Plant-based chimeric HPV-virus-like particles bearing amyloid-ß epitopes elicit antibodies able to recognize amyloid plaques in APP-tg mouse and Alzheimer's disease brains.

The main amyloid-beta (Aß) variants detected in the human brain are full-length Aß1-40 and Aß1-42 peptides; however, a significant proportion of AD brain Aß consists also of N-terminal truncated/modified species. The majority of the previous immunotherapeutic strategies targeted the N-terminal immunodominant epitope of the full-length Aß; however, most of the pathological N-truncated forms of Aß lack this critical B cell epitope. Recently, virus-like particles (VLPs), self-assembled structures with highly ordered repetitive patterns on their surface and capable of inducing robust immune responses, were applied as a promising platform for various antigen expressions. In this study, we expressed in plants two chimeric HPV16 L1 capsid proteins obtained by introduction of the ß-amyloid 11-28 epitope (Aß 11-28) into the h4 helix or into the coil regions of the L1 protein. The Aß 11-28 epitope was chosen because it is present in the full-length Aß 1-42 as well as in the truncated/modified amyloid peptide species. After expression, we assembled the chimerical L1/Aß 11-28 into a VLP in which the Aß 11-28 epitope is exposed at very high density (360 times) on the surface of the VLP. The chimeric VLPs elicited in mice Aß-specific antibodies binding to ß-amyloid plaques in APP-tg mouse and AD brains. Our study is the first to demonstrate a successful production in plants and immunogenic properties in mice of chimeric HPV16 L1 VLPs bearing Aß epitope that may be of potential relevance for the development of multivalent vaccines for a multifactorial disease such as AD.

Intranasal delivery of dexamethasone efficiently controls LPS-induced murine neuroinflammation.

Neuroinflammation is the hallmark of several infectious and neurodegenerative diseases. Synthetic glucocorticoids (GCs) are the first-line immunosuppressive drugs used for controlling neuroinflammation. A delayed diffusion of GCs molecules and the high systemic doses required for brain-specific targeting lead to severe undesirable effects, particularly when lifelong treatment is required. Therefore, there is an urgent need for improving this current therapeutic approach. The intranasal (i.n.) route is being employed increasingly for drug delivery to the brain via the olfactory system. In this study, the i.n. route is compared to the intravenous (i.v.) administration of GCs with respect to their effectiveness in controlling neuroinflammation induced experimentally by systemic lipopolysaccharide (LPS) injection. A statistically significant reduction in interleukin (IL)-6 levels in the central nervous system (CNS) in the percentage of CD45+ /CD11b+ /lymphocyte antigen 6 complex locus G6D [Ly6G+ and in glial fibrillary acidic protein (GFAP) immunostaining was observed in mice from the i.n.-dexamethasone (DX] group compared to control and i.v.-DX-treated animals. DX treatment did not modify the percentage of microglia and perivascular macrophages as determined by ionized calcium binding adaptor molecule 1 (Iba1) immunostaining of the cortex and hippocampus. The increased accumulation of DX in brain microvasculature in DX-i.n.-treated mice compared with controls and DX-IV-treated animals may underlie the higher effectiveness in controlling neuroinflammation. Altogether, these results indicate that IN-DX administration may offer a more efficient alternative than systemic administration to control neuroinflammation in different neuropathologies.

Electric stimulation of the vagus nerve reduced mouse neuroinflammation induced by lipopolysaccharide.

BACKGROUND: Neuroinflammation (NI) is a key feature in the pathogenesis and progression of infectious and non-infectious neuropathologies, and its amelioration usually improves the patient outcome. Peripheral inflammation may promote NI through microglia and astrocytes activation, an increased expression of inflammatory mediators and vascular permeability that may lead to neurodegeneration. Several anti-inflammatory strategies have been proposed to control peripheral inflammation. Among them, electrical stimulation of the vagus nerve (VNS) recently emerged as an alternative to effectively attenuate peripheral inflammation in a variety of pathological conditions with few side effects. Considering that NI underlies several neurologic pathologies we explored herein the possibility that electrically VNS can also exert anti-inflammatory effects in the brain. METHODS: NI was experimentally induced by intraperitoneal injection of bacterial lipopolysaccharide (LPS) in C57BL/6 male mice; VNS with constant voltage (5 Hz, 0.75 mA, 2 ms) was applied for 30 s, 48 or 72 h after lipopolysaccharide injection. Twenty four hours later, pro-inflammatory cytokines (IL-1ß, IL-6, TNFα) levels were measured by ELISA in brain and spleen extracts and total brain cells were isolated and microglia and macrophage proliferation and activation was assessed by flow cytometry. The level of ionized calcium binding adaptor molecule (Iba-1) and glial fibrillary acidic protein (GFAP) were estimated in whole brain extracts and in histologic slides by Western blot and immunohistochemistry, respectively. RESULTS: VNS significantly reduced the central levels of pro-inflammatory cytokines and the percentage of microglia (CD11b/CD45low) and macrophages (CD11b/CD45high), 24 h after the electrical stimulus in LPS stimulated mice. A significantly reduced level of Iba-1 expression was also observed in whole brain extracts and in the hippocampus, suggesting a reduction in activated microglia. CONCLUSIONS: VNS is a feasible therapeutic tool to attenuate the NI reaction. Considering that NI accompanies different neuropathologies VNS is a relevant alternative to modulate NI, of particular interest for chronic neurological diseases.

