治疗性疫苗和免疫治疗

lifevendor

回复 lifevendor 的帖子

表二： 临床前期研究，过去和即将进行的临床测试，和将来的方案

临床前期的在动物模型中的研究

--------------------------------------------------------------------------------------------------------

• Recombinant HBV envelope proteins (HBsAg) withadjuvants in HBV/HBsAg

transgenicmice [74-76]

• DNA vaccines encoding HBV envelope proteins or polymerase in HBsAg/HBV

transgenicmice [49, 77-79]

• Proteins or DNA vaccinescoding for envelope orcore proteins of woodchuck

hepatitis virus or +/- antivirals in chronically infected woodchuck [36, 37, 80]

• DNA vaccines encoding DHBV large envelope protein +/- antivirals inchronically

infectedducks [35, 81]

--------------------------------------------------------------------------------------------------------

Past vaccine trials/以往的临床测试

--------------------------------------------------------------------------------------------------------

• Preventive hepatitis Bvaccines# [82, 83, 27]

• Preventive hepatitis Bvaccines + antiviral treatment(lamivudine) [39-41,43] + Th-

1 adjuvant [42]or IL-2 [45]

• DNA vaccines* +/- antiviral treatment [53, 55]

• HBsAg/anti-HBs immunecomplexes¶ [29]

• Cell-based therapies:CIK° cells [68]; DC+ HBV antigens§ [62, 64]

Ongoing clinical trials/进行中的临床测试

--------------------------------------------------------------------------------------------------------

• Recombinant hepatitis Bproteins (HBsAg+HBcAg), nasal immunization [30, 32];

• Preventive hepatitis B vaccine +/- Clevudine ( NCT00501124)

• DNA vaccines*+ antiviral treatment (NCT00536627; NCT00513968; NCT01189656)

• Preventive hepatitis Bvaccine (preS2/HBsAg vaccine) + IL-7 + Entecavir or

Tenofovir (NCT01027065)

• HBsAg/anti-HBs immunecomplexes¶ [84]

Future approaches/将来的方案

--------------------------------------------------------------------------------------------------------

• Anti-PD-L1 antibodies [15]

• DNA+ viral vectors (prime-boost strategy) [85, 86]

• TCR-transduced T cells with HBV specificity [70, 71]

• HBV-based gene therapy [72]

-----------------------------------------------------------------------------------------------------

# Recombinant HBsAg, preS2/HBsAg orpreS1/preS2/HBsAg with Alum as adjuvant

* DNAs encoding HBV proteins (DNAencoding small and middle envelope proteins

or mixed DNAs encoding envelope,capsid and polymerase proteins and human IL-

12)

¶ HBsAgcomplexed to human anti-HBs immunoglobulins with Alum as adjuvant

°Cytokine-induced killer cells

§ Dendriticcells loaded with HBsAg or HBV-derived peptides

NCT : ClinicalTrials.gov Identifier(http://clinicaltrials.gov)

lifevendor

回复 lifevendor 的帖子

The first Phase I trial of therapeutic DNA immunization for chronichepatitis B was carried out with a DNA vaccine encoding HBV envelope proteins[53] (Table 2). At the time of inclusion, the treated HBV patients presentedviral breakthrough on lamivudine and active HBV replication. Despite thisunfavorable environment, a decrease in viral load and HBeAg seroconversion wereobserved in six and two of 10 patients, respectively. Seroconversion occurredin the two patients with the lowest viral load at the beginning of the trial, highlightingthe importance of HBV DNA titers for successful immunotherapy. DNA vaccinationled to the induction of IFN-γ-secreting T cells specific to severalenvelopederived epitopes and cytotoxic CD8+ T cells, demonstrating the abilityof this approach to induce or to increase the number of HBV-specific T cells.This HBV-specific IFN-γ T-cell response was also correlated with an increase inthe percentage of a specific NK-cell subset known to produce cytokines inabundance, the CD56bright NK-cell population [54]. Nevertheless, despite thestimulation of innate and adaptive immune responses, no severe adverse eventswere reported, with the exception of a transient increase in ALT levels in two patients,probably due to the restoration of T-cell responses. However, T-cell reactivitygradually declined at the end of treatment, reflecting the profound defect inimmune responses observed during chronic HBV infection.

