Karen Zagorski

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.

Immunogenicity of DNA- and recombinant protein-based Alzheimer Disease epitope vaccines

Alzheimer disease (AD) process involves the accumulation of amyloid plaques and tau tangles in the brain, nevertheless the attempts at targeting the main culprits, neurotoxic β-amyloid (Aβ) peptides, have thus far proven unsuccessful for improving cognitive function. Important lessons about anti-Aβ immunotherapeutic strategies were learned from the first active vaccination clinical trials. AD progression could be safely prevented or delayed if the vaccine (1) induces high titers of antibodies specific to toxic forms of Aβ; (2) does not activate the harmful autoreactive T cells that may induce inflammation; (3) is initiated before or at least at the early stages of the accumulation of toxic forms of Aβ. Data from the recent passive vaccination trials with bapineuzumab and solanezumab also indicated that anti-Aβ immunotherapy might be effective in reduction of the AD pathology and even improvement of cognitive and/or functional performance in patients when administered early in the course of the disease. For the prevention of AD the active immunization strategy may be more desirable than passive immunotherapy protocol and it can offer the potential for sustainable clinical and commercial advantages. Here we discuss the active vaccine approaches, which are still in preclinical development and vaccines that are already in clinical trials.

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.

MultiTEP platform-based DNA epitope vaccine targeting N-terminus of tau induces strong immune responses and reduces tau pathology in THY-Tau22 mice.

BACKGROUND By the time clinical symptoms of Alzheimers disease (AD) manifest in patients there is already substantial tau pathology in the brain. Recent evidence also suggests that tau pathology can become self-propagating, further accelerating disease progression. Over the last decade several groups have tested the efficacy of protein-based anti-tau immunotherapeutics in various animal models of tauopathy. Here we report on the immunological and therapeutic potency of the first anti-tau DNA vaccine based on the MultiTEP platform, AV-1980D, in THY-Tau22 transgenic mice. METHODS Starting at 3months of age, mice were immunized intramuscularly with AV-1980D vaccine targeting a tau B cell epitope spanning aa2-18 followed by electroporation (EP). Humoral and cellular immune responses in vaccinated animals were analyzed by ELISA and ELISpot, respectively. Neuropathological changes in the brains of experimental and control mice were then analyzed by biochemical (WB and ELISA) and immunohistochemical (IHC) methods at 9months of age. RESULTS EP-mediated AV-1980D vaccinations of THY-Tau22 mice induced activation of Th cells specific to the MultiTEP vaccine platform and triggered robust humoral immunity response specific to tau. Importantly, no activation of potentially harmful autoreactive Th cell responses specific to endogenous tau species was detected. The maximum titers of anti-tau antibodies were reached after two immunizations and remained slightly lower, but steady during five subsequent monthly immunizations. Vaccinations with AV-1980D followed by EP significantly reduced total tau and pS199 and AT180 phosphorylated tau levels in the brains extracts of vaccinated mice, but produced on subtle non-significant effects on other phosphorylated tau species. CONCLUSIONS These data demonstrate that MultiTEP-based DNA epitope vaccination targeting the N-terminus of tau is highly immunogenic and therapeutically potent in the THY-Tau22 mouse model of tauopathy and indicate that EP-mediated DNA immunization is an attractive alternative to protein-based adjuvanted vaccines for inducing high concentrations of anti-tau antibodies.

