Nonlinear Mixed-Effects Models for HIV Viral Load Trajectories Before and After Antiretroviral Therapy Interruption, Incorporating Left Censoring

Characterizing features of the viral rebound trajectories and identifying host, virological, and immunological factors that are predictive of the viral rebound trajectories are central to HIV cure research. In this paper, we investigate if key features of HIV viral decay and CD4 trajectories during antiretroviral therapy (ART) are associated with characteristics of HIV viral rebound following ART interruption. Nonlinear mixed effect (NLME) models are used to model viral load trajectories before and following ART interruption, incorporating left censoring due to lower detection limits of viral load assays. A stochastic approximation EM (SAEM) algorithm is used for parameter estimation and inference. To circumvent the computational intensity associated with maximizing the joint likelihood, we propose an easy-to-implement threestep method. We evaluate the performance of this method through simulation studies and apply it to data from the Zurich Primary HIV Infection Study. We find that some key features of viral load during ART (e.g., viral decay rate) are significantly associated with important characteristics of viral rebound following ART interruption (e.g., viral set point)

Minority Quasispecies of Drug-Resistant HIV-1 That Lead to Early Therapy Failure in Treatment-Naive and -Adherent Patients

Background.Early virological failure of antiretroviral therapy associated with the selection of drug-resistant human immunodeficiency virus type 1 in treatment-naive patients is very critical, because virological failure significantly increases the risk of subsequent failures. Therefore, we evaluated the possible role of minority quasispecies of drug-resistant human immunodeficiency virus type 1, which are undetectable at baseline by population sequencing, with regard to early virological failure. Methods.We studied 4 patients who experienced early virological failure of a first-line regimen of lamivudine, tenofovir, and either efavirenz or nevirapine and 18 control patients undergoing similar treatment without virological failure. The key mutations K65R, K103N, Y181C, M184V, and M184I in the reverse transcriptase were quantified by allele-specific real-time polymerase chain reaction performed on plasma samples before and during early virological treatment failure. Results.Before treatment, none of the viruses showed any evidence of drug resistance in the standard genotype analysis. Minority quasispecies with either the M184V mutation or the M184I mutation were detected in 3 of 18 control patients. In contrast, all 4 patients whose treatment was failing had harbored drug-resistant viruses at low frequencies before treatment, with a frequency range of 0.07% 2.0%. A range of 1 4 mutations was detected in viruses from each patient. Most of the minority quasispecies were rapidly selected and represented the major virus population within weeks after the patients started antiretroviral therapy. All 4 patients showed good adherence to treatment. Nonnucleoside reverse-transcriptase inhibitor plasma concentrations were in normal ranges for all 4 patients at 2 separate assessment times. Conclusions.Minority quasispecies of drug-resistant viruses, detected at baseline, can rapidly outgrow and become the major virus population and subsequently lead to early therapy failure in treatment-naive patients who receive antiretroviral therapy regimens with a low genetic resistance barrie

Reconciling founder variant multiplicity of HIV-1 infection with the rate of CD4+ decline

HIV-1 transmission precipitates a stringent genetic bottleneck, with 75% of new infections initiated by a single genetic variant. Where multiple variants initiate infection, recipient set point viral load (SpVL) and the rate of CD4+ 4 T cell decline may be elevated, but these findings remain inconsistent. Here, we summarised the evidence for this phenomenon, then tested whether previous studies possessed sufficient statistical power to reliably identify a true effect of multiple variant infection associating with higher SpVL. Next, we combined models of HIV-1 transmission, heritability and disease progression to understand whether available data suggest a faster CD4+ T cell decline would be expected to associate with multiple variant infection, without an explicit dependency between the two. We found that most studies had insufficient power to identify a true significant difference, prompting an explanation for previous inconsistencies. Next, our model framework revealed we would not expect to observe a positive association between multiple variant infections and faster CD4+ T cell decline, in the absence of an explicit dependency. Consequently, while empirical evidence may be consistent with a positive association between multiple variant infection and faster CD4+ T cell decline, further investigation is required to establish a causal basis for this association

