Monique Nijhuis

Current cART strategies

• Current cART is designed to efficiently control HIV replication

• The current arsenal of antiretroviral compounds

affect the steps early in the viral life cycle

• cART can not stop the vicious cycle of

viral production and immune activation

• cART can not eliminate the viral reservoir

Current cART strategies

• Persistence of the viral reservoir and lifelong antiretroviral treatment

• Stop cART: HIV rebound from cellular and/or anatomical reservoirs

1Besson G.J. et al. Clin. Infect. Dis. 2014

Residual replication

Cell-to-cell transfer

Mechanisms of persistence in presence of cART

Long-lived cells

Homeostatic proliferation

Clonal expansion

† †

Passegué, E et al, PNAS, 2003; Barton, K et al, Trends in Microbiology, 2016

The complex nature of HIV reservoirs

Measuring HIV reservoir during cART

• 1 HIV DNA copy per 1,000 CD4 T cells; 1 replication competent virus per million CD4 T cells

Deeks et al, Nature Medicine, 2016

HIV cure strategies

Deeks et al, Nature Medicine, 2016

HIV cure strategies

• • Early HIV therapy

• Anti-latency strategies (kick and kill strategy)

• Stem cell transplantation (CCR5-Δ32/Δ32 or CCR5-WT/WT)

• Gene Therapy (deletion of CCR5 or deletion of HIV)

Deeks et al, Nature Medicine, 2016

Early HIV therapy

Saez Cirion A, et al. Plos Pathogens 2013

• Visconti cohort:

– 14 post treatment controllers (PTCs) were identified in 10 years

– Treated during the acute phase of infection (fiebig I-V)

– Treated for 3 years before therapy was stopped

– No viral rebound after antiretroviral treatment interruption (ATI)

– HIV can be cultured from the cells of the patients: no viral cure

Early HIV therapy

Ananworanich, CROI 2017

• Thai Red Cross study:

– 8 patients identified in fiebig I and treated for >2 years before ATI

– Extremely small HIV reservoir

Early HIV therapy and induced remission of HIV infection

Ananworanich, CROI 2017

Broad application of strategy I: Early HIV therapy

• Most patients are not diagnosed during the acute phase

• No cure

• Post treatment control is only seen in a subgroup of patients

• Early treatment has a favourable effect on the size of the reservoir:

• Ideal candidates for other cure strategies

Anti-latency strategies (kick and kill strategy)

activator

• Protect uninfected cells with cART

• Activate latently infected cells (Latency Reserving Agents, LRAs)

• Activated cells that produces virus can be recognised by cytotoxic T cells

Anti-latency strategies (kick and kill strategy)

• Latency Reserving Agents in clinical studies, LRAs:

• Chromatin environment

• HDAC inhibitors (vorinostat, panobinostat, romidepsin)

• None of the studies demonstrated a significant reduction in the

frequency of infected cells as measured by HIV DNA or quantitative

viral outgrowth assay (QVOA)

Søgaard OS et al, PLOS Pathogens, 2015

Anti-latency strategies (kick and kill strategy)

• New classes LRAs and strategies to boost the immune system:

• NF-κB activation (PKC- agonists, TLR agonist, Maraviroc)

• positive transcription elongation factor b (PTEF-b)

• Chromatin remodelling (BAF inhibitors)

• bnABs

• Therapeutic vaccines

• Study using patients treated during acute infection, being vaccinated

and now receiving again vaccination and the HDACi Romidepsin

Mothe et al, CROI, 2017

Broad application of strategy II: Anti-latency strategies (kick and kill strategy)

• Experimental phase, small clinical studies, follow-up is limited

• No cure: Post treatment control is observed in a few patients

• Combination of different classes of LRAs and immune therapy are needed

• Major safety, toxicity concerns, especially in the central nervous system

• More specific compounds are needed

• Berlin Patient: transplanted CCR5𝚫32/𝚫32 donor cells1

-Viruses in the patient are dependent on CCR5 for replication2

-Off cART since 2007 -Only person ever cured of HIV

• Essen patient: transplanted CCR5𝚫32/𝚫32 donor cells3

-Successful engraftment -Treatment interruption 7 days before transplantation -Rebound of pre-existing CXCR4-tropic virus

