• Ex Vivo/In vivo Gene Editing in Hepatocytes Using "All-in-One" CRISPR-Adeno-Associated Virus Vectors with a Self-Linearizing Repair Template.

      Krooss, Simon Alexander; Dai, Zhen; Schmidt, Florian; Rovai, Alice; Fakhiri, Julia; Dhingra, Akshay; Yuan, Qinggong; Yang, Taihua; Balakrishnan, Asha; Steinbrück, Lars; et al. (Cell Press/Elsevier, 2020-01-24)
      Adeno-associated virus (AAV)-based vectors are considered efficient and safe gene delivery systems in gene therapy. We combined two guide RNA genes, Cas9, and a self-linearizing repair template in one vector (AIO-SL) to correct fumarylacetoacetate hydrolase (FAH) deficiency in mice. The vector genome of 5.73 kb was packaged into VP2-depleted AAV particles (AAV2/8ΔVP2), which, however, did not improve cargo capacity. Reprogrammed hepatocytes were treated with AIO-SL.AAV2ΔVP2 and subsequently transplanted, resulting in large clusters of FAH-positive hepatocytes. Direct injection of AIO-SL.AAV8ΔVP2 likewise led to FAH expression and long-term survival. The AIO-SL vector achieved an ∼6-fold higher degree of template integration than vectors without template self-linearization. Subsequent analysis revealed that AAV8 particles, in contrast to AAV2, incorporate oversized genomes distinctly greater than 5.2 kb. Finally, our AAV8-based vector represents a promising tool for gene editing strategies to correct monogenic liver diseases requiring (large) fragment removal and/or simultaneous sequence replacement.
    • Fate of the UPR marker protein Kar2/Bip and autophagic processes in fed-batch cultures of secretory insulin precursor producing Pichia pastoris.

      Roth, Gustavo; Vanz, Ana Letícia; Lünsdorf, Heinrich; Nimtz, Manfred; Rinas, Ursula; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-08-09)
      Secretory recombinant protein production with Pichia (syn. Komagataella) pastoris is commonly associated with the induction of an unfolded protein response (UPR) usually apparent through increased intracellular levels of endoplasmic reticulum (ER) resident chaperones such as Kar2/Bip. During methanol-induced secretory production of an insulin precursor (IP) under industrially relevant fed-batch conditions the initially high level of intracellular Kar2/Bip after batch growth on glycerol unexpectedly declined in the following methanol fed-batch phase misleadingly suggesting that IP production had a low impact on UPR activation. Analysis of the protein production independent level of Kar2/Bip revealed that high Kar2/Bip levels were reached in the exponential growth phase of glycerol batch cultures followed by a strong decline of Kar2/Bip during entry into stationary phase. Ultra-structural cell morphology studies revealed autophagic processes (e.g. ER phagy) at the end of the glycerol batch phase most likely responsible for the degradation of ER resident chaperones such as Kar2/Bip. The pre-induction level of Kar2/Bip did not affect the IP secretion efficiency in the subsequent methanol-induced IP production phase. During growth on methanol intracellular Kar2/Bip levels declined in IP producing and non-producing host cells. However, extracellular accumulation of Kar2/Bip was observed in IP-producing cultures but not in non-producing controls. Most importantly, the majority of the extracellular Kar2/Bip accumulated in the culture supernatant of IP producing cells as truncated protein (approx. 35 kDa). Rapid growth leads to higher basal levels of the major UPR marker protein Kar2/Bip independent of recombinant protein production. Entry into stationary phase or slower growth on poorer substrate, e.g. methanol, leads to a lower basal Kar2/Bip level. Methanol-induced secretory IP production elicits a strong UPR activation which counteracts the reduced UPR during slow growth on methanol. The major ER chaperone Kar2/Bip is found together with recombinant IP in the culture medium where full-length Kar2/Bip accumulates in addition to large amounts of truncated Kar2/Bip. Thus, for judging UPR activating properties of the produced protein it is important to additionally analyze the medium not only for intact Kar2/Bip but also for truncated versions of this UPR reporter protein.
    • FKBPs in bacterial infections.

