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

Testing a MultiTEP-based combination vaccine to reduce Aβ and tau pathology in Tau22/5xFAD bigenic mice.

BackgroundAlzheimer disease (AD) is characterized by the accumulation of beta-amyloid (Aβ) plaques and neurofibrillary tangles composed of hyperphosphorylated tau, which together lead to neurodegeneration and cognitive decline. Current therapeutic approaches have primarily aimed to reduce pathological aggregates of either Aβ or tau, yet phase 3 clinical trials of these approaches have thus far failed to delay disease progression in humans. Strong preclinical evidence indicates that these two abnormally aggregated proteins interact synergistically to drive downstream neurodegeneration. Therefore, combinatorial therapies that concurrently target both Aβ and tau might be needed for effective disease modification.MethodsA combinatorial vaccination approach was designed to concurrently target both Aβ and tau pathologies. Tau22/5xFAD (T5x) bigenic mice that develop both pathological Aβ and tau aggregates were injected intramuscularly with a mixture of two MultiTEP epitope vaccines: AV-1959R and AV-1980R, targeting Aβ and tau, respectively, and formulated in AdvaxCpG, a potent polysaccharide adjuvant. Antibody responses of vaccinated animals were measured by ELISA, and neuropathological changes were determined in brain homogenates of vaccinated and control mice using ELISA and Meso Scale Discovery (MSD) multiplex assays.ResultsT5x mice immunized with a mixture of Aβ- and tau-targeting vaccines generated high Aβ- and tau-specific antibody titers that recognized senile plaques and neurofibrillary tangles/neuropil threads in human AD brain sections. Production of these antibodies in turn led to significant reductions in the levels of soluble and insoluble total tau, and hyperphosphorylated tau as well as insoluble Aβ42, within the brains of bigenic T5x mice.ConclusionsAV-1959R and AV-1980R formulated with AdvaxCpG adjuvant are immunogenic and therapeutically potent vaccines that in combination can effectively reduce both of the hallmark pathologies of AD in bigenic mice. Taken together, these findings warrant further development of this vaccine technology for ultimate testing in human AD

Primary 4T1 tumor resection provides critical "window of opportunity" for immunotherapy.

It is believed that primary tumor resection modulates host-tumor immune interaction, but this has not been characterized in a stringent breast cancer tumor model. This report, using the 4T1 murine mammary tumor model, characterizes for the first time the dynamic longitudinal changes in immunosuppressive and effector components of the immune system after resection of an established orthotopic primary tumor with a defined natural history of developing lung metastases. More specifically, we analyzed changes of absolute numbers and frequencies of MDSC, regulatory T cells (Treg), as well as activated CD4 and CD8 positive T cells in spleens and, in some studies, lungs of 4T1 tumor-bearing mice and mice after primary tumor resection. Importantly, using mathematical analyses we established that primary resection of an orthotopic tumor had created a "window of opportunity" with decreased tumor-associated immune suppression that existed for approximately 10 days. Although tumor resection did slightly prolong survival, it did not affect the ultimate development of metastatic disease since animals with resected tumors or intact primary tumors eventually died by day 47 and 43, respectively. This window of opportunity likely occurs in humans providing a rationale and parameters for integration and testing of immunotherapeutic strategies in this critical "window of opportunity" to combat the development of metastatic disease

Primary 4T1 tumor resection provides critical "window of opportunity" for immunotherapy.

It is believed that primary tumor resection modulates host-tumor immune interaction, but this has not been characterized in a stringent breast cancer tumor model. This report, using the 4T1 murine mammary tumor model, characterizes for the first time the dynamic longitudinal changes in immunosuppressive and effector components of the immune system after resection of an established orthotopic primary tumor with a defined natural history of developing lung metastases. More specifically, we analyzed changes of absolute numbers and frequencies of MDSC, regulatory T cells (Treg), as well as activated CD4 and CD8 positive T cells in spleens and, in some studies, lungs of 4T1 tumor-bearing mice and mice after primary tumor resection. Importantly, using mathematical analyses we established that primary resection of an orthotopic tumor had created a "window of opportunity" with decreased tumor-associated immune suppression that existed for approximately 10 days. Although tumor resection did slightly prolong survival, it did not affect the ultimate development of metastatic disease since animals with resected tumors or intact primary tumors eventually died by day 47 and 43, respectively. This window of opportunity likely occurs in humans providing a rationale and parameters for integration and testing of immunotherapeutic strategies in this critical "window of opportunity" to combat the development of metastatic disease

Immunogenicity of MultiTEP-Platform-Based Recombinant Protein Vaccine, PV-1950R, Targeting Three B-Cell Antigenic Determinants of Pathological α-Synuclein.

