【作者】 杨红；

【导师】 张志新；

【作者基本信息】 四川大学 ， 人类重大疾病生物治疗， 2020， 博士

【摘要】 1、研究背景抗体作为体液免疫反应中重要的效应分子,在机体的免疫防御中发挥着重要作用。抗体为了完成分泌,两个重链多肽和两个轻链多肽必须完成配对形成四聚体,这种组装方式已经成为抗体分泌的必要条件。但是重链抗体(Heavy chain only antibody,HCAb)的发现打破了这一经典的抗体分泌的理论,即重链抗体可以在轻链缺失的条件下独立表达且分泌。已经发现的重链抗体主要来自两大类群:重链病患者和驼类。重链病基于重链的类型,分为ɑ-HCD、γ-HCD、μ-HCD。重链病中的HCAb长期被认为是疾病的衍生产物,但是骆驼血清中高达50-75%的重链抗体说明HCAb在骆驼科动物的免疫防御体系发挥重要的防御作用。这两大来源的重链抗体,普遍存在结构上的缺失,骆驼的HCAb缺乏C_H1结构域,重链病中的HCAb缺失的部分包括C_H1以及V_H。在抗体的合成过程中,重链多肽进入内质网后其C_H1结构域和分子伴侣Bi P紧密结合,从而阻止重链二聚体的分泌,而重链病和驼类HCAb中C_H1结构域的缺失被认为C_H1区对重链抗体的分泌有重要影响。有研究显示,一类从系统性红斑狼疮患者浆母细胞克隆的IgH基因可以在哺乳动物细胞中单独表达且分泌,同驼类和重链病中HCAb不同的是,这类重链抗体具有完整的V_H和C_H1结构域,同时具有自身反应性,这一研究发现为重链抗体提供了重要补充。人类免疫缺陷病毒(Human immunodeficiency virus,HIV)在世界范围内,已经造成超过7800多万人感染,3900多万感染者死于艾滋病以及其他相关疾病。作为一种危害性极大的传染病,至今尚未研制出根治的药物以及预防的疫苗。设计有效的疫苗去诱导广谱高效中和抗体仍旧是预防HIV传播最根本的方法。近年来,HIV感染者出现自身免疫临床症状的报道越来越多,HIV感染者体内也检测到了自身反应性抗体,说明HIV感染过程伴随自身免反应。研究发现,部分典型的中和抗体也表现出多反应性/自身反应性。近年来,多反应抗体成为抗HIV感染领域研究热点,分析多反应抗体是否具有中和HIV病毒的能力,能否提供高效的免疫防护,从而以多反应抗体做为诱导靶点进行疫苗的设计研发。综上所述,HIV感染伴随自身免疫疾病,导致患者体内出现自身反应性抗体。在B细胞的发育过程中,不依赖Ig L表达分泌的IgH可能会绕开Ig L基因编辑的阴性筛选过程,最终以重链二聚体的形式分泌。我们的研究目的是:探究HIV感染者中是否存在HCAb,以及是否具有生物学功能,探讨这类重链抗体分泌表达的潜在影响因子以及分泌机制;探究多反应性抗体和中和抗体之间是否存在关联,自身反应性HCAb是否具有中和能力,探讨这类HCAb在HIV感染过程中的潜在作用机制及应用价值。2、主要研究方法(1)蛋白免疫印迹:检测从HIV感染者克隆的IgH基因是否独立表达及表达水平,并分析种系基因型对重链抗体分泌的影响。(2)ELISA:用ds DNA、ss DNA、LPS、insulin四种抗原检测HCAb的多反应性;利用gp140T、gp41检测对HIV抗原的亲和性;利用DWEYS检测对神经抗原的识别性。(3)细胞免疫荧光:以Hep-2细胞作为抗原,借助细胞免疫荧光实验分析重链抗体的自身反应性;以神经SH-SY5Y细胞作为抗原,分析HCAb对神经细胞的自身反应性。(4)CCK8实验和流式细胞术:利用SH-SY5Y细胞分析HCAb和NMDAR-DWEYS之间的相互作用。(5)分子构建:通过敲除重链基因CDR3片段分析CDR3结构域对HCAb多反应性/自身反应性影响;通过将CD4i中和抗体中重链可变区连接到表达载体,分析中和抗体重链基因表达的重链抗体的生物性能。(6)Hi Trap Lambda Fab Select和Hi Trap Kappaselect磁珠的应用:利用这两种磁珠可以特异性识别并结合抗体轻链恒定区的特性,从HIV感染者血清中分离重链抗体。3、结果(1)IgH基因的表达分析:对从慢性HIV感染者浆母细胞中克隆的重链基因进行单独表达,发现重链抗体可以在轻链缺失的情况下独立表达且分泌。经过对两组重链基因分泌率统计后发现,相比于对照组23.23%的可分泌率,HIV组82个重链抗体中,可分泌率达到74.39%。(2)基因型分析:在这些IgH基因中,重链抗体的分泌主要由可变区基因决定。对基因型和分泌率统计分析,发现VH3-23基因型在可分泌重链抗体中出现频数最多,属于重链抗体优先分泌基因型。(3)亚基组成及质谱结果:变性非还原电泳结果显示重链抗体蛋白分子量约为110k Da;变性还原电泳显示重链抗体解离成55k Da大小的重链多肽,说明HCAb是由两条相同的重链多肽通过二硫键连接组装而成的同源二聚体。变性还原电泳也显示重链抗体多肽和完整抗体重链多肽大小一致,说明这类HCAb具有完整的结构。质谱鉴定结果显示重链抗体具有C_H1结构域。(4)ELISA:HIV组HCAbs对ds DNA、ss DNA、LPS及insulin四种抗原具有明显的亲和性,ELISA实验显示73.3%的HCAbs具有多反应性;HCAbs对HIV抗原gp140T亲和性较低,对gp41亲和性较高,ELISA结果显示HIV组73.3%的HCAbs抗gp140。HCAbs对神经抗原也具有亲和性,ELISA结果显示HIV组73.3%的HCAbs抗DWEYS抗原。统计发现,HCAbs多反应性和抗gp140性存在强相关性。在HIV组,有66.7%的重链抗体对以上抗原的亲和性高于对应完整抗体。(5)细胞免疫荧光结果:HCAbs能够识别Hep-2细胞核和细胞质组分抗原,且细胞免疫反应较强,HIV组80%的HCAbs显示为自身抗体。重链抗体能够识别SH-SY5Y细胞膜表面抗原。(6)CCK8及流式细胞术:重链抗体和NMDAR-DWEYS能够发生交叉反应,并能够诱导SH-SY5Y细胞凋亡。(7)CDR3敲除实验:CDR3的敲除导致HCAbs对抗原的亲和性严重降低甚至消失。(8)HIV CD4i质粒构建:CD4i中和抗体中IgH基因表达的重链抗体具有较低的多反应性/自身反应性以及对gp140的亲和性。(9)HIV感染者中重链抗体发现:从HV感染者血清中分离到重链抗体,ELISA结果显示血清中HCAbs具有多反应性/自身反应性,以及明显的对gp140、gp41的亲和性。比对发现,HIV患者血清中HCAbs的抗gp140性质要高于重组HCAbs。4、结论(1)从慢性HIV感染者浆母细胞中克隆的IgH基因能够在缺失轻链的条件下在哺乳动物细胞中独立表达且分泌出具有完整结构的重链抗体,这种含有C_H1结构域的人源HCAb,不仅打破了细胞生物学中经典的抗体分泌理论,同时也对驼类重链抗体只能在缺失C_H1结构域的条件下才能分泌至胞外提出了挑战,说明影响重链抗体分泌的结构不仅限于C_H1区。这种HCAb可能以一种特殊的方式进行分泌。(2)从HIV感染者浆母细胞中克隆表达的重链抗体具有多反应性/自身反应性,这些具有生物活性功能的自身重链抗体进一步说明HIV感染过程伴随着自身免疫应答。(3)本实验证实了HIV感染者血清中存在全长的重链抗体,具有多反应性,且抗gp140的能力明显高于重组重链抗体。天然状态下全长重链抗体的发现对于重链抗体而言是非常重要的补充,不仅突破了C_H1区存在条件下重链抗体的分泌理论,对抗原的亲和性也说明这种全长人源HCAb参与免疫应答。多反应性以及明显的抗gp140性,则为研究多反应性抗体和中和抗体相关性提供了一种全新的研究样本,同时也为HIV疫苗设计提供了一种新的研究思路。更多还原

