Nucleotides, vaccines comprising same, and complex immune adjuvants

Abstract

The present application relates to a kind of nucleotide, vaccine comprising it and composite vaccine adjuvant.The sequence of the nucleotide is shown as SEQ ID No:1.Relative to conventional technology, the beneficial effects of the present application include: the present application adopts new optimized sequence, provides a kind of nucleotide, the nucleotide can be as CpG oligonucleotide adjuvant, induces vaccine antigen to produce stronger humoral immunity, cellular immunity level and anti-tumor immune effect etc., has obvious clinical application value and vaccine development value.Moreover, using the immunoadjuvant of the present application helps to reduce the dose of clinical use vaccine, improves safety.

Description

Technical Field

[0001] This invention relates to the field of medical biopharmaceuticals, and in particular to a nucleotide, a vaccine containing the nucleotide, and a combined vaccine adjuvant. Background Technology

[0002] Adjuvants are substances added to vaccines to stimulate and enhance the strength and duration of the immune response. They can enhance the body's immune response to antigens or alter the type of immune response. They are non-specific immune enhancers and are not antigenic themselves. Ideal adjuvants not only enhance the immune response but also enable the body to acquire optimal protective immunity.

[0003] In 1939, the U.S. Food and Drug Administration approved aluminum adjuvant (alum) for the first vaccine adjuvant in human history for use in diphtheria toxin vaccines. For decades afterward, aluminum adjuvant remained the only choice for immune adjuvants in the vaccine industry. It wasn't until the 1990s that the first non-aluminum adjuvant in human history—MF59 oil-emulsion adjuvant—was approved for use in vaccines in Europe. Due to limitations in side effects, stability, and efficacy, currently only a few adjuvants are approved for use in human vaccines, including aluminum adjuvants, MF59, AS series adjuvants, and CpG oligonucleotide adjuvants.

[0004] CpG oligonucleotides, as agonists of the pattern recognition receptor TLR9, can simultaneously activate and enhance both cellular and humoral immunity, and are considered a vaccine adjuvant with great potential. TLR9 has three classes of CpG oligonucleotide ligands, which can be distinguished by their different nucleotide sequence motifs and their ability to stimulate IFNα in plasma cell-like dendritic cells (DCs). The TLR9 agonist CpG 1018 is a 22-mer unmethylated CpG-B oligonucleotide and a potent TH1 cell adjuvant that stimulates strong B cell and NK cell activation. CpG 1018 is currently being evaluated in clinical trials as a potential vaccine adjuvant for COVID-19 vaccines. Furthermore, this molecule is a component of Heplisav-B, a modified HBV vaccine approved for adults (age >18 years). While other HBV vaccines have been licensed and widely used, they are typically administered in a three-dose regimen, whereas Heplisav-B's main advantage is that it offers a simplified two-dose regimen. In preclinical studies in mice, CpG nucleotides induced significantly higher antibody responses to various antigens, including the surface antigen of HBV and HBsAg, compared to formulations without adjuvant or with alum adjuvant. However, the expression pattern of TLR9 in mice differs from that in humans and macaques. In mice, many cell types, including cells from the monocyte / macrophage lineage, express TLR9, while in humans and macaques, TLR9 expression is strictly limited to plasmacytoid dendritic cells (DCs) and B cells, highlighting the importance of understanding the molecular mechanisms of action of novel adjuvants. Finally, the different immunostimulatory capacities of the aforementioned classes of CpG molecules appear to depend on whether they are present in monomeric or multimeric form. Thus, monomeric CpG-B oligonucleotides localize to lysosome-associated membrane protein 1 (LAMP1)-positive endosomes and promote plasmacytoid DC maturation, but produce little or no IFNα; however, when complexed into microparticles, CpG-B localizes to transferrin receptor 1 (TFR1)-positive endosomes and leads to IFNα production via a TLR9-dependent mechanism. Furthermore, monomeric CpG-B can be readily absorbed by B cells and stimulate their activation. In contrast, CpG-A, which spontaneously forms nanoparticle-like complexes due to its palindromic structure, also localizes to TFR1+ endosomes in plasmacytoid dendritic cells (DCs) and stimulates IFNα production, but cannot be absorbed by B cells. B cells do not internalize larger DNA complexes, such as CpG-A. The innate mechanisms and cell types that stimulate adaptive immunity in response to CpG plus antigen also appear to depend on the form of CpG. Therefore, immunization of mice with soluble CpG-B plus antigen induces TH1 CD4+ T cell and antibody responses via a mechanism dependent on TLR9-mediated MyD88 activation in DCs.Conversely, immunization using polymeric CpG-B plus antigen does not appear to heavily rely on MyD88 signaling in dendritic cells (DCs), suggesting that other innate cells play a role in sensing polymeric CpG and initiating adaptive immunity. Although CpG1018 adjuvants have achieved very promising clinical results, some drawbacks remain, most notably the excessively high clinical dosage, posing certain safety risks. Therefore, optimizing CpG motif design and conducting research on novel CpG oligonucleotide adjuvants has significant application value and social implications. Summary of the Invention

