Duration of Postvaccination Neutralizing Antibodies to SARS-CoV-2 and Medication Effects: Results from the Safety and Immunogenicity of COVID-19 Vaccination in Systemic Immune-Mediated Inflammatory Diseases Cohort Study.

OBJECTIVE: In the face of the ongoing circulation of SARS-CoV-2, the durability of neutralization post-COVID-19 vaccination in immune-mediated inflammatory disease (IMID) is a key issue, as are the effects of medications. METHODS: Adults (n = 112) with inflammatory bowel disease, psoriasis/psoriatic arthritis, rheumatoid arthritis, spondylarthritis, and systemic lupus were recruited from participating Canadian medical centers from 2021 to 2023. We focused on log-transformed neutralization (lentivirus methods) as a continuous outcome, with separate models for wild-type and Omicron strains BA.1 and BA.5. RESULTS: Compared with 30 to 120 days postvaccination, subsequent periods were associated with greater neutralization in unadjusted models for wild-type, BA.1, and BA.5 strains and against the BA.1 strain in adjusted models. Rituximab was associated with lower neutralization for the BA.1 strain in adjusted models, with a similar trend for BA.5. In methotrexate users, there were trends for less neutralization of BA.1 and BA.5 in all unadjusted models, whereas in adjusted models, there was significantly lower neutralization only for the wild type. Three or more doses and Omicron-specific vaccines were both independently associated with better neutralization ability for all three strains. A COVID-19 infection within six months before sampling was associated with higher neutralization of wild type and BA.1 in adjusted analyses. Anti-tumor necrosis factor agents were associated with lower neutralization ability for BA.5 in adjusted analyses. CONCLUSION: Neutralization responses in immunosuppressed individuals with IMID were durable over time and were augmented by more than three doses and Omicron-specific vaccines. Less neutralization was seen with certain medications. Our work clarifies the joint effects of vaccine history, infection, and medications on COVID-19 immunity.

SARS-CoV-2 infection modifies the transcriptome of the megakaryocytes in the bone marrow.

ABSTRACT: Megakaryocytes (MKs), integral to platelet production, predominantly reside in the bone marrow (BM) and undergo regulated fragmentation within sinusoid vessels to release platelets into the bloodstream. Inflammatory states and infections influence MK transcription, potentially affecting platelet functionality. Notably, COVID-19 has been associated with altered platelet transcriptomes. In this study, we investigated the hypothesis that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection could affect the transcriptome of BM MKs. Using spatial transcriptomics to discriminate subpopulations of MKs based on proximity to BM sinusoids, we identified ∼19 000 genes in MKs. Machine learning techniques revealed that the transcriptome of healthy murine BM MKs exhibited minimal differences based on proximity to sinusoid vessels. Furthermore, at peak SARS-CoV-2 viremia, when the disease primarily affected the lungs, MKs were not significantly different from those from healthy mice. Conversely, a significant divergence in the MK transcriptome was observed during systemic inflammation, although SARS-CoV-2 RNA was never detected in the BM, and it was no longer detectable in the lungs. Under these conditions, the MK transcriptional landscape was enriched in pathways associated with histone modifications, MK differentiation, NETosis, and autoimmunity, which could not be explained by cell proximity to sinusoid vessels. Notably, the type I interferon signature and calprotectin (S100A8/A9) were not induced in MKs under any condition. However, inflammatory cytokines induced in the blood and lungs of COVID-19 mice were different from those found in the BM, suggesting a discriminating impact of inflammation on this specific subset of cells. Collectively, our data indicate that a new population of BM MKs may emerge through COVID-19-related pathogenesis.

SARS-CoV-2 rapidly evolves lineage-specific phenotypic differences when passaged repeatedly in immune-naïve mice.

