Clinical trial NCT02154061
Conditions: Influenza
Keywords: Flu Vaccine, Intestinal Microbiome, Antibiotics
Sponsor: Emory University
Start date: 2015-04-01; Completion date: 2017-05-26
Enrollment: 33 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of the study is to better understand how the use of antibiotics changes micro-organisms in the intestines. The change to micro-organisms in the intestines may change the body's vaccine immune response and alter the effectiveness of the IIV (seasonal flu) vaccine.

In particular, the investigators will be looking at certain markers in blood after vaccination with IIV (seasonal flu vaccine) with or without the use of antibiotics before vaccination.

There will be two randomized groups. Group A will start taking antibiotic by mouth 3 days prior to vaccination and continue taking antibiotics the day of vaccination and one day after vaccination for a total of 5 days. Group B will only receive the IIV (seasonal flu vaccine) and will not take any antibiotics.

The investigators hope to enroll 50 subjects at Emory.

Detailed description:
We will study using a system biology approach the immune responses to IIV alone vs. IIV with antibiotic administration

Arms: arm 1: This arm will receive antibiotics prior and after IIV administration. arm 2: This arm will not take antibiotics in conjunction with IIV.
Interventions: intervention 1: IIV Flu Vaccine intervention 2: Metronidazole intervention 3: Neomycin intervention 4: Vancomycin
Intervention descriptions: intervention 1: This is an FDA approved and tested Inactivated Flu Vaccine. intervention 2: This is a standard antibiotic intervention 3: This is a standard antibiotic. intervention 4: This is a standard antibiotic.

Protocol documents:
Prot_SAP_000.pdf: 
VAX-002 
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Systems Biology of Inactivated Influenza Vaccine (IIV) in healthy 
adults with or without use of broad spectrum antibiotics. 
Principal Investigator: Nadine Rouphael  
 
IRB00074239 
Protocol Number 
VAX-002 
VERSION 7.0 
5.13.2016 
 
 
 
 


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PRINCIPAL INVESTIGATOR 
Nadine Rouphael, MD 
Hope Clinic, 500 Irvin Court, Suite 200 
Decatur, GA 30030 
Office: 404-712-1435 
Cell : 404-822-1411 
Pager : 404-686-5500 (PIC 16509) 
Fax : 404-499-9727 
Email:nroupha@emory.edu 
 
 
CO-INVESTIGATOR(S) 
 
Mark J. Mulligan, MD  
Hope Clinic, 500 Irvin Court, Suite 200  
Decatur, GA 30030 
Office:404-712-9046 
Pager : 404 686 5500 (PIC 13632) 
Fax : 404-499-9727 
Email: mmulli2@emory.edu 
 
Aneesh Mehta, MD  
2101 Woodruff Memorial Research Building, 101 Woodruff 
Circle, , Atlanta, GA 30322 
Office: 404-727-8435 
Lab: 404-727-5854 
Cell: 678-428-6728 
Pager: 404-686-5500 (PIC 16927) 
Fax: 404-712-2278 
Email aneesh.mehta@emory.edu 
 
Srilatha Edugupanti, MD, MPH 
Hope Clinic, 500 Irvin Court, Suite 200  
Decatur, GA 30030 
Office: 404-712-1434 
Pager : 404 686 5500 (PIC 13721) 
Fax : 404-499-9727 
Email: sedupug@emory.edu 
 
Colleen Kelley, MD 
Hope Clinic, 500 Irvin Court, Suite 200  
Decatur, GA 30030 
Office: 404-712-1370 
Pager : 404 686 5500 (PIC 15205) 
Fax : 404-499-9727 
Email: colleen.kelley@emory.edu 
 
Sarah Kabbani, MD 
Hope  Clinic, 500 Irvin Court, Suite 200  Decatur, GA 30030 
Office: 404-712-1370 
Fax: 404-499-9727 
Email: sarah.kabbani@emory.edu 
 
Allison Beck, PA 
Hope Clinic, 500 Irvin Court, Suite 200  
Decatur, GA 30030 
Office: 404-712-1408 
Fax: 404-499-9727 
Email: aabeck@emory.edu 
 


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BIOSTATISTICIAN 
Tianwei Yu, PhD 
Department of Biostatistics and 
Bioinformatics 
Rollins School of Public Health 
1518 Clifton Rd., N.E. 
Atlanta, GA 30322 
Office: 404-727-7671 
Fax: 404-727-1370 
Email: tyu8@emory.edu 
INDEPENDENT  SAFETY MONITOR 
Primary ISM 
David Rimland, MD 
VA Medical Center 
1670 Clairmont Rd, Decatur, GA 30033 
Phone: 404-321-6111 ext. 6165 
 
Blackberry: 404-771-8601 
Fax: 404-728-7782 
Email: david.rimland@va.gov 
 
Back up ISM 
Bruce Ribner, MD, MPH 
Emory University Hospital 
1364 Clifton Road, NE Suite B-705, 
Atlanta, GA 30322    
 
Phone: 404-727-1580 
Pager : 404 686 5500 (PIC 15326) 
Fax:404-712-4361 
Email: bribner@emory.edu 
 
  
Confidentiality Statement 
The information contained within this document is not to be disclosed in any way without the prior 
permission of the Principal Investigator  
 
 
 
 
 
 
 
 
 
 
 
 


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INVESTIGATOR SIGNATURE PAGE 
Protocol 
Systems Biology of Inactivated Influenza vaccine 
(IIV) in healthy adults with or without use of 
broad spectrum antibiotics. 
Version/Date: 
 7.0/05/13/2016 
 
Principal Investigator: 
Nadine Rouphael, MD 
Short Title:   Responses to IIV in adults with or without antibiotics. 
INSTRUCTIONS:  The Principal Investigator will print, sign, and date at the indicated location 
below. A copy should be kept in the investigator’s records. 
I confirm that I have read the above protocol in the latest version. I understand it, and I will work 
according to principles of Good Clinical Practice (GCP) as described in the United States Code of 
Federal Regulations (CFR) 45 CFR part 46 and 21 CFR parts 50, 56, and 312 , and the  
International Conference on Harmonization (ICH) document “Guidance for Industry:  E6 Good 
Clinical Practice:  Consolidated Guidance” dated April 19961  and the spirit of the Declaration of 
Helsinki,  Further, I will conduct the study in keeping with local, legal, and regulatory 
requirements.  
 
As the Principal Investigator, I agree to conduct VAX-002:   Systems Biology of  Inactivated  Influenza 
vaccine (IIV) in healthy adults with or without use of broad spectrum antibiotics. I agree to carry out the 
study by the criteria written in the protocol and understand that no changes can be made to this 
protocol without written permission by the local IRB. 
 
 
 
___NADINE ROUPHAEL, MD_______________ 
Principal Investigator (Print) 
 
 
 
___________________________________________                                             ____________ 
Principal Investigator (Signature)                                                                              Date 
 
 
 
 
 


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Synopsis 
Title 
Systems Biology of Inactivated Influenza Vaccine (IIV) in healthy 
adults with or without use of broad spectrum antibiotics 
Short Title 
Responses to IIV in adults with or without antibiotics 
Rationale 
Use of antibiotics can significantly impact the microbiota of patients. 
The status of the intestinal microbiome may impact the immune 
responses to vaccinations and alter functional protective immunity in 
humans. Here we hypothesize that the changes in the fecal 
microbiome induced by antibiotics translates into suboptimal 
Hemagglutination Inhibition Assay (HAI) titers. 
Clinical Phase 
N/A 
Mechanistic Study 
Yes 
Principal Investigator  
Nadine Rouphael, MD (PI)  
Participating Site(s) 
The Hope Clinic of the Emory Vaccine Center 
The Hope Clinic is a community-based vaccine research clinic and is 
the clinical arm of the Emory Vaccine Center at Emory University. 
Accrual Objective 
N=50 total (age 18-40) 
Primary Study Objective 
To compare HAI titers after vaccination with IIV in adults with or 
without use of antibiotics 
Study Design 
Single center, open mechanistic study in which subjects will be 
randomized to receive IIV per label at day 4 of a 5 day course of a 
specific antibiotic regimen (Group A) or IIV alone (Group B) 
Blood samples for immunologic testing will be collected at screening  
(from D -21 to D -1), on D0 (at vaccination), D1, D3, D7 (+/- 1 day), 
D30 (+/- 5 days), D90 (+/-14 days), D180 (+-/14 days), D365 (+/-28 
days) post vaccination for both groups to study innate and/or 
adaptive immune responses.  
Stool samples will be collected in both groups at screening (from D -
21 to D -1), on vaccination (D0), D1, D3, D7 (+/- 1 day) and D30 (+/- 
5 days), D90 (+/-14 days), D180 (+/-14 days), D365 (+/- 28 days) to 
study the gut microbiome in both groups. For Group A, additional 
visit on D-6 to D-3 will occur with stool and blood samples collection.   
Study will be conducted outside the 2014-2015 and 2015-2016 
influenza seasons. 


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Antibiotics received by Group A will be started 3 days prior to 
vaccination (D-3) and continued on day of vaccination (D0) and for 
one day after vaccination (D1) for a total of 5 days 
 
Flagyl® 500 mg po tid  
 
Vancocin® 125 mg po qid 
 
Neomycin sulfate ® 500mg po tid 
The dosage of each antibiotic is taken from their respective package 
inserts and does not exceed the maximum dose allowed for each 
antibiotic.  
The antibiotic regimen is a broad spectrum regimen covering all types 
of fecal flora (anaerobes, gram positive, and gram negative bacteria); 
has a good safety record; has poor systemic absorption for part of the 
regimen (Vancocin® and Neomycin sulfate® ); and part of the 
regimen is used to treat Clostridium difficile infections (Vancocin® and 
Flagyl®).  
Subjects in Group A are asked to avoid all ethanol and any ethanol-
containing drugs while taking antibiotics and for 48h before and after 
taking the antibiotics. 
Blood samples for safety laboratory testing (including CBC with 
differential, creatinine, potassium) will be collected at screening (from 
D-21 to D -1) and D 7 (+/- 1 day) for both groups.  
For Group A, stools will be screened for Clostridium difficile carriage 
by PCR at (from D-21 to D-1).  
Study Duration 
Approximately 12 months 
Primary Endpoints 
Comparison of HAI titers at D30 post vaccination in both groups 
Secondary Endpoints 
 
Frequency, severity, and causality of all adverse events in Group A 
subjects. 
Exploratory Endpoints 
 
Analyses of microbiome at D0, D1, D3, D7,  D30, D90, 
D180, D365 and comparison to baseline screening in 
both groups 
 
Analysis of the repertoire and monoclonal antibodies from 
plasmablasts in a subset of vaccinees at D7 in both groups 
 Identification of innate immune signatures (traditional 
immune parameters measurements + array-based gene 
expression) at D1, D3 and D7 in both groups 
 
Correlation of innate immune signatures at days D1, D3 and D7 
with HAI titers at D30 in both groups 
 
Comparison of HAI titers at D90, D180 and D365 post vaccination 
in both groups. 


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Inclusion Criteria 
1. Healthy individuals aged 18-40 years 
2. Able to understand and give informed consent 
3. Women of child-bearing potential (not surgically sterile via tubal 
ligation, bilateral oophorectomy or hysterectomy or who are not 
postmenopausal for ≥1 year) must agree to practice adequate 
contraception that may include, but is not limited to, abstinence, 
monogamous relationship with vasectomized partner, barrier 
methods such as condoms, diaphragms, spermicides, intrauterine 
devices, and licensed hormonal methods* for 30 days before and 30 
days after IIV vaccination. 
* Women of child-bearing potential using licensed hormonal methods 
must also use a second form of contraception. 
4. Men must agree not get a sexual partner pregnant for 1 month 
after enrollment. 
 
Exclusion Criteria 
1. Receipt of the following:  
 
Receipt of blood products 3 months prior to vaccination or 
expected receipt through 6 months after vaccination. 
 
Receipt of any live virus vaccines within 4 weeks prior to 
vaccination or expected receipt within 4 weeks after 
vaccination.* 
 
Receipt of any inactivated vaccine within 2 weeks or expected 
receipt within 2 weeks after vaccination.* 
 
Receipt of any influenza vaccine in the past 3 seasons.  
 
Receipt of any systemic antibiotic 6 months prior to vaccination 
or expected receipt 1 month after vaccination. 
 
Receipt of probiotics and prebiotics 3 months prior to 
vaccination or expected receipt 1 month after vaccination. 
 
Regular receipt of proton pump inhibitors, H2 receptor blockers, 
or antacids 3 months prior to vaccination or expected receipt 1 
month after vaccination. 
2. Documented influenza infection during the 2014-2015 or 2015-
2016 influenza season. However, volunteers with prior upper 
respiratory infections during the 2014-2015 or 2015-2016 
influenza illness will not be excluded from the study. 
3.  Presence of co-morbidities or immunosuppressive states such as:  
 
Chronic medical problems including (but not limited to) insulin 
dependent diabetes, severe heart disease (including 
arrhythmias), severe lung disease, auto immune diseases, and 
grade 4 hypertension**. 


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 
Chronic neurologic conditions including seizure disorder, 
Parkinson’s disease, myasthenia gravis, neuropathy, or history 
of encephalopathy, meningitis or ototoxicity.  
 
Any history of gastrointestinal disease including (but not only): 
documented bacterial gastroenteritis or gastroenteritis 
associated with fever or associated with presence of 
blood/mucus in stools in the last 3 months; inflammatory bowel 
disease, and/or gastrointestinal surgery. 
 
