An Exploration of a Prime-Boost Approach for Universal Influenza Vaccination: Immunogenicity and Safety Study of Inactivated Subunit H5N1 Influenza Vaccine in Prior Recipients of Live Attenuated H2N2, H6N1 and H9N2 Influenza Vaccines and in H5N1 and Live Attenuated Vaccine Naïve Individuals
Phase: PHASE1
Conditions: Influenza
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

An Exploration of a Prime-Boost Approach for Universal Influenza Vaccination: 
Immunogenicity and Safety Study of Inactivated Subunit H5N1 Influenza Vaccine 
in Prior Recipients of Live Attenuated H2N2, H6N1 and H9N2 Influenza Vaccines 
and in H5N1 and Live Attenuated Vaccine Naïve Individuals 
 
 
 
CIR Protocol Number  
 
CIR 327 
JHSPH IRB Protocol Number 
00008909 
FDA IND Number  
 
 
IND 14793 
Ionizing Radiation  
 
 
None  
Multi-institution  
 
 
No  
Project Assurance:  
 
 
FWA #00000287 
Protocol Version  
 
 
3.0 
Version Date    
 
 
12 April 2019  
 
 
 
 
 
 
 
 
 
IND Sponsored By: 
Office of Clinical Research Policy and Regulatory Operations (OCRPRO) 
Division of Clinical Research (DCR) 
National Institute of Allergy and Infectious Diseases (NIAID)  
National Institutes of Health (NIH)  
 
Study Funded By: 
The Bill and Melinda Gates Foundation 
NIAID, National Institutes of Health 
 

TABLE OF CONTENTS 
TABLE OF CONTENTS .................................................................................................... 2 
LIST OF TABLES .............................................................................................................. 5 
LIST OF APPENDICES ..................................................................................................... 5 
TEAM ROSTER ................................................................................................................. 6 
ABBREVIATIONS ............................................................................................................ 9 
PROTOCOL SUMMARY ................................................................................................ 10 
 
Background: Pandemic Influenza ................................................................................. 11 
 
Vaccine Description ...................................................................................................... 12 
 
Clinical Experience with the H5N1 inactivated vaccine .............................................. 12 
 
Rationale ....................................................................................................................... 13 
 
Participation of Children ............................................................................................... 15 
 
Statement of Compliance .............................................................................................. 15 
 
Hypothesis ..................................................................................................................... 16 
2. 
OBJECTIVES ........................................................................................................... 16 
 
Primary Objective: ........................................................................................................ 16 
 
Secondary Objective: .................................................................................................... 16 
 
Exploratory Objectives: ................................................................................................ 16 
3. 
STUDY DESIGN...................................................................................................... 16 
 
Summary ....................................................................................................................... 16 
 
Study Schedule .............................................................................................................. 17 
 
Study Procedures ........................................................................................................... 17 
 
Sample Size ................................................................................................................... 17 
 
Treatment assignment ................................................................................................... 17 
 
Duration of Participation ............................................................................................... 17 
4. 
SELECTION OF STUDY SUBJECTS .................................................................... 17 
 
Inclusion Criteria ........................................................................................................... 17 
 
Exclusion Criteria ......................................................................................................... 18 
 
Access to Medical Records ........................................................................................... 19 
 
Enrollment ..................................................................................................................... 19 
 
Subject Withdrawal/Termination Criteria ..................................................................... 20 
 
Replacement of Subjects ............................................................................................... 20 
 
Special Situations .......................................................................................................... 20 
5. 
VACCINE PREPARATION .................................................................................... 21 
 
Supplies ......................................................................................................................... 21 
 
Vaccine Storage and Accountability ............................................................................. 21 
 
Disposition of Used/Unused Supplies ........................................................................... 22 
6. 
STUDY PROCEDURES .......................................................................................... 22 
 
Recruitment and Screening (Up to 60 Days Prior to Vaccination) ............................... 22 
 
Recruitment ................................................................................................................... 22 
 
CIR 327 Study-Specific Screening and Informed Consent .......................................... 24 
 
Immunization Procedure ............................................................................................... 25 
 
Clinical Evaluation ........................................................................................................ 25 
 
Treatments that Could Potentially Interfere with Vaccine-Induced Immunity ............. 25 

 
Concomitant Medications ............................................................................................. 26 
 
Study Procedures By Day ............................................................................................. 26 
Study Day 0 (Day of Vaccination/Enrollment) ..................................................................... 26 
Study Day 7 ±1 Day ............................................................................................................... 26 
Study Day 14 ±2 Day ............................................................................................................. 27 
Study Day 28 ±7 Days ........................................................................................................... 27 
Study Day 56 ±7 Days ........................................................................................................... 27 
Study Day 90 ±14 Days ......................................................................................................... 27 
Study Day 360 ±30 Days ....................................................................................................... 27 
 
Clinical and Research Laboratory Testing .................................................................... 27 
 
Testing of previous samples .......................................................................................... 28 
7. 
RETENTION OF SPECIMENS FOR FUTURE USE ............................................. 28 
8. 
SAFETY AND ADVERSE EVENT REPORTING ................................................. 29 
 
Definitions ..................................................................................................................... 29 
8.1.1 
Adverse Events ......................................................................................................... 29 
8.1.2 
Solicited Adverse Events (Protocol-Specified Adverse Events) .............................. 30 
8.1.3 
Unsolicited Events .................................................................................................... 30 
8.1.4 
Adverse Reaction (AR) ............................................................................................. 30 
8.1.5 
Suspected Adverse Reaction (SAR) ......................................................................... 30 
8.1.6 
Serious Adverse Events ............................................................................................ 30 
8.1.7 
Unexpected Adverse Events ..................................................................................... 31 
8.1.8 
Suspected Unexpected Serious Adverse Reaction (SUSAR) ................................... 31 
8.1.9 
Unanticipated Problems ............................................................................................ 31 
8.1.10 
Serious Unanticipated Problem ............................................................................ 31 
8.1.11 
Unanticipated Problem that is Not an Adverse Event (UPnonAE) ...................... 31 
8.1.12 
Serious Protocol Deviation ................................................................................... 32 
8.1.13 
Protocol Deviation ................................................................................................ 32 
8.1.14 
Non-compliance [Definition is IRB dependent]: ................................................. 32 
 
Recording Period for Adverse Events ........................................................................... 32 
 
Association of Adverse Events with Receipt of Study Vaccine ................................... 33 
 
Assessment of Severity ................................................................................................. 33 
 
Investigator Reporting Responsibilities ........................................................................ 34 
8.5.1 
Adverse Event Reporting .......................................................................................... 34 
8.5.2 
Unanticipated Problems ............................................................................................ 35 
8.5.3 
Pregnancy ................................................................................................................. 36 
8.5.4 
Reporting Responsibilities to the JHSPH IRB ......................................................... 36 
 
Sponsor’s Reporting Responsibilities ........................................................................... 36 
 
Halting Rules ................................................................................................................. 36 
 
Safety Review and Communications Plan .................................................................... 37 
 
Sponsor Medical Monitor ............................................................................................. 37 
9. 
DATA COLLECTION AND MONITORING ......................................................... 37 
 
Source Documentation .................................................................................................. 37 
 
Study Documentation .................................................................................................... 38 
 
Retention of Records ..................................................................................................... 38 
 
Protocol Revisions ........................................................................................................ 39 
 
Study Monitoring .......................................................................................................... 39 
10. 
STATISTICAL CONSIDERATIONS.................................................................. 39 
 
Description of Statistical Methods ................................................................................ 39 

 
Sample Size Reasoning ................................................................................................. 40 
 
Primary Objective: ........................................................................................................ 40 
 
Primary Endpoint: ......................................................................................................... 40 
 
Secondary Objective ..................................................................................................... 40 
 
Secondary Endpoints: ................................................................................................... 40 
 
Exploratory Objectives: ................................................................................................ 40 
 
Exploratory Endpoints: ................................................................................................. 41 
11. 
HUMAN SUBJECT PROTECTIONS AND ETHICAL OBLIGATIONS .......... 41 
 
Institutional Review Board ........................................................................................... 41 
 
Informed Consent Process ............................................................................................ 42 
 
Participant Confidentiality ............................................................................................ 42 
 
Risks .............................................................................................................................. 43 
11.4.1 
Venipuncture ........................................................................................................ 43 
11.4.2 
Nasal Wash ........................................................................................................... 43 
11.4.3 
Immunization ........................................................................................................ 43 
 
Benefits ......................................................................................................................... 43 
 
Compensation ................................................................................................................ 43 
12. 
REFERENCES ..................................................................................................... 45 
Appendix A: Schedule of Study Procedures ..................................................................... 47 
Appendix B:  Local and Systemic Adverse Event Grading Systems ............................... 48 
Appendix C: H5N1 Vaccine Product Insert ...................................................................... 50 
Appendix D: H5N1 Vaccine Product label ....................................................................... 51 
 
 
 

 
LIST OF TABLES 
Table 1: Antibody Titers After pLAIV pISV Prime Boost Studies ------------------------- 14 
Table 2: Adverse Event Grading System ------------------------------------------------------- 34 
Table 3: Fever Grading System ------------------------------------------------------------------ 34 
Table 4: Local Reaction Grading System ------------------------------------------------------- 48 
Table 5: Vital Signs and Systemic Reactions Grading System ------------------------------ 49 
 
LIST OF APPENDICES 
Appendix A: Schedule of Study Procedures ---------------------------------------------------- 47 
Appendix B:  Local and Systemic Adverse Event Grading Systems------------------------ 48 
Appendix C: H5N1 Vaccine Product Insert ---------------------------------------------------- 50 
Appendix D: H5N1 Vaccine Product label ----------------------------------------------------- 51 
 
 
 

TEAM ROSTER 
 
Principal Investigator:  
 
Kawsar R. Talaat, MD 
Center for Immunization Research  
Johns Hopkins Bloomberg School of Public Health  
624 N. Broadway, Baltimore, MD 21205 
 
 
410-502-9627 (phone) 
email: ktalaat@jhu.edu 
 
Clinical Investigators: 
 
Ruth A. Karron, MD 
 
 
 
 
 
Brittany Feijoo, MSN, FNP-BC 
 
 
 
 
 
Katie Chang, BSN, RN, CCRP 
Center for Immunization Research  
Johns Hopkins Bloomberg School of Public Health  
 
 
Scientific Investigators: 
 
Kanta Subbarao, MD 
World Health Organization (WHO) Collaborating 
Centre for Reference and Research on Influenza 
 
 
 
 
 
 
Jeffrey Cohen, MD 
Yumiko Matsuoka, DVM, PhD 
 
 
 
 
 
Laboratory of Infectious Diseases 
National Institute of Allergy and Infectious 
Diseases 
National Institutes of Health 
 
 
 
 
 
 
Adrian McDermott MSc, PhD 
 
 
 
 
 
Sarah Andrews, PhD 
Vaccine Research Center 
National Institute of Allergy and Infectious 
Diseases 
National Institutes of Health  
 
 
 
 
 
 
Florian Krammer, PhD 
 
 
 
 
 
Raffael Nachbagauer, MD, PhD 
Department of Microbiology 
Icahn School of Medicine at Mount Sinai 
 
John McGready, PhD 
Department of Biostatistics 
Johns Hopkins Bloomberg School of Public Health 
 
RCHSPB Regulatory Affairs: 
John Tierney, RN, MPM 
Office of Clinical Research Policy and Regulatory 
Operations, Division of Clinical Research 
National Institute of Allergy and Infectious 

Diseases 
National Institutes of Health 
5601 Fishers Lane, Room 4B11 MSC 9820, 
Bethesda, MD 20892 
Phone: 301-451-5136 
Email: jtierney@niaid.nih.gov 
 
 
Sponsor Medical Monitor: 
Marc J. Teitelbaum, MD, MS 
Clinical Monitoring Research Program 
Frederick National Laboratory for Cancer Research 
Leidos Biomedical Research, Inc. 
Support to: Regulatory Compliance and Human 
Subjects Protection Program  
National Institute of Allergy and Infectious 
Diseases 
National Institutes of Health  
5705 Industry Lane 
Frederick, Maryland 21704 
Phone: 301-228-4707 
Fax: 301-846-6224 
Email: Marc.Teitelbaum@fnlcr.nih.gov 
 
 
Clinical Trial Site:  
Johns Hopkins Bloomberg School of Public Health 
Center for Immunization Research  
624 N. Broadway  
Baltimore, MD 21205  
410-955-1622 (phone) 
 
Virology/Immunology  
Laboratory: 
Center for Immunization Research Laboratories  
E5402 Hygiene Building  
615 N. Wolfe Street, Baltimore, MD 21205  
410-955-7230 (phone) 
 
Vaccine Immunogenicity Program 
Vaccine Research Center 
National Institute of Allergy and Infectious 
Diseases 
National Institutes of Health 
40 Convent Drive, RM 3613B 
Bethesda, MD 20892 
240-669-5558 (phone) 
  
Icahn School of Medicine at Mount Sinai 
One Gustave L. Levy Place 

New York, 10024 NY 
212-241-8166 (phone) 
 
Adaptive Biotechnologies 
San Francisco, CA 
(855) 466-8667 (phone) 
 
Clinical Laboratories: 
 
Quest Diagnostics  
1901 Sulphur Spring Road  
Baltimore, MD 21227  
410-247-0001 (phone)  
 
Johns Hopkins Medical Laboratories  
600 N. Wolfe Street / Meyer B-130  
Baltimore, MD 21287-7070  
410-614-0455 (fax) 
 
Institutional Review Board:  
Johns Hopkins Bloomberg School of Public Health 
IRB Office  
615 N. Wolfe Street, Suite E1100 
Baltimore, Maryland 21205 
Phone: 410-955-3193 
Toll-free: 1-888-262-3242 
Fax: 410-502-0584 
Email: JHSPH.irboffice@jhu.edu 
 