Phospholipid membranes promote the early stage assembly of α-synuclein aggregates

Development of Parkinson’s disease is associated with spontaneous self-assembly of α-synuclein (α-syn). Efforts aimed at understanding this process have produced little clarity and the mechanism remains elusive. We report a novel effect of phospholipid bilayers on the catalysis of α-syn aggregation from monomers. We directly visualized α-syn aggregation on supported lipid bilayers using time-lapse atomic force microscopy. We discovered that α-syn assemble in aggregates on bilayer surfaces even at the nanomolar concentration of monomers in solution. The efficiency of the aggregation process depends on the membrane composition, being highest for a negatively charged bilayer. Furthermore, assembled aggregates can dissociate from the surface, suggesting that on-surface aggregation can be a mechanism by which pathological aggregates are produced. Computational modeling revealed that interaction of α-syn with bilayer surface changes the protein conformation and its affinity to assemble into dimers, and these properties depend on the bilayer composition. A model of the membrane-mediated aggregation triggering the assembly of neurotoxic aggregates is proposed.

A molecular model of the surface-assisted protein aggregation process

The importance of cell surfaces in the self-assembly of proteins is widely accepted. One biologically significant event is the assembly of amyloidogenic proteins into aggregates, which leads to neurodegenerative disorders like Alzheimer’s and Parkinson’s. The interaction of amyloidogenic proteins with cellular membranes appears to dramatically facilitate the aggregation process. Recent findings indicate that, in the presence of surfaces, aggregation occurs at physiologically low concentrations, suggesting interaction with surfaces plays a critical role in the disease-prone aggregation process. However, the molecular mechanisms behind on-surface aggregation remain unclear. Here we provide a theoretical model that offers a molecular explanation. According to this model, monomers transiently immobilized to surfaces increase the local monomer protein concentration and thus work as nuclei to dramatically accelerate the entire aggregation process. This theory was verified by experimental studies, using mica surfaces, to examine the aggregation kinetics of amyloidogenic-synuclein protein (α-Syn) and non-amyloidogenic cytosine deaminase APOBEC3G (A3G).

MultiTEP platform–based DNA vaccines for alpha-synucleinopathies: preclinical evaluation of immunogenicity and therapeutic potency

We have previously demonstrated that anti-beta amyloid DNA vaccine (AV-1959D) based on our proprietary MultiTEP platform technology is extremely immunogenic in mice, rabbits, and monkeys. Importantly, MultiTEP platform enables development of vaccines targeting pathological molecules involved in various neurodegenerative disorders. Taking advantage of the universality of MultiTEP platform, we developed DNA vaccines targeting 3 B-cell epitopes (amino acids [aa]85-99, aa109-126, and aa126-140) of human alpha-synuclein (hα-Syn) separately or all 3 epitopes simultaneously. All 4 DNA vaccines (1) generate high titers of anti-hα-Syn antibodies and (2) induce robust MultiTEP-specific T-helper cell responses without activation of potentially detrimental autoreactive anti-hα-Syn T-helper cells. Generated antibodies recognize misfolded hα-Syn produced by neuroblastoma cells, hα-Syn in the brain tissues of transgenic mouse strains and in the brain tissues of dementia with Lewy body cases. Based on these results, the most promising vaccine targeting 3 B-cell epitopes of hα-Syn simultaneously (PV-1950D) has been chosen for ongoing preclinical assessment in mouse models of hα-Syn with the aim to translate it to the human clinical trials.

THE FIRST MULTITEP-BASED DNA VACCINE TARGETING N-TERMINUS OF PATHOLOGICAL TAU INDUCES STRONG IMMUNE RESPONSES AND REDUCES TAU PATHOLOGY IN TAU TRANSGENIC MICE

TARGETING N-TERMINUS OF PATHOLOGICAL TAU INDUCES STRONG IMMUNE RESPONSES AND REDUCES TAU PATHOLOGY IN TAU TRANSGENIC MICE Hayk Davtyan, Wesley W. Chen, Karen Zagorski, Joy Davis, Irina Petrushina, David H. Cribbs, Michael G. Agadjanyan, Mathew Blurton-Jones, Anahit Ghochikyan, Institute for Molecular Medicine, Huntington Beach, CA, USA; University of California Irvine, Irvine, CA, USA; 3 University of California, Irvine, Irvine, CA, USA. Contact e-mail: hayk.davtyan@immed.org