A Phase 1 Double-blind, Placebo-controlled, Dose Escalating Study of Intramuscular Detoxified Shigella Flexneri 2a Artificial Invasin Complex (Invaplex[AR-DETOX]) Vaccine
Phase: PHASE1
Conditions: Diarrhea
Sponsor: PATH

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A Phase 1 Double-blind, Placebo-controlled, Dose Escalating Study of Intramuscular 
Detoxified Shigella flexneri 2a Artificial Invasin Complex (InvaplexAR-DETOX) Vaccine 
1. 
Title Page 
Sponsor 
PATH Vaccine Solutions (PVS) 
455 Massachusetts Ave NW 
Suite 1000 
Washington, DC 20001 
Telephone: 202-822-0033 
Fax: 202-457-1466 
Funding Agency 
PATH 
Research Monitor 
Michael Koren, MD 
Walter Reed Army Institute of Research (WRAIR) 
503 Robert Grant Avenue 
Silver Spring, MD 20910 
Telephone: 301-319-9904 
E-mail: michael.a.koren2.mil@mail.mil 
Research Monitor (alternate) 
James Moon, MD 
WRAIR 
Telephone: 301-319-9176 
E-mail: james.e.moon.mil@mail.mil 
Principal Investigator 
Ramiro Gutierrez, MD, MPH 
Enteric Diseases Department (EDD) 
Naval Medical Research Center (NMRC) 
503 Robert Grant Avenue 
Silver Spring, MD 20910 
Telephone: 301-319-3193 
E-mail: ramiro.l.gutierrez.mil@mail.mil 
Subinvestigators 
Tida Lee, MD, PhD 
EDD, NMRC 
Telephone: 301-319-9260 
E-mail: tida.k.lee.mil@mail.mil 
Chad Porter, PhD, MPH 
EDD, NMRC 
Telephone: 301-319-7505 
E-mail: chad.k.porter2.civ@mail.mil 
Robert Kaminski, PhD 
Department of Subunit Enteric Vaccines and Immunology 
(SEVI), WRAIR 
503 Robert Grant Avenue 
Silver Spring, MD 20910 
Telephone: 301-319-9803 
E-mail: robert.w.kaminski.civ@mail.mil 

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Alison Lane, MD, MS 
Walter Reed National Military Medical Center (WRNMMC) 
Telephone: 206-354-4337 
E-mail: alison.b.lane.mil@mail.mil 
Melinda Hamer, MD, MPH 
WRAIR 
Telephone: 301-319-3136 
E-mail: melinda.j.hamer.mil@mail.mil 
Laura Gilbert, MD 
WRNMMC 
Telephone: 301-295-6400 
E-mail: laura.j.gilbert.mil@mail.mil 
Daniel Selig, MD 
WRAIR 
Telephone: 240-475-6111 
E-mail: daniel.j.selig.mil@mail.mil 
Jack Hutter, MD 
WRAIR 
Telephone: 301-319-3095 
E-mail: jack.n.hutter.mil@mail.mil 
Christine Lee, MD 
WRAIR 
Telephone: 301-319-9850 
E-mail: christine.e.lee12.ctr@mail.mil 
Clinical Trial Site 
Walter Reed Army Institute of Research 
Clinical Trials Center (CTC) 
Department of Clinical Trials 
503 Robert Grant Avenue 
Silver Spring, MD 20910 
Telephone: 301-319-9660 
Qualified Physician Responsible for All Trial-Site-Related 
Medical Decisions 
Ramiro Gutierrez, MD, MPH 
EDD, NMRC 
Telephone: 301-319-3193 
E-mail: ramiro.l.gutierrez.mil@mail.mil 
Clinical Laboratories and Other Departments/Institutions 
Involved in the Trial 
 
Site Investigational Product Accountability 
Kate DeTizio 
EDD, NMRC 
Telephone: 301-319-7123 
E-mail: katherine.j.detizio.ctr@mail.mil 
Clinical Laboratory 
Clinical Trials Center (CTC) 
Walter Reed Army Institute of Research 
Quest Diagnostics Incorporated 
1901 Sulphur Spring Road 
Baltimore, MD 21227-0580 
Research Laboratory 
Robert Kaminski, PhD  
SEVI, WRAIR 

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Statistician 
Chad Porter, PhD, MPH  
EDD, NMRC  
Telephone: 301-319-7505 
Fax: 301-319-7679 
E-mail: chad.k.porter2.civ@mail.mil 
PATH Medical Officer 
Rahsan Erdem, MD 
PATH 
455 Massachusetts Avenue NW 
Suite 1000 
Washington, DC 20001 
Telephone: 202-540-4546 
Fax: 202-457-1466 
E-mail: rerdem@path.org 
Data Management 
The EMMES Corporation 
401 N Washington Street, Suite 700 
Rockville, MD 20850 
Telephone: 301-251-1161 (Administrative Coordinator) 
Institutional Review 
Naval Medical Research Center Institutional Review Board 
Research Services Directorate 
Office of Research Administration 
Code 025, Building 500, Room 004 
Silver Spring, MD 20910 
Telephone: 301-319-7276 
Western Institutional Review Board (WIRB) 
1019 39th Avenue SE  
Puyallup, WA 98374 
Phone: 800-562-4789 
Fax: 360-252-2498 

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Table 1: 
Emergency Contacts 
Role in Study 
Name 
Contact Information 
Principal 
Investigator 
Ramiro Gutierrez, MD, MPH 
Telephone: 301-319-3193 
E-mail: ramiro.l.gutierrez.mil@mail.mil 
Subinvestigators 
Tida Lee, MD, PhD 
Telephone: 301-319-9260 
E-mail: tida.k.lee.mil@mail.mil 
Chad Porter, PhD, MPH 
Telephone: 301-319-7505 
E-mail: chad.k.porter2.civ@mail.mil 
Robert Kaminski, PhD 
Telephone: 301-319-9803 
E-mail: robert.w.kaminski.civ@mail.mil 
Alison Lane, MD, MS 
Telephone: 206-354-4337 
E-mail: alison.b.lane.mil@mail.mil 
Melinda Hamer, MD, MPH 
Telephone: 301-319-3136 
E-mail: melinda.j.hamer.mil@mail.mil 
Laura Gilbert, MD 
Telephone: 301-295-6400 
E-mail: laura.j.gilbert.mil@mail.mil 
Daniel Selig, MD 
Telephone: 240-475-6111 
E-mail: daniel.j.selig.mil@mail.mil 
Jack Hutter, MD 
Telephone: 301-319-3095 
E-mail: jack.n.hutter.mil@mail.mil 
Christine Lee, MD 
Telephone: 301-319-9850 
E-mail: Christine.e.lee12.ctr@mail.mil 
Research Monitor 
Michael Koren, MD 
Telephone: 301-319-9904 
E-mail: michael.a.koren2.mil@mail.mil 
Research Monitor 
(alternate) 
James Moon, MD 
Telephone: 301-319-9176 
E-mail: james.e.moon@mail.mil 
Sponsor’s 
Representative 
The EMMES Corporation 
401 N. Washington St., Suite 700 
Rockville, MD 20850 
Institutional 
Review Board 
Naval Medical Research Center 
Institutional Review Board 
Research Services Directorate  
Office of Research Administration 
Code 025, Building 500, Room 004 
Silver Spring, MD 20910 
Telephone: 301-319-7276 

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2. 
Synopsis 
Investigational Product 
Detoxified Shigella flexneri 2a Artificial Invaplex (InvaplexAR-DETOX) (Lot 1972) 
Short Protocol Title 
Safety and immunogenicity of Detoxified Artificial Invaplex (Shigella flexneri 
2a InvaplexAR-DETOX) 
Clinical Phase: 1 
IND Number: 18707 
Sponsor 
PATH 
Vaccine Manufacturer 
Walter Reed Army Institute of Research (WRAIR) Pilot BioProduction Facility 
(PBF) 
Study Sites 
WRAIR Clinical Trials Center (CTC) 
Principal Investigator 
Ramiro Gutierrez, MD, MPH 
Study Duration 
Screening (up to 60 days); vaccination to final blood draw (70 days); telephone 
follow-up: 6 months after final dose; immunology analysis (12 weeks); data 
analysis and report writing (2 months). Advancement between cohorts will be 
reviewed by the Protocol Safety Review Team (PSRT) and occur upon review of 
safety data through 7 days after the 3rd dose (approximately 2-4 weeks between 
cohorts). The clinical phase of the study will last for approximately 1 year 
followed by another year of data analysis and report writing. 
Study Objectives 
Primary: Evaluate the safety of detoxified Shigella flexneri 2a InvaplexAR 
(InvaplexAR-DETOX) administered by intramuscular (IM) immunization. 
Secondary: Evaluate immune responses (serum IgG/IgA and ALS IgG/IgA) 
following IM immunization with InvaplexAR-DETOX. 
Exploratory: 
 
Collect samples from vaccinated subjects for exploratory 
immunological assays. 
 
Perform secondary immunology analyses. 
 
Collect samples for evaluation of C-reactive protein (CRP) responses to 
the vaccine and placebo. 

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Study Design 
This is a randomized, double-blind, placebo-controlled, Phase 1 clinical trial in 
which a total of 60 subjects will receive one of three doses of InvaplexAR-DETOX or 
placebo (saline), as shown in the table below. The vaccine will be administered 
via IM injection on study days 1, 22, and 43. Each subject will receive the same 
formulation at each vaccination dependent upon group assignment. 
The study will be initiated with the lowest dose level (2.5 µg) and will proceed to 
the next highest dose in an escalating fashion. A dose-level with no occurrence of 
stopping criteria in the 7 days following the third vaccine dose will prompt 
moving to the next higher level. All safety data will be summarized and reviewed 
by the PSRT prior to dose-escalation. 
For the initial cohort, enrollment will be limited initially to 4 subjects receiving 
2.5 µg of InvaplexAR-DETOX alone and 1 subject receiving placebo. If no 
unexpected symptoms occur within 7 days following initial vaccination, 
enrollment of the remaining subjects in the cohort may proceed. 
Cohort 
Group 
n 
Route 
InvaplexAR-DETOX (µg) 
A 
A-1 
16 
IM 
2.5 
A-2 
4 
IM 
Placebo 
B 
B-1 
16 
IM 
10 
B-2 
4 
IM 
Placebo 
C 
C-1 
16 
IM 
25 
C-2 
4 
IM 
Placebo 
Specimens will be collected at prescribed intervals to examine systemic and 
mucosal immune responses. Vaccine safety will be actively monitored during 
vaccination and for 28 days following the third vaccine dose. Additionally, a 
telephone follow-up will be performed at 6 months following receipt of the final 
vaccination to assess for adverse events. 
Primary Clinical 
Outcome  
The primary clinical outcome for this study is the assessment of safety and 
tolerability of InvaplexAR-DETOX. All subjects receiving investigational product 
will be included in the safety assessment. For safety laboratory data, including 
hematology, serology, and chemistry parameters, blood will be collected at 
screening and study days as per the Time and Events Schedule (Table 8). Post-
vaccination safety monitoring will include laboratory and clinical evaluations 
following each vaccine/placebo administration, including assessment of post-
vaccination local and systemic reactions through targeted physical exams, 
symptom surveys, symptom diaries, and other adverse event (AE) monitoring. 
Immunology Outcomes  
Primary Immunology Outcomes: The primary immunological outcomes will 
be Antibodies in Lymphocyte Supernatant (ALS) and antibody (IgG and IgA) 
titers against Invaplex using methods previously established. Seroconversion will 
be defined as a ≥ 4-fold increase in endpoint titer between pre-and post-
vaccination samples. Peripheral Blood Mononuclear Cells (PBMCs) will be 
collected to determine antigen-specific IgG and IgA titers in ALS samples. High-
titer specimens will be included on each plate to track day to day inter-assay 
variation. For each antigen, pre- and post-vaccination serum samples will be 
assayed side-by-side. The antibody titer assigned to each sample will represent 
the geometric mean of duplicate tests. Reciprocal endpoint titers less than the 
starting dilution of the assay will be assigned a value of half the starting dilution 
for computational purposes. 
Secondary and Exploratory Evaluations: Serologic and ALS (IgG and IgA) 
responses to additional antigens (S. flexneri 2a LPS, IpaB, IpaC) may be assessed 
depending on sample availability.  IgM and IgG subclass responses (serum and 
ALS) to key antigens may also be assessed.  Serum bactericidal (SBA) titers 
against S. flexneri 2a 2457T will be determined for pre- and post-vaccination 
samples. An SBA responder will be defined as ≥ 9-fold increase in SBA titer as 

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compared to baseline SBA titers. Samples will be collected, processed, and 
archived for analysis. PBMCs will be collected for antigen-specific memory B 
and T cell analysis, antibody secreting cell assay and α4β7+ B cells. Stool 
samples will be collected to assess antigen-specific fecal IgA and IgG responses. 
A fecal response will be defined as a ≥ 4-fold increase from baseline. 
Concurrently, saliva samples may be collected to assess mucosal IgA and IgG 
antibody responses.  
Hypotheses 
Safety: One or more of the proposed dosing regimens will have an acceptable 
frequency and severity of adverse events. 
Immunogenicity: 
 
IM administration of InvaplexAR-DETOX will induce serum IgG antibody 
responses to S. flexneri 2a Invaplex in ≥50% of subjects in at least one 
of the study groups. 
 
IM administration of InvaplexAR-DETOX will induce antibody responses to 
either IpaB or IpaC in ≥50% of subjects in at least one of the study 
groups. 
Estimated Number of 
Subjects Screened 
Based on prior studies with similar study designs, sample collections and 
inclusion/exclusion criteria, we anticipate screening approximately 5 subjects for 
every one subject that is enrolled in the study. Therefore we anticipate 
approximately 300 individuals will be screened. 
Maximum Number of 
Subjects Enrolled 
A maximum of 60 subjects are planned for this study. Up to four alternates per 
group will be selected that may be enrolled in the event a subject becomes 
ineligible prior to receipt of the investigational product. Subjects receiving at 
least one dose of the vaccine (or placebo) will not be replaced. 
Eligibility Criteria 
Inclusion Criteria: 
 
Healthy, adult, male or female, age 18 to 50 years (inclusive) at the time of 
enrollment. 
 
Completion and review of comprehension test (achieved ≥ 70% accuracy, 
two attempts allowed). 
 
Provide written informed consent before initiation of any study procedures. 
 
Agrees to complete all study visits and procedures and to provide a 
screening stool sample. 
 
Women of childbearing capacity: Negative pregnancy test with 
understanding (through informed consent process) to not become pregnant 
during the study or within three (3) months following the last vaccine dose. 
Exclusion Criteria: 
General health 
 
Health problems (for example, chronic medical conditions such as 
psychiatric conditions, diabetes mellitus, hypertension, or any other 
conditions that might place the subjects at increased risk of adverse events)- 
study clinicians, in consultation with the PI, will use clinical judgment on a 
case-by-case basis to assess safety risks under this criterion. The PI will 
consult with the Research Monitor as appropriate. 
 
History of autoimmune disorders, cardiovascular and renal disease. 
 
Use of immunosuppressive medications (systemic corticosteroids or 
chemotherapeutics that may influence antibody development), or 
immunosuppressive illness, including IgA deficiency (defined by serum 
IgA <7mg/dL). 
 
Women who are pregnant or planning to become pregnant during the study 
period plus 3 months beyond the last vaccine dose and currently nursing 
women. 

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 
Participation in research involving another investigational product (defined 
as receipt of an investigational product or exposure to an invasive 
investigational device) 30 days before planned date of first vaccination or 
anytime throughout the duration of the study until the last in-clinic study 
safety visit. 
 
Positive blood test for HBsAG, HCV Ab, HIV-1/HIV-2 Ab. 
 
Clinically significant abnormalities on basic laboratory screening tests. 
 
Systemic antimicrobial treatment (i.e., topical treatments are not an 
exclusion) within 1 week before administration of the first vaccine dose. 
Research specific 
 
Allergies that may increase the risk of AEs. 
 
Regular use (weekly or more often) of antidiarrheal, anti-constipation, or 
antacid therapy. 
 
Abnormal stool pattern (fewer than 3 stools per week or more than 3 stools 
per day) on a regular basis; loose or liquid stools on other than an 
occasional basis. 
 
Personal or family history of an inflammatory arthritis. 
 
Positive blood test for HLA-B27 (associated with increased risk of reactive 
arthritis secondary to Shigella infection) 
 
History of allergy to any vaccine. 
 
Exclusionary skin disease history/findings that would confound assessment 
or prevent appropriate local monitoring of AEs, or possibly increase the risk 
of a local AE. 
Prior Exposure to Shigella 
 
Serum IgG titer > 2500 to Shigella flexneri 2a LPS.  
 
History of microbiologically confirmed Shigella infection. 
 
Received previous licensed or experimental Shigella vaccine or live 
Shigella challenge. 
 
Travel to countries where Shigella or other enteric infections are endemic 
(most of the developing world) within two years prior to dosing (clinician 
judgement). 
 
Occupation involving handling of Shigella bacteria currently, or in the past 
3 years. 
Sample Size 
The sample size of this study is limited by the early stage (Phase 1) of the 
product concept/testing and is designed to evaluate preliminary safety data but 
not designed to show statistically significant differences between groups. Given 
the small number of subjects per group, the precision of our estimate for adverse 
events is limited. For example, using binomial probability formula for no 
observed adverse events within the 16 subjects per group yields a 95% 
confidence interval of 0-21%. Follow-on studies evaluating seemingly safe and 
immunogenic doses will be required with larger numbers of volunteers in order 
to better define the safety profile. 
Statistical Methods 
Rates of all adverse events will be analyzed by Pearson’s Chi-square test (or 
Fisher’s exact test if assumptions are not met for Pearson’s Chi-square), to 
compare groups, if applicable. Summary tables will be created to indicate the 
number of subjects who experience events. Vaccine-related events (definitely, 
probably, or possibly related) will be tabulated by study group. In addition, tables 
will be prepared to list each adverse event, the number of subjects in each 
treatment group who experienced an event at least once, and the rate of subjects 
with adverse event(s). Adverse events will be divided into defined severity 
grades (mild, moderate, severe, potentially life-threatening). The tables will also 

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divide the adverse events by relationship to the investigational product if 
applicable. All immunized subjects will be included in the safety analysis. 
For immunology responses, both qualitative (responder rates) and quantitative 
(e.g., log10 transformed values) results will be analyzed. Geometric mean titers 
will be calculated for serologic and ALS responses along with their 95% 
confidence intervals (or standard deviation). If appropriate, between groups 
comparisons will be examined with nonparametric median tests (Kruskal-Wallis 
for continuous data and Fisher’s exact tests for categorical data) unless 
assumptions are fulfilled for Student’s t or 2.   
Additional comparisons may be made using repeated measures analysis of 
variance with study group as the between subject factor and sample collection 
time-points as the repeated factor. All statistical tests will be interpreted in a two-
tailed fashion using an alpha = 0.05 to represent statistical significance. 
 

