INVESTIGACION BASICA: curación del VIH

POST-CROI2020

Javier Martínez-PicadoICREA Research Professor at IrsiCaixa

Associate Professor Uvic-UCC

An update on the 27th Conference onRetrovirus and Opportunistic Infections

March 8-11

Gupta et al. 2020. Lancet HIV & CROI #346LB

Estimated probability of long-termremission in the London patient

Image: Toni Kelleher

Nixon et al. 2020 Nature; McBrien et al. 2020. Nature

Figure: Lichertfeld 2020 Nature

Reactivation of latent virus in animals

McBrien et al. 2020. Nature

N-803 IL-15 superagonist + CD8 depletion

Intravenousadministration

SIV and HIV reactivation after CD8 depletion combined with N-803 treatment

CD8 depletion combined with N-803 administration does not decrease thesize of the latent SIV viral reservoir

Nixon et al. 2020 Nature

AZD5582 reverses viral latency in vivovia non-canonical NF-κB signalling

Nature | www.nature.com | 3

activat ion and inflammation21 (Supplementary Table 6). Together, these

results demonstrate that AZD5582 does not cause generalized toxicity

or activation of the immune system in the BLT model.

Latency reversal in rhesus macaques

We next evaluated the latency-reversal act ivit y of AZD5582 in 21

MamuB*08− and MamuB*17− rhesus macaques infected with SIVmac239

and t reated with a pot ent ART regimen comprising t enofovir diso-

proxil fumarate, emtricitabine and dolutegravir init iated 8 weeks after

infect ion (Fig. 3a and Supplementar y Table 7)22,23. Suppression of SIV

viraemia below 60  copies per ml (standard assay limit of detect ion)

was achieved in all macaques in 2–20  weeks and ART was cont inued for

55–67 weeks before fur ther t reatment (Fig. 3b). On the basis of phar-

macokinet ic and pharmacodynamic data from uninfected macaques

(Extended Data Fig. 4a) as well as protocols for SMAC mimet ics used in

oncology, int ravenous infusions o f 0 .1 mg kg−1 AZD5582 were admin-

istered weekly t o 12 SIV-infected ART-suppressed rhesus macaques

for 3 or 10  weeks (Fig. 3a). Nine SIV-infected ART-suppressed rhesus

macaques served as cont rols (Fig. 3a). Plasma concent rat ions of

AZD5582 measured after the f irst , third, sixth and tenth dose showed

that drug exposures in SIV-infected ART-suppressed macaques were

consistent across the t reatment per iod and comparable to t hose

observed in uninfected rhesus macaques (Extended Data Fig. 4b).

Latency reversal, def ined as on-ART viraemia increasing from less

than 60  copies per ml of plasma to more than 60  copies per ml of

plasma after AZD5582 t reatment , was observed as ear ly as 96 h after

the f irst dose and reached levels as high as 1,390  copies per ml in SIV-

infected rhesus macaques (Fig. 3c, d). On-ART viraemia >60  copies per

ml of plasma was observed in 5 out of 12 rhesus macaques (42%), cor-

responding to 5 out of 9 rhesus macaques (55%) that received 10  doses

of AZD5582 (Fig. 3c, d). Mult iple instances of sustained viraemia >60

copies per ml between AZD5582 doses were observed. Out of 140 viral

load measurements performed on the 5 macaques that exhibited on-

ART viraemia during AZD5582 t reatment , 64 were >60 copies per ml

(46%); in the macaque with the greatest frequency of react ivat ion, this

proport ion was 15 out of 28 (53%). Longitudinal examinat ion of plasma

virus by single-genome sequencing analysis of the SIVmac239 env gene

in all rhesus macaques that experienced AZD5582-induced on-ART

viraemia was performed at four selected t ime points: 2 weeks after

infect ion (near peak viraemia), 8 weeks after infect ion (immediately

before ART init iat ion), and at 2 t ime points s eparated by 26–42 days

during AZD5582 t reatment . Phylogenet ic analyses showed several

pat terns of virus react ivat ion (Extended Data Fig. 5). In two rhesus

macaques (RDl16 and RKn16), most of the react ivated virus sequences

were phylogenet ically closer to sequences at eight  weeks after infect ion

rather than peak viraemia and were unique, indicat ing that the variants

produced during AZD5582 t reatment or iginated from mult iple cells

that were seeded at the t ime o f ART init iat ion24. In two other rhesus

macaques (RKl16 and RDm16), a large fract ion of the viruses produced

dur ing AZD5582 t reatment showed ident ical sequences, suggest ing

that latency reversal occurred from a single cell or a clonally expanded

populat ion of infected cells. These clones clustered with both peak

and pre-ART t ime points and were accompanied by addit ional unique

sequences. In one rhesus macaque (RLy15), a single virus sequence was

amplif ied at each t ime point during AZD5582 t reatment and these were

both phylogenet ically similar to sequences found before ART treat-

ment . Taken together, these results indicate that AZD5582 induced

virus react ivat ion from a diverse populat ion of cells, some of which

may be clonally expanded25,26.

