Nileena Velappan

As emerging pathogens and antimicrobial resistance are increasing in prevalence, pathogen-agnostic, broad-spectrum therapeutics are urgently needed to address growing threats to public health. Here, we report a novel immune response-enhancing biomaterial that targets dendritic cells to activate innate immunity and strengthen the host defense to any potential pathogen. This biomaterial with therapeutic potential consists of a previously demonstrated dendritic cell-binding peptide (DC) conjugated to nanofibers formed by the self-assembling peptide, Q11. Antibody-based binding assays and computational modeling demonstrated that incorporation of DC-Q11 at 5% relative to unconjugated Q11 optimizes the surface presentation and allowed maximum accessibility of the DC peptide on these nanofibers for biological applications. DC-functionalized Q11 nanofibers promoted dendritic cell maturation, as evidenced by an increase in cell surface receptor expression profile, elevated proinflammatory cytokine production, and a decrease in anti-inflammatory cytokines. Further in vivo evaluation of this material construct could provide a more comprehensive understanding of its potential as a broad-spectrum therapeutic agent. Collectively, utilization of supramolecular materials platform, antibody engineering, and cellular assay techniques described herein might facilitate future research toward the development of broad-spectrum immunotherapeutic applications.

Proteins are the most common targets for antibody discovery and vaccine development, but their sequence variability can limit the breadth of resulting antigens. Lipids represent an alternative class of antigens due to their structural conservation and roles in host–pathogen interactions. Here, we describe the development and optimization of an in vitro antibody selection workflow using lipid-containing nanodiscs as antigen presentation platforms to enable phage and yeast display selections under conditions adapted for these non-protein targets. Lipopolysaccharide (LPS) nanodiscs were first used as a model system to evaluate selection strategies, including competitive and subtractive approaches to reduce non-specific binders, yielding peptide and single-chain variable fragment (scFv) binders that were affinity matured to improve binding signals. The same approach was subsequently used to select scFv antibodies that recognize lipid nanodiscs prepared from Yersinia pestis membrane lipid extracts. These antibodies show binding to lipid nanodiscs derived from Y. pestis, with evidence of selectivity relative to control nanodiscs. Overall, this work establishes a workflow for antibody selection against lipid-containing nanodisc antigens and highlights practical considerations associated with these targets. The approach may be useful for generating affinity reagents to membrane-associated lipids, although further characterization is required to define antigen specificity and functional activity.

Introduction: Evolution or emergence of a new viral variant is a significant public health concern. Alphaviruses, such as Venezuelan equine encephalitis virus (VEEV), are mosquito-borne viruses which are becoming more prevalent due to expansion of vector habitats. Despite this, there are currently no antiviral therapies or FDA-approved vaccines available to treat or prevent VEEV infection. The increased prevalence of such viruses provides opportunities for novel variants to evolve. Key therapeutic molecules that could be developed against viral pathogens are recombinant antibodies or antibody fragments, such as the variable heavy domain of heavy chain antibodies (V _H Hs). Methods: In vitro selections offer a promising pathway for identification of therapeutic antibodies, here we explored isolation of V _H Hs using phage and yeast display methodology with three antigen formats 1) recombinant E2, 2) linear peptides of E2, selected based on molecular dynamics analysis, and 3) UV inactivated virus. Results: Here we report four novel “human” V _H Hs which bind to the VEEV E2 protein selected using different strategies that include both computational and biochemical design of suitable antigens and whole virus selections. These V _H Hs have distinct complementarity-determining regions (CDRs). Multiple VHHs bind to the VEEV viral particles in ELISAs, and we report the peptide epitope recognized by these V _H Hs. Discussion: Though non-neutralizing, these V _H Hs bind to and sequester VEEV viral particles preventing infection, demonstrating the potential of these V _H Hs to perform viral “sponging” which represents a novel therapeutic approach. The selection strategies we report may have applications to further antibody developments against other viruses.

Evolution or emergence of a new viral variant is a significant public health concern. Alphaviruses, such as Venezuelan equine encephalitis virus (VEEV), are mosquito-borne viruses which are becoming more prevalent due to expansion of vector habitats. The increased prevalence of such viruses provides opportunities for novel variants to evolve. Key therapeutic molecules that could be developed against viral pathogens are recombinant antibodies or antibody fragments, such as the variable heavy domain of heavy chain antibodies (V _H Hs). These proteins can neutralize or sequester viral particles, preventing or reducing infection. However, due to the evolution of viruses, there is a need to isolate new antibodies and direct their binding to particular epitopes on the virus. In vitro selections offer a promising pathway for the selection of therapeutic antibodies, but as we demonstrate, the choice of a target for these selections is key to obtaining the desired viral binding characteristics. Here we report four novel “human” V _H Hs which bind to the VEEV E2 protein selected using different strategies that include both computational and biochemical design of suitable antigens and whole virus selections. These V _H Hs have distinct complementarity-determining regions (CDRs). Multiple V _H Hs bind to the VEEV viral particles in ELISAs, and we report the peptide epitope recognized by these V _H Hs. Though non-neutralizing, when immobilized, these V _H Hs bind to and sequester VEEV viral particles preventing infection, demonstrating the potential of these V _H Hs to perform viral “sponging”. The selection strategies we report may have applications to further antibody developments against other viruses. Significance/Importance: Alphaviruses, and in particular Venezuelan equine encephalitis virus (VEEV), are recognized for their ability to cause severe disease and for their potential to be used as a biothreat. Despite this, there are currently no antiviral therapies or FDA-approved vaccines available to treat or prevent VEEV infection. This study reports on a novel antibody selection pipeline to produce antibody fragments against VEEV. Antibodies produced via this method showed strong affinity and high specificity to the VEEV E2 glycoprotein in multiple conformations. Additionally, while not neutralizing, the antibody fragments described were shown to be effective as “viral sponges”, having the ability to bind, sequester, and remove VEEV virions from solution, which represents a novel therapeutic approach.

