Method and use for promoting antibody generation and vaccine protection

Abstract

A method and use of a CGRP signaling pathway activator for promoting antibody generation and vaccine protection, and a related pharmaceutical composition and vaccine composition.

Description

Methods and uses to promote antibody production and vaccine protection TECHNICAL FIELD

[0001] The present invention relates generally to the prophylactic and therapeutic fields. More specifically, the present invention relates to methods and uses to promote germinal center responses, antibody production and vaccine protection, as well as related pharmaceutical combinations and vaccine compositions. BACKGROUND

[0002] High-affinity neutralizing antibodies are an important weapon for the body to effectively resist pathogenic organisms, and are the basis for most vaccines to work. Germinal centers are specific structures that transiently exist in peripheral lymphoid organs (such as the spleen) or lymph nodes when the body encounters antigenic stimulation to mount an immune response, and are the sites of B lymphocyte clonal proliferation and affinity maturation, where the final selection of high-affinity antibody-producing plasma cells is made. Currently, the regulation of germinal centers is mainly focused on the interaction of B cells with follicular helper T cells (Tfh) and follicle dendritic cells (FDC), with emphasis on how Tfh and FDC regulate B cell maturation, germinal center formation, antibody class switching, and somatic hypermutation through the secretion of cytokines and the binding of cell surface ligand receptors. However, more and more studies have shown that the nervous system and the immune system are closely linked and interact in information transmission. In recent years, it has been found that human Tfh can secrete dopamine to act on B cells to promote germinal center responses. Studies have also found that adenosine acts on adenosine receptors on Tfh to inhibit Tfh differentiation and germinal center B cell formation. Recently, a brain-spleen neuro-immune regulatory axis has also been found to promote plasma cell production and antibody immune responses induced by vaccination through neurotransmitters such as norepinephrine and acetylcholine.

[0003] CGRP (calcitonin gene-related peptide) is a neuropeptide associated with immune regulation. It is mainly produced by neurons in the dorsal root ganglia (DRG) and acts on immune cells to affect the function of the immune system. CGRP binds to CGRP receptors on the surface of immune cells, and then activates a series of signaling pathways, ultimately leading to changes in immune cell activity. This change can enhance or inhibit the immune response, thereby achieving the purpose of regulating the immune response. Therefore, CGRP can be considered an important factor in immune regulation. However, there have been no reports of CGRP being involved in germinal center responses and antibody production.

[0004] In view of the importance of the adaptive immune response in anti-infective as well as anticancer immunotherapy, there is still a need in the art for methods and means that can provide improved ways of enhancing adaptive immunity, in particular methods and medicaments that can facilitate antigen-specific antibody generation upon vaccine administration and thereby increase vaccine protection.

[0005] SUMMARY

[0006] Thus, in a first aspect, the present application provides a method for enhancing an antigen-specific humoral immune response, comprising administering to a subject in need thereof an effective amount of a CGRP signaling pathway activator. In some embodiments, the subject has been exposed to the antigen prior to administration of the CGRP signaling pathway activator according to the present application. In other embodiments, the subject has not been exposed to the antigen prior to administration of the CGRP signaling pathway activator according to the present application. In some embodiments, the CGRP signaling pathway activator according to the present application is a CGRP receptor agonist, in particular a CGRP peptide or a functional analogue thereof, preferably the CGRP peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6. In other embodiments, the CGRP signaling pathway activator according to the present application is a CGRP release promoter, preferably administration of the CGRP release promoter results in an increase in CGRP content in the spleen of the subject. In some embodiments, the method further comprises administering to the subject the antigen and optionally an immunomodulator. Preferably, the antigen is a viral, bacterial, fungal and / or parasitic pathogen antigen, or the antigen is a cancer antigen. In some embodiments, the subject is suffering from, or at risk of suffering from, an infectious disease or a cancer. In some embodiments, the subject has been exposed to the infectious disease. In some embodiments, the subject will be exposed to or at risk of being exposed to the infectious disease. In some embodiments, the infectious disease is a viral, bacterial, fungal and / or parasitic infection. In some embodiments, the infectious disease is an influenza virus infection, and preferably the antigen is an influenza virus HA antigen. In some embodiments, the method is for preventing infection. In other embodiments, the method is for treating infection.

[0007] In some embodiments, the method according to the present application comprises one or more of the following:

[0008] (i) the CGRP signaling pathway activator is administered and the antigen is administered in separate compositions, and optionally, the CGRP signaling pathway activator and the antigen are administered at different time points and / or at different sites in the subject;

[0009] (ii) administering one or more doses of the antigen for immunization, and administering multiple doses of the CGRP signaling pathway activator after each dose of the antigen administration, and optionally administering the multiple doses at a frequency of once daily, once every two days, or once every three days;

[0010] (iii) at least one dose of the CGRP signaling pathway activator, administered prior to administration of the antigen;

[0011] (iv) the administration of the CGRP signaling pathway activator is separated from the administration of the antigen (and adjuvant, if administered) by at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours; and / or

[0012] (iv) the CGRP signaling pathway activator is administered continuously for at least 3 days, 5 days, 1 week, 2 weeks, or 3 weeks, or longer. In other embodiments, the method further comprises administering at least one dose of the CGRP signaling pathway activator within 2-72 hours, 2-48 hours, 4-24 hours, 4-18 hours, or 6-12 hours prior to or after administration of the antigen, or concurrently with the antigen.

[0013] The CGRP signaling pathway activator according to the present application can be administered by any suitable route known in the art, including, but not limited to, topical, systemic, parenteral, intramuscular, intraperitoneal, intravenous, oral, nasal, transdermal, transmucosal, and subcutaneous routes, and the like. In some embodiments, the CGRP signaling pathway activator according to the present application is a CGRP receptor agonist, and is preferably administered by intravenous or intraperitoneal injection. In other embodiments, the CGRP signaling pathway activator according to the present application is a CGRP release promoter, and is preferably administered by intragastric or oral administration.

[0014] In some embodiments, the CGRP signaling pathway activator according to the present application is administered in an amount effective to promote germinal center responses in the subject. In some embodiments, the CGRP signaling pathway activator according to the present application is administered in an amount effective to increase the ratio of germinal center B cells to plasma cells in the subject. In other embodiments, the CGRP signaling pathway activator according to the present application is administered in an amount effective to increase the titer of antigen-specific antibodies in the serum of the subject.

[0015] In some embodiments, germinal center responses in the secondary lymphoid organs of the subject are promoted by administration of the CGRP signaling pathway activator according to the present application. In some embodiments, the ratio of germinal center B cells to plasma cells is increased; and / or the titer of antigen-specific antibodies in the serum of the subject is increased, by administration of the CGRP signaling pathway activator according to the present application.

[0016] In a second aspect, the present application provides a method for preventing or treating an infection or a cancer, comprising administering to a subject in need thereof an effective amount of a CGRP signaling pathway activator according to the present application, in particular a CGRP receptor agonist according to the present application. In some embodiments, the method further comprises administering to the subject a vaccine composition comprising an antigen. In some embodiments, the infection is a viral, bacterial, fungal or parasitic infection, in particular a viral infection, such as an influenza virus infection.

[0017] In a third aspect, the present application provides a pharmaceutical combination product comprising components: (a) a CGRP signaling pathway activator according to the present application, in particular a CGRP receptor agonist according to the present application; and (b) a vaccine composition comprising an antigen. In some embodiments, the pharmaceutical combination product is for immunization. In some embodiments, the product comprises a plurality of unit doses of component (a) and a plurality of unit doses of component (b), preferably each unit dose is individually packaged. In some embodiments, the vaccine composition comprises or does not comprise an adjuvant. In some embodiments, the antigen is an influenza virus antigen, such as an influenza virus HA antigen.

[0018] In a fourth aspect, the present application provides a vaccine composition comprising a CGRP signaling pathway activator according to the present application and one or more antigens. In some embodiments, the vaccine composition comprises or does not comprise an adjuvant. In some embodiments, the antigen is an influenza virus antigen, such as an influenza virus HA antigen.

[0019] In a fifth aspect, the present application provides the use of a CGRP signaling pathway activator according to the present application, a pharmaceutical combination product or a vaccine composition according to the present application for use as a medicament, or in the manufacture of a medicament. In some embodiments, the medicament is for use in promoting germinal center responses and / or antibody production in a subject in need thereof, and / or for improving adaptive immune responses, in particular antigen-specific humoral immune responses, in a subject, and / or for enhancing vaccine-induced humoral immune responses, or for preventing or treating an infectious disease or a cancer.

[0020] BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 shows the FACS-gating strategy for flow analysis of the ratio of splenic germinal center B cells and plasma cells. In the figure, live cell staining (FVS) is performed using Fixable Viabilty Dye; total B cells are analyzed by detecting cell surface expression of the B cell lineage-specific marker B220 (CD45R); germinal center B cells are analyzed by detecting cell surface expression of the B220 (CD45R) and the B220 (CD45R) and the B220 (CD45R) lo CD138 hiMarker expression, analyzing the proportion of plasma cells; and by detecting B220 (CD45R) + GL7 + FAS + Marker expression, analyzing the proportion of germinal center B cells in total B cells.

[0022] Figure 2 shows the effect of CGRP injection on the proportion of splenic germinal center B cells and plasma cells after immunization of wild type mice with NP-KLH antigen, compared to PBS group. The proportion of splenic germinal center B cells and plasma cells were detected according to the FACS-gating strategy shown in Figure 1 after PBS treatment and CGRP treatment of mice, respectively. (A): Representative flow cytometry plots; (B): Bar graph, where the proportion of germinal center cells and plasma cells showed significant difference between PBS group and CGRP group (p values were 0.0002 and 0.0007, respectively).

[0023] Figure 3 shows the increase of total and high affinity NP antigen-specific antibody levels in serum of CGRP-injected mice compared to PBS group mice after immunization of wild type mice with NP-KLH antigen (p values were <0.0001, respectively).

[0024] Figure 4 shows the proportion of splenic B cells in conditional Calcrl knockout mice Cd19 Cre / + Calcrl fl / fl mice and their parental Cd19 Cre / + mice, the mRNA expression level of CGRP receptor CALCRL in splenic B cells.

[0025] Figure 5 shows the effect of CGRP injection on the proportion of splenic germinal center B cells and plasma cells after immunization of conditional knockout mice (Cd19 Cre / + Calcrl fl / fl mice) and parental mice (Cd19 Cre / + mice) with NP-KLH antigen. The proportion of splenic germinal center B cells and plasma cells were detected by flow cytometry according to the FACS-gating strategy shown in Figure 1 after CGRP treatment of mice. (A): Representative flow cytometry plots; (B): Bar graph, where the proportion of germinal center cells and plasma cells showed significant difference between conditional knockout mice and parental (p values were 0.0014 and 0.0020, respectively).

[0026] Figure 6 shows the increase of total and high affinity NP antigen-specific antibody levels in serum of conditional knockout mice (Cd19 Cre / + Calcrl Cre / + mice) compared to parental mice (Cd19 fl / flThe effect of CGRP injection on the levels of total and high-affinity NP antigen-specific antibodies in the serum of mice (p values < 0.0001).

[0027] Figure 7 shows that, after immunization of wild-type mice with NP-KLH antigen, the level of CGRP in the spleen of mice fed with capsaicin was significantly increased compared to control mice (p value < 0.0203).

[0028] Figure 8 shows the effect of capsaicin feeding on the proportion of germinal center B cells and plasma cells in the spleen of mice after immunization of wild-type mice with NP-KLH antigen. The proportion of germinal center B cells and plasma cells in the spleen was measured by flow cytometry according to the FACS-gating strategy shown in Figure 1. Compared to the control, capsaicin treatment significantly increased the proportion of germinal center B cells and plasma cells in the spleen (p values 0.0011 and 0.0049, respectively).

[0029] Figure 9 shows the effect of capsaicin feeding on the levels of total and high-affinity NP antigen-specific antibodies in the serum of mice after immunization of wild-type mice with NP-KLH antigen (p values < 0.0001).

[0030] Figure 10 shows a schematic diagram of the experimental protocol for studying the effect of oral capsaicin on the vaccine antigen-induced anti-viral adaptive immune response. The day of the first immunization with HA antigen was set as day D0. Capsaicin feeding was continued throughout the immunization process, from day D-7 to day D28. Virus challenge was performed on day D30.

[0031] Figure 11 shows the titers of antigen HA-specific IgG antibodies in the serum of immunized mice measured on day 14 and day 28, respectively, after the first and second vaccine antigen immunization in animal experiments performed according to the protocol shown in Figure 10. Compared to the control, mice fed with capsaicin had significantly higher serum HA-specific IgG antibody levels (after the first immunization: p value < 0.0082; after the second immunization: p value < 0.0002).

