CN114796476B - Novel subunit vaccine nucleic acid adjuvant system and application thereof - Google Patents
Novel subunit vaccine nucleic acid adjuvant system and application thereof Download PDFInfo
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
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Abstract
The invention relates to a novel subunit vaccine nucleic acid adjuvant system and application thereof, and belongs to the technical field of vaccine adjuvants. The novel subunit vaccine nucleic acid adjuvant system comprises a nucleic acid adjuvant, wherein the nucleic acid adjuvant comprises single-stranded oligodeoxynucleotide fragments (CpG ODN) of GC, and the single-stranded oligodeoxynucleotide fragments of GC are 2395 and BW006. The novel nucleic acid adjuvant CpG ODN system and the proper immune program can stimulate a mouse organism to obtain higher humoral immunity and cellular immunity level, and the immunity is biased to TH1 type, namely biased to the cellular immune response type. The method avoids the risks of potential antibody-dependent infection enhancement of the partially inactivated vaccine and subunit vaccine and the risks of serious lung immunopathology featuring eosinophil infiltration, and can ensure that the vaccine partially dependent on the cellular immune response level obtains better protection effect.
Description
Technical Field
The invention belongs to the technical field of vaccine adjuvants, relates to an adjuvant system of a vaccine, and in particular relates to a novel nucleic acid adjuvant system of a subunit vaccine and application thereof. Particularly when subunit vaccines are used, where the vaccine elicits a weaker immune response in the body or where the protective effect of the vaccine has a great correlation with the strength of the cellular immune response, for example: a bullous subunit vaccine. The novel nucleic acid adjuvant system and the proper immunization program can stimulate the organism of the mouse to obtain higher humoral immunity and cellular immunity level, and the immunity is biased to TH1 type, namely biased to cellular immune response type.
Background
Modern biological technology, such as synthetic peptide vaccines, gene recombinant subunit vaccines, anti-idiotype antibody vaccines, nucleic acid vaccines, etc., has been developed. The novel vaccine has the advantages of high antigen purity, small relative molecular weight and low adverse reaction of the vaccine, but the novel vaccine cannot be denied that the immunogenicity is low and the effective immune response of an organism cannot be stimulated. In order to achieve satisfactory immunostimulatory immunogenicity, and to enhance the ability of a vaccine to induce an immune response in the body, it is necessary to add substances to the vaccine that have an adjuvant effect. The vaccine adjuvant is an important field of research of novel vaccines at present, and the ideal vaccine adjuvant has the characteristics of safety, effectiveness, definite chemical composition, capability of inducing cellular immunity, degradability, no immunogenicity and the like. Currently, there are few vaccine adjuvants approved for use in humans, with classical aluminum adjuvants being the most widely used. Aluminum adjuvants are capable of inducing the body to exert a remarkable immune enhancing effect by means of an immunostimulation effect and a depot effect, and are the only worldwide accepted vaccine adjuvants for humans. However, when the aluminum adjuvant and a plurality of recombinant or synthesized polypeptide vaccines are immunized together, effective immune response cannot be stimulated, freeze-drying cannot be carried out, effective cellular immunity cannot be induced, and local severe reactions such as erythema, nodules, granulomatous inflammation, contact allergy and the like can occur at injection sites, so that the requirements of development of novel vaccines are difficult to meet.
Several nucleic acid materials have been used in medicine for a long time, such as small molecule nucleotide nucleoside analogs, for the treatment of viral infections and cancers because of their ability to interfere with DNA replication and repair, transcription and DNA or RNA stability, directly or by affecting the functional pathways of enzymes, receptors and structural proteins involved therein.
CpG ODN is DNA containing unmethylated CpG ODN motif, and can induce natural immunity and acquired immunity. CpG ODNs have a broad range of immunomodulatory effects, including promoting B cell proliferation and secretion of immunoglobulins; up-regulating synergistic stimulus and MH CII molecular expression to enhance antigen presentation; inducing dendritic cells, monocytes and macrophages to secrete IL-26, IL-12, GM-CSF and tumor necrosis factor (TNF-a) and indirectly activating NK cells to secrete IFN-gamma. The synthetic CpG ODN has the dual functions of enhancing cellular immunity and humoral immunity and is a potential novel adjuvant.
Nucleic acid adjuvants stimulate the body to produce a stronger cellular immune response than conventional Alum adjuvants, which is more important than humoral immune response for long-term incubation and reinfection of the virus in the protective effect of the partial vaccine. For example, the herpes zoster vaccine has been developed: the protection rate of Merk attenuated seedlings Zostavax to 50-59, 60-69 and people over 70 years old is about 70%,64% and 38% respectively. This decrease in protection rate with age is mainly due to the weakening of the cellular immune response that occurs with aging of the immune system. The herpes zoster genetic engineering subunit vaccine Shingrix of GSK uses the conserved viral glycoprotein E (gE) expressed by Chinese hamster ovary Cells (CHO) AS an antigen, and uses an adjuvant AS01B to effectively enhance the specific cellular immune response to the VZV-gE, so that the protection rate of the vaccine in healthy people over 50 years old is AS high AS 97.2 percent (96.6 percent, 97.3 percent and 91.3 percent for people over 50-59, 60-69 and 70 years old respectively) and the vaccine shows good safety and effectiveness in immunodeficiency people including HIV carriers.
