AADvac1, an Active Immunotherapy for Alzheimer's Disease and Non Alzheimer Tauopathies: An Overview of Preclinical and Clinical Development.

Neurofibrillary tau protein pathology is closely associated with the progression and phenotype of cognitive decline in Alzheimer's disease and other tauopathies, and a high-priority target for disease-modifying therapies. Herein, we provide an overview of the development of AADvac1, an active immunotherapy against tau pathology, and tau epitopes that are potential targets for immunotherapy. The vaccine leads to the production of antibodies that target conformational epitopes in the microtubule-binding region of tau, with the aim to prevent tau aggregation and spreading of pathology, and promote tau clearance. The therapeutic potential of the vaccine was evaluated in transgenic rats and mice expressing truncated, non mutant tau protein, which faithfully replicate of human tau pathology. Treatment with AADvac1 resulted in reduction of neurofibrillary pathology and insoluble tau in their brains, and amelioration of their deleterious phenotype. The vaccine was highly immunogenic in humans, inducing production of IgG antibodies against the tau peptide in 29/30 treated elderly patients with mild-to-moderate Alzheimer's. These antibodies were able to recognise insoluble tau proteins in Alzheimer patients' brains. Treatment with AADvac1 proved to be remarkably safe, with injection site reactions being the only adverse event tied to treatment. AADvac1 is currently being investigated in a phase 2 study in Alzheimer's disease, and a phase 1 study in non-fluent primary progressive aphasia, a neurodegenerative disorder with a high tau pathology component.

Immunosenescence - the role in the immunotherapy of older population.

With ageing of their populations, many societies are challenged with serious systemic diseases. One of the causes of these diseases could be the age-related defects in immune system termed immunosenescence. Immunosenescence is characterized by accumulation of memory and non-functional immune cells, impaired signalling due to restricted repertoire of receptors, overall pro-inflammatory environment and complete dysregulation of the immune system. Consequences of immunosenescence are serious, older people are not able to respond to new stimuli, including infections and vaccinations and are more prone to oncologic, neurodegenerative and autoimmune diseases (Ref. 49).

Prion protein prevents heavy metals overloading of cells and thus protects them against their toxicity.

Physiological function of a prion protein (PrP) is not known yet. Regarding the relation of PrP to heavy metals it is known that PrP is able to bind divalent ions of copper, zinc, manganese and nickel through its octarepeat region. It has been hypothesized but not yet confirmed that PrP could play a role in copper metabolism. In this study, cells expressing human full-length PrP (HuPrP1) and PrP-knockout (PrP0/0/1) cells were incubated with various concentrations of copper, zinc, manganese and nickel for 4 days and then were assayed for intracellular content of these metals and cell viability. The results showed that HuPrP1 cells accumulated less heavy metals than PrP0/0/1 cells when concentrations of heavy metals exceeded physiological level. In conclusion, HuPrP1 cells are more resistant to chronic overload with copper, manganese, zinc or nickel than PrP0/0/1 cells. The resistance to metals overload is caused solely by the presence of PrP, since HuPrP1 and PrP0/0/1 cells differ only in the expression of PrP. These results indicate that one of the functions of PrP can be the modulation of trace heavy metal concentrations in cells and protection of cells against heavy metals overload and subsequent oxidative stress.

Establishment of the cell line expressing human prion protein on PrP0/0 background.

In this work we described preparation of the novel cell lines expressing human prion protein on PrP0/0-background. Prepared cell lines originated from the Nagasaki mice (PrP0/0) and showed a fibroblast phenotype. The expression level of human prion protein in the developed cell lines was comparable to the physiological expression measured in GT1-7 cells. A great advantage of the prepared cell lines was their short doubling time that allowed obtaining of a large amount of cells for the proteomic experiments. Newly established cell lines open a broad spectrum of applications in the prion research. Besides the study of physiological function of the prion protein or its interactome, the new cell lines could be successfully employed as a unique tool for the better understanding of key events in the pathogenesis of prion diseases.

Chronic wasting disease.

