Patent Application
20160279236
The present invention belongs to the field of immunology and pharmacy; in particular, the present invention relates to the use of rapamycin as a vaccine adjuvant and the preparation method thereof.
Application of an adjuvant is an important means to optimize the effect of an antigen. Adjuvants can not only quantitatively enhance the immune response induced by a vaccine, increase the titer of neutralizing antibody, but also qualitatively optimize the immune response, for example, improving the quantity of memory B cells and T cells and prolonging their survival time, so as to achieve superior protective effects compared with the immune response induced by “natural infection” itself. So far there are only three kinds of adjuvants for human, which are aluminum adjuvant, MF59 and mono-phosphate lipid A (MPL), and they can not meet the needs for so many vaccine researches. Therefore, the research and development for new adjuvants are imperative. Aluminum adjuvant mainly enhances Th2-type antibody response, which is effective in clearing extracellular pathogens and parasitic infections; however, it is of little effect for treating RSV infection. MPL is only valid in the vaccine strategy against human papilloma virus HPV and hepatitis B virus HBV. Studies have shown that the stimulation of dendritic cells by MPL can only induce limited amounts of IL-12, which makes it of limited capacity in enhancing Th1 response. The T cell-mediated immune responses and the secretion of IFN-γ have been proved to play a key role in clearing HIV, HCV and TB infections, and treating cancers. Therefore, the research and development for vaccine adjuvant strategies which can successfully induce T cell response is particularly urgent.
mTOR signaling pathway is a key pathway in the regulation of cell growth and proliferation. Through integrating signals from nutritious, energy states and growth factors, mTOR can regulate a large number of physiological processes. The disorder of mTOR pathway relates to a variety of human diseases, including cancer, diabetes and cardiovascular disease. Recent studies have shown that mTOR is also involved in the regulation of innate immunity and adaptive immunity, thus becoming a multi-functional regulatory molecule. Rapamycin is a small molecular compound capable of specifically inhibiting mTOR, which has been approved by the United States FAD in 1999 for the anti-rejection therapy of renal transplantation, and it is also being studied for the use in treating cancer and other diseases.
The object of the present invention is to provide a use of rapamycin as a vaccine adjuvant, and the preparation method thereof.
In the first aspect of the present invention, an immune enhancer composition is provided, comprising rapamycin or derivatives thereof, and Toll-like receptor (TLR) agonist adjuvant.
In a preferred embodiment, the Toll-like receptor agonist adjuvant comprises: Toll-like receptor 3 (TLR3), Toll-like receptor 4 (TLR4), Toll-like receptor 7 (TLR7), or combinations thereof.
In another preferred embodiment, the Toll-like receptor agonist adjuvant comprises: Monophosphoryl lipid A, lipopolysaccharide, poly I:C, R848, or combinations thereof.
In another preferred embodiment, in the immune enhancer composition, the weight ratio between rapamycin or derivatives thereof and Toll-like receptor agonist adjuvant is (0.2-20):1; preferably (0.5-15):1; more preferably (2-12):1.
In another preferred embodiment, the immune enhancer composition further comprises: a pharmaceutically acceptable carrier (such as PBS or saline).
In another preferred embodiment, the amount of rapamycin or derivatives thereof and Toll-like receptor agonist adjuvant in the composition is an effective amount, e.g., 0.01-80% (W/W) of the composition; preferably 0.1-20% (w/w).
In another preferred embodiment, the immune enhancer composition further comprises antigens; preferably, the antigens include (but not limited to) inactivated or attenuated virus particles, virus-like particles (VLP) or polypeptide antigens, protein antigens.
In another preferred embodiment, the weight ratio between the antigens and Toll-like receptor agonist adjuvant is (0.2-20):1; preferably (0.5-10):1; for example 1:1, 2:1, 1:2.
In another preferred embodiment, the amount of antigens in the composition is an effective amount, e.g., 0.01-80% (w/w) of the composition; preferably 0.1-20% (w/w).
In another preferred embodiment, the antigens include (but not limited to): surface antigen of hepatitis B virus, antigen of hepatitis C virus, HIV antigens, tumor antigens or ovalbumins.
