NANT COVID Vaccine Cross-Reactivity

Abstract

Recombinant SARS-CoV2 vaccine compositions and methods are presented that have significant reactivity against SARS-CoV2A, as well as unexpected cross-reactivity against a variety of other coronaviruses, particularly SARS-CoV1, MERS-CoV, OC43-CoV, and HKU1-CoV. Furthermore, the vaccine compositions presented herein also generated cross-reactive memory B cells and cross-reactive memory T cells with cross-reactivity across a relatively wide range of different coronaviruses.

Description

[Technical field]

[0001] This application claims the benefit of co-pending U.S. Provisional Patent Application No. 63 / 284,203, filed November 30, 2021, which is incorporated by reference in its entirety herein.

[0002] The field of the invention is vaccine compositions and methods, particularly as it relates to cross-reactive vaccine compositions effective against various coronaviruses. [Background technology]

[0003] The Background Description contains information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In the event that a definition or use of a term in an incorporated reference is inconsistent with or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0005] Although SARS-CoV2 diagnostic tests have become available in a relatively short time, numerous attempts to treat the disease have so far shown conflicting or inconclusive results. Most typically, patients with severe symptoms are treated to maintain breathing / blood oxygenation. More recently, vaccination efforts and the use of antibody cocktails (e.g., casirivimab and imvimab), as well as newly developed antivirals such as paxolobid (Pfizer) or molnupiravir (Merck), have reduced hospitalization and mortality rates. Nevertheless, COVID19 mortality rates have remained significant, especially in the elderly, the immunocompromised, and individuals with cardiac, pulmonary, or diabetes. Despite improvements in acute care, it has become clear that disease containment is crucial, as social distancing and other public health mitigation measures can only provide moderate relief. The need for such containment is particularly pressing, as it is expected that new viral mutants will inevitably evolve over time, and that at least some of these mutants may evade currently known immunotherapies.

[0006] Moreover, the protection of the recently introduced SARS-CoV2 RNA vaccines is not equally effective against SARS-CoV2 wild virus variants, as can be seen in Figure 1. And even when individuals, such as first responders and healthcare workers, are vaccinated early, the protection against new infections begins to wane after a relatively short period of time, as can be seen in Figure 2.

[0007] To address this urgent need, a number of candidate anti-SARS-CoV2 vaccine compositions have been developed, targeting one or more proteins of the virus (see, for example, FIMMU 2020, 11:602256). For example, Sinovac and Sinopharm are currently testing inactivated virus vaccine preparations. Cansino Biologics, Janssen Pharma, Oxford University, and Garnaleya have developed vaccines based on non-replicating adenoviral vectors encoding one or more viral proteins. Novamax has produced a protein subunit-based vaccine. More recently, RNA-based vaccines from Moderna and Pfizer have been approved in several jurisdictions. Most of these vaccines induce at least some (typically non-sterile) immunity against disease-causing infection, but it is unclear whether protection is effective across different variants or even strains, whether protection is effective over months, and / or whether sufficient immune memory protects vaccinated individuals over an extended period of time. Furthermore, it is unclear whether such vaccines would generate clinically meaningful T cell-based responses.Unfortunately, and despite the relatively large number of vaccine formulations in development and use, none of the known vaccine compositions have been shown to be cross-reactive to other coronaviruses, such as MERS-CoV, OC43-CoV, or HKU1-CoV, thereby limiting the usefulness of such vaccines, nor have they been shown to elicit durable memory B and T cell populations.

[0008] Thus, although various vaccine compositions and methods for targeting coronaviruses are known in the art, all or almost all of them have some drawbacks, especially when the vaccine is highly specific for only a single variant of a particular strain. Thus, there remains a need for improved coronavirus compositions and methods that are effective against various coronavirus strains and their variants. Summary of the Invention [Means for solving the problem]

[0009] The subject matter of the present invention relates to various vaccine compositions and methods for generating immune responses against multiple coronaviruses, including SARS-CoV1, SARS-CoV2, MERS-CoV, OC43-CoV, and HKU1-CoV. Notably, the vaccine compositions presented herein, which target both the S (spike protein) and N (nucleocapsid) of SARS-CoV2, showed unexpected cross-reactivity to various other coronaviruses, in addition to SARS-CoV2, particularly SARS-CoV1, MERS-CoV, OC43-CoV, and HKU1-CoV. Even more notably, the vaccine compositions presented herein also generated cross-reactive memory B cells and cross-reactive memory T cells with cross-reactivity across a relatively wide range of different coronaviruses.

[0010] In one aspect of the present subject matter, the inventors contemplate a method of eliciting a cross-reactive immune response against a coronavirus in a subject, comprising administering a recombinant vaccine composition to the subject in prime and / or boost doses. In such a method, the recombinant vaccine composition has (a) a first portion encoding a severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein (N) fused to an endosomal targeting sequence (N-ETSD), the first portion being operably linked to one or more regulatory elements enabling N-ETSD expression, and (b) a second portion encoding a SARS virus spike protein (S), the second portion being operably linked to one or more regulatory elements enabling S expression. The vaccine composition is administered to the subject in an amount that elicits a cross-reactive immune response, the cross-reactive immune response extending from SARS-CoV2 to serologically distinct variants of SARS-CoV2 and / or to coronaviruses other than SARS-CoV2. Most typically, the coronavirus other than SARS-CoV2 is SARS-CoV1, MERS-CoV, OC43-CoV, and / or HKU1-CoV.

[0011] In some embodiments, the immune response is the production of antibodies that bind to at least two of serologically distinct variants of SARS-CoV2 and / or SARS-CoV2 and at least one coronavirus other than SARS-CoV2, while in other embodiments, the immune response is the production of cytotoxic T cells that are cytotoxic to different cells each carrying a serologically distinct variant of SARS-CoV2 and / or cells carrying SARS-CoV2 and cells carrying a coronavirus other than SARS-CoV2. In further embodiments, the immune response is the production of cross-reactive memory T cells, while in still other embodiments, the immune response is the production of cross-reactive memory B cells.

[0012] Preferably, the N protein is from SARS-CoV-2, and it is contemplated that the endosomal targeting sequence of the N-ETSD is encoded at the 5' end of the first portion or at the 3' end of the first portion. Furthermore, it is preferred that the first and second portions are arranged in a bicistronic sequence. For example, the N-ETSD may have an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:1, or may have the amino acid sequence of SEQ ID NO:1. In another example, the first portion may have the nucleotide sequence of SEQ ID NO:2.

[0013] With respect to the S protein, it is contemplated that the S protein may have an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or that the S protein has the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. For example, the second portion may have the nucleotide sequence of SEQ ID NO: 5 or the nucleotide sequence of SEQ ID NO: 6.

