Vaccine

Abstract

The present invention relates to immunogenic protein sequences, and polynucleotides, compositions, and vaccine compositions thereof, which have been optimised for the prophylactic or therapeutic treatment of an infection caused by Betacoronaviruses, and have the ability to confer a broad immunity against multiple Betacoronavirus subgenera, species, and viral variants.

Description

[0001] VACCINE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to vaccine compositions optimised for the prophylactic or therapeutic treatment of an infection caused by Betacoronaviruses, such as human coronavirus OC43, human coronavirus HKU1 , SARS-CoV-1 , SARS-CoV-2, and MERS.

[0004] BACKGROUND

[0005] Coronaviridae is a family of enveloped, positive-strand RNA viruses that can infect a wide variety of animals. Orthocoronavirinae are a subfamily of Coronaviridae consisting of four genera, one of which are Betacoronaviruses. Species of Betacoronavirus subgenera include severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Zmasek et al. 2022, Virology 570:123-133). The coronavirus virion comprises the structural proteins spike (S), envelope (E), membrane (M), and nucleocapsid (N). The N proteins encapsulate the positive sense, singlestranded RNA, which are enveloped by the E and M proteins. The S proteins exist on the outside of the viral envelope and facilitate infection by binding to cell receptors to enter the cell, the distribution of such receptors affecting viral tropism and pathogenicity (V’kovski et al. 2021 , Nat Rev Microbiol 19, 155-170).

[0006] SARS-CoV-1 , MERS-CoV, and SARS-CoV-2 have all resulted in either deadly endemics or pandemics of their caused diseases (SARS, MERS, and COVID-19 respectively). Most recently, the outbreak of SARS-CoV-2 that caused COVID-19 and its rapid worldwide transmission resulted in its declaration as a pandemic by the World Health Organisation (WHO) in March 2020. Whilst the majority of COVID-19 cases result only in mild symptoms including fever, cough, or shortness of breath, a significant number of cases progress to viral pneumonia and multiorgan failure (Hui et al. 2020, Int J Infect Dis 91 : 264-66). The rapid rise in the number of infections and deaths around the globe highlights the urgent need for better therapeutic and prophylactic interventions to combat the disease and an effective vaccine has been hailed by many as a crucial cornerstone in our potential fight against the SARS-CoV-2 virus. Vaccination has been established as an effective form of epidemiological control, and vaccines have had significant success in aiding the decline of infections and mortalities associated with viral infections such as smallpox and polio. Other infections, however, have proven harder to vaccinate against. Much of the global efforts to develop Coronaviridae vaccines to date have focused primarily on stimulating an antibody response against the S-protein that helps facilitate viral entry, since it is the most exposed structural protein on the virus.

[0007] Vaccines for SARS-CoV-1 and MERS-CoV were also in development before the respective outbreaks of the viruses came to an end, removing the need for vaccines (Tseng et al., 2012, PLoS ONE, 7(4): e35421 ; Modjarrad et al., 2019, The Lancet Infectious Diseases, 19(9), 1013-1022). However, it is clear from the widespread outbreaks of MERS-CoV, SARS-CoV-1 and SARS-CoV-2 that viruses of the Betacoronavirus genus present a critical threat to humanity which requires both prophylactic and therapeutic treatment.

[0008] During the containment effort of any disease outbreak, the emergence of viral variants posed a great threat to keeping the disease under control. As evidenced by the COVID-19 pandemic, variants of SARS-CoV-2 emerged quickly and rapidly propagated throughout the population, many with increased severity, transmissibility, and immune evasion properties. One of the first viral variants identified was the D614G substitution in the S-protein. Viral variants arise from the constant generation of diverse viral genomes, mainly by mutation rate (dependent on the fidelity of the virus’ polymerase enzyme) but also through recombination with genomes of genetically distinct viruses. The first divergent SARS-CoV-2 lineages appeared 8 months after identification of the virus in humans, with the Alpha and Gamma variants of concern (VOC) containing 14 and 11 additional non-synonymous mutations compared to their ancestral lineages respectively. During 2021 and 2022, SARS-CoV-2 evolution comprised an increase in divergence within major lineages with additional increase within these major lineages, accelerating the overall evolutionary rate (Markov et al. 2023 Nat Rev Microbiol 21 , 361-379).

[0009] To this day, new SARS-CoV-2 variants are continuing to emerge in the human population. JN.1 , a descendent of the BA.2.86 Omicron variant, constituted over 60% of US infections in January 2024, having first been identified in August 2023 (CDC, 2024). Mutation of certain traits that affect the intrinsic transmissibility of the virus include evading the immune system, survival outside of the host (resistance to pH, temperature, general stability), and favourable interactions with the human host cell receptor for SARS-CoV-2 (ACE2). These impact certain epidemiological parameters including infection incidence, infection prevalence, epidemic growth rate, and overall burden of disease. How quickly a virus can be controlled in a population is a function of the rate new and sufficiently distinct viral variants emerge, the virulence of these viral variants, and the speed at which new vaccines can be manufactured and administered.

[0010] However, once an individual has been vaccinated as to achieve immunity, while they will initially be protected against the one or more viral variants targeted by the specific immunogen administered, subsequent mutations and consequent emergence of new viral variants confer on the individual susceptibility to infection by the virus again. Omicron strains showed a potent ability to evade the immune system in those recovering from COVID-19 and those vaccinated, owing to mutations that abolished the effectiveness of neutralising antibodies directed towards that region (Xia et al. 2022, Sig Transduct Target Ther 7, 241).

[0011] The emergence of in silico methods for use in biological experiments vastly increased the speed of discovery in the field and reduced the need for laborious and time-intensive laboratory work. In silico assays include virtual screening, molecular dynamics simulations, molecular modelling, drug design, and phylogenetic analysis. These assays have been essential in combatting Coronaviridae and contributed to the speed of developing effective vaccines for SARS-CoV-2 (Moradi et al. 2022, Inform Med Unlocked 2022;28: 100862). In silico approaches have allowed the design of more potent and refined vaccine components, such as multi-epitope compositions. Reverse vaccination technology, wherein analysis of the genomic sequence can predict new effective antigens, has helped reduce the period of vaccine target detection and evaluation around one to two years, and these epitope-based vaccines are considered to be more safe than previous vectored or attenuated live vaccines (Kanampalliwar 2020, Mol Biol. 2131 :1-16).

[0012] Even with the new approaches to vaccine design, there exists a constant situation of research institutions and manufacturers playing ‘catch-up’ with the evolution of a virus. Once a new viral variant emerges through the analysis of the viral genome, it must first be assessed if existing vaccinations are sufficient in combatting the new viral variant. If not, vaccines must be updated to encompass the new viral variant and minimise its spread and overall burden on the population. Typical development of vaccines can take upwards often years, due to the lengthy processes of identifying vaccine candidates, preclinical trials, extensive safety, dosing, and efficacy assessments in clinical trials, and finally achieving regulatory approval. The development of an initial vaccine for SARS-CoV-2 took just one year, with the United Kingdom approving BNT162 from Pfizer and BioNTech in December 2020 (Kashte et al. 2021 , Hum Cell 34(3):711-733). SARS-CoV-2 vaccine development was able to be expedited due to global investment, cooperation, and existing knowledge of the coronavirus family (such as viruses causing the common cold, SARS, and MERS). However, due to the continuing evolution of coronavirus species, such as SARS-CoV-2, and current vaccines proving ineffective against present viral variants, there is a continued effort to develop effective immunisations against this virus, particularly those that offer broad protection against multiple viral variants, and more broadly coronavirus subgenera. As such, searching for regions of pathogens’ genomes that are highly conserved either between species or strains may constitute a strategy for selecting broadly protective vaccines.

[0013] In addition to the dangers posed by variants of viruses that infect humans, spillover infections to humans from animals can be devastating if the pathogen adapts to render humans as a new stable reservoir. This can result in widespread human- to-human transmission, causing epidemics and pandemics in turn. Such events occurred in the cases of both the HIV virus and the SARS-CoV-2 virus. As such, it is desirable to generate vaccines that can pre-emptively protect against viruses that have yet to infect humans. Multiple deadly spillover infection events have already been caused by viruses of the Betacoronavirus subgenera, indicating a prudent need for vaccines designed to elicit an immune response of sufficient breadth to provide effective protection against Betacoronaviruses that are yet to infect humans.

[0014] Lymphocytes are a type of white blood cell and are central components of the immune system of most vertebrates. They include B cells, T cells and natural killer (NK) cells. Their function is to identify and protect against pathogens, such as bacteria, viruses, or malignant cancer cells that harbour mutated protein antigens. Both B- and T-cells express surface receptors which identify specific molecular components on pathogens called antigens (Ags). The successful recognition of an Ag triggers a wider adaptive immune response which ultimately may eliminate the potentially pathogenic threat.

[0015] Antibodies (Abs), also known as immunoglobulins (Ig), are glycosylated protein molecules present on the surface of B cells (surface immunoglobulins) or B cell eluted Ig molecules, that act as antigen specific B cell receptors (BCR). Upon successful Ag recognition, the Ig or membrane-bound BCRs will bind on specific Ag amino acid contact points, collectively named here as a B-cell epitope (BCE). This binding event may trigger the activation of an immune response that includes a clonal B cell expansion which will produce a vast amount of the Ag-specific Ig serum eluted molecules or effector B cells harbouring Ag-specific BCRs. The secreted soluble Igs and effector BCRs, also called antibodies (Abs), will have the same binding specificity as the original Ig-Ag binding interaction event. Abs will then circulate into the serum and bind to the same BCEs to mediate the elimination of the pathogenic or cell-stressed cells that correspond to the identified Ags.

[0016] Ab-based vaccines have the potential to prepare or educate the immune system of each individual against potentially harmful Ags sourced from infectious pathogenic threats or stressed malignant cancer cells. The accurate identification or prediction of B-cell epitopes on antigens is therefore of great importance not only for vaccine design, but also in the domains of molecular diagnostics, immune- monitoring and immunotherapy.

[0017] BCEs can be divided into two main categories: linear B cell epitopes (LBCEs) and conformational B cell epitopes (CBCEs). Both LBCEs and CBCEs can be bound by Abs and, thus, trigger an immune response. It is estimated that > 90% of the BCEs are conformational, while the rest are linear. As such, CBCEs represent vaccine candidates of very high importance.