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3. 
Table of Contents, List of Tables and List of Figures 
Table of Contents 
1. 
Title Page ............................................................................................................................... 1 
2. 
Synopsis ................................................................................................................................. 6 
3. 
Table of Contents, List of Tables and List of Figures ......................................................... 11 
4. 
List of Abbreviations and Definitions of Terms .................................................................. 15 
5. 
Introduction ......................................................................................................................... 17 
5.1. 
InvaplexNAT ............................................................................................................. 18 
5.1.1. Synopsis: Human S. flexneri 2a Invaplex IN Investigations ..................... 18 
5.2. 
Artificial Invaplex .................................................................................................. 23 
5.2.1. Synopsis: S. flexneri 2a InvaplexAR ........................................................... 23 
5.2.2. Comparative Efficacy of S. flexneri 2a InvaplexAR (research grade) and S. 
flexneri 2a InvaplexNAT .............................................................................. 24 
5.3. 
Development of S. flexneri 2a InvaplexAR-DETOX .................................................... 25 
5.4. 
Nonclinical Toxicology .......................................................................................... 26 
6. 
Trial Objectives ................................................................................................................... 28 
7. 
Hypotheses .......................................................................................................................... 29 
8. 
Clinical Study Design .......................................................................................................... 29 
8.1. 
Screening and Study Schedule ............................................................................... 33 
8.2. 
Randomization ........................................................................................................ 34 
8.3. 
Blinding .................................................................................................................. 35 
8.4. 
Unblinding .............................................................................................................. 35 
8.5. 
Vaccination ............................................................................................................. 36 
8.5.1. Vaccination Follow-Up ............................................................................. 36 
8.6. 
Endpoints ................................................................................................................ 36 
8.6.1. Safety ......................................................................................................... 36 
8.6.2. Immunology .............................................................................................. 37 
8.7. 
Investigational products.......................................................................................... 37 
8.7.1. InvaplexAR-DETOX ........................................................................................ 37 
8.7.2. Investigational Product Preparation .......................................................... 38 
8.7.3. Investigational Product Accountability ..................................................... 38 
8.8. 
Duration of Subject Participation ........................................................................... 38 
8.9. 
Dose Escalation Criteria ......................................................................................... 38 
8.10. 
Stopping Criteria .................................................................................................... 39 
8.11. 
Identification of Data to Be Recorded on Case Report Forms ............................... 39 
8.12. 
Known and Potential Risks and Benefits to Human Subjects ................................ 40 
8.12.1. Risks/Discomfort to Subjects and Precautions to Minimize Risk ............. 40 
8.12.1.1. 
Local Reactions...................................................................... 40 
8.12.1.2. 
Systemic Reactions ................................................................ 40 
8.12.1.3. 
Pregnancy .............................................................................. 40 
8.12.1.4. 
Lactation ................................................................................ 41 
8.12.1.5. 
Venipuncture .......................................................................... 41 
8.12.1.6. 
Allergic Reaction ................................................................... 41 
8.12.1.7. 
Theoretical Risks ................................................................... 41 
8.12.1.8. 
Unknown Risks ...................................................................... 41 
8.12.2. Alternatives to This IND Product or Study ............................................... 41 

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8.12.3. Intended Benefit for Subjects .................................................................... 42 
8.12.4. Risks to Study Personnel and the Environment ........................................ 42 
8.13. 
Route of Administration, Dosage Regimen, Treatment Period, and Justification .. 42 
8.14. 
Compliance Statement ............................................................................................ 42 
8.15. 
Study Population .................................................................................................... 42 
8.16. 
Study Site ............................................................................................................... 43 
9. 
Selection and Withdrawal of Subjects ................................................................................. 43 
9.1. 
Recruitment of Subjects/Study Population ............................................................. 43 
9.2. 
Informed Consent Process ...................................................................................... 43 
9.3. 
Eligibility Screening ............................................................................................... 44 
9.4. 
Subject Inclusion Criteria ....................................................................................... 44 
9.5. 
Subject Exclusion Criteria ...................................................................................... 44 
9.5.1. General Health ........................................................................................... 45 
9.5.2. Research specific ....................................................................................... 45 
9.5.3. Prior Exposure to Shigella ......................................................................... 45 
9.6. 
Subject Withdrawal Criteria ................................................................................... 46 
9.6.1. When and How to Withdraw Subjects ...................................................... 46 
9.6.2. Follow-up for Withdrawn Subjects ........................................................... 46 
9.6.3. Data Collected for Withdrawn Subjects .................................................... 46 
9.6.4. Replacement of Subjects ........................................................................... 47 
10. 
Treatment of Subjects .......................................................................................................... 47 
10.1. 
Dose Administration/Vaccination and Follow-up Periods/Study Visits ................ 47 
10.1.1. Administration of Vaccination .................................................................. 47 
10.1.2. Handling of Study Samples ....................................................................... 47 
10.2. 
Concomitant Medications ....................................................................................... 47 
10.3. 
Procedures for Monitoring Subject Compliance .................................................... 48 
11. 
Laboratory and Clinical Assessment ................................................................................... 48 
11.1. 
Blood Sample Collection ........................................................................................ 48 
11.2. 
Stool Sample Collection ......................................................................................... 48 
11.3. 
Saliva Sample Collection ....................................................................................... 48 
11.4. 
Study Safety Management ...................................................................................... 48 
11.4.1. Protocol Safety Review Team ................................................................... 48 
11.4.2. Research Monitor ...................................................................................... 49 
11.5. 
Specification of Safety Endpoints .......................................................................... 49 
11.5.1. Local and Systemic Reactions ................................................................... 49 
11.5.2. Vital Signs ................................................................................................. 49 
11.5.3. Physical Examination ................................................................................ 50 
11.5.4. Clinical Laboratory Assessments .............................................................. 50 
11.5.4.1. 
Hematology ............................................................................ 50 
11.5.4.2. 
Blood Chemistry .................................................................... 51 
11.5.4.3. 
C-Reactive Protein ................................................................. 52 
11.5.4.4. 
Virus Testing ......................................................................... 52 
11.5.4.5. 
HLA-B27 Screen ................................................................... 53 
11.5.4.6. 
Drug Screen ........................................................................... 53 
11.5.4.7. 
Pregnancy Screen ................................................................... 53 
11.6. 
IND Safety Reporting ............................................................................................. 53 
11.6.1. Adverse Event or Suspected Adverse Reaction ........................................ 53 
11.6.2. Solicited Adverse Events ........................................................................... 54 
11.6.3. Serious Adverse Event or Serious Suspected Adverse Reaction .............. 54 

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11.6.4. Unexpected Adverse Event or Unexpected Suspected Adverse Reaction 55 
11.6.5. Other Adverse Event ................................................................................. 55 
11.7. 
Relationship to Investigational Product (Assessment of Causality) ....................... 55 
11.8. 
Recording Adverse Events ..................................................................................... 56 
11.8.1. Methods/Timing for Assessing, Recording, and Analyzing Safety 
Endpoints ................................................................................................... 56 
11.8.2. Duration of Follow-up of Subjects After Adverse Events ........................ 56 
11.8.3. Severity Assessment .................................................................................. 56 
11.9. 
Reporting Adverse Events ...................................................................................... 57 
11.9.1. Reporting Serious and Adverse Events ..................................................... 57 
11.9.1.1. 
Reporting to the Sponsor ....................................................... 57 
11.9.1.2. 
Reporting to the IRB .............................................................. 58 
11.9.2. Reporting Additional Immediately Reportable Events to the Sponsor and 
the NMRC IRB .......................................................................................... 60 
11.9.2.1. 
Pregnancy .............................................................................. 60 
11.9.2.2. 
Adverse Event-related Withdrawal of Consent ..................... 60 
11.9.2.3. 
Pending Inspections/Issuance of Reports............................... 60 
11.9.3. IND Annual Report to the FDA ................................................................ 60 
11.9.4. Final Report ............................................................................................... 60 
12. 
Statistics ............................................................................................................................... 61 
12.1. 
Description of Statistical Methods ......................................................................... 61 
12.1.1. Analysis Addressing the Primary Study Objective (Safety Analysis) ...... 61 
12.1.2. Analysis Addressing the Secondary Study Objective ............................... 62 
12.1.3. Clinical Laboratory Data Analyses ........................................................... 62 
12.2. 
Planned Enrollment and Reason for Sample Size .................................................. 62 
12.3. 
Accounting for Missing, Unused, and Spurious Data ............................................ 63 
12.4. 
Procedures for Reporting Deviations from the Original Statistical Plan ................ 63 
12.5. 
Selection of Subjects to Be Included in Analyses .................................................. 63 
13. 
Direct Access to Source Data/Documents ........................................................................... 63 
13.1. 
Study Monitoring ................................................................................................... 63 
13.2. 
Audits and Inspections ........................................................................................... 63 
13.3. 
Institutional Review Boards ................................................................................... 64 
14. 
Quality Control and Quality Assurance............................................................................... 64 
15. 
Ethics ................................................................................................................................... 64 
15.1. 
Ethics Review ......................................................................................................... 64 
15.1.1. Review/Approval of Study Protocol ......................................................... 65 
15.1.2. Protocol Modifications .............................................................................. 65 
15.1.3. Protocol Deviation Procedures .................................................................. 65 
15.2. 
Ethical Conduct of the Study .................................................................................. 66 
15.2.1. Confidentiality ........................................................................................... 66 
15.2.2. Compensation for Participation ................................................................. 67 
15.2.3. Redress of Research-Related Injury .......................................................... 68 
15.3. 
Written Informed Consent ...................................................................................... 69 
16. 
Data Handling and Recordkeeping ...................................................................................... 70 
16.1. 
Inspection of Records ............................................................................................. 70 
16.2. 
Retention of Records .............................................................................................. 71 
17. 
Publication Policy ................................................................................................................ 71 

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18. 
List of References ................................................................................................................ 72 
 
List of Tables 
Table 1: 
Emergency Contacts .............................................................................................................. 5 
Table 2: 
Abbreviations ...................................................................................................................... 15 
Table 3: 
Frequency (%) of Immune Responses to Immunizing Antigen(s) Following First in Human 
Phase 1 Clinical Trial of S. flexneri 2a InvaplexNAT (Lot 0994) .......................................... 19 
Table 4: 
Frequency (%) of Immune Responses to Immunizing Antigen(s) Following Second Phase 
1 Clinical Trial of S. flexneri 2a InvaplexNAT Lot 1307 ....................................................... 20 
Table 5: 
Number (%) of Subjects with Vaccine Related Solicited Signs and Symptoms ................. 22 
Table 6: 
Nonclinical Toxicology Study Design ................................................................................ 26 
Table 7: 
Clinical Trial Design for the Proposed Phase 1 Study with S. flexneri 2a InvaplexAR-
DETOX .................................................................................................................................... 29 
Table 8: 
Clinical Protocol Time and Events Schedule ...................................................................... 31 
Table 9: 
Reference Ranges and Adverse Event Coding for Vital Sign Parameters .......................... 49 
Table 10: 
Reference Ranges and Adverse Event Coding for Clinical Hematology Parameters ......... 51 
Table 11: 
Reference Ranges and Adverse Event Coding for Blood Chemistry Parameters ............... 52 
Table 12: 
Study Contacts for Reporting Serious Adverse Events ....................................................... 57 
Table 13: 
SAE Information to Be Reported to the Sponsor ................................................................ 58 
 
List of Figures 
Figure 1: 
Comparison of S. flexneri 2a InvaplexAR and S. flexneri 2a InvaplexNAT by SDS-PAGE ... 24 
Figure 2: 
Comparative Immune Responses in Mice Receiving S. flexneri 2a InvaplexAR or S. flexneri 
2a InvaplexNAT ..................................................................................................................... 25 
Figure 3: 
Label from a vial of InvaplexAR-DETOX, Lot 1972 ................................................................. 37 
 
 

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4. 
List of Abbreviations and Definitions of Terms 
Table 2: 
Abbreviations 
Abbreviation 
Explanation 
µg 
Microgram(s) 
µL 
Microliter(s) 
AE 
Adverse event, adverse experience 
ALS 
Antibody lymphocyte supernatant 
ASC 
Antibody-secreting cell 
C 
Celsius 
CCHMC 
Cincinnati Children’s Hospital Medical Center 
CFR 
Code of Federal Regulations 
cfu 
Colony forming units 
cGMP 
Current good manufacturing practice(s) 
CHNRI 
Child Health and Nutrition Research Initiative 
CRP 
C-Reactive Protein 
CTC 
Clinical Trials Center 
dmLT 
Double mutant heat labile toxin 
DoD 
Department of Defense 
eCRF 
Electronic case report form 
EDC 
Electronic data capture 
ETEC 
Enterotoxigenic Escherichia coli 
F 
Fahrenheit 
FDA 
US Food and Drug Administration 
GCP 
Good clinical practice(s) 
GLP 
Good laboratory practice(s) 
GMP 
Good manufacturing practice(s) 
HBsAG 
Hepatitis B surface antigen 
HCV 
Hepatitis C virus 
HIV 
Human immunodeficiency virus 
HRPO 
Human Research Protections Office 
IBS 
Irritable bowel syndrome 
ICH 
International Council on Harmonisation 
ID 
Intradermal 
IgA 
Immunoglobulin A 
IgG 
Immunoglobulin G 
IgM 
Immunoglobulin M 
IM 
Intramuscular(ly) 
IN 
Intranasal(ly) 
IND 
Investigational New Drug 
InvaplexAR 
Artificial Invaplex 
InvaplexAR-DETOX 
Detoxified Artificial Invaplex 
InvaplexNAT 
Native Invaplex 
IRB 
Institutional Review Board 

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Abbreviation 
Explanation 
LPS 
Lipopolysaccharide antigen 
mL  
Milliliter(s) 
ml 
Milliliters 
MOP 
Manual of Procedures 
NMRC 
Naval Medical Research Center 
PBF 
Pilot BioProduction Facility 
PBMC 
Peripheral Blood Mononuclear Cells 
PI 
Principal investigator 
PSRT 
Protocol Safety Review Team 
PVS 
PATH Vaccine Solutions 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SBA 
Serum bactericidal activity 
SDS-PAGE 
Sodium dodecyl sulfate – polyacrylamide gel electrophoresis 
SEC-HPLC 
Size exclusion chromatography – high pressure liquid chromatography 
SOP 
Standard operating procedures 
SOS 
Salimetrics Oral Swab 
T3SS 
Type-III Secretion System 
TD 
Travelers’ diarrhea 
USAMRMC 
US Army Medical Research and Materiel Command 
WIRB 
Western Institutional Review Board 
WRAIR 
Walter Reed Army Institute of Research 
WRNMMC 
Walter Reed National Military Medical Center 
 

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5. 
Introduction 
There are approximately 4.6 billion global annual diarrheal cases [1]. Diarrhea is a leading cause 
of mortality in children under 5 years and negatively impacts growth and cognitive development. 
Furthermore, bacillary diarrhea is a continuing problem for American military personnel and 
civilian travelers regions where Shigella is endemic.  
Shigellosis or bacillary dysentery is a food- and waterborne disease predominantly implicated in 
children < 5 years of age. Estimates of Shigella-associated morbidity and mortality range from 
0.1 to 1.1 million deaths (60% in children under 5) and from 90 to 165 million cases of dysentery 
annually [2, 3]. In military populations, Shigella species are a common cause of travelers’ 
diarrhea (TD) responsible for approximately 7% of TD cases [4]. In addition to the acute 
morbidity of diarrhea in this population, shigellosis is linked to several post-infectious sequelae 
including irritable bowel syndrome (IBS) and reactive arthritis [5-7]. 
As Shigella species invade the intestinal mucosa, the disease is less amenable to the salutary 
effects of oral rehydration compared to enterotoxigenic pathogens that cause dehydrating 
diarrhea [8]. Therefore, antibiotics are frequently required for treatment; however, increasing 
resistance to fluoroquinolones and cephalosporins (extended spectrum β-lactamases) is 
undermining efficacy. As therapeutic options narrow, the need for a safe and effective Shigella 
vaccine becomes more pressing. An expert panel convened by the Child Health and Nutrition 
Research Initiative (CHNRI) of the World Bank identified Shigella as one of the highest 
priorities for long-term vaccine development [2]. Furthermore, the Department of Defense 
(DoD) has issued directives for the development of vaccines against Campylobacter, 
enterotoxigenic Escherichia coli (ETEC), and Shigella (DoD Directive 6205.3; BUMEDINSTR 
5450.171). 
There are 4 different species [and 47 antigenically distinct serotypes of Shigella, divided on the 
basis of differences in their outer membrane lipopolysaccharide antigen (LPS)], which are 
Shigella dysenteriae (13 serotypes), S. flexneri (16 serotypes), S. boydii (18 serotypes), and 
S. sonnei (1 serotype) [2]. The multiple and evolving serotypes require the development of 
polyvalent vaccines [9]. Recent vaccine development efforts have targeted S. flexneri 2a, 3a, and 
6 as well as S. sonnei in an effort to provide broad coverage against the most predominant 
Shigella strains.  
Shigella pathogenesis and immunity are attributed to the organism's ability to invade, replicate 
intra-cellularly, and spread inter- and intra-cellularly within the colonic epithelium [8]. This 
invasion eventually results in extensive inflammation and tissue destruction of the villous 
epithelium adjacent to lymphoid follicles resulting in the formation of mucosal micro-abscesses 
and ulcerations [8]. As contiguous epithelial cells die, additional areas of mucosal ulceration and 
hemorrhage are formed. 
To initiate infection, Shigella utilize a Type-III Secretion System (T3SS). The T3SS consists of 
proteins that comprise the needle complex (> 12 proteins) and nonstructural secreted proteins 
forming a conduit for the passage of virulence proteins from the pathogen to the enterocyte. The 
needle apparatus consists of a membrane-embedded basal anchor-like structure, an external 
needle that protrudes from the bacterial surface (which allows passage of effector proteins), and 
a tip complex that caps the needle and functions to sense the presence of eukaryotic cells (also 
serving as a platform of the translocon and regulating the effector protein secretion). Upon host 

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cell contact, a translocon is assembled between the needle tip complex and the host cell, serving 
as a gateway for translocation of effector proteins by creating a pore in the host cell membrane. 
IpaD, located at the needle tip senses the environment and the location in the gastrointestinal 
tract, recruiting IpaB to the needle tip upon host cell recognition. The needle complex then 
interacts with the host cell and inserts itself to deliver Shigella effector proteins. Because the Ipa 
proteins are conserved across all Shigella spp. and are directly responsible for initiating 
infection, immune defenses against the TTSS effector proteins may confer broad-spectrum 
protection against multiple serotypes overcoming the homologous protection afforded by an LPS 
only strategy [10]. 
Immunization strategies for Shigella vaccines to date have included killed whole-cell bacteria, 
live-attenuated bacteria, recombinant carrier organisms, polysaccharide conjugates, and LPS-
protein mixtures. These approaches have often proven either too reactogenic or poorly 
immunogenic in human testing. Oral vaccines may activate multiple effector arms of the immune 
system; however, they are difficult to administer in doses that are immunogenic, safe, and well 
tolerated and suffer from delivery restrictions [erratic absorption, degradation in acidic 
environment], particularly for inactivated or subunit vaccines compromising reproducible 
responses. Development of an immunogenic, safe intramuscular (IM) Shigella vaccine is crucial 
to harmonize immunization with other vaccines administered globally, particularly as use of 
alternate routes would necessitate specialized training for administering personnel.  
5.1. 
InvaplexNAT 
The Shigella invasin complex (termed, Invaplex) containing IpaB, IpaC and LPS underwent 
manufacturing and initial preclinical and clinical testing in the early 2000s. That formulation, 
termed S. flexneri 2a native Invaplex or S. flexneri 2a InvaplexNAT was a macromolecular 
complex isolated from wild-type S. flexneri 2a via FPLC Ion-exchange chromatography 
purification. In addition to IpaB, IpaC, and LPS, this formulation contained other invasins (IpaA, 
IpaD), the VirG protein, two non-virulence specific proteins of 72 and 84 kDa molecular weight 
(DnaK and elongation factor-G, respectively) and several other proteins of various molecular 
weights and limited immunogenicity. 
S. flexneri 2a InvaplexNAT was safely administered intranasally (IN) in small animals and 
stimulated robust mucosal and serum intestinal and pulmonary immune responses [humoral, Th1 
and Th2 directed at IpaB, IpaC, and LPS and intestinal IgA responses] [11, 12]. The S. flexneri 
2a InvaplexNAT vaccine was protective in guinea pigs using the keratoconjunctivitis model and in 
mice using the lethal pneumonia model against both S. sonnei and S. flexneri [11, 13-15]. The 
optimal immunization schedule in mice (5 μg per dose) and guinea pigs (25 μg per dose) was 3 
doses given at 2-week intervals. Immunological memory was observed in mice 15 weeks after 
the final immunization. 
5.1.1. 
Synopsis: Human S. flexneri 2a Invaplex IN Investigations 
Two lots of S. flexneri 2a InvaplexNAT were administered to 117 human subjects in 3 
investigations at doses ranging from 10 to 690 µg [12, 16]. The first clinical trial of S. flexneri 2a 
InvaplexNAT was a dose-escalation study conducted using doses ranging from 10 to 480 µg with 
an initial lot of S. flexneri 2a InvaplexNAT (Lot 0994) [12, 16]. A total of 3 doses (2 weeks apart) 
of vaccine were administered IN to each subject using a pipette. The most commonly reported 