Chronic wasting disease (CWD) is the only known prion disease affecting free-ranging animals and has become a serious epidemic in North America. Although any case was reported from Europe, the spread of the disease to other continents and regions cannot be excluded, because the transmission of CWD is the most efficient among prion diseases. This article reviews the host range of CWD including experimentally infected animals, models for potential transmissibility to humans, clinical signs of the disease, pathogenesis, and methods for CWD detection.

PrP gene polymorphism in sheep breeds in Slovakia and susceptibility to scrapie.

We analyzed the prion protein (PrP) genotype based on the codons 136, 154 and 171 and assigned to five risk groups (R1-R5) in healthy and scrapie-affected sheep in Slovakia. In healthy (asymptomatic) population, 119 Merino, 106 Improved Valachian, 117 Tsigai, and 48 Suffolk breeds were tested. Among the asymptomatic sheep, the low-risk genotypes R1 and R2 were most abundant in Suffolk (94%) and Merino (84%) breeds, followed by Tsigai (58%) and Improved Valachian (40%) breeds. The medium-risk group R3 was most frequent in Improved Valachian (31%) breed, followed by Tsigai (21%), Merino (10%), and Suffolk (6%) breeds. The occurrence of high-risk groups R4 and R5 was none in Suffolk breed, followed by Merino (6%), Tsigai (21%), and Improved Valachian (30%) breeds. Since 2003, altogether 48 cases of scrapie have been confirmed in Tsigai (38), Merino (4), Improved Valachian (2), Improved Valachian x Tsigai (3), and Suffolk (1) breeds. Among sheep with scrapie, Merino breed belonged to the medium-risk group R3. The majority of scrapie-affected Tsigai sheep were classified into high-risk R5 (50%) and medium-risk R3 (42%) groups. We showed an association of scrapie with medium- and high-risk groups of PrP genotype in Slovakia. In particular, the glutamine at position 171 appears to be of major importance for the susceptibility to scrapie.

Antigenic relationship between five isolates of murine gammaherpesvirus analysed with monoclonal antibodies.

A panel of six monoclonal antibodies (mAbs) specific for murine gammaherpesvirus (MHV) was used for analysis of the antigenic relationship between five MHV-isolates (MHV 68, MHV 72, MHV 76, MHV 78, MHV S). Two mAbs raised against MHV 72 and four raised against MHV S were used in the study. Antibody-virus interactions were tested using immunochemical (ELISA, Western blot, immunofluorescence) and biological (virus-neutralization) assays. Immunoanalysis by ELISA showed a close antigenic relationship between the five viruses, nevertheless, some antigenic individuality of the isolate MHV S was observed. This isolate originated from a geographically distinct area in Czechia relative to the other four isolates from Slovakia. In Western blot analysis, antibodies to MHV 72 recognized viral antigens with the relative molecular mass about 116,000. Of four mAbs against MHV S, only two reacted with denatured viral antigen in Western blot and showed specificity for the 50-55,000 protein. These findings suggested that both isolates, besides of minor antigenic variability, could differ also in immunodominant proteins. Mabs to MHV S exhibited much stronger virus-neutralizing potency than mAbs to MHV 72, indicating thus that the 50-55,000 antigen might be more relevant for the infectivity of MHV-virus. Immunofluorescence with mAbs allowed specific localization of antigens in virus-infected VERO cells.

The human interferon system: characterization and classification after discovery of novel members.

The human interferon (IFN) system is the best characterized of all animal IFN systems. Until recently it is thought that all IFNs and IFN-related genes and proteins have been discovered. However, in the last three years, the discovery and characterization of IFNs including IFN-epsilon (IFN-epsilon), IFN-kappa (IFN-kappa) and a novel IFN-lambda (IFN-lambda) family, in particular, substantially changed this opinion. In this article, we attempt to review recent developments in the field of interferon discovery and present an overview of current classification of the human IFN system. Characterization of the constituent parts of the human IFN system including ligands, receptors and players involved in the signal transduction pathway are discussed.

