HIV-1自然感染中的中和抗体反应

中和抗体(Nab)可以防止I型人类免疫缺陷病毒(HIV-1)侵入靶细胞.HIV-1感染数周后即可诱导产生Nab,这些早期抗体只能特异性地中和自体病毒但不能中和异源性病毒.在一些慢性感染者体内则可以检测到可同时中和同源性和异源性病毒的广谱中和抗体(BNab).BNab的靶点通常位于包膜蛋白的保守区域.HIV-1 BNab的产生还受到病毒变异及结构遮盖等因素的限制,同时Nab的中和广度与病毒载量具有相关性.     

HIV-1包膜糖蛋白gp120 C3～C4区中和抗体保守表位氨基酸变异研究

目的：研究HIV-1包膜糖蛋白gp120 C3～CA区中和抗体保守表位氨基酸变异特征，探讨中和抗体表位变异与疾病进展关系，为开展中和抗体免疫治疗及疫苗设计奠定理论基础。方法：RT-PCR及nest-PCR扩增无症状HIV感染者、AIDS患者及疾病长期不进展者HIV-1 gp120 env C3～CA区基因，双脱氧终止法进行核酸序列测定，翻译为氨基酸序列，与HIV-1 Sequence Database参考毒株比对识别中和抗体保守表位氨基酸的变异。结果：HIVgp120 C3～CA区，无症状HIV感染者／AIDS患者CD4结合位点（CD4BS）、CD4诱导（CD4i）、2G12中和抗体保守表位氨基酸均存在变异，长期不进展者2G12中和抗体表位氨基酸存在变异，三组病例表位突变率差异未达显著性（P〉0．05）；CD4BS、CD4i．2G12变异表位构成比分别为15．8％、31．6％、52．6％，2G12表位变异高于CD4BS和CD4i表位，差异具有显著性（P〈0．05）；CD4BS表位变异见于K421R，CIMi表位变异见于R419S／K，K421R，均为单位点氨基酸变异，2G12表位变异见于S334N，N332E／Q，N386D，N392S／T，N448K／I，单、双位点氨基酸变异各半。结论：HIV-1包膜糖蛋白gp120 C3～CA区，无症状HIV感染者／AIDS患者CD4BS、CD4i、2G12中和抗体保守表位氨基酸均存在变异，长期不进展者表位氨基酸相对稳定，目前发现2G12表位存在变异。中和抗体表位中，2G12表位变异较CD4BS和CD4i表位多见。不同类型中和抗体保守表位氨基酸位点变异程度存在差异。     

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.     

SARS-CoV-2广谱中和抗体性质鉴定

目的：对严重急性呼吸综合征冠状病毒2(SARS-CoV-2)广谱中和抗体XMA09进行性质鉴定,探究其广谱中和突变株的潜在机制,为SARS-CoV-2的疫苗设计与广谱中和抗体筛选提供参考。方法：通过ExpiCHO真核表达系统与Protein A层析柱表达纯化XMA09蛋白;冷冻电镜技术确定XMA09识别的受体结构域（RBD）上的关键氨基酸位点;间接ELISA与表面等离子共振（SPR）技术检测XMA09对SARS-CoV-2及其突变株Spike蛋白的亲和力;采用基于水疱性口炎病毒（VSV）的假病毒系统检测XMA09对SARS-CoV-2野生型及突变株的中和能力。结果：本研究发现XMA09识别的表位较为保守,对多种突变株Spike蛋白均具有强结合能力,能广谱中和关切突变株（VOCs）,包括广泛流行的Omicron亚突变株BA.4/5。结论：XMA09是SARS-CoV-2广谱中和抗体,具有作为SARS-CoV-2治疗性抗体的潜力,并可为SARS-CoV-2的广谱疫苗设计与抗体药物开发提供参考意义。     

HIV-1B′亚型毒株抵抗VRC01抗体中和作用的机制研究

目的:探讨具有广谱中和活性的患者DRVI01血浆来源的HIV-1B′亚型毒株抵抗VRC01抗体中和的机制。方法:比较同期感染相同亚型且对VRC01抗体敏感的病毒序列,结合文献报道筛选可能影响VRC01中和作用的关键氨基酸,通过定点突变、不同来源膜蛋白序列交换,验证这些位点氨基酸对VRC01抗体中和作用的影响。结果:位于...     

