Treatment and prevention of neuropathology associated with neurodegenerative diseases

Abstract

Administering a live, attenuated Bordetella pertussis-based vaccine to a subject at risk for developing a neurodegenerative disease featuring Aβ brain plaques can prevent or reduce the amount of Aβ brain plaques that would have developed in the subject without such treatment.

Claims

1. A method for preventing or reducing β-amyloid plaque in the brain of a subject having or at risk for developing Alzheimer's disease, the method comprising the step of administering to the subject a composition comprising a live, attenuated Bordetella pertussis strain which is able to colonize the subject and induce a protective response in the subject that reduces the amount of 3-amyloid plaque that would have formed or would have been present in the brain of the subject if the subject were not administered the composition.

2. The method of claim 1, wherein the live, attenuated Bordetella pertussis strain is capable of colonizing infection of the subject's respiratory tract.

3. The method of claim 1, wherein the live attenuated Bordetella pertussis strain comprises a mutated pertussis toxin gene, a deleted or mutated dermonecrotic gene, and a heterologous ampG gene which replaces the Bordetella pertussis ampG gene.

4. The method of claim 3, wherein the live attenuated Bordetella pertussis strain is a BPZE1 strain deposited with the Collection Nationale de Culture Microorganismes (C.N.C.M.) on Mar. 9, 2006, under accession number 1-3585.

5. The method of claim 1, wherein the live attenuated Bordetella pertussis strain is non-virulent.

6. The use of claim 1, wherein the subject has been diagnosed with Alzheimer's disease.

7. The method of claim 1, wherein the subject has been diagnosed with mild cognitive impairment.

8. The method of claim 1, wherein the subject has mutations in at least one of the genes consisting of the group of genes encoding amyloid precursor protein, presenilin I, and presenilin II.

9. The method of claim 1, wherein the subject has an apolipoprotein E allotype that features one or two epsilon-4 alleles.

10. The method of claim 1, wherein the subject has a subclinical Bordetella pertussis infection.

Description

DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is a schematic image showing an experimental protocol for assessing the effect of BP infection and/or vaccination with a live, attenuated strain of BP in APP/PS1 mice.

[0016] FIG. 2 is a graph showing hippocampal plaque areas in APP/PS1 mice following the protocol shown in FIG. 1.

DETAILED DESCRIPTION

[0017] Described herein are methods for preventing, treating, or slowing the progression of a neurodegenerative disease such as AD, by preventing or reducing BP clinical infection or subclinical BP colonizing infection of a subject or neutralizing a BP toxin which causes or contributes to the neurodegenerative disease. The below described embodiments illustrate representative examples of these methods. Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below.

General Methodology

[0018] Methods involving 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 described in methodology treatises such as Current Protocols in Immunology, Coligan et al., ed., John Wiley & Sons, New York. Techniques of molecular biology are described in detail in treatises 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.

Subjects

[0019] The methods described herein are applicable to any subject having, or at risk for developing a neurodegenerative disease such as AD. Diagnosing AD in human patients can be performed by clinical assessment. A “subject at risk for developing AD” is one diagnosed with mild cognitive impairment (MCI), a person at least 65 years old who has had a parent or sibling with AD, a person having a risk gene associated with developing AD (e.g., APOE-ε4), or a person having a deterministic gene associated with developing AD (e.g., a gene encoding a mutant amyloid precursor protein, presenilin-1, or presenilin-2). Other subjects who might be treated as described herein are those diagnosed with subclinical BP colonizing infection or BP clinical infection, and/or those at risk of acquiring a subclinical BP colonizing infection. The methods described herein are also applicable to subjects having tau tangles and/or beta amyloid plaques.

