An Open-label Trial to Evaluate the Effects of BCG Immunization on Biomarkers of Inflammation/Immune Response and Alzheimer's Disease in Adults With Mild Cognitive Impairment and Mild-to-Moderate Dementia Due to Alzheimer's Disease
Phase: PHASE2
Conditions: Mild Cognitive Impairment, Mild Dementia, Moderate Dementia, Alzheimer Disease
Sponsor: Steven E Arnold, MD

An Open Label Trial to Evaluate the Effects of BCG Immunization on Biomarkers of Inflammation/Immune Response and Alzheimer’s Disease in Adults with
Mild Cognitive Impairment and Mild-to-Moderate Dementia due to Alzheimer’s Disease 
Version Date/#: October 13, 2022/Version 3.3 
Page 1 of 43 
 
 
 
 
 
 
 
An Open Label Trial to Evaluate the Effects of BCG Immunization on Biomarkers 
of Inflammation/Immune Response and Alzheimer’s Disease in Adults with Mild 
Cognitive Impairment and Mild-to-Moderate Dementia due to Alzheimer’s 
Disease 
 
NCT05004688 
October 13, 2022 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

An Open Label Trial to Evaluate the Effects of BCG Immunization on Biomarkers of Inflammation/Immune Response and Alzheimer’s Disease in Adults with
Mild Cognitive Impairment and Mild-to-Moderate Dementia due to Alzheimer’s Disease 
Version Date/#: October 13, 2022/Version 3.3 
Page 2 of 43 
 
 
 
 
 
 
 
An Open Label Trial to Evaluate the Effects of BCG Immunization on Biomarkers 
of Inflammation/Immune Response and Alzheimer’s Disease in Adults with Mild 
Cognitive Impairment and Mild-to-Moderate Dementia due to Alzheimer’s 
Disease 
 
 
 
 
 
 
Steven E. Arnold MD 
Principal Investigator 
Massachusetts General Hospital 
 
Denise Faustman MD, PhD 
Investigator 
Massachusetts General Hospital 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Version Number/Date: 3.3/October 13, 2022 
 

An Open Label Trial to Evaluate the Effects of BCG Immunization on Biomarkers of Inflammation/Immune Response and Alzheimer’s Disease in Adults with
Mild Cognitive Impairment and Mild-to-Moderate Dementia due to Alzheimer’s Disease 
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The information contained herein is confidential and proprietary in nature and will not be disclosed to any third 
party without written approval of authorized designee. This document may be disclosed to the appropriate 
institutional review boards or to duly authorized representatives of the US Food and Drug Administration or national 
regulatory authority under the condition that they maintain confidentiality. 

An Open Label Trial to Evaluate the Effects of BCG Immunization on Biomarkers of Inflammation/Immune Response and Alzheimer’s Disease in Adults with
Mild Cognitive Impairment and Mild-to-Moderate Dementia due to Alzheimer’s Disease 
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STATEMENT OF COMPLIANCE 
 
This study will be conducted in compliance with the protocol, International Conference on Harmonisation of 
Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), Good Clinical Practice (GCP), 
and the applicable regulatory requirements, United States Code of Federal Regulations (CFR) Title 45 CFR Part 46 
and Title 21 CFR Parts 50, 56, and 312. 

An Open Label Trial to Evaluate the Effects of BCG Immunization on Biomarkers of Inflammation/Immune Response and Alzheimer’s Disease in Adults with
Mild Cognitive Impairment and Mild-to-Moderate Dementia due to Alzheimer’s Disease 
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LIST OF ABBREVIATIONS 
AD 
Alzheimer’s Disease 
ADC 
Alzheimer’s Disease Center 
ADRD 
Alzheimer’s Disease and Related Dementias 
BCG 
Bacillus Calmette-Guérin 
CDR 
Clinical Dementia Rating 
CFR 
Code of Federal Regulations 
CN 
Normal cognition 
C-SSRS 
Columbia Suicide Severity Rating Scale 
CRF 
Case Report Form 
CSF 
Cerebrospinal Fluid 
CMP 
Comprehensive Metabolic Panel 
CNS 
Central Nervous System 
CRP 
C-Reactive Protein 
eCRF 
Electronic Case Report Form 
EDC 
Electronic Data Capture 
ET 
Early Termination 
ESR 
Erythroid Sedimentation Rate 
FAQ 
Functional Activities Questionnaire 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
IIR 
Inflammation and Immune Response 
IRB 
Internal Review Board 
LP 
Lumbar Puncture 
MADRC 
Massachusetts Alzheimer’s Disease Research Center 
MCI 
Mild Cognitive Impairment 
MDU 
Memory Disorders Unit 
MoCA 
Montreal Cognitive Assessment 
mTB 
Mycobacterium Tuberculosis 
NACC 
National Alzheimer’s Coordinating Center 
NIA 
National Institute on Aging 
NPIQ 
Neuropsychiatric Inventory Questionnaire 
PCP 
Primary Care Physician 
PI 
Principal Instigator 
PPD 
Purified Protein Derivative 
RBANS 
Repeatable Battery for the Assessment of Neuropsychological Status 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SOB 
Sum of Boxes 
SOP 
Standard Operating Procedure 
TSH 
Thyroid Stimulating Hormone 
Treg 
Regulatory T-cells 
UDS 
Uniform Data Sat 
US 
United States 
WOCBP 
Women of Child Bearing Potential 

An Open Label Trial to Evaluate the Effects of BCG Immunization on Biomarkers of Inflammation/Immune Response and Alzheimer’s Disease in Adults with
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Table of Contents 
 
Table of Contents 
STATEMENT OF COMPLIANCE ......................................................................................................................... 4 
LIST OF ABBREVIATIONS .................................................................................................................................. 5 
1 
ETHICS/PROTECTION OF HUMAN SUBJECTS ............................................................................................ 9 
1.1 
Institutional Review Board (IRB)................................................................................................................................... 9 
1.2 
Ethical Conduct of Study .............................................................................................................................................. 9 
1.3 
Subject Information and Consent ................................................................................................................................. 9 
2 
INTRODUCTION: BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE ....................................... 10 
2.1 
Background Information ............................................................................................................................................ 10 
2.1.2 Alzheimer’s Disease and the Immune Response ......................................................................................................... 10 
2.1.3 BCG Rationale ............................................................................................................................................................ 10 
3 
STUDY DESIGN ........................................................................................................................................ 11 
3.1 
Overall Study Design and Plan .................................................................................................................................... 12 
3.2 
Study Objectives ........................................................................................................................................................ 12 
3.3 
Study Outcome Measures .......................................................................................................................................... 12 
3.3.1 Safety Measure Outcomes ......................................................................................................................................... 12 
3.3.2 CSF and Blood Biomarker Outcomes .......................................................................................................................... 13 
3.3.3 Neurocognitive/Behavioral Measure Outcomes ......................................................................................................... 13 
3.3.4 Structural and Functional MRI Measure Outcomes .................................................................................................... 13 
4 
SUBJECT SELECTION ................................................................................................................................ 14 
4.1 
Inclusion/exclusion Criteria ........................................................................................................................................ 14 
4.1.1 Inclusion Criteria ........................................................................................................................................................ 14 
4.1.2 Exclusion Criteria ........................................................................................................................................................ 14 
4.1.2.1 Women of Childbearing Potential (WOCBP) ............................................................................................................... 15 
4.2 
Recruitment ............................................................................................................................................................... 16 
4.2.1 Recruitment of Subjects through Advertising ............................................................................................................. 16 
4.2.2 Recruitment of Subjects from the Massachusetts Alzheimer’s Disease Research Center ............................................ 16 
4.2.3 Recruitment of Subjects from among MGB Providers ................................................................................................. 16 
4.2.4 Recruitment of Subjects from the CTRU Registry ........................................................................................................ 16 
5 
SUBJECT ENROLLMENT ........................................................................................................................... 17 
5.1 
Informed Consent Process .......................................................................................................................................... 17 
5.2 
Remuneration ............................................................................................................................................................ 18 
5.3 
Discontinuation .......................................................................................................................................................... 19 
5.3.1 Study Discontinuation ................................................................................................................................................ 19 
5.3.2 Discontinuation of Study Intervention ....................................................................................................................... 19 
5.3.3 Voluntary Withdrawals .............................................................................................................................................. 19 
5.3.4 Lost to follow-up ........................................................................................................................................................ 19 
5.4 
Early Termination Visit (ET) ........................................................................................................................................ 20 
5.5 
Termination of Study ................................................................................................................................................. 20 
6 
STUDY PROCEDURES ............................................................................................................................... 21 
6.1 
Schedule of Events ..................................................................................................................................................... 21 
6.1.1 RCT roll over participant schedule .............................................................................................................................. 22 
6.2 
Study Visits................................................................................................................................................................. 22 
6.2.1 Screening ................................................................................................................................................................... 22 
6.2.2 Baseline Visit/Visit 2 (Day 1) ...................................................................................................................................... 23 

An Open Label Trial to Evaluate the Effects of BCG Immunization on Biomarkers of Inflammation/Immune Response and Alzheimer’s Disease in Adults with
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6.2.3 Visit 3 (Day 28 + 14 days) ............................................................................................................................................ 24 
6.2.4 Visit 4 (Day 84 ± 5 days) .............................................................................................................................................. 24 
6.2.5 Visit 5 (Day 182 ± 14 days) .......................................................................................................................................... 25 
6.2.6 Phone Check 1 (Day 273 ± 5 days) .............................................................................................................................. 25 
6.2.7 Visit 6 (Day 364 ± 28 days) .......................................................................................................................................... 25 
6.2.8 Phone Check 2 & 3 (Day 546 ± 28 days, Day 728 ± 28 days) ........................................................................................ 25 
6.2.9 Early Termination Visit ............................................................................................................................................... 25 
6.2.10 Protocol Deviations .................................................................................................................................................... 26 
6.2.11 Missed Visits and Procedures ..................................................................................................................................... 26 
6.3 
Clinical Assessments ................................................................................................................................................... 26 
6.3.1 Safety Measures ......................................................................................................................................................... 26 
Table 1. Safety Laboratory Tests ............................................................................................................................................ 26 
6.3.2 Physical and Neurological Examination ...................................................................................................................... 27 
6.3.3 Phlebotomy for Research: Target Engagement, Cytokine Levels and Biomarker Analysis ........................................... 28 
Table 2. Blood Collection for Research ................................................................................................................................... 28 
6.3.4 Lumbar Puncture for Immune and Biomarker Analysis ............................................................................................... 28 
6.3.5 Injection Site Monitoring............................................................................................................................................ 29 
6.3.6 Magnetic Resonance Imaging (MRI) ........................................................................................................................... 29 
6.4 
Neurocognitive Assessments ...................................................................................................................................... 29 
6.4.1 Montreal Cognitive Assessment (MoCA) .................................................................................................................... 29 
6.4.2 Repeatable Battery for the Assessment of Neuropsychological Status Update (RBANS) ............................................. 30 
6.4.3 Clinical Dementia Rating (CDR) Scale .......................................................................................................................... 30 
6.5 
Neuropsychiatric Assessments ................................................................................................................................... 30 
6.5.1 Neuropsychiatric Inventory Questionnaire (NPI-Q) .................................................................................................... 30 
6.5.2 Functional Activities Questionnaire (FAQ) .................................................................................................................. 30 
6.5.3 Hospital Anxiety and Depression Scale (HADS) ........................................................................................................... 31 
7 
STUDY INTERVENTION ADMINISTERED .................................................................................................. 31 
Table 3. Summary of Investigational Products ....................................................................................................................... 31 
7.1 
Study Drug ................................................................................................................................................................. 31 
7.2 
Study Drug Packaging and Labeling ............................................................................................................................ 32 
7.3 
Study Drug Preparation .............................................................................................................................................. 32 
7.4 
Dosage and Administration ........................................................................................................................................ 32 
7.5 
Study Drug Accountability .......................................................................................................................................... 32 
7.6 
Study Drug Handling and Disposal .............................................................................................................................. 32 
7.7 
Contraindications and Warnings ................................................................................................................................ 32 
7.7.1 Prohibited Medications .............................................................................................................................................. 32 
7.7.2 Warnings .................................................................................................................................................................... 33 
8 
BIOSTATISTICAL ANALYSIS ...................................................................................................................... 33 
8.1 
Sample Size ................................................................................................................................................................ 33 
8.2 
Safety Endpoints ........................................................................................................................................................ 33 
8.3 
Biofluid Biomarker, Clinical and Neuroimaging Endpoints .......................................................................................... 34 
9 
RISKS AND DISCOMFORTS ...................................................................................................................... 34 
9.1 
BCG Vaccine Risks ...................................................................................................................................................... 34 
9.2 
Lumbar puncture ........................................................................................................................................................ 35 
9.3 
Magnetic Resonance Imaging (MRI) ........................................................................................................................... 35 
9.4 
Phlebotomy................................................................................................................................................................ 36 
9.5 
Neurocognitive testing ............................................................................................................................................... 36 
9.6 
Neuropsychiatric and Functional Questionnaires ....................................................................................................... 36 
9.7 
Radiation .................................................................................................................................................................... 36 
9.8 
Genetic Research Risks ............................................................................................................................................... 36 

An Open Label Trial to Evaluate the Effects of BCG Immunization on Biomarkers of Inflammation/Immune Response and Alzheimer’s Disease in Adults with
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9.9 
Other Risks ................................................................................................................................................................. 36 
10 POTENTIAL BENEFITS .............................................................................................................................. 36 
11 MONITORING AND QUALITY ASSURANCE............................................................................................... 36 
11.1 
Independent Monitoring of Source Data .................................................................................................................... 37 
11.2 
Safety Monitoring ...................................................................................................................................................... 37 
11.3 
Adverse Event Reporting Guidelines .......................................................................................................................... 37 
11.3.1 Adverse Event ............................................................................................................................................................ 37 
11.3.2 Assessment and Recording of Adverse Events ............................................................................................................ 39 
11.3.3 Assessment of Adverse Events ................................................................................................................................... 39 
11.3.4 Relatedness of Adverse Event to Investigational Protocol .......................................................................................... 39 
11.3.5 Recording of Adverse Events ...................................................................................................................................... 39 
11.3.6 Adverse Events and Serious Adverse Events - Reportable Events ............................................................................... 39 
12 DATA COLLECTION AND MANAGEMENT ................................................................................................ 40 
12.1 
Purpose of EDC ........................................................................................................................................................... 40 
12.2 
Role of Data Management ......................................................................................................................................... 40 
12.3 
Data Entry and Checks ................................................................................................................................................ 40 
12.4 
Data Lock Process ....................................................................................................................................................... 40 
12.5 
Data Handling and Record Keeping ............................................................................................................................ 40 
12.6 
Confidentiality ........................................................................................................................................................... 41 
12.7 
Retention of Records .................................................................................................................................................. 41 
12.8 
Publications ............................................................................................................................................................... 41 
13 REFERENCES ........................................................................................................................................... 42 
 

An Open Label Trial to Evaluate the Effects of BCG Immunization on Biomarkers of Inflammation/Immune Response and Alzheimer’s Disease in Adults with
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1 
ETHICS/PROTECTION OF HUMAN SUBJECTS 
 
1.1 
Institutional Review Board (IRB) 
This study will be conducted in compliance with current Good Clinical Practices (GCP) and Title 21 Part 56 of the 
United States of America Code of Federal Regulations (CFR) relating to IRBs. 
 