In addition, when developing a coronavirus S protein subunit vaccine, after using the full-length S protein as an immunogen and combining with an aluminum adjuvant system for immunization, the vaccine has a certain protection effect on virus attack, but can induce severe immune pathology of the lung, which is characterized by eosinophil infiltration, due to virus infection. This Th2 type immune response presents a fatal risk to vaccinators (such as many elderly people) with underlying respiratory diseases such as asthma, pulmonary obstruction, etc., which has led to clinical phase 1 experimental withdrawal (NCT 01376765) of the S protein-based SARS subunit vaccine in the United states. The novel nucleic acid adjuvant system can excite the organism to generate stronger cellular immune response and more balanced immune response, so that the immune response generated by the organism is biased towards the cellular immune response to be mainly, and the risk of serious immune pathology of the lung with eosinophil infiltration as a characteristic can be effectively reduced.
Therefore, how to overcome the defects of the prior art is a problem which needs to be solved in the technical field of the current vaccine adjuvant.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provide a novel subunit vaccine nucleic acid adjuvant system and application thereof, wherein the novel subunit vaccine nucleic acid adjuvant system and immunogen (such as S1 protein) can be used for immunizing an organism together faster and better to excite the organism to generate immune response so as to generate a large amount of antibodies, and can also stimulate the organism to generate stronger cellular immune response so as to enable the organism to generate protective antibodies rapidly and in a large amount and generate a relatively balanced immune protection effect. This avoids both the risk of potential antibody dependent infection enhancement (ADE) of partially inactivated vaccines and subunit vaccines and the risk of severe immune pathology in the lungs featuring eosinophil infiltration. It also enables a vaccine that depends in part on the level of cellular immune response to achieve better protection.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
The novel subunit vaccine nucleic acid adjuvant system is characterized by comprising a nucleic acid adjuvant, wherein the nucleic acid adjuvant contains CpG ODN, the CpG ODN is a single-chain oligodeoxynucleotide fragment of GC, the single-chain oligodeoxynucleotide fragment of the GC is 2395 and BW006, and the nucleotide sequence is as follows;
2395：5’-tcgtcgttttcggcgcgcgcc-3’；(SEQ ID NO.1)；
BW006：5’-tcgacgttcgtcgttcgtcgttc-3’；(SEQ ID NO.2)。
Further, preferably, at least one of Poly I C, cdi-AMP and Alum is also included.
Further, it is preferred that the subunit vaccine novel nucleic acid adjuvant system comprises an equal amount of the mixed single stranded oligodeoxynucleotide fragment containing GC and PolyI: C, or an equal amount of the mixed single stranded oligodeoxynucleotide fragment containing GC and cdi-AMP, or an equal amount of the mixed single stranded oligodeoxynucleotide fragment containing GC and Alum.
The invention also provides application of the novel subunit vaccine nucleic acid adjuvant system in preparation of subunit vaccines.
The invention also provides a subunit vaccine, wherein in the single injection vaccine, antigen protein is 10-1000ug, and single-chain oligodeoxynucleotide fragment containing GC is 15-1500ug.
The invention further provides a subunit vaccine, in the single injection vaccine, antigen protein is 10-1000ug, and the total content of nucleic acid is 15-1500ug, wherein the nucleic acid adjuvant is equal mass Poly I, C7.5-750 ug and single-chain oligodeoxynucleotide fragment containing GC is 7.5-750ug, or equal mass nucleic acid adjuvant cdi-AMP 7.5-750ug and single-chain oligodeoxynucleotide fragment containing GC is evenly mixed, or equal mass adjuvant Alum 7.5-750ug and single-chain oligodeoxynucleotide fragment containing GC is evenly mixed.
Further, it is preferable that the antigen protein is an S1 protein.
The immune components are administered by intramuscular injection, and are immunized 2-4 times with an interval of 2-4 weeks, for improving the intensity and bias of the immune response of the vaccine.
CpG is an abbreviation for cytosine (C) -phosphate (p) -guanine (G), and CpG exists in two forms in mammals: one is dispersed in the DNA sequence; the other is in a highly aggregated state, known as CpG islands (CPG ISLAND). CpG islands are often located near gene transcription regulatory regions, and the number of CpG islands has a good correspondence with gene density. Many genes, particularly the promoter regions of housekeeping genes, typically exist in regions that are rich in the dinucleotide "CG," known as "CpG islands" (CPG ISLAND). In addition, prokaryotic bacterial DNA contains high frequencies of CpG dinucleotides, bacterial DNA and certain polynucleotides containing unmethylated CpG dinucleotides are able to stimulate murine and human lymphocytes. Higher vertebrates exhibit a frequency of CpG dinucleotides of 1/50 and are mostly methylated, eukaryotic cells and methylated polynucleotides being unable to stimulate murine and human lymphocytes. CpG ODN DNA can directly stimulate B cells, macrophages and DCs to secrete cytokines. In particular TH 1-like cytokines such as IL-12 and IL-18; cells express a costimulatory factor molecule, which is shown to enhance antigen presentation. The strong TH 1-type response induced by DNA CpG ODN suggests that these molecules can be used as adjuvants for vaccines against various infectious substances, tumor antigens, allergens, etc.