Any history of kidney or liver diseases. 
 
Alcohol abuse, drug abuse, or psychiatric conditions that in the 
opinion of the investigator would preclude compliance with the 
trial or interpretation of safety or endpoint data. 
 
Impaired immune function or known chronic infections 
including, but not limited to, known HIV,  hepatitis B or C; 
organ transplantation; immunosuppression due to cancer; 
current and/or expected receipt of chemotherapy, radiation 
therapy, steroids* (i.e., more than 20 mg of prednisone given 
daily or on alternative days for 2 weeks or more in the past 90 
days , or high dose inhaled corticosteroids***); and any other 
immunosuppressive therapies (including anti-TNF therapy),  
functional or anatomic asplenia, or congenital 
immunodeficiency. 
 
Pregnancy or breast feeding 
4.  Conditions that could affect the safety of the volunteers, such as: 
 
Severe reactions to prior vaccination with IIV, including 
anaphylaxis 
 
History of Guillain Barré syndrome 
 
History of bleeding disorders or current use of warfarin 
 
Use of anticonvulsants 
 
Use of digoxin or other forms of digitalis 
 
Any allergy to any component of the vaccine including egg 
allergy  
 
Allergy to vancomycin, metronidazole or neomycin as well as 
other aminoglycosides (gentamicin, tobramycin, amikacin, 
streptomycin) 
5. Volunteers with any acute illness, including any fever (> 100.4 F 
[> 38.0C], regardless of the route) within 3 days prior to vaccination 
*.   
6. Social, occupational, or any other condition that in the opinion of 
the investigator might interfere with compliance with the study and 
vaccine evaluation. 
7. Positive C difficile testing by PCR at screening or history of C 
difficile infection. 
8. Any grade 2 safety lab test results at screening. 
 


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Note:  
*An individual who initially is excluded from study participation 
based on one or more of the time-limited exclusion criteria (e.g., 
acute illness, receipt or expected receipt of live or inactivated 
vaccines ) may be reconsidered for enrollment once the condition 
has resolved as long as the subject continues to meet all other entry 
criteria.  
Subjects receiving > 20 mg/day of prednisone or its equivalent daily 
or on alternate days for more than 2 weeks may enter the study after 
therapy has been discontinued for more than 3 months. 
** Grade 4 hypertension per CTCAE criteria is defined as Life-
threatening consequences (e.g., malignant hypertension, transient or 
permanent neurologic deficit, hypertensive. 
*** Subjects are excluded if on high dose intranasal steroids defined 
as > 960 mcg/day of beclomethasone dipropionate or equivalent. 
 
Investigational 
Product(s)/ Intervention(s)  
Licensed Inactivated Influenza vaccine (IIV): 2014-2015 and 2015-
2016 Fluzone® (Sanofi Pasteur, Swiftwater, PA, USA). 
Antibitoics:  
Flagyl ®(Pfizer, New York, NY) 
Vancocin® (ViroPharma , Exton, PA) 
Neomycin sulfate® (X-Gen Pharmaceuticals, Big flats, NY) 
Study Procedures 
Antibiotic administration, vaccination, targeted physical 
examination, urine pregnancy testing, phlebotomy, stool collection, 
signs/symptom assessment (local and systemic) and vaccination site 
evaluation. 
Statistical Considerations 
Based on previous studies performed at the Hope Clinic, the rate of 
subjects that become ineligible during the study (i.e. do not meet 
exclusion/inclusion criteria anymore, lost to follow-ups, unable to 
have blood draws) is between 5 to 10%. Assuming a 10% attrition 
rate, 50 healthy adults will be recruited for the study, resulting in an 
expected 10 subjects per arm for data analysis.  
 
 


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TABLE OF CONTENTS 
Table of Contents ...................................................................................................................... 10 
Glossary of Abbreviations ......................................................................................................... 13 
1 
Background and Rationale ............................................................................................... 15 
1.1 
Background .......................................................................................................... 15 
1.2 
Immune Response to IIV Given With Antiobiotics ................................................. 16 
1.3 
Rationale for Selection of Study Population .......................................................... 19 
1.4 
Investigational Product(s)/Intervention (s) ............................................................. 19 
1.5 
Rationale for Selection of Investigational Product(s)/Intervention(s) and 
Regimen ............................................................................................................... 19 
1.6 
Risks ..................................................................................................................... 21 
1.6.1 
Risks of Investigational Product(s)/Intervention(s) ..................................... 21 
1.6.2 
Risk of Study Procedure ............................................................................ 23 
1.6.3 
Risk of Concomitant Medications, Prophylactic Medications and Rescue 
Medications ............................................................................................... 24 
1.7 
Benefits................................................................................................................. 24 
1.7.1 
Benefits of Investigational Product(s)/Intervention(s) ................................. 24 
1.7.2 
Benefits of Study Procedure(s) .................................................................. 24 
2 
Objectives ........................................................................................................................ 25 
2.1 
Primary Objective(s) ............................................................................................. 25 
2.2 
Secondary Objectives ........................................................................................... 25 
2.3 
Exploratory Objectives .......................................................................................... 25 
3 
Study Design .................................................................................................................... 26 
3.1 
Study Endpoints .................................................................................................... 26 
3.1.1 
Primary Endpoint(s) ................................................................................... 26 
3.1.2 
Secondary Endpoint(s) .............................................................................. 26 
3.1.3 
Exploratory Endpoint(s) ............................................................................. 26 
3.2 
Study Completion ................................................................................................. 27 
4 
SeLection of Study Subjects ............................................................................................. 28 
4.1 
Subject Inclusion Criteria ...................................................................................... 28 
4.2 
Subject Exclusion Criteria ..................................................................................... 28 
4.3 
Early Study Termination ........................................................................................ 31 
5 
Investigational Product(s)/intervention material(s),  other study products 
(controls/placebos) .................................................................................................................... 32 
5.1 
Investigational Product(s)/Intervention(s) .............................................................. 32 
5.2 
Formulation, Packaging, and Labeling .................................................................. 32 
5.3 
Preparation, Administration, and Dosage .............................................................. 32 
5.4 
Accountability of Investigational Product(s)/Intervention(s) ................................... 33 
5.5 
Assessment of Compliance with Investigational Product(s)/Intervention 
Material(s) ............................................................................................................. 33 
5.6 
Modification or Discontinuation of Investigational Product(s)/Intervention 
Material(s) ............................................................................................................. 34 
5.6.1 
Modification of Investigational Product(s)/Intervention(s) ........................... 34 
5.6.2 
Premature Discontinuation of Investigational Product(s)/Intervention(s) .... 34 


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6 
Other medications ............................................................................................................ 35 
6.1 
Concomitant Medications ...................................................................................... 35 
6.2 
Prophylactic Medications ...................................................................................... 35 
6.3 
Rescue Medications.............................................................................................. 35 
6.4 
Concomitant Study Medications ............................................................................ 35 
7 
Study visits and procedures ............................................................................................. 37 
7.1 
Enrollment and Randomization ............................................................................. 37 
7.2 
Screening Visit(s) (From D-21 until D-1) ............................................................... 37 
7.3 
BASELINE VISITS ................................................................................................ 38 
7.4 
Main Study Visits .................................................................................................. 39 
7.5 
Visit Windows ....................................................................................................... 41 
7.6 
Study Procedures ................................................................................................. 41 
7.7 
Study Arm Assignment Procedures ...................................................................... 41 
7.7.1 
Blinding and Randomization ...................................................................... 41 
7.7.2 
Securing Randomization Information ......................................................... 41 
7.7.3 
Requirements for Unblinding ..................................................................... 41 
7.7.4 
Documentation of Unblinding .................................................................... 41 
8 
Safety Procedures ............................................................................................................ 42 
8.1 
Stopping Rules ..................................................................................................... 42 
8.1.1 
Study Stopping Rules ................................................................................ 42 
8.1.2 
Individual Subject Stopping Rules ............................................................. 42 
8.1.3 
EARLY TERMINATION FROM STUDY VACCINES/ PROCEDURES 
WITH CONTINUED STUDY PARTICIPATION/FOLLOW-UP .................... 43 
8.1.4 
FOLLOW-UP AFTER EARLY STUDY TERMINATION ............................. 43 
8.1.5 
SUBJECT REPLACEMENT ...................................................................... 43 
8.2 
Adverse Events ..................................................................................................... 44 
8.2.1 
Safety Reporting ....................................................................................... 44 
8.2.2 
Collecting and Recording Adverse Events and Pregnancy ........................ 47 
8.2.3 
Grading and Attribution of Adverse Events ................................................ 48 
8.2.4 
Reporting Serious Adverse Events to the Independent Safety Monitor ...... 51 
8.2.5 
Reporting Serious Adverse Events to the FDA .......................................... 51 
8.2.6 
Notifying Institutional Review Board .......................................................... 51 
8.3 
Protocol Deviations ............................................................................................... 51 
9 
Sample size calculations and statistical plan .................................................................... 53 
9.1 
Sample Size and Power Calculations ................................................................... 53 
9.1.1 
Primary and Secondary Endpoints ............................................................ 53 
9.2 
Data Analysis ........................................................................................................ 54 
9.2.1 
Analyses of Primary Endpoint ................................................................... 54 
9.2.2 
Analyses of Secondary Endpoint ............................................................... 55 
9.2.3 
Analysis of Exploratory Endpoints ............................................................. 55 
9.2.4 
Patient Populations ................................................................................... 57 
9.2.5 
Study Subject Baseline Characteristics and Demographics ....................... 57 
9.2.6 
Status of Study Subjects ........................................................................... 57 
9.3 
Interim Analyses ................................................................................................... 57 


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9.4 
Deviations from Statistical Plan ............................................................................. 57 
10 
Identification and access to source data ........................................................................... 59 
10.1 
Identifying Source Data ......................................................................................... 59 
10.2 
Updating Source Documentation .......................................................................... 59 
10.3 
Permitting Access to Source Data ......................................................................... 59 
11 
Quality Control and Quality Assurance ............................................................................. 61 
12 
Ethical considerations and compliance with good clinical practice .................................... 63 
12.1 
Statement of Compliance ...................................................................................... 63 
12.2 
Informed Consent Process ................................................................................... 63 
13 
Publications ...................................................................................................................... 64 
14 
Literature References ....................................................................................................... 65 
15 
AppendiceS ...................................................................................................................... 70 
15.1 
Appendix A: Schedule of Events ........................................................................... 70 
15.2 
Appendix B. Severity Scale for Local Vaccine Reactions (local reactogenicity 
events) .................................................................................................................. 72 
15.3 
Appendix C. Severity Scale for Generalized Vaccine Reactions (systemic 
reactogenicity events) ........................................................................................... 73 
 


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GLOSSARY OF ABBREVIATIONS 
 
 
ACIP 
Advisory Committee on Immunization Practices 
AADCRC 
Asthma and Allergic Diseases Cooperative Research Center 
AE 
Adverse Event 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
CTCAE 
Common Terminology Criteria for Adverse Events 
DAIT 
Division of Allergy, Immunology, and Transplantation 
cGCP 
Current Good Clinical Practice 
GLP 
Good Laboratory Practice 
HAI 
Hemagglutination Inhibition Assay 
ICH 
International Conference on Harmonization 
IDE 
Investigational Device Exemption 
IFNg 
Interferon gamma 
IIV 
Inactivated Influenza Vaccine 
IL 
InterLeukin 
IM 
IntraMuscular 
ISM 
Independent Safety Monitor 
IND 
Investigational New Drug 
IRB 
Institutional Review Board 
MOP 
Manual of Procedures 
NCI 
National Cancer Institute 
NIAID 
National Institute of Allergy and Infectious Diseases 
NF kB 
Nuclear Factor kappa-light-chain-enhancer of activated B cells 
NSAIDs 
NonSteroidal Anti-Inflammatory Drugs 
PI 
Principal Investigator 


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QID 
Quater In Die (four times a day) 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SOP 
Standard Operating Procedure 
Th17 
T Helper 17 Cells 
TID 
Ter In Die (three times a day) 
TLR 
Toll-Like Receptor 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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1 
BACKGROUND AND RATIONALE 
1.1 
Background  
The human commensal flora is composed of diverse and complex communities of 
microorganisms, which outnumber host cells by a factor of ten (1). They inhabit barrier surfaces 
of major organs (e.g. skin) and tissues (e.g. gastrointestinal tract) and have evolved beneficial 
properties for the host that permits a symbiotic relationship to be maintained with the host (2). 
These include an array of functions ranging from enhancing digestion and metabolism to 
occupying a niche that competes against colonization with invasive pathogens (3-7). Moreover, 
associations between alterations or dysbiosis of the microbiota and susceptibility to metabolic and 
pro-inflammatory diseases such as obesity, diabetes, and inflammatory bowel diseases in 
humans have been widely reported (8-10). Thus, interactions between the host and microbiota 
are crucial factors supporting the balance of their symbiotic relationship. 
Evidence emerging from the past decade highlights additional roles for the microbiota, most 
notably in influencing the development and homeostasis of the host immune system. Studies of 
germ-free animals, which are devoid of any detectable microorganisms, have revealed that 
commensal-derived signals are critical for maintaining a balance of pro-inflammatory and 
regulatory immune mechanisms (11-16). Germ-free mice exhibit reduced capacity of T cells to 
mount IL-17A and IFNg responses in both gut-associated tissues and the skin. Furthermore, 
these mice also exhibit increased frequency of regulatory CD4 T cells. Recent studies using 
animal model systems have also identified specific commensal species that can either promote 
pro-inflammatory or immunoregulatory pathways in the host. For example, colonization of mouse 
gut with segmented filamentous bacteria results in enhanced levels of Th17 cells in the gut (17), 
while Bacteroidetes fragilis or Clostridium subspecies results in the induction IL-10 responses and 
regulatory CD4+ T cells (18-20). Also, previous studies in mice have empirically demonstrated 
that the depletion of host microbiota – via orally administered broad-spectrum antibiotics can 
influence the development and homeostasis of the immune system (11-13, 17-19, 21-25). 
Together, these observations indicate that specific composition of microbiota is an important 
factor influencing the host immune system. Thus, it also suggests that any alterations to the gut 
microbiota can influence the capacity of the host immune system to mount or regulate responses 
against pathogens appropriately. Accordingly, several reports using mice suggest that in the 
absence of the microbiota, host susceptibility to infection is enhanced and the capacity to control 
the infection is compromised. 