The persistence of SARS-CoV-2 despite the development of vaccines and a degree of herd immunity is partly due to viral evolution reducing vaccine and treatment efficacy. Serial infections of wild-type (WT) SARS-CoV-2 in Balb/c mice yield mouse-adapted strains with greater infectivity and mortality. We investigate if passaging unmodified B.1.351 (Beta) and B.1.617.2 (Delta) 20 times in K18-ACE2 mice, expressing the human ACE2 receptor, in a BSL-3 laboratory without selective pressures, drives human health-relevant evolution and if evolution is lineage-dependent. Late-passage virus causes more severe disease, at organism and lung tissue scales, with late-passage Delta demonstrating antibody resistance and interferon suppression. This resistance co-occurs with a de novo spike S371F mutation, linked with both traits. S371F, an Omicron-characteristic mutation, is co-inherited at times with spike E1182G per Nanopore sequencing, existing in different within-sample viral variants at others. Both S371F and E1182G are linked to mammalian GOLGA7 and ZDHHC5 interactions, which mediate viral-cell entry and antiviral response. This study demonstrates SARS-CoV-2's tendency to evolve with phenotypic consequences, its evolution varying by lineage, and suggests non-dominant quasi-species contribution.

Pharmacological targeting of the hyper-inflammatory response to SARS-CoV-2-infected K18-hACE2 mice using a cluster of differentiation 36 receptor modulator.

The scientific and medical community faced an unprecedented global health hazard that led to nearly 7 million deaths attributable to the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In spite of the development of efficient vaccines against SARS-CoV-2, many people remain at risk of developing severe symptoms as the virus continues to spread without beneficial patient therapy. The hyper-inflammatory response to SARS-CoV-2 infection progressing to acute respiratory distress syndrome remains an unmet medical need for improving patient care. The viral infection stimulates alveolar macrophages to adopt an inflammatory phenotype regulated, at least in part, by the cluster of differentiation 36 receptor (CD36) to produce unrestrained inflammatory cytokine secretions. We suggest herein that the modulation of the macrophage response using the synthetic CD36 ligand hexarelin offers potential as therapy for halting respiratory failure in SARS-CoV-2-infected patients.

The immediate-early protein 1 of human herpesvirus 6B interacts with NBS1 and inhibits ATM signaling.

Viral infection often trigger an ATM serine/threonine kinase (ATM)-dependent DNA damage response in host cells that suppresses viral replication. Viruses evolved different strategies to counteract this antiviral surveillance system. Here, we report that human herpesvirus 6B (HHV-6B) infection causes genomic instability by suppressing ATM signaling in host cells. Expression of immediate-early protein 1 (IE1) phenocopies this phenotype and blocks homology-directed double-strand break repair. Mechanistically, IE1 interacts with NBS1, and inhibits ATM signaling through two distinct domains. HHV-6B seems to efficiently inhibit ATM signaling as further depletion of either NBS1 or ATM do not significantly boost viral replication in infected cells. Interestingly, viral integration of HHV-6B into the host's telomeres is not strictly dependent on NBS1, challenging current models where integration occurs through homology-directed repair. Given that spontaneous IE1 expression has been detected in cells of subjects with inherited chromosomally-integrated form of HHV-6B (iciHHV-6B), a condition associated with several health conditions, our results raise the possibility of a link between genomic instability and the development of iciHHV-6-associated diseases.

COVID-19 Vaccine in Immunosuppressed Adults with Autoimmune rheumatic Diseases (COVIAAD): safety, immunogenicity and antibody persistence at 12 months following Moderna Spikevax primary series.

