ProjectsProject 1 - David Topham
Project 2 - Andrea Sant
Project 3 - Tim Mosmann
Project 4 - Gary Whittaker
Project 5 - Toru Takimoto
Project 1: David Topham
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.
Project 2: Andrea Sant
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.
Project 3: Tim Mosmann
The role of human influenza-specific IL-2, IL-4 and CD4+ T helper cells in helping B cell
antibody secretion to promote an immune reaction is critical. T cells that activate
memory B cells to secrete immunoglobulin and therefore enhance rapid antibody responses
are important in vaccine responses. As Thpp are the major T cell population induced by
protein antigen vaccinations, which include very effective vaccines with >95% seroconversion
rates, IL-2+IFN?-IL-4 Thpp cells may be effective B cell helpers. This project will test
the helper abilities of Thpp and other subsets by isolating rare antigen-specific T cells
directly ex vivo and measuring help for B cell proliferation and antibody secretion.
Previous experiments have shown antibody production from B cells incubated with antigen and tetanus-specific Th1, Th2 or Thpp cells isolated from ex vivo peripheral blood mononuclear cell stimulation cultures using the Cytokine Secretion Assay (Miltenyi). However, specificity controls indicated that the “antigen-specific” B cell Elispot was in fact detecting polyclonal antibody secretion. Together with the demonstration by Amanna and Slifka that tetanus-specific B cells were present at only about 1 in 10,000 memory B cells, this indicated that our helper system did not involve cognate antigen-specific help. Because of this, and also because we wished to measure help for B cells at an early time point, before the Thpp cells could differentiate into effector T cells, we established a FACS-based human B cell proliferation assay as a readout for antigen-specific B cells.
Project 4: Gary Whittaker
Influenza virus fusion is well characterized to occur in the low pH environment of
the endosomes. In combination with cell biological assays of endocytosis, we are
examining influenza virus fusion – using structural, computational and
spectrofluorometric analyses . From a structural point of view we are interested in
the changes that occur in the viral hemagglutinin (HA) molecule that might account
for different pathogenic properties as viruses emerge into new species (e.g
avian-equine or avian-human) and well as how the virus adapts to its new host.
While influenza HA has been extensively studied from a structure-function perspective, most studies have focused on the H3 influenza subtype. As there are 16 different HA subtypes in nature, our structure-function studies on the influenza hemagglutin have the goal of identifying novel mechanisms of HA activation across a range of different subtypes, with a focus on human H1 and H2 viruses (including the 1918 pandemic virus), equine and avian H7 viruses and avian H9 viruses. We are investigating novel mechanisms of fusion activation and proteolytic priming that may account for novel pathogenic properties and/or establishment of high pathogenicity avian influenza viruses in new mammalian hosts.
Project 5: Toru Takimoto
The outbreak of avian H5N1 in humans confirms that some avian viruses can directly
infect humans. These incidences highlight the potential for the emergence of
pandemic viruses directly derived from avian influenza A viruses. However, the
molecular determinants and related mechanisms that make certain influenza viruses
highly pathogenic for mammalian species, including humans, remain poorly understood.
Some studies suggest that mutations in viral polymerase proteins play a major role
in adaptation to mammalian species. We therefore hypothesize that polymerase
mutations may contribute to mammalian host adaptation by enhancing virus growth
in mammalian cells or increasing virus pathogenicity. We further hypothesize
that enhanced viral replication or pathogenicity in mammalian hosts may be
associated with changes in
(i) the interaction between the virus RNA polymerase and mammalian cell cofactors required for full polymerase activity and/or
(ii) the enzymatic activity of the purified polymerase. In this project, we will identify the residues in the viral polymerase associated with changes in catalytic activity, interaction with cellular cofactors, and pathogenicity to r eveal the molecular mechanism of mammalian host adaptation and virus mutability. These studies are expected to contribute to the identification of avian viruses that may be associated with pandemic potential, as well as potentially leading to new strategies to develop antivirals and other control measures.
Get in Touch
For general questions, call:
Email: Donna Neu