Immunological response after stopping NUCs

Andre Boonstra, PhD

Erasmus Medical Center

Rotterdam, the Netherlands

Nov 6, 2019

Disclosures

Research funding: Gilead, Roche, Fujirebio, Viroclinics, Janssen

www.escalon.eu

Host mechanisms that promote persistence of HBV in the liver

Regulatory T cells

IL-10 and/or TGF-β

Regulatory B cells

Active elimination of T cells

Impaired NK cells

Myeloid derived suppressor cells

Impaired dendritic cells

High viral load leading to T cell exhaustion

Mitochondrial dysfunction

Cellular Immunityto hepatitis

IT IA IC ENEG

HBV DNA

ALT

EntecavirTenofovir

HBV DNA

weeks months years

Time

100

50

0

Inc

rea

se

of

ma

xim

um

(%)

Initiation of

therapy

Efficient HBV DNA decline on NUC treatment

Typical course of events after NUC discontinuation

What causes or is the consequence of the virological relapse?

What causes or is the consequence of the clinical relapse?

What makes that some patients experience HBsAg loss?

Can we predict the outcome of NUC stopping?

What do we want to understand?

2016 2018

2018

15 HBeAg-negative patients on NUCExclusion: co-infections, cirrhosis, HCC etcHBV genotypes: B (n=3), C (n=1), D (n=10) Medium age: 49

Retreatment in case of a virological relapse

Study design

13 out of 15 patients relapsed with HBV DNA peak at week 8,

and ALT peak at week 12

Honer zu Siederdissen, JID 2016

Global, moderate increase of serum cytokines after NUC cessation

Honer zu Siederdissen, JID 2016

- Increased levels already at week 4

- Statistical significance found for IL-12p70, IL-10, TNF (week 4) and IP-10 (week 8)

Significantly augmented levels of IL-10, IL-12p70, TNF after 4 weeks,

and IP-10 after 12 weeks

Honer zu Siederdissen, JID 2016

NUC STOP has no effect on NK cell frequencies in blood

Zimmer, JID 2018

NUC STOP has minimal effect on NK cell phenotype

Zimmer, JID 2018

CD56-dim

Increased CD56dim NK cell functionality after stopping NUC treatment was

associated with hepatocyte damage

Zimmer, JID 2018

T cell phenotype was only slightly changed after NA discontinuation

Rinker, J Hepatol 2018

Minor, albeit significant, changes NUC STOP were observed for

PD-1 on CD4+ and CD8+ T cells,

TCF-1 on CD4+,

CD161 on CD8+ T cells.

PD-L1 blockade can enhance the core-specific T cell responses

NUC discontinuation resulted in a higher responsiveness to in vitro

peptide stimulation of HBV core-specific blood CD4+ and CD8+ T cells

Rinker, J Hepatol 2018

Heterogenous patterns of core-specific IFNg responses by CD4+ cells after

NUC STOP

Rinker, J Hepatol 2018

2018

Cohort 1: 29 HBeAg-neg, mixedCohort 2: 27 HBeAg-neg, Asian

- Identify immunological biomarkers to predict the occurence of flares after NUC STOP

13 non-flares, 6 flares (2xULN)

Prior to NUC STOP, patients who eventually do not flare have

increased HBV-specific T cell frequencies

core polym.

10 day cultures with 15-mer pools Rivino, JCI 2018

Functional HBV-spec T cells are enriched in the PD1+ T cell

fraction

Nanostring and CyTOF analysis

Rivino, JCI 2018

- PD1 is highest in non-flare HBV-spec CD8

- Core/polym spec T are enriched for PD1

- CD8+PD1+ cells from patients that do not flare

had higher cytokine producing capacity

Non cirrhotic CaucasiansHBeAg-negative

Microarray on PBMC of 3 NUC STOP in remission3 NUC STOP relapse5 chronic HBV patients (IC)

361 DEG

21 immune genes of cytokine-cytokine pathway

Validation by qPCR on 7 NUC STOP remission10 NUC STOP relapse4 chronic HBV

Low expression of IFNg, IL-8, FASLG, CCL4associated with off-treatment remission

Kranidioti, JVH 2019

qPCR

Differentially expressed genes by PCR of PBMC at baseline

between patients in remission versus relapse

Kranidioti, JVH 2019

Asian cohortN=100

39 clinical relapsers6 seroclearance

A high anti-HBc levels at time of NUC STOP is associated with

lower risk of clinical relapse

Chi, 2019

Patients with a clincal relapse show an increase of anti-HBc levels

Chi, 2019

Recap

More pronounced effects are seen with ALT flare, less pronouced with virological

relapse

All studies have been done in peripheral compartment, not liver

After NUC discontinuation the studies point towards involvement during relapse of

- increase of serum cytokines (IL-12p70, IL-10, TNF, IP-10)

- Freq of IFNg+ NK dim cells associate with ALT

- Increase of core/polym specific T cells, but lower than in non-flare patients

- Increase of anti-HBcore levels

Baseline parameters to predict outcome after NUC discontinuation

- no flare: higher baseline freq of core/polym specific T cells (PD-1+),

>40 SPC/105 cells for core/polym T cells, then no flare

- lower risk of relapse: higher baseline anti-Hbcore levels

- remission: lower ccl4, il8, faslg, ifng (full blood qPCR)

Recap

Validation

Larger cohorts

also especially to address the s-loss question

More mechanistic studies

what causes the flares – TRAIL-expressing NK cells?

what triggers immune control?

what is the involvement of non-HBV T cells?

Monitor the liver (fine-needle aspirates)

Conclusions

technicians:

Anthonie Groothuismink

Gertine van Oord

Kubra Koten

Project managers:

Judith Verhagen

Angela Bastidas Coral

post-docs:

Thomas Vanwolleghem (hepatologist)

José Debes (hepatologist)

PhD students:

Lauke Boeijen

Gulce Sari

Boris Beudeker

Ruben Hoogeveen

Noe Rico Montanari

Immunology of Viral Hepatitis, Dept of Gastroenterology & Hepatology, Erasmus MC,

University Medical Centre Rotterdam, The Netherlands