ESAT-6 undergoes self-association at phagosomal pH and an ESAT-6-specific nanobody restricts M. tuberculosis growth in macrophages.

Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6's mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH-dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.

ESAT-6 undergoes self-association at phagosomal pH and an ESAT-6 specific nanobody restricts M. tuberculosis growth in macrophages.

Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6's mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.

Preventing suicide in refugees and asylum seekers: a rapid literature review examining the role of suicide prevention training for health and support staff.

BACKGROUND: Refugees and asylum seekers are exposed to a unique set of circumstances and experiences that are associated with an increased suicide risk. Suicide prevention training has been recognised as a central component supporting a comprehensive approach to suicide prevention. Limited literature exists exploring the role of suicide prevention training for health and support staff working with refugee and asylum seeker consumers. METHODS: To determine the impact suicide prevention training for health staff may have in supporting refugee and asylum seeker suicide prevention, researchers undertook a rapid literature review exploring what elements should be considered when developing suicide prevention training for health and support staff working with refugee and asylum seeker consumers. RESULTS: Results of academic and grey literature screening identified 14 studies exploring suicide prevention training for health and support staff working with refugee and asylum seeker consumers. Findings of the literature review suggest suicide prevention training for health and support staff working with refugee and asylum seekers should consider the inclusion of content which increases participant competence and confidence to identify and respond to suicide risk; provide staff with an understanding of cultural differences and its impact on refugees and asylum seekers recognition of mental health and suicide as a health matter; highlight the importance trauma informed practices in care and consider the lived experience of refugees and asylum seekers. CONCLUSIONS: Inclusion of specific content in refugee and asylum seeker suicide prevention training may provide health and support staff increased competence and confidence to identify and respond to suicide risk in refugees and asylum seekers.

Quality Measurement and Patient Outcomes in Inpatient Behavioral Health: Assessing the Current Framework.

ABSTRACT: Quality measurement across healthcare is undertaken with a goal of improving care and outcomes for patients; however, the relationship between quality measurement and patient outcomes remains largely untested, particularly in inpatient behavioral health. Using a retrospective quantitative design, we assessed 142 behavioral health organizations' quality data submitted to the Hospital-Based Inpatient Psychiatric Services and Inpatient Psychiatric Facility Quality Reporting programs from 2017 to 2018 and tested relationships between compliance on 16 quality measures and symptom improvement on patient self-report outcomes (SROs) at the facility level. Performance on many quality measures was negatively skewed (at least four have almost no room for improvement on average), and there was high interrelatedness between most quality measures. Nine of the assessed measures correlated with patient SROs but not in clear groupings. Findings indicate that an underlying organizational construct may be driving compliance rates on quality measures, but the measures are not linked to treatment outcomes as expected. We encourage an expansion of the current framework of behavioral health quality measurement beyond process and organization and suggest the addition of patient outcomes such as SROs as quality measures to directly assess patient improvement.

Immune Response to Enterococcus gallinarum in Lupus Patients Is Associated With a Subset of Lupus-Associated Autoantibodies.

Interactions between gut microbes and the immune system influence autoimmune disorders like systemic lupus erythematosus (SLE). Recently, Enterococcus gallinarum, a gram-positive commensal gut bacterium, was implicated as a candidate pathobiont in SLE. The present study was undertaken to evaluate the influence of E. gallinarum exposure on clinical parameters of SLE. Since circulating IgG antibodies to whole bacteria have been established as a surrogate marker for bacterial exposure, anti-E. gallinarum IgG antibodies were measured in banked serum samples from SLE patients and healthy controls in the Oklahoma Cohort for Rheumatic Diseases. The associations between anti-E. gallinarum antibody titers and clinical indicators of lupus were studied. Antibodies to human RNA were studied in a subset of patients. Our results show that sera from both patients and healthy controls had IgG and IgA antibodies reactive with E. gallinarum. The antibody titers between the two groups were not different. However, SLE patients with Ribosomal P autoantibodies had higher anti-E. gallinarum IgG titers compared to healthy controls. In addition to anti-Ribosomal P, higher anti-E. gallinarum titers were also significantly associated with the presence of anti-dsDNA and anti-Sm autoantibodies. In the subset of patients with anti-Ribosomal P and anti-dsDNA, the anti-E. gallinarum titers correlated significantly with antibodies to human RNA. Our data show that both healthy individuals and SLE patients were sero-reactive to E. gallinarum. In SLE patients, the immune response to E. gallinarum was associated with antibody response to a specific subset of lupus autoantigens. These findings provide additional evidence that E. gallinarum may be a pathobiont for SLE in susceptible individuals.

