Irina Petrushina

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.

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.

Delivery of a DNA vaccine for Alzheimer's disease by electroporation versus gene gun generates potent and similar immune responses.

Background: Induction of a humoral response against amyloid-β peptide may be beneficial for Alzheimer’s disease (AD) patients and may alleviate the onset and progression of AD. DNA-based vaccination provides a unique alternative method of immunization for treatment and prevention of AD. Currently, the two major delivery methods used for enhancing DNA uptake and immune responses to DNA vaccines in humans are electroporation (EP) and gene gun (GG). Objective: The goal of this translational study was to evaluate the efficacy of an AD DNA epitope vaccine (DepVac) delivered intramuscularly by EP or intradermally by GG. Methods: Humoral and cellular immune responses to immunization with DepVac were evaluated by ELISA and ELISPOT, respectively. Functional activity of the antibodies was also assessed. Results: EP- and GG-mediated immunizations with DepVac induced similar anti-amyloid-β (Aβ) antibody and T cell responses. Anti-Aβ antibodies bound to amyloid plaques in AD brain tissue and to toxic forms of Aβ42 peptide. Conclusion: Both delivery methods are effective at promoting potent antibodies specific for Aβ.

Alzheimer’s disease AdvaxCpG- adjuvanted MultiTEP-based dual and single vaccines induce high-titer antibodies against various forms of tau and Aβ pathological molecules

Although β-amyloid (Aβ) may be the primary driver of Alzheimer’s disease (AD) pathology, accumulation of pathological tau correlates with dementia in AD patients. Thus, the prevention/inhibition of AD may require vaccine/s targeting Aβ and tau simultaneously or sequentially. Since high antibody titers are required for AD vaccine efficacy, we have decided to generate vaccines, targeting Aβ (AV-1959R), Tau (AV-1980R) or Aβ/tau (AV-1953R) B cell epitopes, based on immunogenic MultiTEP platform and evaluate the immunogenicity of these vaccines formulated with AdvaxCpG, delta inulin, Alhydrogel®, Montanide-ISA51, Montanide-ISA720, MPLA-SM pharmaceutical grade adjuvants. Formulation of AV-1959R in AdvaxCpG induced the highest cellular and humoral immune responses in mice. The dual-epitope vaccine, AV-1953R, or the combination of AV-1959R and AV-1980R vaccines formulated with AdvaxCpG induced robust antibody responses against various forms of both, Aβ and tau pathological molecules. While anti-Aβ antibody titers after AV-1953R immunization were similar to that in mice vaccinated with AV-1959R or AV-1959R/AV-1980R combination, anti-tau titers were significantly lower after AV-1953R injection when compared to the AV-1980R or AV-1959R/AV-1980R. In silico 3D-modeling provided insight into the differences in immunogenicity of these vaccine constructs. In sum, AV-1959R and AV-1980R formulated with AdvaxCpG adjuvant were identified as promising immunogenic vaccines for ongoing pre-clinical assessment and future human clinical trials.

Low Concentrations of Anti-Aβ Antibodies Generated in Tg2576 Mice by DNA Epitope Vaccine Fused with 3C3d Molecular Adjuvant Do Not Affect AD Pathology

