulfide bond formed as a result of the substitution of the residues corresponding to amino acid numbers 580 and 60 of SEQ ID NO: 1 with cysteine, and a non-endogenous inter-protomer disulfide bond formed as a result of the substitution of the residues corresponding to amino acid numbers 429 and 1059 of SEQ ID NO: 2 with cysteine (AF).

[0268] In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 362 and 803 of SEQ ID NO: 1 with cysteine (B7). In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 362 and 803 of SEQ ID NO: 2 with cysteine (B7).

[0269] In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 364 and 808 of SEQ ID NO: 1 with cysteine (C44). In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 364 and 808 of SEQ ID NO: 2 with cysteine (C44).

[0270] In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 637 and 1042 of SEQ ID NO: 1 with cysteine (D17). In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 637 and 1042 of SEQ ID NO: 2 with cysteine (D17).

[0271] In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 365 and 930 of SEQ ID NO: 1 with cysteine (E9). In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 365 and 930 of SEQ ID NO: 2 with cysteine (E9).

[0272] In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 509 and 435 of SEQ ID NO: 1 with cysteine (G44). In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 509 and 435 of SEQ ID NO: 2 with cysteine (G44).

[0273] In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 765 and 950 of SEQ ID NO: 1 with cysteine (H2). In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 765 and 950 of SEQ ID NO: 2 with cysteine (H2).

[0274] In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 636 and 1042 of SEQ ID NO: 1 with cysteine (11). In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 636 and 1042 of SEQ ID NO: 2 with cysteine (11).

[0275] In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 781 and 857 of SEQ ID NO: 1 with cysteine (J23). In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 781 and 857 of SEQ ID NO: 2 with cysteine (J23).

[0276] In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 1114 and 1104 of SEQ ID NO: 1 with cysteine (K35). In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 1114 and 1104 of SEQ ID NO: 2 with cysteine (K35).

[0277] In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 1199 and 988 of SEQ ID NO: 1 with cysteine (L21). In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 1199 and 988 of SEQ ID NO: 2 with cysteine (L21).

[0278] In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 1182 and 966 of SEQ ID NO: 1 with cysteine (M2). In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 1182 and 966 of SEQ ID NO: 2 with cysteine (M2).

[0279] In some embodiments, the each protomer of the MERS-CoV S protein trimer can be modified with either one or more of the aforementioned inter-protomer disulfide bond(s). The inter-protomer disulfide bond modifies one or more of the: structure, stability or function of the molecule it is introduced into. In some embodiments, modifying the stability increases the melting temperature of the S protein trimer compared to an identical S protein trimer lacking the interprotomer disulfide bond. In some embodiments, modifying the function increases the immunogenicity of the S protein trimer compared to an identical S protein trimer lacking the interprotomer disulfide bond. In an embodiment, the S-protein trimer is modified to comprise one interprotein disulfide bond. In an embodiment, the S-protein trimer as described herein is modified to comprise one or more inter-protomer disulfide binds. In an embodiment, the S-protein trimer is modified to comprise two inter-protomer disulfide bonds. In an embodiment, the S-protein trimer is modified to comprise three inter-protomer disulfide bonds. In an embodiment, the S-protein trimer is modified to comprise four inter-protomer disulfide bonds.

[0280] In some embodiments, having at least one inter-protomer disulfide bond in the MERS-CoV S protein trimer increases the trimer melting temperature by at least about 1,5°C compared to an identical trimer lacking the inter-protomer disulfide bond. In some embodiments, having at least one inter-protomer disulfide bond in the MERS-CoV S protein trimer increases the trimer melting temperature by at least about 2°C compared to an identical trimer lacking the interprotomer disulfide bond. In some embodiments, having at least one inter-protomer disulfide bond in the MERS-CoV S protein trimer increases the trimer melting temperature by at least about 3°C compared to an identical trimer lacking the inter-protomer disulfide bond. In some embodiments, having at least one inter-protomer disulfide bond in the MERS-CoV S protein trimer increases the trimer melting temperature by at least about 4°C compared to an identical trimer lacking the inter-protomer disulfide bond. In some embodiments, having at least one inter-protomer disulfide bond in the MERS-CoV S protein trimer increases the trimer melting temperature by at least about 5°C compared to an identical trimer lacking the inter-protomer disulfide bond. In some embodiments, having at least one inter-protomer disulfide bond in the MERS-CoV S protein trimer increases the trimer melting temperature by at least about 6°C compared to an identical trimer lacking the inter-protomer disulfide bond. In some embodiments, having at least one interprotomer disulfide bond in the MERS-CoV S protein trimer increases the trimer melting temperature by at least about °C compared to an identical trimer lacking the inter-protomer disulfide bond. In some embodiments, having at least one inter-protomer disulfide bond in the MERS-CoV S protein trimer increases the trimer melting temperature by at least about 8°C compared to an identical trimer lacking the inter-protomer disulfide bond. In some embodiments, having at least one inter-protomer disulfide bond in the MERS-CoV S protein trimer increases the trimer melting temperature by at least about 10°C compared to an identical trimer lacking the inter-protomer disulfide bond. In some embodiments, having at least one inter-protomer disulfide bond in the MERS-CoV S protein trimer increases the trimer melting temperature by at least about 12.5°C compared to an identical trimer lacking the inter-protomer disulfide bond. In some embodiments, having at least one inter-protomer disulfide bond in the MERS-CoV S protein trimer increases the trimer melting temperature by at least about 15°C compared to an identical trimer lacking the inter-protomer disulfide bond. In some embodiments, having at least one interprotomer disulfide bond in the MERS-CoV S protein trimer increases the trimer melting temperature by at least about 20°C compared to an identical trimer lacking the inter-protomer disulfide bond.

[0281] In some embodiments, the melting temperature of the CoV vaccine antigen is about 38°C to about 71 °C, or about 38°C to about 60°C, or about 38°C to about 55°C, or about 40°C to about 55°C, or about 40°C to about 71 °C, or about 42°C to about 71 °C, or about 45°C to about 71 °C, or about 50°C to about 71 °C, or about 55°C to about 71 °C, or about or about 60°C to about 71 °C, or about 51 °C to about 58°C, or about 51°C to about 57°C, or about 51 °C to about 55°C, or about 52°C to about 58°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 38°C to about 71 °C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 38°C to about 60°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 38°C to about 55°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 40°C to about 55°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 40°C to about 71 °C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 42°C to about 71 °C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 45°C to about 71 °C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 50°C to about 71 °C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 51 °C to about 58°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 51 °C to about 57°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 51 °C to about 55°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 52°C to about 58°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 55°C to about 71 °C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 60°C to about 71 °C.

[0282] In some embodiments, the melting temperature of the CoV vaccine antigen is about 40°C, about 41 °C, about 42°C, about 43°C, about 44°C, about 44.5°C, about 45°C, about 46°C, about 47°C, about 48°C, about °C, about 49°C, about 50°C, about 51 °C, or about 52°C, or about 53°C, or about 54°C, or about 55°C, or about 55.8°C, or about 56°C, or about 57°C, or about 57.5°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 40°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 41 °C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 42°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 43°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 44°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 44.5°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 45°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 46°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 47°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 48°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 49°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 50°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 51°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 52°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 53°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 54°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 55°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 55.8°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 56°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 57°C. In some embodiments, the melting temperature of the CoV vaccine antigen is about 57.5°C

[0283] In some embodiments, having at least one inter-protomer disulfide bond in the CoV S protein trimer increases the trimer melting temperature by about 5°C to about 20°C, or by about 5°C to about 15°C, or by about 5°C to about 12.5°C, or by about 5°C to about 10°C, or by about 5°C to about 7°C, compared to an identical trimer lacking the inter-protomer disulfide bond. In some embodiments, having at least one inter-protomer disulfide bond in the CoV S protein trimer increases the trimer melting temperature by about 5°C to about 20°C compared to an identical trimer lacking the inter-protomer disulfide bond. In some embodiments, having at least one interprotomer disulfide bond in the CoV S protein trimer increases the trimer melting temperature by about 5°C to about 15°C compared to an identical trimer lacking the inter-protomer disulfide bond. In some embodiments, having at least one inter-protomer disulfide bond in the CoV S protein trimer increases the trimer melting temperature by about 5°C to about 12.5°C compared to an identical trimer lacking the inter-protomer disulfide bond. In some embodiments, having at least one inter-protomer disulfide bond in the CoV S protein trimer increases the trimer melting temperature by about 5°C to about 10°C compared to an identical trimer lacking the interprotomer disulfide bond. In some embodiments, having at least one inter-protomer disulfide bond in the CoV S protein trimer increases the trimer melting temperature by about 5°C to about 7°C compared to an identical trimer lacking the inter-protomer disulfide bond.

[0284] In an embodiment, the S protein trimer comprises a sequence selected from: SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27.

[0285] In an embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 3. In an embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 5. In an embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 6. In an embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 7. In an embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 8. In an embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 9. In an embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 15. In an embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 16. In an embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 17. In an embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 18. In an embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 19. In an embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 20. In an embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 21. In an embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 22. In an embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 23. In an embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 24. In an embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 25. In an embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 26. In an embodiment, the S protein trimer comprises the sequence of SEQ ID NO: 27.

[0286] Truncations

[0287] In an embodiment, the C-terminal truncation is between residues corresponding to 1220 to 1296 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is between residues corresponding to 1220 to 1275 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is between residues corresponding to 1240 to 1275 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is between residues corresponding to 1220 to 1270 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is between residues corresponding to 1240 to 1270 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is between residues corresponding to 1240 to 1265 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is between residues corresponding to 1220 to 1296 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is between residues corresponding to 1220 to 1275 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is between residues corresponding to 1240 to 1275 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is between residues corresponding to 1220 to 1270 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is between residues corresponding to 1240 to 1270 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is between residues corresponding to 1240 to 1265 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is after a residue corresponding to 1220, 1240, 1248, 1258, 1265, 1270 or 1275 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is after a residue corresponding to 1220, 1240, 1248, 1258, 1265, 1270 or 1275 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1220 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1240 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1248 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is aftera residue corresponding to residue 1258 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1265 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1270 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1275 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1220 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1240 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is aftera residue corresponding to residue 1248 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1258 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1265 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1270 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1275 of SEQ ID NO: 2.

[0288] In an embodiment, the C-terminal truncation is after a residue corresponding to 1220, 1240, 1248, 1258, 1265, 1270 or 1275 of SEQ ID NO: 1 and before the residue corresponding to 1296 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is after a residue corresponding to 1220, 1240, 1248, 1258, 1265, 1270 or 1275 of SEQ ID NO: 2 and before the residue corresponding to 1296 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1220 of SEQ ID NO: 1 and before the residue corresponding to 1296 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1240 of SEQ ID NO: 1 and before the residue corresponding to 1296 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1248 of SEQ ID NO: 1 and before the residue corresponding to 1296 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1258 of SEQ ID NO: 1 and before the residue corresponding to 1296 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1265 of SEQ ID NO: 1 and before the residue corresponding to 1296 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1270 of SEQ ID NO: 1 and before the residue corresponding to 1296 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1275 of SEQ ID NO: 1 and before the residue corresponding to 1296 of SEQ ID NO: 1. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1220 of SEQ ID NO: 2 and before the residue corresponding to 1296 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1240 of SEQ ID NO: 2 and before the residue corresponding to 1296 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1248 of SEQ ID NO: 2 and before the residue corresponding to 1296 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1258 of SEQ ID NO: 2 and before the residue corresponding to 1296 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1265 of SEQ ID NO: 2 and before the residue corresponding to 1296 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1270 of SEQ ID NO: 2 and before the residue corresponding to 1296 of SEQ ID NO: 2. In an embodiment, the C-terminal truncation is after a residue corresponding to residue 1275 of SEQ ID NO: 2 and before the residue corresponding to 1296 of SEQ ID NO: 2.

[0289] In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 1 to 1221, or residues 1 to 1240, or residues 1 to 1248, or residues 1 to 1258, or residues 1 to 1265, or residues 1 to 1270, or residues 1 to 1275 of SEQ ID NO: 1 or SEQ ID NO: 2. In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 1 to 1221 of SEQ ID NO: 1 or SEQ ID NO: 2. In an embodiment, the monomer ofthe S protein trimer comprises residues corresponding to 1 to 1240 of SEQ ID NO: 1 or SEQ ID NO: 2. In an embodiment, the monomer ofthe S protein trimer comprises residues corresponding to 1 to 1248 of SEQ ID NO: 1 or SEQ ID NO: 2. In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 1 to 1258 of SEQ ID NO: 1 or SEQ ID NO: 2. In an embodiment, the monomer ofthe S protein trimer comprises residues corresponding to 1 to 1265 of SEQ ID NO: 1 or SEQ ID NO: 2. In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 1 to 1270 of SEQ ID NO: 1 or SEQ ID NO: 2. In an embodiment, the monomer ofthe S protein trimer comprises residues corresponding to 1 to 1275 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0290] In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1221, or residues 18 to 1240, or residues 18 to 1248, or residues 18 to 1258, or residues 18 to 1265, or residues 18 to 1270, or residues 18 to 1275 of SEQ ID NO: 1 or SEQ ID NO: 2. In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1221 of SEQ ID NO: 1 or SEQ ID NO: 2. In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1240 of SEQ ID NO: 1 or SEQ ID NO: 2. In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1248 of SEQ ID NO: 1 or SEQ ID NO: 2. In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1258 of SEQ ID NO: 1 or SEQ ID NO: 2. In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1265 of SEQ ID NO: 1 or SEQ ID NO: 2. In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1270 of SEQ ID NO: 1 or SEQ ID NO: 2. In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1275 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0291] In an embodiment, when expressed in a recombinant expression system the trimer is produced at a higher level compared to an identical S protein trimer lacking the truncation expressed in the same recombinant expression system.

[0292] In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased about 100% to about 1200% compared to the level of an identical S protein trimer lacking the truncation. In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased about 100% to about 600% compared to the level of an identical S protein trimer lacking the truncation. In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased about 600% to about 1200% compared to the level of an identical S protein trimer lacking the truncation. In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased about 200% to about 1200% compared to the level of an identical S protein trimer lacking the truncation. In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased about 400% to about 1000% compared to the level of an identical S protein trimer lacking the truncation. In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased about 200% to about 1100% compared to the level of an identical S protein trimer lacking the truncation. In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased about 400% to about 700% compared to the level of an identical S protein trimer lacking the truncation. In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased about 500% to about 1000% compared to the level of an identical S protein trimer lacking the truncation. In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased about 500% to about 800% compared to the level of an identical S protein trimer lacking the truncation. In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased about 600% to about 800% compared to the level of an identical S protein trimer lacking the truncation.

[0293] In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased by about 2 fold to about 10 fold compared to the level of an identical S protein trimer lacking the truncation. In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased by about 3 fold to about 9 fold compared to the level of an identical S protein trimer lacking the truncation. In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased by at least about 2 fold compared to the level of an identical S protein trimer lacking the truncation. In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased by at least about 3 fold compared to the level of an identical S protein trimer lacking the truncation. In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased by at least about 4 fold compared to the level of an identical S protein trimer lacking the truncation. In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased by at least about 5 fold compared to the level of an identical S protein trimer lacking the truncation. In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased by at least about 6 fold compared to the level of an identical S protein trimer lacking the truncation. In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased by at least about 7 fold compared to the level of an identical S protein trimer lacking the truncation. In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased by at least about 8 fold compared to the level of an identical S protein trimer lacking the truncation. In an embodiment, when expressed in a recombinant expression system the level of the S protein trimer is increased at least about 8.75 compared to the level of an identical S protein trimer lacking the truncation.

[0294] In an embodiment, the S protein trimer is produced at a level greater than about 300 pg / 50 mL. In an embodiment, the S protein trimer is produced at a level of about 300 pg / 50 mL to about 2000 pg / 50 mL. In an embodiment, the S protein trimer is produced at a level of about 300 pg / 50 mL to about 1900 pg / 50 mL. In an embodiment, the S protein trimer is produced at a level of about 300 pg / 50 mL to about 1100 pg / 50 mL.

[0295] In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1258 of SEQ ID NO: 1 or SEQ ID NO: 2, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to 429 and 1059 of SEQ ID NO: 1 or SEQ ID NO: 2 (F14). In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1258 of SEQ ID NO: 1 or SEQ ID NO: 2, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to 580 and 60 of SEQ ID NO: 1 or SEQ ID NO: 2 (A1). In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1258 of SEQ ID NO: 1 or SEQ ID NO: 2, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to 580 and 60 of SEQ ID NO: 1 or SEQ ID NO: 2, and a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to 429 and 1059 of SEQ ID NO: 1 or SEQ ID NO: 2 (AF). In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1258 of SEQ ID NO: 1 or SEQ ID NO: 2, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to 580 and 60 of SEQ ID NO: 1 or SEQ ID NO: 2 (A1) or 429 and 1059 of SEQ ID NO: 1 or SEQ ID NO: 2 (F14), and wherein the S protein trimer comprises a melting temperature of about 40°C to about 55°C.

[0296] In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1258 of SEQ ID NO: 1 or SEQ ID NO: 2, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to 362 and 803 of SEQ ID NO: 1 or SEQ ID NO: 2 (B7). In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1258 of SEQ ID NO: 1 or SEQ ID NO: 2, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to 364 and 808 of SEQ ID NO: 1 or SEQ ID NO: 2 (C44). In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1258 of SEQ ID NO: 1 or SEQ ID NO: 2, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to 637 and 1042 of SEQ ID NO: 1 or SEQ ID NO: 2 (D17). In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1258 of SEQ ID NO: 1 or SEQ ID NO: 2, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to 365 and 930 of SEQ ID NO: 1 or SEQ ID NO: 2 (E9). In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1258 of SEQ ID NO: 1 or SEQ ID NO: 2, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to 509 and 435 of SEQ ID NO: 1 or SEQ ID NO: 2 (G44). In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1258 of SEQ ID NO: 1 or SEQ ID NO: 2, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to 765 and 950 of SEQ ID NO: 1 or SEQ ID NO: 2 (H2). In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1258 of SEQ ID NO: 1 or SEQ ID NO: 2, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to 636 and 1042 of SEQ ID NO: 1 or SEQ ID NO: 2 (11). In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1258 of SEQ ID NO: 1 or SEQ ID NO: 2, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to 781 and 857 of SEQ ID NO: 1 or SEQ ID NO: 2 (J23). In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1258 of SEQ ID NO: 1 or SEQ ID NO: 2, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to 1114 and 1104 of SEQ ID NO: 1 or SEQ ID NO: 2 (K35). In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1258 of SEQ ID NO: 1 or SEQ ID NO: 2, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to 1119 and 988 of SEQ ID NO: 1 or SEQ ID NO: 2 (L21). In an embodiment, the monomer of the S protein trimer comprises residues corresponding to 18 to 1258 of SEQ ID NO: 1 or SEQ ID NO: 2, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to 1182 and 966 of SEQ ID NO: 1 or SEQ ID NO: 2 (M2).

[0297] In an embodiment, the monomer of the S protein trimer comprises a sequence selected from: SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9. In an embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 3. In an embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 5. In an embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 6. In an embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 7. In an embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 8. In an embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 9.

[0298] In an embodiment, the monomer of the S protein trimer comprises a sequence selected from: SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27.

[0299] In an embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 15. In an embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 16. In an embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 17. In an embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 18. In an embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 19. In an embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 20. In an embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 21. In an embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 22. In an embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 23. In an embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 24. In an embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 25. In an embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 26. In an embodiment, the monomer of the S protein trimer comprises the sequence of SEQ ID NO: 27.

[0300] Non-endogenous inter-protomer disulfide bond increases stability

[0301] In an embodiment, the S protein trimer as described herein comprising the at least one non-endogenous inter-protomer disulfide bond results in stabilization of the S protein trimer. In an embodiment, the S protein trimer as described herein comprising the at least one non-endogenous inter-protomer disulfide bond results in stabilization of the S protein trimer in the presence of an adjuvant, compared to the S protein trimer lacking the non-endogenous interprotomer disulfide bond. As used herein “stabilization” or “stabilised” refers to increasing one or more of the: thermal stability, longevity, immunogenicity and production stability, yield or homogeneity of the S protein trimer, and denaturation stability. In an embodiment, the stability is increased in vitro and / or in vivo stability. In an embodiment, the in vivo stability is increased (when administered to a subject or upon assembly in a subject e.g. after translation from an mRNA vaccine). In an embodiment, the stability is increased in vitro (e.g. during production processes). As used herein, “thermal stability” refers to the efficacy and potency of the vaccine antigen being retained when exposed to a variety of different temperatures. As used herein, “production stability” refers to the stability of the vaccine antigen during the manufacturing process, as opposed to changes in stability of the vaccine antigen over time. For example, some vaccine antigens when produced have poor stability and thus are less desirable for use in vaccines. As disclosed herein, the inventors have found particular modified vaccine antigens that have advantageously high stability during production, thus making them desirable for vaccine development.

