| Vaccines play an important role in preventing various infectious diseases.With the development of biotechnology,live attenuated,inactivated and subunit vaccines have effectively improved the purity and safety of vaccines.However,its single component or live attenuated component reduces the immune activity,which requires the addition of adjuvants to achieve protective immune response.As an important component of vaccines,the demand for adjuvants is constantly increasing.A single adjuvant is sometimes not enough to support the vaccine to produce a sustained and protective immune response.For example,aluminum adjuvant approved by the FDA mainly induces antibody-mediated Th2 immune responses.The lack of Th1 responses limit its longer-lasting cellular immunity.Especially in the development of vaccines such as SARS and MERS,it has been demonstrated that a platform with T cell-mediated cellular immune activation and antibody response coordination will more effectively prevent the occurrence of diseases.Therefore,it is very necessary to develop an adjuvant platform with coordinated regulation.It is known that,CpG ODN adjuvant,which mainly induce Th1 immune respomses,could produce a synergistic effect when combined with aluminum adjuvants to promote cellular immune response.But the current research on the compound method and immune mechanism is still insufficient.Therefore,this study constructed a combination adjuvant containing both CpG ODN and aluminum oxyhydroxide nanoparticles,with the exposure of different ends of CpG to explore the dufferences in the activation of immune responses.For this,a variety of experiments in vivo and in vitro were performed to evaluate the adjuvant effect,and established the structure-activity relationship of the combination adjuvant at the molecular level to guide the development of related vaccines.The specific research content is as follows:First of all,the combination adjuvant was analyzed using various characterization methods such as Zeta PALS instrumen,transmission electron microscope,fourier infrared spectroscopy,etc.,and the results showed that the combination adjuvant was successfully conjugated.The positive（40 m V）changes obviously to the negative（-30 m V）,and the conjugation of small molecules does not change the physical morphology of the nanorods,with a good conjugation efficiency.Secondly,the toxicity and immune activation in vitro was explored,and the results showed that the combination adjuvants did not affect cell viability,and effectively increased the activation of TLR9.They stimulated the activation of antigen presenting cells and the adjuvant endocytosis,and promoted the secretion of cytokines for immune regulation.Interestingly,combination adjuvants showed different immune efficacies,and 5’-CpG-free has a better immune activation effect.At the same time,mice injected with5’-CpG-free intraperitoneally caused more immune cell recruitment and higher cytokine secretion.Moreover,the S-RBD protein was used as the immunogen to evaluate the systemic immune response.The results showed that compared with the pristine Al OOH,the combination adjuvants induced higher Ig G titers and the highest in 5’-CpG-free-treated mice,which promoted the secretion of Ig G2a,and presented a Th1 type immune tendency.In addition,combination adjuvants,especially 5’-CpG-free,significantly enhanced the secretion of IFN-γand IL-4 in splenocytes,and promoted cellular immune responses.At the same time,a higher expression level of CTL principal killing mediators and effector memory T cells were activated,indicating that 5’-CpG-free combination adjuvant has a balanced immune enhancement effect.In summary,this study has successfully constructed a combination adjuvant platform of aluminum oxyhydroxide and CpG ODN,then fully evaluated the immune efficacy in vivo and in vitro,and explored the relationship between the conjugation method and the immune effect,thereby contributing to the development of vaccines with both Th1 and Th2 responses. |
