Clinical Trial of Safety (Reactogenicity) and Immunogenicity of Needle-free Jet Injection of Reduced-dose, Intradermal Influenza Vaccine (INF) Administered to >= 6 to < 24 Month-old Infants and Toddlers in the Dominican Republic
Phase: PHASE1
Conditions: Influenza
Sponsor: Pedro Moro

COVER PAGE for document upload to Record 
NCT00386542 at https://ClinicalTrials.gov  
 
PROTOCOL 
 
“Needle-free Jet Injection of Reduced-
dose, Intradermal, Influenza Vaccine in 
>= 6 to < 24-month-old Children”  
 
Filename:  4785-ID-INF-Prot-Engl-2008Jun24.pdf (prefixed with “Cover-Page+”)  
Full Study Title - English:  Clinical Trial of Safety (Reactogenicity) and 
Immunogenicity of Needle-free Jet Injection of Reduced-dose, 
Intradermal, Influenza Vaccine (INF) Administered to ≥6 -to- <24 
Month-old Infants and Toddlers in the Dominican Republic 
Full Study Title - Español:  Ensayo Clínico de Seguridad y Respuesta 
Inmunológica de Inmunización Administrada sin Aguja con Inyector 
a Chorro en Dosis Reducida por vía Intradérmica con Vacuna 
Influenza (Gripe) (INF) en Niños de ≥6 - <24 Meses de Edad en la 
República Dominicana 
Document Type:  Protocol  [English, final, dated “2008Jun24”] 
Document Description:  Final protocol, dated 2008-06-24, as originally submitted 
on 2006-03-13 to IRB “G” at the U.S. Centers for Disease Control 
and Prevention, originally approved on 2006-06-23, and 
subsequently modified by six investigator-requested and IRB-
approved amendments before and during study implementation, 
with last protocol amendment approved on 2008-06-20.  Subsequent 
IRB-approved study revisions on 2008-08-13 and 2009-04-02 did not 
require changes in the protocol itself.  
 

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[This document is for the use of the investigators, collaborators, participating institutions, members of the 
IRBs and DSMB, and consultants named herein.  It should not be disseminated to other parties or to the 
public without the consent of the investigators.] 
 
PROTOCOL 
 
Clinical Trial of Safety (Reactogenicity) and Immunogenicity of Needle-free Jet 
Injection of Reduced-dose, Intradermal, Influenza Vaccine (INF) Administered to 
≥6 -to- <24 Month-old Infants and Toddlers in the Dominican Republic 
 
Ensayo Clínico de Seguridad  y Respuesta Inmunológica de Inmunización Administrada sin Aguja con Inyector 
a Chorro en Dosis Reducida por vía Intradérmica con Vacuna Influenza (Gripe) (INF) en Niños de ≥6  - <24 
Meses de Edad en la República Dominicana 
 
Centers for Disease Control and Prevention & Hospital Infantil Dr. Robert Reid Cabral 
1. 
Title Page 
Study Title: 
Clinical Trial of Safety (Reactogenicity) and Immunogenicity of Needle-free 
Jet Injection of Reduced-dose, Intradermal Influenza Vaccine (INF) 
administered to ≥6 -to- <24 Month-old Infants and Toddlers in the 
Dominican Republic    [Ensayo Clínico de Seguridad y Respuesta 
Inmunológica de Inmunización Administrada sin Aguja con Inyector a 
Chorro en Dosis Reducida por vía Intradérmica con Vacuna Influenza 
(Gripe) (INF) en Niños de ≥6 - <24 Meses de Edad en la República 
Dominicana] 
Brief Title: 
HIRRC/CDC Needle-free Intradermal Influenza Study   [HIRRC/CDC 
Estudio Influenza Intradérmica sin Aguja]   
Protocol 
Number: 
CDC-ISO-4785   
Investigational 
Product(s): 
Vaxigrip® influenza vaccine (INF) (Sanofi Pasteur, Lyon, France), a 
Dominican-registered product (in prefilled syringes, but not yet in vials), to 
be administered “off label” by intradermal injection using the U.S.-licensed 
Biojector® 2000 jet injector with investigational intradermal spacer (not 
Dominican-registered) 
Indication: 
Induce protective levels of serum antibodies to influenza virus antigens after 
two jet-injected, intradermal, reduced volume/antigen doses of trivalent 
vaccine, spaced at least one month apart, in infants ≥6 to <24 months of age, 
as a correlate of protection from influenza disease 
Sponsor: 
Immunization Safety Office, Centers for Disease Control and Prevention 
(D-26), 1600 Clifton Road, Atlanta, GA  30333 
Phase(s): 
Phase I, followed by Phase II (depending on phase I safety results) 
Sponsor's Responsible 
Medical Officer: 
Bruce G. Weniger, M.D., M.P.H. 
NOTE: This protocol is not longer restricted, and
may be disseminated freely (2023-11-10). 

