RESUMEN
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Human immunodeficiency virus (HIV) persists indefinitely in individuals with HIV who receive antiretroviral therapy (ART) owing to a reservoir of latently infected cells that contain replication-competent virus1-4. Here, to better understand the mechanisms responsible for latency persistence and reversal, we used the interleukin-15 superagonist N-803 in conjunction with the depletion of CD8+ lymphocytes in ART-treated macaques infected with simian immunodeficiency virus (SIV). Although N-803 alone did not reactivate virus production, its administration after the depletion of CD8+ lymphocytes in conjunction with ART treatment induced robust and persistent reactivation of the virus in vivo. We found viraemia of more than 60 copies per ml in all macaques (n = 14; 100%) and in 41 out of a total of 56 samples (73.2%) that were collected each week after N-803 administration. Notably, concordant results were obtained in ART-treated HIV-infected humanized mice. In addition, we observed that co-culture with CD8+ T cells blocked the in vitro latency-reversing effect of N-803 on primary human CD4+ T cells that were latently infected with HIV. These results advance our understanding of the mechanisms responsible for latency reversal and lentivirus reactivation during ART-suppressed infection.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Interleucina-15/agonistas , Virus de la Inmunodeficiencia de los Simios/fisiología , Replicación Viral , Animales , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Humanos , Interleucina-15/inmunología , Depleción Linfocítica , Macaca mulatta , Ratones , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Latencia del Virus , Replicación Viral/inmunologíaRESUMEN
Vaginal HIV transmission accounts for the majority of new infections worldwide. Currently, multiple efforts to prevent HIV transmission are based on pre-exposure prophylaxis with various antiretroviral drugs. Here, we describe two novel nanoformulations of the reverse transcriptase inhibitor rilpivirine for pericoital and coitus-independent HIV prevention. Topically applied rilpivirine, encapsulated in PLGA nanoparticles, was delivered in a thermosensitive gel, which becomes solid at body temperature. PLGA nanoparticles with encapsulated rilpivirine coated the reproductive tract and offered significant protection to BLT humanized mice from a vaginal high-dose HIV-1 challenge. A different nanosuspension of crystalline rilpivirine (RPV LA), administered intramuscularly, protected BLT mice from a single vaginal high-dose HIV-1 challenge one week after drug administration. Using transmitted/founder viruses, which were previously shown to establish de novo infection in humans, we demonstrated that RPV LA offers significant protection from two consecutive high-dose HIV-1 challenges one and four weeks after drug administration. In this experiment, we also showed that, in certain cases, even in the presence of drug, HIV infection could occur without overt or detectable systemic replication until levels of drug were reduced. We also showed that infection in the presence of drug can result in acquisition of multiple viruses after subsequent exposures. These observations have important implications for the implementation of long-acting antiretroviral formulations for HIV prevention. They provide first evidence that occult infections can occur, despite the presence of sustained levels of antiretroviral drugs. Together, our results demonstrate that topically- or systemically administered rilpivirine offers significant coitus-dependent or coitus-independent protection from HIV infection.