[0005] Based on this, the purpose of this application includes providing a nucleotide that can serve as an adjuvant for CpG oligonucleotide vaccines, assisting the antigen in inducing a stronger specific immune response in the body.

[0006] In a first aspect of this application, a nucleotide is provided, the sequence of which is shown in SEQ ID No:1.

[0007] In a second aspect of this application, the use of the nucleotides described in the first aspect of this application as immune adjuvants is provided.

[0008] In a third aspect of this application, a vaccine is provided, the vaccine comprising an antigen and an immune adjuvant, the immune adjuvant comprising the nucleotides described in the first aspect of this application or further comprising other immune adjuvants.

[0009] In some embodiments of this application, the vaccine satisfies one or more of the following conditions:

[0010] (1) The vaccine is a recombinant protein vaccine; and,

[0011] (2) The other immune adjuvants are selected from one or more of aluminum adjuvants and CpG1018.

[0012] In some embodiments of this application, the antigen is a recombinant protein of the novel coronavirus.

[0013] In some embodiments of this application, the antigen is a recombinant human papillomavirus type 16 protein.

[0014] In some embodiments of this application, the mass ratio of the antigen to the nucleotide is (2.5-5):1.

[0015] In some embodiments of this application, the mass ratio of the antigen, the nucleotide, and the other immune adjuvant is (2.5-5):1:(45-55).

[0016] In some embodiments of this application, the mass ratio of the antigen, the nucleotide, and the other immune adjuvant is (2.5-5):1:(49-51).

[0017] In a fourth aspect of this application, a composite immune adjuvant is provided, the composite immune adjuvant comprising the nucleotide and aluminum adjuvant described in the first aspect;

[0018] In some embodiments of this application, the mass ratio of the nucleotide to the aluminum adjuvant is 1:(45-55).

[0019] In some embodiments of this application, the mass ratio of the nucleotide to the aluminum adjuvant is 1:(49-51).

[0020] Compared to traditional technologies, the beneficial effects of this application include:

[0021] This application employs a novel optimized sequence to provide a nucleotide that can act as a CpG oligonucleotide adjuvant, inducing stronger humoral, cellular, and antitumor immune responses in vaccine antigens, demonstrating significant clinical application and vaccine development value. Furthermore, because this application induces stronger immunity in vaccines compared to traditional technologies (such as CpG1018), using this immune adjuvant can help reduce clinical vaccine dosage and improve safety. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The results show the specific antibody levels of the recombinant novel coronavirus protein in Example 1; where A is the experimental group of the novel coronavirus protein plus adjuvant; B is the vaccine group of novel coronavirus protein alone; and C is the control group of CpG0620 adjuvant alone.

[0024] Figure 2 The results show the specific cellular immune levels of the recombinant novel coronavirus protein in Example 1; where A is the experimental group of the novel coronavirus protein plus adjuvant; B is the vaccine group of novel coronavirus protein alone; and C is the control group of CpG0620 adjuvant alone.

[0025] Figure 3The results show the specific antibody levels of the recombinant novel coronavirus protein in Example 2; where A is the experimental group of novel coronavirus protein vaccine plus CpG0620 aluminum adjuvant system; B is the group of novel coronavirus protein vaccine plus aluminum adjuvant alone; and C is the control group of CpG0620 aluminum adjuvant system alone.

