【作者】 王哲；

【导师】 于永利；

【作者基本信息】 吉林大学 ， 免疫学， 2022， 硕士

【摘要】 疫苗佐剂是指能非特异性地提高机体对疫苗特异性免疫应答的一类物质。目前主要的人用疫苗佐剂为铝佐剂、水包油乳化剂（MF59和AS03）、混合佐剂（AS04和AS01）和CpG ODN。铝佐剂作为最为常用的疫苗佐剂,自上世纪20年代以来,就被用于制备破伤风疫苗和白喉类毒素疫苗。直至上世纪90年代,新的疫苗佐剂—水包油乳化剂MF59才获批作为老年流感疫苗佐剂投入使用,克服了铝盐不能快速激起体内免疫应答的问题。随后相继批准了AS03佐剂用于禽流感疫苗佐剂以及AS04作为人乳头瘤病毒疫苗佐剂。2017年,TLR9激动剂CpG ODN因其作为新型重组乙肝疫苗佐剂能产生良好的免疫增效作用而获批使用。但其同时与铝佐剂进行配伍时存在不能快速激起免疫应答的问题,而与水包油乳化剂进行配伍时不能产生持久的免疫效应以长期抵御病原微生物的入侵。CpG ODN通过TLR9通路激活B细胞的同时,免疫抑制性信号也随之产生。因此,推测若将CpG ODN与免疫抑制性分子的反义寡核苷酸联合应用作为疫苗佐剂,通过刺激免疫激活信号并抑制免疫激活后产生的免疫抑制信号,可能使机体在疫苗接种后产生持久免疫应答。此外,从理论上说,体内外来源的危险信号分子均具有成为疫苗佐剂的潜力。那么,一些来源于细胞生命活动过程中产生的代谢产物是否也可以作为疫苗佐剂是一个值得研究探索的问题。病毒疫苗的种类很多,包括减毒活疫苗、灭活疫苗、亚单位疫苗等。亚单位疫苗的抗原成分单一、免疫原性较差,相较于灭活疫苗和减毒活疫苗更加需要佐剂的参与以提高其疫苗效应。因此,本研究选取两种病毒抗原重组蛋白,即SARS-CoV-2重组S蛋白（S蛋白）和猪圆环病毒重组PCV-2b衣壳蛋白（ΔCP蛋白）,作为亚单位疫苗抗原,并将其分别与水包油佐剂配伍,制备成亚单位疫苗（SV和PV）,以此探究肠道代谢产物丁酸的模拟化合物丁酸钠和CpG ODN联合免疫抑制性分子的反义寡核苷酸作为重组亚单位疫苗佐剂时的免疫增效作用,具体内容如下:1.丁酸钠作为重组亚单位疫苗佐剂的初探丁酸是一种在结肠中经厌氧菌发酵膳食纤维而形成的四碳短链脂肪酸,其化合物丁酸钠作为去乙酰化酶抑制剂能显著增强巨噬细胞的抗菌活性且促进记忆性CD8⁺T细胞的产生,推测其具有作为疫苗佐剂的潜力。1.1丁酸钠作为疫苗佐剂的探究为检测丁酸钠能否在重组亚单位疫苗中发挥佐剂效应,本部分实验将丁酸钠以不同的剂量与重组亚单位疫苗PV混合并制备成新的亚单位疫苗于0、14天免疫小鼠,二免后7、28天进行小鼠尾静脉采血,并通过ELISA检测血清中的抗体水平。实验结果表明,丁酸钠作为重组蛋白疫苗佐剂,不仅不能提高抗体产生水平,反而明显抑制了抗体的产生,且在浓度为1000μg时对抗体产生的抑制效果最为明显,提示丁酸钠不能作为疫苗佐剂。为了探究丁酸钠不能对疫苗起增效作用是否因其对小鼠免疫系统有抑制作用,将上述各组小鼠经腹腔注射大肠杆菌（E.coli）（浓度为0.8×10⁸ CFUs/m L）,观察小鼠的生存期。结果表明,含丁酸钠疫苗免疫的小鼠生存期明显相比不含丁酸钠的对照组小鼠生存期更短,提示丁酸钠与疫苗配伍应用可能在小鼠体内发挥了免疫抑制作用,导致小鼠抵御大肠杆菌感染的能力下降。1.2丁酸钠体外对B细胞活化的影响为了探究丁酸钠不能作为疫苗佐剂增效疫苗效应的可能机制,我们首先用丁酸钠作用于小鼠脾细胞,检测B淋巴细胞活化情况;随后将丁酸钠和CpG ODN联合作用于小鼠脾细胞,检测丁酸钠对CpG ODN诱导B细胞活化的抑制作用。结果表明丁酸钠能显著抑制脾细胞中CD19⁺B细胞表面CD40⁺/CD86⁺的表达,且抑制作用与丁酸钠剂量在一定范围内呈正相关,提示丁酸钠对B细胞具有免疫抑制作用;CpG ODN可以使B细胞中CD19^high细胞的比例显著升高,而丁酸钠的作用则体现为显著下调由CpG ODN上调的CD19^high细胞比例,表明丁酸钠可抑制被CpG ODN活化的B细胞。综上,通过体内外实验证明,丁酸钠单独应用或作为疫苗佐剂均发挥了免疫抑制作用,其对B细胞活化的抑制可能使其不能发挥疫苗佐剂的免疫增效作用。本研究为丁酸钠作为免疫抑制剂提供了实验证据。2.CpG ODN联合免疫抑制分子反义寡核苷酸作为重组亚单位疫苗佐剂的研究本实验室一直致力于CpG ODN的研究,CpG5805是本实验室自行设计的CpG ODN,作为疫苗佐剂具有良好的免疫增效作用,但在免疫持续期延长方面还有进一步优化的空间。考虑到免疫激活后可产生免疫抑制性分子,因此探究CpG ODN与免疫抑制性分子反义寡核苷酸联合作为疫苗佐剂是否能更好地发挥免疫增效作用,选择实验室自行设计的两种免疫抑制性分子反义寡核苷酸CMD-1与TIO3分别与CpG5805联合作为疫苗佐剂,与SV亚单位疫苗配伍应用。CMD-1是靶向免疫细胞表面抑制性分子CTLA-4的反义寡核苷酸,其作为重组假病毒颗粒疫苗佐剂能显著增强抗体的产生。TIO3是靶向免疫抑制性细胞因子TGF-β2 m RNA3’UTR的反义寡脱氧核苷酸,其作为微生物疫苗佐剂具有促进抗体水平升高的作用。2.1探究CpG5805与CMD-1联合作为疫苗佐剂的效果将小鼠分为五组进行免疫,即CpG5805、CMD-1和CpG5805+CMD-1分别与SV混合（C+SV、M+SV、C+M+SV）及SV单独,单独S蛋白作为空白对照（S）。于第0、14天免疫小鼠,于二免后第7、14、28天进行小鼠尾静脉采血,通过ELISA检测血清中抗SARS-CoV-2-S蛋白的抗体水平。结果表明,C+M+SV组小鼠与SV组相比,抗体产生水平有升高趋势,但与C+SV组的抗体产生水平基本一致;C+SV组与SV组相比小鼠抗体产生水平升高,而M+SV组小鼠的抗体水平则无明显变化。实验结果表明CpG5805联合CMD-1作为疫苗佐剂时,其效果并不好于单独应用CpG5805作为疫苗佐剂,因此,二者联合使用不适用于SARS-CoV-2重组S蛋白疫苗佐剂进行使用。2.2探究CpG5805与TIO3联合作为疫苗佐剂的效果将CpG5805、TIO3单独和CpG5805+TIO3与SV混合制备成重组蛋白疫苗（C+SV、T+SV、C+T+SV）及SV单独,于0、14天免疫小鼠,于二免后7、14、28天进行小鼠尾静脉采血,通过ELISA检测血清中的抗体水平。实验结果表明,C+T+SV组与对照组SV相比,其抗体产生水平显著升高,抗体产生水平提高一倍左右,且抗体效价显著高于单独应用CpG5805/TIO3组。T+SV组与SV组相比其抗体产生水平也具有上调趋势。因此CpG5805联合TIO3明显优于单独应用CpG5805的免疫效果,具有作为SARS-CoV-2重组S蛋白疫苗佐剂的潜力。