Additional file 1: Figure S1. of Early antiretroviral treatment (eART) limits viral diversity over time in a long-term HIV viral suppressed perinatally infected child

Neighborg phylogenetic tree constructed on the pol gene sequences of 400 isolates and additional 163 HIV-1 subtype references. The bar at the bottom indicating 0.01 nucleotide substitution per site. Bootstrap support >90% were showed along the branches. Isolates of sutypes C are shown in red. The sequences involved in mother to child transmission chain are in bold red. (PPTX 160 kb)

Patient data at enrolment.

<p>ART signifies antiretroviral treatment with PI = protease inhibitors, NRTI = nucleoside reverse transcriptase inhibitors, NNRTI = non nucleoside reverse transcriptase inhibitors.</p

Cellular immune responses to HIV-1 antigens.

<p>^AUCarea under the curve.</p>a<p>week 0–96.</p><p>^bweek 0–60.</p

Safety Profile of the PEDVAC trial: distribution of adverse events during the 96 weeks follow-up of the study.

<p>The black dashed line shows total number of solicited and unsolicited adverse events in control Group A. The gray dashed line indicates total number of solicited and unsolicited adverse events in vaccine Group B, while the black solid line indicates all local and systemic adverse events which were vaccine related. Black arrows show vaccinations (weeks 0, 4, 12 and 36).</p

Lymphoproliferative responses are shown as Stimulation Index (SI) to virion HIV-1 MN (A), recombinant Gag p24 (B) and recombinant RT (C) viral antigens among vaccinated (n = 10; black dots and lines) and controls (n = 10; grey squares and lines).

<p>Black arrows show vaccinations (weeks 0, 4, 12 and 36).</p

Viral dynamics during the vaccination schedule for the control Group A (n = 10) (A) and vaccine Group B (n = 10) (B).

<p>HIV-RNA with a limit of detection of 50 copies/ml is shown as black dots, HIV-RNA ultrasensitive assay with a limit of detection of 1 copy/ml plasma is shown as circles and cell associated HIV-DNA of PBMC shown as triangles indicating number of total HIV-proviral DNA copies/10⁶ PBMC. Black arrows show vaccinations (weeks 0, 4, 12 and 36).</p

Functional profiles of between groups HIV-specific CD4+ and CD8+ T-cells analyzed by ICS at weeks 0, 16, 20 and 60.

<p>The panels show the percentage of T-cells releasing IFN-γ (A) and IL-2 (B) for CD4+ T-cells, and IFN-γ (C) and perforin (D) for CD8+ T-cells for each patient in control Group A (n = 10, circles), and vaccine Group B (n = 10, black dots). T-cell functions were analyzed after stimulation for 16 h with a pool of proteins representing the vaccine sequences. Lines represent median values. In panel (E) CD8+ cells releasing perforin at baseline week 0 and after 60 weeks in vaccine and control groups are shown. Differences within groups between baseline and week 60 levels in panel (E) are indicated for each individual, and the p-value calculated by Wilcoxon signed rank test.</p

CONSORT 2010 flow diagram.

<p>CONSORT 2010 flow diagram.</p

Table_6_Low unspliced cell-associated HIV RNA in early treated adolescents living with HIV on long suppressive ART.docx

IntroductionInitiation of antiretroviral treatment (ART) in patients early after HIV-infection and long-term suppression leads to low or undetectable levels of HIV RNA and cell-associated (CA) HIV DNA and RNA. Both CA-DNA and CA-RNA, overestimate the size of the HIV reservoir but CA-RNA as well as p24/cell-free viral RNA can be indicators of residual viral replication. This study describes HIV RNA amounts and levels of cytokines/soluble markers in 40 well-suppressed adolescents who initiated ART early in life and investigated which viral markers may be informative as endpoints in cure clinical trials within this population.MethodsForty adolescents perinatally infected with HIV on suppressive ART for >5 years were enrolled in the CARMA study. HIV DNA and total or unspliced CA-RNA in PBMCs were analyzed by qPCR/RT-qPCR and dPCR/RT-dPCR. Cell-free HIV was determined using an ultrasensitive viral load (US-VL) assay. Plasma markers and p24 were analyzed by digital ELISA and correlations between total and unspliced HIV RNA and clinical markers, including age at ART, Western Blot score, levels of cytokines/inflammation markers or HIV CA-DNA, were tested.ResultsCA-RNA was detected in two thirds of the participants and was comparable in RT-qPCR and RT-dPCR. Adolescents with undetectable CA-RNA showed significantly lower HIV DNA compared to individuals with detectable CA-RNA. Undetectable unspliced CA-RNA was positively associated with age at ART initiation and Western Blot score. We found that a higher concentration of TNF-α was predictive of higher CA-DNA and CA-RNA. Other clinical characteristics like US-VL, time to suppression, or percent CD4+ T-lymphocytes were not predictive of the CA-RNA in this cross-sectional study.ConclusionsLow CA-DNA after long-term suppressive ART is associated with lower CA-RNA, in concordance with other reports. Patients with low CA-RNA levels in combination with low CA-DNA and low Western Blot scores should be further investigated to characterize candidates for treatment interruption trials. Unspliced CA-RNA warrants further investigation as a marker that can be prioritized in paediatric clinical trials where the sample volume can be a significant limitation.</p

Table_5_Low unspliced cell-associated HIV RNA in early treated adolescents living with HIV on long suppressive ART.docx

IntroductionInitiation of antiretroviral treatment (ART) in patients early after HIV-infection and long-term suppression leads to low or undetectable levels of HIV RNA and cell-associated (CA) HIV DNA and RNA. Both CA-DNA and CA-RNA, overestimate the size of the HIV reservoir but CA-RNA as well as p24/cell-free viral RNA can be indicators of residual viral replication. This study describes HIV RNA amounts and levels of cytokines/soluble markers in 40 well-suppressed adolescents who initiated ART early in life and investigated which viral markers may be informative as endpoints in cure clinical trials within this population.MethodsForty adolescents perinatally infected with HIV on suppressive ART for >5 years were enrolled in the CARMA study. HIV DNA and total or unspliced CA-RNA in PBMCs were analyzed by qPCR/RT-qPCR and dPCR/RT-dPCR. Cell-free HIV was determined using an ultrasensitive viral load (US-VL) assay. Plasma markers and p24 were analyzed by digital ELISA and correlations between total and unspliced HIV RNA and clinical markers, including age at ART, Western Blot score, levels of cytokines/inflammation markers or HIV CA-DNA, were tested.ResultsCA-RNA was detected in two thirds of the participants and was comparable in RT-qPCR and RT-dPCR. Adolescents with undetectable CA-RNA showed significantly lower HIV DNA compared to individuals with detectable CA-RNA. Undetectable unspliced CA-RNA was positively associated with age at ART initiation and Western Blot score. We found that a higher concentration of TNF-α was predictive of higher CA-DNA and CA-RNA. Other clinical characteristics like US-VL, time to suppression, or percent CD4+ T-lymphocytes were not predictive of the CA-RNA in this cross-sectional study.ConclusionsLow CA-DNA after long-term suppressive ART is associated with lower CA-RNA, in concordance with other reports. Patients with low CA-RNA levels in combination with low CA-DNA and low Western Blot scores should be further investigated to characterize candidates for treatment interruption trials. Unspliced CA-RNA warrants further investigation as a marker that can be prioritized in paediatric clinical trials where the sample volume can be a significant limitation.</p