Examination of SARS-CoV-2 serological test results from multiple commercial and laboratory platforms with an in-house serum panel.

Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) is a novel human coronavirus that was identified in 2019. SARS-CoV-2 infection results in an acute, severe respiratory disease called coronavirus disease 2019 (COVID-19). The emergence and rapid spread of SARS-CoV-2 has led to a global public health crisis, which continues to affect populations across the globe. Real time reverse transcription polymerase chain reaction (rRT-PCR) is the reference standard test for COVID-19 diagnosis. Serological tests are valuable tools for serosurveillance programs and establishing correlates of protection from disease. This study evaluated the performance of one in-house enzyme linked immunosorbent assay (ELISA) utilizing the pre-fusion stabilized ectodomain of SARS-CoV-2 spike (S), two commercially available chemiluminescence assays Ortho VITROS Immunodiagnostic Products Anti-SARS-CoV-2 Total Reagent Pack and Abbott SARS-CoV-2 IgG assay and one commercially available Surrogate Virus Neutralization Test (sVNT), GenScript USA Inc., cPass SARS-CoV-2 Neutralization Antibody Detection Kit for the detection of SARS-CoV-2 specific antibodies. Using a panel of rRT-PCR confirmed COVID-19 patients' sera and a negative control group as a reference standard, all three immunoassays demonstrated high comparable positivity rates and low discordant rates. All three immunoassays were highly sensitive with estimated sensitivities ranging from 95.4-96.6 %. ROC curve analysis indicated that all three immunoassays had high diagnostic accuracies with area under the curve (AUC) values ranging from 0.9698 to 0.9807. High positive correlation was demonstrated among the conventional microneutralization test (MNT) titers and the sVNT inhibition percent values. Our study indicates that independent evaluations are necessary to optimize the overall utility and the interpretation of the results of serological tests. Overall, we demonstrate that all serological tests evaluated in this study are suitable for the detection of SARS-CoV-2 antibodies.

Evaluation of a Multiplex Bead Assay against Single-Target Assays for Detection of IgG Antibodies to SARS-CoV-2.

Serological assays for SARS-CoV-2 antibodies must be validated for performance with a large panel of clinical specimens. Most existing assays utilize a single antigen target and may be subject to reduced diagnostic specificity. This study evaluated a multiplex assay that detects antibodies to three SARS-CoV-2 targets. Human serum specimens (n = 323) with known previous SARS-CoV-2 exposure status were tested on a commercially available multiplex bead assay (MBA) measuring IgG to SARS-CoV-2 spike protein receptor-binding domain (RBD), nucleocapsid protein (NP), and RBD/NP fusion antigens. Assay performance was evaluated against reverse transcriptase PCR (RT-PCR) results and also compared with test results for two single-target commercial assays. The MBA had a diagnostic sensitivity of 89.8% and a specificity of 100%, with serum collection at >28 days following COVID-19 symptom onset showing the highest seropositivity rates (sensitivity: 94.7%). The MBA performed comparably to single-target assays with the ability to detect IgG against specific antigen targets, with 19 (5.9%) discrepant specimens compared to the NP IgG assay and 12 (3.7%) compared to the S1 RBD IgG assay (kappa coefficients 0.92 and 0.88 compared to NP IgG and S1 RBD IgG assays, respectively. These findings highlight inherent advantages of using a SARS-CoV-2 serological test with multiple antigen targets; specifically, the ability to detect IgG against RBD and NP antigens simultaneously. In particular, the 100.0% diagnostic specificity exhibited by the MBA in this study is important for its implementation in populations with low SARS-CoV-2 seroprevalence or where background antibody reactivity to SARS-CoV-2 antigens has been detected. IMPORTANCE Reporting of SARS-CoV-2 infections through nucleic acid or antigen based diagnostic tests severely underestimates the true burden of exposure in a population. Serological data assaying for antibodies against SARS-CoV-2 antigens offers an alternative source of data to estimate population exposure, but most current immunoassays only include a single target for antibody detection. This report outlines a direct comparison of a multiplex bead assay to two other commercial single-target assays in their ability to detect IgG against SARS-CoV-2 antigens. Against a well-defined panel of 323 serum specimens, diagnostic sensitivity and specificity were very high for the multiplex assay, with strong agreement in IgG detection for single targets compared to the single-target assays. Collection of more data for individual- and population-level seroprofiles allows further investigation into more accurate exposure estimates and research into the determinants of infection and convalescent responses.

