Two-dimensional multiplexed assay for rapid and deep SARS-CoV-2 serology profiling and for machine learning prediction of neutralization capacity.

Antibody responses serve as the primary protection against SARS-CoV-2 infection through neutralization of viral entry into cells. We have developed a two-dimensional multiplex bead binding assay (2D-MBBA) that quantifies multiple antibody isotypes against multiple antigens from a single measurement. Here, we applied our assay to profile IgG, IgM and IgA levels against the spike antigen, its receptor-binding domain and natural and designed mutants. Machine learning algorithms trained on the 2D-MBBA data substantially improve the prediction of neutralization capacity against the authentic SARS-CoV-2 virus of serum samples of convalescent patients. The algorithms also helped identify a set of antibody isotype-antigen datasets that contributed to the prediction, which included those targeting regions outside the receptor-binding interface of the spike protein. We applied the assay to profile samples from vaccinated, immune-compromised patients, which revealed differences in the antibody profiles between convalescent and vaccinated samples. Our approach can rapidly provide deep antibody profiles and neutralization prediction from essentially a drop of blood without the need of BSL-3 access and provides insights into the nature of neutralizing antibodies. It may be further developed for evaluating neutralizing capacity for new variants and future pathogens.

Antibody isotype diversity against SARS-CoV-2 is associated with differential serum neutralization capacities.

Understanding antibody responses to SARS-CoV-2 is indispensable for the development of containment measures to overcome the current COVID-19 pandemic. Recent studies showed that serum from convalescent patients can display variable neutralization capacities. Still, it remains unclear whether there are specific signatures that can be used to predict neutralization. Here, we performed a detailed analysis of sera from a cohort of 101 recovered healthcare workers and we addressed their SARS-CoV-2 antibody response by ELISA against SARS-CoV-2 Spike receptor binding domain and nucleoprotein. Both ELISA methods detected sustained levels of serum IgG against both antigens. Yet, the majority of individuals from our cohort generated antibodies with low neutralization capacity and only 6% showed high neutralizing titers against both authentic SARS-CoV-2 virus and the Spike pseudotyped virus. Interestingly, higher neutralizing sera correlate with detection of -IgG, IgM and IgA antibodies against both antigens, while individuals with positive IgG alone showed poor neutralization response. These results suggest that having a broader repertoire of antibodies may contribute to more potent SARS-CoV-2 neutralization. Altogether, our work provides a cross sectional snapshot of the SARS-CoV-2 neutralizing antibody response in recovered healthcare workers and provides preliminary evidence that possessing multiple antibody isotypes can play an important role in predicting SARS-CoV-2 neutralization.

Multiplex bead binding assays using off-the-shelf components and common flow cytometers.

The ability to quantify protein-ligand interactions in an accurate and high-throughput manner is important in diverse areas of biology and medicine. Multiplex bead binding assays (MBBAs) are powerful methods that allow for simultaneous analysis of many protein-ligand interactions. Although there are a number of well-established MBBA platforms, there are few platforms suitable for research and development that offer rapid experimentation at low costs and without the need for specialized reagents or instruments dedicated for MBBA. Here, we describe a MBBA method that uses low-cost reagents and standard cytometers. The key innovation is the use of the essentially irreversible biotin-streptavidin interaction. We prepared a biotin-conjugated fluorescent dye and used it to produce streptavidin-coated magnetic beads that are labeled at distinct levels of fluorescence. We show the utility of our method in characterization of phage-displayed antibodies against multiple antigens of SARS-CoV-2, which substantially improves the throughput and dramatically reduces antigen consumption compared with conventional phage ELISA methods. This approach will make MBBAs more broadly accessible.

The ACE2-binding Interface of SARS-CoV-2 Spike Inherently Deflects Immune Recognition.

The COVID-19 pandemic remains a global threat, and host immunity remains the main mechanism of protection against the disease. The spike protein on the surface of SARS-CoV-2 is a major antigen and its engagement with human ACE2 receptor plays an essential role in viral entry into host cells. Consequently, antibodies targeting the ACE2-interacting surface (ACE2IS) located in the receptor-binding domain (RBD) of the spike protein can neutralize the virus. However, the understanding of immune responses to SARS-CoV-2 is still limited, and it is unclear how the virus protects this surface from recognition by antibodies. Here, we designed an RBD mutant that disrupts the ACE2IS and used it to characterize the prevalence of antibodies directed to the ACE2IS from convalescent sera of 94 COVID-19-positive patients. We found that only a small fraction of RBD-binding antibodies targeted the ACE2IS. To assess the immunogenicity of different parts of the spike protein, we performed in vitro antibody selection for the spike and the RBD proteins using both unbiased and biased selection strategies. Intriguingly, unbiased selection yielded antibodies that predominantly targeted regions outside the ACE2IS, whereas ACE2IS-binding antibodies were readily identified from biased selection designed to enrich such antibodies. Furthermore, antibodies from an unbiased selection using the RBD preferentially bound to the surfaces that are inaccessible in the context of whole spike protein. These results suggest that the ACE2IS has evolved less immunogenic than the other regions of the spike protein, which has important implications in the development of vaccines against SARS-CoV-2.

The ACE2-binding interface of SARS-CoV-2 Spike inherently deflects immune recognition.

