Lo mejor del año en investigación básica2017.congresogesida.es/images/site/ponencias/4... ·...
Transcript of Lo mejor del año en investigación básica2017.congresogesida.es/images/site/ponencias/4... ·...
Lo mejor del año en investigación básica
Mayte Coiras
Unidad de Inmunopatología del Sida
Centro Nacional de Microbiología
Instituto de Salud Carlos III
G E S I D A 2 0 1 7
Estrategias para curar la infección por VIH
1. Erradicación: TMO (paciente de Berlín)
2. Remisión prolongada:
• Tratamiento durante la fase aguda:
• Cohorte VISCONTI: long-term functional remission
• Pacientes de Boston: prolonged remission
• Pacientes controladores
Principal obstáculo: reservorios latentes escondidos en tejidos
G E S I D A 2 0 1 7
CD4
Transcripción reversa
ADN lineal doble cadena
Núcleo
Síntesis de proteínas virales
Citoplasma
CCR5/CXCR4
Ensamblaje
Provirus
ARN viral
Regulación transcripcional
Impedir la formación del
reservorio
Estrategias para curar la infección por VIH
Detectar células infectadas en
latencia
Interferencia con el provirus
G E S I D A 2 0 1 7
CD4
Transcripción reversa
ADN lineal doble cadena
Núcleo
Síntesis de proteínas virales
Citoplasma
CCR5/CXCR4
Ensamblaje
Provirus
ARN viral
Impedir la formación del
reservorio
Estrategias para curar la infección por VIH
G E S I D A 2 0 1 7
Tratamiento temprano
Early start TAR: does it prevent the formation of the reservoir?
Henrich et al., PLoS Med 14 (11): e1002417, Univ California
Early ART is not enough to prevent the formation of the reservoir
A very small number of lingering infected cells is enough to reactivate the infection
G E S I D A 2 0 1 7
Tratamiento temprano
CHER trial: infants with early ART
• DNA PCR positive at 32 days of life
• Initiated ART at 8.5 weeks of life
• Interrupted ART after 40 weeks (age 1 year),
• 3-monthly clinical evaluation
• Undetectable VL after 8.75 years off ART
• First case of sustained virological control from a randomized ART interruption trial following early infant treatment
• CHER trial: 353 children
Host factors, in addition to early ART, likely contribute to long-term remission
Violari et al., IAS 2017 TUPDB0106LB, Baragwanath Hospital (South Africa)
G E S I D A 2 0 1 7
Tratamiento temprano
Early ART preserves immune homeostasis
• Central role of innate lymphoid cells (ILCs): immune regulation and tissue homeostasis
• Human blood ILCs were severely depleted during acute viremic HIV-1 infection
• ILC numbers are not recovered after resolution of peak viremia
• ILC numbers are preserved by ART only if initiated during acute infection
Early ART during acute infection preserves many aspects of the immune system
Kløverpris et al., 2016, Immunity 44, 391–405, University of KwaZulu-Natal (South Africa)
G E S I D A 2 0 1 7
Impedir la formación del reservorio
ccr5 edition by CRISPR/Cas9: induce resistance to infection by HIV in CD4+ T cells
ccr5 gene editing by CRISPR/Cas9 in human HSCs CD34+ significantly reduced viremia and increased CD4+ number in NOD/SCID mice
Xu et al., Mol Ther. 2017 Aug 2;25(8):1782-1789
G E S I D A 2 0 1 7
Impedir la formación del reservorio
Sites of proviral integration: relationship with HIV persistence during ART
Zhyvoloup et al., PLoS Pathog. 2017 Jul 20;13(7):e1006460, University College London
Symons et al., Lewin S, IAS 2017 MOAA0104, University of Melbourne
• Biological meaning of proviral integration?
