Laboratory Projects Antigenic evolution and immunity to influenza PI: David Topham, Ph.D. The overall goal of the clinical core is to provide samples from carefully characterized subjects after influenza infection or vaccination, to support the conduct of studies under projects 1 (Determinants of heterosubtypic immunity against influenza); 2 (Specificity of the CD4 T cell immune response to influenza); and 3 (Effector functions of influenza-specific T cells induced by immunization and infection). The clinical core provides for collection of appropriately timed samples from individuals receiving routine vaccination or after natural infection and from individuals participating in clinical trials supported by other mechanisms, and provides sample processing, storage, and tracking. In support of this mission, four clinical protocols have been generated and these protocols are summarized briefly. Studies of the immune response to influenza are usually limited to assessment of secondary infections, because primary exposure to influenza occurs early in life, typically before the age of nine years. Assessment of these responses is complicated by lack of knowledge of the specific influenza virus responsible for earlier exposures, and the timing of previous exposures. These problems is compounded as persons age and have experienced multiple infections. One approach to obtaining samples from primary or near primary infection is to perform prospective surveillance in populations of children with relatively high attack rates. Therefore, this study involves prospective surveillance conducted in families where at least one child is under the age of four years. Although the family study is ideal for assessing primary, or near primary infections, a disadvantage of this approach is that limited numbers of samples, and limited amounts of blood, are feasible to be obtained in the context of a family surveillance study. As a result, we are supplementing these measurements by obtaining serial measurements of immunity in college students and other young adults who are likely to remain in contact with the study center for at least 3 influenza seasons. The primary objective of this surveillance activity is to obtain relatively larger amounts of cells, including pre-influenza season samples. The purpose of the third protocol is to provide cells and plasma from normal donors to develop and validate additional assays of influenza immunity. These assays will then be used in studies to assess immune responses to vaccination and infection in children, adults, and the elderly. Healthy donors undergo medical screening and rapid assessment of hematocrit. The last study is built on previous studies that demonstrated the ability of previous vaccination with a clade 0 H5N1 vaccine to prime for responses to a single dose of clade 1 H5N1 vaccine administered 8 years later. That study was limited by a relatively small study sample size, and the lack of concurrent testing of a vaccine naïve group. The primary objectives of this study are to describe the kinetics of the cellular immune response to subvirion H5N1 vaccination in primed and unprimed individuals, to determine the effects of priming on the repertoire and phenotypes of B and T cells generated in response to vaccination, and to generate hypotheses regarding the relationship between the peptide specificity of CD4 T cells prior to vaccination and subsequent humoral and cellular responses. This will be a randomized, subject and laboratory blinded assessment of the cellular immune response to A/Indonesia/5/05 vaccine in two populations: (1) healthy adults who have previously received an H5 vaccine in a DMID sponsored study; and (2) healthy adults with no previous H5 vaccination and who are not at risk for H5 exposures. Subjects will be randomized to either a low dose (15 mcg) or high dose (90 mcg) of vaccine, and peripheral blood mononuclear cells will be obtained before and at several time points after each dose of vaccine, for assessment of peptide-specific responses of CD4 T cells, cytokine and cell marker phenotypes of responding helper CD4 T cells, numbers of antigen-specific antibody secreting cells by B cell ELISPOT, the presence and quantity of antigen specific memory B cells, and flow cytometric correlates of B cell and antibody responses. Targeting B cell responses to provide broad protection against influenza PI: Patrick Wilson, Ph.D. The defining feature of the immune system is its ability to distinguish self from non-self, a function mediated by antigen-specific T lymphocytes. T cell receptors can only recognize antigens derived from pathogens or transformed cells if these antigens if the derived peptide fragments of pathogenic proteins combine with Major Histocompatibility Complex (MHC) molecules. The assembly of the antigenic peptide-MHC complex takes place in intracellular compartments, by a series of molecular events collectively referred to as "MHC-restricted antigen presentation". The research in my laboratory centers around the molecular events that regulate MHC class II-restricted antigen presentation and CD4 T cell activation in vivo. Our long term goal is to make connections between the mechanisms involved in peptide acquisition by class II