Eradication of HIVBonaventura ClotetHospital Universitàri Germans Trias i PujolBadalona. Barcelona. Catalonia

HAART blocks viral replication but fails eradicating the HIV-1 infection once started

Vira

l Loa

d

Time after infectionStrain JID 2005

¿Es posible erradicar la infección?

Transient Increase in Episomal Viral cDNA after Intensification with Raltegravir

Buzon, Massanella et al. Nat Med 2010Llibre, Buzon, Massanella et al. Antiv Ther 2011

1-RAL will decrease the number of cells activelyproducing viruses 2-Homeostatic proliferation

Very Early Initiation of ART May Limit the HIV ReservoirDecay of integrated HIV DNA during ART by Fiebig

The frequency of PBMCs harbouring integrated HIV DNA decreases rapidly upon ART initiation in FII to FV individuals, whereas no decay is noted in subjects who started ART during chronic infection.Fiebig I remain below limit of detection.

Geometric mean with 95% CI

ART initiation

CD8-T cells are very important and have

implications for cure strategies and vaccine development

Bruce Walker. CROI 2016. Boston (USA)

How do HIV-specific CD8+ T cellslimit virus production?

Infected cell

More virus made, worse a person doesGoal: limit virus productionPreferentially infects CD4 cells, monocytes, macrophages

Infected cell

Viralproteins

HLA Class I

Infected cell

Infected cell

ViralPeptides

(epitopes)HLAClass Ialleles

CD8

Infected cell

HIV-specific CD8+ T cell(Cytotoxic T lymphocyte, CTL)

CD8

Infected cell

T cellReceptor

CD8receptor

CD8

CD8Antiviralcytokines

and chemokines

1. Require only one to a few viral peptide-HLA complexes for recognition

2. Can kill infected cells before progeny virions produced

Lytic granules

Question

• What are the characteristics of HIV-specific CD8+ T cells that contribute to immune control?

Infected cell

“Protective” and “Risk” HLA Alleles

HLAB*3503

HLAB*5701

Kaslow Nat Med 1996Gao NEJM 2001Migueles PNAS 2000Kiepiela Nature 2004Felay Science 2007Dalmasso Plos One 2008Limou JID 2009Pereyra Science 2010Pelak JID 2010Herbeck JID 2010

Infected cell

Viral Peptides Targeted

Viralpeptides

Klein JEM 1995Edwards JV 2002Novitsky JV 2003Masemola JV 2004Zuniga JV 2006Geldmacher JV 2007Kiepiela Nature 2007Pereyra JV 2014Migueles Ebio 2015

CD8

Day Nature 2006Trautmann Nat Med 2006Petrovas JEM 2006

HIV-specific CD8+ T cell exhaustion

PD-1

Continous exposure to viral antigenspromotes PD-1 expression

CD8Viral mutation/immune escape

Phillips Nature 1991Goulder Nat Med 1997Borrow Nat Med 1997Goulder Nature 2001Brockman JV 2007Kawashima Nature 2009Goonetilike JEM 2009Liu JCI 2013Carlson Science 2014Redebe AIDS 2015Roberts Nat Gen 2015

Immune Escape

Conclusions I1. HIV-specific CD8+ T cells can kill virus infected

cells before progeny virions are produced2. HLA and the viral peptides presented

influence viral load3. HIV-specific CD8+ T cells differ in antiviral

efficacy4. Viral mutations in CD8+ T cell epitopes lead to

immune escape5. Persistent viremia leads to dysfunction of

these cells

Latency reversing

agent (LRA)

Shan Immunity 2012Sung JID 2015

Latentlyinfected cell

CD8

Latency reversing

agent (LRA)

Shan Immunity 2012Sung JID 2015

Latentlyinfected cell

CD8

Latency reversing

agent (LRA)

Shan Immunity 2012Sung JID 2015

CD137: Activation marker

CD8+ T cells as biosensors to define effective

latency reversing agents in vitro

RB Jones, unpublished

Infected cell

LRA

CD8

Escape mutation

Deng et al, Nature, 2015

Target non-escaped epitopes

Deng et al, Nature, 2015

Infected cell

CD8

Escape mutation

Subdominant epitope

Subdominant CD8+ T cell response

Deng et al, Nature, 2015Hancock PLoS Path 2015Sung JID 2015

Conclusions III

• Early treatment of hyperacute HIV infection enhances therapeutic vaccine responses

• HIV cure strategies will likely require CD8+ T cell-mediated elimination of infected cells, through targeting of subdominant epitopes

• HIV-specific CD8+ T cells can contribute to HIV vaccine efficacy, in conjunction with other immune responses

Post-CROI 2015

Kick and Kill Strategy to EliminateReservoirs of Latent HIV

Modified ImmunoglobulinsHet IL15

IMMUNOTHERAPY

Mascola JR. CROI 2016, Boston MA. #15.