• Functional connection between HIV-1 integration and T cell activation
• Enrichment of integration in genes upregulated in TCR-activated CD4+ T cells is consistent with latency being established by reversion of activated infected T cells to a resting state
Objective of the integration: to couple the early HIV gene expression with the activation status of the CD4 + T cell
• CA point mutations N74D is independent of Nup358, Nup153, CPSF6 and TNPO3 for infection
• WT HIV-1 integrates more frequently than N74D within or near genes involved in T-cell activation and cell metabolism
A method to assess HIV integration sites
G E S I D A 2 0 1 7
CD4
Transcripción reversa
ADN lineal doble cadena
Núcleo
Síntesis de proteínas virales
Citoplasma
CCR5/CXCR4
Ensamblaje
Provirus
ARN viral
Regulación transcripcional
Estrategias para curar la infección por VIH
Reactivar el provirus
Silenciar el provirus
G E S I D A 2 0 1 7
Regulación transcripcional
Darcis et al. and van Lint, Trends Immunol. 2017 Mar;38(3):217-228, Univ Bruxelles
PTEFb: processive elongation
Tat: processive elongation
G E S I D A 2 0 1 7
Regulación transcripcional
Lücking et al., ChemMedChem 2017, 12, 1776 – 1793, Bayer
BAY 1143572, Atuveciclib:
• Potent, highly selective, oral PTEFb/CDK9 inhibitor
• First selective PTEFb/CDK9 inhibitor that entered clinical evaluation for the treatment of cancer
Elongation interference: potential mechanism to avoid viral replication and reservoir reseeding
G E S I D A 2 0 1 7
Regulación transcripcional
Kessing et al. Valente S, IAS 2017 WEAA0205, Scripps Research institute, CA Mousseau et al., and Valente S, MBio. 2015 Jul 7;6(4):e00465, Scripps Research institute, CA
• 2015: Tat inhibitor didehydro-cortistatin A (dCA):
• Reduces residual levels of Tat-mediated viral transcription in models of HIV latency
• Establishes a nearly permanent state of latency, impeding virus reactivation.
dCA inhibits HIV reactivation after TCR stimulation of CD4+ T cells isolated from virally suppressed subjects
• 2017: dCA
• Ex vivo: prior treatment of CD4+T cells from suppressed infected individuals with dCA significantly reduces viral rebound up to 25 days of treatment interruption
• BLT mice: adding dCA to ART suppressed mice reduced 1.5 to 10.5 fold viral RNA production in all tissues.
dCA may ultimately reduce reservoir replenishment and latent reservoir size
n = 9
G E S I D A 2 0 1 7
CD4
Transcripción reversa
ADN lineal doble cadena
Núcleo
Síntesis de proteínas virales
Citoplasma
CCR5/CXCR4
Ensamblaje
Provirus
ARN viral
Interferencia con el provirus
Estrategias para curar la infección por VIH
Reactivar el provirus
Silenciar el provirus
G E S I D A 2 0 1 7
Interferencia con el provirus
Onafuwa-Nuga & Telesnitsky Microbiol Mol Biol Rev. 2009 Sep;73(3):451-80
Disrupt latency using LRAs CRISPR/Cas9 Gene Editing of the provirus G E S I D A 2 0 1 7
Interferencia con el provirus
Disrupting latency using latency reversing agents (LRAs)
Chomont et al., AIDS. 2017 Nov 10 [Epub ahead of print], Univ Montreal
LRAs during suppressive ART + low CTLs + stable latent HIV reservoir = limited effect on reservoir size and viral rebound
LRAs during ART initiation + normal CTLs + less stable latent HIV reservoir = decrease in reservoir size and delay of viral rebound
G E S I D A 2 0 1 7
Interferencia con el provirus
• Disrupting latency using latency reversing agents (LRAs)
• Combination of more than one LRA: synergy or antagonism?
• LRAs should activate proviral reservoir transcription while avoiding global T-cell activation, which leads to cytokine release and toxicity
López-Huertas et al., Sci Rep. 2017 May 24;7(1):2385, Hosp Ramón y Cajal
HIV-1 replication in rCD4+ T cells treated with MVC alone or with Bryostatin-1
Cell proliferation in rCD4+ T treated with MVC alone or with Bryostatin-1
MVC: new LRA with potency similar as Bryostatin-1
G E S I D A 2 0 1 7
Interferencia con el provirus
• Disrupting latency using latency reversing agents (LRAs)
• LRAs + increased cytotoxicity = reservoir reduction
Lee et al., and Kent S J Virol. 2017 Jul 12;91(15), Univ Melbourne
Panobinostat cannot induce ADCC immediately and it is lost after 2 months
Panobinostat for 8 weeks, followed by ART interruption
for 3 weeks
G E S I D A 2 0 1 7
Interferencia con el provirus
• Disrupting latency using latency reversing agents (LRAs)
• But in vivo LRAs do not reduce the reservoir: because of reseeding?