A nanobody suite for yeast scaffold nucleoporins provides details of the nuclear pore complex structure.

Nuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 or inner ring complex. Working in S. cerevisiae, and to study the assembly of these two essential subcomplexes, we here develop a set of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. These nanobodies all bind specifically and with high affinity. We present structures of several nup-nanobody complexes, revealing their binding sites. Additionally, constitutive expression of the nanobody suite in S. cerevisiae detect accessible and obstructed surfaces of the Y complex and Nic96 within the NPC. Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.

Neoleukin-2 enhances anti-tumour immunity downstream of peptide vaccination targeted by an anti-MHC class II VHH.

Cancer-specific mutations can lead to peptides of unique sequence presented on MHC class I to CD8 T cells. These neoantigens can be potent tumour-rejection antigens, appear to be the driving force behind responsiveness to anti-CTLA-4 and anti-PD1/L1-based therapies and have been used to develop personalized vaccines. The platform for delivering neoantigen-based vaccines has varied, and further optimization of both platform and adjuvant will be necessary to achieve scalable vaccine products that are therapeutically effective at a reasonable cost. Here, we developed a platform for testing potential CD8 T cell tumour vaccine candidates. We used a high-affinity alpaca-derived VHH against MHC class II to deliver peptides to professional antigen-presenting cells. We show in vitro and in vivo that peptides derived from the model antigen ovalbumin are better able to activate naive ovalbumin-specific CD8 T cells when conjugated to an MHC class II-specific VHH when compared with an irrelevant control VHH. We then used the VHH-peptide platform to evaluate a panel of candidate neoantigens in vivo in a mouse model of pancreatic cancer. None of the candidate neoantigens tested led to protection from tumour challenge; however, we were able to show vaccine-induced CD8 T cell responses to a melanoma self-antigen that was augmented by combination therapy with the synthetic cytokine mimetic Neo2/15.

Improved Antitumor Efficacy of Chimeric Antigen Receptor T Cells that Secrete Single-Domain Antibody Fragments.

Chimeric antigen receptor (CAR) T-cell therapy is effective in the treatment of cancers of hematopoietic origin. In the immunosuppressive solid tumor environment, CAR T cells encounter obstacles that compromise their efficacy. We developed a strategy to address these barriers by having CAR T cells secrete single-domain antibody fragments [variable heavy domain of heavy chain antibodies (VHH) or nanobodies] that can modify the intratumoral immune landscape and thus support CAR T-cell function in immunocompetent animals. VHHs are small in size and able to avoid domain swapping when multiple nanobodies are expressed simultaneously-features that can endow CAR T cells with desirable properties. The secretion of an anti-CD47 VHH by CAR T cells improves engagement of the innate immune system, enables epitope spreading, and can enhance the antitumor response. CAR T cells that secrete anti-PD-L1 or anti-CTLA-4 nanobodies show improved persistence and demonstrate the versatility of this approach. Furthermore, local delivery of secreted anti-CD47 VHH-Fc fusions by CAR T cells at the tumor site limits their systemic toxicity. CAR T cells can be further engineered to simultaneously secrete multiple modalities, allowing for even greater tailoring of the antitumor immune response.

Noninvasive imaging of tumor progression, metastasis, and fibrosis using a nanobody targeting the extracellular matrix.

