Compositions for alzheimer's disease vaccines

Abstract

Described herein is an active Alzheimer's Disease (AD) immunotherapy based on a nanoparticle vaccine comprising a plurality of Aβ peptides and / or a plurality of tau peptides. These peptides may correspond to both soluble and aggregated targets and are displayed on the surface of immunogenic liposomes in an orientation that maintains reactivity with epitope-specific monoclonal antibodies. Also provided are methods of making and using same.

Description

COMPOSITIONS FOR ALZHEIMER’S DISEASE VACCINESCROSS REFERENCE TO RELATED APPLICATION

[0001] The instant application claims the benefit of priority to U.S. Provisional Application No. 63 / 676,431, filed on July 28, 2024, the disclosure of which is incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant number R41 AG082620 awarded by National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 28, 2025, is named “011520_01971_ST26.xml”, and is 9,876 bytes in size.BACKGROUND OF THE DISCLOSURE

[0004] Alzheimer’s disease (AD) is the most common irreversible, progressive neurodegenerative disease, characterized by progressive loss of memory and other cognitive functions. At present, there are no effective treatments available to slow or halt disease progression. The two histopathological hallmarks in the brains of AD patients are extracellular senile plaques comprising amyloid-P (AP) peptides and intracellular neurofibrillary tangles (NFTs) comprising hyperphosphorylated tau protein. Both pathologies trigger neuronal dysfunction, neurotoxicity, synaptic loss and inflammation, leading to cognitive impairment, affecting memory and behavior, culminating with severe dementia and death.

[0005] Harnessing the immune system to prevent or clear Ap and tau aggregates is an active area of research. Based on the Ap cascade hypothesis, most therapeutic approaches focus on reducing Ap plaques in the brain. However, prior Ap active or passive immunotherapies targeting different Ap epitopes did not show cognitive benefit in moderate to severe AD patients. Therefore, treatment during the early stages of clinical symptoms may be more effective in delaying downstream events, such as neuroinflammation and tau pathology. Immunotherapy for AD is being investigated for the prevention and earlytreatment of AD. Indeed, the Ap monoclonal antibody Aducanumab has been approved for patients with mild to moderate AD as well as Lecanemab for early AD. At the same time, multivariate analyses indicate that the density of NFTs, neuronal loss and synapse loss, but not amyloid load, correlate with cognitive impairment in patients with AD. Targeting tau pathology could be just as efficacious as Ap-directed clearance therapy for patients with AD. Immunotherapies targeting different tau epitopes, including non-phosphorylated, phosphorylated, oligomeric, and truncated tau, have shown beneficial effects in preclinical and ongoing clinical AD studies.

[0006] The epitopes selected for Ap and tau immunotherapy play a role in therapeutic outcomes and also unforeseen side effects. AN1792, the first Ap vaccine consisted of Api- 42, caused aseptic meningoencephalitis in 6% of treated patients, possibly due to Ap-specific T-cell mediated pro-inflammatory responses. Epitope mapping studies clarified that central part of full-length Ap, AP16-33, mediated the T-cell response. Several Ap immunotherapies targeting the N-terminus of Ap have shown beneficial effects in preclinical studies, but clinical trials did not demonstrate improvement in cognitive performance in patients with moderate to severe AD. A liposome-based vaccine, ACI-24.060 targeting AP1-15 (Clinical Trials.gov # NCT05462106), developed by AC Immune was well-tolerated and facilitated antibody production against of Ap variants with completed phase I and ongoing phase lb / 2a trail in patients with mild-staged AD. Similarly, studies indicate that N-terminal-truncated and pyroglutamate-modified Ap peptides at residue 3 (pE3-AP) are abundant in cored and diffuse Ap deposits in the brains of AD patients. Thus, immunotherapy against the N- terminus of Ap, including pE3-Ap may present a promising approach for AD treatment and indeed the antibody Donanemab, which recognizes pE3-Ap, is currently in large scale clinical testing.

[0007] While Ap is involved in the pathogenesis of AD, Tau pathology is also predominantly correlated with cognitive impairment in AD patients. Unlike normal tau that interacts with tubulin and promotes its assembly in microtubules, abnormally hyperphosphorylated tau is prone to form oligomers and consequently neurofibrillary tangles, the hallmark brain pathology of tauopathies. Thus, tau immunotherapy may be more effective in improving dementia symptoms and cognitive function in AD patients than Ap immunotherapy. Immunotherapies targeting different epitopes and species of tau have also shown beneficial effects in preclinical studies, and in clinical trials of these immunotherapies for AD are ongoing. Clearance of extracellular tau might be the primary mechanism of tau immunotherapy. Studies has indicated that tau can be actively released into the extracellularspace under physiological conditions and be passively released during neurodegeneration. Hyperphosphorylated tau at the site threonine 181 (pTaul81) in cerebrospinal fluid (CSF) has been extensively validated as a biomarker of AD and is currently widely used as one of the diagnostic criteria in clinical practice and for patient selection in clinical trials. Recent studies indicate that hyperphosphorylated tau at threonine 217 in CSF is even better than pTaul81 as a biomarker of AD. Additionally, phosphorylation of tau protein at sites Serine 396 / 404 is one of the earliest events in AD. In preclinical studies, active and passive immunotherapy targeting pTau396 / 404 with different monoclonal antibodies decreased tau pathology and improved cognitive function in other AD mouse models. Additionally, passive immunization with antibody 77E9 targeting tau 184-195 also decreased tau pathology and improved cognitive function in 3xTg-AD mice. Importantly, active immunotherapy with ACI-35 vaccine targeting pTau396 / 404 is in Phase II clinical trials in early AD patients (Clinical Trial #NCT04445831), and passive immunization with Lu AF87908 against tau pS396 / S404 is in phase I and II trials in healthy and early AD patients (Clinical Trial #NCT04149860 and NCT04619420). Passive immunization with JNJ-63733657 against pTau212 / 217 is in phase I clinical trials in healthy participants (Clinical Trial #NCT05407818).

[0008] Some recent studies have targeted multiple epitopes from Ap and tau as a proof-of-concept active immunotherapy. However, most immunotherapy studies have only targeted either Ap or tau, focusing on single epitopes within Ap or tau. Considering the pivotal role of both Ap and tau pathologies in the pathogenesis and progression of AD, a novel immunotherapeutic strategy targeting simultaneously multiple epitopes of both Ap and tau proteins is expected to maximumly clear the Ap and tau aggregates and inhibit the formation and propagation of Ap and tau pathologies in the brain. Several studies have shown the efficacy of A > level reduction in response to P-secretase (B ACE) inhibitors in both preclinical and clinical trial (e.g., Clinical Trial # NCT01978548). Therefore, facilitating prophylactic therapies for early stage of AD are also encouraged before patients have progressed into an overt dementia.

[0009] An adjuvant-based liposome containing cobalt-porphyrin-phospholipids (CoPoP) promotes spontaneous biostable binding of his-tagged antigens that incorporate into a bilayer due to the interaction with intrabilayer chelated cobalt. Modified CoPoP liposome with Phosphorylated HexaAcyl Disaccharide 3D6A (abbreviated herein as PHAD) elicits high immunogenicity of functional antibody responses against several pathogens due to highly antigen presenting cells (APCs) uptake. At present, CoPoP / PHAD formulated vaccine technology has been successfully used for a human-tested COVID-19 vaccine, and preclinicalvaccines for cancer immunotherapy, malaria, lyme disease, and influenza. CoPoP / PHAD liposome can be further co-formulated with another adjuvant, such as QS-21, that is well- known for stimulating Thl and Th2 response to enhance immune response.SUMMARY OF THE PRESENT DISCLOSURE

[0010] Amyloid-P (AP) and hyperphosphorylated tau protein are targets for Alzheimer’s Disease (AD) immunotherapies, which are generally focused on single epitopes within Ap or tau. However, due to the complexity of both Ap and tau in AD pathogenesis, a multipronged approach simultaneously targeting multiple epitopes of both proteins could overcome limitations of monotherapies. Described herein is an active AD immunotherapy based on a nanoparticle vaccine comprising two Ap peptides (1-14 and pyroglutamate pE3- 14) and three tau peptides (centered on phosphorylated pT181, pT217 and pS396 / 404). These correspond to both soluble and aggregated targets and are displayed on the surface of immunogenic liposomes in an orientation that maintains reactivity with epitope-specific monoclonal antibodies. Intramuscular immunization of mice with individual epitopes resulted in minimally cross-reactive antibody induction, while simultaneous co-display of 5 antigens (“5-plex”) induced antibodies against all epitopes without immune interference. Post-immune sera recognized plaques and neurofibrillary tangles from human AD brain tissue. Vaccine administration to 3xTg-AD mice using a prophylactical dosing schedule inhibited tau and amyloid pathologies and resulted in improved cognitive function. Immunization was well tolerated and did not induce antigen-specific cellular responses or persistent inflammatory responses in the peripheral or central nervous system. Antibody levels could be reversed by halting monthly vaccinations. Altogether, these results indicate that active immune therapies based on nanoparticle formulations of multiple Ap and tau epitopes warrant further study for treating early-stage AD.

[0011] Some or all of the cobalt porphyrins in the monolayer or bilayer can non- covalently bind polyhistidine-tagged molecules, such that at least part of the polyhistidine tag resides within the bilayer and the tagged molecule is presented on the surface of the bilayer. In the present bilayers or monolayers, it is considered that one or more histidine residues in the polyhistidine tag are coordinated to the cobalt metal core within the bilayer, thereby providing stability to the structure. The histidine residues of a polyhistidine tag may be coordinated to the cobalt metal in the core of the porphyrin in the membrane. The entire histidine tag may reside within the bilayer. A porphyrin phospholipid conjugate which has cobalt metal conjugated thereto is referred to herein as CoPoP. Liposomes wherein thebilayer comprises CoPoP are referred to herein as CoPoP liposomes. The CoPoP liposomes can be functionalized with histidine tagged molecules. The term “his-tagged molecules” as used herein means molecules - such as, for example, peptides, polypeptides, or proteins - which have a histidine tail. For example, a peptide with a histidine tail is a his-tagged molecule. Such his-tag containing CoPoP liposomes are referred to herein as his-tagged CoPoP liposomes or his-tagged CoPoP.

[0012] The CoPoP monolayers or bilayers functionalized with his-tagged presentation molecules of the present disclosure provide a platform for presentation of various molecules of interest in the circulation or for delivery to desired locations or for generation of specific immune responses to those his-tagged molecules. These molecules are referred to herein as presentation molecules (PMs). Structures containing his-tagged CoPoP bilayers, which have PMs attached to the histidine tag exhibit desirable stability. The his-tagged molecules are non-covalently attached to (coordinated to) the CoPoP and can be prepared by an incubation process. Therefore, the process does not need removal of reactive moieties - such as maleimide and the like - or exogenous catalysts or non-natural amino acids that are used in other types of conjugation chemistries.

[0013] In one example, the PoP is pyropheophorbide-phospholipid. The structure of pyropheophorbide-phospholipid is shown below:

[0014] The liposomes may have various peptide fragments chelated thereto. For example, the peptide fragments may be peptide fragments related to amyloid P and / or tau. In various examples, the peptide fragment is a portion of amyloid P and / or tau, a functionalized variant thereof, a peptide / protein having at least 70% identity thereto (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto), or afunctionalized variant of a protein having at least 70% identity thereto (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto). For example, the peptide fragment may be A0i-i4, A0PE3-i4, tauni-22, taupnsi, tauPT2i7, tauPs396 / s404, a peptide having at least 70% identity to any one or more of A01-14, A0PE3-14, tauni-22, tauPTi8i, tauPT2i7, tauPs396 / s404, or any combination thereof, wherein each of the aforementioned peptides has a polyHis-tag. For example, a peptide fragment of the present disclosure is His-tagged A0i-i4 (DAEFRHDSGYEVHHHH (SEQ ID NO:1)), A0pE3-i4([pyro- E]FRHDSGYEVHHHH, N-terminal pyroglutamate-modified (SEQ ID NO:2)), tauni-22 (HHHHMAEPRQEFEVMEDHAGTYGLGD (SEQ ID NO:3)), taupTi8i (HHHHIPAKTPPAPK[phospho-T]PPSSGEPPKS (SEQ ID NO:4)), taupT2i7 (HHHHGSRSRTPSLP[phospho-T]PPTREPKKVA (SEQ ID NO:5)), or (HGAEIVY[phosphor-K]SPVVSGDTS[phospho-P]RHHHH (SEQ ID NO:6)). The peptide fragments on the liposome may be the same or different. For example, there may be 2, 3, 4, 5, 6, 7, 8, 9, or 10 different peptide fragments chelated thereto. In various examples, any of the aforementioned sequences may be a portion of longer peptide (e.g., a peptide comprising any one of the following sequences).

[0015] In an aspect, the present disclosure provides methods to create antibodies against amyloid 0 and / or tau, and / or a method to reduce plaques formed from amyloid 0 and / or tau.BRIEF DESCRIPTION OF THE FIGURES

[0016] For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.

[0017] Figure 1. Admixture of his-tagged A0 / Tau peptides spontaneously form nanoparticles with C0P0P / PHAD liposomes. (A) Schematic representation of particle formation of 5-plex displaying on C0P0P / PHAD liposomes. (B) Binding percentage of individual antigens and 5-plex to C0P0P / PHAD or PoP / PHAD liposomes (served as a control) after 3 h incubation at 37°C, using a microcentrifugal filtration assay. Error bars show mean ± SD of triplicated tests. Statistical analysis was performed by unpaired t-test (two tailed), ****p<0.0001. (C) Immuno-slot blot assay showing specific immunoreaction of 10 ng / mL C0P0P / PHAD liposome-bound antigens, which was detected by using A0 or Tau epitope-specific antibodies as indicated on the left side of the blot. (D) Cryo-electronmicroscopy image of CoPoP / PHAD liposomes after incubation with (right) or without (left) 5-plex peptides at 37°C for 3 h. A 50 nm scale bar is shown. (E) Reversed Phase High Performance Liquid Chromatography (RP-HPLC) chromatographs of peptide elution with UV detection at 220 nm. The analysis was made using C8 reverse phase column with 0.065% TFA in water and 0.05% TFA in ACN.

[0018] Figure 2. Multivalent vaccine elicits antibodies in mice that recognize Ap plaques and NFTs. (A) IgG antibody titers were monitored and assessed by ELISA. The serum was collected from ICR mice (n = 5) immunized with immunogens of Ap or tau or 5- plex with CoPoP / PHAD liposome or alum on days 0 and 21, and bled on day 42. Vaccine was incubated with liposomes in either “mosaic” way displaying multiple antigens per liposome or “cocktail” way displaying one antigen per liposome on the surface. 5-plex “mosaic” shown in green box was the vaccine used in further 3xTg-AD mice immunization. Error bars show mean ± SD for n = 5. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, as determined by one-way ANOVA followed by Tukey’s test using log-transformed values. (B) Representative IHC staining of serial AD brain tissue sections with anti-APi-14, anti- tauPs396 / s404, 2-plex, and control sera targeted at parahippocampal gryus regions from an 83- y ear-old male AD patient. Scale bar: 500 pm. (C) Enlarged views of 100 pm scale bar indicate the neurofibrillary tau tangles (red arrows) and amyloid plaques (blue arrows) stained with anti-APi-i4, anti-tauPs396 / s404, 5-plex, and control sera in temporal cortex. (D) Staining of amyloid-beta and phosphorylated tau using control, anti-APi-i4, anti-ApPE3-i4, anti- tauPTi8i, anti-tauPT2i7, anti-tauPs396 / s404, 2-plex and 5-plex antibodies were observed in the amygdala section with 100 pm scale bars.

[0019] Figure 3. 5-plex vaccine induces specific antibodies in 3xTg-AD and WT mice. (A) Immunization schedule for mice vaccinated with the 5-plex vaccine. Groups of WT (aged 13-14 weeks / 2 months) and 3xTg-AD mice (aged 15-16 weeks / 3 months) were vaccinated, quarantined, and sent out for behavior testing at the times indicated. Prime vaccination (week 0) at mice aged 3 or 4 months is performed. Submandibular blood was collected prior to the next immunization to examine the antibody levels by ELISA coating with (B) APi -42 fibrils, (C) ApPE3-i4, (D) tauPTi8i, (E) tauPT2i7, (F) tauPs396 / 404. Error bars show mean ± SD for n=20 per group for WT / vaccinated, WT / control, 3xTg-AD / vaccinated, and 3xTg-AD / control groups initially. **P<0.01 and ****P<0.0001, as determined by unpaired two-tailed t-test using log-transformed values of IgG titers on week 12 and week 39.

