scholarly journals A Genetic System for Rhesus Monkey Rhadinovirus: Use of Recombinant Virus To Quantitate Antibody-Mediated Neutralization

2006 ◽  
Vol 80 (3) ◽  
pp. 1549-1562 ◽  
Author(s):  
John P. Bilello ◽  
Jennifer S. Morgan ◽  
Blossom Damania ◽  
Sabine M. Lang ◽  
Ronald C. Desrosiers
Keyword(s):  
B Cells ◽  
Rhesus Monkey ◽  
Rhesus Monkeys ◽  
Fluorescent Protein ◽  
Kaposi Sarcoma ◽  
Genetic System ◽  
Cell Types ◽  
Neutralization Assay ◽  
Neutralizing Activity ◽  
Virus Neutralization Assay

ABSTRACT Rhesus monkey rhadinovirus (RRV), a simian gamma-2 herpesvirus closely related to the Kaposi sarcoma-associated herpesvirus, replicates lytically in cultured rhesus monkey fibroblasts and establishes persistence in B cells. Overlapping cosmid clones were generated that encompass the entire 130-kilobase-pair genome of RRV strain 26-95, including the terminal repeat regions required for its replication. Cloned RRV that was produced by cotransfection of overlapping cosmids spanning the entire RRV26-95 genome replicated with growth kinetics and to titers similar to those of the parental, uncloned, wild-type RRV26-95. Expression cassettes for secreted-engineered alkaline phosphatase (SEAP) and green fluorescent protein (GFP) were inserted upstream of the R1 gene, and the cosmid-based system for RRV genome reconstitution was used to generate replication-competent, recombinant RRV that expressed either the SEAP or GFP reporter gene. Using the SEAP and GFP recombinant RRVs, assays were developed to monitor RRV infection, neutralization, and replication. Heat-inactivated sera from rhesus monkeys that were naturally or experimentally infected with RRV were assayed for their ability to neutralize RRV-SEAP and RRV-GFP infectivity using rhesus monkey fibroblasts. Sera from RRV-positive monkeys, but not RRV-negative monkeys, were consistently able to neutralize RRV infectivity when assayed by the production of SEAP activity or by the ability to express GFP. The neutralizing activity was present in the immunoglobulin fraction. Of the 17 rhesus monkeys tested, sera from rhesus monkey 26-95, i.e., the monkey that yielded the RRV 26-95 isolate, had the highest titer of neutralizing activity against RRV26-95. This cosmid-based genetic system and the reporter virus neutralization assay will facilitate study of the contribution of individual RRV glycoproteins to entry into different cell types, particularly fibroblasts and B cells.

scholarly journals Suitability of Two Rapid Lateral Flow Immunochromatographic Assays for Predicting SARS-CoV-2 Neutralizing Activity of Sera

2020 ◽  
Author(s):  
Arantxa Valdivia ◽  
Ignacio Torres ◽  
Victor Latorre ◽  
Clara Frances-Gomez ◽  
Josep Ferrer ◽  
...  
Keyword(s):  
Predictive Value ◽  
Fluorescent Protein ◽  
Neutralizing Antibody ◽  
Antibody Test ◽  
Neutralization Assay ◽  
Positive Control ◽  
Lateral Flow ◽  
Control Line ◽  
Assay Sensitivity ◽  
Neutralizing Activity

