The Adjuvant Effect Of Inulin In The Mycobacterium Tuberculosis And Zika Virus Subunit Vaccines

Adjuvants have become an indispensable part of subunit vaccines and an important factor to limit the development of subunit vaccines.A few adjuvants are currently used with different degrees of adverse reactions.Therefore,it is urgent to develop more safe and effective adjuvants for subunit vaccines.As a kind of plant polysaccharide with immunomodulatory activity,inulin has a wide range of sources with high safety and low price.Therefore,inulin has a good potential as the adjuvant of subunit vaccines.In order to study the adjuvant effect of inulin on subunit protein vaccines,inulin was chemically modified with Mycobacterium tuberculosis and Zika virus protein antigens.The adjuvant effects of inulin on the bacterial vaccine and virus vaccines were investigated.At present,adjuvants mainly load antigens by the method of physical mixing,according to the principle of electrostatic adsorption or microsphere encapsulation.However,since inulin is electrically neutral,it cannot directly absorb the antigens,and the addition of materials to encapsulate inulin and antigens may cause non-specific immune response and safety problems.In order to overcome the problems caused by the above traditional methods,this paper adopts the chemical modification method to achieve the same APCs after the covalent binding of antigen and adjuvant,so as to enhance the antigen uptake of DC cells and stimulate the body to produce a stronger immune response.As a new antigen delivery system,covalently conjugation of inulin with the protein antigen could form a subunit vaccine with a prolonged half-life.The interaction mechanism between inulin and the immune system was explored.The adjuvant effect of inulin on the vaccine was studied.The main research results are as follows:?1?Inulin shows great adjuvant effect in mycobacterium tuberculosis subunit protein vaccineMycobacterium tuberculosis CFP10-TB10.4 fusion protein?CT?was expressed and purified as an antigen.Inulin was first covalently conjugated with CRM₁₉₇.The conjugate?CRM-inu?was then conjugated with CT to form a subunit vaccine.The immunogenicity and pharmacokinetic characteristics of the subunit vaccine were investigated.The CT-specific IgG antibody titers of the vaccine group was significantly high,where the BALB/c mice were used as the model animals.As compared with the CT group on days14,21 and 28,the vaccine group showed 11.0-,8.6-and 20.6-fold?P<0.05?increases in the CT-specific IgG titers,respectively.As compared with the CT group,the expression of Th1-type cytokine levels?IFN-?and TNF-??of the vaccine group showed 24.1-and 2.4-fold increases?P<0.05?,respectively.In addition,the Th2-type cytokines?IL-5 and IL-10?of the vaccine group showed 7.8-and 3.5-fold increases?P<0.05?.This suggested that conjugation with CRM-inu significantly increased the CT-specific humoral and cellular immune response through the synergistic adjuvant interaction of inulin and CRM₁₉₇.Importantly,studies revealed that anti-inulin antibodies were not detected in the serum samples of mice,and inulin has a great adjuvant potential with extremely low immunogenicity.Moreover,the vaccine showed no apparent toxicity to cardiac,hepatic and renal functions.To further explore the adjuvant mechanism of inulin,the pharmacokinetic characteristics of the vaccine were studied in the SD rats.The results showed that the plasma half-life of CT protein was increased from 7.0±0.2 h to 26.4±0.1 h by conjugation of inulin-CRM.As compared with the CT group,the AUC_0-144-144 of the vaccine group increased from 1228.5±323.5 g mL^-11 h^-1 to 7284.4±535.1 g mL^-11 h^-1.The clearance rate of bioavailability?Cl/F?decreased from 0.32±0.02 g/g mL^-11 h^-1 to 0.15±0.02 g/g mL^-11 h^-1.Therefore,inulin-CRM conjugation can significantly prolong the exposure time of CT in the immune system,improve the absorption and utilization of drugs,and reduce the serum clearance rate of CT,to induce a more intense CT-specific immune response.?2?Inulin was efficient as an adjuvant of subunit protein E of zika virus vaccineZika virus?ZIKV?envelope protein?E protein?was expressed and purified.In this paper,TLR7 receptor agonist Loxoribine?Lox?was selected to modify E protein together with inulin to form a novel E protein subunit vaccine,which integrates immune stimulation molecule and antigen delivery system.The immunogenicity and the immune mechanism of the vaccine were studied.Here,an antigen-adjuvant delivery system was developed by conjugation of E protein with loxoribine and inulin.In this vaccine,loxoribine and inulin function as the adjuvants to induce a strong E protein-specific antibody titers and T cell responses.As compared with the E group on days 14,21 and 28,the Lox-E-inu vaccine group showed 4.0-,9.5-and 25.0-fold?P<0.05?increases in the E-specific IgG titers,respectively.The levels of Th1-type cytokines?IFN-?,TNF-?and IL-2?and Th2-type cytokine?IL-10?of the vaccine group showed 2.8-,1.5-,1.9-and 1.8-fold increases?P<0.05?,as compare to the E protein group alone.The number of CD4⁺T and CD8⁺T subsets increased,whereas the CD4⁺T/CD8⁺T ratio did not change significantly.In addition,no anti-inulin antibodies were detected in the serum samples,indicating that inulin has a great adjuvant potential with extremely low immunogenicity.Loxoribine-inulin conjugated protein is capable of significantly increasing antigen uptake of DC.In addition,the vaccine showed no apparent toxicity to cardiac,hepatic and renal functions.These demonstrate that conjugation of E protein with loxoribine-inulin is an effective strategy for induction of strong humoral and cellular immunities.Its main mechanism is to improve the antigen presentation efficiency of DC cells by promoting the maturation of DC cells.