随后进一步探究CpG5805联合TIO3作为疫苗佐剂的细胞活化情况:免疫小鼠后48 h,检测小鼠脾细胞内的B细胞活化情况,实验结果表明CpG5805联合TIO3能显著提高小鼠脾细胞内CD19⁺CD86⁺细胞的活化比例,且与对照组SV相比,B细胞活化比例上调了1/3,同时上调了MFI的水平。表明CPG5805联合TIO3作为SARS-CoV-2重组S蛋白疫苗佐剂能显著增强B细胞的活化,促进抗体产生,有作为SARS-CoV-2重组S蛋白疫苗佐剂的潜力。本研究对重组亚单位疫苗佐剂进行了初步筛选,证明了丁酸钠不适用于重组亚单位疫苗佐剂,其不仅不能有效提高抗体产生水平反而对抗体的产生具有免疫抑制作用,且能明显抑制B细胞活化。同时证实了CPG5805与TIO3联合作为SARS-CoV-2的重组S蛋白疫苗佐剂能显著增强抗体产生水平,具有成为新型疫苗佐剂的潜力,为新冠疫苗佐剂的研发提供了理论基础。更多还原

【Abstract】 Vaccine adjuvants refer to a class of substances that can non-specifically improve the specific immune response to vaccines.Currently,the main human vaccine adjuvants are aluminum adjuvant,oil-in-water emulsifier（MF59 and AS03）,mixed adjuvant（AS04 and AS01）and CpG ODN.As the most commonly used vaccine adjuvants,Aluminum adjuvants have been used since the 1920s to prepare tetanus and diphtheria toxoid vaccines.It was not until the 1990s that a new vaccine adjuvant,MF59,was approved for use as an adjuvant for influenza vaccine in the elderly,overcoming the problem that aluminum salts could not quickly stimulate the immune response in vivo.Subsequently,AS03 adjuvant was approved as an adjuvant for avian influenza vaccine and AS04 as an adjuvant for human papillomavirus vaccine.In 2017,TLR9 agonist CpG ODN was approved for use as a novel recombinant hepatitis B vaccine adjuvant,which produced a good immunoenhancing effect.However,when it was compatible with aluminum adjuvant,it could not quickly stimulate the immune response,and when it was compatible with oil-in-water emulsifier,it could not produce a lasting immune effect to resist the invasion of pathogenic microorganisms.When CpG ODN activates B cells through TLR9 pathway,immunosuppressive signals are also generated.Therefore,it is speculated that CpG ODN combined with antisense oligonucleotides of immunosuppressive molecules as a vaccine adjuvant may induce a lasting immune response after vaccination by stimulating immune activation signals and inhibiting the immunosuppressive signals generated after immune activation.Furthermore,in theory,danger signaling molecules from both in vivo and in vitro sources may have potential as vaccine adjuvants.Therefore,whether some metabolites produced in the process of cell life can also be used as vaccine adjuvants is a question worthy of study and exploration.There are many types of viral vaccines,including live attenuated vaccines,inactivated vaccines,subunit vaccines,etc.Compared with inactivated vaccine and live attenuated vaccine,the subunit vaccine requires more adjuvants to improve its vaccine effect due to its single antigen component and poor immunogenicity.Therefore,in this study,two recombinant viral antigens,namely SARS-CoV-2 recombinant S protein（S protein）and porcine circovirus recombinant PCV-2b capsid protein（δCP protein）,were