不同治疗效果的艾滋病患者抗病毒治疗前HIV-1gp120准种特征差异

目的 探讨HIV-1 gp120准种在不同治疗效果的艾滋病患者抗病毒治疗前的特征差异.方法 回顾性收集治疗方案为AZT+NVP+3TC的艾滋病患者在抗病毒治疗前的血浆样本,包括病毒抑制(VS)组12例,治疗失败(TF)组12例.采用单基因组扩增技术获得gp120准种序列,分析比较遗传多样性、氨基酸长度、潜在糖基化位点及特征性氨基酸的特点.结果 本研究共获得gpl20序列365条序列,其中VS组168条(6-20条),TF组197条(7-28条).TF组的gp120准种复杂度高于VS组,差异有统计学意义(P=0.003);TF组的gp120准种dS及dN中位数均高于VS组(P=0.017;P=0.002).TF组HIV-1准种gpl20氨基酸长度长于VS组(P=0.00l).TF与VS组的gpl20准种相比共有9个氨基酸位点存在明显差异,多数分布在V1/V2区(6/9,66.6％).结论 不同治疗效果的艾滋病患者治疗前的HIV-1 gp120准种基因特征存在差异,TF组患者来源的HIV-1准种遗传多样性更高.     

自噬对HIV-1 gp120V3环介导的海马神经元L型钙离子通道电流的影响

 目的:观察哺乳动物雷帕雷素靶蛋白（mTOR）依赖性自噬信号转导通路对HIV-1 gp120V3环介导的海马神经元L-型钙离子通道电流的影响。方法：取出生1 d内的乳鼠海马神经元原代培养7 d后用于实验。实验分为2个平行实验组,即空白对照组、gp120V3组、自噬阻断剂3-甲基腺嘌呤（3-MA）组和gp120V3+3-MA组;空白对照组、gp120V3组、自噬激动剂雷帕雷素（rapamycin)组和gp120V3+rapamycin组。以膜片钳技术记录海马神经元的L型钙离子通道电流。结果：gp120V3组及3-MA组与空白对照组比较,L型钙离子通道电流密度增大（P<0.05）;gp120V3+3-MA组与gp120V3组比较,L-型钙离子通道电流密度增大（P<0.05）;rapamycin组与空白对照组比较,L型钙离子通道电流密度减小（P<0.05）;gp120V3+rapamycin组与gp120V3组比较,L型钙离子通道电流密度减小（P<0.05）。结论：从电生理角度证明,自噬可能参与了HIV-1gp120V3环介导的海马神经元L型钙离子通道电流的改变。     

HIV-1广谱中和性单克隆抗体的研究进展

从HIV感染患者血清中分离的针对HIV-1包膜上保守表位的中和性单克隆抗体(mAb)很多,识别的表位分别是HIV-1gp120上CD4结合位点(CD4bs),gp41的跨膜前域MPER,gp120表面包膜的高甘露聚糖,gp120上V2/V3环,这些抗体对HIV-1疫苗免疫原的研究设计至关重要.本文主要综述了各中和性单克隆抗体的结构特征,识别的表位,抗体与病毒包膜之间的相互作用.     

Ⅰ型人类免疫缺陷病毒包膜糖蛋白CD4结合位点修饰诱导小鼠体液免疫反应的实验研究

目的 探讨Ⅰ型人类免疫缺陷病毒(HIV-1)包膜糖蛋白(Env) gp120的CD4结合位点(CD4BS)核心区第423、425和431位氨基酸三联突变ING/MKE对其诱导体液免疫反应的影响.方法 构建HIV-1原代毒株06044包膜gp120 ING/MKE三联突变表达载体pcT22-06044 gp120T-ING/MKE(gp120T-ING/MKE),在体外转染的HEK293T细胞表达三聚化野生型gp120Twt蛋白和突变体gp120T-ING/MKE蛋白.免疫BALB/c小鼠后检测结合抗体、中和抗体和骨髓抗原特异性浆细胞.结果 获得真核表达载体gp120T-ING/MKE.转染后,在293T细胞培养上清中检测到了三聚化重组蛋白.末次免疫后14 d,gp120Twt和gp120T-ING/MKE免疫血清中结合抗体滴度都大于1∶1 000,但两组血清抗体滴度无显著差异.突变体组骨髓特异性浆细胞分泌水平和非特异浆细胞水平均低于野生型gp120免疫组.野生型和突变体免疫诱导血清抗体的中和活性均不强.结论 HIV-1包膜蛋白CD4结合区423、425和431位三联突变没有改善gp120蛋白的免疫原性.     

