Changes in the brain transcriptome after DNA Aß42 trimer immunization in a 3xTg-AD mouse model.

Alzheimer's disease (AD) has been associated with accumulation of amyloid beta (Aß) peptides in brain, and immunotherapy targeting Aß provides potential for AD prevention. We have used a DNA Aß42 trimer construct for immunization of 3xTg-AD mice and found previously significant reduction of amyloid and tau pathology due to the immunotherapy. We show here that DNA Aß42 immunized 3xTg-AD mice showed better performance in nest building activities and had a higher 24 months survival rate compared to the non-treated AD controls. The analysis of differently expressed genes in brains from 24 months old mice showed significant increases transcript levels between non-immunized AD mice and wild-type controls for genes involved in microglia and astrocyte function, cytokine and inflammatory signaling, apoptosis, the innate and adaptive immune response and are consistent with an inflammatory phenotype in AD. Most of these upregulated genes were downregulated in the DNA Aß42 immunized 3xTg-AD mice due to the vaccine. Transcript numbers for the immediate early genes, Arc, Bdnf, Homer1, Egr1 and cfos, involved in neuronal and neurotransmission pathways which were much lower in the non-immunized 3xTg-AD mice, were restored to wild-type mouse brain levels in DNA Aß42 immunized 3xTg-AD mice indicating positive effects of DNA Aß42 immunotherapy on synapse stability and plasticity. The immune response after immunization is complex, but the multitude of changes after DNA Aß42 immunization shows that this response moves beyond the amyloid hypothesis and into direction of disease prevention.

DNA Aß42 immunization via needle-less Jet injection in mice and rabbits as potential immunotherapy for Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia found in the elderly and disease progression is associated with accumulation of Amyloid beta 1-42 (Aß42) in brain. An immune-mediated approach as a preventive intervention to reduce amyloid plaques without causing brain inflammation is highly desirable for future clinical use. Genetic immunization, in which the immunizing agent is DNA encoding Aß42, has great potential because the immune response to DNA delivered into the skin is generally non-inflammatory, and thus differs quantitatively and qualitatively from immune responses elicited by peptides, which are inflammatory with production of IFNγ and IL-17 cytokines by activated T cells. DNA immunization has historically been proven difficult to apply to larger mammals. A potential barrier to use DNA immunization in large mammals is the method for delivery of the DNA antigen. We tested jet injection in mice and rabbits and found good antibody production and safe immune responses (no inflammatory cytokines). We found significant reduction of amyloid plaques and Aß peptides in brains of the DNA Aß42 immunized 3xTg-AD mouse model. This study was designed to optimize DNA delivery for possible testing of the DNA Aß42 vaccine for AD prevention in a clinical trial.

Glial cell transcriptome analyses in 3xTg-AD mice: Effects of aging, disease progression, and anti-Aß immunotherapy.

Background: To investigate how changes in expression of glial genes relate to a progression of Alzheimer's disease (AD) pathology, and how anti-Aß immunotherapy impact these changes, we conducted a transcriptomic analysis for brains from cohorts of 2-, 10-, and 20 month old 3xTg-AD mice, and a cross-sectional study in groups of 20 month-old mice treated with active DNA Aß42 immunization, passive immunotherapy, untreated, and wild-type (wt) controls. Methods: Twenty-four Formalin-Fixed Paraffin-Embedded (FFPE) mouse brain sections were used for the gene expression analyses (nanostring). Adjacent sections from these and additional mouse brains were stained for microglia using antibodies detecting IbaI and Gal3. For a semi-quantitative analysis of increased tau and amyloid pathology with aging and disease progression, a comparison of ELISA results from brains of 12 and 20 months old 3xTg-AD mice were shown. Results: Based on the different comparisons of transcript numbers found the 3xTg-AD age groups with the senescent 20 months old wt control mouse brains, and the 20 months old 3xTg-AD mouse brains with the 20 months old wt control mouse brains, genes were assigned as upregulated due to aging, or due to disease progression, or due to both. The immunohistochemistry of microglia markers revealed that Gal3 might be an important marker for phagocytosing microglia around amyloid plaques. The comparison of the two anti-Aß immunotherapy approaches showed a differential downregulation of inflammatory glial genes. Conclusion: These results are relevant for future clinical trials using active anti-amyloid immunotherapy.