[0026] Figure 4 The results show the specific cellular immune levels of the recombinant novel coronavirus protein in Example 2; where A is the experimental group of novel coronavirus protein vaccine plus CpG0620 aluminum adjuvant system; B is the group of novel coronavirus protein vaccine plus aluminum adjuvant alone; and C is the control group of CpG0620 aluminum adjuvant system alone.

[0027] Figure 5 The results show the specific antibody levels of the recombinant novel coronavirus protein in Example 3; where A is the experimental group of novel coronavirus protein vaccine plus CpG0620 aluminum adjuvant system; B is the experimental group of novel coronavirus protein vaccine plus CpG01018 aluminum adjuvant system; C is the control group of CpG0620 aluminum adjuvant system alone; D is the control group of CpG1018 aluminum adjuvant system alone.

[0028] Figure 6 The results show the specific cellular immune levels of the recombinant novel coronavirus protein in Example 3; where A is the experimental group of novel coronavirus protein vaccine plus CpG0620 aluminum adjuvant system; B is the experimental group of novel coronavirus protein vaccine plus CpG01018 aluminum adjuvant system; C is the control group of CpG0620 aluminum adjuvant system alone; D is the control group of CpG1018 aluminum adjuvant system alone.

[0029] Figure 7 The results show the specific anti-tumor antigen cellular immunity levels of the human papillomavirus type 16 recombinant protein vaccine in Example 4; where A is the experimental group of human papillomavirus type 16 recombinant protein plus CpG0620 adjuvant; B is the human papillomavirus type 16 recombinant protein vaccine alone group; and C is the control group of CpG0620 adjuvant alone.

[0030] Figure 8 The results of the tumor growth inhibition test of the human papillomavirus type 16 recombinant protein vaccine in Example 5 are as follows: A. Human papillomavirus type 16 recombinant protein plus CpG0620 adjuvant experimental group; B. Human papillomavirus type 16 recombinant protein plus CpG1018 adjuvant experimental group; C. Human papillomavirus type 16 recombinant protein vaccine alone group; D. Blank control group. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the invention. The purpose of providing these embodiments and examples is to enable a more thorough and complete understanding of the disclosure of the present invention. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present invention, and the equivalent forms obtained also fall within the protection scope of this application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for descriptive purposes only and is not intended to be limiting of the invention.

[0033] the term

[0034] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0035] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0036] In this invention, terms such as "multiple", "multi-items", "multiple times", and "multi-components" are used, and unless otherwise specified, they refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0037] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0038] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this invention, solve the technical problem of this invention, and achieve the expected technical effect of this invention.

[0039] In this article, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this invention.

[0040] In this invention, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this invention.

[0041] In this invention, "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent.

[0042] In this invention, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on the quantity.

[0043] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0044] In this invention, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0045] Unless otherwise specified, the temperature parameters in this invention can be either constant temperature treatment or variations within a certain temperature range. It should be understood that the constant temperature treatment allows temperature fluctuations within the precision range controlled by the instrument. Fluctuations are permitted within ranges such as ±5℃, ±4℃, ±3℃, ±2℃, and ±1℃.

[0046] In this invention, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0047] All references to this invention 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, the referenced documents involved in this invention are incorporated in their entirety and for all purposes. When references are made in this invention, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When references are made in this invention, examples and preferred embodiments of the relevant technical features cited may also be incorporated herein by reference, but only to the extent that they enable the implementation of this invention. 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 adaptively based on the description in this application.

[0048] Vaccines are biological products made from various pathogenic microorganisms and used for immunization. Vaccines are generally divided into two categories: preventative vaccines and therapeutic vaccines. Preventative vaccines are mainly used for disease prevention, and recipients are healthy individuals or newborns; therapeutic vaccines are mainly used for individuals who are already ill, and recipients are patients.

[0049] Immunoadjuvants, or nonspecific immunoproliferators, are auxiliary substances that, when injected into the body along with or beforehand, enhance the body's immune response to antigens or alter the type of immune response. Adjuvants can be immunogenic or non-immunogenic. There are many types of adjuvants, and there is no unified classification method; Freund's adjuvants and cytokine adjuvants are the most commonly used. The immunobiological effects of adjuvants include enhancing immunogenicity, increasing antibody titers, altering the type of antibody produced, and inducing or enhancing delayed-type hypersensitivity reactions. However, the mechanisms of action of adjuvants are not fully understood, and different adjuvants have different mechanisms of action.

