醫(yī)學(xué)免疫學(xué)課件：Toll樣受體激動劑佐劑研究進(jìn)展

1、Toll樣受體激動劑佐劑研究進(jìn)展01020304免疫佐劑TLR及其信號轉(zhuǎn)導(dǎo)TLR激動劑佐劑作用機制簡介CONTENT05前景與展望免疫佐劑免疫佐劑被廣泛應(yīng)用于疫苗中，是能夠提高機體對抗原的適應(yīng)性免疫應(yīng)答，減少抗原使用量的物質(zhì)。1 http:/photos/view/8059449714/Glass%20Medicine%20Vials.htmlFig. 1. 疫苗1Table 1. 常見佐劑類型舉例優(yōu)點缺點鋁鹽氫氧化鋁凝膠等有效誘導(dǎo)體液免疫應(yīng)答不能有效誘導(dǎo)細(xì)胞免疫應(yīng)答乳劑類佐劑水包油和油包水類乳劑，例如弗氏不完全佐劑，MF59，AS03等較好的安全性，有效誘導(dǎo)體液免疫應(yīng)答副作用，如注射部位的炎

2、癥反應(yīng)、肉芽腫、潰瘍等脂質(zhì)體類DDA，DOTAP誘導(dǎo)體液免疫或細(xì)胞免疫穩(wěn)定性低、 成本高，可能有副作用皂苷QS-21誘導(dǎo)強烈的細(xì)胞免疫反應(yīng)毒性大Toll樣受體激動劑佐劑MPL，GLA等等種類多，安全性好，可誘導(dǎo)體液免疫或細(xì)胞免疫副作用Toll-like receptorToll樣受體（TLR）識別微生物的疾病相關(guān)分子模式（PAMP）的模式識別受體激活天然免疫反應(yīng)促進(jìn)建立獲得性免疫TLR是I型跨膜結(jié)構(gòu)域蛋白質(zhì)，具有三分結(jié)構(gòu)：氨基（N）端結(jié)構(gòu)域，折疊為獨特的馬蹄形結(jié)構(gòu)并負(fù)責(zé)配體識別單個跨膜結(jié)構(gòu)域羧基（C）端球狀的Toll/白介素-1（IL-1）受體（TIR）結(jié)構(gòu)域激動劑：對受體有親和力和內(nèi)在活性，

3、能與受體結(jié)合而興奮受體、發(fā)揮作用的物質(zhì)。Table 2. TLR localisation and ligands.TLRLocalisationMicrobial ligandsEndogenous ligandsTLR1Plasma membraneTriacyl lipoproteinsUnknownTLR2Plasma membraneLipoproteins, zymosan, mannan, peptidoglycan, lipoteichoic acidVersicanTLR3Endolysosomal membraneViral dsRNAmRNATLR4Plasma and

4、endolysosomal membraneLPSOxidised low-density lipoprotein, Amyloid-betaTLR5Plasma membraneFlagellinUnknownTLR6Plasma membraneDiacyl lipoproteins, lipotechoic acid, zymosanOxidised low-density lipoprotein, Amyloid-beta, versicanTLR7Endolysosomal membraneViral and bacterial ssRNAImmune complexes, self

5、 RNATLR8Endolysosomal membraneViral and bacterial ssRNAImmune complexes, self RNATLR9Endolysosomal membraneViral and bacterial CpG DNA, DNA:RNA hybridsChromatin IgG immune complexes, self DNATLR10Plasma membraneUnknownUnknownFig. 2. Crystal structure of TLR receptors. A) Crystal structure of hTLR2ED

6、/hTLR1ED (human TLR2 ectodomain/human TLR1 ectodomain) in complex with Pam3CSK4. hTLR2ED is shown in blue, hTLR1ED in green. Pam3CSK4 is in red. This figure was generated from PDB code .Maximiliano Javier Jimnez-Dalmaroni, M. Eric Gerswhin, Iannis E. AdamopoulosThe critical role of toll-like recepto

7、rs From microbial recognition to autoimmunity: A comprehensive reviewAutoimmunity Reviews, Volume 15, Issue 1, 2016, 18TLR信號轉(zhuǎn)導(dǎo) TLR的激活導(dǎo)致胞質(zhì)內(nèi)TIR結(jié)構(gòu)域銜接蛋白的招募，以便聯(lián)絡(luò)受體到下游效應(yīng)蛋白。 五類銜接蛋白在TLR信號通路中發(fā)揮作用： MyD88、TRIF、MAL、TRAM和SARM。 銜接蛋白觸發(fā)依賴于MyD88或TRIF的兩條主要途徑，導(dǎo)致：上調(diào)吞噬相關(guān)基因的表達(dá)，增強吞噬細(xì)胞吞噬與殺傷能力。誘導(dǎo)I型干擾素（如IFN/）的產(chǎn)生上調(diào)細(xì)胞因子和趨化因子表達(dá)

8、，募集免疫細(xì)胞活化NK細(xì)胞，DC等免疫細(xì)胞，增強抗原提呈。Fig. 3. Overview of TLR signalling pathways. The signalling cascades downstream of TLR3, TLR4 and TLR9 are shown as examples. Activation of TLR4 on the cell surface or TLR9 in endosomes leads to the recruitment of the adaptor MyD88 via TIRTIR interactions and.Dominic De

9、NardoToll-like receptors: Activation, signalling and transcriptional modulationCytokine, Volume 74, Issue 2, 2015, 181189TLR1/2/6激動劑佐劑TLR1/2/6識別脂蛋白脂蛋白Pam3CSK4（屬于三酰脂肽），R4Pam2Cys，rlipoA-RBD，MALP-2（支原體的脂肽）等TLR1/2/6激動劑佐劑 R4Pam2Cys在小鼠中用脂肽R4Pam2Cys投送抗原可以促進(jìn)T細(xì)胞增殖和分化為CTL，以及促進(jìn)記憶T細(xì)胞的產(chǎn)生，表現(xiàn)出比鋁佐劑和弗式完全佐劑更好的佐劑活性1。

10、單酰脂肽一種經(jīng)改良的、高度水溶性的單酰脂肽被合成作為人類TLR2特異性激動劑，并在家兔實驗中誘導(dǎo)強烈的IgG響應(yīng)，表現(xiàn)出突出的佐劑活性2。 rlipoA-RBD在小鼠、家兔和倉鼠實驗中，試驗者重組合成脂蛋白rlipoA-RBD作為佐劑，與B-rRBD配合使用，在艱難梭菌感染實驗中取得了良好的免疫保護效果3。1 Chua, B.Y., et al., The use of a TLR2 agonist-based adjuvant for enhancing effector and memory CD8 T-cell responses. Immunol Cell Biol, 2014. 92

11、(4): p. 377-83.2 Salunke, D.B., et al., Design and Development of Stable, Water-Soluble, Human Toll-like Receptor 2 Specific Monoacyl Lipopeptides as Candidate Vaccine Adjuvants. Journal of Medicinal Chemistry, 2013. 56(14): p. 5885-5900.3 Huang, J., et al., Recombinant lipoprotein-based vaccine can

12、didates against C. difficile infections. Journal of Biomedical Science, 2015. 22(1)TLR3激動劑佐劑TLR3識別病毒雙鏈RNA病毒雙鏈RNA類似物PolyIC（聚肌胞），RGC100，Poly I: Poly C12UI：次黃嘌呤核苷（肌苷）TLR3激動劑佐劑Poly I: Poly C12U鼻內(nèi)用流感疫苗聯(lián)合Poly I: Poly C12U在人體實驗中是耐受良好的，產(chǎn)生針對H5N1，H7N9和H7N3病毒的更好的黏膜IgA響應(yīng)1。RGC100相較于傳統(tǒng)的polyIC佐劑，RGC100有確定的化學(xué)結(jié)構(gòu)和良好的

13、可溶性，可以通過TLR3激活髓系樹突狀細(xì)胞，導(dǎo)致促炎細(xì)胞因子的釋放，和提高CD1c+樹突狀細(xì)胞（CDs）刺激T細(xì)胞增殖的能力2。1 Overton, E.T., et al., Intranasal seasonal influenza vaccine and a TLR-3 agonist, rintatolimod, induced cross-reactive IgA antibody formation against avian H5N1 and H7N9 influenza HA in humans. Vaccine, 2014. 32(42): p. 5490-5495.2 Na

