Generation of nanobodies from transgenic 'LamaMice' lacking an endogenous immunoglobulin repertoire.

Due to their exceptional solubility and stability, nanobodies have emerged as powerful building blocks for research tools and therapeutics. However, their generation in llamas is cumbersome and costly. Here, by inserting an engineered llama immunoglobulin heavy chain (IgH) locus into IgH-deficient mice, we generate a transgenic mouse line, which we refer to as 'LamaMouse'. We demonstrate that LamaMice solely express llama IgH molecules without association to Igκ or λ light chains. Immunization of LamaMice with AAV8, the receptor-binding domain of the SARS-CoV-2 spike protein, IgE, IgG2c, and CLEC9A enabled us to readily select respective target-specific nanobodies using classical hybridoma and phage display technologies, single B cell screening, and direct cloning of the nanobody-repertoire into a mammalian expression vector. Our work shows that the LamaMouse represents a flexible and broadly applicable platform for a facilitated selection of target-specific nanobodies.

CD38-specific nanobodies allow in vivo imaging of multiple myeloma under daratumumab therapy.

Rationale: Recent studies have demonstrated the feasibility of CD38-specific antibody constructs for in vivo imaging of multiple myeloma. However, detecting multiple myeloma in daratumumab-pretreated patients remains difficult due to overlapping binding epitopes of the CD38-specific imaging antibody constructs and daratumumab. Therefore, the development of an alternative antibody construct targeting an epitope of CD38 distinct from that of daratumumab is needed. We report the generation of a fluorochrome-conjugated nanobody recognizing such an epitope of CD38 to detect myeloma cells under daratumumab therapy in vitro, ex vivo, and in vivo. Methods: We conjugated the CD38-specific nanobody JK36 to the near-infrared fluorescent dye Alexa Fluor 680. The capacity of JK36AF680 to bind and detect CD38-expressing cells pretreated with daratumumab was evaluated on CD38-expressing tumor cell lines in vitro, on primary myeloma cells from human bone marrow biopsies ex vivo, and in a mouse tumor model in vivo. Results: Fluorochrome-labeled nanobody JK36AF680 showed specific binding to CD38-expressing myeloma cells pretreated with daratumumab in vitro and ex vivo and allowed for specific imaging of CD38-expressing xenografts in daratumumab-pretreated mice in vivo. Conclusions: Our study demonstrates that a nanobody recognizing a distinct, non-overlapping epitope of CD38 allows the specific detection of myeloma cells under daratumumab therapy in vitro, ex vivo, and in vivo.

Half-Life Extended Nanobody-Based CD38-Specific Bispecific Killercell Engagers Induce Killing of Multiple Myeloma Cells.

CD38 is a target for immunotherapy of multiple myeloma. Llama-derived CD38-specific nanobodies allow easy reformatting into mono-, bi- and multispecific proteins. To evaluate the utility of nanobodies for constructing CD38-specific nanobody-based killer cell engagers (nano-BiKEs), we generated half-life extended nano-BiKEs (HLE-nano-BiKEs) by fusing a CD38-specific nanobody to a CD16-specific nanobody for binding to the Fc-receptor on NK cells and further to an albumin-specific nanobody to extend the half-life in vivo. HLE-nano-BiKEs targeting three different epitopes (E1, E2, E3) of CD38 were expressed in transiently transfected HEK-6E cells. We verified specific and simultaneous binding to CD38 on myeloma cells, CD16 on NK cells, and to albumin. We tested the capacity of these HLE-nano-BiKEs to mediate cytotoxicity against CD38-expressing multiple myeloma cell lines and primary myeloma cells from human bone marrow biopsies in bioluminescence and flowcytometry assays with NK92 cells as effector cells. The results revealed specific time- and dose-dependent cytolysis of CD38+ myeloma cell lines and effective depletion of CD38-expressing multiple myeloma cells from primary human bone marrow samples. Our results demonstrate the efficacy of CD38-specific HLE-nano-BiKEs in vitro and ex vivo, warranting further preclinical evaluation in vivo of their therapeutic potential for the treatment of multiple myeloma.

Bitter taste signaling in tracheal epithelial brush cells elicits innate immune responses to bacterial infection.

Constant exposure of the airways to inhaled pathogens requires efficient early immune responses protecting against infections. How bacteria on the epithelial surface are detected and first-line protective mechanisms are initiated are not well understood. We have recently shown that tracheal brush cells (BCs) express functional taste receptors. Here we report that bitter taste signaling in murine BCs induces neurogenic inflammation. We demonstrate that BC signaling stimulates adjacent sensory nerve endings in the trachea to release the neuropeptides CGRP and substance P that mediate plasma extravasation, neutrophil recruitment, and diapedesis. Moreover, we show that bitter tasting quorum-sensing molecules from Pseudomonas aeruginosa activate tracheal BCs. BC signaling depends on the key taste transduction gene Trpm5, triggers secretion of immune mediators, among them the most abundant member of the complement system, and is needed to combat P. aeruginosa infections. Our data provide functional insight into first-line defense mechanisms against bacterial infections of the lung.

