There is an urgent need for the ability to rapidly develop effective countermeasures for emerging biological threats, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a generalized computational design strategy to rapidly engineer de novo proteins that precisely recapitulate the protein surface targeted by biological agents, like viruses, to gain entry into cells. The designed proteins act as decoys that block cellular entry and aim to be resilient to viral mutational escape. Using our novel platform, in less than ten weeks, we engineered, validated, and optimized de novo protein decoys of human angiotensin-converting enzyme 2 (hACE2), the membrane-associated protein that SARS-CoV-2 exploits to infect cells. Our optimized designs are hyperstable de novo proteins (∼18-37 kDa), have high affinity for the SARS-CoV-2 receptor binding domain (RBD) and can potently inhibit the virus infection and replication in vitro. Future refinements to our strategy can enable the rapid development of other therapeutic de novo protein decoys, not limited to neutralizing viruses, but to combat any agent that explicitly interacts with cell surface proteins to cause disease.

We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo human angiotensin-converting enzyme 2 (hACE2) decoys to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The best monovalent decoy, CTC-445.2, bound with low nanomolar affinity and high specificity to the receptor-binding domain (RBD) of the spike protein. Cryo-electron microscopy (cryo-EM) showed that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, showed ~10-fold improvement in binding. CTC-445.2d potently neutralized SARS-CoV-2 infection of cells in vitro, and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge.

Discovering potent human monoclonal antibodies (mAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on sporozoites (SPZ) and elucidating their mechanisms of neutralization will facilitate translation for passive prophylaxis and aid next-generation vaccine development. Here, we isolated a neutralizing human mAb, L9 that preferentially bound NVDP minor repeats of PfCSP with high affinity while cross-reacting with NANP major repeats. L9 was more potent than six published neutralizing human PfCSP mAbs at mediating protection against mosquito bite challenge in mice. Isothermal titration calorimetry and multiphoton microscopy showed that L9 and the other most protective mAbs bound PfCSP with two binding events and mediated protection by killing SPZ in the liver and by preventing their egress from sinusoids and traversal of hepatocytes. This study defines the subdominant PfCSP minor repeats as neutralizing epitopes, identifies an in vitro biophysical correlate of SPZ neutralization, and demonstrates that the liver is an important site for antibodies to prevent malaria.

Many viral surface glycoproteins and cell surface receptors are homo-oligomers, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.

Influenza is a ubiquitous and recurring infection that results in approximately 500 000 deaths globally each year. Commercially available rapid diagnostic tests are based upon detection of the influenza nucleoprotein, which are limited in that they are unable to differentiate by species and require an additional viral lysis step. Sample preprocessing can be minimized or eliminated by targeting the intact influenza virus, thereby reducing assay complexity and leveraging the large number of hemagglutinin proteins on the surface of each virus. Here, we report the development of a paper-based influenza assay that targets the hemagglutinin protein; the assay employs a combination of antibodies and novel computationally designed, recombinant affinity proteins as the capture and detection agents. This system leverages the customizability of recombinant protein design to target the conserved receptor-binding pocket of the hemagglutinin protein and to match the trimeric nature of hemagglutinin for improved avidity. Using this assay, we demonstrate the first instance of intact influenza virus detection using a combination of antibody and affinity proteins within a porous network. The recombinant head region binder based assays yield superior analytical sensitivity as compared to the antibody based assay, with lower limits of detection of 3.54 × 107 and 1.34 × 107 CEID50/mL for the mixed and all binder stacks, respectively. Not only does this work describe the development of a novel influenza assay, it also demonstrates the power of recombinant affinity proteins for use in rapid diagnostic assays.

Broadly neutralizing antibodies targeting a highly conserved region in the hemagglutinin (HA) stem protect against influenza infection. Here, we investigate the protective efficacy of a protein (HB36.6) computationally designed to bind with high affinity to the same region in the HA stem. We show that intranasal delivery of HB36.6 affords protection in mice lethally challenged with diverse strains of influenza independent of Fc-mediated effector functions or a host antiviral immune response. This designed protein prevents infection when given as a single dose of 6.0 mg/kg up to 48 hours before viral challenge and significantly reduces disease when administered as a daily therapeutic after challenge. A single dose of 10.0 mg/kg HB36.6 administered 1-day post-challenge resulted in substantially better protection than 10 doses of oseltamivir administered twice daily for 5 days. Thus, binding of HB36.6 to the influenza HA stem region alone, independent of a host response, is sufficient to reduce viral infection and replication in vivo. These studies demonstrate the potential of computationally designed binding proteins as a new class of antivirals for influenza.