第一个临床I期的治疗性针对慢性乙肝的DNA疫苗已经展开，DNA疫苗是带有表达HBV封装包膜蛋白的基因【53】（表二）在时间上的包容（At the time ofinclusion），拉米夫定治疗的HBV患者出现了病毒突破和病毒重新活跃复制。除了这个不友好的环境外，10人中六人病毒的载量下降，10人中的两人发生了HbeAg血清转换。发生血清转换的两个病人开始试验前含有最少的病毒载量，提示HBV DNA的定量是免疫治疗成功的关键。DNA疫苗导致IFN-γ分泌针对几个由病毒外壳延伸的抗原表位特异性的T细胞和细胞毒素CD8+ T细胞，显示这种方法有能力导致或者增加HBV特异性的T细胞。这些HBV特异的IFN—γ分泌的T细胞的响应也和NK-细胞的子集（一种叫做CD56 brightNK细胞的群体，他们可以产生丰富的细胞因子）的增加有关。尽管如此，不考虑先天和后天获得性免疫反应的激活，没有发生严重的副作用，其中有两个患者的转氨酶有预期的短暂的升高，可能是因为T细胞响应的恢复导致的。然而，在实验的最后T细胞的活性缓慢的下降，反应了慢性HBV感染中导致的免疫反应的深度的破坏。

The first Phase I trial of therapeutic DNA immunization for chronichepatitis B was carried out with a DNA vaccine encoding HBV envelope proteins[53] (Table 2). At the time of inclusion, the treated HBV patients presentedviral breakthrough on lamivudine and active HBV replication. Despite thisunfavorable environment, a decrease in viral load and HBeAg seroconversion wereobserved in six and two of 10 patients, respectively. Seroconversion occurredin the two patients with the lowest viral load at the beginning of the trial, highlightingthe importance of HBV DNA titers for successful immunotherapy. DNA vaccinationled to the induction of IFN-γ-secreting T cells specific to severalenvelopederived epitopes and cytotoxic CD8+ T cells, demonstrating the abilityof this approach to induce or to increase the number of HBV-specific T cells.This HBV-specific IFN-γ T-cell response was also correlated with an increase inthe percentage of a specific NK-cell subset known to produce cytokines inabundance, the CD56bright NK-cell population [54]. Nevertheless, despite thestimulation of innate and adaptive immune responses, no severe adverse eventswere reported, with the exception of a transient increase in ALT levels in two patients,probably due to the restoration of T-cell responses. However, T-cell reactivitygradually declined at the end of treatment, reflecting the profound defect inimmune responses observed during chronic HBV infection.第一个临床I期的治疗性针对慢性乙肝的DNA疫苗已经展开，DNA疫苗是带有表达HBV封装包膜蛋白的基因【53】（表二）在时间上的包容（At the time ofinclusion），拉米夫定治疗的HBV患者出现了病毒突破和病毒重新活跃复制。除了这个不友好的环境外，10人中六人病毒的载量下降，10人中的两人发生了HbeAg血清转换。发生血清转换的两个病人开始试验前含有最少的病毒载量，提示HBV DNA的定量是免疫治疗成功的关键。DNA疫苗导致IFN-γ分泌针对几个由病毒外壳延伸的抗原表位特异性的T细胞和细胞毒素CD8+ T细胞，显示这种方法有能力导致或者增加HBV特异性的T细胞。这些HBV特异的IFN—γ分泌的T细胞的响应也和NK-细胞的子集（一种叫做CD56 brightNK细胞的群体，他们可以产生丰富的细胞因子）的增加有关。尽管如此，不考虑先天和后天获得性免疫反应的激活，没有发生严重的副作用，其中有两个患者的转氨酶有预期的短暂的升高，可能是因为T细胞响应的恢复导致的。然而，在实验的最后T细胞的活性缓慢的下降，反应了慢性HBV感染中导致的免疫反应的深度的破坏。
图二通常的疫苗和抗病毒治疗的组合方案示意图在组合治疗的测试中，慢性乙肝患者被分为两组（A和B）。两组患者都接受抗病毒治疗。在抗病毒治疗的初级，当病毒的含量低于检测限的时候，A组将多次注射疫苗。HBVDNA病毒载量，HbeAg和HbsAg血清转换率被评价。在抗病毒治疗期间，病毒和抗原的载量下降并且功能障碍的HBV特异性T细胞预期应该恢复。在临床实验的末期（注射完最后疫苗之后），两组同时停止治疗，转氨酶水平被连续监测用于保证快速的检测肝脏损伤。B组中预期会有病毒反弹，但是A组应该可以得到血清病毒的控制。在A组患者中，HBV特异性的疫苗激活的T细胞被预期能够施展他们的抗病毒功能包括分泌细胞因子（IFN-γ， IL-2和TNF-α）并且实现扩增。

lifevendor

回复 lifevendor 的帖子

Similarly, DNA vaccines encoding multiple HBV proteins and amodified human IL-12 molecule were administered once monthly, for 12 months, incombination with lamivudine treatment, to improve the efficacy of DNAvaccination and to broaden immune responses[55]. IL-12 promotes Th1 celldevelopment, cell-mediated cytotoxicity and IFN-γ production and is involved inthe control of viremia in HBeAg-positive chronic hepatitis B carriers [56]. Avirological response was observed in half the treated patients at the end ofcombination therapy and this was significantly higher compared to lamivudinemonotherapy in historical control groups. Memory T cells were detected invirological responders, persisted for 40 weeks after vaccination and wereclosely associated with the suppression of viral rebound after treatment wasstopped. Two of the virological responders still had undetectable viral load 3years after cessation of combined therapy [57]. The presence of modified IL-12as a genetic adjuvant may have contributed as well to the induction oflong-lasting memory T cells. Whether the HBV-specific T cells were present inpatients at the beginning of the trial and were reactivated by the combinedtreatment or were de novo activated by the vaccine is not known as pretreatmentsamples were not analyzed.

相同的，每月给予一次带有多种HBV蛋白和修饰过的人的IL-12分子的DNA疫苗，在12个月内，结合拉米夫定治疗，用于增加DNA疫苗的有效性和扩大免疫反应【55】。IL-12 促使Th1 细胞的发展，细胞调节的细胞毒素和IFN-γ的产生，参与到控制HbeAg阳性乙肝患者的血清病毒中【56】。
在半数接受联合治疗结束时的患者中出现病毒学响应
，这个比率比历史上单独拉米夫定治疗的效果要高很多。在病毒学响应的患者体内可以检测到记忆T细胞，并且记忆T细胞在疫苗之后持续大概40周，并且紧密的和停止治疗后的对病毒反弹的压制相关。在停止治疗后的3年，其中两个患者依然检测不到病毒含量【57】。使用修饰过的IL-12作为基因佐剂可能也对导致长期持续的记忆T细胞有贡献。HBV特异性的T细胞是一开始随着实验出现在患者体内还是由于组合治疗而激活的，或者是由疫苗重新（de novo）激活的还不清楚，因为样本在接受治疗前没有进行分析。

Finally, DNA vaccines may be used to prime immunity before boostingwith viral vectors, such as poxviruses or adenoviruses encoding the sameantigenic proteins. Such combinations result in very large numbers ofmultifunctional T cells, as demonstrated in trials of vaccines for malaria andtuberculosis [58].

最后，DNA疫苗可能可以用于初始免疫，后面可以通过和病毒载体结合提高效果，载体可以包括编译相同的抗原蛋白的水痘病毒（poxviruses）或者是腺病毒。这种组合可以导致大量的多功能的T细胞，这在疟疾和肺结核（tuberculosis）疫苗测试中显示出来【58】。

