Compounds and their use as vaccine adjuvants

Abstract

This article provides a series of compounds and their uses as adjuvants. These compounds, compositions comprising the compounds, and their uses are provided. These compounds can be used as adjuvants for vaccines, significantly enhancing cellular and humoral immune responses to vaccines compared to conventional aluminum adjuvants. As adjuvants, these compounds can increase broad-spectrum protection against various coronaviruses such as SARS, influenza, and HIV, and significantly enhance the durability of vaccine-mediated immune protection.

Description

[0001] priority

[0002] This application claims priority to PCT application No. PCT / CN2021 / 095498, filed on May 24, 2021, entitled “Compounds and their use as vaccine adjuvants,” which is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates to the biomedical field, specifically to a series of small molecule STING agonists. Background Technology

[0004] Vaccines are considered the most powerful weapon against the spread of infectious diseases. Following the outbreak of Coronavirus Disease 2019 (COVID-19), multiple vaccines targeting the SARS-CoV-2 coronavirus rapidly entered clinical trials, including mRNA vaccines, DNA vaccines, inactivated vaccines, and viral vector vaccines. The main components of these vaccines are the coronavirus's spike protein or receptor-binding domain (RBD). Currently, many COVID-19 vaccines have demonstrated protective effects against SARS-CoV-2 infection in human ACE2 transgenic mice and non-human primate models. Although more than ten vaccines have been approved globally, several problems remain to be solved. For example, as the virus spreads in the human population, coronaviruses continuously mutate. These new mutated viral strains pose a significant challenge to currently available vaccines. Current research has shown that SARS-CoV-2 mutant strains in the UK and South Africa have exhibited immune evasion against some existing vaccines on the market (Wang et al., Mrna vaccine-elicited antibodies to SARS-CoV-2 and circulating variants, medRxiv, 2021). Furthermore, over the past 20 years, three highly pathogenic coronaviruses, including SARS-CoV, SARS-CoV-2, and MERS-CoV, have emerged and caused outbreaks. Several SARS-like coronaviruses from bats, such as RS3367 and WIV1 strains, have been discovered, suggesting that SARS-related coronaviruses may still suddenly emerge and spread in the human population, similar to SARS-CoV-2. Currently, only a few studies have shown that vaccines based on SARS-CoV-2 antigens can produce weak cross-neutralizing antibody protection against SARS-CoV and SARS-related coronavirus infections (Liu Z et al., RBD-Fc-based COVID-19 vaccine candidate induces highly potent SARS-CoV-2 neutralizing antibody response, Signal TransductTarget). (Ther, 2020). Whether serum from patients who have recovered from SARS-CoV infection can neutralize SARS-CoV-2 is also controversial.Studies have shown that serum from patients infected with SARS-CoV can cross-neutralize SARS-CoV-2, but the neutralizing activity is weak (Zhu, Y. et al. Cross-reactive neutralization of SARS-CoV-2 by serum antibodies from recovered SARS patients and immunized animals. Sci Adv, 2020). Another study showed that serum from patients who have recovered from SARS-CoV infection cannot effectively neutralize the SARS-CoV-2 virus (Wang, Y. et al. Kinetics of viral load and antibody response in relation to COVID-19 severity. J Clin Invest, 2020). These results indicate that it is difficult to generate a broad-spectrum, durable, and strong protective immune response against SARS-related coronaviruses in vivo through common vaccination strategies or natural infection routes. Therefore, developing a broad-spectrum, long-acting, and potent universal vaccine against SARS-related viruses is crucial for combating current SARS-CoV-2 (including SARS-CoV-2 mutants) and potential future SARS-related virus epidemics.

[0005] Adjuvants are crucial for enhancing the immunoprotective effect of protein subunit vaccines or inactivated vaccines. Currently, aluminum adjuvants are the most commonly used. Recently, aluminum adjuvants have been used in various protein subunit vaccines in clinical trials. While aluminum adjuvants are safe, they primarily enhance humoral immune responses, while cellular immune responses also play a vital role in combating viral infection. Therefore, developing novel adjuvants, especially small-molecule immunostimulants, to enhance the immunoprotective effect of subunit or inactivated vaccines against SARS-CoV-2, induce more diverse immune responses, and prolong the duration of immunity is currently a cutting-edge research area. Summary of the Invention

[0006] In recent years, STING agonists have been found to have the potential for use as vaccine adjuvants. The inventors previously used nano-encapsulated STING agonist cGAMP lung biomimetic particles as an adjuvant for influenza vaccines. Intranasal administration of the vaccine produced strong and broad-spectrum immune protection against influenza virus in mice (Wang, J. et al. Pulmonary surfactant-biomimetic nanoparticles potentiate heterosubtypic influenza immunity. Science, 2020). However, intramuscular injection of cGAMP is less effective.

[0007] In this disclosure, a novel STING small molecule agonist is used as an adjuvant for a SARS-CoV-2 protein subunit RBD-Fc vaccine. In animal models using mice, rabbits, and rhesus monkeys, the STING agonist adjuvant RBD-Fc strongly activated cellular and humoral immune responses, generating broad-spectrum, potent, and durable immune protection against SARS-CoV-2, its mutants, SARS-CoV, and various SARS-related viruses. Compared to conventional aluminum adjuvant vaccines against SARS-CoV-2, the novel STING small molecule agonist significantly enhances cellular and humoral immune responses to the vaccine, strengthens broad-spectrum protection against SARS and other coronaviruses, and significantly improves the durability of the vaccine's immune protection against SARS-CoV-2.

[0008] In one respect, this application provides a compound having formula (I) or a pharmaceutically acceptable salt thereof:

[0009]

[0010] Where R1 is CR1', and R1' is H, -OMe, or -O(CH2). n NR2'R3', where n is an integer from 1 to 6, preferably 2 or 3, R2' and R3' together with the nitrogen atom to which they are attached form a substituted or unsubstituted 5-6 membered heterocyclic alkyl group or a substituted or unsubstituted 5-6 membered heteroaryl group; wherein the substituted 5-6 membered heterocyclic alkyl group or the substituted 5-6 membered heteroaryl group is independently substituted with one or more halogens, OH, amines, CN, CF3 or unsubstituted C1-C4 saturated alkyl groups; preferably, the heterocyclic alkyl group is one of the following:

[0011]

[0012] R2 and R3 are each independently N or NR4', where R4' is H or C. 1-4 Saturated alkyl groups; and

[0013] R4 and R5 are each independently N or NH.

[0014] In one embodiment, the compound has the following structure:

[0015]

[0016]

[0017] In one aspect, this application provides a pharmaceutical composition comprising a compound of this application or a pharmaceutically acceptable salt thereof; and at least one of a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, and a pharmaceutically acceptable diluent.

[0018] In one aspect, this application provides the use of the compound of this application or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of this application, in the preparation of an adjuvant. In one embodiment, the adjuvant is an adjuvant for use in a vaccine.

[0019] In one embodiment, the vaccine comprises an antigen. In one embodiment, the antigen is selected from the group consisting of cancer antigens, viral antigens, bacterial antigens, parasitic antigens, and fungal antigens. In one embodiment, the vaccine is effective in preventing infection with any of the strains described in the embodiment.

[0020] In one embodiment, the viral antigen is selected from the group consisting of HIV antigen, influenza antigen, and coronavirus antigen. In one embodiment, the viral antigen is derived from one or more of the following: HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV, varicella-zoster virus (VZV), and SARS-CoV-2 such as the SARS-CoV-2 Omeprone mutant. In one embodiment, the viral antigen is the SARS-CoV-2 RBD-Fc protein or the gE protein of varicella-zoster virus.

[0021] In one aspect, this application provides a vaccine comprising the compound of this application or a pharmaceutically acceptable salt thereof; and an antigen. In one embodiment, the vaccine is an intramuscular, intradermal, or inhaled vaccine. In one embodiment, the vaccine comprises an antigen. In one embodiment, the antigen is selected from the group consisting of cancer antigens, viral antigens, bacterial antigens, parasitic antigens, and fungal antigens. In one embodiment, the viral antigen is selected from the group consisting of HIV antigens, influenza antigens, and coronavirus antigens. In one embodiment, the antigen is derived from one or more of the following: HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV, varicella-zoster virus (VZV), and SARS-CoV-2 such as the SARS-CoV-2 Omeprón mutant. In one embodiment, the viral antigen is the SARS-CoV-2 RBD-Fc protein or the gE protein of varicella-zoster virus.

[0022] In one aspect, this application provides a method for producing the vaccine of this application, which includes mixing the compound of this application with the antigen.

[0023] In one aspect, this application provides the compound of this application or a pharmaceutically acceptable salt thereof as an adjuvant. In one embodiment, the adjuvant is an adjuvant for a vaccine. In one embodiment, the vaccine comprises an antigen. In one embodiment, the antigen is selected from the group consisting of cancer antigens, viral antigens, bacterial antigens, parasitic antigens, and fungal antigens. In one embodiment, the viral antigen is selected from the group consisting of HIV antigens, influenza antigens, and coronavirus antigens. In one embodiment, the antigen is derived from one or more of the following: HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV, varicella-zoster virus (VZV), and SARS-CoV-2 such as the SARS-CoV-2 Omeprón mutant. In one embodiment, the viral antigen is the SARS-CoV-2 RBD-Fc protein or the gE protein of varicella-zoster virus.

[0024] In one aspect, this application provides a method for treating or preventing infectious diseases or cancer, comprising administering an effective amount of the vaccine of this application to a subject in need of such treatment. In one embodiment, the vaccine is an intramuscular, intradermal, or inhaled vaccine. In one implementation scheme, infectious diseases are selected from the group consisting of: AIDS, Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), COVID-19, varicella-zoster virus, and influenza, and cancers are selected from the group consisting of: HPV-related cancers, HBV-related cancers, ovarian cancer, prostate cancer, breast cancer, brain cancer, head and neck cancer, laryngeal cancer, lung cancer, liver cancer, pancreatic cancer, kidney cancer, bone cancer, melanoma, metastatic cancer, HTERT-related cancers, FAP antigen-related cancers, non-small cell lung cancer, hematologic malignancies, esophageal squamous cell carcinoma, cervical cancer, bladder cancer, colorectal cancer, gastric cancer, anal cancer, synovial sarcoma, testicular cancer, recurrent respiratory papilloma, skin cancer, glioblastoma, liver cancer, gastric cancer, acute myeloid leukemia, triple-negative breast cancer, and primary cutaneous T-cell lymphoma.

[0025] In one aspect, this application provides a kit containing the compounds of this application, antigens, and instructions for treating or preventing infectious diseases or cancer.

[0026] The benefits of this disclosure include:

[0027] (1) This application provides a new series of compounds that can enhance the immune response to antigens.

[0028] (2) The compounds in this application, as adjuvants to the protein subunit RBD vaccine against SARS-CoV-2, can strongly activate cellular and humoral immune responses in animal models including mice, rabbits and rhesus monkeys, and generate broad-spectrum, potent and durable immune protection against SARS-CoV-2, SARS-Co-2 mutants, SARS-CoV and various SARS-related viruses.

[0029] (3) Compared with traditional aluminum adjuvants used in COVID-19 vaccines, the compounds of this application can significantly enhance cellular and humoral immune responses to vaccines, enhance broad-spectrum protection against coronaviruses such as SARS, and significantly improve the durability of immune protection against COVID-19 vaccines.

[0030] (4) The compounds of this application can effectively enhance the immune response to HIV peptide antigens.

[0031] (5) The compounds of this application can enhance the immune response to influenza virus vaccines. Attached Figure Description

[0032] Figure 1The results show the activation levels of various cytokines in mouse lymph nodes at 6 hours after mice were immunized with the STING agonist CF501 or cGAMP.

[0033] Figure 2 The results show the activation of mouse cytokine IFnB at 6, 24 and 48 hours after mice were immunized with the STING agonist CF501 and SARS-CoV-2RBD-Fc protein.

[0034] Figure 3 The results show the activation of mouse cytokine CXCL-10 at 6, 24 and 48 hours after mice were immunized with the STING agonist CF501 and SARS-CoV-2RBD-Fc protein.

[0035] Figure 4 The results show the activation of mouse cytokine CCL-2 at 6, 24 and 48 hours after mice were immunized with the STING agonist CF501 and SARS-CoV-2RBD-Fc protein.

[0036] Figure 5 The results show the activation of mouse cytokine CXCL-9 at 6, 24 and 48 hours after mice were immunized with the STING agonist CF501 and SARS-CoV-2RBD-Fc protein.

[0037] Figure 6 The results show the activation of mouse cytokine IL-1b at 6, 24 and 48 hours after mice were immunized with the STING agonist CF501 and SARS-CoV-2RBD-Fc protein.

[0038] Figure 7 The results show the activation of mouse cytokine IL-6 at 6, 24 and 48 hours after mice were immunized with the STING agonist CF501 and SARS-CoV-2RBD-Fc protein.

[0039] Figure 8 The results show the activation of mouse cytokine TNF-α at 6, 24 and 48 hours after mice were immunized with the STING agonist CF501 and SARS-CoV-2RBD-Fc protein.

[0040] Figure 9 The vaccination protocol for mice is shown.

[0041] Figure 10 The results show the SARS-CoV-2 RBD-specific IgG antibody titers in mice on day 21 post-vaccination in each group.

[0042] Figure 11 The results show the SARS-CoV-2RBD-specific IgG1 antibody titers in mice on day 21 post-vaccination in each group.

[0043] Figure 12 The results show the SARS-CoV-2RBD-specific IgG2a antibody titers in mice on day 21 post-vaccination in each group.

[0044] Figure 13 The results show the SARS-CoV-2 RBD-specific IgG antibody titers in mice on day 35 post-vaccination in each group.

[0045] Figure 14 The results show the SARS-CoV-2RBD-specific IgG1 antibody titers in mice on day 35 post-vaccination in each group.

[0046] Figure 15 The results show the SARS-CoV-2RBD-specific IgG2a antibody titers in mice on day 35 post-vaccination in each group.

[0047] Figure 16 The results show the results of using the ELISPOT assay to detect IFN-γ secretion from the spleen of mice in each group on day 35 post-vaccination.

[0048] Figure 17 The results show the results of using the ELISPOT assay to detect IFN-γ secretion from the lungs of mice in each group on day 35 post-vaccination.

[0049] Figure 18 The results show the results of using the ELISPOT assay to detect TNF-α secretion from the spleen of mice in each group on day 35 post-vaccination.

[0050] Figure 19 The results show the results of using the ELISPOT assay to detect TNF-α secretion from the lungs of mice in each group on day 35 post-vaccination.

[0051] Figure 20 The results show the results of using the ELISPOT assay to detect IL-4 secreted from the spleen of mice in each group on day 35 post-vaccination.

[0052] Figure 21 The results show the results of using the ELISPOT assay to detect IL-4 secretion from the lungs of mice in each group on day 35 post-vaccination.

[0053] Figure 22The results show the neutralizing titers against SARS-CoV-2 pseudovirus in each group of mice on day 21.

[0054] Figure 23 The results show the neutralizing titers against SARS-CoV-2 pseudovirus in each group of mice on day 35.

[0055] Figure 24 The results show the correlation between RBD-specific antibodies and neutralizing antibodies in the serum of each group of mice on day 21.

[0056] Figure 25 The results show the correlation between RBD-specific antibodies and neutralizing antibodies in the serum of each group of mice on day 35.

[0057] Figure 26 The results show the results of using the plaque reduction assay to detect the neutralizing activity against live SARS-CoV-2 virus in each group of mice on day 21.

[0058] Figure 27 The results show the results of using the plaque reduction assay to detect the neutralizing activity against live SARS-CoV-2 virus in each group of mice on day 35.

[0059] Figure 28 The results show the results of using immunofluorescence assay to detect the neutralizing activity against live SARS-CoV-2 virus in each group of mice on day 21.

[0060] Figure 29 The results show the results of using immunofluorescence assay to detect the neutralizing activity against live SARS-CoV-2 virus in each group of mice on day 35.

[0061] Figure 30 Serum from mice vaccinated with CF501 and RBD-Fc showed that SARS-CoV-2-mediated membrane fusion could be inhibited.

[0062] Figure 31 The results show the serum SARS-CoV RBD-specific antibody titers in each group of mice on day 35.

[0063] Figure 32 The results show the neutralizing activity against SARS-CoV pseudovirus in each group of mice on day 35.

[0064] Figure 33 The correlation between neutralizing antibodies against SARS-CoV pseudovirus and SARS-CoV RBD-specific antibodies was shown in each group of mice on day 35.

[0065] Figure 34The results show the neutralizing activity against WIV1 pseudovirus in each group of mice on day 35.

[0066] Figure 35 The results show the neutralizing activity against the Rs3367 pseudovirus in each group of mice on day 35.

[0067] Figure 36 The results show the changes in body weight of mice after attacking the vaccine.

[0068] Figure 37 The results show the viral load in the lungs of mice on day 4 after attacking the vaccinated mice.

[0069] Figure 38 The results show the viral load in the brains of mice on day 4 after they were attacked with the vaccine.

[0070] Figure 39 The results show the viral load in the intestines of mice on day 4 after attacking the vaccinated mice.

[0071] Figure 40 The vaccination schedule for New Zealand white rabbits is shown.

[0072] Figure 41 The results show the SARS-CoV-2RBD-specific IgG antibody titers in New Zealand white rabbits on day 21 after vaccination in each group.

[0073] Figure 42 The results show the SARS-CoV-2RBD-specific IgG antibody titers in New Zealand white rabbits on day 35 after vaccination in each group.

[0074] Figure 43 The results show the neutralizing activity of serum against SARS-CoV-2 pseudovirus in New Zealand white rabbits on day 21 after vaccination in each group.

[0075] Figure 44 The results show the neutralizing activity of serum against SARS-CoV-2 pseudovirus in New Zealand white rabbits on day 35 after vaccination in each group.

[0076] Figure 45 The results show the correlation between neutralizing antibodies and SARS-CoV-2 RBD-specific antibodies in the serum of New Zealand white rabbits on day 21 after vaccination in each group.

[0077] Figure 46 The results show the correlation between neutralizing antibodies and SARS-CoV-2 RBD-specific antibodies in the serum of New Zealand white rabbits on day 35 after vaccination in each group.

[0078] Figure 47The results show the results of using a plaque reduction method to detect the neutralizing activity of serum against live SARS-CoV-2 virus on day 21 post-vaccination in New Zealand white rabbits in each group.

[0079] Figure 48 The results show the results of using a plaque reduction method to detect the neutralizing activity of serum against live SARS-CoV-2 virus in New Zealand white rabbits on day 35 after vaccination in each group.

[0080] Figure 49 The results show the results of using immunofluorescence assay to detect the neutralizing activity of serum against live SARS-CoV-2 virus in New Zealand white rabbits on day 21 after vaccination in each group.

[0081] Figure 50 The results show the results of using immunofluorescence assay to detect the neutralizing activity of serum against live SARS-CoV-2 virus in New Zealand white rabbits on day 35 after vaccination in each group.

[0082] Figure 51 Serum from New Zealand white rabbits vaccinated with CF501 and RBD-Fc vaccines showed that SARS-CoV-2-mediated membrane fusion could be inhibited.

[0083] Figure 52 The results show the neutralizing activity of serum against SARS-CoV-2 pseudovirus in New Zealand white rabbits on day 49 after vaccination in each group.

