The 2010 TRIAD Toolkit Conference, hosted by the Institute for Immunology and Informatics at the University of Rhode Island, was a two-day gathering that provided an opportunity for researchers from a variety of disciplines to collaborate and critique each other’s research, and to familiarize themselves with iVAX,a leading software toolkit that allows researchers to reliably and efficiently make predictions in silico. A host of researchers from around New England, other parts of the United States and distant continents participated in the exchange of knowledge and the progression of science, addressing the application of informatics tools to topics ranging tick vaccines to the investigation of feline immunodeficiency virus as a model for HIV.
In addition, the informatics tools were demonstrated, as were several complementary laboratory protocols, each important to the validation and application of informatics predictions. Day 1 of the conference started with an introduction from Anne De Groot, CEO of EpiVax, Inc. and Professor and Director of I’Cubed at the University of Rhode Island, followed by presentations from Dr. Dale Greiner, Dr. Stephen Gregory, Dr. Paul Knopf, Dr. Wendy Coy, Dr. Denice Spero and Dr. Lenny Moise who explained some of the many applications of TRIAD informatics software and procedures used to validate the prediction tools.
Dr. Stephen Gregory, Brown University: Dendritic Cell-Based Vaccination Against Hepatitis C
Dr. Gregory presented his research on the development of new therapeutics and vaccines against Hepatitis C virus (HCV) and other emerging infectious diseases. HCV infects over 170 million people worldwide, causing serious liver pathology including cirrhosis, hepatitis and carcinoma. Dr. Gregory’s research focuses on developing new strategies to treat HCV by creating a DNA-based vaccine that encodes the highly conserved epitopes; delivering the vaccine through transfected dendritic cells; and elucidating the mechanisms that mediate immune response to HCV. The poor efficacy rate and harmful side effects of current treatments underscores the need for therapeutic or prophylactic vaccines. Thus far, a successful vaccine candidate has been elusive due to the diversity of HCV genotypes, which in turn is a function of the virus’ rapid, low-fidelity replication.
Dr. Gregory utilized iVAX software tools to predict T-cell epitopes that are conserved among multiple quasi-species of HCV, focusing on the most prevalent genotypes in order to develop a vaccine. These predictions will be validated by comparing T-cell responses in blood collected from HCV-infected patients who successfully cleared their infections relative to patients that were infected but were unable to clear the virus. Dendritic cells will be transfected with DNA constructs that encode the epitopes and used to immunize transgenic mice, which will later be challenged with recombinant, HCV protein-expressing vaccinia virus. Additional experiments will determine the immune mechanisms that underlie the response to HCV by assaying cytolytic activity and cytokine production in vitro and in vivo. Dr. Gregory’s research applied iVAX software, not only to the development of hepatitis C therapies, but also to the construction of novel vaccine delivery platforms, and to the extension of our understanding of CD4+ and CD8+ immune responses.
Dr. Lenny Moise, Development of a Non-Tolerogenic aDEC-205 Vaccine Delivery Platform
Dr. Moise discussed a project aimed at devising a novel vaccine delivery method based on targeting natural dendritic cell antigen-presentation pathways. The dendritic cell surface receptor Dec-205 has been identified as a viable target because of its contribution to antigen processing and presentation. When coupled to a costimulatory signal, antigens that bind to Dec-205 are processed more quickly and presented in higher concentrations. It has been well-documented that anti-Dec-205 antibodies effectively carry out this function, and can be linked to the desired target antigen to produce a specific response, while non-co-stimulated antibodies induced tolerance, a result thought to be caused by the presence of regulatory T-cell epitopes in immunoglobulin. Since nonspecific activation of the immune system is extremely dangerous, Dr. Moise’s research proposes to suppress tolerance instead of providing co-stimulation signals. His lab uses informatics tools to identify and modify the regulatory T-cell epitopes, and is in the process of linking these de-tolerized antibodies to antigens of interest.
