Article

Inclusion of Vpr accessory gene in a plasmid vaccine cocktail markedly reduces Nef vaccine effectiveness in vivo resulting in CD4 cell loss and increased viral loads in rhesus macaques

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  • Gene One Life Science Inc
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Abstract

We compared the immunogenicity of plasmid vaccines containing multiple human immunodeficiency virus (HIV) antigens and found that covaccination with plasmids expressing HIV-1 14 kDa vpr gene product profoundly reduces antigen-specific CD8-mediated cytotoxic T-cell activity (CTL). Interestingly, Th1 type responses against codelivered antigens (pGag-Pol, pNef, etc.) encoded by the plasmid vaccines were suppressed. This suggested that vpr might compromise CD8 T-cell immunity in vivo during infection. A pilot primate vaccine study was designed to test the hypothesis to compare the following groups: unvaccinated controls, animals vaccinated without simean immunodeficiency virus (SIV)-Nef antigen plasmid, and animals covaccinated with the identical plasmid antigen and a plasmid construct encoding SIV Vpr/Vpx. Animals were subsequently challenged intrarectally with pathogenic SIVmac251 after the final vaccination of a multiple immunization protocol. Control animals were all infected and exhibited high viral loads and rapid CD4+ T-cell loss. In contrast, the Nef plasmid-vaccinated animals were also infected but exhibited preservation of CD4+ T-cells and a multilog reduction in viral load compared with controls. Animals covaccinated multiple times with the Nef vaccine and pVpr/Vpx plasmid suffered rapid and profound loss of CD4+ T-cells. These results have important implications for the design of multicomponent and particle vaccines for HIV-1 as well as for our understanding of HIV/SIV pathogenesis in vivo.

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... In another immunization study, vaccination of monkeys with Tat peptides encompassing Tat B-cell epitopes has provided evidence for a role of anti-Tat Abs in containing plasma viremia [58].Table 1). More recently, vaccination with SIV nef DNA was shown to be safe and to protect macaques against SIV-induced disease [60] (Table 2). The safety and immunogenicity of HIV-1 Nef delivered as plasmid DNA or as recombinant viral vector (modified vaccinia Ankara, MVA) as a therapeutic vaccine was also proven in HIV-infected individuals [34,61] (Table 3 ). ...
... In a monkey pilot study, Vpr suppressed the benefits of nef DNA vaccination by compromising antigen-specific T-cell immunity [60,65] (Table 2 ). In fact, co-immunization with SIV nef and vpr/vpx (SIV protein with Vpr-like functions) abolished the protective effects of vaccination with nef alone against intrarectal challenge with pathogenic SHIV [60] (Table 2). ...
... In a monkey pilot study, Vpr suppressed the benefits of nef DNA vaccination by compromising antigen-specific T-cell immunity [60,65] (Table 2 ). In fact, co-immunization with SIV nef and vpr/vpx (SIV protein with Vpr-like functions) abolished the protective effects of vaccination with nef alone against intrarectal challenge with pathogenic SHIV [60] (Table 2). By contrast, immunization with tat, rev and/or nef DNA elicited humoral and cellular responses in mice [66] (Table 1 ). ...
Article
By the end of 2004, more than 20 HIV-1 vaccine candidates will have entered clinical testing in at least 30 trials worldwide. Almost half of these vaccines include nonstructural HIV-1 gene products. This represents an important innovation in the HIV vaccine field, because until 9 years ago not even preclinical testing in small animal models had been carried out with such immunogens. This review briefly discusses the experimental evidence that provides the rationale for the use of nonstructural HIV-1 gene products as vaccine antigens, and summarizes the current status and the future development of these novel vaccines.
... The loss in number of effective CD8+ T cells in HIV-1 infected patients has been correlated with reduced antiviral effects and disease progression in parallel with deterioration of immune system [159,160]. It has been reported that HIV-1 Vpr interferes with the development of antigen specific immunity [161]. It specifically inhibits the development of strong CD8+ CTL response and suppresses the Th1 immune responses by down-regulating IFN-production. ...
... It specifically inhibits the development of strong CD8+ CTL response and suppresses the Th1 immune responses by down-regulating IFN-production. In the presence of Vpr, there is an isotype shift towards Th2 response [161]. Moreover, Vpr reduces the efficacy of DNA and SIV-Nef vaccination in vivo, suggesting that Vpr may aid in evasion of immune response during HIV-1 [161,162]. ...
... In the presence of Vpr, there is an isotype shift towards Th2 response [161]. Moreover, Vpr reduces the efficacy of DNA and SIV-Nef vaccination in vivo, suggesting that Vpr may aid in evasion of immune response during HIV-1 [161,162]. The mechanism of immune dysfunction caused by Vpr appears to involve the induction of apoptosis and cell cycle arrest in bystander T cells, contributing to the depletion of immune cells. ...
Article
Full-text available
HIV exploits the T-cell signaling network to gain access to downstream cellular components, which serves as effective tools to break the cellular barriers. Multiple host factors and their interaction with viral proteins contribute to the complexity of HIV-1 pathogenesis and disease progression. HIV-1 proteins gp120, Nef, Tat and Vpr alter the T-cell signaling pathways by activating multiple transcription factors including NF-ĸB, Sp1 and AP-1. HIV-1 evades the immune system by developing a multi-pronged strategy. Additionally, HIV-1 encoded proteins influence the apoptosis in the host cell favoring or blocking T-cell apoptosis. Thus, T-cell signaling hijacked by viral proteins accounts for both viral persistence and immune suppression during HIV-1 infection. Here, we summarize past and present studies on HIV-1 T-cell signaling with special focus on the possible role of T cells in facilitating viral infection and pathogenesis.
... Vpr has profound inhibitory effects on many members of the immune system involved in adaptive response (Figure 2). Consequently, Vpr reduces the efficacy of DNA and SIV-Nef vaccination in vivo, suggesting that Vpr may aid in evasion of immune response during HIV-1 [183,184] . The mechanism of immune dysfunction caused by Vpr appears to involve the induction of apoptosis and cell cycle arrest in bystander T-cells, contributing to the depletion of immune cells. ...
... Further, an accidental infection of a lab worker with HIV-1 containing a frame shift mutation in codon 73 of the Vpr gene as well as infection of rhesus macaques with Vpr mutated virus resulted in spontaneous reversion of the Vpr defective virus to the WT phenotype, which implies that Vpr containing virus obtained a selective advantage over the Vpr mutant [134,215]. Vpr has also been shown to reduce the efficacy of DNA and SIV-Nef vaccination in vivo, suggesting that in the absence of Vpr a more effective immune response to HIV would be possible [183,184]. Finally, a recent study of six vertically infected children that presented as long-term nonprogressors reported that every patient had a mutated Vpr gene in addition to mutations in other genes that were not present in all patients [216]. ...
Article
Full-text available
The HIV protein, Vpr, is a multifunctional accessory protein critical for efficient viral infection of target CD4+ T cells and macrophages. Vpr is incorporated into virions and functions to transport the preintegration complex into the nucleus where the process of viral integration into the host genome is completed. This action is particularly important in macrophages, which as a result of their terminal differentiation and non-proliferative status, would be otherwise more refractory to HIV infection. Vpr has several other critical functions including activation of HIV-1 LTR transcription, cell-cycle arrest due to DCAF-1 binding, and both direct and indirect contributions to T-cell dysfunction. The interactions of Vpr with molecular pathways in the context of macrophages, on the other hand, support accumulation of a persistent reservoir of HIV infection in cells of the myeloid lineage. The role of Vpr in the virus life cycle, as well as its effects on immune cells, appears to play an important role in the immune pathogenesis of AIDS and the development of HIV induced end-organ disease. In view of the pivotal functions of Vpr in virus infection, replication, and persistence of infection, this protein represents an attractive target for therapeutic intervention.