The invention is based on the nucleic acid adjuvant system CpG ODN and antigen protein (S1 protein) according to the specific proportion mixing, to the experimental mice to carry out the intramuscular injection immunization (immune rule) of the specific immunization process, then through the detection of the antibody level in the serum of the mice after immunization, comprising: total antibody titer IgG, typed antibody titer IgG1, igG 2a, ratio of IgG1/2 a. And further determining the total antibody level and the typed antibody titer level of serum after immunization, and finally determining the immune response bias generated by the immunized mice according to the ratio of IgG1/2 a. The invention relates to a method for preparing a composite material, which comprises the following steps: the nucleic acid adjuvant system is used for mixing target antigen proteins, and after intramuscular injection immunization of mice is carried out according to a preset immunization program, higher total antibody IgG titer levels can be obtained, and the total antibody IgG titer levels are not lower than that of the traditional Alum adjuvant group, so that the nucleic acid adjuvant system can induce the mice to generate stronger humoral immune response and also can obtain stronger cellular immune response. Furthermore, we have shown that immunization of mice with an adjuvant system of mixed components achieved a strong humoral and cellular immune response and an immune bias of TH1 type, i.e.a ratio of IgG1/2a <1, by mixing equal amounts of Cp G ODN with equal amounts of other nucleic acid adjuvants PolyI: C, cdi-AMP or Alum adjuvant. The use of different adjuvant components and different immunization programs has a crucial influence on the level of antibodies generated by an organism and the deviation of antibody response, while a CpG ODN nucleic acid adjuvant system can induce the organism to generate higher humoral immune response and generate stronger cellular immune response, i.e. the CpG ODN nucleic acid adjuvant system is superior to the traditional Alum adjuvant system in terms of antibody titer level and immune balance which are excited after immunization; when single-component CpG ODN or CpG ODN is mixed with other adjuvants, higher humoral immunity and cellular immunity level can be obtained, and the immunity bias is TH1 type, namely the cellular immunity response type. The invention provides a more effective and safer adjuvant system for future subunit vaccine research.
Compared with the prior art, the invention has the beneficial effects that:
The novel nucleic acid adjuvant CpG ODN system or CpG ODN is respectively mixed with one of PolyI C, cdi-AMP and Alum in equal quantity, and can stimulate a mouse organism to obtain higher humoral immunity and cellular immunity level by combining with a proper immunization program, and the immunity is biased to TH1 type, namely biased to the cellular immune response type. The method avoids the risks of potential antibody-dependent infection enhancement (ADE) of the partially inactivated vaccine and subunit vaccine and the risks of serious lung immune pathology characterized by eosinophil infiltration, and can ensure that the vaccine partially dependent on the cellular immune response level obtains better post-immune protection effect. For example, the herpes zoster vaccine has been developed: the protection rate of Merk attenuated seedlings Zostavax to 50-59, 60-69 and people over 70 years old is about 70%,64% and 38% respectively. This decrease in protection rate with age is mainly due to the weakening of the cellular immune response that occurs with aging of the immune system. The herpes zoster genetic engineering subunit vaccine Shingrix of GSK uses the conserved viral glycoprotein E (gE) expressed by Chinese hamster ovary Cells (CHO) AS an antigen, and uses an adjuvant AS01B to effectively enhance the specific cellular immune response to the VZV-gE, so that the protection rate of the vaccine in healthy people over 50 years old is AS high AS 97.2 percent (96.6 percent, 97.3 percent and 91.3 percent for people over 50-59, 60-69 and 70 years old respectively) and the vaccine shows good safety and effectiveness in immunodeficiency people including HIV carriers.
In the present invention, the total antibody titer IgG nucleic acid adjuvant CpG ODN group obtained under the 3-needle immunization procedure is not lower than the antibody level of the Alum group, which indicates that the single component nucleic acid adjuvant CpG ODN system can stimulate the organism to generate humoral immune response stronger than that of the traditional Alum system, as shown in fig. 1. In addition, the highest detection value of the parting antibody titer IgG2a of the CpG ODN nucleic acid adjuvant system indicates that the parting antibody titer IgG2a can stimulate the organism to generate stronger cellular immune response. Thus, the nucleic acid adjuvant system can excite the organism to generate stronger cellular immune response, and the immune response generated by the organism is induced to be more balanced;
As can be seen from the results of the total antibody titer IgG detection of fig. 4-B and 4-E, the mixed adjuvants gave very high antibody titers after the immunization procedure, and the nucleic acid adjuvant group (IgG 64000) was significantly higher than the Alum adjuvant group (IgG 16000); in addition, after immunization of all test groups at 3 rd needle, at 2 weeks interval, i.e. from day 42 of starting immunization, significant enhancement of IgG antibodies occurred, with 5-7 groups from no more than 3200 to 64000 in table 1, 10-20 fold improvement. The CpG ODN is added into the adjuvant component for immunization, so that the mouse organism can be stimulated to rapidly produce a large amount of antibodies, and in a nucleic acid adjuvant system, the 3-time immunization program is stronger than the 2-time immunization program, and the mouse organism can be effectively stimulated to obtain the improvement of the antibody titer by 10-20 times. As can be seen from FIG. 4-C, the IgG2a type antibodies were all generated after immunization with the mixed adjuvant supplemented with the nucleic acid CpG ODN component, demonstrating that CpG ODN stimulated a strong cellular immune response in the body. As can be seen from fig. 4-F, mixed component adjuvant immunization program 3: using groups 5-7 of Table 1, intramuscular injections were performed 3 times, each time at 2 week intervals, tail vein blood was taken before each immunization from needle 2 until the mice die, and mice were sacrificed at 4 week intervals after the end of the immunization at needle 3. The IgG1/2a value of each group for detecting immune response bias is less than 1, which indicates that the adjuvant of the mixed components can stimulate the organism to generate TH1 type immune response bias, namely, the cell immune response is mainly.
The characteristics show that the CpG ODN system of the single-component nucleic acid adjuvant or the CpG ODN is respectively mixed with one of the Poly I C, cdi-AMP and Alum in equal quantity for use, and the requirements of pursuing a more effective and safer adjuvant system of the subunit vaccine in the future are met.
