Adjuvant composition, respiratory syncytial virus vaccine composition, and use thereof

Abstract

The present invention relates to the technical field of biomedicine, and in particular to an adjuvant composition, a respiratory syncytial virus (RSV) vaccine composition, and a use thereof. An aluminum adjuvant and a CpG ODN adjuvant are combined in proportion to form an adjuvant composition. An RSV vaccine composition comprising the adjuvant composition has better immunostimulatory activity, can enable early induction of a high humoral immune response, can maintain a strong cellular immune response for a long time, can produce an immune response comparable to that of a licensed vaccine Arexvy, and is a safe, effective and promising human RSV candidate vaccine.

Description

An adjuvant composition, a respiratory syncytial virus vaccine composition and their applications

[0001] This application claims priority to Chinese Patent Application No. CN202411545269.8, filed on November 1, 2024, entitled "An Adjuvant Composition, a Respiratory Syncytial Virus Vaccine Composition and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application belongs to the field of biomedical technology, specifically relating to an adjuvant composition, a respiratory syncytial virus vaccine composition, and their applications. Background Technology

[0003] Respiratory syncytial virus (RSV) is a leading cause of respiratory diseases worldwide. It can infect people of all ages, but infants have the highest incidence of severe illness. It is estimated that RSV causes a significant number of severe cases and deaths among children each year. RSV can also exacerbate conditions such as chronic obstructive pulmonary disease (COPD), asthma, and chronic heart failure in the elderly, leading to serious consequences.

[0004] To reduce the impact of RSV on human health, immunization is primarily achieved through vaccination. As RSV research deepens and new technologies are applied, candidate vaccines from different technology platforms have entered clinical and non-clinical development stages, including live attenuated vaccines, recombinant vector vaccines, subunit vaccines, particle-based vaccines, chimeric vaccines, and nucleic acid vaccines.

[0005] Infants, pregnant women, immunocompromised adults, and the elderly are considered the primary target groups for RSV vaccination. To meet the needs and characteristics of each group, the selection of appropriate adjuvants plays a crucial role in the immunogenicity and efficacy of the vaccine. Adjuvants are molecules that accompany the vaccine, particularly in immunocompromised individuals, acting as transporters and / or immune inducers. Currently, only one adjuvant-containing RSV vaccine is available globally: GlaxoSmithKline's (GSK) Arexvy, which contains the AS01E adjuvant system. This vaccine is used to prevent RSV-induced lower respiratory tract disease (LRTD) in individuals aged 60 and older.

[0006] Several researchers have conducted non-clinical and clinical studies on adjuvanted RSV vaccines. One non-clinical study evaluated the effects of different adjuvants on vaccine efficacy and immune responses in animal models. The results showed that oil-in-water adjuvants (Sigma Adjuvant System (SAS) and Carbopol) induced the highest RSV neutralizing antibody responses compared to DS-Cav1 (a stable pre-F protein) alone, 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 to unadjuvanted vaccines. Another phase I study showed a dose-dependent human immune response to stable RSV F protein in elderly subjects, with the adjuvant (GLA-SE) significantly enhancing humoral and cellular immunity.

[0007] However, a Phase I clinical trial (NCT02298179) conducted by GSK in healthy non-pregnant men aged 18 to 45 years showed that aluminum hydroxide adjuvant or MF59 adjuvant had limited efficacy in RSV F subunit vaccines. Furthermore, a Phase 1 / 2 clinical trial by Pfizer in healthy individuals aged 65-85 years showed that CpG / Al(OH)3 adjuvant did not further enhance the response to the stable RSV pre-F subunit (RSVpreF) candidate vaccine. A Phase III clinical trial by Novavax in pregnant women showed that the RSV fusion (F) protein recombinant nanoparticle candidate vaccine (ResVax) with aluminum phosphate adjuvant failed to meet the pre-defined efficacy targets and thus failed.

[0008] Currently, the licensed monoclonal antibody palivizumab can be administered to extremely premature infants with severe underlying heart or lung disease, immunosuppression, or other critical illnesses. To prevent severe RSV disease (https: / / www.who.int / publications / i / item / WHO-IVB-17.11). However, due to the high cost of antibody therapy, its use is limited, and the only currently marketed adjuvant RSV vaccine, Arexvy, is only used for people over 60 years of age, limiting the number of people who will benefit. Furthermore, 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. Moreover, given the failure of several adjuvant RSV vaccines in clinical trials, there is still a need to develop adjuvant vaccines to prevent RSV disease in order to meet the needs and characteristics of different populations. Summary of the Invention

[0009] The purpose of this application is to provide an adjuvant composition, a respiratory syncytial virus vaccine composition and their application, wherein the vaccine composition including the adjuvant composition has better immunostimulatory activity, can generate higher humoral immunity earlier, and maintain a stronger and longer-lasting cellular immune response.

[0010] This application 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 sequence of the CpG ODN adjuvant is shown in any one of SEQ ID NO.1 to SEQ ID NO.6.

