以假病毒为基础的HIV-1中和抗体检测体系的建立以及初步应用

目的 建立以假病毒为基础的HIV-1中和抗体检测方法,并对其进行初步应用.方法 从重组质粒中扩增出gp160基因片段,并克隆到pcDNA3.1质粒上,酶切鉴定得到阳性克隆.将阳性克隆分别和pSG3△env质粒共转染获得假病毒.用假病毒分别检测单克隆抗体和HIV-1抗体阳性血清的中和活性.结果 成功地获得了4株假病毒CHB01、CHB02、CHBC03和CHAE04.单克隆抗体4E10可以中和4株假病毒;单克隆抗体2F5不能中和CHBC03假病毒株,但可以中和CHB01、CHB02和CHAE04假病毒株;单克隆抗体IgG1b12 可以中和CHBC03、CHB01和CHB02假病毒株,则不能中和CHAE04假病毒株.43份HIV-1抗体阳性血清中针对不同假病毒的中和抗体明显不同.结论 所获得的假病毒可以用于中和抗体的检测,但不同假病毒株的中和特性不同.     

HIV-1膜抗原部分糖基化位点改造对免疫原性及假病毒形成的影响

目的 研究HIV-1膜蛋白(Env)特定糖基化位点改造对Env免疫原性及功能性假病毒形成能力的影响.方法 采用环形诱变,DpnⅠ筛选的方法对Env进行定点突变,单周期感染试验检测功能性假病毒形成能力,免疫小鼠,利用假病毒中和试验与ELISPOT分别检测突变对中和抗体和T细胞分泌Env特异的IFN-γ的影响.结果 N197Q突变体使Env诱导中和抗体的能力提高而诱导T细胞分泌Env特异的IFN-γ的能力降低,并使Env不能形成功能性假病毒;G2突变体(N624Q,N637Q)诱导的中和抗体能更好地中和假病毒74-2,仉中和假病毒Wt的能力下降,对Env诱导T细胞分泌Env特异的IFN-γ的能力和功能性假病毒形成无明显影响.结论 特定糖基化位点的改造影响假病毒的形成及Env的免疫原性.     

新型冠状病毒614D和614G假病毒生物学特性研究

目的利用HIV慢病毒包装系统构建新型冠状病毒(2019 novel coronavirus, 2019-nCoV)原始株614D和突变株614G假病毒, 并初步研究其生物学特性。方法将重组表达质粒pCDNA3.1-614D和pCDNA3.1-614G分别与慢病毒质粒psPAX2和pLenti CMV Puro LUC瞬时共转染293T细胞, 72 h后收集上清, 进行20%蔗糖垫层超速离心, 检测假病毒的滴度、形态、S蛋白表达和中和活性。结果间接免疫荧光检测可见S蛋白特异性荧光, Western blot分析可见2019-nCoV 614D和614G假病毒S蛋白表达, 透射电镜下可见假病毒颗粒具有明显刺突。614D和614G假病毒的滴度分别为1.12×104和2.52×104 TCID50/ml, 均能够中和S蛋白兔多克隆抗体, 表明假病毒具有特异性。结论本研究成功构建了2019-nCoV 614D和614G假病毒, 为建立基于假病毒的体外中和抗体检测平台奠定基础。     

HIV-1包膜蛋白V2区突变对V3区特异的中和抗体的影响

目的 研究HIV-1包膜蛋白V2区突变对V3区特异中和抗体与HIV-1中和作用的影响.方法 使用假病毒中和试验测定L175P突变对于V3区中和抗体对HIV-1中和能力的影响,ELISA方法测定上述中和抗体对gp120蛋白单体的结合力.结果 V2区突变L175P可以改变多个V3区特异性中和抗体对病毒的中和能力,但这些中和抗体对gp120蛋白单体的结合力不因V2区突变而改变.结论 V2区的L175P突变改变了gp120蛋白天然三聚体的结构,促进了V3区抗体的中和能力.