CSF-Derived CD4+ T-Cell Diversity Is Reduced in Patients With Alzheimer Clinical Syndrome.

BACKGROUND AND OBJECTIVES: Patients with Alzheimer dementia display evidence of amyloid-related neurodegeneration. Our focus was to determine whether such patients also display evidence of a disease-targeting adaptive immune response mediated by CD4+ T cells. To test this hypothesis, we evaluated the CSF immune profiles of patients with Alzheimer clinical syndrome (ACS), who display clinically defined dementia. METHODS: Innate and adaptive immune profiles of patients with ACS were measured using multicolor flow cytometry. CSF-derived CD4+ and CD8+ T-cell receptor repertoire genetics were measured using next-generation sequencing. Brain-specific autoantibody signatures of CSF-derived antibody pools were measured using array technology or ELISA. CSF from similar-age healthy controls (HCs) was used as a comparator cohort. RESULTS: Innate cells were expanded in the CSF of patients with ACS in comparison to HCs, and innate cell expansion increased with age in the patients with ACS, but not HCs. Despite innate cell expansion in the CSF, the frequency of total CD4+ T cells reduced with age in the patients with ACS. T-cell receptor repertoire genetics indicated that T-cell clonal expansion is enhanced, and diversity is reduced in the patients with ACS compared with similar-age HCs. DISCUSSION: Examination of CSF indicates that CD4+ T cell-mediated adaptive immune responses are altered in patients with ACS. Understanding the underlying mechanisms affecting adaptive immunity will help move us toward the goal of slowing cognitive decline.

Lymph node-derived donor encephalitogenic CD4+ T cells in C57BL/6 mice adoptive transfer experimental autoimmune encephalomyelitis highly express GM-CSF and T-bet.

Experimental autoimmune encephalomyelitis (EAE) is a relevant animal model for the human demyelinating inflammatory disorder of the central nervous system (CNS), multiple sclerosis (MS). Induction of EAE by adoptive transfer allows studying the role of the donor T lymphocyte in disease pathogenesis. It has been challenging to reliably induce adoptive transfer EAE in C57BL/6 (H-2b) mice. The goal of this study was to develop a reproducible and high yield protocol for adoptive transfer EAE in C57BL/6 mice. A step-wise experimental approach permitted us to develop a protocol that resulted in a consistent relatively high disease incidence of ~70% in recipient mice. Donor mice were immunized with myelin oligodendrocyte glycoprotein (MOG)p35-55 in complete Freund's adjuvant (CFA) followed by pertussis toxin (PT). Only lymph node cells (LNC) isolated at day 12 post immunization, and restimulated in vitro for 72 hours with 10 µg/mL of MOGp35-55 and 0.5 ng/mL of interleukin-12 (IL-12) were able to transfer disease. The ability of LNC to transfer disease was associated with the presence of inflammatory infiltrates in the CNS at day 12. Interferon gamma (IFNγ) was produced at comparable levels in cell cultures prepared from mice at both day 6 and day 12 post immunization. By contrast, there was a trend towards a negative association between IL-17 and disease susceptibility in our EAE model. The amount of GM-CSF secreted was significantly increased in the culture supernatants from cells collected at day 12 post immunization versus those collected at day 6 post-immunization. Activated CD4+ T cells present in the day 12 LNC cultures maintained expression of the transcription factor T-bet, which has been shown to regulate the expression of the IL-23 receptor. Also, there was an increased prevalence of MOGp35-55-specific CD4+ T cells in day 12 LNC after in vitro re-stimulation. In summary, encephalitogenic LNC that adoptively transfer EAE in C57BL/6 mice were not characterized by a single biomarker in our study, but by a composite of inflammatory markers. Our data further suggest that GM-CSF expression by CD4+ T cells regulated by IL-23 contributes to their encephalitogenicity in our EAE model.

DNA immunization against amyloid beta 42 has high potential as safe therapy for Alzheimer's disease as it diminishes antigen-specific Th1 and Th17 cell proliferation.