[0302] In an embodiment, the non-endogenous inter-protomer disulfide bond increases the stability of the S protein trimer compared to the S protein trimer lacking the non-endogenous inter-protomer disulfide bond. In an embodiment, the S protein trimer is stable in the absence of at least part or all of an endogenous trimerization sequence (a trimerization sequence is not required to achieve stability). In an embodiment, the S protein trimer is stable in the absence of a non-endogenous trimerization sequence (a trimerization sequence is not required to achieve stability). In an embodiment, the S protein trimer is stable in the absence of at least part or all of an endogenous trimerization sequence (a trimerization sequence is not required to achieve stability) and in the absence of a non-endogenous trimerization sequence.

[0303] In an embodiment, the non-endogenous inter-protomer disulfide bond increases the melting temperature of the S protein trimer compared to the S protein trimer lacking the non-endogenous inter-protomer disulfide bond. In an embodiment, the non-endogenous interprotomer disulfide bond increases the thermal stability of the S protein trimer. In an embodiment, the non-endogenous inter-protomer disulfide bond increases the melting temperature of the S protein trimer by about 5°C to about 25°C. In an embodiment, the non-endogenous interprotomer disulfide bond increases the melting temperature of the S protein trimer by about 5°C to about 23°C. In an embodiment, the non-endogenous inter-protomer disulfide bond increases the melting temperature of the S protein trimer by about 5°C to about 23°C. In an embodiment, the non-endogenous inter-protomer disulfide bond increases the melting temperature of the S protein trimer by about 10°C to about 23°C. In an embodiment, the non-endogenous interprotomer disulfide bond increases the melting temperature of the S protein trimer by about 10°C to about 20°C. In an embodiment, the non-endogenous inter-protomer disulfide bond increases the melting temperature of the S protein trimer by about 5°C to about 15°C. In an embodiment, the non-endogenous inter-protomer disulfide bond increases the melting temperature of the S protein trimer by about 5°C to about 10°C.

[0304] In an embodiment, the non-endogenous inter-protomer disulfide bond increases the stability of the S protein trimer to denaturing conditions. Denaturing conditions include for example boiling in in the presence of detergent (e.g. sodium dodecyl sulfate) or treating with detergent (e.g. sodium dodecyl sulfate) with and without 2 betamercaptoethanol at room temperature.

[0305] In an embodiment, the present specification enables a method of improving the stability and / or expression of Middle East respiratory syndrome coronavirus (MERS-CoV) S antigen.

[0306] In an embodiment, the MERS-CoV vaccine antigen is soluble. In an embodiment the CoV vaccine antigen is stabilised in a pre-fusion S protein trimer conformation. In an embodiment, the receptor binding domain (RBD) of the S protein trimer is stabilised in an up (RBD-up) orientation. In an embodiment, the MERS-CoV vaccine antigen is able to be bound by RBD-up directed neutralising antibodies. In an embodiment, the receptor binding domain (RBD) of the S protein trimer is stabilised in a down (RBD-down) orientation. In an embodiment, the MERS-CoV vaccine antigen is able to be bound by RBD-down directed neutralising antibodies.

[0307] In an embodiment, the MERS-CoV vaccine antigen lacks a trimerization sequence. In an embodiment, the MERS-CoV vaccine antigen lacks a transmembrane domain. In an embodiment, the MERS-CoV vaccine antigen lacks a foldon sequence / domain.

[0308] In an embodiment, the MERS-CoV vaccine antigen is suitable for intra-dermal administration. In an embodiment, the MERS-CoV vaccine antigen is suitable for oral administration. In an embodiment, the MERS-CoV vaccine antigen is suitable for pulmonary administration. In an embodiment, the MERS-CoV vaccine antigen is suitable for nasal administration.

[0309] Immunogenicity and antigenicity The S protein is the main protein used as a target antigen in available coronavirus vaccines such as SARS-CoV-2. Thus, the S protein is a potential major target antigen for MERS-CoV vaccines. Theoretically, antibodies can target the S protein to inhibit virus infection at multiple stages during the virus entry process. The RBD is the major target for neutralising antibodies (NAbs) that interfere with viral receptor binding. Conserved neutralization sites in S2 include the fusion peptide and stem region. In addition, NAbs targeting the N-terminal domain have been reported in MERS-CoV infection making it another potential target for inclusion in a vaccine. The S2 subunit is also a potential target for neutralising antibodies that interfere with the structural rearrangement of the S protein and the insertion of the fusion protein required for virushost membrane fusion.

[0310] As used herein, “neutralising antibodies” or “NAbs” is a type of antibody that binds an antigen, pathogen or toxin and neutralises its functional activity. NAbs are referred to as functional antibodies because they have a functional anti-virus effect. In an embodiment, the neutralising antibody response inhibits binding to the DPP4 receptor. In an embodiment, the neutralising antibody inhibits binding of the RBD to the DPP4 receptor. In an embodiment, the neutralising antibody binds to a non-RBD epitope. As used herein, a “neutralising antibody response” refers to the production of NAbs in a subject after exposure to an antigen.

[0311] The ability of vaccines to elicit NAbs or effective immune responses against heterologous strains or emerging variants of concern is a major factor influencing the successful roll out of a vaccine program against MERS-CoV.

[0312] The ability of vaccines to elicit NAbs or effective immune responses against homologous and heterologous strains or emerging variants, including variants of concern is a major factor influencing the successful roll out of a vaccine program against MERS-CoV.

[0313] In an embodiment, the vaccine antigens as described herein are pan-coronavirus vaccine antigens. As used herein, “a pan-coronavirus vaccine antigen” is an antigen that produces a neutralising antibody response against more than one merbecovirus, sarbecoviruses and / or more than one betacoronavirus. In an embodiment, the pan-coronavirus vaccine antigen produces a neutralising antibody response against more than one merbecovirus. In an embodiment, the pan-coronavirus vaccine antigen produces a neutralising antibody response against at least two merbecoviruses. In an embodiment, the pan-coronavirus vaccine antigen produces a neutralising antibody response against at least three merbecoviruses. In an embodiment, the merbecovirus is a bat merbecovirus. In an embodiment, the bat merbecovirus is NeoCoV. In an embodiment, the merbecovirus is a HKU4 clade virus. In an embodiment, the merbecovirus is a HKU5 clade virus. In an embodiment, the merbecovirus is a mink respiratory coronavirus (MRCoV). In an embodiment, the pan-coronavirus vaccine antigen produces a neutralising antibody response against MERS-CoV. In an embodiment, the pan-coronavirus vaccine antigen produces a neutralising antibody response against MERS-CoV and NeoCoV. In an embodiment, the pan-coronavirus vaccine antigen produces a neutralising antibody response against NeoCoV and MRCoV. In an embodiment, the pan-coronavirus vaccine antigen produces a neutralising antibody response against MERS-CoV and MRCoV. In an embodiment, the pancoronavirus vaccine antigen produces a neutralising antibody response against MERS-CoV, NeoCoV and MRCoV. In an embodiment, the pan-coronavirus vaccine antigen produces a neutralising antibody response against a merbecovirus HKU4 clade virus. In an embodiment, the pan-coronavirus vaccine antigen produces a neutralising antibody response against a merbecovirus HKU5 clade virus. In an embodiment, the pan-coronavirus vaccine antigen produces a neutralising antibody response against one or more merbecoviruses selected from the group consisting of: a merbecovirus HKU4 clade virus, a merbecovirus HKU5 clade virus, MERS-CoV and MRCoV. In an embodiment, the pan-coronavirus vaccine antigen produces a neutralising antibody response against a merbecovirus HKU4 clade virus, MERS-CoV, NeoCoV and MRCoV. In an embodiment, the pan-coronavirus vaccine antigen produces a neutralising antibody response against a merbecovirus HKU5 clade virus, MERS-CoV, NeoCoV and MRCoV. In an embodiment, the pan-coronavirus vaccine antigen produces a neutralising antibody response against a merbecovirus HKU4 clade virus, a merbecovirus HKU5 clade virus, MERS-CoV, NeoCoV and MRCoV. In an embodiment, the pan-coronavirus vaccine antigen produces a neutralising antibody response against more than one sarbecovirus. In an embodiment, the pancoronavirus vaccine antigen produces a neutralising antibody response against at least two sarbecoviruses. In an embodiment, the pan-coronavirus vaccine antigen produces a neutralising antibody response against a clade 1b sarbecovirus and a clade 1a sarbecovirus. In an embodiment, the pan-coronavirus vaccine antigen produces a neutralising antibody response against a clade 1b sarbecovirus and a clade 3 sarbecovirus. In an embodiment, the pancoronavirus vaccine antigen produces a neutralising antibody response against two or more of a clade 1b sarbecovirus, a clade 1a sarbecovirus and a clade 3 sarbecovirus. Members of the sarbecovirus clades are described, for example, in Khaledian et al. (2022) and Sallard et al. (2021). In an embodiment, the pan-coronavirus vaccine antigen produces a neutralising antibody response against more the one betacoronavirus. In an embodiment, the pan-coronavirus vaccine antigen produces a neutralising antibody response against at least two betacoronavirus.

[0314] In an embodiment, the vaccine antigens as described herein have altered immunogenicity, when delivered to a subject, compared to a vaccine antigen lacking i) an interprotomer disulfide bond as described herein, ii) C-terminal truncation in the stem region as described herein or i) and ii). In an embodiment, the vaccine antigens as described herein have altered immunogenicity, when delivered to a subject, compared to a vaccine antigen lacking an inter-protomer disulfide bond as described herein. In an embodiment, the vaccine antigens as described herein have altered immunogenicity, when delivered to a subject, compared to a vaccine antigen lacking the C-terminal truncation in the stem region as described herein. In an embodiment, the vaccine antigens as described herein have altered immunogenicity, when delivered to a subject, compared to a vaccine antigen lacking an inter-protomer disulfide bond as described herein and lacking the C-terminal truncation in the stem region as described herein.

[0315] As used herein, “immunogenicity” refers to the ability of a substance to induce cellular and humoral immune response. In an embodiment, the altered immunogenicity is increased immunogenicity. In an embodiment, the humoral immune response comprises neutralising antibodies. Immunogenicity can be measured using any method known to a person skilled in the art including measuring antibody titre.

[0316] In an embodiment, the neutralising antibody response is a RBD neutralising antibody response. In an embodiment, the neutralising antibody response comprises antibodies directed to an epitope or epitopes that include(s) part or all of RBM. In an embodiment, the neutralising antibody response is not directed at the RBM.

[0317] In an embodiment, wherein the neutralising antibody response is a non-RBD neutralising antibody response. In an embodiment, the non-RBD neutralising antibody response comprises a neutralising antibody response directed to an epitope or epitopes that include(s) part or all of the stem region. In an embodiment, the non-RBD neutralising antibody response comprises a neutralising antibody response directed to an epitope or epitopes that include(s) part or all of the NTD.

[0318] In an embodiment, the vaccine antigens as described herein elicit antibody responses to the core stem 1138-1165 region. In an embodiment, the vaccine antigens as described herein elicit antibody responses to the core stem 1138-1165 region and beyond the core stem region.

[0319] In an embodiment, the vaccine antigens as described herein have altered antigenicity, when delivered to a subject, compared to a vaccine antigen lacking i) an inter-protomer disulfide bond as described herein, ii) C-terminal truncation in the stem region as described herein or i) and ii). In an embodiment, the vaccine antigens as described herein have altered antigenicity, when delivered to a subject, compared to a vaccine antigen lacking an inter-protomer disulfide bond as described herein. In an embodiment, the vaccine antigens as described herein have altered antigenicity, when delivered to a subject, compared to a vaccine antigen lacking the C-terminal truncation in the stem region as described herein. In an embodiment, the vaccine antigens as described herein have altered antigenicity, when delivered to a subject, compared to a vaccine antigen lacking an inter-protomer disulfide bond as described herein and lacking the C-terminal truncation in the stem region as described herein. As used herein, “antigenicity” refers to the ability of a particular substance to be recognized by antibodies produced as a result of a specific immune response. In an embodiment, the altered antigenicity is increased immunogenicity.

[0320] In an embodiment, the S protein trimer is able to be bound by an antibody that binds an epitope or epitopes that include(s) part or all of the N-terminal domain (NTD). In an embodiment, the antibody is selected from: G2 and an antibody that binds an epitope bound by G2. In an embodiment, the antibody is G2. In an embodiment, the antibody that binds the NTD is an antibody that binds an epitope bound by G2. In an embodiment, the antibody binds an epitope that includes one or more amino acids bound by G2. As used herein, an “epitope bound by G2” comprises the NTD residues corresponding to K27, S28, S191, N193, A197, and N199 of SEQ ID NO: 1 and SEQ ID NO: 2.

[0321] In an embodiment, the S protein trimer is able to be bound by any antibody that binds an epitope or epitopes that include(s) part or all of the RBD. In an embodiment, the antibody is selected from: DPP4-FC, CDC2-C2, KNIH90-F1, MERS4, an antibody that binds an epitope bound by DPP4-Fc, an antibody that binds an epitope bound by CDC2-C2 an antibody that binds an epitope bound by KNIH90-F1 and an antibody that binds an epitope bound by MERS4. As used herein, “DPP4-Fc” refers to a fusion protein wherein the ectodomain of the DPP4 receptor is fused to human IgG 1 Fc. In an embodiment, the antibody binds an epitope that includes one or more amino acids bound by DPP4-Fc. In an embodiment, the antibody binds an epitope that includes one or more amino acids bound by CDC2-C2. In an embodiment, the antibody the binds an epitope that includes one or more amino acids bound by KNIH90-F1. As used herein, an “epitope bound by DPP4-Fc” comprises the residues corresponding to Y499, N501, K502, D510, R511, E513, P515, W535, E536, D539, Y540, R542, W553 and V555 of SEQ ID NO: 1 and SEQ ID NO: 2. As used herein, an “epitope bound by CDC2-C2” comprises the residues corresponding to N501, K502, S504, F506, D510, R511, T512, E513, W535, E536, D537, G538, D539, Y540, Y541, R542, W553, V555, A556 and S557 of SEQ ID NO: 1 and SEQ ID NO: 2. As used herein, an “epitope bound by KNIH90-F1” comprises the residues corresponding to R505, L506, L507, D509, R511, T512, E513, V514, P515, and L517 of SEQ ID NO: 1 and SEQ ID NO: 2. As used herein, an “epitope bound by MERS4” comprises the residues corresponding to S508, N519, N521, Q522 and L548 of SEQ ID NO: 1 and SEQ ID NO: 2.

[0322] In an embodiment, the S protein trimer is able to be bound by any antibody that binds an epitope or epitopes that include(s) part or all of the RBM. In an embodiment, the antibody is selected from: CDC2-C2, KNIH90-F1, an antibody that binds an epitope bound by CDC2-C2, an antibody that binds an epitope bound by KNIH90-F1. In an embodiment, the antibody that binds the RBM is CDC2-C2. In an embodiment, the antibody that binds the RBM is KNIH90-F1. In an embodiment, the antibody that binds the RBM is an antibody that binds an epitope bound by CDC2-C2. In an embodiment, the antibody that binds the RBM is an antibody that binds an epitope bound by KNIH90-F1. In an embodiment, the antibody binds an epitope that includes one or more amino acids bound by CDC2-C2. In an embodiment, the antibody binds an epitope that includes one or more amino acids bound by KNIH90-F1.

[0323] In an embodiment, the S protein trimer is able to be bound by any antibody that binds an epitope or epitopes that include(s) part or all of the stem. In an embodiment, the antibody is selected from: S2P6, CC95-108 and CC99-103. In an embodiment, the antibody is S2P6. As used herein, an “epitope bound by S2P6” comprises the residues corresponding to 11228-V1241 of SEQ ID NO: 1 and SEQ ID NO: 2. In an embodiment, the antibody is CC95-108. In an embodiment, the antibody binds the epitope bound by CC95-108. In an embodiment, the antibody binds an epitope that includes one or more amino acids bound by CC95-108. As used herein, an “epitope bound by CC95-108” comprises the residues corresponding to F1231-F1238 of SEQ ID NO: 1 and SEQ ID NO: 2. In an embodiment, the antibody is CC99-103. In an embodiment, the antibody binds the epitope bound by CC99-103. In an embodiment, the antibody binds an epitope that includes one or more amino acids bound by CC99-103. As used herein, an “epitope bound by CC99-103” comprises the residues corresponding to F1231-F1238 of SEQ ID NO: 1 and SEQ ID NO: 2.

[0324] In an embodiment, the S protein trimer is able to be bound by any antibody that binds an epitope or epitopes that include(s) part or all of the S2 domain. In an embodiment, the antibody is selected from: S2P6, CC95-108 and CC99-103. In an embodiment, the antibody is S2P6. In an embodiment, the antibody is CC95-108. In an embodiment, the antibody binds the epitope bound by CC95-108. In an embodiment, the antibody binds an epitope that includes one or more amino acids bound by CC95-108. In an embodiment, the antibody is CC99-103. In an embodiment, the antibody binds the epitope bound by CC99-103. In an embodiment, the antibody binds an epitope that includes one or more amino acids bound by CC99-103.

[0325] In an embodiment, the S protein trimer is able to be bound by any antibody that binds an epitope or epitopes that include(s) part or all of the fusion peptide. In an embodiment, the antibody that binds an epitope or epitopes that include(s) part or all of the fusion peptide is DPP4-Fc. In an embodiment, the antibody binds the epitope bound by DPP4-Fc. In an embodiment, the antibody binds an epitope that includes one or more amino acids bound by DPP4-Fc.

[0326] In an embodiment, the S protein trimer is able to be bound by any antibody that binds an RBM-independent epitope or epitopes that include(s) part or all of the “up”-oriented RBD. In an embodiment, the antibody that binds the RBM-independent epitope or epitopes that include(s) part or all of the “up”-oriented RBD is MERS4. In an embodiment, the antibody binds the epitope bound by MERS4. In an embodiment, the antibody binds an epitope that includes one or more amino acids bound by MERS4. Further modifications / further stabilising modifications

[0327] In an embodiment, the MERS-CoV vaccine antigen as described herein comprises one or more further modifications to enhance one or more of the: stability, immunogenicity, expression and purification of the S protein trimer.

[0328] In an embodiment, the ribonucleic acid encoding a MERS-CoV vaccine as described herein comprises one or more further modifications to enhance one or more of the: stability, immunogenicity, expression and purification of the S protein trimer.

[0329] In an embodiment, the further modification is selected from a proline stabilisation, furin cleavage site, a trimerization sequence, a repeat or a spacer, or nucleotide sequences encoding same.

[0330] In an embodiment, the proline stabilisation modification is 1060P and / or 1061 P. The presence of both 1060P and 1061 P in the S protein trimer is referred to as the “2P” modification.

[0331] In an embodiment, the further modification is the deletion of a furin cleavage site. In an embodiment, the mutation A748SVG is introduced to delete a furin cleavage site (e.g. A748SVG replaces R748SVR) In an embodiment, the further modification is the addition of a purification tag.

[0332] Antigen combinations

[0333] In an embodiment, the subject modified S antigen elicits neutralising immune responses against the strain from which it is derived and one or more other strains circulating in the community. In another aspect, the antigen or vaccine comprising the antigen or nucleic acid encoding the antigen, delivers one or multiple antigens of interest to a subject and induces an effective functional and polyfunctional immune response against homologous or heterologous strains including for example T-cell and antibody responses. In an embodiment, coronavirus antigens from one or more strains are selected from one or two or three or four of spike, nucleocapsid, membrane and envelope proteins. In an embodiment, amino acid and / or nucleotide sequences encoding MERS-CoV proteins two, three or four of N, M, E and S antigens are employed. In an illustrative embodiment, N, M, E and S antigens are employed. In an embodiment, one or two or multiple different variants of MERS-CoV are combined. In an embodiment, one or two or multiple different variants of MERS-CoV are combined. In an embodiment, multiple variants and multiple antigens are employed. In an embodiment, the antigen or vaccine comprising the antigen or encoding sequence is administered with one or more B-cell and / or T-cell epitopes.

[0334] Cell lines capable of expressing the herein disclosed modified S antigen together with one or more of N, M, E antigens, or their encoding sequences are contemplated herein. Methods of producing the vaccine antigen

[0335] Antigens as described herein may be produced by recombinant or synthetic routes as known in the art. In an embodiment, the antigen is produced from a deoxyribonucleic acid encoding the coronavirus vaccine antigen as described herein. In an embodiment, the antigen is produced from vector comprising a deoxyribonucleic acid encoding the coronavirus vaccine antigen as described herein.

[0336] In an aspect, the present invention provides a host cell comprising the deoxyribonucleic acid as described herein or the vector as described herein.

[0337] In an aspect, the present invention provides a method of producing the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen as described herein comprising culturing the host cell as described herein in culture medium to produce the vaccine antigen. In an embodiment, the method further comprising isolating the vaccine antigen from the cell and / or cell culture medium.