ABBREVIATIONS 
 
Abbreviation 
Definition 
Ab 
antibody 
AE 
adverse event 
AI 
avian influenza 
BARDA 
Biomedical Advanced Research and Development Authority 
ca 
cold-adapted (virus phenotype) 
CBC 
complete blood count 
CFR 
Code of Federal Regulations 
CIR 
Center for Immunization Research 
CRIMSON 
Clinical Research Information Management System of the 
NIAID 
CSO 
Clinical Safety Office 
ELISA 
enzyme-linked immunosorbent assay 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
H1N1, etc. 
hemagglutinin and neuraminidase subtypes of influenza virus 
HA 
hemagglutinin (influenza virus surface glycoprotein) 
HAI 
hemagglutination inhibition assay 
HBsAg 
Hepatitis B virus surface antigen 
HCV 
hepatitis C virus 
HIV 
human immunodeficiency virus 
IDS 
Investigational Drug Service 
IgA 
immunoglobulin A 
IgG 
immunoglobulin G 
IM 
intramuscular 
IND 
investigational new drug 
IRB 
Institutional Review Board 
ISV 
inactivated subvirion vaccine 
LAIV 
live attenuated influenza vaccine 
LID 
Laboratory of Infectious Diseases 
MN 
microneutralization assay 
NA 
neuraminidase (influenza virus surface glycoprotein) 
NIAID 
National Institute of Allergy and Infectious Diseases 
NIH  
National Institutes of Health 
OCRPRO 
Office of Clinical Research Policy and Regulatory Operations 
PI 
Principal Investigator 
pISV 
pandemic inactivated subvirion vaccine 
pLAIV 
pandemic live attenuated influenza vaccine 
rRT-PCR 
real-time reverse transcriptase polymerase chain reaction 
SAE 
serious adverse event 
SUSAR 
suspected unexpected serious adverse reaction 
TCID50 
50% tissue culture infectious dose 
b-HCG test 
test for human choriogonadotropin, an indicator of pregnancy 

PROTOCOL SUMMARY 
 
Protocol Title: 
An Exploration of a Prime-Boost Approach for Universal 
Influenza Vaccination: Immunogenicity and Safety Study 
of a H5N1 Influenza Vaccine in Prior Recipients of Live 
Attenuated H2N2, H6N1 and H9N2 Influenza Vaccines 
and in H5N1 and Live Attenuated Vaccine Naïve 
Individuals  
 
Protocol Version: 
 
V. 3.0 
April 12, 2019 
 
Revision History: 
Protocol Version 
Date 
1.0 
6 November 2018 
1.1 
19 November 2018 
1.2 
10 December 2018 
2.0 
27 February 2019 
 
Subjects: 
 
 
Healthy male and non-pregnant female subjects 18-60  
 
 
 
 
years of age 
 
Number of Subjects: 
Approximately 32-35 
  
 
Trial Design: 
Open-label outpatient trial of 1 dose of H5N1 pISV 
 
Immunization Schedule: 
 
Cohort Number of 
Subjects 
Previous Vaccine 
Number of doses 
of inactivated 
H5N1 vaccine 
1 
12-15 
2 doses of pandemic live attenuated influenza 
vaccine (pLAIV) H2N2, H6N1 or H9N2 
1 
2 
20 
Naive 
1 
 
Product Description: 
Monovalent Influenza H5N1 Subvirion Vaccine 
(A/VietNam/1203/04 (H5N1, clade 1) vaccine, lot number 
UD08916 
 
 
 
 

1. INTRODUCTION 
 Background: Pandemic Influenza 
Influenza is a negative-sense, single-stranded RNA virus belonging to the family 
Orthomyxoviridae that consists of 4 genera: influenza A, influenza B, influenza C, and 
Thogoto viruses. The proteins of influenza A are encoded by genes on 8 RNA segments. 
Influenza A viruses are widely distributed in nature and can infect a wide variety of birds 
and mammals, including humans. Influenza A virus subtypes are classified on the basis 
of the antigenicity of their surface glycoproteins, hemagglutinin (HA) and neuraminidase 
(NA). 
 
Sixteen HA subtypes and 9 NA subtypes of influenza A viruses have been identified that 
infect birds. Waterfowl are the natural reservoir of avian influenza (AI) viruses. Most AI 
infections in birds are not associated with overt disease, and the viruses are generally 
thought to be in evolutionary stasis in these hosts. This is in contrast to influenza A 
viruses in the human population where relatively few subtypes have caused sustained 
outbreaks of disease: viruses bearing H1, H2, and H3 HA and N1 and N2 NA genes have 
circulated in the human population throughout the 20th century. H1N1 viruses first 
appeared in 1918 and circulated until 1957 when they were replaced by H2N2 viruses. 
These in turn were replaced in 1968 by H3N2 viruses that continue to circulate at the 
present time. In 1977, H1N1 viruses reappeared and have continued to co-circulate with 
the H3N2 viruses. The H1N1 virus circulating previously was replaced by the 2009 
pandemic H1N1 virus. Influenza A and B viruses cause epidemics in humans each 
winter.  
 
As seen in 2009, in addition to the seasonal influenza epidemics, the potential also exists for 
an influenza pandemic at any time. This occurs when an influenza strain with a novel HA 
subtype (with or without a novel NA subtype) appears in the human population. In the 20th 
century, pandemics occurred in 1918, 1957, and 1968, and were associated with significant 
morbidity and mortality (1). The 2009 H1N1 pandemic caused approximately 10,000 deaths 
in the United (2), 95% of whom were under the age of 65. It is estimated that, in the United 
States alone, the next influenza pandemic could cause 89,000 to 207,000 deaths, and 
314,000 to 734,000 hospitalizations, as well as tens of millions of outpatient visits and 
illnesses, depending on the severity of the pandemic (3). 
 
During the 2009 H1N1 pandemic, robust antibody responses to the HA stalk epitope were 
detected following infection or vaccination. This led Li and colleagues to propose that 
rare B cells directed against the conserved stalk epitope were amplified because the 
immunodominant head region of the HA was novel (4). Pandemic H1N1 influenza 
vaccine induces a recall response in humans that favors broadly cross-reactive memory 
cells. Subsequently, Krammer and Palese have proposed that sequential immunization 
with influenza viruses bearing chimeric HAs with a conserved HA stalk and different HA 
head domains will amplify the antibody response to the HA stalk (5). 
 
In three independent clinical trials, we have demonstrated that pandemic live attenuated 
influenza vaccine (pLAIV) establishes long lasting immune memory that is detected 

when pLAIV recipients receive a dose of pandemic inactivated subvirion vaccine (pISV) 
(6-8).  
 
However, we have not administered a pISV bearing group 1 HA to pLAIV recipients of a 
mismatched group 1 pLAIV. We have administered a group 1 pISV (H5N1 VN04) to 
recipients of an H7N3 pLAIV and a group 2 pISV (H7N7 Netherlands03) to recipients of 
an H2N3 pLAIV(6, 7). We have previously administered group 1 pLAIVs (H2N2, H6N1, 
H9N2) to study subjects in Baltimore (9-11). This study will examine the effect of a 
group 1 pISV (H5N1 VN04) vaccine to previous recipients of a different group 1 pLAIV 
recipients (H2N2, H6N1 and H9N2). 
 
 Vaccine Description 
Monovalent Influenza Subvirion Vaccine (H5N1) is an inactivated unadjuvanted 
influenza vaccine. The vaccine is a sterile suspension prepared from influenza virus 
propagated in embryonated chicken eggs. The vaccine is manufactured by Sanofi Pasteur 
Inc., Swiftwater, PA. The virus-containing fluids are harvested and inactivated with 
formaldehyde. The influenza virus is concentrated and purified in a linear sucrose density 
gradient solution using a continuous flow centrifuge. The virus is then chemically 
disrupted using a nonionic surfactant, polyethylene glycol p-isooctylphenyl ether (Triton® 
X-100), producing a “split virus”. The split virus is then further purified by chemical 
means and suspended in sodium phosphate-buffered isotonic sodium chloride solution. 
The vaccine is formulated to contain 90  µg HA per 1.0 mL of the influenza virus strain 
A/Viet Nam/1203/04 (H5N1, clade 1). Porcine gelatin (500 µg per 1.0 mL) is added as a 
stabilizer. Thimerosal, a mercury derivative, is added as a preservative to a concentration 
of not more than 98.2 µg thimerosal per 1.0 mL (approximately 50 µg mercury per 1.0 
mL). The vaccine may also contain residual amounts of formaldehyde (not more than 200 
µg per 1.0 mL), polyethylene glycol p-isooctylphenyl ether (not more than 0.05%), and 
sucrose (not more than 2.0%). We will be administering 0.5 mL, or 45 µg HA per each 
subject. 
 
The vaccine is provided as a suspension in 5 mL multi-dose vials and should be 
administered as an intramuscular (IM) injection. The vaccine should be stored between 2 
to 8°C. The vaccine should not be frozen.  
 
The vaccine is licensed for active immunization of persons 18-64 years of age at 
increased risk of exposure to the H5N1 influenza virus subtype contained in the vaccine. 
The clinical trial material for this study is a lot prepared specifically for investigational 
use, lot number UD08916. The vaccine will be provided by the Biomedical Advanced 
Research and Development Authority (BARDA), U.S. Department of Health and Human 
Services (DHHS). The product label is provided in Appendix D.  
 
 
 Clinical Experience with the H5N1 inactivated vaccine 
A prospective, randomized, double-blinded, placebo-controlled, dose-ranging Phase 1-2 
study was conducted in 451 healthy subjects aged 18-64 years (12). The vaccine was well 
tolerated. Mild pain at the injection site was the most common adverse event (AE) for all 

doses of vaccine evaluated (90, 45, 15, or 7.5 µg). The number of subjects with an 
antibody response was highest among those who received two doses of 90 µg of vaccine: 
HI titers of ≥1:40 were observed in 58% of subjects in this group, and 54% of subjects in 
this group had microneutralization assay (MN) titers of ≥1:40. This vaccine was 
subsequently licensed for use in healthy adults aged 18-64 years of age at increased risk 
of H5N1 influenza infection (

Version 3.0, 12April2019 
Appendix C: H5N1 Vaccine Product Insert). 
 
 Rationale 
Unadjuvanted inactivated H5N1 vaccines have been shown to be poorly immunogenic in 
humans, whether administered as inactivated or live attenuated vaccines (12, 13). 
Previously, it has been demonstrated that subjects who were primed with an inactivated 
H5N1 vaccine and then several years later receive another inactivated H5N1 vaccine 
developed significantly greater HI and MN antibody titers than unprimed subjects (14, 
15).  
 
In 2006 and 2007, the safety, infectivity and immunogenicity of 2 live attenuated H5N1 
vaccines, H5N1 VN 04 cold-adapted (ca) and H5N1 HK 03 ca, were evaluated in healthy 
adults (13). Nineteen subjects received 2 doses of 107.5 50% tissue culture infectious dose 
(TCID50) of H5N1 VN 04 ca. Of these subjects, 15 had vaccine virus detected by real-
time reverse transcriptase polymerase chain reaction (rRT-PCR) after either dose, 
however, only 10% of subjects had measurable antibody responses by HI assay, and 5% 
by MN assay. Fifty two percent of subjects had measurable anti-H5 serum 
immunoglobulin A (IgA) antibodies. Sixteen subjects received 2 doses of 107.5 TCID50 of 
H5N1 HK 03 ca. Vaccine virus was detected by rRT-PCR in 12 subjects after either 
dose, and none of the subjects had measurable HI or MN antibodies after vaccination. 
Three subjects had increases in serum anti-H5 serum IgA. 
 
When the inactivated, unadjuvanted H5N1 subvirion vaccine was evaluated in healthy 
adults, 2 doses of 90 µg of vaccine were required to elicit a 4-fold or greater rise in either 
HI or MN titer against the A/Vietnam/1203/2004 (H5N1) virus in the majority of subjects 
(12). Other unadjuvanted H5N1 vaccines were also poorly immunogenic (reviewed in 
(16)). Inactivated and virosomal H5N1 vaccines, while poorly immunogenic, do elicit 
stalk antibody responses (17, 18). 
 
Although initial immunization with the unadjuvanted inactivated H5N1 vaccine or with 
live attenuated H5N1 vaccines generally induced inadequate antibody responses, some 
data suggest that immune priming may occur which is only revealed when individuals are 
revaccinated with H5 vaccine. For example, Goji et al. demonstrated that subjects who 
had received a clade 0 inactivated H5N1 vaccine (derived from A/Hong Kong/156/97) 
were primed to respond years later to a clade 1 H5N1 vaccine (derived from 
A/VietNam/1203/04), and that their antibody responses to the clade 1 vaccine were 
substantially greater than those observed in H5-naïve subjects (14).  
 
Previously we sought to determine whether the H5N1 VN 04 ca and H5N1 HK 03 ca 
vaccines induced subclinical priming that could be revealed when subjects who received 
these vaccines are immunized with the inactivated H5N1 subvirion vaccine. We found 
that use of the H5NI subvirion vaccine increased frequency of antibody response 82% by 
the hemagglutination inhibition assay and displayed a significantly higher antibody titer 
of 112 compared to 76 in naïve subjects. The affinity of antibody and breadth of cross-
clade neutralization was also enhanced in the primed subjects.  

Version 3.0, 12April2019 
  
15 
We found (Table 1 below) that the head-specific antibody (Ab) response in pLAIV 
prime-pISV boost scenarios is not affected by the interval between pLAIV prime and 
pISV boost. We saw similar percent seroconversion and geometric mean titers (GMTs) in 
3 independent clinical trials with different pLAIV/pISV pairs administered at different 
intervals, from 3 months to 56 months (6-8). 
 