ABSTRACT 32LB

Passive administration of bnAbs (VRC01) prevents HIV-1 transmission in humanized mice, macaques.Transiently reduces plasma viral loads by ~1-3 logs in HIV infected humans

Results: Viral rebound

A5340

• Majority of participants rebounded by week 5• 2 participants with delayed rebound at 8, 11 weeks

• Time to rebound not associated with VRC01 level, age, nadir or entry CD4 ct, time on ARTBar KJ. CROI 2016, Boston, MA. #32LB.

Mascola JR. CROI 2016, Boston MA. #15.

Combined Antibodies:Improved Potency and

Breadth

Kong, Montefiori Korber et al. J. Virol (2015)

2 mAbs > 98% coverage

Highly potent multifunctional antibody-based molecules against Human Immunodeficiency Virus.

Julià Blanco, Jorge Carrillo, Bonaventura Clotet

Recombinant antibodies with enhanced antiviral activity: The eCD4-Ig molecule

Highly potent multifunctional antibody-based molecules against HIV

IC50 (ng/ml)NL43 BAL AC10 Description

MOLECULE-0 10,820 27,390 >100 1st GenerationMOLECULE-1 0,108 0,050 >100 REFERENCE (Farzan’s)MOLECULE-5 0,037 0,007 0,375MOLECULE-7 0,029 0,004 0,189MOLECULE-8 0,035 0,028 1,327

MOLECULE-10 0,793 2,924 >100MOLECULE-11 0,110 0,026 1,454

RATIO MOLECULE-7/ MOLECULE-1

x5 x12 >100

1) Antiviral activity

Recombinant antibodies: IrsiCaixa moleculesHighly potent multifunctional antibody-based molecules against HIV

AlbaJuna molecules

2) Antibody Dependent Cellular Cytotoxicity (ADCC)

Highly potent multifunctional antibody-based molecules against HIV

Recombinant antibodies: IrsiCaixa molecules

THERAPEUTIC VACCINE

Main obstacles to HIV vaccine and eradication research

Huge viral diversity around the world—rapid evolution

+

HIV destroys host’s immune system

+

HIV establishes a long-life reservoir very early in infection

in resting memory cells not accessible to drug/immune

system if not reactivated

Vaccinecandidate

Vectors(vehicles)

Immunogens (active component)

Need to be safeNot induce anti-vector immunity

Feasible to produce

Able to induce antibodies& cellular responses effective to contain with viral diversity

To design a focused immunogen sequence displaying the major viral targets for protective/beneficial HIV-1 specific T cell responses able to break immunodominance to

non-beneficial targets ensuring a broad HLA class I and class II coverage.

Immunogenicity data from >1,000 infected individuals screens for T cell responses to the entire HIV proteome yielded26 regions identified as “beneficial” (PR>1) in HIV-1 Gag, Pol, Vif and Nef proteins that were:

i) preferentially targeted by individuals with low viral loads, ii) turned out to be more conserved and iii) elicited responses of higher functional avidity and broader cross-reactivity

Mothe B et al, PLos One 2012Mothe B et al, JTM 2012

HIVACAT T-cell Immunogen design (HTI)3

0

2

4

6

8

2x pDNA 2x pDNA1 MVA

3x pDNA 3x pDNA1 MVA

# o

f IF

Ng

resp

on

ses

Ga

g-p

17

Ga

g-p

24

Ga

g-p

15

Po

l-P

rot

Po

l-R

T

Po

l-In

t

Vif

Ne

f0

250

500

750

1000

1250

IFN

g S

FC

/10

6sp

len

ocyte

s

MVApDNA: 100µg IM / dose MVA: 10^6pfu IM / dose

GagPolVifNef

3x pDNA + 1 MVA3x pDNA

CD4CD8

G ag P o l V if N ef0.00

0.25

0.50

Mean and SD is shown

% IF

Nγ+

CD

3+ s

plen

ocyt

es

6 out of 6 mice

Ga

g-p

17

Ga

g-p

24

Ga

g-p

15

Po

l-P

rot

Po

l-R

T

Po

l-In

t

Vif

Ne

f0

250

500

750

1000

1250

IFN

g S

FC

/10

6sp

len

ocyte

s

MVA boost increases responses broadly after 3 pDNA vaccinations

0

1000

2000

3000

4000

5000

# of

HIV

supr

otei

ns (n

=8)

with

IFN

g re

spon

seHIVACAT T-cell Immunogen design (HTI)

3

Two DNA immunizations only elicited responses in 3/5 animals.

