Mutation of HIV-1 Genomes in a Clinical Population Treated with the Mutagenic Nucleoside KP1461

The deoxycytidine analog KP1212, and its prodrug KP1461, are prototypes of a new class of antiretroviral drugs designed to increase viral mutation rates, with the goal of eventually causing the collapse of the viral population. Here we present an extensive analysis of viral sequences from HIV-1 infected volunteers from the first “mechanism validation” phase II clinical trial of a mutagenic base analog in which individuals previously treated with antiviral drugs received 1600 mg of KP1461 twice per day for 124 days. Plasma viral loads were not reduced, and overall levels of viral mutation were not increased during this short-term study, however, the mutation spectrum of HIV was altered. A large number (N = 105 per sample) of sequences were analyzed, each derived from individual HIV-1 RNA templates, after 0, 56 and 124 days of therapy from 10 treated and 10 untreated control individuals (>7.1 million base pairs of unique viral templates were sequenced). We found that private mutations, those not found in more than one viral sequence and likely to have occurred in the most recent rounds of replication, increased in treated individuals relative to controls after 56 (p = 0.038) and 124 (p = 0.002) days of drug treatment. The spectrum of mutations observed in the treated group showed an excess of A to G and G to A mutations (p = 0.01), and to a lesser extent T to C and C to T mutations (p = 0.09), as predicted by the mechanism of action of the drug. These results validate the proposed mechanism of action in humans and should spur development of this novel antiretroviral approach.Koronis Pharmaceutical

Development and Optimization of Oligonucleotide Ligation Assay (OLA) Probes for Detection of HIV-1 Resistance to Dolutegravir

The WHO currently recommends dolutegravir (DTG)-based ART for persons living with HIV infection in resource-limited-settings (RLS). To expand access to testing for HIV drug resistance (DR) to DTG in RLS, we developed probes for use in the oligonucleotide ligation assay (OLA)-Simple, a near-point of care HIV DR kit. Genotypic data from clinical trials and case reports were used to determine the mutations in HIV-1 integrase critical to identifying individuals with DTG-resistance at virologic failure of DTG-based ART. Probes to detect G118R, Q148H/K/R, N155H and R263K in HIV-1 subtypes A, B, C, D and CRF01_AE were designed using sequence alignments from the Los Alamos database and validated using 61 clinical samples of HIV-1 subtypes A, B, C, D, CRF01_AE genotyped by PacBio (n = 15) or Sanger (n = 46). Initial OLA probes failed to ligate for 16/244 (6.5%) codons (9 at G118R and 7 at Q148H/K/R). Probes revised to accommodate polymorphisms interfering with ligation at codons G118R and Q148R reduced indeterminates to 3.7% (5 at G118R and 4 at Q148H/K/R) and detected DTG-mutations with a sensitivity of 96.5% and 100% specificity. These OLA DTG resistance probes appear highly sensitive and specific across HIV-1 subtypes common in RLS with high burden of HIV infection

Assessment of minority frequency pretreatment HIV drug-resistant variants in pregnant women and associations with virologic non-suppression at term.

ObjectiveTo assess in ART-naïve pregnant women randomized to efavirenz- versus raltegravir-based ART (IMPAACT P1081) whether pretreatment drug resistance (PDR) with minority frequency variants (DesignA case-control study design compared PDR minority variants in cases with virologic non-suppression (plasma HIV RNA >200 copies/mL) at delivery to randomly selected ART-suppressed controls.MethodsHIV pol genotypes were derived from pretreatment plasma specimens by Illumina sequencing. Resistance mutations were assessed using the HIV Stanford Database, and the proportion of cases versus controls with PDR to their ART regimens was compared.ResultsPDR was observed in 7 participants (11.3%; 95% CI 4.7, 21.9) and did not differ between 21 cases and 41 controls (4.8% vs 14.6%, p = 0.4061). PDR detected only as minority variants was less common (3.2%; 95% CI 0.2, 11.7) and also did not differ between groups (0% vs. 4.9%; p = 0.5447). Cases' median plasma HIV RNA at delivery was 347c/mL, with most (n = 19/22) showing progressive diminution of viral load but not ≤200c/mL. Among cases with viral rebound (n = 3/22), none had PDR detected. Virologic non-suppression at term was associated with higher plasma HIV RNA at study entry (pConclusionsWe observed a moderate frequency of PDR that did not significantly contribute to virologic non-suppression at term. Rather, higher pretreatment plasma HIV RNA, randomization to efavirenz-based ART, and shorter duration of ART were associated with non-suppression. These findings support early prenatal care engagement of pregnant women and initiation of integrase inhibitor-based ART due to its association with more rapid suppression of plasma RNA levels. Furthermore, because minority variants appeared infrequent in ART-naïve pregnant women and inconsequential to ART-suppression, testing for minority variants may be unwarranted

