Attenuated signaling associated with immune activation in HIV-1-infected individuals

Chronic immune activation is associated with impaired signal transduction. Since such activation is commonly found during HIV-1 infection, we studied cellular responses to non-specific T-cell receptor stimulation of PBMC obtained from 20 HIV-1 non-infected individuals and 23 highly or partially immune activated HIV-1 infected individuals. PBMC proliferation and ERK-1/2 phosphorylation following anti-CD3 stimulation, and constitutive levels of Cbl-b, were determined. Increased levels of Cbl-b, decreased proliferation, and lower ERK-1/2 phosphorylation were found in PBMC of highly immune activated HIV-1 infected individuals. The elevated expression of Cbl-b and impaired phosphorylation of ERK-1/2 associated with immune activation probably contribute to the attenuated proliferative and cellular responses characteristic of HIV-1 infection. Therefore, targeting immune negative modulators, such as Cbl-b, may serve as a novel approach for controlling HIV-1 disease progression.     

HIV-1中和抗体和基于抗体的疫苗设计

研制一种安全有效并能广泛使用的HIV疫苗对于预防和控制HIV的流行具有重要的意义。尽管人类在HIV-1病原学和免疫学方面的认识不断取得新的进步,对于HIV-1而言,普遍认为诱导保护性中和抗体的科学障碍很难逾越。在抗击HIV-1的感染中传统的疫苗策略不能提供保护。然而,近来的研究揭示在小部分HIV-1感染病人的血清中存在的某些抗体能够中和大多数的HIV-1毒株,对这些血清抗体的深入分析有助于人们揭示抗体识别的病毒表位,这些研究表明自然产生的能够中和HIV-1的中和抗体的发现可能引导未来疫苗设计的思路。高分辨率的结构信息将揭示Env 和中和抗体(Nab)结合区原子水平的结构,这些信息能够帮助设计更好的免疫原。     

A Fusion Intermediate gp41 Immunogen Elicits Neutralizing Antibodies to HIV-1

The membrane-proximal external region (MPER) of the human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein subunit gp41 is targeted by potent broadly neutralizing antibodies 2F5, 4E10, and 10E8. These antibodies recognize linear epitopes and have been suggested to target the fusion intermediate conformation of gp41 that bridges viral and cellular membranes. Anti-MPER antibodies exert different degrees of membrane interaction, which is considered to be the limiting factor for the generation of such antibodies by immunization. Here we characterize a fusion intermediate conformation of gp41 (gp41_int-Cys) and show that it folds into an elongated ∼12-nm-long extended structure based on small angle x-ray scattering data. Gp41_int-Cys was covalently linked to liposomes via its C-terminal cysteine and used as immunogen. The gp41_int-Cys proteoliposomes were administered alone or in prime-boost regimen with trimeric envelope gp140_CA018 in guinea pigs and elicited high anti-gp41 IgG titers. The sera interacted with a peptide spanning the MPER region, demonstrated competition with broadly neutralizing antibodies 2F5 and 4E10, and exerted modest lipid binding, indicating the presence of MPER-specific antibodies. Although the neutralization potency generated solely by gp140_CA018 was higher than that induced by gp41_int-Cys, the majority of animals immunized with gp41_int-Cys proteoliposomes induced modest breadth and potency in neutralizing tier 1 pseudoviruses and replication-competent simian/human immunodeficiency viruses in the TZM-bl assay as well as responses against tier 2 HIV-1 in the A3R5 neutralization assay. Our data thus demonstrate that liposomal gp41 MPER formulation can induce neutralization activity, and the strategy serves to improve breadth and potency of such antibodies by improved vaccination protocols.     

Chemically induced infection of CD4-negative HeLa cells with HIV-1

Infection with human immunodeficiency virus type-1 (HIV-1) requires the presence of a CD4 molecule and chemokine receptors such as CXCR4 or CCR5 on the surface of target cells. However, it is still not clear how the virus enters the cells. Although CD4 was initially identified as the primary receptor for HIV-1, the expression of CD4 or one of the chemokine receptors alone is not sufficient to render susceptibility to infection with the virus. To ascertain whether or not adsorption of the virus needs charge-to-charge interaction between viral envelope and host cell membrane protein(s) and if binding alone promotes penetration of the virus into the cells, we have developed a chemically induced infection system targeting a CD4-negative and CXCR4-positive HeLa cell clone (N7 HeLa) which is usually not susceptible to infection with the LAI strain of HIV-1. Use of a poly-L-lysine (PLL)-coated culture plate to enhance the attachment of the virus to the cells made N7 HeLa cells infectable with HIV-1 at very low efficiency. PLL alone cannot fully substitute for the function of the CD4 molecule. However, trypsin-treated viruses, which have largely lost infectivity to CD4-positive MT-4 cells that are highly susceptible to HIV-1 infection, enhanced infectivity against N7 HeLa cells when the PLL-coated plate was used. These results provide evidence that infection with HIV-1 requires both high binding affinity between viruses and cells, and then needs a modification of the viral envelope such as cleavage of gp120/160 to enhance the infection, probably resulting in exposure of the hydrophobic fusion domain of gp41. HIV-1 infection of N7 HeLa cells was also enhanced by treatment with low pH, 12-O-tetradecanoylphorbol-13-acetate (TPA) and some factor(s) from the MT-4 cell culture supernatant. Not only tight viral adsorption with cleavage of the viral envelope but also some activated status of the cells may be required for sufficient HIV-1 infection in this artificial condition.     

