Engineering coronavirus spike proteins as vaccine antigens, their design and uses

Abstract

To provide a method of vaccinating a mammal subject against infection from at least one group of CoV.SOLUTION: The present invention provides a method that includes separating a broad group of CoV into homology groups, creating a modified S protein containing at least one modification at its SI region, and identifying at least one consensus sequence for each homology group which has a sequence identity of greater than 60% to all other members of the homology group. The consensus sequence is a protein sequence for the modified S protein, a DNA sequence encoding the modified S protein, and an RNA sequence encoding the modified S protein.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Provisional Application No. 63 / 023,599, filed May 12, 2020, the entire contents of which are hereby incorporated by reference, and are not a provisional application.

[0002] The present disclosure relates to a vaccine platform for developing coronavirus vaccines, and more particularly to vaccines containing engineered coronavirus spike proteins. In another embodiment, the present disclosure relates to methods for engineering coronavirus spike proteins and methods for making vaccines containing engineered coronavirus spike proteins.

[0003] Submission of sequence listing The contents of the electronic submission of the Sequence Listing, a text file entitled "Sequence_Listing", created on May 11, 2021, of size 54kb, is hereby incorporated by reference in its entirety. [Background technology]

[0004] Coronaviruses (CoVs) are classified into four genera: alpha-, beta-, gamma- and delta-coronaviruses. β-CoVs are enveloped, positive-stranded RNA viruses that can infect mammals, typically bats and rodents, although many β-CoVs are known to also infect humans. Infections with CoVs in humans and animals generally cause a short-lived, mild to moderate upper respiratory tract disease. Exceptions are Severe Acute Respiratory Syndrome (SARS-1), Middle East Respiratory Syndrome (MERS), and the Wuhan-origin SARS-CoV-2 (SARS-2) (also known as COVID-19), which are characterized by severe and often fatal symptoms. The first cases of SARS-2 infection were seen in December 2019. As of April 16, 2020, in the United States alone, there were an estimated 632,000 cases and an estimated 31,000 deaths reported by the Centers for Disease Control and Prevention (CDC), resulting in a case fatality rate of 4.9%. SARS-2 is highly infectious to humans. On January 30, 2020, the World Health Organization (WHO) declared the SARS-2 global pandemic a Public Health Emergency of International Concern.

[0005] No specific treatment for SARS-2 has been established but is under investigation. The best approach to prevent further spread of the disease is the development of a specific vaccine. Herd immunity against SARS-2 is better achieved with immunization with a benign vaccine rather than natural infection with active SARS-2 virus. One explanation for the low level of immune response seen in recovering patients could be a function of exhausting immunosuppression by SARS-2. However, animal studies with traditional vaccines that use inactive forms of the virus suggest that inactivated virus vaccines may be particularly susceptible to the induction of antibody-dependent enhancement (ADE) of disease. In the case of these vaccinations, Th2-type disease enhancement may be driven by anti-nucleocapsid (NP) responses. It would be desirable to develop a SARS-2 vaccine that does not stimulate ADE in vaccine recipients.

[0006] Although social distancing has successfully contained the aggressive spread of SARS-2, the reopening of society is expected to lead to a short-term surge in infections as well as possible seasonal outbreaks. Some regions have already seen a surge in infections caused by mutated SARS-2 viruses. The overall mutation rate of SARS-related β-CoV (SARSr) has been calculated at a low of 0.1 mutations per generation. Despite the emergence of recent mutations, the SARS-2 virus appears to be stable as well. It would be desirable for any SARS-2 vaccine to also provide protection against short-term variants.

[0007] Numerous animal tests and clinical trials with the related SARS- and MERS-CoVs suggest that it may be possible to produce effective vaccines against the more common β-CoV infections. SARS-2 (COVID-19) is the third deadly β-CoV to jump from an animal host to humans. Considering that 1,800 SARSr have already been identified in animals, some of which may eventually infect humans, it would be desirable to also create group-specific SARSr vaccines to avoid future pandemics.

[0008] There are further factors supporting the need for a broadly reactive vaccine to provide protection against SARS-2 variants and SARSr virus strains. Analysis of SARSr animal sequences reveals that small changes in the S protein receptor binding domain (RBD) sequence enhance viral binding to human ACE2 and thus such changes may facilitate the jump of β-CoV to human populations. The RBD sequence is located in a highly variable region of the S protein. The virus is also known to mutate. As billions of people have been infected with SARS-2, a large number of mutations are expected. A vaccine that induces an immune response against the more conserved regions of the S protein would provide broader protection against infection with different SARSr. Summary of the Invention

[0009] In one embodiment, the present disclosure provides a vaccine for preventing CoV infection. According to an embodiment of the present disclosure, the vaccine for preventing CoV infection comprises at least one sequence selected from the group consisting of a CoV DNA sequence encoding at least a portion of a modified S protein of the CoV, a CoV RNA sequence encoding at least a portion of a modified S protein of the CoV, and a protein sequence of at least a portion of a modified S protein of the CoV, wherein the modified S protein comprises at least one modification.

