[Show abstract][Hide abstract] ABSTRACT: Multiple system atrophy (MSA) is a neurodegenerative disease characterized by parkinsonism, ataxia and dysautonomia. Histopathologically, the hallmark of MSA is the abnormal accumulation of alpha-synuclein (α-syn) within oligodendroglial cells, leading to neuroinflammation, demyelination and neuronal death. Currently, there is no disease-modifying treatment for MSA. In this sense, we have previously shown that next-generation active vaccination technology with short peptides, AFFITOPEs®, was effective in two transgenic models of synucleinopathies at reducing behavioral deficits, α-syn accumulation and inflammation.
In this manuscript, we used the most effective AFFITOPE® (AFF 1) for immunizing MBP-α-syn transgenic mice, a model of MSA that expresses α-syn in oligodendrocytes. Vaccination with AFF 1 resulted in the production of specific anti-α-syn antibodies that crossed into the central nervous system and recognized α-syn aggregates within glial cells. Active vaccination with AFF 1 resulted in decreased accumulation of α-syn, reduced demyelination in neocortex, striatum and corpus callosum, and reduced neurodegeneration. Clearance of α-syn involved activation of microglia and reduced spreading of α-syn to astroglial cells.
This study further validates the efficacy of vaccination with AFFITOPEs® for ameliorating the neurodegenerative pathology in synucleinopathies.
[Show abstract][Hide abstract] ABSTRACT: Recent evidence suggests Alzheimer-Disease (AD) to be driven by aggregated Aß. Capitalizing on the mechanism of molecular mimicry and applying several selection layers, we screened peptide libraries for moieties inducing antibodies selectively reacting with Aß-aggregates. The technology identified a pool of peptide candidates; two, AFFITOPES AD01 and AD02, were assessed as vaccination antigens and compared to Aβ1-6, the targeted epitope. When conjugated to Keyhole Limpet Hemocyanin (KLH) and adjuvanted with aluminum, all three peptides induced Aß-targeting antibodies (Abs). In contrast to Aß1-6, AD01- or AD02-induced Abs were characterized by selectivity for aggregated forms of Aß and absence of reactivity with related molecules such as Amyloid Precursor Protein (APP)/ secreted APP-alpha (sAPPa). Administration of AFFITOPE-vaccines to APP-transgenic mice was found to reduce their cerebral amyloid burden, the associated neuropathological alterations and to improve their cognitive functions. Thus, the AFFITOME-technology delivers vaccines capable of inducing a distinct Ab response. Their features may be beneficial to AD-patients, a hypothesis currently tested within a phase-II-study.
PLoS ONE 01/2015; 10(1):e0115237. DOI:10.1371/journal.pone.0115237 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Low Density Lipoprotein (LDL) hypercholesterolemia, and its associated cardiovascular diseases, are some of the leading causes of death worldwide. The ability of proprotein convertase subtilisin/kexin 9 (PCSK9) to modulate circulating LDL cholesterol (LDLc) concentrations made it a very attractive target for LDLc management. To date, the most advanced approaches for PCSK9 inhibition are monoclonal antibody (mAb) therapies. Although shown to lower LDLc significantly, mAbs face functional limitations because of their relatively short in vivo half-lives necessitating frequent administration. Here, we evaluated the long-term efficacy and safety of PCSK9-specific active vaccines in different preclinical models.
PCSK9 peptide-based vaccines were successfully selected by our proprietary technology. To test their efficacy, wild-type (wt) mice, Ldlr+/- mice, and rats were immunized with highly immunogenic vaccine candidates. Vaccines induced generation of high-affine PCSK9-specific antibodies in all species. Group mean total cholesterol (TC) concentration was reduced by up to 30%, and LDLc up to 50% in treated animals. Moreover, the PCSK9 vaccine-induced humoral immune response persisted for up to one year in mice, and reduced cholesterol levels significantly throughout the study. Finally, the vaccines were well tolerated in all species tested.
Peptide-based anti-PCSK9 vaccines induce the generation of antibodies that are persistent, high-affine, and functional for up to one year. They are powerful and safe tools for long-term LDLc management, and thus may represent a novel therapeutic approach for the prevention and/or treatment of LDL hypercholesterolemia-related cardiovascular diseases in humans.