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A major implication of these findings for global public health is the possibility that the microbiota 
also plays a role in immune responses to vaccines. If so, the status of the host microbiota may be 
a critical determinant of vaccine efficacy and alteration of mirobiota through antibiotic exposure 
could negatively impact vaccine efficacy. Currently, there is no published information in both 
humans and animal models whether the microbiota impacts the magnitude and/or quality of 
vaccine-induced immune responses. Examining the relationship between microbiota and vaccine 
immunogenicity is a novel concept, and understanding how such a phenomenon operates is a 
critical field to study. In this regard, the seasonal influenza vaccine would be an important area to 
focus upon given that it is one of the most widely and frequently administered vaccines worldwide. 
The goal of our study is to determine whether alteration of microbiota by antibiotic exposure can 
negatively impact the immunogenicity of IIV, and to assess the innate and adaptive immune 
mechanisms responsible for that phenomenon. 
 
 
1.2 
Immune Response to IIV Given With Antibiotics 
Recent work from our group at the Emory Vaccine Center (Pulendran Laboratory) on the systems 
biology of the influenza vaccine has revealed a network of genes centered around TLR5 (26), 
which were found to be induced robustly in humans 3 days after vaccination with IIV. Intriguingly, 
this early expression of TLR5 exhibited a striking level of correlation with the magnitude of HAI 
titers at day 28 post vaccination. This was an unexpected observation given that TLR5 is not 
known to be a sensor of viral stimuli, but rather of bacterial flagellin. We addressed whether IIV 
was capable of signaling through TLR5 by utilizing a fibroblast cell line transfected with TLR5 and 
NF-Kb reporter gene, and, indeed, we found no indication that IIV was able to do so. 
 
Thus, we subsequently determined whether TLR5 was essential for the induction of antibody 
responses to IIV vaccination. TLR5-deficient and wild-type littermate control mice were vaccinated 
with IIV and antigen specific antibody titers were measured at various time points following 
vaccination. We observed a striking reduction in the magnitude of antigen specific antibody 
response in TLR5-deficient mice. Given that we previously established that IIV itself does not 
signal through TLR5, we hypothesized that the microbiota-derived factors contribute to TLR5 
mediated augmentation of the immunogenicity of IIV. To test this hypothesis, wild-type  mice were 
administered a cocktail of broad-spectrum antibiotics (Neomycin, Ampicillin, Vancomycin, 


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Metronidazole) via their drinking water to eliminate the microbiota as previously described (12, 
13, 20, 21, 25). Antibiotics were administered for 4 weeks prior to vaccination with IIV and 
continued without cessation for periods following vaccination at which antibody responses were 
measured. As early as Day 7 post vaccination, mice treated with antibiotics exhibited greater than 
50% reduction in antigen-specific antibody titers in the serum when compared to untreated mice 
.We observed differences in antibody titers over periods exceeding 12 weeks following the 
primary vaccination. These observations were rigorously tested across 6 independent 
experiments with a total of 100 mice treated with antibiotics and 80 untreated control mice. 
Ongoing mechanistic studies indicate that boost/recall antibody responses are also reduced in 
antibiotic-treated mice. 
In addition, we conducted longitudinal microbiome sampling in both untreated and antibiotic-
treated animal groups to determine whether the impact of antibiotics on the humoral immune 
response can be attributed to compositional differences in the gut microbiota. The microbiome 
was analyzed using bacterial genomic DNA extracted from fecal samples and the 454 
pyrosequencing platform to identify specific bacterial communities. A sequence clustering 
software originally developed by Robert Knight's group was used to determine the composition of 
the microbiota as previously described (27). The details of analysis methods are highlighted in 
9.2.3.Analysis of exploratory endpoints. Our characterization of the microbiota in mice revealed 
that antibiotic treatment indeed reduced the bacterial load in the gut by a factor greater than ten 
and caused dramatic shifts in major bacterial communities of the gut. Specifically, greatest 
changes were observed in bacterial phyla, Fermicutes and Proteobacteria. The microbiota of 
untreated mice exhibited a greater presence of bacterial communities belonging to the Fermicutes 
phylum; whereas in treated mice, majority of these communities were eliminated and instead 
exhibited a predominance of species from the Proteobacteria phylum. 
This indicated to us that the effects antibiotics and the status of the microbiota around the time of 
vaccination significantly impacted the antibody response. Collectively, our studies demonstrate 
that antibiotic treatment dramatically alters the composition of the host microbiota and results in 
dramatic reduction of antigen-specific antibody titers similar to the phenotype we previously 
observed in TLR5-deficient mice. 
It is unclear how antibiotic exposure affects the human microbiota. Historical evidence on the 
effects of antibiotics in vivo is largely based on biologic assays that require culturing microbes. As 
the majority of microbial communities in the gut are anaerobes and, thus, difficult to culture, the 


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accuracy of our understanding of the microbiota remains limited (2). However, recent advances 
in pyrosequencing technologies are enabling a more detailed analysis of the human microbiome 
to be assessed (28-30). A few reports suggested that some antibiotics such as ciprofloxacin do 
alter the human microbiota, but revealed that only as little as a third of the bacterial taxa in the 
gut were affected by antibiotic treatment (31, 32). Moreover, analyses of the microbiome in these 
studies were limited to only an examination on the relative abundance of microbial communities 
and lacked quantifiable information such as overall bacterial load in the gut. Thus, given the 
relatively nascent stage of our capacity to characterize the human gut microbiome, our 
understanding of how it is influenced by antibiotics remains starkly unclear. 
Using the data generated from our series of clinical flu vaccination trials (21), we found that the 
early induction of TLR5 expression (Day0 vs Day3 post vaccination) positively correlated to the 
levels of HAI titers measured on Day28 post vaccination. We have since demonstrated a causal 
link between TLR5 signaling and induction of antibody responses to IIV in mice. Determining 
whether a functional link can be established in humans is very challenging to address. However, 
one potential approach is to determine whether polymorphisms within the TLR5 gene contribute 
to responsiveness of a given individual to vaccination. We are specifically interested in the 
commonly-known TLR5 stop codon polymorphism (Hawn et al., 2003. J. Exp. Med.). This 
polymorphism refers to a SNP at nucleotide position 1174 (C -> T) resulting in a stop codon 
replacing arginine at amino acid position 392. This allele has been characterized as a dominant 
loss-of-function mutation as humans that are heterozygotes fail to respond to flagellin. Therefore, 
this is a highly intriguing and relevant SNP that will enable us to address a role of TLR5 expression 
in humans to vaccine-induced antibody responses. 
In summary, the data from our experiments in mice suggest a novel role for commensal microbiota 
in enhancing vaccine-elicited immunity. Determining whether these effects are seen in humans 
after vaccination is vital to improve and optimize vaccine efficacy. Thus, we propose to apply our 
experimental data from mice to examine whether exposure to antibiotics given around vaccination 
in humans will negatively impact the ensuing immune response. 
 
 


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1.3 
Rationale for Selection of Study Population 
Twenty two healthy adults with no co-morbidities between the ages of 18-40 years will be recruited 
from the general population of metro Atlanta and enrolled at the Hope Clinic, located in downtown 
Decatur. In previous studies done at the Hope Clinic, 60% of subjects were female and 40% were 
male with good racial representation (55% Caucasian, 35% African American and 10% Asian). 
We chose the above age range to avoid the effect of immune senescence on vaccine efficacy 
since immune responses to IIV are known to decrease with age (33). Additionally, discharge rate 
for Clostridium difficile infection (CDI) from US short-stay hospitals is the lowest among adults 
between the ages of 18 to 44 and is less than 2.5 cases per 1000 discharges (42). This rate does 
not seem to be affected by the emergence of a hypervirulent strain of Clostridium difficile 
(NAP1/BI/027) resistant to quinolones (40). Therefore our study subjects will be healthy adults 
between the ages of 18-40 with no co-morbidities. 
1.4 
Investigational Product(s)/Intervention (s) 
The vaccine used in the study will be the seasonal 2014-2015 and 2015-2016 Fluzone® 
manufactured by Sanofi Pasteur (Swiftwater, PA  USA)(34). Subjects in the study will receive the 
routine adult dose of 0.5 ml given by the intramuscular (IM) route. Fluzone® will be obtained from 
the Emory University pharmacy. 
The antibiotic regimen will be given 3 days prior to vaccination, on the vaccination day, and one 
day after vaccination for a total for 5 days.  The regimen will include the following antibiotics: 
Flagyl ®(35) Pfizer, New York, NY) 500 mg po tid 
Vancocin® (36) (ViroPharma , Exton, PA) 125 mg po qid 
Neomycin sulfate® (37) (X-Gen Pharmaceuticals, Big flats, NY) 500 mg po tid 
All antibiotics will be obtained from the Emory University pharmacy. 
1.5 
Rationale for Selection of Investigational 
Product(s)/Intervention(s) and Regimen 
The majority of studies demonstrating that antibiotic-mediated depletion of host microbiota 
influence the development and homeostasis of the immune system employed a specific cocktail 


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of antibiotics consisting of Ampicillin (1 g/L), Neomycin (1 g/L), Vancomycin (0.5 g/L), and 
Metronidazole (0.5 g/L).  
The basis for using this particular combination of antibiotics is to enable a wide spectrum of 
microbial communities to be targeted for depletion. Indeed, at the indicated doses, this cocktail 
has been shown to effectively deplete more than 90% of the existing flora in the gut and drastically 
alter the composition of the microbiota (22, 25). Specifically, the greatest changes were observed 
in bacterial phyla, Fermicutes and Proteobacteria as seen also in our independent experiments.  
For this study we will use a broad spectrum antibiotic regimen affecting all bacteria in the gut flora 
based on our mice experiments that includes:  Vancocin® (with activity against gram positive 
bacteria), Flagyl® (with activity against anaerobes) and Neomycin sulfate® (with activity against 
gram negative bacteria).  
Additional characteristics of antibiotics selected in the regimen include: 
- 
Poor systemic absorption for Vancocin® and Neomycin sulfate® (3%) when given orally 
which should result in less systemic side effects. 
- 
Good safety profile for all 3 of these antibiotics used for more than half a century 
(Flagyl® was developed in 1960, Vancocin® in 1953 and Neomycin sulfate® in 1949). 
- 
Although any antibiotic can cause Clostridium difficile-associated colitis, the current 
regimen includes Vancocin® and Flagyl®, both used at the same cumulative daily 
dosage to treat Clostridium difficile infection but administered in a shorter course of 5 
days. For Neomycin sulfate®, the cumulative daily dosages are less than half of the 
minimum daily dosage used to treat adult hepatic encephalopathy and a 5-day course is 
same as that required for treating adult hepatic encephalopathy. 
This antibiotic regimen (Vancocin®, Flagyl® and Neomycin sulfate®) will be administered three 
days before vaccination with IIV, on the day of vaccination and one day post-vaccination in 
subjects randomized to Group A (subjects in Group B will only receive IIV). This short course 
reduces the potential risk of bacterial and fungal superinfection, as well as preventing the 
development of antibiotic resistance. 
 


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1.6 
Risks 
1.6.1 Risks of Investigational Product(s)/Intervention(s) 
IIV 
Routine influenza vaccination is now recommended for all persons aged ≥6 months.  Fluzonex® 
is an FDA approved IIV manufactured Sanofi Pasteur (Swiftwater, PA  USA) and indicated for 
active immunization of persons 6 months of age and older against influenza disease caused by 
influenza virus subtypes A and type B contained in the vaccine. The most common (>10%) local 
adverse reactions and general adverse events observed with Fluzone® when given to adults were 
pain and redness at the injection site, and muscle aches, fatigue, and headache, respectively 
(34).  
Acute and potentially life-threatening allergic reactions are also possible, especially in recipients 
of influenza vaccines with a history of severe egg allergy (34). As noted in the exclusion criteria, 
for safety considerations, individuals with an allergy to eggs will be excluded from this study. In 
addition, vaccinations will be administered per label and in clinical settings equipped to manage 
any potential allergic reactions that may occur following vaccine administration. 
Associated with the 1976 flu vaccine, a few patients experienced temporary paralysis, a condition 
known as Guillain-Barré syndrome. However, this syndrome has not been observed with the more 
modern influenza vaccine preparations. Most individuals who develop Guillain-Barré Syndrome 
recover completely, although the recovery period may vary from weeks to a few years. Of those 
who develop Guillain-Barré Syndrome, approximately 30% continue to exhibit residual weakness 
after 3 years, and approximately 3% suffer a relapse of muscle weakness and tingling sensations 
years after the initial attack. Intensive surveillance of Guillain-Barré Syndrome following 
administration of inactivated influenza vaccines since 1976 has shown a slight increase in risk 
over background cases (more than one additional case of Guillain-Barré Syndrome per million 
persons) following vaccination, typically with onset within 6 weeks after vaccination (38). 
Interestingly, although vaccination rates have increased in the last 10 years, the numbers of 
reported cases of vaccine-associated Guillain-Barré Syndrome have declined (39). 
Randomized, controlled clinical trials of the inactivated seasonal influenza vaccine in patients with 
asthma indicate that there is no significant increase in asthma exacerbations immediately after 
vaccination.  