OBJECTIVE: To assess the safety, immunogenicity and cellular responses following the Moderna Spikevax primary series in rheumatic disease. METHODS: We conducted a 12-month, prospective, non-randomised, open-label, comparative trial of adults with either rheumatoid arthritis (RA, n=131) on stable treatment; systemic lupus erythematosus (SLE, n=23) on mycophenolate mofetil (MMF); other rheumatic diseases on prednisone ≥10 mg/day (n=8) or age-matched/sex-matched controls (healthy control, HC, n=58). Adverse events (AEs), humoral immune responses (immunogenicity: IgG positivity for anti-SARS-CoV-2 spike protein and its receptor binding domain, neutralising antibodies (NAbs)), cellular responses (ELISpot) and COVID-19 infection rates were assessed. RESULTS: Frequency of solicited self-reported AEs following vaccination was similar across groups (HC 90%, RA 86%, SLE 90%); among them, musculoskeletal AEs were more frequent in RA (HC 48% vs RA 66% (Δ95% CI CI 3 to 32.6)). Disease activity scores did not increase postvaccination. No vaccine-related serious AEs were reported. Postvaccination immunogenicity was reduced in RA and SLE (RA 90.2%, SLE 86.4%; for both, ΔCIs compared with HC excluded the null). Similarly, NAbs were reduced among patients (RA 82.6%, SLE 81.8%). In RA, age >65 (OR 0.3, 95% CI 0.1 to 0.8) and rituximab treatment (OR 0.003, 95% CI 0.001 to 0.02) were negative predictors of immunogenicity. ELISpot was positive in 16/52 tested RA and 17/26 HC (ΔCI 11.2-53.3). During the study, 11 HC, 19 RA and 3 SLE patients self-reported COVID-infection. CONCLUSION: In COVID-19 Vaccine in Immunosuppressed Adults with Autoimmune Diseases, the Moderna Spikevax primary series was safe. MMF, RA age >65 and rituximab were associated with reduced vaccine-induced protection.

Excision of Integrated Human Herpesvirus 6A Genomes Using CRISPR/Cas9 Technology.

Human herpesviruses 6A and 6B are betaherpesviruses that can integrate their genomes into the telomeres of latently infected cells. Integration can also occur in germ cells, resulting in individuals who harbor the integrated virus in every cell of their body and can pass it on to their offspring. This condition is termed inherited chromosomally integrated HHV-6 (iciHHV-6) and affects about 1% of the human population. The integrated HHV-6A/B genome can reactivate in iciHHV-6 patients and in rare cases can also cause severe diseases including encephalitis and graft-versus-host disease. Until now, it has remained impossible to prevent virus reactivation or remove the integrated virus genome. Therefore, we developed a system that allows the removal of HHV-6A from the host telomeres using the CRISPR/Cas9 system. We used specific guide RNAs (gRNAs) targeting the direct repeat region at the ends of the viral genome to remove the virus from latently infected cells generated in vitro and iciHHV-6A patient cells. Fluorescence-activated cell sorting (FACS), quantitative PCR (qPCR), and fluorescence in situ hybridization (FISH) analyses revealed that the virus genome was efficiently excised and lost in most cells. Efficient excision was achieved with both constitutive and transient expression of Cas9. In addition, reverse transcription-qPCR (RT-qPCR) revealed that the virus genome did not reactivate upon excision. Taken together, our data show that our CRISPR/Cas9 approach allows efficient removal of the integrated virus genome from host telomeres. IMPORTANCE Human herpesvirus 6 (HHV-6) infects almost all humans and integrates into the telomeres of latently infected cells to persist in the host for life. In addition, HHV-6 can also integrate into the telomeres of germ cells, which results in about 80 million individuals worldwide who carry the virus in every cell of their body and can pass it on to their offspring. In this study, we develop the first system that allows excision of the integrated HHV-6 genome from host telomeres using CRISPR/Cas9 technology. Our data revealed that the integrated HHV-6 genome can be efficiently removed from the telomeres of latently infected cells and cells of patients harboring the virus in their germ line. Virus removal could be achieved with both stable and transient Cas9 expression, without inducing viral reactivation.

SARS-CoV-2 Nsp2 Contributes to Inflammation by Activating NF-κB.

