Highly Attenuated Infection With a Vpr-Deleted Molecular Clone of Human Immunodeficiency Virus-1.

A 48-year-old woman was infected with a vpr-defective human immunodeficiency virus (HIV)-1 molecular clone. Seroconversion was markedly delayed, and without treatment she had durably suppressed viremia and normal T-cell levels. Neutralizing antibody and CD8+ T-cell immune responses against HIV-1 were unremarkable. Viral sequences confirmed the source but evolved defective nef, suggesting an unknown mechanistic link to vpr. There were subtle qualitative defects in T and B cells. To our knowledge, this is the only case of human infection with a characterized defective HIV-1 molecular clone, which furthermore recapitulated live-attenuated vaccination in macaque models of HIV-1 vaccine research.

Simian immunodeficiency virus-specific CD8+ T cells recognize Vpr- and Rev-derived epitopes early after infection.

The kinetics of CD8(+) T cell epitope presentation contribute to the antiviral efficacy of these cells yet remain poorly defined. Here, we demonstrate presentation of virion-derived Vpr peptide epitopes early after viral penetration and prior to presentation of Vif-derived epitopes, which required de novo Vif synthesis. Two Rev epitopes exhibited differential presentation kinetics, with one Rev epitope presented within 1 h of infection. We also demonstrate that cytolytic activity mirrors the recognition kinetics of infected cells. These studies show for the first time that Vpr- and Rev-specific CD8(+) T cells recognize and kill simian immunodeficiency virus (SIV)-infected CD4(+) T cells early after SIV infection.

Induction of tumor-specific CTL responses using the C-terminal fragment of Viral protein R as cell penetrating peptide.

The discovery of tumor-associated antigens recognized by T lymphocytes opens the possibility of vaccinating cancer patients with defined antigens. However, one of the major limitation of peptide-based vaccines is the low immunogenicity of antigenic peptides. Interestingly, if these epitopes are directly delivered into the cytoplasm of antigen presenting cells, they can be efficiently presented via the direct MHC class I presentation pathway. To improve antigen entry, one promising approach is the use of cell penetrating peptides (CPPs). However, most studies use a covalent binding of the CPP with the antigen. In the present study, we focused on the C-terminal domain of Vpr which was previously demonstrated to efficiently deliver plasmid DNA into cells. We provide evidence that the peptides Vpr55-91 and Vpr55-82 possess the capacity of delivering proteins and epitopes into cell lines as well as into human primary dendritic cells, without the necessicity for a chemical linkage. Moreover, immunization of HLA-A2 transgenic mice with Vpr55-91 as the sole adjuvant is able to induce antigen-specific cytotoxic T lymphocytes against multiple tumor epitopes.

A comprehensive analysis of the naturally occurring polymorphisms in HIV-1 Vpr: potential impact on CTL epitopes.

The enormous genetic variability reported in HIV-1 has posed problems in the treatment of infected individuals. This is evident in the form of HIV-1 resistant to antiviral agents, neutralizing antibodies and cytotoxic T lymphocytes (CTLs) involving multiple viral gene products. Based on this, it has been suggested that a comprehensive analysis of the polymorphisms in HIV proteins is of value for understanding the virus transmission and pathogenesis as well as for the efforts towards developing anti-viral therapeutics and vaccines. This study, for the first time, describes an in-depth analysis of genetic variation in Vpr using information from global HIV-1 isolates involving a total of 976 Vpr sequences. The polymorphisms at the individual amino acid level were analyzed. The residues 9, 33, 39, and 47 showed a single variant amino acid compared to other residues. There are several amino acids which are highly polymorphic. The residues that show ten or more variant amino acids are 15, 16, 28, 36, 37, 48, 55, 58, 59, 77, 84, 86, 89, and 93. Further, the variant amino acids noted at residues 60, 61, 34, 71 and 72 are identical. Interestingly, the frequency of the variant amino acids was found to be low for most residues. Vpr is known to contain multiple CTL epitopes like protease, reverse transcriptase, Env, and Gag proteins of HIV-1. Based on this, we have also extended our analysis of the amino acid polymorphisms to the experimentally defined and predicted CTL epitopes. The results suggest that amino acid polymorphisms may contribute to the immune escape of the virus. The available data on naturally occurring polymorphisms will be useful to assess their potential effect on the structural and functional constraints of Vpr and also on the fitness of HIV-1 for replication.