[0032] Figure 12 shows the mRNA expression level of viral core protein NP in the lung tissue of mice measured on day 35 after influenza virus challenge in animal experiments performed according to the protocol shown in Figure 10. Compared to control mice, mice that ingested capsaicin had significantly reduced NP protein expression levels in the lung tissue after virus infection (p value < 0.0001).

[0033] Figure 13 shows immunohistochemical staining sections of lung tissue of mice on day 35 after influenza virus challenge in animal experiments performed according to the protocol shown in Figure 10. Compared to control mice, mice that ingested capsaicin had significantly reduced immune cell infiltration in the lung tissue after virus infection.

[0034] Figure 14 shows the body weight changes of control and capsaicin-fed experimental mice measured on the day of influenza virus challenge and for 10 days thereafter in an animal experiment performed according to the protocol shown in Figure 10. Capsaicin treatment effectively prevented the body weight loss in mice compared to the control group.

[0035] Figure 15 shows the survival rates of control and capsaicin-treated experimental mice 10 days after influenza virus challenge in an animal experiment performed according to the protocol shown in Figure 10. The survival rate was significantly different between the two groups by day 10 post-virus infection, p-value = 0.0190.

[0036] Figure 16 shows the effect of ablation of splenic TRPV1 neurons by intranodal injection of resiniferatoxin (RTX) on the splenic CGRP content of mice relative to control mice injected with solvent.

[0037] Figure 17 shows a schematic of the experimental protocol to investigate the dependence of capsaicin-promoted antigen-specific humoral immune responses on TRPV1, in mice immunized with NP-KLH antigen.

[0038] Figure 18 shows the effect of feeding or not feeding capsaicin on the proportion of splenic germinal center B cells and plasma cells in mice injected with RTX, relative to control mice injected with solvent, after immunization of mice with NP-KLH antigen. The proportion of splenic germinal center B cells and plasma cells was flow cytometrically determined according to the FACS-gating strategy shown in Figure 1. The p-values for comparisons between different treatment groups are shown on the figure.

[0039] Figure 19 shows a schematic of the experimental protocol to investigate the dependence of capsaicin-feeding enhanced vaccine-induced humoral immune responses on TRPV1 nerves. Solvent or resiniferatoxin (RTX) was injected into the T8-T13 dorsal root ganglia of mice to ablate splenic TRPV1 nerves at day -35 (D-35) before the first immunization with HA antigen protein. Four weeks after recovery of the animals, capsaicin diet or control diet was fed to the mice daily from day -7 to day 28. Immunization with HA antigen was performed at day 0 and day 21; and virus challenge was performed at day 30.

[0040] Figure 20 shows the changes in body weight and survival rate of RTX-injected mice fed (RTX+Cap) or not fed (RTX+Ctrl) capsaicin relative to solvent-injected control mice (Veh+Ctrl) 10 days after influenza virus challenge in an animal experiment performed according to the protocol shown in Figure 19. By day 10 post-virus infection, there was no significant difference in body weight and survival rate between the two groups of RTX-injected mice fed or not fed capsaicin (survival rate: p-value = 0.5960), both of which were significantly lower than the solvent-injected control mice (survival rate: p-value = 0.0116).

[0041] Figure 21 shows a schematic of an experimental protocol to study the effect of CGRP receptor knock-out on vaccine immunity.

[0042] Figure 22 shows that in animal experiments performed according to the protocol shown in Figure 21, 10 days after challenge with influenza virus, Cd19 Cre / + Calcrl fl / fl Changes in body weight and survival of mice.

[0043] Figure 23A shows a sequence alignment of exemplary CGRP-alpha and CGRP-beta from humans, rats, and mice.

[0044] Figure 23B shows a sequence alignment between CGRP sequences of various species, with dark grey boxes showing conserved regions and light grey boxes showing regions with similar physicochemical parameters.

[0045] DETAILED DESCRIPTION

[0046] DEFINITIONS

[0047] In this document, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0048] In this document, the term "optional" or "optionally" means that the subsequent description is applicable or can be applicable, but that this description also includes instances where the event, situation, or element by itself is not present, and instances in which it occurs. In this document, the term "comprising" or "comprise" means that the subsequently described event, situation, or element can be present, but that it is not required, and that the description also includes instances where the event or situation or element is not present.

[0049] In this document, a numerical range recited by endpoints includes all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0050] In this document, the term "about" or "approximately," when used in reference to a measurable value such as a parameter, amount, duration, and the like, is intended to encompass variations that can occur such as variations that would occur due to experimental error, measurement techniques, differences in measurement techniques, and the like; for example, + / -10% or less, + / -1-5% or less, + / -1% or less, and + / -0.1% or less of the stated value, as long as such variations are appropriate in light of the particular technology with which the disclosed technology is concerned. It is to be understood that the particular value to which the term "about" or "approximately" refers is itself also specifically and explicitly disclosed.

[0051] In this document, the terms "protein," "peptide," and "polypeptide" are used interchangeably and refer to polymers of amino acid monomers that are not of a specific length, wherein the amino acid monomers are linked by peptide bonds. This definition also encompasses peptides that have been post-translationally modified, such as peptides with covalent modifications of glycosylation, amidation, acetylation, phosphorylation, lipid groups, and the like.

[0052] In the present context, the term "compound" refers to a chemical compound (e.g. a small molecule organic compound) or a biochemical compound (e.g. a peptide). The CGRP signaling pathway activator according to the present application can be a chemical compound or a biochemical compound. In some aspects, preferably, the activator is a biochemical compound, and it is also preferred that the activator is a peptide.

[0053] In the present context, the terms "subject", "individual" and "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal, and more preferably a human. Mammals include, but are not limited to, primates (e.g., humans and non-human primates), laboratory animals (e.g., rodents, such as mice and rats), farm animals (e.g., cows, pigs, sheep, and horses), sport animals, and pets (e.g., dogs and cats).

[0054] In the present context, the term "CGRP" is used interchangeably with "calcitonin gene-related peptide". The term encompasses not only any naturally occurring CGRP-alpha peptide and CGRP-beta peptide, but also functional variants, functional fragments thereof, or any mammalian orthologs thereof. In some exemplary embodiments, CGRP also includes peptides that are post-translationally modified, e.g. peptides with amidation, PEGylation, etc. covalent modifications.

[0055] In the present context, the terms "activity", "function", "biological activity" and "biological function" are synonymous for the purposes of the present application and have their generally known meaning in the art. Preferably, the biological activity of CGRP in a subject is the biological activity of binding to and activating a CGRP receptor-mediated signaling pathway, wherein the CGRP receptor is in particular a CGRP receptor expressed on B cells in secondary lymphoid organs, such as the spleen.

[0056] In the present context, "CGRP receptor" refers to a heterodimeric protein formed by the binding of receptor activity-modifying protein 1 (RAMP1) to a CALCRL protein (also known as CRLR). An example of RAMP1 is the receptor protein described under NCBI: NP_005846.1. An example of CALCRL is the receptor protein described under NCBI: NP_005786.1. In the present context, the CGRP receptor does not include a CALCRL protein bound to receptor activity-modifying protein 2 (RAMP2) or RAMP3.

[0057] In the present context, the term "isolated" when used as a modifier of a compound (e.g., a CGRP peptide, etc.) means that the compound is produced by the hand of man or that the compound is completely or at least partially separated from the natural environment in which the compound naturally occurs. It is understood that, unless indicated to the contrary, the compounds used in the present application, whether chemical compounds or biochemical compounds, are isolated. In some instances, an isolated compound is "purified" or "partially purified" from the environment in which it is produced or prepared and thereby free of at least some of the components present in the environment in which the compound is produced or prepared. In some embodiments, an isolated compound, e.g., an isolated CGRP peptide, has a purity of at least 90%. A "purified" or "partially purified" compound can be combined with one or more other compounds and thereby form a composition that achieves a particular purpose. Thus, "purified" or "partially purified" does not exclude the combined situation of such a composition, e.g., a combination of a CGRP peptide with a vaccine component (e.g., an antigen) for immunization, an adjuvant, and / or a second active agent.

[0058] In the present context, a "humoral immune response", also referred to as antibody-mediated immune response. During a humoral immune response, B cells will differentiate into plasma cells and memory B cells capable of producing antibodies against a specific antigen and elicit an antibody-mediated immune reaction with the help of helper T cells. Typically, the humoral immune system deals with antigens of pathogens free in the circulation or outside infected cells.

[0059] In the present context, "germinal center" is used interchangeably with GC (Germinal Center) and has the meaning commonly understood in the art. Generally, a germinal center is a dynamic microstructure that appears in secondary lymphoid tissues after antigenic stimulation or microbial infection. A mature GC consists of two functionally distinct microenvironments, the dark zone (DZ) and the light zone (LZ). In the GC, antigen-activated B cells, with the help of follicular helper T cells and dendritic cells, undergo clonal expansion, hypermutation, and go through affinity selection and maturation, eventually forming antibody-secreting plasma cells or memory B cells. The GC reaction plays an important role in the humoral immune response and the formation of long-term immune memory.

[0060] In the present context, "promoting germinal center responses" means stimulating, enhancing, inducing and / or increasing the process of development of germinal center B cells into antibody-secreting plasma cells and memory B cells in secondary lymphoid organs. In some embodiments, the promotion of germinal centers can be characterized by measuring the proportion of germinal center B cells and / or plasma cells in the secondary lymphoid organs of a subject. In other embodiments, the promotion of germinal centers can be characterized by the proportion of output cells producing antigen-specific antibodies (or producing antigen-specific high-affinity antibodies) in the secondary lymphoid organs. In other embodiments, the promotion of germinal centers can be characterized by measuring the amount of antigen-specific antibodies (or antigen-specific high-affinity antibodies) in the serum of a subject.

[0061] During a germinal center response, antibodies undergo affinity maturation, resulting in antigen-specific antibodies with increased affinity. The affinity of an antibody can be characterized by the binding affinity of the antibody to its cognate antigen. In some cases, the binding affinity can be expressed in terms of an affinity constant. The affinity constant (also known as the association constant) K D is a numerical constant used to describe the binding affinity of two molecules at equilibrium. Binding affinity can be measured using a variety of methods known to those skilled in the art, including immunoblotting, immunoprecipitation assays, radioimmunoassays, ELISA, antibody determination by immunofluorescence microscopy, surface plasmon resonance (BiaCORE).

[0062] In the present context, "antigen", sometimes also referred to as "vaccine antigen", used as a component of a vaccine, means a substance that can be utilized as a vaccine for the purpose of treatment and / or prevention of a disease, and that elicits an immune response, in particular a B cell humoral immune response, in an organism. As some examples of vaccine antigens, one can mention, without limitation, peptides, proteins, glycoproteins, glycolipids, lipids, carbohydrates, nucleic acids, polysaccharides and viruses, bacteroids, allergenic substances, tissues, cells, etc. comprising them. In some aspects according to the present application, the adaptive humoral immune response elicited by an antigen can be potentiated by combining said antigen with a CGRP signaling pathway activator according to the present application. In some embodiments, the vaccine antigen is a pathogen or a pathogen antigen. In other embodiments, the vaccine antigen is a cancer antigen.

[0063] In the present context, the term "treating" a disorder and / or a disease in a mammal means (i) preventing the disorder or disease, i.e. avoiding any clinical symptoms of the disease or disorder; (ii) inhibiting the disorder or disease, i.e. arresting the development or progression of clinical symptoms; and / or (iii) relieving the disorder or disease, i.e. causing the regression of clinical symptoms. The term encompasses therapeutic and prophylactic treatment.

[0064] In the present context, the term "prevention" or "preventative treatment" refers to reducing or decreasing the likelihood of a subject developing a disease or a symptom of a disease (e.g. a symptom of an infection). Thus, in some aspects, a CGRP signaling pathway activator according to the present application can be administered prophylactically to prevent the onset of an infection, or to prevent the recurrence of an infection in a subject.

[0065] In the present context, "effective amount" means the predetermined amount of an active agent that will elicit the desired biological or medical response of a tissue, system, animal, or human that is of present interest, e.g. an amount sufficient to stimulate a germinal response or antigen-specific antibody production, to enhance the protective effect of a vaccine, and / or to prevent, hinder, retard or reverse the progression of a disease state or any other undesirable symptom or otherwise ameliorate the disease state or symptom to achieve the desired therapeutic effect.

