Vaccines against viral pathogens

Abstract

The present invention provides viral peptide (VP) conjugates (VP-hC) or VP oligomers (VPhC) for producing vaccines to treat and / or prevent viral diseases and viral infections. [Solution] A viral peptide (VP) conjugate (VP-hC) or VP oligomer (VPhC) is provided, comprising one or more viral peptides (VP) covalently bonded to a hapten carrier (hC), wherein hC comprises a monomeric peptide having a specific amino acid sequence, the VP conjugate comprises one or more VPs conjugated to hC, and the VP oligomer comprises one or more VPs incorporated into hC.

Description

[[Technical Field]]

[0001] Cross-Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 019,654, filed on May 4, 2020, which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing Information A sequence listing related to the present application is contained in a computer-readable text file with a file size of approximately 64.4 KB entitled "H197-0006PCT_ST25.txt", created on or about May 3, 2021, which is hereby incorporated by reference in its entirety.

[0003] Technical Field The present disclosure describes vaccines against viral pathogens. [[Background Art]]

[0004] Background Pathogens are infectious agents that cause disease. Infectious agents can be microorganisms such as viruses. Viruses are small particles about 20 to 300 nanometers in length that contain RNA or DNA. They infect all types of living organisms, including humans, animals, plants, and other microorganisms such as bacteria and archaea. They only self-replicate inside the living cells of an organism, and cause infectious diseases ranging from the common cold, influenza, mild to severe diseases such as smallpox, influenza, mumps, measles, chickenpox, polio and rubella.

[0005] Vaccines have been developed for many infectious diseases and have successfully reduced the incidence of influenza, mumps, measles, smallpox, varicella, polio, and rubella. Expression of recombinant proteins in hosts such as bacteria (mainly Escherichia coli (E. coli)), yeast, insect cells, and mammalian cells is currently the most common method for producing subunit vaccines, and such methods have been very successful and remain an important method of vaccine production. Typically, the infectious agent protein is identified by genomic analysis, functional assays, in silico analysis (e.g., functional prediction, structural analysis, epitope identification, etc.), or a combination of these three. Expression trials are initiated to evaluate yield and solubility with respect to immunogenicity trials. Next, subunits that produce high-titer antibodies against the disease target are advanced to protective studies where the vaccine is tested for its ability to protect the host against infection and / or the signs and progression of the disease. Next, subunits that meet all these criteria proceed to vaccine production optimization, stability, and toxicity / safety / dosage studies. Expression optimization studies are also crucial in determining production scale and feasibility. The entire process is time-consuming, labor-intensive, and very costly. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Therefore, there is a need to develop more efficient and cost-effective methods for producing vaccines to treat and / or prevent viral diseases and infections. [Means for solving the problem]

[0007] overview This summary of the invention is provided to introduce the selection of concepts in the simplified forms further described below in the detailed description. This summary of the invention is not intended to identify all of the important or essential features of the claimed subject matter, nor is it intended to be used alone as an aid in determining the scope of the claimed subject matter.

[0008] This disclosure describes hMP polypeptides comprising a hapten (h) conjugated to a monomeric peptide (MP). This disclosure also describes conjugates comprising a hapten conjugated to a hapten carrier (hC). The hapten may be a target protein or a target antigen. In some embodiments, the hapten is a viral peptide (VP), the hMP polypeptide comprises the viral peptide (VP) and MP (VPMP), and the conjugate is VP-hC. In some embodiments, since the hMP may have the same secondary, tertiary, or quaternary structure, the hMP may function as an oligomeric hC such as hC or HhC (hexamer hC) after self-assembly. In some embodiments, when a T cell epitope is conjugated to the MP in addition to the viral peptide, the VMP may function similarly to the VP-hC conjugate but without the conjugated viral peptide. VP is a peptide derived from a virus such as SARS-CoV-2 virus, respiratory syncytial virus, influenza A virus, West Nile virus, yellow fever virus, human papillomavirus, and dengue virus. In some embodiments, VP is the S1, S2, S3, S4, S5, S6, or M1 peptide derived from SAR2-CoV-2 virus. In some embodiments, VP includes an amino acid sequence such as that described in SEQ ID NO: 136, SEQ ID NO: 120, SEQ ID NO: 132, SEQ ID NO: 119, SEQ ID NO: 118, SEQ ID NO: 117, SEQ ID NO: 130, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 73, SEQ ID NO: 57, SEQ ID NO: 69, SEQ ID NO: 115, SEQ ID NO: 55, SEQ ID NO: 54, or SEQ ID NO: 67.

[0009] The hC described herein includes a monomeric peptide that is an amphiphilic alpha-helix containing two or more heptad repeats that self-assemble into a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nocumer, or decamer. Each heptad contains an amino acid sequence such as that described in SEQ ID NO: 1. In some embodiments, the monomeric peptide self-assembles onto a hexameric hapten carrier (HhC). The hC may also contain a target peptide such as VP, in which case the hC is a VPMP (VPhC oligomer) that self-assembles onto an oligomer. In some embodiments, the VPMP self-assembles onto a hexamer, for example, an hC containing VP conjugated to HhC (VPHhC). In some embodiments, the conjugate described herein includes a VP conjugated onto HhC (VP-HhC).

[0010] Furthermore, this disclosure describes VPhC oligomers or VP-hC conjugates that contain a T cell epitope at the N and / or C terminus of an amphipathic alpha helix of hC that is part of a monomeric peptide or covalently bonded to either VPhC or VP-hC.

[0011] In some embodiments, this disclosure describes a composition comprising a VP-hC conjugate or VPhC oligomer as described herein and an excipient. In some embodiments, the composition is a pharmaceutical composition which may be used to treat a subject in need, for example, to prevent or reduce the risk of a subject developing a viral disease. The subject was potentially susceptible to viral infection. It is also possible to administer the pharmaceutical composition to the subject before viral infection to prevent the subject from developing a severe or fatal viral disease and / or to alleviate the symptoms of a viral disease. The VPhC oligomer or VP-hC conjugate in the pharmaceutical composition may generate antibodies in the subject to inhibit or reduce the function of the virus, thereby protecting the subject from developing a severe or fatal viral disease or a severe or fatal viral infection. It is also possible to administer the pharmaceutical composition to a subject in need to neutralize the virus and generate antibodies to alleviate the symptoms of a viral infection or viral disease. In one embodiment, a pharmaceutical composition comprising VPhC oligomers and VP-hC conjugates may be used as a vaccine to prevent and / or treat viral diseases or infections.

[0012] In one embodiment, this disclosure describes a method for inducing a robust and sustained immune response in a subject using VPhC oligomers or VP-hC conjugates described herein as therapeutic agents or vaccines, such as immunogens.

[0013] In one embodiment, VP is a SARS-CoV-2 (CoV) peptide, in which case hC is a self-assembled CoVMP (CoVhC oligomer) oligomer. In one embodiment, CoVMP self-assembles into a hexamer, for example, hC (CoVHhC) containing a CoV peptide conjugated to HhC. In one embodiment, the conjugate described herein includes CoV (CoV-HhC) conjugated to HhC.

[0014] In one embodiment, the viral infection or viral disease to be treated and / or prevented is SARS-CoV-2. Brief explanation of the drawing [Brief explanation of the drawing]