The PDBBind database has been widely utilized for the computational prediction of protein–protein binding affinities. While the accuracy of the PDBBind-curated equilibrium dissociation constants (KD) has been reported for the protein–ligand subset of the PDBBind database, the curation accuracy has not been reported for the protein–protein subset. Here, we present a detailed manual analysis for the subset of PDBBind records with PubMed Central Open Access primary publications and find that ~19% of these records had KD values that were not supported by their primary publications. The impact of these putative curation errors on the machine learning-based prediction of KD from experimental protein–protein 3D structures was evaluated and correcting the curation errors improved the Pearson correlation coefficient between measured and random forest-predicted log10(KD) values by ~8 percentage points. This finding underscores the importance of dataset accuracy for computational modelling and highlights the need for more stringent curation processes when extracting information from the scientific literature.

Background: Introducing unnatural base pairs into a natural, double-stranded DNA construct is a powerful tool within synthetic biology. Accordingly, the ability to detect these unnatural base pairs has many applications, including the study and detection of semisynthetic organisms. Objective and Methods: The work described here aimed to select human antibodies for the specific recognition of Hirao’s base pair dDs–dPn in various natural DNA contexts by using a combination of phage and yeast display technologies. Results: We selected a total of six antibodies in yeast-displayed scFv format, and further tested three of these antibodies in soluble form as minibodies and IgGs. We also describe an assay that can be used to detect plasmids containing dDs–dPn pair. Conclusions: Our antibodies did not afford the desired specificity or sensitivity for detection of a single unnatural base pair among thousands of natural. However, our data indicate not only that such detection is possible but also that these antibodies may be candidates for further affinity and specificity maturation.

Yersinia pestis, the causative agent of plague and a biological threat agent, presents an urgent need for novel medical countermeasures due to documented cases of naturally acquired antibiotic resistance and potential person-to-person spread during a pneumonic infection. Immunotherapy has been proposed as a way to circumvent current and future antibiotic resistance. Here, we describe the development and characterization of two affinity matured human antibodies (αF1Ig AM2 and αF1Ig AM8) that promote survival of mice after exposure to aerosolized Y. pestis. We share details of the error prone PCR and yeast display technology-based affinity maturation process that we used. The resultant matured antibodies have nanomolar affinity for Y. pestis F1 antigen, are produced in high yield, and are resilient to 37°C stress for up to 6 months. Importantly, in vitro assays using a murine macrophage cell line demonstrated that αF1Ig AM2 and αF1Ig AM8 are opsonic. Even more importantly, in vivo studies using pneumonic plague mouse models showed that 100% of the mice receiving 500 μg of IgGs αF1Ig AM2 and αF1Ig AM8 survived lethal challenge with aerosolized Y. pestis CO92. Combined, these results provide evidence of the quality and robustness of αF1Ig AM2 and αF1Ig AM8 and support their development as potential medical countermeasures against plague.

Antibodies are important reagents for research, diagnostics, and therapeutics. Many examples of chimeric proteins combining the specific target recognition of antibodies with complementing functionalities such as fluorescence, toxicity or enzymatic activity have been described. However, antibodies selected solely on the basis of their binding specificities are not necessarily ideal candidates for the construction of chimeras. Here, we describe a high throughput method based on yeast display to directly select antibodies most suitable for conversion to fluorescent chimera. A library of scFv binders was converted to a fluorescent chimeric form, by cloning thermal green protein into the linker between VH and VL, and directly selecting for both binding and fluorescent functionality. This allowed us to directly identify antibodies functional in the single chain TGP format, that manifest higher protein expression, easier protein purification, and one-step binding assays.

Here, we describe the isolation of 18 unique anti SARS-CoV-2 human single-chain antibodies from an antibody library derived from healthy donors. The selection used a combination of phage and yeast display technologies and included counter-selection strategies meant to direct the selection of the receptor-binding motif (RBM) of SARS-CoV-2 spike protein’s receptor binding domain (RBD2). Selected antibodies were characterized in various formats including IgG, using flow cytometry, ELISA, high throughput SPR, and fluorescence microscopy. We report antibodies’ RBD2 recognition specificity, binding affinity, and epitope diversity, as well as ability to block RBD2 binding to the human receptor angiotensin-converting enzyme 2 (ACE2) and to neutralize authentic SARS-CoV-2 virus infection in vitro. We present evidence supporting that: 1) most of our antibodies (16 out of 18) selectively recognize RBD2; 2) the best performing 8 antibodies target eight different epitopes of RBD2; 3) one of the pairs tested in sandwich assays detects RBD2 with sub-picomolar sensitivity; and 4) two antibody pairs inhibit SARS-CoV-2 infection at low nanomolar half neutralization titers. Based on these results, we conclude that our antibodies have high potential for therapeutic and diagnostic applications. Importantly, our results indicate that readily available non immune (naïve) antibody libraries obtained from healthy donors can be used to select high-quality monoclonal antibodies, bypassing the need for blood of infected patients, and offering a widely accessible and low-cost alternative to more sophisticated and expensive antibody selection approaches (e.g. single B cell analysis and natural evolution in humanized mice). Graphical Abstract

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