Adjuvant composition, respiratory syncytial virus vaccine composition and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an adjuvant composition, a respiratory syncytial virus vaccine composition and application thereof. In the invention, the aluminum adjuvant and the specific CpG ODN adjuvant form the adjuvant composition according to a specific proportion, and the respiratory syncytial virus (RSV) vaccine composition comprising the adjuvant composition has better immunostimulatory activity, can generate higher humoral immune response earlier, and can maintain higher cellular immune response for a long time. The human RSV vaccine can generate immune response which is comparable to that of a marketed vaccine Arexvy, and is a safe, effective and promising human RSV candidate vaccine.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to an adjuvant composition, a respiratory syncytial virus vaccine composition and applications thereof. Background Art

[0002] Respiratory Syncytial Virus (RSV) is the main pathogenic virus of respiratory diseases worldwide. The virus can cause infection in people of all ages, but the incidence of severe illness is highest in infants. It is estimated that RSV causes a large number of severe illnesses and deaths in children every year. At the same time, RSV can aggravate diseases such as chronic obstructive pulmonary disease (COPD), asthma and chronic heart failure in the elderly, and can lead to serious consequences.

[0003] In order to reduce the impact of RSV virus on human health, immunization is mostly carried out through vaccination. With the continuous deepening of RSV research and the application of new technologies, candidate vaccines of different technology platforms have entered the clinical and non-clinical research and development stages, including live attenuated vaccine, recombinant vector vaccine, subunit vaccine, particle-based vaccine, chimeric vaccine and nucleic acid vaccine.

[0004] Four groups of people are considered to be the main target populations for RSV vaccination: infants, pregnant women, immunocompromised adults, and the elderly. In order to meet the needs and characteristics of each population, the selection of appropriate adjuvants plays a vital role in the immunogenicity and effectiveness of the vaccine. Adjuvants are molecules that accompany vaccines, especially in immunocompromised individuals, acting as transporters and / or immune inducers. Currently, there is only one adjuvanted RSV vaccine on the market worldwide, namely GlaxoSmithKline (GSK)'s Arexvy, which contains the AS01E adjuvant system and is used to prevent lower respiratory tract disease (LRTD) caused by RSV in people aged 60 and above.

[0005] Some researchers have conducted nonclinical and clinical studies on adjuvanted RSV vaccines. A nonclinical study evaluated the effects of different adjuvants on vaccine efficiency and immune response in animal models. The results showed that compared with DS-Cav1 (stable pre-F protein) alone, water-in-oil adjuvants (Sigma Adjuvant System (SAS) and Carbopol) induced the highest RSV neutralizing antibody response, followed by Alum, SAS alone, AdjuPlex and Poly (I: C). A clinical trial showed that AS04 and MF59 adjuvants enhanced the immunogenicity of viral vaccines in humans compared with vaccines without adjuvants. Another phase I study showed that elderly subjects had a dose-dependent human immune response to stable RSV F protein, and the adjuvant (GLA-SE) significantly enhanced humoral immunity and cellular immunity.

[0006] However, the results of a Phase I clinical trial (NCT02298179) conducted by GSK in healthy non-pregnant women and men aged 18 to 45 years showed that aluminum hydroxide adjuvants or MF59 adjuvants have limited effects in RSVF subunit vaccines. In addition, the results of a Phase 1 / 2 clinical trial conducted by Pfizer in healthy people aged 65-85 years showed that CpG / Al(OH)3 adjuvant did not further enhance the response of the stable RSV pre-F subunit (RSVpreF) candidate vaccine. The results of a Phase III clinical trial conducted by Novavax in pregnant women showed that the RSV fusion (F) protein recombinant nanoparticle candidate vaccine (ResVax) with aluminum phosphate as an adjuvant failed to achieve the pre-specified efficacy target.

[0007] Currently, the licensed monoclonal antibody palivizumab can be administered to extremely premature, immunosuppressed, or otherwise critically ill infants with severe underlying cardiac or pulmonary disease To prevent severe RSV disease (https: / / www.who.int / publications / i / item / WHO-IVB-17.11). However, considering the high cost of antibody treatment, its use is limited, and the only adjuvant RSV vaccine currently on the market, Arexvy, is used by people over 60 years old, and the beneficiaries are limited. Arexvy also has local adverse reactions such as pain, erythema, and swelling, as well as systemic adverse reactions such as fatigue, myalgia, headache, joint pain, and fever. At the same time, in view of the failure of many adjuvant RSV vaccines in clinical trials, in order to meet the needs and characteristics of different populations, it is still necessary to develop adjuvant vaccines to prevent RSV disease. Summary of the invention

[0008] The object of the present invention is to provide an adjuvant composition, a respiratory syncytial virus vaccine composition and applications thereof. The vaccine composition comprising the adjuvant composition has better immunostimulatory activity, can produce higher humoral immunity earlier, and maintain stronger and longer-term cellular immune response.

[0009] The present invention provides an adjuvant composition, comprising an aluminum adjuvant and a CpG ODN adjuvant, wherein the mass ratio of the aluminum adjuvant to the CpG ODN adjuvant is (4-50):(0.4-300); the nucleotide of the CpG ODN adjuvant is shown in any one of SEQ ID NO.1 to SEQ ID NO.6.

[0010] Preferably, the CpG ODN adjuvant comprises a nucleotide phosphorothioate-esterified CpG ODN adjuvant.

[0011] Preferably, the aluminum adjuvant includes one or more of aluminum hydroxide adjuvant, aluminum phosphate adjuvant and amorphous aluminum hydroxyphosphate adjuvant.