[0015] [Figure 1] This figure shows the subunits of the SARS-CoV-2 spike glycoprotein (S protein). The positions of candidate antigen peptides within the S protein are indicated. [Figure 2A] This figure shows the cation ESI LC / MS / MS spectrum of an unconjugated scaffold (expected size 6,767 Da). [Figure 2B] This figure shows the cation ESI LC / MS / MS spectrum of the scaffold + S5 peptide conjugate (HS5). The absence of ions at m / z = 6,768 suggests highly efficient and nearly quantitative coupling of S5 to the scaffold. [Figure 3A]This figure shows the mouse IgG titers on day 14 after immunization as described in Table 3. S1 (S-1), S2 (S-2), S3 (S-3), S4 (S-4), S5 (S-5), and S6 (S-6) are antigenic peptides derived from the S glycoprotein. The S4, S5, and S6 peptides are located within the receptor-binding domain, while the remaining peptides contain epitopes important for viral function, such as proteolytic cleavage sites (S2, S3) or sites proximal to proteolytic cleavage sites (S1). S6 is an epitope present in the serum of convalescent COVID-19 patients. M1 contains an epitope on the SARS-CoV-2 membrane protein. These peptides were conjugated into mouse scaffolds (hC) before immunization of mice. Group 9 contained mice immunized with the same □g amounts of scaffold, S1 peptide, S2 peptide, S4 peptide, S6 peptide, and M1 peptide, but the peptides did not covalently couple to the scaffold. Group 9 was included to evaluate the requirement of covalent coupling of antigen peptides to the scaffold for an immune response. To measure immunogenicity, ELISA plates were separately coated with S1 peptide, S2 peptide, S4 peptide, S6 peptide, or M7 peptide so that binding specificity for each peptide could be measured. 9-1, 9-2, 9-4, 9-6, and 9-7 refer to antibody titers in Group 9 measured by ELISA with S1 peptide (9-1), S2 peptide (9-2), S4 peptide (9-4), S6 peptide (9-6), and M1 peptide (9-7) as coating reagents. Groups 10-1 to 10-7 are identical to group 9, but first, each peptide (S1, S2, S4, S6, and M1) was conjugated to a mouse scaffold peptide to produce HS1, HS2, HS4, HS6, or HM1. These were then mixed in the specified pre-immunization dose of □g.The controls included mouse scaffolds alone, as well as PBS+ adjuvant serum incubated with S1, S2, S3, S4, S5, and S6 peptides coated on ELISA plates, comprising PBS-S1, PBS-S2, PBS-S3, PBS-S4, PBS-S5, and PBS-S6. Figure 3A shows IgG titers at d14. The prime was at d0, and boosts were at d14 and d28. These results indicate that each of the tested vaccine candidates produced titers increased to varying degrees compared to the control. Mice immunized with HS2, HS3, HS4, or HM1 consistently produced lower titers than HS1, HS5, and HS6 or the serum of groups 9 and 10. The results for group 9 mice clearly demonstrate that covalent coupling to the scaffold is not required to produce a robust immune response. [Figure 3B]This figure shows the mouse IgG titers on day 28 after immunization as shown in Table 3. S1 (S-1), S2 (S-2), S3 (S-3), S4 (S-4), S5 (S-5), and S6 (S-6) are antigenic peptides derived from the S glycoprotein. The S4, S5, and S6 peptides are located within the receptor-binding domain, while the remaining peptides contain epitopes important for viral function, such as proteolytic cleavage sites (S2, S3) or sites proximal to proteolytic cleavage sites (S1). S6 is an epitope present in the serum of convalescent COVID-19 patients. M1 contains an epitope on the SARS-CoV-2 membrane protein. These peptides were conjugated to mouse scaffolds (hC) before immunization of mice. Group 9 contained mice immunized with the same □g amounts of scaffold, S1 peptide, S2 peptide, S4 peptide, S6 peptide, and M1 peptide, but the peptides did not covalently couple to the scaffold. Group 9 was included to evaluate the requirement of covalent coupling of antigen peptides to the scaffold for an immune response. To measure immunogenicity, ELISA plates were separately coated with S1 peptide, S2 peptide, S4 peptide, S6 peptide, or M7 peptide so that binding specificity for each peptide could be measured. 9-1, 9-2, 9-4, 9-6, and 9-7 refer to antibody titers in Group 9 measured by ELISA with S1 peptide (9-1), S2 peptide (9-2), S4 peptide (9-4), S6 peptide (9-6), and M1 peptide (9-7) as coating reagents. Groups 10-1 to 10-7 are identical to group 9, but first, each peptide (S1, S2, S4, S6, and M1) was conjugated to a mouse scaffold peptide to produce HS1, HS2, HS4, HS6, or HM1. These were then mixed in the specified pre-immunization dose of □g.The controls included mouse scaffolds alone, as well as PBS+ adjuvant serum incubated with S1, S2, S3, S4, S5, and S6 peptides coated on ELISA plates, comprising PBS-S1, PBS-S2, PBS-S3, PBS-S4, PBS-S5, and PBS-S6. Figure 3B shows IgG titers at d28. The prime was at d0, and boosts were at d14 and d28. These results indicate that each of the tested vaccine candidates produced titers increased to varying degrees compared to the control. Mice immunized with HS2, HS3, HS4, or HM1 consistently produced lower titers than HS1, HS5, and HS6 or the serum of groups 9 and 10. The results for group 9 mice clearly demonstrate that covalent coupling to the scaffold is not required to produce a robust immune response. [Figure 3C]Table 3 shows the mouse IgG titers on day 42 after immunization. S1 (S-1), S2 (S-2), S3 (S-3), S4 (S-4), S5 (S-5), and S6 (S-6) are antigenic peptides derived from the S glycoprotein. The S4, S5, and S6 peptides are located within the receptor-binding domain, while the remaining peptides contain epitopes important for viral function, such as proteolytic cleavage sites (S2, S3) or sites proximal to proteolytic cleavage sites (S1). S6 is an epitope present in the serum of convalescent COVID-19 patients. M1 contains an epitope on the SARS-CoV-2 membrane protein. These peptides were conjugated to mouse scaffolds (hC) before immunization of mice. Group 9 contained mice immunized with the same □g amounts of scaffold, S1 peptide, S2 peptide, S4 peptide, S6 peptide, and M1 peptide, but the peptides did not covalently couple to the scaffold. Group 9 was included to evaluate the requirement of covalent coupling of antigen peptides to the scaffold for an immune response. To measure immunogenicity, ELISA plates were separately coated with S1 peptide, S2 peptide, S4 peptide, S6 peptide, or M7 peptide so that binding specificity for each peptide could be measured. 9-1, 9-2, 9-4, 9-6, and 9-7 refer to antibody titers in Group 9 measured by ELISA with S1 peptide (9-1), S2 peptide (9-2), S4 peptide (9-4), S6 peptide (9-6), and M1 peptide (9-7) as coating reagents. Groups 10-1 to 10-7 are identical to group 9, but first, each peptide (S1, S2, S4, S6, and M1) was conjugated to a mouse scaffold peptide to produce HS1, HS2, HS4, HS6, or HM1. These were then mixed in the specified pre-immunization dose of □g.The controls included mouse scaffolds alone, as well as PBS+ adjuvant serum incubated with S1, S2, S3, S4, S5, and S6 peptides coated on ELISA plates, comprising PBS-S1, PBS-S2, PBS-S3, PBS-S4, PBS-S5, and PBS-S6. Figure 3C shows IgG titers at d42. The prime was at d0, and boosts were at d14 and d28. These results indicate that each of the tested vaccine candidates produced titers increased to varying degrees compared to the control. Mice immunized with HS2, HS3, HS4, or HM1 consistently produced lower titers than HS1, HS5, and HS6 or the serum of groups 9 and 10. The results for group 9 mice clearly demonstrate that covalent coupling to the scaffold is not required to produce a robust immune response. [Figure 3D]Table 3 shows the mouse IgG titers on day 56 after immunization. S1 (S-1), S2 (S-2), S3 (S-3), S4 (S-4), S5 (S-5), and S6 (S-6) are antigenic peptides derived from the S glycoprotein. The S4, S5, and S6 peptides are located within the receptor-binding domain, while the remaining peptides contain epitopes important for viral function, such as proteolytic cleavage sites (S2, S3) or sites proximal to proteolytic cleavage sites (S1). S6 is an epitope present in the serum of convalescent COVID-19 patients. M1 contains an epitope on the SARS-CoV-2 membrane protein. These peptides were conjugated to mouse scaffolds (hC) before immunization of mice. Group 9 contained mice immunized with the same □g amounts of scaffold, S1 peptide, S2 peptide, S4 peptide, S6 peptide, and M1 peptide, but the peptides did not covalently couple to the scaffold. Group 9 was included to evaluate the requirement of covalent coupling of antigen peptides to the scaffold for an immune response. To measure immunogenicity, ELISA plates were separately coated with S1 peptide, S2 peptide, S4 peptide, S6 peptide, or M7 peptide so that binding specificity for each peptide could be measured. 9-1, 9-2, 9-4, 9-6, and 9-7 refer to antibody titers in Group 9 measured by ELISA with S1 peptide (9-1), S2 peptide (9-2), S4 peptide (9-4), S6 peptide (9-6), and M1 peptide (9-7) as coating reagents. Groups 10-1 to 10-7 are identical to group 9, but first, each peptide (S1, S2, S4, S6, and M1) was conjugated to a mouse scaffold peptide to produce HS1, HS2, HS4, HS6, or HM1. These were then mixed in the specified pre-immunization dose of □g.The controls included mouse scaffolds alone, as well as PBS+ adjuvant serum incubated with S1, S2, S3, S4, S5, and S6 peptides coated on ELISA plates, comprising PBS-S1, PBS-S2, PBS-S3, PBS-S4, PBS-S5, and PBS-S6. Figure 3D shows IgG titers at d56. The prime was at d0, and boosts were at d14 and d28. These results indicate that each of the tested vaccine candidates produced titers increased to varying degrees compared to the controls. Mice immunized with HS2, HS3, HS4, or HM1 consistently produced lower titers than HS1, HS5, and HS6 or the serum of groups 9 and 10. The results for group 9 mice clearly demonstrate that covalent coupling to the scaffold is not required to produce a robust immune response. [Figure 3E]Table 3 shows the mouse IgG titers on day 84 after immunization. S1 (S-1), S2 (S-2), S3 (S-3), S4 (S-4), S5 (S-5), and S6 (S-6) are antigenic peptides derived from the S glycoprotein. The S4, S5, and S6 peptides are located within the receptor-binding domain, while the remaining peptides contain epitopes important for viral function, such as proteolytic cleavage sites (S2, S3) or sites proximal to proteolytic cleavage sites (S1). S6 is an epitope present in the serum of convalescent COVID-19 patients. M1 contains an epitope on the SARS-CoV-2 membrane protein. These peptides were conjugated to mouse scaffolds (hC) before immunization of mice. Group 9 contained mice immunized with the same □g amounts of scaffold, S1 peptide, S2 peptide, S4 peptide, S6 peptide, and M1 peptide, but the peptides did not covalently couple to the scaffold. Group 9 was included to evaluate the requirement of covalent coupling of antigen peptides to the scaffold for an immune response. To measure immunogenicity, ELISA plates were separately coated with S1 peptide, S2 peptide, S4 peptide, S6 peptide, or M7 peptide so that binding specificity for each peptide could be measured. 9-1, 9-2, 9-4, 9-6, and 9-7 refer to antibody titers in Group 9 measured by ELISA with S1 peptide (9-1), S2 peptide (9-2), S4 peptide (9-4), S6 peptide (9-6), and M1 peptide (9-7) as coating reagents. Groups 10-1 to 10-7 are identical to group 9, but first, each peptide (S1, S2, S4, S6, and M1) was conjugated to a mouse scaffold peptide to produce HS1, HS2, HS4, HS6, or HM1. These were then mixed in the specified pre-immunization dose of □g.Controls include mouse scaffold alone, as well as PBS-S1, PBS-S2, PBS-S3, PBS-S4, PBS-S5 and PBS-S6, which are PBS + adjuvant serum incubated with S1 peptide, S2 peptide, S3 peptide, S4 peptide, S5 peptide and S6 peptide coated on an ELISA plate. Figure 3E shows the IgG titer at d84. Priming was at d0, and boosting was at d14 and d28. These results indicate that each of the tested vaccine candidates produced increased titers to varying degrees relative to the controls. Mice immunized with HS2, HS3, HS4 or HM1 consistently produced lower titers than the sera of HS1, HS5 and HS6 or group 9 and group 10. The results from group 9 mice clearly demonstrate that covalent coupling to the scaffold is not required to generate a robust immune response. [Figure 4A] It is a figure showing that the adjuvant vaccine induces isotype switching and balances Th1 / Th2 immunity at d14. [Figure 4B] It is a figure showing that the adjuvant vaccine induces isotype switching and balances Th1 / Th2 immunity at d28. [Figure 4C] It is a figure showing that the adjuvant vaccine induces isotype switching and balances Th1 / Th2 immunity at d42. [Figure 4D] It is a figure showing that the adjuvant vaccine induces isotype switching and balances Th1 / Th2 immunity at d84. [Figure 5A] It is a plot showing the time course of IgG titer up to d84. The induced immune response was robust and long-lasting up to d84, which indicates that the combination of T cell epitopes in the scaffold combined with an adjuvant has the capability of long-term immunity against SARS-CoV-2. [Figure 5B]This figure shows a time-course plot of IgG titers up to day 84. The induced immune response was robust and long-lasting up to day 84, indicating that the combination of T cell epitopes in the scaffold combined with the adjuvant has the ability to provide long-term immunity against SARS-CoV-2. [Figure 5C] This figure shows a time-course plot of IgG titers up to day 84. The induced immune response was robust and long-lasting up to day 84, indicating that the combination of T cell epitopes in the scaffold combined with the adjuvant has the ability to provide long-term immunity against SARS-CoV-2. [Figure 5D] This figure shows a time-course plot of IgG titers up to day 84. The induced immune response was robust and long-lasting up to day 84, indicating that the combination of T cell epitopes in the scaffold combined with the adjuvant has the ability to provide long-term immunity against SARS-CoV-2. [Figure 5E] This figure shows a time-course plot of IgG titers up to day 84. The induced immune response was robust and long-lasting up to day 84, indicating that the combination of T cell epitopes in the scaffold combined with the adjuvant has the ability to provide long-term immunity against SARS-CoV-2. [Figure 5F] This figure shows a time-course plot of IgG titers up to day 84. The induced immune response was robust and long-lasting up to day 84, indicating that the combination of T cell epitopes in the scaffold combined with the adjuvant has the ability to provide long-term immunity against SARS-CoV-2. [Figure 5G] This figure shows a time-course plot of IgG titers up to day 84. The induced immune response was robust and long-lasting up to day 84, indicating that the combination of T cell epitopes in the scaffold combined with the adjuvant has the ability to provide long-term immunity against SARS-CoV-2. [Figure 5H]This figure shows a time-course plot of IgG titers up to day 84. The induced immune response was robust and long-lasting up to day 84, indicating that the combination of T cell epitopes in the scaffold combined with the adjuvant has the ability to provide long-term immunity against SARS-CoV-2. [Figure 6] This figure shows d56 serum from mice immunized by a pool of non-conjugate (group 9) or conjugate (group 10) binding (to varying degrees) to HS1, HS2, HS3, HS4, HS5, HS6, or native S-glycoproteins produced in human cells. This demonstrates that, in addition to binding to antigen peptides, antibodies in serum can also bind to native epitopes in S proteins. [Figure 7] This figure shows the inhibition of entry of viable SARS-CoV-2 viruses into human cells by a vaccine (VP-HhC) at a serum dilution ratio of 1:400. [Modes for carrying out the invention]

[0016] Detailed explanation Viral diseases or infections are caused by viruses that infect the subject. Examples of viral diseases include AIDS, caused by human immunodeficiency virus (HIV); hepatitis B, caused by hepatitis B virus (HBV); dengue fever, caused by dengue virus; influenza, caused by influenza virus; yellow fever, caused by yellow fever virus (YFV); smallpox, caused by smallpox virus; SARS, caused by severe acute respiratory syndrome (SARS) coronavirus; COVID-19 (SARS-CoV-2), caused by SARS-CoV-2 virus; RSV infection, caused by respiratory syncytial virus (RSV); Zika fever, caused by Zika virus; chikungunya fever, caused by chikungunya virus; West Nile fever, caused by West Nile virus (WNV); and human papillomavirus (HPV) infection. Vaccines have been developed for some (but not all) of these viral diseases. One method for developing vaccines against viral diseases is to obtain peptides from viral proteins or polypeptides and conjugate them to a carrier. However, in many cases, viral peptides or polypeptides are not sufficiently antigenic.

[0017] Haptens are molecules that lack antigenic determinants, usually because they are small molecules. To become antigenic, they must be coupled to a carrier protein. As used herein, the term “hapten” refers to any molecule that lacks antigenic determinants until it is covalently or non-covalently coupled to a carrier protein, or a molecule whose antigenicity is increased by covalent or non-covalent coupling to a carrier protein. Similar to haptens, small peptides (i.e., usually less than 5,000 daltons) also lack antigenic determinants that would induce a robust immune response, and therefore they too must be coupled to a larger carrier protein that would make them immunogenic.

[0018] This disclosure describes hapten carriers (hCs) for peptides such as viral peptides (VPs). Depending on their size, VPs are haptens. When VPs bind to hCs as described herein, VPs can induce a robust immune response. VPs are obtained from viral proteins. VPs consist of small peptides or polypeptides. If a VP is sufficiently long, for example, 15-20 or more residues, coimmunization with hCs that do not covalently bind to hCs may be sufficient to induce a robust and persistent immune response, insofar as the hCs contain T cell epitopes capable of mobilizing T cell assistance.

[0019] In some embodiments, the disclosure also describes a VP-hC conjugate containing VP covalently bound to hC, and a vaccine containing a VPhC oligomer containing VP and monomeric peptides. The vaccines described herein are for preventing and / or treating viral infections or viral diseases. The VPhC oligomer may also contain a T cell epitope. Thus, VP vaccines containing VP-hC conjugates and VPhC oligomers can induce a robust and persistent immune response via both natural and adaptive pathways to produce high-titer, high-affinity antibodies targeting endogenous VP. For example, VP can induce a robust and persistent immune response via T cell activation, dendritic cell maturation, B cell activation, proliferation, maturation, establishment of a robust memory response, and other pathways.

[0020] After the initial prime / boost, antibody titers are expected to be greater than 10⁵. Booster injections were available to maintain or increase the therapeutic efficacy of the vaccine. Vaccine side effects are minimal due to the presence of a precise number of well-characterized and safe T-cell epitopes, the absence of immunodominant epitopes on hC, and the complete synthetic (non-biological) production of the vaccine. Multiple conformations on the carrier and precise spatial and stoichiometric arrangement of linear VP B-cell epitopes result in a potent vaccine capable of preventing and / or treating viral diseases or infections.

[0021] Furthermore, this disclosure describes a novel method for producing VP vaccines comprising VP-hC conjugates or VPhC oligomers. This method eliminates many of the highly sacrificial and time-consuming steps of traditional subunit vaccine development. Instead of producing subunits in a recombinant expression host, the flexible and modular system utilizes hC and viral components synthetically produced by solid-phase peptide synthesis (SPPS). The method described herein includes the step of designing hC components comprising monomeric peptides that self-assemble on an amphiphilic alpha-helix to form a carrier complex large enough to induce a robust immune response after one or more VPs are coupled to hC. In some embodiments, the monomeric peptides may self-assemble on a hexameric hC (HhC) core, and the VPs may be covalently bonded to the HhC core. In some embodiments, the HhC core may also contain T cell epitopes at the N and / or C-terminus of the amphiphilic alpha-helix.

[0022] The HhC (hexameric oligomer) described herein contains a central region that forms a hexameric core after hydration, and the lysine in this region functions as a conjugation site for any antigenic peptide or hapten, such as VP, to form VP-HhC. The size of HhC may vary depending on the T cell epitope length. At the time of hexamerization, the unconjugated hexamer is 38.5 kDa. The conjugated hexamer becomes larger depending on the length and size of the conjugated hapten.