[Virokines and viroceptors--viral immunomodulators with clinical and therapeutic implications]. / Virokíny a viroceptory--imunomodulátory vírusov s klinickým a terapeutickým dosahom.

During evolution viruses have developed variety of sophisticated strategies for interactions with the immune system of the host. One of the defense strategies that counteract the immune responses of the infected organism exploits viral proteins that directly interfere with the host's cytokine system. Among such immunomodulatory molecules are classed also viral homologs of cytokines (virokines) and viral homologs of cytokine receptors (viroceptors), produced and secreted by the virus-infected cell. Virokines and viroceptors are encoded by large DNA viruses--herpesviruses and poxviruses. The respective genes have been obviously "stolen" by viruses from the host genomes and then have been modified. Detailed characterization of these viral elements, which induce or subvert the host's cytokine responses against viral infection, may contribute to a better understanding of the mechanisms which help the viruses to escape immune surveillance. Such knowledge have potential implications for viral epidemiology, treatment or prevention of viral and inflammatory diseases, and for the development of safer vaccines. Examples of viruses indicate, that "capturing" of the immunomodulatory genes may be a more general strategy used also by other types of pathogenic or parasitic organisms to evade the immune responses of their hosts. (Tab. 2, Fig. 2, Ref. 73.)

Engineered acid-stabile human interferon gamma.

Loss of anti-viral potency upon pH2-treatment is an inherent feature of interferon (IFN)-gamma. The phenomenon seems to be caused by dissociation of IFN-gamma homodimer into subunits upon acidification and subsequent self-association of monomers into aggregates with reduced activity after neutralization. We demonstrated that acid-stability could be engineered into human IFN-gamma without affecting its specific activity. An artificial intra-monomer disulphide bond E7C/S69C stabilizes the dimeric form of the cytokine, which retained its full bioactivity after exposure to pH2. Acidification did not modify the antigenic structure of IFN-gamma as proved by a panel of mouse anti-human IFNgamma antibodies.

[New findings on the ecology and epidemiology of murine herpes virus isolated in Slovakia]. / Nové poznatky o ekológii a epidemiológii mysieho herpetického vírusu izolovaného na Slovensku.

Rodents are important reservoir animals of many human microbial pathogens. Of small rodents, trapped on Slovakia territory, were several strains of murine herpes virus (MHV) isolated. Our purpose was to complete the existing knowledge about circulation of MHV in rodents and to find out, whether also other animal species including man are MHV sensitive or not. The presence of antibodies against MHV in serum of the tested animals and men was followed by virus neutralization test (VNT) and ELISA. Pathological changes in differential white blood cell count of the trapped rodents, were also observed because it is known, that MHV induces them in laboratory mice. A total of 627 small terrestrial mammals of nine species were collected in four localities of western and eastern Slovakia during 1984-1988. Neutralizing antibodies to MHV were detected in five species of rodents in 130 cases (20.7%). Antibodies were most frequently detected in Apodemus flavicollis (34.9%). Pathological changes in differential white blood cell count of trapped rodents were detected in 37% (34/92). Neutralizing antibodies were found also in serum of fallow deers (Dama dama), wild boars (Sus scrofa), deers (Cervus elaphus) and sheep but not in serum of pheasants (Phasianus colchicus) and muflons (Ovis musimon). ELISA and VNT tests were used to investigate 20 serums of employees of the Institute of Virology, Slovak Academy of Sciences and Faculty of Natural Sciences of Comenius University. There were eight samples positive (40%). The titers of antibodies were 4-32 in VNT and 1000 in ELISA.

The carboxyterminal domains of human IFN-alpha2 and IFN-alpha8 are antigenically homologous.

