LAMP-Based DNA Vaccines How LAMP Chimeras Access the MHC-II Pathway Immunomic Therapeutics, Inc. www.immunomix.com info@immunomix.com +1-240-731-5232 www.immunomix.com
A Unique Immunotherapy Solution ITI’s LAMPvax DNA vaccines are composed of 3 major elements: the luminal domain of LAMP, the antigenic protein sequence, and the Transmembrane / Cytoplasmic signaling domain of LAMP.
When this unique chimeria is synthesized in the cell, the luminal domain of LAMP and the antigenic protein are localized inside the Golgi and the cytoplasmic targeting sequence remains external to the Golgi and facilitates subcellular localization.
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LAMP Chimera Co-Localizes with MHC-II
From Arruda, et al, 2006
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The LAMP chimera protein has been shown to localize in the MHC-II compartment in many studies by confocal microscopy. In the figure to the left, cells were transfected with a LAMP/gag chimera and stained for anti-Gag (red) and anti-MHCII (green). The coincidental location of both LAMP and Gag are shown in yellow. Virtually all of the Gag protein is found coincidentally with MHC-II.
Dendritic Cells Are Key to Immune System Presentation A fundamental first step in vaccination is that the vaccine proteins must reach the internal compartment of dendritic cells and then be processed and presented to the immune system. Following injection of a plasmid DNA vaccine, the encoded gene is expressed inside the cell and then processed for MHC presentation.
Source: wikimedia, dendritic cell
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Vaccines that include the LAMP gene sequences present through the MHC-II , accessing the primary immune system presentation pathway.
LAMPvax Activation of Dendritic Cells The next few slides diagram the process of DNA vaccination and presentation to the immune system mediated by LAMP. Step 1. DNA is taken up by the DC. This can be naked DNA or DNA complexed to lipids or other carriers.
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Step 2. The DNA enters the nucleus of the cell and begins to prepare mRNA. It is also possible to deliver mRNA directly.
Step 3. The mRNA directs the synthesis of the chimeric LAMP – Antigen protein which is localized first in the Golgi and then in the maturing MHC-II+ lysosomes.
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The Lysosome Compartment is Key to Immune System Activation The MHC-II molecules, essential to antigen presentation to the immune system, are found in the lysosomes. These vesicles receive imported antigens that are then processed (i.e., digested into peptides) for binding to the MHC-II complex. The lysosomes naturally contain the highly glycosylated protein, Lysosomal Associated Membrane Protein or “LAMP.� ITI’s vaccines take advantage of the natural intracellular localization of LAMP to bring the antigen synthesized by the cell to this key immune processing center. www.immunomix.com
Lysosome Compartment MHC-II LAMP – Antigen Chimera In this expanded view of the lysosome compartment from a LAMPvax transfected cell, the two of the key components of the immune response are diagramed. The LAMP – antigen chimera is anchored inside the vesicle with the antigen protein completely sequestered from the cell exterior providing a unique safety element for LAMPvax applications.
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Digestion by Proteases Release Antigen Peptides
As the lysosome matures, the pH of the internal environment drops and enzymes digest the proteins inside the lysosome.
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Peptides “Load� on the MHC-II Peptides released during the digestion process then bind to the MHC-II molecule in a very specific binding cleft in the protein.
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At the Cell Surface, the Antigenic Peptide is Presented to the Immune System Following the antigen peptide digestion, the mature lysosome moves to the cell surface and presents the MHC-II to the immune system. Shown at the left is an expanded view of the MHC-II molecule containing the antigenic peptide. Thus, little or no free antigen is ever exposed outside the cell. The only presentation of antigen is through the very controlled presentation in the binding cleft of the MHC-II molecule.
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