[0338] In an aspect, the present invention provides a vaccine comprising the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen as described herein, or the protein nanoparticle as described herein, or the virus-like particle as described herein, or the deoxyribonucleic acid as described herein, or the vector as described herein. In an embodiment, vaccine is selected from an: inactivated vaccine; live attenuated vaccine; and a protein subunit vaccine.

[0339] In an embodiment, the vaccine further comprises at least one further CoV vaccine antigen or at least one further ribonucleic acid encoding an S protein monomer of a further coronavirus (CoV) vaccine antigen.

[0340] In an embodiment, the vaccine further comprises at least one further MERS-CoV vaccine antigen as described herein.

[0341] In an embodiment, the further MERS-CoV vaccine antigen is selected from one or more of: NeoCoV, MRCoV, MjHKU4rCoV1, merbecovirus HKU4 clade and merbecovirus HKU5 clade vaccine antigens. In an embodiment, the further MERS-CoV vaccine antigen comprises a NeoCoV vaccine antigen. In an embodiment, the further MERS-CoV vaccine antigen comprises a MRCoV vaccine antigen. In an embodiment, the further MERS-CoV vaccine antigen comprises a MjHKU4r CoV1 vaccine antigen. In an embodiment, the further MERS-CoV vaccine antigen comprises a merbecovirus HKU4 vaccine antigen. In an embodiment, the further MERS-CoV vaccine antigen comprises a merbecovirus HKU5 vaccine antigen.

[0342] In an embodiment, the vaccine further comprises at least one further CoV vaccine antigen as described herein. In an embodiment, the further CoV vaccine antigen is selected from one or more of: a sarbecovirus, a bat sarbecovirus (such as PRD-0038 and WIV1), OC43, a SARS-CoV and a SARS-CoV2 vaccine antigen. In an embodiment, the further CoV vaccine antigen comprises a sarbecovirus vaccine antigen. In an embodiment, the further CoV vaccine antigen comprises a bat sarbecovirus vaccine antigen. In an embodiment, the further CoV vaccine antigen comprises a bat divergent sarbecovirus vaccine antigen. In an embodiment, the further CoV vaccine antigen comprises SARS-CoV2, WIV1 and / or PRD-0038 vaccine antigens. In an embodiment, the further CoV vaccine antigen comprises a SARS-CoV2 vaccine antigen. In an embodiment, the further CoV vaccine antigen comprises a WIV1 vaccine antigen. In an embodiment, the further CoV vaccine antigen comprises a PRD-0038 vaccine. In an embodiment, the further CoV vaccine antigen comprises a SARS-CoV2 and WIV1 vaccine antigens. In an embodiment, the further CoV vaccine antigen comprises SARS-CoV2 and PRD-0038 vaccine antigens. In an embodiment, the further CoV vaccine antigen comprises WIV1 and PRD-0038 vaccine antigens. In an embodiment, the further CoV vaccine antigen comprises SARS-CoV2, WIV1 and PRD-0038 vaccine antigens.

[0343] In an embodiment, the vaccine is a multivalent vaccine. In an embodiment, the vaccine is a bivalent vaccine. In an embodiment, the vaccine is a trivalent vaccine. In an embodiment, the vaccine is a tetravalent vaccine. In an embodiment, the vaccine is a pentavalent vaccine.

[0344] In an embodiment, the vaccine comprises a merbecovirus vaccine antigen, a SARS-CoV2 vaccine antigen, a clade 1a sarbecovirus vaccine antigen and a clade 3 sarbecovirus vaccine antigen.

[0345] In an embodiment, the tetravalent vaccine comprises a merbecovirus vaccine antigen, a SARS-CoV2 vaccine antigen, a clade 1a bat sarbecovirus vaccine antigen and a clade 3 bat sarbecovirus vaccine antigen.

[0346] In an embodiment, the merbecovirus vaccine antigen, SAR-CoV2 vaccine antigen, clade 1a sarbecovirus vaccine antigen and the clade 3 sarbecovirus vaccine antigen comprise at least one non-endogenous inter-protomer disulfide bond and / or a C-terminal truncation in the stem region.

[0347] In an embodiment, the merbecovirus vaccine antigen, SAR-CoV2 vaccine antigen, clade 1a sarbecovirus vaccine antigen and the clade 3 sarbecovirus vaccine antigen comprise at least one non-endogenous inter-protomer disulfide bond and a C-terminal truncation in the stem region.

[0348] In an embodiment, the merbecovirus vaccine antigen is a MERS-CoV vaccine antigen. In an embodiment, the SARS-CoV2 vaccine antigen is an omicron variant.

[0349] In an embodiment, the omicron variant is BA.2.86.

[0350] In an embodiment, the clade 1a sarbecovirus is a clade 1a bat sarbecovirus. In an embodiment, the clade 3 sarbecovirus is a clade 3 bat sarbecovirus.

[0351] In an embodiment, the clade 1 a bat sarbecovirus vaccine antigen is WIV1.

[0352] In an embodiment, the clade 3 bat sarbecovirus vaccine antigen is PRD-0038.

[0353] In an embodiment, the SARS-CoV2 vaccine antigen, the clade 1a bat sarbecovirus vaccine antigen and / or the clade 3 bat sarbecovirus vaccine antigen is a S protein trimer. In an embodiment, the SARS-CoV2 vaccine antigen and the clade 1 a bat sarbecovirus vaccine antigen is a S protein trimer. In an embodiment, the clade 1a bat sarbecovirus vaccine antigen and the clade 3 bat sarbecovirus vaccine antigen is a S protein trimer. In an embodiment, the SARS-CoV2 vaccine antigen and the clade 3 bat sarbecovirus vaccine antigen is a S protein trimer. In an embodiment, the SARS-CoV2 vaccine antigen, the clade 1a bat sarbecovirus vaccine antigen and the clade 3 bat sarbecovirus vaccine antigen is a S protein trimer.

[0354] In an embodiment, the SARS-CoV2 vaccine antigen, the clade 1a bat sarbecovirus vaccine antigen and / or the clade 3 bat sarbecovirus vaccine antigen comprises at least one non-endogenous inter-protomer disulfide bond. In an embodiment, the SARS-CoV2 vaccine antigen comprises at least one non-endogenous inter-protomer disulfide bond. In an embodiment, the clade 1a bat sarbecovirus vaccine antigen comprises at least one non-endogenous interprotomer disulfide bond. In an embodiment, the clade 3 bat sarbecovirus vaccine antigen comprises at least one non-endogenous inter-protomer disulfide bond. In an embodiment, the SARS-CoV2 vaccine antigen and the clade 1a bat sarbecovirus vaccine antigen comprises at least one non-endogenous inter-protomer disulfide bond. In an embodiment, the clade 1a bat sarbecovirus vaccine antigen and the clade 3 bat sarbecovirus vaccine antigen comprises at least one non-endogenous inter-protomer disulfide bond. In an embodiment, the SARS-CoV2 vaccine antigen, and the clade 3 bat sarbecovirus vaccine antigen comprises at least one non-endogenous inter-protomer disulfide bond. In an embodiment, the SARS-CoV2 vaccine antigen, the clade 1a bat sarbecovirus vaccine antigen and the clade 3 bat sarbecovirus vaccine antigen comprises at least one non-endogenous inter-protomer disulfide bond.

[0355] In some embodiments, the non-endogenous inter-protomer disulfide bond is formed as a result of the substitution of the residues corresponding to amino acid numbers 637 and 1042 of SEQ ID NO: 1 with cysteine (D17).

[0356] In an embodiment, the MERS-CoV vaccine antigen is a MERS-CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond and / or a C-terminal truncation in the stem region. In an embodiment, the MERS-CoV vaccine antigen is a MERS-CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond. In an embodiment, the MERS-CoV vaccine antigen is a MERS-CoV S protein trimer with a C-terminal truncation in the stem region. In an embodiment, the MERS-CoV vaccine antigen is a MERS-CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond and a C-terminal truncation in the stem region.

[0357] In an embodiment, the MERS-CoV vaccine antigen is a soluble S protein trimer.

[0358] In an embodiment, the MERS-CoV vaccine antigen is as defined in an embodiment of the present disclosure.

[0359] In an embodiment, the vaccine elicits a neutralising antibody response to bat merbecovirus capable of binding to ACE2 and / or DPP4. In an embodiment, the vaccine elicits a neutralising antibody response to bat merbecovirus capable of binding to ACE2. In an embodiment, the ACE2 is human ACE2. In an embodiment, the vaccine elicits a neutralising antibody response to bat merbecovirus capable of binding to DPP4. In an embodiment, the vaccine elicits a neutralising antibody response to bat merbecovirus capable of binding to ACE2 and DPP4. In an embodiment, the bat merbecovirus is NeoCoV.

[0360] NeoCoV is a pre-emergent bat Merbecovirus that can use human ACE2 as an entry receptor (Xiong et al., 2022).

[0361] In an embodiment, the vaccine elicits a neutralising antibody response to mink respiratory coronavirus (MRCoV).

[0362] In an embodiment, the vaccine elicits a neutralising antibody response to MERS-CoV. In an embodiment, the vaccine elicits a neutralising antibody response to SARS-CoV. In an embodiment, the SARS-CoV is SARS-CoV Tor2.

[0363] In an embodiment, the vaccine elicits a neutralising antibody response to SARS-CoV2. In an embodiment, the SARS-CoV2 is a SARS-CoV2 omicron variant.

[0364] In an embodiment, the SARS-CoV2 is a variant of concern (VOC).

[0365] In an embodiment, the VOC is JN.1 and / or XEC.

[0366] In an embodiment, the vaccine elicits a neutralising antibody response against MERS-CoV and NeoCoV. In an embodiment, the vaccine elicits a neutralising antibody response against NeoCoV and MRCoV. In an embodiment, the vaccine elicits a neutralising antibody response against MERS-CoV and MRCoV. In an embodiment, the vaccine elicits a neutralising antibody response against MERS-CoV, NeoCoV and MRCoV.

[0367] In an embodiment, the vaccine elicits a neutralising antibody response against one or more of: SARS-CoV, SARS-CoV2, MERS-CoV, NeoCoV and MRCoV. In an embodiment, the vaccine elicits a neutralising antibody response against SARS-CoV, MERS-CoV, NeoCoV and MRCoV. In an embodiment, the vaccine elicits a neutralising antibody response against SARS-CoV2, MERS-CoV, NeoCoV and MRCoV. In an embodiment, the vaccine elicits a neutralising antibody response against SARS-CoV, SARS-CoV2 and MERS-CoV. In an embodiment, the vaccine elicits a neutralising antibody response against SARS-CoV, SARS-CoV2 and NeoCoV. In an embodiment, the vaccine elicits a neutralising antibody response against SARS-CoV, SARS-CoV2 and MRCoV. In an embodiment, the vaccine elicits a neutralising antibody response against SARS-CoV, SARS-CoV2, MERS-CoV and NeoCoV. In an embodiment, the vaccine elicits a neutralising antibody response against SARS-CoV, SARS-CoV2, MERS-CoV, NeoCoV and MRCoV.

[0368] In an embodiment, the vaccine elicits a neutralising antibody response against more than one merbecovirus. In an embodiment, the vaccine elicits a neutralising antibody response against at least two merbecoviruses. In an embodiment, the vaccine elicits a neutralising antibody response against at least three merbecoviruses. In an embodiment, the merbecovirus is a bat merbecovirus. In an embodiment, the bat merbecovirus is NeoCoV. In an embodiment, the merbecovirus is a HKU4 clade virus. In an embodiment, the merbecovirus is a HKU5 clade virus. In an embodiment, the merbecovirus is a mink respiratory coronavirus (MRCoV). In an embodiment, the vaccine elicits a neutralising antibody response against MERS-CoV. In an embodiment, the vaccine elicits a neutralising antibody response against MERS-CoV and NeoCoV. In an embodiment, the vaccine elicits a neutralising antibody response against NeoCoV and MRCoV. In an embodiment, the vaccine elicits a neutralising antibody response against MERS-CoV and MRCoV. In an embodiment, the vaccine elicits a neutralising antibody response against MERS-CoV, NeoCoV and MRCoV. In an embodiment, the vaccine elicits a neutralising antibody response against a merbecovirus HKU4 clade virus. In an embodiment, the vaccine elicits a neutralising antibody response against a merbecovirus HKU5 clade virus. In an embodiment, the vaccine elicits a neutralising antibody response against one or more merbecoviruses selected from the group consisting of: a merbecovirus HKU4 clade virus, a merbecovirus HKU5 clade virus, MERS-CoV and MRCoV. In an embodiment, the vaccine elicits a neutralising antibody response against a merbecovirus HKU4 clade virus, MERS-CoV, NeoCoV and MRCoV. In an embodiment, the vaccine elicits a neutralising antibody response against a merbecovirus HKU5 clade virus, MERS-CoV, NeoCoV and MRCoV. In an embodiment, the vaccine elicits a neutralising antibody response against a merbecovirus HKU4 clade virus, a merbecovirus HKU5 clade virus, MERS-CoV, NeoCoV and MRCoV. In an embodiment, the vaccine elicits a neutralising antibody response against more than one sarbecoviruses. In an embodiment, the vaccine elicits a neutralising antibody response against at least two sarbecoviruses. In an embodiment, the vaccine elicits a neutralising antibody response against a clade 1b sarbecovirus and a clade 1a sarbecovirus. In an embodiment, the vaccine elicits a neutralising antibody response against a clade 1b sarbecovirus and a clade 3 sarbecovirus. In an embodiment, the vaccine elicits a neutralising antibody response against two or more of a clade 1b sarbecovirus, a clade 1a sarbecovirus and a clade 3 sarbecovirus. Members of the sarbecovirus clades are described, for example, in Khaledian et al. (2022) and Sallard et al. (2021). In an embodiment, the vaccine elicits a neutralising antibody response against more the one betacoronavirus. In an embodiment, the vaccine elicits a neutralising antibody response against at least two betacoronavirus.

[0369] In an embodiment, the vaccine comprises a MERS-CoV vaccine antigen, a SARS-CoV2 BA.2.86 vaccine antigen, a WIV1 vaccine antigen and a PRD-0038 vaccine antigen. In an embodiment, the vaccine comprises a MERS-CoV vaccine antigen as defined in the present disclosure, a SARS-CoV2 BA.2.86 vaccine antigen, a WIV1 vaccine antigen and a PRD-0038 vaccine antigen. In an embodiment, the tetravalent vaccine comprises a MERS-CoV vaccine antigen, a SARS-CoV2 BA.2.86 vaccine antigen, a WIV1 vaccine antigen and a PRD-0038 vaccine antigen. In an embodiment, the tetravalent vaccine comprises a MERS-CoV vaccine antigen as defined in the present disclosure, a SARS-CoV2 BA.2.86 vaccine antigen, a WIV1 vaccine antigen and a PRD-0038 vaccine antigen.

[0370] In an embodiment, the vaccine comprises a MERS-CoV vaccine antigen, a SARS-CoV2 BA.2.86 vaccine antigen, a WIV1 vaccine antigen and a PRD-0038 vaccine antigen, wherein the MERS-CoV vaccine antigen is a MERS-CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond, wherein the non-endogenous inter-protomer disulfide bond is formed between cysteines selected from: i) cysteines at a position corresponding to amino acid numbers 429 and 1059 of SEQ ID NO: 1 (F14); and / or ii) cysteines at a position corresponding to amino acid numbers 580 and 60 of SEQ ID NO: 1 (A1).

[0371] In an embodiment, the vaccine comprises a MERS-CoV vaccine antigen, a SARS-CoV2 BA.2.86 vaccine antigen, a WIV1 vaccine antigen and a PRD-0038 vaccine antigen, wherein the MERS-CoV vaccine antigen is a MERS-CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond, wherein the non-endogenous inter-protomer disulfide bond is formed between cysteines at a position corresponding to amino acid numbers 429 and 1059 of SEQ ID NO: 1 (F14).

[0372] In an embodiment, the vaccine comprises a MERS-CoV vaccine antigen, a SARS-CoV2 BA.2.86 vaccine antigen, a WIV1 vaccine antigen and a PRD-0038 vaccine antigen, wherein the MERS-CoV vaccine antigen is a MERS-CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond, wherein the non-endogenous inter-protomer disulfide bond is formed between cysteines at a position corresponding to amino acid numbers 580 and 60 of SEQ ID NO: 1 (A1).

[0373] In an embodiment, the vaccine comprises a MERS-CoV vaccine antigen, a SARS-CoV2 BA.2.86 vaccine antigen, a WIV1 vaccine antigen and a PRD-0038 vaccine antigen, wherein the MERS-CoV vaccine antigen is a MERS-CoV S protein trimer with a C-terminal truncation in the stem region, wherein the C-terminal truncation is after a residue corresponding to 1258 of SEQ ID NO: 1 and before the residue corresponding to 1296 of SEQ ID NO: 1. In an embodiment, the vaccine comprises a MERS-CoV vaccine antigen, a SARS-CoV2 BA.2.86 vaccine antigen, a WIV1 vaccine antigen and a PRD-0038 vaccine antigen, wherein the MERS-CoV vaccine antigen is a MERS-CoV S protein trimer with a C-terminal truncation in the stem region, wherein the protomer of the S protein trimer comprises or consists of residues corresponding to 18 to 1258 of SEQ ID NO: 1.

[0374] In an embodiment, the vaccine comprises a MERS-CoV vaccine antigen, a SARS-CoV2 BA.2.86 vaccine antigen, a WIV1 vaccine antigen and a PRD-0038 vaccine antigen, wherein the MERS-CoV vaccine antigen is a MERS-CoV S protein trimer comprising or consisting of residues corresponding to 18 to 1258 of SEQ ID NO: 1, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to S429C and D1059C of SEQ ID NO: 1 (F14) or D580 and Q60C of SEQ ID NO: 1 (A1).

[0375] In an embodiment, the vaccine comprises a MERS-CoV vaccine antigen, a SARS-CoV2 BA.2.86 vaccine antigen, a WIV1 vaccine antigen and a PRD-0038 vaccine antigen, wherein the MERS-CoV vaccine antigen is a MERS-CoV S protein trimer comprising or consisting of residues corresponding to 18 to 1258 of SEQ ID NO: 1, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to S429C and D1059C of SEQ ID NO: 1 (F14).

[0376] In an embodiment, the vaccine comprises a MERS-CoV vaccine antigen, a SARS-CoV2 BA.2.86 vaccine antigen, a WIV1 vaccine antigen and a PRD-0038 vaccine antigen, wherein the MERS-CoV vaccine antigen is a MERS-CoV S protein trimer comprising or consisting of residues corresponding to 18 to 1258 of SEQ ID NO: 1, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to D580 and Q60C of SEQ ID NO: 1 (A1).

[0377] In an embodiment, the vaccine comprises a MERS-CoV vaccine antigen, a SARS-CoV2 BA.2.86 vaccine antigen, a WIV1 vaccine antigen and a PRD-0038 vaccine antigen, wherein the MERS-CoV vaccine antigen is a MERS-CoV S protein trimer comprising or consisting of residues corresponding to 18 to 1258 of SEQ ID NO: 1, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to S429C and D1059C of SEQ ID NO: 1 (F14), wherein:

[0378] i) the SARS-CoV BA.2.86 vaccine antigen is a SARS-CoV BA2.86 S protein trimer comprising a non-endogenous inter-protomer disulfide bond formed as a result of the substitution of the residues corresponding to amino acid numbers 637 and 1042 of SEQ ID NO: 1 with cysteine (D17),

[0379] ii) the WIV1 vaccine antigen is a WIV1 S protein trimer comprising a non-endogenous inter-protomer disulfide bond formed as a result of the substitution of the residues corresponding to amino acid numbers 637 and 1042 of SEQ ID NO: 1 with cysteine (D17), and / or

[0380] iii) the PRD-0038 vaccine antigen is a PRD-0038 S protein trimer comprising a non-endogenous inter-protomer disulfide bond formed as a result of the substitution of the residues corresponding to amino acid numbers 637 and 1042 of SEQ ID NO: 1 with cysteine (D17). In an embodiment, the vaccine comprises a MERS-CoV vaccine antigen, a SARS-CoV2 BA.2.86 vaccine antigen, a WIV1 vaccine antigen and a PRD-0038 vaccine antigen, wherein the MERS-CoV vaccine antigen is a MERS-CoV S protein trimer comprising or consisting of residues corresponding to 18 to 1258 of SEQ ID NO: 1, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to S429C and D1059C of SEQ ID NO: 1 (F14), wherein:

[0381] i) the SARS-CoV BA.2.86 vaccine antigen is a SARS-CoV BA2.86 S protein trimer comprising a non-endogenous inter-protomer disulfide bond formed as a result of the substitution of the residues corresponding to amino acid numbers 637 and 1042 of SEQ ID NO: 1 with cysteine (D17),

[0382] ii) the WIV1 vaccine antigen is a WIV1 S protein trimer comprising a non-endogenous inter-protomer disulfide bond formed as a result of the substitution of the residues corresponding to amino acid numbers 637 and 1042 of SEQ ID NO: 1 with cysteine (D17), and

[0383] iii) the PRD-0038 vaccine antigen is a PRD-0038 S protein trimer comprising a non-endogenous inter-protomer disulfide bond formed as a result of the substitution of the residues corresponding to amino acid numbers 637 and 1042 of SEQ ID NO: 1 with cysteine (D17).

