CN112791181B - Manganese nanoadjuvant, preparation method and application thereof - Google Patents
Manganese nanoadjuvant, preparation method and application thereof Download PDFInfo
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- CN112791181B CN112791181B CN202110160471.9A CN202110160471A CN112791181B CN 112791181 B CN112791181 B CN 112791181B CN 202110160471 A CN202110160471 A CN 202110160471A CN 112791181 B CN112791181 B CN 112791181B
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- manganese
- antigen
- nanoadjuvant
- vaccine
- adjuvant
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to the technical field of biological medicine and vaccine, in particular to a manganese nanoadjuvant, a preparation method and application thereof. The manganese nanoadjuvant comprises manganous-manganic oxide nano particles and template molecules wrapped outside the manganous-manganic oxide nano particles, wherein the molar ratio of the template molecules to manganese elements is 1: (10-1000), the template molecule comprises a template protein and fragments or polypeptides thereof. The manganese nanoadjuvant provided by the invention can effectively carry immune antigens, and can obtain more excellent immune treatment effects when the antigen carrying amount is small and the injection use amount is low; can effectively deliver the immune antigen to lymph node tissues, greatly enhance the cell internalization of the immune antigen and activate the immune cells with high efficiency.
Description
Technical Field
The invention relates to the technical field of biological medicine and vaccine, in particular to a manganese nanoadjuvant, a preparation method and application thereof.
Background
In addition to optimizing immune antigens, the use of suitable nanoadjuvants to boost the immunogenicity of new crown antigens, reduce vaccination times and antigen doses, and induce effective neutralizing antibodies and cell-mediated immune responses is one of the potential strategies to effectively prevent new crowns from continuing to be pandemic.
Aluminum adjuvants are well-known safe adjuvants that promote a better immune response to an antigen than to a free antigen, but the simultaneous lack of cellular immunity is a natural shortcoming of alum formulated vaccines. Currently, the design and construction of vaccine systems that achieve satisfactory antigen delivery (targeting Lymph Nodes (LN) and efficient cell membrane permeability) and activation of immune cells (dendritic cells and B cells) remains a major problem faced by protein subunit-based vaccines. The size limiting nature of the lymph nodes makes it difficult to deliver the vaccine specifically to immune cells. The unique size and surface properties of the functional nanomaterial help deliver vaccine components (antigens and adjuvants) to critical immune cells or lymphoid tissues and improve immune responses to prevent infection.
Aiming at the dilemma faced by the development of the vaccine, a novel and universal nano adjuvant capable of simultaneously realizing efficient antigen transfer to lymph nodes is designed, and the method for activating adaptive immunity and innate immunity is an effective means. The use of albumin may confer vaccine targeting ability, for example, delivery of an adjuvant such as evans blue or lipid-CpG to lymph nodes, thereby facilitating induction of an effective immune response. Meanwhile, albumin is also a good template for preparing inorganic nano particles by biomineralization.
Manganese adjuvants are new adjuvants developed in recent years. Currently, there have been reports on the application of divalent and tetravalent manganese to vaccine adjuvants. The use of a divalent manganese for the manufacture of a medicament for improving innate or/and adaptive immunity is disclosed as publication number CN107412260 a. Another example is publication No. CN111821316a which discloses a manganese composition comprising divalent manganese for immune enhancement. The publication number CN107456575A discloses a manganese dioxide nanoadjuvant, and a preparation method and application thereof. However, the immunopotentiating effect of a simple divalent manganese adjuvant or tetravalent manganese adjuvant is still to be improved.
Disclosure of Invention
In view of the above, the invention provides a manganese nanoadjuvant, a preparation method and application thereof. The manganese nanoadjuvant provided by the invention can effectively carry immune antigens, and can obtain more excellent immune treatment effects when the antigen carrying amount is small and the injection use amount is low; can effectively deliver the immune antigen to lymph node tissues, greatly enhance the cell internalization of the immune antigen and activate the immune cells with high efficiency.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an application of manganous-manganic oxide particles in preparing a manganese nanoadjuvant, wherein the manganese nanoadjuvant comprises the manganous-manganic oxide nanoparticles and template molecules wrapped outside the manganous-manganic oxide nanoparticles, and the molar ratio of the template molecules to manganese elements is 1: (10-1000), the template molecule comprises template protein and fragments or polypeptides thereof.
