Peak Viral Levels Research Articles

Early Metabolomic and Immunologic Biomarkers as Prognostic Indicators for COVID-19.

This prospective study in Hong Kong aimed at identifying prognostic metabolomic and immunologic biomarkers for Coronavirus Disease 2019 (COVID-19). We examined 327 patients, mean age 55 (19-89) years, in whom 33.6% were infected with Omicron and 66.4% were infected with earlier variants. The effect size of disease severity on metabolome outweighed others including age, gender, peak C-reactive protein (CRP), vitamin D and peak viral levels. Sixty-five metabolites demonstrated strong associations and the majority (54, 83.1%) were downregulated in severe disease (z score: -3.30 to -8.61). Ten cytokines/chemokines demonstrated strong associations (p < 0.001), and all were upregulated in severe disease. Multiple pairs of metabolomic/immunologic biomarkers showed significant correlations. Fourteen metabolites had the area under the receiver operating characteristic curve (AUC) > 0.8, suggesting a high predictive value. Three metabolites carried high sensitivity for severe disease: triglycerides in medium high-density lipoprotein (MHDL) (sensitivity: 0.94), free cholesterol-to-total lipids ratio in very small very-low-density lipoprotein (VLDL) (0.93), cholesteryl esters-to-total lipids ratio in chylomicrons and extremely large VLDL (0.92);whereas metabolites with the highest specificity were creatinine (specificity: 0.94), phospholipids in large VLDL (0.94) and triglycerides-to-total lipids ratio in large VLDL (0.93). Five cytokines/chemokines, namely, interleukin (IL)-6, IL-18, IL-10, macrophage inflammatory protein (MIP)-1b and tumour necrosis factor (TNF)-a, had AUC > 0.8. In conclusion, we demonstrated a tight interaction and prognostic potential of metabolomic and immunologic biomarkers enabling an outcome-based patient stratification.

Neuroinvasion and Encephalitis Following Intranasal Inoculation of SARS-CoV-2 in K18-hACE2 Mice.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection can cause neurological disease in humans, but little is known about the pathogenesis of SARS-CoV-2 infection in the central nervous system (CNS). Herein, using K18-hACE2 mice, we demonstrate that SARS-CoV-2 neuroinvasion and encephalitis is associated with mortality in these mice. Intranasal infection of K18-hACE2 mice with 105 plaque-forming units of SARS-CoV-2 resulted in 100% mortality by day 6 after infection. The highest virus titers in the lungs were observed on day 3 and declined on days 5 and 6 after infection. By contrast, very high levels of infectious virus were uniformly detected in the brains of all the animals on days 5 and 6. Onset of severe disease in infected mice correlated with peak viral levels in the brain. SARS-CoV-2-infected mice exhibited encephalitis hallmarks characterized by production of cytokines and chemokines, leukocyte infiltration, hemorrhage and neuronal cell death. SARS-CoV-2 was also found to productively infect cells within the nasal turbinate, eye and olfactory bulb, suggesting SARS-CoV-2 entry into the brain by this route after intranasal infection. Our data indicate that direct infection of CNS cells together with the induced inflammatory response in the brain resulted in the severe disease observed in SARS-CoV-2-infected K18-hACE2 mice.

Mathematical modelling of multiple target cells with delay

Target cells contagion has sparked the necessity of a multiple target cell model with chronic infection and intracellular delay (MTC-CI-ID). This paper seeks to advance a viral dynamical model with multiple target cells and corroborate the global stability of their steady states. Hence, the affiliation of the virus with two classes of target cells, CD4 + Tcells and macrophage. The time delay amidst viral entry and the output of virions were intimated and the basic reproductive number was procured, as the long term behavior of the model and less than unity. The contagious free equilibrium E0 was unveiled as locally and globally asymptotically stable. The conditions of an infected CD4 + Tcell and macrophage on viral production were engaged. It was ascertained that CD4 + Tcell accounts for larger amounts of virions in the blood as per macrophage cells. Hence, varying time delay has no impact on peak viral levels, but only shelves the viral peaking time. The adopted model has unrestrained bearing on HIV-1 therapy.