[0066] In the present context, "therapeutically effective amount" and "prophylactically effective amount" refer to an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic result or prophylactic result. The therapeutically effective amount and the prophylactically effective amount can vary according to factors such as the disease to be treated or prevented, the age, sex and weight of the individual, and the like. The therapeutically effective amount and the prophylactically effective amount are amounts that have no toxic or detrimental effects that outweigh the beneficial effects of treatment / prevention. The "therapeutically effective amount" and the "prophylactically effective amount" preferably increase a measurable parameter (e.g. germinal center B cell proportion and / or plasma cell proportion, or serum antigen-specific antibody titer) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and further more preferably by at least about 80%, relative to a subject not administered the drug. The ability of a CGRP signaling pathway activator of the present application to enhance a measurable parameter can be evaluated in animal model systems predictive of therapeutic efficacy in humans. Typically, prophylactic administration is used prior to the onset of symptoms of a disease, or prior to an earlier stage of a disease, or at an earlier stage of a disease.

[0067] In the present text, "infectious disease" and "infection" are used interchangeably and include respiratory infections such as upper respiratory tract infections, lower respiratory tract infections, thoracic infections, and the like; urinary system infections such as pyelonephritis, urethral infections, asymptomatic bacteriuria, and the like; digestive system infections such as gastroenteritis, hepatitis, abdominal cavity infections, and the like; bone and joint infections such as osteomyelitis, joint infections, and the like; central nervous system infections such as intracranial infections, intraspinal abscesses, and the like; cardiovascular system infections such as endocarditis, pericarditis, and the like; blood system infections such as transfusion-related hepatitis, bacteremia, and the like; reproductive system infections such as pelvic infections, prostatitis, and the like; skin and soft tissue infections such as pressure ulcers, necrotizing conjunctivitis, and the like; surgical site infections such as surgical incision infections and deep incision infections, and the like; other site infections such as oral infections, otitis media, sinusitis, conjunctivitis, and the like; and multiple site infections such as multisystem infections, multiple organ infections, and the like. In some aspects according to the present application, the methods and uses according to the present application are for the prevention or treatment of an infectious disease. In some embodiments, the infectious disease is a respiratory tract infection. In other embodiments, the infectious disease is a pulmonary infection. In other embodiments, the infectious disease is an infection in a tissue other than the lungs, for example, a digestive system infection, a gastrointestinal tract infection, a cardiovascular system infection. DETAILED DESCRIPTION

[0068] In mammals, primary lymphoid tissues include the thymus and bone marrow, whose main role is to regulate the development of immature precursor immune cells. Secondary lymphoid tissues include the spleen, lymph nodes, the gut and other specialized sites in mucosal tissues, whose role is to orchestrate the encounter of antigen with antigen-specific lymphocytes and to develop them into effector and memory cells. B lymphocytes (B cells) are derived from hematopoietic stem cells in the bone marrow. Pre-B cells, which are formed in the bone marrow, circulate in the blood and secondary lymphoid tissues after leaving the bone marrow. At this stage, B cells are immature. During a primary humoral response, immature B cells that enter the secondary lymphoid tissues can develop into mature B cells by interacting with T helper cells in the secondary lymphoid tissues. If they encounter and are activated by antigen, these B cells will enter primary lymphoid follicles and proliferate extensively to form germinal centers (GCs). Activated B cells that enter the GCs will then undergo a series of developmental processes in the GCs, including clonal expansion, somatic hypermutation, affinity maturation and differentiation, and eventually develop into antibody-secreting plasma cells and memory B cells, and leave the GCs. In general, only B cells with high affinity to cognate antigens will interact with CD4+ T helper (Th) cells specific to the same antigen in the GCs, and thus be selected and differentiated into memory B cells or plasma cells; in contrast, GC B cells with poor affinity to cognate antigens or with affinity to self-antigens (self-reactivity) will undergo negative selection and be removed. Plasma cells, which are differentiated, divide at a slower rate than GC B cells and secrete high-affinity immunoglobulins such as IgG. Memory B cells, which are differentiated, have a long lifespan and, upon encountering antigen, can become effective antigen-presenting cells (APCs) to induce rapid activation and expansion of Th cell populations, and thus accelerate the generation of plasma cells and memory B cells, thereby mediating a rapid and strong secondary antibody response. Therefore, promoting the GC response will help to improve the humoral immune response and long-term immune memory of the host.

[0069] The present application is based, in part, on the discovery that activators of the CGRP signaling pathway (CGRP neuropeptides or analogs thereof and substances that promote the release of CGRP) can promote germinal center responses and the production of antigen-specific antibodies in secondary lymphoid organs in vivo and enhance the protective effects of vaccines. The present application is also based, in part, on the discovery that the therapeutic activity associated with activators of the CGRP signaling pathway is associated with binding to and activation of CGRP receptors on B cells in secondary lymphoid organs. This activation leads to an increase in the number of germinal center B cells and plasma cells and an increase in specific antibody titers in serum.

[0070] In some aspects, the present application thus provides CGRP signaling pathway activators for use in improving the immune response, particularly the adaptive immune response, in a host. In other aspects, the present application provides CGRP signaling pathway activators for use in enhancing the efficacy of vaccination of a host. In yet other aspects, the present application provides the use of CGRP signaling pathway activators in combination with a pathogen vaccine antigen to enhance the host's ability to defend against infection, or in combination with a cancer vaccine antigen to enhance the host's cancer immunotherapy effect.

[0071] The various aspects of the present application are described in detail below. It will be appreciated by those skilled in the art that any of the technical features of the aspects can be combined unless the context clearly dictates otherwise. Furthermore, it will be appreciated by those skilled in the art that the methods, medicaments, pharmaceutical combinations and uses according to any of the aspects of the present application can comprise any such combination of features unless the context clearly dictates otherwise.

[0072] CGRP signaling pathway activators

[0073] Calcitonin gene-related peptide (CGRP) is a neuropeptide produced primarily by neurons in the dorsal root ganglion (DRG) and is widely expressed in the peripheral and central nervous systems. CGRP exerts its biological effects by interacting with its receptors. The receptor for CGRP, sometimes also simply referred to as CGRP receptor herein, is a heterodimer formed by calcitonin receptor-like receptor (CALCRL, also known as CRLR) and receptor activity-modifying protein 1 (RAMP1). CALCRL is a G protein-coupled seven-transmembrane domain receptor encoded by the calcitonin receptor-like receptor gene Calcrl (also known as crlr). RAMP1 is a single-transmembrane domain protein. RAMP1 is a co-receptor necessary for the cell surface expression of CALCRL and binding to the CGRP peptide ligand. Binding of the CGRP receptor to the ligand CGRP results in the activation of downstream signaling pathways.

[0074] As used herein, a "CGRP signaling pathway activator" refers to an active agent (compound or composition) that is capable of promoting downstream signaling and / or at least one biological response resulting therefrom that is normally associated with the binding of a CGRP receptor to its natural ligand, CGRP. For example, in some cases, a CGRP signaling pathway activator is an active agent that is capable of interacting with a CGRP receptor present on a B cell in a secondary lymphoid tissue and promoting a germinal center response. An example of such an active agent is an agonist of a CGRP receptor, such as a CGRP peptide or a functional variant or analog thereof. In other cases, a CGRP signaling pathway activator is an active agent that is capable of otherwise increasing the level of CGRP in a secondary lymphoid tissue and thereby enhancing CGRP receptor activation on B cells and promoting a germinal center response. An example of such an active agent is, for example, a compound that is capable of promoting the release of CGRP from a nociceptor neuron, such as a capsaicin or a functional analog thereof. The binding interaction between a CGRP receptor agonist and a CGRP receptor can be determined by the ability of the agonist to compete with CGRP (e.g., radiolabeled CGRP) for binding to the receptor and / or using other competition assays. For active agents that promote the release of CGRP, the CGRP release-promoting ability of the active agent can be determined by detecting the amount of CGRP release resulting from contacting the active agent with, for example, a nociceptor neuron, and optionally comparing to capsaicin. Preferably, a CGRP signaling pathway activator according to the present application stimulates a subject to form more germinal center B cells and / or antigen-specific plasma cells upon exposure to an antigen.

[0075] In some aspects according to the present application, a CGRP signaling pathway activator for use in the present application is a CGRP receptor agonist. As is well known in the art, a receptor agonist can be a natural ligand of the receptor, or it can be a compound that is capable of mimicking the action of the natural ligand. More specifically, a receptor agonist is capable of binding to and activating the receptor and triggering the same type of biochemical responses as the natural ligand, particularly the modulation of the same second messengers. CGRP receptor agonists useful in the present application include a variety of types of compounds, including polypeptides, peptides, peptidomimetics, and non-peptide compounds such as small molecule organic molecules (e.g., molecular weight < 1000). Suitable agonists can be identified using the methods described herein, and / or by methods apparent to one of skill in the art in light of the present disclosure. In some aspects according to the present application, a CGRP receptor agonist for use in the present application is a CGRP peptide or a functional analog thereof.

[0076] The natural calcitonin gene-related peptide (CGRP) is a 37 amino acid neuropeptide. The neuropeptide is produced by alternative splicing of the calcitonin gene. There are two main forms of CGRP, referred to as CGRP-alpha and CGRP-beta or in some species as CGRP-I and CGRP-II. CGRP-alpha and CGRP-beta have >90% homology between them and only 3 amino acid differences exist in humans; thus, CGRP-alpha and CGRP-beta have similar biological activities. CGRP has been found to be highly conserved within species. See FIG. 23A, where a sequence alignment of exemplary CGRP-alpha and CGRP-beta from human, rat, and mouse is shown; FIG. 23B, where a sequence alignment between CGRP sequences of multiple species is shown, with dark gray boxes showing conserved regions and light gray boxes showing regions with similar physicochemical parameters.

[0077] Natural CGRP typically has an N-terminal disulfide loop important for biological activity, an alpha helical region, and a C-terminal portion. Using the 37 amino acid peptide of CGRP-alpha as an example, similar to beta CGRP, it comprises a disulfide bridge-linked N-terminal loop structure (residues 1-7) required for receptor activation; an alpha-helical region (residues 8-18); a region comprising a beta-turn (residues 19-27); and a C-terminal region with two corner regions thought to form a binding epitope (residues 28-37). Species differences and structure-activity relationships of CGRP have been extensively studied, and key amino acids that play a critical role in receptor binding and activation have been identified. See, e.g., Watkins HA et al. Structure-activity relationships for alpha calcitonin gene-related peptide. Br J Pharmacol 170: 1308-1322, 2013; and F.A. Russell et al. Calcitonin Gene-Related Peptide: Physiology and Pathophysiology, Physiol Rev. 2014 Oct;94(4): 1099-1142, doi:10.1152 / physrev.00034.2013.

[0078] On the basis of native CGRP, a number of CGRP functional analogs with agonistic activity have been developed. See, e.g., WO2016183479A1, US2003 / 0204063, US8835379B2, US20040038861A1, US20160207975A1, US4804742A, US20040091452A1, and WO2020120480A1. For purposes of the present invention, these documents are hereby incorporated by reference in their entirety into the present application.

[0079] In some aspects, the CGRP peptide according to the present invention is a CGRP compound having the following general formula I:

[0080] X1CX2TX3TCX4TX5RLAX6X7LX8RSGGX9X 10 X 11 X 12 X 13 FVPTX 14 VX 15 X 16 X 17 X 18 F,

[0081] wherein X1is Ala or Ser, X2is Asp or Asn, X3is Ala or Ser, X4is Val or Ala, X5is His or Gin, X6is Gly or Asp, X7is Leu or Phe, X8is Ser, Asn or Arg, X9is Val, lie, Met or Leu, X 10 is Val, Ala, Leu or Gly, X 11 is Lys, Ser, Asn or His, X 12 is Ser, Asn, Asp, Pro, X 13 is Asp or Asn, X 14 is Asp or Asn, X 15 is Gly or Ser, X 16 is Ala or Ser, X 17 is Glu, Gin, Lys or Asn, X 18 is Ala or Ser, and the carboxyl group in the C-terminal amino acid residue is optionally amidated.

[0082] In other aspects, the CGRP peptide according to the present invention is a CGRP compound having the following general formula II:

[0083] X1CX2TATCVTHRLAX6LLX’8RSGGX’9X’ 10 KX’ 12 NFVPTX 14VGSX' 17 AF,

[0084] wherein X1is Ala or Ser, X2is Asp or Asn, X6is Asp or Gly, X'8is Arg or Ser, X'9is Val or Met, X 10 is Val or Leu, X 12 is Asp, Asn or Ser, X 14 is Asp or Asn, X 17 is Glu or Lys, and the carboxyl group in the C-terminal amino acid residue is optionally amidated.

[0085] CGRP compounds included in the above general formula I include, for example, α and β type CGRP peptides from the following species: pig, sheep, Xenopus laevis, human, mouse, horse, dog, rat, Phyllomedusa bicolor, Oryzias latipes, Takifugu rubripes, Paralichthys olivaceus, Carassius auratus, Salmo salar, and Danio rerio. CGRP compounds included in the above general formula II include, for example, α and β type CGRP peptides from the following species: pig, mouse, human, dog, and rat.

