Short Epitope Research Articles

Combining poly-epitope MoonTags and labeled nanobodies for signal amplification in cell-specific PET imaging in vivo

Immunorecognition provides an excellent basis for targeted imaging techniques covering a wide range from basic research to diagnostics and from single cells to whole organisms. Fluorescence- or radioisotope-labeled antibodies, antibody fragments or nanobodies enable a direct signal readout upon binding and allow for versatile imaging from microscopy to whole-body imaging. However, as the signal intensity directly correlates with the number of labeled antibodies bound to their epitopes (1:1 binding), sensitivity for low-expressing epitopes can be limiting for visualization. For the first time, we developed poly-epitope tags with multiple copies (1 to 7) of a short peptide epitope, specifically the MoonTag, that are recognized by a labeled nanobody and aimed at signal amplification in microscopy and cell-specific PET imaging. In transiently transfected HeLa cells or stably transduced A4573 cells we characterized complex formation and in vitro signal amplification. Indeed, using fluorescently and radioactively labeled nanobodies we found an approximately linear signal amplification with increasing numbers of epitope copies in vitro. To test the poly-epitope approach in vivo, A4573 tumor cells were injected subcutaneously into the shoulder of NSG mice, with A4573 tumor cells expressing a poly-epitope of 7 MoonTags on one side and WT cells on the other side. Using a [68Ga]-labeled NODAGA-conjugated MoonTag nanobody, we performed PET/CT imaging at day 8–9 after tumor implantation. Specific binding of a [68Ga]-labeled NODAGA-conjugated MoonTag nanobody was observed in 7xMoonTag tumors (1.7 ± 0.5%ID/mL) by PET imaging, showing significantly higher radiotracer accumulation compared to the WT tumors (1.1 ± 0.3%ID/mL; p < 0.01). Ex vivo gamma counter measurements confirmed significantly higher uptake in 7xMoonTag tumors compared to WT tumors (p < 0.001). In addition, MoonTag nanobody binding was detected by autoradiography which was spatially matched with histological analysis of the tumor tissues. In conclusion, we expect nanobody-based poly-epitope tag strategies to be widely applicable for multimodal imaging techniques given the advantageous properties of nanobodies and their amenability to genetic and chemical engineering.

Molecularly imprinted nanogels as synthetic recognition materials for the ultrasensitive detection of periodontal disease biomarkers

Periodontal disease affects supporting dental structures and ranks among one of the top most expensive conditions to treat in the world. Moreover, in recent years, the disease has also been linked to cardiovascular and Alzheimer’s diseases. At present, there is a serious lack of accurate diagnostic tools to identify people at severe risk of periodontal disease progression. Porphyromonas gingivalis is often considered one of the most contributing factors towards disease progression. It produces the Arg- and Lys-specific proteases Rgp and Kgp, respectively. Within this work, a short epitope sequence of these proteases is immobilised onto a magnetic nanoparticle platform. These are then used as a template to produce high-affinity, selective molecularly imprinted nanogels, using the common monomers N-tert-butylacrylamide (TBAM), N-isopropyl acrylamide (NIPAM), and N-(3-aminopropyl) methacrylamide hydrochloride (APMA). N,N-Methylene bis(acrylamide) (BIS) was used as a crosslinking monomer to form the interconnected polymeric network. The produced nanogels were immobilised onto a planar gold surface and characterised using the optical technique of surface plasmon resonance. They showed high selectivity and affinity towards their template, with affinity constants of 79.4 and 89.7 nM for the Rgp and Kgp epitope nanogels, respectively. From their calibration curves, the theoretical limit of detection was determined to be 1.27 nM for the Rgp nanogels and 2.00 nM for the Kgp nanogels. Furthermore, they also showed excellent selectivity against bacterial culture supernatants E8 (Rgp knockout), K1A (Kgp knockout), and W50-d (wild-type) strains in complex medium of brain heart infusion (BHI).

Immunotherapy targeting tumor-associated antigen in a mouse model of head and neck cancer.

