How does the reservoir influence the clinician's decisions

Prof. Carlo Federico Perno

Acknowledgements

University of Milan, Milan Italy: C. Alteri.

Policlinic of Rome Tor Vergata, Rome, Italy: M. Andreoni, L. Sarmati, M. Viscione, S. Gini, C. Cerva, V. Malagnino, K. Stingone,

T. Guenci, F. Stazi, S. Giannella, V. Serafini, M. Ciotti, P. Paba. S. Grelli.

INMI L Spallanzani, Rome, Italy: A. Antinori, R. Bellagamba, C. Pinnetti, S. Cicalini, R. Gagliardini, A. Mondi, A. Vergori, A.

Sanpaoloesi, G. De Carli, F.M. Fusco, L. Lo Iacono, M.L. Giancola, G. Liuzzi, R. Acinapura, P. Scognamiglio, N. Orchi, E. Girardi,

M.R. Capobianchi, C. Gori, F. Forbici, G. Berno, D. Pizzi, A. Giannetti, P. Lorenzini, A. Navarra, R. Libertone, G. Ippolito.

San Gallicano Hospital, Rome, Italy: A. Latini, M. Colafigli, M. Giuliani, A. Pacifici, A. Cristaudo. General Hospital Umberto I:

V. Vullo, G. D’Ettorre, F. Falasca, O. Turriziani, G. Antonelli. San Giovanni Addolorata Hospital, Rome, Italy: F. Montella, F. Di

Sora, W. Leti, F. Iebba. Sant’Andrea Hospital, Sapienza University, Rome, Italy: A. Pennica. Rebibbia, Rome, Italy: S. Marcellini.

Bambin Gesù Hospital, Rome Italy: S. Bernardi, H Tchidjou Kuekou. Polo Pontino, Sapienza University, Rome, Italy: C.

Mastroianni, M. Lichtner, V.S. Mercurio, C. Del Borgo, R. Marrocco. Frosinone Hospital, Frosinone, Italy: G. Farinelli, E.

Anzalone, M. Limodio, L. Sarracino. Rieti Hospital, Italy: G. Natalini Raponi, M.E. Bonaventura. Viterbo Hospital, Viterbo, Italy:

G. Maffongelli, G. Bernardini, A. Caterini, F. Ferri, A. Ialungo, E. Liguori, D. Migliorini, R. Monarca, R. Preziosi, E. Rastrelli, G.

Starnini, G. Sebastiani.

University of Turin, Turin, Italy: G. Di Perri, S. Bonora, A. Calcagno, V. Ghisetti, G. Vandemmiati, T. Allice.

Modena Hospital, Modena, Italy: C. Mussini, V. Borghi, W. Gennari, A. Cossarizza, M. Nasi, M. Di Gaetano.

Pescara General Hospital, Pescara, Italy: G. Parruti, F. Vadini, F. Sozio, E. Mazzott, T. Ursini, E. Polilli, P. Di Stefano, M.

Tontodonati, G. Calella. San Salvatore, L’Aquila, Italy: A. Grimaldi, A. Cellini. Ancona Hospital, Ancona, Italy: A. Mataloni

Paggi. Giuseppe Mazzini Hospital, Teramo, Italy: Di Giammartino, L. Falconi, P. Tarquini. San Salvatore – Muraglia- Hospital,

Pesaro, Italy: E. Petrelli, G. Corbelli, P. Tarquini. Avezzano Hospital, Avezzano, Italy: M. Paoloni, R. Mariani. AO Papa Giovanni

XXIII, Bergamo, Italy: F. Maggiolo, AP Callegaro. AO Careggi, Florence, Italy: K. Sterrantino.

Cotugno Hospital, Naples, Italy: A. Chirianni, M. Gargiulo. University of Campania Vanvitelli, Italy: S. Marini, N. Coppola.