Antigenic variability: Obstacles on the road to vaccines against traditionally difficult targets.

Despite the impressive impact of vaccines on public health, the success of vaccines targeting many important pathogens and cancers has to date been limited. The burden of infectious diseases today is mainly caused by antigenically variable pathogens (AVPs), which escape immune responses induced by prior infection or vaccination through changes in molecular structures recognized by antibodies or T cells. Extensive genetic and antigenic variability is the major obstacle for the development of new or improved vaccines against "difficult" targets. Alternative, qualitatively new approaches leading to the generation of disease- and patient-specific vaccine immunogens that incorporate complex permanently changing epitope landscapes of intended targets accompanied by appropriate immunomodulators are urgently needed. In this review, we highlight some of the most critical common issues related to the development of vaccines against many pathogens and cancers that escape protective immune responses owing to antigenic variation, and discuss recent efforts to overcome the obstacles by applying alternative approaches for the rational design of new types of immunogens.

Novel amyloid-beta specific scFv and VH antibody fragments from human and mouse phage display antibody libraries.

Anti-amyloid immunotherapy has been proposed as an appropriate therapeutic approach for Alzheimer's disease (AD). Significant efforts have been made towards the generation and assessment of antibody-based reagents capable of preventing and clearing amyloid aggregates as well as preventing their synaptotoxic effects. In this study, we selected a novel set of human anti-amyloid-beta peptide 1-42 (Abeta1-42) recombinant monoclonal antibodies in a single chain fragment variable (scFv) and a single-domain (VH) format. We demonstrated that these antibody fragments recognize in a specific manner amyloid-beta deposits in APP/Tg mouse brains, inhibit toxicity of oligomeric Abeta1-42 in neuroblastoma cell cultures in a concentration-dependent manner and reduced amyloid deposits in APP/Tg2576 mice after intracranial administration. These antibody fragments recognize epitopes in the middle/C-terminus region of Abeta, which makes them strong therapeutic candidates due to the fact that most of the Abeta species found in the brains of AD patients display extensive N-terminus truncations/modifications.

[Characterizing of parameters of shape memory and elasticity of nickel-titanium wires by electro thermal influence].

Optimal parameters of electro thermal influence on nickel-titanium wires for giving them new shape (bends) without losing basic physical properties - shape memory, elasticity are determined in terms of clinic. It is confirmed that by thermal influence of electric current on nickel-titanium wires it's possible to achieve new shape without changing the parameters of elasticity. Optimal parameters of continuous current intensity (strength) and interaction time are determined for: Nitinol 0.016, 0.016 x 0.022, NeoSentalloy 0.016 x 0.022 (GAC International, Inc). It was established that during heat treatment of nickel-titanium arch wires the shape of wire changes earlier than the elasticity.

Immunodominant epitope and properties of pyroglutamate-modified Abeta-specific antibodies produced in rabbits.

N-truncated and N-modified forms of amyloid beta (Abeta) peptide are found in diffused and dense core plaques in Alzheimer's disease (AD) and Down's syndrome patients as well as transgenic mouse models of AD. Although the pathological significance of these shortened forms Abeta is not completely understood, previous studies have demonstrated that these peptides are significantly more resistant to degradation, aggregate more rapidly in vitro and exhibit similar or, in some cases, increased toxicity in hippocampal neuronal cultures compared to the full length peptides. In the present study we further investigated the mechanisms of toxicity of one of the most abundant N-truncated/modified Abeta peptide bearing amino-terminal pyroglutamate at position 3 (AbetaN3(pE)). We demonstrated that AbetaN3(pE) oligomers induce phosphatidyl serine externalization and membrane damage in SH-SY5Y cells. Also, we produced AbetaN3(pE)-specific polyclonal antibodies in rabbit and identified an immunodominant epitope recognized by anti-AbetaN3(pE) antibodies. Our results are important for developing new immunotherapeutic compounds specifically targeting AbetaN3(pE) aggregates since the most commonly used immunogens in the majority of vaccines for AD have been shown to induce antibodies that recognize the N-terminal immunodominant epitope (EFRH) of the full length Abeta, which is absent in N-amino truncated peptides.

