Transcript
INTRODUCTION
Construction of Listeria monocytogenes vaccine candidate expressing CMV specific antigens
Shane Klumpp, Alejandro Alice, Gwen Kramer, Shelly Bambina and Keith Bahjat
We constructed 4 unique Lm strains producing CMV epitopes from pp65 and/or IE-1 genes. Our macrophage and DC infections showed that our strains caninvade and grow within these cells. Our B3Z assays show antigen (SIINFEKL) presentation and CD8+ T cells activation through the MHC I.These results demonstrate that the new strains can secrete the desired proteins and the SIINFEKL epitope can be presented by DC making them good candidatesfor future studies using PBMC samples from CMV+ individuals.
Human Cytomegalovirus (CMV) is a major cause of mortality in immunocompromised patients: including pregnant women and in organ transplant cases. CMV-seropositive donors (D+) to CMV-seronegative recipients (R-) only comprises 20% of total transplant cases, but generates 50-70% of CMV related transplantdisease (Bij & Speich, 2001).
CMV has also been shown to be a factor in glioblastomas (GBM). CMV related antigens have been detected in GBM, and may be exploitable for potentialtreatments of GBM. Traditional treatment of GBM has a median survival of 7-15 months, creating a need for new treatments to enhance and lengthensurvivability of the disease (Cobbs, 2013)
The creation of a CMV vaccine may save up to $4 Billion in annual cost savings, and has therefore been rated as a highest priority treatment in the health caresystem (Griffiths, 2009)
CONCLUSIONS
RESULTS
FUTURE STUDIES1. Testing strains within human CMV+ samples to demonstrate if strains can activate CMV specific CD8+ T cells2. Mouse studies using A2-HHD-II mice
Earle A. Chiles Research Institute, Portland, Oregon, USA
AIM OF RESEARCH
Current CMV vaccines under construction elicit primarily a humoral immunity. The goal of this project was to generate vaccine candidates that elicits a potent andlong-lived CMV-specific CD8+ T cell response that would be used in prophylactic treatment prior to CMV D+/R- organ transplantation and in therapeutic treatmentof CMV+ GBM patients.
Hypothesis
Listeria monocytogenes (Lm) background
• Gram positive bacterium• Lm produces a strong immune response• Recombinant Lm can be made to secrete tumor-associated antigens (TAA’s) • Specifically infects antigen presenting cells (APC’s)• Infection of APC’s allows effective antigen presentation to MHC I & II for CTL’s (CD8+ T cells) and Helper T cells (CD4+ T cells) generating an immune
response• Important genes
• actA• Cell to Cell infection• Deletion from bacterial chromosome attenuates virulence but still allows for initial infection and immune
response• gcpE
• γδ T cell activation that can kill Lm by recognizing HMBPP compound, inhibiting the specific immune response to the TAA
Cytomegalovirus (CMV) background
• Relevant genes• pp65
• IRF-3 inhibitor (Abate et al, 2004)• IE-1
• Transcriptional regulator (Paulus & Nevels, 2009)• Both genes are immunologically significant antigens that can be recognized by CD8+ T cells
• Significant pp65/IE-1 antigen presence in glioblastomas (Lucas et al, 2011)• 100% of seropositive CMV GBM patients had pp65/IE-1 antigen specific T cells in blood serum (Ghazi et al, 2012)• pp65 and IE-1 associated antigens are detectable in CMV+ patients (Koenig et all, 2014)
Vaccine model
Figure 1: 1) Lm infects antigen presenting cells (DC). 2) Lm escapes phagolysosome. 3) Lm secretes recombinant (TAA) and other bacterial proteins. 4) Through antigen presentation, pp65 and IE-1 will be presented in MHC I to CD8+ T cells, producing a specific immune response
Two vaccine candidates, actA-pp65 and actA-duo CMV (Fig. 2), were constructed and conjugated into either Lm ΔactA or Lm ΔactA ΔgcpE strains. Antigenpresentation and CD8+ T cell activation (SIINFEKL) was measured through a B3Z assay. We also evaluated intracellular growth by infecting macrophages anddendritic cells (DC).
METHODS
Listeria secreting pp65 and/or IE-1 protein antigens within cytoplasm of APC’s will induce strong, lasting protective immune responses through specific CD8+ T cell activation
Figure 2 Figure 3
Figure 1
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Macrophage and DC Infection: Lm can grow effectively in macrophage and dendritic cells
B3Z assay: Lm strains activate CD8+ cell responses
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