      Ünal, Can M; Steinert, Michael; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2015-10)
      FK506-binding proteins (FKBPs) contain a domain with peptidyl-prolyl-cis/trans-isomerase (PPIase) activity and bind the immunosuppressive drugs FK506 and rapamycin. FKBPs belong to the immunophilin family and are found in eukaryotes and bacteria.
    • Functional and immunogenic characterization of diverse HCV glycoprotein E2 variants.

      Khera, Tanvi; Behrendt, Patrick; Bankwitz, Dorothea; Brown, Richard J P; Todt, Daniel; Doepke, Mandy; Ghafoor Khan, Abdul; Schulze, Kai; Law, John; Logan, Michael; et al. (Elsevier, 2018-11-12)
      Induction of cross-reactive antibodies targeting conserved epitopes of the envelope proteins E1E2 is a key requirement for an HCV vaccine. Conserved epitopes like the viral CD81-binding site are targeted by rare broadly neutralizing antibodies. However, these viral segments are occluded by variable regions and glycans. We aimed to identify antigens exposing conserved epitopes and to characterize their immunogenicity. We created HCV variants with mutated glycosylation sites and/or hypervariable region 1 (HVR1). Exposure of the CD81 binding site and conserved epitopes was quantified by soluble CD81 and antibody interaction and neutralization assays. E2 or E1-E2 heterodimers with mutations causing epitope exposure were used to immunize mice. Vaccine-induced antibodies were examined and compared with patient-derived antibodies. Mutant viruses bound soluble CD81 and antibodies targeting the CD81 binding site with enhanced efficacy. Mice immunized with E2 or E1E2 heterodimers incorporating these modifications mounted strong, cross-binding, and non-interfering antibodies. E2-induced antibodies neutralized the autologous virus but they were not cross-neutralizing. Viruses lacking the HVR1 and selected glycosylation sites expose the CD81 binding site and cross-neutralization antibody epitopes. Recombinant E2 proteins carrying these modifications induce strong cross-binding but not cross-neutralizing antibodies.
    • Functional design of pH-responsive folate-targeted polymer-coated gold nanoparticles for drug delivery and in vivo therapy in breast cancer

      Mahalunkar, Sneha; Yadav, Amit Singh; Gorain, Mahadeo; Pawar, Vinay; Braathen, Ranveig; Weiss, Siegfried; Bogen, Bjarne; Gosavi, Suresh W.; Kundu, Gopal C.; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2019-01-01)
      Background: Curcumin has been widely used owing to its various medicinal properties including antitumor effects. However, its clinical application is limited by its instability, poor solubility and low bioavailability. Folic acid (FA)-functionalized nanoformulations may enhance the sustained release of an anticancer drug (curcumin) by tumor-specific targeting to improve therapeutic benefit. This study aims to design a nanoconjugate (NC) comprised of folate–curcumin-loaded gold–polyvinylpyrrolidone nanoparticles (FA–CurAu-PVP NPs) for targeted delivery in breast cancer model systems. Methods: We developed curcumin-loaded FA-functionalized Au-PVP NCs by layer-by-layer assembly. The folic acid–curcumin Au-PVP NCs (FA–CurAu-PVP NCs) were characterized by ultraviolet–visible spectra, Fourier transform infrared spectroscopy, X-ray powder diffraction and thermogravimetric analysis. In vitro anticancer and antimigratory effects of NCs were examined by performing MTT and wound migration assays. The in vivo antitumor efficacy of NCs was investigated using a preclinical breast cancer orthotopic mouse model. Results: Curcumin (40 µg/mL) was loaded along with conjugation of folate onto Au-PVP NPs to form FA–CurAu-PVP NCs. The size and charge of the NCs were increased gradually through layer-by-layer assembly and showed 80% release of curcumin at acidic pH. The NC did not show aggregation when incubated with human serum and mimicked an intrinsic peroxidase-like property in the presence of 3,3ʹ,5,5ʹ-tetramethylbenzidine substrate. The MTT data using these NCs showed efficient anticancer activity at lower doses in estrogen/ progesterone receptor (ER/PR)-negative cells compared with ER/PR-positive cells. Furthermore, the NCs did not show cytotoxicity at the investigated concentration in human breast epithelial and mouse fibroblast cell lines. They showed inhibitory effects on cell migration and high antitumor efficacy in in vivo analysis. Conclusion: These results suggest that folate-based tumor targeting using CurAu-PVP NCs is a promising approach for tumor-specific therapy of breast cancer without harming normal cells.
    • Functional metagenomics unveils a multifunctional glycosyl hydrolase from the family 43 catalysing the breakdown of plant polymers in the calf rumen.

