CN112791181A - Manganese nano adjuvant, preparation method and application thereof - Google Patents
Manganese nano adjuvant, preparation method and application thereof Download PDFInfo
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- CN112791181A CN112791181A CN202110160471.9A CN202110160471A CN112791181A CN 112791181 A CN112791181 A CN 112791181A CN 202110160471 A CN202110160471 A CN 202110160471A CN 112791181 A CN112791181 A CN 112791181A
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Abstract
The invention relates to the technical field of biomedicine and vaccines, in particular to a manganese nano adjuvant, a preparation method and application thereof. The manganese nano adjuvant comprises manganic manganous oxide nano particles and template molecules coated outside the manganic manganous 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 a fragment or polypeptide thereof. The manganese nano adjuvant provided by the invention can effectively carry immune antigens, and can obtain more excellent immunotherapy effect when the antigen carrying amount is less and the injection using amount is lower; can effectively deliver immune antigen to lymph node tissue, greatly enhance the cellular internalization of the immune antigen and activate immune cells with high efficiency.
Description
Technical Field
The invention relates to the technical field of biomedicine and vaccines, in particular to a manganese nano adjuvant, a preparation method and application thereof.
Background
In addition to optimizing immune antigens, the use of suitable nano-adjuvants to boost the immunogenicity of neocorona antigens, reduce vaccination times and antigen doses and induce effective neutralizing antibodies and cell-mediated immune responses is one of the potential counter-strategies that effectively prevent the continued pandemic of neocorona.
Aluminum adjuvants are well-established safety adjuvants that promote a better immune response to an antigen than free antigens, but lack of cellular immunity at the same time is a natural drawback of alum formulated vaccines. At present, the design and construction of vaccine systems that achieve satisfactory antigen delivery (targeting to 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 lymph nodes makes it difficult to deliver vaccines specifically to immune cells. The unique size and surface properties of the functional nanomaterials help deliver vaccine components (antigens and adjuvants) to critical immune cells or lymphoid tissues and improve the immune response to prevent infection.
Aiming at the dilemma faced by the development of the vaccine, a novel and universal nano adjuvant which can simultaneously realize the efficient antigen transfer to lymph nodes is designed, and the adaptive immunity and the innate immunity are activated, so that the method is an effective means. The use of albumin can confer targeting capabilities to the vaccine, e.g., delivering an adjuvant such as evans blue or lipid-CpG to lymph nodes, thereby facilitating the induction of an effective immune response. Meanwhile, albumin is also a good template for preparing inorganic nanoparticles by biomineralization.
Manganese adjuvant is a new adjuvant developed in recent years. At present, there have been reports on the use of divalent and tetravalent manganese in vaccine adjuvants. For example, publication No. CN107412260A discloses the use of bivalent manganese for the preparation of a medicament for improving innate immunity or/and adaptive immunity. Also disclosed is a manganese composition for immune enhancement comprising divalent manganese as disclosed in publication No. CN 111821316A. For example, publication No. CN107456575A discloses a manganese dioxide nano adjuvant, a preparation method and application thereof. However, the immune enhancement effect of the simple bivalent manganese adjuvant or tetravalent manganese adjuvant still needs to be improved.
Disclosure of Invention
In view of the above, the invention provides a manganese nano adjuvant, a preparation method and an application thereof. The manganese nano adjuvant provided by the invention can effectively carry immune antigens, and can obtain more excellent immunotherapy effect when the antigen carrying amount is less and the injection using amount is lower; can effectively deliver immune antigen to lymph node tissue, greatly enhance the cellular internalization of the immune antigen and activate 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 the preparation of a manganese nano adjuvant, wherein the manganese nano adjuvant comprises the manganous-manganic oxide nanoparticles and template molecules coated outside the manganous oxide nanoparticles, and the molar ratio of the template molecules to manganese elements is 1: (10-1000), the template molecule comprises a template protein and a fragment or polypeptide thereof.
Preferably, in the manganese nano adjuvant, 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 polypeptide comprises the derived fragment of the template protein and the antigen polypeptide.
Preferably, the average particle size of the manganese nano adjuvant is 1-100 nm.
