RESUMEN
Productively infected cells are generally thought to arise from HIV infection of activated CD4+ T cells, and these infected activated cells are thought to be a recurring source of latently infected cells when a portion of the population transitions to a resting state. We discovered and report here that productively and latently infected cells can instead originate from direct infection of resting CD4+ T cell populations in lymphoid tissues in Fiebig I, the earliest stage of detectable HIV infection. We found that direct infection of resting CD4+ T cells was correlated with the availability of susceptible target cells in lymphoid tissues largely restricted to resting CD4+ T cells in which expression of pTEFb enabled productive infection, and we documented persistence of HIV-producing resting T cells during antiretroviral therapy (ART). Thus, we provide evidence of a mechanism by which direct infection of resting T cells in lymphoid tissues to generate productively and latently infected cells creates a mechanism by which the productively infected cells can replenish both populations and maintain two sources of virus from which HIV infection can rebound, even if ART is instituted at the earliest stage of detectable infection.
Asunto(s)
Infecciones por VIH , Humanos , Latencia del Virus , Replicación Viral , Linfocitos T CD4-PositivosRESUMEN
Anti-retroviral therapy (ART) generally suppresses HIV replication to undetectable levels in peripheral blood, but immune activation associated with increased morbidity and mortality is sustained during ART, and infection rebounds when treatment is interrupted. To identify drivers of immune activation and potential sources of viral rebound, we modified RNAscope in situ hybridization to visualize HIV-producing cells as a standard against which to compare the following assays of potential sources of immune activation and virus rebound following treatment interruption: (i) envelope detection by induced transcription-based sequencing (EDITS) assay; (ii) HIV-Flow; (iii) Flow-FISH assays that can scan tissues and cell suspensions to detect rare cells expressing env mRNA, gag mRNA/Gag protein and p24; and (iv) an ultrasensitive immunoassay that detects p24 in cell/tissue lysates at subfemtomolar levels. We show that the sensitivities of these assays are sufficient to detect one rare HIV-producing/env mRNA+/p24+ cell in one million uninfected cells. These high-throughput technologies provide contemporary tools to detect and characterize rare cells producing virus and viral antigens as potential sources of immune activation and viral rebound. IMPORTANCE Anti-retroviral therapy (ART) has greatly improved the quality and length of life for people living with HIV, but immune activation does not normalize during ART, and persistent immune activation has been linked to increased morbidity and mortality. We report a comparison of assays of two potential sources of immune activation during ART: rare cells producing HIV and the virus' major viral protein, p24, benchmarked on a cell model of active and latent infections and a method to visualize HIV-producing cells. We show that assays of HIV envelope mRNA (EDITS assay), gag mRNA, and p24 (Flow-FISH, HIV-Flow. and ultrasensitive p24 immunoassay) detect HIV-producing cells and p24 at sensitivities of one infected cell in a million uninfected cells, thereby providing validated tools to explore sources of immune activation during ART in the lymphoid and other tissue reservoirs.
Asunto(s)
Infecciones por VIH , VIH-1 , ARN Viral , Tropismo Viral , Activación Viral , Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/uso terapéutico , Antígenos Virales/análisis , Antígenos Virales/genética , Antígenos Virales/metabolismo , Linfocitos T CD4-Positivos , Proteína p24 del Núcleo del VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/crecimiento & desarrollo , VIH-1/inmunología , Humanos , Inmunoensayo , Hibridación Fluorescente in Situ , ARN Mensajero/análisis , ARN Viral/análisis , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Combination anti-retroviral drug therapy (ART) potently suppresses HIV-1 replication but does not result in virus eradication or a cure. A major contributing factor is the long-term persistence of a reservoir of latently infected cells. To study this reservoir, we established a humanized mouse model of HIV-1 infection and ART suppression based on an oral ART regimen. Similar to humans, HIV-1 levels in the blood of ART-treated animals were frequently suppressed below the limits of detection. However, the limited timeframe of the mouse model and the small volume of available samples makes it a challenging model with which to achieve full viral suppression and to investigate the latent reservoir. We therefore used an ex vivo latency reactivation assay that allows a semiquantitative measure of the latent reservoir that establishes in individual animals, regardless of whether they are treated with ART. Using this assay, we found that latently infected human CD4 T cells can be readily detected in mouse lymphoid tissues and that latent HIV-1 was enriched in populations expressing markers of T cell exhaustion, PD-1 and TIGIT. In addition, we were able to use the ex vivo latency reactivation assay to demonstrate that HIV-specific TALENs can reduce the fraction of reactivatable virus in the latently infected cell population that establishes in vivo, supporting the use of targeted nuclease-based approaches for an HIV-1 cure.IMPORTANCE HIV-1 can establish latent infections that are not cleared by current antiretroviral drugs or the body's immune responses and therefore represent a major barrier to curing HIV-infected individuals. However, the lack of expression of viral antigens on latently infected cells makes them difficult to identify or study. Here, we describe a humanized mouse model that can be used to detect latent but reactivatable HIV-1 in both untreated mice and those on ART and therefore provides a simple system with which to study the latent HIV-1 reservoir and the impact of interventions aimed at reducing it.
