Research in this laboratory is focused on achieving an understanding of the immune response by measuring immune activity to develop disease intervention strategies against a number of new and emerging infectious diseases, including influenza virus, SARS, arena viruses, and variola virus the agent of smallpox.

Our goals are to investigate and determine cytotoxic and helper T cell epitopes derived from these infectious agents and presented by murine, non-human primate and human MHC molecules. To accomplish these goals the team is using a multidisciplinary approach based on genome wide scanning of the viruses genome, high throughput binding assays utilizing purified MHC molecules, in vitro cellular assays and in vivo assays, as feasible and appropriate. The identification and characterization of cytotoxic and helper T cell epitopes is key to the development of diagnostic reagents necessary to rigorously evaluate T cell responses associated with infection in humans, and also enable the evaluation, at the level of T cell immunity, of the performance of different vaccine candidates.

The concept of HLA supertypes and its implications for epitope identification

T cells recognize a complex between a specific MHC type and a particular pathogen-derived epitope. Thus, a given epitope will elicit a response only in individuals that express an MHC molecule capable of binding that particular epitope. MHC molecules are extremely polymorphic, with several hundred different variants known in humans. Therefore, selecting multiple peptides with different MHC binding specificities will afford increased coverage of the patient population targeted as vaccine recipient. However, the issue of population coverage in relation to MHC polymorphism is further complicated by the fact that different MHC types are expressed at dramatically different frequencies in different ethnicities. Thus, without careful consideration, a vaccine with ethnically biased population coverage could result in decreased efficacy. One means of circumventing the problem of MHC restriction in the study of immune responses in humans to infections and vaccination relies on the selection of epitopes restricted by MHC types that can be grouped in broad families or supertypes. These supertypes are characterized by largely overlapping peptide repertoires and expressed at high frequencies in all major ethnicities. Specifically, by targeting the A1, A2, A3 and A24 supertypes, population coverage in excess of 90% can be achieved, irrespective of the ethnicity of the target population. Inclusion of the B7 and B44 supertypes allows coverage of virtually 100% of the population.

A number of studies have shown that 85 to 100% of peptides which bind multiple HLA molecules with high affinity are recognized by recall responses detected in Peripheral Blood Mononuclear Cells (PBMC) from immune or infected individuals. Recognition by memory recall responses demonstrates that the epitope is not only generated in vivo in the course of natural infection, but also that a TCR repertoire exists which is capable of recognizing that particular epitope.

Laboratory Projects:

I. Immune Epitope Database and Analysis Program

The NIAID supports research related to basic understanding of immune responses leading to the development of vaccines and novel therapeutic agents for the prevention and treatment of infectious and immune-mediated diseases, and improvements in public health. This includes support of various reagents facilities, repositories, and databases that provide resources for biomedical researchers. As a part of the research program to improve defense against biological terrorism and emerging/re-emerging infectious diseases, the NIAID seeks to establish a comprehensive database of molecular structures recognized by the immune system. These structures, termed epitopes, attach with lock and key precision to receptors of the immune system, namely B cell antibodies and T cell receptors. The immune system is able to respond to an enormous number of sa epitopes. An unlimited number of antibody epitopes and 5 x 1011 T cell-epitopes are estimated to exist. The number of known epitopes currently available for incorporation into the database is many times smaller, but new epitopes are continually being discovered and large numbers can be identified by current techniques. Creation of a standardized database, with a priority for epitopes associated with bioterrorism agents and emerging/re-emerging infectious diseases, will greatly facilitate utilization, for biodefense purposes, of the growing bank of antibody and T cell epitope information. In addition to enhancing research aimed at understanding epitoep generation and immune recognition, creation of an Immune Epitope Database and accompanying Analysis Resource will expedite the development of improved vaccines and immunotherapeutic agents. The purpose of the project is two-fold:

A. The primary purpose of this project is to design, develop, populate, and maintain a publicly accessible, comprehensive Immune Epitope Database containing linear and conformational antibody epiteops and T cell epitoeps composed of MHC-binding peptides and ligands ((e.g., carbohydrates, lipids, and modified peptides) with a priority for epitopes associated with NIAID category A-C potential bioterrorism pathogens and their toxins (listed at http://http://www.niaid.nih.gov/dmid/biodefense/bandc_priority.htm). The Immune Epitope Database will be freely accessible to the scientific community via Internet website.

B. An Analysis Resource will be developed and maintained by Dr. Sette’s group, which will include access to: (1) tools to help researchers locate and analyze information contained in the Immune Epitope Database; (2) other relevant databases and related information; (3) data mining algorithms, mathematical models, and other sophisticated analytical tools to help researchers identify novel antibody and T cell epitopes from genome or protein sequence information, predict the immunogenicity and/or antigenicity of epitopes, and predict host immune responses to particular epitopes; and (4) a quarterly newsletter for the scientific community and an annual compendium of data in the Immune Epitope Database and analytical tools provided at the web site during the previous year. Dr. Sette’s team will develop improved algorithms, models, and other analytical tools for use in discovering new knowledge from the database. All of information contained within the Analysis Resource, including analysis tools and algorithms, will be made freely available to the scientific community.

