HIV-1包膜蛋白2G12和2F5中和表位的改造对假病毒形成及中和活性的影响

目的 研究HIV-1膜蛋白(Env)特定中和表位的改造对功能性假病毒形成及中和活性的影响.方法 采用环形诱变及Dpn I筛选的方法对Env进行定点突变,将2G12和2F5两个中和表位整合入不含该表位的BC亚型的Env上,比较改造对假病毒的形成情况及对2G12和2F5单抗的中和活性的影响.结果 对5株假病毒(BC02、BE03、BC04、BC05和BC12)的Env特定中和表位进行改造,其中BC04和BCl2的2G12表位改造后,不能形成假病毒,BC02、BC03和BC05增加2G12和2F5两个表位后,仍能够形成假病毒,且假病毒滴度较改造前无明显变化,改造后的BC03假病毒较改造前对单抗2G12和2175的中和活性均有所提高,而改造后的BE02和BC05假病毒较改造前对单抗2F5的中和活性增强,而对单抗2G12的中和活性无变化.结论 2G12中和表位部分位点的改造影响假病毒的形成,中和表位的增加能够提高单抗2G12的中和活性,为免疫原的优化提供了新思路.     

SFTSV重组假病毒的构建及Gc糖基化对病毒感染力的影响

目的为探究发热伴血小板减少综合征病毒(SFTSV)中Gc及其N-糖基化位点与病毒感染性的关系, 构建了含有SFTSV Gc糖基化位点突变体的重组假病毒。方法利用定点突变和同源重组技术构建了SFTSV Gc及3个N-糖基化位点突变的真核表达载体pcDNA3.1(+)-Gc、pcDNA3.1(+)-Gc(N291Q)、pcDNA3.1(+)-Gc(N352Q)和pcDNA3.1(+)-Gc(N374Q)。验证其在293T细胞中成功表达后, 感染VSVΔG-Fluc*G假病毒, 构建4株重组假病毒并检测其对细胞感染力的影响。结果双酶切鉴定和序列测定证实成功构建真核表达载体pcDNA3.1(+)-Gc、pcDNA3.1(+)-Gc(N291Q)、pcDNA3.1(+)-Gc(N352Q)和pcDNA3.1(+)-Gc(N374Q)。间接免疫荧光和Western blot结果表明4个重组质粒均成功表达。SFTSV Gc重组假病毒对Vero细胞有感染特异性。糖基化位点突变后假病毒感染能力明显降低, 且352位糖基化位点突变株感染水平最低(P<0.001、P=0.001)。结论 SFTSV G...     

埃博拉假病毒突变体感染性及靶向抗体中和作用评价

目的：利用埃博拉假病毒（EBOV）报告系统，体外评价埃博拉病毒突变体对4种细胞系的相对感染力及3株单抗对突变体的中和能力。方法：构建表达埃博拉假病毒及其突变体GP蛋白的重组质粒GP-pcDNA3.1，与含有萤火虫荧光素酶报告基因的HIV骨架载体pSG3.ΔEnv.CMV.Fluc共转染HEK293T细胞获得假病毒上清。利用Western blot法及HIV p24 ELISA抗原定量试剂盒对假病毒进行鉴定及定量，采用等量的假病毒感染HEK293T、Huh7、A549及THP-1细胞，36 h后，裂解细胞测荧光素酶活性（RLU），计算突变体与母本的相对感染力。将mAb114、ADI-15946、rEBOV-520与EBOV及其突变体假病毒预孵育1 h，将假病毒上清和抗体混合液感染HEK293T细胞，36 h后测RLU值，计算抑制率。结果：相对于母本，所有突变体感染细胞的能力均有不同程度增强。单抗mAb114、ADI-15946能有效中和母本及14株突变体，rEBOV-520对N107D-P330S-G480D中和作用减弱。结论：体外实验证明14株突变体入侵靶细胞能力增强。3株中和抗体对突...     

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.     