1.2 
Ethical Conduct of Study 
The study will be conducted in accordance with GCP defined by the International Conference on Harmonisation 
(ICH) and the ethical principles of the Declaration of Helsinki. 
 
1.3 
Subject Information and Consent 
This study will be conducted in compliance with Title 21 Part 50 of the United States of America Code of Federal 
Regulations (CFR), Federal Regulations, and ICH Guidance Documents pertaining to informed consent. At the first 
visit, prior to initiation of any study-related procedures, subjects will be informed about the nature and purpose of 
the study, participation/termination conditions, and risks and benefits. Subjects will be given adequate time to ask 
questions and become familiar with the study prior to providing consent to participate. Subjects will give their 
written consent to participate in the study and will be provided with a copy of the fully executed consent form for 
their records. 
 
Some of the subjects in this study may be unable to provide informed consent due to cognitive impairment. The 
capacity to consent is gauged by an experienced licensed clinician investigator during the consent process through 
an evaluation of the participant’s understanding of their condition, their options for treatment, their understanding 
of the potential benefits and risks of participating in the clinical trial and of their understanding regarding the 
voluntary nature of their participation in the trial. In addition, participants are evaluated for their level of orientation 
and understanding of what will happen during the trial. This process is recorded as part of the consent discussion 
documentation. 
 
Subjects who are deemed unable to provide consent will be given the opportunity to assent, and consent will be 
obtained from a legally authorized representative. Subjects who are unable to assent or who object to participation 
in the study will not be enrolled. 

An Open Label Trial to Evaluate the Effects of BCG Immunization on Biomarkers of Inflammation/Immune Response and Alzheimer’s Disease in Adults with
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2 
INTRODUCTION: BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE 
 
2.1 Background Information 
2.1.1 
Alzheimer’s Disease and Mild Cognitive Impairment 
Alzheimer’s disease (AD) is the most prevalent form of dementia affecting more than 5,000,000 people in the US 
and an estimated 20,000,000 people worldwide, according to the Alzheimer’s Association 1.  AD causes 
progressive neuronal degeneration, resulting in progressive memory loss, dementia and death. AD has no known 
cure or preventative as yet; however, treatments exist that can temporarily and modestly at best ameliorate 
dementia symptoms.  
Mild cognitive impairment (MCI) is the term used when there is clinical concern about cognitive decline and 
evidence of impairment(s) in cognitive testing, more than expected for the patient's age and educational 
background, but not so severe that the person cannot manage their daily functioning without more than minimal 
assistance. MCI is frequently, though not always, a transitional, prodromal phase of dementia due to Alzheimer’s 
disease. "Dementia" is present when independent daily functioning is affected. 
AD is characterized by the presence of amyloid-β (Aβ) plaques, tau neurofibrillary tangles and loss of neurons 
and synapses in the cerebral cortex, particularly in association areas that subserve cognition. Aβ has been the 
target of many recent clinical trials; however, these studies have failed to find that reducing amyloid meaningfully 
slows progression of dementia. It is increasingly evident that AD is a complex disorder with multiple metabolic, 
inflammatory, vascular and other pathophysiological processes beyond amyloid and tau proteinopathies 
contributing to neurodegeneration. There is now increasing attention to intervening in these non-amyloid 
pathways to stem the tide of neurodegeneration in AD. 
2.1.2 
Alzheimer’s Disease and the Immune Response 
Inflammation and immune responses (IIR) have major roles in the pathophysiology of Alzheimer's disease (AD). 
IIR encompasses many complex and highly regulated biological processes with which immune local tissue cells 
respond to tissue injury and maintain organ function and organismal homeostasis. AD's pathognomonic amyloid-
β (Aβ) peptide itself is hypothesized to be a dysregulated ancient innate immune response to allo- or auto- 
stimuli in the brain.2 Aβ oligomers and aggregates in AD are thought to elicit or exacerbate IIR,3 promoting 
vicious cycles of protein misfolding, endoplasmic reticulum stress, deranged metabolic signaling and 
mitochondrial function, oxidative/nitrosative stress, and cell death.  There is tremendous interest and rapidly 
growing academic, biotech and pharma research on IIR therapeutic approaches in AD and AD-related disorders 
as similar vicious cycles have been proposed for a-synuclein pathologies in LBDs, 4,5 tau pathologies in FTD-tau 
and other tauopathies,6,7 and TDP-43 pathology in FTD-TDP and ALS.8,9  
Peripheral inflammatory markers may serve as biomarkers of AD disease state and distinguish healthy older 
adults from middle-aged adults, and healthy older adults from those with mild cognitive impairment (MCI) or 
Alzheimer’s Disease10. Inflammatory biomarkers may even predict those who will convert from MCI to AD over 
a two-year period 11-13. Innate immune function is affected by progression of AD; Monocytes of subjects with 
MCI or AD induce a greater NLRP3 and NLRP1 inflammasome response to extrinsically applied amyloid beta 
than do healthy controls 14. The adaptive immune system is also involved in AD progression: T-cell reactivity to 
amyloid beta increases with age as well as progression of AD 10, and with worsening disease, clonal populations 
of CD4+ and CD8+ T-cells can be found in the cerebrospinal fluid15,16. Regulatory T cells (Tregs) are of 
particularly significance to AD. While augmenting Tregs increases the number and function of microglia 
associated with amyloid beta plaques, and improves cognitive functions12,17-19, they are fewer in number over the 
course of disease20. 
There is tremendous and rapidly growing academic, biotech and pharma research on IIR therapeutic approaches 
in AD and AD-related disorders as similar vicious cycles have been proposed for -synuclein pathologies in 
LBDs, 5,21 tau pathologies in FTD-tau and other tauopathies,22,23 and TDP-43 pathology in FTD-TDP and 
ALS.24,25  
2.1.3 
BCG Rationale 

An Open Label Trial to Evaluate the Effects of BCG Immunization on Biomarkers of Inflammation/Immune Response and Alzheimer’s Disease in Adults with
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A potentially transformative IIR approach to AD prevention and disease modification that is safe, cheap and ready 
for testing now is Bacillus Calmette-Guérin (BCG) vaccination. Live BCG vaccination has been the mainstay of 
prevention of tuberculosis for almost 100 years, administered in billions of people in more than 180 countries, 
from infants to the elderly. Its only significant contraindication is immunocompromise. "Off-target" benefits of 
BCG vaccination were recognized early in its 40% reduction of all-cause mortality in children and its prevention 
of non-mycobacterial infections,26,27 and BCG is now being explored in clinical trials as a prophylaxis against 
COVID-19.28 BCG has also demonstrated utility in treating non-infectious diseases. One major use is as a 
treatment for non-muscle-invasive bladder cancer, where high-dose intravesicular infusion is a treatment of 
choice,29 and there has been interest in BCG's beneficial associations and effects in other cancers,30-32 multiple 
sclerosis33-35 and type 136,37 and type 2 diabetes.38-41  
In the CNS, ‘sterile inflammation’ resulting from autoimmunity, misfolded proteins, or cell damage or death can 
result in a neurotoxic environment if not adequately kept in balance. T-regulatory cells, or Tregs can regulate 
immunologic balance by controlling microglial and antigen presenting cell (APC) activation, inhibiting effector T 
(Teff) cells, and inducing astrocytic release of neurotrophins, thus effectively transforming a neurotoxic 
environment to a neurotrophic state. For example, two studies noted decreased neuronal cell death in a MPTP 
model of Parkinson’s Disease, finding that the magnitude of dopaminergic preservation correlated with the 
magnitude of Treg response to BCG treatment.42,43 BCG’s ability to induce and/or restore Treg immune balance is 
of great interest for treatment in AD,44,45 where preclinical models find that augmenting Tregs increases the 
number and function of microglia associated with plaques, and improves cognitive functions.18,46,47 In AD, Tregs 
may be overwhelmed due to their low numbers or dysfunction;48 BCG may increase Treg number and function to 
rebalance the neurotoxic state to one of neuroprotection.5  
Limited but provocative epidemiological analyses and in silico trial data support a benefit of BCG for AD. A 
large retrospective analysis found that older, non-demented adults treated with intravesicular BCG for bladder 
cancer had a significant 4-fold lower risk of subsequent AD over a median 8-year follow-up period than those 
treated with other means.45   
BCG, a highly safe, live, attenuated vaccine used throughout the world to prevent tuberculosis and confers many 
health-promoting non-specific effects, from preventing infectious disease to treating cancers. These effects rely on 
its induction of trained and heterologous immune responses in the host. Such immune effects directly overlap and 
influence immune responses known to occur in CNS disease; non-human studies have found benefit to BCG 
vaccination on models of Parkinson’s and Alzheimer’s diseases, including cognitive improvement. These studies 
provide the impetus for our initiating clinical trials with BCG oriented towards primary prevention of AD in older 
adults or disease modification and secondary or tertiary prevention in adults whom AD pathological changes are 
already in motion. This pilot study initiates this program, with focus on the immune consequences of treatment 
and potential changes in AD-specific biomarkers and cognition after vaccination. The BCG strain used in this 
study is not approved in the US for immunization against tuberculosis or for the treatment of AD. 
 
A pilot, open-label study of BCG immunization (Protocol#2020P002042) in both healthy older adults and adults 
with Mild Cognitive Impairment is currently underway with a focus on the immune consequences of BCG vaccine 
and potential changes in AD-specific biomarkers and cognition after vaccination. The 3-month, open-label study 
has so far demonstrated that repeated administration of BCG vaccine is safe and tolerable in healthy adults and 
adults with mild cognitive impairment between the ages of 55 and 80 years old. Following two intradermal 
vaccinations to participants in the unblinded study, there have been no serious adverse events, no clinically 
significant abnormalities detected in safety lab (eg. metabolic, hematologic, and immune labs) or vital sign 
measurements, and only mild-to-moderate, expected adverse events related to the lumbar puncture and blood draw 
procedures in the protocol. This small-scale study has informed the trial design and validated the assays included 
in the proposed in this protocol. 
 
3 
STUDY DESIGN 
 

An Open Label Trial to Evaluate the Effects of BCG Immunization on Biomarkers of Inflammation/Immune Response and Alzheimer’s Disease in Adults with
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3.1 
Overall Study Design and Plan 
This single-site, open-label trial will investigate the effects of BCG vaccination on IIR and AD biofluid biomarkers, 
magnetic resonance imaging (MRI) biomarkers, and neurocognitive/behavioral functioning over a one year period 
in older adults with mild cognitive impairment (MCI) to mild-to-moderate dementia due to AD. This study will also 
gather data on tolerability and safety. Safety will continue to be followed in the second year by phone. 
Up to twenty five participants meeting eligibility criteria will be enrolled. Each participant will receive two intradermal 
BCG vaccinations spaced 4-6 weeks apart and will be evaluated 12, 26, and 52 weeks after the initial immunization. 
Cerebrospinal fluid (CSF), blood, MRI (in a subset of patients), and neurocognitive/behavioral assessments will be 
collected at Screening (CSF, blood, and cognitive/behavioral), Week 1 (MRI and cognitive/behavioral), Week 4 
(blood), Week 12 (CSF, blood, and cognitive/behavioral), Week 26 (Blood and cognitive/behavioral) and Week 52 
(all) to assess biomarkers and safety. Safety measures, including updated medical history and adverse events, will be 
collected at Week 78 and Week 104. 
3.2 
Study Objectives 
The primary objectives of this study are: 
1. To assess the safety and tolerability of BCG vaccination in older adults with MCI and mild to moderate 
dementia due to AD. 
2. To investigate the potential of BCG vaccination as an immunomodulatory and CNS-protective agent for 
AD by evaluating changes from baseline in IIR and AD biomarkers in blood and CSF following BCG 
vaccination; 
3. To measure changes in neurocognitive/behavioral functioning following BCG vaccination (MoCA, 
RBANS, CDR-SB, NPI-Q, HADS, FAQ, DCT Clock). 
The secondary objective of the study is: 
1. To measure changes in structural and functional neuroimaging following BCG vaccination; 
 
3.3 
Study Outcome Measures 
3.3.1 Safety Measure Outcomes 
Medical history, adverse events, and classic metabolic, hematologic, coagulation, and immune blood labs will be 
collected at Screening to determine eligibility. Adverse events will be solicited at every visit. Hematologic and 
coagulation blood labs will be repeated at Week 12 and Week 52 visits for LP safety. Vital signs will be measured 
at all in-person visits. Additionally, four weeks after the first injection, we will assess the injection site and evaluate 
for any signs of systemic infection prior to second BCG vaccination in the other arm. Vaccination sites will also be 
assessed at Weeks 12, 26, and 52. We will use the FDA Toxicity Grading Scale, as performed in vaccine trials, to 
denote changes from baseline. Safety measures, including updated medical history and adverse events, will also be 
collected at Week 78 and Week 104. 