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3. 
Synopsis 
Name of Sponsor/Company: 
Immunization Safety Office, Office of Chief Science Officer, Centers for Disease Control 
and Prevention (E-61), 1600 Clifton Road, Atlanta, GA 30333, USA 
Study number: 
CDC-ISO-4785   
Name of Finished Products (§10.4): 
Vaxigrip® Inactivated Influenza Vaccine Trivalent Types A and B (Split Virion), Sanofi 
Pasteur, Lyon, France 
Biojector® 2000 Needle-free Injection Management System [jet injector] with investigational 
intradermal spacer, Bioject Medical Technologies, Inc., Portland, OR, USA 
Title of study: 
Clinical Trial of Safety (Reactogenicity) and Immunogenicity of Needle-free Jet Injection of 
Reduced-dose, Intradermal Influenza Vaccine (INF) Administered to ≥6 -to- <24 Month-old 
Infants and Toddlers in the Dominican Republic     [Ensayo Clínico de Seguridad y 
Respuesta Inmunológica de Inmunización Administrada sin Aguja con Inyector a Chorro en 
Dosis Reducida por vía Intradérmica con Vacuna Influenza (Gripe) (INF) en Niños de ≥6 - 
<24 Meses de Edad en la República Dominicana] 
Investigators (§8): 
Bruce G. Weniger, M.D., M.P.H.;  Virgen Gómez, M.D.;  Pedro L. Moro, M.D., M.P.H.;  
Josefina Fernández, M.D.;  Glenny Gúzman, M.D., C. Sarah Mota T., M.D., Jesús M. Feris 
Iglesias, M.D.; Carolyn Bridges, M.D.; Martin Friede, Ph.D.,  John K. Iskander, M.D.  
Study site (§10.2): 
Hospital Infantil Dr. Robert Reid Cabral [national children’s hospital] 
Santo Domingo, Dominican Republic 
Cooperating Organizations (§8): 
• World Health Organization, Geneva, Switzerland 
• Program for Appropriate Technology in Health, Seattle, WA, USA 
• Bioject Medical Technologies, Inc., Portland, OR, USA [donor of injection device] 
• Sanofi Pasteur SA [donor of vaccine product]  
• Laboratory facility [to be determined by WHO tender among experienced and qualified 
research level institutions meeting cGLP or equivalent standards]  
 
Study period: 2006 – 2009 
Phase(s) of development:  Phase I and phase II  
Study Description (§10.1):  
A sequential phase I and II, controlled, double-blinded study to determine whether immune 
responses suggesting protection against influenza can safely be induced in young children by 
two reduced doses one month apart of 0.1 mL of a trivalent inactivated influenza vaccine 
(INF) administered by the intradermal (ID) route with an investigational ID spacer on a U.S.-
licensed needle-free jet injector (JI), compared to two standard intramuscular (IM) 0.25 mL 
doses by needle-syringe (N-S) in this age group.  The locale is a developing country where 
financial restraints for the use of full-dose influenza vaccine would limit protection from 
influenza pandemic threat, where N-Ss pose dangers and drawbacks in clinical use, and 

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where Mantoux-type N-S ID injections are difficult to administer during mass campaigns. 
Objectives (§9.6):  
The primary endpoint of this study is to measure the percentage of participants recruited at 
age >6 -to- <24-months with seroconversion on hemagglutination inhibition (HI) assay of 
serum collected at least one month after two doses of influenza vaccine (INF) administered 
in a reduced-dose volume of 0.1 mL intradermally (ID) by needle-free jet injector and 
intramuscularly (IM) by conventional needle-syringe (N-S), compared to standard IM 
injection of full 0.25 mL doses.   
 
