The discovery of an HIV-1 cure remains a medical challenge because the virus rebounds quickly after the cessation of combination antiretroviral drug therapy (cART). Here, we investigate the potential of an engineered tandem bi-specific broadly neutralizing antibody (bs-bnAb) as an innovative product for HIV-1 prophylactic and therapeutic interventions. We discovered that by preserving two scFv binding domains of each parental bnAb, a single-gene-encoded tandem bs-bnAb, namely BiIA-SG, displayed significantly improved breadth and potency. BiIA-SG neutralized all 124 HIV-1 pseudotyped viruses tested, including global subtypes/recombinant forms, transmitted/founder viruses, and variants less or not susceptible to parental and many bnAbs, with an average IC50 value of 0.073 µ/ml (range < 0.001 to 1.03 µg/ml). In humanized mice, an injection of BiIA-SG conferred sterile protection when administered prior to challenges with diverse live HIV-1 stains. Moreover, while BiIA-SG delayed viral rebound in a short-term therapeutic setting when combined with cART, a single injection of AAV-transferred BiIA-SG gene resulted dose-dependently in prolonged in vivo expression of BiIA-SG, which was associated with complete viremia control and subsequent elimination of infected cells in humanized mice. These results warrant the clinical development of BiIA-SG as a promising bs-bnAb-based biomedical intervention for prevention and treatment of HIV-1 infections.
Xilin Wu, Jia Guo, Mengyue Niu, Minghui An, Li Liu, Hui Wang, Xia Jin, Qi Zhang, Ka Shing Lam, Tongjin Wu, Hua Wang, Qian Wang, Yanhua Du, Jingjing Li, Lin Cheng, Hang Ying Tang, Hong Shang, Linqi Zhang, Paul Zhou, Zhiwei Chen
The immune system is tightly controlled by regulatory processes that allow for the elimination of invading pathogens, while limiting immunopathological damage to the host. In the present study, we found that conditional deletion of the cell surface receptor Toso on B cells unexpectedly resulted in impaired proinflammatory T cell responses, which led to impaired immune protection in an acute viral infection model, while, in a chronic inflammatory context, was associated with reduced immunopathological tissue damage. Toso exhibited its B cell-inherent immunoregulatory function by negatively controlling the pool of IL-10-competent B1 and B2 B cells, which were characterized by a high degree of self-reactivity and were shown to mediate immunosuppressive activity on inflammatory T cell responses in vivo. Our results indicate that Toso is involved in the differentiation/maintenance of regulatory B cells by fine-tuning B cell receptor (BCR)-activation thresholds. Furthermore, we showed that during influenza A-induced pulmonary inflammation the application of Toso-specific antibodies selectively induced IL-10-competent B cells at the site of inflammation and resulted in decreased proinflammatory cytokine production by lung T cells. These findings suggest that Toso may serve as a novel therapeutic target to dampen pathogenic T cell responses via the modulation of IL-10-competent regulatory B cells.
Jinbo Yu, Vu Huy Hoang Duong, Katrin Westphal, Andreas Westphal, Abdulhadi Suwandi, Guntram A. Grassl, Korbinian Brand, Andrew C. Chan, Niko Föger, Kyeong-Hee Lee
The lack of defined correlates of protection hampers development of vaccines against tuberculosis (TB). In vitro mycobacterial outgrowth assays are thought to better capture the complexity of the human host/Mycobacterium tuberculosis (Mtb) interaction. We used a PBMC-based “mycobacterial-growth-inhibition-assay” (MGIA) to investigate the capacity to control outgrowth of Bacille Calmette-Guérin (BCG). Interestingly, strong control of BCG outgrowth was observed almost exclusively in individuals with recent exposure to Mtb, but not in (long-term) latent TB infection, and only modestly in BCG vaccinees. Mechanistically, control of mycobacterial outgrowth strongly correlated with the presence of a CD14dim monocyte population, but also required the presence of T cells. The nonclassical monocytes produced CXCL10, and CXCR3-receptor blockade inhibited the capacity to control BCG outgrowth. Expression of CXCR3 splice variants was altered in recently Mtb exposed individuals. Cytokines previously associated with trained immunity were detected in MGIA supernatants, and CXCL9, CXCL10, and CXCL11 represent new markers of trained immunity. These data indicate that CXCR3-ligands are associated with trained immunity and critical factors in controlling mycobacterial outgrowth.In conclusion, control of mycobacterial outgrowth early after exposure to Mtb is the result of trained immunity mediated by a CXCL10-producing non-classical CD14dim monocyte subset.