COVID-19 is associated with robust inflammation and partially impaired antiviral responses. The modulation of inflammatory gene expression by SARS-CoV-2 is not completely understood. In this study, we characterized the inflammatory and antiviral responses mounted during SARS-CoV-2 infection. K18-hACE2 mice were infected with a Wuhan-like strain of SARS-CoV-2, and the transcriptional and translational expression interferons (IFNs), cytokines, and chemokines were analyzed in mouse lung homogenates. Our results show that the infection of mice with SARS-CoV-2 induces the expression of several pro-inflammatory CC and CXC chemokines activated through NF-κB but weakly IL1ß and IL18 whose expression are more characteristic of inflammasome formation. We also observed the downregulation of several inflammasome effectors. The modulation of innate response, following expressions of non-structural protein 2 (Nsp2) and SARS-CoV-2 infection, was assessed by measuring IFNß expression and NF-κB modulation in human pulmonary cells. A robust activation of the NF-κB p65 subunit was induced following the infection of human cells with the corresponding NF-κB-driven inflammatory signature. We identified that Nsp2 expression induced the activation of the IFNß promoter through its NF-κB regulatory domain as well as activation of p65 subunit phosphorylation. The present studies suggest that SARS-CoV-2 skews the antiviral response in favor of an NF-κB-driven inflammatory response, a hallmark of acute COVID-19 and for which Nsp2 should be considered an important contributor.

Impact of Host Telomere Length on HHV-6 Integration.

Human herpesvirus 6A and 6B are two closely related viruses that infect almost all humans. In contrast to most herpesviruses, HHV-6A/B can integrate their genomes into the telomeres during the infection process. Both viruses can also integrate in germ cells and subsequently be inherited in children. How HHV-6A/B integrate into host telomeres and the consequences of this remain a subject of active research. Here, we developed a method to measure telomere length by quantitative fluorescence in situ hybridization, confocal microscopy, and computational processing. This method was validated using a panel of HeLa cells having short or long telomeres. These cell lines were infected with HHV-6A, revealing that the virus could efficiently integrate into telomeres independent of their length. Furthermore, we assessed the telomere lengths after HHV-6A integration and found that the virus-containing telomeres display a variety of lengths, suggesting that either telomere length is restored after integration or telomeres are not shortened by integration. Our results highlight new aspects of HHV-6A/B biology and the role of telomere length on virus integration.

Cytokines and Lipid Mediators of Inflammation in Lungs of SARS-CoV-2 Infected Mice.

Coronavirus disease 19 (COVID-19) is the clinical manifestation of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) infection. A hallmark of COVID-19 is a lung inflammation characterized by an abundant leukocyte infiltrate, elevated levels of cytokines/chemokines, lipid mediators of inflammation (LMI) and microthrombotic events. Animal models are useful for understanding the pathophysiological events leading to COVID-19. One such animal model is the K18-ACE2 transgenic mice. Despite their importance in inflammation, the study of LMI in lung of SARS-CoV-2 infected K18-ACE2 mice has yet to be studied to our knowledge. Using tandem mass spectrometry, the lung lipidome at different time points of infection was analyzed. Significantly increased LMI included N-oleoyl-serine, N-linoleoyl-glycine, N-oleoyl-alanine, 1/2-linoleoyl-glycerol, 1/2-docosahexaenoyl-glycerol and 12-hydroxy-eicosapenatenoic acid. The levels of prostaglandin (PG) E1, PGF2α, stearoyl-ethanolamide and linoleoyl-ethanolamide were found to be significantly reduced relative to mock-infected mice. Other LMI were present at similar levels (or undetected) in both uninfected and infected mouse lungs. In parallel to LMI measures, transcriptomic and cytokine/chemokine profiling were performed. Viral replication was robust with maximal lung viral loads detected on days 2-3 post-infection. Lung histology revealed leukocyte infiltration starting on day 3 post-infection, which correlated with the presence of high concentrations of several chemokines/cytokines. At early times post-infection, the plasma of infected mice contained highly elevated concentration of D-dimers suggestive of blood clot formation/dissolution. In support, the presence of blood clots in the lung vasculature was observed during infection. RNA-Seq analysis of lung tissues indicate that SARS-CoV-2 infection results in the progressive modulation of several hundred genes, including several inflammatory mediators and genes related to the interferons. Analysis of the lung lipidome indicated modest, yet significant modulation of a minority of lipids. In summary, our study suggests that SARS-CoV-2 infection in humans and mice share common features, such as elevated levels of chemokines in lungs, leukocyte infiltration and increased levels of circulating D-dimers. However, the K18-ACE2 mouse model highlight major differences in terms of LMI being produced in response to SARS-CoV-2 infection. The potential reasons and impact of these differences on the pathology and therapeutic strategies to be employed to treat severe COVID-19 are discussed.