selected as subunit vaccine antigens and combined with oil-in-water adjuvants respectively to prepare subunit vaccines（SV and PV）.To explore the immune synergistic effect of sodium butyrate,a simulated compound of intestinal metabolite butyric acid,and CpG ODN combined with antisense oligonucleotide of immunosuppressive molecule as adjuvant of recombinant subunit vaccine,the specific contents are as follows:1.Sodium butyrate as adjuvant of recombinant subunit vaccineButyric acid is a four-carbon short-chain fatty acid formed by dietary fiber fermentation by anaerobic bacteria in the colon.The compound sodium butyrate,as a deacetylase inhibitor,can significantly enhance the antibacterial activity of macrophages and promote the production of memory CD8⁺T cells,suggesting that it has the potential as a vaccine adjuvant.1.1 Study on sodium butyrate as vaccine adjuvantTo test whether sodium butyrate can play an adjuvant effect in recombinant subunit vaccine,sodium butyrate was mixed with the recombinant subunit vaccine PV in different doses to prepare a new subunit vaccine.Mice were immunized at day 0 and14,and tail vein blood samples were collected at day 7 and 28 after the second immunization,and serum antibody levels were detected by ELISA.The results showed that sodium butyrate,as the adjuvant of recombinant protein vaccine,not only could not improve the level of antibody production,but significantly inhibited the production of antibody,and the inhibition effect was the most obvious when the concentration was1000μg,suggesting that sodium butyrate should not be used as vaccine adjuvant.To investigate whether sodium butyrate was not synergistic to the vaccine because it inhibited the immune system of mice,E.coli（0.8×10⁸ CFUs/m L）was intraperitoneally injected into the mice in the above groups,and the survival of the mice was observed.The results showed that the survival time of mice immunized with sodium butyrate vaccine was significantly shorter than that of control mice without sodium butyrate vaccine,suggesting that the combination of sodium butyrate vaccine and sodium butyrate vaccine may play an immunosuppressive role in mice,leading to a decreased ability to resist E.coli infection in mice.1.2 Effect of sodium butyrate on B cell activation in vitroIn order to explore the possible mechanism of sodium butyrate not being used as vaccine adjuvant to enhance vaccine effect,we first treated mouse spleen cells with sodium butyrate to detect the activation of B lymphocytes.Then the spleen cells of mice were treated with sodium butyrate and CpG ODN to detect the inhibitory effect of sodium butyrate on CpG ODN induced B cell activation.The results showed that sodium butyrate could significantly inhibit the expression of CD40⁺/CD86⁺on the surface of CD19⁺B cells