Abstract：

Objective To study the impact of V2 mutations on neutralizing ability of HIV-1-specific neutralizing antibodies. Methods We tested the influence of L175P mutation to the neutralizing ability of V3-specific antibodies by pseudotype virus and the binding affinity of those V3-spesific antibodies to gpl20 monomer by ELISA. Results We found L175P mutation changed the neutralizing ability of V3-specific antibodies. However, L175P mutation showed no effects on the binding affinity of these antibodies to gpl20 monomer. Conclusion Our results revealed the L175P mutation at V2 loop changed the natural trimmer structure of gp120 and enhanced the neutralizing ability of V3-specific antibodies.     

基于H5N1假病毒的体内中和试验模型的建立

目的建立H5N1假病毒的体内感染模型, 并对抗体FHA3的体内中和活性进行鉴定。方法依据A/Anhui/1/2005/H5N1毒株血凝素(hemagglutinin, HA)和神经氨酸酶(neuraminidase, NA)的序列信息, 构建重组表达质粒pcDNA3.1-HA5和pcDNA3.1-NA1, 并与质粒pNL4-3.Luc.R-E-共转染293T细胞制备H5N1假病毒上清, 电镜观察上清中假病毒颗粒形态。假病毒上清感染MDCK细胞后, 测定病毒滴度;经腹腔注射入BALB/c小鼠体内, 在感染后2、5、8、12 d进行生物发光成像, 检测假病毒体内感染情况。利用建立的小鼠感染模型, 评价抗体FHA3的体内功能活性。结果重组表达质粒pcDNA3.1-HA5和pcDNA3.1-NA1构建正确, 与pNL4-3.Luc.R-E-质粒共转染293T细胞可制备出高滴度的假病毒上清, 电镜下可见圆形的病毒颗粒。H5N1假病毒感染后, 小鼠体内发出较强的荧光, 而攻毒前给予抗体FHA3处理可减弱其荧光信号。结论成功构建出H5N1假病毒体内感染模型, 并证实抗体FHA3对假病毒的感染具有体内...     

中国92例HIV/AIDS患者HIV-1 gp41 2F5和4E10中和抗体表位变异研究

目的 探讨92例HIV/AIDS患者HIV-1病毒近膜端(membrane proximal external re-gion,MPER)中和抗体2F5和4E10保守表位ELDKWA、NWFDIT氨基酸变异特点,为中国HIV/AIDS患者免疫治疗以及疫苗设计提供数据.方法 Nest-PCR扩增HIV-1 env区gp41段基因,核酸序列测定,翻译为氨基酸与HIV-1 Sequence Database HXB Ⅱ参考株中和抗体表位数据比对,分析2F5、4E10中和表位氨基酸变异情况.结果 92例HIV/AIDS患者HIV-1外膜蛋白env gp41段中和抗体2F5、4E10保守表位氨基酸均存在突变;2F5中和抗体表位主要有E662A(14.1%)、K665S(17.4%)、A667K(16.3%)突变;4E10中和抗体表位主要有N671S(13.0%)、D674S(3.3%)、T676S(16.3%)突变;CRF_B'C亚型与B'亚型的2F5和4E10表位氨基酸突变差异具有统计学意义(P<0-05);CRF_B'C与CRF01_AE亚型2F5表位突变差异具有统计学意义(P<0.05);B'亚型缓慢进展者、HIV感染者和AIDS患者的4E10表位氨基酸突变差异具有统计学意义(P<0.05).结论 92例HIV/AIDS患者HIV.1包膜蛋白env gp41段中和抗体2F5、4E10中和表位氨基酸存在突变,且变异多样化;不同亚型中和抗体保守表位氨基酸位点变异有差异;B'亚型4E10中和抗体表位变异可能与疾病进展有一定联系.     

HIV-1 Tat蛋白对U87细胞活性及氧化应激的影响

目的:研究HAD和非HAD的艾滋病患者不同部位来源的Tat蛋白氨基酸位点变异及其对U87细胞氧化应激的影响。方法:采用BLAST和MEGA6对一例HAD患者(H)和一例非HAD患者(N)的基底节(BG)、脾脏(SPL)共4个部位来源的HIV-1 Tat蛋白进行序列分析,研究其氨基酸位点变异,并将 tat基因...     