Preferably, in the manganese nanoadjuvant, the molar ratio of the template molecule to the manganese element is 1 (200-400).
Preferably, the template protein comprises one or more of bovine serum albumin, human serum albumin, mouse serum albumin, transferrin and antigen protein; the polypeptides include the above-described template protein-derived fragments and antigenic polypeptides.
Preferably, the average particle size of the manganese nanoadjuvant is 1-100 nm.
The invention also provides a preparation method of the manganese nanoadjuvant, which comprises the following steps:
(1) Mixing a divalent manganese salt aqueous solution with a template molecule solution to obtain a composite solution;
(2) Regulating the pH value of the composite solution to 9 or more than 9, stirring, dialyzing, purifying and freeze-drying to obtain the manganese nanoadjuvant;
the template molecule includes a template protein and fragments or polypeptides thereof.
Preferably, the divalent manganese salt is selected from one or more of manganese chloride, manganese nitrate, manganese acetate and manganese sulfate; however, the present invention is not limited thereto, and the types of divalent manganese salts recognized by those skilled in the art are within the scope of the present invention.
Preferably, the reagent for regulating the pH value of the composite solution is an alkaline reagent, and the alkaline reagent is one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia water, triethylamine, pyridine, N-methylmorpholine and tetramethyl ethylenediamine;
preferably, the concentration of the divalent manganese salt in the composite solution is 0.1-0.5 mol/L, and the concentration of the template protein is 1-50 g/L.
Preferably, the stirring temperature is 30-37 ℃, the stirring time is 1-10 hours, and the stirring rotating speed is 200-1000 r/min.
The invention also provides the manganese nanoadjuvant prepared by the method.
The invention also provides application of the manganese nanoadjuvant in preparing vaccines, wherein the vaccines comprise the manganese nanoadjuvant.
The invention also provides a vaccine, which comprises an antigen and the manganese nanoadjuvant, wherein the mass ratio of manganese element in the antigen and the manganese nanoadjuvant is 1: (0.025 to 40).
Preferably, the mass ratio of the antigen to the manganese element in the manganese nanoadjuvant is 1: (0.1 to 10).
Preferably, the antigen is a protein antigen and/or a polypeptide antigen.
Preferably, the antigen is one or more of a novel coronavirus antigen, an HIV antigen, a tubercle bacillus antigen, a malaria antigen, a human papilloma virus antigen or a tumor-associated antigen.
The invention also provides a preparation method of the vaccine, which comprises the step of incubating the manganese nanoadjuvant and the antigen in a buffer solution at 20-37 ℃ for 30-120 minutes.
Preferably, the buffer is one of PBS buffer, tris-HCl buffer or citric acid buffer.
The invention provides a manganese nanoadjuvant, a preparation method and application thereof. The manganese nanoadjuvant comprises manganous-manganic oxide nano particles and template molecules wrapped outside the manganous-manganic oxide nano particles, wherein the molar ratio of the template molecules to manganese elements is 1: (10-1000), the template molecule comprises template protein and fragments or polypeptides thereof. The invention has the technical effects that:
the invention provides a preparation method of manganese nanoadjuvant based on manganous-manganic oxide nano particles prepared by protein mineralization and a strategy for constructing nano vaccine medicaments based on the manganese nanoadjuvant, which can effectively prevent virus infection. The provided manganese nanoadjuvant is a programmable platform, is simple and convenient to synthesize, and can efficiently enhance the delivery efficiency of antigen molecular lymph nodes and the immunogenicity of antigens. The constructed manganese nanoadjuvant in the nano vaccine can stably and effectively carry antigen molecules, and efficiently enhance antigen-specific antibody and T cell reaction. The natural targeting property of albumin and the size effect of the nano vaccine make the manganese nano adjuvant suitable for effective drainage and retention in lymph nodes, greatly increase the uptake of cells and the activation of immune cells, thereby enhancing antigen-specific immune response.
Experimental results show that the nano vaccine medicine constructed by using the manganese nano adjuvant in mice can efficiently induce humoral and cellular immune responses even under the condition of reducing antigen dose and injection times. More importantly, the constructed nano vaccine drug efficiently induces a neutralizing antibody reaction, and also shows that the nano vaccine drug has potential of providing satisfactory protective immunity against novel coronaviruses and other types of viruses. Finally, each component for constructing the nano vaccine medicament has good biocompatibility and lower cost, and the possibility of the next clinical research is greatly increased.