[0014] In further contemplated embodiments, the recombinant vaccine composition can be formulated as a recombinant virus, most preferably an adenovirus having an E1 gene region deletion and an E2b gene region deletion. Alternatively, or in addition, the recombinant vaccine composition is formulated as a recombinant RNA, preferably a polycistronic RNA comprising a first and a second portion. Optionally, the recombinant vaccine composition can also be formulated as a recombinant DNA, preferably comprising a first and a second portion.

[0015] It is further contemplated that the recombinant vaccine composition is administered in prime and boost doses. Preferably, although not necessarily, the recombinant vaccine composition is formulated as an adenovirus vaccine composition.

[0016] In yet other embodiments, the recombinant vaccine composition is administered only in a boost administration. In such cases, the boost administration may be followed by a prime vaccination using a vaccine such as an RNA vaccine, a DNA vaccine, a viral vaccine, or a subunit vaccine. An exemplary RNA vaccine prime vaccination may be a self-amplifying, self-adjuvanting RNA vaccine (preferably comprising RNA encoding the coronavirus S protein and / or the coronavirus N protein), and an exemplary viral vaccine prime vaccination may include an adenovirus viral vaccine (preferably comprising a recombinant nucleic acid encoding only the coronavirus S protein).

[0017] In another aspect of the inventive subject matter, the inventors contemplate a method of generating memory B cells and / or memory T cells that are cross-reactive against multiple different coronaviruses, comprising administering to a subject a recombinant vaccine composition in a prime and / or boost dose, the recombinant vaccine composition having (a) a first portion encoding a severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein (N) fused to an endosomal targeting sequence (N-ETSD), the first portion being operably linked to one or more regulatory elements that allow for N-ETSD expression, and (b) a second portion encoding a SARS virus spike protein (S), the second portion being operably linked to one or more regulatory elements that allow for S expression. It is contemplated that the memory B cells produce antibodies that are cross-reactive. Most typically, the vaccine composition is administered in an amount that induces the formation of cross-reactive memory B cells and / or memory T cells. Most typically, the multiple different coronaviruses include SARS-CoV1, SARS-CoV2, MERS-CoV, OC43-CoV, and HKU1-CoV.

[0018] It is further generally preferred that the nucleocapsid protein N is derived from SARS-CoV-2, which may further comprise an endosomal targeting sequence at the 5' or 3' end. In a further preferred embodiment, the first and second parts are arranged in a bicistronic arrangement. For example, the N-ETSD may have an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, or may have the amino acid sequence of SEQ ID NO: 1. Thus, the first part has the nucleotide sequence of SEQ ID NO: 2.

[0019] The spike S protein preferably has an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or has the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. Thus, the second portion may have the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

[0020] As will be readily understood, the recombinant vaccine composition may be formulated as a recombinant virus (e.g., an adenovirus having an E1 gene region deletion and an E2b gene region deletion), or may be formulated as recombinant RNA (e.g., a polycistronic RNA comprising a first and a second portion), or may be formulated as recombinant DNA (e.g., comprising a first and a second portion).

[0021] Viewed from a different perspective, the inventor also contemplates a kit comprising a first recombinant vaccine composition having (a) a first portion encoding a severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein (N) fused to an endosomal targeting sequence (N-ETSD), the first portion being operably linked to one or more regulatory elements enabling N-ETSD expression, and (b) a second portion encoding a SARS virus spike protein (S), the second portion being operably linked to one or more regulatory elements enabling S expression. The kit would also include (a) a recombinant viral vaccine comprising a recombinant nucleic acid encoding a SARS virus spike protein (S) operably linked to one or more regulatory elements enabling S expression; or (b) a self-amplifying, self-adjuvanting RNA vaccine comprising a recombinant nucleic acid encoding a SARS virus spike protein (S) operably linked to one or more regulatory elements enabling S expression, and optionally further encoding a Severe Acute Respiratory Syndrome (SARS) coronavirus nucleocapsid protein (N) operably linked to one or more regulatory elements enabling N expression; or (c) a subunit vaccine comprising a coronavirus recombinant protein; or (d) a second recombinant vaccine composition having a heat-inactivated coronavirus vaccine composition.

[0022] Accordingly, the inventors contemplate a recombinant vaccine composition for use as a vaccine to elicit a cross-reactive immune response against coronavirus in a subject, characterized in that the recombinant vaccine composition comprises: (a) a first portion encoding a Severe Acute Respiratory Syndrome (SARS) coronavirus nucleocapsid protein (N) fused to an endosomal targeting sequence (N-ETSD), the first portion being operably linked to one or more regulatory elements enabling N-ETSD expression; and (b) a second portion encoding a SARS virus spike protein (S), the second portion being operably linked to one or more regulatory elements enabling S expression. Preferably, the cross-reactive immune response extends from SARS-CoV2 to serologically distinct variants of SARS-CoV2 and / or from SARS-CoV2 to coronaviruses other than SARS-CoV2.

[0023] Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings in which like numerals represent like elements. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 is a schematic showing the differential efficacy of SARS-CoV2 RNA vaccines against various strains of SARS-CoV2. [Diagram 2] FIG. 1 is a schematic showing the reduced protective efficacy of SARS-CoV2 RNA vaccines. [Diagram 3] FIG. 1 shows a schematic diagram of an exemplary recombinant hAd5 virus used in the cross-reactive vaccine compositions and methods presented herein. [Figure 4A-D] Figure 4 shows exemplary results for antibody cross-reactivity in individuals following vaccination with the recombinant hAd5 virus of Figure 3. Figure 4A shows cross-reactivity results for MERS-CoV, Figure 4B shows cross-reactivity results for HCoV-HKU1, Figure 4C shows cross-reactivity results for HCoV-OC43, and Figure 4D shows the time course for cross-reactivity. [Diagram 5] FIG. 3 shows exemplary results for memory B cells generated in non-human primates after vaccination with recombinant hAd5 virus, demonstrating that hAd5 S+N induces cross-reactive memory B cells to N of SARS-CoV-2. [Figure 6] FIG. 3 shows exemplary results for memory B cells generated in healthy human subjects after vaccination with recombinant hAd5 virus, demonstrating that hAd5 S+N induces cross-reactive memory B cells against SARS-CoV-2 N. [Figure 7] FIG. 3 shows exemplary results for memory T cells generated in healthy human subjects after vaccination with recombinant hAd5 virus, demonstrating that hAd5 S+N induces cross-reactive memory B cells against SARS-CoV-2 N. [Figure 8] FIG. 4 shows one exemplary prime-boost vaccine regimen using the recombinant hAd5 virus of FIG. [Figure 9] FIG. 4 shows an exemplary SASA vaccine composition suitable for use in a prime-boost vaccine regimen using the recombinant hAd5 virus of FIG. [Figure 10] FIG. 4 shows another exemplary prime-boost vaccine regimen using the recombinant hAd5 virus of FIG. [Figure 11] 1 shows exemplary B and T cell cross-reactivity for a universal COVID vaccine. [Figure 12] FIG. 1 shows an exemplary validation of the requirement of S+N to induce long-term memory B&T cells for a universal second generation vaccine. [Figure 13] 1 shows the exemplary importance of N in generating T cell responses. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The inventors have discovered that various SARS-CoV2 vaccine compositions containing nucleocapsid components unexpectedly elicit cross-reactive immune responses in human and non-human subjects upon administration, particularly as boost doses. Notably, the cross-reactivity extended not only across different SARS-CoV2 strains, but also to other members of the coronaviridae family, including SARS-CoV1, MERS-CoV, OC43-CoV, and / or HKU1-CoV. Even more notably, the cross-reactivity was a durable response in which cross-reactive memory T cells and memory B cells were observed, as described in more detail below.

