In vivo bioluminescence imaging in preclinical trials of genetic vaccines

Abstract

DNA immunization is a rapidly developing vaccine platform for infectious diseases, cancer and allergies. The efficiency of DNA vaccination is largely determined by the efficiency of delivery and subsequent expression of genes encoding microbial and tumor antigens or allergens in the cells of vaccine recipients. DNA immunogens are generally administered by intramuscular or intradermal injections, followed by electroporation to enhance the DNA uptake into the cells. An intense debate on the pros and cons of different routes of DNA delivery is still ongoing.

The aim of this work was to develop in vivo imaging applications for improvement of DNA immunization. The first aim was to optimize delivery techniques in order to increase the efficacy of in vivo delivery of DNA vaccines and subsequent immune response. Using model DNA immunogens encoding luciferase, and HIV-derived immunogens encoding protease (PR) and reverse transcriptase (RT), we defined the differences in the strength and type of immune responses induced by them when administered by intradermal or intramuscular injection routes followed by electroporation. Furthermore, we determined the extent to which the method of DNA delivery influences the immune response to Th1 and Th2 type immunogens, represented by plasmids encoding PR and RT of HIV-1. Finally, we developed imaging applications for the in vivo assessment of the effector/lytic potential of the immune response in tumor and surrogate pathogen challenge models.

We immunized mice with DNA immunogens mixed with a gene encoding a bioluminescent reporter. Bioluminescence imaging (BLI) served as a tool to monitor the expression of delivered reporter genes in vivo. By combining the readouts form BLI and immunoassays we defined a set of delivery parameters that led to the best immunization outcome in terms of both immunogen expression and subsequent immune response. After optimizing the delivery conditions we tested different immunization routes to determine the one that ensures maximal immunogenicity of DNA immunogen. Here we show that intradermal administration resulted in a significant enhancement of both cellular and humoral immune responses as compared to intramuscular delivery. This was evident regardless of the nature of the immunogen (Th1 vs. Th2). The kinetics of the loss of co-delivered reporter gene expression was found to correlate with the antigen-specific production of IFN-γ and IL-2 and could thus be used as in vivo correlate of the strength of specific immune responses. Thus, non-invasive imaging allowed to assess the immunogenicity of DNA vaccines in vivo. Using the same parameters we developed a surrogate method that could assess effector memory responses. Finally, we applied BLI to study the growth of luciferase-labeled tumors in luciferase-immunized animals, which provided a functional measure of vaccine efficacy.

Overall, the use of BLI allowed us to establish a methodology to increase the efficacy of delivery, define optimal regimens and test the effector capacity of the immune response induced by DNA vaccination. The application of this technique made it possible to significantly refine and reduce animal experimentation in gene vaccine development.