... The possibility that Vpr contributes to viral immune-evasion is supported by two papers showing that expression of Vpr hinders effective cellular immune responses against Vpr itself as well as against co-expressed viral antigens [78,79]. The role of Vpr could thus be to blunt cellular immune responses directed against HIV. ...
... A. Amino acid sequence alignment of HIV1 Vpr and HIV2 Vpr/Vpx. HIV1 Vpr shares about 50% and 25% protein sequence identity with HIV2/SIV mac Vpr and Vpx, respectively [78,81]. The region of HIV1 Vpr and HIV2 Vpr that overlaps with Tat is shaded in purple and the region of HIV1 Vpr and HIV2 Vpx that overlaps with Vif is shaded in grey. ...
Article
Full-text available
Among the proteins encoded by human and simian immunodeficiency viruses (HIV and SIV) at least three, Vif, Vpu and Vpr, subvert cellular ubiquitin ligases to block the action of anti-viral defenses. This review focuses on Vpr and its HIV2/SIV counterparts, Vpx and Vpr, which all engage the DDB1.Cullin4 ubiquitin ligase complex through the DCAF1 adaptor protein. Here, we discuss the multiple functions that have been linked to Vpr expression and summarize the current knowledge on the role of the ubiquitin ligase complex in carrying out a subset of these activities.
... In vivo Vpr can interfere with the de novo immune response generation both in mice (30) and in non-human primates (39). The mechanism(s) by which Vpr suppresses these CD4 + T cell responses are still unknown. ...
... Previously, we and others have shown that Vpr also exerts robust anti-inflammatory effects (13,17,18,46,47). Additionally, co-immunization of pVpr with other HIV antigens diminishes their immune potency and limits vaccine control of viral load (39). Therefore, it is likely that Vpr could inhibit the cellular immune response within an HIV infection setting. ...
... An antagonistic interaction of immunogens under the conditions inducing cellular responses was demonstrated when studying a multigene HIV-1 vaccine composed of different plasmids each carrying a separate gene (env, gag, rev, or RT) (Brave et al., 2006). Similarly, a profoundly reduced HIV-specific CD8+ and CD4+ T-cell responses were recorded after a coimmunization with the plasmid expressing HIV-1 14 kDa vpr gene product (Muthumani et al., 2002). These results have important implications for the design of promising combinations of multicomponent vaccines against HIV-1. ...
Article
Immunogenic properties of the combined vaccine CombiHIVvac, comprising polyepitope HIV-1 immunogens, one being the artificial polyepitope protein TBI, containing the T- and B-cell epitopes from Env and Gag proteins, and the DNA vaccine construct pcDNA-TCI coding for the artificial protein TCI, carrying over 80 T-cell epitopes (both CD4+ CTL and CD8+ Th) from Env, Gag, Pol, and Nef proteins, are studied in this work. The data reported demonstrate clearly that a combination of two B- and T-cell immunogens (TBI and TCI) in one construct results in a synergistic increase in the antibody response to both TBI protein and the proteins from HIV-1 lysate. The level of antibodies induced by immunization with the constructs containing either immunogen alone (TBI protein or the plasmid pcDNA-TCI) was significantly lower as compared to that induced by the combined vaccine. The analysis performed suggests that the presence of CD4+ T-helper epitopes, which can be presented by MHC class II, in the protein TCI may be the main reason underlying the increased synthesis of antibodies to TBI protein due to a CD4-mediated stimulation of B-cell proliferation and differentiation.
... Further, HIV-1 infected macrophages have been reported to possess a Type 1 phenotype that is more responsive to LPS (Brown et al., 2008). Therefore, we believe that the differential regulation of the NF-κB pathway by Vpr may promote activation of virus replication in macrophages and perhaps infected T-cells, but inhibition of NF-κB in other bystander cells, ultimately suppression of the immune response as reported in previous work without preventing viral replication (Ayyavoo et al., 1997;Ayyavoo et al., 2002;Muthumani et al., 2002). However, the potential selectivity of the inhibitory effects for bystander cells warrants extensive further investigation. ...
Article
Numerous studies have reported that Vpr alters NF-κB signaling in various cell types, however, the findings have been largely conflicting with reports of both stimulatory and inhibitory effects of Vpr. Our aim was to investigate the role of Vpr signaling in myeloid cells using an adenovirus based expression and indicator system. Our results show that Vpr is inhibitory to NF-κB, however, this effect is dependent on the particular manner of NF-κB stimulation. Consistent with this notion, we report that Vpr has inhibitory effects that are specific to the TNF-α pathway, but not affecting the LPS pathway, suggesting that differential targets of Vpr may exist for NF-κB regulation. Further, we identify VprBP as one possible cellular component of Vpr's regulation of IκBα in response to TNF-α stimulation. We did not identify such a role for HSP27, which instead seems to inhibit Vpr functions. Chronically HIV-1 infected U1 cells with knockdown constructs for Vpr were unexpectedly less responsive to TNF-α mediated viral replication, perhaps suggesting that other HIV-1 components may antagonize these anti-NF-κB effects in infected cells. We hypothesize that Vpr may serve an important role in the context of viral infection and immune function in vivo, through its selective inhibition of NF-κB pathways. J. Cell. Physiol. 228: 781–790, 2013. © 2012 Wiley Periodicals, Inc.
... This immunosuppressive effect was confirmed in rhesus ma-caques immunized with either DNA plasmids encoding Nef and Vpr together or Nef alone, and challenged with SIVmac251. Macaques receiving the combination of Nef and Vpr exhibited higher viral loads and increased CD4 + T cell depletion, closely resembling the control group, whereas the group vaccinated with Nef alone had a significantly improved disease course [55]. ...
Article
Full-text available
The HIV-1 Vpr protein is a viral accessory protein that plays a number of important roles during HIV infection. The activities of Vpr are numerous and include the induction of apoptosis, the modulation of cell cycle arrest, as well as control of viral transcription. Study of HIV clones lacking Vpr in vitro and analysis of HIV variants isolated from longterm nonprogressors in vivo highlight the importance of Vpr for viral replication as well as immune suppression and cell death. Vpr may therefore be considered among the most important accessory proteins encoded by HIV.
... HIV-2 is less pathogenic than HIV-1, likely because the two viruses employ different replication strategies. HIV-1 Vpr, HIV-2/SIV Vpr and Vpx are obviously critical to their respective viruses in vivo as they are all conserved and their absence or alteration produces milder diseases both in humans and simian models [53][54][55][56]. We propose that HIV-2 is better, or at least differently, adapted to its host than HIV-1. ...
Article
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The search for the role(s) that HIV-1 Vpr and its HIV2/SIV paralogs Vpr and Vpx play in viral infection and pathogenesis showed that all three engage CRL4 ubiquitin ligase complexes. This association triggers ubiquitination and degradation of cellular substrates. The identity of the ubiquitin ligase substrates is only now beginning to be revealed. This review focuses on recent work that has identified one such substrate and exposed new cellular restrictions to infection. Three groups have now described cellular factors that restrict HIV-1 infection in cells of the myeloid lineage. One of these factors, sterile alpha motif- and metal-dependent phosphohydrolase domain-containing protein 1 (SAMHD1), was shown to be depleted through the CRL4 ubiquitin ligase complex in the presence of HIV-2/SIV Vpx. The other restriction can be defeated by Vpx in the absence of at least one part of the ubiquitin ligase complex that triggers SAMHD1 depletion.Another group has shown that the previously described upregulation of natural killer-cell ligands on the surface of HIV-1-infected cells requires the actions of both the cytidine deaminase APOBEC3G and uracil-N-glycosylase 2 in association with HIV-1 Vpr. As more cellular interaction partners are identified for HIV-1 Vpr and its paralogs from other viruses, details are emerging about Vpr function. The recent findings have highlighted the existence of two new human proteins that can act to combat HIV infection and have revealed how HIV-1 proteins act in concert to modulate the interaction between natural killer cells and HIV-1 infected cells.