Simone A. Joosten, Krista E. van Meijgaarden, Sandra M. Arend, Corine Prins, Fredrik Oftung, Gro Ellen Korsvold, Sandra V. Kik, Rob J.W. Arts, Reinout van Crevel, Mihai G. Netea, Tom H.M. Ottenhoff
HLA-B*57 control of HIV involves enhanced CD8+ T cell responses against infected cells, but extensive heterogeneity exists in level of HIV control among B*57+ individuals. Using whole genome sequencing of untreated B*57+ HIV-1 infected controllers and non-controllers, we identified a single variant (rs643347A/G) encoding an isoleucine to valine substitution at position 47 (I47V) of the inhibitory killer cell immunoglobulin-like receptor, KIR3DL1, as the only significant modifier of B*57 protection. The association replicated in an independent cohort and across multiple outcomes. The modifying effect of I47V was confined to B*57:01, and was not observed for the closely related B*57:03. Positions 2, 47, and 54 track one another nearly perfectly, and two KIR3DL1 allotypes differing only at these three positions showed significant differences in binding B*57:01 tetramers, where the protective allotype showed lower binding. Thus, variation in an immune natural killer cell receptor that binds B*57:01 modifies its protection. These data speak to exquisite specificity of KIR-HLA interactions in human health and disease.
Maureen P. Martin, Vivek Naranbhai, Patrick R. Shea, Ying Qi, Veron Ramsuran, Nicolas Vince, Xiaojiang Gao, Rasmi Thomas, Zabrina L. Brumme, Jonathan M. Carlson, Steven M. Wolinsky, James J. Goedert, Bruce D. Walker, Florencia P. Segal, Steven G. Deeks, David W. Haas, Stephen A. Migueles, Mark Connors, Nelson Michael, Jacques Fellay, Emma Gostick, Sian Llewellyn-Lacey, David A. Price, Bernard A. Lafont, Phillip Pymm, Philippa M. Saunders, Jacqueline Widjaja, Shu Cheng Wong, Julian P. Vivian, Jamie Rossjohn, Andrew G. Brooks, Mary Carrington
BACKGROUND. Sporadic vascular malformations (VMs) are complex congenital anomalies of blood vessels that lead to stroke, life-threatening bleeds, disfigurement, overgrowth, and/or pain. Therapeutic options are severely limited and multi-disciplinary management remains challenging, particularly for high-flow arteriovenous malformations (AVM). METHODS. To investigate the pathogenesis of sporadic intracranial and extracranial VMs in 160 children in which known genetic causes had been excluded, we sequenced DNA from affected tissue and optimised analysis for detection of low mutant allele frequency. RESULTS. We discovered multiple mosaic activating variants in four genes of the RAS-MAPK pathway, KRAS, NRAS, BRAF, and MAP2K1, a pathway commonly activated in cancer and responsible for the germ-line RAS-opathies. These variants were more frequent in high-flow than low-flow VMs. In vitro characterisation and two transgenic zebrafish AVM models which recapitulated the human phenotype validated the pathogenesis of the mutant alleles. Importantly, treatment of AVM-BRAF mutant zebrafish with the BRAF inihibitor, Vemurafinib, restored blood flow in AVM. CONCLUSIONS. Our findings uncover a major cause of sporadic vascular malformations of different clinical types, and thereby offer the potential of personalised medical treatment by repurposing existing licensed cancer therapies. FUNDING. This work was funded or supported by grants from AVM Butterfly Charity, the Wellcome Trust (UK), the Medical Research Council (UK), the UK National Institute for Health Research, L’Oreal-Melanoma Research Alliance, the European Research Council, and the National Human Genome Research (US).