A suboptimal 5' splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication.

BACKGROUND: Inefficient alternative splicing of the human immunodeficiency virus type 1(HIV-1) primary RNA transcript results in greater than half of all viral mRNA remaining unspliced. Regulation of HIV-1 alternative splicing occurs through the presence of suboptimal viral 5' and 3' splice sites (5' and 3'ss), which are positively regulated by exonic splicing enhancers (ESE) and negatively regulated by exonic splicing silencers (ESS) and intronic splicing silencers (ISS). We previously showed that splicing at HIV-1 3'ss A2 is repressed by ESSV and enhanced by the downstream 5'ss D3 signal. Disruption of ESSV results in increased vpr mRNA accumulation and exon 3 inclusion, decreased accumulation of unspliced viral mRNA, and decreased virus production. RESULTS: Here we show that optimization of the 5'ss D2 signal results in increased splicing at the upstream 3'ss A1, increased inclusion of exon 2 into viral mRNA, decreased accumulation of unspliced viral mRNA, and decreased virus production. Virus production from the 5'ss D2 and ESSV mutants was rescued by transient expression of HIV-1 Gag and Pol. We further show that the increased inclusion of either exon 2 or 3 does not significantly affect the stability of viral mRNA but does result in an increase and decrease, respectively, in HIV-1 mRNA levels. The changes in viral mRNA levels directly correlate with changes in tat mRNA levels observed upon increased inclusion of exon 2 or 3. CONCLUSION: These results demonstrate that splicing at HIV-1 3'ss A1 is regulated by the strength of the downstream 5'ss signal and that suboptimal splicing at 3'ss A1 is necessary for virus replication. Furthermore, the replication defective phenotype resulting from increased splicing at 3'ss A1 is similar to the phenotype observed upon increased splicing at 3'ss A2. Further examination of the role of 5'ss D2 and D3 in the alternative splicing of 3'ss A1 and A2, respectively, is necessary to delineate a role for non-coding exon inclusion in HIV-1 replication.

Development and characterization of ten monoclonal anti-Vpr antibodies.

HIV-1 Vpr is a 96-amino acid auxiliary protein that performs numerous activities during viral infection. In the present study, 10 antibodies were generated after mice immunization with either the N- or the C-terminus domain of Vpr, respectively, Vpr(1-51) and Vpr(52-96). ELISA and immunoblot experiments using pure synthetic overlapping Vpr peptides suggested that these anti-Vpr antibodies could be classified into five groups and that they recognized conformational or linear Vpr epitopes. Further analysis revealed the effect of C-terminal arginine mutations on the antibody binding. Two of the antibodies precipitated Vpr expressed after transfection of a Vpr-encoding vector in human cells. More importantly, one of them was able to detect Vpr in HIV-1-infected U1 cells and in HIV-1-infected human PBMC. Surface plasmon resonance experiments demonstrated that some of these antibodies prevented the interaction between Vpr and one of its cellular partners, the adenine nucleotide translocator. Thus, these anti-Vpr monoclonal antibodies may be useful to any laboratory working on the molecular mechanism of HIV-1 infection.

HIV and inflammation.

A main feature of HIV infection is the expression of several proinflammatory cytokines. Proinflammatory cytokines expressed as soluble factors or membrane-bound molecules regulate both HIV replication and T cell apoptosis. Proinflammatory cytokines have key roles in the HIV lifecycle, especially at the level of transcription, favouring the ability of HIV to establish latent reservoirs. In addition, proinflammatory cytokines are involved in both CD4+ T cell and CD8+ T cell apoptosis, resulting in immune suppression. Moreover, several HIV proteins such as Nef, Tat, and Vpr hijack proinflammatory cytokine signaling, further underlining the potential importance of inflammation in HIV pathogenesis. In vivo chronic inflammatory conditions have been correlated to increased levels of viremia and accelerated disease progression. This article raises the possibility that inflammation plays a crucial role in both immune suppression and the formation of viral reservoirs during HIV infection. Understanding the role of inflammation in HIV infection could lead to new therapeutic strategies that could ultimately enhance immune restoration and limit the formation of viral reservoirs in HIV-infected patients.

