CN113797329A - Vaccine adjuvant composition of bivalent manganese adjuvant and CpG adjuvant and preparation method thereof - Google Patents
Vaccine adjuvant composition of bivalent manganese adjuvant and CpG adjuvant and preparation method thereof Download PDFInfo
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- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A61K2039/55511—Organic adjuvants
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Abstract
The invention discloses a vaccine adjuvant composition of a bivalent manganese adjuvant and a CpG adjuvant, which comprises the bivalent manganese adjuvant and the CpG adjuvant, wherein the CpG adjuvant comprises: k-type ODN, D-type ODN, C-type ODN. The manufacturing method comprises the following steps: vaccine antigen, bivalent manganese adjuvant and CpG adjuvant are combined in a stable form. The immune enhancement effect of the bivalent manganese adjuvant can be improved by combining the CpG adjuvant and the bivalent manganese adjuvant.
Description
Technical Field
The invention relates to the technical field of biomedicine and vaccines, in particular to a vaccine adjuvant composition of a bivalent manganese adjuvant and a CpG adjuvant and a preparation method thereof.
Background
Adjuvants are a class of substances added to vaccines to increase the immunogenicity of antigens. Adjuvants can be divided into two classes, depending on the mechanism of action: immunopotentiators (immunopotentiators) and delivery systems (delivery systems). Immunopotentiators include: some activators of pathogen-associated molecular patterns (PAMPs) or synthetic pattern-recognition receptors (PPRs) that activate innate immune responses and subsequently induce cytokine and chemokine production. Still others include immune cells or cytokines such as Dendritic Cells (DCs), IL-12 or GM-CSF, which are involved directly in the immune response and act more rapidly. Delivery systems include liposomes (liposomes), micelles (micelles), viroids (virosomes), nanoparticles (nanoparticules), microspheres (microspheres), Oil/Water emulsions (Oil/Water emulsions), virus-like particles (VLPs), and immunostimulatory complexes (ISCOMs), which act by carrying antigens to target cells and facilitating antigen presentation by Antigen Presenting Cells (APCs).
The aluminum adjuvant is an inorganic salt adjuvant which is firstly discovered, can activate Th2 immune response, namely humoral immune response, for nearly 100 years since being discovered in 1926 to be used up to now, but has the limitation that cellular immunity cannot be activated. Cellular immunity is mainly involved in the immune response to intracellular parasitic pathogenic microorganisms and in the immune response to tumor cells, and thus plays an important role in the resistance to viral infection and tumor treatment.
The CpG adjuvant is a deoxynucleotide sequence containing unmethylated cytosine guanine, can activate TLR9 receptor, generate multiple cytokines, enhance the processing and presentation of antigen, and induce Th1 immune response. Since CpG acts on different animals through a Toll-like receptor 9 (TLR 4, TLR 9) and expression of different cell tissues is greatly different, CpG is classified into three classes, namely, K-type ODN, D-type ODN, and C-type ODN. The K-type ODN activates Dendritic Cells (DC) to secrete TNF-alpha and IL-6, activates B cells and generates antibodies; d-type ODN stimulates Antigen Presenting Cells (APC) to mature and secrete IFN-alpha; type C ODN stimulate IL-6 and IFN- α secretion from B cells and DC cells.
The prior art discloses divalent manganese colloids or divalent manganese nascent precipitates for enhancing immunity and their use for enhancing immunity, such as for use as immunoadjuvants, antiviral or antitumor. However, in further studies, it was found that the immune activating effect of colloidal manganese adjuvant could be further improved by combining with CpG adjuvant, and is more suitable for vaccine development. When the immune enhancing effect of the manganese adjuvant is researched, the manganese adjuvant can generate immune enhancing effect in the body, but the effect is not strong enough.
Disclosure of Invention
The invention aims to provide a vaccine adjuvant composition of a bivalent manganese adjuvant and a CpG adjuvant and a preparation method thereof, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a vaccine adjuvant composition of a divalent manganese adjuvant and a CpG adjuvant comprising the steps of:
the adjuvant comprises a divalent manganese adjuvant and a CpG adjuvant, wherein the mass ratio of manganese element to CpG is 1: 0.001-1: 0.1, wherein the CpG adjuvant comprises: k-type ODN, D-type ODN, C-type ODN.
The invention is further improved in that: the CpG adjuvant is ODN D-SL01 with the sequence 5'-tcg cga cgt tcg ccc gac gtt cgg ta-3' (26 mer) and is modified by sulfo.