      Ferrer, Manuel; Ghazi, Azam; Beloqui, Ana; Vieites, José María; López-Cortés, Nieves; Marín-Navarro, Julia; Nechitaylo, Taras Y; Guazzaroni, María-Eugenia; Polaina, Julio; Waliczek, Agnes; et al. (2012)
      Microbial communities from cow rumen are known for their ability to degrade diverse plant polymers at high rates. In this work, we identified 15 hydrolases through an activity-centred metagenome analysis of a fibre-adherent microbial community from dairy cow rumen. Among them, 7 glycosyl hydrolases (GHs) and 1 feruloyl esterase were successfully cloned, expressed, purified and characterised. The most striking result was a protein of GH family 43 (GHF43), hereinafter designated as R_09-02, which had characteristics very distinct from the other proteins in this family with mono-functional β-xylosidase, α-xylanase, α-L-arabinase and α-L-arabinofuranosidase activities. R_09-02 is the first multifunctional enzyme to exhibit β-1,4 xylosidase, α-1,5 arabinofur(pyr)anosidase, β-1,4 lactase, α-1,6 raffinase, α-1,6 stachyase, β-galactosidase and α-1,4 glucosidase activities. The R_09-02 protein appears to originate from the chromosome of a member of Clostridia, a class of phylum Firmicutes, members of which are highly abundant in ruminal environment. The evolution of R_09-02 is suggested to be driven from the xylose- and arabinose-specific activities, typical for GHF43 members, toward a broader specificity to the glucose- and galactose-containing components of lignocellulose. The apparent capability of enzymes from the GHF43 family to utilise xylose-, arabinose-, glucose- and galactose-containing oligosaccharides has thus far been neglected by, or could not be predicted from, genome and metagenome sequencing data analyses. Taking into account the abundance of GHF43-encoding gene sequences in the rumen (up to 7% of all GH-genes) and the multifunctional phenotype herein described, our findings suggest that the ecological role of this GH family in the digestion of ligno-cellulosic matter should be significantly reconsidered.
    • Future Organization of Clinical Research in Germany: The Road to the "German Centre for Digestive Health" (GCDH).

      Manns, Michael P; Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School (MHH), Hannover, Germany. (2016-12)
    • Gain of function in Jak2V617F-positive T-cells.

      Nishanth, G; Wolleschak, D; Fahldieck, C; Fischer, T; Mullally, A; Perner, F; Schnöder, T M; Just, S; Heidel, F H; Schlüter, D; et al. (2017-04)
    • Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica.

      Kube, Michael; Chernikova, Tatyana N; Al-Ramahi, Yamal; Beloqui, Ana; Lopez-Cortez, Nieves; Guazzaroni, María-Eugenia; Heipieper, Hermann J; Klages, Sven; Kotsyurbenko, Oleg R; Langer, Ines; et al. (2013-07-23)
      Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis-the paradigm of mesophilic hydrocarbonoclastic bacteria-O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.
    • Genomewide identification of genetic determinants of antimicrobial drug resistance in Pseudomonas aeruginosa.