人类免疫缺陷病毒-1包膜糖蛋白进化对抗原表位及病毒亲嗜性的影响

Objective To investigate the evolution of HIV-1 envelope (env) gene from the individuals infected by the virus from one donor, the entry mediated by the envelope glycoprotein and the variation in the main neutralizing epitopes of envelope. Methods The genetic distances of the HIV-1 envelope genes derived from previous studies were analyzed. A series of envelope-pseudotyped viruses were constructed by co-transfecting HEK293T cells with a HIV-1 plasmid bearing the firefly luciferase reporter gene and an envelope expression plasmid. The entry ability of the envelope-pseudotyped viruses into U87. CD4. CCR5 or U87. CD4. CXCR4 cell lines was examined. The ami-no acid sequences representing the epitopes to the broad-neutralizing antibodies within the envelope glycoproteins were also investigated. Results It was found that the genetic distance of the 24 env genes with complete open reading frame was (7.91 ±0.78)% towards HIV-1 CNHN24, and (6.90 ±0.79)% towards RL42. Among the variable regions, the genetic distance of V1/V2 showed the biggest distance, and that of V3 showed the smallest distance. There were CCR5-tropic, CXCR4-tropic and CCR5/CXCR4-dual-tropic Env-pseudoviruses. Furthermore, in these envelopes, the epitopes to IgG1 b12 2F5 and 4E10 antibody were conserved, while the epitope to 447-52D was variable. Conclusion There is definite env gene variation among the viruses derived from the same donor. The variation influences the entry ability and tropism of emelope pseudoviruses. The epitopes to the main broad-neutralizing antibodies are conserved.     

人类免疫缺陷病毒-1包膜糖蛋白进化对抗原表位及病毒亲嗜性的影响

Objective To investigate the evolution of HIV-1 envelope (env) gene from the individuals infected by the virus from one donor, the entry mediated by the envelope glycoprotein and the variation in the main neutralizing epitopes of envelope. Methods The genetic distances of the HIV-1 envelope genes derived from previous studies were analyzed. A series of envelope-pseudotyped viruses were constructed by co-transfecting HEK293T cells with a HIV-1 plasmid bearing the firefly luciferase reporter gene and an envelope expression plasmid. The entry ability of the envelope-pseudotyped viruses into U87. CD4. CCR5 or U87. CD4. CXCR4 cell lines was examined. The ami-no acid sequences representing the epitopes to the broad-neutralizing antibodies within the envelope glycoproteins were also investigated. Results It was found that the genetic distance of the 24 env genes with complete open reading frame was (7.91 ±0.78)% towards HIV-1 CNHN24, and (6.90 ±0.79)% towards RL42. Among the variable regions, the genetic distance of V1/V2 showed the biggest distance, and that of V3 showed the smallest distance. There were CCR5-tropic, CXCR4-tropic and CCR5/CXCR4-dual-tropic Env-pseudoviruses. Furthermore, in these envelopes, the epitopes to IgG1 b12 2F5 and 4E10 antibody were conserved, while the epitope to 447-52D was variable. Conclusion There is definite env gene variation among the viruses derived from the same donor. The variation influences the entry ability and tropism of emelope pseudoviruses. The epitopes to the main broad-neutralizing antibodies are conserved.     