• Sequencing of independent clonal isolates of replication-competent virus revealed that 57% had env sequences identical to other isolates from the same patient
Hosmane et al., and Siliciano R J Exp Med. 2017 Apr 3;214(4):959-972
A substantial fraction of the latent reservoir arises by cell proliferation of a smaller number of infected cells rather than infection of multiple cells by a dominant viral species
G E S I D A 2 0 1 7
Interferencia con el provirus
• Disrupting latency using latency reversing agents (LRAs)
• But in vivo LRAs do not reduce the reservoir: because of reseeding?
• Measurement of cell-associated HIV RNA (CA RNA) estimates the increase in HIV reactivation rate, but HIV reactivation is only one of the mechanisms of loss and renewal that regulate the reservoir size
Petravic et al., and Lewin S, J Virol. 2017 Apr 13;91(9)
The final reduction of the latent reservoir caused by increasing HIV reactivation can be estimated only when the relative contributions of all other mechanisms are also considered
G E S I D A 2 0 1 7
Interferencia con el provirus
• CRISPR/Cas9 gene editing of the provirus
• Proof of concept: lentiviral-delivered CRISPR/Cas9 removed the entire HIV proviral DNA spanning between the 5’ and 3’LTRs: Kaminski et al., Sci Rep 2016;6:22555
• Major issues:
• Generation of resistance due to mutations at the targeted site that will be no longer recognized by CRISPR/Cas9
• Safer and more effective mechanisms of delivery
• Specific gRNA candidate evaluation for HIV provirus eradication
• How to get access to all tissues and cells potentially harboring the HIV provirus, including hidden reservoirs as the central nervous system?
Soriano V., AIDS Rev. 2017 Oct-Dec;19(3):167-172, Hosp La Paz
Ledford H, Nature. 2016 Mar 10;531(7593):156-9
G E S I D A 2 0 1 7
Interferencia con el provirus
• CRISPR/Cas9 gene editing of the provirus
• Screening method of single guided RNAs (gRNAs) targeting different regions of HIV provirus
Huang & Nair, Sci Rep. 2017 Jul 20;7(1):5955, Univ Florida
gLTR
Control
gLTR and gpol
gLTR and gtat
gnef and gpol
gnef and gtat
Astrocytes
Bright field RFP Bright field RFP
CRISPR/Cas9 research is still in early phases and ongoing
G E S I D A 2 0 1 7
Sensing IFN e integración del provirus
Vermeire et al, 2017 Cell Reports 17, 413–424, Univ Ghent
HIV triggers IFN-I response in CD4+ T cells upon proviral integration
HIV-1-infected CD4+ T cells are potential contributors to IFN-I production and harmful chronic inflammation
• Cytosolic DNA receptor cGAS senses HIV-1 infection and induces type I interferon (IFN-I) production in primary CD4+ T cells
• Effective IFN-I induction requires proviral integration and expression of Vpr
• Vpu efficiently suppresses IFN-I induction in HIV-1-infected CD4+ T cells
G E S I D A 2 0 1 7
CD4
Transcripción reversa
ADN lineal doble cadena
Núcleo
Síntesis de proteínas virales
Citoplasma
CCR5/CXCR4
Ensamblaje
Provirus
ARN viral
Estrategias para curar la infección por VIH
Detectar células infectadas en
latencia
G E S I D A 2 0 1 7
Células que componen el reservorio
• The expression of CD32a, a low-affinity receptor for IgG Fc fragment, is highly induced in the surface of HIV-infected quiescent CD4 T cells
• CD32a expression is not linked to T cell activation mediated by the TCR, suggesting that CD32a induction is dependent on the cell quiescence
Descours et al., Nature. 2017 Mar 23;543(7646):564-567, IGH Montpellier
CD4+ CD32a+ T cells might represent the latently infected cells of the reservoir
n = 12 n = 7
One major barrier to eradication is the infection of multiple cell types that individually contribute to HIV persistence, forming the reservoir G E S I D A 2 0 1 7
Células que componen el reservorio