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There may be other unknown side effects. 
Antibiotics 
The most common risk factor for Clostridium difficile infection (CDI) is antibiotic use. CDI typically 
results in mild illness with diarrhea, fever, and abdominal pain. Severe cases of CDI have resulted 
in ICU admissions, colectomy, and deaths, mostly occurring in elderly patients or patients with 
co-morbidities (41).  However, hospital discharge rate for CDI from US short-stay hospitals is the 
lowest among adults between the ages of 18 to 44 and is less than 2.5 cases per 1000 discharges 
(42). This rate does not seem to be affected by the emergence of a hypervirulent strain of 
Clostridium difficile (NAP1/BI/027) resistant to quinolones (40). Therefore our study subjects will 
be healthy adults between the ages of 18-40 with no co-morbidities. Although CDI can occur with 
any antibiotic exposure, we specifically excluded quinolones from our antibiotic regimen as it has 
been the antibiotic most frequently associated with the epidemic strain (43).   
Flagyl®  
The most serious adverse reactions reported in patients treated with Flagyl® have been 
convulsive seizures, encephalopathy, aseptic meningitis, optic and peripheral neuropathy (35).  
Peripheral neuropathy is characterized mainly by numbness or paresthesia of an extremity (35). 
The most common adverse reactions reported have been referable to the gastrointestinal tract, 
particularly nausea, reported by approximately 12% of patients, and can be accompanied by 
headache, anorexia, and occasionally vomiting; diarrhea; epigastric distress; and abdominal 
cramping (35).  
Vancocin®  
Nephrotoxicity (renal failure, renal impairment, blood creatinine increased) has occurred following 
oral VANCOCIN therapy in randomized controlledclinical studies, and can occur either during or 
after completion of therapy. The risk of nephrotoxicity is increased in patients >65 years of age 
(36). 
Ototoxicity has occurred in patients receiving vancomycin (36). It may be transient or permanent, 
but has been reported mainly in patients who have been given excessive intravenous doses, who 
have an underlying hearing loss, or who are receiving concomitant therapy with another ototoxic 
agent, such as an aminoglycoside (36). 


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The most common (>10%) adverse reactions associated with the use of Vancocin® in clinical 
trials included nausea (17%), abdominal pain (15%) and hypokalemia (13%). Other adverse 
events (>5%) included peripheral edema, fatigue, fever, diarrhea, vomiting, flatulence, urinary 
tract infection, back pain and headache. Also important/life-threatening side effects are rare (<1%) 
and include vasculitis, thrombocytopenia, nephrotoxicity, neurotoxicity and ototoxicity (36).  
Neomycin sulfate®  
Nephrotoxicity, ototoxicity, and neuromuscular blockage have been reported with the use of 
Neomycin sulfate® (37). Manifestations of neurotoxicity may include numbness, skin tingling, 
muscle twitching, and convulsions (37). The risk of hearing loss continues after drug withdrawal 
(37). Subjects concurrently receiving other neurotoxic and/or nephrotoxic drugs may have can 
have a possible enhancement of the nephrotoxicity and/or ototoxicity of neomycin (37). 
Concurrent or serial use of other aminoglycosides and polymyxins may enhance neomycin’s 
nephrotoxicity and/or ototoxicity and potentiate neomycin sulfate’s neuromuscular blocking 
effects (37). 
The most common adverse reactions to oral neomycin sulfate are nausea, vomiting and diarrhea 
(37).  
All three antibiotics in this regimen can lead to the overgrowth of non-susceptible bacteria. 
1.6.2 Risk of Study Procedure 
The discomforts of this study include having blood drawn, intramuscular (IM) injection, and 
possible allergic reactions to the components of IIV and the antibiotics.  
Drawing blood causes transient discomfort and may cause fainting. Bruising at the blood draw 
site may occur but can be prevented or lessened by applying pressure to the draw site for several 
minutes. Intramuscular injection may also cause transient discomfort. The use of aseptic 
technique will make infection at the site where blood will be drawn or where the vaccination is 
given extremely unlikely. Vaccines used in the study will be a single dose syringe preventing the 
risk of over or under dosing and the potential risk of contamination. 


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1.6.3 Risk of Concomitant Medications, Prophylactic Medications and 
Rescue Medications 
We do not anticipate the use of any other medication; however, should anaphylactic or 
hypersensitivity reactions occur, epinephrine (1:1000) and diphenhydramine injections are readily 
available at the Hope Clinic during vaccine use.  
Epinephrine injection can be associated with high blood pressure, arrhythmia, lightheadedness, 
nervousness, restlessness, tremor, shortness of breath and diaphoresis. The frequency of these 
side effects is not defined. Diphenhydramine injection may be required to treat possible allergic 
reactions and its use can be associated with low blood pressure, arrhythmia, confusion, dizziness, 
sedation, restlessness, diarrhea, nausea and urinary retention. The frequency of these side 
effects is also not defined. 
When facing a medical emergency, the clinic staff will follow the institutional SOP by calling 911 
first. If needed, the subject will be transferred to Emory University Emergency Department for 
further care. 
Subjects are allowed to use acetaminophen or NSAIDs if they experience a moderate to severe 
local or systemic side effects after vaccine administration. 
1.7 
Benefits 
1.7.1 Benefits of Investigational Product(s)/Intervention(s) 
None.  
1.7.2 Benefits of Study Procedure(s) 
None. 


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2 
OBJECTIVES 
Use of antibiotics can significantly impact the microbiota of patients. The status of the intestinal 
microbiome may impact the immune responses to vaccinations and alter functional protective 
immunity in humans. Here we hypothesize that the changes in the fecal microbiome induced by 
antibiotics translates into suboptimal Hemagglutination Inhibition Assay (HAI) titers. 
2.1 
Primary Objective(s) 
Compare HAI titers to IIV at D30 post vaccination in the group receiving antibiotics to 
the group not receiving antibiotics.  
2.2 
Secondary Objectives 
Evaluate the safety profile of the different antibiotics given in the study. 
2.3 
Exploratory Objectives 
Analyze the microbiome in both groups. 
 
Analyze the repertoire and monoclonal antibodies from plasmablasts in a subset of 
vaccinees in both groups.  
 
Identify innate immune signatures (traditional immune parameters measurements + 
array-based gene expression) and correlate signatures with HAI titers at D30 in both 
groups. 
 
Compare HAI titers to IIV at D90, D180 and D365 post vaccination in the group receiving 
antibiotics to the group not receiving antibiotics. 
 
 
 


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3 
STUDY DESIGN 
This is a single center, mechanistic study in which 50 healthy subjects will be randomized to 
receive IIV with antibiotics (Group A) or IIV without antibiotics (Group B).  
Study volunteers will be recruited from the general population of metropolitan Atlanta. The study 
will be conducted at the Hope Clinic of the Emory Vaccine Center (Decatur, GA). The expected 
duration of subject participation is one year. 
 
3.1 
Study Endpoints 
3.1.1 Primary Endpoint(s) 
 
Comparison of HAI titers at D30 post vaccination in both groups. 
3.1.2  Secondary Endpoint(s) 
 
Frequency, severity, and causality of all adverse events in Group A subjects. 
3.1.3 Exploratory Endpoint(s) 
 
Analyses of microbiome at D0, D1, D3, D7 and D30, D90, D180, D365 and comparison 
to baseline screening in both groups. 
 
Analysis of the repertoire and monoclonal antibodies from plasmablasts in a subset of 
vaccinees at D7 in both groups. 
 
Identification of innate immune signatures (traditional immune parameters 
measurements + array-based gene expression) at D1, D3 andD7 in both groups.  
 
Correlation of innate immune signatures at days D1, D3 and D7 with HAI titers at D30 
in both groups. 
 
Comparison of HAI titers at D90, D180 and D365 post vaccination in both groups. 
 


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3.2 
Study Completion 
This study will be considered “completed” when the primary and secondary objectives have been 
met. After the study is completed, the Principal Investigator or Data Center will compile a final 
study report as per ICH E6 and 21CFR312. The study report will be submitted to the local IRB 
and ISM. 


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4 
SELECTION OF STUDY SUBJECTS 
4.1 
Subject Inclusion Criteria 
 
Subjects must meet all of the following inclusion criteria: 
1. Healthy individuals between the ages of 18 and 40 years. 
2. Able to understand and give informed consent.  
3. Women of childbearing potential (not surgically sterile via tubal ligation, bilateral 
oophorectomy or hysterectomy or who are not postmenopausal for ≥ 1 year) must 
agree to practice adequate contraception that may include, but is not limited to, 
abstinence, monogamous relationship with vasectomized partner, barrier methods 
such as condoms, diaphragms, spermicides, intrauterine devices, and licensed 
hormonal methods* for 30 days before and 30 days after IIV vaccination. 
 
* Women of child-bearing potential using licensed hormonal methods must also use a 
second form of contraception. 
4. Men must agree not get a sexual partner pregnant for 1 month after enrollment. 
 
4.2 
Subject Exclusion Criteria 
 
Subjects who meet any of the following exclusion criteria will be excluded from study 
participation: 
1. Receipt of the following:  
 Receipt of blood products 3 months prior to vaccination or expected receipt 
through 6 months after vaccination. 
 Receipt of any live virus vaccines within 4 weeks prior to vaccination or 
expected receipt within 4 weeks after vaccination.* 
 Receipt of any inactivated vaccine within 2 weeks or expected receipt within 2 
weeks after vaccination.* 
 Receipt of any influenza vaccine in the past 3 seasons. 
 Receipt of any systemic antibiotic 6 months prior to vaccination or expected 
receipt 1 month after vaccination. 


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 Receipt of probiotics and prebiotics 3 months prior to vaccination or expected 
receipt 1 month after vaccination. 
 Regular receipt of proton pump inhibitors, H2 receptor blockers, or antacids 3 
months prior to vaccination or expected receipt 1 month after vaccination. 
2. Documented influenza infection during the 2014-2015 or 2015-2016 influenza 
season. Not excluded from the study, volunteers with prior upper respiratory 
infections during the 2014-2015 or 2015-2016 influenza illness. 
3.  Presence of co-morbidities or immunosuppressive states such as:  
 Chronic medical problems including, but not limited to, insulin dependent 
diabetes, severe heart disease including arrhythmias, severe lung disease, 
auto immune diseases, and grade 4 hypertension**. 
 Chronic neurologic conditions including seizure disorder, Parkinson’s disease, 
myasthenia gravis, neuropathy, or history of encephalopathy, meningitis or 
ototoxicity.  
 Any history of gastrointestinal disease including (but not only): documented 
bacterial gastroenteritis or gastroenteritis associated with fever or associated 
with presence of blood/mucus in stools in the last 3 months; inflammatory 
bowel disease, and/or gastrointestinal surgery. 
 Any history of kidney or liver diseases. 
 Alcohol or drug abuse and psychiatric conditions that in the opinion of the 
investigator would preclude compliance with the trial or interpretation of safety 
or endpoint data. 
 Impaired immune function or known chronic infections including, but not 
limited, to known HIV,  hepatitis B or C; organ transplant; immunosuppression 
due to cancer; current and/or expected receipt of chemotherapy, radiation 
therapy, steroids* (i.e., more than 20 mg of prednisone given daily or on 
alternative days for 2 weeks or more in the past 90 days , or high dose 
inhaled corticosteroids*** or any other immunosuppressive therapies 
(including anti-TNF therapy),  functional or anatomic asplenia and congenital 
immunodeficiency. 
 Pregnancy or breast feeding. 


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4.  Conditions that could affect the safety of the volunteers, such as: 
 Severe reactions to prior vaccination with IIV, including anaphylaxis. 
 History of Guillain Barré syndrome. 
 History of bleeding disorders or current use of warfarin. 
 Use of anticonvulsants. 
 Use of digoxin or other forms of digitalis. 
 Any allergy to any component of the vaccine, including egg allergy.  
 Allergy to vancomycin, metronidazole or neomycin as well as other 
aminoglycosides (gentamicin, tobramycin, amikacin, streptomycin). 
5. Volunteers with any acute illness, including any fever (> 100.4 F [> 38.0C], 
regardless of the route) within 3 days prior to vaccination *.   
6. Social, occupational, or any other condition that in the opinion of the investigator 
might interfere with compliance with the study and vaccine evaluation. 
7. Positive C difficile testing by PCR at screening or a history of C difficile infection. 
8. Any grade 2 safety lab test results at screening. 
 
