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1.
Sci Rep ; 10(1): 15866, 2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32985522

RESUMO

HIV-1 cell entry is mediated by binding to the CD4-receptor and chemokine co-receptors CCR5 (R5) or CXCR4 (X4). R5-tropic viruses are predominantly detected during early infection. A switch to X4-tropism often occurs during the course of infection. X4-tropism switching is strongly associated with accelerated disease progression and jeopardizes CCR5-based HIV-1 cure strategies. It is unclear whether host immunological factors play a causative role in tropism switching. We investigated the relationship between immunological factors and X4-tropism in a cross-sectional study in HIV-1 subtype C (HIV-1C)-infected patients and in a longitudinal HIV-1 subtype B (HIV-1B) seroconverter cohort. Principal component analysis identified a cluster of immunological markers (%HLA-DR+ CD4+ T-cells, %CD38+HLA-DR+ CD4+ T-cells, %CD38+HLA-DR+ CD8+ T-cells, %CD70+ CD4+ T-cells, %CD169+ monocytes, and absolute CD4+ T-cell count) in HIV-1C patients that was independently associated with X4-tropism (aOR 1.044, 95% CI 1.003-1.087, p = 0.0392). Analysis of individual cluster contributors revealed strong correlations of two markers of T-cell activation (%HLA-DR+ CD4+ T-cells, %HLA-DR+CD38+ CD4+ T-cells) with X4-tropism, both in HIV-1C patients (p = 0.01;p = 0.03) and HIV-1B patients (p = 0.0003;p = 0.0001). Follow-up data from HIV-1B patients subsequently revealed that T-cell activation precedes and independently predicts X4-tropism switching (aHR 1.186, 95% CI 1.065-1.321, p = 0.002), providing novel insights into HIV-1 pathogenesis and CCR5-based curative strategies.


Assuntos
Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1/fisiologia , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Tropismo Viral , Adulto , Estudos Transversais , Feminino , Humanos , Masculino
2.
RSC Adv ; 10(31): 18062-18072, 2020 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-35517228

RESUMO

On-chip sample preparation in self-contained microfluidic devices is a key element to realize simple, low-cost, yet reliable in vitro diagnostics that can be carried out at the point-of-care (POC) with minimal training requirements by unskilled users. To address this largely unmet POC medical need, we have developed an optimized polysaccharide matrix containing the reagents which substantially improves our fully printed POC CD4 counting chambers for the monitoring of HIV patients. The simply designed counting chambers allow for capillary-driven filling with unprocessed whole blood. We carefully tailored a gellan/trehalose matrix for deposition by inkjet printing, which preserves the viability of immunostains during a shelf life of at least 3 months and enables controlled antibody release for intense and homogeneous immunofluorescent cell staining throughout the complete 60 mm2 image area within 30 min. Excellent agreement between CD4 counts obtained from our fully printed CD4 counting chambers and the gold standard, flow cytometry, is demonstrated using samples both from healthy donors and HIV-infected patients.

3.
PLoS Pathog ; 14(12): e1007432, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30521629

RESUMO

CCR5 plays immune functions and is the coreceptor for R5 HIV-1 strains. It exists in diverse conformations and oligomerization states. We interrogated the significance of the CCR5 structural diversity on HIV-1 infection. We show that envelope glycoproteins (gp120s) from different HIV-1 strains exhibit divergent binding levels to CCR5 on cell lines and primary cells, but not to CD4 or the CD4i monoclonal antibody E51. This owed to differential binding of the gp120s to different CCR5 populations, which exist in varying quantities at the cell surface and are differentially expressed between different cell types. Some, but not all, of these populations are antigenically distinct conformations of the coreceptor. The different binding levels of gp120s also correspond to differences in their capacity to bind CCR5 dimers/oligomers. Mutating the CCR5 dimerization interface changed conformation of the CCR5 homodimers and modulated differentially the binding of distinct gp120s. Env-pseudotyped viruses also use particular CCR5 conformations for entry, which may differ between different viruses and represent a subset of those binding gp120s. In particular, even if gp120s can bind both CCR5 monomers and oligomers, impairment of CCR5 oligomerization improved viral entry, suggesting that HIV-1 prefers monomers for entry. From a functional standpoint, we illustrate that the nature of the CCR5 molecules to which gp120/HIV-1 binds shapes sensitivity to inhibition by CCR5 ligands and cellular tropism. Differences exist in the CCR5 populations between T-cells and macrophages, and this is associated with differential capacity to bind gp120s and to support viral entry. In macrophages, CCR5 structural plasticity is critical for entry of blood-derived R5 isolates, which, in contrast to prototypical M-tropic strains from brain tissues, cannot benefit from enhanced affinity for CD4. Collectively, our results support a role for CCR5 heterogeneity in diversifying the phenotypic properties of HIV-1 isolates and provide new clues for development of CCR5-targeting drugs.


