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Aim of this project is the development of a CTL assay to evaluate and measure the extent of cell-mediated immune responses induced by the vaccine


Academic year: 2021

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Vaccination is based on the stimulation of natural defense mechanisms of the host immune system to generate resistance against various pathogenic agents. Most of the currently used vaccines are able to elicit mainly humoral immune responses. However, in case of intracellular chronic infections, such as retrovirus infections, the cell-mediated response is the most important in controlling viral replication and conferring a strong protective immunity. Live attenuated vaccines have been shown to be the most effective in inducing cytotoxic T cell (CTL) activation, but a valid and safer alternative represented by DNA vaccines has recently emerged. This approach is particularly effective when DNA is used to prime the immune system and is followed by a boost with a recombinant vector, through a prime-boost protocol.

In our laboratory we are developing a prime-boost vaccination protocol against the feline immunodeficiency virus (FIV), which represents a well kwnown model system for developing anti-AIDS vaccines. The prime consists in administration of a plasmid DNA carrying FIV env in association with the feline cytokine granulocyte macrophage colony stimulating factor (fGM-CSF), a strong natural adjuvant.

The boost, whose purpose is to potentiate the cell-mediated immune response, consists in the administration of autologous dendritic cells transduced in vitro with a FIV-derived vector vehiculating env and feline IL15.

The latter cytokine is used to stimulate the differentiation and proliferation of immune response effector cells.

Aim of this project is the development of a CTL assay to evaluate and measure the extent of cell-mediated immune responses induced by the vaccine.




This assay is based on the use of feline autologous fibroblasts, previously trasformed to generate a stable cell line which can be maintened in culture for long periods of time and is viable after repeated freeze-thaw cycles.

These cell lines have been stably transduced with a bicistronic FIV-vector expressing the antigen of interest and a reporter gene, the green fluorescent protein (GFP).

Transformed and transduced fibroblasts generated from each cat are used as target cells in a CTL assay throught incubation with autologous peripheral blood mononuclear cells (PBMC) collected and purified from the vaccinated animals.

If vaccination has induced and sensitised Env-specific CTL, they can recognize the antigen expressed by the fibroblasts and can lyse the antigen- expressing cells. Such a lysis can be measured through flowcytometry by registering the decrease in the number of GFP-expressing fibroblasts. The percentage of lysis allows us to evaluate the extent of cell-mediated immune response developed by the vaccinated animal.

Compared to conventional techniques, this assay shows several advantages such as ease of application, low cost and low risk as there is no need of toxic or radioactive reagents. If opportunely modified, this method can be easily applied to other vaccination protocols and can be transferred to humans or other experimental models.



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