Anahit Ghochikyan

Prototype Alzheimer’s Disease Vaccine Using the Immunodominant B Cell Epitope from β-Amyloid and Promiscuous T Cell Epitope Pan HLA DR-Binding Peptide

Immunization of amyloid precursor protein transgenic mice with fibrillar β-amyloid (Aβ) prevents Alzheimer’s disease (AD)-like neuropathology. The first immunotherapy clinical trial used fibrillar Aβ, containing the B and T cell self epitopes of Aβ, as the immunogen formulated with QS21 as the adjuvant in the vaccine. Unfortunately, the clinical trial was halted during the phase II stage when 6% of the participants developed meningoencephalitis. The cause of the meningoencephalitis in the patients that received the vaccine has not been definitively determined; however, analysis of two case reports from the AN-1792 vaccine trial suggest that the meningoencephalitis may have been caused by a T cell-mediated autoimmune response, whereas production of anti-Aβ Abs may have been therapeutic to the AD patients. Therefore, to reduce the risk of an adverse T cell-mediated immune response to Aβ immunotherapy we have designed a prototype epitope vaccine that contains the immunodominant B cell epitope of Aβ in tandem with the synthetic universal Th cell pan HLA DR epitope, pan HLA DR-binding peptide (PADRE). Importantly, the PADRE-Aβ1–15 sequence lacks the T cell epitope of Aβ. Immunization of BALB/c mice with the PADRE-Aβ1–15 epitope vaccine produced high titers of anti-Aβ Abs. Splenocytes from immunized mice showed robust T cell stimulation in response to peptides containing PADRE. However, splenocytes from immunized mice were not reactivated by the Aβ peptide. New preclinical trials in amyloid precursor protein transgenic mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse events that occurred in the first clinical trial.

Reducing AD-Like Pathology in 3xTg-AD Mouse Model by DNA Epitope Vaccine — A Novel Immunotherapeutic Strategy

Background The development of a safe and effective AD vaccine requires a delicate balance between providing an adequate anti-Aβ antibody response sufficient to provide therapeutic benefit, while eliminating an adverse T cell-mediated proinflammatory autoimmune response. To achieve this goal we have designed a prototype chemokine-based DNA epitope vaccine expressing a fusion protein that consists of 3 copies of the self-B cell epitope of Aβ42 (Aβ1–11) , a non-self T helper cell epitope (PADRE), and macrophage-derived chemokine (MDC/CCL22) as a molecular adjuvant to promote a strong anti-inflammatory Th2 phenotype. Methods and Findings We generated pMDC-3Aβ1–11-PADRE construct and immunized 3xTg-AD mouse model starting at age of 3–4 months old. We demonstrated that prophylactic immunizations with the DNA epitope vaccine generated a robust Th2 immune response that induced high titers of anti-Aβ antibody, which in turn inhibited accumulation of Aβ pathology in the brains of older mice. Importantly, vaccination reduced glial activation and prevented the development of behavioral deficits in aged animals without increasing the incidence of microhemorrhages. Conclusions Data from this transitional pre-clinical study suggest that our DNA epitope vaccine could be used as a safe and effective strategy for AD therapy. Future safety and immunology studies in large animals with the goal to achieve effective humoral immunity without adverse effects should help to translate this study to human clinical trials.

Anti-Aβ1–11 Antibody Binds to Different β-Amyloid Species, Inhibits Fibril Formation, and Disaggregates Preformed Fibrils but Not the Most Toxic Oligomers

Different strategies proposed as therapy for Alzheimer disease (AD) have aimed to reduce the level of toxic forms of Aβ peptide in the brain. Here, we directly analyze the therapeutic utility of the polyclonal anti-Aβ1–11 antibody induced in 3xTg-AD mice vaccinated with the second generation prototype epitope vaccine. Substoichiometric concentrations of purified anti-Aβ1–11 antibody prevented aggregation of Aβ42 and induced disaggregation of preformed Aβ42 fibrils down to nonfilamentous and nontoxic species. Anti-Aβ1–11 antibody delayed Aβ42 oligomer formation but ultimately appeared to stabilize nonfibrillar conformations, including oligomer-like assemblies. The reduced oligomer-mediated cytotoxicity observed upon preincubation of Aβ oligomers with the anti-Aβ1–11 antibody in the absence of oligomer disaggregation suggests a possible oligomer rearrangement in the presence of the antibody. These in vitro observations suggest that preventive vaccination may protect from AD or may delay the onset of the disease, whereas therapeutic vaccination cannot disrupt the toxic oligomers and may only minimally alleviate preexisting AD pathology.