molecules and those aspects of immunology that critically depend on the specific peptides presented by the class II molecule. Our laboratory has a long-standing interest in the mechanisms involved in establishment of immunodominance in CD4 T cell responses. During immune responses pathogens or to protein antigens, T lymphocytes only respond to a limited number of peptide epitopes contained in the immunogens. These peptides are termed "immunodominant". Our experiments seek to understand the elements in vivo that dictate the narrowed selection of specificities in CD4 T cells during protective immune response. Most recently, we have begun to explore the hierarchy of CD4 T cells responses to influenza virus, a human pathogen that poses unique challenges for vaccine design because of its high degree of genetic variability and because of the recent threat of avian influenza. The research area in our laboratory focuses on understanding the molecular and cellular events that shape and refine the repertoire of CD4 T cells that are specific for influenza virus, using both animal models and analyses of human immune responses. We also seek to understand the specificity of human CD4 responses to influenza virus and vaccines during the primary response to influenza infection,, during the establishment of long term memory and during challenge with vaccines or live viruses. Or research on influenza has the long-term goal of rational vaccine design to develop vaccines that promote heterosubtypic immunity in the CD4 T cell compartment, by developing strategies to focus the CD4 T cells towards the most biologically active and genetically conserved epitopes. We are developing strategies to dissect MHC class II restricted presentation of influenza antigens by B cells in order to understand the mechanisms that control recruitment of CD4 T cell help by B cell during influenza infection, a requisite event for the production of high affinity neutralizing antibodies to influenza virus. Links between specificity and function of influenza specific CD4 T cells PI: Andrea Sant, Ph.D. There are several critical voids in our knowledge of influenza specific immunity which is due in part to the complexity of CD4 T cell specificity and function. There is still uncertainty regarding predictors and correlates of protection and a need distinguish the individuals whose immune system is competent to withstand a challenge from those that require boosting or de novo vaccination. We also need to know which vaccines are optimal for eliciting the most effective B cells and T cells. Finally, for pandemic preparedness, it is critical to identify the individuals that can mount a robust response and those whose immune status requires boosts or adjuvant. Our projects will provide the needed new insight into B cell recognition of viral antigen, delineation of the key subsets and antigen specificity of CD4 T cells that convey the needed function and will evaluate newly developed vaccines that may offer significant advantages in elicitation of protective immunity to influenza in human populations. The core hypothesis that underlies this proposal is that distinct functions of CD4 T cells are carried by cells of different peptide specificities. In some cases, such as help for antibody responses, linkage of function with specificity may be due to physical coupling of T and B cell epitopes. In other cases, functionality in CD4 T cells may be linked to the context or history of antigen encounter. We will develop the strategies needed to characterize antigen handling/class II processing/presentation by influenza-specific B cells. We will analyze transgenic animals that express the cell surface Ig specific for HA and will characterize antigen recognition, peptide:class II epitopes display and how HA-specific B cells specific for the stalk vs. the head of HA compete for viral antigen acquisition. We will also evaluate the links between immunological memory and potential to respond to seasonal and novel influenza viruses and vaccines. We will determine whether the functional potential of CD4 T cells is correlated with specificity for particular viral proteins, with a focus on provision of help for antibody production, cytokine production, and cytotoxic potential. We will compare immune potential and responses to infection and vaccination in children and adults to determine if maturation of the adapted immune response to influenza potentiates or compromises protective immunity. Finally, there have been new advances in vaccine design for influenza that may offer considerable advantages to protective immunity. We will evaluate the ability of these vaccines to elicit CD4 T cell and B cell responses in human subjects and to better poise individuals for protection from potentially pandemic influenza viruses. Pandemic research plan and response PI: Luis Martinez-Sobrido, Ph.D. The pandemic plan will include both a pre-pandemic risk assessment component, as well as an emergency response plan. The focus of the pre-pandemic risk will be to use age-stratified, banked samples of sera and PBMC, including pre- and post-seasonal vaccine samples, to comprehensively assess the level of population baseline immunity to candidate pandemic threat virus. The emergency