Luis J. Sigal, DVM, PhD
Office Phone: 215-728-7061
Lab Phone: 215-728-7065,7067,7020
Specific Projects on Antigen Presentation
1. MHC Class I Antigen Presentation in Viral Infections
When cells are infected with viruses, they present at the cell surface short viral peptides bound to MHC class I molecules (MHC I). These peptide-MHC I complexes are the antigen (Ag; plural, Ags) recognized by anti-viral CD8+ T lymphocytes. For a long time, it was thought that every cell displaying viral peptides with MHC I could elicit (prime) anti-viral CD8+ T lymphocyte (TCD8+) responses. However, several years ago we showed that only bone marrow-derived cells can prime TCD8+ to several viruses. Bone marrow-derived cells capable of priming T cells are known as professional antigen presenting cells (pAPC) and traditionally include dendritic cells (DC) and macrophages (Mf). Two of the most outstanding characteristics of pAPC are that they express costimulatory molecules, and that they are capable of migrating from tissues into secondary lymphoid organs (SLO) such as lymph nodes (LN). Therefore, it is thought that pAPC are required for T cell priming because: 1) they are the only cell type that can bring Ags from infected tissues to SLO where T cell responses are initiated; 2) they can provide costimulatory signals required for the activation of naïve T cells. However, the effects of viral infection on pAPC migration have not been studied. Therefore, some of our work is geared to understand how viral infection affects pAPC migration.
In recent years it has become widely accepted that only DC are capable of priming T cells. However, direct evidence that DC are responsible for the priming of anti-viral TCD8+ lymphocyte responses in vivo is scarce. In addition, it is still possible that Mf have a role in priming CD8+ T lymphocytes. Hence, another important aspect of our work is to elucidate which are the pAPC that prime TCD8+ in vivo.
For most cells, MHC I presentation is restricted to antigenic determinants derived from endogenously synthesized proteins (direct presentation, DP). However, we have also shown that, in vivo, pAPC can prime anti viral CD8+ T cell responses by presenting Ag acquired from other infected cells (cross-presentation, CP). Additional work in our laboratory has characterized some of the mechanisms of CP during VACV infection and how they differ from DP. However, the overall contribution of CP and DP in the induction of anti-viral CD8+ T lymphocyte responses remains unsolved and is a matter of heated debate. Our very recent work shows that DP is the main mechanisms for the priming of anti-VACV TCD8+.Top
Specific Projects in Viral Pathogenesis
1. Orthopoxvirus Pathogenesis and Vaccines
OPVs are large DNA viruses that can be highly lethal to their natural hosts. Smallpox was produced by the human-specific OPV variola virus (VARV) and was eradicated through vaccination with live VACV, a mildly pathogenic OPV. Despite this success, we still know little about the reasons for the high pathogenicity of OPVs in their natural hosts and the mechanisms whereby the smallpox vaccine protects. Studying OPVs is important for human health for several reasons: 1) OPVs are common in many animal species and some of these viruses could jump the species barrier and become human pathogens. 2) There is fear that VARV could be used as a weapon. 3) The vaccine based on live VACV is not safe by current standards and killed VACV does not protect. 4) Since the smallpox vaccine is so effective, understanding how it protects may be valuable for developing vaccines for other viruses including other large DNA viruses such as herpesviruses.
For many years we have worked with VACV and other viruses to study different aspects of the immune response such as the role of CD28-CD80/86 costimulation and bone marrow-derived antigen presenting cells in CD8+ T cell responses, and the mechanisms whereby antigens from VACV-infected cells are transferred to antigen presenting cells for CP. However, VACV is not the best model to investigate the mechanisms of pathogenesis and acquired resistance to disease because VACV is not very pathogenic to the mouse. ECTV is the agent of mousepox, a disease remarkably similar to human smallpox. Furthermore, ECTV is genetically and antigenically very similar to VARV and VACV. Mouse strains such as BALB/c are highly susceptible to mousepox experiencing 100% morbidity and 80-100% mortality. Similar to smallpox in humans, immunization of BALB/c mice with live VACV results in acquired resistance to mousepox. Therefore, studies with susceptible mouse strains can be very useful to dissect the mechanism involved in pathogenesis of lethal OPV infections, determine the mechanisms of acquired protection induced by primary infection or vaccination and to test OPV vaccines. In the relatively short time since we initiated our ECTV program we have made several important observations: 1) We demonstrated that antibodies and B-lymphocytes are indispensable for long-term resistance to primary mousepox. 2) We have shown that a subunit vaccine using a recombinant viral protein of the outer envelope (EVM135) protects susceptible mice from fatal mousepox but not from disease. 3) We demonstrated that both antibodies and memory TCD8+ can independently protect susceptible but immunocompetent mice from mousepox but not from infection.
