Sigal Research

Research in the Sigal laboratory revolves around viral pathogenesis, immunology, and vaccines. Our papers have been published in high-impact journals such as Immunity, Cell Host & Microbe, Journal of Experimental Medicine, PNAS, Molecular Therapy, Cell Reports, PLoS Pathogens, and others. One of our main interests is the study of the pathogenesis, immunobiology, and mechanism of vaccine-induced protection of Orhtopoxviruses (OPVs), a genus that includes the human pathogens variola virus (the virus of smallpox) and Mpox virus (MPXV), which is currently causing a major human outbreak in the Democratic Republic of Congo, and vaccinia virus (VACV), which is the smallpox vaccine. To study OPVs in a natural host, we use ectromelia virus (ECTV), a mouse-specific OPV. ECTV is also an outstanding model to study acute systemic viral infections as they spread in their biological host following infection through the periphery. Our ECTV papers have looked at various mechanisms of intrinsic and acquired resistance to viral disease. A major aspect of our research has focused on the immune responses in the lymph nodes draining the sites of viral infection and vaccination. As examples, our work in this area has elucidated how Langerhans cells, conventional dendritic cells, inflammatory monocytes, Natural Killer cells, cytotoxic CD4, CD8 T-cells, and B-cells, as well as some of their products, including TLR9, cGAS, STING, Type I and Type interferons, the chemokines CCL2, CCL7, and CXCL9, the effector molecule granzyme B, the integrin alpha2beta1, NKG2D and its ligands, the non-classical MHC class I molecule Qa-1b and certain antibodies, curtail the systemic spread of ECTV from the lymph node or clear the infection in the liver, which is ECTV’s main target organ. We have also studied the protection mechanisms by protein, live, and mRNA-LNP vaccines. More recently, our lab has expanded its scope to study the pathogenesis, immunology, and mechanisms of vaccine-mediated protection from SARS-CoV-2 and the enteroviruses Coxsackievirus B3 (CVB3), a major cause of Type I diabetes and myocarditis, and enterovirus D68 (EV-D68) which causes respiratory illness and in some cases polio-like flaccid paralysis and is considered a pandemic risk. We also use CRISPR/Cas9 technology to make novel mouse strains deficient in specific genes to understand their role in anti-viral protection.

Gudrun Debes and Luis J. Sigal, PIs (NIH-NIAID R21 AI173837 2022-2025).

Natural Killer (NK) cells and innate lymphoid cells (ILC) type I (ILC1) are key effectors in host defense against skin-borne viruses and in cutaneous anti-tumor responses; they also modulate inflammatory skin diseases. Despite their critical role, little is known about the migratory patterns of skin-homing NK cells and ILC1, their organ selectivity or functions, and whether they can be selectively targeted. In contrast, organ-selective homing to skin and intestines of T cell subsets is harnessed for vaccine strategies and in the treatment of organ-specific autoimmunity. This exploratory proposal is based on the overall hypothesis that there is a distinct population of skin-homing NK cells and ILC1 that is is key to cutaneous host defense. To study skin-trafficking of NK cells and ILC1, we propose to revisit the classic model of afferent lymph cannulation in the sheep, which allows to collect NK cells during their physiological recirculation through skin. By targeted analysis of skin-recirculating lymph- borne NK cells, we will assess expression of canonical skin-homing receptors and effector molecules. In addition, we will use Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE Seq) to determine gene expression profiles of skin-recirculating NK and ILC1 cells and assess overlap with NK cells in control sites (intestinal lymph and blood). These studies will be complemented by genetic mouse models and viral skin infection mouse models utilizing the poxviruses vaccinia virus (VACV) and ectromelia virus (ECTV). Our preliminary studies discovered that skin-recirculating NK cells express high levels of α4β1-integrin, whose ligand VCAM-1 is constitutively expressed by skin vasculature. Therefore, we will test the role of α4β1-integrin in NK cell skin homing and relevance for resistance to skin-borne VACV and ECTV. This will also establish a pipeline to test the significance of additional molecules expressed by skin-recirculating NK cell in future studies. In summary, the proposed studies will greatly enhance our understanding of skin-homing NK cells and ILC1, and our ability to manipulate skin-specific immune responses in cutaneous pathologies ranging from infection and cancer to inflammation.

