Characterisation of the antigenic and immunogenic properties of bacterially expressed, sexual stage antigens of the coccidian parasite, Eimeria maxima

Coccidiosis in poultry is caused by the intestinal parasite Eimeria; it causes significant financial losses to the commercial poultry industry worldwide. CoxAbic® is the first commercially available subunit vaccine against coccidiosis. The vaccine consists of affinity purified sexual stage (gametocyte) antigens (APGA) isolated from Eimeria maxima. Production of this vaccine is time-consuming and laborious and, therefore, a recombinant subunit vaccine substitute for CoxAbic® is desirable. The genes encoding the two immunodominant components of CoxAbic®, gam56 and gam82, were cloned into the bacterial expression vector, pTRCHisB, and the proteins expressed and purified. Both recombinant proteins were recognised by protective chicken antibodies that were raised to APGA, by immunoblotting. In a competitive ELISA, a combination of the recombinant proteins inhibited the binding of anti-APGA antibodies to APGA by 76%, which was comparable to the inhibition of 98% observed when APGA was used as the competing protein in the assay. In two breeds of chicken (Australorp and Cobb500), the recombinant proteins alone, or in combination, elicited a dose-dependent, antibody response that recognised APGA by ELISA, and gametocytes by immunoblotting. Together, the results suggested that the development of a recombinant subunit vaccine that maintains the antigenic and immunogenic properties of the native protein vaccine, CoxAbic®, is feasible. © 2004 Elsevier Ltd. All rights reserved

lentiMPRA and MPRAflow for high-throughput functional characterization of gene regulatory elements

Massively parallel reporter assays (MPRAs) can simultaneously measure the function of thousands of candidate regulatory sequences (CRSs) in a quantitative manner. In this method, CRSs are cloned upstream of a minimal promoter and reporter gene, alongside a unique barcode, and introduced into cells. If the CRS is a functional regulatory element, it will lead to the transcription of the barcode sequence, which is measured via RNA sequencing and normalized for cellular integration via DNA sequencing of the barcode. This technology has been used to test thousands of sequences and their variants for regulatory activity, to decipher the regulatory code and its evolution, and to develop genetic switches. Lentivirus-based MPRA (lentiMPRA) produces 'in-genome' readouts and enables the use of this technique in hard-to-transfect cells. Here, we provide a detailed protocol for lentiMPRA, along with a user-friendly Nextflow-based computational pipeline-MPRAflow-for quantifying CRS activity from different MPRA designs. The lentiMPRA protocol takes ~2 months, which includes sequencing turnaround time and data processing with MPRAflow

Polygenic burden associated to oligodendrocyte precursor cells and radial glia influences the hippocampal volume changes induced by aerobic exercise in schizophrenia patients

Hippocampal volume decrease is a structural hallmark of schizophrenia (SCZ), and convergent evidence from postmortem and imaging studies suggests that it may be explained by changes in the cytoarchitecture of the cornu ammonis 4 (CA4) and dentate gyrus (DG) subfields. Increasing evidence indicates that aerobic exercise increases hippocampal volume in CA subfields and improves cognition in SCZ patients. Previous studies showed that the effects of exercise on the hippocampus might be connected to the polygenic burden of SCZ risk variants. However, little is known about cell type-specific genetic contributions to these structural changes. In this secondary analysis, we evaluated the modulatory role of cell type-specific SCZ polygenic risk scores (PRS) on volume changes in the CA1, CA2/3, and CA4/DG subfields over time. We studied 20 multi-episode SCZ patients and 23 healthy controls who performed aerobic exercise, and 21 multi-episode SCZ patients allocated to a control intervention (table soccer) for 3 months. Magnetic resonance imaging-based assessments were performed with FreeSurfer at baseline and after 3 months. The analyses showed that the polygenic burden associated with oligodendrocyte precursor cells (OPC) and radial glia (RG) significantly influenced the volume changes between baseline and 3 months in the CA4/DG subfield in SCZ patients performing aerobic exercise. A higher OPC- or RG-associated genetic risk burden was associated with a less pronounced volume increase or even a decrease in CA4/DG during the exercise intervention. We hypothesize that SCZ cell type-specific polygenic risk modulates the aerobic exercise-induced neuroplastic processes in the hippocampus