... The reduced cellular immunity may be explained by the blocking of IL-12 secretion by antigen presenting cells such as DC expressing Vpr. This idea is supported by a study in rhesus macaques where inclusion of the Vpr accessory gene in an experimental plasmid-based vaccine containing Nef antigen markedly reduced Th1 type responses compared to the vaccine containing only the Nef antigen, which led to decreased CD4 T cell counts and increased viral loads [25]. Negative effects attributed to Vpr manifested by downregulation of costimulatory molecules and suppression of IL-12 secretion have been reported. ...
Article
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A hallmark of AIDS progression is a switch of cytokines from Th1 to Th2 in the plasma of patients. IL-12, a critical Th1 cytokine secreted by antigen presenting cells (APCs) is suppressed by Vpr, implicating it as an important virulence factor. We hypothesize that Vpr protein packaged in the virion may be required for disabling APCs of the first infected mucosal tissues. Consistent with this idea are reports that defects in the C-terminus of Vpr are associated with long-term non-progression. Vpr RNA amplified from various sources was electroporated into monocyte-derived DC and IL-12 levels in supernatants were analyzed. The analysis of previously reported C-terminal Vpr mutations demonstrate that they do not alleviate the block of IL-12 secretion. However, a novel single conservative amino acid substitution, R90K, reverses the IL-12 suppression. Analysis of 1226 Vpr protein sequences demonstrated arginine (R) present at position 90 in 98.8%, with other substitutions at low frequency. Furthermore, none of sequences report lysine (K) in position 90. Vpr clones harboring the reported substitutions in position 90 were studied for their ability to suppress IL-12. Our data demonstrates that none of tested substitutions other than K relieve IL-12 suppression. This suggests a natural selection for sequences which suppress IL-12 secretion by DC and against mutations which relieve such suppression. Further analyses demonstrated that the R90K, as well as deletion of the C-terminus, directs the Vpr protein for rapid degradation. This study supports Vpr as an HIV virulence factor during HIV infection and for the first time provides a link between evolutionary conservation of Vpr and its ability to suppress IL-12 secretion by DC. DC activated in the presence of Vpr would be defective in the production of IL-12, thus contributing to the prevailing Th2 cytokine profile associated with progressive HIV disease. These findings should be considered in the design of future immunotherapies that incorporate Vpr as an antigen.
... In an attempt to induce broad immunity, all SIVmac239 genes were included in the SIV DNA and rAd/SIV vaccines with the exception of vpr gene which was reported to suppress immune responses to co-delivered antigen [38,39]. The SIV DNA vaccine contained DNA vectors expressing a mutated form of human IL-12 gene (hlL-12N222L) as a genetic adjuvant in addition to SIV Gag-Env, sPol, sVif-Nef, and sTat-Vpx genes (Fig. 1A). ...
Article
In this study, we investigated the ability of a multigenic SIV DNA prime/replication-defective adenovirus serotype 5 (rAd/SIV) boost regimen to induce SIV-specific immune responses and protection against intrarectal challenge with SIVmac251 in rhesus macaques. Four rhesus macaques were immunized intramuscularly three times at 8-week intervals with SIV DNA vaccine and boosted once with rAd/SIV vaccine Four control macaques received the same amount of mock plasmid DNA and mock adenovirus vector. While the SIV DNA vaccine included plasmids expressing a mutated human IL-12 gene (IL-12N222L) as well as SIVmac239 structural and regulatory genes, the rAd/SIV vaccine contained rAd vectors expressing SIVmac239 genes only. Immunization with SIV DNA vaccine alone induced SIV-specific IFN-gamma ELISPOT responses in only two of four vaccinated macaques, whereas all animals developed SIV-specific T-cell responses and Env- and Tat-specific antibody responses following the rAd/SIV vaccine boost. Upon intrarectal challenge with pathogenic SIVmac251, strong anamnestic Env-specific binding and neutralizing antibody responses were detected in the vaccinated macaques. Overall, the immunized macaques had lower peak and set-point viral loads than control macaques, suggesting that the induced immune responses play a role in the control of viremia. In addition, the loss of CD4+ T cells was delayed in the vaccinated macaques after challenge. These results indicate that the multigenic DNA prime-adenovirus boost immunization may be a promising approach in developing an effective AIDS vaccine.
... In addition to the viral structural and enzymatic proteins, Env, Gag, and Pol, viral regulatory, and accessory proteins are important potential vaccine components (Robert-Guroff, 2002). Tat, Rev, and Nef, in particular, have been targeted by several laboratories for vaccine development ( Makitalo et al., 2004; Mossman et al., 2004; Muthumani et al., 2002; Negri et al., 2004; Osterhaus et al., 1999; Patterson et al., 2002; Richardson et al., 2002; Tikhonov et al., 2003; Verrier et al., 2002). Because of its critical role in viral pathogenesis and infectivity (Chang et al., 1995), the transactivator protein, 0042-6822/$ -see front matter. ...
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Among candidate antigens for human immunodeficiency virus (HIV) prophylactic vaccines, the regulatory protein Tat is a critical early target, but has a potential for immune suppression. Adenovirus (Ad) recombinants encoding wild-type HIV Tat (Tat-wt) and a transdominant negative mutant HIV Tat (Tat22) were constructed and administered to mice separately or together with Ad-SIVgag. Immunogenicity and effects on immune responses to the co-administered Gag immunogen were evaluated. Wild-type and mutant Tat recombinants elicited similar Tat-specific cellular and humoral immune responses. Co-administration of either Tat immunogen with Ad-SIVgag induced modest but significant enhancement of Gag-specific interferon-gamma secreting T cells and lymphoproliferative responses. Neither the Ad-recombinant encoding Tat-wt nor Tat22 suppressed induction of anti-Tat or anti-Gag antibodies. Based on the immune responses observed in mice, both recombinants appear to be suitable vaccine candidates. Their contribution to protective efficacy remains to be determined in a non-human primate model.
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The human immunodeficiency virus type 1 (HIV-1) particles consists of two molecules of genomic RNA as well as molecules originating from gag, pol, and env products, all synthesized as precursor proteins. The 96-amino-acid Vpr protein, the only virion-associated HIV-1 regulatory protein, is not part of the virus polyprotein precursors, and its incorporation into virus particles must occur by way of an interaction with a component normally found in virions. To investigate the mechanism of incorporation of Vpr into the HIV-1 virion, Vpr- proviral DNA constructs harboring mutations or deletions in specific virion-associated gene products were cotransfected with Vpr expressor plasmids in COS cells. Virus released from the transfected cells was tested for the presence of Vpr by immunoprecipitation with Vpr-specific antibodies. The results of these experiments show that Vpr is trans-incorporated into virions but at a lower efficiency than when Vpr is expressed from a proviral construct. The minimal viral genetic information necessary for Vpr incorporation was a deleted provirus encoding only the pr55gag polyprotein precursor. Incorporation of Vpr requires the expression but not the processing of gag products and is independent of pol and env expression. Direct interaction of Vpr with the Pr55gag precursor protein was demonstrated by coprecipitation experiments with gag product-specific antibodies. Overall, these results indicate that HIV-1 Vpr is incorporated into the nascent virion through an interaction with the Gag precursor polyprotein and demonstrate a novel mechanism by which viral protein can be incorporated into virus particles.