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symptoms were rhinorrhea and nasal congestion seen in 50% and 47%, respectively. Mild nasal 
discharge and nasal turbinate edema were observed on clinical exam in 63% and 50% of 
volunteers. There were no significant differences in the frequency of these adverse events across 
dose levels or by dose. The majority (99%) of symptoms/signs were mild. High pre-vaccination 
findings were also observed (nasal discharge: 46%; nasal edema: 38%; rhinorrhea: 21%; and 
nasal congestion: 17%). Safety laboratory tests showed no significant changes from baseline 
among any of the study groups. Doses ≥ 240 μg induced increasing frequency of immune 
responses (Table 3); however, the magnitude of immune responses was moderate. 
Table 3: 
Frequency (%) of Immune Responses to Immunizing Antigen(s) Following 
First in Human Phase 1 Clinical Trial of S. flexneri 2a InvaplexNAT 
(Lot 0994) 
Antigen 
Assay 
Study Group 
A (n = 7) 
B (n = 8) 
C (n = 7) 
D (n = 8) 
10 μg 
50 μg 
240 μg 
480 μg 
S. flexneri 2a 
InvaplexNAT 
IgA ASC 
0.0 
25.0 
42.9 
25.0 
IgG ASC 
0.0 
25.0 
100 
50.0 
Serum IgA 
0.0 
12.5 
14.3 
0.0 
Serum IgG 
0.0 
0.0 
14.3 
0.0 
Fecal IgA 
0.0 
12.5 
28.6 
37.5 
LPS 
IgA ASC 
0.0 
12.5 
42.9 
25.0 
IgG ASC 
0.0 
12.5 
71.4 
37.5 
Serum IgA 
0.0 
12.5 
0.0 
0.0 
Serum IgG 
0.0 
0.0 
28.6 
0.0 
Fecal IgA 
0.0 
12.5 
28.6 
50.0 
Note: Analysis limited to subjects receiving at least 2 doses of S. flexneri 2a InvaplexNAT and having post-Dose 2 
blood samples collected. 
The results of this study led to a second in human clinical investigation, a randomized, double-
blind, placebo-controlled study with four groups [3 groups with 12 subjects each were 
randomized to 1 of 3 S. flexneri 2a InvaplexNAT dose levels—240 µg, 480 µg, or 690 µg]. The 
test article was administered with the Dolphin™ nasal delivery device [purported to optimize IN 
delivery via nasal mucosal deposition subsequently improving the immune response]. 
Additionally, a cohort of 8 subjects received 240 μg of the new vaccine Lot 1307 via pipette as a 
bridge to the first in human clinical trial [to compare the relative superiority in the delivery 
devices (Dolphin™ to pipette)]. The 690 μg dose of S. flexneri 2a InvaplexNAT (Lot 1307) was 
selected based on data from the phase 1 clinical trial. Similar to the first-in-human clinical trial, 
the most commonly reported symptoms were rhinorrhea (31%) and nasal congestion (33%). 
Sneezing and nasal itching were also observed relatively frequently (both seen in 28% of the 
subjects). The majority (98%) of all vaccine-associated adverse events were classified as mild. 
No subjects reported any related severe or serious adverse events. Table 4 shows the frequency 
of individual immune responses to the immunizing antigens (S. flexneri 2a InvaplexNAT, LPS). 
Serologic responses were relatively uncommon with no more than 20% of the vaccine recipients 
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InvaplexNAT and LPS). Antibody-secreting cell (ASC) responses were more common and seen in 
50%-60% of the vaccine recipients depending on the antigen evaluated. Positive antigen-specific 
ASC responses were detected in volunteers from all study groups. In general, antigen-specific 
IgA and IgG ASCs peaked following the second vaccination in the majority (75%–77%) of 
volunteers regardless of vaccine dose (data not shown). The 690 μg dose elicited the greatest 
response rate (maximum with IgA ASC—75%) and magnitude of response. Of importance, 
vaccination with comparable doses of the S. flexneri 2a InvaplexNAT LPS/protein subunit 
complex via the Dolphin™ resulted in higher plasma and ASC immune responses as compared 
to pipette delivery confirming the importance of optimizing IN deposition in eliciting optimal 
immune responses.   
Table 4: 
Frequency (%) of Immune Responses to Immunizing Antigen(s) Following 
Second Phase 1 Clinical Trial of S. flexneri 2a InvaplexNAT Lot 1307 
Group 
S. flexneri 2a InvaplexNAT 
LPS 
Serology 
ASC 
Serology 
ASC 
IgA 
IgG 
IgA 
IgG 
IgA 
IgG 
IgA 
IgG 
A 
(240 µg) 
n = 11 
0.0 
27.3 
18.2 
27.3 
9.1 
45.5 
27.3 
36.4 
B 
(480 µg) 
n = 10 
10.0 
30.0 
20.0 
20.0 
10.0 
40.0 
20.0 
20.0 
C 
(690 µg) 
n = 12 
50.0 
16.7 
75.0 
58.3 
25.0 
41.7 
58.3 
58.3 
D 
(240 µg) 
n = 8 
0.0 
0.0 
14.3 
14.3 
0.0 
0.0 
14.3 
14.3 
Note: Analysis limited to subjects receiving at least 2 doses of S. flexneri 2a native InvaplexNAT and having post-
Dose 2 blood samples collected. 
Groups A, B, and C were immunized with nasal delivery device; group D was immunized with a micropipette 
In a follow-on double-blind, placebo-controlled, vaccination-challenge study, a total of 52 
subjects were randomized to receive 690 μg of S. flexneri 2a InvaplexNAT (n=31) or placebo 
(n = 21). Other than sneezing, which was more common in vaccine recipients (Chi-square 
p = 0.03), there were no significant differences in the frequency of adverse events between the 
2 study groups. Additionally, the majority of all adverse events were of mild severity and no 
adverse events were coded as severe. Further, there were no adverse events that met the 
vaccination stopping criteria, nor were there any vaccine-related serious adverse events. Based 
on a priori subject selection criteria, 10 vaccinees and 12 placebo recipients advanced to the 
challenge phase of the study. All 22 subjects were admitted and challenged with 800 cfu of live 
S. flexneri 2a strain 2457T. There were no significant differences in the rate of fever, diarrhea, or 
any of the diarrhea severity indices between vaccine recipients selected to advance to challenge 
and placebo recipients. 
Throughout the course of several clinical trials with S. flexneri 2a InvaplexNAT, there were no 
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serious adverse events. Table 5 details the vaccine-related, surveyed signs and symptoms of 
subjects across the 2 lots and the range of doses. The most common adverse symptoms include 
nasal congestion (33.3%), rhinorrhea (29.9%), and sneezing (25.6%), all of mild severity. 
Placebo recipients have experienced similar rates of solicited symptoms. The only solicited local 
physical exam finding or solicited symptom that appeared to be significantly different between 
vaccinees and placebo recipients (18.8% and 0.0%, respectively; p = 0.03) was mild nasal 
itching; however, there was no significant increase in the rate of nasal itching with increasing 
vaccine doses (chi-square test for trend p = 0.70). 

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Table 5: 
Number (%) of Subjects with Vaccine Related Solicited Signs and Symptoms 
Lot Number 
0994 
0994 
0994 
0994 
1307 
1307 
1307 
1307 
Placebo 
Delivery Device 
Pipet 
Pipet 
Pipet 
Pipet 
Pipet 
Dolphin™ 
Dolphin™ 
Dolphin™ 
Dolphin™ 
Dose 
10 μg 
50 μg 
240 μg 
480 μg 
240 μg 
240 μg 
480 μg 
690 μg 
-- 
N 
8 
8 
8 
8 
8 
12 
12 
53
a 
28
b 
Malaise 
0 (0.0) 
1 (12.5) 
2 (25.0) 
0 (0.0) 
1 (12.5) 
1 (8.3) 
0 (0.0) 
6 (11.3) 
2 (7.1) 
Headache 
0 (0.0) 
4 (50.0) 
2 (25.0) 
4 (50.0) 
1 (12.5) 
1 (8.3) 
1 (8.3) 
8 (15.1) 
7 (25.0) 
Rhinorrhea 
2 (25.0) 
4 (50.0) 
5 (62.5) 
5 (62.5) 
1 (12.5) 
4 (33.3) 
3 (25.0) 
11 (20.8) 
5 (17.9) 
Nasal congestion 
3 (37.5) 
3 (37.5) 
3 (37.5) 
6 (75.0) 
2 (25.0) 
7 (58.3) 
2 (16.7) 
13 (24.5) 
7 (25.0) 
Nasal burning 
0 (0.0) 
2 (25.0) 
2 (25.0) 
1 (12.5) 
1 (12.5) 
1 (8.3) 
0 (0.0) 
8 (15.1) 
2 (7.1) 
Nasal itching 
1 (12.5) 
2 (25.0) 
1 (12.5) 
2 (25.0) 
1 (12.5) 
1 (8.3) 
4 (33.3) 
10 (18.9) 
0 (0.0) 
Sore throat 
2 (25.0) 
0 (0.0) 
1 (12.5) 
4 (50.0) 
1 (12.5) 
2 (16.7) 
0 (0.0) 
10 (18.9) 
4 (14.3) 
Postnasal drip 
2 (25.0) 
0 (0.0) 
5 (62.5) 
3 (37.5) 
2 (25.0) 
2 (16.7) 
2 (16.7) 
10 (18.9) 
5 (17.9) 
Cough 
0 (0.0) 
2 (25.0) 
1 (12.5) 
1 (12.5) 
0 (0.0) 
1 (8.3) 
0 (0.0) 
8 (15.1) 
1 (3.6) 
Sinus pain 
0 (0.0) 
0 (0.0) 
1 (12.5) 
0 (0.0) 
0 (0.0) 
0 (0.0) 
0 (0.0) 
4 (7.5) 
1 (3.6) 
Sneezing 
2 (25.0) 
2 (25.0) 
3 (37.5) 
3 (37.5) 
0 (0.0) 
1 (8.3) 
4 (33.3) 
15 (28.3) 
4 (14.3) 
Itching eyes 
0 (0.0) 
0 (0.0) 
0 (0.0) 
2 (25.0) 
0 (0.0) 
1 (8.3) 
1 (8.3) 
5 (9.4) 
2 (7.1) 
Nose bleed 
0 (0.0) 
0 (0.0) 
0 (0.0) 
2 (25.0) 
0 (0.0) 
0 (0.0) 
0 (0.0) 
4 (7.5) 
0 (0.0) 
Nasal mucosa 
hyperemia 
2 (25.0) 
5 (62.5) 
0 (0.0) 
1 (12.5) 
1 (12.5) 
1 (8.3) 
0 (0.0) 
15 (28.3) 
7 (25.0) 
Nasal discharge 
3 (37.5) 
7 (87.5) 
5 (62.5) 
5 (62.5) 
1 (12.5) 
1 (8.3) 
0 (0.0) 
6 (11.3) 
7 (25.0) 
Nasal edema 
6 (75.0) 
6 (75.0) 
1 (12.5) 
3 (37.5) 
0 (0.0) 
1 (8.3) 
0 (0.0) 
6 (11.3) 
1 (3.6) 
Pharyngeal 
erythema 
2 (25.0) 
0 (0.0) 
0 (0.0) 
0 (0.0) 
0 (0.0) 
0 (0.0) 
0 (0.0) 
3 (5.7) 
0 (0.0) 
Epistaxis 
1 (12.5) 
0 (0.0) 
0 (0.0) 
1 (12.5) 
0 (0.0) 
0 (0.0) 
0 (0.0) 
0 (0.0) 
0 (0.0) 
a Data pooled from a phase 1 safety and immunogenicity study (N = 12) [16], a placebo controlled vaccination study (M. Riddle, personal communication, N = 
10) and a placebo controlled vaccination-challenge study (C. Harro, personal communication, N = 31). 
b Data pooled from a placebo controlled vaccination study (M. Riddle, personal communication, N=7) and a placebo controlled vaccination-challenge study (C. 
Harro, personal communication, N = 21) 

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5.2. 
Artificial Invaplex 
Despite the excellent safety profile of S. flexneri 2a InvaplexNAT, inconsistent immune responses 
and lack of protection from disease in a human challenge model highlighted the need for further 
vaccine development. The composition of InvaplexNAT includes a high molecular weight 
complex and several undefined proteins. The high molecular weight complex contains the known 
major Shigella-virulence factors and immunogens, including IpaB, IpaC, and LPS and is 
associated with protective activity of S. flexneri 2a InvaplexNAT. Other, undefined proteins are 
not considered active ingredients of the vaccine and, therefore, may have diluted the potency of 
key antigens (IpaB, IpaC and LPS) [11] in the high molecular weight complex and thus 
undermining the optimal potential immune response solicited. In addition, the undefined 
components may contribute destabilizing activities, such as protease activity, that may have 
contributed to inconsistent immunogenicity of the S. flexneri 2a InvaplexNAT vaccine observed 
over time. Subsequently, to improve the overall definition and immunogenicity of the high 
molecular weight component of Invaplex the development of a more refined vaccine [S. flexneri 
2a artificial Invaplex (InvaplexAR)] commenced.  
5.2.1. 
Synopsis: S. flexneri 2a InvaplexAR 
Briefly, recombinant IpaB and IpaC proteins were purified from separate recombinant 
Escherichia coli [17, 18]. LPS was purified from S. flexneri 2a strain 2457T by the Westphal 
procedure [19]. The individual components (IpaB, IpaC and LPS) were assembled into the high 
molecular weight S. flexneri 2a InvaplexAR complex, which was subsequently purified by ion-
exchange size exclusion chromatography using conditions similar to that used to purify S. 
flexneri 2a InvaplexNAT. The vaccine was then sterile filtered, stored frozen, and available for 
vaccine studies. Initial studies compared S. flexneri 2a InvaplexNAT to S. flexneri 2a InvaplexAR. 
Figure 1 shows the overall composition of each vaccine and demonstrates that the research grade 
S. flexneri 2a InvaplexAR has only 3 components (IpaB, IpaC, and LPS) at much higher 
proportions on a weight basis as compared to S. flexneri 2a InvaplexNAT. 

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Figure 1: 
Comparison of S. flexneri 2a InvaplexAR and S. flexneri 2a InvaplexNAT by 
SDS-PAGE 
 
S. flexneri 2a InvaplexAR is assembled from purified IpaB (lane 1, 1 µg), purified IpaC (lane 2, 1 µg), and purified 
S. flexneri 2a LPS (lane 5, 2.5 µg). After assembly the complex is isolated by ion-exchange chromatography. The 
purified InvaplexAR (5 µg) is in lane 3. For comparison purposes InvaplexNAT (5 µg) is also run on these gels (lane 
4). The panel on the left is a Coomassie stained SDS-PAGE gel, the middle panel is a western blot probed with 
monoclonal antibodies specific for IpaB and IpaC and the panel on the right is a silver-stained gel that is used to 
show both the protein and LPS components of the Invaplex product. In this gel, HTIpaC (HisTag-IpaC) is used; in 
later stages of InvaplexAR development, only non-his-tagged IpaC was used. The InvaplexNAT preparation consists of 
many proteins in addition to IpaC and IpaB thereby reducing the proportion of the key antigens (IpaB, IpaC, and 
LPS) in the overall product composition. 
5.2.2. 
Comparative Efficacy of S. flexneri 2a InvaplexAR (research grade) and S. 
flexneri 2a InvaplexNAT 
To demonstrate that the increased proportions of the key antigens in S. flexneri 2a InvaplexAR 
resulted in improved immunogenicity groups of mice (5 per group) were intranasally immunized 
on Day 0, 14, and 28 with a research grade lot of S. flexneri 2a InvaplexNAT (0.1, 1, 2.5, 5, or 
10 µg), and a research grade lot of S. flexneri 2a InvaplexAR (0.1, 1, 2.5, or 5 µg) or saline. Blood 
was collected on Day 42 and analyzed by enzyme-linked immunosorbent assay for anti-LPS, 
IpaB and IpaC IgG and IgA endpoint titers. The mean endpoint titers + 1 standard error are 
shown in Figure 2. The results clearly demonstrate that lower dose amounts of InvaplexAR could 
be used to stimulate higher titers than that possible with S. flexneri 2a InvaplexNAT. 

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Figure 2: 
Comparative Immune Responses in Mice Receiving S. flexneri 2a InvaplexAR 
or S. flexneri 2a InvaplexNAT 
 
 
5.3. 
Development of S. flexneri 2a InvaplexAR-DETOX 
While InvaplexAR has demonstrated immunogenicity when administered by IN route, there is 
concern that the LPS moiety of the vaccine could lead to excessive reactogenicity when used 
parenterally. Additionally, intranasal immunization may not be practical or acceptable as a route 
of administration to infants and children. In light of this concern, the “detoxified” InvaplexAR-

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DETOX was developed using genetically-attenuated Shigella flexneri lacking late acyl transferases 
(ΔmsbB), resulting in lipid A mutants that are underacylated. This underacylated LPS is used in 
combination with recombinant IpaB and IpaC in similar fashion as InvaplexAR under cGMP. 
Underacylated LPS induces less pro-inflammatory cytokine release from macrophages in vitro, 
and InvaplexAR-DETOX has been demonstrated to induce lower reactogenicity as compared to 
InvaplexAR after ID vaccination in mice, while still inducing the same levels of Shigella-specific 
antibodies in mice and guinea pigs. 
5.4. 
Nonclinical Toxicology  
A nonclinical toxicology study was conducted in New Zealand White (NZW) rabbits to evaluate 
the toxicity and local site reactogenicity of InvaplexAR-DETOX when administered intramuscularly 
at up to 25 µg protein content. This study also included an adjuvant, dmLT, which is not 
included in the clinical protocol due to formulation challenges. This study was conducted in 
compliance with Good Laboratory Practices. 
As shown in Table 6, a total of 88 (44 male and 44 female) NZW rabbits were randomized into 
each designated group. Animals were administered four biweekly intramuscular doses of saline 
(Group 1), 10 µg InvaplexAR-DETOX + dmLT (Group 2), 25 µg InvaplexAR-DETOX (Group 3), or 25 
µg InvaplexAR-DETOX + dmLT (Group 4) on days 1, 15, 29, and 43. Animals were followed for 
standard vaccine toxicology endpoints, including detailed physical exams, ophthalmological 
exams, body weight measurements, evaluation of body temperature and assessments of dose site 
inflammation following each dose, and clinical pathology. Six animals of each sex from each 
group were euthanized and underwent full necropsy on study day 45 (2 days post final 
vaccination). The remaining five animals per sex from each group were euthanized and 
underwent a full necropsy on day 73 following a 28 day recovery period. Microscopic evaluation 
of major organ systems and local injection sites was conducted on all animals sacrificed on study 
day 45. Only injection sites were evaluated microscopically from those animals sacrificed on 
study day 73. 
Table 6: 
Nonclinical Toxicology Study Design 
Group 
InvaplexAR-DETOX Dose 
dmLT Dose 
Number of Animals 
Termination 
Day 45 
Termination 
Day 73 
1 
N/A 
N/A 
6M/6F 
5M/5F 
2 
10 µg 
0.1 µg 
6M/6F 
5M/5F 
3 
25 µg 
N/A 
6M/6F 
5M/5F 
4 
25 µg 
0.1 µg 
6M/6F 
5M/5F 
InvaplexAR-DETOX was well-tolerated following four bi-weekly doses, with no adverse effects 
observed in NZW rabbits at either 10 or 25 μg. Non-adverse apparent effects of treatment 
included decreased food consumption without correlating changes in body weights, gross and 
microscopic inflammation at the injection sites, temporary shifts in clinical pathology indicating 
an immune and/or inflammatory response following vaccination, increased relative and absolute 
spleen weights, and microscopic changes in the spleen and sciatic nerve. These findings were 
consistent with the pharmacology of the vaccine and/or were not associated with a chronic or 

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disease effect. While InvaplexAR-DETOX appears to induce an acute systemic inflammatory 
response, this response appears to be in a dose-related manner. 
 