[0384] In an embodiment, the MERS-CoV vaccine antigen comprises or consists of the sequence selected from the group consisting of: SEQ ID NO: 5-9, 15 and 20.

[0385] In an embodiment, the SARS-CoV2 BA.2.86 vaccine antigen comprises or consists of the sequence selected from the group consisting of: SEQ ID NO: 59, 60, 61, 65 and 66.

[0386] In an embodiment, the WIV1 vaccine antigen comprises or consists of the sequence of SEQ ID NO: 73 or 74.

[0387] In an embodiment, the PRD-0038 vaccine antigen comprises or consists of the sequence of SEQ ID NO: 69 or 70.

[0388] In an embodiment, the vaccine comprises a MERS-CoV vaccine antigen, a SARS-CoV2 BA.2.86 vaccine antigen, a WIV1 vaccine antigen and a PRD-0038 vaccine antigen, wherein:

[0389] i) the MERS-CoV vaccine antigen comprises or consists of the sequence selected from the group consisting of: SEQ ID NO: 5-9, 15 and 20;

[0390] ii) the SARS-CoV2 BA.2.86 vaccine antigen comprises or consists of the sequence selected from the group consisting of: SEQ ID NO: 59, 60, 61, 65 and 66;

[0391] iii) the WIV1 vaccine antigen comprises or consists of the sequence of SEQ ID NO: 73 or 74; and

[0392] iv) the PRD-0038 vaccine antigen comprises or consists of the sequence of SEQ ID NO: 69 or 70.

[0393] In an embodiment, the vaccine comprises a nucleic acid encoding a merbecovirus vaccine antigen, a nucleic acid encoding a SARS-CoV2 vaccine antigen, a nucleic acid encoding a clade 1a bat sarbecovirus vaccine antigen and a nucleic acid encoding a clade 3 bat sarbecovirus vaccine antigen. In an embodiment, the tetravalent vaccine comprises a nucleic acid encoding a merbecovirus vaccine antigen, a nucleic acid encoding a SARS-CoV2 vaccine antigen, a nucleic acid encoding a clade 1a bat sarbecovirus vaccine antigen and a nucleic acid encoding a clade 3 bat sarbecovirus vaccine antigen.

[0394] In an embodiment, the vaccine comprises a nucleic acid encoding a MERS-CoV vaccine antigen, a nucleic acid encoding a SARS-CoV2 BA.2.86 vaccine antigen, a nucleic acid encoding a WIV1 vaccine antigen and a nucleic acid encoding a PRD-0038 vaccine antigen. In an embodiment, the tetravalent vaccine comprises a nucleic acid encoding a MERS-CoV vaccine antigen, a nucleic acid encoding a SARS-CoV2 BA2.86 vaccine antigen, a nucleic acid encoding a WIV1 vaccine antigen and a nucleic acid encoding a PRD-0038 vaccine antigen.

[0395] In an embodiment, the MERS-CoV vaccine antigen is encoded by a sequence selected from the group consisting of: SEQ ID NO: 10-14, 28 and 33.

[0396] In an embodiment, the SARS-CoV2 BA.2.86 vaccine antigen is encoded by a sequence selected from the group consisting of: SEQ ID NO: 62, 63, 64, 67 and 68.

[0397] In an embodiment, the WIV1 vaccine antigen is encoded by the sequence of SEQ ID NO: 75 or 76.

[0398] In an embodiment, the PRD-0038 vaccine antigen is encoded by the sequence of SEQ ID NO: 71 or 72.

[0399] In an embodiment, the sequences of two of the vaccine antigens are about 30-40% identical. In an embodiment, the sequences of two of the vaccine antigens are about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or about 40% identical. In an embodiment, the sequences of two of the vaccine antigens are about 30% identical. In an embodiment, the sequences of two of the vaccine antigens are about 31% identical. In an embodiment, the sequences of two of the vaccine antigens are about 32% identical. In an embodiment, the sequences of two of the vaccine antigens are about 33% identical. In an embodiment, the sequences of two of the vaccine antigens are about 34% identical. In an embodiment, the sequences of two of the vaccine antigens are about 35% identical. In an embodiment, the sequences of two of the vaccine antigens are about 36% identical. In an embodiment, the sequences of two of the vaccine antigens are about 37% identical. In an embodiment, the sequences of two of the vaccine antigens are about 38% identical. In an embodiment, the sequences of two of the vaccine antigens are about 39% identical. In an embodiment, the sequences of two of the vaccine antigens are about 40% identical.

[0400] In an embodiment, the sequences of two of the vaccine antigens are about 70-80% identical. In an embodiment, the sequences of two of the vaccine antigens are about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or about 80% identical. In an embodiment, the sequences of two of the vaccine antigens are about 70% identical. In an embodiment, the sequences of two of the vaccine antigens are about 71% identical. In an embodiment, the sequences of two of the vaccine antigens are about 72% identical. In an embodiment, the sequences of two of the vaccine antigens are about 73% identical. In an embodiment, the sequences of two of the vaccine antigens are about 74% identical. In an embodiment, the sequences of two of the vaccine antigens are about 75% identical. In an embodiment, the sequences of two of the vaccine antigens are about 76% identical. In an embodiment, the sequences of two of the vaccine antigens are about 77% identical. In an embodiment, the sequences of two of the vaccine antigens are about 78% identical. In an embodiment, the sequences of two of the vaccine antigens are about 79% identical. In an embodiment, the sequences of two of the vaccine antigens are about 80% identical.

[0401] In an embodiment, the sequence of the merbecovirus vaccine antigen and the sequence of the clade 1a bat sarbecovirus vaccine antigen is about 30-40% identical. In an embodiment, the sequence of the merbecovirus vaccine antigen and the sequence of the clade 3 bat sarbecovirus vaccine antigen is about 30-40% identical. In an embodiment, the sequence of the Merbecovirus vaccine antigen and the sequence of the SARS-CoV2 vaccine antigen is about 30-40% identical. In an embodiment, the sequence of the clade 1 a bat sarbecovirus vaccine antigen and the sequence of the clade 3 bat sarbecovirus vaccine antigen is about 70-80% identical. In an embodiment, the sequence of the clade 1a bat sarbecovirus vaccine antigen and the sequence of the SARS-CoV2 vaccine antigen is about 70-80% identical. In an embodiment, the sequence of the clade 3 bat sarbecovirus vaccine antigen and the sequence of the SARS-CoV2 vaccine antigen is about 70-80% identical.

[0402] In an embodiment, the vaccine is a veterinary vaccine. In an embodiment, the vaccine is a human vaccine.

[0403] In an embodiment, a nanoparticle is provided comprising an antigen as described herein fused to a polyhedrin targeting peptide from cytoplasmic polyhedrosis virus (CPV) or other suitable virus. Other nanoparticles are known in the art and include stimuli-responsive nanoparticles (SOR), luminazine synthase particles, pyruvate dehydrogenase particles.

[0404] The antigen may be linked to a carrier or nanoparticle for increased immunogenicity. Suitable carriers are known in the art.

[0405] Viral like particles offer some of the structural complexity / advantages of viral surface proteins to antigens and may be derived from any suitable viruses. As used herein “a virus like particle” refers to vaccines comprising viral surface proteins but lack the viral genome and one or more structural proteins. Human and hepadnavirus hepatitis B virus (HBV) are good examples. VLPs comprising the antigen may form for example spontaneously upon recombinant expression of the protein and may be characterised using conventional technology. In an aspect, the present invention provides a method of increasing S protein trimer yield comprising modifying the MERS-CoV S protein trimer to comprise a stem region C-terminal truncation.

[0406] In an aspect, the present invention provides a method of stabilizing a MERS-CoV S protein trimer in a prefusion conformation comprising modifying the MERS-CoV S protein trimer to comprise at least one non-endogenous inter-protomer disulfide bond.

[0407] In an aspect, the present invention provides a method of increasing the melting temperature of a MERS-CoV S protein trimer comprising modifying the MERS-CoV S protein trimer to comprise a stem region C-terminal truncation.

[0408] In an aspect, the present invention provides a method of increasing the melting temperature of a MERS-CoV S protein trimer stabilised in the prefusion conformation comprising modifying the MERS-CoV S protein trimer to comprise a stem region C-terminal truncation.

[0409] In an aspect, the present invention provides a method of enhancing neutralising antibody responses comprising modifying the MERS-CoV S protein trimer to comprise at least one interprotomer disulfide bond and / or modifying the MERS-CoV S protein trimer to comprise a stem region C-terminal truncation.

[0410] In some embodiments, a ribonucleic acid encoding the antigen is administered to a subject and the antigen is produced in the subject.

[0411] RNAs encoding the vaccine antigen and methods of production thereof

[0412] The RNA as described herein may be modified by stabilizing sequences, capping, and polyadenylation. RNA may be delivered in a vector as described herein. RNA may be delivered as plasmids to express antigen and induce immune responses. The RNA may be modified to enhance delivery via a lipid nanoparticle. The RNA may be modified to increase stability of the RNA molecule. RNA-based approaches can include amplifying or non-self amplifying constructs. In an embodiment, the RNA is a messenger RNA (mRNA).

[0413] In some embodiments, an RNA encoding the antigen as described herein is administered. In some embodiments, the RNA encodes an antigen lacking a coronavirus transmembrane domain as described herein. In some embodiments, the RNA encodes an antigen comprising a trimerization domain as described herein. In some embodiments, the RNA encodes an antigen lacking a trimerization domain as described herein.

[0414] In some embodiments the RNA to be administered by transient in vivo transfection is a chemically modified RNA in which a proportion (e.g., 10%, 30%, 50%, or 100%) of at least one type of nucleotide, e.g., cytosine, is chemically modified to increase its stability in vivo. For example, in some cases modified cytosines are 5 methylcytosines. Such RNAs are particularly useful for delivery / transfection to cells in vivo, especially when combined with a transfection / delivery agent. In some cases, a chemically modified RNA is a chemically modified RNA in which a majority of (e.g., all) cytosines are 5-methylcytosines, and where a majority (e.g., all) of uracils are pseudouracils. In some embodiments, non-native cysteines are engineered to create di-sulphide bonds (e.g. via recombinant genetic technologies). The synthesis and use of such modified RNAs are described in, e.g., WO 2011 / 130624. Methods for in vivo transfection of RNA polynucleotides are known in the art.

[0415] The term " RNA" relates to a molecule which comprises ribonucleotide residues and preferably being entirely or substantially composed of ribonucleotide residues. " Ribonucleotide" relates to a nucleotide with a hydroxyl group at the 2'-position of a p-D-ribofuranosyl group. The term includes double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of a RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

[0416] Accordingly, in an embodiment, the G / C content of the coding region is modified, particularly increased, compared to the G / C content of the coding region of its particular wild type coding sequence, i.e. the unmodified mRNA. The encoded amino acid sequence of the mRNA is preferably not modified compared to the coded amino acid sequence of the particular wild type mRNA.

[0417] An optimised mRNA based composition could comprise a 5' and 3' non translated region (5'-UTR, 3'-UTR) that optimise translation efficiency and intracellular stability as known in the art and an open reading frame encoding the S protein. In an embodiment, removal of uncapped 5'-triphosphates can be achieved by treating RNA with a phosphatase. RNA may have modified ribonucleotides in order to increase its stability and / or decrease cytotoxicity. For example, in an embodiment, in the RNA, 5-methylcytidine is substituted partially or completely, for cytidine. Alternatively, or additionally, pseudouridine is substituted partially or completely, preferably completely, for uridine. These modifications may also reduce indiscriminate immune inactivation which may hinder translation of the RNA. In an embodiment, the term "modification" relates to providing an RNA with a 5'-cap or 5'-cap analog. The term "5'-cap" refers to a cap structure found on the 5'-end of an mRNA molecule and generally consists of a guanosine nucleotide connected to the mRNA via an unusual 5' to 5' triphosphate linkage. In an embodiment, this guanosine is methylated at the 7-position. The term "conventional 5'-cap" refers to a naturally occurring RNA 5'-cap, preferably to the 7-methylguanosine cap. The term "5'-cap" includes a 5'-cap analog that resembles the RNA cap structure and is modified to possess the ability to stabilize RNA and / or enhance translation of RNA.

[0418] A further modification of RNA may be an extension ortruncation of the naturally occurring UTR such as the X-region tail or an alteration of the 5'- or 3 '-untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA, for example, the exchange of the existing 3'-UTR with or the insertion of one or more, preferably two copies of a 3'-UTR derived from a globin gene, such as alpha2-globin, alphal-globin, beta-globin. RNA having an unmasked poly-A sequence is translated more efficiently than RNA having a masked poly-A sequence.

[0419] The term "poly(A) tail" or "poly-A sequence" relates to a sequence of adenyl (A) residues which may be located on the 3'-end of a RNA molecule and "unmasked poly-A sequence" means that the poly-A sequence at the 3' end of an RNA molecule ends with an A of the poly-A sequence and is not followed by nucleotides other than A located at the 3' end, i.e. downstream, of the poly-A sequence. Furthermore, a long poly-A sequence of about 120 base pairs results in an optimal transcript stability and translation efficiency of RNA.

[0420] Therefore, in order to increase stability and / or expression of the RNA it may be modified so as to be present in conjunction with a heterologous poly-A sequence, preferably having a length of 10 to 500, more preferably 30 to 300, even more preferably 65 to 200 and especially 100 to 150 adenosine residues. In an especially preferred embodiment, the poly-A sequence has a length of approximately 120 adenosine residues. To further increase stability and / or expression of the RNA used according to the invention, the poly-A sequence can be unmasked.

[0421] In addition, incorporation of a 3'-non translated region (UTR) into the 3'-non translated region of an RNA molecule can result in an enhancement in translation efficiency. A synergistic effect may be achieved by incorporating two or more of such 3'-non translated regions. The 3'-non translated regions may be autologous or heterologous to the RNA into which they are introduced. In one particular embodiment the 3'-non translated region is derived from the human p-globin gene.

[0422] A combination of the above described modifications, i.e. optionally incorporation of a poly-A sequence, unmasking of the poly-A sequence and incorporation of one or more 3 '-non translated regions, has a synergistic influence on the stability of RNA and increase in translation efficiency.

[0423] In order to increase expression of the RNA it may be modified within the coding region so as to increase the GC-content to increase mRNA stability and to perform a codon optimization and, thus, enhance translation in cells. Modified mRNA may be synthesised enzymatically and packaged into nanoparticles such as lipid nanoparticles and administered, for example intramuscularly. Self-replicating RNA or protamine complexed RNA approaches have also been shown to generate immune responses against viral infections.

[0424] The RNA molecule can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are known in the art and disclosed in Remington, the Science and Practice of Pharmacy, 20th Edition, Remington, J., ed. (2000).

[0425] Various approaches for systemic administration of RNA as nanoparticles or colloidal systems are known. In non-viral approaches, cationic liposomes are used to induce RNA condensation and to facilitate cellular uptake. The cationic liposomes usually consist of a cationic lipid, like DOTAP, and one or more helper lipids, like DOPE. So-called 'lipoplexes' can be formed from the cationic (positively charged) liposomes and the anionic (negatively charged) RNA. In the simplest case, the lipoplexes form spontaneously by mixing the RNA with the liposomes with a certain mixing protocol, however various other protocols may be applied. In an embodiment, nanoparticulate RNA formulations such as RNA lipoplexes, are produced with defined particle size wherein the net charge of the particles is close to zero or negative. For example, electroneutral or negatively charged lipoplexes from RNA and liposomes lead to substantial RNA expression in spleen or immune cells after systemic administration as disclosed in WO2013 / 143683. In an embodiment, the nanoparticles comprise at least one lipid. In an embodiment, the nanoparticles comprise at least one cationic lipid. The cationic lipid can be monocationic or polycationic. Any cationic amphiphilic molecule, e.g., a molecule which comprises at least one hydrophilic and lipophilic moiety is a cationic lipid within the meaning of the present invention. In an embodiment, the positive charges are contributed by the at least one cationic lipid and the negative charges are contributed by the RNA. In an embodiment, the nanoparticles comprises at least one helper lipid. The helper lipid may be a neutral or an anionic lipid. The helper lipid may be a natural lipid, such as a phospholipid or an analogue of a natural lipid, or a fully synthetic lipid, or lipid-like molecule, with no similarities with natural lipids. In an embodiment, the cationic lipid and / or the helper lipid is a bilayer forming lipid.

[0426] In an embodiment, the at least one cationic lipid comprises l,2-di-0-octadecenyl-3-trimethylammonium propane (DOTMA) or analogs or derivatives thereof and / or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or analogs or derivatives thereof.

[0427] In an embodiment, the at least one helper lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or analogs or derivatives thereof, cholesterol (Choi) or analogs or derivatives thereof and / or l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or analogs or derivatives thereof. In an embodiment, the molar ratio of the at least one cationic lipid to the at least one helper lipid is from 10:0 to 3:7, preferably 9: 1 to 3:7, 4: 1 to 1: 2, 4: 1 to 2: 3, 7: 3 to 1: 1, or 2: 1 to 1: 1, preferably about 1: 1. In an embodiment, in this ratio, the molar amount of the cationic lipid results from the molar amount of the cationic lipid multiplied by the number of positive charges in the cationic lipid. In the nanoparticles described herein the lipid may form a complex with and / or may encapsulate the RNA. In an embodiment, the nanoparticles comprise a lipoplex or liposome. In an embodiment, the lipid is comprised in a vesicle encapsulating said RNA. The vesicle may be a multilamellar vesicle, an unilamellar vesicle, or a mixture thereof. The vesicle may be a liposome.

[0428] A lipid nanoparticle (LNP) is generally known as a nanosized particle composed of a combination of different lipids (an aqueous volume is encapsulated by amphipathic lipid bilayers e.g., single; unilamellar or multiple; multilamellar). Many different types of lipids may be included in LNP. In some embodiments, the lipids may be one or more of an ionisable lipid, a phospholipid, a structural lipid, neutral lipid and a PEG lipid. For example, the mRNA is encapsulated in a LNP. In another example, the mRNA is bound to the LNP. For example, the mRNA is absorbed on the LNP.

[0429] Methods of preparing LNP are known to a person skilled in that art and are described, for example, in Huang et al., 2021 and Schoenmaker et al., 2021. As used herein, the term “ionisable lipid” or “ionisable lipids” shall refer to a lipid having at least one protonatable or deprotonatable group. For example, the lipid is positively charged at a pH at or below physiological pH (e.g. pH 7.4), and neutral at a second pH (e.g. at or above physiological pH), n an embodiment, the lipid nanoparticle comprises an ionisable lipid as described in Table 1 of Schoenmaker et al., 2021.

[0430] Suitable ionisable lipids can have an anionic, cationic or zwitterionic hydrophilic head group. Exemplary phospholipids (anionic or zwitterionic) for use in the present disclosure include, for example, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, and phosphatidylglycerols. In one example, the lipid is a cationic lipid. Exemplary cationic lipids include, but are not limited to, dioleoyl trimethylammonium propane (DOTAP), l,2-distearyloxy-N, N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy- N, Ndimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N, N-dimethyl-3- aminopropane (DLinDMA), 2,5-bis((9z,12z)-octadeca-9,12,dien-1-yloxyl)benzyl-4-(dimethylamino)butanoate (LKY750). In one example, the phospholipid is 2,5-bis((9z,12z)-octadeca-9,12,dien-1-yloxyl)benzyl-4- (dimethylamino)butanoate (LKY750). Exemplary zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids, such as dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC) and dodecylphosphocholine. The lipids can be saturated or unsaturated. In an embodiment, the lipid nanoparticle does not comprise a cationic lipid.

[0431] A person skilled person in the art will appreciate that reference to a PEGylated lipid is a lipid that has been modified with polyethylene glycol. Exemplary PEGylated lipids include, but are not limited to, PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols. For example, a PEG lipid includes PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, a PEG-DSPE lipid and combinations thereof.

[0432] Suitable neutral or zwitterionic lipids for use in the present disclosure will be apparent to the skilled person and include, for example, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1 -glycerol) sodium salt (DOPG), and sphingomyelin. The lipids can be saturated or unsaturated.

[0433] Exemplary structural lipids include, but are not limited to, cholesterol fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid and alpha-tocopherol. In an embodiment, the structural lipid is a sterol. In an embodiment, the structural lipid is cholesterol. In an embodiment, the structural lipid is campesterol.

[0434] Vaccines in the form of liposomes are encompassed. The term "liposome" herein refers to uni- or multilamellar lipid structures enclosing an aqueous interior. Lipids which are capable of forming liposomes include all substances having fatty or fat-like properties. Dynamic laser light scattering is a method used to measure the size of liposomes well known to those skilled in the art. An extensive description of adjuvants can be found in Cox and Coulter, " Advances in Adjuvant Technology and Application", in Animal Parasite Control Utilizing Biotechnology, Chapter 4, Ed. Young, W. K., CRC Press 1992, and in Cox and Coulter, Vaccine 15(3): 248- 256, 1997. In an aspect, the present invention provides a host cell comprising the ribonucleic acid as described herein or the vector as described herein.

