Neuartige Salmonellen mit in vivo amplifizierbaren Plasmiden für die Vakzinierung und Tumortherapie

Live attenuated Salmonella are highly potential vectors for the delivery of proteins and DNA with applications in vaccination and tumor therapy. Conventional expression systems are based on maintenance of the plasmid at multi-copy numbers representing a high metabolic burden for the bacteria that results in a decreased performance of the vector. In this work, a system for in vivo inducible amplification of expression plasmids was established in Salmonella enterica serovar Typhimurium strain SL7207. Amplifiable plasmids contain two replication origins. The first replicon ensures the minimal-copy number state under non-inducing conditions. A second medium-copy replicon was placed under control of Salmonella-specific in vivo inducible promoters PsifA, Pstm1630 and PphoN or the PBAD promoter. PsifA exhibited strong amplification of the plasmid copy number from 1-5 to 50-100 copies and enhanced expression of plasmid-encoded antigen in induction medium, upon infection of macrophages and mice. However, in vivo imaging demonstrated that PsifA is activated already early in the mouse intestine resulting in impaired invasion efficiency of orally administered plasmid amplifying strains. Nevertheless, after intravenous immunization, plasmid amplification resulted in substantial ovalbumin-specific CD4+ T cell responses. In the second part of this work, plasmid amplification could be activated by the L-arabinose inducible PBAD promoter. This remote control was possible in culture, upon infection of macrophages and in tumor, spleen and liver of tumor-bearing mice. Administration of the sugar resulted in high reporter activity especially when plasmid amplification and reporter expression were simultaneously driven by PBAD. In addition, repeated induction of reporter expression could be demonstrated in vivo. In summary, inducible plasmid amplification should contribute to the optimization of Salmonella-mediated delivery of prophylactic and therapeutic molecules.Lebende attenuierte Salmonellen sind vielversprechende Überträger von Proteinen und DNA für die Vakzinierung und Tumortherapie. Konventionelle Expressionssysteme basieren auf Multicopy Plasmiden, die eine starke metabolische Belastung für die Transformanten darstellen und deren Effizienz verringern. In dieser Arbeit wurde ein in vivo induzierbares Plasmidamplifikationssystem in Salmonella enterica serovar Typhimurium SL7207 etabliert. Die amplifizierbaren Plasmide enthalten zwei Replikationsursprünge. Das erste Replikon stellt eine minimale Kopiezahl der Plasmide unter uninduzierten Bedingungen sicher. Ein zweites Mediumcopy Replikon wurde durch die Salmonellen-spezifischen in vivo induzierbaren Promotoren PsifA, Pstm1630 und PphoN oder den PBAD Promotor kontrolliert. Durch den PsifA Promotor konnte eine Amplifikation der Plasmidkopiezahl von 1-5 auf 50-100 und eine starke Expression von plasmidkodierten Antigen im Induktionsmedium und nach einer Infektion von Makrophagen und Mäusen induziert werden. Der PsifA Promotor wird allerdings schon im Darm von Mäusen aktiviert, was die Invasionseffizienz von oral verabreichten Stämmen herabsetzt. Nach intravenöser Gabe konnten substantielle Ovalbumin-spezifische CD4+ T Zell Antworten gemessen werden. Im zweiten Teil dieser Arbeit wurde die Plasmidamplifikation durch den L-Arabinose induzierbaren PBAD Promotor aktiviert. Die Fernsteuerung des Systems wurde in Kultur, in infizierten Makrophagen sowie in Tumor, Milz und Leber von tumortragenden Mäusen demonstriert. Dabei führte die Gabe des Zuckers zu einer starken Reporteraktivität, insbesondere bei simultaner Kontrolle von Plasmidampifikation und Reporterexpression durch PBAD. Außerdem war eine wiederholte Induktion der Reporterexpression in vivo möglich. Zusammenfassend sollte das in vivo induzierbare Plasmidamplifikationssystem zu einer Optimierung der Salmonellen-vermittelten Übertragung von prophylaktischen und therapeutischen Molekülen führen

Local and systemic effect of transfection-reagent formulated DNA vectors on equine melanoma