lifevendor

回复 lifevendor 的帖子

基于细胞的治疗是不是另一个吸引人的可以替代疫苗的方案？

As an alternative to vaccination, DCs loaded ex vivo with HBVantigens could also be used to stimulate T cells (Table 2). The use ofautologous DCs pulsed with tumor-related antigens as immunotherapy for cancerhas been largely documented [59]. Fewstudies have reported the use of DCs for diseases related to viral infections[60], [61]. A pilot study was carried out in five patients with chronic hepatitisB, to evaluate the safety of HBsAg-pulsed autologous DCs [62]. Monocyte-derivedDCs were isolated from peripheral blood from a given patient by a classicalmethod using granulocyte macrophage colony-stimulating factor and IL-4. Theywere then pulsed with purified HBsAg and re-injected into the patient. This approachhad previously been shown to induce anti-HBs antibodies when administered to vaccine-non-responders[63]. Administration was safe and no exacerbation of liver damage was observedin patients with chronic hepatitis B. In another recent trial, autologous DCs pulsedwith HLA-A2-restricted peptides derived from HBcAg (core 18-27 epitope) andfrom the N-terminal part of the middle envelope protein were re-injected on 9occasions into 380 chronic hepatitis B patients in an autologous manner [64].Interestingly, a significant response, as demonstrated by a decrease in HBV DNAlevels to below 103cp/ml and transaminase normalization, was observed in halfthe HBeAg-negative/anti-HBe-positive patients, whereas only 13% of theHBeAg-positive patients displayed complete responses. This treatment was mostsuccessful in HBeAg-negative patients and patients with a viral load <105cp/ml, some of whom had cleared HBsAg. A transient increase in ALT levels was observedin some patients during the first few weeks of treatment, possibly reflectingan effect of the treatment on infected hepatocytes, but ALT levels returned tonormal by the end of treatment. However, in that study, the immuneresponse-mediated mechanisms underlying the observed serologic and virologicaleffects were not documented. An increase in the number of CD8+ T cells wasreported, but it was not determined whether these T cells are specific for theHBV peptides loaded onto DCs. Furthermore, it has been reported that the core18-27 peptide used to load DCs frequently induces T cell responses in HLA-A0201Caucasian patients, but is rarely able to induce CD8+ T cells in HLA-A0206 orHLA-A0203 Chinese patients [65]. Moreover, the sequences of the two peptidesused in the former study vary according to HBV genotype, raising questions aboutthe specificity of this DC-mediated therapy.

作为疫苗的替换方案，在HBV抗原之后装载DCs可能也可以用于激发T细胞（表二）。使用自身获取的（autologous）DCs和调节癌症-相关的抗原振荡混合（pulsed）一起作为癌症的免疫治疗方法已经被大量的记录【59】。少量的研究报告记录使用DCs细胞治疗病毒相关的感染【60】【61】。
在对五位慢性乙肝感染患者的一个前期试探性的实验，用于评价HbsAg-导致的自身获取的DCs细胞的安全性【62】。单核白血球（Monocyte）衍生的DCs首先用传统的有粒细胞（granulocyte）巨噬细胞克隆刺激因子和IL-4方法从病人的外周血中分离出来。这些细胞然后和HbsAg一起混合振荡，然后再注射到病人体内。这种方法以前显示可以对疫苗不响应的患者导致抗-Hbs的抗体产生【63】。在慢性乙肝患者中，实验是安全的而且没有任何加剧肝脏损伤（exacerbation）。再另一项最近的实验中，自身获取的DCs和从HbcAg（核18-27抗原受体）和病毒中间包膜蛋白的N端得到的HLA-A2-限制性多肽一起振荡混合，然后分9次以一种自身获取的方式，再注射回到慢性乙肝患者中【64】。有趣的是，一个显著的反应可以由以下来证实，一半的HbeAg阴性/抗Hbe阳性患者显示HBVDNA水平下降低于10^3cp/ml, 转氨酶正常化，然而仅仅有13% HbeAg阳性患者显示完全的响应。这项治疗在HbeAg阴性患者中病毒数量<10^5 cp/ml 非常成功，他们中的某些人清除了HbsAg。在前几周的治疗中，有些患者出现了短暂的ALT水平升高，可能反应了治疗方法在受感染的肝细上面的作用，但是alt在治疗的后期回到正常水平。然而，在那项研究中，血清学和病毒学效果下的免疫调节机制并没有记载。有报道说CD8+的T细胞有增加，但是不能确定的是这些T细胞是不是特异性的针对DCs上的HBV多肽。此外，据报道在HLA-A0201白种人（Caucasian）病人身上用于装载DCs的核心18-27多肽频繁的导致T细胞响应，但是在HLA-A0206或者HLA-A0203中国病人身上，却很少能产生CD8+T细胞【65】。另外，由于HBV的基因型的不同，前两项试验的多肽的序列也不同，提出了DC调节的治疗的特异性的问题。