PLoS ONE 12/2014; 9(12):e114469. DOI:10.1371/journal.pone.0114469 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutations in Leucine-rich repeat kinase 2 (LRRK2) are frequently involved in both familial and sporadic forms of Parkinson’s disease (PD). Until now, no functional murine LRRK2 transgenic model is publicly available that fully recapitulates the clinical symptoms of PD. We recently developed a novel mouse model of Parkinson’s disease over-expressing human wild-type LRRK2 under the control of the neuron specific Thy1.2 promoter.
In order to determine whether neuron-specific over-expression of human wild-type LRRK2 would lead to phenotypic alterations in this novel mouse model for PD, we started to implement a battery of classical behavioural tests.
Mice were subjected to behavioural testing at 4, 8, 12 and 16 months of age. After completing a modified SHIRPA screen for general health and reflexes, animals were tested for cognitive, functional and motoric alterations assessing anosmia, nesting behaviour, memory deficits, and deficits in grip strength, endurance and motor-coordination.
We found that expression of human LRRK2 under the control of the neuron specific Thy1.2 promoter results in viable offspring, which develops age dependent, progressive phenotypical alterations at around 8 months of age. Results depicting these deficits will be discussed.
Based on the initial findings presented, this novel transgenic model over-expressing LRRK2 could be instrumental to further elucidate the biologic and pathologic role of Leucine repeat rich kinase-2 (LRRK2) in the nervous system and could thus be a suitable tool for studying the effects of therapeutic drugs for PD in animals.
10th International Conference AD/PD, Barcelona, March 2011; 03/2011
[Show abstract][Hide abstract] ABSTRACT: Neurodegenerative diseases are still an area of unmet medical need. This is in contrast to our increasing knowledge on their pathology (e.g., Alzheimer's- (AD), Parkinson's (PD) disease). They are driven by the cerebral accumulation and aggregation of specific proteins (e.g., β-amyloid and hyperphosphorylated tau in the case of AD) in defined brain regions and, as a consequence, death of neurons. Accordingly, removal of given protein aggregates is expected to modify the course of the respective neurodegenerative disease. This has been convincingly demonstrated in animal models of human diseases. However, not every technology that can be used and proves successful in animal models can be translated to the human situation. As highlighted by recent progress in the field of AD research, specific immunotherapy is a viable option in this regard. Given the fact that the aggregates are composed of self-proteins, immunotherapeutic approaches have to consider the issue of potential autoimmunity. This is especially true in case of vaccines. An innovative solution to this problem is offered by the so called AFFITOME® technology, which relies on the use of "doubles" of native molecules, functionally mimotopes or AFFITOPES® if identified by AFFiRiS, as the antigenic vaccine component.
Human vaccines 11/2010; 6(11):948-52. DOI:10.4161/hv.6.11.13217 · 3.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Subcutaneous injection of GM-CSF-expressing cancer cells into experimental animals results in protective cancer immunity. To delineate the mode of action of such vaccines, we used trinitrophenyl, the antigenic moiety of the contact allergen trinitrochlorobenzene, as surrogate Ag. Trinitrophenyl-derivatized bone marrow-derived dendritic cells were found to elicit a contact hypersensitivity response in syngeneic, but not in allogeneic recipients, compatible with their expected mode of direct Ag presentation. When expressing GM-CSF, haptenized M3 melanoma cells were also able to induce a contact hypersensitivity response but, in contrast to bone marrow-derived dendritic cells, not only in syngeneic but also in allogeneic recipients. This argues for a critical role of host APC. To identify their nature, we introduced the beta-galactosidase (betagal) gene into M3-GM cells. Their administration activated betagal-specific, L(d)-restricted CTL in syngeneic BALB/c mice. Evaluation of lymph nodes draining M3-GM-betagal injection sites revealed the presence of cells presenting the respective L(d)-binding betagal peptide epitope. Based on their capacity to activate betagal-specific CTL, they were identified as being CD11c(+) dendritic cells. These experiments provide a rational basis for the use of GM-CSF-based melanoma cell vaccines in an allogeneic setting.