The existing preparation methods of the inorganic manganous manganic oxide nanoparticles are mostly thermal decomposition methods, high-temperature water/solvent thermal methods, oil emulsion methods and the like, and the preparation process needs high temperature, is complex in flow and is unfavorable for clinical transformation application as a vaccine adjuvant. The preparation method of the nano adjuvant is simple.
Drawings
FIG. 1 is a flow chart of the preparation of the manganese nanoadjuvant of example 1 of the present invention;
FIG. 2 is an electron transmission microscope image and an X-ray diffraction pattern of the manganese nanoadjuvant prepared in example 1 of the present invention;
FIG. 3 is a TEM spectrum of the manganese nanoadjuvant prepared in example 2 of the present invention;
FIG. 4 shows the results of an experiment for the biotoxicity, cell internalization and promotion of DC maturation in vitro of the manganese nanoadjuvant (MnARK) obtained in example 1;
FIG. 5 is a schematic diagram of manganese nanoadjuvant preparation and nanovaccine drug construction;
FIG. 6 is a graph showing the data of the construction of nano-vaccine drug using the manganese nanoadjuvant obtained in example 1;
FIG. 7 is a nano-vaccine drug in vivo accumulation and lymph node targeted fluorescence imaging;
FIG. 8 is a nanovaccine drug-activated B cell constructed in example 4;
FIG. 9 is a comparison of the effects of nano-vaccine drug induced immunity constructed in example 4;
FIG. 10 is a comparison of the in vitro immune effects of the nanovaccine drug constructed in example 4;
FIG. 11 is a comparison of neutralizing antibody production effects of the nanovaccine constructed in example 4 and manganese chloride.
Detailed Description
The invention discloses a manganese nanoadjuvant, a preparation method and application thereof, and a person skilled in the art can properly improve the technological parameters by referring to the content of the manganese nanoadjuvant. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
The manganese nanoadjuvant, the preparation method and the raw materials or reagents used in the application of the manganese nanoadjuvant can be purchased from the market.
The invention is further illustrated by the following examples:
example 1
The procedure for preparing the manganese nanoadjuvant is as follows (fig. 1):
a) 5mL of a manganese chloride aqueous solution with the concentration of 0.1mol/L is added into 10mL of a Bovine Serum Albumin (BSA) solution with the concentration of 10mg/mL, and the mixture is stirred and mixed uniformly to obtain a composite solution.
B) And (3) adding 0.1mL of 2mol/L NaOH aqueous solution into the composite solution in the step (A), continuously stirring at 34 ℃ and 900rpm for 2 hours, and dialyzing, purifying and freeze-drying to obtain a manganese nanoadjuvant (MnARK) product.
See fig. 2. Fig. 2 is an electron transmission microscope image and an X-ray diffraction pattern of the manganese nanoadjuvant prepared in example 1 of the present invention.
As can be seen from fig. 2, the average particle size of the manganese nanoadjuvant prepared in example 1 of the present invention is 9.77 nm, and the manganese nanoadjuvant has good dispersibility and uniform size; XRD data show that the manganese nanoadjuvant prepared in the embodiment 1 of the invention is of a tetragonal crystal phase manganous oxide structure.
Example 2
The preparation method of the manganese nanoadjuvant comprises the following steps:
a) 10mL of a manganese nitrate aqueous solution with a concentration of 0.2mol/L was added to 5mL of a Human Serum Albumin (HSA) solution with a concentration of 50mg/mL, and the mixture was stirred and mixed to obtain a composite solution.
B) And (3) adding 0.5mL of 0.1mol/L NaOH aqueous solution into the composite solution in the step (A), continuously stirring at 700rpm at 30 ℃ for 5 hours, and dialyzing, purifying and freeze-drying to obtain the manganese nanoadjuvant product.
See fig. 3. Fig. 3 is a TEM image of the manganese nanoadjuvant prepared in example 2 of the present invention.
As can be seen from FIG. 3, the average particle size of the manganese nanoadjuvant prepared in example 2 of the present invention is 28.43 nm, and the manganese nanoadjuvant has good dispersibility and uniform size.
Example 3
The manganese nanoadjuvant (MnARK) obtained in example 1 was tested for biotoxicity and activation of immunocyte activity.
