- Research article
- Open Access
GM-CSF-loaded chitosan hydrogel as an immunoadjuvant enhances antigen-specific immune responses with reduced toxicity
© Noh et al.; licensee BioMed Central Ltd. 2014
- Received: 19 May 2014
- Accepted: 10 October 2014
- Published: 18 October 2014
The application of vaccine adjuvants has been vigorously studied for a diverse range of diseases in order to improve immune responses and reduce toxicity. However, most adjuvants have limited uses in clinical practice due to their toxicity.
Therefore, to reduce health risks associated with the use of such adjuvants, we developed an advanced non-toxic adjuvant utilizing biodegradable chitosan hydrogel (CH-HG) containing ovalbumin (OVA) and granulocyte-macrophage colony-stimulating factor (GM-CSF) as a local antigen delivery system.
After subcutaneous injection into mice, OVA/GM-CSF-loaded CH-HG demonstrated improved safety and enhanced OVA-specific antibody production compared to oil-based adjuvants such as Complete Freund’s adjuvant (CFA) or Incomplete Freund’s adjuvant (IFA). Moreover, CH-HG system-mediated immune responses was characterized by increased number of OVA-specific CD4+ and CD8+ INF-γ+ T cells, leading to enhanced humoral and cellular immunity.
In this study, the improved safety and enhanced immune response characteristics of our novel adjuvant system suggest the possibility of the extended use of adjuvants in clinical practice with reduced apprehension about toxic side effects.
- Immune response
Vaccine adjuvants have been extensively studied in order to enhance their safety and improve the immune responses elicited by the accompanying antigen for immunotherapy -. However, conventional adjuvants evoke serious side effects due their toxicity . This has limited their use in clinical trials in recent years and poses as a serious hurdle to effective adjuvant-based immunotherapy, resulting in side effects such as boil and pyrexia occurring in clinical trials. Mineral oil-based media, Complete Freund’s adjuvant (CFA) and Incomplete Freund’s adjuvant (IFA) ,, are available as commercialized products and are potent adjuvants commonly used in animal research, as they can augment both humoral and cellular immune responses to a wide range of antigens. However, they are toxic to human subjects, and their use in animals are now also discouraged or banned by many institutional animal ethics committees due to their noxious side effects . These limitations enforce the need for novel and improved adjuvant systems to replace the existing ones.
Here, we have developed an improved chitosan hydrogel (CH-HG) system as a vaccine adjuvant to enhance induced immune responses and reduce toxicity ,. Chitosan is particularly attractive for clinical and biological applications due to its low toxicity, low immunogenicity, biocompatibility, and biodegradability ,. In addition, CH-HG displays a liquid-solid phase transition depending on temperature, allowing CH-HG to simply be injected at the diseased site without a requirement for surgical procedure. Therefore, CH-HG may lead to enhanced safety and immune responses at disease sites. Moreover, CH-HG can be gradually degraded by enzymes in the body after the antigen has been completely released.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been used as a vaccine adjuvant, which stimulates macrophage differentiation and proliferation, and to activate antigen presenting cells (APCs) such as dendritic cells (DCs) or macrophages. These qualities make GM-CSF important and relevant to developing vaccine therapies -. GM-CSF can augment the primary immune response of murine spleen cells to sheep red blood cells and increase interleukin-1 secretion . In addition, GM-CSF can increase the immunogenicity of tumors in animal models and stimulate both humoral and cellular immune responses, thus serving as an effective adjuvant for DNA or peptide based vaccines ,. This increased immune response is presumed to be a result of the GM-CSF-mediated increase in maturation and function of APCs . Further studies, using microspheres of encapsulated GM-CSF mixed with irradiated tumor cells that were injected subcutaneously also demonstrated increased tumor immunogenicity . These studies motivated us to ask whether GM-CSF function could be improved in prolonged immune responses at a local adjuvant site. Here, we present a novel immunization strategy using CH-HG-loaded GM-CSF/OVA as an adjuvant system to increase immunogenicity in both humoral and cellular immune responses in a mouse model as a proof-of-concept, and in order to approach a clinically relevant study of GM-CSF in CH-HG as an enhanced adjuvant for vaccination that evokes an improved immune response, while exhibiting lower toxicity compared to existing adjuvants.
