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1.
Nat Med ; 26(2): 222-227, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-32015556

RESUMEN

Combination antiretroviral therapy (ART) is highly effective in controlling human immunodeficiency virus (HIV)-1 but requires lifelong medication due to the existence of a latent viral reservoir1,2. Potent broadly neutralizing antibodies (bNAbs) represent a potential alternative or adjuvant to ART. In addition to suppressing viremia, bNAbs may have T cell immunomodulatory effects as seen for other forms of immunotherapy3. However, this has not been established in individuals who are infected with HIV-1. Here, we document increased HIV-1 Gag-specific CD8+ T cell responses in the peripheral blood of all nine study participants who were infected with HIV-1 with suppressed blood viremia, while receiving bNAb therapy during ART interruption4. Increased CD4+ T cell responses were detected in eight individuals. The increased T cell responses were due both to newly detectable reactivity to HIV-1 Gag epitopes and the expansion of pre-existing measurable responses. These data demonstrate that bNAb therapy during ART interruption is associated with enhanced HIV-1-specific T cell responses. Whether these augmented T cell responses can contribute to bNAb-mediated viral control remains to be determined.

2.
J Virol ; 2020 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-31941771

RESUMEN

Along with other immune checkpoints, T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is expressed on exhausted CD4+ and CD8+ T cells and is upregulated on the surface of these cells upon infection by Human Immunodeficiency Virus Type 1 (HIV-1). Recent reports have suggested an antiviral role for Tim-3. However, the molecular determinants of HIV-1 which modulate cell surface Tim-3 levels have yet to be determined. Herein, we demonstrate that HIV-1 Vpu downregulates Tim-3 from the surface of infected primary CD4+ T cells, thus attenuating HIV-1-induced upregulation of Tim-3. We also provide evidence that the transmembrane domain of Vpu is required for Tim-3 downregulation. Using immunofluorescence microscopy, we determined that Vpu is in close proximity to Tim-3 and alters its subcellular localization by directing it to Rab 5+ vesicles and targeting it for sequestration within the trans- Golgi network (TGN). Intriguingly, Tim-3 knockdown and Tim-3 blockade increased HIV-1 replication in primary CD4+ T cells, thereby suggesting that Tim-3 expression might represent a natural immune mechanism limiting viral spread.IMPORTANCE HIV infection modulates surface expression of Tim-3 but the molecular determinants remain poorly understood. Here, we show that HIV-1 Vpu downregulates Tim-3 from the surface of infected primary CD4+ T cells through its transmembrane domain and alters its subcellular localization. Tim-3 blockade increases HIV-1 replication, suggesting a potential negative role of this protein in viral spread that is counteracted by Vpu.

3.
Nat Immunol ; 20(8): 1059-1070, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31308541

RESUMEN

Dysfunction of virus-specific CD4+ T cells in chronic human infections is poorly understood. We performed genome-wide transcriptional analyses and functional assays of CD4+ T cells specific for human immunodeficiency virus (HIV) from HIV-infected people before and after initiation of antiretroviral therapy (ART). A follicular helper T cell (TFH cell)-like profile characterized HIV-specific CD4+ T cells in viremic infection. HIV-specific CD4+ T cells from people spontaneously controlling the virus (elite controllers) robustly expressed genes associated with the TH1, TH17 and TH22 subsets of helper T cells. Viral suppression by ART resulted in a distinct transcriptional landscape, with a reduction in the expression of genes associated with TFH cells, but persistently low expression of genes associated with TH1, TH17 and TH22 cells compared to the elite controller profile. Thus, altered differentiation is central to the impairment of HIV-specific CD4+ T cells and involves both gain of function and loss of function.


Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Expresión Génica/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Células TH1/patología , Células Th17/patología , Perfilación de la Expresión Génica , Infecciones por VIH/virología , Humanos , Receptores CXCR5/metabolismo , Células TH1/citología , Células TH1/inmunología , Células Th17/citología , Células Th17/inmunología , Carga Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacos
4.
mBio ; 10(3)2019 06 18.
Artículo en Inglés | MEDLINE | ID: mdl-31213558

RESUMEN

The HIV-1 accessory protein Vpu enhances viral release by counteracting the restriction factor BST-2. Furthermore, Vpu promotes NK cell evasion by downmodulating cell surface NTB-A and PVR, known ligands of the NK cell receptors NTB-A and DNAM-1, respectively. While it has been established that Vpu's transmembrane domain (TMD) is required for the interaction and intracellular sequestration of BST-2, NTB-A, and PVR, it remains unclear how Vpu manages to target these proteins simultaneously. In this study, we show that upon upregulation, BST-2 is preferentially downregulated by Vpu over its other TMD substrates. We found that type I interferon (IFN)-mediated BST-2 upregulation greatly impairs the ability of Vpu to downregulate NTB-A and PVR. Our results suggest that occupation of Vpu by BST-2 affects its ability to downregulate other TMD substrates. Accordingly, knockdown of BST-2 increases Vpu's potency to downmodulate NTB-A and PVR in the presence of type I IFN treatment. Moreover, we show that expression of human BST-2, but not that of the macaque orthologue, decreases Vpu's capacity to downregulate NTB-A. Importantly, we show that type I IFNs efficiently sensitize HIV-1-infected cells to NTB-A- and DNAM-1-mediated direct and antibody-dependent NK cell responses. Altogether, our results reveal that type I IFNs decrease Vpu's polyfunctionality, thus reducing its capacity to protect HIV-1-infected cells from NK cell responses.IMPORTANCE The restriction factor BST-2 and the NK cell ligands NTB-A and PVR are among a growing list of membrane proteins found to be downregulated by HIV-1 Vpu. BST-2 antagonism enhances viral release, while NTB-A and PVR downmodulation contributes to NK cell evasion. However, it remains unclear how Vpu can target multiple cellular factors simultaneously. Here we provide evidence that under physiological conditions, BST-2 is preferentially targeted by Vpu over NTB-A and PVR. Specifically, we show that type I IFNs decrease Vpu's polyfunctionality by upregulating BST-2, thus reducing its capacity to protect HIV-1-infected cells from NK cell responses. This indicates that there is a hierarchy of Vpu substrates upon IFN treatment, revealing that for the virus, targeting BST-2 as part of its resistance to IFN takes precedence over evading NK cell responses. This reveals a potential weakness in HIV-1's immunoevasion mechanisms that may be exploited therapeutically to harness NK cell responses against HIV-1.

5.
Front Immunol ; 10: 1062, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31139189

RESUMEN

Genetic and immunologic analyses of epidemiologically-linked HIV transmission enable insights into the impact of immune responses on clinical outcomes. Human vaccine trials and animal studies of HIV-1 infection have suggested immune correlates of protection; however, their role in natural infection in terms of protection from disease progression is mostly unknown. Four HIV-1+ Cameroonian individuals, three of them epidemiologically-linked in a polygamous heterosexual relationship and one incidence-matched case, were studied over 15 years for heterologous and cross-neutralizing antibody responses, antibody binding, IgA/IgG levels, antibody-dependent cellular cytotoxicity (ADCC) against cells expressing wild-type or CD4-bound Env, viral evolution, Env epitopes, and host factors including HLA-I alleles. Despite viral infection with related strains, the members of the transmission cluster experienced contrasting clinical outcomes including cases of rapid progression and long-term non-progression in the absence of strongly protective HLA-I or CCR5Δ32 alleles. Slower progression and higher CD4/CD8 ratios were associated with enhanced IgG antibody binding to native Env and stronger V1V2 antibody binding responses in the presence of viruses with residue K169 in V2. ADCC against cells expressing Env in the CD4-bound conformation in combination with low Env-specific IgA/IgG ratios correlated with better clinical outcome. This data set highlights for the first time that V1V2-directed antibody responses and ADCC against cells expressing open, CD4-exposed Env, in the presence of low plasma IgA/IgG ratios, can correlate with clinical outcome in natural infection. These parameters are comparable to the major correlates of protection, identified post-hoc in the RV144 vaccine trial; thus, they may also modulate the rate of clinical progression once infected. The findings illustrate the potential of immune correlate analysis in natural infection to guide vaccine development.

