A Prospective, Observational, Cohort, Multicenter Study of Early Anti-Retroviral Treatment in HIV- Infected Infants
Conditions: HIV Infections
Sponsor: PENTA Foundation

EARTH v3, DECEMBER 2021 
1 
Study Title: A prospective, observational, cohort, multicenter study of Early Anti-
Retroviral Treatment in HIV- infected Infants. 
 
 
Short Title: Early Anti-Retroviral Treatment in HIV- infected Children. 
 
Acronym: EARTH 
 
 
 
 
 
 
 
 
 
 
 
 
Version 3, Date DECEMBER 2021 
 
 
Sponsor: Penta (Child Health Research)  
Funded by ViiV. 
 
 
 
 
 

EARTH v3, DECEMBER 2021 
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TABLE OF CONTENTS 
 
Study Title: A prospective, observational, cohort, multicentre study of Early Anti-
Retroviral Treatment in HIV- infected Infants. 
1 
1 
STUDY PERSONNEL 
3 
2 
ABBREVIATIONS 
7 
3 
SUMMARY 
9 
4 
BACKGROUND 
15 
5 
RATIONALE 
17 
6 
HYPOTHESIS 
19 
7 
OBJECTIVES 
20 
8 
DESIGN 
22 
9 
ENDPOINTS 
23 
10 
SELECTION AND WITHDRAWAL OF PARTICIPANTS 
25 
11 
CLINICAL PROCEDURES AND METHODS 
27 
12 
DRUG REGIMEN 
34 
13 
POTENTIAL ADVERSE EVENTS REPORTING 
35 
14 
LABORATORY TESTING ASSAYS 
37 
15 
STATISTICAL ANALYSIS AND ACCESS TO SOURCES OF INFORMATION 
40 
16 
REGULATORY, ETHICAL AND LEGAL ISSUES 
44 
17 
ADMINISTRATIVE MATTERS AND PUBLICATION POLICY 
48 
18 
REFERENCES 
50 
 
 
 

EARTH v3, DECEMBER 2021 
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1 
STUDY PERSONNEL 
 
Sponsor Chairs: 
Carlo Giaquinto ( Penta (Child Health Research), Padova, Italy), carlo.giaquinto@unipid.it, 
+39 0498213585 
Paolo Rossi (Hospital Bambino Gesù, Roma, Italy), paolo.rossi@opbg.net  
 
Clinical Chairs, Protocol Implementation:  
Alfredo Tagarro (Hospital 12 de Octubre, Madrid, Spain, Universidad Europea de Madrid), 
alfredotagarro@gmail.com, +34 606194888 
Co-chairs 
Pablo Rojo (Hospital 12 de Octubre, Madrid, Spain), pablorojoconejo@aim.com, +34 
615513222). 
Louise Kuhn (Columbia University, NYC, USA), lk24@cumc.columbia.edu, +1 212 3052398) 
 
Immunology Chair 
Paolo Palma   
 
 
 
 
paolo.palma@opbg.net 
Childrens9 Hospital <Bambino Gesu=  
 
Tel: +39 0668592455 
P.ZZA Sant9Onofrio 4 00165 
Rome, Italy  
 
 
 
Virology Chair 
Dr Mathias Lichterfeld 
Ragon Institute of MGH, MIT and Harvard 
400 Technology Square, Cambridge, MA 02139, USA 
Tel: (617) 724-6454 
 
Ethics Chair  
Viviana 
Giannuzzi 
(Gianni 
Benzi 
Pharmacological 
Research 
Foundation), 
vg@benzifoundation.org +393201172913 
Alessandra Nardone (Fondazione Penta Onlus) alessandra.nardone@pentafoundation.org 
Co-chair: 
Maria 
Grazia 
Lain 
(F. 
Ariel 
Glaser 
contra 
o 
SIDA 
Pediatrico), 
mlain@arielglaser.org.mz 
 
In-country responsible of Ethics and Export / Import Permits 
South Africa: Shaun Barnabas, barnabas@sun.ac.za, Anita Janse Van Rensburg 
ajvanr@sun.ac.za 
Mozambique: Maria Grazia Lain, mlain@arielglaser.org.mz 
Italy: Ilaria Pepponi, ilaria.pepponi@opbg.net 
United Kingdom: Moira Spyer, moira.spyer@ucl.ac.uk 
Spain: Alfredo Tagarro, alfredotagarro@gmail.com 
USA: Louise Kuhn, lk24@cumc.columbia.edu 
 
Person in charge of oversight of sample management and shipping of samples:  
Dr Moira Spyer; moira.spyer@ucl.ac.uk (UCL Great Ormond Street Institute of Child Health) 

EARTH v3, DECEMBER 2021 
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Ilaria Pepponi, OPBG, Rome, ilaria.pepponi@opbg.net 
 
Field Sites Leaders: 
1. Tygerberg Children9s Hospital (TCH), Stellenbosch University (SU), Cape Town, South 
Africa.  
Clinical Leader: Shaun Barnabas, barnabas@sun.ac.za, Anita Janse Van Rensburg 
ajvanr@sun.ac.za 
Lab leader: Dr. Gert van Zyl; gert guvz@sun.ac.za 
 
2. Perinatal HIV Research Unit, Chris Hani Baragwaneth Hospital, University of the 
Witwatersrand, Soweto, South Africa. 
Clinical leader: Avy Violari, violaria@phru.co.za  
Lab leader (subcontracted): Fiona Shahim, Central Laboratory Services (CLS), Johannesburg. 
fshahim@cls.co.za 
 
3. Africa Health Research Institute , KwaZulu Natal, South Africa 
Clinical leader Nigel Klein, n.klein@ucl.ac.uk, Osse Behuhuma Obehuhuma@gmail.com 
Lab leader: Nigel Klein, n.klein@ucl.ac.uk,  
Thumbi N9dungu: thumbi.ndungu@ahri.org 
 
4. Centro de Investigaçao em Saude de Manhiça (CISM) and  Barcelona Institute for Global 
Health - ISGlobal 
Clinical leader: Elisa Lopez Varela, elisa.lopez@isglobal.org and  Tacilta Nhampossa, 
Tacilta.Nhampossa@manhica.net  Sheila Fernández, Sheila.fernandeez@isglobal.org  
Lab leader: Anelsio Cossa, Anelsio.Cossa@manhica.net 
 
5. Fundação Ariel Glaser contra o SIDA Pediátrico (F.Ariel) 
Clinical 
leader: 
Maria 
Grazia 
Laín, 
mlain@arielglaser.org.mz, 
Paula 
Vaz, 
pvaz@arielglaser.org.mz 
Lab 
leader: 
Dulce 
Bila, 
dbila@arielglaser.org.mz; 
Nadia 
Sitoe 
(INS-MOH) 
nadia_sitoe@yahoo.com.br  
 
6. Centre Hospitalier Universitaire Gabriel Touré 
Clinical leader: Mariam Sylla dr_mame@yahoo.fr  
Laboratory leader: anne-genevieve.marcelin@aphp.fr,  
Almoustapha Issiaka: almoustapha@gmail.com, amaiga@icermali.org 
 
 
Clinical, immunological, virological and statistical co-investigators and advisors:  
Dr. Denise Naniche 
Barcelona Institute for Global Health – ISGlobal     denise.naniche@isglobal.org 
Dr. Nicola Cotugno   
cotugnonicola@yahoo.it 
Children9 Hospital <Bambino Gesu=  
Dr.ssa Paola Zangari  
paola.zangari@opbg.net 
Children9 Hospital <Bambino Gesu= Tel: +39 06685925 
Kennedy Otwombe, otwombek@phru.co.za 

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Perinatal HIV Research Unit, Chris Hani Baragwaneth Hospital, University of the 
Witwatersrand, Soweto, South Africa 
Dr. Savita Pahwa,  
University of Miami Miller School of Medicine in Miami, USA; spahwa@med.miami.edu 
Anne-Genevieve Marcelin; Pierre et Marie Curie University in Paris; anne-
genevieve.marcelin@aphp.fr  
Dr. Camille Brehin, SERMAS-Fundación 12 de Octubre; brehin.c@chu-toulouse.fr 
Dr Moira Spyer; UCL Great Ormond Street Institute of Child Health; moira.spyer@ucl.ac.uk 
Dr. Eleni Nastuoli; UCL Great Ormond Street Institute of Child Health; e.nastouli@ucl.ac.uk 
Dr Anita de Rossi, University of Padova; anita.derossi@unipd.it 
 
 
Statistics: Sara Dominguez (Fundación 12 de Octubre), Kennedy Otwombe (Perinatal HIV 
Research Unit, Chris Hani Baragwaneth Hospital, University of the Witwatersrand, Soweto, 
South Africa). 
 
Data Management Board: 
Andrea 
Oletto 
(Penta 
(Child 
Health 
Research), 
Padova, 
Italy), 
andrea.oletto@pentafoundation.org 
Sara 
Domínguez 
(Hospital 
Universitario 
12 
de 
Octubre, 
Madrid, 
Spain); 
sara.dominguez.r@gmail.com 
Kennedy Otwombe (Perinatal HIV Research Unit, Chris Hani Baragwaneth Hospital, 
University of the Witwatersrand, Soweto, South Africa), otwombek@phru.co.za 
Alfredo 
Tagarro 
(Hospital 
Universitario 
12 
de 
Octubre, 
Madrid, 
Spain), 
alfredotagarro@gmail.com 
Dickman Gareta (AHRI, Kwa-Zulu Natal, ZA), Brian Grey and Lerato Mohale (Tygerberg, Cape 
Town, ZA), Juan Erasmus (PHRU, Johannesburg, ZA), Josue Togo (CH Bamako, Mali), Aderito 
magumane, (Manhiça, Mozambique), Loide Cardoso (Ariel Glaser, Maputo, Mozambique). 
 
Social Sciences Substudy Chair and researchers: 
Lead: Holly Peay, hpeay@rti.org 
RTI International  
Miriam Hartmann, mhartmann@rti.org 
RTI International  
Katherine Porter, kmporter@rti.org 
RTI International  
Danielle Toth, datoth@rti.org 
RTI International 
Gail Henderson, gail_henderson@med.unc.edu 
University of North Carolina at Chapel Hill 
Substudy 2: Strategies to improve viral suppression (SIVS) Substudy leaders 
Shaun Barnabas 
Tygerberg Children9s Hospital (TCH), Stellenbosch University (SU), Cape Town, South Africa.  
Clinical Leader: Shaun Barnabas, barnabas@sun.ac.za 
 
Elisa López Varela 

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Elisa Lopez Varela, elisa.lopez@isglobal.org 
 
 
Financial management:  
Daniel Gomez-Peña (Penta Foundation) daniel.Gomezpena@pentafoundation.org, +39 
320322600 
 

EARTH v3, DECEMBER 2021 
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2 
ABBREVIATIONS 
ABV:   
Abacavir 
AHRI:                African Health Research Institute 
AIDS:   
Acquired Immunodeficiency Syndrome 
ART:   
Antiretroviral Therapy 
ARV:                  Antiretroviral 
AUC:   
Area Under the Curve 
AZT:   
Zidovudine 
CAF:   
Clinical Assessment Form  
CCR:   
High risk pediatric clinic (from Portuguese: <consulta de criança em  
                      
risco=) 
CISM:   
Centro de Investigaçao em Saude de Manhiça 
CMV:   
Cytomegalovirus 
CTZ:   
Cotrimoxazole 
CRF:   
Case Report Form 
D4T:   
Stavudine  
DBS 
 
Dried Blood Spot 
DDI:   
Didanosine 
DNA:   
Deoxyribonucleic acid 
DSMB:  
Data Safety and Monitoring Board 
EFV:   
Efavirenz 
EIA:  
 
Enzyme immunoassay  
EPIICAL:  
Early treated Perinatally HIV Infected individuals: Improving Children9s Actual     
Life 
F.Ariel:  
Fundação Ariel Glaser contra o SIDA Pediátrico 
FLIPS:                Full Length Individual Sequencing  
FTC:   
Emtricitabine 
GCP:   
Good Clinical Practice 
GLP: 
 
Good Laboratory Practice 
HF: 
 
