Design and Immunological Validation of Macaca fascicularis Papillomavirus Type 3 Based Vaccine Candidates in Outbred Mice: Basis for Future Testing of a Therapeutic Papillomavirus Vaccine in NHPs

Persistent human papillomavirus (HPV) infections are causative for cervical neoplasia and carcinomas. Despite the availability of prophylactic vaccines, morbidity and mortality induced by HPV are still too high. Thus, an efficient therapy, such as a therapeutic vaccine, is urgently required. Herein, we describe the development and validation of Macaca fascicularis papillomavirus type 3 (MfPV3) antigens delivered via nucleic-acid and adenoviral vectors in outbred mouse models. Ten artificially fused polypeptides comprising early viral regulatory proteins were designed and optionally linked to the T cell adjuvant MHC-II-associated invariant chain. Transfected HEK293 cells and A549 cells transduced with recombinant adenoviruses expressing the same panel of artificial antigens proved proper and comparable expression, respectively. Immunization of outbred CD1 and OF1 mice led to CD8+ and CD4+ T cell responses against MfPV3 antigens after DNA- and adenoviral vector delivery. Moreover, in vivo cytotoxicity of vaccine-induced CD8+ T cells was demonstrated in BALB/c mice by quantifying specific killing of transferred peptide-pulsed syngeneic target cells. The use of the invariant chain as T cell adjuvant enhanced the T cell responses regarding cytotoxicity and in vitro analysis suggested an accelerated turnover of the antigens as causative. Notably, the fusion-polypeptide elicited the same level of T-cell responses as administration of the antigens individually, suggesting no loss of immunogenicity by fusing multiple proteins in one vaccine construct. These data support further development of the vaccine candidates in a follow up efficacy study in persistently infected Macaca fascicularis monkeys to assess their potential to eliminate pre-malignant papillomavirus infections, eventually instructing the design of an analogous therapeutic HPV vaccine

Analyse und Charakterisierung von ausgewählten signifikanten Sense- und Antisense-Transkripten bei chronischer Pankreaitis und Bauchspeicheldrüsenkrebs

Ziel der Diplomarbeit ist die Analyse und Identifizierung signifikant differentiell exprimierter Sense- und Antisense-Transkripte zur Erforschung und Entwicklung geeigneter diagnostischer Tumormarker für Bauchspeicheldrüsenkrebs. Mit Hilfe des von der GenXPro GmbH entwickelten und patentierten SuperSAGE-Verfahrens können RNA-Transkripte, die sowohl aus gesundem als auch aus an Krebs erkranktem Pankreasgwebe isoliert wurden, quantifiziert und entsprechende Genexpressionprofile erstellt werden. Auf Grundlage dieser Daten sollen genetische Tumormarker entwickelt werden, wobei vor allem stark differentiell exprimierte RNA-Transkripte im Vordergrund stehen - insbesondere Antisense-RNA. Die quantitative Echtzeit-PCR (qRT-PCR oder qPCR) ist dabei ein geeignetes Mittel zum Test fluoreszens-markierter Sonden auf Eignung als Tumormarker sowie zu Validierung der SuperSAGE-Daten. Dabei wurden bekannte Verfahren wie TaqMan™ und SYBR® Green Asssays angewandt

No relevant alterations in the adult cerebellum as a result of RNAi activation in the early cerebellar primordium.

<p>(A-B) Low magnification DAPI stainings of postnatal day 60 cerebella. Normal lobulation in the RNAi-expressing cerebellum (<i>Ebf2</i>^<i>Cre</i>::<i>Rosa</i>^{<i>26RNAi/+</i>}, panel B), compared to a Cre- control (A). (C-D) Sagittal sections of adult cerebella stained for the Purkinje cell (PC)-specific marker calbindin (CaBP, red) and for Gfp. RNAi-expressing PCs (Gfp⁺, panel D) develop normal somata and dendritic arbors positive for CaBP. Panel E (normal control) shows sagittally sectioned CaBP⁺ PCs from an <i>Ebf2</i>^<i>Cre</i>::<i>Rosa26</i>^{<i>YFP/YFP</i>} mouse. Size bars: B, 200 µm; E, 25 µm.</p

No leakiness, high inducibility and strong expression of the transgene <i>in</i><i>vivo</i>.

<p>(A) Single cell suspensions from bone marrow of mice with the indicated genotypes were analysed for the expression of Gfp and the B cell marker B220. Gfp-positive cells are observed only in combination of the <i>vav</i>^<i>Cre</i> and the transgenic <i>Rosa26</i>^<i>RNAi</i> genotypes. All other genotypes show expression of B220, but a lack of Gfp. Note the slight shift in expression of Gfp from <i>Rosa26</i>^{<i>RNAi/+</i>} to <i>Rosa26</i>^{<i>RNAi/RNAi</i>}, due to the presence of two alleles of the transgene. Cell have been gated for FSC/SSC and as PI^-. (B) Statistical analysis of bone marrow cells stained and analysed as in A. Significant differences based on genotypes are observed only in combination of the <i>vav</i>^<i>Cre</i> and <i>Rosa26</i>^<i>RNAi</i> transgenes compared to all other genotypes; n=3-4; error bars=SD; ** p<0.01, *** p<0.001.</p

The transgene shows no leakiness and high inducibility in murine ES cells.