Note:  
*An individual who initially is excluded from study participation based on one or more of 
the time-limited exclusion criteria (e.g., acute illness, receipt or expected  receipt of live 
or inactivated vaccines ) may be reconsidered for enrollment once the condition has 
resolved as long as the subject continues to meet all other entry criteria.  
Subjects receiving > 20 mg/day of prednisone or its equivalent daily or on alternate days 
for more than 2 weeks may enter the study after therapy has been discontinued for more 
than 3 months. 
** Grade 4 hypertension per CTCAE criteria is defined as Life-threatening consequences 
(e.g., malignant hypertension, transient or permanent neurologic deficit, hypertensive). 
*** Subjects are excluded if on high dose intranasal steroids defined as > 960 mcg/day of 
beclomethasone dipropionate or equivalent. 
 


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4.3 
Early Study Termination 
Subjects may be terminated prior to study completion from the study for the following reasons: 
A. The subject elects to withdraw consent from all future study activities, including follow-
up. 
B. The subject is considered by the PI to be “lost to follow-up” (i.e., no further follow-up is 
possible because attempts to reestablish contact with the subject have failed). 
C. The subject dies. 
D. The subject develops a medical condition or is started on new medication(s) not 
previously mentioned in the list of prohibited medications that, in the opinion of the 
investigator, may pose additional risks from participation in the study, may interfere with 
the subject’s ability to comply with study requirements, or may impact the quality of the 
data obtained from the study. 
E. Blood is not able to be drawn (for technical or other reasons) or the subject does not 
tolerate multiple blood draw attempts. 
F. As deemed necessary by the PI or her designee for noncompliance of any nature.  
G. As deemed necessary by the PI after development of a related AE/SAE. 
H. The subject meets one or more of the individual stopping rules delineated in section 8. 
Subjects with early termination status are replaced as needed to preserve the statistical power 
needed to substantiate the primary endpoint (Refer to section 9.1). 
Note:  
Up to the discretion of the PI, subjects receiving concomitant medications may still continue with 
scheduled study blood draws. Subjects receiving concomitant medications along with subjects 
with early termination from this study for any reason may be replaced as needed to preserve the 
statistical power needed to substantiate the primary endpoint. (Refer to section 9.1) 
 
 


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5 
INVESTIGATIONAL PRODUCT(S)/INTERVENTION 
MATERIAL(S), OTHER STUDY PRODUCTS 
(CONTROLS/PLACEBOS) 
5.1 
Investigational Product(s)/Intervention(s) 
The Emory Investigational Drug Service will purchase Fluzone® manufactured by Sanofi Pasteur 
(Swiftwater, PA  USA) from Emory Pharmacy.  The Emory Investigational Drug Service will store 
the vaccines and will monitor temperatures of the refrigerator(s) containing the vaccines. All 
antibiotics (Flagyl ®(Pfizer, New York, NY); Vancocin® (ViroPharma , Exton, PA); Neomycin 
sulfate® (X-Gen Pharmaceuticals, Big flats, NY)) will be purchased from Emory Pharmacy as 
well.  Refer to section 1.6, and applicable product labeling, for known and potential risks to human 
subjects associated with the investigational product(s) intervention(s). 
5.2 
Formulation, Packaging, and Labeling 
IIV 
Fluzone® is a suspension available in 5-mL multi-dose vials. Fluzone® is formulated to contain 
45 micrograms (mcg) hemagglutinin (HA) per 0.5-mL dose, in the recommended ratio of 15 mcg 
HA of each of the following 3 strains: A/Christchurch/16/2010 NIB-74XP (H1N1) (an 
A/California/7/2009-like virus), A/Texas/50/2012 NYMC X-223A (H3N2) (an A/Victoria/361/2011-
like virus), and 287 B/Massachusetts/2/2012 NYMC BX-51B. 
Fluzone® is formulated without preservatives. Fluzone® does contain thimerosal. Each 5mL dose 
also contains sodium phosphate-buffered isotonic sodium chloride solution, gelatin, octylphenol 
ethoxylate, formaldehyde. Fluzone® does not contain natural rubber latex. 
5.3 
Preparation, Administration, and Dosage 
IIV adult dosing is 0.5 ml and is given via the intramuscular route in the deltoid muscle at D0. It 
does not require any preparation and will come as a single dose in a prefilled syringe. IIV is stored 
between 2 to 8oC (36°F to 46°F) as per the manufacturers’ instructions at the pharmacy. In the 
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Antibiotics tablets are provided from IDS at room temperature (20º-25ºC (68º-77ºF)) to subjects 
in Group A, once they are deemed eligible to participate in the study at the screening visit (from 
D-21 to D-1). All antibiotics will be self-administered by the subject by mouth from D-3 until D1 
(total of 5 days) as stated below: 
• 
Flagyl® 500 mg po tid  
• 
Vancocin® 125 mg po qid 
• 
Neomycin sulfate ® 500mg po tid 
5.4 
Accountability of Investigational Product(s)/Intervention(s) 
Under Title 21 of the Code of Federal Regulations (21CFR §312.62), the investigator will maintain 
adequate records of the disposition of the investigational product(s)/intervention material(s), 
including the date and quantity of the drug received, to whom the drug was dispensed (subject-
by-subject accounting), and a detailed accounting of any investigational product(s)/intervention 
material(s) accidentally or deliberately destroyed. 
Records for receipt, storage, use, and disposition will be maintained by the study site. A 
dispensing log will be kept current for each subject. This log will contain the identification of each 
subject, the name of the vaccine administered to the subject, the lot of vaccine received by the 
subject and the date and quantity of vaccine dispensed. A similar log is available for antibiotics 
dispensed for Group A subjects. A pill count is performed on D0, D1, and D7 to record compliance 
to the antibiotic regimen. All records regarding the disposition of the investigational 
product(s)/intervention material(s) will be available for inspection by the site monitor and the 
health authorities. 
5.5 
Assessment of Compliance with Investigational 
Product(s)/Intervention Material(s) 
The number of used vaccine syringes will be tracked and reconciled by the nursing staff at the 
Hope Clinic prior to sending any vials of unused  vaccine back to the Emory Investigational Drug 
Service. 


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5.6 
Modification or Discontinuation of Investigational 
Product(s)/Intervention Material(s) 
5.6.1 Modification of Investigational Product(s)/Intervention(s) 
Unless IIV or any of the antibiotics are recalled by the manufacturers, there will be no 
discontinuation of administration of study vaccine and antibiotics. 
5.6.2 Premature Discontinuation of Investigational 
Product(s)/Intervention(s) 
Refer to sections 4.3 for possible causes of early study termination. 


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6 
OTHER MEDICATIONS 
6.1 
Concomitant Medications 
In accordance with exclusion criteria, subjects expected to receive prohibited medications (see 
section 6.4) will be considered ineligible for the study. All medications, therapies, or vaccines 
administered to study subjects after study entry will be documented at each visit. 
6.2 
Prophylactic Medications 
Prophylactic medications will not be administered before vaccination or any other study 
procedures. 
6.3 
Rescue Medications 
We do not anticipate the use of any rescue medication; however, should anaphylactic or 
hypersensitivity reactions occur, epinephrine (1:1000) and diphenhydramine injections are readily 
available at the Hope Clinic during vaccine use. 
When facing a medical emergency, the clinic staff will follow the institutional SOP by calling 911 
first. If needed, subject will be transferred to Emory University Emergency department for further 
care. 
Subjects are allowed to use acetaminophen or NSAIDs if they experience a moderate to severe 
local or systemic side effect after vaccine administration. 
6.4 
Concomitant Study Medications 
All medications and vaccines received by study subjects after administration of study vaccine 
should be reported to the study staff and recorded.  This includes, but is not limited to, the 
following: 
• 
blood products, chemotherapy, immunosuppressive therapy (including anti-INF 
therapy), and radiation therapy (administered at any time after study vaccination)  
• 
inactivated vaccines (administered before the day 30 blood draw) 
• 
live-attenuated vaccine (administered before the day 30 blood draw) 


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• 
any antibiotic aside from the study antibiotic (taken before the day 30 blood draw). 
• 
any probiotics and prebiotics (taken before the day 30 blood draw). 
• 
any proton pump inhibitors, H2 receptor blockers, or antacids (taken before the day 
30 blood draw). 
Any of the above medications could affect the innate and adaptive assay results and should not 
be used unless medically indicated.  
Upon the discretion of the PI, subjects receiving concomitant medications may still continue with 
scheduled study blood draws. Subjects receiving concomitant medications and early terminated 
subjects may be replaced as needed to preserve the statistical power needed to substantiate the 
primary endpoint. (Refer to section 9.1.). 


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7 
STUDY VISITS AND PROCEDURES 
7.1 
Enrollment and Randomization 
This research study will be explained in lay terms to each potential research subject. The potential 
subject will sign an informed consent form before undergoing any screening study procedures. 
Subjects who are deemed eligible for the study (see sections 4.1 and 4.2) will be enrolled and 
assigned a unique subject number. Enrollment will occur over an approximate 6-month time span.  
The duration of participation for each subject is approximately 1 year 
Twenty-two subjects will be randomized to Group A (receiving antibiotics) or to Group B (not 
receiving antibiotics) in a 1:1 ratio. All eligible subjects will receive IIV at D0. 
7.2 
Screening Visit(s) (From D-21 until D-1) 
Subjects responding to study ads will call the Hope Clinic for a telephone screening where the 
eligibility criteria of the subject will be reviewed. If the subject is found to be eligible and continues 
to be interested in participating after reading the informed consent (emailed or mailed to him/her), 
a screening and enrollment appointment will be scheduled. Study staff will review the informed 
consent form with the subject and will answer all questions related to the study.  
Once the subject signs the informed consent, a study participation number will be assigned. The 
volunteer will be asked to provide demographic information and information related to his/her 
medical history, including current medication use and vaccination history. The subject’s vital signs 
will be recorded, and a targeted physical exam will be conducted as indicated, based on review 
of the subject’s health status. For female volunteers who are of childbearing potential, a urine 
pregnancy test will be performed. Only females with a negative urine pregnancy test will be 
enrolled in the study. All subjects will have a stool sample collected from them. A stool kit will be 
mailed prior to the visit. 
The subjects will be randomized to be in Group A or Group B in a 1:1 ratio and  will have blood 
drawn (to measure CBC with differential, creatinine, and potassium) and immunologic assays.  
This visit will last approximately 60 minutes. 


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7.3 
BASELINE VISITS 
Antibiotic initiation visit (Group A only) (From D-6 until D-3) 
Subjects in Group A will also be given the three antibiotics and instructed on when and how to 
start taking the antibiotic pills (D-3 until D1). Subjects in Group A will provide stool and blood 
samples as well and should have a negative pregnancy test. 
Subjects in Group A are asked to call the Hope Clinic for any symptom while on antibiotics-see 
below.   
Vaccination (D0) 
Study personnel will first review the subject’s current health status, list of medications being taken, 
and note any change since the screening visit. The subject’s vital signs will be recorded as well 
as height and weight and type of diet typically followed and a targeted physical exam will be 
conducted, as indicated by a review of the subject’s health status. For female volunteers of 
childbearing potential, a urine pregnancy test will be performed. Only females with a negative 
urine pregnancy test will receive the vaccine. Inclusion/exclusion criteria will be also verified 
before vaccination. Subjects in Group A will be asked if they have had any symptoms since 
starting antibiotics, and a pill count is performed. All subjects will have a stool sample collected. 
Blood for immunological assays will be drawn at this visit before administration of the vaccine. To 
ensure safety, each subject will be observed for a minimum of 15 minutes following vaccination 
to note the occurrence of any immediate hypersensitivity reactions. After 15 minutes, the injection 
site will be examined by either a RN or a MD and subject will be asked for systemic adverse 
reactions.  
The subject will be provided with a written description of local and systemic vaccine reactions of 
mild, moderate and severe intensity (Refer to Appendix B and Appendix C). Subjects will be 
instructed to notify the study center by telephone if they develop any severe reactions within 1 
week following vaccination.  
At the end of the visit, the subject will also be instructed to promptly call the site if he/she develops 
any of the following: 
• 
Illness or treatment from a physician or emergency department; and/or hospitalization 
due to any illness throughout the entire duration of the study; 


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• 
Development of any adverse event that limits self-care activities of daily living (e.g.  
bathing, dressing and undressing, feeding self, using the toilet, taking medications, and 
not bed ridden) even if he/she decides not to seek medical care; 
• 
For Group A only: development of any of the following symptoms while on antibiotics 
and throughout D90: 
o 
diarrhea (≥ 3 unformed stools/24h) for more than 72h with either fever or 
abdominal pain* 
o 
tinnitus** 
o 
vertigo** 
o 
decrease hearing** 
o 
numbness/tingling** 
• 
Beginning or discontinuing any medications/therapies during enrollment in the study. 
Refer to section 8.2 for safety data that must be recorded and reported. 
Note:  Subjects calling the site for any of the above will receive further instructions on the proper 
course of action, including a return to the clinic for immediate evaluation, if appropriate. 
This visit will last approximately 60 minutes. 
7.4 
Main Study Visits 
All subjects will return for the study-related blood draws on Days 1, 7(+1), 21(+3), and 30 (+5) 
after immunization. All subjects will have stool collected at D1, D3, D7 D30, D90, D180 and D365. 
Additionally, for subjects, safety labs are obtained at D7.  
On days 1, 3, 7, 30, 90, 180 and 365 study personnel will review current health status, including: 
• 
Evaluation of local REs and systemic REs of grade 3 or higher severity developing after 
the previous visit (only until Day 7 Visit). 