[0086] In some aspects, the CGRP peptides according to the present application can be derived from the following species: Homo sapiens, Equus caballus, Rattus norvegicus, Mus musculus, Gallus gallus, Sus scrofa, Ovis aries, Bos taurus, Canis lupus familiaris, Callithrix jacchus, Monodelphis Domestica, Gekko japonicas, Phyllomedusa bicolor, Takifugu rubripes, Paralichthys olivaceus, Carassius auratus, Salmo salar, Oryzias latipes, and Danio rerio, or functional variants thereof.

[0087] In some preferred aspects, the CGRP peptides for use in the present application are α and β type CGRP peptides from human, rat or mouse, or functional variants thereof.

[0088] An example of a human CGRP-α peptide can be found in UniProtKB / Swiss-Prot ref.: P06881, which is encoded by the human Calca gene and has the sequence set forth in SEQ ID NO: 1 : ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF-NH2. In some embodiments, the CGRP receptor activator according to the application is a human CGRP-α peptide. In some embodiments, the CGRP receptor activator according to the application is SEQ ID NO: 1 or a functional variant thereof.

[0089] An example of a human CGRP-β peptide can be found in UniProtKB / Swiss-Prot ref.: P10092, which is encoded by the human Calcb gene and has the sequence set forth in SEQ ID NO: 2: ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF-NH2. In some embodiments, the CGRP receptor activator according to the application is a human CGRP-β peptide. In some embodiments, the CGRP receptor activator according to the application is SEQ ID NO: 2 or a functional variant thereof.

[0090] An example of a rat CGRP-α peptide can be found in UniProtKB / Swiss-Prot ref.: P01256, which is encoded by the rat Calca gene and has the sequence set forth in SEQ ID NO: 3: SCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSEAF-NH2. In some embodiments, the CGRP receptor activator according to the application is a rat CGRP-α peptide. In some embodiments, the CGRP receptor activator according to the application is SEQ ID NO: 3 or a functional variant thereof.

[0091] An example of a rat CGRP-β peptide can be found in UniProtKB / Swiss-Prot ref.: P10093, which is encoded by the rat Calcb gene and has the sequence set forth in SEQ ID NO: 4: SCNTATCVTHRLAGLLRRSGGVVKDNFVPTNVGSKAF-NH2. In some embodiments, the CGRP receptor activator according to the application is a rat CGRP-β peptide. In some embodiments, the CGRP receptor activator according to the application is SEQ ID NO: 4 or a functional variant thereof.

[0092] An example of a mouse CGRP-α peptide can be found in UniProtKB / Swiss-Prot ref.: Q99JA, which is encoded by the mouse Calca gene, and has the sequence of SEQ ID NO: 5: SCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSEAF-NH2. In some embodiments, the CGRP receptor activator according to the application is a mouse CGRP-α peptide. In some embodiments, the CGRP receptor activator according to the application is SEQ ID NO: 5 or a functional variant thereof.

[0093] An example of a mouse CGRP-β peptide can be found in UniProtKB / Swiss-Prot ref.: Q99MP3, which is encoded by the mouse Calcb gene, and has the sequence of SEQ ID NO: 6: SCNTATCVTHRLADLLSRSGGVLKDNFVPTDVGSEAF-NH2. In some embodiments, the CGRP receptor activator according to the application is a mouse CGRP-β peptide. In some embodiments, the CGRP receptor activator according to the application is SEQ ID NO: 6 or a functional variant thereof.

[0094] In the present context, a functional variant of CGRP refers to a peptide whose amino acid sequence differs from the wild-type CGRP, but which generally retains the biological activity of the wild-type CGRP. In embodiments according to the application, the functional variant of CGRP is a ligand that binds to and activates the CGRP receptor. The functional variant can also include a peptide with chemical modifications, or a peptide with non-natural amino acid residues, or a fusion protein (e.g. a fusion with another protein or polypeptide, such as a fusion with an immunoglobulin or a fragment thereof). In some cases, the functional variant can have an extended residence time in the body fluids. In certain embodiments, the variant of CGRP has at least 80%, 85%, 90%, 95%, 99% of the biological activity of CGRP. Preferably, the functional variant of CGRP-α differs from this CGRP-α by no more than 5 amino acid residues, preferably no more than 3 amino acid residues, e.g. by only 1, 2 or 3 amino acid residues. Preferably, the functional variant of CGRP-β differs from this CGRP-β by no more than 5 amino acid residues, preferably no more than 3 amino acid residues, e.g. by only 1, 2 or 3 amino acid residues. The amino acid residue difference is selected from an amino acid substitution, a deletion or an insertion, preferably an amino acid substitution, more preferably a conservative amino acid substitution.

[0095] In the present context, the term "conservative amino acid substitution" refers to a change in the amino acid composition of a protein, polypeptide or peptide, wherein a residue is replaced by another amino acid residue that is structurally similar and that does not significantly alter the activity of the protein, polypeptide or peptide. Tables of conservative substitution of amino acids that provide functionally similar amino acids are well known in the art. The following six groups each contain amino acid residues that are conservative substitutions for one another: 1) Alanine, Serine, and Threonine, 2) Aspartic acid and Glutamic acid, 3) Asparagine and Glutamine, 4) Arginine and Lysine, 5) Isoleucine, Leucine, Methionine, and Valine, and 6) Phenylalanine, Tyrosine, and Tryptophan.

[0096] In some aspects, the present application also contemplates agonists that correspond to or consist of a fragment of a CGRP polypeptide (e.g., at least 6 residues, more often at least about 20, 25, 30 or 35 residues), including agonists having chimeric CGRP peptide sequences in which one or more amino acids in the native sequence from one species (e.g., human) are replaced with different amino acids at the corresponding sequence position from CGRP peptide of one or more different species (e.g., rat).

[0097] Preferably, the CGRP peptides and functional variants thereof for use in the present application retain the conventional intramolecular disulfide bridge between the two Cys residues (typically at positions 2 and 7 corresponding to SEQ ID NO: 1) and / or C-terminal amidation.

[0098] As understood by the skilled person, the CGRP peptides and functional variants thereof of the present application can have chemical modifications and not affect their desired biological function, such as amidation, PEGylation and / or polymer attachment for enhancing their metabolic stability and / or plasma half-life. See, e.g., CN102666579A, WO2021022117A1, WO2020130927A1, WO2009142727A2, WO2011051312A1, WO2011051312A1. For the purposes of the present application, these documents are hereby incorporated by reference in their entirety into the present application.

[0099] In some aspects, the CGRP signaling pathway activator according to the present application is an active agent capable of increasing the level of CGRP in secondary lymphoid tissue and thereby enhancing CGRP receptor activation on B cells and promoting germinal center responses. In some embodiments, the active agent is a CGRP release promoter that promotes the release of CGRP from nerve terminals, e.g., a compound capable of promoting the release of CGRP from nociceptive sensory neurons, such as capsaicin or a functional analog thereof.

[0100] In humans, CGRP is produced by selective splicing of the calcitonin gene in peripheral and central neurons. CGRP-containing central neurons are complex pattern nociceptors that can be activated by chemical / thermal / mechanical stimuli, release CGRP and transmit pain to the cortex. CGRP can also be released from peripheral nerve terminals, changing the plasma levels of CGRP.

[0101] A variety of CGRP release promoters are known in the art, see, e.g., F. A. Russell et al., Calcitonin Gene-Related Peptide: Physiology and Pathophysiology, Physiol Rev. 2014 Oct;94(4): 1099-1142, doi:10.1152 / physrev.00034.2013; X-F Huang et al., Capsaicin and its analogues: structure-activity relationship study, Curr Med Chem. 2013, 20(21): 2661-72, doi:10.2174 / 0929867311320210004; and CN109293660A.

[0102] In some embodiments, the CGRP release-promoter for use in the present application is a TRPV1 agonist. Transient receptor potential vanilloid subfamily 1 (TRPV1), also known as vanilloid receptor 1 (VR1), is a ligand-gated non-selective cation channel protein. TRPV1 is mainly distributed in the nervous system and expressed in the neural tissue of various internal organs such as the digestive tract and the spleen. TRPV1 can be activated by capsaicin, which makes TRPV1+ neurons release CGRP. Agonists of TRPV1 include, for example, vanilloid compounds such as capsaicin, resiniferatoxin as a capsaicin derivative, cannabidiol, MD-652, etc.; and TRPV1 agonist molecules reported in the following documents: CN101827816B; Wei Xinmiao et al., Application of TRPV1 Channel Function, Gating Mechanism and Modulators in Drug Research and Development [J]. Progress in Biochemistry and Biophysics, 2023, 50(03):421-436, DOI:10.16476 / j.pibb.2022.0198; Siena U D SD, Calabria U D. Biaryl amide or urea derivatives as TRPV1 ligands: USA, WO2015162216. 2015-10-29; CN105198786; WO2023247962A1; US7,446,226; and US7,429,673. For the purposes of the present application, these documents are hereby incorporated by reference in their entirety.

[0103] In some embodiments, the TRPV1 agonist for use in the present application is a vanilloid compound. In other embodiments, the TRPV1 agonist is selected from the group consisting of capsaicin, RTX, piperine, gingerol, shogaol, zingerone, oleyl vanillamide, cis-capsaicin, CA-008, biaryl amide derivatives, and spirofuranone derivatives.

[0104] In some embodiments, the CGRP release-promoter according to the present application is a TRPV1 agonist, and preferably the TRPV1 agonist is administered orally or intragastrically. In other embodiments, the CGRP release-promoter according to the present application is not capsaicin. In other embodiments, the CGRP release-promoter according to the present application is not a TRPV1 agonist.

[0105] In addition to the known activators of the CGRP signaling pathway described above, one skilled in the art can also design and identify variants or analogs and test their agonist activity or CGRP release-promoting effect under the guidance of the present disclosure. A method for identifying non-natural agonists or CGRP release-promoters useful as therapeutic agents, in one embodiment, can comprise: (i) obtaining a variant of a naturally occurring CGRP peptide or an analog of a capsaicin; (ii) testing the ability of the variant to bind to a CGRP receptor, or the ability of the analog to promote the release of CGRP from peripheral nerve terminals; (iii) testing the ability of the variant or analog to stimulate one or more activities mediated by the CGRP signaling pathway (e.g., the number of germinal center B cells and plasma cells in the spleen or lymph nodes, or serum antigen-specific antibody titers); and (iv) identifying variants or analogs that can agonize the CGRP signaling pathway. In another embodiment, the method can comprise screening a peptide library or a compound library for the CGRP receptor agonists or CGRP release-promoters.

[0106] Methods and uses

[0107] Upon exposure to foreign antigens, a mammalian individual will induce a germinal center response in secondary lymphoid tissues, leading to a specific humoral immune response. The present inventors have surprisingly found that the neuropeptide CGRP, based on its immunomodulatory effects, can act on CGRP receptors on B cells in secondary lymphoid tissues and promote germinal center B cell development. Based at least in part on this finding, the present invention proposes the use of activators of the CGRP signaling pathway in anti-infective or anticancer immunotherapy.

[0108] For the purposes of the present invention, a CGRP signaling pathway activator according to the present invention can be administered by any route of administration, e.g., orally, parenterally, topically, for promoting a germinal center response and / or antibody production in a subject, and / or for improving the adaptive immune response, especially the antigen-specific humoral immune response, of a host, as can be appreciated by one skilled in the art. Moreover, for facilitating said administration, in some cases, it can be advantageous to formulate the CGRP signaling pathway activator of the present invention into a pharmaceutical preparation, as can be appreciated by one skilled in the art.

[0109] Thus, in one aspect, the present application provides the use of a CGRP signaling pathway activator according to the present application, or of a pharmaceutical composition comprising a CGRP signaling pathway activator according to the present application and a pharmaceutically acceptable carrier or excipient, for promoting germinal center responses and / or antibody production in a subject, and / or for improving the adaptive immune response, in particular the antigen-specific humoral immune response, of a host, and / or for enhancing a vaccine-induced humoral immune response. The pharmaceutical composition according to the present application is advantageously formulated specifically for the intended route of administration. According to the present application, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" means a compound or combination of compounds, typically a pharmaceutically acceptable carrier / excipient, included in a pharmaceutical composition which does not cause a detrimental reaction in a recipient and, for example, serves one or more of the following: facilitates administration of an active compound, prolongs the life and / or effectiveness of an active compound in the body of a living organism, increases the solubility of an active compound in a solution or improves its storage. Such pharmaceutically acceptable carriers and excipients are well known and can be adapted by a person skilled in the art depending on the nature of the active compound selected and the route of administration.