The pathogenesis of Alzheimer's disease (AD) has been strongly associated with the accumulation of amyloid beta (Aß) peptides in brain, and immunotherapy targeting Aß provides potential for AD prevention. A clinical trial in which AD patients were immunized with Aß42 peptide was stopped when 6% of participants showed meningoencephalitis, apparently due to an inflammatory Th1 immune response. Previously, we and other have shown that Aß42 DNA vaccination via gene gun generates a Th2 cellular immune response, which was shown by analyses of the respective antibody isotype profiles. We also determined that in vitro T cell proliferation in response to Aß42 peptide re-stimulation was absent in DNA Aß42 trimer-immunized mice when compared to Aß42 peptide-immunized mice. To further characterize this observation prospectively and longitudinally, we analyzed the immune response in wild-type mice after vaccination with Aß42 trimer DNA and Aß42 peptide with Quil A adjuvant. Wild-type mice were immunized with short-term (1-3× vaccinations) or long-term (6× vacinations) immunization strategies. Antibody titers and isotype profiles of the Aß42 specific antibodies, as well as cytokine profiles and cell proliferation studies from this longitudinal study were determined. Sufficient antibody titers to effectively reduce Aß42, but an absent T cell proliferative response and no IFNγ or IL-17 secretion after Aß42 DNA trimer immunization minimizes the risk of inflammatory activities of the immune system towards the self antigen Aß42 in brain. Therefore, Aß42 DNA trimer immunization has a high probability to be effective and safe to treat patients with early AD.

DNA beta-amyloid(1-42) trimer immunization for Alzheimer disease in a wild-type mouse model.

CONTEXT: DNA beta-amyloid(1-42) (Abeta42) trimer immunization was developed to produce specific T helper 2 cell (T(H)2)-type antibodies to provide an effective and safe therapy for Alzheimer disease (AD) by reducing elevated levels of Abeta42 peptide that occur in the brain of patients with AD. OBJECTIVE: To compare the immune response in wild-type mice after immunization with DNA Abeta42 trimer and Abeta42 peptide. DESIGN AND INTERVENTION: Wild-type mice received either 4 microg of DNA Abeta42 trimer immunization administered with gene gun (n = 8) or intraperitoneal injection of 100 microg of human Abeta42 peptide with the adjuvant Quil A (n = 8). Titers, epitope mapping, and isotypes of the Abeta42-specific antibodies were analyzed. MAIN OUTCOME MEASURES: Antibody titers, mapping of binding sites (epitopes), isotype profiles of the Abeta42-specific antibodies, and T-cell activation. RESULTS: DNA Abeta42 trimer immunization resulted in antibody titers with a mean of 15 microg per milliliter of plasma. The isotype profile of the antibodies differed markedly. A predominant IgG1 antibody response was found in the DNA-immunized mice, indicating a T(H)2 type of immune response (IgG1/IgG2a ratio of 10). The peptide-immunized mice showed a mixed T(H)1/T(H)2 immune response (IgG1/IgG2a ratio of 1) (P < .001). No increased T-cell proliferation was observed in the DNA-immunized mice (P = .03). CONCLUSION: In this preliminary study in a wild-type mouse model, DNA Abeta42 trimer immunization protocol produced a T(H)2 immune response and appeared to have low potential to cause an inflammatory T-cell response.

Active full-length DNA Aß42 immunization in 3xTg-AD mice reduces not only amyloid deposition but also tau pathology.

BACKGROUND: Alzheimer's disease (AD) is the most well-known and most common type of age-related dementia. Amyloid deposition and hyperphosphorylation of tau protein are both pathological hallmarks of AD. Using a triple-transgenic mouse model (3xTg-AD) that develops plaques and tangles in the brain similar to human AD, we provide evidence that active full-length DNA amyloid-ß peptide 1-42 (Aß42) trimer immunization leads to reduction of both amyloid and tau aggregation and accumulation. METHODS: Immune responses were monitored by enzyme-linked immunosorbent assay (ELISA) (antibody production) and enzyme-linked immunospot (cellular activation, cytokine production). Brains from 20-month-old 3x Tg-AD mice that had received DNA Aß42 immunotherapy were compared with brains from age- and gender-matched transgenic Aß42 peptide-immunized and control mice by histology, Western blot analysis, and ELISA. Protein kinase activation and kinase levels were studied in Western blots from mouse hemibrain lysates. RESULTS: Quantitative ELISA showed a 40% reduction of Aß42 peptide and a 25-50% reduction of total tau and different phosphorylated tau molecules in the DNA Aß42 trimer-immunized 3xTg-AD mice compared with nonimmunized 3xTg-AD control animals. Plaque and Aß peptide reductions in the brain were due to the anti-Aß antibodies generated following the immunizations. Reductions of tau were likely due to indirect actions such as less Aß in the brain resulting in less tau kinase activation. CONCLUSIONS: The significance of these findings is that DNA Aß42 trimer immunotherapy targets two major pathologies in AD-amyloid plaques and neurofibrillary tangles-in one vaccine without inducing inflammatory T-cell responses, which carry the danger of autoimmune inflammation, as found in a clinical trial using active Aß42 peptide immunization in patients with AD (AN1792).