 

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6. 
Trial Objectives 
Primary: Evaluate the safety of a detoxified Shigella flexneri 2a InvaplexAR (InvaplexAR-DETOX) 
administered by intramuscular immunization. 
Secondary: Evaluate immune responses (serum IgG/IgA and ALS IgG/IgA) following IM 
immunization with InvaplexAR-DETOX. 
Exploratory:  
 Collect samples from vaccinated subjects for exploratory immunological assays. 
 Perform secondary immunology analyses. 
 Collect samples for evaluation of C-reactive protein (CRP) responses to vaccine and 
placebo. 

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7. 
Hypotheses 
Safety: One or more of the proposed dosing regimens will have an acceptable frequency and 
severity of adverse events (AEs). 
Immunogenicity: 
 IM administration of InvaplexAR-DETOX will induce serum IgG antibody responses to S. 
flexneri 2a Invaplex in ≥50% of subjects in at least one of the study groups. 
 IM administration of InvaplexAR-DETOX will induce antibody responses to either IpaB or 
IpaC in ≥50% of subjects in at least one of the study groups. 
8. 
Clinical Study Design 
This is a double-blinded, placebo-controlled, dose-escalating study in which a total of 60 
subjects will receive three vaccinations (Days 1, 22 and 43) of one of three doses of InvaplexAR-
DETOX (2.5, 10, or 25 µg) or placebo as per Table 7. A complete 3-dose series will be completed 
for an entire cohort at one dose level prior to advancing to the next dose level. Prior to advancing 
to the next dose level, a safety analysis (including events up to 7 days after the third dose) will be 
completed and reviewed by the Protocol Safety Review Team (PSRT). If no halting criteria are 
met, the next cohort and dose level will be initiated.  
As an added precaution in the event of severe reactogenicity when InvaplexAR-DETOX is 
administered intramuscularly, five subjects of Cohort A will be enrolled in a pilot group prior to 
enrolling the remaining subjects. In this pilot group, four subjects will receive 2.5 µg of 
InvaplexAR-DETOX and one subject will receive placebo (saline). These subjects will be monitored 
for 7 days post vaccination for solicited and systemic reactions, unsolicited adverse events, and 
serious adverse events. If no unexpected symptoms occur within 7 days of vaccination, 
enrollment of the remaining subjects in Cohort A may proceed. 
Table 7: 
Clinical Trial Design for the Proposed Phase 1 Study with S. flexneri 2a 
InvaplexAR-DETOX 
Cohort 
Group 
n 
Route 
InvaplexAR-DETOX (µg) 
A 
A-1 
16 
IM 
2.5 
A-2 
4 
IM 
Placebo 
B 
B-1 
16 
IM 
10 
B-2 
4 
IM 
Placebo 
C 
C-1 
16 
IM 
25 
C-2 
4 
IM 
Placebo 
Blood and stool specimens will be collected at prescribed intervals to examine systemic and 
mucosal immune responses. Vaccine safety will be actively assessed at vaccination and for 28 
days following receipt of the third vaccine dose (see Table 8). The decision to advance to the 
next cohort (higher dose level) will be based on the safety assessment (not immunogenicity). A 
dose level with no occurrence of stopping criteria will prompt moving to the next cohort. All 
safety data will be summarized and reviewed by the PSRT prior to dose-escalation. 

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The study consists of 11 visits not including the screening visit or the Day -7 visit. Additionally, 
subjects are expected to be available for a telephone follow-up approximately 6 months after 
receipt of the last vaccine dose. The study time and events schedule is shown in Table 8. 
 

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Table 8: 
Clinical Protocol Time and Events Schedule 
Study Event 
Screening 
-71 
1 
2 
8 
22 
23 
29 
43 
44 
50 
57 
712 
223 
Compliance Ranges (days) 
-60 to -9 
-14 to -2 
N/A 
N/A 
±1 
±2 
N/A 
±2 
±2 
N/A 
±3 
±3 
±4 
± 28 
Study Briefing 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Comprehension Assessment 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Informed Consent (study 
participation) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Informed Consent (HIV testing) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Screening Medical History/ 
Physical Examination 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
CBC and Serum Chemistry3 
X 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
 
C-Reactive Protein 
 
 
X 
X 
X 
 
 
 
 
 
 
 
 
 
Anti-HIV-1/2 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
HLA-B27 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
HBs Ag 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Anti-HCV 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Serum IgA 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Anti LPS Antibody Screening 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vital signs (BP, HR, T) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Urine pregnancy test 
X 
 
X 
 
 
X 
 
 
X 
 
 
 
X 
 
Vaccination 
 
 
X 
 
 
X 
 
 
X 
 
 
 
 
 
Clinical check4 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Symptom Diary5 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Serology6 
 
 
X 
 
 
X 
 
 
X 
 
X 
X 
X 
 
Peripheral Blood Mononuclear 
Cells (PBMCs)7 
 
X 
X 
 
X 
 
 
X 
 
 
X 
 
X 
 
Fecal IgA 
 
X 
X 
 
 
 
 
X 
 
 
X 
 
 
 
Additional sample collections8 
 
X 
X 
 
 
 
 
X 
 
 
X 
 
 
 
Study completion 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Post-study safety assessment9 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Blood volume (mL) by study day 
25 
82 
74 
4 
70 
10 
0 
67 
10 
0 
77 
10 
70 
0 
1Day -7 stool range is -14 to -2. 
2 The data will be locked following entry of the Day 71 data 
3 Chemistry includes serum electrolytes, glucose, BUN, creatinine, AST, and ALT. CBC and chemistry will not be repeated if the screening visit occurs during 
the day -14 to -9 window. 

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4 Clinical checks on vaccination days include pre- and post-vaccination complete assessments including targeted physical exams (volunteers will be observed for 
30 minutes post-vaccination), baseline exam, and physical assessment. 
5 Volunteer diaries post-vaccination will begin day of vaccination through the 7-day post-vaccination follow-up for each dose. 
6 Samples taken from blood draw for that day, serum samples will be assayed for antibody (IgG and IgA) titers against S. flexneri 2a LPS, IpaB, IpaC, and S. 
flexneri 2a Invaplex by previously established methods. 
7 PBMCs include collection for Antibody Secreting Cells (ASC), Antibody Lymphocyte Supernatant (ALS), memory B and T cells and α4β7+ B cells. A missed 
PBMC collection on Day -7 will not be considered a protocol deviation. 
8 Additional sample collection may include saliva. 
9Day 223 assessments will be performed via telephone.

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8.1. 
Screening and Study Schedule 
The screening visit is scheduled -60 to -9 days before the first vaccination. Fully informed, 
written consent must be obtained from each subject prior to conducting any study procedure. 
Two consent forms will be signed; a consent form for study participation and a separate consent 
form for HIV testing. To assess understanding, a comprehension assessment will be administered 
after the subjects sign the study participation written consent. The following 
evaluations/procedures will be carried out: 
 Oral and/or video presentation of clinical trial design, risks and study schedule 
 Review of informed consent document 
 One-on-one discussions with principal investigator or subinvestigator 
 Signature of informed consent document (see section 9.2 for informed consent 
process) 
 Comprehension test (minimum of 70% accuracy required for participation, two 
attempts allowed) 
 Medical history – between Days -60 to -9 
 Physical examination – between Days -60 to -9 
 Vital Signs – between Days -60 to -9 
 Screening laboratory analysis (allowable time period for accepting test): 
 Complete blood count (between Days -60 to -9) 
 Serum transaminases (ALT/AST) (between Days -60 to -9) 
 Na, K+, CL-, HCO3, glucose, BUN and creatinine (between Days -60 to -9) 
 Total serum IgA (between Days -60 to -9) 
 Pregnancy test (urine -hCG) for women, as well as 0-24 hours before each 
vaccination 
 HLA-B27 (between Days -60 to -9) 
 Serology screening (Anti LPS Antibody) for prior Shigella exposure (between 
days -60 to -9)  
 Serum HIV 1/2 antibody (between days -14 to -2) 
 Hepatitis B surface antigen (HBsAg) (between days -14 to -2) 
 Hepatitis C virus Ab (between days -14 to -2) 
Additionally, approximately 7 days prior to vaccination (allowable range: Days -14 to -2), 
subjects will have a follow-up medical history and brief physical exam to ensure ongoing 
eligibility.  If the initial screening is within the -14-day window, it can count as both the initial 
screening and pre-vaccination visit (and all screening activities will be conducted at the one 
visit). Blood, saliva, and stool will be collected for baseline serology testing. Additional 

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instruction will be reviewed with each subject to include review of the study visit schedule, 
preparation procedures for saliva collection, and procedures for stool collection and storage. The 
following screening laboratory analyses will be carried out (below tests will not be repeated if 
the initial screening visit occurs during the day -14 to -9 window): 
 Repeat complete blood count 
 Repeat serum transaminases (ALT/AST) 
 Repeat Na, K+, CL-, HCO3, glucose, BUN and creatinine 
 Serum HIV 1/2 antibody (between Days -14 to -2) 
 Hepatitis B surface antigen (HBsAG) (between Days -14 to -2) 
 Hepatitis C virus (anti-HCV antibody) (between Days -14 to -2) 
A screening test for any of the above assessments may be repeated in the event of a laboratory 
error (i.e., hemolyzed sample) or in the event the screening physician believes: 1) the laboratory 
test has identified a normal variant of a healthy state (i.e., below normal hemoglobin in a 
menstruating female, or cyclic reductions in absolute neutrophil count in subjects of African 
descent) and 2) the variant is no more than a Grade 1 abnormality. This repeat screening applies 
to the initial screening tests [(executed between -60 to -9 days) and the repeat screening labs if 
executed (between days -14 to -2)]. If a repeat screening blood test is performed, only the results 
of the second test will be used to determine eligibility.  
The WRAIR Clinical Trials Center (CTC) may use a screening protocol in recruiting volunteers 
for this study. All screening procedures outlined in this section may be completed under this 
screening protocol. 
During this screening process, the laboratory assays will be reviewed to determine if any clinical 
laboratory abnormalities exist that would preclude study participation. Subjects who have only 
1 mild (grade 1) abnormality may be included if the PI determines that their participation will not 
present undue risk to the subject. Subjects with 2 or more mild abnormalities may be included in 
the study only with the consensus of the PI and the research monitor. Subjects with clinical 
laboratory abnormalities of greater than mild severity will not participate in this clinical trial. 
The clinical toxicity grading scale that will be used as a guideline is based on the scale used by 
the Division of AIDS (DAIDS) for adverse events and the guidance from the US Food and Drug 
Administration (FDA) Center for Biologics Evaluation and Research. If any additional safety 
labs are performed, either scale may be utilized. 
8.2. 
Randomization 
Within each cohort, 20 subjects will be randomly assigned to one of 2 different treatment groups 
using block sizes of 5. The first cohort will be enrolled in a staggered manner in order to evaluate 
the safety and tolerability of the vaccine in a limited number of subjects before exposing a larger 
number of subjects to the study product. 
The pilot group will assign subjects to each treatment group with a ratio of 4:1, so that 4 subjects 
will receive InvaplexAR-DETOX and 1 subject will receive placebo. 
After review of the safety data through Day 7 of this pilot group, if no safety concerns are 
identified, the remaining 15 subjects will be randomly assigned to one of the two treatment 

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groups in the same ratio (4:1) with 12 subjects receiving InvaplexAR-DETOX and 3 subjects 
receiving placebo. 
Subsequent dose-escalating cohorts will be randomized after review of safety data including 7 
days after the final vaccination of the previous cohort. Depending on the safety profile, the PSRT 
will make a recommendation to the sponsor and investigator to proceed or not with the 
vaccination of the next cohort at a higher dose level. 
Participant randomization will be generated from the Advantage eClinical system based on the 
enrollment sequence in the treatment table, therefore the randomization plan will also provide the 
maximum number of sequence numbers by site. An Emmes statistician will write code using 
SAS and the seed(s) for the random number generator will be documented. When a participant is 
randomized in Advantage eClinical only the cohort, coded treatment assignment and the 
sequence number will display. A second Emmes statistician will conduct and document quality 
control (QC) of the randomization plan, treatment table and all other related documents using the 
statistical considerations in the protocol. Once the treatment table has been finalized, the Emmes 
Systems Programmer will transfer the treatment table into the Advantage eClinical environment 
and the Emmes Project Manager will approve the treatment table. The person performing 
vaccine formulation will be provided a list of the coded treatment numbers with their 
corresponding unblinded treatment assignments, as well as the sealed back-up manual 
randomization list. 
8.3. 
Blinding 
The study is designed to keep subjects and investigators blinded until completion of the clinical 
phase of the trial and monitoring of the clinical data. Members of the study staff not involved in 
clinical outcome assessment will perform formulation of the test articles. During the course of 
the study, all efforts will be made to keep subjects and investigators unaware of subject 
assignment. 
8.4. 
Unblinding 
In the event of emergency, the site Investigator may require that the blind be broken for the 
subject experiencing the emergency when knowledge of the subject's treatment assignment may 
influence the subject's clinical care. Emergency unblinding can occur by contacting Emmes 
during business hours or by contacting the unblinded vaccine formulation expert after business 
hours. Every effort will be made not to unblind the subject unless it is considered necessary for 
the welfare of the subject. Prior to unblinding, the site Investigator is encouraged (to the extent 
possible, without jeopardizing the subject's health) to contact the Sponsor (or designee) to 
discuss the decision to break the blind. The site PI will be expected to provide a rationale for the 
necessity of unblinding based on the expectation that knowledge of the subject's treatment 
assignment will have a meaningful impact on the subject's medical care in the short term. If a 
subject's treatment assignment is unblinded, the subject will remain in the study and continue 
with protocol-defined study visits, but not receive further vaccinations. The decision to unblind 
will be communicated to any regulatory bodies (e.g., institutional review boards [IRBs]) as 
required. At the end of the study, documentation of all unblinded subjects (and the rationale for 
unblinding) will be incorporated into the Trial Master File. 

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8.5. 
Vaccination 
After initial screening and informed consent, eligible volunteers will be vaccinated with S. 
flexneri 2a InvaplexAR-DETOX Lot 1972 or placebo (saline). The vaccine will be administered IM 
on Days 1, 22, and 43. The investigational product administration procedure is outlined in detail 
in the Manual of Procedures (MOP). Briefly, the IM vaccination site (deltoid muscle) will be 
pre-treated with an alcohol pad and the vaccine will be administered using a 1-inch needle. 
Approximately 100 µL of appropriately formulated vaccine product or placebo will be 
administered. Subjects will be observed for at least 30 minutes post-vaccination and given 
symptom diaries to record local and systemic reactions for 7 days post vaccination for each dose. 
After the 30-minute observation period, an assessment will be performed including vital signs 
and the recording of all adverse events. Subjects will return approximately 24 hours after 
vaccination for a follow-up examination. For each subsequent immunization, the injection will 
be given in alternate deltoid regions from the previous vaccination. An exception may be needed 
if there is pain/tenderness, erythema, edema, pruritus, induration, or rash at the planned site of 
vaccination. In this case, the Principal Investigator, following discussion with the Independent 
Research Monitor, may exercise the discretion to administer the vaccination on the same arm as 
the previous vaccination. The site of vaccine administration will be recorded in the source 
documents. 
8.5.1. 
Vaccination Follow-Up 
The timing and number of follow-up visits are delineated in the Time and Events Schedule 
(Table 8) and are scheduled to coincide with vaccination or specimen collection visits. These 
visits may include a brief history, targeted exam, blood, stool, and salivary specimen collection, 
and a reminder to the volunteer to contact the investigator if s/he experiences any medical 
problems. Subjects will return approximately 24 hours and 7 days after vaccination for follow-
up.  
Approximately 6 months from final vaccination all subjects will be contacted via telephone to 
assess for a final safety assessment. A history for any additional serious new diagnoses or 
hospitalizations will be solicited.  Three attempts will be made and documented; after 3 
unsuccessful attempts a certified letter and/or email will be used to request the subject to contact 
the study team. 
8.6. 
Endpoints 
8.6.1. 
Safety 
Safety monitoring will be undertaken using in-person symptom surveillance, symptom diaries, 
and targeted physical exams. Solicited adverse event monitoring will survey and specifically 
inquire about symptoms listed in section 11.6.2. Abnormalities will be described in detail in 
order to monitor progress. This will include the name of the adverse event, the date and time of 
onset and offset (duration), severity, and Principal Investigator determination of relationship to 
the investigational vaccine. Safety monitoring labs will also be performed on Days 8, 29, and 50. 
Clinical outcomes will be prospectively defined and actively surveyed during the study. The 
study procedures are detailed in Table 8. 