in spleen cells,and the inhibitory effect was positively correlated with the dose of sodium butyrate in a certain range,suggesting that sodium butyrate had immunosuppressive effect on B cells.CpG ODN could significantly increase the proportion of CD19^high in B cells,while sodium butyrate significantly down-regulated the proportion of CD19^high up-regulated by CpG ODN,suggesting that sodium butyrate could inhibit the B cells activated by CpG ODN.In conclusion,in vivo and in vitro experiments have proved that sodium butyrate can exert immunosuppressive effect either alone or as a vaccine adjuvant,and its inhibition of B cell activation may make it unable to exert immunoenhancing effect of vaccine adjuvant.This study provides experimental evidence for sodium butyrate as an immunosuppressant.2.CpG ODN combined with immunosuppressive molecular antisense oligonucleotide as recombinant subunit vaccine adjuvantOur laboratory has been committed to the study of CpG ODN.CpG5805 is a CpG ODN designed by our laboratory.As a vaccine adjuvant,CpG ODN has a good immunosynergistic effect,but it still needs to be further optimized in prolonging the duration of immunization.Considering that immunosuppressive molecules can be generated after immune activation,in order to explore whether the combination of CpG ODN and immunosuppressive molecule antisense oligonucleotide can play a better immunosynergistic effect as a vaccine adjuvant,we selected CMD-1 and TIO3,two immunosuppressive molecular antisense oligonucleotides designed by our laboratory,to be combined with CpG5805,respectively,as vaccine adjuvants for compatibility with SV subunit vaccine.CMD-1 is an antisense oligonucleotide targeting the inhibitory molecule CTLA-4 on the surface of immune cells,and previous work in our laboratory has shown that CMD-1 can significantly enhance antibody production as an adjuvant of recombinant pseudovirus particle vaccine.TIO3 is an antisense oligodeoxynucleotidetargeting the immunosuppressive cytokine TGF-β2 m RNA3’UTR.Our laboratory’s previous experiments have proved that TIO3 can promote the increase of antibody level as an adjuvant of microbial vaccine.2.1 The effect of CpG5805 combined with CMD-1 as vaccine adjuvantTo investigate whether CpG5805 combined with CMD-1 can improve the immune effect of vaccine,we divided mice into five groups for immunization,namely CpG5805,CMD-1 and CpG5805+CMD-1 mixed with SV（C+SV,M+SV,C+M+SV）and SV alone.Mice were immunized on day 0 and 14,and blood samples from tail vein were collected on day 7,14 and 28 after the second immunization.The serum anti-SARS-CoV-2 S protein antibody level was detected by ELISA.The results showed that the antibody production level of mice in C+M+SV group increased compared with that in SV group,but was basically the same