Lara Al-Olabi, Satyamaanasa Polubothu, Katherine Dowsett, Katrina A. Andrews, Paulina Stadnik, Agnel P. Joseph, Rachel Knox, Alan Pittman, Graeme Clark, William Baird, Neil Bulstrode, Mary Glover, Kristiana Gordon, Darren Hargrave, Susan M. Huson, Thomas S. Jacques, Gregory James, Hannah Kondolf, Loshan Kangesu, Kim M. Keppler-Noreuil, Amjad Khan, Marjorie J. Lindhurst, Mark Lipson, Sahar Mansour, Justine O'Hara, Caroline Mahon, Anda Mosica, Celia Moss, Aditi Murthy, Juling Ong, Victoria E. Parker, Jean-Baptiste Rivière, Julie C. Sapp, Neil J. Sebire, Rahul Shah, Branavan Sivakumar, Anna Thomas, Alex Virasami, Regula Waelchli, Zhiqiang Zeng, Leslie G. Biesecker, Alex Barnacle, Maya Topf, Robert K. Semple, E. Elizabeth Patton, Veronica A. Kinsler
Genetic forms of vitamin D–dependent rickets (VDDRs) are due to mutations impairing activation of vitamin D or decreasing vitamin D receptor responsiveness. Here we describe two unrelated patients with early-onset rickets, reduced serum levels of the vitamin D metabolites 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, and deficient responsiveness to parent and activated forms of vitamin D. Neither patient had a mutation in any genes known to cause VDDR, however, using whole exome sequence analysis we identified a recurrent de novo missense mutation c.902T>C (p.I301T) in CYP3A4 in both subjects that alters the conformation of substrate-recognition-site 4 (SRS-4). In vitro, the mutant CYP3A4 oxidized 1,25-dihydroxyvitamin D with 10-fold greater activity than wild-type CYP3A4 and 2-fold greater activity than CYP24A1, the principal inactivator of vitamin D metabolites. As CYP3A4 mutations have not previously been linked to rickets, these findings provide new insight into vitamin D metabolism, and demonstrate that accelerated inactivation of vitamin D metabolites represents a previously undescribed mechanism for vitamin D deficiency.
Jeffrey D. Roizen, Dong Li, Lauren O'Lear, Muhammad K. Javaid, Nicholas J. Shaw, Peter R. Ebeling, Hanh H. Nguyen, Christine P. Rodda, Kenneth E. Thummel, Tom D Thacher, Hakon Hakonarson, Michael A. Levine
Synthetic lethality is an efficient mechanism-based approach to selectively target DNA repair defects. ERCC1 deficiency is frequently found in non-small cell lung cancers, making this DNA repair protein an attractive target for exploiting synthetic lethal approaches in this disease. Using unbiased proteomic and metabolic high-throughput profiling on a unique in-house generated isogenic model of ERCC1 deficiency, we found marked metabolic rewiring of ERCC1-deficient populations, including decreased levels of the metabolite NAD+ and reduced expression of the rate-limiting NAD+ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT). We further evidenced reduced NAMPT expression in NSCLC samples with low levels of ERCC1. These metabolic alterations were a primary effect of ERCC1 deficiency, and caused selective exquisite sensitivity to small molecule NAMPT inhibitors, both in vitro — ERCC1-deficient cells being approximately 1000 times more sensitive — and in vivo. Using transmission electronic microscopy and functional metabolic studies, we found that ERCC1-deficient cells harbor mitochondrial defects. We propose a model where NAD+ acts as a regulator of ERCC1-deficient NSCLC fitness. These findings open therapeutic opportunities that exploit a yet undescribed nuclear — mitochondrial synthetic lethal relationship in cancer cells, and highlight the potential for targeting DNA repair/metabolic crosstalks for cancer therapy.