Agents which Prevent or Reduce BP Subclinical Colonizing Infection

[0020] To prevent or treat AD, a subject can be administered an agent which prevents or reduces clinical BP infection or subclinical BP colonizing infection. The agent can be a BP vaccine that induces potent mucosal immunity against BP such as a vaccine including the live attenuated BPZE1 strain described in U.S. Pat. No. 9,119,804, or derivatives thereof such as the adenylate cyclase deficient BPAL10 strain described in U.S. Pat. No. 9,655,959; the fusion protein-expressing BP strains described in U.S. Pat. No. 9,528,086; the serotype 3 fimbrae-expressing BP strains described in WO2019077028A1; the pertactin-deficient BP strains described in U.S. Pat. No. 10,682,377; and the BP strain deficient in adenylate cyclase catalytic domain activity described in WO2020/049133A1. Other suitable attenuated BP strains might be used as the agent. Attenuation might be achieved by mutating a BP strain to reduce its production of one or more (e.g., 1, 2, 3, 4, 5 or more) of the following: pertussis toxin (PTX), dermonecrotic toxin (DNT), tracheal cytotoxin (TCT), adenylate cyclase (AC), lipopolysaccharide (LPS), filamentous hemagglutinin (FHA), pertactin, or any of the bvg-regulated components. Methods for making such mutants are described herein and in U.S. Pat. No. 9,119,804 and U.S. patent application Ser. No. 15/472,436. The agent can also be an antibiotic (e.g., an intranasal antibiotic) which can clear or prevent subclinical Bordetella pertussis infections, clinical Bordetella pertussis infection, or whooping cough. The antibiotic can, for example be, erythromycin, clarithromycin, or azithromycin.

Agents which Neutralize a BP Toxin

[0021] To prevent or treat AD, a subject can be administered an agent which targets and neutralizes one or more BP toxins (e.g., Bordetella pertussis toxin). Such agents can be antibodies that specifically bind an antigen expressed by Bordetella pertussis bacteria, or vaccines which induce the production of such antibodies.

Formulations/Dosage/Administration

[0022] The BP strains described above can be formulated as a vaccine for administration to a subject. A suitable number of live bacteria are mixed with a pharmaceutically suitable excipient or carrier such as phosphate buffered saline solutions, distilled water, emulsions such as an oil/water emulsions, various types of wetting agents, sterile solutions and the like. In some cases, the vaccine can be lyophilized and then reconstituted prior to administration. The use of pharmaceutically suitable excipients or carriers which are compatible with mucosal (particularly nasal, bronchial, or lung) administration are preferred for the purpose of exposing the respiratory tract to BP strains. See Remington's Pharmaceutical Sciences, a standard text in this field, and in USP/NF.

[0023] When formulated for mucosal administration, each dose of the vaccine can include a sufficient number of live Bordetella bacteria to result in a non-virulent subclinical BP colonizing infection of the respiratory tract, e.g., approximately (i.e., +/−50%) 5×10.sup.5 to 5×10.sup.10 bacteria, depending on the weight and age of the mammal receiving it. For administration to human subjects, the dose can include approximately 1×10.sup.5, 1×10.sup.6, 5×10.sup.6, 1×10.sup.7, 5×10.sup.7, 1×10.sup.8, 5×10.sup.8, 1×10.sup.9, 5×10.sup.9, or 1×10.sup.10 live BP bacteria. The dose may be given once or on multiple (2, 3, 4, 5, 6, 7, 8 or more) occasions 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. Generally, sufficient amounts of the vaccine are administered to result in infection and the protective response. Additional amounts can be administered after the induced protective response wanes.

Methods of Eliciting Protective Responses

[0024] The vaccines described herein can be administered to a mammalian subject (e.g., a human being) by any suitable method that deposits the bacteria within the vaccine in the respiratory tract. For example, the vaccines may be administered by inhalation or intranasal introduction, e.g., using an inhaler, a syringe, an insufflator, a spraying device, etc. While administration of a single dose of between 1×10.sup.5 to 1×10.sup.9 (e.g., 1×10.sup.5, 5×10.sup.5, 1×10.sup.6, 5×10.sup.6, 1×10.sup.6, 5×10.sup.6, 1×10.sup.7, 5×10.sup.7, 1×10.sup.8, 5×10.sup.8, 1×10.sup.9+/−10, 20, 30, 40, 50, 60, 70, 80, or 90%) live bacteria is typically sufficient to induce a protective response, one or more (1, 2, 3, 4, or more) additional booster doses might be administered in intervals of 4 or more days (e.g., 4, 5, 6, or 7 days; or 1, 2 3, 4, 5, 6, 7, or 8 weeks) until a sufficiently protective response has developed. The development of a protective response can be evaluated by methods known in the art such as quantifying Bordetella-specific antibody titers and measuring of Bordetella antigen-specific T cells responses (e.g., using an ELISPOT assay). Neuroimaging (e.g., functional MRI and positron emission tomography (PET) studies of cerebral metabolism with fluoro-deoxy-d-glucose (FDG) and amyloid tracers such as Pittsburgh Compound-B (PiB)) can be used to evaluate the progression of neurodegeneration. In cases were a vaccine-induced protective response has waned (e.g., after 1, 2, 3, 4, 5, 10 or more years from the last vaccination) a subject may again be administered the vaccine in order to boost the protective response.

