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Measuring the entropic properties of polymers such as proteins is critical to accurate prediction of their functional properties. However, the measurement of configurational entropy is possible only by low throughput techniques such as calorimetry, NMR and CD spectroscopy. Moreover, to our knowledge no system exists that allows molecular selection/enrichment based on the molecules configurational entropy. We tested the ability of the scalable tripartite GFP system to offer fine resolution of differences in configurational entropy in molecules and to isolate molecules based on their configurational entropy. The system was able to both finely resolve molecules with different configurational entropies, as well as capture them for isolation. We were able to tune the sensitivity of the system by using different mutations of the protein components. Lastly, we were able to apply the system to polypeptoid molecules and posit that the system may be applied to any other hydrophilic polymer of up to 10^3 repeating units.

Vaccines based on proteins and peptides may be safer and more broad-spectrum than other approaches Physicochemical property consensus (PCPcon) alphavirus antigens from the B-domain of the E2 envelope protein were designed and synthesized recombinantly. Those based on individual species (eastern or Venezuelan equine encephalitis (EEEVcon, VEEVcon), or chikungunya (CHIKVcon) viruses generated species-specific antibodies. Peptides designed to surface exposed areas of the E2-A-domain were added to the inocula to provide neutralizing antibodies against CHIKV. EVCcon, based on the three different alphavirus species, combined with E2-A-domain peptides from AllAV, a PCPcon of 24 diverse alphavirus, generated broad spectrum antibodies. The abs in the sera bound and neutralized diverse alphaviruses with less than 35% amino acid identity to each other. These included VEEV and its relative Mucambo virus, EEEV and the related Madariaga virus, and CHIKV strain 181/25. Further understanding of the role of coordinated mutations in the envelope proteins may yield a single, protein and peptide vaccine against all alphaviruses.

Vaccines based on proteins and peptides may be safer and if calculated based on many sequences, more broad-spectrum than those designed based on single strains. Physicochemical Property Consensus (PCPsubcon/sub) alphavirus (AV) antigens from the B-domain of the E2 envelope protein were designed, synthesized recombinantly and shown to be immunogenic (i.e. sera after inoculation detected the antigen in dotspots and ELISA). Antibodies in sera after inoculation with B-region antigens based on individual AV species (eastern or Venezuelan equine encephalitis (EEEVsubcon/sub, VEEVsubcon/sub), or chikungunya (CHIKVsubcon/sub) bound only their cognate protein, while those designed against multiple species (Mosaiksubcon/suband EVCsubcon/sub) recognized all three serotype specific antigens. The VEEVsubcon/suband EEEVsubcon/subsera only showed antiviral activity against their related strains (in plaque reduction neutralization assays (PRNTsub50/80/sub). Peptides designed to surface exposed areas of the E2-A-domain of CHIKVsubcon/subwere added to CHIKVsubcon/subinocula to provide anti-CHIKV antibodies. EVCsubcon/sub, based on three different alphavirus species, combined with E2-A-domain peptides from AllAVsubcon/sub, a PCPcon of 24 diverse AV, generated broad spectrum, antiviral antibodies against VEEV, EEEV and CHIKV, AV with less than 35% amino acid identity to each other (gt;65% diversity). This is a promising start to a molecularly defined vaccine against all AV. Further study with these antigens can illuminate what areas are most important for a robust immune response, resistant to mutations in rapidly evolving viruses. The validated computational methods can also be used to design broad spectrum antigens against many other pathogen families.

There is an urgent need for inexpensive, rapid and specific antigen-based assays to test for vaccine efficacy and detect infection with SARS-CoV-2 and its variants. We have identified a small, synthetic protein (JS7), representing a region of maximum variability within the receptor binding domain (RBD), which binds antibodies in sera from nine patients with PCR-verified COVID-19 of varying severity. Antibodies binding to either JS7 or the SARS-CoV-2 recombinant RBD, as well as those that disrupt binding between a fragment of the ACE2 receptor and the RBD, are proportional to disease severity and clinical outcome. Binding to JS7 was inhibited by linear peptides from the RBD interface with ACE2. Variants of JS7, such as E484 K or N501Y, can be quickly synthesized in a pure form in large quantities by automated methods. JS7 and related synthetic antigens can provide a basis for specific diagnostics for SARS-CoV-2 infections.