Drawings
FIG. 1 is a graph of immunization program and detection of antibody titers using the Elisa method; wherein, the left of fig. 1 is a flow chart of the immunization program. The single component novel nucleic acid adjuvants CpG15ug, poly I: C15ug, cdi-AMP15ug system and traditional Alum adjuvant 15ug system were administered by intramuscular injection 3 times, each at 2 week intervals, and the immunization program of mice was sacrificed at 2 week intervals after the end of the 3 rd needle immunization. FIG. 1 shows the results of detection of antibody titer by the Elisa method, followed by total antibody titer IgG from left to right; the ratio of the typed antibody titers IgG1, gG2a and IgG1/2 a;
FIG. 2 shows the results of detection of antibody titer using the Elisa method, followed by total antibody titer IgG from left to right; the ratio of the typed antibody titers IgG1, igG2a and IgG1/2 a;
FIG. 3 is a graph of the results of a method test using a flow assay; FIG. 3-A is a graph showing the results of detecting CD4+ T cells (%) using a flow assay; FIG. 3-B is a graph showing the results of detecting CD8+ T cells (%) using a flow assay;
FIG. 4 is a graph of immunization program and immunization results; wherein, FIG. 4-A is a flow chart of the immunization program; firstly, carrying out intramuscular injection for 2 times by using single-component Poly I (C15 ug), cdi-AMP (adenosine triphosphate) 15ug and single-component Alum adjuvant 15ug, wherein each time is separated by 2 weeks, then replacing an adjuvant system of mixing C7.5ug and CpG ODN7.5ug uniformly in equal quantity, mixing cdi-AMP7.5ug and CpG ODN7.5ug uniformly in equal quantity, mixing Alum 7.5ug and CpG ODN7.5ug uniformly in equal quantity, carrying out intramuscular injection for the subsequent 2-needle immunization, and killing mice at 2-week intervals after the 4 th-needle immunization is ended; FIG. 4-B shows the results of total antibody titres IgG after immunization following the 4-A protocol, at 14, 28, 42, 56 days after immunization initiation, respectively, in various adjuvant combinations. FIG. 4-C shows the results of post-immunization isotype antibody titres IgG1, igG2a for the various adjuvant combinations at 14, 28, 42, 56 days after immunization according to the 4-A protocol; FIG. 4-D is a flow chart of immunization procedure, using the mixed component adjuvant PolyI: C7.5ug and CpG ODN7.5ug equally mixed, cdi-AMP7.5ug and CpG ODN7.5ug equally mixed, alum 7.5ug and CpG ODN7.5ug equally mixed and intramuscular injection performed 3 times, each time interval being 2 weeks, and the immunization procedure of killing mice at 4 weeks after the end of the 3 rd needle immunization. FIG. 4-E shows the results of total antibody titres IgG after immunization following the 4-B protocol, at 14, 28, 42, 56 days after immunization initiation, respectively, in various adjuvant combinations. FIG. 4-F shows the ratio of the bias of the immune response towards IgG1/2a after immunization according to the 4-A and 4-B protocols, respectively 28 days, 42 days, and 56 days after the start of immunization, in combination with various adjuvants.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The materials or equipment used are conventional products available from commercial sources, not identified to the manufacturer.
Example 1
BALB/c mice (6-8 weeks old, 14-20 g, purchased from animal center of institute of medical biology, academy of medical sciences, china) were used in the experiment, and the use amount of each component was 1/10 of the human body amount in the mouse immunization experiment. The specific content of each component of the immune antigen protein and adjuvant system (per injection) is shown in table 1.
TABLE 1
Materials: immune antigen protein, S1 protein, purchased from Beijing Yiqiao Shenzhou (cat: 40591-V08H); a low molecular weight PolyI: C double stranded polycytidylic acid fragment purchased from (Inviv oGen, inc. san Diego, calif., USA); the GC-rich single-stranded oligodeoxynucleotide fragment (CpG ODN) comprises 2 nucleic acid sequences:
2395：5’-tcgtcgttttcggcgcgcgcc-3’；(SEQ ID NO.1)；
BW006：5’-tcgacgttcgtcgttcgtcgttc-3’；(SEQ ID NO.2)。
when in use, the quality of 2395, BW006 and the like are evenly mixed for use;
Cdi-AMP cyclic diadenosine monophosphate is available from InvivoGen, inc.San Diego, CA, USA (cat: tlrl-nacda-5); alum aluminum adjuvant was purchased from thermo (cat: 77161).
EXAMPLE 2 immunization procedure for different Components
Single component immunization procedure 1: immunization compositions intramuscular injections were performed 3 times, 2 weeks apart, using the sequence numbers 1-4 of Table 1 above, and mice were sacrificed 2 weeks apart after the end of the 3 rd immunization, see FIG. 1-A for immunization procedures.
Mixed component immunization program 2: immunization compositions intramuscular injections were performed 3 times, 2 weeks apart, using the sequence numbers 5-7 of Table 1 above, and mice were sacrificed 2 weeks apart after the end of the 3 rd immunization, see FIG. 1-A for immunization procedures.
Mixed component immunization procedure 3: immunization compositions intramuscular injections were performed 3 times, 2 weeks apart, using the sequence numbers 5-7 of Table 1 above, and mice were sacrificed 4 weeks apart after the end of the 3 rd immunization, see FIG. 4-D for immunization procedures.