[0012] Preferably, the mass ratio of the aluminum adjuvant to the CpG ODN adjuvant is (30-50):(1-300) or 4:(0.4-5).

[0013] The present invention also provides a respiratory syncytial virus vaccine composition, which comprises respiratory syncytial virus pre-fusion F glycoprotein and the adjuvant composition described in the above technical solution.

[0014] Preferably, the amino acid sequence of the respiratory syncytial virus pre-fusion F glycoprotein is as shown in SEQ ID NO.7.

[0015] Preferably, the mass ratio of the respiratory syncytial virus prefusion F glycoprotein to the CpG ODN adjuvant is (0.5-240): (4-3000).

[0016] Preferably, the mass ratio of the respiratory syncytial virus prefusion F glycoprotein to the CpG ODN adjuvant is (30-240): (10-3000) or (0.5-5): (4-50).

[0017] Preferably, in each dose of the respiratory syncytial virus vaccine composition, the content of the respiratory syncytial virus prefusion F glycoprotein is 30-240 μg, the content of the CpG ODN adjuvant is 10-3000 μg, and the content of the aluminum adjuvant is 0.3-0.5 mg;

[0018] Or, the content of the respiratory syncytial virus pre-fusion F glycoprotein is 0.5-5 μg, the content of the CpG ODN adjuvant is 4-50 μg, and the content of the aluminum adjuvant is 0.04 mg.

[0019] The present invention also provides the use of the adjuvant composition described in the above technical solution or the respiratory syncytial virus vaccine composition described in the above technical solution in the preparation of medicines for preventing and treating respiratory syncytial virus infection and / or respiratory syncytial virus-mediated diseases.

[0020] The present invention also provides the use of the adjuvant composition described in the above technical solution or the respiratory syncytial virus vaccine composition described in the above technical solution in the preparation of a medicine for producing humoral immunity and / or cellular immune response to respiratory syncytial virus in the body.

[0021] Preferably, the respiratory syncytial virus includes one or more of human respiratory syncytial virus subtype A, a mutant of human respiratory syncytial virus subtype A, a variant of human respiratory syncytial virus subtype A, human respiratory syncytial virus subtype B, a mutant of human respiratory syncytial virus subtype B, and a variant of human respiratory syncytial virus subtype B.

[0022] Beneficial effects:

[0023] The present invention provides an adjuvant composition, comprising an aluminum adjuvant and a CpG ODN adjuvant, wherein the mass ratio of the aluminum adjuvant to the CpG ODN adjuvant is (4-50): (0.4-300); the nucleotide of the CpG ODN adjuvant is as shown in any one of SEQ ID NO.1 to SEQ ID NO.6. In the present invention, the aluminum adjuvant and the specific CpG ODN adjuvant form an adjuvant composition in a specific ratio, and the respiratory syncytial virus (RSV) vaccine composition comprising the adjuvant composition has better immunostimulatory activity, can produce a higher humoral immune response earlier, and can maintain a stronger cellular immune response for a longer period of time, and can produce an immune response comparable to the marketed vaccine Arexvy, and is a safe, effective and promising human RSV candidate vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0025] Figure 1 The RSVPreF3-specific antibody titer in serum detected by ELISA method in Example 2;

[0026] Figure 2 For detecting the neutralizing antibody titer against RSVA2 or RSVB virus in serum in Example 2;

[0027] Figure 3 This is the result of detecting the number of IL-2 positive cell spots by solid phase enzyme-linked immunosorbent spot method (ELISPOT) in Example 2. DETAILED DESCRIPTION

[0028] The present invention provides an adjuvant composition, comprising an aluminum adjuvant and a CpG ODN adjuvant, wherein the mass ratio of the aluminum adjuvant to the CpG ODN adjuvant is (4-50):(0.4-300); the nucleotide of the CpG ODN adjuvant is shown in any one of SEQ ID NO.1 to SEQ ID NO.6.

[0029] The nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.6 of the present invention are as follows:

[0030] SEQ ID NO.1, specifically: 5'-tcgcgaacgttcgccgcgtacgtacgcgg-3';

[0031] SEQ ID NO. 2, specifically: 5'-tcgcaacgttgccttcgaagg-3';

[0032] SEQ ID NO. 3, specifically: 5'-tcgacgttcgtcgttcgtcgtcgttc-3';

[0033] SEQ ID NO. 4, specifically: 5'-tcgcgacgttcgccgacgttcgta-3';

[0034] SEQ ID NO. 5, specifically: 5'-tgactgtgaacgttcgagatga-3';

[0035] SEQ ID NO. 6, specifically: 5'-tcgtcgttttgtcgttttgtcgtt-3'.

[0036] As an embodiment, the aluminum adjuvant of the present invention can be one or more of aluminum hydroxide adjuvant, aluminum phosphate adjuvant and amorphous aluminum hydroxyphosphate adjuvant.

[0037] As an embodiment, the mass ratio of the aluminum adjuvant to the CpG ODN adjuvant of the present invention can be 4:(0.4-5); as another embodiment, the mass ratio of the aluminum adjuvant to the CpG ODN adjuvant can be 4:(0.4-2). The mass ratio of the aluminum adjuvant to the CpG ODN adjuvant in the adjuvant composition of the present invention is preferably the mass ratio when applied on a mouse model.

