Alumina Microemulsion As A New Pulmonary Mucosal Vaccine Adjuvant

Objective: Nano-aluminum oxide(AN),pharmaceutical grade Tween80 and squalene were used to construct an alumina microemulsion(ANME)as a new pulmonary mucosal vaccine adjuvant.The ANME was characterized by various methods and its safety was tested.After clarifying the immune potential of ANME in vitro,We used ovalbumin(OVA)as a model antigen to construct a new lung vaccine,and investigated the immune efficacy of the vaccine in mice by lung inhalation,and systematically evaluated the potential of the vaccine to induce cellular immunity,humoral immunity and mucosal immunity.Methods:(1)The emulsion was prepared by ultrasonic method and the prescription was optimized,the optimum preparation conditions were selected,and the stable ANME was prepared.The particle size,PDI and ζ potential of the emulsion were detected by Malvin particle size,and the morphology of the sample was observed by transmission electron microscope and Laser confocal microscope(LSCM).The cell safety of the emulsion was detected by MTT assay.Flow cytometry(FACS)was used to detect the uptake of ANME by antigen-presenting cells(APCs)and the activation of APCs by vaccine formulation.The relative intake of different vaccine adjuvants by APCs and the presence of lysosome escape were observed by LSCM.(2)KM mice were immunized by pulmonary inhalation(p.i.)with two different vaccine formulations,AN-OVA and ANME-OVA,as well as a negative control group(mice were not treated)and a positive control group(MF59-OVA).The mice were immunized again at 6 weeks after the first immunization.After immunizing mice,orbital blood was collected,supernatant was collected by centrifugation,and OVA specific Ig G,Ig G1 and Ig G2 a antibodies in mouse serum were detected by enzyme-related immunosorbent assay(ELISA)IL-4,IFN-γ levels;Oral flushing solution,nasal flushing solution,vaginal flushing solution and alveolar lavage solution were collected at the 4th week after secondary immunization,and s Ig A secreted by mucous membrane was detected by ELISA.At the fourth week of secondary immunization,spleen cells were extracted aseptically from each group,and spleen cell typing was detected by FACS and proliferation was detected by MTT method.Results:(1)After prescription optimization,3 mg/m L ANs,0.8% Tween80 and 10%(V/V)squalene were selected to construct oil-in-water alumina microemulsion(ANME).The average particle size of ANME was 279.76 ± 13.99 nm,and the ζ potential was-9 ±1.06 m V.ANs was adsorbed on the oil-water interface of the emulsion,and the ANME was spherical and uniformly dispersed with good safety.(2)APCs have a higher intake of ANME,and there is an obvious lysosome escape phenomenon.ANME loaded with antigen can significantly promote the expression of CD40 and CD86 on the surface of BMDCs and stimulate the maturation of BMDCs.(3)After loading OVA with ANME,OVA specific Ig G level in serum and s Ig A expression in oral,vaginal and pulmonary mucosa were significantly increased.Conclusions:(1)This study successfully prepared ANME with good stability and safety,which can be used as an effective immune adjust-delivery system(VADS).(2)After loading antigen with immune adjuvant,lung inoculation can induce the body to produce strong and balanced mucosal immunity,humoral immunity and cellular immunity,with triple immune response.