      Dötsch, Andreas; Becker, Tanja; Pommerenke, Claudia; Magnowska, Zofia; Jänsch, Lothar; Häussler, Susanne; Chronic Pseudomonas Infections Research Group, Helmholtz Center for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. (2009-06)
      The emergence of antimicrobial drug resistance is of enormous public concern due to the increased risk of delayed treatment of infections, the increased length of hospital stays, the substantial increase in the cost of care, and the high risk of fatal outcomes. A prerequisite for the development of effective therapy alternatives is a detailed understanding of the diversity of bacterial mechanisms that underlie drug resistance, especially for problematic gram-negative bacteria such as Pseudomonas aeruginosa. This pathogen has impressive chromosomally encoded mechanisms of intrinsic resistance, as well as the potential to mutate, gaining resistance to current antibiotics. In this study we have screened the comprehensive nonredundant Harvard PA14 library for P. aeruginosa mutants that exhibited either increased or decreased resistance against 19 antibiotics commonly used in the clinic. This approach identified several genes whose inactivation sensitized the bacteria to a broad spectrum of different antimicrobials and uncovered novel genetic determinants of resistance to various classes of antibiotics. Knowledge of the enhancement of bacterial susceptibility to existing antibiotics and of novel resistance markers or modifiers of resistance expression may lay the foundation for effective therapy alternatives and will be the basis for the development of new strategies in the control of problematic multiresistant gram-negative bacteria.
    • Global genotype-phenotype correlations in Pseudomonas aeruginosa.

      Pommerenke, Claudia; Müsken, Mathias; Becker, Tanja; Dötsch, Andreas; Klawonn, Frank; Häussler, Susanne; Chronic Pseudomonas Infections, Helmholtz Center for Infection Research, Braunschweig, Germany. (2010)
      Once the genome sequence of an organism is obtained, attention turns from identifying genes to understanding their function, their organization and control of metabolic pathways and networks that determine its physiology. Recent technical advances in acquiring genome-wide data have led to substantial progress in identifying gene functions. However, we still do not know the function of a large number of genes and, even when a gene product has been assigned to a functional class, we cannot normally predict its contribution to the phenotypic behaviour of the cell or organism--the phenome. In this study, we assessed bacterial growth parameters of 4030 non-redundant PA14 transposon mutants in the pathogenic bacterium Pseudomonas aeruginosa. The genome-wide simultaneous analysis of 119 distinct growth-related phenotypes uncovered a comprehensive phenome and provided evidence that most genotypes are not phenotypically isolated but rather define specific complex phenotypic clusters of genotypes. Since phenotypic overlap was demonstrated to reflect the relatedness of genotypes on a global scale, knowledge of an organism's phenome might significantly contribute to the advancement of functional genomics.
    • Glutathione Restricts Serine Metabolism to Preserve Regulatory T Cell Function.

      Kurniawan, Henry; Franchina, Davide G; Guerra, Luana; Bonetti, Lynn; -Baguet, Leticia Soriano; Grusdat, Melanie; Schlicker, Lisa; Hunewald, Oliver; Dostert, Catherine; Merz, Myriam P; et al. (Elsevier (Cell Press), 2020-03-25)
      Regulatory T cells (Tregs) maintain immune homeostasis and prevent autoimmunity. Serine stimulates glutathione (GSH) synthesis and feeds into the one-carbon metabolic network (1CMet) essential for effector T cell (Teff) responses. However, serine's functions, linkage to GSH, and role in stress responses in Tregs are unknown. Here, we show, using mice with Treg-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), that GSH loss in Tregs alters serine import and synthesis and that the integrity of this feedback loop is critical for Treg suppressive capacity. Although Gclc ablation does not impair Treg differentiation, mutant mice exhibit severe autoimmunity and enhanced anti-tumor responses. Gclc-deficient Tregs show increased serine metabolism, mTOR activation, and proliferation but downregulated FoxP3. Limitation of cellular serine in vitro and in vivo restores FoxP3 expression and suppressive capacity of Gclc-deficient Tregs. Our work reveals an unexpected role for GSH in restricting serine availability to preserve Treg functionality.
    • Groundwater, soil and compost, as possible sources of virulent and antibiotic-resistant Pseudomonas aeruginosa.