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• 
Any solicited adverse event (AE) or serious adverse event (SAE) which may not have 
been reported by the subject by calling the site as directed at a prior visit. 
• 
Development of any adverse event that limits self-care activities of daily living (e.g.  
bathing, dressing and undressing, feeding self, using the toilet, taking medications, and not 
bed ridden) even if he/she decides not to seek medical care. 
• 
Any medications administered after vaccination. 
 
Pill count for Group A ( D1 and D3) 
Subjects in Group A will also be instructed to call the site if he/she: 
• 
Develops any symptoms while taking antibiotics  
• 
Develops any of the following symptoms until D90: 
o 
diarrhea (≥ 3 unformed stools/24h) for more than 72h with either fever or abdominal 
pain* 
o 
tinnitus** 
o 
vertigo** 
o 
decrease hearing** 
o 
numbness/tingling** 
A focused physical exam will be conducted (if indicated) based on results of the review of the 
subject’s health status as outlined above. 
Note:  
* if C difficile infection is suspected, C diff. PCR testing will be performed. If positive, treatment 
and follow up for C difficile infection will be facilitated. 
** if tinnitus, vertigo, decrease hearing, numbness/tingling are present, medical referral will be 
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7.5 
Visit Windows 
Study visits should take place within the time limits below: 
D0, D1,D3,  D7 (+/-1), D30 (+/-5), D90 (+/-14), D180 (+/-14), D365 (+/-28). 
7.6 
Study Procedures 
Refer to sections 7.1, 7.2, 7.3, and 7.4. 
 
 
7.7 
Study Arm Assignment Procedures 
7.7.1 Blinding and Randomization 
In this study 50 healthy subjects will be randomized to be in either Group A or Group B in a 1:1 
ratio. 
The randomization will be performed by the Emory Investigational Drug Service. 
7.7.2 Securing Randomization Information 
The information on randomization is kept at the Emory Investigational Drug Service. 
7.7.3 Requirements for Unblinding 
N/A. 
7.7.4 Documentation of Unblinding 
N/A. 
 


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8 
SAFETY PROCEDURES 
8.1 
Stopping Rules 
8.1.1 Study Stopping Rules 
Study vaccination will be suspended pending expedited review of all pertinent data by the 
institutional review board and the ISM, if Fluzone® or antibiotics (Vancocin®, Flagyl®, and 
Neomycin sulfate®) were recalled by the manufacturer.  
Also, vaccination will be suspended pending review of all pertinent data by the PI  and the ISM 
after the occurrence of any of the following:  
1 SAE  
1 AE of Grade 4 severity. 
2 AEs of Grade 3 severity of similar type other than expected events. 
2 grade 2 AEs of abnormal laboratory values at D7. 
2 cases of C difficile infections in Group A subjects. 
1 case of ototoxicity. 
1 case of neuropathy. 
1  case of Guillain-Barré Syndrome. 
8.1.2 Individual Subject Stopping Rules 
Early study termination will occur in individual subjects due to any of the following circumstances: 
• 
Development of a related serious adverse event. 
• 
Development of a condition for which continued participation, in the opinion of the 
investigator, would pose a risk to the subject. 
• 
Development of a condition for which continued participation, in the opinion of the 
investigator, would be likely to confound interpretation of the results of the study. 


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• 
Participation in the study may be terminated as deemed necessary by the PI or 
her designee for noncompliance with study procedures.   
• 
Participation in the study may be terminated when blood is not able to be drawn 
(for technical or other reasons) at time points required for the primary endpoint, 
or the subject does not tolerate multiple blood draw attempts.  
• 
Pregnancy 
8.1.3 EARLY TERMINATION FROM STUDY VACCINES/ PROCEDURES 
WITH CONTINUED STUDY PARTICIPATION/FOLLOW-UP 
Refer to section 4.3. 
8.1.4 FOLLOW-UP AFTER EARLY STUDY TERMINATION 
Subjects in Group A who are prematurely terminated from the study for reasons other than safety 
events will still be followed to monitor safety for 365 days after the last administered dose of 
antibiotics at the same intervals used for the Group A subjects who remain in the study.  
 Subjects in Group A and B who are prematurely terminated from the study due to an AE will be 
followed until resolution of the AE or until 30 days after a subject terminates from the study, 
whichever comes later. (Note: subjects in Group A terminate study participation 90 days +/-14 
days after the last dose of antibiotics is administered; subjects in Group B terminate study 
participation at Day 30 +/- 5 days)Resolution of an AE is defined as the return to baseline status 
or as stabilization of the condition with the expectation that it will remain chronic. 
After assessing terminated subjects for safety under the provisions stated above, the subject will 
be seen in clinic, if necessary. 
8.1.5 SUBJECT REPLACEMENT 
In the case that premature termination causes the attrition rate to be higher than 10%, we will 
recruit extra patients to maintain the target sample size of 10 subjects/group and the statistical 
power to substantiate primary endpoint.  Please refer to section 9 - Statistical Analysis. 


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8.2 
Adverse Events 
This section defines the types of adverse events that may occur, and outlines the procedures for 
appropriately adverse event collecting, grading, recording, and reporting.   
Information in this section complies with 21CFR 312; ICH Guideline E2A: Clinical Safety Data 
Management: Definitions and Standards for Expedited Reporting and ICH E6: Guideline for Good 
Clinical Practice; and applies the standards set forth in the National Cancer Institute (NCI), 
Common Terminology Criteria for Adverse Events Version 4.0 [Published: May 28, 2009; revised 
version 4.03; June 14. 2010, http://evs.nci.nih.gov/ftp1/CTCAE/About.html] 
These criteria have been reviewed by the study investigators and have been determined to be 
appropriate for this study population. 
8.2.1 Safety Reporting 
8.2.1.1 
Adverse Events (AE) 
Adverse event means any untoward medical occurrence associated with the use of a drug in 
humans, whether or not considered drug related. 
An adverse event (also referred to as an adverse experience) can be any unfavorable and 
unintended sign (e.g., an abnormal laboratory finding), symptom, or disease temporally 
associated with the use of a drug, without any judgment about causality. An adverse event can 
arise from any use of the drug (e.g., off-label use, use in combination with another drug) and from 
any route of administration, formulation, or dose, including an overdose. 
8.2.1.2 
Suspected Adverse Reaction (SAR) 
Suspected adverse reaction means any adverse event for which there is a reasonable possibility 
that the drug caused 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. 
SARs after vaccine administration may include: 


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• 
Local reactions: hardening of the skin at the injection site, injection site redness, 
injection site swelling, pain at the site of injection, bruising of the skin at the 
injection site. 
• 
Systemic reactions: aching muscles, headache, joint pain, fatigue, fever, and 
shivering.   
8.2.1.3 
SERIOUS ADVERSE EVENT (SAE) OR SERIOUS SUSPECTED 
ADVERSE REACTION 
An adverse event or suspected adverse reaction is considered ‘‘serious’’ if, in the view of the 
investigator, it results in any of the following outcomes:  
• 
Death 
• 
A life-threatening adverse event 
• 
Inpatient hospitalization or prolongation of existing hospitalization 
• 
A persistent or significant incapacity or substantial disruption of the ability to 
conduct normal life functions 
• 
A congenital anomaly/birth defect.  
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 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 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. 
8.2.1.4 
Life-Threatening Adverse Event or Life-Threatening Suspected 
Adverse Reaction 
An adverse event or suspected adverse reaction is considered ‘‘life-threatening’’ if, in the view of 
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It does not include an adverse event or suspected adverse reaction that, had it occurred in a more 
severe form, might have caused death. 
8.2.1.5 
Unexpected Adverse Event or Unexpected Suspected Adverse 
Reaction 
An adverse event or suspected adverse reaction is considered ‘‘unexpected’’ if it is not listed in 
the Summary of Product Characteristics or is not listed at the specificity or severity that has been 
observed; or, if the Summary of Product Characteristics is not required or available, 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 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 brochure listed only cerebral vascular accidents. 
‘‘Unexpected,’’ as used in this definition, also refers to adverse events or suspected adverse 
reactions that are mentioned in the Summary of Product Characteristics 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. 
8.2.1.6 
Independent Safety Monitoring 
The ISM is a physician with relevant expertise in vaccine trials whose primary responsibility will 
be to provide independent safety monitoring in a timely fashion and to provide recommendations 
regarding the safe continuation of this study.  
The ISM will evaluate safety data generated from study subjects including all SAEs against the 
known safety profile of the study vaccine to assess for possible changes to the overall risk of the 
study.   
Contact information for the ISM is listed on page 3 of this protocol.  
 The ISM will communicate with the PI as needed to discuss any safety events of special interest 
developing during the study and when conducting the review of the monthly reports of cumulative 
safety data. The study has provisions for a back-up ISM to ensure that independent safety 


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monitoring happens at all times during the study. Additional roles and responsibilities of the ISM 
are described in section 8.2.4 below. 
8.2.2 Collecting and Recording Adverse Events and Pregnancy 
 
Adverse events may be identified during this study through any of these methods: 
1.  Examination of the subject during study visits. 
2.  Questioning the subject during study visits. 
3.  Receiving a safety report from the subject at any time during the study  
Note: subjects will be asked to call the site if they develop any of the following: 
• 
Illness or treatment from a physician or emergency department the entire duration of 
the study; 
• 
Any adverse event that limits self-care activities of daily living (e.g.  bathing, dressing 
and undressing, feeding self, using the toilet, taking medications, and not bed ridden) 
even if he/she decides not to seek medical care; 
• 
Local REs and/or systemic REs of grade 3 or higher severity after the previous visit 
(only until Day 7 Visit). 
4. Receiving a safety report from subject in Group A if they: 
• 
Develop any symptoms while on antibiotics  
• 
Develop any of the following until D90: 
o 
diarrhea (≥ 3 unformed stools/24h) for more than 72h with either fever or 
abdominal pain 
o 
tinnitus 
o 
vertigo* 
o 
decrease hearing 
o 
numbness/tingling 
 
A complete recording of safety events in the CRF will include event term, date(s) of onset and 


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resolution/stabilization, 
assessment 
of 
severity, 
relationship 
to 
study 
vaccine 
or 
procedures/intervention(s) such as phlebotomy, expectedness, determination of whether the AE 
qualifies as serious or non-serious, treatment required, action taken with study participation, and 
outcome. AEs qualifying as serious also require a narrative of the event. Updates in safety events 
will be recorded as additional information becomes available. 
Adverse events will be followed until they are resolved. Resolution of an AE is defined as the 
return to baseline status or as stabilization of the condition with the expectation that it will remain 
chronic. 
Information on pregnancies will be collected from the time a subject signs the consent until the 
subject completes study participation.  Cumulatively monthly reports of safety data will capture 
any pregnancies and pregnancy outcomes at least on a quarterly basis. 
 If a subject becomes pregnant after study entry, the investigator will discuss with the subject 
and/or the treating physician the known possible risks to the fetus. 
Subjects becoming pregnant after study entry will be withdrawn from the study and followed until 
the end of the pregnancy.  A pregnancy resulting in congenital anomaly/birth defect will be 
considered a SAE. Any premature termination of the pregnancy will also be reported and 
assessed as an SAE as needed. 
8.2.3 Grading and Attribution of Adverse Events 
8.2.3.1 
Grading Criteria 
Adverse events will be graded according to the criteria set forth in the National Cancer Institute’s 
Common Terminology Criteria for Adverse Events Version 4.0 [May 28, 2009;revised version 
4.03; June 14. 2010,  http://evs.nci.nih.gov/ftp1/CTCAE/About.html] 
This document (referred to herein as the NCI-CTCAE manual) provides a common language to 
describe levels of severity, to analyze and interpret data, and to articulate the clinical significance 
of all adverse events.   
All adverse events whether or not listed in the NCI-CTCAE will be graded on a scale from 1 to 5 
according to the following standards in the NCI-CTCAE manual (A semi-colon indicates ‘or’ within 
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Grade 1 Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only; 
intervention not indicated.  
Grade 2 Moderate; minimal, local or noninvasive intervention indicated; limiting age-appropriate 
instrumental ADL*. 
Grade 3 Severe or medically significant but not immediately life-threatening; hospitalization or 
prolongation of hospitalization indicated; disabling; limiting self-care ADL**. 
Grade 4 Life-threatening consequences; urgent intervention indicated. 
Grade 5 Death related to AE. 
*Instrumental Activities of Daily Living (ADL) refer to preparing meals, shopping for 
groceries or clothes, using the telephone, managing money, etc. 
**Self-care  Activities of Daily Living (ADL)   refer to bathing, dressing and undressing, 
feeding self, using the toilet, taking medications, and not bedridden. 
 