Easy and accurate reconstruction of whole HIV genomes from short-read sequence data with shiver

Studying the evolution of viruses and their molecular epidemiology relies on accurate viral sequence data, so that small differences between similar viruses can be meaningfully interpreted. Despite its higher throughput and more detailed minority variant data, next-generation sequencing has yet to be widely adopted for HIV. The difficulty of accurately reconstructing the consensus sequence of a quasispecies from reads (short fragments of DNA) in the presence of large between-and within-host diversity, including frequent indels, may have presented a barrier. In particular, mapping (aligning) reads to a reference sequence leads to biased loss of information; this bias can distort epidemiological and evolutionary conclusions. De novo assembly avoids this bias by aligning the reads to themselves, producing a set of sequences called contigs. However contigs provide only a partial summary of the reads, misassembly may result in their having an incorrect structure, and no information is available at parts of the genome where contigs could not be assembled. To address these problems we developed the tool shiver to pre-process reads for quality and contamination, then map them to a reference tailored to the sample using corrected contigs supplemented with the user's choice of existing reference sequences. Run with two commands per sample, it can easily be used for large heterogeneous data sets. We used shiver to reconstruct the consensus sequence and minority variant information from paired-end short-read whole-genome data produced with the Illumina platform, for sixty-five existing publicly available samples and fifty new samples. We show the systematic superiority of mapping to shiver's constructed reference compared with mapping the same reads to the closest of 3,249 real references: median values of 13 bases called differently and more accurately, 0 bases called differently and less accurately, and 205 bases of missing sequence recovered. We also successfully applied shiver to whole-genome samples of Hepatitis C Virus and Respiratory Syncytial Virus. shiver is publicly available from https://github.com/ChrisHIV/shiver.Peer reviewe

Reappearance of Minority K103N HIV-1 Variants after Interruption of ART Initiated during Primary HIV-1 Infection

BACKGROUND: In the Zurich Primary HIV infection study (ZPHI), minority drug-resistant HIV-1 variants were detected in some acutely HIV-1-infected patients prior to initiation of early antiretroviral therapy (ART). Here, we investigated the reappearance of minority K103N and M184V HIV-1 variants in these patients who interrupted efficient early ART after 8-27 months according to the study protocol. These mutations are key mutations conferring drug resistance to reverse transcriptase inhibitors and they belong to the most commonly transmitted drug resistance mutations. METHODOLOGY/PRINCIPAL FINDINGS: Early ART was offered to acutely HIV-1-infected patients enrolled in the longitudinal prospective ZPHI study. Six patients harboring and eleven patients not harboring drug-resistant viruses at low frequencies prior to ART were included in this substudy. Minority K103N and M184V HIV-1 variants were quantified in longitudinal plasma samples after treatment interruption by allele-specific real-time PCR. All 17 patients were infected with HIV-1 subtype B between 04/2003 and 09/2005 and received LPV/r+AZT+3TC during primary HIV-1 infection (PHI). Minority K103N HIV-1 variants reappeared after cessation of ART in two of four patients harboring this variant during PHI and even persisted in one of those patients at frequencies similar to the frequency observed prior to ART (<1%). The K103N mutation did not appear during treatment interruption in any other patient. Minority M184V HIV-1 variants were detected in two patients after ART interruption, one harboring and one not harboring these variants prior to ART. CONCLUSION: Minority K103N HIV-1 variants, present in acutely HIV-1 infected patients prior to early ART, can reappear and persist after interruption of suppressive ART containing two nucleoside/nucleotide analogue reverse transcriptase inhibitors and a ritonavir-boosted protease inhibitor. TRIAL REGISTRATION: Clinicaltrials.gov NCT00537966