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by the aberrant accumulation of intracytoplasmic misfolded and aggregated α-synuclein (α-Syn), resulting in neurodegeneration associated with inflammation. The propagation of α-Syn aggregates from cell to cell is implicated in the spreading of pathological α-Syn in the brain and disease progression. We and others demonstrated that antibodies generated after active and passive vaccinations could inhibit the propagation of pathological α-Syn in the extracellular space and prevent/inhibit disease/s in the relevant animal models. We recently tested the immunogenicity and efficacy of four DNA vaccines on the basis of the universal MultiTEP platform technology in the DLB/PD mouse model. The antibodies generated by these vaccines efficiently reduced/inhibited the accumulation of pathological α-Syn in the different brain regions and improved the motor deficit of immunized female mice. The most immunogenic and preclinically effective vaccine, PV-1950D, targeting three B-cell epitopes of pathological α-Syn simultaneously, has been selected for future IND-enabling studies. However, to ensure therapeutically potent concentrations of α-Syn antibodies in the periphery of the vaccinated elderly, we developed a recombinant protein-based MultiTEP vaccine, PV-1950R/A, and tested its immunogenicity in young and aged D-line mice. Antibody responses induced by immunizations with the PV-1950R/A vaccine and its homologous DNA counterpart, PV-1950D, in a mouse model of PD/DLB have been compared

Immunogenicity of MultiTEP-Platform-Based Recombinant Protein Vaccine, PV-1950R, Targeting Three B-Cell Antigenic Determinants of Pathological α-Synuclein

Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are characterized by the aberrant accumulation of intracytoplasmic misfolded and aggregated α-synuclein (α-Syn), resulting in neurodegeneration associated with inflammation. The propagation of α-Syn aggregates from cell to cell is implicated in the spreading of pathological α-Syn in the brain and disease progression. We and others demonstrated that antibodies generated after active and passive vaccinations could inhibit the propagation of pathological α-Syn in the extracellular space and prevent/inhibit disease/s in the relevant animal models. We recently tested the immunogenicity and efficacy of four DNA vaccines on the basis of the universal MultiTEP platform technology in the DLB/PD mouse model. The antibodies generated by these vaccines efficiently reduced/inhibited the accumulation of pathological α-Syn in the different brain regions and improved the motor deficit of immunized female mice. The most immunogenic and preclinically effective vaccine, PV-1950D, targeting three B-cell epitopes of pathological α-Syn simultaneously, has been selected for future IND-enabling studies. However, to ensure therapeutically potent concentrations of α-Syn antibodies in the periphery of the vaccinated elderly, we developed a recombinant protein-based MultiTEP vaccine, PV-1950R/A, and tested its immunogenicity in young and aged D-line mice. Antibody responses induced by immunizations with the PV-1950R/A vaccine and its homologous DNA counterpart, PV-1950D, in a mouse model of PD/DLB have been compared

Targeting TLR-4 with a novel pharmaceutical grade plant derived agonist, Immunomax®, as a therapeutic strategy for metastatic breast cancer.

BackgroundPreviously we demonstrated that the resection of primary 4T1 tumors only slightly prolongs mouse survival, but importantly, creates a "window of opportunity" with attenuated suppressor cell and increased activated T cell populations. This suggests that additional activation of the immune system by immunostimulatory agents during this period may enhance anti-tumor immunity and potentially eradicate micro-metastatic disease in this stringent model. We hypothesized that the immunostimulator Immunomax®, which is comprised of a plant-derived polysaccharide, is non-toxic in humans and stimulates immune defense during the infectious diseases treatment, may have also anti-tumor activity and be beneficial in the adjuvant setting when endogenous anti-tumor responses are present and during the "window of opportunity" in post-resection metastatic breast cancer model. Here we provide the initial report that Immunomax® demonstrates the capacity to eliminate micro-metastatic disease in the post-resection, 4T1 mouse model of breast cancer.MethodsThe efficacy of Immunomax® was evaluated by analyzing survival rate and the number of spontaneous clonogenic tumor cells in the lung homogenates of mice. The frequencies of activated NK, CD4(+) and CD8(+) cells as well as myeloid-derived suppressor cells and Treg cells were evaluated using flow cytometry. Highly purified mouse and human dendritic and NK cells were sorted and the effect of Immunomax® on activation status of these cells was assessed by flow cytometry. The property of Immunomax® as TLR-4 agonist was determined by NF-κB/SEAP reporter gene assay, WB, RT-PCR.ResultsImmunomax® injections significantly prolonged overall survival and cured 31% of mice. This immunostimulator activates DCs via the TLR-4, which in turn stimulates tumoricidal NK cells and in vitro, completely inhibits growth of 4T1 cells. Incubation of PBMC from healthy donors with Immunomax® activates NK cells via activation of plasmacytoid DC leading significantly higher efficacy in killing of human NK-target cells K562 compared with non-treated cells.ConclusionThis is the first demonstration that Immunomax® is a TLR-4 agonist and the first report of a documented role for this pharmaceutical grade immunostimulator in augmenting anti-tumor activity, suggesting that incorporation of Immunomax® into developing breast cancer therapeutic strategies may be beneficial and with less potential toxicity than checkpoint inhibitors