[0435] In an aspect, the present invention provides a method of producing a Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine comprising culturing the host cell as described herein in culture medium to produce the ribonucleic acid described herein. In an embodiment, the ribonucleic acid as described herein is isolated and formulated into a pharmaceutical composition.

[0436] Cell culture

[0437] A skilled person would appreciate that the vaccine antigen as described herein, deoxyribonucleic acid as described herein, ribonucleic acid as described herein, or vector as described herein can be produced in cell culture. In one example, the cells are prokaryotic or eukaryotic. In one example, the cells are of mammalian, avian, bacterial or Arthropoda origin. In one example, the cells are mammalian. In one example, the cells are from a continuous cell line. In one example, the cells are from a primary cell line. In one example, the cells are from an immortalized cell line. In one example, the cells are adherent cells. In one example, the cells are non-adherent cells (suspension cells). In one example, the cells are immune cells.

[0438] In one example, the mammalian cells are HEK, CHO or HeLa cells. In one example, the cells are HeLa cells.

[0439] The cells can be cultured in any cell culture medium that allows the expansion of the cells in vitro. Such mediums and processes will be known to the skilled person. Exemplary cell culture mediums for culturing the population of cells of the present invention include, but are not limited to: Iscove’s medium, UltraCHO, CD Hybridoma serum free medium, episerf medium, MediV SF103 (serum free medium), Dulbecco’s modified eagle medium (DMEM), Eagles Modified Eagle Medium (EMEM), Glasgow’s modified eagle medium (GMEM), SMIP-8, modified eagle medium (MEM), VP-SFM, DMEM based SFM, DMEM / F12, DMEM / Ham’s F12, VPSFM / William’s medium E, ExCell 525(SFM), adenovirus expression medium (AEM) and Excell 65629. It will be appreciated by persons skilled in the art that such mediums may be supplemented with additional growth factors, for example, but not limited, amino acids, hormones, vitamins and minerals. Optionally, such mediums may be supplemented with serum, for example fetal calf serum.

[0440] In one example, the cells are cultured using the batch cell culture process. In one example, the cells are cultured using the perfusion cell culture process. In one example, the cells are cultured in a seed medium and a production medium. In one example, the cells are cultured in a stirred-tank bioreactor. In one example, the volume of the bioreactor is from about 1L to about 2500L. Vectors

[0441] Antigen may be delivered in the form of viral or non-viral DNA vectors. The term "vector" as used herein, includes any transmitting moiety into which the antigen encoding sequence at least is inserted, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, virus-like particles, viral vectors such as such as adenoviruses, adeno-associated viruses (AAV), alphaviruses, flaviviruses, herpes simplex viruses (HSV), measles viruses, CMV, rhabdoviruses, retroviruses, lentiviruses, Newcastle disease virus (NDV), paramyxoviruses, rhabdoviruses, poxviruses such as vaccinia virus (VV) or modified vaccinia Ankara (MVA), and picornaviruses or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or PI artificial chromosomes (PAC). Vectors include expression as well as cloning vectors.

[0442] Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments.

[0443] In an embodiment, the vector is a viral vector or a non-viral vector. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses and lentiviruses, and attenuated forms thereof, each of which have their own advantages and disadvantages as known in the art. Viral vectors specifically include without limitation an adenoviral vector and a poxviral vector. Typically, for viral vectors, about 5 x 107to 5 x 1012viral particles are administered, typically about 5 x 109to 5 x 1010viral particles. Vectors may be replicating or non-replicating.

[0444] Compositions, routes of delivery and doses

[0445] A person skilled in the art will appreciate that the vaccine antigen as described herein, deoxyribonucleic acid encoding a MERS-CoV antigen as described herein, ribonucleic acid as described herein, or vector as described herein may be formulated into a pharmaceutical composition. In an embodiment, the pharmaceutical composition is a vaccine.

[0446] Such compositions may include one or more pharmaceutically acceptable carriers. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed immunogens. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable carriers such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional nontoxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions (such as immunogenic compositions) to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In particular embodiments, suitable for administration to a subject the carrier may be sterile, and / or suspended or otherwise contained in a unit dosage form containing one or more measured doses of the composition suitable to induce the desired immune response. It may also be accompanied by medications for its use for treatment purposes. The unit dosage form may be, for example, in a sealed vial that contains sterile contents or a syringe for injection into a subject, or lyophilized for subsequent solubilization and administration or in a solid or controlled release dosage.

[0447] In an embodiment, the lyophilized vaccine comprises one or more lyoprotectants.

[0448] In an embodiment, the lyoprotectant includes but is not necessarily limited to, glucose, trehalose, sucrose, maltose, lactose, mannitol, inositol, hydroxypropyl-P-cyclodextrin, and / or polyethylene glycol.

[0449] In an embodiment, the lyophilized vaccine comprises a poloxamer, potassium sorbate, a sugar such as sucrose, or any combination thereof.

[0450] In an embodiment, the lyophilized vaccine comprises about 0.1 to about 5.0 mg / mL of the vaccine antigen.

[0451] In an embodiment, the lyophilized vaccine comprises about 0.1 to about 10 % w / v of a sugar.

[0452] In an embodiment, the sugar is sucrose, raffinose, or trehalose.

[0453] In some embodiments, the lyophilized vaccine comprises about 0.01 to about 1.0% w / w of a poloxamer. In some embodiments, the lyophilized vaccine comprises about 1.0 to about 5.0% w / w of potassium sorbate.

[0454] In an embodiment, the composition comprises a vaccine antigen as described herein. In an embodiment, the composition comprises a vector as described herein. In an embodiment, the vector comprises a deoxyribonucleic acid as described herein. In an embodiment, the composition comprises a ribonucleic acid as described herein. In an embodiment the composition comprises a lipid nanoparticle as described herein. In an embodiment, the lipid nanoparticle encapsulates a ribonucleic acid as described herein.

[0455] In an embodiment, the composition may comprise one or more other epitopes for eliciting an immune response e.g. B-cell and / or T-cell epitopes. In an embodiment, the composition may comprise one or more other RNAs encoding epitopes for eliciting an immune response e.g. B-cell and / or T-cell epitopes.

[0456] In an embodiment, the composition is formulated to be compatible with its intended route of administration, e.g., local or systemic. Examples of routes of administration include intradermal, subcutaneously, intravenously, intra-arterially, intraperitoneal, intranasal, sublingual, tonsillar, orally, pulmonary, topical or other parenteral and mucosal routes.

[0457] In an embodiment, the composition is formulated to be stable at refrigerator temperature. In an embodiment, the composition is formulated so that it is suitable for transportation and / or storage at refrigerator temperature. In an embodiment, refrigerator temperature is about 3°C to about 17°C, or about 4°C to about 10°C, or about 4°C. In an embodiment, the composition is formulation to be stable at room temperature. In an embodiment, room temperature is about 18°C to about 24°C, or about 20°C to about 23°C, or about 23°C. In an embodiment, the composition is formulated so that is suitable for non-cold chain transportation and / or storage. In an embodiment, the composition is formulated so that is suitable for room temperature storage and / or transpiration. In an embodiment, the composition is formulated so that is suitable for transportation and / or storage at temperatures higher than room temperature e.g. about 25°C to 40°C (for countries where cold chain and low temperature storage and transportation trains are not available.

[0458] Oral, nasal and pulmonary administration include administration via inhalation and sprays delivered to the aforementioned sites. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0459] Compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, non-aqueous solutions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N. J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride can also be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, such as aluminum monostearate or gelatin.

[0460] Sterile injectable solutions can be prepared by incorporating the required amount in an appropriate solvent or buffer with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, suitable methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0461] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration excipients suitable for use in sprays, tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The sprays, tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds ofa similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavouring agent such as peppermint, methyl salicylate, or orange flavouring.

[0462] Formulations suitable for administration by nasal inhalation include where the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 1 to about 500 microns, which is administered in the manner via a spray, nebuliser, inhaler or snuffed. Suitable formulations wherein the carrier is a liquid for administration by nebulizer, include aqueous or oily solutions of the agent. For administration by inhalation, the agent(s) can also be delivered in the form of drops or an aerosol spray from a pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U. S. 6,468,798.

[0463] Formulations suitable for administration by oral inhalation include where the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns, which is administered by oral inhalation from a container holding the powder held close to the mouth or where the carrier is a liquid for administration by nebulizer, which can include aqueous or oily solutions of the agent.

[0464] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays, drops, or suppositories.

[0465] Intra dermal delivery of vaccines by needle or needle free approaches offers advantages in terms of ease of administration and intradermal administration approaches that effectively target immunocompetent cells are contemplated. Liquid formulations may be provided in prefilled or non-prefilled syringes or needs such disposable-syringe jet injectors, hollow microneedles mounted on syringes, and needles adapted for intra-dermal delivery. Prefilled syringes with a single ID needle are commercially available. Alternatively, solid or biodegradable microneedles coated or impregnated with vaccine such as patches or other mini-needle / spike devices, or composed of vaccine may be employed. These are inserted into the dermal layers of the skin where eitherthe vaccine coating is dissolved, orthe microneedle itself dissolves in place. The formulation may be provided as a liquid or semi liquid formulation, or as a solid or powdered formulation. Jet-injectors operate by generating a high pressured stream, which flushes a liquid vaccine formulation into the deeper skin layers. However, approaches that deliver vaccines in a solid form may also prove to be promising. One such method is the ballistic approach, in which solid vaccine particles or vaccine-coated gold particles are accelerated towards the skin by needle-free devices, so that the particles are deposited in the epidermal and dermal layers of the skin.

[0466] Intramuscular administration, can be via any intramuscular method known by a person skilled in the art, including for example, intramuscular injection.

[0467] The compositions can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0468] Compositions may include adjuvants. Immune responses to antigens can be enhanced if administered as a mixture with one or more adjuvants. Immune adjuvants typically function in one or more of the following ways: (1) immunomodulation (2) enhanced presentation (3) CTL production (4) targeting; and / or (5) depot generation.

[0469] Illustrative adjuvants that may or may not be included include: particulate or nonparticulate adjuvants, complete Freund's adjuvant (CFA), aluminum salt-based adjuvant, emulsion based adjuvant, TLR agonists, ISCOMS, LPS derivatives such as MPL and derivatives thereof such as 3D-MPL also GLA, and AGP, mycobacterial derived proteins such as muramyl di- or tri-peptides, particular saponins from Quillaja saponaria, such as QS21, QS7, and ISCOPREP™ saponin, ISCOMATRIX™ adjuvant, and peptides, such as thymosin alpha 1. In addition to the saponin component, the adjuvant may comprises a sterol such as beta-sitosterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. In some embodiments, the adjuvant is presented in the form of an oil-in-water emulsion, e.g. comprising squalene, alpha-tocopherol and a surfactant or in the form of a liposome. In some embodiments, the adjuvant is Addavax. AddaVax is a squalene based oil in water nano emulsion based on the formulation of MF-59 that has been found useful in flu vaccines. In some embodiments, the adjuvant is MF-59. The adjuvants AS03, MF59, and CpG 1018 have already been used in licensed vaccines. Other suitable adjuvants include lecithin and caromer homopolymers, Matrix M, ASO1, ALFQ. CpG mofits and co-stimulatory molecules including TLR agonists, B7, OX-40L, G-CSF are contemplated. Adjuvants are discussed in Liang et al., 2020.

[0470] In an embodiment, the composition comprises an adjuvant selected from one or more of an aluminium salt-based adjuvant, emulsion adjuvant, or a TLR agonist. Examples of such adjuvants are described for example in Liang et al., 2020.

[0471] Treatment can be by a single dose schedule or a multiple dose schedule. A subject may receive one dose of the composition or two or three doses of the composition at scheduled intervals. Multiple doses may be used in a primary immunisation schedule and / or in a booster immunisation schedule. In a multiple dose schedule the various doses may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. Administration of more than one dose (typically two doses) is particularly useful in immunologically naive patients e.g. for subjects who have never received a coronavirus vaccine before, or for coronavirus vaccines including a new vaccine antigen. Multiple doses will typically be administered at least 1 week apart (e.g. about 7 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 12 weeks, about 16 weeks, etc.).

[0472] Antibodies generated against the subject antigen may be used in therapy or for screening. Antibody include an immunoglobulin, antigen-binding fragment, or derivative thereof, that specifically binds and recognises the antigen or an antigenic fragment thereof, or a dimer or multimer of the antigen. The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multi (and bi) specific antibodies, and antibody fragments. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab').sub.2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (Reference may be made to Kontermann and Dubel (Ed), Antibody Engineering, Vol 1-2, Ed., Springer Press, 2010).

[0473] The term epitope refers to particular peptide sequences on a molecule that are antigenic, such that they elicit a specific immune response. An epitope is the region of an antigen to which B and / or T cells respond. An antibody can bind to a particular antigenic epitope which may be formed both from contiguous amino acids or noncontiguous amino acids.

[0474] Methods of producing a CoV vaccine

[0475] In an aspect, the present invention provides a method of producing a coronavirus (CoV) vaccine, the method comprising: (a) providing (i) a sugar; (ii) at least one buffering agent selected from one or more of: acetate, succinate, citrate, prolamine, arginine, glycine, histidine, borate, carbonate and phosphate and (iii) at least one CoV vaccine antigen; (b) combining (a) together to create a liquid formulation; (c) cooling the liquid formulation to below a freezing state in (b) to create a frozen formulation; and (d) lyophilizing the frozen formulation in (c) to create the CoV vaccine.

[0476] In an aspect, the present invention provides a method of producing a coronavirus (CoV) vaccine, the method comprising: (a) providing (i) a sugar; (ii) at least one buffering agent selected from one or more of: acetate, succinate, citrate, prolamine, arginine, glycine, histidine, borate, carbonate and phosphate and (iii) at least one CoV vaccine antigen comprising at least one non-endogenous inter-protomer disulfide bond and / or a C-terminal truncation; (b) combining (a) together to create a liquid formulation; (c) cooling the liquid formulation to below a freezing state in (b) to create a frozen formulation; and (d) lyophilizing the frozen formulation in (c) to create the CoV vaccine.

[0477] In an aspect, the present invention provides a method of producing a coronavirus (CoV) vaccine, the method comprising: (a) providing (i) a sugar; (ii) at least one buffering agent selected from one or more of: acetate, succinate, citrate, prolamine, arginine, glycine, histidine, borate, carbonate and phosphate and (iii) at least one CoV vaccine antigen comprising at least one non-endogenous inter-protomer disulfide bond; (b) combining (a) together to create a liquid formulation; (c) cooling the liquid formulation to below a freezing state in (b) to create a frozen formulation; and (d) lyophilizing the frozen formulation in (c) to create the CoV vaccine. In an aspect, the present invention provides a method of producing a coronavirus (CoV) vaccine, the method comprising: (a) providing (i) a sugar; (ii) at least one buffering agent selected from one or more of: acetate, succinate, citrate, prolamine, arginine, glycine, histidine, borate, carbonate and phosphate and (iii) at least one CoV vaccine antigen comprising a C-terminal truncation; (b) combining (a) together to create a liquid formulation; (c) cooling the liquid formulation to below a freezing state in (b) to create a frozen formulation; and (d) lyophilizing the frozen formulation in (c) to create the CoV vaccine.

[0478] In an aspect, the present invention provides a method of producing a Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine, the method comprising: (a) providing (i) a sugar; (ii) at least one buffering agent selected from one or more of: acetate, succinate, citrate, prolamine, arginine, glycine, histidine, borate, carbonate and phosphate and (iii) at least one MERS-CoV vaccine antigen; (b) combining (a) together to create a liquid formulation; (c) cooling the liquid formulation to below a freezing state in (b) to create a frozen formulation; and (d) lyophilizing the frozen formulation in (c) to create the CoV vaccine.

[0479] In an embodiment, the at least one CoV vaccine antigen is the CoV vaccine antigen as described herein.

[0480] In an embodiment, the at least one MERS-CoV vaccine antigen is the MERS-CoV vaccine antigen as described herein.

[0481] In an embodiment, the sugar is sucrose, raffinose, or trehalose. In an embodiment, the sugar is sucrose. In an embodiment, the sugar is raffinose. In an embodiment, the sugar is trehalose.

[0482] In an embodiment, the sucrose is provided in a solution at a concentration of about 0.1 to about 10 % w / v, based on the total volume of the sucrose solution. In an embodiment, the sucrose is provided in a solution at a concentration of about 3 to about 10 % w / v, based on the total volume of the sucrose solution. In an embodiment, the sucrose is provided in a solution at a concentration of about 0.1 to about 6 % w / v, based on the total volume of the sucrose solution. In an embodiment, the sucrose is provided in a solution at a concentration of about 4 to about 6 % w / v, based on the total volume of the sucrose solution.

[0483] In an embodiment, the sucrose is provided in a solution at a concentration of about 0.1 % w / v, based on the total volume of the sucrose solution. In an embodiment, the sucrose is provided in a solution at a concentration of about 1 % w / v, based on the total volume of the sucrose solution. In an embodiment, the sucrose is provided in a solution at a concentration of about 2 % w / v, based on the total volume of the sucrose solution. In an embodiment, the sucrose is provided in a solution at a concentration of about 3 % w / v, based on the total volume of the sucrose solution. In an embodiment, the sucrose is provided in a solution at a concentration of about 4 % w / v, based on the total volume of the sucrose solution. In an embodiment, the sucrose is provided in a solution at a concentration of about 5 % w / v, based on the total volume of the sucrose solution. In an embodiment, the sucrose is provided in a solution at a concentration of about 6 % w / v, based on the total volume of the sucrose solution. In an embodiment, the sucrose is provided in a solution at a concentration of about 7 % w / v, based on the total volume of the sucrose solution. In an embodiment, the sucrose is provided in a solution at a concentration of about 8 % w / v, based on the total volume of the sucrose solution. In an embodiment, the sucrose is provided in a solution at a concentration of about 9 % w / v, based on the total volume of the sucrose solution. In an embodiment, the sucrose is provided in a solution at a concentration of about 10 % w / v, based on the total volume of the sucrose solution.

[0484] In an embodiment, the sucrose is provided in phosphate buffered solution (PBS).

[0485] In an embodiment, the CoV vaccine antigen is provided in a solution at a concentration of about 0.1 mg / mL to about 5 mg / mL, based on the total volume of the CoV vaccine antigen solution. In an embodiment, the CoV vaccine antigen is provided in a solution at a concentration of about 0.1 mg / mL to about 1 mg / mL, based on the total volume of the CoV vaccine antigen solution. In an embodiment, the CoV vaccine antigen is provided in a solution at a concentration of about 0.1 mg / mL to about 2 mg / mL, based on the total volume of the CoV vaccine antigen solution. In an embodiment, the CoV vaccine antigen is provided in a solution at a concentration of about 1 mg / mL to about 2 mg / mL, based on the total volume of the CoV vaccine antigen solution. In an embodiment, the CoV vaccine antigen is provided in a solution at a concentration of about 2 mg / mL to about 5 mg / mL, based on the total volume of the CoV vaccine antigen solution. In an embodiment, the CoV vaccine antigen is provided in a solution at a concentration of about 3 mg / mL to about 5 mg / mL, based on the total volume of the CoV vaccine antigen solution.

[0486] In an embodiment, the MERS-CoV vaccine antigen is provided in a solution at a concentration of about 0.1 mg / mL to about 5 mg / mL, based on the total volume of the MERS-CoV vaccine antigen solution. In an embodiment, the MERS-CoV vaccine antigen is provided in a solution at a concentration of about 0.1 mg / mL to about 1 mg / mL, based on the total volume of the MERS-CoV vaccine antigen solution. In an embodiment, the MERS-CoV vaccine antigen is provided in a solution at a concentration of about 0.1 mg / mL to about 2 mg / mL, based on the total volume of the MERS-CoV vaccine antigen solution. In an embodiment, the MERS-CoV vaccine antigen is provided in a solution at a concentration of about 1 mg / mL to about 2 mg / mL, based on the total volume of the MERS-CoV vaccine antigen solution. In an embodiment, the MERS-CoV vaccine antigen is provided in a solution at a concentration of about 2 mg / mL to about 5 mg / mL, based on the total volume of the MERS-CoV vaccine antigen solution. In an embodiment, the MERS-CoV vaccine antigen is provided in a solution at a concentration of about 3 mg / mL to about 5 mg / mL, based on the total volume of the MERS-CoV vaccine antigen solution.

