How does the reservoir influence the clinician's decisions

Prof. Carlo Federico Perno Acknowledgements The Patients University of Milan, Milan : C. Alteri. University of “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. 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. 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- 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. 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) Among 9014 isolates from cART failing patients the prevalence of M184V dramatically decreased from 56% in 1999 to 21% in 2013. In the last 5 years the M184V prevalence is stably settled at 17%.

Prevalence of M184V among isolates from cART failing patients over time

100% 90% 80% 70% 60% 60% 56% 57% 60% 51% 54% 45% 46% 50% 41% 40% 33% 27% % of % isolates 30% 23% 21% 17% 16% 19% 17% 18% 20% P<0.001 P=0.705 10% 0%

Analysis performed on 9014 isolates from cART failing patients (Update August 2018) Armenia & Santoro, Unpublished data 2019 In the past M184V was mainly detected in isolates under suboptimal regimens based on NRTIs or unboosted PIs

Prevalence of regimens used at the moment of M184V detection over time Suboptimal 100% 90% 80%

detection 70% 60% 59%

50% M184V M184V

at 40% 30% 20% P<0.001 10%

regimens 0%

0% % of of %

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 229 varied autopsy tissues from 20 ART- treated patients with low or undetectable plasma 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 findings were identified in all partecipants (brain, spleen, lung, lymph node, liver, aorta and kidney).

Tissues assayed with the number of HIV+ (red) S.L. Lamers 2016; 20:8968-83 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. Defining total-body AIDS-virus burden with implications for curative strategies

In the quest for a functional cure or eradication of HIV infection, we need to know how large 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-human primates and lymphoid tissue (LT) biopsies from infected humans. Before antiretroviral therapy (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 and their persistence was associated with relatively low drug concentrations in LT compared to peripheral blood. Prolonged ART also reduced the level of SIV and HIV-DNA+ cells, but the estimated size of the residual tissue burden of 108 vDNA+ cells that potentially harbor replication competent proviruses, along with the evidence for continuing virus production in LT despite ART, identify two important sources for rebound following treatment interruption. The large sizes of these tissue reservoirs underscore the challenges in developing “HIV cure” 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

During ART the numbers of virus (v) RNA+ cells substantially decreased but remained detectable. Graphical representation of the proportion of vRNA+ cells in each organ 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 decreased but remained detectable. Graphical representation of the proportion of vRNA+ cells in each organ system before and during suppressive ART.

Jacob D Estes Nature Medicine 2017 « Size » of the HIV reservoir

The « real reservoir » ?

• The vast majority of proviruses that persist on ART are defective.

Ho et al. Cell 2013 • 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.

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.

Bruner et al. Nature 2019 2018 The Remarkable Frequency of Human Immunodeficiency Virus Type 1 Genetic Recombination A Onafuwa-Nuga and A Telesnitsky Microbiology and Molecular Biology Reviews - 2009

The vast majority of acute transforming retroviruses are replication defective, with the oncogene-containing genome being transmissible 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 recombining with its replication-competent “helper.” In fact, there is some evidence that Rous sarcoma virus, possibly the only naturally arising replication-competent retrovirus containing a host oncogene, was replication defective initially Infectivity of recombinant viruses generated following transfection of 8E5 cells with defective molecular clones of HIV.

As we observed for 8E5 in this study, genetic recombination could generate replication-competent viruses from such a collection of defective proviral sequences.

• The HIV-1 sanctuary: the meaning of compartmentalization • This is the first study to evaluate GALT and LN tissue concentrations in patients receiving RAL and 800 mg daily DRV.

• Tissue:plasma ratios were higher in ileum>rectum as shown previously, and lowest in lymph node.

• In a limited number of participants, concentrations of RAL were significantly lower in lymph nodes vs. GALT, supporting prior observations.

• These results support the current limited data on tissue ART drug concentrations and have potential implications on HIV cure strategies.

Lee, et al Abstr 407, CROI 2017 Untreated Treated

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

Ceccherini-Silberstein et al. , JAC Dec 2018 By considering the 397 patients achieving virological suppression, the probability of experiencing virological rebound, defined by 2 confirmed plasma HIV-RNA >50 copies/mL, was 12% (95% CI: 8.6-15.5). By stratifying patients for the 3 different pre-ART HIV-DNA levels, increasing rates of virological rebound were found by increasing pre-ART HIV-DNA.

