Th2 and Th17-associated immunopathology following SARS-CoV-2 breakthrough infection in Spike-vaccinated ACE2-humanized mice.

Vaccines have demonstrated remarkable effectiveness in protecting against COVID-19; however, concerns regarding vaccine-associated enhanced respiratory diseases (VAERD) following breakthrough infections have emerged. Spike protein subunit vaccines for SARS-CoV-2 induce VAERD in hamsters, where aluminum adjuvants promote a Th2-biased immune response, leading to increased type 2 pulmonary inflammation in animals with breakthrough infections. To gain a deeper understanding of the potential risks and the underlying mechanisms of VAERD, we immunized ACE2-humanized mice with SARS-CoV-2 Spike protein adjuvanted with aluminum and CpG-ODN. Subsequently, we exposed them to increasing doses of SARS-CoV-2 to establish a breakthrough infection. The vaccine elicited robust neutralizing antibody responses, reduced viral titers, and enhanced host survival. However, following a breakthrough infection, vaccinated animals exhibited severe pulmonary immunopathology, characterized by a significant perivascular infiltration of eosinophils and CD4+ T cells, along with increased expression of Th2/Th17 cytokines. Intracellular flow cytometric analysis revealed a systemic Th17 inflammatory response, particularly pronounced in the lungs. Our data demonstrate that aluminum/CpG adjuvants induce strong antibody and Th1-associated immunity against COVID-19 but also prime a robust Th2/Th17 inflammatory response, which may contribute to the rapid onset of T cell-mediated pulmonary immunopathology following a breakthrough infection. These findings underscore the necessity for further research to unravel the complexities of VAERD in COVID-19 and to enhance vaccine formulations for broad protection and maximum safety.

Comparative analysis of fusion tags used to functionalize recombinant antibodies.

Recombinant antibodies can be expressed as fusion constructs in combination with tags which simplify their engineering into reliable and homogeneous immunoreagents by allowing site-specific, 1:1 functionalization. Several tags and corresponding reagents for recombinant protein derivatization have been proposed but benchmarking surveys for the evaluation of their effect on the characteristics of recombinant antibodies have not been reported. In this work we evaluated the impact on expression yields, shelf-stability, thermostability and binding affinity of a set of C-terminal tags fused to the same anti-Her2 nanobody. Furthermore, we assessed the efficiency of the derivatization process. The constructs always bore a 6xHis tag plus either the controls (EGFP and C-tag) or CLIP, HALO, AviTag, the LEPTG sequence recognized by Sortase A (Sortase tag), or a free cysteine. The advantages and drawbacks of the different systems were analyzed and discussed.

Programmable polyproteams built using twin peptide superglues.

Programmed connection of amino acids or nucleotides into chains introduced a revolution in control of biological function. Reacting proteins together is more complex because of the number of reactive groups and delicate stability. Here we achieved sequence-programmed irreversible connection of protein units, forming polyprotein teams by sequential amidation and transamidation. SpyTag peptide is engineered to spontaneously form an isopeptide bond with SpyCatcher protein. By engineering the adhesin RrgA from Streptococcus pneumoniae, we developed the peptide SnoopTag, which formed a spontaneous isopeptide bond to its protein partner SnoopCatcher with >99% yield and no cross-reaction to SpyTag/SpyCatcher. Solid-phase attachment followed by sequential SpyTag or SnoopTag reaction between building-blocks enabled iterative extension. Linear, branched, and combinatorial polyproteins were synthesized, identifying optimal combinations of ligands against death receptors and growth factor receptors for cancer cell death signal activation. This simple and modular route to programmable "polyproteams" should enable exploration of a new area of biological space.

Whole-cell biopanning with a synthetic phage display library of nanobodies enabled the recovery of follicle-stimulating hormone receptor inhibitors.

Antibodies are essential reagents that are increasingly used in diagnostics and therapy. Their specificity and capacity to recognize their native antigen are critical characteristics for their in vivo application. Follicle-stimulating hormone receptor is a GPCR protein regulating ovarian follicular maturation and spermatogenesis. Recently, its potentiality as a cancer biomarker has been demonstrated but no antibody suitable for in vivo tumor targeting and treatment has been characterized so far. In this paper we describe the first successful attempt to recover recombinant antibodies against the FSHR and that: i) are directly panned from a pre-immune library using whole cells expressing the target receptor at their surface; ii) show inhibitory activity towards the FSH-induced cAMP accumulation; iii) do not share the same epitope with the natural binder FSH; iv) can be produced inexpensively as mono- or bivalent functional molecules in the bacterial cytoplasm. We expect that the proposed biopanning strategy will be profitable to identify useful functional antibodies for further members of the GPCR class.