一例未经抗病毒治疗的HIV-1 CRF07BC患者中和敏感性时序进化特征

本研究追踪了一例长期不进展的HIV-1 CRF07_BC感染者体内病毒进化的特征, 并分析了该病毒中和敏感性的变化。在2016年至2020年间采集患者四个时点的血浆, 所有血浆样本对Global panel假病毒的中和宽度均为100%。基于四个时点血浆, 扩增了59个全长env基因片段, 并成功构建了24株功能性假病毒。患者的env基因序列显示, V1和V5中潜在的N-连锁糖基化位点(PNGS)随着时间的推移显著增加。所有24株假病毒对同期和后期的自体血浆以及bNAbs(包括10E8、VRC01和12A21)敏感, 但自体血浆和bNAbs对后期采样时点构建的假病毒的中和敏感性下降。本研究发现Env的V5区S465T突变与VRC01中和敏感性下降相关。     

CD45RO N-糖基化位点对galectin-3结合CD45RO突变体转染细胞的影响

目的 构建CD45RO不同N-糖基化位点突变的T细胞株,并检测其与galectin-3的结合情况.方法 采用N→Q定点突变技术分别去除CD45RO的11个N-糖基化位点,制备单个N-糖基化位点缺失的CD45RO,然后将其导入慢病毒表达载体pWPXL中,11个携带单个N-糖基化位点缺失的CD45RO重组慢病毒质粒与慢病毒包装质粒psPAX2和MD2.G共转染293T细胞,将包装重组慢病毒感染CD45-的J45.01细胞,流式分选后获得11株分别表达CD45RO单个N-糖基化位点缺失的J45.01 T细胞株.通过RT-PCR和流式细胞术分别从RNA水平和蛋白水平验证CD45RO突变体的转录和表达,应用流式细胞术检测CD45RO突变细胞株与galectin-3的结合情况.结果 成功构建了1 1个CD45RO单个N-糖基化位点缺失的T细胞株,各突变细胞株能稳定表达突变基因,经测序再次证实突变位点正确,CD45 RO突变体能稳定表达于细胞表面.galectin-3与N327Q突变细胞的结合明显增强,与N36Q突变细胞和N217Q突变细胞的结合明显减弱.结论 CD45RON-糖基化位点对galectin-3与CD45 RO-J45.01细胞的结合有调节作用.     

融合肽抑制剂T20抵抗株包膜蛋白热变异位点547对人类免疫缺陷病毒-1生物学功能的影响

目的 分析人类免疫缺陷病毒(HIV)的融合肽抑制剂T20抵抗突变株热变异位点547对病毒生物学功能的影响.方法 采用PCR和定点突变方法构建HIV包膜蛋白(Env)突变体pSM-HXB2-DIV/DIM/SIM和PSM-LAI-DIV四种表达质粒,并构建了含有547D突变并置换HXB2或LAI株gp120部分的Env质粒,pSM-LAI gp120/HXB2 gp41-DIV (L/H-DIV)和pSM-HXB2 gp120/LAI gp41-DIV(H/L-DIV).假病毒感染实验和细胞融合实验分析Env的功能;Western blot检测Env表达.圆二色谱(CD)分析6螺旋束(6HB)的稳定性;PyMOL软件分析含有547D的6HB结构.结果 HXB2为骨架的547DIV、DIM及SIM和LAI为骨架的DIV假病毒的感染性低于其野生型(wt),前者的各变异Env的融合活性也明显降低;L/H-DIV假病毒感染性与HXB2 wt相似,但融合活性明显低于wt;H/L-DIV假病毒的感染性低于两种野生型,融合活性与LAI wt相似,但低于HXB2 wt.CD结果证明547D突变降低6HB的稳定性;结构分析显示,547D突变没有改变NHR中该位点与CHR中Q652位点之间的氢键,但可能通过增加NHR的负电荷从而减少病毒与T20的结合.结论 547D突变降低LAI与HXB2株的感染性和HXB2株的融合活性;该突变通过降低6HB稳定性和增加gp41所带负电荷来减少病毒与T20的结合.     