The Journal of Immunology 12/2003; 171(10):5180-7. DOI:10.4049/jimmunol.171.10.5180 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The s.c injection of tumor Ag-derived, MHC class I-binding peptides together with cationic poly-amino acids (e.g., poly-L-arginine; pR) has been shown to protect animals against a challenge with tumor cells expressing the respective peptide(s). Given our only restricted knowledge about immunogenic tumor-associated peptides, we sought to determine whether this pR-based vaccination protocol would also induce protective cancer immunity if large proteins were used instead of peptide epitopes. We found that the intracutaneous administration of the model Ag beta-galactosidase (beta-gal) together with pR (referred to as pR-based protein vaccine; pR-PV) was significantly more potent in protecting mice against the growth of beta-gal-expressing RENCA cells than the protein alone. Coadministration of pR enhanced both the beta-gal-induced specific humoral and CD8 response. The protective effect required CD8(+), but neither CD4(+) T lymphocytes nor beta-gal-specific Abs. beta-Gal priming of protective CD8(+) T lymphocytes was found to be CD4(+) T cell-independent, to take place within the draining lymph nodes, and to be accomplished by day 5 after vaccination. Ablation of the injection sites as early as 1.5 h after pR-PV administration still led to protection in a large proportion of the animals, indicating that certain protein Ags administered intradermally in the context of polycations are quickly transported to the draining nodes, where they induce molecular and cellular events resulting in the helper-independent priming and expansion of Tc1 cells. However, optimal protection required the prolonged presence of the injection site, suggesting that pR-PV injection facilitates the formation of a cutaneous depot of Ag-charged cells capable of migration and T cell activation.
The Journal of Immunology 12/2002; 169(9):5217-26. DOI:10.4049/jimmunol.169.9.5217 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study describes an entirely synthetic vaccine composed of antigenic peptides (T cell epitopes), oligodeoxynucleotides containing CpG-motifs (CpG-ODN) and poly-L-arginine (pR). CpG-ODN are known to be potent inducers of predominantly type 1-like immune responses, while polycationic amino acids, like pR, facilitate the uptake of antigens into antigen presenting cells (APCs). We demonstrate that the application of peptides and pR/CpG-ODN results in strongly enhanced peptide-specific immune responses as compared to the application of peptides with either of the immunomodulators alone. High numbers of antigen-specific T cells can be observed even after only one injection of the vaccine for a remarkably long period of time (at least 372 days). Furthermore, the potentially harmful systemic release of pro-inflammatory cytokines induced upon injection of CpG-ODN is inhibited. Thus, the combined application of CpG-ODN and pR may represent a novel vaccine strategy in humans.
[Show abstract][Hide abstract] ABSTRACT: For the design of potent subunit vaccines, it is of paramount importance to identify all antigens immunologically recognized by a patient population infected with a pathogen. We have developed a rapid and efficient procedure to identify such commonly recognized antigens, and here we provide a comprehensive in vivo antigenic profile of Staphylococcus aureus, an important human pathogen. S. aureus peptides were displayed on the surface of Escherichia coli via fusion to one of two outer membrane proteins (LamB and FhuA) and probed with sera selected for high Ab titer and opsonic activity. A total of 60 antigenic proteins were identified, most of which are located or predicted to be located on the surface of the bacterium or secreted. The identification of these antigens and their reactivity with individual sera from patients and healthy individuals greatly facilitate the selection of promising vaccine candidates for further evaluation. This approach, which makes use of whole genome sequence information, has the potential to greatly accelerate and facilitate the formulation of novel vaccines and is applicable to any pathogen that induces Abs in humans and/or experimental animals.
Proceedings of the National Academy of Sciences 06/2002; 99(10):6573-8. DOI:10.1073/pnas.092569199 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vaccines that induce high numbers of sustained T cell responses are urgently needed for the treatment of numerous diseases including cancer. Antigen-presenting cells (APCs), the most important of which are dendritic cells, orchestrate antigen-dependent T cell responses in that they present antigens to T cells in an appropriate environment. Here we present evidence that after vaccination with a simple mixture of the cationic poly-amino acid poly-L-arginine and tumor antigen-derived peptide antigens, large numbers of antigen-specific T cells are induced and APCs mediate the generation of T lymphocytes. We observe that after s.c. injection, MHC class II(+) cells infiltrate injection sites and are loaded with large amounts of antigen in vivo under the influence of poly-L-arginine. Consequently, numerous antigen-charged APCs can be detected in draining lymph nodes of vaccinated animals. Antigen-specific T cell responses induced are systemic and were readily detected more than 4 months after the last vaccination, the latest time point we measured. By contrast, even after repeat injections, we were consistently unable to detect antibody responses against poly-L-arginine, allowing this compound to be used for numerous booster injections. Clinical trials in cancer patients using poly-L-arginine as immunostimulant will be carried out in the near future.