The antigenic properties of human hybrid IFN-alpha8(60)/alpha1(92)/alpha8 were compared with those of human IFN-alpha1 and IFN-alpha2 using monoclonal antibodies (mAb). Hybrid IFN demonstrated a significantly closer antigenic relationship to the subtype alpha2 than to the subtype alpha1. In particular, high homology was observed between antigenic structures located in the C-terminal domains (93-166) of IFN-alpha8 and IFN-alpha2, whereas the corresponding N-terminal receptor-binding domains (30-53) showed distinct antigenic characteristics. The 100% homology between IFN-alpha8 and IFN-alpha2 in the region 114-131 (helix D) indicated the role of this region in formation of the common antigenic structure. In IFN-alpha8/1/8, this shared antigenic structure was important for antiviral activity and exhibited immunodominant properties, consistent with functional and antigenic properties of the corresponding structure in IFN-alpha2. Based on this antigenic homology, we suggest that IFN-alpha8 and IFN-alpha2 are evolutionarily more closely related to each other than to IFN-alpha1. This study will contribute to a better understanding of evolutionary events in the human IFN-alpha family.

Radioiodination of human interferon-alpha2 interferes with binding of C-terminal specific antibodies.

Radioimmunoassays based on reactivity between a monoclonal antibody (mAb) and human 125I-interferon (IFN)-alpha2 are frequently exploited in interferon research. In general, epitopes of antibodies specific for human IFN-alpha2 are located on the two immunodominant structures formed in the N- and C-terminal domains, respectively. We found that labelling of IFN-alpha2 with Na(125)I by the chloramine-T method did not affect the binding of antibodies recognising the N-terminal region 30-53. In contrast, radioiodination of IFN was associated with a dramatic decrease in IFN reactivity with mAbs specific for the C-terminus (residues approximately 120-145 approximately ). We suggest that steric hindrance araising from the incorporation of 125I into the tyrosine residues at positions 123, 130 and 136 may be responsible for the change in immunoreactivity. The adverse effect of radioiodination of IFN-alpha2 on the binding potency of C-terminal specific mAbs must be taken into consideration in experiments based on the interaction of such antibodies (i.e. NK2) with the labelled antigen.

Functional blockade of tyrosine kinase A in the rat basal forebrain by a novel antagonistic anti-receptor monoclonal antibody.

We have exploited a new monoclonal antibody against the tyrosine kinase A (TrkA) nerve growth factor (NGF) receptor to block the NGF-TrkA interaction in the rat basal forebrain. The monoclonal antibody MNAC13 is a potent antagonist that prevents the binding of NGF to TrkA in a variety of systems. This antibody was used to study the maintenance of the cholinergic phenotype in the rat basal forebrain in vivo, by the implant of antibody-secreting cells. Basal forebrain cholinergic neurons (BFCNs) are greatly affected by the antibody treatment, both in terms of cell number and of cell soma size. When antibody-secreting cells are implanted at postnatal day 2 (P2), the effects observed at P8 are as severe as those obtained with anti-NGF antibodies and, interestingly, are observed also if anti-TrkA cells are implanted at P8, when anti-NGF antibodies, delivered by the same route, are no longer effective (). The effects induced by anti-TrkA, as those induced by anti-NGF, are reversible, but the time required for recovery and the critical period in the sensitivity of BFCNs to the functional inactivation of TrkA is twice as long than that observed when NGF is intercepted. These results demonstrate that BFCNs are more sensitive to the block of TrkA activation than they are to the block of NGF. The cloning of MNAC13 variable regions and their assembly into a functional polypeptide of reduced size (single chain Fv fragment) will allow its use in gene transfer applications.

Structural and functional heterogeneity of the amino-terminal receptor-binding domain of human interferon-alpha 2.

Structural immunoanalysis of human interferon (IFN)-alpha 2c revealed antigenic and functional heterogeneity in its N-terminal receptor-binding domain (loop AB). Monoclonal antibodies (mAbs) mapped to the region 30-53 of IFN-alpha 2 defined three partially overlapping antigenic sites designated here as 'a', 'b' and 'c'. For the high-affinity binding of IFN-alpha 2c to the cellular receptor, site b located in segment 34-41 and site c (residues 43-53) appeared to be most important. Only the part of site a (amino acids 30-33) seemed to be involved in the interaction with receptor. The segment of residues 30-46 forms a relatively straight structure on the protein surface, according to the three-dimensional model of human IFN-alpha 2.

Immunogenicity of interferon-alpha 2 in therapy: structural and physiological aspects.

