CORRELATION BETWEEN HIV-1 MOLECULAR CHARACTERISTICS AND RESPONSE TO HAART IN PEDIATRIC POPULATION FROM SAO PAULO, BRAZIL Cristina Mendes de Oliveira, Rosangela Rodrigues, Carla M P Vazquez, Maria do Socorro Carneiro Ferrão, Alexandre Campeas, Márcia Crozatti, Silvia Marques, Luis Fernando de Macedo Brígido. Laboratório de Retrovírus, Instituto Adolfo Lutz, São Paulo, Brasil Instituto de Infectologia Emílio Ribas, são Paulo, Brasil

Decision on when to start ARV therapy is a matter of controversy even for adults Children pose additional challenges even in virological failure due to specific issues, including: –Potential interference with normal growth and development –Toxicity; Pharmaco-dynamics –Limited ARV options; posology limitations –Adherence

What is the contribution of molecular information to the management of children living with HIV/AIDS ARV drug resistance test were the fist molecular characterization brought into clinical practice routine, subsidizing salvage ARV therapy. Additional molecular characteristics of HIV-1 that provide surrogate markers do determine when and how to treat HIV infected children can be useful in subsidizing decision on starting as well as changing ARV therapy.

Methodology Inclusion criteriaInclusion criteria Informed consent from parents or gardian and additional informal consent for older children at inclusion visit Informed consent from parents or gardian and additional informal consent for older children at inclusion visit Naïve : samples collected before initiation of ARV ( ) Treated : children or adolescent using EFV or LPV based HAART ( collected from 6 / 2005 to 11 / 2006 ) Viral load bDNA (Bayer); TCD4 ( BD), performed at certified National Network Laboratory HIV sequencing performed at Retrovirus Laboratory, Adolfo Lutz Institute

Samples collected at baseline EDTA blood collected at service and processed within 6 hs Cell lysade and plasma maintained at -70 °C Extraction plasma RNA and/or Cell DNA (Qiagem/GFX) RNA retrotranscrition (Superscript II, Invitrogen, USA) Nested PCR Sequencing (ABI 3100) with Big Dye chemistry (ABI,USA) Edition : Sequencher (Gene codes corp.,USA) Phylogeny : REGA, NCBI, SIMPLOT, PAUP Drug Resistance Mutations (DRM), Stanford HIV Database

Baseline Characteristics Naïve Treated n=16 n=79 Age (mean years ) Gender (% males) 44% 54% Mean number of ARV regimens TCD4 ( cels/mm 3 ) Plasma Viremia (log) % undetectable 0% 24%

Clinical outcome of ARV treated cases 78 cases with follow up information 79.5% Asymptomatic 7% Bacterial Pneumonia 4% TB Other complications One death (adenocarcinoma) One loss to follow up (change town) - Citopenias in 6% - Pancreatitis - CMV

Outcome categories defined based on : (i)Viral load at last observation (end first semester of 2008) (ii)Clinical observation (patients’ pediatrician) at last documented visit and TCD4 cell counts (cells / mm3) at last observation GOOD RESPONDERS TCD4 counts over 350 and clinically asymptomatic POOR RESPONDERS TCD4 counts under 350 or clinical Aids related condition

Viral Load and TCD4 outcome TCD4 mean 650 cels/mm3 TCD4 mean 691cels/mm3 61% with TCD4 over % % below % aviremic - among viremic Log % aviremic - among viremic Log At baseline At last visit

Treatment at last evaluation NNRTIs + EFV and/or LPV based HAART ARVAvir GR LPV 65%41%62% EFV 21%23%67% EFV+LPV 12%40%80%

Age (mean) % males number regimens (mean) Time on ARV (weeks) Time of follow up (weeks) % avir at baseline * Baseline Viremia among viremic Avir (<50c/ML) Vir 2-4 Log Vir> 4 Log Baseline characteristics by Virological outcome * P< 0.002

Presence of DRMs by virological strata TAM NRTI. DRM NNRTI DRM PI. DRM PI. sec/acc PI Major DRM Vir> vir Avir Vir>4 x Avir P= 0.43 p=0.14 p=0.9 P<0.001 p<0.05 p<0.01 Mean number of DRM at RT and PT DRM Drug Resistance Mutations RT reverse transcriptase gene PT –Protease gene Tam -Tymidine Analogue mutations NRTI -Nucleoside analogue ARV class NNRTI - Non analogue ARV class PI -Protease inhibitor class Sec/acc -Secondary/accessory mutations Major- Principal mutations to PI

Proportion of DRM to NRTI class in ARV resistance codons at Reverse transcriptase (RT) by treatment outcome PR poor response; GR good response

Proportion of DRM to NNRTI class in ARV resistance codons at Reverse transcriptase (RT) by treatment outcome PR poor response; GR good response

Proportion of DRM to Protease Inhibitors (PI) at ARV resistance codon at PROTEASE by treatment outcome

Proportion of DRM to Protease Inhibitors (PI) at ARV resistance codon at PROTEASE by treatment outcome PR poor response; GR good response

Proportion of DRM to Protease Inhibitors (PI) at ARV resistance codon at PROTEASE by clinical outcome

Impact of DRM on treatment outcome V82A/F/T/SL90M GR 9% PR 45% 30% Proportion of selected major DRM at PR p< P=0.067

Impact of DRM on treatment outcome Among cases currently using LPV based HAART P< p=0.2 NNRTINRTI ALL NRTI TAM IP Acc /sec IP major GR PR GR Good responders ; PR Poor responders Mean number of DRM at RT and PT BY outcome

Impact of DRM on treatment outcome among cases using EFV based HAART NNRTINRTI ALL NRTI TAM IP Acc /sec IP major GR PR Mean number of DRM at RT and PT GR Good responders ; PR Poor responders

Protease polimorphisms by pol clade

V3 loop signatures among naïve and treated cases stratified by poor responders (PR) and good responders (GR)

PR GR

Recombination patterns at gag ( ), pol ( ) and env ( ), positions related to HXB2 ( K03455). HIV-1 B HVI-1 F HIV-1 C BF mosaics

HIV-1 clades by virological outcome and clinical outcome

Predicted HIV tropism by virological and clinical outcome

V3 loop AA alignment of paired cell and plasma

Impact V3 mixtures on treatment outcome Proportion of V3 with mixtures Proportion of nu Mixtures N GR49% PR44% Mean number of Mixtures at V3 by outcome Most sequences ( 51% among GR and 54% among PR) do not show mixtures at V3 P<0.031

DIVERSIDADE - DIVERGENCIA intra- hospedeiro Arts et al. 2007

Conclusions Albeit treated for over a mean of 449 weeks (8.6 years) over one third of these children had viral load< 50 c/ml at last evaluation Protease inhibitors mutations, both major as well as accessory/ Secondary DRM, correlated to viremia and poor clinical outcome; some polymorphisms common among naïve cases and may have an impact in resistance evolution Presence of mixtures at V3 codons were associated to good clinical outcome