A nanoparticle-based COVID-19 vaccine candidate elicits broad neutralizing antibodies and protects against SARS-CoV-2 infection.

A vaccine candidate to SARS-CoV-2 was constructed by coupling the viral receptor binding domain (RBD) to the surface of the papaya mosaic virus (PapMV) nanoparticle (nano) to generate the RBD-PapMV vaccine. Immunization of mice with the coupled RBD-PapMV vaccine enhanced the antibody titers and the T-cell mediated immune response directed to the RBD antigen as compared to immunization with the non-coupled vaccine formulation (RBD + PapMV nano). Anti-RBD antibodies, generated in vaccinated animals, neutralized SARS-CoV-2 infection in vitro against the ancestral, Delta and the Omicron variants. At last, immunization of mice susceptible to the infection by SARS-CoV-2 (K18-hACE2 transgenic mice) with the RBD-PapMV vaccine induced protection to the ancestral SARS-CoV-2 infectious challenge. The induction of the broad neutralization against SARS-CoV-2 variants induced by the RBD-PapMV vaccine demonstrate the potential of the PapMV vaccine platform in the development of efficient vaccines against viral respiratory infections.

Live imaging of platelets and neutrophils during antibody-mediated neurovascular thrombosis.

Immune complexes form in systemic disorders such as rheumatological, autoimmune, and allergic diseases or in response to infections or medications. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) adenoviral vector vaccines have been associated with rare yet serious thrombotic complications in the brain due to the formation of immune complexes that activate platelets. There are currently no data visualizing the interplay of platelets with leukocytes and the brain vasculature endothelium in response to immune complexes. This is in part due to the absence of FcγRIIA in mice, a receptor for immune complexes implicated in these thrombotic incidents. Here, we describe and illustrate events at the cellular level that take place in the brain vasculature in response to systemic administration of surrogate immune complexes. We used Ly6gCre+/-::Rosa26-TdT+/-::CD41-YFP+/- mice expressing the FcγRIIA transgene and fluorescence in neutrophils and platelets. Using real-time videomicroscopy to capture high-velocity events in conjunction with unbiased computer-assisted analyses, we provide images and quantifications of the cellular responses downstream of FcγRIIA stimulation. We observed transient and stable platelet-neutrophil interactions, platelets forming thrombi, and neutrophil adhesion to blood vessel walls. This imaging approach in a quadruple transgenic animal model can be used for the study of the pathogenic roles of immune complexes in disease.

Platelet activation by SARS-CoV-2 implicates the release of active tissue factor by infected cells.