[0084] Figure 53 The results show the results of using a plaque reduction method to detect the neutralizing activity of serum against live SARS-CoV-2 virus in New Zealand white rabbits on day 49 after vaccination in each group.

[0085] Figure 54 The results show the neutralizing activity of serum against SARS-CoV pseudovirus in New Zealand white rabbits on day 35 after vaccination in each group.

[0086] Figure 55 The results show the neutralizing activity of serum against WIV1 pseudovirus in New Zealand white rabbits on day 35 after vaccination in each group.

[0087] Figure 56 The results show the SARS-CoV RBD-specific IgG antibody titers in New Zealand white rabbits on day 49 after vaccination in each group.

[0088] Figure 57 The results show the neutralizing activity against SARS-CoV pseudovirus in New Zealand white rabbits on day 49 after vaccination in each group.

[0089] Figure 58The results show the correlation between SARS-CoV RBD-specific antibodies and neutralizing antibodies against SARS-CoV pseudovirus in the serum of New Zealand white rabbits on day 49 after vaccination in each group.

[0090] Figure 59 The results show the neutralizing activity against WIV1 pseudovirus in New Zealand white rabbits on day 49 after vaccination in each group.

[0091] Figure 60 The results show the neutralizing activity against the Rs3367 pseudovirus in New Zealand white rabbits on day 49 after vaccination in each group.

[0092] Figure 61 The results show the neutralizing activity against pseudoviruses of various SARS-CoV-2 mutants in New Zealand white rabbits on day 49 after vaccination in each group.

[0093] Figure 62 The vaccination protocol for rhesus monkeys is shown.

[0094] Figure 63 The results show the results of IgG antibodies bound to SARS-CoV-2RBD in the serum of rhesus monkeys in each group on day 14 after vaccination.

[0095] Figure 64 The results show the serum IgG antibody titers against SARS-CoV-2RBD in rhesus monkeys in each group on day 14 post-vaccination.

[0096] Figure 65 The results show the IgG antibodies bound to SARS-CoV-2RBD in the serum of rhesus monkeys in each group on day 28 after vaccination.

[0097] Figure 66 The results show the serum IgG antibody titers against SARS-CoV-2RBD in rhesus monkeys in each group on day 28 post-vaccination.

[0098] Figure 67 The results show the IFN-γ secretion levels of PBMCs (peripheral blood mononuclear cells) on day 14 after vaccination in each group of rhesus monkeys.

[0099] Figure 68 The results show the IFN-γ secretion levels of PBMCs (peripheral blood mononuclear cells) on day 28 after vaccination in each group of rhesus monkeys.

[0100] Figure 69 The results show the inhibition of SARS-CoV-2 pseudovirus infection by serum in rhesus monkeys on day 14 after vaccination in each group.

[0101] Figure 70 The results show the inhibition of SARS-CoV-2 pseudovirus infection by serum in rhesus monkeys on day 28 after vaccination in each group.

[0102] Figure 71 The results show the correlation between the serum SARS-CoV-2RBD-specific antibody titer and the neutralizing antibody titer on day 28 post-vaccination in rhesus monkeys in each group.

[0103] Figure 72 The results show the inhibition of SARS-CoV-2 live virus infection in rhesus monkey serum on day 28 post-vaccination in each group using a plaque reduction method.

[0104] Figure 73 The results show the results of using the plaque reduction method to detect the titers of SARS-CoV-2 live virus neutralizing antibodies in serum at different time points from 14 to 191 days post-vaccination in rhesus monkeys.

[0105] Figure 74 The neutralizing activity of serum from each group of rhesus monkeys against pseudoviruses of SARS-CoV-2 variants and single-point mutants was shown.

[0106] Figure 75 The IgG endpoint titer of RBD-specific antibodies against the Omeprone strain was shown in the serum of rhesus monkeys in each group from day 28 to day 191 post-vaccination.

[0107] Figure 76 The results show the serum neutralizing antibody titers against Omeprone pseudovirus in each group of rhesus monkeys from day 28 to day 191 post-vaccination.

[0108] Figure 77 Serum from rhesus monkeys on day 122 post-vaccination showed neutralizing activity against live Omeprone virus.

[0109] Figure 78 The results show the inhibition of SARS-CoV pseudovirus infection by serum in rhesus monkeys on day 28 after vaccination in each group.

[0110] Figure 79 The results show the correlation between the serum SARS-CoV-2RBD-specific antibody titer and the SARS-CoV neutralizing antibody titer on day 28 after vaccination in each group of rhesus monkeys.

[0111] Figure 80 The results show the inhibition of WIV1 pseudovirus infection by serum in rhesus monkeys on day 28 after vaccination in each group.

[0112] Figure 81The results show the correlation between serum SARS-CoV-2RBD-specific antibody titers and WIV1 neutralizing antibody titers on day 28 post-vaccination in rhesus monkeys in each group.

[0113] Figure 82 The results show the inhibition of Rs3367 pseudovirus infection by serum in rhesus monkeys on day 28 after vaccination in each group.

[0114] Figure 83 The results show the correlation between the serum SARS-CoV-2RBD-specific antibody titer and the Rs3367 neutralizing antibody titer on day 28 post-vaccination in each group of rhesus monkeys.

[0115] Figure 84 The study shows the SARS-CoV-2 viral load in nasal swabs on days 3, 5, and 7 after challenge in rhesus monkeys treated with PBS.

[0116] Figure 85 The study showed the SARS-CoV-2 viral load in nasal swabs on days 3, 5, and 7 after challenge with aluminum / RBD-Fc rhesus monkeys.

[0117] Figure 86 The study shows the SARS-CoV-2 viral load in nasal swabs on days 3, 5, and 7 after challenge to rhesus monkeys immunized with CF501 / RBD-Fc.

[0118] Figure 87 The SARS-CoV-2 viral load in the lung lobe of an immunized rhesus monkey shown on day 7 post-infection is displayed.

[0119] Figure 88 The study showed the SARS-CoV-2 viral load in the nasal turbinates of immunized rhesus monkeys on day 7 post-attack.

[0120] Figure 89 The study showed the SARS-CoV-2 viral load in the nasal mucosa of immunized rhesus monkeys on day 7 post-attack.

[0121] Figure 90 The results show the N3G protein-specific antibodies in the serum of mice vaccinated with CF501 and HIV N3G protein or HIV N3G protein alone.

[0122] Figure 91 The results show the H1N1 HA-specific antibodies in the serum of mice 14 days after vaccination with a quadrivalent influenza virus vaccine with different adjuvants.

[0123] Figure 92The results show the H3N2 HA-specific antibodies in the serum of mice 14 days after vaccination with a quadrivalent influenza virus vaccine with different adjuvants.

[0124] Figure 93 The results show the serum of mice 14 days after vaccination with a quadrivalent influenza virus vaccine with different adjuvants, indicating the presence of B / Victoria HA-specific antibodies.

[0125] Figure 94 The results show the serum of mice 14 days after vaccination with a quadrivalent influenza virus vaccine with different adjuvants, indicating the presence of B / Yamagata HA-specific antibodies.

[0126] Figure 95 The results show the H1N1 HA-specific antibodies in the serum of mice 21 days after vaccination with a quadrivalent influenza virus vaccine with different adjuvants.

[0127] Figure 96 The results show the H3N2 HA-specific antibodies in the serum of mice 21 days after vaccination with a quadrivalent influenza virus vaccine with different adjuvants.

[0128] Figure 97 The results show the serum of mice 21 days after vaccination with a quadrivalent influenza virus vaccine with different adjuvants, indicating the presence of B / Victoria HA-specific antibodies.

[0129] Figure 98 The results show the serum of mice 21 days after vaccination with a quadrivalent influenza virus vaccine with different adjuvants, indicating the presence of B / Yamagata HA-specific antibodies.

[0130] Figure 99 The detection of H1N1 HA-specific antibodies in the serum of mice on days 14 and 21 after vaccination with a quadrivalent influenza virus vaccine with different adjuvants is shown.

[0131] Figure 100 The detection of H3N2 HA-specific antibodies in the serum of mice on days 14 and 21 after vaccination with a quadrivalent influenza virus vaccine with different adjuvants is shown.

[0132] Figure 101 The detection of B / Victoria HA-specific antibodies in the serum of mice on days 14 and 21 after vaccination with a quadrivalent influenza virus vaccine with different adjuvants is shown.

[0133] Figure 102 The detection of B / Yamagata HA-specific antibodies in the serum of mice on days 14 and 21 after vaccination with a quadrivalent influenza virus vaccine with different adjuvants is shown.

[0134] Figure 103 The study showed the detection of varicella-zoster virus gE protein-specific antibodies in the serum of mice immunized with an inactivated varicella-zoster virus vaccine plus different adjuvants on day 21 after the first immunization.

[0135] Figure 104 The study showed the detection of varicella-zoster virus gE protein-specific antibodies in the serum of mice immunized with an inactivated varicella-zoster virus vaccine plus different adjuvants on day 35 after the first immunization. Invention Details

[0137] The term "halogen" or "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0138] Unless otherwise stated, the term "alkyl" itself, or as part of another substituent, refers to a straight (i.e., unbranched) or branched carbon chain (or carbon) or combination thereof, which may be fully saturated, monounsaturated, or polyunsaturated, and may contain monovalent, divalent, and polyvalent radicals. Alkyl groups may contain a specified number of carbons (e.g., C1-C1). 10 (Representing 1 to 10 carbon atoms). In embodiments, the alkyl group is fully saturated. In embodiments, the alkyl group is monounsaturated. In embodiments, the alkyl group is polyunsaturated. The alkyl group is an uncyclic chain. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, methyl, for example, n-pentyl, n-hexyl, n-heptyl, and n-octyl, and their homologues and isomers. In embodiments, the term "alkyl" refers to a straight or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, without unsaturation (i.e., saturated alkyl); having 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms; and being attached to the rest of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, and 1,1-dimethylethyl (tert-butyl), etc.

[0139] In the embodiments, the term "heterocyclic alkyl" refers to a monocyclic, bicyclic, or polycyclic heterocyclic alkyl ring system. In the embodiments, the heterocyclic alkyl group is fully saturated. In the embodiments, a bicyclic or polycyclic heterocyclic alkyl ring system refers to a fused polycyclic system wherein at least one of the fused rings is a heterocyclic alkyl ring, and wherein the plurality of rings are attached to the parent molecule moiety by any atoms contained within the heterocyclic alkyl rings of the plurality of rings.

[0140] In the embodiments, heterocyclic alkyl groups are "heterocyclic groups," which refer to stable 3- to 15-membered ring groups consisting of a carbon atom and 1-5 heteroatoms selected from nitrogen, oxygen, and sulfur. In one embodiment, the heterocyclic system group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen or sulfur atom in the heterocyclic ring system group may optionally be oxidized; the nitrogen atom may optionally be quaternized; and the heterocyclic group may be partially or fully saturated or aromatic. The heterocyclic ring system can be attached to the host structure at any heteroatom or carbon atom, resulting in the formation of stable compounds. Exemplary heterocyclic radicals include azetidinyl, benzopyranonyl, benzopyranyl, benzotetrahydrofuranyl, benzotetrahydrothienyl, chromanyl, chromonyl, coumarinyl, decahydroisoquinolinyl, dibenzofuranyl, dihydrobenzisothiazinyl, dihydrobenzisoxazinyl, dihydrofuryl, dihydropyranyl, and dioxopentaneyl. lanyl), dihydropyrazinyl, dihydropyridinyl, dihydropyrazolyl, dihydropyrimidinyl, dihydropyrrolyl, dioxolanyl, 1,4-dithianyl, isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isochromanyl, isocoumarinyl, benzo[1,3]dioxol-5-yl, benzodioxolyl, 1,3-dioxol-2-yl (1,3-dioxolan-2-yl), dioxolanyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, tetrahydrofuran, oxazolidin-2-onyl, oxazolidinonyl, piperidinyl, piperazinyl, pyranyl, tetrahydroiuryl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydrothienyl, pyrrolidinonyl, oxathiolanyl, and pyrrolidinyl.

[0141] Unless otherwise stated, the term "aromatic group" refers to a polyunsaturated, aromatic hydrocarbon substituent, which may be monocyclic or fused together (i.e., fused-ring aromatic group) or covalently linked polycyclic (preferably 1 to 3 rings). In embodiments, a fused-ring aromatic group refers to a plurality of fused rings, wherein at least one of the fused rings is an aromatic ring, and wherein the plurality of rings are attached to a parent molecule moiety via any carbon atom contained within the aromatic ring of the plurality of rings. The term "heteroaryl" refers to an aromatic group (or ring) containing at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. In embodiments, the term "heteroaryl" comprises a fused-ring heteroaryl group (i.e., a plurality of fused rings, wherein at least one of the fused rings is a heteroaryl ring, and wherein the plurality of rings are attached to a parent molecule moiety via any atom contained within the heteroaryl ring of the plurality of rings). 5,6-fused-ring heteroarylene refers to two fused rings, one ring having a 5-membered ring and the other having a 6-membered ring, wherein at least one ring is a heteroaryl ring. Similarly, 6,6-fused-ring heteroarylene refers to two fused rings, one of which has a 6-membered ring and the other has a 6-membered ring, and at least one of the rings is a heteroaryl ring. 6,5-fused-ring heteroarylene refers to two fused rings, one of which has a 6-membered ring and the other has a 5-membered ring, and at least one of the rings is a heteroaryl ring. The heteroaryl group can be attached to the remainder of the molecule via carbon or heteroatom. Non-limiting examples of aromatic and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purine, oxazolyl, isoxazolyl, thiazolyl, furanyl, thiophene, pyridinyl, pyrimidinyl, benzothiazole, benzoxazolyl, benzimidazolyl, benzofuran, isobenzofuranyl, indoleyl, isoindoleyl, benzothiophenyl, isoquinolinyl, quinoxalinyl, quinolinyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl. 3-Pyrroloyl, 3-Pyrazolyl, 2-Imidazolyl, 4-Imidazolyl, Pyrazinyl, 2-Oxazolyl, 4-Oxazolyl, 2-Pheny-4-Oxazolyl, 5-Oxazolyl, 3-Isooxazolyl, 4-Isooxazolyl, 5-Isooxazolyl, 2-Thiazolyl, 4-Thiazolyl, 5-Thiazolyl, 2-Furanyl, 3-Furanyl, 2-Thienyl, 3-Thienyl, 2-Pyridinyl, 3-Pyridinyl, 4-Pyridinyl, 2-Pyrimidinyl, 4-Pyrimidinyl, 5-Benzothiazolyl, Purinyl, 2-Benzimidazolyl, 5-Indoleyl, 1-Isoquinolinyl, 5-Isoquinolinyl, 2-Quinoxolinyl, 5-Quinoxolinyl, 3-Quinolinyl, and 6-Quinolinyl.

[0142] As used in this article, symbols This means that two atoms can be connected by a single bond or a double bond, as long as the valence states of the two atoms are appropriate.

[0143] The term "antigen" refers to any substance that can be used as a target for an immune response. An immune response can be a cellular immune response or a humoral immune response. In one embodiment, the vaccine contains an antigen. In one embodiment, the antigen is selected from the group consisting of cancer antigens, viral antigens, bacterial antigens, parasitic antigens, and fungal antigens.

[0144] In one embodiment, the viral antigen is selected from the group consisting of HIV antigen, influenza antigen, and coronavirus antigen. In one embodiment, the viral antigen is derived from one or more of the following antigens: HCOV-229E, HCOV-OC43, SARS-COV, HCOV-NL63, HCOV-HKU1, MERS-COV, varicella-zoster virus, and SARS-COV-2 such as the SARS-COV-2 Omeprone mutant. In one embodiment, the viral antigen is the SARS-COV-2 RBD-Fc protein or the gE protein of varicella-zoster virus (VZV).

[0145] In one aspect, this disclosure provides a vaccine comprising the compounds and antigens of this disclosure. In one embodiment, the vaccine is an intramuscular, intradermal, or inhaled vaccine. The antigen may be a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, and / or a fungal antigen. For example, the viral antigen may be selected from the group consisting of HIV antigen, influenza antigen, and coronavirus antigen. The viral antigen may be derived from one or more of the following antigens: HCOV-229E, HCOV-OC43, SARS-COV, HCOV-NL63, HCOV-HKU1, MERS-COV, varicella-zoster virus, and SARS-COV-2 such as the SARS-COV-2 Omeprone mutant. In one embodiment, the viral antigen is the SARS-COV-2 RBD-Fc protein or the gE protein of varicella-zoster virus (VZV).

[0146] The term "vaccine" in this document refers to a preparation of an antigen that typically contains some part of an infectious agent and elicits an immune response in a subject upon injection. The antigenic portion of a vaccine preparation can be a microorganism or a natural product purified from a microorganism, a synthetic product, or a genetically engineered protein, peptide, polysaccharide, etc. Preferably, the vaccine is an inactivated vaccine, a live attenuated vaccine, a subunit vaccine, or a nucleic acid vaccine such as an mRNA or DNA vaccine.

[0147] As used herein, the term "adjuvant" refers to any substance that, when mixed with an injected immunogen, can increase or improve an immune response. In this document, an adjuvant can be one or more of the following compounds:

[0148]

[0149]

[0150]

[0151] Effective vaccines must elicit an appropriate response to the antigen. Several unique types of immune responses exist, each with different protective capabilities against specific diseases. For example, antibodies offer protection against bacterial infections, but cell-mediated immunity is required to eliminate many viral infections and tumors. A variety of unique antibody and cell-mediated immune responses exist. Cell-mediated responses fall into two basic groups: 1) delayed-type hypersensitivity reactions, in which T cells act indirectly through macrophages and other cells or cell products, and 2) cytotoxic reactions, in which specialized T cells specifically and directly attack and kill infected cells.

[0152] There are five main classes of antibodies: IgM, IgG, IgE, IgA, and IgD. These antibodies have unique functions in immune responses. IgG is the dominant class of antibodies in the blood and can be further subdivided into several different subtypes or isotypes. In mice, the isotypes are IgG1, IgG2a, IgG2b, and IgG3. In humans, the isotypes are IgG1, IgG2, IgG3, and IgG4. IgG isotypes have different protective capabilities against specific infections. Mouse IgG2a and IgG2b can activate complement and mediate antibody-mediated cytotoxicity and cell-mediated cytotoxicity. They are particularly effective against many bacterial, viral, and parasitic infections. Similar isotypes in humans are represented by IgG1 and IgG3. In contrast, mouse IgG3 has particularly effective protection against bacteria with polysaccharide membranes, such as pneumococcus. The human isotype can be IgG4. Isotypes such as mouse IgG1 do not bind to complement and cannot effectively neutralize toxins. They have low activity against many bacterial and viral infections. Because different IgG isotypes have significantly different immune functions, it is important to induce the most suitable isotype for a specific infection with a vaccine. Although the nomenclature differs, valid evidence and previous theories suggest that the immunogens that determine antibody isotypes across mammalian species are similar in nature. In other words, immunogens that can activate IgG antibodies in delayed-type hypersensitivity and complement-fixing responses in one species can stimulate similar responses in other species.

[0153] In some embodiments, this application provides a kit containing an immunogenic composition or vaccine and instructions for use. The kit may contain the immunogenic composition or vaccine in a suitable container, along with various buffer solutions well known in the art. In some embodiments, the kit contains one or more compounds of this application. Therefore, in some embodiments, the immunogenic composition or vaccine and these compounds are in the same vial. In some embodiments, the immunogenic composition or vaccine and these compounds are in separate vials.