[0011] Preferably, the CpG ODN adjuvant comprises a nucleotide-thiophosphated CpG ODN adjuvant.

[0012] Preferably, the aluminum adjuvant includes one or more of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, and amorphous aluminum hydroxyphosphate adjuvant.

[0013] Preferably, the mass ratio of the aluminum adjuvant to the CpG ODN adjuvant is (30-50):(1-300).

[0014] Preferably, the mass ratio of the aluminum adjuvant to the CpG ODN adjuvant is 4:(0.4-5).

[0015] This application also provides a respiratory syncytial virus (RSV) vaccine composition comprising pre-fusion F glycoprotein of RSV and the adjuvant composition described in the above-described technical solution.

[0016] Preferably, the amino acid sequence of the pre-fusion F glycoprotein of the respiratory syncytial virus is shown in SEQ ID NO.7.

[0017] Preferably, the mass ratio of the pre-fusion F glycoprotein of the respiratory syncytial virus to the CpGODN adjuvant in the adjuvant composition is (0.5-240):(4-3000).

[0018] Preferably, the mass ratio of the pre-fusion F glycoprotein of the respiratory syncytial virus to the CpGODN adjuvant in the adjuvant composition is (30-240):(10-3000).

[0019] Preferably, the mass ratio of the pre-fusion F glycoprotein of the respiratory syncytial virus to the CpGODN adjuvant in the adjuvant composition is (0.5-5):(4-50).

[0020] Preferably, in each dose of the respiratory syncytial virus vaccine composition, the content of the pre-fusion F glycoprotein of the respiratory syncytial virus is 30-240 μg, the content of CpG ODN adjuvant in the adjuvant composition is 10-3000 μg, and the content of aluminum adjuvant is 0.3-0.5 mg;

[0021] Preferably, in each dose of the respiratory syncytial virus vaccine composition, the content of the pre-fusion F glycoprotein of the respiratory syncytial virus is 0.5-5 μg, the content of CpG ODN adjuvant in the adjuvant composition is 4-50 μg, and the content of aluminum adjuvant is 0.04 mg.

[0022] This application also provides the use of the adjuvant composition or the respiratory syncytial virus vaccine composition described in the above-described technical solutions in the preparation of pharmaceuticals for the prevention and / or treatment of respiratory syncytial virus infection and / or respiratory syncytial virus-mediated diseases.

[0023] This application also provides the use of the adjuvant composition or the respiratory syncytial virus vaccine composition described above in the preparation of a pharmaceutical product that generates humoral and / or cellular immune responses against respiratory syncytial virus in the body.

[0024] Preferably, the respiratory syncytial virus includes one or more of the following: 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.

[0025] This application also provides a method for preventing and / or treating respiratory syncytial virus (RSV) infection and / or RSV-mediated diseases, characterized in that it includes immunizing a subject with the RSV vaccine composition described in the above technical solution.

[0026] This application also provides a method for generating humoral and / or cellular immune responses against respiratory syncytial virus in a treated subject, characterized by comprising: immunizing the treated subject with the respiratory syncytial virus vaccine composition described in the above technical solution.

[0027] Preferably, the immunization method includes: administering the respiratory syncytial virus vaccine composition via intramuscular injection to the treated subject, with an injection volume of 0.5 mL per subject. Beneficial effects:

[0028] This application 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 sequence of the CpG ODN adjuvant is shown in any one of SEQ ID NO.1 to SEQ ID NO.6. In this application, the aluminum adjuvant and the CpG ODN adjuvant are proportioned to form an adjuvant composition. The respiratory syncytial virus (RSV) vaccine composition comprising the adjuvant composition exhibits better immunostimulatory activity, can generate a higher humoral immune response earlier, and can maintain a strong cellular immune response for a longer period of time, producing an immune response comparable to the marketed vaccine Arexvy. It is a safe, effective, and promising human RSV vaccine candidate. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0030] Figure 1 shows the results of ELISA detection of RSVReF3 specific antibody titer in serum in Example 2;

[0031] Figure 2 shows the titer of neutralizing antibodies against RSV A2 or RSV B viruses in serum detected in Example 2;

[0032] Figure 3 shows the results of detecting the number of IL-2 positive cell spots using the solid-phase enzyme-linked immunospot (ELISPOT) method in Example 2. Detailed Implementation

[0033] This application 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 sequence of the CpG ODN adjuvant is shown in any one of SEQ ID NO.1 to SEQ ID NO.6.

[0034] The nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.6 described in this application are as follows:

[0035] SEQ ID NO.1, specifically: 5'-tcgcgaacgttcgccgcgtacgtacgcgg-3';

[0036] SEQ ID NO.2, specifically: 5'-tcgcaacgttgccttcgaagg-3';

[0037] SEQ ID NO.3, specifically: 5'-tcgacgttcgttcgtcgttcgttc-3';

[0038] SEQ ID NO.4, specifically: 5'-tcgcgacgttcgccgacgttcgta-3';

[0039] SEQ ID NO.5, specifically: 5'-tgactgtgaacgttcgagatga-3';

[0040] SEQ ID NO.6, specifically: 5'-tcgtcgttttgtcgttttgtcgtt-3'.

