Expression of complete SIV p27 Gag and HIV gp120 engineered outer domains targeted by broadly neutralizing antibodies in live rubella vectors

Infection with HIV or SIV often elicits a potent immune response to viral antigens. This includes T cells and antibodies specific for Gag and Env antigens. In contrast, when given as a vaccine, the same antigens have been weak immunogens, unable to elicit antibodies with comparable titer, durability, or neutralizing activity. We have used the live attenuated rubella vaccine strain RA27/3 as a viral vector to express HIV and SIV antigens. By mimicking an HIV infection, these vectors could elicit stronger and more durable immunity to HIV antigens. The vectors are based on the licensed rubella vaccine strain, which has demonstrated safety and potency in millions of children. One or two doses protect for life against rubella infection. The question was whether rubella vectors could similarly enhance the immunogenicity of a foreign vaccine insert.We have previously reported that rubella vectors can express small protein antigens in vitro and in vivo, where they elicit a strong immune response to the vaccine insert. The vectors have now expressed larger vaccine inserts that include epitope-rich fragments of the Gag matrix and capsid proteins (aa 41-211) or the complete p27 capsid protein with p2 (aa 136-381). These vectors have elicited a robust and durable immune response to Gag in rhesus macaques. This size range also encompasses the engineered outer domain (eOD) of HIV envelope gp120 (172 amino acids). The rubella/eOD-GT6 and GT8 vectors stably expressed glycoproteins that bind germline precursors and mature forms of VRC01-class broadly neutralizing antibodies. These vectors potentially could be used as part of a sequential immunization strategy to initiate the production of broadly neutralizing antibodies.     

CEMx174细胞中SAMHD1蛋白表达与SIVmac239病毒水平的相关性研究

目的研究SIV在感染CEMx174细胞过程中,SAMHD1在基因和蛋白水平的变化与病毒水平之间的相关性。方法 SIVmac239病毒感染CEMx174细胞后,每天收集上清和细胞,应用Taq Man探针实时荧光定量RT-PCR方法,检测细胞内SAMHD1 mRNA的相对表达量的变化;用Western blot检测SAMHD1蛋白与SIVmac239P27蛋白表达量的变化,并分析其相关性。结果 CEMx174细胞感染SIVmac239后,细胞中的SAMHD1 mRNA基于内参基因GAPDH的相对表达量逐渐增加,前3 d分别增加了14%,16%,42%,随后开始迅速增加,第4天达到了200%,第6天更是达到了890%;细胞中SAMHD1蛋白的表达量在感染后第1、2天时变化不大,随后逐渐降低,到第5、6天时,几乎检测不到,呈现逐渐下降的趋势。结论 CEMx174细胞感染SIVmac239后,SAMHD1基因表达上调,与上清中病毒载量水平正相关,细胞内SAMHD1蛋白水平与SIVmac239 P27蛋白水平呈负相关。     

Restricted access to myeloid cells explained

The lentiviral accessory protein, Vpx, is known to counteract a restriction factor that is specific to myeloid cells, such as macrophages and dendritic cells. This review summarizes the findings in two seminal studies that identify SAMHD1 as the cellular protein that is responsible for myeloid cell restriction, and establish the existence of other types of restriction in these cells.     

Immunogenicity of viral vector,prime-boost SIV vaccine regimens in infant rhesus macaques: Attenuated vesicular stomatitis virus (VSV) and modified vaccinia Ankara (MVA) recombinant SIV vaccines compared to live-attenuated SIV

In a previously developed infant macaque model mimicking HIV infection by breast-feeding, we demonstrated that intramuscular immunization with recombinant poxvirus vaccines expressing simian immunodeficiency virus (SIV) structural proteins provided partial protection against infection following oral inoculation with virulent SIV. In an attempt to further increase systemic but also local antiviral immune responses at the site of viral entry, we tested the immunogenicity of different orally administered, replicating vaccines. One group of newborn macaques received an oral prime immunization with a recombinant vesicular stomatitis virus expressing SIVmac239 gag, pol and env (VSV-SIVgpe), followed 2 weeks later by an intramuscular boost immunization with MVA-SIV. Another group received two immunizations with live-attenuated SIVmac1A11, administered each time both orally and intravenously. Control animals received mock immunizations or non-SIV VSV and MVA control vectors. Analysis of SIV-specific immune responses in blood and lymphoid tissues at 4 weeks of age demonstrated that both vaccine regimens induced systemic antibody responses and both systemic and local cell-mediated immune responses. The safety and immunogenicity of the VSV-SIVgpe + MVA-SIV immunization regimen described in this report provide the scientific incentive to explore the efficacy of this vaccine regimen against virulent SIV exposure in the infant macaque model.     