Asunto(s)
VIH-1/inmunología , Latencia del Virus/inmunología , Latencia del Virus/fisiología , Animales , Antirretrovirales/farmacología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Modelos Animales de Enfermedad , Infecciones por VIH/virología , Seropositividad para VIH/tratamiento farmacológico , VIH-1/patogenicidad , Humanos , Ratones , Receptor de Muerte Celular Programada 1/inmunología , Receptores Inmunológicos/inmunología , Nucleasas de los Efectores Tipo Activadores de la Transcripción/inmunología , Activación Viral , Replicación ViralRESUMEN
BACKGROUND: Notwithstanding 1 documented case of HIV-1 cure following allogeneic stem cell transplantation (allo-SCT), several subsequent cases of allo-SCT in HIV-1 positive individuals have failed to cure HIV-1 infection. The aim of our study was to describe changes in the HIV reservoir in a single chronically HIV-infected patient on suppressive antiretroviral therapy who underwent allo-SCT for treatment of acute lymphoblastic leukemia. METHODS AND FINDINGS: We prospectively collected peripheral blood mononuclear cells (PBMCs) by leukapheresis from a 55-year-old man with chronic HIV infection before and after allo-SCT to measure the size of the HIV-1 reservoir and characterize viral phylogeny and phenotypic changes in immune cells. At day 784 post-transplant, when HIV-1 was undetectable by multiple measures-including PCR measurements of both total and integrated HIV-1 DNA, replication-competent virus measurement by large cell input quantitative viral outgrowth assay, and in situ hybridization of colon tissue-the patient consented to an analytic treatment interruption (ATI) with frequent clinical monitoring. He remained aviremic off antiretroviral therapy until ATI day 288, when a low-level virus rebound of 60 HIV-1 copies/ml occurred, which increased to 1,640 HIV-1 copies/ml 5 days later, prompting reinitiation of ART. Rebounding plasma HIV-1 sequences were phylogenetically distinct from proviral HIV-1 DNA detected in circulating PBMCs before transplantation. The main limitations of this study are the insensitivity of reservoir measurements, and the fact that it describes a single case. CONCLUSIONS: allo-SCT led to a significant reduction in the size of the HIV-1 reservoir and a >9-month-long ART-free remission from HIV-1 replication. Phylogenetic analyses suggest that the origin of rebound virus was distinct from the viruses identified pre-transplant in the PBMCs.