Table 1: Antibody Titers After pLAIV pISV Prime Boost Studies 
pLAIV 
pISV 
Interval 
pLAIV-
pISV 
HAI Ab titer following receipt of pISV 
Vaccine 
# doses 
n 
GMT 
(range) 
Responders 
% 
GMT 
H5N1 
VN04 
2 
H5N1 
VN04 
56 m 
11 
48 
(5-1280) 
64 
165 
H7N7 
NL03 
2 
H7N7 
NL03 
19-24 m 
13 
34 
(2-1024) 
69 
119 
H7N9 
Anhui/13 
2 
H7N9 
Anhui/13 
3 m 
14 
74  
(2-512) 
79 
175 
HAI = hemagglutination inhibition assay 
 
We have evidence that pLAIV primes for a stalk-specific response (19, 20). However, in 
each of our prime-boost studies, the inactivated vaccine boost that recalled the response 
primed by the pLAIV was matched for the HA head and as Halliley et. al. and Henry 
Dunand et. al. have shown, this approach boosts the head-specific antibodies to a much 
greater extent than the stalk-specific antibodies (19, 20). In the proposed study, our 
objective is to determine whether boosting with an inactivated vaccine bearing a 
mismatched head, but shared stalk will boost the stalk response preferentially. Preclinical 
studies have demonstrated that prime-boost strategies using influenza viruses with 
mismatched HA heads and conserved stalks boost the antibody response to the HA stalk 
(21, 22).  
 
All participants in the study will have been exposed to group 1 stalk via exposure to 
H1N1pdm09 viruses since 2009. However, the subjects that received pLAIV 5-10 years 
ago differ from the control cohort in having received two doses of a different group 1 
virus (H2N2 or H6N1 or H9N2) followed by interim exposure to H1N1pdm09 viruses 
and vaccines.  
 
The first-time people were vaccinated against H1N1pdm09, they had a very robust stalk-
specific response, presumably because the HA head was novel, and the stalk was 
conserved. However, on revaccination, the head-specific response became dominant and 
the stalk-specific response was not detected (23). These findings are consistent with 
Ellebedy et. al. findings that there was no rise in stalk-specific antibody titers over a 4-
year period from 2010 to 2014 (18). The data from these studies indicate that (1) although 
stalk-specific immunoglobulin G (IgG) + memory B cells are detectable, they are 
minimally boosted by seasonal influenza vaccine that contains the H1N1pdm09 HA and 
(2) over time and re-exposure, the head-specific response dominates over the stalk-
specific response.  
 

Version 3.0, 12April2019 
  
16 
The data from Ellebedy et. al. (18) suggest that it is likely that the H5N1 pISV will 
induce a stalk-specific response in the control cohort as well as the pLAIV primed 
subjects, but we hypothesize that recipients of the H2N2, H6N1 and H9N2 pLAIVs will 
have more memory B cells against the group 1 stalk than the control cohort because they 
differ from the control cohort in having received two doses of a different group 1 virus 
(H2N2 or H6N1 or H9N2). It is now 9 years since the emergence of the 2009 pandemic 
H1N1 virus and people would have been exposed to the virus or vaccine more than once 
so they will have a predominantly head-specific response to H1N1pdm09 as reported by 
Andrews et. al. (23).  
 
Additionally, Yassine et. al. (24), reported that not all broadly neutralizing antibodies are 
created equal; using stalk stabilized probes against H1 seasonal, H1 pdm09, H2, H5 and 
H9 HA stems, they found that ~45% of 202 people had antibodies that bound the stalks 
of non-circulating viruses (H2, H5, H9) and 30% had antibodies that bound 4 of the 5 
probes. They reported that sera collected either prior to the 2009 pandemic or afterwards 
showed no differences in cross-reactivity and they observed no difference in the profiles 
of cross-reactive sera from vaccinated and non-vaccinated subjects collected after the 
H1N1 pandemic emergence. However, there was a statistically significant boost in 
specific stalk antibody titers to H1 CA 09 HA SS probe in subjects that received 
pandemic H1N1 influenza vaccine compared to those that did not receive the vaccine 
within the same time period. They also noted a higher rate of cross-reactive antibodies in 
the older population, especially those born within or before the period of H2N2 pandemic 
(24).  
We will use similar probes (25) to detect stalk antibodies in the sera from this trial and 
hypothesize that on administration of an H5N1 inactivated vaccine, we will see a stalk-
specific antibody response in both cohorts but the magnitude of the response and the 
breadth of the stalk antibody response in the pLAIV primed subjects will be greater than 
in the controls. 
In summary, we hypothesize that people that have been primed with pLAIVs bearing 
novel HAs will develop antibodies that react more broadly than people whose only 
exposure to a novel HA head was H1N1pdm09.  
 
 Participation of Children 
It is felt that insufficient data are available to judge the potential risk in children. As such, 
children will not be enrolled in this study. In addition, to date, children have not received 
the H2, H6 or H9 ca vaccines. 
 
 Statement of Compliance 
This trial will be conducted in compliance with the protocol, International Conference on 
Harmonisation/Good Clinical Practice (ICH/GCP) guidelines, Food and Drug 
Administration (FDA) regulations (21 Code of Federal Regulations [CFR]), and the 
Johns Hopkins Bloomberg School of Public Health Institutional Review Board.  
 
 Hypothesis 

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Recipients of the H2N2, H6N1 and H9N2 pLAIVs will have more memory B cells 
against the group 1 stalk than the control cohort, whose only exposure to a novel HA 
head was H1N1pdm09. Therefore, the magnitude of the response and the breadth of the 
stalk antibody response in the pLAIV primed subjects will be greater than in the controls 
following administration of an H5N1 inactivated vaccine.  
 
2. OBJECTIVES 
 Primary Objective:  
To explore the difference in the stalk antibody response against Influenza A group 1 
stalks as measured by enzyme-linked immunosorbent assay (ELISA) and neutralization 
titer after receipt of the Monovalent Influenza Subvirion Vaccine (H5N1) in previous 
recipients of H2N2, H6N1 and H9N2 pLAIVs compared with healthy, pLAIV-naïve 
individuals. 
 
 Secondary Objective: 
Assess the reactogenicity of the Monovalent Influenza Subvirion Vaccine (H5N1) in 
previous recipients of live attenuated influenza vaccine (LAIV) compared with healthy, 
pLAIV-naïve individuals.  
Adverse events will be assessed, graded and recorded for 28 days after vaccination. 
Serious Adverse Events (SAEs) will be assessed for 90 days after the immunization. 
 
 Exploratory Objectives: 
• To assess the biological activity of antibodies to the HA stalk region by passive 
transfer of serum from study subjects to mice to assess their ability to protect the mice 
from lethal challenge with an influenza virus. 
• To correlate ELISA and neutralizing stalk antibody titers with functional ability of the 
post vaccine serum to protect mice from lethal challenge. 
• To assess the breadth of the vaccine induced antibody response. 
 
• To assess plasmablast and memory B cell responses to the pISV in the previous 
pLAIV recipients compared with naïve individuals. 
• To assess the longevity of the stalk antibody response. 
• To determine the titer of anti-H5 head antibodies in the 2 cohorts of subjects. 
• To determine the titer of antibodies directed to the HA head received in the pLAIV 
doses. 
3. STUDY DESIGN 
 Summary 
This open-label, outpatient, exploratory study will recall study subjects who previously 
received a group 1 pLAIV (H2N2, H6N1 or H9N2) and administer 1 dose of pISV (e.g. 
H5N1 pISV) to determine whether the HA stalk antibody response is amplified following 
administration of pISV H5N1 with a mismatched HA head and conserved HA stalk.  
 
A control cohort of naive subjects will receive H5N1 pISV.  
 
All subjects will be followed for approximately 360 days after receipt of the H5N1 pISV. 

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 Study Schedule 
The study will be divided into 3 phases: 
1. Screening, eligibility determination and consent up to 60 days prior to 
vaccination/enrollment (usually 2 visits initiated under a separate institutional 
review board (IRB)-approved screening protocol) 
2. Vaccination/Enrollment (Day 0)  
3. Outpatient follow-up at 7, 14, 28, 56, 90, and 360 days post vaccination 
 
 Study Procedures  
(See Section 6  for detailed description of study procedures) 
 
 Sample Size 
Up to 35 healthy adults will be selected to receive 1 dose of H5N1 pISV. Up to 15 of 
these subjects will have previously received group 1 pLAIVs (H2N2, H6N1 or H9N2), 
and 20 of these subjects will be naïve. More than 35 subjects may be consented, 
vaccinated and enrolled (see Replacement of Subjects) if necessary to achieve enrollment 
goals. The Medical Monitor/Safety Office and the Clinical Trials Monitor will be notified 
promptly by email in advance if the study team anticipates that study vaccinations will 
exceed 38 subjects per replacement procedure. 
 
 Treatment assignment 
All subjects enrolled in the study will receive 1 dose of H5N1pISV 
 
 Duration of Participation 
Duration of individual subject participation in the trial is approximately 360 days from 
time of vaccination (not including 1 – 2 screening visits prior to enrollment) and will 
consist of a vaccination/enrollment visit and 6 follow up visits.  The duration of the 
study, from the time of the first subject vaccination to the end of the last subject study 
visit, is estimated to be approximately 18 months. 
4. SELECTION OF STUDY SUBJECTS 
Individuals who previously received H2N2, H6N1, or H9N2 pLAIV as participants in 
CIR vaccine studies and who indicated they would be willing to participate in future 
studies, will be contacted and invited to participate in this study, as described in 
Section 6. Healthy subjects will be recruited from the general population for the LAIV 
negative controls.  
 
 Inclusion Criteria 
All the following criteria must be met for a subject to be enrolled in this trial: 
1. Adult males and non-pregnant females between 18 years and 60 years of age 
inclusive.  
2. General good health, without significant medical illness, physical examination 
findings, or significant laboratory abnormalities as determined by the investigator.  
3. Available for the duration of the trial.  

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4. Able to demonstrate understanding of key study concepts, study rationale, and 
study participation requirements by scoring ≥ 70% on a written comprehension 
assessment in ≤ 3 attempts. 
5. Willingness to participate in the study as evidenced by signing the informed 
consent document. 
6. Willingness to allow storage and testing of laboratory samples for future research. 
7. Received 2 doses of live attenuated H2N2, H6N1, or H9N2 vaccine in a prior trial 
(Cohort 1) or H2N2, H6N1 and H9N2 naïve (Cohort 2) 
8. Willingness to forego seasonal influenza virus vaccination from 1 month before 
vaccination until 3 months after vaccination. 
9. Female subjects of childbearing potential must agree to have used effective birth 
control methods beginning at least one month prior to vaccination, and continuing 
with ‘per label/fully effective use’ for the chosen method for duration of the 
study, from amongst these: 
• pharmacologic/hormonal contraceptives, including oral, parenteral, 
subcutaneous,  and transcutaneous delivery; 
• condoms with spermicide; 
• diaphragm with spermicide; 
• intrauterine device; 
• absolute abstinence from heterosexual intercourse as a matter of normal 
preferred lifestyle; 
 
or must be surgically sterile or must be age 50 AND have had no menses at all 
for at least one full year. 
 
All females must provide urine for pregnancy testing prior to enrollment 
(immediately prior vaccination), as well as a statement of menstrual history 
and a summary of all potentially reproductive sexual activity for the month 
prior to vaccination, and at each study contact throughout the study, and report 
known or suspected pregnancy immediately.  
10. Willingness to refrain from blood donation during the course of the study. 
 
 Exclusion Criteria 
The presence of any one of the following criteria is sufficient to exclude a prospective 
subject from enrolling in this study: 
1. Pregnancy as determined by a positive test for human choriogonadotropin (b-
HCG), an indicator of pregnancy or history of recent unprotected intercourse in a 
woman of reproductive capacity. 
2. Currently breast-feeding. 
3. Evidence of clinically significant neurologic, cardiac, pulmonary, hematologic, 
hepatic, rheumatologic, autoimmune, or renal disease by history, physical 
examination, and/or laboratory studies.  
4. Behavioral or cognitive impairment or psychiatric disease that in the opinion of 
the investigator affects the ability of the subject to understand and cooperate with 
the study protocol. 

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5. Have medical, occupational, or family problems as a result of alcohol or illicit 
drug use during the past 12 months. 
6. Other condition that in the opinion of the investigator would jeopardize the safety 
or rights of a subject participating in the trial or would render the subject unable 
to comply with the protocol. 
7. History of anaphylaxis in response to any vaccine or vaccine component. 
8. History of life-threatening reaction to prior influenza vaccine. 
9. Positive enzyme-linked immunosorbent assay (ELISA) and confirmatory test for 
human immunodeficiency virus type 1 (HIV-1). 
10. Positive ELISA and confirmatory test (e.g., HCV RNA PCR) for hepatitis C virus 
(HCV). 
11. Positive hepatitis B virus surface antigen (HBsAg) by ELISA. 
12. Known immunodeficiency syndrome or history suggestive of impaired immune 
function. 
13. History of Guillain-Barré syndrome. 
14. Use of chronic oral or intravenous administration (≥14 days) of 
immunosuppressive doses of steroids, i.e., prednisone >10 mg per day, 
immunosuppressants or other immune-modifying drugs within 30 days of starting 
this study. 
15. Receipt of a live vaccine within 4 weeks or a killed vaccine within 2 weeks prior 
to study vaccination. 
16. Receipt of blood or blood-derived products (including immunoglobulin) within 6 
months prior to study vaccination. 
17. Receipt of another investigational vaccine or drug within 30 days prior to study 
vaccination. 
 
In addition to the above, participants in Cohort 1 (pLAIV recipients) and Cohort 2 
(naïve cohort) must also not experience any of the following: 
1. Previous enrollment in an H5N1 influenza vaccine trial.  
 
Participants in Cohort 2 (naïve cohort) will be excluded if they are:  
1. Seropositive to the H5N1 influenza A virus (serum hemagglutination inhibition 
assay (HAI) titer >1:8). 
2. Have previously received an investigational LAIV in another study. 
 
 Access to Medical Records 
A medical history will be obtained directly from each subject. Medical records will not 
be requested unless there is a need to clarify a question in the subject’s medical history or 
to document an AE during the study. Medical records will not be requested without a 
signed medical release and the informed consent of the subject. 
 
 Enrollment 
Subjects will be scheduled for vaccination when they complete the screening process, are 
determined to be eligible, and provide consent for study participation. Subjects will be 
considered enrolled in the study upon receipt of the study agent. 
 