In another preferred embodiment, the derivative of rapamycin comprises (but not limited to): temsirolimus, everolimus, Deforolimus (AP23573), Zotarolimus, Ridaforolimus, CCI-779, RAD001, ABT-578, SDZ RAD, SAR 943, 1139-52 Biolimus A9 or other types of mTOR inhibitors which comprise (but not limited to) Ku0063794, PP242, PI-103, AZD8055, Palomid 529, AZD2014, Torin1, SNS-032.
In another aspect of the present invention, a kit is provided, which comprises the immune enhancer composition; or the kit comprises separately packaged rapamycin or derivatives thereof and Toll-like receptor agonist adjuvant.
In another preferred embodiment, the kit further comprises antigens.
In another aspect of the present invention, the use of rapamycin or derivatives thereof in the preparation of formulations, in which the activity of Toll-like receptor agonist adjuvant is enhanced, is provided.
In another aspect of the present invention, a method for producing the immune enhancer composition is provided, which comprises: mixing rapamycin or derivatives thereof and a Toll-like receptor agonist adjuvant so as to obtain the immunity enhancer composition.
In a preferred embodiment, rapamycin or derivatives thereof and Toll-like receptor agonist adjuvant are mixed at weight ratio of (0.2-20):1; preferably (0.5-15):1; more preferably (2-12):1.
Other aspects of the present invention are obvious for those skilled in the art based on the disclosure herein.
(a) The influences of rapamycin on the transcriptional expression of cytokines induced by LPS in BMDMs;
(b) The influences of rapamycin on the transcriptional expression of costimulatory molecules induced by LPS in BMDMs;
(c) The influences of rapamycin on the transcriptional expression of proinflammatory cytokines and chemokines induced by LPS in BMDMs;
(a) rapamycin enhanced the OVA-specific Th1 and CD8+ T cell responses;
(b) rapamycin enhanced the OVA-specific IgG2b and IgG2c antibody titers;
(a) rapamycin increased the HBsAg-specific antigen response enhanced by MPL;
(b) rapamycin increased the HBsAg-specific CD4+ and CD8+ T cell response enhanced by MPL;
(c) rapamycin increased the HBsAg-specific Th2 cell response enhanced by MPL;
(d) rapamycin increased the HBsAg-specific Th17 cell response enhanced by MPL.
(a) Different amounts of rapamycin increased the HBsAg-specific antibody responses enhanced by MPL;
(b) Different amounts of rapamycin increased the HBsAg-specific CD4+ and CD8+ T cell response enhanced by MPL.
A, rapamycin and MPL synergistically induced high level of T cell immune responses against HCV E2.
B, rapamycin and MPL synergistically induced high level of antibody responses against HCV E2.
C and D, rapamycin and MPL synergistically induced persistent antibody responses against HCV E2.
Upon long and extensive research, the present inventors have surprisingly found that rapamycin possesses adjuvant activity which can promote the generation of some immune parameters. Moreover, rapamycin will show synergistic effects when applied together with Toll-like receptor agonist adjuvant, such as a monophosphoryl lipid A (MPL), thereby greatly enhancing the immune response against the exogenous antigens in vivo. The present invention is completed based on the above findings.
Rapamycin and Uses Thereof
The molecular formula of rapamycin is C51H79NO13, and the structure formula thereof is formula (I):
Derivatives of rapamycin can also be included in the present invention. The derivatives of the rapamycin include but not limited to: Temsirolimus, everolimus, Deforolimus (AP23573), Zotarolimus, Ridaforolimus, CCI-779, RAD001, ABT-578, SDZ RAD, SAR 943, 1139-52 Biolimus A9, and other types of mTOR inhibitors, including but not limited to: Ku0063794, PP242, PI-103, AZD8055, Palomid 529, AZD2014, Torin1, SNS-032. These derivatives have similar structures to rapamycin (e.g., having similar molecular structure) or similar mechanism, thus can be used in the present invention. For example, Koichi Araki 1 et al. (Nature. 2009 Jul. 2; 460(7251): 108-112. doi: 10.1038/nature08155.mTOR regulates memory CD8 T cell differentiation) and Amiel E et al. (J Immunol. 2012 Sep. 1; 189(5):2151-8. doi: 10.4049/jimmunol.1103741. Epub 2012 Jul. 23. Inhibition of mechanistic target of rapamycin promotes dendritic cell activation and enhances therapeutic autologous vaccination in mice) have shown that mTOR inhibitors have similar mechanism.