[0023] This disclosure includes a peptide with at least 14 amino acid residues in length, where each heptad is in the pattern hwxhxyz (SEQ ID NO: 1) (In the formula, h is a hydrophobic or nonpolar residue. w is a positively charged, loaded, polar uncharged, or nonpolar aliphatic residue. x is a load charge, positive charge, nonpolar aliphatic, polar uncharged residue, or any native or unnatural residue for epitope coupling to a hapten or any other molecule. y is any natural or unnatural residue for epitope coupling to a hapten or any other molecule. z is a loaded, positively charged, polar uncharged, nonpolar aliphatic residue, or any native or unnatural residue for epitope coupling to a hapten or any other molecule. We describe the core region of hC that contains at least two heptad repeats having the following characteristics.

[0024] In one embodiment, the hC core region is pattern (hwxhxyz)n (sequence number 2) (In the formula, h is I, L, V, F, W, Y, M, G, or A. w is G, R, A, N, Q, H, S, D, E, K, or T. x is R, S, N, Q, A, G, T, D, E, K, H, or C. y is a non-natural amino acid or molecule containing K, H, C, D, E, R, W, Y, Q, N, or a reactive group suitable for covalent coupling. z is A, D, H, S, E, R, N, Q, K, or G. n is an integer greater than 1. It contains peptides.

[0025] In one embodiment, the exemplary heptad described herein has the following amino acid sequence: LRSIGKD (Sequence ID 3), LRSIGRD (Sequence ID 4), IREISRA (Sequence ID 5), IREVAQS (Sequence ID 6), IRDIAKA (Sequence ID 7), IRDIGRA (Sequence ID 8), IRDVGQS (Sequence ID 9), IRDLAKG (Sequence ID 10), VKDVARG(SEQ ID NO: 11), IRDIGNS (Sequence ID 12), IKDLARG (Sequence ID 13), IKKLKKK (Sequence ID 14), IRSIGKE (SEQ ID NO: 15), IRSIGRE (Sequence ID 16), IKSIGRE (SEQ ID NO: 17), or IRSIGRG (Sequence ID 18) It has.

[0026] In one embodiment, the core region of hC includes one or more heptads as described herein (wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11).

[0027] This disclosure describes a core region of hC containing a peptide of at least 14 residues. In some embodiments, the peptide comprises 14 to 18 residues in length and contains 2 to 11 heptad repeats. In some embodiments, the hC core region contains a peptide comprising 20 to 70 residues, 25 to 60 residues, 28 to 50 residues, 28 to 40 residues, or 28 to 30 residues. Peptides comprising 14 to 80 residues in length are monomers.

[0028] The terms “monomer peptide (MP)” and “monomer hC (MhC) peptide” are used interchangeably to refer to the monomer peptides described herein. In some embodiments, the exemplary monomer peptides or monomer hC peptides described herein have the following amino acid sequence: LRSIGKDLRSIGKDLRSIGKDLRSIGKD (Sequence ID 19), LRSIGKDLRSIGKDLRSIGKDLRSIGKDS (Sequence ID 20), LRSIGKDLRSIGRDLRSIGKDLRSIGRD (Sequence ID 21), IREISRAIREVAQSIRDIAKAIREIGKS (Sequence ID 22), IRDIGRAIRDVGQSIRDLAKGIRDISKG (Sequence No. 23), VKDVARGIRDIGNSIKDLARGIRDIGRG(SEQ ID NO: 24), LRSIGKDLRSIGRDLRSIGKDLRSIGRD (Sequence ID 25), IREISRAIREVAQSIRDIAKAIREIGKS (Sequence ID 26), IRDIGRAIRDVGQSIRDLAKGIRDISKG (Sequence No. 27), VKDVARGIRDIGNSIKDLARGIRDIGRG(Sequence ID 28), IRSIGKEIRSIGREIKSIGREIRSIGRG (Sequence No. 29), IRSIGKEIRSIGREIRSIGKEIRSIGRE (Sequence ID 30), or IRSIGKEIRSIGREIRSIGREIRSIGRE (Sequence No. 31) Includes.

[0029] The peptides described herein can be modified to include one or more substitutions, insertions, and / or deletions while maintaining the hwxhxyz (SEQ ID NO: 1) pattern described above. The heptad repeat or modifications at each position within the peptide must maintain the amphiphilic alpha-helix structure, stability, and oligomer-forming state of the peptide.

[0030] In one embodiment, the peptides described herein include (SEQ ID NO: 3)n, (SEQ ID NO: 4)n, (SEQ ID NO: 5)n, (SEQ ID NO: 6)n, (SEQ ID NO: 7)n, (SEQ ID NO: 8)n, (SEQ ID NO: 9)n, (SEQ ID NO: 10)n, (SEQ ID NO: 11)n, (SEQ ID NO: 12)n, (SEQ ID NO: 13)n, (SEQ ID NO: 14)n, (SEQ ID NO: 15)n, (SEQ ID NO: 16)n, (SEQ ID NO: 17)n, (SEQ ID NO: 18)n, (SEQ ID NO: 19)n, (SEQ ID NO: 20 The peptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with (21)n, (SEQ ID NO: 22)n, (SEQ ID NO: 23)n, (SEQ ID NO: 24)n, (SEQ ID NO: 25)n, (SEQ ID NO: 26)n, (SEQ ID NO: 27)n, (SEQ ID NO: 28)n, (SEQ ID NO: 29)n, (SEQ ID NO: 30)n, or (SEQ ID NO: 31)n (wherein n is an integer from 2 to 11). In some embodiments, the peptides described herein include peptides comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31. Sequence identity refers to the degree of agreement between two sequences in an alignment, often expressed as a percentage. The difference between two sequences may be determined by methods routinely used in the art to determine identity, designed to grant the greatest possible match between the sequences being examined. Methods for determining sequence identity may be determined by using publicly available computer programs. BLASTP is an example of a computer program method for determining identity between two sequences. Programs in the BLASTP family are publicly available from NCBI and other suppliers.

[0031] In some embodiments, one or more residues may be added to the N-terminus or C-terminus of monomeric peptides described herein to increase the peptide's stability in vivo. For example, V (valine), M (methionine), G (glycine), I (isoleucine), D (aspartic acid), or P (proline), or combinations thereof, may be added to the N-terminus or C-terminus of a peptide. Furthermore, protecting groups may be added to residues to protect peptides from degradation and, in particular, increase their stability in vivo. Examples of such protecting groups include acetyl, acrylic, 9-fluorenylmethoxycarbonyl, tert-butyloxycarbonyl, allyloxycarbonyl, benzyloxycarbonyl, and PEG (polyethylene glycol), and amides on the N-terminus or C-terminus. In some embodiments, an amide group protects the C-terminus.

[0032] The peptides described herein may be monomeric hC peptides, but because monomeric hC peptides self-assemble, they may self-assemble into an oligomer hC composed of dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nocumerers, or decamers. In some embodiments, the monomeric peptide self-assembles into a hexamer having six amphiphilic alpha-helices. In some embodiments, hC is a hexameric oligomer.

[0033] In some embodiments, the disclosure describes an hC containing one or more residues for conjugating a hapten such as a VP. The optimal site on the hC for conjugating a hapten is the y residue in the heptad repeat, but VP coupling can also occur on the w, x, and z residues, since the w, x, and z residues are accessible to the solvent and the VP can be covalently bonded to the hC containing the HhC using any residue that can covalently bond to the HhC. In some embodiments, the y residue is K, H, C, D, E, R, W, Y, Q, N, or a non-natural amino acid containing a reactive group suitable for covalent coupling. In some embodiments, there are 2 to 4 y residues on one side of each of the six amphiphilic alpha-helices to provide a coupling site. In some embodiments, the y residue is lysine (K).

[0034] In some embodiments, one or more VP peptides may be conjugated to MP using a y residue in SPPS, or after MP has been assembled into an oligomer such as a hexamer. VP conjugated to hC is a conjugate and is referred to as a VP-hC conjugate or VP-oligomer conjugate. In some embodiments, hC is linked to 1 to 100, 10 to 90, 20 to 80, 30 to 70, 40 to 60, or 50 viral peptides (VP). In some embodiments, hC is HhC, and the conjugate is VP-HhC.

[0035] In one embodiment, VP may be added to the N and / or C-terminus of monomeric peptides in SPPS (before self-assembly) to form VPMPs. Subsequently, VPMPs may self-assemble into VP oligomers, or more specifically, oligomers such as VP HhC (VP hexamer hC or VPHhC).

[0036] In one embodiment, hMP (hapten bound to a monomeric peptide) can self-assemble into hhC (hapten bound at the N or C terminus of a hapten carrier). If the hapten is VP, the hMP is VPMP, which self-assembles into a VPhC oligomer, for example, VPHhc (VP bound to a hexameric hapten carrier).

[0037] A vaccine for producing a vaccine to prevent and / or treat SARS-CoV-2 infection contains one or more antigenic peptides of the SARS-CoV-2 virus. Antigenic peptides derived from the SARS-CoV-2 virus may include amino acid sequences SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, or SEQ ID NO: 136. The antigenic peptides are S-glycoprotein (spike glycoprotein) derived antigenic peptides, such as SARS-CoV-2 virus-derived peptides S1, S2, S3, S4, S5, or S6. The antigenic peptides may also include SARS-CoV-2 virus-derived membrane protein (M1). The antigenic peptide for producing a vaccine against SARS-CoV-2 includes S1 (SEQ ID NO: 136), S2 (SEQ ID NO: 120), S3 (SEQ ID NO: 132), S4 (SEQ ID NO: 119), S5 (SEQ ID NO: 118), S6 (SEQ ID NO: 117), and / or M1 (SEQ ID NO: 130). In one embodiment, the antigenic peptide includes S1 (SEQ ID NO: 136), S5 (SEQ ID NO: 118), and / or S6 (SEQ ID NO: 117).

[0038] The vaccine for producing a vaccine to prevent and / or treat RSV infection contains one or more amino acid sequences derived from RSV, such as SEQ ID NO: 74 or SEQ ID NO: 75.

[0039] A vaccine for producing a vaccine to prevent and / or treat influenza caused by influenza A virus comprises one or more amino acid sequences derived from influenza A virus: SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82 and / or SEQ ID NO: 83.

[0040] The vaccine for producing a vaccine to prevent or treat West Nile fever comprises one or more amino acid sequences derived from WNV: SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 and / or SEQ ID NO: 87.

[0041] The VP for producing a vaccine to prevent or treat yellow fever contains one or more amino acid sequences derived from YFV: SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 and / or SEQ ID NO: 91.

[0042] The VP for producing a vaccine to prevent or treat human papillomavirus infection comprises one or more amino acid sequences derived from HPV, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95 and / or SEQ ID NO: 96.

[0043] A vaccine for producing a vaccine to prevent or treat dengue fever comprises one or more amino acid sequences derived from the dengue virus: SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 and / or SEQ ID NO: 102.

[0044] In some embodiments, the wild-type VP described herein can be modified to produce useful peptide immunogens. The wild-type VP may be modified by substitution, deletion, or insertion to include residues that make the peptide more useful and easier to use as a peptide immunogen. The modification does not alter the functional properties of the VP, and as a result, it can still be used as a peptide immunogen. For example, a C (cysteine) residue can be replaced with an S (serine) residue because S has similar polarity and shape to C, but its hydroxyl group does not react with maleimide-activated hC, thereby facilitating conjugation with hC. In some embodiments, the modified VP of the SARS-CoV2 peptide includes the S4 peptide (SEQ ID NO: 139) and the S6 peptide (SEQ ID NO: 140).

[0045] In some embodiments, one or more VPs that can bind to hC may be the same VP or different VPs. For example, one or more S peptides, e.g., S1, S2, S3, S4, S5, S6, or M1, may bind to the same hC. As another example, VPs from different SARS-CoV-2 virus strains may bind to the same hC for the purpose of producing a SARS-CoV-2 vaccine. Different VPs may bind to hC simultaneously or separately and then be combined.

[0046] In the context of conjugates or oligomers, the terms “conjugated,” “linked,” or “coupled” are interchangeable to refer to conjugation to a self-assembled oligomer (hC), or addition to or incorporation of a monomeric peptide in SPPS prior to self-assembly to an oligomer hC.

[0047] One or more residues may be added to the N-terminus or C-terminus of the VP described herein. One or more residues may make the VP more stable. For example, one or more residues increase the in vivo half-life of the VP. In some embodiments, by adding one or more residues to the N-terminus of the VP, the in vivo half-life of the VP may be increased to be longer than that of the VP peptide without the one or more residues added to its N-terminus or C-terminus, by more than about 5 times and more than about 100 times. In some embodiments, one or more residues may increase the in vivo half-life of the VP to be longer than that of the VP without the residues added to its N-terminus or C-terminus, by more than 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times. In some embodiments, residues such as G (glycine), V (valine), M (methionine), or A (alanine), or combinations thereof, may be added to the N or C terminus of VP for stability. In some embodiments, V is added to the N terminus of VP to improve its in vivo half-life from about 60 minutes to 100 hours.

[0048] In some embodiments, the N-terminus of VP can be protected by an acetylating group, and / or the C-terminus can be protected by an amide group. In some embodiments, the in vivo half-life can be increased to more than 100 hours by adding valine to the N-terminus of VP and protecting valine with an acetyl group.

