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

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/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 Advax(CpG), delta inulin, Alhydrogel(®), Montanide-ISA51, Montanide-ISA720, MPLA-SM pharmaceutical grade adjuvants. Formulation of AV-1959R in Advax(CpG) 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 Advax(CpG) 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 Advax(CpG) adjuvant were identified as promising immunogenic vaccines for ongoing pre-clinical assessment and future human clinical trials

Potential of Core-Collapse Supernova Neutrino Detection at JUNO

JUNO is an underground neutrino observatory under construction in Jiangmen, China. It uses 20kton liquid scintillator as target, which enables it to detect supernova burst neutrinos of a large statistics for the next galactic core-collapse supernova (CCSN) and also pre-supernova neutrinos from the nearby CCSN progenitors. All flavors of supernova burst neutrinos can be detected by JUNO via several interaction channels, including inverse beta decay, elastic scattering on electron and proton, interactions on C12 nuclei, etc. This retains the possibility for JUNO to reconstruct the energy spectra of supernova burst neutrinos of all flavors. The real time monitoring systems based on FPGA and DAQ are under development in JUNO, which allow prompt alert and trigger-less data acquisition of CCSN events. The alert performances of both monitoring systems have been thoroughly studied using simulations. Moreover, once a CCSN is tagged, the system can give fast characterizations, such as directionality and light curve

Detection of the Diffuse Supernova Neutrino Background with JUNO

As an underground multi-purpose neutrino detector with 20 kton liquid scintillator, Jiangmen Underground Neutrino Observatory (JUNO) is competitive with and complementary to the water-Cherenkov detectors on the search for the diffuse supernova neutrino background (DSNB). Typical supernova models predict 2-4 events per year within the optimal observation window in the JUNO detector. The dominant background is from the neutral-current (NC) interaction of atmospheric neutrinos with 12C nuclei, which surpasses the DSNB by more than one order of magnitude. We evaluated the systematic uncertainty of NC background from the spread of a variety of data-driven models and further developed a method to determine NC background within 15\% with {\it{in}} {\it{situ}} measurements after ten years of running. Besides, the NC-like backgrounds can be effectively suppressed by the intrinsic pulse-shape discrimination (PSD) capabilities of liquid scintillators. In this talk, I will present in detail the improvements on NC background uncertainty evaluation, PSD discriminator development, and finally, the potential of DSNB sensitivity in JUNO

Real-time Monitoring for the Next Core-Collapse Supernova in JUNO

Core-collapse supernova (CCSN) is one of the most energetic astrophysical events in the Universe. The early and prompt detection of neutrinos before (pre-SN) and during the SN burst is a unique opportunity to realize the multi-messenger observation of the CCSN events. In this work, we describe the monitoring concept and present the sensitivity of the system to the pre-SN and SN neutrinos at the Jiangmen Underground Neutrino Observatory (JUNO), which is a 20 kton liquid scintillator detector under construction in South China. The real-time monitoring system is designed with both the prompt monitors on the electronic board and online monitors at the data acquisition stage, in order to ensure both the alert speed and alert coverage of progenitor stars. By assuming a false alert rate of 1 per year, this monitoring system can be sensitive to the pre-SN neutrinos up to the distance of about 1.6 (0.9) kpc and SN neutrinos up to about 370 (360) kpc for a progenitor mass of 30$M_{\odot}$ for the case of normal (inverted) mass ordering. The pointing ability of the CCSN is evaluated by using the accumulated event anisotropy of the inverse beta decay interactions from pre-SN or SN neutrinos, which, along with the early alert, can play important roles for the followup multi-messenger observations of the next Galactic or nearby extragalactic CCSN.Comment: 24 pages, 9 figure

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

BackgroundBy the time clinical symptoms of Alzheimer's 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.MethodsStarting 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.ResultsEP-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.ConclusionsThese 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

Humanized monoclonal antibody armanezumab specific to N-terminus of pathological tau: characterization and therapeutic potency.