[0110] In another aspect, the present application also provides a method for promoting germinal center responses and / or antibody production in a subject in need thereof, and / or for improving the adaptive immune response, in particular the antigen-specific humoral immune response, of a subject, and / or for enhancing a vaccine-induced humoral immune response, wherein an effective amount of a CGRP signaling pathway activator according to the present application, or of a pharmaceutical composition comprising the same, is administered to said subject.

[0111] The methods and uses according to the present application can comprise administering a CGRP signaling pathway activator of the present application, or a pharmaceutical composition comprising the same, before, during and / or after the subject is exposed to an antigen, such as a pathogen, a pathogen antigen or a cancer antigen. In some embodiments, the antigen is a vaccine antigen or a composition comprising the same, and the method comprises administering said vaccine antigen or composition thereof to said subject. In some embodiments, the methods and uses further comprise administering to the subject an immunomodulator, such as an adjuvant. According to the present application, the antigen or composition thereof and optionally the immunomodulator administered to the subject for prophylactic or therapeutic purposes, can be formulated separately from the CGRP signaling pathway activator of the present application, in different pharmaceutical compositions, or combined in a single pharmaceutical composition; and can be administered simultaneously or separately from the CGRP signaling pathway activator of the present application, including separately at any suitable interval and order, depending on the therapeutic purpose desired. In some preferred embodiments, the antigen or composition thereof and optionally the immunomodulator administered to the subject, are administered at least 12 hours, at least 24 hours, at least 36 hours, at least 72 hours or more apart from the CGRP signaling pathway activator of the present application.

[0112] In some embodiments, the methods and uses according to the application are for providing or enhancing anti-infective or anti-tumor immunity in a subject. In some embodiments, the infection is a viral, bacterial, fungal and / or pathogen infection. In some embodiments, the infection is a viral infection. In some embodiments, the infection is an influenza virus infection.

[0113] Thus, in one aspect, the application also provides methods for preventing or treating an infection or cancer, comprising administering to a subject in need thereof a CGRP signaling pathway activator of the application or a pharmaceutical composition comprising the same. In some embodiments, the methods further comprise administering to the subject a vaccine antigen against the infection or the cancer. In some embodiments, the methods are methods for preventing or treating an infection, wherein the methods comprise administering a pathogen or a pathogen antigen. In some embodiments, the methods are methods for preventing or treating a cancer, wherein the methods comprise administering a cancer antigen. In some embodiments, the pathogen antigen or cancer antigen induces a B cell humoral immune response against the infection or cancer. In some embodiments, the pathogen antigen or cancer antigen is a T cell dependent antigen. In some embodiments, the methods and uses further comprise or do not comprise administering to the subject an immunomodulator, such as an adjuvant. In some aspects, the methods and uses of the application can be used for preventing or treating (e.g., preventing, ameliorating, alleviating or mitigating) in a subject one or more conditions, pathologies, diseases or symptoms caused by or associated with a pathogen infection or cancer.

[0114] In the present disclosure, pathogens and pathogen antigens that can be used for administration to a subject to induce an immune response include any pathogen or pathogen antigen that is suitable for administration as a vaccine or immunizing agent, and generally can or can potentially provide a protective effect against the pathogen. Such pathogen or pathogen antigen can be provided in the form of a whole pathogen vaccine, a subunit vaccine, or a nucleic acid vaccine. Non-limiting examples of whole pathogen vaccines include inactivated vaccines produced by killing pathogens with chemicals, heat, or radiation; live attenuated vaccines comprising live attenuated pathogens; and chimeric virus vaccines comprising genetic information from different parental viruses and exhibiting biological properties of the different parental viruses. Subunit vaccines contain only the components or antigens that most stimulate the immune system, which design can make the vaccine safer and easier to produce, but often requires incorporation of immunopotentiators to elicit a strong protective immune response. Vaccine antigens used in subunit vaccines can be in various forms such as polysaccharides, proteins, recombinant proteins, polysaccharide-protein conjugates, toxoids, virus-like particles (VLPs), and the like. Nucleic acid vaccines include DNA vaccines and mRNA vaccines that are capable of introducing genetic material encoding one or more antigens that can induce an immune response to a subject. Preferably, the pathogen and pathogen antigens used in the present application are capable of inducing a B cell humoral immune response upon administration to a subject. In some embodiments, the antigens used in the present application are provided in the form of a subunit vaccine, e.g., in the form of a protein antigen.

[0115] In the present disclosure, cancer antigens that can be used for administration to a subject to induce an immune response include any antigen of a cancer or tumor that is suitable for use in treating or enhancing the therapeutic effect of vaccination. Examples of cancer antigens include antigenic substances derived from proteins that are specifically expressed in cancer cells, and that can exert a cancer treatment and / or prevention effect through an immune response by being administered from outside of an organism into the organism. In some aspects, the CGRP signaling pathway activator of the present application can be used as a vaccine for treating and / or preventing cancer by being combined with a cancer antigen.

[0116] Specific examples of pathogen antigens useful in the present application include, but are not limited to, antigens derived from influenza virus, antigens derived from hepatitis A virus, antigens derived from hepatitis B virus, antigens derived from hepatitis C virus, antigens derived from hepatitis D virus, antigens derived from hepatitis E virus, antigens derived from hepatitis F virus, antigens derived from HIV virus, antigens derived from herpes virus (HSV-1, HSV-2), antigens derived from anthrax bacteria, antigens derived from chlamydia, antigens derived from pneumococcus, antigens derived from Japanese encephalitis virus, antigens derived from measles virus, antigens derived from rubella virus, antigens derived from tetanus bacteria, antigens derived from varicella virus, antigens derived from SARS virus, antigens derived from Epstein-Barr virus, antigens derived from papilloma virus, antigens derived from Helicobacter pylori bacteria, antigens derived from rabies virus, antigens derived from West Nile virus, antigens derived from Hanta virus, antigens derived from streptococcus, antigens derived from staphylococcus, antigens derived from Bordetella pertussis bacteria, antigens derived from Mycobacterium tuberculosis, antigens derived from Plasmodium, antigens derived from polio virus, and the like.

[0117] Specific examples of tumor antigens useful in the present application include, but are not limited to, telomerase components; multidrug resistance proteins, such as P-glycoprotein; MAGE-1, alpha-fetoprotein, carcinoembryonic antigen, mutant P53, immunoglobulins of B-cell derived malignancies, fusion polypeptides expressed from genes juxtaposed by chromosomal translocation, human chorionic gonadotropin, calcitonin, tyrosinase, papilloma virus antigens, gangliosides or other carbohydrate-containing components of melanoma or other tumor cells.

[0118] In some preferred embodiments of the application, the methods and uses of the application are for the prevention and / or treatment of a pathogen infection selected from the group consisting of viral infection, bacterial infection, fungal infection, and parasitic infection. Non-limiting examples of viral infections include poxvirus, herpesvirus, hepadnavirus, immunodeficiency virus, flavivirus, papillomavirus (PV), polyomavirus, rhabdovirus, orthomyxovirus, arenavirus, coronavirus, adenovirus, reovirus, picornavirus, togavirus, bunyavirus, parvovirus, and retrovirus. Non-limiting examples of orthomyxovirus include influenza virus, such as influenza A, influenza B, and influenza C. Non-limiting examples of bacterial pathogen infection include Bordetella, Borellia, Brucella, Burkholderia, Campylobacter, Chlamydia, Heliobacter, Legionella, Listeria, Mycoplasma, Mycobacterium, Neisseria, Pseudomonas, Rickettsia, Staphlyococcus, Streptococcus, Salmonella, Shigella, Treponema, Vibrio, and Yersenia. Non-limiting examples of fungal pathogen infection include yeast and mold. More specific examples include Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocysti, and Stachybotrys. Non-limiting examples of parasitic pathogen infection include protozoa. More specific examples include Plasmodium, which causes malaria, Leishmania, Toxoplasma, and Trypanosoma.

[0119] In some particularly preferred embodiments of the application, the methods and uses of the application are for the prevention and / or treatment of a viral infection, particularly an influenza virus infection. In some embodiments, the influenza virus is an H1N1 influenza A virus. In some embodiments, the antigen used in conjunction with the CGRP signaling pathway activator of the application is an influenza virus HA protein antigen.

[0120] Mode of administration

[0121] In some embodiments of the methods and uses of the application, the subject is an individual afflicted with an infection, e.g., an individual afflicted with a chronic or acute infection. The infection can have been ongoing for days, months, or years. In other embodiments of the methods and uses of the application, the subject is an individual at risk for an infection, or an individual who has been exposed recently or will be exposed to an infectious agent. For example, the exposure to the infectious agent can occur days before (e.g., 1-7 days), at the same time as, or days or weeks after (e.g., 1-7 days or 1-2 weeks after the last immunization) administration of the CGRP signaling pathway activator of the application and the vaccine antigen for immunization. Alternatively, the exposure can last for days, weeks, or months or more.

[0122] In some embodiments, the methods of the application are used to prevent or treat an infection, e.g., a viral infection, particularly an influenza virus infection, the methods comprising administering to a subject who has not been exposed to the infection, a CGRP signaling pathway activator of the application and an anti-infective vaccine antigen. In some embodiments, the CGRP signaling pathway activator of the application can be administered continuously for at least 3 days, at least 5 days, at least 7 days, at least 14 days, at least 21 days, or more. In some embodiments, the CGRP signaling pathway activator of the application is administered continuously throughout the entire immunization process of the vaccine antigen. Continuous administration herein can include administration at any suitable dosing frequency for any suitable length of time, e.g., once a day, once every two days, or once every three days, for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks. In some embodiments, administration of the CGRP signaling pathway activator of the application continues for days, weeks, or more after the subject is exposed to the viral infection. In some embodiments, the subject can receive continuous administration of the CGRP signaling pathway activator of the application until 6 hours, 12 hours, 18 hours, or 24 hours after, or 1 day, 2 days, 3 days, 4 days, or 5 days after the symptoms of the infection have disappeared or lessened.

[0123] The above administration regimens are merely illustrative in nature and are not intended to be limiting. Depending on the prophylactic or therapeutic treatment application purposes, the CGRP signaling pathway activator (e.g., CGRP and / or CGRP receptor agonist) according to the present application can also include a one-time administration, e.g., prior to infection, or multiple administrations at intervals or continuously, e.g., considering long-term prophylactic or therapeutic purposes. As an example, the methods and uses according to the present application can include administration of the CGRP signaling pathway activator of the present application or a pharmaceutical composition comprising the same to a subject not more than 2-72 hours, 2-48 hours, 4-24 hours, 4-18 hours, or 6-12 hours before or after, or simultaneously with, or more than 12 hours, 24 hours, 32 hours, 48 hours, 64 hours, 72 hours, contacting a pathogenic antigen or a cancer antigen, or contacting a vaccine antigen. As another example, it can also be contemplated that the CGRP signaling pathway activator of the present application or a pharmaceutical composition comprising the same is administered multiple times, and at least one administration is separated from the time of contacting a pathogenic antigen or a cancer antigen and / or an adjuvant, or contacting a vaccine composition according to the present application, by at least 12 hours, 24 hours, 32 hours, 48 hours, 64 hours, 72 hours or more.

[0124] In some embodiments, the CGRP signaling pathway activator of the present application is administered at least or at most 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 10 hours, or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days (or any range derivable therein) before or after the vaccine antigen. In some embodiments, the CGRP signaling pathway activator of the present application is administered simultaneously with the vaccine antigen. In some embodiments, the CGRP signaling pathway activator of the present application is administered within 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, or 10 hours (or any range derivable therein) of each other of the vaccine antigen. In some embodiments, the CGRP signaling pathway activator of the present application is administered locally to the same site or different sites of the subject as the vaccine antigen. In some embodiments, the CGRP signaling pathway activator of the present application is administered to the subject in the same composition as the vaccine antigen. In some embodiments, the CGRP signaling pathway activator of the present application is administered to the subject in a different composition.

[0125] In some embodiments, the methods and uses according to the present application can comprise: administering a plurality of doses of the vaccine antigen, wherein each subsequent dose of the vaccine antigen is separated from its immediately preceding dose of the vaccine antigen by at least 1 week, at least 2 weeks or at least 3 weeks, and wherein the CGRP signaling pathway activator according to the present application is administered continuously after the first dose of the vaccine antigen, preferably until at least 3 days, at least 5 days or at least 7 days after the last dose of the vaccine antigen. In some embodiments, the methods and uses further comprise: administering the CGRP signaling pathway activator according to the present application continuously for at least 3 days, at least 5 days or at least 7 days prior to the first vaccine antigen.

