RESUMO
Given the high variability and drug-resistance problem by human immunodeficiency virus type 1 (HIV-1), the development of bispecific or multi-specific inhibitors targeting different steps of HIV entry is highly appreciated. We previously generated a very potent short-peptide-based HIV fusion inhibitor 2P23. In this study, we designed and characterized a bifunctional inhibitor termed 2P23-iMab by genetically conjugating 2P23 to the single-chain variable fragment (scFv) of ibalizumab (iMab), a newly approved antibody drug targeting the cell receptor CD4. As anticipated, 2P23-iMab could bind to the cell membrane through CD4 anchoring and inhibit HIV-1 infection as well as viral Env-mediated cell-cell fusion efficiently. When tested against a large panel of HIV-1 pseudoviruses with different subtypes and phenotypes, 2P23-iMab exhibited dramatically improved inhibitory activity than the parental inhibitors; especially, it potently inhibited the viruses not being susceptible to iMab. Moreover, 2P23-iMab had a dramatically increased potency in inhibiting two panels of HIV-1 mutants that are resistant to T-20 or 2P23 and the infections of HIV-2 and simian immunodeficiency virus (SIV). In conclusion, our studies have provided new insights into the design of novel bispecific HIV entry inhibitors with highly potent and broad-spectrum antiviral activity.
Assuntos
Inibidores da Fusão de HIV , Infecções por HIV , HIV-1 , Vírus da Imunodeficiência Símia , Animais , Antirretrovirais , Inibidores da Fusão de HIV/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-2/fisiologia , Proteínas Virais de FusãoRESUMO
The discovery of the G-protein coupled-receptor (GPCR) CXCR4 as a major coreceptor of HIV-1 entry about three decades ago explained why the chemokine SDF-1/CXCL12 inhibits specific viral strains. The knowledge that RANTES, MlP-1α, and MlP-1ß specifically inhibit other primary HIV-1 strains allowed the rapid discovery of CCR5 as second major viral coreceptor and explained why individuals with deletions in CCR5 are protected against sexual HIV-1 transmission. Here, we provide an update on endogenous ligands of GPCRs that act as endogenous inhibitors of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) entry. In addition, we summarize the development of optimized derivatives of endogenous GPCR ligands and their perspectives as antiviral agents and beyond. Finally, we provide examples for other endogenous peptides that may contribute to our innate immune defense against HIV-1 and other viral pathogens and offer prospects for preventive or therapeutic development.
Assuntos
Inibidores da Fusão de HIV , Infecções por HIV , HIV-1 , Animais , Inibidores da Fusão de HIV/farmacologia , Inibidores da Fusão de HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/metabolismo , HIV-1/fisiologia , HIV-2/metabolismo , HIV-2/fisiologia , Humanos , Ligantes , Peptídeos/uso terapêutico , Receptores CCR5 , Receptores Acoplados a Proteínas G/uso terapêutico , Transdução de Sinais , Vírus da Imunodeficiência SímiaRESUMO
Emerging studies indicate that infusion of HIV-resistant cells could be an effective strategy to achieve a sterilizing or functional cure. We recently reported that glycosylphosphatidylinositol (GPI)-anchored nanobody or a fusion inhibitory peptide can render modified cells resistant to HIV-1 infection. In this study, we comprehensively characterized a panel of newly isolated HIV-1-neutralizing antibodies as GPI-anchored inhibitors. Fusion genes encoding the single-chain variable fragment (scFv) of 3BNC117, N6, PGT126, PGT128, 10E8, or 35O22 were constructed with a self-inactivating lentiviral vector, and they were efficiently expressed in the lipid raft sites of target cell membrane without affecting the expression of HIV-1 receptors (CD4, CCR5 and CXCR4). Significantly, transduced cells exhibited various degrees of resistance to cell-free HIV-1 infection and cell-associated HIV-1 transmission, as well as viral Env-mediated cell-cell fusion, with the cells modified by GPI-10E8 showing the most potent and broad anti-HIV activity. In mechanism, GPI-10E8 also interfered with the processing of viral Env in transduced cells and attenuated the infectivity of progeny viruses. By genetically linking 10E8 with a fusion inhibitor peptide, we subsequently designed a group of eight bifunctional constructs as cell membrane-based inhibitors, designated CMI01â¼CMI08, which rendered cells completely resistant to HIV-1, HIV-2, and simian immunodeficiency virus (SIV). In human CD4+ T cells, GPI-10E8 and its bifunctional derivatives blocked both CCR5- and CXCR4-tropic HIV-1 isolates efficiently, and the modified cells displayed robust survival selection under HIV-1 infection. Therefore, our studies provide new strategies for generating HIV-resistant cells, which can be used alone or with other gene therapy approaches.