Extracellular matrix (ECM) deposition is a hallmark of many diseases, including cancer and fibroses. To exploit the ECM as an imaging and therapeutic target, we developed alpaca-derived libraries of "nanobodies" against disease-associated ECM proteins. We describe here one such nanobody, NJB2, specific for an alternatively spliced domain of fibronectin expressed in disease ECM and neovasculature. We showed by noninvasive in vivo immuno-PET/CT imaging that NJB2 detects primary tumors and metastatic sites with excellent specificity in multiple models of breast cancer, including human and mouse triple-negative breast cancer, and in melanoma. We also imaged mice with pancreatic ductal adenocarcinoma (PDAC) in which NJB2 was able to detect not only PDAC tumors but also early pancreatic lesions called pancreatic intraepithelial neoplasias, which are challenging to detect by any current imaging modalities, with excellent clarity and signal-to-noise ratios that outperformed conventional 2-fluorodeoxyglucose PET/CT imaging. NJB2 also detected pulmonary fibrosis in a bleomycin-induced fibrosis model. We propose NJB2 and similar anti-ECM nanobodies as powerful tools for noninvasive detection of tumors, metastatic lesions, and fibroses. Furthermore, the selective recognition of disease tissues makes NJB2 a promising candidate for nanobody-based therapeutic applications.

Nanobody-based CAR T cells that target the tumor microenvironment inhibit the growth of solid tumors in immunocompetent mice.

Chimeric antigen receptor (CAR) T cell therapy has been successful in clinical trials against hematological cancers, but has experienced challenges in the treatment of solid tumors. One of the main difficulties lies in a paucity of tumor-specific targets that can serve as CAR recognition domains. We therefore focused on developing VHH-based, single-domain antibody (nanobody) CAR T cells that target aspects of the tumor microenvironment conserved across multiple cancer types. Many solid tumors evade immune recognition through expression of checkpoint molecules, such as PD-L1, that down-regulate the immune response. We therefore targeted CAR T cells to the tumor microenvironment via the checkpoint inhibitor PD-L1 and observed a reduction in tumor growth, resulting in improved survival. CAR T cells that target the tumor stroma and vasculature through the EIIIB+ fibronectin splice variant, which is expressed by multiple tumor types and on neovasculature, are likewise effective in delaying tumor growth. VHH-based CAR T cells can thus function as antitumor agents for multiple targets in syngeneic, immunocompetent animal models. Our results demonstrate the flexibility of VHH-based CAR T cells and the potential of CAR T cells to target the tumor microenvironment and treat solid tumors.

Targeting small molecule drugs to T cells with antibody-directed cell-penetrating gold nanoparticles.

We sought to develop a nanoparticle vehicle that could efficiently deliver small molecule drugs to target lymphocyte populations. The synthesized amphiphilic organic ligand-protected gold nanoparticles (amph-NPs) were capable of sequestering large payloads of small molecule drugs within hydrophobic pockets of their ligand shells. These particles exhibit membrane-penetrating activity in mammalian cells, and thus enhanced uptake of a small molecule TGF-ß inhibitor in T cells in cell culture. By conjugating amph-NPs with targeting antibodies or camelid-derived nanobodies, the particles' cell-penetrating properties could be temporarily suppressed, allowing targeted uptake in specific lymphocyte subpopulations. Degradation of the protein targeting moieties following particle endocytosis allowed the NPs to recover their cell-penetrating activity in situ to enter the cytoplasm of T cells. In vivo, targeted amph-NPs showed 40-fold enhanced uptake in CD8+ T cells relative to untargeted particles, and delivery of TGF-ß inhibitor-loaded particles to T cells enhanced their cytokine polyfunctionality in a cancer vaccine model. Thus, this system provides a facile approach to concentrate small molecule compounds in target lymphocyte populations of interest for immunotherapy in cancer and other diseases.

Sortase A: A Model for Transpeptidation and Its Biological Applications.

Molecular biologists and chemists alike have long sought to modify proteins with substituents that cannot be installed by standard or even advanced genetic approaches. We here describe the use of transpeptidases to achieve these goals. Living systems encode a variety of transpeptidases and peptide ligases that allow for the enzyme-catalyzed formation of peptide bonds, and protein engineers have used directed evolution to enhance these enzymes for biological applications. We focus primarily on the transpeptidase sortase A, which has become popular over the past few years for its ability to perform a remarkably wide variety of protein modifications, both in vitro and in living cells.