Recombinant human interferon (rIFN)-alpha 2 has been approved for therapeutic application in a range of human oncological and viral diseases. However, some patients can develop strictly specific antibody response to rIFN-alpha 2, which may diminish its therapeutic potential. Such humoral response appears to be quite complex and obviously depends on multiple parameters. Our review is aimed primarily to factors associated with structural modifications of rIFN-alpha 2 that we consider crucial for formation of therapy-induced antibodies. These factors are either related to inherent conformational differences between three IFN-alpha 2 subvariants or to immunogenically active contaminating derivatives resulting from production, purification and storage of this recombinant protein. In addition, the role of treatment regimen and physiological variables modulating the immune response to rIFN-alpha 2 in the challenged organism are mentioned.

[Present views on interferons]. / Súcasný pohl'ad na interferón.

The nature of interferons and mechanisms of their action have been studied for more than four decades. The review article brings the current view of interferon, putting emphasis on historical transitions in its definition and changes in the understanding of the physiological role of interferon in organism. The recent classification of interferons together with the more detailed characterization of human interferons are presented. (Tab. 1, Ref. 40.)

Therapy-induced antibodies to interferon-alpha 2a recognise its receptor-binding site.

Fifty-eight patients with chronic hepatitis B (HB) or C (HC) were treated with recombinant human interferon (rIFN)-alpha 2 and their sera were assayed for antibodies to rIFN-alpha 2c. Twelve of these patients produced low titres and two high titres of the antibodies. We localized the region which was recognised by the high-titre therapy-induced antibodies on the IFN molecule by testing the antibodies with a set of murine monoclonal antibodies (MoAbs) to IFN-alpha 2 in a competitive radioimmune assay (RIA). Only MoAbs with epitopes located in the amino-terminal portion of IFN-alpha 2 could inhibit the binding of radiolabelled IFN-alpha 2 by patients' sera. Our data indicate that the therapy-induced antibodies were directed to the receptor-binding domain of IFN-alpha 2 formed by amino acids (aa) 30-53. In accordance with this observation, human anti-IFN sera inhibited the binding of rIFN-alpha 2 to human cells.

Structural immuno-analysis of human and porcine interferon gamma: identification of shared antigenic domain.

We examined the antigenic resemblance between human (h) and porcine (p) interferon (IFN)-gamma by binding (ELISA) and neutralization assays. The murine polyclonal antisera and sets of murine monoclonal antibodies (mAbs) raised against either IFN were tested in confrontation with recombinant IFNs of either species, and with site-specific mutants of hIFN-gamma. Several of the mAbs raised against pIFN-gamma cross-reacted in ELISA with hIFN-gamma. In contrast, none of the anti-hIFN-gamma mAbs cross-reacted. By employing site-specific mutants of recombinant hIFN-gamma as antigens in ELISA we succeeded in identifying the C-terminal portion 97-111 as the antigenic site in hIFN-gamma recognized by the cross-reactive anti-pIFn-gamma mAbs. None of the mAbs recognizing the common antigenic structure had neutralizing potency, although His111 was determined by others as the residue important for bioactivity of hIFN-gamma. Mutations in the domain 97-111 had no or little influence on homospecific reactivity of anti-hIFN-gamma mAbs, indicating that this domain, while being mouse-immunodominant in the case of pIFn-gamma was poorly immunogenic in the case of hIFN-gamma. The epitopes of three out of five anti-hIFN-gamma mAbs mapped in the N-terminal region 1-23, indicating immunodominance of this region in hIFN-gamma. Another mAb (D9D10), also directed to the N-terminus of hIFN-gamma, apparently recognized a conformational epitope. This antibody lacked ELISA-reactivity with the wild-type hIFN-gamma but strongly bound mutant protein with an engineered disulfide bridge Cys7-Cys69. Surprisingly, D9D10 showed high reactivity also with the wild type hIFN-gamma produced by baculovirus construct coding for the mature protein with signal sequence or with wild type protein possessing residues Cys-Tyr-Cys from the signal sequence.