[0018] LBCEs are made up of continuous (sequential) sequences of amino acids. These BCEs can be discovered by identifying positions on the unfolded Ag sequence which can be bound by Abs. LBCEs are often catalogued at the immune epitope database (IEDB) sourced from a large variety of experimental assays. Each assay provides information about the protein sequence tested (that is, the Ag), the LBCE sequence as well as its coordinates on the Ag, and the Ab which the current LBCE was tested on. Moreover, they provide an indicator as to whether the LBCE was bound by the given Ab. The binding information is sometimes described in the IEDB in the form of human readable labels, that is; positive, positive-high, positiveintermediate, positive-low and negative. The simplicity of LCBE as linear molecular entities, and the well-established experimental methods that are used to characterise them has led to a relatively large amount of available LBCE data compared to CBCE data. Nevertheless, annotation errors are frequent in this type of data, since many amino acids of the identified BCEs are not actually interacting with the Abs under the tertiary conformation of the Ag.

[0019] CBCEs are made up of short segments of amino-acids with potential diverse cis, trans, short or distal coordinate distances between each of the amino acid contact points that constitute the BCE. These segments come into proximity during the original folding of the Ag and their relative 3D coordinates can be further altered in the act of binding to the Ab. CBCEs can be experimentally characterized using X-ray crystallography on Ag-Ab structure complexes. This data is usually catalogued in the Protein Data Bank (PDB) in the form of 3D structures, and typically consist of one or more Ag structures bound by at least one Ab structure. PDB structure data provides information about the 3D coordinates of each molecule in the structure. However, the complexity of this method makes the CBCE data somewhat more sparce compared to that of LBCEs. Moreover, the 3D structures may not accurately reflect true CBCEs. For example, Abs on the complexes may be missing their heavy or light chains, or the 3D structure may only provide Ag-Ab contacts outside the CDR regions. Further, undiscovered CBCEs on any Ag, and the immensely sparse information available for negative epitopes on an Ag, makes the prediction of true CBCEs extremely challenging. CBCEs generally provide a higher level of specificity in immune recognition than LBCEs, allowing antibodies to distinguish between closely related molecules with subtle structural differences. As such, vaccine design using CBCEs is desirable, but has been traditionally difficult to execute.

[0020] The current lack of an approved vaccine composition which is efficacious across multiple subgenera of Betacoronaviruses creates significant danger for at-risk populations, including health care workers and patients in acute danger of nosocomial or community-transmitted infections. This risk is compounded by the rapid mutational rate of Betacoronavirus subgenera, which has led to immune evasion and reduced efficacy in vaccines with narrow antigenic targets, as observed in the recent COVID-19 pandemic. Viruses of the Betacoronavirus genus have caused multiple devastating spillover infection events to date and it is difficult to predict which virus may adapt to infect humans next.

[0021] Thus, there exists an urgent need for a safe and effective vaccine that targets CBCEs for use in the therapeutic or prophylactic treatment of conditions associated with Betacoronavirus.

[0022] SUMMARY OF INVENTION

[0023] This invention is based on the surprising discovery of immunogenic protein sequences that confer broad immunity to Betacoronavirus subgenera, species, and viral variants thereof. A vaccine comprising such sequences has the potential to stimulate a broad adaptive immune response to multiple Betacoronaviruses subgenera, species, and viral variants thereof, and future Betacoronavirus spillover to human host events, variants of concern that are yet to emerge, for the therapeutic or prophylactic treatment of infection with Betacoronavirus subgenera, species, and viral variants thereof in humans across the global population.

[0024] In a first aspect of the invention, there is provided a polypeptide comprising the amino acid sequences of any one of SEQ ID Nos 1 to 4, or a variant thereof with at least 70% sequence identity, wherein the one or more epitope sequence comprises at least the following residues, or, in a variant sequence, residues at equivalent positions: a. N373, P409, G410 and Q411 of SEQ I D No 1 , or b. D361 , T369, E370, F371. S372 and T373 of SEQ ID No 2, or c. S370, F371 , S372, T373, F374, K375, A381 , T382 and K383 of SEQ ID no 3, or d. D361 , S372, K373 and K375 of SEQ ID no 4.

[0025] In a second aspect of the invention, there is provided a polypeptide comprising the amino acid sequences of any one or more of SEQ ID Nos 5 to 8, or a variant thereof with at least 70% sequence identity, wherein the polypeptide is for use in therapy or prophylaxis.

[0026] In a third aspect of the invention, there is provided a polynucleotide encoding a polypeptide according to the first or second aspects of the invention. The polynucleotide may be DNA, RNA, or mRNA. The polynucleotide may include synthetic non-natural nucleotides, as would be understood by a person of skill in the art.

[0027] In a fourth aspect of the invention, there is provided a vector comprising one or more polynucleotides according the third aspect of the invention, wherein the vector may further comprise regulatory elements capable of driving transcription and / or translation of the polynucleotide in a host cell. The vector may be a plasmid or a viral vector or any other suitable vector. The plasmid vector may comprise DNA or RNA (such as mRNA). The viral vector may comprise DNA or RNA (such as mRNA). In a fifth aspect of the invention, there is provided a microorganism comprising one or more polypeptides according to the first or second aspects of the invention. The microorganism may be bacteria.

[0028] In a sixth aspect of the invention, there is provided a microorganism comprising one or more polynucleotides according to the third aspect of the invention. The microorganism may be bacteria.

[0029] In a seventh aspect of the invention, there is provided one or more polynucleotides according to the third aspect of the invention, or a vector according to the fourth aspect of the invention, formulated in a nanoparticle or lipid formulation.

[0030] In an eighth aspect of the invention, there is provided a composition comprising: i) one or more polypeptides according to the first or second aspects of the invention, or ii) one or more polynucleotides according to the third aspect of the invention.

[0031] In a ninth aspect of the invention, there is provided a polypeptide, polynucleotide, vector, microorganism or composition according to the first, second, third, fourth, fifth, sixth, seventh or eighth aspects of the invention for therapeutic or prophylactic use.

[0032] In a tenth aspect of the invention, there is provided a polypeptide, polynucleotide, vector, microorganism or composition according to the first, second, third, fourth, fifth, sixth, seventh, eighth or ninth aspects of the invention, for use in the treatment or prophylaxis of an infection caused by a Betacoronavirus.

[0033] In an eleventh aspect of the invention, there is provided a polypeptide, polynucleotide, vector, microorganism or composition for use according to the tenth aspect of the invention, wherein the infection is caused by a member of the Embecovirus, Hibecovirus, Merbecovirus, Nobecovirus or Sarbecovirus subgenera, or by MERS-CoV, SARS-CoV-1 , human coronavirus OC43, human coronavirus HKU1 , SARS-CoV-2, or variants thereof.

[0034] In a twelfth aspect of the invention, there is provided a vaccine composition comprising a polypeptide, polynucleotide, vector or microorganism according to the first, second, third, fourth, fifth, sixth or seventh aspects of the invention. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 shows the number of Betacoronavirus species that successfully had the SARS-CoV-2 RBD mapped onto them.

[0036] Figure 2 shows the number of Betacoronavirus species each tested Permutation ID was positive for, at a confidence level of 90%. A Permutation ID represents a conformational B cell epitope on an amino acid sequence. Specifically, the Permutation ID is on the full length Betacoronavirus spike protein sequence in question. A positive Permutation ID indicates the presence of a true epitope. The most positive Permutation ID is predicted to be a true epitope on >200 Betacoronavirus species.

[0037] Figure 3 shows the mutation policy carried out on the Betacoronavirus species amino acid sequences to make the conformational epitope “TPGQ” appear on the amino acid sequences of the Betacoronavirus species. This formed a common baseline to compare the effects of mutating the amino acids of the conformational epitope. “TPGQ” was already a positive permutation on 193 amino acid sequences, and 97 sequences were mutated either by shifting the RBD + / - 5 amino acids (y-axis) or by mutating up to 3 amino acids (x-axis). The mutation policy method first involved the in silico substitution of each amino acid on each position of a conformational epitope to all the other known amino acids. Then, for each in silico substitution, the candidate conformational epitope was re-predicted for all the spike proteins from the Betacoronavirus species with the substituted sequence. The total species that became positive following the substitutions were then counted.

[0038] Figure 4 shows the result of mutating the amino acid sequences of 97 Betacoronavirus species to make the conformational epitope “TPGQ” appear on the amino acid sequences of the Betacoronavirus species, as detailed in Figure 3. N = negative for the permutation and therefore not a true epitope for “TPGQ”; P = positive for the permutation and therefore a true epitope for “TPGQ”. By making “TPGQ” appear on the amino acid sequences of the Betacoronavirus species, the Betacoronavirus species can either become more positive (a true epitope for “TPGQ”), more negative, or unchanged. Once “TPGQ” appears on the amino acid sequence of each Betacoronavirus species, it is then possible to compare the results of mutating the amino acids of the conformational epitope “TPGQ” to predict if the mutated conformational epitope has increased immunodominance, or is reactive against more Betacoronavirus species, or both.

[0039] Figure 5 shows the mutation policy carried out on the Betacoronavirus species amino acid sequences to make the conformational epitope “DTFFST” appear on the amino acid sequences of the Betacoronavirus species. This formed a common baseline to compare the effects of mutating the amino acids of the conformational epitope. “DTFFST” was already a positive permutation on 134 amino acid sequences, and 156 sequences were mutated either by shifting the RBD + / - 5 amino acids (y-axis) or by mutating up to 3 amino acids (x-axis).

[0040] Figure 6 shows the result of mutating the amino acid sequences of 156 Betacoronavirus species to make the conformational epitope “DTFFST” appear on the amino acid sequences of the Betacoronavirus species, as detailed in Figure 5. N = negative for the permutation and therefore not a true epitope for “DTFFST”; P = positive for the permutation and therefore a true epitope for “DTFFST”. By making “DTFFST” appear on the amino acid sequences of the Betacoronavirus species, the Betacoronavirus species can either become more positive (a true epitope for “DTFFST”), more negative, or unchanged. Once “DTFFST” appears on the amino acid sequence of each Betacoronavirus species, it is then possible to compare the results of mutating the amino acids of the conformational epitope “DTFFST” to predict if the mutated conformational epitope has increased immunodominance, or is reactive against more Betacoronavirus species, or both.