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Recently, immunization techniques in which DNA constructs are introduced directly into mammalian tissue in vivo have been developed. In theory, gene inoculation should result in the production of antigenic proteins in a natural form in the immunized host. Here we present the use of such a technique for the inoculation of mice with a human immunodeficiency virus type 1 (HIV-1) envelope DNA construct (pM160). Mice were injected intramuscularly with pM160 and were subsequently analyzed for their anti-HIV envelope immune responses. Antisera collected from inoculated animals reacted with the recombinant HIV-1 envelope in ELISA and immunoprecipitation assays. The antisera also contained antibodies that were able to neutralize HIV-1 infection and inhibit HIV-1-mediated syncytium formation in vitro. Furthermore, splenic lymphocytes derived from pM160-inoculated animals demonstrated HIV-envelope-specific proliferative responses. The gene inoculation technique mimics features of vaccination with live attenuated viruses and, therefore, may ultimately prove useful in the rapid development of safe and efficacious vaccines as it provides for production of relevant antigen in vivo without the use of infectious agents.
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Convincing data on experimental vaccines against AIDS have been obtained in the simian immunodeficiency virus (SIV) macaque model by preinfection with a virus attenuated by a nef deletion. To investigate the efficacy of a nef deletion mutant of SIVmac32H called pC8 as a live-attenuated vaccine after shorter preinfection periods and to learn more about the nature of the immune protection induced, eight rhesus monkeys were infected intravenously with the pC8 virus. All monkeys became persistently infected, exhibiting low cell-associated viral loads, but strong cellular and, in terms of binding antibodies, strong humoral antiviral responses. Two of eight pC8-infected monkeys developed an immunodeficiency and were not challenged. Sequence analysis of their nef revealed complete replenishment of the deletion. The other six monkeys, two preinfected for 42 weeks and four for 22 weeks, were challenged with pathogenic spleen-derived SIV. Complete protection was achieved in four vaccinees. Virus was consistently detected in two vaccinees from the 22-week-group challenge, however, they remained clinically healthy over a prolonged period. Protection from challenge virus infection or a delayed disease development seemed to be associated with a sustained SIV-specific T helper cell response after challenge. Thus, a sterilizing immunity against superinfection with pathogenic SIV can be induced even after a relatively short waiting period of 22 weeks. Nevertheless, such a vaccine raises severe safety concerns because of its potential to revert to virulence.
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Despite a strong cytotoxic T-lymphocyte (CTL) response directed against viral antigens, untreated individuals infected with the human immunodeficiency virus (HIV-1) develop AIDS. We have found that primary T cells infected with HIV-1 downregulate surface MHC class I antigens and are resistant to lysis by HLA-A2-restricted CTL clones. In contrast, cells infected with an HIV-1 in which the nef gene is disrupted are sensitive to CTLs in an MHC and peptide-specific manner. In primary T cells HLA-A2 antigens are downmodulated more dramatically than total MHC class I antigens, suggesting that nef selectively downmodulates certain MHC class I antigens. In support of this, studies on cells expressing individual MHC class I alleles have revealed that nef does not downmodulate HLA-C and HLA-E antigens. This selective downmodulation allows infected cells to maintain resistance to certain natural killer cells that lyse infected cells expressing low levels of MHC class I antigens. Downmodulation of MHC class I HLA-A2 antigens occurs not only in primary T cells, but also in B and astrocytoma cell lines. No effect of other HIV-1 accessory proteins such as vpu and vpr was observed. Thus Nef is a protein that may promote escape of HIV-1 from immune surveillance.
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Human immunodeficiency virus type 1 (HIV-1) Vpr participates in nuclear targeting of the viral preintegration complex in nondividing cells and induces G2 cell cycle arrest in proliferating cells, which creates an intracellular milieu favorable for viral replication. Vpr also activates the transcription of several promoters and enhancers by a poorly understood mechanism. Vpr enhances glucocorticoid receptor (GR) signaling and may mediate the effects of steroids on HIV replication. More specifically, recombinant Vpr can potentiate virion production from U937 cells, downregulate NF-κB induction, and enhance programmed cell death, all effects also mediated by glucocorticoids. Vpr has been proposed to act as a GR coactivator, although other studies suggest that these enhancing effects are merely a consequence of G2 cell cycle arrest. We now demonstrate that Vpr functions as a GR coactivator and that this activity is independent of cell cycle arrest. In addition, we show that the Vpr-induced coactivation requires an intact glucocorticoid response element, that it is dependent on the presence of hormone and the corresponding receptor, and that it is mediated by the two highly conserved leucine-rich domains within Vpr that resemble the GR coactivator signature motif.
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In an effort to evaluate the feasibility of developing a safe DNA vaccine for acquired immunodeficiency syndrome (AIDS), we have prepared a plasmid-based immunogen modeled after a naturally occurring noninfectious mutant of the simian immunodeficiency virus (SIV). The mutant SIV genome produces defective virus particles that are noninfectious in vitro and nonpathogenic in vivo in rhesus macaques. Analysis of the mutant genome revealed a 1.6 kb deletion that is in frame and spans integrase, vif, vpx, and most of vpr and results in a pol/vpr gene fusion. This deletion was introduced into the parental pathogenic molecular clone and the U3 region of the 5' LTR was replaced with a cytomegalovirus promoter to produce a candidate DNA vaccine, pIV. After transfection with this plasmid, SIV gag and envelope proteins are expressed and properly processed in vitro. When injected into rabbits, pIV elicited an antibody response to SIV gp130 envelope glycoprotein with titers reaching 1:2048, and a strong lymphoproliferative response to SIV gp130 and whole SIV. The potential to produce defective virus particles in vivo without integrating into the host genome should result in both a strong humoral and cellular immune response in rhesus macaques. In addition, this approach offers a safe alternative to live attenuated vaccines and DNA vaccines that are capable of integration.
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All primate lentiviruses known to date contain one or two open reading frames with homology to the human immunodeficiency virus type 1 (HIV-1) vpr gene. HIV-1 vpr encodes a 96-amino-acid protein with multiple functions in the viral life cycle. These functions include modulation of the viral replication kinetics, transactivation of the long terminal repeat, participation in the nuclear import of preintegration complexes, induction of G2 arrest, and induction of apoptosis. The simian immunodeficiency virus (SIV) that infects African green monkeys (SIVagm) contains a vpr homologue, which encodes a 118-amino-acid protein. SIVagm vpr is structurally and functionally related to HIV-1 vpr. The present study focuses on how three specific functions (transactivation, induction of G2 arrest, and induction of apoptosis) are related to one another at a functional level, for HIV-1 and SIVagm vpr. While our study supports previous reports demonstrating a causal relationship between induction of G2 arrest and transactivation for HIV-1 vpr, we demonstrate that the same is not true for SIVagm vpr. Transactivation by SIVagm vpr is independent of cell cycle perturbation. In addition, we show that induction of G2 arrest is necessary for the induction of apoptosis by HIV-1 vpr but that the induction of apoptosis by SIVagm vpr is cell cycle independent. Finally, while SIVagm vpr retains its transactivation function in human cells, it is unable to induce G2 arrest or apoptosis in such cells, suggesting that the cytopathic effects of SIVagm vpr are species specific. Taken together, our results suggest that while the multiple functions of vpr are conserved between HIV-1 and SIVagm, the mechanisms leading to the execution of such functions are divergent.
