Treatment and prevention of neuropathologies associated with neurodegenerative diseases

Abstract

Administration of a live attenuated Bordetella pertussis-based vaccine to a subject at risk of developing a neurodegenerative disease characterized by Aβ brain plaques can prevent or reduce the amount of Aβ brain plaques that would develop in this subject in the absence of such treatment.

Description

[Technical Field]

[0001] Related technologies This application claims priority to U.S. Provisional Patent Application No. 63 / 114,909, filed on 17 November 2020.

[0002] Description of federally funded research and development. Not applicable.

[0003] Technical field This disclosure relates, in general, to the fields of microbiology, vaccinology, neurology, and medicine. More specifically, this disclosure relates to the prevention or reduction of neuropathologies associated with neurodegenerative diseases such as Alzheimer's disease (AD) in the subject by preventing or reducing Bordetella pertussis (BP) or asymptomatic BP colonization infection in the subject. [Background technology]

[0004] Alzheimer's disease (AD) is a neurodegenerative disease characterized by slowly progressive cognitive and behavioral impairment in individuals with intracellular neurofibrillary tangles (NFTs) composed of abnormal tau protein and extracellular plaques composed of amyloid-beta (Aβ) peptide. Current treatments only serve the symptoms of the disease, and despite considerable effort, no treatment has been approved to halt or reverse its progression.

[0005] The causes and progression of Alzheimer's disease (AD) are not well understood. Most cases of AD are sporadic and occur after age 65. The risk of developing the disease is most predictive with age. Genetic characteristics also play an important role in susceptibility to AD. Mutations at several different loci have been identified as influencing the onset and progression of AD. These mutations are found in genes including those encoding amyloid precursor proteins, presenilin I and presenilin II, as well as apolipoprotein E allotypes. For example, the presence of the APOEε-4 allele in subjects confers a relative risk of developing the disease that is 30 times higher than in non-carriers and 3.7 times higher in ε-3 / ε-4 heterozygotes (Myers, RH, et al. “Apolipoprotein E element 4 association with dementia in a population-based study: The Framingham Study.” Neurology 46.3(1996):673-677). The relationship between other factors (e.g., low hormone levels, metal exposure) and AD is under investigation, but a clear causal relationship has not been established.

[0006] Most experts believe that for decades, pathologically produced amyloid-beta (Aβ) fibrils and plaques accumulate in brain tissue, activating microglia and astrocytes, leading to neuroinflammation and ultimately toxicity to synaptic function and neuronal death. Other experts have suggested that systemic inflammatory diseases may cause neurodegeneration in AD, and that prions may be a cause of AD.

[0007] Another assumption, largely dismissed by the scientific community, is that Alzheimer's disease (AD) is caused by a bacterial infection. Clinical trials aimed at eliminating Chlamydia pneumoniae colonization in patients with mild to moderate AD have shown no statistically significant effect of antibiotic treatment compared to placebo. Molloy et al., Int J Geriat Psychiatry, 28:463-70, 2013. Other microorganisms that have been suggested to contribute to the pathogenesis of AD include human herpesviruses 1-6, hepatitis C virus, Helicobacter pylori, Borrelia burgdorferi, Treponema pallidum, Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Candida albicans, and Toxoplasma gondii. Sochocka et al., Curr Neuropharmacol, 15:996-1009, 2017. Nevertheless, since no specific pathogen has been definitively proven to cause AD, the concept that AD has an infectious source remains debatable. [Overview of the project]

[0008] It was discovered that administering a live attenuated BP-based vaccine to subjects at risk of developing neurodegenerative diseases characterized by Aβ plaques prevented or reduced the amount of β plaques that would have developed in these untreated subjects. Notably, a protective response to vaccination was observed even in subjects that were not subsequently infected with pathogenic strains of BP.