[0154] The container may include at least one vial, tube, flask, bottle, syringe, or other container device that may contain an immunogenic composition or vaccine. If other components are provided, the kit may contain additional containers to contain those components. The kit may also include tools for containing the immunogenic composition or vaccine, as well as any other sealed reagent containers for commercial sale. Such containers may contain injection-molded or blow-molded plastic containers that retain the desired vials therein. The container and / or kit may include labels with instructions and / or warnings. In some embodiments, this application provides a kit containing a container that includes a vaccine containing an immunogenic composition, optionally a pharmaceutically acceptable carrier, and a packaging insert with instructions for vaccination to treat or prevent a disease in a subject. In some embodiments, the kit further includes the compound of this application and instructions for administering the compound to treat or prevent a disease in a subject.

[0155] The present application is further illustrated by the following examples. These examples should not be construed as limiting. Example

[0156] The following compounds were used in the examples, and their synthesis is described below. The starting reagents were commercially available.

[0157] (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazol[4,5-b]pyridine-6-carboxamide((E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido) ido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (which is called "STING agonist 502" or CF502).

[0158]

[0159] (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carbamoyl)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carbamoyl)-3H-imidazol[4,5-b]pyridine-6-carbamoyl((E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carbamoyl)-3H-imidazol[4,5-b]pyridine-6-carbamoyl((E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5- (carboxamido)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (which is called "STING agonist 501" or CF501).

[0160]

[0161] (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carbamoyl)-7-(3-(piperazin-1-yl)propoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carbamoyl)-3H-imidazol[4,5-b]pyridine-6-carbamoyl((E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-c) arboxamido)-7-(3-(piperazin-1-yl)propoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (which is called "STING agonist 508" or CF508).

[0162]

[0163] (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carbamoyl)-7-(2-morpholinoethoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carbamoyl)-3H-imidazol[4,5-b]pyridine-6-carbamoyl((E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carbamoyl)-3H-imidazol[4,5-b]pyridine-6-carbamoyl((E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5- (carboxamido)-7-(2-morpholinoethoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (which is known as "STING agonist 510" or CF510).

[0164]

[0165] (E)-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-3-methyl-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-1-methyl-2,3-dihydro-1H-imidazol[4,5-b]pyridine-6-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino) ino)-3-methyl-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H -pyrazole-5-carbonyl)imino)-1-methyl-2,3-dihydro-1H-imidazo[4,5-b]pyridine-6-carboxamide) (which is called "STING Agonist 512" or CF512).

[0166]

[0167] Compound CF502 and its synthesis

[0168] (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazol[4,5-b]pyridine-6-carboxamide

[0169]

[0170] Synthesis scheme

[0171] Step 1:

[0172] (E)-3-(4-((4-carbamoyl-2-nitrophenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide

[0173] A solution of (E)-3-(4-aminobut-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (intermediate 2, 6.00 g, 14.342 mmol, 1.00 eq, HCl), 4-fluoro-3-nitro-benzamide (2.64 g, 14.3 mmol, 1.00 eq), DIPEA (7.40 g, 57.3 mmol, 9.98 mL, 4.00 eq) and NaHCO3 (4.81 g, 57.30 mmol, 2.23 mL, 4 eq) in EtOH (60 mL) was stirred at 110 °C under N2 for 16 h to provide a yellow suspension. LC-MS showed a main peak, and the desired MS peak was found. The reaction mixture was diluted with H2O (60 mL) and filtered to provide (E)-3-(4-((4-carbamoyl-2-nitrophenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (5.0 g, 9.14 mmol, 63.80% yield, 99.9% purity) as a yellow solid, which was used directly in the next step without further purification.

[0174] 1 HNMR (400MHz, DMSO-d6): δ (ppm) 12.83 (br s, 1H), 8.72 (d, J = 1.50Hz, 1H), 8.62 (d, J = 2.00Hz, 1H), 8.49 (t, J = 5.94Hz, 1H), 8.13 (br s,2H),7.86-8.01(m,2H),7.51(brs,1H),7.25(br s,1H),6.95(d,J=9.13Hz,1H),6.59(s,1H),5.86-5.96(m,1H),5.72-5.83(m,1H),4.81(br d,J=4.88Hz,2H),4.55(q,J=7.05Hz,2H),4.05(br s, 2H), 2.15 (s, 3H), 1.31 (t, J = 7.07Hz, 3H). LCMS: m / z 547.2 (M+1).

[0175] Step 2

[0176] (E)-3-(4-((2-amino-4-carbamoylphenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide

[0177]

[0178] A solution of (E)-3-(4-((4-carbamoyl-2-nitrophenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (5.00 g, 9.15 mmol, 1.00 eq) in DMF (50 mL) and H₂O (25 mL) was supplemented with NH₃·H₂O (12.8 g, 91.4 mmol, 14.0 mL, 25% purity, 10.0 eq), followed by disodium bis(2-ethylhexyl) and BLAH (4.78 g, 27.4 mmol, 5.97 mL, 3.00 eq). The reaction mixture was stirred at 25 °C for 1 hour to provide a yellow suspension. LC-MS showed the expected MS peak. The reaction mixture was diluted with H₂O (500 mL) and lyophilized. The residue was diluted with DMF (400 mL) and filtered. The filtrate was concentrated under vacuum. (E)-3-(4-((2-amino-4-carbamoylphenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (4.5 g, crude) was obtained as a pale yellow solid and was used directly in the next step without further purification. LCMS: m / z 517.3 (M+1).

[0179] Step 3

[0180] (E)-3-(4-(2-amino-5-carbamoyl-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazol[4,5-b]pyridine-6-carboxamide

[0181]

[0182] BrCN (2.77 g, 26.13 mmol, 1.92 mL, 3 eq) was added to a solution of (E)-3-(4-((2-amino-4-carbamoylphenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (4.5 g, 8.71 mmol, 1 eq) in DMF (45 mL) and MeOH (90 mL), and the reaction mixture was stirred at 50 °C under N2 for 2 h to provide a yellow suspension. LC-MS showed the desired MS peak. The reaction mixture was concentrated under reduced pressure. The residue was ground together with EtOAc / i-PrOH (30 / 10 mL) to provide (E)-3-(4-(2-amino-5-carbamoyl-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (3.20 g, 5.06 mmol, 58.1% yield, 98.5% purity, HBr), a yellow solid. LCMS: m / z 542.4 (M+1).

[0183] Step 4

[0184] (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazol[4,5-b]pyridine-6-carboxamide

[0185]

[0186] HATU (794 mg, 2.09 mmol, 1.30 eq) was added to a solution of (E)-3-(4-(2-amino-5-carbamoyl-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (1.00 g, 1.61 mmol, 1.00 eq, HBr), 1-ethyl-3-methyl-1H-pyrazole-5-carboxylic acid (297 mg, 1.93 mmol, 1.20 eq), and DIEA (1.04 g, 8.03 mmol, 1.40 mL, 5.00 eq) in DMF (15.0 mL), and the reaction mixture was stirred at 50 °C under N2 for 16 h to provide a yellow solution. LC-MS showed the desired MS peak. The reaction mixture was filtered. The filter cake was washed with cold DMF (5 mL x 2) and lyophilized. (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (1.40 g) was a pale yellow solid.

[0187] 1 HNMR (400MHz, DMSO-d6): δ (ppm) 12.64-13.11 (m, 2H), 8.71 (s, 1H), 8.12 (s, 2H), 7.85-8.00 (m, 2H) 7.71 (d, J = 8.13Hz, 1H), 7.26 -7.58(m,3H),6.55(s,2H),5.79-6.07(m,2H),4.89-4.76(m,4H),4.38-4.59(m,4H),2.12(s,6H),1.06-1.35(m,6H).LCMS:m / z 678.5(M+1). HPLC: 93% purity.

[0188] Compound CF501 and its synthesis

[0189] (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide

[0190]

[0191] Synthesis scheme

[0192] Step 1: 4-Chloro-3-methoxy-5-nitrobenzamide

[0193]

[0194] 4-Chloro-3-methoxy-5-nitrobenzoate (25 g, 10.1 mmol) was stirred in NH4OH (250 mL, 1.9 mmol) at room temperature for 24 h. The reaction temperature was then increased to 50 °C for 2 h. Another 50 mL (~3.7 eq) of NH4OH was added to the container. After stirring at 50 °C for another 2 hours, the reaction mixture was cooled to room temperature. The solid was filtered and washed with cold water. The solid was dried under house vacuum and lyophilized to provide 4-chloro-3-methoxy-5-nitrobenzoamide (13 g, 68% yield) as a brown solid.

[0195] 1 H NMR (400MHz, DMSO-d6): δ (ppm): 8.31 (br.s., 1H), 8.06 (d, J = 1.8Hz, 1H), 7.88 (d, J = 1.8Hz, 1H), 7.81 (br.s., 1H), 4.02 (s, 3H). LCMS: m / z230.9 (M+1).

[0196] Step 2: 4-Chloro-3-hydroxy-5-nitrobenzamide

[0197]

[0198] 4-Chloro-3-methoxy-5-nitrobenzamide (16 g, 69.3 mmol) was suspended in dry DCM (250 mL) and stirred at room temperature. BBr3 (280 mL, 1 M, in DCM) was added dropwise to the reaction. A slurry was rapidly formed and stirred overnight at room temperature under nitrogen. The reaction mixture was poured into ice water (3 L) and stirred vigorously for 30 min. The resulting suspension was filtered and the solid was dried to provide 4-chloro-3-hydroxy-5-nitrobenzamide (11.6 g, 77% yield). 1 H NMR(400MHz,DMSO-d6):δ(ppm):11.53(br.s.,1H),8.17(br.s.,1H),7.92(s,1H),7.72(s,1H),7.66(br.s.,1H).LC-MS:[M+H] + =217.LCMS:m / z217.0(M+1).

[0199] Step 3: 4-Chloro-3-(3-morpholinopropoxy)-5-nitrobenzamide

[0200]

[0201] A mixture of 4-chloro-3-hydroxy-5-nitrobenzamide (11.6 g, 53.5 mmol), 4-(3-chloropropyl)morpholine (10.5 g, 64.2 mmol), and K₂CO₃ (9.6 g, 69.6 mmol) in DMF (100 mL) was stirred overnight at 70 °C. The solvent was removed under vacuum to provide a crude solid product, which was purified by silica gel chromatography (MeOH:DCM = 1:10) to provide 4-chloro-3-(3-morpholinopropoxy)-5-nitrobenzamide (10.5 g, 57% yield) as a yellow solid. 1 H NMR (400MHz, DMSO-d6): δ (ppm): 8.30 (s, 1H), 8.05 (d, J = 1.8Hz, 1H), 7.88 (d, J = 1.8Hz, 1H), 7.80 (s, 1H), 4.28 (t, J = 6.2Hz ,2H),3.57(t,J=4.6Hz,4H),2.41-2.47(m,2H),2.37(br.s.,4H),1.97(dd,J=13.94,7.35Hz,2H); LCMS: m / z344.1(M+1).

[0202] Step 4(E)-6-((4-((4-carbamoyl-2-(3-morpholinopropoxy)-6-nitrophenyl)amino)but-2-en-1-yl)amino)-5-nitronicotinamide

[0203]

[0204] A solution of (E)-6-((4-aminobut-2-en-1-yl)amino)-5-nitronicotinamide (intermediate 3,220 mg, 0.87 mmol), 4-chloro-3-(3-morpholinopropoxy)-5-nitrobenzamide (200 mg, 0.58 mmol), i-PrOH (5 mL), and DIEA (1.12 g, 8.7 mmol) in a microwave-safe vial was irradiated at 120 °C for 6 hours. Upon cooling, the resulting solid was separated by filtration, washed with i-PrOH (2 x 1 mL), and dried to provide (E)-6-((4-((4-carbamoyl-2-(3-morpholinopropoxy)-6-nitrophenyl)amino)but-2-en-1-yl)amino)-5-nitronicotinamide (113 mg, 35%) as a red solid.

[0205] Step 5(E)-5-amino-6-((4-((2-amino-4-carbamoyl-6-(3-morpholinopropoxy)phenyl)amino)but-2-en-1-yl)amino)nicotinamide

[0206]

[0207] (E)-6-((4-((4-carbamoyl-2-(3-morpholinopropoxy)-6-nitrophenyl)amino)but-2-en-1-yl)amino)-5-nitronicotinamide (2.7 g, 4.8 mmol) in MeOH (40.0 mL) was added to sodium hydrosulfite (11.7 g, 67.2 mmol) in water (45 mL). After 15 min, solid sodium bicarbonate (24 g) was added. After 10 min, the reaction mixture was filtered and the solid was washed with MeOH (4 x 20 mL). The combined filtrate was concentrated onto diatomaceous earth (Celite) and purified by preparative HPLC to provide (E)-5-amino-6-((4-((2-amino-4-carbamoyl-6-(3-morpholinopropoxy)phenyl)amino)but-2-en-1-yl)amino)nicotinamide (1.81 g, 3.26 mmol, 49% yield) as a dark yellow solid. 1 H NMR (400MHz, DMSO-d6): δ (ppm): 7.93 (s, 1H), 7.60 (m, 1H), 7.10 (s, 1H), 6.96 (br s,1H),6.85(m,2H),6.77(s,1H),6.14(m,1H),5.73(m,2H),4.81(m,2H),4.66(m 2H), 3.96 (m, 4H), 3.83 (m, 1H), 3.54 (m, 6H), 2.39 (t, J = 7.2Hz, 2H), 2.32 (br s, 4H), 1.84 (t, J = 6.4Hz, 2H). LCMS: m / z 499.3 (M+1).

[0208] Step 6(E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide:

[0209]

[0210] At 0°C, 0.4M 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (intermediate 4, 6 mL, 3.89 mmol) in dioxane was added to (E)-5-amino-6-((4-((2-amino-4-carbamoyl-6-(3-morpholinopropoxy)phenyl)amino)but-2-en-1-yl)amino)nicotinamide (812 mg, 1.62 mmol) in DMF (20 mL). After approximately 10 min, another portion of 0.4M 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (intermediate 3, 2 mL, 0.48 mmol) in dioxane was added, followed by the final portion (2 mL, 0.48 mmol) after approximately 15 min. After a total reaction time of 35 min, EDC (1.087 g, 5.67 mmol) was added, followed by triethylamine (656 mg, 6.48 mmol). The mixture was warmed to room temperature and stirred overnight. The reaction was quenched with a 3:1 water:saturated NH4Cl aqueous solution (10 mL) and extracted with a 3:1 chloroform:ethanol solution (2 x 40 mL). The combined organic phases were washed with water (10 mL), dried over magnesium sulfate, and concentrated. The resulting residue was purified by preparative HPLC, and the desired eluent was lyophilized to provide (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide, as a white solid (445 mg, yield 45%; HPLC purity: 97.7% (254 nm)).

[0211] 1 H-NMR (400MHz, DMSO-d6): δ (ppm): 8.70 (s, 1H), 8.14 (s, 1H), 7.66 (s, 1H), 7.33 (s, 1H), 6.51 (d, J = 12.0Hz 2H),5.95(m,1H),5.74(m,1H),4.94(d,J=4.0Hz,2H),4.79(d,J=4.8Hz,2H),4.52(m,4H),4.13(t,J=11.2Hz 2H),3.98(m,2H),3.67(s,2H),3.38(t,J=12.4Hz,2H),3.24(m,2H),3.05(m,2H),2.09(s,6H),2.06(m,2H),1.26(t,J=14.0Hz,6H).LCMS:m / z 821.4(M+1),819.3(M-1).

[0212] Compound CF508 and its synthesis

[0213] (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-7-(3-(piperazin-1-yl)propoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide

[0214]

[0215] Synthesis scheme

[0216] Step 14 - (3-(5-carbamoyl-2-chloro-3-nitrophenoxy)propyl)piperazine-1-carboxylic acid tert-butyl ester

[0217]

[0218] A mixture of 4-chloro-3-hydroxy-5-nitrobenzamide (prepared as in Example 3, 3.20 g, 14.8 mmol, 1.00 eq), 4-(3-chloropropyl)piperazine-1-carboxylic acid tert-butyl ester (4.66 g, 17.7 mmol, 1.20 eq), and K₂CO₃ (2.65 g, 19.2 mmol, 1.30 eq) in DMF (32 mL) was stirred at 70 °C under a N₂ atmosphere for 16 hours to produce a yellow solution. LC-MS showed complete consumption of the starting material and a main peak with the desired MS signal was detected. The mixture was concentrated under reduced pressure to produce a crude product. The crude product was ground together with H2O (100 mL) at 25 °C for 16 hours to provide 4-(3-(5-carbamoyl-2-chloro-3-nitrophenoxy)propyl)piperazine-1-carboxylic acid tert-butyl ester (6.00 g, 13.6 mmol, 91.7% yield) as a yellow solid.

[0219] HNMR (400MHz, DMSO-d6): δ8.34(br s,1H),8.05(s,1H),7.89(s,1H),7.80(br s,1H),4.28(br t,J=5.4Hz,2H),3.37(br s,12H),2.17-2.38(m,5H),1.86-2.08(m,2H),1.39(s,12H); LCMS: m / z(ES+)[M+H]+=443.5.

[0220] Step 2

[0221] (E)-4-(3-(5-carbamoyl-2-((4-((5-carbamoyl-3-nitropyridin-2-yl)amino)but-2-en-1-yl)amino)-3-nitrophenoxy)propyl)piperazine-1-carboxylic acid tert-butyl ester

[0222]

[0223] A solution of (E)-6-((4-aminobut-2-en-1-yl)amino)-5-nitronicotinamide (intermediate 3, 9.74 g, 33.9 mmol, 1.50 eq, HCl), 4-(3-(5-carbamoyl-2-chloro-3-nitrophenoxy)propyl)piperazine-1-carboxylic acid tert-butyl ester (10.0 g, 22.6 mmol, 1.00 eq), DIEA (11.7 g, 90.3 mmol, 15.7 mL, 4.00 eq), and NaHCO3 (7.59 g, 90.3 mmol, 3.51 mL, 4.00 eq) in EtOH (110 mL) was stirred at 110 °C in a sealed N2 tube for 16 h to provide a yellow suspension. LC-MS showed complete consumption of the starting material and a main peak with the desired MS was detected. The mixture was concentrated to provide a crude product. The residue was subjected to rapid silica gel chromatography (… 120g SilicaFlash Column, 5-10% methanol / dichloromethane @ 100 mL / min, elution buffer of dichloromethane / methanol = 10:1, product R f =0.50, UV 254nm) purification to produce (E)-4-(3-(5-carbamoyl-2-((4-(((5-carbamoyl-3-nitropyridin-2-yl)amino)but-2-en-1-yl)amino)-3-nitrophenoxy)propyl)piperazine-1-carboxylic acid tert-butyl ester (5.50 g, 7.51 mmol, 33.2% yield, 89.8% purity), as a reddish-brown solid.

[0224] 1HNMR (400MHz, DMSO-d6): δ8.73-8.99(m,3H),7.99-8.23(m,3H),7.71-7.85(m,1H),7.41-7.60(m,2H),7.34(br s,1H),5.56-5.87(m,2H),4.38(br s,1H),4.08-4.26(m,4H),4.05(br t,J=6.1Hz,2H),3.53-3.69(m,1H),3.03-3.49(m,15H),2.19-2.46(m,6H),1.90(br s, 2H), 1.39 (s, 9H), 1.17-1.31 (m, 5H), 1.06 (t, J = 7.0Hz, 3H); LCMS: m / z (ES+) [M+H]+ = 658.2.