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We have generated simplified simian immunodeficiency virus (SIV) constructs lacking the nef, vpr, vpx, vif, tat, and rev genes (Delta6 viruses). To accomplish this, we began with an infectious molecular clone of SIV, i.e. SIVmac239, and replaced the deleted segments with three alternate elements: (i) a constitutive transport element (CTE) derived from simian retrovirus type 1 to replace the Rev/Rev-responsive element (RRE) posttranscriptional regulation system, (ii) a chimeric SIV long terminal repeat (LTR) containing a cytomegalovirus (CMV) promoter to augment transcription and virus production, and (iii) an internal ribosome entry site (IRES) upstream of the env gene to ensure expression of envelope proteins. This simplified construct (Delta6CCI) efficiently produced all viral structural proteins, and mature virions possessed morphology typical of wild-type virus. It was also observed that deletion of the six accessory genes dramatically affected both the specificity and efficiency of packaging of SIV genomic RNA into virions. However, the presence of both the CTE and the chimeric CMV promoter increased the specificity of viral genomic RNA packaging, while the presence of the IRES augmented packaging efficiency. The Delta6CCI virus was extremely attenuated in replication capacity yet retained infectiousness for CEMx174 and MT4 cells. We also generated constructs that retained either the rev gene or both the rev and vif genes and showed that these viruses, when complemented by the CMV promoter, i.e., Delta5-CMV and Delta4-CMV, were able to replicate in MT4 cells with moderate and high-level efficiency, respectively. Long-term culture of each of these constructs over 6 months revealed no potential for reversion. We hope to shortly evaluate these simplified constructs in rhesus macaques to determine their long-term safety as well as ability to induce protective immune responsiveness as proviral DNA vaccines.
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Tat-specific cytotoxic T cells have previously been shown to exert positive Darwinian selection favoring amino acid replacements of an epitope of simian immunodeficiency virus (SIV). The region of thetat gene encoding this epitope falls within a region of overlap between the tat and vpr reading frames, and nonsynonymous nucleotide substitutions in thetat reading frame were found to occur disproportionately in such a way as to cause synonymous changes in the vprreading frame. Comparison of published complete SIV genomes showed Tat to be the least conserved at the amino acid level of nine proteins encoded by the virus, while Vpr was one of the most conserved. Numerous parallel amino acid changes occurred within the Tat epitope independently in different monkeys, and purifying selection on thevpr reading frame, by limiting acceptable nonsynonymous substitutions in the tat reading frame, evidently has enhanced the probability of parallel evolution.
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The ability to monitor vaccine-elicited CD8+ cytotoxic T-lymphocyte (CTL) responses in simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys has been limited by our knowledge of viral epitopes predictably presented to those lymphocytes by common rhesus monkey MHC class I alleles. We now define an SIV and SHIV Nef CTL epitope (YTSGPGIRY) that is presented to CD8+ T lymphocytes by the common rhesus monkey MHC class I molecule Mamu-A*02. All seven infectedMamu-A*02 + monkeys evaluated demonstrated this response, and peptide-stimulated interferon gamma Elispot assays indicated that the response represents a large proportion of the entire CD8+ T-lymphocyte SIV- or SHIV-specific immune response of these animals. Knowledge of this epitope and MHC class I allele substantially increases the number of available rhesus monkeys that can be used for testing prototype HIV vaccines in this important animal model.
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Highly pathogenic simian/human immunodeficiency virus chimeric viruses are known to induce a rapid, irreversible depletion of CD4+ T lymphocytes in the peripheral blood of acutely infected macaque monkeys. To more fully assess the systemic effects of this primary virus infection, specimens were collected serially between days 3 and 21 postinfection from variety of lymphoid tissues (lymph nodes, thymus, and spleen) and gastrointestinal tract and examined by DNA and RNA PCR, in situ hybridization, and immunohistochemical assays. In addition, the lymphoid tissues were evaluated by fluorescence-activated cell sorting. Virus infection was initially detected by DNA PCR on day 3 postinfection in lymph node samples and peaked on day 10 in the T-lymphocyte-rich areas of this tissue. CD4+ T-cell levels remained stable through day 10 in several lymphoid tissue specimens examined but fell precipitously between days 10 and 21. In situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays revealed the accumulation of apoptotic cells during the second week of infection in both lymph nodes and thymus, which colocalized, to a large extent, to sites of both virus replication and CD4+ T-lymphocyte loss.
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Cytotoxic T-lymphocyte (CTL) responses are thought to control human immunodeficiency virus replication during the acute phase of infection. Understanding the CD8(+) T-cell immune responses early after infection may, therefore, be important to vaccine design. Analyzing these responses in humans is difficult since few patients are diagnosed during early infection. Additionally, patients are infected by a variety of viral subtypes, making it hard to design reagents to measure their acute-phase immune responses. Given the complexities in evaluating acute-phase CD8(+) responses in humans, we analyzed these important immune responses in rhesus macaques expressing a common rhesus macaque major histocompatibility complex class I molecule (Mamu-A*01) for which we had developed a variety of immunological assays. We infected eight Mamu-A*01-positive macaques and five Mamu-A*01-negative macaques with the molecularly cloned virus SIV(mac)239 and determined all of the simian immunodeficiency virus-specific CD8(+) T-cell responses against overlapping peptides spanning the entire virus. We also monitored the evolution of particular CD8(+) T-cell responses by tetramer staining of peripheral lymphocytes as well as lymph node cells in situ. In this first analysis of the entire CD8(+) immune response to autologous virus we show that between 2 and 12 responses are detected during the acute phase in each animal. CTL against the early proteins (Tat, Rev, and Nef) and against regulatory proteins Vif and Vpr dominated the acute phase. Interestingly, CD8(+) responses against Mamu-A*01-restricted epitopes Tat(28-35)SL8 and Gag(181-189)CM9 were immunodominant in the acute phase. After the acute phase, however, this pattern of reactivity changed, and the Mamu-A*01-restricted response against the Gag(181-189)CM9 epitope became dominant. In most of the Mamu-A*01-positive macaques tested, CTL responses against epitopes bound by Mamu-A*01 dominated the CD8(+) cellular immune response.
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Deletion of the nef gene from simian immunodeficiency virus (SIV) strain SIVmac239 yields a virus that undergoes attenuated growth in rhesus macaques and offers substantial protection against a subsequent challenge with some SIV wild-type viruses. We used a recently described model to identify sites in which the SIVDeltanef vaccine strain replicates and elicits immunity in vivo. A high dose of SIVDeltanef was applied to the palatine and lingual tonsils, where it replicated vigorously in this portal of entry at 7 days. Within 2 weeks, the virus had spread and was replicating actively in axillary lymph nodes, primarily in extrafollicular T-cell-rich regions but also in germinal centers. At this time, large numbers of perforin-positive cells, both CD8(+) T cells and CD3-negative presumptive natural killer cells, were found in the tonsil and axillary lymph nodes. The number of infected cells and perforin-positive cells then fell. When autopsy studies were carried out at 26 weeks, only 1 to 3 cells hybridized for viral RNA per section of lymphoid tissue. Nevertheless, infected cells were detected chronically in most lymphoid organs, where the titers of infectious virus could exceed by a log or more the titers in blood. Immunocytochemical labeling at the early active stages of infection showed that cells expressing SIVDeltanef RNA were CD4(+) T lymphocytes. A majority of infected cells were not in the active cell cycle, since 60 to 70% of the RNA-positive cells in tissue sections lacked the Ki-67 cell cycle antigen, and both Ki-67-positive and -negative cells had similar grain counts for viral RNA. Macrophages and dendritic cells, identified with a panel of monoclonal antibodies to these cells, were rarely infected. We conclude that the attenuated growth and protection observed with the SIVDeltanef vaccine strain does not require that the virus shift its characteristic site of replication, the CD4(+) T lymphocyte. In fact, this immunodeficiency virus can replicate actively in CD4(+) T cells prior to being contained by the host, at least in part by a strong killer cell response that is generated acutely in the infected lymph nodes.
