Glowing tumor marker hampers mouse cancer studies.

Response to protein complicates immunotherapy research.

A Promising Intracellular Protein-Degradation Strategy: TRIMbody-Away Technique Based on Nanobody Fragment.

Most recently, a technology termed TRIM-Away has allowed acute and rapid destruction of endogenous target proteins in cultured cells using specific antibodies and endogenous/exogenous tripartite motif 21 (TRIM21). However, the relatively large size of the full-size mAbs (150 kDa) results in correspondingly low tissue penetration and inaccessibility of some sterically hindered epitopes, which limits the target protein degradation. In addition, exogenous introduction of TRIM21 may cause side effects for treated cells. To tackle these limitations, we sought to replace full-size mAbs with the smaller format of antibodies, a nanobody (VHH, 15 kDa), and construct a new type of fusion protein named TRIMbody by fusing the nanobody and RBCC motif of TRIM21. Next, we introduced enhanced green fluorescent protein (EGFP) as a model substrate and generated αEGFP TRIMbody using a bispecific anti-EGFP (αEGFP) nanobody. Remarkably, inducible expression of αEGFP TRIMbody could specifically degrade intracellular EGFP in HEK293T cells in a time-dependent manner. By treating cells with inhibitors, we found that intracellular EGFP degradation by αEGFP TRIMbody relies on both ubiquitin-proteasome and autophagy-lysosome pathways. Taken together, these results suggested that TRIMbody-Away technology could be utilized to specifically degrade intracellular protein and could expand the potential applications of degrader technologies.

Small p53 derived peptide suitable for robust nanobodies dimerization.

Bivalent VHHs have been shown to display better functional affinity compared with their monovalent counterparts. Bivalency can be achieved either by inserting a hinge region between both VHHs units or by using modules that lead to dimerization. In this report, a small self-associating peptide originating from the tetramerization domain of p53 was developed as a tool for devicing nanobody dimerization. This E3 peptide was evaluated for the dimerization of an anti-eGFP nanobody (nano-eGFP-E3) whose activity was compared to a bivalent anti-eGFP constructed in tandem using GS rich linker. The benefit of bivalency in terms of avidity and specificity was assessed in different in vitro and in cellulo assays. In ELISA and SPR, the dimeric and tandem formats were nearly equivalent in terms of gain of avidity compared to the monovalent counterpart. However, in cellulo, the nano-eGFP-E3 construct showed its superiority over the tandem format in terms of specificity with a highest and better ratio signal-to-noise. All together, the E3 peptide provides a universal suitable tool for the construction of dimeric biomolecules, in particular antibody fragments with improved functional affinity.

Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of membrane proteins.

GFP fusion-based fluorescence-detection size-exclusion chromatography (FSEC) has been widely employed for membrane protein expression screening. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins, depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP for FSEC analysis. FSEC-Nb enables the evaluation of the expression, monodispersity and thermostability of membrane proteins without the need for purification but does not require direct GFP fusion to targeted proteins. Our results show FSEC-Nb as a powerful tool for expression screening of membrane proteins for structural and functional studies.

Rapid and reliable hybridoma screening method that is suitable for production of functional structure-recognizing monoclonal antibody.

Monoclonal antibodies are extremely valuable functional biomaterials that are widely used not only in life science research but also in antibody drugs and test drugs. There is also a strong need to develop high-quality neutralizing antibodies as soon as possible in order to stop the rapid spread of new infectious diseases such as the SARS-CoV-2 virus. This study has developed a membrane-type immunoglobulin-directed hybridoma screening (MIHS) method for obtaining high-quality monoclonal antibodies with high efficiency and high speed. In addition to these advantages, this paper demonstrates that the MIHS method can selectively obtain monoclonal antibodies that specifically recognize the functional structure of proteins. The MIHS method is a useful technology that greatly contributes to the research community because it can be easily introduced in any laboratory that uses a flow cytometer.

Designing a Single Protein-Chain Reporter for Opioid Detection at Cellular Resolution.