<p>(A) Schematic depiction of the transgene inserted into the <i>Rosa26</i> locus. Expression is driven by the strong CAG promoter, but aborted due to the presence of the transcriptional Stop sequence. Upon Cre mediated deletion, the stop cassette is removed and a polycistronic mRNA encoding for Gfp and short-hairpin sequences against <i>Ebf3</i>, <i>Ebf2</i> and <i>Ebf1</i> gets expressed. (B) ES cell clones tested by Southern blot as correctly targeted for either the RNAi-a or RNAi-b construct were transfected with the pGK-Cre-bpA plasmid encoding for the Cre recombinase or with the empty parental vector (mock). 48 h after transfection, cells were analysed by FACS for the expression of Gfp and for cell death by propidium iodide. Cells have been gated for FSC/SSC.</p

Generation of <i>EbfmiRNA</i> transgenic murine ES cells.

<p>(A) Schematic representation of the <i>Rosa26</i> wild-type locus. A solid line represents regions involved in the targeting construct, while a dashed line depicts genomic regions beyond the targeting construct. The targeting strategy consists of inserting the transgene into the XbaI site in intron 1, which is flanked by homology arms including exons1, 2 and 3. The transgene consists of the synthetic CAG-promoter, a <i>PGK::Neo</i> cassette with a transcriptional Stop sequence flanked by <i>loxP</i> sites, the RNAi insert as a black box, and a polyA sequence. In the third line the insert is detailed as consisting of the <i>Gfp</i> gene, and DNA sequences encoding for short hairpin mRNA against <i>Ebf3</i>, <i>Ebf2</i> and <i>Ebf1</i>. These sequences are surrounded by the flanking region of miR155, which is recognised by the ribonuclease Drosha. The locus after recombination is depicted in line 4, also indicating the direction of transcription from the CAG promoter and the <i>PGK::Neo-Stop</i> cassette. Endogenous and newly introduced restriction sites are indicated and the resulting fragment lengths for wild-type and targeted allele with specific restriction enzymes are given. Southern blot probes corresponding to the <i>5</i>’ region and to the <i>Gfp</i> gene are indicated. (B) Southern blot of genomic DNA from one wild-type and four mutant ES cell clones digested with <i>Eco</i>RI and <i>Sca</i>I as indicated, and hybridised to the <i>5</i>´ probe (as shown in A). Bands corresponding to the wild-type (wt) and the targeted (int) allele are indicated. (C) Southern blot of genomic DNA as in B, hybridised to the internal <i>Gfp</i> probe. Bands of the correct size appear in all four targeted ES cell clones as indicated.</p

No alterations in early B cell fractions mediated by expression of the transgene.

<p>(A) Single cell suspensions from bone marrow of mice with the indicated genotypes were analysed for the percentage of early B cell fractions. Cells were stained with propidium iodide, B220, CD43, BP-1, HSA and CD24. Propidium iodide negative cells are gated as B220/CD43 double positive (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080312#pone.0080312.s003" target="_blank">Figure S3</a>), and analysed for expression of HSA/CD24 allowing to distinguish fractions A (B220⁺CD43⁺CD24^-BP-1^-), B (B220⁺CD43⁺CD24⁺BP-1^-) and C (B220⁺CD43⁺CD24⁺BP-1⁺) of B cell development. Representative examples of the indicated genotypes are shown. Cells were further gated for FSC/SSC. (B) Statistical analysis of bone marrow cells stained and analysed as in A. No significant differences based on genotypes are observed; n=3-4; error bars=SD.</p

Expression of the transgene does not interfere with Ebf1 levels <i>in</i><i>vivo</i>.

<p>(A) Single cell suspensions from bone marrow of mice with the same genotypes as before were prepared and stained for B220/CD43 and CD24/BP1. Cells were gated in flow cytometry for FSC/SSC and as B220⁺CD43⁺ as indicated in the left panel in a representative example. The right panel shows the gates used for sorting of cells from fraction A (B220⁺CD43⁺CD24^-BP-1^-), B (B220⁺CD43⁺CD24⁺BP-1^-) and C (B220⁺CD43⁺CD24⁺BP-1⁺). These B cell fractions were sorted from all the genotypes used throughout this study. (B) Sorted cells from different B cell fractions of the indicated genotypes were subjected to qPCR analysis of Ebf1. All values were normalised to <i>HPRT</i> and the expression of Ebf1 in wild-type cells was set to 1 in each case; n=3-4; error bars=SD. (C) B220⁺CD43⁺ cells were sorted from total bone marrow of mice with the indicated genotypes and used for analysis of expression of Ebf1 by Western blot (Fractions A-C). Bone marrow from wild-type mice was depleted of B220⁺ cells (B220^- BMMNC) and used as negative control, β-actin is used as loading control. (D) Measurement of the amount of protein present in the B cell fractions and control as depicted in C using ImageJ; n=3-4; error bars=SD, ** p<0.01.</p