Interestingly, MVA-HTI vaccine was able to boost responses (both in breadth and magnitude,) in the two groups analyzed, with significantly increased magnitudes of responses in the group that obtained 3 doses of pDNA-HTI

(median total IFNg positive responses of 777 vs 2865 SFC/million splenocytes after 3xpDNA and 3xpDNA followed by 1xMVA respectively, p= 0.0087).

MVA-HIVACAT vaccine Elicits CD8 effector memory response (which has been associated with HIV control in Elite Controllers and in the animal model(Picker et al. Nature 2011)

Journal of Translational Medicine. Bea Mothe et al 2015

HIVACAT T-cell Immunogen design (HTI)

Currently the 3 most interesting clinical trials for HIV eradication combining vaccine and HDACs are:

1-REDUC (Vax4x + RMD) chronically infected HIV patients (Danish group)2-RIVER (HIVconsv + SAHA) early treated does not include stop of ARVs (Londres/Oxford)3-BCN02-Romi (HIVconsv + RMD) early treated includes Stop of ARVS (irsicaixa-Hivacat/Barcelona)

ABSTRACT 26LB

VACC-4

• Vacc-4x consist of 4 peptides (20-27aa each one) corresponding to conserved regions on the p24 protein slightly modified to optimize HLA presentation.

• The vaccine is administered with GM-CSF as adjuvant for DCs.

• The vaccine is intended to induce, sustain or recover CD4+ and CD8+ T cell responses to p24, so that the immune cells recognize and remove infected cells presenting the native versions of such p24 peptides

ABSTRACT 26LB

ABSTRACT 26LB

ABSTRACT 26LB

ABSTRACT 26LB

ABSTRACT 26LB

REDUC TRIAL (1)• The latent HIV reservoir was significantly reduced by 40%

Total HIV DNA and qVOA (quantitative viral outgrowth assay). The impact on proviral DNA after RMD could have been mediated by NK or ADCC???

• 8 out of 17 (47%) had detectable VL (but low levels < 100 c/ml) during RMD infusion

• Reactivation of the latent HIV reservoir was confirmed by increases in histone acetylation levels and viral expressions.

• After ARV treatment interruption median time to viral rebound > 50c/ml, 14 days

• The combination of Vacc-4x and RMD was safe and well tolerated

REDUC TRIAL (2)

• The possible explanation for not impacting the viral rebound could be:1-The DNA decay observed was low (0.5 log) in a

group of patients that started therapy at chronic stages with high proviral DNA at BL (1000 c/Milion of CD4s). Despite the decay achieved still quite high viral reservoir was present at the time of ATI. As a reference point in elite or post-treatment controllers viral reservoir is usually much lower (

REDUC TRIAL (3)

• The possible explanation for not impacting the viral rebound could be:2-This vaccine did not induce specific CD8 responses3-In vitro studies has not shown suppressor activity4-Even in the phase II study showing 0,5 lower viral set

point after ATI (Pollard Lancet Infect Dis), there was not delay in the viral rebound

5-Timing of vaccine and RMD and ATI (between last vaccination and ATI-14 weeks) was not the optimal and if any response was achieved, it was light and could have disappeared

REDUC TRIAL (4)• How to overcome the problem:• A better vaccine stimulating significantly and

long-lasting the CD8 could impact the rebound and delay it

• There is a need to achieve lower levels of the viral reservoir before ATI in all vaccine trials (select very early treated patients or use more potent vaccine+LRA or use more shoots of LRA but with lower doses?)

• These suggestions need to be assessed in the upcoming clinical trials

B CELL FOLLICLE SANCTUARY

CD8 can not enter into the B-cell follicle

• Lymphocyte expansion and differentiation

• Enhanced activation and cytotoxicity

• Migration via increase of CXCR5

259LB Increased Effector CD8 Lymphocyte Trafficking to Lymph Nodes Induced by hetIL-15 (heterodimeric IL-15)

259LB Increased Effector CD8 Lymphocyte Trafficking to Lymph Nodes Induced by hetIL-15 Conclusions: We explore the potential of IL-15 as a viral reservoir reducing agent in ART-treated SIV infected macaques. hetIL-15 treatment enhances the number, activation and cytotolytic potential of CD8 cells in LN and germinal centers, a known site of virus persistence. hetIL-15 treatment in combination with pDNA vaccine targeting the “Achilles’ heel” of the virus, i.e., the highly conserved regions (CE), in virus sanctuary areas (germinal centers) is a promising HIV eradication strategy.