[0225] Step 3

[0226] (E)-4-(3-(3-amino-2-((4-((3-amino-5-carbamoylpyridin-2-yl)amino)but-2-en-1-yl)amino)-5-carbamoylphenoxy)propyl)piperazine-1-carboxylic acid tert-butyl ester

[0227]

[0228] To a solution of (E)-4-(3-(5-carbamoyl-2-((4-(((5-carbamoyl-3-nitropyridin-2-yl)amino)but-2-en-1-yl)amino)-3-nitrophenoxy)propyl)piperazine-1-carboxylic acid tert-butyl ester (5.50 g, 8.36 mmol, 1.00 eq) in MeOH (110 mL) and H₂O (55.0 mL), NaHCO₃ (42.0 g, 500 mmol, 19.5 mL, 59.8 eq) was added, followed by the addition of disodium benzoate; BLAH (20.4 g, 117 mmol, 25.5 mL, 14.0 eq) at 0 °C. The reaction mixture was then stirred at 20 °C for 2 hours. A pale yellow suspension was obtained. LCMS showed that reactant 1 was completely consumed and a main peak with the desired MS was detected. The reaction mixture was filtered, and the filter cake was washed with MeOH (100 mL * 2). Concentrate the filtrate. The crude product (E)-4-(3-(3-amino-2-((4-(((3-amino-5-carbamoylpyridin-2-yl)amino)but-2-en-1-yl)amino)-5-carbamoylphenoxy)propyl)piperazine-1-carboxylic acid tert-butyl ester (5.00 g, 8.37 mmol, 100% yield) was used directly for the next step without purification. LCMS: m / z(ES+)[M+H]+=598.2;

[0229] Step 4

[0230] (E)-4-(3-((5-carbamoyl-1-(4-(6-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carbamoyl)-3H-imidazo[4,5-b]pyridin-3-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carbamoyl)-1H-benzo[d]imidazo-7-yl)oxy)propyl)piperazine-1-carboxylic acid tert-butyl ester

[0231]

[0232] A solution of tert-butyl 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (intermediate 4, 4.41 g, 22.6 mmol, 2.70 eq) in dioxane was added to a solution of (E)-4-(3-(3-amino-2-((4-(((3-amino-5-carbamoylpyridin-2-yl)amino)but-2-en-1-yl)amino)-5-carbamoylphenoxy)propyl)piperazine-1-carboxylic acid tert-butyl ester (5.00 g, 8.37 mmol, 1.00 eq) in DMF (100 mL) at 0 min (3 mL), 10 min (3 mL), and 15 min (3 mL), respectively. The reaction mixture was stirred at 0 °C for 30 min, then EDCI (5.61 g, 29.3 mmol, 3.50 eq) and Et3N (6.77 g, 66.9 mmol, 9.31 mL, 8.00 eq) were added at 0 °C, followed by heating to 25 °C and stirring for 2 h to provide a yellow solution. LC-MS showed that the starting material was completely consumed and the desired compound was detected. The reaction mixture was quenched with saturated NH4Cl aqueous solution (50 mL). The solution was filtered, and the filtrate was concentrated. The crude product was purified by preparative HPLC (column: Phenomenex Gemini YMC Triart C18 250*50mm*7um; mobile phase: [water (10mM NH4OAc)-ACN, 30-49, 30min)) to provide (E)-4-(3-((5-carbamoyl-1-(4-(6-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazol-5-carbamoyl)-3H-imidazo[4,5-b]pyridin-3-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carbamoyl)-1H-benzo[d]imidazo-7-yl)oxy)propyl)piperazine-1-carboxylic acid tert-butyl ester (2.50 g, 2.42 mmol, 28.9% yield, 89.0% purity). LCMS:m / z(ES+)[M+H] + =920.5.

[0233] Step 5

[0234] (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-7-(3-(piperazin-1-yl)propoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide

[0235]

[0236] A solution of (E)-4-(3-((5-carbamoyl-1-(4-(6-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridin-3-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazo-7-yl)oxy)propyl)piperazine-1-carboxylic acid tert-butyl ester (900 mg, 978 μmol, 1.00 eq) in MeOH (15 mL) and dioxane (15 mL) was added to HCl / dioxane (4 M, 30 mL, 123 eq), and the mixture was stirred at 25 °C under N2 for 16 h to provide a yellow solution. LC-MS showed that the starting material was completely consumed and the desired compound was detected. The mixture was concentrated under reduced pressure to provide the residue. The crude product was purified by preparative-HPLC (column: Phenomenex Gemini YMC Triart C18 250x50mm x7μm; mobile phase: [water (0.05% HCl)-ACN, 20-80 30min)) to provide (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-7-(3-(piperazin-1-yl)propoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (150 mg, 0.176 mmol, 17.9% yield, 96.0% purity), as a white solid.

[0237] 1HNMR (400MHz, DMSO-d6): δ11.89-12.32(m,1H),9.57-10.02(m,2H),8.78(d,J=1.8Hz,1H),8.09-8.33(m,2H),7.99(br s,1H),7.66(d,J=1.0Hz,1H),7.56(br s,1H),7.26-7.49(m,2H),6.53(s,1H),6.48(s,1H),6.00(dt,J=15.5,5.1Hz,1H),5.73(dt,J=15.6,5.6Hz,1H),4.98(br d,J=3.6Hz,2H),4.81(br d,J=5.3Hz,2H),4.39-4.58(m,4H),3.21-3.80(m,9H),2.13-2.30(m,3H),2.09( d, J=4.9Hz, 6H), 1.25ppm (td, J=7.1, 3.9Hz, 6H); LCMS: m / z (ES+) [M+H]+ = 920.4.

[0238] Compound CF510 and its synthesis

[0239] (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-7-(2-morpholinoethoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide

[0240]

[0241] Synthesis scheme

[0242] Step 1: 4-Chloro-3-(2-morpholinoethoxy)-5-nitrobenzamide

[0243]

[0244] A mixture of 4-chloro-3-hydroxy-5-nitrobenzamide (prepared as in Example 3, 1.00 g, 4.62 mmol, 1.00 eq), 4-(2-chloroethyl)morpholine (1.03 g, 5.54 mmol, 1.20 eq, HCl), and K₂CO₃ (1.28 g, 9.23 mmol, 2.00 eq) in DMF (15.0 mL) was stirred at 70 °C under a N₂ atmosphere for 16 h to provide a yellow solution. LC-MS showed that the starting material was completely consumed and a main peak with the desired MS was detected. The mixture was concentrated under reduced pressure to produce a crude product. The crude product was milled with H₂O (50.0 mL) at 25 °C for 16 h. 4-chloro-3-(2-morpholinoethoxy)-5-nitrobenzamide (2.60 g, 7.65 mmol, 82.8% yield, 97.0% purity) was obtained as a yellow solid. LCMS: m / z(ES+)[M+H]+=330.1.

[0245] Step 2

[0246] (E)-6-((4-((4-carbamoyl-2-(2-morpholinoethoxy)-6-nitrophenyl)amino)but-2-en-1-yl)amino)-5-nitronicotinamide

[0247]

[0248] A solution of 4-chloro-3-(2-morpholinoethoxy)-5-nitrobenzamide (1.05 g, 3.64 mmol, 1.20 eq, HCl), NaHCO3 (1.02 g, 12.1 mmol, 472 μL, 4.00 eq), and 4-chloro-3-(2-morpholinoethoxy)-5-nitrobenzamide (intermediate 3, 1.00 g, 3.03 mmol, 1.00 eq), DIEA (1.57 g, 12.1 mmol, 2.11 mL, 4.00 eq) in EtOH (10.0 mL) was stirred at 110 °C under N2 for 16 h to provide a yellow suspension. LC-MS showed that the starting material was completely consumed. The residue was subjected to rapid silica gel chromatography (…). 330g Silica Flash Column, 2-5% methanol / dichloroethane @ 100 mL / min - dichloroethane / methanol = 10:1 eluent, product R f =0.27, UV 254nm) purification. (E)-6-((4-((4-carbamoyl-2-(2-morpholinoethoxy)-6-nitrophenyl)amino)but-2-en-1-yl)amino)-5-nitronicotinamide (1.34 g, 2.12 mmol, 69.8% yield) was obtained as a reddish-brown solid.

[0249] 1 HNMR (400MHz, DMSO-d6): δ0.77-0.85 (m, 6H) 1.11 (t, J = 7.13Hz, 4H) 3.49 (br d,J=6.13Hz,2H)3.75-4.25(m,12H)5.56-5.74(m,2H)6.82-6.96(m,1H)7.78(br t,J=6.00Hz,1H)7.93-8.15(m,3H)8.72-8.91(m,3H); LCMS:m / z(ES+)[M+H] + =545.2.

[0250] Step 3

[0251] (E)-5-amino-6-((4-((2-amino-4-carbamoyl-6-(2-morpholinoethoxy)phenyl)amino)but-2-en-1-yl)amino)nicotinamide

[0252]

[0253] To MeOH (20 mL) and H₂O (15.0 mL), (E)-6-((4-((4-carbamoyl-2-(2-morpholinoethoxy)-6-nitrophenyl)amino)but-2-en-1-yl)amino)-5-nitronicotinamide (1.00 g, 1.84 mmol, 1.00 eq) was added, followed by the addition of disodium benzoate; BLAH (4.48 g, 25.7 mmol, 5.60 mL, 14.0 eq) at 0 °C. The reaction mixture was then stirred at 20 °C for 2 hours. A pale yellow suspension was obtained. LCMS showed that the starting material was completely consumed and detected a main peak with the desired MS. The reaction mixture was filtered, and the filter cake was washed with MeOH (50.0 mL). The filtrate was concentrated. The crude product was used directly for the next step without purification. (E)-5-amino-6-((4-((2-amino-4-carbamoyl-6-(2-morpholinoethoxy)phenyl)amino)but-2-en-1-yl)amino)nicotinamide (889 mg, 1.84 mmol, 100% yield) was obtained as a yellow solid. LCMS: m / z(ES+)[M+H] + =484.3.

[0254] Step 4

[0255] (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-7-(2-morpholinoethoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide

[0256]

[0257] A solution of (E)-5-amino-6-((4-((2-amino-4-carbamoyl-6-(2-morpholinoethoxy)phenyl)amino)but-2-en-1-yl)amino)nicotinamide (889 mg, 1.84 mmol, 1.00 eq) in DMF (23.0 mL) was added at 0 min (3.00 mL), 10 min (3.00 mL), and 15 min (3.00 mL) to a solution of 2-ethyl-5-methyl-pyrazole-3-carbonyl isothiocyanate (intermediate 4,968 mg, 4.96 mmol, 2.70 eq) in dioxane (5.30 mL). The reaction mixture was stirred at 0 °C for 35 min, then EDCI (1.23 g, 6.43 mmol, 3.50 eq) and Et3N (1.49 g, 14.7 mmol, 2.04 mL, 8.00 eq) were added at 0 °C, followed by heating to 25 °C and stirring for 16 h to provide a yellow solution. LCMS showed that reactant 1 was completely consumed and the desired compound was detected. The reaction mixture was quenched with saturated NH4Cl aqueous solution (50.0 mL). The solution was filtered and the filtrate was concentrated. The crude product was purified by preparative-HPLC (column: Phenomenex Gemini C18 250x 50mm x 7um; mobile phase: [water (10mM HCl)-ACN]; B%: 10%-40%, 20 min) to provide (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-7-(2-morpholinoethoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (52.0 mg, 0.06 mmol, 3.27% yield), as a white solid.

[0258] 1HNMR (400MHz, DMSO-d6): δ1.19-1.36(m,8H)2.03-2.25(m,9H)2.26-2.35(m,5H)2.68(br s,1H)3.45-3.53(m,3H)4.11(br t,J=5.63Hz,2H)4.45-4.61(m,4H)4.79(br s,2H)4.97(br s,2H)5.87-5.99(m,2H)6.54(s,2H)7.34(s,2H)7.53(br s,1H)7.64(s,1H)7.93(br s,1H)8.12-8.19(m,2H)8.71(s,1H); LCMS: m / z(ES+)[M+H]+=807.3.

[0259] Compound CF512 and its synthesis

[0260]

[0261] Synthesis scheme

[0262] A solution of MeI (3.05 g, 21.5 mmol, 1.34 mL, 44.1 eq) in DMF (0.8 mL) was added to a solution of (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazol-5-carboxamido)-3H-imidazol[4,5-b]pyridine-6-carboxamide (Example 4, 400 mg, 487 μmol, 1.00 eq) and K2CO3 (148 mg, 1.07 mmol, 2.20 eq) in DMF (8 mL), and the reaction mixture was stirred at 25 °C for 16 hours to provide a yellow solution. The mixture was concentrated under reduced pressure to provide the residue. The crude product was passed through a preparative HPLC system (column: Phenomenex Gemini C18 150*25mm*10um; mobile phase: [water (0.05% NH3H2O ​​+ 10mM)]). [NH4HCO3)-ACN]; B%: 10%-40%, 20 min) Purification to produce (E)-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-3-methyl-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-1-methyl-2,3-dihydro-1H-imidazo[4,5-b]pyridine-6-carboxamide (120 mg, 0.138 mmol, 28.4% yield, 97.9% purity), as a white solid.

[0263] 1HNMR (400MHz, DMSO-d6): δ8.74(d,J=1.6Hz,1H),8.35(d,J=1.6Hz,1H),8.14(br s,1H),8.04(br s,1H),7.71(s,1H),7.61(br s,1H),7.36-7.49(m,2H),6.40(s,1H),6.35(s,1H),5.75-5.88(m,1H),5.60-5.75(m,1H),4.80(br dd,J=16.4,5.3Hz,4H),4.30-4.54(m,4H),4.07(br t,J=6.3Hz,2H),3.40-3.58(m,10H),2.18-2.33(m,6H),2.10(d,J=13.3Hz,6H),1 .73(q,J=6.5Hz,2H), 1.21(dt,J=8.9,7.2Hz,6H); LCMS: m / z(ES+)[M+H]+=849.6.

[0264] Example 1: Detection of innate immune response activated by STING agonists in mice

[0265] 1. Materials:

[0266] Eight-week-old SPF C57 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. STING agonist and 3'-3' cGAMP (hereinafter referred to as cGAMP) were purchased from Invivogen. Trizol was purchased from Takara Bio Inc. A reverse transcription kit (PrimeScript) was used. TM The RT reagent kit with gDNA Eraser (Perfect Real Time) was purchased from Takara Bio Inc. The quantitative PCR kit (TB) was also purchased. Premix DimerEraser TM (Perfect Real Time) was purchased from Takara BioInc.

[0267] 2.1 Detection of innate immune response in mice activated by intramuscular injection of STING agonist

[0268] 2.1.1 Experimental Methods

[0269] (1) Twelve SPF C57 mice were randomly divided into three groups of four each. The mice were injected intramuscularly with 20 μg of the STING agonist CF501, cGAMP, or an equal volume of PBS.

[0270] (2) Six hours later, the mice were euthanized, the draining lymph nodes were isolated, and the total RNA of the lymph nodes was extracted by the Trizol method.

[0271] (3) Use a reverse transcription kit to reverse transcribe the extracted RNA into cDNA.

[0272] (4) IFNb, CXCL-10, CXCL-9, CCL-2, TNFα, IL-1β and IL-6 in mouse lymph nodes were detected using a real-time PCR kit.

[0273] like Figure 1 As shown, the STING agonist CF501, along with cGAMP, can activate a large production of cytokines 6 hours after intramuscular injection. However, CF501 can detect the production of these cytokines more effectively than cGAMP.

[0274] 2.2 Monitor the innate immune response in mice activated by intramuscular injection of CF501 and SARS-CoV-2RBD-Fc proteins at different time points.

[0275] 2.2.1 Experimental Methods

[0276] (1) Twenty-one SPF C57 mice were randomly assigned. In Group 1, three mice were euthanized, and their draining lymph nodes were collected as a control before administration. In Group 2, nine mice were injected intramuscularly with 5 μg of SARS-CoV-2 RBD-Fc protein (KactusBiosystems Co. Ltd, catalog number: COV-VM5BD; hereinafter also abbreviated as RBD-Fc or RBD-Fc protein), and three mice were euthanized at 6, 24, and 48 hours post-injection to collect draining lymph nodes. In Group 3, nine mice were injected intramuscularly with 5 μg of RBD-Fc protein and 20 μg of CF501, and three mice were euthanized at 6, 24, and 48 hours to collect draining lymph nodes.

[0277] (2) The Trizol method was used to extract RNA from the collected lymph nodes.

[0278] (3) The reverse transcription kit and the quantitative fluorescence kit were used to detect the dynamic changes in the level of each cytokine.

[0279] like Figure 2-8As shown, intramuscular injection of CF501 and SARS-CoV-2 RBD-Fc protein into mice strongly activated the innate immune response at hour 6, while RBD-Fc alone did not effectively activate the innate immune response. Although mice vaccinated with SARS-CoV-2 RBD-Fc protein mixed with CF501 exhibited strongly increased levels of various cytokines at hour 6, these levels tended to normalize by hour 48. This indicates that CF501 only transiently activates the innate immune response in mice and does not induce inflammation. The transient activation of the innate immune response was strong, but the compound did not sustainably upregulate cytokines, suggesting a superior effect on activating the innate immune response and that it is safe.

[0280] Example 2: Detection of antibody immune response in mice vaccinated with SARS-CoV-2 RBD-Fc protein using STING agonist as adjuvant.

[0281] 1. Experimental Materials

[0282] Six-week-old SPF Balb / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Aluminum adjuvant was purchased from Thermo Scientific. cGAMP was purchased from Invivogen.

[0283] 2. Experimental methods.

[0284] The vaccination schedule for mice is as follows: Figure 9 As shown.

[0285] The steps are as follows:

[0286] (1) Fifty-four mice were randomly divided into nine groups of six each.

[0287] (2) Mice in group 1 were injected intramuscularly with 5 μg of RBD-Fc protein. Mice in group 2 were injected intramuscularly with 5 μg of RBD-Fc protein and an equal volume of aluminum adjuvant. Mice in group 3 were injected intramuscularly with 5 μg of RBD-Fc protein and 20 μg of CF501 (RBD-Fc+CF501). Mice in group 4 were injected with 5 μg of RBD-Fc protein and 20 μg of the STING agonist CF502. Mice in group 5 were injected with 5 μg of RBD-Fc protein and 20 μg of the STING agonist CF508. Mice in group 6 were injected intramuscularly with 5 μg of RBD-Fc protein and 20 μg of the STING agonist CF510. Mice in group 7 were injected with 5 μg of RBD-Fc protein and 20 μg of the STING agonist CF512. Mice in group 8 were injected with 5 μg of RBD-Fc protein and 20 μg of cGAMP. Mice in group 9 were injected with an equal volume of PBS.

[0288] (3) A second booster immunization was administered 14 days after the first immunization. Blood was collected from mice on day 21 after the first immunization to obtain serum. A third booster immunization was administered 28 days after the first immunization. Blood was collected from mice on day 35 after the first immunization to obtain serum.