The invention also provides a preparation method of the manganese nano adjuvant, which comprises the following steps:
(1) mixing a divalent manganese salt aqueous solution with a template molecule solution to obtain a composite solution;
(2) adjusting the pH value of the composite solution to be more than 9 or 9, stirring, dialyzing, purifying and freeze-drying to obtain the manganese nano adjuvant;
the template molecule includes a template protein and a fragment or polypeptide 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 divalent manganese salt species recognized by those skilled in the art are within the scope of the present invention.
Preferably, the reagent for adjusting 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 tetramethylethylenediamine;
preferably, in the composite solution, the concentration of the divalent manganese salt 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 speed is 200-1000 r/min.
The invention also provides the manganese nano adjuvant prepared by the method.
The invention also provides application of the manganese nano adjuvant in preparation of vaccines, wherein the vaccines comprise the manganese nano adjuvant.
The invention also provides a vaccine, which comprises an antigen and the manganese nano adjuvant, wherein the mass ratio of the antigen to manganese in the manganese nano adjuvant is 1: (0.025 to 40).
Preferably, the mass ratio of the antigen to the manganese element in the manganese nano adjuvant is 1: (0.1-10).
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 Mycobacterium tuberculosis antigen, a malaria antigen, a human papilloma virus antigen or a tumor associated antigen.
The invention also provides a preparation method of the vaccine, and the manganese nano adjuvant and the antigen are incubated in the buffer solution at the temperature of 20-37 ℃ for 30-120 minutes.
Preferably, the buffer is one of a PBS buffer, a Tris-HCl buffer or a citrate buffer.
The invention provides a manganese nano adjuvant, a preparation method and application thereof. The manganese nano adjuvant comprises manganic manganous oxide nano particles and template molecules coated outside the manganic manganous 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 a fragment or polypeptide thereof. The invention has the technical effects that:
the invention provides a preparation method of a manganese nano adjuvant of manganomanganic oxide nano particles prepared based on protein mineralization and a strategy for constructing a nano vaccine medicament based on the manganese nano adjuvant, which can effectively prevent virus infection. The provided manganese nano adjuvant is a programmable platform, is simple and convenient to synthesize, and can efficiently enhance the lymph node delivery efficiency of antigen molecules and the immunogenicity of antigens. The manganese nano adjuvant in the constructed nano vaccine can stably and effectively carry antigen molecules, and efficiently enhance the reaction of antigen specific antibodies and T cells. Due to the natural targeting property of albumin and the size effect of the nano vaccine, the manganese nano adjuvant is suitable for effective drainage and retention in lymph nodes, and the uptake of cells and the activation of immune cells are greatly increased, so that the antigen-specific immune response is enhanced.
The experimental result shows that even under the condition of reducing antigen dose and injection times, the nano vaccine medicament constructed by using the manganese nano adjuvant in the mouse can also efficiently cause humoral and cellular immune response. More importantly, the constructed nano vaccine drug can efficiently induce a neutralizing antibody reaction, and the nano vaccine drug also has the potential of providing satisfactory protective immunity against novel coronaviruses and other viruses. Finally, each component for constructing the nano vaccine medicament has good biocompatibility and lower cost, and greatly increases the possibility of the next clinical research.
Most of the existing preparation methods of inorganic manganous manganic oxide nanoparticles are thermal decomposition method, high-temperature water/solvent thermal method, oil emulsion method and the like, the preparation process needs high temperature and complicated flow, and the preparation method is not beneficial to 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 manganese nano adjuvant according to example 1 of the present invention;
FIG. 2 is an electron transmission microscope image and an X-ray diffraction spectrum of the manganese nano adjuvant prepared in example 1 of the present invention;
FIG. 3 is a TEM image of the manganese nano adjuvant prepared in example 2 of the present invention;
FIG. 4 shows the results of experiments on biotoxicity, cell internalization and promotion of DC maturation in vitro of the manganese nano adjuvant (MnARK) obtained in example 1;
FIG. 5 is a schematic diagram of preparation of a manganese nano adjuvant and construction of a nano vaccine drug;
FIG. 6 is a data map of the construction of the nano vaccine medicament by the manganese nano adjuvant obtained in example 1;
FIG. 7 shows fluorescence imaging of nano-vaccine drug live accumulation and lymph node targeting;
FIG. 8 shows that the nano-vaccine drug constructed in example 4 activates B cells;
FIG. 9 is a comparison of the effect of inducing immunity by the nano-vaccine drug constructed in example 4;
FIG. 10 is a comparison of the in vitro immune effects of the nano-vaccine drugs constructed in example 4;
fig. 11 is a graph comparing the effect of neutralizing antibodies generated by the nano vaccine constructed in example 4 and manganese chloride.
