Establishment of an indirect ELISA-based method involving the use of a multiepitope recombinant S protein to detect antibodies against canine coronavirus.

Here, we report the development of an indirect enzyme-linked immunosorbent assay (ELISA) method that involves using multiepitope recombinant S protein (rSP) as the coating antigen to detect antibodies against canine coronavirus (CCoV). rSP was designed by arranging its four S fragments (91-135 aa, S1 gene; 377-434 aa, S2 gene; 647-671 aa, S3 gene; 951-971 aa, S4 gene; 207-227 aa) and two T-cell epitopes in tandem: T-E1-E2-E3-E4-T. This multiepitope antigen, which has a molecular weight of approximately 25 kDa and contains a His-tag, was recognized by a CCoV-positive serum in a Western blot assay. The optimal concentration of rSP as a coating antigen in the ELISA was 2 µg/mL, and the optimal dilution of enzyme-labeled secondary antibody was 1:10,000. The cutoff OD450 value was established at 0.2395. No reactivity was observed with antisera against canine distemper virus, canine parvovirus, or feline calicivirus, indicating that this assay is highly specific. We also tested 64 clinical serum samples using our newly established method, and the positive rate was found to be 82.8%. In conclusion, our assay was found to be highly sensitive and specific for the detection of antibodies against CCoV, and it can therefore serve as a new, efficient diagnostic method.

Individualized regimen of low-dose rituximab monotherapy for new-onset AChR-positive generalized myasthenia gravis.

BACKGROUND: Generalized AChR-MG is an archetype of B cell-mediated autoimmune disorders, and use of biologic agent rituximab (RTX) for B cell depletion is generally limited to immunosuppressive therapy-refractory cases. However, benefit of RTX monotherapy and individualized regimen with optimal dosage in early stage of new-onset generalized AChR-MG still remains to be elucidated. In this retrospective study, we explore the efficacy and safety of personalized regimen of 100 mg low-dose rituximab monotherapy in treating new-onset generalized AChR-MG. METHODS: Thirteen new-onset generalized AChR-MG patients were enrolled for the study, initiating RTX treatment from November 2017 to August 2020. The individualized low-dose RTX monotherapy protocol consisted of 100 mg induction treatment weekly with no more than three circles, followed by reinfusion (100 mg once) sequentially according to whether achieving primary endpoint and peripheral CD19 + B-cell repopulation ≥ 1% of total lymphocytes at each visit (every 3 months). Outcome measures included MGFA-PIS Minimal Manifestation (MM) or better status (primary endpoint), changes in QMG, MMT, MG-ADL and MGQOL-15 scores (secondary endpoint), as well as cholinesterase inhibitors dosage. RESULTS: All 13 patients achieved the primary endpoint in parallel with significant improvement of QMG, MMT, MG-ADL MGQOL-15 scores, and reduction of cholinesterase inhibitors dose. A total of 52 visits were performed during follow-up, and only 10 assessments presenting peripheral CD19 + B-cell repopulation (≥ 1%) without "MM or better status" were followed by RTX reinfusions (100 mg once) for clinical remission. The total dosage of RTX was only 346.15 ± 96.74 mg (including 269.23 ± 63.04 mg for induction and 76.92 ± 59.91 mg for reinfusion), which seemed to be much lower than those dosages used in new-onset generalized AChR-MG as described previously. Moreover, compared with patients without thymoma, thymectomy markedly delayed initiation of RTX for patients with thymoma (log-rank test, p = 0.0002), but the delaying treatments showed no influence on the time for achieving primary outcome (log-rank test, p = 0.2517). CONCLUSION: Our study firstly showed that individualized regimen of low-dose RTX monotherapy is effective and safe for early treatment of new-onset generalized AChR-MG, and practicable for directing RTX reinfusion and withdrawal. Moreover, the monotherapy protocol was also indicated to be extensively applicable in both new-onset AChR-MG with thymoma (thymectomy) and without thymoma.

Case report: MOG-IgG-associated encephalitis with Epstein-Barr virus infection and Alzheimer's pathologic change in cerebrospinal fluid.

Immunoglobulin G antibodies to myelin oligodendrocyte glycoprotein (MOG-IgG) associated disease is a rare, demyelinated disease in the central nerve system (CNS) predominately involving optic nerve, spinal cord, and brain leading to optic neuritis (ON), transverse myelitis (TM), encephalitis. The phenotype of MOG-IgG-associated encephalitis is similar to acute disseminated encephalomyelitis (ADEM) presenting with seizures, abnormal behavioral and psychological symptoms, and cognitive impairment. A few brain biopsies show multiple sclerosis (MS) pattern histopathology with T cells, macrophages, and complement activation. To date, how MOG-IgG is produced is unknown. Herein, we report a case of a 32-year-old male with MOG-IgG-associated encephalitis presenting MOG-IgG in cerebrospinal fluid (CSF) but seronegative, as well as Epstein-Barr virus (EBV) infection and Alzheimer's pathologic change in CSF (Aß42 = 317 pg/ml, T-Tau = 538 pg/ml, p-Tau =10.09 pg/ml). With a combination treatment of administering intravenous immunoglobulin (0.4 mg/kg/d, 5 days) with a low dose of methylprednisolone (80 mg/d, 5 days) and rituximab (100 mg/week, 3 weeks), the patient recovered significantly after 3 months follow-up. This case provides us with new thoughts into the production of MOG-IgG and the possible pathologic mechanism of MOG-IgG-associated disease (MOG-AD) and simultaneously further confirms the interaction between EBV and changes of CSF biomarkers of Alzheimer's disease (AD).