The identification of epitope peptides from tumor-associated antigens (TAAs) is informative for developing tumor-specific immunotherapy. However, only a few epitopes have been detected in mouse TAAs of head and neck cancer (HNSCC). Novel mouse c-Met-derived T-cell epitopes were predicted by computer-based algorithms. Mouse HNSCC cell line-bearing mice were treated with a c-Met peptide vaccine. The effects of CD8 and/or CD4 T-cell depletion, and vaccine combination with immune checkpoint inhibitors (ICIs) were evaluated. Tumor re-inoculation was performed to assess T-cell memory. We identified c-Met-derived short and long epitopes that elicited c-Met-reactive antitumor CD8 and/or CD4 T-cell responses. Vaccination using these peptides showed remarkable antitumor responses via T cells in which ICIs were not required. The c-Met peptide-vaccinated mice rejected the re-inoculated tumors. We demonstrated that novel c-Met peptide vaccines can induce antitumor T-cell response, and could be a potent immunotherapy in a syngeneic mouse HNSCC model.

Analysis of Tau heterogeneity and proteome‐wide changes in Alzheimer’s Disease at the single‐molecule level

AbstractBackgroundThe molecular heterogeneity of Tau is a critical contributor to Alzheimer’s disease (AD) progression and pathology. Currently, very little is known about the prevalence or impact of the diverse collection of Tau proteoforms. Additionally, individual proteoforms may have different interactions or frequencies within the broader proteome. Here, we detected and analyzed Tau proteoforms in the context of the broader proteome in model systems using a novel single‐molecule proteomic analysis platform with future applicability in AD samples.MethodWe used a single‐molecule proteomic analysis platform that leverages a set of target isoform specific and target PTM specific affinity reagents combined with novel instrumentation, single‐molecule biochemistry, and machine learning bioinformatics to enable deep proteoform and broad proteome analysis. In this study, we first evaluated the proteoform distribution of Tau using commercially available antibody reagents. Proteoform heterogeneity was compared (specifically splicing and phosphorylation variants) in both healthy and diseased samples. Then, total protein counts were assessed across the proteome using Protein Identification by Short‐epitope Mapping (PrISM), which leverages proprietary multi‐affinity probes designed to recognize short epitopes and a machine learning algorithm that decodes binding of hundreds of multi‐affinity probes into protein quantifications.ResultThe approach was evaluated by first measuring defined mixtures of recombinant Tau proteins. Next, we examined Tau enriched from induced pluripotent stem cell (iPSC)‐derived neurons and tau‐expressing cell lines. The platform revealed the molecular heterogeneity of Tau proteoforms missed by bulk measurements and peptide‐centric proteomics approaches. The proteoform data was then supplemented with broader proteome analysis in order to identify proteome‐wide changes – differences in proteins and pathways due to or in combination with specific Tau proteoforms and disease.ConclusionUnderstanding the biology of complex diseases like AD and other Tauopathies depends on understanding both the specific Tau proteoforms that exist at baseline and in disease as well as the pathways and processes impacted in the proteome. Single‐molecule tools that can analyze proteoforms in depth and the proteome broadly and link molecular signatures to disease states are essential. These tools will enable improved biomarkers, improved understanding of disease, and improved therapies for Alzheimer’s disease in the future.

Development of T cell receptor-engineered T cells targeting the sarcoma-associated antigen papillomavirus binding factor.

We previously identified papillomavirus binding factor (PBF) as an osteosarcoma antigen recognized by an autologous cytotoxic T lymphocyte clone. Vaccination with PBF-derived peptide presented by HLA-A24 (PBF peptide) elicited strong immune responses. In the present study, we generated T cell receptor-engineered T cells (TCR-T cells) directed against the PBF peptide (PBF TCR-T cells). PBF TCR was successfully transduced into T cells and detected using HLA-A*24:02/PBF peptide tetramer. PBF TCR-T cells generated from a healthy donor were highly expanded and recognized T2-A24 cells pulsed with PBF peptide, HLA-A24+ 293T cells transfected with PBF cDNA, and sarcoma cell lines. To establish an adoptive cell therapy model, we modified the PBF TCR by replacing both α and β constant regions with those of mice (hybrid PBF TCR). Hybrid PBF TCR-T cells also showed reactivity against T2-A24 cells pulsed with PBF peptide and to HLA-A24+ 293T cells transfected with various lengths of PBF cDNA including the PBF peptide sequence. Subsequently, we generated target cell lines highly expressing PBF (MFH03-PBF [short] epitope [+]) containing PBF peptide with invivo tumorigenicity. Hybrid PBF TCR-T cells exhibited antitumor effects compared with mock T cells in NSG mice xenografted with MFH03-PBF (short) epitope (+) cells. CD45+ T cells significantly infiltrated xenografted tumors only in the hybrid PBF TCR T cell group and most of these cells were CD8-positive. CD8+ T cells also showed Ki-67 expression and surrounded the CD8-negative tumor cells expressing Ki-67. These findings suggest that PBF TCR-T cell therapy might be a candidate immunotherapy for sarcoma highly expressing PBF.