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3.3.2 
CSF and Blood Biomarker Outcomes 
CSF and blood will be collected at Screening, Week 4 (blood only), Week 12, Week 26 (blood only), and Week 52 
visits to examine biochemical biomarkers of: 
1. Target engagement: BCG, purified protein derivative (PPD) and / or mycobacterium tuberculosis (heat- 
killed, mTB) – induced cytokine response in peripheral blood cells will be used to evaluate for BCG 
vaccine-induced immunity. 
2. Pharmacodynamic response: a) Circulating cytokines and other related blood- and CSF-based IIR 
biomarkers relating to innate and adaptive immune responses; b) Epigenetic changes in peripheral blood 
cells and / or CSF- derived cell populations as indicators that BCG can lead to long term changes in 
peripheral and central nervous system immune cells. 
3. AD pathophysiology: Amyloid-β42/40, phospho-tau, total tau and / or neurofilament light protein 
biomarkers (ATN) and other exploratory AD-relevant synaptic and IIR biomarkers may be compared within 
subjects over time and between subjects and groups. 
4. BCG effects on the interactions of IIR and amyloid: We will perform PBMC stimulation with Aβ42 and 
positive-control antigens (e.g. lipopolysaccharide) and measure the molecular response in the supernatant 
at multiple timepoints after BCG /treatment. Aβ42 has immunostimulatory / inflammatory effects that 
increase with AD progression and may be modulated by an immune-provoking stimulus such as BCG. 
5. Additional related Alzheimer’s Disease and immune measures in plasma, CSF, and their cell populations 
at the transcriptional, protein, or epigenetic levels. 
We have several robust platforms in the Arnold lab (which serves as the Biomarker Core of the Massachusetts 
Alzheimer's Disease Research Center) to conduct these assays, including highly validated Euroimmun assays, 
Fujirebio Lumipulse assays, Meso Scale Discovery with plates run on a new Tecan EV0200 multi-functional robotic 
liquid handling platform, and Quanterix SR-X for ultrasensitive Simoa assays. We have exhaustively validated 
many assays specifically for use in short-term clinical trials49, and all assays that we propose demonstrate good 
precision and biotemporal stability. Change in a composite score from Baseline will be used as an outcome of 
response to BCG. 
 
3.3.3 Neurocognitive/Behavioral Measure Outcomes 
We will apply several robust neurocognitive tests to measure the effects of BCG vaccination on mentation, including 
the MoCA, RBANS, CDR-SB, NPI-Q, HADS, DCT Clock and FAQ. 
 
3.3.4 Structural and Functional MRI Measure Outcomes 
Up to 12 participants over 60 years old will be invited to undergo 3T structural and functional MRI at Week 1 and 
Week 52 visits to assess the effect of the study vaccine on regional brain volumes, cerebral perfusion, brain 
connectivity, and safety. Each scan will include high-resolution T1-weighted multi-echo MPRAGE for volumetric 
analysis, task-free blood oxygen level dependent (BOLD) sequence to measure resting neural connectivity, 
diffusion tensor imaging (DTI) to quantify white matter structural connectivity, T2-weighted fluid attenuated 
inversion recovery (FLAIR) sequence, and susceptibility- weighted imaging (SWI). 

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4 
SUBJECT SELECTION 
Each Investigator must adhere to the protocol detailed in this document and agree that any changes to the protocol 
must be approved by Institutional Review Board (IRB). Each investigator will be responsible for enrolling only 
those study subjects who have met protocol eligibility criteria. 
 
4.1 
Inclusion/exclusion Criteria 
Investigators will use their best clinical judgment when selecting potential research subjects for this study and will 
not enroll any individuals who are frail or in questionable health, even if they meet all inclusion/exclusion criteria. 
 
4.1.1 Inclusion Criteria 
Study subjects meeting all of the following criteria will be allowed to enroll in the study: 
1. Individuals between the ages of 55-85; 
2. MCI or moderate dementia due to AD as defined by the 2011 NIA-AA Workgroup recommendations; 
3. MoCA ≥ 8 at screening; 
4. Global CDR between 0.5-2 (inclusive) at screening; 
5. Amyloid and/or tau biomarkers indicative of AD pathology; 
6. Education level, English language skills and literacy indicates subject will be able to complete all 
assessments; 
7. Has a study partner who, in the investigator’s judgement, has frequent, direct contact with the participant 
at least several days a week, can accompany the participant to all visits, and is also able to provide 
information to study investigator/staff; 
8. Willing and able to complete all assessment and study procedures, including blood and lumbar punctures, 
and clinical assessments; 
9. If on cholinesterase inhibitor and/or memantine, doses are stable for 3 months prior to baseline; 
10. Negative test results for HIV antibody and Tuberculosis (QuantiFERON or T-spot TB test) at screening; 
11. No prior BCG exposure either through birth vaccinations (born in North American) or BCG bladder cancer 
treatment. 
4.1.2 
Exclusion Criteria 
Subjects meeting any of the following criteria during the screening evaluation will be excluded: 
1. History of chronic infectious disease, such as HIV or untreated or active hepatitis; 
2. History of tuberculosis, positive interferon-gamma release assay (IGRA, also known as the QuantiFERON- 
TB test) or T-spot TB test, including a test with a high reactivity to mycobacteria of non-tuberculosis 
variety; 
3. Prior BCG vaccination, positive T-spot tuberculosis test or a T-spot test showing significant Mycobacteria 
exposure; 
4. A positive SARS-CoV-2 PCR result within 3 months of screening, or known close contact with a confirmed 
COVID-19 positive person or symptoms highly suspicious for COVID-19 (per CDC guidelines) within 1 
month of screening, including fever, cough, shortness of breath, chills, muscle pain, new loss of taste or 
smell, vomiting or diarrhea, and/or sore throat, based on clinician’s judgment; 
5. History of treatment with metformin within the past one year; 
6. Treatment with other investigational agents which, at the discretion of the investigator, interfere with safety 
and/or study outcomes; 
7. Current treatment with immunosuppressants (calcineurin inhibitors, corticosteroids, or biological or 
cytotoxic immunosuppressants, or disease or condition likely to require high dose steroid or 

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immunosuppressive therapy); 
8. Other conditions or treatments associated with increased risk of infections or treatment with 
immunosuppressive medications for any reason; 
9. Current treatment with aspirin > 160 mg/day or chronic, daily NSAIDs; 
10. Current (as of time of study screening) or chronic use of antibiotics; 
11. History of keloid formation; 
12. Living with someone who is immunosuppressed and/or at high risk for infectious diseases (for example, 
HIV+ or taking immunosuppressive medications for any reason), or in a job (e.g. healthcare) in which the 
subject works with immunosuppressed populations; 
13. Other/confounding neurological or psychiatric condition, unstable medical or psychiatric conditions, 
contraindications to BCG use and lab abnormalities or concurrent medication use posing risk for BCG or 
study procedures; 
14. Laboratory abnormalities in B12, Folate, TSH, or other common laboratory parameters that may contribute 
to cognitive dysfunction per clinician judgment; 
15. Laboratory abnormalities in CBC, electrolytes, LFTs, BUN, Cr, total serum immunoglobulins, ESR, CRP, 
or urinalysis posing risk to treatment with BCG per clinician judgment; 
16. Laboratory abnormalities in PT-INR, which would pose a risk to performing the lumbar puncture 
procedure; 
17. Discontinuation of cholinesterase inhibitor or memantine within one month (28 days) prior to baseline visit; 
18. Females who are pregnant, lactating or of child-bearing potential; 
19. If male with female partner(s) of childbearing potential, unwilling or unable to adhere to contraception 
requirements specified in the protocol. 
20. Increased intracranial pressure as determined on a fundoscopy/neurological examination performed 
within 30 days of LP; 
21. Administration of live vaccine within 30 days of screening visit or BCG immunizations 
22. Administration of non-live vaccine within 14 days of screening visit or BCG immunizations 
23. If participating in optional MRI: Existing contraindication to MRI per MGH Athinoula A. Martinos Center 
research guidelines 
 
4.1.2.1 
Women of Childbearing Potential (WOCBP) 
For the purposes of this study, women of childbearing potential are defined as all women who are capable of 
becoming pregnant, unless they meet one of the following criteria: 
1. 
12-months post-menopausal. 
2. 
Post-hysterectomy. 
3. 
Surgically sterile. 
If a female subject does not meet these criteria and is considered of childbearing potential, they will be excluded 
from the clinical trial. 
 
Male subjects with female partners of child-bearing potential must use at least 1 of the following contraceptive 
methods: hormonal contraceptives (oral, injectable, patch, intrauterine devices), a barrier method (such as condoms 
or diaphragm) used with a spermicide (a foam, cream, or gel that kills sperm), male sterilization, or total abstinence 
from heterosexual intercourse, when this is the preferred and usual lifestyle of the subject. Abstinence is defined as 
refraining from heterosexual intercourse during the entire period of risk associated with the study. Note that periodic 
abstinence (calendar, symptothermal, post-ovulation methods), withdrawal (coitus interruptus), spermicides only, 

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and lactational amenorrhoea method are not acceptable methods of contraception. 
4.2 
Recruitment 
Up to twenty five subjects will be screened and enrolled in the study, and after screening we expect 12 subjects  to 
complete the study. 
 
4.2.1 
Recruitment of Subjects through Advertising 
Advertisement flyers will be posted on bulletin boards around MGB to advertise for the study as well as an 
advertisement on Partners Rally for Research. A phone number will be provided that will ring directly to the research 
coordinator, and voice messages can be left for the coordinator on a password-protected voice mailbox. The study 
coordinator will contact the subject and explain study in further detail and if the subject is interested, potentially 
complete a telephone prescreening. A listing of the study will also be posted on the MGH ACTRU website. 
Subjects may also be recruited through the distribution of IRB approved recruitment materials at community 
outreach initiatives including informational sessions to various community partners such as city-based and non- 
profit organizations. 
 
4.2.2 
Recruitment of Subjects from the Massachusetts Alzheimer’s Disease Research Center 
Subjects will also be recruited from an observational study that follows a longitudinal research cohort (LC) of 
approximately 400 active research participants in the Massachusetts Alzheimer's Disease Research Center 
(MADRC) recruited from the MGH’s Memory Disorders Unit clinic and other diverse sources. LC subjects are 
followed-up on an approximately annual basis, either in-person at the MGH or by means of a telephone follow-up 
‘visit’. Study staff will only contact subjects that have indicated to the MADRC that they are interested in hearing 
about/participating in other studies. 
 
4.2.3 
Recruitment of Subjects from among MGB Providers 
Subjects will be recruited from the outpatient clinical practices of the principal investigator (Dr. Steven Arnold). If 
the potential subject is the investigator’s patient, another member of the study or clinic staff will introduce the study 
to the patient and determine if they are interested in learning more about the study. An NP investigator will be made 
available to explain the study to the subject at their request. If the potential subject is not the investigator’s patient, 
the investigators will not directly approach the patient regarding possible participation in the study. 
 
Other providers across MGB will also be made aware of the study (including key eligibility criteria) via an IRB-
approved email and a link to the Rally ad. These providers (not participating directly in the study) may discuss the 
study directly with their patients, if they choose to do so. If the prospective participant is interested, their provider 
will provide them with a link to the Rally ad, which contains the research coordinator’s contact information. 
 
4.2.4 
Recruitment of Subjects from the CTRU Registry 
A research volunteer registry (P2021000916) was established in 2021 to maintain a list of people interested in 
participating in clinical research with the MGH Clinical and Translational Research Unit (CTRU). The goal of the 
CTRU, located in CNY 149, is to serve the clinical and research departments, institutes, centers, programs and labs 
that comprise the MGH Neuroscience community and to be a foundational component and research core facility 
within MGH’s emerging translational programs. Eligible participants who have consented to be contacted for future 
research may be identified using data stored within the volunteer registry. These participants will be contacted by 
phone or email and evaluated for interest and eligibility using the study-specific phone screen. 

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5 
SUBJECT ENROLLMENT 
 
5.1 
Informed Consent Process 
This study will be conducted in compliance with Title 21 Part 50 of the United States of America Code of Federal 
Regulations (CFR), Federal Regulations, and ICH Guidance Documents pertaining to informed consent. 
 
Potential subjects will be given general information about the research (e.g., through informational sheets, email, 
online Rally ad, or discussion with their treating physicians). If they are interested in learning more about the study, 
they will then contact the research coordinator. The research coordinator will obtain verbal consent in accordance 
with Mass General Brigham’s Prescreening Guidelines prior to performing a telephone prescreening interview. 
Information collected during the telephone prescreen will be entered directly into a REDCap survey for review by 
clinicians to determine eligibility for the study.  If the subject meets pre-screening criteria and wishes to continue 
the screening process, an in-person screening visit will be scheduled. 
 