[0049] Other residues may also be added to the N-terminus or C-terminus of the VP to aid in conjugation to hC. For example, one or more residues can be added to the N-terminus or C-terminus of the VP to sufficiently reduce the pI (isoelectric point) by decreasing the electrostatic repulsion with hC. For example, residues GEDC (SEQ ID NO: 53), DGEGC (SEQ ID NO: 137), or DDEDC (SEQ ID NO: 116) can be added to the C-terminus or N-terminus as linkers for conjugation and, if necessary, to modify the pI of the VP. For example, if hC has a pI value greater than 7, DDEDC can be used to lower the pI of the VP and reduce the charge repulsion, which can significantly affect the efficiency of conjugation. If the scaffold pI is acidic (approximately 4 or 5), DDEDC is converted to RRKR (SEQ ID NO: 138) to increase the pI of the peptide. The residues added depend on the sequence of the core region and the T cell epitopes at the N and C terminals, because they affect the pI of hC. If modification of the pI of VP is not necessary, a linker containing a G residue, such as GGGC (SEQ ID NO: 103), is added.

[0050] Optionally, other molecules may be directly conjugated with VP to hC oligomers such as HhC. Other molecules may also be conjugated to VP and subsequently to hC. Furthermore, as described herein, VP can be conjugated to the N or C-terminus of the hC monomer peptide during SPPS to form VPMP before self-assembly to VPhC oligomers such as VPHhC. One or more other molecules may also be conjugated to the N or C-terminus of the hC monomer (MhC) peptide during SPPS, in addition to VP, before self-assembly to the hC oligomer.

[0051] Other molecules that can bind to hC oligomers or MPs include any active agents that can induce the production of antibodies useful in treating, preventing, or mitigating symptoms of viral disease or infection, or in reducing the risk of developing viral disease or infection in a subject. Examples of other molecules, in addition to VP, include immunomodulators and haptens. Examples of immunomodulators, including adjuvant molecules, include T-cell epitope peptides, nucleic acids, lipids, lipopeptides, lipoproteins, carbohydrates, and short peptides. Peptides that can be used as haptens, including VP and B-cell epitopes, include naturally occurring or unnaturally produced D- or L-amino acids, synthetically produced or recombinantly produced, or natural peptides or proteins.

[0052] Haptens other than VP described herein may be conjugated with VP to hC such as HhC. As previously explained, the term “hapten” refers to molecules that are not good immunogens on their own, but become immunogenic when conjugated to another molecule, such as a larger molecule. Haptens may be, for example, small organic molecules, monosaccharides, disaccharides, oligosaccharides, lipids, nucleic acids, peptides, or polypeptides. Haptens may be able to bind to antibodies, but hapten-mediated immunization does not usually induce a potent antibody response. However, immunogenicity can be achieved when haptens are covalently bonded by being linked or conjugated to a larger carrier molecule, such as a hapten-carrier conjugate greater than 5,000 daltons.

[0053] Other haptens that can be conjugated to hC include any active substances that induce the production of antibodies useful in preventing or treating viral infections, thereby reducing or eliminating the symptoms of viral diseases. Haptens can also reduce the risk for patients to develop diseases or disorders caused by viral infections. In addition to VP, examples of haptens include peptides, nucleic acids, lipids, lipopeptides, lipoproteins, carbohydrates, and small molecules. Examples of peptides that can be used as haptens include T cell epitopes and VP B cell epitopes. Peptides that can be used as haptens, including VP, T cell epitopes, and B cell epitopes, include synthetically produced, recombinant, or natural peptides or proteins containing natural or non-natural D- or L-amino acids.

[0054] T cell epitopes (for T cells to assist B cells) that can be used to activate the T cell response are found in the extracellular proteins of Clostridium botulinum, Clostridium perfringens, and Staphylococcus aureus, as well as in the extracellular solute-binding proteins of Mycobacterium and Clostridium tetani. T cell epitopes are also present in Mycobacterium tuberculosis, Mumps rubulavirus, Plasmodium falciparum, human immunodeficiency virus 1, hepatitis C virus, and influenza A virus. Examples of such T cell epitopes include amino acid sequence SEQ ID NO: 32 or SEQ ID NO: 33 (derived from extracellular protein of Clostridium botulinum, GenBank: STC78113.1), SEQ ID NO: 34 (derived from extracellular protein of Clostridium perfringens, GenBank: SUY45886.1), SEQ ID NO: 35 (derived from extracellular protein of Staphylococcus aureus, GenBank: SAO03917.1), SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40 (derived from extracellular solute-binding proteins of various species of Mycobacterium, NCBI reference sequence: WP_055398728.1), SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43 (extracellular solute-binding proteins of Mycobacterium tetanus). Examples include protein-binding proteins (GenBank: CDI50554.1), SEQ ID NO: 44 (derived from ESAT-6-like protein EsxB of Mycobacterium tuberculosis), SEQ ID NO: 45 (derived from alpha-crystalline protein of Mycobacterium tuberculosis), SEQ ID NO: 46 (derived from mumps virus protein of mumps virus), SEQ ID NO: 47 (derived from DNAJ of Plasmodium falciparum), SEQ ID NO: 48 (derived from Gag-Pol polyprotein of human immunodeficiency virus 1), SEQ ID NO: 49 (derived from genomic polyprotein of hepatitis C virus), SEQ ID NO: 50 (derived from substrate protein 1 of influenza A virus), and SEQ ID NO: 51 (derived from hemagglutinin of influenza A virus).

[0055] Lipids that can bind to hC include those that induce innate immune responses through binding to Toll-like receptors (TLRs). Lipids can also function as adjuvants. Examples of such lipids include monophosphoryl lipid-A, squalene, lipopolysaccharides (LPS), lipoproteins, or lipopeptides. Carbohydrates that can function as haptens include glucose, disaccharides, trisaccharides, and larger sugars, including complex carbohydrates.

[0056] Examples of TLR-binding peptides that may utilize haptens include TLR ligands, such as TLR-4 agonists. These peptides act as adjuvant peptides. In one embodiment, the adjuvant peptide comprises the amino acid sequence APPHALS (SEQ ID NO: 52).

[0057] Other molecules may include haptens, such as B cell epitopes. Examples of B cell epitopes that can be used as haptens include those on any peptide for which an immune response is desired. Further VPs of the same target as peptide immunogens may be added as other molecules. As used herein, the same target refers to treating and preventing diseases or infections caused by the same virus.

[0058] If a hapten is a small peptide, the entire peptide can be used as the hapten. If a hapten is a protein, only a portion of it can be used as the hapten. The portion of the protein to be used as a hapten can be determined using well-known methods such as in silico prediction algorithms or peptide-based epitope mapping of the entire protein. Many T cell and B cell epitopes have been determined using these methods.

[0059] Haptens that can enhance the immunogenicity of VP or enhance the duration or extent of the immune response to VP can be conjugated with hC along with VP. For example, a hapten that functions to bind TLRs may have an adjuvant function and enhance the immunogenicity of VP. In some embodiments, the VP-HhC conjugate may contain one or more different or the same VP, as well as other haptens or peptides.

[0060] One or more residues may be added to the N-terminus or C-terminus of the haptens described herein to increase the in vivo stability of the peptide. For example, V (valine), M (methionine), G (glycine), I (isoleucine), D (aspartic acid), or P (proline), or combinations thereof, may be added to the N-terminus or C-terminus of the peptide.

[0061] This disclosure describes VP immunogens comprising VP-hC conjugates and VPhC oligomers. These conjugates and oligomers may also comprise other molecules. Peptides used to prepare VP immunogens include monomeric peptides, VP, other molecules including T cell epitopes, haptens, and adjuvant peptides, as described herein. These can be chemically synthesized by manual techniques or automated procedures. For example, solid-phase polypeptide synthesis (SPPS) has been practiced since the early 1960s. Over many years, improvements have been made to early SPPS methods, and many methods have been automated.

[0062] Peptides, including those for generating hC, VP, and other haptens described herein, can be chemically synthesized by manual techniques or automated procedures. For example, solid-phase polypeptide synthesis (SPPS) has been practiced since the early 1960s. Over many years, improvements have been made to early SPPS, and many methods have been automated. Chemical components have been developed to protect terminals and other reactive groups. The terminals of the peptides described herein can be protected, for example, by an acetyl group, benzyloxycarbonyl group, biotin group, cinnamic acid group, FMOC group, tBOC group, formyl group, or N-methyl group at the N-terminus, and / or an amide group at the C-terminus. Linkers, such as proteolytic cleavage sites, spacers, and / or haptens, such as T-cell epitopes, can be added to monomeric peptides before the addition of protecting groups.

[0063] Peptides, particularly the longer peptides described herein, can be produced by native chemical ligation (NCL). Using NCL, larger peptides (polypeptides) can be formed by ligating (or coupling) two or more smaller peptides. In some embodiments, a polypeptide comprising a monomeric peptide and two or more haptens can be prepared from two or more smaller peptide fragments and assembled together using NCL techniques. For example, a polypeptide comprising a monomeric peptide and two haptens (at the N and C-terminuses of the monomeric peptide) can be synthesized from two smaller peptides, which are covalently bonded by NCL. Using NCL, C (cysteine) is added to the N-terminus of one of the two smaller peptides, and a thioester functional group is added to the C-terminus of the other of the two smaller peptides, and then these two peptides are ligated into a full-length polypeptide. In some embodiments, residues are added to the peptides described herein for the ease of synthesis of longer polypeptides.

[0064] In some embodiments, a spacer may be added between the monomeric peptide and VP or one or more other haptens during synthesis. Examples of one or more residues that can be inserted as a spacer include G (glycine), D (aspartic acid), S (serine), C (cysteine), or combinations thereof. In some embodiments, D, GD, or GSG may also be used as spacers.

[0065] The peptides and haptens described herein may also be produced biologically or recombinantly in heterologous expression systems. Any heterologous expression system may be used to produce the peptides described herein. In some embodiments, the expression system comprises Escherichia coli, which lacks mechanisms for post-translational modification, making the expression system a suitable host for producing the peptides described herein.

[0066] Other molecules containing VP may be conjugated to hC using click chemistry or any known method including homo- or heterobifunctional crosslinking reagents or peptide bond formation. In some embodiments, the hapten may be conjugated to hC using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) / NHS (N-hydroxysuccinimide) or NHS / maleimide crosslinking chemistry, which are routinely used in conjugation reactions. A y residue, such as lysine, is arranged to provide a clearly defined hapten configuration and coupling stoichiometry.

[0067] Other molecules containing VP may also be bound to hC via any suitable linker moiety. Examples of linkers include those forming amide bonds, ester bonds, and disulfide bonds. The linker may be a cleavable linker, e.g., a protease-cleavable peptide linker, a nuclease-sensitive nucleic acid linker, a lipase-sensitive lipid linker, a glycosidase-sensitive carbohydrate linker, a pH-sensitive linker, a hypoxia-sensitive linker, a photocleavable linker, a thermally unstable linker, or an enzymatically cleavable linker, e.g., a proteolytic cleavage site. For example, a proteolytic cleavage site may include the amino acid sequence YR. The linker may also be non-cleavable. Linkers can be bound to hC using any known method, e.g., click chemistry, passive adsorption, polyvalent chelation, high-affinity non-covalent bonding, or covalent bond formation. Haptens may also be bound to hC without a linker.

[0068] Furthermore, other molecules, including VP, can be conjugated to hC through other molecules. For example, VP or another B cell or T cell epitope can first be conjugated to HhC after being bound to a carrier for displaying the desired epitope. Examples of such carriers include proteins, peptides, nanoparticles, virus-like particles, or anything that can function as a carrier for displaying VP or another desired epitope.

[0069] Furthermore, this disclosure describes VP-hC conjugates or VPhC oligomers that optionally include one or more other molecules, such as those described herein. One or more other molecules include immunomodulators and / or haptens. In some embodiments, one or more other molecules include T cell epitopes, B cell epitopes, short peptides, such as VP peptides, or combinations thereof. In some embodiments, one or more other molecules are ligated to the N and / or C-terminuses of one or more helices in the core of hC. In some embodiments, one or more other molecules are ligated to the N-terminuses of one or more helices in the core of hC. In some embodiments, one or more molecules are ligated to the C-terminuses of one or more helices in the core of hC.

[0070] In some embodiments, T cell epitopes located at the N and / or C-terminuses of one or more helices in the core of hC mobilize T helper cells, induce B cells to yield maximum IgG titers for a robust immune response, and promote affinity maturation and class switching. Methods for selecting T cell epitope peptides are well known. For example, T cell epitopes may be selected by experimental methods known in the art, identified from chemical literature, predicted using bioinformatics tools, designed de novo, or a combination thereof. In some embodiments, the T cell epitopes at the N-terminus and C-terminus may be the same or different. In some embodiments, the T cell epitopes may be, for example, CD4+ T cell epitopes known to enhance the development of memory B cells and plasma cells that produce high-affinity antibodies. In some embodiments, T cell epitopes that may be contained in the N and / or C-terminus of one or more helices of hC include TCE1, TCE2, TCE3, TCE4, TCE5, or combinations thereof. For example, T cell epitopes include amino acid sequences SEQ ID NOs. 44, 45, 46, 47, 48, 49, 50, 51, 105, 106, 107, or 108. In some embodiments, a T cell epitope containing amino acid sequence SEQ ID NOs. 105 or 106 binds to the N-terminus, and a T cell epitope containing amino acid sequence SEQ ID NOs. 107 or 108 binds to the C-terminus. One or more of these T cell epitopes may bind to hC or VP.

[0071] One or more T cell epitopes and / or B cell epitopes can also be conjugated to VP before conjugating to hC. Furthermore, these epitopes recruit T helper cells and induce B cells to yield maximum IgG titer, as well as promote affinity maturation and class switching.