lifevendor

回复 lifevendor 的帖子

基于细胞的治疗是不是另一个吸引人的可以替代疫苗的方案？

As an alternative to vaccination, DCs loaded ex vivo with HBVantigens could also be used to stimulate T cells (Table 2). The use ofautologous DCs pulsed with tumor-related antigens as immunotherapy for cancerhas been largely documented [59]. Fewstudies have reported the use of DCs for diseases related to viral infections[60], [61]. A pilot study was carried out in five patients with chronic hepatitisB, to evaluate the safety of HBsAg-pulsed autologous DCs [62]. Monocyte-derivedDCs were isolated from peripheral blood from a given patient by a classicalmethod using granulocyte macrophage colony-stimulating factor and IL-4. Theywere then pulsed with purified HBsAg and re-injected into the patient. This approachhad previously been shown to induce anti-HBs antibodies when administered to vaccine-non-responders[63]. Administration was safe and no exacerbation of liver damage was observedin patients with chronic hepatitis B. In another recent trial, autologous DCs pulsedwith HLA-A2-restricted peptides derived from HBcAg (core 18-27 epitope) andfrom the N-terminal part of the middle envelope protein were re-injected on 9occasions into 380 chronic hepatitis B patients in an autologous manner [64].Interestingly, a significant response, as demonstrated by a decrease in HBV DNAlevels to below 103cp/ml and transaminase normalization, was observed in halfthe HBeAg-negative/anti-HBe-positive patients, whereas only 13% of theHBeAg-positive patients displayed complete responses. This treatment was mostsuccessful in HBeAg-negative patients and patients with a viral load <105cp/ml, some of whom had cleared HBsAg. A transient increase in ALT levels was observedin some patients during the first few weeks of treatment, possibly reflectingan effect of the treatment on infected hepatocytes, but ALT levels returned tonormal by the end of treatment. However, in that study, the immuneresponse-mediated mechanisms underlying the observed serologic and virologicaleffects were not documented. An increase in the number of CD8+ T cells wasreported, but it was not determined whether these T cells are specific for theHBV peptides loaded onto DCs. Furthermore, it has been reported that the core18-27 peptide used to load DCs frequently induces T cell responses in HLA-A0201Caucasian patients, but is rarely able to induce CD8+ T cells in HLA-A0206 orHLA-A0203 Chinese patients [65]. Moreover, the sequences of the two peptidesused in the former study vary according to HBV genotype, raising questions aboutthe specificity of this DC-mediated therapy.

作为疫苗的替换方案，在HBV抗原之后装载DCs可能也可以用于激发T细胞（表二）。使用自身获取的（autologous）DCs和调节癌症-相关的抗原振荡混合（pulsed）一起作为癌症的免疫治疗方法已经被大量的记录【59】。少量的研究报告记录使用DCs细胞治疗病毒相关的感染【60】【61】。
在对五位慢性乙肝感染患者的一个前期试探性的实验，用于评价HbsAg-导致的自身获取的DCs细胞的安全性【62】。单核白血球（Monocyte）衍生的DCs首先用传统的有粒细胞（granulocyte）巨噬细胞克隆刺激因子和IL-4方法从病人的外周血中分离出来。这些细胞然后和HbsAg一起混合振荡，然后再注射到病人体内。这种方法以前显示可以对疫苗不响应的患者导致抗-Hbs的抗体产生【63】。在慢性乙肝患者中，实验是安全的而且没有任何加剧肝脏损伤（exacerbation）。再另一项最近的实验中，自身获取的DCs和从HbcAg（核18-27抗原受体）和病毒中间包膜蛋白的N端得到的HLA-A2-限制性多肽一起振荡混合，然后分9次以一种自身获取的方式，再注射回到慢性乙肝患者中【64】。有趣的是，一个显著的反应可以由以下来证实，一半的HbeAg阴性/抗Hbe阳性患者显示HBVDNA水平下降低于10^3cp/ml, 转氨酶正常化，然而仅仅有13% HbeAg阳性患者显示完全的响应。这项治疗在HbeAg阴性患者中病毒数量<10^5 cp/ml 非常成功，他们中的某些人清除了HbsAg。在前几周的治疗中，有些患者出现了短暂的ALT水平升高，可能反应了治疗方法在受感染的肝细上面的作用，但是alt在治疗的后期回到正常水平。然而，在那项研究中，血清学和病毒学效果下的免疫调节机制并没有记载。有报道说CD8+的T细胞有增加，但是不能确定的是这些T细胞是不是特异性的针对DCs上的HBV多肽。此外，据报道在HLA-A0201白种人（Caucasian）病人身上用于装载DCs的核心18-27多肽频繁的导致T细胞响应，但是在HLA-A0206或者HLA-A0203中国病人身上，却很少能产生CD8+T细胞【65】。另外，由于HBV的基因型的不同，前两项试验的多肽的序列也不同，提出了DC调节的治疗的特异性的问题。

lifevendor

回复 lifevendor 的帖子

将来还有哪些途径可以用于处理T细胞?