Asunto(s)
Infecciones por VIH/terapia , Carga Viral/efectos de los fármacos , Antirretrovirales/uso terapéutico , VIH/genética , Infecciones por VIH/virología , VIH-1/genética , Humanos , Leucocitos Mononucleares , Masculino , Persona de Mediana Edad , Filogenia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Trasplante de Células Madre/métodos , Carga Viral/fisiologíaRESUMEN
In the quest for a functional cure or the eradication of HIV infection, it is necessary to know the sizes of the reservoirs from which infection rebounds after treatment interruption. Thus, we quantified SIV and HIV tissue burdens in tissues of infected nonhuman primates and lymphoid tissue (LT) biopsies from infected humans. Before antiretroviral therapy (ART), LTs contained >98% of the SIV RNA+ and DNA+ cells. With ART, the numbers of virus (v) RNA+ cells substantially decreased but remained detectable, and their persistence was associated with relatively lower drug concentrations in LT than in peripheral blood. Prolonged ART also decreased the levels of SIV- and HIV-DNA+ cells, but the estimated size of the residual tissue burden of 108 vDNA+ cells potentially containing replication-competent proviruses, along with evidence of continuing virus production in LT despite ART, indicated two important sources for rebound following treatment interruption. The large sizes of these tissue reservoirs underscore challenges in developing 'HIV cure' strategies targeting multiple sources of virus production.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH/aislamiento & purificación , Carga Viral , ADN Viral/análisis , VIH/genética , Infecciones por VIH/sangre , Humanos , Tejido Linfoide/virología , ARN Viral/análisisRESUMEN
A human immunodeficiency virus (HIV) infection cure requires an understanding of the cellular and anatomical sites harboring virus that contribute to viral rebound upon treatment interruption. Despite antiretroviral therapy (ART), HIV-associated neurocognitive disorders (HAND) are reported in HIV-infected individuals on ART. Biomarkers for macrophage activation and neuronal damage in cerebrospinal fluid (CSF) of HIV-infected individuals demonstrate continued effects of HIV in brain and suggest that the central nervous system (CNS) may serve as a viral reservoir. Using a simian immunodeficiency virus (SIV)/macaque model for HIV encephalitis and AIDS, we evaluated whether infected cells persist in brain despite ART. Eight SIV-infected pig-tailed macaques were virally suppressed with ART, and plasma and CSF viremia levels were analyzed longitudinally. To assess whether virus persisted in brain macrophages (BrMΦ) in these macaques, we used a macrophage quantitative viral outgrowth assay (MΦ-QVOA), PCR, and in situ hybridization (ISH) to measure the frequency of infected cells and the levels of viral RNA and DNA in brain. Viral RNA in brain tissue of suppressed macaques was undetectable, although viral DNA was detected in all animals. The MΦ-QVOA demonstrated that the majority of suppressed animals contained latently infected BrMΦ. We also showed that virus produced in the MΦ-QVOAs was replication competent, suggesting that latently infected BrMΦ are capable of reestablishing productive infection upon treatment interruption. This report provides the first confirmation of the presence of replication-competent SIV in BrMΦ of ART-suppressed macaques and suggests that the highly debated issue of viral latency in macrophages, at least in brain, has been addressed in SIV-infected macaques treated with ART.IMPORTANCE Resting CD4+ T cells are currently the only cells that fit the definition of a latent reservoir. However, recent evidence suggests that HIV/SIV-infected macrophages persist despite ART. Markers of macrophage activation and neuronal damage are observed in the CSF of HIV-infected individuals and of SIV-infected macaques on suppressive ART regimens, suggesting that the CNS has continued virus infection and latent infection. A controversy exists as to whether brain macrophages represent a latent source of replication-competent virus capable of reestablishing infection upon treatment interruption. In this study, we demonstrated the presence of the latent macrophage reservoir in brains of SIV-infected ART-treated macaques and analyzed the reservoir using our established outgrowth assay to quantitate macrophages harboring replication-competent SIV genomes. Our results support the idea of the existence of other latent reservoirs in addition to resting CD4+ T cells and underscore the importance of macrophages in developing strategies to eradicate HIV.
Asunto(s)
Encéfalo/virología , Macrófagos/virología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/fisiología , Latencia del Virus , Animales , Antirretrovirales/administración & dosificación , Antirretrovirales/uso terapéutico , Encéfalo/inmunología , Macaca mulatta , Reacción en Cadena de la Polimerasa , ARN Viral/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunología , Carga Viral , Activación Viral , Replicación ViralRESUMEN
OBJECTIVE: Resting CD4 T cells have been recognized as the major cell reservoir of latent HIV-1 during antiretroviral therapy (ART). Using an simian immunodeficiency virus (SIV)/macaque model for AIDS and HIV-related neurocognitive disorders we assessed the contribution of the brain to viral latency and reactivation. DESIGN: Pigtailed macaques were dual inoculated with SIVDeltaB670 and SIV17E-Fr and treated with an efficacious central nervous system-penetrant ART. After 500 days of viral suppression animals were treated with two cycles of latency reversing agents and increases in viral transcripts were examined. METHODS: Longitudinal plasma and cerebrospinal fluid (CSF) viral loads were analyzed by quantitative and digital droplet PCR. After necropsy, viral transcripts in organs were analyzed by PCR, in-situ hybridization, and phylogenetic genotyping based on env V1 loop sequences. Markers for neuronal damage and CSF activation were measured by ELISA. RESULTS: Increases in activation markers and plasma and CSF viral loads were observed in one animal treated with latency reversing agents, despite ongoing ART. SIV transcripts were identified in occipital cortex macrophages by in-situ hybridization and CD68 staining. The most abundant SIV genotype in CSF was unique and expanded independent from viruses found in the periphery. CONCLUSION: The central nervous system harbors latent SIV genomes after long-term viral suppression by ART, indicating that the brain represents a potential viral reservoir and should be seriously considered during AIDS cure strategies.