[0026] For example, one vaccine composition containing both S and N components is shown in Figure 3, where the vaccine composition is formulated as a recombinant human adenovirus, specifically hAd5, with deletions in E1, E2b, and E3. A recombinant nucleic acid is inserted into the viral genome, having a first segment encoding an S-fusion protein (comprising the S protein of SARS-CoV2 fused to a segment that enhances the expression of the fusion protein) and a second segment encoding an N-ETSD (comprising the N protein of SARS-CoV2 and an endosomal targeting segment). As can be seen in Figure 3, both the S-fusion and the N-ETSD are under the control of a strong constitutive CMV promoter to drive the expression of the recombinant SARS-CoV2 protein in cells infected with the recombinant virus.

[0027] The above-mentioned adenovirus-based vaccines containing hAd5 S-fusions + N-ETSD used a unique, clinically available, only human adenovirus (hAd5) vector technology without adenovirus fiber production due to deletion of E1, E2b, and E3 genes, allowing for potent and sustained protein production for maximum cellular and humoral immunity. Moreover, such recombinant adenoviruses showed a proven safety profile in 13 Phase I / II clinical trials in over 125 elderly and immunocompromised cancer patients. Furthermore, the recombinant adenovirus in Figure 3 produced antigen-specific CD4+ and CD8+ T cells in patients even with prior adenovirus immunity. Thus, it should be appreciated that the recombinant adenovirus technology provided a unique vaccine construct that maximizes cell-mediated immunogenicity and reduces the risk of antibody-dependent enhancement. It should also be further recognized that such recombinant viruses can be prepared in large quantities using established cell lines, and such vaccines are stable under simple refrigeration (2-8 °C).

[0028] While this recombinant virus vaccine construct is generally preferred for the contemplated uses and methods, it should be recognized that numerous modifications can be made so long as the vaccine construct contains the N-protein components. Thus, it should be understood that recombinant constructs include recombinant viruses and recombinant yeast, each of which contains recombinant nucleic acids that will result in expression of the N protein (or modifications and / or portions thereof) and the S protein (or modifications and / or portions thereof).

[0029] In one embodiment, the N-ETSD polypeptide can comprise a sequence having at least 80% identity to SEQ ID NO:1. In other embodiments, the identity value is at least 85%. In yet other embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%. It is further contemplated that the N-ETSD fusion protein comprises a linker between the N-ETSD domain and the nucleocapsid protein. For example, the linker can be a 16 amino acid linker having the sequence (G3S)4. In certain embodiments, methods are disclosed herein for enhancing the immunogenicity of intracellular antigens, the methods comprising tagging the antigen with ETSD and expressing the tagged antigen in an antigen-presenting cell (e.g., a dendritic cell).

[0030] In some embodiments, a fusion protein comprising N-ETSD and a CoV-2 nucleocapsid protein may be encoded by a nucleic acid sequence having at least 80% identity to SEQ ID NO:2. In some embodiments, the identity value is at least 85%. In some embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%.

[0031] It is contemplated that the CoV-2 spike protein has at least 85% identity to SEQ ID NO:3. In some embodiments, the identity value is at least 85%. In some embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%. The nucleic acid encoding the CoV-2 spike protein has at least 85% identity to SEQ ID NO:5. In some embodiments, the identity value is at least 85%. In some embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%.

[0032] It is contemplated that the CoV-2 spike fusion protein has at least 85% identity to SEQ ID NO:4. In some embodiments, the identity value is at least 85%. In some embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%. The nucleic acid encoding the CoV-2 spike fusion protein has at least 85% identity to SEQ ID NO:6. In some embodiments, the identity value is at least 85%. In some embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%.

[0033] In a second aspect of the disclosure, provided herein is a recombinant yeast comprising a nucleic acid encoding a protein selected from the group consisting of a coronavirus 2 (CoV-2) nucleocapsid protein, a CoV2 N-ETSD protein, a CoV2 spike protein, a CoV2 spike-fusion protein, and combinations thereof. Additionally, each of these encoded proteins may be further modified, as described in more detail below. Preferably, the recombinant yeast is Saccharomyces cerevisiae.

[0034] In some embodiments of this second aspect, the CoV-2 nucleocapsid protein or variant thereof comprises a sequence having at least 80% identity to SEQ ID NO:1 or SEQ ID NO:7. In other embodiments, the identity value is at least 85%. In still other embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%.

[0035] In some embodiments of this second aspect, the CoV-2 spike protein or spike fusion protein comprises a sequence having at least 80% identity to SEQ ID NO:3 or SEQ ID NO:4. In other embodiments, the identity value is at least 85%. In still other embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%.

[0036] In some embodiments, the nucleic acid encoding a CoV-2 spike protein or spike fusion protein comprises a sequence having at least 80% identity to SEQ ID NO:5 or SEQ ID NO:6. In other embodiments, the identity value is at least 85%. In still other embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%.

[0037] Most preferably, the recombinant virus is administered via subcutaneous or subdermal injection. However, in other contemplated embodiments, administration can also be intravenous or intramuscular. In another embodiment, the recombinant virus can be administered intranasally, for example, via an intranasal spray. Alternatively or additionally, antigen-presenting cells can be isolated or grown from the patient's cells, infected in vitro, and then injected into the patient.

[0038] In one aspect of any of the embodiments described above or elsewhere herein, the composition is formulated in a pharma- ceutically acceptable excipient suitable for administration to a subject.

[0039] The immunotherapeutic compositions disclosed herein can be either "prophylactic" or "therapeutic." When provided prophylactically, the compositions of the disclosure are provided prior to or upon detection of an outbreak of coronavirus disease, with the goal of preventing, inhibiting, or delaying the onset of coronavirus disease; and / or generally preventing or inhibiting the progression of coronavirus disease in an individual. Thus, prophylactic compositions can be administered to individuals believed to be free of coronavirus disease (healthy, or normal individuals) or individuals in whom coronavirus has not yet been detected. Individuals at high risk of developing coronavirus disease can be treated prophylactically with the compositions of the disclosure.