Secondary objectives of this study are to determine for the above comparison seroprotection 
rates, geometric mean titers, and the extent and frequencies of local and systemic reactions.  
 
 
Methods (§10): 
Randomized, observer-blinded, clinical pilot (phase I) trial of safety, followed by a clinical 
(phase II) trial of safety and non-inferiority of immune response to the standard route and 
dose for the merged participants from both phases.   
 
Phase I - Influenza-vaccine naïve children (n=48) aged ≥6 -to- <24 months will be 
randomized in a 1:1:1 ratio to the following three study arms, each to receive two doses on 
days 0 and 28 of trivalent inactivated influenza (INF) vaccine (Vaxigrip®, Sanofi Pasteur, 
Lyon, France) into the left thigh (<12 months) or left deltoid (≥12 months):  
 
• 
Group “ID-JI-0.1” (n=16)  -  reduced 0.1 mL INF doses administered intradermally (ID) 
by needle-free jet injector (JI) (Biojector® 2000 subcutaneous syringe no. 2 (green color 
code), with 2cm investigational spacer, Bioject Medical Technologies, Inc., Portland, 
OR, USA)  
 
• 
Group “IM-NS-0.1” (n=16)  -  reduced 0.1 mL INF doses administered intramuscularly 
(IM) needle-syringe (NS) (via 22-25 gauge needle, minimum 25mm/1-inch length)  
 
• 
Group “IM-NS-0.25” (controls) (n=16)  -  full 0.25 mL INF doses administered 
intramuscularly (IM) by needle-syringe (NS) (22-25 gauge needle, minimum 25mm/1-
inch length)  
 
Phase II - Upon assessment of the safety profile from phase I by the unblinded Data Safety 
Monitoring Board (DSMB), with its approval an additional 402 children will be recruited and 
randomized (134 per group) as in phase I above.  Total participants in phase I and II = 450 
(150 in each of three study arms).   
 
Adverse Event Diaries (§10.6.4):  Parents will be trained to complete a diary form to 
observe, measure, and record solicited local reactions for the injection site and systemic signs 
and symptoms for the child for days 0 through 7 after vaccination, plus unsolicited 
symptoms, illness, and medications for days 0 through 28.   
 

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Followup (§10.6.1):  Return clinic visits will be scheduled on days 2, 7, and 28 after INF 
dose 1, at which times the diary card data will be recorded by staff and the card collected on 
day 28.  Telephone calls and/or home visits on days 3, 9, and 29 will occur for participants 
not returning as scheduled on the preceding day.  Upon receiving dose 2 of vaccine, 
participants will be scheduled again to return to the study center 2, 7, and 28 days afterwards.  
The same procedures as for dose 1 regarding diary cards, telephone followup, and home 
visits will apply after dose 2.  Upon returning to clinic on day 28 after dose 2 (day 56 after 
dose 1), the children in reduced dose groups ID-JI-0.1 and IM-NS-0.1 will receive an 
unblinded, “insurance”, full-volume, 0.25 mL dose (#3) of influenza vaccine by NS IM.  
Group IM-NS-0.25 will receive a blinded mock 3rd injection.  All groups will return 6 
months later (8 months after dose 1, 7 after dose 2) for a “bonus” booster vaccination for the 
next influenza season, at which time late adverse events since the last visit will be queried.  
 
Serum and Virus Collection (§10.6.10):  Blood specimens to measure serologic responses 
will be collected three times, just prior to vaccination on day 0 (INF dose 1), on day 28 (INF 
dose 2), and on day 56 (INF “insurance” dose 3).  Participants with influenza-like illness will 
have nasopharyngeal/throat swabs analyzed by rapid influenza test.  Those with positive 
influenza A results will have specimens sent for viral culture. 
 
Stopping rules (§10.9):  Vaccination will be suspended for all participants, pending 
unblinded review by the DSMB and its clearance or denial to resume, as well as 
simultaneous IRB guidance, upon occurrences deemed possibly- or probably-related to the 
study vaccinations by any of the Dominican or U.S. Principal or Senior Investigators of 
serious adverse events in any 3 (6%) phase I vaccinees or any 16 (4%) phase II vaccinees, or 
upon incidents of entire-limb swelling or other severe local reactions at the INF injection site 
in any 5 (10%) phase I vaccinees or any 20 (5%) phase II vaccinees.  
 