Mu-opioid receptor (MOR) signaling regulates multiple neuronal pathways, including those involved in pain, reward, and respiration. To advance the understanding of MOR's roles in pain modulation, there is a need for high-throughput screening methods of opioids in vitro and high-resolution mapping of opioids in the brain. To fill this need, we designed and characterized a genetically encoded fluorescent reporter, called Single-chain Protein-based Opioid Transmission Indicator Tool for MOR (M-SPOTIT). M-SPOTIT represents a new and unique mechanism for fluorescent reporter design and can detect MOR activation, leaving a persistent green fluorescence mark for image analysis. M-SPOTIT showed an opioid-dependent signal to noise ratio (S/N) up to 12.5 and was able to detect as fast as a 30-second opioid exposure in HEK293T cell culture. Additionally, it showed an opioid-dependent S/N up to 4.6 in neuronal culture and detected fentanyl with an EC50 of 15 nM. M-SPOTIT will potentially be useful for high-throughput detection of opioids in cell cultures and cellular-resolution detection of opioids in vivo. M-SPOTIT's novel mechanism can be used as a platform to design other G-protein-coupled receptor-based sensors.

Application of dual Nr4a1-GFP Nr4a3-Tocky reporter mice to study T cell receptor signaling by flow cytometry.

This protocol uses Nr4a1-GFP Nr4a3-Tocky mice to study T cell receptor (TCR) signaling using flow cytometry. It identifies the optimal mouse transgenic status and fluorochromes compatible with the dual reporter. This protocol has applications in TCR signaling, and we outline how to obtain high-quality datasets. It is not compatible with cellular fixation, and cells should be analyzed immediately after staining. For complete details on the use and execution of this protocol, please refer to Jennings et al., 2020.

Integrating quantitative proteomics with accurate genome profiling of transcription factors by greenCUT&RUN.

Genome-wide localization of chromatin and transcription regulators can be detected by a variety of techniques. Here, we describe a novel method 'greenCUT&RUN' for genome-wide profiling of transcription regulators, which has a very high sensitivity, resolution, accuracy and reproducibility, whilst assuring specificity. Our strategy begins with tagging of the protein of interest with GFP and utilizes a GFP-specific nanobody fused to MNase to profile genome-wide binding events. By using a GFP-nanobody the greenCUT&RUN approach eliminates antibody dependency and variability. Robust genomic profiles were obtained with greenCUT&RUN, which are accurate and unbiased towards open chromatin. By integrating greenCUT&RUN with nanobody-based affinity purification mass spectrometry, 'piggy-back' DNA binding events can be identified on a genomic scale. The unique design of greenCUT&RUN grants target protein flexibility and yields high resolution footprints. In addition, greenCUT&RUN allows rapid profiling of mutants of chromatin and transcription proteins. In conclusion, greenCUT&RUN is a widely applicable and versatile genome-mapping technique.

Nanobody-mediated control of gene expression and epigenetic memory.

Targeting chromatin regulators to specific genomic locations for gene control is emerging as a powerful method in basic research and synthetic biology. However, many chromatin regulators are large, making them difficult to deliver and combine in mammalian cells. Here, we develop a strategy for gene control using small nanobodies that bind and recruit endogenous chromatin regulators to a gene. We show that an antiGFP nanobody can be used to simultaneously visualize GFP-tagged chromatin regulators and control gene expression, and that nanobodies against HP1 and DNMT1 can silence a reporter gene. Moreover, combining nanobodies together or with other regulators, such as DNMT3A or KRAB, can enhance silencing speed and epigenetic memory. Finally, we use the slow silencing speed and high memory of antiDNMT1 to build a signal duration timer and recorder. These results set the basis for using nanobodies against chromatin regulators for controlling gene expression and epigenetic memory.

NOD Mice-Good Model for T1D but Not Without Limitations.

The nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D) was discovered by coincidence in the 1980s and has since been widely used in the investigation of T1D and diabetic complications. The current in vivo study was originally designed to prospectively assess whether hyperglycemia onset is associated with physical destruction or functional impairment of beta cells under inflammatory insult during T1D progression in diabetes-prone female NOD mice. Prediabetic 16- to 20-wk-old NOD mice were transplanted with green fluorescent protein (GFP)-expressing reporter islets in the anterior chamber of the eye (ACE) that were monitored longitudinally, in addition to glycemia, with and without immune modulation using anti-CD3 monoclonal antibody therapy. However, there was an early and vigorous immune reaction against the GFP-expressing beta cells that lead to their premature destruction independent of autoimmune T1D development in progressor mice that eventually became hyperglycemic. This immune reaction also occurred in nonprogressor NOD recipients. These findings showed a previously unknown reaction of NOD mice to GFP that prevented achieving the original goals of this study but highlighted a new feature of the NOD mice that should be considered when designing experiments using this model in T1D research.

Inducible Degradation of Target Proteins through a Tractable Affinity-Directed Protein Missile System.