[0040] When provided therapeutically, the immunotherapeutic composition is provided to an individual diagnosed with a coronavirus disease with the objective of ameliorating or curing the coronavirus disease; increasing the individual's survival rate; or preventing, inhibiting, reversing or delaying the onset of the coronavirus disease in the individual.

[0041] In yet another embodiment, disclosed herein is a vaccine composition comprising the adenovirus or yeast disclosed above, wherein the composition is formulated for injection. The vaccine composition can be used to induce immunity against CoV-2 in a patient in need thereof by administering the vaccine composition to the patient.

[0042] Also disclosed herein are methods for preventing and / or treating coronavirus diseases, particularly COVID-19. Preferably, the methods include using a virus or yeast vector encoding a wild-type or modified form of the coronavirus nucleocapsid protein and / or a wild-type or modified form of the spike protein in an immunogenic composition administered to a subject individual. The virus and / or yeast vaccine so administered will infect the individual with the wild-type or modified form of CoV-2, nucleocapsid or spike protein. Once that is in place, the individual will have an immune response against it and will be vaccinated. In particular, since the nucleocapsid protein and the spike protein are relatively conserved polypeptides, an immune response can be elicited against diverse members of the coronavirus family.

[0043] In the case where the recombinant vector is an adenovirus, the adenovirus vector can be modified to encode wild-type or modified forms of nucleocapsid protein and / or spike protein. Similarly, in the case of yeast, the yeast vector can also be modified to encode wild-type or modified forms of nucleocapsid protein and / or spike protein. As shown in more detail below, after administration of an immunogenic composition comprising a virus and / or yeast vector in a patient in need thereof, a positive immune response is obtained for cell-mediated immunity. Thus, in one embodiment, the present disclosure contemplates creating expression of coronavirus spikes on the yeast surface. In such an embodiment, the yeast functions as an avatar coronavirus to stimulate B cells, which then provide humoral immunity.

[0044] As disclosed herein, a next-generation bivalent human adenovirus serotype 5 (hAd5) vaccine capable of inducing immunity in patients with pre-existing adenovirus immunity, which contains both an S sequence optimized for cell surface expression (S-fusion) and a conserved nucleocapsid (N) antigen designed to be trafficked to the endosomal intracellular compartment, has the potential to generate durable immune protection. As further described herein, such a bivalent vaccine has been found to be optimized for immunogenicity as evidenced by the following findings: 1) The optimized S-fusions showed improved S receptor binding domain (RBD) cell surface expression compared to S-WT, which had barely detectable surface expression; 2) the expressed RBD from the S-fusions retained conformational integrity and recognition by ACE2-Fc; 3) viral N proteins modified with enhanced T cell stimulatory domains (ETSDs) localized to endosomal / lysosomal intracellular compartments for MHC I / II presentation; and 4) Optimization of these S and N (S-fusions and N-ETSDs) generated enhanced de novo antigen-specific B cell and CD4+ and CD8+ T cell responses in antigen-native preclinical models.

[0045] Both T cell and antibody immune responses to the S and N components showed a T-helper 1 (Th1) bias. The antibody responses were neutralizing as shown by an independent SARS-CoV-2 neutralization assay. Thus, in one embodiment, the next generation bivalent hAd5 S-fusion + N-ETSD vaccine provides robust and durable cell-mediated and humoral immunity against SARS-CoV-2 infection. Additionally, and as further described in more detail below, the vaccine constructs can be administered orally, intranasally, or sublingually. Thus, in one embodiment, the present disclosure also provides vaccine constructs in oral, intranasal, and sublingual formulations, in addition to injectable formulations (e.g., SC or IM), to induce mucosal immunity in addition to cell-mediated and humoral immunity. Viewed from another perspective, substantial immunity can be generated by injection, oral / mucosal administration, alone or in combination. In one embodiment, the COVID-19 vaccine disclosed herein generates long-term T and B cell memory. Further aspects, advantages and considerations suitable for use herein are disclosed in the applicants' co-pending international publication having Publication No. WO 2021 / 183665 (PCT / US21 / 21737), the entirety of which is incorporated herein by reference.

[0046] Using the above-mentioned adenoviral hAd5 S+N vaccine composition as shown in FIG. 3 in a prime and boost regimen in humans (healthy volunteers), the inventors discovered that not only did the vaccine composition after boost induce a strong immune response against S and N of SARS-CoV2, but the antibodies of vaccinated humans also had significant cross-reactivity against other coronaviruses, particularly MERS-CoV, HcoV-HKU1 and HCoV-OC43, as exemplarily shown in FIG. 4A, FIG. 4B and FIG. 4C, respectively. When observing the time course of antibody production in vaccinated volunteers, it was observed that anti-N antibodies increased rapidly compared to anti-S antibodies, as seen in FIG. 4D. Such a finding was completely unexpected and is due to the presence of N as a component in the vaccine, and possibly also due to the ETSD sequence bound to the N protein, which directs the N protein to the endosomal presentation pathway via MHC-II, thereby inducing a strong CD4+ response.

[0047] Following these results, the inventors then sought to identify whether the vaccine composition presented herein also induces cross-reactive memory B cells against N in response to vaccination. Notably, the hAd5 S+N vaccine again induced the production of cross-reactive memory B cells, as shown in the exemplary data in FIG. 5. Here, cross-reactivity was observed against MERS-CoV, HcoV-HKU1, and HCoV-OC43. Similarly, when healthy human volunteers were subjected to prime and boost vaccination with the hAd5 S+N vaccine, the vaccine induced the formation of memory B cells, as shown in the exemplary data in FIG. 6. Again, cross-reactivity was observed against MERS-CoV, HcoV-HKU1, and HCoV-OC43.

[0048] A further set of experiments was then performed to determine whether the hAd5 S+N vaccine induces the formation of cross-reactive memory T cells in healthy human volunteers. Exemplary results are shown in Figure 7. As is readily apparent, the vaccine was effective not only against the wild type variant, but across a broad range of variants.

[0049] While the above experimental data was obtained under a protocol using hAd5 S+N vaccine in both prime and boost administrations, it should be understood that the vaccine formulations presented herein are suitable for either prime or boost. However, it is specifically contemplated that the vaccine compositions presented herein are particularly beneficial when used in a boost administration following a prime administration that may or may not include an N-component. Thus, contemplated prime vaccine administrations that may be followed by the vaccine compositions presented herein include those that target the S protein, fragments of the S protein (and particularly fragments that include the RBD of the S protein), and / or fusion proteins of the S protein or fragments thereof.