Cancer Research 04/2002; 62(5):1477-80. · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We present a novel strategy, termed CISTEM, which allows direct in vivo screening of polypeptides displayed on the surface of E. coli cells by a combination of ligand-mediated protection and phage-mediated selection. The effectiveness of this new approach was demonstrated by displaying the T7.tag on the surface of E. coli as a fusion with the outer membrane protein A, the receptor for bacteriophage K3. A monoclonal T7.tag antibody was used as protective ligand for T7.tag-displaying cells and phage K3 for the elimination of unprotected cells. When populations of bacteria, containing between 6 to 10,000 cells displaying the T7.tag and approximately 10(8) cells displaying an unrelated OmpA fusion protein, were infected with phage K3, specific and antibody-dependent survival of T7.tag displaying cells was observed, yielding an enrichment factor of up to 10(7)-fold. The CISTEM technology was used to select sequences from a T7.tag-based, randomised library and the results were compared to those obtained from selection by MACS with the same library. Together, these results reveal a novel in vivo screening strategy in which an E. coli phage receptor is used as display plafform and selection is performed in suspension upon addition of a protective ligand and a bacteriophage. Extentions and modifications of the basic strategy should lead to novel applications for the identification of protein-ligand interactions.
[Show abstract][Hide abstract] ABSTRACT: We performed a phase I trial to evaluate the safety and tolerability of repeated skin injections of IL-2-transfected autologous melanoma cells into patients with advanced disease. Cell suspensions, propagated from excised metastases, were IL-2 gene transfected by adenovirus-enhanced transferrinfection and X-irradiated prior to injection. Vaccine production was successful in 54% of the patients. Fifteen patients (37%) received two to eight skin vaccinations of either 3 x 10(6) (intradermal) or 1 x 10(7) (half intradermal, half subcutaneous) transfected melanoma cells per vaccination (secreting 140-17,060 biological response modifier program units of IL-2/10(6) cells/24 hr). Analyses of safety and efficacy were carried out in 15 and 14 patients, respectively. Overall, the vaccine was well tolerated. All patients displayed modest local reactions (erythema, induration, and pruritus) and some experienced flu-like symptoms. Apart from newly appearing (4 of 14) and increasing (5 of 14) anti-adenovirus and newly detectable anti-nuclear antibody titers (1 of 15), recipients developed de novo or exhibited increased melanoma cell-specific delayed-type hypersensitivity (DTH) reactions (8 of 15) and vitiligo (3 of 15) and showed signs of tumor regression (3 of 15). This supports the idea of a vaccine-induced or -amplified anti-cancer immune response. None of the patients exhibited complete or partial regressions, but five of them experienced periods of disease stabilization. Three of these individuals received more than the four planned vaccinations and their mean survival time was 15.7 +/- 3.5 months as compared to 7.8 +/- 4.6 months for the entire patient cohort. These data indicate that IL-2-producing, autologous cancer cells can be safely administered to stage IV melanoma patients and could conceivably be of benefit to patients with less advanced disease.