Criteria for proceeding to phase II (§10.10):  Either or both of the study’s investigational 
arms (ID-JI-0.1 or IM-NS-0.1) will not proceed to phase II if the observed frequency of the 
following events in that arm exceeds the frequency observed in the control IM-NS-0.25 
group by the designated excess:    
 
 
      (Invest. Arm) – (Control Arm) 
• Any serious adverse event, possibly- or probably-related:  exceeds by ≥3 
• Fever >40º C possibly- or probably-related:  
 
exceeds by ≥4 
• Injection site whole-limb swelling: 
 
exceeds by ≥4 
• Injection site induration >5 cm diameter:  
 
exceeds by ≥5 
• Injection site erythema >5 cm diameter:  
 
exceeds by ≥5 
• Injection site pain level 3 (cries upon moving limb): 
exceeds by ≥5 
 
Number of participants planned: 
Phase I = 48;  phase II = 402  (total participants = 450) 
Participant characteristics and main criteria for inclusion and exclusion (§10.3): 
Healthy, full-term infants of age ≥6 -to- <24 months (before second birthday) who are 
recruited as prior or current patients, or siblings of patients, attending a public outpatient 
clinic at the national children’s hospital.  Excluded will be infants not up-to-date on 
immunizations who need other injected vaccines recommended routinely at younger ages, or 

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those with chronic or acute clinical illness contraindicating vaccination, or residence outside 
the metropolitan area (Distrito Nacional) whence return visits would be inconvenient. 
Investigational vaccine(s), antigen content, dosage, route of administration (§10.4.2): 
Inactivated Influenza Vaccine Trivalent Types A and B (Split Virion) (INF) (Vaxigrip®a, 
Sanofi-Pasteur, Lyon, France), containing 15 µg hemagglutin (HA) per 0.5 mL for each of 
three virus strains (2006 Southern Hemisphere formulation):   
 
•  A/New Caledonia/20/99 (H1N1) [IVR-116] 
 
•  A/New York/55/2004 (H3N2) [NYMC X-157] (an A/California/7/2004-like strain) 
 
•  B/Victoria/2/87 lineage (a B/Malaysia/2506/2004-like strain) 
When the initial season formulation expires, new participants will be vaccinated with either 
the 2006-2007 Northern Hemisphere and/or the succeeding 2007 Southern Hemisphere 
formulations.  If these formulations differ in their virus strains, recruitment will be deferred 
to ensure each participant receives unexpired doses from the same formulation (serologic 
assays will be segregated accordingly). 
The investigational regimen will be a reduced dose of 0.1 mL (3 µg HA per virus strain) 
injected into the left thigh (or left deltoid for age ≥12 months) by either routine intramuscular 
(IM) needle-syringe (N-S) injection, or by needle-free Biojector® 2000 jet injector (JI) with 
investigational spacer for intradermal (ID) delivery (Bioject Medical Technologies, Portland, 
OR, USA)b.  Same dose repeated one month later.  One month after that, all participants 
receive unblinded third “insurance” dose of full 0.25 mL by N-S IM.   
Reference vaccine, antigen content, dosage regimen, route of administration: 
For the control group, the same INF vaccine as above will be administered in standard 0.25 
mL dosage (7.5 µg HA per virus strain) for this age group into the left thigh (or left deltoid 
for age ≥12 months) IM by conventional N-S.  Same dose repeated one month later.  One 
month after that, all participants receive unblinded third “insurance” dose of full 0.25 mL by 
N-S IM.   
Concomitant vaccines (§10.4.7): 
No other routine or investigational vaccines will be administered within 28 days before or 
after either of the study’s investigational INF doses 1 and 2 described above.  Participants 
who may have received, or are due to receive recommended vaccines such as POLOPV and 
DTPw-HBV-HIB (recommended at 6 and 18 months) and MMR (12 months) will not be 
recruited during appropriate intervals before and after these routine doses.  
Measurement of Immunogenicity (§10.5.1): 
(See primary and secondary endpoints in Objectives section, above.)   
Tenders for performance of serologic assays will be solicited by WHO from the Laboratory 
for Specialized Clinical Studies, Division of Infectious Diseases, Cincinnati Children’s 
                                                 