[0050] For example, a suitable prime / boost regime is shown diagrammatically in FIG. 8, where the prime vaccination uses a recombinant adenovirus (here Ad26) containing a nucleic acid encoding the S protein. The boost vaccination uses a hAd5 S+N vaccine, as diagrammatically shown in FIG. 3. Alternatively, the prime vaccination does not have to be based on a recombinant virus as described above, but may use a SASA type vaccine composition in which the nucleic acid encoding the S and / or N proteins is bound to a lipid carrier to form a self-amplifying, self-adjuvanting RNA or DNA vaccine, as exemplarily shown in FIG. 9. SASA type vaccines have various advantages over nanoparticle-based RNA vaccines (such as those provided by Pfizer or Moderna). The table below shows exemplary advantages for SASA type vaccines as opposed to nanoparticle-based RNA vaccines.

[0051] [Table 1]

[0052] Therefore, the inventors also contemplate the use of SASA prime vaccination as exemplarily shown in FIG. 10 followed by recombinant virus boost vaccination using hAd5 S+N vaccine as exemplarily shown in FIG. 3. In this context, it should be understood that heterologous prime boost ("Mix and Match") vaccine regimes have been shown to induce some of the strongest and potentially most durable immune responses against COVID. In particular, "prime" vaccination with RNA vaccines results in strong antibody responses, while "boost" vaccination with recombinant adenovirus vaccines results in strong cellular immune responses. Such a vaccine strategy, exemplarily outlined in FIG. 10, is believed to deliver strong antibody responses: strong Th1 antibodies against both wild type and β variants, as well as strong immune cell responses: strong CD8+ Tt cells against both wild type and β variants S and N, as well as strong CD4+ T cells against both wild type and β variants S and N.

[0053] It is therefore contemplated that any given prime vaccination against SARS-CoV2 can be substantially enhanced with a boost vaccination using a hAd5 S+N vaccine (or other vaccine formulations containing an N component) as exemplarily shown in Figure 3. Indeed, the hAd5 S+N vaccine may also be suitable when an individual has already received a prime and boost vaccination (e.g., Pfizer, Moderna, or Johnson & Johnson vaccines). Such an additional boost would confer the same advantages in terms of cross-reactivity and memory B and T cell formation.

[0054] In further contemplated aspects of the present subject matter, particularly when recombinant S and / or N proteins are expressed in yeast or another suitable expression system, the recombinant proteins can be combined with the adjuvant 3M-052-Alum (developed by IDRI and 3M) as a subunit vaccine. As unexpectedly observed, the 3M-052-Alum adjuvant also elicited significant cross-reactivity against other SARS-CoV variants and even other coronaviruses. Thus, the N / N-ETSD and S / S fusion sequences presented herein are particularly contemplated for such subunit vaccines with the 3M-052-Alum adjuvant.

[0055] The key new findings of B and T cell cross-reactivity for a universal COVID vaccine are shown in Figure 11. Hicks J et al. (Serologic cross-reactivity of SARS-CoV-2 with endemic and seasonal Betacoronaviruses. J Clin Immunol. 2021 Mar 16, incorporated herein by reference) disclose possible cross-reactivity of SARS-CoV-2 antibodies with the complete spike proteins of four other betacoronaviruses that cause disease in humans: MERS-CoV, SARS-CoV, HCoV-OC43 and HCoV-HKU1. It was found that there is potential cross-reactivity of antibodies against SARS-CoV-2 with the other four coronaviruses, with the strongest cross-recognition between SARS-CoV-2 and SARS / MERS-CoV antibodies (as would be expected based on the sequence homology of their respective spike proteins).

[0056] The results disclosed herein support the inclusion of non-spike antigens in second generation vaccines. In particular, T cells induced by common cold coronaviruses play a protective role against SARS-COV2 infection. These T cells provide protection by attacking proteins within the virus, rather than the spike protein on the surface of the virus. The spike protein is under strong immune pressure from vaccine-induced antibodies that drive the evolution of vaccine escape variants. In contrast, there are far fewer mutations in the internal proteins that T cells target. As a result, they are highly conserved among the various SARS-CoV-2 variants, including omicron. Thus, the vaccines disclosed herein that induce broadly protective T cell responses provide better protection against current and future SARS-CoV-2 variants.

[0057] FIG. 12 validates the necessity for the presence of both S+N to induce long-term memory B and T cells for a universal second generation vaccine. SARS-CoV-2 infected patients are protected by cross-reactive T cells without antibodies. hAd5 S+N vaccination induces memory B cells with full protection after viral challenge in NHPs. hAd5 S+N vaccination induces both T cells and cross-reactive memory B cells in healthy subjects. The importance of N in generating T cell responses is further disclosed in FIG. 13. As can be seen from this figure, the hAd5 S+N vaccine prime+boost schedule disclosed herein provides better and longer protection compared to spike-based vaccines. As a result, the inventors have surprisingly found that the vaccine composition presented herein, which targets both S and N of SARS-CoV2, shows unexpected cross-reactivity against various other coronaviruses in addition to SARS-CoV2, in particular SARS-CoV1, MERS-CoV, OC43-CoV and HKU1-CoV. EXAMPLES

[0058] Embodiments of the present disclosure are further described in the following examples, which are merely illustrative and do not in any way limit the scope of the invention as claimed.

[0059] Example 1 For the experiments performed and data presented, the following reagents and methods were used in addition to well-known protocols.

[0060] Peptide pool (Pepmix™): 15-mer peptides overlapping by 11 amino acids and spanning the entire protein sequence of the spike of SARS-CoV-2 (Wuhan, Alpha, Epsilon, Gamma and Beta) were purchased from JPT (JPT Peptide Technologies GmbH, Berlin, Germany).

[0061] ELISpot assay: ELISpot plates were coated with human IFNγ and IL-4 antibodies (ImmunoSpot, Cleveland, USA) overnight at 4°C. Then, 300,000 PBMCs were seeded per well and stimulated with SARS-CoV-2 Pepmix™ (2.5 μg / ml / peptide, JPT, Germany) for 44-48 h, and subsequently, plates were developed according to the kit instructions (hIFNgIL4-2M / 2, Immunospot). Plates were scanned and spot-forming units (SFU) were quantified using an ImmunoSpot S6 Universal-V Analyzer with ImmunoSpot MultiSet AutoCount™ software.