EXAMPLES

Example 1—Materials and Methods

[0025] APP/PS1 mice transfected with human genes that cause early onset human AD were used in the experiments described below. See, Holcomb et al. Nat. Med., 4:97-100, 1998. These mice form β-amyloid-containing plaque in their brains, including in the hippocampus, as is seen in human AD. Wild-type (WT) mice were used as controls.

[0026] BPZE1, a live attenuated intranasal vaccine derived from Bordetella pertussis, from which three of its hallmark virulent toxins have been either inactivated or removed was used in the experiments described below. See, U.S. Pat. No. 9,730,995.

[0027] Forty WT and forty APP/PS1 8-week old mice were used in the study. They were assigned to the Groups shown in Table 1 below:

TABLE-US-00001 TABLE 1 The treatment groups (10 groups, 10 mice/group) are as follows: Mice, Sacrifice for 8 BPZE1 BP BPZE1 Pathology/ Group weeks pre-BP challenge post BP Assays 1 WT No no no 64 weeks APP/PS 2 1 No no no 64 weeks 3 WT 6 and 32 weeks no no 64 weeks APP/PS 4 1 6 and 32 weeks no no 64 weeks 5 WT No 40 weeks no 64 weeks APP/PS 6 1 No 40 weeks no 64 weeks 7 WT 6 and 32 weeks 40 weeks no 64 weeks APP/PS 8 1 6 and 32 weeks 40 weeks no 64 weeks 9 WT No 40 weeks 44 weeks 64 weeks APP/PS 10 1 No 40 weeks 44 weeks 64 weeks

[0028] The experimental protocol is shown in FIG. 1. Mice were treated with BPZE1 or vehicle at 0 and 2 weeks. At 40 weeks mice were infected with BP or mock infected. At 44 weeks Groups 9 and 10 were again administered BPZE1. At 64 weeks, the hippocampus was removed from each animal and assessed for Aβ plaques using Congo Red staining.

Example 2—Results

[0029] Referring to FIG. 2, hippocampal Aβ plaque was assessed by Congo Red staining. The mean plaque area as a percentage of the controls was determined for plaques 21-50, 51-80, 90-120, and >120 pixels in size. Infection with BP alone tended to increase hippocampal area covered by Aβ plaque compared to controls. BPZE1 pre-vaccination reduced hippocampal area covered by Aβ plaque in APP/PS1 mice subsequently exposed to BP, compared with BP exposed mice not pre-vaccinated with BPZE1 (mixed p-value for repeated measures across multiple β-amyloid plaque sizes, 0.003). BPZE1 vaccination reduced hippocampal area coverage with β-amyloid plaque in the combined group of APP/PS1 mice subsequently exposed and not exposed to BP, compared with control mice not exposed to BP (mixed p-value for repeated measures across multiple β-amyloid plaque sizes, 0.001). BPZE1 exhibited a strong trend to reduce the hippocampal area covered by β-amyloid plaque in APP/PS1 mice that have not been exposed to BP, compared with control unvaccinated mice not exposed to BP (mixed p-value for repeated measures across multiple β-amyloid plaque sizes, 0.055).

[0030] BPZE1 has protective benefits vs. controls in mice genetically predisposed to produce Aβ brain plaques, as seen in human AD. BPZE1 vaccination significantly reduces Aβ plaque hippocampal coverage in mice subsequently exposed to BP, and is associated with less hippocampal Aβ plaque in the combined group of mice that are subsequently exposed and not exposed to BP.

OTHER EMBODIMENTS

[0031] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not 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.