A) And (3) activity detection experiment: DC2.4 cells were seeded in 96-well plates and incubated with varying concentrations (0 to 0.5 mmol/L) of manganese chloride or MnARK, respectively, for 24 hours. Cell viability was assessed using the CCK-8 kit.
B) Flow cytometry analysis experiments: DC2.4 cells were plated at 3X 10 cells per well 5 The density of individual cells was seeded in 6-well plates and cultured for 12 hours, then the cells were treated with manganese chloride or MnARK and incubated for 24 hours, after which the cells were collected and stained with anti-CD 11C, anti-CD 80 and anti-CD 86 (all available from TONBO Biosciences) for flow cytometry analysis (BD Accuri C6, BD, united states).
See fig. 4. FIG. 4 shows the results of the experiment of the biotoxicity of the manganese nanoadjuvant (MnARK) obtained in example 1, internalization of the nanoadjuvant by DC2.4 cells and promotion of DC maturation in vitro.
As can be seen from FIG. 4, those MnARK nanoparticles were not significantly cytotoxic to DC cells even at a concentration of 0.5mmol/L compared to free manganese ions (manganese chloride). At the same time MnARK can also induce higher levels of DC cell maturation.
The following examples are: aiming at the novel coronavirus, the manganese nanoadjuvant obtained in the example 1 is applied to the construction of nano vaccine medicaments and immunotherapy.
Example 4
The manganese nanoadjuvant obtained in example 1 was used to bind antigen to construct nanovaccine drug, the procedure was as follows (fig. 5):
a) The novel coronavirus antigen RBD is selected as an immune antigen study object. The fluorescently labeled antigen and the manganese nanoadjuvant obtained in example 1 were added to PBS and incubated at 25 ℃ for 60 minutes. The mass ratio of the added antigen molecules to the manganese element in the manganese nanoadjuvant is 1:5.
b) And C, centrifuging the solution obtained in the step A at a speed of 10,000 g for 20 minutes, and collecting the centrifuged precipitate to obtain the nano vaccine medicament constructed by combining the manganese nano adjuvant with the antigen. The concentration of unbound antigen in the supernatant was then measured by fluorescence. Fluorescence measurements were performed using a Perkin Elmer fluorescent plate reader.
See fig. 6. FIG. 6 shows adsorption data of the manganese nanoadjuvant obtained in example 1 on antigen molecules of different concentrations; a plot of real-time measurements of association and dissociation of RBD antigen and manganese nanoadjuvant interactions; and change in hydrated particle size after RBD combined with manganese nanoadjuvant (MnARK).
As can be seen from fig. 6, the nano probe obtained in example 1 can effectively adsorb and carry antigen molecules; the RBD antigen is stably combined with the manganese nanoadjuvant with high affinity; compared with the manganese nanoadjuvant, the hydration particle size of the nano vaccine drug formed after the RBD antigen is combined is obviously increased, which proves that the RBD antigen and the manganese nanoadjuvant are effectively combined.
Example 5
A) On the premise of following national animal health care protocol, selecting BALB/c mice of 6-8 weeks of age into 3 groups, and injecting the following reagent (1) and 25 mug manganese nanoadjuvant with 10 mug fluorescent molecule Cy5 modified RBD antigen into thigh muscle of right rear leg to construct nano vaccine medicine; (2) 10 mug of RBD antigen modified by fluorescent molecule Cy5 is carried; (3) physiological saline.
B) The mice were analyzed for fluorescence imaging at injection time points of 0 hours and at 12, 24, 48, 72 hours. And after 12, 24, 48, 72 hours of fluorescence imaging, 3 mice each were collected for fluorescence imaging analysis of axilla and inguinal lymph nodes.
C) DC cells in lymph node tissues were analyzed using a flow cytometer, and the internalization of RBD antigen in DC cells was counted.
See fig. 7. FIG. 7 is a nano-vaccine drug living body accumulation and lymph node targeting fluorescence imaging.
As can be seen from fig. 7, the nanovaccine drug constructed with the manganese nanoadjuvant induced greater RBD antigen accumulation at the injection site compared to RBD alone, and the antigen lasted for more than 3 days at the site. Meanwhile, the nano vaccine medicine induces stronger fluorescent signals in lymph nodes. Quantitative analysis showed that 12 h after injection, the nano vaccine drug accumulated about 2 times more efficiently in lymph nodes than free RBD antigen, indicating that the nano vaccine drug constructed with manganese nanoadjuvant promoted in vivo antigen delivery and efficient accumulation to lymph nodes.
