Studies On The Preparation Of Curdlan-quaternized Chitosan Nanoparticles Used As A Nasal Mucosal Adjuvant For Subunit Vaccine Against The H1N1 Influenza Virus

Influenza,a kind of seasonal epidemic,seriously threatens human health due to that influenza virus is generally susceptible to all people,and can spread rapidly.Vaccination is an important method to prevent the spread of influenza virus all over the world.Currently,influenza vaccines administered subcutaneously or intramuscularly are poorly tolerated and have poor immunity.In order to increase the compliance and effectiveness of vaccination,people are trying to develop a nasal vaccination rout.Nasal vaccination is not only easy to operate and painless,but also simulates the pathway that the influenza virus infects the humans or animals and produces better and more comprehensive immune effects.However,due to the nasal mucosal barrier,it is difficult for the antigens of influenza virus to pass through the nasal mucosa and induce the immune responses.Therefore,people have to find new nasal mucosal adjuvants to help the influenza antigens to exert their immune effects.In our previous study ewe have found that the nanoparticles consisting of curdlan sulfate and 6-O-2-hydroxypropyl trimethyl ammonium chloride chitosan(OHTCC),CS/OHTCC nanoparticles,could activate antigen presenting cells(APCs),stimulate the expression of cytokines such as NO,TNF?,IL-6,and IL-1?,and cause cross-presentation.Nanoparticles loaded with OVA could induce the activation and presentation of APCs,promote T cell proliferation,and induce systemic immune response and mucosal immune response.It was proved that CS/OHTCC nanoparticles is a good candidate as immunoadjuvant.However,its application was limited because of its poor stability.Therefore,curdlan-OHTCC conjugate(C-O)was synthesized and the C-O nanoparticles were prepareds.Then the application of C-O nanoparticles as nasal mucosal vaccine adjuvant was studied by detecting the physical and chemical properties and evaluating the immune activities in vitro.Afterwards,the effects of C-O nanoparticles as a recombinant subunit vaccine adjuvant of H1N1 influenza virus was investigated.The main research contents include:1.The EDC/NHS reaction system was used to prepare C-O by optimizing the reaction conditions.The structure of C-O was identified by IR spectrum,1H NMR spectrum and elemental analysis.2.The C-O nanoparticles were prepared by ultrasonic method.The particle size and Zeta potential of the nanoparticles were detected by layer particle size analyzer,and the antigen loading efficiency and release in vitro were determined by BCA method.3.To evaluate the immune activity of C-O nanoparticles in vitro,the effects on the proliferation and phagocytosis of macrophages was detected as well as the secretion of inflammatory factors including NO,IL-6,and IL-1?.The expressions of CD40,CD80,CD86,MHC II and MHC 1 on the surface of dendritic cells were evaluated.At the same time,the proliferation of spleen lymphocytes was detected.4.The immune activity in vivo of C-O nanoparticles as a nasal mucosal adjuvant for influenza virus subunit vaccine was evaluated.The concentrations of H1N1 specific antibodies in the serum and mucosal lavage fluid of the immunized mice were detected by ELISA and the surface markers of immune cells were evaluated by flow cytometry(FCM).The results and conclusions are as follows:1.C-O was successfully prepared as a white flocculent solid with a yield of 75.1%,and the ratio of succinyl curdlan to OHTCC was 1:6.14.The preparation process is relatively simple,safe and low toxicity.2.The prepared C-O nanoparticles are uniform and spherical with a nean particle size of 190.53 nm and a Zeta potential of 15.93 mV.The C-O nanoparticles could be stably stored for about 6 weeks at 4?.Its OVA antigen carrier efficiency was 60.02%,and it could release OVA slowly resulting in the prolongation of antigen effect.So,C-O nanoparticles have the identity to carry negative charge materials such as the proteins3.In the experiment in vitro,C-O nanoparticles could promote the activation of APCs and stimulate the proliferation of spleen lymphocytes.It could promote the phagocytosis of macrophages,increase the secretion of the inflammatory factors such as NO,IL-6,and IL-1?,and enhance the expression of downstream COX-2.The expression of CD40,CD80,CD86 and MHC ? on the surface of DCs were up-regulated.It demonstrated that the activation and maturation of DCs was promoted.What is more,C-O nanoparticles could cause cross-presentation due to up-regulating the expression of MHC I.C-O nanoparticles also could stimulate spleen lymphocyte proliferation directly.It was proved that C-O nanoparticles could promote the activation of APCs and stimulate the proliferation of spleen lymphocytes.4.In the experiment in vivo,the C-O nanoparticles used as nasal mucosal adjuvant of H1N1 influenza vaccine could improve the immunogenicity of recombinant H1N1 HA,promote the activation of APCs and lymphocytes,and induce the production of H1N1 specificity antibodies.And the C-O nanoparticles were able to cause the secretion of sIgA antibodies in oral cavity and vaginal mucosa,which certify that C-O nanoparticles cause local mucosal immunization.