Immunomodulatory Mechanisms Of Chitosan And Its Derivatives And Its Application As Adjuvant In Newcastle Disease Vaccine

Immune adjuvants are substances which promote the potency of antigen-specific immune responses.When added to vaccines,they can delay antigen degradation,enhance immune intensity and reduce the amount of antigen.Furthermore,adjuvants may enhance the ability of antigen presenting cells to process and present antigens,stimulate lymphocyte proliferation and differentiation,or promote the release of cytokines.Therefore,adjuvants have a broad market prospect in vaccines.An ideal immune adjuvant is supposed to produce an efficient immune response at a low dosage without side effects,and is easy to be produced and be applied.The existing veterinary adjuvants are increasingly unable to meet market requirements due to disadvantages such as difficulty in metabolism along with serious adverse reactions,thus it is urgent to develop new adjuvants with no toxic side effects.Chitosan is a natural polysaccharide with certain immunological activity,it is biocompatible,non-toxic and rich in resources,but its low solubility in neutral environment limits its application,while its derivatives such as quaternized chitosan and sulfated chitosan showed better water solubility.Therefore,in this article,we prepared ?-and ?-chitosan with different molecular weights and the water-soluble derivatives – sulfated chitosan and quaternized chitosan.Then the infrared spectrum,nuclear magnetic resonance(NMR)one-dimensional and two-dimensional spectrum,high performance liquid chromatography(HPLC)were determined to characterize the derivatives.Finally,the immunostimulatory effects of the derivatives were compared and the screened derivative with highest effect were used to prepare antigen-loaded nanoparticles to develop new types of adjuvant with immune stimulation and antigen delivery function.The main results are as follows:1)?-and ?-chitosan,chitosan sulfates and quaternized chitosan with different molecular weights were successfully prepared.RAW 264.7 cells were used as a model for in vitro to study the immune mechanisms of the derivatives.Firstly,through the test of nitric oxide production and cytotoxicity of cells,it was found that the immune effect of ?-chitosan is better than that of ?-chitosan,the immunological effects of chitosan sulfate and quaternized chitosan are better than low molecular weight chitosan,and ?-quaternized chitosan showed the best activity.Quantitative RT-PCR,flow cytometry and western blot were used to study the immune mechanisms of three kinds of samples.The results showed that 100 ?g/mL chitosan sulfate could activate the PI3K-Akt pathway in RAW 264.7 cells and promote the translocation of NF-?B and AP-1 into the nucleus,thereby increasing the expression of cytokines such as TNF-?.100 ?g/mL chitooligosaccharide can activate the MAPK and PI3K/Akt pathways in RAW 264.7 cells,and promote the entry of NF-?B into the nucleus,thereby increasing the expression of IL-6 and other cytokines.The quaternized chitosan at 50 ?g/mL can activate the MAPK,JAK/STAT and PI3K/Akt pathways in RAW 264.7 cells,and promote the the translocation of NF-?B and AP-1 into the nucleus,thereby increasing the expression of IL-6 and other cytokines.2)Newcastle disease(ND)is an avian infectious disease caused by Newcastle disease virus(NDV)infection.ND has caused great losses to the poultry industry since it was first discovered in 1926.Therefore,it has attracted much attention and is now mainly prevented by vaccination.Nano-drug delivery system can prolong the drug release time and achieve a sustained release effect.As an adjuvant,it can release antigens slowly and thus reduce the usage amount of antigen.In this study,NDV was used as the antigen,and NDV-loaded nanoparticles were prepared by ion-crosslinking method using quaternized chitosan and chitosan as adjuvants.The average particle size of the nanoparticles was 320±0.84 nm,the polydispersity index was 0.36±0.02,the zeta potential was +18.3±0.5 mV,and the encapsulation efficiency and drug loading were 37.4±2.69% and 16.16±1.84%,respectively.Scanning electron microscopy(SEM)results showed that the nanoparticles were almost spherical,with smooth surface and no obvious aggregation,and the particle size was similar to that of the particle size meter,which further proved the successful preparation of nanoparticles.In vitro MTT assay and in vivo animal experiments showed that the nanoparticles had no obvious cytotoxicity.Then the NDV-loaded nanoparticles were used to immunize chickens.It was found that the humoral immunity level of the nanoparticle immunized group was slightly lower than that of the oil emulsion vaccine group,but its cellular immunity level was significantly higher than that of the white oil group,and the protection rate of nanoparticle immunized chickens could reach 100%.In summary,chitosan,chitosan sulfate and quaternized chitosan can activate the signaling pathways of RAW 264.7 cells and regulate the immune response.Quaternized chitosan nanoparticles as adjuvants for Newcastle disease vaccine can significantly enhance humoral and cellular immunity of chickens,protect immune chickens from Newcastle disease virus infection,and provide a basis for the development of quaternized chitosan/chitosan as new vaccine adjuvants.