SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus.

Development of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines

California serogroup viruses: Gb mediated fusion and entry

Members of the California serogroup of orthobunyaviruses, including La Crosse (LAC) and Tahyna (TAH) viruses, are significant human pathogens in areas where their mosquito vectors are endemic. Previous studies using wild-type LAC and TAH181/57, a highly neurovirulent strain with low neuroinvasiveness have demonstrated that the neuroinvasive phenotype maps to the M segment. To further define the role of the M segment in fusion and entry, a panel of recombinant M segment constructs were prepared using LAC, TAH181/57, and V22F, a monoclonal-resistant variant of LAC with deficient fusion function. These M segment constructs were tested in two surrogate assays for virus entry: a cell-to-cell fusion assay based on T7-luciferase expression, and a pseudotype transduction assay based on the incorporation of the bunyavirus glycoproteins on an Murine Leukemia Virus (MLV) backbone. Both assays demonstrated that Gc is the principal determinant of virus fusion and cell entry. Further, the region delineated by amino acids 860-1442, which corresponds to the membrane proximal two-thirds of Gc, is key to these processes. These results, coupled with structural modeling suggesting homologies between the carboxy region of Gc and the fusion protein (E1) from two alphaviruses (Semliki Forest virus and Sindbis virus), suggest that the LAC Gc functions as a type II fusion protein. Using the predicted structural model of Gc, an amino acid region (1066-1087) that shares a homologous structural location with the fusion domain of SFV E1 was identified as the putative fusion domain for LAC Gc. To examine the role of this region in fusion and entry, a panel of LAC point mutant M segments were prepared and examined for total and surface expression by IFA and flow cytometry. These constructs were then examined in our cell-to-cell fusion and pseudotype transduction assays. The results demonstrated mutations within this putative fusion domain either disrupted or abrogated fusion. In total, these results combined with computational modeling, suggest the hydrophobic region (1066-1087) of LAC Gc functions as the fusion peptide. These studies provided a better understanding of LAC virus entry and highlighted similarities between disparate viral fusion proteins

California serogroup viruses: Gb mediated fusion and entry

Members of the California serogroup of orthobunyaviruses, including La Crosse (LAC) and Tahyna (TAH) viruses, are significant human pathogens in areas where their mosquito vectors are endemic. Previous studies using wild-type LAC and TAH181/57, a highly neurovirulent strain with low neuroinvasiveness have demonstrated that the neuroinvasive phenotype maps to the M segment. To further define the role of the M segment in fusion and entry, a panel of recombinant M segment constructs were prepared using LAC, TAH181/57, and V22F, a monoclonal-resistant variant of LAC with deficient fusion function. These M segment constructs were tested in two surrogate assays for virus entry: a cell-to-cell fusion assay based on T7-luciferase expression, and a pseudotype transduction assay based on the incorporation of the bunyavirus glycoproteins on an Murine Leukemia Virus (MLV) backbone. Both assays demonstrated that Gc is the principal determinant of virus fusion and cell entry. Further, the region delineated by amino acids 860-1442, which corresponds to the membrane proximal two-thirds of Gc, is key to these processes. These results, coupled with structural modeling suggesting homologies between the carboxy region of Gc and the fusion protein (E1) from two alphaviruses (Semliki Forest virus and Sindbis virus), suggest that the LAC Gc functions as a type II fusion protein. Using the predicted structural model of Gc, an amino acid region (1066-1087) that shares a homologous structural location with the fusion domain of SFV E1 was identified as the putative fusion domain for LAC Gc. To examine the role of this region in fusion and entry, a panel of LAC point mutant M segments were prepared and examined for total and surface expression by IFA and flow cytometry. These constructs were then examined in our cell-to-cell fusion and pseudotype transduction assays. The results demonstrated mutations within this putative fusion domain either disrupted or abrogated fusion. In total, these results combined with computational modeling, suggest the hydrophobic region (1066-1087) of LAC Gc functions as the fusion peptide. These studies provided a better understanding of LAC virus entry and highlighted similarities between disparate viral fusion proteins

La Crosse Virus Nonstructural Protein NSs Counteracts the Effects of Short Interfering RNA