[0038] As an embodiment, the mass ratio of the aluminum adjuvant and the CpG ODN adjuvant of the present invention can be (30-50): (1-300); as another embodiment, the mass ratio of the aluminum adjuvant and the CpG ODN adjuvant can be (30-50): (2-200); as another embodiment, the mass ratio of the aluminum adjuvant and the CpG ODN adjuvant can be (30-50): (5-50). The mass ratio of the aluminum adjuvant and the CpG ODN adjuvant in the adjuvant composition of the present invention is preferably the mass ratio for human use.

[0039] As an embodiment, the CpG ODN adjuvant of the present invention can be a CpG ODN adjuvant with nucleotide thiophosphate esterification, that is, all the nucleotide phosphodiester bonds in the CpG ODN adjuvant are replaced with thiophosphate bonds. Phosphorothioate backbone modification can reduce the sensitivity of oligonucleotides to nucleases and is more stable under certain conditions compared to natural phosphodiester backbone nucleic acids.

[0040] In the adjuvant composition of the present invention, the aluminum adjuvant has a "depot effect" and an "immunostimulatory effect", and can also promote the activation and maturation of antigen-presenting cells, improve their ability to take up antigens, and activate inflammasomes to participate in immune responses. The CpG ODN adjuvant triggers cells expressing Toll-like receptor 9 (including human plasmacytoid dendritic cells and B cells) to produce an innate immune response, which is characterized by the production of Th1 and pro-inflammatory cytokines. CpG ODN improves the function of professional antigen-presenting cells and promotes the generation of humoral and cellular vaccine-specific immune responses, and the combination of the two exerts a synergistic effect.

[0041] The present invention also provides a respiratory syncytial virus vaccine composition, which comprises respiratory syncytial virus pre-fusion F glycoprotein and the adjuvant composition described in the above technical solution.

[0042] As an embodiment, the amino acid sequence of the respiratory syncytial virus prefusion F glycoprotein of the present invention is shown in SEQ ID NO.7, specifically MEWSWVFLFFLSVTTGVHSQNITEEFYQ STCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIM CIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCY GKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0043] As an embodiment, the mass ratio of the respiratory syncytial virus pre-fusion F glycoprotein and the CpG ODN adjuvant of the present invention can be (0.5-240): (4-3000).

[0044] As one embodiment, the mass ratio of the respiratory syncytial virus prefusion F glycoprotein and the CpG ODN adjuvant can be (0.5-5): (4-50); as another embodiment, the mass ratio of the respiratory syncytial virus prefusion F glycoprotein and the CpG ODN adjuvant can be (0.5-2): (4-50); as another embodiment, the mass ratio of the respiratory syncytial virus prefusion F glycoprotein and the CpG ODN adjuvant can be (0.5-2): (4-20). The mass ratio of the respiratory syncytial virus prefusion F glycoprotein and the CpG ODN adjuvant of the present invention is preferably the mass ratio of the respiratory syncytial virus prefusion F glycoprotein and the CpG ODN adjuvant in the respiratory syncytial virus vaccine composition on a mouse model.

[0045] As an embodiment, the mass ratio of the respiratory syncytial virus prefusion F glycoprotein and the CpG ODN adjuvant can be (30-240): (10-3000); as another embodiment, the mass ratio of the respiratory syncytial virus prefusion F glycoprotein and the CpG ODN adjuvant can be (60-120): (10-3000). The mass ratio of the respiratory syncytial virus prefusion F glycoprotein and the CpG ODN adjuvant of the present invention is preferably the mass ratio of the respiratory syncytial virus prefusion F glycoprotein and the CpG ODN adjuvant in the respiratory syncytial virus vaccine composition on humans. The mass ratio of the respiratory syncytial virus prefusion F glycoprotein and the CpG ODN adjuvant in the respiratory syncytial virus vaccine composition on humans of the present invention is based on the amount used in the mouse model, with reference to the amount of antigen and adjuvant in the commercially available respiratory syncytial virus vaccine Arexvy and the literature [Bouzya B, Rouxel RN, Sacconnay L, Mascolo R, Nols L, Quique S, L,Atas A,Warter L,Dezutter N,Lorin C.Immunogenicity of an AS01-adjuvanted respiratory syncytialvirus prefusion F(RSVPreF3)vaccine in animal models.NPJ Vaccines.2023Sep 29; 8(1):143.doi:10.1038 / s41541-023-00729-4.PMID:37773185;PMCID:PMC10541443.】The dosage of RSVPreF3 antigen in the commercially available respiratory syncytial virus vaccine Arexvy is 120 μg, the dosage of RSVPreF3 antigen in the mouse model experiment in the above-mentioned literature is 0.5 μg, and the dosage of adjuvant AS01E in the respiratory syncytial virus vaccine Arexvy (i.e. the dosage of adjuvant AS01E in human respiratory syncytial virus vaccine) is 10 times the dosage of mice in the non-clinical experiments in the above-mentioned literature.

[0046] As an embodiment, in each dose of respiratory syncytial virus vaccine composition, the content of the respiratory syncytial virus pre-fusion F glycoprotein of the present invention is 30-240 μg; as another embodiment, the content of the respiratory syncytial virus pre-fusion F glycoprotein is 60-120 μg. As an embodiment, in each dose of respiratory syncytial virus vaccine composition, the content of the CpG ODN adjuvant of the present invention is 10-3000 μg; as another embodiment, the content of the CpGODN adjuvant is 20-2000 μg; as another embodiment, the content of the CpG ODN adjuvant is 50-500 μg. As an embodiment, in each dose of respiratory syncytial virus vaccine composition, the content of the aluminum adjuvant is 0.3-0.5 mg. As an embodiment, the respiratory syncytial virus vaccine composition of the present invention is a human respiratory syncytial virus vaccine composition.