Example 6
A) The nanovaccine drug obtained in example 4 was subjected to ELISPOT analysis using commercial kit of R & D system (mouse IFN-. Gamma.ELISA kit for ELISA spot (ELISPOT) device). The specific procedure is as follows, cytokine capture antibodies against mouse IFN- γ (200-fold diluted with sterile PBS) were coated onto polyvinylidene fluoride membranes (PVDF) in 96-well plates and incubated overnight at 4 ℃. 96-well plates were blocked with complete 1640 medium containing 10% fetal bovine serum for 2 hours at room temperature. Dividing into three groups, and respectively adding RBD protein antigens (1) and 5 mug/mL; (2) physiological saline; (3) the nanovaccine drug constructed in example 4 containing RBD protein antigen at 5 μg/mL was followed immediately by fresh preparation of mouse spleen cells (5×10 5 Cells/wells) were added to the plates. The plates were incubated at 37℃with 5% CO 2 Incubate for 18 hours and wash four times with PBS (PBST) supplemented with 0.05% tween 20. The plates were then incubated with 2. Mu.g/mL of biotinylated detection antibody against mouse IFN-. Gamma.for 2 hours. ELISPOT development was performed by incubation with avidin-HRP complex in PBST for one hour, followed by four washes with PBS. Finally, the plate was incubated with peroxidase substrate AEC for 30 minutes. The ELISPOT points were enumerated using an automated ELISPOT reader system (Bio-Red).
Referring to fig. 8, fig. 8 is a nano vaccine drug-activated B cell constructed in example 4.
As can be seen from FIG. 8, the expression of three activation markers (MHC-II, CD69 and CD 86) on the surface of the B cells of mice receiving the nanovaccine drug constructed in example 4 was significantly increased compared to mice receiving RBD protein or physiological saline alone (control group), indicating that the nanovaccine drug constructed in example 4 can promote the maturation of B cells in vivo.
Example 7
The nanovaccine drug constructed in example 4 was vaccinated.
A) On the premise of following the national animal health care protocol, selecting BALB/c mice of 6-8 weeks of age for 3 times, wherein the number of each group of mice is 6, and three groups are respectively (1) a nano vaccine drug constructed by 25 mug manganese nano adjuvant carrying 10 mug RBD antigen; (2) 175 μg aluminum adjuvant (purchased by Invivogen) loaded with 50 μg RBD antigen; (3) 50 μg RBD antigen, without nanoadjuvant. The first mouse thigh intramuscular inoculation was used as day 0, the second inoculation was used as day 21, the third inoculation was used as day 42, and serum samples were collected on day 57.
B) On the premise of following the national animal health care protocol, selecting BALB/c mice of 6-8 weeks of age for inoculation, wherein the number of each group of mice is 6, and three groups are totally provided, namely (1), a nano vaccine medicament constructed by 25 mug manganese nano adjuvant carrying 50 mug RBD antigen is respectively inoculated for two times; (2) three inoculations were performed with 175 μg of aluminum adjuvant (purchased by Invivogen) loaded with 50 μg of RBD antigen; (3) 50 μg RBD antigen without nanoadjuvant, three vaccinations were performed. The first mouse thigh intramuscular inoculation was used as day 0, the second inoculation was used as day 21, the third inoculation was used as day 42, and serum samples were collected on day 57.
C) The levels of IgG and IgM in the vaccine-induced mouse serum in step a and step B were assessed by conventional enzyme-linked immunosorbent assay (ELISA). First, 96-well microtiter plates were pre-coated with RBD antigen, incubated overnight at 4 ℃ and blocked with 2% bsa for 2 hours at 37 ℃. The mouse serum collected in step a and step B was then added to a 96-well plate after gradient dilution, then incubated at 37 ℃ for 1 hour, and then washed four times with PBS. The bound antibody was then reacted with HRP conjugated goat anti-mouse IgG for 1 hour at 37 ℃. After four washes with PBS, the substrate 3,3', 5' -Tetramethylbenzidine (TMB) was added to a 96-well plate and the reaction was stopped by adding 0.05% sulfuric acid. Absorbance at 450 nm and 630 nm was measured in an ELISA plate reader (Tecan, san Jose, CA).
See fig. 9, fig. 9 is a comparison of the immune induction effects of the nanovaccine drug constructed in example 4.