[0072] When haptens or immunomodulators, such as T cell and B cell epitopes, are linked to VPs for conjugation to hC or to the N and / or C-terminus of monomeric peptides, one or more spacers may be inserted between the hapten and the VP, or between the hapten and the monomeric peptide. Spacers are added for precise processing of the T cell epitope, ensuring proteolytic trimming that results in a size suitable for the MHC II junction gap, for immunomodulators, such as T cell epitopes. Examples of such spacers include residues D (aspartic acid), G (glycine), P (proline), S (serine), or combinations thereof. In some embodiments, the spacers include one or more of D, GD, PGP, GSG, GPGP (SEQ ID NO: 109), GPGPG (SEQ ID NO: 104), GPGPGC (SEQ ID NO: 110), SGPGPG (SEQ ID NO: 111), or HAA. In one embodiment, the spacer for precise processing of the T-cell epitope includes GPGPG (SEQ ID NO: 104).

[0073] Haptens or immunomodulators described herein, which are small peptides, may be linked at the N and / or C-terminus of one or more helices of the core of an hC. They may be incorporated into monomer peptides so that they are covalently bonded to the N and / or C-terminus of the monomer peptide using solid-phase synthesis or native chemical ligation (NCL). Haptens may be covalently bonded to the N and / or C-terminus using homo- or hetero-bifunctional crosslinking agents or click chemistry reagents, which are well-known reagents for coupling molecules. In some embodiments, immunomodulators or haptens, such as T cell epitopes and / or B cell epitopes, are already bound to the N and / or C-terminus before self-assembly to an hC core, such as an HhC core, and the VP may be conjugated after self-assembly to the hC core.

[0074] The hapten or immunomodulator at the N and / or C terminus may also be linked to or conjugated to hC via either a reactive small molecule or a large molecule intermediate functional reagent. Examples of such small molecules include catalysts, stable intermediates, or salts. Examples of such large molecules include multiantigenic peptides, proteins, or enzymes.

[0075] Furthermore, the conjugation of haptens containing VP and / or other molecules to the hC core can be carried out using any type of linker. The linker may be cleavable or incleavable. Examples of cleavable linkers include protease-cleavable peptide linkers, nuclease-sensitive nucleic acid linkers, lipase-sensitive lipid linkers, glycosidase-sensitive carbohydrate linkers, pH-sensitive linkers, enzyme-cleavable linkers, thermally unstable linkers, and photocleavable linkers. Crosslinkers may also be used by activating side-chain or terminal atoms for covalent reactions with intermediate or final molecular atoms to form covalent bonds.

[0076] This disclosure describes a scaffold peptide (hapten carrier (hC)) comprising an hC monomer peptide and one or more haptens and / or immunomodulators, such as one or more T cell epitopes and / or VPs linked to its N or C terminus. As described herein, the scaffold peptide may also include one or more spacers, for example, one or more residues for precise processing of the T cell epitope or for stabilizing the hapten and / or hC monomer peptide. Table 1 discloses exemplary scaffold peptides.

[0077] [Table 1]

[0078] The mouse scaffold peptide (hC) contains a T cell epitope at the N-terminus (SEQ ID NO: 105), an hC monomer peptide (SEQ ID NO: 30), and another T cell epitope at the C-terminus (SEQ ID NO: 107). It also contains a stabilizing residue valine (V) and a linker (SEQ ID NO: 104) inserted between the T cell epitope and the hC monomer peptide. Residue D is added as a spacer. In this example, the T cell epitope binds to the hC monomer peptide before self-assembly into a hexameric core for binding VP. The linker (SEQ ID NO: 104) is added to enhance precise T cell epitope processing.

[0079] The human scaffold peptide contains a T cell epitope at the N-terminus (SEQ ID NO: 106), an hC monomer peptide (SEQ ID NO: 30), and another T cell epitope at the C-terminus (SEQ ID NO: 108). It also contains one or more stabilizing residues, valine (V) and aspartic acid (D), as well as linker / proteolytic cleavage sites (HAA and YR) inserted between the T cell epitope and the hC monomer peptide. The linker (HAA) is added to enhance precise T cell epitope processing, and the linker (YR) is a proteolytic cleavage site. In this example, the T cell epitope is bound to the hC monomer peptide before self-assembly into a hexameric core for binding to VP. The human scaffold peptide (hC) can be used in human subjects as well as other mammalian subjects, including mice and rabbits.

[0080] These scaffold peptides can be protected at the N-terminus and / or C-terminus by protecting groups to prevent peptide proteolysis. For example, an acetyl group can be added to the N-terminus, and an amino group can be added to the C-terminus.

[0081] The exemplary scaffold peptides shown in Table 1 self-assemble to form a hexameric core (Hex) or hexameric scaffold.

[0082] This disclosure also describes peptide immunogens containing VP for conjugating to hC to form VP-hC conjugates for vaccine preparation. As described herein, peptide immunogens may also contain one or more other residues for stabilizing VP or for precise processing of T cell epitopes. Peptide immunogens may be based on viral peptides, e.g., wild-type peptide sequences. However, wild-type peptide sequences may also be modified to produce peptides that are more useful or easier to use as peptide immunogens. Table 2 discloses exemplary peptide immunogens for conjugating to or binding to hC.

[0083] [Table 2]

[0084] For example, peptide immunogens S4 and S6 have been modified by substituting C (cysteine) with S (serine) in the wild-type peptide because C may interfere with the conjugation to hC.

[0085] The peptide immunogens described herein may optionally include one or more further haptens, such as one or more immunomodulators, including T cell epitopes.

[0086] These peptide immunogens may be protected at the N-terminus and / or C-terminus by protecting groups to prevent peptide proteolysis. For example, an acetyl group can be added to the N-terminus, and an amide group can be added to the C-terminus.

[0087] Furthermore, this disclosure describes VP-hC conjugates. Exemplary VP-hC conjugates include S1 peptide + scaffold peptide (S1 peptide immunogen conjugated to a hexamer scaffold (H) containing the scaffold peptide shown in Table 1), S2 peptide + scaffold, S3 peptide + scaffold, S3 peptide + scaffold, S4 peptide + scaffold, S5 peptide + scaffold, S6 peptide + scaffold, and M1 peptide + scaffold.

[0088] In some embodiments, when a virus-derived peptide immunogen such as S1, S2, S3, S4, S5, S6, or M1 is bound to (incorporated into) the scaffold peptide, the monomeric peptides described herein may also be used as immunogens. One or more peptide immunogens may be inserted during the synthesis of the scaffold peptide such that the last peptide contains both the scaffold peptide and one or more peptide immunogens. The last peptide may also self-assemble into an oligomer such as a hexamer. Examples of scaffolds containing VP immunogen (VPHhC) include S1HexhC, S2HexhC, S3HexhC, S4HexhC, S5HexhC, S6HexhC, and M1HexhC, where S1 is the peptide immunogen and HexhC is the hexamer core. Short peptides and residues described herein may be added to provide stability. Optionally, additional haptens may be conjugated to the oligomeric cores of these scaffold peptides.

[0089] The VP-hC conjugates and VPhC oligomers described herein are used to prepare compositions such as pharmaceutical compositions. Pharmaceutical compositions comprising one or more VP-hC conjugates and one or more VPhC oligomers may be used as therapeutic agents or vaccines or vaccine or therapeutic compositions. The pharmaceutical compositions described herein are also immunogenic compositions that contain immunomodulators to enhance the immunogenicity of VP. The pharmaceutical compositions described herein are also therapeutic compositions that may be used to treat patients who need them.

[0090] This disclosure describes compositions comprising a VP-hC conjugate, a VPhC oligomer as described herein, and one or more excipients. In some embodiments, hC is conjugated to one or more VPs and optionally comprises other haptens, such as one or more T cell epitopes at the N and / or C-terminuses of one or more amphiphilic helices of the core of hC. In some embodiments, the composition is a pharmaceutical composition, and the excipients are pharmaceutically acceptable excipients. In some embodiments, hC is HhC (hexameric hapten carrier).

[0091] The term "excipient" refers to a diluent, adjuvant, or vehicle administered together with hC. Examples of adjuvants include complete and incomplete Freund's adjuvants, which are used with animals, especially research animals. Pharmaceutically acceptable excipients may be sterile liquids such as water and oil, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. When the pharmaceutical composition is administered intravenously, water is a preferred excipient. Saline solutions, dextrose water, and glycerol solutions may also be used as liquid excipients, particularly with respect to injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, white flour, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, and ethanol. Examples of pharmaceutically acceptable adjuvants include those based on monophosphoryl lipid-A (MPL A) mixed with oil to form a stable emulsion, such as squalene.

[0092] The composition or pharmaceutical composition may also optionally contain small amounts of wetting agents or emulsifiers, or pH buffers. These compositions may take the form of liquids, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may contain standard excipients, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such formulations contain a therapeutically effective amount of purified hC along with an amount of excipients suitable for providing a form for appropriate administration to the subject.

[0093] The pharmaceutical compositions described herein may be administered by any convenient method, including by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein may also be administered to a subject orally, topically, intranasally, enterally, rectally, orally, transvaginally, sublingually, subcutaneously, intradermally, intratumorally, intranodally, intramedullarily, intramuscularly, intravenously, intracranially, intraperitoneally, or in combination thereof. The pharmaceutical compositions may be administered in any manner effective in delivering a therapeutically and / or prophylactically effective amount of the conjugate described herein to a subject requiring delivery.

[0094] The compositions described herein include immunogenic compositions. In some embodiments, the compositions described herein are therapeutic agents or vaccines. This disclosure describes a method for preparing a vaccine, comprising the steps of designing and preparing a monomeric peptide for the core of hC described herein, oligomerizing the monomeric peptide, and conjugating one or more VPs to the oligomerized hC to obtain a VP-hC conjugate. In some embodiments, hC is hexamer hC (Hhc). The one or more VPs may be the same or different. Furthermore, this disclosure describes a method for preparing a vaccine or therapeutic agent, comprising the steps of designing and preparing a monomeric peptide for the core of hC described herein, covalently bonding VPs to the monomeric peptide, and oligomerizing the monomeric peptide to obtain a VPHhC oligomer such as an S1HhC oligomer. As described above, monomeric peptides can be synthesized by SPPS, which includes supplying prepared monomeric peptides in lyophilized form. Hydration of the lyophilized monomeric peptides leads to oligomerization. The lyophilized monomeric peptides can be hydrated using PBS containing salt and buffering capacity. In one embodiment, the oligomerized hC is HhC.

[0095] The methods described herein include increasing the immunogenicity of VP. The methods include conjugating one or more VPs to hC as described herein. The methods may further include a step of synthesizing monomeric peptides using one or more other haptens or immunomodulators, for example, T cell or B cell epitopes located at the N and / or C-terminus of one or more helices of the core of hC. In some embodiments, the monomeric peptides are synthesized using T cell and / or B cell epitopes located at the N and / or C-terminus. The increase in the immunogenicity of VP is compared to the immunogenicity of VP that is not conjugated to hC or excipients, or that is not bound to hC or excipients. Furthermore, the methods described herein also include a step of conjugating one or more other haptens or immunomodulators to increase the immunogenicity of VP. Examples of such haptens and immunomodulators include small molecules, lipids, lipoproteins, and TLR-4 agonists.

[0096] In some embodiments, this disclosure describes an immunogenic composition comprising a VP-hC conjugate as described herein. The VP-hC conjugate optionally comprises one or more T-cell and / or B-cell epitopes and / or one or more further haptens other than VP. In some embodiments, hC is HhC. The immunogenic composition comprises one or more pharmaceutically acceptable excipients. The excipients may be adjuvants used to improve or enhance the immune response to the VP-hC conjugate in a therapeutically effective manner. The immunogenic composition may be administered to a subject requiring it by any route described herein for delivering an effective amount of the VP vaccine to that subject.

[0097] The dosages for administering the pharmaceutical and immunogenic compositions described herein vary depending on the exact nature of the condition being treated and the recipient of the treatment. Dosage scaling for human administration may be performed by physicians in accordance with accepted practices in the art, depending on various factors.

[0098] The pharmaceutical compositions or immunogenic compositions described herein may be formulations. In some embodiments, the pharmaceutical compositions or immunogenic compositions may be formulated for immediate release, or for sustained-release or prolonged-release. Such formulations may be prepared using well-known techniques. Sustained-release formulations may contain the conjugate described herein, dispersed in an excipient matrix and / or contained in a reservoir surrounded by a rate-limiting membrane. The excipients for use in such formulations are biocompatible and / or biodegradable. The formulations provide a relatively constant level of active ingredient release. The amount of conjugate contained in a sustained-release formulation depends on the site of implantation, the rate and expected duration of release, and the nature of the condition to be treated or prevented.

[0099] This disclosure also describes kits having unit doses of the conjugates described herein. Such kits may include a container containing the unit dose, an informational document with instructions for use to treat or prevent a disease or disorder of interest using the kit, and optionally an appliance or device for delivering the composition.

[0100] Furthermore, this disclosure describes methods for enhancing the immunogenicity of VP. In some embodiments, the method includes the steps of obtaining a monomeric peptide as described herein, self-assembling the monomeric peptide into an oligomer (hC) such as a hexamer, and conjugating a hapten such as VP into an oligomer (hexamer hC) to obtain a hapten-hC such as VP-HhC. Immunomodulators can also be conjugated into oligomers. In some embodiments, the method also includes the steps of synthesizing a monomeric peptide (MP) such that it contains a VP peptide at its N and / or C-terminus, and self-assembling the VPMP (VPMP) into an oligomer such as a hexamer to obtain a VPhC oligomer such as a VPHhC oligomer. As described herein, a VP-hC conjugate or VPhC oligomer may comprise one or more further haptens or immunomodulators, for example, one or more T-cell epitopes or B-cell epitopes. As described herein, a VP-hC conjugate or VPhC oligomer may further comprise one or more residues for stabilizing the hapten, one or more residues for proper processing of the T-cell epitope, and / or one or more spacers inserted between the hapten and the monomeric peptide. The methods described herein may be used to prepare a vaccine or therapeutic agent, or a composition comprising a vaccine or therapeutic agent, for administration to a subject that requires it to prevent or treat the subject, for example, a VP immunogenicity therapeutic composition.