Bisceglie-Trani Hospital, Bisceglie, Italy: R. Losappio. Catania Hospital, Catania, Italy: R. La Rosa. Enna Hospital, Enna, Italy:

L. Guarneri. Palermo Hospital, Palermo, Italy: F. Di Lorenzo T. Prestileo, A. Cascio.

The Patients

University of Rome “Tor Vergata”, Rome Italy: F. Ceccherini Silberstein, V. Svicher, A. Bertoli, M.C. Bellocchi, L. Fabeni, B. Yagai

Romeo, R. Salpini, R. Scutari, S. Barbaliscia, M. Brugneti, A. Biddittu, M. Bruni, L. Carioti, P. Saccomandi.

Unicamillus, Rome Italy: D. Armenia.

Thanks to the modern therapies, today around

95% of HIV infected individuals achieve

virological suppression

The success of antiretroviral therapy

allowed to a consequent dropping of

resistance development (at least in high income countries)

56%60% 60% 57%

51% 54%45%

41%46%

33%23%

27%21%

17% 16% 19% 17% 18%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

% o

f is

ola

tes

P<0.001 P=0.705

Among 9014 isolates from cART failing patients the prevalence of M184Vdramatically decreased from 56% in 1999 to 21% in 2013. In the last 5 years theM184V prevalence is stably settled at 17%.

Analysis performed on 9014 isolates from cART failing patients (Update August 2018)

Prevalence of M184V among isolates from cART failing patients over time

Armenia & Santoro, Unpublished data 2019

59%

0%0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

% o

f re

gim

en

sat

M1

84

V d

ete

ctio

n

Suboptimal

Prevalence of regimens used at the moment of M184V detection over time

In the past M184V was mainly detected in isolates under suboptimal regimensbased on NRTIs or unboosted PIs

P<0.001

Analysis performed on 3475 isolates for whom GRT revealed M184V from 2388 cART failing patients (Update August 2018)

Armenia & Santoro, Unpublished data 2019

• A new era for antiretroviral therapy is approaching

• New strategies are needed to maintain virus under control for decades, and preserve immune functions• Long-acting therapies• Simpler drug regimens

• while….

• Cure options are under study and clinical assessment

• The lower is the total viral burden, the higher is the chance that the patient could be eligible for a cure approach

The HIV hiding places

2016

S.L. Lamers 2016; 20:8968-83

229 varied autopsy tissues from 20 ART-treated patients with low or undetectableplasma viremia and cerebral fluid (CSF) VL prior to death, were analysed. HIV-DNA (>200 cp/106 cell) was identified in 48/87 brain tissues and 82/142 non-brain tissues. Abnormal histological findingswere identified in all partecipants (brain, spleen, lung, lymph node, liver, aorta and kidney).

Tissues assayed with the number of HIV+ (red) and HIV- (green) tissues identified

2019

HIV DNA was detected in most body tissues despite long-term ART and despite confirmed undetectable

HIV RNA in plasma at the time of death. The majority of full-length (FL) HIV-env sequences appeared to

be intact.

In the quest for a functional cure or eradication of HIV infection, we need to know howlarge the reservoirs are from which infection rebounds when treatment is interrupted.To that end, we quantified SIV and HIV tissue burdens in tissues of infected non-humanprimates and lymphoid tissue (LT) biopsies from infected humans. Before antiretroviraltherapy (ART), LTs harbor more than 98 percent of the SIV RNA+ and DNA+ cells.While ART substantially reduced their numbers, vRNA+ cells were still detectable andtheir persistence was associated with relatively low drug concentrations in LT comparedto peripheral blood. Prolonged ART also reduced the level of SIV and HIV-DNA+ cells, butthe estimated size of the residual tissue burden of 108 vDNA+ cells that potentiallyharbor replication competent proviruses, along with the evidence for continuing virusproduction in LT despite ART, identify two important sources for rebound followingtreatment interruption.The large sizes of these tissue reservoirs underscore the challenges in developing “HIVcure” strategies that target multiple sources of virus production

Jacob D Estes Nature Medicine 2017

Defining total-body AIDS-virus burden with implications for curative strategies

Graphical representation of the proportion of vRNA+ cells in eachorgan system before and during suppressive ART.