抗VEGF中和抗体对Ⅱ型胶原诱导的关节炎形成的影响

目的：了解抗血管内皮生长因子（VEGF）中和抗体对Ⅱ型胶原（CoⅡ）诱导的关节炎（CIA）形成的影响。方法：于8～10wk龄的DBA／1J小鼠尾根部皮内注射鸡CoⅡ进行被动免疫，建立小鼠CIA模型，以CIA发生率、关节炎指数及关节组织的病理变化为指标，评价抗VEGF抗体对CIA形成的影响。结果：与对照组相比较．在CIA形成的早期，注射抗VEGF中和抗体能有效地抑制关节炎的产生及减轻其严重程度（P〈0．05）；而在CIA已形成后再注射抗VEGF中和抗体对关节炎的严重程度则无明显影响（P〉0．05）。结论：抗VEGF中和抗体明显抑制滑膜细胞增殖、血管新生及血管炎，因此有望成为类风湿性关节炎（RA）治疗的新型制剂。     

新型冠状病毒刺突蛋白的变异及其对中和活性的影响

2019年底发生的COVID-19疫情迅速传播至世界各地, 使全球公共卫生体系面临严峻考验。随着疫情的持续, 新型冠状病毒(severe acute respiratory syndrane coronavirus 2, SARS-CoV-2)变异株正在不断涌现。特别是病毒刺突蛋白的突变, 可以引起病毒感染性和抗原性改变, 从而导致病毒传染性增加, 以及现有疫苗保护效力的下降, 由此引起了流行毒株的替换。这也是目前疫情未得到有效控制的原因之一。目前主要的流行变异株都出现了一定程度的特性改变, 部分变异株与原始株相比对中和单抗、免疫血清及恢复期血清的中和敏感性出现了一定程度的下降。变异株的产生既与病毒本身特点有关, 也与传播宿主改变、免疫低下人群的慢性感染有关。应密切关注和监测不断出现的变异株, 并对其功能特性进行系统研究, 为疫苗研发和免疫策略的制定提供参考。     

Structural insights into key sites of vulnerability on HIV-1 Env and influenza HA

Human immunodeficiency virus-1 (HIV-1) envelope protein (Env) and influenza hemagglutinin (HA) are the surface glycoproteins responsible for viral entry into host cells, the first step in the virus life cycle necessary to initiate infection. These glycoproteins exhibit a high degree of sequence variability and glycosylation, which are used as strategies to escape host immune responses. Nonetheless, antibodies with broadly neutralizing activity against these viruses have been isolated that have managed to overcome these barriers. Here, we review recent advances in the structural characterization of these antibodies with their viral antigens that defines a few sites of vulnerability on these viral spikes. These broadly neutralizing antibodies tend to focus their recognition on the sites of similar function between the two viruses: the receptor-binding site and membrane fusion machinery. However, some sites of recognition are unique to the virus neutralized, such as the dense shield of oligomannose carbohydrates on HIV-1 Env. These observations are discussed in the context of structure-based design strategies to aid in vaccine design or development of antivirals.     

The impact of envelope glycoprotein cleavage on the antigenicity, infectivity, and neutralization sensitivity of Env-pseudotyped human immunodeficiency virus type 1 particles

Endoproteolytic processing of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoproteins is an obligate part of the biosynthetic pathway that generates functional, fusion-competent Env complexes, which are then incorporated into infectious virions. We have examined the influence of cleavage on Env-specific antibody reactivity, Env incorporation into pseudovirions, and the infectivity and neutralization sensitivity of Env-pseudotyped viruses. To do so, we have used both incompletely processed wild-type (Wt) Env and engineered, cleavage-defective Env mutants. We find that there is no simple association between antibody reactivity to cell surface-expressed Env, and the ability of the same antibody to neutralize virus pseudotyped with the same Env proteins. One explanation for the absence of such an association is the diverse array of Env species present on the surface of transiently transfected cells. We also confirm that cleavage-defective mutants are antigenically different from Wt Env. These findings have implications for the use of Env binding assays as predictors of neutralizing activity, and for the development of cleavage-defective Env trimers for use as subunit immunogens.     