[0041] As one implementation method, the aluminum adjuvant in this application may be one or more of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, and amorphous aluminum hydroxyphosphate adjuvant.

[0042] In one embodiment, the mass ratio of aluminum adjuvant to CpG ODN adjuvant in this application can be 4:(0.4-5); in another embodiment, the mass ratio of aluminum adjuvant to CpG ODN adjuvant can be 4:(0.4-2). The preferred mass ratio of aluminum adjuvant to CpG ODN adjuvant in the adjuvant composition of this application is the mass ratio used in mouse models.

[0043] In one embodiment, the mass ratio of aluminum adjuvant to CpG ODN adjuvant in this application can be (30-50):(1-300); in another embodiment, the mass ratio of aluminum adjuvant to CpG ODN adjuvant can be (30-50):(2-200); in yet another embodiment, the mass ratio of aluminum adjuvant to CpG ODN adjuvant can be (30-50):(5-50). The preferred mass ratio of aluminum adjuvant to CpG ODN adjuvant in the adjuvant composition of this application is the mass ratio used for human application.

[0044] As one implementation method, the CpG ODN adjuvant described in this application can be a nucleotide thiophosphate-modified CpG ODN adjuvant, that is, all phosphate diester bonds in the nucleotides of the CpG ODN adjuvant are replaced with thiophosphate bonds. Thiophosphate backbone modification can reduce the sensitivity of oligonucleotides to nucleases and, compared with natural phosphate diester backbone nucleic acids, make them more stable under certain conditions.

[0045] In the adjuvant composition described in this application, the aluminum adjuvant has a "reservoir effect" and an "immunostimulatory effect," and can also promote the activation and maturation of antigen-presenting cells, enhance their ability to take up antigens, and activate inflammasomes to participate in the immune response. The CpG ODN adjuvant triggers innate immune responses in cells expressing Toll-like receptor 9 (including human plasmacytoid dendritic cells and B cells), 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; the combination of the two exerts a synergistic effect.

[0046] This application also provides a respiratory syncytial virus (RSV) vaccine composition comprising pre-fusion F glycoprotein of RSV and the adjuvant composition described in the above-described technical solution.

[0047] As one embodiment, the amino acid sequence of the pre-fusion F glycoprotein of the respiratory syncytial virus described in this application is as shown in SEQ ID NO.7, specifically as follows.

[0048] As one implementation method, the mass ratio of the pre-fusion F glycoprotein of respiratory syncytial virus and CpG ODN adjuvant described in this application can be (0.5-240):(4-3000).

[0049] In one embodiment, the mass ratio of the pre-fusion F glycoprotein of respiratory syncytial virus (RSV) to CpG ODN adjuvant can be (0.5–5):(4–50); in another embodiment, the mass ratio can be (0.5–2):(4–50); and in yet another embodiment, the mass ratio can be (0.5–2):(4–20). The preferred mass ratio of the pre-fusion F glycoprotein of RSV to CpG ODN adjuvant in this application is the mass ratio of the pre-fusion F glycoprotein of RSV to CpG ODN adjuvant in the RSV vaccine composition used in mouse models.

[0050] In one embodiment, the mass ratio of the pre-fusion F glycoprotein of respiratory syncytial virus (RSV) to CpG ODN adjuvant can be (30-240):(10-3000); in another embodiment, the mass ratio of the pre-fusion F glycoprotein of RSV to CpG ODN adjuvant can be (60-120):(10-3000). The preferred mass ratio of the pre-fusion F glycoprotein of RSV to CpG ODN adjuvant in this application is the mass ratio of the pre-fusion F glycoprotein of RSV to CpG ODN adjuvant in the RSV vaccine composition for humans. The mass ratio of the pre-fusion F glycoprotein of RSV to CpG ODN adjuvant in the RSV vaccine composition for humans described in this application is based on the dosage in a mouse model, referring to the dosage of antigen and adjuvant in the commercially available RSV 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 syncytial virus 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 conversion yields that the dose of RSVPreF3 antigen in the commercially available respiratory syncytial virus vaccine Arexvy is 120μg, while the dose of RSVPreF3 antigen in the mouse model experiment in the above literature is 0.5μg. The dose of adjuvant AS01E in the respiratory syncytial virus vaccine Arexvy (i.e., the dose of adjuvant AS01E in the human respiratory syncytial virus vaccine) is 10 times the dose in the non-clinical experimental mice in the above literature.