Impact of a University-Wide Interdisciplinary Mind-Body Skills Program on Student Mental and Emotional Well-Being.

BACKGROUND: Positive effects of mind-body skills programs on participant well-being have been reported in health professions students. The success seen with medical students at this university led to great interest in expanding the mind-body skills program so students in other disciplines could benefit from the program. OBJECTIVE: The purpose of this study was to assess the effects of a 9-week mind-body skills program on the mental and emotional well-being of multidisciplinary students compared to controls. We also sought to determine if the program's effects were sustained at 1-year follow-up. METHODS: A cross-sectional pre-post survey was administered online via SurveyMonkey to participants of a 9-week mind-body skills program and a control group of students from 7 colleges at a public university from 2017-2019. Students were assessed on validated measures of stress, positive/negative affect, resilience, depression, anxiety, fatigue, sleep disturbance, mindfulness, empathy, and burnout. Scores were analyzed between-groups and within-groups using bivariate and multivariate analyses. A 1-year follow-up was completed on a subset of participants and controls. RESULTS: 279 participants and 247 controls completed the pre-survey and post-survey (79% response rate; 71% female, 68% white, mean age = 25 years). Participants showed significant decreases in stress, negative affect, depression, anxiety, sleep disturbance, and burnout, while positive affect, resilience, mindfulness, and empathy increased significantly (P < .05). Only sleep disturbance showed a significant decrease in the control group. Follow-up in a subset of participants showed that only mindfulness remained elevated at 1-year (P < .05), whereas the significant changes in other well-being measures were not sustained. CONCLUSION: Participation in a 9-week mind-body skills program led to significant improvement in indicators of well-being in multidisciplinary students. A pilot 1-year follow-up suggests that effects are only sustained for mindfulness, but not other parameters. Future programming should focus on implementing mind-body skills booster sessions to help sustain the well-being benefits.

The alternative cap-binding complex is required for antiviral defense in vivo.

Cellular response to environmental challenges requires immediate and precise regulation of transcriptional programs. During viral infections, this includes the expression of antiviral genes that are essential to combat the pathogen. Transcribed mRNAs are bound and escorted to the cytoplasm by the cap-binding complex (CBC). We recently identified a protein complex consisting of NCBP1 and NCBP3 that, under physiological conditions, has redundant function to the canonical CBC, consisting of NCBP1 and NCBP2. Here, we provide evidence that NCBP3 is essential to mount a precise and appropriate antiviral response. Ncbp3-deficient cells allow higher virus growth and elicit a reduced antiviral response, a defect happening on post-transcriptional level. Ncbp3-deficient mice suffered from severe lung pathology and increased morbidity after influenza A virus challenge. While NCBP3 appeared to be particularly important during viral infections, it may be more broadly involved to ensure proper protein expression.

Current pharmacotherapy for the treatment of dyslipidemia associated with HIV infection.

Introduction: Cardiovascular disease is an important cause of morbidity and mortality in persons with human immunodeficiency virus (PWH). The risk of atherosclerotic cardiovascular disease (ASCVD) is higher in PWH compared to uninfected persons. Dyslipidemia is a critical link in the pathogenesis of ASCVD in PWH. Chronic inflammation associated with HIV infection may drive both dyslipidemia and ASCVD. Areas covered: The authors review the evidence for using lipid-lowering therapy in PWH and includes an overview of the utility and complexity of using statins in PWH, in particular, drug interactions, safety, and efficacy. In addition, data covering alternate therapies like omega-3 fatty acids, fibrates, niacin, ezetimibe, and PCSK-9 inhibitors are reviewed. Expert opinion: Dyslipidemia is a common problem in PWH. The risk of ASCVD is higher in PWH. Lipid-lowering therapy reduces the risk of ASCVD, but clinical endpoint trials are lacking in PWH. Statin therapy is the mainstay of primary prevention for ASCVD. The timing of when to initiate primary prevention with statins in PWH is unclear. Beyond statins, there are limited data that other lipid-lowering agents have utility in PWH. Ongoing trials like the REPRIEVE trial will inform the community about the optimal approach to lipid-lowering therapy in PWH.

Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a phosphoprotein that plays key, yet poorly defined, roles in both virus genome replication and virion assembly/release. It has been proposed that differential phosphorylation could act as a switch to regulate the various functions of NS5A; however, the mechanistic details of the role of this posttranslational modification in the virus life cycle remain obscure. We previously reported (D. Ross-Thriepland, J. Mankouri, and M. Harris, J Virol 89:3123-3135, 2015, doi:10.1128/JVI.02995-14) a role for phosphorylation at serine 225 (S225) of NS5A in the regulation of JFH-1 (genotype 2a) genome replication. A phosphoablatant (S225A) mutation resulted in a 10-fold reduction in replication and a perinuclear restricted distribution of NS5A, whereas the corresponding phosphomimetic mutation (S225D) had no phenotype. To determine the molecular mechanisms underpinning this phenotype we conducted a label-free proteomics approach to identify cellular NS5A interaction partners. This analysis revealed that the S225A mutation disrupted the interactions of NS5A with a number of cellular proteins, in particular the nucleosome assembly protein 1-like protein 1 (NAP1L1), bridging integrator 1 (Bin1, also known as amphiphysin II), and vesicle-associated membrane protein-associated protein A (VAP-A). These interactions were validated by immunoprecipitation/Western blotting, immunofluorescence, and proximity ligation assay. Importantly, small interfering RNA (siRNA)-mediated knockdown of NAP1L1, Bin1 or VAP-A impaired viral genome replication and recapitulated the perinuclear redistribution of NS5A seen in the S225A mutant. These results demonstrate that S225 phosphorylation regulates the interactions of NS5A with a defined subset of cellular proteins. Furthermore, these interactions regulate both HCV genome replication and the subcellular localization of replication complexes.IMPORTANCE Hepatitis C virus is an important human pathogen. The viral nonstructural 5A protein (NS5A) is the target for new antiviral drugs. NS5A has multiple functions during the virus life cycle, but the biochemical details of these roles remain obscure. NS5A is known to be phosphorylated by cellular protein kinases, and in this study, we set out to determine whether this modification is required for the binding of NS5A to other cellular proteins. We identified 3 such proteins and show that they interacted only with NS5A that was phosphorylated on a specific residue. Furthermore, these proteins were required for efficient virus replication and the ability of NS5A to spread throughout the cytoplasm of the cell. Our results help to define the function of NS5A and may contribute to an understanding of the mode of action of the highly potent antiviral drugs that are targeted to NS5A.

Discrimination of Self and Non-Self Ribonucleic Acids.

Most virus infections are controlled through the innate and adaptive immune system. A surprisingly limited number of so-called pattern recognition receptors (PRRs) have the ability to sense a large variety of virus infections. The reason for the broad activity of PRRs lies in the ability to recognize viral nucleic acids. These nucleic acids lack signatures that are present in cytoplasmic cellular nucleic acids and thereby marking them as pathogen-derived. Accumulating evidence suggests that these signatures, which are predominantly sensed by a class of PRRs called retinoic acid-inducible gene I (RIG-I)-like receptors and other proteins, are not unique to viruses but rather resemble immature forms of cellular ribonucleic acids generated by cellular polymerases. RIG-I-like receptors, and other cellular antiviral proteins, may therefore have mainly evolved to sense nonprocessed nucleic acids typically generated by primitive organisms and pathogens. This capability has not only implications on induction of antiviral immunity but also on the function of cellular proteins to handle self-derived RNA with stimulatory potential.

The Hepatitis E virus intraviral interactome.

Hepatitis E virus (HEV) is an emerging virus causing epidemic acute hepatitis in developing countries as well as sporadic cases in industrialized countries. The life cycle of HEV is still poorly understood and the lack of efficient cell culture systems and animal models are the principal limitations for a detailed study of the viral replication cycle. Here we exhaustively examine all possible intraviral protein-protein interactions (PPIs) of HEV by systematic Yeast two-hybrid (Y2H) and LuMPIS screens, providing a basis for studying the function of these proteins in the viral replication cycle. Key PPIs correlate with the already published HEV 3D structure. Furthermore, we report 20 novel PPIs including the homodimerization of the RNA dependent RNA polymerase (RdRp), the self-interaction of the papain like protease, and ORF3 interactions with the papain-like protease and putative replicase components: RdRp, methylase and helicase. Furthermore, we determined the dissociation constant (Kd) of ORF3 interactions with the viral helicase, papain-like protease and methylase, which suggest a regulatory function for ORF3 in orchestrating the formation of the replicase complex. These interactions may represent new targets for antiviral drugs.

mRNA export through an additional cap-binding complex consisting of NCBP1 and NCBP3.

The flow of genetic information from DNA to protein requires polymerase-II-transcribed RNA characterized by the presence of a 5'-cap. The cap-binding complex (CBC), consisting of the nuclear cap-binding protein (NCBP) 2 and its adaptor NCBP1, is believed to bind all capped RNA and to be necessary for its processing and intracellular localization. Here we show that NCBP1, but not NCBP2, is required for cell viability and poly(A) RNA export. We identify C17orf85 (here named NCBP3) as a cap-binding protein that together with NCBP1 forms an alternative CBC in higher eukaryotes. NCBP3 binds mRNA, associates with components of the mRNA processing machinery and contributes to poly(A) RNA export. Loss of NCBP3 can be compensated by NCBP2 under steady-state conditions. However, NCBP3 becomes pivotal under stress conditions, such as virus infection. We propose the existence of an alternative CBC involving NCBP1 and NCBP3 that plays a key role in mRNA biogenesis.