Background Equine melanoma has a high incidence in grey horses. Xenogenic DNA vaccination may represent a promising therapeutic approach against equine melanoma as it successfully induced an immunological response in other species suffering from melanoma and in healthy horses. In a clinical study, twenty- seven, grey, melanoma-bearing, horses were assigned to three groups (n = 9) and vaccinated on days 1, 22, and 78 with DNA vectors encoding for equine (eq) IL-12 and IL-18 alone or in combination with either human glycoprotein (hgp) 100 or human tyrosinase (htyr). Horses were vaccinated intramuscularly, and one selected melanoma was locally treated by intradermal peritumoral injection. Prior to each injection and on day 120, the sizes of up to nine melanoma lesions per horse were measured by caliper and ultrasound. Specific serum antibodies against hgp100 and htyr were measured using cell based flow- cytometric assays. An Analysis of Variance (ANOVA) for repeated measurements was performed to identify statistically significant influences on the relative tumor volume. For post-hoc testing a Tukey-Kramer Multiple-Comparison Test was performed to compare the relative volumes on the different examination days. An ANOVA for repeated measurements was performed to analyse changes in body temperature over time. A one-way ANOVA was used to evaluate differences in body temperature between the groups. A p–value < 0.05 was considered significant for all statistical tests applied. Results In all groups, the relative tumor volume decreased significantly to 79.1 ± 26.91% by day 120 (p < 0.0001, Tukey-Kramer Multiple-Comparison Test). Affiliation to treatment group, local treatment and examination modality had no significant influence on the results (ANOVA for repeated measurements). Neither a cellular nor a humoral immune response directed against htyr or hgp100 was detected. Horses had an increased body temperature on the day after vaccination. Conclusions This is the first clinical report on a systemic effect against equine melanoma following treatment with DNA vectors encoding eqIL12 and eqIL18 and formulated with a transfection reagent. Addition of DNA vectors encoding hgp100 respectively htyr did not potentiate this effect

Differential effect of auxotrophies on the release of macromolecules by Salmonella enterica vaccine strains.

Attenuated Salmonella enterica strains have been widely used as live carriers for vaccines and therapeutic molecules. Appropriate attenuation has been introduced into such bacteria for safety reasons and the improvement of strain properties. Here, we compared two strains that were rendered auxotroph for diaminopimelic acid or thymidine monophosphate precursors by deletion of the genes asd or thyA, respectively. Upon removal of the complementing compound from bacterial cultures, both strains quickly lose their property to form colonies. However, while the Deltaasd bacteria lysed almost immediately under such conditions, DeltathyA bacteria remained physically intact during the observation period. As a consequence, the Deltaasd bacteria released their intracellular content such as proteins or plasmids into the supernatant. In contrast, no intracellular component, either proteins or plasmids, could be recovered from the supernatants of DeltathyA bacteria upon depletion of thymidine. Thus, the release of macromolecules from the bacterial carrier occurs as a consequence of appropriate lethal attenuation. This might substitute for sophisticated secretion systems

Remote control of tumour-targeted Salmonella enterica serovar Typhimurium by the use of L-arabinose as inducer of bacterial gene expression in vivo.

We have used Salmonella enterica serovar Typhimurium (S. typhimurium) which are able to colonize tumours besides spleen and liver. Bacteria were equipped with constructs encoding green fluorescent protein or luciferase as reporters under control of the promoter PBAD that is inducible with L-arabinose. Reporter genes could be induced in culture but also when the bacteria resided within the mouse macrophages J774A.1. More important, strong expression of reporters by the bacteria could be detected in mice after administration of L-arabinose. This was especially pronounced in bacteria colonizing tumours. Histology demonstrated that the bacteria had accumulated in and close to necrotic areas of tumours. Bacterial gene induction was observed in both regions. PBAD is tightly controlled also in vivo because gene E of bacteriophage PhiX174 could be introduced as inducible suicide gene. The possibility to deliberately induce genes in bacterial carriers within the host should render them extremely powerful tools for tumour therapy

Drug-inducible remote control of gene expression by probiotic Escherichia coli Nissle 1917 in intestine, tumor and gall bladder of mice.

The probiotic bacterium Escherichia coli Nissle 1917 (EcN) constitutes a prospective vector for delivering heterologous therapeutic molecules to treat several human disorders. To add versatility to this carrier system, bacteria should be equipped with expression modules that can be regulated deliberately in a temporal and quantitative manner. This approach is called in vivo remote control (IVRC) of bacterial vectors. Here, we have evaluated promoters P(araBAD), P(rhaBAD) and P(tet), which can be induced with L-arabinose, L-rhamnose or anhydrotetracycline, respectively. EcN harboring promoter constructs with luciferase as reporter gene were administered either orally to healthy mice or intravenously to tumor bearing animals. Subsequent to bacterial colonization of tissues, inducer substances were administered via the oral or systemic route. By use of in vivo bioluminescence imaging, the time course of reporter gene expression was analyzed. Each promoter displayed a specific in vivo induction profile depending on the niche of bacterial residence and the route of inducer administration. Importantly, we also observed colonization of gall bladders of mice when EcN was administered systemically at high doses. Bacteria in this anatomical compartment remained accessible to remote control of bacterial gene expression

Combination of MIDGE-Th1 DNA vaccines with the cationic lipid SAINT-18: Studies on formulation, biodistribution and vector clearance