Intradermal active full-length DNA Aß42 immunization via electroporation leads to high anti-Aß antibody levels in wild-type mice.

Aß immunotherapies with anti-Aß antibody responses have high potential as possible prevention treatment for Alzheimer's disease. We have previously shown that active DNA Aß1-42 immunization via gene gun delivery led to a non-inflammatory immune response resulting in decreased Aß levels in brains of an immunized AD mouse model. To make DNA vaccination more applicable for clinical use, we used here intradermal electroporation. With fine tuning of the electropulse parameters, high antibody levels and low levels of inflammatory cytokines in the cellular immunoassays were observed. Full-length DNA Aß1-42 immunization delivered via electroporation has potential to be used in the clinical setting.

Cerebrospinal fluid B cells from multiple sclerosis patients are subject to normal germinal center selection.

Previous findings from our laboratory demonstrated that some clonally expanded cerebrospinal fluid (CSF) B cells from MS patients exhibit diminished mutation targeting patterns in comparison to typical B cells selected in the context of germinal centers (GCs). In order to determine whether the overall CSF B cell repertoires adhered to mutation patterns typical of GC-selected B cells, we analyzed the immunoglobulin repertoires from CSF B cells of 8 MS patients for mutation characteristics typical of GC-derived B cells. Mutation targeting was preserved. Thus, clonal expansion of some CSF B cells may occur independently of GC, but the CSF B cell pool is governed by typical GC selection. Interestingly, the heavy chain CDR3's of CSF B cells from MS patients had a net acidic charge, similar to GC-derived B cells, but a tendency towards longer CDR3's, consistent with autoreactive B cells. How these findings may support current hypotheses regarding the origin of CSF B cells is discussed.

Antigen specificity of clonally expanded and receptor edited cerebrospinal fluid B cells from patients with relapsing remitting MS.

We re-engineered the immunoglobulin rearrangements from clonally expanded CSF B cells of three Multiple Sclerosis patients as Fab fragments, and used three methods to test for their antigen (Ag) specificity. Nine out of ten Fab fragments were reactive to Myelin Basic Protein (MBP). The one Fab that did not react to MBP was a product of receptor editing. Two of the nine MBP reactive Fabs were also reactive to GFAP and CNPase, indicating that these clones were polyreactive. Targeting the mechanisms that allow these self-reactive B cells to reside in the CSF of MS patients may prove to be a potent immunotherapeutic strategy.

Evaluation of a DNA Aß42 vaccine in adult rhesus monkeys (Macaca mulatta): antibody kinetics and immune profile after intradermal immunization with full-length DNA Aß42 trimer.