a As of 8 June 2006, Sanofi Pasteur’s Vaxigrip® influenza vaccine was licensed by the regulatory authority of the 
Dominican Republic, the Dirección General de Drogas y Farmacias, for routine sale and use only in its prefilled 
syringe packaging format, but not in its 5 mL multidose vials.  Vaxigrip® is licensed in Canada and many other 
countries of Latin America, the Caribbean, Europe, Africa, and Asia, but not in the United States.   
b As of 8 June 2006, Bioject’s Biojector® 2000 jet injector was licensed as a medical device in the U.S., Canada, 
and other countries of the Americas, Europe, and Asia, but not in the Dominican Republic.   

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Hospital Medical Center, Cincinnati, OH, USA; from the National Institute for Biological 
Standards and Controls, Potters Bar, Hertfordshire, UK; and from other qualified research 
laboratories via public solicitation.   
Hemagglutination inhibition (HI) assays against viral strains similar to those included in the 
vaccine will be performed according to current Good Laboratory Practices (cGLP) and 
standard assay protocol.  Assay antigens will be selected for suitability with strains in the 
vaccine formulations.  After treatment to eliminate nonspecific inhibitors, specimens will be 
diluted 2-fold through a final dilution of 1:1024 or higher.  Additional serum controls diluted 
from each specimen and red blood cell controls will be used on each plate.   Viral strain 
antigens -- obtained from external reference laboratories or prepared on site -- will be 
prepared daily when assays are performed.   
Measurements of safety (§10.5.2): 
• Investigator-observed local injection site and systemic symptoms at 0 and 30-60 
minutes, plus 2, 7, and 28 days after both the injections.  Home telephone calls and 
home visits, if needed, by study staff on days 3, 9, and 29 for participants failing to 
return to clinic as scheduled.  
• Prompted daily measurement and recording on parental diary form for 8 days 
following both study injections of local (tenderness, erythema, induration, 
ecchymosis, circumference) and systemic reactions (fever, anti-fever medication, loss 
of appetite, sleepiness, irritability, unusual or inconsolable crying, vomiting, diarrhea, 
convulsions, other to be specified).  
• Unprompted reports on parental diary form or by telephone of these or other adverse 
events or symptoms occurring up to 28 days after each injection, plus report upon 
return for “bonus” booster vaccination 6 months after dose 2 + 28 days.  
 
Statistical power considerations (§10.8): 
For each of three treatment arms in the combined phases I and II, 150 participants will be 
studied, resulting in a total sample size of 450.  This number results from rounding up, in 
anticipation of modest loss-to-followup, from a calculated 142 participants required per arm. 
The sample size calculation is based on a non-inferiority model using an expected 
seroconversion rate of 86.4% derived from a mean from four prior clinical studies of 
Vaxigrip® in the similar age of range of 6 to 36 months.  A twelve percent decrement, i.e., 
0.104, is selected, corresponding to an acceptable seroconversion rate as low as 76.0% in the 
investigational study arms to demonstrate they are not worse than the control arm by more 
than this amount.  Sample size parameters are α = 0.05, Power = 0.80 (β = 0.2).  