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 Subject Withdrawal/Termination Criteria 
A subject will have completed the study if he/she receives the specified study vaccination 
and completes at least the Day 90 visit after the vaccination. A subject will be withdrawn 
from the study and not be considered to have completed the trial if any of the following 
apply:  
 
1. Research terminated by Sponsor or investigator: applies to the situation where the 
entire study is terminated by the Sponsor or investigator for any reason.  
 
2. Withdrawal of consent: applies to a subject who withdraws consent to participate 
in the study for any reason.  
 
3. Noncompliant with protocol: applies to a subject who does not comply with 
protocol specific visits, evaluations, or procedures on a consistent basis, such that 
adequate follow-up is not possible, the subject’s safety would be compromised by 
continuing in the trial, or the integrity of the study data could potentially be 
harmed.  
 
4. Withdrawal by PI: may occur if the investigator believes that it is in the best 
interest of the subject to withdraw the subject.  
 
5. Other: a category used when previous categories do not apply and requires an 
explanation.  
 
 Replacement of Subjects 
Subjects who are enrolled, vaccinated and subsequently withdraw, are withdrawn, or are 
taken off study prior to completion of Day 28 follow up visit, may be replaced subject to 
the principle investigator’s discretion in order to ensure sufficient data to achieve study 
objectives.  All vaccinated subjects will be followed for safety if subject circumstances 
allow. All vaccinated subjects will be followed for safety if subject circumstances allow.  
See ‘Sample Size’, Section 3.4, for sponsor/safety notification requirement related to 
replacement.     
 
 Special Situations 
1. Pregnancy: If a subject becomes pregnant, she will be advised to notify her 
obstetrician of study agent exposure. The subject will not be included in the 
immunogenicity evaluations from her estimated date of conception even if the pregnancy 
ends early. If the pregnancy occurs prior to Study Day 90, she will be encouraged to 
remain in the study for periodic safety evaluations and will be followed until completion 
of her pregnancy or the end of the study. The subject will be asked to sign a release of 
medical information form so that records can be obtained from her pregnancy provider 
regarding the outcome of the pregnancy. If a subject becomes pregnant after Study Day 
90, but before Study Day 360, she will be withdrawn from the study.  (See Investigator 
Reporting Responsibilities – Section 8.5). 
 
2. Lost to follow-up: A subject who cannot be contacted by telephone or other means of 
communication, including U.S. mail, is considered lost to follow-up. A subject may be 

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considered lost to follow-up and withdrawn from the study once 3 attempts to contact the 
subject by any means and a certified letter sent to the last known address have failed to 
elicit a response from the subject. 
 
3. Incarceration: If a subject becomes incarcerated during the course of the study, he or 
she may be terminated from the study if the period of incarceration renders him or her 
unable to complete the study. Alternatively, a subject who received the investigational 
product and was subsequently incarcerated may be taken “off treatment” rather than 
withdrawn to facilitate continued safety monitoring upon release. 
 
5. VACCINE PREPARATION 
 Supplies 
Monovalent Influenza Subvirion Vaccine (H5N1) vaccine, lot number UD08916, will be 
supplied in multiuse vials by BARDA. Vaccine for this protocol will be stored at the 
Division of Microbiology and Infectious Diseases (DMID), National Institutes of Health 
(NIH), repository until requested by the Center for Immunization Research (CIR). 
Vaccine will be formally requested to be transferred to the Pharmacy and Investigational 
Drug Service (IDS), Room 100 Osler at the Johns Hopkins Hospital by the Principal 
Investigator (PI) after IRB and FDA approval for the study has been granted. Vaccine 
will then be stored at the IDS. On the day of vaccination, the PI or disignated clinical 
investigator will supply to the pharmacy individual participant specific vaccine request 
form (prescription) for each subject to be vaccinated.  Each individual subject 
prescription will include the protocol number, the vaccine name, the vaccine dose, the 
investigational new drug (IND) number, and the number and name of the subject to be 
vaccinated. The IDS requires that the name be included and keeps these requests in a 
locked file as part of the study documents. The Pharmacist will draw up the vaccine from 
the multi-dose vials into syringes, 0.5 mL vaccine/syringe (45 μg dose, 10 per vial) based 
on the number of subjects to be vaccinated. The pharmacist will label each syringe with 
the study product name, PI initials, study number, subject number, preparer and study 
product verifier’s initials and expiration date and time. He or she will supply the 
appropriate number of syringes to the investigator. The vaccine will be transported to the 
investigator on wet ice or using cool packs for administration at the vaccination site on 
the same day. 
 
 Vaccine Storage and Accountability 
Vaccine will be stored at 2° to 8°C. Vaccine should not be frozen. Vaccine will be 
shipped to the IDS from the DMID repository with cool packs and a temperature log to 
ensure that the vaccine remained within the acceptable range. Vaccine cannot be used 
until it is verified by the Sponsor that the temperature has stayed within range. If during 
shipping, the vaccine falls out of the acceptable range, it will be quarantined until 
BARDA has been contacted and has determined whether or not it is acceptable to use the 
shipment. If not, it will be disposed of per BARDA’s instruction, and a new shipment 
will be requested. Once at the IDS, vaccine will be stored in temperature-monitored 
refrigerator, and the logs will be available for the monitors, if they wish to see them. In 
transport from the IDS, a study specific procedure re: “The transport of Investigational 
Agent for administration to study subjects” will be followed. 

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 Disposition of Used/Unused Supplies  
At the time of study product administration, the study product administrator and verifier 
will initial the label and remove one of two detachable labels from the syringe and place 
it on a piece of paper (labeled with the CIR study number, the subject’s study ID number, 
and the time of virus administration). The paper with the labels will be kept in the Study 
Binder. The other label will be placed in the subject chart as source documentation. The 
study product administration time will be documented on the Study Product 
Administration Record (SPAR). A copy of the completed SPAR will be placed in the 
Study Binder. The original will be returned to the CIR laboratory personnel along with 
any unused supply. The SPAR will be maintained in the laboratory and filed in the 
accountability binder. Used syringes will be disposed of in a sharps container. Any 
unused syringes will be returned to the pharmacy with the labels intact and will be 
documented on the accountability log by the pharmacist as unused. The Pharmacist will 
also document the use of multi-dose vials. All open multi-dose vials and unused syringes 
will be destroyed by the pharmacy according to pharmacy specific procedures. Unopened 
vials will be kept by the pharmacy until instructed by the Sponsor. In this manner, 
monitoring personnel will be able to account for all vials and syringes used for the study.  
 
6. STUDY PROCEDURES  
 Recruitment and Screening (Up to 60 Days Prior to Vaccination) 
Subjects will be evaluated, initially, through a JHBSPH IRB-approved general screening 
protocol – JH200 (IRB# H22040219A2). This general screening protocol is used to 
identify subjects who may be eligible to participate in a vaccine trial.  
 
 Recruitment 
The CIR disseminates information about opportunities to participate in its vaccine 
clinical trials via IRB-approved content on its own website 
(centerforimmunizationresearch.org), social media platforms, YouTube, and in web, 
newspaper, radio and television advertisements. Study specific IRB-approved fliers and 
brochures are distributed locally.  
 
Cohort 1 will consist of prior recipients of Live Attenuated H2N2, H6N1 and H9N2 
Influenza Vaccines as participants in previous CIR studies (CIR 211, CIR 247, CIR 251)  
With each initial and subsequent telephone contact, subjects are asked if they will allow 
us to call them about opportunities to participate in future studies.  The response to this 
question is documented on a pre-screening assessment form and in the CIR Adult Studies 
Data Base.  Study staff has compiled a list of approximately 63 volunteers who 
participated in these three studies from 2005 - 2009.  They will be contacted via phone, 
and/or IRB-approved letter, and invited to participate in our general screening study to 
see if they would be eligible and willing to participate in this study as recalled subjects.  
After this initial contact, subjects will be referred to the telephone recruiter.  The recruiter 
will administer a brief qualifying phone screen after obtaining verbal consent and eligible 
recruits will be scheduled for in-person screening visits according to their interests and 
availability.  
 

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Cohort 2 – the control cohort consisting of LAIV-naïve/H5 antibody negative subjects, 
will be identified as individuals who make inquiries by telephone call or email to the 
clinical trial site. A telephone recruiter will respond to calls and emails and provide brief 
scripted descriptions of currently accruing CIR trials as specified in the CIR’s general 
screening protocol. The recruiter will administer a brief qualifying phone screen after 
obtaining verbal consent and eligible recruits will be scheduled for in-person screening 
visits according to their interests and availability. 
 
The screening evaluation outlined below will be completed within 60 days prior to 
vaccination. During the initial in-person screening visit trained study staff will: 
 
1. Provide a copy of the screening study consent form and encourage the volunteer 
to read the consent and ask questions  
2. Administer a brief, written comprehension assessment to confirm understanding 
of the screening study procedures and requirements  
3. Obtain written, informed consent from the subject indicating willingness to 
participate in the CIR JH200 Screening Protocol as evidenced by subject 
signature, date and time on the informed consent document. The staff person 
obtaining consent will sign, date and time the ICF document. A copy of the signed 
document will be given to the subject. The original will be kept in the subject 
chart. 
 
4. Elicit a complete medical history, including current medications, and, for females, 
menstrual and contraceptive history, assessment of childbearing potential, and/or 
history of surgical sterility or menopausal status  
 
5. Provide human immunodeficiency virus (HIV) pretest counseling, including 
information about HIV testing, transmission, and prevention, explanation of test 
results, post-test counseling, results reporting, and that testing is voluntary, but 
necessary for study participation  
 
6. Provide pregnancy prevention counseling for females focused on the 
importance of preventing pregnancy during study participation, potential risks 
associated with pregnancy while taking investigational drugs, effective 
contraceptive options, and local resources for obtaining family planning 
services. 
 
7. After review of medical history, the study nurse or nurse practitioner will 
offer at no cost the regular dose quadrivalent seasonal flu vaccine – 
Fluzone.  
 
Additionally, the following activities may occur or be deferred to a subsequent screening 
visit depending on study requirements: 
 
1. Administer a complete physical examination  
 

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2. Obtain blood for complete blood count (CBC) with differential and platelet count, 
hepatitis B surface antigen, hepatitis C antibody, HIV antibody (will be completed 
within 30 days of vaccination) and H5N1 HAI assay. Urine will be collected from 
females for ß-HCG testing.    
 
 CIR 327 Study-Specific Screening and Informed Consent 
After the initial screening process has been completed, Subjects will be notified of their 
eligibility for further screening. All clinically significant abnormalities will be reviewed 
with the subjects and referrals for follow-up care will be provided. Subjects who test 
positive for hepatitis B, hepatitis C, or HIV) will be notified and referred to the Baltimore 
City Health Department according to Maryland reporting requirements. Eligible and 
willing subjects will be asked to return to the CIR for study-specific screening and 
consent.  
 
Informed consent is an ongoing process that includes the informed consent document and 
continues throughout the trial. Trained study staff will initiate the consenting process as 
follows 
 
1. Potential study participants will be given a copy of the IRB-approved study 
consent document to read.   
 
2. Subjects will be encouraged take as much time as they need and to ask questions  
 
3. Subjects will be asked to complete multiple-choice/fill in the blank questionnaire 
to evaluate comprehension of study aims, procedures, requirements, and risks.  
 
4. Subjects will meet privately with study staff to review the completed 
comprehension assessments to identify gaps in subjects’ understanding of study 
rationale, requirements, and risks, and their role as study participants.  
 
5. Study staff will review the pertinent study information with the subject until the 
subject verbalizes understanding. The subject may be offered up to 2 more 
attempts to answer 70% or more of the questions correctly. 
 
6. Subjects achieving a score of 70% or better and verbalizing willingness to 
participate in the study will be asked to indicate their consent in writing by 
signing the informed consent document and recording date and time that consent 
was given.  
 
7. The staff person obtaining consent will sign, date and time the ICF 
document. A copy of the signed document will be given to the subject. The 
original will be kept in the subject chart. 
 
After obtaining informed consent the following procedures may be completed 
 

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1. A complete physical exam if not done during the general screening visit or at the 
discretion of the PI or designee. 
 
2. No cost flu vaccine will be offered to subjects who declined flu vaccination 
during initial screening. Subjects who continue to decline the flu vaccine will be 
counseled as to the benefits of annual seasonal flu vaccine and risk of illness due 
to declining or delaying vaccination.  Subjects will be offered the option of 
deferring study enrollment for 30 days if they choose to accept the flu vaccine. 
 
3. Any laboratory assays or screening procedures mentioned above that were not 
completed during the first screening visit (or if assays need to be repeated), may 
be completed during the study specific screening visit.  Urine will be collected 
from females for ß-HCG testing. 
 
 Immunization Procedure 
Syringes prepared by the IDS and placed in cold box (as per Section 5.1 above) will be 
removed in order as subjects receive vaccination. The syringe will be warmed briefly, the 
subject number will be verified, and then the vaccine will be administered 
intramuscularly (IM) in the deltoid muscle to eligible and consenting subjects. Empty 
syringes will be disposed of in an appropriate sharps container. 
 
 Clinical Evaluation 
Vital signs (temperature, heart rate, respiratory rate, and blood pressure) will be assessed 
during screening, pre and 30 minutes post vaccination, Day 7 and Day 14. Focused 
physical examinations (examination of head, eyes, ears, nose, throat, heart and lungs) will 
be performed on the day of vaccination and at Day 7 and as indicated by interim history 
to day 360. 
 
 Treatments that Could Potentially Interfere with Vaccine-Induced 
Immunity 
The following criteria will be reviewed with the subjects during each follow up visit up to 
and including day 90. If any of these become applicable during the study, it will be noted 
in the subject’s record. The subject will not be included in further immunogenicity 
evaluations as of the visit in which the exclusionary treatment was reported. Subjects will 
be encouraged to participate in the safety evaluations, including blood draws at the 
discretion of the investigator for the duration of the study. 
 
1. Use of any investigational drug or investigational vaccine other than the study 
article within 90 days of receipt of the study agent. 
 