Single component immunization followed by replacement of mixed components, immunization program 4: the immune component was used in groups 1-4 of Table 1 above. Firstly, using single-component nucleic acid adjuvant Poly I C, cdi-AMP and Alum adjuvant respectively 15 ug/needle to be uniformly mixed with 10 ug/needle S1 protein, dissolving in 50 ul/needle sterile PBS, and performing intramuscular injection for 2 times, wherein each time is 2 weeks apart; then adding equal amount of CpG ODN adjuvant into the adjuvant component Poly I C, cdi-AMP and Alum adjuvant respectively, and replacing the adjuvant component with mixed adjuvant component, namely: poly I is an adjuvant system in which C7.5 ug and CpG ODN7.5ug are uniformly mixed in equal quantity, cdi-AMP 7.5ug and CpG ODN7.5ug are uniformly mixed in equal quantity, alum 7.5ug and CpG ODN7.5ug are uniformly mixed in equal quantity, then the subsequent intramuscular injection immunization is carried out for 2 weeks at intervals, mice are killed at intervals of 2 weeks after the 4 th immunization is finished, and the immunization program is shown in figure 4-A.
Example 3 animal experiments and detection sampling
Each group was immunized by intramuscular injection with BALB/c mice (6-8 weeks old, 14-20 g, purchased from animal center of institute of medical biology, national academy of medical sciences) as in example 2. Cardiac puncture blood collection after expiration of immunization or tail vein blood collection is performed before each immunization, starting from the 2 nd needle after immunization. The immune serum was then separated by centrifugation at 3000rpm for 20 minutes after standing overnight at 4℃and subjected to ELisa method for detection of total antibody titer IgG, the detection results are shown in FIG. 1 left, FIG. 2 left, FIG. 4-B, FIG. 4-E, the detection results of the typing antibody titers IgG1, igG2a are shown in FIG. 1, FIG. 2, FIG. 4-C and the ratios of the immune responses to IgG1/2a are shown in FIG. 1 right, FIG. 2 right, FIG. 4-F. Meanwhile, spleen lymphocyte separation was performed on mice immunized with expired, and after the mice were anesthetized with the anesthetic tribromoethanol (sigma), the spleen was dissected and removed and placed in a sterilized centrifuge tube. A50 mL centrifuge tube (Kogyo) was prepared, the cell filter was placed on the centrifuge tube (CELL STRAIN ER um Falcon), the spleen was placed on the filter, the spleen was ground with a 2.5mL syringe core, the spleen cells were wetted and rinsed with 3mL 1640 medium, and the filter was rinsed with 3mL sterile PBS. The collected 6mL of cell suspension was transferred to a 15mL pointed bottom centrifuge tube (Kogyo), centrifuged at 1800rpm for 5min. 2mL of RBC Lysis added and the reaction is carried out for 5min at room temperature, 6mL of PBS is added and terminated, at 1800rpm for 5min at 4 ℃, then 5mL of PBS is used for resuspension counting, centrifugation is carried out again at 1800rpm for 5min, finally, the cells are resuspended with 1640 complete medium until the concentration reaches 10 7/mL, 100 mu L of 96-well cell culture plate (purchased from Corning) is added to the final cell quantity of 1X 10 6 cells/well, and flow cytometry detection is carried out. The results of the flow assay are shown in FIG. 3-A, CD for CD 4-T cells and FIG. 3-B for CD 4-T cells.
Example 4 antibody titre detection
The immunogenic protein S1 was dissolved as a capture antibody in 2ug/ml sterile PBS, and after thorough mixing, 50 ul/well was plated, 96 well ELISA plate (Corning) and incubated overnight at 4 ℃. Plates were washed 200 ul/well x 2 times using TBST (0.05% (V/V) Tween-20 in PBS) as wash solution; then adding a sealing solution (5% (W/V) skimmed milk dissolved in PBS), and incubating at 37deg.C for 1 hr; TBST wash plates after removal of blocking solution, 200 ul/well X4 times. Then, 50. Mu.L/well of serum samples were added and incubated with a 2-fold serial dilution of 8 dilutions (8-11 dilutions of test serum were selected depending on serum titer level) using a dilution (1% (W/V) of skim milk in PBS) at 37℃for 1h, and the plates were washed 200 ul/well X5 times with TBST. Then, according to the detected antibody type, adding: horse radish peroxidase HRP-labeled secondary anti-goat anti-mouse IgG at 1:10000, horse radish peroxidase HRP-labeled secondary anti-goat anti-mouse IgG1 at 1:500, and horse radish peroxidase-labeled secondary anti-goat anti-mouse gG2a (Sieimer fly) at 1:2000, and incubated for 1h at 37 ℃. The plates were then washed 200 ul/well x5 times with TBST. Finally, 50. Mu.L of a color-developing solution (from BD) was added to each well, and after 10 minutes of standing at room temperature in the dark, 50. Mu.L of 2M sulfuric acid was added to each well to terminate the reaction, and the light absorption was measured at 450 nm. The critical serum dilution of OD450-Blank >0.1 was taken as antibody titer.