[0126] For the purposes of the present application, the CGRP signaling pathway activator according to the present application or the pharmaceutical composition comprising the same can be administered by any route of administration, for example oral, parenteral (e.g. intramuscular, intraperitoneal, intravenous, ICV, cisterna injection or infusion, subcutaneous injection or implant), inhalative spray, nasal, vaginal, rectal, sublingual or topical administration. In some embodiments, preferably, the CGRP signaling pathway activator according to the present application, in particular the peptide compound according to the present application, such as a CGRP peptide or a functional analogue thereof, is administered by parenteral (e.g. intravenous or subcutaneous) injection. In some embodiments, preferably, the CGRP signaling pathway activator according to the present application, in particular the small molecule chemical compound according to the present application, such as a TPRV1 agonist, such as a capsaicin or a functional analogue thereof, is administered by gastric or oral route.

[0127] CGRP can induce migraine attacks in some cases due to its vasodilatory properties. When applying the CGRP signaling pathway activator of the present application for prophylaxis and treatment, it can be envisaged by the person skilled in the art to reduce these side effects by adjusting the administration regimen, including but not limited to adjusting the administration interval when administered continuously, the administered dose, etc. It can also be envisaged to reduce the side effects of the administered CGRP signaling pathway activator by supplementally using a compound that reduces said side effects, for example a compound with vasoconstrictive properties. In some cases, at least one vasoconstrictor can thus be added to the pharmaceutical composition of the present application or administered in combination with the pharmaceutical composition of the present application.

[0128] While the specific dose depends on the molecular structure and chemical properties of the particular compound of the present application that is administered, the person skilled in the art of pharmacology will appreciate from the disclosure herein that the appropriate dose can be determined using routine techniques. For example, the dose or dosage regimen of a compound of the present application can be determined in various ways. Preferably, the dose or dosage regimen does not cause or only minimally causes undesired side effects (e.g. migraine) in the mammal.

[0129] The amount of a compound of the application to be administered to a subject to achieve a target level or concentration of the compound of the application depends on many factors well known to medical practitioners, e.g., the compound's half-life (e.g., serum half-life) and frequency and mode of administration. To illustrate and without limitation, in the case of CGRP peptides, a dose of a compound of the application in the range of 20 picograms to 1 gram, or a dose of 3 nanograms to 50 micrograms per day can be administered. In various embodiments, the unit dose (in some cases, the daily dose) is less than about 10 micrograms, less than about 1 microgram, less than about 100 nanograms, less than about 10 nanograms, less than about 1 nanogram, less than about 100 picograms, or less than about 10 picograms.

[0130] In the case of naturally occurring CGRP-alpha polypeptides (e.g., human CGRP-alpha), in some cases, pharmaceutical formulations that result in plasma or serum concentrations of CGRP-alpha in the EC 50 range of CGRP receptors (e.g., in the range of 10 -11 M to 10 -13 M) are useful. Exemplary doses for CGRP-alpha and functional variants thereof include amounts that can result in plasma or serum levels of between about 10 -15 M and about 10 -10 M, between about 10 -15 M and about 10 -11 M, or between about 10 -15 M and about 10 -12 M. Useful plasma or serum levels are typically < 10 -10 M. It will be appreciated that these ranges are exemplary and not limiting. Plasma and serum levels of compounds (including non-peptide compounds) can be measured by routine methods, e.g., ELISA, RIA, spectroscopy, enzymatic assays, or other methods.

[0131] It will be appreciated that for any particular subject, specific dosage levels and frequency of administration depend on a variety of factors, including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, and the severity of the particular condition. In some embodiments, administration of a compound of the application (e.g., a CGRP peptide or functional analog thereof) is contemplated on a daily or weekly basis. Appropriate dosage ranges for a compound of the application will be readily apparent to those skilled in the art based upon initial dose-response curves and other data that can be obtained by routine methods.

[0132] In some embodiments, the amount of CGRP peptide or functional analog thereof administered to a human or non-human primate subject corresponds to a dose in a mouse of equal to or greater than 50 pg / kg, 100 pg / kg, 150 pg / kg, 200 pg / kg, 250 pg / kg, 300 pg / kg, 350 pg / kg, 400 pg / kg, 450 pg / kg, or 500 pg / kg. In some embodiments, the amount of CGRP peptide or functional analog thereof administered to a human or non-human primate subject corresponds to a dose in a mouse of greater than or less than 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 125 pg, 150 pg, 175 pg, 200 pg, 225 pg, 250 pg, 275 pg, 300 pg, 325 pg, 350 pg, 375 pg, 400 pg, 425 pg, 450 pg, 475 pg, 500 pg, 525 pg, 550 pg, 575 pg, 600 pg, 625 pg, 650 pg, 675 pg, 700 pg, 725 pg, 750 pg, 775 pg, 800 pg, 825 pg, 850 pg, 875 pg, or 900 pg (or any derivable range therein). In some embodiments, the CGRP peptide or functional analog thereof is administered by intraperitoneal or intravenous injection. In some embodiments, the CGRP peptide or functional analog thereof is administered continuously for at least 5 days, 7 days, 9 days, 11 days, 14 days, 17 days, 21 days, or more. In some embodiments, the CGRP peptide or functional analog thereof is administered beginning no more than 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 10 hours or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after administration of a vaccine antigen. In some embodiments, the administration is prophylactic. In some embodiments, the method is used to prevent viral infection, such as influenza virus infection.

[0133] In some embodiments, the amount of capsaicin or functional analog thereof administered to a human or non-human primate subject corresponds to a dose in a mouse of equal to or greater than 50 μg / kg, 100 μg / kg, 150 μg / kg, 200 μg / kg, 250 μg / kg, 300 μg / kg, 350 μg / kg, 400 μg / kg, 450 μg / kg, or 500 μg / kg. In some embodiments, the amount of CGRP peptide or functional analog thereof administered to a human or non-human primate subject corresponds to a dose in a mouse of greater than or less than 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 125 μg, 150 μg, 175 μg, 200 μg, 225 μg, 250 μg, 275 μg, 300 μg, 325 μg, 350 μg, 375 μg, 400 μg, 425 μg, 450 μg, 475 μg, 500 μg, 525 μg, 550 μg, 575 μg, 600 μg, 625 μg, 650 μg, 675 μg, 700 μg, 725 μg, 750 μg, 775 μg, 800 μg, 825 μg, 850 μg, 875 μg, or 900 μg (or any derivable range therein). In some embodiments, the capsaicin or functional analog thereof is administered orally. In some embodiments, the capsaicin or functional analog thereof is administered continuously for at least 5 days, 7 days, 9 days, 11 days, 14 days, 17 days, 21 days, 25 days, 30 days, 35 days, or more. In some embodiments, the capsaicin or functional analog thereof is administered continuously for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 9 days, 11 days, or 14 days prior to administration of a vaccine antigen, and preferably, the continuous administration is continued for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after administration of the vaccine antigen. In some embodiments, the capsaicin or functional analog thereof is continued to be administered continuously for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after exposure of the subject to a pathogen. In some embodiments, the administration is prophylactic. In some embodiments, the method is used to prevent a viral infection, such as an influenza virus infection.

[0134] Administration of vaccine antigens according to the disclosure can be by any common route, so long as the target tissue can be reached by this route in order to maximize antigen delivery to the site for maximum (or in some cases minimum) immune response. Administration is typically by in situ, intradermal, mucosal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Other delivery areas include: oral, nasal, buccal, rectal, vaginal, or topical. Vaccines of the invention are preferably administered parenterally, e.g., by subcutaneous or intramuscular injection. Vaccine antigens according to the disclosure can be given in a single dose regimen or a multiple dose regimen. A multiple dose regimen can include, for example, 1 to 10 separate doses, followed by other doses at subsequent time intervals as needed to maintain and / or boost the immune response, e.g., a second dose in 1 to 4 months, and if needed, subsequent dose(s) months later. Periodic boosting is expected every 1 to 5 years (typically 3 years) to maintain the desired level of protective immunity.

[0135] Pharmaceutical combination and vaccine composition

[0136] In one aspect, the present invention provides a pharmaceutical combination comprising (a) a CGRP signaling pathway activator of the present invention or a pharmaceutical composition containing the same; and (b) a vaccine composition comprising a vaccine antigen (e.g., a pathogen, a pathogen antigen, or a cancer antigen).

[0137] In another aspect, the present invention provides a vaccine composition comprising (a) a CGRP signaling pathway activator of the present invention and (b) a vaccine antigen (e.g., a pathogen, a pathogen antigen, or a cancer antigen) formulated together therewith.

[0138] In some embodiments, the pharmaceutical combinations and vaccine compositions according to the present application further comprise or do not comprise an immunomodulator, e.g., an adjuvant. The immunomodulator can be present independently of the (a) component or the (b) component described above, or in combination with (a) or (b) or both. Adjuvants that can be used in the methods, uses, pharmaceutical combinations, and vaccines of the present application include, but are not limited to, aluminum salts, Freund’s complete or incomplete adjuvant, MF59, AS03, CpG, AddaVax, LPS, CpG ODN 1018, MPLA, Pam3CSK4, Pam2CSK4, R848, 2BXy, QS-21, AS01B, or combinations thereof. For a description of adjuvants, see, e.g., An Overview of Vaccine Adjuvants: Current Evidence and Future Perspectives, Vaccines (Basel), 2022 May; 10(5): 819, doi:10.3390 / vaccines10050819. In some embodiments, the adjuvant is an aluminum adjuvant.

[0139] In related aspects, the present application also provides unit dosage forms of the compounds of the present application for administration to a subject. As used herein, "unit dosage form" means a composition administered singly for treatment of a subject suffering from a disease or medical condition. Each unit dosage form typically contains each of the compounds of the present application plus pharmaceutically acceptable excipients. Examples of unit dosage forms are single tablets, single capsules, bulk powders, liquid solutions, suppositories, emulsions or suspensions. Treatment of a disease or condition requires periodic administration of unit dosage forms, e.g., one unit dosage form two or more times a day, one unit dosage form with each meal, one unit dosage form every four hours or other intervals, or only one unit dosage form per day. Injection preparations of unit dosage forms can be provided, e.g., in vials or multiple dose containers. Unit dosage forms of the present application contain a therapeutically effective dose of a compound of the present application. In one embodiment, administration of a unit dosage form produces a suitable level of a compound of the present application (e.g., a CGRP peptide or functional analog thereof) in a mammal.

[0140] The pharmaceutical compositions and vaccine compositions according to the present application can be in any formulation form suitable for administration of the compounds and vaccine antigens of the present application, e.g., in the form of sterile aqueous injection solutions or oleaginous suspensions. Such suspensions can be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents. The sterile injection can also be a sterile injection solution or suspension in a non-toxic, parenterally acceptable diluent or solvent.

[0141] If desired (e.g., to maintain a particular plasma concentration), the compounds of the application (e.g., CGRP peptides or functional analogs thereof) can be administered to a subject in a controlled delivery formulation. Various suitable controlled delivery systems are known, including formulations suitable for oral, parenteral, and other routes of administration. Excipients used in the preparation of drug delivery systems are described in various publications known to those skilled in the art. The U.S. Pharmacopeia provides examples of many modified release oral dosage forms. This publication also provides general chapters and specific tests for determining the drug release capabilities of delayed and extended release tablets and capsules.

[0142] In one aspect of the application, the compounds of the application (e.g., CGRP peptides or functional analogs thereof) are administered in conjunction with an immunization regimen in which a vaccine antigen is administered, thereby enhancing the antigen-specific humoral immune response and / or long-term immune memory in the subject.

[0143] Examples

[0144] Experimental animals

[0145] Wild-type C57BL / 6 mice used in the following examples were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd., weighing about 20 g, 8-12 weeks old.

[0146] Cd19Cre / +Calcrl fl / fl mice, by Calcrl fl / fl Mice (S-CKO-11816, Sibiongen) and Cd19-Cre (Jax 006785, The Jackson Laboratory) were mated to produce offspring.

[0147] Experimental methods

[0148] 1. Immunization of mice with NP-KLH

[0149] NP-KLH, a hapten conjugate of 4-hydroxy-3-nitrophenyl acetyl hapten (NP) and keyhole limpet hemocyanin (KLH), was used to immunize mice. Briefly, 25 mg of NP-KLH (N-5060-25, Biosearch Technologies) was added to 25 mL of autoclaved deionized water, and the final concentration of the antigen was 1 mg / mL. After the antigen was completely dissolved, it was aliquoted in the dark. When immunizing mice, 100 μg of NP-KLH was added to 100 μL of aluminum adjuvant (77161, ThermoFisher Scientific), and the mixture was thoroughly mixed and then injected intraperitoneally into mice for a single immunization.