[0041] Figure 7 shows the mutation policy carried out on the Betacoronavirus species amino acid sequences to make the conformational epitope “FFSTFKATK” appear on the amino acid sequences of the Betacoronavirus species. This formed a common baseline to compare the effects of mutating the amino acids of the conformational epitope. “FFSTFKATK” was already a positive permutation on 134 amino acid sequences, and 156 sequences were mutated either by shifting the RBD + / - 5 amino acids (y-axis) or by mutating up to 3 amino acids (x-axis). Figure 8 shows the result of mutating the amino acid sequences of 156 Betacoronavirus species to make the conformational epitope “FFSTFKATK” appear on the amino acid sequences of the Betacoronavirus species, as detailed in Figure 7. N = negative for the permutation and therefore not a true epitope for “FFSTFKATK”; P = positive for the permutation and therefore a true epitope for “FFSTFKATK”. By making “FFSTFKATK” appear on the amino acid sequences of the Betacoronavirus species, the Betacoronavirus species can either become more positive (a true epitope for “FFSTFKATK”), more negative, or unchanged. Once “FFSTFKATK” appears on the amino acid sequence of each Betacoronavirus species, it is then possible to compare the results of mutating the amino acids of the conformational epitope “FFSTFKATK” to predict if the mutated conformational epitope has increased immunodominance, or is reactive against more Betacoronavirus species, or both.

[0042] Figure 9 shows the mutation policy carried out on the Betacoronavirus species amino acid sequences to make the conformational epitope “DSTK” appear on the amino acid sequences of the Betacoronavirus species. This formed a common baseline to compare the effects of mutating the amino acids of the conformational epitope. “DSTK” was already a positive permutation on 186 amino acid sequences, and 104 sequences were mutated either by shifting the RBD + / - 5 amino acids (y-axis) or by mutating up to 3 amino acids (x-axis).

[0043] Figure 10 shows the result of mutating the amino acid sequences of 104 Betacoronavirus species to make the conformational epitope “DSTK” appear on the amino acid sequences of the Betacoronavirus species, as detailed in Figure 9. N = negative for the permutation and therefore not a true epitope for “DSTK”; P = positive for the permutation and therefore a true epitope for “DSTK”. By making “DSTK” appear on the amino acid sequences of the Betacoronavirus species, the Betacoronavirus species can either become more positive (a true epitope for “DSTK”), more negative, or unchanged. Once “DSTK” appears on the amino acid sequence of each Betacoronavirus species, it is then possible to compare the results of mutating the amino acids of the conformational epitope “DSTK” to predict if the mutated conformational epitope has increased immunodominance, or is reactive against more Betacoronavirus species, or both.

[0044] Figure 11 shows an increase in the number of Betacoronavirus species a Permutation ID was positive for when it was mutated from “TPGQ” to “NPGQ”. A positive Permutation ID indicates the presence of a true epitope. The hatched bar indicates the number of positive Betacoronavirus species after the mutation of “TPGQ” to “NPGQ”, whereas the white bar indicates the number of positive Betacoronavirus species before the mutation.

[0045] Figure 12 shows the change in positivity in the Permutation ID for the mutation of the conformational epitope “TPGQ” to “NPGQ”. N = negative for the permutation and therefore not a true epitope for “NPGQ”; P = positive for the permutation and therefore a true epitope for “NPGQ”. 229 species of Betacoronavirus became more positive due to the mutation and 26 species increased from negative to positive permutations, indicating that “NPGQ” is a true epitope on 257 Betacoronavirus species.

[0046] Figure 13 shows an increase in the number of Betacoronavirus species a Permutation ID was positive for when it was mutated from “DTFFST” to “DTEFST”. A positive Permutation ID indicates the presence of a true epitope. The hatched bars indicate the number of positive Betacoronavirus species after the mutation of “DTFFST” to “DTEFST”, whereas the white bar indicates the number of positive Betacoronavirus species before the mutation.

[0047] Figure 14 shows the change in positivity in the Permutation ID for the mutation of the conformational epitope “DTFFST” to “DTEFST”. N = negative for the permutation and therefore not a true epitope for “DTEFST”; P = positive for the permutation and therefore a true epitope for “DTEFST”. 224 species of Betacoronavirus became more positive due to the mutation and 7 species increased from negative to positive permutations, indicating that “DTEFST” is a true epitope on 231 Betacoronavirus species. Figure 15 shows an increase in the number of Betacoronavirus species a Permutation ID was positive for when it was mutated from “FFSTFKATK” to “SFSTFKATK”. A positive Permutation ID indicates the presence of a true epitope. The hatched bar indicates the number of positive Betacoronavirus species after the mutation of “FFSTFKATK” to “SFSTFKATK”, whereas the white bar indicates the number of positive Betacoronavirus species before the mutation.

[0048] Figure 16 shows the change in positivity in the Permutation ID for the mutation of the conformational epitope “FFSTFKATK” to “SFSTFKATK”. N = negative for the permutation and therefore not a true epitope for “SFSTFKATK”; P = positive for the permutation and therefore a true epitope for “SFSTFKATK”. 213 species of Betacoronavirus became more positive due to the mutation and 8 species increased from negative to positive permutations, indicating that “SFSTFKATK” is a true epitope on 221 Betacoronavirus species.

[0049] Figure 17 shows an increase in the number of Betacoronavirus species a Permutation ID was positive for when it was mutated from “DSTK” to “DSKK”. A positive Permutation ID indicates the presence of a true epitope. The hatched bar indicates the number of positive Betacoronavirus species after the mutation of “DSTK” to “DSKK”, whereas the white bar indicates the number of positive Betacoronavirus species before the mutation.

[0050] Figure 18 shows the change in positivity in the Permutation ID for the mutation of the conformational epitope “DSTK” to “DSKK”. N = negative for the permutation and therefore not a true epitope for “DSKK”; P = positive for the permutation and therefore a true epitope for “DSKK”. 213 species of Betacoronavirus became more positive due to the mutation and 11 species increased from negative to positive permutations, indicating that “DSKK” is a true epitope on 224 Betacoronavirus species.

[0051] Figure 19 shows the number of Betacoronavirus species that share at least one positive permutation with another Betacoronavirus species. The y-axis represents the total number of species that have at least one positive permutation in common with another Betacoronavirus species. Figure 20 shows the neutralizing antibody titers in the sera of mice against the SARS-CoV-2 Lelystad strain. Mice have been vaccinated with mRNA LNPs for the SARS-CoV-2 ancestral spike or NEC constructs. Every dot represents one mouse.

[0052] Figure 21 shows the neutralizing antibody titers in the sera of mice against the SARS-CoV-2 BA.5 Omicron strain. Mice have been vaccinated with mRNA LNPs for the SARS-CoV-2 Ancestral spike or NEC constructs. Every dot represents one mouse.

[0053] Figure 22 shows the neutralizing antibody titers in the sera of mice against the SARS-CoV-2 XBB.1 .5 Omicron strain. Mice have been vaccinated with mRNA LNPs for the SARS-CoV-2 Ancestral spike or NEC constructs. Every dot represents one mouse.

[0054] Figure 23 shows the neutralizing antibody titers in the sera of mice against the SARS-CoV HKU39849 strain. Mice have been vaccinated with mRNA LNPsforthe SARS-CoV-2 Ancestral spike or NEC constructs. A comparison with the Comirnaty BA.4 / BA.5 vaccine is also provided. Every dot represents one mouse.

[0055] Figure 24 shows the neutralizing antibody titers in the sera of mice against BtKY72 Coronavirus strain. Mice have been vaccinated with mRNA LNPs of the SARS- CoV-2 Ancestral spike or NEC constructs. Every dot represents one mouse.

[0056] Figure 25 shows the neutralizing antibody titers in the sera of mice against the NeoCov strain. Mice have been vaccinated with mRNA LNPs of the SARS-CoV-2 Ancestral spike or NEC constructs. Every dot represents one mouse.

[0057] Figure 26 shows the neutralizing antibody titers in the sera of mice against the Pangolin Coronavirus GX strain. Mice have been vaccinated with mRNA LNPs of the SARS-CoV-2 Ancestral spike or NEC constructs. Every dot represents one mouse. Figure 27 shows the neutralizing antibody titers in the sera of mice against the SHC014 coronavirus strain. Mice have been vaccinated with mRNA LNPs of the SARS-CoV-2 Ancestral spike or NEC constructs. Every dot represents one mouse.

[0058] DETAILED DESCRIPTION

[0059] This invention is predicated on the identification of Betacoronavirus shared conformational epitopes that have a high probability of effectively stimulating a broad adaptive humoral immune response to multiple Betacoronavirus subgenera, species and variants thereof when used in a vaccine composition. Thus, the incorporation of such shared conformational epitopes into polypeptides, polynucleotides, compositions, microorganisms, vectors, ora vaccine composition may allow for the therapeutic or prophylactic treatment of infection with various Betacoronaviruses, including those that do not yet infect humans. Unlike prior Betacoronavirus vaccination approaches, the polypeptide, polynucleotide, vector, microorganism, composition, or vaccine composition of the present invention is designed to stimulate a broad adaptive humoral immune response with widespread cross-reactivity across Betacoronavirus subgenera. To achieve this, the inventors sought the identification of native and mutated conformational epitopes shared by multiple Betacoronavirus subgenera, and in turn synthesised polypeptides capable of priming immune responses across said subgenera. It is expected that this will lead to the generation of a more broad, substantial and sustained level of immunity.

[0060] The inventors have surprisingly found that polypeptides identified with a predictor platform show in silico and in vivo immunogenicity to a broad range of Betacoronavirus subgenera. To arrive at the amino acid sequences of SEQ ID Nos 1 to 4, the inventors first mapped the receptor binding domain (RBD) of SARS-CoV-2 to the amino acid sequences of 290 Betacoronavirus species. The “receptor binding domain” retains its meaning as the skilled person would understand, namely that it is a part of a virus located on the spike domain that allows it to dock to body receptors to gain entry into cells. The inventors then generated 450,000 permutations which were then placed inside the RBD of each species. The term “permutation” refers to a configuration of amino acids that defines the contact position of a candidate epitope with an antibody, allowing for a reliable prediction as whether the epitope binds to an antibody. The permutations were then compared to each of the 290 Betacoronavirus species sequences to predict whether that permutation is a true epitope, namely one that is immunogenic, on each species. The result of predicting the immunogenicity of an epitope was a binary output, which was then filtered to retain predictions with a probability of being an epitope of >90%. The result of this analysis produced 569,230 true epitopes.