Jacob D Estes Nature Medicine 2017

Defining total-body AIDS-virus burden with implications for curative strategies

During ART the numbers of virus (v) RNA+ cells substantially decreasedbut remained detectable.

Graphical representation of the proportion of vRNA+ cells in eachorgan system before and during suppressive ART.

Jacob D Estes Nature Medicine 2017

Defining total-body AIDS-virus burden with implications for curative strategies

During ART the numbers of virus (v) RNA+ cells substantially decreasedbut remained detectable.

« Size » of the HIV reservoir

Ho et al. Cell 2013

The « real reservoir » ?

• The vast majority of

proviruses that persist on

ART are defective.

• These “ZOMBIE” proviruses (Imamichi, H. et al., International AIDS

Conference, 2014) lack the ability to produce intact viruses but can

inflict harm by producing foreign nucleic acids and proteins.

Persistence of these proviruses may explain the persistent

seropositivity to HIV-1 and persistent immune activation seen in

patients with "undetectable" virus.

• Of the minority proviruses that are intact (~2%), the

fractions that are latent or replicative competent are not

known.

Bruner et al. Nature 2019

DNA PCR assays predominantly measure defective proviruses. Proviruses persisting in CD4+ T cells of individuals

on suppressive ART as detected by nFGS (near full genome sequencing). The near full genome sequencing (nFGS) are

methods used identify defects throughout the genome except the 5′ long terminal repeat (LTR). Defects include internal

stop codons, deletions not attributable to normal length polymorphisms, and APOBEC3G/F mediated hypermutation

(G→A). Most deletions were large except for those in the packaging signal (ψ) or major splice donor site. Analysis is

based on 211 sequences from individuals initiating ART during chronic infection.

2018

The vast majority of acute transforming retroviruses are replicationdefective, with the oncogene-containing genome beingtransmissible only during mixed infection with a replication-competent virus. A defective retrovirus that relies on complementing functions can, in some instances, become replication competent by recombiningwith its replication-competent “helper.” In fact, there is some evidence that Rous sarcoma virus, possiblythe only naturally arising replication-competent retrovirus containing a host oncogene, was replication defective initially

The Remarkable Frequency of Human ImmunodeficiencyVirus Type 1 Genetic RecombinationA Onafuwa-Nuga and A Telesnitsky

Microbiology and Molecular Biology Reviews - 2009

As we observed for 8E5 in this study, genetic recombination

could generate replication-competent viruses from such a

collection of defective proviral sequences.

Infectivity of recombinant viruses

generated following transfection

of 8E5 cells with defective

molecular clones of HIV.

• The HIV-1 sanctuary: the meaning of

compartmentalization

Lee, et al Abstr 407, CROI 2017

• This is the first study to evaluate GALTand LN tissue concentrations in patientsreceiving RAL and 800 mg daily DRV.

• Tissue:plasma ratios were higher inileum>rectum as shown previously, andlowest in lymph node.

• In a limited number of participants,concentrations of RAL were significantlylower in lymph nodes vs. GALT, supportingprior observations.

• These results support the current limiteddata on tissue ART drug concentrations andhave potential implications on HIV curestrategies.

UntreatedTreated

RALT: rectum

COMPARATIVE

LYMPHOID

TISSUE

PHARMACOKINE

TICS OF

INTEGRASE

INHIBITORS.

C. V. Fletcher

CROI 2018

In the contest of long-term strategies, a deepen

evaluation of viral reservoir with biomarkers easy to be

measured in clinical practice is today fundamental!

In this frame, an evaluation of quantity of HIV-DNA (as a

measure of HIV reservoir) and the archived resistance

needs to be considered.

The quantification of total HIV-DNA in PBMCs provides

a reliable and easy way of measuring the size of the

cellular reservoirs of HIV.