[0047] As an embodiment, in each dose of respiratory syncytial virus vaccine composition, the content of the respiratory syncytial virus pre-fusion F glycoprotein of the present invention is 0.5-5 μg; as another embodiment, the content of the respiratory syncytial virus pre-fusion F glycoprotein is 0.5-2 μg. As an embodiment, in each dose of respiratory syncytial virus vaccine composition, the content of the CpG ODN adjuvant of the present invention is 4-50 μg; as another embodiment, the content of the CpG ODN adjuvant is 4-20 μg. As an embodiment, in each dose of respiratory syncytial virus vaccine composition, the content of the aluminum adjuvant is 0.04 mg. As an embodiment, the respiratory syncytial virus vaccine composition of the present invention is a mouse model respiratory syncytial virus vaccine composition.

[0048] In the present invention, the aluminum adjuvant and the specific CpG ODN adjuvant are formed into the adjuvant composition in a specific ratio. The respiratory syncytial virus (RSV) vaccine composition including the adjuvant composition has better immunostimulatory activity, can produce a higher humoral immune response earlier, and can maintain a stronger cellular immune response for a longer period of time.

[0049] Based on the above advantages, the use of the adjuvant composition described in the above technical scheme or the respiratory syncytial virus vaccine composition described in the above technical scheme in the preparation of medicines for preventing and treating respiratory syncytial virus infection and / or respiratory syncytial virus-mediated diseases, as well as the use of the adjuvant composition described in the above technical scheme or the respiratory syncytial virus vaccine composition described in the above technical scheme in the preparation of medicines for producing humoral immunity and / or cellular immune response to respiratory syncytial virus in the body both fall within the protection scope of the present invention.

[0050] As an embodiment, the respiratory syncytial virus described in the present invention includes one or more of human respiratory syncytial virus subtype A, a mutant of human respiratory syncytial virus subtype A, a variant of human respiratory syncytial virus subtype A, human respiratory syncytial virus subtype B, a mutant of human respiratory syncytial virus subtype B, and a variant of human respiratory syncytial virus subtype B.

[0051] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0052] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0053] General Technical and Definition Notes:

[0054] The practice of the present application will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art.

[0055] As used herein, the phrase "comprising" is open ended, indicating that such embodiments may include additional elements. In contrast, the phrase "consisting of" is closed ended, indicating that such embodiments do not include additional elements (except for trace impurities). The phrase "consisting essentially of" is partially closed ended, indicating that such embodiments may further include elements that do not materially change the basic characteristics of such embodiments.

[0056] As used in the present invention, the term "vaccine" is a vaccine well known to those of ordinary skill in the art, and generally refers to any biological product that can induce the body to produce specific antibodies and / or cellular immunity against a specific pathogen after injection or mucosal inoculation, thereby enabling the body to obtain protection or eliminate the pathogen, including proteins, polysaccharides, nucleic acids, live vectors or infectious factors, etc. Vaccines are autoimmune preparations for preventing infectious diseases made by artificially attenuating, inactivating or using genetic engineering methods for pathogenic microorganisms (such as bacteria, rickettsia, viruses, etc.) and their metabolites. Vaccines retain the characteristics of pathogens that stimulate the body's immune system. When the body comes into contact with this harmless pathogen, the immune system will produce certain protective substances, such as immune hormones, active physiological substances, special antibodies, etc.; when the body comes into contact with this pathogen again, the body's immune system will follow its original memory and produce more protective substances to prevent the harm of the pathogen.

[0057] As used herein, the term "immunization" refers to the process of increasing a subject's response to an antigen and thereby improving its ability to resist or overcome infection and / or resist disease.

[0058] As used herein, the term "vaccination" refers to the introduction of a vaccine into the body of a subject.

[0059] As used in the present invention, the term "antigen" (Ag) refers to all substances that can induce an immune response in the body. That is, substances that can be specifically recognized and bound by antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activate T / B cells, cause them to proliferate and differentiate, produce immune response products (sensitized lymphocytes or antibodies), and can specifically bind to corresponding products in vivo and in vitro. Including but not limited to heterologous antigens, such as pathogenic microorganisms, viruses, toxoids and other antigens between different races; alloantigens, self-antigens and heterophilic antigens.

[0060] As used in the present invention, the term "RSV" is a non-segmented negative-stranded single-stranded RNA virus with an envelope, belonging to the genus Pneumovirus of the family Pneumoviridae, with 10 genes encoding 11 proteins, of which surface glycoprotein G is related to antigenic variation, and fusion protein F is an important antigenic epitope of RSV, and F protein has two conformations before fusion (Pre-fusion, pre-F)) and after fusion (Post-fusion, post-F), wherein the F protein in the pre-fusion conformation can activate more efficient neutralizing antibodies. According to the different G protein gene sequences, it is divided into two subtypes, A and B, and can still be subdivided into different genotypes within the subtype (Zu Xiangyang, Gao Weina, Du Zhe, et al. Research progress of respiratory syncytial virus vaccine [J]. Chinese Vaccine and Immunity, 2018, 24 (2): 237-42.). Subtype A strains include A2, Long, ON1, etc.; subtype B strains include CH-18537, 9320, BA9, etc.

[0061] As used in the present invention, "adjuvant" refers to some substances that are injected before or simultaneously with the antigen, and can enhance the immunogenicity of the antigen in the body.