Structures and formulas of some derivatives of rapamycin are listed hereinafter:
The molecular formula of Temsirolimus is C56H87NO16, and the structure formula thereof is formula (II):
The molecular formula of Temsirolimus is C56H87NO16, and the structure formula thereof is formula (III):
The molecular formula of everolimus is C53H83NO14, and the structure formula thereof is formula (IV):
The molecular formula of 42-(dimethyl sulfoxide phosphonomethyl) rapamycin is C53H84NO14P, and the structure formula thereof is formula (V):
The molecular formula of 42-(dimethyl sulfoxide phosphonomethyl) rapamycin is C53H84NO14P, and the structure formula thereof is formula (VI):
The molecular formula of Zotarolimus is C52H79N5O12, and the structure formula thereof is formula (VII):
The molecular formula of Olcorolimus is C51H81NO12, and the structure formula thereof is formula (VIII):
The molecular formula of Umirolimus is C55H87NO14, and the structure formula thereof is formula (VIIII):
The molecular formula of Ku0063794 is C25H31N5O4, and the structure formula thereof is formula (X):
The molecular formula of PP242 is C16H16N6O, and the structure formula thereof is formula (XI):
The molecular formula of PI-103 is C19H16N4O3, and the structure formula thereof is formula (XII):
The molecular formula of AZD8055 is C25H31N5O4, and the structure formula thereof is formula (XIII):
The molecular formula of Palomid 529 is C24H22O6, and the structure formula thereof is formula (XIIII):
The molecular formula of AZD2014 is C25H30N6O3, and the structure formula thereof is formula (XV):
The molecular formula of Torin1 is C35H28F3N5O2, and the structure formula thereof is formula (XVI):
The molecular formula of SNS-032 is C17H24N4O2S2, and the structure formula thereof is formula (XVII):
Currently, in the prior art, rapamycin or derivatives thereof are used as immunosuppressive drugs. They exert their effects by inhibiting G0 and G1 phase of cell cycle and blocking G1 phase entering into S phase through combining with the corresponding immunophilin RMBP, and their effects are listed as follows: {circle around (1)} inhibiting the proliferation of T and B cells; {circle around (2)} inhibiting lymphocyte proliferation induced by IL-1, IL-2, IL 6 and IFN-γ; {circle around (3)} inhibiting the production of IgG and donor-specific antibody (cytotoxic antibodies); {circle around (4)} inhibiting the proliferation of monocyte. They can be used in the anti-transplant rejection, and in the treatment of rheumatoid arthritis, lupus and other autoimmune diseases.
In the present invention, it was found that rapamycin or a derivative thereof can be used as an immune adjuvant. It has exhibited strong feasibility in terms of safety since it has been widely used in patients. The present invention has also demonstrated that rapamycin or derivatives thereof can significantly optimize the activity of Toll-like receptor agonist adjuvant; and in particular, it can enhance MPL induced T cell responses.
Rapamycin or derivatives thereof are a type of commercial products, which is commercially available to the skilled person in the art. In addition, it can also be obtained through chemical synthesis.
Uses of rapamycin or derivatives thereof, or a pharmaceutically acceptable salt thereof are provided in the present invention for preparing immune enhancer; preferably, for preparing formulations for enhancing of activities of Toll-like receptor agonist adjuvants.
In order to confirm the above uses of rapamycin, the inventors, through in vitro cell tests, verified that rapamycin can promote dendritic cells to produce IL-12 and IL-23, inhibit the production of IL-10; promote the transcript expression of IL-12a, IL-12b and IL-23a; and inhibit the expression of IL-10 and PD-L1. Furthermore, animal tests have confirmed that rapamycin can enhance the activity of a Toll-like receptor agonist adjuvant.