Asunto(s)
Encéfalo/virología , Trastornos Neurocognitivos/virología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/fisiología , Activación Viral , Latencia del Virus , Animales , Antirretrovirales/uso terapéutico , Líquido Cefalorraquídeo/virología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Productos del Gen env/genética , Técnicas de Genotipaje , Hibridación in Situ , Macaca , Trastornos Neurocognitivos/tratamiento farmacológico , Filogenia , Plasma/virología , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Síndrome de Inmunodeficiencia Adquirida del Simio/complicaciones , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Carga ViralRESUMEN
A primary obstacle to an HIV-1 cure is long-lived viral reservoirs, which must be eliminated or greatly reduced. Cure strategies have largely focused on monitoring changes in T cell reservoirs in peripheral blood (PB), even though the lymphoid tissues (LT) are primary sites for viral persistence. To track and discriminate viral reservoirs within tissue compartments we developed a specific and sensitive next-generation in situ hybridization approach to detect vRNA, including vRNA+ cells and viral particles ("RNAscope"), vDNA+ cells ("DNAscope") and combined vRNA and vDNA with immunohistochemistry to detect and phenotype active and latently infected cells in the same tissue section. RNAscope is highly sensitive with greater speed of analysis compared to traditional in situ hybridization. The highly sensitive and specific DNAscope detected SIV/HIV vDNA+ cells, including duplexed detection of vDNA and vRNA or immunophenotypic markers in the same section. Analysis of LT samples from macaques prior to and during combination antiretroviral therapy demonstrated that B cell follicles are an important anatomical compartment for both latent and active viral persistence during treatment. These new tools should allow new insights into viral reservoir biology and evaluation of cure strategies.
RESUMEN
Macrophages have long been considered to contribute to HIV infection of the CNS; however, a recent study has contradicted this early work and suggests that myeloid cells are not an in vivo source of virus production. Here, we addressed the role of macrophages in HIV infection by first analyzing monocytes isolated from viremic patients and patients undergoing antiretroviral treatment. We were unable to find viral DNA or viral outgrowth in monocytes isolated from peripheral blood. To determine whether tissue macrophages are productively infected, we used 3 different but complementary humanized mouse models. Two of these models (bone marrow/liver/thymus [BLT] mice and T cell-only mice [ToM]) have been previously described, and the third model was generated by reconstituting immunodeficient mice with human CD34+ hematopoietic stem cells that were devoid of human T cells (myeloid-only mice [MoM]) to specifically evaluate HIV replication in this population. Using MoM, we demonstrated that macrophages can sustain HIV replication in the absence of T cells; HIV-infected macrophages are distributed in various tissues including the brain; replication-competent virus can be rescued ex vivo from infected macrophages; and infected macrophages can establish de novo infection. Together, these results demonstrate that macrophages represent a genuine target for HIV infection in vivo that can sustain and transmit infection.
Asunto(s)
Infecciones por VIH/inmunología , VIH-1/inmunología , Macrófagos/inmunología , Linfocitos T/inmunología , Replicación Viral/inmunología , Animales , Femenino , Infecciones por VIH/patología , Humanos , Macrófagos/patología , Macrófagos/virología , Masculino , Ratones , Ratones Endogámicos NOD , Linfocitos T/patologíaRESUMEN
Live attenuated vaccines such as SIV with a deleted nef gene have provided the most robust protection against subsequent vaginal challenge with wild-type (WT) SIV in the SIV-rhesus macaque model of HIV-1 transmission to women. Hence, identifying correlates of this protection could enable design of an effective HIV-1 vaccine. One such prechallenge correlate of protection from vaginal challenge has recently been identified as a system with three components: 1) IgG Abs reacting with the viral envelope glycoprotein trimeric gp41; 2) produced by plasma cells in the submucosa and ectopic tertiary lymphoid follicles in the ectocervix and vagina; and 3) concentrated on the path of virus entry by the neonatal FcR in the overlying epithelium. We now examine the mucosal production of the Ab component of this system after vaginal challenge. We show that vaginal challenge immediately elicits striking increases in plasma cells not only in the female reproductive tract but also at other mucosal sites, and that these increases correlate with low but persistent replication at mucosal sites. We describe vaginal ectopic follicles that are structurally and functionally organized similar to follicles in secondary lymphoid organs, and we provide inferential evidence for a key role of the female reproductive tract epithelium in facilitating Ab production, affinity maturation, and class switch recombination. Vaccination thus accesses an epithelial-immune system axis in the female reproductive tract to respond to exposure to mucosal pathogens. Designing strategies to mimic this system could advance development of an effective HIV-1 vaccine.