as that in C+SV group.The antibody production level of mice in C+SV group was higher than that in SV group,while the antibody level of mice in M+SV group was not significantly changed.The experimental results showed that CpG5805 combined with CMD-1 as a vaccine adjuvant was not better than CpG5805 alone as a vaccine adjuvant.Therefore,the combination of CpG5805 and CMD-1 is not suitable for the use of SARS-COV-2 recombinant S protein vaccine adjuvant,and it cannot play a better immune synergistic effect.2.2 The effect of CpG5805 combined with TIO3 as vaccine adjuvantCpG5805 and TIO3 alone and CpG5805+TIO3 mixed with SV were used to prepare recombinant protein vaccines（C+SV,T+SV,C+T+SV）and SV alone.Mice were immunized at day 0 and 14,and tail vein blood samples were collected at day 7,14 and 28 after the second immunization.Serum antibody levels were detected by ELISA.The results showed that the antibody production level of C+T+SV group was significantly higher than that of the control group SV,and the antibody titer was significantly higher than that of CpG5805/TIO3 group.The antibody production level of T+SV group was also up-regulated compared with that of SV group.Therefore,CpG5805 combined with TIO3 has significantly better immune effect than CpG5805alone,and has the potential as an adjuvant of SARS-COV-2 recombinant S protein vaccine.Then,in order to explore the cell activation of CpG5805 combined with TIO3as vaccine adjuvant,the activation of B cells in mouse spleen cells was detected 48h after immunization.The experimental results showed that CpG5805 combined with TIO3 could significantly improve the activation ratio of CD19⁺CD86⁺cells in mouse spleen cells,and compared with the control group,The proportion of B cell activation was upregulated by 1/3,and the level of MFI was also upregulated.These results indicated that CPG5805 combined with TIO3 as the adjuvant of SARS-CoV-2recombinant S protein vaccine can significantly enhance the activation of B cells and promote the production of antibodies,and has the potential as the adjuvant of SARS-CoV-2 recombinant S protein vaccine.In this study,the recombinant subunit vaccine adjuvant was preliminarily screened,and it was proved that sodium butyrate was not suitable for the recombinant subunit vaccine adjuvant,which not only could not effectively improve the level of antibody production but had immunosuppressive effect on antibody production,and could significantly inhibit the activation of B cells.Meanwhile,CPG5805 and TIO3combined as the recombinant S protein vaccine adjuvant of SARS-CoV-2 can significantly enhance the antibody production level,which has the potential to become a new vaccine adjuvant,providing a theoretical basis for the research and development of novel coronavirus vaccine adjuvant.更多还原