[0050] CpG oligonucleotides can effectively trigger mammalian immune responses through the TLR9 signaling pathway, thus serving as a good immune adjuvant for adjuvant treatment of infections and tumors.

[0051] The first aspect of this application

[0052] This application provides a nucleotide, the sequence of which is shown in SEQ ID No:1.

[0053] It is understood that, provided a stable nucleotide can be obtained, this application does not impose any particular limitation on its synthesis method. For example, it can be synthesized artificially using a synthesizer or through biological synthesis. It is also understood that, during the synthesis of the nucleotide, the base raw materials used can be modified, including but not limited to thiomodification, which can be complete or partial thiomodification.

[0054] Second aspect of this application

[0055] This application provides for the use of the nucleotides described in the first aspect of this application as vaccine adjuvants.

[0056] The nucleotide is a CpG oligonucleotide, which is named CpG0620 in this application embodiment.

[0057] Third aspect of this application

[0058] This application provides a vaccine comprising an antigen and an immune adjuvant, wherein the immune adjuvant comprises the nucleotides described in the first aspect of this application or further comprises other immune adjuvants.

[0059] The nucleotide can be used alone or in combination with other immune adjuvants (such as aluminum adjuvants) during the process of using it as an immune adjuvant.

[0060] This application does not specifically limit the type of vaccine, which may include live attenuated vaccines, inactivated vaccines, antitoxins, subunit vaccines (including peptide vaccines), vector vaccines, nucleic acid vaccines, etc. Optionally, the vaccine may be a recombinant protein vaccine.

[0061] Similarly, this application does not specifically limit the types of antigens in the vaccine, including but not limited to recombinant proteins of the novel coronavirus and recombinant proteins of human papillomavirus type 16.

[0062] It is understood that this application does not impose any particular limitation on the amount of the antigen and the nucleotide used; any appropriate amount can be used. Optionally, the mass ratio of the antigen to the nucleotide is (2.5-5):1, for example, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3.0:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4.0:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1.

[0063] When the nucleotide is used in combination with other immune adjuvants, this application does not particularly limit the types of other immune adjuvants, including but not limited to aluminum adjuvants and CpG1018. It is understood that this application does not particularly limit the amount of the antigen, the nucleotide, and the other immune adjuvants; appropriate amounts are sufficient. Optionally, the mass ratio of the antigen, the nucleotide, and the other immune adjuvants is (2.5-5):1:(45-55). For example, 2.5:1:45, 2.6:1:46, 2.7:1:47, 2.8:1:48, 2.9:1:49, 3.0:1:50, 3.1:1:51, 3.2:1:52, 3.3:1:53, 3.4:1:54, 3.5:1:55, 3.6:1:45, 3.7:1:46, 3.8:1:47, 3.9:1:48, 4.0:1:49, 4.1:1:50, 4.2:1:51, 4.3:1:52, 4.4:1:53, 4.5:1:54, 4.6:1:55, 4.7:1:45, 4.8:1:46, 4.9:1:47, 5:1:48. Further, optionally, the mass ratio of the antigen, the nucleotide, and the other immune adjuvant is (2.5-5):1:(49-51).

[0064] Fourth aspect of this application

[0065] This application provides a composite immune adjuvant comprising the nucleotide and aluminum adjuvant described in the first aspect.

[0066] This application does not impose any particular limitation on the amount of the nucleotide and the aluminum adjuvant; it is understood that any appropriate amount can be selected. Optionally, the mass ratio of the nucleotide to the aluminum adjuvant is 1:(45-55). For example, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, or 1:55. More preferably, the mass ratio of the nucleotide to the aluminum adjuvant is 1:(49-51). Specific Implementation

[0068] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0069] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0070] The Tween 20, phosphate buffer, HRP-labeled goat anti-mouse IgG monoclonal antibody, TMB chromogenic solution, and enzyme-linked dot immunoassay kit used in the following examples are all commercially available products.