Assuntos
Anticorpos Anti-HIV/imunologia , Proteína gp41 do Envelope de HIV/antagonistas & inibidores , Infecções por HIV/terapia , HIV-1/fisiologia , Fragmentos de Peptídeos/farmacologia , Anticorpos de Cadeia Única/imunologia , Fármacos Anti-HIV/farmacologia , Anticorpos Amplamente Neutralizantes/genética , Anticorpos Amplamente Neutralizantes/imunologia , Linfócitos T CD4-Positivos/virologia , Fusão Celular , Linhagem Celular , Terapia Genética , Glicosilfosfatidilinositóis , Anticorpos Anti-HIV/genética , Proteína gp41 do Envelope de HIV/imunologia , Inibidores da Fusão de HIV/farmacologia , Infecções por HIV/virologia , HIV-1/imunologia , HIV-2/imunologia , HIV-2/fisiologia , Humanos , Microdomínios da Membrana , Fragmentos de Peptídeos/genética , Receptores de HIV/metabolismo , Vírus da Imunodeficiência Símia/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Anticorpos de Cadeia Única/genética , Transgenes , Tropismo ViralRESUMO
Dendritic cells (DCs) play a pivotal role in the functional differentiation of CD4+ T cells in response to pathogens. In CD4+ T cells, HIV-1 replicates efficiently, while HIV-2, a related virus of reduced pathogenicity, is better controlled. How the DC response to HIV-1 vs HIV-2 contributes to programming an antiviral state in CD4+ T cells is not known. Here, we identify a transcriptional signature associated with progressive resistance to HIV infection in CD4+ T cells. We developed a model of naïve CD4+ T cell priming by DCs stimulated with a panel of seven viruses or synthetic ligands for the viral nucleic acid sensors cGAS and TLRs. DCs produced a cytokine response to HIV-2 infection more similar to the response to cGAS ligands than TLR ligands. In response to these signals, naive CD4+ T cells acquired a gradual antiviral resistance to subsequent HIV infection. The antiviral state was concomitant with the induction of the TH1 cytokine IFNγ and the type I interferon-stimulated gene (ISG) MX1, while the TFH cytokine IL-21 was not increased. By performing a transcriptional network analysis in T cells, we identified five distinct gene modules with characteristic ISG, TH1, TFH, IFN-I and proliferative signatures. Finally, we leverage this module to assemble a T antiviral signature of 404 genes that correlate with the antiviral state in T cells. Altogether, the study illuminates the programming of the antiviral state in T cells. The T antiviral gene signature in human CD4+ lymphocytes constitutes a resource for genetic screens and genomics analysis.
Assuntos
Linfócitos T CD4-Positivos , Células Dendríticas , Infecções por HIV , HIV-2 , Transcriptoma , Linfócitos T CD4-Positivos/imunologia , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/virologia , Infecções por HIV/genética , Infecções por HIV/imunologia , HIV-2/genética , HIV-2/fisiologia , Humanos , Ligantes , Nucleotidiltransferases/metabolismo , Replicação ViralRESUMO
SAMHD1, a human host factor found in myeloid cells which restricts HIV-1 replication. It depletes the dNTPs pool for viral cDNA syntheses, thus preventing the viral replication in the cells. The viral accessory protein, Vpx, exists only in SIVmac/HIV-2 particles. Vpx in SIVmac can induce proteosomal degradation of SAMHD1, which then leads to a decrease in the cytoplasmic dNTP pool. The protein-protein interaction between Vpx and SAMHD1 and its consequences are still unclear. Methods: In this study, we cloned, for the first time, Vpx gene from a HIV-2 infected patient and found up to 30% sequence variation compared to known HIV-2 strains. We then analyzed the role of SAMHD1 protein expression in transfected THP-1 and U937 cells by transfecting with the Vpx gene derived from SIVmac, HIV-2 from the NIH sample as well as HIV-2 from a Saudi patient. We found that Vpx gene expression led to reduced levels of intracellular SAMHD1. When the supernatants of the transfected cell lines were examined for secreted cytokines, chemokines and growth factors, Vpx expression seemed to be suppressive of pro-inflammatory response, and skewed the immune response towards an anti-inflammatory response. These results suggest that Vpx can act at two levels: clearance of intracellular restriction factor and suppression of cytokine storm: both aimed at long-term latency and host-pathogen stand-off, suggesting that Vpx is likely to be a potential therapeutic target.