Mehdi Touat, Tony Sourisseau, Nicolas Dorvault, Roman M. Chabanon, Marlène Garrido, Daphné Morel, Dragomir B. Krastev, Ludovic Bigot, Julien Adam, Jessica Frankum, Sylvère Durand, Clement Pontoizeau, Sylvie Souquère, Mei-Shiue Kuo, Sylvie Sauvaigo, Faraz Mardakheh, Alain Sarasin, Ken A. Olaussen, Luc Friboulet, Frédéric Bouillaud, Gérard Pierron, Alan Ashworth, Anne Lombès, Christopher J. Lord, Jean-Charles Soria, Sophie Postel-Vinay
Immune evasion and the suppression of anti-tumor responses during cancer progression are considered hallmarks of cancer and are typically attributed to tumor-derived factors. Although the molecular basis for the crosstalk between tumor and immune cells is an area of active investigation, whether host-specific germline variants can dictate immunosuppressive mechanisms has remained a challenge to address. A commonly occurring germline mutation (c.1162G>A/rs351855 G/A) in the FGFR4 (CD334) gene enhances STAT3 signaling and is associated with poor prognosis and accelerated progression of multiple cancer types. Here, using rs351855 single nucleotide polymorphism (SNP) knock-in transgenic mice and Fgfr4 knockout mice, we reveal the genotype-specific gain of immunological function of suppressing the CD8/CD4+FOXP3+CD25+ve regulatory T cell ratio in vivo. Furthermore, using knock-in transgenic mouse models for lung and breast cancers, we establish the host-specific tumor-extrinsic functions of STAT3-enhancing germline variants in impeding the tumor infiltration of CD8 T cells. Thus, STAT3-enhancing germline receptor variants contribute to immune evasion through their pleiotropic functions in immune cells.
Daniel Kogan, Alexander Grabner, Christopher Yanucil, Christian Faul, Vijay Kumar Ulaganathan
Major histocompatibility (MHC) class II molecules are strongly associated with many autoimmune disorders. In type 1 diabetes, the DQ8 molecule is common, confers significant disease risk and is involved in disease pathogenesis. We hypothesized blocking DQ8 antigen presentation would provide therapeutic benefit by preventing recognition of self-peptides by pathogenic T cells. We used the crystal structure of DQ8 to select drug-like small molecules predicted to bind structural pockets in the MHC antigen-binding cleft. A limited number of the predicted compounds inhibited DQ8 antigen presentation in vitro with one compound preventing insulin autoantibody production and delaying diabetes onset in an animal model of spontaneous autoimmune diabetes. An existing drug of similar structure, methyldopa, specifically blocked DQ8 in recent-onset patients with type 1 diabetes along with reducing inflammatory T cell responses toward insulin, highlighting the relevance of blocking disease-specific MHC class II antigen presentation to treat autoimmunity.
David A. Ostrov, Aimon Alkanani, Kristen A. McDaniel, Stephanie Case, Erin E. Baschal, Laura Pyle, Samuel Ellis, Bernadette Pöllinger, Katherine J. Seidl, Viral N. Shah, Satish K. Garg, Mark A. Atkinson, Peter A. Gottlieb, Aaron W. Michels
Insulin resistance and type 2 diabetes are associated with low levels of high-density lipoprotein-cholesterol (HDL-C). The insulin-repressible FoxO transcription factors are potential mediators of insulin’s effect on HDL-C. FoxOs mediate a substantial portion of insulin-regulated transcription, and poor FoxO repression is thought to contribute to the excessive glucose production in diabetes. In this work, we show that mice with liver-specific triple FoxO knockout (L-FoxO1,3,4), which are known to have reduced hepatic glucose production, also have increased HDL-C. This was associated with decreased expression of HDL-C clearance factors, scavenger receptor class B type I (SR-BI) and hepatic lipase, and defective selective uptake of HDL-cholesteryl ester by the liver. The phenotype could be rescued by re-expression of SR-BI. These findings demonstrate that hepatic FoxOs are required for cholesterol homeostasis and HDL-mediated reverse cholesterol transport to the liver.
Samuel X. Lee, Markus Heine, Christian Schlein, Rajasekhar Ramakrishnan, Jing Liu, Gabriella Belnavis, Ido Haimi, Alexander W. Fischer, Henry Ginsberg, Joerg Heeren, Franz Rinninger, Rebecca A. Haeusler
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