6.
Cell Host Microbe ; 25(4): 578-587.e5, 2019 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-30974085

RESUMEN

The HIV-1 envelope glycoprotein (Env) (gp120-gp41)3 is the target for neutralizing antibodies and antibody-dependent cellular cytotoxicity (ADCC). HIV-1 Env is flexible, sampling different conformational states. Before engaging CD4, Env adopts a closed conformation (State 1) that is largely antibody resistant. CD4 binding induces an intermediate state (State 2), followed by an open conformation (State 3) that is susceptible to engagement by antibodies that recognize otherwise occluded epitopes. We investigate conformational changes in Env that induce ADCC in the presence of a small-molecule CD4-mimetic compound (CD4mc). We uncover an asymmetric Env conformation (State 2A) recognized by antibodies targeting the conserved gp120 inner domain and mediating ADCC. Sera from HIV+ individuals contain these antibodies, which can stabilize Env State 2A in combination with CD4mc. Additionally, triggering State 2A on HIV-infected primary CD4+ T cells exposes epitopes that induce ADCC. Strategies that induce this Env conformation may represent approaches to fight HIV-1 infection.


Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Linfocitos T CD4-Positivos/virología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Antígenos CD4/metabolismo , Células Cultivadas , Humanos , Unión Proteica , Conformación Proteica , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química
7.
J Clin Invest ; 129(6): 2463-2479, 2019 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-30912767

RESUMEN

Rationale Tumor infiltrating lymphocytes are widely associated with positive outcomes, yet carry key indicators of a systemic failed immune response against unresolved cancer. Cancer immunotherapies can reverse their tolerance phenotypes, while preserving tumor-reactivity and neoantigen-specificity shared with circulating immune cells. Objectives We performed comprehensive transcriptomic analyses to identify gene signatures common to circulating and tumor infiltrating lymphocytes in the context of clear cell renal cell carcinoma. Modulated genes also associated with disease outcome were validated in other cancer types. Findings Using bioinformatics, we identified practical diagnostic markers and actionable targets of the failed immune response. On circulating lymphocytes, three genes, LEF1, FASLG, and MMP9, could efficiently stratify patients from healthy control donors. From their associations with resistance to cancer immunotherapies and microbial infections, we uncovered not only pan-cancer, but pan-pathology failed immune response profiles. A prominent lymphocytic matrix metallopeptidase cell migration pathway, is central to a panoply of diseases and tumor immunogenicity, correlates with multi-cancer recurrence, and identifies a feasible, non-invasive approach to pan-pathology diagnoses. Conclusions The non-invasive differently expressed genes we have identified warrant future investigation towards the development of their potential in precision diagnostics and precision pan-disease immunotherapeutics.

8.
PLoS Pathog ; 15(2): e1007619, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30811499

RESUMEN

The phenotypic characterization of the cells in which HIV persists during antiretroviral therapy (ART) remains technically challenging. We developed a simple flow cytometry-based assay to quantify and characterize infected cells producing HIV proteins during untreated and treated HIV infection. By combining two antibodies targeting the HIV capsid in a standard intracellular staining protocol, we demonstrate that p24-producing cells can be detected with high specificity and sensitivity in the blood from people living with HIV. In untreated individuals, the frequency of productively infected cells strongly correlated with plasma viral load. Infected cells preferentially displayed a transitional memory phenotype and were enriched in Th17, peripheral Tfh and regulatory T cells subsets. These cells also preferentially expressed activation markers (CD25, HLA-DR, Ki67), immune checkpoint molecules (PD-1, LAG-3, TIGIT, Tim-3) as well as the integrins α4ß7 and α4ß1. In virally suppressed individuals on ART, p24-producing cells were only detected upon stimulation (median frequency of 4.3 p24+ cells/106 cells). These measures correlated with other assays assessing the size of the persistent reservoir including total and integrated HIV DNA, Tat/rev Induced Limiting Dilution Assay (TILDA) and quantitative viral outgrowth assay (QVOA). In ART-suppressed individuals, p24-producing cells preferentially displayed a transitional and effector memory phenotype, and expressed immune checkpoint molecules (PD-1, TIGIT) as well as the integrin α4ß1. Remarkably, α4ß1 was expressed by more than 70% of infected cells both in untreated and ART-suppressed individuals. Altogether, these results highlight a broad diversity in the phenotypes of HIV-infected cells in treated and untreated infection and suggest that strategies targeting multiple and phenotypically distinct cellular reservoirs will be needed to exert a significant impact on the size of the reservoir.