[0062] As used in the present invention, "CpG oligonucleotide (CpG oligonucleotide, CpG ODN)" is an artificially synthesized oligonucleotide (ODN) containing non-methylated cytosine-phosphate-guanine dinucleotide (CpG). As a ligand of Toll-like receptor 9 (TLR9), CpG ODNs can directly activate plasmacytoid dendritic cells (pDCs) and B cells through TLR9, inducing humoral immunity and cellular immunity. CpG ODN includes D-type (also known as A-type), K-type (also known as B-type), C-type, and P-type ODN. K-type ODN encodes multiple CpG motifs on the phosphorothioate backbone. When compared with natural phosphodiester nucleotides, the use of phosphorothioate nucleotides enhances resistance to nuclease digestion, thereby greatly extending its half-life in vivo (30-60 minutes, compared to 5-10 minutes for phosphodiester). K-type ODNs trigger pDC differentiation and TNF-α production, and B cell proliferation and IgM secretion. D-type ODNs are composed of a mixed phosphodiester / phosphorothioate backbone, contain a CpG motif flanked by palindromic sequences, and have poly G tails (a structural motif that favors concatamer formation) at the 3′ and 5′ ends. D-type ODNs trigger pDC maturation and IFN-α secretion, but have no effect on B cells. The different activities of K-type and D-type ODNs have been attributed to the different retention times of the CpG / TLR-9 complex in pDC endosomes. K-type ODNs are rapidly transported through early endosomes to late endosomes, while D-type ODNs are retained in early endosomes for a longer period of time. Here, D-type ODNs interact with the MyD88 / IRF-7 complex and trigger a signaling cascade that supports IFN-α production. C-type ODNs are similar to K-types and are composed entirely of phosphorothioate nucleotides, but similar to D-types, they contain palindromic CpG motifs. This type of ODN stimulates B cells to secrete IL-6 and pDC to produce IFN-α. C-type ODN is present in both early and late endosomes, and therefore has common properties with K-type ODN. P-type ODN contains two palindromic sequences, which enable them to form higher-order structures. P-type ODN activates B cells and pDC. CpG oligonucleotides can contain naturally occurring or modified non-natural bases, and can contain modified sugars, phosphates and / or ends. For example, in addition to phosphodiester bonds, phosphate modifications include, but are not limited to: methyl phosphonates, phosphorothioates, phosphoramidates (bridged or non-bridged), phosphotriesters and dithiophosphates, and can be used in any combination. CpG oligonucleotides can have a homogeneous backbone (e.g., a complete phosphodiester or a complete phosphorothioate) or a heterogeneous (or chimeric) backbone.Phosphorothioate backbone modifications can make oligonucleotides less sensitive to nucleases and thus more stable under certain conditions (compared to natural phosphodiester backbone nucleic acids). Thus, in some embodiments, CpG oligonucleotides have non-naturally occurring backbones. In some embodiments, CpG oligonucleotides have backbones that are entirely phosphorothioate.

[0063] As used in the present invention, "aluminum adjuvant" refers to an adjuvant comprising aluminum. Aluminum adjuvants include, but are not limited to, aluminum hydroxide, aluminum oxyhydroxide, aluminum hydroxide gel, aluminum phosphate, aluminum phosphate gel, hydroxyaluminum phosphate, hydroxyaluminum phosphate sulfate, amorphous hydroxyaluminum phosphate sulfate, potassium aluminum sulfate, aluminum monostearate, or a combination of the above aluminum adjuvants.

[0064] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of the present application, the cited documents involved in the present invention are cited with all contents and all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the content of the citation conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0065] In the following examples, the sources of materials used are as follows:

[0066] PBS phosphate buffered saline dry powder, purchased from Solarbio;

[0067] Aluminum hydroxide (Al(OH)3) adjuvant was purchased from Thermo;

[0068] Respiratory syncytial virus vaccine Arexvy, each dose contains 120μg RSVPreF3 antigen and 0.5mL AS01E adjuvant, produced by GSK; the RSVPreF3 protein sequence is shown in SEQ ID NO.7.

[0069] CB6F1 female mice aged 6-8 weeks were purchased from Weitonglihua Experimental Animal Technology Co., Ltd.

[0070] CpG-B and CpG-C are provided by Huapu Biotechnology (Hebei) Co., Ltd.; wherein the nucleotide sequence of CpG is selected from the sequences shown in SEQ ID NO.1 to SEQ ID NO.6, wherein SEQ ID NO.1 is a CpG-B type nucleotide sequence, and SEQ ID NO.2 to SEQ ID NO.6 are CpG-C type nucleotide sequences.

[0071] Example 1

[0072] Preparation of respiratory syncytial virus vaccine composition

[0073] In order to study the technical effect of the respiratory syncytial virus vaccine composition provided by the present invention. This example prepared the following multiple respiratory syncytial virus vaccine compositions, which contained RSVPreF3 protein, aluminum adjuvant and CpG ODN adjuvant. The specific preparation method is as follows:

[0074] 1. Take an appropriate amount of RSVPreF3 protein (amino acids as shown in SEQ ID NO.7) and dilute it with PBS buffer;

[0075] 2. Add aluminum hydroxide adjuvant to the solution obtained in step 1 and mix thoroughly;

[0076] 3. Before injection, add CpG-B (nucleotide as shown in SEQ ID NO.2) or CpG-C (nucleotide as shown in SEQ ID NO.1) and mix thoroughly. Each dose (50 μL) of the prepared vaccine for immunizing mice contains 0.5 μg of RSVPreF3 protein, 40 μg of Al(OH)3 and 8 μg of CpG ODN.