[0020] Figure 4. Immunization with 5-plex vaccine prevents cognitive impairment in 3xTg-AD mice. (A) Schematic representation of open field test,MWM test and rMWM test.(B) Total distance travelled in apparatus was recorded. (C-H), MWM test. (C), the escape latency (sec) to reach the hidden platform during acquisition phase for 4 consecutive days. Percent time in the quadrant (D), percent time in the platform area (E), number of target crossings (F), latency to first entrance into target zone (G), and swim speed (H) during the probe trial. (I-N), rMWM test. (I), the escape latency (sec) to reach the hidden platform during acquisition phase for 3 consecutive days. Percent time in the quadrant (J), percent time in the platform area (K), number of target crossings (L), latency to first entrance into target zone (M), and swim speed (N) during the probe trial. N = 20 for WT / 5-plex, 17 for WT / control, 17 for 3xTg-AD / 5-plex, and 16 for 3xTg-AD / control. *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA followed by a Bonferroni’s posthoc test.

[0021] Figure 5. Immunization with 5-plex decreases tau pathology in mouse brains. (A) Immunostaining with AT8 antibody of the CAI sector of the hippocampus of mice with or without prior immunization with the 5-plex vaccine. (B) Quantification of AT8 immunostaining intensity in the CAI region was done from 6 sections which were from every 6thsagittal brain section per mouse. N = 13 for 3xTg-AD / control, and 14 for 3xTg-AD / 5- plex. Data are shown as mean ± SEM. **p < 0.01 by unpaired two-tailed t-test. (C) Representative Western blots of the hippocampus developed with 43D against human transgenic tau, R134d against total tau, and with several phosphorylation-dependent and sitespecific tau antibodies. (D) Densitometric quantification of the blots in C after normalization with total tau level (R134d blot). (E) Representative Western blots of sarkosyl-soluble fraction of the cerebrocortex developed with R134d against total tau, and with several phosphorylation-dependent and site-specific tau antibodies. (F) Densitometric quantification of the blots in E after normalization with total tau level (R134d blot). (G) Representative Western blots of sarkosyl insoluble fraction of the cortex developed with R134d against total tau, and with several phosphorylation-dependent and site-specific tau antibodies. (H) Densitometric quantification of the insoluble tau (F) after normalization with the soluble total tau level (R134d blot). Data are the percentage of control -treated animals and are presented as mean ± SEM. *p < 0.05, **p < 0.01 ***p < 0.001 by unpaired two-tailed t-test. For C -F, N = 6 for WT / control, 6 for WT / 5-plex, 10 for 3xTg-AD / control, and 10 for 3xTg-AD / 5- plex.For G and H, N = 6 for WT / control, 6 for WT / 5-plex, 10 for 3xTg-AD / control, and 8 for 3xTg-AD / 5-plex.

[0022] Figure 6. Immunization with 5-plex reduces amyloid plaques in 3xTg-AD mouse brains. (A) Staining of the subiculum of 3xTg-AD mouse brains with thioflavin S. (B) Quantification of the number of thioflavin S positive (Ths+) amyloid plaques (Arrow), and(C) the area of amyloid plaques in the subiculum from 6 sections (every 6thsagittal brain section per mouse). (D) Staining of the subiculum of 3xTg-AD mouse brains with an A0 antibody. (E) Quantification of the area of amyloid plaques in the subiculum from 6 sections (every 6thsagittal brain section per mouse). Data are presented as mean ± SEM *p < 0.05 by unpaired two-tailed t test. N = 13 for 3xTg-AD / control, and 14 for 3xTg-AD / 5-plex.

[0023] Figure 7. developed with antibodies against synaptic proteins and neuroinflammation markers. (B), Densitometric quantification of the blots. Data are the percentage of control-treated animals and are presented as mean ± SEM. N = 6 for WT / control, 6 for WT / 5-plex, 10 for 3xTg-AD / control, and 10 for 3xTg-AD / 5-plex. *p < 0.05, **p < 0.01 by unpaired two-tailed t test.

[0024] Figure 8. Multivalent 5-plex appears to be safe and well-tolerated in mice. (A) Representative ELISpot image of 5 immunogens with CoPoP / PHAD / + / -QS21 liposomes immunized mice and control mice producing IgG antibodies reacted to the specific immunogens. Negative (Medium) and positive (PHA-L) controls were included. (B) Quantifying IFN-g dots from (A) were counted with data shown in mean ± SD, and analyzed by a two-way ANOVA with Tukey’s multiple comparison test, *P<0.05, **P<0.01, ***p<0.001, ****P<0.0001. The samples were performed in n = 5 mice per group. (C) Representative image for Prussian blue staining in WT / control, WT / 5-plex, 3xTg- AD / Control, and 3xTg-AD / 5-plex mice. (D) Quantification of Prussian blue positive profiles. The Prussian blue positive profiles were counted from 6 sections which were from every 6thsagittal brain section per mouse. N = 14 for WT / control, 16 for WT / 5-plex, 13 for 3xTg- AD / control, and 14 for 3xTg-AD / 5-plex. *p < 0.05, ***p < 0.001 by ANOVA followed by a Bonferroni’s posthoc test or by unpaired two-tailed t-test. (E) The level of proinflammatory cytokines on IL-6, IL-12p70, GM-CSF, and IFN-g was analyzed by Luminex multiplex assay. *p < 0.05, ***p < 0.001 by ANOVA followed by a Bonferroni’s posthoc test or by unpaired two-tailed t-test.

[0025] Figure 9. Immuno-slot blot assay of (A) POP / PHAD liposome-bound antigens and (B) soluble antigens reacted with epitope-specific antibodies shown as a control in the study. (C) Liposome diameter and (D) poly dispersity were assessed using dynamic light scattering (DLS), with incubation with A 1-14, A0PE3-i4,tauPTi8i, tauPT2i7, and tauPs396 / s404. Data show mean ± SD of three independent tests.

[0026] Figure 10. IgG titers of antisera from ICR mice immunized with CoPoP / PHAD liposomes containing (A) A0i-i4 (B) A0PE3-14, (C) tauPTi8i, (D) tauPT2i7€nd (E) tauPs396 / s404 with indicated incubation formats. Serum was collected from mice on day 42 afterpriming on day 0 and boosting on day 21. Scale bar = 500 pm. Error bars show mean ± SD for n=5. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, as determined by one-way ANOVA followed by Tukey’s test using log-transformed values.

[0027] Figure 11. Cross-reactivity of antibodies with the non-PTM counterpart of Tau antigens. Mice serum was collected on day 42 after giving two vaccinations of both phosphorylated and non-phosphorylated tau peptides on day 0 and day 21. IgG titers of antisera were examined against (A) tau-pl81 and tau-181, and (B) tau-p217 and tau-217. Error bars show geometric mean ± SD for n=4. Data was analyzed by unpaired t-test (two- tailed), no significant difference observed.

[0028] Figure 12. Behavioral response in the open field test. Central area entries (A), central area time (sec) (B), and percent distance travelled in central area (C) were recorded.*p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA followed by a Bonferroni’s posthoc test.

[0029] Figure 13. The effect of 5-plex on quantified Ap level. The level of total Ap40 (A), AP42 (B) and the ratio of AP42 / AP40 (C) in the hippocampus was quantified by ELISA. N = 13 for 3xTg-AD / control, and 14 for 3xTg-AD / 5-plex.

[0030] Figure 14. Cellular immune responses induced by irreverent antigen immunization as a positive control. ICR mice were injected intramuscularly with BRM28 with CoPoP / PHAD / + / -QS21 liposomes on days 0 and 21, and splenocytes were collected on day 42 to restimulate with BRM28. (A) Images of ELISpot results on IFN-g dots induced by CoPoP / PHAD liposomes with or without QS-21 adjuvant. (B) Quantification of dot numbers is shown in (A). Data were analyzed by two-way ANOVA multiple comparison followed by Tukey’s test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0031] Figure 15. Body weight was monitored after the first immunization, and the changes in body weight were expressed as a percentage of the initial weight. The red arrow represents the boost vaccine on indicated weeks after priming, whereas the blue arrow pointed to the week when applied behavior tests. Error bars show mean ± SEM for n=20 per group.

[0032] Figure 16. Liposome diameter and poly dispersity of 4-plex vaccine were assessed using dynamic light scattering (DLS). Error bars show mean ± SD of triplicated tests.

[0033] Figure 17. Immunization with 4-plex increased the short-term cognitive function in novel object recognition test. (A) The percentage of time spent exploring two identical objects during sample phase. (B) Discrimination index (time spent exploring novelobject / time spent exploring novel and familiar objects) x 100% in test phase. N = 19 for WT / control, 8 for 3xTg-AD / control, and 10 for 3xTg-AD / 4-plex. **p < 0.01, ***p < 0.001. n = 12 for WT mice treated with IgG or 43D + 77E9, 3*Tg-AD mice treated with 43D, 77E9, or 43D + 77E9; and n = 14 for 3*Tg-AD mice treated with IgG. Data are reported as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by ANOVA followed by a Bonferroni post hoc test. AD Alzheimer’s disease, ANOVA Analysis of variance, IgG Immunoglobulin G, 3><Tg Triple-transgenic, WT Wild type. Behavioral studies were conducted on the third day after 5thimmunization. The animals were euthanized at 14.5 weeks (wks) after the initiation of immunization.

[0034] Figure 18. Immunization with 4-plex vaccine improves special learning and memory in Morris water maze test. (A) the escape latency (sec) to reach the hidden platform during acquisition phase for 4 consecutive days. Percent time in the target quadrant (B), number of target crossings (C), latency to first entrance into target zone (D), and swim speed (E) during the probe trial. N =17 for WT / control, 12 for 3xTg-AD / control, and 14 for 3xTg- AD / 4-plex. *p <0.05, **p < 0.01,***p < 0.001 by two-way ANOVA followed by a Bonferroni’ s posthoc test.

[0035] Figure 19. 4-plex vaccine induces specific antibodies in 3xTg-AD / vaccinated mice. Groups of female WT and 3xTg-AD mice (aged 9.5-10.5 months) were vaccinated with control or 4-plex for 5 doses with every 3 -week interval. Female WT mice immunized with CoPoP / EcML liposome was only used for control. Blood was collected after behavioral studies to examine the antibody levels by ELISA coating with AP1-42 fibrils, ApPE3-i4, taupnsi, and tauPs396 / 404. Error bars show mean ± SEM for n=19 per group for WT / control, 8 for 3xTg- AD / control, 10 for 8 for 3xTg-AD / 4-plex. ****P<0.0001, as determined by one-way ANOVA followed by Tukey’s test using log-transformed values.

[0036] Figure 20. 5-plex vaccine with different MPLA type elicits Ap and tau antibodies in mice. Blood was collected on day 42 after two injections to examine the antibody levels by ELISA coating with (A) AP1-42 fibrils, (B) ApPE3-i4, (C) tauPTi8i, (D) tauPs396 / 404, (E) tauni-22. Error bars show mean ± SD for n=4 per group. *P<0.05, **P<0.01, as determined by one-way ANOVA followed by Tukey’s test using scientific values.

[0037] Figure 21. His-tagged Ap / Tau peptides form 5-plex and 4-plex vaccines with CoPoP / PHAD liposomes. Liposome diameter of (A) 5-plex and (B) 4-plex vaccine were assessed using dynamic light scattering (DLS). Error bars show mean ± SD of triplicated tests. (C) Immuno-slot blot assay with 10 ng / mL CoPoP / PHAD liposome-bound 5-plex antigens (5 different freezing cycles) were detected by Ap or Tau epitope-specific antibodies.

[0038] Figure 22. 5-plex improved the spatial learning and memory in 3xTg-AD mice in Morris water maze (MWM) test. (A) The escape latency (sec) to reach the hidden platform during acquisition phase for 4 consecutive days. (B) percent time in the target quadrant. (C) percent time in the platform area, (D) number of target crossings, (E) latency to first entrance into target zone, and (F) swim speed during the probe trial.

[0039] Figure 23. Cognitive function assessment in male and female WT and 3xTg- AD mice treated with or without 4-plex vaccine. In NOR sample phase, all groups of male (A) and female (E) exhibited comparable exploration time for object 1 and 2, showing no innate object preference. In NOR test phase, 3xTg-AD / Control mice showed a decreased discrimination index compared to WT / Control, while 4-plex immunization improved recognition memory in both male (B) and female (F) 3xTg-AD mice. In Morris Water Maze acquisition phase, 3xTg-AD / Control mice showed impaired learning performance compared to WT / Control, while 4-plex vaccine improved learning performance in 3xTg-AD mice in both males (C) and females (G). In MWM probe test phase, 3xTg-AD / Control mice spent significantly less time in the target quadrant, expressing impaired spatial memory. With 4- plex immunization, both male (D) and female (H) 3xTg-AD mice decreased spatial memory deficits.DETAILED DESCRIPTION OF THE DISCLOSURE

[0040] Although claimed subject matter will be described in terms of certain examples, other examples, including examples that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, process step, and electronic changes may be made without departing from the scope of the disclosure.

[0041] As used herein, unless otherwise indicated, “about”, “substantially”, or “the like”, when used in connection with a measurable variable (such as, for example, a parameter, an amount, a temporal duration, or the like) or a list of alternatives, is meant to encompass variations of and from the specified value including, but not limited to, those within experimental error (which can be determined by, e.g., a given data set, an art accepted standard, etc. and / or with, e.g., a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as, for example, variations of + / - 10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value), insofar such variations in a variable and / or variations in the alternatives are appropriate to perform in the instant disclosure. As used herein, the term “about” may mean that the amount or value inquestion is the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, compositions, parameters, and other quantities and characteristics are not and need not be exact but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error, or the like, or other factors known to those of skill in the art such that equivalent results or effects are obtained. In general, an amount, size, composition, parameter, or other quantity or characteristic, or alternative is “about” or “the like,” whether or not expressly stated to be such. It is understood that where “about,” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0042] Ranges of values are disclosed herein. The ranges set out a lower limit value and an upper limit value. Unless otherwise stated, the ranges include the lower limit value, the upper limit value, and all values between the lower limit value and the upper limit value, including, but not limited to, all values to the magnitude of the smallest value (either the lower limit value or the upper limit value) of a range. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “0.1% to 5%” should be interpreted to include not only the explicitly recited values of 0.1% to 5%, but also, unless otherwise stated, include individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5% to 1.1%; 0.5% to 2.4%; 0.5% to 3.2%, and 0.5% to 4.4%, and other possible sub-ranges) within the indicated range. It is also understood (as presented above) that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about, it will be understood that the particular value forms a further disclosure. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0043] The articles “a” and “an” are used in this disclosure to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0044] As used herein, unless otherwise stated or indicated, “s” refers to second(s), “min” refers to minute(s), and “h” refers to hour(s).

[0045] The phrase “therapeutically effective amount” or “effective amount” is used herein to mean an amount sufficient to reduce by at least about 15 percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably prevents oxidative stress in the individual. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the individual.

[0046] As used herein, unless otherwise stated, the term “group” refers to a chemical entity that is monovalent (i.e., has one terminus that can be covalently bonded to other chemical species), divalent, or polyvalent (i.e., has two or more termini that can be covalently bonded to other chemical species). The term “group” also includes radicals (e.g., monovalent and multivalent, such as, for example, divalent radicals, trivalent radicals, and the like).Illustrative examples of groups include:

[0047] Amyloid-P (AP) and hyperphosphorylated tau protein are targets for Alzheimer’s Disease (AD) immunotherapies, which are generally focused on single epitopes within Ap or tau. However, due to the complexity of both A < and tau in AD pathogenesis, a multipronged approach simultaneously targeting multiple epitopes of both proteins could overcome limitations of monotherapies. Described herein is an active AD immunotherapy based on a nanoparticle vaccine comprising two Ap peptides (1-14 and pyroglutamate pE3- 14) and three tau peptides (centered on phosphorylated pT181, pT217 and pS396 / 404). These correspond to both soluble and aggregated targets and are displayed on the surface of immunogenic liposomes in an orientation that maintains reactivity with epitope-specific monoclonal antibodies. Intramuscular immunization of mice with individual epitopes resulted in minimally cross-reactive antibody induction, while simultaneous co-display of 5 antigens (“5-plex”) induced antibodies against all epitopes without immune interference. Post-immune sera recognized plaques and neurofibrillary tangles from human AD brain tissue. Vaccine administration to 3xTg-AD mice using a prophylactical dosing schedule inhibited tau and amyloid pathologies and resulted in improved cognitive function. Immunization was well tolerated and did not induce antigen-specific cellular responses or persistent inflammatory responses in the peripheral or central nervous system. Antibody levels could be reversed by halting monthly vaccinations. Altogether, these results indicate that active immune therapiesbased on nanoparticle formulations of multiple Ap and tau epitopes warrant further study for treating early-stage AD.