Health Facility 
HIV:   
Human Immunodeficiency Virus 
ICH:  
 
International Conference of Harmonization 
ICH-UCL: 
Institute of Child Health, University College London 
ICHT:  
Imperial College Healthcare NHS Trust 
ICF:  
 
Informed consent form 
IEC:  
 
Independent Ethics Committee 
INI:  
 
Integrase Inhibitor 
IPT:  
 
Isoniazide Preventive Therapy 
IQR:   
Interquartile Range 
IRB:  
 
Institutional Review Board 
JCRC  
Joint Clinical Research Centre, Kampala 
LPV/r:   
Lopinavir/ritonavir 
MTCT:   
Mother-to-child transmission 
MRC:  
Medical Research Council 
NDMTs:  
Novel Disease Modifying Therapies 
NNRTI:  
Non-nucleoside reverse transcriptase inhibitors 

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NVP:   
Nevirapine 
PaHIV:  
Perinatally acquired HIV 
PBMC:  
Peripheral Blood Mononuclear Cells 
PI:  
 
Protease Inhibitors 
PCR:   
Polymerase Chain Reaction 
PENTA:  
Pediatric European Network for Treatment of AIDS 
PMTCT:  
Prevention of mother-to-child transmission 
POC:   
Proof of Concept 
RNA:  
Ribonucleic acid 
ROC:   
Receiver Operating Characteristics 
SAE:   
Serious Adverse Event 
SD:  
 
Standard Deviation 
SMG:   
Study Management Group 
SOP:   
Standard Operating Procedure 
SSC:   
Study Steering Committee 
TNA:   
Total DNA 
TDF:   
Tenofovir 
UCL: 
 
University College London 
VL:  
 
Viral load 
WHO:              World Health Organization 
WP:  
 
Work Package 
3TC:   
Lamivudine  
 

EARTH v3, DECEMBER 2021 
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3 
SUMMARY 
 
Background: Early antiretroviral therapy (ART) initiation is correlated with a low 
reservoir (total HIV-DNA) and good immunological outcomes. The EPIICAL (Early treated 
Perinatally HIV Infected individuals: Improving Children9s Actual Life) project is an 
international consortium whose aim is to select promising HIV therapeutic strategies 
candidates for use among early-treated perinatally HIV-positive children. 
Study Design: Prospective cohort study 
Hypothesis: Monitoring HIV-DNA from birth during the first 4 years of age, we can 
identify and characterize a proportion of early-treated children who will have an excellent 
control of HIV at 4 years of age in terms of clinical, viral and immunological features, and 
other who will not have such excellent control. This differentiation will help us to identify 
which participants are more likely to benefit from which novel therapeutic interventions, 
depending on the profile of the patient, the intervention and the endpoint. 
 
Study Participants: Perinatally infected infants who begin ART f 90 days after diagnosis. 
Breastfed infants diagnosed with HIV f90 days of age and starting ART f 90 days after 
diagnosis.  
The diagnosis of HIV transmission by breastmilk will be made from infants who:  
1. Have a negative birth HIV PCR  
2. Are being breastfed  
3. Who subsequently have 2 molecular methods positive for HIV. These tests will be 
conducted selectively among participants with a clinical evaluation suggestive of HIV 
infection, at the discretion of the attending physician.  
Primary aim:  
To monitor clinical, virological and immunological features of HIV-positive, early treated 
children (f90 days after diagnosis), in order to identify participants with excellent viral 
and immunological control, and also other without excellent control, in order to stratify 
potential participants in proof-of-concept trials directed to HIV cure.  
 
Secondary aims  
1. 
To investigate factors associated with mortality, progression to AIDS, suppression 
(including resistance to ART), adherence and retention, and anthropometric measures. 
2. 
To investigate factors associated with low total HIV-1 DNA in African children with 
a suppressed viral load, who started ART very early in life (<6 months of age). 
3. 
To validate models based on from European cohort data predicting which children 
will have excellent virologic control in limited-resource settings.  
4. 
To study the association of HIV reservoir size with an appropriate immunological 
response (exhaustion and activation) at age 2 and 4 in limited-resource settings.  
5. 
To evaluate children with unplanned cessation of ART who persist with a low 
reservoir at age 2 and 4, if any exist. 
6. 
To study potential new virological markers relevant for reservoir, as they arise 
from ongoing protocols of EPIICAL or another ground-breaking research elsewhere. 
7. 
To study adherence issues and parental expectations from cohort membership 
and clinical trials, and to ensure acceptable benefit: risk balance in the trials. 

EARTH v3, DECEMBER 2021 
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8. To know the proportion of infants with HIV that had high-risk of transmission, 
according to HIV-exposed Infants Programmatic Update 2018, WHO, Geneva. 
 
Planned Sample Size:  The size is based on the ability to recruit participants fulfilling the 
inclusion criteria. We expect to recruit a minimum of 300 participants at all sites during 
the recruitment period of up to 3 years. Assuming a 30% loss to follow-up during the 2-
year follow-up period, we will obtain a sample of 200 participants at the end of the study. 
Approximately 30% of participants from European Cohorts had a low reservoir (median 
age, 7 y). Thus, we expect a sample of 60 participants who fulfil the definition of excellent 
controllers and 140 who do not.  
 
Planned Study Period: Up to 4 years from enrolment from the beginning (began in May 
2018), and at least up to 4 years of follow-up for every participant. 
Inclusion Criteria 
• Perinatally infected infants who start ART at f90 days after diagnosis (HIV 
infection diagnosed and confirmed by molecular methods in 2 different samples, 
i.e., positive qualitative HIV DNA/RNA test).  
• Breastfed-infected children found to be infected f 90 days of age and who started 
ART at < 90 days after diagnosis. This group will be younger than six months of 
age at enrolment 
• Able to take ART 
• Caregivers (mother, if alive and available) able to provide informed consent. 
 
Interventions: Up to 12 visits for all participants (enrolment visit + 11 visits), with 11 
blood draws for virological and immunological measurements.  
Stratification:  Infants will be stratified by study site. 
Study procedure:  Prospective, cohort study. Newly diagnosed HIV positive children will 
start early ART as soon as possible, following local standard of care, and will be followed 
for up to 4 years of age.  
Clinical data and blood for viral load, immunology and serology will be collected in the 11 
visits. The HIV reservoir size and HIV-specific immune responses will be analyzed at visit 
1 and at 1, 2, 3 and 4 years after enrolment. We will identify HIV infected children eligible 
to participate in proof-of-concept cure/remission trials.  
A predictive model developed from European participants9 data will help identifying 
children with a low reservoir. These predictions will then be compared with available data 
of participants already enrolled with excellent control to refine the model. 
Primary outcome variable:  Percentage of children with excellent HIV control profile at 2 
and 4 years of age. This excellent control profile includes a composite endpoint of:  
1) Undetectable HIV RNA viral load in peripheral blood in the last year  
2) Reservoir size (Total DNA /million PBMC) below 25th percentile 
3) Good immunological control (defined as a CD4 % >30%) during the last year 
Treatment: Mother-To-Child Transmission Prophylaxis: according to local guidelines. 
Treatment: according to local standard of care: 2 nucleoside reverse transcriptase 
Inhibitors (NRTIs) + 1 non-nucleoside reverse transcriptase inhibitors (NNRTI) or Protease 
inhibitor (PI) or 1 Integrase Inhibitor (INI) (according to national guidelines).  

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Recommended: NVP at birth and LPV/R when >14 days of age.  
Assays 
In-country  
Point of care HIV diagnosis 
Qualitative HIV DNA, RNA or total DNA (TNA) by Polymerase Chain Reaction (PCR).  
HIV RNA quantification (Viral Load) 
CD4 count/percent. 
serology - if 4th generation test (AbbotTM) available  
Hematology and biochemistry 
Malaria slide (where appropriate).   
HIV ART resistance testing 
 
AHRI  
Total HIV DNA / million PBMC (ddPCR/IPDA) 
 
Ragon Institute 
Deep sequencing on selected PBMC (FLIPS) 
 
Stellenbosch 
Detailed viral reservoir profiling on selected EARTH samples, using HIV-1 envelope 
single-genome sequencing (SGS) using Oxford Nanopore Technology (ONT) 
 
Rome (Italy) 
a. To identify cytokine profile associated with viral control in the early phase 
of the infection and longitudinally to define ontology modification of 
proteomic in children living with HIV 
b. To identify cytotoxicity of NK and CD8 in the early phase of the infection 
and correlate it with disease progression and HIV reservoir in later 
timepoints.  
c. To analyze frequency of ICAM+ Ag specific CD8 cells. 
d. Development of neutralizing antibodies and ADCC. 
e. To analyze telomere length as a measurement of immunosenescence. 
f. To define Ab responses upon routine vaccination in HIV infected children. 
Final goal: design booster intervention in HIV infected children.  
g. Investigate whether the presence of protective HLA allele (HLA-B57, -B27, 
-B14, -Cw8, and -DRB1*10) interferes with HIV progression. Furthermore, 
we will define telomer length in association to HIV progression. 
h. To predict bNAb susceptibility for characterized bNAb. Ospedale 
Bambino Gèsu will store plasma samples for susceptibility to bnAbs until 
the final laboratory to do this test is identified. 
 
Miami (USA).  
Transcriptome: RNA sequencing and gene expression profiles on PBMC inhibitory check 
points evaluation on T cells. 
 

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Harvard (USA) 
Metabolomics, untargeted proteomics by mass spectrometry 
 
Cape Town (South Africa) 
ARV drug levels 
Substudies: 2 substudies are proposed:  
Substudy 1: Social/Behavioural Research Substudy (SBR) 
Background 
The social and behavioral aspects of children with HIV are essential areas to take into 
account for preserving their health and involve the adherence to the drugs and to the 
programs. We need to explore attitudes and social context of caregivers to understand 
adherence barriers, expectations of EARTH cohort participation, and attitudes about 
future research options. This is a first and exploratory step that will inform a larger 
social/behavioral research study that will develop, adapt, apply, and evaluate 
supportive interventions (under a separate and later protocol).  
Aims 
To explore adherence, retention, and attitudes about future clinical research.  
Participants: 
Biological mothers, legal guardians, and/or other primary caregivers of children enrolled 
in EARTH. 
Specific Aims: 
To study caregivers9 HIV experiences, ART attitudes, adherence barriers and facilitators 
and parental expectations from cohort membership and future clinical research.  
To compare self-reported and biological measures of adherence in mother and infant. 
Sample size: 
Investigators will recruit the biological mother or another guardian/caregiver for each 
infant enrolled in EARTH. Thus, the anticipated sample size is up to the size of the EARTH 
cohort. 
Inclusion criteria: 
The substudy will include caregivers (biological mothers and/or other primary caretakers) 
of infants enrolled in the EARTH study. Eligibility will be dependent on 1) being a self-
reported primary caregiver of an infant enrolled in the EARTH study who is aware of the 
infant9s HIV+ status and gives ARVs at least occasionally 2) being aged 18 and above, 3) 
willingness to provide informed consent, and 4) ability to participate in the study in the 
local language(s) offered at each site. Only one caregiver will be enrolled in the SBR 
substudy for each infant enrolled in EARTH. 
Intervention: 
Data collection will occur via in-depth interviewing and brief questionnaires that may be 
self- or interviewer-administered. 
Data collection will occur via a brief questionnaire including measures of mental health, 
internalized stigma, social support, and adherence; and an in-depth interview to explore 
perceptions, attitudes, and social context of caregivers of children enrolled in the 
EARTH study to understand adherence barriers, expectations of EARTH cohort 
participation, and attitudes about future research options. The data collection is 
expected to take 60-90 minutes and may occur at one or two study visits. 