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Clinical definitions will be used to grade the severity of symptoms in accordance to the severity 
scale included in section 11.8.3. 
8.6.2. 
Immunology 
A secondary objective is to assess IgG and IgA serologic and Antibody in Lymphocyte 
Supernatant (ALS) responses to S. flexneri 2a Invaplex using methods previously established.  
Seroconversion will be defined as a ≥ 4-fold increase in endpoint titer between pre- and post-
vaccination samples. Peripheral Blood Mononuclear Cells (PBMCs) will be collected to 
determine antigen-specific IgG and IgA titers in ALS samples. An ALS response is defined as ≥ 
4-fold increase in antibody titer at any point post-vaccination as compared to baseline titers. For 
serologic and ALS assays, high-titer specimens will be included on each plate to track day-to-
day interassay variation. For each antigen, pre- and post-vaccination serum samples will be 
assayed side-by-side. The antibody titer assigned to each sample will represent the geometric 
mean of duplicate tests. Reciprocal endpoint titers less than the starting dilution of the assay will 
be assigned a value of half the starting dilution for computational purposes. 
Secondary immunologic evaluations (an exploratory objective) may include serologic and ALS 
responses to additional antigens (S. flexneri 2a LPS, IpaB, IpaC), additional IgG subclasses, IgM 
responses, and serum bactericidal antibody (SBA) titers against S. flexneri 2a 2457T (responders 
defined as ≥ 9-fold increase in SBA titer at any point post-vaccination compared to baseline 
titers). Stool samples will be collected to assess antigen-specific fecal Ig responses. A fecal 
response will be defined as a ≥ 4-fold increase from baseline. 
Samples will also be collected, processed, and archived for potential future analysis as follows. 
PBMCs will be collected for antigen-specific memory B/T cell analyses, antibody secreting cell 
(ASC) assay, and α4β7+ B cells. Concurrently, saliva samples may be collected to assess 
mucosal IgA and IgG antibody responses. CRP responses in serum after initial vaccination will 
be collected for exploratory correlation with immune responses and AEs. 
8.7. 
Investigational product 
8.7.1. 
InvaplexAR-DETOX 
S. flexneri 2a InvaplexAR-DETOX Lot 1972 was produced under cGMP at the WRAIR Pilot 
Bioproduction Facility, Silver Spring, MD. The individual components IpaB (Lot 1757), IpaC 
(Lot 1771) and detoxified LPS (Lot 1902) were assembled into the InvaplexAR-DETOX complex 
and subsequently purified by ion-exchange chromatography yielding the bulk drug product (Lot 
1954). The drug product, designated as Lot 1972, was filled in 2mL glass vials, stoppered and 
sealed, and stored at -80 ± 10°C. 
Figure 3: 
Label from a vial of InvaplexAR-DETOX, Lot 1972 
Shigella flexneri 2a WR30 Artificial  
Invaplex (InvaplexAR-Detox) 
 
BPR No.: BPR-1214-00   
Lot No.: 1972 
 
Contents: 0.6 ml ± 0.06          
Storage: -80 ± 10°C  
Caution: 
New Drug - Limited by Federal (or United States) law to investigational use. 
Date of Mfg.: 15 Jan 16 
Manufactured By: WRAIR, Silver Spring, MD 20910 

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8.7.2. 
Investigational Product Preparation 
On the day of vaccination, the product will be used to formulate the appropriate vaccine 
preparations for the clinical groups. The vaccine will be used within 6 hours of preparation. 
Prepared vaccine or open vials will not be saved for use on another study day. Any remaining 
vaccine product will be properly disposed of in accordance with the MOP. Vaccine formulation 
for each of the groups is described in detail in the MOP. 
8.7.3. 
Investigational Product Accountability  
The sponsor is responsible for authorizing the release for distribution of the investigational 
products to the study site. The NMRC Enteric Diseases Department and/or WRAIR will arrange 
for shipment, under appropriate conditions, of the investigational products to the clinical site. At 
the WRAIR CTC, investigational product inventory logs will be maintained in the accountability 
files. The Release for Use Memo signed by the sponsor’s representative gives permission to the 
WRAIR Pilot Bioproduction Facility to release product to the PI. After the investigational 
product is distributed, the PI is responsible for and will maintain logs of investigational product 
receipt, storage, reconstitution, accountability by subject, and investigational product remaining 
before final disposition. These logs will be maintained in the regulatory files at the WRAIR 
CTC. The PI may delegate, in writing, this responsibility to another individual, but the PI is 
ultimately responsible for the investigational product and its proper storage upon receipt at the 
study site until it is transferred back to the sponsor or designee, utilized as directed by the 
sponsor or destroyed. Any excess formulated vaccine remaining from vaccination day will be 
disposed of or used for research purposes as authorized by the sponsor. All used or partially used 
investigational product and empty vials will be destroyed or used for nonclinical research 
purposes as per the MOP.  
8.8. 
 Duration of Subject Participation 
Screening of volunteers will occur during a period of up to 60 days prior to initial vaccinations. 
Subjects will receive 3 vaccinations on Days 1, 22, and 43. Clinical follow-up will occur as 
delineated in the Time and Events schedule (Table 8). A phone survey will occur at 
approximately 6 months after the final vaccine dose. This phone survey will specifically inquire 
if the subject has experienced any new serious health problems or hospitalizations since their last 
study follow-up. Inclusion criteria require that a subject be available for the required follow-up 
period and scheduled clinic visits. Therefore, no military subjects will be enrolled in the study if 
a known deployment is to conflict with his or her involvement. If there is an unexpected 
deployment, efforts (e.g., telephone, e-mail, command mail) will be undertaken to obtain safety 
follow-up data per the protocol schedule. 
8.9. 
Dose Escalation Criteria 
The decision to advance to the next dose level will be based solely on the safety assessment. A 
dose level with no occurrence of stopping criteria will prompt moving to the next higher level. 
The Protocol Safety Review Team (PSRT) comprised of the Principal Investigator, the PATH 
Medical Officer, and the Independent Research Monitor will review blinded safety data for the 
recommendation to proceed with dose escalation. 

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8.10. 
Stopping Criteria 
The study will use a dose-escalation design to evaluate increasing doses of InvaplexAR-DETOX. 
The PI, along with the PSRT, may determine a subject’s local site reactions warrant 
discontinuation of subsequent vaccinations for that subject. If it is determined that a subject will 
not continue with future vaccine doses, they may remain enrolled in the study for follow-up. 
If any of the following events occurs, administration of investigational product will be 
discontinued for the group until a thorough review of the event is undertaken by the investigators 
and the PSRT: 
 The occurrence of one serious adverse event determined to be related to the 
investigational vaccine (definitely, probably, or possibly) at any time post-vaccination. 
 Severe diarrhea, defined as six or more liquid stools within 24 hours of vaccination. 
 A severe local rash, defined as one that makes the subject unable to perform normal daily 
activities and which is not attributable to another cause. 
 Systemic rash, including but not limited to generalized urticarial, generalized petechiae, 
or erythema multiforme, occurring in two or more subjects in a group will result in 
stopping further vaccination in subject’s group. 
 One vaccine-related serious or unexpected AE evaluated by the principal investigator 
(PI), Independent Research Monitor, and sponsor and determined to be an unacceptable 
risk to the health and safety of other investigational product recipients. 
 If a subject experiences grade 2 or greater hypotension in the 30 minutes following 
vaccination, they will not be eligible to receive subsequent vaccinations. 
Further vaccination, in accordance with the protocol, may be resumed with the concurrence of 
the PRST, sponsor’s representative, the PI, and the US Food and Drug Administration (FDA).  
An interim analysis for each cohort will be prepared and reviewed to include all safety data 
through seven days after the third vaccination dose for each cohort. This report will be provided 
to the PSRT for their review to determine whether the study can continue to enroll for the next, 
dose-escalating cohort or whether the study should be stopped. In addition to the interim safety 
analyses, the PI, sponsor’s representative along with the PSRT will stop the study until further 
review by the PSRT if any of the following criteria that are thought to be definitely, probably, or 
possibly related to the investigational vaccine are identified: 
 One or more SAEs, including grade 4 adverse events or laboratory abnormalities. 
 Two or more grade 3 adverse events or laboratory abnormalities of the same type. 
 One or more grade 3 laboratory abnormalities. 
 Two or more grade 2 laboratory abnormalities. 
8.11. 
Identification of Data to Be Recorded on Case Report Forms 
The electronic case report form (eCRF) data will be entered from source documentation. No 
source data will be recorded directly in eCRF (i.e., without prior record of data). These data will 
be consistent with the source documents or the discrepancies will be explained. No personally 

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identifying data will be recorded on eCRFs. The investigator is ultimately responsible for the 
accuracy of data transcribed on the eCRF. Data monitoring and management will be performed 
in the electronic data capture (EDC) database system by the study monitor and the designated 
data management group. A detailed data management plan will be written and approved by the 
study team and the PI prior to study start. All updates to the data management plan must be 
approved before study closeout and database lock. 
8.12. 
Known and Potential Risks and Benefits to Human Subjects 
8.12.1. 
Risks/Discomfort to Subjects and Precautions to Minimize Risk 
Outlined as follows are anticipated and unexpected adverse reactions with a brief description of 
procedures to ameliorate risks and symptoms. In general, dose escalation is being performed to 
ensure an acceptable safety record for IM administration of the vaccine product in this study. 
Prior to proceeding with the next dose in the next group of subjects, all safety data will be 
reviewed by the PSRT, and a determination will be made as to whether the study can continue to 
enroll subsequent cohorts (at the dose escalation planned) based on the observed safety record 
from the preceding dose level. In addition to this overall safeguard, individual risks will be 
closely monitored and any AEs will be quickly identified and managed. 
8.12.1.1. Local Reactions 
Local reactions at the vaccination site are expected from IM immunization. The IM route of 
vaccination, in general, has been shown to be a safe and effective route of administration. There 
may be some mild discomfort and irritation from an IM vaccination from the initial injection. 
These are typically mild and transient in nature. In addition, there is the possibility of local 
erythema, pruritus, swelling, and/or induration that appears over time. In addition, subjects may 
develop temporary swelling in the lymph nodes under the arm where vaccination occurred.  
8.12.1.2. Systemic Reactions 
Any vaccine may be associated with a wide range of systemic reactions, such as fevers, 
constitutional symptoms (fatigue, malaise, appetite change), and gastrointestinal symptoms 
(diarrhea, abdominal pain) as well as others to include allergic reactions (bronchospasm, 
urticaria, anaphylaxis). The frequency and type of systemic symptoms will be assessed and 
analyzed with respect to study product. 
8.12.1.3. Pregnancy 
During initial screening and prior to each vaccination, a urine pregnancy test will be performed. 
Subjects with a positive test will be excluded from the study. A final urine pregnancy test will be 
conducted during the last clinic visit. Female subjects of childbearing potential must agree to use 
an effective method of birth control (birth control pills, injection hormonal contraceptive, 
implant hormonal contraceptive, hormonal patch, IUD, sterilization by hysterectomy or tubal 
ligation, spermicidal products and barrier methods such as cervical sponge, diaphragm, or 
condom) during the study and 3 months following the last vaccination. Abstinence is acceptable. 
While female subjects will not be required to disclose their method of birth control, they will be 
counseled during the initial screening on acceptable forms of birth control required for study 
enrollment. At each visit, female subjects will be reminded that they must agree to use effective 

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contraception to remain in the study. Female subjects should not become pregnant during the 
study and for at least 3 months after the last vaccine dose. 
8.12.1.4. Lactation 
Risks to nursing infants are unknown at this time; breastfeeding females will be excluded from 
this study. 
8.12.1.5. Venipuncture 
Blood sampling carries a minimal risk of minor discomfort and the possibility of minor bruising 
at the site of the needle puncture and, rarely, the possibility of infection at the needle puncture 
site. 
8.12.1.6. Allergic Reaction 
As with any Investigational New Drug (IND) product administration and no matter what 
precautions are taken, there is always the risk of a serious, or even life-threatening, allergic 
reaction. Although anaphylactic reactions are not anticipated, as with any vaccination, the rare 
possibility exists. For this reason, all subjects will be observed at the study site (WRAIR CTC) 
for a minimum of 30 minutes following vaccine dosing. Appropriate emergency equipment (e.g., 
automated external defibrillator, air-shields manual breathing unit bag) and medication [e.g., 
antihistamines and adrenaline (epinephrine)] for initial treatment of an allergic reaction will be 
available at the WRAIR CTC whenever immunizations are given. This equipment is available to 
handle emergencies such as anaphylaxis, angioedema, bronchospasm, and laryngospasm. 
Existing staff standard operating procedures at the WRAIR CTC are also in place for these 
events. 
8.12.1.7. Theoretical Risks 
Post-infection reactive arthritis, characterized as an asymmetric, oligoarticular arthritis 
commonly involves the knees, ankles, or wrists is known to occur following Shigella infection. 
Some data suggests that the risk of reactive arthritis following shigellosis may be higher in 
persons carrying the Human Leukocyte Antigen (HLA)-B27 antigen [20]. There is enough 
uncertainty as to the etiology of arthritis, that a specific entry criteria related to this theoretical 
risk is included and will exclude volunteers with a personal or immediate family history of 
inflammatory arthritis or HLA-B27 positivity. 
8.12.1.8. Unknown Risks 
As with all research, there is the remote possibility of risks that are unknown or that cannot be 
foreseen based on current information. This would include late effects that have been seen with 
some vaccines. 
8.12.2. 
Alternatives to This IND Product or Study 
At this time, there are no known alternative, licensed vaccines directed at Shigella. An 
alternative choice is not to participate in this study. 

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8.12.3. 
Intended Benefit for Subjects 
There is no benefit that can be guaranteed to volunteers for participating in this research study. 
However, there is potential societal benefit of the development of an effective Shigella vaccine. 
8.12.4. 
Risks to Study Personnel and the Environment 
The principal risk in the clinical setting is in the handling of needles that may be contaminated 
and the attendant risks including hepatitis, human immunodeficiency virus (HIV), and other 
human pathogens. Adherence to standard operating procedures (SOPs) for working with 
infectious agents and universal precautions will reduce the risk of exposure. 
There are no known risks to the environment other than those associated with the generation of 
biohazardous waste attendant to vaccination of humans. All biohazardous waste will be disposed 
of as stipulated by local, state, and federal regulations and in accordance with study-site SOPs. 
8.13. 
Route of Administration, Dosage Regimen, Treatment Period, and Justification 
On days 1, 22, and 43 subjects will be administered InvaplexAR-DETOX or placebo. Each subject 
will receive the same formulation at each vaccination dependent upon group assignment. Dose 
formulations will be prepared each day of immunization at the clinical site using the InvaplexAR-
DETOX drug product. Because InvaplexAR-DETOX has not been previously administered to humans, 
limited information is available to estimate the dose needed to induce suitably robust immune 
responses while maintaining an acceptable safety profile. Guinea pigs have been immunized 
intranasally with 25 µg or intramuscularly with 1, 5, and 25 µg of cGMP InvaplexAR-DETOX. The 
vaccine was well-tolerated and induced a robust immune response. This study is designed as a 
placebo-controlled, dose-escalating study starting with the lowest dose of InvaplexAR-DETOX (2.5 
µg) and progressing upwards through higher doses up to 25 µg. Following completion of each of 
the vaccination series, an interim safety report will be compiled and a dose-escalation decision 
will be made in concert with the PSRT. If the vaccine is safe and immunogenic, future studies 
will evaluate the preliminary efficacy of this vaccine using a homologous enteric S. flexneri 2a 
strain in a human challenge model. The overall goal of the current Shigella development plan is 
to create a broadly protective multivalent Shigella vaccine from a combination of Invaplex 
vaccines encompassing the conserved invasin proteins and the combination of serotype specific 
LPS for each proposed strain to integrate. The proposed multivalent vaccine would integrate the 
major Shigella serotypes responsible for human disease: S. flexneri 2a, S. flexneri 3a, S. flexneri 
6, and S. sonnei.  
8.14. 
Compliance Statement 
The study will be conducted according to the protocol and in compliance with International 
Council on Harmonisation (ICH) Good Clinical Practice (GCP), Belmont Principles, and other 
applicable regulatory and DoD requirements. All identified study personnel will be trained to 
perform their roles and will carry out their responsibilities in accordance with ICH GCP 
guidelines and clinic site study procedures. 
8.15. 
Study Population 
In this initial study, it is important to restrict enrollment to a small number of healthy subjects 
without factors that may limit or confound assessment of AEs and vaccine-induced immune 

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response. The study population will consist of healthy male and nonpregnant, nonlactating 
female subjects, ages 18-50 years (inclusive), from the greater Washington, DC and Baltimore, 
Maryland areas, who can give informed consent and understand the risks and benefits of the 
study and are willing to comply with all protocol procedures and time commitments. A 
maximum of 60 subjects will be enrolled in this study. Four alternates per cohort may be asked 
to go to the clinic on vaccination days in the event a planned subject cannot/does not enroll in the 
study. 
8.16. 
Study Site 
The study will be conducted at the WRAIR CTC, Silver Spring, Maryland. Immunologic assays 
will be conducted at the subunit enteric vaccines and immunology laboratory of the WRAIR, 
Silver Spring, Maryland. 
9. 
Selection and Withdrawal of Subjects 
9.1. 
Recruitment of Subjects/Study Population 
Healthy adults, both males and nonpregnant females, will be recruited from the 
Baltimore/Washington, DC, area through the WRAIR CTC by the use of advertisement in 
multiple media formats, to include but not limited to: newspapers, fliers, e-mails, the WRAIR 
CTC web site, public listservs, social media (such as Facebook), posters, bus ads, and generic 
radio advertisements. E-mail announcements and web site postings (e.g., WRAIR CTC) will 
include information found on recruitment scripts (excluding any compensation information) or 
posters excluding any photos unless attached as a complete flyer. Recruitment may also include 
oral presentations at events, meetings, and briefings wherein the desired recruit population might 
reasonably be expected to attend. All forms of recruitment, printed media, handouts, and briefs 
will have IRB approval prior to being used.  
When a subject calls the WRAIR CTC recruitment office and discloses an interest in the study, 
the recruitment staff will discuss details of the trial from a prepared, IRB-approved script. If the 
subject is still interested, contact information will be obtained and an appointment for 
briefing/screening will be arranged.  
Up to 60 subjects will be recruited for enrollment in this Phase 1 trial. Additionally, there will be 
up to 4 alternates recruited for each cohort.  
9.2. 
Informed Consent Process 
All subjects will undergo an informed consent process consisting of a detailed informational 
presentation of study-related material using a pre-recorded brief. Following the briefing, one of 
the study investigators will reiterate the purpose, procedures, and risks of the study as needed 
and answer any questions. This process will take place at the WRAIR CTC immediately 
preceding the screening procedures.  
During the briefing session, an investigator or designee will brief prospective subjects using the 
briefing slides. The briefing may be provided by a pre-recorded slide set. An investigator will be 
available to answer any questions raised during the session. Following the briefing, the 
coordinator or designee will provide the subject with ample time to read the consent and will 

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answer any questions. The subject will be asked to sign the consent, as will the investigator 
conducting the informed consent discussion. The subject will be allowed to take the consent 
document home to consider and discuss it with others and can return it to the WRAIR CTC at a 
later time to sign it. After signing the consent (to include future use of specimens), the subject 
will take a comprehension test (“open book” format). Any questions missed will be explained to 
the subject, and the subject’s questions will be answered. The test is administered to aid study 
personnel in identifying gaps in understanding. If the subject appears not to understand the 
content of any of the questions, one of the study facilitators will be informed and must document 
whether the subject understands the risks, benefits, procedures, and time commitment as well as 
the answers to the questions that he or she missed. Any subject who, in the opinion of the PI, 
does not understand the study well enough to consider his or her consent truly informed will be 
excluded. The subject will be given one additional opportunity to take the comprehension test. A 
passing score of at least 70% will be required to participate in the study.  
The informed consent process will include a recruitment script, briefing material, and an 
informed consent document. Throughout the study, all questions and concerns of the subject will 
be addressed. If the subject has additional concerns, the research monitor will be available to 
answer any medical questions, and the NMRC Office of Research Administration is available to 
answer any human use-related concerns.  
No study procedures will occur before the subject provides written informed consent. Active 
duty military will not be specifically recruited for this study. Active duty military members 
interested in participating will need to obtain supervisor approval. To minimize any possible 
coercion, supervisors or anyone in the chain of command or in a dominant position will not be 
permitted to consent the subject.  
9.3. 
Eligibility Screening 
Each subject must meet all inclusion and no exclusion criteria. The PI or designee will make the 
final decision of the eligibility. Only eligible subjects will be given the investigational product. 
9.4. 
Subject Inclusion Criteria 
Subjects must meet all of the following criteria to be included in the study: 
1. Healthy, adult, male or female, age 18 to 50 years (inclusive) at the time of enrollment. 
2. Completion and review of comprehension test (achieved ≥ 70% accuracy, two attempts 
allowed). 
3. Provide written informed consent before initiation of any study procedures. 
4. Agrees to complete all study visits and procedures and provide a screening stool sample. 
5. Women of childbearing capacity: Negative pregnancy test with understanding (through 
informed consent process) to not become pregnant during the study or within three (3) 
months following the last vaccine dose. 
9.5. 
Subject Exclusion Criteria 
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9.5.1. 
General Health 
1. Health problems (for example, chronic medical conditions such as psychiatric conditions, 
diabetes mellitus, hypertension or any other conditions that might place the subject at 
increased risk of adverse events) – study clinicians, in consultation with the PI, will use 
clinical judgment on a case-by-case basis to assess safety risks under this criterion. The 
PI will consult with the Research Monitor as appropriate. 
2. History of autoimmune disorders, cardiovascular and renal diseases. 
3. Use of immunosuppressive medications (systemic corticosteroids or chemotherapeutics 
that may influence antibody development), or immunosuppressive illness, including IgA 
deficiency (defined by serum IgA <7mg/dL). 
4. Women who are pregnant or planning to become pregnant during the study period plus 3 
months beyond the last vaccine dose and currently nursing women. 
5. Participation in research involving another investigational product (defined as receipt of 
investigational product or exposure to invasive investigational device) 30 days before 
planned date of first vaccination or anytime through the last in-clinic study safety visit. 
6. Positive blood test for HBsAG, HCV, HIV-1/2. 
7. Clinically significant abnormalities on basic laboratory screening (see section 8.1). 
8. Systemic antimicrobial treatment (i.e., topical treatments are not an exclusion) within 1 
week before administration of the first vaccine. 
9.5.2. 
Research specific 
9. Allergies that may increase the risk of AEs. 
10. Regular use (weekly or more often) of antidiarrheal, anti-constipation, or antacid therapy. 
11. Abnormal stool pattern (fewer than 3 stools per week or more than 3 stools per day) on a 
regular basis; loose or liquid stools on other than an occasional basis. 
12. Personal or family history of inflammatory arthritis. 
13. Positive blood test for HLA-B27 (associated with increased risk of reactive arthritis 
secondary to Shigella infection). 
14. History of allergy to any vaccine. 
15. Exclusionary skin disease history/finding that would confound assessment or prevent 
appropriate local monitoring of AEs, or possibly increase the risk of a local AE. 
9.5.3. 
Prior Exposure to Shigella  
16. Serum IgG titer > 2500 to Shigella flexneri 2a LPS. 
17. History of microbiologically confirmed Shigella infection. 
18. Received previous licensed or experimental Shigella vaccine or live Shigella challenge. 
19. Travel to countries where Shigella or other enteric infections are endemic (most of the 
developing world) within two years prior to dosing (clinician judgment). 