Total HIV DNA is associated with other easier-

to-measure parameters in patients under

successful therapy

• Pre-therapy plasma HIV RNA

– Hocqueloux, JAC 2013; Lambert-Niclot, PLoS ONE 2012

• Residual viremia, even when simply classified as detectable vs.

undetectable

– Chun, JID 2011; Lambert-Niclot, PLoS ONE 2012; Mexas, AIDS

; Parisi et al., JCM 2012; Falasca et al., JAIDS 2015; Parisi, CMI

2015.

• Nadir CD4 counts

– Watanabe, BMCID 2011; Lambert-Niclot, PLoS ONE 2012

• Duration of suppression of plasma HIV RNA

– Watanabe, BMCID 2011

• Earlier treatment start

– Hocqueloux, JAC 2013

Ceccherini-Silberstein et al. , JAC Dec 2018

Pre-ART HIV-DNA correlates with pre-ARTHIV-RNA, CD4+ T-cells, CD4/CD8 ratio.

By considering the 397 patients achieving virological suppression, theprobability of experiencing virological rebound, defined by 2 confirmedplasma HIV-RNA >50 copies/mL, was 12% (95% CI: 8.6-15.5). By stratifyingpatients for the 3 different pre-ART HIV-DNA levels, increasing rates ofvirological rebound were found by increasing pre-ART HIV-DNA.

17.2%

7.4%4.3%

15.2%

4.8%

0%

Ceccherini-Silberstein et al. JAC 2018

HIV-1 DNA may be a strong predictor of :• Immunological progression (CD4 <350) in absence of

cART.

• Immunological progression (CD4 <350) following

treatment interruption.

• Time to viral rebound.

Williams, eLife 2014

Various studies have shown that the level

of baseline HIV-DNA can influence the maintenance

of virological success under simplification therapy

2013

Virological Factors Associated With Outcome of Dual ETR/RAL Therapy (ANRS-163 Trial)

Cathia Soulie, Lambert Assoumou, Sophie Sayon, Thuy Nguyen, Marc-Antoine Valantin, Virginie Ferre, Chakib Alloui, Brigitte Montes,

Véronique Avettand-Fenoel, Constance Delaugerre, Diane Descamps, Esteban Martinez, Jacques Reynes, Gilles Peytavin, Dominique

Costagliola, Christine Katlama, Vincent Calvez, Anne-Geneviève Marcelin.

PS6/4

FACTORS PREDICTING VIROLOGICAL FAILURE DURING DOLUTEGRAVIR MAINTENANCE MONOTHERAPYIngeborg Wijting, Sofie L. Rutsaert, Casper Rokx, David M. Burger, Elrozy Andrinopoulou1, Linos Vandekerckhove, Bart Rijnders

Defining a Total HIV DNA threshold as

guidance for therapy simplification strategies

S. Rutsaert1, I. Wijting2, W. De Spiegelaere3, L. De Clercq1, B. Rijnders2, L. Vandekerckhove1

HIV Cure and Research Symposium, Ghent

Therapy

simplification studies

PROTEA DOMONO

PROTEA▪ Substudy of the PROTEA (NCT01448707)

▪ Randomized clinical trial

HIV-1 infected patients

• First-line ART• VL undetectable• CD4 nadir >100

cells/mm3

• CD4 at baseline ≥200 cells/mm3

Triple therapy

DRV/r 800/100mg +2 NRTIs

Monotherapy

DRV/r 800/100mg N= 77

W48 W96

▪ Substudy of the DOMONO (NCT02401828)

▪ Randomized clinical trial

HIV-1 infected patients

• VL undetectable• CD4 nadir ≥200

cells/mm3

• HIV RNA zenith < 105

copies/ml

Monotherapy

DTG 50mg N= 77

W48

DOMONO

Undetected Detected Failers Undetected Detected Failers

PROTEA DOMONO

RESULTS

*

*

*

*

: p<0,05

Still……. a relevant clinical cut-off of total HIV DNA

needs to be defined in both drug-naïve and

virologically suppressed patients.