Alzheimer's Disease Peptide Epitope Vaccine Reduces Insoluble But Not Soluble/Oligomeric Aβ Species in Amyloid Precursor Protein Transgenic Mice

Active vaccination of elderly Alzheimers disease (AD) patients with fibrillar amyloid-β peptide (Aβ42), even in the presence of a potent Th1 adjuvant, induced generally low titers of antibodies in a small fraction (∼20% responders) of those that received the AN-1792 vaccine. To improve the immunogenicity and reduce the likelihood of inducing adverse autoreactive T-cells specific for Aβ42, we previously tested in wild-type mice an alternative approach for active immunization: an epitope vaccine that selectively initiate B cell responses toward an immunogenic self-epitope of Aβ in the absence of anti-Aβ T cell responses. Here, we describe a second generation epitope vaccine composed of two copies of Aβ1–11 fused with the promiscuous nonself T cell epitope, PADRE (pan human leukocyte antigen DR-binding peptide) that completely eliminates the autoreactive T cell responses and induces humoral immune responses in amyloid precursor protein transgenic 2576 mice with pre-existing AD-like pathology. Based on the titers of anti-Aβ1–11 antibody experimental mice were divided into low, moderate and high responders, and for the first time we report a positive correlation between the concentration of anti-Aβ1–11 antibody and a reduction of insoluble, cerebral Aβ plaques. The reduction of insoluble Aβ deposition was not associated with adverse events, such as CNS T cell or macrophage infiltration or microhemorrhages. Surprisingly, vaccination did not alter the levels of soluble Aβ. Alternatively, early protective immunization before substantial neuropathology, neuronal loss and cognitive deficits have become firmly established may be more beneficial and safer for potential patients, especially if they can be identified in a preclinical stage by the development of antecedent biomarkers of AD.

Immunogenicity, Efficacy, Safety, and Mechanism of Action of Epitope Vaccine (Lu AF20513) for Alzheimer’s Disease: Prelude to a Clinical Trial

The Alzheimers disease (AD) process is understood to involve the accumulation of amyloid plaques and tau tangles in the brain. However, attempts at targeting the main culprits, neurotoxic Aβ peptides, have thus far proven unsuccessful for improving cognitive function. Recent clinical trials with passively administrated anti-Aβ antibodies failed to slow cognitive decline in mild to moderate AD patients, but suggest that an immunotherapeutic approach could be effective in patients with mild AD. Using an AD mouse model (Tg2576), we tested the immunogenicity (cellular and humoral immune responses) and efficacy (AD-like pathology) of clinical grade Lu AF20513 vaccine. We found that Lu AF20513 induces robust “non-self” T-cell responses and the production of anti-Aβ antibodies that reduce AD-like pathology in the brains of Tg2576 mice without inducing microglial activation and enhancing astrocytosis or cerebral amyloid angiopathy. A single immunization with Lu AF20513 induced strong humoral immunity in mice with preexisting memory T-helper cells. In addition, Lu AF20513 induced strong humoral responses in guinea pigs and monkeys. These data support the translation of Lu AF20513 to the clinical setting with the aims of: (1) inducing therapeutically potent anti-Aβ antibody responses in patients with mild AD, particularly if they have memory T-helper cells generated after immunizations with conventional tetanus toxoid vaccine, and (2) preventing pathological autoreactive T-cell responses.

Generation and characterization of the humoral immune response to DNA immunization with a chimeric β‐amyloid‐interleukin‐4 minigene

Active immunization with fibrillar β–amyloid peptide (Aβ42) as well as passive transfer of anti‐Aβ antibodies significantly reduces Aβ plaque deposition, neuritic dystrophy, and astrogliosis in the brain of mutant amyloid precursor protein (APP)‐transgenic mice. Although the mechanism(s) of clearance of Aβ from the brain following active or passive immunization remains to be determined, it is clear that anti‐Aβ antibodies are critical for clearance. DNA immunization provides an attractive alternative to direct peptide and adjuvant approaches for inducing a humoral response to Aβ. We constructed a DNA minigene with Aβ fused to mouse interleukin‐4 (pAβ42‐IL‐4) as a molecular adjuvant to generate anti‐Aβ antibodies and enhance the Th2‐type of immune responses. Gene gun immunizations induced primarily IgG1 and IgG2b anti‐Aβ antibodies. Fine epitope analysis with overlapping peptides of the Aβ42 sequence identified the 1–15 region as a dominant B cell epitope. The DNA minigene‐induced anti‐Aβ antibodies bound to Aβ plaques in brain tissue from an Alzheimers disease patient demonstrating functional activity of the antibodies and the potential for therapeutic efficacy.

Importance of IgG2c isotype in the immune response to β-amyloid in amyloid precursor protein/transgenic mice

A careful analysis of the immune response to immunization of amyloid precursor protein/transgenic (APP/Tg) mice with beta-amyloid (Abeta) may provide insights into why a subset of the patients in a clinical trial receiving Abeta-immunotherapy developed encephalomyelitis. Characterization of isotypic immune responses have been reported in different APP/Tg models. In these studies the relative ratios of IgG1 to IgG2a anti-Abeta antibodies has been used as an indirect measure of T helper 1 (Th1) and Th2 types immune responses. However, it has previously been shown that certain strains of mice, C57Bl/6, C57Bl/10, SJL, and NOD, have an IgG2c rather than an IgG2a gene. Since a substantial number of Abeta-immunization studies rely on APP/Tg mice that have at least one parental C57Bl/6 strain, we have investigated whether antibodies specific for IgG2a can be used for characterization of antibody isotypes in APP/Tg2576 mice. Our results suggest that APP/Tg2576 and major histocompatibilty complex-matched parental strains are not expressing IgG2a, producing instead IgG2c anti-Abeta antibodies.