A preparation method of a vaccine adjuvant composition of a bivalent manganese adjuvant and a CpG adjuvant comprises the steps of preparing a vaccine antigen, the bivalent manganese adjuvant and the CpG adjuvant into a stable form combination, wherein the concentration of the vaccine antigen is 50-100 ug/ml, the concentration of the bivalent manganese adjuvant is 1 mg/ml, the concentration of the CpG adjuvant is 1-100 ug/ml, and the concentration of a stabilizing agent gelatin is 2-3%;
firstly, uniformly stirring and mixing the divalent manganese adjuvant and the CpG adjuvant on a magnetic stirrer for 10-30min, wherein the temperature is room temperature, and the rotating speed is set to be 800-1000 rpm; then adding 10% gelatin solution and stirring for 10-30min at 50-60 deg.C and rotation speed set at 800-; finally, the vaccine antigen is added and stirred for 10-30min, the temperature is room temperature, and the rotating speed is set to be 800-1000 rpm.
The invention is further improved in that: vaccine antigens include inactivated pathogens or extracted pathogen subunit antigens and combinations thereof.
The invention is further improved in that: pathogens include common pairs of viruses, bacteria and/or parasites.
The invention is further improved in that: the virus is selected from: DNA viruses and RNA viruses, the DNA viruses being of the herpesviridae, hepadnaviridae, papillomaviridae, or poxviridae family; the RNA virus is Rhabdoviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Coronaviridae, picornaviridae, Flaviviridae or Retroviridae.
The invention is further improved in that: the bacteria are selected from gram-negative bacteria and gram-positive bacteria.
The invention is further improved in that: the bacteria are selected from Streptococcus pneumoniae, Haemophilus influenzae, Salmonella, Diplococcus meningitidis, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Acinetobacter baumannii.
The invention is further improved in that: the parasite is selected from the group consisting of Plasmodium, Toxoplasma, Trypanosoma, Schistosoma, filarial, and Leishmania.
A method of using a vaccine adjuvant composition of a divalent manganese adjuvant and a CpG adjuvant, comprising intramuscular injection, intradermal injection, subcutaneous injection, intravenous injection, mucosal administration, and any combination thereof.
Compared with the prior art, the invention has the beneficial effects that:
the immune enhancement effect of the bivalent manganese adjuvant can be improved by combining the CpG adjuvant and the bivalent manganese adjuvant.
Drawings
FIG. 1 is a graph of the subcutaneous immunization effect of a divalent manganese adjuvant plus a CpG adjuvant;
FIG. 2 is a graph of muscle immunization effect of divalent manganese adjuvant plus CpG adjuvant.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.
The embodiment provides a technical scheme: a vaccine adjuvant composition of a bivalent manganese adjuvant and a CpG adjuvant comprises the bivalent manganese adjuvant and the CpG adjuvant, wherein the CpG adjuvant comprises: k-type ODN, D-type ODN, C-type ODN. The CpG adjuvant is ODN D-SL01 with the sequence 5'-tcg cga cgt tcg ccc gac gtt cgg ta-3' (26 mer) and is modified by sulfo.
Bivalent manganese adjuvant (CAS number: MS 0002), CpG adjuvant was synthesized by primer company.
A preparation method of a vaccine adjuvant composition of a bivalent manganese adjuvant and a CpG adjuvant comprises the steps of preparing a vaccine antigen, the bivalent manganese adjuvant and the CpG adjuvant into a stable form combination, wherein the concentration of the vaccine antigen is 50-100 ug/ml, the concentration of the bivalent manganese adjuvant is 1 mg/ml, the concentration of the CpG adjuvant is 1-100 ug/ml, and the concentration of a stabilizing agent gelatin is 2-3%; firstly, uniformly stirring and mixing the divalent manganese adjuvant and the CpG adjuvant on a magnetic stirrer for 10-30min, wherein the temperature is room temperature, and the rotating speed is set to be 800-1000 rpm; then adding 10% gelatin solution and stirring for 10-30min at 50-60 deg.C and rotation speed set at 800-; finally, the vaccine antigen is added and stirred for 10-30min, the temperature is room temperature, and the rotating speed is set to be 800-1000 rpm. Vaccine antigens include inactivated pathogens or extracted pathogen subunit antigens and combinations thereof. Pathogens include common pairs of viruses, bacteria and/or parasites. The virus is selected from: DNA viruses and RNA viruses. Preferably, the virus is selected from: herpesviridae, rhabdoviridae, filoviridae, orthomyxoviridae, paramyxoviridae, coronaviridae, picornaviridae, hepadnaviridae, flaviviridae, papilloma viridae, poxviridae, and retroviridae. Further preferably, the virus is selected from: herpes simplex virus, vesicular stomatitis virus, vaccinia virus, HIV and HBV. The bacteria are selected from gram-negative bacteria and gram-positive bacteria. Preferably, the bacteria are selected from the group consisting of Streptococcus pneumoniae, Haemophilus influenzae, Salmonella, Diplococcus meningitidis, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Acinetobacter baumannii. The parasite is selected from the group consisting of Plasmodium, Toxoplasma, Trypanosoma, Schistosoma, filarial, and Leishmania.