Female C57BL/6 mice (6 weeks old, 20 g) were purchased from Daehan Biolink (Chungbuk, Korea) and maintained under a protocol approved by the Korea University Institutional Animal Care and Use Committee (KUIACUC-2013-210). All procedures were performed in accordance with recommendations for the proper animal care and use.
Preparation of OVA and GM-CSF-loaded CH-HG
We have previously reported the physical characteristics of CH-HG as an in vitro or in vivo depot system after subcutaneous (SC) injection ,. We prepared CH-HG containing OVA and/or GM-CSF with mild modification made to our previously reported method. Briefly, chitosan solution (medium molecular weight of 161 kDa, viscosity of 200,000 cps and a degree of deacetylation of 80%) was obtained by dissolving 40 mg of chitosan in 1.8 ml of 0.1 M HCl solution. Glycerol 2-phosphate disodium salt hydrate (β-GP) solution containing 50 μg of OVA and/or 50 ng of GM-CSF was prepared by dissolving 0.2 g of β-GP and predetermined amount of OVA or GM-CSF in 0.2 ml of distilled water. The CH solution was cooled to 4 °C and continuously stirred while adding 0.2 ml of mixed solution was added. The final product is successfully formed hydrogel at body temperature and physiological pH in vivo after subcutaneous (SC) injection into mice. Notably, the mice did not suffer severe side effects, including pus formation or inflammation, and maintained a healthy appearance after CH-HG implantation.
Safety test of CH-HG in mice after SC injection
To confirm the safety of adjuvants, 50 μl of CH-HG, Complete Freund’s adjuvant (CFA), or Incomplete Freund’s adjuvant (IFA) were injected subcutaneously into mice. The external morphologies of the adjuvants were monitored in the mice, and the hydrogel volume was measured using calipers for 14 days. After 14 days from initial administration, we measured body weights to evaluate the toxicity of each adjuvant.
Immune response against CH-HG containing OVA + GM-CSF
Fifty microliters of CH-HG, CFA, and IFA containing OVA + GM-CSF were injected subcutaneously into mice, and the adjuvants were boosted at 7 days using the same injection volume. The IgG, IgG1, and IgG2a levels in serum were measured by ELISA 3 weeks after the first immunization. Briefly, the presence of OVA-specific antibodies in the sera from CH-HG mediated immunization of C57BL/6 mice (five per group) was determined by ELISA using microwell plates coated with OVA protein. Purified OVA protein was diluted to 0.5 μg/ml in PBS buffer (pH 7.4), and 50 μl of that solution was then added to each well of 96-well microtiter plates. Purified OVA protein was used as a negative control. The plates were incubated overnight at 37 °C, followed by three washes with 300 μl of PBS. After washing, 200 μl of blocking solution (1% skim milk) was incubated at 37 °C for 1 hr, and then the plates were washed three additional times using PBS containing 0.05% Tween20 (PBS-T). Serial dilutions of the tested sera were made (0.1 ml/well), and the plates were incubated for 1 hr at 37 °C. The plates were washed with PBS-T and incubated with 0.1 ml of alkaline phosphatase-conjugated rabbit anti-mouse antibodies (Zymed) per well for 1 hr at 37 °C. The plates were again washed with PBS-T and incubated with alkaline phosphatase substrate (100 μl/well, Sigma) according to the manufacturer’s instructions for 1 hr at 37 °C. Plates were read on a MicroElisa reader at a wavelength of 650 nm (Additional file 1).