2. Administration of chronic (defined as more than 14 days) immunosuppressants or 
other immune-modifying drugs (topical and nasal steroids are allowed). 
 
3. Receipt of any licensed vaccine in the 4 weeks after vaccination. 
 
4. Receipt of immunoglobulins and/or any blood products. 

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  Concomitant Medications 
A concomitant medication (con-med) is a drug or biological product, other than a study 
drug, taken by a subject during a clinical trial. Information about routine medication use 
will be documented in the screening medical history. Concomitant medications and 
changes in pre-existing medication regimens indicating new diagnoses or worsening of a 
pre-existing condition will be collected from vaccination to Day 28 to identify solicited 
and unsolicited AE’s, new or changed significant or chronic conditions and treatments 
potentially interfering with vaccine immunity. Between day 28 and 90 concomitant 
medications will only be collected if associated with an SAE or with a treatment that 
interferes with vaccine-induced immunity as above.  
  Study Procedures By Day  
See Appendix A for a tabular representation of study procedures. In the description 
below, Study Day 0 is the day of receipt of vaccine. The days are numbered thereafter. 
  Study Day 0 (Day of Vaccination/Enrollment)  
1. Verify that study-specific Informed Consent was obtained.  
2. Ensure that all inclusion/exclusion criteria are met. 
3. Record vital signs (blood pressure, temperature, heart rate, and respiratory rate). 
4. Perform an abbreviated history and focused physical examination. 
5. For women, obtain urine for b-HCG testing. Ensure the test is negative before 
proceeding; a positive test will exclude the subject from the trial. 
6. Obtain blood for Serum and for PBMCs. 
7. Administer the vaccine. 
8. Observe for at least 30 minutes after vaccination to evaluate for immediate adverse 
reactions. 
9. Record vital signs (blood pressure, temperature, heart rate, and respiratory rate) 30 
minutes after vaccination. 
10. Review pregnancy prevention Counseling (Females only).  
  Study Day 7 ±1 Day 
1. Obtain interim history and perform focused physical examination, 
concentrating on any acute complaints and adverse events after vaccination. 
2. Assess continuing eligibility and for treatments that could potentially interfere 
with vaccine-induced immunity. 
3. Record vital signs. (blood pressure, temperature, heart rate). 
4. Evaluate injection site. 
5. Perform an abbreviated history and focused physical examination. 
6. Obtain blood for serum and for PBMCs. 
7. Review Pregnancy Prevention counseling (Females only). 
  Study Day 14 ±2 Day 
1. Obtain interim history concentrating on any acute complaints.  

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2. Assess continuing eligibility. Review use of any concurrent medications. 
Assess for treatments that could potentially interfere with vaccine-induced 
immunity. 
3. Perform focused physical exam if indicated. 
4. Record vital signs. (blood pressure, temperature, heart rate). 
5. Obtain blood for serum and PBMCs. 
6. Review pregnancy prevention counseling (Females only). 
  Study Day 28 ±7 Days 
1. Obtain interim history concentrating on any acute complaints.  
2. Assess continuing eligibility. Review use of any concurrent medications. 
Assess for treatments that could potentially interfere with vaccine-induced 
immunity. 
3. Perform focused physical exam if indicated. 
4. If female, obtain urine for b-HCG testing.  
5. Obtain blood for serum and PBMCs. 
  Study Day 56 ±7 Days 
1. Perform basic history, concentrating on any acute complaints. 
2. Obtain blood for serum and PBMC’s. 
  Study Day 90 ±14 Days 
1. Perform basic history, concentrating on any acute complaints. 
2. Obtain blood for serum and PBMCs. 
3. If female, obtain urine for b-HCG testing 
  Study Day 360 ±30 Days 
1.  Perform basic history, concentrating on any acute complaints. 
2. Obtain blood for serum and PBMCs. 
 
 Clinical and Research Laboratory Testing 
 
For both cohorts, the maximum volume of blood to be drawn is approximately 755 mL 
over the 12 month study period and not more than 550 ml’s in any 8 week period, which 
should not compromise the health of trial subjects and is consistent with the NIH 
guidelines set forth in the M95-9 (rev) “Guidelines for Limits of Blood Drawn for 
Research Purposes in the Clinical Center .” 
Using standard techniques, and with samples labeled with a unique study identifier for 
each subject, Quest Laboratories will perform the following tests: 
1. Complete blood count plus white blood cell differential and platelets. 
2. Chemistry panel (if indicated). 
3. HIV assay (4th generation ELISA with Western blot confirmation). 
4. HBsAg ELISA 

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5. HCV RNA PCR confirmation. 
6. Serum pregnancy test (if indicated) 
 
Urine b-HCG testing will be performed at the clinical trial site using an FDA-approved 
urine pregnancy test kit.  
 
HAI assays done for the purpose of screening potential subjects will be done by the CIR 
laboratory.  
 
Quantitative ELISA and MN for anti-stalk antibody, the passive serum transfer study, and 
the LD50 challenge study in mice will be assessed at the Icahn School of Medicine at 
Mount Sinai. 
 
Assessment of Plasmablasts and Memory B cells responses will be conducted at the 
Vaccine Research Center, NIH.  
 
De-identified PBMCs may be sent to Adaptive Biotechnology for immune profiling.  
 
Nasal secretions will be obtained by nasal lavage using 20 mL of Lactated Ringer’s 
Solution for Injection (10 mL per nares) with the glottis in a closed position. The 
collected lavage secretions will be used to screen for adventitious agents should subjects 
report URI symptoms during the 28 day post-vaccination AE reporting period. 
 
Specimens will be stored at the clinical trial site or the Laboratory of Infectious Diseases 
(LID), National Institute of Allergy and Infectious Diseases (NIAID). Access to research 
samples will be limited using a locked room. Samples and data will be stored using codes 
assigned by the investigators. Laboratory data will be kept in password-protected 
computers. Only investigators will have access to the samples and data. Samples at the 
clinical trial site are tracked manually on specimen logs that are kept in a separate, locked 
file cabinet.  
 
Any loss or unanticipated destruction of samples that compromises the scientific integrity 
of the data collected for the study (for example, due to freezer malfunction) will be 
reported to the IRB. 
 
 Testing of previous samples 
Samples from the subjects in the previous pLAIV studies are still stored in the NIAID 
repository. With subject consent, pre-and post pLAIV serum samples will be retrieved 
and tested for anti-stalk antibodies, in parallel with the samples collected during this 
study.  
 
7. RETENTION OF SPECIMENS FOR FUTURE USE 
All specimens collected as part of this trial may be stored indefinitely for future research. 
These samples may be used to learn more about influenza infection and other diseases but 
will not be used for genetic testing. These samples will not be sold or used to make 
commercial products. Samples will be stored only with the subject’s permission, which is 

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a requirement for participation in the study. The subject may withdraw permission for 
future use of specimens at any time. If a subject withdraws his or her permission for 
future use of specimens, those specimens will be destroyed. All samples stored will be 
labeled with the subject’s study identification (ID) number, which cannot identify the 
study subject but is linkable to other research databases (e.g., from questionnaires, 
clinical assessments, logbooks, etc.) generated by the main study. The database will 
contain only the study subject’s ID number. A master log linking the study subject ID 
number to the name of the subject will be maintained in a password protected database 
system with access limited to authorized research team members.  
 
At the completion of the protocol (termination), samples and data will either be 
destroyed, or transferred to a repository (JHBSPH IRB protocol R22.05.04.29.A2). In the 
future, other investigators (both at NIH and outside) may wish to study these samples 
and/or data. In that case, IRB approval must be sought prior to any sharing of samples. 
Any clinical information shared about the sample with or without patient identifiers 
would similarly require prior IRB approval. The research use of stored, unlinked, or 
unidentified samples (for example, as a standard for immunological analyses) may be 
exempt from the need for prospective IRB review and approval. Exemption requests will 
be submitted in writing to the NIH Office of Human Subjects Research, which is 
authorized to determine whether a research activity is exempt.  
 
8. SAFETY AND ADVERSE EVENT REPORTING 
 Definitions 
8.1.1 Adverse Events 
An adverse event (AE) is any untoward medical occurrence in an investigational subject 
following receipt of an investigational drug, regardless of causality. Therefore, an AE can 
be any unfavorable and unintended sign (including an abnormal laboratory finding), 
physical exam, symptom, or disease temporally associated with the use of the 
investigational vaccine, whether or not related to it. Exacerbation of pre-existing 
conditions and intercurrent illnesses will be recorded as AEs. Stable chronic conditions 
that are present prior to enrollment and do not worsen are not considered AEs but will be 
accounted for in the subject’s medical history. If a diagnosis is clinically evident or 
subsequently determined, the diagnosis rather than the individual signs and symptoms or 
lab abnormalities will be recorded as the AE. 
 
A laboratory abnormality that is not included in the solicited AEs will be reported as an 
AE if it results in an intervention. Interventions include, but are not limited to, treatment 
discontinuation, dose reduction/delay, additional assessments (including repeat testing at 
a later time point if not specified in this protocol), or concomitant treatment. In addition, 
any medically important laboratory abnormality may be reported as an AE, at the 
discretion of the investigator. This could include a laboratory result for which there is not 
an intervention, but the abnormal value suggests a disease or organ toxicity. If the 
laboratory abnormality is the clinical sequela of a clinical condition, the diagnosis or 

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medical condition should be reported as the AE (e.g., renal failure, hematuria) rather than 
the laboratory abnormality (e.g., elevated creatinine, urine red blood cell increase). 
8.1.2   Solicited Adverse Events (Protocol-Specified Adverse Events) 
AEs will be categorized as Solicited AEs and Other AEs. Solicited AEs are predefined 
AEs that can potentially occur after vaccine administration. Reactogenicity events are 
solicited adverse events that occur in the first 7 days after vaccination. The following will 
be considered solicited AEs for subjects receiving the Monovalent Influenza Subvirion 
Vaccine (H5N1): 
 
1. Fever ³100.4°F (38°C) oral 
2. Headache 
3. Malaise 
4. Nausea 
5. Myalgia 
6. Injection pain 
7. Injection site erythema 
8. Injection site swelling 
9. Injection site induration 
10. Injection site pruritus 
8.1.3   Unsolicited Events  
AEs that are not specifically listed in Section 8.1.2  and not documented in the screening 
medical history as pre-existing conditions will be categorized as “Other AE’s and 
identified by asking subjects about any new diagnoses, acute illnesses, worsening of a 
pre-existing condition, visits to the physician, ER, or hospital, medication use and 
indication. 
8.1.4   Adverse Reaction (AR) 
An AE that is caused by the vaccine. 
8.1.5   Suspected Adverse Reaction (SAR) 
An AE for which there is a reasonable possibility that the vaccine caused the AE. 
‘Reasonable possibility’ means that there is evidence to suggest a causal relationship 
between the vaccine and the AE. A suspected adverse reaction implies a lesser degree of 
certainty about causality than adverse reaction, which implies a high degree of certainty. 
8.1.6   Serious Adverse Events 
A SAE is an AE, whether considered related to the investigational vaccine or not, 
resulting in one of the following outcomes: 
 
1. Death during the period of protocol-defined surveillance. 
2. Life threatening event: defined as an event that places a subject at immediate risk of 
death at the time of the event and does not refer to an event that hypothetically might 
have caused death were it more severe. 

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3. Requires hospitalization or prolongation of a hospitalization during the period of 
protocol-defined surveillance: defined as at least an overnight stay in the hospital for 
treatment that would have been inappropriate if administered in the outpatient setting. 
4. Results in a congenital anomaly or birth defect. 
5. Results in a persistent or significant disability or substantial disruption of the ability 
to conduct normal life functions.  
6. Any other important medical event that may not result in death, be life threatening, or 
require hospitalization, may be considered a serious AE when, based upon 
appropriate medical judgment, the event may jeopardize the subject and may require 
medical or surgical intervention to prevent one of the outcomes listed above. 
8.1.7   Unexpected Adverse Events 
An AE is considered unexpected if it is not listed in the Investigator Brochure or Package 
Insert (for marketed products) or is not listed at the specificity or severity that has been 
observed. “Expected” means that the event has previously been observed with the 
investigational agent and is identified and/or described in the current Investigator’s 
Brochure (IB) or package insert. It does not mean that the event is expected with 
pharmacologically similar drugs, the underlying disease(s), or concomitant medications. 
Such events are considered unexpected for reporting purposes. 
8.1.8   Suspected Unexpected Serious Adverse Reaction (SUSAR) 
A SUSAR is a Suspected Adverse Reaction that is both Serious and Unexpected.  
8.1.9   Unanticipated Problems 
An Unanticipated Problem (UP) is any incident, experience, or outcome that is 
1.  unexpected in terms of nature, severity, or frequency in relation to 
a. the research risks that are described in the IRB-approved research protocol and 
informed consent document; Investigator’s Brochure or other study documents; 
and 
b. the characteristics of the subject population being studied; and 
2. possibly, probably or definitely related to participation in the research; and 
3. places subjects or others at a greater risk of harm (including physical, psychological, 
economic, or social harm) than was previously known or recognized. (Per the IND 
Sponsor, an AE with a serious outcome will be considered increased risk.) 
8.1.10 Serious Unanticipated Problem 
A UP that meets the definition of a serious adverse event or compromises the safety, 
welfare or rights of subjects or others 
8.1.11 Unanticipated Problem that is Not an Adverse Event (UPnonAE) 
A UP that does not fit the definition of an AE, but which may, in the opinion of the 
investigator, involve risk to the subject, affect others in the research study, or 
significantly impact the integrity of research data. For example, report occurrences of 
breaches of confidentiality, accidental destruction of study records, or unaccounted-for 
study drug. 

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8.1.12 Serious Protocol Deviation 
A “Protocol Deviation” [see below] that meets the definition of a Serious Adverse Event 
or compromises the safety, welfare, or rights of subjects or others. 
8.1.13 Protocol Deviation 
Any change, divergence, or departure from the IRB-approved study procedures in a 
research protocol. Protocol deviations are further characterized as: 
 
1.  Those that occur because a member of the research team deviates from the 
protocol. 
2.  Those that are identified before they occur but cannot be prevented. 
3.  Those that are discovered after they occur. 
8.1.14 Non-compliance [Definition is IRB dependent]: 
At a Site Overseen by non-NIH IRB: Investigators must follow the definitions, 
requirements, and reporting procedures per the IRB of record for the study/site where the 
possible non-compliance event occurred.  
 