SpyLigase peptide-peptide ligation polymerizes affibodies to enhance magnetic cancer cell capture.

Individual proteins can now often be modified with atomic precision, but there are still major obstacles to connecting proteins into larger assemblies. To direct protein assembly, ideally, peptide tags would be used, providing the minimal perturbation to protein function. However, binding to peptides is generally weak, so assemblies are unstable over time and disassemble with force or harsh conditions. We have recently developed an irreversible protein-peptide interaction (SpyTag/SpyCatcher), based on a protein domain from Streptococcus pyogenes, that locks itself together via spontaneous isopeptide bond formation. Here we develop irreversible peptide-peptide interaction, through redesign of this domain and genetic dissection into three parts: a protein domain termed SpyLigase, which now ligates two peptide tags to each other. All components expressed efficiently in Escherichia coli and peptide tags were reactive at the N terminus, at the C terminus, or at internal sites. Peptide-peptide ligation enabled covalent and site-specific polymerization of affibodies or antibodies against the tumor markers epidermal growth factor receptor (EGFR) and HER2. Magnetic capture of circulating tumor cells (CTCs) is one of the most promising approaches to improve cancer prognosis and management, but CTC capture is limited by inefficient recovery of cells expressing low levels of tumor antigen. SpyLigase-assembled protein polymers made possible the isolation of cancerous cells expressing lower levels of tumor antigen and should have general application in enhancing molecular capture.

SpyAvidin hubs enable precise and ultrastable orthogonal nanoassembly.

The capture of biotin by streptavidin is an inspiration for supramolecular chemistry and a central tool for biological chemistry and nanotechnology, because of the rapid and exceptionally stable interaction. However, there is no robust orthogonal interaction to this hub, limiting the size and complexity of molecular assemblies that can be created. Here we combined traptavidin (a streptavidin variant maximizing biotin binding strength) with an orthogonal irreversible interaction. SpyTag is a peptide engineered to form a spontaneous isopeptide bond to its protein partner SpyCatcher. SpyTag or SpyCatcher was successfully fused to the C-terminus of Dead streptavidin subunits. We were able to generate chimeric tetramers with n (0 ≤ n ≤ 4) biotin binding sites and 4-n SpyTag or SpyCatcher binding sites. Chimeric SpyAvidin tetramers bound precise numbers of ligands fused to biotin or SpyTag/SpyCatcher. Mixing chimeric tetramers enabled assembly of SpyAvidin octamers (8 subunits) or eicosamers (20 subunits). We validated assemblies using electrophoresis and native mass spectrometry. Eicosameric SpyAvidin was used to cluster trimeric major histocompatibility complex (MHC) class I:ß2-microglobulin:peptide complexes, generating an assembly with up to 56 components. MHC eicosamers surpassed the conventional MHC tetramers in acting as a powerful stimulus to T cell signaling. Combining ultrastable noncovalent with irreversible covalent interaction, SpyAvidins enable a simple route to create robust nanoarchitectures.

Generation and Selection of Synthetic Human Antibody Libraries via Phage Display.

Synthetic antibody libraries enable the development of antibodies that can recognize virtually any antigen, with affinity and specificity profiles that are superior to those of natural antibodies. By using highly stable and optimized frameworks, synthetic antibody libraries can be rapidly generated by precisely designing synthetic DNA, allowing absolute control over the position and chemical diversity introduced while expanding the sequence space for antigen recognition. Here, we describe a detailed protocol for the generation of highly diverse synthetic antibody phage display libraries based on a single framework, with diversity genetically incorporated by using finely designed mutagenic oligonucleotides. This general method enables the facile construction of large antibody libraries with precisely tunable features, resulting in the rapid development of recombinant antibodies for virtually any antigen.

Beyond Natural Immune Repertoires: Synthetic Antibodies.

Synthetic antibody libraries, in which the antigen-binding sites are precisely designed, offer unparalleled precision in antibody engineering, exceeding the potential of natural immune repertoires and constituting a novel generation of research tools and therapeutics. Recent advances in artificial intelligence-driven technologies and their integration into synthetic antibody discovery campaigns hold the promise to further streamline and effectively develop antibodies. Here, we provide an overview of synthetic antibodies. Our associated protocol describes how to develop highly diverse and functional synthetic antibody phage display libraries.