Platelets are hyperactivated in coronavirus disease 2019 (COVID-19). However, the mechanisms promoting platelet activation by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not well understood. This may be due to inherent challenges in discriminating the contribution of viral vs host components produced by infected cells. This is particularly true for enveloped viruses and extracellular vesicles (EVs), as they are concomitantly released during infection and share biophysical properties. To study this, we evaluated whether SARS-CoV-2 itself or components derived from SARS-CoV-2-infected human lung epithelial cells could activate isolated platelets from healthy donors. Activation was measured by the surface expression of P-selectin and the activated conformation of integrin αIIbß3, degranulation, aggregation under flow conditions, and the release of EVs. We find that neither SARS-CoV-2 nor purified spike activates platelets. In contrast, tissue factor (TF) produced by infected cells was highly potent at activating platelets. This required trace amounts of plasma containing the coagulation factors FX, FII, and FVII. Robust platelet activation involved thrombin and the activation of protease-activated receptor (PAR)-1 and -4 expressed by platelets. Virions and EVs were identified by electron microscopy. Through size-exclusion chromatography, TF activity was found to be associated with a virus or EVs, which were indistinguishable. Increased TF messenger RNA (mRNA) expression and activity were also found in lungs in a murine model of COVID-19 and plasma of severe COVID-19 patients, respectively. In summary, TF activity from SARS-CoV-2-infected cells activates thrombin, which signals to PARs on platelets. Blockade of molecules in this pathway may interfere with platelet activation and the coagulation characteristic of COVID-19.

[The importance of telomeres in human herpesvirus-6A/B infections]. / L'importance des télomères dans les infections par les Herpèsvirus humains-6A/B.

Herpesviruses are undisputed masters of disguise. The ability to become invisible to the immune system effectors is a complex process resting on a variety of stealth approaches. Among these, human herpesviruses-6A and -6B (HHV-6A/B) have developed the unique ability to integrate their genome within the ends of chromosomes allowing viral persistence in the absence of viral protein expression. This aptitude, unique to HHV-6A/B among human herpesviruses, requires close interactions between the telomeric regions of chromosomes and the viral genome. In this review article, the biology of telomeres and the mechanisms responsible for viral integration are discussed. In closing, the possible biological consequences of HHV-6A/B integration into chromosomal DNA are discussed.

TITLE: L'importance des télomères dans les infections par les Herpèsvirus humains-6A/B. ABSTRACT: Les Herpèsvirus sont des maîtres incontestés du camouflage. En effet, ces virus utilisent divers moyens pour assurer leur persistance chez l'hôte infecté. Les Herpèsvirus humains-6A et -6B (HHV-6A/B) ont ainsi développé une approche unique, en intégrant l'ensemble de leur génome au sein des extrémités des chromosomes des cellules qu'ils infectent. Cette capacité, propre aux HHV-6A/B parmi les Herpèsvirus humains, requiert des interactions étroites entre les régions télomériques des chromosomes de l'hôte et le génome viral. Dans cette revue, la biologie des télomères et les mécanismes responsables de l'intégration virale seront abordés et les conséquences biologiques de l'intégration des HHV-6A/B au sein de l'ADN chromosomique seront discutées.

Platelet extracellular vesicles in COVID-19: Potential markers and makers.

Platelets and platelet extracellular vesicles (pEV) are at the crossroads of coagulation and immunity. Extracellular vesicles are messengers that not only transmit signals between cells, but also provide information about the status of their cell of origin. Thus, pEVs have potential as both biomarkers of platelet activation and contributors to pathology. Coronavirus Disease-19 (COVID-19), caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a complex disease affecting multiple organs and is characterized by a high degree of inflammation and risk of thrombosis in some patients. In this review, we introduce pEVs as valuable biomarkers in disease with a special focus on their potential as predictors of and contributors to COVID-19.

SARS-CoV-2 deregulates the vascular and immune functions of brain pericytes via Spike protein.