[0077] Example 2

[0078] Effect of CpG ODN on the immunogenicity of respiratory syncytial virus vaccine composition in mice

[0079] In order to evaluate the effect of CpG ODN on the immunogenicity of the respiratory syncytial virus vaccine composition prepared in Example 1, this example carried out an immunogenicity study using 6-8 week old CB6F1 female mice as an animal model. The specific method for evaluating immunogenicity is as follows:

[0080] 1. Mouse immunization

[0081] CB6F1 female mice aged 6-8 weeks were randomly divided into groups, with 8 mice in each group, for a total of 6 groups. Each group of mice was immunized once on D0 (the first immunization day) and D28 days, and different respiratory syncytial virus vaccine compositions were immunized by intramuscular injection, with an injection volume of 50 μL / mouse. The grouping of mice in this example is shown in Table 1, and the preparation and dissolution of the corresponding vaccine for each group of mice is the same as in Example 1.

[0082] Table 1 Grouping of mice

[0083] Grouping Vaccine composition and dosage 1 PBS buffer 2 0.5 μg RSVPreF3 3 <![CDATA[0.5μgRSVPreF3+8μgCpG-B+40μgAl(OH)3]]> 4 <![CDATA[0.5μgRSVPreF3+8μgCpG-C+40μgAl(OH)3]]> 5 Arexvy (0.5 μg RSVPreF3 + AS01E (1 / 10), i.e. 0.05 mL) 6 <![CDATA[0.5μgRSVPreF3+40μgAl(OH)3]]>

[0084] 2. Humoral Immunity Testing

[0085] 2.1ELISA method to detect antibody titer in serum

[0086] Blood was collected from each group of mice on the 14th, 28th, 35th and 56th day after the first immunization. The collected whole blood was centrifuged at 3000 rpm for 15 min at 4°C to separate the serum, which was stored at -80°C and the antibody titer in the serum was detected by ELISA. 200 ng / well of RSVPreF3 protein was coated on a 96-well plate and incubated overnight at 4°C. The next day, the plate was blocked with a 2% BSA-PBST solution at 37°C for 2 hours, and then the serum samples diluted 5 times with complete culture medium were added to the 96-well plate and incubated at 37°C for 1 hour. HPR-labeled IgG antibody was then added and incubated at 37°C for 1 hour. The plate was washed 4 times, then color was developed at 37°C in the dark for 10 minutes, and the OD was detected using an enzyme reader. 450nm The absorbance value below.

[0087] The results are as follows Figure 1 As shown, in Figure 1 D28, D35 and D56 represent serum samples on the 28th, 35th and 56th days, respectively. Figure 1 It can be concluded that neither the PBS group nor the RSVPreF3 protein group could produce strong PreF-specific antibodies; on day 28, RSVPreF3 protein plus CpG ODN combined with aluminum adjuvant produced very high levels of antibodies, while Arexvy's original drug, RSVPreF3 protein plus AS01E, produced lower levels of antibodies. This indicates that the combination of RSVPreF3 protein, CpG-B / C, and aluminum adjuvant produced a significant specific humoral immune response earlier than Arexvy, producing higher levels of IgG. On days 35 and 56, there was no significant difference in the specific antibodies produced by the combination of RSVPreF3 protein, CpG-B / C, and aluminum adjuvant and the combination of RSVPreF3 and AS01E, but the combination of RSVPreF3 protein, CpG-B / C, and aluminum adjuvant had a higher trend, and the CpG-B group had the same effect as the CpG-C group. It can be seen that CpG ODN combined with aluminum adjuvant effectively enhanced the immunogenicity of the respiratory syncytial virus vaccine composition and could produce high levels of antigen-specific binding antibodies earlier.

[0088] 2.2 Detection of neutralizing antibodies in serum samples

[0089] For the detection of neutralizing antibodies, the following steps were followed: serum samples were incubated in a water bath at 56°C for 30 minutes to inactivate complement cascade proteins. Serum samples to be tested were serially diluted in complete medium, each serum sample was initially diluted 40-fold, and then serially diluted 3-fold, for a total of 8 dilutions, and mixed with an equal volume of 50 plaque-forming units (pfu) of RSVA2 or RSVB virus (from ATCC). The virus-serum mixture was added to the Hep2 cell monolayer in a 96-well culture plate and incubated at 37°C for 1 hour. Subsequently, the plate was rotated on a rotating platform tilted at about 30° for 10 minutes, and then incubated in a CO2 incubator at 37°C for 30 minutes. This cycle lasted for 4 hours. The plate was then incubated in a CO2 incubator at 37°C for 48 hours. At the end of the incubation, 100 μL of fixative reagent was added to fix the cells. Then, the primary antibody (Costain CSD0024) diluted 1:500 in PBS was added and incubated at 37°C for 2 hours, and then HRP-labeled secondary antibody (Boaosen bs-0297-HRP) (1:1000 dilution) was added and incubated at room temperature for 1 hour. Finally, KPL TrueBlue Peroxidase Substrate was added for color development, and the spots were analyzed by enzyme-linked immunosorbent assay.