[0009] Based on these findings, this specification describes methods for preventing or treating pathological features of neurodegenerative diseases such as Alzheimer's disease (AD) in subjects who have or are at risk of developing the disease, by administering to a subject an agent that (a) prevents or reduces asymptomatic BP colonization infection or clinical BP infection, or (b) neutralizes BP toxins that cause or contribute to the pathological features of neurodegenerative diseases such as AD. This specification also describes methods for preventing or reducing β-amyloid plaques in the brain of subjects who have or are at risk of developing AD. The latter method comprises the step of administering to the subject a therapeutically effective amount of a bioattenuated Bordetella pertussis strain (e.g., in a pharmaceutically acceptable composition or vaccine) capable of colonizing the subject, and includes a defensive response in the subject that reduces the amount of β-amyloid plaques that would have formed or been present in the brain of the subject if the subject had not been administered the composition. Similarly, the use of a composition comprising a bioattenuated Bordetella pertussis strain to prevent or reduce β-amyloid plaques in the brain of a subject who has or is at risk of developing Alzheimer's disease, wherein the bioattenuated Bordetella pertussis strain can induce a protective response in the subject that colonizes and reduces the amount of β-amyloid plaques that have formed or existed in the brain of the subject if the subject were not administered the composition, is described herein.

[0010] The drug may be a bio-attenuated Bordetella pertussis strain capable of inducing a non-pathogenic BP asymptomatic colony-forming infection in the subject (e.g., respiratory colony-forming infection) and eliciting a protective response in the subject that prevents or reduces the pathological features of neurodegenerative disease. The bio-attenuated Bordetella pertussis strain may contain a mutant pertussis toxin gene, a deletion or mutant cutaneous necrotizing gene, and a heterologous ampG gene that replaces the natural BPampG gene (e.g., strain BPZE1 deposited with the National Collection of Microorganisms (CNCM) on March 9, 2006, receipt number 1-3585).

[0011] Neurodegenerative diseases may be characterized by the presence of β-amyloid plaques in the brain of the subject, and the step of administering the vaccine to the subject may result in a protective response that reduces or prevents the formation of Aβ plaques in the brain of the subject.

[0012] In the methods described herein, the subjects may be individuals diagnosed with or at risk of developing Alzheimer's disease, or individuals suffering from mild cognitive impairment. Furthermore, the subjects may have mutations in at least one of the genes encoding amyloid precursor protein, presenilin I, and presenilin II; or have an apolipoprotein E allotype characterized by one or two ε-4 alleles. Furthermore, the subjects may have asymptomatic Bordetella pertussis colony-forming infection.

[0013] As used herein, the phrase “Bordetella pertussis clinical infection” or “BP clinical infection” means symptomatic BP infection characterized by frequent coughing followed by a high-pitched “inspiratory whistling” sound. As used herein, the phrase “asymptomatic Bordetella pertussis colonization infection” or “asymptomatic BP colonization infection” means asymptomatic or mildly symptomatic BP infection (e.g., transient cough or runny nose) that does not feature frequent coughing followed by a high-pitched “inspiratory whistling” sound.

[0014] Unless otherwise defined, all technical terms used herein have the same meaning as those commonly understood by those skilled in the art in which the invention pertains. Methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention, but suitable methods and materials are described below. All publications, patents, and patent applications referenced herein are invoked by reference to their entirety. In case of any inconsistency, this specification, including definitions, shall prevail. In addition, the specific embodiments described below are illustrative and not intended to be limiting. [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1 is a schematic diagram showing an experimental protocol for evaluating the effects of BP infection and / or vaccination with live attenuated strains of BP in APP / PS1 mice. [Figure 2] Figure 2 shows the hippocampal Aβ plaque region in APP / PS1 mice following the protocol shown in Figure 1. [Modes for carrying out the invention]

[0016] A method for preventing, treating, or delaying the progression of a neurodegenerative disease such as AD is described herein by preventing or reducing a target BP clinical infection or asymptomatic BP colonization infection or neutralizing a BP toxin that causes or contributes to a neurodegenerative disease. The following embodiments illustrate representative examples of these methods. Nevertheless, based on the descriptions provided below, other aspects of the present invention can be made and / or implemented from the descriptions of these embodiments.