[0289] (4) The titer of RBD-specific antibodies in serum was detected using an ELISA method. Specifically, 1 μg / ml of RBD-His protein (Kactus Biosystems Co. Ltd., catalog number: COV-VM4BD) was coated onto an ELISA plate. After blocking the plate with 5% skim milk powder, serum was serially diluted 3 or 4 times and added to the ELISA plate, and incubated at 37°C for 30 minutes. After washing the plate 5 times with PBST, HRP-labeled rabbit anti-mouse IgG, HRP-labeled rabbit anti-mouse IgG1, and HRP-labeled rabbit anti-mouse IgG2a antibodies were added, and the plate was incubated at 37°C for 30 minutes. After washing the plate with PBST, the substrate TMB was added for color development for 15 minutes, and then H2SO4 was added to terminate the reaction. OD450 was detected using a microplate reader. The highest dilution with an OD450 greater than that of the blank control group (no serum but with added PBS) was defined as the serum antibody titer. The serum was initially diluted 100 times. If the antibody titer cannot be measured at a 100-fold dilution, set the serum antibody titer to 1:50.

[0290] like Figure 10-15As shown, mice in each group produced levels of RBD-specific antibodies on days 21 and 35. Total mouse IgG results on days 21 or 35 indicate that mice vaccinated with SARS-CoV-2 RBD-Fc protein mixed with CF501 or CF512 had the highest RBD-specific antibody titers, significantly higher than those produced by mice vaccinated with unadjuvanted RBD-Fc protein, mice vaccinated with SARS-CoV-2 RBD-Fc protein mixed with aluminum adjuvant, and mice vaccinated with SARS-CoV-2 RBD-Fc protein mixed with cGAMP (Tables 1 and 2). Mouse IgG1 results, consistent with the total IgG results, showed that mice vaccinated with SARS-CoV-2 RBD-Fc protein mixed with CF501 or CF512 produced the highest antibody titers (Tables 3 and 4). The results for mouse IgG2a showed that neither adjuvanted nor aluminum-adjuvanted RBD-Fc proteins effectively activated the production of RBD-specific IgG2a antibodies. The STING agonists CF501 and CF512 potently activated the production of RBD-specific IgG2a antibodies in vaccinated mice, significantly higher than in mice vaccinated with SARS-CoV-2 RBD-Fc protein mixed with cGAMP (Tables 5 and 6). IgG1 represents the TH2 immune response, and IgG2a represents the TH1 immune response. A TH1-type immune response is essential for the protective effect of vaccines. Therefore, the STING agonists CF501 and CF512 strongly activated and enhanced the TH1-type immune response.

[0291] Table 1. Serum titers of SARS-CoV-2RBD-specific IgG antibodies in mice on day 21 post-vaccination.

[0292]

[0293] The titers in the table are the mean ± standard error of the mean; significance analysis: one-way ANOVA compared to the CF501 treatment group (RBD-Fc+CF501). Values ​​in the following tables are expressed in the same manner.

[0294] Table 2. Serum titers of SARS-CoV-2RBD-specific IgG antibodies in mice on day 35 post-vaccination.

[0295]

[0296] Table 3. Serum titers of SARS-CoV-2RBD-specific IgG1 antibodies in mice on day 21 post-vaccination.

[0297]

[0298] Table 4. Serum titers of SARS-CoV-2RBD-specific IgG1 antibodies in mice on day 35 post-vaccination.

[0299]

[0300] Table 5. Serum titers of SARS-CoV-2RBD-specific IgG2a antibodies in mice on day 21 post-vaccination.

[0301]

[0302] Table 6. Serum titers of SARS-CoV-2RBD-specific IgG2a antibodies in mice on day 35 post-vaccination.

[0303]

[0304] Compared to aluminum adjuvants, the STING agonists CF501, CF502, CF508, CF510, or CF512 significantly enhanced TH1 and TH2 immune responses in mice. Compared to other adjuvants, CF501 significantly enhanced TH1 and TH2 immune responses in mice.

[0305] Example 3: Detection of cellular immune response in mice activated by CF501

[0306] 1. Materials

[0307] Six-week-old SPF Balb / c mice were purchased from Vital River Pharmaceuticals, Beijing. The ELISPOT kits targeting mouse IFNγ, TNFα, and IL-4 were purchased from Mabtech. The RBD full-length peptide library was synthesized by GL Biochem (Shanghai) Ltd. Mouse IL-2 was purchased from Beijing Dakewe.

[0308] 2. Experimental steps:

[0309] (1) Mice were vaccinated with the same vaccination dose and procedure as in Example 2.

[0310] (2) The mice were euthanized on day 7 after the third immunization in order to collect their spleen and lungs.

[0311] (3) Grind the spleen and lungs of mice to prepare spleen cell suspension and lung cell suspension.

[0312] (4) Use red blood cell lysis buffer to lyse red blood cells in cell suspension.

[0313] (5) After washing the cells with PBS several times, count the cells.

[0314] (6) Aseptically remove the ELISPOT plate, add RPMI 1640 medium with 10% FBS to the plate and block it at 37°C for 2 hours.

[0315] (7) Add 2×10 to each hole of the ELISPOT board 5 Each cell was used, and 2 μg / ml of the full-length RBD peptide library was added to each well simultaneously.

[0316] (8) Incubate the plate in a cell culture incubator at 37°C for 48 hours, remove the cell supernatant and wash the plate 5 times with PBS.

[0317] (9) Add biotinylated antibodies to IFNγ, TNFα and IL-4 respectively, and incubate the plate at room temperature for 2 hours.

[0318] (10) Wash the plate 5 times with PBS, add the ALP-conjugated avidin from the kit to the plate, and then place the plate at room temperature for 1 hour.

[0319] (11) Wash the plate 5 times with PBS and add the substrate from the kit for color development.

[0320] (12) Once obvious spots appear, discard the liquid and rinse the plate to terminate the reaction.

[0321] (13) Use an ELSPOT reader to count the spots.

[0322] The results are as follows Figure 16-21 As shown, CF501 strongly activates T-cell immune responses in mice. IFNγ and TNFα represent the TH1-type immune response, while IL-4 represents the TH2-type immune response. In the spleen cells of mice vaccinated with RBD-Fc protein and aluminum adjuvant, almost no IFNγ and TNFα were produced, but IL-4 was significantly produced, indicating that the aluminum adjuvant only activates the TH2 immune response. CF501 strongly activates the TH1 immune response, and the levels of IFNγ and TNFα produced in spleen and lung cells of mice vaccinated with RBD-Fc protein mixed with CF501 were significantly higher than those in mice vaccinated with RBD-Fc protein mixed with cGAMP. This indicates that CF501 is superior to cGAMP in activating the TH1-biased cellular immune response. Compared to other adjuvants, CF501 strongly activates the cellular immune response in mice.

[0323] Example 4: STING agonists can induce potent neutralizing antibody responses in mice.

[0324] 1. Detection of neutralizing antibody levels in mouse serum using a SARS-CoV-2 pseudovirus detection system.

[0325] (1) Generation of SARS-CoV-2 pseudovirus. HEK293T cells (purchased from the American Type Culture Collection, ATCC) were co-transfected with the constructed PcDNA3.1-SARS-CoV-2-S plasmid (provided by the BEI Resource Center, catalog number NR-52420) and the HIV backbone plasmid (pNL4-3.Luc.RE, from the NIH AIDS Reagent Program, catalog number: 3418). Cell supernatant (containing SARS-CoV-2 pseudovirus) was collected after 48 h.

[0326] (2) The serum collected from the vaccinated mice was inactivated at 56°C for 30 min.

[0327] (3) Huh-7 cells (obtained from ATCC) were introduced at a rate of 1 × 10⁶ cells per well. 4 The cells were seeded onto the plate.

[0328] (4) After 8 hours, the serum was diluted 3 or 4 times with DMEM, and then the same volume of SARS-CoV-2 pseudovirus was added. The mixture of serum and pseudovirus was incubated at 37°C for 30 minutes.

[0329] (5) Add the mixture of serum and pseudovirus to Huh-7 cells (1×10⁻⁶ cells). 4 (cells / well).

[0330] (6) Incubate the plate for 12 hours and replace the medium with fresh DMEM medium containing 2% FBS.

[0331] (7) 48 h after changing the culture medium, add lysis buffer (Promega, luciferase assay kit) and lyse for 30 min, then add luciferase substrate.

[0332] (8) Use a microplate reader to detect luciferase levels.

[0333] (9) Calculate the percentage of viral suppression and NT50 (serum dilution at which 50% of the virus is neutralized).

[0334] The serum was initially diluted at the highest dilution (100x). If the serum could not neutralize 50% of the virus when diluted 100x, the NT50 of the serum was set to 50 by default.

[0335] The neutralizing titer of serum collected on day 21 against SARS-CoV-2 pseudovirus was as follows: Figure 22 As shown in Table 7, mice vaccinated with RBD-Fc protein mixed with CF501 or CF512 had the highest neutralizing antibody titers, significantly higher than those of mice vaccinated with unadjuvanted RBD-Fc protein, mice vaccinated with RBD-Fc protein mixed with aluminum adjuvant, and mice vaccinated with RBD-Fc protein mixed with cGAMP. The neutralizing titers against SARS-CoV-2 pseudovirus in serum collected on day 35 are shown in Table 7. Figure 23 As shown in Table 8, the mean neutralizing titer in the serum of mice vaccinated with RBD-Fc protein mixed with CF501 reached 26730, while the mean neutralizing titer in the serum of mice vaccinated with RBD-Fc protein mixed with aluminum adjuvant was only 863. The neutralizing antibody titers in mice vaccinated with RBD-Fc protein mixed with adjuvant CF501 were also significantly higher than those in mice vaccinated with RBD-Fc protein mixed with cGAMP, indicating that CF501 strongly generates neutralizing antibodies when used as an adjuvant for RBD-Fc protein. The neutralizing antibody titers in these vaccinated mice showed a strong correlation with the titers of RBD-specific IgG antibodies, such as... Figure 24 and Figure 25 As shown in the image.

[0336] Table 7. Detection of neutralizing antibody titers against SARS-CoV-2 pseudovirus in mouse serum 21 days after vaccination

[0337]

[0338] Table 8. Detection of neutralizing antibody titers against SARS-CoV-2 pseudovirus in mouse serum 35 days after vaccination

[0339]

[0340] 2. Plaque reduction method for detecting the inhibitory activity of serum against live SARS-CoV-2 virus.

[0341] (1) Vero-E6 (obtained from ATCC) cells were seeded into 96-well plates with 15,000 cells per well and incubated.

[0342] (2) After 24 hours, the serum from each group of mice was mixed. The serum was then serially diluted with DMEM at a ratio of 3 or 4.

[0343] (3) Add approximately 30 PFU of SARS-CoV-2 (SH-01, operated in the P3 laboratory of Fudan University) and incubate with an equal volume of diluted serum for 30 min.

[0344] (4) Add the mixture to Vero-E6 cells (2 × 10⁻⁶). 4 (1 cell / well), incubate for 2 hours, then add 100 μl of carboxymethyl cellulose.

[0345] (5) Incubate the plate for 48 hours and remove the supernatant. Add 50 μl of 4% paraformaldehyde for fixation and add 50 μl of 1% crystal violet for staining.

[0346] (6) Rinse the plate with tap water and count the empty spots.

[0347] On day 21, the inhibitory activity of serum from vaccinated mice against live SARS-CoV-2 virus was as follows: Figure 26 As shown, the NT50 against live SARS-CoV-2 in mice vaccinated with RBD-Fc protein mixed with CF501 was 3411, while the NT50 in mice vaccinated with RBD-Fc protein mixed with aluminum adjuvant was only 513. The NT50 in mice vaccinated with RBD-Fc protein mixed with cGAMP was 2701.

[0348] On day 35, the inhibitory activity of serum from vaccinated mice against live SARS-CoV-2 virus was as follows: Figure 27 As shown, the NT50 against live SARS-CoV-2 in mice vaccinated with RBD-Fc protein mixed with CF501 was 17032, while the NT50 in mice vaccinated with RBD-Fc protein mixed with aluminum adjuvant was 1032. The NT50 in mice vaccinated with RBD-Fc protein mixed with cGAMP was 6898.

[0349] 3. An immunofluorescence method for detecting the inhibitory activity of serum from vaccinated mice against live SARS-CoV-2 virus.

[0350] (1) Vero-E6 cells were seeded into 96-well plates with 10,000 cells per well.

[0351] (2) After 24 hours, the mixed serum from each group was serially diluted 3 or 4 times with DMEM (the serum from each group of mice was mixed together).

[0352] (3) Add an equal volume (60 μl) of SARS-CoV-2 virus (1 × 10⁻⁶ μL) to the SARS-CoV-2 virus (1 × 10⁻⁶ μL). 5 PFU / ml) was mixed with diluted serum (60 μl) and incubated for 30 min, then added to the cells.

[0353] (4) After 48 hours, the cell supernatant was removed and 50 μl of 4% paraformaldehyde was added for fixation.

[0354] (5) Add 50 μl of 0.2% Tritium for perforation.

[0355] (6) Add rabbit anti-SARS-CoV-2N protein antibody (Sino biological, 1:4000 dilution) to the plate and incubate at 37°C for 1 h.

[0356] (7) After washing with PBST 5 times, add fluorescently labeled donkey anti-rabbit IgG antibody conjugated with fluorescein flour488 (Thermo, 1:3000 dilution) and incubate the plate for 1 hour.

[0357] (8) After washing the plate with PBST 5 times, take pictures using a fluorescence microscope.

[0358] The results are as follows Figure 28 and 29 As shown, on day 21, serum from mice vaccinated with RBD-Fc protein mixed with CF501 effectively inhibited SARS-CoV-2N protein expression at a 2700-fold dilution. On day 35, serum from mice vaccinated with RBD-Fc protein mixed with CF501 still effectively inhibited SARS-CoV-2N protein expression at a 25600-fold dilution.

[0359] 4. Inhibition of SARS-CoV-2S-mediated cell-cell fusion by serum

[0360] (1) Deploy HEK-293T cells (available from ATCC) into 6-well plates.

[0361] (2) After incubation for 24 hours, cells were transfected using Vigofect transfection reagent with the pAAV-SARS-CoV-2-S-GFP plasmid (obtained by inserting the SARS-CoV-2 S protein gene into the plasmid pAAV-IRES-EGFP; constructed by the inventor's laboratory). After transfection, the plasmid could express the SARS-CoV-2 S protein on the surface of HEK293T cells.

[0362] (3) 24 hours after transfection, the cells were digested after GFP fluorescence was fully expressed.

[0363] (4) Coat Huh-7 cells at 20,000 cells per well onto the plate and incubate for 1 day.

[0364] (5) Mix 60 μl of 293T cells expressing SARS-CoV-2S protein with an equal volume of serum of each dilution and incubate for 30 minutes, then add it to Huh-7 cells.

[0365] (6) After incubation for 6 hours, when the cells in the control wells (i.e., serum-free cell wells) showed obvious fusion, paraformaldehyde was added to terminate the fusion.

[0366] (7) Observe the fused cells under a fluorescence microscope.

[0367] The results are as follows Figure 30 As shown, when serum from mice vaccinated with RBD-Fc protein mixed with CF501 was diluted 900-fold, SARS-CoV-2 S protein-mediated cell-cell fusion was significantly inhibited. However, when diluted 900-fold, serum from mice vaccinated with RBD-Fc protein mixed with aluminum adjuvant and serum from mice vaccinated with RBD-Fc protein mixed with cGAMP did not effectively inhibit SARS-CoV-2 S protein-mediated cell-cell fusion, as was the case with serum from mice treated with PBS. All these results indicate that the STING agonists of this disclosure, especially CF501, can effectively activate both cellular and humoral immune responses in mice compared to aluminum adjuvant and cGAMP. When CF501 is used as an adjuvant for RBD-Fc protein, neutralizing antibody levels in vaccinated mice can be significantly increased, and a strong effect against SARS-CoV-2 infection can be observed. Compared to other adjuvants, the use of CF501 as an adjuvant for RBD-Fc protein strongly induces neutralizing antibodies in vaccinated mice.

[0368] Example 5: Evaluation of cross-neutralizing activity against SARS-related coronaviruses in the serum of mice vaccinated with RBD-Fc proteins containing different STING agonists.

[0369] 1. Detection of cross-reactivity of serum from mice in Example 2 to SARS-CoV RBD.

[0370] (1) The SARS-CoV RBD-His (Kactus Biosystems Co. Ltd, catalog number: COV-VM4BD) protein was coated onto an ELISA plate and incubated at 4°C overnight.

[0371] (2) Use PBS-blocked ELISA plates containing 5% skim milk powder.

[0372] (3) The mouse serum from Example 2 was serially diluted and added to an ELISA plate, and then the plate was incubated at 37°C for 30 min.

[0373] (4) Wash the ELISA plate 5 times with PBST and add HRP-labeled rabbit anti-mouse IgG secondary antibody (Dako, 1:2000 dilution).

[0374] (5) Incubate the plate at 37°C for 30 min and wash it 5 times with PBST.

[0375] (6) Add TMB substrate (Sigma) for color development, and then add H2SO4 to terminate the reaction.

[0376] (7) Use a microplate reader to detect OD450.

[0377] The highest dilution with an OD450 greater than that of the blank control group (no serum, but with added PBS) at OD450 × 2.1 is defined as the serum antibody titer. Serum is initially diluted 100-fold. If the OD450 at the 100-fold dilution is still not greater than 2.1 times the OD450 value of the blank control group, the serum antibody titer is set at 1:50.

[0378] The results are as follows Figure 31 As shown in Table 9, serum from mice vaccinated with SARS-CoV-2 RBD-Fc protein also demonstrated binding ability to the SARS-CoV RBD protein. In mice, the group vaccinated with RBD-Fc protein mixed with CF501 produced the most potent cross-binding antibodies. Furthermore, the cross-binding antibody titers in mice treated with both RBD-Fc protein and CF501 were significantly higher than those in mice vaccinated with RBD-Fc protein with aluminum adjuvant or cGAMP.

[0379] Table 9. Detection of cross-neutralizing antibodies against SARS-CoV RBD protein in serum from vaccinated mice on day 35 post-vaccination.

[0380]

[0381] 2. Detection of neutralizing activity against SARS-CoV pseudovirus in serum from vaccinated mice

[0382] (1) Generation of SARS-CoV pseudovirus. HEK-293T cells were co-transfected with plasmid PcDNA3.1-SARS-CoV-S (a gift from Dr. Lanying Du of the New York Blood Center, constructed and preserved in the inventor's laboratory) and HIV backbone plasmid PNL-4-3-luc (provided by the NIH AIDS Research and Reference Reagent Program, catalog number 3418, owned by the inventor's laboratory). Cell supernatant containing SARS-CoV pseudovirus was collected after 48 hours.

[0383] (2) Plate Huh-7 cells onto the plate, 10,000 cells per well.

[0384] (3) After incubation for 8 hours, the serum was diluted 3 times with DMEM, and then an equal volume of SARS-CoV pseudovirus was added and the mixture was incubated at 37°C for 30 minutes.

[0385] (4) A mixture of serum and SARS-CoV pseudovirus was added to Huh-7 cells (1×10⁻⁶ cells). 4 (Cells / well). After incubation for 12 hours, replace the medium with fresh DMEM medium.

[0386] (5) After incubation for 48 hours, the cells were lysed using the cell lysis buffer from the Promega luciferase kit, and the luciferase activity in the lysate was detected.