Asunto(s)
Vacunas contra el SIDA/inmunología , Epitelio/metabolismo , Infecciones por VIH/inmunología , VIH-1/fisiología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/fisiología , Vagina/inmunología , Animales , Anticuerpos Antivirales/metabolismo , Epitelio/inmunología , Femenino , Proteína gp41 de Envoltorio del VIH/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunidad Humoral , Inmunidad Mucosa , Inmunización , Macaca mulatta , Receptores Fc/metabolismo , Vacunas Atenuadas , Internalización del Virus , Replicación ViralRESUMEN
Antiretroviral therapy (ART) suppresses HIV replication in most individuals but cannot eradicate latently infected cells established before ART was initiated. Thus, infection rebounds when treatment is interrupted by reactivation of virus production from this reservoir. Currently, one or a few latently infected resting memory CD4 T cells are thought be the principal source of recrudescent infection, but this estimate is based on peripheral blood rather than lymphoid tissues (LTs), the principal sites of virus production and persistence before initiating ART. We, therefore, examined lymph node (LN) and gut-associated lymphoid tissue (GALT) biopsies from fully suppressed subjects, interrupted therapy, monitored plasma viral load (pVL), and repeated biopsies on 12 individuals as soon as pVL became detectable. Isolated HIV RNA-positive (vRNA+) cells were detected by in situ hybridization in LTs obtained before interruption in several patients. After interruption, multiple foci of vRNA+ cells were detected in 6 of 12 individuals as soon as pVL was measureable and in some subjects, in more than one anatomic site. Minimal estimates of the number of rebounding/founder (R/F) variants were determined by single-gene amplification and sequencing of viral RNA or DNA from peripheral blood mononuclear cells and plasma obtained at or just before viral recrudescence. Sequence analysis revealed a large number of R/F viruses representing recrudescent viremia from multiple sources. Together, these findings are consistent with the origins of recrudescent infection by reactivation from many latently infected cells at multiple sites. The inferred large pool of cells and sites to rekindle recrudescent infection highlights the challenges in eradicating HIV.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , VIH/fisiología , Tejido Linfoide/virología , Adulto , Fármacos Anti-VIH/administración & dosificación , Recuento de Linfocito CD4 , Esquema de Medicación , VIH/genética , Infecciones por VIH/virología , Humanos , Hibridación in Situ , Persona de Mediana Edad , Datos de Secuencia Molecular , Filogenia , ARN Viral/sangre , Carga ViralRESUMEN
Principles to guide design of an effective vaccine against HIV are greatly needed, particularly to protect women in the pandemic's epicenter in Africa. We have been seeking these principles by identifying correlates of the robust protection associated with SIVmac239Δnef vaccination in the SIV-rhesus macaque animal model of HIV-1 transmission to women. We identified one correlate of SIVmac239Δnef protection against vaginal challenge as a resident mucosal system for SIV-gp41 trimer Ab production and neonatal FcR-mediated concentration of these Abs on the path of virus entry to inhibit establishment of infected founder populations at the portal of entry. In this study, we identify blocking CD4(+) T cell recruitment to thereby inhibit local expansion of infected founder populations as a second correlate of protection. Virus-specific immune complex interactions with the inhibitory FcγRIIb receptor in the epithelium lining the cervix initiate expression of genes that block recruitment of target cells to fuel local expansion. Immune complex-FcγRIIb receptor interactions at mucosal frontlines to dampen the innate immune response to vaginal challenge could be a potentially general mechanism for the mucosal immune system to sense and modulate the response to a previously encountered pathogen. Designing vaccines to provide protection without eliciting these transmission-promoting innate responses could contribute to developing an effective HIV-1 vaccine.