Assuntos
Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-2/fisiologia , Interações Hospedeiro-Patógeno , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Clonagem Molecular , Citocinas/metabolismo , Suscetibilidade a Doenças , Regulação Viral da Expressão Gênica , Humanos , Imunofenotipagem , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Ligação Proteica , Análise de Sequência de DNA , Proteínas Virais Reguladoras e Acessórias/genética , Latência ViralRESUMO
The dynamics of T-cell receptor (TCR)selection in chronic HIV-1 infection, and its association with clinical outcome, is well documented for an array of MHC-peptide complexes and disease stages. However, the factors that may contribute to the selection and expansion of CD8+ T-cells in chronic HIV-2 infection, especially at the clonal level remain unclear. To address this question, we undertook a detailed molecular characterization of the clonotypic architecture of an HLA-B*3501 restricted Gag-specific CD8+ T-cell response in donors chronically infected with HIV-2 using a combination of flow cytometry, tetramer-specific CD8+ TCR clonotyping, and in vitro assays. We show that the response to the NY9 epitope is hierarchical and narrow in terms of T-cell receptor-alpha (TCRA) and -beta (TCRB) gene usage yet clonotypically diverse. Furthermore, clonotypic dominance in shared origin CTL clones was associated with a greater magnitude of cytokine production and antigen sensitivity at limiting antigen dilution as well as enhanced cross-reactivity for known HIV-2 variants. Hence, our data suggest that effector mobilization and expansion in human chronic HIV-2 infection may be linked to the qualitative features of specific CD8+ T-cell clonotypes, which could have implications for viral control and disease outcome.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-2/fisiologia , Especificidade do Receptor de Antígeno de Linfócitos T , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Motivos de Aminoácidos , Linfócitos T CD8-Positivos/metabolismo , Doença Crônica , Sequência Conservada , Epitopos de Linfócito T/imunologia , Infecções por HIV/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismoRESUMO
BACKGROUND: The genomes of HIV-2 and some SIV strains contain the accessory gene vpx, which carries out several functions during infection, including the downregulation of SAMHD1. Vpx is also commonly used in experiments to increase HIV-1 infection efficiency in myeloid cells, particularly in studies that investigate the activation of antiviral pathways. However, the potential effects of Vpx on cellular innate immune signaling is not completely understood. We investigated whether and how Vpx affects ISG responses in monocytic cell lines and MDMs during HIV-1 infection. RESULTS: HIV-1 infection at excessively high virus doses can induce ISG activation, although at the expense of high levels of cell death. At equal infection levels, the ISG response is potentiated by the presence of Vpx and requires the initiation of reverse transcription. The interaction of Vpx with the DCAF1 adaptor protein is important for the enhanced response, implicating Vpx-mediated degradation of a host factor. Cells lacking SAMHD1 show similarly augmented responses, suggesting an effect that is independent of SAMHD1 degradation. Overcoming SAMHD1 restriction in MDMs to reach equal infection levels with viruses containing and lacking Vpx reveals a novel function of Vpx in elevating innate immune responses. CONCLUSIONS: Vpx likely has as yet undefined roles in infected cells. Our results demonstrate that Vpx enhances ISG responses in myeloid cell lines and primary cells independently of its ability to degrade SAMHD1. These findings have implications for innate immunity studies in myeloid cells that use Vpx delivery with HIV-1 infection.
Assuntos
HIV-2/genética , Interações Hospedeiro-Patógeno , Imunidade Inata/genética , Proteína 1 com Domínio SAM e Domínio HD/genética , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/imunologia , Linhagem Celular , Células HEK293 , Infecções por HIV/genética , Infecções por HIV/imunologia , HIV-2/fisiologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Leucócitos Mononucleares/virologia , Proteólise , Proteína 1 com Domínio SAM e Domínio HD/imunologia , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Células THP-1 , Replicação ViralRESUMO
Human macrophages are protected by intrinsic antiviral defenses that provide moderate protection against HIV-1 infection. Macrophages that do become infected can serve as long-lived reservoirs, to disseminate HIV-1 to CD4+ T cells. Infection of macrophages with HIV-1 and HIV-2 is inhibited by constitutive mobilization of antioxidant response master transcription regulator Nrf2. The downstream mediator of this restriction was not identified. Among the tens of genes controlled directly by Nrf2 in macrophages, we found that xCT/SLC7A11, a 12-transmembrane, cystine-glutamate antiporter promotes antiretroviral activity. We show here that depletion of xCT mRNA increases HIV-1 infection. Reconstitution of xCT knock out cells with wild-type xCT but not a transport-deficient mutant restores anti-HIV-1 activity. Pharmacological inhibitors of xCT amino acid transport also increase infection. The block is independent of known restriction factors and acts against HIV-1 and HIV-2. Like the block triggered through Nrf2, xCT function impedes infection immediately before 2-LTR circle formation.