Novel Vaccine against Pathological Pyroglutamate-Modified Amyloid Beta for Prevention of Alzheimer's Disease.

Post-translationally modified N-terminally truncated amyloid beta peptide with a cyclized form of glutamate at position 3 (pE3Aβ) is a highly pathogenic molecule with increased neurotoxicity and propensity for aggregation. In the brains of Alzheimer's Disease (AD) cases, pE3Aβ represents a major constituent of the amyloid plaque. The data show that pE3Aβ formation is increased at early pre-symptomatic disease stages, while tau phosphorylation and aggregation mostly occur at later stages of the disease. This suggests that pE3Aβ accumulation may be an early event in the disease pathogenesis and can be prophylactically targeted to prevent the onset of AD. The vaccine (AV-1986R/A) was generated by chemically conjugating the pE3Aβ3-11 fragment to our universal immunogenic vaccine platform MultiTEP, then formulated in AdvaxCpG adjuvant. AV-1986R/A showed high immunogenicity and selectivity, with endpoint titers in the range of 105-106 against pE3Aβ and 103-104 against the full-sized peptide in the 5XFAD AD mouse model. The vaccination showed efficient clearance of the pathology, including non-pyroglutamate-modified plaques, from the mice brains. AV-1986R/A is a novel promising candidate for the immunoprevention of AD. It is the first late preclinical candidate which selectively targets a pathology-specific form of amyloid with minimal immunoreactivity against the full-size peptide. Successful translation into clinic may offer a new avenue for the prevention of AD via vaccination of cognitively unimpaired individuals at risk of disease

Targeting TLR-4 with a novel pharmaceutical grade plant derived agonist, Immunomax®, as a therapeutic strategy for metastatic breast cancer.

BackgroundPreviously we demonstrated that the resection of primary 4T1 tumors only slightly prolongs mouse survival, but importantly, creates a "window of opportunity" with attenuated suppressor cell and increased activated T cell populations. This suggests that additional activation of the immune system by immunostimulatory agents during this period may enhance anti-tumor immunity and potentially eradicate micro-metastatic disease in this stringent model. We hypothesized that the immunostimulator Immunomax®, which is comprised of a plant-derived polysaccharide, is non-toxic in humans and stimulates immune defense during the infectious diseases treatment, may have also anti-tumor activity and be beneficial in the adjuvant setting when endogenous anti-tumor responses are present and during the "window of opportunity" in post-resection metastatic breast cancer model. Here we provide the initial report that Immunomax® demonstrates the capacity to eliminate micro-metastatic disease in the post-resection, 4T1 mouse model of breast cancer.MethodsThe efficacy of Immunomax® was evaluated by analyzing survival rate and the number of spontaneous clonogenic tumor cells in the lung homogenates of mice. The frequencies of activated NK, CD4(+) and CD8(+) cells as well as myeloid-derived suppressor cells and Treg cells were evaluated using flow cytometry. Highly purified mouse and human dendritic and NK cells were sorted and the effect of Immunomax® on activation status of these cells was assessed by flow cytometry. The property of Immunomax® as TLR-4 agonist was determined by NF-κB/SEAP reporter gene assay, WB, RT-PCR.ResultsImmunomax® injections significantly prolonged overall survival and cured 31% of mice. This immunostimulator activates DCs via the TLR-4, which in turn stimulates tumoricidal NK cells and in vitro, completely inhibits growth of 4T1 cells. Incubation of PBMC from healthy donors with Immunomax® activates NK cells via activation of plasmacytoid DC leading significantly higher efficacy in killing of human NK-target cells K562 compared with non-treated cells.ConclusionThis is the first demonstration that Immunomax® is a TLR-4 agonist and the first report of a documented role for this pharmaceutical grade immunostimulator in augmenting anti-tumor activity, suggesting that incorporation of Immunomax® into developing breast cancer therapeutic strategies may be beneficial and with less potential toxicity than checkpoint inhibitors