The results of the antibody titer detection and the IgG1/2a ratio of the immune serum are shown in the right side of FIG. 1, FIG. 2 and FIG. 4. Shown in FIG. 1 are the total antibody titers IgG, typed antibody titers IgG1, igG2a, and IgG1/2a values for detection of immune response bias obtained after immunization of groups 1-4 of Table 1 according to one-component immunization procedure 1 (FIG. 1-A). As can be seen, the total antibody titer IgG nucleic acid adjuvant CpG ODN group obtained under the 3-needle immunization procedure was not lower than the antibody level of the Alum group, which indicates that the nucleic acid adjuvant system can stimulate the organism to generate humoral immune response stronger than that of the traditional Alum system. In addition, the detection value of the parting antibody titer IgG2a can show that the CpG ODN adjuvant group immune serum can obtain higher IgG2a antibody titer, which indicates that the CpG ODN nucleic acid adjuvant system can stimulate the organism to generate stronger cellular immune response. Finally, the ratio of IgG1/2a shows that the nucleic acid adjuvant system has a lower ratio approaching 1, i.e. the nucleic acid adjuvant system can excite the organism to generate stronger cellular immune response, and the immune response generated by the organism is more balanced; the IgG1/2a ratio of the nucleic acid adjuvant CpG ODN immune serum is 0.25, which shows that the immune response is biased to TH1, namely the cellular immune response is mainly, and the nucleic acid adjuvant CpG ODN immune serum can also stimulate the organism to generate stronger humoral immune response, so that the nucleic acid adjuvant CpG ODN immune serum is an ideal adjuvant system, can not only stimulate higher antibody level, but also stimulate the organism to generate more balanced immune response.
FIG. 2 shows the total antibody titer IgG, the typed antibody titers IgG1, igG2a and the IgG1/2a ratio for detecting the bias of the immune response obtained after immunization of components 5-7 of Table 1 according to the mixed component immunization program 2 (FIG. 1-A). As can be seen from the left side of FIG. 2, the total antibody titer IgG of the mouse immune serum obtained under the 3-needle immunization procedure, the mixed component nucleic acid adjuvants PolyI: C7.5ug+CpG ODN 7.5ug, cdi-AMP 7.5ug+CpG ODN 7.5ug all obtained 64000 higher than that of 16000 of the group of Alum7.5ug+CpG ODN 7.5ug, which again demonstrates that the use of the mixed component nucleic acid adjuvant system PolyI: C+CpG ODN, cdi-AMP+CpG ODN can stimulate the organism to generate a stronger humoral immune response than the use of the conventional Alum+CpG ODN system. The level of titre of typed antibodies (in FIG. 2) was less than Ig G2a, indicating that the bias of the immune response generated by the body was shifted after immunization with the adjuvant system mixed with CpG ODN, from the TH2 type, i.e.humoral immunity, which generates antibodies in large amounts, to the TH1 type, i.e.humoral immunity, which is based on cellular immunity, such as intramuscular injection of a single component Alum 15ug adjuvant group, which generates high-intensity humoral immunity, when intramuscular injection is performed after equal amounts of Alum7.5ug and CpG ODN 7.5ug are mixed, the immune serum obtained is based on TH1 type cellular immunity, and the IgG1/2a value is 0.25 (right in FIG. 2). In addition, the ratio of the immune serum obtained from the 5-7 components in the above table 1 is less than 1 in the IgG1/2a value for detecting the bias of immune response, which indicates that the CpG ODN mixed component is used for intramuscular injection according to the immunization program 2, and the immune response is biased to TH1 type and cell immune response type.
Further, groups 5-7 of Table 1 were immunized as mixed components with procedure 3 (FIG. 4-D): intramuscular injections were performed 3 times, 2 weeks apart, with tail vein bleeding from the 2 nd needle until the mice die, and tail vein bleeding from the tail vein before each immunization, and mice were sacrificed 4 weeks apart after the 3 rd needle immunization was completed, as shown in fig. 4-D. Simultaneously, single component immunization was performed and the mixed components were replaced, immunization program 4 (FIG. 4-A): intramuscular injections were first performed 2 times with a single component nucleic acid adjuvant PolyI: C15 ug, cdi-AMP 15ug, and Alum 15ug adjuvant, each time 2 weeks apart, with tail vein blood collection taking place before each immunization, starting with the 2 nd needle immunization until the death of the mice. CpG ODN was then added separately and replaced with mixed components: poly I is an adjuvant system in which C7.5 ug and CpG ODN 7.5ug are uniformly mixed in equal quantity, cdi-AMP 7.5ug and CpG ODN 7.5ug are uniformly mixed in equal quantity, alum 7.5ug and CpG ODN 7.5ug are uniformly mixed in equal quantity, then the subsequent intramuscular injection immunization is carried out for 2 weeks at intervals, mice are killed at intervals of 2 weeks after the 4 th immunization is finished, and the immunization program is shown in figure 4-A.
As can be seen from the results of the total antibody titer IgG detection in fig. 4-B and 4-E, the 5-7 groups of adjuvants all obtained very high antibody titers after the immunization procedure was completed, and the nucleic acid adjuvant group (IgG 64000) was significantly higher than the Alum adjuvant group (IgG 16000); in addition, after immunization of all test groups at 3 rd needle, at intervals of 2 weeks, i.e. from day 42 of starting immunization, significant enhancement of IgG antibodies occurred, and in table 1, 5-7 groups of IgG were raised from no more than 3200 to 64000, by 10-20 fold. The CpG ODN is added into the adjuvant component for immunization, so that the mouse organism can be stimulated to rapidly produce a large amount of antibodies, and in a nucleic acid adjuvant system, the immunization program of 3 times is stronger than that of 2 times, and the mouse organism can be effectively stimulated to obtain the improvement of the antibody titer by 10-20 times.