We have previously shown that the combination of MIDGE-Th1 DNA vectors with the cationic lipid SAINT-18 increases the immune response to the encoded antigen in mice. Here, we report on experiments to further optimize and characterize this approach. We evaluated different formulations of MIDGE-Th1 vectors with SAINT-18 by assessing their influence on the transfection efficiency in cell culture and on the immune response in mice. We found that high amounts of SAINT-18 in formulations with a w/w ratio MIDGE Th1/SAINT-18 of 1:4.8 are beneficial for cell transfection in vitro. In contrast, the formulation of HBsAg-encoding MIDGE-Th1 DNA vectors with the lowest amount of SAINT-18 (w/w ratio MIDGE Thl/SAINT-18 of 1:0.5) resulted in the highest serum IgG1 and IgG2a levels after intradermal immunization of mice. Consequently, latter formulation was selected for a comparative biodistribution study in rats. Following intradermal administration of both naked and formulated MIDGE-Th1 DNA, the vectors localized primarily at the site of injection. Vector DNA levels decreased substantially over the two months duration of the study. When administered in combination with SAINT-18, the vectors were found in significantly higher amounts in draining lymph nodes in comparison to administration of naked MIDGE-Th1 DNA. We propose that the high immune responses induced by MIDGE-Th1/SAINT-18 lipoplexes are mediated by enhanced transfection of cells in vivo, resulting in stronger antigen expression and presentation. Importantly, the combination of MIDGE-Thl vectors with SAINT-18 was well tolerated in mice and rats and is expected to be safe in human clinical applications. (C) 2014 Elsevier Ltd. All rights reserved

Cationic lipid-formulated DNA vaccine against hepatitis B virus: immunogenicity of MIDGE-Th1 vectors encoding small and large surface antigen in comparison to a licensed protein vaccine.

Currently marketed vaccines against hepatitis B virus (HBV) based on the small (S) hepatitis B surface antigen (HBsAg) fail to induce a protective immune response in about 10% of vaccinees. DNA vaccination and the inclusion of PreS1 and PreS2 domains of HBsAg have been reported to represent feasible strategies to improve the efficacy of HBV vaccines. Here, we evaluated the immunogenicity of SAINT-18-formulated MIDGE-Th1 vectors encoding the S or the large (L) protein of HBsAg in mice and pigs. In both animal models, vectors encoding the secretion-competent S protein induced stronger humoral responses than vectors encoding the L protein, which was shown to be retained mainly intracellularly despite the presence of a heterologous secretion signal. In pigs, SAINT-18-formulated MIDGE-Th1 vectors encoding the S protein elicited an immune response of the same magnitude as the licensed protein vaccine Engerix-B, with S protein-specific antibody levels significantly higher than those considered protective in humans, and lasting for at least six months after the third immunization. Thus, our results provide not only the proof of concept for the SAINT-18-formulated MIDGE-Th1 vector approach but also confirm that with a cationic-lipid formulation, a DNA vaccine at a relatively low dose can elicit an immune response similar to a human dose of an aluminum hydroxide-adjuvanted protein vaccine in large animals

Duration of HBV antigen-specific humoral immune responses in individual animals.

<p>Pigs were immunized with MIDGE-Th1 DNA vaccine candidates and Engerix-B (see legend <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101715#pone-0101715-g004" target="_blank">Figure 4</a>) and serum samples were collected and analyzed by ELISA for total S protein-specific antibodies (A), S protein-specific IgG1 (C) and IgG2 (C) on various test days up to 6 months after the third immunization. The animal number is shown in the legend of the graph.</p

Kinetics of HBV antigen-specific humoral immune responses in pigs.

<p>Pigs were immunized with SAINT-18 (Ctrl.), a low, mid or high dose of SAINT-18-formulated MIDGE-Th1 vectors encoding the S protein (low S, mid S, high S), a high dose of SAINT-18-formulated MIDGE-Th1 vectors encoding the L protein (high L) or a human dose of Engerix-B on days 1, 29 and 57 (↓). Serum samples were collected on various test days and analyzed by ELISA for total S protein-specific antibodies (A), PreS1-specific IgG (B), S protein-specific IgG1 (C) and IgG2 (D). Data points represent mean values for all animals in each group (n = 3–5). For graphical purposes, samples without a PreS1-titer were set to 10 in (B). Statistical analysis is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101715#pone.0101715.s001" target="_blank">Tables S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101715#pone.0101715.s004" target="_blank">S4</a>.</p

Expression of HBV antigens in CHO-K1 cells.

<p>Cells were transfected with expression vectors encoding the S or the L protein or eGFP as control (Ctrl.). Expression in cell lysates was detected by Western Blot using polyclonal S protein-specific antibody (A) or PreS1-specific mouse serum (B). rec. HBsAg: recombinant S protein of HBsAg. (C) Cells were transfected with the indicated amounts of single expression vectors or mixtures thereof. S protein expression was analyzed by ELISA in supernatants of cells 72 h after transfection.</p