14、umann, K., et al., Activation of Dendritic Cells by the Novel Toll-Like Receptor 3 Agonist RGC100. Clinical and Developmental Immunology, 2013. 2013: p. 1-11.TLR3激動劑佐劑PDLCPICDOTAP脂質(zhì)體復(fù)合物（PDLC）納米顆粒通過加強TLR3IRF3信號通路、誘導(dǎo)樹突狀細(xì)胞（DCs）成熟和促進(jìn)I型干擾素的產(chǎn)生，穩(wěn)健地提升了癌癥疫苗抗腫瘤活性1。1 Wang, C., et al., Toll-like receptor 3 agon

15、ist complexed with cationic liposome augments vaccine-elicited antitumor immunity by enhancing TLR3IRF3 signaling and type I interferons in dendritic cells. Vaccine, 2012. 30(32): p. 4790-4799.Fig. 4. Graphical abstract1TLR4激動劑佐劑TLR4識別細(xì)菌的脂多糖（LPS）MPLRibi 公司開發(fā)出LPS 無毒衍生物單磷脂A（MPL）佐劑，源于革蘭氏陰性沙門氏菌細(xì)胞壁的LPS。A

16、S01（MPL，QS21，脂質(zhì)體）：MosquirixAS02（MPL，QS21，乳劑）AS04（MPL，鋁鹽佐劑）：Cervarix，F(xiàn)endrix AS15（MPL， QS21，CpG，脂質(zhì)體）吡喃葡萄糖基脂佐劑（GLA）：MPL類似物。TLR4激動劑佐劑AS012015年7月，在歐洲上市的第一種重組瘧疾疫苗（Mosquirix）使用了佐劑AS01。佐劑AS01含有脂質(zhì)體MPL和QS21，其中脂質(zhì)體可以包裹并投放親脂性免疫增強劑MPL1。吡喃葡萄糖基脂（GLA）GLA是MPL的合成類似物，GLA乳劑（GLA-SE）作為佐劑在針對異型H5N1病毒時，可以加速誘導(dǎo)初次免疫應(yīng)答以及增長持久性，誘

17、導(dǎo)抗體應(yīng)答的增強2。1 Morrison, C., Landmark green light for Mosquirix malaria vaccine. Nat Biotechnol, 2015. 33(10): p. 1015-6. O Hagan, D.T. and C.B. Fox, New generation adjuvants From empiricism to rational design. Vaccine, 2015. 33: p. B14-B20.2 Clegg, C.H., et al., Adjuvant solution for pandemic influenz

18、a vaccine production. Proceedings of the National Academy of Sciences, 2012. 109(43): p. 17585-17590.3 /commentary/mosquirix-malaria-vaccine-by-oluwatosin-omole-et-al-2015-09Fig. 5. Mosquirix3TLR5激動劑佐劑TLR5配體是細(xì)菌鞭毛蛋白鞭毛蛋白具有作為佐劑的潛力TLR5激動劑佐劑FljB在鼠傷寒沙門氏菌鞭毛蛋白FljB與PRRSV病毒蛋白GP5的融合蛋白的免疫實驗中，F(xiàn)ljB促進(jìn)誘導(dǎo)小鼠體液免疫反應(yīng)和Ig

19、G抗體的產(chǎn)生1。FliC和m229在非洲綠猴新生兒中，注入鞭毛蛋白佐劑的滅活流感病毒IPR8可以誘導(dǎo)更強的IgG響應(yīng)和T細(xì)胞響應(yīng) 2。1 Xiong, D., et al., A porcine reproductive and respiratory syndrome virus (PRRSV) vaccine candidate based on the fusion protein of PRRSV glycoprotein 5 and the Toll-like Receptor-5 agonist Salmonella Typhimurium FljB. BMC Veterinary

20、 Research, 2015. 11(1).2 Kim, J.R., et al., Inclusion of Flagellin during Vaccination against Influenza Enhances Recall Responses in Nonhuman Primate Neonates. Journal of Virology, 2015. 89(14): p. 7291-7303.TLR7/8激動劑佐劑富含鳥嘌呤核苷和尿嘧啶核苷的單鏈RNA最早分離鑒定的天然TLR7/8激動劑內(nèi)源性核糖核酸酶迅速降解單鏈RNA合成小分子化合物1咪唑喹啉雷西莫特鳥嘌呤核苷和尿嘧啶核