[0062] Example 2: Generation of cytometric bead arrays Conjugation of beads with streptavidin: Cytometric bead arrays (CBAs) used in this analysis were constructed using Spherotech 4um and 5um Carboxy Blue Pak Array Kits (cat PAK-4067-8K and PAK-5067-10K, respectively). The beads were first functionalized by conjugating streptavidin (SA) to the beads via the commonly used 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) chemistry. SA (Southern Biotech cat 7105-01) was buffer exchanged into PBS using a pd-10 column (Cytiva 17-0851-01) and diluted to 2mg / mL. For conjugation, 10e8 Spherotech particles were isolated by centrifugation at 10,000Xg for 3 minutes. After careful removal of the supernatant, the bead pellet was resuspended in 0.5ml of SA in PBS. After thorough resuspension by pipetting, 0.5 ml of 6 mM EDC dissolved in 0.05 M MES buffer PH 5.0 was added and the reaction mixture was rotated overnight at room temperature. After the conjugation reaction was completed, 0.1 ml of 1 M tris PH 8.0 was added to quench the reaction. After 1 hour of incubation rotating at room temperature, the beads were collected by centrifugation as above and washed twice in 1 ml PBS. After the final wash, the beads were resuspended in 1 ml PBS containing 0.25% NaN3 and stored at 4°C until use.

[0063] SA Loading Quality Assurance: Following SA conjugation, quality control experiments were performed to determine the extent and uniformity of labeling (if multiple particle sizes and / or peak identities are used) by staining the SA-conjugated particles with fluorescently labeled biotinylated hemagglutinin (PR8). The individual array components were mixed and diluted to 1e6 of each particle per mL. 40 ml of serial dilutions of PR8 were prepared in 96-well U-bottom plates (costar 3797) ranging from 1ug / mL to 2ng / mL. 5 ml of bead suspension was added, mixed by pipetting, and incubated for 15 minutes at room temperature. 200 ml of PBS was added and the plate was centrifuged at 3000Xg for 5 minutes. The beads were resuspended in 80 ml of PBS. Samples were then analyzed by flow cytometry.

[0064] Recombinant antigen absorption: Following the SA conjugation and quality control procedures described above, biotinylated recombinant array antigens were passively absorbed onto individual particles. For antigens used in this array configuration, a single biotin site was enzymatically added onto the carboxy-terminal AVI tag. SA-conjugated particles were collected by centrifugation as described above and resuspended in 1 mg / mL of biotinylated recombinant protein in 1% BSA in PBS. Antigen loading was performed by rotating overnight at 4°C. After absorption, beads were collected by centrifugation as described and washed twice with 1% BSA in PBS. Finally, antigen-coated beads were resuspended at 1e8 particles / mL in 1% BSA in PBS, 0.25% NaN3 and stored at 4°C until use.

[0065] Ig Standards: To construct indirect standard beads, selected bead peaks for each isotype were combined and biotinylated goat anti-isotype F(ab)2 abs (Southern Biotech: anti-IgM 2022-01, anti-IgA 2052-01, and anti-IgG 2042-01) were added at a concentration of 1 mg / mL. Standard bead preparations were washed, collected, and stored as described for antigen-coated beads.

[0066] Example 3: Generation of recombinant antigens Recombinant antigens used in the CBA: The recombinant antigens used in this array include influenza H1 Ca09 hemagglutinin (HA) and b-coronavirus (CoV) spike (SP), spike subdomains (receptor binding domain (RBD) and N-terminal domain (NTD)), and nucleocapsid protein (N). Recombinant CoV S and N proteins were generated from sequences derived from five known human infectious b-coronaviruses. These include the Wuhan / Washington strains of SARS-CoV-2 (abbreviated as C), SARS1 (abbreviated as S), MERS (abbreviated as M), OC43 (abbreviated as O), and HKU1 (abbreviated as H). The RBD and NTD SP subdomains were produced from sequences derived from the Wuhan / Washington strain of SARS-CoV-2. It is contemplated that an influenza hemagglutinin protein has at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO:26.

[0067] Production of pre-fusion recombinant spike (SP) protein: Ectodomain SP pre-fusion trimers (SARS-CoV-2 S14-1211) were produced by co-transfecting SP-Avi tag and SP-6X-His tag constructs in a 1:2 ratio into FreeStyle 293-F cells. Transfected cells were cultured in FreeStyle 293 medium for 3 days, and recombinant SP trimers were purified from the culture supernatant by FPLC using nickel affinity chromatography. Purified proteins were biotinylated in vitro using BirA enzyme.

[0068] With respect to the CoV spike (SP) ectodomain, it is contemplated that a SARS1-CoV spike ectodomain (S SP) with an AVI tag has at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 8. It is contemplated that a S SP 6His protein has at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 9. It is contemplated that a SARS-CoV2 spike ectodomain (C SP) with an AVI tag has at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 10. The C SP 6 His tagged protein is contemplated to have at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 11. The MERS spike ectodomain (M SP) 6 His tagged protein is contemplated to have at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 12. The M SP AVI tagged protein is contemplated to have at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 13. The OC43 spike ectodomain (O SP) 6 His tagged protein is contemplated to have at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 14. It is contemplated that the OC43 spike ectodomain (OSP)6His tagged protein has at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO:15.It is contemplated that an HKU1 spike ectodomain (HSP) 6His tagged protein has at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 16. It is contemplated that an HSP Avi tagged protein has at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 17.

[0069] Generation of the S subdomain: NTD (SARS-CoV-2 S) with a C-terminal double Avi-tag / 6X-His-tag sequence 14-305 ) and RBD (SARS-CoV-2 S 319-541 ) monomers were generated by transfecting single constructs into FreeStyle 293-F cells. After 3 days of expression, subdomains were purified from culture supernatants by FPLC using nickel affinity chromatography and biotinylated in vitro by addition of BirA.

[0070] With respect to the spike subdomain, it is contemplated that the Sars-CoV-2 receptor binding domain (C RBD) 6HIS with an AVI tag has at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 18. It is contemplated that the Sars-CoV-2 N-terminal domain (C NTD) has at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 19. It is contemplated that the SARS1-CoV receptor binding domain (S RBD) 6HIS AVI tag protein has at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 20.

[0071] Production of CoV nucleocapsid protein (N): Recombinant N containing full-length N and tandem Avi-tag / 6X-His-tag sequences was produced by co-transformation of Rosetta cells with an N expression plasmid and an inducible BirA expression plasmid. Cells were grown in the presence of chloramphenicol, ampicillin and streptomycin, induced with IPTG and supplemented with biotin. Biotinylated N protein was purified by FPLC using a nickel affinity column followed by size exclusion chromatography.

[0072] With respect to the N protein, it is contemplated that the SARS-CoV nucleocapsid protein (S NP) 6HIS AVI tag has at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 21. It is contemplated that the SARS-CoV-2 nucleocapsid protein (C NP) 6HIS AVI tag has at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 22. It is contemplated that the MERS nucleocapsid protein (M NP) 6HIS AVI tag has at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 23. It is contemplated that the OC43 nucleocapsid protein (O NP) 6 HIS AVI tag has at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 24. It is contemplated that the HKU1 nucleocapsid protein (H NP) 6 HIS AVI tag has at least about 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 25.