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20. Occupation involving handling of Shigella bacteria currently, or in the past 3 years. 
9.6. 
Subject Withdrawal Criteria 
Each subject may withdraw consent at any time during the study without penalty. Counseling 
about the subject’s health will be provided if he or she decides to discontinue participation in the 
study. Medical advice regarding what is in the best interest of the subject will be provided. 
The PI may discontinue the subject’s activity without the subject’s consent if any of these criteria 
is met: 
 A subject fails to comply with study procedures 
 A subject’s safety or health may be compromised by further participation 
 It is determined to be in the subject’s best interest 
9.6.1. 
When and How to Withdraw Subjects 
A subject may end his or her participation in the study at any time. If a subject withdraws, the 
investigator will make a reasonable effort to determine the reason for the withdrawal from the 
study and to complete termination procedures. Telephone calls, registered letters, and e-mail 
correspondence are considered reasonable effort. For subjects leaving the study, a targeted 
examination may be performed if medically indicated and if permitted by the subject. 
A subject may be withdrawn for an AE or SAE resulting in a safety concern or for 
noncompliance with protocol requirements. When a subject withdraws or is withdrawn by the PI 
due to an AE that results in a safety concern, the sponsor must be notified within 72 hours. 
Investigators must follow specific policy at each institution regarding the timely reporting of 
AEs and SAEs to the local IRB (section 11.9.1.2). In all cases, the PI will make a reasonable 
effort to complete study termination procedures. Subjects who do not receive subsequent vaccine 
doses but who continue in follow-up for safety and immunogenicity are not considered to be 
withdrawn from the study. 
If a subject meets withdrawal conditions for a concomitant medication violation or 
noncompliance, this should clearly be stated in the source document and the study termination 
eCRF. 
9.6.2. 
Follow-up for Withdrawn Subjects 
Attempts will be made to follow all subjects for safety up to 28 days after receipt of the last 
vaccine dose. Short-term safety follow-up 7-days post vaccination will also be attempted. 
9.6.3. 
Data Collected for Withdrawn Subjects 
All data collected up to the time of withdrawal will be reported, unless consent is withdrawn. If 
the subject proceeds with the specific follow-up procedures for withdrawn subjects, this data will 
also be collected and reported. The study termination eCRF will be completed with the reason 
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9.6.4. 
Replacement of Subjects 
Alternates recruited for each cohort will be asked to come to the day of the first vaccination for 
each cohort. If an assigned subject does not present on the first day of vaccination, elects to 
withdraw, or is found to no longer be eligible, an alternate will be enrolled. Subjects who 
withdraw or are withdrawn after the first vaccination will not be replaced. 
10. 
Treatment of Subjects 
If the subject is acutely ill on the day of first vaccination, the subject will not be vaccinated and 
may be asked to return to be enrolled in a subsequent dose group. If a subject returns to be 
enrolled in a subsequent dose group whose screening was performed more than 60 days prior to 
the vaccination date, he or she will be screened again for eligibility. This screening will include 
the labs required 7 days prior to vaccination (between Days -14 to -2) as delineated in section 8.1 
(Study and Screening Schedule). If the subject is acutely ill on the day of vaccine Dose 2, the 
subject may return for vaccination after recovery from the acute illness if the return visit is 
within the compliance range for receiving that dose.  
10.1. 
Dose Administration/Vaccination and Follow-up Periods/Study Visits 
10.1.1. 
Administration of Vaccination 
The vaccine or placebo will be administered intramuscularly to alternating deltoid regions, per 
the Time and Events Schedule (Table 8). The vaccine or placebo will be administered in 
accordance with formulation and vaccination procedures outlined in the MOP.  
10.1.2. 
Handling of Study Samples 
Samples collected under this protocol will be used to conduct protocol-related safety and 
immunogenicity evaluations. Sera and whole blood will be stored at the WRAIR in quality-
controlled environments. Transport and storage of these biological samples will be handled 
according to appropriate procedures. Any study for the future use of these biological samples 
will undergo appropriate regulatory review. All subjects will consent for the future use of their 
specimens.  
10.2. 
Concomitant Medications 
Subjects taking regular medication (i.e., birth control pills) prior to enrollment in the trial will be 
allowed to continue to take this medication unless it is specifically excluded as part of the 
inclusion/exclusion criteria for the trial. Subjects needing to take non-approved or excluded 
medication will not be eligible for enrollment in this study. Investigators will make 
determinations of continued eligibility throughout the trial. Any medication ordered by the study 
physician during the course of the trial will be documented on appropriate source documents. 
Medications being taken prior to and during the course of the trial will also be documented in 
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10.3. 
Procedures for Monitoring Subject Compliance 
All vaccines will be administered or witnessed by a study investigator, clinical research 
coordinator, or designee. 
11. 
Laboratory and Clinical Assessment  
11.1. 
Blood Sample Collection 
Study serology and immunological analyses will be performed from blood samples drawn at 
several of the follow up clinic appointments. Blood for serologic assays will be obtained per the 
Time and Events Schedule.  
11.2. 
Stool Sample Collection 
Stool will be collected from subjects for immunogenicity testing for fecal IgA. Subjects will be 
provided stool hats to self-collect all stools which will be processed within two hours of receipt 
in the CTC and within 8 hours of collection by the subject, according to study specific 
procedures that have been utilized successfully for other enteric disease studies. 
11.3. 
Saliva Sample Collection 
Collection of saliva samples will be performed utilizing synthetic oral swabs (Oracol Swab; 
Malvern Medical). The subject will gently rub the sponge portion of the Oracol Swab along their 
gum line for approximately one minute. Subjects will be instructed not to eat or drink anything, 
including chewing gum or tobacco, for 10 minutes prior to saliva collection. Subjects will be 
instructed to avoid drinking alcohol or using mouthwash for 24 hours and to avoid caffeinated 
beverages for 12 hours prior to collecting the sample. Saliva collection will proceed regardless of 
subject compliance with these instructions. Noncompliance with these instructions will not be 
considered a protocol deviation but will be documented.  
11.4. 
Study Safety Management 
The IRB, research monitor, and PI will review any safety concerns. 
Safety monitoring will be conducted throughout the study; therefore, safety concerns will be 
identified by continuous review of the data by the PI, clinic staff, Research Monitor, and PSRT. 
11.4.1. 
Protocol Safety Review Team 
The Protocol Safety Review Team (PSRT), comprised of the Principal Investigator, the PATH 
Medical Officer, and the Independent Research Monitor will routinely monitor safety throughout 
the duration of the trial. The PSRT will be chaired by the Independent Research Monitor, and 
may seek additional independent expert medical opinion as dictated by needs. Emmes will 
prepare blinded safety reports for review by the PSRT. These reports will provide at a minimum 
the following information: 1) accrual and participant status data with regard to completion of 
study vaccinations and study visits; and 2) summaries of solicited and unsolicited adverse events 
including safety labs.  The PSRT safety review will be conducted electronically (or by 
teleconference when needed). In addition to safety review, the PSRT may elect to discuss trial 
conduct issues that impact study integrity and participant safety. These may include but are not 
limited to data quality, critical monitoring findings, study product, research specimens, etc. 

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Emmes will also notify the PSRT of ad hoc safety reviews whenever it is aware of SUSAR or 
adverse events that meet pre-specified study pause criteria as per section 8.10. 
In addition to routine review of safety information, a central role of the PSRT is the blinded 
review of safety data for the recommendation of whether to progress to the next dose level in 
each cohort and to the next cohort. Cumulative safety data will be available continuously for 
review by PSRT members, and safety reports will be prepared for each PSRT deliberation of 
dose escalation and progression to subsequent cohorts. 
11.4.2. 
Research Monitor 
The research monitor will function as an independent safety advocate for subjects per Army 
Regulation 70-25 and DoD Instruction 3216.02. An independent research monitor is required to 
review all unanticipated problems involving risk to subjects or others, SAEs, and all subject 
deaths associated with the protocol and provide an unbiased written report of the event. At a 
minimum, the research monitor should comment on the outcomes of the event or problem and, in 
the case of an SAE or death, comment on the relationship to participation in the study. The 
research monitor should also indicate whether he or she concurs with the details of the report 
provided by the study investigator. Reports for events determined by either the investigator or 
research monitor to be possibly or definitely related to participation and reports of events 
resulting in death should be promptly forwarded to the IRB. 
11.5. 
Specification of Safety Endpoints 
11.5.1. 
Local and Systemic Reactions 
Signs and symptoms will be specifically surveyed at each visit as well as by patient self-report 
using a symptom diary. Symptoms that will be specifically solicited are listed in section 11.6.2. 
Additionally, we will record all unsolicited symptoms. These local reactions will be coded for 
severity based on the subject and investigator severity grading scale.  
11.5.2. 
Vital Signs 
Vital signs (temperature, blood pressure, and heart rate) will be obtained at each study visit 
(Table 9). 
Table 9: 
Reference Ranges and Adverse Event Coding for Vital Sign Parameters 
Vital Signs 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening (Grade 4) 
Heart Rate 
Tachycardia 
101–115 
116–130 
> 130 
ER visit or hospitalization for 
arrhythmia 
Bradycardia 
50–54
a
 
45–49 
< 45 
ER visit or hospitalization for 
arrhythmia 
Fever (°C)  
          (°F) 
38.0–38.4 
100.4–101.1 
38.5–38.9 
101.2–102.0 
39.0–40 
102.1–104 
> 40 
> 104 
Blood Pressure 

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Vital Signs 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening (Grade 4) 
Hypertension 
(systolic, mm Hg) 
141–150 
151–155 
> 155 
ER visit/hospitalization for 
malignant hypertension 
Hypertension 
(diastolic, mm Hg) 
91–95 
96–100 
> 100 
ER visit/hospitalization for 
malignant hypertension 
Hypotension (systolic, 
mm Hg)
b
 
85–89 
80–84 
< 80 
ER visit/hospitalization for 
hypotensive shock 
a Grade 1 bradycardia will not be considered an abnormality for this study unless judged to be clinically significant 
by the PI or the PI in consultation with the research monitor and sponsor. 
b If a subject has a baseline systolic blood pressure in the 90s then a decrease in blood pressure < 10 without 
associated clinical symptoms will not be considered an abnormality for this study unless judged to be clinically 
significant by the PI. 
c If a subject visits an emergency room for a non-life-threatening illness or symptoms (i.e., visits emergency 
department on weekend for mild problems because the physician’s office is closed), the severity of the AE will be 
assessed according to the subject’s clinical signs and symptoms. 
11.5.3. 
Physical Examination 
A complete physical exam will be conducted during the screening visit as part of the screening 
process. Subsequent focused clinical examinations will occur at each study visit with specific 
attention to the identification of local, systemic, or other adverse reactions.  
11.5.4. 
Clinical Laboratory Assessments 
Venous blood samples will be collected for chemistries and hematology. Hematology and 
chemistry analyses (BUN, creatinine, AST, ALT, electrolytes, and glucose) will occur per the 
Time and Events Schedule. Hematology and chemistry analyses will be performed by a 
commercial laboratory (Quest Diagnostics, Incorporated). Additional specimens may be 
collected to confirm and evaluate any abnormal values. 
The clinical toxicity grading scale that will be used as a guideline is based on the Guidance for 
Industry: Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in 
Preventive Vaccine Clinical Trials. Final grading determination will be made by the PI based on 
normal lab values for the specific lab and clinical symptoms. If any additional safety labs are 
performed, the FDA Guidance for Industry will be utilized. 
11.5.4.1. Hematology 
The following hematology parameters (Table 10) will be assessed: 
 White blood cell count (and differential) 
 Red blood cell count 
 Hematocrit 
 Platelet count 
 Hemoglobin 

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Table 10: 
Reference Ranges and Adverse Event Coding for Clinical Hematology 
Parameters 
Test 
Quest 
Normal 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening 
(Grade 4) 
Hemoglobin (g/dL) 
(for screening purposes only) 
M: LLN = 13.2 
F: LLN = 11.7 
M: 12.5-13.1 
F: 11.0-11.6 
M: 10.5-12.4 
F: 9.5-10.9 
M: 8.5-10.4 
F: 8.0-9.4 
M: <8.5 
F: <8.0 
Hemoglobin - decrease from 
lower limit of normal 
(used to grade toxicity)a 
0.5-1.5 
1.6-2.0 
2.1-5.0 
> 5.0 
Neutrophils (cells/mm3) 
1,500-7,800 
1,225-1,499 
1,000-1,224 
776-999 
< 776 
Leukocytes (white blood 
cells) (cells/mm3) 
3,800-10,800 
Leukopenia 
2,500-3,799 
1,500-2,499 
1,000-1,499 
< 1,000 
Leukocytosis 
10,801-15,000 
15,001- 
20,000 
20,001- 
25,000 
> 25,000 
Lymphocytes (cells/mm3) 
850-3,900 
750-849 
500-749 
250-499 
< 250 
Eosinophils (cells/mm3) 
15-500 
551-1,500 
1,501-5,000 
> 5,000 
Hypereosinophilic 
Platelets decreased – 
103/mm3 
140-400 
125-139 
100-124 
25-99 
< 25 
a In an instance where a post-vaccination hemoglobin result is below the Quest normal range, an AE is recorded 
when the post-vaccination value represents a decrease of ≥0.5 g/dL from that subject’s baseline value. If a subject 
has a decrease in hemoglobin ≥0.5 g/dL but remains within the Quest normal range, that will not be documented as 
an AE. 
11.5.4.2. Blood Chemistry 
The following clinical chemistry parameters listed in Table 11 will be assessed: 

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Table 11: 
Reference Ranges and Adverse Event Coding for Blood Chemistry 
Parameters 
Test 
Quest 
Normal 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening 
(Grade 4) 
Sodium 
135-146 
(mmol/L) 
 
 
 
 
Hyponatremia 
 
132-134 
130-131 
125-129 
< 125 
Hypernatremia 
 
147-148 
149-150 
151-152 
> 152 
Potassium 
3.5-5.5 
(mmol/L) 
 
 
 
 
Hypokalemia 
 
3.3-3.4 
3.1-3.2 
2.9-3.0 
< 2.9 
Hyperkalemia 
 
5.6-5.7 
5.8-5.9 
6.0-6.1 
≥ 6.2 
Glucose, Random 
65-139 
(mg/dL) 
 
 
 
 
Hyperglycemia 
 
140-155 
156-200 
> 200 
Insulin requirements 
or hyperosmolar coma 
Hypoglycemia 
 
60-64 
55-59 
45-54 
< 45 
SGOT/AST (elevation) 
M: 10-40 U/L 
F: 10-30 U/L 
M: 41-100 
F: 31-75 
M: 101-200 
F: 76-150 
M: 201-400 
F: 151-300 
M: > 400 
F: > 300 
SGPT/ALT (elevation) 
M: 9-60 U/L 
F: 6-40 U/L 
M: 61-150 
F: 41-100 
M: 151-300 
F: 101-200 
M: 301-600 
F: 201-400 
M: > 600 
F: > 400 
BUN (elevation) 
7-25 
26-28 
29-31 
> 31 
Requires dialysis 
Creatinine (elevation) 
M: 0.7-1.4 
F: 0.5-1.1 
M: 1.5-1.7 
F: 1.2-1.7 
M: 1.8-2.0 
F: 1.8-2.0 
M: 2.1-2.5 
F: 2.1-2.5 
M: >2.5 
F: >2.5 or requires 
dialysis 
11.5.4.3. C-Reactive Protein 
C-Reactive Protein (CRP) will be assessed on study days 1, 2, and 8. CRP assessment is for 
exploratory purposes only (immunologic response and safety endpoints) and will not be used as 
an eligibility criterion or safety signal. There are few published reports addressing healthy 
subjects’ CRP responses after vaccination. Both the correlation of CRP to immune responses and 
to systemic AEs (malaise, fever) has been performed in these trials [21-24].  
In this trial, collection of CRP will be limited to baseline and two time points after initial 
vaccination.    
11.5.4.4. Virus Testing 
Testing for evidence of HIV-1/HIV-2, HCV, and HBV infections will be obtained during the 
screening process as delineated in the time and events schedule (Table 8) and no earlier than 14 
days before initial vaccination. Evidence of infection will make a subject ineligible. Additional 
diagnostic testing will not be performed as part of this study. 

