GLRCE Vaccine Development
Research Project 1 - Vaccines and Therapies against Botulism
This research project will continue to develop a subunit vaccine against botulism, characterize the neuronal co-receptors for the clostridial neurotoxins (CNT). These studies will provide insight in the development of immuno-therapies and inhibitors to block BoNT entry into neurons.
There are two specific aims:
1. BONT VACCINES AND RECEPTORS Previous studies established the utility of an E. coli derived hepta-serotype HCR vaccine. Experiments will continue to optimize the HCR based vaccine and characterize the molecular properties of clostridial neurotoxins-receptor interactions.
- Optimize a subunit hepta-serotype BoNT vaccine
- Determine the role of sub-serotype specificity of BoNTs for vaccine development
- Characterize a new serotype of BoNT
- Determine the molecular basis for anti-sera neutralization of BoNT toxicity
- Characterize the synaptic protein complex that is a high affinity receptor for the clostridial neurotoxin co-receptors
2. STRUCTURAL PROPERTIES ON BONT HCRS The structures of the BoNT HCR vaccine candidates will be determined to define the physical relationships between these recombinant derivatives and native BoNT. Crystal structures of HCRs in complex with ganglioside and protein receptors (both binary and ternary complexes) will be determined to develop strategies for therapeutics that inhibit BoNT binding to neurons. We will focus on the structural determinations of:
- HCR/A and HCR/F
- HCR/D, HCR/C
- HCR domain of a new BoNT serotype
- CNT HCRs bound to co-receptors
Research Project 5 - Vaccines against Plague
Yersinia pestis, the highly virulent agent of plague, is a biological weapon. Strategies to prevent plague have been sought for centuries, however neither an FDA approved vaccine or the molecular basis of plague immunity are established. Immunization of animals or humans with live-attenuated (non-pigmented) Y. pestis strains raises protective immunity; however associated side effects prohibit the use of whole cell vaccines in humans. Previous efforts to develop subunit vaccines combined two protein antigens, F1 and LcrV, to prevent bubonic and pneumonic plague. This research project addresses the need for plague vaccines and also seeks to understand the molecular basis of plague immunity. Our work demonstrates that Y. pestis F1 pili are dispensable for the pathogenesis of bubonic or pneumonic plague. During infection, breakthrough mutants emerge that henceforth escape plague immunity derived from either F1 subunit vaccines or live-attenuated strains. Breakthrough mutants carry IS1541 insertions in caf1A (which specifies the usher for pilus assembly), indicating that F1 pili are not a suitable vaccine component. LcrV subunit vaccines were shown to protect mice and non-human primates against bubonic and pneumonic plague. LcrV displays immune modulatory effects. A variant, V10, lacks these properties, but retains the ability to raise protective immunity. LcrV is positioned at the tip of type III needles and antibodies against LcrV protect immune cells from Yersinia type III injection of effector Yops, a virulence mechanism that blocks bacterial phagocytosis and NF-κB activation by host immune cells. Plague bacteria preferentially inject phagocytes and this target selection requires CD14 and TLR6 on the surface of immune cells. LcrV-mediated engagement of CD14/TLR2/TLR6 triggers signal transduction cascades, IL-10 release as well as suppression of proinflammatory cytokines. Goals of this application are to develop subunit vaccines for plague protection and to appreciate plague immunity at a molecular level by determining the nature of protective antibodies and Y. pestis escape variants. Other work will determine the contributions of TLR2, TLR6 and CD14 towards Y. pestis selection of targets for type III injection and unravel the mechanisms whereby the pathogen evades the development of immunity during plague infections.
Research Project 8 : Vaccines against MRSA
Staphylococcus aureus is the leading cause of bloodstream, lower respiratory tract, and skin and soft tissue infections in the United States. Of all individuals that are admitted to US hospitals, 4.6% develop staphylococcal infections. S. aureus strains harboring genetic determinants of antibiotic resistance, especially methicillin-resistant S. aureus (MRSA), are isolated in up to 60% of community and 80% of hospital infections with an aggregate mortality of at least 18,000 lives per year. The search for protective immunity against invasive S. aureus disease has been a premier research goal since the discovery of this microbe. Whole-cell live or killed vaccines have failed to generate protective immune responses in humans and are no longer being evaluated for human vaccine development. Several envelope components and secreted products have also been investigated as vaccine antigens. While some measure of pre-clinical success has been achieved with these approaches, no single antigen has as of yet facilitated the development of long-term protective immunity.
This research project is based on three fundamental discoveries that together serve to define the complexity of the host-pathogen interaction and suggest novel strategies for augmenting the host defense mechanism.
- First, S. aureus modulates immune responses during infection thereby preventing the development of protective immunity. When used as live-attenuated whole cell vaccines, staphylococcal variants are capable of generating protective immunity in animal models of disease. Thus, the genetic basis of protective immunity can now be established by molecular genetic perturbation of the organism (Aim 1).
- Second, the level of protective immunity against S. aureus infection in animal models is increased when two or more protein antigens are introduced as a combination vaccine. The reciprocal phenomenon is also true: genetic subtraction of more than one vaccine antigen (virulence factor) leads to a commensurate reduction of staphylococcal virulence. Combinations of vaccine antigens will be tested in specific models of disease and protection will be correlated with their contribution to virulence (Aim 2).
- Third, specific virulence determinants may be identified to direct organ specific disease processes, as evidenced in staphylococcal pneumonia. The primary importance of α-hemolysin has been demonstrated in this disease (depicted in figures A and B below), enabling the design and testing of unique vaccine and immunotherapeutic strategies (Aim 3).