The affinity-directed protein missile (AdPROM) system utilizes specific polypeptide binders of intracellular proteins of interest (POIs) conjugated to an E3 ubiquitin ligase moiety to enable targeted proteolysis of the POI. However, a chemically tuneable AdPROM system is more desirable. Here, we use Halo-tag/VHL-recruiting proteolysis-targeting chimera (HaloPROTAC) technology to develop a ligand-inducible AdPROM (L-AdPROM) system. When we express an L-AdPROM construct consisting of an anti-GFP nanobody conjugated to the Halo-tag, we achieve robust degradation of GFP-tagged POIs only upon treatment of cells with the HaloPROTAC. For GFP-tagged POIs, ULK1, FAM83D, and SGK3 were knocked in with a GFP-tag using CRISPR/Cas9. By substituting the anti-GFP nanobody for a monobody that binds H- and K-RAS, we achieve robust degradation of unmodified endogenous RAS proteins only in the presence of the HaloPROTAC. Through substitution of the polypeptide binder, the highly versatile L-AdPROM system is useful for the inducible degradation of potentially any intracellular POI.

Characterisation of alternative expression vectors for recombinant Bacillus Calmette-Guérin as live bacterial delivery systems.

BACKGROUND Bacillus Calmette-Guérin (BCG) is considered a promising live bacterial delivery system. However, several proposals for rBCG vaccines have not progressed, mainly due to the limitations of the available expression systems. OBJECTIVES To obtain a set of mycobacterial vectors using a range of promoters with different strengths based on a standard backbone, previously shown to be stable. METHODS Mycobacterial expression vectors based on the pLA71 vector as backbone, were obtained inserting different promoters (PAN, PαAg, PHsp60, PBlaF* and PL5) and the green fluorescence protein (GFP) as reporter gene, to evaluate features such as their relative strengths, and the in vitro (inside macrophages) and in vivo stability. FINDINGS The relative fluorescence observed with the different vectors showed increasing strength of the promoters: PAN was the weakest in both Mycobacterium smegmatis and BCG and PBlaF* was higher than PHsp60 in BCG. The relative fluorescence observed in a macrophage cell line showed that PBlaF* and PHsp60 were comparable. It was not possible to obtain strains transformed with the extrachromosomal expression vector containing the PL5 in either species. MAIN CONCLUSION We have obtained a set of potentially stable mycobacterial vectors with a arrange of expression levels, to be used in the development of rBCG vaccines.

Development and production of nanobodies specifically against green fluorescence protein.

Variable domains of heavy chains of camelid heavy-chain antibodies (VHHs) are known as nanobodies. Nanobodies are approximately 15 kDa in size with high affinity to their antigens. They can be easily manipulated and produced in microorganisms. In this study, an alpaca was immunized with purified green fluorescence protein (GFP) and a VHH library from lymphocytes of the immunized alpaca was constructed with a capacity of 6.7 × 107. The library was biopanned against GFP by phage display technique and four unique DNA sequences coding for anti-GFP nanobodies were identified by enzyme-linked immunosorbent assay, named a12, e6, d5, and b9. The four DNA sequences were then cloned into pADL-10b-6×His or pBAD24-Flag-6×His for expression in bacteria. Purified A12, E6, D5, and B9 were demonstrated to bind GFP specifically both in vitro by enzyme-linked immunosorbent assay and native-PAGE analysis and in vivo by immunofluorescence and immunoprecipitation. Taken together, our results demonstrate that anti-GFP nanobodies are successfully selected from the immune library, are produced in bacteria, and are available for basic research.Key Points• Four different GFP binders were successfully obtained from an immune VHH library.• The four GFP binders were successfully purified from bacteria. • Purified GFP binders can bind GFP both in vitro and in vivo and are ready for use in basic research.

Identification of Common CD8+ T Cell Epitopes from Lassa Fever Survivors in Nigeria and Sierra Leone.