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Accumulating evidence suggests that HIV-specific CD8(+) CTL are dysfunctional in HIV-infected individuals with progressive clinical disease. In the present studies, cytokine production by virus-specific CTL was assessed in the rhesus monkey model for AIDS to determine its contribution to the functional impairment of CTL. CTL from monkeys infected with nonpathogenic isolates of simian and simian-human immunodeficiency virus expressed high levels of IFN-gamma, TNF-alpha, and IL-2 after in vitro exposure to a nonspecific mitogen or the optimal peptide representing a dominant virus-specific CTL epitope. However, similarly performed studies assessing these capabilities in CTL from monkeys infected with pathogenic immunodeficiency virus isolates demonstrated a significant dysfunction in the ability of the CTL to produce IL-2 and TNF-alpha. Importantly, CTL from vaccinated monkeys that effectively controlled the replication of a highly pathogenic simian-human immunodeficiency virus isolate following challenge demonstrated a preserved capacity to produce these cytokines. These experiments suggest that defects in cytokine production may contribute to CTL dysfunction in chronic HIV or SIV infection. Moreover, an AIDS vaccine that confers protection against clinical disease evolution in this experimental model also preserves the functional capacity of these CTL to produce both IL-2 and TNF-alpha.
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HIV-1 viral protein R (Vpr) is a virion-associated gene product that profoundly affects T cell proliferation, induces apoptosis and can affect cytokine production in part through interfering with NF-kappa B-mediated transcription from host cells. Collectively, these effects support that Vpr could influence immune activation in vivo. However, this effect of Vpr has not been explored previously. Here we examined the effect of Vpr expression in an in vivo model system on the induction of antigen-specific immune responses using a DNA vaccine model. Vpr co-vaccination significantly altered the immune response to co-delivered antigen. Specifically, in the presence of Vpr, inflammation was markedly reduced compared to antigen alone. Vpr reduced antigen-specific CD8-mediated cytotoxic T lymphocyte activity and suppressed T(h)1 immune responses in vivo as evidenced by lower levels of IFN-gamma. In the presence of Vpr, there is a profound shift in isotype towards a T(h)2 response as determined by the IgG2a:IgG1 ratio. The data support that Vpr compromises antigen-specific immune responses and ultimately effector cell function, thus confirming a strong selective advantage to the virus at the expense of the host.
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A versatile DNA vaccine (pdIV3) was constructed by replacing the integrase, vif, vpx, and vpr genes of a pathogenic simian immunodeficiency virus (SIV) molecular clone with a linker containing unique cloning sites. The 5' long terminal repeat (LTR) is truncated and transcription is controlled by a cytomegalovirus (CMV) promoter. The construct expresses Gag and Env in vitro and noninfectious virus particles are produced from transfected cells. The ability of pdIV3 to promote cellular and humoral immune responses, along with the flexibility of the linker design to allow insertion of immunostimulatory genes in future constructs, makes this a useful base vector for immunization against primate lentiviruses. We present the construction of a retroviral plasmid designed to serve as a template for the development of safe and effective vaccines against primate immunodeficiency retroviruses. This vaccine component should facilitate the simultaneous induction of cellular and humoral immune responses that protect primates against infection with SIV and human immunodeficiency virus (HIV) and the development of acquired immune deficiency syndrome (AIDS). This plasmid could induce the appropriate immune response required to attack both cell-free and cell-associated viruses. The lack of infectivity, the inability to integrate, and the SIV origin make this construct a safe alternative to attenuated vaccines based on HIV. In addition, we intend to develop this construct as an immunotherapeutic approach to lower the viremia in AIDS patients.
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Recent studies of human immunodeficiency virus type 1 (HIV-1) infection in humans and of simian immunodeficiency virus (SIV) in rhesus monkeys have shown that resolution of the acute viral infection and control of the subsequent persistent infection are mediated by the antiviral cellular immune response. We comparatively assessed several vaccine vector delivery systems-three formulations of a plasmid DNA vector, the modified vaccinia Ankara (MVA) virus, and a replication incompetent adenovirus type 5 (Ad5) vector-expressing the SIV gag protein for their ability to elicit such immune responses in monkeys. The vaccines were tested either as a single modality or in combined modality regimens. Here we show that the most effective responses were elicited by a replication-incompetent Ad5 vector, used either alone or as a booster inoculation after priming with a DNA vector. After challenge with a pathogenic HIV-SIV hybrid virus (SHIV), the animals immunized with Ad5 vector exhibited the most pronounced attenuation of the virus infection. The replication-defective adenovirus is a promising vaccine vector for development of an HIV-1 vaccine.
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HIV-1 viral protein R (Vpr) is a virion-associated gene product that profoundly affects T cell proliferation, induces apoptosis and can affect cytokine production in part through interfering with NF-κB-mediated transcription from host cells. Collectively, these effects support that Vpr could influence immune activation in vivo. However, this effect of Vpr has not been explored previously. Here we examined the effect of Vpr expression in an in vivo model system on the induction of antigen-specific immune responses using a DNA vaccine model. Vpr co-vaccination significantly altered the immune response to co-delivered antigen. Specifically, in the presence of Vpr, inflammation was markedly reduced compared to antigen alone. Vpr reduced antigen-specific CD8-mediated cytotoxic T lymphocyte activity and suppressed Th1 immune responses in vivo as evidenced by lower levels of IFN-γ. In the presence of Vpr, there is a profound shift in isotype towards a Th2 response as determined by the IgG2a:IgG1 ratio. The data support that Vpr compromises antigen-specific immune responses and ultimately effector cell function, thus confirming a strong selective advantage to the virus at the expense of the host.
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We previously constructed a simian immunodeficiency virus/human immunodeficiency virus type 1 (HIV-1) chimeric virus, NM-3rN to generate a pathogenic HIV-1 in macaque monkeys. During the in vivo passage of this virus in several monkeys, a viral strain, R43-56 was obtained which acquired a better replication ability in vivo. MM121, one of the three monkeys inoculated with the R43-56, showed weight loss, diarrhea and a rapid and continuous decrease in CD4(+) lymphocytes at the moribund stage. An autopsy revealed generalized lymphadenopathy, dehydration, and ileocecal intussusception. In situ hybridization showed that the virus infection was in systemic lymphoid organs. We are presently monitoring the survivors to obtain candidates for a more virulent virus. R43-56 may be a better challenge virus and useful tool for human acquired immunodeficiency syndrome research.
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The human immunodeficiency virus 1 (HIV-1) is a complex retrovirus with more genes than most retroviruses. One of these extra genes codes for a protein called Vpr, which has recently been shown to prevent activation of the mitotic cyclin-dependent kinase and thereby prevent infected cells from undergoing mitosis and proliferating. Vpr also plays an important role in another property of HIV-1 that is unusual for a retrovirus – its ability to enter the nucleus of a nondividing cell. Understanding the interactions between HIV-1 and the cell cycle should lead to new insights into both viral pathogenesis and basic cell biology.