Asunto(s)
Infecciones por VIH/prevención & control , Rilpivirina/administración & dosificación , Animales , Fármacos Anti-VIH/administración & dosificación , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Infecciones por VIH/transmisión , Células HeLa , Humanos , Ratones , Nanopartículas/administración & dosificación , Cremas, Espumas y Geles Vaginales/farmacologíaRESUMEN
BACKGROUND: The latent reservoir in resting CD4(+) T cells presents a major barrier to HIV cure. Latency-reversing agents are therefore being developed with the ultimate goal of disrupting the latent state, resulting in induction of HIV expression and clearance of infected cells. Histone deacetylase inhibitors (HDACi) have received a significant amount of attention for their potential as latency-reversing agents. RESULTS: Here, we have investigated the in vitro and systemic in vivo effect of panobinostat, a clinically relevant HDACi, on HIV latency. We showed that panobinostat induces histone acetylation in human PBMCs. Further, we showed that panobinostat induced HIV RNA expression and allowed the outgrowth of replication-competent virus ex vivo from resting CD4(+) T cells of HIV-infected patients on suppressive antiretroviral therapy (ART). Next, we demonstrated that panobinostat induced systemic histone acetylation in vivo in the tissues of BLT humanized mice. Finally, in HIV-infected, ART-suppressed BLT mice, we evaluated the effect of panobinostat on systemic cell-associated HIV RNA and DNA levels and the total frequency of latently infected resting CD4(+) T cells. Our data indicate that panobinostat treatment resulted in systemic increases in cellular levels of histone acetylation, a key biomarker for in vivo activity. However, panobinostat did not affect the levels of cell-associated HIV RNA, HIV DNA, or latently infected resting CD4(+) T cells. CONCLUSION: We have demonstrated robust levels of systemic histone acetylation after panobinostat treatment of BLT humanized mice; and we did not observe a detectable change in the levels of cell-associated HIV RNA, HIV DNA, or latently infected resting CD4(+) T cells in HIV-infected, ART-suppressed BLT mice. These results are consistent with the modest effects noted in vitro and suggest that combination therapies may be necessary to reverse latency and enable clearance. Animal models will contribute to the progress towards an HIV cure.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Linfocitos T CD4-Positivos/virología , ADN Viral/metabolismo , VIH-1/efectos de los fármacos , Ácidos Hidroxámicos/uso terapéutico , Indoles/uso terapéutico , ARN Viral/metabolismo , Latencia del Virus/efectos de los fármacos , Acetilación , Animales , Fármacos Anti-VIH/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/genética , VIH-1/fisiología , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Indoles/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Ratones , Ratones Transgénicos , Panobinostat , ARN Viral/sangre , Activación Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Autologous induced pluripotent stem cells (iPSCs) constitute an unlimited cell source for patient-specific cell-based organ repair strategies. However, their generation and subsequent differentiation into specific cells or tissues entail cell line-specific manufacturing challenges and form a lengthy process that precludes acute treatment modalities. These shortcomings could be overcome by using prefabricated allogeneic cell or tissue products, but the vigorous immune response against histo-incompatible cells has prevented the successful implementation of this approach. Here we show that both mouse and human iPSCs lose their immunogenicity when major histocompatibility complex (MHC) class I and II genes are inactivated and CD47 is over-expressed. These hypoimmunogenic iPSCs retain their pluripotent stem cell potential and differentiation capacity. Endothelial cells, smooth muscle cells, and cardiomyocytes derived from hypoimmunogenic mouse or human iPSCs reliably evade immune rejection in fully MHC-mismatched allogeneic recipients and survive long-term without the use of immunosuppression. These findings suggest that hypoimmunogenic cell grafts can be engineered for universal transplantation.
Asunto(s)
Diferenciación Celular/inmunología , Rechazo de Injerto/inmunología , Antígenos HLA/genética , Células Madre Pluripotentes Inducidas/trasplante , Animales , Diferenciación Celular/genética , Rechazo de Injerto/genética , Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Ratones , Miocitos Cardíacos/química , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Trasplante Homólogo/métodosRESUMEN
The human brain is an important site of HIV replication and persistence during antiretroviral therapy (ART). Direct evaluation of HIV infection in the brains of otherwise healthy individuals is not feasible; therefore, we performed a large-scale study of bone marrow/liver/thymus (BLT) humanized mice as an in vivo model to study HIV infection in the brain. Human immune cells, including CD4+ T cells and macrophages, were present throughout the BLT mouse brain. HIV DNA, HIV RNA, and/or p24+ cells were observed in the brains of HIV-infected animals, regardless of the HIV isolate used. HIV infection resulted in decreased numbers of CD4+ T cells, increased numbers of CD8+ T cells, and a decreased CD4+/CD8+ T cell ratio in the brain. Using humanized T cell-only mice (ToM), we demonstrated that T cells establish and maintain HIV infection of the brain in the complete absence of human myeloid cells. HIV infection of ToM resulted in CD4+ T cell depletion and a reduced CD4+/CD8+ T cell ratio. ART significantly reduced HIV levels in the BLT mouse brain, and the immune cell populations present were indistinguishable from those of uninfected controls, which demonstrated the effectiveness of ART in controlling HIV replication in the CNS and returning cellular homeostasis to a pre-HIV state.