Flow cytometry analysis
To assess OVA specific immune responses, we immunized mice with 50 μl of one of five different solutions; 1) 50 μg of OVA solution, 2) 50 μg of OVA solution with GM-CSF, 3) CH-HG containing 50 μg of OVA, 4) CH-HG containing 50 μg of OVA +50 ng of GM-CSF, or 5) IFA containing 50 μg of OVA +50 ng of GM-CSF. Splenocytes were harvested from the immunized mice (five per group) for 2 weeks after the first immunization. Prior to intracellular cytokine staining, 5×106 pooled splenocytes from each immunization group were incubated overnight with 1 μg/ml OVA peptide containing MHC class I epitope (aa 257-264) or MHC class II epitope (aa 323-339) to detect OVA-specific CD4+ and CD8+ T cell precursors. Intracellular IFN-γ and IL4 staining, as well as flow cytometric analysis were performed using a Becton-Dickinson FACScan with CELLQuest software (Becton Dickinson Immunocytometry Systems, Mountain View, CA). The number of OVA-specific INF-γ or IL4 secreting CD4+ T cells and INF-γ secreting CD8+ T cells were assessed by intracellular cytokine staining and FACScan analysis.
Differences in continuous variables were analyzed by Studen’s t-test for comparing two groups and ANOVA was performed to compare differences between multiple groups. For values that were not normally distributed, the Mann-Whitney rank sum test was used. The statistical package for the Social Sciences (SPSS, Inc.) was used for all statistical analyses. A p value of <0.05 was considered statistically significant.
CH-HG safety after SC injection in mice
CH-HG containing OVA and GM-CSF induced titers of OVA specific IgG
CH-HG/OVA + GM-CSF induction of OVA-specific IgG compared to CFA and IFA
CH-HG/OVA + GM-CSF generated an enhanced OVA-specific cellular immune response
Adjuvant-based vaccination in immunotherapy is highly effective, but it is associated with inherent safety and toxicity problems that need to be overcome before use in clinical settings ,. We demonstrate here that a novel CH-HG based adjuvant system loaded with GM-CSF, a broad immune-modulating cytokine, led to potent both humoral and cellular immune responses without severe side effects. This approach has broad utility for adjuvant mediated therapeutic materials as well as other immunotherapy applications.
Several synthetic biomaterials have been proposed as adjuvants for effective antigen delivery. These include microparticles , nanofibers , metal-based particles , and emulsions . Although many types of compounds can be potentially used as delivery agents, their safety and evoked immune responses cause concerns. The development of adjuvant-based vaccination for immunotherapy, therefore, requires clinically suitable, safe, and effective delivery systems.
Therapeutic materials packaged into CH-HG may provide a clinically viable approach for the development of a vaccine related immunotherapy. The biocompatibility and biodegradability of these systems are key parameters for medical and pharmaceutical applications . Here, we demonstrate that a novel hydrogel-based adjuvant system loaded with OVA and GM-CSF led to potent immune responses in mice. This study implements a novel adjuvant with improved safety and reduced toxicity. In addition, the CH-HG system allows for the co-delivery of therapeutic materials, such as an antigen, which could further enhance the antigen specific immune response without increasing toxicity. Injection of CH-HG is simple because CH-HG is a liquid phase at room temperature and therefore, does not require surgery for insertion at the site of the disease, which would disrupt the disease environment. Moreover, the biodegradation rate of injected hydrogel can be controlled by modifying the injected volume. In this study, we injected 50 μl of chitosan solution, which degrades in approximately 2 weeks, suggesting that this amount can be used for repeated injections. This local depot system may be attractive for many biomedical applications, including administration of anesthetic agents after surgery and treatment of certain skin, breast, or neck cancers. Such an approach may also be useful for adjuvant therapy, or as a local treatment for chronic periodontitis. Although the CH-HG mediated adjuvant platform can be useful for diseases associated with the immune system and with the intent to enhance immune response, additional possibilities of optimized loading for effective cytokine or immune modulator using the CH-HG platform may be explored and developed for research purposes.
In summary, we have developed a novel adjuvant system that yields high immune responses without negative side effects at the local site of interest due to matrix toxicity. Our data demonstrate that CH-HG based local antigen delivery can invoke antigen specific CD8+ T cell immunity. Such CH-HG based adjuvant strategies may have broad potential as antigen delivery platforms in human disease and represent an opportunity for further development of vaccine based immunotherapeutics.