[0071] In the following examples, the average weight of the mice was approximately 20g.

[0072] Example 1: CpG0620 adjuvant alone induces stronger specificity in recombinant protein vaccines against the novel coronavirus. Humoral immunity and cellular immunity

[0073] (1) According to the sequence of CpG0620: 5'-TGACGTTTTCGTTAACGTT-3' (SEQ ID No:1), it was artificially synthesized using a synthesizer. All the nucleotide bases used in the synthesis were thiomodified. The synthesized CpG oligonucleotide sequence was purified by high performance liquid chromatography, which is the required CpG0620 adjuvant.

[0074] (2) The recombinant protein of the novel coronavirus was combined with CpG0620 adjuvant to obtain a vaccine sample containing 50 μg / mL of recombinant protein of the novel coronavirus and 20 μg / mL of CpG0620 adjuvant.

[0075] (3) Six-week-old Babl / c mice were divided into three groups of eight each: the experimental group of recombinant protein of novel coronavirus plus adjuvant (Group A), the vaccine group of recombinant protein of novel coronavirus alone (Group B), and the control group of CpG0620 adjuvant alone (Group C). Each mouse was immunized with 1 ml of the dose. Serum and spleen mononuclear lymphocytes were collected on the 14th day after the first immunization.

[0076] (4) Take the serum collected in step (3), serially dilute it (1:20-1:2560), and add it to an ELISA plate pre-coated with specific SARS-CoV-2 protein antigen at a rate of 100 μL / well. Incubate at 37°C for 60 minutes. Wash the plate three times with phosphate buffer containing 0.05% (v / v) Tween 20, and add 1:2000 diluted horseradish peroxidase (HRP)-labeled goat anti-mouse immunoglobulin (IgG) monoclonal antibody at a rate of 100 μL / well. The antibody was incubated at 37°C for 30 minutes; the plate was washed three times with phosphate buffer containing 0.05% (v / v) Tween 20, and then 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic solution was added for color development; finally, the plate was read at an absorbance wavelength of 450 nm using an ELISA reader, and the serum antibody titer (EU) was calculated based on the maximum dilution factor that was 2.1 times greater than the average absorbance value of the negative control wells. The geometric mean (GMT) of the antibody titers of each group of mice was calculated.

[0077] (5) Collect the spleen mononuclear lymphocytes obtained in step (3), count them, and then dilute them to 5×10⁻⁶. 6 Cells / mL. 100 μL of cell suspension was added to each well of an ELISA plate pre-coated with mouse IFN-γ. A SARS-CoV-2-specific mixed peptide library was used as a stimulant. Each mouse cell was used in duplicate. The number of effector T cells that specifically secrete IFN-γ in the spleen lymphocytes of each mouse was detected according to the ELISA kit instructions.

[0078] like Figure 1 The results showed that, compared with the vaccine group receiving only the recombinant protein of the novel coronavirus (Group B), the use of CpG0620 adjuvant (Group A) could effectively increase the level of specific antibodies against the recombinant protein of the novel coronavirus.

[0079] like Figure 2 The results showed that, compared with the vaccine group receiving the recombinant SARS-CoV-2 protein alone (Group B), the use of CpG0620 adjuvant (Group A) could effectively enhance the specific cellular immune level of the recombinant SARS-CoV-2 protein.

[0080] Example 2: An adjuvant system consisting of CpG0620 and aluminum adjuvant induces the production of a recombinant protein vaccine against the novel coronavirus. Stronger specific humoral and cellular immunity

[0081] (1) After preparing CpG0620 adjuvant according to step (1) in Example 1, it is mixed with aluminum adjuvant to obtain an adjuvant system containing 20 μg / mL CpG0620 adjuvant and 1 mg / mL aluminum adjuvant.

[0082] (2) The recombinant protein of the novel coronavirus and the adjuvant system described in step (1) are mixed in a ratio of 1:1 (v:v) to obtain a vaccine sample containing 50 μg / mL of recombinant protein of the novel coronavirus, 10 μg / mL of CpG0620 adjuvant and 0.5 mg / mL of aluminum adjuvant.