Asunto(s)
Citometría de Flujo/métodos , Infecciones por VIH/inmunología , VIH/fisiología , Adulto , Antirretrovirales , Linfocitos T CD4-Positivos , Reservorios de Enfermedades/virología , Femenino , VIH/patogenicidad , Proteína p24 del Núcleo del VIH , Infecciones por VIH/virología , VIH-1/inmunología , VIH-1/patogenicidad , Humanos , Integrina alfa4beta1/metabolismo , Masculino , Persona de Mediana Edad , Fenotipo , ARN Viral , Análisis de la Célula Individual/métodos , Subgrupos de Linfocitos T , Carga Viral , Latencia del Virus
9.
Trends Mol Med ; 25(1): 1-3, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30473188

RESUMEN

Immunometabolism is important to T cell dysfunction in chronic infections. A recent publication in The Journal of Clinical Investigation (2018;128:5083-5094) [1] shows reduced mitochondrial fitness in regulatory CD4+ T cells (Tregs) of patients with HIV and failed immune restoration on antiretroviral therapy (ART). This defect can be reversed by IL-15, revealing a new immunotherapy target for regulatory T cell restoration.


Asunto(s)
Mitocondrias/metabolismo , Linfocitos T Reguladores/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , Humanos , Interleucina-15/metabolismo
10.
J Immunol ; 201(3): 971-981, 2018 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-29934472

RESUMEN

Immune exhaustion is an important feature of chronic infections, such as HIV, and a barrier to effective immunity against cancer. This dysfunction is in part controlled by inhibitory immune checkpoints. Blockade of the PD-1 or IL-10 pathways can reinvigorate HIV-specific CD4 T cell function in vitro, as measured by cytokine secretion and proliferative responses upon Ag stimulation. However, whether this restoration of HIV-specific CD4 T cells can improve help to other cell subsets impaired in HIV infection remains to be determined. In this study, we examine a cohort of chronically infected subjects prior to initiation of antiretroviral therapy (ART) and individuals with suppressed viral load on ART. We show that IFN-γ induction in NK cells upon PBMC stimulation by HIV Ag varies inversely with viremia and depends on HIV-specific CD4 T cell help. We demonstrate in both untreated and ART-suppressed individuals that dual PD-1 and IL-10 blockade enhances cytokine secretion of NK cells via restored HIV-specific CD4 T cell function, that soluble factors contribute to these immunotherapeutic effects, and that they depend on IL-2 and IL-12 signaling. Importantly, we show that inhibition of the PD-1 and IL-10 pathways also increases NK degranulation and killing of target cells. This study demonstrates a previously underappreciated relationship between CD4 T cell impairment and NK cell exhaustion in HIV infection, provides a proof of principle that reversal of adaptive immunity exhaustion can improve the innate immune response, and suggests that immune checkpoint modulation that improves CD4/NK cell cooperation can be used as adjuvant therapy in HIV infection.