[0051] In one embodiment, the content of the pre-fusion F glycoprotein of respiratory syncytial virus (RSV) described in this application is 30–240 μg in each dose of the respiratory syncytial virus vaccine composition; in another embodiment, the content of the pre-fusion F glycoprotein is 60–120 μg. In one embodiment, the content of the CpG ODN adjuvant described in this application is 10–3000 μg in each dose of the respiratory syncytial virus vaccine composition; in another embodiment, the content of the CpG ODN adjuvant is 20–2000 μg; in yet another embodiment, the content of the CpG ODN adjuvant is 50–500 μg. In one embodiment, the content of the aluminum adjuvant is 0.3–0.5 mg in each dose of the respiratory syncytial virus vaccine composition. In one embodiment, the respiratory syncytial virus vaccine composition described in this application is a human respiratory syncytial virus vaccine composition.

[0052] In one embodiment, the content of the pre-fusion F glycoprotein of respiratory syncytial virus (RSV) described in this application is 0.5–5 μg in each dose of the respiratory syncytial virus vaccine composition; in another embodiment, the content of the pre-fusion F glycoprotein is 0.5–2 μg. In one embodiment, the content of the CpG ODN adjuvant described in this application is 4–50 μg in each dose of the respiratory syncytial virus vaccine composition; in another embodiment, the content of the CpG ODN adjuvant is 4–20 μg. In one embodiment, the content of the aluminum adjuvant is 0.04 mg in each dose of the respiratory syncytial virus vaccine composition. In one embodiment, the respiratory syncytial virus vaccine composition described in this application is a mouse model respiratory syncytial virus vaccine composition.

[0053] In this application, an aluminum adjuvant and a specific CpG ODN adjuvant are combined in a specific ratio to form the adjuvant composition. The respiratory syncytial virus (RSV) vaccine composition including the adjuvant composition has better immunostimulatory activity, can generate a higher humoral immune response earlier, and can maintain a strong cellular immune response for a longer period of time.

[0054] Based on the above advantages, the use of the adjuvant composition or the respiratory syncytial virus vaccine composition described in the above technical solutions in the preparation of pharmaceuticals for the prevention and / or treatment of respiratory syncytial virus infection and / or respiratory syncytial virus-mediated diseases, as well as the use of the adjuvant composition or the respiratory syncytial virus vaccine composition described in the above technical solutions in the preparation of pharmaceuticals that generate humoral and / or cellular immune responses against respiratory syncytial virus in the body, all fall within the scope of protection of this application.

[0055] As one implementation, the respiratory syncytial virus described in this application includes one or more of the following: 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.

[0056] To further illustrate this application, the technical solutions provided by this application will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of this application.

[0057] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0058] General technical and definition descriptions:

[0059] Unless otherwise instructed, the practice of this application will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, all of which are within the scope of the art.

[0060] The phrase “comprising” as used in this application 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 (other than trace impurities). The phrase “substantially consisting of” is partially closed-ended, indicating that such embodiments may further include elements that do not materially alter the fundamental characteristics of such embodiments.

[0061] As used in this application, the term "vaccine" is the same as that known to those skilled in the art, broadly referring to any biological product that, after administration via injection or mucosal route, can induce the body to produce specific antibodies and / or cellular immunity against a specific pathogen, thereby enabling the body to protect against or eliminate that pathogen. This includes proteins, polysaccharides, nucleic acids, live vectors, or infectious agents. A vaccine is an active immunizing agent used to prevent infectious diseases, made from pathogenic microorganisms (such as bacteria, rickettsiae, viruses, etc.) and their metabolites through artificial attenuation, inactivation, or genetic engineering. Vaccines retain the characteristic of pathogens stimulating the body's immune system. When the body comes into contact with this harmless pathogen, the immune system produces certain protective substances, such as immune hormones, active physiological substances, and specific antibodies. When the body comes into contact with the same pathogen again, the immune system, following its previous memory, produces more protective substances to prevent harm from the pathogen.

[0062] As used in this application, the term "immunization" refers to a process that increases a subject's response to an antigen and thus improves their ability to resist or overcome infection and / or disease.

[0063] As used in this application, the term "vaccination" refers to the introduction of a vaccine into the body of a patient.

[0064] As used in this application, the term "antigen" (Ag) refers to any substance that can induce an immune response in the body. Specifically, it refers to substances that can be specifically recognized and bound by antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activating T / B cells, causing them to proliferate and differentiate, producing immune response products (sensitized lymphocytes or antibodies), and that can specifically bind to these products in vivo and in vitro. This includes, but is not limited to, heteroantigens, such as pathogenic microorganisms, viruses, toxoids, and other antigens between different species; allogeneic antigens, autoantigens, and heterophilic antigens.