Through a process known as RNA interference (RNAi), double-stranded short interfering RNAs (siRNAs) silence gene expression in a sequence-specific manner. Recently, several viral proteins, including the nonstructural protein NSs of tomato spotted wilt virus (a plant-infecting bunyavirus), the interferon antagonist protein NS1 of influenza virus, and the E3L protein of vaccinia virus, have been shown to function as suppressors of RNAi, presumably as a counterdefense against cellular mechanisms that decrease viral production. La Crosse virus (LACV), a member of the California serogroup of orthobunyaviruses, has a trisegmented negative-stranded genome comprised of large (L), medium (M), and small (S) segments. To develop a strategy for segment-specific inhibition of transcription, we designed 13 synthetic siRNAs targeting specific RNA segments of the LACV genome that decreased LACV replication and antigen expression in mammalian (293T) and insect (C6/36) cells. Furthermore, NSs, a LACV nonstructural protein, markedly inhibited RNAi directed both against an LACV M segment construct and against a host gene (glyeraldehyde-3-phosphate dehydrogenase), suggesting a possible role for this viral protein in the suppression of RNA silencing. Segment-specific siRNAs will be useful as a tool to analyze LACV transcription and replication and to obtain recombinant viruses. Additionally, NSs will help us to identify molecular pathways involved in RNAi and further define its role in the innate immune system

Time of Initiating Enzyme Replacement Therapy Affects Immune Abnormalities and Disease Severity in Patients with Gaucher Disease

<div><p>Gaucher disease (GD) patients often present with abnormalities in immune response that may be the result of alterations in cellular and/or humoral immunity. However, how the treatment and clinical features of patients impact the perturbation of their immunological status remains unclear. To address this, we assessed the immune profile of 26 GD patients who were part of an enzyme replacement therapy (ERT) study. Patients were evaluated clinically for onset of GD symptoms, duration of therapy and validated outcome measures for ERT. According to DS3 disease severity scoring system criteria, they were assigned to have mild, moderate or severe GD. Flow cytometry based immunophenotyping was performed to analyze subsets of T, B, NK, NKT and dendritic cells. GD patients showed multiple types of immune abnormalities associated to T and B lymphocytes with respect to their subpopulations as well as memory and activation markers. Skewing of CD4 and CD8 T cell numbers resulting in lower CD4/CD8 ratio and an increase in overall T cell activation were observed. A decrease in the overall B cells and an increase in NK and NKT cells were noted in the GD patients compared to controls. These immune alterations do not correlate with GD clinical type or level of biomarkers. However, subjects with persistent immune alterations, especially in B cells and DCs correlate with longer delay in initiation of ERT (ΔTX). Thus, while ERT may reverse some of these immune abnormalities, the immune cell alterations become persistent if therapy is further delayed. These findings have important implications in understanding the immune disruptions before and after treatment of GD patients.</p></div

Correlation between immune alterations and ΔTX, DS3 and combined scores.

<p>GD patients were sub-categorized based on whether or not they show persistent B-cell (A), T-cell (B), NKT cells (CD16/CD56+ T cells) (C) and dendritic cell (D) immune alterations and plotted against the delay in initiation of ERT (ΔTX), disease severity (DS3) and a combined score (ΔTX+DS3).</p

Clinical evaluations and DS3 scoring.

<p>Clinical evaluations and DS3 scoring.</p

Correlation between delay in treatment initiation and disease severity scores.

<p>Delay in initiation of treatment for GD (ΔTX) was plotted against disease severity score (DS3) for each patient. The trend line shows a positive Pearson correlation coefficient between the two values (r value = 0.55, P = 0.0018) (A). Subjects were divided based on their DS3 scores as being mild/moderate (DS3<5) or marked (DS3>6) and their ΔTX values were plotted. Unpaired Student’s t-test revealed P value = 0.0036 (**) indicating very significant difference (B).</p

High‑κ Lanthanum Zirconium Oxide Thin Film Dielectrics from Aqueous Solution Precursors

Metal oxide thin films are critical components in modern electronic applications. In particular, high-κ dielectrics are of interest for reducing power consumption in metal–insulator–semiconductor (MIS) field-effect transistors. Although thin-film materials are typically produced via vacuum-based methods, solution deposition offers a scalable and cost-efficient alternative. We report an all-inorganic aqueous solution route to amorphous lanthanum zirconium oxide (La₂Zr₂O₇, LZO) dielectric thin films. LZO films were spin-cast from aqueous solutions of metal nitrates and annealed at temperatures between 300 and 600 °C to produce dense, defect-free, and smooth films with subnanometer roughness. Dielectric constants of 12.2–16.4 and loss tangents <0.6% were obtained for MIS devices utilizing LZO as the dielectric layer (1 kHz). Leakage currents <10^–7 A cm^–2 at 4 MV cm^–1 were measured for samples annealed at 600 °C. The excellent surface morphology, high dielectric constants, and low leakage current densities makes these LZO dielectrics promising candidates for thin-film transistor devices