Detailed Description
The invention discloses a manganese nano adjuvant, a preparation method and application thereof, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The manganese nano adjuvant, the preparation method and the raw materials or reagents used in the application can be purchased from the market.
The invention is further illustrated by the following examples:
example 1
The steps for preparing the manganese nano adjuvant are as follows (figure 1):
A) 5mL of 0.1mol/L manganese chloride aqueous solution is added into 10mL of 10mg/mL Bovine Serum Albumin (BSA) solution, and the mixture is stirred and mixed uniformly to obtain a composite solution.
B) And C, adding 0.1mL of 2mol/L NaOH aqueous solution into the composite solution in the step A, continuously stirring at 900rpm for 2 hours at 34 ℃, dialyzing, purifying and freeze-drying to obtain a manganese nano adjuvant (MnARK) product.
See fig. 2. Fig. 2 is an electron transmission microscope image and an X-ray diffraction spectrum of the manganese nano adjuvant prepared in example 1 of the present invention.
As can be seen from FIG. 2, the average particle size of the manganese nano adjuvant prepared in the embodiment 1 of the present invention is 9.77 nm, and the manganese nano adjuvant has good dispersibility and uniform size; XRD data show that the manganese nano adjuvant prepared in the embodiment 1 of the invention has a tetragonal phase manganomanganic oxide structure.
Example 2
The preparation method of the manganese nano adjuvant comprises the following steps:
A) 10mL of 0.2mol/L manganese nitrate aqueous solution was added to 5mL of 50mg/mL Human Serum Albumin (HSA) solution, and the mixture was stirred and mixed to obtain a complex solution.
B) And D, adding 0.5mL of 0.1mol/L NaOH aqueous solution into the composite solution in the step A, continuously stirring at 30 ℃ and 700rpm for 5 hours, dialyzing, purifying and freeze-drying to obtain the manganese nano adjuvant product.
See fig. 3. Fig. 3 is a TEM image of the manganese nano adjuvant prepared in example 2 of the present invention.
As can be seen from FIG. 3, the average particle size of the manganese nano adjuvant prepared in example 2 of the present invention is 28.43 nm, and the manganese nano adjuvant has good dispersibility and uniform size.
Example 3
The biotoxicity and activated immune cell activity of the manganese nano adjuvant (MnARK) obtained in example 1 were tested.
A) And (3) activity detection experiment: DC2.4 cells were seeded in 96-well plates and incubated with different 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 per well5The cells were seeded at a density in 6-well plates and cultured for 12 hours, then treated with manganese chloride or MnARK and after 24 hours of incubation, the cells were harvested 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, usa).
See fig. 4. FIG. 4 shows the biological toxicity of the manganese nano adjuvant (MnARK) obtained in example 1, internalization of the nano adjuvant by DC2.4 cells, and the results of experiments for promoting 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, as compared to free manganese ion (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 nano adjuvant obtained in the example 1 is applied to the construction of a nano vaccine medicament and the immunotherapy.
Example 4
The manganese nano adjuvant obtained in example 1 is combined with antigen to construct a nano vaccine medicament, and the steps are as follows (fig. 5):
A) selecting a new coronavirus antigen RBD as an immune antigen research object. The fluorescently labeled antigen and the manganese nano adjuvant 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 nanometer adjuvant is 1: 5.
B) and D, centrifuging the solution obtained in the step A at the speed of 10,000 g for 20 minutes, and collecting the centrifuged precipitate, namely the nano vaccine medicament constructed by the manganese nano adjuvant and the antigen. The concentration of unbound antigen in the supernatant was then measured by fluorescence. Fluorescence measurements were performed using a Perkin Elmer fluorescence plate reader.
See fig. 6. FIG. 6 shows adsorption data of different concentrations of antigenic molecules by the manganese nano adjuvant obtained in example 1; a curve for real-time measurement of the association and dissociation effects of the interaction of the RBD antigen and the manganese nano adjuvant; and change in hydrated particle size after RBD is combined with manganese nano adjuvant (MnARK).