Purpose: Assessment of commercial SARS-CoV-2 immunoassays for their capacity to provide reliable information on sera neutralizing activity is an emerging need. We evaluated the performance of two commercially-available lateral flow immunochromatographic assays (LFIC) (Wondfo SARS-CoV-2 Antibody test and the INNOVITA 2019-nCoV Ab test) in comparison with a SARS-CoV-2 neutralization pseudotyped assay for COVID-19 diagnosis in hospitalized patients, and investigate whether the intensity of the test band in LFIC associates with neutralizing antibody (NtAb) titers. Patients and Methods: Ninety sera were included from 51 patients with moderate to severe COVID-19. A green fluorescent protein (GFP) reporter-based pseudotyped neutralization assay (vesicular stomatitis virus coated with SARS-CoV-2 spike protein) was used. Test line intensity was scored using a 4-level scale (0 to 3+). Results: Overall sensitivity of LFIC assays was 91.1% for the Wondfo SARS-CoV-2 Antibody test, 72.2% for the INNOVITA 2019-nCoV IgG, 85.6% for the INNOVITA 2019-nCoV IgM and 92.2% for the NtAb assay. Sensitivity increased for all assays in sera collected beyond day 14 after symptoms onset (93.9%, 79.6%,93.9% and 93.9%, respectively). Reactivities equal to or more intense than the positive control line (≥2+) in the Wondfo assay had a negative predictive value of 100% and a positive predictive value of 96.4% for high NtAb50 titers (≥1/160). Conclusions: Our findings support the use of LFIC assays evaluated herein, particularly the Wondfo test, for COVID-19 diagnosis. We also find evidence that these rapid immunoassays can be used to predict high SARS-CoV-2-S NtAb50 titers.

scholarly journals Inference of SARS-CoV-2 spike-binding neutralizing antibody titers in sera from hospitalized COVID-19 patients by using commercial enzyme and chemiluminescent immunoassays

2020 ◽  
Author(s):  
Arantxa Valdivia ◽  
Ignacio Torres ◽  
Victor Latorre ◽  
Carla Frances-Gomez ◽  
Eliseo Albert ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Virus Neutralization ◽  
Pseudotyped Virus ◽  
S Protein ◽  
Neutralizing Activity ◽  
Virus Neutralization Assay ◽  
Degree Correlation ◽  
Antibody Titers

Background: Whether antibody levels measured by commercially-available enzyme or chemiluminescent immunoassays targeting the SARS-CoV-2 spike (S) protein can act as a proxy for serum neutralizing activity remains to be established for many of these assays. Objectives: To evaluate the degree of correlation between neutralizing antibodies (NtAb) binding the SARS-CoV-2 Spike (S) protein and SARS-CoV-2-S-IgG levels measured by four commercial immunoassays in sera drawn from hospitalized COVID-19 patients. Patients and Methods: Ninety sera from 51 hospitalized COVID-19 patients were assayed by a pseudotyped virus neutralization assay, the LIAISON SARS-CoV-2 S1/S2 IgG, the Euroimmun SARS-CoV-2 IgG ELISA, the MAGLUMI 2019-nCoV IgG and the COVID-19 ELISA IgG assays. Results: Overall, the results obtained with the COVID-19 ELISA IgG test showed the highest agreement with the NtAb assay (κ, 0.85; 95% CI, 0.63-1). The most sensitive tests were the pseudotyped virus NtAb assay and the COVID-19 ELISA IgG assay (92.2% for both). Overall, the degree correlation between antibody titers resulting in 50% virus neutralization (NtAb50) in the pseudotyped virus assay and SARS-CoV-2 IgG levels was strong for the Euroimmun SARS-CoV-2 IgG ELISA (Rho=0.73) and moderate for the remaining assays (Rho=0.48 to 0.59). The kinetic profile of serum NtAb50 titers could not be reliably predicted by any of the SARS-CoV-2 IgG immunoassays. Conclusions: the suitability of SARS-CoV-2-S-IgG commercial immunoassays for inferring neutralizing activity of sera from hospitalized COVID-19 patients varies widely across tests and is influenced by the time of sera collection after the onset of symptoms.

scholarly journals Flow cytometric detection of receptors for interleukin-6 on bone marrow and peripheral blood cells of humans and rhesus monkeys

Blood ◽  
1993 ◽  
Vol 81 (8) ◽  
pp. 2036-2043
Author(s):  
AW Wognum ◽  
FC van Gils ◽  
G Wagemaker
Keyword(s):  
Bone Marrow ◽  
T Lymphocytes ◽  
Rhesus Monkey ◽  
Interleukin 6 ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Rhesus Monkeys ◽  
Cell Types ◽  
Cd8 T Lymphocytes ◽  
Blast Cells