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11.5.4.5. HLA-B27 Screen 
Serologic evidence of HLA-B27 will be obtained during the screening process (Days -60 to -9). 
Evidence of HLA-B27 positivity will make a subject ineligible. 
11.5.4.6. Drug Screen 
No drug screening is planned for this study described herein. 
11.5.4.7. Pregnancy Screen 
A urine sample for pregnancy testing (female subjects of childbearing capacity) will be collected 
at the screening visit. Pregnancy tests will also be obtained on each day of vaccination and at the 
final in-clinic study visit. A positive pregnancy test prior to vaccination will result in no 
additional study vaccinations being provided. Any subjects who become pregnant during the 
study will be removed from receiving further treatment with InvaplexAR-DETOX and followed until 
the end of their pregnancy. Procedures to be followed in the event a study participant becomes 
pregnant during the study period are outlined in 11.9.2.1. 
11.6. 
IND Safety Reporting 
The following terms, as defined by 21 Code of Federal Regulations (CFR) 312.32, apply to IND 
safety reporting. 
11.6.1. 
Adverse Event or Suspected Adverse Reaction 
An AE, as defined by the ICH guideline for GCP, is “Adverse event means any untoward 
medical occurrence associated with the use of a drug in humans, whether or not considered drug 
related.” 
Suspected adverse reaction means any AE for which there is a reasonable possibility that the 
drug caused the AE. For the purposes of IND safety reporting, “reasonable” possibility means 
there is evidence to suggest a causal relationship between the drug and the adverse event. 
Suspected adverse reaction implies a lesser degree of certainty about causality than adverse 
reaction, which means any adverse event caused by a drug. 
An AE is considered any adverse change or exacerbation from a baseline condition that occurs 
following the initial administration of an investigational product whether or not the event is 
considered to be related to the investigational product. Examples of this include but are not 
limited to the following: 
 Adverse changes including new signs and symptoms, intercurrent illness modifying 
the clinical course, or the worsening of a baseline condition including the increased 
frequency of an event or an increased intensity of a condition 
 Concomitant disease with onset or increased severity after the start of study product 
administration 
 A new pattern in a preexisting condition occurring after the receipt of investigational 
product that may signal a clinically meaningful change 
 Clinically significant changes in laboratory values 

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11.6.2. 
Solicited Adverse Events 
A solicited AE is a predetermined event, identified in the Investigator’s Brochure, which may 
reflect safety concerns related to the investigational product. The solicited AEs for this study 
include: 
 Site pain 
 Site tenderness 
 Swelling 
 Induration (determined by investigator exam) 
 Site redness 
 Pruritus 
 Fever 
 Nausea 
 Vomiting 
 Abdominal pain 
 Diarrhea (loose stools) 
 Appetite change 
 Fatigue 
 Headache 
 Myalgias (general pain or soreness in muscles) 
 Arthralgias (general pain in joints) 
 Malaise 
11.6.3. 
Serious Adverse Event or Serious Suspected Adverse Reaction 
An AE or suspected adverse reaction is considered “serious” if, in the view of either the 
investigator or the sponsor it results in any of the following outcomes: 
 Death 
 Life-threatening AE 
 Inpatient hospitalization or prolongation of existing hospitalization 
 Persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
 Congenital anomaly/birth defect (Abortion, stillbirth, and any malformation/disease 
must be reported as an SAE.) 
An AE or suspected adverse reaction is considered “life threatening” if, in the view of either the 
investigator or sponsor, its occurrence places the patient or subject at immediate risk of death. It 

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does not include an AE or suspected adverse reaction that, had it occurred in a more severe form, 
might have caused death. 
Important medical events that may not result in death, be life threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical judgment, they 
may jeopardize the patient or subject and may require medical or surgical intervention to prevent 
one of the outcomes listed in this definition. Examples of such medical events include allergic 
reactions or bronchospasm requiring intensive treatment in an emergency room or at home, 
blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the development 
of drug dependency or drug abuse. 
11.6.4. 
Unexpected Adverse Event or Unexpected Suspected Adverse Reaction 
An AE or suspected adverse reaction is considered “unexpected” if it is not listed in the 
Investigator’s Brochure or is not listed at the specificity or severity that has been observed. It can 
also be considered “unexpected” if an Investigator’s Brochure is not required or available and is 
not consistent with the risk information described in the general investigational plan or elsewhere 
in the current application, as amended. For example, under this definition, hepatic necrosis 
would be unexpected (by virtue of greater severity) if the Investigator’s Brochure referred only 
to elevated hepatic enzymes or hepatitis. Similarly, cerebral thromboembolism and cerebral 
vasculitis would be unexpected (by virtue of greater specificity) if the Investigator’s Brochure 
listed only cerebral vascular accidents. “Unexpected,” as used in this definition, also refers to 
AEs or suspected adverse reactions that are mentioned in the Investigator’s Brochure as 
occurring with a class of drugs or as anticipated from the pharmacological properties of the drug 
but are not specifically mentioned as occurring with the particular drug under investigation.  
11.6.5. 
Other Adverse Event 
Other AEs will be identified by the PI during the evaluation of safety data. Significant AEs of 
particular clinical importance, other than SAEs and those AEs leading to discontinuation of the 
subject from the study will be classified as other AEs. For each, a narrative may be written and 
included in the clinical study report. 
11.7. 
Relationship to Investigational Product (Assessment of Causality) 
The PI must assign a relationship of each AE to the receipt of the investigational product. The 
investigator will use clinical judgment in conjunction with the assessment of a plausible biologic 
mechanism, a temporal relationship between the onset of the event in relation to receipt of the 
investigational product, and identification of possible alternate etiologies including underlying 
disease, concurrent illness, or concomitant medications. The following guidelines will be used by 
investigators to assess the relationship of an AE to study product administration. 
Not related: No relationship to the study product. Applies to those events for which 
evidence exists that there is an alternate etiology. 
Unlikely: Likely unrelated to the investigational product. Likely to be related to factors other 
than the study product but cannot be ruled out with certainty. 
Possible: An association between the event and the administration of the investigational 
product cannot be ruled out. There is a reasonable temporal association, but there may also 

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be an alternative etiology such as the subject’s clinical status or underlying factors including 
other therapy. 
Probable: There is a high degree of certainty that a relationship to the investigational 
product exists. There is a reasonable temporal association, and the event cannot be explained 
by known characteristics of the subject’s clinical state or factors including other therapy. 
Definite: An association exists between the receipt of the investigational product and the 
event. An association to other factors has been ruled out. 
11.8. 
Recording Adverse Events 
11.8.1. 
Methods/Timing for Assessing, Recording, and Analyzing Safety Endpoints 
AEs, solicited AEs, and SAEs will be assessed at all study visits, documented in the source 
records, and recorded on the eCRFs using accepted medical terms and/or the diagnoses that 
accurately characterize the event. When a diagnosis is known, the AE term recorded on the 
eCRF will be the diagnosis rather than a constellation of symptoms. The investigator will assess 
all AEs for seriousness, relationship to the investigational product, severity, and other possible 
etiologies. When an event has not resolved by the prescribed reporting period, it will be 
documented on the AE eCRF as “unknown.” 
The time frame for the collection of AEs and SAEs begins at the first administration of 
investigational product through 28 days after the last dose of investigational product is 
administered. Additionally, subjects will be contacted by telephone approximately 6 months after 
the final vaccination to assess for any new-onset SAEs since participation in the trial. 
11.8.2. 
Duration of Follow-up of Subjects After Adverse Events 
The PI and/or subinvestigators will follow SAEs to resolution, even if this extends beyond the 
prescribed reporting period. Resolution is the return to baseline status or stabilization of the 
condition with the probability that it will become chronic. SAE outcomes will be reported to the 
sponsor using the supplemental Serious Adverse Event Report Form. 
Investigators are not obligated to actively seek SAEs in former subjects; however, if an SAE, 
considered to be related to the investigational product is brought to the attention of the 
investigator at any time following completion of the study, the event will be reported to the 
sponsor as defined in section 11.9.1.1.  
11.8.3. 
Severity Assessment 
All AEs will be assessed for severity by the investigator. The investigating team will execute the 
investigator severity scale below. Inherent in this assessment is the medical and clinical 
consideration of all information surrounding the event including any medical intervention 
required. Each event will be assigned one of the following categories: mild, moderate, severe, or 
potentially life-threatening. See section 11.5.2 for vital signs and section 11.5.4 for laboratory 
values for further guidance in the assignment of severity. The following criteria may be used for 
any symptom not included in the grading scale. The eCRF for AEs will reflect only the highest 
severity for continuous days an event occurred. 
     Mild 
 
 
 
Grade 1 
Does not interfere with routine activities 

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Minimal level of discomfort 
     Moderate  
 
 
Grade 2 
Interferes with routine activities 
 
 
 
 
 
 
 
Moderate level of discomfort 
     Severe 
 
 
 
Grade 3 
Unable to perform routine activities 
 
 
 
 
 
 
 
Significant level of discomfort 
     Potentially life-threatening 
Grade 4 
Hospitalization or ER visit for potentially  
 
 
 
 
 
 
 
life-threatening event 
FDA guidelines for toxicity will be followed; however, if a subject is evaluated in an emergency 
room for a non-life-threatening illness or symptoms (i.e., visits emergency department on 
weekend for mild problems because the physician’s office is closed), the information from that 
visit will be reviewed, and severity of the AE will be assessed according to the subject’s clinical 
signs and symptoms. 
11.9. 
Reporting Adverse Events 
The PI will report all AEs to the sponsor and the local IRB in the appropriate safety, annual, 
and/or final reports. The investigative team will prepare annual reports and final reports for 
submission to the FDA. 
11.9.1. 
Reporting Serious and Adverse Events 
Contact information for reporting SAEs and unanticipated problems is provided in Table 12. 
11.9.1.1. Reporting to the Sponsor 
All SAEs and unexpected AEs must be reported promptly (within 72 hours) to the sponsor, 
whether or not the event is considered related to the study product. Further, the investigator 
should comply with relevant study-site SOPs on reporting SAEs. 
The information that the investigator will provide to the sponsor is specified in Table 13. The 
sponsor may request additional information for purposes of the study. 
Table 12: 
Study Contacts for Reporting Serious Adverse Events 
Sponsor’s Medical Officer 
Rahsan Erdem, MD 
PATH 
455 Massachusetts Ave NW 
Suite 1000 
Washington, DC 20001 
Telephone: 202-540-4546 
Fax: 202-457-1466 
E-mail: rerdem@path.org 
Institutional Review Board 
Naval Medical Research Center 
Research Services Directorate Office of Research Administration Code 
025, Building 500, Room 004 
Silver Spring, MD Telephone: 301-319-7276 
Fax: 301-319-7277 

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Research Monitor 
Michael Koren, MD 
WRAIR 
Telephone: 301-319-9904 
E-mail: michael.a.koren2.mil@mail.mil 
Research Monitor (alternate) 
James Moon, MD 
WRAIR 
Telephone: 301-319-9176 
E-mail: james.e.moon.mil@mail.mil 
Table 13: 
SAE Information to Be Reported to the Sponsor 
Notification Method 
Information to Be Provided 
E-mail or Telephone 
(within 72 hours) 
IND number, sponsor study number, name of the investigational product, and investigator 
name and contact number 
 
Subject identification number 
 
SAE, onset date, date of investigational product administration, severity, relationship, and 
subject’s current status 
AND 
 
E-mail or Fax 
Cover sheet or letter 
 
Adverse event case report form 
 
Supplemental SAE report forms 
 
Concomitant medication case report form or a list of concomitant medications 
 
Medical record progress notes including pertinent laboratory/diagnostic test results 
NOTE: When submitting SAE reports via e-mail, the subject line of each e-mail notification will read as follows: 
SAFETY REPORT – IND #   
_, Sponsor Study #  
, Subject#   
, Event Term:   
 
To comply with regulations mandating sponsor notification of specified SAEs to the FDA within 
7 calendar days, investigators must submit additional information as soon as it is available. The 
sponsor will report unexpected SAEs associated with the use of the drug to the FDA as specified 
in 21 CFR 312.32 (c). 
Investigators must follow all relevant regulatory requirements as well as specific policy at each 
institution regarding the timely reporting of SAEs to the NMRC IRB, and the research monitor. 
11.9.1.2. Reporting to the IRB 
Unanticipated problems involving risk to subjects or others, SAEs related to participation in the 
study, and all subject deaths should be promptly reported by phone, e-mail, or fax to the local 
IRB. 
It is the NMRC’s policy to report to the IRB within 24 hours after determination of the event. A 
formal written report is due within 5 business days of AE reporting. 
The following AEs should be considered as unanticipated problems that must be reported to the 
IRB:  
 A single occurrence of a serious, unexpected, and uncommon event that is strongly 
associated with exposure to the investigational product. 

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 A single or small number of a serious, unexpected event that is not commonly 
associated with exposure to the investigational product but is uncommon in the study 
population. 
 Multiple occurrences of an AE that, based on aggregate analysis, is determined to be 
an unanticipated problem. There should be a determination that the series of AEs 
represents a signal that the AEs were not just isolated occurrences and involve risk to 
human subjects. 
 An AE that is described in the Investigator’s Brochure, protocol, or informed consent 
documents but occurs at a specificity or severity that is inconsistent with prior 
observations. 
 An SAE that is described in the Investigator’s Brochure, protocol, or informed 
consent documents but for which there is a clinically significant increase in the 
expected rate of occurrence. 
 Any other AE or safety finding, including those based on animal or epidemiologic 
data that would cause the sponsor to modify the Investigator’s Brochure, protocol, or 
informed consent documents or would prompt other action by the IRB to ensure the 
protection of human subjects. 
All SAEs will be reported to the Western Institutional Review Board (WIRB) by the sponsor 
according to the WIRB guidelines and using the WIRB Promptly Reportable Information Form. 
 
WIRB Phone: 800-562-4789, Fax: 360-252-2498 
PATH is required to report SAEs or other events that fit the following criteria within 5 working 
days of the time of becoming aware of them: 
 New or increased risk 
 Protocol deviation that harmed a subject or placed subject at risk of harm 
 Protocol deviation made without prior IRB approval to eliminate an immediate hazard to 
a subject 
 Audit, inspection, or inquiry by a federal agency 
 Written reports of federal agencies (e.g., FDA Form 483) 
 Allegation of Noncompliance or Finding of Noncompliance 
 Breach of confidentiality 
 Unresolved subject complaint 
 Suspension of premature termination by the sponsor, investigator, or institution 
 Incarceration of a subject in a research study not approved to involve prisoners 
 Adverse events or IND safety reports that require a change to the protocol or consent 
 State medical board actions 
 Unanticipated adverse device effect 
 Information where the sponsor requires prompt reporting to the IRB 

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11.9.2. 
Reporting Additional Immediately Reportable Events to the Sponsor and the 
NMRC IRB 
11.9.2.1. Pregnancy 
Each pregnancy must be reported immediately (within 72 hours of identification) by e-mail or 
fax to the sponsor and the NMRC IRB. 
Subjects who become pregnant after receiving the first dose of vaccine or up to 3 months after 
the last vaccination will be followed to term, and information will be gathered for outcome, date 
of delivery, and health status of the mother and child including the child’s gender, length, and 
weight. Complications and/or abnormalities should be reported including any premature 
terminations. A pregnancy is reported as an AE or SAE only when there is suspicion that the 
investigational product may have interfered with the effectiveness of contraception or there was 
a serious complication in the pregnancy including a spontaneous abortion or an elective 
termination for medical rationale. Principal investigator will notify study monitor, the local IRB, 
the research monitor and the sponsor for subjects that become pregnant during the study. The 
subject will be asked to provide serial follow-ups on the status of her pregnancy as well as 
provide health information on her infant following delivery. The subject will be asked to provide 
copies of clinic visits only if needed. The Subjects will receive no further vaccinations but will 
continue safety monitoring and all follow up visits as noted in the study schedule. 
11.9.2.2. Adverse Event-related Withdrawal of Consent 
Any AE-related withdrawal of consent due to a safety concern during the study must be reported 
immediately (within 72 hours of identification) by e-mail or fax to the sponsor and the NMRC 
IRB. 
11.9.2.3. Pending Inspections/Issuance of Reports  
The knowledge of any pending compliance inspection/visit by the FDA, Office for Human 
Research Protections (Department of Health and Human Services), or other government agency 
concerning clinical investigation or research, the issuance of Inspection Reports, Form FDA 483, 
warning letters, or actions taken by any regulatory agency including legal or medical actions, and 
any instances of serious or continuing noncompliance with the regulations or requirements will 
be reported immediately to the NMRC IRB and the sponsor. 
11.9.3. 
IND Annual Report to the FDA 
The PI will be responsible for the preparation of a detailed annual synopsis of clinical activity, 
including AEs, for submission to the sponsor. Each annual report will summarize IND activity 
for 1 year beginning approximately 3 months before the IND FDA anniversary date. The sponsor 
will notify the PI of the due date with sufficient time for the PI to assemble the required 
information. 
11.9.4. 
Final Report 
A final study report will be prepared in accordance with Guidance for Industry: Submission of 
Abbreviated Reports and Synopses in Support of Marketing Applications and ICH E3 Guideline 

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Structure and Content of Clinical Study Reports and provided to the sponsor for review and 
approval. The sponsor will use this report to prepare the submission to the FDA. 
12. 
Statistics 
Detailed statistical procedures, listings, table shells, and figures will be provided in a separate 
statistical analysis plan (SAP) written shortly after protocol approval but before any subject 
enrollment. The SAP will be finalized before study closeout and database lock. The following 
key statistical components will be considered, and a detailed description will be documented in 
the SAP: 
 Primary and secondary endpoints and how they will be measured, 
 Statistical methods and tests that will be used to analyze the endpoints, 
 Strategy that will be used if the statistical test assumptions are not satisfied, 
 Indication of whether the comparisons will be one-tailed or two-tailed (with 
justification of the choice) and the level of significance to be used, 
 Identification of whether any adjustments to the significance level or the overall 
 p value will be made to account for any planned or unplanned subgroup analyses or 
multiple testing, 
 Specification of potential adjusted analyses and a statement of which covariates or 
factors will be included, 
 Planned exploratory analyses and justification with their importance, and 
 Any subgroup effects with biological justification and support from within and 
outside the study. 
12.1. 
Description of Statistical Methods 
12.1.1. 
Analysis Addressing the Primary Study Objective (Safety Analysis) 
The primary objective of this study is safety of the InvaplexAR-DETOX vaccination. All immunized 
subjects will be included in the safety analysis. AE data will be listed individually and 
summarized by system organ class and preferred terms within a system organ class for each 
treatment group. Serious and/or unexpected AEs will also be discussed on a case-by-case basis. 
For the tabulation of AEs by system organ class, a subject will be counted only once in a given 
system organ class. For example, a subject reporting nausea and diarrhea will be reported as one 
subject, but the symptoms will be listed as two separate AEs within the class. Therefore, the total 
number of AEs reported within a system organ class may exceed the number of subjects within 
the system organ class reporting AEs. 
Rates of all adverse events will be analyzed by Pearson’s Chi-square test (or Fisher’s exact test if 
assumptions are not met for Pearson’s Chi-square), to compare groups, if applicable. Summary 
tables will be created to indicate the number of subjects who experienced events. Vaccine-related 
events (definitely, probably, or possibly related) will be tabulated by study group. In addition, 
tables will be prepared to list each adverse event, the number of subjects in each treatment group 

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who experienced an event at least once, and the rate of subjects with adverse event(s). Adverse 
events will be divided into defined severity grades (mild, moderate, severe, potentially life-
threatening). The tables will also divide the adverse events by relationship to the investigational 
product if applicable. All immunized subjects will be included in the safety analysis. 
Clinical and laboratory data regarding safety, which include vital signs and safety laboratory 
measures, will be included in the primary analysis. Changes in pulse rate, systolic and diastolic 
blood pressure, weight, and respiratory rate will be compared within each group and among 
groups using analysis of variance procedures. For hematology and serum chemistry tests, the 
mean, mean change, median, median change, and range of all values for each test for each 
treatment group at baseline and for the final “on therapy” values will be tabulated. The tables 
will also divide the adverse events into those considered at least possibly related to the vaccine 
and those considered not related. 
If serious adverse events occur, they will be listed separately delineating outcome, treatment, 
concomitant medication, relevant medical history, and laboratory measurements. 
12.1.2. 
Analysis Addressing the Secondary Study Objective 
For immunology responses, both qualitative (responder rates) and quantitative (e.g., log10 
transformed values) results will be analyzed. Geometric mean titers will be calculated for 
serological responses along with their 95% confidence intervals (or standard deviation). If 
appropriate, between groups comparisons will be examined with nonparametric median tests 
(Kruskal-Wallis for continuous data and Fisher’s exact test for categorical data) unless 
assumptions are fulfilled for Student’s t or χ2. 
Additional comparisons may be made using repeated measures analysis of variance with study 
group as the between subject factor and sample collection time-points as the repeated factor. 
Only subjects who receive at least 2 vaccine doses will be included in the immunology analysis. 
All statistical tests will be interpreted in a two-tailed fashion using an α=0.05 to represent 
statistical significance. 
12.1.3. 
Clinical Laboratory Data Analyses 
Hematology and serum chemistry test values will be analyzed and evaluated. These evaluations 
will be reviewed by the PI or research monitor to evaluate whether any significant trends in 
laboratory values occurred.  
12.2. 
Planned Enrollment and Reason for Sample Size 
A total of 60 subjects are planned for this study. The sample size for this study is limited by the 
early stage (Phase 1) of the product concept/testing and is designed to evaluate preliminary 
safety data but not designed to show statistically significant differences between groups. Given 
the small number of subjects per group, the precision of our estimate for AEs is limited. For 
example, using binomial probability formulae for no observed adverse events within the 16 
subjects yields a 95% confidence interval of 0-21%. Follow-on studies evaluating seemingly safe 
and immunogenic doses will be required with larger numbers of volunteers to better define the 
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12.3. 
Accounting for Missing, Unused, and Spurious Data 
Non-analyzable data will be documented in the deviations. 
12.4. 
Procedures for Reporting Deviations from the Original Statistical Plan 
Any deviation(s) from the original statistical plan as indicated in the protocol will be described in 
an amendment to the protocol and the SAP. Deviations from the SAP will be documented in 
accordance with NMRC SOPs. 
12.5. 
Selection of Subjects to Be Included in Analyses 
All subjects who receive at least one dose of the investigational product will be included in the 
safety analyses.  All subjects who receive at least two doses of the investigational product will be 
included in the immunological analyses. 
13. 
Direct Access to Source Data/Documents 
Subjects will be identified on eCRFs by a unique subject identification number and on source 
documents by name and date of birth. No personal identifier will be used in any publication or 
communication used to support this research study. The subject identification number will be 
used if it becomes necessary to identify data specific to a single subject. Representatives of the 
sponsor, the local IRB, and the FDA are eligible to review medical and research records related 
to this study as a part of their responsibility to protect human subjects in clinical research. 
Personal identifiers will be removed from photocopied medical and research records. 
13.1. 
Study Monitoring 
Study monitoring will be the responsibility of the clinical monitoring team. Upon successful 
approval of the protocol and establishment of the regulatory file, the clinical monitor will 
establish a clinical monitoring plan. To ensure that the investigator and the study staff understand 
and accept their defined responsibilities, the clinical monitor will maintain regular 
correspondence with the site and may be present during the course of the study to verify the 
acceptability of the facilities, compliance with the investigational plan and relevant regulations, 
and the maintenance of complete records. The clinical monitors may observe study procedures as 
needed to ensure subject safety and rights are protected and verify compliance with the protocol. 
Monitoring visits will be scheduled to take place at the initiation of the study, during the study at 
appropriate intervals, and after the last subject has completed the study. A report of monitoring 
observations will be provided to the PI (for corrective actions), the sponsor, and the product 
manager. 
13.2. 
Audits and Inspections 
Authorized representatives of the sponsor, the FDA, the Independent Ethics Committee or IRB, 
and the Department of the Navy Human Research Protections Program may visit the site to 
perform audits or inspections, including source data verification. The purpose of the audit or 
inspection is to systematically and independently examine all study-related activities and 
documents to determine whether these activities were conducted and data were recorded, 