As can be seen from FIG. 6, the nanoprobe obtained in example 1 can effectively adsorb and carry antigen molecules; the RBD antigen can be stably combined with the manganese nano adjuvant with high affinity; the hydrated particle size of the nano vaccine drug formed after the RBD antigen is combined is obviously increased compared with that of the manganese nano adjuvant, and the effective combination of the RBD antigen and the manganese nano adjuvant is realized.
Example 5
A) On the premise of following the national animal health protocol, selecting BALB/c mice of 6-8 weeks old into 3 groups, each group comprises 12 mice, and injecting a reagent (i) and a nano vaccine medicament constructed by 25 mug of manganese nano adjuvant carrying 10 mug of fluorescent molecule Cy5 modified RBD antigen at the thigh muscle of the right rear leg; ② carrying 10 mu g of fluorescent molecule Cy5 modified RBD antigen; ③ physiological saline.
B) Fluorescence imaging analysis was performed on mice at injection time points of 0 hours, and at 12, 24, 48, 72 hours. And 3 mice were collected for fluorescence imaging analysis of axillary and inguinal lymph nodes after 12, 24, 48, and 72 hours of fluorescence imaging.
C) DC cells in lymph node tissues are analyzed by using a flow cytometer, and the internalization condition of the RBD antigen in the DC cells is counted.
See fig. 7. FIG. 7 shows fluorescence imaging of nano-vaccine drug live accumulation and lymph node targeting.
As can be seen from fig. 7, the nano vaccine drug constructed by the manganese nano adjuvant induces greater accumulation of RBD antigen at the injection site compared to RBD alone, and the antigen persists for more than 3 days at the site. Meanwhile, the nano vaccine medicine induces stronger fluorescence signals in lymph nodes. Quantitative analysis shows that the accumulation efficiency of the nano vaccine medicament in lymph nodes is about 2 times of that of free RBD antigen 12 hours after injection, and the nano vaccine medicament constructed by the manganese nano adjuvant promotes the delivery and effective accumulation of in vivo antigen to lymph nodes.
Example 6
A) The nano-vaccine drug obtained in example 4 was subjected to ELISPOT analysis using a commercial kit of R & D system (a kit for use in a mouse IFN-. gamma.enzyme-linked immunosorbent assay (ELISPOT) device). In a specific procedure, cytokine capture antibodies against mouse IFN- γ (200-fold diluted with sterile PBS) were coated onto polyvinylidene fluoride (PVDF) in 96-well plates and incubated overnight at 4 ℃. The 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 (i) RBD protein antigens of 5 mug/mL; ② normal saline; ③ 5 μ g/mL of the NanoVan drug constructed in example 4 containing RBD protein antigen, and immediately thereafter freshly prepared mouse splenocytes (5X 10)5Cells/well) were added to the plate. The plates were incubated at 37 ℃ and 5% CO2Incubate for 18 hours next 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 plates were incubated with peroxidase substrate AEC for 30 minutes. ELISPOT points were enumerated using an automated ELISPOT reader system (Bio-Red).
Referring to fig. 8, fig. 8 shows that the nano-vaccine drug constructed in example 4 activates B cells.
As can be seen from FIG. 8, the expression of three activation markers (MHC-II, CD69 and CD 86) on the surface of B cells of mice receiving the nano-vaccine drug constructed in example 4 was significantly increased compared to those of mice receiving only RBD protein or physiological saline (control group), indicating that the nano-vaccine drug constructed in example 4 can promote the maturation of B cells in vivo.
Example 7
Nano vaccine drug inoculation constructed in example 4.
A) On the premise of following the national animal health protocol, selecting 6-8 weeks old BALB/c mice to inoculate for 3 times, wherein each group of mice is 6, and the total number of mice is three groups, namely, a nano vaccine medicament constructed by 25 mu g of manganese nano adjuvant carrying 10 mu g of RBD antigen; ② 175. mu.g of aluminum adjuvant (purchased from Invivogen) carrying 50. mu.g of RBD antigen; ③ 50 mug of RBD antigen without nano adjuvant. The first mice were inoculated intramuscularly in the thigh on day 0, the second on day 21, and the third on day 42, and serum samples were collected on day 57.