BACKGROUND: Aggregated amyloid-ß peptide 1-42 (Aß42), derived from the cellular amyloid precursor protein, is one of the pathological hallmarks of Alzheimer's disease (AD). Although active immunization against Aß42 peptide was successful in AD mouse models and led to removal of plaques and improved memory, a similar clinical trial in humans (Aß42 peptide immunization with QS-21 adjuvant) was stopped in phase II, when 6% of the treated patients developed encephalitis. Currently ongoing passive immunizations with the injection of preformed monoclonal antibodies against different epitopes within the Aß1-42 peptide, which do not lead to activation of the immune system, have shown some effects in slowing AD pathology. Active DNA Aß42 immunizations administered with the gene gun into the skin are noninflammatory because they activate a different T-cell population (Th2) with different cytokine responses eliciting a different humoral immune response. We present our findings in rhesus macaques that underwent the DNA Aß42 immunization via gene gun delivery into the skin. METHODS: Six rhesus monkeys received two different doses of a DNA Aß42 trimer vaccine. The humoral immune response was analyzed from blood throughout the study, and cellular immune responses were determined in peripheral blood mononuclear cells (PBMCs) after three and six immunizations. RESULTS: DNA Aß42 trimer immunization led to high titer antibody responses in the nonhuman primate (NHP) model. Antibodies generated in the rhesus monkeys following DNA Aß42 immunization detected amyloid plaques consisting of human Aß42 peptide in the brain of the triple-transgenic AD mouse model. T-cell responses showed no interferon (IFN)-γ- and interleukin (IL)-17-producing cells from PBMCs in Enzyme-Linked ImmunoSpot assays after three immunization time points. At six immunization time points, IFN-γ- and IL-17-producing cells were found in immunized animals as well as in control animals and were thus considered nonspecific and not due to the immunization regimen. IFN-γ and IL-17 secretion in response to Aß42 peptide restimulation became undetectable after a 3-month rest period. CONCLUSIONS: Intradermal DNA Aß42 immunization delivered with the gene gun produces a high antibody response in NHPs and is highly likely to be effective and safe in a clinical AD prevention trial in patients.

Evaluation of a DNA Aß42 Vaccine in Aged NZW Rabbits: Antibody Kinetics and Immune Profile after Intradermal Immunization with Full-Length DNA Aß42 Trimer.

A pathological hallmark of Alzheimer's disease (AD) are amyloid plaques in the brain consisting of aggregated amyloid-ß 42 peptide (Aß42) derived from cellular amyloid-ß protein precursor (AßPP). Based on successful experiments in mouse AD models, active immunization with Aß42 peptide and passive immunizations with anti-Aß42 antibodies were started in clinical trials. Active Aß42 peptide immunization in humans had led to an inflammatory autoimmune response, and the trial was stopped. Passive immunizations had shown some effects in slowing AD pathology. Active DNA Aß42 immunizations administered with the gene gun into the skin elicits a different immune response and is non-inflammatory. While in rodents, good responses had been found for this type of immunization, positive results in larger mammals are missing. We present here results from sixteen New Zealand White Rabbits, which underwent intradermal DNA Aß42 immunization via gene gun. The humoral immune response was analyzed from blood throughout the study, and cellular immune responses were determined from spleens at the end of the study. A good anti-Aß antibody response was found in the rabbit model. The T cell response after re-stimulation in cell culture showed no IFNγ producing cells when ELISPOT assays were analyzed from PBMC, but low numbers of IFNγ and IL-17 producing cells were found in ELISPOTS from spleens (both 5 immunizations). Brains from immunized rabbits showed no signs of encephalitis. Based on these results, DNA Aß42 immunization is highly likely to be safe and effective to test in a possible clinical AD prevention trial in patients.

Laquinimod has no effects on brain volume or cellular CNS composition in the F1 3xTg-AD/C3H mouse model of Alzheimer's disease.

BACKGROUND: Laquinimod is an anti-inflammatory agent with good central nervous system (CNS) bioavailability, and neuroprotective and myelorestorative properties. A clinical trial in patients with multiple sclerosis demonstrated that laquinimod significantly reduced loss of brain volume. The cellular substrate or molecular events underlying that treatment effect are unknown. In this study, we aimed to explore laquinimod's potential effects on brain volume, animal behavior, cellular numbers and composition of CNS-intrinsic cells and mononuclear cells within the CNS, amyloid beta (Aß) accumulation and tau phosphorylation in the F1 3xTg-AD/C3H mouse model of Alzheimer's disease. METHODS: Utilizing a dose response study design, four months old F1 3xTg-AD/C3H mice were treated for 10months between ages 4 and 14months with laquinimod (5, 10, or 25mg/kg), or PBS administered by oral gavage. Brain volumes were measured in a 7 Tesla magnetic resonance imager (MRI) at ages 4 and 14months. Behavioral testing included locomotor and rearing activity and the Morris water maze task. Cell numbers and immunophenotypes were assessed by multiparameter flow cytometry. Aß deposition and tau phosphorylation were determined by immunohistochemistry. RESULTS: In the F1 3xTg-AD/C3H animal model of AD, there was no detectable reduction of brain volume over a period of 10months of treatment, as there was not brain atrophy in any of the placebo or treatment groups. Laquinimod had no detectable effects on most neurobehavioral outcomes. The number or composition of CNS intrinsic cells and mononuclear subsets isolated from the CNS were not altered by laquinimod. CONCLUSION: This is the first demonstration that there are no age-associated brain volume changes in the F1 3xTg-AD/C3H mouse model of Alzheimer's disease. Consequently, laquinimod had no effect on that outcome of this study. Most secondary outcomes on the effects of laquinimod on behavior and the number and composition of CNS-intrinsic cells and mononuclear cells within the CNS were also negative.