Not all Grades are appropriate for all AEs; therefore, some AEs are listed with fewer than five 
options for Grade selection.  
Anaphylaxis is a disorder characterized by an acute inflammatory reaction resulting from the 
release of histamine and histamine-like substances from mast cells, causing a hypersensitivity 
immune response. Clinically, it presents with breathing difficulty, dizziness, hypotension, cyanosis 
and loss of consciousness and may lead to death. 
Severity grading of anaphylaxis as per the NCI-CTCAE manual is as follows: 
Grade 1= not applicable 
Grade 2= not applicable 
Grade 3= Symptomatic bronchospasm, with or without urticaria; parenteral intervention 
indicated; allergy-related edema/angioedema; hypotension 
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Grade 5= Death  
Severity grading of AEs of laboratory abnormalities will be assessed as per the NCI-CTCAE 
manual. 
8.2.3.2 
Definition of Attribution 
The site investigator will initially determine the relationship of an adverse event to the study 
vaccine or study procedures (blood draw). The investigator’s determination of causality will be 
used for FDA reporting purposes.  
The relationship of an AE to study participation will be determined using definitions in the table 
below: 
Code 
Descriptor 
Definition (guidelines) 
UNRELATED CATEGORY  
1 
Unrelated 
The adverse event is clearly not related to study.  The event 
is completely related to an etiology other than the study 
product or study intervention (the alternative etiology must be 
documented in the study subject’s medical record) 
2 
Unlikely 
The adverse event is doubtfully related to study and likely to 
be related to factors other than study product or study 
intervention. 
RELATED CATEGORIES  
 
 
 
3 
Possible 
The adverse event may be related to study.  There is an 
association between the event and the administration of study 
product and there is a plausible mechanism for the event to 
be related to the study product; there may be also an 
alternative etiology, such as characteristics of the subject’s 
clinical status and/or underlying disease 
 
4 
Probable 
The adverse event is likely related to study.  There is (1)  an 
association between the event and the administration of study 
product or study intervention, (2) a plausible mechanism for 
the event to be related to the study product,  and (3) the event 


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could not be reasonably explained by known characteristics 
of the subject’s clinical status and  or an alternative etiology is 
not apparent 
5 
Definite 
The adverse event is clearly related to study.  There is (1) an 
association between the event and the administration of the 
study product or study intervention, (2) a plausible mechanism 
for the event to be related to the related to the study product, 
and (3) causes other than the study product have been ruled 
out and/or the event re-appeared on re-exposure to the study 
product 
8.2.4 Reporting Serious Adverse Events to the Independent Safety 
Monitor 
The Principal Investigator will notify the ISM by email of any SAE within 24 hours of becoming 
aware of the event. The initial SAE CRF should be signed by the PI or co-PI and include as much 
information as possible. 
The SAE case report form will be re-submitted and signed by the PI or co-PI to the ISM with 
updated relevant medical information as needed until the event is considered closed by the ISM.    
8.2.5 Reporting Serious Adverse Events to the FDA 
N/A. 
8.2.6 Notifying Institutional Review Board 
The Principal Investigator will ensure the timely dissemination of SAE information, including SAEs 
requiring expedited review by the ISM and resulting ISM memorandums following evaluations of 
safety data, to the IRB in accordance with IRB regulations and guidelines. 
8.3 
Protocol Deviations 
Deviations occur when the Investigator, site study staff, or subjects fail to adhere to protocol 
requirements or when there is non-adherence to (GCP) as delineated in ICH E6 (R1) guidelines.   


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Upon determination that a protocol deviation has occurred, the study staff will a) notify the 
Principal Investigator, and b) will complete the Protocol Deviation form. There could be a 
discussion between the Principal Investigator and the ISM to determine the effect of the protocol 
deviation on the study subject and his/her further study participation, the effect of the protocol 
deviation on the overall study, and corrective actions.   
The Principal Investigator will complete and sign the Protocol Deviation form and submit it to the 
ISM Monitor and to the site IRB, per IRB regulations.   
Identification of protocol deviations will be done through the quality manager at the study site. 
Reviewing and reporting of minor protocol deviations will be discussed at the Hope Clinic monthly 
meetings. Major protocol deviations will be discussed with the PI and the ISM.  
All study deviations will be captured in monthly reports along with safety data and forwarded to 
the ISM. 


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9 
SAMPLE SIZE CALCULATIONS AND STATISTICAL 
PLAN 
9.1 
Sample Size and Power Calculations 
Based on previous studies performed at the Hope Clinic, the rate of subjects that become 
ineligible during the study (i.e. do not meet exclusion/inclusion criteria anymore, lost to follow-ups, 
unable to have blood draws) is between 5 to 10%. Assuming a 10% attrition rate, 50 healthy 
adults will be recruited for the study, resulting in an expected 10 subjects per arm for data analysis. 
In the case that the sample size of subjects missing any of the 2 draws required for the primary 
endpoint [meaning draws at days 0 and day 30] is higher than 10% of the 50 subjects to be 
recruited for the study, we will recruit extra patients to maintain the target sample size of 10/group 
and the statistical power to prove primary endpoint. 
9.1.1 Primary and Secondary Endpoints 
The comparison of HAI titer will be conducted in three ways: direct comparison of HAI titers, 
comparing seroprotection rate, and comparing seroconversion rate. 
For the comparison of the mean log2 HAI titers between the two groups, with 10 subjects in each 
group, at the alpha level of 0.05, we can reject the null hypothesis of equal means with 80% power 
if the true effect size ((mean0-mean1)/sigma, assuming equal variance) is 1.3 using an unpaired 
one-sided t-test. 
Based on a previous study of Fluzone® in a large cohort, we expect 98% of subjects without 
antibiotics treatment will reach seroprotection, i.e. HAI titers of ≥1:40. Our null hypothesis is the 
two groups have equal proportions of subjects who will reach seroprotection, and the alternative 
hypothesis is the antibiotic treatment lowers the proportion of subjects reaching seroprotection. 
The sample size of 10 subjects per treatment group will allow us 80% power to detect a difference 
in proportions of 0.41 or higher in a one-sided test of two proportions using a significance level of 
0.05.  
Based on a previous study of Fluzone® in a large cohort, we expect 65% of subjects without 
antibiotics treatment will achieve seroconversion, i.e. ≥four-fold increase in HAI titers over 
baseline. The sample size of 10 subjects per treatment group will allow us 80% power to detect 


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a difference in proportions of 0.50 or higher in a one-sided test of two proportions using a 
significance level of 0.05. 
9.2 
Data Analysis 
Data from the study will be analyzed using the statistical software R .  High-throughput data will 
be analyzed using appropriate methods, either by stand-alone software or modules in leading 
statistical softwares, e.g. the Bioconductor in the R framework (44). For cytokine data, missing 
values will be dealt with using multiple imputations. For microarray data, nearest neighbor method 
and local least squares may be used to fill missing values. Unsupervised learning techniques 
such as PCA, PLSDA, clustering, and factor analysis will be used for the visualization and 
identification of global patterns (45). For descriptive endpoints, we will generate summary 
statistics, and visualize the data using histograms and boxplots when applicable. For the 
identification of innate signatures, the two treatment groups will be analyzed separately. 
9.2.1 Analyses of Primary Endpoint 
Comparison of HAI Titers at D30 Post-Vaccination in Both Groups 
Data from the study will be analyzed using the statistical software R. We will take three alternative 
routes for data analysis. The first is directly comparing HAI titers between the treatment groups; 
the second is comparing the proportions of subjects achieving seroprotection from each group; 
and the third is comparing the proportions of subjects achieving seroconversion from each group.  
The first data analysis route is directly comparing the means/medians of the HAI titers between 
the two groups at D30. We will first log2 – transform the HAI titers for each subject. The log2 HAI 
titers of each group will be summarized and presented using histograms and strip charts for visual 
comparison. We will use the t-test to compare the mean log2 HAI titers between the two groups. 
The Wilcoxon test may also be used depending on the distribution of the data.  
In addition, we will compare the two groups based on the proportion of achieving seroprotection 
and seroconversion. For seroprotection, we will dichotomize the outcome into D30 HAI titer ≥1:40 
or D30 HAI titer <1:40. For seroconvertion, we will dichotomize the outcome into D30 HAI titer 
≥four-fold or <four-fold rise relative to baseline titer. If a subject has negative titer at D0, we will 
consider the subject a seroconverter if his/her HAI reaches 1:40 at D30. We will then compare 
the two treatment groups by conducting a Fisher’s exact test on the 2×2 contingency table of 


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treatment versus dichotomized HAI titers. We will also report the estimated proportion of 
seroprotection/seroconversion and its 95% confidence interval for each group. 
9.2.2 Analyses of Secondary Endpoint 
9.2.3 Analysis of Exploratory Endpoints 
Exploratory Endpoint 1: Analyses of microbiome at D0, D7, and D30 in Group A and 
comparison to baseline Screening 
Sequence results will be analyzed using the open source software package called Quantitative 
Insights into Microbial Ecology (QIIME) (46). Primary objective of our analyses will be to 
characterize the composition of microbiota samples and to identify specific microbial communities 
significantly associated with an experimental group. 
Prior to analysis, sequences will undergo quality filtering using QIIME and the sequence quality 
scores annotated to each raw sequence data. The 16s gene sequences will be aligned and 
clustered using UCLUST, which is based on a pair-wise identity threshold of 97% (47). Clustered 
sequences will be assigned to operational taxonomic units (OTUs) (48). A single representative 
sequence for each OTU will be aligned using PyNAST   to enable taxonomic classification and 
generation of phylogenetic trees for subsequent analyses (46, 49, 50). 
OTUs with taxonomic assignments will allow QIIME to assemble a matrix of OTU abundance in 
each sample. This matrix will then be used to summarize communities by taxonomic composition 
and abundance, which can be visualized together as area/bar graphs or heatmaps. Information 
from phylogenetic trees will be used to compute distances or levels of dissimilarity between 
microbial communities using Unifrac (51). This tool will enable compositional comparisons 
between samples (beta diversity) to be conducted. For this purpose, we will use the OTU matrix 
to calculate distance or dissimilarity of samples between experimental groups using weighted and 
unweighted UniFrac metrics. QIIME will then generate 2 and 3 dimensional Principal Coordinate 
Analysis plots to represent the distance matrix data. Furthermore, QIIME codes a tool based on 
G test of Independence to identify OTUs that are differentially represented across experimental 
groups. This tool will also be conducted and applied to the beta diversity Principal Coordinate 
Analyses to help explain which OTUs are significantly associated with a particular experimental 
group. 


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For longitudinal analyses of samples within each group, semivariogram plots will be generated 
using dissimilarity metrics over time (Euclidean) plotted against community dissimilarity (UniFrac) 
(46,52). 
Exploratory endpoint 2: Analysis of the repertoire and monoclonal antibodies from 
plasmablasts in a subset of vaccinees at D7 in both groups 
The repertoire and monoclonal antibody levels will be presented as descriptions of the actual 
values. Summary statistics, boxplots and histograms will be used to summarize the data. 
Exploratory endpoint 3: Identification of innate immune signatures (traditional immune 
parameters measurements + array-based gene expression) at D1, D3 and D7 in both 
groups 
We will take the measurements on the day of vaccination as baseline. For traditional immune 
parameters, we will test if an immune parameter shows significant change over baseline at day 
1, 3, and 7 after each vaccination, using a two-sided paired t-test. Fold-change (ratio between the 
mean values of the two groups) will be combined with the test p-value in a selection criterion as 
appropriate. Based on previous studies similar in nature, the tentative selection criterion is p-value 
≤ 0.01 and fold change ≥ 3.  
For the gene expression data, we will use the method Significance Analysis of Microarrays (SAM) 
with paired design to find differentially expressed genes (53). False discovery rate (FDR) will be 
used as selection criterion (54). The tentative selection criterion is FDR ≤ 0.1. 
Exploratory endpoint 4: Correlation of innate immune signatures at days D1, D3 and D7 
with HAI titers at D30 in both groups 
For traditional immune parameters, we will calculate the correlation coefficient between the 
immune parameter and the adaptive immune response (HAI titers at D30) he p-values associated 
with the correlation coefficients will be used to select traditional immune parameters that are 
associated with the respective adaptive immune responses. The tentative selection criterion is p-
value ≤ 0.01.  
For gene expression data, we will use the method Significance Analysis of Microarrays (SAM) 
with quantitative outcome to identify genes that are significantly associated with the respective 


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adaptive immune responses (HAI titers at D30 ). False discovery rate (FDR) will be used as 
selection criterion. The tentative selection criterion is FDR ≤ 0.1. 
Exploratory endpoint 5: Comparison of HAI Titers at D30 Post-Vaccination in Both Groups 
Analysis similar to 9.2.1. 
9.2.4 Patient Populations 
We will use all subjects who are randomized, receive the vaccine, complete at least one day of 
antibiotic treatment, and have measurements at days 0 and 21 in the data analysis. 
9.2.5 Study Subject Baseline Characteristics and Demographics 
A summary of descriptive statistics for baseline and demographic characteristics will be provided 
for all enrolled subjects. Demographic data will include age, race, sex, and medical history, 
including current medication use and vaccination history. 
9.2.6 Status of Study Subjects 
Status of study subjects (continue in the study vs. left the study) will be incorporated into the 
periodic safety reports. 
9.3 
Interim Analyses 
No interim analysis will be conducted.   
9.4 
Deviations from Statistical Plan 
The principal features of the study design and of the plan for statistical analysis of the data are 
outlined in this protocol. Any changes in these principal features will require a protocol 
amendment and will be described in the final report. These changes will be subject to review by 
the IRB, ISM.  
Although our statistical methods and time-points for measurements will adhere to what is 
proposed herein, our experience with analyses of similar studies in the past has underscored the 
need for flexibility and adaptability in trying different statistical approaches to arrive at the most 
informative results, especially for the high-throughput data.  


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As such, we may use alternative approaches such as the gene set enrichment analyses, or other 
approaches, and run additional statistical analyses of data generated by use of assays at time 
points other than those stated in the primary, secondary and exploratory endpoints, on an ad hoc 
basis (55). 
 