Asunto(s)
Encéfalo/inmunología , Encéfalo/virología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Linfocitos T/inmunología , Animales , Fármacos Anti-VIH/farmacología , Encéfalo/patología , ADN Viral/genética , ADN Viral/metabolismo , Modelos Animales de Enfermedad , Femenino , Infecciones por VIH/tratamiento farmacológico , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , Células Mieloides/inmunología , Células Mieloides/patología , Células Mieloides/virología , ARN Viral/genética , ARN Viral/metabolismo , Linfocitos T/patología , Linfocitos T/virologíaRESUMEN
Despite years of fully suppressive antiretroviral therapy (ART), HIV persists in its hosts and is never eradicated. One major barrier to eradication is that the virus infects multiple cell types that may individually contribute to HIV persistence. Tissue macrophages are critical contributors to HIV pathogenesis; however, their specific role in HIV persistence during long-term suppressive ART has not been established. Using humanized myeloid-only mice (MoM), we demonstrate that HIV infection of tissue macrophages is rapidly suppressed by ART, as reflected by a rapid drop in plasma viral load and a dramatic decrease in the levels of cell-associated viral RNA and DNA. No viral rebound was observed in the plasma of 67% of the ART-treated animals at 7 weeks after ART interruption, and no replication-competent virus was rescued from the tissue macrophages obtained from these animals. In contrast, in a subset of animals (â¼33%), a delayed viral rebound was observed that is consistent with the establishment of persistent infection in tissue macrophages. These observations represent the first direct evidence, to our knowledge, of HIV persistence in tissue macrophages in vivo.
Asunto(s)
Infecciones por VIH/virología , VIH-1/fisiología , Macrófagos/virología , Animales , Fármacos Anti-VIH/uso terapéutico , Terapia Antirretroviral Altamente Activa , Médula Ósea , ADN Viral , Electroforesis en Gel de Campo Pulsado , Infecciones por VIH/tratamiento farmacológico , VIH-1/genética , Trasplante de Células Madre Hematopoyéticas , Humanos , Inmunohistoquímica , Lactonas , Leucocitos Mononucleares , Hígado , Macrófagos Alveolares/virología , Ratones , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Fenoles , ARN Viral , Bazo , Linfocitos T , Carga Viral , Latencia del Virus , Replicación ViralRESUMEN
Novel therapeutic strategies are needed for the treatment of hematologic malignancies; and bispecific antibody-derived molecules, such as dual-affinity re-targeting (DART) proteins, are being developed to redirect T cells to kill target cells expressing tumor or viral antigens. Here we present our findings of specific and systemic human B-cell depletion by a CD19xCD3 DART protein in humanized BLT mice. Administration of the CD19xCD3 DART protein resulted in a dramatic sustained depletion of human CD19(+) B cells from the peripheral blood, as well as a dramatic systemic reduction of human CD19(+) B-cell levels in all tissues (bone marrow, spleen, liver, lung) analyzed. When human CD8(+) T cells were depleted from the mice, no significant B-cell depletion was observed in response to CD19xCD3 DART protein treatment, confirming that human CD8(+) T cells are the primary effector cells in this in vivo model. These studies validate the use of BLT humanized mice for the in vivo evaluation and preclinical development of bispecific molecules that redirect human T cells to selectively deplete target cells.