Asunto(s)
Cuello del Útero/inmunología , Receptores de IgG/inmunología , Vacunas contra el SIDAS/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Vagina/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Linfocitos T CD4-Positivos/inmunología , Movimiento Celular/inmunología , Cuello del Útero/virología , Femenino , Perfilación de la Expresión Génica , Proteína gp41 de Envoltorio del VIH/inmunología , Inmunidad Innata , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Activación de Linfocitos/inmunología , Macaca mulatta , Membrana Mucosa/inmunología , Vacunas contra el SIDAS/administración & dosificación , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Vacunación , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Vagina/virologíaRESUMEN
We sought design principles for a vaccine to prevent HIV transmission to women by identifying correlates of protection conferred by a highly effective live attenuated SIV vaccine in the rhesus macaque animal model. We show that SIVmac239Δnef vaccination recruits plasma cells and induces ectopic lymphoid follicle formation beneath the mucosal epithelium in the rhesus macaque female reproductive tract. The plasma cells and ectopic follicles produce IgG Abs reactive with viral envelope glycoprotein gp41 trimers, and these Abs are concentrated on the path of virus entry by the neonatal FcR in cervical reserve epithelium and in vaginal epithelium. This local Ab production and delivery system correlated spatially and temporally with the maturation of local protection against high-dose pathogenic SIV vaginal challenge. Thus, designing vaccines to elicit production and concentration of Abs at mucosal frontlines could aid in the development of an effective vaccine to protect women against HIV-1.
Asunto(s)
Cuello del Útero/inmunología , Vacunas contra el SIDAS/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios/inmunología , Vagina/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos/inmunología , Cuello del Útero/virología , Femenino , Proteína gp41 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , VIH-1/inmunología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Macaca mulatta , Membrana Mucosa/inmunología , Vacunas contra el SIDAS/administración & dosificación , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Vacunación , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Vagina/virologíaRESUMEN
NK cell responses to HIV/SIV infection have been well studied in acute and chronic infected patients/monkeys, but little is known about NK cells during viral transmission, particularly in mucosal tissues. In this article, we report a systematic study of NK cell responses to high-dose vaginal exposure to SIVmac251 in the rhesus macaque female reproductive tract (FRT). Small numbers of NK cells were recruited into the FRT mucosa following vaginal inoculation. The influx of mucosal NK cells preceded local virus replication and peaked at 1 wk and, thus, was in an appropriate time frame to control an expanding population of infected cells at the portal of entry. However, NK cells were greatly outnumbered by recruited target cells that fuel local virus expansion and were spatially dissociated from SIV RNA+ cells at the major site of expansion of infected founder populations in the transition zone and adjoining endocervix. The number of NK cells in the FRT mucosa decreased rapidly in the second week, while the number of SIV RNA+ cells in the FRT reached its peak. Mucosal NK cells produced IFN-γ and MIP-1α/CCL3 but lacked several markers of activation and cytotoxicity, and this was correlated with inoculum-induced upregulation of the inhibitory ligand HLA-E and downregulation of the activating receptor CD122/IL-2Rß. Examination of SIVΔnef-vaccinated monkeys suggested that recruitment of NK cells to the genital mucosa was not involved in vaccine-induced protection from vaginal challenge. In summary, our results suggest that NK cells play, at most, a limited role in defenses in the FRT against vaginal challenge.
Asunto(s)
Vacunas contra el SIDA/farmacocinética , Inmunidad Mucosa , Células Asesinas Naturales/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios/inmunología , Vagina/inmunología , Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Animales , Femenino , Macaca mulatta , Síndrome de Inmunodeficiencia Adquirida del Simio/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/genética , VacunaciónRESUMEN
Antiretroviral therapy can reduce HIV-1 to undetectable levels in peripheral blood, but the effectiveness of treatment in suppressing replication in lymphoid tissue reservoirs has not been determined. Here we show in lymph node samples obtained before and during 6 mo of treatment that the tissue concentrations of five of the most frequently used antiretroviral drugs are much lower than in peripheral blood. These lower concentrations correlated with continued virus replication measured by the slower decay or increases in the follicular dendritic cell network pool of virions and with detection of viral RNA in productively infected cells. The evidence of persistent replication associated with apparently suboptimal drug concentrations argues for development and evaluation of novel therapeutic strategies that will fully suppress viral replication in lymphatic tissues. These strategies could avert the long-term clinical consequences of chronic immune activation driven directly or indirectly by low-level viral replication to thereby improve immune reconstitution.