Epitope mapping and neuroprotective properties of a human single chain FV antibody that binds an internal epitope of amyloid-beta 1-42.

Active and passive immunotherapy targeted at the amyloid-beta (Abeta) peptide has been proposed as therapeutic approach against Alzheimer's disease (AD), and efforts towards the generation and application of antibody-based reagents that are capable of preventing and clearing amyloid aggregates are currently under active investigation. Previously, we selected and characterized a new anti-Abeta1-42 phage-displayed scFv antibody, designated clone b4.4, using a non-immune human scFv antibody library and demonstrated that a peptide based on the sequence of the Ig heavy chain (VH) complementarity-determining region (HCDR3) of this antibody fragment bound to Abeta1-42)and had neuroprotective potential against Abeta1-42 mediated neurotoxicity in rat hippocampal cultured neurons. In the present study, using novel computational methods and in vitro experiments we demonstrated that b4.4 binds to the central region of Abeta1-42. We also demonstrated that this scFv antibody binds to Abeta-derived diffusible ligands (ADDLs) and neutralizes the toxicity of both fibrillar and oligomeric forms of Abeta1-42 tested in vitro in SH-SY5Y cell cultures.

Further evaluation of the synthetic peptide vaccine S3Pvac against Taenia solium cysticercosis in pigs in an endemic town of Mexico.

Taenia solium cysticercosis is a parasitic disease frequently affecting human health and the pig industry in many developing countries. A synthetic peptide vaccine (designated S3Pvac) against porcine cysticercosis has been developed previously as an aid to interrupt transmission and has been shown to be effective. The results of the present study support the effectiveness of the vaccine under endemic field conditions. However, given the time-frame of the vaccination trial, no changes in the local levels of transmission were detectable before and after vaccination using sentinel pigs. Thus, this investigation shows the limited usefulness of single vaccination as the sole means of interrupting Taenia solium transmission in an endemic region.

Human single chain Fv antibodies and a complementarity determining region-derived peptide binding to amyloid-beta 1-42.

A library of phage-displayed human single-chain Fv (scFv) antibodies was selected against the human amyloid-beta peptide (Abeta42). Two new anti-Abeta42 phage-displayed scFvs antibodies were obtained, and the sequences of their V(H) and Vkappa genes were analyzed. A synthetic peptide based on the sequence of Ig heavy chain (V(H)) complementarity-determining region (HCDR3) of the clone with the highest recognition signal was generated and determined to bind to Abeta42 in ELISA. Furthermore, we showed for the first time that an HCDR3-based peptide had neuroprotective potential against Abeta42 neurotoxicity in rat cultured hippocampal neurons. Our results suggest that not only scFvs recognizing Abeta42 but also synthetic peptides based on the V(H) CDR3 sequences of these antibodies may be novel potential candidates for small molecule-based Alzheimer's disease (AD) therapy.

[Effect of a postsynaptic alpha2-adrenoreceptor antagonist beditin on the rat resistance to seizure and Ca(+)-level in the brain tissue]. / Vliianie antagonista postsinapticheskikh alpha2-adrenoretseptorov beditina na ustoichivost' krys k sudorozhnym vozdeistviiam i soderzhanie Ca(2+) v tkani mozga.

In rats with a hemodynamic disorder caused by acute myocardial ischemia, preliminary administration of beditin in a dose of 25 mg/kg fully prevents a manifold increase in the level of 45Ca2+ in the cytosol of brain cells and leads to enhanced trapping and accumulation of labeled calcium in endoplasmic reticulum. With respect to calcium binding in endoplasmic reticulum, the action of beditin substantially differs from that of verapamil.

Amyloid-beta peptide-specific single chain Fv antibodies isolated from an immune phage display library.

A single-chain fragment variable (scFv) antibody library displayed on phage was constructed using spleen cells from mice immunized with human amyloid-beta peptide (Abeta42). This first anti-Abeta42 scFv immune antibody library was selected against human Abeta42. A number of positive clones were obtained, and sequences of VH and Vkappa genes were analyzed using ExPASy and BLAST computer tools. This analysis revealed that only two unique clones with identical VH and Vkappa complementarity determining region (CDR) (except HCDR2) and identical germline genes were selected, indicating that oligoclonal immune response was occurring in Abeta42-immunized mice. Abeta42-specific scFv antibodies selected from this first immune anti-Abeta42 phage antibody library may be an important tool for the development of therapeutic molecules for Alzheimer's disease (AD).