Early and robust T cell responses have been associated with survival from Lassa fever (LF), but the Lassa virus-specific memory responses have not been well characterized. Regions within the virus surface glycoprotein (GPC) and nucleoprotein (NP) are the main targets of the Lassa virus-specific T cell responses, but, to date, only a few T cell epitopes within these proteins have been identified. We identified GPC and NP regions containing T cell epitopes and HLA haplotypes from LF survivors and used predictive HLA-binding algorithms to identify putative epitopes, which were then experimentally tested using autologous survivor samples. We identified 12 CD8-positive (CD8+) T cell epitopes, including epitopes common to both Nigerian and Sierra Leonean survivors. These data should be useful for the identification of dominant Lassa virus-specific T cell responses in Lassa fever survivors and vaccinated individuals as well as for designing vaccines that elicit cell-mediated immunity.IMPORTANCE The high morbidity and mortality associated with clinical cases of Lassa fever, together with the lack of licensed vaccines and limited and partially effective interventions, make Lassa virus (LASV) an important health concern in its regions of endemicity in West Africa. Previous infection with LASV protects from disease after subsequent exposure, providing a framework for designing vaccines to elicit similar protective immunity. Multiple major lineages of LASV circulate in West Africa, and therefore, ideal vaccine candidates should elicit immunity to all lineages. We therefore sought to identify common T cell epitopes between Lassa fever survivors from Sierra Leone and Nigeria, where distinct lineages circulate. We identified three such epitopes derived from highly conserved regions within LASV proteins. In this process, we also identified nine other T cell epitopes. These data should help in the design of an effective pan-LASV vaccine.

ILC2 activation by keratinocyte-derived IL-25 drives IL-13 production at sites of allergic skin inflammation.

BACKGROUND: Atopic dermatitis skin lesions demonstrate increased expression of IL-25 by keratinocytes and increased numbers of type 2 innate lymphoid cells (ILC2s) that express high levels of IL-25 receptor (IL-25R). IL-13 is expressed in atopic dermatitis skin lesions and plays an important role in pathogenesis of the disease. OBJECTIVE: Our aim was to determine the role of IL-25 and ILC2s in a mouse model of antigen-driven allergic skin inflammation. METHODS: Wild-type mice; mice that express an Il13-driven enhanced green fluorescent protein; and mice that lack IL-25R, IL-25 in keratinocytes, or IL-13 or IL-25R in ILC2s were subjected to acute or chronic epicutaneous sensitization with ovalbumin. Sensitized skin was examined by histology for epidermal thickening. Cellular infiltrates were analyzed for surface markers and intracellular expression of enhanced green fluorescent protein by flow cytometry. Gene expression was quantitated by RT quantitative PCR. RESULT: In both acute and chronic antigen-driven allergic skin inflammation, signaling by keratinocyte-derived IL-25 in ILC2s is important for epidermal hyperplasia, dermal infiltration by CD4+ T cells, and cutaneous expression of Il13 and the IL-13-dependent TH2-cell-attracting chemokines Cc17 and Ccl22. ILCs are the major source of IL-13 in acutely sensitized mouse skin, whereas T cells are its major source in chronically sensitized mouse skin. CONCLUSION: ILC2 activation by IL-25 is essential for IL-13 expression at sites of allergic skin inflammation.

A self-aggregating peptide: implications for the development of thermostable vaccine candidates.

BACKGROUND: The use of biomaterials has been expanded to improve the characteristics of vaccines. Recently we have identified that the peptide PH(1-110) from polyhedrin self-aggregates and incorporates foreign proteins to form particles. We have proposed that this peptide can be used as an antigen carrying system for vaccines. However, the immune response generated by the antigen fused to the peptide has not been fully characterized. In addition, the adjuvant effect and thermostability of the particles has not been evaluated. RESULTS: In the present study we demonstrate the use of a system developed to generate nano and microparticles carrying as a fusion protein peptides or proteins of interest to be used as vaccines. These particles are purified easily by centrifugation. Immunization of animals with the particles in the absence of adjuvant result in a robust and long-lasting immune response. Proteins contained inside the particles are maintained for over 1 year at ambient temperature, preserving their immunological properties. CONCLUSION: The rapid and efficient production of the particles in addition to the robust immune response they generate position this system as an excellent method for the rapid response against emerging diseases. The thermostability conferred by the particle system facilitates the distribution of the vaccines in developing countries or areas with no electricity.

Region-specific transcriptomic and functional signatures of mononuclear phagocytes in the epididymis.