[0090] The results are as follows Figure 2 As shown, no neutralizing antibodies were produced in the PBS group; on day 56, for RSVA and B viruses, RSVPreF3 protein added with CpG-B and CpG-C respectively combined with aluminum adjuvant produced the highest neutralizing antibody level. The RSVA neutralizing antibody level produced by RSVPreF3 protein + CpG-B / C + aluminum adjuvant was more than 3 times higher than Arexvy, and the RSVB neutralizing antibody level produced was more than 2 times higher than Arexvy. Among them, CpG-B and CpG-C had similar effects, and the CpG-C group was slightly higher. It can be seen that CpG ODN combined with aluminum adjuvant effectively enhanced the immunogenicity of the respiratory syncytial virus vaccine composition and could produce higher levels of neutralizing antibodies. Therefore, the combination of RSVPreF3 protein, CpG and aluminum adjuvant can be used as a more preferred RSV candidate vaccine.

[0091] 3. Cellular Immunity Detection

[0092] 7 days and 28 days after the second immunization, spleens were collected under sterile conditions. Specifically, spleen cell suspensions were prepared by the following operations:

[0093] 1. Grinding and centrifugation: Place a 70μm cell sieve on a sterile plate, wash the spleen in PBS solution containing 1% double antibody, and then put it into the sieve. Grind until no red tissue is visible, transfer the suspension to a 50mL centrifuge tube, add about 5mL 1640 culture medium to wash the sieve and the plate, transfer the suspension to the tube, add culture medium to a final volume of 15mL. Centrifuge at 300g in a horizontal rotor at room temperature for 5 minutes, and discard the supernatant.

[0094] 2. Lyse red blood cells: Add 7 mL of red blood cell lysis buffer to resuspend the precipitated cells, let stand at room temperature for 5 minutes, add PBS to a final volume of 40 mL. Centrifuge at 300g in a horizontal rotor at room temperature for 5 minutes and discard the supernatant.

[0095] 3. Washing and filtering: Add 20 mL of sterile PBS to the precipitated cells and mix by pipetting. Prepare a new 50 mL centrifuge tube and place a 70 μm cell sieve on it. Filter the suspension in the original tube into a new tube with the corresponding group number, discard the sieve, and transfer 5 mL of the suspension to a 15 mL centrifuge tube with a pipette. Centrifuge the 15 mL centrifuge tube suspension at 300 g in a horizontal rotor at room temperature for 5 minutes and discard the supernatant. Add 2 mL of CTL serum-free medium + 1% Gln + 1% double antibody serum-free medium (CTL, Cellular Technology Ltd) to resuspend the precipitated cells and mix thoroughly to obtain a spleen cell suspension.

[0096] The cells were counted at 2.0 × 10 5 The experiment was performed at 4 °C for 18-22 h. The cells were stimulated under the above three conditions, namely, culture medium negative control, RSV F protein peptide library (Sino Biological, Cat. No.: PP004) stimulation (final concentration 1 μg / mL), and phorbol fatty acid (PMA) / Ionomycin positive control (final concentration 20 ng / mL). The cells were stimulated under the above three conditions and incubated at 37 °C for 18-22 h. Pour out the liquid in the well and pat dry, add PBS 200 μL / well, pour out and pat dry, wash twice in the same way, add PBST 200 μL / well, and wash twice in the same way. Add 80 μL / well of diluted biotin-labeled IL-2 mouse antibody working solution (1:1000) and incubate at room temperature in the dark for 2 hours. Pour out the liquid in the well and pat dry, add PBST 200 μL / well, wash 3 times and pat dry. Add 80 μL / well of Strep-AP working solution (1:1000) and incubate at room temperature in the dark for 30 minutes. Pour out the liquid in the well and pat dry, wash twice and pat dry, add 200μL of deionized water to each well, wash twice and pat dry, add 80μL / well of colorimetric solution, incubate at room temperature and avoid light for 15 minutes. Pour out the liquid in the well and pat dry, rinse the wells with deionized water until the background is clear. After the well plate is completely dry, scan the plate using the CTL enzyme-linked immunospot analyzer.

[0097] The results are as follows Figure 3As shown in FIG. 1 , where D35 and D56 represent the 35th day and 56th day after the first immunization, respectively. Figure 3 It can be concluded that neither the PBS group nor the RSVPreF3 protein group can induce a cellular immune response; on the 35th day (7 days after the second immunization), the RSVPreF3 protein plus CpG-B or CpG-C combined with aluminum adjuvant produced a significant cellular immune response. The CpG-B and CpG-C groups had equivalent effects. By the 56th day (28 days after the second immunization), the cellular immune response of each group weakened, but the RSVPreF3 protein plus CpG-B or CpG-C combined with aluminum adjuvant group still had a strong cellular immune response, and the CpG-C group was slightly better than the CpG-B group. It can be seen that the combination of CpG ODN and aluminum adjuvant effectively enhanced the immunogenicity of the respiratory syncytial virus vaccine composition and produced a stronger and more lasting cellular immune response. Therefore, the combination of RSVPreF3 protein, CpG ODN and aluminum adjuvant can be used as a more preferred RSV candidate vaccine.

[0098] Example 3

[0099] Preparation of respiratory syncytial virus vaccine composition

[0100] In order to study the technical effect of the respiratory syncytial virus vaccine composition provided by the present invention. This example prepared the following multiple respiratory syncytial virus vaccine compositions, which contained RSVPreF3 protein, aluminum adjuvant and CpG ODN adjuvant. The specific preparation method is as follows:

[0101] 1. Take an appropriate amount of RSVPreF3 protein (amino acids as shown in SEQ ID NO.7) and dilute it with PBS buffer;

[0102] 2. Add aluminum hydroxide adjuvant to the solution obtained in step 1 and mix thoroughly;

[0103] 3. Before injection, add CpG-B (nucleotide as shown in SEQ ID NO.2) or CpG-C (nucleotide as shown in SEQ ID NO.1) and mix thoroughly. Each dose (50 μL) of the prepared vaccine for immunizing mice contains 5 μg of RSVPreF3 protein, 40 μg of Al(OH)3 and 50 μg of CpG ODN.

