Listeria monocytogenes is an intracellular bacterial pathogen that causes serious foodborne illness in pregnant women, the elderly, infants and immunocompromised individuals. L. monocytogenes infections have the highest case fatality rate of all reported foodborne illnesses. L. monocytogenes can be easily cultured outside of host cells under standard laboratory conditions, making L. monocytogenes a significant public health risk and a significant potential threat as a biological weapons agent. Animal models show that protective immunity to L. monocytogenes is mediated by CD8+ effector cells that recognize and eliminate infected host cells. Vaccine studies have demonstrated that stimulation of protective CD8+ effector cells requires subclinical infection with live bacteria. However, immunization of humans with virulent bacteria fully capable of intracellular replication imposes a significant health risk to any population as a vaccine strategy, especially for those individuals inherently at risk to L. monocytogenes infection. We have recently developed a novel strategy for the generation of replication-deficient bacterial vaccine vectors that are capable of stimulating protective CD8+ effector cell responses. The focus of this proposal is to utilize this approach to produce non-replicating L. monocytogenes vaccine strains capable of generating protective CD8+ effector cell responses.
In Specific Aim I, we will construct non-replicating L. monocytogenes vaccine vectors to deliver protective native bacterial antigens to the cytosol of professional and nonprofessional antigen presenting cells (APC) for endogenous processing and MHC Class I presentation.
In Specific Aim II, we will determine the kinetics of antigen delivery to APC and the requirement of bacterial viability for efficient antigen delivery.
In Specific Aim III, we will determine whether uptake of the vaccine constructs sensitizes APC for recognition by L. monocytogenes-specific effector cells.
In Specific Aim I V, we will determine whether antigen specific effector cells are stimulated following immunization with the replication-deficient vaccine constructs, and assess stimulation of protective antilisterial immunity. Our goal is that following completion of the proposed studies, a safe and effective replication-deficient vaccine formulation will be identified that is suitable for clinical trials. It is also envisioned that these studies will provide a foundation for the development of replication-deficient vaccine vectors against other intracellular pathogens using a similar approach.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01AI056446-04
Application #
7012283
Study Section
Special Emphasis Panel (ZAI1-ALR-M (M4))
Program Officer
Mills, Melody
Project Start
2003-08-01
Project End
2008-01-31
Budget Start
2006-02-01
Budget End
2007-01-31
Support Year
4
Fiscal Year
2006
Total Cost
$424,552
Indirect Cost
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Burrack, Laura S; Harper, J Wade; Higgins, Darren E (2009) Perturbation of vacuolar maturation promotes listeriolysin O-independent vacuolar escape during Listeria monocytogenes infection of human cells. Cell Microbiol 11:1382-98
Feng, Xiuhong; Rodriguez-Contreras, Dayana; Buffalo, Cosmo et al. (2009) Amplification of an alternate transporter gene suppresses the avirulent phenotype of glucose transporter null mutants in Leishmania mexicana. Mol Microbiol 71:369-81
Miner, Maurine D; Port, Gary C; Bouwer, H G Archie et al. (2008) A novel prfA mutation that promotes Listeria monocytogenes cytosol entry but reduces bacterial spread and cytotoxicity. Microb Pathog 45:273-81
Smithey, Megan J; Brandt, Suzanne; Freitag, Nancy E et al. (2008) Stimulation of enhanced CD8 T cell responses following immunization with a hyper-antigen secreting intracytosolic bacterial pathogen. J Immunol 180:3406-16
Rodriguez-Contreras, Dayana; Feng, Xiuhong; Keeney, Kristie M et al. (2007) Phenotypic characterization of a glucose transporter null mutant in Leishmania mexicana. Mol Biochem Parasitol 153:9-18
Burrack, Laura S; Higgins, Darren E (2007) Genomic approaches to understanding bacterial virulence. Curr Opin Microbiol 10:4-9
Ortiz, Diana; Sanchez, Marco A; Pierce, Steven et al. (2007) Molecular genetic analysis of purine nucleobase transport in Leishmania major. Mol Microbiol 64:1228-43
Bouwer, H G Archie; Alberti-Segui, Christine; Montfort, Megan J et al. (2006) Directed antigen delivery as a vaccine strategy for an intracellular bacterial pathogen. Proc Natl Acad Sci U S A 103:5102-7