In drug-naïve patients starting a first-line regimen, the risk of virological

rebound was significantly higher in patients with a pre-cART total HIV-1 DNA

>10,000 copies/106 CD4+ T cells than in those with a total HIV-1 DNA ranging from

1,000 to 10,000 copies/106 CD4+ T cells and <1,000copies/106 CD4+ T cells.

Ceccherini-Silberstein et al., JAC 2018

In virologically suppressed patients who switched to a PI-sparing regimen,

higher HIV DNA levels (>226 copies/106 PBMCs) at baseline were independently

associated to an increased risk of virological failure or viral blip.

Sarmati et al., J Med Virol 2007

More data are required to set up specific cut offs relevant for the virological

outcome and significant progress in this area is awaited depending on

availability and widened use of standardized HIV DNA assays.

What about the impact of archived resistance in virologically

suppressed patients that need a therapy switch?

2016

RT resistance mutations detected by population sequencing (PS) and deep sequencing (DS), with indication of the number of patients in whom the mutations were present.

Of the 20 patients selected according to the Sanger sequencing results, 17 of them had valid RNA and DNAdeep sequencing results. The DS results shown are the results after read data processing and quality filteringbut before hypermutation cleanup. Mutations that result from a G-to-A transition are underlined.

Dauwe et al, J Clin Microbiol 2017

A

Patients with pre-existent NRTI+NNRTI resistance had a higher probability of

experiencing VR compared to those harboring pre-existent NRTI or NNRTI

resistance and to those without pre-existent RTI resistance.

Armenia et al., JAC Jan 2017

0 12 24 36 48 60 72

0.0

0.2

0.4

0.6

0.8

1.0

224 197 175 140252

12 24 36 480

Time (Weeks)

No. at risk

0.0

0.2

0.4

0.6

0.8

1.0

Pro

ba

bilit

y o

f e

xp

eri

en

cin

g

vir

olo

gic

al

reb

ou

nd

34 32 30 2839

12 9 7 718

9.4%11.5%

39.2%

p<0.0001

Pre-existent RTI-resistance before switching:

No resistance

NRTI or NNRTI

NRTI + NNRTI

102

60

18

7

75

72

13

6

The presence of historical M184V was associated with the risk ofexperiencing viral blips

Gagliardini et al Open Forum Infectious Diseases2018

Estimated probability of remaining free from VFin dual therapy for different time of viralsuppression

45Gagliardini et al Open Forum Infectious Diseases 2018

In an additional analysis selecting patients with equal to or less than3 years of viral suppression, the respective 1- and 3-year probabilities of remaining free from virological failure were 100.0% and 67.7% in the M184V+ group and 97.3% an 96.2% in theM184V- group (P = .002)

67.7%

96.2%

JAC 2018

Switch Strategies for Virologically Suppressed

Persons

A complete ARV history with HIV-VL,

tolerability issues and cumulative genotypic

resistance history should be analysed prior to

any drug switch……

October 2017

…..Together with duration of viral

suppression and the type of mutations (and

their fitness)!