Changing sensitivity to broadly neutralizing antibodies b12, 2G12, 2F5, and 4E10 of primary subtype B human immunodeficiency virus type 1 variants in the natural course of infection

The conserved nature of the epitopes of the four broadly neutralizing antibodies (BNAbs), b12, 2G12, 2F5, and 4E10, may imply that the sensitivity of HIV-1 for these BNAbs remains fairly constant over the course of infection. Here, we demonstrate that viruses isolated early during the course of infection were mostly sensitive to HIVIg and antibody neutralization, although variation was observed in neutralization sensitivity of coexisting viruses to the different antibodies as well as between viruses from different patients. HIV-1 resistance to HIVIg developed relatively early during follow-up in three out of five patients, while early, b12 sensitive viruses in three out of five patients were replaced by b12 resistant variants relatively late in infection. In contrast, viruses generally remained sensitive to 2F5 and 4E10 neutralization over the course of infection, although 2F5 and/or 4E10 resistant variants did emerge later in infection in four out of five patients. In most patients, HIV-1 resistance to 2F5 or 4E10 did not correlate with mutations at critical amino acid positions in their defined epitopes. Viruses resistant to 2G12-mediated neutralization were present throughout the course of infection. As viral resistance against BNAb-mediated neutralization generally developed when autologous serum neutralizing activity had faded, it seems unlikely that these changes are driven by escape from autologous humoral immunity.     

Biochemical and biophysical comparison of cleaved and uncleaved soluble, trimeric HIV-1 envelope glycoproteins

Human immunodeficiency virus type 1 (HIV-1) entry into host cells is mediated by the trimeric envelope glycoprotein complex (Env). Accordingly, the Env proteins are the targets for neutralizing antibodies (NAbs) and are the focus of vaccines intended to induce NAbs. Because the Env complex is labile, soluble recombinant Env (gp140) trimers require engineering to stabilize them sufficiently for use as immunogens. Trimeric forms of gp140 trimers can be created that are either cleavage-competent or cleavage-defective at the junction between the gp120 and gp41 subunits. As functional trimers are cleaved at this site, the question arises as to whether cleavage affects the antigenic structure of the Env complex in a way that is relevant to vaccine design. Here, we present a comparative analysis of the antigenicity profiles of cleaved and uncleaved gp140 trimers derived from the KNH1144 (subtype A) virus that are otherwise closely sequence-matched. While cleavage did not affect the exposure of NAb epitopes on the gp140 trimers, non-neutralizing antibodies to gp41 epitopes bound much more strongly to uncleaved trimers. Hence cleavage does alter the structure of the HIV-1 Env complex.     

N-terminal substitutions in HIV-1 gp41 reduce the expression of non-trimeric envelope glycoproteins on the virus

The native, functional HIV-1 envelope glycoprotein (Env) complex is a trimer of two non-covalently associated subunits: the gp120 surface glycoprotein and the gp41 transmembrane glycoprotein. However, various non-functional forms of Env are present on virus particles and HIV-1-infected cells, some of which probably arise as the native complex decays. The aberrant forms include gp120-gp41 monomers and oligomers, as well as gp41 subunits from which gp120 has dissociated. The presence of non-functional Env creates binding sites for antibodies that do not recognize native Env complexes and that are, therefore, non-neutralizing. Non-native Env forms (monomers, dimers, tetramers and aggregates) can also arise when soluble gp140 proteins, lacking the cytoplasmic and transmembrane domains of gp41, are expressed for vaccine studies. We recently identified five amino acids in the gp41 N-terminal region (I535, Q543, S553, K567 and R588) that promote gp140 trimerization. We have now studied their influence on the function and antigenic properties of JR-FL Env expressed on the surfaces of pseudoviruses and Env-transfected cells. The 5 substitutions in gp41 reduce the expression of non-trimeric gp160s, without affecting trimer levels. Pseudovirions bearing the mutant Env are fully infectious with similar kinetics of Env-mediated fusion. Various non-neutralizing antibodies bind less strongly to the Env mutant, but neutralizing antibody binding is unaffected. Hence the gp41 substitutions do not adversely affect Env structure, supporting their use for making new Env-based vaccines. The mutant Env might also help in studies intended to correlate antibody binding to virus neutralization. Of note is that the 5 residues are much more frequent, individually or collectively, in viruses from subtypes other than B.     