21、苷的類似物 Fig. 6. Imidazoquinoline21 Gorden KB, Gorski KS, Gibson SJ,et al. Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8 J. J Immunol, 2005, 174: 1259-1268.2 /wiki/ImidazoquinolineTLR7/8激動劑佐劑Aldara和瑞喹莫德TLR7激動劑Aldara（5%咪喹莫特乳膏）早已上市，被用于治療HPV導(dǎo)致的外生殖器疣，表淺性基底細(xì)胞癌和光化性角化病；瑞喹莫德和

22、咪喹莫特被測試于許多關(guān)于慢性病毒感染和癌癥的臨床試驗中，可以激活產(chǎn)生I型干擾素的固有免疫1。咪唑喹啉類似物Hybrid-2TLR8激動劑可以誘導(dǎo)人類新生兒臍帶血單核細(xì)胞產(chǎn)生成人水平的Th1極化細(xì)胞因子。咪唑喹啉類似物Hybrid-2選擇性激活TLR7和TLR8，相較于R848在體外實驗中更有能力誘導(dǎo)成年人相似水平的TNF-和IL-12。咪唑喹啉衍生物CL097TLR7/8激動劑CL097聯(lián)接HBV-Ag后應(yīng)用于對乙肝病毒抗原免疫耐受的小鼠中可以誘導(dǎo)抗原特異性Th1免疫反應(yīng)，說明其有應(yīng)用于免疫耐受個體的能力3。1 Vasilakos JP, Tomai MA. The use of Toll-l

23、ike receptor 7/8 agonists as vaccine adjuvants. Expert Rev Vaccines. 2013 Jul;12(7):809-19.2 Ganapathi, L., et al., The Imidazoquinoline Toll-Like Receptor-7/8 Agonist Hybrid-2 Potently Induces Cytokine Production by Human Newborn and Adult Leukocytes. PLOS ONE, 2015. 10(8): p. e0134640.3 Wang, Y.,

24、et al., Immunizations with hepatitis B viral antigens and a TLR7/8 agonist adjuvant induce antigen-specific immune responses in HBV-transgenic mice. International Journal of Infectious Diseases, 2014. 29: p. 31-36.TLR9激動劑佐劑TLR9識別含有CpG基序的DNA。含CpG序列的DNA有佐劑潛能，如1018，1826等等。Fig. 7. 寡脫氧核苷酸（CpG ODN）11 http

25、:/tlr9-agonistTLR9激動劑佐劑CpG ODN 1018CpG ODN 1018作為佐劑，對健康的1870歲成年人使用2倍劑量的HBsAg-1018相較于3倍劑量的含鋁佐劑的Engerix-B (HBsAg-Eng)誘導(dǎo)更高的血清保護率1。慢性腎病并發(fā)II型糖尿病的患者對HBsAg-Eng響應(yīng)度很低，對這樣的患者使用3倍劑量的HBsAg-1018相較于4倍劑量的含鋁佐劑的Engerix-B在安全的前提下可以誘導(dǎo)更高的血清保護率2。1 Janssen, J.M., et al., Immunogenicity of an investigational hepatitis B va

26、ccine with a toll-like receptor 9 agonist adjuvant (HBsAg-1018) compared with a licensed hepatitis B vaccine in subpopulations of healthy adults 1870 years of age. Vaccine, 2015. 33(31): p. 3614-3618.2 Janssen, J.M., et al., Immunogenicity and safety of an investigational hepatitis B vaccine with a Toll-like receptor 9 agonist adjuvant (HBsAg-1018) compared with a licensed hepatitis B vaccine in patients with chronic kidney disease and type 2 diabetes mellitus. Vaccine, 2015. 33(7): p. 833-837.TLR9激動劑佐劑CpG ODN 1826在小鼠中使用CpG ODN 1826和鋁佐劑的海洛因疫苗相較于不使用CpG ODN 1826的海洛因疫苗展示出高度轉(zhuǎn)變的劑量-響應(yīng)曲線1。1 Bremer,