In vivo bioluminescence imaging in preclinical trials of genetic vaccines

DNA immunization is a rapidly developing vaccine platform for cancer, infectious disease, and allergies. The efficiency of DNA vaccination is largely determined by the efficiency of delivery and subsequent expression of the HIV-1 genes in the cells. 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. A number of studies have compared the effect of the delivery methods on the amount and quality of DNA-directed immunogen expression, as well as on the magnitude and specificity of the immune response they generate. Several studies were based on post mortem studies of the tissues, or on indirect expression monitored by techniques such as in vivo imaging of reporter genes co-delivered or fused to immunogens. The aim of this work was to develop in vivo imaging applications for DNA immunization. The first aim was to optimize delivery techniques in order to increase the efficacy and immunogenicity of DNA vaccines. Furthermore we set out to use the differences in the strength and type of immune response induced by DNA immunogens administered by intradermal (ID) or intramuscular (IM) injection routes, each followed by electroporation. In particular, the task was to determine the extent to which the method of DNA delivery influences the immune response to Th1 and Th2 type immunogens, represented by the viral protease (PR) and reverse transcriptase (RT) of HIV-1, respectively. Our final objective was to use the acquired results in an attempt to model immune responses induced by DNA immunogens in silico. BALB/c mice were immunized with DNA immunogens mixed with a gene encoding a bioluminescent reporter. We used bioluminescence imaging (BLI) as a tool to monitor the expression of delivered reporter genes in vivo. By combining the readouts form BLI and immunoassays we were able to produce a set of delivery parameters that result in the best immunization outcome in terms of expression and immunogenicity. Upon the optimization of delivery conditions we exploited different immunization routes to determine the one that is best suited and providing maximal immunogenicity for DNA vaccines. Here we show that ID administration of DNA immunogens results in a significant enhancement of both cellular and humoral immune responses in mice as compared to IM. The increase in the magnitude of immune responses 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 to characterize the strength of specific immune responses against the delivered immunogen. Thus, we were able to assess the immunogenicity of a DNA vaccine by non-invasive imaging of bioluminescence from the co-delivered reporters. The use of bioluminescent reporters is a new strategy to assess the delivery of DNA immunogens and their expression from the start to the completion of the immunization experiment. The level of reporter expression in the presence of the DNA immunogens reflects the in vivo immunogenicity of the construct, presenting anon-invasive method (technique) to assess the dynamics of the immune responses in individual DNA immunogen recipients useful for determination of the study end-points. The application of this technique allows us to significantly refine and reduce animal experimentation in gene vaccine development. LIST OF SCIENTIFIC PAPERS I. Petkov S, Heuts F, Krotova O, Kilpelainen A, Engstrom G, Starodubova E, Isaguliants M. Evaluation of immunogen delivery by DNA immunization using non-invasive bioluminescence imaging. Hum. Vaccin. Immunother 2013, Oct; 9(10): 2228-2236. II. Krotova O, Starodubova E, Petkov S, Kostic L, Agapkina J, Hallengard D, Viklund A, Latyshev O, Gelius E, Dillenbeck T, Karpov V, Gottikh M, Belyakov I, Lukashov V, Isaguliants M. Consensus HIV-1 FSU-A integrase gene variants electroporated into mice induce polyfunctional antigen-specific CD4+ and CD8+ T cells. PLoS ONE 201, 8(5): e62720. III. Petkov S, Latanova A, Starodubova E, Kilpelainen A, Isaguliants M. Expression localization determines the level of expression and the strength but not the type of immune responses to DNA immunogens in mice. Manuscript. CONTENTS