Recommendations on data sharing in HIV drug resistance research

• Human immunodeficiency virus (HIV) drug resistance has implications for antiretroviral treatment strategies and for containing the HIV pandemic because the development of HIV drug resistance leads to the requirement for antiretroviral drugs that may be less effective, less well-tolerated, and more expensive than those used in first-line regimens.  • HIV drug resistance studies are designed to determine which HIV mutations are selected by antiretroviral drugs and, in turn, how these mutations affect antiretroviral drug susceptibility and response to future antiretroviral treatment regimens.  • Such studies collectively form a vital knowledge base essential for monitoring global HIV drug resistance trends, interpreting HIV genotypic tests, and updating HIV treatment guidelines.  • Although HIV drug resistance data are collected in many studies, such data are often not publicly shared, prompting the need to recommend best practices to encourage and standardize HIV drug resistance data sharing.  • In contrast to other viruses, sharing HIV sequences from phylogenetic studies of transmission dynamics requires additional precautions as HIV transmission is criminalized in many countries and regions.  • Our recommendations are designed to ensure that the data that contribute to HIV drug resistance knowledge will be available without undue hardship to those publishing HIV drug resistance studies and without risk to people living with HIV

Efficacy and safety of two neutralising monoclonal antibody therapies, sotrovimab and BRII-196 plus BRII-198, for adults hospitalised with COVID-19 (TICO): a randomised controlled trial

BACKGROUND: We aimed to assess the efficacy and safety of two neutralising monoclonal antibody therapies (sotrovimab [Vir Biotechnology and GlaxoSmithKline] and BRII-196 plus BRII-198 [Brii Biosciences]) for adults admitted to hospital for COVID-19 (hereafter referred to as hospitalised) with COVID-19. METHODS: In this multinational, double-blind, randomised, placebo-controlled, clinical trial (Therapeutics for Inpatients with COVID-19 [TICO]), adults (aged ≥18 years) hospitalised with COVID-19 at 43 hospitals in the USA, Denmark, Switzerland, and Poland were recruited. Patients were eligible if they had laboratory-confirmed SARS-CoV-2 infection and COVID-19 symptoms for up to 12 days. Using a web-based application, participants were randomly assigned (2:1:2:1), stratified by trial site pharmacy, to sotrovimab 500 mg, matching placebo for sotrovimab, BRII-196 1000 mg plus BRII-198 1000 mg, or matching placebo for BRII-196 plus BRII-198, in addition to standard of care. Each study product was administered as a single dose given intravenously over 60 min. The concurrent placebo groups were pooled for analyses. The primary outcome was time to sustained clinical recovery, defined as discharge from the hospital to home and remaining at home for 14 consecutive days, up to day 90 after randomisation. Interim futility analyses were based on two seven-category ordinal outcome scales on day 5 that measured pulmonary status and extrapulmonary complications of COVID-19. The safety outcome was a composite of death, serious adverse events, incident organ failure, and serious coinfection up to day 90 after randomisation. Efficacy and safety outcomes were assessed in the modified intention-to-treat population, defined as all patients randomly assigned to treatment who started the study infusion. This study is registered with ClinicalTrials.gov, NCT04501978. FINDINGS: Between Dec 16, 2020, and March 1, 2021, 546 patients were enrolled and randomly assigned to sotrovimab (n=184), BRII-196 plus BRII-198 (n=183), or placebo (n=179), of whom 536 received part or all of their assigned study drug (sotrovimab n=182, BRII-196 plus BRII-198 n=176, or placebo n=178; median age of 60 years [IQR 50-72], 228 [43%] patients were female and 308 [57%] were male). At this point, enrolment was halted on the basis of the interim futility analysis. At day 5, neither the sotrovimab group nor the BRII-196 plus BRII-198 group had significantly higher odds of more favourable outcomes than the placebo group on either the pulmonary scale (adjusted odds ratio sotrovimab 1·07 [95% CI 0·74-1·56]; BRII-196 plus BRII-198 0·98 [95% CI 0·67-1·43]) or the pulmonary-plus complications scale (sotrovimab 1·08 [0·74-1·58]; BRII-196 plus BRII-198 1·00 [0·68-1·46]). By day 90, sustained clinical recovery was seen in 151 (85%) patients in the placebo group compared with 160 (88%) in the sotrovimab group (adjusted rate ratio 1·12 [95% CI 0·91-1·37]) and 155 (88%) in the BRII-196 plus BRII-198 group (1·08 [0·88-1·32]). The composite safety outcome up to day 90 was met by 48 (27%) patients in the placebo group, 42 (23%) in the sotrovimab group, and 45 (26%) in the BRII-196 plus BRII-198 group. 13 (7%) patients in the placebo group, 14 (8%) in the sotrovimab group, and 15 (9%) in the BRII-196 plus BRII-198 group died up to day 90. INTERPRETATION: Neither sotrovimab nor BRII-196 plus BRII-198 showed efficacy for improving clinical outcomes among adults hospitalised with COVID-19. FUNDING: US National Institutes of Health and Operation Warp Speed