Therefore, rapamycin or derivatives thereof are a type of adjuvant which can effectively enhance antigen-specific T-cell and antibody response, and a combination of rapamycin or derivatives thereof and Toll-like receptor agonist adjuvant is even better than MPL and Aluminum Adjuvant which are the best in the present stage.
Composition
Based on the novel discovery of the present invention an immune enhancer composition comprising rapamycin or derivatives thereof and a Toll-like receptor agonist adjuvant is provided. The amounts of rapamycin or derivatives thereof and Toll-like receptor agonist adjuvant in the composition are both at safe and effective amount. As a preferred embodiment of the present invention, in the composition, the weight ratio between rapamycin or derivatives thereof and Toll-like receptor agonist adjuvant is (0.2-20):1; preferably (0.5-15):1; more preferably (2-12):1. The composition can further comprise pharmaceutically acceptable carriers or excipients.
In the present invention, the “immune enhancer” means non-specific immune enhancer, which is also known as “adjuvant”. When injected together with an antigen, or pre-injected into a body, the adjuvant can enhance the immune response against an antigen or change the type of immune response in the body.
In the present invention, the term “comprising” or “including” means that the various components can be used together in the mixture or composition of the present invention. The terms “mainly consisting of . . . ” and “consisting of . . . ” are included in the scope of the term “comprise”.
In the present invention, the term “pharmaceutically acceptable” component is a substance which is suitable for applying to humans and/or animals without undue undesired side reactions (such as toxicity, stimulation or allergy), that is, a substance of reasonable benefit/risk ratio.
In the present invention, the term “pharmaceutically acceptable carrier” refers to a solvent, suspending agent or excipient acceptable in pharmaceutical or food which is used to deliver rapamycin or a derivative, or a physiologically acceptable salt thereof of the present invention to an animal or human. The carrier can be liquid or solid.
Dosage forms of the pharmaceutical composition according to the present invention can be varied, so long as the active ingredient can effectively get to the mammal. For example, it can be selected from: solutions, suspensions, powders, granules, tablets, capsules, syrups or aerosols, wherein rapamycin or derivatives thereof and/or Toll-like receptor agonist adjuvant may be present in a suitable solid or liquid carrier or dilution.
In the present invention, rapamycin or derivatives thereof and/or Toll-like receptor agonist adjuvant, and the compositions thereof may also be stored in a disinfected device suitable for injection or infusion.
The dose and mode for administering rapamycin or derivatives thereof and/or a Toll-like receptor agonist adjuvant may be varied. Toll-like receptor agonist adjuvant can be administered at 0.03-150 mg/60 kg of body weight, preferably 3-60 mg/60 kg of body weight; and rapamycin or derivatives thereof can be administered at 0.03-900 mg/60 kg of body weight; preferably 0.3-600 mg/60 kg of body weight. Generally, satisfying immune response can be induced by one treatment of the compounds of the present invention and the antigen, and even better effects can be achieved by repeating immunization for one time after a month. Booster immunization can be repeated for one time every 1-3 months, if necessary, or repeated 2-3 times so as to achieve the best immunity.
Rapamycin or derivatives thereof and/or Toll-like receptor agonist adjuvant may be comprised in a vaccine composition. The vaccine composition further comprises antigens to form a complete vaccine composition capable of inducing the immune response in the body.
The present invention will be further illustrated below with reference to the specific examples. It should be understood that these examples are only to illustrate the invention but not to limit the scope of the invention. The experimental methods with no specific conditions described in the following examples are generally performed under the conventional conditions, for example, according to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002, or according to the manufacture's instructions. Unless indicated otherwise, parts and percentage are calculated by weight.
Cytokines IL-12, which is secreted by dendritic cells, is a major regulatory factor for inducing Th1 and CD8 responses. MPL, as a ligand for TLR4, merely stimulate dendritic cells to induce limited IL-12. The inventors firstly tested by in vitro experiment whether rapamycin can enhance IL-12 production induced by activation of TLR4 signaling in dendritic cells.