In addition to treatments designed to stimulate HBV-specific T cellsin vivo by vaccination strategies or ex vivo with cytokines or DCs, a novelapproach is based on the genetic modification of T cells by T-cell receptor(TCR) gene transfer [69]. This involves (1) isolation of the HBV-specific TCRfrom T cells obtained from individuals with self-resolved hepatitis, (2) theinsertion of this TCR into a DNA or viral vector and (3) the transfer of this TCRspecificity to T cells from patients with chronic HBV infection. Retroviralvectors have been used to introduce HLA-A2-restricted HBV-specific TCR into Tcells from chronic patients. The TCR-transduced T cells produced IFN-γ, TNF-α andIL-2 following target cell recognition and lysed hepatocyte-like cell linesexpressing cognate HBV antigens. In vivo functionality was also assessed by theadoptive transfer of TCR-redirected T cells in immunodeficient mice, leading tothe rejection of grafted tumor cells expressing HBV antigens. This strategyshould make it possible to reconstitute functional HBV-specific T cells inpatients with chronic infection [28, 70]. Alternatively, artificial chimericTCRs composed of a single-chain antibody fragment recognizing HBV envelopeproteins, and cytoplasmic regions of the costimulatory CD28 molecule followedby the CD3-ζ signaling domain have

用于设计激发HBV特异性的T细胞可以在活体中通过疫苗策略，或者在活体之外中利用细胞因子或DCs，此外还有一个基于基因转移T细胞的受体（TCR）基因来达到修改T细胞的新颖方法【69】。这包括（1）从自愈的患者身上的T细胞中分离HBV特异性的TCR，（2）将这段TCR放入到一个DNA或者病毒载体里面（3）把这些TCR转译到慢性HBV携带者身上的T细胞内。逆转录病毒载体已经用于把HLA-A2限制性的HBV特异性的TCR引入到慢性患者的T细胞内。经TCR变换过的T细胞，在识别靶位细胞后，可以生产IFN-γ，TNF-α和IL-2并且可以溶解掉表达HBV同源的抗原的类肝细胞的细胞群。在活体中，其功能通过接受性的转移TCR-从新导向过的T细胞到有免疫缺陷的小鼠身上得到评价，可以导致排除小鼠内移植的表达HBV抗原的癌细胞。这些策略让在慢性患者身上重组功能性的HBV特异性的T细胞成为可能【28,70】。此外，临床由识别HBV膜蛋白的单链抗体片段和细胞液区域的共刺激CD28分子，另外还有CD3 信号区域组成的人工嵌合（chimeric）TCRs被构造。这些嵌合的受体，当被逆转录病毒传递并在细胞表面表达的时候，使人的主要T细胞识别HbsAg阳性的肝细胞，释放IFN-γ和IL-2，更为重要的是，溶解掉含有HBV复制的细胞【71】。然而，是否这些从新注入的TCR-从新导向的T细胞在体内可以扩增和可以接触到靶位器官还不清楚，是否新处理过的T细胞是否会处于一个可容忍（tolerizing）环境。

If anti-HBV immunotherapy is to be efficient, the activated T-cellresponse must specifically reach the liver. Deng et al. [72] therefore designeda recombinant HBV (rHBV) containing a modified viral core gene for the specificdelivery of foreign (i.e. non-HBV) epitopes to the liver. This recombinantvirus was engineered to self-maintain only in hepatocytes already infected withHBV through capsid complementation. The activated non-HBV specific CD8+ T-cellresponse is expected to be functional and to compensate for the deficiencies ofHBV-specific anti-viral immunity. It should target infected liver cells and wouldnot be subject to functional exhaustion during chronic hepatitis B.Proof-of-concept for this approach was demonstrated through a protocol forrHBV-based active immunization in HLA-A2/DR1-transgenic mice (see Fig 3).