A notable feature of all OPVs is their expression of secreted, non-structural proteins that mimic host factors important for the immune response (immune response modifiers, IRMs). Among them, several genes target the production or function of interferons (IFNs). It is thought that OPVs use these IRMs to facilitate replication and spread which should result in increased pathology. However, on a few occasions only, the role of these proteins in pathogenesis has been studied in natural hosts. A focus of our current work is to make ECTV mutants in genes coding for various IRMs and compare them with wild type virus for their growth in tissue culture; their ability to spread and induce pathology in immunodeficient and immunocompetent mice; and the strength and type of humoral and cellular immune response that they induce. Moreover, we are interested in elucidating whether these IRMs constitute natural targets of protective immune responses and whether they can be used as vaccines. Our recent work in this area showed that the Type I IFN binding protein of ECTV is essential for pathogenesis, is a natural target of the Ab response and can be used as a vaccine. Current work is aimed at understanding how the Type I IFN bp contributes to pathogenesis and how Abs counteract it. In further work related to ECTV pathogenesis we have shown that Natural Killer (NK) cells protect from mousepox using a direct cytolytic function as well as their ability to boost the T cell response. Furthermore, we show that the activating receptor NKG2D is required for optimal NK cell-mediated resistance to disease and lethality.Top
2. Mousepox in Aged Mice
It is well known that resistance to viral infections and the ability to generate protective immunity following vaccination declines with age. While most of us have been vaccinated and are probably life-long immune to many common viral diseases, we have recently become more aware that emerging infectious diseases are possible, that pandemics with new strains of known viruses such as influenza are likely to occur, and that highly pathogenic microorganisms could be used as weapons. In each of these cases, the non-immune elderly would be at a much higher risk of disease and death than the young. Because the elderly represent a sizable proportion of the world population, understanding the reasons and trying to overcome the consequences of the age-dependent loss of natural and acquired resistance to viral diseases is of major public health interest. However, a direct detailed analysis of the loss of resistance to viral diseases in humans is not possible and appropriate animal models must be used. Some strains of mice, such as C57BL/6 (B6), are known to be naturally resistant to mousepox. However, we have recently found that B6 mice lose this resistance as they age. Thus, comparing the immune functions in response to primary ECTV infection of young and aged B6 mice offers an excellent model to understand the age-dependent loss of resistance to viral disease. Furthermore, because mousepox can be prevented with the smallpox vaccine, our new finding opens the opportunity to determine whether aged mice can be protected by vaccination. The overarching goal of this exploratory project is to pinpoint the specific immune functions affected by age that correlate with the loss of natural and acquired resistance to mousepox. The long-term objective for this project is to understand the underpinnings behind any defective function discovered during this exploratory phase. Furthermore, we intend to find new methods to manipulate the immune response to restore resistance and improve vaccine efficacy in aged mice as a first approach to improve vaccine efficacy in elderly people.Top
Immune Mechanisms That Control Ectromelia Virus Infection
This is a multi-investigator cooperative agreement in which I am the PI, and the Program Leader for Project B, the Bioreagents Core and the Administrative Core. In addition, Dr. Christopher Norbury at Pennsylvania State University is the Program Leader in one research project and the Imaging Core and Dr. Laurence Eisenlohr at Thomas Jefferson University is the program leader for a third project.
1. Project B: Mechanisms of Natural and Acquired Resistance to Mousepox
There are two major mechanisms that prevent mousepox. A) Natural or innate resistance (not to be confused with the innate immune system), is the genetically determined ability of some mouse strains such as B6 and 129 to survive a first encounter with wild type (WT) ECTV without major symptoms of mousepox. B) Acquired resistance is the ability of mousepox susceptible (i.e. non-genetically resistant) mice that normally die during primary ECTV infection such as BALB/c and DBA/2 mice, to survive an encounter with WT ECTV without major symptoms of the disease if they have previously been exposed to ECTV, cross-reactive viruses, or certain OPV antigens (Ags). During the past few years we have made substantial contributions to the identification of components and mechanisms of the innate and adaptive immune system that contribute to natural and acquired resistance to mousepox. In this project we are unveiling the specific mechanisms whereby many of these components contribute to natural and acquired resistance to mousepox. In the context of natural immunity, we are specifically investigating the mechanisms whereby Type I IFNs (T1-IFNs) contribute to natural resistance to lethal mousepox. Death from mousepox is due to uncontrolled ECTV replication in the liver. Work by us and others has shown that T1-IFNs are essential for natural resistance to mousepox.
The central hypotheses to be tested are that T1-IFNs provide resistance to mousepox by limiting virus spread from the draining lymph node (D-LN), by helping orchestrate the innate and adaptive immune response, and by directly providing an anti-viral state to liver cells. In addition, we will investigate differential roles in resistance of the various T1-IFN subtypes and identify the sensing mechanisms and the key cells important for T1-IFN production during ECTV infection. In the context of acquired immunity, we are specifically investigating the mechanisms whereby memory TCD8+ and humoral immunity provide acquired resistance to mousepox. We have already demonstrated that not only Abs but also memory TCD8+ (TCD8+) can afford acquired resistance to mousepox. Furthermore, we have shown that not only Abs directed to structural viral proteins but also Abs directed to non-structural virulence factors can protect. In this project we are trying to identify specific mechanisms whereby these two arms of the adaptive immune system protect. This project should provide a deep knowledge regarding specific mechanisms whereby mice control ECTV infections to resist mousepox as an invaluable model to understand how humans control periphery→systemic viral infections in general and OPVs in particular to resist viral disease.Top
2. Bioreagents Core
This Core supports the three projects in the Program by supplying genetically manipulated ECTV. In addition, the Core’s own research project will require the deletion of two ECTV genes. The genomes of poxviruses can be manipulated by homologous recombination to either eliminate genes or to add foreign sequences and the procedures involved are relatively simple. While most of the techniques for the genetic manipulation of poxviruses were developed for vaccinia virus (VACV), we have successfully used homologous recombination to manipulate the ectromelia virus (ECTV) genome. In addition, Project 2 will need to create transgenic mice, which requires expertise in genetic engineering and design of genetically modified mice with which we also have strong expertise. In addition, this core has a pilot project where we are studying whether the ECTV immune response modifiers (IRMs) EVM043 and EVM145 play a role in virulence. The prediction is that these viruses will also be highly attenuated. If so, they may constitute excellent targets for antiviral therapy.Top