The clinical outcome of SARS-CoV-2 infections, which can range from asymptomatic to lethal, is crucially shaped by the concentration of antiviral antibodies and by their affinity to their targets. However, the affinity of polyclonal antibody responses in plasma is difficult to measure. Here we used microfluidic antibody affinity profiling (MAAP) to determine the aggregate affinities and concentrations of anti–SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 19 of which were healthy donors, 20 displayed mild symptoms, and 3 were critically ill. We found that dissociation constants, Kd, of anti–receptor-binding domain antibodies spanned 2.5 orders of magnitude from sub-nanomolar to 43 nM. Using MAAP we found that antibodies of seropositive individuals induced the dissociation of pre-formed spike-ACE2 receptor complexes, which indicates that MAAP can be adapted as a complementary receptor competition assay. By comparison with cytopathic effect–based neutralisation assays, we show that MAAP can reliably predict the cellular neutralisation ability of sera, which may be an important consideration when selecting the most effective samples for therapeutic plasmapheresis and tracking the success of vaccinations.

The formation of amyloid fibrils and oligomers is a hallmark of several neurodegenerative disorders, including Alzheimer’s disease (AD), and contributes to the disease pathway. To progress our understanding of these diseases at a molecular level, it is crucial to determine the mechanisms and rates of amyloid formation and replication. In the context of AD, the self-replication of aggregates of the Aβ42 peptide by secondary nucleation, leading to the formation of new aggregates on the surfaces of existing ones, is a major source of both new fibrils and smaller toxic oligomeric species. However, the core mechanistic determinants, including the presence of intermediates, as well as the role of heterogeneities in the fibril population, are challenging to determine from bulk aggregation measurements. Here, we obtain such information by monitoring directly the time evolution of individual fibrils by TIRF microscopy. Crucially, essentially all aggregates have the ability to self-replicate via secondary nucleation, and the amplification of the aggregate concentration cannot be explained by a small fraction of “superspreader” fibrils. We observe that secondary nucleation is a catalytic multistep process involving the attachment of soluble species to the fibril surface, followed by conversion/detachment to yield a new fibril in solution. Furthermore, we find that fibrils formed by secondary nucleation resemble the parent fibril population. This detailed level of mechanistic insights into aggregate self-replication is key in the rational design of potential inhibitors of this process.

The formation of amyloid fibrils from soluble peptide is a hallmark of many neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Characterization of the microscopic reaction processes that underlie these phenomena have yielded insights into the progression of such diseases and may inform rational approaches for the design of drugs to halt them. Experimental evidence suggests that most of these reaction processes are intrinsically catalytic in nature and may display enzymelike saturation effects under conditions typical of biological systems, yet a unified modeling framework accounting for these saturation effects is still lacking. In this paper, we therefore present a universal kinetic model for biofilament formation in which every fundamental process in the reaction network can be catalytic. The single closed-form expression derived is capable of describing with high accuracy a wide range of mechanisms of biofilament formation and providing the first integrated rate law of a system in which multiple reaction processes are saturated. Moreover, its unprecedented mathematical simplicity permits us to very clearly interpret the effects of increasing saturation on the overall kinetics. The effectiveness of the model is illustrated by fitting it to the data of in vitro Aβ40 aggregation. Remarkably, we find that primary nucleation becomes saturated, demonstrating that it must be heterogeneous, occurring at interfaces and not in solution.

The formation of ordered protein and peptide assemblies is a phenomenon related to a wide range of human diseases. However, the mechanism of assembly at the molecular level remains largely unknown. Minimal models enable the exploration of the underlying interactions that are at the core of such self-assembly processes. In particular, the ability of phenylalanine, a single aromatic amino acid, to form an amyloid-like structure has challenged the previous dogma viewing a peptide backbone as a prerequisite for assembly. The driving forces controlling the nucleation and assembly in the absence of a peptide backbone remain to be identified. Here, aiming to unravel these forces, we explored the kinetics and thermodynamics of three phenylalanine-containing molecules during their assembly process: the amino acid phenylalanine, which accumulates in phenylketonuria patients, the diphenylalanine core-motif of the amyloid β peptide related to Alzheimer's disease, and the extended triphenylalanine peptide which forms a range of distinct nanostructures in vitro. We found that the aggregation propensity, regarding the critical monomer concentration, strongly increases with size, with triphenylalanine being the most aggregation-prone species under our experimental conditions. In the context of classical nucleation theory, this increase in aggregation propensity can be attributed to the larger free energy decrease upon aggregation of larger peptides and is not due to the presence/absence of a peptide bond per se. Taken together, this work provides insights into the aggregation processes of chemically simple systems and suggests that both backbone-containing peptides and backbone-lacking amino acids assemble through a similar mechanism, thus supporting the classification of amino acids in the continuum of amyloid-forming building blocks.

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