At the screening visit, prior to starting the consenting process, one of our licensed Nurse Practitioners, Dr. Alison 
McManus, DNP, Kelly Devitte-McKee, NP, Amanda DeSenna, NP or one of our physicians Dr. Steven Arnold, 
MD and Dr. Marc Weinberg, MD will evaluate capacity to consent. These clinicians are experienced in evaluating 
capacity to consent in cognitively impaired patient populations and perform this assessment regularly. Like 
informed consent, evaluation of consent capacity is a process. The consenting clinician begins with an informal 
conversation prior to the start of the consent discussion with a prospective subject and the study partner, including 
simple questions to determine if the prospective subject may have problems that could affect decision making.  
 
Following the initial informal discussion, an investigator will discuss with the potential subject and their study 
partner the details of the study using the informed consent document as a guide. This discussion will include all the 
required elements of informed consent, including the purpose of the research, the procedures to be followed, the 
risks and discomforts, as well as potential benefits associated with participation, and alternative procedures to study 
participation. Their questions will be answered to their satisfaction. The subject will be provided with adequate time 
to reflect on the potential benefits and risks and possible discomforts of participation, and to make an informed 
decision. The capacity to consent again is gauged by an experienced licensed clinician investigator during the 
consent process through an evaluation of the participant’s understanding of their condition, their options for 
treatment, their understanding of the potential benefits and risks of participating in the clinical trial and of their 
understanding regarding the voluntary nature of their participation in the trial. In addition, participants are evaluated 
for their level of orientation and understanding of what will happen during the trial. Following this process, a 
discussion with the participant’s study partner will also inform the determination of the patient’s capacity to consent. 
All study staff will report any changes in function of the participant to the investigators, who will then reassess and 
document any changes in the patient's capacity to consent. 
 
If the clinician determines that the subject has adequate consent capacity, the participant will be able to consent for 
themselves and may enroll into the study. If it is determined by the clinician that the participant is unable to consent, 
but is willing and able to assent, then the LAR will provide surrogate consent to enroll the participant in the study. 
In either case, a study partner is required for participation in the study and they will be required to sign the consent 
form as such.  
 
Prior to proceeding with surrogate consent for assenting subjects, the clinician will also ensure that the surrogate 
LAR understands that his or her decisions should be based on "substituted judgment," which means that the decision 
reflects a potential subject's own views when s/he had the capacity to express them. If a potential subject did not 

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previously express a view on the matter, the surrogate should make the decision based on the potential subject's best 
interests. 
 
A licensed  Nurse Practitioner Investigator or Physician Investigator will then obtain written informed consent or 
surrogate consent from each participant prior to the initiation of any study procedures. We have included Dr. Alison 
McManus, DNP, Kelli Devitte-McKee, NP, and Amanda DeSenna, NP as study staff. All are experienced research 
nurse practitioners who will be consenting participants for this study in addition to the physician investigators. They 
currently consent on several other IRB-approved interventional trials run by the group, both industry and 
investigator sponsored. All potential subjects will be given the opportunity to speak with the Physician Investigator 
should a licensed Nurse Practitioner be involved in obtaining informed consent. 
 
If a participant is deemed unable to consent, either at the start of the extension study or while participating in the 
study, then consent will be obtained from a legally authorized representative. The relationship of the representative 
to the subject will be documented and kept for our records. The list of eligible legally authorized representatives, in 
order of preference, is as follows: 
1. Court appointed guardian with specific authority to consent to participation in research or authority to 
make health care decisions for a class of diagnostic and therapeutic decisions inclusive of the proposed 
research 
2. Health care proxy/person with durable power of attorney with specific authority for making health care 
decisions inclusive of the proposed research 
3. Spouse, adult child, or adult sibling. 
Subjects who cannot give informed consent must be able to give assent. If the individual objects to participation, 
they will not be enrolled. Investigators will document capacity to give assent during the consenting process. 
5.2 
Remuneration 
All subjects will receive: 
 
Parking fees paid for all in-person visits 
 
Lunch voucher provided for subject at each study visit that exceeds two hours, which can be used 
at the cafeteria at MGH CNY 149 
 
If appropriate, travel expenses will be reimbursed, including mileage at the current IRS travel rate for 
medical purposes, up to $150 per visit 
 
Honoraria: Up to $540 if subject completes the study (up to $740 including optional MRIs): 
o $15 for screening (SVa and SVb, inclusive) 
o $25 for each subsequent visit to the screening visit ($125 total) 
o $100 for each lumbar puncture ($300 total) 
o $100 upon study completion 
 
Optional study activities: 
o $100 for each optional MRI ($200 total) 
 
If for any reason the subject stops the study, they will be compensated for only the visits that they have completed. 
 
Payment will be made in the form of a check mailed to the subject's home address. They should receive the check 
within 2-3 weeks from the date of the study visit or study completion. Their social security number (SSN) will be 
required to process the payments. 

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5.3 
Discontinuation 
5.3.1 
Study Discontinuation 
A study subject will be discontinued from participation in the study if: 
1. Any clinical adverse event (AE), concurrent illness, or other medical condition or situation occurs such that 
continued participation in the study would not be in the best interest of the subject. 
2. The subject meets any exclusion criteria (either newly developed or not previously recognized) and/or fails 
to continue to meet inclusion criteria which the study physician deems to be a risk to continued participation. 
5.3.2 
Discontinuation of Study Intervention  
Individual subjects will be allowed to continue in the study, but administration of the second immunization will 
be withheld if, after the first injection, any of the following criteria are met: 
1. Any of the following serious adverse events attributable to BCG: 
 
Life-threatening adverse event 
 
Permanent or severe disability 
 
Important BCG-specific medical events that do not result in death, are not life-threatening, and/or 
do not require hospitalization will be considered as serious if, based on appropriate medical 
judgment, they jeopardize the participant and would require medical or surgical intervention to 
prevent a serious adverse event. 
2. Presentations of symptoms of disseminated BCG infection including persistent fevers, night sweats, 
weight loss. These symptoms may not occur within the timeline of the current study (they usually present 
4 months to 2 years after vaccination), however should such symptoms occur prior to the second 
immunization, the study vaccine would be stopped. 
3. The occurrence of any serious local skin reactions (≥ grade 3 ulceration, abscess formation, skin necrosis 
at the site of injection, or the development of suppurative lymphadenitis) as characterized by the standard 
FDA Toxicity grading scale (https://www.fda.gov/regulatory-information/search-fda-guidance- 
documents/toxicity-grading-scale-healthy-adult-and-adolescent-volunteers-enrolled-preventive-vaccine- 
clinical). 
5.3.3 
Voluntary Withdrawals 
A participant may choose to discontinue participation in the study at any time. Participants may be asked to 
complete an Early Termination Visit when they withdraw consent, i.e. withdrawing consent for participation in 
future study procedures. 
 
5.3.4 
Lost to follow-up 
The following actions must be taken in relation to a subject who fails to attend the clinic for a required study visit: 
 
The site must attempt to contact the subject and re-schedule the missed visit as soon as possible. 
 
The site must counsel the subject on the importance of maintaining the assigned visit schedule 
and ascertain whether or not the subject wishes to and/or should continue in the study. 
 
In cases where the subject is deemed ‘lost to follow up’, the Investigator or designee must make 
every effort to regain contact with the subject (where possible, 3 telephone calls and, if 
necessary  an email with read receipt). These contact attempts should be documented in the 
subject’s study record. 
 
Should the subject continue to be unreachable, only then will he/she be considered to have 

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withdrawn from the study with a primary reason of “Lost to Follow-up”. 
 
5.4 
Early Termination Visit (ET) 
Subjects who decide to withdraw from the study at any point after receiving the initial BCG injection  may be 
invited to come in for an Early Termination Visit. 
5.5 
Termination of Study 
This study may be prematurely terminated if, in the opinion of the investigator, there is sufficient reasonable 
cause. 
Circumstances that may warrant termination include, but are not limited to: 
1. Determination of unexpected, significant, or unacceptable risk to subjects; 
2. Unsatisfactory enrollment; 
3. Insufficient adherence to protocol requirements; 
4. Data that are not sufficiently complete and/or evaluable; 
5. If any grade 3 “severe” or higher adverse event occurs in seven or more participants, or if any participants 
have severe systemic reactions to the antigenicity of the vaccination with respiratory problems, the study 
protocol will be paused for review. The FDA, and all other relevant oversight bodies will be notified in a 
timely fashion if a batch of BCG is problematic. 
6. If during the study any subject develops a systemic BCG infection in the absence of an underlying 
immunocompromised state, the study will be stopped for a study of this side effect. 
 
If the study is prematurely terminated or suspended, the investigators will promptly inform the institution and 
regulatory authorities of the termination or suspension and the reason(s) for the termination or suspension. The 
IRB will also be informed promptly and provided the reason(s) for the termination or suspension by the 
investigator, as specified by applicable regulatory requirement(s). 

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6 
STUDY PROCEDURES 
6.1 
Schedule of Events 
 
Activity 
 
Phone 
Screen 
Visit 1 
Screen 
Visit 
Visit 2 
Baseline 
Visit 
Visit 
 3  
 
Visit  
4 
 
Visit 
5 
 
Phone 
Check 
1 
 
Visit 
6/ET 
 
Phone 
Check 
2 
 
Phone 
Check 
3 
 
 
Day ≥ 
-45
Week 1 
Week 4 
Week 
12 
Week 
26 
Week 
39 
Week 
52 
Week 
78 
Week 
104 
 
 
SVa 
SVb 
Day 1 
Day 28 + 
14 
Day 84 
±5 
Day 
182 
±14 
Day 
273 ±5 
Day 
364 ± 
28 
Day 
546 ± 
28 
Day 
728 ± 
28 
Informed Consent 
 
x 
 
 
 
 
 
 
 
 
 
Inclusion/Exclusion Review 
x 
x 
 
x 
 
 
 
 
 
 
 
Demographics 
x 
x 
 
 
 
 
 
 
 
 
 
Medical History Review 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Concomitant Medication 
Review 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Family History Review 
 
x 
 
 
 
 
 
 
 
 
 
Vital Signs, Height, and 
Weight 1
 
x 
x 
x 
x 
x 
x 
 
x 
 
 
Physical & Neurological Exam 
2 
 
x 
 
 
 
 
 
 
 
 
 
Fundoscopy 
 
 
 
 
x 
 
 
x 
 
 
Screening Labs  3 
 
x 
 
 
 
 
 
 
 
 
 
BCG Vaccination  
 
 
x
x
 
 
 
 
 
 
Injection Site 
Monitoring 4
 
 
 
 
x 
x 
x 
 
x 
 
 
LP Safety Labs 5 
 
x 
 
 
 
x 
 
 
x 
 
 
Blood Draw for 
Biomarkers 
 
 
x 
x 
x 
x 
x 
 
x 
 
 
CSF Draw for Eligibility 
& Biomarkers 
 
 
 
 
x 
 
 
 
x 
 
 
 
x 
 
 
Optional MRI 6 
 
 
 
x 
 
 
 
 
x 
 
 
RBANS, NPI-Q, HADS 
 
 
 
x 
 
x 
x 
 
x 
 
 
MoCA, FAQ 
 
x 
 
 
 
x 
x 
 
x 
 
 
CDR 
 
 
 
x 
 
 
 
 
x 
 
 
DCT Clock 
 
 
 
x 
 
 
x 
 
x 
 
 
Adverse Event  Review 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
[1] Height measured at screening only. Weight measured at screening, week 26, and week 52 
[2] Physical and neurological examinations will be performed at screening and at any other time if indicated per clinician investigator judgement 
[3] Eligibility lab tests include CBC w/ diff, CMP, TSH, Folate, B12, HIV antibodies, serum immunoglobulins, ESR, CRP, 
QuantiFERON-TB Gold Test/T-spot TB test, urinalysis, PT/INR 
[4] Vaccination site monitoring may include photographs of the injection site if indicated per clinician investigator judgement 
[5] LP safety labs include PT/INR and CBC w/ diff 
[6] Multi-sequence MRI for structural and functional measures include T1 MP-RAGE, T2 FLAIR, DWI, DTI, BOLD, ASL 

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6.1.1  RCT roll over participant schedule 
Participants enrolled in the original randomized-controlled BCG trial will be invited to roll into the open-label trial at 
their scheduled Visit 3 using a Consent addendum. After unblinding the randomization schedule, participants will be 
told which study vaccine they received at their Baseline Visit.  
 
If at Baseline, the participant received BCG, they will proceed in the study along the same schedule outlined in the 
Schedule of Events, receiving their second BCG vaccine at Visit 3/Day 28.  
 
If at Baseline, the participant received placebo, their Day 28 visit will be become their new Day 1 visit. At this Day 1 
visit we will collect vitals and administer the first BCG vaccine. No additional blood or CSF will be collected, nor 
will any cognitive testing be performed. The participant will receive their second BCG vaccine approximately 1 month 
after the first BCG vaccine, and continue on the Schedule of Events outlined after the Day 1 visit in Section 6.1. The 
total time in the study will be extended by 1 month for participants originally assigned to the placebo arm of the 
randomized controlled trial. 
 