B) On the premise of following the national animal health protocol, selecting 6-8 weeks old BALB/c mice for inoculation, wherein each group of mice is 6, and the total number of the mice is three, namely, a nano vaccine medicament which is constructed by 25 mu g of manganese nano adjuvant carrying 50 mu g of RBD antigen is inoculated twice; ② inoculation was carried out three times with 175. mu.g of aluminum adjuvant (purchased from Invivogen) carrying 50. mu.g of RBD antigen; ③ 50 mug of RBD antigen, without nano adjuvant, three times of inoculation. The first mice were inoculated intramuscularly in the thigh on day 0, the second on day 21, and the third on day 42, and serum samples were collected on day 57.
C) The levels of IgG and IgM in the mouse sera induced by the vaccine in steps a and B were assessed by a 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 at 37 ℃ for 2 hours. The mouse sera collected in step a and step B were then added to 96-well plates after gradient dilution, and then incubated at 37 ℃ for 1 hour, followed by four washes 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',5,5' -Tetramethylbenzidine (TMB) was added to a 96-well plate and the reaction was stopped by adding 0.05% sulfuric acid. The absorbance at 450 nm and 630 nm was measured in an ELISA plate reader (Tecan, San Jose, Calif.).
Referring to fig. 9, fig. 9 is a graph comparing the effect of inducing immunity by the nano vaccine drug constructed in example 4.
As can be seen from FIG. 9, the nano-vaccine drug obtained in example 4 can induce about 5 times of IgG and IgM response intensity even at a lower antigen-carrying amount (10. mu.g) than a larger amount of RBD alone (50. mu.g) or RBD carried in a commercial aluminum adjuvant (50. mu.g); under the condition of the same antigen carrying amount, the nano vaccine medicament constructed in the embodiment 4 after two times of injection can generate 10 times of IgG signals and 5 times of IgM signal intensity compared with RBD induced by RBD alone or commercial aluminum adjuvant carried three times of injection. These results indicate that the nano-vaccine drug constructed in example 4 can generate stronger immune response after receiving less antigen injection amount (antigen carrying amount and/or injection times).
Example 8
A) Four groups of mouse serum samples obtained in step a of example 7 were subjected to a pseudovirus infection neutralization test, as follows: supernatants containing pseudovirus (50. mu.l; purchased from Sino Biological) were preincubated with serially diluted mouse serum at 37 ℃ for 1 hour and then added to 293T cells (5X 10) expressing ACE24Cells). 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 efficiencies were calculated and expressed as 50% and 90% neutralizing antibody titers.
B) Four groups of mouse serum samples from step a of example 7 were subjected to a novel neutralization assay for live coronavirus infection, specifically as follows: mouse sera were diluted in 2-fold gradients and mixed with live virus, incubated for 1 hour at 37 ℃ and added in triplicate to 293T cells expressing ACE 2. Cytopathic effect (CPE) was observed daily in each well and recorded one week post infection. The neutralizing titer of mouse antiserum that could completely prevent CPE was calculated.
Referring to fig. 10, fig. 10 is a graph showing the comparison of the in vitro immune effect of the nano vaccine drug constructed in example 4 against the pseudovirus and the novel coronavirus live virus.
As can be seen from fig. 10, the nano-vaccine drug obtained in example 4 can induce a significantly enhanced neutralizing antibody response against the novel coronavirus even at a relatively low antibody loading amount.
The following example is to compare the effect of the manganese nano adjuvant obtained by the present invention on the generation of neutralizing antibodies by using bivalent manganese ions (manganese chloride) and hyaluronic acid coated manganese dioxide particles (Mn @ HA) as a neocorona vaccine adjuvant.
Example 9
The manganese nano adjuvant obtained in the example 1, manganese chloride salt and hyaluronic acid coated manganese dioxide combined antigen prepared in a laboratory are used for constructing a nano vaccine medicament to carry out animal immunity experiments:
A) preparation of hyaluronic acid-coated manganese dioxide particles, 1.0g of HA was weighed into a flask, and ultrapure water (1 g: 25mL of water) was refluxed with stirring in a fume hood at 102 ℃ and 560rpm, and MnCl was injected2The solution (1 g/ml) was mixed well and added with sodium hydroxide (1M, 15 ml), stirred (700 rpm) and refluxed for 2h, followed by dialysis in ultra pure water for 3 days to give tan Mn @ HA particle adjuvant.
