Titers do not depend on the number of variant antigens encountered in each B cell—FDC interaction.

(DOCX)</p

Mean panel titers as a function of the number of mutations in panel antigens.

Mutations occur either in any variable residues (All variable residues) or in the same residues that are mutated in the sequences 1–6 (Seq1-6 mutated residues). Mutated residues take on values of -4. B cells encounter either all antigens at a time (All-antigen) or one antigen at a time (One-antigen) on the FDC. (TIF)</p

Residues making up the conserved region in the spike S2 domain.

Numbering is based on the SARS-CoV-2 spike protein (PDB ID: 6VXX). (DOCX)</p

Mean panel titers as a function of the number of mutations in panel antigens.

Mutations occur either in any variable residues (All variable residues) or in the same residues that are mutated in the sequences 1–6 (Seq1-6 mutated residues). Panel titers are calculated against panels of 100 antigens and 1000 antigens. Mutated residues take on a value of -4. B cells are assumed to encounter all antigens at a time on the FDC. (TIF)</p

Spike protein structures colored by conservation fraction with varying weights of the structural conservation fraction (F_struc) and biochemical conservation fraction (F_bio) using SARS-CoV-2 as a reference.

Green residues have conservation fractions above 0.8, blue residues have conservation fractions below 0.8 and are not in the RBD, and red residues have conservation fractions below 0.8 and are in the RBD. (TIF)</p

Collection and processing of GISAID SARS-CoV-2 sequences.

(DOCX)</p

Choice of number of escape mutations considered.

(DOCX)</p

Simulations of Pure Ceramide and Ternary Lipid Mixtures as Simple Interior <i>Stratum Corneum</i> Models

The barrier function of the <i>stratum corneum</i> (SC) is intimately related to the structure of the lipid matrix, which is composed of ceramides (Cer), cholesterol (Chol), and free fatty acid (FFA). In this study, the all-atom CHARMM36 (C36) force field is used to simulate bilayers of <i>N</i>-palmitoylsphingosine (Cer16), <i>N</i>-lignoceroylsphingosine (Cer24), Chol, and lignoceric acid (LA) as simple models of the SC. Equimolar mixtures of Cer, Chol, and LA are replicated from experiment for comparison and validation of the C36 force field, and the effects of lipid diversity and temperature are studied. The presence of Chol and LA have effects on nearly all membrane properties including surface area per lipid, area compressibility moduli, chain order, Chol tilt, bilayer thickness, interdigitation, hydrogen bonding, and lipid clustering, while temperature has a more moderate effect. In systems containing Cer16, there is a profound difference in interdigitation between pure Cer and mixed systems, while systems containing Cer24 are relatively unaffected. Increasing temperature has the potential to shift hydrogen bonding pairs rather than uniformly decrease bonding, which can lead to greater Cer–Cer bonding at higher temperatures. Comparison with deuterium order parameter experiments demonstrates good agreement, which supports further use of this class of lipids and fatty acids for development of more complex SC models

Molecular Dynamics Simulations of Ceramide and Ceramide-Phosphatidylcholine Bilayers

Recent studies in lipid raft formation and stratum corneum permeability have focused on the role of ceramides (CER). In this study, we use the all-atom CHARMM36 (C36) force field to simulate bilayers using <i>N</i>-palmitoylsphingosine (CER16) or α-hydroxy-<i>N</i>-stearoyl phytosphingosine (CER­[AP]) in 1,2-dimyristoyl-<i>sn</i>-glycero-3-phosphocholine (DMPC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), which serve as general membrane models. Conditions are replicated from experimental studies for comparison purposes, and concentration (<i>X</i>_CER) is varied to probe the effect of CER on these systems. Comparisons with experiment based on deuterium order parameters and bilayer thickness demonstrate good agreement, thus supporting further use of the C36 force field. CER concentration is shown to have a profound effect on nearly all membrane properties including surface area per lipid, chain order and tilt, area compressibility moduli, bilayer thickness, hydrogen bonding, and lipid clustering. Hydrogen bonding in particular can significantly affect other membrane properties and can even encourage transition to a gel phase. Despite CER’s tendency to condense the membrane, an expansion of CER lipids with increasing <i>X</i>_CER is possible depending on how the balance between various hydrogen-bond pairs and lipid clustering is perturbed. Based on gel phase transitions, support is given for phytosphingosine’s role as a hydrogen-bond bridge between sphingosine ordered domains in the stratum corneum