Studies On Vaccine Adjuvants To Promote Protein Antigen-Specific CD8~+CTL Response

CD8⁺CTL response play a critical role in immunotherapies against tumors and chronicinfections. Protein or peptides can induce effective CD8⁺CTL response throughcross-presentation and cross-priming by engagement of adjuvants. Preliminary resultsshowed that C57BL/6mice immunized intramuscularly with chicken ovalbumin (OVA) as amodel antigen and Al(OH)₃+CpG-X1as a composite adjuvant can induce effective CD8⁺CTL response. In the present study, IL-2, a T cell growth factor, was used as an adjuvant onthe basis of Al(OH)₃+CpG-X1to further enhance the effect on CD8⁺CTL responses throughcross-presentation and cross-priming of protein antigens.C57BL/6mice were immunized intramuscularly with OVA as a model antigen, andAl(OH)₃+CpG-X1as a composite adjuvant. Low-dose IL-2was injected at different time pointsbetween4to6days after immunization. The intracellular cytokine staining assay showed thatIL-2can further enhance CD8⁺CTL response through cross-presentation and cross-primingof protein antigens on the basis of Al(OH)₃+CpG-X1composite adjuvant. In addition, theinjection time, doses and dosage of IL-2were optimized after priming and boostingimmunization. The results showed that the optimal injection condition for IL-2was daily10,000IU/injection between5to7days after priming and2to4days after boosting.To determine the necessity of Al(OH)₃+CpG-X1+IL-2as a composite adjuvant,C57BL/6mice were immunized intramuscularly with OVA as a model antigen andAl(OH)₃+CpG-X1as a composite adjuvant, and IL-2was administered as mentioned above.The intracellular cytokine staining assay was used to detect specific CD8⁺CTL response. Theresults showed that the adjuvant effect of Al (OH)3+CpG-X1+IL-2was the strongest,followed by CpG-X1+IL-2, and Al(OH)₃+IL-2was the weakest.Preliminary results showed that both CpG-X1and Al(OH)₃+CpG-X1can promote CD8⁺CTL responses through cross-presentation and cross-priming. However, in this study, the resultsdemonstrated that both CpG-X1+IL-2and Al (OH)3+CpG-X1+IL-2can also be effective inpromoting protein antigen through cross-presentation and cross-priming to induce CD8⁺CTL response. Therefore, the effect of the four adjuvants on inducing CD8⁺CTL responsewere comprehensively compared using intracellular cytokine staining assay and in vivo CTL killing assay. The results showed that the adjuvant effect of Al (OH)3+CpG-X1+IL-2wasthe strongest,followed by CpG-X1+IL-2, CpG-X1and Al(OH)₃+CpG-X1were the weakest.To study the in vivo tumor treatment effect of CD8⁺CTL response induced by differentadjuvants through cross-presentation and cross-priming, C57BL/6mice were inoculated withMO5melanoma or EG7thymoma. When the tumors were visible5-7days after inoculation,the mice were immunized intramuscularly with OVA, OVA+Al(OH)₃, OVA+CpG-X1andOVA+Al(OH)₃+CpG-X1respectively, and part of the mice were administered with low doseIL-25-7days after immunization. The results of tumor growth showed thatAl(OH)₃+CpG-X1+IL-2, CpG-X1+IL-2, Al(OH)₃+CpG-X1or CpG-X1as adjuvants cansubstantially inhibit tumor growth and prolong mice survival time, compared with the othergroups.In conclusion, IL-2can further promote CD8⁺CTL response through cross-presentation andcross-priming of protein antigens on the basis of Al(OH)₃+CpG-X1composite adjuvant.These results provide the adjuvant basis for the research and development of therapeuticvaccines against tumors and chronic infections.