Modified chimeric coronavirus spike protein for enhancing viral titer

Abstract

The present disclosure relates to the production of viral particles, in particular adenoviral particles, for use in vaccines. Modified chimeric coronavirus spike antigens are disclosed along with polynucleotides encoding the antigens, viral particles, and methods of producing / formulating the viral particles.

Description

[Technical field]

[0001] The present invention relates to constructs and methods used to improve the production of viral particles, particularly viral particles expressing antigens for use in vaccines. The present invention particularly relates to modified antigens, in which mutations have been made to the antigen in order to target the antigen to the plasma membrane and / or to reduce membrane fusion / syncytium formation.

[0002] For example, the present invention relates to modified forms of the SARS-CoV-2 spike protein. The present invention provides polynucleotides encoding such modified spike proteins, together with viral particles comprising the polynucleotides and pharmaceutical compositions / vaccines comprising the viral particles. The present invention also relates to methods of producing the viral particles and vaccines. [Background technology]

[0003] SARS-CoV-2 (also known as COVID-19 or 2019-nCoV) has become a major global health problem since cases first emerged in late 2019. As of March 2021, there have been over 120 million cases and over 2.7 million deaths worldwide. Vaccination against SARS-CoV-2 is considered critical to overcoming the pandemic.

[0004] Many vaccines have been approved, including viral vector vaccines, nucleic acid vaccines (mRNA and DNA), subunit vaccines, virus-like particle vaccines, inactivated vaccines, and attenuated vaccines, with others in development. Vaccines typically utilize the SARS-CoV-2 spike protein as the antigen.

[0005] There remains a need to provide not only vaccines with enhanced immunogenicity, but also vaccines that can be produced more efficiently to meet the global demand. This applies not only to vaccines targeting SARS-CoV-2 but also other viral infections. Summary of the Invention

[0006] The present invention provides polynucleotides encoding antigens derived from viruses, where the antigens have been modified to (i) target the antigen to the plasma membrane and / or (ii) reduce membrane fusion and / or syncytia formation.

[0007] The present invention also provides a viral vector comprising a polynucleotide of the present invention.

[0008] The present invention further provides polypeptides encoded by the polynucleotides of the present invention and viral particles comprising the polynucleotides of the present invention.

[0009] The present invention also provides a vaccine comprising the viral particle.

[0010] Finally, the present invention provides a method for producing a viral particle of the present invention, comprising culturing a host cell containing a vector of the present invention under conditions allowing the production of virus, and recovering the virus produced by the host cell. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows an adenoviral vector of the invention encoding a modified SARS-CoV-2 spike protein of the invention. [Diagram 2] Prism graph showing GEN1 and GEN3 yields (IFU / ml) in infected HEK-293 cells (each flask contained the same cell density, was infected at the same MOI, and harvested at the same time). GEN1 and GEN3 titers were measured using AdenoX (Takara kit). Bars represent the average titers from three AdenoX dilutions. Note. AdenoX was performed in a 96-well plate format using a 10-fold dilution series. [Diagram 3] FIG. 1 shows immunological responses (IgG) against GEN1 and GEN3 antigens.

[0012] Brief Description of the Sequence Listing SEQ ID NO:1 shows the sequence of the SARS-CoV-2 spike protein from UniProt entry P0DTC2. SEQ ID NO:2 shows the IBV canonical dilysine endoplasmic reticulum retrieval signal. SEQ ID NO:3 shows the sequence of the IBV cytoplasmic tail. SEQ ID NO: 4 shows the coronavirus dibasic motif sequence. SEQ ID NO:5 shows the IBV tail in which the di-lysine motif has been replaced with an alanine. SEQ ID NO:6 shows the IBV tail in which the di-lysine motif is replaced with an alanine and the tyrosine in the internalization motif is replaced with an alanine. SEQ ID NO:7 shows an exemplary cytoplasmic tail sequence. SEQ ID NO:8 shows an exemplary cytoplasmic tail sequence. SEQ ID NO: 9 shows the complete vector sequence (nucleic acid) of FIG. SEQ ID NO: 10 shows the aromatic stretch of SARS-CoV-2. SEQ ID NO:11 shows an exemplary aromatic stretch. SEQ ID NO:12 shows an aromatic stretch of SEQ ID NO:11 in which tryptophan has been replaced with alanine. SEQ ID NO: 13 shows an exemplary aromatic stretch. SEQ ID NO:14 shows a modified version of SEQ ID NO:13. SEQ ID NO:15 shows a modified version of SEQ ID NO:13. SEQ ID NO: 16 shows an exemplary sequence of the invention having modifications of the cytoplasmic tail and transmembrane region. SEQ ID NO: 17 shows the SARS-CoV-2 spike protein sequence of the present invention. SEQ ID NO: 18 shows the optimized SARS-CoV-2 spike protein sequence of FIG. 1 including the modified STM-IBV tail. SEQ ID NO: 19 shows the SwTMF2aEnv4b_1_F sequence of FIG. SEQ ID NO: 20 shows the SwTMF2aEnv4b_1_R sequence of FIG. SEQ ID NO: 21 shows the SwTMF2aEnv4b_2_F sequence of FIG. SEQ ID NO: 22 shows the SwTMF2aEnv4b_2_R sequence of FIG. SEQ ID NO: 23 shows the SwTMF2aEnv4b_3_F sequence of FIG. 1. SEQ ID NO: 24 shows the SwTMF2aEnv4b_3_R sequence of FIG. SEQ ID NO: 25 shows the SwTMF2aEnv4b_4_F sequence of FIG. 1. SEQ ID NO: 26 shows the SwTMF2aEnv4b_4_R sequence of FIG. 1. SEQ ID NO: 27 shows the SwTMF2aEnv4b_5_F sequence of FIG. 1. SEQ ID NO: 28 shows the SwTMF2aEnv4b_5_R sequence of FIG. 1. SEQ ID NO: 29 shows the SwTMF2aEnv4b_6_F sequence of FIG. 1. SEQ ID NO: 30 shows the SwTMF2aEnv4b_6_R sequence of FIG. 1. SEQ ID NO: 31 shows the SwTMF2aEnv4b_7_F sequence of FIG. 1. SEQ ID NO: 32 shows the SwTMF2aEnv4b_7_R sequence of FIG. 1. SEQ ID NO: 33 shows the SwTMF2aEnv4b_8_F sequence of FIG. 1. SEQ ID NO: 34 shows the SIBV_FOR sequence of FIG. SEQ ID NO: 35 shows the SwTMF2aEnv4b_8_R sequence of FIG. 1. SEQ ID NO: 36 shows the SIBV_REV sequence of FIG. SEQ ID NO: 37 shows the V5 tag of FIG. SEQ ID NO: 38 shows the AmpR promoter and gene sequence of FIG. SEQ ID NO:39 shows the reverse strand of SEQ ID NO:9. SEQ ID NO: 40 shows the polypeptide sequence of AmpR in FIG. SEQ ID NO:41 shows the Factor Xa site of FIG. SEQ ID NO:42 shows SEQ ID NO:16 but without the initial KT residue. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] It is to be understood that different applications of the disclosed products and methods may be tailored to the particular needs of the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0014] Moreover, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0015] Thus, for example, reference to "an amino acid sequence" includes two or more such sequences, and so forth.

[0016] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0017] Polynucleotides and Polypeptides The present invention provides a polynucleotide encoding an antigen from a virus, the antigen being modified to target the antigen to the plasma membrane and / or to reduce membrane fusion / syncytium formation. The virus may be any virus where infection proceeds by fusion of the viral membrane with the membrane of the target cell. The virus may be an enveloped virus. The virus may be a virus that forms syncytia.

[0018] In the present invention, the virus is typically a coronavirus (particularly a SARS coronavirus, more particularly SARS-CoV-2). The virus may also be of the family Orthomyxoviridae, Paramyxoviridae, Filoviridae, Retroviridae, Togaviridae, Rhabdoviridae, Arenaviridae, Flaviviridae, Bunyaviridae, Poxviridae, Hepadnaviridae or Herpesviridae. The virus may be, for example, influenza virus, respiratory syncytial virus (RSV), HIV, herpes simplex virus (HSV), Ebola virus, human T-cell leukemia virus (HTLV), West Nile virus, dengue virus, or measles morbillivirus (MeV). As discussed further below, the present invention typically relates to SARS-CoV-2, although the modifications disclosed herein may be applied to other viruses.