6.2 
Study Visits 
6.2.1 
Screening 
6.2.1.1 
Telephone Pre-Screening 
Subjects will be screened over the phone by study staff to determine eligibility and to ensure that the subject is 
safe to undergo all study assessments. These procedures include: 
 
Obtain verbal pre-screening informed consent from subject 
 
Assess inclusion and exclusion criteria 
 
Collect brief demographics 
 
Obtain neurocognitive disease diagnosis history and medical history, including COVID-19 diagnosis and 
previous exposure risk 
 
Review and document concomitant medications and therapies 
 
During this call, study procedures will be discussed in detail and the subject will be given an opportunity to ask 
questions about the study. All other study procedures will take place after signing an IRB-approved consent form 
in-person. For those that have impaired decision making, the phone screening process will be coordinated with 
the study partner and will be conducted in the presence of both the study partner and the potential participant. 
 
6.2.1.2 
Screening Visits (Visit 1; Day ≥-45) 
All subjects who are eligible after a telephone pre-screening will be invited to MGH CNY for Screening that 
will include 2 separate visits.  
 
The first visit (SVa) will take approximately 2 hours and following procedures will be performed: 
 
Obtain written informed consent from subject 
 
Review inclusion and exclusion criteria 
 
Obtain medical and family history including COVID-19 history, concomitant medications and therapies 
 
Vital signs, height, and weight 
 
Obtain demographic information 
 
Neurocognitive and behavioral assessments: MoCA (version 8.1), CDR, FAQ 
 
Perform a physical, including inspection for BCG scar on either deltoid 
 
Perform a neurological examination 
 
Blood draw for screening labs 

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o CBC with differential 
o Comprehensive metabolic panel (CMP) 
o Thyroid stimulating hormone (TSH) 
o B12 
o Folate 
o HIV antibodies 
o Tuberculosis (QuantiFERON-TB Gold Test/T-Spot TB Test) 
o C-Reactive Protein (CRP) 
o Erythrocyte sedimentation rate (ESR) 
o Serum immunoglobins (IgA, IgG, IgM) 
o PT/INR 
 
Urinalysis 
 
Assess and document adverse events (AEs) after subject signs ICF 
 
If the participant is still eligible after the first Screening visit (SVa), they will be invited to come back for a second 
Screening visit (SVb) which will take approximately 1 hour. The following procedures will be performed: 
 
Vital signs 
 
Blood draw for biomarkers 
 
Fasting CSF draw for screening (if required) and baseline biomarkers 
 
Assess for changes to medical history, concomitant medications and/or adverse events 
 
6.2.1.3 
Screen Failures 
Any subject who signs consent will be considered enrolled in the study. If a subject fails screening, at a minimum, 
the following information should be captured and entered into the Electronic Data System (EDC). 
 
Inclusion/Exclusion Criteria 
 
Demographics 
 
Reason for screen failure 
6.2.1.4 
Re-screening 
One re-screen is allowed ≥30 days after the initial screening. All screening procedures will be repeated at the re- 
screen visit. 
 
6.2.2 
Baseline Visit/Visit 2 (Day 1) 
All subjects who pass screening will return to MGH CNY for the Baseline visit. At this visit, baseline clinical 
measures will be collected, and the first BCG injection will be administered. The Baseline visit will occur within 
45 days of the screening visit. Baseline measures may be collected up to 3 days before the first BCG dose is 
administered. The date of the first BCG injection will be considered “Day 1”. The visit will take approximately 3-
4 hours and will include: 
 
Vital signs 
 
Review inclusion and exclusion criteria 
 
Assess for changes to medical history, concomitant medications and/or adverse events 
 
Neurocognitive and behavioral assessments: RBANS (form a), NPI-Q, HADS, DCT Clock 
 
Blood draw for biomarkers 
 
MRI (optional) 
 
First BCG administration followed by 30 minutes of monitoring 

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6.2.3 
Visit 3 (Day 28 + 14 days) 
Subjects will be asked to return to MGH CNY for a second BCG injection visit 28 (+14) days after the first 
vaccination. This visit will take approximately one hour and will include: 
 
Vital signs 
 
Assess any changes to medical history, concomitant medications and/or adverse events 
 
Blood draw for biomarkers 
 
Injection site monitoring 
 
Second BCG dose administered followed by 30 minutes of monitoring 
 
6.2.4 
Visit 4 (Day 84 ± 5 days) 
Subjects will be asked to return to MGH CNY 84 (± 5) days after the first injection. This visit will take 
approximately three hours and will include: 
 
Vital signs 
 
Assess any changes to medical history, concomitant medications and/or adverse events 
 
Blood draw for safety 
 
Blood draw for biomarkers 
 
Focused Exam: Fundoscopy 
 
Neurocognitive and behavioral assessments: RBANS (form b), NPI-Q, HADS, MoCA (version 8.2), 
FAQ  
 
Blood draw for LP safety labs  
 
Blood draw for biomarkers  
 
Fasting CSF draw for biomarkers  
 
Injection site monitoring  

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6.2.5 
Visit 5 (Day 182 ± 14 days) 
Subjects will be asked to return to MGH CNY 182 (± 14) days after the first injection. This visit will take 
approximately two hours and will include: 
 
Vital signs 
 
Assess any changes to medical history, concomitant medications and/or adverse events 
 
Blood draw for biomarkers 
 
Neurocognitive and behavioral assessments: MoCA (version 8.3), FAQ, RBANS (form c), NPI-
Q, HADS, DCT Clock 
 
Injection site monitoring 
 
6.2.6 
Phone Check 1 (Day 273 ± 5 days) 
Subjects will be asked to complete a check-in phone call 273 (± 5) days after the first injection. This will take 
approximately fifteen minutes and will include: 
 
Assess any changes to medical history, concomitant medications and/or adverse events 
 
6.2.7 
Visit 6 (Day 364 ± 28 days) 
Subjects will be asked to return to MGH CNY for the final in-person visit at Day 364 (± 14 days) to assess study 
outcomes. This visit will take approximately four hours and will include: 
 
Vital signs 
 
Assess any changes to medical history, concomitant medications and/or adverse events 
 
Neurocognitive and behavioral assessments: FAQ, MoCA (version 8.1), CDR, RBANS (form a), 
NPI-Q, HADS, DCT Clock 
 
Blood draw for biomarkers 
 
Blood draw for LP safety 
 
Focused Exam: Fundoscopy 
 
Fasting CSF draw for biomarkers 
 
MRI (optional) 
 
Injection site monitoring 
 
6.2.8 
Phone Check 2 & 3 (Day 546 ± 28 days, Day 728 ± 28 days) 
Subjects will be asked to complete a check-in phone call approximately 18 months and 2 years after the first 
injection to monitor safety. This will take about fifteen minutes and will: 
 
Collect any changes to medical history, concomitant medications, and/or adverse events 
If the subject reports any new adverse events at the Phone Check that may require follow up, they will be 
referred to their primary care provider for further evaluation. 
 
6.2.9 
Early Termination Visit 
If the subject withdraws from the study before completion of all study visits, they may be invited to return to 
MGH CNY for a final visit. This visit will take approximately four hours and will include the following measures: 
 
Vital signs 
 
Assess any changes to medical history, concomitant medications and/or adverse events 
 
Neurocognitive and behavioral assessments: FAQ, MoCA (version 8.1), CDR, RBANS (form a), 
NPI-Q, HADS, DCT Clock 
 
Blood draw for biomarkers 
 
Blood draw for LP safety 

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 
Fasting CSF draw for biomarkers 
 
MRI (optional) 
 
Injection site monitoring 
The option to decline any of the early termination procedures or the entire visit will be given to all participants 
withdrawing from the study. 
6.2.10 Protocol Deviations 
A protocol deviation is any noncompliance with the current clinical trial protocol. The noncompliance may be on 
the part of the subject, the PI or Co-Is, or the study staff. As a result of deviations, corrective actions will be 
developed by the PI and implemented promptly. All deviations from the protocol must be addressed in the subject’s 
documents. Protocol deviations will be sent to the IRB per their guidelines and entered in the Protocol Deviations 
Log. 
6.2.11 Missed Visits and Procedures 
Missed visits and any procedures not performed (not attempted) for reasons other than illness, injury, or progressive 
disability (i.e.: a subject is physically unable to perform them) will be reported as protocol deviations. 
Procedures or visits not performed due to illness, injury, or disability, including procedures that were attempted but 
failed (i.e.: blood samples unable to be drawn after multiple attempts or weight unable to be obtained due to subject 
immobility) will not be reported as protocol deviations. 
 
6.3 
Clinical Assessments 
Assessments will be performed at designated time-points throughout the study for clinical evaluation. In addition 
to the assessments evaluated below, subjects will provide information on their demographics, past medical and AD 
history, family history, medication usage, and vaccination history. 
 
6.3.1 
Safety Measures 
6.3.1.1 
Vital Signs, Height, Weight 
Vital signs, including systolic and diastolic blood pressure, pulse rate (radial artery)/minute, respiratory rate/minute, 
and temperature will be assessed at all in-person visits. Height will be measured at the screening visit only. Weight 
will be measured at screening, week 26, and week 52 visits. Verbal weight may be documented for those subjects 
utilizing a wheelchair. 
 
6.3.1.2 
Clinical Laboratory Assessments 
Study participants will be asked to provide the volumes of blood and urine detailed in Table 1 for eligibility (at 
screening) and safety lab analyses. The participant will have their whole blood collected by either a nurse or 
phlebotomist from a peripheral vein (or finger prick for point of care coagulation test). Blood will be handled, 
processed, and analyzed in accordance with regulations set forth by the American Society for Clinical Pathology 
and the College of American Pathologists. 
 
Table 1. Safety Laboratory Tests 

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Visit 
Labs 
Volume 
required 
 
Screening (SV1a) 
PT/INR, Complete Blood Count w/ differential, Complete Metabolic Panel, C- 
Reactive Protein, Erythrocyte Sedimentation Rate, HIV Antibody, 
QuantiFERON-TB Gold test/T-spot TB test, Folate, B12, Serum 
immunoglobins (IgG, IgA, 
IgM), TSH, Urinalysis
 
33 mL blood, 
10 mL urine 
Screening (SV1b)
N/A 
N/A 
Week 1 
N/A 
N/A 
Week 4 
N/A 
N/A 
Week 12 
Complete Blood Count w/ differential and PT/INR 
6 mL 
Week 26 
N/A 
N/A 
Week 52 
Complete Blood Count w/ differential and PT/INR 
6 mL 
 
All subjects will have safety laboratory tests at the designated visits outlined in the protocol. These samples will be 
analyzed at MGH Core Laboratory. The SI may order additional testing, if thought to be necessary, to further assess 
an adverse event (AE), or if there is any suspicion that a subject may be pregnant, throughout the course of the 
study. 
 
Tests for Complete Blood Count (CBC), Comprehensive Metabolic Panel (CMP), and International Normalized 
Ratio (INR) may be performed with CLIA-waived point of care devices: Sysmex XW-100 CBC Analyzer, Piccolo 
Xpress Chemistry Analyzer, and the Roche CoaguChek XS Plus PT/INR Analyzer, respectively. 
 
6.3.2 
Physical and Neurological Examination 
A physical and neurological examination will be performed at the Screening visit. The following systems will be 
examined: general appearance, head, eyes, ears, nose, throat, neck, chest, heart, abdomen, extremities, edema, 
peripheral vascular, skin and appendages, musculoskeletal, central nervous system and back. A physical and/or 
neurological exam may be repeated at any visit if indicated per clinician judgement. 
A focused fundoscopy examination will be repeated before the second and third lumbar punctures to assess for 
increased intracranial pressure. 

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6.3.3 
Phlebotomy for Research: Target Engagement, Cytokine Levels and Biomarker Analysis 
Subjects will provide additional blood samples for biomarker analysis at all in person visits. Target engagement 
will determine whether the subject’s immune system has responded to BCG. In general, whole blood will be either 
collected and utilized for biochemical / metabolic assays, or spun in EDTA-containing tubes with plasma being 
frozen for cytokine analysis of IIR and / or AD biomarkers. Remaining plasma may be used for other biochemical 
analyses. Peripheral blood mononuclear cells (PBMCs) will be isolated from the cell pellets, some of which will be 
frozen for later culturing and cell-stimulation studies. Other PBMCs will be further isolated into subpopulations of 
cells and frozen for epigenetic analyses (e.g. ATACseq) and possibly subject to other immunostaining or 
quantitative biochemical assays. 
 
In addition to these analyses, blood will also be collected for routine blood tests that will be used for research 
purposes including Complete Blood Count with differential (CBC w/ diff), C-Reactive Protein (CRP), and 
Erythrocyte Sedimentation Rate (ESR). CRP and ESR tests will be performed at a central laboratory. CBC w/ diff 
may be performed either on-site using a CLIA-waived point-of-care device (Sysmex XW-100 CBC Analyzer) or at 
a central laboratory. Blood volumes collected at each visit are detailed in Table 2. 
 
Table 2. Blood Collection for Research 
 
Visit 
Blood volume required 
for biomarker analyses 
(mL) 
Volume required for 
routine tests (mL) 
TOTAL 
(mL) 
Screening 
(SV1a)
0 
0 
0 
Screening 
(SV1b)
75 
0 
75 
Week 1 
50 
0 
50 
Week 4 
75 
3 for CBC w/ diff 
78 
Week 12 
125 
9 for CBC w/ diff, ESR, CRP 
134 
Week 26 
125 
9 for CBC w/ diff, ESR, CRP 
134 
Week 52 
125 
6 for ESR, CRP 
131 
Total 
575 
27 
602 
 
 
Blood processing will occur on site at MGH. Blood samples will be de-identified, codified, and stored on-site. 
Blood samples will be stored for research until they are used, damaged, or otherwise unfit for analysis. Some 
biomarker analyses may be done at Harvard-affiliated core labs. 
 