Gene-mutated HIV-1/SIV chimeric viruses as AIDS live attenuated vaccines for potential human use.

To develop an AIDS vaccine for human use as well as a suitable animal model for AIDS research, we constructed a series of HIV-1/SIVmac chimeric viruses (SHIVs). We successfully generated a SHIV (designated as NM-3rN) having the HIV-1 env gene, which enabled the evaluation of the efficacy of HIV-1 Env-targeted vaccines in macaque monkeys instead of chimpanzees. Two NM-3rN derivatives (NM-3 and NM-3n) induced long-term anti-virus immunities without manifesting the disease. The monkeys vaccinated with NM-3 or NM-3n became resistant to a challenge inoculation with NM-3rN. Serum from a monkey vaccinated with NM-3 neutralized not only the parental HIV-1 (NL432), but also an antigenically different HIV-1 (MN). In vivo experiments confirmed the heterologous protection against an SHIV having the HIV-1 (MN) env. In addition to specific immunity including neutralizing antibodies and cytotoxic T lymphocyte activity, nonspecific immunity such as natural killer activity is associated with this protection. These data suggest that the live vaccine has the ability to protect individuals against various types of HIVs. These SHIVs should contribute to the development of future anti-HIV-1 live vaccines in humans.

Packageable antiviral therapeutics against human immunodeficiency virus type 1: virion-targeted virus inactivation by incorporation of a single-chain antibody against viral integrase into progeny virions.

To determine their activities as an antiviral agent packageable within virions and suitable for continued expression in cells, we tested a single-chain antibody (scAb) against human immunodeficiency virus type 1 (HIV-1) integrase and its three fusion proteins: fused to viral protein R (scab-Vpr), a double-cassette of the WXXF motif binding to Vpr (scAb-WXXF), and viral major capsid protein (scAb-CA), respectively. Cotransfection of human 293T cells with expression plasmid for scAb-Vpr or -WXXF along with HIV-1 clone pLAI resulted in the production of a normal amount of progeny virions with infectivity decreased by more than 10(3)-fold. Immunoblot analyses showed that scAb-Vpr or -WXXF was associated with virions, whereas scAb or scAb-CA was not, suggesting that scAb-Vpr or -WXXF was incorporated into virions. The incorporation of scAb-WXXF appeared to be Vpr dependent, because the fusion protein was associated with the wild-type but not with Vpr-truncated HIV-1 virions. Since G418-selected HeLa clones carrying expression plasmid for scAb-WXXF were obtained much more frequently than those for scAb-Vpr, scAb-WXXF was inferred to be less toxic to cells than scAb-Vpr. These results suggest that scAb-WXXF may serve as a novel class of antiviral therapeutic that inactivates progeny HIV virions from within.

Induction of neutralizing antibodies against human immunodeficiency virus type 1 using synthetic peptide constructs containing an immunodominant T-helper cell determinant from vpr.

Identification of immunodominant T-helper-cell determinants after natural infection is an important step in the design of immunogens for potential use in vaccination. Using cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals and a panel of peptides encompassing the sequence of the regulatory protein vpr from HIV-1, we identified the T-helper determinant QLLFIHFRIGCRHSR, which is active in 37.5% of these individuals. To gain insight on the efficacy of this peptide in helping induce neutralizing antibodies against a B-cell determinant (BD), we synthesized constructs containing B- and T-cell determinants and tested them in BALB/c mice, the highest responders to the T-cell determinant moiety among several strains tested. These immunogens induced antibodies against two chosen B-cell determinants from HIV-1IIIB gp160 (amino acids 310-322 from the V3 loop of gp120 and 736-751 from gp41) that were able to neutralize HIV-1 infection in vitro. The highest neutralization titer against HIV-1IIIB was obtained by immunization with the homopolymer of the construct containing the T-cell epitope from vpr and the B-cell epitope from the V3 loop. We believe that the immunodominant T-cell determinant from vpr is a promising epitope to consider in the design of future peptide vaccines.

Recombinant Vpr (rVpr) causes augmentation of HIV-1 p24 Ag level in U1 cells through its ability to induce the secretion of TNF.