[0073] Antibodies and Standards: Detection of IG fluorescent goat polyclonal anti-IG F(ab')2 secondary antibody, SouthernBiotech (IgM cat 2022-02, IgG cat# 2062-09, IgA cat# 2052-09) isotype standards were generated by running arrays against a mixture of 0.75x serial dilutions of purified human antibodies Southern Biotech (IgG cat# 0150-01, IgM cat# 0158L-01, IgA cat# 0155L-01) ranging from 1ug / mL to 1.3ng / mL with IG capture beads.

[0074] Example 4: CBA Assay Serum samples were diluted in PBS (1 / 7150 for IgG detection or 1 / 500 for IgM and IgA detection) and arrayed in 96-well u-bottom plates. 5 μl of suspension containing 5×1e5 of each antigen-coated microparticle was added to the sample. For Ig standards, anti-IgM, anti-IgA, and anti-IgG beads were added to 50 ml of serial dilutions of standard Ab. The suspension was mixed by pipetting and incubated for 15 min at room temperature. The beads were washed by adding 200 μl of PBS and centrifuged at 3000 g for 5 min at room temperature. CBA particles were resuspended in secondary staining solution consisting of the appropriate secondary diluted 1 / 400 in 1% BSA in PBS. The suspension was incubated for 15 min at room temperature in the dark. The beads were washed by adding 200 μl of PBS and pelleted by centrifugation at 3000 g for 5 min at room temperature. Particles were resuspended in 80 μl of PBS and analyzed directly on a BD Cytoflex flow cytometer in plate mode at a sample rate of 100 ml / min. Sample collection was stopped after acquisition of 75 μL. After acquisition, the resulting FCS files were processed using the software described below.

[0075] Example 5: Sample Analysis FCS processing: FCS files obtained from samples were automatically processed using custom software to rapidly quantify and analyze antibody reactivity in serum samples. The software was developed in Matlab (The Mathworks, Inc. Natick MA, USA) version R2020a on MacOS. It requires Statistics and Machine Learning Toolbox, Curve Fitting Toolbox, Signal Processing Toolbox, and additional code from Matlab Central (www.mathworks.com / matlabcentral / ).

[0076] Concentration determination: MFI data is extracted from the FCS files and transformed using a hyperbolic arcsine. Next, a forward scatter vs. side scatter plot is used to distinguish different sizes of beads and intensity as a density of points in the APC-cy7 channel. These are automatically detected and events within these gates are annotated as separate populations of beads. Finally, events from each bead gate are evaluated on a secondary isotype flow channel r each bead feature and isotype.

[0077] Standard samples for each isotype and bead size are treated similarly and the resulting data is used to calculate a four parameter logistic (4PL) fit for each bead size / isotype / dilution. Finally, the 4PL fit is used to back-calculate concentration units of the MFI data across the entire data set as a single tabular text file containing the calculated Ig concentration data for all features in the array.

[0078] In some embodiments, the numbers expressing properties such as amounts of ingredients, concentrations, reaction conditions, etc., used to describe and claim certain embodiments of the present invention should be understood as being modified in some instances by the term "about". Thus, in some embodiments, the numerical parameters set forth in the written description and the appended claims are approximations that may vary depending on the desired properties sought to be obtained by a particular embodiment. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein.

[0079] As used herein, the term "administering" a pharmaceutical composition or drug refers to both direct and indirect administration of a pharmaceutical composition or drug, where direct administration of a pharmaceutical composition or drug is typically performed by a medical professional (e.g., a doctor, a nurse, etc.), and indirect administration includes providing or making available a pharmaceutical composition or drug to a medical professional for direct administration (e.g., by injection, infusion, oral delivery, topical delivery, etc.). It should be further noted that the term "predicting" or "predicting" a condition, susceptibility to development of a disease, or response to an intended treatment is intended to cover the act or prediction (but not treatment or diagnosis) of predicting a condition, susceptibility, and / or response, including the rate of progression, improvement, and / or persistence of a condition in a subject.

[0080] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided with respect to specific embodiments herein, or the use of exemplary language (e.g., "etc.") are intended merely to better clarify the invention and do not otherwise limit the scope of the invention as claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0081] As used throughout this description and the claims that follow, the meanings of "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Also, as used in this description, the meaning of "in" includes "in" and "on," unless the context clearly dictates otherwise. Also, as used herein, unless the context dictates otherwise, the term "coupled to" is intended to include both direct coupling (wherein the two elements that are coupled to each other are in contact with each other) and indirect coupling (wherein at least one additional element is located between the two elements). Thus, the terms "coupled to" and "coupled with" are used interchangeably.

[0082] It will be apparent to those skilled in the art that many more modifications beyond those already described are possible without departing from the inventive concept herein. Thus, the subject matter of the present invention should not be limited, except as set forth in the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprise" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced element, component, or step may be present, utilized, or combined with other elements, components, or steps not expressly referenced. When the specification or claims refer to at least one selected from the group consisting of A, B, C... and N, the sentence should be interpreted as requiring only one element from the group, not A+N, or B+N, etc.

Claims

1. A recombinant vaccine composition for use in inducing an immune response against serologically distinct variants of SARS-CoV-2 or coronaviruses other than SARS-CoV-2 in a subject, comprising: A first portion encoding a severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein (N) (N-ETSD) fused to an endosome targeting sequence, the first portion being operably linked to one or more regulatory elements enabling N-ETSD expression; A second portion encoding a SARS virus spike protein (S), the second portion being operably linked to one or more regulatory elements enabling S expression; comprising The vaccine composition is formulated to be administered as a prime and / or boost vaccination in an amount that induces the immune response.

2. The composition according to claim 1, wherein the immune response is the production of antibodies that bind to at least two serologically distinct variants of SARS-CoV-2 and / or SARS-CoV-2, and at least one coronavirus other than SARS-CoV-2.

3. The composition according to claim 1, wherein the immune response is the production of cytotoxic T cells that are cytotoxic to different cells carrying each serologically distinct variant of SARS-CoV-2 and / or cells carrying SARS-CoV-2, and cells carrying coronaviruses other than SARS-CoV-2.

4. The composition according to claim 1, wherein the immune response is the production of memory T cells.

5. The composition according to claim 1, wherein the immune response is the production of memory B cells.

6. The composition according to claim 1, wherein the coronavirus other than SARS-CoV-2 is SARS-CoV-1, MERS-CoV, OC43-CoV, and / or HKU1-CoV.

7. The composition according to claim 1, wherein the N is derived from SARS-CoV-2.

8. The composition according to claim 7, wherein the endosome targeting sequence of the N-ETSD is encoded at the 5' end of the first portion.

9. The composition according to claim 7, wherein the endosome targeting sequence of the N-ETSD is encoded at the 3' end of the first portion.