Samples drawn for biomarker research may also be used for genome analyses in the future. Subjects will not 
receive the results of these studies if they are done and genetic research results will not be placed in the subjects’ 
medical records. 
6.3.3.1   Sending Blood Samples to Research Collaborators Outside Mass General Brigham 
Blood samples may be sent to Merck & Co. and Active Motif, Inc. for analyses. Samples may include isolated 
PBMCs or their derivatives. Samples will be de-identified with a study code and visit date. 
6.3.4 
Lumbar Puncture for Immune and Biomarker Analysis 

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Lumbar punctures (LP) will occur at Screening, Week 12, and Week 52. Lumbar punctures will be performed after 
a minimum of an 8 hour fast. The subject will be positioned seated or lying on their side on the examination table. 
Standard protocols will be used employing palpation to identify the L3-4, L4-5 or L5-S1 vertebral interspaces, 
sterile conditions, local lidocaine anesthesia, and use of standard of care spinal needles. Clinical judgement will be 
used to determine the optimal spinal needle type/size for each participant. Lumbar punctures will be performed by 
qualified, experienced practitioners. Approximately 25 mL of CSF at each visit will be collected. A small portion 
of this volume will be used for analysis of cell count with differential, total protein, and glucose. The remainder 
will be spun and cells isolated from supernatant. Supernatant will be frozen for later immunoassays. Cells will be 
frozen for later biochemical analyses. Subjects will also complete a brief REDCap survey after the LP in which they 
will answer questions about the tolerability of the procedure. After the LP is complete, subjects will be given time 
to eat breakfast or have a snack before continuing with the visit. 
If a subject has difficult back anatomy or is particularly overweight, and the physician or nurse practitioner does 
not feel comfortable performing the LP because they are not able to locate the proper lumbar landmarks, the subject 
will be scheduled to undergo the LP under fluoroscopy at MGH Interventional Radiology. Fluoroscopy time will 
vary person to person. The average radiation dose is 0.18 millisieverts (mSv), or the equivalent of 21 days of natural 
background radiation, for each lumbar puncture done under fluoroscopy. 
CSF samples will be de-identified, codified, and stored on-site at the MADRC/MIND biorepository at the Arnold 
Lab. CSF samples will be stored for research until they are used, damaged, or otherwise unfit for analysis. Some 
biomarker analyses may be done at Harvard-affiliated core labs. 
6.3.4.1   Sending CSF Samples to Research Collaborators Outside Mass General Brigham 
CSF cell samples may be sent to Merck & Co. and Active Motif, Inc. for analyses. Samples will be de-identified 
with a study code and visit date. 
6.3.5 
Injection Site Monitoring 
Prior to the first injection, both deltoids will be inspected to confirm there is no evidence of previous BCG 
vaccination and no obvious symptoms that would contraindicate vaccine injection. At the Week 4 visit, prior to the 
second injection, the vaccination site will be inspected for any signs of local infection. At Week 12, Week 26, and 
Week 52 and if indicated by subject-reported AEs, the site(s) of vaccination will be examined, and any visible 
reactivity will be recorded. In addition, photographs of the injection site may be taken using an MGB-approved and 
encrypted device. Photographs will be stored on the MGB Dropbox. 
6.3.6 
Magnetic Resonance Imaging (MRI) 
Based on investigator discretion and cognitive ability, up to 12 participants over 60 years old may be invited to 
participate in an optional 3T structural and functional MRI at Week 1 and Week 52 to assess the effect of the study 
vaccine on regional brain volumes, cerebral perfusion, and structural and functional brain connectivity. Each scan 
will include high resolution T1- weighted multi-echo MPRAGE for volumetric analysis, task-free blood oxygen 
level dependent (BOLD) sequence to measure resting neural connectivity, diffusion tensor imaging (DTI) to quantify 
white matter structural connectivity, T2-weighted fluid attenuated inversion recovery (FLAIR) sequence to identify 
brain lesions, including cortical and periventricular lesions, and susceptibility-weighted imaging (SWI) to identify 
micro-hemorrhages and venous connectivity in lesions, and pseudo-continuous arterial spin labeling (pCASL) to 
quantify regional cerebral  blood flow (a marker of resting cerebral perfusion). 
 
6.4 
Neurocognitive Assessments 
6.4.1 
Montreal Cognitive Assessment (MoCA) 
The Montreal Cognitive Assessment (MoCA) is a commonly utilized questionnaire in clinical trials and research 
settings to measure levels of cognitive impairment.50 The MoCA measures five areas of cognitive function: 
orientation, visuospatial, attention and calculation, recall, and language. The MoCA (version 8.1) will take 
approximately 15 minutes to complete. The MoCA will be administered at the Screening visit (version 8.1) to 
determine subject eligibility (MoCA >8) and repeated at Week 12 (version 8.2), Week 26 (version 8.3), and Week 
52 (version 8.1). 

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6.4.2 
Repeatable Battery for the Assessment of Neuropsychological Status Update (RBANS) 
The RBANS51 is a commonly used 25-minute, standardized neurocognitive battery. The RBANS measures five 
neurocognitive domains, with twelve subtests measuring cognitive decline or improvement with immediate memory 
(List Learning and Story Memory), visuospatial/constructional (Figure Copy and Line Orientation), language 
(Picture naming and Semantic Fluency), attention (Digit Span and Coding), and delayed memory (List Recall, List 
Recognition, Story Memory, and Figure Recall). The RBANS has been shown to be effective at both detecting and 
characterizing forms of dementia. The Delayed Memory domain has been shown to be particularly sensitive to 
discriminating mild cognitive impairment (MCI) due to Alzheimer's disease from controls, and also is predictive of 
cerebral amyloid burden. The RBANS will be administered at Week 1 (form a), Week 12 (form b), Week 26 (form 
c), and Week 52 (form a). 
 
6.4.3 
Clinical Dementia Rating (CDR) Scale 
The CDR is a 5-point scale used to characterize six domains of cognitive and functional performance applicable to 
Alzheimer disease and related dementias using a 30-minute semi-structured interview: Memory, Orientation, 
Judgment & Problem Solving, Community Affairs, Home & Hobbies, and Personal Care. The necessary 
information to make each rating is obtained through a semi-structured interview of the patient and an informant 
(study partner). The CDR table provides descriptive anchors that guide the clinician in making appropriate ratings 
based on interview data and clinical judgment. In addition to ratings for each domain, an overall CDR score may 
be calculated using an algorithm. This score is useful for characterizing and tracking a patient's level of 
impairment/dementia: 0 = Normal; 0.5 = Very Mild Dementia; 1 = Mild Dementia; 2 = Moderate Dementia; 3 = 
Severe Dementia. The CDR scores will be converted into a ‘sum of boxes (CDR-SOB)’ score, which is more 
sensitive in distinguishing between individuals with milder declines in functioning as it increases the range of 
possible scores 52. The CDR will be administered at Screening to determine subject eligibility (CDR between 0.5-2 
inclusive) and will be repeated at Week 52. The portion of the CDR completed with the study partner may be 
completed over the phone at Week 52.  
 
6.4.4   DCT Clock 
DCT clock, a digitized version of the standard pen and paper neuropsychological clock drawing test, is a non-
invasive, computer-based cognitive assay. The DCT clock application is preloaded onto the iPad. The test involves 
participants drawing two clock faces on an iPad, with a digital pen that precisely tracks and records drawing 
behavior. The positional data generated during this assessment is then analyzed by DCT’s proprietary algorithms 
that evaluate hundreds of features captured in the pen stroke information. By comparing test results to normative 
data, the system then determines whether the test was within normal limits and provides a detailed breakdown of 
performance on the various cognitive tasks evaluated during the test. 
 
6.5 
Neuropsychiatric Assessments 
6.5.1 
Neuropsychiatric Inventory Questionnaire (NPI-Q) 
The NPI-Q53 will rate symptoms in 12 sub-domains of behavioral functioning including: hallucinations, delusions, 
agitation, dysphoria, anxiety, euphoria, apathy, disinhibition, irritability, aberrant motor activity, eating 
abnormalities, and night-time behavioral alternations. The NPI-Q is administered to an informant (study partner) 
and will be done at Week 1, Week 12, Week 26, and Week 52. The NPIQ may be completed over the phone at 
Week 12, 26, and 52.  
 
 
6.5.2 
Functional Activities Questionnaire (FAQ) 

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The FAQ12,54is a brief rating scale used to determine a subjects’ level of functional independence when performing a 
range of instrumental activities of daily living (IADLs), with repeat assessments useful for monitoring performance 
in these areas over time. The FAQ total score (ranging from 0-30) reflects the sum of ordinal ratings (0 = fully 
independent, 1 = has difficulty but does by self, 2 = requires assistance, and 3 = dependent) across ten items 
assessing a variety of functional activities (i.e., preparing a balanced meal, financial management skills, and 
shopping), with higher scores indicating increasing levels of dependence. For activities not normally undertaken by 
a person, a score of 1 is assigned if the subject would be unable to complete the task if required, or a score of 0 is 
assigned if the subject believes they could successfully carry out the task if needed. Overall, the FAQ is a sensitive 
marker of functional impairment among individuals with varying dementia severity and has been shown to 
differentiate mild cognitive impairment from early dementia due to Alzheimer’s Disease with 80% sensitivity and 
87% specificity. The FAQ demonstrates high reliability (exceeding 0.90), takes about 5 minutes to complete, and 
requires limited rater training to administer. The FAQ is administered to an informant (study partner) and will be 
done at Screening, Week 12, Week 26, and Week 52. 
 
6.5.3 Hospital Anxiety and Depression Scale (HADS) 
 
The HADS (Hospital Anxiety and Depression Scale) aims to measure symptoms of anxiety and depression. HADS 
is a 14-item scale with seven items each for anxiety and depression subscales. Scoring for each item ranges from 
zero to three. A subscale score >8 denotes anxiety or depression. HADS will be administered at Week 1, Week 12, 
Week 26, and Week 52. 
 
 
7 
STUDY INTERVENTION ADMINISTERED 
 
Table 3. Summary of Investigational Products 
 
 
Investigational Products 
Product Name: 
Bacillus Calmette-Guérin (BCG) vaccine 
[Tokyo BCG (JBL, Tokyo, Japan)] 
Diluent (0.9% sodium chloride for 
injection) 
Dosage Form: 
For intradermal injection 
For intradermal injection 
Unit Dose 
0.36-3.9 x 106 colony forming 
units(CFU)/injection of 0.1ml
0.1 mL 
Route of Administration 
Intradermal injection 
Intradermal injection 
Physical Description 
sterile, freeze-dried 
BCG diluent 
Manufacturer 
Japan BCG Laboratory (JBL) 
Supplied by MGH clinical trials 
pharmacy (manufacturer varies)
 
7.1 
Study Drug 
Japan BCG is an approved drug in Japan and has been sanctioned by UNICEF and WHO as a standard vaccine for 
the prevention of tuberculosis. BCG has been administered for this purpose for over 100 years on a global basis, 
with typical distributions of over 50,000,000 doses to newborns per year. The vaccine has also been approved and 
utilized for treatment of bladder cancer for the past 30 years. 

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JBL is the manufacturer of BCG products produced from the BCG strain of Tokyo 172-1 and Dr Faustman and 
MGH oversee and regulate this manufacturing. This protocol will be reviewed by the Japan BCG Laboratory ethics 
committee prior to commencement of study activities. Drug-related contracting will occur through Dr. Faustman 
who maintains the Master Drug File, coordinates various IND filing around this vaccine and will interface with 
regulatory affairs management. 
The study drugs (BCG vaccine and diluent) are manufactured by Japan BCG Laboratory and supplied by the MGH 
Clinical Trials Pharmacy (manufacturers vary), respectively, and will be managed and stored by the study 
pharmacist in the Faustman lab according to the directions provided by the manufacturer in  the approved product 
labeling. The same study pharmacist and study drug have been used for the past 6 years in Dr. Faustman’s protocol 
#2013P002633 in addition to the pilot protocol #2020P002042. 
Japan BCG is a freeze-dried preparation made from the Tokyo 172 strain of Mycobacterium bovis Bacillus Calmette 
and Guérin (BCG). Japan BCG is an attenuated BCG product derived from the original attenuated BCG generated 
by Albert Calmette and Camille Guerin at the Pasteur Institute. 
7.2 
Study Drug Packaging and Labeling 
The Japan BCG drug product is a sterile, freeze-dried preparation of BCG.  
This drug is stored at 2-8oC. 
7.3 
Study Drug Preparation 
The BCG vaccine is supplied as a 10-dose ampoule. For reconstitution, 1.0 ml of sterile saline is added to the 10- 
dose ampoule. Each 0.1ml dose is drawn by syringe from the ampoule following reconstitution and administered 
intradermally. Once reconstituted, the pharmacy provides the study clinic with the pre-filled syringe in a covered 
surgical tray to prevent light exposure. If the prepared syringe cannot be immediately administered, it will be 
temporarily stored at 4oC until administration (< 1 hour from reconstitution). The BCG vaccine will be prepared by 
the study pharmacist at MGH. 
Each ampoule may be used for a maximum of two study participants in this study. The ampoule will be reconstituted, 
and two syringes may be drawn at the same time. As above, the prepared syringes  will be covered and stored at 4oC 
until administration within 1 hour of reconstitution. The remaining unused volume  in the ampoule will not be used 
for additional patients in this trial. 
7.4 
Dosage and Administration 
All subjects will be consented by a study clinician before any of the following procedures are done. The skin site 
(deltoid region, alternating between first and second dose) is first cleansed with an alcohol or acetone sponge and 
allowed to dry thoroughly. Next, 0.36-3.9 x 106 colony forming units (CFU), reconstituted by the study pharmacist 
in 0.9% saline diluent within 1 hour, will be administered intradermally via a 15mm, 26-gauge needle. 
Dose selection is based on published data illustrating that two doses of BCG in autoimmune type 1 diabetes confer 
the immune regulatory/inflammation modulation properties that are of interest in the present study. Specifically, 
administration of two doses in type 1 diabetes is associated with short term changes in biomarkers with 22 weeks 
of observations.36  Moreover, two doses of BCG resulted in a clinical correction of HbA1c in type 1 diabetes with 
stable correction of disease process for 8 years.37 
7.5 
Study Drug Accountability 
Vaccine accountability will be maintained by the study pharmacist. 
7.6 
Study Drug Handling and Disposal 
Vaccine handling and disposal will be managed by the study pharmacist. 
7.7 
Contraindications and Warnings 
7.7.1 
Prohibited Medications 

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Treatment with the following medications during the study is prohibited: 
 Metformin 
 Aspirin > 160 mg/day 
 Chronic daily NSAIDS 
 Immunosuppressants including calcineurin inhibitors, corticosteroids, or biological or cytotoxic 
immunosuppressants (e.g. imuran, methotrexate, cyclosporine, etanercept, infliximab) 
 Antibiotics within 14 days of immunizations 
 Administration of live vaccine within 30 days of screening visit or BCG immunizations 
 Administration of non-live vaccine within 14 days of screening visit or BCG immunizations 
 
7.7.2 
Warnings 
BCG contains live, attenuated mycobacteria, and carries risk for adverse consequences particularly in 
immunocompromised individuals for whom the attenuated strain could become pathogenic as described below 
(section 9.1, BCG vaccine risks). 
 