[0010] In one embodiment, the S protein has an S1 region and the at least one modification is selected from the group consisting of a complete deletion of the S1 region, a partial deletion of the S1 region, a complete replacement of the S1 region, and a partial replacement of the S1 region. In a further embodiment, the S1 region has at least one variable region and the at least one modification is selected from the group consisting of a complete deletion of at least one variable region, a partial deletion of at least one variable region, a complete replacement of at least one variable region, and a partial replacement of at least one variable region. In yet a further embodiment, the CoV is SARS-2 β-CoV and the at least one variable region is between amino acid residues 342 and 533. In another embodiment, the at least one variable region comprises a receptor binding domain (RBD). In one embodiment, the at least one modification is a partial replacement of the variable region or a complete replacement of the variable region, wherein the variable region is replaced by a peptide linker.

[0011] In one embodiment, the CoV is an α-CoV. In another embodiment, the CoV is a β-CoV. In a further embodiment, the sequence is a CoV DNA sequence or a β-CoV DNA sequence. In another embodiment, the sequence is a CoV RNA sequence or a β-CoV RNA sequence. In one embodiment, the RNA is mRNA. In yet another embodiment, the sequence is a CoV protein sequence or a β-CoV protein sequence. In a further embodiment, the sequence is a SARS-2 β-CoV sequence.

[0012] In one embodiment, the present disclosure provides a modified CoV S protein. According to an embodiment of the present disclosure, the modified CoV S protein comprises an S1 region and at least one modification in the S1 region selected from the group consisting of a partial deletion of the S1 region, a complete deletion of the S1 region, a partial replacement of the S1 region, and a complete replacement of the S1 region.

[0013] In one embodiment, the S1 region has at least one variable region and the at least one modification is selected from the group consisting of a complete deletion of at least one variable region, a partial deletion of at least one variable region, a complete replacement of at least one variable region, and a partial replacement of at least one variable region. In a further embodiment, the CoV is SARS-2 β-CoV and the at least one variable region is between amino acid residues 342 and 533. In yet a further embodiment, the at least one variable region comprises a receptor binding domain (RBD). In another embodiment, the at least one modification is a partial replacement of a variable region or a complete replacement of a variable region, wherein the variable region is replaced by a peptide linker.

[0014] In one embodiment, the present disclosure provides a method of making a vaccine for protecting against infection by at least one CoV. According to an embodiment of the present disclosure, the method of making a vaccine for protecting against infection by at least one CoV comprises generating a modified S protein comprising an S1 region and at least one modification in the S1 region, wherein the at least one modification is selected from the group consisting of a partial deletion of the S1 region, a complete deletion of the S1 region, a partial replacement of the S1 region, and a complete replacement of the S1 region.

[0015] In one embodiment, the disclosure provides a method of vaccinating a mammalian subject against infection from at least one group of CoVs. According to an embodiment of the disclosure, the method of vaccinating a mammalian subject against infection from at least one group of CoVs includes separating a broad group of CoVs into homology groups based on similarity of the CoV S proteins, generating a modified S protein for each homology group, the modified S protein comprising an S1 region and at least one modification selected from the group consisting of a partial deletion of the S1 region, a complete deletion of the S1 region, a partial replacement of the S1 region, and a complete replacement of the S1 region, identifying at least one consensus sequence for each homology group having greater than 60% sequence identity with all other members of the homology group, the at least one consensus sequence being selected from the group consisting of a protein sequence of the modified S protein, a DNA sequence encoding the modified S protein, and an RNA sequence encoding the modified S protein.

[0016] In another embodiment, the method further comprises preparing a viral vector comprising at least one consensus sequence. In another embodiment, separating the broad group of CoVs into homologous groups comprises obtaining at least one CoV from a mammal. In another embodiment, the mammal is a human.

[0017] In a further embodiment, the S1 region comprises at least one variable region and the at least one modification is selected from the group consisting of a complete deletion of at least one variable region, a partial deletion of at least one variable region, a complete replacement of at least one variable region, and a partial replacement of at least one variable region.

[0018] In one embodiment, the CoV is SARS-2 β-CoV and the at least one variable region is between amino acid residues 342 and 533. In a further embodiment, the at least one variable region comprises a receptor binding domain (RBD). In yet a further embodiment, the at least one modification is a partial or complete replacement of the variable region, wherein the variable region is replaced by a peptide linker.

[0019] In another embodiment, the CoV is an α-CoV. In yet a further embodiment, the CoV is a β-CoV.

[0020] In one embodiment, the consensus sequence is a CoV DNA sequence, or a β-CoV DNA sequence. In yet another embodiment, the consensus sequence is a CoV RNA sequence, or a β-CoV RNA sequence. In one embodiment, the RNA is mRNA. In a further embodiment, the consensus sequence is a CoV protein sequence, or a β-CoV protein sequence. In one embodiment, the sequence is a SARS-2 β-CoV sequence.