[0017] General methodology Methods including conventional microbiological, immunological, molecular biological, and medical techniques are described herein. Microbiological methods are described in Methods for General and Molecular Microbiology (3d Ed), Reddy et al., ed., ASM Press. Immunological methods are generally known in the art and are described in methodology textbooks such as Current Protocols in Immunology, Coligan et al., ed., John Wiley & Sons, New York. Molecular biological techniques are described in detail in textbooks such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Sambrook et al., ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; and Current Protocols in Molecular Biology, Ausubel et al., ed., Greene Publishing and Wiley-Interscience, New York. General methods of medical treatment are described in McPhee and Papadakis, Current Medical Diagnosis and Treatment 2010, 49th Edition, McGraw-Hill Medical, 2010; and Fauci et al., Harrison’s Principles of Internal Medicine, 17th Edition, McGraw-Hill Professional, 2008.

[0018] subject The methods described herein are applicable to any subject who has or is at risk of developing neurodegenerative diseases such as Alzheimer's disease (AD). Diagnosis of AD in human patients can be made by clinical evaluation. "Subjects at risk of developing AD" include those diagnosed with mild cognitive impairment (MCI), those at least 65 years of age with a parent or sibling who had AD, those with a risk gene associated with AD (e.g., APOE-ε4), or those with a definitive gene associated with AD (e.g., genes encoding mutated amyloid precursor protein, presenilin I, or presenilin 2). Other subjects who may be treated as described herein include those diagnosed with asymptomatic BP colonization infection or clinical BP infection, and / or those at risk of contracting asymptomatic BP colonization infection. The methods described herein are also applicable to subjects with tau tangles and / or β-amyloid plaques.

[0019] Drugs that prevent or reduce asymptomatic BP colony-forming infections To prevent or treat AD, agents can be administered to prevent or reduce clinical BP infections or asymptomatic BP colonization infections. Such agents may include bio-attenuated BPZE1 strain described in U.S. Patent No. 9,119,804, or its derivatives such as adenylate cyclase-deficient BPAL10 strain described in U.S. Patent No. 9,655,959; fusion protein-expressing BP strains described in U.S. Patent No. 9,528,086; serotype 3 described in International Publication No. 2019 / 077028(A1); partactin-deficient BP strains described in U.S. Patent No. 10,682,377; and BP vaccines that induce potent mucosal immunity against BP, such as vaccines containing BP strains lacking adenylate cyclase catalytic domain activity described in International Publication No. 2020 / 049133(A1). Other suitable attenuated BP strains may also be used as agents. Attenuation may be achieved by mutations in BP strains that reduce the production of one or more of the following (e.g., 1, 2, 3, 4, 5 or more) of: pertussis toxin (PTX), cutaneous necrotizing toxin (DNT), tracheal cytotoxin (TCT), adenylyl cyclase (AC), lipopolysaccharide (LPS), filamentous hemagglutinin (FHA), partactin, or bvg regulatory components. Methods for producing such variants are described herein and in U.S. Patent No. 9,119,804 and U.S. Patent Application Publication No. 15 / 472,436. The agent may be an antibiotic (e.g., an intranasal antibiotic) that eliminates or prevents asymptomatic Bordetella pertussis infection, clinical Bordetella pertussis infection, or pertussis. The antibiotic may be, for example, erythromycin, clarithromycin, or azithromycin.

[0020] Drugs that neutralize BP toxin To prevent or treat AD, an agent that neutralizes one or more BP toxins (e.g., Bordetella pertussis toxin) targeting it can be administered to a subject. Such an agent can be an antibody that specifically binds to an antigen expressed by Bordetella pertussis bacteria, or a vaccine that induces the production of such an antibody.