The expression of receptors for interleukin-6 (IL-6) on human and rhesus monkey peripheral blood and bone marrow (BM) cells was examined by multiparameter flow cytometry after staining with biologically active, biotin-labeled human IL-6 and phycoerythrin-conjugated streptavidin. Consistent with the multiple biologic effects of IL-6 in stimulating immune functions and hematopoiesis, IL-6 receptors were detectable on a wide variety of cell types. In peripheral blood, IL-6 receptors were detectable on monocytes, granulocytes, and on CD4+ T lymphocytes but not on resting, CD19+ B lymphocytes and CD56+ natural killer (NK) cells. CD8+ T lymphocytes also expressed IL-6 receptors but at lower levels than CD4+ cells. The IL-6 receptors on granulocytes were only detectable after staining with high concentrations of biotin- IL-6, suggesting that most IL-6 receptors on these cells represent low- affinity sites. In contrast, IL-6 receptors on both CD4+ and CD8+ T lymphocytes were detectable at biotin-IL-6 concentrations as low as 10 pmol/L, indicating that these cells bind IL-6 with high affinity. IL-6 receptor expression patterns on rhesus monkey and human blood cells were very similar except that receptor levels on granulocytes were lower in humans than in rhesus monkeys. Similar differences in expression levels were observed for IL-6 receptors that were detectable on most granulocyte precursors in the mononuclear fraction of rhesus monkey and human bone marrow. In addition to these relatively mature cell types, IL-6 receptors were detectable on a large fraction of human and rhesus monkey BM blast cells that express the CD34 antigen. The presence of IL-6 receptors on CD34+ BM blast cells, which are the precursor cells of most, if not all, BM-derived blood cells, is consistent with the ability of IL-6, in conjunction with other cytokines, to stimulate immature hemopoietic cells in vitro and to promote blood cell production when administered in vivo.

Relationship between numbers of retinal ganglion cells and lateral geniculate neurons in the rhesus monkey

1996 ◽  
Vol 13 (1) ◽  
pp. 199-203 ◽  
Author(s):  
Peter D. Spear ◽  
Charlene B. Y. Kim ◽  
Aneeq Ahmad ◽  
Bryony W. Tom
Keyword(s):  
Rhesus Monkey ◽  
Retinal Ganglion Cells ◽  
Lateral Geniculate Nucleus ◽  
Rhesus Monkeys ◽  
Ganglion Cells ◽  
Cell Types ◽  
Retinal Ganglion ◽  
Average Ratio ◽  
Lateral Geniculate ◽  
Unbiased Estimates

AbstractStudies of the numbers of retinal ganglion cells and lateral geniculate nucleus (LGN) neurons in primates suggest that the numbers of both types of neurons may vary over a two-fold range from one individual to another. This raises the question of whether the numbers of ganglion cells and LGN neurons are related or vary independently from individual to individual. We used stereological procedures to obtain unbiased estimates of the numbers of both cell types in seven rhesus monkeys. We found no significant correlation (rs. = −0.21) between the numbers of retinal and LGN cells in the same animals. In agreement with previous studies, the average ratio of the number of retinal ganglion cells that project to the LGN and the number of LGN cells was approximately 1:1. However, this ratio varied over a two-fold range, from 0.78:1 to 1.64:1, in individual animals. These results have important implications for understanding the mechanisms of retino-geniculate development and for understanding the connectional wiring between the retina and LGN.

scholarly journals mRNA-1273 and BNT162b2 mRNA vaccines have reduced neutralizing activity against the SARS-CoV-2 Omicron variant

2021 ◽  
Author(s):  
Venkata-Viswanadh Edara ◽  
Kelly E Manning ◽  
Madison Ellis ◽  
Lilin Lai ◽  
Kathryn M Moore ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Antibody Responses ◽  
Spike Protein ◽  
Live Virus ◽  
Neutralizing Activity ◽  
Virus Neutralization Assay ◽  
Fold Reduction

The BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) vaccines generate potent neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the global emergence of SARS-CoV-2 variants with mutations in the spike protein, the principal antigenic target of these vaccines, has raised concerns over the neutralizing activity of vaccine-induced antibody responses. The Omicron variant, which emerged in November 2021, consists of over 30 mutations within the spike protein. Here, we used an authentic live virus neutralization assay to examine the neutralizing activity of the SARS-CoV-2 Omicron variant against mRNA vaccine-induced antibody responses. Following the 2nd dose, we observed a 30-fold reduction in neutralizing activity against the omicron variant. Through six months after the 2nd dose, none of the sera from naive vaccinated subjects showed neutralizing activity against the Omicron variant. In contrast, recovered vaccinated individuals showed a 22-fold reduction with more than half of the subjects retaining neutralizing antibody responses. Following a booster shot (3rd dose), we observed a 14-fold reduction in neutralizing activity against the omicron variant and over 90% of boosted subjects showed neutralizing activity against the omicron variant. These findings show that a 3rd dose is required to provide robust neutralizing antibody responses against the Omicron variant.

scholarly journals Epstein-Barr Virus gH Is Essential for Penetration of B Cells but Also Plays a Role in Attachment of Virus to Epithelial Cells

2000 ◽  
Vol 74 (14) ◽  
pp. 6324-6332 ◽  
Author(s):  
Sara J. Molesworth ◽  
Cathleen M. Lake ◽  
Corina M. Borza ◽  
Susan M. Turk ◽  
Lindsey M. Hutt-Fletcher
Keyword(s):  
B Cells ◽  
Epithelial Cells ◽  
Epstein Barr Virus ◽  
Fluorescent Protein ◽  
Cell Types ◽  
Class Ii ◽  
Hla Class Ii ◽  
Kinase Gene ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Entry of Epstein-Barr virus (EBV) into B cells is initiated by attachment of glycoprotein gp350 to the complement receptor type 2 (CR2). A complex of three glycoproteins, gH, gL, and gp42, is subsequently required for penetration. Gp42 binds to HLA class II, which functions as an entry mediator or coreceptor and, by analogy with other herpesviruses, gH is then thought to be involved virus-cell fusion. However, entry of virus into epithelial cells is thought to be different. It can be initiated by attachment by an unknown glycoprotein in the absence of CR2. There is no interaction between gp42 and HLA class II and instead a distinct complex of only the two glycoproteins gH and gL interacts with a novel entry mediator. Again, by analogy with other viruses gH is thought to be critical to fusion. To investigate further the different roles of gH in infection of the two cell types and to examine its influence on the assembly of the gH-gL-gp42 complex, we constructed two viruses, one in which the gH open reading frame was interrupted by a cassette expressing a neomycin resistance gene and the gene for green fluorescent protein and one as a control in which the neighboring nonessential thymidine kinase gene was interrupted with the same cassette. Virus lacking gH exited from cells normally, although loss of gH resulted in rapid turnover of gL and gp42 as well. The virus bound normally to B lymphocytes but could not infect them unless cells and bound virus were treated with polyethylene glycol to induce fusion. In contrast, virus that lacked the gH complex was impaired in attachment to epithelial cells and the effects of monoclonal antibodies to gH implied that this resulted from loss of gH rather than other members of the complex. These results suggest a role for gH in both attachment and penetration into epithelial cells.

scholarly journals Microglia: An Intrinsic Component of the Proliferative Zones in the Fetal Rhesus Monkey (Macaca mulatta) Cerebral Cortex