Detection of Antibodies against Endemic and SARS-CoV-2 Coronaviruses with Short Peptide Epitopes.

(1) Background: Coronavirus proteins are quite conserved amongst endemic strains (eCoV) and SARS-CoV-2. We aimed to evaluate whether peptide epitopes might serve as useful diagnostic biomarkers to stratify previous infections and COVID-19. (2) Methods: Peptide epitopes were identified at an amino acid resolution that applied a novel statistical approach to generate data sets of potential antibody binding peptides. (3) Results: Data sets from more than 120 COVID-19 or eCoV-infected patients, as well as vaccinated persons, have been used to generate data sets that have been used to search in silico for potential epitopes in proteins of SARS-CoV-2 and eCoV. Peptide epitopes were validated with >300 serum samples in synthetic peptide micro arrays and epitopes specific for different viruses, in addition to the identified cross reactive epitopes. (4) Conclusions: Most patients develop antibodies against non-structural proteins, which are useful general markers for recent infections. However, there are differences in the epitope patterns of COVID-19, and eCoV, and the S-protein vaccine, which can only be explained by a high degree of cross-reactivity between the viruses, a pre-existing immune response against some epitopes, and even an alternate processing of the vaccine proteins.

Abstract 5305: Detection and quantification of proteins using protein identification by short-epitope mapping (PrISM)

Abstract Introduction: Here, we demonstrate Protein Identification by Short-epitope Mapping (PrISM), which aims to provide comprehensive proteome analysis with broad dynamic range at single-molecule resolution by interrogating immobilized, intact proteins in parallel using multi-affinity probes. Improving the dynamic range and scale for protein analyses will enable a deeper understanding of low abundant protein in all stages of cancer progression. In addition, enabling the interrogation of single-molecules will provide a deeper understanding of protein diversity, such as the proteoforms resulting from non-canonical post-translational modifications inherent to cancer1. The combination of single-molecule sensitivity with comprehensive proteome coverage could also open the door for highly sensitive and specific diagnostics. Methods: PrISM uses non-traditional affinity reagents with high affinity and low specificity that bind to short epitopes in multiple proteins. Sample proteins were conjugated to DNA nanoparticles and deposited on a high-density patterned flow cell at optically resolvable locations. Multi-affinity probes were applied to sample proteins over multiple cycles to generate binding patterns for each single-molecule protein, which are translated to protein identifications and quantities using a custom machine learning approach. We acquired PrISM data on native biological and control samples using dozens of multi-affinity probes targeting trimer or tetramer sequences. Results: We report single-molecule deposition of over 1 billion DNA nanoparticle complexes on a flow cell. We demonstrate how the PrISM methodology identifies individual protein molecules through iterative probing with our multi-affinity probes. Further, we provide an analytical assessment of the sensitivity and specificity of PrISM and demonstrate the ability to accurately estimate the false identification rate of these proteins using a target-decoy based statistical approach. Conclusions: Combining single-molecule analysis, intact (non-digested) proteins, and iterative probing, PrISM provides a new tool for quantitative proteomics. We demonstrate linear and reproducible quantification of proteins using PrISM, potentially enabling detection of low abundant proteins and proteoforms associated with cancer. The ability to make comprehensive measurements of intact proteins at single-molecule resolution could accelerate basic cancer research through to the clinic.

A pan-variant mRNA-LNP T cell vaccine protects HLA transgenic mice from mortality after infection with SARS-CoV-2 Beta.

