PI susceptibilities of pre-PI and failure virus from two patients demonstrate changes in phenotypic susceptibility over time.

<p>Full-length Gag-Protease sequences from pre-PI therapy and failure time-points was amplified or synthesised, and single round phenotypic susceptibility testing performed. PIs tested were Lopinavir (LPV) and Darunavir (DRV). (A) Susceptibility data from patient 5 demonstrates the reduction in susceptibility conferred by major mutations I84V (black) and I54V (grey) in our system in comparison with the patient virus at pre-PI therapy (white). Error bars represent standard deviation of two independent experimental repeats. (B) Virus derived pre-PI therapy (white bar) and at time of failure (black bar) from patient 6 demonstrated a difference in susceptibility to LPV in the absence of the development of major resistance mutations.</p

Viral load and patient treatment information.

<p>Treatment history of each patient is shown, from switch to second-line therapy during the DART trial (week -24, BL) and their enrollment in SARA (week 0), shown for most patients by simplification to LPV/r monotherapy. All patients were randomised to the LPV/r monotherapy arm of the SARA trial except for patients 1 and 7, who were randomised to continue on LPV/r containing triple therapy (CT). Available viral load measurements are shown; these were performed retrospectively and did not inform treatment decisions. The limit of detection for the assay (<50 copies/mL) is denoted with a red dashed line. The time from which the pre-PI (baseline, BL) and failure (Fail) samples included in this study were derived is highlighted by grey bars. For patient 7, the pre-PI sample was derived from week 0 of the DART trial before the week -24 of SARA timepoint, hence is not shown on this graph.</p

Maximum Likelihood phylogeny of virus from pre-PI therapy and treatment failure timepoints.

<p>Phylogenetic reconstruction was performed using the GTR model of nucleotide substitution. Phylogeny for the three patients for whom multiple sequences from both baseline and failure timepoints is shown: (a) patient 1, (b) patient 5 and (c) patient 3. Pre-PI virus sequences are denoted with circles and failure virus sequences with triangles. For patient 5 the drug resistance positions Gag 431 and Protease 54 and 84 were stripped from the alignment before phylogenetic re-construction. Nodes separating pre-PI and failure variants that supported by >75% bootstrapping are depicted with an asterisk (*).</p

PI susceptibility and single round infectivity of patient Gag-Protease derived pre-PI therapy and at the time of treatment failure.

<p>Full-length Gag-Protease from pre-PI therapy and failure time-points was amplified or synthesised, and cloned into our Gag-Pol expression vector p8.9NSX+. PI susceptibility of VSV-g pseudotyped viruses from patients experiencing virological failure in the absence of major resistance mutations was measured in a cell-based, single-round, phenotypic assay to the PIs A) Lopinavir (LPV) and B) Darunavir (DRV). The patient numbers are shown for each data point and data are means of two independent repeats. Our data demonstrate no significant difference in susceptibility between pre-PI therapy and failure viruses to the PIs LPV (t test, p = 0.91) and DRV (t test, p = 0.78), in patients failing in the absence of major resistance mutations. (C) Single-round infectivity of viruses was measured in the absence of drug in HEK 293T cells and compared in patients failing without major resistance mutations. No significant decrease in single-round infectivity was present (t test, p = 0.07).</p

Amino acid changes in <i>Gag-protease</i> at time of treatment failure relative to pre-PI Gag-Protease sequence in study participants.

<p>*I54V and I84V were not present in the same viral clone, but were present on separate clones. A431V developed only in the clones with I54V. ‘-‘) denotes when no changes in protease were present at failure.</p><p>Amino acid changes in <i>Gag-protease</i> at time of treatment failure relative to pre-PI Gag-Protease sequence in study participants.</p

Study profile of the Highly Active Antiretroviral therapy as Prevention (HAARP) Study.

<p>Study profile of the Highly Active Antiretroviral therapy as Prevention (HAARP) Study.</p

Characteristics of couples where seroconversion occurred when HIV participant was receiving ART for more than 3 months.

<p>Notes:</p><p>¹ Adherence is a composite of two questions: “How often do you use miss taking your ARVs?” and “How many pills have you missed in the last week?”. If participant reported any response other than “never” and “0”, respectively, was reported as non-adherent</p><p>² Condom use is a composite of two questions: “How often do you use condoms?” and “Did you use a condom the last time you had sex?”. If participant reported any response other than “always” and “yes”, respectively, condom use is reported as inconsistent</p><p>³ VL result taken on the day of serconversion = 176 copies/ mL. Reported value of 235,789 copies/mL was prior to ART initiation, 6 months prior to seroconversion.</p><p>Characteristics of couples where seroconversion occurred when HIV participant was receiving ART for more than 3 months.</p

Summary of baseline characteristics for the 3 lakeshore districts surveyed in 5 fishing communities between 2009–2011.

<p>Summary of baseline characteristics for the 3 lakeshore districts surveyed in 5 fishing communities between 2009–2011.</p

Heat map depicting clustering proportions of individuals sequenced 34 clusters (n = 283) stratified by baseline characteristics.

<p>The x-axis represents district, uc is un-clustered, c is clustered. The columns list on the y-axis are individual characteristics at study initiation and subsequent columns are the proportions clustered or un-clustered by district. Proportions are shown as a heat map to represent increasing proportions. Cells were colour coded with sliding colours as follows: blue corresponds to a low number of clustering, yellow corresponds to an intermediate amount of clustering and red corresponds to high level of clustering. Note that individuals in Masaka, Wakiso and Mukono; 2,2,1 had missing age, 25,3,1, had duration resident in area missing, 2,2,1 had missing marital status, 24,3,1 had missing occupation, 7,3,1 had alcohol use missing, 11,9,1 had partnerships missing, 7,3,1 had away 3 months missing and the remaining characteristics had none missing.</p

Characteristics of 34 clusters identified in <i>gag</i> or <i>env</i> gene regions for 283 HIV-infected participants 2009–2011.

<p>Characteristics of 34 clusters identified in <i>gag</i> or <i>env</i> gene regions for 283 HIV-infected participants 2009–2011.</p