A method of using a vaccine adjuvant composition of a divalent manganese adjuvant and a CpG adjuvant, comprising intramuscular injection, intradermal injection, subcutaneous injection, intravenous injection, mucosal administration, and any combination thereof.
Determination of RVG-specific IgG antibodies by ELISA method: diluted sera from immunized mice were incubated on ELISA plates coated with 2. mu.g/ml RVG. After washing, bound corresponding antibodies were detected using the HRP-conjugated antibody mouse IgG (Proteintech, Cat # SA 00001). The plate was then incubated with the substrate TMB (eBioscience) with 1M H3PO4The reaction was stopped and then the absorbance at absorption wavelengths of 450 nm and 650nm was measured.
Example 1 Effect of bivalent manganese adjuvant plus CpG adjuvant on subcutaneous immunization (FIG. 1)
CTL group: 200 ul of injection is added with 10 ug of protein, and the rest is supplemented with normal saline to immunize mice. Alhy group: 200 ul of injection is added with 100 ug of aluminum adjuvant and 10 ug of protein, and the rest is supplemented with normal saline. MnJ group: 200 ul of injection is added with 100 ug of bivalent manganese adjuvant and 10 ug of protein, and the rest is supplemented with normal saline. MnJ + CpG group: 200 ul injection is added with 100 ug of bivalent manganese adjuvant, 10 ug of CpG adjuvant, 10 ug of protein, and the rest is supplemented with physiological saline. Mice were immunized subcutaneously on days 0 and 14, respectively, and specific antibodies were detected on day 21. The antibody production enhancing effect of the divalent manganese adjuvant and CpG adjuvant group is obviously higher than that of the divalent manganese adjuvant group and the aluminum adjuvant group.
FIG. 1 shows four immunization protocols, CTL stands for 10 ug of the rabies G protein alone, Alhy stands for 100 ug of the aluminum adjuvant plus 10 ug of the rabies G protein, MnJ stands for 100 ug of the bivalent manganese adjuvant plus 10 ug of the rabies G protein, MnJ + CpG stands for 100 ug of the bivalent manganese adjuvant plus 10 ug of the CpG adjuvant plus 10 ug of the rabies G protein. The route of immunization is subcutaneous immunization. The immunized animals were C57BL/6 mice. The immunization time was measured by ELISA method for specific IgG antibody in serum on day 0, 14 and 21.
Example 2 divalent manganese adjuvant plus CpG adjuvant muscle immunization Effect (FIG. 2)
CTL group: 200 ul of injection is added with 10 ug of protein, and the rest is supplemented with normal saline to immunize mice. Alhy group: 200 ul of injection is added with 100 ug of aluminum adjuvant and 10 ug of protein, and the rest is supplemented with normal saline. MnJ group: 200 ul of injection is added with 100 ug of bivalent manganese adjuvant and 10 ug of protein, and the rest is supplemented with normal saline. MnJ + CpG group: 200 ul injection is added with 100 ug of bivalent manganese adjuvant, 10 ug of CpG adjuvant, 10 ug of protein, and the rest is supplemented with physiological saline. Mice were muscle immunized on day 0 and 14, respectively, and specific antibodies were detected on day 21. The antibody production enhancing effect of the divalent manganese adjuvant and CpG adjuvant group is obviously higher than that of the divalent manganese adjuvant group and the aluminum adjuvant group.
FIG. 2 shows four immunization protocols, CTL stands for 10 ug of the rabies G protein alone, Alhy stands for 100 ug of the aluminum adjuvant plus 10 ug of the rabies G protein, MnJ stands for 100 ug of the bivalent manganese adjuvant plus 10 ug of the rabies G protein, MnJ + CpG stands for 100 ug of the bivalent manganese adjuvant plus 10 ug of the CpG adjuvant plus 10 ug of the rabies G protein. The route of immunization is muscle immunization. The immunized animals were C57BL/6 mice. The immunization time was measured by ELISA method for specific IgG antibody in serum on day 0, 14 and 21.
The bivalent manganese nano-particles can directly activate immune response, both bivalent manganese adjuvant and CpG adjuvant have immune effect, and the original immune effect can be improved by adding the CpG adjuvant in the bivalent manganese nano-particles.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
It should be noted that the terms "first," "second," and the like in the description and claims of this application and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances such that the embodiments of the application described herein are, for example, capable of operation in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, article, or apparatus.