[0019] Targeting of antigen to plasma membrane may be determined using techniques known in the art, such as fluorescent labeling and visualization of antigen. The presence of antigen in plasma membrane increases compared to unmodified antigen. Similarly, membrane fusion or syncytium formation may be observed using standard techniques appropriate for the virus in question.

[0020] In the present invention, as discussed below, targeting to the plasma membrane is typically increased by modification of the cytoplasmic end of the antigen, and reduction of membrane fusion / syncytia formation is typically achieved by substituting tryptophan residues in the antigen. Such modifications are discussed below with respect to the SARS-CoV-2 spike protein. However, such modifications may be equally applied to antigens from other viruses.

[0021] The antigen is typically a viral membrane fusion protein. Three different classes of viral membrane fusion proteins have been identified based on structural criteria. The antigen can be, for example, a class I, class II, or class III fusion protein.

[0022] In some instances where the virus is an orthomyxoviridae, the antigen may be a hemagglutinin (HA) antigen. For retroviridae, the antigen may be Env (a subunit thereof). For paramyxoviridae, the antigen may be F or HN. For coronoviridae, the antigen may be the S protein. For filoviridae, the antigen may be the GP protein. For arenaviridae, the antigen may be GP or SSP. For togaviridae, the antigen may be E1 / E2. For Flaviviridae, the antigen may be E or E1 / E2. For bunyaviridae, the antigen may be G or HN. N / G C For rhabdoviridae, the antigen can be G. For herpesviridae, the antigen can be gB, gD, or gH / L. For hepadnaviridae, the antigen can be S or L.

[0023] The antigen may be RSF-V, influenza HA, West Nile Virus / Dengue E or HIV gp41.

[0024] In the present invention, the antigen is typically a coronavirus spike antigen (S), preferably a SARS-CoV-2 spike antigen. Coronaviruses are enveloped viruses that assemble at the intracellular membrane of the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) of infected cells. Coronaviruses are typically classified into groups 1, 2 or 3 by sequence homology.

[0025] Coronaviruses use the homotrimeric spike glycoprotein (S protein) on their envelope to bind to coronavirus cellular receptors. Such binding triggers a cascade of events that results in fusion between the cellular and viral membranes for cell entry. The SARS-CoV-2 spike protein is thought to bind to the ACE2 receptor on target cells.

[0026] Each monomer of the spike protein is believed to contain a subunit called S1, which functions to bind to a receptor on a target cell, and a subunit called S2, which functions to fuse the viral and host cell membranes. The S1 region contains the N-terminal domain (NTD) and the receptor binding domain (RBD). The S2 region then contains the fusion peptide (FP), heptad repeats (HR1 and 2), the transmembrane domain (TM), and the cytoplasmic tail (CT). A protease cleavage site is present at the interface between the S1 and S2 subunits.

[0027] The sequence of the SARS-CoV-2 spike protein from UniProt entry P0DTC2 is as follows: JPEG2024511213000001.jpg167158

[0028] A number of variants of the spike protein have already been identified, including D614G, which may be associated with increased viral load. Other mutations include Q493N, Q493Y and N501T.

[0029] Below is an overview of the main spike variants as of March 2022. [Table 1]

[0030] Although the present invention is generally described with respect to the above sequences, the modifications described below may equally apply to other variants of spike protein. Such variants may have, for example, 80, 90, 95, 96, 97, 98, or 99% amino acid sequence identity with the above sequences over the length of the sequence. Such variants may also have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid substitutions, deletions, or insertions compared to the above sequences (in addition to the changes described below).

[0031] Where residue numbering is provided for the above sequences, one of skill in the art will be able to readily identify the corresponding residues in variant sequences using standard techniques, such as the use of sequence alignment algorithms.

[0032] With respect to amino acid sequences, "sequence identity" refers to sequences having the recited value when assessed using ClustalW (Thompson et al., 1994, supra) with the following parameters: Pairwise alignment parameters-method: exact, matrix: PAM, gap opening penalty: 10.00, gap extension penalty: 0.10; Multiple alignment parameters - matrix: PAM, gap open penalty: 10.00, % identity delayed: 30, penalize end gaps: on, gap separation distance: 0, negative matrix: no, gap extension penalty: 0.20, residue specific gap penalty: on, hydrophilic gap penalty: on, hydrophilic residues: G / P / S / N / D / Q / E / K / R. Sequence identity at specific residues is intended to include identical residues that have simply been derivatized.

[0033] Previous studies have shown that coronavirus S proteins are concentrated in the Golgi region, but some S proteins appear to be transported to the cell surface.

[0034] We investigated modifications of the SARS-CoV-2 S protein to promote its expression and retention at the cell membrane (to enhance immunogenicity). First, we mutated the cytoplasmic tail.

[0035] Lontok et al. (2004) Journal of Virology, 78, 5913-5922 discuss the intracellular targeting signals that contribute to the localization of the coronavirus spike protein. In particular, Lontok et al. demonstrated that the S protein from the group 3 coronavirus infectious bronchitis virus (IBV) contains a canonical dilysine endoplasmic reticulum retrieval signal (-KKXX-COOH; SEQ ID NO: 2) at its cytoplasmic end. The IBV cytoplasmic end is encoded by the sequence JPEG2024511213000003.jpg11144 The di-lysine motif is highlighted and a potential tyrosine internalization signal is italicized, which causes retention of the protein in the ERGIC but, when mutated, allows transport of the S protein to the plasma membrane.

[0036] Such dibasic motifs have also been shown to be present in other types of coronaviruses (eg, KXHXX-COOH (SEQ ID NO: 4) in the S protein from group 1 coronaviruses).

[0037] As shown above, the SARS-CoV-2 spike protein contains a KXHXX motif at the C-terminus (positions 1269-1273).

[0038] The present invention provides polynucleotides encoding SARS-CoV-2 spike proteins (or antigens from alternative viruses) in which the cytoplasmic tail is modified to target the protein to the plasma membrane. For example, the polynucleotide may encode an antigen (SARS-CoV-2 spike protein) in which a dibasic motif in the cytoplasmic tail is mutated to another amino acid residue. The dibasic motif is typically mutated to an alanine residue, but can be, for example, a conservative substitution for alanine. For example, the dibasic motif can be mutated to valine, glycine, leucine, or isoleucine (or any combination of A, V, G, L, or I).

[0039] The dibasic motif may be a KXHXX motif of the above sequence in which the K and H residues are mutated, typically to alanine, as described above.

[0040] In some instances, the motif may also be a tribasic or polybasic motif, for example having additional basic amino acid residues. Basic amino acids are lysine, arginine and histidine.

[0041] For example, influenza hemagglutinin contains a tribasic motif. Such substitutions can be for any amino acid residue that has the desired function. Such residues are typically mutated to alanine (or conservative alanine substitutions as described above).

[0042] In the present invention, the polynucleotide preferably encodes an antigen (SARS-CoV-2 spike protein), in which the cytoplasmic tail is replaced with the cytoplasmic tail from IBV, and the di-lysine motif (positions -3 and -4) is replaced with another amino acid. The amino acid is typically an alanine residue, but can be, for example, the conservative alanine substitutions (any combination) described above. The sequence of such a cytoplasmic tail is shown below: JPEG2024511213000004.jpg11144

[0043] In some instances, the two tyrosine residues in the putative tyrosine internalization sequence may be specifically mutated to alanine residues (or optionally any combination of V, G, L, or I). Sequences with these modifications are shown below. JPEG2024511213000005.jpg11144

[0044] In some instances, the cytoplasmic tail may contain further modifications to the sequence presented above (typically retaining the alanine mutations above). Modifications may be substitutions, deletions or insertions.