We have found that an HIV-1 accessory gene product Vpr enhanced HIV-1 reproduction in U1 cells chiefly by the induction of TNF, a proinflammatory cytokine, which was also known to be an activator of HIV-1 reproduction. We have generated the functional HIV-1 accessory gene product Vpr in bacterial cells. Vpr was generated in an Escherichia coli system (rVpr), purified with antibodies (Ab) to the 16 C-terminal amino acids of Vpr. The purified rVpr of 15 kDa was examined for its ability to upregulate HIV-1 reproduction in U1 cells, which is a reported function of the authentic Vpr. rVpr upregulated HIV-1 reproduction in U1 cells in a dose-dependent manner and induced the secretion of TNF. The upregulation of HIV-1 by rVpr was completely inhibited not only by anti-Vpr antibodies but also by anti-TNF antibody. These findings suggested that Vpr caused an HIV-1 reproduction in U1 cells through the induction of TNF.

Generation of monoclonal antibodies specifically directed against the proximal zinc finger of HIV type 1 NCp7.

The HIV-1 NCp7 contains two spatially close zinc fingers, required for the production of infectious particles. To investigate in more detail the function of the zinc finger domain, monoclonal antibodies were generated with a cyclic analog of the NCp7 proximal zinc finger. This analog was shown to bind zinc ions and to preserve the highly folded structure of the native peptide (Dong C-Z et al.: J Am Chem Soc 1995;117:2726-2731). We report here two monoclonal antibodies (2B10 and 4D3), which are the first monoclonal antibodies directed against CCHC NCp7 zinc fingers. Dot-blot experiments revealed that a few nanograms of synthetic NCp7 can be detected on a nitrocellulose membrane. Whereas 2B10 appears specific for an epitope located in sequence 19-27 of NCp7, 4D3 appears to be structurally specific. Immunocomplex affinities were evaluated, using BIAcore technology, to be up to 1 and 10 nM, respectively, for 2B10 and 4D3 in 100 mM NaCl. These antibodies were able to recognize NCp7 in the Gag polyprotein precursor and were shown to immunoprecipitate NCp7 from a cell supernatant. Moreover, NCp7-Vpr interaction mediated by the zinc fingers is inhibited by 2B10, emphasizing the role of these domains in the protein-protein complex. These results indicate that 2B10 and 4D3 behave as useful tools for studying both NC protein functions during the course of virion morphogenesis and the role played by its zinc finger domain at various steps in the retroviral life cycle.

Antibodies to Tat and Vpr in the GRIV cohort: differential association with maintenance of long-term non-progression status in HIV-1 infection.

The HIV-1 regulatory protein Tat and the accessory protein Vpr are thought to stimulate viral replication and contribute to viral pathogenesis as extracellular proteins. Humoral immune responses to these early viral proteins may therefore be beneficial. We examined serum anti-Tat and anti-Vpr IgG by ELISA in the GRIV cohort of HIV-1 seropositive slow/non-progressors (NP) and fast-progressors (FP), and in seronegative controls. Based on information obtained during a brief follow-up period (median = 20 months), NPs were sub-grouped as those maintaining non-progression status and therefore stable (NP-S), and those showing signs of disease progression (NP-P). As the primary comparison, initial serum anti-Tat and anti-Vpr IgG (prior to follow-up) were analyzed in the NP sub-groups and in FPs. Anti-Tat IgG was significantly higher in stable NP-S compared to unstable NP-P (P = 0.047) and FPs (P < 0.0005); the predictive value of higher anti-Tat IgG for maintenance of non-progression status was 92% (P = 0.029). In contrast, no-difference was observed in anti-Vpr IgG between NP-S and NP-P, although both were significantly higher than FPs (P

Human immunodeficiency virus (HIV) type 1 Vpr induces differential regulation of T cell costimulatory molecules: direct effect of Vpr on T cell activation and immune function.