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In order to examine the efficiency of an AIDS vaccine potentially acceptable for human use we have investigated a split vaccine. Since such vaccines are safe and efficient, they have been in use for many years to protect man against enveloped RNA viruses, e.g., influenza and measles. Seven rhesus monkeys were immunized at Week 0, 4, 8, and 16 by im injection of 2 ml of vaccine containing 140 micrograms of Tween-ether-disrupted SIVmac251/32H adsorbed onto aluminum hydroxide. The immunized animals and three nonvaccinated control monkeys were challenged 2 weeks after the last immunization by iv injection of 10 to 50 minimal monkey infectious doses of SIVmac251/32H. Four of seven immunized animals did not show any signs of virus replication and therefore appeared to be protected. Nonvaccinated control animals and the vaccine failures showed a rise in their urinary neopterin concentrations 1 to 2 weeks after infection. At the end of the second week and thereafter, cocultures and polymerase chain reaction of their peripheral blood lymphocytes were positive. After the challenge, control animals and infected vaccinees showed a primary or secondary antibody response while antibody titers declined in virus-negative animals. Specific cytotoxic T-lymphocytes were not present prior to challenge, but were present in some animals thereafter. Therefore, these seem to reflect a response to viral replication rather than to immunization. Prior to challenge the CD4-positive lymphocytes of the peripheral blood of the four virus-negative animals only proliferated after exposure to the immunizing antigen. Thus, this reaction appears to predict protection.
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In studies of the genetics of human immunodeficiency virus type 1 (HIV-1), the product of the nef gene, formerly known as F, 3'-orf, or B-ORF, was a negative regulator of HIV-1 replication. Proviruses with mutations in the nef gene replicated better than their standard counterparts during transient expression, and the mutant virus maintained its enhanced replication even after serial passages in T lymphocytes. The nef protein trans-suppressed, in a dose-dependent manner, the replication of wild-type and nef mutant proviruses and the expression of reporter genes linked to the HIV-1 long terminal repeat (LTR). The repression induced by the nef protein was mediated by inhibition of transcription from the HIV-1 LTR, which contains a far upstream cis element (previously recognized to be a negative regulatory element) between 340 and 156 nucleotides upstream of the RNA initiation site.
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Cell lines from rhabdomyosarcomas, which are tumors of muscle origin, have been used as models of CD4-independent HIV infection. These cell lines can be induced to differentiate in vitro. We report here that the vpr gene of HIV1 is sufficient for the differentiation of the human rhabdomyosarcoma cell line TE671. Differentiated cells are characterized by great enlargement, altered morphology, lack of replication, and high level expression of the muscle-specific protein myosin. We have also observed the morphological differentiation and inhibition of proliferation of two other transformed cell lines. vpr-transfected cells remain fully viable in culture for extended periods. These observations elucidate a potential role for vpr in the virus life cycle and raise the possibility that some aspects of HIV-induced pathologies may be caused by a disturbance of cells by vpr.
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The human immunodeficiency virus 1 (HIV-1) is a complex retrovirus with more genes than most retroviruses. One of these extra genes codes for a protein called Vpr, which has recently been shown to prevent activation of the mitotic cyclin-dependent kinase and thereby prevent infected cells from undergoing mitosis and proliferating. Vpr also plays an important role in another property of HIV-1 that is unusual for a retrovirus - its ability to enter the nucleus of a nondividing cell. Understanding the interactions between HIV-1 and the cell cycle should lead to new insights into both viral pathogenesis and basic cell biology.
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Internally controlled RT-PCR methods (QC-RT-PCR) for quantification of SIV RNA are effective, but are relatively cumbersome, expensive, and time and labor intensive. For greater throughput and efficiency, we have developed a method for quantification of plasma SIV RNA levels by real-time RT-PCR using the Applied Biosystems Prism 7700 sequence detection system. This assay format allows real-time kinetic analysis of PCR product generation, providing a broad linear dynamic range and ensuring that quantification is based on analysis during the exponential phase of amplification, regardless of the input template copy number. Simultaneous amplification and analysis eliminates any requirement for handling amplified products, increasing throughput and eliminating a potential source of assay contamination. The assay we have developed for quantification of SIV RNA has a nominal threshold sensitivity of 300 copy Eq/ml of plasma, although as little as 10 copy Eq/reaction of SIV RNA template can be detected. The linear dynamic range is in excess of 5 logs. Interassay reproducibility averages 25% (coefficient of variation), based on studies of extraction and analysis of replicate aliquots of the same plasma specimens. The combination of sensitivity, precision, and broad dynamic range allows reliable quantification of viral load even during dynamic phases of SIV infection, such as through the onset and resolution of primary infection, or during treatment with antiretroviral agents. The primer-probe combinations we have developed allow quantification of SIV isolates most commonly used for experimental studies. Availability of this assay should greatly facilitate studies of basic pathogenesis and evaluation of therapeutic and prophylactic approaches in the SIV-infected macaque.
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We previously constructed a simian immunodeficiency virus+human immunodeficiency virus type 1 (HIV-1) chimeric virus, NM-3rN to generate a pathogenic HIV-1 in macaque monkeys. During the in vivo passage of this virus in several monkeys, a viral strain, R43-56 was obtained which acquired a better replication ability in vivo. MM121, one of the three monkeys inoculated with the R43-56, showed weight loss, diarrhea and a rapid and continuous decrease in CD4+ lymphocytes at the moribund stage. An autopsy revealed generalized lymphadenopathy, dehydration, and ileocecal intussusception. In situ hybridization showed that the virus infection was in systemic lymphoid organs. We are presently monitoring the survivors to obtain candidates for a more virulent virus. R43-56 may be a better challenge virus and useful tool for human acquired immunodeficiency syndrome research.
Article
Expression of human immunodeficiency virus–type 1 (HIV-1) Vpr after productive infection of T cells induces cell cycle arrest in the G2 phase of the cell cycle. In the absence of de novo expression, HIV-1 Vpr packaged into virions still induced cell cycle arrest. Naturally noninfectious virus or virus rendered defective for infection by reverse transcriptase or protease inhibitors were capable of inducing Vpr-mediated cell cycle arrest. These results suggest a model whereby both infectious and noninfectious virions in vivo, such as those surrounding follicular dendritic cells, participate in immune suppression.
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To develop an HIV-1 accessory gene immunogen using a DNA vaccine approach. HIV-1 accessory genes vif, vpu and nef were modified to express under the control of a single promoter with cellular proteolytic cleavage sites between the coding sequences (VVN-P). Immune responses induced by these constructs were evaluated in mice. DNA vaccine construct (pVVN-P) expressing Vif, Vpu and Nef was processed and the fusion protein was cleaved appropriately. Vif, Vpu and Nef as a fusion protein with proteolytic cleavage sites (VVN-P) is able to induce a significant level of cellular immune responses. We also observed that accessory genes Vif, Vpu and Nef (VVN-P) induced an effective T helper 1 proliferative response measured by cytokine production. Furthermore, expression cassette pVVN-P was able to induce cytotoxic T lymphocyte (CTL) responses against diverse HIV-1 viruses in infected target cells. We conclude that cell-mediated immune responses induced by accessory gene constructs from clade B may have a broader recognition of divergent HIV-1 viruses and should be further examined for both prophylactic and therapeutic vaccination schemes against HIV-1.
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A definition of the specific cell types that support HIV replication early in the course of infection will be important for understanding AIDS pathogenesis and designing strategies for preventing infection. Observations have indicated that the population of lymphocytes susceptible to productive infection extends beyond activated CD4(+) T cells. To explore this issue, we have employed laser scanning cytometry technology and the techniques of lymphocyte surface immunophenotyping followed by fluorescent in situ hybridization to detect simian immunodeficiency virus of macaques (SIVmac) RNA in phenotypically defined rhesus monkey lymphocytes. The immunophenotype of productively infected cells in either a rhesus monkey T cell line or in PBMCs infected in vitro with SIVmac was remarkably similar to that observed in productively infected PBMCs obtained from monkeys during primary infection. We observed low levels or no detectable expression of CD4 on cells infected in vitro or on PBMCs of infected monkeys. However, a substantial number of SIVmac-infected PBMCs both in cultured lymphocytes and sampled directly from infected monkeys expressed CD8 but not CD4. These observations are consistent with the possibility that the CD4 molecule may be modulated off the surface of CD4(+)CD8(-) or CD4(+)CD8(+) lymphocytes after infection or that infection occurred via a CD4-independent mechanism. Moreover, there was no preferential expression of CD25 on cells positive for SIVmac RNA, which might have been predicted if replication of the virus was occurring selectively in activated lymphocytes. These results broaden the range of lymphocytes that support productive SIVmac infection to include CD4(-)CD8(-) and CD4(-)CD8(+) subsets, and are consistent with virus replication occurring in nonactivated cells.
