Tissue-culture adaptation of viruses can modulate infection. Laboratory passage and bacterial artificial chromosome (BAC)mid cloning of human cytomegalovirus, HCMV, resulted in genomic deletions and ...rearrangements altering genes encoding the virus entry complex, which affected cellular tropism, virulence, and vaccine development. Here, we analyse these effects on the reference genome for related betaherpesviruses, Roseolovirus, human herpesvirus 6A (HHV-6A) strain U1102. This virus is also naturally "cloned" by germline subtelomeric chromosomal-integration in approximately 1% of human populations, and accurate references are key to understanding pathological relationships between exogenous and endogenous virus. Using whole genome next-generation deep-sequencing Illumina-based methods, we compared the original isolate to tissue-culture passaged and the BACmid-cloned virus. This re-defined the reference genome showing 32 corrections and 5 polymorphisms. Furthermore, minor variant analyses of passaged and BACmid virus identified emerging populations of a further 32 single nucleotide polymorphisms (SNPs) in 10 loci, half non-synonymous indicating cell-culture selection. Analyses of the BAC-virus genome showed deletion of the BAC cassette via loxP recombination removing green fluorescent protein (GFP)-based selection. As shown for HCMV culture effects, select HHV-6A SNPs mapped to genes encoding mediators of virus cellular entry, including virus envelope glycoprotein genes gB and the gH/gL complex. Comparative models suggest stabilisation of the post-fusion conformation. These SNPs are essential to consider in vaccine-design, antimicrobial-resistance, and pathogenesis.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
New antivirals are required to prevent rising antimicrobial resistance from replication inhibitors. The aim of this study was to analyse the range of emerging mutations in herpesvirus by whole genome ...deep sequencing. We tested human herpesvirus 6 treatment with novel antiviral K21, where evidence indicated distinct effects on virus envelope proteins. We treated BACmid cloned virus in order to analyse mechanisms and candidate targets for resistance. Illumina based next generation sequencing technology enabled analyses of mutations in 85 genes to depths of 10,000 per base detecting low prevalent minority variants (<1%). After four passages in tissue culture the untreated virus accumulated mutations in infected cells giving an emerging mixed population (45–73%) of non-synonymous SNPs in six genes including two envelope glycoproteins. Strikingly, treatment with K21 did not accumulate the passage mutations; instead a high frequency mutation was selected in envelope protein gQ2, part of the gH/gL complex essential for herpesvirus infection. This introduced a stop codon encoding a truncation mutation previously observed in increased virion production. There was reduced detection of the glycoprotein complex in infected cells. This supports a novel pathway for K21 targeting virion envelopes distinct from replication inhibition.
•Whole genome deep sequencing used to analyse herpesvirus targets novel antiviral K21.•Polymorphic mixtures identified in reconstituted BAC cloned virus removed by K21 treatment.•Targets virus envelope glycoprotein genes affecting infection by cell fusion.•Treated virus had reduced glycoprotein complex for infection.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Human herpesvirus-6A and B (HHV-6A, HHV-6B) have recently defined endogenous genomes, resulting from integration into the germline: chromosomally-integrated "CiHHV-6A/B". These affect approximately ...1.0% of human populations, giving potential for virus gene expression in every cell. We previously showed that CiHHV-6A was more divergent than CiHHV-6B by examining four genes in 44 European CiHHV-6A/B cardiac/haematology patients. There was evidence for gene expression/reactivation, implying functional non-defective genomes. To further define the relationship between HHV-6A and CiHHV-6A we used next-generation sequencing to characterize genomes from three CiHHV-6A cardiac patients. Comparisons to known exogenous HHV-6A showed CiHHV-6A genomes formed a separate clade; including all 85 non-interrupted genes and necessary cis-acting signals for reactivation as infectious virus. Greater single nucleotide polymorphism (SNP) density was defined in 16 genes and the direct repeats (DR) terminal regions. Using these SNPs, deep sequencing analyses demonstrated superinfection with exogenous HHV-6A in two of the CiHHV-6A patients with recurrent cardiac disease. Characterisation of the integration sites in twelve patients identified the human chromosome 17p subtelomere as a prevalent site, which had specific repeat structures and phylogenetically related CiHHV-6A coding sequences indicating common ancestral origins. Overall CiHHV-6A genomes were similar, but distinct from known exogenous HHV-6A virus, and have the capacity to reactivate as emerging virus infections.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Our understanding of how checkpoint inhibitors (CPI) affect T cell evolution is incomplete, limiting our ability to achieve full clinical benefit from these drugs. Here we analyzed peripheral T cell ...populations after one cycle of CPI and identified a dynamic awakening of the immune system revealed by T cell evolution in response to treatment. We sequenced T cell receptors (TCR) in plasma cell-free DNA (cfDNA) and peripheral blood mononuclear cells (PBMC) and performed phenotypic analysis of peripheral T cell subsets from metastatic melanoma patients treated with CPI. We found that early peripheral T cell turnover and TCR repertoire dynamics identified which patients would respond to treatment. Additionally, the expansion of a subset of immune-effector peripheral T cells we call T
cells correlated with response. These events are prognostic and occur within 3 weeks of starting immunotherapy, raising the potential for monitoring patients responses using minimally invasive liquid biopsies."