[0101] This disclosure also describes the use of the conjugates, oligomers, pharmaceutical compositions, therapeutic agents, therapeutic compositions, and vaccines described herein for treating subjects that require them. Vaccines, like the compositions, include the conjugates or oligomers described herein. This disclosure also describes methods for treating subjects that require them.

[0102] The methods described herein include steps for treating subjects such as humans, veterinary animals (dogs, cats, reptiles, birds, etc.), livestock (horses, cattle, goats, pigs, chickens, etc.), and research animals (monkeys, rats, fish, etc.). Subjects requiring treatment are those having a disease or disorder that needs to be treated with a VP vaccine or immunogenic composition that induces an immune response in the subject, which is sufficient to prevent or treat the subject's disease or disorder, or is therapeutically effective. Subjects requiring such treatment may also be susceptible to or at risk of developing a viral disease or infection. Subjects requiring such treatment may also be individuals infected with a virus that causes a viral disease or infection. The viral disease or infection may be of a mild, severe, or fatal nature.

[0103] For example, antibodies induced by vaccination with VP-hC conjugates or VPhC oligomers described herein can neutralize, prevent, or treat viral diseases or infections. The immune response produced by VP-hC conjugates or VPhC oligomers is sufficient to prevent or treat viral diseases or infections. In some embodiments, the methods described herein may be used to prevent a subject from developing a viral disease or to treat a subject infected with a virus.

[0104] As used herein, prevention refers to preventing or reducing the risk that a subject requiring them will develop a viral disease or viral infection. Prevention includes inhibiting, reducing, or attenuating the function of the virus, for example, weakening the virus to make it difficult for the virus to fuse with and enter the membrane of a host (subject) cell. In some embodiments, the VP-hC conjugate or VPhC oligomer described herein induces an immune response in the subject to produce antibodies that reduce the ability of the virus to invade host cells. The antibodies remain in the subject and inhibit, reduce, or attenuate the generation of new viruses or viral particles. In some embodiments, the VP-hC conjugate or VPhC oligomer is administered to the subject before infection to prevent the subject from developing the disease. In some embodiments, the VP-hC conjugate or VPhC oligomer administered to the subject according to herein is administered after infection to prevent the subject from developing a serious viral disease or viral infection.

[0105] The treatment of subjects requiring the use thereof as described herein includes reducing the symptoms that a viral disease or viral infection would normally cause in a subject if the subject were not administered a VP vaccine before or after infection. In some embodiments, the VP-hC conjugate or VPhC oligomer described herein induces an immune response in a subject, generates antibodies that neutralize the virus and reduce the symptoms of a viral disease or viral infection, and as a result the subject does not suffer from a mild, severe, or fatal viral disease or viral infection. In some embodiments, the VP-hC conjugate or VPhC oligomer may be administered as a treatment after infection to inhibit, reduce, or attenuate the function of the virus and reduce the symptoms of a viral disease or viral infection.

[0106] The methods described herein include prophylactic measures for subjects requiring them. The methods described herein include protecting subjects from viral diseases by inducing an immune response in the subjects that is sufficient to protect the subjects from viral diseases, reduce the symptoms of viral diseases, or is therapeutically effective.

[0107] The preventive and therapeutic methods described herein involve administering an effective amount of the conjugate described herein or a composition comprising an effective amount of the conjugate described herein. “Effective amount” is the amount of the activator, e.g., the conjugate or composition described herein, required to produce a desired physiological change in vivo or in vitro. A therapeutically effective amount encompasses the amount that provides an effective amount.

[0108] An effective vaccine contains components capable of inducing both innate and adaptive immune responses after immunization. While innate immunity is induced using an adjuvant, in some embodiments, the vaccine described herein is a VP-hC conjugate or VPhC oligomer containing adaptive B and T cell epitopes. The VP-hC conjugate contains a minimal exogenous sequence for a more focused and robust immune response to the VP B cell epitope. In some embodiments, for CD4+ and CD8+ T cell activation, the N and C-terminuses of each of the six helices and / or cores of the HhC contain species-specific CD4+ and CD8+ T cell epitopes required as part of the adaptive immune system to generate pathogen-specific memory for immune defense, mobilize T cell assistance, and produce long-lived plasma cells and high-titer / high-affinity antibodies to lead to robust and persistent protective memory. These epitopes are located at the terminals of the HhC, and as a result, these epitopes do not interfere with hapten coupling. These epitopes are selected to lack lysine and cysteine ​​residues, and as a result, they are not haptenized or uncontrollably crosslinked during the B-cell epitope coupling process. Lysine haptenization in T-cell epitopes has been shown to greatly reduce their activity and function. A wide variety of species-derived T-cell epitopes can be obtained from the IEDB database and selected based on positive T and B-cell assays, including MHC ligand binding assays, the ability to mobilize T-cell aids, and induction of B-cell proliferation. The modular nature of the vaccine technologies described herein simplifies the transfer of vaccine constructs between species, as when various diseases or conditions are targeted, it is simply a matter of replacing T-cell epitopes and modifying B-cell epitopes.

[0109] Similar to VP-hC conjugates, VPhC oligomers contain minimal exogenous sequences for a more intensive and robust immune response against VP B-cell epitopes. VPhC oligomers can contain T and / or B-cell epitopes because these epitopes can bind to hC, i.e., the oligomer core, via covalent coupling.

[0110] The apparent advantage of the HhC core region described herein is its reduced immunogenicity, which minimizes the presentation of unproductive or unprotective immunodominant epitopes. Therefore, the combination of presentation of multiple VP B-cell epitopes and multiple T-cell epitopes, accompanied by a reduction in unproductive immunodominant epitopes, produces a highly effective vaccine.

[0111] The advantages of using fully synthetic VP-hC conjugates or VPhC oligomer vaccines are numerous. Modern SPPS routinely produce peptides up to 70–75 residues in length. HhCs described herein range in size from 55–65 residues, with the length of the T cell epitope determining how much longer the core region of the HhC is compared to 28–30 residues. VP may contain additional amino acids to provide spacing or to confer specific chemistry, enabling the total synthetic construction of vaccines. Producing kilogram quantities of vaccine peptides in a cGMP facility eliminates the costly, time-consuming, and resource-intensive industrial production and purification of recombinant proteins, and eliminates the need for subsequent experiments for virus elimination, endotoxin removal, or the presence of infective agents. Peptide synthesis is generally perceived as very costly for large-scale vaccine production. However, when high-nanogram to low-μg doses can be used, peptide vaccines are several times more cost-effective than VP conjugated into recombinant subunit vaccines.

[0112] The terms “residue” and “amino acid” are used interchangeably throughout this disclosure and refer to “amino acid.”

[0113] As those skilled in the art will understand, each embodiment disclosed herein may include, essentially consist of, or be composed of, its particular described elements, processes, components, or constituents. Therefore, the terms “includes” or “contains” should be interpreted as “includes, consists of, or essentially consists of.” The transitional phrases “includes (plural)” or “includes (singular)” mean to include, but not limited to, unspecified elements, processes, components, or constituents, even in major quantities, and enable their inclusion. The transitional phrase “consists of” excludes any elements, processes, components, or constituents not described. The transitional phrase “essentially consists of” limits the scope of the embodiment to the specified elements, processes, components, or constituents, and those that do not substantially affect the embodiment. In some embodiments, those that do not substantially affect the embodiment are, for example, elements, processes, components, or constituents that do not reduce the embodiment’s ability to perform a function in a statistically significant manner in vitro or in vivo, such as providing immunity to a disease or producing an immune response. In some embodiments, the components of the conjugates and oligomers described herein, such as VP, hC, or T cell epitopes, may consist essentially of or be composed of specific sequences. In some embodiments, a vaccine or vaccine composition may consist essentially of a VP-hC conjugate or VPhC oligomer and an excipient, or may consist of a VP-hC conjugate or VPhC oligomer and an excipient.

[0114] Furthermore, unless otherwise noted, numbers representing quantities of components, constituents, reaction conditions, etc., used in the specification and claims should be understood to be modified with the term "approximately." Therefore, unless otherwise indicated, the numerical parameters described in the specification and accompanying claims are approximations that may vary depending on the desired properties to be obtained by the subject matter presented herein. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the claims, each numerical parameter should be interpreted in light of the number of significant figures reported and by applying the usual rounding technique. Although the numerical ranges and parameters describing a broad range of the subject matter presented herein are approximations, the numerical values ​​described in specific examples are reported as accurately as possible. However, any numerical value inherently contains certain errors due to the standard deviation observed in each of its experimental measurements.

[0115] Where further clarity is required, the term “approximately” when used in conjunction with a stated number or range has a meaning reasonably attributable to those skilled in the art, namely, a little more or a little less than the stated value or range, ±20% of the stated value, ±15% of the stated value, ±10% of the stated value, ±5% of the stated value, ±4% of the stated value, ±3% of the stated value, ±2% of the stated value, ±1% of the stated value, or any percentage between 1% and 20% of the stated value.

[0116] In the contexts describing the present invention (particularly in the contexts of the claims below), “a,” “an,” “the,” and similar references should be interpreted as encompassing both singular and plural forms unless otherwise specified herein or unless clearly contradicted by the context.

[0117] The enumeration of value ranges in this specification is intended to serve simply as an abbreviation for referring individually to each separate value that falls within that range. Unless otherwise noted herein, each individual value is incorporated into the specification as if it were individually listed herein. It should be understood that the range formatting is for convenience and conciseness only and should not be interpreted as an inflexible limitation to the scope of this disclosure. Therefore, the range description should be considered to specifically disclose all possible subranges and the individual numerical values ​​within those ranges. For example, a range description such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and the individual numbers within those ranges, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the range width.

[0118] All methods described herein may be carried out in any appropriate order, unless otherwise specified herein or unless it is clearly contrary to the circumstances.

[0119] Any examples or illustrative language (e.g., “etc.”) provided herein are intended solely to facilitate a better understanding of the invention and do not impose any limitation on the scope of the invention as otherwise claimed. Language in this specification should not be construed as indicating any unclaimed elements essential to the practice of the invention.

[0120] The classification of alternative elements or embodiments of the Invention disclosed herein should not be construed as limiting. Each member of the group may be referred to and claimed individually or optionally in combination with other members or elements of the group as found herein. It is anticipated that one or more members of the group may be included in or excluded from the group for convenience and / or patentability reasons. In any case where such inclusion or exclusion is made, the specification shall be deemed to contain the group as modified and therefore shall perform a written description of all Markush groups used in the accompanying claims.

[0121] The following exemplary embodiments and examples are provided herein. These exemplary embodiments and examples are not intended to limit the scope of the disclosure, nor should they be construed as limiting the scope of the disclosure. It will be apparent that the methods can be performed in ways other than those specifically described herein. Numerous modifications and variations are possible in consideration of the teachings herein and are therefore within the scope of the disclosure.

[0122] Exemplary Embodiments 1. A viral peptide (VP) conjugate (VP-hC) or VP oligomer (VPhC) comprising one or more viral peptides (VPs) covalently bound to a hapten carrier (hC), wherein the hC has the following amino acid sequence: (hwxhxyz)n (Sequence ID 2) (In the formula, h is a hydrophobic or nonpolar residue. w is a positively charged, loaded, polar uncharged, or nonpolar aliphatic residue. x is a loaded, positively charged, nonpolar aliphatic, or polar uncharged residue. y is a residue for epitope coupling, z is a loaded, positively charged, polarly uncharged, or nonpolar aliphatic residue. n is an integer greater than 1. It contains an oligomer containing monomeric peptides, A viral peptide (VP) conjugate (VP-hC) or VP oligomer (VPhC) is a viral peptide (VP) conjugate (VP-hC) or VP oligomer (VPhC) in which the VP conjugate contains one or more VPs that are conjugated to hC, and the VP oligomer contains one or more VPs that are incorporated into hC.

[0123] 2. The monomeric peptide has amino acid sequence number 2 (In the formula, h is I, L, V, F, W, Y, M, G, or A. w is G, R, A, N, Q, H, S, D, E, K, or T. x is R, S, N, Q, A, G, T, D, E, K, H, or C. y is a non-natural amino acid or molecule containing K, H, C, D, E, R, W, Y, Q, N, or a reactive group suitable for covalent coupling. z is A, D, H, S, E, R, N, Q, K, or G. n is between 2 and 10. A VP conjugate or VP oligomer of Embodiment 1, including the above.

[0124] 3. A VP conjugate or VP oligomer of Embodiment 1 or 2, wherein the monomeric peptide comprises the amino acid sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0125] 4. A VP conjugate or VP oligomer according to any one of Embodiments 1 to 3, wherein the monomeric peptide comprises the amino acid sequence SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31.

[0126] 5. A VP conjugate or VP oligomer according to any one of Embodiments 1 to 4, wherein the monomeric peptide further comprises V, M, G, I, D, P, C, S, C, or a combination thereof at its N-terminus and / or C-terminus.

[0127] 6. A VP conjugate or VP oligomer according to any one of Embodiments 1 to 5, wherein the oligomer is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, noumer, or decaper.

[0128] 7. A VP conjugate or VP oligomer according to any one of Embodiments 1 to 6, wherein the oligomer is a hexamer.

[0129] 8. A VP conjugate or VP oligomer according to any one of Embodiments 1 to 7, wherein one or more VPs are obtained from coronavirus, influenza virus, respiratory syncytial virus (RSV), human papillomavirus (HPV), dengue virus, yellow fever virus (YFV), or West Nile virus (WNV).