[0045] For example, the cytoplasmic terminus may include up to 10 additional modifications or up to 5 additional modifications. In some examples, the cytoplasmic terminus includes 1, 2, 3, 4, or 5 modifications. In some examples, the substitution is a conservative substitution. For example, an amino acid may be replaced with an alternative amino acid having similar properties, such as another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid. Some properties of the 20 main amino acids that can be used to select suitable replacements are as follows: [Table 2]

[0046] The amino acids used in the sequences of the invention may also be derivatized or modified, e.g., labeled.

[0047] In some instances, the cytoplasmic tail has at least 90% identity or at least 95% identity over the length of the sequence with the above sequence, the percent identity being as described above. Again, the alanine mutations described above are typically retained.

[0048] In some instances, the cytoplasmic tail comprises the following sequence: JPEG2024511213000007.jpg11150 or JPEG2024511213000008.jpg11168

[0049] The cytoplasmic terminus may also be as shown in Figure 1 (from positions 1124 to 1167 of STM-IBV).

[0050] In some instances, the cytoplasmic tail may comprise a variant of the above sequence (or the sequence shown in Figure 1). Such variants are as described above. Such variants typically retain AA residues (or alternative substitutions) at positions -3 and -4, and retain an AA residue (or alternative substitution) in the putative tyrosine internalizing sequence.

[0051] The polynucleotides of the invention may also encode additional mutations in the antigen, such as mutations designed to reduce / prevent membrane fusion and / or syncytium formation when the protein is present on the host cell surface, thus enhancing antigen availability. Such mutations are described for SARS-CoV in Corver et al. (2009), Virology Journal, 6.

[0052] As described in Corver et al., the TM domain of the SARS-CoV spike protein consists of three domains: (1) a highly conserved N-terminal aromatic (tryptophan)-rich stretch, (2) a hydrophobic core sequence, and (3) a C-terminal cysteine-rich domain. These domains are highly conserved in all coronaviruses. The aromatic stretch of the TM domain is particularly thought to be involved in viral entry into target cells, and Corver et al. investigated the effects of mutations in this region on membrane fusion and viral entry.

[0053] In the present invention, the polynucleotide may code for mutations in the antigen, in particular in the TM domain of the antigen. These mutations may, for example, be in the aromatic stretch of the TM domain. The aromatic stretch of SARS-CoV-2 occurs from residues 1209 to 1217 (YIKWPWYIW; SEQ ID NO: 10).

[0054] In some instances, the aromatic stretch is deleted. In other instances, the tryptophan residues in the aromatic stretch are replaced with other amino acid residues. The replacement amino acid may be, for example, phenylalanine. Preferably, the replacement amino acid is alanine (or a conservative substitution such as V, G, L or I). All combinations are contemplated.

[0055] In some instances, a single tryptophan residue is mutated. Preferably, at least two tryptophan residues are mutated. More preferably, all three tryptophan residues are mutated. As above, the substituted amino acid is preferably alanine, but may be any suitable amino acid (in some instances, V, G, L, or I).

[0056] For example, as described above, the aromatic stretch of SARS-CoV-2 occurs from residues 1209 to 1217 of SEQ ID NO:1 (YIKWPWYIW; SEQ ID NO:10).

[0057] In some examples, a polynucleotide of the invention may encode a protein in which residues 1209 to 1217 of the above sequence (SEQ ID NO:1) are deleted.

[0058] In some examples, the polynucleotides of the invention encode a W1212 mutation, typically a W1212A mutation (numbering according to the SARS-CoV-2 sequence above). In some examples, the polynucleotides of the invention encode a W1214 mutation, typically a W1214A mutation. In some examples, the polynucleotides of the invention encode a W1217 mutation, typically a W1217A mutation. In some examples, the polynucleotides of the invention encode mutations at two or three positions (e.g., W1212A / W1214A, W1212A / W1217A, or W1214A / W1217A). Typically, the polynucleotides encode mutations at all three of these positions in the above sequence (e.g., W1212A / W1214A / W1217A). As mentioned above, the substituted amino acid is typically an alanine, but may be a conservative alanine substitution (all combinations are contemplated).

[0059] In the present invention, the aromatic stretch WXWXXW (SEQ ID NO: 11) may be replaced with AXAXXA (SEQ ID NO: 12) (or conservative substitutions of A residues in any combination). For example, the sequence WPWYIW (SEQ ID NO: 13) may be replaced with APAYIA (SEQ ID NO: 14) or in some instances APAYVA (SEQ ID NO: 15).

[0060] A polynucleotide of the invention may encode an antigen having the cytoplasmic tail sequence and the transmembrane domain alterations described above. In addition, other alterations may be present, also as described above.

[0061] In one example, the antigen (specifically the SARS-CoV-2 spike protein antigen) may be JPEG2024511213000009.jpg14157 or JPEG2024511213000010.jpg14157 SEQ ID NO: 42 is the same as SEQ ID NO: 16, but does not include the initial KT residue. In some examples, the polynucleotide encodes a sequence from position 1094 of FIG. 1 (designated STM-IBV). In some examples, the antigen may also include variants of these sequences. Such variants may, for example, have at least 90% identity, or may be variants having up to 10, and in some examples up to 5, deletions, substitutions or additions (see above). Variant sequences typically retain the AXAXXA (SEQ ID NO: 12) motif (or alternative substitutions) described above, as well as AA residues (or alternative amino acids) at positions -3 and -4. Variants also typically retain AA residues (or alternative amino acids) in the tyrosine internalization sequence. Again, the A residues may, for example, be A, V, G, L or I (any combination).

[0062] In some examples, the polynucleotide of the invention encodes a SARS-CoV-2 spike protein comprising the following sequence: JPEG2024511213000011.jpg168160

[0063] In some examples, the polynucleotide encodes the spike protein (SEQ ID NO:18) shown in FIG. 1 (including the STM-IBV region).

[0064] In some examples, the polynucleotide encodes a spike protein having at least 90% identity to the sequence above or the sequence of FIG. 1. Percent identity is also described above. In other examples, the polynucleotide encodes a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid substitutions, deletions, or insertions. In all cases, the variant sequence typically retains the AXAXXA (SEQ ID NO: 12) motif, the AA substitutions in the tyrosine internalization sequence, and the AA residues at positions -3 and -4 of the sequence. Again, substitutions other than A are contemplated (see above).

[0065] The variant sequences of the invention preferably retain any advantageous properties of the parent sequence. For example, as discussed below, the modified antigens of the invention preferably provide increased viral titers when compared to the unmodified sequence.

[0066] In some examples, the polynucleotide of the invention may be as shown in FIG.

[0067] The polynucleotides of the invention may be RNA or DNA, but are typically DNA.

[0068] The polynucleotide sequence encoding the antigen of the present invention can be determined by methods well known to those skilled in the art.

[0069] The invention also provides polypeptides encoded by the polynucleotide sequences of the invention. For example, the invention provides polypeptides of the spike protein sequences set out above or the spike protein of Figure 1. As noted above, the polypeptides may be variants of these sequences.

[0070] Vectors and viral particles The present invention provides a viral vector comprising a polynucleotide of the present invention together with a viral particle carrying the polynucleotide / vector.

[0071] The present invention is typically carried out using adenovirus particles / vectors.However, other suitable viruses are adeno-associated viruses, lentiviruses and oncolytic viruses, such as HSV and vaccinia viruses.The adenovirus is preferably serotype 5, such as human serotype 5.

[0072] In the vectors of the invention, the polynucleotide of the invention is inserted into a viral backbone vector, preferably an adenoviral vector. The adenoviral vectors used in the invention may be either replication-competent or replication-incompetent. Such vectors are well known. For example, in replication-deficient vectors, the E1 region may be deleted and replaced with an expression cassette with an exogenous promoter driving the expression of a heterologous antigen. Usually, the E3 region is also deleted. The deletion of E3 allows for a larger insert into the E1 region. As discussed below, such vectors may be propagated in a suitable cell line, such as HEK293 cells, which retain and express E1A and E1B proteins. Later generation vectors also lack the E4 region, and some vectors additionally lack the E2 region. E2 and E4 vectors must be propagated on a cell line that complements the E1, E4 and E2 deletions.