As can be seen from FIG. 4-C, the IgG2a type antibodies were all generated after immunization with the adjuvant mixed with the nucleic acid CpG ODN component, indicating that CpG ODN stimulated the body to generate a stronger cellular immune response and also enhanced the humoral immune response. As can be seen from fig. 4-F, mixed component immunization program 3: using groups 5-7 of Table 1, intramuscular injections were performed 3 times, each time at 2 week intervals, tail vein blood was taken before each immunization from needle 2 until the mice die, and mice were sacrificed at 4 week intervals after the end of the immunization at needle 3. The IgG1/2a value of each group of detection immune response bias is less than 1, which indicates that the body generates TH1 type immune response bias, namely, the cell immune response is mainly. However, immunization program 4 was performed when using groups 2-7 of table 1: intramuscular injections were first performed 2 times, each 2 weeks apart, using a single component nucleic acid adjuvant PolyI: C15 ug, cdi-AMP 15ug, and Alum 15ug adjuvant. And then adding an adjuvant system in which CpG ODN is replaced by a mixed component PolyI, wherein C7.5 ug and CpG ODN 7.5ug are uniformly mixed in an equal amount, cdi-AM P7.5ug and CpG ODN 7.5ug are uniformly mixed in an equal amount, alum 7.5ug and CpG ODN 7.5ug are uniformly mixed in an equal amount, performing subsequent intramuscular injection immunization for 2 needles, performing tail vein blood sampling before each immunization from the beginning of the 2 nd needle until the death of the mouse, and killing the mouse after the end of the 4 th needle at an interval of 2 weeks. On day 28, 42 and 56 days, the immune response bias IgG1/2a values were all greater than 1, which indicated that the body developed a TH 2-type immune response bias, i.e., a humoral immune response, when mice were immunized with the first 2-needle single-component adjuvant, and cdi-AMP and Alum adjuvant, indicating that the body could not be altered in immune response bias by adding the same mass of CpG ODN, i.e., the 5-7-group mixed adjuvant of Table 1, after the TH 2-type immune response bias was developed using the 2-4-group single-component adjuvant of Table 1, during the subsequent 2-needle intramuscular injection.
Flow analysis:
S1 protein solution was added to the isolated spleen lymphocytes to a final concentration of 10ug/mL, with 10 6 spleen cells per well. Incubate at 37℃for 2h. BrefeldinA. Mu.l (Biolegend) was added and incubated overnight at 37 ℃. The following day, the cell suspension was transferred to an EP tube, centrifuged at 400g for 5min, the supernatant was discarded, washed once at 400. Mu. L STAINING buffer (Biolegend), and after adding a final concentration of 5ug/mL of 50ulCD16/32 (Biolegend) antibody for resuspension, the Fc receptor was blocked by incubation at 4℃for 10 min. Surface staining antibodies percp/CYani ne 5.5.5 anti-murine CD4 and FTTC anti-murine CD8a 50ul were added and stained in the dark at 4℃for 30min. 400ul staining buffer (Biolegend) was added directly for 1 wash, 200ul fixation buffer (Biolegend) was added to resuspend cells and incubated at room temperature for 20min in the dark. 400ul permeabilization wash buffer (Biolegend) was added and washed 2 times, 100ul of intracellular antibody was added: PE is anti-murine IFN-gamma (Biolegend) and APC is anti-murine IL-2 (Bi olegend). Incubate at room temperature for 30min in the dark. Directly 400uL permeabilization wash buffer (Biolegend) was added and washed 1 pass, and after 300uL PBS was resuspended, the detection was performed by an up-flow cytometer (beckman-cytoFLEX).
In groups 5-7 of Table 1, the adjuvant system of the equivalent mixing of Poly I: C7.5 ug with CpG ODN 7.5ug, cdi-AMP 7.5ug with CpG ODN 7.5ug, alum 7.5ug with CpG OD N7.5 ug was followed by mixed component immunization procedure 2 (FIG. 1-A), and the single component nucleic acid adjuvants of groups 2-4 of Table 1, poly I: C15 ug, cdi-AMP 15ug and conventional Alum 15ug adjuvants were compared by single component immunization procedure 1 (FIG. 1-A). Intramuscular injections were performed 3 times, each at 2 weeks intervals, and the immunization program of mice was sacrificed at 2 weeks intervals after the end of the 3 rd needle immunization. After expiration sampling CD4+ T cells (%) were detected using flow analysis as shown in FIG. 3-A and CD8+ T cells (%) as shown in FIG. 3-B. As can be seen from FIG. 3-A and FIG. 3-B, CD4+ T cells (%) and CD8+ T cells (%) are significantly improved after the mixed use of CpG ODN components, and it is also demonstrated that CpG ODN can effectively enhance cellular immune response of mouse body when used as an adjuvant component in combination with other components, and has an important influence on immune bias.
Example 5
A novel subunit vaccine nucleic acid adjuvant system comprises a nucleic acid adjuvant, wherein the nucleic acid adjuvant contains CpG ODN, the CpG ODN is a single-chain oligodeoxynucleotide fragment of GC, the single-chain oligodeoxynucleotide fragment of the GC is 2395 and BW006, and the nucleotide sequence is as follows;
2395：5’-tcgtcgttttcggcgcgcgcc-3’；(SEQ ID NO.1)；
BW006：5’-tcgacgttcgtcgttcgtcgttc-3’；(SEQ ID NO.2)。
Example 6
A novel subunit vaccine nucleic acid adjuvant system comprises a nucleic acid adjuvant, wherein the nucleic acid adjuvant contains CpG ODN, the CpG ODN is a single-chain oligodeoxynucleotide fragment of GC, the single-chain oligodeoxynucleotide fragment of the GC is 2395 and BW006 with equal mass, and the nucleotide sequence is as follows;
2395：5’-tcgtcgttttcggcgcgcgcc-3’；(SEQ ID NO.1)；
BW006：5’-tcgacgttcgtcgttcgtcgttc-3’；(SEQ ID NO.2)。
Also comprises Poly I and C.