[0130] 9. One or more VPs obtained from the SARS-CoV-2 virus, a VP conjugate or VP oligomer of any one of Embodiments 1 to 8.

[0131] 10. A VP conjugate or VP oligomer of any one of Embodiments 1 to 9, wherein one or more VPs comprise one or more S peptides and / or membrane proteins (M) of the SARS-CoV-2 virus spike glycoprotein.

[0132] 11. One VP conjugate or VP oligomer from any one of Embodiments 1 to 10, wherein one or more VPs include S1, S2, S3, S4, S5, S6, and / or M1.

[0133] 12. A VP conjugate or VP oligomer of any one of Embodiments 1 to 11, wherein one or more VPs include one or more amino acid sequences of SEQ ID NO: 136, SEQ ID NO: 120, SEQ ID NO: 132, SEQ ID NO: 119, SEQ ID NO: 118, SEQ ID NO: 117, and / or SEQ ID NO: 130.

[0134] 13. A VP conjugate or VP oligomer of any one of Embodiments 1 to 11, wherein one or more VPs comprise one or more modified peptides.

[0135] 14. A VP conjugate or VP oligomer of Embodiment 13, wherein the modified peptide comprises one or more cysteine ​​(C) molecules in its sequence, which are replaced by serine (S).

[0136] 15. The VP conjugate or VP oligomer of Embodiment 14, wherein the modified peptide comprises amino acid sequence number 139 or sequence number 140.

[0137] 16. A conjugate of any one of Embodiments 1 to 8, 13, or 14, wherein one or more VPs include one or more amino acid sequences: SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83 (Influenza A virus), SEQ ID NO: 74, or SEQ ID NO: 75 (RSV), SEQ ID NO: 82, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, or SEQ ID NO: 96 (HPV), SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, or SEQ ID NO: 102 (Dengue fever virus), SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, or SEQ ID NO: 91 (YFV), or SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87 (WNV).

[0138] 17. A VP conjugate or VP oligomer of any one of Embodiments 1 to 16, wherein one or more VPs contain further amino acids at their N and / or C-terminus.

[0139] 18. The VP conjugate or VP oligomer of Embodiment 17, wherein the further amino acid is V (valine) and / or DDEDC (SEQ ID NO: 116).

[0140] 19. A VP conjugate or VP oligomer of any one of Embodiments 1 to 18, wherein one or more VPs include a protecting group at their N and / or C-terminus.

[0141] 20. A VP conjugate or VP oligomer of Embodiment 19, wherein the protecting group comprises an acetyl group and / or an amide group.

[0142] 21. Any one of the VP conjugates or VP oligomers of Embodiments 1 to 20, comprising two or more VPs and / or the VPs being obtained from different sources of viruses.

[0143] 22. A VP conjugate or VP oligomer of Embodiment 21, wherein two or more VPs are derived from different strains of SARS-CoV-2.

[0144] 23. A VP conjugate or VP oligomer from any one of Embodiments 1 to 22, wherein one or more VPs are conjugated to hC through a y residue on a monomeric peptide.

[0145] 24. Any one of the VP conjugates or VP oligomers of Embodiments 1 to 23, further comprising one or more immunomodulators or further haptens.

[0146] 25. A VP conjugate or VP oligomer of any one of Embodiments 1 to 24, wherein one or more immunomodulators or further haptens are covalently fused (incorporated) to the N and / or C-terminus of a monomeric peptide, or covalently bonded to one or more N and / or C-terminuses of an oligomer helix.

[0147] 26. Any one of Embodiments 1 to 25, comprising one or more spacers or linkers between a hapten or immunomodulator and a monomeric peptide, VP conjugate or VP oligomer.

[0148] 27. A VP conjugate or VP oligomer of Embodiment 26, wherein one or more spacers or linkers include G (glycine), D (aspartic acid), S (serine), C (cysteine), or a combination thereof.

[0149] 28. One or more spacers comprising D, GD, and / or GSG, a VP conjugate or VP oligomer of Embodiment 26 or 27.

[0150] 29. A VP conjugate or VP oligomer from any one of Embodiments 1 to 28, wherein one or more haptens or immunomodulators comprise one or more further VPs, one or more T cell epitopes, and / or one or more B cell epitopes.

[0151] 30. A VP conjugate or VP oligomer of Embodiment 29, wherein one or more T cell epitopes include a CD4+ T cell epitope.

[0152] 31. A VP conjugate or VP oligomer of Embodiment 29 or 30, wherein one or more T cell epitopes include amino acid sequences SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107 and / or SEQ ID NO: 108.

[0153] 32. Any one of the VP conjugates or VP oligomers of Embodiments 24 to 31, further comprising one or more residues for precise processing of one or more T cell epitopes.

[0154] 33. A VP conjugate or VP oligomer of Embodiment 27, wherein one or more residues include D, G, P, or S, or a combination thereof.

[0155] 34. A VP conjugate or VP oligomer of any one of embodiments 24 to 33, wherein one or more immunomodulators or further haptens enhance the immunogenicity of one or more VPs, or enhance the duration or width of the immune response of one or more VPs.

[0156] 35. Any one of the VP conjugates or VP oligomers of Embodiments 24 to 34, wherein one or more immunomodulators or further haptens comprises a lipid, peptide, nucleic acid, or a combination thereof, and one or more immunomodulators or further haptens are conjugated to hC or covalently bonded to or incorporated at the N and / or C-terminus of one or more N and / or C-terminuses of the oligomer's helix.

[0157] 36. One VP conjugate or VP oligomer of any one of Embodiments 24 to 35, wherein one or more immunomodulators or further haptens comprises monophosphoryl lipid-A, squalene, lipopolysaccharide (LPS), lipoprotein, lipopeptide, or APPHALS (SEQ ID NO: 52).

[0158] 37. A VP conjugate or VP oligomer from any one of Embodiments 1 to 36, comprising an oligomer hC (scaffold) peptide containing amino acid sequence SEQ ID NO: 112 or SEQ ID NO: 113.

[0159] 38. One VP conjugate or VP oligomer from any one of Embodiments 1 to 37, wherein one or more VPs include one or more amino acid sequences SEQ ID NO: 73, SEQ ID NO: 57, SEQ ID NO: 69, SEQ ID NO: 115, SEQ ID NO: 55, SEQ ID NO: 54, and / or SEQ ID NO: 67, and optionally, one or more VPs include SEQ ID NO: 73(S1), SEQ ID NO: 55(S5), and / or SEQ ID NO: 54(S6).

[0160] 39. A composition comprising one VP conjugate or VP oligomer from any one of Embodiments 1 to 38 and an excipient.

[0161] 40. The composition of Embodiment 39, wherein the composition is a pharmaceutical composition, the excipient is a pharmaceutically acceptable excipient, and optionally the pharmaceutical composition contains an adjuvant such as MPL A.

[0162] 41. A method for treating a subject having a viral disease or viral infection and / or preventing the subject from developing a viral disease or viral infection, comprising the step of administering to the subject an effective amount of one VP conjugate or VP oligomer from any one of Embodiments 1 to 38, or a composition of Embodiment 39 or 40, wherein the VP induces an immune response in the subject, thereby treating the subject having a viral disease or viral infection or preventing the subject from developing a viral disease or viral infection.

[0163] 42. The method of Embodiment 41, wherein the subject is a mammal.

[0164] 43. The method of Embodiment 41 or 42, wherein the subject is a human.

[0165] 44. Any one of embodiments 41 to 43, wherein a viral disease or viral infection is caused by SARS-CoV-2.

[0166] 45. A method for enhancing the immunogenicity of VP, A step to obtain one monomeric peptide from any one of Embodiments 1 to 38, The process involves self-assembling the monomer peptide onto hC, A step of conjugating one of the VPs from Embodiments 1 to 38 to the hC to obtain a VP-hC conjugate. A method that includes [this].

[0167] 46. ​​The method of Embodiment 45, wherein the VP-hC conjugate is a VP hexamer (VP-HhC) conjugate.

[0168] 47. A method for enhancing the immunogenicity of VP, A step of synthesizing a VP monomer peptide (VPMP), wherein the VPMP comprises one monomer peptide (MP) from any one of Embodiments 1 to 38 and one VP peptide from any one of Embodiments 1 to 38; and a step of self-assembling the VPMP into a VPhC oligomer. A method that includes [this].

[0169] 48. The method of Embodiment 47, wherein the VP oligomer is a VP hexamer oligomer (VPHhC).

[0170] 49. A method for preparing a VP treatment drug or vaccine, A step to obtain one monomeric peptide from any one of Embodiments 1 to 38, A process of self-assembling monomeric peptides into hC, The process involves conjugating one of the VPs from Embodiments 1 to 38 with hC to obtain a VP-hC conjugate, thereby obtaining a VP therapeutic agent or vaccine. A method that includes this.

[0171] 50. The method of Embodiment 49, wherein the VP-hC conjugate is a VP-HhC conjugate.

[0172] 51. A method for preparing a VP treatment drug or vaccine, A step of synthesizing a VP monomer peptide (VPMP), wherein the VPMP comprises one monomer peptide from any one of Embodiments 1 to 38 and one VP from any one of Embodiments 1 to 38. A process to obtain a VP therapeutic agent or vaccine by self-assembling the VPMP into a VPhC oligomer. A method that includes [this].

[0173] 52. The method of Embodiment 51, wherein the VPhC oligomer is a VP hexamer oligomer (VPHhC).

[0174] 53. A peptide immunogen comprising any one VP from Embodiments 1 to 38.

[0175] 54. The peptide immunogen of Embodiment 53, wherein the peptide immunogen comprises S1, S2, S3, S4, S5, or S6 of the SARS-CoV-2 S glycoprotein, and optionally, the peptide immunogen comprises S1, S5, or S6.

[0176] 55. The peptide immunogen of Embodiment 53 or 54, wherein the VP comprises amino acid sequence number 73, sequence number 57, sequence number 69, sequence number 115, sequence number 55, sequence number 54, or sequence number 67, and optionally the peptide immunogen comprises sequence number 73(S1), sequence number 55(S5), or sequence number 54(S6).

[0177] 56. A peptide immunogen according to any one of embodiments 53 to 55, wherein the N-terminus and C-terminus of the peptide immunogen contain protecting groups, and optionally the N-terminal protecting group is an acetyl group and the C-terminal protecting group is an amide group.

[0178] 57. A peptide scaffold (hC) according to any one of Embodiments 1 to 38.

[0179] 58. A peptide scaffold of Embodiment 57 comprising amino acid sequence number 112 or sequence number 113.

[0180] 59. A peptide scaffold of Embodiment 57 or 58, wherein the N-terminus and C-terminus of hC contain protecting groups, and optionally the N-terminus protecting group is an acetyl group and the C-terminus protecting group is an amide group.

[0181] 60. A composition comprising one or more peptide immunogens from any one of Embodiments 53 to 56 and at least one peptide scaffold from any one of Embodiments 57 to 59.

[0182] 61. The composition of Embodiment 60, wherein one or more peptide immunogens are bound to at least one peptide scaffold of any one of Embodiments 57 to 59.

[0183] 62. The composition of Embodiment 60, wherein one or more peptide immunogens are not bound to the peptide scaffold. [Examples]

[0184] (Example 1) Development of VP vaccine Each hexameric carrier for hapten conjugation has 24 coupling sites, but due to steric hindrance, it is unlikely that conjugation will occur at all sites. Previous studies have shown that saturating the carrier with haptens does not necessarily produce the most robust immune response, and that there is a trade-off between coupling density, epitope spatial / steric availability for accurate B cell epitope presentation, and antibody titer. Therefore, three separate hexameric conjugation reactions are performed to obtain conjugates with different epitope loading levels. For example, one reaction is performed using 3–5 molar equivalents of VP, resulting in the conjugation of only 3–4 peptides; another reaction uses 8–10 molar equivalents to form a conjugate with 6–10 peptides; and a third reaction is performed using 25–50 molar equivalents (saturation conditions) to conjugate as many epitopes as possible.

[0185] VP is designed so that the N-terminal residue is acetylated to protect the N-terminal amine from derivatization by crosslinking agents. The pI of VP can be adjusted by adding a residue (GEDC, SEQ ID NO: 53).

[0186] Tryptophan fluorescence, gel filtration chromatography, undenatured PAGE, and SELDI-TOF (a MALDI-type MS instrument ideally suited for determining the molecular weight of protein-peptide conjugates) are methods used to quantify peptide epitope coupling efficiency. Calculating the number of VP conjugated to hexamer carriers and to BSA is relatively straightforward (VP-BSA is used as a coating reagent in ELISA assays). KLH was used as a positive immunization control because it is an antigenic "gold-standard" hapten carrier. However, because KLH is so large, it may only be possible to confirm successful conjugation without accurately calculating the number of conjugated peptides.

[0187] (Example 2) Characterization of VP-hC structures Adjuvants: Adjuvants were used in all immunizations to enhance adaptive B and T cell responses, modulate the degree of protective immunity, and maximize the VP-specific antibody response. The best adjuvants directly stimulate dendritic cell maturation, and the most effective way to achieve this is through TLR-mediated activation. Synthetic TLR-4 based adjuvants are among the most effective, and therefore, at least two of these were tested. Monophosphoryl lipid A (MPL) is a potent TLR4 agonist that can function as a major adjuvant. MPL was emulsified with squalene (Sq) to form MPL-Sq. The emulsion efficiently primes CD4+ T cells, which are crucial for inducing both memory and long-lived VP antibody responses. Adjuvants E6020 and GLA, which are approved for use in humans, were also tested. All adjuvants can induce VP-hC-specific Th1 CD4+ T cells to bind to T cell epitopes by assisting CD4+-inducible VP-hC uptake into dendritic cells. To evaluate adjuvant function, CD4+ T cells and IgG isotype class switching were quantified in immunized mouse serum. Another important benefit of adjuvants is the potential for antigen dose saving, which is also a matter to be examined. Dose saving can reduce the amount of VP-hC conjugate per immunization and increase the number of doses that can be obtained from a synthetic peptide batch, which is a key determinant in reducing the manufacturing cost of synthetic VP-hC conjugates.