[0073] The vector may also be a helper-dependent vector that lacks most or all adenoviral genes but retains cis-acting sequences such as inverted terminal repeats, as well as packaging sequences necessary for the genome to be packaged and replicated. These vectors are propagated in the presence of the helper adenovirus, which must be eliminated. Again, such systems are well known in the art.

[0074] In the present invention, the adenoviral vector is typically a replication-deficient vector (e.g., an E1 / E3 deleted vector (ΔE1, ΔE3)), but the vector can also be helper-dependent or replication-competent. Replication-competent vectors are well known. One skilled in the art will be able to readily select an appropriate vector system.

[0075] The expression of the transgene can be driven via operably linking to a suitable promoter. The vectors used in the present invention may use other suitable expression control elements such as enhancers and regulators. Enhancers are broadly defined as cis-acting agents that increase the transcription of a gene sequence when operably linked to a promoter / gene sequence. Enhancers can function from positions much farther away from the sequence of interest than other expression control elements (e.g., promoters) and may operate when placed in either orientation relative to the sequence of interest. Enhancers have been identified from several viral sources, including adenovirus.

[0076] Adenovirus vectors may use highly active promoters such as the cytomegalovirus (CMV) promoter, the SV40 large T antigen promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (MLP), the mouse mammary tumor virus LTR promoter, or the SV40 early promoter.

[0077] The vector of the present invention may be produced using any suitable method known in the art. For example, the polynucleotide of the present invention may be introduced into the vector using homologous recombination between a backbone vector and a second vector (e.g., a shuttle plasmid) containing the polynucleotide. Such homologous recombination can be achieved using commercially available products, kits and protocols. Alternatively, a cassette carrying the polynucleotide of the present invention can be directly inserted (cloned) into the vector without the need for homologous recombination. Again, such techniques are known in the art.

[0078] Where the antigen is the SARS-CoV-2 spike protein, the vector of the invention may further encode other SARS-CoV-2 antigens, such as the SARS-CoV-2 nucleocapsid.

[0079] The vector of the present invention is shown in FIG.

[0080] Method for producing virus particles The present invention also provides a method for preparing / manufacturing viral particles. The method includes culturing a host cell containing the vector of the present invention under conditions that allow virus production and replication, and harvesting the virus produced by the host cell. The method may also include formulating the particles into a composition. The method may also optionally include drying the composition and packaging the composition into a capsule tablet for oral administration to a patient. These methods are further described below.

[0081] The vectors of the present invention may be introduced into host cells using any suitable technique. When the virus is an adenovirus, suitable cells include human embryonic kidney (HEK) 293 cells or HEK293 derived cell clones, appropriate for the type of adenovirus being used. The cells may be cultured under any suitable conditions that result in the production of viral particles. The culture may include an initial production phase, followed by an amplification phase.

[0082] Harvesting and purification of the virus may be accomplished by methods known in the art. For example, the virus may be cultured in HEK293 cells and purified by double cesium chloride density gradient centrifugation followed by column desalting (e.g., PD-10) into an excipient formulation, which is described below. The formulation may then be dried as described below.

[0083] The modified antigens of the present invention preferably result in an increased viral titer after culture in a host cell compared to an antigen that does not contain the modifications of the present invention. For example, the examples of the present application show that the use of the modified SARS-CoV-2 spike protein of the present invention resulted in a 40-fold increase in viral titer. Viral titer may be determined using methods known in the art.

[0084] In some examples, the modified antigens of the present invention may result in a 10-fold, 20-fold, 30-fold or 40-fold increase in viral titer.

[0085] Pharmaceutical Compositions / Vaccines The present invention also provides a pharmaceutical composition comprising a viral particle of the invention. The pharmaceutical composition is in particular a vaccine.

[0086] Pharmaceutical compositions are usually sterile and typically include a pharma- ceutically acceptable carrier and / or adjuvant. As used herein, a "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The pharmaceutical composition / vaccine may be suitable for parenteral administration, e.g., by injection or infusion, e.g., intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration. Alternatively, the carrier may be suitable for non-parenteral administration, e.g., topical, epidermal or mucosal routes of administration. The carrier may be particularly suitable for oral administration.

[0087] Pharmaceutical compositions for oral administration of viral particles are described in WO 2018 / 127702. As described in WO 2018 / 127702, the viral particles are formulated with excipients that provide thermal stability, including stabilizing the viral particles against damage caused by freezing, lyophilization, and thawing.

[0088] In the present invention, the viral particles may be formulated in a composition comprising a compound of formula (I) or a physiologically acceptable salt or ester thereof and / or a compound of formula (II) or a physiologically acceptable salt or ester thereof, and optionally one, two or more sugars. [ka]

[0089] The viral particles are typically contacted with a compound of formula (I) or a physiologically acceptable salt or ester thereof and / or a compound of formula (II) or a physiologically acceptable salt or ester thereof, and optionally one or more sugars in an aqueous solution, and then the resulting solution in which the viral particles are present is dried to form a composition incorporating the viral particles.

[0090] Thus, the viral particles may be mixed with an aqueous solution of a compound of formula (I) or a physiologically acceptable salt or ester thereof and / or a compound of formula (II) or a physiologically acceptable salt or ester thereof, and optionally one or more sugars. The resulting solution is then dried to form a composition incorporating the viral particles. The dried composition may take the form of a cake or a powder. The cake may be ground into a powder if necessary.

[0091] The virus particles are stored in the aqueous solution prior to the drying step, which allows the aqueous solution to be stored after preparation for a time period during which the drying step can be carried out without undue loss of virus activity.

[0092] The compounds of formulae (I) and (II) may exist as their physiologically acceptable salts or esters.

[0093] Salts are typically salts with a physiologically acceptable acid and thus include those formed with inorganic acids such as hydrochloric or sulphuric acid, or organic acids such as citric, tartaric, malic, maleic, mandelic, fumaric or methanesulphonic acid. Hydrochloride salts are preferred.

[0094] The esters are typically 1-6 Alkyl esters, preferably C 1-4 It is an alkyl ester. Thus, the ester may be a methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl ester. The ethyl ester is preferred.

[0095] As used herein, C 1-6 The alkyl group is preferably C 1-4 Alkyl groups. Preferred alkyl groups are selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl. Methyl and ethyl are particularly preferred.

[0096] For the avoidance of doubt, the definition of the compounds of formula (I) and formula (II) also includes compounds in which the carboxylate anion is protonated to give -COOH and the ammonium or sulfonium cation is associated with a pharma- ceutically acceptable anion.Further, for the avoidance of doubt, the compounds defined above may be used in any tautomeric or enantiomeric form.

[0097] Compounds of formula (I) Typically, R1 is hydrogen or C 1-6 R represents hydrogen. Typically, R represents hydrogen or C 1-6 Preferably, R1 is hydrogen or C 1-6 R represents hydrogen; R represents hydrogen or C 1-6 More preferably, R1 represents hydrogen or C 1-6 R represents hydrogen; and R represents C. 1-6 Represents alkyl.

[0098] Preferably, the compound of formula (I) is 1-6 Alkyl-, N,N-di(C 1-6 Alkyl)- or N,N,N-tri(C 1-6 alkyl)-glycine or a physiologically acceptable salt or ester thereof, more preferably N,N-di(C 1-6 Alkyl)- or N,N,N-tri(C 1-6 The alkyl group is typically C 1-4Alkyl groups. Preferred alkyl groups are selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl. Methyl and ethyl are particularly preferred.

[0099] The preferred compounds of formula (I) are N-methylglycine, N,N-dimethylglycine or N,N,N-trimethylglycine or physiologically acceptable salts or esters thereof. N-methyl-glycine is also called sarcosine. N,N-dimethylglycine is also called dimethylglycine (DMG) or 2-(dimethylamino)-acetic acid. N,N,N-trimethylglycine is called trimethylglycine (TMG). The most preferred compound of formula (I) is DMG.