Bone marrow stem cells from 6-8 weeks aged C57BL/6 mice were taken, and erythrocytes were removed by using ammonium chloride lysate (0.15 M NH4Cl, 1 mM KHCIO3, 0.1 mM Na2EDTA, pH7.3), and then suspension-cultured at a cell density of 106 cells/mL in a dendritic cell (bone-marrow derived dendritic cell, BMDC) differentiation medium (formulation of which was: RPMI 1640 complete medium (comprising 5% (v/v) FBS and 1% (v/v) pen strep), with 20 ng/ml GM-CSF, 10 ng/ml IL-4, 2 mM L-glutamine and 200 μM β-mercaptoethanol being added). After differentiation for 7 days, the cells were purified with CD11c magnetic beads to obtain BMDCs. The purified BMDCs were resuspended in a fresh dendritic cell culture medium (106 cells/ml), and inoculated into 24-well plate with 500 μL per well 100 nM of rapamycin or DMSO (control) were used to pre-treat the cells for 1 hour, and then the cells were stimulated with 100 ng/ml LPS for 24 hours. The supernatant was taken and the amount of IL-12 was measured by ELISA.
The results were shown in
Bone marrow stem cells of 6-8 weeks aged C57BL/6 mice were taken, and the cells are cultivated at 106 cells/mL cell density in bone marrow-derived macrophage (BMDM) differentiation medium (formulation of which was: RPMI 1640 complete medium added with 30% (v/v) L929 conditioned medium and 1% (v/v) pen/strep). After differentiation for 4 days, the old culture medium (comprising suspended cells) was removed and fresh BMDM differentiation medium was added. The adhered BMDMs were collected on the 7th day of differentiation and used in in vitro experiment.
The BMDMs were pre-treated with 100 nM of rapamycin or DMSO for 1 hour, and then the cells were stimulated with 100 ng/ml LPS for 24 hours. The cells were collected at 1, 2, 4 and 8 hours, and were cryopreserved in a refrigerator at −80° C. for the extraction and preparation of RNA.
TRIZOL (Invitrogen) method was used to extract RNA of BMDM, and Superscript™ III Reverse Transcriptase Kit (Invitrogen) was used to respectively reverse transcribe 1 μg RNA so as to synthesize the first-strand cDNA for Real-time quantitative PCR determine of gene expression levels. The primers for Real-time quantitative PCR are shown in table 1. ΔΔ method was used to calculate the relative expression level of the determined gene compared with GAPDH.
The results of real-time quantification PCR were shown in
Furthermore, the inventors have also found that some costimulatory molecules and cytokines involved in adaptive immune response were also affected by rapamycin, and exhibited different expression patterns within the time frame of the process.
Moreover, rapamycin did not affect the transcription and expression of pro-inflammatory cytokines or chemokines such as TNFα and CXCL2 (
Rapamycin could significantly enhance the production of IL-12(p70) induced by LPS both in dendritic cells and macrophages, but how about it's ability to regulate antigen-specific adaptive immune response? 6-8 weeks aged mice were primarily immunized by the inventors using intraperitoneal injection method, wherein Ovalbumin (10 μg/mouse) was used as antigen and PBS (negative control), MPL (10 μg/mouse, positive control), rapamycin (100 μg/mouse), rapamycin (100 μg/mouse)+MPL (10 μg/mouse) as adjuvant respectively, and were immunized again with ovalbumin (10 μg/mouse, without adjuvant) at 3 weeks. The mice were killed 1 week after the secondary immunization, and blood was taken to determine the content of OVA specific IgG1, IgG2b, IgG2c; meanwhile, 10 μg/ml of OVA-1 (SIINFEKL) or OVA-II (ISQAVHAAHAEINEAGR, Invitrogen) was used to stimulate splenic cells (5×105 cells). After 48 hours, enzyme linked immunosorbent assay (ELISA) was used to determine the content of IFNγ in the supernatant.