The COVID-19 pandemic remains a global threat, and host immunity remains the main mechanism of protection against the disease. The spike protein on the surface of SARS-CoV-2 is a major antigen and its engagement with human ACE2 receptor plays an essential role in viral entry into host cells. Consequently, antibodies targeting the ACE2-interacting surface (ACE2IS) located in the receptor-binding domain (RBD) of the spike protein can neutralize the virus. However, the understanding of immune responses to SARS-CoV-2 is still limited, and it is unclear how the virus protects this surface from recognition by antibodies. Here, we designed an RBD mutant that disrupts the ACE2IS and used it to characterize the prevalence of antibodies directed to the ACE2IS from convalescent sera of 94 COVID19-positive patients. We found that only a small fraction of RBD-binding antibodies targeted the ACE2IS. To assess the immunogenicity of different parts of the spike protein, we performed in vitro antibody selection for the spike and the RBD proteins using both unbiased and biased selection strategies. Intriguingly, unbiased selection yielded antibodies that predominantly targeted regions outside the ACE2IS, whereas ACE2IS-binding antibodies were readily identified from biased selection designed to enrich such antibodies. Furthermore, antibodies from an unbiased selection using the RBD preferentially bound to the surfaces that are inaccessible in the context of whole spike protein. These results suggest that the ACE2IS has evolved less immunogenic than the other regions of the spike protein, which has important implications in the development of vaccines against SARS-CoV-2.

ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference.

ATP-dependent chromatin remodellers regulate access to genetic information by controlling nucleosome positions in vivo. However, the mechanism by which remodellers discriminate between different nucleosome substrates is poorly understood. Many chromatin remodelling proteins possess conserved protein domains that interact with nucleosomal features. Here we used a quantitative high-throughput approach, based on the use of a DNA-barcoded mononucleosome library, to profile the biochemical activity of human ISWI family remodellers in response to a diverse set of nucleosome modifications. We show that accessory (non-ATPase) subunits of ISWI remodellers can distinguish between differentially modified nucleosomes, directing remodelling activity towards specific nucleosome substrates according to their modification state. Unexpectedly, we show that the nucleosome acidic patch is necessary for maximum activity of all ISWI remodellers evaluated. This dependence also extends to CHD and SWI/SNF family remodellers, suggesting that the acidic patch may be generally required for chromatin remodelling. Critically, remodelling activity can be regulated by modifications neighbouring the acidic patch, signifying that it may act as a tunable interaction hotspot for ATP-dependent chromatin remodellers and, by extension, many other chromatin effectors that engage this region of the nucleosome surface.

Selective recognition of histone crotonylation by double PHD fingers of MOZ and DPF2.

Recognition of histone covalent modifications by 'reader' modules constitutes a major mechanism for epigenetic regulation. A recent upsurge of newly discovered histone lysine acylations, such as crotonylation (Kcr), butyrylation (Kbu), and propionylation (Kpr), greatly expands the coding potential of histone lysine modifications. Here we demonstrate that the histone acetylation-binding double PHD finger (DPF) domains of human MOZ (also known as KAT6A) and DPF2 (also known as BAF45d) accommodate a wide range of histone lysine acylations with the strongest preference for Kcr. Crystal structures of the DPF domain of MOZ in complex with H3K14cr, H3K14bu, and H3K14pr peptides reveal that these non-acetyl acylations are anchored in a hydrophobic 'dead-end' pocket with selectivity for crotonylation arising from intimate encapsulation and an amide-sensing hydrogen bonding network. Immunofluorescence and chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) showed that MOZ and H3K14cr colocalize in a DPF-dependent manner. Our studies call attention to a new regulatory mechanism centered on histone crotonylation readout by DPF family members.

Molecular Coupling of Histone Crotonylation and Active Transcription by AF9 YEATS Domain.

Recognition of histone covalent modifications by chromatin-binding protein modules ("readers") constitutes a major mechanism for epigenetic regulation, typified by bromodomains that bind acetyllysine. Non-acetyl histone lysine acylations (e.g., crotonylation, butyrylation, propionylation) have been recently identified, but readers that prefer these acylations have not been characterized. Here we report that the AF9 YEATS domain displays selectively higher binding affinity for crotonyllysine over acetyllysine. Structural studies revealed an extended aromatic sandwiching cage with crotonyl specificity arising from π-aromatic and hydrophobic interactions between crotonyl and aromatic rings. These features are conserved among the YEATS, but not the bromodomains. Using a cell-based model, we showed that AF9 co-localizes with crotonylated histone H3 and positively regulates gene expression in a YEATS domain-dependent manner. Our studies define the evolutionarily conserved YEATS domain as a family of crotonyllysine readers and specifically demonstrate that the YEATS domain of AF9 directly links histone crotonylation to active transcription.

Influence of global fluorination on chloramphenicol acetyltransferase activity and stability.

Varied levels of fluorinated amino acid have been introduced biosynthetically to test the functional limits of global substitution on enzymatic activity and stability. Replacement of all the leucine (LEU) residues in the enzyme chloramphenicol acetyltransferase (CAT) with the analog, 5',5',5'-trifluoroleucine (TFL), results in the maintenance of enzymatic activity under ambient temperatures as well as an enhancement in secondary structure but loss in stability against heat and denaturants or organic co-solvents. Although catalytic activity of the fully substituted CAT is preserved under standard reaction conditions compared to the wild-type enzyme both in vitro and in vivo, as the incorporation levels increase, a concomitant reduction in thermostability and chemostability is observed. Circular dichroism (CD) studies reveal that although fluorination greatly improves the secondary structure of CAT, a large structural destabilization upon increased levels of TFL incorporation occurs at elevated temperatures. These data suggest that enhanced secondary structure afforded by TFL incorporation does not necessarily lead to an improvement in stability.