[0188] At least three sets of experiments were conducted for each VP-hC conjugate. Mice were conjugated with prime-boost immunization (IM), and B and T cell function was measured at several post-immunization time points. Three dose levels of the VP-hC conjugate were compared to determine the level at which maximum anti-VP IgG titer was obtained. The hexamer was maximally loaded with VP and formulated with the MPL-Sq adjuvant before immunization. Three dose levels (e.g., VP-hC 0.1 μg, 1 μg, and 10 μg) were tested and optimized according to anti-VP IgG titer. This experiment also tested anti-VP IgG specificity by measuring the IgG response to hC alone, VP alone, and VP+hC (not conjugated, but combined).

[0189] Mouse immunization with VP-hC: Inbred mice were immunized with either adjuvant-treated VP-hC or a control (VP-KLH conjugate) as prime / boost immunization. The first set of mice in the study provided the optimal VP-hC dose, and anti-VP IgG titers were measured at each dose level. Serum was collected 14 days after both prime and boost (d35) immunization, and antibody midpoint titers were measured. Mouse blood was used to perform B and T cell assays.

[0190] B-cell function: Vaccine efficacy was measured by VP-specific antibody titers in collected mouse serum using a standard ELISA. ELISA plates were coated with VP-BSA conjugate, and eight sequential 10-fold dilutions (1:10) of serum in blocking buffer were used. 3 ~1:10 10 A solution was prepared and added to the ELISA plate wells. HRP-labeled anti-mouse secondary antibody was added, the plate was developed with a colorimetric substrate, and measured using an ELISA plate reader. The data were plotted, curves were fitted, and statistical analysis was performed using Prism Graph Pad software to calculate the midpoint and endpoint titers.

[0191] T cell function: T cell epitope and adjuvant function were measured by well-established T cell ELISA assays. Commercially available coating reagents and primary / secondary antibodies were purchased and used according to the manufacturer's protocol. IFN-γ, IL-2, IL-4, and TNF-α were quantified in mouse serum as readouts of T cell function in VP-hC immunized mice. These targets were readily extended to include other markers of T cell function, including IL-5, IL-8, IL-10, IL-12p70, and IL-13. VP-hC-induced T cell-dependent isotype class switching was assayed by ELISA using reagents specific to IgG, IgG1, and IgG2a as a whole.

[0192] VP-hC Safety: Initial safety assessments were conducted in a non-GLP setting to ensure that mice did not exhibit adverse reactions to the vaccine components (VP-hC, adjuvant). This initial assessment provided several key readouts to guide vaccine dose, adjuvant dose, and immunization schedule. Potential local and systemic toxicity was assessed by observing injection site reactions and signs of inflammation, as well as mouse behavior (e.g., signs of coma). If toxicity was observed, different adjuvants and / or T cell epitopes were evaluated.

[0193] (Example 3) Vaccine development for SARS-CoV-2 Antigen Peptide Selection: Antigen peptides for testing as vaccine candidates were selected based on a combination of several criteria. The first was based on the presence of S protein peptides in the serum of convalescent SARS-CoV-2 patients who had been infected with the virus but had a protective immune response. The second was based on in silico analysis of the three-dimensional structure of the SARS-CoV-2 S protein, focusing on analyzing the S1 region of the holoprotein. The receptor-binding domain of the S protein was given special consideration because it is particularly important for binding to human cells by the ACE2 receptor and entry into host cells. Other functional regions tested included two proteolytic cleavage sites important for converting the S protein into fusion-competent isoforms, and one non-S protein peptide representing the SARS-CoV-2 membrane protein (M1). Figure 1 shows the selected peptides within the S1 region of the S protein. For simplification, only one of the three subunits is shown.

[0194] Table 3 shows peptide immunogens derived from the wild-type S peptide of SARS-CoV-2. Residues and protecting groups were added to each S peptide to protect them from proteolysis and increase their serum half-lives in vivo. Furthermore, the carbon (C) in the S4 and S6 peptides was replaced with sulfur (S) to avoid interference with HhC coupling.

[0195] [Table 3]

[0196] The peptide immunogens in Table 3 are conjugated into hC (scaffolds) for immunizing mice. Table 4 shows mouse and human hCs. Each hC contains a linker / spacer and one or more residues such as V and D. Each hC also contains a T cell epitope and protecting groups at the N-terminus and C-terminus.

[0197] [Table 4]

[0198] Solid-phase peptide synthesis: The peptides in Table 3 and Table 4 were synthesized by solid-phase peptide synthesis. The peptides were synthesized with an N-terminal acetyl protecting group and a C-terminal amide group to increase their stability in serum (in vivo). The purity and identity of the synthesized peptides were confirmed by HPLC-UV and MALDI, respectively. The peptides were delivered as lyophilized powder and stored at -20°C until use. Before use (in conjugation or immunoassay experiments), the peptides were dissolved in water and stored at -20°C.

[0199] Covalent coupling of antigen peptide to scaffold peptide: The cysteinyl sulfhydryl group of the antigen peptide was covalently coupled to two lysine residues in the scaffold peptide using a heterobifunctional crosslinking agent. Each coupling reaction was performed separately. The scaffold peptide was incubated with 25 molar equivalents of sulfo-GMBS (N-γ-maleimidobutyryl-oxysulfosuccinimide ester) in 50 mM potassium phosphate buffer, pH 6.0, at room temperature, in the dark, for 2 hours. The activated peptide was separated from the excess (unreacted) sulfo-GMBS by gel filtration through Sephadex G-10 resin equilibrated in 50 mM potassium phosphate buffer, pH 6.6. The antigen peptide containing the C-terminal cysteine ​​was incubated with 5 mM DTT in 50 mM potassium phosphate buffer, pH 6.6, at room temperature, for 30 minutes. DTT was separated from the peptide using gel filtration through Sephadex G-10 resin equilibrated in 50 mM potassium phosphate, pH 6.6. 2.5 molar equivalents of the reduced peptide were incubated in a sulfo-GMBS activated scaffold for 16 hours at room temperature in the dark. Using these conditions, LC / MS / MS and SDS-PAGE experiments showed that the coupling of the peptide to the scaffold was almost quantitative (i.e., the unconjugated scaffold was undetectable by LC / MS / MS; Figures 2A and 2B).

[0200] Mouse immunization: 60 male BALB / cJ mice (5 per group) were immunized (intramuscularly) with 10 μg of adjuvant scaffold conjugate peptides (HS1 (S1 peptide conjugated to hexamer (H)hC), HS2, HS3, HS4, HS5, HS6, and HM1) or two separate pooled mixtures. The first pooled mixture contained the scaffold, S1 peptide, S2 peptide, S4 peptide, S6 peptide, and M1 peptide, which were mixed in equal amounts of μg prior to immunization (group 9). The second pool consisted of a mixture of equal amounts of μg of HS1, HS2, HS4, HS6, and HM1 prior to immunization. The immunization schedule was prime-boost-boost. Mice were immunized on d0 and boosted on d14 and d28. Blood samples were collected on days 14, 28, 42, 56, and 84 for antibody titer determination and SARS-CoV-2 virus neutralization assay. Table 5 summarizes the immunization schedule.

[0201] [Table 5]

[0202] Antibody titer determination: NUNC Maxisorp plates were coated with a capture reagent containing the peptides listed in Table 3, covalently coupled to bovine serum albumin. 96-well plates were coated with 200 ng of BSA-conjugated peptide and incubated overnight at 4°C in 50 mM potassium phosphate buffer (pH 7.4). The plates were washed twice with washing buffer (1× Tris-buffered saline, 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 0.01% Tween20). The plates were blocked for 1 hour in blocking buffer (3% BSA + Tris-buffered saline) and then washed as described above.

[0203] Initial dilutions of mouse serum were prepared (1:100 for the d14 bred, 1:500 for all other breds), and seven sequential 5-fold dilutions were prepared in 0.1 ml of blocking buffer. The diluted serum was transferred to a blocked ELISA plate and incubated for 1 hour with gentle shaking. The plate was washed four times using an automated plate washer with tris-buffered saline + 0.01% Tween 20. Goat anti-mouse IgG-HRP was diluted 1:8000 in blocking buffer and then added to the washed ELISA plate (0.1 ml per well). After incubation for 1 hour with gentle shaking, the plate was washed as described above, and then TMB substrate solution was added. The plate was allowed to develop color at room temperature for 30 minutes, and then the reaction was inhibited by adding 0.05 ml of 2 M sulfuric acid. Absorbance at 450 nm was measured in all wells of the plate. The endpoint force value was calculated as the dilution at which the ELISA signal in the diluted serum was greater than the cutoff value. The cutoff value was calculated by diluting untreated mouse serum in the same manner as when diluting immunized serum, and then running these controls together with the immunized serum. (Untreated serum + (2)) * The average absorbance (SD) was defined as the cutoff value.

[0204] The results, shown in Figures 3 to 5, demonstrate that the peptide immunogen conjugated to hC (scaffold) enhanced IgG titer compared to the control.

[0205] SARS-CoV-2 virus neutralization assay: Dilute mouse serum immunized with HS1, HS5, HS6, or pooled (HS1, HS2, HS4, HS6, and HM1) in MEM medium at a ratio of 1:400 and allow to stand at room temperature for 30 minutes. 4 The SARS-CoV-2 virus particles were incubated with 10 samples. Then, the virus + serum mixture (pre-seeded in 96-well plates and grown overnight at 37°C) was incubated with 10 samples. 4The drug was added to individual Vero-E6 human kidney cells and incubated at 37°C for 48 hours. The cells were fixed, permeabilized, and incubated with fluorescent IgG specific to SARS-CoV-2 protein. Viral infection was measured by reading the fluorescence on a plate reader. The data were compared 10 times without any prior incubation with mouse serum. 4 10 virus particles incubated with 4 The viral inhibition rate was expressed as a percentage (%) compared to individual Vero-E6 cells. Figure 7 shows the inhibition of entry of live SARS-CoV-2 virus into human cells by VP-HhC at a serum dilution of 1:400. The VP in VP-HhC is S1, S5, or S6. Group 9 is a mixture of S1 peptide, S2 peptide, S4 peptide, and S6 peptide, as well as hC (mouse scaffold) without conjugation.

[0206] SARS-CoV-2 Plaque Assay: To measure the ability of adjuvant SARS-CoV-2 peptide antigen to inhibit viral entry, self-replication, and plaque formation (showing virus-induced cell lysis) in human cells, serum from immunized mice in groups 1, 5, and 6 (HS1, HS5, and HS6, Table 4) was tested. 4 The mixture was mixed with 100% live SARS-CoV-2 virus particles, incubated for 30 minutes, and then added to human Vero-E6 kidney cells grown in vitro (3 times). After 72 hours of incubation at 37°C, the number of plaques on each plate was quantified. Table 6 shows the number of plaques formed after incubation of mouse-derived serum immunized with adjuvant HS1, HS5, or HS6 vaccine and then added to human Vero-E6 kidney cells. The data shown in Table 6 were obtained from three replicates.

[0207] [Table 6]

[0208] In summary, antibodies produced in vaccinated mouse studies were present in the serum, and these antibodies prevented live SARS-CoV-2 virus from entering human cells (detectable using fluorescence analysis) and prevented live virus from forming plaques within human cells (biological readout). The results demonstrate that scaffold peptides (hC), including peptide immunogens and human scaffold peptides (Table 1), as described herein, are useful for treating and preventing virus-induced diseases in human and other subjects.

[0209] Certain embodiments of the Invention, including the best mode known to the inventors for carrying out the Invention, are described herein. Naturally, variations of these described embodiments will be apparent to those skilled in the art in interpretation of the foregoing description. The inventors anticipate that those skilled in the art will use such variations as appropriate, and the inventors intend that the Invention will be carried out in ways other than those specifically described herein. Accordingly, this disclosure encompasses all modifications and equivalents of the subject matter enumerated in the accompanying claims, to the extent permitted by applicable law. Furthermore, any combination of the elements described above in all possible variations is encompassed by the Invention unless otherwise specified herein or unless it is clearly contrary to the circumstances.

[0210] All publications, patents, and patent applications cited herein are incorporated herein by reference in whole, as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. While the foregoing has been described in relation to various embodiments, it will be understood by those skilled in the art that various modifications, substitutions, omissions, and changes can be made without departing from the spirit of the invention.

[0211] References [Table 7-1] [Table 7-2] Table 7-3 Table 7-4

Claims

[Claim 1] A viral peptide (VP) conjugate (VP-hC) or VP oligomer (VPhC) comprising one or more viral peptides (VPs) covalently bound to a hapten carrier (hC), wherein the hC has the following amino acid sequence: (hwxhxyz)n (Sequence ID 2) (In the formula, h is a hydrophobic or nonpolar residue. w is a positively charged, loaded, polar uncharged, or nonpolar aliphatic residue. x is a loaded, positively charged, nonpolar aliphatic, or polar uncharged residue. y is a residue for epitope coupling, z is a loaded, positively charged, polarly uncharged, or nonpolar aliphatic residue. n is an integer greater than 1. It contains monomeric peptides, A viral peptide (VP) conjugate (VP-hC) or VP oligomer (VPhC) is a viral peptide (VP) conjugate (VP-hC) or VP oligomer (VPhC) in which the VP conjugate contains one or more VPs that are conjugated to hC, and the VP oligomer contains one or more VPs that are incorporated into hC.

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