野生型SARS-CoV-2感染康复者血清中SARS-CoV-2 Omicron突变株BA.1.1.529、BA.2和BA.3的中和抗体评估

目的 分析野生型SARS-CoV-2(2019新型冠状肺炎病毒)感染康复者血清中SARS-CoV-2 Omicron变异株BA.1.1.529、BA.2和BA.3的中和抗体滴度。方法 以8名已从SARS-CoV-2新冠肺炎中恢复的康复者血清为研究对象,采用ELISA方法检测野生型SARS-CoV-2感染康复者血清中SARS-CoV-2 Omicron变异株BA.1.1.529、BA.2和BA.3的中和抗体滴度,并将其与野生株SARS-CoV-2的中和抗体滴度进行对比分析。结果 在野生型SARS-CoV-2感染康复者血清中,SARSCoV-2 Omicron变异株BA.1.1.529中和抗体滴度显著低于野生株SARS-CoV-2中和抗体滴度（P<0.001）,而SARS-CoV-2Omicron的BA.1.1.529、BA.2和BA.3 3个亚系之间的中和抗体滴度无差异。结论 野生型SARS-CoV-2感染康复者血清中SARS-CoV-2 Omicron变异株BA.1.1.529、BA.2和BA.3的中和抗体滴度均较少。提示感染野生株SARS-CoV-2患者康复后可能对Omicro...     

针对SARS⁃CoV2不同突变株广谱特异性中和抗体的筛选与鉴定

目的 筛选对严重急性呼吸综合征冠状病毒2(SARS?CoV2)不同突变株具有广谱特异性的、高亲和性中和抗体,并进行功能验证.方法 用脂质体包被的SARS?CoV2刺突蛋白(S蛋白)mRNA疫苗对Balb/c小鼠进行2次免疫,测定小鼠血清中特异性抗体含量,选取抗体含量较高的小鼠进行冲击免疫;取冲击免疫后小鼠脾细胞与骨髓瘤...     

HIV-1中和抗体b12的研究进展

HIV-1由于其外膜糖蛋白结构多变可以很容易逃避抗体介导的中和作用.b12抗体是目前少数几种被证明具有广谱抗HIV-1活性的中和抗体,近来关于gp120结构研究表明在gp120上有一个保守的中和位点,且b12抗体可以和这个位点结合从而阻断靶细胞CD4与gp120作用,这一发现预示b12抗体对于新一代艾滋病疫苗设计具有重要的作用.本文即对b12抗体的有关研究进展作一综述.     

HIV-1中和抗体b12的研究进展

HIV-1由于其外膜糖蛋白结构多变可以很容易逃避抗体介导的中和作用.b12抗体是目前少数几种被证明具有广谱抗HIV-1活性的中和抗体,近来关于gp120结构研究表明在gp120上有一个保守的中和位点,且b12抗体可以和这个位点结合从而阻断靶细胞CD4与gp120作用,这一发现预示b12抗体对于新一代艾滋病疫苗设计具有重要的作用.本文即对b12抗体的有关研究进展作一综述.     

HIV-1中和抗体b12的研究进展

HIV-1由于其外膜糖蛋白结构多变可以很容易逃避抗体介导的中和作用.b12抗体是目前少数几种被证明具有广谱抗HIV-1活性的中和抗体,近来关于gp120结构研究表明在gp120上有一个保守的中和位点,且b12抗体可以和这个位点结合从而阻断靶细胞CD4与gp120作用,这一发现预示b12抗体对于新一代艾滋病疫苗设计具有重要的作用.本文即对b12抗体的有关研究进展作一综述.     

In vivo alteration of humoral responses to HIV-1 envelope glycoprotein gp120 by antibodies to the CD4-binding site of gp120

The binding of antibodies to the CD4-binding site (CD4bs) of the HIV-1 envelope glycoprotein gp120 has been shown to induce gp120 to undergo conformational changes that can expose and/or shield specific epitopes on gp120. Here, we study alterations in the antigenicity and immunogenicity of gp120 when complexed with human monoclonal antibodies (mAbs) specific for the CD4bs of gp120. The data showed that gp120 bound by anti-CD4bs mAbs had enhanced reactivity with mAbs to the V3 and N-terminal regions, but not with mAb to the C terminus. Moreover, mice immunized with the gp120/anti-CD4bs mAb complexes produced higher titers of gp120-specific serum IgG and IgA than mice immunized with uncomplexed gp120 or other gp120/mAb complexes. Notably, the enhanced antibody production was directed against V3 and correlated with better exposure of V3 on the gp120/anti-CD4bs mAb complexes. The higher antibody reactivity was evident against the homologous V3(LAI) peptide, but not against heterologous V3 peptides. Potent neutralization activity against HIV-1(LAI) was also observed in the sera from mice immunized with gp120/anti-CD4bs mAb complexes, although the sera exhibited poor neutralizing activities against other viruses tested. These results indicate that the anti-CD4bs antibodies alter the antigenicity and immunogenicity of gp120, leading to enhanced production of anti-gp120 antibodies directed particularly against the V3 region.     