Spatially relative terms, such as "above … …," "above … …," "above … …," "above," and the like, may be used herein for ease of description to describe one device or feature's spatial relationship to another device or feature as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is turned over, devices described as "above" or "on" other devices or configurations would then be oriented "below" or "under" the other devices or configurations. Thus, the exemplary term "above … …" can include both an orientation of "above … …" and "below … …". The device may be otherwise variously oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.
Exemplary embodiments according to the present application will now be described in more detail with reference to the accompanying drawings. These exemplary embodiments may, however, be embodied in many different forms and should not be construed as limited to only the embodiments set forth herein. It is to be understood that these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the exemplary embodiments to those skilled in the art, in the drawings, it is possible to enlarge the thicknesses of layers and regions for clarity, and the same devices are denoted by the same reference numerals, and thus the description thereof will be omitted.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A vaccine adjuvant composition of a divalent manganese adjuvant and a CpG adjuvant, characterized in that: the adjuvant comprises a divalent manganese adjuvant and a CpG adjuvant, wherein the mass ratio of manganese element to CpG is 1: 0.001-1: 0.1, wherein the CpG adjuvant comprises: k-type ODN, D-type ODN, C-type ODN.
2. The vaccine adjuvant composition of a divalent manganese adjuvant and a CpG adjuvant according to claim 1, wherein: the CpG adjuvant is ODN D-SL01 with the sequence 5'-tcg cga cgt tcg ccc gac gtt cgg ta-3' (26 mer) and is modified by sulfo.
3. A method of making a vaccine adjuvant composition of a divalent manganese adjuvant and a CpG adjuvant according to claim 1, characterized in that: the vaccine antigen, the bivalent manganese adjuvant and the CpG adjuvant are made into a stable form combination, the concentration of the vaccine antigen is 50-100 ug/ml, the concentration of the bivalent manganese adjuvant is 1 mg/ml, the concentration of the CpG adjuvant is 1-100 ug/ml, and the concentration of the stabilizer gelatin is 2-3%;
firstly, uniformly stirring and mixing the divalent manganese adjuvant and the CpG adjuvant on a magnetic stirrer for 10-30min, wherein the temperature is room temperature, and the rotating speed is set to be 800-1000 rpm; then adding 10% gelatin solution and stirring for 10-30min at 50-60 deg.C and rotation speed set at 800-; finally, the vaccine antigen is added and stirred for 10-30min, the temperature is room temperature, and the rotating speed is set to be 800-1000 rpm.
4. The method of claim 3, wherein the vaccine adjuvant composition comprises a divalent manganese adjuvant and a CpG adjuvant, wherein the divalent manganese adjuvant comprises: the vaccine antigens include inactivated pathogens or extracted pathogen subunit antigens and combinations thereof.
5. The method of making a vaccine adjuvant composition of a divalent manganese adjuvant and a CpG adjuvant according to claim 4, wherein: pathogens include common pairs of viruses, bacteria and/or parasites.
6. The method of claim 5, wherein the vaccine adjuvant composition comprises a divalent manganese adjuvant and a CpG adjuvant, wherein the divalent manganese adjuvant comprises: the virus is selected from: DNA viruses and RNA viruses, said DNA viruses being of the herpesviridae, hepadnaviridae, papillomaviridae, or poxviridae family; the RNA virus is Rhabdoviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Coronaviridae, picornaviridae, Flaviviridae or Retroviridae.
7. The method of claim 5, wherein the vaccine adjuvant composition comprises a divalent manganese adjuvant and a CpG adjuvant, wherein the divalent manganese adjuvant comprises: the bacteria are selected from gram-negative bacteria and gram-positive bacteria.
8. The method of claim 5, wherein the vaccine adjuvant composition comprises a divalent manganese adjuvant and a CpG adjuvant, wherein the divalent manganese adjuvant comprises: the bacteria are selected from Streptococcus pneumoniae, Haemophilus influenzae, Salmonella, Diplococcus meningitidis, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Acinetobacter baumannii.
9. The method of claim 5, wherein the vaccine adjuvant composition comprises a divalent manganese adjuvant and a CpG adjuvant, wherein the divalent manganese adjuvant comprises: the parasite is selected from the group consisting of Plasmodium, Toxoplasma, Trypanosoma, Schistosoma, filarial and Leishmania.
10. A method of using a vaccine adjuvant composition of a divalent manganese adjuvant and a CpG adjuvant, characterized in that: including intramuscular injection, intradermal injection, subcutaneous injection, intravenous injection, mucosal administration, and any combination thereof.
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