Assuntos
Sistema y+ de Transporte de Aminoácidos/imunologia , Infecções por HIV , HIV-1 , HIV-2 , Células HEK293 , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/fisiologia , HIV-2/imunologia , HIV-2/fisiologia , Células HeLa , Humanos , Leucócitos , Células THP-1RESUMO
Chronic immune activation is an important driver of human immunodeficiency virus type 1 (HIV-1) pathogenesis and has been associated with the presence of tumor necrosis factor-α converting enzyme (TACE) in extracellular vesicles (EVs) circulating in infected individuals. We have recently shown that activation of the Src-family tyrosine kinase hematopoietic cell kinase (Hck) by HIV-1 Nef can trigger the packaging of TACE into EVs via an unconventional protein secretion pathway. Using a panel of HIV-1 Nef mutants and natural HIV-2 and simian immunodeficiency virus (SIV) Nef alleles, we now show that the capacity to promote TACE secretion depends on the superior ability of HIV-1-like Nef alleles to induce Hck kinase activity, whereas other Nef effector functions are dispensable. Strikingly, among the numerous Src-family downstream effectors, serine/threonine kinase Raf-1 was found to be necessary and alone sufficient to trigger the secretion of TACE into EVs. These data reveal the involvement of Raf-1 in regulation of unconventional protein secretion and highlight the importance of Raf-1 as a cellular effector of Nef, thereby suggesting a novel rationale for testing pharmacological inhibitors of the Raf-MAPK pathway to treat HIV-associated immune activation.IMPORTANCE Chronic immune activation contributes to the immunopathogenesis of human immunodeficiency virus type 1 (HIV-1) infection and is associated with poor recovery of the immune system despite potent antiretroviral therapy, which is observed in 10% to 40% drug-treated patients depending on the definition of immune reconstitution. We have previously shown that the HIV pathogenicity factor Nef can promote loading of the proinflammatory protease TACE into extracellular vesicles (EVs), and the levels of such TACE-containing EVs circulating in the blood correlate with low CD4 lymphocyte counts in HIV patients receiving antiretroviral therapy. Here, we show that Nef promotes uploading of TACE into EVs by triggering unconventional secretion via activation of the Hck/Raf/mitogen-activated protein kinase (MAPK) cascade. We find that several pharmaceutical inhibitors of these kinases that are currently in clinical use for other diseases can potently suppress this pathogenic deregulation and could thus provide a novel strategy for treating HIV-associated immune activation.
Assuntos
Proteína ADAM17/metabolismo , Vesículas Extracelulares/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Proteínas Proto-Oncogênicas c-raf/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Células HEK293 , HIV-2/fisiologia , Humanos , Proteínas Proto-Oncogênicas c-hck/metabolismo , Vírus da Imunodeficiência Símia/fisiologia , Células THP-1 , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
HIV-2, a human pathogen that causes acquired immunodeficiency syndrome, is distinct from the more prevalent HIV-1 in several features including its evolutionary history and certain aspects of viral replication. Like other retroviruses, HIV-2 packages two copies of full-length viral RNA during virus assembly and efficient genome encapsidation is mediated by the viral protein Gag. We sought to define cis-acting elements in the HIV-2 genome that are important for the encapsidation of full-length RNA into viral particles. Based on previous studies of murine leukemia virus and HIV-1, we hypothesized that unpaired guanosines in the 5' untranslated region (UTR) play an important role in Gag:RNA interactions leading to genome packaging. To test our hypothesis, we targeted 18 guanosines located in 9 sites within the HIV-2 5' UTR and performed substitution analyses. We found that mutating as few as three guanosines significantly reduce RNA packaging efficiency. However, not all guanosines examined have the same effect; instead, a hierarchical order exists wherein a primary site, a secondary site, and three tertiary sites are identified. Additionally, there are functional overlaps in these sites and mutations of more than one site can act synergistically to cause genome packaging defects. These studies demonstrate the importance of specific guanosines in HIV-2 5'UTR in mediating genome packaging. Our results also demonstrate an interchangeable and hierarchical nature of guanosine-containing sites, which was not previously established, thereby revealing key insights into the replication mechanisms of HIV-2.