[0021] In one embodiment, the method further comprises injecting the vaccine into a mammalian subject. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 is a schematic diagram showing functional portions of the SARS-2 β-CoV RNA segment encoding the S protein, along with the portions that are most variable and that elicit the greatest immune response, according to an embodiment of the present disclosure. [Diagram 2] FIG. 1 shows the design of a modified S protein according to an embodiment of the present disclosure. [Diagram 3] FIG. 1 shows the activity of MERS-CoV vaccines. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Before describing any embodiment of the present disclosure in detail, it should be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and can be practiced or carried out in various ways. It is also understood that the phraseology and terminology used herein are for the purpose of description and should not be considered as limiting. The use of "including", "comprising" or "having" and variations thereof herein means to include the items recited thereafter and equivalents thereof as well as additional items. The use of "including essentially" and "consisting essentially of" and variations thereof herein means to cover the items recited thereafter as well as equivalents and additional items that provide such equivalents and additional items so as not to substantially alter the overall properties, use or manufacture. The use of "consisting" and variations thereof herein means to include only the items recited thereafter and those items only.

[0024] With reference to the drawings, like numbers refer to like elements throughout. Terms such as first, second, etc. may be used herein to describe various elements, components, regions, and / or sections, but it will be understood that these elements, components, regions, and / or sections should not be limited by these terms. These terms are used only to distinguish one element, component, region, and / or section from another element, component, region, and / or section. Thus, a first element, component, region, or section can be referred to as a second element, component, region, or section without departing from this disclosure.

[0025] Numerical ranges in this disclosure are approximations and may include values ​​outside the range unless otherwise indicated. Numerical ranges include all values ​​from and including the lower and higher values ​​in increments of one unit (unless otherwise stated), provided that there is a separation of at least two units between any lower value and any higher value. As an example, if a composition, physical or other property, such as the amount of an ingredient by weight, is 10-100, all individual values ​​such as 10, 11, 12, etc., and subranges such as 10-44, 55-70, 97-100, etc., are intended to be explicitly recited. Ranges that include explicit values ​​(e.g., ranges from 1, or 2, or 3 to 5, or 6, or 7) include any subranges between any two explicit values ​​(e.g., the range 1-7 above includes the subranges 1-2, 2-6, 5-7, 3-7, 5-6, etc.). For ranges containing values ​​less than one or containing fractional ranges greater than one (e.g., 1.1, 1.5, etc.), one unit is considered to be 0.0001, 0.001, 0.01 or 0.1, as appropriate. In ranges containing single digit numbers less than ten (e.g., 1 to 5), one unit is typically considered to be 0.1. These are only examples of what is specifically intended, and all possible combinations of numerical values ​​between the lowest and highest values ​​recited should be considered to be expressly set forth in this disclosure.

[0026] Spatial terms such as "beneath," "below," "lower," "above," and "upper" may be used herein for ease of description to describe the relationship of one element or feature to another element or feature as shown in the figures. It will be understood that spatially relative terms are intended to encompass different orientations depending on the orientation in use or illustration. For example, if a device in the figures is turned over, an element described as being "below" or "below" another element or feature would be oriented "above" the other element or feature. Thus, the exemplary term "below" can encompass both an orientation of above and below. The device may be otherwise oriented (rotated 90 degrees or to other orientations) and the spatially relative descriptors used herein would be interpreted accordingly.

[0027] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. For example, when used in a phrase such as "A and / or B," the term "and / or" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" when used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0028] In one embodiment, the present disclosure provides a method for preparing a vaccine for preventing at least one CoV infection or at least one β-CoV infection in a subject, particularly a mammalian subject, more particularly a human subject.

[0029] Identification of β-CoV According to an embodiment of the present disclosure, a method for preparing a vaccine for preventing at least one β-CoV infection in a subject, particularly a mammalian subject, more particularly a human subject, comprises identifying at least one β-CoV from an animal host, particularly a mammalian host. In particular, in one embodiment, a method for preparing a vaccine for preventing at least one β-CoV infection in a subject, particularly a mammalian subject, more particularly a human subject, comprises identifying at least one β-CoV from a mammalian host selected from the group consisting of bat, rat, human, and combinations thereof. In one embodiment, the at least one β-CoV comprises at least one SARSr. In another embodiment, the at least one β-CoV comprises at least one SARS-2 β-CoV.

[0030] Identification of homologous groups According to an embodiment of the present disclosure, a method for preparing a vaccine for preventing at least one β-CoV infection in a subject, particularly a mammalian subject, more particularly a human subject, comprises separating identified β-CoVs, e.g., β-CoVs identified from an animal host, into homology groups based on genetic sequence similarity and preparing at least one consensus sequence for each homology group. The homology groups can be based on similarity of the entire β-CoV' genetic sequence, multiple portions of the β-CoV' genetic sequence, or a single portion of the β-CoV' genetic sequence. The genetic sequence is selected from the group consisting of a DNA sequence, an RNA sequence, a protein sequence, and combinations thereof. It will be understood that if a single β-CoV is identified, it is the only member of a single homology group.

[0031] In certain embodiments, the β-CoV comprises multiple SARSr, and the multiple SARSr are separated into one, or at least two, or at least three, or at least four, or at least five homology groups. In one embodiment, the homology groups are based on at least a portion, or at least two or more portions, or all of the gene sequences associated with the spike protein (S protein), the SARS receptor binding domain (RBD), the envelope protein, the nucleoprotein, and combinations thereof.