[0065] As used in this application, the term "RSV" refers to a non-segmented, negative-sense, single-stranded RNA virus with an envelope, belonging to the genus *Pneumovirus* of the family Pneumoviridae. It has 10 genes encoding 11 proteins, among which the surface glycoprotein G is associated with antigenic variation, and the fusion protein F is an important antigenic epitope of RSV. The F protein has two conformations: pre-fusion (pre-F) and post-fusion (post-F). The pre-fusion conformation of the F protein can activate more efficient neutralizing antibodies. Based on different G protein gene sequences, it is divided into two subtypes, A and B, and within each subtype, it can be further subdivided into different genotypes (Zu Xiangyang, Gao Weina, Du Zhe, et al. Research progress of respiratory syncytial virus vaccines [J]. Chinese Journal of Vaccines and Immunization, 2018, 24(2):237-42.). Subtype A strains include A2, Long, ON1, etc.; subtype B strains include CH-18537, 9320, BA9, etc.

[0066] As used in this application, "adjuvant" refers to substances that are injected prior to or simultaneously with the antigen and can enhance the immunogenicity of the antigen in the body.

[0067] As used in this application, “CpG oligonucleotide (CpG ODN)” is a synthetically produced oligonucleotide (ODN) containing unmethylated cytosine-phosphate-guanine dinucleotide (CpG). CpG ODNs act as ligands for Toll-like receptor 9 (TLR9), directly activating plasmacytoid dendritic cells (pDCs) and B cells via TLR9, inducing humoral and cellular immunity. CpG ODNs include D-type (also known as A-type), K-type (also known as B-type), C-type, and P-type ODNs. K-type ODNs encode multiple CpG motifs on a phosphate thioester backbone. Compared to native phosphodiester nucleotides, the use of phosphate thioester nucleotides enhances resistance to nuclease digestion, thereby significantly extending their half-life in vivo (30-60 minutes, compared to 5-10 minutes for phosphodiester nucleotides). K-type ODN triggers pDC differentiation and TNF-α production, and triggers B cell proliferation and IgM secretion. D-type ODN is composed of a mixed phosphodiester / phosphothiophosphate backbone, containing a CpG motif with palindromic sequences on its flanks, and poly G tails (a structural motif that facilitates concatamer formation) at the 3′ and 5′ ends. D-type ODN triggers pDC maturation and IFN-α secretion, but has no effect on B cells. The different activities of K-type and D-type ODN are attributed to the different retention times of the CpG / TLR-9 complex in pDC endosomes. K-type ODN is rapidly transported to late endosomes via early endosomes, while D-type ODN is retained in early endosomes for a longer period. Here, D-type ODN interacts with the MyD88 / IRF-7 complex, triggering a signaling cascade that supports IFN-α production. C-type ODN is similar to K-type, consisting entirely of phosphothiophosphate nucleotides, but like D-type, it contains a palindromic CpG motif. These types of ODNs stimulate B cells to secrete IL-6 and stimulate pDCs to produce IFN-α. C-type ODNs are present in both early and late endosomes, thus sharing characteristics with K-type ODNs. P-type ODNs contain two palindromic sequences, enabling them to form higher-order structures. P-type ODNs activate B cells and pDCs. CpG oligonucleotides may contain naturally occurring or modified non-naturally occurring bases, and may contain modified sugars, phosphate esters, and / or terminals. For example, in addition to phosphodiester bonds, phosphate ester modifications include, but are not limited to: methyl phosphonate, thiophosphate, aminophosphate (bridged or non-bridged), triphosphate, and dithiophosphate, and can be used in any combination. CpG oligonucleotides may have a homogeneous backbone (e.g., a complete phosphodiester or a complete thiophosphate) or a heterogeneous (or chimeric) backbone.Modification of the phosphate thioester backbone can reduce the sensitivity of oligonucleotides to nucleases, thereby making them more stable under certain conditions (compared to native phosphodiester backbone nucleic acids). Therefore, in some embodiments, CpG oligonucleotides have a non-naturally occurring backbone. In some embodiments, CpG oligonucleotides have a backbone that is entirely composed of phosphate thioesters.

[0068] As used in this application, "aluminum adjuvant" refers to an adjuvant that includes aluminum. Aluminum adjuvants include, but are not limited to, aluminum hydroxide, aluminum hydroxide gel, aluminum phosphate, aluminum phosphate gel, aluminum hydroxyphosphate, aluminum hydroxyphosphate sulfate, amorphous aluminum hydroxyphosphate sulfate, potassium aluminum sulfate, aluminum monostearate, or combinations of the above aluminum adjuvants.

[0069] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0070] The materials used in the following embodiments are from the following sources:

[0071] PBS phosphate buffer powder, purchased from Solarbio;

[0072] Aluminum hydroxide (Al(OH)3) adjuvant, purchased from Thermo;

[0073] Arexvy, a respiratory syncytial virus vaccine, contains 120 μg RSVPreF3 antigen and 0.5 mL AS01E adjuvant per dose and is manufactured by GSK; the RSVPreF3 protein sequence is shown in SEQ ID NO.7.

[0074] Six- to eight-week-old female CB6F1 mice were purchased from Vital River Laboratory Animal Technology Co., Ltd.