10. The composition according to claim 7, wherein the first and second portions are arranged in a bicistronic sequence.

11. The composition according to claim 7, wherein the N-ETSD has an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

7.

12. The composition according to claim 7, wherein the N-ETSD has the amino acid sequence of SEQ ID NO:

1.

13. The composition according to claim 1, wherein the first portion has the nucleotide sequence of SEQ ID NO:

2.

14. The composition according to claim 7, wherein the S protein has an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:

4.

15. The composition according to claim 7, wherein the S protein has the amino acid sequence of SEQ ID NO:

3.

16. The composition according to claim 7, wherein the S protein has the amino acid sequence of SEQ ID NO:

4.

17. The composition according to claim 1, wherein the second portion has the nucleotide sequence of SEQ ID NO:

5.

18. The composition according to claim 1, wherein the second portion has the nucleotide sequence of SEQ ID NO:

6.

19. The composition according to claim 1, wherein the recombinant vaccine composition is formulated as a recombinant virus.

20. The composition according to claim 19, wherein the recombinant virus is an adenovirus having a deletion in the E1 gene region and a deletion in the E2b gene region.

21. The composition according to claim 1, wherein the recombinant vaccine composition is formulated as recombinant RNA.

22. The composition according to claim 21, wherein the recombinant RNA is a polycistronic RNA comprising the first and second portions.

23. The composition according to claim 1, wherein the recombinant vaccine composition is formulated as recombinant DNA.

24. The composition according to claim 23, wherein the recombinant DNA comprises the first and second portions.

25. The composition according to claim 1, wherein the recombinant vaccine composition is administered by the prime administration and the boost administration.

26. The composition according to claim 25, wherein the recombinant vaccine composition is formulated as an adenovirus vaccine composition.

27. The composition according to claim 1, wherein the recombinant vaccine composition is administered only in the boost administration.

28. The composition according to claim 27, wherein the boost administration follows a prime vaccination selected from the group consisting of an RNA vaccine, a DNA vaccine, a viral vaccine, and a subunit vaccine.

29. The composition according to claim 28, wherein the RNA vaccine prime vaccination comprises a self-amplifying self-adjuvant RNA vaccine.

30. The composition according to claim 29, wherein the self-amplifying self-adjuvant RNA vaccine comprises RNA encoding a coronavirus S protein and / or a coronavirus N protein.

31. The composition according to claim 27, wherein the viral vaccine prime vaccination comprises an adenovirus viral vaccine, and the adenovirus viral vaccine comprises a recombinant nucleic acid encoding a coronavirus S protein.

32. A recombinant vaccine composition for use in the production of memory B cells having specificity for a plurality of different coronaviruses or serologically different variants thereof, comprising: A first portion encoding a severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein (N) (N-ETSD) fused to an endosome-targeting sequence, the first portion being operably linked to one or more regulatory elements that enable N-ETSD expression; A second portion encoding a SARS virus spike protein (S), the second portion being operably linked to one or more regulatory elements that enable S expression; and the vaccine composition is formulated to be administered as a prime and / or boost vaccination in an amount that induces the production of the memory B cells.

33. A recombinant vaccine composition for use in the production of memory T cells having specificity for a plurality of different coronaviruses or serologically different variants thereof, comprising: A first portion encoding a severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein (N) (N-ETSD) fused to an endosome-targeting sequence, the first portion being operably linked to one or more regulatory elements that enable N-ETSD expression; A second portion encoding a SARS virus spike protein (S), the second portion being operably linked to one or more regulatory elements that enable S expression; and The vaccine composition is formulated to be administered as a prime and / or boost vaccination in an amount that induces the production of the memory T cells.

34. The composition according to claim 32, wherein the plurality of different coronaviruses are selected from the group consisting of SARS-CoV1, SARS-CoV2, MERS-CoV, OC43-CoV, and / or HKU1-CoV.

35. The composition according to claim 32, wherein the N is derived from SARS-CoV-2.

36. The composition according to claim 35, wherein the endosomal targeting sequence of the N-ETSD is encoded at the 5' end of the first portion.

37. The composition according to claim 35, wherein the endosomal targeting sequence of the N-ETSD is encoded at the 3' end of the first portion.

38. The composition according to claim 35, wherein the first and second portions are arranged in a bicistronic sequence.

39. The composition according to claim 35, wherein the N-ETSD has an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

1.

40. The composition according to claim 35, wherein the N-ETSD has the amino acid sequence of SEQ ID NO:

1.

41. The composition according to claim 32, wherein the first portion has the nucleotide sequence of SEQ ID NO:

2.

42. The composition according to claim 32, wherein the S protein has an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:

4.

43. The composition according to claim 42, wherein the S protein has the amino acid sequence of SEQ ID NO:

3.

44. The composition according to claim 42, wherein the S protein has the amino acid sequence of SEQ ID NO:

4.

45. The composition according to claim 32, wherein the second portion has the nucleotide sequence of SEQ ID NO:

5.

46. The composition according to claim 32, wherein the second portion has the nucleotide sequence of SEQ ID NO:

6.

47. The composition according to claim 32, wherein the recombinant vaccine composition is formulated as a recombinant virus.

48. The composition according to claim 47, wherein the recombinant virus is an adenovirus having a deletion in the E1 gene region and a deletion in the E2b gene region.

49. The composition according to claim 32, wherein the recombinant vaccine composition is formulated as recombinant RNA.

50. The composition according to claim 49, wherein the recombinant RNA is a polycistronic RNA comprising the first and second parts.

51. The composition according to claim 32, wherein the recombinant vaccine composition is formulated as recombinant DNA.

52. The composition according to claim 51, wherein the recombinant DNA comprises the first and second parts.

53. A kit for use in inducing an immune response against serologically distinct variants of SARS-CoV-2 or coronaviruses other than SARS-CoV-2 in a subject, comprising: A first part encoding a severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein (N) (N-ETSD) fused to an endosome targeting sequence, functionally linked to one or more regulatory elements enabling N-ETSD expression; A second part encoding a SARS virus spike protein (S), functionally linked to one or more regulatory elements enabling S expression; A first recombinant vaccine composition having; A recombinant viral vaccine comprising a recombinant nucleic acid encoding a SARS virus spike protein (S) functionally linked to one or more regulatory elements enabling S expression; or A self-amplifying self-adjuvant RNA vaccine comprising a recombinant nucleic acid encoding a SARS virus spike protein (S) functionally linked to one or more regulatory elements enabling S expression and optionally further functionally linked to one or more regulatory elements enabling N expression, encoding a severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein (N); or A subunit vaccine comprising a recombinant protein of a coronavirus; or A heat-inactivated coronavirus vaccine composition A second recombinant vaccine composition having Comprising, a kit.

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