Assuntos
Regiões 5' não Traduzidas , Guanosina/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-2/fisiologia , RNA Viral , Empacotamento do Genoma Viral , Sequência de Bases , Linhagem Celular , Regulação Viral da Expressão Gênica , Genoma Viral , Humanos , Mutação , Conformação de Ácido Nucleico , Montagem de Vírus , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The antiviral activity of nucleoside reverse transcriptase inhibitors is often hampered by insufficient phosphorylation. Nucleoside triphosphate analogues are presented, in which the γ-phosphate was covalently modified by a non-bioreversible, lipophilic 4-alkylketobenzyl moiety. Interestingly, primer extension assays using human immunodeficiency virus reverse transcriptase (HIV-RT) and three DNA-polymerases showed a high selectivity of these γ-modified nucleoside triphosphates to act as substrates for HIV-RT, while they proved to be nonsubstrates for DNA-polymerases α, ß, and γ. In contrast to d4TTP, the γ-modified d4TTPs showed a high resistance toward dephosphorylation in cell extracts. A series of acyloxybenzyl-prodrugs of these γ-ketobenzyl nucleoside triphosphates was prepared. The aim was the intracellular delivery of a stable γ-modified nucleoside triphosphate to increase the selectivity of such compounds to act in infected versus noninfected cells. Delivery of γ-ketobenzyl-d4TTPs was proven in T-lymphocyte cell extracts. The prodrugs were potent inhibitors of HIV-1/2 in cultures of infected CEM/0 cells and more importantly in thymidine kinase-deficient CD4+ T-cells.
Assuntos
Fármacos Anti-HIV/química , Pró-Fármacos/química , Fármacos Anti-HIV/farmacologia , Antivirais/química , Antivirais/farmacologia , Células Cultivadas , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , HIV-2/efeitos dos fármacos , HIV-2/fisiologia , Humanos , Polifosfatos/química , Polifosfatos/farmacologia , Pró-Fármacos/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/fisiologia , Linfócitos T/virologia , Tiamina/química , Tiamina/farmacologiaRESUMO
HIV transactivator protein (Tat) plays a pivotal role in viral replication through modulation of cellular transcription factors and transactivation of viral genomic transcription. The effect of HIV-1 Tat on reverse transcription has long been described in the literature, however, that of HIV-2 is understudied. Sequence homology between Tat proteins of HIV-1 and 2 is estimated to be less than 30%, and the main difference lies within their N-terminal region. Here, we describe Y44A-inactivating mutation of HIV-2 Tat, studying its effect on capsid production, reverse transcription, and the efficiency of proviral transcription. Investigation of the mutation was performed using sequence- and structure-based in silico analysis and in vitro experiments. Our results indicate that the Y44A mutant HIV-2 Tat inhibited the activity and expression of RT (reverse transcriptase), in addition to diminishing Tat-dependent LTR (long terminal repeat) transactivation. These findings highlight the functional importance of the acidic domain of HIV-2 Tat in the regulation of reverse transcription and transactivation of the integrated provirions.
Assuntos
Repetição Terminal Longa de HIV , HIV-2/genética , Mutação de Sentido Incorreto , Transcrição Reversa , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , HIV-2/fisiologia , Domínios Proteicos , Replicação Viral , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
The Vpx and Vpr proteins of human immunodeficiency virus type 2 (HIV-2) are important for virus replication. Although these proteins are homologous, Vpx is expressed at much higher levels than Vpr. Previous studies demonstrated that this difference results from the presence of an HHCC zinc-binding site in Vpx that is absent in Vpr. Vpx has another unique region, a poly-proline motif (PPM) of seven consecutive prolines at the C-terminus. Using PPM point mutants of Vpx, this study demonstrated that these seven consecutive prolines are critical for suppressing proteasome degradation of Vpx in the absence of Gag. Both the PPM and the zinc-binding site stabilize Vpx but do so via different mechanisms. PPM and zinc-binding site mutants overexpressed in Escherichia coli aggregated readily, indicating that these motifs normally prevent exposure of the hydrophobic region outside the structure. Furthermore, introduction of the zinc-binding site and the PPM into Vpr increased the level of Vpr expression so that it was as high as that of Vpx. Intriguingly, HIV-2 has evolved to express Vpx at high levels and Vpr at low levels based on the presence and absence of these two motifs with distinct roles.