2018 ◽  
Vol 29 (7) ◽  
pp. 2782-2796 ◽  
Author(s):  
Nicole Barger ◽  
Janet Keiter ◽  
Anna Kreutz ◽  
Anjana Krishnamurthy ◽  
Cody Weidenthaler ◽  
...  
Keyword(s):  
Rhesus Monkey ◽  
Fluorescent Protein ◽  
Lentiviral Vector ◽  
Ventricular Zone ◽  
Cortical Cell ◽  
Cell Types ◽  
Precursor Cells ◽  
Microglial Cells ◽  
Cellular Interactions ◽  
Green Fluorescent

Abstract Microglial cells are increasingly recognized as modulators of brain development. We previously showed that microglia colonize the cortical proliferative zones in the prenatal brain and regulate the number of precursor cells through phagocytosis. To better define cellular interactions between microglia and proliferative cells, we performed lentiviral vector-mediated intraventricular gene transfer to induce enhanced green fluorescent protein expression in fetal cerebrocortical cells. Tissues were collected and counterstained with cell-specific markers to label microglial cells and identify other cortical cell types. We found that microglial cells intimately interact with the radial glial scaffold and make extensive contacts with neural precursor cells throughout the proliferative zones, particularly in the rhesus monkey fetus when compared to rodents. We also identify a subtype of microglia, which we term ‘periventricular microglia’, that interact closely with mitotic precursor cells in the ventricular zone. Our data suggest that microglia are structural modulators that facilitate remodeling of the proliferative zones as precursor cells migrate away from the ventricle and may facilitate the delamination of precursor cells. Taken together, these results indicate that microglial cells are an integral component of cortical proliferative zones and contribute to the interactive milieu in which cortical precursor cells function.

scholarly journals Flow cytometric detection of receptors for interleukin-6 on bone marrow and peripheral blood cells of humans and rhesus monkeys

Blood ◽  
1993 ◽  
Vol 81 (8) ◽  
pp. 2036-2043 ◽  
Author(s):  
AW Wognum ◽  
FC van Gils ◽  
G Wagemaker
Keyword(s):  
Bone Marrow ◽  
T Lymphocytes ◽  
Rhesus Monkey ◽  
Interleukin 6 ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Rhesus Monkeys ◽  
Cell Types ◽  
Cd8 T Lymphocytes ◽  
Blast Cells

Abstract The expression of receptors for interleukin-6 (IL-6) on human and rhesus monkey peripheral blood and bone marrow (BM) cells was examined by multiparameter flow cytometry after staining with biologically active, biotin-labeled human IL-6 and phycoerythrin-conjugated streptavidin. Consistent with the multiple biologic effects of IL-6 in stimulating immune functions and hematopoiesis, IL-6 receptors were detectable on a wide variety of cell types. In peripheral blood, IL-6 receptors were detectable on monocytes, granulocytes, and on CD4+ T lymphocytes but not on resting, CD19+ B lymphocytes and CD56+ natural killer (NK) cells. CD8+ T lymphocytes also expressed IL-6 receptors but at lower levels than CD4+ cells. The IL-6 receptors on granulocytes were only detectable after staining with high concentrations of biotin- IL-6, suggesting that most IL-6 receptors on these cells represent low- affinity sites. In contrast, IL-6 receptors on both CD4+ and CD8+ T lymphocytes were detectable at biotin-IL-6 concentrations as low as 10 pmol/L, indicating that these cells bind IL-6 with high affinity. IL-6 receptor expression patterns on rhesus monkey and human blood cells were very similar except that receptor levels on granulocytes were lower in humans than in rhesus monkeys. Similar differences in expression levels were observed for IL-6 receptors that were detectable on most granulocyte precursors in the mononuclear fraction of rhesus monkey and human bone marrow. In addition to these relatively mature cell types, IL-6 receptors were detectable on a large fraction of human and rhesus monkey BM blast cells that express the CD34 antigen. The presence of IL-6 receptors on CD34+ BM blast cells, which are the precursor cells of most, if not all, BM-derived blood cells, is consistent with the ability of IL-6, in conjunction with other cytokines, to stimulate immature hemopoietic cells in vitro and to promote blood cell production when administered in vivo.

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