8 
BIOSTATISTICAL ANALYSIS 
8.1 
Sample Size 
A sample size of approximately 15 subjects was chosen based on feasibility and is not based on statistical 
considerations. The primary objective of this early-phase trial is to measure safety and target engagement. While 
there is no ‘best practice’ method of determining target engagement of the BCG vaccine, the method that best takes 
advantage of the collected biomaterials is the in-vitro IFN-gamma release assay. PBMCs are stimulated and after 
several days the culture media is tested for presence of the IFN-gamma cytokine. In a previously published study, 
it was demonstrated a significant IFN-gamma release difference associated with BCG treatment in n = 14 subjects 
55.  
 
8.2 
Safety Endpoints 
The primary safety endpoint will be the incidence of treatment emergent Grade II-IV adverse events. 
Other safety endpoints are: 
 
Incidence and severity of treatment emergent adverse events (AEs) 
 
Clinical laboratory tests 
 
Vital signs 
 
Physical examinations 
 
Use of concomitant medications for treatment of AEs 
All concomitant medications will be tabulated according to drug class and preferred term using the WHO 
dictionary. The safety data will be summarized by treatment group. Treatment AEs will be coded and graded 
using MedDRA grading criteria. The treatment groups will be compared with respect to occurrence of each 
adverse event and incidence of Grade II/IV adverse events. Withdrawal, abnormal laboratory tests, vital signs and 
use of concomitant medications used for treatment of AEs will be assessed to characterize the safety profile of 
BCG. Descriptive statistics denoting the changes from baseline to the final assessment visit with respect to key 
laboratory parameters and vital signs will also be provided. 
Adverse events (AE) occurring after the start of study drug dosing at Baseline will be summarized descriptively. 
All AEs will be coded according to system organ class (SOC) and preferred term (PT) using a Medical Dictionary 
for Regulatory Activities (MedDRA) dictionary. Summary tables showing the number of subjects and percent 
within each category will be generated for each of the following types of adverse events and its relationship to 
study treatment (related to study treatment): 

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 
All events 
 
Serious events 
 
Deaths 
 
Events leading to withdrawal 
 
Severe events 
 
Treatment AEs will be coded and graded using MedDRA grading criteria. and compared with respect to 
occurrence of each adverse event and incidence of Grade II/IV adverse events. Total number of adverse events 
will be compared between groups using Fisher's exact test. Any treatment AE still present upon completion of 
treatment (including early discontinuation) should be monitored until resolution or until the AE is declared a 
chronic condition. AEs will be monitored until they become chronic or have completely resolved. 
Laboratory parameters will be summarized by visit; descriptive statistics denoting the changes from baseline to 
the final assessment visit with respect to key laboratory parameters and vital signs will also be provided. 
Frequencies of high and low values with respect to the normal range will be displayed, as will shift tables 
comparing each treatment visit and Baseline visit by time point.  
Vital signs will be summarized across groups by visit using descriptive statistics, and at each outcome visit and at 
end of study. Physical examination findings and number of subjects will be summarized as the count and 
percentage of subjects by eCRF pre-defined categories at last visit. Change from baseline at last visit will be 
summarized in a shift table comparing baseline and last visit results. Concomitant medications will be 
summarized, drug class and preferred term. 
8.3 
Biofluid Biomarker, Clinical and Neuroimaging Endpoints 
The change in the following assessments from Screening and/or Baseline to each post-baseline visit as described 
below based on the Schedule of Activities after BCG vaccination: 
 
Biochemical Biomarkers in Blood (Screening/Baseline, Weeks 4, 12, 26, 52) and CSF (Screening/Baseline, 
Weeks 12, 52) : a) Target engagement (e.g., PPD-induced PBMC cytokine release); b) Pharmacodynamic 
response (e.g., circulating cytokines in blood and CSF); c) AD pathophysiology (e.g., amyloid-b, tau and 
neurofilament light in CSF and blood); d) PBMC stimulation with AD-relevant immunogens (e.g., amyloid- 
b); e) Other novel biochemical biomarkers 
 
Clinical Neurocognitive and Behavioral Measures (Baseline, Week 12, 26 and 52): MoCA, RBANS, CDR-
SB, NPI-Q, HADS, DCT Clock and FAQ 
 
Multi-Sequence Structural and Functional MRI (Baseline, Week 52) 
 
Mixed-effects Model for Repeated Measures models will be used for biofluid and clinical endpoints to calculate 
differences at each post-baseline visit in which the biomarker is collected using baseline assessment as a covariate. 
Observed Case analysis will be used for differences in change of MRI measures over 52 weeks. 
 
9 
RISKS AND DISCOMFORTS 
9.1 
BCG Vaccine Risks 
The BCG injection is associated with minimal, brief discomfort. The following risks may also be associated with 
BCG vaccination per the manufacturer product insert. 

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Although BCG vaccination often causes local reactions, serious or long-term complications are rare. Reactions that 
can be expected after vaccination include moderate axillary or cervical lymphadenopathy and induration and 
subsequent pustule formation at the injection site; these reactions can persist for as long as 3 months after 
vaccination. The intensity and duration of the local reaction depends on the depth of penetration of the puncture 
device and individual variations in patients’ tissue reactions. Slight tenderness at the puncture site may be 
encountered as well as some itching. The initial skin lesions usually appear within 10–14 days and consist of small 
red papules at the site. The papules reach maximum diameter (about 3 mm) after 4 to 6 weeks, after which they may 
scale and then slowly subside. More serious local reactions include ulceration at the vaccination site, regional 
suppurative lymphadenitis with draining sinuses, and caseous lesions or purulent draining at the puncture site. These 
manifestations might occur up to 5 months after vaccination and could persist for several weeks. 
Acute, localized irritative toxicities of BCG may be accompanied by systemic manifestations, consistent with a 
“flu-like” syndrome. Systemic adverse effects of 1–2 days’ duration such as fever, anorexia, myalgia, and neuralgia, 
often reflect hypersensitivity reactions. However, symptoms such as fever of 103 °F or greater, or acute localized 
inflammation persisting longer than 2–3 days suggest active infections, and evaluation for serious infectious 
complication should be considered. 
Rare adverse events (mostly suppurative adenopathy) have been reported in Europe and other locales, mostly in the 
setting of mistakenly subcutaneous, rather than intradermal vaccination. The frequency of more severe cutaneous 
reactions, such as an ulcer >1 cm, axillary adenopathy which may be suppurative, or other localized skin reactions, 
is approximately 0.02% for first vaccinations and 0.04% for second vaccinations. Suppurative adenopathy is not 
treated but observed. 
The most serious complication of BCG vaccination is disseminated BCG infection. The most frequent disseminated 
infection is BCG osteomyelitis (0.01 to 43 cases per million doses of vaccine administered) which usually occurs 4 
months to 2 years after vaccination. Fatal disseminated BCG infection has occurred at a rate of 0.06–1.56 cases per 
million doses; these deaths occurred primarily among immunocompromised persons (tested for and excluded from 
this study). 
9.2 
Lumbar puncture 
Pain may occur during the procedure. This is usually temporary, confined to the lower back, and minimized with 
the cutaneous and soft tissue administration of 1% lidocaine as a local anesthetic. Short-lived LP associated 
headaches occur in <1%-36% of subjects with an incidence that decreases with age. Less commonly, a persistent 
low-pressure headache may develop as a result of a post-LP CSF leakage. Potentially more serious, but very rare 
risks, include infection, damage to radicular nerves and bleeding into the lumbar CSF space. The risk of these 
procedure related complications is much less than 1%. There is also a very rare risk of an allergic reaction to the 
lidocaine. 
 
9.3 
Magnetic Resonance Imaging (MRI) 
MRI is a safe procedure for participants who do not have metal implants or other contraindications. Individuals who 
have electrically, magnetically, or mechanically-activated implants (such as heart pacemakers) or those who have 
clips on blood vessels in their brain will not be allowed to participate in the study due to risks associated with MRI 
scanning. The MRI will be operated in a manner accepted by the Food and Drug Administration (FDA). The 
protocol requires participants to remain still within a relatively confined space during the scanning session, and the 
scanner makes loud knocking and beeping sounds as it takes images. While participants with severe claustrophobia 
are excluded from the study, some participants may find the physical confinement or noise uncomfortable. Every 
effort will be made to enhance each subject’s comfort level, and participants will be given earplugs to reduce 
discomfort due to noise. If a subject notices any discomfort while in the MRI scanner, they should notify the 
administrator immediately. If the discomfort cannot be stopped, the scanning session will be stopped. The MRI has 
the potential, during normal routine use, to cause localized warming of the skin and underlying tissues. Participants 
should immediately inform the study staff if they experience discomfort due to warming of the skin and the 
procedure will be stopped. 

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9.4 
Phlebotomy 
The risks associated with having blood drawn include bruising and local discomfort. Rarely an infection may occur 
at this site, and if an infection does occur it will be assessed and treated by the study physician. 
 
9.5 
Neurocognitive testing 
The neurocognitive tests that will be administered to assess mental performance may be stressful and potentially 
cause anxiety, fatigue, and frustration. In our prior experience with similar protocols, risks have occurred 
infrequently and very few subjects have terminated testing. However, testing will be discontinued immediately upon 
any request by the subject to do so. 
 
9.6 
Neuropsychiatric and Functional Questionnaires 
Questionnaires administered during the protocol may cause participants to feel sad or upset about their diagnosis 
and daily functioning or how it affects their quality of life. Study staff is experienced with such evaluations and 
sensitive to these issues. Any question can be omitted per the subject’s request. 
 
9.7 
Radiation 
In general, exposure to ionizing radiation may have health risks. Participants who require fluoroscopy-guided LP 
will be exposed to a maximum of approximately 0.18 millisieverts (mSv) for each LP. This is the same amount of 
radiation as a person would normally get in 21 days from natural background radiation and is comparable to the 
dose from a single chest x-ray. 
9.8 
Genetic Research Risks 
The study includes the potential to generate genetic data from patient samples. Genetic information that results from 
this study does not have medical or treatment importance at this time. Genetic information obtained in this study 
will not be placed in the medical record. Taking part in a genetic study may also have a negative impact on family 
or other relationships. We will inform subjects that there may be new technologies that may be developed in the 
future that may allow the linking of genetic or medical information back to subjects. 
To maximize confidentiality, all samples and the information associated with the samples will be coded to prevent 
the exposure of the participant’s information and identity. In addition, sample information is stored in one secured 
database while genetic data is stored in an independent secured database. 
9.9 
Other Risks 
Reviewing health-related information might be stressful or make the participant feel uncomfortable. Participants do 
not have to answer any questions they do not want to. In addition, there may be incidental medical findings as a 
result of the clinical examinations. Standard lab tests completed through MGH Core Lab will be placed in subjects’ 
medical records but no additional research results will be placed in their medical records. Coded, de-identified 
data/samples may be provided to authorized federal data repositories for broad-sharing with approved researchers. 
As with all database systems, there is a slight risk that there could be a breach in the security of these data banks 
resulting in the access of information. Safeguards are in place to minimize privacy risks. 
 
10 POTENTIAL BENEFITS 
The aim of this trial is to further understand the effects of BCG on IIR and AD biofluid and MRI biomarkers and 
neurocognitive/behavioral functioning over one year. We do not know what the potential benefits may be to 
participants who choose to participate in this study. There may be no benefit at all. This trial is assessing multiple 
biomarkers in concert with clinical outcomes, which will provide a detailed understanding of the effect of BCG and 
provide a well-curated data set for the Alzheimer’s research community to improve our understanding of the disease 
with an eye towards better treatments and prevention. 
 