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10 
IDENTIFICATION AND ACCESS TO SOURCE DATA 
 
10.1 Identifying Source Data 
The investigator will keep accurate records to ensure that the conduct of the study is fully 
documented. Data forms are either considered source or protocol-specific CRFs as detailed in 
the MOPs. 
10.2 Updating Source Documentation 
Documents describing the safety profile of investigational products, such as the investigator’s 
brochure and the package insert, will be amended as needed by the investigational products 
manufacturer to ensure that the description of safety information adequately reflects any new 
clinical findings.  
The Principal Investigator (or co-PI) will provide the ISM and the IRB with the most up-to-date 
versions of the above documents as soon as the Principal Investigator (or co-PI) becomes aware 
of any changes. For purchased investigational products, the Principal Investigator (or co-PI) will 
confirm that there are no changes to the package insert every 3 months. In case of package insert 
changes, the Principal Investigator (or co-PI) will notify the ISM and the IRB.     
 
10.3 Permitting Access to Source Data 
The investigational site participating in this study will maintain the highest degree of confidentiality 
for the clinical and research information obtained from the subjects in this study.  Medical and 
research records will be maintained at the study site in the strictest confidence.  However, as a 
part of the quality assurance and legal responsibilities of an investigation, the investigational site 
will permit authorized representatives of regulatory authorities to examine (and when required by 
applicable law, to copy) clinical records for the purpose of quality assurance reviews, audits, and 
evaluations of the study safety and progress.  Unless required by the laws that permit copying of 
records, only the coded identity associated with documents or with other subject data may be 
copied (and all personally identifying information will be removed).  Authorized representatives 


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described above are bound to maintain the strict confidentiality of medical and research 
information that is linked to identify individuals.   


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11 
QUALITY CONTROL AND QUALITY ASSURANCE 
The Principal Investigator (or co-PI) will keep accurate records to ensure that the conduct of the 
study is fully documented.  The investigator will ensure that all CRFs and subject study files are 
legible and complete for every subject. 
When the CRFs are complete, they will be reviewed and signed by the Principal Investigator or 
co-PI.  All discrepancies identified will be reviewed, and any resulting queries will be resolved with 
the Principal Investigator (or co-PI) and the CRFs will be amended as needed.  
The Principal Investigator (or co-PI), through the use of an internal Quality Management Plan, 
appropriate site quality control, and quality assurance monitoring staff, will be responsible for the 
regular review of the conduct of the trial, for verifying adherence to the protocol, and for confirming 
the completeness, consistency, and accuracy of all documented data and accuracy of source 
documentation verification.  The reports of the internal site monitor will be submitted to the 
Principal Investigator (or co-PI).  
As per the clinical monitoring plan, site monitoring will be conducted by the independent site 
monitor in accordance with established Good Clinical Practices (ICH GCP 5.1.1, 5.2, 5.18.1) and 
the Code of Federal Regulations, as applicable.  The overall objectives of site monitoring visits 
are to ensure: 
1. 
Site compliance with the current version of the approved protocol, consent, 
documents and local Institutional Review Board requirements. 
2. 
Accuracy and completeness of data entry. 
3. 
Required regulatory documents are current and maintained in the protocol-specific 
regulatory binder. 
4. 
A designated percent of signed consent forms, inclusion/exclusion criteria, primary, 
secondary, and tertiary endpoints, safety monitoring parameters, deviations, and 
serious adverse events are documented. 
5. 
Procedures are in place to administer and monitor study drug / product accountability 
and documentation of destruction policies. 


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6. 
The research staff is adequately trained with respect to GCP and GLP. 
7. 
All observed anomalies or protocol deviations are reported and identify an action 
plan to minimize study dropouts and non-compliance with defined study procedures. 
The results of the site monitoring report will be discussed with the PI, the staff to ensure 
compliance with the monitor’s findings.  
 
 


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12 
ETHICAL CONSIDERATIONS AND COMPLIANCE WITH 
GOOD CLINICAL PRACTICE 
12.1 Statement of Compliance 
This study was designed to ensure the protection of subjects according to the ethical principles 
of the Declaration of Helsinki and amendments concerning medical research in human subjects. 
This clinical study will be conducted using current good clinical practice (cGCP), as delineated in 
Guidance for Industry: E6 Good Clinical Practice Consolidated Guidance (1), and according to the 
criteria specified in this study protocol. Before study initiation, the protocol the informed consent 
documents will be reviewed and approved by IRB, as well as any other appropriate health 
authorities. Any amendments to the protocol or to the consent materials will also be approved by 
the appropriate bodies listed above prior to implementation. 
12.2 Informed Consent Process 
The informed consent form will provide information about the study to a prospective subject or 
subject’s legal representative to allow for an informed decision about participation in the study. 
Prospective subject or subject’s legal representative must be given ample opportunity to review 
the informed consent and inquire about the results of the study. All subjects (or their legally 
acceptable representative) must read, sign, and date a consent form prior to study participation. 
Consent materials for subjects who do not speak or read English will be translated into the 
subjects’ appropriate language. 
The informed consent form will be revised and receive IRB approval whenever important new 
safety information is available, whenever the protocol is amended, and/or whenever any new 
information becomes available that may affect participation in the trial. 
A copy of the informed consent form will be given to a prospective subject for review prior to any 
study procedure. The Principal Investigator or an approved designee will discuss the consent with 
the prospective subject and answer questions. The prospective subject will be told that being in 
the trial is voluntary and that he or she may withdraw from the study at any time, for any reason. 


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13 
PUBLICATIONS 
Publication of any data from this study must be carried out in agreement of sponsor and 
investigators. 
 
 


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Lozupone, C. and R. Knight, UniFrac: a new phylogenetic method for comparing 
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Carvalho, F.A., et al., Transient inability to manage proteobacteria promotes chronic gut 
inflammation in TLR5-deficient mice. Cell Host Microbe. 12(2): p. 139-52(2012). 
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Tusher, V.G., R. Tibshirani, and G. Chu, Significance analysis of microarrays applied to 
the ionizing radiation response. Proc Natl Acad Sci U S A. 98(9): p. 5116-21 (2001). 
54. 
Storey, J.D. and R. Tibshirani, Statistical significance for genomewide studies. Proceedings 
of the National Academy of Sciences of the United States of America. 100(16): p. 9440-5 (2003).  
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A. Subramanian et al., Gene set enrichment analysis: a knowledge-based approach for 
interpreting genome-wide expression profiles. Proceedings of the National Academy of 
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Responses to IIV in adults with or without antibiotics. 
         Version 6. 0
 
May 13, 2016 
56. Oh JZ, Ravindran R, Chassaing B, Carvalho FA, Maddur MS, Bower M, Hakimpour P, Gill 
KP, Nakaya HI, Yarovinsky F, Sartor RB, Gewirtz AT, Pulendran B. TLR5-mediated sensing of 
gut microbiota is necessary for antibody responses to seasonal influenza vaccination. Immunity. 
2014 Sep 18;41(3):478-92 


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15 
APPENDICES 
15.1 Appendix A: Schedule of Events 
 
Day 
D-21 
D-1 
D-6 
D-3f 
D0 
D1 
D3 
D7 
+/-1 
D30 
+/-5 
D90 
+/-14 
D180 
+/-14 
Visit 
1 
 
2 
3 
4 
5 
6 
7 
 
D
+
Informed consent & HIPPA  
X 
 
 
 
 
 
 
 
 
Demographic and Medical History (including 
medication and vaccine history). 
X 
 
 
 
 
 
 
 
 
Verify eligibility 
X 
 
X 
 
 
 
 
 
 
PID assignment 
X 
 
 
 
 
 
 
 
 
Vital signs, height and weight 
X 
 
X 
 
 
 
 
 
 
Focused physical exam (only if indicated based on 
review of health status) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Urine pregnancy test (for female volunteers of 
childbearing potential) 
X 
X 
X 
 
 
 
 
 
 
Stool sample collection 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Randomization 
X 
 
 
 
 
 
 
 
 
Assessment of health status while on antibiotics 
(Group A only)a 
 
 
X 
X 
 
 
 
 
 
Distribute assigned antibiotics and instruct subject 
when and how to take (Group A only)  
 
X 
 
 
 
 
 
 
 
Vaccination 
 
 
X 
 
 
 
 
 
 
Study drug accountability (Group A only) 
 
 
X 
X 
X 
 
 
 
 


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 ____________________________________________________________________________________________ 
 
Day 
D-21 
D-1 
D-6 
D-3f 
D0 
D1 
D3 
D7 
+/-1 
D30 
+/-5 
D90 
+/-14 
D180 
+/-14 
Visit 
1 
 
2 
3 
4 
5 
6 
7 
 
D
+
Instruction given to study subjects on safety events and 
concomitant medications.b 
 
X 
 
X 
X 
X 
X 
X 
 
 
Assessment of health status c and concomitant 
medications after administration of study vaccine 
 
 
X 
X 
X 
X 
X 
X 
X 
Blood draw for Innate Assays and Adaptive Assays d 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Blood draw for safety labse 
X 
 
 
 
 
X 
 
 
 
 
 
Footnotes: 
 
a  Subjects in Group A are asked to report any symptoms experienced while on antibiotics (from D-3 until D1).  
b  Any Adverse Event including – but not limited to- vaccine reactions and local or systemic Reactogenicity Events of grade 3 or higher severity or serious adverse 
event (SAE) occurring after vaccination while the subjects is still at the clinical site will be recorded and reported. 
c  Instruction includes the following: 
1. Subjects will be provided (on Day 0 only) with a written description of local and systemic vaccine reactions of mild, moderate and severe intensity and 
instructed to call the site to report reactogencity events of grade 3 (severe) or higher severity within 1 week following vaccination. 
2. Subjects will also be instructed to promptly call the site if he/she develops any of the following: 
• 
Illness or treatment from a physician or emergency department; and/or hospitalization due to any illness throughout the entire duration of the 
study; 
• 
Any adverse event that limits self-care activities of daily living (e.g.  bathing, dressing and undressing, feeding self, using the toilet, taking 
medications, and not bed ridden) even if he/she decides not to seek medical care; or 
• 
Any of the following symptoms until D90 (Group A subject only): 
o 
diarrhea (≥ 3 unformed stools/24h) for more than 72h with either fever or abdominal pain* 
o 
tinnitus** 
o 
vertigo** 
o 
decrease hearing** 
o 
numbness/tingling** 
• 
he/she starts/stops medications during enrollment in the study. 
d  Innate assays will not be conducted at day 30. Adaptive assays will not be conducted at day 1. 
e  Safety labs include CBC with differential, creatinine, and potassium.  
F Only for Group A 


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15.2 Appendix B. Severity Scale for Local Vaccine Reactions 
(local reactogenicity events) 
 
INJECTION SITE REACTIONS 
 
Grade 
 
1 
2 
3 
Swelling/ 
Induration/ 
Mild induration, able to 
move skin parallel to 
plane (sliding) and 
perpendicular to skin 
(pinching up) 
Moderate induration, 
able to slide skin, 
unable to pinch skin; 
limiting instrumental 
activities of daily living 
Severe induration, unable to 
slide or pinch skin; limiting arm 
movement limiting self-care 
activities of daily living 
Redness/ 
erythema 
Asymptomatic or mild 
symptoms; intervention 
not indicated 
Moderate; minimal, 
local; limiting age-
appropriate 
instrumental activities 
of daily living 
Severe but not immediately 
life-threatening; hospitalization 
or prolongation of existing 
hospitalization indicated; 
disabling;  limiting self-care 
activities of daily living 
Pain/tenderness 
Mild 
Moderate pain; limiting 
instrumental activities 
of daily living 
Severe pain; limiting self-care 
activities of daily living 
Note: 
Instrumental Activities of Daily Living refer to preparing meals, shopping for groceries or clothes,  
using the telephone, managing money, etc. 
Self-care Activities of Daily Living refer to bathing, dressing and undressing, feeding self,  
using the toilet, taking medications, and not bedridden. 
  
 
 
 
 
 
 
 
 
 


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15.3 Appendix C. Severity Scale for Generalized Vaccine 
Reactions (systemic reactogenicity events) 
 
GENERAL ADVERSE REACTIONS  
 
Grade 
 
1 
2 
3 
Fatigue/astheni
a 
Fatigue relieved by 
rest 
Fatigue not relieved by 
rest; 
limiting instrumental 
activities of daily living 
Fatigue not relieved by rest, 
limiting self-care activities of 
daily living 
Body 
ache/myalgia 
Mild pain 
Moderate pain; limiting 
instrumental activities of 
daily living 
Severe pain; limiting self-care 
ADL 
Headache 
Mild pain 
Moderate pain; limiting 
instrumental activities of 
daily living 
Severe pain; limiting self-care 
activities of daily living 
Joint pain 
Mild pain 
Moderate pain; limiting 
instrumental activities of 
daily living 
Severe pain; limiting self-care 
activities of daily living 
Chills 
Mild sensation of 
cold; 
shivering; chattering 
of teeth 
Moderate tremor of the 
entire 
body; narcotics indicated 
Severe or prolonged, not 
responsive to narcotics 
Fever 
38.0 - 39.0 degrees 
C (100.4 - 102.2 
degrees F) 
>39.0 - 40.0 degrees C 
(102.3 - 104.0 degrees F) 
>40.0 degrees C (>104.0 
degrees F) for <=24 hrs 
Note: 
Instrumental Activities of Daily Living refer to preparing meals, shopping for groceries or clothes,  
using the telephone, managing money, etc. 
Self-care Activities of Daily Living refer to bathing, dressing and undressing, feeding self, using the  
toilet, taking medications, and not bedridden