[0061] The inventors then analysed the positive permutations to see how many Betacoronavirus species each permutation was predicted to be reactive to. Some permutations showed reactivity in over 200 Betacoronavirus species, indicating they were promising candidates for treatments that confer broad immunity to Betacoronaviruses that are both currently infectious in humans as well as those which may become infectious through mutation in the future. From this result, the inventors sought to identify the conformational epitopes that most commonly appeared across the broadest range of species, to design polypeptides and further vaccines that cause the highest immunogenicity to the largest number of Betacoronavirus species. The term “conformational epitope” refers to a sequence of amino acids composing an antigen that comes into direct contact with a receptor of the immune system. The sequence of amino acids may be discontinuous, whereby the amino acids are not adjacent in the amino acid sequence, but come together during folding to form an epitope.

[0062] Shared conformational epitope identification was first accomplished by creating sequence logos from the positive permutations that were reactive to the highest number of Betacoronavirus species. The term “sequence logos” refers to collections of amino acids predicted to form a conformational epitope together. This process predicted the individual amino acids that were involved in creating a conformational epitope against the virus species, which resulted in the identification of the discontinuous sequence “TPGQ” as the conformational epitope that appears in the highest number of Betacoronavirus species. The next step was to mutate the identified discontinuous sequence to see whether mutations caused the sequence to be immunogenic to more Betacoronavirus species, cause higher immunogenicity in the species it is immunogenic to, or both simultaneously. However, to prepare the sequences for this analysis, the inventors amended the Betacoronavirus species sequences that were not immunogenic to the sequence “TPGQ” by shifting the RBD mapped to them or by systematically mutating individual amino acids as to force the sequence “TPGQ” onto the sequences in question. This step increased the number of sequences that the conformational epitope appeared on from 193 to all 290 species, which could then act as a common baseline for mutating the sequence “TPGQ”. It was discovered that mutating the sequence “TPGQ” to “NPGQ” caused a significant increase in the number of species the conformational epitope was reactive to as well as a positive increase in the immunogenicity of said conformational epitopes. This analysis was repeated with the next 3 top permutations: the conformational epitope “DTFFST” was mutated to “DTEFST”; the conformational epitope “FFSTFKATK” was mutated to “SFSTFKATK”; the conformational epitope “DSTK” was mutated to “DSKK”. The mutated conformational epitopes were applied in turn to the spike protein of Omicron B.A.2 to produce the amino acid sequences represented by SEQ ID Nos 1 to 4.

[0063] Naturally occurring spike proteins from the 290 Betacoronavirus species sequences that were predicted to be the most immunodominant and cross reactive, and consequently potentially confer broad protection against other viruses, were identified using the true positive permutations, these included the spike proteins of Pangolin coronavirus (taxid: 2708335), Rhinolophus affinis coronavirus (taxid: 1487703), Betacoronavirus HKU24 (taxid: 1590370) and Bat coronavirus RaTG13 (taxid: 2709072), which are represented by SEQ ID Nos 5 to 8 respectively.

[0064] Thus, in a first aspect of the invention, there is provided a polypeptide comprising the amino acid sequences of any one of SEQ ID Nos 1 to 4, or a variant thereof with at least 70% sequence identity, wherein the polypeptide comprises at least the following residues, or in a variant sequence, residues at equivalent positions: a. N373, P409, G410 and Q411 of SEQ ID No 1 , or b. D361 , T369, E370, F371 , S372 and T373 of SEQ ID No 2, or c. S370, F371 , S372, T373, F374, K375. A381 , T382 and K383 of SEQ ID no 3, or d. D361 , S372, K373 and K375 of SEQ ID no 4.

[0065] In a second aspect of the invention, there is provided a polypeptide comprising the amino acid sequences of any one or more of SEQ ID Nos 5 to 8, or a variant thereof with at least 70% sequence identity, wherein the polypeptide is for use in therapy or prophylaxis.

[0066] It is envisaged that the polypeptide may comprise one of any of SEQ ID No 1 to 8, or a variant thereof.

[0067] The wild-type or naturally occurring amino acid sequences of the species identified and represented by SEQ ID No 5 to 8 were predicted to be the most immunodominant and cross reactive across the highest number of species, while covering the most subgenera of Betacoronavirus. The term “immunodominant” refers to the probability that an epitope can be recognised by a cognate antibody or antibody receptor. The higher the probability hit of an epitope when tested against a true positive permutation, the higher the immunodominance.. The term “cross reactive” refers to epitope sequences that share the maximum number of high probability hits when tested against a true positive permutation, across the maximum number of Betacoronavirus subgenera and species.

[0068] It is envisaged that the polypeptides of the first and second aspects of the invention may be combined in the preparation of a composition. The polypeptides of the first and second aspects of the invention may be combined in the preparation of a vaccine composition. It is also envisaged that the polypeptides of the first and second aspects of the invention may be combined in the preparation of a composition for use in the treatment or prophylaxis of an infection caused by a Betacoronavirus. It is envisaged that the composition or vaccine composition comprising the polypeptides of the first and second aspects of the invention may comprise polypeptides having the amino acid sequence of at least one of any of SEQ ID No 1 to 8. It is also envisaged that the composition of vaccine composition may comprise at least 2, 3, 4, 5, 6, 7, 8 or more polypeptides having the amino acid sequence of at least one of any of SEQ ID No 1 to 8. The composition or vaccine composition may comprise any combination of polypeptides having the amino acid sequence of any of SEQ ID No 1 to 8, including multiple copies of any polypeptide having the amino acid sequence of any of SEQ ID No 1 to 8. For example, a composition or vaccine composition may comprise three polypeptides having the amino acid sequence of SEQ ID No 2, one polypeptide having the amino acid sequence of SEQ ID No 4 and two polypeptides having the amino acid sequence of SEQ ID No 6. It is envisaged that through the specification of certain amino acid residues in SEQ ID No 1 to 4, conformational epitopes will be created. The amino acid residues specified in SEQ ID No 1 to 4 form conformational epitopes that directly contact receptors of the immune system to generate effective immune responses. The term “vaccine composition”, or “vaccine”, which from herein may be referred to interchangeably as the “composition”, relates to a biological preparation that induces active acquired immunity to a particular infectious agent , in this case a Betacoronavirus. Typically, the vaccine contains an agent, or “foreign” agent, that resembles the infection-causing pathogen, or part of the infection causing pathogen, which within the prior art has often been a weakened or killed form of said pathogen, or recombinant protein or protein fragment from such pathogen, or polynucleotide encoding such a protein or protein fragment (Williamson et al. 1995, FEMS Immunology and Medical Microbiology 12 (3-4): 223-230). Such a foreign agent, protein or protein fragment would be recognised by a vaccine-receiver’s immune system, which in turn would destroy said agent and develop “memory” against the pathogen, inducing a level of lasting protection against future infections from the same or similar pathogenic sub-species. Through the route of vaccination, including those vaccine compositions of the present invention, it is envisaged that once the vaccinated subject again encounters the same or similar pathogen of which said subject was vaccinated against, the individual’s immune system may thereby recognise said pathogen and elicit a more effective defence against infection. A more in-depth description of types of vaccines within the art can be found in US6541003 B1. The vaccine composition may comprise an adjuvant, a pharmaceutically acceptable carrier or excipient. In an embodiment of the invention, each of the one or more amino acid sequences of the polypeptide, composition or vaccine composition have at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID No 1 to SEQ ID No 8, or a variant thereof.

[0069] The term “polypeptide” is used to mean a molecule that is composed of two or more amino acids, which combine to make a protein / polypeptide. As used herein, the term “polypeptide” denotes the claimed polypeptide, whereas the term “epitope” is used to denote potentially shorter amino acid sequences making up the polypeptide, including conformational epitopes. Though of course, the “epitopes” are also polypeptides as denoted in the art and as would be understood by the skilled person. The term “epitope” as used herein also refers to any part of an antigen that is specifically recognised by any antibodies or B cells, as is its natural meaning. The term “amino acid” retains its meaning as used in the art and would be understood by the skilled person. The term “variant” is used to refer to amino acid sequences that differ from the indicated SEQ ID No by one or more amino acid residues. This could be a substitution, addition or deletion of one or more amino acids, other than those amino acids expressly stated herein as required. An example would be the amino acid sequences ACDEF and ACDEE, whereby the latter is a variant of the former as one amino acid has been substituted. Thus, variants may have amino acids inserted, deleted or substituted from the indicated sequence. The term “variant” is also intended to encompass amino acid sequences that differ substantially from the indicated SEQ ID No, including by way of truncation. Thus, a variant of an amino acid sequence indicated by SEQ ID No may be a variant of full length that is the same as the indicated sequence, or a variant that has fewer amino acids than the indicated sequence. Any variant with differing amino acids may affect the specific residues predicted to form conformational epitopes according to the first aspect of the invention. This effect may be particularly potent in truncated polypeptides. The term “equivalent positions”, therefore refers to amino acid residues that comprise the conformational epitope and have retained the same or similar amino acids residues, but wherein the amino acid residues appear at a different location on the polypeptide. For example, a truncation to the amino acid sequence of SEQ ID No 1 may remove the first 100 aa from the polypeptide. The resulting polypeptide may therefore comprise the residues N273, P309, G310 and Q311. While the truncated polypeptide would no longer have the amino acid residues N373, P409, G410 and Q411 of SEQ ID No 1 , the residues of the truncated polypeptide are equivalent to those specified in SEQ ID No 1 , and are thus encompassed by the present invention.

[0070] As used herein, the term “sequence identity” and “sequence homology” are interchangeable and refers to the number of identical residues over a defined length into a given alignment. To calculate % sequence identity of any of the sequences herein disclosed, sequence comparison software may be used, for example, using the default settings on the BLAST software package (V2.10.1). The skilled person would understand that the composition or vaccine composition of the invention may therefore comprise combinations of polypeptides with amino acid sequences selected from SEQ ID Nos 1 to 8 having any % identity with the respective SEQ ID No. An example of this would be a composition or vaccine composition comprising polypeptide with 100% identity to SEQ ID No 1 and 90% identity to SEQ ID No 8. This means that the composition or vaccine composition may retain its intended qualities and uses thereof if the amino acid sequence changes. Substitutions, deletions and insertions of amino acids may comprise potential manipulations of the composition or vaccine composition of the invention.