As can be seen from fig. 9, the nano vaccine drug obtained in example 4 was able to induce about 5-fold intensity of IgG and IgM response than more RBD alone (50 μg) or RBD with commercial aluminum adjuvant (50 μg) even at lower amount of immunogen loading (10 μg); under the condition of the same antigen carrying amount, the injection of the nano vaccine medicine constructed in the example 4 for two times can enhance the IgG signal by 10 times and the IgM signal intensity by 5 times compared with the induction of RBD carried by three independent RBDs or commercial aluminum adjuvant. These results indicate that the nanovaccine drug constructed in example 4 can produce a stronger immune response after receiving a smaller amount of antigen injection (antigen loading and/or number of injections).
Example 8
A) The four groups of mouse serum samples obtained in step A of example 7 were subjected to a pseudovirus infection neutralization assay, as follows: supernatants containing pseudovirus (50 μl; purchased from Sino Biological Co.) were preincubated with serial dilutions of mouse serum at 37deg.C for 1 hour and then added to 293T cells expressing ACE2 (5×10) 4 Cells). After 24 hours fresh medium was added and the cells were lysed using commercially available cell lysis buffer. After addition of the luciferase substrate, the relative luciferase activity was determined in a luminometer (Bio-Tech). Pseudovirus neutralization efficiency was calculated and expressed as 50% and 90% neutralizing antibody titers.
B) The four groups of mouse serum samples obtained in step a of example 7 were subjected to a novel coronavirus live virus infection neutralization assay, specifically as follows: mouse serum was diluted in 2-fold gradients and mixed with live virus, incubated at 37 ℃ for 1 hour, and added in triplicate to 293T cells expressing ACE 2. Cytopathic effect (CPE) was observed daily for each well and recorded one week after infection. The neutralizing titers of the mouse antisera that were fully capable of preventing CPE were calculated.
See fig. 10, fig. 10 is a comparison of the in vitro immune effects of the nanovaccine drug constructed in example 4 against pseudoviruses and novel coronavirus live viruses.
As can be seen from fig. 10, the nano vaccine drug obtained in example 4 can induce significantly enhanced neutralizing antibody response against novel coronavirus even at a relatively low antibody loading.
The following examples are presented to compare the neutralizing antibody production effect of the obtained manganese nanoadjuvants of the present invention with divalent manganese ions (manganese chloride), hyaluronic acid coated manganese dioxide particles (mn@ha) as a novel corona vaccine adjuvant.
Example 9
Animal immunization experiments were carried out with the manganese nanoadjuvant, manganese chloride salt, and laboratory-prepared hyaluronic acid-coated manganese dioxide-conjugated antigen-constructing nanovaccine drug obtained in example 1:
a) Hyaluronic acid coated manganese dioxide particles were prepared, HA 1.0g was weighed into a flask, and ultrapure water (1 g:25mL of water), to a fume hood, stirring and refluxing at 102℃and 560rpm, and injecting MnCl 2 The solution (1 g/ml) was mixed and then poured into sodium hydroxide (1M, 15 ml), stirred (700 rpm) and refluxed for 2 hours, followed by dialysis against ultra pure water for 3 days, to give a tan Mn@HA particle adjuvant.
B) The novel coronavirus antigen RBD is selected as an immune antigen study object. 10 mug RBD antigen is respectively added into PBS together with the manganese nanoadjuvant obtained in the example 1, the Mn@HA particles obtained in the step A) and manganese chloride, and the mixture is incubated for 60 minutes at 25 ℃ to obtain different manganese adjuvant vaccines. The mass ratio of the antigen molecules to the manganese element added in each group is 1:5.
c) The vaccine obtained in the step B) is selected to inoculate BALB/c mice of 6-8 weeks of age for 3 times on the premise of following national animal health care protocol, the number of each group of mice is 6, five groups are totally divided into (1), and the nanometer vaccine medicament constructed by 25 mug manganese nanometer adjuvant carrying 10 mug RBD antigen is respectively; (2) a vaccine constructed with 25 μg Mn@HA adjuvant carrying 10 μg RBD antigen; (3) 10 μg of RBD antigen, 25 μg of manganese chloride constructed vaccine; (4) 10 μg RBD antigen, without nanoadjuvant; (5) and physiological saline group. The first mouse thigh intramuscular inoculation was used as day 0, the second inoculation was used as day 21, the third inoculation was used as day 42, and serum samples were collected on day 57. The IgG levels in the vaccine-induced mouse serum in step a and step B were assessed by conventional enzyme-linked immunosorbent assay (ELISA).