Neutralizing activity of antibodies to the V3 loop region of HIV-1 gp120 relative to their epitope fine specificity

The V3 loop of HIV-1 gp120 is considered occluded on many primary viruses. However, virus sensitivity to neutralization by different V3 mAbs often varies, indicating that access to V3 is not restricted equally for all antibodies. Here, we have sought to gain a better understanding of these restrictions by determining the neutralizing activities of 7 V3 mAbs (19b, 39F, CO11, F2A3, F530, LA21, and LE311) against 15 subtype B primary isolates and relating these activities to the fine specificity of the mAbs. Not surprisingly, we found that most mAbs neutralized the same 2-3 viruses, with only mAb F530 able to neutralize 2 additional viruses not neutralized by the other mAbs. Epitope mapping revealed that positively-charged residues in or near the V3 stem are important for the binding of all the mAbs and that most mAbs seem to require the Pro residue that forms the GPGR β hairpin turn in the V3 tip for binding. Based on the mapping, we determined that V3 sequence variation accounted for neutralization resistance of approximately half the viruses tested. Comparison of these results to those of select V3 mAbs with overall better neutralizing activities in the light of structural information illustrates how an antibody's mode of interaction with V3, driven by contact residue requirements, may restrict the antibody from accessing its epitope on different viruses. Based on the data we propose an angle of interaction with V3 that is less stringent on access for antibodies with cross-neutralizing activity compared to antibodies that neutralize relatively fewer viruses.     

Identification of amino acids in the V3 region of gp120 critical for virus neutralization by human HIV-1-specific antibodies.

The importance of the dependence on single amino acids in the V3 region of HIV-1 gp120 was evaluated for virus neutralization and antibody-dependent cellular cytotoxicity (ADCC). Synthetic overlapping 15-mer peptides and a set of omission peptides covering amino acids 301-317 were used. Sera from 29 HIV-1-infected individuals at different stages of disease were tested for neutralization, ADCC and specific IgG reactivity. Six HIV-1 neutralizing monoclonal antibodies (mAb) acted as controls. All mAb reacted with a region (amino acids 304-318) of gp120, previously shown to induce neutralizing antibodies. The amino acids essential for reactivity were identified to be within the sequence GPGR (amino acids 312-315). The importance of this region for occurrence of neutralizing antibodies in infected humans was investigated using the same set of peptides. Out of 29 individuals, 21 were found to have neutralizing antibodies in titres between 100 and 1000. Among the neutralization-positive sera, 17/21 (81%) reacted with amino acids 304-318, compared with only one of eight sera (13%) negative in neutralization. When any of the four amino acids G, P, G or R were deleted, the seroreactivity decreased considerably. The conserved sequence GPGR was therefore considered to be the most important for neutralization in this region in human sera as well. Thus, the conserved sequence GPGR in the V3 region of gp120 is critical for virus neutralization by human HIV-1-specific antibodies.     

Focusing the immune response on the V3 loop, a neutralizing epitope of the HIV-1 gp120 envelope

Rabbits were immunized with a novel regimen designed to focus the immune response on a single neutralizing epitope of HIV-1 gp120 and thereby preferentially induce neutralizing antibodies (Abs). Animals were primed with gp120 DNA from a clade A Env bearing the GPGR V3 motif and/or a clade C Env bearing the GPGQ V3 motif, and boosted with one or more fusion proteins containing V3 sequences from clades A, B and/or C. Immune sera neutralized three of four Tier 1 primary isolates, including strains heterologous to the immunizing strains, and potent cross-clade-neutralizing activity was demonstrated against V3 chimeric pseudoviruses carrying in a Tier 1 Env, the consensus V3 sequences from clades A1, AG, B, AE, or F. The broadest and most potent neutralizing responses were elicited with the clade C gp120 DNA and a combination of V3-fusion proteins from clades A, B and C. Neutralizing activity was primarily due to V3-specific Abs. The results demonstrate that the immune response can be focused on a neutralizing epitope and show that the anti-V3 Abs induced recognize a diverse set of V3 loops.     

Ontogeny-based immunogens for the induction of V2-directed HIV broadly neutralizing antibodies

The development of a preventative HIV vaccine able to elicit broadly neutralizing antibodies (bNAbs) remains a major challenge. Antibodies that recognize the V2 region at the apex of the HIV envelope trimer are among the most common bNAb specificities during chronic infection and many exhibit remarkable breadth and potency. Understanding the developmental pathway of these antibodies has provided insights into their precursors, and the viral strains that engage them, as well as defined how such antibodies mature to acquire breadth. V2-apex bNAbs are derived from rare precursors with long anionic CDR H3s that are often deleted in the B cell repertoire. However, longitudinal studies suggest that once engaged, these precursors contain many of the structural elements required for neutralization, and can rapidly acquire breadth through moderate levels of somatic hypermutation in response to emerging viral variants. These commonalities in the precursors and mechanism of neutralization have enabled the identification of viral strains that show enhanced reactivity for V2 precursors from multiple donors, and may form the basis of germline targeting approaches. In parallel, new structural insights into the HIV trimer, the target of these quaternary antibodies, has created invaluable new opportunities for ontogeny-based immunogens designed to select for rare V2-bNAb precursors, and drive them toward breadth.     