• Boston Patients: transplanted with CCR5WT donor cells4

-Successful engraftment, GVHD -No HIV detected in blood and rectal mucosa -Treatment was interrupted (ATI) -Viral rebound was observed after 12, 32 weeks

-

1Hutter et al, NEJM, 2008; 2Symons et al, CID, 2014 ; Kordelas et al, NEJM, 2014; Henrich et al, Ann. Intern. Med., 2014

Stem cell transplantation (CCR5Δ32/Δ32 or CCR5WT/WT)

• Which factors contributed to the cure of the Berlin Patient? • CCR5Δ32Δ32 donor cells • Absence of CXCR4 tropic viruses • Immune suppressive treatment (ATG levels) • Graft Versus Host Disease • Total body irradiation • Patient was CCR5 heterozygote

• Were was the virus hiding in the Boston Patients? • Blood vs tissue • Biomarker for the size of the reservoir (viral, immunological marker) • Biomarker for the rebound of HIV after ATI

-

Stem cell transplantation (CCR5Δ32/Δ32 or CCR5WT/WT)

Stem cell transplantation (CCR5Δ32/Δ32 or CCR5WT/WT) IciStem consortium

International collaboration to guide and investigate the potential for HIV cure in HIV-infected

patients requiring allogeneic stem cell transplantation for hematological disorders

AIM 1

To guide clinicians involved in allogeneic SCT

procedures in HIV infected individuals

AIM 2

To better understand the underlying biological

processes leading to viral reservoir reduction

and potential cases of HIV-1

eradication/remission.

www.icistem.org

Principal Investigators:

Javier Martinez Picado

Annemarie Wensing

Stem cell transplantation (CCR5Δ32/Δ32 or CCR5WT/WT) IciStem consortium

• 29 patients registered from 8 different countries

• 22 patients transplanted

• Mean follow-up: 630 days -12 patients alive in active follow-up -7 patients beyond 2nd year post-SCT -all patients are still on cART

Patient 5

100

200

300

600

500

400

Weeks

-3

Pro

vira

l D

NA

(co

pie

s/m

illio

n P

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-2 -1 0 1 2 3 4 5 6 7 8 9 10

1000

700

800

900

a n t i r e t r o v i r a l t h e r a p y

Donor engraftment

SCT

(CCR5Δ32/Δ32) 100%

274

27 22 18

45

22

0

20

40

260

280

terminalileum

spleen lung (right)HIV

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mill

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Patient 5: HIV DNA in post-mortem biopsies

Death

Stem cell transplantation (CCR5Δ32/Δ32 or CCR5WT/WT) IciStem consortium

Broad application of strategy III: Stem cell transplantation

• Only HIV cure so far

• Stem cell transplantation has a high mortality risk

• Only suitable for patients with a haematological malignancy

• Will provide insight in the viral reservoir dynamics and characteristics

Gene Therapy

• Tools for gene-editing:

• Recombinase1

• Nucleases (molecular scissors)

• zinc finger nucleases (ZFNs)2

• transcription activator-like effector nucleases (TALENs)3

• clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 nuclease4-6

• Target CCR5 (HIV co-receptor), prevent infection of new cells:

• -cleavage of CCR5 with ZFNs may result in viral control7

• Target HIV, delete the viral reservoir and prevent infection of new cells

1Karpinski J et al, Nat Biotechn, 2016; 2Urnov FD et al, Nature, 2005; 3Mahfouz MM et al, PNAS, 2011; 4Horvath P et al, Science, 2010; 5Doudna J et al, Science, 2014; 6Mali P et al, Science, 2013; 7Tebas et al, NEJM, 2014

Gene Therapy

Repair by the error-prone

non-homologous end joining

(NHEJ) machinery.

