Ad5mir122 shows reduced E1A activity in primary hepatocytes.

<p>Primary human hepatocytes were infected with either Ad5mir122luc or Ad5WTluc at 1 vp/cell. Luciferase levels are shown 3 days post infection (n = 3) as relative light units per mg total protein. Error bars represent standard deviation.</p

Pharmacodynamics led dose escalation study to determine the optimal treatment dose with Ad5mir122 and Ad5WT in tumour bearing mice.

<p>A) Serum Alanine Transaminase (ALT) levels from mice receiving Ad5mir122, Ad5WT or PBS. ALT values for each group are shown at days 2, 5 and 8 (n = 3) after the first injection. Data is presented as ALT units per litre using the equation in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016152#s2" target="_blank">materials and methods</a>. B) Luciferase imaging 2 days after the first injection of Ad5mir122 (left panel) or Ad5WT (right panel). C) Luciferase imaging eight days after the first injection. Mice which received a single injection of Ad5WT are shown in the right panel and mice receiving three injections of Ad5mir122 are shown in the left panel. Images are all presented on the same scale. D) Dosage and treatment schedule for Ad5mir122 and Ad5WT. Viral doses are indicated above each line and include 10% of a Luciferase reporter virus (Ad5mir122luc). All mice were treated with bisphosphonate liposomes at day -1.</p

MicroRNA mediated knockdown of both E1A mRNA and protein measured by RT-QPCR and western blot after intravenous injection of 2×10¹⁰ vp of Ad5mir122.

<p>A) RT QPCR for the 13S E1A mRNA transcript in the livers of mice 48 hrs after intravenous injection with 2×10¹⁰ vp of Ad5mir122, Ad5WT, Ad5Luc or PBS. Ad5mir122 shows significantly reduced E1A mRNA during liver infection when compared to Ad5WT (N = 3). Statistical analysis was performed using a two tailed student T-Test (*P = <0.05). B) Western blot to confirm that all E1A proteins variants are knocked down. Each lane represents protein extracted from an individual mouse. Control lanes containing liver from mice treated with either an E1A deleted Ad5Luc vector or PBS show no E1A signal. Ad5WT treatment shows 3 clearly defined bands corresponding to proteins produced from the 13S (36 kDa), 12S (26 kDa) and a smaller fainter band which may represent the 11S or the 10S E1A transcript product. Treatment with Admir122 shows significant knockdown in E1A protein levels for all splice variants. The blot was exposed for 1, 5 and 10 minutes with the 5 minute exposure presented here. Molecular weights were calculated against a dual colour molecular weight ladder (Bio-Rad).</p

The level and activity of mature mir122 <i>in vivo</i> is unaffected by Ad5mir122.

<p>Mice were injected with 2×10¹⁰ vp of either Ad5mir122, Ad5WT, an E1A deleted Ad5luc vector or PBS. Quantification of mir122 mature RNA levels was performed using a Taqman microRNA assay specific for mir122. CT values were corrected against the levels of the microRNA, let7a, as a reference gene using the method published by Pfaffl M <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016152#pone.0016152-Pfaffl1" target="_blank">[25]</a>. A) RT-QPCR for mir122 showing nine superimposed amplification curves (from three mice) in each treatment group, before correction against let7a. Samples were reverse transcribed using equal amounts of total RNA (5 ng) and RT-PCR was performed using equal amounts of cDNA. CT values shown here represent the average of the reactions from three mice plus or minus standard deviation. B) The total number of mRNA changes recorded by genome wide mRNA profiling of extracted murine hepatic RNA. Positive signals are those in which the median mRNA level changed by ≧2-fold from all mice in each group in comparison to median mRNA level in mice treated with PBS (n = 3 for all groups). The total number of genes altered is calculated using the average of the three independent replicates and therefore no error bars are shown. C) Western blot analysis of the mir122 regulated protein Aldolase A in mice treated as above. Liver protein extracts were subjected to a BCA protein assay and equal loading was confirmed by Ponceau stain (data not shown).</p

Ad5mir122 kills mir122 negative cells with Ad5WT potency.

<p>Comparison of Ad5mir122 and Ad5WT in cancer cell lines incubated at a multiplicity of infection of 100 viral particles per cell. The percentage cell survival is shown 6 days post infection (N = 5) using an MTS cell survival assay. Statistical analysis was performed using one way ANOVA (* = p<0.05, NS  =  not significant).</p

A Rapid-Response Humoral Vaccine Platform Exploiting Pre-Existing Non-Cognate Populations of Anti-Vaccine or Anti-Viral CD4+ T Helper Cells to Confirm B Cell Activation

<div><p>The need for CD4+ T cell responses to arise <i>de novo</i> following vaccination can limit the speed of B cell responses. Populations of pre-existing vaccine-induced or anti-viral CD4+ T cells recognising distinct antigens could be exploited to overcome this limitation. We hypothesise that liposomal vaccine particles encapsulating epitopes that are recognised, after processing and B cell MHCII presentation, by pre-existing CD4+ T cells will exploit this pre-existing T cell help and result in improved antibody responses to distinct target antigens displayed on the particle surface. Liposomal vaccine particles were engineered to display the malaria circumsporozoite (CSP) antigen on their surface, with helper CD4+ epitopes from distinct vaccine or viral antigens contained within the particle core, ensuring the B cell response is raised but focused against CSP. <i>In vivo</i> vaccination studies were then conducted in C57Bl/6 mice as models of either vaccine-induced pre-existing CD4+ T cell immunity (using ovalbumin—OVA) or virus-induced pre-existing CD4+ T cell immunity (murine cytomegalovirus—MCMV). Following the establishment of pre-existing by vaccination (OVA in the adjuvant TiterMax^® Gold) or infection with MCMV, mice were administered CSP-coated liposomal vaccines containing the relevant OVA or MCMV core CD4+ T cell epitopes. In mice with pre-existing anti-OVA CD4+ T cell immunity, these vaccine particles elicited rapid, high-titre, isotype-switched CSP-specific antibody responses—consistent with the involvement of anti-OVA T helper cells in confirming activation of anti-CSP B cells. Responses were further improved by entrapping TLR9 agonists, combining humoral vaccination signals ‘one’, ‘two’ and ‘three’ within one particle. Herpes viruses can establish chronic infection and elicit significant, persistent cellular immune responses. We then demonstrate that this principle can be extended to re-purpose pre-existing anti-MCMV immunity to enhance anti-CSP vaccine responses—the first description of a strategy to specifically exploit anti-cytomegalovirus immunity to augment vaccination against a target antigen.</p></div