Dendritic cells from HIV-1-infected patients naturally express HIV-1 gp120 V3 loop-derived peptide ligands

Little is known of the peptide ligands expressed in vivo on antigen-presenting cells (APC) or of the APC lineages involved. In this study we have addressed this question using HLA-DRβ1*0101-restricted CD4 T cell clones (TLC) specific for a synthetic peptide based on the HIV-1 gp120 V3 loop consensus sequence for the Clade B isolates predominantly found in European and North American patients. These TLC were found to respond, in a dose-dependent manner, to freshly isolated HIV-infected patient APC in the absence of exogenously added peptides. Further APC purification showed that the naturally expressed peptide ligands were present in both the APC lineages shown to be infected with the virus and were most strongly detectable on purified blood dendritic cells. Peptides based on consensus sequences of viruses isolated from one of the patients over the period when naturally expressed peptide ligands could be detected were all found to stimulate TLC proliferation. These studies, therefore, show that peptide ligands derived from natural infection are detectable on APC lineages, particularly on dendritic cells which play an important role in the immune response to viruses. Even small differences in sequence between the vaccine isolate and the natural infection, if they occur in the key residues of protective T cell epitopes, could therefore have a profound effect on the efficacy of vaccines against viruses with high rates of mutation.     

Characterization of antibodies that inhibit HIV gp120 antigen processing and presentation

Antibodies to the CD4-binding site (CD4bs) of HIV-1 envelope gp120 have been shown to inhibit MHC class II presentation of this antigen, but the mechanism is not fully understood. To define the key determinants contributing to the inhibitory activity of these antibodies, a panel of anti-CD4bs monoclonal antibodies with different affinities was studied and compared to antibodies specific for the chemokine receptor-binding site or other gp120 regions. Anti-CD4bs antibodies that completely obstruct gp120 presentation exhibit three common properties: relatively high affinity for gp120, acid-stable interaction with gp120, and the capacity to slow the kinetics of gp120 proteolytic processing. None of these antibodies prevents gp120 internalization into APC. Notably, the broadly virus-neutralizing anti-CD4bs IgG1b12 does not block gp120 presentation as strongly, because although IgG1b12 has a relatively high affinity, it dissociates from gp120 more readily at acidic pH and only moderately retards gp120 proteolysis. Other anti-gp120 antibodies, regardless of their affinities, do not affect gp120 presentation. Hence, high-affinity anti-CD4bs antibodies that do not dissociate from gp120 at endolysosomal pH obstruct gp120 processing and prevent MHC class II presentation of this antigen. The presence of such antibodies could contribute to the dearth of anti-gp120 T helper responses in chronically HIV-1-infected patients.     

Functional and immunochemical cross-reactivity of V2-specific monoclonal antibodies from HIV-1-infected individuals

The recent analysis of the first successful RV144 vaccine trial revealed that a high titer of plasma anti-V2 antibodies (Abs) correlated with a decreased risk of HIV-1 infection in vaccine recipients. To understand the mechanism of immune correlates, we studied seven anti-V2 monoclonal Abs (mAbs) developed from HIV-1 infected individuals. The V2 mAbs target conserved epitopes, including the binding site for α4β7 integrin, and are broadly cross-reactive with various gp120 proteins. Preferential usage of the VH1-69 gene by V2 mAbs may depend on selection by the same antigenic structure. Six of seven V2 mAbs weakly neutralized four to eight of the 41 pseudoviruses tested and resistance to neutralization was correlated with longer V2 domains. The data suggest the presence of shared, conserved structural elements in the V2 loop, and these can be used in the design of vaccine immunogens inducing broadly reactive Abs with anti-viral activities.     

Deletion of T lymphocytes in human CD4 transgenic mice induced by HIV-gp120 and gp120-specific antibodies from AIDS patients

CD4, a T cell receptor for major histocompatibility complex class II antigen, is a key regulator of immunological reactivities. When engaged together with the T cell antigen receptor, CD4 enhances immune reactions, whereas when ligated independently of the antigen receptor CD4 inhibits the activation of T cells or initiates their deletion. CD4 serves also as a receptor for the human immunodeficiency virus (HIV), which binds the receptor with high avidity through its envelope molecule, gp120. Studies in tissue culture have shown that its affinity to CD4 gives the virus opportunities to utilize CD4-mediated signaling and to manipulate immunocytes. We show here in human CD4 transgenic mice that appropriately cross-linked HIV envelope protein causes massive deletion of HIV-reactive T cells in vivo.