While the cost of DNA sequencing has dropped by five orders of magnitude in the past decade, DNA synthesis remains expensive for many applications. Although DNA microarrays have decreased the cost of oligonucleotide synthesis, the use of array-synthesized oligos in practice is limited by short synthesis lengths, high synthesis error rates, low yield and the challenges of assembling long constructs from complex pools. Toward addressing these issues, we developed a protocol for multiplex pairwise assembly of oligos from array-synthesized oligonucleotide pools. To evaluate the method, we attempted to assemble up to 2271 targets ranging in length from 192-252 bases using pairs of array-synthesized oligos. Within sets of complexity ranging from 131-250 targets, we observed error-free assemblies for 90.5% of all targets. When all 2271 targets were assembled in one reaction, we observed error-free constructs for 70.6%. While the assembly method intrinsically increased accuracy to a small degree, we further increased accuracy by using a high throughput 'Dial-Out PCR' protocol, which combines Illumina sequencing with an in-house set of unique PCR tags to selectively amplify perfect assemblies from complex synthetic pools. This approach has broad applicability to DNA assembly and high-throughput functional screens.

Computational protein design is used to create a protein that binds the steroid digoxigenin (DIG) with high affinity and selectivity; the computational design methods described here should help to enable the development of a new generation of small molecule receptors for synthetic biology, diagnostics and therapeutics. Current approaches to the design of ligand-binding proteins for medical or biotechnological applications involve raising antibodies against a target antigen in immunized animals and/or performing directed evolution experiments on proteins with pre-existing, low affinity for the desired ligand. This paper describes a general method for the computational design of small molecule binding proteins that the authors use to design high-affinity and highly selective binding sites for the steroid digoxigenin, a cardiac glycoside used to treat heart disease. Using this method it should be possible to rapidly create small molecule receptors for synthetic biology applications, therapeutic scavengers for toxic compounds in vivo and robust ligand-binding domains for diagnostic devices. The ability to design proteins with high affinity and selectivity for any given small molecule is a rigorous test of our understanding of the physiochemical principles that govern molecular recognition. Attempts to rationally design ligand-binding proteins have met with little success, however, and the computational design of protein–small-molecule interfaces remains an unsolved problem1. Current approaches for designing ligand-binding proteins for medical2 and biotechnological uses rely on raising antibodies against a target antigen in immunized animals3,4 and/or performing laboratory-directed evolution of proteins with an existing low affinity for the desired ligand5,6,7, neither of which allows complete control over the interactions involved in binding. Here we describe a general computational method for designing pre-organized and shape complementary small-molecule-binding sites, and use it to generate protein binders to the steroid digoxigenin (DIG). Of seventeen experimentally characterized designs, two bind DIG; the model of the higher affinity binder has the most energetically favourable and pre-organized interface in the design set. A comprehensive binding-fitness landscape of this design, generated by library selections and deep sequencing, was used to optimize its binding affinity to a picomolar level, and X-ray co-crystal structures of two variants show atomic-level agreement with the corresponding computational models. The optimized binder is selective for DIG over the related steroids digitoxigenin, progesterone and β-oestradiol, and this steroid binding preference can be reprogrammed by manipulation of explicitly designed hydrogen-bonding interactions. The computational design method presented here should enable the development of a new generation of biosensors, therapeutics and diagnostics.

An apparatus for concentrating macromolecules and removing macro molecules from nonionic solutes electrophoretically has been developed. Typical applications and techniques are outlined. Concentrations from 24- to 50-fold were achieved in a short period of time. The nondisruptive nature and versatility of the system is shown by the concentration of bovine serum albumin and three different enzymes.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTProteins of TuberculinFlorence B. Seibert and J. Walter NelsonCite this: J. Am. Chem. Soc. 1943, 65, 2, 272–278Publication Date (Print):February 1, 1943Publication History Published online1 May 2002Published inissue 1 February 1943https://doi.org/10.1021/ja01242a040RIGHTS & PERMISSIONSArticle Views30Altmetric-Citations16LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

An intensive study has been made in recent years of tuberculous sera, with the objective of detecting the presence of antigen, antibody or any substance which may be characteristic of the disease. The primary approach has been based upon observation of the electrophoretic diagrams of sera by means of the Tiselius technic.It was shown1 that during the development of tuberculosis in the rabbit the beta globulin fraction increased in proportion to the extent of the disease. In the human being, on the other hand, the consistent change1 was a progressive increase in the gamma globulin. Increases in alpha and beta globulin fractions also frequently occurred but not with the same degree of regularity as was the case with the gamma globulin.An explanation of the cause of the rise in any of these components would be of value in the understanding of the disease. The possibility of the presence of antibodies immediately presents itself in connection with the rise in the gamma globulin, especially in view of the fact...

Antibody to tuberculin protein has been identified in serum as a component with an electrophoretic mobility close to α-globulin. This component increases in sera following sensitization to the protein, and is removed in part with the specific precipitate produced by adding antigen to the immune serum.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThe Preparation of OrthoestersS. M. McElvain and J. Walter NelsonCite this: J. Am. Chem. Soc. 1942, 64, 8, 1825–1827Publication Date (Print):August 1, 1942Publication History Published online1 May 2002Published inissue 1 August 1942https://pubs.acs.org/doi/10.1021/ja01260a024https://doi.org/10.1021/ja01260a024research-articleACS PublicationsRequest reuse permissionsArticle Views2009Altmetric-Citations90LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts