A Comparison of Red Fluorescent Proteins to Model DNA Vaccine Expression by Whole Animal In Vivo Imaging

DNA vaccines can be manufactured cheaply, easily and rapidly and have performed well in pre-clinical animal studies. However, clinical trials have so far been disappointing, failing to evoke a strong immune response, possibly due to poor antigen expression. To improve antigen expression, improved technology to monitor DNA vaccine transfection efficiency is required. In the current study, we compared plasmid encoded tdTomato, mCherry, Katushka, tdKatushka2 and luciferase as reporter proteins for whole animal in vivo imaging. The intramuscular, subcutaneous and tattooing routes were compared and electroporation was used to enhance expression. We observed that overall, fluorescent proteins were not a good tool to assess expression from DNA plasmids, with a highly heterogeneous response between animals. Of the proteins used, intramuscular delivery of DNA encoding either tdTomato or luciferase gave the clearest signal, with some Katushka and tdKatushka2 signal observed. Subcutaneous delivery was weakly visible and nothing was observed following DNA tattooing. DNA encoding haemagglutinin was used to determine whether immune responses mirrored visible expression levels. A protective immune response against H1N1 influenza was induced by all routes, even after a single dose of DNA, though qualitative differences were observed, with tattooing leading to high antibody responses and subcutaneous DNA leading to high CD8 responses. We conclude that of the reporter proteins used, expression from DNA plasmids can best be assessed using tdTomato or luciferase. But, the disconnect between visible expression level and immunogenicity suggests that in vivo whole animal imaging of fluorescent proteins has limited utility for predicting DNA vaccine efficacy

DNA vaccination against Respiratory Syncytial Virus [RSV]

Despite years of research, a vaccine for RSV is currently not available. One attractive platform for RSV vaccines is DNA vaccination, however to date DNA vaccines have been poorly immunogenic in humans. More understanding of the interplay between route of delivery, expression and immunogenicity of DNA vaccines is required. The aim of this project was to use the novel linear DNA construct developed by Touchlight Genetics Ltd to investigate the immune response to DNA vaccines. These constructs, or doggybones (dbDNA) because of their characteristic shape, are faster and easier to produce than conventional plasmid DNA vaccines, can be produced synthetically and only contain elements needed for gene expression. Initial studies investigated the relationship between expression and immunogenicity. dbDNA and circular plasmid vaccines were compared by evaluating the visible expression of a range of red fluorescent protein using in vivo imaging; immunogenicity was compared using vaccination models. Expression of dbDNA and plasmid vaccines was similar but there was a disconnect between expression and immunogenicity. dbDNA and plasmid vaccines of the RSV F, M2-1 and G genes were produced and efficacy evaluated after intramuscular, subcutaneous and tattoo delivery. The distinct immune profiles produced by RSV genes allowed for examination of immune response generated after delivery by various routes. dbDNA and plasmid vaccines had comparable immune responses, but no viable vaccine candidates were identified due to priming for vaccine enhanced disease. The route of delivery did not alter the immune phenotype of the antigen, but it did alter the magnitude. Intramuscular delivery produced strongest immune responses, followed by the tattooing route and with limited responses observed after subcutaneous delivery. The DNA vaccines generated in the project were immunogenic, generating strong T cell responses, but not protective. Better correlates of anti-RSV cellular protection are needed, therefore, the contribution of T cells to protection was investigated. Natural RSV infection induces tissue resident memory (TRM) populations in the airways which were not produced after intramuscular immunisation with DNA vaccines. Airway T cells from RSV exposed mice were able to protect naïve mice from RSV disease in a novel model of cell transfer. These studies provide evidence of protective roles of TRM cells and support the need to induce TRM with DNA vaccine strategies for protective immunogenicity.Open Acces

Adjuvanted influenza vaccines

In spite of current influenza vaccines being immunogenic, evolution of the influenza virus can reduce efficacy and so influenza remains a major threat to public health. One approach to improve influenza vaccines is to include adjuvants; substances that boost the immune response. Adjuvants are particularly beneficial for influenza vaccines administered during a pandemic when a rapid response is required or for use in patients with impaired immune responses, such as infants and the elderly. This review outlines the current use of adjuvants in human influenza vaccines, including what they are, why they are used and what is known of their mechanism of action. To date, six adjuvants have been used in licensed human vaccines: Alum, MF59, AS03, AF03, virosomes and heat labile enterotoxin (LT). In general these adjuvants are safe and well tolerated, but there have been some rare adverse events when adjuvanted vaccines are used at a population level that may discourage the inclusion of adjuvants in influenza vaccines, for example the association of LT with Bell's Palsy. Improved understanding about the mechanisms of the immune response to vaccination and infection has led to advances in adjuvant technology and we describe the experimental adjuvants that have been tested in clinical trials for influenza but have not yet progressed to licensure. Adjuvants alone are not sufficient to improve influenza vaccine efficacy because they do not address the underlying problem of mismatches between circulating virus and the vaccine. However, they may contribute to improved efficacy of next-generation influenza vaccines and will most likely play a role in the development of effective universal influenza vaccines, though what that role will be remains to be seen

Comparison of luciferase expression after DNA by various routes.