[0071] For the avoidance of doubt, the composition or vaccine composition of the invention, according to any embodiment, may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or more than eight polypeptides or polynucleotides having the amino acid sequences of any of SEQ ID Nos 1 to 8, or any variant thereof having at least 70% sequence identity thereto. As described herein, it will be appreciated that any number and / or combination of the amino acid sequences of any of SEQ ID Nos 1 to 8 may be utilised. The amino acid sequences of SEQ ID Nos 1 to 8 of the invention are as follows In

[0072] Table 1 :

[0073] Table 1 : The amino acid sequences of the invention, identified to be broadly immunogenic across multiple Betacoronavirus subgenera. Amino acids that comprise the conformational B cell epitopes of interest are highlighted in bold and underlined.

[0074] The term “therapy” is to be used interchangeably with the terms “therapeutic treatment” and “therapeutic use” and refer to a medical procedure with the purpose of treating or curing a viral infection or the associated symptoms thereof, as would be appreciated within the art. The term “therapy” also encompasses the treatment of a disease in general or to a curative treatment in the narrow sense as well as the alleviation of the symptoms of pain or suffering. The terms “prophylaxis” and “prophylactic use” are used interchangeably and, as used herein, refer to a medical procedure whose purpose is to prevent or reduce the morbidity or duration of (rather than treat or cure) a disease, such as an infection.

[0075] In a third aspect of the invention, there is provided a polynucleotide encoding a polypeptide according to the first or second aspects of the invention. The polynucleotide may be DNA, RNA or mRNA. The term “polynucleotide” refers to a linear polymer whose molecule is composed of many nucleotide units, constituting a section of a nucleic acid molecule. The terms “DNA”, “RNA” and “mRNA” retain their meaning as used in the art and would be familiar to the skilled person. In a fourth aspect of the invention, there is provided a vector comprising one or more polynucleotides according to the third aspect of the invention, wherein the vector further comprises regulatory elements capable of driving transcription and / or translation of the polynucleotide in a host cell. The vector may also be a viral vector. By “vector” we intend any vehicle that is capable of supporting (i.e. carrying, encapsulating, incorporating and / or protecting) the polynucleotide of the invention and facilitating stable or transient transfection of a target host cell with the polynucleotide of the invention. The vector may also include the indicated regulatory elements to drive expression of a polypeptide encoded by the polynucleotide in the host cell. Suitable vectors may be, for example, plasmids, viral particles (e.g. lentiviruses, adenoviruses, adeno-associated viruses and any number of other viruses, as would be understood by a person of skill in the art), nanoparticles, lipid- nanoparticles etc. An example of a vector suitable for the invention is a lentivirus, capable of inserting DNA into host cell genomes. Examples may also include one or more adenovirus vectors, vesicular stomatis virus vectors, influenza virus vectors or measles virus vectors. When viral particles are used as vectors, they are typically modified from their wild-type form to remove viral antigen encoding nucleic acid and / or to prevent viral replication in the host cell. The vector may be a naked DNA such as a plasmid or a virus, among other vectors used in the art and that would be familiar to the skilled person. The term “regulatory elements” refers to nucleotide sequences of genes that are involved in regulation of transcription.

[0076] In another embodiment, the vector is a nanoparticle. The nanoparticle may be a lipid nanoparticle ora polymer nanoparticle. The polynucleotide of the third aspect of the invention or vector of the fourth aspect of the invention may be formulated within a nanoparticle for delivery to and subsequent transfection of a target host cell, such as a cell of a subject suffering from a Betacoronavirus-related disease or Betacoronavirus infection. For example, there may be the delivery of mRNA via a lipid nanoparticle. The polypeptide of the first or second aspects of the invention may be formulated within a nanoparticle. In a fifth aspect of the invention, there is provided a microorganism comprising one or more polypeptides according to the first or second aspects of the invention. The microorganism may be bacteria.

[0077] In a sixth aspect of the invention, there is provided a microorganism comprising one or more polynucleotides according to the third aspect of the invention. The microorganism may be bacteria. The term “microorganism” refers to a microscopic organism, such as a bacterium, virus or fungus. Microorganisms may be used to further propagate / replicate the polynucleotide or polypeptide of the invention or to be administered as part of therapeutic or prophylactic use in order to deliver the polynucleotide or polypeptide to a specific tissue or cell type such as an antigen presenting cell. A microorganism comprising a polypeptide of the invention may be suited to industrial production of the polypeptide, and may include: Escherichia coli, Bacillus subtilis, Streptomyces spp., Corynebacterium glutamicum, Pseudomonas putida, Clostridium spp. And Lactobacillus. The microorganism of the invention may also be genetically modified. The polypeptide of the invention may also be produced by synthetic methods, of which the skilled person would be aware. The skilled person would be aware of both recombinant and synthetic methods (e.g., chemical or de novo synthesis) of polypeptide synthesis.

[0078] In a seventh aspect of the invention, there is provided one or more polynucleotides according to the third aspect of the invention, or a vector according to the fourth aspect of the invention, formulated in a nanoparticle or lipid formulation. The term “nanoparticle” refers to a nanoscale particle between 1 and 100 nanometres in diameter, often with a very high surface area to volume ratio. Formulation in a nanoparticle may improve delivery of the polynucleotide or vector of the invention to a subject and / or the immune system of a subject. The nanoparticle may be a lipid nanoparticle, designed to facilitate encapsulation and delivering of the polynucleotide or vector of the invention.

[0079] In an eighth aspect of the invention, there is provided a composition comprising: i) one or more polypeptides according to the first or second aspects of the invention, or ii) one or more polynucleotides according to the third aspect of the invention.

[0080] In a ninth aspect of the invention, there is provided a polypeptide, polynucleotide, vector, microorganism or composition according to the first, second, third, fourth, fifth, sixth, seventh or eighth aspects of the invention for therapeutic or prophylactic use. The polypeptide, polynucleotide, vector, microorganism or composition according to the first, second, third, fourth, fifth, sixth, seventh or eighth aspects of the invention may be for use in medicine. The terms “therapeutic treatment” and “therapeutic use” retain their meaning as defined above. The terms “prophylaxis” and “prophylactic use” retain their meaning as defined above.

[0081] In a tenth aspect of the invention, there is provided a polypeptide, polynucleotide, vector, microorganism or composition according to the first, second, third, fourth, fifth, sixth, seventh, eighth or ninth aspects of the invention, for use in the treatment or prophylaxis of an infection caused by Betacoronavirus. The terms “treatment” and “prophylaxis” are to be used interchangeably with “therapeutic treatment” and “prophylactic treatment” respectively. It is envisaged that the polypeptide, polynucleotide, vector, microorganism or composition of the present invention may be used against any Betacoronavirus infection.

[0082] Betacoronaviruses, from the family Coronaviridae, are a group of enveloped, positive-sense single-stranded RNA ((+)ssRNA) viruses which can cause respiratory tract infections in human hosts. Mild Betacoronavirus infections include some cases of the common cold, whilst more lethal species of Betacoronavirus such as severe acute respiratory syndrome-related coronavirus (SARS-CoV-1), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), can cause the more serious diseases SARS, MERS, and COVID-19, respectively. It is proposed that SARS-CoV-2 shares zoonotic origins and close genetic similarity with SARS- CoV-1 , and as such much of our understanding of COVID-19, as well as the research and development of potential prophylactic and therapeutic treatments, has come from the analysis of such other coronaviruses. SARS-CoV-2 is the causative viral agent behind the 2019-2020 pandemic of COVID-19, a respiratory syndrome characterised by high fever, malaise, rigors, headache, dry cough, lymphopenia and progression to interstitial infiltration in lungs with an eventual mortality of greater than 10% in many countries. SARS related pathologies of the lungs involve the subsequent stages of viral replication, immune system hyperactivation, and pulmonary destruction (Weis & Navas- Martin 2005, Microbiol Mol Biol Rev. 69 (4): 635-64) and inflammatory exudates in the lungs.

[0083] Betacoronaviruses, such as SARS-CoV-2, attach to their specific cellular receptors via the viral spike protein-invading cells lining the respiratory tract. The receptor for the SARS-CoV-2 virus, a positive single stranded RNA ((+)ssRNA) coronavirus, was identified as angiotensin-converting enzyme 2 (ACE2): a zinc metalloprotease (Li et al. 2003, Nature 426: 450-454). Diseased lungs show diffuse alveolar damage, epithelial cell proliferation, and an increased number of macrophages. Further, multinucleate giant-cell infiltrates of macrophage or epithelial cells with syncytium-like cell formation have been described. In addition to hemophagocytosis in the lung, lymphopenia and white-pulp atrophy of the spleen have been observed in SARS patients. At present, most COVID-19 patients receive traditional supportive care such as breathing assistance and / or steroid therapy.

[0084] It is envisaged that the polypeptide, polynucleotide, vector, microorganism or composition of the present invention may aid in the therapeutic or prophylactic treatment of a Betacoronavirus infection.

[0085] In an eleventh aspect of the invention, there is provided a polypeptide, polynucleotide, vector, microorganism or composition for use according to the tenth aspect of the invention, wherein the infection is caused by a member of the Embecovirus, Hibecovirus, Merbecovirus, Nobecovirus or Sarbecovirus subgenera, or by MERS-CoV, SARS-CoV-1 human coronavirus OC43, human coronavirus HKU1 , SARS-CoV-2, or variants thereof. It is envisaged that the virus species may comprise Betacoronavirus 1 , Bovine coronavirus, Human coronavirus OC43, China Rattus coronavirus HKU24, Human coronavirus HKU1 , Murine coronavirus, Myodes coronavirus 2JL14, SARS-CoV-1 , SARS-CoV-2, Bat SARS-like coronavirus WIV1 , Bat coronavirus RaTG13, Hedgehog coronavirus 1 , MERS-CoV, Pipistrellus bat coronavirus HKU5, Tylonycteris bat coronavirus HKU4, Eidolon bat coronavirus C704, Rousettus bat coronavirus GCCDC1 , Rousettus bat coronavirus HKU9, Bat Hp-betacoronavirus Zhejiang2013 or any other bat coronavirus. The skilled person would understand that this is not an exhaustive list of virus species and that in any embodiment that a “Betacoronavirus” is referred to may comprise one of the above virus species.

[0086] The virus species may also comprise any selected from Table 2.

[0087] Table 2: The Betacoronavirus species that the amino acid sequences of the invention were tested against for immunogenicity.