Unexpected roles for UPF1 in HIV-1 RNA metabolism and translation

The HIV-1 ribonucleoprotein (RNP) contains the major structural protein, pr55(Gag), viral genomic RNA, as well as the host protein, Staufen1. In this report, we show that the nonsense-mediated decay (NMD) factor UPF1 is also a component of the HIV-1 RNP. We investigated the role of UPF1 in HIV-1-expressing cells. Depletion of UPF1 by siRNA resulted in a dramatic reduction in steady-state HIV-1 RNA and pr55(Gag). Pr55(Gag) synthesis, but not the cognate genomic RNA, was efficiently rescued by expression of an siRNA-insensitive UPF1, demonstrating that UPF1 positively influences HIV-1 RNA translatability. Conversely, overexpression of UPF1 led to a dramatic up-regulation of HIV-1 expression at the RNA and protein synthesis levels. The effects of UPF1 on HIV-1 RNA stability were observed in the nucleus and cytoplasm and required ongoing translation. We also demonstrate that the effects exerted by UPF1 on HIV-1 expression were dependent on its ATPase activity, but were separable from its role in NMD and did not require interaction with UPF2.     

核酸定量检测试验应用于HIV-1感染实验室诊断的探讨

目的:探讨核酸定量检测在HIV-1感染实验室诊断中的应用。方法:选取145例第四代抗原/抗体联合诊断筛查试验为阳性反应的血浆样本,分别用Western印迹和HIV-1核酸定量方法进行检测,综合对比分析2种方法检测结果。结果:Western印迹检出阳性样本120例,不确定样本17例,阴性样本8例;HIV-1核酸定量试验检出结果大于检测限样本131例,其中包括12例Western印迹不确定样本、2例Western印迹阴性样本;有3例Western印迹阳性样本用HIV-1核酸定量检测试验未能检出。结论:核酸定量检测试验对于HIV-1感染阳性样本是一种有效的实验室诊断方法;对HIV-1核酸定量检测结果为"TND"的样本,建议加做Western印迹或结合其他补充试验结果进行综合诊断。     

A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.     

Antiretroviral (HIV-1) activity of azulene derivatives

The antiretroviral activity of azulene derivatives was detected for the first time. A series of eighteen diversely substituted azulenes was synthesized and tested in vitro using HIV-1 based virus-like particles (VLPs) and infectious HIV-1 virus in U2OS and TZM-bl cell lines. Among the compounds tested, the 2-hydroxyazulenes demonstrated the most significant activity by inhibiting HIV-1 replication with IC₅₀ of 2–10 and 8–20 μM for the VLPs and the infectious virus, respectively. These results indicate that azulene derivatives may be potentially useful candidates for the development of antiretroviral agents.     

Glutamylation of an HIV-1 protein inhibits the immune response by hijacking STING

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International NeuroAIDS: prospects of HIV-1 associated neurological com-plications

INTRODUCTION AND NEUROEPIDEMIO- LOGY OF HIV/AIDS AIDS was first recognized as a new and distinct clini- cal entity in 1981 [1] and the HIV-1 as their casual agent in 1983 [2]. Since then, the HIV/AIDS epidemic has reached epidemic proportions with a total accumulative number of more than 60 million people, according to the Joint United Nations Programme on HIV/AIDS (UNAIDS) and WHO. The extensive spread of HIV-1 epidemics in Asia was not appreciated in the 1980s,…     

A hollow-fiber bioreactor for expanding HIV-1 in human lymphocytes used in preparing an inactivated vaccine candidate

An inactivated HIV vaccine intended to elicit broadly neutralizing antibodies is designed to use a pool of population-prevalent HIV-1 from plasma (PHIV), isolated before evolution of antibody-mediated genetic mutations. A suitable cell substrate (CS) for isolating such PHIV is peripheral blood mononuclear cells (PBMC) after stimulating with phytohemagglutinin (PHA) and interleukin-2 (IL-2). Feasibility of employing a hollow-fiber bioreactor under optimized conditions was investigated for large-scale expansion and efficient recovery of concentrated PHIV. Each CS batch was infected in vitro with a prototype PHIV, the infected cells were introduced into the bioreactor for 7-10 days in co-culture, and the cell-free supernatants were assayed for p24 antigen as an index of HIV synthesis. PBMC versus CD8-depleted (CD8D) CS, 20kDa versus 5kDa molecular weight cut-off (MWCO) bioreactor cartridges, 7- versus 10-day culture periods, and varying concentrations of IL-2, fetal bovine serum (FBS) and glucose content in the medium were functionally evaluated for p24 yield. PBMC cultures in 20kDa MWCO cartridges with 15% FBS, 80IU/mL IL-2 and 2.0g/L glucose produced the highest p24 yield; however, CD8D-CS, 20-30% FBS and 80 IU/mL IL-2 within 5kDa cartridges and 2.0 g/L glucose in the circulating medium was more cost-effective for synthesis of virion p24.     