Control of HIV-1 replication in vitro by vaccine-induced human CD8+ T cells through conserved subdominant Pol epitopes

ObjectiveThe specificity of CD8⁺ T cells is critical for early control of founder/transmitted and reactivated HIV-1. To tackle HIV-1 variability and escape, we designed vaccine immunogen HIVconsv assembled from 14 highly conserved regions of mainly Gag and Pol proteins. When administered to HIV-1-negative human volunteers in trial HIV-CORE 002, HIVconsv vaccines elicited CD8⁺ effector T cells which inhibited replication of up to 8 HIV-1 isolates in autologous CD4⁺ cells. This inhibition correlated with interferon-γ production in response to Gag and Pol peptide pools, but direct evidence of the inhibitory specificity was missing. Here, we aimed to define through recognition of which epitopes these effectors inhibit HIV-1 replication.DesignCD8⁺ T-cells from the 3 broadest HIV-1 inhibitors out of 23 vaccine recipients were expanded in culture by Gag or Pol peptide restimulation and tested in viral inhibition assay (VIA) using HIV-1 clade B and A isolates.MethodsFrozen PBMCs were expanded first using peptide pools from Gag or Pol conserved regions and tested on HIV-1-infected cells in VIA or by individual peptides for their effector functions. Single peptide specificities responsible for inhibition of HIV-1 replication were then confirmed by single-peptide expanded effectors tested on HIV-1-infected cells.ResultsWe formally demonstrated that the vaccine-elicited inhibitory human CD8⁺ T cells recognized conserved epitopes of both Pol and Gag proteins. We defined 7 minimum epitopes, of which 3 were novel, presumably naturally subdominant. The effectors were oligofunctional producing several cytokines and chemokines and killing peptide-pulsed target cells.ConclusionsThese results implicate the use of functionally conserved regions of Pol in addition to the widely used Gag for T-cell vaccine design. Proportion of volunteers developing these effectors and their frequency in circulating PBMC are separate issues, which can be addressed, if needed, by more efficient vector and regimen delivery of conserved immunogens.     

SIV Infection of Macaques as a Model for AIDS Pathogenesis

Simian immunodeficiency virus (SIV) infection of macaques is the best available animal model for studying the pathogenesis of AIDS. Experimental inoculation of macaques with SIV results in a persistent infection that leads to immunodeficiency, opportunistic infections, and death. Most aspects of the illness, including immunologic and virologic parameters, are easily quantified. Furthermore, pathologic processes can be evaluated throughout the course of experimental infection. Recently, molecular clones of SIV proviral DNA have been used to study genetic variation and specific viral determinants of pathogenesis. Considered together, these observations support the continued detailed study of SIV infection of macaques as a model for human AIDS.     

猴免疫缺陷病毒培养特性及温度敏感性的研究

SIV感染CEMx-174和HUT-78细胞后,显示出SIV对CEMx-174细胞敏感,产生明显的CPE并能杀死该细胞;SIV感染HUT-78细胞亦能引起CPE,但CPE不易与正常HUT-78细胞中的大细胞相区分;60℃加热30分钟能杀死SIV,4℃可存放2周,22℃可存放1周,-20℃存放3周滴度由1:8192分别下降到1:1024、1:16和1:2048,-70℃存放2周滴度由1:8192下降到1:4096并维持此水平2个月。     

Homotypical ipsilateral cortical projections between somatosensory areas I and II in the cat

In 11 cats, small quantities of horseradish peroxidase conjugated to wheat germ agglutinin were placed into cortical zones of somatosensory area I representing the distal digits (n = 3), distal toes (n = 2), toes and digits (n = 1), proximal forelimb (n = 1), proximal hindlimb (n = 1), trunk (n = 2), and the face and nose (n = 1). Reconstruction of the pattern of retrograde labeling in somatosensory area II revealed dense, heavily labeled patches of cells in regions that were precisely homotypical to the injection site as determined by electrophysiological recordings. This dense, homotypical patch of labeled cells was usually surrounded by a less densely populated fringe of labeled cells that bordered, but did not appear to enter, heterotypical zones. In two animals, however, some retrogradely labeled cells were found in the cortex representing somatotopic zones adjacent to the sites injected with horseradish peroxidase. These results indicated that somatosensory area II primarily sends homotypical projections to somatosensory area I. In a few cases, however, some retrogradely labeled cells may represent either homo-or heterotypical projections depending on how receptive field sizes and the areal extent of labeling in somatosensory areas I and II are interpreted.