[0088] The virus classification system differs from that used for cellular organisms, but still places viruses in a taxonomic system as with cellular organisms by using the International Committee on Taxonomy of Viruses (ICTV classification). These classifications used herein will be apparent to the skilled person. As used herein, the term “subgenus” or “subgenera” refers to the viral taxonomic rank directly below genus. Examples of subgenera of the family Betacoronavirus include Embecovirus, Hibecovirus, Merbecovirus, Nobecovirus, and Sarbecovirus. As used herein, the term “species” refers to the viral taxonomic rank directly below subgenus. Examples of species include SARS-CoV, SARS-CoV-2 (from the Sarbecovirus subgenus), Human coronavirus OC43 (from the Embecovirus subgenus) and MERS-CoV (from the Merbecovirus subgenus). As used herein, the term “viral variant” refers to both strains and variants of a certain viral species which will be apparent to the skilled person and define those which belong to the same species but have stable and heritable biological, serological and / or molecular characteristics. While the subgenera of the family Betacoronavirus are distinct from one another and encompass a variety of species, the inventors have identified amino acid sequences that are common across many species and subgenera which in turn form CBCEs that can be targeted to generate broad immunity in human populations.

[0089] In a twelfth aspect of the invention, there is provided a vaccine composition comprising a polypeptide, polynucleotide, vector or microorganism according to the first, second, third, fourth, fifth, sixth or seventh aspects of the invention. In an embodiment of the invention, the vaccine composition is an RNA vaccine. In a preferred embodiment of the invention, the vaccine composition is an mRNA vaccine. In a further embodiment of the invention, the vaccine composition comprises a pharmaceutically acceptable carrier, diluent, excipient and / or adjuvant. In yet a further embodiment of the invention, the vaccine composition is formulated for parenteral, oral, sublingual, nasal, naso-oral, or pulmonary administration. In a still further embodiment of the invention, said parenteral administration is subcutaneous, intradermal, intramuscular, subdermal, intraperitoneal, or intravenous administration. In a preferred embodiment of the invention, the vaccine composition may be for administration to a subject via one or more intramuscular injections. In a further embodiment of the invention, the vaccine composition may be for administration to a subject via one or more intradermal injections. The term “subject” refers to a person subjected to treatment, observation or experiment and would be understood by the skilled person as such.

[0090] The term “mRNA vaccine” describes a vaccine that uses a copy of an mRNA molecule to produce an immune response. The vaccine delivers molecules of antigen-encoding mRNA into host immune cells, which used the designed mRNA as a blueprint to build the encoded foreign protein, or fragment of a protein, that would normally be produced by a pathogen such as a virus. These protein molecules stimulate an adaptive immune response that teaches the body to identify and destroy the corresponding pathogen. In an embodiment of the invention, the foreign protein produced is of a Betacoronavirus. The mRNA may be delivered by a co-formulation of the mRNA encapsulated in lipid nanoparticles. The mRNA may also be formulated for nasal or intratracheal delivery via a nanoparticle-delivery-based system. This system may comprise a biodegradable poly(amine-co-ester) polymer that forms polyplexes with mRNA so that the vaccine is inhalable.

[0091] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a human, as appropriate. The preparation of a pharmaceutical composition that contains the vaccine composition of the present invention will be known to those of skill in the art in light of the present disclosure. Moreover, for human administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards. Examples include, but are not limited to disodium hydrogen phosphate, soya peptone, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, magnesium sulphate, calcium chloride, sucrose, borate buffer, sterile saline solution (0.9 % NaCI) and sterile water.

[0092] Suitable aqueous and non-aqueous carriers that may be employed in the vaccine compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0093] As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives {e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavouring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington’s Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329).

[0094] Examples of adjuvants which may be effective include but are not limited to: unmethylated cytosine-guanine dinucleotide (CpG) motifs, granulocytemacrophage colony-stimulating factor (GM-CSF), aluminium hydroxide, N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N acetylmuramyl-L-alanyl-Disoglutaminyl-L-alanine-2-(1 '-2'-dipalmitoyl-sn-glycero- 3-hydroxyphosphoryloxy)-ethylamine (CGP I9835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene / Tween 80 emulsion. Further examples of adjuvants and other agents include aluminium hydroxide, aluminium phosphate, aluminium potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAB-dextran, blocked copolymers or other synthetic adjuvants. Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund’s Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.).

[0095] The invention also provides for the use of the polypeptide, polynucleotide, vector, microorganism, composition or vaccine composition of the invention in the manufacture of a medicament for the treatment of a Betacoronavirus infection.

[0096] The present invention provides a method of treating a Betacoronavirus infection, comprising administering to a subject an effective amount of the polypeptide, polynucleotide, vector, microorganism, composition or vaccine composition of the present invention.

[0097] It is envisaged that the polypeptide, polynucleotide, vector, microorganism, composition or vaccine composition of the present invention may aid in the therapeutic or prophylactic treatment of a Betacoronavirus infection in a human subject, wherein said composition comprises one or more epitopes of the present invention.

[0098] The active acquired immunity that may be induced by the invention may be humoral. Humoral immunity refers to a response involving B cells which produce antibodies that specifically bind to antigens, or any future antigens, corresponding to those within the administered vaccine composition. B cells, each expressing a unique B cell receptor (BCR), recognise antigens in their native form. Upon this recognition and further interaction with other cells of the immune system, the activated B cell can differentiate into a plasma cell specialised to secrete antibodies against the encountered antigen. The term antibody refers to an immunoglobulin (Ig) that is produced by the immune system to specifically identify and neutralise foreign antigens. A subset of these B-cell derived plasma cells become long-lived antigen-specific memory B cells, as would be well understood by the skilled person.

[0099] It is envisaged that the vaccine composition of the present invention may be an epitope-based vaccine, or in other words, is comprised of one or more epitopes. Epitope-based vaccines (eVs) make use of short antigen-derived peptides corresponding to immune epitopes, which are administered to trigger a protective humoral immune response. eVs potentially allow for precise control over the immune response activation by focusing on the most relevant — immunogenic and conserved — antigenic regions. Experimental screening of large sets of peptides is time-consuming and costly; therefore, in silico methods that facilitate B-cell epitope mapping of protein antigens are paramount for eV development.

[0100] An “antigen” refers to a molecule capable of being bound by an antibody or a B cell. As such, the terms epitope and antigen may be used interchangeably herein. Epitopes may also be referred to by the molecule for which they bind, such as “B cell epitopes”.

[0101] In some embodiments, the polypeptide, polynucleotide, vector, microorganism, composition or vaccine composition may be used in the therapeutic or prophylactic treatment of any Betacoronavirus infection. In a preferred embodiment, the Betacoronavirus infection may be caused by members of the Embecovirus, Hibecovirus, Merbecovirus, Nobecovirus, Sarbecovirus subgenera, or by MERS- CoV, SARS-CoV-1 , human coronavirus OC43, human coronavirus HKU1 , SARS- CoV-2, or variants thereof.

[0102] The one or more compositions of the present invention may be formulated for parenteral, oral, sublingual, nasal, naso-oral, or pulmonary administration. In a preferred embodiment, the parenteral administration may be subcutaneous, intradermal, intramuscular, subdermal, intraperitoneal or intravenous.

[0103] It is envisaged that administration of the polypeptide, polynucleotide, vector, microorganism, composition or vaccine composition according to the present invention would be carried out following an appropriate immunisation regimen. The term “appropriate immunisation regimen” is to be construed as a schedule or timescale of one or more administrations of the compositions of the present invention, which may resultantly yield the most effective results in consideration of immunisation efficacy and safety of the subject to which the composition is being administered. For example, for the therapeutic or prophylactic treatment of a Betacoronavirus infection, an immunisation regimen should be chosen that yields as effective immunisation against the Betacoronavirus as possible, whilst still maintaining suitable safety for the subject. The immunisation regimen may act to "prime” “condition”, “boost”, “amplify”, “enhance”, “improve”, “augment” or “promote” (used interchangeably) an immune response in the subject receiving the compositions of the present invention. The immune response may be, amongst others, a systemic immune response, a local immune response, an innate immune response, an adaptive immune response, a memory immune response, a primary and / or secondary immune response, a specific and / or nonspecific immune response, immune cell activation, proliferation, and / or differentiation or the like, or any combinations thereof.

[0104] In some embodiments of the present invention, the immunisation regimen may comprise a single administration. In other embodiments, the immunisation regimen may comprise multiple administrations, either concomitantly or over an appropriate period of time.

[0105] It is envisaged that the appropriate dosage regimen may be repeated for a subject at a suitable time.

[0106] There exists further the possibility to further administer boost immunisations after a more extended period of time. This may be selected as an appropriate measure if a subject’s antibody levels or titre, or B-cell response, falls below determined protective levels. The boost immunisations may be administered if a subject’s immunoglobulin G (IgG) antibody levels fall below determine protective levels. Thus, in some embodiments, an appropriate dosage regimen may be given as a “boost immunisation” after 6 months. In some embodiments of the present invention, the polypeptide, polynucleotide, vector, microorganism, composition or vaccine composition may be administered for the treatment or prevention of infections caused by a virus in combination with one or more other antiviral therapies or other appropriate therapies such as stem cell therapies. Such antiviral therapies may include administration of oseltamivir phosphate (Tamiflu ®), zanamivir (Relenza ®), peramivir (Rapivab ®), baloxavir marboxil (Xofluza ®), or lopinavir / ritonavir (Aluvia ®). Antiviral therapies may include administration of nucleos(t)ide analogues, such as remdesivir (Veklury ®). Other therapies may include, but are not limited to, pegylated interferons, interferon alpha (Intron A), small interfering RNAs (siRNA), tenofovir prodrugs, entry inhibitors, capsid inhibitors, smoothened agonist inhibitors, cccDNA inhibitors, CRISPR-Cas and transcription activator-like effector nucleases, toll-like agonists, STING, second mitochondrial-derived activator of caspases mimetics, and cyclophilin inhibitors. Any combination of these therapies may be administered with the polypeptide, polynucleotide, vector, microorganism, composition, or vaccine composition of the present invention.

[0107] Such therapies may be administered simultaneously, separately, or sequentially with the polypeptide, polynucleotide, vector, microorganism, composition, or vaccine composition of the present invention. In a further embodiment, the antiviral therapy may be administered via the same or different route of administration as the polypeptide, polynucleotide, vector, microorganism, composition, or vaccine composition of the present invention, for example via intradermal injection.

[0108] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the indefinite articles "a" or "an" should be understood to refer to "one or more" of any recited or enumerated component.