在鸡痘病毒中共表达HIV-1 gp120与IL-2基因

外膜蛋白gp120是HIV—1主要结构蛋白之一，它的V3区存在着中和抗体决定簇、CTL决定簇，以及对巨噬细胞和脑组织纤维特异性识别的区，与HIV—1可诱导中和抗体应答、特异性细胞毒反应及分子致病机制密切相关。针对V3环的抗体可干扰gp120和CCR5之间的结合，这一发现为艾滋病的预防和治疗提供了新的途径。现已证明，多数细胞因子具有良好的免疫佐剂性能，对免疫应答具有刺激作用。本研究拟利用我国鸡痘病毒疫苗株为载体，构建可共表达HIV-1 gp120与IL-2基因的重组鸡痘病毒载体，为开发HIV重组病毒活载体疫苗提供一种安全的新途径。     

5株不同亚型HIV-1毒株gp120序列特征分析及其蛋白的表达

目的 分析我国HIV-1主要亚型毒株的gp120区域的序列特征,并表达出gp120糖基化蛋白,为研究gp120的致病机制和设计疫苗奠定基础.方法 利用巢式PCR从来自于不同省份的5名HIV-1感染者外周血单个核细胞DNA中扩增全长gp120基因并测序,对序列特征进行分析,并将获得的gp120基因克隆入真核表达载体,体外表达获得gp120糖基化蛋白.结果 序列分析显示,此5条gp120序列分属HIV-1 Thai-B、BC重组和AE重组哑型,虽然亚型不同,但这些gp120序列在相同的位置都存在着一些保守的糖基化位点,且都具备相同的Furin蛋白酶第一切割位点,与参考序列比较发现,不同的HIV亚型序列中,除V3序列长度较为保守外,V1、V2、V4、V5各区域都有不同程度的缺失现象,与BC重组和AE重组亚型相比,Thai-B亚型V3的顶端环呈现出多种组合.最终将这5条gpl20序列都克隆人真核表达载体并表达出糖基化的gp120蛋白.结论 在设计疫苗和检测试剂时应考虑到膜区的高变性,gp120糖蛋白的体外表达有利于进一步开展针对我国主要HIV流行毒株膜蛋白致病机制和疫苗学的研究.     

gp120mAb对gp120抑制大鼠海马脑片CA1区LTP作用的研究

目的探讨人类免疫缺陷病毒Ⅰ型(HIV-1)的包膜糖蛋白gp120特异抗体gp120mAb对gp120引起大鼠海马脑片CA1区的突触传递及可塑性变化的影响.方法应用离体脑片记录技术,记录大鼠海马CA1区的兴奋性突触后电位(EPSP),研究gp120mAb对gp120抑制高频电刺激Schaffer侧支引起的鼠海马长时程增强效应(LTP)作用的影响.结果gp120对高频电刺激(HFS,100 Hz,1 000 ms×2,串间隔20秒,共2次)Schaffer侧支引起的大鼠海马CA1区LTP产生抑制作用,而对其基础EPSP没有影响.用浓度为200 pmol/L的gp120灌流脑片,可引起LTP的维持发生抑制.这种抑制作用可被gp120特异抗体gp120mAb(50 ng/ml)所拮抗.结论gp120mAb可能是通过拮抗gp120抑制海马CA1区的LTP诱发和维持而参与艾滋病痴呆(HIV-1 associated dementia,HAD)的形成.     

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。     

High titer HIV-1 V3-specific antibodies with broad reactivity but low neutralizing potency in acute infection and following vaccination