[0075] CpG-B and CpG-C were provided by Huapu Biotechnology (Hebei) Co., Ltd.; the nucleotide sequences of CpG were selected from the sequences shown in SEQ ID NO.1 to SEQ ID NO.6, where SEQ ID NO.1 is the nucleotide sequence of CpG-C type and SEQ ID NO.2 to SEQ ID NO.6 are the nucleotide sequences of CpG-B type.

[0076] Example 1

[0077] Preparation of respiratory syncytial virus vaccine composition

[0078] To investigate the technical effects of the respiratory syncytial virus (RSV) vaccine compositions provided in this application, this embodiment prepared several RSVreF3 vaccine compositions containing RSVPreF3 protein, aluminum adjuvant, and CpG ODN adjuvant. The specific preparation methods are as follows:

[0079] 1. Take an appropriate amount of RSVPreF3 protein (amino acids as shown in SEQ ID NO.7) and dilute it with PBS buffer;

[0080] 2. Add aluminum hydroxide adjuvant to the solution obtained in step 1 and mix thoroughly;

[0081] 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), mix thoroughly, and 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.

[0082] Example 2

[0083] Effect of CpG ODN on the immunogenicity of respiratory syncytial virus vaccine composition in mice

[0084] To evaluate the effect of CpG ODN on the immunogenicity of the respiratory syncytial virus vaccine composition prepared in Example 1, this example uses 6-8 week old female CB6F1 mice as an animal model to conduct immunogenicity studies. The specific methods for evaluating immunogenicity are as follows:

[0085] 1. Mouse immunization

[0086] Six- to eight-week-old female CB6F1 mice were randomly divided into six groups of eight mice each. Each group was immunized once on day 0 (the day of the first immunization) and again on day 28, using different respiratory syncytial virus (RSV) vaccine compositions administered intramuscularly at a volume of 50 μL per mouse. The mouse grouping in this example is shown in Table 1. The preparation and dissolution of the corresponding vaccines for each group were the same as in Example 1.

[0087] Table 1. Grouping of mice

[0088] 2. Humoral immunity testing

[0089] 2.1 ELISA method for detecting antibody titers in serum

[0090] Blood samples were collected from mice on days 14, 28, 35, and 56 after the initial immunization. Whole blood was centrifuged at 3000 rpm for 15 min at 4°C to obtain serum, which was stored at -80°C. Antibody titers in the serum were detected by ELISA. 200 ng / well of RSVPreF3 protein was coated onto 96-well plates and incubated overnight at 4°C. The next day, the plates were blocked with 2% BSA in PBST solution at 37°C for 2 hours. Serum samples serially diluted 5-fold with complete culture medium were then added to the 96-well plates and incubated at 37°C for 1 hour. HPR-labeled IgG antibody was then added, and the plates were incubated at 37°C for 1 hour. The plates were washed four times, and then developed at 37°C in the dark for 10 minutes. OD was detected using a microplate reader. 450nm The absorbance value at that point.

[0091] The results are shown in Figure 1, where D28, D35, and D56 represent serum samples taken on days 28, 35, and 56, respectively. Figure 1 shows that neither the PBS group nor the RSVPreF3 protein group produced strong PreF-specific antibodies. On day 28, RSVPreF3 protein combined with CpG ODN and aluminum adjuvant produced high levels of antibodies, while the original drug of Arexvy, RSVPreF3 protein combined with AS01E, produced lower antibody levels. This indicates that the combined use of RSVPreF3 protein, CpG-B / C, and aluminum adjuvant elicits 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 combined use of RSVPreF3 protein, CpG-B / C, and aluminum adjuvant compared to the combined use of RSVPreF3 and AS01E. However, the combined use of RSVPreF3 protein, CpG-B / C, and aluminum adjuvant showed a higher trend, with the CpG-B group showing comparable efficacy to the CpG-C group. This indicates that the combination of CpG ODN and aluminum adjuvant effectively enhances the immunogenicity of the respiratory syncytial virus vaccine composition, leading to earlier production of high levels of antigen-specific binding antibodies.

[0092] 2.2 Detection of neutralizing antibodies in serum samples

[0093] For the detection of neutralizing antibodies, follow these steps: Incubate serum samples in a 56°C water bath for 30 minutes to inactivate complement cascade proteins. Serially dilute the serum samples to be tested with complete culture medium, initially diluting 40-fold, then 3-fold, for a total of 8 dilutions, and mix with an equal volume of 50 plaque-forming units (pfu) of RSV A2 or RSV B virus (derived from ATCC). Add the virus-serum mixture to a Hep2 cell monolayer in a 96-well plate and incubate at 37°C for 1 hour. Then rotate the plate on a rotating platform at approximately 30° for 10 minutes, followed by incubation at 37°C in a CO2 incubator for 30 minutes; repeat this cycle for 4 hours. Finally, incubate the plates at 37°C in a CO2 incubator for 48 hours. After incubation, add 100 μL of fixative to fix the cells. Subsequently, the primary antibody (KOSTAN CSD0024) diluted 1:500 in PBS was added and incubated at 37°C for 2 hours. Then, the secondary antibody (Boao Sen bs-0297-HRP) (1:1000 diluted) was added and incubated at room temperature for 1 hour. Finally, KPL TrueBlue Peroxidase Substrate was added for color development, and the speckle analysis was performed using an ELISA speckle analyzer.