As can be seen from FIG. 11, all three Mn-based vaccine adjuvants showed a significant increase in RBD-specific IgG levels compared to free RBD. Notably, the highest IgG levels were found in the manganese nanovaccine group, suggesting that manganese nanovaccines can elicit strong immune responses in vivo and are superior to MnCl 2 And Mn@HA.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (5)
1. The manganese nano vaccine adjuvant is characterized by comprising manganese tetraoxide nano particles and template molecules wrapped outside the manganese tetraoxide nano particles;
the manganese nanometer vaccine adjuvant is prepared according to the following method:
a) Stirring and uniformly mixing a manganese chloride aqueous solution with the concentration of 0.1mol/L and a bovine serum albumin solution with the concentration of 10mg/mL to obtain a composite solution, wherein the volume ratio of the manganese chloride aqueous solution to the bovine serum albumin solution is 1:2;
b) Mixing the composite solution in the step A) with 2mol/L NaOH aqueous solution, continuously stirring at 34 ℃ and 900rpm for 2 hours, and dialyzing, purifying and freeze-drying to obtain the manganese nano vaccine adjuvant;
the volume ratio of the NaOH aqueous solution to the manganese chloride aqueous solution is 0.1:5.
2. A method for preparing the manganese nano-vaccine adjuvant according to claim 1, comprising the steps of:
a) Stirring and uniformly mixing a manganese chloride aqueous solution with the concentration of 0.1mol/L and a bovine serum albumin solution with the concentration of 10mg/mL to obtain a composite solution, wherein the volume ratio of the manganese chloride aqueous solution to the bovine serum albumin solution is 1:2;
b) Mixing the composite solution in the step A) with 2mol/L NaOH aqueous solution, continuously stirring at 34 ℃ and 900rpm for 2 hours, and dialyzing, purifying and freeze-drying to obtain the manganese nano vaccine adjuvant;
the volume ratio of the NaOH aqueous solution to the manganese chloride aqueous solution is 0.1:5.
3. Use of a manganese nanovaccine adjuvant in the preparation of a vaccine, characterized in that the vaccine comprises a manganese nanovaccine adjuvant according to claim 1.
4. A vaccine comprising an antigen and the manganese nanovaccine adjuvant of claim 1, wherein the mass ratio of the antigen to manganese element in the manganese nanovaccine adjuvant is 1: (0.025 to 40).
5. The method for preparing the vaccine according to claim 4, wherein the manganese nano vaccine adjuvant according to claim 1 and the antigen are incubated in a buffer solution at 20-37 ℃ for 30-120 minutes.
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| CN113797329A (en) * | 2021-10-19 | 2021-12-17 | 启锰生物科技(江苏)有限公司 | Vaccine adjuvant composition of bivalent manganese adjuvant and CpG adjuvant and preparation method thereof |
| CN114177283B (en) * | 2021-12-08 | 2023-11-17 | 湖南中医药大学 | Magnetic drive nano motor Fe 3 O 4 /Ca@MnCO 3 Is prepared from (A) and its application in DC vaccine |
| CN114432437A (en) * | 2022-01-07 | 2022-05-06 | 广东粤港澳大湾区国家纳米科技创新研究院 | Application of mangano-manganic oxide particles in preparation of vaccine adjuvant |
| CN114366809A (en) * | 2022-01-12 | 2022-04-19 | 广东粤港澳大湾区国家纳米科技创新研究院 | Aluminum nanocrystal delivery system and combined vaccine antigen molecule self-assembled particle adjuvant vaccine thereof |
| CN114848810B (en) * | 2022-05-11 | 2023-10-10 | 江南大学 | Chiral nanometer vaccine and preparation method and application thereof |
| WO2024119524A1 (en) * | 2022-12-10 | 2024-06-13 | 苏州大学 | Diagnosis and treatment integrated manganese oxide protein nanoparticles, preparation method therefor, and use thereof |
| CN117752812B (en) * | 2023-12-11 | 2024-07-12 | 山东第一医科大学(山东省医学科学院) | Manganese-arsenic virus-like particle modified by protein crown bionic modification and preparation method thereof |
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| WO2022165980A1 (en) | 2022-08-11 |
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