[0048] Some or all of the cobalt porphyrins in the monolayer or bilayer can non- covalently bind polyhistidine-tagged molecules, such that at least part of the polyhistidine tag resides within the bilayer and the tagged molecule is presented on the surface of the bilayer. In the present bilayers or monolayers, it is considered that one or more histidine residues in the polyhistidine tag are coordinated to the cobalt metal core within the bilayer, thereby providing stability to the structure. The histidine residues of a polyhistidine tag may be coordinated to the cobalt metal in the core of the porphyrin in the membrane. The entire histidine tag may reside within the bilayer. A porphyrin phospholipid conjugate which has cobalt metal conjugated thereto is referred to herein as CoPoP. Liposomes wherein the bilayer comprises CoPoP are referred to herein as CoPoP liposomes. The CoPoP liposomes can be functionalized with histidine tagged molecules. The term “his-tagged molecules” as used herein means molecules - such as, for example, peptides, polypeptides, or proteins - which have a histidine tail. For example, a peptide with a histidine tail is a his-tagged molecule. Such his-tag containing CoPoP liposomes are referred to herein as his-tagged CoPoP liposomes or his-tagged CoPoP.

[0049] The CoPoP monolayers or bilayers functionalized with his-tagged presentation molecules of the present disclosure provide a platform for presentation of various molecules of interest in the circulation or for delivery to desired locations or for generation of specific immune responses to those his-tagged molecules. These molecules are referred to herein as presentation molecules (PMs). Structures containing his-tagged CoPoP bilayers, which have PMs attached to the histidine tag exhibit desirable stability. The his-tagged molecules are non-covalently attached to (coordinated to) the CoPoP and can be prepared by an incubation process. Therefore, the process does not need removal of reactive moieties - such as maleimide and the like - or exogenous catalysts or non-natural amino acids that are used in other types of conjugation chemistries.

[0050] The cobalt-porphyrin may be in a bilayer in self-assembling liposomes enclosing therewithin an aqueous compartment. Alternatively, it may be in a single layer or bilayer coating that coats other nanoparticles. Cobalt-porphyrin phospholipid (CoPoP) behaves like a conventional lipid with respect to its amphipathic nature. Therefore, monolayers or bilayers comprising CoPoP can be used for coating of nanoparticles by methods that are known to those skilled in the art. In one example, the bilayer or monolayer of the present disclosure may be present on other nanoparticles, such as, for example, in theform of a coating. In one example, the bilayer or monolayer containing cobalt-porphyrin (e.g., cobalt porphyrin-phospholipid) is present as a coating on gold or silica nanoparticles, or other nanoparticles with a hydrophilic surface. In one example, the coating may be in the form of monolayers. In one example the monolayer or bilayer containing cobalt-porphyrin (e.g., cobalt porphyrin-phospholipid) is present as a coating on hydrophobic surfaces such as carbon nanotubes. In one example, the monolayers may form micelles surrounding one or more hydrophobic molecules.

[0051] This disclosure provides a nanostructure comprising a monolayer or a bilayer, wherein the monolayer or bilayer comprises: i) optionally, phospholipids and ii) porphyrin which has cobalt coordinated thereto forming cobalt-porphyrin. Optionally, the nanostructure also has one or more polyhistidine-tagged presentation molecule. At least a portion of the polyhistidine tag resides in the hydrophobic portion of the monolayer or the bilayer and one or more histidines of the polyhistidine tag are coordinated to the cobalt in the cobalt- porphyrin. At least a portion of the polyhistidine-tagged presentation molecule is exposed to the outside of the nanostructure. The nanostructure can be in the form of a liposome that encloses an aqueous compartment. However, the nanostructure may also coat a hydrophilic or hydrophobic material such as a gold or silica nanoparticle. The cobalt porphyrin may be conjugated to a phospholipid to form a cobalt porphyrin-phospholipid conjugate. The cobalt porphyrin can make up from 1 to 100 mol % of the monolayer or the bilayer, including 0.1 mol% values and ranges therebetween. For example, the cobalt porphyrin can make up from 1 to 20 mole %, or from 5 to 10 mol% of the monolayer or the bilayer. If the cobalt porphyrin makes up 100% of the monolayer or the bilayer, then there are no phospholipids present that are not conjugated to cobalt porphyrin. The bilayer or the monolayer can also comprise sterol and / or polyethylene glycol. The sterol can be cholesterol.

[0052] The number of histidines in the polyhistidine-tag (polyHis-tag) in the monolayer or bilayer can be from 2 to 20. For example, the number of histidines can be 2, 3, or 4. The polyHis-tag may be on the N- or C-terminus of the peptide fragment.

[0053] The liposomes may be spherical or non- spherical. The size of the liposomes can be from 50 to 1000 nm or more. In one example, the liposomes have a size (e.g., a longest dimension such as, for example, a diameter) of 50 to 1000 nm, including all integer nm values and ranges therebetween. For example, the size may be from 50 to 200 nm or from 20 to 1000 nm. If the liposomes are not spherical, the longest dimension can be from 50 to 1000 nm. These dimensions can be achieved while preserving the nanostructure width of the monolayer of the bilayer. The liposomes can carry cargo in the aqueous compartment. Thecargo, or part thereof, can also, or alternatively, be incorporated in the monolayer or the bilayer.

[0054] In one example, this disclosure provides a liposome comprising: a monolayer or a bilayer, wherein the monolayer or bilayer comprises cobalt-porphyrin phospholipid conjugate, optionally phospholipids that are not conjugated to cobalt porphyrin, and a polyhistidine-tagged presentation molecule, wherein at least a portion of the polyhistidine tag resides in the hydrophobic portion of the monolayer or the bilayer and one or more histidines of the polyhistidine tag are coordinated to the cobalt in the cobalt-porphyrin phospholipid conjugates. At least a portion of the polyhistidine-tagged presentation molecule is exposed to the outside of the nanostructure. The nanostructure, such as a liposome, can enclose an aqueous compartment. The monolayer or the bilayer need not contain any phospholipids that are not conjugated to cobalt porphyrin and in this case only has cobalt porphyrin phospholipid conjugates. Cargo can be present in the aqueous compartment. The cargo need not reside exclusive in the aqueous compartment and a part thereof can reside in the monolayer or the bilayer.

[0055] The disclosure also provides a monolayer or a bilayer, wherein the monolayer or bilayer comprises phospholipid monomers and porphyrin having cobalt coordinated thereto (forming cobalt-porphyrin). The monolayer or the bilayer has associated therewith one or more polyhistidine-tagged presentation molecules, wherein at least a portion of the polyhistidine tag resides in the hydrophobic portion of the monolayer or the bilayer. One or more histidines of the polyhistidine tag are coordinated to the cobalt in the cobalt-porphyrin and at least a portion of the polyhistidine-tagged presentation molecule is outside of the bilayer or the monolayer. In various examples, the monolayer or the bilayer encloses an aqueous compartment or forms a coating on a nanoparticle - such as a gold or silica nanoparticle.

[0056] The disclosure provides a nanostructure comprising a core, and a monolayer or a bilayer coating on the core, wherein the monolayer or bilayer comprises phospholipids, and porphyrin having cobalt coordinated thereto forming cobalt-porphyrin. The nanostructure can have one or more polyhistidine-tagged presentation molecules, wherein at least a portion of the polyhistidine tag resides in the hydrophobic portion of the monolayer or the bilayer and one or more histidines of the polyhistidine tag are coordinated to the cobalt in the cobalt- porphyrin. At least a portion of the polyhistidine-tagged presentation molecule is exposed to the outside of the nanoparticle. The core of the nanostructure can be a nanoparticle such as a gold or silica nanoparticle.

[0057] The liposomes, or nanoparticles having a coating or monolayer or bilayer, as described herein can have presentation molecules thereon, which can be antigenic molecules and / or targeting molecules. The presentation molecules can also provide targeting ability and / or imaging or other functionalities.

[0058] Liposomes or other nanostructures comprising his-tagged polypeptides and CoPoP compositions exhibit high serum-stability with respect to binding of the his-tagged polypeptide to the liposome. In one example, when incubated with serum (such as diluted serum) at room temperature, more than 60% of the his-tagged peptide remains bound to the CoPoP-containing bilayer after 24 hours incubation. In one example, more than 85% of the his-tagged peptide remains bound to the CoPoP layer after incubation with serum for 24 hours.

[0059] The CoPoP liposomes or the His-tagged CoPoP liposomes can be loaded with cargo - which typically resides in the aqueous compartment but may reside entirely or partially embedded in the bilayer - if it is hydrophobic or has a hydrophobic component. In addition to having presentation molecules on the surface, these structures can be used to load cargo in the aqueous compartment within the structures, or in the bilayer. The release of cargo from the CoPoP -liposomes can be triggered by near infrared (NIR) light. The cargo can be released at desired locations - such as by being internalized in targeted cells or by light triggered release.

[0060] The cobalt-porphyrin of the monolayers or bilayers is a porphyrin having a cobalt (Co) cation conjugated to the porphyrin. The porphyrin can be conjugated to a phospholipid (referred to herein as a cobalt porphyrin-phospholipid or cobalt porphyrinphospholipid conjugate).

[0061] The porphyrin portion of the cobalt-porphyrin or cobalt-porphyrin conjugate making at least part of some of the bilayer of the liposomes or other structures comprise porphyrins, porphyrin derivatives, porphyrin analogs, or combinations thereof. Exemplary porphyrins include hematoporphyrin, protoporphyrin, and tetraphenylporphyrin. Exemplary porphyrin derivatives include pyropheophorbides, bacteriochlorophylls, Chlorophyll A, benzoporphyrin derivatives, tetrahydroxyphenyl chlorins, purpurins, benzochlorins, naphthochlorins, verdins, rhodins, keto chlorins, azachlorins, bacteriochlorins, tolyporphyrins, and benzobacteriochlorins. Exemplary porphyrin analogs include expanded porphyrin family members (such as texaphyrins, sapphyrins and hexaphyrins) and porphyrin isomers (such as porphycenes, inverted porphyrins, phthalocyanines, and naphthalocyanines). For example, the cobalt-porphyrin can be a vitamin B12 (cobalamin) or derivative.

[0062] In one example, the PoP is pyropheophorbide-phospholipid. The structure of pyropheophorbide-phospholipid is shown below:

[0063] In one example, the layer (monolayer or bilayer) has only CoPoP which has his-tagged presentation molecules embedded therein. In this example, the only phospholipid in the layer is CoPoP (i.e., CoPoP is 100 mol %). In one example, the layer (monolayer or bilayer) has only CoPoP and porphyrin conjugated phospholipids (PoP), wherein CoPoP has histidines embedded therein, with the histidines having a peptide or other presentation molecules attached thereto. In certain examples, there are no other phospholipids, but the layer (monolayer or bilayer) may optionally contain sterols and / or PEG-lipid.

[0064] In one example, in addition to the CoPoP, the bilayer or monolayer also has phospholipids which are not conjugated to porphyrin and therefore, not coordinated with Co. Such phospholipids may be referred to herein as “additional phospholipids”. The bilayer or monolayer may also comprise sterol and PEG-lipid. In one example, the bilayer or monolayer consists essentially of, or consists of CoPoP, phospholipids that are not conjugated to porphyrins, and optionally sterol and / or PEG, wherein the PEG may be conjugated to lipid. In one example, the only metal-PoP in the bilayer is CoPoP, which has his-tagged presentation molecules embedded therein. In one example, the only metal in the bilayer is Co.

[0065] In one example, the bilayer of the liposomes comprises CoPoP and PoP. In addition to the CoPoP and the PoP, the bilayer can have additional phospholipids. The bilayer or monolayer may further comprise sterol and / or PEG. The PEG may be conjugated to lipid. In one example, the bilayer consists essentially of, or consists of CoPoP, PoP, additional phospholipids, and optionally sterol and / or PEG, wherein the PEG may be conjugated to lipid. In one example, the only metal-PoP in the bilayer is CoPoP. In one example, the only metal in the bilayer is Co.

[0066] In one example, the CoPoP is present in the nanoparticles from 0.1 to 20 mol % with the remainder 99.9 to 80 mol % being made up by additional lipids, with the percent being of the entire bilayer lipids. For example, the combination of CoPoP can be present from 0.1 to 10 mol %, sterol can be present from 0.1 to 50 mol %, optionally, attenuated lipid A derivatives such as monophosphoryl lipid A or 3 -deacylated monophosphoryl lipid A or a related analog that is a toll-like-receptor 4 (TLR4) lipid agonist can be present from 0 to 20 mol % or 0.1 to 20 mol %, and the remainder can be made up by additional phospholipids. The phospholipids are DOPC, DSPC, DMPC or combinations thereof, and sterol, if present, can be cholesterol.

[0067] In one example, the combination of CoPoP and PoP may be present in the nanoparticles from 0.1 to 20 mol % with the remaining 99.9 to 80 mol% being made up by additional phospholipids. For example, the combination of CoPoP and PoP can be present from 0.1 to 10 mol %, sterol can be present from 0 to 50 mol % or 0.1 to 50 mol%, and the remainder can be made up by phospholipids. The phospholipids can be DOPC, DSPC, DMPC or combinations thereof and sterol, if present, can be cholesterol.

[0068] As used herein, “phospholipid” is a lipid having a hydrophilic head group having a phosphate group connected via a glycerol backbone to a hydrophobic lipid tail. The phospholipid comprises an acyl side chain of 6 to 22 carbons, including all integer number of carbons and ranges therebetween. In certain examples, the phospholipid in the porphyrin conjugate is l-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine. The phospholipid of the porphyrin conjugate may comprise, or consist essentially of phosphatidylcholine, phosphatidylethanoloamine, phosphatidylserine and / or phosphatidylinositol.

[0069] In certain examples, the porphyrin is conjugated to the glycerol group on the phospholipid by a carbon chain linker of 1 to 20 carbons, including all integer number of carbons therebetween.

[0070] In various examples, in addition to the porphyrin conjugates disclosed herein, the bilayer of the liposomes also comprises other phospholipids. The fatty acid chains of these phospholipids may contain a suitable number of carbon atoms to form a bilayer. For example, the fatty acid chain may contain 12, 14, 16, 18 or 20 carbon atoms. In different examples the bilayer comprises phosphatidylcholine, phosphatidylethanoloamine, phosphatidyl serine and / or phosphatidylinositol.

[0071] The present bilayers and monolayers may also comprise sterols. The sterols may be animal sterols or plant sterols. Examples of sterols include cholesterol, sitosterol, stigmasterol, and cholesterol. In examples, cholesterol may be from 0 mol % to 50 mol %, or0.1 to 50 mol %. In other examples, cholesterol may be present from 1 to 50 mol%, 5 to 45 mol%, 10 to 30 mol%.

[0072] The vaccine composition may further comprise various adjuvants. The adjuvant can be administered as a separate component in the compositions, or it can be incorporated into the liposome. Examples of adjuvants include complete Freund’s adjuvant, incomplete Freund’s adjuvant, monophosphoryl lipid A (MPLA), aluminum phosphate, aluminum hydroxide, alum, phosphorylated hexaacyl disaccharide (PHAD), Sigma adjuvant system (SAS), AddaVax (Invitrogen), MF59, QS21, saponin, and combinations thereof. Other carriers like wetting agents, emulsifiers, fillers, and the like may also be used. MPLA can be used in the present compositions and methods, including PHAD, PHAD-504, and 3D6A-PHAD or other related forms of lipid TLR4 agonist. In various examples, the liposomes have the following mass ratio DOPC:CHOL THAD: CoPoP: [20:5:X:Y], where X is a range from 0.1 to 1, and Y is a range from 1 to 10.

[0073] An adjuvant can be used as a 0.001 to 50 wt % solution in phosphate buffered saline, and the antigen is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, such as about 0.0001 to about 1 wt %, or such as about 0.0001 to about 0.05 wt %. The antigen can be present in an amount in the order of micrograms to milligrams, or, about 0.001 to about 20 wt %, such as about 0.01 to about 10 wt %, or about 0.05 to about 5 wt %.