EARTH v3, DECEMBER 2021 
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Substudy 2: Strategies to improve viral suppression (SIVS) 
Background 
Early antiretroviral (ARV) initiation is associated with low HIV reservoir and good 
immunological and virological outcomes in children. The Early Anti-Retroviral Treatment 
in HIV- infected Children (EARTH) cohort initiates HIV-infected infants on ARV treatment 
within the first 3 months after diagnosis (always within 6 months of life) and aims to 
establish a cohort of infants with good immunological and viral markers who may 
subsequently benefit from novel cure/remission therapeutic intervention trials. 
However, the poor viral suppression seen in the EARTH cohort, of 36% at 24 weeks across 
all sites, will likely limit the inclusion of the majority of EARTH participants in these future 
studies.  
Aim  
The overarching goal is to contribute to the understanding of the barriers to viral 
suppression to improve suppression rates in the EARTH study participants 
Hypothesis 
Low or undetectable ARV concentrations are associated with unsuppressed viral loads 
Primary Objective  
• To determine exposure to ARV drugs in infants enrolled on EARTH through the 
quantification of plasma ARV concentrations 
Secondary objectives 
• To determine exposure to ARV drugs in mothers of infants enrolled on EARTH through 
the quantification of plasma ARV concentrations 
Study Design  
Longitudinal study performed at 24, 48, 72 and 96 weeks after treatment initiation in the 
infant. 
Cross-sectional sampling from their mothers at 48 weeks.  
Study Participants 
All participants and their mothers enrolled in EARTH at TBH-CT and CISM Mozambique 
sites 
Interventions 
Adherence assessments in infants: 
• Indicators of adherence such as dispensing records, pill counts and self- reported 
adherence. 
• CD4 count and viral loads coupled to adherence measures. 
• Plasma concentrations of lopinavir or nevirapine  
Adherence assessments in mothers of infants: 
• Simultaneous (with the infant) DBS of tenofovir diphosphate (weeks of adherence) 
as a longer-term marker of adherence (sub-study).  
Assays 
• DBS of tenofovir diphosphate as a longer-term marker of adherence; 
• Plasma nevirapine and/or lopinavir/ritonavir concentrations (above level of 
detection and above therapeutic minimum 
 
 
 

EARTH v3, DECEMBER 2021 
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EARTH v3, DECEMBER 2021 
15 
4. BACKGROUND 
 
There is currently no cure for HIV infection. An estimated 3-4 million children are still living 
with HIV, more than 90% in sub-Saharan Africa. Almost all of these infections are acquired 
through mother-to-child transmission (MTCT).1,2 Despite a 52% decline in the number of 
newly infected children from 2001 to 2012, there are still about 250,000 infants newly 
infected with HIV every year. 2  
 
Antiretroviral therapy (ART) is very effective in preventing mortality when initiated early in 
infancy.3 Currently, international guidelines recommend initiation of ART in all HIV-positive 
infants aged < 5 year, regardless of clinical and immunological parameters.4–6 However, 
although ART has revolutionized survival, it is unable to cure as latent viral reservoirs are 
immediately established in HIV-1 infection7,8. This latent HIV-1 reservoir in memory CD4+ T 
cells forms a major barrier to cure by providing a lifetime of viral persistence, necessitating 
life-long ART.9  
 
Lifelong ART has disadvantages. Toxicity is frequent and concerns around the in-utero 
effects of ART persist including the possible long-term effects on mitochondrial and 
immunologic function10. Lifelong treatment adherence and retention in care can also be 
challenging. Poor adherence is associated with increased viral resistance to ART and more 
than 20% of vertically HIV-positive children develop triple-class virological failure after 5 
years on the same ART regimen possibly increasing the prevalence of primary drug 
resistance at treatment initiation.11–13 Finally, the economic burden for families and 
countries is significant and concerns exist about the feasibility of treating all participants for 
life in limited resource settings.14 Thus, there is an urgent need to define new treatment 
strategies that provide long-term viral suppression off ART in this population.15 
 
Currently, the viral reservoir is considered the main barrier to achieving HIV cure. Initiation 
of ART soon after infection seems to minimize the size of the long-lived HIV reservoir in 
resting memory CD4+ T cells and limits the evolution of HIV-1 in viral reservoirs.16 In most 
early treated cases that interrupt ART, plasma viral rebound is observed in less than 2-4 
weeks.17,18 However, the Mississippi baby case showed that long-term viral control can be 
maintained months after ART interruption in newborns after very early ART. This child had 
an unplanned ART interruption at the age of 18 months followed by 27 months with 
undetectable plasma HIV RNA, no replication competent virus in CD4+ T cells, with only 
traces of HIV DNA in peripheral blood cells. This case provides proof-of-concept that limiting 
the size of the HIV-1 latent reservoir in perinatal infection may allow a subsequent time-
limited period of remission off ART. However, at the age of 4 years, the child had a rebound 
of HIV RNA to nearly 20,000 copies/ml and ART was resumed.19 This single case is consistent 
with the notion that targeting acute HIV infection could lead to a significant reduction of 
HIV reservoirs. 20,21 
 
Children with prolonged excellent control may be the best candidates to try new treatment 
strategies that target ART withdrawal and a functional cure of HIV. At the moment, broadly 
neutralizing antibodies (bNAbs), eCD4-Ig and therapeutic vaccines, among others, are 
possible future choices for children after ART interruption. 

EARTH v3, DECEMBER 2021 
16 
The EPIICAL project is an international consortium whose aim is to select promising HIV 
therapeutic strategies candidates among early-treated perinatally HIV-positive children. 
 
A previous study from the EPIICAL Group with European cohorts of perinatally infected 
children demonstrated that early initiation ART of life in limits the size of the viral 
reservoir.22 A model is being built to predict which children will have a low reservoir with 
data from these studies.  
 
Several questions remain. What immunological and virological factors are associated with 
excellent HIV control? Do factors, such as malaria, cytomegalovirus infection or latent TB 
infection impact HIV control? Are we able to predict which children will achieve good viral 
control and a low reservoir? Are models built in high-resource countries applicable to 
limited-resource settings? How is good immunological and viral response elicited in children 
with early ART? Are these excellent controller potential participants for proof-of-concept 
(POC) studies aimed at curing HIV infection?.21 
 
 
Substudies considerations  
 
During the first months of EARTH, some questions became evident: 
 
Substudy 1: Social/Behavioural Research Substudy (SBR) 
Are there barriers or facilitators to adherence experienced by caregivers that may be 
amenable to intervention? To what extent are caregivers willing to enroll their children in 
POC studies, and what may make them more willing? What do caregivers expect from their 
children9s participation in EARTH? 
A social sciences/behavioural substudy was designed to answer these questions.  
 
Substudy 2: Strategies to improve viral suppression (SIVS) 
Also, an interim analysis of EARTH patients shows that only 36% reach suppression at 24 
weeks after ART, across all sites, which prompted a second substudy proposal to test if low 
or undetectable ARV concentrations are associated with unsuppressed viral load. 
 
 

EARTH v3, DECEMBER 2021 
17 
5. RATIONALE 
 
Perinatally HIV-positive individuals represent a unique population to examine how early 
ART (alone or in combination with new Novel Disease Modifying Therapies [NDMTs]) affect 
the HIV reservoir and on-treatment long-term viral control.  
 
This protocol is part of the EPIICAL consortium (Early treated Perinatally HIV Infected 
individuals: Improving Children9s Actual Life). EPIICAL is a multi-center, multi-cohort global 
collaboration coordinated by Penta (Child Health Research).  
 
EPIICAL overall goals are: 
1. To 
characterize 
the 
virological, 
immunological 
and 
transcriptomic 
correlates/profiles of viral control in existing cohorts of early treated HIV-positive children. 
2. Using these data, to develop a predictive model for positive response to ART. 
3. Conducting proof of concept clinical studies of disease modifying therapies in the 
setting of early treated HIV-positive children using and validating endpoints derived from 
the above predictive platform. 
 
The EARTH study is nested in the first and second goals of EPIICAL and intends to prepare 
the ground for responding to the third.  
 
This protocol intends to develop a multicenter cohort study of children with perinatally 
acquired HIV infection. We intend to establish a well characterized cohort of newly infected 
infants who will start on suppressive ART soon after diagnosis, ideally in the first weeks of 
life. We intend to gain knowledge about factors involved in excellent control (viral, host and 
external factors). Participants with excellent control may be considered for 
immunotherapeutic interventions aimed at achieving ART-free HIV control (i.e. functional 
cure or remission). However, not only participants with excellent control may benefit from 
future clinical trials. Different participants with varying phenotypes, including those not 
suppressed, might benefit from different approaches.  
 
More participants than the initially 30 estimated participants may benefit from proof-of-
concepts trials, hence the increase in sample size.  
 
 
Substudies proposals rationale: 
 
Substudy 1: Social/Behavioural Research Substudy (SBR) 
 
The experience to date in EARTH has indicated that adherence to ART is a challenge. In other 
populations, pediatric adherence has been shown to have a broad range of predictors 
including characteristics of the child, the caregivers and family, socio-cultural, 
organizational and community characteristics.  Understanding and supporting efforts to 
improve adherence will advance the health of the child and result in a larger cohort of 
children who may be eligible for future POC trials. Further, maintaining high retention rates 

EARTH v3, DECEMBER 2021 
18 
is a key goal for all cohorts and is especially important and relevant to the EARTH cohort, 
where maintenance in the cohort may help protect the child9s health.  
 
The objective is to explore perceptions, attitudes, and social context of caregivers of 
children enrolled in the EARTH study to understand adherence barriers, expectations of 
EARTH cohort participation, and attitudes about future research options. This is the first 
and exploratory step that will inform a larger social/behavioral research study that will 
develop or adapt, apply, and evaluate supportive interventions related to adherence, 
retention, and future clinical research opportunities. Results from this first data collection 
will be used to determine the longer-term aims and procedures. The larger SBR substudy 
will be described later in a separate protocol.  
 
Substudy 2: Strategies to improve viral suppression (SIVS) 
Early antiretroviral (ARV) initiation is associated with low HIV reservoir and good 
immunological and virological outcomes in children. The Early Anti-Retroviral Treatment in 
HIV- infected Children (EARTH) cohort initiates HIV-infected infants on ARV treatment 
within the first 3 months after diagnosis (always within 6 months of life) and aims to 
establish a cohort of infants with good immunological and viral markers who may 
subsequently benefit from novel cure/remission therapeutic intervention trials. However, 
the poor viral suppression seen in the EARTH cohort, of 36% at 24 weeks across all sites, will 
likely limit the inclusion of the majority of EARTH participants in these future studies.  
The overarching goal of this substudy is to contribute to the understanding of the barriers 
to viral suppression to improve suppression rates in the EARTH study participants 
 
 
 

EARTH v3, DECEMBER 2021 
19 
6. HYPOTHESIS 
Monitoring HIV-DNA from birth during the first 4 years of age, we can identify and 
characterize a proportion of early-treated children who will have an excellent control of HIV 
at 4 years of age in terms of clinical, viral and immunological features, and other who will 
not have such excellent control. This differentiation will help us to identify which 
participants are more likely to benefit from which novel therapeutic interventions, 
depending on the profile of the patient, the intervention and the endpoint. 
 
 
Substudies hypothesis: 
 
Substudy 1: Social/Behavioural Research Substudy (SBR) 
The collection and analysis of study data will generate hypotheses to test in a subsequent 
study. 
 
 
Substudy 2: Strategies to improve viral suppression (SIVS) 
Hypothesis: 
Low or undetectable ARV concentrations are associated with unsuppressed viral loads, even 
in children with good adherence records.

EARTH v3, DECEMBER 2021 
20 
7. OBJECTIVES 
 
Primary aim 
To monitor clinical, virological and immunological features of HIV-positive, early treated 
children (f90 days after diagnosis), in order to identify participants with excellent viral and 
immunological control, and also other without excellent control, in order to stratify 
potential participants in proof-of-concept trials directed to HIV cure.  
 