Specificity of private mutational change over time in the treated (panel A) and control (panel B) groups.

<p>Open bars are from day 0, shaded bars are from day 56 and black bars are from day 125. The y-axis shows the total number of private mutations in the two groups of 10 subjects.</p

Genetic diversity of 1.0 kb segments of HIV-1 <i>gag</i> genes through time in individuals treated and untreated with KP1461.

<p>Average population diversity of samplings of 105 1.0 kb <i>gag</i> gene segments through time is shown for treated (panel A) and untreated (panel B) groups.</p

Differences in private site mutations observed in treated and control groups.

1<p>Mean number of private site mutations over all participants at time 0.</p>2<p>Difference (treated minus control) after subtracting the difference at baseline.</p>3<p>All p-values are two-sided and based on permutations tests with 1000 repetitions.</p>4<p>Difference (treated minus control) at day 124 minus difference at day 56.</p

Private mutations detected in HIV-1 <i>gag</i> genes in treated and control groups.

<p>The average number of private mutations (found in only one of 105 sequences in each sample) per kilobase of sequence are shown for treated (Panel A) and control (Panel C) subjects at three time points and in Phase I trial participants (Clay et al, manuscript in preparation). In panels A and C, open bars are from day 0 (t1), shaded bars are from day 56 (t2) and black bars are from day 125 (t3). Patient 1 underwent 2 courses of treatment, pt1 and pt1.2, with a 9-day gap in between. The trajectory lines drawn on top of each 3-bar data set (for each individual) track the accumulation or loss of mutations over the study. In Panel A, 8 of 11 comparisons revealed an accumulation of mutations, whereas in control participants depicted in Panel C, only 2 0f 10 had an accumulation of mutations over the study. Panel B shows the number of private mutations per subject at each of the three time points. Data from treated subjects are shown with diamond symbols and controls with circles. Phase I trial participants sampled at day 0 (Panel B) are also shown with triangle symbols. Median levels for each group are shown with a horizontal line. Panel D shows the overall change in the number of private mutations over the 125-day sampling period in the treated group and over comparable intervals in the untreated control subjects. Wilcoxon ranked sum tests were used to assess differences in total private site counts within subjects and between treated and untreated groups. In panel B, p values for treated vs. phase I subjects  = 0.894; phase I subjects vs. phase 2 controls  = 0.549.</p

Number of SARS2 rtPCR tests, weekly surveys, and SARS2 cases by calendar week.

The study was conducted across a total of 6,411 unvaccinated person weeks, 1,268 partially vaccinated person weeks (<14 days from complete vaccination), and 6,845 fully vaccinated weeks (≥14 days from complete vaccination). SARS2 cases denoted with red arrows. Most cases had a discrete risk factor: black star = health care worker; blue star = family member of SARS2(+) healthcare worker; green star = household SARS2(+) exposure; purple star = attending in person school. No stars = no identified risk factor.</p

Demographics of treated and control groups.

<p>*At Day 0 of treatment.</p

Chemical structures of KP1212 and its prodrug, KP1461, administered in the Phase IIa trial.

<p>Chemical structures of KP1212 and its prodrug, KP1461, administered in the Phase IIa trial.</p