Identifying the earliest neutralizing antibody specificities that are elicited following infection or vaccination by HIV-1 is an important objective of current HIV/AIDS vaccine research. We have shown previously that transplantation of HIV-1 V3 epitopes into an HIV-2 envelope (Env) scaffold provides a sensitive and specific means to detect and quantify HIV-1 V3 epitope specific neutralizing antibodies (Nabs) in human sera. Here, we employ this HIV-2/HIV-1 V3 scaffolding strategy to study the kinetics of development and breadth of V3-specific Nabs in longitudinal sera from individuals acutely infected with clade C or clade B HIV-1 and in human subjects immunized with clade B HIV-1 immunogens. HIV-2/HIV-1 chimeras containing V3 sequences matched to virus type (HIV-2 or HIV-1), subtype (clade B or C), or strain (autologous or heterologous) were used as test reagents. We found that by 3-8 weeks post infection, 12 of 14 clade C subjects had a median IC₅₀ V3-specific Nab titer of 1:700 against chimeric viruses containing a heterologous clade C V3. By 5 months post-infection, all 14 subjects were positive for V3-specific Nabs with median titers of 1:8000 against heterologous clade C V3 and 1:1300 against clade B V3. Two acutely infected clade B patients developed heterologous clade B V3-specific Nabs at titers of 1:300 and 1:1800 by 13 weeks of infection and 1:5000 and 1:11000 by 7 months of infection. Titers were not different against chimeras containing autologous clade B V3 sequences. Each of 10 uninfected normal human volunteers who were immunized with clade B HIV-1 Env immunogens, but none of five sham immunized control subjects, developed V3-specific Nabs titers as high as 1:3000 (median 1:1300; range 1:700-1:3000). None of the HIV-1 infected or vaccinated subjects had antibodies that neutralized primary HIV-1 virus strains. These results indicate that high-titer, broadly reactive V3-specific antibodies are among the first to be elicited during acute and early HIV-1 infection and following vaccination but these antibodies lack neutralizing potency against primary HIV-1 viruses, which effectively shield V3 from antibody binding to the functional Env trimer.     

Improvement of neutralizing activity of human scFv antibodies against hepatitis B virus binding using CDR3 V(H) mutant library

CDR3 of the heavy-chain variable region of immunoglobulin is a region in which somatic mutation occurs heavily after secondary antibody response, resulting in an affinity maturation of antibodies in vivo. The aim of this study was to improve the affinity of a human single-chain variable fragment (scFv) specific for pre-S1 of hepatitis B virus (HBV) by introducing random mutagenesis in CDR3 variable region of heavy chain (V(H)) of the parental scFv clone 1E4. By using a BIAcore for panning and screening, we have selected three clones (A9, B2, and B9) with lower highest affinity (K(D)) than 1E4. Affinities of selected clones ranged from 1.7 x 10(7) mol/L to 6.3 x 10(8) mol/L, which were increased by factors of 1.4 to 4.0, respectively, compared to the parental clone. Binding inhibition assay using flow cytometry and polymerase chain reaction revealed that B2 (6.4 x 10(8) mol/L) had a higher neutralizing activity against pre-S1 or HBV virion binding to liver cell line. This anti-pre-S1 scFv can be considered as a potential therapeutic tool for a passive immunotherapy for HBV infection.     

Combinatorial library cloning of human antibodies to Streptococcus pneumoniae capsular polysaccharides: variable region primary structures and evidence for somatic mutation of Fab fragments specific for capsular serotypes 6B, 14, and 23F

Antibodies specific for capsular polysaccharides play a central role in immunity to encapsulated Streptococcus pneumoniae, but little is known about their genetics or the variable (V) region polymorphisms that affect their protective function. To begin to address these issues, we used combinatorial library cloning to isolate pneumococcal polysaccharide (PPS)-specific Fab fragments from two vaccinated adults. We determined complete V region primary structures and performed antigen binding analyses of seven Fab fragments specific for PPS serotype 6B, 14, or 23F. Fabs were of the immunoglobulin G2 or A isotype. Several V(H)III gene segments (HV 3-7, 3-15, 3-23, and 3-11) were identified. V(L) regions were encoded by several kappa genes (KV 4-1, 3-15, 2-24, and 2D-29) and a lambda gene (LV 1-51). Deviation of the V(H) and V(L) regions from their assigned germ line counterparts indicated that they were somatically mutated. Fabs of the same serotype specificity isolated from a single individual differed in affinity, and these differences could be accounted for either by the extent of mutation among clonal relatives or by usage of different V-region genes. Thus, functionally disparate anti-PPS antibodies can arise within individuals both by activation of independent clones and by intraclonal somatic mutation. For one pair of clonally related Fabs, the more extensively mutated V(H) was associated with lower affinity for PPS 14, a result suggesting that somatic mutation could lead to diminished protective efficacy. These findings indicate that the PPS repertoire in the adult derives from memory B-cell populations that have class switched and undergone extensive hypermutation.     