Asunto(s)
Antirretrovirales/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Células Asesinas Naturales/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Línea Celular Tumoral , Estudios de Cohortes , Infecciones por VIH/inmunología , VIH-1/efectos de los fármacos , VIH-1/inmunología , Humanos , Interferón gamma/inmunología , Interleucina-10/inmunología , Interleucina-2/inmunología , Células K562 , Células Asesinas Naturales/inmunología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Receptor de Muerte Celular Programada 1/inmunología , Carga Viral/efectos de los fármacos , Carga Viral/inmunología
11.
Viruses ; 10(6)2018 06 19.
Artículo en Inglés | MEDLINE | ID: mdl-29921828

RESUMEN

Passive administration of broadly neutralizing antibodies (bNAbs) capable of recognizing a broad range of viral strains to non-human primates has led to protection from infection with chimeric SIV/HIV virus (SHIV). This data suggests that generating protective antibody responses could be an effective strategy for an HIV vaccine. However, classic vaccine approaches have failed so far to induce such protective antibodies in HIV vaccine trials. HIV-specific bNAbs identified in natural infection show high levels of somatic hypermutations, demonstrating that they underwent extensive affinity maturation. It is likely that to gain ability to recognize diverse viral strains, vaccine-induced humoral responses will also require complex, iterative maturation. T follicular helper cells (Tfh) are a specialized CD4+ T cell subset that provides help to B cells in the germinal center for the generation of high-affinity and long-lasting humoral responses. It is therefore probable that the quality and quantity of Tfh responses upon vaccination will impact development of bNAbs. Here, we review studies that advanced our understanding of Tfh differentiation, function and regulation. We discuss correlates of Tfh responses and bNAb development in natural HIV infection. Finally, we highlight recent strategies to optimize Tfh responses upon vaccination and their impact on prophylactic HIV vaccine research.


Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/metabolismo , Linfocitos B/inmunología , Descubrimiento de Drogas/métodos , Anticuerpos Anti-VIH/metabolismo , Infecciones por VIH/prevención & control , Linfocitos T Colaboradores-Inductores/inmunología , Vacunas contra el SIDA/aislamiento & purificación , Humanos
12.
Front Immunol ; 9: 628, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29632541

RESUMEN

Using 5' rapid amplification of cDNA ends, Illumina MiSeq, and basic flow cytometry, we systematically analyzed the expressed B cell receptor (BCR) repertoire in 14 healthy adult PBMCs, 5 HIV-1+ adult PBMCs, 5 cord blood samples, and 3 HIS-CD4/B mice, examining the full-length variable region of µ, γ, α, κ, and λ chains for V-gene usage, somatic hypermutation (SHM), and CDR3 length. Adding to the known repertoire of healthy adults, Illumina MiSeq consistently detected small fractions of reads with high mutation frequencies including hypermutated µ reads, and reads with long CDR3s. Additionally, the less studied IgA repertoire displayed similar characteristics to that of IgG. Compared to healthy adults, the five HIV-1 chronically infected adults displayed elevated mutation frequencies for all µ, γ, α, κ, and λ chains examined and slightly longer CDR3 lengths for γ, α, and λ. To evaluate the reconstituted human BCR sequences in a humanized mouse model, we analyzed cord blood and HIS-CD4/B mice, which all lacked the typical SHM seen in the adult reference. Furthermore, MiSeq revealed identical unmutated IgM sequences derived from separate cell aliquots, thus for the first time demonstrating rare clonal members of unmutated IgM B cells by sequencing.


Asunto(s)
Linfocitos B/inmunología , Sangre Fetal/fisiología , Infecciones por VIH/inmunología , VIH-1/fisiología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Inmunoglobulina M/genética , Receptores de Antígenos de Linfocitos B/genética , Región de Flanqueo 5'/genética , Adulto , Animales , Enfermedad Crónica , Células Clonales , Biología Computacional , Voluntarios Sanos , Humanos , Ratones , Ratones SCID , Mutación/genética
13.
mBio ; 9(2)2018 03 20.
Artículo en Inglés | MEDLINE | ID: mdl-29559570