【Abstract】 1.Introduction As an important effector molecule in the humoral immune response,antibody plays an important role in body’s immune defense.In order to complete the secretion of antibody,two heavy chain polypeptides and two light chain polypeptides must be paired to form a tetramer.This assembly method has become a necessary condition for antibody secretion.However,the discovery of heavy chain antibody broke this classic theory of antibody secretion: Heavy chain antibody can be secreted independently in the absence of light chains.Heavy chain antibody mainly comes from two major groups: patients with heavy chain disease and camels.Heavy chain diseases are divided into ɑ-HCD,γ-HCD,and μ-HCD based on the type of heavy chain.HCAb in heavy chain diseases has long been regarded as a derivative product.Until the discovery of HCAb in camel serum,the proportion of HCAb in total camel serum antibody is as high as 50-75%,which is enough to show that HCAb plays a role in the immune defense system of camelid animals.Heavy chain antibody from the two sources generally has structural deletions.Camel HCAb lacks the CH1 domain.The missing parts of HCAb in heavy chain diseases include CH1 and VH domain.In the process of antibody synthesis,after the heavy chain polypeptide enters the endoplasmic reticulum,CH1 domain and its molecular chaperone Bi P are tightly bound,thereby preventing the secretion of heavy chain dimers.The deletion of CH1 domain in heavy chain diseases and camel HCAb is believed to affect the secretion of heavy chain antibody.Studies have shown that a type of IgH gene cloned from plasmablasts of patients with systemic lupus erythematosus can be independently expressed and secreted in mammalian cells.Unlike HCAb in camels and heavy chain diseases,this type of heavy chain antibody has complete VH and CH1 domains.Human immunodeficiency virus has infected more than 78 million people worldwide,and more than 39 million infected people have died of AIDS and other related diseases.As a very harmful infectious d isease,no radical cure or preventive vaccine has been developed so far.In recent years,there have been more and more reports of autoimmune clinical symptoms in HIV-infected persons.Autoreactive antibody has also been detected in HIV-infected persons,indicating that the process of HIV infection is accompanied by autoimmune diseases.At the same time,typical neutralizing antibody also exhibits polyreactivity/autoreactivity.Whether there is a connection between polyreactivity and neutralization ability,whether polyreactive antibody can provide high-efficiency immune protection,and whether polyreactive antibody could be used as neutralizing antibody for vaccine induction,polyreactive antibody have become a research hotspot in the field of anti-HIV infection.In summary,HIV infection is accompanied by autoimmune diseases,thus leading to the appearance of autoreactive antibody in patients.During the development of B cells,IgH that does not rely on Ig L expression and secretion may bypass the negative screening process of Ig L gene editing,and is finally secreted in the form of heavy chain dimers.The