Viral GPCR US28 can signal in response to chemokine agonists of nearly unlimited structural degeneracy.

Human cytomegalovirus has hijacked and evolved a human G-protein-coupled receptor into US28, which functions as a promiscuous chemokine 'sink' to facilitate evasion of host immune responses. To probe the molecular basis of US28's unique ligand cross-reactivity, we deep-sequenced CX3CL1 chemokine libraries selected on 'molecular casts' of the US28 active-state and find that US28 can engage thousands of distinct chemokine sequences, many of which elicit diverse signaling outcomes. The structure of a G-protein-biased CX3CL1-variant in complex with US28 revealed an entirely unique chemokine amino terminal peptide conformation and remodeled constellation of receptor-ligand interactions. Receptor signaling, however, is remarkably robust to mutational disruption of these interactions. Thus, US28 accommodates and functionally discriminates amongst highly degenerate chemokine sequences by sensing the steric bulk of the ligands, which distort both receptor extracellular loops and the walls of the ligand binding pocket to varying degrees, rather than requiring sequence-specific bonding chemistries for recognition and signaling.

Exploiting Nanobodies' Singular Traits.

The unique class of heavy chain-only antibodies, present in Camelidae, can be shrunk to just the variable region of the heavy chain to yield VHHs, also called nanobodies. About one-tenth the size of their full-size counterparts, nanobodies can serve in applications similar to those for conventional antibodies, but they come with a number of signature advantages that find increasing application in biology. They not only function as crystallization chaperones but also can be expressed inside cells as such, or fused to other proteins to perturb the function of their targets, for example, by enforcing their localization or degradation. Their small size also affords advantages when applied in vivo, for example, in imaging applications. Here we review such applications, with particular emphasis on those areas where conventional antibodies would face a more challenging environment.

Anti-CTLA-4 therapy requires an Fc domain for efficacy.

Ipilimumab, a monoclonal antibody that recognizes cytotoxic T lymphocyte antigen (CTLA)-4, was the first approved "checkpoint"-blocking anticancer therapy. In mouse tumor models, the response to antibodies against CTLA-4 depends entirely on expression of the Fcγ receptor (FcγR), which may facilitate antibody-dependent cellular phagocytosis, but the contribution of simple CTLA-4 blockade remains unknown. To understand the role of CTLA-4 blockade in the complete absence of Fc-dependent functions, we developed H11, a high-affinity alpaca heavy chain-only antibody fragment (VHH) against CTLA-4. The VHH H11 lacks an Fc portion, binds monovalently to CTLA-4, and inhibits interactions between CTLA-4 and its ligand by occluding the ligand-binding motif on CTLA-4 as shown crystallographically. We used H11 to visualize CTLA-4 expression in vivo using whole-animal immuno-PET, finding that surface-accessible CTLA-4 is largely confined to the tumor microenvironment. Despite this, H11-mediated CTLA-4 blockade has minimal effects on antitumor responses. Installation of the murine IgG2a constant region on H11 dramatically enhances its antitumor response. Coadministration of the monovalent H11 VHH blocks the efficacy of a full-sized therapeutic antibody. We were thus able to demonstrate that CTLA-4-binding antibodies require an Fc domain for antitumor effect.

Targeting Cytokine Therapy to the Pancreatic Tumor Microenvironment Using PD-L1-Specific VHHs.

Cytokine-based therapies for cancer have not achieved widespread clinical success because of inherent toxicities. Treatment for pancreatic cancer is limited by the dense stroma that surrounds tumors and by an immunosuppressive tumor microenvironment. To overcome these barriers, we developed constructs of single-domain antibodies (VHHs) against PD-L1 fused with IL-2 and IFNγ. Targeting cytokine delivery in this manner reduced pancreatic tumor burden by 50%, whereas cytokines fused to an irrelevant VHH, or blockade of PD-L1 alone, showed little effect. Targeted delivery of IL-2 increased the number of intratumoral CD8+ T cells, whereas IFNγ reduced the number of CD11b+ cells and skewed intratumoral macrophages toward the display of M1-like characteristics. Imaging of fluorescent VHH-IFNγ constructs, as well as transcriptional profiling, demonstrated targeting of IFNγ to the tumor microenvironment. Many tumors and tumor-infiltrating myeloid cells express PD-L1, rendering them potentially susceptible to this form of targeted immunotherapy. Cancer Immunol Res; 6(4); 389-401. ©2018 AACR.