Licensed COVID-19 vaccines ameliorate viral infection by inducing production of neutralizing antibodies that bind the SARS-CoV-2 Spike protein and inhibit viral cellular entry. However, the clinical effectiveness of these vaccines is transitory as viral variants escape antibody neutralization. Effective vaccines that solely rely upon a T cell response to combat SARS-CoV-2 infection could be transformational because they can utilize highly conserved short pan-variant peptide epitopes, but a mRNA-LNP T cell vaccine has not been shown to provide effective anti-SARS-CoV-2 prophylaxis. Here we show a mRNA-LNP vaccine (MIT-T-COVID) based on highly conserved short peptide epitopes activates CD8+ and CD4+ T cell responses that attenuate morbidity and prevent mortality in HLA-A*02:01 transgenic mice infected with SARS-CoV-2 Beta (B.1.351). We found CD8+ T cells in mice immunized with MIT-T-COVID vaccine significantly increased from 1.1% to 24.0% of total pulmonary nucleated cells prior to and at 7 days post infection (dpi), respectively, indicating dynamic recruitment of circulating specific T cells into the infected lungs. Mice immunized with MIT-T-COVID had 2.8 (2 dpi) and 3.3 (7 dpi) times more lung infiltrating CD8+ T cells than unimmunized mice. Mice immunized with MIT-T-COVID had 17.4 times more lung infiltrating CD4+ T cells than unimmunized mice (7 dpi). The undetectable specific antibody response in MIT-T-COVID-immunized mice demonstrates specific T cell responses alone can effectively attenuate the pathogenesis of SARS-CoV-2 infection. Our results suggest further study is merited for pan-variant T cell vaccines, including for individuals that cannot produce neutralizing antibodies or to help mitigate Long COVID.

A small non-interface surface epitope in human IL18 mediates the dynamics and self-assembly of IL18-IL18BP heterodimers

Interleukin 18 (IL18) is a pro-inflammatory cytokine that modulates innate and adaptive immune responses. IL18 activity is tightly controlled by the constitutively secreted IL18 binding protein (IL18BP). PDB structures of human IL18 showed that a short stretch of amino acids between 68 and 81 adopted a disordered conformation in all IL18-IL18BP complexes while adopting a 310 helical structure in other IL18 structures, including the receptor complexes. The C74 of human IL18, which was reported to form a novel intermolecular disulfide bond in the human tetrameric assembly, is also located in this short epitope. These observations reflected the importance of this short surface epitope for the structure and dynamics of the IL18-IL18BP heterodimers. We have analyzed all known IL18-IL18BP complexes in the PDB by all-atom MD simulations. The analysis also included two computed complex models adopting a helical structure for the surface epitope. Heterodimer simulations showed a stabilizing impact of the small surface region at the helical form by reducing flexibility of the complex backbone. Analysis of the symmetry-related human IL18-IL18BP tetramer showed that the unfolding of this small surface region also contributed to the IL18-IL18BP stability through a completely exposed C74 sidechain to form an intermolecular disulfide bond in the self-assembled human IL18-IL18BP dimer. Our findings showed how the conformation of the short IL18 epitope between amino acids 68 and 81 would affect IL18 activity by mediating the intermolecular interactions of IL18.

Stimulation of the immune system by a tumor antigen-bearing adenovirus-inspired VLP allows control of melanoma growth

Virus-like particles (VLPs) are versatile protein-based platforms that can be used as a vaccine platform mainly in infectiology. In the present work, we compared a previously designed, non-infectious, adenovirus-inspired 60-mer dodecahedric VLP to display short epitopes or a large tumor model antigen. To validate these two kinds of platforms as a potential immuno-stimulating approach, we evaluated their ability to control melanoma B16-ovalbumin (OVA) growth in mice. A set of adjuvants was screened, showing that polyinosinic-polycytidylic acid (poly(I:C)) was well suited to generate a homogeneous cellular and humoral response against the desired epitopes. In a prophylactic setting, vaccination with the VLP displaying these epitopes resulted in total inhibition of tumor growth 1month after vaccination. A therapeutic vaccination strategy showed a delay in grafted tumor growth or its total rejection. If the "simple" epitope display on the VLP is sufficient to prevent tumor growth, then an improved engineered platform enabling display of a large antigen is a tool to overcome the barrier of immune allele restriction, broadening the immune response, and paving the way for its potential utilization in humans as an off-the-shelf vaccine.

Adenovirus-Inspired Virus-Like-Particles Displaying Melanoma Tumor Antigen Specifically Target Human DC Subsets and Trigger Antigen-Specific Immune Responses.