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Control of the worldwide AIDS epidemic will only be achieved with a safe and effective prophylactic HIV-1 vaccine. DNA vaccination has recently emerged as a promising vaccine modality that can elicit both humoral and cellular immune responses. HIV-1- and SIV-specific immune responses have been elicited by DNA vaccines in both mice and nonhuman primates. However, these immune responses have not been capable of protecting nonhuman primates against pathogenic AIDS virus challenges. A number of approaches are therefore being investigated to augment DNA vaccine-elicited immune responses.
Article
DNA vaccination techniques have been recently under intensive investigation both preclinically and in human studies aimed at impacting viral infection. Collectively, DNA vaccines expressing viral antigens induce both antigen-specific humoral and cellular immune responses which in model systems are capable of impacting viral infection. However, in clinical settings the potency of this approach is still under investigation. Efficacy is improved in specific circumstances through the addition of immunomodulatory molecules including cytokines as plasmid cassettes or through modification of the numbers of specific CpG sequences present in the backbone. Furthermore, combined vaccination schemes have been an important research focus for generating enhanced immunogenicity against viral infections. The ultimate utility of these approaches to prevent viral infection will require more work. However, improvements in the potency and focus of DNA vaccines present us with new opportunities for both basic research into protective immunity as well as novel strategies for immune therapy and prophylaxis.
Article
Heterologous prime/boost regimens have the potential for raising high levels of immune responses. Here we report that DNA priming followed by a recombinant modified vaccinia Ankara (rMVA) booster controlled a highly pathogenic immunodeficiency virus challenge in a rhesus macaque model. Both the DNA and rMVA components of the vaccine expressed multiple immunodeficiency virus proteins. Two DNA inoculations at 0 and 8 weeks and a single rMVA booster at 24 weeks effectively controlled an intrarectal challenge administered 7 months after the booster. These findings provide hope that a relatively simple multiprotein DNA/MVA vaccine can help to control the acquired immune deficiency syndrome epidemic.
Article
Multicomponent DNA vaccines were used to elicit immune responses, which can impact viral challenge in three separate rhesus macaque models. Eight rhesus macaques were immunized with DNA vaccines for HIV env/rev and SIV gag/pol and were challenged intravenously with 10 animal infective doses (AID(50)) of cell-free SHIV IIIB. Three of eight immunized rhesus macaques were protected, exhibiting no detectable virus. Animals protected from nonpathogenic SHIVIIIB challenge were rested for extended periods of time and were rechallenged first with pathogenic SIV(mac239) and subsequently with pathogenic SHIV89.6P viruses. Following the pathogenic challenges, all three vaccinated animals were negative for viral coculture and antigenemia and were negative by PCR. In contrast, the control animals exhibited antigenemia by 2 weeks postchallenge and exhibited greater than 10 logs of virus/10(6) cells in limiting dilution coculture. The control animals exhibited CD4 cell loss and developed SIV-related wasting with high viral burden and subsequently failed to thrive. Vaccinated animals remained virus-negative and were protected from the viral load, CD4 loss, disease, and death. We observed strong Th1-type cellular immune responses in the protected macaques throughout the study, suggesting their important roles in protection. These studies support the finding that multicomponent DNA vaccines can directly impact viral replication and disease in a highly pathogenic challenge system, thus potentially broadening our strategies against HIV.
Article
We developed an AIDS vaccine based on attenuated VSV vectors expressing env and gag genes and tested it in rhesus monkeys. Boosting was accomplished using vectors with glycoproteins from different VSV serotypes. Animals were challenged with a pathogenic AIDS virus (SHIV89.6P). Control monkeys showed a severe loss of CD4+ T cells and high viral loads, and 7/8 progressed to AIDS with an average time of 148 days. All seven vaccinees were initially infected with SHIV89.6P but have remained healthy for up to 14 months after challenge with low or undetectable viral loads. Protection from AIDS was highly significant (p = 0.001). VSV vectors are promising candidates for human AIDS vaccine trials because they propagate to high titers and can be delivered without injection.
Article
Human immunodeficiency virus type 1 (HIV-1) Vpr, a 14-kDa virion-associated protein, plays an important role in the viral life cycle. Using a panel of truncated HIV-1 LTR-CAT constructs and Vpr expression plasmid, we have identified sequences from nucleotide -278 to -176 in LTR as Vpr-mediated transactivation domain. This region includes the glucocorticoid response element (GRE) in HIV-1 LTR. Transactivation by Vpr was noted with the HIV-1 LTR reporter constructs containing CAT or luciferase. A similar effect was also observed with a construct in which the GRE motif was linked to CAT. Studies involving Vpr mutants identified that helical domains I and III, and amino acid residues at G75 and C76, are responsible for GRE-mediated LTR transactivation. The transactivation function of Vpr is independent of its cell cycle arrest activity. Further, viral replication studies indicated that Vpr-mediated increase in viral replication is directly correlated with the ability of Vpr to transactivate HIV-1 LTR. The results presented here demonstrate that Vpr activates HIV-1 LTR through the host GR pathway and suggest that an intact GRE in the LTR is critical for Vpr activity.
Article
Although CTL escape has been well documented in pathogenic simian immunodeficiency virus (SIV) infection, there is no information on CTL escape in nonpathogenic SIV infection in nonhuman primate hosts like the sooty mangabeys. CTL responses and sequence variation in the SIV nef gene were evaluated in one sooty mangabey and one rhesus macaque inoculated together with the same stock of cloned SIVmac239. Each animal developed an immunodominant response to a distinct CTL epitope in Nef, aa 157-167 in the macaque and aa 20-28 in the mangabey. Nonsynonymous mutations in their respective epitopes were observed in both animals and resulted in loss of CTL recognition. These mutations were present in the majority of proviral DNA sequences at 16 weeks post infection in the macaque and >2 years post infection in the mangabey. These results document the occurrence of CTL escape in a host that does not develop AIDS, and adds to the growing body of evidence that CTL exert significant selective pressure in SIV infection.
Article
Potent virus-specific cytotoxic T lymphocyte (CTL) responses elicited by candidate AIDS vaccines have recently been shown to control viral replication and prevent clinical disease progression after pathogenic viral challenges in rhesus monkeys. Here we show that viral escape from CTL recognition can result in the eventual failure of this partial immune protection. Viral mutations that escape from CTL recognition have been previously described in humans infected with human immunodeficiency virus (HIV) and monkeys infected with simian immunodeficiency virus (SIV). In a cohort of rhesus monkeys that were vaccinated and subsequently infected with a pathogenic hybrid simian-human immunodeficiency virus (SHIV), the frequency of viral sequence mutations within CTL epitopes correlated with the level of viral replication. A single nucleotide mutation within an immunodominant Gag CTL epitope in an animal with undetectable plasma viral RNA resulted in viral escape from CTLs, a burst of viral replication, clinical disease progression, and death from AIDS-related complications. These data indicate that viral escape from CTL recognition may be a major limitation of the CTL-based AIDS vaccines that are likely to be administered to large human populations over the next several years.
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