The results have shown that (
Virus-like particles (VLP) consisting of (HBV) outer membrane protein antigens is one of the few successful recombinant vaccines clinically used. Aluminum adjuvant (Alum) can only enhance the immune response of VLP in health populations, while it is of limited effects in the elderly people and patients with renal dysfunction. However, Alum together with MPL can significantly increase the activity of HBV vaccine. The previous results of the inventors have shown that rapamycin can effectively enhance the adjuvant effect of MPL. In order to further analyze the adjuvant activity of rapamycin, C57BL/6 mice were immunized by the inventors using HBsAg VLP (5 μg/mouse) (Shanghai Yemin Biotechnology Co., Ltd., Item number YM-101) as antigen in mixture with Alum (200 μg/mouse), Alum (200 μg/mouse)+MPL (10 μg/mouse), MPL (10 μg/mouse)+rapamycin (100 μg/mouse) respectively, and was immunized with HBsAg (without MPL or rapamycin) 3 weeks after the primary immunization. The mice were killed 2 weeks after the secondary immunization, and blood was taken to determine the content of HBsAg-specific IgG1, IgG2b, IgG2c. To test HBsAg-specific T cell responses, the inventors have determined HBsAg-specific induced Th1, Th17 and Th2 responses by Elispot (Elispot Assay).
The specific operations are listed as follows: spleen cells were inoculated at 5×105 cells per-well into Elispot 96-wells plate of IFNγ, IL-17 or IL-4, and treated or untreated (control) with HBsAg (10 μg/ml) or CD8 peptides (10 μg/ml, ILSPFLPLL, VWLSVIWM, China Peptides Co., LTD., Shanghai), incubated under 37° C., 5% CO2 for two days. The cells and supernatants were removed. The plates were washed with PBST for 5 times, then incubated with 50 μl of 2 μg/ml biotin-coupled IFNγ (ebioscience, cat No: 13-7312), IL-17 (ebioscience, cat No: 13-7177) or IL-4 (Mabtech, cat No: 3311-6-250) detecting antibody under 37° C. for 2 hours, washed with PBST for 5 times, then incubated with 100 μl of 1000-fold diluted Streptavidin-alkaline phosphatase (Mabtech, cat No: 3310-10) under 37° C. for 1 hour. After this incubation, plates washed with PBST for 3 times, and then washed with PBS for two times. 100 μl of NBT/CPIT substrate (Mabtech, cat No: 3650-10) was added and incubated at room temperature for 0.5 hour. Plates were washed with deionized water for 3 times, and then naturally dried in darkness. The numbers of positive spots was calculated with Elispot Reader, and then statistically analyzed.
The results (
Based on the above results, rapamycin is a type of adjuvant which could effectively enhance the antigen-specific T cell and antibody responses, while the combination of rapamycin and MPL is even better than the MPL and aluminum adjuvant, which is the best at this stage.
Composition 1, comprising (dosage for one mouse): rapamycin 5 μg, MPL 10 μg, and PBS 0.2 ml;
Composition 2, comprising (dosage for one mouse): rapamycin 20 μg, MPL 10 μg, and PBS 0.2 ml;
Composition 3, comprising (dosage for one mouse): rapamycin 100 μg, MPL 10 μg, and PBS 0.2 mL.
Mice were immunized with Compositions 1-3 in mixture with HBsAg (5 μg, dosage for one mouse) respectively, and were boosted 3 weeks after the primary immunization. The mice were killed after one week, and the serum was prepared to determine the content of HBsAg-specific IgG1, IgG2b and IgG2c (
HBsAg (5 μg/mouse) was respectively mixed with the following adjuvants or adjuvant combinations: rapamycin (100 μg/mouse)+MPL (10 μg/mouse), MPL (10 μg/mouse), rapamycin (100 μg/mouse) or Alum (200 μg/mouse)+MPL (10 μg/mouse) to immunize (primary immunization) C57BL6 mice. After 4 weeks, each group of mice was boosted according to the same program as the primary immunization. The mice were killed after two weeks, and serum was prepared for determining the content of HBsAg-specific IgG1, IgG2b and IgG2c.