如果抗HBV免疫疗法有效，激活的T细胞的响应必须特异性的抵达肝部。Deng et al. [72]因此设计了一个从组的HBV（rHBV）包含一个修改过的病毒核基因用于特异性的传递外来（比如说，非HBV）抗原表位到肝脏。这种组合过的病毒被处理成仅能够在已经被HBV感染的肝细胞内可以自我维持，因为它可以共享HBV的外壳。激活的非HBV特异性的CD8+T细胞响应被认为是功能性的而且可以作为HBV特异性的抗病毒免疫丧失的补充。它应该以受感染的肝细胞为靶位，应该不会导致慢性乙肝的功能性耗竭。为了测试这个概念，一个基于rHBV激活免疫的实验方案选择在HLA-A2/DR1-转基因小鼠上完成了。
（见图 Fig3）

图三:慢性乙肝的基因治疗一个免疫治疗的方案成立了，结合了疫苗和肝脏基因治疗。
设计了一个重组的HBV病毒（rHBV），即作为基因传递的载体也是一个抗原携带物，为在肝内表达一种外来抗原的多重表位。在这项策略中，在使用rHBV基因治疗之前，患者体内首先产生功能性的多表位特异性的T细胞响应。rHBV被构造，通过插入一段外来抗原序列来破坏rHBV病毒的核基因。这个核缺失的病毒没有复制的能力，除非宿主的肝细胞可以反向提供野生型的病毒衣壳蛋白。rHBV特异性的感染人的肝部细胞，和野生型的病毒（wtHBV）一样。然而，rHBV不能再健康的患者中复制，仅能够活在肝细胞长期被野生型wtHBV感染的慢性感染患者身上。在有HBV 肝脏特异性的启动因素出现的时候，rHBV蛋白（聚合酶，L,M,S 包膜蛋白，Hbx）同时还有外来抗原性的多表位被产生，合成并以多肽的形式存在肝细胞内。这些外来的抗原表位可以招来很强的功能性的针对肝细胞的T细胞响应，这些响应可以弥补慢性感染患者身上的有缺陷的HBV特异性的T细胞响应。

lifevendor

回复 lifevendor 的帖子

A strong polyepitope-specific T-cell response was first primed inthe periphery by DNA immunization. In absence of a mouse model susceptible toHBV infection, hydrodynamic injection was used to mimic rHBV replication andgene expression in the mouse liver. This method allows direct in vivotransfection of hepatocytes and bypass the strict host-range of the virus forhuman hepatocytes [73]. The expression of

foreign antigenic epitopes in hepatocytesrecruited a vigorous T-cell response in situ. Most liver-infiltratingpolyepitope-specific CD8+ T cells proved to be functional effectors. FollowingDNA priming and hydrodynamic injection, the rHBV-based expression of HBsAg inmouse liver was completely inhibited without major liver injury. Studies inHLAA2/DR1/HBsAg-transgenic mice as a surrogate model for HBV chronic infectionfurther validated this approach. In these mice the polyepitope-specific functionalT cell response not only controlled the rHBV expression but also controlled HBVtransgene expression in the liver. Thus, rHBV and foreign antigen-based activeimmunotherapy constitute a promising strategy for the treatment of persistentHBV infection. This strategy could potentially be generalized and extended toother chronic viral diseases.

在外围，由于DNA的免疫，一个很强的多表位特异性的T细胞响应首先发生。在缺少一个易于（susceptible）被HBV感染的老鼠模型，水动力注射被用于模拟在老鼠肝部rHBV的复制和基因表达。这个方法允许直接的活体转染肝细胞并且让这种有严格宿主范围的病毒感染人的肝细胞【73】。由于在肝部表达的外来抗原的表位导致了一个非常强的原位（in situ）T细胞响应。大部分肝部渗透（infiltrating）的多抗原表位特异的CD8+T细胞被证明是功能性的效应器。在DNA 结合和水动力注射后，基于rHBV的老鼠肝部的HBsAg表达被彻底的抑制了，而且肝部没有主要的伤害。在HLAA2/DR1/HbsAg转基因小鼠的研究可以作为代表性的HBV慢性感染模型用于进一步的核实这种方案。在这些老鼠中，多抗原表位特异性的功能T细胞的反应不仅可以控制rHBV的表达，同时也可以控制HBV在转基因小鼠肝部的表达。因此，rHBV和外来抗原激活的免疫治疗组成了一个有希望的策略用于治疗持续的HBV感染。这种方法有潜力扩展和一般化到其他的慢性病毒导致的疾病。

