Primer ID Ultra-Deep Sequencing Reveals Dynamics of drug Resistance-Associated Variants in Breakthrough Hepatitis C Viruses: Relevance to Treatment Outcome and Resistance Screening

Barnard, Richard; Chopra, Abha; James, Ian; Blinco, J.; Watson, Mark W.; Jabara, Cassandra B.; Hazuda, Daria J.; Lemon, Stanley M.; Mallal, S.; Gaudieri, Silvana

doi:10.3851/imp3056

Cited by 5 publications

(3 citation statements)

References 43 publications

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“…The use of large size amplicons to cover much of the HIV genome, as described in this study, precludes the use of unique primer identifiers (PIDs) to directly quantitate RNA species as previously described [51, 52]. However, in order to determine if the approach described here identified variants at a frequency that correlates to the original RNA species population (no PCR bias), five subjects were selected for PID analysis using subject-specific primers for a smaller amplicon in Nef.…”

Section: Methodsmentioning

confidence: 99%

Deep sequence analysis of HIV adaptation following vertical transmission reveals the impact of immune pressure on the evolution of HIV

et al. 2019

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Human immunodeficiency virus (HIV) can adapt to an individual’s T cell immune response via genomic mutations that affect antigen recognition and impact disease outcome. These viral adaptations are specific to the host’s human leucocyte antigen (HLA) alleles, as these molecules determine which peptides are presented to T cells. As HLA molecules are highly polymorphic at the population level, horizontal transmission events are most commonly between HLA-mismatched donor/recipient pairs, representing new immune selection environments for the transmitted virus. In this study, we utilised a deep sequencing approach to determine the HIV quasispecies in 26 mother-to-child transmission pairs where the potential for founder viruses to be pre-adapted is high due to the pairs being haplo-identical at HLA loci. This scenario allowed the assessment of specific HIV adaptations following transmission in either a non-selective immune environment, due to recipient HLA mismatched to original selecting HLA, or a selective immune environment, mediated by matched donor/recipient HLA. We show that the pattern of reversion or fixation of HIV adaptations following transmission provides insight into the replicative cost, and likely compensatory networks, associated with specific adaptations in vivo. Furthermore, although transmitted viruses were commonly heavily pre-adapted to the child’s HLA genotype, we found evidence of de novo post-transmission adaptation, representing new epitopes targeted by the child’s T cell response. High-resolution analysis of HIV adaptation is relevant when considering vaccine and cure strategies for individuals exposed to adapted viruses via transmission or reactivated from reservoirs.

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Section: Methodsmentioning

confidence: 99%

Deep sequence analysis of HIV adaptation following vertical transmission reveals the impact of immune pressure on the evolution of HIV

et al. 2019

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“…Primers were synthesized (Integrated DNA Technologies, USA). Accordingly, each cDNA template was synthesized with a unique primer ID (barcode tag), allowing accurate estimation of the viral species in the sample pool and the elimination of PCR artifacts in the analysis pipeline ( 40 ). Following cDNA synthesis, the reaction product was treated with RNase H (Invitrogen, USA) and purified with AMPure XP (Agencourt, Beckman Coulter).…”

Section: Methodsmentioning

confidence: 99%

Escape of Hepatitis C Virus from Epitope I Neutralization Increases Sensitivity of Other Neutralization Epitopes