[0387] The results are as follows Figure 32 As shown in Table 10, sera from mice vaccinated with either SARS-CoV-2 RBD-Fc protein or SARS-CoV-2 RBD-Fc protein mixed with aluminum adjuvant showed almost no neutralizing activity against SARS-CoV pseudoviruses. Conversely, sera from mice vaccinated with SARS-CoV-2 RBD-Fc protein mixed with CF501 produced significant cross-neutralizing activity against SARS-CoV pseudoviruses. The mean neutralizing antibody titer in the CF501-treated mouse group was approximately 1000, significantly higher than the cross-neutralizing antibody titer produced in the cGAMP-treated mouse group. Furthermore, we found that the neutralizing antibody titer against SARS-CoV pseudovirus infection produced by these mice was correlated to some extent with the titer of SARS-CoV RBD-specific antibodies (…). Figure 33 ).

[0388] Table 10. Detection of neutralizing antibody titers against SARS-CoV in mouse serum on day 35 post-vaccination.

[0389]

[0390] 3. Detection of neutralizing activity of serum from vaccinated mice against bat-derived SARS-like viruses WIV1 and Rs3367

[0391] (1) Generation of WIV1 and Rs3367 pseudoviruses. HEK293T cells were co-transfected with plasmids PcDNA3.1-WIV1-S or PcDNA3.1-Rs3367-S (obtained by inserting the gene sequences of the S proteins of WIV1 and Rs3367 into the PcDNA3.1 vector, respectively; both plasmids were constructed by the inventors' laboratory) and the HIV backbone pNL4-3.Luc.RE (from the NIH AIDS Research and Reference Reagent Program, catalog number: 3418). After incubation for 48 h, the cell supernatant (containing WIV1 or Rs3367 pseudoviruses) was collected.

[0392] (2) Plate Huh-7 cells onto the plate, 10,000 cells per well.

[0393] (3) Serum was serially diluted 3-fold using DMEM, and then equal volumes of WIV1 pseudovirus and Rs3367 pseudovirus were added. After incubation at 37°C for 30 min, the serum and pseudovirus were added to Huh-7 cells (1×10⁻⁶ cells). 4 (cells / pores).

[0394] (4) After incubation for 12 hours, replace the medium with fresh DMEM medium.

[0395] (5) After incubation for 48 hours, the cells were lysed using the cell lysis buffer from the Promega luciferase kit, and the luciferase activity in the lysate was detected.

[0396] The results are as follows Figure 34 As shown in Tables 11-1 and 11-2, serum from mice vaccinated with SARS-CoV-2 RBD-Fc protein alone showed no inhibitory activity against WIV1 pseudovirus, and serum from mice vaccinated with SARS-CoV-2 RBD-Fc protein mixed with aluminum adjuvant showed only weak inhibitory activity against WIV1 pseudovirus, with a mean neutralizing antibody titer of 170. Serum from mice vaccinated with SARS-CoV-2 RBD-Fc protein mixed with CF501 showed strong cross-neutralizing activity against WIV1 pseudovirus, with a mean neutralizing titer of 838, which was significantly higher than the cross-neutralizing antibody titers produced by mice treated with cGAMP as an adjuvant.

[0397] The results regarding cross-inhibition activity against the Rs3367 pseudovirus are consistent with the trend observed against the WIV1 pseudovirus. Figure 35(See Table 11-2). Serum from mice vaccinated with SARS-CoV-2 RBD-Fc protein alone showed almost no detectable inhibitory activity against Rs3367 pseudovirus. Serum from mice vaccinated with SARS-CoV-2 RBD-Fc protein and aluminum adjuvant showed weak cross-neutralizing activity against Rs3367 pseudovirus, with a mean neutralizing antibody titer of 387. Serum from mice vaccinated with SARS-CoV-2 RBD-Fc protein mixed with CF501 showed strong cross-neutralizing activity against Rs3367 pseudovirus, with a mean neutralizing titer of 3308, which was also significantly higher than the cross-neutralizing antibody titers produced by mice treated with cGAMP. When aluminum adjuvant is used, broadly neutralizing antibodies against SARS-related viruses can be induced in vaccinated mice with little or no effect. Compared with aluminum adjuvant, the STING agonist CF501 can potently induce cross-neutralizing antibodies against SARS-related viruses in mice as an adjuvant.

[0398] Table 11-1. Detection of neutralizing antibody titers against SARS-related virus WIV1 pseudovirus in mouse serum on day 35 post-vaccination.

[0399]

[0400] Table 11-2. Detection of neutralizing antibody titers against SARS-related virus Rs3367 pseudovirus in mouse serum on day 35 post-vaccination.

[0401]

[0402]

[0403] Example 6: Challenge test of human ACE2 transgenic mice vaccinated with RBD-Fc protein mixed with the STING agonist CF501

[0404] 1. Materials: 8-week-old SPF ACE2 transgenic mice were purchased from Shanghai Model Organisms Center, Inc.

[0405] (1) Twelve ACE2 transgenic mice were randomly divided into two groups of six each.

[0406] (2) In Group 1, mice were intramuscularly injected with SARS-CoV-2 RBD-Fc protein and CF501 according to the vaccination dosage and vaccination protocol of Example 2. In Group 2, mice were given an equal volume of PBS.

[0407] (3) Two weeks after the third immunization, SARS-CoV-2 virus (1×10⁻⁶) was administered via nasal drops.6 Mice were attacked with PFU.

[0408] (4) Record the weight changes of the mice daily after the attack.

[0409] (5) On the 4th day after the attack, the mice were euthanized and their lungs, intestines and brains were removed.

[0410] (6) RNA was extracted from the tissue using Trizol (Takara), and the viral load in the mouse tissue was detected using the RT-qPCR detection kit (Takara).

[0411] The results are as follows Figure 36 As shown, no weight loss was observed in mice vaccinated with RBD-Fc protein mixed with CF501 after challenge. However, more severe weight loss was observed in mice treated with PBS. Regarding viral load in the lungs, significantly higher viral loads (10-1) were detected in the lungs of mice treated with PBS. 8 (Copies / ml). Conversely, in 6 mice treated with CF501, no viral load was detected in the lungs of 5 mice. The SARS-CoV-2N gene was detectable in only one lung, with a titer of 10. 4 ( Figure 37 Similarly, significantly higher viral loads, reaching 10, were detected in the brains of mice treated with PBS. 10 In contrast, mice vaccinated with STING agonist and RBD-Fc protein had a viral load of only about 10 in their brains. 3 ( Figure 38 In the intestines of mice, the viral load in the intestines of mice treated with PBS reached 10. 6 In six mice vaccinated with the STING agonist CF501 and RBD-Fc protein, the N gene of the virus was not detected in three mice, and low viral load was detected in the other three mice. Figure 39 ).

[0412] These results strongly suggest that vaccination of mice with CF501 and RBD-Fc proteins can effectively protect them from SARS-CoV-2 infection.

[0413] Example 7: Detection of immune response activated by STING agonist in a New Zealand white rabbit animal model

[0414] 1. Experimental Materials

[0415] The New Zealand White Rabbit was purchased from Shanghai Zeyu Biological Technology Co., Ltd.

[0416] 2. Experimental Methods

[0417] 2.1 The vaccination protocol for New Zealand white rabbits is shown in Figure 40 The steps are as follows.

[0418] (1) Divide 54 New Zealand white rabbits into 9 groups of 6 each.

[0419] (2) Group 1 was vaccinated with 10 μg of RBD-Fc protein. Group 2 was vaccinated with 10 μg of RBD-Fc protein and an equal volume of aluminum adjuvant. Group 3 was vaccinated with 10 μg of RBD-Fc protein and 40 μg of CF501. Group 4 was vaccinated with 10 μg of RBD-Fc protein and 40 μg of the STING agonist CF502. Group 5 was vaccinated with 10 μg of RBD-Fc protein and 40 μg of the STING agonist CF508. Group 6 was vaccinated with 10 μg of RBD-Fc protein and 40 μg of the STING agonist CF510. Group 7 was vaccinated with 10 μg of RBD-Fc protein and 40 μg of the STING agonist CF512. Group 8 was vaccinated with 10 μg of RBD-Fc protein and 40 μg of cGAMP. Group 9 was injected with an equal volume of PBS.

[0420] (3) New Zealand white rabbits were vaccinated on days 1, 14, 28 and 42, and rabbit serum was collected on days 21, 35 and 49.

[0421] 2.2 Evaluation of antibody immune response in New Zealand white rabbits after vaccination.

[0422] (1) The obtained serum was inactivated at 56℃ for 30 min.

[0423] (2) The SARS-CoV-2 RBD-His protein (Kactus Biosystems Co. Ltd, catalog number: COV-VM4BD) was coated on an ELISA plate and incubated overnight at 4°C.

[0424] (3) Use PBS containing 5% skim milk powder to seal the plate for 2 hours.

[0425] (4) Use PBST to dilute rabbit serum 3 or 4 times, and add the diluted serum to the ELISA plate. Incubate the ELISA plate at 37°C for 30 min.

[0426] (5) Wash the ELISA plate with PBST 5 times.

[0427] (6) Add HRP-labeled goat anti-rabbit IgG enzyme-labeled secondary antibody (Dako, 1:2000 dilution).

[0428] (7) Incubate the ELISA plate at 37°C for 30 min and wash it with PBST.

[0429] (8) Add TMB substrate (Sigma) for color development and add H2SO4 to terminate the reaction.

[0430] (9) OD450 was measured using a microplate reader. The highest dilution with an OD450 greater than 2.1 times that of the blank control group (no serum but with added PBS) was defined as the serum antibody titer. Serum was initially diluted 100-fold. If the OD450 at the 100-fold dilution was still not greater than 2.1 times that of the blank control group, the serum antibody titer was set to 1:50.

[0431] The results are as follows Figure 41 and 42 As shown in Tables 12 and 13. On day 21 post-vaccination, serum from rabbits vaccinated with RBD-Fc protein mixed with CF501 showed the highest antibody titers. The specific antibody titers against SARS-CoV-2 RBD produced by rabbits vaccinated with RBD-Fc protein mixed with CF501 were significantly higher than those in rabbit serum vaccinated with unadjuvanted RBD-Fc protein and rabbit serum vaccinated with RBD-Fc protein mixed with aluminum adjuvant or cGAMP.

[0432] On day 35 post-vaccination, rabbits vaccinated with RBD-Fc protein mixed with CF501 still exhibited the highest titers of SARS-CoV-2 RBD-specific antibodies, significantly higher than those vaccinated with RBD-Fc protein mixed with aluminum adjuvant or cGAMP. Notably, other STING agonists with structures similar to CF501, such as CF512, CF510, CF508, and CF502, failed to induce more potent binding antibodies relative to aluminum in rabbits, demonstrating that small structural changes can significantly affect adjuvant activity.

[0433] Table 12. Detection of specific antibody titers against SARS-CoV-2RBD in rabbit serum on day 21 post-immunization

[0434]

[0435] Table 13. Detection of specific antibody titers against SARS-CoV-2RBD in rabbit serum on day 35 post-immunization.

[0436]

[0437] Example 8: Detection of neutralizing antibodies against SARS-CoV-2 in serum from vaccinated rabbits

[0438] 1. A method for detecting neutralizing antibody levels in serum using SARS-CoV-2 pseudoviruses.

[0439] (1) The generation of SARS-CoV-2 pseudovirus is the same as in Example 4.

[0440] (2) Huh-7 cells were seeded onto the plate at a density of 10,000 cells per well.

[0441] (3) After incubation for 8 hours, the serum was continuously diluted with DMEM at 3 or 4 times, SARS-CoV-2 pseudovirus was added to the serum and incubated for 0.5 hours.

[0442] (4) Add the mixture of pseudovirus and serum into Huh-7 cells.

[0443] (5) After incubation for 12 hours, replace the medium with fresh DMEM medium.

[0444] (6) After incubation for 48 hours, the cells were lysed and the luciferase activity in the lysate was detected.

[0445] The serum is initially diluted 100-fold. If the serum cannot neutralize 50% of the pseudovirus when diluted 100-fold, the NT50 of the serum is set to 50 by default.

[0446] The results are as follows Figure 43 , Figure 44 As shown in Tables 14 and 15. Figure 43 The results show the neutralizing antibody titers against SARS-CoV-2 pseudovirus on day 21 post-vaccination in rabbits. The neutralizing activity of serum from vaccinated rabbits differed from that from vaccinated mice. It can be seen that serum from rabbits vaccinated with RBD-Fc protein mixed with aluminum adjuvant or cGAMP showed only low levels of neutralizing antibodies after two immunizations, while serum from rabbits vaccinated with RBD-Fc protein mixed with CF501 still showed high levels of neutralizing antibodies, indicating that the neutralizing antibody immune response was effectively activated in rabbits vaccinated with both RBD-Fc protein and CF501. Figure 44The titers of neutralizing antibodies in serum were shown on day 35 after the first immunization. It can be seen that the levels of neutralizing antibodies produced in rabbits vaccinated with RBD-Fc protein mixed with cGAMP or with unadjuvanted RBD-Fc protein were similar, indicating that cGAMP does not activate a neutralizing antibody immune response in vaccinated rabbits as strongly as it does in vaccinated mice. Rabbits vaccinated with RBD-Fc protein mixed with aluminum adjuvant produced some levels of neutralizing antibodies on day 35, but the titers of neutralizing antibodies produced in rabbits vaccinated with RBD-Fc protein mixed with CF501 were significantly higher than those in rabbits vaccinated with RBD-Fc protein mixed with aluminum adjuvant or other STING agonists. Notably, other STING agonists showing structural similarity to CF501, such as CF512, CF510, CF508, and CF502, failed to induce more potent neutralizing antibodies relative to aluminum in rabbits, demonstrating that small structural changes can significantly affect adjuvant effects. The levels of neutralizing antibodies in serum from vaccinated rabbits showed a strong correlation with RBD-specific IgG titers. Figure 45 and Figure 46 Rabbits vaccinated with RBD-Fc protein mixed with the STING agonist CF501 as an adjuvant produced the highest levels of neutralizing antibodies.

[0447] Table 14. Detection of neutralizing activity against SARS-CoV-2 pseudovirus in serum on day 21 post-vaccination in rabbits.

[0448]

[0449] Table 15. Detection of neutralizing activity against SARS-CoV-2 pseudovirus in serum on day 35 post-vaccination in rabbits.

[0450]

[0451] 2. Plaque reduction assay for detecting the inhibitory activity of serum from vaccinated rabbits against live SARS-CoV-2 virus.

[0452] (1) Vero-E6 cells were seeded into 96-well plates, 100 μl per well for a total of 15,000 cells.

[0453] (2) Dilute the serum 3 or 4 times sequentially and incubate it with about 30 PFU of live SARS-CoV-2 virus for 30 min.

[0454] (3) Add 100 μl of the mixture of virus and serum to the plated Vero-E6 cells and incubate for 2 h.

[0455] (4) Add 50 μl of carboxymethyl cellulose.

[0456] (5) After incubation for 48 hours, add 50 μl of paraformaldehyde for fixation. Add 50 μl of 1% crystal violet for staining.

[0457] (6) Count the empty spots.

[0458] The results are as follows Figure 47 and Figure 48 As shown. Figure 47 The inhibitory activity of rabbit serum against live SARS-CoV-2 virus was demonstrated on day 21 post-vaccination. The NT50 of rabbit serum against SARS-CoV-2 was 183 in rabbits vaccinated with RBD-Fc protein mixed with aluminum adjuvant, and 673 in rabbit serum vaccinated with RBD-Fc protein mixed with CF501. Figure 48 The study demonstrated the inhibitory activity of rabbit serum against live SARS-CoV-2 virus on day 35 post-primary immunization. The NT50 of serum from rabbits vaccinated with RBD-Fc protein mixed with aluminum adjuvant was 1172, while the NT50 of serum from rabbits vaccinated with RBD-Fc protein mixed with cGAMP was 393. The highest NT50 was 6485 from serum from rabbits vaccinated with RBD-Fc protein mixed with CF501.

[0459] 3. An immunofluorescence assay for detecting the inhibitory activity of serum from vaccinated rabbits against SARS-CoV-2 virus.

[0460] (1) Plate Vero-E6 cells into 96-well plates, 10,000 cells per well.

[0461] (2) Rabbit serum vaccinated with RBD-Fc vaccine containing a mixture of STING agonist 501 was diluted 3- or 4-fold with DMEM. The diluted serum was then mixed with an equal volume of SARS-CoV-2 (1 × 10⁻⁶). 5 The mixture (PFU / ml) was incubated at 37°C for 30 min and then added to Vero-E6 cells.

[0462] (3) After incubation for 48 hours, remove the cell supernatant and add 4% paraformaldehyde for fixation. Add 0.02% Triton for perforation.

[0463] (4) Add rabbit anti-SARS-CoV-2N protein antibody (Sino biological, 1:3000 dilution) and incubate the plate for 30 min.

[0464] (5) Add the goat anti-rabbit secondary antibody labeled with fluoresceinflour488 (Thermo, 1:3000 dilution) and incubate the plate for 30 min.

[0465] (6) Clean the plate with PBST and take pictures using a fluorescence inverted microscope.

[0466] The results are as follows Figure 49 and Figure 50 As shown, on day 21, serum from vaccinated rabbits at a 900-fold dilution significantly inhibited the expression of SARS-CoV-2N protein. On day 35, serum from vaccinated rabbits at a 600-fold dilution still effectively inhibited the expression of SARS-CoV-2N protein.

[0467] 4. Inhibition of SARS-CoV-2S-mediated cell-cell fusion by serum

[0468] (1) The PAAV-SARS-CoV-2-S plasmid was transfected into HEK-293T cells. After fluorescence appeared, the cells were 293T cells expressing SARS-CoV-2S fluorescent protein.

[0469] (2) After incubation for 24 hours, when the fluorescence becomes obvious, the cells are collected and used as effector cells.

[0470] (3) Rabbit serum was serially diluted 3-fold with DMEM and co-incubated with 293T cells expressing SARS-CoV-2S fluorescent protein.

[0471] (4) Add serum and effector cells to the plated Huh-7 cells.

[0472] (5) After incubation for 6 hours, when fusion cells are formed (in wells of 293T cells expressing SARS-CoV-2S protein without added serum), paraformaldehyde is added for fixation and to terminate the fusion reaction.

[0473] (6) Take pictures using a fluorescence inverted microscope.

[0474] The results are as follows Figure 51 As shown, serum from rabbits vaccinated with RBD-Fc protein mixed with CF501, even at a 900-fold dilution, effectively inhibited SARS-CoV-2S protein-mediated cell-cell fusion. However, serum from rabbits vaccinated with RBD-Fc protein mixed with aluminum adjuvant or cGAMP, as well as serum from control rabbits, did not effectively inhibit SARS-CoV-2S protein-mediated cell-cell fusion.

[0475] 5. Neutralizing activity against SARS-CoV-2 in serum from vaccinated rabbits after the fourth immunization.

[0476] Serum was collected one week after the fourth immunization of rabbits. The neutralizing activity of these sera against SARS-CoV-2 was evaluated using a pseudovirus detection system. Results are as follows: Figure 52As shown in Table 16, after four immunizations, rabbits vaccinated with RBD-Fc protein mixed with CF501 still produced the highest neutralizing antibody titers, and the neutralizing antibody levels in rabbits vaccinated with RBD-Fc protein mixed with CF501 were still significantly higher than those in rabbits vaccinated with RBD-Fc protein mixed with aluminum adjuvant or cGAMP. Plaque reduction assays were further used to detect the inhibition of these sera against live SARS-CoV-2 virus, showing that the NT50 of serum from rabbits vaccinated with RBD-Fc protein mixed with CF501 was 9720, while the NT50 of serum from rabbits vaccinated with RBD-Fc protein mixed with cGAMP was only 534. The NT50 of serum from rabbits vaccinated with RBD-Fc protein mixed with aluminum adjuvant was 2205 (…). Figure 53 ).