[0083] (3) Six-week-old Babl / c mice were divided into three groups of eight each: the novel coronavirus recombinant protein plus CpG0620 aluminum adjuvant system experimental group (Group A), the novel coronavirus recombinant protein plus aluminum adjuvant group (Group B), and the CpG0620 aluminum adjuvant system control group (Group C). Each mouse was immunized with 1 ml of the dose. Serum and spleen mononuclear lymphocytes were collected on the 14th day after the first immunization.

[0084] (4) Take the serum collected in step (3), serially dilute it (1:40-1:5120), and add it to an ELISA plate pre-coated with specific SARS-CoV-2 protein antigen at a rate of 100 μL / well. Incubate at 37°C for 60 minutes. Wash the plate three times with phosphate buffer containing 0.05% (v / v) Tween 20, and add 1:2000 diluted horseradish peroxidase (HRP)-labeled goat anti-mouse immunoglobulin (IgG) monoclonal antibody at a rate of 100 μL / well. The antibody was incubated at 37°C for 30 minutes; the plate was washed three times with phosphate buffer containing 0.05% (v / v) Tween 20, and then 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic solution was added for color development; finally, the plate was read at an absorbance wavelength of 450 nm using an ELISA reader, and the serum antibody titer (EU) was calculated based on the maximum dilution factor that was 2.1 times greater than the average absorbance value of the negative control wells. The geometric mean (GMT) of the antibody titers of each group of mice was calculated.

[0085] (5) Collect the spleen mononuclear lymphocytes obtained in step (3), count them, and then dilute them to 5×10⁻⁶. 6 Cells / mL. 100 μL of cell suspension was added to each well of an ELISA plate pre-coated with mouse IFN-γ. A SARS-CoV-2-specific mixed peptide library was used as a stimulant. Each mouse cell was used in duplicate. The number of effector T cells that specifically secrete IFN-γ in the spleen lymphocytes of each mouse was detected according to the ELISA kit instructions.

[0086] like Figure 3The results showed that, compared with the group consisting of recombinant SARS-CoV-2 protein plus aluminum adjuvant alone (Group B), the adjuvant system consisting of CpG0620 and aluminum adjuvant (Group A) could induce stronger levels of specific antibodies against recombinant SARS-CoV-2 protein.

[0087] like Figure 4 The results showed that, compared with the group receiving recombinant SARS-CoV-2 protein plus aluminum adjuvant alone (Group B), the adjuvant system consisting of CpG0620 and aluminum adjuvant (Group A) induced a stronger level of specific cellular immunity against the recombinant SARS-CoV-2 protein.

[0088] Example 3: Comparison of CpG0620 and the marketed CpG1018-induced recombinant protein vaccine for novel coronavirus production Specific humoral and cellular immunity levels

[0089] (1) After preparing the CpG0620 adjuvant according to step (1) in Example 1, it was mixed with an aluminum adjuvant to obtain a CpG0620 aluminum adjuvant system containing 20 μg / mL CpG0620 adjuvant and 1 mg / mL aluminum adjuvant. At the same time, the commercially available CpG1018 adjuvant was also mixed with the aluminum adjuvant to obtain a CpG1018 aluminum adjuvant system containing 20 μg / mL CpG1018 and 1 mg / mL aluminum adjuvant.

[0090] (2) The recombinant protein of the novel coronavirus was mixed with the two adjuvant systems in step (1) at a ratio of 1:1 (v:v) to obtain a vaccine sample containing 50 μg / mL of recombinant protein of the novel coronavirus, 10 μg / mL of CpG adjuvant and 0.5 mg / mL of aluminum adjuvant.

[0091] (3) Six-week-old Babl / c mice were divided into four groups of eight each: the novel coronavirus recombinant protein plus CpG0620 aluminum adjuvant system experimental group (group A), the novel coronavirus recombinant protein plus CpG01018 aluminum adjuvant system experimental group (group B), the CpG0620 aluminum adjuvant system control group alone (group C) and the CpG1018 aluminum adjuvant system control group alone (group D). Each mouse was immunized with a dose of 1 ml. Serum and spleen mononuclear lymphocytes were collected on the 14th day after the first immunization.