人类免疫缺陷病毒1型(HIV-1)糖蛋白gp120部分糖基化位点突变对其抗原性影响

目的 研究HIV-1包膜糖蛋白gp120特定糖基化位点突变后对gp120抗原性的影响,并推测糖基化位点突变对结构的影响.方法 采用连接PCR方法,将获得的野生型gp120中选定N糖基化位点的Asn定点突变为Cln,使该位点去糖基化,构建DNA疫苗,免疫小鼠,ELISA检测鼠血清中的抗体,进行统计学分析,推测糖基化位点突变对gp120抗原性和结构的影响.结果 2组糖基化位点突变的gp120诱发非V3特异性抗体的能力较之野生型gp120有明显提高,但诱发V3特异性抗体能力没有显著提高,7个位点突变的gp120诱发V3特异性抗体能力有很大降低.结论 gp120部分糖基化位点的突变在一定程度上能提高或改变gp120的抗原性,这可能由突变导致的构象变化和/或糖基化寡糖链移除使原来被遮盖的抗原表位暴露引起.     

大肠杆菌麦芽糖结合蛋白（MBP）诱导小鼠Th1细胞的活化作用

目的:探讨MBP对小鼠T细胞的调节作用.方法:采用淋巴细胞分层液分离小鼠脾脏淋巴细胞,在体外用MBP刺激淋巴细胞,通过MTT法测定淋巴细胞增殖反应;ELISA检测脾细胞培养上清中细胞因子的分泌水平;MBP免疫小鼠后,ELSPOT检测MBP刺激脾淋巴细胞分泌IFN-γ的细胞频数;免疫组化检测MBP与T细胞的结合作用.结果:MBP以剂量依赖关系促进小鼠淋巴细胞的增殖,促进淋巴细胞分泌IL-2和IFN-γ,轻微抑制IL-4的分泌;ELSPOT检测细胞分泌IFN-γ结果显示,MBP可诱导特异性Th1活化,也可刺激非特异性Th1活化;免疫组化结果显示,抗MBP抗体可与经MBP刺激的淋巴细胞发生反应,阳性细胞占总淋巴细胞的37.7%,当MBP采用 1∶2 000抗MBP抗体中和后,再与淋巴细胞反应,结果未见阳性细胞.结论:MBP可作用于淋巴细胞诱导非特异性Th1活化,也可通过免疫诱导大量特异性Th1活化;MBP可作为新的免疫增强剂开发利用.     

糖基化修饰的DC疫苗特异性抗骨髓瘤活性的体外研究

目的：探讨经过糖基化修饰改造过的肿瘤相关糖抗原冲击树突状细胞（DC）制备的DC疫苗特异性抗骨髓瘤作用。方法：采用化学方法及肿瘤细胞的生物工程法使骨髓瘤细胞表达新肿瘤相关抗原N-丙酰多聚唾液酸（NPrPSA）；在无血清培养条件下，采用GM—CSF／IFN-α及TNF-α从MM患者外周血单核细胞诱导培养DC。然后用表达新抗原的肿瘤细胞冲击DC制备DC疫苗，将其刺激T细胞后，通过LDH释放法观察其对骨髓瘤细胞的杀伤效力，并采用ELISA法测定了T细胞分泌细胞因子IFN-γ的能力。结果：糖基化修饰的CD138^＋Npr—DC组LDH释放率较CD138^＋未修饰DC组明显增高（P〈0．01）。CD138^＋Npr—DC组T细胞分泌IFN-γ与CD138^＋未修饰DC组相比亦明显增加（P〈0．05），而CD138^＋Npr—DC组与CD138^-未修饰DC组之间无统计学意义（P〉0．05）。结论：糖基化修饰的DC疫苗可以有效地激活Th1细胞免疫应答，分泌高水平的细胞因子IFN-γ；可诱导出较普通DC疫苗更明显的抗骨髓瘤作用，并具有一定的安全性。     

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.     