Gene Therapy

• Target HIV: delete/destroy the viral reservoir

Methods: • T-cell line latently infected with HIV-GFP reporter virus (JLAT-FL) • Transduced with the CRISPR/Cas9 and gRNAs (single or

combination)

LTR6 frequency (%)J.Lat FL T G T A C T G G G T C T C T C T G G T T A G A C C A G A T C T G A G C C T G G G A G C T C T C T G G C

variant 1 32 . . . . . . . . . . . . . . . . . C G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

variant 2 8.6 . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

variant 3 7.7 . . . . . . . . . . . . . . . . . . A C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

variant 4 2.7 . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .variant 5 2.2 . . . . . . . . . . . . . . . . . . T G T G - - - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .variant 6 2 . . . . . . . . . . . . . . . . . . A G C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

MA3 frequency (%)J.Lat FL A G C G T C A G T A T T A A G C G G G G G A G A A T T A G A T C G A T G G G A A A A A A T T C G G Tvariant 1 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - - . . . . . . . . . . . . . . .variant 2 1.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T C G A T G - - - - . . . . . . . . . . . . . .variant 3 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - - - - - - - . . . . . . . . . . . . . - .

IN5 frequency (%)J.Lat FL G A C A A T G G C A G C A A T T T C A C C A G T G C T A C G G T T A A G G C C G G C C G C C T G T T G

variant 1 2.9 . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . .

variant 2 2.5 . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . .variant 3 2.2 . . . . . . . . . . . . . . . . . . . . . . . . . G T T T - - - - - - - - - - - . . . . . . . . . . . . . . .variant 4 2.1 . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . .

variant 5 1.9 . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . .variant 6 1.6 . . . . . . . . . . . . . . . . . . . . . . . . - - - - - - - - - - - - . . . . . . . . . . . . . . . . . . .

2 gRNAs:

>98% loss of HIV reactivation

Lebbink et al, Nature Scientific Reports, 2017

Gene Therapy

• Target HIV: delete/destroy the viral reservoir

• Two gRNAs: >98% loss of HIV reactivation • In line with current literature1,2,3,4,5

• Target HIV: Prevent infection of new cells? • Inhibit short-term HIV replication (2-4 days)2,4 • Long-term viral escape?

1Ebina et al, Sci Rep, 2013; 2Hu et al, PNAS, 2014; 3Zhu et al, Retrovirology, 2015; 4Liao et al, Nat Commun, 2015; 5Kaminski et al, Sci Rep, 2016.

Gene Therapy

• Generated T-cells stably expressing single gRNAs and Cas9 • Infection with HIV reporter virus (luciferase) (n=4)

• Amplified and deep-sequenced the CRISPR/Cas9 target regions of the viral RNA from the culture supernatants: RT2: wt virus; other gRNAs: escape

Viral breakthrough/escape

variants?

Lebbink et al, Nature Scientific Reports, 2017

Gene Therapy

• `

• These observations have questioned the feasibility of the CRISPR/Cas9 based gene-editing approach1,2

1Callaway, Nature News, 2016; 2Liang et al, Retrovirology, 2016.

Gene Therapy

• Generated T-cells stably expressing two gRNAs and Cas9

• Infection with HIV reporter virus (luciferase) (n=4)

• Combining two potent gRNAs can successfully prevent viral replication and

escape

Lebbink et al, Nature Scientific Reports, 2017

Broad application of strategy IV: Gene therapy

• No cure, one case of viral control after CCR5 editing

• Delivery of gene therapy

• Specificity, off-targeting

Conclusion

• One person has been cured of HIV infection and we have seen several remissions

• Cure strategies (will) focus on combination of several approaches

• If you don't try you'll never know and it will always seem impossible and out of reach.1

1 Nelson Mandela

Final remarks

Acknowledgement

Translational Virology, UMCU

Virology, UMCU Emmanuel Wiertz

Robert Jan Lebbink

Acknowledgement