Identification of a spike-specific CD8+ T cell epitope following vaccination against the Middle East respiratory syndrome coronavirus in humans.

Licensed vaccines against the Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging pathogen of concern, are lacking. The Modified Vaccinia virus Ankara vector-based vaccine MVA-MERS-S, expressing the MERS-CoV-spike glycoprotein (MERS-S), is one of three candidate vaccines in clinical development and elicits robust humoral and cellular immunity. Here, we identified for the first time a MERS-S-specific CD8+ T-cell epitope in an HLA-A*03:01/HLA-B*35:01-positive vaccinee using a screening assay, intracellular cytokine staining, and in silico epitope prediction. As evidence from MERS-CoV infection suggests a protective role of long-lasting CD8+ T-cell responses, the identification of epitopes will facilitate longitudinal analyses of vaccine-induced T-cell immunity.

Increased neutralization and IgG epitope identification after MVA-MERS-S booster vaccination against Middle East respiratory syndrome.

Vaccine development is essential for pandemic preparedness. We previously conducted a Phase 1 clinical trial of the vector vaccine candidate MVA-MERS-S against the Middle East respiratory syndrome coronavirus (MERS-CoV), expressing its full spike glycoprotein (MERS-CoV-S), as a homologous two-dose regimen (Days 0 and 28). Here, we evaluate the safety (primary objective) and immunogenicity (secondary and exploratory objectives: magnitude and characterization of vaccine-induced humoral responses) of a third vaccination with MVA-MERS-S in a subgroup of trial participants one year after primary immunization. MVA-MERS-S booster vaccination is safe and well-tolerated. Both binding and neutralizing anti-MERS-CoV antibody titers increase substantially in all participants and exceed maximum titers observed after primary immunization more than 10-fold. We identify four immunogenic IgG epitopes, located in the receptor-binding domain (RBD, n = 1) and the S2 subunit (n = 3) of MERS-CoV-S. The level of baseline anti-human coronavirus antibody titers does not impact the generation of anti-MERS-CoV antibody responses. Our data support the rationale of a booster vaccination with MVA-MERS-S and encourage further investigation in larger trials. Trial registration: Clinicaltrials.gov NCT03615911.

Perilipin-2 is critical for efficient lipoprotein and hepatitis C virus particle production.

In hepatocytes, PLIN2 is the major protein coating lipid droplets (LDs), an organelle the hepatitis C virus (HCV) hijacks for virion morphogenesis. We investigated the consequences of PLIN2 deficiency on LDs and on HCV infection. Knockdown of PLIN2 did not affect LD homeostasis, likely due to compensation by PLIN3, but severely impaired HCV particle production. PLIN2-knockdown cells had slightly larger LDs with altered protein composition, enhanced local lipase activity and higher ß-oxidation capacity. Electron micrographs showed that, after PLIN2 knockdown, LDs and HCV-induced vesicular structures were tightly surrounded by ER-derived double-membrane sacs. Strikingly, the LD access for HCV core and NS5A proteins was restricted in PLIN2-deficient cells, which correlated with reduced formation of intracellular HCV particles that were less infectious and of higher density, indicating defects in maturation. PLIN2 depletion also reduced protein levels and secretion of ApoE due to lysosomal degradation, but did not affect the density of ApoE-containing lipoproteins. However, ApoE overexpression in PLIN2-deficient cells did not restore HCV spreading. Thus, PLIN2 expression is required for trafficking of core and NS5A proteins to LDs, and for formation of functional low-density HCV particles prior to ApoE incorporation.This article has an associated First Person interview with the first author of the paper.

Sensitive detection of idiotypic platelet-reactive alloantibodies by an electrical protein chip.

To prevent and treat immune-mediated platelet disorders (e.g. neonatal allo-immune thrombocytopenia and platelet transfusion refractoriness) the causative idiotypic platelet-reactive antibodies have to be detected with high sensitivity and specificity. The "Monoclonal Antibody Immobilization Platelet Assay" (MAIPA) is the diagnostic gold standard for immunotyping sera with respect to alloantibodies against human platelet antigens (HPA). However, it is labor-intensive and time-consuming. In this work, an automated protein chip assay (enzyme-linked sandwich immunoassay) based on interdigitated gold microelectrodes in combination with an electrical read-out system was developed and optimized. For this purpose, specific capture antibodies were immobilized on the gold electrodes. The binding of the target is detected via an enzyme-labeled detection antibody by a redox-recycling process that corresponds to the amount of bound target molecule. With this electrical chip assay it is possible to detect antibodies against HPA-1a, HPA-5b and HLA with high sensitivity and specificity in less than half the duration of the MAIPA protocol with similar intra- and interassay variance.