Monoclonal antibodies differentially affect the interaction between the hemagglutinin of H9 influenza virus escape mutants and sialic receptors

To determine the receptor binding properties of various H9 influenza virus escape mutants in the presence and absence of antibody, sialyloligosaccharides conjugated with biotinylated polyacrylamide were used. A mutant virus with a L226Q substitution showed an increased affinity for the Neu5Acalpha2-3Galbeta1-4Glc. Several escape mutants viruses carrying the mutation N193D bound to Neu5Acalpha2-6Galbeta1-4GlcNAc considerably stronger than to Neu5Acalpha2-6Galbeta1-4Glc. Several monoclonal antibodies unable to neutralize the escape mutants preserved the ability to bind to the hemagglutinin as revealed by enzyme-linked immunosorbent assay. In each case, the bound monoclonal antibodies did not prevent the binding of the mutant HA to high affinity substrates and did not displace them from the virus binding sites. Together, these data suggest that amino acid changes selected by antibody pressure may be involved in the specificity of host-cell recognition by H9 hemagglutinin and in the ability of viruses with these mutations to escape the neutralizing effect of antibodies in a differential way, depending on the specificity of the host cell receptor. It may be important in the natural evolution of the H9 subtype, a plausible candidate for the agent likely to cause a future pandemic.     

Fine characterization of a V3-region neutralizing epitope in human immunodeficiency virus type 2

We have previously identified two distinct antigenic sites in the third variable region (V3) of human immunodeficiency virus type 2 (HIV-2) corresponding to the principal neutralizing determinant (PND) of HIV-1, the conserved Phe-His-Ser-Gln and Trp-Cys-Arg motifs (positions 315-318 and 329-331), which possibly interact to form a discontinuous antigenic site. The aim of this study was to further identify and characterize the immunogenic sites in the V3-loop of HIV-2 that are important in the binding of neutralizing antibodies and to study in detail the importance of different configurations of peptides corresponding to this region. Peptides representing modifications of the V3-region of HIV-2(SBL6669-ISY) were used for immunization of guinea pigs. With one exception, both the Phe-His-Ser-Gln and the Trp-Cys-Arg motifs were required in the peptide sequences to obtain neutralizing hyperimmune guinea pig sera, and the highest titers were obtained after immunization with 20-27 amino acids (aa) long peptides. Neither substitutions nor deletions of residues between the two motifs, nor the addition of peptide sequences representing a T-helper epitope improved the induction of neutralizing antibodies. Computer simulation modeling revealed that the Phe-315, His-316, Trp-329 and Cys-330 are likely to participate in the formation of a discontinuous epitope. Taken together, these data support the hypothesis that the well conserved motifs FHSQ (positions 315-318) and WCR (positions 329-331) of the HIV-2(SBL6669) V3 region are important targets for neutralizing antibodies, and this may have implications for the design of a future HIV-2 vaccine.     

Conservation of the DENV-2 type-specific and DEN complex-reactive antigenic sites among DENV-2 genotypes

The envelope (E) protein is composed of three domains (ED1, ED2 and ED3) with ED3 targeted by the most potent neutralizing antibodies. DENV-2 strains can be divided into six genotypes. Comparison of ED3 of representative strains of the six genotypes revealed that there are nine variable residues that are specific to a given genotype. Recombinant ED3s (rED3s) of six different DENV-2 strains representing all nine variable residues were expressed, and their reactivity against a panel of two DENV-2 type-specific and three DENV complex-reactive monoclonal antibodies (mAbs) were compared. The differences in binding affinity to the rED3s representing different DENV-2 genotypes were relatively small, with the exception of type-specific-mAb 3H5 that showed up to 10-fold differences in binding between genotypes. Overall the binding differences did not lead to detectable differences in neutralization. Based on these results, DENV-2 ED3-specific neutralizing antibodies will likely be effective against DENV-2 strains from all six genotypes.     

Dual specificity of a human neutralizing monoclonal antibody, specific for the V3 loop of GP120 (HIV-1).