SUN-127 LONGITUDINAL STUDY OF BKV OUTCOMES, RISK FACTORS, AND KINETICS IN RENAL TRANSPLANTATION PATIENTS

In immunocompromised conditions following renal transplantation, BK virus can reactivate and cause BK virus associated nephropathy (BKVN), potentially leading to renal dysfunction and allograft loss. Increased BK viral loads and extended duration of infection have been linked to the risk of BKVN development. The current major treatment option against BK virus is reduction of immunosuppression. The aim of this study was to observe the incidence of BKV infection and BKVN, their risk factors, and the kinetics of infection and disease in renal transplantation recipients. The effectiveness of intravenous immunoglobin (IVIG) on treatment of viremia was also assessed. This is a longitudinal cohort observational study of 139 renal transplantation patients treated at a single clinic from 2014 to 2018. Serum samples were collected from patients within one month after transplantation and at 1–2 month intervals following, for at least 6 and up to 40 months post-transplantation. Quantitative PCR assay was conducted to assess longitudinal BK viral loads. High BK viremia was defined as serum BKV DNA ≥ 4 log10 copies/ml for two or more consecutive readings. BKVN was confirmed by biopsy and defined as an allograft biopsy revealing typical viral cytopathic changes and positive staining for SV40 large T antigen. Distance correlation analysis of patient clinical characteristics was performed to determine risk factors for BKV infection and associated disease. A total of 1785 patient serum samples were assessed, with a mean patient follow-up duration of 19.4 ± 7.3 months. Within our cohort of 139 patients, 110 (79.1%) did not develop high BK viremia. Of the 29 (20.9%) patients who developed high BK viremia, 9 (6.5%) were transient cases (≥ 4 log10 copies/ml for 4 or less months), 13 (9.4%) were persistent cases (≥ 4 log10 copies/ml for more than 4 months), and 7 (5.0%) developed biopsy-confirmed BKVN. Peak viral levels of the BKVN group were significantly higher compared to both the BK viremia transient (6.96 vs. 4.63 log10 copies/ml, p < 0.001) and BK viremia persistent groups (6.96 vs. 5.42 log10 copies/ml, p < 0.001). Time to reach peak viral load was delayed in BKVN (13.55 vs. 3.19 months, p < 0.001) and BK persistent patients (7.43 vs. 3.19 months, p = 0.026) compared to patients with transient BK infection. Clinical parameters associated with diabetes (FBS, HbA1c) and hyperlipidemia (TG, TC, LDL-C) were found to be correlated with development of high BK viremia or BKVN. Significant differences in BK viral loads and kidney function (eGFR) developed between BK viremic patients and BKVN patients by 6–9 months post-transplantation. In 3 of 4 patients receiving IVIG treatment, BK viral loads were reduced by at least 1 log within 3 months of administration. BK viremia and BKVN incidence was in line with previously published figures. Diabetes and hyperlipidemia were identified as potential risk factors for development of high BK viremia and/or BKVN. IVIG treatment was seen to be effective in reducing viral titers. The period 0–9 months post-transplantation was identified as important for the development of BKVN from high BK viremia. Clinicians should pay close attention to patient BKV levels during this time period and respond with the appropriate reduction in immunosuppression and/or treatment with IVIG.

Rhinovirus C infection of human airway epithelial cells causes transient replication‐dependent changes in cellular architecture and barrier function