Evaluation of anti-factor VIII antibody levels in patients with haemophilia A receiving immune tolerance induction therapy or bypassing agents.

INTRODUCTION: Bleeding episodes in patients who have haemophilia A (HA), a hereditary bleeding disorder caused by a deficiency in factor VIII (FVIII), are treated or prophylactically prevented with infusions of exogenous FVIII. Neutralizing antibodies, referred to as inhibitors, against infusion products are a major complication experienced by up to 30% of patients who have severe HA. Bypassing agents (BPA), a class of therapeutics given to patients who have inhibitors, bypass the need for FVIII in the coagulation cascade, and long-term inhibitor eradication is accomplished using immune tolerance induction therapy (ITI). Data examining the antibody levels in patients receiving BPA and ITI are limited. AIM: Measure anti-FVIII antibody levels in specimens from patients receiving ITI or BPA in order to evaluate the anti-FVIII antibody response in those patients. METHODS: Specimens were tested using the CDC-modified Nijmegen-Bethesda assay (NBA) and the CDC fluorescence immunoassay (FLI) for anti-FVIII IgG1 and IgG4 . RESULTS: NBA-negative specimens from patients undergoing ITI or receiving BPAs have a higher frequency of anti-FVIII IgG4 positivity compared with the previously published level for NBA-negative HA patients. Analysis of anti-FVIII antibody levels in serial samples from patients undergoing ITI reveals that antibodies can persist even after the patient's NBA result falls into the negative range. CONCLUSIONS: Measurement of anti-FVIII antibodies may be a useful means to better contextualize NBA results in specimens from patients receiving BPA or ITI. In addition, assessment of anti-FVIII antibody levels has the potential to improve inhibitor surveillance and clinical decision-making related to the progress of ITI.

An Observational Study from Long-Term AAV Re-administration in Two Hemophilia Dogs.

Adeno-associated virus (AAV) vectors have been successfully applied in hemophilia clinical trials. However, this approach is limited to patients without AAV-neutralizing antibodies (NAbs). In this study, we explored the feasibility of AAV re-administration in hemophilia A dogs treated initially 8 years ago with AAV8.canine FVIII. After the re-administration in two NAb-negative dogs with AAV8 vectors carrying human factor VIII (hFVIII), along with the proteasome inhibitor bortezomib, we observed a phenotypic improvement in both dogs that persisted in one dog. Phenotypic improvement disappeared at 59 days after re-administration in the other dog, and specific cytotoxic T lymphocytes (CTLs) to the capsid were detected at day 17, but not to hFVIII. hFVIII inhibitors were observed at day 59 and gradually increased. Mechanistic studies demonstrated an increase in pro-inflammatory cytokines, a decrease in immunomodulatory cytokines, as well as lower Tregs after re-administration. These results suggest that hFVIII inhibitor development may contribute to the therapeutic failure via immune response activation. Interestingly, it takes about 30-50 days for AAV NAb titers to decrease by half. Collectively, this study suggests that re-administration of the same AAV serotype after long-term follow-up is feasible and that the study of AAV NAb kinetics will provide important information for predicating the efficacy of re-administration.

AAV liver expression of FIX-Padua prevents and eradicates FIX inhibitor without increasing thrombogenicity in hemophilia B dogs and mice.

Emerging successful clinical data on gene therapy using adeno-associated viral (AAV) vector for hemophilia B (HB) showed that the risk of cellular immune response to vector capsid is clearly dose dependent. To decrease the vector dose, we explored AAV-8 (1-3 × 10(12) vg/kg) encoding a hyperfunctional factor IX (FIX-Padua, arginine 338 to leucine) in FIX inhibitor-prone HB dogs. Two naïve HB dogs showed sustained expression of FIX-Padua with an 8- to 12-fold increased specific activity reaching 25% to 40% activity without antibody formation to FIX. A third dog with preexisting FIX inhibitors exhibited a transient anamnestic response (5 Bethesda units) at 2 weeks after vector delivery following by spontaneous eradication of the antibody to FIX by day 70. In this dog, sustained FIX expression reached ∼200% and 30% of activity and antigen levels, respectively. Immune tolerance was confirmed in all dogs after challenges with plasma-derived FIX concentrate. Shortening of the clotting times and lack of bleeding episodes support the phenotypic correction of the severe phenotype, with no clinical or laboratory evidence of risk of thrombosis. Provocative studies in mice showed that FIX-Padua exhibits similar immunogenicity and thrombogenicity compared with FIX wild type. Collectively, these data support the potential translation of gene-based strategies using FIX-Padua for HB.