Assuntos
Infecções por HIV/metabolismo , HIV-1/fisiologia , Receptores CCR5/química , Receptores CCR5/metabolismo , Internalização do Vírus , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Fenótipo , Ligação Proteica
4.
Biosens Bioelectron ; 117: 659-668, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-30005387

RESUMO

We demonstrate the fabrication of fully printed microfluidic CD4 counting chips with complete on-chip sample preparation and their applicability as a CD4 counting assay using samples from healthy donors and HIV-infected patients. CD4 counting in low-income and resource-limited point-of-care settings is only practical and affordable, if disposable tests can be fabricated at very low cost and all manual sample preparation is avoided, while operation as well as quantification is fully automated and independent of the skills of the operator. Here, we show the successful use of (inkjet) printing methods both to fabricate microfluidic cell counting chambers with controlled heights, and to deposit hydrogel layers with embedded fluorophore-labeled antibodies for on-chip sample preparation and reagent storage. The maturation process of gelatin after deposition prevents antibody wash-off during blood inflow very well, while temperature-controlled dissolution of the matrix ensures complete antibody release for immunostaining after the inflow has stopped. The prevention of antibody wash-off together with the subsequent complete antibody release guarantees a homogeneous fluorescence background, making rapid and accurate CD4 counting possible. We show the successful application of our fully printed CD4 counting chips on samples from healthy donors as well as from HIV-infected patients and find an excellent agreement between results from our method and from the gold standard, flow cytometry, in both cases.


Assuntos
Contagem de Linfócito CD4/instrumentação , Contagem de Linfócito CD4/métodos , Técnicas Analíticas Microfluídicas , Sistemas Automatizados de Assistência Junto ao Leito , Contagem de Linfócito CD4/normas , Citometria de Fluxo/normas , Humanos , Reprodutibilidade dos Testes
5.
Sci Signal ; 9(452): ra107, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27803285

RESUMO

Chemokines stimulate signals in cells by binding to G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors. These chemoattractant cytokines also interact with heparan sulfate (HS), which provides positional information within tissues in the form of haptotactic gradients along which cells can migrate directionally. To investigate the mechanism by which HS modulates chemokine functions, we used the CXC chemokine CXCL12, which exists in different isoforms that all signal through CXCR4 but have distinct HS-binding domains. In experiments with both cell-associated and solubilized CXCR4, we found that although CXCL12γ bound to CXCR4 with a higher affinity than did CXCL12α, CXCL12γ displayed reduced signaling and chemotactic activities. These properties were caused by the specific carboxyl-terminal region of CXCL12γ, which, by interacting with CXCR4 sulfotyrosines, mediated high-affinity, but nonproductive, binding to CXCR4. HS prevented CXCL12γ from interacting with the CXCR4 sulfotyrosines, thereby functionally presenting the chemokine to its receptor such that its activity was similar to that of CXCL12α. HS had no effects on the binding of CXCL12α to CXCR4 or its biological activity, suggesting that this polysaccharide controls CXCL12 in an isoform-specific manner. These data suggest that the HS-dependent regulation of chemokine functions extends beyond the simple process of immobilization and directly modulates receptor ligation and activation.


Assuntos
Movimento Celular , Quimiocina CXCL12/metabolismo , Heparitina Sulfato/metabolismo , Receptores CXCR4/metabolismo , Linfócitos T/metabolismo , Humanos , Isoformas de Proteínas/metabolismo , Linfócitos T/citologia
6.
Front Immunol ; 4: 385, 2013 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-24312095

RESUMO

By targeting cells that provide protection against infection, HIV-1 causes acquired immunodeficiency syndrome. Infection starts when gp120, the viral envelope glycoprotein, binds to CD4 and to a chemokine receptor usually CCR5 or CXCR4. As many microorganisms, HIV-1 also interacts with heparan sulfate (HS), a complex group of cell surface associated anionic polysaccharides. It has been thought that this binding, occurring at a step prior to CD4 recognition, increases infectivity by pre-concentrating the virion particles at the cell surface. Early work, dating from before the identification of CCR5 and CXCR4, showed that a variety of HS mimetics bind to the gp120 V3 loop through electrostatic interactions, compete with cell surface associated HS to bind the virus and consequently, neutralize the infectivity of a number of T-cell line-adapted HIV-1 strains. However, progress made to better understand HIV-1 attachment and entry, coupled with the recent identification of additional gp120 regions mediating HS recognition, have considerably modified this view. Firstly, the V3 loop from CXCR4-using viruses is much more positively charged compared to those using CCR5. HS inhibition of cell attachment is thus restricted to CXCR4-using viruses (such as T-cell line-adapted HIV-1). Secondly, studies aiming at characterizing the gp120/HS complex revealed that HS binding was far more complex than previously thought: in addition to the V3 loop of CXCR4 tropic gp120, HS interacts with several other cryptic areas of the protein, which can be induced upon CD4 binding, and are conserved amongst CCR5 and CXCR4 viruses. In view of these data, this review will detail the present knowledge on HS binding to HIV-1, with regards to attachment and entry processes. It will discuss the perspective of targeting the gp120 co-receptor binding site with HS mimetic compounds, a strategy that recently gave rise to entry inhibitors that work in the low nanomolar range, independently of co-receptor usage.