RATIONALEInfections with human rhinovirus (HRV)‐C strains are associated with more severe exacerbations and increased hospitalizations in children with asthma. Although the replication cycles of HRV‐A and B strains have been extensively studied using cells of non‐airway origin (e.g. HeLa cells), HRV‐C replication within human airway epithelial cells (HAE) remains poorly understood as HRV‐C exclusively infects highly‐differentiated HAE. We have recently shown that HRV infections can be completely cleared from HAE without any requirement for immune cells. Our data suggest that mechanism of epithelial clearance involves a combination of shedding of infected cells, and antiviral responses, culminating in re‐differentiation. We are now seeking to elucidate the mechanisms by which HRV‐C remodels HAE cellular architecture during replication. We have focused on the modulation of cytoskeletal signaling pathways and resulting transient loss of barrier function.METHODSHighly differentiated air‐liquid interface (ALI) cultures of HAE, isolated from normal non‐transplanted human lungs, were infected with HRV‐C15 and monitored at 24, 48, 72, 96, 120, 144 hours post‐infection. HRV‐C15 replication kinetics were evaluated by serial apical washes every 24h to assess shed virus levels and cell lysates to quantify intracellular viral RNA by RT‐PCR. Studies of changing cytoskeletal architecture and intercellular junctions were performed using immunofluorescence staining for ZO‐1 and f‐actin. Other stimuli include replication‐deficient HRV, poly(I:C) (a mimic of the double‐stranded RNA produced during HRV replication), ROCK inhibitors, cytochalasin D (actin disruptor), and paclitaxel (microtubule stabilizer). Barrier function studies in Ussing chambers used FITC‐dextran (permeability) and measured real‐time transepithelial electrical resistance (TEER).RESULTSHRV‐C15 causes dramatic remodeling of highly‐differentiated HAE that follows the time course of viral replication and clearance from 24h to 144h. These changes include co‐localization of dsRNA‐positive cells with apically expressed ZO‐1 (indicative of infected cell shedding). Transient loss of barrier function is prominent at peak viral levels 24h post‐infection, but recovers with re‐differentiation within 144h. Preliminary data suggests that cytoskeletal inhibitors diminish the ability of HRV‐C15 to replicate and HAE shedding to occur; indicating a critical role for cytoskeletal signaling in HRV‐C15 replication.CONCLUSIONWe conclude that a HRV‐C serotype induces altered cellular architecture in HAE that is associated with disruption of the actin cytoskeleton and intercellular junctions. Future studies will evaluate the role of Rho GTPase signaling in HRV‐C replication or HAE shedding.Support or Funding InformationThis study was supported by grant number PJT‐159635 from the Canadian Institutes of Health Research (CIHR).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract B74: A phase I trial and viral clearance study of reovirus (Reolysin) in children with relapsed or refractory extra-cranial solid tumors: A Children's Oncology Group phase I consortium report

Abstract Purpose: Reovirus (Respiratory Enteric Orphan virus) Serotype 3 – Dearing Strain is a naturally occurring, ubiquitous, non-enveloped human virus. Reovirus has been shown to replicate specifically in, and be cytopathic to, transformed cells possessing an activated Ras signaling pathway. We conducted a Phase I trial of Reolysin, a manufactured, purified reovirus, in children with relapsed or refractory extracranial solid tumors to define a maximum tolerated or recommended phase 2 dose (PP2D), as well as its toxicities and pharmacokinetic properties. Experimental Design: Reovirus was administered intravenously, daily for 5 consecutive days every 28 days for up to 12 cycles. Using a 3+3 design, two planned dose levels were evaluated: 3 x 108 TCID50/kg (∼60% of the adult dose) and 5 x 108 TCID50/kg. The cohort was expanded at the RP2D to obtain additional single agent safety and viral clearance data. Additionally, a 3rd cohort received the RP2D ( 5 x 108 TCID50/kg Reolysin) plus oral cyclophosphamide (50 mg/m2/day x 21 days). Twice weekly quantitative real time PCR of reovirus genomic RNA from serum, stool and saliva, and antiviral immune response by ELISA was evaluated after the first two courses. Results: Twenty-nine patients were enrolled, one was subsequently found to be ineligible. Of the 28 eligible patients, 5 are not evaluable (one is still on study) for dose-limiting toxicity (DLT). Six evaluable patients were enrolled on dose level 1; one with extensive pulmonary metastases experienced grade 5 respiratory failure attributed to their underlying disease. Given the severity of the event, the cohort was expanded to six without further DLTs. None of the six evaluable patients enrolled on dose levels 2 and 3, respectively, had a DLT. One patient in the single agent expansion cohort experienced a grade 5 thromboembolic event, grade 4 hypokalemia, grade 3 hypocalcemia, grade 4 hypophosphatemia, grade 3 hyponatremia, and grade 3 hypoalbuminemia possibly attributed to the Reolysin. With twice weekly monitoring by quantitative real time PCR, the median time to a clear the reovirus viremia was 6.5 days. Seven patients were viremic beyond the five days of therapy. No patient had detectable virus in their serum beyond day 17. Only one patient had detectable viral RNA in saliva or stool (day 6 in saliva). Four patients receiving the RP2D had stable disease at the end of one cycle, all others had progressive disease. All patients developed an anti-reovirus antibody during the first cycle of treatment. In three patients receiving a second cycle of reovirus, peak viral levels were reduced relative to cycle 1. There were no objective responses. Conclusions: Reovirus at a dose of 5 x 108 TCID50/kg daily for 5 days is well tolerated alone and in combination with oral cyclophosphamide. Virus is cleared rapidly from the serum of most patients, even when given with cyclophosphamide, and shedding in stool and saliva is not seen. Citation Format: Edward A. Kolb, Valerie B. Sampson, Deborah Stabley, Alexa Walter, Timothy P. Cripe, Pooja Hingorani, Charlotte Hsieh Ahern, Ashish M. Ingle, Brenda J. Weigel, Susan B. Blaney. A phase I trial and viral clearance study of reovirus (Reolysin) in children with relapsed or refractory extra-cranial solid tumors: A Children's Oncology Group phase I consortium report. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B74.