lifevendor

回复 lifevendor 的帖子

结论

For chronic infections (with HBV, HCV, HIVetc.), therapeutic interventions aim to counteract the negative effects of theimmunosuppressive environment and high antigen load on T cells. The combinationof therapeutic vaccination with other types of immunotherapy and classicalantiviral treatments may prove the most effective strategy for additive or synergisticefficacy. However, the natural course of the disease, the duration of infectionand the immune tolerance status of the targeted patients must be taken intoaccount. Most of the early therapeutic vaccine trials were performed inpatients with persistent infection over several decades, patients with highviral loads or patients who did not respond to classical antiviral treatments.This may accounted for efficacy being lower than initially expected. Indeed,most of the observed beneficial effects concerned patients with HBV viral loadsbelow 106 cp/ml, patients with high ALT levels and HBeAg-negative patients. Thedefinition of subgroups of patients on the basis of biomarkers predictive ofthe efficacy of therapeutic vaccination or other immunotherapy-based strategieswould make it possible to advance toward more sophisticated combinations. Thereconstitution of virus-specific T-cell responses is an important safety issuethat must be carefully evaluated before each new therapeutic intervention. Manyobstacles remain to be overcome before the ideal therapeutic vaccine can bedeveloped on a large scale. However, a growing understanding of the mechanisms underlyingthe immune defects occurring during chronic HBV infection and the experimentaldata collected in previous clinical studies in patients suggest that this “HolyGrail” of modern medicine may be attainable.

对于慢性感染者（包括HBV，HCB，HIV等），治疗性的插入目的是为了抵消免疫抑制的环境带来的消极作用和T细胞上面的高的抗原载量。将治疗性疫苗和其他的免疫疗法还有传统的抗病毒疗法结合起来可能是最有效的策略因为有添加效果或者是协同效果。然而，目标患者的疾病的自然发展，感染持续的时间，免疫耐受的状态都必须考虑进来。大部分的早期的治疗性的疫苗都针对数十年感染的慢性患者，患者有高的病毒载量或者是患者对传统的抗病毒治疗没有响应。这个可以解释为什么治疗性疫苗的效果比预期的要低。事实上，所有观察到的有利的效果都是发生在那些病毒的载量在10^6cp/ml以下，高的转氨酶和HbeAg阴性患者。基于生物标记物来定义不同次小组的患者可以预测治疗型疫苗或者是其他基于免疫治疗的策略的有效性有可能可以升级到更多复杂的组合。每一次治疗的介入（intervention）都必须仔细的评价从组病毒特异性的T细胞响应的安全问题。在理想的治疗性疫苗大规模开发出来之前，还有许多障碍仍然需要克服。然而，越来越多的慢性乙肝感染导致的免疫缺陷的背后机制被了解，并且先前从患者临床收集到的数据提示这块现代医学的圣地有可能可以得到。

Key messages/核心信息:

• 当前的针对免疫介入疗法和治疗型疫苗的有效性的测量方法缺少统一标准。定义病毒学上的和免疫学上的终点（end-point,应该指治疗指标的终点）可以用于改善分析比较所有临床试验得到的结果。Current measurement ofclinical efficacy following immune-intervention or

therapeuticvaccination lack standardization. Definition of virological and immunologicalend-points that could be used across all clinical studies would improvecomparative analysis between studies.

• 并不是所有的慢性乙肝患者适合治疗介入。仔细的选择患者对基于免疫的治疗非常有利（成年感染，低病毒载量<10^6 cp/ml, 高的转氨酶）可以增加成功率并且让我们可以更好的理解持续清除HBV的一个潜在机制。Not all patients withchronic hepatitis are good candidates for therapeuticintervention. Carefulselection of patients in which the immune-based therapies would be mostbeneficial (patients infected in adulthood, with low viral load <106 cp/ml, high ALT) would increase the rate of success andallow a better understanding of the mechanisms underlying sustained clearanceof HBV.

• 定义一个更好的免疫参数可以和基于免疫的或者是疫苗治疗的有效率相关已经被保证，这将毫无疑问的改善现有的正在处理的方案。A better definition ofimmune parameters that correlate with the efficacy of immune-based or vaccinetherapy is yet warranted and will undoubtedly improve the current approaches.

kennyu

lifevendor 发表于 2011-5-30 12:34
Therapeutic vaccines and immune-basedtherapies for thetreatment of chronichepatitis B: perspectives  ...

你翻译的这个东西，可以放到译言上面去。

StephenW

真的是很难和费时的工作。很专业，细致的翻译. 非常感谢.