We quant if ied cell-associated SIV RNA and SIV DNA in rest ing CD4+

T cells sor ted f rom SIV-infected ART-suppressed rhesus macaques

t reated or not with AZD5582. Cell-associated SIV RNA levels in rest ing

CD4+ T cells isolat ed from lymph nodes were signif icant ly higher in

macaques who received ten doses of AZD5582 compared with controls

(P = 0.0148) (Fig. 3e). A similar t rend was observed in rest ing CD4+ T cells

isolated from the spleens of a subgroup of six macaques that were

euthanized. Levels of cell-associated SIV DNA in rest ing CD4 + T cells

were similar in each compartment across groups (Fig. 3e). To fur ther

understand whether latency reversal induced by AZD5582 resulted in a

per turbat ion of the overall level of infected CD4+ T cells, we performed

longitudinal measurements of cell-associated SIV DNA in total (rather

than rest ing) CD4+ T cells isolated f rom lymph nodes and blood as

well as quant itat ive viral outgrowth assays using CD4+ T cells f rom

lymph nodes and spleen at the end of the t reatment period (Extended

b

c

0 24 48

Control (n = 6) AZD5582 (n = 6) Control (n = 4)

Time after treatment (h )

AZD5582 (n = 4)

dP = 0.0145P = 0.0201 P = 0.0004P = 0.0038 P = 0.0029P = 0.0426

BM Org LN Spleen Liver Lung

a

Rela

tive g

ag

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Expose BLT mice

to HIV-1 JR-CSF

Inject AZD5582 (3 mg kg –1)

or control vehicle

Bleed to monito r

plasma viral load

0 h 24 h 48 h

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Time (week after expsour e)

PBMCs

Co

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Female reproductive tract Brain

P = 0.3095P = 0.0152 P = 0.0147

HIV-1 (TDF, FTC, RAL)HIV-1 JR-CSF

101

103

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control(n = 6)

0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 1214

ART

AZD5582(n = 6)

ARTcontrol(n = 4)

ARTAZD5582

(n = 4)

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Control AZD Control AZD Control AZD Control AZD Control AZD Control AZD

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Control AZD Control AZD Control AZD

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Fig. 2 | AZD5582 induces HIV RNA expression in resting CD4+ T cells from

tissues of HIV-infected ART-suppressed BLT mice. a, BLT mice were infected

with HIV-1JR-CSF. Af ter 10  weeks of ART t reatment , mice received vehicle cont rol

or AZD5582. FTC, emt r icit abine; RAL, raltegravir ; TDF, tenofovir disoproxil

fumarate. b, HIV RNA copies per ml−1 of plasma of HIV-infected ART-t reated BLT

mice before t reatment with vehicle cont rol (lef t ; blue lines) or AZD5582 (r ight ;

red lines). Two independent exper iments were per formed (lef t , n = 6 mice per

group; r ight , n = 4 mice per group). Grey shading, per iod of ART administ rat ion.

c, Plasma HIV RNA levels in HIV-infected ART-suppressed mice f rom b t reated

with vehicle cont rol or AZD5582. d, HIV viral RNA (vRNA) levels in rest ing CD4+

T cells isolated from the bone marrow (BM), thymic organoid (Org), lymph

nodes (LN), spleen, l iver and lung of cont rol or AZD5582-t reated mice (cells

pooled from n = 6 mice per group for each t issue) were analysed in t r iplicate.

Data are mean ± s.e.m. St at ist ical signif icance was determined using a t wo-

sided Student ’s t-test . e, Cell-associated HIV RNA copies in the blood (n = 6),

female reproduct ive t ract (n = 6) and brain (n = 3). PBMCs, per ipheral-blood

mononuclear cells. St at ist ical signif icance was determined using a two-sided

Mann–Whitney test (per ipheral-blood mononuclear cells and female

reproduct ive t ract) or Student ’s t-test (brain). Colours indicate samples f rom

the same mice. Data are mean ± s.e.m.