[0074] The liposomes of the present disclosure (without the his-tagged molecules) can be substantially spherical and have a size (e.g., a longest dimension such as, for example, a diameter) of 30 nm to 250 nm, including all integers to the nm and ranges therebetween. In one example, the size of the liposomes is from 100-175 nm. In one example, at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% of the liposomes in the composition have a size of from 30 to 250 nm or from 100 to 175 nm. The liposomes or nanostructures can be more than 200 nm. In one example, the nanostructures are more than 1000 nm. In one example, the nanostructures are from 200 to 1000 nm. The liposomes or nanostructures may be spherical or non-spherical. In one example, the largest dimensions of the nanostructure are less than 200 nm, while preserving the nanostructure width of the monolayer or bilayer. In one example, the size of the nanostructure exceeds 200 nm in some dimensions, while preserving the nanostructure width of the monolayer or bilayer. In one example, the size of the nanostructure exceeds 1000 nm in some dimensions, while preserving the nanostructure width of the monolayer or bilayer.

[0075] The structures formed by the layers of the present disclosure are serum stable. For example, in vitro, the his-tag binding stability to the CoPoP bilayers is stable when incubated in 50% bovine serum at room temperature for 24 hours. Thus, these structures can be stable under serum or concentrated or diluted serum conditions.

[0076] The liposomes may have various peptide fragments chelated thereto. For example, the peptide fragments may be peptide fragments related to amyloid P and / or tau. In various examples, the peptide fragment is a portion of amyloid P and / or tau, a functionalized variant thereof, a peptide / protein having at least 70% identity thereto (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto), or a functionalized variant of a protein having at least 70% identity thereto (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto). For example, the peptide fragment may be AP1-14, ApPE3-i4, tauni-22, taupnsi, tauPT2i7, tauPs396 / s404, a peptide having at least 70% identity to any one or more of AP1-14, ApPE3-i4, tauni-22, tauPTi8i, tauPT2i7, tauPs396 / s404, or any combination thereof, wherein each of the aforementioned peptides has a polyHis-tag. For example, a peptide fragment of the present disclosure is His-tagged APi-14 (DAEFRHDSGYEVHHHH (SEQ ID NO:1)), AppE3-i4([pyro- E]FRHDSGYEVHHHH, N-terminal pyroglutamate-modified (SEQ ID NO:2)), tauni-22 (HHHHMAEPRQEFEVMEDHAGTYGLGD (SEQ ID NO:3)), taupTi8i (HHHHIPAKTPPAPK[phospho-T]PPSSGEPPKS (SEQ ID NO:4)), taupT2i7 (HHHHGSRSRTPSLP[phospho-T]PPTREPKKVA (SEQ ID NO:5)), HGAEIVY[phosphor-K]SPVVSGDTS[phospho-P]RHHHH (SEQ ID NO: 6), or tauPs396 / s404 (HGAEIVYK[phospho-S]PVVSGDT[phospho-S]PRHHHH (SEQ ID NO:7). The peptide fragments on the liposome may be the same or different. For example, there may be 2, 3, 4, 5, 6, 7, 8, 9, or 10 different peptide fragments chelated thereto. In various examples, any of the aforementioned sequences may be a portion of longer peptide (e.g., a peptide comprising any one of the following sequences).

[0077] The peptide fragments may contain substitutions to modify the immunogenicity. The modified peptide fragments may have only naturally occurring amino acids or may be a mixture of naturally occurring and non-naturally occurring amino acids. In some examples, the sequences or variants thereof may be the only amino acid sequences present in the liposomes.

[0078] In an aspect, the disclosure provides a composition comprising liposomes or other structures of the present disclosure or a mixture of different liposomes or other structures. The compositions can also comprise a sterile, suitable carrier for administration to subjects including humans, such as, for example, a physiological buffer such as sucrose, dextrose, saline, pH buffering (such as from pH 5 to 9, from pH 7 to 8, from pH 7.2 to 7.6, (e.g., 7.4)) element such as citrate or phosphate. In an example, the composition comprises at least 0.1% (w / v) CoPoP liposomes or his-tagged-CoPoP liposomes or other structures. In various examples, the composition comprises from 0.1 to 100 mol% CoPoP liposomes or his- tagged CoPoP liposomes or other structures such as bilayer coated nanoparticles. In an example, the composition comprises from 0.1 to 99 mol% CoPoP liposomes having his- tagged presentation molecules associated therewith.

[0079] In an example, the peptides of the present disclosure are free of mal eimide or succinimidyl ester reactive groups. In an example, the peptides of the present disclosure are free of covalent modification with any lipid moiety. In one example, the tagged molecule to be attached to the membrane does not have a non-natural amino acid.

[0080] The present disclosure also provides methods for using structures bearing the bilayers as described herein. In one example, this disclosure provides a method of eliciting an immune response in a host. The immune response may generate antibodies. The method comprises administering to a subject a composition comprising a structure bearing CoPoP bilayers to which is conjugated a histidine tagged antigen. The compositions may be administered by any standard route of immunization including subcutaneous, intradermal, intramuscular, intratumoral, or any other route. The compositions may be administered in a single administration or may be administered in multiple administrations including booster shots. Antibody titres can be measured to monitor the immune response.

[0081] The vaccine compositions may comprise one or more adjuvants. The vaccine compositions can comprise additives, such as diluents, adjuvants, excipients, or carriers. Such additives can be liquids, such as water, oils, saline, glucose or the like, and auxiliary, stabilizing, thickening, or lubricating agents, wetting or emulsifying agents, or pH buffering agents, gelling or viscosity enhancing additives, detergents and solubilizing agents (e.g., TWEEN® 20, TWEEN® 80 also referred to as polysorbate 20 or 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzyl alcohol), bulking substances (e.g., lactose, mannitol), flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. See Remington: The Science and Practice of Pharmacy (2012) 22nd Edition. Non-aqueous solvents or vehicles can be used such aspropylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations may be lyophilized and redissolved or resuspended just before use. The formulation may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.

[0082] In an aspect, the present disclosure provides methods to create antibodies against amyloid P and / or tau, and / or a method to reduce plaques formed from amyloid P and / or tau.

[0083] The method may be performed on or in a subject in need of treatment. The subject to be treated by the method of the disclosure may be human or non-human (e.g., mammal). Non-human animals include ungulates such as bovines. Additional on-limiting examples of non-human mammals include pigs, mice, rats, rabbits, cats, dogs, or other agricultural mammals, pet, or service animals, and the like.

[0084] The compositions can be introduced or administered into a subject using any suitable administration route, including, but not limited to parenteral, subcutaneous, intraperitoneal, intramuscular, intravenous, mucosal, topical, intradermal, and oral administration.

[0085] Immunization can be done by way of a single dose, or it can be done by multiple doses that are spaced apart. For example, an initial administration and subsequent booster doses can be used. The compositions can be administered alone or can be coadministered or sequentially administered with other prophylactic (such as, for example, other immunogenic compositions) or therapeutic compositions (such as, for example, antiviral agents).

[0086] The dose of peptide fragment (e.g., His-tagged AP1-14 (DAEFRHDSGYEVHHHH (SEQ ID NO: 1)), AppE3-i4 ( EFRHDSGYEVHHHH (SEQ ID NO:2), N-terminal pyroglutamate-modified), tauPTi8i (HHHHIPAKTPPAPK[phospho- T]PPSSGEPPKS (SEQ ID NO:4)), taupT2i7 (HHHHGSRSRTPSLP[phospho- T]PPTREPKKVA (SEQ ID NO: 5)), or tauPs396 / s404 (HGAEIVYK[phospho- S]PVVSGDT[phospho-S]PRHHHH (SEQ ID NO:7)) needed for mice immunization are in a range from 100 ng to 5 pg, including all 0.1 ng values and ranges therebetween. The expected human dosing of the peptide fragment (e.g., His-tagged AP1-14 (DAEFRHDSGYEVHHHH (SEQ ID NO: 1)), AppE3-i4 ( EFRHDSGYEVHHHH (SEQ ID NO:2), N-terminal pyroglutamate-modified), taupTi8i (HHHHIPAKTPPAPK[phospho-T]PPSSGEPPKS (SEQ ID NO:4)), tauPT2i7 (HHHHGSRSRTPSLP[phospho-T]PPTREPKKVA (SEQ ID NO:5)), ortaupS396 / s404 (HGAEIVYK[phospho-S]PVVSGDT[phospho-S]PRHHHH (SEQ ID N0:7)) may be 0.1-5000 pg, total peptide dose. In examples, the total peptide dose may be 1 pg to 2.0 mg. For example, the dose may be 100 pg to 1 mg. In examples, the dose may be 200 to 800 pg or 250 to 750 pg or about 500 pg.

[0087] The steps of the method described in the various examples and examples disclosed herein are sufficient to carry out the methods of the present invention. Thus, in an example, the method consists essentially of a combination of the steps of the methods disclosed herein. In another example, the method consists of such steps.

[0088] The following Statements provide various examples of the present disclosure. They are not intended to limit the scope of the present disclosure.Statement 1. A vaccine composition comprising: a) a plurality of liposomes, each liposome comprising i) a bilayer, wherein the bilayer comprises A) one or more phospholipids andwherein cobalt is chelated to the porphyrin or B) one or more phospholipids a porphyrin having cobalt chelated thereto; and ii) one or more amyloid P fragments and / or tau peptide fragments, each fragment having a polyHis-tag attached to a terminus of the fragments, wherein each His-tag comprises two or more histidine residues and at least a portion of the polyHis tag resides in the hydrophobic portion of the bilayer and one or more histidines of the polyHis tag are coordinated to the cobalt in the cobalt-porphyrin, and wherein at least a portion of the amino acid sequence is exposed to the outside of the liposome; and b) a pharmaceutical carrier.Statement 2. A vaccine composition according to Statement 1, wherein the one or more amyloid P fragments and / or tau peptide fragments are chosen from AP1-14 (DAEFRHDSGYEVHHHH (SEQ ID NO: 1)), AppE3-i4 ([pyro-E]FRHDSGYEVHHHH, N- terminal pyroglutamate-modified (SEQ ID NO:2)), tauni-22(HHHHMAEPRQEFEVMEDHAGTYGLGD (SEQ ID N0:3)), tauPTi8i (HHHHIPAKTPPAPK[phospho-T]PPSSGEPPKS (SEQ ID N0:4)), tauPT2i7 (HHHHGSRSRTPSLP[phospho-T]PPTREPKKVA (SEQ ID N0:5)), HGAEIVY[phosphor- K]SPVVSGDTS[phospho-P]RHHHH (SEQ ID N0:6), tauPs396 / s404 (HGAEIVYK[phospho- S]PVVSGDT[phospho-S]PRHHHH (SEQ ID N0:7), a peptide have at least 80% identity to any one or more of A 1-14 (DAEFRHDSGYEVHHHH (SEQ ID NO: 1)), AppE3-i4 ([pyro- E]FRHDSGYEVHHHH, N-terminal pyroglutamate-modified (SEQ ID NO:2)), tauni-22 (HHHHMAEPRQEFEVMEDHAGTYGLGD (SEQ ID NO:3)), taupTi8i (HHHHIPAKTPPAPK[phospho-T]PPSSGEPPKS (SEQ ID NO:4)), taupT2i7 (HHHHGSRSRTPSLP[phospho-T]PPTREPKKVA (SEQ ID NO:5)), HGAEIVY[phosphor- K]SPVVSGDTS[phospho-P]RHHHH (SEQ ID NO:6), tauPs396 / s404 (HGAEIVYK[phospho- S]PVVSGDT[phospho-S]PRHHHH (SEQ ID NO:7), and any combination thereof.Statement 3. A vaccine composition according to Statement 2, wherein the composition comprises a plurality (e.g., two, three, four, or five) of different amyloid P fragments and / or a plurality (e.g., two, three, four, or five) tau peptide fragments.Statement 4. A vaccine composition according to Statement 2, wherein the composition comprises AP1-14, APE3-14, tauPTi8i, tauPT2i7, and tauPs396 / s404.Statement 5. A vaccine composition according to Statement 2, wherein the composition comprises A01-14, ApPE3-i4, tauni-22, tauPTi8i, and tauPs396 / s404.Statement 6. A vaccine composition according to Statement 2, wherein the composition comprises A0i-i4, ApPE3-i4, tauni-22, and tauPTi8i.Statement 7. A vaccine composition according to Statement 2, wherein the composition comprises A0i-i4, ApPE3-i4, tauPs396 / s404, and tauPTi8i.Statement 8. A vaccine composition according to Statement 2, wherein the composition comprises A01-14, ApPE3-i4, tauni-22, and tauPs396 / s404.Statement 9. The vaccine composition according to any one of the preceding Statements, wherein the cobalt porphyrin-phospholipid conjugate makes up from 0.1 to 25 mol % of the bilayer, including all 0.01 mol% values and ranges therebetween (e.g., 5 to 10 mol% of the bilayer).Statement 10. A vaccine composition according to any one of the preceding Statements, wherein the cobalt porphyrin-phospholipid conjugate makes up from 5 to 10 mol % of the bilayer.Statement 11. A vaccine composition according to any one of the preceding Statements, wherein the polyHis-tag comprises 2, 3, or 4 histidine residues.Statement 12. A vaccine composition according to any one of the preceding Statement, wherein size of the liposome is 50 nm to 200 nm, including all 0.01 nm values and ranges therebetween.Statement 13. A vaccine composition according to any one of the preceding Statements, wherein the liposomes further comprise one or more adjuvants.Statement 14. A vaccine composition according to any one of the preceding Statements, wherein the one or more adjuvants are attenuated lipid A derivatives, phosphorylated hexaacyl disaccharides (e.g., phosphorylated hexaacyl disaccharide (PHAD)), and / or QS21.Statement 15. A vaccine composition according to any one of the preceding Statements, further comprising one or more adjuvants that are not associated with the liposomes.Statement 16. A vaccine composition according to any one of the preceding Statements, further comprising one or more sterols.Statement 17. A vaccine composition according to any one of the preceding Statements, wherein the one or more sterols are cholesterol.Statement 18. A method for generating antibodies against amyloid P and / or tau comprising administering to a subject a vaccine composition according to any one of the preceding Statements.Statement 19. A method according to Statement 18, wherein the administration does not induce an inflammatory response.Statement 20. A method according to Statement 18 or Statement 19, wherein the vaccine composition is administered two or more times.Statement 21. A method according to any one of Statements 18 to 20, wherein native aggregation of amyloid P and / or tau is reduced and / or inhibited in the subject.Statement 22. A method according to any one of Statements 18 to 21, wherein the amount of amyloidogenic plaques associated with amyloid P and / or tau in the subject’s brain is reduced.

[0089] The following examples are presented to illustrate the present disclosure. They are not intended to be limiting in any matter.EXAMPLE 1

[0090] This example provides a description of compositions of the present disclosure.

[0091] Described herein is a vaccine, termed 5-plex, by admixture of his-tagged N- terminal A0 (Api-14), pyroglutamate-modified Ap (APpE3-14), taul71-191 (tau-pT181), tau207-227 (tau-pT217), and tau388-407 (tau-pS396 / S404) peptides with liposomes containing CoPoP / PHAD to assess its prophylactical effect on AD-like pathologies and cognitive function in 3xTg-AD mice at early stage. The epitopes of the 5-plex vaccine were selected on the basis of previous preclinical studies of individual single-epitope immunotherapy approach. The first vaccination was initiated at 3 months (prior to the onset of amyloid plaques that start at ~6 months of age and of neurofibrillary tangles that start at ~12 months of age in 3xTg-AD mice), followed with three boosters at a 3 -week interval, performed behavioral tests, and euthanized the animals 32 weeks after completion of the vaccination. It was found that the 5-plex vaccine induced high titers of antibodies against their specific antigens without induction of detectable inflammatory response in the peripheral or central nervous system. Active immunization with 5-plex was well tolerated, decreased AD-like pathologies, and improved cognitive function in 3xTg-AD mice.

[0092] Nanoparticle Formulation of Multivalent Synthetic A0 / tau Epitopes. CoPoP / PHAD liposome promotes a biostable vaccine platform displaying multivalent antigens on the surface and rapidly forms nanoparticles through a simple admixture. The synthetic peptides (A ni4, A0PE3-14, taupnsi, tauPT2i7, and tauPs396 / s404) were modified with 2, 3 or 4 exogenous histidine residues, so that they would contain 4 consecutive histidine residues which are sufficient for interaction with CoPoP, for stably display on liposome surfaces as depicted in Figure 1A. The binding of individual peptide or multivalent peptides to CoPoP / PHAD liposomes or PoP / PHAD liposomes, which lack cobalt, was assessed. After incubating with liposomes for 3 h at 37 °C, over 85% of peptides bound to CoPoP / PHAD liposomes in both individual and multiplexed formats, whereas minimal percentage of peptides bound to cobalt-free PoP liposomes (Figure IB). To examine the conformational integrity of peptides bound to the liposomes, an immuno-slot blot assay was accessed after incubation. Epitope-specified monoclonal antibodies (mAbs) or polyclonal antibodies (pAbs) were found to target their specific antigens in particle form, as shown in a chemiluminescence blot (Figure 1C). The weak detection for A0PE3-I4 against 6E10 appearedto be due to the similar peptide sequence as A ni4, except pyroglutamate-modified on 3rdamino acid position. The 5-plex vaccine was effectively recognized by all antibodies, indicating that each antigen was bound to liposomes and retained peptide epitope accessibility. When CoPoP / PHAD was replaced with PoP / PHAD liposomes or PBS, the soluble peptides would easily pass through the nitrocellulose membrane and became less apparent to be accessible with antibodies (Figure 9A, 9B).