Secondary aims  
1. To investigate factors associated with mortality, progression to AIDS, suppression, 
adherence and retention, and anthropometric measures. 
2. To investigate factors associated with low total HIV-1 DNA in African children with a 
suppressed viral load, who started ART very early in life (<6 months of age). 
3. To validate models based on from European cohort data predicting which children 
will have excellent virologic control in limited-resource settings.  
4. To study the association of HIV reservoir size with an appropriate immunological 
response (exhaustion and activation). Specific immunological aims are: 
a. To identify cytokine profile associated with viral control in the early phase of 
the infection and longitudinally to define ontology modification of proteomic 
in children living with HIV 
b. To identify cytotoxicity of NK and CD8 in the early phase of the infection and 
correlate it with disease progression and HIV reservoir in later timepoints.  
c. To analyze frequency of ICAM+ Ag specific CD8 cells. 
d. Development of neutralizing antibodies and ADCC. 
e. To analyze telomere length as a measurement of immunosenescence. 
f. To define Ab responses upon routine vaccination in HIV infected children. 
Final goal: design booster intervention in HIV infected children.  
g. Investigate whether the presence of protective HLA allele (HLA-B57, -B27, -
B14, -Cw8, and -DRB1*10) interferes with HIV progression. Furthermore, we 
will define telomer length in association to HIV progression. 
h. To predict bNAb susceptibility for characterized bNAb. Ospedale Bambino 
Gèsu will store plasma samples for susceptibility to bnAbs until the final 
laboratory to do this test is identified. 
i. RNA sequencing and gene expression profiles on PBMC inhibitory check 
points evaluation on T cells. 
j. To identify metabolomics and untargeted proteomics by mass specrometry 
profile associated with viral control in the early phase of the infection. 
5. To evaluate children with unplanned cessation of ART who persist with a low 
reservoir at age 2 and 4, if any exist. 
6. To study potential new virological markers relevant for reservoir, as they arise from 
ongoing protocols of EPIICAL or other ground-breaking research elsewhere. 
7. To study adherence issues and parental expectations from cohort membership and 
clinical trials, and to ensure acceptable benefit: risk balance in the trials. 
8. To know the proportion of infants actually with HIV that had high-risk of 
transmission, according to HIV-exposed Infants Programmatic Update 2018, WHO, 
Geneva. 

EARTH v3, DECEMBER 2021 
21 
 
Substudies objectives 
 
Substudy 1: Social/Behavioural Research Substudy (SBR) 
 
a. To study caregivers9 HIV experiences, ART attitudes, adherence barriers and 
facilitators and parental expectations from cohort membership and future clinical 
research.  
b. To compare self-reported and biological measures (obtained in substudy of barriers 
to viral suppression) of adherence in mother and infant. 
Substudy 2: Strategies to improve viral suppression (SIVS) 
 
Primary Objective 
• To determine exposure to ARV drugs in infants enrolled on EARTH through the 
quantification of plasma ARV concentrations. 
 
Secondary objectives 
• To determine exposure to ARV drugs in mothers of infants enrolled on EARTH through 
the quantification of plasma ARV concentrations 
 

EARTH v3, DECEMBER 2021 
22 
8. DESIGN 
 
Design: Prospective cohort study.  
 
Sites:   
The participants will be enrolled at 6 rural and urban sites in 3 different countries (1 in Mali, 
2 in Mozambique, 3 in South Africa).  
 
The biological samples will be collected at the clinical sites, stored initially at the site 
laboratories, with some testing done in-country and some samples shipped and analyzed in 
specialized laboratories in Mozambique, South Africa, Italy and USA (the latter including 
Massachusetts, San Francisco and Miami). 
 
*See Appendix 13: LOCAL SPECIFITIES PER SITE AND COUNTRY about clinical procedures 
and methods.  
 
Main outcome variable   
Proportion of children with excellent HIV control (see endpoint definition).  
 
Newly-diagnosed HIV-positive children will start ART as soon as possible, following local 
standard of care. Children on early ART will be followed up to 4 years after enrolment. 
 
Clinical data and blood for viral load, immunology and serology will be collected in up to 12 
visits. The HIV reservoir size and HIV-specific immune responses will be analyzed at visit 1 
and 1, 2, 3 and 4 years after enrolment. We will identify HIV-positive children eligible for 
proof-of-concept trials intended towards HIV cure.  
 
 
Substudies design: 
 
Substudy 1: Social/Behavioural Research Substudy (SBR) 
Mixed methods study comprising a set of quantitative measures and an in-depth, semi-
structured interview. 
 
Substudy 2: Strategies to improve viral suppression (SIVS) 
Longitudinal study performed at 24, 48, 72 and 96 weeks after treatment initiation in the 
infant. 
Cross-sectional sampling from their mothers at 48 weeks. 
This study is intended to be developed currently only in 2 sites: Cape Town and CISM. 
 
 
 
 

EARTH v3, DECEMBER 2021 
23 
9. ENDPOINTS 
 
Primary outcome variable 
Proportion of children with excellent HIV control profile at 2 and 4 years of age. This 
excellent control profile includes a composite endpoint of all of the following:  
1) Undetectable HIV RNA viral load in peripheral blood in the last year (blips, spikes and 
suboptimal viral control allowed, see below for definitions), 
2) Reservoir size (Total DNA /million PBMC) below 25th percentile with ddPCR / IPDA. 
3) Good immunological control (defined as a CD4 >30% and a good NK or CTL response) 
during the last year 
 
If in the next years, any more sensitive and specific immunological assessments arise from 
specific humoral or cellular response assays or transcriptome, lack of antigenic stimulation 
might be substituted by a new test after proper amendments.  
 
Secondary endpoints: 
CLINICAL 
- Mortality 
- Viral load suppression 
- WAZ, HAZ, WHZ (anthropometric measures) 
- Adherence issues. 
- HIV Drug Resistance Profiles: Resistance on non-supressed participants, will be 
performed, to try and identify causes of viral failure in children otherwise with 
acceptable adherence. Children will benefit of adjustment of ART if resistance is 
found. The tests will be done as per standard of care at National Health Systems 
laboratories.  
- Progression to AIDS (WHO clinical stage 3 or 4, CD4 < 25%) 
- High risk of vertical transmission, based on maternal history (viral load and ART) 
- High risk of vertical transmission, based on maternal history (viral load and ART) 
- ART resistance testing 
- ARV levels 
 
VIROLOGICAL 
- Reservoir (total DNA/million PBMC) and intact proviral DNA assay (IPDA). 
- Full length individual sequencing (FLIPS)/ Deep sequencing on PBMC 
- Detailed reservoir profiling using HIV-1 envelope single-genome sequencing (SGS).  
- bNAb susceptibility for characterized bNAbs. 
 
IMMUNOLOGICAL 
 
- Cytokine profile  
- To identify cytotoxicity of NK and CD8.  
- ICAM+ Ag specific CD8 T cells 
- Neutralizing antibodies and ADCC 
- Telomere length 
- Ab responses upon routine vaccination  

EARTH v3, DECEMBER 2021 
24 
- Protective HLA allele (HLA-B57, -B27, -B14, -Cw8, and -DRB1*10)  
- RNA sequencing and gene expression profiles on PBMC inhibitory check points 
evaluation on T cells. 
- Metabolomic profile 
- Proteomic profile 
 
 
Definitions of blips, spikes and suboptimal viral control  
o Blips: viral load 50 to 399c/ml after viral suppression <50 c/mL, with subsequent 
return to <50c/ml on repeat sampling in next visit (within 6 months and same treatment).  
o Spikes: defined as single viral load measurements of 400 -1000 c/mL with 
subsequent return to <50 c/mL at next sampling (within 6 months and same treatment). 
o Suboptimal viral control: g2 viral load measurements 50 to 1000 c/mL without any 
change in treatment.  
 
Potential factors associated with control: Variables to be explored as potential factors 
associated with excellent control will be recorded (see Master File). 
 
 
Substudies outcomes 
 
Substudy 1: Social/Behavioural Research Substudy (SBR) 
This is an exploratory study to generate hypotheses and thus does not have a primary and 
secondary outcome variable. Participants will take part in a brief questionnaire and an in-
depth interview in a private setting. This data collection point will capture the caretaker9s 
mental health using the WHO SRQ-20; internalized stigma using the AIDS-related stigma 
scale, social support using the ACTG social support measure; self-reported caregiver 
adherence (for HIV+ caregivers) using the Morisky simple medication adherence 
questionnaire; and self-reported pediatric adherence using the ICAMP measure with an 
additional purposively developed item set, and caregiver adherence (as relevant) using 
items modified from Knobel et al. (2002). Measures will be self-administered or 
administered by the interviewer. The in-depth interviews will then be conducted in English 
or a local language using a standardized, semi-structured interview guide. In-depth 
interviews will be audio-recorded. 
 
Substudy 2: Strategies to improve viral suppression (SIVS) 
 
Adherence assessments in infants: 
• DBS of tenofovir diphosphate (which is a proxy to weeks of adherence) as a longer-
term marker of adherence. 
Indicators of adherence such as dispensing records, pill counts and self- reported 
adherence. 
• CD4 count and viral loads coupled to adherence measures. 
• Plasma concentrations of lopinavir or nevirapine  
 

EARTH v3, DECEMBER 2021 
25 
Adherence assessments in mothers of infants: 
• Simultaneous (with the infant) DBS of tenofovir diphosphate (weeks of adherence) as 
a longer-term marker of adherence.  
 
Assays 
• DBS of tenofovir diphosphate as a longer-term marker of adherence; 
• Plasma nevirapine and/or lopinavir/ritonavir concentrations (above level of detection 
and above therapeutic minimum). 
 
 
10. SELECTION AND WITHDRAWAL OF PARTICIPANTS 
 
Inclusion Criteria 
1. 
Perinatally infected infants who start ART at f90 days after diagnosis (HIV infection 
diagnosed and confirmed by molecular methods in 2 different samples, i.e., positive 
qualitative HIV DNA/RNA test).  
2. 
Breastfed-infected children found to be infected f 90 days of age and who started 
ART at < 90 days after diagnosis. This group will be younger than six months of age at ART 
initiation. 
3. 
Caregivers (mother, if alive and available) able to provide informed consent. 
4. 
Able to take ART. 
 
Exclusion/Withdrawal Criteria 
1. 
Second and successive RNA PCR negative  
2. 
Malignancy  
3. 
Current concomitant immunosuppressive therapy (including>15 days and >2 
mg/kg/day of prednisone-equivalent).  
4. 
Caregivers withdraw consent 
5. 
Age >180 days.  
 
 
Substudies eligibility 
 
Substudy 1: Social/Behavioural Research Substudy (SBR) 
 
Inclusion criteria: 
1. self-reported primary caregiver of an infant enrolled in the EARTH study  
2. aware of the infant9s HIV+ status and give infant ARVs at least occasionally 
3. Age 18 and above 
4. ability to participate in the study in the local language(s) offered at each site 
Only one caregiver will be enrolled in the SBR substudy for each infant enrolled in EARTH. 
 
Exclusion/withdrawal criteria: 

EARTH v3, DECEMBER 2021 
26 
1. Determination of the interviewer or social/behavioral research team that the 
caregiver participant is unable to provide consent or to continue with the 
protocol, for reasons related to capacity  
2. Caregiver under 18 years of age 
3. Caregiver will not provide consent or withdraws from the substudy 
 
Substudy 2: Strategies to improve viral suppression (SIVS) 
 
Study Participants 
All participants and their mothers enrolled in EARTH at TBH-CT and CISM Mozambique 
sites. 

EARTH v3, DECEMBER 2021 
27 
11. 
 CLINICAL PROCEDURES AND METHODS 
 
Planned Study Period: Up to 4 years from enrolment from the beginning (began in May 
2018), and at least up to 4 years of follow-up for every participant. 
Interventions: Up to 12 visits for all participants (enrolment visit + 11 visits), with 11 blood 
draws for virological and immunological measurements.  
Recruitment 
Eligible children who are meet inclusion criteria will be identified by clinical and/or research 
staff under the leadership of the local EARTH principal investigator.  
 