Coronavirus disease 19 (COVID-19) is a respiratory illness caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). COVID-19 pathogenesis causes vascular-mediated neurological disorders via elusive mechanisms. SARS-CoV-2 infects host cells via the binding of viral Spike (S) protein to transmembrane receptor, angiotensin-converting enzyme 2 (ACE2). Although brain pericytes were recently shown to abundantly express ACE2 at the neurovascular interface, their response to SARS-CoV-2 S protein is still to be elucidated. Using cell-based assays, we found that ACE2 expression in human brain vascular pericytes was increased upon S protein exposure. Pericytes exposed to S protein underwent profound phenotypic changes associated with an elongated and contracted morphology accompanied with an enhanced expression of contractile and myofibrogenic proteins, such as α-smooth muscle actin (α-SMA), fibronectin, collagen I, and neurogenic locus notch homolog protein-3 (NOTCH3). On the functional level, S protein exposure promoted the acquisition of calcium (Ca2+) signature of contractile ensheathing pericytes characterized by highly regular oscillatory Ca2+ fluctuations. Furthermore, S protein induced lipid peroxidation, oxidative and nitrosative stress in pericytes as well as triggered an immune reaction translated by activation of nuclear factor-kappa-B (NF-κB) signaling pathway, which was potentiated by hypoxia, a condition associated with vascular comorbidities that exacerbate COVID-19 pathogenesis. S protein exposure combined to hypoxia enhanced the production of pro-inflammatory cytokines involved in immune cell activation and trafficking, namely macrophage migration inhibitory factor (MIF). Using transgenic mice expressing the human ACE2 that recognizes S protein, we observed that the intranasal infection with SARS-CoV-2 rapidly induced hypoxic/ischemic-like pericyte reactivity in the brain of transgenic mice, accompanied with an increased vascular expression of ACE2. Moreover, we found that SARS-CoV-2 S protein accumulated in the intranasal cavity reached the brain of mice in which the nasal mucosa is deregulated. Collectively, these findings suggest that SARS-CoV-2 S protein impairs the vascular and immune regulatory functions of brain pericytes, which may account for vascular-mediated brain damage. Our study provides a better understanding for the mechanisms underlying cerebrovascular disorders in COVID-19, paving the way to develop new therapeutic interventions.

Variation in human herpesvirus 6B telomeric integration, excision, and transmission between tissues and individuals.

Human herpesviruses 6A and 6B (HHV-6A/6B) are ubiquitous pathogens that persist lifelong in latent form and can cause severe conditions upon reactivation. They are spread by community-acquired infection of free virus (acqHHV6A/6B) and by germline transmission of inherited chromosomally integrated HHV-6A/6B (iciHHV-6A/6B) in telomeres. We exploited a hypervariable region of the HHV-6B genome to investigate the relationship between acquired and inherited virus and revealed predominantly maternal transmission of acqHHV-6B in families. Remarkably, we demonstrate that some copies of acqHHV-6B in saliva from healthy adults gained a telomere, indicative of integration and latency, and that the frequency of viral genome excision from telomeres in iciHHV-6B carriers is surprisingly high and varies between tissues. In addition, newly formed short telomeres generated by partial viral genome release are frequently lengthened, particularly in telomerase-expressing pluripotent cells. Consequently, iciHHV-6B carriers are mosaic for different iciHHV-6B structures, including circular extra-chromosomal forms that have the potential to reactivate. Finally, we show transmission of an HHV-6B strain from an iciHHV-6B mother to her non-iciHHV-6B son. Altogether, we demonstrate that iciHHV-6B can readily transition between telomere-integrated and free virus forms.