[0487] In an embodiment, the CoV vaccine antigen is provided in a solution at a concentration of about 0.1 mg / mL, based on the total volume of the CoV vaccine antigen solution. In an embodiment, the CoV vaccine antigen is provided in a solution at a concentration of about 0.5 mg / mL, based on the total volume of the CoV vaccine antigen solution. In an embodiment, the CoV vaccine antigen is provided in a solution at a concentration of about 1 mg / mL, based on the total volume of the CoV vaccine antigen solution. In an embodiment, the CoV vaccine antigen is provided in a solution at a concentration of about 1.5 mg / mL, based on the total volume of the CoV vaccine antigen solution. In an embodiment, the CoV vaccine antigen is provided in a solution at a concentration of about 2 mg / mL, based on the total volume of the CoV vaccine antigen solution. In an embodiment, the CoV vaccine antigen is provided in a solution at a concentration of about 3 mg / mL, based on the total volume of the CoV vaccine antigen solution. In an embodiment, the CoV vaccine antigen is provided in a solution at a concentration of about 4 mg / mL, based on the total volume of the CoV vaccine antigen solution. In an embodiment, the CoV vaccine antigen is provided in a solution at a concentration of about 5 mg / mL, based on the total volume of the CoV vaccine antigen solution.

[0488] In an embodiment, the MERS-CoV vaccine antigen is provided in a solution at a concentration of about 0.1 mg / mL, based on the total volume of the MERS-CoV vaccine antigen solution. In an embodiment, the MERS-CoV vaccine antigen is provided in a solution at a concentration of about 0.5 mg / mL, based on the total volume of the MERS-CoV vaccine antigen solution. In an embodiment, the MERS-CoV vaccine antigen is provided in a solution at a concentration of about 1 mg / mL, based on the total volume of the MERS-CoV vaccine antigen solution. In an embodiment, the MERS-CoV vaccine antigen is provided in a solution at a concentration of about 1.5 mg / mL, based on the total volume of the MERS-CoV vaccine antigen solution. In an embodiment, the MERS-CoV vaccine antigen is provided in a solution at a concentration of about 2 mg / mL, based on the total volume of the MERS-CoV vaccine antigen solution. In an embodiment, the MERS-CoV vaccine antigen is provided in a solution at a concentration of about 3 mg / mL, based on the total volume of the MERS-CoV vaccine antigen solution. In an embodiment, the MERS-CoV vaccine antigen is provided in a solution at a concentration of about 4 mg / mL, based on the total volume of the MERS-CoV vaccine antigen solution. In an embodiment, the MERS-CoV vaccine antigen is provided in a solution at a concentration of about 5 mg / mL, based on the total volume of the MERS-CoV vaccine antigen solution.

[0489] In an embodiment, step (c) is conducted at a temperature of about -80°C.

[0490] In an embodiment, step (d) comprises drying the frozen formulation under vacuum. In an embodiment, step (d) is conducted for a period of about 1 hour to about 10 hours. In an embodiment, step (d) is conducted for a period of about 2 hour to about 10 hours. In an embodiment, step (d) is conducted for a period of about 3 hour to about 8 hours. In an embodiment, step (d) is conducted for a period of about 4 hour to about 6 hours. In an embodiment, step (d) is conducted for a period of about 5 hour to about 10 hours.

[0491] In an embodiment, step (d) is conducted for a period of about 1 hour. In an embodiment, step (d) is conducted for a period of about 2 hours. In an embodiment, step (d) is conducted for a period of about 3 hours. In an embodiment, step (d) is conducted fora period of about 4 hours. In an embodiment, step (d) is conducted for a period of about 5 hours. In an embodiment, step (d) is conducted for a period of about 6 hours. In an embodiment, step (d) is conducted for a period of about 7 hours. In an embodiment, step (d) is conducted for a period of about 8 hours. In an embodiment, step (d) is conducted for a period of about 9 hours. In an embodiment, step (d) is conducted for a period of about 10 hours.

[0492] In an embodiment, step (d) is conducted at a temperature of about -20°C to -30°C. In an embodiment, step (d) is conducted at a temperature of about -25°C to -30°C. In an embodiment, step (d) is conducted at a temperature of about -20°C to -25°C. In an embodiment, step (d) is conducted at a temperature of about -22°C to -27°C.

[0493] In an embodiment, step (d) is conducted at a temperature of about -20°C. In an embodiment, step (d) is conducted at a temperature of about -22°C. In an embodiment, step (d) is conducted at a temperature of about -25°C. In an embodiment, step (d) is conducted at a temperature of about -27°C. In an embodiment, step (d) is conducted at a temperature of about -30°C.

[0494] In an embodiment, the vaccine remains efficacious for at least 2 weeks, at least 1 month, at least 2 months, at least 6 months or at least 12 months, at room temperature or at a temperature of about 20°C to about 40°C. In an embodiment, the vaccine remains efficacious for at least 1 month at room temperature or at a temperature of about 20°C to about 40°C. In an embodiment, the vaccine remains efficacious for at least 1 month at room temperature or at a temperature of about 25°C to about 35°C. In an embodiment, the vaccine remains efficacious for at least 1 month at room temperature or at a temperature of about 30°C to about 35°C. In an embodiment, the vaccine remains efficacious for at least 2 months at room temperature or at a temperature of about 20°C to about 25°C. In an embodiment, the vaccine remains efficacious for at least 2.5 months at room temperature or at a temperature of about 20°C to about 25°C. In an embodiment, the vaccine remains efficacious for at least 3 months at room temperature or at a temperature of about 20°C to about 25°C. In an embodiment, the vaccine remains efficacious for at least 6 months at room temperature or at a temperature of about 22°C to about 27°C. In an embodiment, the vaccine remains efficacious for at least 12 months at room temperature or at a temperature of about 20°C to about 25°C.

[0495] As used herein, the term “efficacious” refers to the ability of the vaccine to be useful in a method as described herein, for example, to induce an immune response, enhance an immune response, prevent or reduce the likelihood of a CoV infection, prevent or reduce the likelihood or severity of a symptom of a CoV infection, reduce the severity and / or duration of a CoV infection, preventing or reducing viral shedding in a subject infected with a CoV infection, prevent or reduce the likelihood ofa MERS-CoV infection, prevent or reduce the likelihood or severity ofa symptom of a MERS-CoV infection, reduce the severity and / or duration of a MERS-CoV infection, or preventing or reducing viral shedding in a subject infected with a MERS-CoV infection.

[0496] In an embodiment, the vaccine remains stable for at least 2 weeks, at least 1 month, at least 2 months, at least 6 months or at least 12 months, at room temperature or at a temperature of about 20°C to about 40°C. In an embodiment, the vaccine remains stable for at least 1 month at room temperature or at a temperature of about 20°C to about 40°C. In an embodiment, the vaccine remains stable for at least 1 month at room temperature or at a temperature of about 25°C to about 35°C In an embodiment, the vaccine remains stable for at least 1 month at room temperature or at a temperature of about 30°C to about 35°C. In an embodiment, the vaccine remains stable for at least 2 months at room temperature or at a temperature of about 20°C to about 25°C. In an embodiment, the vaccine remains stable for at least 6 months at room temperature or at a temperature of about 20°C to about 25°C. In an embodiment, the vaccine remains stable for at least 12 months at room temperature or at a temperature of about 20°C to about 25°C.

[0497] As used herein, “stable” or “stability” with reference to a vaccine provided herein refers to one in which the vaccine antigens and components thereof essentially retains their physical and chemical stability and integrity. One measure of stability is whether the vaccine antigen remains predominantly in trimeric form. One measure indicating that the integrity of the vaccines is intact is whether the vaccines maintain biological activity such as the efficacy of the vaccine antigens. Another measure may be the substantial lack of aggregation, denaturation or fragmentation of the vaccine antigens over the period of time being measured.

[0498] In an embodiment, the vaccine remains predominantly in trimeric form for at least 2 weeks, at least 1 month, at least 2 months, at least 6 months or at least 12 months, at room temperature or at a temperature of about 20°C to about 40°C. In an embodiment, the vaccine remains predominantly in trimeric form for at least 1 month at room temperature or at a temperature of about 20°C to about 40°C. In an embodiment, the vaccine remains predominantly in trimeric form for at least 1 month at room temperature or at a temperature of about 25°C to about 35°C. In an embodiment, the vaccine remains predominantly in trimeric form for at least 1 month at room temperature or at a temperature of about 30°C to about 35°C. In an embodiment, the vaccine remains predominantly in trimeric form for at least 2 months at room temperature or at a temperature of about 20°C to about 25°C. In an embodiment, the vaccine remains predominantly in trimeric form for at least 2.5 months at room temperature or at a temperature of about 20°C to about 25°C. In an embodiment, the vaccine remains predominantly in trimeric form for at least 3 months at room temperature or at a temperature of about 20°C to about 25°C. In an embodiment, the vaccine remains predominantly in trimeric form for at least 6 months at room temperature or at a temperature of about 22°C to about 27°C. In an embodiment, the vaccine remains predominantly in trimeric form for at least 12 months at room temperature or at a temperature of about 20°C to about 25°C.

[0499] As used herein, the term "room temperature" or “(RT)” refers to a temperature typically comprised between about 20°C and about 25°C. For example, room temperature may refer to a temperature of about 20°C, about 22.5°C, about 25°C. Those of skill in the art appreciate that room temperature varies by location and prevailing conditions. For example, room temperatures can be higher in warmer climates.

[0500] In an aspect, the present invention provides a CoV vaccine produced by the method as described herein.

[0501] In an aspect, the present invention provides a MERS-CoV vaccine produced by the method as described herein.

[0502] Methods of prevention and / or treatment

[0503] In an aspect, the present invention provides a method of preventing and / or treating a coronavirus infection in a subject.

[0504] As used herein, the term "prevention" or “prophylaxis” refers to reducing the likelihood of contracting or developing infection or a symptom thereof. Prevention need not be complete and does not imply that a subject will not eventually contract or develop the infection or a symptom thereof.

[0505] As used herein, the terms "treating" or "treatment" refers to at least partially obtaining a desired therapeutic outcome. In an embodiment, treatment comprises preventing or delaying the appearance of one or more symptoms of a Middle East respiratory syndrome coronavirus (MERS-CoV) infection. In an embodiment, treatment comprises arresting or reducing the development of one or more symptoms of a MERS-CoV infection.

[0506] Reference to "subject" or "subjects" includes a subject susceptible to a coronavirus infection, or at risk of exposure to a coronavirus. The subject may be infected or uninfected, and may be symptomless or in need of treatment. In an embodiment, the subject is susceptible, or at risk of exposure to a MERS-CoV infection. For example, the subject can be a mammal, avian, arthropod, chordate, camelid, amphibian or reptile. Exemplary subjects include but are not limited to humans, primates, livestock (e.g. sheep, cow, chicken, horse, donkey, pig, mink), companion animals (e.g. dogs, cats), laboratory test animals (e.g. mice, rabbits, rats, guinea pigs, hamsters), captive wild animals (e.g. fox, deer), zoo animals (e.g. lion, tiger, bear), and reservoir animals (e.g. bat, camel, pangolin).

[0507] In an embodiment, the subject is a non-human animal.

[0508] In an embodiment, the non-human animal is selected from one or more the group consisting of: a non-human primate, a mammal, a livestock animal, a domestic animal, a laboratory test animal, a captive wild animal, a zoo animal, and a reservoir animal. In an embodiment, the non-human animal is a primate. In an embodiment, the non-human animal is a mammal. In an embodiment, the non-human animal is a livestock animal. In an embodiment, the non-human animal is a domestic animal. In an embodiment, the non-human animal is a laboratory test animal. In an embodiment, the non-human animal is a captive wild animal. In an embodiment, the non-human animal is a zoo animal. In an embodiment, the non-human animal is a reservoir animal.

[0509] In an embodiment, the subject is a camelid. In an embodiment, the camelid is selected from one or more of: a dromedary or Arabian camel (Camelus dromedarius), a Bactrian camel (Camelus bactrianus), a wild Bactrian camel (Camelus ferus), a llama (Lama glama), an alpaca (Lama pacos), a vicuna (Lama vicugna), and a guanaco (Lama guanicoe).

[0510] In an embodiment, the subject is a pig. In an embodiment, the pig is a domestic pig (Sus scrofa domesticus).

[0511] In an embodiment, the subject is a sheep. In an embodiment, the sheep is a domestic sheep (Ovis aries).

[0512] In an embodiment, the subject is an ungulate. In an embodiment, the ungulate is a pig, a cow, a sheep, a deer, a goat or a camel.

[0513] In an embodiment, the subject is from the family Mustelidae. In an embodiment, the subject is a mink, a weasel, a badger or an otter. In an embodiment, the subject is a mink.

[0514] MERS-CoV is a zoonotic virus, which means that is transmitted between animals and people. Studies have shown that humans are infected through direct or indirect contact with infected dromedary camels, although the exact route of transmission remains unclear. MERS-CoV has been identified in dromedary camels in the Middle East, Africa and South Asia. Despite a limited number of human infections reported outside the Middle East, recent studies in human populations with occupational exposure to dromedary camels in a number of Member States indicate that there is also zoonotic transmission occurring in the African continent.

[0515] As used herein, the term “permissible host” refers to a subject or host in which MERS-CoV or a variant thereof is able to infect, and potentially pass on the MERS-CoV or variant thereof to a subsequent animal or human. MERS-CoV utilizes DPP4 as its host receptor to infect cells, and the ability of MERS-CoV to infect a permissible host depends on its ability to bind the hostspecific DPP4.

[0516] In an embodiment, the subject is a permissible host. In an embodiment, the subject expresses a dipeptidyl peptidase IV (DPP4) capable of being recognized by the MERS-CoV.

[0517] In an embodiment, the subject is a mammal. In an embodiment, the subject is human. In an embodiment, the subject is a camel. In an embodiment, the human is a fetus, infant, child, early adult and adult. In an embodiment, the adult is an elderly adult. In an embodiment, the adult is one or more of greater than 60 years in age, greater than 65 years in age, greater than 70 years in age, greater than 75 years in age, greater than 80 years in age, greater than 85 years in age, greater than 90 years in age. In an embodiment, the subject has had a prior coronavirus infection. In an embodiment, the subject has had a prior MERS-CoV infection. In an embodiment, the subject has received a primary coronavirus treatment regimen as described herein. In an embodiment, the subject has received a primary and a secondary coronavirus treatment regimen. In an embodiment, the subject has received a primary coronavirus treatment regimen, a secondary coronavirus treatment regimen and a tertiary coronavirus treatment regimen. In an embodiment, the subject is immunocompromised. In an embodiment, the subject has a respiratory condition.

[0518] In an aspect, the present invention provides a method of inducing an immune response to a Middle East respiratory syndrome coronavirus (MERS-CoV) in a subject, the method comprising delivering the vaccine as described herein to a subject.

[0519] In an aspect, the present invention provides a method of enhancing the immune response to a Middle East respiratory syndrome coronavirus (MERS-CoV) in a subject, the method comprising delivering the vaccine antigen as described herein, or vaccine as described herein to a subject.

[0520] In an aspect, the present invention provides a method of preventing or reducing the likelihood of a Middle East respiratory syndrome coronavirus (MERS-CoV) infection in a subject, the method comprising delivering the vaccine antigen as described herein, or vaccine as described herein to a subject.

[0521] In an aspect, the present invention provides a method of preventing, or reducing the likelihood or severity of a symptom of a Middle East respiratory syndrome coronavirus (MERS-CoV) infection in a subject, the method comprising delivering the vaccine antigen as described herein, or vaccine as described herein to a subject.

[0522] In an aspect, the present invention provides a method of reducing the severity and / or duration of a Middle East respiratory syndrome coronavirus (MERS-CoV) infection in a subject, the method comprising delivering the vaccine antigen as described herein, or vaccine as described herein to a subject.

[0523] In an aspect, the present invention provides a method of preventing or reducing viral shedding in a human individual infected with a Middle East respiratory syndrome coronavirus (MERS-CoV), the method comprising delivering the vaccine antigen as described herein, or vaccine as described herein to a subject.

[0524] In an embodiment, the vaccine is delivered intramuscularly, intradermal, subcutaneously, intravenously, intra-arterially, intraperitoneal, intranasal, sublingual, tonsillar, orally, pulmonary, topical or other parenteral and mucosal routes.

[0525] In an aspect, the present invention provides vaccine antigen as described herein or the vaccine as described herein for use in one or more of: i) inducing an immune response to a MERS-CoV in a subject; ii) enhancing the immune response to a MERS-CoV in a subject; iii) preventing or reducing the likelihood of a MERS-CoV infection in a subject; iv) preventing or reducing the likelihood of severity of a MERS-CoV symptom in a subject; v) reducing the severity and / or duration of a MERS-CoV infection in a subject; vi) preventing or reducing viral shedding in a subject; and vii) treating a MERS-CoV infection in a subject.

[0526] In an aspect, the present invention provides kit, device, surface or strip comprising the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen as described herein.

[0527] In an aspect, the present invention provides use of the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen as described herein in the manufacture of a medicament for one or more of: i) inducing an immune response to a CoV in a subject; ii) enhancing the immune response to a CoV in a subject; iii) preventing or reducing the likelihood of a CoV infection in a subject; iv) preventing or reducing the likelihood of severity of a CoV symptom in a subject; v) reducing the severity and / or duration of a CoV infection in a subject; vi) preventing or reducing viral shedding in a subject; and vii) treating a CoV infection in a subject.

[0528] In an aspect, the present invention provides a method of inducing an immune response to a Middle East respiratory syndrome coronavirus (MERS-CoV) in a subject, the method comprising delivering the ribonucleic acid as described herein or RNA vaccine as described herein to a subject.

[0529] In an aspect, the present invention provides a method of enhancing the immune response to a Middle East respiratory syndrome coronavirus (MERS-CoV) in a subject, the method comprising delivering the ribonucleic acid as described herein or RNA vaccine as described herein to a subject.

[0530] In an aspect, the present invention provides a method of preventing or reducing the likelihood of a Middle East respiratory syndrome coronavirus (MERS-CoV) infection in a subject, the method comprising delivering the ribonucleic acid as described herein or RNA vaccine as described herein to a subject.

[0531] In an aspect, the present invention provides a method of preventing, or reducing the likelihood or severity of a symptom of a Middle East respiratory syndrome coronavirus (MERS-CoV) infection in a subject, the method comprising delivering the ribonucleic acid as described herein or RNA vaccine as described herein to a subject.

[0532] In an aspect, the present invention provides a method of reducing the severity and / or duration of a Middle East respiratory syndrome coronavirus (MERS-CoV) infection in a subject, the method comprising delivering the vaccine ribonucleic acid as described herein or RNA vaccine as described herein to a subject.

[0533] In an aspect, the present invention provides a method of preventing or reducing viral shedding in a human individual infected with a Middle East respiratory syndrome coronavirus (MERS-CoV), the method comprising delivering the ribonucleic acid as described herein or RNA vaccine as described herein to a subject.

[0534] In an embodiment, the ribonucleic acid or RNA vaccine is delivered intramuscularly, intradermal, subcutaneously, intravenously, intra-arterially, intraperitoneal, intranasal, sublingual, tonsillar, orally, pulmonary, topical or other parenteral and mucosal routes.

[0535] In an aspect, the present invention provides a ribonucleic acid as described herein or the vaccine as described herein for use in one or more of: i) inducing an immune response to a MERS-CoV in a subject; ii) enhancing the immune response to a MERS-CoV in a subject; iii) preventing or reducing the likelihood of a MERS-CoV infection in a subject; iv) preventing or reducing the likelihood of severity of a MERS-CoV symptom in a subject; v) reducing the severity and / or duration of a MERS-CoV infection in a subject; vi) preventing or reducing viral shedding in a subject; and vii) treating a MERS-CoV infection in a subject.

[0536] In an aspect, the present invention provides a kit, device, surface or strip comprising the Middle East respiratory syndrome coronavirus (MERS-CoV) ribonucleic acid as described herein.

[0537] In an aspect, the present invention provides use of the Middle East respiratory syndrome coronavirus (MERS-CoV) ribonucleic acid as described herein in the manufacture of a medicament for one or more of: i) inducing an immune response to a MERS-CoV in a subject; ii) enhancing the immune response to a MERS-CoV in a subject; iii) preventing or reducing the likelihood of a MERS-CoV infection in a subject; iv) preventing or reducing the likelihood of severity of a MERS-CoV symptom in a subject; v) reducing the severity and / or duration of a MERS-CoV infection in a subject; vi) preventing or reducing viral shedding in a subject; and vii) treating a MERS-CoV infection in a subject.

[0538] As used herein, the phrase “reducing the severity of an infection”, or similar phrases, includes reducing one or more of the following in an individual: titre of a virus, duration of the virus infection, the harshness or duration of one or more symptoms of a coronavirus infection in a subject. As used herein, the phrase “duration of a coronavirus infection” refers to the time in which an individual has a MERS-CoV infection or a symptom caused by a MERS-CoV infection.

[0539] In an embodiment, the present invention provides a vaccine that is a primary vaccine regimen. As used herein a “primary vaccine regimen” is the first vaccine regimen administered to a subject to produce a response to a specific pathogen. In the context of MERS-CoV, a primary vaccine is the first vaccine regimen administered to a subject to produce an immune response to the ancestral strain and / or a variant thereof.