Non-cognate anti-viral CD4+ T cell populations in mice chronically infected with murine cytomegalovirus can enhance antibody production against target antigens.

<p>Mice (n = 4) were infected with MCMV. IFNγ and IL-4 responses to the MCMV m09 peptide were measured by ELISPOT assay in splenocytes from mice infected with MCMV for 8 weeks, or non-MCMV-infected matched controls and compared using unpaired, two-tailed t tests (a). 8 weeks post-infection, mice—including uninfected matched controls—were vaccinated with CSP-coated liposomes containing the m09 peptide. Each vaccine dose contained 0.5 μg of CSP and 0.1 μg of m09 peptide. The total anti-CSP response and CSP-specific IgG1 (c), IgG2b (d), and IgG2c (e) were quantified. Responses were compared using two-way ANOVA with Bonferroni’s post-test. The IgG1/IgG2c profile of the anti-MCMV response was also assessed by ELISA (n = 2) and is presented alongside anti-CSP IgG1 and anti-CSP IgG2c levels from vaccinated infected and uninfected mice (n = 4) with levels compared using unpaired, two-tailed t tests (f).</p

Vaccine mechanism of action.

<p>Pre-existing non-cognate CD4+ T cell help is exploited to enhance antibody responses to target antigens. B-cells with BCRs specific for the antigen on the particle surface are engaged by vaccine particles. Following internalisation, endosomal proteases generate peptide fragments from both the surface antigen and the helper antigen entrapped within the vaccine particle. These may be presented on MHC class II to cognate T cells (i.e. those that recognise the same antigen as the BCR—which may be rare for weak antigens) and non-cognate T cells (i.e. those that recognise the strong helper MHCII epitopes). Pre-existing CD4+ T cells that recognise the helper antigen (generated by pre-vaccination or infection) can provide costimulatory signals to the B-cell, resulting in confirmation of antibody production. These signals drive B-cell proliferation, differentiation, antibody production, somatic hypermutation, and isotype switching. Because the specificity of this antibody response was determined at the point of BCR-mediated antigen recognition, the antibody produced will be directed against the target antigen on the vaccine particle surface, and not against the core helper components.</p

Pre-existing CD4+ T cell immunity to epitopes contained within the core of liposomal vaccine particles enhances the antibody response to non-cognate target antigens on the particle surface.

<p>Mice (n = 4) were pre-vaccinated with either PBS or OVA_323-339 in TMG at week 0 and week 2, prior to vaccination with CSP(OVA_323-339) liposomes at week 4. The effect of this pre-existing immunity to OVA_323-339 on anti-CSP antibody responses was measured over weeks 4 to 8 and compared using two-way ANOVA with Bonferroni’s post-test. Asterisks indicate the statistical significance of the difference between OVA_323-339 + TMG (black circles, solid black line) and PBS + TMG (grey squares, solid grey line) pre-vaccinated groups. This difference was found reproducibly in multiple experiments and a typical result is shown (b). Anti-OVA_323-339 responses were also measured in both PBS and OVA_323-339 pre-vaccinated mice administered CSP(OVA_323-339) liposomes (c). The effect of liposomal particle dose was examined by vaccinating mice (n = 4) with five-fold dilutions of CSP(OVA_323-339) vaccine (the highest concentration containing 50 μg of CSP and 10 μg of OVA_323-339). Anti-CSP responses were compared using a one-way ANOVA with Tukey’s post-test for multiple comparisons (d). Pre-vaccination with PBS or OVA_323-339 in TMG was then performed either intramuscularly or subcutaneously. Subsequently, CSP(OVA_323-339) liposomal vaccines were then administered either subcutaneously (e) or intramuscularly (f) and mean anti-CSP responses were compared over time using two-way ANOVA with Bonferroni’s post test. Asterisks indicate the difference between groups shown in blue and grey groups at the indicated time points.</p

Assessment of hepatotoxicity of wild-type Ad5 modified with microRNA binding sites.

<p>(A) Measurement of serum ALT (black bars) and AST (grey bars) 72 h following intravenous administration of 5×10¹⁰ viral particles of wild-type Ad5 and Ad5-mir122. Analysis was performed as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000440#s4" target="_blank">Methods</a> section. (B) Adenovirus genomes in murine liver were measured by real time PCR 72 h following intravenous administration of 5×10¹⁰ viral particles of wild-type Ad5 WT and Ad5-mir122, as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000440#s4" target="_blank">Methods</a> section. (C) Assessment of liver histology. The left liver lobe from each mouse was immersed in 10% buffered formalin overnight at room temperature, embedded in wax and sectioned using a vibratome. Sections were stained with haematoxylin and eosin and analysed by light microscopy at ×40 magnification. Mice were treated with PBS, non-replicating E1, E3-deleted Ad5 expressing GFP (Ad5-GFP), wild-type Ad5, or wild-type Ad5 modified to contain 4 mir-122 binding sites, as indicated. (*** P<0.0005).</p