HIV-1 acute infection env glycomutants designed from 3D model: effects on processing, antigenicity, and neutralization sensitivity

The human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) has evolved to limit its overall immunogenicity by extensive glycosylation. Only a few studies dealing with glycosylation sites have taken into account available 3D data in a global approach. We compared primary env sequences from patients with acute HIV-1 infection. Conserved N-glycosylation sites were placed on the gp120-3D model. Based on vicinity, we defined glycosylation clusters. According to these clusters, we engineered plasmids encoding deglycosylated gp160 mutants. We also constructed mutants corresponding to nonclustered glycans or to the full deglycosylation of the V1 or V2 loop. After in vitro expression, mutants were tested for functionality. We also compared the inhibition of pseudotyped particles infection by human-neutralizing sera. Generally, clustered and nonclustered mutants were affected similarly. Silencing of more than one glycan had deleterious effects, independently of the type of sugar removed. However, some mutants were moderately affected by glycans removal suggesting a distinct role for these N-glycans. Additionally, compared to the wild-type pseudovirus, two of these mutants were neutralized at higher sera dilutions strengthening the importance of the location of specific N-glycans in limiting the neutralizing response. These results could guide the selection of env mutants with the fewest antigenic and functional alterations but with enhanced neutralization sensitivity.     

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.     

Comparison between env-specific T-cell epitopic responses in HIV-1-uninfected adults immunized with combination of ALVAC-HIV(vCP205) plus or minus rgp160MN/LAI-2 and HIV-1-infected adults

In this study, we investigated the CD4 T-helper response induced by ALVAC-HIV(vCP205) +/- rgp160MN/LAI-2 using a series of 15 overlapping amino acid peptides spanning the entire gp160MN/LAI-2 antigen. CD4 Env-specific T-cell lines were established from three groups of HIV-1-negative HIV vaccine recipients: vCP205 + gp160MN/LAI-2, vCP205 only, and gp160MN/LAI-2 only. CD4 Env-specific T-cell lines established from individuals who received the prime-boost vCP205 + rgp160MN/LAI-2 generated strong and broad T-helper responses scattered across the Env sequence, whereas Env-specific T-cell lines from individuals receiving the vCP205 vaccine alone generated reactivity to only a few peptides. CD4 -specific T-cell lines were also established from HIV-1-infected individuals and demonstrated poor reactogenicity to Env peptides in both breadth and amplitude of response. These results highlight the complexity of major histocompatibility complex class II presentation and CD4 antigen-specific reactivity, emphasizing the need to better understand these crucial T-helper cell responses in the setting of HIV infection and HIV vaccine development.     

Production and characterization of high-affinity human monoclonal antibodies to human immunodeficiency virus type 1 envelope glycoproteins in a mouse model expressing human immunoglobulins

Human (Hu) monoclonal antibodies (MAbs) against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) are useful tools in the structural and functional analysis of Env, are under development both as potential prophylaxis and as therapy for established HIV-1 infection, and have crucial roles in guiding the design of preventative vaccines. Despite representing more than 50% of infections globally, no MAbs have been generated in any species against C clade HIV-1 Env. To generate HuMAbs to a novel Chinese C clade Env vaccine candidate (primary isolate strain HIV-1(97CN54)), we used BAB5 mice that express a human immunoglobulin (Ig) M antibody repertoire in place of endogenous murine immunoglobulins. When immunized with HIV-1(97CN54) Env, these mice developed antigen-specific IgM antibodies. Hybridoma fusions using splenocytes from these mice enabled the isolation of two Env-specific IgM HuMAbs: N3C5 and N03B11. N3C5 bound to HIV-1 Env from clades A and C, whereas N03B11 bound two geographically distant clade C isolates but not Env from other clades. These HuMAbs bind conformational epitopes within the immunodominant region of the gp41 ectodomain. N3C5 weakly neutralized the autologous isolate in the absence of complement and weakly enhanced infection in the presence of complement. N03B11 has no effect on infectivity in either the presence or the absence of complement. These novel HuMAbs are useful reagents for the study of HIV-1 Env relevant to the global pandemic, and mice producing human immunoglobulin present a tool for the production of such antibodies.     