 Recording Period for Adverse Events 
• All non-serious AEs will be assessed, recorded and reported for 28 days 
beginning at study vaccination, and will be followed to resolution, even if more 
than 28 days, or until the PI deems the event to be chronic or the subject to be 
stable.    
o Solicited non-serious AEs may be assessed/graded per a different table, if 
specified herein, and may be recorded and reported separately for tracking 
purposes.  
o Reactogenicity AEs/events are solicited AEs that are collected in the first 
7 days after vaccination and may be recorded and reported separately for 
tracking purposes.  
• SAEs, regardless of causality, will be assessed, recorded and reported for 90 days 
beginning at study vaccination, and will be followed to resolution, even if more 
than 90 days, or until the PI deems the event to be chronic or the subject to be 
stable.  
• SUSARs, UPs, and UPnonAEs will be assessed, recorded and reported through 
the end of the study, beginning at study ENROLLMENT, and will be followed to 
resolution, even if beyond the end of the study (as may be permissible and 
possible in consultation with the IRB and sponsor) or until the PI deems the event 
to be chronic or the subject to be stable.  
 
Assessment of safety will include clinical observation and monitoring of clinical and 
immunologic parameters. Safety will be evaluated by monitoring of subjects for local and 
systemic adverse reactions within the first 30 minutes post vaccination. Subjects will 
return to the clinic on Day 7 following vaccination for in-person clinical assessment.  
 

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All AEs will be graded for severity and assessed for relationship to the study product. A 
study clinician will be available 24 hours a day during the study evaluation period. 
Should a subject call a study clinician to report an AE, it will be fully documented in the 
subject’s study chart and discussed with the PI. Interim follow-up visits (between 
protocol scheduled visits) may be scheduled at the PI’s discretion if additional assessment 
is indicated. 
 
All AEs will be captured in the source documents and in Clinical Research Information 
Management System of the NIAID (CRIMSON). Those assessed as serious will be 
further reported on the Sponsor’s SAE-UP report form. The AEs judged to be possibly, 
probably, or definitely related to the study product will be followed as specified above, 
generally until resolution. 
 
 Association of Adverse Events with Receipt of Study Vaccine 
All AEs will have their possible relationship to study vaccine.  Causality will be assessed 
by the PI or designee using the following definition: 
 
Definitely related: 
Clear-cut temporal association, and no other possible cause. 
Probably related: 
Reasonable temporal association and a potential alternative cause 
 
 
 
is not apparent. 
Possibly related: 
Less clear temporal association; other causes also possible. 
Unlikely related: 
Temporal association between the AE and the vaccine or the nature 
of the event is such that the vaccine is not likely to have had any 
reasonable association with the observed illness/event (cause and 
effect relationship improbable but not impossible). 
Not Related:  
The AE is completely independent of vaccine administration; 
and/or evidence exists that the event is definitely related to another 
cause. 
 
The degree of certainty with which an AE can be attributed to administration of the study 
vaccine will be determined by how well the event can be understood in terms of 1 or 
more of the following: 
 
1. The event being temporally related with vaccination. 
2. A reaction of similar nature having previously been observed with this type of 
vaccine and/or formulation. 
3. The event having often been reported in the literature for similar types of 
vaccines. 
 
 Assessment of Severity 
All AEs will be graded for severity.  
• Solicited AEs will be assessed using the grading system shown in Appendix 
B:  Local and Systemic Adverse Event Grading Systems. This table is based 
on the NIAID “Division of AIDS (DAIDS) Table for Grading the Severity of 
Adult and Pediatric Adverse Events,” Version 2.1, March 2017: 
https://rsc.tech-res.com/docs/default-source/safety/daids-ae-grading-table-

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mar2017.pdf.   
 
• All AEs will be assessed by the study investigators using the grading system 
shown in Table 2, below:  
Table 2: Adverse Event Grading System 
Severity 
Grade 
Defined 
None 
0 
N/A 
Mild 
1 
No effect on activities of daily living, over the counter 
treatment given ≤2x/day 
Moderate 
2 
Partial limitation in activities of daily living 
(can complete ≥50% baseline, or treatment given 
>2x/day) 
Severe) 
3 
Activities of daily living limited to <50% of baseline. 
Medical intervention often required. 
Life-threatening 
4 
Inability to perform basic self-care functions OR 
severity of illness requires hospitalization 
 
Severity of fever will also be assessed by degree 
 
Table 3: Fever Grading System 
 
Intensity  
Grade 
Defined 
<100.4°F (<38.0°C) 
0 
Fever (oral) 
Temperatures ≥100.4°F, will be confirmed 
by repeating temperature after waiting 20 
minutes 
≥100.4°F – 101.4°F (≥38.0°C – 38.6°C) 
1 
≥101.5°F – 102.4°F (38.7°C – 39.1°C) 
2 
>102.4°F (>39.1°C) 
3 
 
 Investigator Reporting Responsibilities 
8.5.1  Adverse Event Reporting 
The Office of Clinical Research Policy and Regulatory Operations (OCRPRO) 
(DCR/NIAID/NIH) is the Sponsor for the IND filed at the U.S. FDA. AE data will be 
submitted to the IND Sponsor when requested for periodic safety assessments, review of 
IND annual reports, review of IND safety reports, and preparation of final study reports.  
 
In the interest of subject safety in OCRPRO-sponsored studies and to fulfill regulatory 
requirements, any deaths and life-threatening SAEs due to any cause that occur during 
the course of the study must be reported to the OCRPRO Clinical Safety Office (CSO) by 
the PI within 1 business day after the clinical site becomes aware of the event, and all 
other SAEs must be reported as soon as possible, but no later than 3 business days after 
site awareness. SAEs (regardless of relationship and whether or not they are also UPs) 
must be reported on the Safety Expedited Report Form and sent to the CSO by fax or 
email attachment. 

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SAEs that have not resolved by the end of the 3-month follow-up period are followed 
until final outcome is known. If it is not possible to obtain a final outcome for an SAE 
(e.g., the subject is lost to follow- up), the reason a final outcome could not be obtained 
will be recorded by the investigator on the AE case report form (CRF) and the Safety 
Expedited Report Form (SERF). 
 
SAEs will be reported to: 
• Sponsor, OCRPRO, CSO: Mailing address: Clinical Safety Office, 5705 Industry 
Lane, Frederick MD 21704, Phone 301-846-5301, Fax: 301-846-6224, Email: 
rchspsafety@mail.nih.gov 
 
• Dr. Jeffrey Cohen, Phone: 301-496-5265; Email: jcohen@niaid.nih.gov 
 
• JHSPH IRB Phone: 410-955-3193, Toll-Free: 1-888-262-3242, Fax: 410-502-
0584, Email: JHSPH.irboffice@jhu.edu. JHSPH IRB will be notified according 
to its guidelines, and unexpected AEs that are probably, possibly, or definitely 
related to the vaccine and meet other JHSPH IRB reporting criteria will be 
reported in the manner specified by JHSPH IRB. 
 
• The Johns Hopkins University (JHU) Institutional Biosafety Committee (IBC): 
Phone: 410-955-5918, Fax: 410-955-5929. The JHU IBC will be notified of all 
SAEs by telephone (followed by written report), email, or fax within 1 working 
day of notification of the SAE occurrence. 
 
Following notification from the PI, the OCRPRO CSO, as the representative of the IND 
Sponsor, will report AEs that are serious, unexpected, and related (possibly, probably, or 
definitely) to the vaccine to the FDA within 15 calendar days of such determination. The 
FDA will also be notified by phone or fax of all deaths and life-threatening SAEs that are 
unexpected and related to the vaccine within 7 calendar days, followed by a formal 
Safety Report within 15 days after being informed of the event. All SAEs and non-
serious AEs will be reported to the FDA at least annually in a summary format. 
8.5.2  Unanticipated Problems 
Non-Serious AEs that are UPs must also be reported on the SAE/UP Report Form and 
sent to the CSO by fax or e-mail attachment no later than 7 calendar days of PI awareness 
of the event. The UPs that are not AEs are not reported to the Sponsor CSO. 
 
 
 
8.5.3   Pregnancy 
All pregnancies through study day 90 will be reported on the Pregnancy 
Notification/Outcome Form to the CSO within 1 business day from site awareness.  
 

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Pregnancy itself is not an AE. However, complications of pregnancies are AEs and may 
be Serious Adverse Events (SAEs). Events that meet SAE criteria during pregnancy, 
delivery, or in the neonate (e.g., congenital anomaly/birth defect) are reportable on the 
SERF.  
 
Pertinent obstetrical information for all pregnancies will be reported to the Clinical Safety 
Office (CSO) via fax or email within 3 business days from site awareness of the 
pregnancy.  
 
Pregnancy outcome data (e.g., delivery outcome, spontaneous or elective termination of 
the pregnancy) will be reported to the CSO within 3 business days of the site’s awareness 
on a protocol-specified form.  
 
Pregnancy events will also be reported to the JHSPH IRB. 
8.5.4  Reporting Responsibilities to the JHSPH IRB 
 In accordance with JHSPH IRB policy, all unexpected SAEs will be reported by the PI 
within 10 working days of being detected; expected SAEs will be reported annually. All 
local and systemic reactions not meeting the criteria for SAEs will be captured on the 
appropriate source documents and entered in CRIMSON, followed to resolution, and 
reported annually to the FDA and JHSPH IRB. 
 
  Sponsor’s Reporting Responsibilities 
Suspected unexpected serious adverse reactions (SUSARs) as defined in 21 CFR 312.32 
and determined by the Sponsor will be reported to FDA as IND Safety Reports. 
 
The Sponsor will also submit an IND Annual Report of the progress of the investigation 
to the FDA as defined in 21 CFR 312.33.  
 
 Halting Rules 
Halting the study requires immediate discontinuation of study agent administered for all 
subjects and suspension of enrollment until a decision is made whether or not to continue 
enrollment and study agent administration. 
 
The PI will closely monitor study data as they become available and will make 
determinations regarding the presence and grading of AEs. The AEs will be evaluated 
with regard to the known complications associated with administration of vaccine 
components. If any of the following events occur, vaccinations will be suspended until 
the events are reviewed with the study Sponsor (OCRPRO): 
 
1. One or more subjects experience an SAE as defined in Section 8.1.6 of this protocol 
that is determined to be possibly, probably, or definitely related to the vaccine, or 
2. One or more subjects experience a hypersensitivity reaction that is probably or 
definitely related to the vaccine, or 

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3. Any severe clinical illness occurs that is not explained by a diagnosis that is unrelated 
to vaccination, or 
4. Two or more subjects in any cohort experience any Grade 3 systemic AE that is 
determined to be possibly, probably, or definitely related to the vaccine as defined in 
this protocol.  
The IRB, the NIAID, The Gates Foundation, the FDA, or other government agencies, 
may discontinue the study at any time. Subsequent review of serious, unexpected, and 
related AEs by the IRB, the IND sponsor, the FDA, and other regulatory authorities may 
also result in suspension of further administration of vaccine at the clinical site. The 
FDA, other regulatory authorities, and the study sponsor(s) retain the authority to suspend 
additional enrollment and administration of vaccine for the entire study as applicable. 
 
The IND Sponsor, in collaboration with the PI will determine if it is safe to resume the 
study. 
 
  Safety Review and Communications Plan 
A safety review and communications plan (SRCP) has been developed for the protocol. 
The SRCP is an internal communications document between the PI and the CSO, which 
delineates the safety oversight responsibilities of the PI, the CSO, and other stakeholders. 
The SRCP also includes the overall plan for conducting periodic safety surveillance 
assessments. 
 
  Sponsor Medical Monitor 
A Medical Monitor, representing the IND Sponsor (OCRPRO), has been appointed to 
provide safety oversight of this clinical study. The Sponsor Medical Monitor will be 
responsible for performing safety assessments as outlined in a Safety Review and 
Communication Plan. 
 
The PI is responsible for ensuring that the Safety Monitor is aware of all new safety 
information during the course of the clinical trial.  
 
9. DATA COLLECTION AND MONITORING 
   Source Documentation 
Complete source documentation (laboratory test reports and/or medical records) is 
required for every study subject for the duration of the study. Specified data from source 
documentation for subjects enrolled in the study will be entered into the CRIMSON data 
system. The data entry is to be completed on an ongoing basis during the study. Data 
entered into CRIMSON shall be performed by authorized individuals. Corrections to the 
data system shall be tracked electronically (password protected) with time, date, 
individual making the correction, and what was changed. Source documentation should 
support the data collected in CRIMSON and must be signed and dated by the person 
recording and/or reviewing the data. 
 
The PI is responsible for the accuracy, completeness, and timeliness of the data reported 
to the Sponsor through the CRIMSON Data System. All data entered into CRIMSON 

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should be reviewed by an Investigator and signed as required with written or electronic 
signature, as appropriate. Data reported in CRIMSON should be consistent with source 
documents or the discrepancies should be explained. Source documentation will be made 
available for review or audit by the Sponsor or designee and any applicable Federal 
authorities. 
 
   Study Documentation 
Study-related documentation will be completed as required by the IRB, the Sponsor, and 
regulatory authorities. Continuing review documentation will be submitted by the PI to 
the IRB on the anniversary date of initial review as specified by the IRB. An annual 
report will be submitted by the Sponsor to the FDA within 60 days after the anniversary 
date that the IND for this study went into effect. The annual report will provide a brief 
description of the progress of the investigation as outlined in 21 CFR 312.33, and will 
discuss any revisions that have been made to or are planned for the protocol. 
 
The PI will maintain adequate records of the disposition of the investigational product, 
including dates of receipt and disposition, administration, quantity, and use by subjects. If 
the study is terminated, suspended, or completed, all unused supplies of the 
investigational product will be destroyed by the investigational pharmacy staff.  
 