[0100] Alternatively, the compound of formula (I) is typically a glycine derivative of formula (IA) or a physiologically acceptable salt or ester thereof. [ka] (Wherein, R5 and R6 are independently C 1-6 Alkyl, e.g., methyl or ethyl, 1-4 alkyl, R7 is C 1-6 Alkyl, e.g., methyl or ethyl, 1-4 Alkyl, or -(CH2) 2-5 NHC(O)(CH2) 5-15 CH3. Preferred compounds of formula (IA) are trimethylglycine (TMG) and cocamidopropyl betaine (CAPB) or a physiologically acceptable salt or ester thereof. Trimethylglycine is preferred.

[0101] Alternatively, the compound of formula (I) is typically a proline derivative of formula (IB) or a physiologically acceptable salt or ester thereof. [ka] (Wherein, R8 and R9 are independently C1-6 Alkyl, e.g., methyl or ethyl 1-4 Preferably, R8 and R9 both represent methyl. This compound is known as proline betaine. S-proline betaine or a physiologically acceptable salt or ester thereof is particularly preferred. [ka]

[0102] Compounds of formula (IA) or a physiologically acceptable salt or ester thereof are preferred.

[0103] Preferably, the compound of formula (I) is N,N-dimethylglycine or N,N,N-trimethylglycine or a physiologically acceptable salt or ester thereof. Most preferably, the compound of formula (I) is N,N-dimethylglycine or a physiologically acceptable salt or ester thereof.

[0104] Compound of formula (II) Typically, R c The carboxylate and amine substituents of R c are attached to the same carbon atom of the alkyl moiety. Typically, R c is C 2-4 Or C 2-3 It is an alkyl portion.

[0105] The compound of formula (II) is typically a sulfone compound of formula (IIA) or a physiologically acceptable salt or ester thereof. [ka] (In the formula, R c and R d is independently 1-6 Alkyl, e.g. C 1-4represents alkyl.) Preferred alkyl groups are selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl. Methyl and ethyl are particularly preferred. The most preferred sulfone compound is methylsulfonylmethane (MSM), also known as dimethylsulfone (DMSO2).

[0106] The compound of formula (II) may be a compound of formula (IIB) or a physiologically acceptable salt or ester thereof. [ka] (In the formula, R e and R f is independently 1-6 Alkyl, e.g., methyl or ethyl, 1-4 represents alkyl, R g is a C substituted with a carboxylate anion and an amine (-NH2) moiety. 1-6 Alkyl, e.g., methyl or ethyl, 1-4 (IIB) represents an alkyl group.) Preferably, the carboxylate and amine substituents are attached to the same carbon atom. A preferred compound of formula (IIB) is S-methyl-L-methionine (SMM) or a physiologically acceptable salt or ester thereof.

[0107] Most preferably, in the present invention, the compound of formula (I) is DMG or a physiologically acceptable salt or ester thereof, and the compound of formula (II) is MSM or a physiologically acceptable salt or ester thereof.

[0108] sugar Sugars suitable for use in the present invention include reducing sugars such as glucose, fructose, glyceraldehyde, lactose, arabinose and maltose, preferably non-reducing sugars such as sucrose and raffinose, more preferably sucrose. Sugars may be monosaccharides, disaccharides, trisaccharides or other oligosaccharides. The term "sugar" includes sugar alcohols. Thus, in one embodiment, the use of non-reducing sugars or sugar alcohols is preferred.

[0109] Monosaccharides such as galactose and mannose; dissaccharides such as sucrose, lactose and maltose; trisaccharides such as raffinose; tetrasaccharides such as stachyose are contemplated. Trehalose, umbelliferose, verbascose, isomaltose, cellobiose, maltulose, turanose, melezitose and melibiose are also suitable for use in the present invention. A preferred sugar alcohol is mannitol. The use of mannitol can result in cakes with improved appearance upon freeze drying.

[0110] The presence of sugar may act to improve stability. The addition of sugar may also provide other benefits, such as altered lyophilization cake for more rapid reconstitution and improved solubility. Generally, when lyophilization is used, one or more sugars are present. When one sugar is used, the sugar is preferably sucrose or mannitol.

[0111] Preservation of viral activity is particularly effective when two or more sugars are used in the preservation mixture. Two, three or four sugars may be used. Preferably, the aqueous solution is a solution of sucrose and raffinose. Sucrose is a disaccharide of glucose and fructose. Raffinose is a trisaccharide composed of galactose, fructose and glucose.

[0112] In the present invention, the compound of formula (I) is preferably DMG or a physiologically acceptable salt or ester thereof, and the compound of formula (II) is preferably MSM or a physiologically acceptable salt or ester thereof. The composition also preferably includes sucrose.

[0113] Other components of the aqueous composition In the present invention, the aqueous solution containing the virus particles, optionally one or more sugars and the compound of formula (I) or its physiologically acceptable salt or ester and / or the compound of formula (II) or its physiologically acceptable salt or ester is typically dried. Any suitable aqueous solution may be used. The solution may be buffered. The solution may be HEPES, phosphate buffer, Tris buffer or pure water.

[0114] The solution may have a pH of 2 to about 12 and may be buffered. The solution may be buffered with HEPES buffer, phosphate buffer, Tris buffer, sodium citrate buffer, bicine buffer (i.e., N,N-bis(2-hydroxyethyl)glycine buffer) or MOPS buffer (i.e., 3-(N-morpholino)propanesulfonic acid buffer). The solution may or may not contain NaCl. Thus, the solution may be a saline sodium citrate (SSC) buffer.

[0115] Generally, a preparation of viral particles is mixed with a preservation mixture, i.e., an aqueous solution of a compound of formula (I) or a physiologically acceptable salt or ester thereof and / or a compound of formula (II) or a physiologically acceptable salt or ester thereof, and optionally one, two or more sugars. The preservation mixture itself may be buffered. The preservation mixture may be HEPES, phosphate buffer, Tris buffer or pure water solution.

[0116] Alternatively, the aqueous solution may typically consist or consist essentially of virus particles, a compound of formula (I) or a physiologically acceptable salt or ester thereof and / or a compound of formula (II) or a physiologically acceptable salt or ester thereof, and optionally one or more sugars.

[0117] The concentration of the compound of formula (I) or its physiologically acceptable salt or ester and / or the compound of formula (II) or its physiologically acceptable salt or ester, and each optional sugar can be determined by routine experiment.Therefore, the optimized concentration that brings about the best stability can be selected.The compound of formula (I) or its physiologically acceptable salt or ester and / or the compound of formula (II) or its physiologically acceptable salt or ester compounds may act synergistically to improve stability.

[0118] The concentration of the sugar, if present in the aqueous drying solution, is at least 0.01M, typically up to saturation. Generally, the sugar concentration, if present, is at least 0.1M, at least 0.2M or at least 0.5M up to saturation, for example up to saturation at room temperature, or up to 3M, 2.5M or 2M. The sugar concentration may therefore be, for example, in the range of 0.1M to 3M or 0.2M to 2M. Preferably, one sugar is present. Alternatively, if more than one sugar is present, the sugar concentration or the total sugar concentration may therefore be in the range of 0.08M to 3M, 0.15M to 2M or 0.2M to 1M. A preferred range is 0.05 to 1M.

[0119] When multiple sugars are present, preferably one of the sugars is sucrose, which may be present at a concentration of from 0.05M, 0.1M, 0.25M or 0.5M up to saturation, for example up to saturation at room temperature, or up to 3M, 2.5M or 2M.

[0120] The ratio of the molar concentration of sucrose to the molar concentration of the other sugar is typically 1:1 to 20:1, such as 5:1 to 15:1. Thus, when two sugars are present, in particular when sucrose and raffinose are present, the ratio of the molar concentrations of sucrose is typically 1:1 to 20:1, such as 5:1 to 15:1, preferably about 10:1.

[0121] The concentration of each compound of formula (I) or a physiologically acceptable salt or ester thereof, or the compound of formula (II) or a physiologically acceptable salt or ester thereof in the aqueous solution for drying is generally in the range of 0.001 M to 2.5 M, more specifically, 0.01 M to 2.5 M. For example, the concentration range may be 0.1 M to 2.5 M.