Recombinant Mycobacterium bovis bacillus Calmette-Guerin elicits human immunodeficiency virus type 1 envelope-specific T lymphocytes at mucosal sites

A successful vaccine vector for human immunodeficiency virus type 1 (HIV-1) should induce anti-HIV-1 T-cell immune responses at mucosal sites. We have constructed recombinant Mycobacterium bovis bacillus Calmette-Guérin (rBCG) expressing an HIV-1 group M consensus envelope (Env) either as a surface, intracellular, or secreted protein as an immunogen. rBCG containing HIV-1 env plasmids engineered for secretion induced optimal Env-specific T-cell gamma interferon enzyme-linked immunospot responses in murine spleen, female reproductive tract, and lungs. While rBCG-induced T-cell responses to HIV-1 envelope in spleen were lower than those induced by adenovirus prime/recombinant vaccinia virus (rAd-rVV) boost, rBCG induced comparable responses to rAd-rVV immunization in the female reproductive tract and lungs. T-cell responses induced by rBCG were primarily CD4(+), although rBCG alone did not induce anti-HIV-1 antibody. However, rBCG could prime for a protein boost by HIV-1 envelope protein. Thus, rBCG can serve as a vector for induction of anti-HIV-1 consensus Env cellular responses at mucosal sites.     

Substantial envelope-specific CD8 T-cell immunity fails to control siv disease

It is unknown which HIV proteins to target by vaccination in order to generate the most effective CD8 T-cell immunity. We recently immunized SIV_mac251-infected pigtail macaques with Gag peptides or a cocktail of peptides spanning all SIV proteins, including SIV Env. High-level SIV Env-specific CD8 T-cell responses were generated and 7 novel Env-specific CD8 T-cell epitopes in 10 animals were mapped. Env-specific CD8 T-cell responses were significantly inferior to Gag-specific responses, and no better than unvaccinated control animals, in the control of SIV replication and prevention of disease. Escape mutations emerged within several Env-specific CTL epitopes, suggesting at least some pressure imparted by the Env CTL responses, but this did not correlate with significantly reduced SIV replication. We conclude Env-specific CTL may not be the most effective response to induce by vaccination.     

Human Endogenous Retrovirus K(HML-2) Gag- and Env-Specific T-Cell Responses Are Infrequently Detected in HIV-1-Infected Subjects Using Standard Peptide Matrix-Based Screening

T-cell responses to human endogenous retrovirus (HERV) K(HML-2) Gag and Env were mapped in HIV-1-infected subjects using 15mer peptides. Small peptide pools and high concentrations were used to maximize sensitivity. In the 23 subjects studied, only three bona fide HERV-K(HML-2)-specific responses were detected. At these high peptide concentrations, we detected false-positive responses, three of which were mapped to an HIV-1 Gag peptide contaminant. Thus, HERV-K(HML-2) Gag- and Env-specific T-cell responses are infrequently detected by 15mer peptide mapping.     

The importance of antibody isotype in HIV-1 virus capture assay and in TZM-bl neutralization

The binding of murine IgM mAbs to five different clades of HIV-1 was examined using a modified ELISA-based virus capture assay. Two murine multispecific IgM mAbs that exhibit both lipid and gp41 epitope specificities, and one murine IgM mAb that exhibits lipid-binding specificity, were utilized. The binding of the IgG and the IgM isotypes of human mAb 2F5 to clades A through AE were also evaluated. The binding of 2F5 to HIV-1 was dependent upon the antibody isotype. Monoclonal IgM antibodies bound significantly lower amounts of HIV-1 than the corresponding IgG isotype. Although murine IgM mAbs bound HIV-1 to varying degrees in the virus capture assay, they failed to neutralize HIV-1 in a TZM-bl pseudovirus assay. In contrast, 2F5-IgM mAb bound certain HIV-1 isolates, and also neutralized them, although not as efficiently as the 2F5-IgG isotype. Studies on the relationship between virus binding and neutralization in a TZM-bl pseudovirus assay indicated that in most cases, mAbs that exhibited neutralization also bound the virus.