[0092] (4) Take the serum collected in step (3), serially dilute it (1:40-1:5120), and add it to an ELISA plate pre-coated with specific SARS-CoV-2 protein antigen at a rate of 100 μL / well. Incubate at 37°C for 60 minutes. Wash the plate three times with phosphate buffer containing 0.05% (v / v) Tween 20, and add 1:2000 diluted horseradish peroxidase (HRP)-labeled goat anti-mouse immunoglobulin (IgG) monoclonal antibody at a rate of 100 μL / well. The antibody was incubated at 37°C for 30 minutes; the plate was washed three times with phosphate buffer containing 0.05% (v / v) Tween 20, and then 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic solution was added for color development; finally, the plate was read at an absorbance wavelength of 450 nm using an ELISA reader, and the serum antibody titer (EU) was calculated based on the maximum dilution factor that was 2.1 times greater than the average absorbance value of the negative control wells. The geometric mean (GMT) of the antibody titers of each group of mice was calculated.

[0093] (5) Collect the spleen mononuclear lymphocytes obtained in step (3), count them, and then dilute them to 5×10⁻⁶. 6 Cells / mL. 100 μL of cell suspension was added to each well of an ELISA plate pre-coated with mouse IFN-γ. A SARS-CoV-2-specific mixed peptide library was used as a stimulant. Each mouse cell was used in duplicate. The number of effector T cells that specifically secrete IFN-γ in the spleen lymphocytes of each mouse was detected according to the ELISA kit instructions.

[0094] like Figure 5 The results showed that, compared with the adjuvant system consisting of CpG1018 and aluminum adjuvant (Group B), the adjuvant system consisting of CpG0620 and aluminum adjuvant (Group A) induced stronger levels of specific antibodies against the recombinant protein of the novel coronavirus.

[0095] like Figure 6 The results showed that there was no significant difference in the level of specific cellular immunity against the recombinant protein of the novel coronavirus induced by the CpG0620 aluminum adjuvant system (Group A) and the CpG1018 aluminum adjuvant system (Group B) (P>0.05).

[0096] Example 4: CpG0620 adjuvant alone induces stronger specificity in recombinant human papillomavirus type 16 protein vaccine. Sexual antitumor antigen cellular immune level

[0097] (1) Prepare CpG0620 adjuvant according to step (1) in Example 1.

[0098] (2) The recombinant human papillomavirus type 16 protein was combined with CpG0620 adjuvant to obtain a vaccine sample containing 500 μg / mL recombinant human papillomavirus type 16 protein and 200 μg / mL CpG0620 adjuvant.

[0099] (3) Six-week-old C57BL / c mice were divided into three groups of eight each: human papillomavirus type 16 recombinant protein plus CpG0620 adjuvant experimental group (Group A), human papillomavirus type 16 recombinant protein vaccine alone group (Group B), and CpG0620 adjuvant alone control group (Group C). Each mouse was injected into the thigh muscle with 0.1 ml of the vaccine. Splenic mononuclear lymphocytes were collected on the 14th day after the first immunization.

[0100] (4) Collect the spleen mononuclear lymphocytes obtained in step (3), count them, and then dilute them to 5×10⁻⁶. 6 Cells / mL. 100 μL of cell suspension was added to each well of an ELISA plate pre-coated with mouse IFN-γ. A human papillomavirus type 16 recombinant protein-specific mixed peptide library was used as a stimulus. Each mouse cell was used in duplicate. The number of effector T cells that specifically secrete IFN-γ in the spleen lymphocytes of each mouse was detected according to the ELISA kit instructions.

[0101] like Figure 7 The results showed that, compared with the group receiving the recombinant human papillomavirus type 16 vaccine alone (Group B), the use of CpG0620 adjuvant (Group A) could effectively enhance the level of cellular immunity against specific anti-tumor antigens of the recombinant human papillomavirus type 16 protein.

[0102] Example 5: CpG0620 adjuvant alone induces stronger inhibition of human papillomavirus type 16 recombinant protein vaccine. The ability of tumors to grow

[0103] (1) Prepare CpG0620 adjuvant according to step (1) in Example 1.