Assuntos
Motivos de Aminoácidos , HIV-2/fisiologia , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/química , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Sítios de Ligação , Células HEK293 , HIV-2/genética , Células HeLa , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Peptídeos , Mutação Puntual , Prolina/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Proteólise , Proteínas Virais Reguladoras e Acessórias/genética , Zinco/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genéticaRESUMO
The antiviral efficacy of many nucleoside analogues is strongly dependent on their intracellular activation by host cellular kinases to yield ultimately the bioactive nucleoside analogue triphosphates (NTP). The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. We developed a nucleoside triphosphate (NTP) delivery system (the TriPPPro approach), in which the γ-phosphate is covalently modified by two different biodegradable masking units, one is the acyloxybenzyl (AB) moiety and the other is the alkoxycarbonyloxybenzyl (ACB) group. Such compounds formed NTPs with high selectivity by an enzyme-triggered mechanism in human T-lymphocyte CEM cell extracts loosing first the AB moiety, followed by the ACB group. This enables the bypass of all steps of the intracellular phosphorylation. This approach was applied here to convert some modestly active or even inactive nucleoside analogues into powerful biologically active metabolites. Potent antiviral activity profiles were obtained depending on the lipophilicity of the TriPPPro-NTP prodrugs against HIV-1 and HIV-2 replication in cultures of infected wild-type CD4+ CEM T-cells and more importantly in thymidine kinase-deficient CD4+ T-cells (CEM/TK-). This TriPPPro strategy offers high potential for future antiviral and antitumoral chemotherapies.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Nucleosídeos/química , Nucleosídeos/metabolismo , Polifosfatos/química , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , HIV-2/efeitos dos fármacos , HIV-2/fisiologia , Humanos , Nucleosídeos/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: In remote settings, timely plasma separation and transportation to testing laboratories is an impediment to the access of HIV viral load (VL) testing. Potential solutions are whole blood testing through point of care (POC) assays or dried blood spots (DBS). METHODS: We evaluated the performance of a prototype Alere q whole blood VL protocol and compared it against plasma (Abbott RealTime HIV-1) and DBS VL (Abbott RealTime HIV-1 DBS revised prototype protocol and Roche CAP/CTM HIV-1 v2.0 DBS free virus elution protocol). Virological failure (VF) was defined at >1000 copies/ml. RESULTS: Of 299 samples, Alere q correctly classified VF in 61% versus 87% by Abbott DBS and 76% by Roche FVE. Performance varied across plasma VL categories. Alere q showed 100% sensitivity. Below 1000 copies/ml of plasma, Alere q demonstrated over-quantification, with 19% specificity. Abbott DBS had 91% sensitivity and the best overall correlation with plasma (r2 = 0.72). Roche FVE had the best specificity of 99% but reduced sensitivity of 52%, especially between 1000-10,000 copies/ml of plasma. Correlation was best for all assays at >10,000 copies/ml. CONCLUSION: Variability was prominent between the assays. Each method requires optimization to facilitate the implementation of a cut-off with optimal sensitivity and specificity for VF. Although Alere q whole blood assay exhibited excellent sensitivity, the poor specificity of only 19% would lead to unnecessary switching of regimens. Thus any VF detected would need to be confirmed by a more specific assay. Both the Abbott DBS and Roche FVE protocols showed good specificity, however sensitivity was reduced when the plasma VL was 1000-10,000 copies/ml. This could result in delays in detecting VF and accumulation of drug resistance. Field evaluation in settings that have adopted these DBS protocols are necessary.
Assuntos
Teste em Amostras de Sangue Seco/métodos , HIV-1/fisiologia , HIV-2/fisiologia , Carga Viral , Humanos , Limite de Detecção , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
Follicular helper T cells (Tfh), CD4 lymphocytes critical for efficient antibody responses, have been shown to be key human immunodeficiency virus (HIV)-1 reservoirs. Human immunodeficiency virus-2 infection represents a unique naturally occurring model for investigating Tfh role in HIV/acquired immune deficiency syndrome, given its slow rate of CD4 decline, low to undetectable viremia, and high neutralizing antibody titers throughout the disease course. In this study, we investigated, for the first time, Tfh susceptibility to HIV-2 infection by combining in vitro infection of tonsillar Tfh with the ex vivo study of circulating Tfh from HIV-2-infected patients. We reveal that Tfh support productive HIV-2 infection and are preferential viral targets in HIV-2-infected individuals.