[0032] In a further embodiment, at least one SARS-2 β-CoV is identified and separated into at least one homology group.

[0033] In one embodiment, within each homology group, the gene sequence has 60% or more, or 65% or more, or 70% or more, or 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more sequence identity to all other members of the homology group.

[0034] In one embodiment, within each homology group, the gene sequence has at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85% to at most 90%, or 95%, or 96%, or 97%, or 98%, or 99%, or at most 100% sequence identity with all other members of the homology group.

[0035] In one embodiment, the gene sequence of each homology group defines a distinct protein sequence of the homology group. In one embodiment, the distinct protein is selected from the group consisting of an S protein, an envelope protein, a nucleoprotein, and combinations thereof. In a further embodiment, the distinct protein is an S protein.

[0036] In a particular embodiment, multiple SARSrs are analyzed and separated into five homology groups, and within each homology group, the gene sequences have greater than 65% to 99% sequence identity.

[0037] Exemplary processes for identifying homology groups and consensus sequences are provided herein.

[0038] SARS-2 β-CoV has a positive-sense single-stranded RNA genome of about 30 kb and four structural proteins. One of the structural proteins is the spike (S) peplomer. These S proteins are found on the surface of SARS-2 β-CoV and mediate cellular receptor binding, thus determining the host tropism of the virus. The protein portion of the RNA encoding the S protein is divided into S1 and S2 strands, with S1 strand 10 and S2 strand 20 separated by a franc cleavage site 25, as shown in Figure 1. The RBD 30 is located in the S1 strand 10. It was discovered that variation in the RBD affects the binding of the virus to angiotensin-converting enzyme 2 (ACE2), and this enhanced binding is due to facilitating the jump of the virus from an animal host to a human host. The membrane fusion portion 40 is located within the S2 strand 20. Further shown in Figure 1 are the heptapeptides HR1 and HR2, the transmembrane TM and the cytoplasmic domain of the S protein.

[0039] The SARS-2 β-CoV S protein is split into S1 strand 10 and S2 strand 20. A conformational change in S2 strand 20 leads to viral fusion within the host cell. This makes the S protein-encoding RNA sequence, in combination with the RBD-containing S protein-encoding RNA sequence, an important candidate for use in anti-SARS-2 β-CoV vaccine regimens.

[0040] FIG. 1 also shows the S protein portions of SARS-2 β-CoV that elicit a greater immune response (60). As shown, portion 60b overlaps with RBD 30 and is a fairly large portion, meaning that there is a significant immune response associated with RBD 30. Portions 60d and 60e overlap with the less variable membrane fusion portion 40, but are smaller and therefore elicit a less robust immune response. In analyzing the immune response to different β-CoVs, such as SARS-CoV-1 and MERS-CoV, it was observed that antibodies capable of neutralizing the activity of these coronaviruses can also bind to more conserved regions of the S protein, namely, within the S protein stem region within the S2 domain 20. As further illustrated in FIG. 1, aligning the S protein coding sequences of RNAs from various SARSr shows significant differences throughout the gene (50). Therefore, the SARS-2 β-CoV RNA-based vaccine of the present invention may not be able to efficiently protect against infections caused by other SARSr. However, analysis of the S protein coding sequences of RNA from multiple SARSrs allows separation of the SARSrs into homologous groups, as shown in Table 1. JPEG2025032108000002.jpg57170

[0041] To obtain the information in Table 1, sequences were found using ViPR and NCBI. Global alignments were performed using Clustal Omega. Related alignments (>92%) were extracted to create groupings, which were aligned using Clustal Omega and confirmed using BLAST multiple sequence alignment.

[0042] For the SARS-CoV group, 1130 sequences from GenBank and ViPR covering the original SARS-CoV-1 were analyzed. Some sequences contained random inserts likely responsible for gaps, but all small variants maintained antibody binding. For the SARS-CoV 2 group, over 3000 sequences were analyzed, including the new clades 20H, 201 and 20J (corresponding to the South African, California and UK variants, respectively). WIV-1 is the prominent SARSr in bats, but has been shown to replicate in human cells. 56 WIV-1 strains were analyzed, including the RaTG13 strain that is thought to have given rise to the SARS-2 β-CoV. Only 16 strains had a complete CDS. The structure appeared stable across variants, as indicated by the NCBI conserved protein domain families cd21477 and Cn3D. For the YNLF group, 71 sequences (39 with complete CDS) were obtained from bats, pangolins and camels. These SARSr strains are less similar to SARS-2 β-CoV than to the WIV1 family, but have some strong similarities to the SARS-CoV and SARS-CoV 2 groups in certain regions. The overall spike alignment is moderate, but the RBD alignment shows strong similarities. For the bat 2013 group, 19 samples with high similarity were analyzed. The bat 2013 group shows higher variability than the other groups, but many strains show cross-reactivity to the same antibodies.

[0043] Consensus sequence According to an embodiment of the present disclosure, a method for preparing a vaccine for preventing at least one CoV, preferably at least one β-CoV infection in a subject, particularly a mammalian subject, more particularly a human subject, comprises identifying at least one consensus sequence for each homology group. A consensus sequence is a DNA, RNA or protein sequence developed for a group that contains the statistically most frequent residue at each position in the sequence. In one embodiment, a consensus sequence for a homology group has at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% commonality with each member of the corresponding homology group.

