Early Combination Antiretroviral Therapy in Infants

Impact of Early Initiation of Combination Antiretroviral Therapy on Measures of Virus in Peripheral Blood of Vertically HIV-1-Infected

Children

Jason Brophy1, Tae-Wook Chun2, Lindy Samson1, Fatima Kakkar3, Hugo Soudeyns3, Mario Ostrowski4, S. Mujib5, John Kim6, Paul Sandstrom6, Richard Harrigan7, Stanley E. Read8, Ari Bitnun8

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 1 Case 2 Case 3* Case 4*

Maternal Characteristics

Age 30 years 32 years 29 years 29 years

Viral load pre-delivery 97,701 c/mL Unknown** 6326 c/mL 6326 c/mL

CD4 count pre-delivery 190 cells/L 10 cells/L 61 cells/µL 61 cells/µL

Clade G (CRF 6) Unknown C C

Infant Characteristics

Mode of delivery Emergency C/S Spontaneous Spontaneous Spontaneous

Gestational age at birth 34 weeks 27 weeks 36 weeks 36 weeks

Birth weight 2980 grams 1070 grams 2270 grams 1640 grams

HIV PCR (age) Positive (day 1) Positive (day 2) Positive (day 1) 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 loadCD4 count

3940 (55%)

Case 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


CD4 count 2663 cells/L (63%, day 15)

· Now 3 years old· Remain on same ART combination and have sustained virologic suppresSion


CD4 count 1997 cells/L (41%, day 17)

795 c/mL (day 12)

Case 1 Case 2 Case 3 Case 4Age/Sex 7 years/F 7 years/F 2.5 years/F 2.5 years/M

Serology (ELISA, WB) Negative Negative Negative Negative

HIV-specific T-cell responses (Gag, Nef)

Undetectable Undetectable Undetectable Undetectable

Plasma viremia ‡ < 1.5 copies/mL < 1.5 copies/mL < 1.5 copies/mL < 1.5 copies/mL

Cell-associated proviral DNA

< 2.6 copies/g < 2.6 copies/g < 2.6 copies/g < 2.6 copies/g

Cell-associated RNA § 24.9 copies/1.5g RNA

20.0 copies/1.5g RNA

19.5 copies/1.5g RNA

130 copies/1.5g RNA

HIV RNA in stimulated CD4 T-cells

Not detected (5.4 million cells)




Quantitative CD4 T-cell co-culture

Not detected 0.1 infectious units/10^6 CD4 T-cells

Not detected Not done

CCR-5 32 status Wild type Wild type Wild type 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

A*01–A*66; B*55–B*58; C*03–C*03

HLA-B variation ¥ 67CM; 70K/S; 97R/T 67S; 70N; 97R 67Y/M; 70Q/S; 97R/T 67Y/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 TI Case 5

Age/Sex/Clade 3 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/1.5g RNA

HIV RNA in stimulated T-cells Not detected (6.7 million cells)

CCR-5 32 status Wild type

HLA typingHLA-B variation ¥

A*01-A*02; B*08-B*27; C*01-C*0767C/F; 70N/Q; 97N/S

Case 5

HIV DNA PCR positive

(Day 4)

cART started (day 1) - AZT/3TC/NVP

CD4 1995 (38.3%)

cART changed (day 21) - AZT/3TC/LPV

CD4 2568 (46.2%)

CD4 2145 (40.0%)

CD4 2299(37.3%)

CD4 2584 (39.3%)

-70 -5 21 42 77 112 14010

100

1000

10000

100000

1000000

<40

779711358

9944

102172

3915

Days After Stopping Therapy

Vira

l Loa

d Lo

g 10

HIV ELISA,WB,DNA PCR: POSITIVE

CMI TESTING:gag/nef POSIT-

IVEAZT/3TC/LPV

Re-started

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 2010Lazaryan, J Virology 2006; Lazaryan, J Virology 2010

Discussion Our 5th 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 (EPIC4) 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 EPIC4 study

QUESTIONS?

Reservoir, Immunologic Responses and Genetics

Level of cell-associated HIV-1 DNA in CD4+ T cellsreal-time PCR

Level of cell-associated HIV RNACobas Ampliprep/Cobas Taqman HIV 1 assay‐

Residual plasma viremiamodified Cobas Ampliprep/Cobas Taqman HIV 1 assay‐

Presence of replication competent viruslevel of virion-associated HIV RNA in culture supernatant after mitogenic stimulation

co-culture assayHIV serology; HIV-specific cell-mediated immune responses; HLA typing and CCR5 delta 32 genotyping

Case 1 Case 2 Case 3 Case 4Age/Sex 7 years/F 7 years/F 2.5 years/F 2.5 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

A*01–A*66; B*55–B*58; C*03–C*03

HLA-B variation ¥ 67CM; 70K/S; 97R/T 67S; 70N; 97R 67Y/M; 70Q/S; 97R/T 67Y/M; 70Q/S; 97R/T

CCR-5 32 status Wild type Wild type Wild type Wild type

Serology (ELISA, WB) Negative Negative Negative Negative

HIV-specific T-cell responses (Gag, Nef)

Undetectable Undetectable Undetectable Undetectable

Plasma viremia ‡ < 1.5 copies/mL < 1.5 copies/mL < 1.5 copies/mL < 1.5 copies/mL

Cell-associated proviral DNA

< 2.6 copies/g < 2.6 copies/g < 2.6 copies/g < 2.6 copies/g

Cell-associated RNA § 24.9 copies/1.5g RNA

20.0 copies/1.5g RNA 19.5 copies/1.5g RNA

130 copies/1.5g RNA

HIV RNA in stimulated CD4 T-cells





Quantitative CD4 T-cell co-culture

Not detected 0.1 infectious units/10^6 CD4 T-cells

Not detected Not 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

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Early Combination Antiretroviral Therapy in Infants