[0109] As used herein, "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, "about" can mean a range from the nearest whole number and up to 20%. When particular values are provided in the application and claims, unless otherwise stated, the meaning of "about" should be assumed to be within an acceptable error range for that particular value.

[0110] The identification of the polypeptides comprising conformational epitope sequences disclosed herein was made partly on the development of a machine learning platform trained using bidirectional long short-term memory neural networks (BLSTMs). The platform accurately predicts conformational B cell epitopes (CBCEs) on a given antigen (Ag) from its primary sequence in combination with one or more 3D structure and / or surface characteristics of the Ag, with no requirement for the full 3D structure of the Ag as an input into the predictor. As the platform is trained using BLSTMs, it allows for the processing of the whole Ag sequence as one data-point, processed from both directions without the need for k-mer segmentation. Each amino-acid of theAg is treated as a timestep in the network, whose characteristics can be affected by both preceding and succeeding amino-acids that flank the amino-acid in question, thus, allowing for the discovery of the contextual relationships between spatially distant amino-acids which could be important parts of conformational CBCEs.

[0111] The machine learning platform is capable of discovering separate or independent CBCEs that may exist on a single Ag protein sequence. A feature of the platform is that it takes as input an Ag protein sequence and a binary “permutation vector” or “encoding” representing each single candidate CBCE, as well as the one or more 3D structure and / or surface characteristics of the Ag. The output of the platform is a single probability indicating the likelihood that the input permutation vector is a bone fide CBCE. In other words, the user asks the question to the platform if the specific query CBCE, encoded as a binary permutation vector on the given Ag sequence, is a true CBCE or not. However, the platform may be used to provide a second output in the form of a probability vector with a probability for each amino-acid in the Ag sequence. The second output can be seen as the contribution of each amino acid to the CBCE in question, and may be used to compare the current approach with conventional platforms that predict on an “amino acid by amino acid” basis. The first machine learning algorithm is preferably trained on unbound 3D Ag protein structures prior to the Ag-Ab binding event, to learn the properties of BCEs on the Ag prior to the Ab binding event, in order to capture the bone fide (“true”) BCEs on the query Ag protein sequence.

[0112] Structural characteristics of the Ag sequence are important for predicting CBCEs. However, the platform negates the need for the complete 3D structure of the protein sequence to be experimentally measured or predicted, and then used as input into the algorithm. The machine learning algorithm does not require the coordinates of each atom in each amino acid in the 3D protein sequence (i.e. it does not require the full 3D structure), and instead may predict BCEs using one or more structure and / or surface characteristics of the query protein, such as relative solvent accessibility (RSA), upper half-sphere exposure (HSE), lower halfsphere exposure (LHSE), and secondary structure (SS).

[0113] The “top-level” most important features such as RSA, HSE and SS, may be predicted with one or more trained machine learning algorithms, typically receiving as input the amino acid sequence of the protein. In this way, structural and / or surface characteristics of the protein under investigation may be computed directly from the amino acid sequence, with no requirement for extra information to be sourced or input by the user. Those algorithms use BLSTM networks also and are shown to have a high performance.

[0114] In this way, the machine learning platforms together form a “pipeline” which requires as input only the amino acid sequence of a query protein in order to predict whether that query protein comprises a true B-cell epitope.

[0115] To predict a likely CBCE, the amino acid sequence of a protein to be analysed for the presence of B-cell epitopes is first accessed. Typically, the protein under investigation will comprise an antigen. Various techniques known to the skilled person may be used to access the amino acid sequence, for example the sequence may be downloaded from a bioinformatics repository such as UniProt. Then one or more (e.g. three-dimensional) structure and / or surface characteristics of the protein in an unbound state are accessed. Typically, these characteristics of the protein are predicted using one or more trained machine learning platform(s) that receives the amino acid sequence accessed above as input. The structure and / or surface characteristics typically include the secondary structure of the query protein and relative solvent accessibility. The half sphere exposure (both upper and lower half sphere exposure) may also be used. Values or categories for each of these characteristics are typically assigned to each amino acid of the sequence. However, other techniques for accessing the three-dimensional structure and surface characteristics of the query protein may be employed, such as database entries, X-ray crystallography or computational approaches to predicting full 3D protein folding structures (from which the structure and / or surface characteristics can be obtained).

[0116] Candidate encodings of one or more candidate CBCEs on the query protein are then generated. The candidate encodings are each typically in the form of a binary vector, with each data element in the binary vector corresponding to an amino acid of the amino acid sequence forming the respective candidate CBCE. In such a binary “permutation vector” encoding, each amino acid of the sequence forming part of the candidate CBCE is assigned a “1” and each amino acid of the sequence not forming part of the candidate CBCE is assigned a “0”. For example, for the exemplary amino acid sequence “ABCDEFG”, a candidate conformational B-cell epitope constituted by the amino acids B, D, F and G may be encoded as the binary vector (0101011). In this way, a candidate encoding for each possible candidate CBCE on the protein can be generated. However, in order to reduce computational load, the search space may be limited based on physical priors (e.g. knowledge of a mutation point on a neoantigen of the input protein or receptor binding domain on the spike protein of SARS-CoV-2, etc) or on techniques such as deep reinforcement learning or related generative platforms.

[0117] The amino acid sequence and the one or more structure and / or surface characteristics previously accessed, as well as the candidate encodings generated, are input into a trained machine learning platform, which has been trained using BLSTMs. One or more physiochemical characteristics assigned to each amino acid of the amino acid sequence may also be used as input to the trained machine learning platform. The output of the trained machine learning platform is a probability that each of the candidate encoding(s) represents a true B-cell epitope on the query protein.

[0118] The S protein was chosen as a starting point for the identification of common motifs due to its impact in infection, pathogenicity and transmission, and somewhat conserved nature across coronaviruses. The term “motif’ refers to a common three-dimensional structure that appears in multiple molecules. This protein is exposed on the surface of coronaviruses due to its role in membrane fusion, and this makes it an ideal target for neutralising antibodies. The S protein consists of two subunits, S1 and S2, which are cleaved in two by target cell proteases to activate a membrane fusion domain to facilitate viral entry. S1 contains the receptor binding domain (RBD) which allows cell binding of the virus, such as to the Angiotensin Converting Enzyme 2 (ACE2) receptor for SARS-CoV- 2. Genetic divergence of the S genes is consistent with that of full-length coronavirus genomes, and compared to other regions of the S protein, the RBD is less conserved (Zhu et al. 2021 , J Med Virol. 2021 Oct;93(10):5729-5741). However, identical residues between SARS-CoV-2 and SARS-CoV RBD exist, even in the more variable receptor-binding motif (RBM). For example, the SARS- CoV and SARS-CoV-2 are 73%-76% similar in sequence in their respective RBMs) (Huang et al. 2020, Acta Pharmacol Sin 41 , 1141-1149). Accounting for this and the essential role of the RBD in S-protein function, the RBD constitutes a desirable target to identify common epitopes across Betacoronavirus subgenera to develop therapeutic agents that can facilitate a broad and effective adaptive immune response towards them.

[0119] The invention is now described with reference to the following Examples:

[0120] Example 1 The receptor binding domain (RBD) of SARS-CoV-2 was first mapped to the amino acid sequences of 290 Betacoronavirus species (chosen from Table 2). 450,000 permutations were generated which were then placed inside the RBD of each species (Figure 1). The permutations were then compared to each of the 290 Betacoronavirus species sequences to predict whether that permutation is a true epitope, namely one that is immunogenic, on each species. The result of predicting the immunogenicity of an epitope was a binary output (0 = negative, 1 - positive), which was then filtered to retain predictions with a probability of being an epitope of >90%. The result of this analysis produced 569,230 true epitopes. The results of this prediction process are shown in Table 3:

[0121] Table 3: Total predictions made for the SARS-CoV-2 RBD on the sequences of Betacoronavirus species listed in Table 2. The 0.9 cutoff represents epitopes predicted to be true epitopes at a 90% confidence level.

[0122] The positive permutations were then analysed to see how many Betacoronavirus species each permutation was predicted to be reactive to. Some permutations showed reactivity in over 200 Betacoronavirus species (Figure 2), indicating they were promising candidates for treatments that confer broad immunity to Betacoronaviruses that are both currently infectious as well as those which may become infectious through mutation in the future.

[0123] From this result, the most cross reactive conformational epitopes were identified i.e. those that were predicted to be reactive across the broadest range of species, and subsequently used to design polypeptides and vaccines that in theory should generate the highest immunogenicity to the largest number of Betacoronavirus species. Conformational epitope identification was first accomplished by creating sequence logos from the positive permutations that were reactive to the highest number of Betacoronavirus species. This process predicted the individual amino acids that were involved in creating a conformational epitope against the virus species, which resulted in the identification of the discontinuous sequence “TPGQ” as the conformational epitope that appears in the highest number of Betacoronavirus species. The second most prolific conformational epitope was “DTFFST”. The third most prolific conformational epitope was “FFSTFKATK”. The fourth most prolific conformational epitope was “DSTK”. The next step was to mutate the identified discontinuous sequence to see whether mutations caused the sequence to be immunogenic to more Betacoronavirus species, cause higher immunogenicity in the species it is immunogenic to, or both simultaneously. However, to prepare the sequences for this analysis, the Betacoronavirus species sequences that were not immunogenic to the sequence “TPGQ” were amended by shifting the RBD mapped to them or by manually mutating individual amino acids as to force the sequence “TPGQ” onto the sequences in question. This was done by either mutating the positions of actively contacting amino acids in the permutation in order to match the sequence, or shifting the permutation by -5 / +5 amino acids to check for a match, or both approaches and then choose the pair that leads to fewer mutations per sequence (Figure 3 and Figure 4). This step increased the number of sequences that the conformational epitope appeared on from 193 to all 290 species, which could then act as a common baseline for mutating the sequence “TPGQ”. These steps were repeated for the other conformational epitopes identified (Figures 5-10). It was discovered that mutating the sequence “TPGQ” to “NPGQ” caused a significant increase in the number of species the conformational epitope was reactive to as well as a positive increase in the immunogenicity of said conformational epitopes (Figure 11 and Figure 12).

[0124] This analysis was repeated with the next 3 most cross reactive conformational epitopes: the conformational epitope “DTFFST” was mutated to “DTEFST”; the conformational epitope “FFSTFKATK” was mutated to “SFSTKATK”; the conformational epitope “DSTK” was mutated to “DSKK”. All the mutations caused increases in immunodominance and the number of Betacoronavirus species positive for the conformational epitopes (Figures 13-18). The mutated conformational epitopes were applied in turn to the amino acid sequence of the spike protein of Omicron B.A.2 to produce the amino acid sequences represented by SEQ ID Nos 1 to 4.

