IgG3 and IgM Identified as Key to SARS-CoV-2 Neutralization in Convalescent Plasma Pools.

Analysis of convalescent plasma derived from individuals has shown that IgG3 has the most important role in binding to SARS-CoV-2 antigens; however, this has not yet been confirmed in large studies, and the link between binding and neutralization has not been confirmed. By analyzing plasma pools consisting of 247-567 individual convalescent donors, we demonstrated the binding of IgG3 and IgM to Spike-1 protein and the receptor-binding domain correlates strongly with viral neutralization in vitro. Furthermore, despite accounting for only approximately 12% of total immunoglobulin mass, collectively IgG3 and IgM account for approximately 80% of the total neutralization. This may have important implications for the development of potent therapies for COVID-19, as it indicates that hyperimmune globulins or convalescent plasma donations with high IgG3 concentrations may be a highly efficacious therapy.

Trafficking of prion proteins through a caveolae-mediated endosomal pathway.

To understand the posttranslational conversion of the cellular prion protein (PrPC) to its pathologic conformation, it is important to define the intracellular trafficking pathway of PrPC within the endomembrane system. We studied the localization and internalization of PrPC in CHO cells using cryoimmunogold electron microscopy. At steady state, PrPC was enriched in caveolae both at the TGN and plasma membrane and in interconnecting chains of endocytic caveolae. Protein A-gold particles bound specifically to PrPC on live cells. These complexes were delivered via caveolae to the pericentriolar region and via nonclassical, caveolae-containing early endocytic structures to late endosomes/lysosomes, thereby bypassing the internalization pathway mediated by clathrin-coated vesicles. Endocytosed PrPC-containing caveolae were not directed to the ER and Golgi complex. Uptake of caveolae and degradation of PrPC was slow and sensitive to filipin. This caveolae-dependent endocytic pathway was not observed for several other glycosylphosphatidyl inositol (GPI)-anchored proteins. We propose that this nonclassical endocytic pathway is likely to determine the subcellular location of PrPC conversion.

Coexistence of multiple PrPSc types in individuals with Creutzfeldt-Jakob disease.

BACKGROUND: The molecular typing of sporadic Creutzfeldt-Jakob disease (CJD) is based on the size and glycoform ratio of protease-resistant prion protein (PrP(Sc)), and on PRNP haplotype. On digestion with proteinase K, type 1 and type 2 PrP(Sc) display unglycosylated core fragments of 21 kDa and 19 kDa, resulting from cleavage around amino acids 82 and 97, respectively. METHODS: We generated anti-PrP monoclonal antibodies to epitopes immediately preceding the differential proteinase K cleavage sites. These antibodies, which were designated POM2 and POM12, recognise type 1, but not type 2, PrP(Sc). FINDINGS: We studied 114 brain samples from 70 patients with sporadic CJD and three patients with variant CJD. Every patient classified as CJD type 2, and all variant CJD patients, showed POM2/POM12 reactivity in the cerebellum and other PrP(Sc)-rich brain areas, with a typical PrP(Sc) type 1 migration pattern. INTERPRETATION: The regular coexistence of multiple PrP(Sc) types in patients with CJD casts doubts on the validity of electrophoretic PrP(Sc) mobilities as surrogates for prion strains, and questions the rational basis of current CJD classifications.

Studies on the dissociation of botulinum neurotoxin type A complexes.

UNLABELLED: The neurotoxins produced by the various strains of the anaerobic bacterium Clostridium botulinum naturally occur associated with complexing proteins which serve to protect the neurotoxins from the harsh environment of the mammalian gastrointestinal tract during bacterial invasion of the host. Three different complex species with the discrete sizes 19S (900 kDa, LL complex), 16S (500 kDa, L complex) and 12S (300 kDa, M complex) may be isolated from C. botulinum type A cultures. However, to affect their target cells these complexes must dissociate releasing the free 150 kDa neurotoxin. This study assesses the stability of these Clostridium botulinum neurotoxin serotype A (BoNT/A) complexes and identifies factors which influence their dissociation. The knowledge gained with purified toxin complexes was subsequently employed to analyze the presence of such complexes in the freeze or spray-dried commercial BoNT/A products Botox and Dysport in comparison to the complexing protein free product Xeomin. Purified 900 kDa and 500 kDa toxin complex preparations show a pH and time dependent release of the 150 kDa neurotoxin with a half-life of less than a minute at pH values >7.0. At pH values of 6.25 or less, the complexes are stable. Furthermore, dilution of concentrated 900 kDa complexes leads to dissociation into 500 kDa, neurotoxin containing complexes. Addition of sodium chloride as contained in isotonic saline leads to further disruption of these complexes resulting in the release of the free 150 kDa neurotoxin. Examination of the commercial botulinum neurotoxin products Botox and Dysport using the same analytical procedures leads to the same conclusion: the dilution, drying and reconstitution processes of these products lead to a complete dissociation of 900 kDa complexes and 85% or more of neurotoxin are present in free form. CONCLUSION: BoNT A toxin complexes have evolved to quickly respond to specific environmental changes by efficient release of the neurotoxin. During pharmaceutical production and reconstitution of BoNT A products, the same principles effect the quantitative dissociation of 900 kDa complexes and release of free neurotoxin prior to injection into target tissues.

The POM monoclonals: a comprehensive set of antibodies to non-overlapping prion protein epitopes.

PrP(Sc), a misfolded and aggregated form of the cellular prion protein PrP(C), is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrP(C) in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrP(C) and PrP(Sc). Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrP(C). Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrP(C). Some of these antibodies recognize epitopes that are resilient to protease digestion in PrP(Sc). Other antibodies immunoprecipitate PrP(C), but not PrP(Sc). A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrP(C) and PrP(Sc). Amino-proximal antibodies were found to react with repetitive PrP(C) epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays.

Diagnosis of human prion disease.

With the discovery of the prion protein (PrP), immunodiagnostic procedures were applied to diagnose Creutzfeldt-Jakob disease (CJD). Before development of the conformation-dependent immunoassay (CDI), all immunoassays for the disease-causing PrP isoform (PrPSc) used limited proteolysis to digest the precursor cellular PrP (PrPC). Because the CDI is the only immunoassay that measures both the protease-resistant and protease-sensitive forms of PrPSc, we used the CDI to diagnose human prion disease. The CDI gave a positive signal for PrPSc in all 10-24 brain regions (100%) examined from 28 CJD patients. A subset of 18 brain regions from 8 patients with sporadic CJD (sCJD) was examined by histology, immunohistochemistry (IHC), and the CDI. Three of the 18 regions (17%) were consistently positive by histology and 4 of 18 (22%) by IHC for the 8 sCJD patients. In contrast, the CDI was positive in all 18 regions (100%) for all 8 sCJD patients. In both gray and white matter, approximately 90% of the total PrPSc was protease-sensitive and, thus, would have been degraded by procedures using proteases to eliminate PrPC. Our findings argue that the CDI should be used to establish or rule out the diagnosis of prion disease when a small number of samples is available as is the case with brain biopsy. Moreover, IHC should not be used as the standard against which all other immunodiagnostic techniques are compared because an immunoassay, such as the CDI, is substantially more sensitive.