[0044] In certain embodiments, a consensus sequence is a DNA sequence that has 60% or more, or 65% or more, or 70% or more, or 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more sequence identity with all other members of the corresponding homology group.

[0045] In certain embodiments, a consensus sequence is an RNA sequence that has 60% or more, or 65% or more, or 70% or more, or 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more sequence identity with all other members of the corresponding homology group.

[0046] In certain embodiments, a consensus sequence is a protein sequence that has 60% or more, or 65% or more, or 70% or more, or 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more sequence identity with all other members of the corresponding homology group.

[0047] In one embodiment, the consensus sequence of each homology group is selected from the group consisting of a DNA sequence, an RNA sequence, a protein sequence, and combinations thereof. In one embodiment, at least one consensus sequence of a homology group is RNA. In a further embodiment, the RNA is mRNA.

[0048] In one embodiment, the β-CoV analyzed is SARSr. In a further embodiment, the SARSr comprises at least one SARS-2 β-CoV separated into at least one homology group, and the consensus sequence of the at least one homology group is a DNA sequence, an RNA sequence, or a protein sequence. In an embodiment where a single SARSr, e.g. a single SARS-2 β-CoV, is identified and where the single SARSr is the only member of the homology group, it will be understood that the consensus sequence may be a DNA sequence, and the RNA sequence or the protein sequence has 100% commonality with SARSr.

[0049] In one embodiment, the consensus sequence is a SARS-2 β-CoV DNA sequence, which is at least a portion of the S protein coding sequence.

[0050] In one embodiment, the consensus sequence is a SARS-2 β-CoV RNA sequence, which is at least a portion of the S protein coding sequence.

[0051] In one embodiment, the consensus sequence is a SARS-2 β-CoV protein sequence, and the SARS-2 β-CoV protein sequence is at least a portion of the S protein.

[0052] Spike protein modification In one embodiment, the consensus sequence is a protein sequence of at least a part of the S protein of a CoV, or a CoV DNA or RNA sequence encoding at least a part of the S protein, where the S protein comprises at least one modification. That is, when the consensus sequence is a protein sequence, it is for a modified S protein. Similarly, when the consensus sequence is a DNA or RNA sequence, it encodes a modified S protein.

[0053] FIG. 2A is a schematic diagram showing the full-length S protein sequence, and FIGS. 2B-2D are schematic diagrams showing exemplary modifications of the S protein sequence.

[0054] In one embodiment, S1 region 10 of the S protein is defined as the region of the protein beginning at the amino terminus of the protein and ending with an amino acid sequence corresponding to peptidase recognition site 694 found in the SARS-2 S protein. In one embodiment, at least one modification is within S1 region 10 (i.e., at least one modification is in all or at least a portion of the protein sequence from the amino terminus through the 694 amino acid sequence).

[0055] In one embodiment, the at least one modification is a deletion of the entire S1 region 10, e.g., a 694 amino acid sequence from the amino terminus of the S protein. In another embodiment, the at least one modification is a deletion of one or more sequences less than all of S1 region 10, as shown in Figures 2B and 2D.

[0056] In one embodiment, a "variable region" of the S protein is defined as a region of the S protein that exhibits high sequence variability when comparing the SARSr sequences of naturally occurring SARS-CoV viruses and / or SARS-2 variants. Exemplary variable regions of the S protein are shown in Figure 1, with the hypermutated region shown at 50 being an exemplary variable region. In one embodiment, the S protein has a single highly variable region referred to as the "variable region."

[0057] In one embodiment, at least one modification is a complete or partial deletion of at least one variable region or variable regions as shown in Figure 2B.

[0058] In certain embodiments, the variable region of the S protein is located between amino acid residues corresponding to SARS-2 amino acid residues 342 and 533 (NC_045512.2). In such embodiments, at least one modification is a full or partial deletion of the variable region located between amino acid residues corresponding to amino acid residues 342 and 533.

[0059] In certain embodiments, the S protein has a variable region corresponding to most or all of the RDB region. In such embodiments, at least one modification is a complete or partial deletion of the RDB region. In one embodiment, the RDB region is located between amino acid residues corresponding to amino acid residues 342 and 533 of SARS-2, or even between amino acid residues 446 and 520 (NC_045512.2).

[0060] In one embodiment, at least one modification is a complete or total replacement of at least one variable region, or a variable region according to any embodiment or combination of embodiments described above. At least one variable region, or a variable region, can be replaced with a peptide linker sequence designed to bridge the gap created by the deletion, as shown in Figure 2C.

[0061] Viral Vectors According to an embodiment of the present disclosure, a method for preparing a vaccine for preventing at least one CoV infection, preferably β-CoV infection, in a subject, particularly a mammalian subject, more particularly a human subject, comprises inserting at least one consensus sequence into a viral vector. In one embodiment, the viral vector is an adenoviral vector component.