II. Class I and class II restricted epitopes from a representative sample of the different arenavirus species pathogenic in humans.

This project addresses the discovery and validation of cytotoxic and helper T cell epitopes presented by HLA class I and class II MHC molecules, respectively, that are derived from a group of prevalent arenaviruses (Junin, Guanarito, Sabia, Machupo, Lassa, LCMV and Whitewater arroyo) with known potential for causing disease in humans, and representative of a diverse set of arenavirus phylogenetic groups. We also plan to investigate and determine epitopes presented by murine, non-human primate and human MHC molecules. To accomplish these goals we plan to utilize a multidisciplinary approach, based on bioinformatic scanning of viral genomes, high throughput binding assays utilizing purified MHC molecules, in vitro cellular assays and in vivo assays. These studies should lead to the definition of a broad range of epitopes, facilitate development of diagnostic reagents allowing a rigorous evaluation of T cell responses associated with infection in humans, and enable the evaluation of the performance of different vaccine candidates. Little is known about the degree to which cellular responses in humans are cross-reactive amongst various arenavirus species. Thus, for each virus, we will map the epitopes recognized within each viral antigen by class I and class II molecules and determine their degree of cross-reactivity with homologous sequences from other arenaviruses. We will also pursue the development of a multi-epitope vaccine construct, encompassing epitopes cross-reactive with multiple arenaviruses, as well as a collection of non-cross-reactive epitopes derived from each distinct virus, so that a single vaccine entity might be able to protect from, or ameliorate the disease course caused by, several major arenavirus species.

III. Identification of class I and class II restricted epitopes derived from variola and vaccinia viruses.

This study addresses the discovery and validation of cytotoxic and helper T cell epitopes derived from the vaccinia and variola viruses and presented by murine, non-human primate and human MHC molecules. To accomplish these goals we plan to utilize a multidisciplinary approach based on genome wide scanning of the viruses genome, high throughput binding assays utilizing purified MHC molecules, in vitro cellular assays and in vivo assays, as feasible and appropriate. In the first part of our studies, we will 1) determine the immunodominant antigens recognized by class I and class II restricted responses in humans immunized with vaccinia virus, 2) map the epitopes recognized within each antigen, and 3) determine their degree of cross-reactivity with homologous variola virus-derived sequences.

IV. Epitope-based Multi-peptide Vaccines for Expanded Coverage Against Inter-pandemic Influenza. (In collaboration with Dr. Mbawuike, Baylor College of Medicine, Houston, TX)

The CDC estimates that influenza epidemics kill about 20-40,000 persons annually in the U.S, most of them elderly. Currently licensed inactivated subunit influenza virus vaccines (IVVs) show reduced effectiveness against variant interpandemic drift influenza A viruses because of the inevitably poor match between vaccine-elicited antibodies and the virus. Influenza subunit vaccines are also ineffective in inducing influenza virus-specific CD8+ cytotoxic T lymphocytes (CTL) which appear to be critical in mediating the clearance of virus infection, thus promoting recovery and lowering rates of morbidity and mortality. The inadequacy of subunit influenza vaccine is obviously due to the narrow spectrum of response it elicits and frequent mutation of influenza virus. We hypothesize that a vaccine based on reproducing the breath of responses induced by the whole organism, including T cells, B cells and appropriate cytokines, should provide better protection against influenza disease. Such optimally effective vaccine should include epitopes derived from all viral antigens recognized by most of the genetically diverse human population.

The goal of this proposal is to identify and characterize immunodominant and subdominant CTL and helper T lymphocyte (HTL) epitopes contained in all ten influenza protein segments restricted by major human HLA supertypes.

V. Human T-cell Epitopes in SARS. (In collaboration with Dr. Buchmeier, The Scripps Research Institute, San Diego, CA).

The discovery and validation of HLA-restricted cytotoxic and helper T cell epitopes derived from the Severe Acute Respiratory Syndrome coronavirus (SARS CoV) represents a significant challenge because of the large genome size and because of the extreme polymorphism of HLA alleles. Herein, we propose to combine bioinformatic approaches and high throughput MHC-peptide binding assays to identify epitopes restricted by HLA class I and class II molecules representative of >95% of the general population, irrespective of ethnicity. These epitopes will be further validated with in vivo and in vitro immunogenicity studies utilizing HLA transgenic mice and human PBMC from healthy unexposed donors. Animal models are likely to play a vital role in the development of SARS specific vaccines and diagnostics. Accordingly, we also plan to identify epitopes presented by mouse and non-human primate MHC molecules.