      Kaszab, Edit; Radó, Júlia; Kriszt, Balázs; Pászti, Judit; Lesinszki, Virág; Szabó, Ádám; Tóth, Gergő; Khaledi, Ariane; Szoboszlay, Sándor; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Taylor & Francis, 2019-11-18)
      Pseudomonas aeruginosa is a major public health concern all around the world. In the frame of this work, a set of diverse environmental P. aeruginosa isolates with various antibiotic resistance profiles were examined in a Galleria mellonella virulence model. Motility, serotypes, virulence factors and biofilm-forming ability were also examined. Molecular types were determined by pulsed-field gel electrophoresis (PFGE). Based on our results, the majority of environmental isolates were virulent in the G. mellonella test and twitching showed a positive correlation with mortality. Resistance against several antibiotic agents such as Imipenem correlated with a lower virulence in the applied G. mellonella model. PFGE revealed that five examined environmental isolates were closely related to clinically detected pulsed-field types. Our study demonstrated that industrial wastewater effluents, composts, and hydrocarbon-contaminated sites should be considered as hot spots of high-risk clones of P. aeruginosa.
    • Guidelines for Small-Scale Production and Purification of Hepatitis B Surface Antigen Virus-Like Particles from Recombinant Pichia pastoris.

      Zahid, Maria; Rinas, Ursula; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Humana Press, 2019-01-01)
      Virus-like particle (VLP)-based vaccines have been in the market since decades for preventing viral infection and have proven their usefulness also in other areas of biotechnology. Here, we describe in detail simple small-scale production and purification procedures for the generation of hepatitis B surface antigen (HBsAg) VLPs using Pichia pastoris as expression host. This protocol may also be applicable with variations to other HBsAg-based VLPs additionally carrying antigens of other pathogens.
    • HBV evolution and genetic variability: Impact on prevention, treatment and development of antivirals.

      Glebe, Dieter; Goldmann, Nora; Lauber, Chris; Seitz, Stefan; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Elsevier, 2020-11-06)
      Hepatitis B virus (HBV) poses a major global health burden with 260 million people being chronically infected and 890,000 dying annually from complications in the course of the infection. HBV is a small enveloped virus with a reverse-transcribed DNA genome that infects hepatocytes and can cause acute and chronic infections of the liver. HBV is endemic in humans and apes representing the prototype member of the viral family Hepadnaviridae and can be divided into 10 genotypes. Hepadnaviruses have been found in all vertebrate classes and constitute an ancient viral family that descended from non-enveloped progenitors more than 360 million years ago. The de novo emergence of the envelope protein gene was accompanied with the liver-tropism and resulted in a tight virus-host association. The oldest HBV genomes so far have been isolated from human remains of the Bronze Age and the Neolithic (~7000 years before present). Despite the remarkable stability of the hepadnaviral genome over geological eras, HBV is able to rapidly evolve within an infected individual under pressure of the immune response or during antiviral treatment. Treatment with currently available antivirals blocking intracellular replication of HBV allows controlling of high viremia and improving liver health during long-term therapy of patients with chronic hepatitis B (CHB), but they are not sufficient to cure the disease. New therapy options that cover all HBV genotypes and emerging viral variants will have to be developed soon. In addition to the antiviral treatment of chronically infected patients, continued efforts to expand the global coverage of the currently available HBV vaccine will be one of the key factors for controlling the rising global spread of HBV. Certain improvements of the vaccine (e.g. inclusion of PreS domains) could counteract known problems such as low or no responsiveness of certain risk groups and waning anti-HBs titers leading to occult infections, especially with HBV genotypes E or F. But even with an optimal vaccine and a cure for hepatitis B, global eradication of HBV would be difficult to achieve because of an existing viral reservoir in primates and bats carrying closely related hepadnaviruses with zoonotic potential.
    • Helicobacter pylori vacA genotype is a predominant determinant of immune response to Helicobacter pylori CagA.