[0540] In an embodiment, the present invention provides a booster vaccine for a primary coronavirus vaccine regimen. In an embodiment, the present invention provides a booster vaccine for instances where a subject has received more than one prior coronavirus vaccine regimen. In an embodiment, the booster acts by enhancing the immune response elicited by the primary vaccine regimen. In an embodiment, the booster acts by enhancing the immune response to a variant such as a VOC or VOI or VHCs to which a lesser, little or no protective immune response is generated by the primary vaccine regimen. In an embodiment, the booster is administered at leasts months, or at least 12 months, or at least 18 months, or at least 2 years, or at least 3 years or at least 5 years, or at least 6 years, or at least 7 years after the primary vaccine regimen. In an embodiment, the booster is administer sequentially or in combination with one or more other booster vaccines.

[0541] Combination treatments

[0542] A Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen or vaccine or ribonucleic acid as described herein may be administered to a subject in combination with one or more further ribonucleic acids, vaccine antigens or vaccines. The further ribonucleic acids, vaccine antigens or vaccines may produce an immune response against an infectious pathogenic agent that may be the same or different to MERS-CoV. In an embodiment, the pathogen is selected from influenza, Respiratory syncytial virus or a specific CoV variant such as a VOC, VOI or VHC thereof. In some embodiments, the CoV is selected from one or more of: a bat sarbecovirus (such as PRD-0038 and WIV1), a OC43, a SARS-CoV, a SARS-CoV2 and variants thereof. In an embodiment, the MERS-CoV vaccine antigen is administered in combination with one or more: a SARS-CoV2 vaccine antigen, a WIV1 vaccine antigen and / or a PRD-0038 vaccine antigen, or a nucleic acid encoding the vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen is administered in combination with a SARS-CoV2 vaccine antigen, or a nucleic acid encoding the SARS-CoV2 vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen is administered in combination with a WIV1 vaccine antigen, or a nucleic acid encoding the WIV1 vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen is administered in combination with a PRD-0038 vaccine antigen, or a nucleic acid encoding the PRD-0038 vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen is administered in combination with a SARS-CoV2 vaccine antigen and a WIV1 vaccine antigen, or a nucleic acid encoding the SARS-CoV2 vaccine antigen and a nucleic acid encoding the WIV1 vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen is administered in combination with a PRD-0038 vaccine antigen, or a nucleic acid encoding the PRD-0038 vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen is administered in combination with a SARS-CoV2 vaccine antigen and a PRD-0038 vaccine antigen, or a nucleic acid encoding the SARS-CoV2 vaccine antigen and a nucleic acid encoding the PRD-0038 vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen is administered in combination with a WIV1 vaccine antigen and a PRD-0038 vaccine antigen, or a nucleic acid encoding the WIV1 vaccine antigen and a nucleic acid encoding the PRD-0038 vaccine antigen. In an embodiment, the MERS-CoV vaccine antigen is administered in combination with a SARS-CoV2 vaccine antigen, a WIV1 vaccine antigen and a PRD-0038 vaccine antigen, or a nucleic acid encoding the SARS-CoV2 vaccine antigen, a nucleic acid encoding the WIV1 vaccine antigen and a nucleic acid encoding the PRD-0038 vaccine antigen. Administration may be in combination (at the same time) or sequential in either order. Exemplary SARS-CoV-2 vaccine antigens are described previously in PCT / AU2022 / 050429.

[0543] Kit, device, surface or strip

[0544] The subject Middle East respiratory syndrome coronavirus (MERS-CoV) antigen, deoxyribonucleic acid or ribonucleic acid as described herein is captured on solid or semi-solid surfaces for assay purposes, including epidemiological, diagnostic, purification, drug-screening, vaccine screening applications etc. Many such applications and methods of immobilising antigen to surfaces are known in the art and encompassed. EXAMPLES

[0545] Example 1 - Materials and methods

[0546] Recombinant spike proteins. M2P-1275 and C-terminal truncation mutants thereof. A synthetic gene encoding the MERS CoV England 1 (strain as defined in GenBank accession number KC164505.2) spike ectodomain (spike protein as defined in Genbank accession number AFY13307) was obtained from Genscript. The gene encodes S amino acids 1 to 1275, an l-to V substitution at position 2, the potential furin site mutation, R748SVR-> ASVG, and the ‘2P’ mutation (V1060P / L1061P; Pallesen et al., 2017). The C-terminus of M2P-1275 was appended with a Gly-Ser-Gly-Ser linker, a hexa-His affinity tag, a Gly-Ser-Gly-Ser linker and an avitag (GLNDIFEAQKIEWHE) sequence (SEQ ID NO: 47). The synthetic M2P-1275 gene was ligated into the polycloning site of pcDNA3 (Invitrogen). DNA fragments encoding C-terminal truncation mutations were prepared by polymerase chain reaction using Phusion DNA polymerase (ThermoFisher), M2P-1275 DNA as template, the forward primer, 5’GCTCTGTCTGCTCAGCTGGCC (SEQ ID NO: 41), and reverse primers designed to encode a Gly-Ser-His6 sequence and stop codon after the desired C-terminal spike residue:

[0547] (SEQ ID NO: 42) K1240

[0548] 5’-CGCCGCTCTAGATTAGTGATGATGATGGTGATGAGAGCCAGATCCCTTGAAGAACTCGTCCAGCTC (SEQ ID NO: 43) N1248

[0549] 5’-CGCCGCTCTAGATTAGTGATGATGATGGTGATGAGAGCCAGATCCGTTGGGAATGCTGGTGGACAC (SEQ ID NO: 44) T1258

[0550] 5’-CGCCGCTCTAGATTAGTGATGATGATGGTGATGAGAGCCAGATCCTGTGGTGTTGATCTGTGTCAGG (SEQ ID NO: 45) E1265

[0551] 5’-CGCCGCTCTAGATTAGTGATGATGATGGTGATGAGAGCCAGATCCCTCGTAGGTCAGGTCCAGCAG (SEQ ID NO: 46) Q1270

[0552] 5’-CGCCGCTCTAGATTAGTGATGATGATGGTGATGAGAGCCAGATCCCTGCAGGGACAGCATCTCGTAG The PCR products encoding the C-terminal truncations were used to replace the DNA sequence encompassed by BspEI and Xba\ restriction sites within the pcDNA3-M2P-1275 expression vector. All sequences were verified by fluorescent Sanger sequencing (BigDye Terminator v3.1, ABI).

[0553] Cysteine substitution mutants. Cysteine substitution mutations were introduced into the M2P-1258 expression vector using synthetic genes (Genscript) encoding the paired Cys substitution mutations listed in Table 1.

[0554] MERS CoV spike trimer interfacial amino acids that mediate inter-monomer contacts as determined using the PDB PISA (Proteins, Interfaces, Structures and Assemblies) server and the protein coordinates in PDB ID 5X5F. Only polar contacting amino acids that are not involved in electrostatic interactions nor glycosylation sequons are shown. The available and buried surface area of contacting amino acids in the chain B-C interface as well as the Ca-Ca and Cp-Cp distances between contacting amino acids in the chains B-C and chains A-B interfaces are shown. The contacting amino acid pairs were replaced with Cys residues in the context of M2P-1258. The mutant code is to the left of the mutational targets.

[0555] Expression and purification of recombinant spike proteins. Spike expression vectors were transfected into Expi293F cells using Expifectamine, as recommended by the manufacturer (ThermoFisher Scientific). The cells were cultured for 7 days at 34°C after which the transfection supernatants were clarified by centrifugation and filtration through 0.45 pm nitrocellulose filters. The MERS CoV glycoproteins were then purified by divalent cation affinity chromatography using TALON resin (Merck) followed by size exclusion chromatography (SEC) using a Superose 6 Increase 10 / 300 column linked to an AKTApure instrument (Cytiva). All proteins were concentrated using Amicon centrifugal filter units. The protein solutions were filter-sterilized using 0.45 pm nitrocellulose filters and protein aliquots stored at -80°C. The buffer was phosphate buffered saline pH 7.4. Protein purity was assessed by SDS-PAGE and SEC. SDS-PAGE was performed using NuPAGE Bis-Tris and Tris-Acetate precast gels and an XCell SureLock Mini-Cell Electrophoresis System (Thermo Fisher) as recommended by the manufacturer. Precision plus prestained protein standards (Bio-Rad) were the molecular weight markers.

[0556] Recombinant spike ligands. 1) DPP4-Fc is a recombinant fusion protein comprising amino acids 19-615 of the human dipeptidyl peptidase 4 (DPP4) isoform 1 (NCBI Reference Sequence: NP_001926.2) linked to the Fc domain of human lgG1 via a GS linker and is based on the ACE2-Fc construct described previously (Poumbourios et al., 2023). 2) Recombinant monoclonal neutralizing antibodies (mNAbs). pCDNA3-based human lgG1 heavy and kappa and lambda light chain expression vectors (Center et al., 2020) containing the variable regions of MERS CoV directed mNAbs CDC2-C2 (Wang et al., 2018), MERS-4 (Zhang et al., 2018), KNIH90-F1 (Jang et al., 2022), 7D10 (Zhou et al., 2019), G2 (Wang et al., 2019), CC95.108 and CC99.103 (Zhou et al., 2023), and S2P6 (Pinto et al., 2021) were produced in-house as described in (Poumbourios et al., 2023).

[0557] Differential scanning fluorimetry (DSF). Differential scanning fluorimetry was used to assess protein thermostability (Niesen et al., 2007). Protein (10 pg) was diluted into 25pL with 5x concentration SYPRO Orange Protein Gel Stain (Sigma Aldrich) in duplicate. The samples were then heated in an Quantstudio 7 Real-time qPCR System in 0.5°C increments from 25°C to 95°C for 1 minute per increment. Measurements of fluorescence were taken at the end of each increment. Excitation was at 492nm, and emission at 610nm. The Tm was determined to be the minimum of the negative first derivative of the melting curve. Table 1. Shows MERS CoV spike trimer interfacial amino acids that mediate inter-monomer contacts

[0558] Chain B^C Chain B“C Chain A*S Chain A-B Contacting Available Buried Calpha-Calphs Cbeta^Cbeta 5x6f Contacting C&lpha~Calpha Cbcta-Cheta Mutant

[0559] Chain B amine acid Bar face surface distance di s tance Chain A amine acid distance distance

[0560] Coda,

[0561] aiDln© acid in 3x5f squared ft squared k (chain B:3^. Ddcwn { chain B: ES&dcranJ ajElnja acid in 3x5f (chain A: RB&df>wn> (chain AiSBBdo’wnJ (chain Ci RBDup) (chain c^aBDnp) (chain B: RBibdnwn> (chain a: aBDdownJ

[0562] Al D580 chain £t£60 101, 81 69.93 4.6A 3. 7 A £>680 chain B:g6G 7, -iA 7, 8 A

[0563] B7 3362 chain C:^803 1O2.0S 76.38 '. ■)!■ 3362 chain B-T803 7. -^-. _ 1

[0564] C44 SW chain C^SQB 71.36 S9.66 S564 chain B ggQB 6.? A <; >,

[0565] 017 #637 chain C: K1042 n / d n / d 7 _ 7 A / : 2A K637 chain B: K1042 7 / 7A $, £ A

[0566]

[0567] S3 3363 Cham C ft930 24.18 24.03 6,8A,:5 / < 3363 chain B::ft93Q ^. SA 4. '■ A

[0568] F14 S429 chain C. D1S253 46. -32 39.46 4.1A x - i- i=423 chain h: D1059 7 -SA:. cA

[0569] G44 D5G9 chain C:, B435 86.62 35.22 i-. OA?:, 1 A £>609 chain B: S435. LS, OA i 2 • k

[0570] Hl #763 <&ain C:8930 113. QX 91,98 9, -iA 6, H763 chain

[0571] 11 #636 chain C. K1S242 n / d n / d v S #636 chain h: M1042 6.1 A 4 4 ><

[0572] J23 3701 chain CXQ897 40.07 36.9 5-.1A, 2 k 3781 chain 0: $857; 8.1A

[0573] K35 81114 chain c: S1104 77. 97 63,05 i;, 8A 31114 chain

[0574] £21 £1188 chain Cr£W 69 / ?2 32. t <, i- SI 189 chain B:2988 ".3A. Jiri

[0575] M2 D1182 chain C: 5966 6,61 S.64 iA3A '■ i i' D1182 chain a: 5966 A

[0576]

[0577] Biolayer interferometry. BLI-based measurements were determined using an OctetRED96 System (ForteBio, Fremont CA). Antibodies were diluted in kinetic bufferto 10 pg / ml and immobilized onto anti-human IgG Fc capture biosensors (AHC, ForteBio). Binding assays were carried out at 30 °C using standard kinetics acquisition rate settings (5.0 Hz, averaging by 20) at a sample plate shake speed of 1,000 rpm. The binding experiments included five steps: (a) baseline (180 s); (b) antibody loading (300 s); (c) second baseline (180 s); (d) association of antigen (300 s), and (e) dissociation of antigen (300 s). Fitting curves were constructed using ForteBio Data Analysis 10.0 software using a 1: 1 binding model, and double reference subtraction was used for correction.

[0578] S6P. BA286-1192 glycoprotein expression vectors. The SARS CoV-2 ancestral Hu-1 numbering system (Genbank accession number YP_009724390) is used when referring to sarbecovirus spikes. Synthetic genes encoding amino acids 16-1192 of the SARS CoV-2 omicron BA.2.86 isolate were obtained from Genscript. The synthetic genes encode the hexaPro or ‘6P’ mutation F817P, A892P, A899P, A942P, V986P, K987P (Hsieh et al., (2020), the furin cleavage site mutation: R681RRAR-> P681GSAS, and in some cases the VI mutation: A1016V / A1020I (Poumbourios et al., 2023). The C-terminus of the S6P-1192 spikes was appended with a Gly-Ser linker and a hexa-His affinity tag. The synthetic genes were ligated downstream of a DNA sequence encoding the tissue plasminogen activator leader via Nhe\, within pcDNA3 (Invitrogen). Cysteine substitution mutations were introduced into the S6P-1192 expression vectors using synthetic gene fragments (Genscript) encoding paired Cys substitution mutations corresponding to D17 (D571C / S967C) and 11 (A570C / S967C). The glycoproteins were expressed in Expi293T cells and purified as described for the M2P-1258 constructs.

[0579] S6P. BA286-1192 glycoprotein stability experiments. Purified, trimeric parental, D17 and 11+VI -mutated S6P. BA286-1192 glycoproteins in PBS were adjusted to 1 mg / mL and supplemented with 0.02% w / v sodium azide. The samples were incubated at 37°C for up to 28 days in a humidified tissues culture incubator in the presence of 5% CO2. At 7-day intervals, the samples were assessed by Superose 6 SEC, DSF in the presence of SYPRO orange, reactivity with ACE2-Fc (Poumbourios et al., 2023) and NAbs S2H97, directed to the RBD flanks (Starr et al., 2021) and S2P6, directed to the stem (Pinto et al., 2021) in BLI, and SDS-PAGE under reducing and non-reducing conditions.

[0580] Sarbecovirus soluble spike expression vectors. Synthetic DNA encoding parental and / or D17-mutated S6P-1192 sequences derived from omicron BA.2.68, PRD-0038 and WIV1 (Genbank accession numbers: OR775659, QTJ30153, AGZ48828, respectively) were obtained from Genscript. The ‘D17’ mutation corresponds to the SARS CoV-2 ancestral spike mutations D571C / S967C. The Sarbecovirus spikes contain the hexa-Pro mutation, the corresponding to the SARS CoV-2 ancestral spike mutations F817P, A892P, A899P, A942P, V986P, K987P designed to stabilize the pre-fusion conformation (Hsieh et al., 2020). The ancestral spike Genbank accession number is YP_009724390. All Spike DNA sequences were codon-optimised for expression in human cells and verified by Sanger sequencing with ABI BigDye Terminator 3.

[0581] RBD expression vectors. Synthetic genes encoding the receptor binding domain (RBD) corresponding to the amino acids EAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQIS PAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPL KYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVAS GSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEY of MERS CoV Englandl, amino acids VTNLCPFHEVFNATRFASVYAWNRTRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTN VYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRSFR KSKLKPFERDISTEIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAP ATVCGPKKSTN of SARS CoV-2 Omicron JN.1, amino acids ITNLCPFGEVFNATTFPSVYAWERKRISNCVADYSVYNSTSFSTFKCYGVSATKLNDLCFSNVY ADSFWKGDDVRQIAPGQTGVIADYNYKLPDDFTGCVLAWNTRNIDATQTGNYNYKYRSLRH GKLRPFERDISNVPFSPDGKPCTPPAFNCYWPLNDYGFYITNGIGYQPYRVWLSFELLNAPAT VCGPKLSTD of bat sarbecovirus WIV1 and amino acids ITNLCPFGQVFNASKFPSVYAWERLRISDCVADYSVLYNSSSSFSTFKCYGVSPTKLNDLCFSS VYADYFVVKGDDVRQIAPAQTGVIADYNYKLPDDFTGCVLAWNTNSVDSKQGNNFYYRLFRH GKIKPYERDISNVLYNSAGGTCSSTSQLGCYEPLKSYGFTPTVGVGYQPYRVVVLSFELLNAP ATVCGPKKSTE of bat sarbecovirus PRD-0038 were obtained from GenSCRIPT and ligated to the tissue plasminogen activator leader via Nhel in pcDNA3. The genes encode a C-terminal hexa-His tag and Avitag sequence.

[0582] Stem synthetic peptide and MBP-stem (1138-1208) chimeric protein. The MBP-stem(1138-1208) chimera comprises E. coli MBP linked to the ancestral stem amino acids 1138-1208 via a tri-alanine linker (Kobe et al., 1999) and was expressed in E. coli and purified as described (Center et al., 2008). A synthetic peptide corresponding to the stem (amino acids 1142-1165, QPELDSFKEELDKYFKNHTSPDVD), was synthesized by Genscript. The peptide contains an N-terminal biotin moiety and C-terminal amide.

[0583] Expression and purification of recombinant spike proteins. Spike expression vectors were transfected into Expi293F cells using Expifectamine, as recommended by the manufacturer (ThermoFisher Scientific). The cells were cultured for 7 days at 34°C after which the transfection supernatants were clarified by centrifugation and filtration through 0.45 pm nitrocellulose filters. The SARS CoV-2 glycoproteins were then purified by divalent cation affinity chromatography using TALON resin (Merck) followed by size exclusion chromatography using a Superose 6 Increase 10 / 300 column linked to an AKTApure instrument (Cytiva). All proteins were concentrated using Amicon centrifugal filter units. The protein solutions were filter-sterilized using 0.45 pm nitrocellulose filters and protein aliquots stored at -80°C. The buffer was phosphate buffered saline pH 7.4. Protein purity was assessed by SDS-PAGE and SEC. SDS-PAGE was performed using NuPAGE Bis-Tris and Tris-Acetate precast gels and an XCell SureLock MiniCell Electrophoresis System (Thermo Fisher) as recommended by the manufacturer. Precision plus prestained protein standards (Bio-Rad) were the molecular weight markers.

[0584] S-HIV pseudovirus neutralizing assay. S-HIV luciferase reporter viruses were produced by cotransfecting 293T cells with pNL4.3LucR-E- and pcDNA3 containing DNA inserts encoding the S open reading frames from MERS CoV Englandl (GenBank accession number AFY13307), NeoCoV (AGY29650.2), SARS CoV Tor2 (AAP41037.1), WIV1 (AGZ48828), PRD-0038 (QTJ30153) and BtKY72 (APO40579); the BtKY72 spike contains the K482Y / T487W and is called BtKY72. YW. The viruses were harvested 72 h post transfection and were stored at -80°C until needed. Heat inactivated sera (56°C for 30 minutes) were serially diluted in DMF10 and each dilution mixed with an equal volume of S-pseudotyped HIV luciferase reporter viruses and incubated for 1h at 37°C. Virus-serum mixtures were added to monolayers of huh7.5 cells (for MERS CoV), or HAT24 cells (for NeoCoV, SARS CoV Tor2 and WIV1) or HAT 24 cells transfected with a Rhinolophus alcyone expression vector (for PRD-0038 and BtKY72. YW) attached to 96 well plates the day prior at 10,000 cells / well. After 3 days, tissue culture fluid was removed, monolayers were washed once with PBS and lysed with cell culture lysis reagent (Promega) and luciferase measured using luciferase substrate (Promega) in a CLARIOstar plate reader (BMG LabTechnologies). The mean percentage entry was calculated as (RLU plasma+virus) / (RLU medium+virus)*100. The percentage entry was plotted against the reciprocal dilution of plasma in Prism v9.3.0 and curves fitted using the One-site specific binding with Hill slope model. The reciprocal dilution of plasma required to prevent 90% or 50% virus entry was calculated from the non-linear regression line (ID90, ID50 respectively).