Enrolling sites: 
Mozambique: 
- 
Fundaçao Ariel: maternity ward and High-Risk Pediatric Clinic at Boane Health 
Centre, Ndlavela HC, Matola Gare HC, Machava 2, Matola 2 HC and Matola Provincial 
Hospital, Beleluane HC, Campoane HC, Matola Rio HC, Tsalala HC, Liberdade HC 
- 
Manhiça CISM sites: maternity ward and High Risk Pediatric Clinic at Manhiça District 
Hospital and HC and Xinavane Rural Hospital, Centro de Saúde de Maragra and Centro de 
Saúde de Palmeira (Malavela) Taninga, Ilha Josina, 3 de Fevereiro, Nwamatibjana, Maluana, 
Munguine, Chibucutso e Calanga  
 
 
South Africa: 
- 
Tygerberg Children9s hospital (Cape Town): Family Center for Research with Ubuntu 
will be a research site in this international, multicenter study. FAMCRU, located in Cape 
Town, South Africa, conducts clinical trials in infectious diseases in adults and children with 
a focus on HIV and TB. FAMCRU forms part of the clinical trials unit of Stellenbosch 
University, whose medical school serves the tertiary level care hospital, Tygerberg. 
Recruitment will be done at Tygerberg Hospital in: the pediatric ward, FAMCRU (and its 
satellites) and the infectious diseases clinic. 
- 
Chris Hani Baragwanath Hospital (Johannesburg) at the Perinatal HIV Research Unit. 
- 
AHRI (Durban): maternity and pediatric wards and infectious diseases clinic. 
*See APPENDIX: LOCAL SPECIFITIES PER SITE AND COUNTRY for Mozambique and Cape 
Town specificities.  
 
Mali:  
- Centre Hospitalier Universitaire Gabriel Touré 
Centers associated to Centre Hospitalier Gabriel Touré: 
a) 
Centre d9Ecoute, de Soins, d9Animation et de Conseils (CESAC de Bamako) 
b) 
Unité de Soins, d9Animation et de Conseil, USAC Commune V, ARCAD-SIDA, Mali 
 
Screening 
Participants will be assessed against the above inclusion and exclusion criteria and the 
parent/guardian will be asked if they wish to participate if they are eligible for the study. An 
explanation of the study will be provided along with a copy of the participant information 
sheet. They will be given as much time as they need to consider their participation and will 
be informed that participation is voluntary and a decision not to participate will not affect 

EARTH v3, DECEMBER 2021 
28 
their clinical care. Written informed consent will be obtained from parent or legal guardian 
before they undergo any research procedures.  
 
The caregiver will also be asked to give consent for access to their own and their child9s past 
clinical notes and laboratory results for verification of ART history, viral load and CD4 
measurements and concomitant medication and medical history. 
 
The study will prospectively collect clinical information and infant9s blood samples to 
determine HIV reservoirs and HIV-specific immune responses. Participants can be 
recruited from 0 to 6 months of age.  
 
All children should have 12 visits (enrolment + 11 additional visits). 
 
In special circumstances where consent cannot be obtained for logistical reasons prior to 
ART initiation, the participant may still be enrolled retrospectively. No study procedure will 
be carried out before informed consent.  
 
1) 
*See Appendix 13: APPENDIX: LOCAL SPECIFITIES PER SITE AND COUNTRY about 
clinical procedures and methods. 
 
Follow-up 
Follow up in the first month after starting ART will be performed according to the National 
guidelines. Study visits are scheduled every three months (see Figure 1) across all 
recruitment sites.  
 
The study includes 1 enrolment visit and up to 11 visits for clinical data and blood collection 
from the child to characterize the HIV reservoir size and replication as well as the HIV-
specific immune responses. The baseline visit will occur at diagnosis. Reasonable efforts will 
be made to contact participants and re-schedule missed visits if the participant does attend 
the scheduled visit. The minimum visits required are enrolment, and twice a year for two 
years.   
 
The study visits will be scheduled periodically after ART initiation to evaluate serology, viral 
load and CD4.  
At the visit 1 and visit 5, 7, 9 and 11 (corresponding to baseline, 48 weeks~12 months, 96 
weeks~2 years, at ~3 years and at ~4 years) blood for reservoir and immunological features 
will be collected and shipped. 
If there is not enough blood for performing immunological/virological assays from baseline 
visit 1, and there is stored blood remaining from visit 0, 2 or 3, suitable for these assays, 
these samples can be used as baseline, as time of those visits are within 12 weeks after visit 
1. 
At the visit 4, 6, 8, 10 (corresponding to 24 weeks~6 months, 72 weeks~18 months, at ~2.5 
years, ~3.5 years), blood will be obtained for standard of care and for in-site promoted sub-
studies.  
 
 

EARTH v3, DECEMBER 2021 
29 
 
 
 
 
Figure 1: Blood withdraw timeline and tests. In red, new visits included in amendment v2. 
*: Visit 2 and 3 can be skipped if visit 1 is delayed. 
**: In some instances, plasma and PBMC can be recovered from VL sample if enough blood. 
***: Ideally, these volumes should be collected. For CD4+FBC, 1 mL may be acceptable. For 
Viral Load, 1.5 to 3 mL might be acceptable. For WP3 cell storage, 1 mL might be acceptable. 
For WP4 Cell Storage, 3 mL may be acceptable. 
(^): Only CD4 and Viral Load is Standard of Care in Johannesburg, only these tests will be 
performed at this timepoint in this site. 
(^^): In the sites where technique is available, immunophenotype in-site will be done in this 
visit. 
(^^^): Where these tests are standard of care. Otherwise, Viral Load will be done as a 
Research Sample and FBC and Chemistry will not be performed. 
 
Details of study visits 
The study has 12 visits scheduled for all children enrolled: the enrolment visits and up to 11 
study visits. The majority of participants will follow one of two profiles: participants who 
test positive at birth, and those who test positive around 4-6 weeks of age. The actions, 
material and storage that the physician or researcher needs to achieve in each visit are 
spelled out and detailed in Appendix 1 as check lists. 
 
Windows of visits: 
Visits windows are:  
• _Regular window for visit 1 should be 1 week before and 2 weeks after planned visit. It 
is possible to widen the window up to 4 weeks in exceptional cases given the relevance of 
visit 1.  
 

EARTH v3, DECEMBER 2021 
30 
Note: In these cases (wider window up to 4 weeks), we will have to skip visit 2 and go 
directly from visit 1 to visit 3, to regularize the schedule.  
• _Regular window for visit 2 should be 2 weeks before and 3 weeks after planned visit 
• _Regular window for visit 3 should be 3 weeks before and 6 weeks after planned visit 
• _Regular window for visit 4 should be 6 weeks before and 12 weeks after planned visit 
• _Regular window for visit 5 to 11 should be 12 weeks before and after planned visit 
 
If some visit is scheduled too late and overlaps to the next timeframe, we should skip this 
visit and go to the next one.  
Also, given the relevance of visit 4, if visit 3 is scheduled too late and overlaps with visit 4, 
it's better to skip visit 3 and go directly from visit 2 to visit 4, to regularize the schedule.  
If needed, inside the window for any visit it is possible to perform 2 analytics/extractions 
corresponding to only one visit. 
If needed and justified (for example in case it is not possible to draw the required blood 
volume for technical reasons), 1 visit might split extractions into 2 separate days, if 
mother/responsible adult agrees, and if both extractions are inside the window. For 
example, in visit 3, 3 weeks before and 6 weeks after planned visit. However, this should 
be avoided as much as possible, and all necessary blood should be taken in 1 extraction/1 
day.  
If this situation this happens, a comment should be added in the free field of the forms, to 
report the date of the second extraction.  
 
A WHO review on blood volumes in pediatric research recommends limiting blood volume 
withdraw to between 1% and 5% of total blood volume on a single draw or over 24h and of 
up to 10% of total blood volume over 8 weeks.24  
 
The blood volumes taken from children for clinical and research purposes should not 
exceed maximum allowable limits. Based on the WHO publication, a limit ranging from 1% 
to 5% of total blood volume over 24 hours and up to 10% of total blood volume over 4 
weeks was considered. For healthy children, estimation will be 3% total blood volume in a 
24 hour period (approx. 2.5 mL/kg); see Appendix XI for blood draw volumes per kg.  
The amount of blood in all the visits will be in any case <5% of the total blood.  
 
Visit 
Numb
er 
Week 
numb
er 
Yea
r 
Loca
l VL 
WP3 
Virolog
y 
WP4 
Immunolo
gy 
In site 
storag
e 
Total 
For 
researc
h 
Standar
d of 
care 
Tota
l  
0   
0 
1 
3ml 
NC 
NC 
NC 
3 mL 
2.5 mL 
5.5 
mL 
1 
2 
1 
NC 
2ml 
4ml 
NC 
6 mL 
NC 
6 
mL 
2 
6 
1 
3ml 
NC 
NC 
NC 
3 mL 
NC 
3 
mL 
3 
12 
1 
3ml 
NC 
NC 
NC 
3 mL 
NC 
3 
mL 

EARTH v3, DECEMBER 2021 
31 
4pb 
24 
1 
NC 
NC 
NC 
3ml 
3 mL 
4.5 mL 
7.5 
mL 
5 
48 
1 
NC 
3ml 
4ml 
3ml 
10 mL 
3.5 mL 
13.5 
mL 
6 
72 
2 
NC 
NC 
NC 
5ml 
5 mL 
3.5 mL 
8.5 
mL 
7 
96 
2 
NC 
3ml 
6ml 
4ml 
13 mL 
3.5 mL 
16.6 
mL 
8 
120 
3 
NC 
NC 
NC 
NC 
 
3.5 mL 
3.5 
mL 
9 
144 
3 
NC 
5ml 
6ml 
4ml 
15 mL 
3.5 mL 
18.5 
mL 
10 
168 
4 
NC 
NC 
NC 
NC 
 
3.5 mL 
3.5 
mL 
11 
192 
4 
NC 
5ml 
6ml 
4ml 
16 mL 
3.5 mL 
18.5 
mL 
 
 
 
 
Substudies procedures 
 
Substudy 1: Social/Behavioural Research Substudy (SBR) 
 
One to two data collection points within the EARTH study period 
Data collection will occur via an investigator-administered brief questionnaire and 
an in-depth interview. The data collection is expected to take 60-90 minutes and may be 
split over two visits if needed.  
Study participants will be recruited by site staff during the child9s study visits for 
EARTH. Site staff will explain the purpose of the study and procedures and if the participant 
is interested, undergo a written informed consent process in the language of the 
participant9s choice (English, or a s local language). 
Mothers or other caregivers will be assessed against the substudy inclusion and 
exclusion criteria and, if eligible, will be invited to participate. An explanation of the study 
will be provided along with a copy of the participant information sheet. Potential 
participants will be informed that participation is voluntary and a decision not to participate 
will not affect their child9s continued enrollment in EARTH or their own or their child9s 
clinical care. Written informed consent will be obtained from participant.  
The SBR substudy will include one to two study visits. In cases where the data cannot 
be obtained in one visit, such as if the participant become fatigued and requests a break, or 
if there is a disturbance or interruption (e.g., the child becomes too demanding of the 
caregiver for the data collection to continue), then the data collection may be spread across 
two visits. If after conducting several interviews we find that the data collection takes too 
long and becomes burdensome to the participants or sites, we will reduce the number of 
questions/measures used in the interviews. 

EARTH v3, DECEMBER 2021 
32 
Data collection will occur in person and in a private setting after substudy 
participants provide consent. Data collection will be conducted in the local language(s) and 
is expected to take 60-90 minutes. As much as possible, consistency will be maintained 
throughout the sub-study such that the same interviewer will interview a given participant 
at each data collection point. All interviewers will be trained in prior to the initiation of data 
collection. 
In-depth interviews and measurement of factors influencing adherence  
Participants will take part in a brief questionnaire and an in-depth interview in a private 
setting. This data collection point will capture the caretaker9s mental health using the 
WHO SRQ-20; internalized stigma using the AIDS-related stigma scale, social support using 
the ACTG Social Support measure; self-reported adherence for HIV+ caregivers (as 
relevant) using the Morisky scale; and self-reported pediatric adherence using the ICAMP 
measure with an additional purposively developed item set, and caregiver adherence (as 
relevant) using items modified from Knobel et al. (2002). Measures will be self-
administered or administered by the interviewer. The in-depth interviews will then be 
conducted in English or a local language using a standardized, semi-structured interview 
guide, with slight modifications as necessary at each site to make the questions culturally 
acceptable and understandable. Such adjustments are standard practice for semi-
structured interviewing across multiple countries and cultures. 
Recruitment will begin after the modification to the protocol is approved and will 
be conducted on a rolling basis—that is, at their child9s next study visit to EARTH, the 
mother or caregiver will be invited to participate in the substudy, and if they consent, an 
appointment for the substudy will be scheduled. The timing of the substudy data 
collection will depend on the child9s progress through the EARTH protocol. The preferred 
time for caregivers of children newly enrolled in EARTH to have their SBR substudy visit is 
after EARTH visit 3, but the study team will accommodate caregivers9 scheduling needs as 
much as possible. Parents of children already enrolled in EARTH who agree to participate 
will have their SBR-substudy visits anytime between visit 3 and the termination of their 
child9s EARTH enrollment.  
Informed consent:  
A specific information sheet for caregivers will be developed for this substudy, and a 
checkbox will be added to the general informed consent form.  
 