Dr. Paul M. Knopf, Brown University; EpiVax, Inc. Complement Component C3d
Dr. Knopf, a talisman of immunology and cell biology as well as the discoverer of the polyribosome, presents his findings on the paradoxical discovery of self-reactive antigens in the complement pathway. One complement pathway is antibody-mediated, the other two are activated by the innate immune system, responding to microbial domains and moieties such as LPS and peptidoglycan. Its effector functions include lysis of target cells, opsonization of pathogens, and activation of inflammatory response. Complement protein C3 is crucial to the efficient function of the immune system, as it is a part of all three complement pathways. Moreover, Knopf’s findings indicate that it may act as a connection between the innate immune system and adaptive immune system that assists in classifying antigens according to their potential hazard.
EpiMatrix analysis predicts auto-reactive T-cell epitopes on protein C3d, a proteolytic fragment of C3 that has been found to bind covalently to antigens as well as enhance antibody responses to antigens lacking T-cell epitopes. These findings suggest that the immune system has evolved pseudo-autoimmune pathways that are useful in clearing infection. Moreover, the increase in antibody response was retained in CD21 knockout mice, therefore it appears to be independent of signal 1, which suggests an alternative pathway of B-cell activation. Hence, not only were iVax software tools instrumental in improving our understanding of T-cell epitopes, but they also provided insights which revealed new approaches to investigating innate immunity and B-cell activation
Dr. Dale Grenier, UMass Medical School. HuSCID Mice and Their Use in the Study of Infectious Disease
Dr. Greiner discussed the history and contemporary role of the uses of humanized mice for immunological research and explained recent genetic advances that allow for new in vivo tests that in turn may lead to the development of new therapies. Humanized mice, in other words those engrafted with functional human cells or expressing human proteins, must have severely restricted immune system, so that the graft is not rejected. A number of mutations have been identified that interfere with normal immune development, including scid, which disrupts B and T cells but results in high numbers of NK cells; NOD, which reduces NK cell number, disrupts dendritic cell maturation, macrophage activation and hemolytic complement; and mutations to IL-2r?, which disrupt cytokine receptors, thereby suppressing both innate and adaptive immunity. Various breeding programs have produced mouse strains that combine these traits, allowing for improved results in transgenic grafting, and a number of grafting techniques have improved the homology between the model and real human immune systems. Dr. Grenier’s lab has used these mice as models for HIV, salmonella, dengue, ebola and Typhoid.
Day 2 of the TRIAD Conference saw presentations from Dr. Janet Yamamoto, Matt Ardito, Joe Desrosiers, Dr. Loren Fast and Nick Trotter-Mayo, discussing the applications of the TRIAD toolkit, as well as demonstrating some of the lab techniques and instruments used to validate and apply its findings
Dr. Janet Yamamoto, University of Florida. Feline Immunodeficiency Virus/Cat Model: for T-Cell-Based HIV/FIV Vaccines
Dr. Yamamoto evaluated the validity of feline immunodeficiency virus (FIV) as a model for researching HIV and developing a vaccine. FIV vaccine has been commercially available for use in domestic cats since 2002. Its high rate of protection against a variety of viral genotypes (100% vs subtype A FIV-Pet, 89% vs B FIV-FC1 (Florida), 61% vs recombinant subtype A/B FIV-Bang (Massachusetts), 44% vs recombinant F’/C FIV-NZ1 (New Zealand), and 40% vs A FIV-Uk8 (Glasgow)) indicates that the FIV/cat model may be useful in developing an HIV-1 vaccine. This model may help illuminate the immune mechanism required for protection, and can provide helpful insights into the search for evolutionarily conserved protective epitopes.
Matt Ardito, Gary Kurczy & Bill Martin, EpiVax, Inc.