purpose of our research is to explore whether there are HCAbs in HIV-infected persons,and whether they have biological functions,to explore the potential factors affecting the secretion and expression of such heavy chain antibody and the secretion mechanism,to explore whether there is a relationship between polyreactive antibody and neutralizing antibody and whether autoreactive HCAb has the neutralizing ability,and to explore the potential mechanism and application value of HCAb during HIV infection.2.Methods(1)Western blotting: To detect the expression and expression level of IgH gene cloned from HIV-infected patients,and to analyze the effects of germline genotype on heavy chain antibody secretion.(2)ELISA: ds DNA,ss DNA,LPS,and insulin were used to detect the polyreactivity of HCAb;gp140T and gp41 were used to detect the affinity to HIV antigen.DWEYS was used to detect the recognition of neural antigens.(3)Immunofluorescence Staining: Hep-2 cells were used as antigens to analyze the autoreactivity of heavy chain antibody;neural SH-SY5 Y cells were used as antigens to analyze the autoreactivity of HCAb to nerve cells.(4)CCK8 experiment and Flow cytometry: SH-SY5 Y cells were used to analyze the interaction between HCAb and NMDAR-DWEYS.(5)Molecular construction: Analyzing the effects of CDR3 domain polyreactivity and autoreactivity by knocking out the CDR3 fragment of the heavy ch ain gene;by linking the heavy chain variable region of the CD4 i neutralizing antibody to the expression vector to analyze the neutralizing antibody biological properties of HCAbs.(6)Application of Hi Trap Lambda Fab Select and Hi Trap Kappa select magnetic beads: Using the characteristics of the two magnetic beads that can specifically recognize the bind to the constant region of the antibody light chain,and separate heavy chain antibody from the serum of HIV patients.3.Results(1)IgH gene expression analysis: The heavy chain genes cloned from plasmablasts of chronic HIV-infected patients were separately expressed,and it was found that heavy chain antibody can be independently expressed and secreted in the absence of light chain.After statistics on the secretion rate of heavy chain genes in the two groups,it was found the secretion rate of the control group is 23.23%,the HIV group is 74.39%.(2)Genotype analysis: Among these IgH genes,the secretion of heavy chain antibody is mainly determined by variable region genes.Statistical analysis of the genotype and secretion rate revealed that the VH3-23 genotype appeared the most frequently in secreted heavy chain antibody.(3)Subunit composition and mass spectrometry results: Denaturing non-reducing electrophoresis results showed that the molecular weight of the heavy chain antibody protein is about 110 k Da;denaturing reduction electrophoresis showed that the heavy chain antibody dissociates into a 55 k Da heavy chain polypeptide,indicating that HCAb is composed of two identical heavy chain polypeptides.Denaturing reduction electrophoresis also showed that the heavy chain antibody polypeptide and the complete antibody heavy chain polypeptide had the same size,indicating that this type of HCAb has a complete structure.The results of mass spectrometry showed that the heavy chain antibody has CH1 domain.