Generation of Ca2+-independent sortase A mutants with enhanced activity for protein and cell surface labeling.

Sortase A, a calcium-dependent transpeptidase derived from Staphylococcus aureus, is used in a broad range of applications, such as the conjugation of fluorescent dyes and other moieties to proteins or to the surface of eukaryotic cells. In vivo and cell-based applications of sortase have been somewhat limited by the large range of calcium concentrations, as well as by the often transient nature of protein-protein interactions in living systems. In order to use sortase A for cell labeling applications, we generated a new sortase A variant by combining multiple mutations to yield an enzyme that was both calcium-independent and highly active. This variant has enhanced activity for both N- and C-terminal labeling, as well as for cell surface modification under physiological conditions.

Localized CD47 blockade enhances immunotherapy for murine melanoma.

CD47 is an antiphagocytic ligand broadly expressed on normal and malignant tissues that delivers an inhibitory signal through the receptor signal regulatory protein alpha (SIRPα). Inhibitors of the CD47-SIRPα interaction improve antitumor antibody responses by enhancing antibody-dependent cellular phagocytosis (ADCP) in xenograft models. Endogenous expression of CD47 on a variety of cell types, including erythrocytes, creates a formidable antigen sink that may limit the efficacy of CD47-targeting therapies. We generated a nanobody, A4, that blocks the CD47-SIRPα interaction. A4 synergizes with anti-PD-L1, but not anti-CTLA4, therapy in the syngeneic B16F10 melanoma model. Neither increased dosing nor half-life extension by fusion of A4 to IgG2a Fc (A4Fc) overcame the issue of an antigen sink or, in the case of A4Fc, systemic toxicity. Generation of a B16F10 cell line that secretes the A4 nanobody showed that an enhanced response to several immune therapies requires near-complete blockade of CD47 in the tumor microenvironment. Thus, strategies to localize CD47 blockade to tumors may be particularly valuable for immune therapy.

PD-L1 is an activation-independent marker of brown adipocytes.

Programmed death ligand 1 (PD-L1) is expressed on a number of immune and cancer cells, where it can downregulate antitumor immune responses. Its expression has been linked to metabolic changes in these cells. Here we develop a radiolabeled camelid single-domain antibody (anti-PD-L1 VHH) to track PD-L1 expression by immuno-positron emission tomography (PET). PET-CT imaging shows a robust and specific PD-L1 signal in brown adipose tissue (BAT). We confirm expression of PD-L1 on brown adipocytes and demonstrate that signal intensity does not change in response to cold exposure or ß-adrenergic activation. This is the first robust method of visualizing murine brown fat independent of its activation state.Current approaches to visualise brown adipose tissue (BAT) rely primarily on markers that reflect its metabolic activity. Here, the authors show that PD-L1 is expressed on brown adipocytes, does not change upon BAT activation, and that BAT volume in mice can be measured by PET-CT with a radiolabeled anti-PD-L1 antibody.

Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation.

Strategies for site-specific protein modification are highly desirable for the construction of conjugates containing non-genetically-encoded functional groups. Ideally, these strategies should proceed under mild conditions, and be compatible with a wide range of protein targets and non-natural moieties. The transpeptidation reaction catalyzed by bacterial sortases is a prominent strategy for protein derivatization that possesses these features. Naturally occurring or engineered variants of sortase A from Staphylococcus aureus catalyze a ligation reaction between a five-amino-acid substrate motif (LPXTG) and oligoglycine nucleophiles. By pairing proteins and synthetic peptides that possess these ligation handles, it is possible to install modifications onto the protein N- or C-terminus in site-specific fashion. As described in this unit, the successful implementation of sortase-mediated labeling involves straightforward solid-phase synthesis and molecular biology techniques, and this method is compatible with proteins in solution or on the surface of live cells. © 2017 by John Wiley & Sons, Inc.