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.     

Effect of Morphine on the Neuropathogenesis of SIVmac Infection in Indian Rhesus Macaques

Morphine is known to prevent the development of cell-mediated immune (CMI) responses and enhance expression of the CCR5 receptor in monocyte macrophages. We undertook a study to determine the effect of morphine on the neuropathogenesis and immunopathogenesis of simian immunodeficiency virus (SIV) infection in Indian Rhesus Macaques. Hypothetically, the effect of morphine would be to prevent the development of CMI responses to SIV and to enhance the infection in macrophages. Sixteen Rhesus Macaques were divided into three experimental groups: M (morphine only, n = 5), VM (morphine + SIV, n = 6), and V (SIV only, n = 5). Animals in groups M and VM were given 2.5 mg/kg of morphine sulfate, four times daily, for up to 59 weeks. Groups VM and V were inoculated with SIVmacR71/17E 26 weeks after the beginning of morphine administration. Morphine prevented the development of enzyme-linked immunosorbent spot-forming cell CMI responses in contrast to virus control animals, all of which developed CMI. Whereas morphine treatment had no effect on viremia, cerebrospinal fluid viral titers or survival over the time course of the study, the drug was associated with a tendency for greater build-up of virus in the brains of infected animals. Histopathological changes in the brains of animals that developed disease were of a demyelinating type in the VM animals compared to an encephalitic type in the V animals. This difference may have been associated with the immunosuppressive effect of the drug in inhibiting CMI responses.     

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.     

A critical analysis of the cynomolgus macaque,Macaca fascicularis,as a model to test HIV-1/SIV vaccine efficacy

The use of a number of non-rhesus macaque species, but especially cynomolgus macaques as a model for HIV-1 vaccine development has increased in recent years. Cynomolgus macaques have been used in the United Kingdom, Europe, Canada and Australia as a model for HIV vaccine development for many years. Unlike rhesus macaques, cynomolgus macaques infected with SIV show a pattern of disease pathogenesis that more closely resembles that of human HIV-1 infection, exhibiting lower peak and set-point viral loads and slower progression to disease with more typical AIDS defining illnesses. Several advances have been made recently in the use of the cynomolgus macaque SIV challenge model that allow the demonstration of vaccine efficacy using attenuated viruses and vectors that are both viral and non-viral in origin. This review aims to probe the details of various vaccination trials carried out in cynomolgus macaques in the context of our modern understanding of the highly diverse immunogenetics of this species with a view to understanding the species-specific immune correlates of protection and the efficacy of vectors that have been used to design vaccines.     

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.     

Viral vectored granulocyte-macrophage colony stimulating factor inhibits vaccine protection in an SIV challenge model: protection correlates with neutralizing antibody

In a previous vaccine study, we reported significant and apparently sterilizing immunity to high-dose, mucosal, simian immunodeficiency virus (SIV) quasi-species challenge. The vaccine consisted of vectors based on vesicular stomatitis virus (VSV) expressing simian immunodeficiency virus (SIV) gag and env genes, a boost with propagating replicon particles expressing the same SIV genes, and a second boost with VSV-based vectors. Concurrent with that published study we had a parallel group of macaques given the same doses of vaccine vectors, but in addition, we included a third VSV vector expressing rhesus macaque GM-CSF in the priming immunization only. We report here that addition of the vector expressing GM-CSF did not enhance CD8 T cell or antibody responses to SIV antigens, and almost completely abolished the vaccine protection against high-dose mucosal challenge with SIV. Expression of GM-CSF may have limited vector replication excessively in the macaque model. Our results suggest caution in the use of GM-CSF as a vaccine adjuvant, especially when expressed by a viral vector. Combining vaccine group animals from this study and the previous study we found that there was a marginal but significant positive correlation between the neutralizing antibody to a neutralization resistant SIV Env and protection from infection.     

An optimized SIV DNA vaccine can serve as a boost for Ad5 and provide partial protection from a high-dose SIVmac251 challenge

One limitation in the development of an improved cellular response needed for an effective HIV-vaccine is the inability to induce robust effector T-cells capable of suppressing a heterologous challenge. To improve cellular immune responses, we examined the ability of an optimized DNA vaccine to boost the cellular immune responses induced by a highly immunogenic Ad5 prime. Five Chinese rhesus macaques received pVax encoding consensus (con) gag/pol/env intramuscularly (IM) with electroporation followed by the Merck Ad5 gag/pol/nef vaccine. A second group of five animals were vaccinated with Merck Ad5 gag/pol/nef followed by pVax gag/pol/env. One year following vaccination, Ad5-prime DNA-boosted monkeys and four unvaccinated controls received an intrarectal challenge with 1000 ID50 SIV(mac)251. The quality and magnitude of the T-cell response was analyzed by ELISpot and polyfunctional flow cytometry. We observed that an Ad5-prime DNA-boost resulted in significantly elevated SIV-specific T-cell responses even compared with animals receiving a DNA-prime Ad5-boost. Ad5 prime DNA boosted animals were capable of suppressing a pathogenic SIV(mac)251 challenge. Peak control correlated with the expansion of HLA-DR(+) CD8(+) T-cells two weeks post-infection. These data illustrate that high optimization of a DNA vaccine can drive of immune responses primed by a robust vector system. This previously unachievable feature of these newly optimized DNAs warrants future studies of this strategy that may circumvent issues of serology associated with viral vector prime-boost systems.     

Divergent HIV and simian immunodeficiency virus surveillance, Zaire

Recent HIV infection or divergent HIV or simian immunodeficiency virus (SIV) strains may be responsible for Western blot-indeterminate results on 70 serum samples from Zairian hospital employees that were reactive in an enzyme immunoassay. Using universal polymerase chain reaction HIV-1, HIV-2, and SIV primers, we detected 1 (1.4%) HIV-1 sequence. Except for 1 sample, no molecular evidence for unusual HIV- or SIV-like strains in this sampling was found.     

蛋脑啡肽对猴免疫缺陷病毒感染CEM×174细胞kappa阿片受体表达的调节作用(英文)

目的：研究蛋脑啡肽对猴免疫缺陷病毒（ＳＩＶ）感染ＣＥＭ×１７４ 细胞ｋａｐｐａ 阿片受体表达的调节作用。方法：用ＳＩＶ 感染ＣＥＭ×１７４ 细胞并加入不同浓度蛋脑啡肽。在２４ ｈ 提取ＲＮＡ,用ＲＴ－ＰＣＲ 方法扩增ｋａｐｐａ 阿片受体ｍ ＲＮＡ 并且进行定量分析。结果：显示ＳＩＶ 能够抑制ＣＥＭ×１７４ 细胞的生长。在１０－ ７ｍ ｏｌ／Ｌ蛋脑啡肽存在下,ＳＩＶ 对细胞的损害减轻。１０－ ７ｍ ｏｌ／Ｌ和１０－ ６ｍ ｏｌ／Ｌ蛋脑啡肽不能改变正常细胞ｋａｐｐａ 阿片受体的表达,但是在ＳＩＶ 感染组的表达显著增高。结论：实验结果提示蛋脑啡肽能够维持正常淋巴细胞的生长。Ｋａｐｐａ 阿片受体表达的改变可能与蛋脑啡肽对免疫细胞的调节机理有关