Genomics of Alzheimer Disease: A Review.

IMPORTANCE: To provide a comprehensive review of knowledge of the genomics of Alzheimer disease (AD) and DNA amyloid ß 42 (Aß42) vaccination as a potential therapy. OBSERVATIONS: Genotype-phenotype correlations of AD are presented to provide a comprehensive appreciation of the spectrum of disease causation. Alzheimer disease is caused in part by the overproduction and lack of clearance of Aß protein. Oligomer Aß, the most toxic species of Aß, causes direct injury to neurons, accompanied by enhanced neuroinflammation, astrocytosis and gliosis, and eventually neuronal loss. The strongest genetic evidence supporting this hypothesis derives from mutations in the amyloid precursor protein (APP) gene. A detrimental APP mutation at the ß-secretase cleavage site linked to early-onset AD found in a Swedish pedigree enhances Aß production, in contrast to a beneficial mutation 2 residues away in APP that reduces Aß production and protects against the onset of sporadic AD. A number of common variants associated with late-onset AD have been identified including apolipoprotein E, BIN1, ABC7, PICALM, MS4A4E/MS4A6A, CD2Ap, CD33, EPHA1, CLU, CR1, and SORL1. One or 2 copies of the apolipoprotein E ε4 allele are a major risk factor for late-onset AD. With DNA Aß42 vaccination, a Th2-type noninflammatory immune response was achieved with a downregulation of Aß42-specific effector (Th1, Th17, and Th2) cell responses at later immunization times. DNA Aß42 vaccination upregulated T regulator cells (CD4+, CD25+, and FoxP3+) and its cytokine interleukin 10, resulting in downregulation of T effectors. CONCLUSIONS AND RELEVANCE: Mutations in APP and PS-1 and PS-2 genes that are associated with early-onset, autosomal, dominantly inherited AD, in addition to the at-risk gene polymorphisms responsible for late-onset AD, all indicate a direct and early role of Aß in the pathogenesis of AD. A translational result of genomic research has been Aß-reducing therapies including DNA Aß42 vaccination as a promising approach to delay or prevent this disease.

A Single Amino Acid Substitution Prevents Recognition of a Dominant Human Aquaporin-4 Determinant in the Context of HLA-DRB1*03:01 by a Murine TCR.

BACKGROUND: Aquaporin 4 (AQP4) is considered a putative autoantigen in patients with Neuromyelitis optica (NMO), an autoinflammatory disorder of the central nervous system (CNS). HLA haplotype analyses of patients with NMO suggest a positive association with HLA-DRB1* 03:01. We previously showed that the human (h) AQP4 peptide 281-300 is the dominant immunogenic determinant of hAQP4 in the context of HLA-DRB1*03:01. This immunogenic peptide stimulates a strong Th1 and Th17 immune response. AQP4281-300-specific encephalitogenic CD4+ T cells should initiate CNS inflammation that results in a clinical phenotype in HLA-DRB1*03:01 transgenic mice. METHODS: Controlled study with humanized experimental animals. HLA-DRB1*03:01 transgenic mice were immunized with hAQP4281-300, or whole-length hAQP4 protein emulsified in complete Freund's adjuvant. Humoral immune responses to both antigens were assessed longitudinally. In vivo T cell frequencies were assessed by tetramer staining. Mice were followed clinically, and the anterior visual pathway was tested by pupillometry. CNS tissue was examined histologically post-mortem. Flow cytometry was utilized for MHC binding assays and to immunophenotype T cells, and T cell frequencies were determined by ELISpot assay. RESULTS: Immunization with hAQP4281-300 resulted in an in vivo expansion of antigen-specific CD4+ T cells, and an immunoglobulin isotype switch. HLA-DRB1*03:01 TG mice actively immunized with hAQP4281-300, or with whole-length hAQP4 protein were resistant to developing a neurological disease that resembles NMO. Experimental mice show no histological evidence of CNS inflammation, nor change in pupillary responses. Subsequent analysis reveals that a single amino acid substitution from aspartic acid in hAQP4 to glutamic acid in murine (m)AQP4 at position 290 prevents the recognition of hAQP4281-300 by the murine T cell receptor (TCR). CONCLUSION: Induction of a CNS inflammatory autoimmune disorder by active immunization of HLA-DRB1*03:01 TG mice with human hAQP4281-300 will be complex due to a single amino acid substitution. The pathogenic role of T cells in this disorder remains critical despite these observations.