Assuntos
DNA Viral/metabolismo , Infecções por HIV/imunologia , HIV-1/fisiologia , HIV-2/fisiologia , Linfócitos T Auxiliares-Indutores/virologia , Feminino , Infecções por HIV/virologia , Humanos , Pessoa de Meia-Idade , Tonsila Palatina/imunologia , Tonsila Palatina/patologia , Cultura Primária de Células , RNA Mensageiro/metabolismo , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Tropismo Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
Human immunodeficiency virus type 2 (HIV-2) infection results in a milder course of disease and slower progression to AIDS than does HIV-1. We hypothesized that this difference may be due to degradation of the sterile alpha motif and HD domain 1 (SAMHD1) host restriction factor by the HIV-2 Vpx gene product, thereby diminishing abortive infection and pyroptotic cell death within bystander CD4 T cells. We have compared CD4 T cell death in tonsil-derived human lymphoid aggregate cultures (HLACs) infected with wild-type HIV-2, HIV-2 ΔVpx, or HIV-1. In contrast to our hypothesis, HIV-2, HIV-2 ΔVpx, and HIV-1 induced similar levels of bystander CD4 T cell death. In all cases, cell death was blocked by AMD3100, a CXCR4 entry inhibitor, but not by raltegravir, an integrase, indicating that only early life cycle events were required. Cell death was also blocked by a caspase-1 inhibitor, a key enzyme promoting pyroptosis, but not by a caspase-3 inhibitor, an important enzyme in apoptosis. HIV-1-induced abortive infection and pyroptotic cell death were also not reduced by forced encapsidation of HIV-2 Vpx into HIV-1 virions. Together, these findings indicate that HIV-2 and HIV-1 support similar levels of CD4 T cell depletion in vitro despite HIV-2 Vpx-mediated degradation of the SAMHD1 transcription factor. The milder disease course observed with HIV-2 infection likely stems from factors other than abortive infection and caspase-1-dependent pyroptosis in bystander CD4 T cells.IMPORTANCE CD4 T cell depletion during HIV-1 infection involves the demise of bystander CD4 T cells due to abortive infection, viral DNA sensing, inflammasome assembly, and death by caspase-1-dependent pyroptosis. HIV-2 infection is associated with milder disease and lower rates of CD4 T cell loss. We hypothesized that HIV-2 infection produces lower levels of pyroptosis due to the action of its Vpx gene product. Vpx degrades the SAMHD1 restriction factor, potentially reducing abortive forms of infection. However, in tonsil cell cultures, HIV-2, HIV-2 ΔVpx, and HIV-1 induced indistinguishable levels of pyroptosis. Forced encapsidation of Vpx into HIV-1 virions also did not reduce pyroptosis. Thus, SAMHD1 does not appear to play a key role in the induction of bystander cell pyroptosis. Additionally, the milder clinical course of HIV-2-induced disease is apparently not explained by a decrease in this inflammatory form of programmed cell death.
Assuntos
Linfócitos T CD4-Positivos/virologia , Infecções por HIV/imunologia , HIV-2/fisiologia , Piroptose/fisiologia , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Morte Celular , Células HEK293 , Infecções por HIV/virologia , HIV-1/fisiologia , HIV-2/genética , Interações Hospedeiro-Patógeno , Humanos , Piroptose/genética , Células THP-1 , Proteínas Virais Reguladoras e Acessórias/genética , Vírion/metabolismoRESUMO
Schools are considered as one of the most effective vectors for education on sexually transmitted diseases among young people. We report here the results of a study of HIV infection as presented in school textbooks in Ivory Coast, conducted in June 2018 as part of the development of a communication strategy to increase demand for HIV infection testing, especially among young people. Surprisingly, even though the textbooks studied were published between 2007 and 2017, almost all of them stated that HIV infection leads to death, with no mention of the existence of treatments that make it possible to live in good health. Some textbooks even stated that no treatment is available. These findings highlight one cause - perhaps major - for the reluctance of young people to get tested. The misrepresentation of HIV infection, reinforced by education, hampers incentive campaigns for HIV testing, for which the best argument is that awareness of a positive HIV status makes it possible to benefit from treatment. The present study included textbooks from the French educational program, used throughout French-speaking Africa, and from the Ivory Coast program. It would be interesting to conduct a similar study in other French-speaking and English-speaking African countries. Adapting the presentation of HIV in school textbooks to reflect the current situation in the fight against HIV infection should be a priority.