[0585] Authentic virus neutralization assay. The neutralizing activity of sera against authentic SARS-CoV-2 JN.1 and XEC was determined with the rapid high-content SARS-CoV-2 microneutralization assay described by Aggarwal et al., 2022. Briefly, Hoechst-33342-stained HAT-24 cells were seeded in 384-well plates (Corning, CLS3985). Serially diluted heat-inactivated (56°C, 30 min) vaccinal sera were co-incubated with an equal volume of SARS-CoV-2 virus solution at twice the median lethal dose for 1 h at 37 °C. 40 pl of serum-virus mixtures were added to an equal volume of pre-plated cells, incubated for 20 h and then directly imaged on an ImageXpress Pico Automated Cell Imaging System (Molecular Devices). Cellular nuclei counts were obtained with CellReporterXpress Image Acquisition and Analysis software (Molecular Devices), and the percentage of virus neutralization was calculated as described in Aggarwal et al. 2022. The neutralization ID50 was the last consecutive dilution reaching >50% neutralization.

[0586] Chemiluminescence Immunoassay (ELISA). Nunc Maxisorp 384-well white plates were coated with 1 pg / ml of S glycoproteins, NTD proteins, RBD proteins, stem proteins or synthetic peptides overnight at 4°C, washed with PBS and blocked with BSA (10 mg / ml, PBS) at room temperature for 1 h. The plates were again washed and incubated with serially diluted serum samples or mNAbs for 2 h at room temperature. Antibody binding was detected using horseradish peroxidase-labelled rabbit anti-guinea pig antibody (Dako, Glostrup, Denmark). Light signals were detected using SuperSignal ELISA Pico Chemiluminescent Substrate (ThermoFisher Scientific) and measured immediately for 0.5 seconds using CLARIOstar (BMG Lab Technologies). Relative light units (RLUs) were plotted against the reciprocal dilution in GraphPad Prism 10.1.0 and curves fitted using Specific binding with Hill slope. The binding titre was defined as the reciprocal dilution of serum giving RLUs fifty-times that of background, as defined by binding to BSA.

[0587] M2P-1258 glycoprotein stability experiments. Purified, trimeric parental or F14-mutated M2P-1258 glycoproteins in PBS were adjusted to 1 mg / mL and supplemented with 0.02% w / v sodium azide. The samples were incubated at 37°C for up to 28 days in a humidified tissue culture incubator in the presence of 5% CO2. At various intervals, the samples were assessed ...

Claims

CLAIMS1. A Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen comprising a MERS-CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond.

2. The MERS-CoV vaccine antigen of claim 1, wherein the non-endogenous inter-protomer disulfide bond is formed between cysteines selected from:i) cysteines at a position corresponding to amino acid numbers 429 and 1059 of SEQ ID NO: 1 (F14); and / orii) cysteines at a position corresponding to amino acid numbers 580 and 60 of SEQ ID NO: 1 (A1).

3. The MERS-CoV vaccine antigen of claim 1 or claim 2, wherein the non-endogenous interprotomer disulfide bond is formed between cysteines at a position corresponding to amino acid numbers 429 and 1059 of SEQ ID NO: 1 (F14).

4. The MERS-CoV vaccine antigen of claim 1 or claim 2, wherein the non-endogenous interprotomer disulfide bond is formed between cysteines at a position corresponding to amino acid numbers 580 and 60 of SEQ ID NO: 1 (A1).

5. The MERS-CoV vaccine antigen of any one of claims 1 to 4, wherein the S protein trimer melting temperature is increased by at least about 5°C compared to a S protein trimer lacking the non-endogenous inter-protomer disulfide bond.

6. The MERS-CoV vaccine antigen of any one of claims 1 to 5, wherein the S protein trimer melting temperature is increased by about 5°C to about 20°C, or by about 5°C to about 15°C, or by about 5°C to about 12.5°C, or by about 5°C to about 10°C, or by about 5°C to about 7°C, compared to a S protein trimer lacking the non-endogenous inter-protomer disulfide bond.

7. The MERS-CoV vaccine antigen of any one of claims 1 to 6, wherein two or more protomers of the S protein trimer are bonded by a non-endogenous inter-protomer disulfide bond.

8. The MERS-CoV vaccine antigen of any one of claims 1 to 7, comprising one or more of the following features:i) the S protein trimer comprises an endogenous or a non-endogenous membrane spanning sequence;ii) the S protein trimer comprises an endogenous or a non-endogenous membrane spanning sequence, wherein the endogenous membrane spanning sequence comprises a sequence selected from: SEQ ID NO: 52, SEQ ID NO: 53 SEQ ID NO: 54 and SEQ ID NO: 55;iii) the protomer of the S protein trimer comprises a sequence selected from: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 15 and SEQ ID NO: 20; andiv) the S protein trimer is stabilised in the prefusion conformation.

9. A Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen comprising a MERS-CoV S protein trimerwith a C-terminal truncation in the stem region between residues corresponding to 1220 to 1275 of SEQ ID NO: 1.

10. The MERS-CoV vaccine antigen of any one of claims 1 to 8, wherein the S protein trimer has a C-terminal truncation in the stem region between residues corresponding to 1220 to 1275 of SEQ ID NO: 1.I I. The MERS-CoV vaccine antigen of claim 9 or 10, wherein the C-terminal truncation is between residues corresponding to 1220 to 1265 of SEQ ID NO: 1.

12. The MERS-CoV vaccine antigen of any one of claims 9 to 11, wherein the C-terminal truncation is after a residue corresponding to 1220, 1240, 1248, 1258, 1265 or 1270 of SEQ ID NO: 1 and before the residue corresponding to 1276 of SEQ ID NO: 1.

13. The MERS-CoV vaccine antigen of any one of claims 9, 10 and 12, wherein the C-terminal truncation is after a residue corresponding to 1220, 1240, 1248, 1258, 1265, 1270 of SEQ ID NO: 1.

14. The MERS-CoV vaccine antigen of any one of claims 9 to 13, wherein the protomer of the S protein trimer comprises or consists of residues corresponding to 18 to 1221, or residues 18 to 1240, or residues 18 to 1248, or residues 18 to 1258, or residues 18 to 1265, or residues 18 to 1270, or residues 18 to 1275 of SEQ ID NO: 1.

15. The MERS-CoV vaccine antigen of claim 14, wherein the protomer of the S protein trimer comprises or consists of residues corresponding to 18 to 1248 of SEQ ID NO: 1.

16. The MERS-CoV vaccine antigen of claim 14, wherein the protomer of the S protein trimer comprises or consists of residues corresponding to 18 to 1258 of SEQ ID NO: 1.

17. The MERS-CoV vaccine antigen of any one of claims 1 to 16, wherein when administered to a subject the S protein trimer elicits a neutralising antibody response.

18. The MERS-CoV vaccine antigen claim 17, wherein the neutralising antibody response is a neutralising antibody response directed to an epitope or epitopes that include(s) part or all of the receptor binding domain (RBD).

19. The MERS-CoV vaccine antigen claim 17, wherein the neutralising antibody response is a non-RBD neutralising antibody response.

20. The MERS-CoV vaccine antigen of claim 19, wherein the non-RBD neutralising antibody response comprises a neutralising antibody response directed to an epitope or epitopes that include(s) part or all of the stem region.

21. The MERS-CoV vaccine antigen of claim 19 or claim 20, wherein the non-RBD neutralising antibody response comprises a neutralising antibody response directed to an epitope or epitopes that include(s) part or all of the N-terminal domain region (NTD).

22. The MERS-CoV vaccine antigen of any one of claims 19 to 21, wherein the neutralising antibody response comprises a neutralising antibody response directed to an epitope or epitopes that include(s) part or all of the S2 domain.

23. The MERS-CoV vaccine antigen of claim 18, wherein the neutralising antibody response comprise eliciting neutralising antibodies to an epitope or epitopes that include(s) part or all of the receptor binding motif (RBM).

24. The MERS-CoV vaccine antigen of any one of claims 1 to 18, wherein the S protein trimer does not elicit neutralising antibody responses to the receptor binding motif (RBM).

25. The MERS-CoV vaccine antigen of any one of claims 1 to 24, wherein the S protein trimer is able to be bound by an antibody that binds an epitope or epitopes that include(s) part or all the N-terminal domain (NTD).

26. The MERS-CoV vaccine antigen of any one of claims 1 to 25, wherein the S protein trimer is able to be bound by any antibody that binds an epitope or epitopes that include(s) part or all of the receptor binding domain (RBD).

27. The MERS-CoV vaccine antigen of any one of claims 1 to 26, wherein the S protein trimer is able to be bound by any antibody that binds an epitope or epitopes that include(s) part or all the RBM.

28. The MERS-CoV vaccine antigen of any one of claims 1 to 27, wherein the S protein trimer is able to be bound by any antibody that binds an epitope or epitopes that include(s) part or all the stem region.

29. The MERS-CoV vaccine antigen of any one of claims 1 to 28, wherein the S protein trimer is able to be bound by an antibody that binds an epitope or epitopes that include(s) part or all the S2 domain.

30. The MERS-CoV vaccine antigen of any one of claims 1 to 29, wherein the S protein trimer is able to be bound by an antibody that binds an epitope or epitopes that include(s) part or all the fusion peptide (FP) domain.

31. The MERS-CoV vaccine antigen of any one of claims 1 to 30, wherein the S protein trimer is able to be bound by any antibody that binds an RBM-independent epitope or epitopes that include(s) part or all of the “up”-oriented RBD.

32. The MERS-CoV vaccine antigen of any one of claims 1 to 31, wherein the melting temperature of the S protein trimer is about 35°C to about 60°C, or about 40°C to about 60°C, or about 42°C to about 60°C, or about 45°C to about 60°C, or about 50°C to about 60°C, or about 40°C to about 55°C, or about 40°C to about 50°C.

33. The MERS-CoV vaccine antigen of claim 32, wherein the melting temperature is about 40°C to about 60°C.

34. The MERS-CoV vaccine antigen of anyone of claims 1 to 33, comprising one or more of the following features:i) the S protein trimer is soluble;ii) the vaccine antigen additionally comprises the 2P modification;iii) the vaccine antigen is a pan-coronavirus vaccine antigen; andiv) the S protein trimer lacks a signal sequence.

35. The MERS-CoV vaccine antigen of any one of claims 9 to 34, wherein the S protein trimer comprises or consists of residues corresponding to 18 to 1258 of SEQ ID NO: 1, a non-endogenous inter-protomer disulfide bond is formed between residues corresponding to S429C and D1059C of SEQ ID NO: 1 (F14) or D580 and Q60C of SEQ ID NO: 1 (A1), and wherein the S protein trimer comprises a melting temperature of about 40°C to about 55°C.

36. The MERS-CoV vaccine antigen of any one of claims 9 to 35, wherein the S protein trimer comprises or consists of a sequence selected from: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

37. The MERS-CoV vaccine antigen of any one of claims 9 to 36, wherein the S protein trimer comprises or consists of a sequence selected from SEQ ID NO: 15 and SEQ ID NO: 20.

38. The MERS-CoV vaccine antigen of any one of claims 9 to 37, wherein the C-terminal truncation is between residues corresponding to 1240 to 1275 of SEQ ID NO: 1.

39. The MERS-CoV vaccine antigen of any one of claims 9 to 38, wherein the C-terminal truncation is after a residue selected from a residue corresponding to 1240, 1248, 1258, 1265, 1270 of SEQ ID NO: 1.

40. A protein nanoparticle comprising the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen of any one of claims 1 to 39.

41. A virus-like particle comprising the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen of any one of claims 1 to 39.

42. A deoxyribonucleic acid or a ribonucleic acid encoding the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen of any one of claims 1 to 39.

43. A ribonucleic acid encoding a S protein monomer of a Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen wherein the vaccine antigen comprises:i) a MERS-CoV S protein trimer with at least one non-endogenous inter-protomer disulfide bond; and / orii) a C-terminal truncation in the stem region.

44. A vector comprising the deoxyribonucleic acid or ribonucleic acid of claim 42 or 43.

45. A host cell comprising the deoxyribonucleic acid or ribonucleic acid of claim 42 or 43, or the vector of claim 44.

46. A method of producing the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen of any one of claims 1 to 39, the ribonucleic acid or the deoxyribonucleic acid of claim 42 or 43, comprising culturing the host cell of claim 45 in culture medium to produce the vaccine antigen, the ribonucleic acid or the deoxyribonucleic acid.

47. A vaccine comprising the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen of any one of claims 1 to 39, or the protein nanoparticle of claim 40, or the viruslike particle of claim 41.

48. The vaccine of claim 47, wherein the vaccine further comprises at least one further CoV vaccine antigen which is an S protein monomer of a further CoV vaccine antigen.

49. The vaccine of claim 48, wherein the further CoV vaccine antigen is selected from one or more of: a sarbecovirus, a bat sarbecovirus (such as PRD-0038 and WIV1), a OC43, a SARS-CoV and a SARS-CoV2 vaccine antigen.

50. The vaccine of any one of claims 47 to 49, wherein the vaccine is a tetravalent vaccine.

51. A vaccine comprising a merbecovirus vaccine antigen, a clade 1b vaccine antigen, a clade 1a sarbecovirus vaccine antigen, and a clade 3 sarbecovirus vaccine antigen.

52. The vaccine of claim 51, wherein the merbecovirus vaccine antigen, clade 1b vaccine antigen, clade 1a sarbecovirus vaccine antigen and the clade 3 sarbecovirus vaccine antigen comprise at least one non-endogenous inter-protomer disulfide bond and a C-terminal truncation in the stem region.

53. The vaccine of claim 51 or 52, wherein the merbecovirus vaccine antigen is a MERS-CoV vaccine antigen and / or the clade 1b vaccine antigen is a SARS-CoV2 vaccine antigen.

54. The vaccine of claim 53, wherein the SARS-CoV2 vaccine antigen is an omicron variant and / or a BA.2.86 omicron variant.

55. The vaccine of any one of claims 51 to 54, wherein the bat sarbecovirus vaccine antigen is WIV1 and / or PRD-0038.

56. The vaccine of any one of claims 47 to 55, wherein the vaccine elicits a neutralising antibody response to SARS-CoV Tor2.

57. The vaccine of any one of claims 47 to 56, wherein the vaccine elicits a neutralising antibody response to a SARS-CoV2 omicron variant.

58. The vaccine of claim 57, wherein the SARS-CoV2 is a variant of concern (VOC), wherein the VOC is JN.1 and / or XEC.

59. The vaccine of any one of claims 47 to 58, wherein the vaccine is a human vaccine or a non-human animal (veterinary) vaccine.

60. The vaccine of claim 59, wherein the non-human animal is selected from one or more of: a non-human primate, a mammal, a livestock animal, a domestic animal, a laboratory test animal, a captive wild animal, a zoo animal, and a reservoir animal.

61. The vaccine of claim 60, wherein the non-human animal is a mammal, and one or more of the following apply:i) the mammal is a camelid, a pig, a sheep or a mink;ii) the mammal is a camelid selected from one or more of: a dromedary or Arabian camel (Camelus dromedaries), a Bactrian camel (Camelus bactrian us), a wild Bactrian camel (Camelus ferus), a llama (Lama glama), an alpaca (Lama pacos), a vicuna (Lama vicugna), and a guanaco (Lama guanicoe);iii) the mammal is a domestic pig (Sus scrofa domesticus); andiv) the mammal is a domestic sheep (Ovis aries).

62. A method of one or more of:i) inducing an immune response to a Middle East respiratory syndrome coronavirus (MERS-CoV) in a subject;ii) enhancing the immune response to Middle East respiratory syndrome coronavirus (MERS-CoV) in a subject;iii) preventing or reducing the likelihood of a Middle East respiratory syndrome coronavirus (MERS-CoV) infection in a subject;iv) preventing or reducing the likelihood or severity of a symptom of a Middle East respiratory syndrome coronavirus (MERS-CoV) infection in a subject;v) reducing the severity and / or duration of a Middle East respiratory syndrome coronavirus (MERS-CoV) infection in a subject, andvi) preventing or reducing viral shedding in a subject infected with a Middle East respiratory syndrome coronavirus (MERS-CoV),the method comprising delivering the vaccine antigen of any one of claims 1 to 39, the protein nanoparticle of claim 40, the virus-like particle of claim 41 or the deoxyribonucleic acid of claim 42, or the ribonucleic acid of claim 43, or the vaccine of any one of claims 47 to 61 to a subject.

63. The method of claim 62, wherein delivery is intramuscular, intradermal, subcutaneous, intravenous, intra-arterial, intraperitoneal, intranasal, sublingual, tonsillar, oral, pulmonary, topical or another parenteral mucosal route.

64. The MERS-CoV vaccine antigen of any one of claims 1 to 39, or the deoxyribonucleic acid of claim 42, or the ribonucleic acid of claim 43, or the vaccine of any one of claims 47 to 61 for use in one or more of:i) inducing an immune response to a MERS-CoV in a subject;ii) enhancing the immune response to a MERS-CoV in a subject;iii) preventing or reducing the likelihood of a MERS-CoV infection in a subject;iv) preventing or reducing the likelihood of severity of a MERS-CoV symptom in a subject; v) reducing the severity and / or duration of a MERS-CoV infection in a subject;vi) preventing or reducing viral shedding in a subject; andvii) treating a MERS-CoV infection in a subject.

65. A kit, device, surface or strip comprising the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen of any one of claims 1 to 39, or the ribonucleic acid of claim 43.

66. Use of the Middle East respiratory syndrome coronavirus (MERS-CoV) vaccine antigen of any one of claims 1 to 39, the protein nanoparticle of claim 40, or the virus-like particle of claim 41, orthe deoxyribonucleic acid of claim 42, orthe ribonucleic acid of claim 43, in the manufacture of a medicament for one or more of:i) inducing an immune response to a MERS-CoV in a subject;ii) enhancing the immune response to a MERS-CoV in a subject;iii) preventing or reducing the likelihood of a MERS-CoV infection in a subject;iv) preventing or reducing the likelihood of severity of a MERS-CoV symptom in a subject; v) reducing the severity and / or duration of a MERS-CoV infection in a subject;vi) preventing or reducing viral shedding in a subject; andvii) treating a MERS-CoV infection in a subject.

67. A method of one or more of:i) increasing a MERS-CoV S protein trimer yield;ii) stabilizing a MERS-CoV S protein trimer in a prefusion conformation;iii) increasing the melting temperature of a MERS-CoV S protein trimer;iv) increasing the melting temperature of a MERS-CoV S protein trimer stabilised in the prefusion conformation;v) enhancing neutralising antibody responses of a MERS-CoV S protein trimer; the method comprising modifying the MERS-CoV S protein trimer to comprise at least one interprotomer disulfide bond and / or modifying the MERS-CoV S protein trimer to comprise a stem region C-terminal truncation.

68. The vaccine of any one of claims 47 to 61, wherein the vaccine is lyophilized.

69. The vaccine of claim 68, wherein one or more of the following apply:i) the lyophilized vaccine comprises one or more lyoprotectants;ii) the lyophilized vaccine comprises a poloxamer, potassium sorbate, sucrose, or any combination thereof;iii) the lyophilized vaccine comprises about 0.1 to about 5.0 mg / mL of the vaccine antigen; iv) the lyophilized vaccine comprises about 0.1 to about 10 % w / v of a sugar; and v) the lyophilized vaccine comprises about 0.1 to about 10 % w / v of a sugar, wherein the sugar is sucrose.

70. A method of producing a CoV vaccine, the method comprising:(a) providing (i) a sugar; (ii) at least one buffering agent selected from one or more of: acetate, succinate, citrate, prolamine, arginine, glycine, histidine, borate, carbonate and phosphate and (iii) at least one CoV vaccine antigen;(b) combining (a) together to create a liquid formulation;(c) cooling the liquid formulation to below a freezing state in (b) to create a frozen formulation; and(d) lyophilizing the frozen formulation in (c) to create the CoV vaccine.

71. The method of claim 70, wherein the vaccine antigen comprises a non-endogenous interprotomer disulfide bond and / or a C-terminal truncation.

72. The method of claim 70 or 71, wherein the at least one CoV vaccine antigen is the MERS-CoV vaccine antigen according to any one of claims 1 to 39.

73. The method of any one of claims 70 to 72, wherein one or more of the following apply:i) the sugar is sucrose;ii) the sugar is sucrose, wherein the sucrose is provided in a solution at a concentration of about 0.1 to about 10 % w / v, based on the total volume of the sucrose solution; iii) the CoV vaccine antigen is provided in a solution at a concentration of about 0.1 mg / mLto about 5 mg / mL, based on the total volume of the CoV vaccine antigen solution; iv) step (c) is conducted at a temperature of about -80°C;v) step (d) comprises drying the frozen formulation under vacuum;vi) step (d) is conducted for a period of about 1 hour to about 10 hours;vii) step (d) is conducted at a temperature of about -20°C to -30°C;viii)the vaccine remains efficacious for at least 2 weeks, at least 1 month, at least 2 months, at least 6 months or at least 12 months, at room temperature or at a temperature of about 22°C to about 40°C;ix) the vaccine remains stable for at least 2 weeks, at least 1 month, at least 2 months, at least 6 months or at least 12 months, at room temperature or at a temperature of about 22°C to about 40°C; andx) the vaccine remains stable for at least 1 month, at a temperature of about 30°C.

74. A CoV vaccine produced by the method according to any one of claims 70 to 73.

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