[0062] To minimize pre-existing and induced interfering anti-adenovirus immune responses, all endogenous genes are deleted from the adenovirus vector component, i.e., in one embodiment, the adenovirus vector component is a fully deleted (fd) adenovirus vector.

[0063] In one embodiment, the consensus sequence is a SARS-2 β-CoV DNA sequence that is at least a portion of a modified S protein coding sequence, a SARS-2 β-CoV RNA sequence that is at least a portion of a modified S protein coding sequence, or a SARS-2 β-CoV DNA sequence, wherein the SARS-2 β-CoV protein sequence is at least a portion of a modified S protein, and the modified S protein comprises one or more modifications according to any embodiment or combination of embodiments provided herein.

[0064] Vaccine Composition In one embodiment, the present disclosure provides a vaccine, more particularly a composition of a vaccine, for preventing infection from a CoV, preferably a β-CoV, more preferably SARSr.

[0065] According to embodiments of the present disclosure, the vaccine comprises one or more consensus sequences derived from one or more CoVs, or β-CoVs, or preferably one or more SARSr, carried on at least one viral vector. The consensus sequences can be according to any embodiment or combination of embodiments described herein.

[0066] In one embodiment, the one or more consensus sequences are β-CoV DNA sequences, RNA sequences, protein sequences, or combinations thereof, preferably SARSr DNA sequences, RNA sequences, protein sequences, or combinations thereof.Furthermore, in one embodiment, the one or more consensus sequences are for a modified S protein.

[0067] According to embodiments of the present disclosure, the one or more consensus sequences include at least one SARSr DNA or RNA sequence, or preferably at least one SARS-2 β-CoV DNA or RNA sequence, hi one embodiment, the SARSr DNA or RNA sequence or the SARS-2 β-CoV DNA or RNA sequence is at least a portion of a modified S protein coding sequence.

[0068] The modified S protein is according to any embodiment or combination of embodiments provided herein. In certain embodiments, the S protein comprises at least one modification which is a partial or complete deletion or replacement of at least one variable region, preferably in the S1 region. In certain embodiments, the one or more consensus sequences comprise at least one SARSr DNA or RNA sequence, preferably at least one SARS-2 β-CoV DNA or RNA sequence, which is at least a portion of the modified S protein coding sequence, and at least one modification is a partial or complete removal or replacement of the RBD.

[0069] In one embodiment, expression of the consensus sequence is driven by a promoter. The promoter may be specific to the consensus sequence, the vaccinated animal, and the particular composition of the vaccine. In one embodiment, the promoter is selected from the group consisting of human cytomegalovirus immediate early promoter / enhancer, polyadenylation site from human growth gene, elongation factor 1-alpha, phosphoglycerate kinase, ubiquitin C, beta actin gene, and combinations thereof. In an embodiment, the activity of the promoter may be affected by chemicals, such as, but not limited to, antibiotics. Tetracycline is a non-limiting example of an antibiotic that affects the activity of the promoter.

[0070] In certain embodiments, the vaccine is specifically designed to prevent infection from at least SARS-2 β-CoV. In such embodiments, the one or more consensus sequences comprise at least one SARS-2 β-CoV DNA or RNA sequence. Preferably, the SARS-2 β-CoV DNA or RNA sequence is a DNA or RNA sequence encoding a modified S protein, the modified S protein being an S protein containing at least one modification, the at least one modification being at least one deletion or substitution in the S1 region. In further embodiments, the SARS-2 β-CoV DNA or RNA sequence is an RNA sequence that is a modified S protein coding sequence (partial or complete), the S protein having at least one modification being a deletion or substitution of all or part of the RBD.

[0071] In an embodiment where the consensus sequence is a SARS-2 β-CoV RNA sequence (partial or complete) encoding an S protein with at least a portion of the variable region of the S1 region deleted or replaced, the SARS-2 β-CoV DNA sequence is human codon-optimized and expression of the specific RNA is driven by the human cytomegalovirus immediate early promoter / enhancer followed by a polyadenylation site derived from the human growth gene. In other embodiments, expression of the SARS-2 β-CoV RNA is driven by other promoters, including but not limited to promoters derived from elongation factor 1-alpha, phosphoglycerate kinase, ubiquitin C, beta-actin genes, and combinations thereof. In another embodiment, expression of the SARS-2 β-CoV RNA is driven by a promoter whose activity can be affected by chemicals, including but not limited to the antibiotic tetracycline.

[0072] In further embodiments, the vaccine comprises two or more consensus sequences in one or more viral vectors. According to embodiments of the present disclosure, one consensus sequence is a SARS-2 β-CoV DNA, RNA or S protein sequence and the vaccine comprises at least one additional consensus sequence that is a SARSr DNA, RNA or protein sequence.

[0073] How to vaccinate In one embodiment, the present disclosure provides a method of vaccinating an animal subject, preferably a mammalian subject, more preferably a human subject, against infection from at least one group of CoV, preferably β-CoV.