Identification of peptide sequences specific for serum antibodies from human papillomavirus-infected patients using phage display libraries.

Three random phage display peptide libraries were screened with sera from human papillomavirus (HPV)-infected patients to characterize the specificities of antibodies present in patients' sera and to identify molecules that correspond to or mimic natural epitopes; 141 phage clones were randomly selected in three rounds of bioselection and their binding properties were analyzed in ELISA using sera from 36 patients with confirmed HPV 16 infection and 24 healthy control female blood donors. Sixteen of 36 (44%) patients' sera reacted with at least 1 phage clone, and only 2 of 24 female donors' sera showed positive reaction with 1 of the selected clones. We conclude that the combination of various disease-specific epitopes generated by screening of phage display peptide libraries may potentially lead to a multicomponent diagnostic assay for the early detection of HPV infection and precancerous cervical lesions, making possible the prevention of one of the most common cancers in women.

Phage displayed biomolecules as preventive and therapeutic agents.

Phage display is a powerful technology for selecting and engineering peptides and proteins expressed on the surface of filamentous bacteriophage. The advantages of phage display technology over other research tools and its' great potential have been demonstrated by successful application of phage display in diverse fields of biomedical/clinical research. In this review we will describe some recent developments in phage display, including new expression vectors, display formats, bioselection strategies and applications in pharmaceutical biotechnology. We highlight some important applications of phage display to identify disease- and pathogen-specific biomolecules, making particular emphasis on development of phage display-derived preventive and therapeutic vaccines.

Synthetic peptide vaccine against Taenia solium pig cysticercosis: successful vaccination in a controlled field trial in rural Mexico.

Taenia solium cysticercosis seriously affects human health when localised in the central nervous system (CNS) and causes great economic loss in pig husbandry in rural areas of endemic countries. Increasing the resistance to the parasite in the obligatory host pig may help in curbing transmission. Three synthetic peptides based on protein sequences of the murine parasite Taenia crassiceps, which had previously been shown to induce protection in mice against homologous challenge, were tested as a vaccine against T. solium cysticercosis in pigs. Vaccinated and unvaccinated piglets (240 in all) were distributed in pairs among the peasants' households of two rural villages in Mexico in which 14% of the native pigs were cysticercotic. Ten to twelve months later, the effect of vaccination was evaluated at necropsy. Vaccination decreased the total number of T. solium cysticerci (98.7%) and reduced the prevalence (52.6%). The natural challenge conditions used in this field trial strengthen the likelihood of successful transmission control to both pig and human through a large-scale pig vaccination program. We believe this is a major contribution in anticysticercosis vaccine development as these rather simple yet protective peptides are potentially more cost-effective to produce and less variable in results than antigens that are more complex.

Intraspleen DNA inoculation elicits protective cellular immune responses.

DNA immunization or inoculation is a recent vaccination method that induces both humoral and cellular immune responses in a range of hosts. Independent of the route or site of vaccination, the transfer of antigen-presenting cells (APC) or antigens into lymphoid organs is necessary. The aim of this investigation was to test whether intraspleen (i.s.) DNA inoculation is capable of inducing a protective immune response. We immunized mice by a single i.s. injection of a DNA construct expressing the immunoglobulin (Ig) heavy-chain variable domain (VH) in which the complementarity-determining regions (CDR) had been replaced by a Taenia crassiceps T-cell epitope. In these mice, immune responses and protective effects elicited by the vaccine were measured. We have shown here for the first time that i.s. DNA inoculation can induce protective cellular immune responses and activate CD8(+) T cells. Also, Ig V(H) appeared to be the minimal delivery unit of "antigenized" Ig capable of inducing T-cell activation in a lymphoid organ. The strategy of introducing T-cell epitopes into the molecular context of the V(H) domain in combination with i.s. DNA immunization could have important implications and applications for human immunotherapy.

Two epitopes shared by Taenia crassiceps and Taenia solium confer protection against murine T. crassiceps cysticercosis along with a prominent T1 response.