In the epididymis, prevention of autoimmune responses against spermatozoa and simultaneous protection against pathogens is important for male fertility. We have previously shown that mononuclear phagocytes (MPs) are located either in the epididymal interstitium or in close proximity to the epithelium. In the initial segments (IS), these 'intraepithelial' MPs extend slender luminal-reaching projections between epithelial cells. In this study, we performed an in-depth characterisation of MPs isolated from IS, caput-corpus and cauda epididymis of CX3CR1EGFP+/- mice that express EGFP in these cells. Flow cytometry analysis revealed region-specific subsets of MPs that express combinations of markers traditionally described in 'dendritic cells' or 'macrophages'. RNA sequencing identified distinct transcriptomic signatures in MPs from each region and revealed specific genes involved in inflammatory and anti-inflammatory responses, phagosomal activity and antigen processing and presentation. Functional fluorescent in vivo labelling assays showed that higher percentages of CX3CR1+ MPs that captured and processed antigens were detected in the IS compared to other regions. Confocal microscopy showed that in the IS, caput and corpus, circulatory antigens were internalised and processed by interstitial and intraepithelial MPs. However, in the cauda only interstitial MPs internalised and processed antigens, while intraepithelial MPs did not take up antigens, indicating that all antigens have been captured before they reached the epithelial lining. Cauda MPs may thus confer a stronger protection against blood-borne pathogens compared to proximal regions. By identifying immunoregulatory mechanisms in the epididymis, our study may lead to new therapies for male infertility and epididymitis and identify potential targets for immunocontraception.

Characterisation of alternative expression vectors for recombinant Bacillus Calmette-Guérin as live bacterial delivery systems

BACKGROUND Bacillus Calmette-Guérin (BCG) is considered a promising live bacterial delivery system. However, several proposals for rBCG vaccines have not progressed, mainly due to the limitations of the available expression systems. OBJECTIVES To obtain a set of mycobacterial vectors using a range of promoters with different strengths based on a standard backbone, previously shown to be stable. METHODS Mycobacterial expression vectors based on the pLA71 vector as backbone, were obtained inserting different promoters (PAN, PαAg, PHsp60, PBlaF* and PL5) and the green fluorescence protein (GFP) as reporter gene, to evaluate features such as their relative strengths, and the in vitro (inside macrophages) and in vivo stability. FINDINGS The relative fluorescence observed with the different vectors showed increasing strength of the promoters: PAN was the weakest in both Mycobacterium smegmatis and BCG and PBlaF* was higher than PHsp60 in BCG. The relative fluorescence observed in a macrophage cell line showed that PBlaF* and PHsp60 were comparable. It was not possible to obtain strains transformed with the extrachromosomal expression vector containing the PL5 in either species. MAIN CONCLUSION We have obtained a set of potentially stable mycobacterial vectors with a arrange of expression levels, to be used in the development of rBCG vaccines.

Clade F AAVHSCs cross the blood brain barrier and transduce the central nervous system in addition to peripheral tissues following intravenous administration in nonhuman primates.

The biodistribution of AAVHSC7, AAVHSC15, and AAVHSC17 following systemic delivery was assessed in cynomolgus macaques (Macaca fascicularis). Animals received a single intravenous (IV) injection of a self-complementary AAVHSC-enhanced green fluorescent protein (eGFP) vector and tissues were harvested at two weeks post-dose for anti-eGFP immunohistochemistry and vector genome analyses. IV delivery of AAVHSC vectors produced widespread distribution of eGFP staining in glial cells throughout the central nervous system, with the highest levels seen in the pons and lateral geniculate nuclei (LGN). eGFP-positive neurons were also observed throughout the central and peripheral nervous systems for all three AAVHSC vectors including brain, spinal cord, and dorsal root ganglia (DRG) with staining evident in neuronal cell bodies, axons and dendritic arborizations. Co-labeling of sections from brain, spinal cord, and DRG with anti-eGFP antibodies and cell-specific markers confirmed eGFP-staining in neurons and glia, including protoplasmic and fibrous astrocytes and oligodendrocytes. For all capsids tested, 50 to 70% of glial cells (S100-ß+) and on average 8% of neurons (NeuroTrace+) in the LGN were positive for eGFP expression. In the DRG, 45 to 62% of neurons and 8 to 12% of satellite cells were eGFP-positive for the capsids tested. eGFP staining was also observed in peripheral tissues with abundant staining in hepatocytes, skeletal- and cardio-myocytes and in acinar cells of the pancreas. Biodistribution of AAVHSC vector genomes in the central and peripheral organs generally correlated with eGFP staining and were highest in the liver for all AAVHSC vectors tested. These data demonstrate that AAVHSCs have broad tissue tropism and cross the blood-nerve and blood-brain-barriers following systemic delivery in nonhuman primates, making them suitable gene editing or gene transfer vectors for therapeutic application in human genetic diseases.