[0122] Alternatively, for example, when the compound of formula (I) is DMG or a salt or ester, the concentration of each compound of formula (I) or its physiologically acceptable salt or ester, or the compound of formula (II) or its physiologically acceptable salt or ester in the aqueous solution for drying is generally in the range of 0.1 mM to 3 M or 1 mM to 2 M. The concentration may be 1 mM to 1.5 M, or 5 mM to 1 M, or 0.07 M to 0.7 M. A preferred concentration is 7 mM to 1.5 M or 0.07 M to 1.2 M. Another more preferred range is 0.5 to 1.5 M, especially when the compound of formula (I) is an N-alkylated glycine derivative such as DMG.

[0123] The particular concentration of the compound of formula (I) or a physiologically acceptable salt or ester thereof or the compound of formula (II) or a physiologically acceptable salt or ester thereof used will depend on several factors, including the type of virus particle being preserved; the particular compound being used; whether one or two additional sugars are present and the identity of the sugars; and the drying procedures and conditions. Thus, The concentration of the compound of formula (II) or the compound of formula (IIA), where -X represents -S(O)2-, such as MSM, or a physiologically acceptable salt or ester thereof, is preferably from 0.2 mM to 1 M, such as from 0.35 mM to 1 M, 3.5 mM to 0.5 M, 0.035 M to 0.5 M, or 0.035 M to 0.25 M. The compound of formula (I) or compound of formula (IA) or formula (IB), such as TMG, or a physiologically acceptable salt or ester thereof, is preferably used at a concentration of 0.01M to 2M, for example 0.07M to 2M, 0.2M to 1.5M, 0.23M to 1.5M or 0.07M to 0.7M. -X is -S + (R c The concentration of the compound of formula (II) or the compound of formula (IIB), for example S-methyl-L-methionine, or a physiologically acceptable salt or ester thereof, representing S-methyl-L-methionine, is preferably 0.005M to 2M, for example, 0.007M to 2M, 0.02M to 2M, 0.023M to 1.5M, or 0.07M to 1M. The concentration of the compound of formula (I) in the absence of -sugar, such as N,N-dimethylglycine (DMG) or a physiologically acceptable salt or ester thereof, is 5 mM to 1.5 M, or 70 mM to 1.5 M or 1.2 M, or 7 mM to 1 M. More preferred concentrations are 0.023 M to 0.7 M or 1 M, or 0.07 M to 0.7 M or 1 M, for example about 0.7 M. The concentration of the compound of formula (I) when one or more sugars are present, such as N,N-dimethylglycine (DMG) or a physiologically acceptable salt or ester thereof, is generally lower, in the range of 1 mM to 1 M or 1.5 M, or 5 mM to 1 M. More preferred concentrations are 0.007 M to 0.7 M or 1 M, for example about 0.007 M. An especially preferred range is 0.5 to 1.5 M.

[0124] When a compound of formula (I) or a physiologically acceptable salt or ester thereof and a compound of formula (II) or a physiologically acceptable salt or ester thereof are present, preferably when an N-alkylated glycine derivative or a salt or ester thereof and a sulfone compound of formula (IIA) or (IIC) are present, the compounds may be present in synergistic amounts, for example: The concentration of the N-alkylated glycine derivative or its salt or ester in the aqueous solution for drying is generally in the range of 0.1 mM to 3 M or 1 mM to 2 M. The concentration may be 1 mM to 1.5 M or 5 mM to 1 M. The preferred concentration is 0.1 M to 1.5 M or 0.5 M to 1.25 M. The concentration of the sulfone compound of formula (IIA) or (IIC) in the aqueous solution for drying is generally in the range of 0.1 mM to 3 M, 1 mM to 2 M, or 0.2 mM to 1 M. The concentration may be 0.1 M to 1.5 M or 0.5 M to 1.25 M.

[0125] The compositions may also contain other preservatives such as antioxidants, lubricants and binders that are well known in the art.

[0126] The composition may be dried as described below.

[0127] Drying Typically, drying is accomplished by freeze-drying, vacuum drying, fluidized bed drying, or spray drying. Freeze-drying is preferred. By reducing the water in the material and sealing the material in a vial, the material can be easily stored, shipped, and later reconstituted to its original form. Drying conditions can be suitably optimized by routine experimentation.

[0128] Upon drying, a composition is formed that incorporates the viral particles. A matrix is ​​produced that incorporates the viral particles. The composition is typically an amorphous solid. Thus, a solid matrix is ​​typically formed that is typically an amorphous solid matrix. "Amorphous" means unstructured and does not have an observable regular or repeating organization of molecules (i.e., non-crystalline).

[0129] When present, one or more sugars provide an amorphous matrix in the dry composition. The compound of formula (I) or its physiologically acceptable salt or ester and / or the compound of formula (II) or its physiologically acceptable salt or ester are dispersed in the sugar matrix. Thus, the compound of formula (I) or its physiologically acceptable salt or ester and / or the compound of formula (II) or its physiologically acceptable salt or ester are incorporated in the sugar matrix. The virus particles are also incorporated in the sugar matrix. Thus, the drying procedure can be carried out, for example, by freeze-drying to form an amorphous cake in which the virus particles are incorporated.

[0130] Drying step is generally carried out immediately after the aqueous solution is prepared or immediately thereafter.Alternatively, the aqueous solution is typically stored before drying step.The virus particles in the aqueous solution are preserved by the compound of formula (I) or its physiologically acceptable salt or ester and / or the compound of formula (II) or its physiologically acceptable salt or ester, and optionally one or more sugars during storage.

[0131] The aqueous solution or bulk intermediate solution is generally stored for up to 5 years, such as up to 4 years, 3 years, 2 years or 1 year. Preferably, the solution is stored for up to 6 months, more preferably up to 3 months or up to 2 months, such as 1 day to 1 month or 1 day to 1 week. Before drying, the solution is typically stored in a refrigerator or freezer. The temperature of the refrigerator is typically 2 to 8°C, preferably 4 to 6°C, or for example about 4°C. The temperature of the freezer is typically -10 to -80°C, preferably -10 to -30°C, such as about -20°C.

[0132] The solution is typically stored in a sealed container, preferably a sealed inert plastic container such as a bag or bottle. The container is typically sterile. The volume of the bulk intermediate solution is typically 0.1 to 100 liters, preferably 0.5 to 100 liters, for example 0.5 to 50 liters, 1 to 20 liters, or 5 to 10 liters. The container typically has a volume of 0.1 to 100 liters, preferably 0.5 to 100 liters, for example 0.5 to 50 liters, 1 to 20 liters, or 5 to 10 liters.

[0133] When a stored bulk intermediate solution is lyophilized, the bulk intermediate solution is typically poured into lyophilization trays prior to the drying step.

[0134] Lyophilization has three main stages: freezing, primary drying and secondary drying. Freezing is typically performed using a freeze dryer. In this process, it is important to cool the biological material below its eutectic point (Teu) for simple crystalline products or below the glass transition temperature (Tg') for amorphous products, i.e. below the lowest temperature at which the solid and liquid phases of the material can coexist. This ensures that sublimation rather than melting occurs in the following primary drying stage.

[0135] During primary drying, pressure is controlled by applying an appropriate level of vacuum while sufficient heat is supplied to sublimate the water. At this stage, at least 50%, typically 60-70%, of the water in the material is sublimated. Primary drying may be slow, as excessive heat can degrade or alter the structure of the biological material. The low temperature condenser chamber and / or condenser plate provide a surface where water vapor is captured by re-solidification.

[0136] In the secondary drying process, the water of hydration is removed by application of additional heat. Typically, the pressure is also reduced to facilitate further drying. After completion of the freeze-drying process, the vacuum can be broken with an inert gas such as nitrogen before sealing, or the material can be sealed under vacuum.

[0137] In a particular embodiment, vacuum drying is performed using a vacuum dryer at about 1300 Pa. However, vacuum drying is not essential to the invention, and in other embodiments, the preservation mixture contacted with the viral particles is tumbled (i.e., rotary dried) or freeze-dried. Advantageously, the method of the invention further comprises subjecting the preservation mixture containing the viral particles to a vacuum. Conveniently, the vacuum is applied at a pressure of 20,000 Pa or less, preferably 10,000 Pa or less. Advantageously, the vacuum is applied for a period of at least 10 hours, preferably 16 hours or more. As known to the skilled person, the duration of vacuum application depends on the size of the sample, the machine used and other parameters.

