In Vitro Vaccination Site

The MIMIC™ system (Modular IMmune In vitro Constructs) is as much a process as it is a replicative immune system, and the vaccination site (VS) represents the first part of that process. When an outside pathogen threatens the body's immune system, it does so at some point of entry into the body. Your body responds to the threat by sending antibodies and other immune responses to that location. Within the MIMIC™ system, the VS represents that point of entry and pathogen and/or vaccine interaction.

The goal of the vaccination site is the creation of a three-dimensional peripheral tissue model that reliably mimics the immune process. An important component of the immune response is the capture of antigens by antigen-presenting cells (APCs). The APCs engulf and process antigen and may then traffic to the closest lymph node(s), where they interact with T and/or B cells to initiate antigen-specific immune responses. An essential aspect of the VS is the reproduction of this process, by allowing autonomous generation of resident macrophages and APCs such as migratory dendritic cells (DC). The DCs have the capacity to migrate out of the site of antigen presentation and reach localized T and B cells to initiate antigen-specific immune responses. Simple tissue constructs based on endothelial cells and a 3D matrix have shown the in vitro potential for autonomous generation of monocyte-derived DCs and macrophages. In a model based on one monolayer of endothelial cells grown to confluency over a 3D collagen matrix, monocytes from total PBMCs selectively extravasate in and differentiate into either resident macrophages or migratory DCs with potent antigen-presenting capacity.

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VaxDesign has found that the vaccination site module provides a good representation of what occurs in vivo due to the following characteristics:

• Autonomous DC generation from monocytes without exogenous factors being added;
• Similar DC development kinetics;
• A wide array of DC differentiation and maturation states;
• A natural selection of hematopoietic DC precursors from whole peripheral blood mononuclear cells (PBMCs) due to the presence of an endothelium;
• Presence of a 3D extracellular matrix environment;

VaxDesign has focused its initial efforts on a skin equivalent version of the VS to test the immunogenicity of injectable immunopotentiators and adjuvants.

Vaccination Site Structure

We have shown that monocytes can selectively extravasate through the endothelium into the collagen matrix, and spontaneously differentiate into antigen presenting cells, such as macrophages and dendritic cells without the addition of any exogenous factors. We can create a quiescent endothelium on top of a collagen matrix in a 96 well format.

The endothelium can be activated with various inflammatory signals such as interluekin-1β or TNFα. This leads to neutrophil migration into the endothelium. We show that the confluent endothelium on the collagen-membrane is quiescent via a neutrophil migration assay; thus it does not have a high inflammatory background. When endothelial cells are grown in the presence of an inflammatory mediator, they upregulate adhesion molecules like E-selectin, VCAM-1, and ICAM-1. Chemokines such as IL-8 are also produced, an attractant for the body’s main inflammatory cell, the neutrophil. However, when endothelial cells are cultured in a non-inflammatory environment or with constructs that have a low endotoxin level such as LPS, they do not express these molecules that promote neutrophil transendothelial trafficking.

In vivo, neutrophils are only recruited into inflamed tissues. If neutrophil migration across the endothelium is evident in vitro, the endothelium must have upregulated neutrophil migration-promoting molecules in response to any one of a number of possible inflammatory mediators in the environment. Representative pictures of the activated (TNFα) and non-activated vaccination site structures are shown below. The collagen-membrane VS structures are not activated in their native form, precisely what is desired.

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