Virus-like particles constitute versatile vectors that can be used as vaccine platforms in many fields from infectiology and more recently to oncology. We previously designed non-infectious adenovirus-inspired 60-mer dodecahedric virus-like particles named ADDomers displaying on their surface either a short epitope or a large tumor/viral antigen. In this work, we explored for the first time the immunogenicity of ADDomers exhibiting melanoma-derived tumor antigen/epitope and their impact on the features of human dendritic cell (DC) subsets. We first demonstrated that ADDomers displaying tumor epitope/antigen elicit a strong immune-stimulating potential of human DC subsets (cDC2s, cDC1s, pDCs), which were able to internalize and cross-present tumor antigen, and subsequently cross-prime antigen-specific T-cell responses. To further limit off-target effects and enhance DC targeting, we engineered specific motifs to de-target epithelial cells and improve DCs' addressing. The improved engineered platform making it possible to display large antigen represents a tool to overcome the barrier of immune allele restriction, broadening the immune response, and paving the way to its potential utilization in humans as an off-the-shelf vaccine.

Breasi-CRISPR: an efficient genome-editing method to interrogate protein localization and protein-protein interactions in the embryonic mouse cortex.

In developing tissues, knowing the localization and interactors of proteins of interest is key to understanding their function. Here, we describe the Breasi-CRISPR approach (Brain Easi-CRISPR), combining Easi-CRISPR with in utero electroporation to tag endogenous proteins within embryonic mouse brains. Breasi-CRISPR enables knock-in of both short and long epitope tag sequences with high efficiency. We visualized epitope-tagged proteins with varied expression levels, such as ACTB, LMNB1, EMD, FMRP, NOTCH1 and RPL22. Detection was possible by immunohistochemistry as soon as 1 day after electroporation and we observed efficient gene editing in up to 50% of electroporated cells. Moreover, tagged proteins could be detected by immunoblotting in lysates from individual cortices. Next, we demonstrated that Breasi-CRISPR enables the tagging of proteins with fluorophores, allowing visualization of endogenous proteins by live imaging in organotypic brain slices. Finally, we used Breasi-CRISPR to perform co-immunoprecipitation mass-spectrometry analyses of the autism-related protein FMRP to discover its interactome in the embryonic cortex. Together, these data demonstrate that Breasi-CRISPR is a powerful tool with diverse applications that will propel the understanding of protein function in neurodevelopment.

Enhanced Proliferation of Human Mesenchymal Stem Cells by Self-Assembled Peptide Hydrogel Modified with Heparin Mimetic Peptide

The development of three-dimensional (3D) microenvironments that mimic the role of native extracellular matrix (ECM) components is very crucial. Self-assembling peptide (SAP) hydrogels have been utilized as scaffolds for encapsulation, 3D culture, proliferation, and differentiation of cells and also for repairing defects in cartilage tissue. SAPs can be biofunctionalized with short peptide epitopes to form biomimetic scaffolds. Herein, KLD (KLDLKLDLKLDL) SAP was functionalized with a short bioactive motif, EGDK, to fabricate KLD-HM (KLDLKLDLKLDL-EGDK) SAP hydrogel and self-assembled. In this study, it was aimed to investigate the effect of developed KLD-HM SAP hydrogels on the viability and proliferation of human mesenchymal stem cells (hMSCs). For ensuring the stability of SAPs, the rheological properties and degradation profile of produced SAP hydrogels were assessed. After the encapsulation of hMSCs in SAP hydrogels, MTT assay and Live and Dead staining assay were conducted. We showed that these biomimetic peptide hydrogel scaffolds provided a proper microenvironment for encapsulated hMSCs and the developed SAP hydrogels promoted the adhesion, viability, and proliferation of hMSCs. Our results suggest that designed bioactive SAP hydrogel scaffolds might be useful for promoting the regeneration of cartilage tissue.

Peptidomimetic Small-Molecule Inhibitors of 3CLPro Activity and Spike-ACE2 Interaction: Toward Dual-Action Molecules against Coronavirus Infections.

The development of molecules able to target protein–protein interactions (PPIs) is of interest for the development of novel therapeutic agents. Since a high percentage of PPIs are mediated by α-helical structure at the interacting surface, peptidomimetics that reproduce the essential conformational components of helices are useful templates for the development of PPIs inhibitors. In this work, the synthesis of a constrained dipeptide isostere and insertion in the short peptide epitope EDLFYQ of the angiotensin-converting enzyme 2 (ACE2) α1 helix domain resulted in the identification of a molecule capable of inhibiting the SARS-CoV-2 ACE2/spike interaction in the micromolar range. Moreover, inhibition of SARS-CoV-2 3CLPro main protease activity was assessed as an additional inhibitory property of the synthesized peptidomimetics, taking advantage of the C-terminal Q amino acid present in both the ACE2 epitope and the Mpro recognizing motif (APSTVxLQ), thus paving the way to the development of multitarget therapeutics toward coronavirus infections.