Immune Epitopes of SARS-CoV-2 Spike Protein and Considerations for Universal Vaccine Development.

Despite the success of global vaccination programs in slowing the spread of COVID-19, these efforts have been hindered by the emergence of new SARS-CoV-2 strains capable of evading prior immunity. The mutation and evolution of SARS-CoV-2 have created a demand for persistent efforts in vaccine development. SARS-CoV-2 Spike protein has been the primary target for COVID-19 vaccine development, but it is also the hotspot of mutations directly involved in host susceptibility and virus immune evasion. Our ability to predict emerging mutants and select conserved epitopes is critical for the development of a broadly neutralizing therapy or a universal vaccine. In this article, we review the general paradigm of immune responses to COVID-19 vaccines, highlighting the immunological epitopes of Spike protein that are likely associated with eliciting protective immunity resulting from vaccination in humans. Specifically, we analyze the structural and evolutionary characteristics of the SARS-CoV-2 Spike protein related to immune activation and function via the TLRs, B cells, and T cells. We aim to provide a comprehensive analysis of immune epitopes of Spike protein, thereby contributing to the development of new strategies for broad neutralization or universal vaccination.

Phage-Displayed SH2 Domain Libraries: From Ultrasensitive Tyrosine Phosphoproteome Probes to Translational Research.

Tyrosine phosphorylation is a critical regulator of cell signaling. A large fraction of the tyrosine phosphoproteome, however, remains uncharacterized, largely due to a lack of robust and scalable methods. The Src homology 2 (SH2) domain, a structurally conserved protein domain present in many intracellular signal-transducing proteins, naturally binds phosphorylated tyrosine (pTyr) residues, providing an ideal scaffold for the development of sensitive pTyr probes. Its modest affinity, however, has greatly limited its application. Phage display is an in vitro technique used for identifying ligands for proteins and other macromolecules. Using this technique, researchers have been able to engineer SH2 domains to increase their affinity and customize their specificity. Indeed, highly diverse phage display libraries have enabled the engineering of SH2 domains as affinity-purification (AP) tools for proteomic analysis as well as probes for aberrant tyrosine signaling detection and rewiring, and represent a promising class of novel diagnostics and therapeutics. This review describes the unique structure-function characteristics of SH2 domains, highlights the fundamental contribution of phage display in the development of technologies for the dissection of the tyrosine phosphoproteome, and highlights prospective uses of SH2 domains in basic and translational research.

Ubiquitin Engineering for Interrogating the Ubiquitin-Proteasome System and Novel Therapeutic Strategies.

Protein turnover, a highly regulated process governed by the ubiquitin-proteasome system (UPS), is essential for maintaining cellular homeostasis. Dysregulation of the UPS has been implicated in various diseases, including viral infections and cancer, making the proteins in the UPS attractive targets for therapeutic intervention. However, the functional and structural redundancies of UPS enzymes present challenges in identifying precise drug targets and achieving target selectivity. Consequently, only 26S proteasome inhibitors have successfully advanced to clinical use thus far. To overcome these obstacles, engineered peptides and proteins, particularly engineered ubiquitin, have emerged as promising alternatives. In this review, we examine the impact of engineered ubiquitin on UPS and non-UPS proteins, as well as on viral enzymes. Furthermore, we explore their potential to guide the development of small molecules targeting novel surfaces, thereby expanding the range of druggable targets.

Engineering SH2 Domains with Tailored Specificities and Affinities.

The Src Homology 2 (SH2) domain is an emerging biotechnology with applications in basic science, drug discovery, and even diagnostics. The SH2 domains rapid uptake into different areas of research is a direct result of the wealth of information generated on its biochemical, biological, and biophysical role in mammalian cell biology. Functionally, the SH2 domain binds and recognizes specific phosphotyrosine (pTyr) residues in the cell to mediate protein-protein interactions (PPIs) that govern signal transduction networks. These signal transduction networks are responsible for relaying growth and stress state signals to the cell's nucleus, ultimately effecting a change in cell biology. Protein engineers have been able to increase the affinity of SH2 domains for pTyr while also tailoring the domains' specificity to unique amino acid sequences flanking the pTyr residue. In this way, it has been possible to develop unique SH2 variants for use in affinity-purification coupled to mass spectrometry (AP-MS) experiments, microscopy, or even synthetic biology. This chapter outlines methods to tailor the affinity and specificity of virtually any human SH2 domain using a combination of rational engineering and phage-display approaches.