• Enrichment for HIV DNA in CD32+ CD4+ T cells can be found in tissue and blood of patients treated during primary infection
• There is no correlation between CD32 expression on CD4 T cells and either HIV DNA levels or time to rebound viraemia following treatment interruption
• CD32+ CD4+ T cells have a more differentiated memory phenotype
Martin et al., bioRxiv 169342; IAS 2017 MOAA0106LB, University of Oxford
The enrichment of proviral HIV DNA in CD32+ CD4+ T cells seems to be more related to preferential infection or survival than up-regulation
n = 20
G E S I D A 2 0 1 7
Células que componen el reservorio
• HIV-infected tissue macrophages have a short half-life
• ART alone can significantly reduce the levels of infected tissue macrophages
• Viral rebound can be observed in 33% of the infected, virally suppressed mice: macrophages may represent a persistent viral reservoir for HIV
• Specialized, long-lived macrophages, such as microglia, may long persist in the CNS, contributing to viral persistence over time
Honeycutt et al., and García JV Nat Med. 2017 May;23(5):638-643
Critical contribution of T cells to the viral reservoir
HIV infection of tissue macrophages is rapidly suppressed by ART in humanized myeloid-only mice (MoM).
ART-treated n = 13
Untreated n = 5
G E S I D A 2 0 1 7
Células que componen el reservorio
Banga et al., and Pantaleo G, Nat Med. 2016 Jul;22(7):754-61, University of Lausanne
• Germinal centers within lymph nodes serve as primary sites for HIV-1 replication
• PD-1+ cells are the major CD4 T cell compartment in the blood and lymph nodes for:
• production of replication-competent and infectious HIV-1
• active and persistent virus transcription in long-term-ART treated aviremic individuals
PD-1+ cells may represent a major obstacle to finding a functional cure for HIV-1 infection
Blood
Lymph nodes
G E S I D A 2 0 1 7
Tratamiento con anticuerpos
Nishimura et al., and Martin MA, Nature. 2017 Mar 23;543(7646):559-563, NIH
• Early administration of bNAbs in a SHIV model is associated with very low levels of persistent viraemia
• This leads to the establishment of T-cell immunity and long-term infection control.
G E S I D A 2 0 1 7
Tratamiento con anticuerpos
• Administration of CD8 T-cell-depleting mAb to 6 controller macaques produced a burst of plasma viraemia
• Viraemia declined to baseline in all of the monkeys except one
Passive immunotherapy during acute SHIV infection facilitates a potent CD8+ T-cell immunity able to durably suppress virus replication.
Nishimura et al., and Martin MA, Nature. 2017 Mar 23;543(7646):559-563, NIH
G E S I D A 2 0 1 7
Conclusiones
• TAR temprano es importante para proteger la respuesta inmune pero no es suficiente para impedir la formación del reservorio
• La remisión prolongada en algunos pacientes podría ser dependiente de factores específicos del propio paciente
• La edición génica por CRISPR/Cas9 podría tener aplicaciones tanto en impedir la formación del reservorio (ccr5) y en la eliminación del reservorio (provirus)
• El sitio de integración del provirus es esencial para sincronizar la replicación viral con el estatus de activación de la célula
G E S I D A 2 0 1 7
Conclusiones
• La interferencia en la transcripción y elongación del provirus mediante fármacos específicos (atuveciclib, dCA) impide la replicación viral y el mantenimiento del reservorio
• Continúan los estudios de combinación de distintos LRAs y el análisis de la actividad citotóxica en presencia de los LRAs
• Identificacion de las células que component el reservorio: CD4+ CD32a+, macrófagos, células PD-1+
• La inmunoterapia pasiva contra SHIV con combinaciones de bNAbs induce una potente respuesta CD8 que induce un control prolongado de la viremia
G E S I D A 2 0 1 7