[0074] According to an embodiment of the present disclosure, the method comprises modifying an S protein of a CoV, preferably a β-CoV, more preferably a SARS-2 β-CoV, to generate a modified S protein. The modified S protein comprises at least one modification that is a full deletion, a partial deletion, a full replacement or a partial replacement of the S1 region of the S protein. In a particular embodiment, the modified S protein comprises at least one deletion or replacement of the S1 sequence from the amino terminus to position 694. In a further embodiment, the modified S protein has a partial deletion of the variable region, a full deletion of the variable region, a partial replacement of the variable region, or a full replacement of the variable region. In one embodiment, the variable region is amino residues 342 to 533 of the S protein. In a further embodiment, the variable region corresponds to a part or all of the RBD. When the modification is a full or partial replacement of the variable region, the variable region may be replaced by a peptide linker.

[0075] The modified S protein can be used directly in a vaccine, for example, alone or in combination with a viral vector, hi a further embodiment, a consensus sequence of CoV DNA or RNA encoding the modified S protein can be used.

[0076] According to an embodiment of the present disclosure, the method includes providing a vaccine comprising at least one viral vector comprising at least one CoV consensus sequence, or at least one β-CoV consensus sequence, or at least one SARSr consensus sequence, or at least one SARS-2 β-CoV consensus sequence, where the consensus sequence is for a modified S protein. The at least one consensus sequence may be included in the at least one viral vector. The vaccine may further comprise a plasmid. The viral vector (including the consensus sequence) and the plasmid are transfected into a packaging cell, and the packaging cell is encapsidated into a capsid.

[0077] In one embodiment, the at least one CoV consensus sequence is according to any embodiment or combination or plurality of embodiments described herein. In a particular embodiment, the at least one CoV consensus sequence is a β-CoV DNA or RNA sequence encoding at least a portion of a modified S protein.

[0078] The method further comprises injecting the consensus sequence into an animal subject, preferably a mammalian subject, such as a human. In one embodiment, the single dose is sufficient to provide protection against at least one CoV, preferably at least one β-CoV, and more specifically, any CoV that shares 60% or more, or 65% or more, or 70% or more, or 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more sequence identity with at least one of the consensus sequences included in the vaccine.

[0079] In other embodiments, more than one dose may be required to provide protection. In particular, two, three, or four doses are sufficient to provide protection against at least one CoV, more particularly against any CoV that has 60% or more, or 65% or more, or 70% or more, or 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more sequence identity with at least one of the consensus sequences included in the vaccine. EXAMPLES

[0080] Example 1 A control group of 5 mice (BALB / c mice) will be vaccinated with placebo. An experimental group of 5 mice (BALB / c mice) will be vaccinated with vector suspension buffer (PBS, MgCl 2 3 × 10 cells suspended in 5 mM NaCl, 0.1 mM EDTA, 5% sucrose 7 Mice were vaccinated with a genome equivalent of GreMERSfl vaccine (containing a viral vector with a consensus sequence with at least 60% commonality with EMX / 2012 MERS-CoV). The consensus sequence is the full-length S protein of MERS-CoV. The control and experimental groups received a first intramuscular injection on day 1, followed by a booster dose with the same respective preparation on day 17. On day 19, both groups were infected intranasally with an LD50 of MERS. Both groups were mixed on day 21. Mice were tested for the presence of antibodies that neutralized infection of test cells with EMX / 2012 MERS-CoV. As shown in Figure 3, control mice did not show antibodies, while the experimental group showed significant virus neutralization.

[0081] Although several embodiments of the viral vectors and associated vaccines have been described in detail herein, it is clear that modifications and variations thereof are possible, all of which fall within the true spirit and scope of the invention. In particular, the present viral vectors and vaccines have been described in detail with respect to β-CoV, and more specifically SARS-2 and SARSr viruses, but it will be understood that the viral vectors and vaccines can be modified according to the art for application to other classes of coronaviruses, such as α-CoV, γ-CoV, and δ-CoV. Furthermore, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and described, and therefore, all suitable modifications and equivalents may be resorted to, within the scope of the present disclosure.

Claims

1. A vaccine for preventing CoV infection, comprising: A vaccine comprising at least one sequence selected from the group consisting of a CoV DNA sequence encoding at least a portion of a modified S protein of the CoV, a CoV RNA sequence encoding at least a portion of a modified S protein of the CoV, and a protein sequence of at least a portion of a modified S protein of the CoV, wherein the modified S protein comprises at least one modification.

2. 2. The vaccine of claim 1, wherein the S protein has an S1 region and the at least one modification is selected from the group consisting of a complete deletion of the S1 region, a partial deletion of the S1 region, a complete replacement of the S1 region, and a partial replacement of the S1 region.

3. 3. The vaccine of claim 2, wherein the S1 region has at least one variable region and the at least one modification is selected from the group consisting of a complete deletion of at least one variable region, a partial deletion of at least one variable region, a complete replacement of at least one variable region, and a partial replacement of at least one variable region.

4. 4. The vaccine of claim 3, wherein the CoV is SARS-2 β-CoV and at least one variable region is between amino acid residues 342 and 533.

5. The vaccine of claim 4, wherein at least one variable region comprises a receptor binding domain (RBD).

6. 6. The vaccine of claim 1, wherein at least one modification is a partial or complete replacement of the variable region, the variable region being replaced by a peptide linker.