[0138] In another embodiment, drying is accomplished by spray drying or spray freeze drying the virus particles mixed with the preservation mixture of the present invention. These techniques are well known to those skilled in the art and include methods of drying a liquid feed via a gas, such as air, oxygen-free gas or nitrogen, or liquid nitrogen in the case of spray freeze drying. The liquid feed is atomized into a spray of droplets. The droplets are then dried by contact with the gas or liquid nitrogen in a drying chamber.

[0139] In a further embodiment, drying is achieved by fluidized bed drying of the virus particles mixed with the preservation mixture. This technique is well known to those skilled in the art and typically involves passing a gas (e.g. air) through the product bed under controlled velocity conditions to create a fluidized state. This technique may include the stages of drying, cooling, agglomeration, granulation and coating of the granular product material. Heat may be provided by a fluidizing gas and / or by other heated surfaces (e.g. panels or tubes) immersed in the fluidized bed. Cooling can be achieved using cold gas and / or cooling surfaces immersed in the fluidized bed. The agglomeration and granulation steps are well known to those skilled in the art and can be carried out in various ways depending on the product characteristics to be achieved. Coating of granular products such as powders, granules or tablets can be achieved by spraying a liquid onto the fluidized particles under controlled conditions.

[0140] This allows a composition with low residual moisture content to be obtained. A level of residual moisture content is achieved that provides long-term storage at temperatures above refrigeration temperatures, e.g. in the range of 40°C to 56°C or higher, or below refrigeration temperatures, e.g. in the range of 0°C to 70°C or lower. Thus, the dry composition may have a residual moisture content of 10% or less, 5% or less, 2% or less, or 1% or less by weight. Preferably, the residual moisture content is 0.5% or more and 1% or more. Typically, the dry composition has a residual moisture content of 0.5 to 10% (by weight), preferably 1 to 5% (by weight).

[0141] The composition can be obtained in a dry powder form. For example, the cake obtained from freeze-drying can be ground into a powder form. Thus, the solid composition according to the present invention can be in the form of free-flowing particles.

[0142] In the present invention, the powder may be compressed into tablet form, which is described below. The powder may also be filled into capsules, which are again described below.

[0143] Tablets and capsules The pharmaceutical compositions described herein may be administered in the form of an aqueous suspension or solution or a lozenge, but are typically administered as a tablet or capsule. The compositions may also be administered as gelatin wafers. Tablets may be coated or uncoated. Preferably, the compositions are incorporated into a capsule, such as a gelatin capsule.

[0144] Pharmaceutically compatible binders and / or adjuvant materials can be included in the oral formulations. Tablets, capsules, troches, etc. can contain any of the following ingredients, or compounds of a similar nature: binders such as microcrystalline cellulose, tragacanth or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, primogel, or corn starch; lubricants such as magnesium stearate or sterote; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavorings such as peppermint, methyl salicylate, or flavorings.

[0145] Capsules and tablets are typically enteric coated. A person skilled in the art would be readily able to select and apply an appropriate enteric coating depending on the transgene to be delivered using methods known in the art. For example, the enteric coating may target delivery to the duodenum. An example of such a coating is poly(methacyclic acid-co-thyl acrylate) 1:1 copolymer. The enteric coating may have a threshold pH of 5.8 to 6.8.

[0146] Treatment method The invention also includes methods of treatment comprising administering the viral particles or pharmaceutical compositions comprising the viral particles to a patient in need thereof, which is typically a human, but can also be an animal, such as a domestic animal, a companion animal (such as a dog or cat), or a livestock animal (sheep, pig, cow).

[0147] The administration of the viral particles may be for either prophylactic or therapeutic purposes. However, the viral particles of the present invention are typically used in vaccines intended to prevent the occurrence of disease or to reduce the severity of such disease. In the case of viral infections, prevention of disease typically means preventing infection from occurring in a patient. Reduction of the severity of disease may mean that a patient is asymptomatic for the disease or that a reduction in the severity of symptoms is observed.

[0148] The dosage and frequency of administration will depend on the disease and the desired outcome, and may be readily ascertained by one skilled in the art.

[0149] The present invention particularly provides a method for preventing or reducing the severity of a SARS-CoV-2 infection, said method comprising administering a viral particle or composition / vaccine of the invention to a patient in need thereof.

[0150] The present invention also provides a viral particle / vaccine of the invention for use in the manufacture of a medicament for preventing or reducing the severity of a SARS-CoV-2 infection.

[0151] Furthermore, the present invention provides a viral particle or a vaccine of the invention for use in a method for preventing or reducing the severity of a SARS-CoV-2 infection.

[0152] In all of these cases, the viral particles express SARS-CoV-2 antigens, in particular the S protein, as described above.

[0153] The following examples are presented below so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention. The examples are not intended to limit the scope of what the inventors regard as their invention.

[0154] example Experiments were performed using the SARS-CoV-2 unmodified spike protein ("GEN1") and the modified spike protein ("GEN3") with the sequence shown in Figure 1. Adenoviral vectors (serotype 5, ΔE1, ΔE3) were prepared and transduced into HEK293 cells.

[0155] Example 1 - Virus yield Plated HEK-293 cells were infected with adenoviral vectors encoding the GEN1 and GEN3 spike proteins (0.1 ppc per virus - two T175 flasks) for expansion (and comparison of viral yields): The following was observed: GEN3=>0.1ppc×10×10 6 cells=10×10 5 Particles / 5.4×10 7 pfu / ml = 18.5μl GEN1 => 0.1ppc x 10 x 10 6 cells=10×10 5 Particle / 1.0×10 8 pfu / ml = 10μl GEN3 and GEN1 infected T175 flasks of HEK-293 cells were checked (0.1 ppc 96 hours). For both GEN3 and GEN1, additional areas of CPE were observed. An additional 10 ml of DMEM was added to each flask.

[0156] After 2 days, the GEN3 / GEN1 infected HEK-293 cells were checked again. More plaques / CPE formed and more cells detached from the flask. The GEN3 and GEN1 infected HEK-293 cells were then harvested by centrifugation at 4100 rpm for 1 hour at 4°C and resuspending the cell pellet in 4 ml of supernatant. The remaining supernatant was retained and stored at -80°C.

[0157] The titers of the recovered GEN1 and GEN3 viruses were then tested using AdenoX. First, serial dilutions of the samples were performed by adding 20 μl of the stock to DMEM (10 -1 Dilution ~10 -12 100 μl of each dilution was added to HEK-293 cells and incubated at 37° C. for 48 hours.

[0158] The results were as follows: GEN3(HEK-293): 2 pieces (10 -7 Dilution) => 2 particles in 100 μl (10 of 100 μl-7 Dilutions added to 100 μl of HEK-293 cells for AdenoX) => 2 x 10 in 100 μl of original sample 7 particles => 2×108 pfu / ml 4 pieces (10 -6 Dilution) => 4 particles in 100 μl (10 -6 Dilutions added to 100 μl of HEK-293 cells for AdenoX) => 4 x 10 in 100 μl of original sample 6 particles => 4×10 7 pfu / ml 20 pieces (10 -5 Dilution) => 20 particles in 100 μl (10 -5 Dilutions added to 100 μl of HEK-293 cells for AdenoX) => 20 x 10 in 100 μl of original sample 5 particles => 20×10 6 pfu / ml Average GEN3 titer => 8.67 x 10 7 pfu / ml GEN1(HEK-293): 5 pieces (10 -5 Dilution) => 5 particles in 100 μl (10 -5 Dilutions added to 100 μl of HEK-293 cells for AdenoX) => 5 x 10 in 100 μl of original sample 5 particles => 5 10 6 pfu / ml 11 pieces (10 -4 Dilution) => 11 particles in 100 μl (10 particles in 100 μl -4 Dilutions added to 100 μl of HEK-293 cells for AdenoX) => 11 x 10 in 100 μl of original sample 4 particles => 11 x 10 5 pfu / ml 69 pieces (10 -3 Dilution) => 69 particles in 100 μl (10 -3 Dilutions added to 100 μl of HEK-293 cells for AdenoX) => 69 x 10 in 100 μl of original sample 3 particles => 69×10 4 pfu / ml Average GEN1 titer => 2.26 x 10 6 pfu / ml

[0159] Thus, the average titer of GEN3 was approximately 40-fold higher than that of GEN1.