Substudy 2: Strategies to improve viral suppression (SIVS) 
 
Adherence assessments in infants: 
• Indicators of adherence such as dispensing records, pill counts and self- reported 
adherence; included already in the database. 
• CD4 count and viral loads coupled to adherence measures; 
• Plasma concentrations of lopinavir or nevirapine  
 
Adherence assessments in mothers of infants: 
• Simultaneous (with the infant) DBS of tenofovir diphosphate (weeks of adherence) as 
a longer-term marker of adherence (sub-study).  
Assays 

EARTH v3, DECEMBER 2021 
33 
• DBS of tenofovir diphosphate as a longer-term marker of adherence; 
• Plasma nevirapine and/or lopinavir/ritonavir concentrations (above level of detection 
and above therapeutic minimum) 
 
Informed consent:  
A specific information will be added in the information sheet for caregivers, relating drugs 
levels in blood of the child, and the mother. A separate consent will be signed to provide 
the possibility to join the main study and not the sub-study. 
 
The amount of extra blood for this substudy will be <0.5 mL, since it9s a dried spot test and 
will be kept within the accepted limits stated above.  
 
 

EARTH v3, DECEMBER 2021 
34 
12. 
DRUG REGIMEN 
 
ART choice and any further medical management will be according to standard of care at 
the treating site and not a part of this research protocol. However, a 3- drug regimen 
including 2 reverse transcriptase inhibitor backbone plus 1 NNRTI or protease inhibitor or 
will be required as standard ART. The drug regimen will be recorded for analysis purposes. 
*See APPENDIX: LOCAL SPECIFITIES PER SITE AND COUNTRY for Mozambique specifics. 
 
 
 
Substudies regimen 
No comments relating this section. 

EARTH v3, DECEMBER 2021 
35 
13. 
 POTENTIAL ADVERSE EVENTS REPORTING 
 
Since all children will be managed according to the local standard of care in place, adverse 
events relating to HIV progression or ART toxicity or other aspects of the medical 
management of these children will be reported in compliance with the national rules and 
included in the case report forms (CRFs).  
Adverse events related to the research will also be reported.  These include but are not 
limited to loss of confidentiality, adverse events related to blood draws, stigmatization etc. 
For serious adverse events (SAEs), the investigators will report these into the next-visit CRF.   
 
All adverse events will also be reported to the local regulatory boards following applicable 
rules. 
 
Potential Risks and Protection against Risk 
The major risks associated with participation include those associated with phlebotomy. 
The risks when drawing a blood sample may include some discomfort, bleeding or bruising 
at the site of entry of the needle, rarely the formation of a small blood clot or swelling of 
the vein, bleeding from the puncture site, and on rare occasion infection. Risks related to 
blood drawing are not anticipated to be serious and are of a mild and transient nature. All 
phlebotomists are trained and experienced in drawing blood on newborns, infants and 
toddlers. Every effort will be made to minimize any adverse experience by the study team.  
 
Potential benefits of the proposed research to the subjects and others 
In the consent form, it will be explained this benefit and that there is no other benefit to 
the subject. However, the information generated from this study may help us to better 
understand the HIV reservoirs and form the basis for strategies aimed to cure HIV. 
The close monitoring of immunological and virological response to ART will allow prompt 
identification of infants who do not respond to ART and warrant early investigation of 
possible causes and eventually ART regimen switch. 
 
*See APPENDIX: LOCAL SPECIFITIES PER SITE AND COUNTRY for Mozambique.  
 
 
Substudies specificities adverse events 
 
Substudy 1: Social/Behavioural Research Substudy (SBR) 
 
Potential Risks and Protection against Risk 
 
Substudy risks include distress or other adverse psychological reaction, 
identification of an urgent caregiver or child risk during data collection, and risk of loss of 
privacy and confidentiality. 
Caregivers will be informed that participation in the substudy is voluntary and their decision 
will not impact their child9s enrollment in the EARTH study. Further, they will be informed 
that they can refuse to answer any question or stop participation at any time. Interviewers 
will be trained to respond to emotional responses during data collection. Caregivers will be 

EARTH v3, DECEMBER 2021 
36 
referred to mental health services if investigators have concerns about the caregiver 
psychological wellbeing. We will only enroll adult participants in the SBR substudy. 
 
The complete transcripts and recordings of the qualitative data will be maintained in secure 
files that are separated from the EARTH data. Only social/behavioral investigators engaged 
in the data collection and analysis will have access to the complete individual transcripts. 
Other EARTH investigators will not have access to the complete individual transcripts. They 
will only have access to aggregated reports with identifiers removed. In this way, SBR 
substudy participants9 personal accounts will be maintained as private except in cases 
where the SBR investigators have urgent concerns about the mental or physical health of 
the participating adult or the infant enrolled in EARTH. Such concerns will be reported to 
the EARTH clinical team to facilitate appropriate referrals or responses. These exceptions 
to participant privacy are noted in the substudy consent form. Analysis of the SBR substudy 
data will be conducted at each EARTH site and at RTI International and University of North 
Carolina at Chapel Hill. Data analyzed at RTI and UNC will not include any identifiers. 
 
The information sheet explains that there are no benefits to participation. The 
information will be used to better understand barriers and facilitators to adherence to ART 
and caregivers9 thoughts about EARTH and other clinical research studies. 
 
Substudy 2: Strategies to improve viral suppression (SIVS) 
 
 
No further risks besides those stated in the main protocol 

EARTH v3, DECEMBER 2021 
37 
14. 
 LABORATORY TESTING ASSAYS 
 
Comments on samples 
 
One of the aims of the EPIICAL consortium is to strengthen the link between local research 
centers and European/US centers. Efforts will be made for transfer of knowledge from 
European/US labs to local sites to implement or improve techniques if necessary, including 
the possibility of human resources mobility. 
 
The biological samples will be collected at each clinical site.  Some testing will be done in-
country and others in centralized, specialized laboratories in Italy,  South Africa (Cape Town, 
Durban), and USA (Miami, Massachusetts, San Francisco), as detailed below. 
 
In particular, in Mozambique samples collected at CISM supported Health Facility will be 
sent to the laboratory at CISM, and samples collected at F. Ariel supported –Health Facility 
will be sent to the National Health Institute (INS) Lab. 
 
In South Africa, samples collected at Tygerberg Hospital will be processed, in the Hospital 
and sent to the Division of Medical Virology, Faculty of Medicine, Tygerberg Campus.  
Samples collected at Chris Hani Baragwaneth Hospital will be sent to Clinical Laboratory 
Services in Johannesburg. Samples collected at AHRI will be sent and processed at AHRI 
Laboratory in Durban.  
 
In Mali, samples collected, at the Centre Hospitalier Universitaire Gabriel Touré will be 
processed at the Bacteriology Laboratory, Centre Hospitalier Universitaire Gabriel Touré, 
Bamako, Mali, Faculté de Pharmacie, Université des Sciences, des Techniques et des 
Technologies de Bamako, Bamako, Mali. 
 
Haematology, biochemistry, CD4 count, and viral load will always be done on site. Real-time 
performance of tests relevant for clinical care will be done always on-site, including viral 
load, and CD4 count. 
 
Samples workflow: 
At the visit 1 and visit 5, 7, 9 and 11 (corresponding to baseline, 48 weeks~12 months, 96 
weeks~2 years, at ~3 years and at ~4 years) blood for reservoir and immunological features 
will be collected and shipped. 
If there is not enough blood for performing immunological/virological assays from baseline 
visit 1, and there is stored blood remaining from visit 0, 2 or 3, suitable for these assays, 
these samples can be used as baseline, as time of those visits are within 12 weeks after visit 
1. 
These samples from baseline, v5, v7, v9 and v11 will be split in aliquots and shipped for:  
i) 
AHRI (Durban, SA): Reservoir size (ddPCR/IPDA):  
ii) 
Massachusetts, USA (Ragon Institute): Sequencing (FLIPS) 
iii) 
Selected samples: from CISM and Stellenbosch to University Cape Town. 
iv) 
Immunology tests: Samples for USA will be shipped to Rome first, and then 
from Rome to the USA. Final destines are: 

EARTH v3, DECEMBER 2021 
38 
a. Rome (Italy) Cytokine via proteomics, cytotoxicity of NK and CD8, 
transcriptome, transcriptional analysis on  HIV-specific T and B cell subsets, 
specifically including intracellular cytokine production, cytotoxic and 
degranulation activity of NK and CD8 T cells, ICAM+ specific CD8 T cells, 
ADCC and Abs profile, HLA typing, telomere length, Ab responses to 
vaccination. Store samples for susceptibility to bnAbs. 
b. Massachusetts, USA (The Children9s Hospital Corporation d/b/a Boston 
Children's Hospital): Metabolomics and untargeted proteomics by mass 
spectrometry 
c. Miami University (USA): RNA sequencing and gene expression profiles on 
PBMC inhibitory check points evaluation on T cells 
 
At the visit 4, 6, 8, 10 (corresponding to 24 weeks~6 months, 72 weeks~18 months, at ~2.5 
years, ~3.5 years), blood will be obtained for standard of care and for in-site promoted 
sub-studies, including: 
- ART resistance in standardized in-country laboratories such as NHS (all sites). 
- Detailed viral reservoir profiling on selected EARTH samples, using HIV-1 envelope 
single-genome sequencing (SGS) using Oxford Nanopore Technology (ONT): Stellenbosch 
University only. 
- ART levels (as per substudy 2): Stellenbosch University, Manhiça. 
 
These samples will also be stored during 10 years for future use if the informed consent 
form has been obtained, as proposed by EPIICAL researchers and approved by EPIICAL 
governance, including new, high complexity virological assays. 
Any new use of the samples will only be done after approval by the competent IECs/IRBs.  
 
Blood samples for virology: 
Extraction of 2 mL of whole blood at visit 1, 3 mL at 1 year, 3 mL at 2 years, 5 mL at 3 years, 
and at 18.5 mL at 4 years after enrolment.  
All PBMCs should be stored in Liquid Nitrogen (LN2), or in a minus 150 degrees freezer. 
 
Blood samples for immunology: 
Blood draws will be 4 ml of whole blood at visit 1, 6 ml at year one and 6 ml at year 2, 3 and 
4 after enrolment).  
All PBMCs should be stored in Liquid Nitrogen (LN2), or in a minus 150 degrees freezer. 
The blood for Rome/Miami/Massachusetts will be equally separated in vials each containing 
5 million PBMCs that will be stored in liquid Nitrogen and shipped using a dry shipper in a 
timely manner as detailed in SOPs.  
Plasma samples will be stored at -80ºC, or -20ºC if this is not possible. 
 
In-site 
3 mL-samples for storage and research on site will be obtained and stored at 6 months and 
18 months, and 5 mL at 72 weeks, 4 mL at 144 and 192 weeks. 
 
 
 
Comments on Samples for Genetics Testing 

EARTH v3, DECEMBER 2021 
39 
Ongoing research strongly suggests that genetic factors have a very relevant influence in 
control of HIV. This is why we will ask the permission of the participants to store some 
material for a 10 year period for possible genetic testing. A future genetics assay could 
possibly revel the key for HIV excellent control. One of the assays recently suggested is HLA, 
as mentioned in page 18 and 29. A specific checkbox in the informed consent is now 
available for genetic testing.  
  
Shipping of the Samples 
 
Samples for USA will be shipped to Rome first, and then from Rome to USA 
 
1st shipping When first 150 fulfilled visit 1 year of age. 
2nd shipping (estimated, 24 months after the 1st shipping): in 2023 
 
Substudies specificities about tests 
 
Substudy 1: Social/Behavioural Research Substudy (SBR) 
Not applicable. 
 