Characterization of a Nanobody-Epitope Tag Interaction and Its Application for Receptor Engineering.

Peptide epitope tags offer a valuable means for detection and manipulation of protein targets for which high quality detection reagents are not available. Most commonly used epitope tags are bound by conventional, full-size antibodies (Abs). The complex architecture of Abs complicates their application in protein engineering and intracellular applications. To address these shortcomings, single domain antibodies (nanobodies, Nbs) that recognize short peptide epitopes have become increasingly prized. Here, we characterize the interaction between a Nb (Nb6E) and a 14-mer peptide epitope. We identify residues in the peptide epitope essential for high affinity binding. Using this information in combination with computational modeling we propose a mode of interaction between Nb6E and this epitope. We apply this nanobody-epitope pair to augment the potency of a ligand at an engineered adenosine A2A receptor. This characterization of the nanobody-epitope pair opens the door to diverse applications including mechanistic studies of the G protein-coupled receptor function.

Abstract LB225: Toward comprehensive, single-molecule proteomics: Protein identification by short-epitope mapping

Abstract Protein Identification by Short-epitope Mapping (PrISM) is a novel proteomics method with the goal of comprehensive proteome quantification at single-molecule sensitivity. In this work, we present computational modeling demonstrating the feasibility of this goal and experimental application of the approach leveraging single-molecule protein measurements to identify a small number of model proteins. The implementation of PrISM as presented works by acquiring sequential affinity reagent binding measurements on single protein molecules. Counterintuitively, using affinity reagents with “poor” specificity (i.e. cross-reacting to many proteins) enables identification of tens of thousands of proteins with only hundreds of affinity reagents. To experimentally demonstrate PrISM, we use a small number of affinity reagents to quantify a larger number of proteins. In simulations, PrISM is able to identify more than 98% of proteins in human, mouse, yeast, and E. coli using 300 affinity reagents targeting short trimer epitopes. PrISM is robust to experimental confounders including non-specific binding and noisy, incomplete, or inaccurate estimation of affinity reagent to protein binding characteristics. Simulations of the approach with a chip containing 10 billion protein molecules measured in parallel show a dynamic range of detection of up to 9.5 and 11.5 orders of magnitude for HeLa cells and plasma, respectively. Using such a chip, the PrISM method can identify and quantify over 90% of the human proteome in a single experiment, potentially revolutionizing biomarker discovery and cancer research. Citation Format: Jarrett D. Egertson, Cassandra Stawicki, Taryn E. Gillies, James H. Joly, Noah Steiner, Kevin Cardenas, Jonathan B. Leano, Kevin Chen, Sophie Watts, Hunter B. Boyce, Dan DiPasquo, Alana Killeen, Vadim Lobanov, Greg Kapp, Sujal Patel, Parag Mallick. Toward comprehensive, single-molecule proteomics: Protein identification by short-epitope mapping [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB225.

Epitope-Based Vaccines against the Chlamydia trachomatis Major Outer Membrane Protein Variable Domain 4 Elicit Protection in Mice

Chlamydia trachomatis (Ct) is the most common bacterial sexual transmitted pathogen, yet a vaccine is not currently available. Here, we used the immunogenic bacteriophage MS2 virus-like particle (VLP) technology to engineer vaccines against the Ct major outer membrane protein variable domain 4 (MOMP-VD4), which contains a conserved neutralizing epitope (TTLNPTIAG). A previously described monoclonal antibody to the MOMP-VD4 (E4 mAb) is capable of neutralizing all urogenital Ct serovars and binds this core epitope, as well as several non-contiguous amino acids. This suggests that this core epitope may require conformational context in order to elicit neutralizing antibodies to Ct. In order to identify immunogens that could elicit neutralizing antibodies to the TTLNPTIAG epitope, we used two approaches. First, we used affinity selection with a bacteriophage MS2-VLP library displaying random peptides in a constrained, surface-exposed loop to identify potential E4 mAb mimotopes. After four rounds of affinity selection, we identified a VLP-displayed peptide (HMVGSTKWTN) that could bind to the E4 mAb and elicited serum IgG that bound weakly to Ct elementary bodies by ELISA. Second, two versions of the core conserved TTLNPTIAG epitope (TTLNPTIAG and TTLNPTIAGA) were recombinantly expressed on the coat protein of the MS2 VLP in a constrained, surface-exposed loop. Mouse immune sera IgG bound to Ct elementary bodies by ELISA. Immunization with these MS2 VLPs provided protection from vaginal Chlamydia infection in a murine challenge model. These data suggest that short peptide epitopes targeting the MOMP-VD4 could be appropriate for Ct vaccine design when displayed on an immunogenic bacteriophage VLP vaccine platform.