Decreased hematopoietic progenitor cell mobilization in pearl mice.

Neutropenia is common to both Hermansky-Pudlak syndrome type 2 and canine cyclic hematopoiesis (CH) which are caused by mutations in the AP3B1 gene. The purpose of this study was to determine if pearl mice were neutropenic. Complete blood counts (CBCs) and bone marrow differential counts, colony forming unit (CFU) assay, bone marrow lineage negative (lin(-)), Sca(+) and c-kit(+) cells (LSK cells), bone marrow elastase, myeloperoxidase, and cathepsin G enzyme activity were compared in C57Bl6 (Bl/6) and pearl mice. Stress granulopoiesis was evaluated following 200 mg/kg cyclophosphamide or 1 mg/kg bortezomib administration and by limiting dilution bone marrow transplantation. The CBCs and CFUs were determined in Bl/6 and pearl mice following AMD3100 or granulocyte colony-stimulating factor (G-CSF) administration. Pearl mice were not neutropenic and did not have cyclic neutropenia. Bone marrow elastase, myeloperoxidase, and cathepsin G enzyme activity were similar in pearl and Bl/6 mice. The numbers of CFU-G, CFU-GEMM, and LSK cells were increased moderately in pearl mice. Stress granulopoiesis was similar in Bl/6 and pearl mice. CFU assays and CBCs performed on Bl/6 and pearl mice administered AMD3100 resulted in similar results. However, normal mice administered G-CSF had higher peripheral blood neutrophil counts and greater CFU numbers compared with pearl mice. Unlike patients with HPS-2 and dogs with CH, pearl mice did not have neutropenia or CH but had decreased hematopoietic progenitor cell and granulocyte mobilization in response to G-CSF.

Neutrophil elastase-processing defect in cyclic hematopoietic dogs.

OBJECTIVE: Canine cyclic hematopoiesis (CH), a model of human cyclic neutropenia and severe congenital neutropenia, is characterized by a periodic reduced neutrophil count and decreased neutrophil elastase (NE) enzymatic activity. Canine CH is caused by a mutation of AP3B1 encoding the beta3A subunit of adaptor protein complex-3 (AP-3). It has been proposed that trafficking of elastase is affected by AP-3. The aim of this study was to study intracellular sorting/trafficking of NE in CH dogs using antibodies specific to canine NE. MATERIALS AND METHODS: Polyclonal and monoclonal antibodies were generated to immunogenic epitopes in the middle (aa85-98) and C-terminal (aa269-282) regions of NE. The antibodies to canine NE were characterized by Western immunoblotting and immunocytochemistry. RESULTS: Antibody ELA85 (antibody to canine NE aa 85-98) specifically recognized mature 28-kD NE. Immunocytochemical analysis using ELA85 and an antibody to myeloperoxidase demonstrated colocalizaton of NE and myeloperoxidase in primary granules of normal dogs. Antibody ELA269 (antibody to canine NE aa 269-282) reacted exclusively with the 33-kD NE presumptive precursor form. Immunocytochemical analysis demonstrated that the NE precursor was not colocalized with myeloperoxidase in the primary granules of normal or CH dogs. Western immunoblotting using these antibodies demonstrated that CH dogs contained reduced mature NE, but accumulated a large amount of the NE precursor protein that was not enzymatically active. CONCLUSION: Antibodies ELA85 and ELA269 were found to be useful reagents for studying the biosynthesis, processing, and trafficking of NE during normal myelopoiesis. Neutrophils from CH dogs accumulated large amounts of higher molecular weight elastase precursors compared to normal dogs.

Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy.