[0477] All these results indicate that using CF501 as an adjuvant for RBD-Fc protein vaccination in rabbits produces the most potent neutralizing antibody response. The immune response generated in rabbits vaccinated with RBD-Fc protein mixed with cGAMP was very weak. Although rabbits vaccinated with RBD-Fc protein mixed with aluminum adjuvant also produced some levels of neutralizing antibodies, these levels were significantly lower than those produced in rabbits vaccinated with RBD-Fc protein mixed with CF501. Therefore, neutralizing antibodies are most effectively generated in mice vaccinated with RBD-Fc protein mixed with CF501 and in rabbits vaccinated with RBD-Fc protein mixed with CF501 compared to mice or rabbits treated with other adjuvants.

[0478] Table 16. Detection of neutralizing activity against SARS-CoV-2 pseudovirus in serum on day 49 post-rabbit vaccination

[0479]

[0480] Example 9: Evaluation of the broad-spectrum antiviral properties of rabbit antiserum against SARS-related viruses

[0481] 1. Packaging of SARS-related pseudoviruses (SARS-CoV, WIV1, and Rs3367).

[0482] (1) HEK-293T cells were co-transfected with plasmids PCDNA-3.1-SARS-CoV-S, PCDN-3.1-WIV1-S or PCDNA-3.1-Rs3367-S (which were obtained by inserting the S gene sequence of SARS-CoV-2, WIV1 or Rs3367 into the PcDNA3.1 vector, respectively) and HIV backbone plasmid PNL-4-3-Luc.

[0483] (2) After incubation for 48 hours, collect the cell supernatant containing the corresponding pseudovirus.

[0484] 2. Evaluation of serum inhibition against SARS-CoV, WIV1, and Rs3367 pseudoviruses

[0485] (1) The serum of rabbits immunized 3 and 4 times was serially diluted 3 times with DMEM, and then incubated with an equal volume of the corresponding pseudovirus (SARS-CoV, WIV1 or Rs3367) at 37°C for 30 min.

[0486] (2) Add the mixture of pseudovirus and serum to Huh-7 cells that have been plated in the plate.

[0487] (3) After incubation for 12 hours, replace the culture medium with fresh DMEM.

[0488] (4) After incubation for 48 hours, the cells were lysed using the cell lysis solution from the Promega luciferase assay kit and the luciferase activity in the lysate was detected.

[0489] The results are as follows Figure 54 , Figure 55 As shown in Table 17. Figure 54 Results of neutralizing titers against SARS-CoV pseudovirus in rabbit serum on day 35 post-vaccination are shown. It can be seen that no cross-neutralization against SARS-CoV pseudovirus was detectable at the highest dilution (1:100) in serum from rabbits vaccinated with unadjuvanted RBD-Fc protein or with RBD-Fc protein mixed with cGAMP. Serum from rabbits vaccinated with RBD-Fc protein mixed with aluminum adjuvant showed a mean neutralizing titer of 137 against SARS-CoV pseudovirus. Serum from rabbits vaccinated with RBD-Fc protein mixed with the STING agonist CF501 showed the highest cross-neutralizing antibody titer against SARS-CoV pseudovirus at 502, which was significantly higher than the cross-neutralizing antibody titers in serum from rabbits vaccinated with RBD-Fc protein mixed with aluminum adjuvant or cGAMP. Figure 55Table 18 shows the results of neutralizing titers against WIV1 pseudovirus in rabbit serum on day 35 post-vaccination. It can be seen that serum from rabbits vaccinated with unadjuvanted RBD-Fc protein showed only very weak cross-neutralizing activity against WIV1 pseudovirus, with a mean neutralizing titer of 10⁵. Serum from rabbits vaccinated with RBD-Fc protein mixed with cGAMP also showed very weak cross-neutralizing activity, with a mean neutralizing titer of 10⁷. Serum from rabbits vaccinated with RBD-Fc protein mixed with aluminum adjuvant showed weak neutralizing activity against WIV1 pseudovirus, with a mean neutralizing titer of 280. Conversely, serum from rabbits vaccinated with RBD-Fc protein mixed with CF501 showed very high levels of cross-neutralizing antibodies, with a mean neutralizing antibody titer of 1567.

[0490] We further examined antibodies against SARS-CoV RBD on day 45 post-vaccination in rabbits and found that the level of SARS-CoV RBD-specific antibodies in the serum of rabbits vaccinated with RBD-Fc protein mixed with CF501 was significantly higher than that in the serum of rabbits vaccinated with RBD-Fc protein mixed with cGAMP and rabbits vaccinated with RBD-Fc protein mixed with aluminum adjuvant (Table 19). Figure 56 Using a pseudovirus detection system, serum from rabbits vaccinated with unadjuvanted RBD-Fc protein showed no neutralizing activity against SARS-CoV pseudovirus at a 1:100 dilution. Serum from rabbits vaccinated with RBD-Fc protein mixed with cGAMP showed weak cross-neutralizing activity, with an average neutralizing titer of 178. Serum from rabbits vaccinated with RBD-Fc protein mixed with aluminum adjuvant still showed weak cross-neutralizing antibody activity against SARS-CoV, with an average neutralizing titer of 242. Rabbits vaccinated with RBD-Fc protein mixed with CF501 produced a cross-neutralizing antibody titer reaching 1472. Figure 57 (and Table 20). This shows a certain correlation between SARS-CoV RBD-specific antibody titers and neutralizing antibody titers (and Table 20). Figure 58 On day 49 post-vaccination in rabbits, the serum neutralizing antibody titers against pseudoviruses WIV1 and Rs3367 were as follows: Figure 59As shown in Table 21. For the WIV1 pseudovirus, the neutralizing titer of serum from rabbits vaccinated with RBD-Fc protein mixed with CF501 was 1487, which was significantly higher than the neutralizing titers from serum from rabbits vaccinated with RBD-Fc protein mixed with cGAMP and RBD-Fc protein mixed with aluminum adjuvant. For the Rs3367 pseudovirus, the neutralizing titer of serum from rabbits vaccinated with RBD-Fc protein mixed with CF501 reached 20299, which was significantly higher than the neutralizing titers from serum from rabbits vaccinated with RBD-Fc protein mixed with cGAMP and RBD-Fc protein mixed with aluminum adjuvant. Figure 60 (and Table 22). When used as an adjuvant for a COVID-19 vaccine, CF501 can potently activate a broad-spectrum immune response against SARS-related viruses in vaccinated rabbits.

[0491] Table 17. Detection of neutralizing activity against SARS-CoV pseudovirus in serum on day 35 post-vaccination in rabbits.

[0492]

[0493] Table 18. Detection of neutralizing activity against SARS-CoV-related virus WIV1 pseudovirus in serum on day 35 post-vaccination in rabbits.

[0494]

[0495] Table 19. Titer of SARS-CoV RBD-specific antibodies in rabbit serum on day 49 post-vaccination.

[0496]

[0497] Table 20. Detection of neutralizing antibody titers against SARS-CoV pseudovirus in serum on day 49 post-rabbit vaccination

[0498]

[0499] Table 21. Detection of neutralizing antibody titers against SARS-CoV-related virus WIV1 pseudovirus in serum on day 49 post-vaccination in rabbits.

[0500]

[0501] Table 22. Detection of neutralizing antibody titers against SARS-CoV-related virus Rs3367 pseudovirus in serum on day 49 post-rabbit vaccination.

[0502]

[0503] 3. Detection of neutralizing activity of antiserum against SARS-CoV-2 mutant virus in rabbits after vaccination

[0504] Currently, SARS-CoV-2 continues to mutate. To verify whether antibodies produced in rabbits immunized with adjuvanted vaccines still possess strong neutralizing activity against current SARS-CoV-2 mutant viruses, we performed site-directed mutagenesis on wild-type SARS-CoV-2 plasmids. This generated more than 40 pseudoviruses of currently identified SARS-CoV-2 mutants. Neutralizing activity against these mutant viruses in rabbit serum was measured after four immunizations using RBD-Fc proteins mixed with aluminum adjuvant, cGAMP, or CF501. Results are as follows... Figure 61 As shown, serum from rabbits vaccinated with RBD-Fc protein mixed with CF501 exhibited the highest neutralizing activity against pseudoviruses of these 40 SARS-CoV-2 mutants, with NT50 values ​​between 3323 and 14188. Although serum from rabbits vaccinated with RBD-Fc protein mixed with aluminum adjuvant also showed neutralizing activity against these mutant strains, this activity was weaker than that of serum from rabbits vaccinated with RBD-Fc protein mixed with CF501. Serum from rabbits vaccinated with RBD-Fc protein mixed with cGAMP showed the weakest activity.

[0505] These results indicate that serum from rabbits vaccinated with RBD-Fc protein mixed with CF501 can exhibit broad-spectrum and potent neutralizing activity against SARS-CoV-2 mutants and SARS-related viruses.

[0506] Example 10: Comparison of immune responses in rhesus monkeys vaccinated with CF501 mixed with SARS-CoV-2 RBD-Fc protein or with aluminum adjuvant

[0507] 1. Materials

[0508] Nine two-year-old rhesus monkeys were purchased from Beijing Xierxin Biological Co., Ltd., including five females and four males.

[0509] 2. Evaluation of humoral immune response in rhesus monkeys after vaccination

[0510] 2.1 The vaccination schedule for rhesus monkeys is as follows: Figure 62 As shown, the specific steps are as follows:

[0511] (1) Nine rhesus monkeys were randomly divided into three groups of three.

[0512] (2) Group 1 rhesus monkeys were vaccinated intramuscularly with 100 μg of RBD-Fc protein and an equal volume of aluminum adjuvant.

[0513] (3) Group 2 rhesus monkeys were vaccinated intramuscularly with 100 μg of RBD-Fc protein and 400 μg of CF501.

[0514] (4) The third group of rhesus monkeys were vaccinated intramuscularly with an equal volume of PBS.

[0515] (5) Boost the immunization of rhesus monkeys on day 21.

[0516] (6) Antibody evaluation was performed on the collected rhesus monkey serum on days 14 and 28.

[0517] 2.2 Detection of SARS-CoV-2RBD-specific antibody titers in serum of vaccinated rhesus monkeys

[0518] (1) Coat SARS-CoV-2RBD onto ELISA plates and incubate overnight at 4°C.

[0519] (2) Add PBS containing 5% skim milk powder to the plate at 37°C and seal for 2 hours.

[0520] (3) Use PBST to continuously dilute the serum 3 or 4 times, and add the diluted serum to the ELISA plate. Incubate the plate at 37°C for 30 min.

[0521] (4) Add HRP-labeled goat anti-monkey IgG enzyme-labeled secondary antibody (Abcam, 1:10000 dilution) to the plate and incubate the plate at 37°C for 30 min.

[0522] (5) After washing the plate with PBST 5 times, add TMB substrate for color development, and then add H2SO4 to terminate the reaction.

[0523] (6) Read OD450 in the microplate reader.

[0524] The results are as follows Figures 63-66As shown in Tables 23 and 24, after the first vaccination, the serum titer of SARS-CoV-2 RBD-specific antibodies in rhesus monkeys vaccinated with RBD-Fc protein mixed with CF501 reached 218,776, significantly higher than that in rhesus monkeys vaccinated with RBD-Fc protein mixed with aluminum adjuvant and those vaccinated with PBS. After the second vaccination, the serum titer of SARS-CoV-2 RBD-specific antibodies in rhesus monkeys vaccinated with RBD-Fc protein mixed with CF501 reached 4,130,475, also significantly higher than that in rhesus monkeys vaccinated with RBD-Fc protein mixed with aluminum adjuvant and those vaccinated with PBS. This indicates that CF501 can still strongly activate humoral immune responses in primates as it does in mice and rabbits. Compared to aluminum adjuvant, when CF501 is used as an adjuvant for a COVID-19 vaccine, it can generate a potent antibody humoral immune response in vaccinated monkeys.

[0525] Table 23. Detection of SARS-CoV-2RBD-specific antibodies in serum of rhesus monkeys on day 14 after vaccination.

[0526]

[0527] Table 24. Detection of SARS-CoV-2RBD-specific antibodies in serum of rhesus monkeys on day 28 after vaccination.

[0528]

[0529] Example 11: Evaluation of cellular immune response in vaccinated rhesus monkeys

[0530] (1) Whole blood of rhesus monkeys was collected during week 1 of the first and second vaccinations with RBD-Fc protein mixed with aluminum adjuvant or CF501 or with PBS.

[0531] (2) PBMCs were isolated from whole blood.

[0532] (3) The ELISPOT kit was used to detect the cellular immune response in rhesus monkeys.

[0533] The results are as follows Figure 67As shown, after the first vaccination of rhesus monkeys, CF501 significantly activated the cellular immune response, and the IFN-γ levels produced by rhesus monkeys vaccinated with RBD-Fc protein mixed with CF501 were significantly higher than those produced by rhesus monkeys vaccinated with RBD-Fc protein mixed with aluminum adjuvant and those vaccinated with PBS. Aluminum adjuvant was not effective in activating the cellular immune response in vaccinated rhesus monkeys. After the second vaccination of rhesus monkeys, CF501 further activated the cellular immune response in vaccinated monkeys, while aluminum adjuvant was still not effective in activating the cellular immune response in vaccinated monkeys. Figure 68 This indicates that CF501 can effectively activate humoral and cellular immune responses in rhesus monkeys after a single vaccination. Compared to aluminum adjuvants, when CF501 is used as an adjuvant for a COVID-19 vaccine to immunize monkeys, it produces a stronger cellular immune response in the vaccinated monkeys.

[0534] Example 12: Detection of neutralizing antibody titers in the serum of vaccinated rhesus monkeys

[0535] 1. Detection of neutralizing antibody titers against SARS-CoV-2 in serum of vaccinated rhesus monkeys using SARS-CoV-2 pseudovirus.

[0536] (1) The generation of SARS-CoV-2 pseudovirus is the same as in Example 4.

[0537] (2) Huh-7 cells were seeded onto the plate at a density of 10,000 cells per well.

[0538] (3) After incubation for 8 hours, the serum was diluted 3 or 4 times with DMEM and an equal volume of SARS-CoV-2 pseudovirus was added. The mixture of pseudovirus and serum was incubated for 0.5 hours.

[0539] (4) Add a total of 100 μl of the mixture of pseudovirus and serum to the plated Huh-7 cells.

[0540] (5) After incubation for 12 hours, replace the medium with fresh DMEM medium.

[0541] (6) After incubation for 48 hours, the cells were lysed using the cell lysis solution in the Promega luciferase assay kit, and the luciferase activity in the lysate was detected.

[0542] The results are as follows Figure 69As shown. After the first vaccination of rhesus monkeys, the serum of three rhesus monkeys vaccinated with RBD-Fc protein mixed with CF501 showed NT50 values ​​against SARS-CoV-2 pseudovirus of 1494, 3281, and 262, respectively, while the serum of rhesus monkeys vaccinated with RBD-Fc protein mixed with aluminum adjuvant showed NT50 values ​​against SARS-CoV-2 pseudovirus of 333, 314, and 150, respectively. After the second vaccination of rhesus monkeys, the serum of three rhesus monkeys vaccinated with RBD-Fc protein mixed with CF501 showed NT50 values ​​against SARS-CoV-2 pseudovirus of 19949, 26031, and 9746, respectively. The serum of three rhesus monkeys vaccinated with RBD-Fc protein mixed with aluminum adjuvant showed NT50 values ​​against SARS-CoV-2 pseudovirus of 3268, 1889, and 2744, respectively. Figure 70 We found that the level of SARS-CoV-2RBD-specific antibodies in serum on day 28 showed a very high correlation with the level of neutralizing antibodies. Figure 71 ).

[0543] 2. Plaque reduction assay for detecting the inhibitory activity of rhesus monkey serum against live SARS-CoV-2 virus.

[0544] (1) Vero-E6 cells were seeded into 96-well plates with 15,000 cells per well.

[0545] (2) Serum was serially diluted 4-fold with DMEM and incubated with approximately 30 PFU of live SARS-CoV-2 virus for 30 min.

[0546] (3) Add the mixture to Vero-E6 cells.

[0547] (4) After incubating for 2 hours, add 50 μl of carboxymethyl cellulose.

[0548] (5) After incubation for 48 hours, add 50 μl of paraformaldehyde for fixation. Add 50 μl of 1% crystal violet for staining.

[0549] (6) Count the empty spots.

[0550] The results are as follows Figure 72As shown in the figure, the results of the plaque reduction test showed that the serum of three rhesus monkeys vaccinated with RBD-Fc protein mixed with CF501 showed NT50 values ​​of 55948, 67654, and 21569 against live SARS-CoV-2 virus, respectively. The serum of three rhesus monkeys vaccinated with RBD-Fc protein mixed with aluminum adjuvant showed NT50 values ​​of 1843, 1553, and 2200 against live SARS-CoV-2 virus, respectively. These results indicate that when CF501 is used as an adjuvant for RBD-Fc protein, high levels of neutralizing antibodies can be induced, and the levels of neutralizing antibodies in the serum of rhesus monkeys vaccinated with RBD-Fc protein mixed with CF501 are tens of times higher than those in the serum of rhesus monkeys vaccinated with RBD-Fc protein mixed with aluminum adjuvant. Compared with aluminum adjuvant, when CF501 is used as an adjuvant for COVID-19 vaccines, a more potent neutralizing antibody immune response is generated in vaccinated monkeys.

[0551] Following vaccination, the titers of neutralizing antibodies in the serum of rhesus monkeys were continuously monitored. It was found that in the serum of rhesus monkeys immunized with CF501 / RBD-Fc, the neutralizing antibody titer against live SARS-CoV-2 virus remained at 4696 mmol / L 113 days after the first vaccination. Conversely, in the serum of rhesus monkeys immunized with aluminum / RBD-FC, the neutralizing antibody titer against live SARS-CoV-2 virus was only 439 mmol / L 113 days after the first vaccination. Figure 73 ).

[0552] Rhesus monkeys were vaccinated a third time 115 days after their first vaccination, and then the titer of neutralizing antibodies against live SARS-CoV-2 virus in their serum was measured 122 days after the first vaccination. The titer of neutralizing antibodies against live SARS-CoV-2 virus in the serum of rhesus monkeys vaccinated with RBD-Fc protein mixed with CF501 reached 134,827. Conversely, the titer of neutralizing antibodies against live SARS-CoV-2 virus in the serum of rhesus monkeys vaccinated with RBD-Fc protein mixed with aluminum adjuvant reached 9,771. The titer of neutralizing antibodies in the serum was continuously monitored for 191 days after the first vaccination. Even on day 191 after vaccination with RBD-Fc protein mixed with CF501, the titer of neutralizing antibodies against live SARS-CoV-2 virus in the serum remained at 39,746. Conversely, after vaccinating rhesus monkeys with RBD-Fc protein mixed with aluminum adjuvant, the serum neutralizing antibody titer against live SARS-CoV-2 virus was 2153 ( Figure 73 These data strongly demonstrate the robust and durable immune protection produced by the RBD-Fc protein mixed with CF501.

