Impact of Early Initiation of Combination Antiretroviral Therapy on Measures of Virus in Peripheral Blood of Vertically HIV-1-Infected Children Jason Brophy 1, Tae-Wook Chun 2, Lindy Samson 1, Fatima Kakkar 3, Hugo Soudeyns 3, Mario Ostrowski 4, S. Mujib 5, John Kim 6, Paul Sandstrom 6, Richard Harrigan 7, Stanley E. Read 8, Ari Bitnun 8 1. Children’s Hospital of Eastern Ontario, University of Ottawa; 2. National Institute of Allergy & Infectious Diseases, National Institutes of Health, U.S.A.; 3. CHU Sainte-Justine, Université de Montréal; 4. Department of Immunology and Medicine, University of Toronto, 5. University of Toronto, Institute of Medical Sciences, Department of Medicine; 6. National HIV & Retrovirology Laboratories, Public Health Agency of Canada; 7. University of British Columbia, British Columbia Centre for Excellence in HIV/AIDS; 8. Hospital for Sick Children, University of Toronto

Early Combination Antiretroviral Therapy in Infants  The “Mississippi baby” received early cART – experienced a 2-year “viral remission” after stopping treatment until a recent viral rebound  raises the possibility of this as an intervention to limit reservoir establishment and enable “viral remission”  Triple cART as HIV-post exposure prophylaxis has been routinely administered to newborns at high risk for HIV infection in our centres for many years  SickKids (Toronto), CHEO (Ottawa), and CHU Ste-Justine (Montreal)

Objectives  To investigate HIV-1 reservoirs in peripheral blood of HIV-1-infected children with SVS following initiation of cART within 72 hours of birth  SVS: defined as absence of detectable virus in standard viral load (VL) assay subsequent to having achieved an undetectable VL (< 50 copies/mL)

Methods  Retrospective review at our 3 centres of all children born to HIV-infected mothers who received triple cART within 72 hours of birth  Evaluation of HIV reservoir, immune responses, and genetic characteristics in those infected children with SVS after cART

Results – Retrospective Review  136 infants received triple cART  HIV infection in 12 (8.8%)  In utero infection probable in at least 50% (n=6; HIV PCR positive within 48 hours of birth)  Timing uncertain in 50% (n=6) as testing done after 48 hrs  Four HIV-infected children achieved SVS (Cases 1-4)  Eight HIV-infected children did not achieve SVS  6 of 8 did not achieve virologic suppression due to poor adherence  2 of 8 initially suppressed for 2-3 years, then experienced viral rebound after poor adherence (one during the course of our study – Case 5)

Reservoir Evaluation of 4 Early-Treated, HIV-Infected Children  Cases 1 to 4 had SVS from 2-7 years  All 4 remained on their original cART regimen of zidovudine, lamivudine, and nevirapine

Case 1Case 2Case 3*Case 4* Maternal Characteristics Age30 years32 years29 years Viral load pre-delivery97,701 c/mLUnknown**6326 c/mL CD4 count pre-delivery 190 cells/  L10 cells/  L 61 cells/µL CladeG (CRF 6)UnknownCC Infant Characteristics Mode of deliveryEmergency C/SSpontaneous Gestational age at birth34 weeks27 weeks36 weeks Birth weight2980 grams1070 grams2270 grams1640 grams HIV PCR (age)Positive (day 1)Positive (day 2)Positive (day 1) Maternal Characteristics and Infant Diagnostic Testing * Non-identical twins; ** mother died soon after delivery from OI

Case 1 cART initiated (day 1) HIV DNA PCR positive (day 1)  Now 7.5 years old  Remains on same ART combination  Has maintained an undetectable viral load CD4 count 3940 (55%)

Case 2 cART initiated (day 1) HIV DNA PCR positive (day 2) CD4 count 1447 cells/  L (28.9%; day 20)  Now 7.5 years old  Remains on same ART combination  Undetectable viral load

Cases 3 & 4 cART initiated (day 1) HIV DNA PCR positive (day 1) CD4 count 2663 cells/  L (63%, day 15)  Now 3 years old  Remain on same ART combination and have sustained virologic suppresSion cART initiated (day 1) HIV DNA PCR positive (day 1) CD4 count 1997 cells/  L (41%, day 17) 795 c/mL (day 12)

Case 1Case 2Case 3Case 4 Age/Sex7 years/F 2.5 years/F2.5 years/M Serology (ELISA, WB)Negative HIV-specific T-cell responses (Gag, Nef) Undetectable Plasma viremia ‡< 1.5 copies/mL Cell-associated proviral DNA < 2.6 copies/  g Cell-associated RNA § 24.9 copies/1.5  g RNA 20.0 copies/1.5  g RNA 19.5 copies/1.5  g RNA 130 copies/1.5  g RNA HIV RNA in stimulated CD4 T- cells Not detected (5.4 million cells) Not detected (7.2 million cells) Not detected (8.0 million cells) Quantitative CD4 T- cell co-culture Not detected 0.1 infectious units/10^6 CD4 T-cells Not detectedNot done CCR-5  32 status Wild type HLA typing A*01–A*02; B*27– B*58; C*02–C*03 A*30–A*66; B*44– B*45; C*03–C*04 A*01–A*66; B*55– B*58; C*03–C*03 HLA-B variation ¥67CM; 70K/S; 97R/T67S; 70N; 97R67Y/M; 70Q/S; 97R/T ‡ Limit of detection: 1.5 copies/mL; each assay performed on 6 mL of whole blood; § Limit of detection: 1.5  g RNA; performed in duplicate; ¶ Limit of detection; 20 copies/ml; ¥ HLA-B variation at positions associated with better virologic control (lower set-point) and specific protective amino acid substitutions are indicated in bold