KHN and YMP performed the preparation and characterization of the hydrogel. HSK, THK, KHS, YHL, YB, and HNJ participated in the animal experiment and T cell immune response. IDJ, BCS, KML, and SYS conceive of the study and participated in data analysis. HDH, and TWK participated in its design and coordination. All authors read and approved the final manuscript.
This work was supported by the National Research Foundation of Korea (NRF) grant funded the Korea government (NRF-2012R1A2A1A03008433) (Y.M.P.), (NRF-2013M3A9D3045881), and (NRF-2012R1A2A2A01007527). This work was supported by Basic Research Laboratory Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (No.2013R1A4A1069575) (H.D.H.). This work was supported by a grant of the Korea Healthcare technology R&D Project, Ministry of Health and Welfare, Republic of Korea (A062260) (T.W.K.).
- Wilson-Welder JH, Torres MP, Kipper MJ, Mallapragada SK, Wannemuehler MJ, Narasimhan B: Vaccine adjuvants: current challenges and future approaches. J Pharm Sci. 2009, 98 (4): 1278-1316. 10.1002/jps.21523.PubMedView ArticleGoogle Scholar
- Riese P, Schulze K, Ebensen T, Prochnow B, Guzman CA: Vaccine adjuvants: key tools for innovative vaccine design. Curr Top Med Chem. 2013, 13 (20): 2562-2580. 10.2174/15680266113136660183.PubMedView ArticleGoogle Scholar
- Reed SG, Orr MT, Fox CB: Key roles of adjuvants in modern vaccines. Nat Med. 2013, 19 (12): 1597-1608. 10.1038/nm.3409.PubMedView ArticleGoogle Scholar
- Chapel HM, August PJ: Report of nine cases of accidental injury to man with Freund's complete adjuvant. Clin Exp Immunol. 1976, 24 (3): 538-541.PubMedPubMed CentralGoogle Scholar
- Bijker MS, van den Eeden SJ, Franken KL, Melief CJ, Offringa R, van der Burg SH: CD8+ CTL priming by exact peptide epitopes in incomplete Freund's adjuvant induces a vanishing CTL response, whereas long peptides induce sustained CTL reactivity. J Immunol. 2007, 179 (8): 5033-5040. 10.4049/jimmunol.179.8.5033.PubMedView ArticleGoogle Scholar
- Stewart-Tull DE: Freund's complete and incomplete adjuvants, preparation, and quality control standards for experimental laboratory animals use. Methods Mol Biol. 2010, 626: 59-72. 10.1007/978-1-60761-585-9_5.PubMedView ArticleGoogle Scholar
- Seo SH, Han HD, Noh KH, Kim TW, Son SW: Chitosan hydrogel containing GMCSF and a cancer drug exerts synergistic anti-tumor effects via the induction of CD8+ T cell-mediated anti-tumor immunity. Clin Exp Metastasis. 2009, 26 (3): 179-187. 10.1007/s10585-008-9228-5.PubMedView ArticleGoogle Scholar
- Han HD, Mora EM, Roh JW, Nishimura M, Lee SJ, Stone RL, Bar-Eli M, Lopez-Berestein G, Sood AK: Chitosan hydrogel for localized gene silencing. Cancer Biol Ther. 2011, 11 (9): 839-845. 10.4161/cbt.11.9.15185.PubMedPubMed CentralView ArticleGoogle Scholar
- Han HD, Nam DE, Seo DH, Kim TW, Shin BC: Preparation and biodegradation of thermosensitive chitosan hydrogel as a function of pH and temperature. Macromol Res. 2004, 12: 507-511. 10.1007/BF03218435.View ArticleGoogle Scholar
- Zhang C, Wang B, Wang M: GM-CSF and IL-2 as adjuvant enhance the immune effect of protein vaccine against foot-and-mouth disease.Virol J 2011, 8:7.,Google Scholar