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analyzed, and accurately reported according to the protocol, ICH GCP, and any applicable 
regulatory requirements. 
The investigator should contact the sponsor and the NMRC IRB immediately (within 24 hours) if 
contacted by a regulatory agency about an inspection. 
13.3. 
Institutional Review Boards 
The PI must obtain IRB approval for the study. Initial IRB approval and all materials approved 
by the IRB for this study, including the subject consent form and recruitment materials, must be 
maintained by the investigator and made available for inspection. 
The PI will be responsible for preparing and submitting continuing review reports per institution 
and IRB policies. The PI or a designee will submit the approved continuing review reports and 
the local IRB approval notifications to HRPO (if applicable) as soon as the documents are 
available. 
WIRB will provide sponsor level review for PATH Vaccine Solutions (PVS) for this study. PVS 
will submit the AE/SAE to WIRB only if it is considered unanticipated and related to study 
product(s) according to the WIRB defined procedures. 
14. 
Quality Control and Quality Assurance 
During the study, the investigator will maintain complete and accurate documentation for the 
study, including records detailing the progress of the study for each subject; laboratory reports; 
eCRFs; a signed informed consent form for each study subject; drug disposition records; 
correspondence with the IRB, the study monitor and the sponsor; AE reports; and information 
regarding subject discontinuation and completion of the study. 
All required study data will be recorded clearly and accurately by authorized study personnel in 
the eCRFs. Only designated study-site personnel shall record or change data in the eCRFs. 
During the study, the investigator will be responsible for ensuring the quality of data recorded in 
the eCRFs as per written eCRF completion guidelines. 
To ensure compliance with GCP and all applicable regulatory requirements, the sponsor may 
conduct quality assurance audits.  
Auditing of the clinical trial may be conducted at any time during the study to ensure continued 
compliance with regulations, policies, and procedures. Auditing will be undertaken, as needed, 
by independent personnel designated by the sponsor. Audit findings will be documented in a 
formal audit report that will detail the conduct of the audit and summarize the observations 
noted. 
15. 
Ethics 
15.1. 
Ethics Review 
The study is based on adequately performed laboratory and animal experimentation and will be 
conducted under a protocol reviewed by the NMRC IRB. The study is to be conducted by 
scientifically and medically qualified persons. The rights and welfare of the subjects will be 

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respected, the physicians conducting the study will ensure that the hazards do not outweigh the 
potential benefits, the results to be reported will be accurate, subjects will give their informed 
consent and will be competent to do so and not under duress, and all study staff will comply with 
the ethical principles in 21 CFR Part 50 and the Belmont Principles. 
15.1.1. 
Review/Approval of Study Protocol 
Before a clinical study can be initiated, the study protocol and other required documents will be 
submitted to the following for review and/or approval. Additionally, the protocol may be 
presented to the USAMRMC Commanding General prior to study initiation. The timing of that 
presentation will vary depending on other extraneous factors. 
 NMRC Scientific Review Board 
 PATH 
 NMRC IRB 
 Commander, WRAIR 
 FDA 
 WIRB (sponsor level review) 
Enrollment in this protocol may not begin until all approvals have been obtained and the sponsor 
has authorized study start. 
The investigator will not take actions that require protocol amendment for any reason without 
obtaining appropriate review/approval, except in cases of medical emergencies.  
15.1.2. 
Protocol Modifications 
All modifications to the protocol and supporting documents (informed consent, MOP, SOPs, 
recruitment materials, etc.) must be reviewed and approved prior to implementation. Any 
protocol amendment will be agreed upon by the sponsor prior to submission to the local IRB and 
prior to implementation of said change or modification. The informed consent document must be 
revised to concur with any amendment as appropriate and must also be reviewed and approved 
with the amendment. Any subject already enrolled in the study will be informed about the 
revision and asked to sign the revised informed consent document if the modification directly 
affects the individual’s participation in the study. A copy of the revised, signed, and dated 
informed consent document will be given to the subject. All original versions of the informed 
consent document will be retained in the protocol regulatory file, and a copy will be retained in 
the clinic medical record. 
Any modification that could potentially increase risk to subjects must be submitted to the NMRC 
IRB, WIRB, and the FDA for approval prior to implementation. Documentation that the local 
IRB reviewed and approved the modifications also will be submitted. All other amendments will 
also be submitted to the NMRC IRB for inclusion in the HRPP study file. 
15.1.3. 
Protocol Deviation Procedures 
All subject-specific deviations from the protocol (e.g., failure to return for follow-up visits or 
blood collection within the time indicated in the protocol) are to be documented. The PI or 

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designee will be responsible for identifying and reporting all deviations, which are defined as 
isolated occurrences involving a procedure that did not follow the study protocol or MOP. 
Deviations will be reported annually in the continuing review report to the IRB and HRPO, if 
appropriate. The PI or subinvestigator will assess action taken in response to the deviation and 
the impact of the deviation. 
Any protocol deviation that adversely affects the safety or rights of a subject or the scientific 
integrity of the study will be reported immediately to the sponsor and the NMRC IRB. 
15.2. 
Ethical Conduct of the Study 
This study will be conducted in accordance with all applicable federal and DoD human research 
protections requirements and the Belmont Principles of respect for persons, beneficence, and 
justice. 
The procedures set out in this study are designed to ensure that the sponsor and all study 
personnel abide by the principles of the ICH GCP Guideline and the CFR. The PI confirms this 
by signing this study protocol and Form FDA 1572. 
15.2.1. 
Confidentiality 
In this research, the subject’s health information will be collected and used to conduct the study; 
monitor the subject’s health status; measure effects of the investigational product; determine 
research results; and possibly to develop new tests, procedures, and commercial products. Health 
information is used to report results of research to the sponsor and federal regulators and may be 
reviewed during study audits for compliance with study plans, regulations, and research policies. 
After the study ends, each subject has the right to see and receive a copy of his or her 
information. 
The sponsor, the DoD, and the FDA are eligible to photocopy and review records related to this 
protocol as a part of their responsibility to protect the participants of this treatment protocol. No 
personal identifier will be used in any publication or communication used to support this 
research study. The subject’s identification number will be used in the event it becomes 
necessary to identify data specific to a single subject. 
All study procedures will be conducted per GCP guidelines, and every effort will be made to 
protect volunteer privacy and confidentiality to the extent possible. 
All study-related information will be stored securely at the study site or at a designated, secure 
off-site location. While not in use and under immediate control of study staff, all volunteer 
information will be stored in locked areas with access limited to study staff. Data collection, 
process and administrative forms, laboratory specimens, and other reports will be identified 
exclusively by a coded number to maintain volunteer confidentiality. All local databases will be 
secured with password-protected access systems. Volunteers’ study information will not be 
released without their written permission, except as necessary for monitoring or compliance with 
legal or regulatory requirements. Additionally, the study team does not foresee any commercial 
value for the samples collected in this study. 
Medical records containing identifying information may be made available for review when the 
sponsor or an authorized regulatory agency monitors the study. Direct access may include 

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examining, analyzing, verifying, and reproducing any records and reports that are important to 
the evaluation of the study. 
15.2.2. 
Compensation for Participation 
Compensation will occur at the time of each designated visit. Compensation will be provided 
only for completed study procedures designated for compensatory payment. Subjects will only 
be eligible for compensation outlined in the informed consent documents at the time their 
consent is obtained. If a subject is eligible to participate in the investigation protocol after 
screening, he or she will receive the following compensation: 
Civilian/off-duty military compensation: 
 Screening: $25 
 Day -7: $100 
 Day 1: $150 
 Day 2: $100 
 Day 8: $100 + $20 (if subjects return and complete symptom diary) 
 Day 22: $150 
 Day 23: $100 
 Day 29: $100 + $20 (if subjects return and complete symptom diary) 
 Day 43: $150 
 Day 44:$100 
 Day 50: $100 +$20 (if subjects return and complete symptom diary) 
 Day 57: $100 
 Day 71: $160 
Total: $1,495 (maximum if subjects return all symptom diaries) 
If subjects do not complete the study, their compensation will be less in proportion to the amount 
they did not complete. 
Visits that are not specifically planned in the protocol, such as may be required to repeat labs to 
verify/clarify results or labs drawn to better evaluate abnormal lab values or adverse events may 
be compensated at the discretion of the PI relative to the severity of the event and scope of 
evaluation. 
Subjects asked to come to the clinic as an alternate on the first vaccination day of a cohort may 
be compensated $50 if they are not enrolled in the cohort. 
Federal Employee/on-duty military compensation: 
By regulation, active duty personnel and federal employees can be compensated only for visits in 
which blood draws occur, and then only $50 per visit, unless the visits occur during off-duty 
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the same as non-military/non-federal personnel. The total amount of compensation may vary 
depending on the number of visits completed. 
 Screening: $25 
 Day -7: $50 
 Day 1: $50 
 Day 2: $50 
 Day 8: $50 
 Day 22: $50 
 Day 23: $0 
 Day 29: $50 
 Day 43: $50 
 Day 44: $0 
 Day 50: $50 
 Day 57: $50 
 Day 71: $50 
Total: $525 
15.2.3. 
Redress of Research-Related Injury 
All study-related medical care will be provided without cost. Should a subject be injured as a 
direct result of participating in this research project, he or she will be provided medical care by 
the staff at the Walter Reed National Military Medical Center (or other military-affiliated 
medical center), at no cost to the subject, for that injury. Subjects will not receive any injury or 
disability compensation, only medical care. Subjects will not be compensated for care if they 
choose to seek care from their own physicians. 
All nonexempt research involving human subjects shall, at a minimum, meet the requirement of 
32 CFR 219.116(a)(6). 
If a subject is injured because of participation in this research and is a DoD health care 
beneficiary (e.g., active duty in the military, military spouse or dependent), the subject is entitled 
to medical care for that injury within the DoD health care system as long as the subject remains a 
DoD health care beneficiary. This care includes, but is not limited to, free medical care at Army 
hospitals or clinics. 
If a subject is injured because of participation in this research and is not a DoD health care 
beneficiary, the subject is entitled to free medical care for that injury at an Army hospital or 
clinic. It cannot be determined in advance which Army hospital or clinic will provide care. If the 
subject receives care for research-related injuries outside of an Army hospital or clinic, the 
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For all subjects: Transportation to and from Army hospitals or clinics will not be provided. No 
reimbursement is available if the subject incurs long-term medical expenses to treat research-
related injuries. No compensation is available for research-related injuries or related disability. 
The subject is not waiving any legal rights. The subject should contact the PI if the subject 
believes he or she has sustained a research-related injury. The subject should contact the PI for 
any questions. 
Requests for other benefits, such as compensation for lost time from work, are processed 
independently of this protocol. Military members retain the right to pursue military disability 
benefits, and federal civilian employees retain the right to pursue relief through established 
workers’ compensation processes, but neither military disability benefits nor workers’ 
compensation benefits are guaranteed. The right of other parties to seek redress against the US 
Government is limited to that set forth by existing agency regulations and the Federal Tort 
Claims Act. The subject should understand that this does not constitute a waiver or release of 
legal rights. This issue is addressed in the informed consent and will be discussed with the 
subject by the investigator or designee before the subject signs the informed consent to 
participate in the study. 
15.3. 
Written Informed Consent 
The informed consent process and document(s) will be reviewed and approved by the IRB and 
sponsor prior to initiation of the study. The consent document(s) contains a full explanation of 
the possible risks, advantages, and alternate treatment options, and availability of treatment in the 
case of injury, in accordance with 21 CFR 50. The consent document indicates that by signature, 
the subject, or where appropriate, legal guardian, permits access to relevant research records by 
the sponsor and by representatives of the FDA. The sponsor will submit a copy of the initial 
IRB- and sponsor- approved consent form to the FDA and will maintain copies of revised 
consent documents that have been reviewed and approved by the IRB/ethics committee. 
A written informed consent document, in compliance with 21 CFR Part 50, 32 CFR Part 219 and 
the Belmont Principles will be signed by the subject before any study-related procedures are 
initiated for that subject. This consent document must be retained by the investigator as part of 
the study records. The investigators or their designees will present the protocol in lay terms to 
individual subjects. Questions on the purpose of the protocol, protocol procedures, and risks to 
the subjects will then be solicited. Any question that cannot be answered will be referred to the 
PI. The subject will be allowed to take the consent document home to consider and discuss it 
with others and can return to the CTC later to sign it. The subject should understand that the 
study product is an investigational drug and is not licensed by the FDA for commercial use but is 
permitted to be used in this clinical research. Informed consent includes the principle that it is 
critical the subject be informed about the principal potential risks and benefits. This information 
will allow the subject to make a personal risk versus benefit decision and understand the 
following: 
 Participation is entirely voluntary, 
 Subjects may withdraw from participation at any time, 
 Refusal to participate involves no penalty, and 

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 The individual is free to ask any questions that will allow him or her to understand the 
nature of the protocol. 
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required 
by US law. 
Should the protocol be modified, the subject consent document must be revised to reflect the 
changes to the protocol. If a previously enrolled subject is directly affected by the change, the 
subject will receive a copy of the revised informed consent document. The approved revision 
will be read, signed, and dated by the subject. 
Military personnel may be recruited by CTC personnel. No recruitment of military personnel will 
occur in the presence of his or her supervisor. There is no benefit to military personnel for 
participating in this study. There will be no coercion or disciplinary actions for not participating 
or withdrawing if enrolled. 
16. 
Data Handling and Recordkeeping 
The primary source document for this study will be the subject’s research record. If separate 
medical records are maintained by the investigator(s), the medical record and the research 
records will be considered the source documents for the purposes of auditing the study. The 
source documents will be retained at the site. 
For this study, an EDC database system will be used for the collection of study data in an 
electronic format. The EDC database system will be designed based on the protocol 
requirements, the approved eCRF layouts and specifications, and in accordance with 21 CFR 
Part 11. The eCRF layouts and specifications define and identify the applicable source data that 
will be collected and captured into the EDC database system. The applicable source data will be 
electronically transcribed by the site designee onto the eCRF (data entry screens) in the EDC 
database system. The investigator is ultimately responsible for the accuracy of data transcribed 
on the eCRF. Data monitoring and management will be performed in the EDC database system 
by the study monitor and the designated data management group. 
A detailed data management plan will be written and approved by the study team and the PI prior 
to study start, with approval by the sponsor. All updates to the data management plan must be 
approved before study closeout and database lock. 
16.1. 
Inspection of Records 
The sponsor or designee will be allowed to conduct site visits to the investigation facilities for 
the purpose of monitoring any aspect of the study. The investigator agrees to allow the monitor 
to inspect the drug storage area, investigational product stocks, drug accountability records, 
subject charts, study source documents, and other records relative to study conduct. 
Subjects’ health information is used to report results of research to the sponsor and federal 
regulators and may be reviewed during study audits for compliance with study plans, regulations, 
and research policies. The consent document indicates that, by signature, the subject permits 
access to relevant research records by the sponsor and by representatives of the FDA. 

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The investigator must be aware that representatives from regulatory authorities or the IRB may 
wish to inspect the eCRFs and associated study records. The investigator will notify the sponsor 
within 24 hours following contact by a regulatory agency. The investigator and study coordinator 
must make the relevant records available for inspection and will be available to respond to 
reasonable requests and audit queries made by authorized representatives of regulatory agencies. 
The investigator will provide the sponsor with copies of all correspondence that may affect the 
review of the current study or his or her qualification as an investigator in clinical studies 
conducted by the sponsor. The sponsor will provide needed assistance in responding to 
regulatory audits or correspondence. 
16.2. 
Retention of Records 
The PI must maintain all documentation relating to the study for a period of 2 years after the last 
marketing application approval or, if not approved, for 2 years following the discontinuance of 
the investigational product for investigation. If it becomes necessary for the sponsor, their 
designee, or the FDA to review any documentation relating to the study, the investigator must 
permit access to such records. 
Completed, monitored records will be stored in a secure location.  
The PI will be responsible for retaining sufficient information about each subject, that is, name, 
address, telephone number, social security number, and subject identifier in the study, so that the 
sponsor, the local IRB, the FDA, employees of USAMRMC, or other regulatory authorities may 
have access to this information should the need arise. 
It is the policy of USAMRMC that data sheets are to be completed for all subjects participating 
in research (Form 60-R, Volunteer Registry Data Sheet). The data sheets will be entered into this 
Command’s Volunteer Registry Database. The information to be entered into this confidential 
database includes the subject’s name, address, and social security number; study title; and dates 
of participation. The intent of this database is twofold: first, to readily answer questions 
concerning an individual’s participation in research sponsored by USAMRMC; and second, to 
ensure that USAMRMC can exercise its obligation to ensure research subjects are adequately 
warned (duty to warn) of risks and provided new information as it becomes available. The 
information will be stored at USAMRMC for a minimum of 75 years. The Volunteer Registry 
Database is a separate entity and is not linked to the study database. 
17. 
Publication Policy 
All data collected during this study will be used to support this IND. All publications and 
presentations are governed by the standards and norms detailed in NAVMEDRSCHCENINST 
5721.1. All authors will submit the proposed publication/presentation at least 30 days prior to the 
submission date. Prior to submission, the directorate will conduct a substantive scientific and 
professional review. The document is routed to the Office of Research Administration (ORA) for 
review and routing for Command review and approval, ultimately by the NMRC Public Affairs 
Officer (PAO). Once it is cleared at NMRC, it may be forwarded to BUMED through NMSC, if 
appropriate. Prior to publication, an author must have a completed Publication Clearance Request 
Submission Form with signatures from all approving and reviewing authorities. 

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influenza vaccine with and without Escherichia coli heat-labile toxin in adult 
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Riddle, M.S., et al., Safety and immunogenicity of an intranasal Shigella flexneri 2a 
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Gaston, J.S., How much HLA-B27 expression is needed for spondyloarthritis? J 
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Treanor, J.J., et al., Safety and immunogenicity of a recombinant hemagglutinin 
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Madhi, S.A., et al., Usefulness of C-reactive protein to define pneumococcal 
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Korczowski, B., Procalcitonin and C-reactive protein in vaccination-associated 
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Madhi, S.A., et al., Use of procalcitonin and C-reactive protein to evaluate vaccine 
efficacy against pneumonia. PLoS Med, 2005. 2(2): p. e38.