Metabolic modulation of tumours with engineered bacteria for immunotherapy.

The availability of L-arginine in tumours is a key determinant of an efficient anti-tumour T cell response1-4. Consequently, increases of typically low L-arginine concentrations within the tumour may greatly potentiate the anti-tumour responses of immune checkpoint inhibitors, such as programmed death-ligand 1 (PD-L1)-blocking antibodies5. However, currently no means are available to locally increase intratumoural L-arginine levels. Here we used a synthetic biology approach to develop an engineered probiotic Escherichia coli Nissle 1917 strain that colonizes tumours and continuously converts ammonia, a metabolic waste product that accumulates in tumours6, to L-arginine. Colonization of tumours with these bacteria increased intratumoural L-arginine concentrations, increased the number of tumour-infiltrating T cells and had marked synergistic effects with PD-L1 blocking antibodies in the clearance of tumours. The anti-tumour effect of these bacteria was mediated by L-arginine and was dependent on T cells. These results show that engineered microbial therapies enable metabolic modulation of the tumour microenvironment leading to enhanced efficacy of immunotherapies.

Clonal analysis of immunodominance and cross-reactivity of the CD4 T cell response to SARS-CoV-2.

The identification of CD4+ T cell epitopes is instrumental for the design of subunit vaccines for broad protection against coronaviruses. Here, we demonstrate in COVID-19-recovered individuals a robust CD4+ T cell response to naturally processed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein and nucleoprotein (N), including effector, helper, and memory T cells. By characterizing 2943 S-reactive T cell clones from 34 individuals, we found that the receptor-binding domain (RBD) is highly immunogenic and that 33% of RBD-reactive clones and 94% of individuals recognized a conserved immunodominant S346-S365 region comprising nested human leukocyte antigen DR (HLA-DR)- and HLA-DP-restricted epitopes. Using pre- and post-COVID-19 samples and S proteins from endemic coronaviruses, we identified cross-reactive T cells targeting multiple S protein sites. The immunodominant and cross-reactive epitopes identified can inform vaccination strategies to counteract emerging SARS-CoV-2 variants.

Rationally designed Human Cytomegalovirus gB nanoparticle vaccine with improved immunogenicity.

Human cytomegalovirus (HCMV) is the primary viral cause of congenital birth defects and causes significant morbidity and mortality in immune-suppressed transplant recipients. Despite considerable efforts in vaccine development, HCMV infection still represents an unmet clinical need. In recent phase II trials, a MF59-adjuvanted gB vaccine showed only modest efficacy in preventing infection. These findings might be attributed to low level of antibodies (Abs) with a neutralizing activity induced by this vaccine. Here, we analyzed the immunogenicity of each gB antigenic domain (AD) and demonstrated that domain I of gB (AD5) is the main target of HCMV neutralizing antibodies. Furthermore, we designed, characterized and evaluated immunogenic responses to two different nanoparticles displaying a trimeric AD5 antigen. We showed that mice immunization with nanoparticles induces sera neutralization titers up to 100-fold higher compared to those obtained with the gB extracellular domain (gBECD). Collectively, these results illustrate with a medically relevant example the advantages of using a general approach combining antigen discovery, protein engineering and scaffold presentation for modern development of subunit vaccines against complex pathogens.

P2X7 Receptor Activity Limits Accumulation of T Cells within Tumors.

Extracellular ATP (eATP) is a signaling molecule that variably affects all cells of the immune system either directly or after hydrolysis to adenosine. Although eATP is virtually absent in the interstitium of normal tissues, it can be present in the hundreds of micromolar range in tumors, a concentration compatible with activation of the ATP-gated ionotropic P2X7 receptor. Here, we show that P2X7 activity in tumor-infiltrating lymphocytes (TIL) induces cellular senescence and limits tumor suppression. P2X7 stimulation affected cell cycling of effector T cells and resulted in generation of mitochondrial reactive oxygen species and p38 MAPK-dependent upregulation of cyclin-dependent kinase inhibitor 1A (Cdkn1a, encoding for p21Waf1/Cip1). Lack of P2X7 promoted a transcriptional signature that correlated with enhanced cytotoxic T-cell response in human solid tumors. In mice, transfer of tumor-specific T cells with deletion of P2rx7 significantly reduced tumor growth and extended survival. Collectively, these findings uncover a purinergic checkpoint that can be targeted to improve the efficacy of cancer immunotherapy strategies. SIGNIFICANCE: These findings suggest that the purinergic checkpoint P2X7 may be targeted to enhance T-cell-mediated cancer immunotherapy and improve T effector cell accumulation in the tumor microenvironment. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/18/3906/F1.large.jpg.

Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a worldwide public health concern for which no vaccine is available. Elucidation of the prefusion structure of the RSV F glycoprotein and its identification as the main target of neutralizing antibodies have provided new opportunities for development of an effective vaccine. Here, we describe the structure-based design of a self-assembling protein nanoparticle presenting a prefusion-stabilized variant of the F glycoprotein trimer (DS-Cav1) in a repetitive array on the nanoparticle exterior. The two-component nature of the nanoparticle scaffold enabled the production of highly ordered, monodisperse immunogens that display DS-Cav1 at controllable density. In mice and nonhuman primates, the full-valency nanoparticle immunogen displaying 20 DS-Cav1 trimers induced neutralizing antibody responses ∼10-fold higher than trimeric DS-Cav1. These results motivate continued development of this promising nanoparticle RSV vaccine candidate and establish computationally designed two-component nanoparticles as a robust and customizable platform for structure-based vaccine design.

Virus-Like Particles and Nanoparticles for Vaccine Development against HCMV.

Human cytomegalovirus (HCMV) infects more than 70% of the human population worldwide. HCMV is responsible for high morbidity and mortality in immunocompromised patients and remains the leading viral cause of congenital birth defects. Despite considerable efforts in vaccine and therapeutic development, HCMV infection still represents an unmet clinical need and a life-threatening disease in immunocompromised individuals and newborns. Immune repertoire interrogation of HCMV seropositive patients allowed the identification of several potential antigens for vaccine design. However, recent HCMV vaccine clinical trials did not lead to a satisfactory outcome in term of efficacy. Therefore, combining antigens with orthogonal technologies to further increase the induction of neutralizing antibodies could improve the likelihood of a vaccine to reach protective efficacy in humans. Indeed, presentation of multiple copies of an antigen in a repetitive array is known to drive a more robust humoral immune response than its soluble counterpart. Virus-like particles (VLPs) and nanoparticles (NPs) are powerful platforms for multivalent antigen presentation. Several self-assembling proteins have been successfully used as scaffolds to present complex glycoprotein antigens on their surface. In this review, we describe some key aspects of the immune response to HCMV and discuss the scaffolds that were successfully used to increase vaccine efficacy against viruses with unmet medical need.

An Unbiased Screen for Human Cytomegalovirus Identifies Neuropilin-2 as a Central Viral Receptor.

Characterizing cell surface receptors mediating viral infection is critical for understanding viral tropism and developing antiviral therapies. Nevertheless, due to challenges associated with detecting protein interactions on the cell surface, the host receptors of many human pathogens remain unknown. Here, we build a library consisting of most single transmembrane human receptors and implement a workflow for unbiased and high-sensitivity detection of receptor-ligand interactions. We apply this technology to elucidate the long-sought receptor of human cytomegalovirus (HCMV), the leading viral cause of congenital birth defects. We identify neuropilin-2 (Nrp2) as the receptor for HCMV-pentamer infection in epithelial/endothelial cells and uncover additional HCMV interactors. Using a combination of biochemistry, cell-based assays, and electron microscopy, we characterize the pentamer-Nrp2 interaction and determine the architecture of the pentamer-Nrp2 complex. This work represents an important approach to the study of host-pathogen interactions and provides a framework for understanding HCMV infection, neutralization, and the development of novel anti-HCMV therapies.

TRPM7 kinase activity is essential for T cell colonization and alloreactivity in the gut.

The melastatin-like transient-receptor-potential-7 protein (TRPM7), harbouring a cation channel and a serine/threonine kinase, has been implicated in thymopoiesis and cytokine expression. Here we show, by analysing TRPM7 kinase-dead mutant (Trpm7 R/R ) mice, that the enzymatic activity of the receptor is not essential for thymopoiesis, but is required for CD103 transcription and gut-homing of intra-epithelial lymphocytes. Defective T cell gut colonization reduces MHCII expression in intestinal epithelial cells. Mechanistically, TRPM7 kinase activity controls TGF-ß-induced CD103 expression and pro-inflammatory T helper 17, but not regulatory T, cell differentiation by modulating SMAD2. Notably, we find that the TRPM7 kinase activity promotes gut colonization by alloreactive T cells in acute graft-versus-host disease. Thus, our results unravel a function of TRPM7 kinase in T cell activity and suggest a therapeutic potential of kinase inhibitors in averting acute graft-versus-host disease.