Asunto(s)
Fármacos Anti-VIH/farmacocinética , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/fisiología , Tejido Linfoide/metabolismo , Replicación Viral , Adolescente , Adulto , Niño , Preescolar , Femenino , Semivida , Humanos , Masculino , Adulto JovenRESUMEN
Antiretroviral therapy (ART) can reduce HIV levels in plasma to undetectable levels, but rather little is known about the effects of ART outside of the peripheral blood regarding persistent virus production in tissue reservoirs. Understanding the dynamics of ART-induced reductions in viral RNA (vRNA) levels throughout the body is important for the development of strategies to eradicate infectious HIV from patients. Essential to a successful eradication therapy is a component capable of killing persisting HIV infected cells during ART. Therefore, we determined the in vivo efficacy of a targeted cytotoxic therapy to kill infected cells that persist despite long-term ART. For this purpose, we first characterized the impact of ART on HIV RNA levels in multiple organs of bone marrow-liver-thymus (BLT) humanized mice and found that antiretroviral drug penetration and activity was sufficient to reduce, but not eliminate, HIV production in each tissue tested. For targeted cytotoxic killing of these persistent vRNA(+) cells, we treated BLT mice undergoing ART with an HIV-specific immunotoxin. We found that compared to ART alone, this agent profoundly depleted productively infected cells systemically. These results offer proof-of-concept that targeted cytotoxic therapies can be effective components of HIV eradication strategies.
Asunto(s)
Antirretrovirales/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , VIH-1/metabolismo , Inmunotoxinas/farmacología , ARN Viral/sangre , Animales , Antirretrovirales/inmunología , Infecciones por VIH/sangre , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Inmunotoxinas/inmunología , Ratones , Ratones Endogámicos NOD , Ratones SCID , ARN Viral/inmunologíaRESUMEN
One pathological hallmark of HIV-1 infection is chronic activation of the immune system, driven, in part, by increased expression of proinflammatory cytokines. The host attempts to counterbalance this prolonged immune activation through compensatory mediators of immune suppression. We recently identified a gene encoding the proinflammatory cytokine IL-32 in microarray studies of HIV-1 infection in lymphatic tissue (LT) and show in this study that increased expression of IL-32 in both gut and LT of HIV-1-infected individuals may have a heretofore unappreciated role as a mediator of immune suppression. We show that: 1) IL-32 expression is increased in CD4(+) T cells, B cells, macrophages, dendritic cells, and epithelial cells in vivo; 2) IL-32 induces the expression of immunosuppressive molecules IDO and Ig-like transcript 4 in immune cells in vitro; and 3) in vivo, IL-32-associated IDO/Ig-like transcript 4 expression in LT macrophages and gut epithelial cells decreases immune activation but also may impair host defenses, supporting productive viral replication, thereby accounting for the correlation between IL-32 levels and HIV-1 replication in LT. Thus, during HIV-1 infection, we propose that IL-32 moderates chronic immune activation to avert associated immunopathology but at the same time dampens the antiviral immune response and thus paradoxically supports HIV-1 replication and viral persistence.