[0021] Formulation / Dosage / Administration The above BP strain can be formulated as a vaccine for administration to a subject. An appropriate number of live bacteria are mixed with a pharmaceutically appropriate excipient or carrier such as phosphate buffered saline, distilled water, an emulsion such as an oil / water emulsion, various types of wetting agents, a sterile solution, and the like. In some cases, the vaccine is lyophilized and then reconstituted prior to administration. Pharmaceutically appropriate excipients or carriers compatible with mucosal (especially nasal, bronchial, or pulmonary) administration are preferred for the purpose of exposing the airway to the BP strain. See Remington’s Pharmaceutical Sciences, a standard text in this field and in USP / NF.

[0022] When formulated for mucosal administration, each dose of the vaccine contains an adequate number of live Bordetella bacteria to cause a non-pathogenic asymptomatic BP colony-forming infection in the airway, e.g., approximately (i.e., ±50%) 5×10 5 ~5×10 10 bacteria, depending on the weight and age of the mammal receiving it. For administration to human subjects, the dose is about 1×10 5 、1×10 6 、5×10 6 、1×10 7 、5×10 7 、1×10 8 、5×10 8 、1×10 9 、5×10 9 、or 1×10 10Live BP bacteria may be included. The dose may be administered once or multiple times (2, 3, 4, 5, 6, 7, 8 or more times) at intervals of 1, 2, 3, 4, 5, or 6 days or 1, 2, 3, 4, 5, or 6 weeks or 1, 2, 3, 4, 5, 6, or 12 months. In general, a sufficient amount of vaccine is administered to induce infection and a protective response. Additional doses may be administered after the induced protective response has subsided.

[0023] Methods for inducing defensive reactions The vaccines described herein can be administered to mammalian subjects (e.g., humans) by any suitable method for depositing the vaccine's bacteria in the airway. For example, the vaccine may be administered by inhalation or intranasal introduction using, for example, an inhaler, syringe, insuffler, sprayer, or the like. 1 × 10 5 ~1 × 10 9 (For example, 1 × 10 5 , 5×10 5 , 1 x 10 6 , 5×10 6 , 1 x 10 6 , 5×10 6 , 1 x 10 7 , 5×10 7 , 1 x 10 8 , 5×10 8 , 1 x 10 9A single dose of live bacteria in an area of ​​±10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% is usually sufficient to induce a protective response. However, one or more additional booster doses (1, 2, 3, 4, or more) may be administered at intervals of four days or more (e.g., 4, 5, 6, or 7 days; or 1, 2, 3, 4, 5, 6, 7, or 8 weeks) until a sufficient protective response develops. The development of a protective response can be evaluated by methods known in the art, such as the quantification of Bordetella-specific antibody titers and the measurement of Bordetella antigen-specific T-cell responses (e.g., using the ELISPOT assay). Neuroimaging can be used to assess the progression of neurodegeneration (for example, using fluorodeoxy-d-glucose (FDG) for functional MRI and positron emission tomography (PET) studies of brain metabolism, as well as amyloid tracers such as Pittsburgh compound B (PiB)). If the vaccine-induced protective response has weakened (for example, 1, 2, 3, 4, 5, or more than 10 years after the last vaccination), the vaccine may be re-administered to boost the protective response. [Examples]

[0024] Example 1 - Materials and Method

[0025] APP / PS1 mice transfected with a human gene that induces early onset of human Alzheimer's disease (AD) were used in the following experiment. (See Holcomb et al. Nat. Med., 4:97-100, 1998.) These mice produce β-amyloid-containing plaques in their brains, including in the hippocampus, as seen in human AD. Wild-type (WT) mice were used as a control.

[0026] In the following experiments, we used a live attenuated intranasal vaccine derived from Bordetella pertussis, BPZE1, from which three of its characteristic pathogenic toxins had been inactivated or removed. See U.S. Patent No. 9,730,995.