   Retention of Records 
Trial-related documents will be maintained by the PI. All trial-related documents will be 
stored securely to maintain the confidentiality of subjects’ personal and health-related 
information. All records pertaining to this protocol will be stored in a locked cabinet at 
JHU or at an offsite, locked storage facility per CIR process and regulations.  
 
The FDA requires study records to be retained for up to 2 years after marketing approval 
or disapproval (21 CFR 312.62), or until at least 2 years have elapsed since the formal 
discontinuation of clinical development of the investigational agent for a specific 
indication. These records are also to be maintained in compliance with IRB/Ethics 
Committee (EC), state, and federal medical records retention requirements, whichever is 
longest. All stored records are to be kept confidential to the extent provided by federal, 
state, and local law. It is the investigator’s responsibility to retain copies of source 
documents until receipt of written notification to the contrary from the 
OCRPRO/DCR/NIAID. No study document should be destroyed without prior written 
agreement between OCRPRO/DCR/NIAID and the PI. Should the investigator wish to 
assign the study records to another party and/or move them to another location, the 
investigator must provide written notification of such intent to OCRPRO/DCR/NIAID 
with the name of the person who will accept responsibility for the transferred records 
and/or their new location. NIAID must be notified in writing and written NIAID 
permission must be received by the site prior to destruction or relocation of research 
records other than as per CIR SOP. 
 
   Protocol Revisions 
No revisions to this protocol will be permitted without documented approval from the 
Sponsor and the IRB that granted the original approval for the study. This does not apply 

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to changes made to reduce discomfort or avert risk to study subjects. Furthermore, in the 
event of a medical emergency, the PI shall perform any medical procedures that are 
deemed medically appropriate. The PI must notify the Sponsor of all such occurrences. 
 
   Study Monitoring 
As per ICH GCP 5.18 and FDA 21 CFR 312.50, clinical protocols are required to be 
adequately monitored by the study sponsor. This study monitoring will be conducted 
according to the “NIAID Intramural Clinical Monitoring Guidelines.” Monitors under 
contract to the OCRPRO/DCR/NIAID will visit the clinical research site to monitor all 
aspects of the study in accordance with the appropriate regulations and the approved 
protocol. The objectives of a monitoring visit will be: 1) to verify the existence of signed 
informed consent documents for each monitored subject; 2) to verify the prompt and 
accurate recording of all monitored data points and prompt reporting of all SAEs; 3) to 
compare abstracted information (CRIMSON pulls) with individual subject’s records and 
source documents (supporting data, laboratory specimen records, clinical notes); and 4) 
to ensure study subject protection, investigators’ compliance with the protocol, and study 
record completeness and accuracy. The monitors will also inspect the clinical site 
regulatory files to ensure that regulatory requirements (Office for Human Research 
Protections), FDA and applicable guidelines (ICH GCP) are being followed. During the 
monitoring visits, the PI (and/or designee) and other study personnel will be available to 
discuss the study progress and monitoring visit.  
 
A specific protocol monitoring plan will be discussed with the PI and study staff prior to 
enrollment. The plan will outline the frequency of monitoring visits based on such factors 
as study enrollment, data collection status, and regulatory obligations. 
 
10. STATISTICAL CONSIDERATIONS 
  Description of Statistical Methods 
This study is exploratory rather than confirmatory; its purpose is to estimate AE rates and 
patterns of immune responses rather than to test formal statistical hypotheses. Estimates 
will be presented with their 95% confidence intervals. Sample size was determined based 
on similar exploratory vaccine trials, and also based on the sizes of the previous cohorts 
to be recruited into the current study. We would like to enroll at least 12 people who were 
H2N2, H6N1 and H9N2 pLAIV recipients. In addition, we would like to enroll at least 20 
negative controls. 
 
Descriptive approaches will be used to meet the protocol objectives as stated in Section 2 
of this protocol, as well as formal statistical tests as outlined below. Results will be 
presented in tabular format, as well as graphically where appropriate. 
 
  Sample Size Reasoning 
The null hypothesis for the study is that the HA stalk antibody response after receipt of 
the H5N1 pISV in the pLAIV recipients (treatment cohort, T) will be the same as in the 
naïve (control, C) cohort. The alternative is that the recipients of the pLAIV will have a 
higher stalk antibody response. Based on the previous studies of H5N1 stalk response in 
naïve individuals (17, 18), we assume that the naïve cohort will have a 4-fold increase in 

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antibodies targeting the stalk after the H5N1 pISV. A total of 15 subjects in the pLAIV 
cohorts will allow for 80% power to detect a 2-fold higher response (8-fold increase in 
antibody titers), with an alpha of 0.05. 12 subjects in the pLAIV cohort will allow for 
80% power to detect a 2.1-fold higher response: an 8.2-fold increase in antibody titers. 
 
  Primary Objective:  
To explore the difference in the stalk antibody response against Influenza A group 1 
stalks as measured by ELISA and neutralization titer after receipt of the Monovalent 
Influenza Subvirion Vaccine (H5N1) in previous recipients of H2N2, H6N1 and H9N2 
pLAIVs compared with healthy, pLAIV-naïve individuals. 
 
  Primary Endpoint: 
1. The fold rise in titer of anti-group 1 stalk-antibodies measured by ELISA and 
microneutralization assay (MN) using the chimeric cH6/N1 probe, in recipients of the 
pLAIVs (H2N2, H6N1 and H9N2) compared to the control pLAIV-naïve cohort. In 
addition to testing sera following administration of the H5N1 subunit vaccine, if possible, 
this analysis will include sera collected before and after receipt of pLAIV. 
 
Analysis plan: Antibody titers will be converted to a log scale, and comparisons of the 
antibody titers will be made using a t-test or ANOVA as appropriate.  
If more than 2 cohorts are being compared, appropriate post-hoc, pairwise (as applicable) 
comparisons will be made with appropriate alpha-adjustments if the null hypothesis is 
rejected.  
 
  Secondary Objective 
Assess the reactogenicity of the Monovalent Influenza Subvirion Vaccine (H5N1) in 
previous recipients of live attenuated influenza vaccine (LAIV) compared with healthy, 
pLAIV-naïve individuals.  
Adverse events will be assessed, graded and recorded for 28 days after vaccination. 
Serious Adverse Events (SAEs) will be assessed for 3 months after the immunization. 
 
  Secondary Endpoints: 
• The number of vaccine-related adverse events in the recipients of the pLAIV 
compared to the control cohort.  
 
  Exploratory Objectives: 
• To assess the biological activity of antibodies to the HA stalk region by passive 
transfer of serum from study subjects to mice to assess their ability to protect the mice 
from lethal challenge with an influenza virus 
• To correlate ELISA and neutralizing stalk antibody titers with functional ability of the 
post vaccine serum to protect mice from lethal challenge 
• To assess the breadth of the vaccine induced antibody response by using a panel of 
HAs that include HA stalks from a variety of group 1 and group 2. 
 
• To assess B- cell responses to the pISV in the previous pLAIV recipients compared 
with naïve individuals. 

Version 3.0, 12April2019 
  
42 
• To assess the level of serum stalk antibodies after the initial pLAIV (from stored 
samples) and compare to the post-pISV response. 
• To assess the longevity of the stalk antibody response 
• To determine the titer of anti-H5 antibodies in the 2 cohorts of subjects. 
• To determine the titer of antibodies directed to the HA received in the pLAIV doses. 
 
  Exploratory Endpoints: 
• Difference in survival rates between mice challenged with H5N1 after passive serum 
from the subjects previously vaccinated with pLAIV compared to those receiving 
serum from the control cohort after vaccination with H5N1 pISV. 
• The correlation of the serum stalk antibody titers by neutralization assay and ELISA 
with the ability of the sera to protect against lethal challenge. 
• Measurement of cross-reactivity of antibody responses in pLAIV primed vs. naïve 
individuals to a variety of viruses representing multiple HA stalks. 
• The percentage of memory B cells against group 1 stalk in the recipients of the 
pLAIV compared to the control cohort. 
• The level of stalk antibodies generated by the pLAIV – tested in sera obtained shortly 
after receipt of pLAIV and compared to the stalk antibodies generated after receipt of 
the H5N1 pISV.  
• HAI titer against H5 in the pLAIV primed vs. naïve individuals. 
• HAI titer against H2, H6 and H9 in the pLAIV primed individuals.  
 
11. HUMAN SUBJECT PROTECTIONS AND ETHICAL OBLIGATIONS 
This research will be conducted in compliance with the protocol, GCP, and all applicable 
regulatory requirements. 
 
  Institutional Review Board 
The PI will be responsible for obtaining IRB approval for the study. Before the start of 
the study, the appropriate documents (including the protocol, informed consent form, 
information sheets, and advertisements) will be submitted to the reviewing IRB for 
written approval. The PI must submit and obtain approval from the IRB for all 
subsequent amendments to the protocol, informed consent documents, and other study 
documentation referenced above. The PI will be responsible for obtaining IRB approval 
of the annual continuing review throughout the duration of the study. A copy of the study 
approval (including approval of the informed consent form) is to be maintained in the 
PI’s study document binder and a copy will be supplied to the Sponsor. During the study, 
the PI is responsible for providing the IRB with all documents subject to review (i.e., 
protocol amendments, informed consent form updates, advertisements, and any written 
information that may be provided to the subject). The PI will also provide annual reports 
on the study’s progress to the IRB in accordance with IRB guidelines and government 
regulations. 
 
  Informed Consent Process 
Informed consent is a process that is initiated prior to obtaining consent from a potential 
participant and continues throughout the individual’s study participation. The volunteer’s 

Version 3.0, 12April2019 
  
43 
consent for study participation will be documented on an Informed Consent Form (ICF). 
The ICF must be approved by the JHSPH IRB prior to use. The ICF provides detailed 
information about the study agent/intervention(s), study procedures, and risks. The 
subject will be asked to read and review the document. Study staff will engage volunteers 
in discussion of study aims and procedures, and the risks and possible benefits of study 
participation, encourage them to ask questions and verbalize their understanding of study 
requirements and administer a written comprehension tool. Subjects will not be rushed or 
coerced into study participation. Subjects may withdraw consent at any time throughout 
the course of the trial. The informed consent process will be documented in the subject’s 
research chart, as required by 21 CFR 312.62. The informed consent form will be signed 
and personally dated by the subject and the individual who conducted the informed 
consent discussion. The original signed informed consent form will be retained in the 
subject’s chart, and a signed and dated copy will be given to the subject. (For a more 
detailed description consent procedures, refer to the study procedures Section 6) 
 
  Participant Confidentiality 
Subjects will not be identified in any publicly released reports of this study. All records 
will be kept confidential to the extent provided by federal, state, and local law. The study 
monitors and other authorized representatives of the Sponsor may inspect all documents 
and records required to be maintained by the Investigator, including but not limited to, 
medical records. The investigator will inform the subjects that the above-named 
representatives will review their study-related records without violating the 
confidentiality of the subjects. All laboratory specimens, evaluation forms, reports, and 
other records that leave the site will be identified only by a coded number in order to 
maintain subject confidentiality. All records will be kept locked and all computer entry 
and networking programs will be done with coded numbers only.  
 
Subjects’ study information will not be released without the written permission of the 
subject, except as required, these groups may include:  
 
1. Audit and Compliance Officers and Legal Counsel  
2. OHRP  
3. The U.S. Food and Drug Administration (FDA) and other similar regulatory 
agencies  
4. The study sponsor or designee 
5. Study monitors, Medical Monitors  
6. Department of Health and Human Services (DHHS) agencies  
7. Governmental agencies to which HIV and hepatitis testing must be reported  
8. The Johns Hopkins University Bloomberg School of Public Health  
 
  Risks 
Risks to the subjects are associated with study procedures and immunization. Female 
subjects will be cautioned of the unknown risk of study vaccines to the fetus and must 
agree to use effective birth control methods for the duration of the study. 

Version 3.0, 12April2019 
  
44 
11.4.1 Venipuncture 
Risks occasionally associated with venipuncture include pain and bruising at the site of 
venipuncture, bleeding, infection, lightheadedness, and syncope (rarely). 
11.4.2 Nasal Wash 
 
Risks occasionally associated with nasal wash include pain or discomfort, and very 
rarely, epistaxis. 
11.4.3 Immunization 
Possible local vaccine reactions include pain, swelling, erythema, induration or pruritus at 
the injection site. Systemic reactions such as fever, chills, headache, fatigue, malaise, and 
myalgia may also possibly occur, with some reactions moderate or severe. A placebo- 
controlled trial of the H5N1 inactivated vaccine (12) showed that overall, the vaccine was 
generally well tolerated. The most common AE following immunization was mild pain at 
the injection site. Tenderness was also commonly reported. Less frequently, subjects 
reported malaise, myalgia or headache, although those symptoms were not statistically 
greater in the vaccinated group compared to the placebo group. Most reported AEs were 
mild (84%), some were moderate, and few were severe. 
 
It is possible that a subject may experience a reaction that is severe or even life 
threatening, including, but not limited to an immune/allergy mediated response. 
 
  Benefits 
Subjects will not receive any direct benefit from participation in this study. It is hoped 
that information gained in this study will contribute to the development of a safe and 
effective vaccine for the prevention of pandemic influenza. 
 
  Compensation 
Volunteers who are enrolled in the study will be paid $80.00 for screening and $80.00 for 
each follow up visit completed on time. On vaccination day, the volunteers will be 
compensated $125.00 because they will be required to stay in the clinic for a substantially 
longer period of time. Volunteers, who attend as alternates on the day of vaccination but 
are not enrolled, will receive $80.00 for screening and $80.00 for serving as an alternate. 
Enrolled subjects will receive a $250 bonus at the end of the study for complying with all 
study requirements and completing all study visits on time. Subjects will be compensated 
$80 each time they are asked to return to the CIR for an extra outpatient visit.  
 
