Inhibition of phosphatidylserine recognition heightens the immunogenicity of irradiated lymphoma cells in vivo.

Strategies to enhance the immunogenicity of tumors are urgently needed. Although vaccination with irradiated dying lymphoma cells recruits a tumor-specific immune response, its efficiency as immunogen is poor. Annexin V (AxV) binds with high affinity to phosphatidylserine on the surface of apoptotic and necrotic cells and thereby impairs their uptake by macrophages. Here, we report that AxV preferentially targets irradiated lymphoma cells to CD8+ dendritic cells for in vivo clearance, elicits the release of proinflammatory cytokines and dramatically enhances the protection elicited against the tumor. The response was endowed with both memory, because protected animals rejected living lymphoma cells after 72 d, and specificity, because vaccinated animals failed to reject unrelated neoplasms. Finally, AxV-coupled irradiated cells induced the regression of growing tumors. These data indicate that endogenous adjuvants that bind to dying tumor cells can be exploited to target tumors for immune rejection.

Blending of a conventional Mycoplasma hyopneumoniae vaccine with a positive marker: tracking of immunised pigs by peptide-specific antibodies raised to the marker component.

Highly immunodominant marker antigens simply blended to existing veterinary vaccines may represent a smart approach for addressing the still open issue of vaccination compliance. This approach was evaluated by blending a widely deployed Mycoplasma hyopneumoniae vaccine with a peptide-KLH (Keyhole Limpet Hemocyanin) conjugate as marker. Piglets were vaccinated twice with: (i) a combination of the M. hyopneumoniae-specific vaccine and the marker, (ii) M. hyopneumoniae-specific vaccine, (iii) marker alone or (iv) placebo dose only. All piglets which received the M. hyopneumoniae-specific vaccine/marker formulation or, as control, the marker blended with Montanide IMS1313 adjuvant responded to the respective immunisation from day 21 to 77 post vaccination as seropositive for the appropriate peptide and KLH. However, the responder rate to M. hyopneumoniae of piglets administered with M. hyopneumoniae-specific vaccine/marker was slightly reduced at day 35 and 49 post immunisation in comparison with piglets vaccinated with M. hyopneumoniae-specific vaccine alone. Accordingly, we conclude that this marker technology could be successfully applied to label a whole set of vaccines prevented that the blending process will be optimised.

Rapid screening method for antisense oligonucleotides against human growth factor receptor p185(erbB-2).

The aim of this study was the development of an indirect cell proliferation assay as screening tool for antisense oligonucleotides. Unmodified and phosphorothioate-modified oligonucleotides with different amounts of sulfur in the DNA backbone were examined for biologic activity. The human growth factor receptor p185(erbB-2) was chosen as cellular target. High-level expression of this protein can be related to an early event in tumor development and cell proliferation. We correlated the expression of p185(erbB-2) with the cell proliferation of BT-474. Additionally a control cell line (MCF-7) with very low p185(erbB-2) expression was cultivated. Antisense oligonucleotides were transfected as a liposome formulation (Lipofectin), GIBCO-BRL, Eggenstein, Germany). Cell count was correlated with a total protein quantification assay (BCA method). Stability against nuclease digestion was determined with a DNase I assay. Sequence-specific antisense effects on the p185(erbB-2) protein level were determined by Western blot. An antisense phosphorothioate oligonucleotide was identified to inhibit the cell proliferation in comparison to a random control and a negative control oligonucleotide sequence. The comparison of fully thioated, partly thioated, and unmodified oligonucleotides verified the correlation between the enzymatic stability and the biologic activity of the different modifications. Using the unstable oligonucleotides, more treatments were necessary to achieve an antiproliferative effect. In our study, the indirect proliferation assay was found to be a reliable and potent tool for an antisense oligonucleotide screening by targeting the p185(erbB-2) protein.

ImmunoTrack: the novel antibody-based technology for tracing in animal health.

This paper describes a novel antibody-based livestock movement control tool and method of meat allocation, both in livestock husbandry as well as during the meat-processing chain. Immuno Track fulfills diverse prerequisites and meets regulatory demands which are substantial for a successful monitoring technology: (i) the induction of long-lasting antibody responses detectable onsite throughout the whole mast period of pigs, (ii) a single immunization injection with protein derivatives is sufficient to evoke a strong epitope-specific antibody response, and (iii) the complete degradation of the protein markers after the antibody response has been triggered in meatproducing animals such as cattle or pigs. There are diverse fields of application for the Immuno-Track marker technology, such as in quality meat programs, as compliance markers for animal vaccines or as a tool for verification of origin. Combination of this monitoring technology with the husbandry and identification databases for cattle and pigs within the European Community will lead to greater transparency in meat production, thereby regaining consumers' trust in concomitant structures of the meat-producing industry.

Development of a vaccine marker technology: display of B cell epitopes on the surface of recombinant polyomavirus-like pentamers and capsoids induces peptide-specific antibodies in piglets after vaccination.

Highly immunogenic capsomers (pentamers) and virus-like particles (VLPs) were generated through insertion of foreign B cell epitopes into the surface-exposed loops of the VP1 protein of murine polyomavirus and via heterologous expression of the recombinant fusion proteins in E. coli. Usually, complex proteins like the keyhole limpet hemocyanin (KLH) act as standard carrier devices for the display of such immunogenic peptides after chemical linkage. Here, a comparative analysis revealed that antibody responses raised against the carrier entities, KLH or VP1 pentamers, did not significantly differ up to 18 weeks, demonstrating the highly immunogenic nature of VP1-based particulate structures. The carrier-specific antibody response was reproducibly detected in the meat juice after processing. More importantly, chimeric VP1 pentamers and VLPs carrying peptides of 12 and 14 amino acids in length, inserted into the BC2 loop, induced a strong and long-lasting humoral immune response against VP1 and the inserted foreign epitope. Remarkably, the epitope-specific antibody response was only moderately decreased when VP1 pentamers were used instead of VLPs. In conclusion, we identified polyomavirus VP1-based structures displaying surface-exposed immunodominant B cell epitopes as being an efficient carrier system for the induction of potent peptide-specific antibodies. The application of this approach in vaccine marker technology in livestock holding and the meat production chain is discussed.