Development of a high-throughput bead based assay system to measure HIV-1 specific immune signatures in clinical samples

The monitoring and assessment of a broadly neutralizing antibody (bnAb) based HIV-1 vaccine require detailed measurements of HIV-1 binding antibody responses to support the detection of correlates of protection. Here we describe the development of a flexible, high-throughput microsphere based multiplex assay system that allows monitoring complex binding antibody signatures. Studying a panel of 13 HIV-1 antigens in a parallel assessment of different IgG subclasses (IgG1, IgG2 and IgG3) we demonstrate the potential of our strategy. The technical advances we describe include means to improve antigen reactivity using directed neutravidin-biotin immobilization of antigens and biotin saturation to reduce background. A particular emphasis of our study was to provide tools for the assessment of reproducibility and stability of the assay system and strategies to control for variations allowing the application in highthroughput assays, where reliability of single measurements needs to be guaranteed

Drug resistance mutations during structured treatment interruptions

BACKGROUND: We assessed whether treatment interruptions induce selection of mutations associated with drug resistance in the Swiss-Spanish Intermittent Treatment Trial (SSITT). Patients had been on HAART without previous failure and had undetectable viraemia for at least 6 months. Their HAART was interrupted for 2 weeks and restarted for 8 weeks. After four of these cycles, treatment was definitively interrupted at week 40. METHODS: Genotypic resistance testing was performed in 87/97 Swiss patients: in those failing treatment before week 40, at the time of first viral rebound > 500 copies/ml off treatment and preceding failure to reach RNA 1000 copies/ml after week 40. RESULTS: Mutations associated with drug resistance were detected in 9/25 (36%) patients with virological failure during the first 40 weeks and in 6/59 (10%) patients after week 40. Overall, drug resistance mutations were detected in 17% of patients, all but two with the 184V/I mutation. Among the 74 patients receiving lamivudine, the M184V/I mutation was detected in 13/74 (17.6%) patients. A wild-type codon at position 184 was detected in previous samples in all but two. The relative risk for virological failure was 2.55-fold higher in patients with the M184V/I mutation than in patients without detectable mutation (P=0.007). CONCLUSIONS: The M184V/I mutation is frequently selected during repeated treatment interruptions

Proviral HIV-DNA predicts viral rebound and viral setpoint after structured treatment interruptions

In HIV-1-infected patients with long-term undetectable viraemia on highly active antiretroviral treatment (HAART), we found that pre-HAART plasma viraemia and the baseline proviral DNA level were significantly associated with the viraemia setpoint during scheduled treatment interruptions. In long-term treated patients, pre-HAART viraemia may not be available, and in these circumstances proviral DNA, measured at the time of scheduled treatment interruption, can help to identify patients likely to reach a low viraemia setpoint after treatment interruption