      Link, Alexander; Langner, Cosima; Schirrmeister, Wiebke; Habendorf, Wiebke; Weigt, Jochen; Venerito, Marino; Tammer, Ina; Schlüter, Dirk; Schlaermann, Philipp; Meyer, Thomas F; et al. (2017-07-14)
      To evaluate the frequency of Helicobacter pylori (H. pylori) CagA antibodies in H. pylori infected subjects and to identify potential histopathological and bacterial factors related to H. pylori CagA-immune response.
    • Heparin: role in protein purification and substitution with animal-component free material.

      Bolten, Svenja Nicolin; Rinas, Ursula; Scheper, Thomas; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer, 2018-10-01)
      Heparin is a highly sulfated polysaccharide which belongs to the family of glycosaminoglycans. It is involved in various important biological activities. The major biological purpose is the inhibition of the coagulation cascade to maintain the blood flow in the vasculature. These properties are employed in several therapeutic drugs. Heparin's activities are associated with its interaction to various proteins. To date, the structural heparin-protein interactions are not completely understood. This review gives a general overview of specific patterns and functional groups which are involved in the heparin-protein binding. An understanding of the heparin-protein interactions at the molecular level is not only advantageous in the therapeutic application but also in biotechnological application of heparin for downstreaming. This review focuses on the heparin affinity chromatography. Diverse recombinant proteins can be successfully purified by this method. While effective, it is disadvantageous that heparin is an animal-derived material. Animal-based components carry the risk of contamination. Therefore, they are liable to strict quality controls and the validation of effective good manufacturing practice (GMP) implementation. Hence, adequate alternatives to animal-derived components are needed. This review examines strategies to avoid these disadvantages. Thereby, alternatives for the provision of heparin such as chemical synthesized heparin, chemoenzymatic heparin, and bioengineered heparin are discussed. Moreover, the usage of other chromatographic systems mimetic the heparin effect is reviewed.
    • Hepatitis D virus in Africa: several unmet needs.

      Manns, Michael P; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-10)
    • Hepatitis E Virus (HEV)-Specific T Cell Receptor Cross-Recognition: Implications for Immunotherapy.

      Soon, Chai Fen; Zhang, Shihong; Suneetha, Pothakamuri Venkata; Antunes, Dinler Amaral; Manns, Michael Peter; Raha, Solaiman; Schultze-Florey, Christian; Prinz, Immo; Wedemeyer, Heiner; Sällberg Chen, Margaret; et al. (Frontiers, 2019-01-01)
      T cell immunotherapy is a concept developed for the treatment of cancer and infectious diseases, based on cytotoxic T lymphocytes to target tumor- or pathogen-specific antigens. Antigen-specificity of the T cell receptors (TCRs) is an important selection criterion in the developmental design of immunotherapy. However, off-target specificity is a possible autoimmunity concern if the engineered antigen-specific T cells are cross-reacting to self-peptides in-vivo. In our recent work, we identified several hepatitis E virus (HEV)-specific TCRs as potential candidates to be developed into T cell therapy to treat chronic hepatitis E. One of the identified TCRs, targeting a HLA-A2-restricted epitope at the RNA-dependent RNA polymerase (HEV-1527: LLWNTVWNM), possessed a unique multiple glycine motif in the TCR-β CDR3, which might be a factor inducing cross-reactivity. The aim of our study was to explore if this TCR could cross-recognize self-peptides to underlay autoimmunity. Indeed, we found that this HEV-1527-specific TCR could also cross-recognize an apoptosis-related epitope, Nonmuscle Myosin Heavy Chain 9 (MYH9-478: QLFNHTMFI). While this TCR had dual specificities to both viral epitope and a self-antigen by double Dextramer binding, it was selectively functional against HEV-1527 but not activated against MYH9-478. The consecutive glycine motif in β chain may be the reason promoting TCR binding promiscuity to recognize a secondary target, thereby facilitating cross-recognition. In conclusion, candidate TCRs for immunotherapy development should be screened for autoimmune potential, especially when the TCRs exhibit unique sequence pattern.