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4. 
Table of Contents 
1. 
Title Page..............................................................................................................................1 
2. 
Signatures ............................................................................................................................2 
3. 
Synopsis...............................................................................................................................3 
4. 
Table of Contents.................................................................................................................8 
5. 
Study and Events Timeline ...............................................................................................11 
6. 
List of Abbreviations and Definition of Terms................................................................12 
7. 
Ethics ..................................................................................................................................13 
7.1 
Institutional Review Boards or Independent Ethics Committees .................................13 
7.2 
Ethical Conduct of the Study .......................................................................................14 
7.3 
Participant Information and Consent ...........................................................................14 
7.4 
Obligation to Participants for Related Adverse Consequences...................................14 
7.5 
Insurance.....................................................................................................................14 
7.6 
Level of Risk for Research on Children .......................................................................15 
7.7 
Regulatory Oversight of Investigational Products........................................................15 
8. 
Investigators and Study Administrative Structure .........................................................15 
8.1 
U.S. Investigators ........................................................................................................16 
8.1.1 U.S. Principal Investigator, Sponsor Representative.............................................16 
8.1.2 U.S. Co-Investigators ............................................................................................16 
8.1.3 U.S. Senior Investigator.........................................................................................16 
8.2 
Dominican Investigators ..............................................................................................17 
8.2.1 Dominican Principal Investigator ...........................................................................17 
8.2.2 Dominican Co-investigators...................................................................................17 
8.2.3 Dominican Senior Investigator...............................................................................18 
8.3 
World Health Organization ..........................................................................................18 
8.4 
Program for Appropriate Technology in Health ..........................................................18 
8.5 
Biojector® 2000 Injector Manufacturer .........................................................................18 
8.6 
Vaxigrip® Influenza Vaccine Manufacturer ..................................................................19 
8.7 
Consultants..................................................................................................................19 
8.8 
Data and Safety Monitoring Board...............................................................................20 
9. 
Introduction and Background...........................................................................................21 
9.1 
Pandemic Preparedness – Utility of Intradermal Jet Injection .....................................21 
9.2 
Dangers of Unsafe Needle-syringe Injections .............................................................23 
9.3 
Needle-free Jet Injection Technology ..........................................................................24 
9.3.1 Description and Clinical History .............................................................................24 
9.3.2 MUNJIs vs. DCJIs..................................................................................................25 
9.3.3 Jet Injectors in Children .........................................................................................25 
9.3.4 Immunogenicity of Jet-injected Vaccines...............................................................26 
9.3.4.1 
Non-adjuvanted Vaccines Delivered by Jet Injection ....................................26 
9.3.5 Reactogenicity of Jet-injected IM and SC Vaccines ..............................................27 
9.3.5.1 
Alum-adjuvanted Vaccines Delivered by Jet Injection...................................27 
9.4 
Intradermal Route of Vaccination ................................................................................30 
9.4.1 Vaccines other than Influenza ...............................................................................30 

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9.4.2 Influenza by Intradermal Needle Injection .............................................................30 
9.4.3 Reduced-dose Influenza Vaccination by Different Routes ....................................31 
9.4.4 Influenza by Intradermal Needle-free Jet Injection ................................................31 
9.5 
Influenza Disease Burden............................................................................................31 
9.5.1 Epidemiology of Influenza in Dominican Republic and Caribbean ........................33 
9.6 
Objectives and Rationale for Study .............................................................................34 
9.6.1 Study Objectives....................................................................................................34 
9.6.2 Rationale................................................................................................................34 
9.6.3 Intended / Potential Use of Study Findings............................................................34 
9.6.4 Research Questions ..............................................................................................35 
10. Investigational Plan ...........................................................................................................35 
10.1 Overall Study Design and Plan....................................................................................35 
10.1.1 Study Arms ............................................................................................................36 
10.1.2 Phases...................................................................................................................36 
10.1.3 Endpoints...............................................................................................................37 
10.1.3.1 Primary Immunologic.....................................................................................37 
10.1.3.2 Primary Safety...............................................................................................37 
10.2 Physical Facilities ........................................................................................................38 
10.2.1 Description.............................................................................................................38 
10.2.2 Participant Recruitment .........................................................................................39 
10.2.3 Study Center..........................................................................................................39 
10.3 Selection of Study Population......................................................................................40 
10.3.1 Inclusion Criteria....................................................................................................40 
10.3.2 Exclusion Criteria...................................................................................................40 
10.3.3 Removal of Participants from Therapy or Assessment..........................................42 
10.4 Treatments...................................................................................................................42 
10.4.1 Treatments Administered.......................................................................................42 
10.4.2 Identity of Investigational Vaccine (Vaxigrip®, Sanofi-Pasteur)..............................42 
10.4.2.1 Product Characteristics .................................................................................43 
10.4.2.2 Storage..........................................................................................................44 
10.4.2.3 Directions for Vaccine Administration............................................................44 
10.4.2.4 Vaccine Immunogenicity Profile ....................................................................46 
10.4.2.5 Vaccine Efficacy ............................................................................................48 
10.4.2.6 Vaccine Safety Profile ...................................................................................49 
10.4.2.7 Guillain-Barré Sequelae of Influenza Vaccination .........................................51 
10.4.3 Identity of Injection Device (Biojector® 2000, Bioject, Inc.) ....................................52 
10.4.3.1 Preclinical and Veterinary Studies in Animals ...............................................53 
10.4.3.2 Clinical Studies of Licensed Drugs and Vaccines .........................................53 
10.4.3.3 Clinical Studies of Investigational Drugs and Vaccines.................................54 
10.4.3.4 Investigational Spacer ...................................................................................56 
10.4.3.5 Preclinical Studies of the Biojector ID Spacer ...............................................57 
10.4.3.6 Clinical Studies of the Biojector ID Spacer....................................................63 
10.4.4 Method of Assigning Participants to Treatment Groups ........................................67 
10.4.4.1 Equal Allocation by Sextuplet Blocks ............................................................67 
10.4.4.2 Avoiding Block Counting................................................................................68 
10.4.4.3 Allocation Group Randomization Cards ........................................................68 
10.4.4.4 Allocation Group Randomization Envelopes .................................................71 
10.4.5 Timing of Doses for Each Participant ....................................................................72 