[0093] Additionally, the morphology of liposomes was studied by using cryotransmission electron microscopy (cryo-TEM). It was observed that the liposomes were spherical with liposome sizes of -100 nm in diameter after being bound with or without antigens (Figure ID). This cryo-TEM result was also confirmed by dynamic light scattering (DLS) with measured size -100-120 nm in diameter and poly dispersity index less than 0.20 (Figure 9C, 9D), indicating there were no significant liposome aggregations appeared during particleization. The analytical RP-HPLC revealed different elution times for individual peptides absorbed at UV 220 nm, and consequently, all corresponding five peptide peaks were quantified and observed separately in the 5-plex (Figure IE). Taken together, both individual and multivalent A0 and tau peptides displayed on CoPoP / PHAD liposomes remained intact with evidence of unaltered antigen conformations.

[0094] Antibody Induction by Multivalent Vaccine and Immunoreactivities to AD pathology. To elucidate antigen efficacy when particleized, monovalent and multivalent antigens incubated with either CoPoP / PHAD liposome or alum were used to immunize mice intramuscularly on days 0 and 21, with serum collected on day 42 for ELISA testing of immunoglobulin G (IgG) titers (Figure 2A). Compared to the titer levels from mice immunized with alum admixture or untreated control mice, CoPoP / PHAD liposome- displayed A01-14, A0pE3-i4,tauPTi8i, tauPT2i7, and tauPs396 / s404 generated higher levels of antibodies against their own antigen sequences (apart from A0i-i4 antisera against to fibrillar A01-42). Due to overlapped sequence, there was cross-reactivity observed at lower IgG titer levels between A0i-i4 and A0PE3-I4, and statistical analysis showed a substantial difference in targeting its own specified antigen. In contrast, the multivalent antigens with alum induced no significant antibody induction. Similarly, monovalent liposomal antigen immunization did not induce antibodies against other antigens excluded from the liposomal formulation. The 5- plex vaccine revealed robust antibody responses against all A0 and tau coated antigens. The immunogenicity among different immunogen formats were also compared and found no significant differences in IgG titers between the monovalent and the 5-plex vaccines in either mosaic or cocktail format against A0ni4, A0PE3-i4,tauPTi8i, tauPT2i7, (Figure 10A-D). Anexception was observed for vaccines against tauPs396 / 404, where a small difference in the titers was found between the monovalent and the mosaic form (Figure 10E). However, no significant titer difference was observed in cross-reactivity of phosphorylated and nonphosphorylated tau peptides. (Figure 11A,B)

[0095] AD brain tissue immunohistochemical staining was further assessed with those anti-sera from mice immunized with individual, duplex (2-plex), or multiplexed (5- plex) antigen liposomal vaccine. Serial sections of AD brains were immunostained with enhanced anti-Ap antibodies for Ap plaques detection or anti-Tau antibodies for NFT detection. In the amygdala and its adjacent areas, parahippocampal gyrus and temporal cortex, a significant number of Ap plaques (diameter >30 pm) and tau tangles (diameter >10 pm) indicated the apparent immunoreactive responses with anti-APi-14, anti-ApPE3-i4, anti- tauPTi8i, anti-tauPT2i7, anti-tauPs396 / s404, 2-plex and 5-plex antibodies, whereas negligible pathologic hallmarks were observed from control mouse antisera (Figure 2B-D). With its specified view at amygdala and temporal cortex, Ap plaques (pointed by blue arrows) and tau tangles (pointed by red arrows) were appreciably stained by those indicated antisera (Figure 2C, D). Multiple diffused Ap plaques were detected by with anti-APi-14, and anti-ApPE3-i4, while neurofibrillary tau tangles were detected by anti-tauPsi8i, anti-tauPT2i7, and anti- tauPs396 / 404 antibodies in amygdala sections. Correspondingly, multivalent liposomal vaccines containing five antigens successfully induced antibodies to recognize both hallmarks (Figure 2C, D). Compared to the monovalent liposomal vaccines that only induced either anti-Ap or anti-Tau antibodies, the 5-plex vaccine not only promoted a wider targeting site against APi- 14, ApPE3-i4, tauPTi8i, tauPT2i7, and tauPs396 / 404 simultaneously, but also elicited strong antibodies that detected pathological Ap plaques and tau tangles in human brain sections.

[0096] Evaluation of Antibody Generation after Immunization of Mice with 5- plex Vaccine. To evaluate the functional antibodies induced by the liposomal 5-plex vaccine, both B6129SF2 / J-WT and 3xTg-AD mice were intramuscularly administered with four doses of 5-plex immunizations to access anti-Ap or anti-Tau IgG titers. In contrast, no immunization was performed on control WT and 3xTg-AD mice. After the initial priming on week 0, vaccinated mice at aged 3 or 4 months were administered the boost every 3 weeks until week 9, and 100 pL blood was collected from all mouse groups prior to subsequent immunization through submandibular bleeding until week 12 for assessing the immunogenicity by ELISA assay. Mice were quarantined at Charles River Lab for 9 weeks before the behavior tests started (Figure 3A). A serological analysis of serum IgG was measured against AP42 fibrillar (Figure 3B), ApPE3-i4 (Figure 3C), tauPTi8i(Figure 3D),tauPT2i7 (Figure 3E), tauPs396 / 404 (Figure 3F) at every 3 weeks after post-immunizations. Marked generation of specific antibodies against all the five expected epitopes were found starting three weeks after initial immunization (Figure 3B-F). The antibody titers increased remarkably with boosting doses and appeared to reach the peaks after three doses of vaccines. There was no difference in the immunogenicity of this vaccine between the 3xTg-AD mice and WT mice. As expected, mice without receiving any vaccine injections showed only the background levels of IgG titers at all time points (Figure 3A-F). At week 37 when the mice had not received further boosting doses, the specific antibody titers reduced to 21-34% of the highest titers, but the antibody levels were still significantly higher than the background levels. Taken together, these results indicated that the immunization with 4 doses of multivalent 5-plex vaccine can induce high levels of specific anti-Ap and anti-tau antibodies for a reasonable period of time (weeks to months) in mice.

[0097] Prevention of Cognitive Impairment of 3xTg-AD mice by Immunization with 5-plex Vaccine. To investigate whether immunization with the 5-plex vaccine can prevent cognitive impairment in 3*Tg-AD mice, open field tests, Morris water maze (MWM), and reversal Morris water maze (rMWM) tasks were conducted to assess the special learning and reference memory (Figure 4A). Since anxiety can adversely affect the cognitive function, whether there is a difference in anxiety among WT and 3xTg-AD mice immunized with the 5-plex vaccine or placebo was first assessed. It was found that the immunization with the 5-plex vaccine reduced the locomotive activity in the 3xTg-AD mice but not in the WT mice (Figure 4B). 3xTg-AD mice travelled similar distances in the apparatus compared with the WT mice, suggesting no difference in the locomotive activity between the 3xTg-AD and WT mice. Additionally, the 3xTg-AD mice exhibited similar central entries (Figure 12A), time spent in the central area (Figure 12B), and central distance travelled (%) compared to the WT mice (Figure 12C), which indicates that there is no significant difference in the anxiety between the 3xTg-AD and WT mice. The 3xTg-AD and WT mice vaccinated with 5-plex have similar central entries and time in the central area compared to their control animals (Figure 12A-C), which suggests that immunization with 5-plex does not affect animals’ anxiety.

[0098] During MWM test, the 3xTg-AD mice took a significantly longer time than the WT mice to find the hidden platform in the acquisition phase (Figure 4C). They also spent less time in the target quadrant (Figure 4D) and platform area (Figure 4E) and had fewer target crossings (Figure 4F) than the WT mice in the probe test phase. These results indicate that the 3xTg-AD mice exhibited special learning and memory impairments. Nosignificant difference was observed in the latency to the first entry into the platform area (Figure 4G). However, immunization with the 5-plex vaccine did not significantly affect the escape latency in the acquisition phase or the time in the target quadrant and platform area in 3xTg-AD and WT animals during the probe trial test.

[0099] During the rMWM test, we observed that the 3xTg-AD mice after prior immunization with the 5-plex vaccine not only took less time than the control 3xTg-AD mice to find the hidden platform in the acquisition phase (Figure 41), but also spent more extended time in the platform area (Figure 4K), had more target crossings (Figure 4L), and took much less time to enter the target quadrant (Figure 4M) during probe trial test. Unlike the unvaccinated 3xTg-AD mice that showed clear cognitive impairment, the performance of the vaccinated 3xTg-AD mice in the rMWM was indistinguishable from the WT control mice. These results indicate that prior immunization with the 5-plex vaccine can prevent the deficits in special learning and memory of the 3xTg-AD mice. No significant difference was observed in the swim speed among the groups of mice (Figure 4H and N), which otherwise could affect the performance of mice in the water maze.

[0100] Reduction of Tau Pathology in the Brains of 3xTg-AD Mice after Immunization with 5-plex Vaccine. The 3xTg-AD mice develop AD tau pathology in the brain gradually. It was next investigated whether immunization of these mice with the 5-plex vaccine before the appearance of tau pathology can delay or prevent tau pathology in the mouse brain. Monoclonal antibody AT8, which recognizes tau hyperphosphorylated at Ser202 / Thr205 as well as NFTs, is widely used for tau pathology studies and thus was used in this study to assess tau pathology through immunohistochemical studies. It was observed that immunostaining with AT8 antibody in 3xTg-AD mice but not in WT animals (Figure 5 A), which indicated that it is the specific AT8 staining observed in 3xTg-AD animals. Importantly, it was observed a clear overall reduction of AT8 immunoreactivity throughout the brains of 3xTg-AD mice after prior immunization with the 5-plex vaccine compared to those without immunization. The AT8 immunostaining was abundant in the hippocampus where its reduction with immunization was clearly seen (Figure 5A) and quantified (Figure 5B) These results indicate that immunization with the 5-plex vaccine can delay or reduce tau pathology in the brains of 3xTg-AD mice.

[0101] It was also determined the level and phosphorylation state of tau protein in the mouse brains by semi-quantitative western blots of the hippocampal tissue homogenates. The 43D blot confirmed tau transgene of the 3xTg-AD mice since this antibody recognizes only human tau but not mouse tau (Figure 5C). Polyclonal antibody R134d was used to measurethe total tau level in the tissue homogenates, as this antibody recognizes both human tau and mouse tau independent of its phosphorylation state. Surprisingly, this analysis revealed that more tau level, as measured with antibody R134d, is in the hippocampi of 3xTg-AD mice after prior immunization with 5-plex vaccine than those without prior immunization (Figure 5C,D). However, contrary to our expectations, we did not observe a marked reduction in tau band intensities with phosphorylation-dependent tau antibodies in the vaccinated 3xTg-AD mice group compared to those of non-vaccinated group (Figure 5C), suggesting a reduction in the net phosphorylation level of tau proteins (i.e., the ratio of pTau / total tau) in the vaccinated group. Quantifications of the net tau phosphorylation levels by normalization of pTau intensity with the total tau level confirmed that the prior immunization of 3xTg-AD mice with the 5-plex vaccine reduced the net phosphorylation of tau (pTau / total tau) at T181, S202 / T205 (AT8 sites) and T217 (Figure 5D). These results are consistent with our immunohistochemical findings that immunization with the 5-plex vaccine can delay or decrease tau pathology in the brains of 3xTg-AD mice. In addition, reduction of net tau phosphorylation level at T181, S199, S202 / T205, T212, T217 and Ser396 / Ser404 (PHF1 sites) was also observed in WT mice after immunization with the 5-plex vaccine.

[0102] The levels of sarkosyl-soluble and sarkosyl-insoluble tau in the cerebrocortical tissue were investigated. Consistent with the results in the hippocampus, it was also found significantly more soluble tau level in the cortex of 3xTg-AD mice after prior immunization with the 5-plex vaccine than those without prior immunization (Figure 5E,F).Quantifications of the net tau phosphorylation levels by normalization of pTau intensity with the total tau level indicated that the prior immunization with the 5-plex vaccine reduced the net phosphorylation of tau at T181, T217, and S396 / S404 (PHF1 sites) in 3xTg-AD mice, and at T181 and T217 in WT mice (Figure 5E,F). Importantly, immunization with 5-plex significantly reduced the ratio of insoluble to soluble total tau, as determined by using antibody R134d (Figure 5G,H), suggesting that the vaccine can prevent or reduce the aggregation of tau in the mouse brain. The levels of phosphorylated tau at T181, T217, and S396 / S404 (PHF1 sites) were also found to be decreased significantly after immunization with the vaccine in 3xTg-AD mice (Figure 5G,H). In wild-type control mice, immunization with 5-plex only reduced the level of sarkosyl-insoluble tau phosphorylated tau at S396 / S404 (PHF1 sites) significantly. Altogether, these results suggest that immunization with 5-plex can prevent and / or reduce hyperphosphorylation and aggregation of tau in the brains of 3xTg- AD mice.

[0103] Reduction of Amyloid Pathology in the Brains of 3xTg-AD Mice after Immunization with 5-plex Vaccine. The 5-plex vaccine was designed to attack both tau and amyloid pathologies in the brain. To investigate whether early vaccination can prevent or reduce the development of amyloid pathology in the 3xTg-AD mouse brains, Ap pathology was studied by both Thioflavin S (Ths) and Ap staining of the brain tissue sections and ELISA of the brain tissue extracts. Amyloid plaques scattering in the brains of 3xTg-AD mice was found, as stained by Ths that typically stains the P-pleated sheet conformation of mature amyloid plaques. The majority of Ths-positive plaques were observed in the subiculum of the mouse brains (Figure 6A). As expected, a noticeable reduction in the number of amyloid plaques was seen in the brains of 3xTg-AD mice after immunization with the 5-plex vaccine compared to the mice without prior immunization, which was confirmed by quantification analysis (Figure 6B). These results suggest that immunization with the 5- plex vaccine can delay or reduce amyloid pathology in the brains of 3xTg-AD mice.However, when the areas of the Ths-positive staining were quantified, such a reduction did not reach statistical significance (Figure 6C). Since Ths stains mature amyloid fibrils with a P-sheet conformation only, the brain tissue sections were stained with an Ap antibody. As expected, the Ap immunofluorescent staining revealed more and larger amyloid plaques than the Ths staining in the subiculum (Figure 6 C,E). Importantly, immunization with 5-plex significantly reduced the amyloid plaque area in the subiculum (Figure 6 D,E). The amyloid plaques tested by both Ths and Ap antibody staining were found to be restricted in the subiculum area of 3xTg-AD mice. In addition, the ELISA assays of Ap40 and Ap42 levels in the brain extracts did not show significant differences between the 3xTg-AD mice with prior immunization with the 5-plex vaccine and those without prior immunization (Figure 13A-C).

[0104] Effects of Immunization of 3xTg-AD Mice with 5-plex Vaccine on Neuronal and Glial Proteins. Neuronal and synaptic loss are among the pathological features of AD and correlates strongly with the progression of the disease. The neuronal and synaptic proteins in the brains of mice post immunization with the 5-plex vaccine were studied. Significant increase in the level of presynaptic protein synapsin 1, postsynaptic protein PSD95, and neuronal marker NeuN was found in the 3xTg-AD mice after immunization as compared to those without immunization (Figure 7). These results suggest that the cognitive benefits of the vaccine observed in this study might partially be attribute to the protection of neurons and synapses with the immunization. It was also determined that the astrocyte marker GFAP and microglia marker Ibal, as elevations of these two markers in the brain indicate neuroinflammation. A slight decrease in the GFAP level and no change in theIbal level with the 5-plex vaccination was found (Figure 7). These results indicate that immunization with the 5-plex vaccine does not induce chronic neuroinflammation.