B) Selecting a new coronavirus antigen RBD as an immune antigen research object. Adding 10 mu g of RBD antigen, the manganese nano adjuvant obtained in the example 1, the Mn @ HA particles obtained in the step A) and manganese chloride into PBS, and incubating for 60 minutes at 25 ℃ to obtain adjuvant vaccines with different manganese. The mass ratio of the added antigen molecules to the manganese elements in each group is 1: 5.
C) b) on the premise of following the national animal health protocol, selecting 6-8 weeks old BALB/c mice to inoculate for 3 times, wherein each group of mice is 6, and each group of mice has five groups, namely, a nano vaccine medicament constructed by 25 mu g of manganese nano adjuvant carrying 10 mu g of RBD antigen; ② a vaccine constructed by 25 mug of Mn @ HA adjuvant carrying 10 mug of RBD antigen; ③ 25 mug of manganese chloride of 10 mug of RBD antigen; fourthly, 10 mu g of RBD antigen without nano adjuvant; fifthly, normal saline group. The first mice were inoculated intramuscularly in the thigh on day 0, the second on day 21, and the third on day 42, and serum samples were collected on day 57. The IgG levels in the serum of the mice induced by the vaccine in step a and step B were evaluated by a conventional enzyme-linked immunosorbent assay (ELISA).
As can be seen in 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 nano-vaccine group, indicating that the manganese nano-vaccine can elicit a robust immune response in vivo and is superior to MnCl2And Mn @ HA.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. The manganese nano adjuvant is characterized by comprising the manganous-manganic oxide nano particles and template molecules coated outside the manganous-manganic oxide nano particles, wherein the molar ratio of the template molecules to manganese elements is 1: (10-1000), wherein the template molecule comprises a template protein and a fragment or polypeptide thereof.
2. The use of claim 1, wherein the template protein comprises one or more of bovine serum albumin, human serum albumin, mouse serum albumin, transferrin, and antigenic protein; the polypeptide comprises the derived fragment of the template protein and the antigen polypeptide.
3. The use according to claim 1 or 2, wherein the average particle size of the manganese nano adjuvant is 1-100 nm.
4. The preparation method of the manganese nano adjuvant is characterized by comprising the following steps of:
(1) mixing a divalent manganese salt aqueous solution with a template molecule solution to obtain a composite solution;
(2) adjusting the pH value of the composite solution to be more than 9 or 9, stirring, dialyzing, purifying and freeze-drying to obtain the manganese nano adjuvant;
the template molecule includes a template protein and a fragment or polypeptide thereof.
5. The preparation method according to claim 4, wherein the divalent manganese salt is selected from one or more of manganese chloride, manganese nitrate, manganese acetate and manganese sulfate; the reagent for adjusting 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 tetramethylethylenediamine; in the composite solution, the concentration of the divalent manganese salt is 0.1-0.5 mol/L, and the concentration of the template protein is 1-50 g/L.
6. The method according to any one of claims 4 to 5, wherein the stirring temperature is 30 to 37 ℃, the stirring time is 1 to 10 hours, and the stirring speed is 200 to 1000 r/min.
7. A manganese nano adjuvant prepared by the method of any one of claims 4 to 6.
8. Use of a manganese nano-adjuvant in the preparation of a vaccine, wherein said vaccine comprises a manganese nano-adjuvant according to any one of claims 1 to 7.
9. A vaccine, which is characterized by comprising an antigen and the manganese nano adjuvant of any one of claims 1 to 7, wherein the mass ratio of the antigen to manganese element in the manganese nano adjuvant is 1: (0.025 to 40).
10. The method for preparing the vaccine according to claim 9, wherein the manganese nano adjuvant and the antigen are incubated in the buffer solution at 20-37 ℃ for 30-120 minutes.
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| WO2023131356A3 (en) * | 2022-01-07 | 2023-08-24 | 广东粤港澳大湾区国家纳米科技创新研究院 | Use of trimanganese tetraoxide particles in preparation of vaccine adjuvant |
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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 |
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| WO2023134783A3 (en) * | 2022-01-12 | 2023-09-14 | 广东粤港澳大湾区国家纳米科技创新研究院 | Aluminum nanocrystal delivery system, and self-assembled particle adjuvant vaccine based on binding of aluminum nanocrystal delivery system and vaccine antigen molecule |
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