El-Turabi, A. | Search bioRxiv

bioRxiv2023

Structural basis of epitope recognition by anti-alpha synuclein antibodies MJFR14-6-4-2

Intraneuronal -synuclein inclusions in the brain are hallmarks of so-called Lewy body diseases - Parkinsons disease and Dementia with Lewy bodies. Lewy bodies are cytoplasmic inclusions, containing mainly aggregated -synuclein together with some other proteins including ubiquitin, neurofilament protein, and alpha B crystallin. In its monomeric form, -synuclein is predominantly localized in nerve terminals, regulating neuronal transmission and synaptic vesicle trafficking. Monomeric -synuclein lacks a well-defined three-dimensional structure and is considered an intrinsically disordered protein. However, in diseased cells -synuclein aggregates into oligomeric and fibrillar amyloid species, which can be detected using aggregate-specific antibodies. Here we investigate the aggregate specificity of rabbit monoclonal MJFR14-6-4-2 antibodies, preferentially recognizing aggregated -synuclein species. We conclude that partial masking of epitope in unstructured monomer in combination with a high local concentration of epitopes instead of distinct epitope conformation is the main reason for apparent selectivity towards various aggregates, including oligomers, fibrils, and artificial virus-like particle constructs bearing multiple copies of the MJFR14-6-4-2 epitope. Based on the structural insight, we were able to express mutant -synuclein that when fibrillated are unable to bind MJFR14-6-4-2. Using these "stealth" fibrils as a tool for seeding cellular -synuclein aggregation, provides superior signal/noise ratio for detection of cellular -synuclein aggregates by MJFR14-6-4-2 immunocytochemistry. Our data provide a molecular level understanding of specific recognition of toxic amyloid oligomers, which is critical for the development of inhibitors against synucleinopathies.

neuroscience↗

bioRxiv2020

Dissection-independent production of a protective whole-sporozoite malaria vaccine

Complete protection against human malaria challenge has been achieved using infected mosquitoes as the delivery route for immunization with Plasmodium parasites. Strategies seeking to replicate this efficacy with either a manufactured whole-parasite or subunit vaccine, however, have shown only limited success. A major roadblock to whole parasite vaccine progress and understanding of the human infective sporozoite form in general, is reliance on manual dissection for parasite isolation from infected mosquitoes. We report here the development of a four-step process based on whole mosquito homogenization, slurry and density-gradient filtration, combined with free-flow electrophoresis that is able to rapidly produce a pure, aseptic sporozoite inoculum from hundreds of mosquitoes. Murine P. berghei or human-infective P. falciparum sporozoites produced in this way are 2-3-fold more infective with in vitro hepatocytes and can confer sterile protection when immunized intravenously with subsequent challenge using a mouse malaria model. Critically, we can also demonstrate for the first time 60-70% protection when the same parasites are administered via intramuscular (i.m.) route. In developing a process amenable to industrialisation and demonstrating efficacy by i.m. route these data represent a major advancement in capacity to produce a whole parasite malaria vaccine at scale. One-Sentence SummaryA four-step process for isolating pure infective malaria parasite sporozoites at scale from homogenized whole mosquitoes, independent of manual dissection, is able to produce a whole parasite vaccine inoculum that confers sterilizing protection.

microbiology↗