[0105] Safety of the 5-plex Vaccine. The most significant adverse effects of the amyloid immunotherapy for AD are the increased risk for microhemorrhages and cerebral amyloid angiopathy, which are probably caused by vaccine-induced overactivation of immune response such as T-cell response. Therefore, the T-cell immune response was investigated by using IFN-y ELISpot. No activation of the T-cell response was found after the post-immune spleen were restimulated with Ap or Tau antigens used for the 5-plex vaccine, as assessed by counting the IFN-y dots (Figure 8A,B) On the contrary, robust stimulation of the T-cell response was observed in mice immunized with BRM28, an unrelated antigen, along with QS-21 as an adjuvant under the same immunization conditions. The elevated spot count implies that the adjuvant enhances the T-cell response (Figure 14A,B), hinting at the existence of a T-cell epitope within the BRM28 peptide, unlike in Ap or Tau peptides. These results suggest that immunization with our multivalent liposomal vaccine does not induce heightened Ap-specific or Tau-specific cellular responses in mice.

[0106] It was investigated whether immunization of mice with the 5-plex vaccine causes microhemorrhage by Prussian blue staining of the mouse brain tissue sections. The Prussian blue staining was only sparsely and randomly distributed in the brain. Minimal number of Prussian blue staining in the brain sections of WT mice was found (Figure 8C). This number was even smaller in 3xTg-AD mice than in WT mice (Figure 8D). Immunization with the 5-plex vaccine did not increase the Prussian blue staining in WT mice. A moderate increase in the Prussian blue staining was seen in 3xTg-AD after immunization, but the Prussian blue staining of these brain sections was still less than it in the WT mice (Figure 8D). These results suggest that immunization with the 5-plex vaccine has no or minimal risk for inducing brain microhemorrhages.

[0107] To investigate whether immunization with the 5-plex vaccine causes any peripheral immune / inflammatory activation in mice, blood samples were collected when the mice were sacrificed for measurements of the serum cytokine and chemokine levels. Some differences in the cytokine and chemokine levels between the 3xTgAD mice and WT control mice were observed, suggesting the role of the transgenes in the immune / inflammatory homeostasis in mice. Importantly, no serum cytokine or chemokine, except for GM-CSF in WT mice, showed higher levels in the vaccinated mice as compared to the unvaccinated mice (Figure 8E, Table 1A-C). Instead, the vaccination was found to significantly reduce the level of IL-P, TNFa, and MCP-1 / CCL2 in the WT mice (Table 1A) and of IL-6, IL-12p70, andGM-CSF in the 3xTg-AD mice (Figure 8E). These results, together with the absence of neuroinflammation in the brains of the vaccinated mice observed above, suggest that immunization with the 5-plex vaccine does not cause any detectable chronic inflammatory response in the brain or in the periphery.

[0108] The general conditions of the mice during and after immunization with the 5- plex vaccine was monitored. We did not observe any noticeable changes in general health conditions or behaviors with the vaccination. The growth curves were not significantly different between the vaccinated and unvaccinated mice, although the 3xTg-AD mice showed moderately heavier body weights than the WT control mice (Figure 15). The small, temporary body weight losses seen during weeks 32-37 correlated to the stress induced by behavioral tests performed on these mice.

[0109] Ap and tau play a pivotal role in the pathogenesis and progression of AD; therefore, the development of clinically effective immunotherapy targeting Ap and / or tau is a promising disease-modifying approach for treating AD. In this study, we developed and characterized a novel AD vaccine, the 5-plex, which targets two epitopes of Ap peptide and three epitopes of tau proteins simultaneously. The high binding efficiency and conformational rigidity of the 5-plex vaccine were confirmed by the filtration assays and epitope-specific antibody in slot blots, indicating that the his-tagged antigens readily particlelized with CoPoP liposomes, but not PoP liposomes. HPLC was performed to separate peptides in 5-plex, which is useful for eventual AD vaccine component quantification. Both individual and multivalent antigens from the 5-plex effectively exhibited enhanced antibodies in mice when complexed with CoPoP / PHAD liposomes, showing the rapid cellular uptake of smaller particle-based antigens in draining lymph nodes to trigger immunogenicity. The 5-plex vaccine elicited high titers of specific antibodies against both Ap and tau in mice and reacted to amyloid plaques and NFTs in IHC staining without significant immune interference effects. Mice after vaccination can produce high levels of specific antibodies for a relative long period of time, since 21-34% of the highest antibody titers was still observed 28 weeks after the last boosting dose.

[0110] To assess the efficacy of this novel vaccine in treating AD, it was tested in 3xTg-AD mice, a commonly used transgenic mouse model for AD by initial immunization at the age of 3-4 months (prior to the development of AD-like brain pathology and cognitive deficits), followed by 3 boosters with a 3-week interval. It is interesting to note that the 3xTg- AD mice after immunization with 5-plex showed reduced locomotive activity in the open field test. However, the anxiety level of the mice did not appear to be altered by the vaccinesince the central entries, the times spent in the central area, and the central distances travelled in the open filed test were not significantly differed between the two groups. No significant differences were observed between 3xTg-AD mice and the WT control mice either. The reduced locomotive activity in the open field did not cause bias for MWM assessment because 3xTg-AD mice immunized with or without 5-plex exhibited similar swimming speed during both MWM and rMWM tests. When the spatial memory was assessed in MWM at the age of 11 months, no significant effects of the vaccine were observed. Thus, the rM WM test was performed, which is more sensitive than the standard MWM test for assessing the cognitive function in rodents. It was found that the 3xTg-AD mice immunized with the 5- plex vaccine displayed better spatial learning and reference memory than the control -treated 3xTg-AD mice in rMWM. These results suggest that the 5-plex vaccine can prevent or slow down the development of cognitive impairment in this AD mouse model. This prophylactic efficacy of the vaccine is consistent with the reduction of tau hyperphosphorylation and of amyloid plaques, as well as the increases in neuronal / synaptic markers, in the brains of mice after immunization, compared with unimmunized mice.[OHl] It was noticed that the antibodies generated by the selected phospho-Tau peptides for this disclosure also reacted to the non-phosphorylated tau peptide counterparts with slightly weaker affinities. However, the cross-reactivity of tau antibodies generated in mouse sera to non-phosphorylated tau peptides is unlikely to raise any concerns or to prevent the development of the multivalent vaccine for clinical use. In AD brain, abnormally hyperphosphorylated tau aggregates together with normal tau to form tau seeds and neurofibrillary tangles. It is currently believed that tau pathology propagates from degenerative neurons to healthy neurons through seeding mechanisms. Therefore, tau immunotherapy may potentially act through blocking the spreading of oligomeric / aggregated tau at the extracellular space. If tau seeds at the extracellular space are cleared by tau antibodies against either normal tau or phosphorylated tau, therapeutic efficacy could be expected. Antibodies against phospho-tau and unphosphorylated tau are both currently under investigation for the development of AD therapeutics.

[0112] Both active and passive immunotherapy might induce over-activation of the innate and adaptive immune systems, resulting in adverse side effects. The development of a clinically effective AD vaccine should avoid unforeseen neuroinflammation in the brain. The clinical trials of AN1792, a vaccine targeting full-length Ap, were terminated due to the occurrence of T cell-mediated meningoencephalitis in 6% of treated AD patients. To avoid Ap-induced specific T-cell response, the vaccine and monoclonal antibodies targetingdifferent epitopes and forms of Ap is absent from T-cell epitopes. Active vaccine with partial sequences selected from AP1-14 can facilitate B-cell receptor (BCR) binding onto a B cell surface, which trigger intracellular signaling pathways and strong B-cell activation without T- cell help in order to induce immune response.

[0113] Therefore, the 5-plex liposomal vaccine containing Api-14 and APpE3-14 without T-cell epitope 16-33 did not activate T-cell immune response. Those animals demonstrated tolerance to the current immunization procedure without significant inflammatory response. Monitoring the cellular immune response of secreted IFN-y using ELISpot assays after sacrificing mice on day 42 provides insight into autoreactive T-cells inactivation in vitro. Importantly, immunization with the 5-plex vaccine decreased the peripheral levels of pro-inflammatory cytokines, including IL-P, TNFa, IL-6, IL-12p70, and GM-CSF. Furthermore, immunization with the 5-plex did not activate the microglia cells in the brain. Taken together, these data indicate that the 5-plex vaccine does not cause any detectable inflammatory response in both WT and 3xTg-AD mice.

[0114] Immunotherapy against Ap can lead to microhemorrhages and cerebral amyloid angiopathy (CAA) in different human APP transgenic mouse models. The exact mechanisms leading to microhemorrhage remain to be elucidated. One hypothesis is that the clearance of amyloid from the brain might cause the accumulation of CAA, and this CAA might result in overall weakening and leakage of vasculature, leading to microhemorrhage. The functional consequences of microhemorrhage remain unclear. Ap immunotherapy significantly improved cognitive function with the presence of microhemorrhages in different mouse models. In the present study, we found that immunization with four doses of the 5- plex vaccine caused only a slight increase in microhemorrhages above the baseline in 3xTg- AD mice, but no increase at all in WT mice. In addition, the level of microhemorrhages in the vaccinated 3xTg-AD mice were still lower than the baseline level of the WT mice. These observations suggest that the 5-plex vaccine is safe and is unlikely to lead to significant microhemorrhages in the brain. Since the 5-plex vaccine did not cause microhemorrhages in WT mice, the slight increase in microhemorrhages observed in 3xTg-AD mice with the vaccination might be due to the amyloid-related abnormalities, which have been observed in human clinical trials. The incidence of amyloid-related imaging abnormalities with microhemorrhage is variable in clinical trials from 4.9% for Solanezumab and Crenezumab, 10.7% for Lecanemab, 16.2% for Gantenerumab, 19.5% for Aducanumab, and 30.5% for Donanemab. Fortunately, amyloid-related imaging abnormalities with microhemorrhage aregenerally not associated with clinical sequelae, similar to microhemorrhages and superficial siderosis when occurring independently from anti-amyloid therapy.

[0115] The present prophylactic study demonstrates the efficacies of the 5-plex vaccine in preventing or reducing the development of cognitive impairment and AD-like brain pathologies in 3xTg-AD mice. The multivalent AD vaccine approach targeting both key AD pathologies simultaneously has a potential to produce better therapeutic efficacy than any single target AD immunotherapies. Future studies are needed to assess the efficacies in reducing and even reversing cognitive impairment and AD-like brain pathologies using a therapeutic approach, i.e., immunization of mice after the onset of cognitive impairment and brain pathologies. Studies incorporating both male and female of transgenic mice should be addressed to explore potential gender-specific differences and enhance the applicability of our results for Alzheimer’s Disease. Finally, more thorough safety and immunological analysis is desired. The immune response against the abbreviated his-tags (3-4 histidine residues used to display each peptide with CoPoP liposomes) was not assessed, but previous clinical studies showed that even full length his-tags (6 histidine residues) did not induce appreciable anti-his tag antibodies in human clinical testing.

[0116] In conclusion, it was found that CoPoP / PHAD liposome rapidly generated a spontaneous particle formation of Ap and tau peptides and induced robust antibody production with high levels of IgG titers through intramuscularly administrated 5-plex in both WT and 3xTg-AD mice. In 3xTg-AD mice, active 5-plex vaccination reduced AD-like pathologies and cognitive impairment. Immunization appeared to be well-tolerated without activating T-cell immune responses for neuroinflammation. This 5-plex multivalent vaccine targeting multiple epitopes of both Ap and tau simultaneously may potentially offer a novel therapeutic opportunity for AD.

[0117] Materials: His-tagged APi-u (DAEFRHDSGYEVHHHH (SEQ ID NO:1)), AppE3-i4 (pEFRHDSGYEVHHHH (SEQ ID NO:2), N-terminal pyroglutamate-modified), tauPTi8i (HHHHIPAKTPPAPK[phospho-T]PPSSGEPPKS (SEQ ID NO:4)), taupT2i7 (HHHHGSRSRTPSLP[phospho-T]PPTREPKKVA (SEQ ID NO:5)), taUPS396 / S404 (HGAEIVYK[phospho-S]PVVSGDT[phospho-S]PRHHHH (SEQ ID NO:7) peptides were synthesized by GenScript. CoPoP was produced as described previously. CoPoP liposome was synthesized using the following lipids: CoPoP, l,2-dipalmitoylsn-glycero-3- phosphocholine (DOPC, Corden # LP-R4-078), cholesterol (PhytoChoi, Wilshire Technologies), Monophosphoryl Hexa-acyl Lipid A, 3 -Deacyl (Synthetic PHAD, Avanti Cat # 699855P). Other reagents used were alhydrogel 2 % aluminum gel (InvivoGen, CAS#21645-51-2), goat anti-mouse IgG HRP (Novex, # A16072), and 3, 3’, 5,5’- tetramethylbenzidine solution (Surmodics, TMBW-0100-01). QS-21 was obtained from Desert King, and ELISpot kit (Mouse IFN-y Single-Color) was obtained from Mabtech.

[0118] Preparation and characterization of liposomes. CoPoP / PHAD liposomes were prepared by ethanol injection, followed by nitrogen-pressurized lipid extrusion in phosphate-buffered saline (PBS) as previously described. The CoPoP / PHAD (CP) liposome formulation had a mass ratio of [DOPC:CHOL:PHAD : CoPoP] [20: 5: 0.4: 1], while PoP / PHAD liposomes served as control liposomes with a formulation of [DOPC:CHOL:PHAD:PoP] [20: 5: 0.4: 1], PoP lipids were similar to CoPoP, but lack of cobalt. Lipids were first dissolved in ethanol at 55 °C for 10 min, and then re-dissolved in PBS at 55 °C for another 10 min. Sonication of dissolved lipid could be used to break up large un dissolved particles. Liposomes were extruded multiple times via 200-, 100-, and 80- nm stacked polycarbonate membrane filters in a lipid extruder [Northern Lipids] under nitrogen pressure. Excess ethanol was removed through dialysis in 1 L PBS at 4 °C overnight. The final concentration was adjusted 320 pg / mL CoPoP or PoP and stored at 4 °C. QS-21 (1 mg / mL) was added at the same mass ratio as PHAD after the liposome has synthesized. Dynamic light scattering (DLS) with a NanoBrook 90Plus PALS instrument was used to measure liposome sizes and polydispersity index (PDI) of samples after 500-fold dilution in PBS.

[0119] Vaccine Preparation. CoPoP / PHAD / + / -QS21 liposomes (320 pg / mL) and peptides (80 pg / mL) were incubated at a 4: 1 mass ratio for 3 h at 37 °C and then stored at 4 °C overnight. Bivalent (2-plex) was carried out by incubating A0i-i4 and tauPs396 / s404 with liposomes, whereas 5-plex was admixed A i-i4, A0PE3-I4, taupTisi, tauPT2i7, and tauPs396 / s404 with liposomes, and was incubated in either way: “mosaic” refers to liposomes that display multiple antigens per liposome (antigens mixed with liposomes), and “cocktail” refers to liposomes that display only one antigen per liposome (each antigen incubated with liposomes first, then mixed before injection). All liposomal vaccines were further reconstituted to a final concentration of 1 pg individual antigen or 3 pg bivalent antigens (1.5 pg for each) or 5 pg multivalent antigens per 50 pL diluted with PBS before injection. Alum vaccine was prepared by mixing indicated five antigens with 1.5 mg / mL of 2% aluminum gel (InvivoGen, CAS# 21645-51-2) diluting within HEPES buffer (10 mM HEPES, 150 mM NaCl, pH 7.0) for 1 h, and was resuspended by pipetting before injection.