The ability to achieve our research goals rests on our ability to recruit and enroll healthy 
subjects who received H2N2, H6N1, or H9N2 in prior CIR studies.  This is a highly 
rarefied subset of healthy individuals and as such has a high value in terms of achieving 
our research goals.  Recalled subjects will receive an additional bonus of $250.00 upon 
completion of the first 28 days of study follow-up to both incentivize enrollment and 
completion of key study time points and acknowledge the important role they play in the 
study. 
 

Version 3.0, 12April2019 
  
45 
 
 
 

Version 3.0, 12April2019 
  
46 
12. REFERENCES  
1. 
Cox NJ, Subbarao K. Global epidemiology of influenza: past and present. Annu 
Rev Med. 2000;51:407-21. 
2. 
Shrestha SS, Swerdlow DL, Borse RH, Prabhu VS, Finelli L, Atkins CY, et al. 
Estimating the burden of 2009 pandemic influenza A (H1N1) in the United States (April 
2009-April 2010). Clin Infect Dis. 2011;52(Suppl 1):S75-82. doi: 10.1093/cid/ciq012. 
3. 
Meltzer MI, Cox NJ, Fukuda K. The economic impact of pandemic influenza in 
the United States: priorities for intervention. Emerging infectious diseases. 
1999;5(5):659-71. 
4. 
Li GM, Chiu C, Wrammert J, McCausland M, Andrews SF, Zheng NY, et al. 
Pandemic H1N1 influenza vaccine induces a recall response in humans that favors 
broadly cross-reactive memory B cells. Proc Natl Acad Sci U S A. 2012;109(23):9047-
52. doi: 10.1073/pnas.1118979109. Epub 2012 May 21. 
5. 
Krammer F, Palese P. Influenza virus hemagglutinin stalk-based antibodies and 
vaccines. Curr Opin Virol. 2013;3(5):521-30. doi: 10.1016/j.coviro.2013.07.007. Epub  
Aug 24. 
6. 
Talaat KR, Luke CJ, Khurana S, Manischewitz J, King LR, McMahon BA, et al. 
A live attenuated influenza A(H5N1) vaccine induces long-term immunity in the absence 
of a primary antibody response. The Journal of infectious diseases. 2014;209(12):1860-9. 
7. 
Babu TM, Levine M, Fitzgerald T, Luke C, Sangster MY, Jin H, et al. Live 
attenuated H7N7 influenza vaccine primes for a vigorous antibody response to 
inactivated H7N7 influenza vaccine. Vaccine. 2014;32(50):6798-804. doi: 
10.1016/j.vaccine.2014.09.070. Epub  Oct 16. 
8. 
Sobhanie M, Matsuoka Y, Jegaskanda S, Fitzgerald T, Mallory R, Chen Z, et al. 
Evaluation of the Safety and Immunogenicity of a Candidate Pandemic Live Attenuated 
Influenza Vaccine (pLAIV) Against Influenza A(H7N9). J Infect Dis. 2016;213(6):922-9. 
doi: 10.1093/infdis/jiv526. Epub 2015 Dec 9. 
9. 
Talaat KR, Karron RA, Liang PH, McMahon BA, Luke CJ, Thumar B, et al. An 
open-label phase I trial of a live attenuated H2N2 influenza virus vaccine in healthy 
adults. Influenza and other respiratory viruses. 2013;7(1):66-73. 
10. 
Talaat KR, Karron RA, Luke CJ, Thumar B, McMahon BA, Chen GL, et al. An 
open label Phase I trial of a live attenuated H6N1 influenza virus vaccine in healthy 
adults. Vaccine. 2011;29(17):3144-8. 
11. 
Karron RA, Callahan K, Luke C, Thumar B, McAuliffe J, Schappell E, et al. A 
live attenuated H9N2 influenza vaccine is well tolerated and immunogenic in healthy 
adults. Journal of Infectious Diseases. 2009;199(5):711-6. 
12. 
Treanor JJ, Campbell JD, Zangwill KM, Rowe T, Wolff M. Safety and 
immunogenicity of an inactivated subvirion influenza A (H5N1) vaccine. N Engl J Med. 
2006;354(13):1343-51. 
13. 
Karron RA, Talaat K, Luke C, Callahan K, Thumar B, DiLorenzo S, et al. 
Evaluation of two live attenuated cold-adapted H5N1 influenza virus vaccines in healthy 
adults. Vaccine. 2009;27(36):4953-60. 
14. 
Goji NA, Nolan C, Hill H, Wolff M, Noah DL, Williams TB, et al. Immune 
responses of healthy subjects to a single dose of intramuscular inactivated influenza 
A/Vietnam/1203/2004 (H5N1) vaccine after priming with an antigenic variant. J Infect 
Dis. 2008;198(5):635-41. 

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15. 
Galli G, Hancock K, Hoschler K, DeVos J, Praus M, Bardelli M, et al. Fast rise of 
broadly cross-reactive antibodies after boosting long-lived human memory B cells primed 
by an MF59 adjuvanted prepandemic vaccine. Proc Natl Acad Sci U S A. 
2009;106(19):7962-7. 
16. 
Subbarao K, Luke C. H5N1 viruses and vaccines. PLoS Pathog. 2007;3(3):e40. 
17. 
Nachbagauer R, Wohlbold TJ, Hirsh A, Hai R, Sjursen H, Palese P, et al. 
Induction of broadly reactive anti-hemagglutinin stalk antibodies by an H5N1 vaccine in 
humans. J Virol. 2014;88(22):13260-8. doi: 10.1128/JVI.02133-14. Epub 2014 Sep 10. 
18. 
Ellebedy AH, Krammer F, Li GM, Miller MS, Chiu C, Wrammert J, et al. 
Induction of broadly cross-reactive antibody responses to the influenza HA stem region 
following H5N1 vaccination in humans. Proc Natl Acad Sci. 2014;111(36):13133-8. 
19. 
Halliley JL, Khurana S, Krammer F, Fitzgerald T, Coyle EM, Chung KY, et al. 
High-Affinity H7 Head and Stalk Domain-Specific Antibody Responses to an Inactivated 
Influenza H7N7 Vaccine After Priming With Live Attenuated Influenza Vaccine. J Infect 
Dis. 2015;212(8):1270-8. doi: 10.093/infdis/jiv210. Epub 2015 Apr 2. 
20. 
Henry Dunand CJ, Leon PE, Huang M, Choi A, Chromikova V, Ho IY, et al. 
Both Neutralizing and Non-Neutralizing Human H7N9 Influenza Vaccine-Induced 
Monoclonal Antibodies Confer Protection. Cell Host Microbe. 2016;19(6):800-13. doi: 
10.1016/j.chom.2016.05.014. 
21. 
Isakova-Sivak I, Korenkov D, Smolonogina T, Kotomina T, Donina S, 
Matyushenko V, et al. Broadly protective anti-hemagglutinin stalk antibodies induced by 
live attenuated influenza vaccine expressing chimeric hemagglutinin. Virology. 
2018;518:313-323.(doi):10.1016/j.virol.2018.03.013. Epub  Mar 22. 
22. 
Nachbagauer R, Kinzler D, Choi A, Hirsh A, Beaulieu E, Lecrenier N, et al. A 
chimeric haemagglutinin-based influenza split virion vaccine adjuvanted with AS03 
induces protective stalk-reactive antibodies in mice. NPJ Vaccines. 
2016;1.(doi):10.1038/npjvaccines.2016.15. Epub  Sep 22. 
23. 
Andrews SF, Huang Y, Kaur K, Popova LI, Ho IY, Pauli NT, et al. Immune 
history profoundly affects broadly protective B cell responses to influenza. Sci Transl 
Med. 2015;7(316):316ra192. doi: 10.1126/scitranslmed.aad0522. 
24. 
Yassine HM, McTamney PM, Boyington JC, Ruckwardt TJ, Crank MC, Smatti 
MK, et al. Use of Hemagglutinin Stem Probes Demonstrate Prevalence of Broadly 
Reactive Group 1 Influenza Antibodies in Human Sera. Sci Rep. 2018;8(1):8628. doi: 
10.1038/s41598-018-26538-7. 
25. 
Nachbagauer R, Choi A, Izikson R, Cox MM, Palese P, Krammer F. Age 
Dependence and Isotype Specificity of Influenza Virus Hemagglutinin Stalk-Reactive 
Antibodies in Humans. MBio. 2016;7(1):e01996-15. doi: 10.1128/mBio.-15. 

Version 3.0, 12April2019 
APPENDICES 
Appendix A: Schedule of Study Procedures 
 
 
  
 
 
 
 
Procedure or Test 
 
 
Day -60 to 
0 
 
 
D0 
 
 
D7 
 
 
D14 
 
 
D28 
 
 
D56 
 
 
D90 
 
D360 
Informed consent  
X 
 
 
 
 
 
 
 
Screening evaluation 
X 
 
 
 
 
 
 
 
Pregnancy Prevention 
Counseling 
X 
X 
X 
X 
 
 
 
 
Complete Physical Exam 
X 
 
 
 
 
 
 
 
Focused Physical Exam 
 
X 
X 
(X) 
(X) 
(X) 
(X) 
(X) 
HbSAg, HCV, HIV 
X 
 
 
 
 
 
 
 
CBC with differential 
X 
 
 
 
 
 
 
 
H5N1 HAI assay 
X 
 
 
 
 
 
 
 
Urine or serum beta-HCG 
(women) 
X 
X 
 
 
X 
 
X 
 
Complete medical history 
X 
 
Review of exclusion/inclusion 
X 
X 
 
 
 
 
 
 
Vital signs 
X 
X 
X 
X 
 
 
 
 
Administer Vaccine 
 
X 
 
 
 
 
 
 
Interim history 
X 
X 
X 
X 
X 
X 
X 
Serology 
 
X 
X 
X 
X 
X 
X 
X 
Whole blood (PBMC) (mL) 
 
X 
X 
X 
X 
X 
X 
X 

Version 3.0, 12April2019 
Appendix B:  Local and Systemic Adverse Event Grading Systems 
These tables are modified versions of the FDA Toxicity Grading Scale for Healthy Adult 
and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials to be used to 
grade adverse events. 
 
Tables for Clinical Abnormalities 
 
Table 4: Local Reaction Grading System 
 
* In addition to grading the measured local reaction at the greatest single diameter, the measurement should 
be recorded as a continuous variable.  
** Induration/Swelling should be evaluated and graded using the functional scale as well as the actual 
measurement. 
 
Local Reaction to 
Injectable Product 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
Potentially Life 
Threatening 
(Grade 4) 
Pain 
Does not 
interfere 
with activity 
Repeated use of non-
narcotic pain reliever 
> 24 hours or 
interferes with 
activity 
Any use of 
narcotic pain 
reliever or 
prevents daily 
activity 
Emergency room 
(ER) visit or 
hospitalization 
Pruritus 
Does not 
interfere 
with activity 
Requiring po or 
topical treatment 
>24 hrs or IV meds 
or steroids for ≤ 24 
hrs 
Requiring IV 
medication or 
steroids for >24 
hours 
ER visit or 
hospitalization 
Erythema/Redness 
* 
2.5 – 5 cm 
5.1 – 10 cm 
> 10 cm 
Necrosis or 
exfoliative 
dermatitis 
Induration/Swellin
g** 
2.5 – 5 cm and 
does not 
interfere 
with activity 
5.1 – 10 cm or 
interferes with 
activity 
> 10 cm or 
prevents daily 
activity 
Necrosis 

Version 3.0, 12April2019 
Table 5: Vital Signs and Systemic Reactions Grading System 
 
Vital Signs * 
/Systemic adverse 
events 
Mild (Grade 1) Moderate(Grade 2) 
Severe (Grade 3) 
Potentially Life 
Threatening 
(Grade 4) 
Tachycardia - beats 
per minute ** 
101 – 115 
116 – 130 
> 130 
ER visit or 
hospitalization for 
arrhythmia 
Bradycardia - beats 
per minute** 
50 – 54 
45 – 49 
< 45 
ER visit or 
hospitalization for 
arrhythmia 
 
Hypertension 
(systolic – mm Hg) 
 
141 – 150 
 
151 – 155 
 
> 155 
ER visit or 
hospitalization for 
malignant 
hypertension 
Hypertension 
(diastolic) - mm Hg 
91 – 95 
96 – 100 
> 100 
ER visit or 
hospitalization for 
malignant 
hypertension 
Hypotension 
(systolic) – mm Hg 
85 – 89 
80 – 84 
< 80 
ER visit or 
hospitalization for 
hypotensive shock 
Respiratory Rate – 
breaths per minute 
17 – 20 
21 – 25 
> 25 
Intubation 
Nausea/vomiting 
No interference 
with activity or 1 
– 2 
episodes/24 
hours 
Some interference 
with activity or > 2 
episodes/24 hours 
Prevents daily 
activity, requires 
outpatient IV 
hydration 
ER visit or 
hospitalization for 
hypotensive shock 
Headache 
No interference 
with 
activity 
Repeated use of non- 
narcotic pain 
reliever > 24 hours or 
some interference 
with activity 
Significant; any 
use of narcotic 
pain reliever or 
prevents daily 
activity 
ER visit or 
hospitalization 
Fatigue/Malaise 
No interference 
with 
activity 
Repeated use of non- 
narcotic pain 
reliever > 24 hours or 
some interference 
with activity 
any use of narcotic 
pain reliever or 
prevents daily 
activity 
ER visit or 
hospitalization 
Myalgia 
No interference 
with 
activity 
Repeated use of non- 
narcotic pain 
reliever > 24 hours or 
some interference 
with activity 
any use of narcotic 
pain reliever or 
prevents daily 
activity 
ER visit or 
hospitalization 
*  Subject should be at rest for all vital sign measurements.  
** When resting heart rate is between 60 – 100 beats per minute. Use clinical judgment when 
characterizing bradycardia among some healthy subject populations, for example, conditioned athletes. 
 
 
 

Version 3.0, 12April2019 
Appendix C: H5N1 Vaccine Product Insert 
Product Insert can be found online at the following site: 
 
https://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProducts/U
CM112836.pdf 
 
 

Version 3.0, 12April2019 
  
52 
Appendix D: H5N1 Vaccine Product label