RESUMEN

The conformation of the HIV-1 envelope glycoprotein (Env) substantially impacts antibody recognition and antibody-dependent cellular cytotoxicity (ADCC) responses. In the absence of the CD4 receptor at the cell surface, primary Envs sample a "closed" conformation that occludes CD4-induced (CD4i) epitopes. The virus controls CD4 expression through the actions of Nef and Vpu accessory proteins, thus protecting infected cells from ADCC responses. However, gp120 shed from infected cells can bind to CD4 present on uninfected bystander cells, sensitizing them to ADCC mediated by CD4i antibodies (Abs). Therefore, we hypothesized that these bystander cells could impact the interpretation of ADCC measurements. To investigate this, we evaluated the ability of antibodies to CD4i epitopes and broadly neutralizing Abs (bNAbs) to mediate ADCC measured by five ADCC assays commonly used in the field. Our results indicate that the uninfected bystander cells coated with gp120 are efficiently recognized by the CD4i ligands but not the bNabs. Consequently, the uninfected bystander cells substantially affect in vitro measurements made with ADCC assays that fail to identify responses against infected versus uninfected cells. Moreover, using an mRNA flow technique that detects productively infected cells, we found that the vast majority of HIV-1-infected cells in in vitro cultures or ex vivo samples from HIV-1-infected individuals are CD4 negative and therefore do not expose significant levels of CD4i epitopes. Altogether, our results indicate that ADCC assays unable to differentiate responses against infected versus uninfected cells overestimate responses mediated by CD4i ligands.IMPORTANCE Emerging evidence supports a role for antibody-dependent cellular cytotoxicity (ADCC) in protection against HIV-1 transmission and disease progression. However, there are conflicting reports regarding the ability of nonneutralizing antibodies targeting CD4-inducible (CD4i) Env epitopes to mediate ADCC. Here, we performed a side-by-side comparison of different methods currently being used in the field to measure ADCC responses to HIV-1. We found that assays which are unable to differentiate virus-infected from uninfected cells greatly overestimate ADCC responses mediated by antibodies to CD4i epitopes and underestimate responses mediated by broadly neutralizing antibodies (bNAbs). Our results strongly argue for the use of assays that measure ADCC against HIV-1-infected cells expressing physiologically relevant conformations of Env to evaluate correlates of protection in vaccine trials.


Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos/fisiología , VIH-1/inmunología , Anticuerpos Neutralizantes/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/genética , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Citometría de Flujo , Granzimas/genética , Granzimas/metabolismo , Células HEK293 , Proteína gp120 de Envoltorio del VIH/inmunología , Humanos
14.
Retrovirology ; 15(1): 18, 2018 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-29394935

RESUMEN

Recent years have seen a substantial increase in the number of tools available to monitor and study HIV reservoirs. Here, we discuss recent technological advances that enable an understanding of reservoir dynamics beyond classical assays to measure the frequency of cells containing provirus able to propagate a spreading infection (replication-competent reservoir). Specifically, we focus on the characterization of cellular reservoirs containing proviruses able to transcribe viral mRNAs (so called transcription-competent) and translate viral proteins (translation-competent). We suggest that the study of these alternative reservoirs provides complementary information to classical approaches, crucially at a single-cell level. This enables an in-depth characterization of the cellular reservoir, both following reactivation from latency and, importantly, directly ex vivo at baseline. Furthermore, we propose that the study of cellular reservoirs that may not contain fully replication-competent virus, but are able to produce HIV mRNAs and proteins, is of biological importance. Lastly, we detail some of the key contributions that the study of these transcription and translation-competent reservoirs has made thus far to investigations into HIV persistence, and outline where these approaches may take the field next.