COMBINATION OF STRATEGIES TO ACHIEVE THE CURE OF HIV

Infectedcell

Infected celldestruction

HIV-1 infected patient HIV-1 free individual

Multiple strategies for eradication

Vaccine (HTI)

CTLCTL

Vaccine (HTI)

T

Infected celldestruction

HIV-1 infected patient HIV-1 free individual

Multiple strategies for eradication

Vaccine (HTI)

CTLCTL

DC (SIGLEC-1) DeliveredHDAC Inhibitors,TLR-7;

Anti-PD1 boosting?

Vaccine (HTI)

Infectedcell

T

Virus inactivationVirus

Neutralizationby AJ-Mabs

New infections protection

Infected celldestruction

HIV-1 infected patient HIV-1 free individual

IgGsAlbaJunaMabs (AJ-Mabs)

(IgG+CCR5+CD4+FX)

Multiple strategies for eradication

Vaccine (HTI)

CTLCTL

Anti-PD1 boosting?

AJ-Mabs

XVaccine (HTI)

NK

Infectedcell

T

Virus inactivationVirus

Neutralizationby AJ-Mabs

New infections protection

Infected celldestruction

HIV-1 infected patient HIV-1 free individual

IgGsAlbaJunaMabs (AJ-Mabs)

(IgG+CCR5+CD4+FX)

Multiple strategies for eradication

Vaccine (HTI)

CTLCTL

Anti-PD1 boosting?

AJ-Mabs

AJ-Mabs

XVaccine (HTI)

NK

Infectedcell

T

Virus inactivationVirus

Neutralizationby AJ-Mabs

New infections protection

Infected celldestruction

HIV-1 infected patient HIV-1 free individual

IgGsAlbaJunaMabs (AJ-Mabs)

(IgG+CCR5+CD4+FX)

Multiple strategies for eradication

Vaccine (HTI)

CTLCTL

DC (SIGLEC-1) DeliveredHDAC Inhibitors,TLR-7,hetIL-15

Anti-PD1 boosting?

AJ-Mabs

AJ-Mabs

XVaccine (HTI)

NK

Infectedcell

T

Virus inactivationVirus

Neutralizationby AJ-Mabs

New infections protection

Infected celldestruction

HIV-1 infected patient HIV-1 free individual

IgGsAlbaJunaMabs (AJ-Mabs)

(IgG+CCR5+CD4+FX)

Multiple strategies for eradication

Vaccine (HTI)

CTLCTL

DC (SIGLEC-1) DeliveredHDAC Inhibitors,TLR-7hetIL-15

Anti-PD1 boosting?

•Therapeutic Vaccine (HTI)•HDACs inhibitors delivered through Siglec-1

mechanism; TLR7; Heterodimeric IL-15•Anti-PD-1 as CD8 and vaccine boosters?•Recombinant proteins (AlbaMab/JunaMab)•Microbiome

AJ-Mabs

AJ-Mabs

XVaccine (HTI)

THANK YOU VERY MUCH

Special thanks to Bruce Walker and to my colleagues Julià BlancoJavier Martinez-Picado, Bea Mothe& Roger Paredes

Dianummer 1Dianummer 2¿Es posible erradicar la infección?Dianummer 4Transient Increase in Episomal Viral cDNA after Intensification with RaltegravirDianummer 6Very Early Initiation of ART May Limit the HIV Reservoir�Decay of integrated HIV DNA during ART by Fiebig CD8-T cells �are very important and have implications for cure strategies and vaccine development� �Bruce Walker. CROI 2016. Boston (USA)�Dianummer 9Dianummer 10Dianummer 11Dianummer 12Dianummer 13Dianummer 14Dianummer 15Dianummer 16Dianummer 17QuestionDianummer 19Dianummer 20Dianummer 21Dianummer 22Conclusions IDianummer 24Dianummer 25Dianummer 26CD8+ T cells as biosensors to define effective �latency reversing agents in vitroDianummer 28Target non-escaped epitopes�Dianummer 30Conclusions IIIDianummer 32Dianummer 33Dianummer 34Dianummer 35Dianummer 36Results: Viral reboundDianummer 38Dianummer 39Dianummer 40Dianummer 41Dianummer 42Dianummer 43Dianummer 44Dianummer 45Dianummer 46Dianummer 47Dianummer 48Dianummer 49Dianummer 50VACC-4Dianummer 52Dianummer 53Dianummer 54Dianummer 55Dianummer 56REDUC TRIAL (1)REDUC TRIAL (2)REDUC TRIAL (3)REDUC TRIAL (4)Dianummer 61Dianummer 62Dianummer 63Dianummer 64Dianummer 65Dianummer 66Dianummer 67Dianummer 68Dianummer 69Dianummer 70Dianummer 71Dianummer 72Dianummer 73Dianummer 74Dianummer 75