HIV-1 Env vaccine comprised of electroporated DNA and protein co-administered with Talabostat

Selection of potent yet low reactogenic adjuvants for protein immunization is important for HIV-1 vaccine development. Immunogenicity of electroporated DNA (HIV env) and recombinant gp120, administered with either QS-21 or the orally administered immunomodulator, Talabostat, was evaluated in BALB/c mice. Electroporation of low dose DNA elicited Th1 cytokines and anti-envelope antibodies. Immunization with gp120 protein alone with or without Talabostat elicited lower Th1 and Th2 cytokine levels but comparable anti-gp120 antibodies to QS-21-formulated protein. Boosting of DNA-primed mice with gp120/Talabostat induced similar anti-gp120 antibody titers and slightly higher levels of Th1 and Th2 cytokines relative to QS-21-formulated protein. Induction of CD8⁺ and CD4⁺ T cells and functional CTL activity was noted. These results highlight the potential use of orally administered Talabostat for efficient protein boosting of antibody and T-cell responses primed by DNA.     

Expression, Purification, and In Vivo Administration of a Promising Anti-Idiotypic HIV-1 Vaccine

To date only a few neutralizing antibodies against HIV-1 exist. Since these neutralizing antibodies are only rarely found in sera of HIV-1 infected individuals an active vaccine is required. We recently developed murine anti-idiotypic antibody Ab2/3H6 against monoclonal antibody (mAb) 2F5, which is one of the most prominent neutralizing antibodies. Anti-idiotypic antibody Ab2/3H6 has been partially humanized and expressed as whole immunoglobulin G in Chinese hamster ovary cells in order to minimize the human anti-mouse antibody response. Here we describe the expression, purification, and immunohistochemical characterization of the chimeric Ab2/3H6 Fab fragment, which was finally used beside the whole IgG1 as an antigen for immunization of guinea pigs. The crude sera were screened for specific antibodies against the epitope of mAb 2F5 ELDKWA as well as for reactivity against HIV-1 gp41.     

Fighting a virus with a virus: a dynamic model for HIV-1 therapy

A mathematical model examined a potential therapy for controlling viral infections using genetically modified viruses. The control of the infection is an indirect effect of the selective elimination by an engineered virus of infected cells that are the source of the pathogens. Therefore, this engineered virus could greatly compensate for a dysfunctional immune system compromised by AIDS. In vitro studies using engineered viruses have been shown to decrease the HIV-1 load about 1000-fold. However, the efficacy of this potential treatment for reducing the viral load in AIDS patients is unknown. The present model studied the interactions among the HIV-1 virus, its main host cell (activated CD4+ T cells), and a therapeutic engineered virus in an in vivo context; and it examined the conditions for controlling the pathogen. This model predicted a significant drop in the HIV-1 load, but the treatment does not eradicate HIV. A basic estimation using a currently engineered virus indicated an HIV-1 load reduction of 92% and a recovery of host cells to 17% of their normal level. Greater success (98% HIV reduction, 44% host cells recovery) is expected as more competent engineered viruses are designed. These results suggest that therapy using viruses could be an alternative to extend the survival of AIDS patients.     

HIV-1 C/B'亚型多价重组MVA病毒疫苗

目的：检测人类免疫缺陷病毒1型（HIV-1） C/B'亚型与痘苗病毒安卡拉株（MVA）的多价重组疫苗的免疫原性,此疫苗中包含HIV-1 C/B'CRF 株的五个基因,分别是env、gag、pol、nef、tat。方法：设105 pfu/mL 、106 pfu/mL、107 pfu/mL ADMVA三个剂量组,用ELISPOT方法检测细胞免疫反应。同时,以Gag蛋白作为包被抗原,使用间接ELISA的方法检测体液免疫反应。结果：免疫后的小鼠对插入基因env、gag、pol和nef所表达蛋白均产生了特异性细胞免疫应答,且免疫效果与免疫剂量呈正相关。ELISA结果表明, MVA重组疫苗免疫诱导产生了特异性体液免疫,抗HIV-1Gag的抗体滴度与免疫次数和免疫剂量都存在正相关性。结论：多价MVA重组疫苗能有效地诱导小鼠产生特异地细胞免疫和体液免疫反应。