Substudy 2: Strategies to improve viral suppression (SIVS) 
 
- Pharmacokinetic measurements of ARVs: 
- 
DBS of tenofovir diphosphate (weeks of adherence) as a longer-term marker of 
adherence (sub-study)  
- 
Plasma nevirapine and/or lopinavir/ritonavir concentrations (above level of 
detection and above therapeutic minimum. 
 
These samples will be shipped to the UCT pharmacology lab in Cape Town, South Africa. 
 
 
 
 
 

EARTH v3, DECEMBER 2021 
40 
15. 
 STATISTICAL ANALYSIS AND ACCESS TO SOURCES OF INFORMATION 
 
Sample Size 
This is a study with a whose size is based on the ability to recruit participants fulfilling the 
inclusion criteria. We expect to recruit a minimum of 300 participants at all sites during the 
recruitment period of up to 3 years.   Assuming a 30% loss to follow-up during the 2-year 
follow-up period, we will obtain a sample of 200 participants at the end of the study. 
Approximately 30% of participants from European Cohorts had a low reservoir (median age, 
7 y). Thus, we expect a sample of 60 participants who fulfil the definition of excellent 
controllers and 140 who do not.  
 
*See APPENDIX: LOCAL SPECIFITIES PER SITE AND COUNTRY for Mozambique and Cape 
Town.  
 
Primary statistical methods 
In summary tables qualitative variables will be presented as counts and frequency 
distribution. Quantitative variables will be presented as mean and standard deviation (± SD) 
when normally distributed and as medians and interquartile range (IQR: P25-P75) when 
non-normally distributed. Shapiro-Wilk test will be performed to test normality. Chi-
squared and Fisher Tests will be performed when categorical variables. For normally 
distributed continuous variables, Student T test will be performed and U-Mann Whitney or 
Kruskal Wallis when non-parametric. All hypothesis testing will be carried out at the 5% 
significance level and p-values will be rounded to three decimal places. In summary tables, 
p-values less than 0.001 will be reported as <0.001.  
To test the association between the primary outcome (presence / absence of excellent 
control) and factors of interest a multivariable logistic regression model will be used.  
Variable selection will be performed using a stepwise Akaike Criterion method and 
interaction between predictors will be tested. A regression diagnostic will be performed to 
ensure that all the model assumptions are met as independence and collinearity. If it were 
the case that the model does not fit the assumptions and the number of predictors highly 
overcome the number of events for the primary outcome, alternative methods as penalized 
or ridge regression models will be used. Finally, in order to evaluate the predictive 
performance of the model, sensitivity, specificity, accuracy and the area under the receiver 
operating characteristics (ROC) curve (AUC).  
 
Secondary statistical methods 
To investigate factors associated with mortality, progression to AIDS, suppression, 
adherence and anthropometric measures, a multivariable logistic regression will be used. 
Variable selection and regression diagnostic will be assessed with the same methodology 
stated in the primary statistical methods. Alternatively, to investigate the association 
between survival and the different predictors time-to-event models will be used with right-
censoring. Survival models as Cox proportional hazards, semiparametric models, or random 
forest survival models will be tested and compared. 

EARTH v3, DECEMBER 2021 
41 
To test the association between HIV DNA-1 reservoir and sociodemographic, clinical, 
virological, and immunological factors (secondary objectives 2, 4, and 6) a Generalized 
Linear Poisson model will be used.  Regression diagnostic will be examined to test for all 
model assumptions including dispersion and zero-inflation. In case of overdispersion or 
zero-inflation, negative binomial distributions and hurdle models will be applied 
respectively.  
European Cohort data models will be validated in our cohort assessing the model 
performance in terms of accuracy, sensitivity, and specificity. The AUC will be calculated 
and compare with our designed models.  
To study the risk-benefit of including these vulnerable infants in clinical trials, adherence 
and parental expectations will be described by means of summary tables and summary 
plots to determine factors potentially associated with poor adherence.  
 
Missing data 
In order to avoid loss of information and statistical power in the association analysis, missing 
data will be imputed. To prevent to many assumptions, only variables with less than 20% of 
missing information will be considered for imputation. Imputation will be performed using 
random forest imputation implemented in missforest R package[2]. Out-of-bag (OOB) 
imputation error estimate will be assessed and error for imputing categorical (proportion 
of falsely classified) and continuous (normalized root mean squared error) will be reported. 
In case higher imputation error (>10%) were reached, alternative methods as multiple 
imputation chained equations will be performed implemented in MICE R package [3]. 
Finally, sensitivity analysis will then be undertaken to assess the robustness of the 
conclusions to assumptions regarding the missing data. 
 
 
All analysis will be performed using R software. The statistically significant threshold will be 
set as p <0.05. We will add to all the report the size effect of the analyses when statistically 
significance differences are reached and statistical power of the analyses when they are 
not.  
 
 
Modelling 
We will build predictive mathematical models with our data. Also, the participants9 data will 
be checked into the mathematical models built using data from European participants to 
check whether or not these models have predictive value for control at two and four years 
of age, these models will be rebuilt and refined if necessary. 
 
Data Analysis 
The analysis will be performed at the Hospital 12 de Octubre, Madrid, with the assistance 
of Perinatal HIV Research Unit, Chris Hani Baragwaneth Hospital, University of the 
Witwatersrand, Soweto, South Africa. 

EARTH v3, DECEMBER 2021 
42 
Interim analysis will be performed after first 150 enrolled participants fulfil visit 1 and 5; 
and after all participants fulfil visit 1 and 5. 
 
Data Management Plan and Data Safety and Monitoring Board  
A Data Management Team (DMT) will be settled with one member of each site, two central 
data managers from Hospital 12 de Octubre, and a data manager member from PENTA. The 
DMT will create a Data Management Plan which will follow for data collection and handling.  
All study data will be collected confidentially by the clinical staff or data entry personnel at 
the research centers using designated source documents. Data will be written down in 
paper case report forms (CRFs) and then introduced in an electronic CRF (eCRFs) 
implemented in REDCap collection data system (https://www.project-redcap.org/). This 
electronic database will be securely allocated at PENTA (study sponsor). This centralized 
database, managed by the DMT, will be the central data repository for all sites participating 
in the study. Each site participating in the trial will be responsible for data entry and first 
cleaning. 
 
User-specific usernames and passwords are required to log onto the database. User rights 
will be provided by PENTA to study staff, PIs, and co-investigators at the level appropriate 
for each individual9s job description. 
This database will be built and managed by the central data managers, with revision and 
approval from the protocol board. Only the protocol chairs, data managers and statisticians 
will have access to the whole pseudonymized data in REDCap.  
 
In summary, the plan for data monitoring will be the following: 
1. In-site researchers will include eligible patients and complete participant forms into 
REDCap eCRFs every time that a participant is enrolled. 
2. Central data managers will produce monthly reports of enrolled participants and 
urgent issues 
3. Quarterly, central data managers will performed standardized quality controls to 
monitor missing values, logical inconsistences, clinical inconsistences, and outliers. This 
report will be sent to the sites to correct or justify each of the queries. 
 
 
Substudies specificities about data management and statistics 
 
Substudy 1: Social/Behavioural Research Substudy (SBR) 
Investigators will recruit the biological mother or another guardian/caregiver for each infant 
enrolled in EARTH. Thus, the anticipated sample size is between 100 participants and up to 
the size of the EARTH cohort. 
 
Qualitative and quantitative data will be collected. Items or measures will be removed if 
they are not applicable to the caregiver being interviewed—for example, if the participant 
is a grandmother who is not HIV positive. Analysis of the SBR substudy data will be 
conducted at each EARTH site and at RTI International and University of North Carolina at 
Chapel Hill. All data (recordings, transcripts, summary reports, translations) will be coded 

EARTH v3, DECEMBER 2021 
43 
with the date of data collection and the EARTH identification number. Any identifiers will 
be removed at each site prior to the data being shared with the RTI/UNC research team. 
Qualitative: An in-depth semi-structured interview will cover four domains: 1) the 
caregiver9s personal history with HIV and treatment adherence, 2) their child9s history with 
HIV and treatment adherence, 3) current EARTH study participation, and 4) future 
engagement in with research studies. The in-depth interview guides may be modestly 
adjusted as needed based on the emerging data provided by the participant and/or based 
on site-specific acceptability of specific questions. These data will be audio-recorded. 
Immediately following each interview, the interviewer will be asked to complete a 
debriefing form in English, or to be translated to English, that will provide information about 
the interview (i.e. duration, mood of interview, participant ID, etc.) as well as a summary 
report of the content, which will include staff9s impressions of key issues related to the main 
outcomes of interest. All debriefing reports will initially be reviewed by local site staff and 
then sent to RTI/UNC via a secure file sharing platform (e.g., sftp). Audio recordings of the 
interviews will be transcribed and translated to English and will also be shared via a secure 
file sharing platform. 
Standardized summary forms will be used to capture major themes and subthemes using 
a rapid analysis approach. Summary forms will be translated into English, if captured in 
another language. The forms will be used to develop a brief and structured analysis 
scheme, comprising scoring of affective and contextual content, that will be applied to 
each narrative. Transcripts of the recordings and translations of the full recordings to 
English will be performed as required for data analysis. Data will be structurally coded, 
and themes compared and summarized across participants and sites. 
Quantitative: Participants will take part in a brief questionnaire to assess the caretaker9s 
mental health, internalized stigma, social support, and child and caregiver adherence. 
Questionnaires will be completed either on paper-based forms or entered directly into an 
online database (REDCap). Quality checks will be conducted by EARTH sites and RTI/UNC. 
Data analysis will include descriptive statistics and simple modeling that includes key data 
from EARTH, for example the duration of the child9s enrollment in EARTH, child age, and 
clinical data relevant to adherence. We will also explore the concordance between self-
reported adherence data provided in this substudy with the biomarker data obtained in the 
viral suppression substudy. 
 
 
Substudy 2: Strategies to improve viral suppression (SIVS) 
Investigators will recruit the biological mother and infants enrolled in EARTH at two sites 
(Manhiça and Cape Town). The anticipated sample size is 80 mothers and 80 infants. 
 
In previous literature, the proportion of participants with virological failure at 24 weeks 
was 21%, in patients with suboptimal levels, and 8% in those with optimal levels (p<0.001)  
(https://academic.oup.com/jac/article/64/1/109/754487).  
For this substudy, we assume viral failure at 48 weeks 30% in those with suboptimal levels 
and 10% in those with optimal levels. For a level of confidence 5% and error beta 20%, 
sample size as is estimated in a minimum of 60 participants. 

EARTH v3, DECEMBER 2021 
44 
16. 
REGULATORY, ETHICAL AND LEGAL ISSUES 
 
Declaration of Helsinki 
The investigator and sponsor will ensure that this study is conducted in full conformity with 
the latest revision of the 1964 Declaration of Helsinki (2013), the 2017 Ethical 
Considerations for clinical trials on medicinal products conducted with minors from the 
European Commission, the International Ethical Guidelines for Biomedical Research 
Involving Human Subjects CIOMS-WHO (2016); and the Additional Protocol of the Oviedo 
Convention (2005). 
 
Independent Ethics Committee/Institutional Review Board Approval  
Initial Approval 
Prior to the enrolment of participants, the competent Independent Ethics 
Committee/Institutional Review Board (IEC/IRB) must provide written approval of the 
conduct of the study at each site, the protocol and any amendments, the Information Sheet 
and Consent Form (and assent if the mother is minor), any other written information that 
will be provided to the mother/parents/legal guardians, any advertisements or additional 
documentation that will be used.  
 
Approval of Amendments 
Proposed amendments to the protocol and aforementioned documents must be submitted 
to the IEC/IRB for approval/notification as required by the applicable law. Amendments 
requiring IEC/IRB approval may be implemented only after written IEC/IRB9s approval has 
been obtained.  
Amendments that are intended to eliminate an apparent immediate hazard to subjects may 
be implemented prior to receiving Sponsor or IEC/IRB approval. However, in this case, 
approval must be obtained as soon as possible after implementation. 
 
End of Study Notification 
The IEC/IRB will be informed about the end of the study, within the required timelines. 
 