Human Gene Therapy 05/1999; 10(6):983-93. DOI:10.1089/10430349950018382 · 3.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Summary With the advent of gene therapy there has been a revival of immunotherapy of cancer. Preclinical studies with the so called tumor vaccines - syngeneic, irradiated tumor cells secreting cytokines - are at present entering clinical trials and hold much promise for efficacious treatment, maybe even cures, of cancer. However, autologous whole cell vaccines which are cytokine gene-modified are expensive and difficult to standardize. In addition, autologous tumor cell cultures, and hence tumor vaccines, cannot be prepared for all patients. For these reasons we have investigated alternative schemes for vaccination against cancer. We have developed vaccines on the basis of tumor antigen- derived peptides using a method, we called "transloading", to transfer peptides efficiently into cells. Such vaccines are chemically well defined and inexpensive to produce. We show in a preclinical mouse model system for mastocytoma, and to a somewhat lesser extent for melanoma, that peptide vaccines give excellent protection against tumor take, provided tumor antigen peptides are injected subcutaneously in conjunction with polycations like polyarginine. Immunohistological investigations on thin sections show that three days after injection, the vaccine bolus is heavily infiltrated by antigen presenting cells which take up peptides. We posit that such antigen presenting cells then migrate into draining lymph nodes where they activate naive T cells to become anti-tumor cytotoxic lymphocytes (CTL). The consequences of strong dependency of peptide sequence on the HLA-type of patients and possible remedies are discussed. One avenue to be tested in mouse models is the injection of mixtures of peptides covering a multiplicity of MHC-specific peptides. Another is the production of recombinant proteins as vaccines whose application is considerably less dependent on HLA-types of patients to be treated. In addition, such proteins would be expected to contain both class I and class II restricted peptides. Alternatively, artificial proteins incorporating many CTL epitopes of known tumor antigens, including members of the MAGE family, thus yielding a SUPERMAGE vaccine with utility for several human HLA- types could be designed. The prophylactic application of peptide/polyepitope vaccines can be envisaged.
[Show abstract][Hide abstract] ABSTRACT: Cancer vaccines are based on the concept that tumors express novel antigens and thus differ from their normal tissue counterparts. Such putative tumor-specific antigens should be recognizable by the immune system. However, malignant cells are of self origin and only poorly immunogenic, which limits their capability to induce an anticancer immune response. To overcome this problem, tumor cells have been isolated, genetically engineered to secrete cytokine gene products and administered as cancer vaccines. We used adenovirus-enhanced transferrinfection (AVET), which allows high-level transient transgene expression, to introduce cytokine gene expression vectors into murine melanoma cells. The efficiency of AVET makes laborious selection and cloning procedures obsolete. We administered such modified tumor cells as cancer vaccines to syngeneic animals and investigated their impact on the induction of anticancer immunity. We found that IL-2 or GM-CSF gene-transfected murine melanoma cells are highly effective vaccines. Both of these cytokine-secreting vaccines cured 80% of animals which bore a subcutaneous micrometastasis prior to treatment, and induced potent antitumor immunity. The generation of antitumor immunity by these cytokine-secreting vaccines requires three different steps: (1) tumor antigen uptake and processing by antigen-presenting cells (APCs) at the site of vaccination; (2) migration of these APCs into the regional lymph nodes where T-cell priming occurs; (3) recirculation of specific, activated T-cells that recognize distinct tumor load and initiate its elimination. Extending our previously reported studies, we have now comprehensively analysed the requirements for effective antitumor vaccination in animals. This may also become the basis for treatment of human cancer patients.
[Show abstract][Hide abstract] ABSTRACT: The presence of about 1.2 M glycerol during transfection with DNA/transferrin-polylysine and DNA/polylysine complexes dramatically increases transgene expression in a variety of cell types, provided that the complexes have an excess of polylysine. We have characterized this phenomenon using a human melanoma cell line (H225). The addition of 1.2 M glycerol to the transfection medium has no influence on the internalization of DNA complexes or on the promoter activity used to direct reporter gene expression. Neither prenor postincubation of the cells with glycerol results in a notable increase in transgene expression. Bafilomycin A1 and chloroquine, two drugs affecting the endosomal pathway, both influenced transgene expression, indicating that glycerol acts on internal vesicles. Glycerol and polylysine synergized in their ability to lyse erythrocytes as well as internal vesicles (microsomes) isolated from H225 cells, indicating that the glycerol effect is due to a labilization of vesicular membranes, which facilitates membrane disruption by polylysine. Our current model suggests that the excess of polylysine in the DNA complexes disrupts vesicular membranes in the presence of glycerol, thus allowing the release of DNA complexes into the cytoplasm.
Experimental Cell Research 05/1997; 232(1):137-45. DOI:10.1006/excr.1997.3486 · 3.25 Impact Factor