[0094] The results are shown in Figure 2. No neutralizing antibodies were produced in the PBS group. On day 56, for RSV A and B viruses, the highest levels of neutralizing antibodies were produced by adding RSVPreF3 protein to CpG-B and CpG-C in combination with aluminum adjuvant, respectively. The RSV A neutralizing antibody level produced by RSVPreF3 protein + CpG-B / C + aluminum adjuvant was more than 3 times higher than that produced by Arexvy, and the RSV B neutralizing antibody level produced was more than 2 times higher than that produced by Arexvy. Among them, CpG-B and CpG-C had comparable effects, with the CpG-C group showing slightly higher levels. It can be seen that CpG ODN combined with aluminum adjuvant effectively enhances the immunogenicity of the respiratory syncytial virus vaccine composition and can produce higher levels of neutralizing antibodies. Therefore, the combined use of RSVPreF3 protein, CpG, and aluminum adjuvant can be considered a better RSV candidate vaccine.

[0095] 3. Cellular immune detection

[0096] Spleens were collected under aseptic conditions 7 and 28 days after the second immunization. Specifically, spleen cell suspensions were prepared through the following procedures.

[0097] 1. Grinding and Centrifugation: Place a 70μm cell sieve on a sterile Petri dish. Wash the spleen in PBS solution containing 1% penicillin and antibiotics, then place it in the sieve. Grind until no red tissue is visible. Transfer the suspension to a 50mL centrifuge tube. Add approximately 5mL of 1640 medium to wash the sieve and Petri dish. Transfer the suspension to the tube and add medium to a final volume of 15mL. Centrifuge at 300g horizontally at room temperature for 5 minutes, then discard the supernatant.

[0098] 2. Lysis of red blood cells: Resuspend the precipitated cells in 7 mL of red blood cell lysis buffer, incubate at room temperature for 5 min, and add PBS to a final volume of 40 mL. Centrifuge at 300 g horizontally at room temperature for 5 min, and discard the supernatant.

[0099] 3. Washing and Filtration: Add 20 mL of sterile PBS to the precipitated cells and mix thoroughly by pipetting. Prepare a new 50 mL centrifuge tube with a 70 μm cell strainer. Filter the suspension from the original tube into the corresponding group-numbered new tube, discard the strainer, and transfer 5 mL of the suspension to a 15 mL centrifuge tube using a pipette. Centrifuge the 15 mL suspension in the centrifuge tube at 300 g horizontally at room temperature for 5 minutes, and discard the supernatant. Resuspend the precipitated cells in 2 mL of serum-free CTL medium + 1% Gln + 1% penicillin-antibiotic medium (CTL, Cellular Technology Ltd.) and mix thoroughly to obtain a spleen cell suspension.

[0100] Cell counting was performed at a concentration of 2.0 × 10⁻⁶. 5 Cells were used for experiments in each well. Three stimulation conditions were set up: a negative control using culture medium, stimulation with an RSV F protein peptide library (Sinochem, catalog number: PP004) (final concentration 1 μg / mL), and a positive control using phorbol ester (PMA) / Ionomycin (final concentration 20 ng / mL). Cells were stimulated under these three conditions and incubated at 37°C for 18-22 h. The liquid in the wells was discarded and the cells were blotted dry. 200 μL of PBS was added to each well, and the cells were washed twice using the same method. 200 μL of PBST was added to each well, and the cells were washed twice using the same method. 80 μL of diluted biotin-labeled IL-2 mouse antibody working solution (1:1000) was added to each well, and the cells were incubated at room temperature in the dark for 2 hours. The liquid in the wells was discarded and the cells were blotted dry. 200 μL of PBST was added to each well, and the cells were washed three times and blotted dry. 80 μL of Strep-AP working solution (1:1000) was added to each well, and the cells were incubated at room temperature in the dark for 30 minutes. Discard the liquid in the wells and blot dry. Wash twice and blot dry. Add 200 μL of deionized water to each well, wash twice and blot dry. Add 80 μL of chromogenic reagent to each well and incubate at room temperature in the dark for 15 minutes. Discard the liquid in the wells and blot dry. Rinse the wells with deionized water until the background is clear. After the plate is completely dry, scan the plate using a CTL enzyme-linked immunosorbent assay (ELISA) analyzer.