AZD5582 induces HIV RNA expression in rCD4+ T cells fromtissues of HIV-infected ART-suppressed BLT mice

AZD5582 induces SIV RNA expression in theplasma and LN of ART-suppressed SIV-infectedRM

No reduction of the viral reservoir and/or delay of viral rebound after ART interruption

Rasmussen et al. #37

Impact of immunological check-point blockers on the viral reservoir

n=33

n=26 αPD-1 (Nivolumab, 3mg/Kg or 240mg)

n=7 αPD-1 + αCTLA-4 (Nivolumb 240mg + Ipilimumab, 1mg/Kg)

Overall no change in the frequency of cells containingreplication competent HIV (maybe with PD-1 + CTLA-4)

Transient decrease in cell-associated HIV DNA

Modest Latency Reversal with combined blockade of PD-1 + CTLA-4

Gruell et al. #38

Impact of the bnAb 3BNC117 and Romidepsin on the viral reservoir

n=20

Randomizedn=11 3BNC117 (30mg/Kg) +RMD (5mg/m2)n=9 RMD (5mg/m2)2 cycles (120 days)

Modest Latency Resersal

No changes in cell-associated HIV DNA

No clear delay on time to viral rebound

SenGupta et al. #40

Impact of vesatolimod (LTR-7a) on the viral rebound in HIV controllers

n=25

PLHIV controllers on ART >6 mothsPre-ART pVL 50-5000 c/ml4-8mg for 20 weeks → ATI (ART restart if 10K c/ml x 4 weeks)

n=17 VES (GS-9620) + n=8 placebo

Modest increased time to viral rebound and decreased viral set point compared to pre-ART

Impact in intact HIV DNA but not in total HIV DNA

Augmented immune responses (also ISG, cytokines)

Discordant data when tested in NHP

Image: Toni Kelleher

Sanvant et al. #26

Ledgins to induce HIV-1 super-latency

Chen et al., Nat Comm. 2016

Transfer plasmid: HIV genome

+packaging plasmid+pVSV-GRandom barcode (20 bp)

High complexity (>1 million barcodes)

Sanvant et al. #26

Ledgins to induce HIV-1 super-latency

LEDGIN treatment retargets integration out of active genes3. Results

42

Figure 1.2. LEDGIN treatment retargets integration out of active genes. A) Integration sites retrieved in each condition in Jurkat cells were divided in four genomic categories and plotted as relative proportions: silent genes (SG), intergenic regions (IR), regulatory elements containing enhancers and promoters (RE) and active genes (AG). B-I) The distance in base pairs (bp) between the integration site and certain features is plotted for each barcode. Each dot represents a unique integration site. Error bars represent median and interquartile range. The lower quartile is not plotted in case it equals ‘0’. Panels B-I plot the distance to: B) H3K36me3, C) H3K79me3, D) H3K79me2, E) H3K27ac, F) H3K4me1, G) RNAPII, H) H3K4me3 and I) H3K9me3 (See also Supplemental Table 1.3 for explanation of different markers). Statistical significance was calculated by the Kruskal-Wallis test, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Two experiments were performed in SupT1 cells and two in Jurkat cells. Results are shown for one representative experiment in Jurkat cells (experiment A).

Silent Genes

Active Genes

Intergenic regions

Regulatory Elements

3. Results

45

Figure 1.3. LEDGIN treatment reduces RNA expression. Two weeks post transduction in the presence of varying concentrations of LEDGIN CX014442, mRNA was extracted and reverse transcribed to cDNA to determine RNA expression. A) The expression score in Jurkat cells (experiment B) is calculated for each unique barcode (dot) by normalizing the barcode counts in the RNA to the corresponding DNA counts. Error bars represent median and interquartile range. B) Median RNA expression scores for two independent experiments in Jurkat cells (experiment A and B) and one in SupT cells (experiment A). C)

LEDGIN treatment reduces RNA expression

Latent HIV Super-Latent HIV

Jiang #73

Summary

▪HIV-1 can be cured (2 new cases in IciStem) but new strategies are needed to make the cure approach scalable

▪ “Shock and kill” strategies are becoming more consistent in inducing viral reactivation in animal models but still of limited efficacy in humans.

▪ “Block and Lock” strategies are moving forward in vitro with some of them entering in vivo phases in NHP