[0160] FIG. 2 shows a comparison of GEN1 and GEN3 yields.

[0161] Example 2 - Immunological response The EUROIMMUN anti-SARS-CoV2 ELISA kit was used to study the immunological response following administration of GEN1 and GEN3 viruses to rats. The EUROIMMUN anti-SARS-CoV2 IgG ELISA kit is designed to provide a semi-quantitative in vitro measurement of human antibodies (IgG) to SARS-CoV-2 in serum or plasma. However, deviations from the kit protocol were made to detect rat IgG antibodies as described below.

[0162] Rat plasma samples were diluted 1:10 (11 μl in 99 μl Euroimmun kit buffer). Samples were mixed by vortexing. 100 μl of diluted samples were added to the relevant wells on the Euroimmun Sars Cov-2 IgG ELISA plate. Plates were covered and incubated for 1.5 h at 37°C. Plates were washed 3 times with 300 μl Euroimmun kit wash buffer (1×). Plates were tapped dry. Anti-Rat IgG (whole molecule) Biotin (SIGMA, B7139, SLBM7228V) was diluted 1:20000 in PBS and added at 100 μl / well. Plates were covered and incubated for 1 h at room temperature. Plates were washed 3 times with 300 μl Euroimmun kit wash buffer (1×). Plates were tapped dry and 100 μl / well Streptavidin-HRP Reagent 1× was added (VIRUSYS, SA-HRP, Lot# 826503, 09 / 2020). Plates were covered and incubated for 30 min at room temperature. Plates were washed 3 times with 300 μl Euroimmun kit wash buffer (1×). Plates were tapped dry. 100 μl / well Euroimmun kit substrate solution was added. Plates were covered and incubated for 15 min at room temperature in the dark. 100 μl / well Euroimmun kit stop solution was added and plates were read at 450 nm.

[0163] The results are shown in Figure 3. Figure 3 shows higher levels of IgG against the GEN3 antigen compared to the GEN1 antigen. [Sequence List Free Text]

[0164] Sequence Listing 2 <223> Xaa can be any naturally occurring amino acid Sequence Listing 4 <223> Xaa can be any naturally occurring amino acid Sequence Tables 5-8 <223> Synthetic Polypeptides Sequence Listing 9 <223> Synthetic Nucleotides Sequence Listing 11 <223> Synthetic Polypeptides Sequence Listing 11 <223> Xaa can be any naturally occurring amino acid Sequence Listing 12 <223> Synthetic Polypeptides Sequence Listing 12 <223> Xaa can be any naturally occurring amino acid Sequence Tables 13-18 <223> Synthetic Polypeptides Sequence Listing 19 <223> SwTMF2aEnv4b_1_F primer Sequence Listing 20 <223> SwTMF2aEnv4b_1_R primer Sequence Listing 21 <223> SwTMF2aEnv4b_2_F primer Sequence Listing 22 <223> SwTMF2aEnv4b_2_R primer Sequence Listing 23 <223> SwTMF2aEnv4b_3_F primer Sequence Listing 24 <223> SwTMF2aEnv4b_3_R primer Sequence Listing 25 <223> SwTMF2aEnv4b_4_F primer Sequence Listing 26 <223> SwTMF2aEnv4b_4_R primer Sequence Listing 27 <223> SwTMF2aEnv4b_5_F primer Sequence Listing 28 <223> SwTMF2aEnv4b_5_R primer Sequence Listing 29 <223> SwTMF2aEnv4b_6_F primer Sequence Listing 30 <223> SwTMF2aEnv4b_6_R primer Sequence Listing 31 <223> SwTMF2aEnv4b_7_F primer Sequence Listing 32 <223> SwTMF2aEnv4b_7_R primer Sequence Listing 33 <223> SwTMF2aEnv4b_8_F primer Sequence Listing 34 <223> SIBV_FOR primer Sequence Listing 35 <223> SwTMF2aEnv4b_8_R primer Sequence Listing 36 <223> SIBV_REV primer Sequence Listing 37 <223> V5 Tag Sequence Listing 38 <223> AmpR promoter and gene Sequence Listing 39 <223> Synthetic Nucleotides Sequence table 40-42 <223> Synthetic Polypeptides

Claims

1. A polynucleotide encoding an antigen derived from a virus, the antigen being modified to (i) target the antigen to the plasma membrane and / or (ii) reduce membrane fusion and / or syncytia formation. (a) the antigen is a class I, class II or class III membrane fusion protein, and / or (b) the targeting to the plasma membrane is increased by modifying the cytoplasmic tail of the antigen, for example by substituting a polybasic motif, optionally a dibasic motif, and further, optionally, the polybasic motif is substituted with an alanine.

3. the cytoplasmic tail is replaced with a cytoplasmic tail from infectious bronchitis virus in which a di-lysine motif has been replaced, e.g., the di-lysine motif has been replaced with an alanine, and optionally (a) the tyrosine internalization sequence is also mutated to optionally replace the tyrosine with an alanine; and / or (b) the cytoplasmic tail comprises one or more additional amino acid substitutions, deletions or insertions; A polynucleotide according to claim 1 or 2.

4. 4. The cytoplasmic end of the modified antigen comprises the following sequence: The polynucleotide of claim 3, comprising:

5. The modification to reduce membrane fusion and / or syncytia formation comprises substitution of a tryptophan residue, and optionally (a) the tryptophan residue is in a transmembrane domain of the antigen, e.g. in an aromatic stretch of the transmembrane domain; and / or 5. The polynucleotide of claim 1, wherein the tryptophan residue is replaced by an alanine and, optionally, the motif WXWXXW (SEQ ID NO: 11) is replaced by AXAXXA (SEQ ID NO: 12).

6. 6. The polynucleotide of claim 1, wherein the antigen is derived from a coronavirus, for example, the antigen is the spike (S) protein of SARS-CoV-2.

7. array or Contains or (a) a variant having at least 90% identity to said sequence, or (b) a variant of said sequence having up to 10 deletions, substitutions or additions. The polynucleotide of claim 6, encoding a SARS-CoV-2 spike protein comprising:

8. 8. The polynucleotide of claim 7, wherein the variant retains the AXXA (SEQ ID NO: 12) motif and AA residues at positions -3 and -4.

9. (a) encoding a SARS-CoV-2 spike protein comprising the sequence 9. The polynucleotide of any one of claims 1 to 8, wherein the polynucleotide (a) encodes a SARS-CoV-2 spike protein having at least 90% identity to the sequence shown in Figure 1, or (b) encodes a SARS-CoV-2 spike protein comprising the sequence shown in Figure 1, or encodes a spike protein having at least 90% identity to the sequence shown in Figure 1.

10. 10. A viral vector comprising the polynucleotide of any one of claims 1 to 9, optionally (a) the viral vector is an adenoviral vector; (b) the viral vector is a type 5 adenoviral vector; or (c) The viral vector is a ΔE1, ΔE3 adenoviral vector. (a) the polynucleotide encoding the modified antigen is under the control of a CMV promoter; and / or (b) the viral vector of claim 10, wherein the viral vector encodes a modified SARS-CoV-2 spike protein and further encodes an additional SARS-CoV-2 antigen, such as a SARS-CoV-2 nucleocapsid.

12. A polypeptide encoded by a polynucleotide according to any one of claims 1 to 9.

13. A viral particle carrying a polynucleotide according to any one of claims 1 to 9.

14. A viral particle comprising the vector of claim 10, optionally comprising: The virus is an adenovirus, and optionally (a) the virus is a type 5 adenovirus, and / or (b) the virus is replication-deficient, such as a ΔE1, ΔE3 virus, the viral particle of claim 13.

15. 14. A vaccine comprising the viral particle of claim 13, optionally for oral administration to a subject in need of the vaccine.

16. A vaccine comprising the viral particles of claim 14, optionally for oral administration to a subject in need of the vaccine.

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