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.

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.

Improving live attenuated bacterial carriers for vaccination and therapy.

Live attenuated bacteria are well established as vaccines. Thus, their use as carriers for prophylactic and therapeutic macromolecules is a logical consequence. Here we describe several experimental applications of bacteria to carry heterologous macromolecules into the murine host. First, Listeria monocytogenes are described that are able to transfer eukaryotic expression plasmids into host cells for gene therapy. High multiplicities of infection are still required for efficient gene transfer and we point out some of the bottlenecks that counteract a more efficient transfer and application in vivo. Then, we describe Salmonella enterica serovar Typhimurium (S. typhimurium) as an expression plasmid transfer vehicle for oral DNA vaccination of mice. We demonstrate that the stabilization of the plasmid transformants results in an improved immune response. Stabilization was achieved by replacing the origin of replication of the original high-copy-number plasmid by a low-copy-number origin. Finally, we describe Salmonella carriers for the improved expression of heterologous proteins. We introduce a system in which the plasmid is carried as a single copy during cultivation but is amplified several fold upon infection of the host. Using the same in vivo inducible promoter for both protein expression and plasmid amplification, a substantial increase in antigen expression in vivo can be achieved. A modification of this approach is the introduction of inducible gene expression in vivo with a low-molecular-weight compound. Using P(BAD) promoter and L-arabinose as inducer we were able to deliberately activate genes in the bacterial carrier. No background activity could be observed with P(BAD) such that an inducible suicide gene could be introduced. This is adding an important safety feature to such live attenuated carrier bacteria.

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.

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 Th1/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-Th1 vectors with SAINT-18 was well tolerated in mice and rats and is expected to be safe in human clinical applications.

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.

Immune response induced by a linear DNA vector: influence of dose, formulation and route of injection.

Previously, minimalistic, immunogenetically defined gene expression (MIDGE) vectors were developed as effective and sophisticated carriers for DNA vaccination. Here we evaluate the influence of dose, formulation and delivery route on the immune response after vaccination with MIDGE-Th1 vectors encoding hepatitis B virus surface antigen (HBsAg). An HBsAg-specific IgG1 and IgG2a antibody response was induced in a dose-dependent manner, whereas the IgG2a/IgG1 ratio was independent of the injected DNA dose. Formulation of MIDGE-HBsAg-Th1 with the cationic pyridinium amphiphile SAINT-18 significantly increased antibody levels of IgG1 and IgG2a compared to the unformulated vector. In contrast, SAINT-18 had neither a significant effect on the IgG2a/IgG1 ratio nor on the type and strength of cellular immunity. Overall, the strongest immune response was generated after intradermal injection, followed by intramuscular and subcutaneous (s.c.) injection. The results show that the formulation of MIDGE-Th1 with SAINT-18 increased the efficacy of the MIDGE-Th1 DNA vaccine and is therefore a suitable approach to improve the efficacy of DNA vaccines also in large animals and humans.

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.

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.