- Ullenhag GJ, Frodin JE, Strigard K, Mellstedt H, Magnusson CG: Induction of IgG subclass responses in colorectal carcinoma patients vaccinated with recombinant carcinoembryonic antigen. Cancer Res. 2002, 62 (5): 1364-1369.PubMedGoogle Scholar
- Disis ML, Bernhard H, Shiota FM, Hand SL, Gralow JR, Huseby ES, Gillis S, Cheever MA: Granulocyte-macrophage colony-stimulating factor: an effective adjuvant for protein and peptide-based vaccines. Blood. 1996, 88 (1): 202-210.PubMedGoogle Scholar
- Morrissey PJ, Bressler L, Park LS, Alpert A, Gillis S: Granulocyte-macrophage colony-stimulating factor augments the primary antibody response by enhancing the function of antigen-presenting cells. J Immunol. 1987, 139 (4): 1113-1119.PubMedGoogle Scholar
- Sun X, Hodge LM, Jones HP, Tabor L, Simecka JW: Co-expression of granulocyte-macrophage colony-stimulating factor with antigen enhances humoral and tumor immunity after DNA vaccination. Vaccine. 2002, 20 (9-10): 1466-1474. 10.1016/S0264-410X(01)00476-5.PubMedView ArticleGoogle Scholar
- Toubaji A, Hill S, Terabe M, Qian J, Floyd T, Simpson RM, Berzofsky JA, Khleif SN: The combination of GM-CSF and IL-2 as local adjuvant shows synergy in enhancing peptide vaccines and provides long term tumor protection. Vaccine. 2007, 25 (31): 5882-5891. 10.1016/j.vaccine.2007.05.040.PubMedView ArticleGoogle Scholar
- Reid CD, Stackpoole A, Meager A, Tikerpae J: Interactions of tumor necrosis factor with granulocyte-macrophage colony-stimulating factor and other cytokines in the regulation of dendritic cell growth in vitro from early bipotent CD34+ progenitors in human bone marrow. J Immunol. 1992, 149 (8): 2681-2688.PubMedGoogle Scholar
- Golumbek PT, Azhari R, Jaffee EM, Levitsky HI, Lazenby A, Leong K, Pardoll DM: Controlled release, biodegradable cytokine depots: a new approach in cancer vaccine design. Cancer Res. 1993, 53 (24): 5841-5844.PubMedGoogle Scholar
- Igartua M, Hernandez RM, Esquisabel A, Gascon AR, Calvo MB, Pedraz JL: Enhanced immune response after subcutaneous and oral immunization with biodegradable PLGA microspheres. J Control Release. 1998, 56 (1-3): 63-73. 10.1016/S0168-3659(98)00077-7.PubMedView ArticleGoogle Scholar
- Chesson CB, Huelsmann EJ, Lacek AT, Kohlhapp FJ, Webb MF, Nabatiyan A, Zloza A, Rudra JS: Antigenic peptide nanofibers elicit adjuvant-free CD8(+) T cell responses. Vaccine. 2014, 32 (10): 1174-1180. 10.1016/j.vaccine.2013.11.047.PubMedView ArticleGoogle Scholar
- Xu Y, Tang H, Liu JH, Wang H, Liu Y: Evaluation of the adjuvant effect of silver nanoparticles both in vitro and in vivo. Toxicol Lett. 2013, 219 (1): 42-48. 10.1016/j.toxlet.2013.02.010.PubMedView ArticleGoogle Scholar
- Vono M, Taccone M, Caccin P, Gallotta M, Donvito G, Falzoni S, Palmieri E, Pallaoro M, Rappuoli R, Di Virgilio F, De Gregorio E, Montecucco C, Seubert A: The adjuvant MF59 induces ATP release from muscle that potentiates response to vaccination. Proc Natl Acad Sci U S A. 2013, 110 (52): 21095-21100. 10.1073/pnas.1319784110.PubMedPubMed CentralView ArticleGoogle Scholar
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