Env length and N-linked glycosylation following transmission of human immunodeficiency virus Type 1 subtype B viruses

Whether there is selection for specific viral Env variants upon HIV-1 transmission is controversial. We examined the V1V2 and V1V4 regions of Env in 10 new and 8 previously described transmission pairs infected with HIV-1 subtype B, including a total of 9 pairs in which the infecting partner had developed substantial viral diversity prior to transmission. We found that during transmission of HIV-1 subtype B, as well as for other subtypes reported in the past, viral populations in recipients undergo substantial genetic bottlenecks, as well as weak evidence for a propensity to replicate viruses with shorter variable loops and fewer potential N-linked glycosylation sites.     

Dominant-negative effect of hetero-oligomerization on the function of the human immunodeficiency virus type 1 envelope glycoprotein complex

The human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein forms trimers that mediate interactions with the CD4 receptor and a co-receptor on the target cell surface, thereby triggering viral fusion with the cell membrane. Cleavage of Env into its surface, gp120, and transmembrane, gp41, moieties is necessary for activation of its fusogenicity. Here, we produced pseudoviruses with phenotypically mixed wild-type (Wt) and mutant, cleavage-incompetent Env in order to quantify the effects of incorporating uncleaved Env on virion infectivity, antigenicity and neutralization sensitivity. We modeled the relative infectivity of three such phenotypically mixed viral strains, JR-FL, HXBc2 and a derivative of the latter, 3.2P, as a function of the relative amount of Wt Env. The data were fit very closely (R(2) > 0.99) by models which assumed that only Wt homotrimers were functional, with different approximate thresholds of critical numbers of functional trimers per virion for the three strains. We also produced 3.2P pseudoviruses containing both a cleavage-competent Env that is defective for binding the neutralizing monoclonal antibody (NAb) 2G12, and a cleavage-incompetent Env that binds 2G12. The 2G12 NAb was not able to reduce the infectivity of these pseudoviruses detectably. Their neutralization by the CD4-binding site-directed agents CD4-IgG2 and NAb b12 was also unaffected by 2G12 binding to uncleaved Env. These results further strengthen the conclusion that only homotrimers consisting of cleaved Env are functional. They also imply that the function of a trimer is unaffected sterically by the binding of an antibody to an adjacent trimer.     

HIV-1 pol抗原HLA-A＊0201限制性低亲和性CTL表位预测及改造分析

目的:初步筛选HIV-1 pol抗原的HLA-A*0201 限制性低亲和性CTL表位,预测并初步鉴定修饰后的表位与HLA-A*0201之间亲和性的变化.方法:采用超基序、蛋白酶解、HLA结合力等预测相结合的办法筛选HLA-A*0201限制性低亲和性CTL表位,通过氨基酸置换适当修饰,并以T2细胞检测HLA-A*0201分子与肽的亲和力和稳定性来评价修饰后表位.结果:筛选出的低亲和性CTL候选表位,经修饰后与HLA-A*0201 之间的亲和性均有不同程度的提高.其中,YVSLSFPQI (pol52-60Y1),YVSQIIEQL(pol673-681Y1),YIQKETWEA(pol548-556Y1)HLA-A*0201呈高亲和力结合,同时肽-HLA-A*0201复合物半数解离时间(Dissociation Complex50,DC50)均大于8 h.结论:预测的pol抗原表位经过修饰可能会成为潜在的HLA-A*0201限制性表位.