11 MONITORING AND QUALITY ASSURANCE 

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11.1 Independent Monitoring of Source Data 
The PI will ultimately be responsible for the validity and integrity of the data collected at the MGH site, and for 
ensuring that the study is conducted in accordance with the IRB-approved protocol. After data is collected and 
recorded on forms, the study coordinator may input the data into the Partners approved StudyTRAX EDC within 7 
days of the study visit. Entries will be reviewed for accuracy and completeness by a second study coordinator. 
Finally, the study team will conduct quarterly reviews to check that data in StudyTRAX accurately reflects the data 
collected on the original data capture forms and the PI will maintain oversight of this process. The research team 
(PI, Co-I, research coordinators) will subsequently meet to discuss the results of this review, as well as case report 
forms and source documentation. 
 
All electronic documentation will be stored on password-protected devices. Paper forms will be stored in locked 
cabinets located in secured areas. 
11.2 Safety Monitoring 
The adverse event (AE) definitions and reporting procedures provided in this protocol comply with all applicable 
United States Food and Drug Administration (FDA) regulations and International Conference on Harmonization 
(ICH) guidelines. The PI will carefully monitor each subject throughout the study for possible adverse events. All 
AEs will be documented on CRFs designed specifically for this purpose. It will be important to report all AEs, 
whether serious or non-serious. 
 
11.3 Adverse Event Reporting Guidelines 
11.3.1 Adverse Event 
An adverse event (AE) is any unfavorable and unintended sign (including a clinically significant abnormal 
laboratory finding, for example), symptom, or disease temporally associated with a study, use of a drug product or 
device, whether or not considered related to the drug product or device. 
 
Examples of adverse events include: new conditions, worsening of pre-existing conditions, clinically significant 
abnormal physical examination signs (i.e. skin rash, peripheral edema, etc.), or clinically significant abnormal test 
results (i.e. lab values or vital signs), with the exception of outcome measure results, which are not being recorded 
as adverse events in this trial (they are being collected, but analyzed separately). The BCG vaccine causes a circular 
and contained inflammatory response with scab followed by scar formation. For this study, this expected BCG 
vaccine site reaction is not considered an adverse event. Stable chronic conditions (e.g. arthritis) that are present 
prior to the start of the study and do not worsen during the trial are NOT considered adverse events. Chronic 
conditions that occur more frequently (for intermittent conditions) or with greater severity are considered as 
worsened and therefore would be recorded as adverse events. Adverse events are generally detected in two ways: 
 
Clinical  symptoms reported by the subject or signs detected on examination. 
 
Ancillary Tests  abnormalities of vital signs, laboratory tests, and other diagnostic procedures 
(other than the outcome measures, the results of which are not being captured as AEs). 
 
If discernible at the time of completing the AE log, a specific disease or syndrome rather than individual associated 
signs and symptoms should be identified by the PI and recorded on the AE log. However, if an observed or reported 
sign, symptom, or clinically significant laboratory anomaly is not considered by the PI to be a component of a 
specific disease or syndrome, then it should be recorded as a separate AE on the AE log. Clinically significant 
laboratory abnormalities, such as those that require intervention, are those that are identified as such by the PI. 
 
Participants will be monitored for adverse events from the time they sign consent until completion of their 
participation in the study (defined as death, consent withdrawal, loss to follow up, early study termination for other 
reasons, or following completion of the entire study). An unexpected adverse event is any adverse event, the 
specificity or severity of which is not consistent with the current product insert. 
 

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The study procedures and the well-being of all participants will be monitored closely by the Principal Investigator, 
Steven Arnold MD, and the Co-Investigators Alison McManus DNP, FNP-BC, and Marc Weinberg M.D., Ph.D. 
Throughout the course of the study, constant feedback with the subject is maintained in order to assess comfort and 
safety and to minimize risks throughout the procedure. The above investigators will be responsible for determining 
if a subject should be removed from the study. Criteria for removal include the following: 1) if a subject is unwilling 
or unable to participate in study procedures 2) if the subject refuses to participate and consent, 3) if the subject 
acquires a medical condition that prohibits further participation, 4) if in the opinion of the MGH principal 
investigator, Dr. Steven Arnold, it is decided that it is not in the subject’s best interest to continue participation. 
 
Unanticipated problems including adverse events will be reported to the PHRC as described in the PHRC policy on 
Unanticipated Problems Involving Risks to Subjects or Others including adverse events. 
 
All data will be managed in compliance with applicable regulatory requirements. The study coordinator, under the 
supervision of the PI, will collect, transcribe, correct, and transmit the data onto source documents, Case Report 
Forms (CRFs), and/or other forms used to report, track, and record clinical research data. 
 
11.3.1.1 Serious Adverse Events 
All adverse events will be reviewed by the Principal Investigator, Dr. Steven Arnold, and will be reported to Partners 
IRB and to the Human Research Committee (HRC) in accordance with HRC Guidelines. A serious adverse event 
(SAE) is defined as an adverse event that meets any of the following criteria: 
1. Results in death. 
2. Is life threatening: that is, poses an immediate risk of death as the event occurs. 
o This serious criterion applies if the study subject, in the view of the PI, is at immediate risk of death 
from the AE as it occurs. It does not apply if an AE hypothetically might have caused death if it 
were more severe. 
3. Requires inpatient hospitalization or prolongation of existing hospitalization. 
o Hospitalization for an elective procedure or a routinely scheduled treatment is not an SAE by this 
criterion because an elective or scheduled “procedure” or a “treatment” is not an untoward medical 
occurrence. 
4. Results in persistent or significant disability or incapacity. 
o This serious criterion applies if the “disability” caused by the reported AE results in a substantial 
disruption of the subject’s ability to carry out normal life functions. 
5. Results in congenital anomaly or birth defect in the offspring of the subject. 
6. Necessitates medical or surgical intervention to preclude permanent impairment of a body function or 
permanent damage to a body structure. 
7. Important medical events that may not result in death, are not life-threatening, or do not require 
hospitalization may also be considered SAEs when, based upon appropriate medical judgment, they 
may jeopardize the subject and may require medical or surgical intervention to prevent one of the 
outcomes listed in this definition. Examples of such medical events include blood dyscrasias or 
convulsions that do not result in inpatient hospitalization. 
 
An inpatient hospital admission in the absence of a precipitating, treatment-emergent, clinical adverse event may 
meet criteria for "seriousness" but is not an adverse experience and will therefore not be considered an SAE. An 
example of this would include a social admission (subject admitted for reasons other than medical, e.g., lives far 
from the hospital, has no place to sleep). 
 
The PI is responsible for classifying adverse events as serious or non-serious and determining if there is reasonable 
possibility that the drug was the cause. 

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11.3.2 Assessment and Recording of Adverse Events 
The PI will carefully monitor each subject throughout the study for possible AEs. All AEs will be documented on 
CRFs designed specifically for this purpose. All AEs will be collected and reported in the EDC system. 
 
11.3.3 Assessment of Adverse Events 
At each visit (including telephone visits), the subject will be asked if they had any problems or symptoms since 
their last visit in order to determine the occurrence of adverse events. If the subject reports an adverse event, the 
Investigator will probe further to determine: 
 
Type of event 
 
Date of onset and resolution (duration) 
 
Severity (mild, moderate, severe) 
 
Seriousness (does the event meet the above definition for an SAE) 
 
Causality, relation to investigational protocol 
 
Outcome 
 
11.3.4 Relatedness of Adverse Event to Investigational Protocol 
 
1. Not Related: 
Concomitant illness, accident, or event with no reasonable association with protocol. 
2. Unlikely: 
The reaction has little or no temporal sequence from administration of the 
investigational protocol, and/or a more likely alternative etiology exists. 
3. Possibly Related: 
The reaction follows a reasonably temporal sequence from administration of the 
investigational protocol and follows a known response pattern to the suspected 
investigational protocol; the reaction could have been produced by the 
investigational protocol or could have been produced by the subject’s clinical state 
or by other modes of therapy administered to the subject. 
4. Probably Related: 
The reaction follows a reasonably temporal sequence from administration of 
investigational protocol; is confirmed by discontinuation of the investigational 
protocol or by re-challenge; and cannot be reasonably explained by the known 
characteristics of the subject’s clinical state. 
5. Definitely Related: 
The reaction follows a reasonable temporal sequence from administration of 
investigational protocol; that follows a known or expected response pattern to the 
investigational protocol; and that is confirmed by improvement on stopping of the 
investigational protocol, and reappearance of the reaction on repeated exposure. 
 
11.3.5 Recording of Adverse Events 
All clinical adverse events are recorded in the Adverse Event (AE) Log in the subject’s study binder. Entries on 
the AE Log (and into the EDC) will include the following: name and severity of the event, the date of onset, the 
date of resolution, relationship to investigational product, action taken, and primary outcome of event. 
 
11.3.6 Adverse Events and Serious Adverse Events - Reportable Events 
All events that meet the above criteria for Serious Adverse Events must be reported to the IRB within 24 hours of 
a study investigator being notified of the event. 

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Any adverse reaction that is serious, unexpected, and associated with the use of the product will be reported to the 
FDA in an IND safety report no more than 15 calendar days after the PI determines that the suspected adverse 
reaction qualifies for reporting, or 7 days for unexpected fatal or life-threatening adverse reaction reports. 
 
12 DATA COLLECTION AND MANAGEMENT 
12.1 Purpose of EDC 
Study data will be collected and managed using Studytrax electronic data capture software. The purpose of 
Studytrax is to track subject enrollment and flow through the study (e.g. Scheduling of visits), capture data 
measures, and facilitate the transfer of data into statistical packages for analysis. In compliance with HIPPA 
regulations, the database security features of Studytrax target multiple levels including the data element (e.g., 
restricted access to fields), user (e.g., password authentication access), application (e.g., role-based access to 
features, access audit trails), and hosting services (e.g., firewall, secure sockets layer). These features ensure access 
control, audit control, data integrity, user authentication, and transmission security. The research project will be set 
up in Studytrax to ensure exported datasets are de-identified as defined in the HIPAA privacy regulation [45 C.F.R. 
§164.514 (b)(2)]. A 21 CFR Part 11 compliance document is available upon request from the creators of the 
software, a company called ScienceTRAX, LLC. 
12.2 Role of Data Management 
All data will be managed in compliance with applicable regulatory requirements. Study personnel will collect, 
transcribe, correct, and transmit the data onto source documents, Case Report Forms (CRFs), and/or other forms 
used to report, track, and record clinical research data. Data management is responsible for developing, testing, and 
managing clinical data management activities. 
 
12.3 Data Entry and Checks 
The study personnel are instructed to enter information into Studytrax within 7 days. Data capture is the 
responsibility of the staff under the supervision of the PI. During the study, the PI must maintain complete and 
accurate documentation for the study. 
 
The Studytrax platform provides password protection. An edit checking and data clarification process will be put 
in place to ensure accuracy of the data. Logic and range checks as well as more sophisticated rules will be built into 
the system to provide immediate error checking of the data entered. The system has the capability to automatically 
create electronic queries for forms that contain data that are out of range, out of window, missing, or not calculated 
correctly. 
 
 
12.4 Data Lock Process 
The platform will have the ability to lock the project-specific visits to prevent any modification of data once the 
project is closed. Once this option is activated, every user will have Read-Only access to the data. 
 
12.5 Data Handling and Record Keeping 
The PI is responsible for ensuring accuracy, completeness, legibility, and timeliness of the data reported. Data 
reported in the eCRF derived from source documents should be consistent with the source documents and 
discrepancies should be explained. 

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12.6 Confidentiality 
Studytrax hosted solutions are fully HIPAA Compliant and ensure access control, audit control, data integrity, user 
authentication, and transmission security. Studytrax uses the data center services of Rackspace, a premier hosting 
company. Rackspace offers top of the line hosting facilities. As a summary of the Rackspace facilities: (1) Access 
to data center is secured by Biometric hand scanners and monitored 24×7 by closed circuit cameras. (2) Public 
access to data center is strictly forbidden. Only level three technicians are permitted in the data center. (3) HVAC 
[Heating Ventilation Air Conditioning] systems are used to completely circulate and filter all the air every 90 
seconds. (4) Continuous UPS [Uninterrupted Power Supply] systems keep all servers up and running in the event 
of a total power outage. (5) Diesel engines are located on-site to provide power for extended power outages. (6) 
Enterprise-class routing equipment used in conjunction with multiple fiber carriers to ensure zero downtime due to 
network access. 
Data is protected from loss by the following: (1) A redundant array of independent disk [RAID] Level 5 is used to 
ensure that data will not be lost if a hard drive fails, (2) full database backups are done nightly, (3) database log file 
backups are done every 15 minutes, (3) database integrity checks and index maintenance are performed nightly, (4) 
the database and log backup files are retained as part of Rackspace’s backup process and also transferred every hour 
to Microsoft’s Azure geographically redundant storage. 
Data security is assured by the following: (1) All server requests are transmitted over SSL using 256-bit encryption, 
(2) a dedicated Cisco router firewall only allows requests to Studytrax, (3) the database is stored on a separate server 
in a private independent subnet with no public IP address, (4) database and log files are encrypted, and (5) database 
and log backups are encrypted. 
12.7 Retention of Records 
Research records will be retained in accordance with IRB policies. 
 
12.8 Publications 
The Principal Investigator, Steven E. Arnold, will be responsible for publication of results from this study. Dr. 
Arnold’s responsibilities will include the following: 
 
Analyze and interpret data gathered in this study and write publications from these data
 
Submit manuscripts to selected journals and address peer reviewers’ comments
 
Submit abstracts to selected meetings and present data at the meetings
 
Determine authorship on the basis of the Uniform Requirements for Manuscripts

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