1. Sarmati L, Nicastri E, Uccella I, et al. 2003. J Clin Microbiol 41:1760-2.

2. Parisi SG, Boldrin C, Cruciani M, et al. 2007. J Clin Microbiol 45:1783-1788.

3. Turriziani O, Bucci M, Stano A, et al. 2007. J Acquir Immune Defic Syndr 44:518-524.

4. Palmisano L, Galluzzo CM, Giuliano M. 2009. J Acquir Immune Defic Syndr 51:233-234.

5. Banks L, Gholamin S, White E, et al. 2012. J AIDS Clin Res 3:141-147.

6. Delaugerre C, Braun J, Charreau I, et al. 2012. HIV Med 13:517-525.

7. Bon I, Turriziani O, Musumeci G, et al. J Med Virol 2015 87:315-322.

8. Fabeni L, Berno G, Svicher V, et al. 2015. J Clin Microbiol 53:2935-41.

9. Gallien S, Charreau I, Nere ML, et al. 2015. J Antimicrob Chemother 70:562-565.

10. Lubke N, Di Cristanziano V, Sierra S, et al. 2015. Intervirology 58:184-189.

11. Gantner P, Morand-Joubert L, Sueur C, et al. 2016. J Antimicrob Chemother 71:751-61.

12. Michelini Z, Galluzzo CM, Pirillo MF, et al. 2016. J Med Virol. doi: 10.1002/jmv.24581.

13. Fernández-Caballero JÁ, Chueca N, Álvarez M, et al. 2016. BMC Infect Dis. 16:197.

14. Zaccarelli M, Santoro MM, Armenia D, et al. 2016. J Clin Virol 82:94-100.

15. Lambert-Niclot S, Allavena C, Grude M, et al. 2016. J Antimicrob Chemother. 71:2248-51.

16. Rodallec A, Le Guen L, Leplat A, et al. 2017. IAS. Abstract MOPEB0270.

17. Allavena C, Rodallec A, Leplat A, et al. J Virol Methods. 2018 Jan;251:106-110.

18. Armenia D, Zaccarelli M, Borghi V et al. J Clin Virol. 2018 Jul;104:61-64.

19. Boukli N, Boyd A, Collot M, et al. J Antimicrob Chemother. 2018 Aug 17.

20. Rodriguez C, Nere ML, et al. J Antimicrob Chemother. 2018 Aug 20.

21. Sotillo A, Sierra O, Martínez-Prats L, et al. J Virol Methods. 2018 Oct;260:1-5.

HIV DNA Genotypic Resistance Test is a good tool for therapy optimization

in both drug-naïve and drug-experienced patients

Journal of Clinical Virology 2016

Proportion of Patients with MRM in PBMCs and Cumulative Plasma(149 Patients with DNA GRT and ≥2 Plasma GRTs, 9 Patients for INSTI)

50.343

30.2

11.1

61.1

38.3

28.2

16.811.1

51

11.4 10.1

1.3 0.3

20.1

0

10

20

30

40

50

60

70

80

90

100

NRTI NNRTI PI INSTI Overall(PI/NNRTI/NRTI)

Re

sist

ance

pre

vale

nce

(%

) Mutations detected only in cumulative plasma

Mutations detected in PBMCs and cumulative plasma

Mutations detected only in PBMCs

1.29 (±1.67) 0.74 (±1.00) 0.77 (±1.39) 0.11 (±0.33) 2.79 (±3.26)

1.03 (±1.66) 0.38 (±0.72) 0.34 (±8.84) 0.44 (±1.33) 1.75 (±2.57)

0.13 (±0.37) 0.14 (±0.47) 0.02 (±0.18) 0.00 (±0.00) 0.29 (±0.71)

Mean (±SD) number of MRM,

Zaccarelli et al., JCV2016

By exploring plasma cumulative resistance for any class andresistance detected in PBMC, 20.1% of patients harboured majorresistance mutations (MRMs) not detected in any of previous GRTsperformed in plasma.

Resistance detected in PBMCs predicts virological rebound in HIV-1

suppressed patients switching treatment

Armenia S et al. J Clin Virol 2018;104:61-64

Probability of viral rebound at month 24

Conclusions (I)

• The construction and management of antiretroviral therapy

is designed to take into account a long-term strategy

finalized to decrease to the lowest possible level of HIV

replication and disease /comorbidity progression.

• In this contest, the evaluation of clinically relevant

virological biomarkers not considered in the past (such as

HIV-DNA) is today crucial to ensure a long term control

replication.

• Moreover, they can also be useful in better understanding

factors assessing off-therapy virological remission, and

thus could be relevant for therapeutic strategies aimed at

achieving HIV cure.

• The improvement of resistance testing, finalized to detect

resistance even at undetectable viremia, may allow

clinicians to optimize therapy in the case of switch for

treatment simplification.

• Further investigation, through ultra-sensitive technology,

is needed to clarify the clinical impact of resistance

present in PBMCs.

• Joint efforts among virologists, immunologists, and

clinicians are necessary in order to properly position these

virological parameters into clinical practice and current

guidelines.

Conclusions (II)

Thank you for

the attention!