[0027] Eight-week-old mice of 40WT and 40APP / PS1 were used in the study. These mice were assigned to the groups shown in Table 1 below. TIFF0007862015000001.tif113170

[0028] The experimental protocol is shown in Figure 1. Mice were treated with BPZE1 or a media at weeks 0 and 2. At week 40, mice were infected with BP or an infection mock. At week 44, groups 9 and 10 were re-administered with BPZE1. At week 64, the hippocampus was removed from each animal and evaluated for Aβ plaques using Congo red staining.

[0029] Example 2 - Results

[0030] Referring to Figure 2, hippocampal Aβ plaques were evaluated by Congo red staining. The mean plaque area was determined as a percentage of the control for plaque sizes 21–50, 51–80, and 90–120, with a size of >120 pixels. Infection with BP alone tended to increase the hippocampal area covered by Aβ plaques compared to the control. Pre-vaccination with BPZE1 reduced the hippocampal area covered by Aβ plaques in APP / PS1 mice subsequently exposed to BP compared to BP-exposed mice that were not pre-vaccinated with BPZE1 (mixed p-value for repeated measures across multiple β-amyloid plaque sizes, 0.003). BPZE1 vaccination reduced the hippocampal area covered by β-amyloid plaques in a composite group of subsequently exposed and unexposed APP / PS1 mice compared to control mice that were not exposed to BP (mixed p-value for repeated measures across multiple β-amyloid plaque sizes, 0.001). BPZE1 showed a strong tendency to reduce the hippocampal area covered by β-amyloid plaques in APP / PS1 mice that were not exposed to BP, compared to unvaccinated control mice that were not exposed to BP (mixed p-value for repeated measures across multiple β-amyloid plaque sizes, 0.055).

[0031] BPZE1 has a preventive effect against controls in mice that are genetically predisposed to producing Aβ brain plaques, as seen in human Alzheimer's disease (AD). BPZE1 vaccination significantly reduces Aβ hippocampal coverage in mice subsequently exposed to BP and is associated with fewer hippocampal Aβ plaques in a composite group of mice subsequently exposed to and unexposed to BP.

[0032] Other Embodiments Although the present invention is described in connection with its detailed description, the foregoing description is intended to illustrate and not to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A composition for preventing or reducing β-amyloid plaques in the brain of a person who has Alzheimer's disease or is at risk of developing it, The composition comprises a bio-attenuated Bordetella pertussis strain, which can colonize in the subject and induce a defense response in the subject that reduces the amount of β-amyloid plaques formed or present in the subject's brain when the subject is not administered the composition, and which comprises a mutant pertussis toxin gene, a deletion or mutant cutaneous necrotizing gene, and a heterologous ampG gene in which the Bordetella pertussis ampG gene is substituted. A composition characterized by the following features.

2. The composition according to claim 1, wherein the live, attenuated Bordetella pertussis strain can form colonies in the target airway.

3. The composition according to claim 1, wherein the aforementioned attenuated Bordetella pertussis strain is strain BPZE1, deposited with the National Collection of Microorganisms (C.N.C.M.) on March 9, 2006, under receipt number 1-3585.

4. The composition according to claim 1, wherein the aforementioned attenuated Bordetella pertussis strain is nonpathogenic.

5. The subject is diagnosed with Alzheimer's disease, and the composition is as described in claim 1.

6. The subject is diagnosed with mild cognitive impairment, and the composition is as described in claim 1.

7. The composition according to claim 1, wherein the subject has a mutation in at least one of the genes comprising the group consisting of a gene encoding amyloid precursor protein, a gene encoding presenilin I, and a gene encoding presenilin II.

8. The composition according to claim 1, wherein the subject has an apolipoprotein E allotype characterized by one or two ε-4 alleles.

9. The composition according to claim 1, wherein the subject is suffering from asymptomatic Bordetella pertussis infection.

Images