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et al. 2018

J Virol

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The hepatitis C virus (HCV) E2 glycoprotein is a major target of the neutralizing antibody (nAb) response, with multiple type-specific and broadly neutralizing antibody (bnAb) epitopes identified. The 412-to-423 region can generate bnAbs that block interaction with the cell surface receptor CD81, with activity toward multiple HCV genotypes. In this study, we reveal the structure of rodent monoclonal antibody 24 (MAb24) with an extensive contact area toward a peptide spanning the 412-to-423 region. The crystal structure of the MAb24–peptide 412-to-423 complex reveals the paratope bound to a peptide hairpin highly similar to that observed with human MAb HCV1 and rodent MAb AP33, but with a different angle of approach. In viral outgrowth experiments, we demonstrated three distinct genotype 2a viral populations that acquired resistance to MAb24 via N415D, N417S, and N415D/H386R mutations. Importantly, the MAb24-resistant viruses exhibited significant increases in sensitivity to the majority of bnAbs directed to epitopes within the 412-to-423 region and in additional antigenic determinants located within E2 and the E1E2 complex. This study suggests that modification of N415 causes a global change in glycoprotein structure that increases its vulnerability to neutralization by other antibodies. This finding suggests that in the context of an antibody response to viral infection, acquisition of escape mutations in the 412-to-423 region renders the virus more susceptible to neutralization by other specificities of nAbs, effectively reducing the immunological fitness of the virus. A vaccine for HCV that generates polyspecific humoral immunity with specificity for the 412-to-423 region and at least one other region of E2 is desirable.IMPORTANCE Understanding how antibodies neutralize hepatitis C virus (HCV) is essential for vaccine development. This study reveals for the first time that when HCV develops resistance to a major class of bnAbs targeting the 412-to-423 region of E2, this results in a concomitant increase in sensitivity to neutralization by a majority of other bnAb specificities. Vaccines for the prevention of HCV infection should therefore generate bnAbs directed toward the 412-to-423 region of E2 and additional bnAb epitopes within the viral glycoproteins.

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“…Primer ID sequencing overcomes several limitations of conventional NGS by eliminating multiple types of PCR-related errors, thereby allowing unprecedented sequencing depth and accuracy (overall error rate ~0.01%) (Jabara et al, 2011; Zhou et al, 2015). Each template consensus sequence (TCS) generated by primer ID sequencing represents an original viral RNA template queried at the very first cDNA synthesis step, making primer ID sequencing an excellent tool to study highly diversified viral populations such as HIV-1 and HCV (Barnard et al, 2016; Jabara et al, 2014; Keys et al, 2015; Zhou et al, 2016). Coupling this approach with a unique set of serial serum samples collected before, during and after five days of elbasvir monotherapy, we show here that gt-1a and gt-1b HCV acquire antiviral resistance through distinct selection pathways, and that genotype-specific differences in RAS linkage and frequency can be linked to differences in cell culture assays of viral fitness.…”

Section: Introductionmentioning

confidence: 99%

Evolutionary pathways to NS5A inhibitor resistance in genotype 1 hepatitis C virus

et al. 2018

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Direct-acting antivirals (DAAs) targeting NS5A are broadly effective against hepatitis C virus (HCV) infections, but sustained virological response rates are generally lower in patients infected with genotype (gt)-1a than gt-1b viruses. The explanation for this remains uncertain. Here, we adopted a highly accurate, ultra-deep primer ID sequencing approach to intensively study serial changes in the NS5A-coding region of HCV in gt-1a- and gt-1b-infected subjects receiving a short course of monotherapy with the NS5A inhibitor, elbasvir. Low or undetectable levels of viremia precluded on-treatment analysis in gt-1b-infected subjects, but variants with the resistance-associated substitution (RAS) Y93H in NS5A dominated rebounding virus populations following cessation of treatment. These variants persisted until the end of the study, two months later. In contrast, while Y93H emerged in multiple lineages and became dominant in subjects with gt-1a virus, these haplotypes rapidly decreased in frequency off therapy. Substitutions at Q30 and L31 emerged in distinctly independent lineages at later time points, ultimately coming to dominate the virus population off therapy. Consistent with this, cell culture studies with gt-1a and gt-1b reporter viruses and replicons demonstrated that Y93H confers a much greater loss of replicative fitness in gt-1a than gt-1b virus, and that L31M/V both compensates for the loss of fitness associated with Q30R (but not Y93H) and also boosts drug resistance. These observations show how differences in the impact of RASs on drug resistance and replicative fitness influence the evolution of gt-1a and gt-1b viruses during monotherapy with an antiviral targeting NS5A.

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Primer ID Ultra-Deep Sequencing Reveals Dynamics of drug Resistance-Associated Variants in Breakthrough Hepatitis C Viruses: Relevance to Treatment Outcome and Resistance Screening

Cited by 5 publications

References 43 publications

Deep sequence analysis of HIV adaptation following vertical transmission reveals the impact of immune pressure on the evolution of HIV

Deep sequence analysis of HIV adaptation following vertical transmission reveals the impact of immune pressure on the evolution of HIV

Escape of Hepatitis C Virus from Epitope I Neutralization Increases Sensitivity of Other Neutralization Epitopes

Evolutionary pathways to NS5A inhibitor resistance in genotype 1 hepatitis C virus

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