Neutralizing antibodies specific for the third variable (V3) domain of gp120, the HIV-1 surface envelope protein, appear early in infection. However, they are usually highly specific for the priming isolate. To identify potential mimotopes of the V3 domain, we have screened a hexapeptide phage library with a human neutralizing mAb, mAb 268, specific for the V3 loop of the viral MN isolate. We have identified two groups of sequences. Within the first group, sequence 268-1 reproduces the linear epitope identified using a conventional epitope mapping approach. The sequence 268-1, H L G P G R, corresponds to amino acids 315-320, localized in the highly conserved tip of the V3 loop. A second group of sequences was identified, including sequence 268-2, K A I H R I. Partial homology with a more variable region of the V3 loop can be found. Using synthetic peptides, we demonstrated that peptides, 268-1 and 268-2, both interact with the same binding site as the V3 region on the 268 mAb. Moreover, both peptides can inhibit the interaction of the 268 mAb with the original immunogen, gp120MN. Peptide 268-1 can compete with peptide 268-2, albeit poorly, for binding of the 268 mAb. When injected into rabbits, KLH conjugated peptide 268-2 elicited antibodies that interact specifically with the initial immunogen gp120MN. These data suggest that peptide 268-2 is both an antigenic and immunogenic mimic of the natural antigen, gp120MN.     

V3 loop region of the HIV-1 gp120 envelope protein is essential for virus infectivity.

The mechanism by which HIV-1 mediates cell fusion and penetrates target cells, subsequent to receptor (CD4) binding, is not well understood. However, neutralizing antibodies, which recognize the principal neutralizing determinants of the gp120 envelope protein (the V3 loop region, residues 296 to 331), have been shown to effectively block cell fusion and virus infectivity independent of the initial gp120-CD4 binding. To investigate the role of the V3 loop in an HIV infection, a series of site-specific mutations were introduced into the HIV-1 envelope gene. Specifically, each residue (312 to 315) in the strongly conserved tetrapeptide sequence, GPGR, which is positioned in the center of the V3 loop domain was individually altered. The processing, transport, and CD4 binding properties of the mutant envelope proteins were comparable to those of the wild-type protein, however, none of the mutants were able to form syncytia in the HeLa-T4 assay. Molecular HIV-1 clones containing mutations altering the G312, G314, or R315 residues produced noninfectious virions, whereas a clone with a P313A mutation was found to be infectious. These results demonstrate that certain V3 loop mutations can be lethal and clearly indicate that this region of the HIV-1 gp120 protein is essential for virus infectivity.     

A broad range of mutations in HIV-1 neutralizing human monoclonal antibodies specific for V2, V3, and the CD4 binding site

The HIV vaccine-induced neutralizing antibodies (Abs) display low rates of mutation in their variable regions. To determine the range of neutralization mediated by similar human monoclonal Abs (mAbs) but derived from unselected chronically HIV-1 infected subjects, we tested a panel of 66 mAbs specific to V3, CD4 binding site (CD4bs) and V2 regions. The mAbs were tested against 41 pseudoviruses, including 15 tier 1 and 26 tier 2, 3 viruses, showing that the neutralization potency and breadth of anti-V3 mAbs were significantly higher than those of the anti-CD4bs and anti-V2 mAbs, and only anti-V3 mAbs were able to neutralize some tier 2, 3 viruses. The percentage of mutations in the variable regions of the heavy (VH) and light (VL) chains varied broadly in a range from 2% to 18% and correlated moderately with the neutralization breadth of tier 2, 3 viruses. There was no correlation with neutralization of tier 1 viruses as some mAbs with low and high percentages of mutations neutralized the same number of viruses. The electrostatic interactions between anti-V3 mAbs and the charged V3 region may contribute to their neutralization because the isoelectric points of the VH CDR3 of 48 anti-V3 mAbs were inversely correlated with the neutralization breadth of tier 2, 3 viruses. The results demonstrate that infection-induced antibodies to CD4bs, V3 and V2 regions can mediate cross-clade neutralization despite low levels of mutations which can be achieved by HIV-1 vaccine-induced antibodies.     

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.