7. The vaccine of any one of claims 1 to 3, wherein the CoV is an α-CoV.

8. The vaccine of any one of claims 1 to 6, wherein the CoV is a β-CoV.

9. 9. The vaccine of any one of claims 1 to 8, wherein the sequence is a CoV DNA sequence.

10. 10. The vaccine of claim 9, wherein the CoV DNA sequence is a β-CoV DNA sequence.

11. 9. The vaccine of claim 1, wherein the sequence is a CoV RNA sequence.

12. 12. The vaccine of claim 11, wherein the CoV RNA sequence is a β-CoV RNA sequence.

13. The vaccine of any one of claims 11 to 12, wherein the RNA is mRNA.

14. 9. The vaccine of any one of claims 1 to 8, wherein the sequence is a CoV protein sequence.

15. 15. The vaccine of claim 14, wherein the sequence is a β-CoV protein sequence.

16. The vaccine of any one of claims 1 to 6 and 8 to 15, wherein the sequence is a SARS-2 β-CoV sequence.

17. A modified CoV S protein comprising an S1 region and at least one modification in the S1 region selected from the group consisting of a partial deletion of the S1 region, a complete deletion of the S1 region, a partial replacement of the S1 region, and a complete replacement of the S1 region.

18. 18. The modified CoV S protein of claim 17, wherein the S1 region has at least one variable region and the at least one modification is selected from the group consisting of a complete deletion of at least one variable region, a partial deletion of at least one variable region, a complete replacement of at least one variable region, and a partial replacement of at least one variable region.

19. 20. The modified CoV S protein of claim 18, wherein the CoV is SARS-2 β-CoV and at least one variable region is between amino acid residues 342 and 533.

20. The modified CoV S protein of claim 19 , wherein at least one variable region comprises a receptor binding domain (RBD).

21. 21. The modified CoV S protein of claim 18, wherein at least one modification is a partial or complete replacement of the variable region, wherein the variable region is replaced by a peptide linker.

22. 1. A method for making a vaccine for protecting against infection with at least one CoV, comprising: A method comprising producing a modified S protein comprising an S1 region and at least one modification in the S1 region, wherein the at least one modification is selected from the group consisting of a partial deletion of the S1 region, a complete deletion of the S1 region, a partial replacement of the S1 region, and a complete replacement of the S1 region.

23. 1. A method of vaccinating a mammalian subject against infection from at least one group of CoVs, comprising: Separating a broad group of CoVs into homologous groups based on similarities in the CoV S protein; generating a modified S protein for each homology group, the modified S protein comprising an S1 region and at least one modification selected from the group consisting of a partial deletion of the S1 region, a complete deletion of the S1 region, a partial replacement of the S1 region, and a complete replacement of the S1 region; identifying at least one consensus sequence for each homology group that has greater than 60% sequence identity with all other members of the homology group, wherein the at least one consensus sequence is selected from the group consisting of a protein sequence of the modified S protein, a DNA sequence encoding the modified S protein, and an RNA sequence encoding the modified S protein; A method comprising:

24. 24. The method of claim 23, further comprising preparing a viral vector comprising at least one consensus sequence.

25. 25. The method of any one of claims 23 to 24, wherein the step of separating the broad group of CoVs into homologous groups comprises obtaining at least one CoV from a mammal.

26. 26. The method of claim 25, wherein the mammal is a human.

27. 27. The method of any one of claims 23 to 26, wherein the S1 region has at least one variable region and the at least one modification is selected from the group consisting of a complete deletion of at least one variable region, a partial deletion of at least one variable region, a complete replacement of at least one variable region, and a partial replacement of at least one variable region.

28. 28. The method of claim 27, wherein the CoV is SARS-2 β-CoV and at least one variable region is between amino acid residues 342 and 533.

29. 29. The method of claim 28, wherein at least one variable region comprises a receptor binding domain (RBD).

30. 30. The method of any one of claims 27 to 29, wherein at least one modification is a partial or complete replacement of the variable region, wherein the variable region is replaced by a peptide linker.

31. 28. The method of any one of claims 23 to 27, wherein the CoV is α-CoV.

32. 31. The method of any one of claims 23 to 30, wherein the CoV is β-CoV.

33. 33. The method of any one of claims 23 to 32, wherein the consensus sequence is a CoV DNA sequence.

34. 34. The method of claim 33, wherein the CoV DNA sequence is a β-CoV DNA sequence.

35. 33. The method of any one of claims 23 to 32, wherein the consensus sequence is a CoV RNA sequence.

36. 36. The method of claim 35, wherein the CoV RNA sequence is a β-CoV RNA sequence.

37. 37. The method of claim 35 or 36, wherein the RNA is mRNA.

38. 33. The method of any one of claims 23 to 32, wherein the consensus sequence is a CoV protein sequence.

39. 39. The method of claim 38, wherein the sequence is a β-CoV protein sequence.

40. 40. The method of any one of claims 23 to 30 and 32 to 39, wherein the sequence is a SARS-2 β-CoV sequence.

41. 41. The method of any one of claims 23 to 40, further comprising injecting the vaccine into a mammalian subject.

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