| Varicella-zoster virus(VZV)can cause two kinds of diseases.Chickenpox leads to centrally distributed papules and blisters which is common among children.Because of the neurotropism of VZV,it can be dormant in human dorsal root ganglion for a long time.The virus can be activated due to hypoimmunity resulting in the distribution of rashes like a band along innervated skins,and the above symptom is named as herpes zoster.The incidence of herpes zoster among the elderly is high.Complications after shingles such as ocular herpes zoster and post-herpetic neuralgia(PHN)bring long-term troubles to senior citizens.Due to lack of specific treatment for shingles,vaccination has become the best and necessary means to prevent shingles.Globally,herpes zoster vaccines are divided into attenuated vaccines and recombinant subunit vaccines.Although the former has a certain effect on the prevention of shingles,the effect will decrease significantly with age.The latter plays a good effect among all ages,which can maintain quite a long time.This subunit vaccine is composed of glycoprotein E,which is with strongest antigenicity and highest content in VZV,and AS01B adjuvant system.Although the vaccine has been marketed in China in 2019,no domestically developed recombinant subunit herpes zoster vaccine has been put into the market yet showing a huge prospect of subunit herpes zoster vaccine in the domestic market.Based on the effectiveness and importance of the existing subunit herpes zoster vaccines,we have designed three recombinant subunit vaccines.Meanwhile,the adjuvants of marketed herpes zoster vaccines are protected by patents,so this thesis plans to find another safe and effective adjuvant system leading to a high immune response and slight side effects providing experimental data for the development of subunit herpes zoster vaccines.Glycoprotein E contains B cell and T cell epitopes,which is an ideal antigen for preparing subunit vaccines.We synthesized the pc DNA3.4-gE-his plasmid which can express gE protein in the Chinese Hamster Ovary Cell(CHO)Expression System and expressed the VZV-gE protein using CHO Expression System.After nickel column purification,target protein with higher purity were harvested.In addition,considering the importance of cellular immunity in preventing the reactivation of VZV,we immunized mice with compound adjuvants and glycoprotein E which are expected to generate strong cellular immune response.Evaluating the safety of subunit vaccines by monitoring the weight and body temperature of mice.The intensity of immune responses of each subunit herpes zoster vaccines was evaluated in terms of antibody level,antibody isotyping,cytokine secretion,IFN-γ~+T cells frequency.The results show that(1)Each subunit herpes zoster vaccine is safe within the scope of the experiment and the adverse effects on animals are weak and reversible;(2)On the 14 days after second immunization,MF59 adjuvant,the adjuvant composed of MF59 and QS-21 or CpG 2006 can induce higher antibody levels which has no significant differences with the positive control group;MF59 adjuvant can induce humoral immune response,while MF59/QS-21 and MF59/CpG 2006 enhance cellular-mediated immune responses,which was similar to the positive control adjuvant.(3)The results of Th1/Th2 cytokine detection shows that compared with the MF59adjuvant alone,the addition of CpG 2006 increases the secretion of IL-2 and QS-21adjuvant increases the secretion of IL-4 cytokines.Both CpG 2006 and QS-21 adjuvants can significantly increase the secretion of IFN-γand IL-10 cytokines;(4)Similarly,the frequency of IFN-γpositive cells at the single-cell level shows the same results.The combination of MF59 and QS-21 or CpG 2006 can induce higher frequency of IFN-γ~+T cells.To sum up,the results show that the MF59 and QS-21 or MF59 and CpG 2006adjuvant can be used as an ideal candidate adjuvant for recombinant subunit herpes zoster vaccines.It is promising that these herpes zoster vaccines are likely to be put into the subsequent production and application. |
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