[0101] The results are shown in Figure 3, where D35 and D56 represent days 35 and 56 after the first immunization, respectively. Figure 3 shows that neither the PBS group nor the RSVPreF3 protein group induced a cellular immune response. On day 35 (7 days after the second immunization), the RSVPreF3 protein combined with CpG-B or CpG-C in combination with aluminum adjuvant produced a significant cellular immune response. The effects of the CpG-B and CpG-C groups were comparable. By day 56 (28 days after the second immunization), the cellular immune responses in all groups weakened, but the RSVPreF3 protein combined with CpG-B or CpG-C in combination with aluminum adjuvant still showed a strong cellular immune response. The CpG-C group was slightly better than the CpG-B group. This demonstrates that CpG ODN combined with aluminum adjuvant effectively enhanced the immunogenicity of the respiratory syncytial virus vaccine composition, producing a stronger and more durable cellular immune response. Therefore, the combined use of RSVPreF3 protein, CpG ODN and aluminum adjuvant can be considered a better RSV candidate vaccine.

[0102] Example 3

[0103] Preparation of respiratory syncytial virus vaccine composition

[0104] To investigate the technical effects of the respiratory syncytial virus (RSV) vaccine compositions provided in this application, this embodiment prepared several RSVreF3 vaccine compositions containing RSVPreF3 protein, aluminum adjuvant, and CpG ODN adjuvant. The specific preparation methods are as follows:

[0105] 1. Take an appropriate amount of RSVPreF3 protein (amino acids as shown in SEQ ID NO.7) and dilute it with PBS buffer;

[0106] 2. Add aluminum hydroxide adjuvant to the solution obtained in step 1 and mix thoroughly;

[0107] 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), mix thoroughly, and 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.

[0108] Example 4

[0109] Effect of CpG ODN on the immunogenicity of respiratory syncytial virus vaccine composition in mice

[0110] To evaluate the effect of CpG ODN on the immunogenicity of the respiratory syncytial virus vaccine composition prepared in Example 3, this example uses 6-8 week old female CB6F1 mice as an animal model to conduct immunogenicity studies. The specific methods for evaluating immunogenicity are as follows:

[0111] 1. Mouse immunization

[0112] Six- to eight-week-old female CB6F1 mice were randomly divided into six groups of eight mice each. Each group was immunized once on day 0 (the day of the first immunization) and again on day 28, using different respiratory syncytial virus (RSV) vaccine compositions administered intramuscularly at a volume of 50 μL per mouse. The mouse grouping in this example is shown in Table 1. The preparation and dissolution of the corresponding vaccines for each group were the same as in Example 3.

[0113] Table 1. Grouping of mice

[0114] From the above embodiments, it can be concluded that the respiratory syncytial virus vaccine composition containing the adjuvant composition described in this application has better immunostimulatory activity, generates higher humoral immunity earlier, and maintains a stronger and longer-lasting cellular immune response.

[0115] Although the above embodiments have provided a detailed description of this application, they are only some embodiments of this application, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of this application.

Claims

1. An adjuvant composition, characterized in that, It includes 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 sequence 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 nucleotide-thiophosphated CpG ODN adjuvant.

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).

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

6. A respiratory syncytial virus vaccine composition, characterized in that, The respiratory syncytial virus vaccine composition comprises respiratory syncytial virus pre-fusion F glycoprotein and the adjuvant composition according to any one of claims 1 to 5.

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

7.

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

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

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

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

12. The respiratory syncytial virus vaccine composition according to claim 10, characterized in that, In each dose of the respiratory syncytial virus vaccine composition, the content of the pre-fusion F glycoprotein of the respiratory syncytial virus is 0.5–5 μg, the content of CpG ODN adjuvant in the adjuvant composition is 4–50 μg, and the content of aluminum adjuvant is 0.04 mg.

13. The use of the adjuvant composition according to any one of claims 1 to 5 or the respiratory syncytial virus vaccine composition according to any one of claims 6 to 12 in the preparation of a pharmaceutical product for the prevention and / or treatment of respiratory syncytial virus infection and / or respiratory syncytial virus-mediated diseases.

14. The use of the adjuvant composition according to any one of claims 1 to 5 or the respiratory syncytial virus vaccine composition according to any one of claims 6 to 12 in the preparation of a pharmaceutical product that generates a humoral and / or cellular immune response against respiratory syncytial virus in the body.

15. The application according to claim 13 or 14, characterized in that, The respiratory syncytial virus includes one or more of the following: 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.

16. A method for preventing and / or treating respiratory syncytial virus (RSV) infection and / or RSV-mediated diseases, characterized in that, include: Immunize the recipient with the respiratory syncytial virus vaccine composition according to any one of claims 6 to 12.

17. A method for generating a humoral and / or cellular immune response against respiratory syncytial virus in a treated subject, characterized in that, include: Immunize the recipient with the respiratory syncytial virus vaccine composition according to any one of claims 6 to 12.

18. The method according to claim 16 or 17, characterized in that, The immunization method includes: administering the respiratory syncytial virus vaccine composition via intramuscular injection to the treated subject, with an injection volume of 0.5 mL per subject.

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