Co-stimulation with TNF receptor superfamily 4/25 antibodies enhances in-vivo expansion of CD4+CD25+Foxp3+ T cells (Tregs) in a mouse study for active DNA Aß42 immunotherapy.

The study was designed to test DNA Aß42 immunization in mice as alternative approach for possible active immunotherapy in Alzheimer patients. As results, we found polarized Th2 immune responses, efficient Aß42 antibody levels, and disappearance of antigen specific T cells. In-vivo TNFRSF4/25 antibody co-stimulation enhanced Aß42 specific T cell responses with initial Th2 expansion and subsequent development of Aß42 specific CD4+CD25+Foxp3+ cells. It showed that Th2 biased responses due to gene gun immunizations propagate the development of regulatory T cells. In conclusion, full-length DNA Aß42 immunization into skin results in a regulatory response with minimal risk of inflammation and autoimmunity.

A noninflammatory immune response in aged DNA Aß42-immunized mice supports its safety for possible use as immunotherapy in AD patients.

Aging in the immune system results in tendency to proinflammatory responses. Intradermal DNA immunization showed Th2 polarized noninflammatory immune responses. We tested here 18-month-old mice which were immunized with Aß42 peptide, DNA Aß42 trimer, or 2 different prime boost protocols identical to previous experiments. High Aß42 antibody levels were found in aged mice which had received peptide immunizations (900 µg/mL plasma), and in mice which had received peptide prime and DNA boost immunizations (500 µg/mL), compared with antibodies in DNA Aß42 immunized mice with 50 µg/mL. Although we found T-cell proliferation and inflammatory cytokines in mice which had received peptide or prime boost immunization, these were not found in DNA-immunized mice. The results are concordant with proinflammatory responses because of immunosenescence and contraindicate the use of Aß42 peptide immunizations or prime boost immunization protocols for the use in elderly Alzheimer's disease patients. DNA Aß42 immunization only on the other hand does lead to effective levels of antibodies without inflammatory cytokine or T-cell responses in the aged animal model tested.

Advances in the development of vaccines for Alzheimer's disease.

One of the challenges of our society is to find a treatment or cure for Alzheimer's disease (AD). AD is the leading form of age-related dementia and with the increase of life expectancy worldwide, the social and economic burden from this disease will increase dramatically. It is a progressive and, in regard to clinical scores, a highly variable disease. AD pathogenesis has been associated with the accumulation, aggregation, and deposition of amyloid beta (Abeta) peptides in the brain. Hallmarks of AD are the amyloid plaques consisting of fibrillar Abeta and neurofibrillary tangles which are intracellular fibrils of hyperphosphorylated tau protein that develop later in this disease. The amyloid cascade hypothesis postulates that Abeta deposition is an initial event in the multifactorial pathogenesis and Abeta deposition may precede AD symptoms in some patients by at least 20 years. Amyloid beta therapy with active and passive immunizations against Abeta has a high possibility to be effective in removing Abeta from brain and might thus prevent the downstream pathology. Since 2000 a number of clinical trials for AD immunotherapy have started, have failed, and are continuing to be pursued. This article will review these clinical trials and ongoing research in this regard.