Asunto(s)
Infecciones por VIH/virología , VIH-1/genética , Proteínas del Virus de la Inmunodeficiencia Humana/análisis , ARN Mensajero/análisis , ARN Viral/análisis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Citometría de Flujo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Humanos , Hibridación in Situ , Provirus/genética , Análisis de la Célula Individual
15.
Curr HIV/AIDS Rep ; 15(1): 39-48, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29374858

RESUMEN

PURPOSE OF REVIEW: The long-lived HIV reservoir remains a major obstacle for an HIV cure. Current techniques to analyze this reservoir are generally population-based. We highlight recent developments in methods visualizing HIV, which offer a different, complementary view, and provide indispensable information for cure strategy development. RECENT FINDINGS: Recent advances in fluorescence in situ hybridization techniques enabled key developments in reservoir visualization. Flow cytometric detection of HIV mRNAs, concurrently with proteins, provides a high-throughput approach to study the reservoir on a single-cell level. On a tissue level, key spatial information can be obtained detecting viral RNA and DNA in situ by fluorescence microscopy. At total-body level, advancements in non-invasive immuno-positron emission tomography (PET) detection of HIV proteins may allow an encompassing view of HIV reservoir sites. HIV imaging approaches provide important, complementary information regarding the size, phenotype, and localization of the HIV reservoir. Visualizing the reservoir may contribute to the design, assessment, and monitoring of HIV cure strategies in vitro and in vivo.


Asunto(s)
Citometría de Flujo/métodos , Infecciones por VIH/virología , Proteínas del Virus de la Inmunodeficiencia Humana/análisis , Hibridación Fluorescente in Situ/métodos , Tomografía de Emisión de Positrones/métodos , Infecciones por VIH/diagnóstico , Infecciones por VIH/tratamiento farmacológico , VIH-1/genética , Humanos , ARN Viral/genética , Latencia del Virus/genética
16.
Virology ; 515: 38-45, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29248757

RESUMEN

Recent analysis of HIV-1 envelope glycoproteins (Env) dynamics showed that the unliganded Env trimer can potentially sample three conformations: a metastable "closed" conformation (State 1), an "open" CD4-bound conformation (State 3), and an intermediate "partially open" conformation (State 2). HIV-1 evolved several mechanisms to avoid "opening" its Env in order to evade immune responses such as antibody-dependent cellular cytotoxicity (ADCC), which preferentially targets Envs in the CD4-bound conformation on the surface of infected cells. Here we took advantage of a well-characterized single-residue change in the gp120 trimer association domain to modify Env conformation and evaluate its impact on ADCC responses. We found that cells infected with viruses expressing Env stabilized in States 2/3 become highly susceptible to ADCC responses by sera from HIV-1-infected individuals. Our results indicate that the conformations spontaneously sampled by the Env trimer at the surface of infected cells has a significant impact on ADCC responses.


Asunto(s)
Infecciones por VIH/inmunología , VIH-1/inmunología , Suero/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos CD4/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/fisiología , Humanos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética
17.
PLoS One ; 12(10): e0186998, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29065175

RESUMEN

The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells.


Asunto(s)
Antígenos/inmunología , Biomarcadores/metabolismo , Linfocitos T CD4-Positivos/inmunología , Activación de Linfocitos/inmunología , Animales , Antígenos CD/inmunología , Efecto Espectador , Estudios de Cohortes , Humanos , Macaca mulatta
18.
AIDS Res Ther ; 14(1): 40, 2017 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-28893281

RESUMEN

Despite the tremendous success of anti-retroviral therapy (ART) no current treatment can eradicate latent HIV reservoirs from HIV-infected individuals or generate, effective HIV-specific immunity. Technological limitations have hampered the identification and characterization of both HIV-infected cells and HIV-specific responses in clinical samples directly ex vivo. RNA-flow cytometric fluorescence in situ hybridisation (RNA Flow-FISH) is a powerful technique, which enables detection of mRNAs in conjunction with proteins at a single-cell level. Here, we describe how we are using this technology to address some of the major questions remaining in the HIV field in the era of ART. We discuss how CD4 T cell responses to HIV antigens, both following vaccination and HIV infection, can be characterized by measurement of cytokine mRNAs. We describe how our development of a dual HIV mRNA/protein assay (HIVRNA/Gag assay) enables high sensitivity detection of very rare HIV-infected cells and aids investigations into the translation-competent latent reservoir in the context of HIV cure.