[0120] Antigen-Liposome Binding Assay. CoPoP / PHAD or PoP / PHAD liposome binding with antigens were admixed and incubated at 37 °C for 3 h with a 4: 1 mass ratio ofliposome to antigen. The binding percentage of liposomes to antigen was carried out by bicinchoninic acid assay (BCA, Fisher catalog# PI23235) using a micro-centrifugal filtration assay. A 50-pL of incubated mixture was diluted to 200 pL with PBS, and centrifuged at 1200 g using a 100 kDa centrifugal tube (PALL) for 60 min at room temperature to allow free peptides in supernatant to be extracted from liposomes. The supernatant was further incubated with BCA reagent at 1 : 1 volume ratio at 60 °C for 30 min, and transferred 100 pL into a 96-well plate in triplicate. The absorbance was measured at 562 nm, and the following equation calculated the binding percentage:% Binding = [1-OD562of filtered liposome + peptides / OD562of filtered peptides in supernatant only] x l()0%

[0121] Slot blot Assay. His-tagged synthetic Ap and phosphorylated Tau peptides were reconstituted to 80 pg / mL with PBS, followed by a 3 h incubation at 37 °C with 320 pg / mL CP or 2HP [4: 1 liposome: antigen]. Peptides alone serve as a negative control. A 48- well slot blot apparatus (Bio-Rad, catalog# 1706542) was set up as described in the manufacturer’s instructions. Fifty microliters of PBS were first applied to each well to investigate any leakage. A hundred microliters of diluted samples (10 ng / mL) were loaded into a prewetted 0.2-pm nitrocellulose membrane (Thermofisher, catalog# 77012). The membrane was further cut into strips and blocked using 5% BSA in PBS on a plate shaker for 1 h at room temperature. After blocking, the strips were incubated with antigen-specific primary antibodies in 5% BSA: anti-Ap clone 6E10 diluted 1 :2500 (Biolegend, catalog# 803004), anti-Glu3 clone 337.48 diluted 1 :2500 (Biolegend, catalog# 822301), anti-Tau Phospho (Thrl81) clone M7004D06 diluted 1 :2500 (Invitrogen, catalog# 710561), anti-Tau Phospho (Thr217) diluted 1 :2500 (Invitrogen, catalog# 44-744), and anti-Tau Phospho (Ser396 / Thr404) clone PHF-1 (from lab of Albert Einstein College) diluted at 1 :5000 for 1 h at room temperature. The strips were separately incubated with anti-mouse (Cell Signaling, catalog #7076S) or anti-rabbit (Cell Signaling, catalog #707 S) HRP-conjugated secondary antibody (1 :2000 dilution) in 5% BSA for 30 min after rinsing with PBS twice. VisiGlo HRP substrate mixture (VWR, catalog# 97063-148) was applied onto the membrane after washing with PBS twice. The membrane was imaged using a Bio-Rad ChemiDoc™ Imager.

[0122] ELISA (Enzyme-linked immunosorbent assay). The 96-well microtiter plates were coated with 2.5 pg / mL fibrillar Api-42, pyroglutamate-modified A.PE3-i4, taupnsi, tauPT2i7, or tauPs396 / s404 peptides in 0.1 M sodium carbonate / bicarbonate buffer, pH 9.6, for 1 h at 37 °C. The preparation of fibrillar Ap42 was performed as described. After washing theplates with PBST (0.05% Tween-20 in PBS) twice, non-specific binding sites were blocked with 2% bovine serum albumin (BSA) in PBST for 1 h at 37 °C. Subsequently, 100 pL of serially diluted mice antisera (collected from blood after centrifuged at 2000 g for 20 min) was added to the wells for 1 h incubation at 37 °C after washing with PBST 3 times, followed by the incubation of 100 pL secondary anti-mouse IgG conjugated HRP at a dilution of 1 :2000 for 30 min at 37 °C. The 3, 3’, 5, 5’- tetramethylbenzidine solution was applied to the wells for signal detection, and the reaction was stopped by IM HC1 after 15 min. The absorbance was read at an optical density (OD) 450 nm. Antigen-specific IgG titers were calculated as the reciprocal serum dilution at which absorbance at 450 nm exceeded background value by 0.5 absorbance unit.

[0123] Reverse-Phase HPLC Analysis. RP-HPLC analysis on peptides was carried out by an analytical column and run as follows: sample peptide concentrations were diluted to 50 pg / mL in DI water. Twenty pL of each sample (run at 10 °C) was injected into a Inertsil ODS-3 column (250 mm x 4.6 mm, 100 A, 37 °C) at 1 mL / min flow rate. Mobile phase A: 0.065% Trifluoroacetic Acid (TFA) in distilled water, mobile phase B: 0.05% Trifluoroacetic Acid (TFA) in Acetonitrile (€). Gradient method: 0 min - 5% B, 25 min - 65% B, 25.1 min - 95% B, 32 min - 95% B, 32.01 min - 5% B, 35 min - 5% B. UV absorption was detected at 220 nm.

[0124] Immunization of Mice. The animal studies were approved by University at Buffalo Institutional Animal Care and Use Committee (IACUC) and New York State Institute for Basic Research in Developmental Disability IACUC. Groups of female CD-I mice (from Envigo RMS LLC) of age 5-6 weeks (n = 5) were immunized by intramuscular injection of 50 pL liposomal vaccines on day 0 and 21. They were bled for serum collection on day 42 under isoflurane anesthesia. The serum was examined by ELISA and sent to the Department of Aging Neurobiology at National Center for further IHC staining analysis. Similarly, adult female triple-transgenic(3xTg)-AD mice (MMRRC: 034830-JAX) of age 3 months and B6129SF2 / J WT mice (strain #101045 from Jackson Laboratory) of age 2 months (n = 20 for wild type / 5-plex, n = 20 for wild type / control, n = 20 for 3xTg-AD / 5- plex, and n = 20 for 3xTg-AD / control) were vaccinated prophylactically beginning at the age of 3 or 4 months. The 3xTg-AD mice develop AD-like pathologies and cognitive impairment in an age-dependent manner, including neuroinflammation, amyloid plaques, and cognitive impairment starting at ~6 months of age, and neurofibrillary tangles starting at ~12 months of age. The pathologies are predominantly restricted to the hippocampus, amygdala, and cerebral cortex. Compared to males, female 3*Tg-AD mice exhibit more consistent brainpathologies and cognitive impairment and thus were employed in this first study. Both B6129SF2 / J wild type and 3xTg-AD transgenic mice were immunized with 5 pg of 5-plex once per month to maintain a steady antibody level, and submandibular blood collection was performed before subsequent immunization. After four injections at three-week intervals, those mice were quarantined at Charles River and sent to the Department of Neurochemistry at New York State Institute for Basic Research for behavior testing and biochemistry analysis. All mice were sacrificed at age 12-12.6 months, i.e., after completion of behavioral tests.

[0125] Splenocytes Collection and ELISpot. Antigen-specific antibody response of IFN-y was measured by enzyme-linked immune absorbent spot (ELISpot) assay. Splenocytes were harvested from immunized mice after being euthanized on day 42. Each spleen was mashed through a 70 pm cell strainer, and splenocytes were collected in a 50 mL centrifuge tube. Cells were centrifuged at 500 g for 5 min and applied 5 mL red blood cell lysis buffer for another 5 min on ice. After incubation, splenocytes in lysis buffer were diluted with 40 mL of PBS and centrifuged at 500 g for 5 min. Cells (1 : 100 dilution) were counted in a hemocytometer after resuspended in 1 mL PBS. ELISpot plate was seeded 3xl05splenocytes and admixed with 10 pg / mL peptides, or PHA-L positive control, or media alone for negative control in each well. Cells were further cultured in a humidified chamber (5% CO2 / 95% air at 37 °C) for 24 h. The spot detection was performed regarding to the manufacturer instructions using Murine IFN-y Single-Color ELISpot kit from Immunospot. The images with number of spots counted were acquired by CTL ImmunoSpot S6 FLuoroCore analyzer.

[0126] Immunohistochemical staining. Paraffin-embedded postmortem brain slides of AD patient were obtained from the Brain Bank for Aging Research (BBAR) at the Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology under the approval of the institutional ethics committees (No.1719-2). After deparaffinization, brain sections were submerged in EDTA buffer (1 mM EDTA, pH 8.0) and microwaved at 500 watts for 15 min. These sections were immersed in 95% formic acid for 30 min for antigen retrieval. Then, endogenous peroxidase activity was quenched by incubating with 3% hydrogen peroxide for 5 min at room temperature. After blocking with Protein Block solution (Dako), sections were incubated with 100-fold diluted each anti-serum at 4 °C overnight. The next day, sections were incubated with anti-mouse HRP-conjugated secondary antibody, and then reacted with diaminobenzidine reaction mixture (ImmPACT DAB substrate kit, Vector Laboratories). Sections were counterstained with Gill’s Haematoxylin II (Muto Pure Chemicals, Tokyo,Japan), mounted by Entellan® new (Merck), and observed under a microscope (BZ-X810, Keyence, Osaka, Japan).

[0127] Cryo-Electron microscopy. Sample vitrification was performed using a Vitrobot Mark IV (Thermo Fisher Scientific). Holey carbon grids (C-Flat 2 / 2-3Cu-T) were washed with chloroform for 2 h before sample vitrification. Grids were treated with negative glow discharge in air at 5 mA for 15 sec, right before the sample was applied. For all samples, a volume of 3.6 pL of the liposome sample was applied to a holey carbon grid and manually blotted using the Vitrobot blotting paper (Standard Vitrobot Filter Paper, 055 / 20 mm, Grade 595). Then a volume of 3.6 pL of the same sample was applied for a second time to the same holey carbon grid, and the grid was blotted once in the Vitrobot for 3 sec using a blot force +1 before plunging it into liquid ethane at —190 °C. The Vitrobot was set at 25 °C and 100% relative humidity. Data was acquired using SerialEM software on the Titan Krios electron microscope at FEMR-McGill, operated at 300kV. The sample was kept at -190 °C during imaging. Images were collected with a Gatan K3 direct electron detector equipped with a Bioquantum imaging filter. The nominal defocus was set at -3.00 pm. Images were collected using a total exposure of 50 e- / A2 at a nominal magnification of 81,000 x corresponding to a calibrated pixel size of 1.09 A.

[0128] General examination of mice. A general examination of all the mice was conducted in the home cages throughout the whole study. Any gross abnormalities in overall health, home cage nesting, sleeping, feeding, grooming, and condition of the fur of animals were noted. Body weight was measured once a week during the period of the study.

[0129] Open field. The Open field test was commonly used to simultaneously locomotion, exploration and anxiety in rodents and was performed as previously described. Briefly, the mice were carried to the test room to habituate 1 h before starting the test. Each mouse was placed in the open field arena (made of opaque white plastic material, 50 cm x 50 cm x 40 cm, length x width x height) by a blinded experimenter and allowed to explore the arena for 10 min. The distance travelled (meters) in open field arena, entries. Time spent in the central area, and distance travelled (%) in the central area (10 cm x 10 cm), were automatically recorded by a video tracking system (Any maze version 4.5 software, Stoelting Co.)

[0130] Morris water maze task. Morris water maze (MWM) task was used to evaluate the spatial learning and memory of the mice (D'Hooge and De Deyn, 2001; Vorhees and Williams, 2006; Zhao et al., 2020). The test was performed in a circular white pool (with a diameter of 180 cm and a height of 60 cm) filled with non-toxic white dye tinted water andmaintained at room temperature (20 ± 1 °C). The maze was designated of two virtual principal axes with each line bisecting the maze perpendicular to the other one to divide the maze into four equal quadrants. The end of each line demarcates four cardinal points: North (N), South (S), East (E) and West (W). A platform was positioned in the middle of one of the quadrants submerged 0.5 cm below the water surface. Each mouse performed 4 trials on for 4 consecutive days from semi-random start positions (Vorhees and Williams, 2006) to find the hidden platform. Each trial was terminated after the mouse climbed onto the hidden platform. If a mouse failed to find the platform within 90 sec, it was gently guided to the platform. At the end of each trial, the mouse was left on the platform for 15 sec, then removed, dried and returned to its home cage. A 90-sec probe test without a platform was performed 24 h after the last trial. Escape latency (sec) in initial training, and latency to 1stentrance into target (platform location area), time spent in target quadrant (sec) and in the platform area, target crossings and swim speed (cm / sec) in probe test were recorded through an automated tracking system (Smart video tracking system, version 2.0.14, Panlab; Harvard Apparatus).

[0131] Reversal Morris water maze test. The reversal Morris water maze (rMWM) task was conducted 9 days after the probe test in the MWM. A similar procedure was used in the MWM, except the invisible platform was moved to the quadrant, which was in an opposite location in the MWM. The probe test was conducted after 3 consecutive days of initial training.

[0132] Tissue processing. All mice were sacrificed by cervical dislocation. Forebrain cortex and hippocampus were detached immediately from the right hemisphere and frozen in dry ice for biochemical analysis. The left hemisphere was fixed in 4% paraformaldehyde in 100 mM phosphate buffered saline (PBS) for 24 h at room temperature. Tissues were then post-fixed in a 30% sucrose solution at 4 °C for overnight. Forty pm sagittal sections of the entire half hemisphere were cut using a freezing microtome. The sections were stored in glycol anti-freeze solution (ethylene glycol, glycerol, and 100 mM PBS in 3:3:4 ratio) at - 20CC until further processing.

[0133] Mouse serum collection. Blood collected via cardiac puncture immediately after cervical dislocation at the experimental endpoint was started at 4 °C for 1 h. The serum was collected by centrifuge at 1000 gx10 min at 4 °C. The serum was aliquoted for antibodies titers and cytokines analysis (Creative Proteomics, NY, USA)

[0134] Mouse cytokines analysis. The levels of preinflammatory cytokines, antiinflammatory cytokines, and preinflammatory chemokines were analyzed by Luminex multiplex assay by Creative Proteomics (New York, USA).

[0135] Western blot analysis. Mouse hippocampus was homogenized in pre-chilled buffer containing 50 mM Tris-HCl (pH 7.4), 100 mM sodium fluoride, and 1 mM sodium othovanadate, 1 mM EGTA, 0.5 mM AEBSF, 10 pg / ml aprotinin, 10 pg / ml leupeptin, 4 pg / ml pepstatin. Each homogenate was boiled in 2* Laemmli’s buffer for 10 min, and protein concentration was measured by Pierce™ 660 nm protein assay (Thermo Scientific, Rockford, IL, USA). The samples were resolved by 10% or 12% SDS-PAGE and electro-transferred onto Immobilon-P membrane (Millipore, Bedford, MA, USA). The blots were then probed with primary antibodies and developed with the corresponding horseradish peroxidase- conjugated secondary antibody and ECL kit (Pierce, Rockford, IL). Densitometrical quantification of protein bands in Western blots were analyzed by using the Multi Gauge V3.0 software (Fuji Photo Film Co., Ltd).

[0136] Sarkosyl-soluble and -insoluble tan. Cortical sarkosyl-soluble and -insoluble tau were prepared by homogenization of cortical tissue from brains of WT / Control. WT / 5- plex, 3xTg-AD / Control, and 3xTg-AD / 5-plex mice with 8 volumes of buffer (50 mM Tris- HCl, 50 mM NaF, 2 mM EGTA, 2 mM Na3VO4, 0.5 mM AEBSF, and 10 pg / ml aprotinin, leupeptin, and pepstatin). The homogenates were centrifuged at 14,000 g for 10 min at 4°C. The supernatant was adjusted to 1% N-lauroylsarcosine and incubated for 1 h at room temperature. After the incubation, supernatant was spun at 200,000 g for 45 min at 25 °C. The supernatant (sarkosyl soluble) and the pellet (sarkosyl insoluble) was dissolved in Laemmli sample buffer and subjected to Western blot analysis (18658135).

[0137] Immunofluorescence and thioflavin S staining. Serial 6 free-floating sagittal sections per mouse from 13-14 mice per group were washed in 10 mM PBS (15 min x 3) and then incubated in 0.3% Triton X-100 for 1 h. The sections were blocked in blocking solution (5% normal goat serum, 0.1% Triton X-100 and 0.05% Tween-20 in PBS) for 1 h. Sections were then incubated with AT8 antibody against phosphorylated tau at Ser202 / Thr205 (0.2 pg / ml) at 4 °C overnight. After washing three times for 15 min each with 10 mM PBS, sections were incubated with Alexa 594-conjugated donkey anti-mouse IgG secondary antibodies (1 :500, Molecular Probes, Carlsbad, CA, USA) in 10 mM PBS with 0.05% Tween-20 for 2 h at room temperature followed by incubating with 0.1 pg / ml DAPI for 10 min. Sections were subsequently washed and mounted on a slide, and dried for overnight at room temperature. Sections were stained with 0.05% thioflavin-S in water in dark for 8 min. Finally, sections were washed in 80% ethanol for 2 min and in water for 3 min, and mounted and cover slipped using prolong gold antifade reagent from Thermo Fisher Scientific Inc (Waltham, MA, USA). For A|3 immunofluorescence staining, serial 6 free-floating sagittalsections per mouse from 13-14 mice per group were washed in 10 mM PBS (15 min x 3) and then incubated in 0.3% Triton X-100 for 1 h. The sections were blocked in blocking solution (5% normal goat serum, 0.1% Triton X-100 and 0.05% Tween-20 in PBS) for 1 h. Sections were then incubated with A antibody (0.25 pg / ml, Cell Signaling, Cat#8243S) at 4 °C overnight. After washing three times for 15 min each with 10 mM PBS, sections were incubated with Alexa 488-conjugated goat anti -rabbit IgG secondary antibodies (1 : 1000, Molecular Probes, Carlsbad, CA, USA) in 10 mM PBS with 0.05% Tween-20 for 2 h at room temperature followed by incubating with 0.1 pg / ml DAPI for 10 min. Sections were subsequently washed and mounted on a slide, and cover slipped using prolong gold antifade reagent (Thermo Fisher Scientific Inc, Waltham, MA, USA). The maximum projection images were taken with Olympus Fluoview FV3000 confocal laser scanning microscope. AT8 positive neurons in the hippocampus CAI area ,thioflavin-S positive plaque load in subiculum, and A positive staining in subiculum were quantified using NIH Fiji (Image J) software.