Taenia crassiceps recombinant antigens KETc1 and KETc12 have been shown to induce high level of protection against experimental murine T. crassiceps cysticercosis, an experimental model successfully used to test candidate antigens for use in vaccination against porcine Taenia solium cysticercosis. Based on the deduced amino acid sequence, KETc1 and KETc12 were chemically synthesized in linear form. Immunization with KETc1 induced 66.7 to 100% protection against murine cysticercosis, and immunization with KETc12 induced 52.7 to 88.1% protection. The elicited immune response indicated that both peptides contain at least one B-cell epitope (as demonstrated by their ability to induce specific antibodies) and one T-cell epitope that strongly stimulated the proliferation of T cells primed with either the free peptide or total cysticercal T. crassiceps antigens. The high percentage of spleen cells expressing inflammatory cytokines points to the likelihood of a T1 response being involved in protection. The protective capacity of the peptides and their presence in all developmental stages of T. solium point to these two epitopes as strong candidates for inclusion in a polyepitopic synthetic vaccine against T. solium pig cysticercosis.

Identification of mimotopes of platelet autoantigens associated with autoimmune thrombocytopenic purpura.

GPIIb/IIIa, the human platelet glycoprotein complex, is the autoantigen most commonly recognized by autoantibodies in autoimmune thrombocytopenic purpura (AITP). Two murine monoclonal antibodies (mAbs), namely Y2/51 and 5B12, directed against gpIIIa and gpIIb/IIIa, respectively, and rabbit anti-human platelet polyclonal antibodies have been used to select AITP-related epitopes from a phage display peptide library expressing random dodecapeptides in the pIII coat protein of M13 phage. The selected phage clones were tested by ELISA for binding to rabbit anti-human platelet antibodies as well as to sera from AITP patients. Seven clones reacted strongly with rabbit anti-human platelet antibodies, and four clones reacted with sera from AITP patients. Some homology between peptide inserts sequences of selected clones and human platelet gpIIIa and gpIb were found.

Characterization of a novel human immunodeficiency virus type 1 neutralizable epitope within the immunodominant region of gp41.

Previously, we generated human monoclonal antibodies using peripheral blood mononuclear cells from an asymptomatic human immunodeficiency virus type 1 (HIV-1)-seropositive donor. One of these monoclonal antibodies (designated clone 3, CL3) recognized 10 amino acids (GCSGKLICTT) within the immunodominant region (cluster I) of the transmembrane envelope glycoprotein gp41 and neutralized infection of target cells with different laboratory isolates. Because the epitope recognized by CL3 has two cysteine residues that could potentially produce a disulfide loop in gp41, we analyzed binding of our monoclonal antibody to the cyclic and linear motif of the peptide sequence IWGCSGKLICTTAVP (residues 600-614). The CL3 antibody did not bind to the synthetic cyclic peptide but did recognize the linear form. Two polyclonal rabbit sera against both the linear and cyclic peptides were then generated. Both antisera bound to viral glycoproteins gp41 and gp160, but neither sera neutralized HIV-1 laboratory isolates. Using a set of alanine-substituted IWGCSGKLICTTAV peptides, we analyzed binding of polyclonal antisera and CL3. The profile of binding of polyclonal antisera to these peptides was different from that of CL3 to the same peptides. This suggests that CL3 recognized a unique neutralizable core epitope, which was not immunogenic in either the cyclic or the linear IWGCSGKLICTTAVP peptides used as immunogens in the rabbits.

Cysticercosis: towards the design of a diagnostic kit based on synthetic peptides.

Cysticercosis caused by Taenia solium is a very common disease in developing countries that seriously affects human health. Diagnosis can only be confirmed with the aid of computerized tomography or nuclear magnetic resonance (NMR) creating obvious difficulties for epidemiological studies. Reliable immunoassays employing cerebrospinal fluid (CSF) have been developed, based on the use of cysticercal antigens. However, the reliance on parasite material is restrictive. Herein, we report the advances in the design of a diagnostic kit based on immunodominant synthetic peptides, targeting four candidate epitopes KETc1, KETc12, 410 and 413 which were identified from three different clones (KETc1, 12 and 4) selected from a cDNA library of Taenia crassiceps. CSF antibodies against T. solium cysticercal antigens (TCA) as well as the four peptides were determined by enzyme-linked immunoabsorbent assays (ELISA) using two panels of CSF from patients with confirmed neurocysticercosis and other neurological diseases. In the first CSF panel which included patients with high level of antibodies against TCA, KETc12 exhibited almost the same sensitivity (87.5%) as TCA (93.7%) and 100% specificity. In the second panel of 110 CSF collected at random, two peptides (KETc1 and KETc12) exhibited sensitivities of 40 and 36% respectively, and were 100% specific.