<p>50μg DNA encoding luciferase was delivered by the i.m., s.c. or tattooing routes. At various time points after transfection, mice were injected i.p. with Luciferin substrate and imaged <i>in vivo</i> using an Imaging System. Representative images from day 7 after transfection by the i.m. (a), s.c. (b) and tattoo (c) routes. Relative expression levels were quantified using image analysis software (d), points represent mean +/- SEM of n = 5 animals.</p

Comparison of red fluorescent proteins <i>in vitro</i>.

<p>CHO-k1 cells were transfected with 1μg DNA complexed with Lipofectamine. At various time points, cells were imaged by fluorescent microscopy—excitation 540–580nm and emission 600–660nm (a). Fluorescence intensity was measured by image analysis software at 550–600 nm (b) or 600–700 nm (c). Images in (a) representative of n = 3 experiments, points in b and c represent mean +/- SEM of 4 repeats, *** p<0.001 comparing tdTomato and other proteins in panel b, ## p<0.01 comparing tdKatushka2 and other proteins in panel c, by 2 way ANOVA.</p

Fluorescent proteins used in the study.

<p>Values are taken from published studies (as indicated) and <a href="http://fccf.salk.edu/fluorochrometable.php" target="_blank">http://fccf.salk.edu/fluorochrometable.php</a>.</p><p>Fluorescent proteins used in the study.</p

Comparison of red fluorescent proteins after subcutaneous DNA delivery.

<p>Mice were injected s.c. with 50μg DNA in the epigastric region and the site electroporated. At days 3, 5 and 7 after transfection, mice were imaged <i>in vivo</i> using an Imaging System at 550nm excitation and 600nm emission. Representative of n = 3 experiments. White circles indicate fluorescent protein expression.</p

Comparison of red fluorescent proteins after intramuscular DNA delivery.

<p>Mice were injected i.m. with 50μg DNA in the anterior tibialis muscle and the site electroporated. At days 3, 5 and 7 after transfection, mice were imaged <i>in vivo</i> using an Imaging System at 550nm excitation and 600nm emission. Representative of n = 3 experiments. White circles indicate fluorescent protein expression.</p

Combined HDAC and BET Inhibition Enhances Melanoma Vaccine Immunogenicity and Efficacy

The combined inhibition of histone deacetylases (HDAC) and the proteins of the bromodomain and extraterminal (BET) family have recently shown therapeutic efficacy against melanoma, pancreatic ductal adenocarcinoma, testicular, and lymphoma cancers in murine studies. However, in such studies, the role of the immune system in therapeutically controlling these cancers has not been explored. We sought to investigate the effect of the HDAC inhibitor romidepsin (RMD) and the BET inhibitor IBET151, both singly and in combination, on vaccine-elicited immune responses. C57BL/6 mice were immunized with differing vaccine systems (adenoviral, protein) in prime-boost regimens under treatment with RMD, IBET151, or RMD+IBET151. The combined administration of RMD+IBET151 during vaccination resulted in a significant increase in the frequency and number of Ag-specific CD8+ T cells. RMD+IBET151 treatment significantly increased the frequency of vaccine-elicited IFN-γ+ splenic CD8+ T cells and conferred superior therapeutic and prophylactic protection against B16-OVA melanoma. RNA sequencing analyses revealed strong transcriptional similarity between RMD+IBET151 and untreated Ag-specific CD8+ T cells except in apoptosis and IL-6 signaling-related genes that were differentially expressed. Serum IL-6 was significantly increased in vivo following RMD+IBET151 treatment, with recombinant IL-6 administration replicating the effect of RMD+IBET151 treatment on vaccine-elicited CD8+ T cell responses. IL-6 sufficiency for protection was not assessed. Combined HDAC and BET inhibition resulted in greater vaccine-elicited CD8+ T cell responses and enhanced therapeutic and prophylactic protection against B16-OVA melanoma. Increased IL-6 production and the differential expression of pro-and anti-apoptotic genes following RMD+IBET151 treatment are likely contributors to the enhanced cancer vaccine responses