15.2% 17.2%

4.8% 7.4% 4.3% 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, PS6/4 Véronique Avettand-Fenoel, Constance Delaugerre, Diane Descamps, Esteban Martinez, Jacques Reynes, Gilles Peytavin, Dominique Costagliola, Christine Katlama, Vincent Calvez, Anne-Geneviève Marcelin.

FACTORS PREDICTING VIROLOGICAL FAILURE DURING DOLUTEGRAVIR MAINTENANCE MONOTHERAPY Ingeborg 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 W48 W96

HIV-1 infected patients Triple therapy DRV/r 800/100mg +2 NRTIs • First-line ART • VL undetectable • CD4 nadir >100 cells/mm3 Monotherapy • CD4 at baseline ≥200 DRV/r 800/100mg cells/mm3 N= 77 DOMONO

▪ Substudy of the DOMONO (NCT02401828) ▪ Randomized clinical trial

W48

HIV-1 infected patients

• VL undetectable Monotherapy • CD4 nadir ≥200 DTG 50mg N= 77 cells/mm3 • HIV RNA zenith < 105 copies/ml RESULTS * * *

Undetected Detected Failers Undetected Detected Failers PROTEA DOMONO

*: 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 DNA deep sequencing results. The DS results shown are the results after read data processing and quality filtering but before hypermutation cleanup. Mutations that result from a G-to-A transition are underlined.

Dauwe et al, J Clin Microbiol 2017 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.

Pre-existent RTI-resistance before switching: No resistance

A 1.0 1.0 NRTI or NNRTI NRTI + NNRTI

0.8 0.8

0.6 0.6 rebound

p<0.0001 of experiencing

0.4 0.4 39.2% 0.2 virological 0.2 11.5% Probability Probability 9.4%

0.0 0.0

00 1212 2424 3636 4848 6060 7272 Time (Weeks) 252 224 197 175 140 102 75 39 34 32 30 28 18 13 18 12 9 7 7 7 6 No. at risk Armenia et al., JAC Jan 2017

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

Gagliardini et al Open Forum Infectious Diseases 2018 Estimated probability of remaining free from VF in dual therapy for different time of viral suppression

96.2%

67.7% In an additional analysis selecting patients with equal to or less than 3 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 the M184V- group (P = .002)

Gagliardini et al 2018 Open Forum Infectious Diseases45

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……

…..Together with duration of viral suppression and the type of mutations (and their fitness)!

October 2017 HIV DNA Genotypic Resistance Test is a good tool for therapy optimization in both drug-naïve and drug-experienced patients 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. Journal of Clinical Virology 2016 By exploring plasma cumulative resistance for any class and resistance detected in PBMC, 20.1% of patients harboured major resistance mutations (MRMs) not detected in any of previous GRTs performed in plasma. Proportion of Patients with MRM in PBMCs and Cumulative Plasma (149 Patients with DNA GRT and ≥2 Plasma GRTs, 9 Patients for INSTI) 100 Mutations detected only in cumulative plasma 90 Mutations detected in PBMCs and cumulative plasma 80 Mutations detected only in PBMCs 70 61.1 60 50.3 51 50 43 38.3 40 28.2 30.2 30 16.8 20.1 20 11.4 10.1 11.111.1 10 1.3

Resistance prevalence (%) prevalence Resistance 0.3 0 NRTI NNRTI PI INSTI Overall (PI/NNRTI/NRTI) Mean (±SD) 1.29 (±1.67) 0.74 (±1.00) 0.77 (±1.39) 0.11 (±0.33) 2.79 (±3.26) number of 1.03 (±1.66) 0.38 (±0.72) 0.34 (±8.84) 0.44 (±1.33) 1.75 (±2.57) MRM, 0.13 (±0.37) 0.14 (±0.47) 0.02 (±0.18) 0.00 (±0.00) 0.29 (±0.71) Zaccarelli et al., JCV2016 Resistance detected in PBMCs predicts virological rebound in HIV-1 suppressed patients switching treatment

Probability of viral rebound at month 24

Armenia S et al. J Clin Virol 2018;104:61-64 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. Conclusions (II)

• 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. Thank you for the attention!