Case 5 – Prior to Treatment Interruption Initial findings before TICase 5 Age/Sex/Clade3 years/M/Clade B Serology (ELISA & WB)Negative Plasma viremia ‡< 1.5 copies/mL Cell-associated proviral DNA < 2.6 copies/  g Cell-associated RNA § 149 copies/1.5  g RNA HIV RNA in stimulated T-cellsNot detected (6.7 million cells) CCR-5  32 status Wild type HLA typing HLA-B variation ¥ A*01-A*02; B*08-B*27; C*01-C*07 67C/F; 70N/Q; 97N/S

Case 5 HIV DNA PCR positive (Day 4) cART started (day 1) - AZT/3TC/NVP CD (38.3%) cART changed (day 21) - AZT/3TC/LPV CD (46.2%) CD (40.0%) CD (37.3%) CD (39.3%)

Discussion  Absence of detectable HIV DNA and absent/very low levels of replication-competent virus in peripheral blood and lack of HIV-specific immune responses demonstrated in a subgroup of children initiated on cART <72 hours of birth  Suggests early cART initiation can greatly reduce HIV reservoir size  Genetic factors may also play an important role - protective HLA genotypes were found in 3 of 4 children  HLA B*58  Sequence variation at HLA-B positions 67, 70 and 97 (associated with superior control of HIV replication)  The child with replication competent virus did not have these protective genotypic features International HIV Controllers Study, Science 2010 Lazaryan, J Virology 2006; Lazaryan, J Virology 2010

Discussion  Our 5 th case with rapid viral rebound after interruption of therapy despite limited reservoir size demonstrates that early cART will not be effective in all patients  Multiple factors may have influenced this patient’s outcome  Baseline NNRTI resistance, sub-optimal initial regimen  Low-level detectable VL after initial suppression  This, along with late relapse of viral replication in Mississippi baby, underscore the need for better understanding of contributing factors to reservoir & viral control  Host and viral genetics  Timing and completeness of initial viral suppression  Selection of components of cART regimen in infants

Conclusions  Based on our study findings and other reports, early cART for HIV-infected infants clearly limits size of viral reservoir  Additional non-blood reservoir sites require investigation  The clinical significance and benefit of this remain to be seen  Accurate estimation of size of HIV viral reservoir in children is significantly impacted by limitations in collection of adequate blood volumes in children compared with adults  A prospective multi-centre observational study (EPIC 4 ) is underway in Canada to determine the impact of early versus later treatment on reservoir size and HIV control in children

Acknowledgments  The children and parents who agreed to participate in this study  The Canadian Institutes for Health Research, Canadian Association for AIDS Research, and the International AIDS Society for funding to carry out EPIC 4 study

QUESTIONS?

Reservoir, Immunologic Responses and Genetics  Level of cell-associated HIV-1 DNA in CD4+ T cells  real-time PCR  Level of cell-associated HIV RNA  Cobas Ampliprep/Cobas Taqman HIV‐1 assay  Residual plasma viremia  modified Cobas Ampliprep/Cobas Taqman HIV‐1 assay  Presence of replication competent virus  level of virion-associated HIV RNA in culture supernatant after mitogenic stimulation  co-culture assay  HIV serology; HIV-specific cell-mediated immune responses; HLA typing and CCR5 delta 32 genotyping

Case 1Case 2Case 3Case 4 Age/Sex7 years/F 2.5 years/F2.5 years/M HLA typing A*01–A*02; B*27– B*58; C*02–C*03 A*30–A*66; B*44– B*45; C*03–C*04 A*01–A*66; B*55– B*58; C*03–C*03 HLA-B variation ¥67CM; 70K/S; 97R/T67S; 70N; 97R67Y/M; 70Q/S; 97R/T CCR-5  32 status Wild type Serology (ELISA, WB)Negative HIV-specific T-cell responses (Gag, Nef) Undetectable Plasma viremia ‡< 1.5 copies/mL Cell-associated proviral DNA < 2.6 copies/  g Cell-associated RNA § 24.9 copies/1.5  g RNA 20.0 copies/1.5  g RNA 19.5 copies/1.5  g RNA 130 copies/1.5  g RNA HIV RNA in stimulated CD4 T- cells Not detected (5.4 million cells) Not detected (7.2 million cells) Not detected (8.0 million cells) Quantitative CD4 T- cell co-culture Not detected0.1 infectious units/10^6 CD4 T-cells Not detectedNot done ‡ Limit of detection: 1.5 copies/mL; each assay performed on 6 mL of whole blood; § Limit of detection: 1.5  g RNA; performed in duplicate; ¶ Limit of detection; 20 copies/ml; ¥ HLA-B variation at positions associated with better virologic control (lower set-point) and specific protective amino acid substitutions are indicated in bold