(4)ELISA: HIV group HCAbs have obvious affinity to the four antigens of ds DNA,ss DNA,LPS and insulin.ELISA experiments showed that 73.3% of HCAbs are polyreactive;HCAbs have low affinity to HIV antigen gp140 T,gp41 has a high affinity,and ELISA results showed that 73.3% of HCAbs in the HIV group were anti-gp140.ELISA results showed that 73.3% of HCAbs in the HIV group were anti-DWEYS antigens.In the HIV group,66.7% of the heavy chain antibody has higher affinity for the above antigens than conventional antibody.(5)Immunofluorescence staining: HCAb can recognize Hep-2 nuclear and cytoplasmic component antigens,and the cellular immune response is s trong.A total of 80% of HCAb in the HIV group showed autoantibody.Heavy chain antibody can recognize SH-SY5 Y cell membrane surface antigens.(6)CCK8 and Flow cytometry: HCAb and NMDAR-DWEYS can cross-react,and can induce SH-SY5 Y cell apoptosis.(7)CDR3 knock-out experiment: CDR3 knock-out led to a severe decrease or even disappearance of the affinity of HCAbs to antigen.(8)HIV CD4 i plasmid construction: HCAbs of CD4 i neutralizing antibody showed low polyreactivity/self-reactivity and affinity for gp140.(9)Discovery of heavy chain antibody in HIV-infected patients: HCAbs were isolated from the serum of HV-infected persons.The ELISA results showed that the HCAbs in the serum have polyreactivity/autoreactivity,and obvious affinity for the gp140 and gp41.The comparison found that the anti-gp140 properties of HCAbs in serum of HIV patients were higher than that of recombinant HCAbs.4.Conclusion(1)The IgH gene cloned from plasmablasts of chronic HIV-infected persons can be independently expressed in mammalian cells without the light chain and secrete heavy chain antibody with a complete structure.This kind of HCAb not only breaks the classic antibody secretion theory in cell biology,but also challenges the secretion of camel heavy chain antibody to the outside of the cell only when the CH1 domain is missing,indicating that it affects the secretion of heavy chain antibody.The structure is not limited to the CH1 area.This HCAb may be secreted in a special way.(2)The heavy chain antibody cloned and expressed from the plasmablasts of HIV-infected persons is polyreactive/self-reactive.Self-HCAbs with biologically active indicate that the HIV infection process is accompanied by an autoimmune response.(3)This experiment confirmed that there is full-length heavy chain antibody in the serum of HIV-infected persons,and it is polyreactive and its anti-gp140 ability is significantly higher than that of recombinant HCAbs.The discovery of full-length HCAbs in the natural state is a very important su pplement to heavy-chain antibody.It not only breaks the secretion theory of heavy-chain antibody in the presence of CH1 region,the affinity for antigens also shows that full-length HCAb participates in the immune response.Polyreactivity and obvious anti-gp140 provide a new research sample for studying the correlation between polyreactive antibody and neutralizing antibody,as well as a new research idea for HIV vaccine design.更多还原