Asunto(s)
Citometría de Flujo/métodos , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , VIH-1/inmunología , Hibridación Fluorescente in Situ/métodos , ARN/análisis , Vacunas contra el SIDA , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/ultraestructura , Linfocitos T CD4-Positivos/virología , Citocinas/genética , Citocinas/inmunología , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Vacunación , Latencia del Virus
19.
Nat Protoc ; 12(10): 2029-2049, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28880280

RESUMEN

Efforts to cure HIV are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in individuals receiving antiretroviral therapy (ART). We describe a protocol for flow cytometric identification of viral reservoirs, based on concurrent detection of cellular HIV Gagpol mRNA by in situ RNA hybridization combined with antibody staining for the HIV Gag protein. By simultaneously detecting both HIV RNA and protein, the CD4 T cells harboring translation-competent virus can be identified. The HIVRNA/Gag method is 1,000-fold more sensitive than Gag protein staining alone, with a detection limit of 0.5-1 Gagpol mRNA+/Gag protein+ cells per million CD4 T cells. Uniquely, the HIVRNA/Gag assay also allows parallel phenotyping of viral reservoirs, including reactivated latent reservoirs in clinical samples. The assay takes 2 d, and requires antibody labeling for surface and intracellular markers, followed by mRNA labeling and multiple signal amplification steps.


Asunto(s)
Linfocitos T CD4-Positivos/virología , Citometría de Flujo/métodos , Infecciones por VIH/diagnóstico , Infecciones por VIH/virología , Hibridación Fluorescente in Situ/métodos , ARN Viral/sangre , Proteínas de Fusión gag-pol/genética , Humanos , Límite de Detección , ARN Mensajero/sangre
20.
J Virol ; 91(16)2017 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-28615199

RESUMEN

HIV-1 Nef clones isolated from a rare subset of HIV-1-infected elite controllers (EC), with the ability to suppress viral load to undetectable levels in the absence of antiretroviral therapy, are unable to fully downregulate CD4 from the plasma membrane of CD4+ T cells. Residual CD4 left at the plasma membrane allows Env-CD4 interaction, which leads to increased exposure of Env CD4-induced epitopes and increases susceptibility of infected cells to antibody-dependent cellular cytotoxicity (ADCC). ADCC is mediated largely by natural killer (NK) cells, which control their activation status through the cumulative signals received through activating and inhibitory receptors. Recently, the activating NKG2D receptor was demonstrated to positively influence ADCC responses. Since HIV-1 Nef has been reported to reduce the expression of NKG2D ligands, we evaluated the relative abilities of Nef from EC and progressors to downmodulate NKG2D ligands. Furthermore, we assessed the impact of EC and progressor Nef on the ADCC susceptibility of HIV-1-infected cells. We observed a significantly increased expression of NKG2D ligands on cells infected with viruses coding for Nef from EC. Importantly, NKG2D ligand expression levels correlated with enhanced susceptibility of HIV-1-infected cells to ADCC. The biological significance of this correlation was corroborated by the demonstration that antibody-mediated blockade of NKG2D significantly reduced ADCC of cells infected with viruses carrying Nef from EC. These results suggest the involvement of NKG2D-NKG2D ligand interactions in the enhanced susceptibility of EC HIV-1-infected cells to ADCC responses.IMPORTANCE Attenuated Nef functions have been reported in HIV-1 isolated from EC. The inability of elite controller Nef to fully remove CD4 from the surface of infected cells enhanced their susceptibility to elimination by ADCC. We now show that downregulation of NKG2D ligands by HIV-1 Nef from EC is inefficient and leaves infected cells susceptible to ADCC. These data suggest a critical role for NKG2D ligands in anti-HIV-1 ADCC responses.


Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Infecciones por VIH/inmunología , VIH-1/fisiología , Interacciones Huésped-Patógeno , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Sobrevivientes de VIH a Largo Plazo , Humanos
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