Asunto(s)
Infecciones por VIH/inmunología , VIH-1/inmunología , Interleucinas/inmunología , Tejido Linfoide/inmunología , Adulto , Femenino , Perfilación de la Expresión Génica , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/genética , VIH-1/fisiología , Interacciones Huésped-Patógeno/inmunología , Humanos , Íleon/inmunología , Íleon/metabolismo , Íleon/virología , Hibridación in Situ , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/virología , Tejido Linfoide/metabolismo , Tejido Linfoide/virología , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Viral/genética , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Recto/inmunología , Recto/metabolismo , Recto/virología , Factores de Tiempo , Replicación Viral/genética , Replicación Viral/inmunologíaRESUMEN
The hallmark of HIV-1 and SIV infections is CD4(+) T cell depletion. Both direct cell killing and indirect mechanisms related to immune activation have been suggested to cause the depletion of T cells. We have now identified a mechanism by which immune activation-induced fibrosis of lymphoid tissues leads to depletion of naive T cells in HIV-1 infected patients and SIV-infected rhesus macaques. The T regulatory cell response to immune activation increased procollagen production and subsequent deposition as fibrils via the TGF-ß1 signaling pathway and chitinase 3-like-1 activity in fibroblasts in lymphoid tissues from patients infected with HIV-1. Collagen deposition restricted T cell access to the survival factor IL-7 on the fibroblastic reticular cell (FRC) network, resulting in apoptosis and depletion of T cells, which, in turn, removed a major source of lymphotoxin-ß, a survival factor for FRCs during SIV infection in rhesus macaques. The resulting loss of FRCs and the loss of IL-7 produced by FRCs may thus perpetuate a vicious cycle of depletion of T cells and the FRC network. Because this process is cumulative, early treatment and antifibrotic therapies may offer approaches to moderate T cell depletion and improve immune reconstitution during HIV-1 infection.
Asunto(s)
Infecciones por VIH/inmunología , VIH-1/genética , Infecciones por Retroviridae/inmunología , Virus de la Inmunodeficiencia de los Simios/metabolismo , Linfocitos T/citología , Adipoquinas , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Proteína 1 Similar a Quitinasa-3 , Fibroblastos/citología , Glicoproteínas/metabolismo , Humanos , Interleucina-7/metabolismo , Lectinas/metabolismo , Linfocitos/citología , Macaca mulatta , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Much effort has been spent recently in identifying host factors required for HIV-1 to effectively replicate in cultured human cells. However, much less is known about the genetic factors in vivo that impact viral replication in lymphatic tissue, the primary anatomical site of virus-host interactions where the bulk of viral replication and pathogenesis occurs. To identify genetic determinants in lymphatic tissue that critically affect HIV-1 replication, we used microarrays to transcriptionally profile and identify host genes expressed in inguinal lymph nodes that were associated determinants of viral load. Strikingly, â¼95% of the transcripts (558) in this data set (592 transcripts total) were negatively associated with HIV-1 replication. Genes in this subset 1) inhibit cellular activation/proliferation (e.g., TCFL5, SOCS5 and SCOS7, KLF10), 2) promote heterochromatin formation (e.g., HIC2, CREBZF, ZNF148/ZBP-89), 3) increase collagen synthesis (e.g., PLOD2, POSTN, CRTAP), and 4) reduce cellular transcription and translation. Potential anti-HIV-1 restriction factors were also identified (e.g., NR3C1, HNRNPU, PACT). Only â¼5% of the transcripts (34) were positively associated with HIV-1 replication. Paradoxically, nearly all of these genes function in innate and adaptive immunity, particularly highlighting heightened gene expression in the IFN system. We conclude that this conventional host response cannot contain HIV-1 replication and, in fact, could well contribute to increased replication through immune activation. More importantly, genes that have a negative association with virus replication point to target cell availability and potentially new viral restriction factors as principal determinants of viral load.
Asunto(s)
Perfilación de la Expresión Génica , Infecciones por VIH/genética , VIH-1/fisiología , Ganglios Linfáticos/virología , Replicación Viral/genética , Adulto , Femenino , Técnica del Anticuerpo Fluorescente , Expresión Génica , Infecciones por VIH/inmunología , Humanos , Ganglios Linfáticos/inmunología , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/análisis , Carga Viral , Replicación Viral/inmunología , Adulto JovenRESUMEN
The enteropathic manifestations of the human immunodeficiency virus (HIV) and the simian immunodeficiency virus (SIV) in late infection are usually due to infection by other microbes, but in early infection the viruses themselves cause an enteropathy by heretofore undetermined mechanisms. Here we report that SIV induces massive apoptosis of intestinal epithelial cells lining the small and large bowel, thus identifying apoptosis as the driving force behind the regenerative pathology of early infection. We found that apoptosis of gut epithelium paralleled the previously documented apoptosis and massive depletion of CD4 T cells in gut lamina propria, triggered by established mechanisms of gut epithelial cell apoptosis and, at peak, possibly by virus interactions with GPR15/Bob, an intestinal epithelial cell-associated alternative coreceptor for SIV and HIV-1. Apoptosis in early SIV infection is thus the common theme of the pathological processes that quickly afflict the innate as well as adaptive arms of the gut immune system.