Informed Consent  
Participants identified as HIV positive who fit the inclusion criteria and their 
parent(s)/legally designated representative(s) will be approached by a member of the 
research team. An explanation of the study will be then provided to parent(s)/legally 
designated representative(s) along with a copy of the information sheet and their level of 
interest will be gauged. The consent will be joint for: enrolment of the baby, for obtaining 
data and samples from the mother.  
*See APPENDIX: LOCAL SPECIFITIES PER SITE AND COUNTRY for Mozambique.  
Parent(s)/legally designated representative(s) will be given the Informed Consent leaflet. 
The trained study doctor will give time to read the leaflet and then go through the aims and 
procedures required to participate and answer any questions. All parent(s)/legally 
designated representative(s) will be informed that their child9s care is not dependent on 
their willingness to participate in the study. If parent(s)/legally designated representative(s) 
agree with the participation of the child, the study doctor will go through the consent form 
with him/her, answer any questions and he/she will then sign the form. 
 

EARTH v3, DECEMBER 2021 
45 
Separate tick boxes will be required to be completed for baby enrolment, caregiver 
enrolment, storage of samples.  
Caregivers will be given as much time as they need to consider the participation of their 
babies and will be informed that participation is voluntary and a decision not to participate 
will not affect their clinical care. Written informed consent will be obtained from 
parent(s)/legally designated representative(s) before subjects undergo any research 
procedures. Informed consent will be asked to the mother in order to collect data. The 
original signed consent forms if agreed will be retained with the source documents and a 
copy included in the participant9s medical notes.  
 
Parent(s)/legally designated representative(s) will be provided with a copy of the signed 
consent form and all consent form updates. The informed consent process must be 
traceable from the available documentation. If the parent(s)/legally designated 
representative(s) is/are unable to write, the consent may be given and recorded through 
appropriate alternative means in the presence of at least one impartial witness, who will 
sign and date the informed consent document, according to the applicable law. 
Furthermore, detailed information to the parent(s)/legally designated representative(s) will 
have to be on the basis of fair compensation in the case of damage, on the rights and 
safeguards dictated by law for the child protection, on the right to refuse or to withdraw 
from the study at any time without reasoning the decision and on the way to ensure respect 
for privacy and confidentiality of the individual data. 
 
The parent(s)/legally designated representative(s) is/are free to withdraw consent at any 
time and for any reason, whether expressed or not. No further data would be collected 
from the subject although data collected from the subject up until withdrawal would remain 
in the study records. The subject would continue standard care given at the specific site, 
without any penalty or loss of benefits to which the subject is otherwise entitled.  
Mothers 16 to 18 years old will follow the same protocol as >18 years old. Mothers <16 
years will have to sing an assent and will need an additional consent form from their 
parent(s)/legally designated representative(s), according to local laws.  
 
Subject Confidentiality 
Subject data protection and confidentiality will be guaranteed following the provisions 
stated in the Directive 95/46/EC of the European Parliament and of the Council as repealed 
by the European General Data Protection Regulation (EU) 2016/679 and in the applicable 
national legislations. The investigator must ensure that the subject9s privacy is maintained. 
On the CRF or other documents submitted to the Sponsors, subjects will be identified by a 
subject identification number only. Documents that are not submitted to the Sponsor (e.g., 
signed informed consent form) should be kept in a strictly confidential file by the 
investigator. Data will be access only by authorized people including principal investigators 
and their staff. Pseudonymized data will be accessed by data managers and statisticians. 
 
The investigator shall permit direct access to subjects9 records and source document for the 
purposes of monitoring, auditing, or inspection by the Sponsor, authorized representatives 
of the Sponsor, Regulatory Authorities and IECs/IRBs. 
 

EARTH v3, DECEMBER 2021 
46 
End of Study 
The End of the Study is defined as the last visit of the last participant. 
 
Study Documentation and Data Storage 
The investigators must retain essential documents until notified by the Sponsor, for at least 
for five years after study completion. The participant identification codes shall be archived 
by the principal investigators and their staff. Pseudonymized data in the REDcap database 
will be stored for 25 years after the completion (or discontinuation) of the study. After the 
end of the storage period, data will be made anonymous. Subject files and other source 
data (including copies of protocols, CRFs, original reports of test results, correspondence, 
records of informed consent, and other documents pertaining to the conduct of the study) 
must be kept for 25 years. Documents, without personal identifiers, should be stored in 
such a way that they can be accessed at a later date after the storage period. Consideration 
should be given to security and environmental risks. 
 
No study document will be destroyed without prior written agreement between the 
Sponsor and the investigator. If the investigator wishes to assign the study records to 
another party or move them to another location, written agreement must be obtained from 
the Sponsor. 
 
Data Sharing and Material Transfer Agreements 
Data sharing and sample transfer agreements will be signed by the receiving institutions, 
the9 providing sites and the sponsor. These documents will be stored by the sponsor.  
 
Storage of specimens  
A maximum volume of 18.5 mL per patient will be stored in-site if available and following 
the applicable local and International regulations.  
 
Specimens will be stored in the sites for 10 years after the study completion and may be 
used by the sites, AHRI, Rome, Massachusetts or Miami Laboratories to evaluate objectives 
consistent with this protocol to advance our understanding of the characterization and 
predictors of favorable HIV reservoirs and HIV-specific immunity profiles.  After the storage 
period samples will be destroyed. Should new techniques rise specifically for evaluation of 
reservoir and excellent control, an amendment to this protocol will be done in order to 
perform these techniques in the stored samples. Specific examples that are in development 
and dependent of further funding are CD32a+ lymphocytes or multiparametric visualization 
of infected cells.25 
 
Informed consent will be asked for storage of samples. Usage of samples for purposes other 
than the ones stated in this protocol and the informed consent requires additional ethical 
approval. Any reasonable effort will be pursued to get consent from the subjects for the use 
of the samples for purposes different from the ones stated in this protocol.  
 
Subject reimbursement 

EARTH v3, DECEMBER 2021 
47 
Subjects will not be paid to take part in the study however the parents/legal guardians could 
be compensated for expenses and loss of earnings directly related to participation in the 
trial, as per site regulation and practice.  
*See APPENDIX: LOCAL SPECIFITIES PER SITE AND COUNTRY for Mozambique.  
 
 
Substudies specificities about regulatory and ethics 
 
Specific and separate consent for the substudies will be obtained from parent(s)/legally 
designated representative(s) to provide the possibility to be enrolled in the study without 
participating in the substudies.  This will be clearly explained to the subject and the subject9s 
parent(s)/legal guardian(s), both verbally and in the information sheet. 
 
Substudy 1: Social/Behavioural Research Substudy (SBR) 
A specific information sheet will be provided for this substudy.  
 
Adult mothers, legal guardians or other caregivers are the participants in the SBR substudy. 
They will complete a separate consent process for their participation that follows a similar 
process and set of protections utilized for consent to EARTH: potential participants will 
receive an information sheet and be informed about the substudy by a member of the 
research team, in particular about the aims and procedures required to participate and 
answer any questions. All potential participants will be informed that their child9s care or 
participation in EARTH is not dependent on their willingness to participate in the substudy. 
Caregivers will be given as much time as they need to consider participation and will be 
informed that participation is voluntary. Written informed consent will be obtained from 
the caregivers. The original signed consent forms if agreed will be retained with the EARTH 
source documents for the related child. Participants will be provided with a copy of the 
consent form and all consent form updates. If the participant is unable to write, the consent 
may be given and recorded through appropriate alternative means in the presence of at 
least one impartial witness, who will sign and date the informed consent document, 
according to the applicable law. 
 
Participants are free to withdraw consent at any time and for any reason. No further data 
will be collected but data collected from the subject up until withdrawal will be maintained 
in the study records. If mental health concerns are identified or suspected in the 
participating caregivers they will be referred for specialty care.  
 
We will not enroll minors in the SBR substudy. 
 
Substudy 2: Strategies to improve viral suppression (SIVS) 
Specific information for this substudy has been added to the general information sheet. A 
specific box has been added to allow blood drawn from the mother.  
 
 
 

EARTH v3, DECEMBER 2021 
48 
17. 
 ADMINISTRATIVE MATTERS AND PUBLICATION POLICY 
 
Source Data 
Source Data will be participant medical records and laboratory reports. 
 
Language 
CRFs will be in French, English, Portuguese and local language if required. Generic names 
for concomitant medications should be recorded in the CRF wherever possible. Consent 
form will be in English or Portuguese and Changana (local language in Maputo Province, 
Mozambique), Afrikaans, Xhosa, Zulu and Sotho. All written material to be used by subjects 
must use vocabulary that is clearly understood and be in the language appropriate for the 
study site. 
 
Study Management Group (SMG) 
This group will oversee the day-to-day running of the study and the members will be 
primarily the clinical, laboratory and data management teams. Minutes of meetings will be 
taken and will form the basis of the progress report to the EPIICAL Steering Committee. The 
SMG will consist of the Principal Co-ordinating Investigator, a representative of the Sponsor, 
a representative from the regulatory and ethics work package 6, a representative of the 
Clinical platform work package 2, 3 and 4 and Site Principle Investigators. The SMG will 
report study data to the EPIICAL steering committee at the end of the study. 
 
Study Steering Committee (SSC) 
The Steering Committee for the EPIICAL project will be responsible for study supervision. 
 
Monitoring  
 
The study will be monitored by the Sponsor to assess the progress of the study, verify 
adherence to the protocol, and other national/international requirements and to review 
the completeness, accuracy and consistency of the data. 
 
Quality Control and Quality Assurance 
Quality Control will be performed according to Sponsor standard operating procedures. The 
study may be audited by a Quality Assurance representative of the Sponsor. All necessary 
data and documents will be made available for inspection. 
 
Disclosure of data and publication 
Information concerning the study, patent applications, processes, scientific data or other 
pertinent information is confidential and remains the property of the Sponsor. The 
investigators may use this information for the purposes of the study only. 
Verbal or written discussion of results prior to study completion and full reporting should 
only be undertaken with written consent from the Sponsor. Therefore, all information 
obtained as a result of the study will be regarded as CONFIDENTIAL, at least until 
appropriate analysis and review by the investigator(s) are completed.  
 
The results may be published or presented by the investigator(s), but the Sponsor must be 
given the opportunity to review and comment on proposed publications or presentations 

EARTH v3, DECEMBER 2021 
49 
for at least 15 days before they are submitted externally. Preparation of a manuscript for 
rapid publication will be the responsibility of the SSC. High priority will be given to this and 
it is anticipated that a report will be completed within six months of the study. Any 
preliminary and final publications will require approval by the SSC and the Sponsor. 
 
Publication Plan 
EPIICAL has a publication policy with clear authorship policies, specified in a separate 
document. In summary, authorship rules will follow BMJ publication rules 
(http://www.bmj.com/about- 
bmj/resources-authors/articlesubmission/authorship-
contributorship). 
 
The main papers (e.g., results of clinical trials) should have a name authorship (e.g. EPIICAL 
XY STUDY) with a writing committee at the bottom of the page and a list of collaborators. 
This is needed because we might need to include a number of authors higher than what 
allowed by the journal. 
 
For publications on the key work of each EPIICAL working group (WG), the following phrase 
should be included immediately after the authorship list: 8on behalf of the EPIICAL 
Consortium9. 
 
Expected Consortium Abstracts/Publications using data from the EARTH study: 
• Report of clinical outcomes at 6, 12, and 24 months of age of first 150 and 300 
participants. Leading, EPIICAL WG2(Clinical). 
• Specific publication on evolution of viral reservoir. Timing, first semester of 2021. 
Leading, EPIICAL WG3 (Virology). 
• Specific publication on immunological response. Timing, first semester of 2021. Leading, 
WG4 (Immunology) 
 
Substudies specificities publication policy 
Substudy 1: Social/Behavioural Research Substudy (SBR) 
 
• Barriers and facilitators to ART adherence and cohort retention 
• Stigma, social support and unmet needs among caregivers of children in the EARTH 
cohort 
 
Substudy 2: Strategies to improve viral suppression (SIVS) 
Report on objectives reported at the end of the main study. 
 
 
 

EARTH v3, DECEMBER 2021 
50 
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