Precision immuno-oncology approaches are needed to improve cancer care. We recently demonstrated that in patients with metastatic melanoma, an increase of clonality or diversity of the T cell ...receptor (TCR) repertoire of peripheral T cells following one cycle of immunotherapy is coincident with response to immune-checkpoint blockade (ICB). We also identified a subset of peripheral CD8+ immune-effector memory T cells (TIE cells) whose expansion was associated with response to ICB and increased overall survival. To improve our understanding of peripheral T cell dynamics, we examined the clinical correlates associated with these immune signatures.
Fifty patients with metastatic melanoma treated with first-line anti-PD-1 ICB were included. We analysed TCR repertoire and peripheral TIE cell dynamics by age before treatment (T0) and after the first cycle of treatment at week 3 (W3).
We observed a correlation between TIE abundance and age at T0 (r = 0.40), which reduced following treatment at W3 (r = 0.07). However, at W3, we observed two significantly opposing patterns (p = 0.03) of TCR repertoire rearrangement in patients who responded to treatment, with patients ≥70 years of age showing an increase in TCR clonality and patients <70 years of age showing an increase in TCR diversity.
We demonstrate that immunotherapy-induced immune-awakening patterns in patients with melanoma are age-related and may impact patient response to ICB, and thus have implications for biomarker development and planning of personalised therapeutic strategies.
•Immune-effector T cell expansion early during immunotherapy predicts tumour shrinkage.•Immune-checkpoint blockade drives age-dependent bifurcated evolution of T cell clonality or diversity.•T cell evolution provides a convenient early biomarker of response to immunotherapy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Distinctions in the biological features of human herpesvirus-6A (HHV-6A) and -6B (HHV-6B) have recently led to their reclassification from variants to distinct viral species. Unique for human ...herpesviruses, these viruses also exist in an endogenous form in ~1% of the human population, resulting from germ-line chromosomal integration. Chromosomally integrated human herpesvirus-6A (CI-HHV-6A) and -6B (CI-HHV-6B) genomes are inherited in a Mendelian manner, leading to the potential for viral gene expression and/or reactivation in every cell of the body. As yet, the effects of these integrated viral genomes on health and their relationship to circulating viral strains remain unclear. To address this, next generation sequencing (NGS) methods were established, to define a readily utilisable pipeline for genomic sequencing of these herpesviruses directly from cell or tissue samples. This was first tested to derive the complete genome sequence of a third HHV-6A strain, AJ. This showed close conservation with a recent North American isolate despite distinct geographic origins, which may reflect highly evolved viral status or recent emergence. Subsequently, using this defined NGS methodology, supplemented by Sanger sequencing, integrated viral genomes were characterised. Integration site analyses revealed a high prevalence of integration on chromosome 17, all with a shared junctional architecture, indicative of a single ancestral germ-line integration event at this chromosome. Genomic analyses revealed CI-HHV-6A retains the full HHV-6A gene complement, with no interrupted open reading frames, and important cis acting signals likely required for any viral replication/reactivation mechanism. Deep sequencing with minority variant analysis suggested a potential reactivation mechanism, resulting from superinfection with circulating viral strains. CI-HHV-6A was also found to diverge at a set of genes which have been used as markers of speciation amongst roseoloviruses, while retaining many of the features of HHV-6A. One of the key viral immunomodulatory genes, the chemokine receptor U51, was investigated further. This showed specific coding changes and a panel of viral-human chimeric and viral mutant receptor expression vectors were generated to investigate this. Initial structural modelling and functional characterisations were made showing distinctions. Overall, the results identify unique characteristics of the integrated genomes compared to known circulating viral strains.
Abnormal excitability in cortical networks has been reported in patients and animal models of Alzheimer's disease (AD), and other neurodegenerative conditions. Whether hyperexcitability is a core ...feature of alpha(α)-synucleinopathies, including dementia with Lewy bodies (DLB) is unclear. To assess this, we used two murine models of DLB that express either human mutant α-synuclein (α-syn) the hA30P, or human wild-type α-syn (hWT-α-syn) mice. We observed network hyperexcitability in vitro in young (2–5 months), pre-symptomatic transgenic α-syn mice. Interictal discharges (IIDs) were seen in the extracellular local field potential (LFP) in the hippocampus in hA30P and hWT-α-syn mice following kainate application, while only gamma frequency oscillations occurred in control mice. In addition, the concentration of the GABAA receptor antagonist (gabazine) needed to evoke IIDs was lower in slices from hA30P mice compared to control mice. hA30P mice also showed increased locomotor activity in the open field test compared to control mice. Intracellular recordings from CA3 pyramidal cells showed a more depolarised resting membrane potential in hA30P mice. Quadruple immunohistochemistry for human α-syn, and the mitochondrial markers, porin and the complex IV enzyme cytochrome c oxidase subunit 1 (COX1) in parvalbumin (PV+)-expressing interneurons showed that 25% of PV+ cells contained human α-syn in hA30P mice. While there was no change in PV expression, COX1 expression was significantly increased in PV+ cells in hA30P mice, perhaps reflecting a compensatory change to support PV+ interneuron activity. Our findings suggest that hippocampal network hyperexcitability may be an important early consequence of α-syn-mediated impairment of neuronal/synaptic function, which occurs without any overt loss of PV interneurons. The therapeutic benefit of targeting network excitability early in the disease stage should be explored with respect to α-synucleinopathies such as DLB.
•Young transgenic α-syn mice exhibit network hyperexcitability in the hippocampus in vitro.•Young transgenic α-syn mice have increased locomotor activity in an open field test.•Hippocampal pyramidal cells are more depolarised in young transgenic α-syn mice.•Increased mitochondrial cytochrome c oxidase (complex IV) function in PV+ interneurons in young transgenic a-syn mice.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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