It has been demonstrated that an active vaccination strategy with protein- or DNA-based epitope vaccines composed of the immunodominant self B cell epitope of amyloid-β₄₂ (Aβ₄₂) and a non-self T helper (Th) cell epitope is an immunotherapeutic approach to preventing or treating Alzheimers disease (AD). As a DNA-based epitope vaccine, we used a plasmid encoding three copies of Aβ(1-11) and Th cell epitope, PADRE (p3Aβ(1-11)-PADRE). We have previously reported that three copies of component of complement C3d (3C3d) acts as a molecular adjuvant significantly enhancing immune responses in wild-type mice of the H2(b) haplotype immunized with p3Aβ(1-11)-PADRE. Here, we tested the efficacy of p3Aβ(1-11)-PADRE and the same vaccine fused with 3C3d (p3Aβ(1-11)-PADRE-3C3d) in a transgenic (Tg) mouse model of AD (Tg2576) of the H2(bxs) immune haplotype. The overall responses to both vaccines were very weak in Tg2576 mice despite the fact that the 3C3d molecular adjuvant significantly enhanced the anti-Aβ response to 3Aβ(1-11)-PADRE. Importantly, generation of low antibody responses was associated with the strain of amyloid precursor protein Tg mice rather than with a molecular adjuvant, as a p3Aβ(1-11)-PADRE-3C3d vaccine induced significantly higher antibody production in another AD mouse model, 3xTg-AD of the H2(b) haplotype. Finally, this study demonstrated that low concentrations of antibodies generated by both DNA vaccines were not sufficient for the reduction of Aβ pathology in the brains of vaccinated Tg2576 animals, confirming previous reports from preclinical studies and the AN-1792 clinical trials, which concluded that the concentration of anti-Aβ antibodies may be essential for the reduction of AD pathology.

BTX AgilePulse TM System is an Effective Electroporation Device for Intramuscular and Intradermal Delivery of DNA Vaccine

DNA vaccines promote immune system activation in small animals and exhibit certain advantages when compared to conventional recombinant protein vaccines. However in clinical trials DNA vaccines are less effective in inducing potent immune responses due to the low delivery efficiency and expression of antigens. Currently, various delivery devices such as gene-guns, bioinjectors and electroporation systems are being used in order to increase the potency of DNA vaccines. However, the optimal delivery parameters are required and must be carefully set to obtain the highest levels of gene expression and strong immune responses in humans. The focus of this study was to optimize electroporation settings (voltage, pulse length, pulse intervals, and number of pulses), as well as the route of administration (intradermal vs. intramuscular) and dosage of the DNA epitope vaccine, AV-1959D, delivered by the BTX AgilePulse(TM) system. As a result, we have chosen the optimal settings for electroporation delivery using different routes of immunization with this vaccine, generating (i) robust antibody production to the B cell epitope (a small peptide, derived from β-amyloid), and (ii) strong cellular immune responses to Th epitopes (a small synthetic peptide and eleven peptides from various pathogens) incorporated into DNA vaccine platform.

Exploiting prime-boost regimen and various delivery strategies to improve Alzhaimer's disease DNA vaccine efficacy

Background: Data from pre-clinical and clinical trials have suggested that the development of a safe and effective Ab-based vaccine for AD may require the generation of high titers of anti-Ab antibodies while avoiding autoreactive proinflammatory Th1 responses. We have previously reported that our DNA vaccine (DepVac) composed of 3 copies of Ab11 fused with the T cell epitope, PADRE and chemokine, MDC (MDC-3Ab11-PADRE) generated strong anti-Ab immune responses in mice. DNA-based vaccination provides a unique alternative method of immunization, it is safe in humans and its large-scale production is easy and inexpensive. However, DNA vaccines that are effective in mice, could induce low immune responses in large animals and humans. Hence, a further enhancement of vaccine immunogenicity is key to effective translation to the clinic. The focus of this report is on bolstering immune responses achieved by selecting the best delivery system and developing an improved DNA vaccination protocol of the primeboost regimen. Methods: In this study we compare the effectiveness of different strategies of DNA vaccine delivery in wild type mice: intradermal injection (i.d.) with conventional syringe, gene gun (GG), or electroporation (EP), and intramuscular injection (i.m.) with syringe or via electroporation. The further enhancement of the anti-Ab antibody responses in mice was achieved by developing a DNA prime/protein boost regimen. Results: In this study we compare the effectiveness of different strategies of DNA vaccine delivery in wild type mice: intradermal injection with conventional syringe, gene gun (GG), or electroporation (EP), and intramuscular injection with syringe or via electroporation. The further enhancement of the antiAb antibody responses in mice was achieved by developing a DNA prime/ protein boost regimen. Conclusions: The DNA vaccine chosen from this translational study will be tested in Rhesus monkeys before moving into the clinic. This DNA vaccination protocol, which allows the generation of high titers of high avidity antibodies over a long period of time, could be very cost effective and advantageous for humans.