Raul Andino (UCSF) and Luis J. Sigal (TJU), PIs (NIH-NIAID R01 AI169460 2022-2027). 

Human enterovirus (HEV) infections primarily affect infant and adolescent populations, causing a wide range of clinical manifestations that commonly include respiratory illness and mucocutaneous lesions, or hand, foot and mouth disease. In some cases, the infection is life-threatening. The clinical manifestations are a function of their tropism. For example, some HEVs, including EV-A71 and EV-D68, are associated with serious neurological symptoms due to invasion and damage of central nervous tissues. Others, such CVB3, are implicated in respiratory symptoms and cardiomyopathy. This proposal aims to take an integrative approach to understand how tissues, and cells within these tissues, respond to infection by CVB3 and the emerging pathogens EV-D68 and EV-A71. We will determine the temporal and spatial dynamics of HEV infection using recent advances in genomics: (i) We will profile single-cell transcriptomes to quantify viral replication levels and the host response to infection across cells and tissues over the course of infection. (ii) Given that intra-host adaptation appears to be important in infection, in parallel, we will map the mutational spectrum of the replicating viruses using a novel ultra-deep sequencing approach. We will use new innovative technologies, such as ultra-deep virus population sequencing, deep learning and single-cell analysis to increase our basic understanding of the pathogenesis of enteroviruses A, B and D. Finally, given that innate immunity is a major determinant of tissue tropism, we will use mice with deletions of specific type-I IFN subtypes to determine the significance of interferon diversity in controlling HEV infections. These data will enable us to determine cell types that HEVs infect, the response that the host mounts against them in each cell and tissue, and the viral mutants that emerge in different tissues. Understanding pathogenesis is critically needed for developing effective and broadly-acting countermeasures and to inform the development of effective and broad-spectrum vaccines and antiviral compounds. 

Project 1: Design and testing of picornavirus vaccine candidates.

Raul Andino (UCSF), PI. Luis J. Sigal (TJU) Co-I and Carolyn Coyne (Duke University), Co-I.

The need for effective vaccines against enterovirus and rhinovirus cannot be overstated. Given the ever-increasing threat of these viral infections and their associated health impacts, it is crucial to develop a general platform for rapid vaccine development. With this in mind, we propose to create such a platform, one that can be easily and quickly adapted to different strains of the viruses as they emerge. We envision a system that combines the latest tools and technologies, including molecular biology, immunology, and vaccine design, to accelerate the discovery and development of new vaccines. By doing so, we hope to make a meaningful contribution towards protecting global populations from the devastating effects of these viruses.

Our approach is to use protective human monoclonal antibodies (Project 2) to design immunogens that effectively protect against these viruses. This is a crucial undertaking, as the five human pathogen prototypes we will focus on- Rhinovirus C, Enteroviruses A71 and D68, as well as Coxsackievirus B3 and Echovirus 11 - are among the most significant viruses causing respiratory, gastrointestinal, and nervous system diseases on a global scale. Our vaccine platform is designed with structurally stabilized virus-like particles (VLPs) that can be used as protein- or nucleic acid-based immunogens. By leveraging the knowledge we extract from protective monoclonal antibodies, we can provide advanced protection from pathogenic picornaviruses and reduce the risk of dangerous outbreaks. In addition, we will combine these immunogens with a self-replicative RNA-based mucosal adjuvant. The adjuvant mimics virus infection and triggers innate local immune responses, ensuring robust T-cell and B-cell adaptive immunity, as well as long-lasting protection. We hope to create a robust defense against these viruses by generating both systemic and mucosal immune responses. 

In the context of these studies, we will develop animal models to optimize vaccination strategies and examine the immunogenicity and protection elicited by our vaccines.