7.
AIDS Res Hum Retroviruses ; 24(5): 743-51, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18507530

RESUMO

CCR5 has preferentially been used by all circulating HIV-1 subtype C viruses for cell entry. Recently, we reported the highest proportion of CXCR4-utilizing primary isolates among a cohort of 20 South African AIDS patients. This study describes and compares the Env genotypic characteristics from these 20 HIV-1 subtype C (and unique CD recombinant) primary isolates. Fourteen primary isolates utilized CCR5, four (including the CD recombinant) used CXCR4, and two were dual tropic. Extensive analysis and comparison of important structural motifs such as the N-linked glycosylation sites, signal sequences, CD4-binding sites, variable loops, cleavage sites, known neutralizing antibody and small molecule inhibitor binding sites confirmed that other than the expected differences in the V3 loop, no sequence motifs distinguished between R5 and X4 tropism. Further correlation of the env genotype to functionally relevant motifs is necessary to elucidate the relationship between biologically and immunologically relevant sites and aid vaccine and novel drug design.


Assuntos
Infecções por HIV/virologia , HIV-1/genética , HIV-1/metabolismo , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Sequência de Aminoácidos , Sítios de Ligação , Antígenos CD4/metabolismo , Glicosilação , Proteína gp120 do Envelope de HIV/genética , HIV-1/classificação , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , África do Sul
8.
AIDS ; 22(7): 896-9, 2008 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-18427209

RESUMO

This study investigated the genotype and phenotype of HIV-1 isolates of 20 South African AIDS patients. We found the highest percentage of CXCR4 usage among primary isolates, in which 30% efficiently utilized CXCR4 and exhibited the syncytium-inducing phenotype. Phylogenetic analysis of env confirmed that 19 of the 20 were subtype C, and syncytium-inducing viruses had genetic changes in the V3 loop, characteristic of CXCR4 usage. Results imply that the frequency of CXCR4-utilizing subtype C is increasing with time.


Assuntos
Síndrome da Imunodeficiência Adquirida/virologia , HIV-1/fisiologia , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Síndrome da Imunodeficiência Adquirida/metabolismo , Adulto , Sequência de Aminoácidos , Sequência de Bases , Estudos de Coortes , DNA Viral/análise , Surtos de Doenças , Feminino , Genes env , Genótipo , Células Gigantes/virologia , HIV-1/genética , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fenótipo , África do Sul
9.
AIDS Res Hum Retroviruses ; 23(2): 322-30, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17331040

RESUMO

HIV-1 Vif, Vpr, and Vpu proteins have a profound effect on efficient viral replication and pathogenesis. This study describes the genotypic characterisation of vif , vpr and vpu from 20 South African HIV-1 subtype C primary isolates, and extensive analysis and comparison of known motifs. All HIV-1 subtype C Vif, Vpr and Vpu proteins revealed the presence of highly conserved structural and functional motifs similar to other sub-types, for example, the Vif-APOBEC3G interaction domains. However, several differences were noted when these sequences were compared to subtype B, such as the presence of the LRLL motif which has been implicated in targeting subtype C Vpu predominantly to the cell surface, instead of the Golgi apparatus. A better understanding of the structure/function relationship of these proteins may lead to the development of new classes of antiviral drugs. These results indicate that antiviral drugs that target the conserved functional domains within Vif, Vpr or Vpu could be active against all circulating subtypes, including HIV-1 subtype C.


Assuntos
Genes vif , Genes vpr , Genes vpu , Infecções por HIV/genética , HIV-1/genética , Produtos do Gene vif/química , Produtos do Gene vpr/química , Proteínas do Vírus da Imunodeficiência Humana , Humanos , Filogenia , Alinhamento de Sequência , Análise de Sequência de Proteína , Proteínas Virais Reguladoras e Acessórias/química , Produtos do Gene vif do Vírus da Imunodeficiência Humana , Produtos do Gene vpr do Vírus da Imunodeficiência Humana
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