Preclinical studies and initial clinical trials have documented the feasibility of adenoassociated virus (AAV)-mediated gene therapy for hemophilia B. In an 8-year study, inhibitor-prone hemophilia B dogs (n = 2) treated with liver-directed AAV2 factor IX (FIX) gene therapy did not have a single bleed requiring FIX replacement, whereas dogs undergoing muscle-directed gene therapy (n = 3) had a bleed frequency similar to untreated FIX-deficient dogs. Coagulation tests (whole blood clotting time [WBCT], activated clotting time [ACT], and activated partial thromboplastin time [aPTT]) have remained at the upper limits of the normal ranges in the 2 dogs that received liver-directed gene therapy. The FIX activity has remained stable between 4% and 10% in both liver-treated dogs, but is undetectable in the dogs undergoing muscle-directed gene transfer. Integration site analysis by linear amplification-mediated polymerase chain reaction (LAM-PCR) suggested the vector sequences have persisted predominantly in extrachromosomal form. Complete blood count (CBC), serum chemistries, bile acid profile, hepatic magnetic resonance imaging (MRI) and computed tomography (CT) scans, and liver biopsy were normal with no evidence for tumor formation. AAV-mediated liver-directed gene therapy corrected the hemophilia phenotype without toxicity or inhibitor development in the inhibitor-prone null mutation dogs for more than 8 years.

Hematopoietic chimerism induces renal and skin allograft tolerance in DLA-identical dogs.

OBJECTIVE: Hematopoietic chimerism, a state where donor and recipient bone marrow cells coexist, is associated with donor-specific tolerance. Nonmyeloablative bone marrow transplantation (BMT) has been shown to induce stable mixed hematopoietic chimerism in dog leukocyte antigen (DLA)-matched dogs. The potential for inducing renal and skin allograft tolerance with nonmyeloablative BMT was investigated in DLA-identical and DLA-haploidentical dogs in this study. MATERIALS AND METHODS: Renal allografts were performed in 8 DLA-identical and 4 DLA-haploidentical dogs with nonmyeloablative conditioning (200 cGy TBI) and transient immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF) with (n = 8) and without (n = 4) simultaneous BMT. Skin allografts were performed in 2 DLA-identical and 4 DLA-haploidentical dogs after stopping CSP and MMF. Two DLA-identical control dogs received renal allografts without TBI, BMT, or immunosuppression with CSP and MMF. Molecular chimerism was determined with a PCR-based DNA microsatellite assay. Serum creatinine (Cr) concentration, urine specific gravity, and sequential renal biopsies were monitored to assess renal allograft function. RESULTS: Donor-type blood cells were first detected 4 weeks posttransplantation in both the myeloid and lymphoid lineages. Donor chimerism was present for at least 76 weeks in the DLA-identical dogs. Mixed chimerism was not observed in the DLA-haploidentical dogs or DLA-identical dogs that did not undergo BMT. The renal allografts were acutely rejected within 14 days in the 2 DLA-identical control dogs. There was long-term (> 5 yrs) renal allograft survival as evidenced by a normal (< 2.0 mg/dL) serum Cr concentration in both the DLA-identical and DLA-haploidentical dogs that underwent 200 cGy TBI and transient immunosuppression with CSP and MMF either with or without simultaneous BMT. Renal allograft inflammation was severe in the control dogs, mild to moderate in the DLA-haploidentical dogs, and minimal in the DLA-identical dogs. Donor-specific skin grafts were accepted in the DLA-identical dogs but rejected in the DLA-haploidentical dogs. Nonmyeloablative conditioning (200 cGy TBI) and transient immunosuppression with CSP and MMF induce renal and skin allograft tolerance in DLA-identical and permit long-term renal allograft survival in DLA-haploidentical dogs. These findings suggest it may possible to obtain long-term allograft survival in DLA-identical and -haploidentical dogs without the need for chronic immunosuppressive therapy.

Renal allograft histopathology in dog leukocyte antigen mismatched dogs after renal transplantation.