Similarly, when derivatives of rapamycin were used to replace rapamycin for combining with MPL as above, it was found that the rapamycin derivatives can also significantly enhance the HBsAg-induced antibody titers, which provided significant effects as compared with MPL alone.
Hepatitis C virus HCV infects about 3-4 million people per year, and there is no effective preventive or therapeutic vaccines at present. Since IFN-γ secreting T cells and broad-spectrum neutralizing antibodies are considered as necessary immune response in preventing the HCV infection, and the previous data have shown that rapamycin can enhance MPL-induced T cell immune response and antibody titers, therefore, the present inventors have further studied whether the combination of rapamycin and MPL can induce an high strength immune response against HCV outer membrane protein E2.
C57BL/6 mice were immunized using purified E2 protein (20 μg/mouse, bought from eEnzyme company, Cat No. HCV-E21b) as antigen in mixture with Alum (200 μg/mouse), MPL (10 μg/mouse), rapamycin (100 μg/mouse), MPL (10 82 g/mouse)+rapamycin (100 μg/mouse) respectively, and after 4 weeks, were boosted by the same antigen+adjuvant program. 8 weeks after the secondary immunization, mice were immunized for one time with E2 protein (without MPL or rapamycin) (5 μg/mouse), and the mice were killed after 2 weeks. Spleen cells were taken to determine immune response of T cells (
These results indicate that rapamycin and MPL as adjuvant was capable of inducing strong and sustained T cell and antibody responses, which is the best candidate adjuvant for new HCV vaccine.
polyI:C, as an immune adjuvant, can effectively induce cytotoxic T cell immune response, therefore, it has been widely used in cancer vaccines. But tumor vaccines with polyI:C as adjuvant has not yet been clinically used. One possible reason is that the strength and durability of induced cytotoxic T cell immune response need to be improved.
The present inventors have firstly studied the influence of rapamycin on IL-12 expression induced by a variety of Toll-like receptors agonist including polyI:C by in vitro experiments. Dendritic cells differentiated from mouse bone marrow cells (BMDCs differentiated for 7 days with 20 ng/ml GM-CSF and 10 ng/ml IL-4) were pre-treated with or without rapamycin (100 nM) for 1 hour, and stimulated by LPS (100 ng/ml), MPL (1 μg/ml), R848 (1 μg/ml) and polyI:C (20 μg/ml) respectively for 24 hours, and cell culture media were taken to determine the content of IL-12 by ELISA. The results have shown that not only can rapamycin enhance the IL-12 expression induced by lipopolysaccharide and MPL (TLR4 ligand), but also can it effectively enhance polyI:C (TLR3 ligand) and R848 (TLR7 ligand) induced IL-12 expression, as shown in
The inventors have further explored the ability of rapamycin to enhance MPL and polyI:C-induced cytotoxic T-cell immune responses and the anti-tumor effect. By intraperitoneal injecting, 6-8 weeks aged mice were immunized using ovalbumin (10 μg/mouse) as antigen and PBS (200 μl, negative control), polyI:C (Invivogen) (50 μg/mouse), rapamycin (100 μg/mouse), MPL (10 μg/mouse), MPL (10 μg/mouse)+rapamycin (100 μg/mouse), polyI:C (50 μg/mouse)+rapamycin (100 μg/mouse) used as adjuvant to conduct primary immunization. After two weeks, the mice were boosted by the same program. 4 weeks after the secondary immunization, melanoma murine cell line B16 OVA expressing OVA was subcutaneous implanted into mice (1×106 cells/mouse), and the tumor growth was measured. In the mice of control group without adjuvant being added, the tumor normally grew and the tumor volume increased with time (
All literatures mentioned in the present application are incorporated herein by reference, as though each one is individually incorporated by reference. Additionally, it should be understood that after reading the above teachings, those skilled in the art can make various changes and modifications to the present invention. These equivalents also fall within the scope defined by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201310320670.7 | Jul 2013 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2014/083111 | 7/28/2014 | WO | 00 |