[0553] Example 13: Evaluation of neutralizing activity against SARS-CoV-2 variants in serum of rhesus monkeys after vaccination

[0554] Pseudoviruses of SARS-CoV-2 variants or mutants were prepared as described in Example 9. Neutralizing activity against nine SARS-CoV-2 variants and 41 SARS-CoV-2 single-point mutants was detected in serum 28 days after vaccination in rhesus monkeys (7 days after the second vaccination). Results are as follows: Figure 74 As shown. Serum from rhesus macaques immunized with RBD-Fc protein mixed with CF501 effectively neutralized nine variants 28 days post-vaccination. These nine variants included Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Iota, and Kappa. The neutralizing titers against these mutants in serum from rhesus macaques immunized with RBD-Fc protein mixed with CF501 ranged from 29,584 to 123,589. In contrast, the neutralizing titers against these mutants in serum from rhesus macaques immunized with RBD-Fc protein mixed with aluminum adjuvant ranged only from 242 to 2016. Serum from rhesus macaques immunized with RBD-Fc protein mixed with CF501 also effectively neutralized pseudoviruses of 41 SARS-CoV-2 mutants with single-point mutations. Figure 74 (Table 25).

[0555] Table 25: Neutralizing antibody titers against SARS-CoV-2 mutant pseudoviruses in serum 28 days after rhesus monkeys were vaccinated with RBD-Fc protein mixed with CF501, RBD-Fc protein mixed with aluminum adjuvant, and PBS.

[0556]

[0557]

[0558] Recently, the Omeprone variant has become the predominant circulating strain. Therefore, the binding capacity to the Omeprone pseudovirus and the titer of neutralizing antibodies in the serum of rhesus monkeys were also tested 28 to 191 days post-vaccination. The results are as follows... Figure 75 and Figure 76As shown, serum from rhesus macaques vaccinated with RBD-Fc protein mixed with CF501 still effectively produced antibodies specifically binding to the RBD of Omeprone and neutralizing antibodies against Omeprone pseudovirus. Twenty-eight days after vaccination with RBD-Fc protein mixed with CF501, the serum neutralizing antibody titer against Omeprone pseudovirus reached 6468. Conversely, for rhesus macaques vaccinated with RBD-Fc protein mixed with aluminum adjuvant, the serum neutralizing antibody titer against Omeprone pseudovirus was only 208. Seven days after the third vaccination of rhesus macaques with RBD-Fc protein mixed with CF501 (122 days after the first vaccination), the serum neutralizing antibody titer against Omeprone pseudovirus was 35066. However, 122 days after the first vaccination with RBD-Fc protein mixed with aluminum adjuvant, the serum neutralizing antibody titer against Omeprone pseudovirus was 2602. Furthermore, from 28 to 191 days after the first vaccination, the serum neutralizing antibody titer against Omeprone pseudovirus in rhesus macaques vaccinated with RBD-Fc protein mixed with CF501 was significantly higher than that in rhesus macaques vaccinated with RBD-Fc protein mixed with aluminum adjuvant.

[0559] The neutralizing activity of live Omeprone virus in the serum of rhesus monkeys was also tested 122 days after the first vaccination. The results were as follows: Figure 77 As shown, 122 days after vaccination with RBD-Fc protein mixed with CF501, the serum neutralizing antibody titer against live Omega virus was 9322. 122 days after vaccination with RBD-Fc protein mixed with aluminum adjuvant, the serum neutralizing antibody titer against live Omega virus was only 615. These results demonstrate that vaccination with RBD-Fc protein mixed with CF501 strongly induces neutralizing antibodies against Omega virus.

[0560] Example 14: Evaluation of the broad-spectrum neutralizing activity of serum from vaccinated rhesus monkeys against SARS-related viruses.

[0561] 1. Packaging of SARS-related pseudoviruses (SARS-CoV, WIV1, and rs3367).

[0562] (1) HEK-293T cells were co-transfected with PCDNA-3.1-SARS-CoV-S, PCDN-3.1-WIV1-S or PCDNA-3.1-Rs3367-S plasmids and HIV backbone plasmid PNL-4-3-Luc plasmid (see above).

[0563] (2) After incubation for 48 hours, collect the cell supernatant containing the corresponding pseudovirus.

[0564] 2. Evaluation of serum inhibition against SARS-CoV, WIV1, and Rs3367 pseudoviruses

[0565] (1) Dilute rhesus monkey serum three times serially, and then add the corresponding pseudovirus (SARS-CoV, WIV1 or Rs3367). Incubate the mixture at 37°C for 30 minutes.

[0566] (2) Add the mixture to the Huh-7 cells that have been plated into the plate.

[0567] (3) After incubation for 12 hours, replace the culture medium with fresh DMEM.

[0568] (4) After incubation for 48 hours, the cells were lysed using the cell lysis solution in the Promega luciferase detection kit, and the luciferase activity in the lysate was detected.

[0569] The results are as follows Figure 78 As shown, this study demonstrates the neutralizing activity of serum from vaccinated rhesus macaques against SARS-CoV pseudovirus. It can be seen that serum from three rhesus macaques vaccinated with RBD-Fc protein mixed with CF501 showed NT50 values ​​of 5430, 4060, and 2243 against SARS-CoV pseudovirus, respectively, while serum from three rhesus macaques vaccinated with RBD-Fc protein mixed with aluminum adjuvant showed NT50 values ​​of 818, 2002, and 1189 against SARS-CoV pseudovirus, respectively. SARS-CoV-2 RBD-specific antibodies in the serum of these nine rhesus macaques showed a strong correlation with SARS-CoV neutralizing antibody titers. Figure 79 ). Figure 80 The study demonstrated the neutralizing activity of serum from vaccinated rhesus macaques against the WIV1 pseudovirus. It was observed that serum from three rhesus macaques vaccinated with RBD-Fc protein mixed with CF501 showed NT50 values ​​of 11972, 12582, and 5950 against the SARS-CoV WIV1 pseudovirus, respectively, while serum from three rhesus macaques vaccinated with RBD-Fc protein mixed with aluminum adjuvant showed NT50 values ​​of 1233, 3185, and 1280 against the SARS-CoV WIV1 pseudovirus, respectively. SARS-CoV-2 RBD-specific antibodies in the serum of these nine rhesus macaques showed a strong correlation with the neutralizing antibody titer against the WIV1 pseudovirus. Figure 81 ).

[0570] Figure 82The study demonstrated the neutralizing activity of serum from vaccinated rhesus macaques against the Rs3367 pseudovirus. It was observed that serum from three rhesus macaques vaccinated with RBD-Fc protein mixed with CF501 showed NT50 values ​​of 4729, 2921, and 1267 against the SARS-CoV pseudovirus, respectively, while serum from three rhesus macaques vaccinated with RBD-Fc protein mixed with aluminum adjuvant showed NT50 values ​​of 231, 458, and 374 against the SARS-CoV pseudovirus, respectively. SARS-CoV-2 RBD-specific antibodies in the serum of these nine rhesus macaques showed a strong correlation with the neutralizing antibody titer against Rs3367. Figure 83 Compared with aluminum adjuvants, CF501 produced a more potent, broadly neutralizing antibody immune response in vaccinated monkeys when used as an adjuvant for a COVID-19 vaccine.

[0571] Example 15: Determining the protective effect of vaccination of rhesus monkeys with RBD Fc protein containing Sting agonist CF501 after SARS-CoV-2 challenge test.

[0572] (1) Rhesus monkeys were challenged with SARS-CoV-2 223 days after their first vaccination. In short, 1 ml of SARS-CoV-2 / WH-09 / human / 2020 / CHN was administered at 10... 6 A concentration of TCID50 / ml was obtained by infecting rhesus monkeys with nasal drops.

[0573] (2) Nasal swabs were collected 3, 5 and 7 days after infection of rhesus monkeys.

[0574] (3) Seven days after infection, the rhesus monkeys were euthanized and their lungs, nasal turbinates and nasal mucosa were collected.

[0575] (4) RNA was extracted from the tissues using Trizol (Takara), and the viral load in the rhesus monkey tissues was detected using an RT-qPCR detection kit (Takara).

[0576] The results of viral load in nasal swabs are as follows Figure 84 , 85As shown in Tables 86 and 26, the viral load was higher in nasal swabs from rhesus monkeys vaccinated with PBS at days 3, 5, and 7 post-infection. In one of the rhesus monkeys vaccinated with RBD-Fc protein mixed with CF501, only low levels of SARS-CoV-2 RNA were detected in nasal swabs at day 3 post-infection, and were undetectable at days 5 and 7. SARS-CoV-2 RNA was detected in nasal swabs from other rhesus monkeys vaccinated with RBD-Fc protein mixed with CF501 at days 3, 5, and 7 post-infection, but the levels were significantly lower than those in the PBS-vaccinated monkeys. At days 3, 5, and 7 post-infection, the copy numbers of SARS-CoV-2 RNA in nasal swabs from rhesus monkeys vaccinated with RBD-Fc protein mixed with aluminum adjuvant were similar to those in the PBS-vaccinated monkeys.

[0577] The results of viral load in the lungs of rhesus monkeys are as follows Figure 87 As shown, high copy numbers of SARS-CoV-2 RNA were detected in the upper left, middle left, lower left, upper right, middle right, lower right, and accessory lobes of the lungs of rhesus monkeys vaccinated with PBS or with RBD-Fc protein mixed with aluminum adjuvant. In one of the rhesus monkeys vaccinated with RBD-Fc protein mixed with CF501, SARS-CoV-2 RNA was undetectable in all lung lobes. In the other rhesus monkeys vaccinated with RBD-Fc protein mixed with CF501, low copy numbers of SARS-CoV-2 RNA were detected in the lower right lung. This indicates that vaccination with RBD-Fc protein mixed with CF501 reduces SARS-CoV-2 infection in the lungs compared to vaccination with PBS or with RBD-Fc protein mixed with aluminum adjuvant.

[0578] Viral load in the nasal mucosa and turbinates of rhesus monkeys as follows Figure 88 and 89 As shown in Tables 27 and 28, the viral load in the nasal turbinates and nasal mucosa of rhesus monkeys vaccinated with RBD-Fc protein mixed with CF501 was significantly lower than that in rhesus monkeys vaccinated with PBS.

[0579] Table 26: Viral load in rhesus monkey nasal swabs at 3, 5 and 7 days after challenge with SARS-CoV-2

[0580]

[0581] Table 27: Viral load in the nasal turbinates of rhesus monkeys 7 days after challenge with SARS-CoV-2

[0582]

[0583] Table 28: Viral load in the nasal mucosa of rhesus monkeys 7 days after challenge with SARS-CoV-2

[0584]

[0585] Example 16: CF501 can enhance the immune response to HIV NHR trimer.

[0586] Balb / C mice were divided into two groups. The first group was vaccinated with HIV NHR trimer (N3G) (from Wang Chao of the Academy of Military Medical Sciences), and the second group was vaccinated with both HIV NHR trimer (N3G) and CF501. Mice were boosted with a vaccine 14 days after the first vaccination. Serum was collected from mice 7 days after the second vaccination. Specific antibodies against HIV-NHR trimer in the mice were tested using an ELISA assay.

[0587] The results are as follows Figure 90 As shown, CF501 can still be used as an adjuvant for HIV NHR trimers and can significantly enhance antibody immune responses in vaccinated mice. CF501 can not only be used in COVID-19 vaccines, but can also effectively enhance immune responses to HIV peptide antigens.

[0588] Example 17: CF501 can enhance the immune response to inactivated quadrivalent influenza virus vaccine.

[0589] 1. Balb / c mice were divided into three groups of six each. Group 1 was vaccinated only with an inactivated quadrivalent influenza virus vaccine (Hualan Bio Co., Ltd.) (containing influenza subtypes H1N1, H3N2, B / Yamagata, and B / Victoria). Group 2 was vaccinated with an aluminum adjuvant and an inactivated quadrivalent influenza virus vaccine. Group 3 was vaccinated with CF501 and an inactivated quadrivalent influenza virus vaccine. Serum was collected 14 days after vaccination. A second booster vaccination was administered 21 days after the first vaccination, and serum was collected 28 days after the first vaccination.

[0590] 2. Evaluation of influenza virus HA-specific antibody titers in the serum of vaccinated mice

[0591] 1) The HA proteins of four influenza virus subtypes (H1N1, H3N2, B / Yamagata and B / Victoria, purchased from Sino Biological Inc.) corresponding to the virus subtypes in the vaccine were coated onto ELISA plates.

[0592] 2) Use PBST to serially dilute mouse serum 3 or 5 times, and then add the diluted serum to the coated ELISA plate.

[0593] 3) After incubating at 37°C for 1 hour, wash the plate 5 times with PBST and add rabbit anti-mouse HRP secondary antibody (Dako, 1:2000 dilution).

[0594] 4) After incubating at 37°C for 1 hour, wash the plate 5 times with PBST, add TMB (Sigma) substrate for color development, and add H2SO4 to terminate the reaction. Read the OD450 on a microplate reader.

[0595] The results are as follows Figures 91-102 As shown in Tables 29-36. Among the three groups of mice, the highest antibody titers against the HA protein of the four influenza virus subtypes were observed in the serum of mice vaccinated with the tetravalent inactivated influenza virus vaccine and CF501 vaccine on both day 14 and day 28. These titers were significantly higher than those in the serum of mice vaccinated with the unadjuvanted tetravalent inactivated influenza virus vaccine and mice vaccinated with the tetravalent inactivated influenza virus vaccine and aluminum adjuvant vaccine.

[0596] Table 29. Antibody titers against H1N1 subtype HA protein in serum of mice 14 days after vaccination with tetravalent inactivated influenza vaccine.

[0597]

[0598] Table 30. Antibody titers against H3N2 subtype HA protein in serum of mice on day 14 after vaccination with tetravalent inactivated influenza vaccine.

[0599]

[0600] Table 31. Antibody titers against HA protein of B / Yamagata subtype in mouse serum on day 14 after vaccination with tetravalent inactivated influenza vaccine.

[0601]

[0602] Table 32. Antibody titers against HA protein of B / Victoria subtype in mouse serum on day 14 after vaccination with tetravalent inactivated influenza vaccine.

[0603]

[0604]

[0605] Table 33. Antibody titers against H1N1 subtype HA protein in serum of mice 28 days after vaccination with tetravalent inactivated influenza vaccine.

[0606]

[0607] Table 34. Antibody titers against H3N2 subtype HA protein in mouse serum on day 28 after vaccination with tetravalent inactivated influenza vaccine.

[0608]

[0609] Table 35. Antibody titers against HA protein of B / Yamagata subtype in mouse serum on day 28 after vaccination with tetravalent inactivated influenza vaccine.

[0610]

[0611] Table 36. Antibody titers against HA protein of B / Victoria subtype in mouse serum on day 28 after vaccination with tetravalent inactivated influenza vaccine.

[0612]

[0613] Example 18: Sting agonist CF501 can enhance the immune response to inactivated varicella-zoster virus (VZV) vaccine.

[0614] Materials: VZV inactivated vaccine can be purchased from Sinovac (Dalian) Biotech Ltd.

[0615] 1) Divide the mice into two groups of 6 each.

[0616] 2) The first group of mice were intramuscularly vaccinated with 20 μg of CF501 and VZV inactivated vaccine containing 10 ng gE protein.

[0617] 3) The second group of mice were intramuscularly vaccinated with 200 μg of aluminum adjuvant and VZV inactivated vaccine containing 10 ng gE protein.

[0618] 4) Mice were vaccinated on days 0, 14 and 28, and serum from mice was collected on days 21 and 35.

[0619] 5) On days 21 and 35, ELISA was used to determine the level of antibodies in mouse serum that specifically bind to VZV gE protein.

[0620] Specifically, the ELISA test is performed as follows.

[0621] A. Coat each well of the ELISA plate with 50 μl of gE protein at a concentration of 1 μg / ml, and incubate the plate overnight at 4°C. Block the plate with PBS containing 5% BSA at 37°C for 2 hours.

[0622] C. Initially, mouse serum was diluted 100-fold, then serially diluted 10-fold and added to ELISA plates. The plates were incubated at 37°C for 45 min.

[0623] D. After washing the wells of the plate five times with PBST, add HRP-labeled rabbit anti-mouse IgG and incubate the plate at 37°C for 45 min.

[0624] E. After washing the wells of the plate five times with PBST, add TMB substrate and develop for 15 min. Add H2SO4 to stop the development.

[0625] F. OD450 was measured using a microplate reader.

[0626] The results are as follows Figure 103 and 104 As shown, compared to VZV inactivated vaccines using aluminum adjuvants, VZV inactivated vaccines using CF501 as an adjuvant elicited a stronger antibody immune response in mice. These results strongly suggest that CF501 can be used as a universal adjuvant to stimulate a stronger immune response against different viral subunits and inactivated vaccines.

Claims

1. Use of compounds of the following formula or pharmaceutically acceptable salts thereof in the preparation of adjuvants for use in vaccines: ， The vaccine contains an antigen, which is one or more of the following: SARS-CoV, varicella-zoster virus, HIV NHR trimer, SARS-CoV-2, and influenza subtypes H1N1, H3N2, B / Yamagata, and B / Victoria.

2. The use of claim 1, wherein the compound or a pharmaceutically acceptable salt thereof is used in combination with at least one of a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, and a pharmaceutically acceptable diluent.

3. The use of claim 1, wherein the antigen is an antigen derived from the SARS-CoV-2 Omecron mutant.

4. The use of claim 1, wherein the antigen is SARS-CoV-2 RBD-Fc protein or gE protein of varicella-zoster virus.

5. The use of claim 1, wherein the vaccine is an inactivated vaccine, a live attenuated vaccine, a subunit vaccine, or a nucleic acid vaccine.

6. The use of claim 5, wherein the nucleic acid vaccine is an mRNA vaccine or a DNA vaccine.

7. The use of claim 1, wherein the vaccine is an intramuscular vaccine, an intradermal vaccine, or an inhaled vaccine.

8. A vaccine, said vaccine comprising: (1) A compound of the following formula or a pharmaceutically acceptable salt thereof: ;and (2) An antigen, wherein the antigen is one or more of the following: SARS-CoV, varicella-zoster virus, HIV NHR trimer, SARS-CoV-2, and influenza subtypes H1N1, H3N2, B / Yamagata, and B / Victoria.

9. The vaccine of claim 8, wherein the vaccine is an intramuscular vaccine, an intradermal vaccine, or an inhaled vaccine.

10. The vaccine of claim 8, wherein the antigen is an antigen derived from the SARS-CoV-2 Omecron mutant.

11. The vaccine of claim 8, wherein the antigen is SARS-CoV-2 RBD-Fc protein or gE protein of varicella-zoster virus.

12. The vaccine of claim 8, wherein the vaccine is an inactivated vaccine, a live attenuated vaccine, a subunit vaccine, or a nucleic acid vaccine.

13. The vaccine of claim 12, wherein the nucleic acid vaccine is an mRNA or DNA vaccine.

14. A method for producing a vaccine according to any one of claims 8-13, comprising mixing the compound or a pharmaceutically acceptable salt thereof with the antigen.

15. Use of the vaccine of any one of claims 8-13 for the preparation of a medicament for the treatment or prevention of an infectious disease, wherein the infectious disease is selected from the group consisting of: severe acute respiratory syndrome (SARS), COVID-19, varicella-zoster virus and influenza.

16. A kit comprising the vaccine of any one of claims 8-13 and instructions for treating or preventing infectious diseases.

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