OBJECTIVE: To evaluate allograft histopathology in dog leukocyte antigen (DLA)-mismatched dogs undergoing renal transplantation, with transient immunosuppression. STUDY DESIGN: Prospective study. ANIMALS: Ten healthy adult mongrel dogs. METHODS: Reciprocal renal transplantation and bilateral nephrectomy were performed. Immune conditioning consisted of nonmyeloablative (200 cGy), total body irradiation (TBI), bone marrow transplantation (BMT; 7 dogs), cyclosporine (CSA; 15 mg/kg every 12 hours), mycophenolate mofetil (MMF; 10 mg/kg every 12 hours) and intermittent prednisone (1 mg/kg every 12-24 hours). Biopsies were collected at transplantation, during full immunosuppression (44-90 days), and once medications were reduced or discontinued (228-580 days). Biopsies were evaluated for interstitial, tubular, vascular, and glomerular lesions. Blood urea nitrogen, creatinine, serum CSA concentrations, and clinical score were determined at each biopsy. RESULTS: Seven dogs survived >200 days (mean, 380 days). Transient CSA toxicity was suspected in 6 dogs. Lymphocytic, plasmacytic interstitial inflammation, and tubulitis progressed when immunosuppressive medications were decreased. All 7 dogs had histologic lesions consistent with some degree of allograft rejection at study end. CONCLUSION: Nonmyeloablative TBI, BMT, and short-term immunosuppression with CSA, MMF, and prednisone allowed renal allograft function and dog survival for >200 days. It appears unlikely that total drug withdrawal will be possible in unrelated DLA-mismatched dogs using this protocol. CLINICAL RELEVANCE: Transient immunosuppression with MMF, CSA, and prednisone along with BMT and nonmyeloablative TBI may make kidney transplantation a clinical reality for treatment of kidney failure in dogs. Initiating both MMF and CSA at lower dosages may potentially eliminate early renal allograft injury.

Correction of a large animal model of type I Glanzmann's thrombasthenia by nonmyeloablative bone marrow transplantation.

OBJECTIVE: The purpose of this study was to determine if nonmyeloablative bone marrow transplantation would induce stable hematopoietic chimerism that would correct the bleeding diathesis associated with type I Glanzmann's thrombasthenia (GT). METHODS: Three young dogs (less than 12 weeks of age) with GT were transplanted with DLA-matched bone marrow from littermates. Recipients received a sublethal dose (200 cGy) of total-body irradiation (TBI) prior to infusion with bone marrow (1-4 x 10(8) cells/kg). Recipient dogs were immunosuppressed with cyclosporine (15 mg/kg) and mycophenolate mofetil (10 mg/kg). Chimerism was determined by quantitation of donor microsatellite repeat polymorphisms in peripheral blood DNA and by flow cytometry to detect the presence of glycoproteins IIb and IIIa on platelets. Platelet function was assessed by a clot retraction test. RESULTS: One dog died one week posttransplant due to hemorrhage. Another dog died four weeks posttransplant from an unrecognized congenital heart defect and complications due to canine distemper virus infection. At the time of death, microsatellite analysis indicated 35 to 50% chimerism. Flow cytometry showed 20% of circulating platelets positive for glycoproteins IIb and IIIa. The third dog is alive and doing well approximately two years posttransplant. Hematopoietic chimerism has been sustained at 35 to 60% with approximately 30% of the platelets positive for glycoproteins IIb and IIIa. Platelet function is normal based on clot retraction. The animal does not have clinical signs of bleeding. CONCLUSIONS: These observations suggest that GT and perhaps other severe inherited platelet disorders can be corrected using nonmyeloablative bone marrow transplantation to establish partial chimerism with normal platelets in the platelet compartment.

Isolation and characterization of multipotential mesenchymal stem cells from feline bone marrow.

OBJECTIVE: Although several types of stem cells have been isolated from rodent and human tissues, very few data exist on stem cell isolation from nonrodent animals, which seriously limits the advancement of stem cell biology and its ultimate translation to human clinical applications. Domestic cats are used frequently in biomedical research and are the preferred species for studies of normal physiology and disease, particularly in neuroscience. Therefore, the objective of this study was to characterize mesenchymal stem cells (MSC) from feline bone marrow for use in research on the application of stem cells to human health problems for which cats are the preferred model. METHODS: Mesenchymal stem cells from feline bone marrow were isolated by standard methodology developed for other species and characterized according to morphology, growth traits, cell-surface antigen profile, and differentiation repertoire in vitro. RESULTS: Feline mesenchymal stem cells exhibit a fibroblast-like morphology with bipolar or polygonal cell bodies and possess a cell-surface antigen profile similar to their rodent and human counterparts. Feline MSC exist at a frequency of 1 in 3.8 x 10(5) bone marrow mononuclear cells and are capable of differentiation to adipocytic, osteocytic, and neuronal phenotypes when exposed to appropriate induction media. CONCLUSIONS: Mesenchymal stem cells isolated from feline bone marrow possess several traits typical of MSC from other species. Characterization of feline mesenchymal stem cells will facilitate future studies of stem cell biology and therapeutics for which the domestic cat is an indispensable model.