Human immunodeficiency virus type 1 (HIV-1) viral proteins disrupt the normal host cellular immune pathways thus exploiting the cellular machinery for replication, survival and to escape host immune attack. Here we evaluated the direct effects of HIV-1 Vpr-mediated immune modulation of infected T cells. Vpr specifically downregulated the expression of CD28 and increased the expression of CTLA-4, whereas no significant difference in the expression of CD25 and HLA-DR was observed. Interferon gamma (IFN-gamma) production in T cells was evaluated as a measure of the downstream effector functions. Results indicate that Vpr significantly inhibited IFN-gamma production and this may, in part, due to Vpr's ability to inhibit the nuclear translocation of NF-kappaB, and its transcriptional regulation. Together these results support that HIV-1 Vpr selectively dysregulates the immune functions at multiple levels and exerts its inhibitory effects in the presence of other viral proteins.

Immunodetection of human immunodeficiency virus type 1 (HIV-1) Vpr in brain tissue of HIV-1 encephalitic patients.

Human immunodeficiency virus (HIV) encephalitis (HIVE), the most severe neurological complication associated with HIV-1 infection, leads to the onset of HIV-1-associated dementia (HAD). Several HIV-1 viral proteins have been implicated in HIVE-associated neurodegeneration. HIV-1 viral protein R (Vpr), a virion associated gene product known to induce apoptosis in nonproliferating cells, including neurons, is thought to contribute to the neuropathogenesis associated with HIVE. Though current research suggests that Vpr plays a significant role in neuropathogenesis, the presence of Vpr in the brain tissue of HIVE patients has not been assessed. Using a panel of HIVE patient brain tissue, the authors have shown that Vpr is present in detectable amounts in both the basal ganglia and frontal cortex of all HIVE brain tissue samples tested. Double immunofluorescence indicated that Vpr was found in the macrophages and neurons, but not in the astrocytes, of HIVE patients. These results for the first time show the presence of Vpr in vivo and further support the role of Vpr in neuropathogenesis.

HIV-1 Viral protein-r (Vpr) protects against lethal superantigen challenge while maintaining homeostatic T cell levels in vivo.

The HIV-1 accessory protein Vpr exhibits many interesting features related to macrophage and T cell biology. As a viral protein or as a soluble molecule it can suppress immune cell activation and cytokine production in vitro in part by targeted inhibition of NF-kappaB. In this regard we sought to test its effects in vivo on an NF-kappaB-dependent immune pathway. We examined the activity of Vpr in a lethal toxin-mediated challenge model in mice. Intravenous delivery of Vpr was sufficient to protect mice from lethal challenge with staphylococcal endotoxin B (SEB). Furthermore, Vpr protected host CD4+ T cells from in vivo depletion likely by preventing induction of AICD of SEB-exposed cells in a post-toxin-binding fashion. Understanding the biology of Vpr's activities in this model may allow for new insight into potential mechanisms of hyperinflammatory disease and into Vpr pathobiology in the context of HIV infection.

The majority of currently circulating human immunodeficiency virus type 1 clade B viruses fail to prime cytotoxic T-lymphocyte responses against an otherwise immunodominant HLA-A2-restricted epitope: implications for vaccine design.

Human immunodeficiency virus type 1 (HIV-1) mutates to escape immune selection pressure, but there is little evidence of selection mediated through HLA-A2, the dominant class I allele in persons infected with clade B virus. Moreover, HLA-A2-restricted responses are largely absent in the acute phase of infection as the viral load is being reduced, suggesting that circulating viruses may lack immunodominant epitopes targeted through HLA-A2. Here we demonstrate an A2-restricted epitope within Vpr (Vpr59-67) that is targeted by acute-phase HIV-1-specific CD8+ T cells, but only in a subset of persons expressing HLA-A2. Individuals in the acute stage of infection with viruses containing the most common current sequence within this epitope (consensus sequence) were unable to mount epitope-specific T-cell responses, whereas subjects infected with the less frequent I60L variant all developed these responses. The I60L variant epitope was a stronger binder to HLA-A2 and was recognized by epitope-specific T cells at lower peptide concentrations than the consensus sequence epitope. These data demonstrate that HLA-A2 is capable of contributing to the acute-phase cytotoxic T-lymphocyte response in infected subjects, but that most currently circulating viruses lack a dominant immunogenic epitope presented by this allele, and suggest that immunodominant epitopes restricted by common HLA alleles may be lost as the epidemic matures.