[0138] Human A]J40 and A|J42 measurements by ELISA. Forty microliter hippocampus homogenate was mixed with 60 pl guanidine hydrochloride buffer (50 mM Tris-HCl, pH 8.0, 8.3 M guanidine hydrochloride) 4 h at room temperature and then stored at -80 °C. For ELISA measurement, each brain homogenate was diluted 1 :25 with ice-cold PBS containing lx protease inhibitor cocktail (Calbiochem, cat#539131) and centrifuged at 16,000xg for 20 min at 4 °C. The final concentration of AEBSF was 1 mM to prevent proteolysis of Ap peptides, and the final concentration of guanidine hydrochloride was 0.1 M. The supernatant was further diluted 1 :4 with standard diluent buffer and assessed using ELISA kit specific for Human Ap40 (Cat #KHB3481) or Ap42 (Cat #KHB3441) and calibrated with synthetic Ap peptides from Invitrogen according to the manufacturer’s instructions. The Ap40 and Ap42 peptide standards were prepared with the same composition of the buffer used to dilute the samples. The concentration of Human AP40 and Human Ap42 was further normalized to the total protein in the hippocampus homogenate measured by Pierce™ 660 nm protein assay.

[0139] Prussian blue staining. Prussian blue reaction is one of the most sensitive histochemical tests for microhemorrhages. Treatment of brain sections with acid solutions of ferrocyanides will liberate loosely bound ferric iron from protein. Ferric ion present in the tissue will combine with the ferrocyanide and form a bright blue pigment called Prussian blue, or ferric ferrocyanide. Six serial sagittal brain sections (40 pm) per mouse from 13 to 16mice per group were washed with PBS, mounted on the slide and dried for overnight at room temperature. On the second day, the sections were hydrated in distilled water for 3 min and then stained for hemosiderin using 5% potassium ferrocyanide (ThermoFhishe Scientific, Cat#424130025) in 5% hydrochloric acid (J.T. Baker, Cat#9535-00) for 15 min. After washing two times with distilled water, the sections were counterstained with nuclear fast red solution (Thermo Scientific, Cat#J61010.AP) for 10 min. The sections were washed twice with distilled water and then dehydrated in 90% alcohol x 3 min, 95% alcohol x 3 min, 100% alcohol for 2 x 3 min. Finally, the slide was cleared with 100% xylene for 2 x 3 min, followed by a coverslip. The numbers of Prussian blue-positive sites were counted on all sections and the average number of sites per section was calculated. All images were acquired using Olympus Fluoview FV3000 confocal laser scanning microscope.

[0140] Statistical Analysis. Statistically significant differences in results were analyzed by unpaired t-tests, one-way or two-way ANOVA analysis with Tukey’s multiple comparisons test as indicated. The p values of <0.05 were considered significant differences. All statistical analyses were performed using GraphPad Prism version 9.0 for MacOS (GraphPad Software, San Diego, CA, USA)

[0141] Data of biochemical data and behavior tests were analyzed using GraphPad Prism version 5.0 (GraphPad Software Inc, La Jolla, CA, USA) and one-way or two-way ANOVA (as appropriate) followed by a Bonferroni’s posthoc test. Further intergroup comparisons were also performed using an un-paired two-tailed Ltest. All data are presented as means ± SEM. P < 0.05 was considered statistically significant.

[0142] Table 1 : Immunization with 5-plex decreases the levels of cytokines and chemokines.Anti-inflammatoryChemokinesData shows mean ± SD of (top) the level of proinflammatory, (middle) the level of anti-inflammatory cytokines, and (bottom) the level of proinflammatory chemokines. N = 14 for WT / control, 16 for WT / 5- plex, 13 for 3xTg-AD / control, and 14 for 3xTg-AD / 5-plex.EXAMPLE 2

[0143] This example provides a description of compositions of the present disclosure.

[0144] Using a vaccine comprising 4 epitopes derived from A0 and Tan. We found that a vaccine comprising four epitopes, as listed below, is also effective in the animal model.

[0145] Methods. EcML is a form of MPLA. CoPoP / EcML liposomes (320 pg / mL) and peptides (80 pg / mL) were incubated at a 4: 1 mass ratio for 3 h at 37 °C and stored at 4 °C overnight. Our 4-plex vaccine was admixed A i-i4, A0PE3-I4, taupnsi, and tauPs396 / s404 with liposomes. This liposomal vaccine was further reconstituted to a final concentration of 2 pg individual antigen per 50 pL diluted with PBS before intramuscularly injected to the mice. Dynamic light scattering (DLS) with a NanoBrook 90Plus PALS instrument was used to measure liposome sizes and poly dispersity index (PDI) of samples after 500-fold dilution in PBS. We followed the immunization schedule by injecting WT / 3xTg-AD control with 50 pL CoPoP / EcML liposome, and 3xTg-AD mice with 50 pL 4-plex vaccine on every 3-week intervals (total 5 injections). Mice were euthanized after conducting behavior tests, and serum were collected for examining ELISA titers.

[0146] Table 2. Formulation table for 4-plex vaccine preparation and injectionEXAMPLE 3

[0147] This example provides a description of compositions of the present disclosure.

[0148] Storing the vaccine in a storage in a frozen state. The vaccine can be frozen and thawed without disrupting size or function. Freezing the vaccine enables long-term storage at temperatures below freezing such as -20 or -80 C.

[0149] Methods. CoPoP / PHAD liposomes (320 pg / mL) and peptides (80 pg / mL) were incubated at a 4: 1 mass ratio for 3 h at 37 °C and stored at 4 °C overnight. Our 4-plex vaccine was admixed with A i-i4, A0PE3-I4, taupnsi, and tauPs396 / s404 with liposomes, while the 5-plex vaccine was admixed with A0i-i4, A0PE3-I4, tauPTisi, tauni-22 and tauPs396 / s404. This liposomal vaccine was further reconstituted to a final concentration of 2 pg individual antigen and diluted to 50 pL with PBS to further access slot blot assay. After incubation, the vaccine was divided into 5 vials, each subjected to a different number of freezing cycles: 1, 2, 3, 4, and 5 cycles respectively. Each cycle consisted of freezing for 1 hour, followed by thawing, and then refreezing. Fifty microliters of PBS were applied to a 48-well slot blot apparatus to check for leakage. A hundred microliters of diluted samples (10 ng / mL) with varying freezing cycles were loaded onto a nitrocellulose membrane, cut into strips, and blocked with 5% BSA in PBS for 1 hour at room temperature. The strips were incubated with primary antibodies in 5% BSA for 1 hour: anti-A0 (1 :2500), anti-Glu3 (1:2500), anti-Tau Phospho (Thrl81, 1 :2500), anti-Tau Phospho (TauNl-22, 1 :2500), and anti-Tau Phospho (Ser396 / Thr404, 1 :5000). After rinsing with PBS twice, strips were incubated with HRP- conjugated anti-mouse or anti-rabbit secondary antibodies (1 :2000 in 5% BSA) for 30 minutes. VisiGlo HRP substrate (VWR #97063-148) was then applied, followed by imaging with a Bio-Rad ChemiDoc™ Imager. WT and 3xTg-AD mice (9- to 10-month-old) were immunized with CoPoP / PHAD liposomes only or 5-plex for 5 doses with a 3-week interval. Behavioral tests were conducted starting 72 hours after 5thvaccination, and all mice were euthanized 48 hours later after completion of behavioral tests.EXAMPLE 4

[0150] This example provides a description of compositions of the present disclosure.

[0151] Various MPLA types are effective. MPLA is a vaccine adjuvant and a TLR4 agonist. Various synthetic or natural MPLAs are available. We found no difference between a synthetic MPLA: PHAD-3D6A (abbreviated as PHAD) or an extract (EcML) with immunization.

[0152] Methods. CoPoP / PHAD liposomes were prepared by ethanol injection, followed by nitrogen-pressurized lipid extrusion in phosphate-buffered saline (PBS). CoPoP / PHAD (CP) and CoPoP / EcML liposome formulation had a mass ratio of [DOPGCHOL HAD: C0P0P] [20: 5: 0.4: 1], while the PHAD (ETOH) were dissolved in ethanol and admixed with C0P0P liposome through 1 hr sonication. Liposome bound with antigens were admixed at 4: 1 mass ratio and incubated at 37 °C for 3 h for immunization.The formulation data with dosage use in the following Table. Group of ICR mice (n=4) were immunized based on the listing below: Group 1-5 immunized with 4-plex, and group 6 immunized with 5-plex. Mice were primed on day 0, boosted on day 21, and euthanize for serum for ELISA titers on day 42.

[0153] Table 3 of AD 4-plex / 5-plex vaccine formulationEXAMPLE 5

[0154] This example provides a description of compositions of the present disclosure.

[0155] Background: Immunotherapy targeting Ap and / or tau proteins represents a promising disease-modifying therapeutic strategy for treating AD. Due to the complexity of both Ap and tau in AD pathogenesis, current immunotherapies which are generally focused on single epitopes within Ap or tau may limit their effectiveness. Therefore, a multipronged approach simultaneously targeting multiple epitopes of both proteins could overcome limitations of monotherapies. Here, we developed a pentavalent peptide vaccine which simultaneously elicits Ap and Tau antibodies and assessed its prophylactic activity in an AD mouse model, 3xTg-AD mice.

[0156] A nanoparticle vaccine comprising two Ap peptides (1-14 and pyroglutamate pE3-14) and three tau peptides (centered on phosphorylated pT181, pT217 and pS396 / 404), named 5-Plex, was formulated with the spontaneous nanoliposome antigen particle (SNAP) platform. The immunogenicity of 5-plex vaccine was assessed by ELISA. The specificity of 5-plex-induced antibodies was investigated in human AD brain tissue by immunohistochemistry. 3-4-month-old 3xTg-AD and age-matched wild type animals were intramuscularly vaccinated for 4 doses of 5-Plex vaccine with a 3-week interval. Twenty-nine weeks after 1stvaccination, the prophylactical effect on cognitive function was assessed by Morris water maze test and reversal Morris water maze test, and on Ap and tau pathologies was analyzed by Western Blotting, immunohistochemistry and ELISA.

[0157] Active immunization with 5-Plex vaccine induced high titers of epitopespecific antibodies which recognized plaques and neurofibrillary tangles from human ADbrain tissue. Importantly, 5-Plex administration to 3xTg-AD mice using a prophylactical dosing schedule inhibited tau and Ap pathologies resulting in improved cognitive function. Additionally, immunization was well tolerated and did not induce microhemorrhages in brain and antigen-specific cellular responses or persistent inflammatory responses in the peripheral or central nervous system.

[0158] These findings indicate that active immune therapies based on nanoparticle formulations of multiple Ap and tau epitopes warrant further study for treating early-stage AD.

[0159] Although the present disclosure has been described with respect to one or more particular embodiments and / or examples, it will be understood that other embodiments and / or examples of the present disclosure may be made without departing from the scope of the present disclosure.

Claims

CLAIMS:

1. A vaccine composition comprising: a) a plurality of liposomes, each liposome comprising i) a bilayer, wherein the bilayer comprises A) one or more phospholipids andwherein cobalt is chelated to the porphyrin or B) one or more phospholipids a porphyrin having cobalt chelated thereto; and ii) one or more amyloid P fragments and / or tau peptide fragments, each fragment having a polyHis-tag attached to a terminus of the fragments, wherein each His-tag comprises two or more histidine residues and at least a portion of the polyHis tag resides in the hydrophobic portion of the bilayer and one or more histidines of the polyHis tag are coordinated to the cobalt in the cobalt-porphyrin, and wherein at least a portion of the amino acid sequence is exposed to the outside of the liposome; and b) a pharmaceutical carrier.

2. The vaccine composition according to claim 1, wherein the one or more amyloid P fragments and / or tau peptide fragments are chosen from AP1-14 (DAEFRHDSGYEVHHHH (SEQ ID NO: 1)), APpE3-i4 ([pyro-E]FRHDSGYEVHHHH, N-terminal pyroglutamate- modified (SEQ ID NO:2)), tauni-22 (HHHHMAEPRQEFEVMEDHAGTYGLGD (SEQ ID NO:3)), tauPTi8i (HHHHIPAKTPPAPK[phospho-T]PPSSGEPPKS (SEQ ID NO:4)), taupT2i7 (HHHHGSRSRTPSLP[phospho-T]PPTREPKKVA (SEQ ID NO:5)), HGAEIVY[phosphor- K]SPVVSGDTS[phospho-P]RHHHH (SEQ ID NO:6), tauPs396 / s404 (HGAEIVYK[phospho- S]PVVSGDT[phospho-S]PRHHHH (SEQ ID NO:7), a peptide have at least 80% identity to any one or more of APi-i4 (DAEFRHDSGYEVHHHH (SEQ ID NO: 1)), AppE3-i4 ([pyro- E]FRHDSGYEVHHHH, N-terminal pyroglutamate-modified (SEQ ID NO:2)), tauni-22(HHHHMAEPRQEFEVMEDHAGTYGLGD (SEQ ID N0:3)), tauPTi8i (HHHHIPAKTPPAPK[phospho-T]PPSSGEPPKS (SEQ ID N0:4)), tauPT2i7 (HHHHGSRSRTPSLP[phospho-T]PPTREPKKVA (SEQ ID N0:5)), HGAEIVY[phosphor-K]SPVVSGDTS[phospho-P]RHHHH (SEQ ID N0:6), tauPs396 / s404 (HGAEIVYK[phospho- S]PVVSGDT[phospho-S]PRHHHH (SEQ ID N0:7), and any combination thereof.

3. The vaccine composition according to claim 2, wherein the composition comprises two, three, four, or five different amyloid P fragments and / or tau peptide fragments.

4. The vaccine composition according to claim 2, wherein the composition comprises A 1-14, APpE3-14, taUPT181, taUPT217, and taUPS396 / S404.

5. The vaccine composition according to claim 2, wherein the composition comprises A 1-14, APpE3-14, taUnl-22, taUPT181, and taUPS396 / S404.

6. The vaccine composition according to claim 2, wherein the composition comprises A 1-14, ApPE3-i4, tauni-22, and tauPTi8i.

7. The vaccine composition according to claim 2, wherein the composition comprises A 1-14, ApPE3-i4, tauPs396 / s404, and taUPT181.

8. The vaccine composition according to claim 2, wherein the composition comprises A 1-14, APPE3-14, taUnl-22, and taUPS396 / S404.

9. The vaccine composition according to claim 1, wherein the cobalt porphyrin-phospholipid conjugate makes up from 0.1 to 25 mol % of the bilayer.

10. The vaccine composition according to claim 9, wherein the cobalt porphyrinphospholipid conjugate makes up from 5 to 10 mol % of the bilayer.

11. The vaccine composition according to claim 1, wherein the polyHis-tag comprises 2, 3, or 4 histidine residues.

12. The vaccine composition according to claim 1, wherein size of the liposome is 50 nm to 200 nm.

13. The vaccine composition according to claim 1, wherein the liposomes further comprise one or more adjuvants.

14. The vaccine composition according to claim 13, wherein the one or more adjuvants are attenuated lipid A derivatives, phosphorylated hexaacyl disaccharides, and / or QS21.

15. The vaccine composition according to claim 1, further comprising one or more adjuvants that are not associated with the liposomes.

16. The vaccine composition according to claim 1, further comprising one or more sterols.

17. The vaccine composition according to claim 16, wherein the one or more sterols are cholesterol.

18. A method for generating antibodies against amyloid P and / or tau comprising administering to the subject a vaccine composition according to claim 1.

19. The method according to claim 18, wherein the administration does not induce an inflammatory response in the subject.

20. The method according to claim 18, wherein the vaccine composition is administered two or more times.

21. The method according to claim 18, wherein native aggregation of amyloid P and / or tau is reduced or inhibited in the subject.

22. The method according to claim 18, wherein the amount of amyloidogenic plaques associated with amyloid P and / or tau in the subject’s brain is reduced.

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