response plan will utilize the expertise of our center’s assessment of the host response to infection to characterize the host response to an emerging pandemic in humans and relevant animal models, including both the innate and adaptive response to infection and vaccination. Identifying the levels of immunity pre pandemic can help to guide public health preparedness activities, including predictions of the populations most at risk as well as the expected benefits, if any, of existing seasonal vaccines, and the predicted need for multiple doses of candidate pandemic vaccines. The emergency response plan will help guide strategies to optimize the immunogenicity of vaccination as well as potentially identifying targets for therapeutic agents including repurposing existing drugs that may modify the innate response. Immune Response Project: Systems Biology of Innate Immunity and Vaccination The innate immune response to influenza virus is a key determinant of disease severity and subsequent adaptive immunity to infection. As such, modern broad-spectrum vaccination strategies against influenza virus should start with an understanding of the innate immune response. Defining innate immune signaling pathways—and the viral and host factors and interactions that trigger and regulate them—is therefore essential to developing effective drugs and vaccines. In this Project, we will use high-throughput molecular profiling and novel computational methods to build network models of innate immune signaling, identify predictors of vaccine immunogenicity and efficacy, and discover targets and candidate drugs for use as adjuvants or host-directed antiviral therapy. In this project, we will define molecular mechanisms of innate immunity and molecular correlates of robust immune responses and protection following either seasonal influenza vaccination or passive exposure to seasonal influenza strains. Using computational approaches, we will determine whether existing small molecules or therapeutics can be repurposed for use as adjuvants or broad-spectrum therapies. These studies will be augmented by targeted in vitro approaches to augment our understanding of how influenza virus engages the human innate immune system. This Project will provide new knowledge regarding the innate immune response to influenza virus infection and vaccination that can be used to develop more effective vaccines (e.g., to elicit a stronger, longer lasting, or more broadly protective immune response). In addition, this Project uses novel computational methods that make use of genomic profiles to rapidly screen small molecules and FDA-approved drugs for repurposing as antiviral therapies. Because many of these compounds have already been evaluated in human subjects, this strategy may significantly reduce the time needed to translate findings into clinical studies. Viral markers of pathogenesis / Cleavage activation of HA PI: Gary Whittaker, Ph. D Our objectives are to understand the cleavage-activation of influenza HA by proteases in the human respiratory tract and how cleavage-activation is modulated during the emergence of new viruses with variant HAs and in the context of bacterial co-infections. Aim 1. Characterization of circulating influenza viruses with modified HA cleavage sites. Aim 2. Determine the interplay between influenza and co-infecting bacteria for HA cleavage-activation Over the past six months, the Whittaker lab has become part of the CEIRS Virus Risk Assessment program. In line with the overall scope of this program, we have begun to include an assessment of the cleavage-activation of emerging avian influenza virus HAs. The Whittaker lab is also a member of the CEIRS Data Standards Working Group. Cross-CEIRS Collaboration Project: Determining Early Genomic Signatures from Ferret Infection Models This project will examine the early functional genomics responses in ferrets infected with influenza strains having known differences in pathogenicity. Tissues harvested from different anatomical compartments will be used to look for early expression signatures associated with high vs. low pathogenesis. We will focus on the analysis of early blood samples to determine those features that provide an early distinction between severe vs. mild outcomes. We will then utilize these signatures in evaluating gene expression data from early peripheral blood samples from human subjects. These samples will have been drawn on presentation with expected differing disease outcomes (severe or mild). Aim 1: Use RNA-seq to examine transcriptional responses in ferrets infected with influenza viruses of varying degrees of virulence. Aim 2: Apply the blood signatures and biomarkers developed from Aim 1 to predict disease outcomes in human subjects. Option Reagent Development PI: Randy Albrecht Ph. D PI: Andy Pekosz Ph. D PI: Andrea Sant Ph. D PI: Stacy Schultz-Cherry Ph. D PI: Paul Thomas Ph. D PI: Mark Tompkins Ph.D Development of reagents for ferret studies: We have also been tasked with the development of appropriate monoclonal antibodies to perform flow cytometry studies in ferrets as part of a CEIRS-wide effort to improve the ability to use this animal model to study the immunology and host response to influenza and influenza vaccination.