[0150] 2. Administration of drugs to mice

[0151] 1) CGRP injection. CGRP stock solution was diluted with sterile PBS, and prepared fresh every day. After NP-KLH immunization, mice were injected intraperitoneally with 200 μL PBS or CGRP (500 μg kg-1, RP11095, GenScript) every day using a 1 mL syringe, and injected continuously for 13 days. This CGRP peptide is a C-terminal amidated peptide with the amino acid sequence shown in SEQ ID NO: 3 (SCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSEAF-NH2).

[0152] 2) Capsaicin (HY-10448, MCE) feeding. Capsaicin was mixed into the mouse feed (100 mg / kg), and mice were fed with it for one week before NP-KLH immunization. After NP-KLH immunization, mice were continuously fed with capsaicin for 13 days.

[0153] 3. Flow cytometry analysis of splenic germinal center B cells and plasma cells

[0154] 1) Mice were sacrificed by cervical dislocation, and the spleen was removed and placed in FACS buffer (PBS + 1% FBS + 5 mM EDTA, pH 7.4).

[0155] 2) A 70 μm blue filter was placed on a 50 mL centrifuge tube, and the spleen was placed on the filter. The spleen was ground with a syringe, and FACS buffer was added to make a single cell suspension.

[0156] 3) 1500 r / min, 4°C, 5 min.

[0157] 4) The supernatant was discarded with a pipette, and the cell pellet in the centrifuge tube was retained. 2 mL ACK red blood cell lysis solution was added to resuspend the cells, and the cells were lysed at room temperature for 3 min.

[0158] 5) 8 mL FACS buffer was added to stop the lysis of red blood cells, and the cell suspension was passed through the filter again to remove the red blood cell clumps, resulting in a single cell suspension.

[0159] 6) 1500 r / min, 4°C, 5 min.

[0160] 7) The cells were resuspended with 400 μL FACS buffer, and the cell density was adjusted to 10 8 / ml.

[0161] 8) 100 μL of the single cell suspension of the spleen was taken, and 1 μg of anti-CD16 / 32 antibody was added to block the IgG antibody receptors, and the mixture was incubated on ice for 10 min.

[0162] 9) The following protocol was used for fluorescent antibody staining:

[0163] Staining protocol for splenic germinal center B cells and plasma cells

[0164] After staining, cells were washed with 1 mL FACS buffer, 3500 r / min, 4°C, 3 min. The supernatant was discarded, and the cells were resuspended with 300 μL PBS, ready for flow cytometry analysis.

[0165] 4. Anti-NP antibody avidity experiment

[0166] NP-KLH immunized mice were bled 13 days after the last immunization. The blood samples were allowed to stand at room temperature for 3 h, and then centrifuged at 6000 r / min, 4°C, 10 min. The serum was collected. NP3-BSA (N-5050XL, Biosearch Technologies) or NP 27 -BSA (N-5050H, Biosearch Technologies) at 5 mg / ml in water was diluted to 12.5 μg / mL, and 100 μL of NP3-BSA or NP 27 -BSA antigen was added to each well of an ELISA-specific 96-well plate and incubated overnight at 4°C. After the plate was washed with washing buffer (PBS + 0.05% Tween-80) four times, 100 μL of blocking solution (PBS + 10% FBS) was added to each well, and the plate was incubated at room temperature for 1 h. After the liquid in the wells was removed, the mouse serum diluted with the blocking solution (eight gradients from 1:4000 or 1:20000) was added, and the plate was incubated at 37°C for 1 h. After the plate was washed with the washing buffer four times, 100 μL of secondary antibody (horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody (115-035-003, Jackson ImmunoResearch, 1:5000) diluted 1:5000 with the blocking solution) was added to each well, and the plate was incubated at 37°C for 1 h. After the plate was washed with the washing buffer four times, 100 μL of tetramethylbenzidine (TMB) substrate was added to each well, and the plate was incubated at 37°C for about 10 min in the dark to develop color. The color development reaction was terminated by adding 100 μL of 1 mol / L HCl to each well, and the OD value of each well was immediately measured at 450 nm using an enzyme-labeled instrument.

[0167] 5. Detection of CGRP content in mouse spleen

[0168] The spleen was weighed and then homogenized using a grinder. After homogenization, the supernatant was collected by centrifugation and used for ELISA analysis. The spleen CGRP level was measured according to the CGRP EIA kit (589001, Cayman Chemical). The final result was expressed as the CGRP mass concentration normalized by the weight of the spleen.

[0169] 6. Immunization of mice with influenza virus HA protein

[0170] 100 μg of HA protein (11684-V08H, Sino Biological) was taken and added to 250 μL of high-pressure deionized water to prepare a solution with a final concentration of 400 μg / mL of antigen. After the antigen was fully dissolved, 1 μg of HA protein was added to the aluminum adjuvant, mixed thoroughly, and then injected intraperitoneally into the mice for immunization. After 21 days, 1 μg of HA protein was again added to the aluminum adjuvant, mixed thoroughly, and then injected intraperitoneally into the mice. Capsaicin (HY-10448, MCE) was fed to the mice during the entire immunization process.

[0171] 7. Influenza virus challenge

[0172] On the ninth day after the second immunization with HA protein, the mice were anesthetized with sodium pentobarbital. The H1N1 influenza A virus was prepared at a virus titer of 5 LD50 or 30 LD50, and then the mice were infected by nasal instillation (40 μl per mouse). LD50 refers to the median lethal dose, defined as the minimum amount of influenza virus required to cause half of the mice of a certain weight to die within a specified time through nasal instillation.

[0173] 8. Detection of indicators after influenza virus challenge

[0174] As shown in Figure 10, on the fourteenth day after the first immunization with HA protein and the seventh day after the second immunization, orbital blood was taken to determine the level of HA-specific IgG antibodies in the mice. After the influenza virus challenge, the body weight and survival of the mice were recorded daily, and a mouse was determined to have died when its body weight was less than 75%. On the fifth day after the influenza virus challenge, the lungs of the mice were taken for HE staining and fluorescent quantitative PCR to detect the level of lung inflammation and virus content in the mice.

[0175] 9. Anti-HA antibody affinity experiment

[0176] The affinity of anti-HA antibody was detected by ELISA. In brief, the mice were taken blood from the orbita on day 14 after the first immunization and day 7 after the second immunization. The blood sample was placed at room temperature for 3 h, then centrifuged at 4°C, 6000 r / min for 10 min, and the serum was collected. The HA protein was diluted to 1 μg / mL with PBS, and 100 μL of 1 μg / mL HA protein antigen was added to each well of the ELISA special 96-well plate, which was coated at 4°C overnight. After the enzyme-labeled plate was washed repeatedly 4 times with washing solution (PBS+0.05% Tween-80), 100 μL of blocking solution (PBS+10% FBS) was added to each well, and the plate was blocked at room temperature for 1 h. After removing the liquid in the wells, 1:200 diluted mouse serum was added to each well, and the plate was incubated at 37°C for 1 h. After the enzyme-labeled plate was washed repeatedly 4 times with washing solution, 100 μL of secondary antibody (horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody (115-035-003, Jackson ImmunoResearch, 1:5000) diluted 1:5000 with blocking solution) was added to each well, and the plate was incubated at 37°C for 1 h. After the enzyme-labeled plate was washed repeatedly 4 times with washing solution, 100 μL of tetramethylbenzidine (TMB) substrate was added to each well, and the plate was incubated at 37°C for about 10 min in the dark for color development. The color development reaction was terminated by adding 100 μL of 1 mol / L HCl to each well, and the OD value of each well was immediately measured at 450 nm wavelength by an enzyme-labeled instrument.

[0177] 10. HE staining to detect the level of inflammation in the lung of mice

[0178] The mice were sacrificed by cervical dislocation 5 days after infection with influenza virus, and their lungs were taken. The separated tissues were placed in 4% paraformaldehyde and fixed at 4°C overnight for paraffin embedding. A paraffin section machine was used to prepare 3-5 μm paraffin sections. After the sections were stained with hematoxylin for the nucleus and neutral resin for mounting, the sections were scanned by NanoZoomer.

[0179] 11. RT-qPCR to detect the expression of B cell Calcrl and influenza virus nucleoprotein NP mRNA in the lung of mice

[0180] The mice were sacrificed by cervical dislocation, and their spleens and lungs were taken. The spleens were ground into single-cell suspensions, and the spleen B cells were sorted by magnetic beads according to the B cell separation kit (130-090-862, Miltenyi Biotec). The total RNA of the spleen B cells and lung tissues was extracted by the TRIzol method, and the cDNA was synthesized using the Prime Script RT Master Mix (TaKaRa) reverse transcription kit. The reaction system was prepared: 500 ng of cDNA, 2 μL of 5x Prime Script Mix (RR036A-1, TaKaRa), and DEPC water was supplemented to 10 μL; and the fluorescence quantitative PCR detection was performed according to the Power Up SYBR Green Master Mix (AB) kit usage method. The Calcrl gene PCR primer sequence F-ATCTCAGCAGAGTCGGAAGAA, R-CAGGTCCTATTGCAGTAAAGGC; and the influenza virus NP gene PCR primer sequence F-TGCTTCAAAACAGCCAAGTG, R-GATGCCCTCTGTTGATTGGT were synthesized by Shenguo Biotechnology Co., Ltd. After the RT-qPCR, the hypoxanthine phosphoribosyltransferase (HPRT) was used as an internal reference gene, and the relative expression of the related genes was calculated by the ΔΔCt method.

[0181] 12. Resin toxin RTX intra-ganglion injection ablates mouse TRPV1 neurons

[0182] To specifically ablate TRPV1 in T8-T13 dorsal root ganglion + Nociceptors, 6-week-old wild-type C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were injected with solvent (0.25% DMSO / 0.02% Tween-80 / 0.05% ascorbic acid / PBS) or resin toxin (RTX, R8756, Sigma, 3 ng dissolved in 200 nl of solvent) into individual dorsal root ganglion. The mice were recovered for 4 weeks before the subsequent experiments were performed.

[0183] Example 1

[0184] Intravenous injection of CGRP promotes the percentage of germinal center B cells and plasma cells

[0185] To verify the effect of CGRP on B cell differentiation, we immunized mice with the immunological classic antigen NP-KLH plus aluminum adjuvant. NP-KLH is a T cell-dependent antigen that can induce germinal center responses in mice. After immunization with NP-KLH, the germinal center response gradually strengthens. By detecting the proportion of germinal center B cells 7 days, 13 days and 21 days after NP-KLH immunization, it has been found that the proportion of germinal center B cells is the highest at 13 days, and then begins to decline (see, Chen, D. et al. Coupled analysis of transcriptome and BCR mutations reveals role of OXPHOS in affinity maturation. Nat Immunol 22, 904-913 (2021). https: / / doi.org / 10.1038 / s41590-021-00936-y). Therefore, we selected the time point of harvesting the spleen after NP-KLH immunization as 13 days after immunization.

[0186] Using the methods described in Experimental Methods Part 1-3, wild-type C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were first injected intraperitoneally with 100 μg of NP-KLH antigen for single immunization. After immunization, the mice were randomly divided into groups and received intraperitoneal injection of PBS (11) or CGRP (10) once a day. After 13 days, the mice were sacrificed and the spleens were harvested, and the proportions of splenic germinal center B cells and plasma cells were analyzed by flow cytometry. The FACS circle gate strategy is shown in Figure 1. As shown in the flow cytometry results in Figure 2, after NP-KLH antigen immunization, compared with the PBS group, CGRP injection significantly promoted the increase in the proportion of splenic germinal center B cells and plasma cells (unpaired T test, p values were 0.0002 and 0.0007, respectively) (Figure 2).

[0187] In vivo injection of CGRP promotes the generation of antigen-specific antibodies

[0188] To determine whether CGRP can promote the production of antigen-specific antibodies in serum while promoting the formation of germinal center B cells and plasma cells. After immunizing wild-type mice with NP-KLH for 13 days as described above, we used the method described in Experimental Methods Part 4 to measure the total (anti-NP 27 -BSA) carrying different numbers of NP hapten groups (NP3-BSA and NP 27) and high affinity (anti-NP3) NP-specific IgG antibody titers. As shown in Figure 3, the levels of both total and high affinity NP antigen-specific antibodies in the sera of the CGRP-injected mice were significantly higher than those of the control mice (Two-way ANOVA test, p values < 0.0001).

[0189] Deletion of B cell CGRP receptor significantly inhibits the percentage of germinal center B cells, plasma cells and antibody production

[0190] We further investigated the mechanism by which CGRP modulates the germinal center response. As shown in Figure 4, interestingly, we found that the total B cells in the spleen expressed high levels of the neuropeptide CGRP receptor CALCRL. Next, we investigated whether the effect of CGRP on the germinal center response was dependent on the receptor CALCRL.