The informatics team from EpiVax, Inc. outlined and demonstrated the TRIAD Vaccine toolkit. As members of the Immunoinformatics teams at EpiVax, Inc., they provided an in-depth , practical demonstration of the TRIAD Toolkit, which consists of a number of proprietary tools for genome screening and protein characterizations, built on a suite of both publically available and proprietary tools related to the fields of Genomics, Proteomics, Bioinformatics, and Computational Biology. These algorithmic tools were developed by EpiVax for the purposes of vaccine design and the demonstration included the following:
1. EpiMatrix : used to screen genomic sequences from genomes of interest for T cell epitope content.
2. ClustiMer: used to identify clusters of Class II T-helper epitopes contained in short protein segments.
3. Conservatrix: used to select putative epitopes that are conserved across a range of variant protein isolates for even the most mutable of protein targets.
4. EpiAssembler: used to knit conserved sequences together into highly immunogenic consensus sequences.
5. Aggregatrix: used to find the minimal set of epitopes that “covers” the maximum number of HLA types and observed strains of the target pathogen.
6. Vaccine CAD: used to convert any epitope set as input material and create an optimized string-of beads while minimizing deleterious “junctional” epitopes.
Dr. Loren Fast, Brown University. Flow Cytometry Overview, Immunophenotyping of Cell Populations
Dr. Fast provided a succinct general overview of the function of the flow cytometer and its applications in examining the various ways it can be used to characterize lymphocyte populations. Flow Cytometry is a technique for counting and examining microscopic particles, such as cells, that allows the researcher to analyze several thousand particles in seconds in real time. Moreover, it can actively separate and isolate particles having specific properties by tagging these properties with fluorescent labels with quantized input and output wavelengths. For example, a CD4+ cell can be labeled by incubation with a fluorescent-coupled anti-CD4 antibody that absorbs violet light and emits green light, as long as the Flow Cytometer is equipped with a violet laser.
Joseph Desrosiers II, URI, demonstrated tools that can be applied to complement the use of the TRIAD Toolkit. These tools (PBMC Separation, ELISpot, FACS) support the underlying theme of the TRIAD toolkit, which is to develop a vaccine development process that produces broad-spectrum vaccines facilitated by informatics tools, and validated using in vitro and in vivo methods. The tools and processes are explained in greater detail below:
1. Human Peripheral Blood Mononuclear Cell (PBMC) separation: A PBMC is any blood cell having a round nucleus, including lymphocytes, monocytes or macrophages, all critical components of the immune system. The lymphocyte population is of particular interest to the design of bioinformatics-based vaccines, and it consists of T cells (CD4 and CD8), B cells and NK cells. These cells were extracted from whole blood using ficoll, a hydrophilic polysaccharide that separates layers of blood, with monocytes and lymphocytes forming a buffy coat under layer of plasma. Alternatively, PBMCs can be extracted from whole blood using a hypotonic lysis which preferentially lyses red blood cells. Flow Cytometric analysis (Cellular phenotypeing) and BD LSRII to illustrate how data is ascertained from assessing cell populations.
2. Enzyme-Linked Immunosorbent Spot (ELISpot) is a variation of the typical sandwich ELISA technique. A detector antibody (often monoclonal) is coated onto a set of microplates. The microplates are then blocked with non-reactive serum proteins, and incubated with cells at various known densities, along with antigen. Following incubation, the cells are washed to remove cells, serum, media, and unbound proteins, and incubated with biotinylated “indicator” antibody. After a wash to remove unbond biotinylated antibodies, the plates are visualized, yielding an accurate assessment of cellular secretion of a desired protein, often a cytokine of interest. The Immunospot S5 UVanalyzer contains software that efficiently counts the biotinylated spots and returns an accurate and fast readout.
3. Fluorescence-Activated Cell Sorting (FACS) is a specialized type of Flow Cytometry (see above) that allows the cells to be separated into separate containers according to physical properties at a high rate and with high precision. As the droplets (each containing a single cell) leave the nozzle, they pass through a charging ring and a fluorescence detector. The charging ring imbues the particular droplet with an electric charge according to the fluorescent readout, and an electromagnetic deflector system guides the droplet to the appropriate collecting vessel.