BackgroundThe experience from clinical trials indicates that anti-Aβ immunotherapy could be effective in early/pre-clinical stages of AD, whereas at the late stages promoting the clearing of Aβ alone may be insufficient to halt the disease progression. At the same time, pathological tau correlates much better with the degree of dementia than Aβ deposition. Therefore, targeting pathological tau may provide a more promising approach for the treatment of advanced stages of AD. Recent data demonstrates that the N-terminal region of tau spanning aa 2-18 termed "phosphatase activation domain" that is normally hidden in the native protein in 'paperclip'-like conformation, becomes exposed in pathological tau and plays an essential role in the inhibition of fast axonal transport and in aggregation of tau. Hence, we hypothesized that anti-Tau2-18 monoclonal antibodies (mAb) may recognize pathological, but not normal tau at very early stages of tauopathy and prevent or decrease the aggregation of this molecule.MethodsMouse mAbs were generated using standard hybridoma methodology. CDR grafting was used for humanization of mouse mAb. Humanized mAb (Armanezumab) was characterized and tested in vitro/ex vivo/in vivo using biochemical and immunological methods (HPLC, Biacore, ELISA, IHC, FRET, etc.). Stable DG44 cell line expressing Armanezumab was generated by clone selection with increased concentrations of methotrexate (MTX).ResultsA panel of mouse mAbs was generated, clone 1C9 was selected based on binding to pathological human tau with high affinity and humanized. Fine epitope mapping revealed conservation of the epitope of human tau recognized by the parent murine mAb and Armanezumab. Importantly, Armanezumab (i) bound to tau with high affinity as determined by Biacore; (ii) bound pathological tau in brains from AD, FTD and Pick's disease cases; (iii) inhibited seeding effect of aggregated tau from brain lysate of P301S Tg mice; (iv) inhibited cytotoxic effect of tau oligomers; (v) reduced total tau (HT7) and AT100, PHF1, AT8, AT180, p212, p214-positive tau species in brains of tau transgenic mice after intracranial injection. A stable CHO cell line producing >1.5 g/l humanized mAb, Armanezumab was generated.ConclusionThese findings suggest that Armanezumab could be therapeutic in clinical studies for treatment of AD

Humanized monoclonal antibody armanezumab specific to N-terminus of pathological tau: characterization and therapeutic potency

Abstract Background The experience from clinical trials indicates that anti-Aβ immunotherapy could be effective in early/pre-clinical stages of AD, whereas at the late stages promoting the clearing of Aβ alone may be insufficient to halt the disease progression. At the same time, pathological tau correlates much better with the degree of dementia than Aβ deposition. Therefore, targeting pathological tau may provide a more promising approach for the treatment of advanced stages of AD. Recent data demonstrates that the N-terminal region of tau spanning aa 2–18 termed “phosphatase activation domain” that is normally hidden in the native protein in ‘paperclip’-like conformation, becomes exposed in pathological tau and plays an essential role in the inhibition of fast axonal transport and in aggregation of tau. Hence, we hypothesized that anti-Tau2–18 monoclonal antibodies (mAb) may recognize pathological, but not normal tau at very early stages of tauopathy and prevent or decrease the aggregation of this molecule. Methods Mouse mAbs were generated using standard hybridoma methodology. CDR grafting was used for humanization of mouse mAb. Humanized mAb (Armanezumab) was characterized and tested in vitro/ex vivo/in vivo using biochemical and immunological methods (HPLC, Biacore, ELISA, IHC, FRET, etc.). Stable DG44 cell line expressing Armanezumab was generated by clone selection with increased concentrations of methotrexate (MTX). Results A panel of mouse mAbs was generated, clone 1C9 was selected based on binding to pathological human tau with high affinity and humanized. Fine epitope mapping revealed conservation of the epitope of human tau recognized by the parent murine mAb and Armanezumab. Importantly, Armanezumab (i) bound to tau with high affinity as determined by Biacore; (ii) bound pathological tau in brains from AD, FTD and Pick’s disease cases; (iii) inhibited seeding effect of aggregated tau from brain lysate of P301S Tg mice; (iv) inhibited cytotoxic effect of tau oligomers; (v) reduced total tau (HT7) and AT100, PHF1, AT8, AT180, p212, p214-positive tau species in brains of tau transgenic mice after intracranial injection. A stable CHO cell line producing >1.5 g/l humanized mAb, Armanezumab was generated. Conclusion These findings suggest that Armanezumab could be therapeutic in clinical studies for treatment of AD

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

Proteins of the Rpf Family: Immune Cell Reactivity and Vaccination Efficacy against Tuberculosis in Mice

It was shown recently that Mycobacterium tuberculosis expresses five proteins that are homologous to Rpf (resuscitation promoting factor), which is secreted by growing cells of Micrococcus luteus. Rpf is required to resuscitate the growth of dormant Micrococcus luteus organisms, and its homologues may be involved in mycobacterial reactivation. Mycobacterial Rpf-like products are secreted proteins, which makes them candidates for recognition by the host immune system and anti-Rpf immune responses potentially protective against reactivated tuberculosis. Here we report that the Rpf protein itself and four out of five of its mycobacterial homologues, which were administered as subunit vaccines to C57BL/6 mice, are highly immunogenic. Rpf-like proteins elicit immunoglobulin G1 (IgG1) and IgG2a responses and T-cell proliferation and stimulate production of gamma interferon, interleukin-10 (IL-10), and IL-12 but not IL-4 or IL-5. Both humoral and T-cell responses against these antigens show a high degree of cross-reactivity. Vaccination of mice with Rpf-like proteins results in a significant level of protection against a subsequent high-dose challenge with virulent M. tuberculosis H37Rv, both in terms of survival times and mycobacterial multiplication in lungs and spleens