Kinetics of Major Histocompatibility Class I Antigen Presentation in Acute Infection

Ag presentation within the regional lymph node is crucial for the initiation of CD8+ T cell responses following viral infection. The magnitude and quality of the CD8+ T cell response are regulated by the interplay between the size of the APC population and duration of Ag presentation. To understand how these parameters are finely regulated during an immune response, we have investigated the dynamics of Ag presentation in influenza A virus and HSV-1 infection. In both infections, APC production was calculated to occur over the first few days of infection, after which there was slow exponential decay over a period of up to 2 wk. This production rate is most likely determined by the Ag availability and recruitment and/or maturation rate of dendritic cells. APC production was found to closely parallel lymph node cell recruitment in both infections. This was greatest in the first 6 h of infection for HSV and over the second and third day for influenza. In HSV infection, the peak production also coincides with peak viral levels. By contrast, in influenza infection, APC production ceased between the third and fourth day despite the presence of high levels of virus until 5 days after infection. These analyses demonstrate that two quite different self-limiting infections generate the APC necessary to drive T cell responses early in infection at different rates. Understanding how such contrasting kinetics of Ag presentation impacts on the growth and size of developing protective T cell populations has important implications for the design of vaccines and immunotherapies.

An Age-Structured Model of HIV Infection that Allows for Variations in the Production Rate of Viral Particles and the Death Rate of Productively Infected Cells

Mathematical models of HIV-1 infection can help interpret drug treatment experiments and improve our understanding of the interplay between HIV-1 and the immune system. We develop and analyze an age- structured model of HIV-1 infection that allows for variations in the death rate of productively infected T cells and the production rate of viral particles as a function of the length of time a T cell has been infected. We show that this model is a generalization of the standard differential equation and of delay models previously used to describe HIV-1 infection, and provides a means for exploring fundamental issues of viral production and death. We show that the model has uninfected and infected steady states, linked by a transcritical bifurcation. We perform a local stability analysis of the nontrivial equilibrium solution and provide a general stability condition for models with age structure. We then use numerical methods to study solutions of our model focusing on the analysis of primary HIV infection. We show that the time to reach peak viral levels in the blood depends not only on initial conditions but also on the way in which viral production ramps up. If viral production ramps up slowly, we find that the time to peak viral load is delayed compared to results obtained using the standard (constant viral production) model of HIV infection. We find that data on viral load changing over time is insufficient to identify the functions specifying the dependence of the viral production rate or infected cell death rate on infected cell age. These functions must be determined through new quantitative experiments.