The subunit vaccine new type nucleic acid adjuvant system includes single-chain oligodeoxynucleotide fragment containing GC and Poly I: C which are uniformly mixed in equal quantity.
Example 7
A novel subunit vaccine nucleic acid adjuvant system comprises a nucleic acid adjuvant, wherein the nucleic acid adjuvant contains CpG ODN, the CpG ODN is a single-chain oligodeoxynucleotide fragment of GC, the single-chain oligodeoxynucleotide fragment of the GC is 2395 and BW006 with equal mass, and the nucleotide sequence is as follows;
2395：5’-tcgtcgttttcggcgcgcgcc-3’；(SEQ ID NO.1)；
BW006：5’-tcgacgttcgtcgttcgtcgttc-3’；(SEQ ID NO.2)。
cdi-AMP is also included.
The subunit vaccine novel nucleic acid adjuvant system comprises single-stranded oligodeoxynucleotide fragments containing GC and cdi-AMP which are uniformly mixed in equal amounts.
Example 8
A novel subunit vaccine nucleic acid adjuvant system comprises a nucleic acid adjuvant, wherein the nucleic acid adjuvant contains CpG ODN, the CpG ODN is a single-chain oligodeoxynucleotide fragment of GC, the single-chain oligodeoxynucleotide fragment of the GC is 2395 and BW006 with equal mass, and the nucleotide sequence is as follows;
2395：5’-tcgtcgttttcggcgcgcgcc-3’；(SEQ ID NO.1)；
BW006：5’-tcgacgttcgtcgttcgtcgttc-3’；(SEQ ID NO.2)。
also include Alum.
The subunit vaccine novel nucleic acid adjuvant system comprises single-stranded oligodeoxynucleotide fragments containing GC and Alum which are uniformly mixed in equal amounts.
Example 9
A subunit vaccine, in single injection vaccine, antigen protein 10ug, contains single-chain oligodeoxynucleotide fragment 15ug of GC.
Example 10
A subunit vaccine, in single injection vaccine, S1 protein 1000ug, contains single-chain oligodeoxynucleotide fragment 1500ug of GC.
Example 11
A subunit vaccine, in single injection vaccine, S1 protein 100ug, contains single-chain oligodeoxynucleotide fragment 300ug of GC.
Example 12
In the single injection vaccine, antigen protein 10ug, poly I: C7.5ug, single-chain oligodeoxynucleotide fragment containing GC 7.5ug. The single-stranded oligodeoxynucleotide fragments of the GC are 2395 and BW006 with equal mass.
Example 13
A subunit vaccine, in single injection vaccine, S1 protein 1000ug, cdi-AMP 750ug, single-chain oligodeoxynucleotide fragment 750ug containing GC. The single-stranded oligodeoxynucleotide fragments of the GC are 2395 and BW006 with equal mass.
Example 14
A subunit vaccine, in single injection vaccine, S1 protein 100ug, alum 100ug, single-chain oligodeoxynucleotide fragment containing GC 100ug. The single-stranded oligodeoxynucleotide fragments of the GC are 2395 and BW006 with equal mass.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
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<120> A novel nucleic acid adjuvant system for subunit vaccine and application thereof
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<210> 2
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<212> DNA
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tcgacgttcg tcgttcgtcg ttc 23
Claims (4)
1. A subunit vaccine comprising a novel nucleic acid adjuvant system for subunit vaccine and an antigenic protein;
The subunit vaccine novel nucleic acid adjuvant system comprises a nucleic acid adjuvant, wherein the nucleic acid adjuvant contains CpG ODN, the CpG ODN is a single-chain oligodeoxynucleotide fragment of GC, the single-chain oligodeoxynucleotide fragment of the GC is 2395 and BW006, and the nucleotide sequence is as follows;
2395：5’-tcgtcgttttcggcgcgcgccg-3’；（SEQ ID NO.1）；
BW006：5’-tcgacgttcgtcgttcgtcgttc-3’；（SEQ ID NO.2）；
also comprises at least one of Poly I C, cdi-AMP and Alum;
the subunit vaccine novel nucleic acid adjuvant system comprises single-stranded oligodeoxynucleotide fragments containing GC and Poly I, which are uniformly mixed in equal quantity, or single-stranded oligodeoxynucleotide fragments containing GC and cdi-AMP, which are uniformly mixed in equal quantity, or single-stranded oligodeoxynucleotide fragments containing GC and Alum, which are uniformly mixed in equal quantity;
the antigen protein is coronavirus S1 protein.
2. The subunit vaccine of claim 1 wherein: in the single injection vaccine, antigen protein 10-1000ug and single-chain oligodeoxynucleotide fragment containing GC 15-1500ug.
3. The subunit vaccine of claim 1, wherein in the single injection vaccine, the antigen protein is 10-1000ug, the total content of nucleic acid is 15-1500ug, and the nucleic acid adjuvant is equal mass Poly I, C7.5-750 ug and single-chain oligodeoxynucleotide fragment containing GC is 7.5-750ug, or equal mass nucleic acid adjuvant cdi-AMP 7.5-750ug and single-chain oligodeoxynucleotide fragment containing GC is equal mass mixed, or equal mass adjuvant Alum 7.5-750ug and single-chain oligodeoxynucleotide fragment containing GC is equal mass mixed.
4. The use of the novel nucleic acid adjuvant system for subunit vaccine of claim 1 in the preparation of subunit vaccine, wherein the subunit vaccine comprises the novel nucleic acid adjuvant system for subunit vaccine and an antigenic protein, wherein the antigenic protein is coronavirus S1 protein.
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