[0104] Example 4

[0105] Effect of CpG ODN on the immunogenicity of respiratory syncytial virus vaccine composition in mice

[0106] In order to evaluate the effect of CpG ODN on the immunogenicity of the respiratory syncytial virus vaccine composition prepared in Example 3, this example carried out an immunogenicity study using 6-8 week old CB6F1 female mice as an animal model. The specific evaluation method of immunogenicity is as follows:

[0107] 1. Mouse immunization

[0108] CB6F1 female mice aged 6-8 weeks were randomly divided into groups, with 8 mice in each group, for a total of 6 groups. Each group of mice was immunized once on D0 (the first immunization day) and D28 days, and different respiratory syncytial virus vaccine compositions were immunized by intramuscular injection, with an injection volume of 50 μL / mouse. The grouping of mice in this example is shown in Table 1, and the preparation and dissolution of the corresponding vaccine for each group of mice is the same as in Example 3.

[0109] Table 1 Grouping of mice

[0110] Grouping Vaccine composition and dosage 1 PBS buffer 2 5 μg RSVPreF3 3 <![CDATA[5μgRSVPreF3+50μgCpG-B+40μgAl(OH)3]]> 4 <![CDATA[5μgRSVPreF3+50μgCpG-C+40μgAl(OH)3]]> 5 Arexvy (5 μg RSVPreF3 + AS01E (1 / 10), i.e. 0.05 mL) 6 <![CDATA[5μgRSVPreF3+40μgAl(OH)3]]>

[0111] It can be concluded from the above examples that the respiratory syncytial virus vaccine composition containing the adjuvant composition of the present invention has better immunostimulatory activity, produces higher humoral immunity earlier, and maintains stronger and longer cellular immune response.

[0112] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An adjuvant composition, characterized in that It comprises an aluminum adjuvant and a CpG ODN adjuvant, wherein the mass ratio of the aluminum adjuvant to the CpG ODN adjuvant is (4-50):(0.4-300); the nucleotide of the CpG ODN adjuvant is shown in any one of SEQ ID NO.1 to SEQ ID NO.

6.

2. The adjuvant composition according to claim 1, characterized in that The CpG ODN adjuvant includes a CpG ODN adjuvant containing nucleotide phosphorothioate.

3. The adjuvant composition according to claim 1, characterized in that The aluminum adjuvant includes one or more of aluminum hydroxide adjuvant, aluminum phosphate adjuvant and amorphous aluminum hydroxyphosphate adjuvant.

4. The adjuvant composition according to claim 1, characterized in that The mass ratio of the aluminum adjuvant to the CpG ODN adjuvant is (30-50):(1-300) or 4:(0.4-5).

5. A respiratory syncytial virus vaccine composition, characterized in that: The respiratory syncytial virus vaccine composition comprises the respiratory syncytial virus prefusion F glycoprotein and the adjuvant composition according to any one of claims 1 to 4.

6. The respiratory syncytial virus vaccine composition according to claim 5, characterized in that The amino acid sequence of the respiratory syncytial virus pre-fusion F glycoprotein is shown in SEQ ID NO.

7.

7. The respiratory syncytial virus vaccine composition according to claim 5 or 6, characterized in that: The mass ratio of the respiratory syncytial virus pre-fusion F glycoprotein to the CpG ODN adjuvant is (0.5-240): (4-3000).

8. The respiratory syncytial virus vaccine composition according to claim 7, characterized in that The mass ratio of the respiratory syncytial virus pre-fusion F glycoprotein to the CpG ODN adjuvant is (30-240): (10-3000) or (0.5-5): (4-50).

9. The respiratory syncytial virus vaccine composition according to claim 7, characterized in that In each dose of respiratory syncytial virus vaccine composition, the content of the respiratory syncytial virus pre-fusion F glycoprotein is 30-240 μg, the content of the CpGODN adjuvant is 10-3000 μg, and the content of the aluminum adjuvant is 0.3-0.5 mg; Or, the content of the respiratory syncytial virus pre-fusion F glycoprotein is 0.5-5 μg, the content of the CpG ODN adjuvant is 4-50 μg, and the content of the aluminum adjuvant is 0.04 mg.

10. Use of the adjuvant composition according to any one of claims 1 to 4 or the respiratory syncytial virus vaccine composition according to any one of claims 5 to 9 in the preparation of a medicament for preventing and treating respiratory syncytial virus infection and / or respiratory syncytial virus-mediated diseases.

11. Use of the adjuvant composition according to any one of claims 1 to 4 or the respiratory syncytial virus vaccine composition according to any one of claims 5 to 9 in the preparation of a medicament for producing humoral immunity and / or cellular immune response to respiratory syncytial virus in a body.

12. The use according to claim 10 or 11, characterized in that: The respiratory syncytial virus includes one or more of human respiratory syncytial virus subtype A, mutants of human respiratory syncytial virus subtype A, variants of human respiratory syncytial virus subtype A, human respiratory syncytial virus subtype B, mutants of human respiratory syncytial virus subtype B and variants of human respiratory syncytial virus subtype B.

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