Immune Epitopes of SARS-CoV-2 Spike Protein and Considerations for Universal Vaccine Development.

Despite the success of global vaccination programs in slowing the spread of COVID-19, these efforts have been hindered by the emergence of new SARS-CoV-2 strains capable of evading prior immunity. The mutation and evolution of SARS-CoV-2 have created a demand for persistent efforts in vaccine development. SARS-CoV-2 Spike protein has been the primary target for COVID-19 vaccine development, but it is also the hotspot of mutations directly involved in host susceptibility and immune evasion. Our ability to predict emerging mutants and select conserved epitopes is critical for the development of a broadly neutralizing therapy or a universal vaccine. In this article, we review the general paradigm of immune responses to COVID-19 vaccines, highlighting the immunological epitopes of Spike protein that are likely associated with eliciting protective immunity resulting from vaccination. Specifically, we analyze the structural and evolutionary characteristics of the SARS-CoV-2 Spike protein related to immune activation and function via the toll-like receptors (TLRs), B cells, and T cells. We aim to provide a comprehensive analysis of immune epitopes of Spike protein, thereby contributing to the development of new strategies for broad neutralization or universal vaccination.

Biological Applications of Synthetic Binders Isolated from a Conceptually New Adhiron Library.

BACKGROUND: Adhirons are small (10 kDa) synthetic ligands that might represent an alternative to antibody fragments and to alternative scaffolds such as DARPins or affibodies. METHODS: We prepared a conceptionally new adhiron phage display library that allows the presence of cysteines in the hypervariable loops and successfully panned it against antigens possessing different characteristics. RESULTS: We recovered binders specific for membrane epitopes of plant cells by panning the library directly against pea protoplasts and against soluble C-Reactive Protein and SpyCatcher, a small protein domain for which we failed to isolate binders using pre-immune nanobody libraries. The best binders had a binding constant in the low nM range, were produced easily in bacteria (average yields of 15 mg/L of culture) in combination with different tags, were stable, and had minimal aggregation propensity, independent of the presence or absence of cysteine residues in their loops. DISCUSSION: The isolated adhirons were significantly stronger than those isolated previously from other libraries and as good as nanobodies recovered from a naïve library of comparable theoretical diversity. Moreover, they proved to be suitable reagents for ELISA, flow cytometry, the western blot, and also as capture elements in electrochemical biosensors.

Correction: Magazine et al. Mutations and Evolution of the SARS-CoV-2 Spike Protein. Viruses 2022, 14, 640.

In the original publication [...].

Th2 and Th17-Associated Immunopathology Following SARS-CoV-2 Breakthrough Infection in Spike-Vaccinated ACE2-humanized Mice.

Vaccines have demonstrated remarkable effectiveness in protecting against COVID-19; however, concerns regarding vaccine-associated enhanced respiratory diseases (VAERD) following breakthrough infections have emerged. Spike protein subunit vaccines for SARS-CoV-2 induce VAERD in hamsters, where aluminum adjuvants promote a Th2-biased immune response, leading to increased type 2 pulmonary inflammation in animals with breakthrough infections. To gain a deeper understanding of the potential risks and the underlying mechanisms of VAERD, we immunized ACE2-humanized mice with SARS-CoV-2 Spike protein adjuvanted with aluminum and CpG-ODN. Subsequently, we exposed them to increasing doses of SARS-CoV-2 to establish a breakthrough infection. The vaccine elicited robust neutralizing antibody responses, reduced viral titers, and enhanced host survival. However, following a breakthrough infection, vaccinated animals exhibited severe pulmonary immunopathology, characterized by a significant perivascular infiltration of eosinophils and CD4+ T cells, along with increased expression of Th2/Th17 cytokines. Intracellular flow cytometric analysis revealed a systemic Th17 inflammatory response, particularly pronounced in the lungs. Our data demonstrate that aluminum/CpG adjuvants induce strong antibody and Th1-associated immunity against COVID-19 but also prime a robust Th2/Th17 inflammatory response, which may contribute to the rapid onset of T cell-mediated pulmonary immunopathology following a breakthrough infection. These findings underscore the necessity for further research to unravel the complexities of VAERD in COVID-19 and to enhance vaccine formulations for broad protection and maximum safety.

Mutations and Evolution of the SARS-CoV-2 Spike Protein.

The SARS-CoV-2 spike protein mediates target recognition, cellular entry, and ultimately the viral infection that leads to various levels of COVID-19 severities. Positive evolutionary selection of mutations within the spike protein has led to the genesis of new SARS-CoV-2 variants with greatly enhanced overall fitness. Given the trend of variants with increased fitness arising from spike protein alterations, it is critical that the scientific community understand the mechanisms by which these mutations alter viral functions. As of March 2022, five SARS-CoV-2 strains were labeled "variants of concern" by the World Health Organization: the Alpha, Beta, Gamma, Delta, and Omicron variants. This review summarizes the potential mechanisms by which the common mutations on the spike protein that occur within these strains enhance the overall fitness of their respective variants. In addressing these mutations within the context of the SARS-CoV-2 spike protein structure, spike/receptor binding interface, spike/antibody binding, and virus neutralization, we summarize the general paradigms that can be used to estimate the effects of future mutations along SARS-CoV-2 evolution.

Panel of Engineered Ubiquitin Variants Targeting the Family of Human Ubiquitin Interacting Motifs.

Ubiquitin (Ub)-binding domains embedded in intracellular proteins act as readers of the complex Ub code and contribute to regulation of numerous eukaryotic processes. Ub-interacting motifs (UIMs) are short α-helical modular recognition elements whose role in controlling proteostasis and signal transduction has been poorly investigated. Moreover, impaired or aberrant activity of UIM-containing proteins has been implicated in numerous diseases, but targeting modular recognition elements in proteins remains a major challenge. To overcome this limitation, we developed Ub variants (UbVs) that bind to 42 UIMs in the human proteome with high affinity and specificity. Structural analysis of a UbV:UIM complex revealed the molecular determinants of enhanced affinity and specificity. Furthermore, we showed that a UbV targeting a UIM in the cancer-associated Ub-specific protease 28 potently inhibited catalytic activity. Our work demonstrates the versatility of UbVs to target short α-helical Ub receptors with high affinity and specificity. Moreover, the UbVs provide a toolkit to investigate the role of UIMs in regulating and transducing Ub signals and establish a general strategy for the systematic development of probes for Ub-binding domains.

Engineered SH2 Domains for Targeted Phosphoproteomics.

A comprehensive analysis of the phosphoproteome is essential for understanding molecular mechanisms of human diseases. However, current tools used to enrich phosphotyrosine (pTyr) are limited in their applicability and scope. Here, we engineered new superbinder Src-Homology 2 (SH2) domains that enrich diverse sets of pTyr-peptides. We used phage display to select a Fes-SH2 domain variant (superFes; sFes1) with high affinity for pTyr and solved its structure bound to a pTyr-peptide. We performed systematic structure-function analyses of the superbinding mechanisms of sFes1 and superSrc-SH2 (sSrc1), another SH2 superbinder. We grafted the superbinder motifs from sFes1 and sSrc1 into 17 additional SH2 domains and confirmed increased binding affinity for specific pTyr-peptides. Using mass spectrometry (MS), we demonstrated that SH2 superbinders have distinct specificity profiles and superior capabilities to enrich pTyr-peptides. Finally, using combinations of SH2 superbinders as affinity purification (AP) tools we showed that unique subsets of pTyr-peptides can be enriched with unparalleled depth and coverage.

Single-domain antibodies that compete with the natural ligand fibroblast growth factor block the internalization of the fibroblast growth factor receptor 1.

Single-domain antibodies in VHH format specific for fibroblast growth factor receptor 1 (FGFR1) were isolated from a phage-display llama naïve library. In particular, phage elution in the presence of the natural receptor ligand fibroblast growth factor (FGF) allowed for the identification of recombinant antibodies that compete with FGF for the same region on the receptor surface. These antibodies posses a relatively low affinity for FGFR1 and were never identified when unspecific elution conditions favoring highly affine binders were applied to panning procedures. Two populations of competitive antibodies were identified that labeled specifically the receptor-expressing cells in immunofluorescence and recognize distinct epitopes. Antibodies from both populations effectively prevented FGF-dependent internalization and nuclear accumulation of the receptor in cultured cells. This achievement indicates that these antibodies have a capacity to modulate the receptor physiology and, therefore, constitute powerful reagents for basic research and a potential lead for therapeutic applications.