A multiplexed epitope barcoding strategy that enables dynamic cellular phenotypic screens.

Pooled genetic libraries have improved screening throughput for mapping genotypes to phenotypes. However, selectable phenotypes are limited, restricting screening to outcomes with a low spatiotemporal resolution. Here, we integrated live-cell imaging with pooled library-based screening. To enable intracellular multiplexing, we developed a method called EPICode that uses a combination of short epitopes, which can also appear in various subcellular locations. EPICode thus enables the use of live-cell microscopy to characterize a phenotype of interest over time, including after sequential stimulatory/inhibitory manipulations, and directly connects behavior to the cellular genotype. To test EPICode's capacity against an important milestone-engineering and optimizing dynamic, live-cell reporters-we developed a live-cell PKA kinase translocation reporter with improved sensitivity and specificity. The use of epitopes as fluorescent barcodes introduces a scalable strategy for high-throughput screening broadly applicable to protein engineering and drug discovery settings where image-based phenotyping is desired.

A Light-Activatable Photocaged Variant of the Ultra-High Affinity ALFA-Tag Nanobody.

Nanobodies against short linear peptide‐epitopes are widely used to detect and bind proteins of interest (POI) in fusion constructs. Engineered nanobodies that can be controlled by light have found very recent attention for various extra‐ and intracellular applications. We here report the design of a photocaged variant of the ultra‐high affinity ALFA‐tag nanobody, also termed ALFA‐tag photobody. ortho‐Nitrobenzyl tyrosine was incorporated into the paratope region of the nanobody by genetic code expansion technology and resulted in a ≥9,200 to 100,000‐fold impairment of the binding affinity. Irradiation with light (365 nm) leads to decaging and reconstitutes the native nanobody. We show the light‐dependent binding of the ALFA‐tag photobody to HeLa cells presenting the ALFA‐tag. The generation of the first photobody directed against a short peptide epitope underlines the generality of our photobody design concept. We envision that this photobody will be useful for the spatiotemporal control of proteins in many applications using cultured cells.

Flagellin/Virus-like Particle Hybrid Platform with High Immunogenicity, Safety, and Versatility for Vaccine Development.

Hepatitis B core (HBc) virus-like particles (VLPs) and flagellin are highly immunogenic and widely explored vaccine delivery platforms. Yet, HBc VLPs mainly allow the insertion of relatively short antigenic epitopes into the immunodominant c/e1 loop without affecting VLP assembly, and flagellin-based vaccines carry the risk of inducing systemic adverse reactions. This study explored a hybrid flagellin/HBc VLP (FH VLP) platform to present heterologous antigens by replacing the surface-exposed D3 domain of flagellin. FH VLPs were prepared by the insertion of flagellin gene into the c/e1 loop of HBc, followed by E. coli expression, purification, and self-assembly into VLPs. Using the ectodomain of influenza matrix protein 2 (M2e) and ovalbumin (OVA) as models, we found that the D3 domain of flagellin could be replaced with four tandem copies of M2e or the cytotoxic T lymphocyte (CTL) epitope of OVA without interfering with the FH VLP assembly, while the insertion of four tandem copies of M2e into the c/e1 loop of HBc disrupted the VLP assembly. FH VLP-based M2e vaccine elicited potent anti-M2e antibody responses and conferred significant protection against multiple influenza A viral strains, while FljB- or HBc-based M2e vaccine failed to elicit significant protection. FH VLP-based OVA peptide vaccine elicited more potent CTL responses and protection against OVA-expressing lymphoma or melanoma challenges than FljB- or HBc-based OVA peptide vaccine. FH VLP-based vaccines showed a good systemic safety, while flagellin-based vaccines significantly increased serum interleukin 6 and tumor necrosis factor α levels and also rectal temperature at increased doses. We further found that the incorporation of a clinical CpG 1018 adjuvant could enhance the efficacy of FH VLP-based vaccines. Our data support FH VLPs to be a highly immunogenic, safe, and versatile platform for vaccine development to elicit potent humoral and cellular immune responses.