[0104] (2) The recombinant human papillomavirus type 16 protein was combined with CpG0620 adjuvant to obtain a vaccine sample containing 500 μg / mL of recombinant human papillomavirus type 16 protein and 200 μg / mL of CpG0620. Simultaneously, the recombinant human papillomavirus type 16 protein was also combined with commercially available CpG1018 to obtain a vaccine sample containing 500 μg / mL of recombinant human papillomavirus type 16 protein and 200 μg / mL of CpG1018.

[0105] (3) Human papillomavirus-specific tumor cells TC-1 were passaged and cultured until they reached approximately 90% confluence. The cells were then digested to prepare a TC-1 tumor cell suspension. The resulting cells were diluted with phosphate-buffered saline to a concentration of 4 × 10⁻⁶ cells / mL. 5 Cells / mL. Six-week-old C57BL / c mice were subcutaneously inoculated with 100 μL of TC-1 tumor cells per mouse in the inner groin area of ​​the left leg to obtain a mouse tumor model.

[0106] (4) Mice inoculated with tumor cells were divided into four groups of 10 each: group A (recombinant human papillomavirus type 16 protein and CpG0620 adjuvant), group B (recombinant human papillomavirus type 16 protein and CpG1018 adjuvant), group C (recombinant human papillomavirus type 16 protein vaccine alone), and group D (blank control group). On the third day after inoculation with tumor cells, mice were immunized by intramuscular injection of 100 μL / mouse in the right thigh.

[0107] (5) Record the tumor formation changes of each mouse regularly.

[0108] like Figure 8 The results showed that, compared with the control group (Group D), the recombinant human papillomavirus type 16 (HPV16) vaccine with CpG0620 adjuvant (Group A), the recombinant HPV16 vaccine with CpG1018 adjuvant (Group B), and the HPV16 vaccine alone (Group C) all significantly inhibited tumor growth in mouse tumor models. The antitumor effect of HPV16 recombinant protein containing CpG0620 or CpG1018 was better than that of HPV16 vaccine alone (Group C). Furthermore, no tumors were observed in mice in the HPV16 recombinant protein vaccine group with CpG0620 adjuvant (Group A), while a few mice in the HPV16 recombinant protein vaccine group with CpG1018 adjuvant (Group B) began to develop tumors on day 21 of tumor growth.

[0109] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0110] The above-described embodiments are merely illustrative of several implementation methods of the present invention, facilitating a detailed and specific understanding of the technical solutions of the present invention. However, they should not be construed as limiting the scope of protection of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Furthermore, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this invention patent should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A nucleotide, characterized in that, The sequence of the nucleotide is shown in SEQ ID No:

1.

2. Use of the nucleotide of claim 1 in the preparation of an immune adjuvant.

3. A vaccine, characterized in that, The vaccine comprises an antigen and an immune adjuvant, wherein the immune adjuvant comprises the nucleotide of claim 1 or further comprises other immune adjuvants.

4. The vaccine according to claim 3, characterized in that, The vaccine meets one or more of the following conditions: (1) The vaccine is a recombinant protein vaccine; and, (2) The other immune adjuvants are selected from one or more of aluminum adjuvants and CpG1018.

5. The vaccine according to claim 4, characterized in that, The antigen is a recombinant protein of the novel coronavirus.

6. The vaccine according to claim 4, characterized in that, The antigen is a recombinant protein of human papillomavirus type 16.

7. The vaccine according to any one of claims 3 to 6, characterized in that, The mass ratio of the antigen to the nucleotide is (2.5-5):

1.

8. The vaccine according to any one of claims 3 to 6, characterized in that, The mass ratio of the antigen, the nucleotide, and the other immune adjuvant is (2.5-5):1:(45-55).

9. The vaccine according to claim 8, characterized in that, The mass ratio of the antigen, the nucleotide, and the other immune adjuvant is (2.5-5):1:(49-51).

10. A compound immune adjuvant, characterized in that, The composite immune adjuvant comprises the nucleotide and aluminum adjuvant as described in claim 1.

11. The compound immune adjuvant according to claim 10, characterized in that, The mass ratio of the nucleotide to the aluminum adjuvant is 1:(45-55).

12. The compound immune adjuvant according to claim 11, characterized in that, The mass ratio of the nucleotide to the aluminum adjuvant is 1:(49-51).

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