L'éducation scolaire est connue comme l'un des vecteurs les plus efficaces d'éducation sur les maladies sexuellement transmissibles chez les jeunes. Nous rapportons ici les résultats d'une étude de la présentation de l'infection à VIH dans les manuels scolaires en Côte d'Ivoire. De façon surprenante, bien qu'ils aient été imprimés entre 2007 et 2017, ces manuels affirment dans leur quasi-totalité que le VIH conduit à la mort, sans mentionner l'existence de traitements qui permettent de vivre en bonne santé, voire pour certains en affirmant qu'il n'existe pas de traitement. Ceci met en lumière une cause, peut-être majeure, de la réticence des jeunes à se faire dépister. La représentation erronée de l'infection à VIH renforcée par l'enseignement entrave les campagnes d'incitation au passage du test de dépistage, le meilleur argument pour celui-ci étant que connaître son statut positif permet de bénéficier des traitements. Certains des livres étudiés sont utilisés dans toute l'Afrique francophone. Il serait intéressant de mener une étude similaire dans d'autres pays d'Afrique francophone et anglophone. Vérifier le contenu scientifique et adapter le discours des manuels scolaires sur le VIH à la situation actuelle de la lutte contre l'infection devraient être une urgence.
Assuntos
Comunicação , Infecções por HIV , Serviços de Saúde Escolar , Livros de Texto como Assunto , Síndrome da Imunodeficiência Adquirida/diagnóstico , Síndrome da Imunodeficiência Adquirida/prevenção & controle , Síndrome da Imunodeficiência Adquirida/transmissão , Atitude Frente a Saúde , Côte d'Ivoire/epidemiologia , Infecções por HIV/diagnóstico , Infecções por HIV/prevenção & controle , Infecções por HIV/transmissão , HIV-1/fisiologia , HIV-2/fisiologia , Promoção da Saúde/métodos , Promoção da Saúde/normas , Humanos , Conhecimento , Idioma , Programas de Rastreamento/psicologia , Serviços de Saúde Escolar/normas , Serviços de Saúde Escolar/estatística & dados numéricos , Educação Sexual/normas , Educação Sexual/estatística & dados numéricos , Infecções Sexualmente Transmissíveis/diagnóstico , Infecções Sexualmente Transmissíveis/prevenção & controle , Infecções Sexualmente Transmissíveis/transmissão , Livros de Texto como Assunto/normasRESUMO
INTRODUCTION: HIV self-testing (HIVST) offers a useful addition to HIV testing services and enables individuals to test privately. Despite recommendations to the contrary, repeat HIV testing is frequent among people already on anti-retroviral treatment (ART) and there are concerns that oral self-testing might lead to false negative results. A study was conducted in Khayelitsha, South Africa, to assess feasibility and uptake of HIVST and linkage-to-care following HIVST. METHODS: Participants were recruited at two health facilities from 1 March 2016 to 31 March 2017. People under 18 years, or with self-reported previously-diagnosed HIV infection, were excluded. Participants received an OraQuick Rapid HIV-1/2 Antibody kit, and reported their HIVST results by pre-paid text message (SMS) or by returning to the facility. Those not reporting within 7 days were contacted by phone. Electronic and paper-based clinical and laboratory records were retrospectively examined for all participants to identify known HIV outcomes, after matching for name, date of birth, and sex. These findings were compared with self-reported HIVST results where available. RESULTS: Of 639 participants, 401 (62.8%) self-reported a negative HIVST result, 27 (4.2%) a positive result, and 211 (33.0%) did not report. The record search identified that of the 401 participants self-reporting a negative HIVST result, 19 (4.7%) were already known to be HIV positive; of the 27 self-reporting positive, 12 (44%) were known HIV positive. Overall, records showed 57/639 (8.9%) were HIV positive of whom 39/57 (68.4%) had previously-diagnosed infection and 18/57 (31.6%) newly-diagnosed infection. Of the 428 participants who self-reported a result, 366 (85.5%) reported by SMS. CONCLUSIONS: HIVST can improve HIV testing uptake and linkage to care. SMS is acceptable for reporting HIVST results but negative self-reports by participants may be unreliable. Use of HIVST by individuals on ART is frequent despite recommendations to the contrary and its implications need further consideration.