Chemokines and eicosanoids fuel the hyperinflammation within the lungs of patients with severe COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can lead to a variety of clinical outcomes, ranging from the absence of symptoms to severe acute respiratory disease and ultimately death. A feature of patients with severe coronavirus disease 2019 (COVID-19) is the abundance of inflammatory cytokines in the blood. Elevated levels of cytokines are predictive of infection severity and clinical outcome. In contrast, studies aimed at defining the driving forces behind the inflammation in lungs of subjects with severe COVID-19 remain scarce. OBJECTIVE: Our aim was to analyze and compare the plasma and bronchoalveolar lavage (BAL) fluids of patients with severe COVID-19 (n = 45) for the presence of cytokines and lipid mediators of inflammation (LMIs). METHODS: Cytokines were measured by using Luminex multiplex assay, and LMIs were measured by using liquid chromatography-tandem mass spectrometry. RESULTS: We revealed high concentrations of numerous cytokines, chemokines, and LMIs in the BAL fluid of patients with severe COVID-19. Of the 13 most abundant mediators in BAL fluid, 11 were chemokines, with CXCL1 and CXCL8 being 200 times more abundant than IL-6 and TNF-α. Eicosanoid levels were also elevated in the lungs of subjects with severe COVID-19. Consistent with the presence chemotactic molecules, BAL fluid samples were enriched for neutrophils, lymphocytes, and eosinophils. Inflammatory cytokines and LMIs in plasma showed limited correlations with those present in BAL fluid, arguing that circulating inflammatory molecules may not be a reliable proxy of the inflammation occurring in the lungs of patients with severe COVID-19. CONCLUSIONS: Our findings indicate that hyperinflammation of the lungs of patients with severe COVID-19 is fueled by excessive production of chemokines and eicosanoids. Therapeutic strategies to dampen inflammation in patients with COVID-19 should be tailored accordingly.

High levels of eicosanoids and docosanoids in the lungs of intubated COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 is responsible for coronavirus disease 2019 (COVID-19). While COVID-19 is often benign, a subset of patients develops severe multilobar pneumonia that can progress to an acute respiratory distress syndrome. There is no cure for severe COVID-19 and few treatments significantly improved clinical outcome. Dexamethasone and possibly aspirin, which directly/indirectly target the biosynthesis/effects of numerous lipid mediators are among those options. Our objective was to define if severe COVID-19 patients were characterized by increased bioactive lipids modulating lung inflammation. A targeted lipidomic analysis of bronchoalveolar lavages (BALs) by tandem mass spectrometry was done on 25 healthy controls and 33 COVID-19 patients requiring mechanical ventilation. BALs from severe COVID-19 patients were characterized by increased fatty acids and inflammatory lipid mediators. There was a predominance of thromboxane and prostaglandins. Leukotrienes were also increased, notably LTB4 , LTE4 , and eoxin E4 . Monohydroxylated 15-lipoxygenase metabolites derived from linoleate, arachidonate, eicosapentaenoate, and docosahexaenoate were also increased. Finally yet importantly, specialized pro-resolving mediators, notably lipoxin A4 and the D-series resolvins, were also increased, underscoring that the lipid mediator storm occurring in severe COVID-19 involves pro- and anti-inflammatory lipids. Our data unmask the lipid mediator storm occurring in the lungs of patients afflicted with severe COVID-19. We discuss which clinically available drugs could be helpful at modulating the lipidome we observed in the hope of minimizing the deleterious effects of pro-inflammatory lipids and enhancing the effects of anti-inflammatory and/or pro-resolving lipid mediators.

Higher-Order Chromatin Structures of Chromosomally Integrated HHV-6A Predict Integration Sites.

Human herpesvirus -6A and 6B (HHV-6A/B) can integrate their genomes into the telomeres of human chromosomes. Viral integration can occur in several cell types, including germinal cells, resulting in individuals that harbor the viral genome in every cell of their body. The integrated genome is efficiently silenced but can sporadically reactivate resulting in various clinical symptoms. To date, the integration mechanism and the subsequent silencing of HHV-6A/B genes remains poorly understood. Here we investigate the genome-wide chromatin contacts of the integrated HHV-6A in latently-infected cells. We show that HHV-6A becomes transcriptionally silent upon infection of these cells over the course of seven days. In addition, we established an HHV-6-specific 4C-seq approach, revealing that the HHV-6A 3D interactome is associated with quiescent chromatin states in cells harboring integrated virus. Furthermore, we observed that the majority of virus chromatin interactions occur toward the distal ends of specific human chromosomes. Exploiting this finding, we established a 4C-seq method that accurately detects the chromosomal integration sites. We further implement long-read minION sequencing in the 4C-seq assay and developed a method to identify HHV-6A/B integration sites in clinical samples.