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

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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


Detectar células infectadas en

latencia

Interferencia con el provirus

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CD4



Núcleo


Citoplasma

CCR5/CXCR4

Ensamblaje

Provirus

ARN viral

Impedir la formación del

reservorio


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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

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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)

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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)

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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

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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

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CD4



Núcleo


Citoplasma

CCR5/CXCR4

Ensamblaje

Provirus

ARN viral



Reactivar el provirus

Silenciar el provirus

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Darcis et al. and van Lint, Trends Immunol. 2017 Mar;38(3):217-228, Univ Bruxelles

PTEFb: processive elongation

Tat: processive elongation

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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

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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

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CD4



Núcleo


Citoplasma

CCR5/CXCR4

Ensamblaje

Provirus

ARN viral



Reactivar el provirus

Silenciar el provirus

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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


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

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• 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

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• 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

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• 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

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• 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

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• 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

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• 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

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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

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CD4



Núcleo


Citoplasma

CCR5/CXCR4

Ensamblaje

Provirus

ARN viral


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


• 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

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• 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

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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

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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.

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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

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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

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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

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Conclusiones

El control a largo plazo de la viremia es consecuencia del equilibrio entre:

• El mantenimiento de los niveles de CD4

• El tamaño del reservorio

• La producción continua de suficientes cantidades de antígeno que generen una respuesta CD8 estable

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