DNA prime–protein boost increased the titer, avidity and persistence of anti-Aβ antibodies in wild-type mice

Recently, we reported that a DNA vaccine, composed of three copies of a self B cell epitope of amyloid-β (Aβ42) and the foreign T-cell epitope, Pan DR epitope (PADRE), generated strong anti-Aβ immune responses in wild-type and amyloid precursor protein transgenic animals. Although DNA vaccines have several advantages over peptide–protein vaccines, they induce lower immune responses in large animals and humans compared with those in mice. The focus of this study was to further enhance anti-Aβ11 immune responses by developing an improved DNA vaccination protocol of the prime–boost regimen, in which the priming step would use DNA and the boosting step would use recombinant protein. Accordingly, we generated DNA and recombinant protein-based epitope vaccines and showed that priming with DNA followed by boosting with a homologous recombinant protein vaccine significantly increases the anti-Aβ antibody responses and do not change the immunoglobulin G1 (IgG1) profile of humoral immune responses. Furthermore, the antibodies generated by this prime–boost regimen were long-lasting and possessed a higher avidity for binding with an Aβ42 peptide. Thus, we showed that a heterologous prime–boost regimen could be an effective protocol for developing a potent Alzheimers disease (AD) vaccine.

Antitumor efficacy of DNA vaccination to the epigenetically acting tumor promoting transcription factor BORIS and CD80 molecular adjuvant

Cancer testis (CT) antigens are promising candidates for tumor vaccines due to their immunogenicity and tissue‐restricted expression. Recently, we identified a novel cancer testis gene, BORIS, whose expression is restricted to male testis after puberty and is strictly absent in non‐malignant female tissue. BORIS encodes a DNA‐binding protein that shares 11 zing finger (ZF) with transcription factor CTCF and differs at the N‐ and C‐termini. CTCF has been implicated in epigenetic regulation of imprinting, X chromosome inactivation, repression, and activation of cancer testis antigens. BORIS expression has been documented in cancers of diverse histological origin, including, but not limited to breast, prostate, ovary, gastric, liver, endometrial, glia, colon, and esophagus. Interestingly, BORIS induces demethylation and subsequent expression of many cancer‐testis genes, including MAGE‐A1 and NY‐ESO‐1, indicating that it is expressed very early in malignancy and might be an attractive candidate for immunotherapy. In this study we tested BORIS as a vaccine in a very aggressive, highly metastatic, and poorly immunogenic murine model of mammary carcinoma. Immunizations with a DNA encoding the mutant form of murine BORIS antigen (pmBORIS lacking DNA‐binding function) significantly prolonged survival, and inhibited tumor growth in BALB/c mice inoculated with 4T1 cells. Priming with pmBORIS mixed with molecular adjuvant and boosting with adenoviral vector expressing mBORIS was generally more effective, suggesting that the vaccination protocol could be further optimized. This is the first report demonstrating the feasibility of vaccination with a cancer associated epigenetic regulator for the induction of tumor inhibition. J. Cell. Biochem. 98: 1037–1043, 2006.

CD80 (B7-1) and CD86 (B7-2) are functionally equivalent in the initiation and maintenance of CD4+ T-cell proliferation after activation with suboptimal doses of PHA

Effective activation of T cells requires engagement of two separate T-cell receptors. The antigen-specific T-cell receptor (TCR) binds foreign peptide antigen-MHC complexes, and the CD28 receptor binds to the B7 (CD80/CD86) costimulatory molecules expressed on the surface of antigen-presenting cells (APC). The simultaneous triggering of these T-cell surface receptors with their specific ligands results in an activation of this cell. In contrast, CTLA-4 (CD152) is a distinct T-cell receptor that, upon binding to B7 molecules, sends an inhibitory signal to T cell activation. Many in vitro and in vivo studies demonstrated that both CD80 and CD86 ligands have an identical role in the activation of T cells. Recently, functions of B7 costimulatory molecules in vivo have been investigated in B7-1 and/or B7-2 knockout mice, and the authors concluded that CD86 could be more important for initiating T-cell responses, while CD80 could be more significant for maintaining these immune responses. In this study, we directly compared the role of CD80 and CD86 in initiating and maintaining proliferation of resting CD4(+) T cells in an in vitro mode system that allowed to provide the first signal-to-effector cells through the use of suboptimal doses of PHA and the second costimulatory signal through cells expressing CD80 or CD86, but not any other costimulatory molecules. Using this experimental system we demonstrate that the CD80 and CD86 molecules can substitute for each other in the initial activation of resting CD4(+) T cells and in the maintenance of their proliferative response.