[0191] Specifically, we generated Cd19 Cre / + Calcrl fl / fl mice to conditionally knock out Calcrl in all B cells. From the generated Cd19 Cre / + Calcrl fl / fl mice and their parental Cd19 Cre / + mice, we harvested the spleens, extracted total B cells, and detected the expression levels of Calcrl by RT-qPCR using the methods described in Experimental Methods Section 11. As shown in Figure 4, compared to the high level of CALCRL expression in the spleen B cells of the parental mice, the Calcrl expression in the spleen B cells of the Cd19 Cre / + Calcrl fl / fl mice was successfully knocked out.

[0192] Subsequently, we immunized the Cd19 Cre / + Calcrl fl / fl mice and their parental Cd19 Cre / + mice with NP-KLH, administered CGRP injection, performed flow cytometry analysis of the spleen germinal center B cells (i.e., GC B cells) and plasma cells, and detected the serum anti-NP antibody titers using the methods described in Experimental Methods Sections 1-4. It was observed that the Cd19 Cre / + Calcrl fl / fl mice had significantly reduced spleen germinal center B cells (GC B cells), spleen plasma cells (SPPC), and the production of antigen NP-specific antibodies compared to the Cd19 Cre / + mice (Figures 5 and 6). These findings indicate that the expression of the receptor CALCRL on B cells is critical for the role of CGRP in promoting the germinal center response.

[0193] Example 2

[0194] Capsaicin feeding promotes germinal center B cells, plasma cells percentage and antibody production in mice

[0195] It is known that CGRP is a neuropeptide released by nociceptive neurons. Based on the role of CGRP in the germinal center reaction, we hypothesized that ingestion of spicy food containing capsaicin, a natural TRPV1 agonist that triggers nociceptive neuron activation and CGRP release, might potentially enhance humoral immunity.

[0196] Wild type C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were intraperitoneally injected with 100 μg NP-KLH antigen for single immunization (10 mice per group) using the methods described in Experimental Procedures Part 1-2. One week before immunization and for 13 consecutive days after immunization, mice were fed with capsaicin-spiked chow (100 mg capsaicin per 1 kg chow). Thirteen days after immunization, mice were sacrificed and their spleens were taken for analysis of CGRP expression relative to control mice not fed with capsaicin using the methods described in Experimental Procedures Part 5. The percentage of germinal center B cells and plasma cells in the spleen and the titer of NP-specific antibodies in the serum of mice were analyzed using the methods described in Experimental Procedures Part 3-4.

[0197] The data showed that capsaicin indeed led to a significant increase in CGRP levels in the spleen of mice (Figure 7, p-value < 0.0203, unpaired T-test relative to control). Moreover, in line with our hypothesis, NP-KLH immunized mice that ingested a capsaicin diet exhibited a significantly enhanced humoral immune response, including increased GC B cells, SPPC, and NP-specific antibody production (Figures 8 and 9).

[0198] Capsaicin feeding enhances vaccine-induced humoral immune responses

[0199] We investigated the effect of capsaicin diet on the protective effect of vaccine antigens against influenza A virus (IAV) infection. The experimental protocol is shown in Figure 10. Vaccine immunization, virus challenge, and readout were performed according to the methods described in Experimental Procedures Part 6-11.

[0200] Briefly, control wild type C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were fed with normal mouse chow; experimental mice were fed with capsaicin-spiked chow (100 mg capsaicin per 1 kg chow). After one week of feeding, control or capsaicin-fed mice were intraperitoneally injected with hemagglutinin (HA) antigen from influenza virus (PR8 strain) for immunization, followed by challenge with 30 LD50 of PR8 strain. PR8 is a H1N1 influenza virus against mice, which is known to cause severe infection in mice.

[0201] The results showed that capsaicin significantly increased the HA-specific IgG antibody titers after immunization (10 mice per group, Fig. 11). Notably, the mice fed with capsaicin had significantly reduced lung viral load and immune cell infiltration after PR8 virus infection (6 mice per group for lung viral load assay, 3 mice per group for HE staining, Fig. 12 and Fig. 13). Importantly, capsaicin treatment effectively prevented the body weight loss and significantly improved the survival rate of mice (10 mice per group, Fig. 14 and Fig. 15). Collectively, these results suggest that the activation of nociceptive neurons by dietary capsaicin, which enhances the germinal center reaction promoted by CGRP in secondary lymphoid organs, can enhance the vaccine-induced humoral immune response and improve the host defense against viral infection.

[0202] Example 3

[0203] Effect of ablation of splenic TRPV1 neurons on capsaicin-promoted antigen-specific humoral immune response

[0204] To further confirm that the capsaicin-promoted antigen-specific humoral immune response is dependent on TRPV1 neurons, we first injected 6-week-old wild-type C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) with intra-ganglion RTX to specifically ablate TRPV1+ nociceptors in T8-T13 dorsal root ganglia, using the method described in Experimental Methods Section 12. As shown in Fig. 16, intra-ganglion injection of RTX significantly reduced splenic CGRP levels in mice with ablated splenic TRPV1 neurons, relative to control mice injected with solvent (Fig. 16, p-value < 0.0001, unpaired T-test).

[0205] Wild-type C57BL / 6 mice (9) injected with intra-ganglion solvent and wild-type C57BL / 6 mice (21) injected with intra-ganglion RTX were immunized with 100 pg NP-KLH antigen intraperitoneally, using the method described in Experimental Methods Sections 1-2. A subset of mice (10) injected with intra-ganglion RTX were fed with capsaicin-spiked chow (100 mg capsaicin per 1 kg chow) for 13 days before and after immunization (Fig. 17). The proportion of splenic germinal center B cells and plasma cells was analyzed by flow cytometry, according to the method described in Experimental Methods Section 3.

[0206] The data showed that the humoral immune response was attenuated in mice injected with intra-ganglion RTX, including reduced GC B cell and SPPC production. Meanwhile, the data showed that capsaicin failed to promote the humoral immune response in mice injected with intra-ganglion RTX (Fig. 18). This indicates that the capsaicin-promoted antigen-specific humoral immune response is dependent on TRPV1 neurons.

[0207] Dependence of capsaicin feeding-enhanced vaccine-induced humoral immune response on TRPV1 neurons

[0208] To further confirm that capsaicin feeding enhances vaccine-induced humoral immune responses depend on TRPV1 nerves, the experimental protocol as shown in Figure 19 was implemented. The vaccine immunization, virus challenge and index detection were performed according to the methods described in Experimental Methods Section 6-11.

[0209] Briefly, wild type C57BL / 6 mice (10, Beijing Vital River Laboratory Animal Technology Co., Ltd.) injected with solvent in the node and half of wild type C57BL / 6 mice (10, Beijing Vital River Laboratory Animal Technology Co., Ltd.) injected with RTX in the node were fed with normal mouse feed; the other half of wild type C57BL / 6 mice (10) injected with RTX in the node were fed with feed mixed with capsaicin (100 mg capsaicin / 1 kg feed). After one week of feeding, all mice were immunized with hemagglutinin (HA) antigen of influenza virus (PR8 strain) by intraperitoneal injection, and then challenged with 5LD50 of influenza virus PR8 strain.

[0210] The results showed that the weight loss and survival rate of mice injected with RTX in the node were more obvious than those of mice injected with solvent in the node. At the same time, the phenotype of weight loss and survival rate could not be alleviated by eating capsaicin after RTX injection in the node (Figure 20). Overall, these results suggest that the activation of nociceptive neurons by dietary capsaicin enhances the germinal center response promoted by CGRP in secondary lymphoid organs, which can enhance vaccine-induced humoral immune responses and improve the host's defense against viral infection.

[0211] Effect of CGRP receptor knockout on vaccine immunization

[0212] To confirm the role of CGRP receptor Calcrl in vaccine-induced humoral immune responses, Cd19 Cre / + Calcrl fl / fl Mice, the animal experiment as shown in Figure 21 was implemented. The vaccine immunization, virus challenge and index detection were performed according to the methods described in Experimental Methods Section 6-11.

[0213] Briefly, control Cd19 Cre / + mice (10) and Cd19 Cre / + Calcrl fl / fl mice (10) were immunized with hemagglutinin (HA) antigen of influenza virus (PR8 strain) by intraperitoneal injection, and then challenged with 5LD50 of influenza virus PR8 strain. It was observed that Cd19 Cre / + Calcrl fl / fl mice with Cd19 Cre / +The weight loss was more pronounced and the survival rate was lower in mice compared to the controls (Figure 22). These findings further suggest that the expression of the receptor CALCRL on B cells is essential for the promotion of vaccine-elicited humoral immune responses by CGRP.

Claims

1. A method for enhancing an antigen-specific humoral immune response, comprising administering an effective amount of a CGRP signaling pathway activator to a subject in need thereof.

2. The method of claim 1, wherein the subject has been exposed to the antigen, or has not been exposed to the antigen, prior to administration of the CGRP signaling pathway activator.

3. The method of claim 1 or 2, wherein the CGRP signaling pathway activator is a CGRP receptor agonist.

4. The method according to any one of claims 1 to 3, wherein the CGRP signaling pathway activator is a CGRP peptide or a functional analogue thereof, preferably the CGRP peptide has an amino acid sequence selected from SEQ ID NOs: 1-6.

5. The method of any one of claims 1-4, wherein the CGRP signaling pathway activator is a CGRP release promoter.

6. The method of any one of claims 1-5, wherein the method further comprises administering the antigen and, optionally, an immunomodulator to the subject.

7. The method of any one of claims 1 to 6, wherein the antigen is a viral, bacterial, fungal and / or parasitic pathogen antigen, or the antigen is a cancer antigen.

8. The method of any one of claims 1-7, wherein the subject has, or is at risk of having, an infectious disease or cancer.

9. The method of claim 8, wherein the infectious disease is a viral, bacterial, fungal and / or parasitic infection.

10. The method of claim 9, wherein the infectious disease is influenza virus infection.

11. The method of any one of claims 1 to 10, wherein the method comprises one or more of the following: (i) administering the CGRP signaling pathway activator and the antigen in separate compositions, and optionally, administering the CGRP signaling pathway activator and the antigen at different time points and / or at different sites in the subject; (ii) administering one or more doses of the antigen for immunization, and administering multiple doses of the CGRP signaling pathway activator after each dose of the antigen, and optionally administering the multiple doses once a day, once every two days, or once every three days; (iii) at least one dose of the CGRP signaling pathway activator, administered before administering the antigen; (iv) the interval between the administration of the CGRP signaling pathway activator and the administration of the antigen is at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours; and / or (iv) continuously administering the CGRP signaling pathway activator for at least 3 days, 5 days, 1 week, 2 weeks, or 3 weeks, or longer.

12. The method of any one of claims 1 to 11, wherein: - the CGRP signaling pathway activator is a CGRP receptor agonist, and is preferably administered by intravenous or intraperitoneal injection; or - The CGRP signaling pathway activator is a CGRP release promoter, and is preferably administered intragastrically or orally.

13. The method of any one of claims 1 to 12, wherein the method is used for: - Promotes germinal center responses in the subject's secondary lymphoid organs; - increasing the ratio of germinal center B cells and plasma cells; and / or - increasing the titer of antigen-specific antibodies in the serum of the subject.

14. A method for preventing or treating an infection or a cancer, comprising administering to a subject in need thereof an effective amount of a CGRP signaling pathway activator as defined in any one of claims 1 or 3-5.

15. The method of claim 14, further comprising administering to the subject a vaccine composition comprising an antigen.

16. The method of claim 14 or 15, wherein the infection is a viral, bacterial, fungal or parasitic infection, in particular a viral infection, preferably an influenza virus infection.

17. A pharmaceutical combination comprising components (a) a CGRP signaling pathway activator as defined in any one of claims 1 or 3-5 and (b) a vaccine composition comprising an antigen.

18. A vaccine composition comprising a CGRP signaling pathway activator of any one of claims 1 or 3-5 and one or more antigens, and optionally an immunomodulator.

19. The pharmaceutical combination of claim 17 or the vaccine composition of claim 18, wherein the antigen is an influenza virus antigen.

20. A CGRP signaling pathway activator of any one of claims 1 or 3-5, a pharmaceutical combination of any one of claims 17-19 or the use of a vaccine composition for use as a medicament, or for the manufacture of a medicament, wherein preferably the medicament is for use in promoting a germinal center response and / or antibody production in a subject in need thereof, and / or for improving the adaptive immune response, in particular the antigen-specific humoral immune response, of a subject, and / or for enhancing a vaccine-induced humoral immune response.

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