[0125] Example 2

[0126] Using the data generated when predicting the positivity of permutations across the 290 Betacoronavirus species (Figure 2), it was possible to predict the total positive permutations each Betacoronavirus species shares with others of the 290 species investigated (Figure 19). The species that shared at least one positive permutation with the most other species were: Pangolin coronavirus, Rhinolophus affinis coronavirus, Betacoronavirus HKU24 and Bat coronavirus RaTG13 (amino acid sequences represented by SEQ ID Nos 5 to 8 respectively). As these species share positive permutations with the highest number of other Betacoronavirus species across the most Betacoronavirus subgenera, they are predicted to be the best candidates for a vaccine that confers immunity to the highest number and variety of Betacoronavirus species.

[0127] Example 3

[0128] To evaluate the breath of protection of vaccines designed using the sequences of the invention, two mRNA vaccines were created, each encapsulating in a lipid nanoparticle SEQ ID NOs: 2 or 3 (the mutated spike sequences) as the antigenic payload. These two vaccines were then tested in BALB / c mice and evaluated the breadth of neutralization of such vaccine for different strains of virus including three strains of SARS-CoV-2 and one strain of SARS-CoV. Sequences were compared to a vaccine designed using a wild-type spike antigen sequences from the ancestral SARS-CoV-2.

[0129] In total, four groups of 9-week-old BALB / c mice (n = 8 per group) were immunized as follows:

[0130] 1 . Negative control: immunization with GFP LNP (data not shown)

[0131] 2. Control: SARS-CoV-2 Spike

[0132] 3. Test Group 2: Predicted mutated epitope 2 (NEC-B-2; SEQ ID NO: 2) 4. Test Group 3: Predicted mutated epitope 3 (NEC-B-3; SEQ ID NO: 3)

[0133] Five days before immunization, a 100 pl blood sample was collected to assess virus-neutralizing antibody titers and exclude pre-existing immunity.

[0134] Vaccines were administered on day 0 and again 28 days later using 10 pg of LNP- formulated mRNA in a total volume of 50 pl via intramuscular injection. On day 42 (two weeks after the second vaccination), the mice were euthanized, and a 600 pl terminal blood sample was collected for neutralization assay. Blood samples were allowed to clot at room temperature for at least 1 hour. Subsequently, they were centrifuged at 2,500 x g for 10 minutes, and the serum was isolated. The sera aliquots were frozen and stored at -75°C ± 10°C until further use.

[0135] In the following neutralisation assay, a two-fold serial dilution series of heat- inactivated sera were prepared in duplicate (starting dilution 1 :10*) and were mixed and incubated with a calculated dose of 100 TCID50 of SARS-CoV-2 or SARS-CoV strain for 1 .5 hours at ambient temperature before infecting adherent VERO-hSLAM (VHS) cells in 96-well plates. After 2 days of incubation, neutralizing antibody titers and virus dose (back titration) were determined using an immunoperoxidase monolayer assay (IPMA) readout, specifically visualizing SARS-CoV-2 Spike protein or SARS-CoV Nucleoprotein. The titer of each mouse serum was defined as the reciprocal (Iog2) value of the highest serum dilution capable of neutralizing the virus (no staining) in 50% of the wells.

[0136] Immunogenicity and protective efficacy were evaluated using a live virus neutralization assay against the following strains:

[0137] • SARS-CoV-2 ancestral strain (Lelystad)

[0138] • SARS-CoV-2 Omicron XBB.1 .5 strain

[0139] • SARS-CoV-2 Omicron BA.5 strain

[0140] • SARS-Cov- HKU39849 BtKY72 Coronavirus strain

[0141] • NeoCov coronavirus strain

[0142] • Pangolin coronavirus GX strain SHC014-CoV strain

[0143] Figures 20-27 show the neutralizing antibody titers in the sera of mice against various coronavirus strains. Each dot represents one mouse, who have been vaccinated with either mRNA LNPs- of the SARS-CoV-2 Ancestral spike, NEC-B- 2 (SEQ ID NO: 2) or NEC-B-3 (SEQ ID NO: 3).

[0144] The ability of NEC-B-2 (SEQ ID NO: 2) and NEC-B-3 (SEQ ID NO: 3) to produce neutralizing antibody titers against the SARS-CoV-2 Lelystad (ancestral) strain in immunised mice is shown in Figure 20, with the induction of antibody responses shown for both constructs.

[0145] Responses in mice against both the SARS-CoV-2 BA.5 and XBB.1.5 Omicron strains (Figures 21 and 22 respectively) showed a major improvement compared with those displayed by mice immunised with the SARS-CoV-2 ancestral spike. Responses were around 10-fold and 25-fold higher for NEC-B-2 (SEQ ID NO: 2) and NEC-B-3 (SEQ ID NO: 3) respectively for the BA.5 strain compared to the SARS-CoV-2 ancestral spike. Immunisation with the SARS-CoV-2 ancestral spike did not produce neutralising antibodies against the XBB.1 .5 strain at all.

[0146] For responses against the SARS-CoV HKU39849 strain, shown in Figure 23, both NEC-B-2 (SEQ ID NO: 2) and NEC-B-3 (SEQ ID NO: 3) produced greatly improved responses compared to the SARS-CoV-2 ancestral spike.

[0147] Figure 24 shows that a major improvement in antibody response was obtained through NEC-B-2 (SEQ ID NO: 2) and NEC-B-3 (SEQ ID NO: 3) vaccination against the BtKY72 strain compared to the SARS-CoV-2 ancestral spike, with the magnitude of improvement being around 9-fold and 8-fold respectively.

[0148] Responses against the Neo-CoV and Pangolin CoV-GX strains were also achieved through NEC-B-2 (SEQ ID NO: 2) and NEC-B-3 (SEQ ID NO: 3) vaccination (Figures 25 and 26 respectively), similar to those achieved by the SARS-CoV-2 ancestral spike. Finally, the responses against the SHC014 coronavirus strain were tested, with both NEC-B-2 (SEQ ID NO: 2) and NEC-B-3 (SEQ ID NO: 3) showing improved outcomes compared to the SARS-CoV-2 ancestral spike, particularly for NEC-B- 3 (SEQ ID NO: 3).

[0149] These results therefore show that the constructs of NEC-B-2 (SEQ ID NO: 2) and NEC-B-3 (SEQ ID NO: 3) can provide a large breath of protection against coronavirus strains in vivo when administered as a vaccine. Therefore, these sequences are proven to be suitable for immunisation to produce a broad adaptive humoral immune response to multiple coronavirus subgenera, species and variants thereof when used in a vaccine composition.

Claims

CLAIMS1 . A polypeptide comprising the amino acid sequences of any one of SEQ ID Nos 1 to 4, or a variant thereof with at least 70% sequence identity, wherein the polypeptide comprises at least the following residues, or, in a variant sequence, residues at equivalent positions: a. N373, P409, G410 and Q411 of SEQ ID No 1 , or b. D361 , T369, E370, F371 , S372 and T373 of SEQ ID No 2, or c. S370, F371 , S372, T373, F374, K375, A381 , T382 and K383 of SEQ ID no 3, or d. D361 , S372, K373 and K375 of SEQ ID no 4.

2. A polypeptide comprising the amino acid sequences of any one of SEQ ID Nos 5 to 8, or a variant thereof with at least 70% sequence identity, wherein the polypeptide is for use in therapy or prophylaxis.

3. The polypeptide, or the variant thereof, according to any preceding claim, comprising any one of SEQ ID Nos 1 to 8, or a sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

4. A polynucleotide encoding a polypeptide as defined in any preceding claim.

5. The polynucleotide according to claim 4, wherein the polynucleotide is DNA.

6. The polynucleotide according to claim 4, wherein the polynucleotide is RNA, preferably mRNA.

7. A vector comprising one or more polynucleotides according to any one of claims 4 to 6, preferably wherein the vector further comprises regulatory elementscapable of driving transcription and / or translation of the one or more polynucleotides in a host cell.

8. The vector according to claim 7, wherein the vector is a viral vector.

9. A microorganism comprising one or more polypeptides according to any one of claims 1 to 3, preferably wherein said microorganism is bacteria.

10. A microorganism comprising one or more polynucleotides according to any one of claims 4 to 6, preferably wherein said microorganism is bacteria.11 . The one or more polynucleotides according to any one of claims 4 to 6 or a vector according to claim 7, formulated in a nanoparticle or lipid formulation.

12. A composition comprising: i) one or more polypeptides as defined in any of claims 1 to 3, or ii) one or more polynucleotides as defined in any of claims 4 to 6.

13. The polypeptide, polynucleotide, vector, microorganism or composition according to any preceding claim, for therapeutic or prophylactic use.

14. The polypeptide, polynucleotide, vector, microorganism or composition according to any one of claims 1 to 13, for use in the treatment or prophylaxis of an infection caused by a Betacoronavirus.

15. The polypeptide, polynucleotide, vector, microorganism or composition for use according to claim 14, wherein the infection is caused by a member of the Embecovirus, Hibecovirus, Merbecovirus, Nobecovirus or Sarbecovirus subgenera, or by MERS-CoV, SARS-CoV-1 , human coronavirus OC43, human coronavirus HKU1 , SARS-CoV-2, or variants thereof.

16. A vaccine composition comprising one or more polypeptides, polynucleotides, vectors or microorganisms according to any one of claims 1 to17. The vaccine composition according to claim 16, wherein the vaccine is an mRNA vaccine.

18. The vaccine composition according to claim 16 or claim 17, further comprising a pharmaceutically acceptable carrier, diluent, excipient and / or adjuvant.

19. The vaccine composition according to any one of claims 16 to 18, wherein said vaccine composition is formulated for a parenteral, oral, sublingual, nasal, naso-oral, or pulmonary administration.

20. The vaccine composition according to claim 19, wherein said parenteral administration is subcutaneous, intradermal, intramuscular, subdermal, intraperitoneal, or intravenous administration.

21. The vaccine composition according to claim 20, wherein said vaccine composition is for administration to a subject via one or more intramuscular injections.

22. The vaccine composition according to claim 20, wherein said vaccine composition is for administration to a subject via one or more intradermal injections.

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