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10.4.6 Blinding..................................................................................................................72 
10.4.6.1 Premature “Breaking” of the Allocation Code................................................73 
10.4.6.2 Separation of Parent from Child during Injection...........................................73 
10.4.6.3 Caching of Immediate Injection Site Observations by Nurses.......................75 
10.4.7 Prior and Concomitant Therapy - Other Vaccinations ...........................................75 
10.4.8 Treatment Compliance and Participant Incentives ................................................76 
10.5 Efficacy and Safety Variables......................................................................................77 
10.5.1 Efficacy ..................................................................................................................77 
10.5.1.1 Immunogenicity .............................................................................................77 
10.5.1.2 Primary Efficacy Variable ..............................................................................77 
10.5.1.3 Secondary Efficacy Variables........................................................................77 
10.5.1.4 Subsequent Specimen Storage and Laboratory Assay(s).............................78 
10.5.2 Safety.....................................................................................................................78 
10.5.2.1 Local and Systemic Reactions ......................................................................78 
10.5.2.2 Safety Variables and Monitoring....................................................................79 
10.5.2.3 Determination of Followup Period for Adverse Events ..................................82 
10.5.3 Appropriateness of measurements........................................................................83 
10.5.4 Pharmacokinetics ..................................................................................................83 
10.6 Study Procedures ........................................................................................................83 
10.6.1 Time and Events Table..........................................................................................83 
10.6.2 Training of Study Personnel ..................................................................................84 
10.6.2.1 Stage 1: Logistics and Practice .....................................................................84 
10.6.2.2 Phase 2: Pilot Actual Recruitment and Study Initiation .................................89 
10.6.3 Screening and Recruitment ...................................................................................90 
10.6.4 Informed Consent Process ....................................................................................91 
10.6.5 Monitoring of Informed Consent Process ..............................................................91 
10.6.6 Parent Training Procedure.....................................................................................92 
10.6.7 Venipuncture Procedure........................................................................................93 
10.6.8 Vaccination Procedure...........................................................................................93 
10.6.9 Rapid Diagnosis and Virus Culture for Influenza-Like Illness ................................94 
10.6.10 
Specimen Preparation, Labeling, Storage, and Shipping................................94 
10.6.10.1 Serum Specimens .........................................................................................95 
10.6.10.2 Virus Culture Specimens...............................................................................96 
10.6.11 
Data Coding, Handling, and Analysis..............................................................96 
10.6.12 
Laboratory Assays...........................................................................................97 
10.6.12.1 Serology ........................................................................................................97 
10.6.12.2 Rapid Test for Influenza Illness .....................................................................98 
10.6.12.3 Influenza Virus Culture ..................................................................................98 
10.6.13 
Termination Visit..............................................................................................98 
10.7 Data Quality Assurance...............................................................................................98 
10.7.1 Clinical Procedures................................................................................................98 
10.7.2 Good Clinical Practices Monitoring........................................................................99 
10.7.3 Data Handling........................................................................................................99 
10.8 Statistical Methods and Determination of Sample Size...............................................99 
10.8.1 Statistical and Analytical Plans ..............................................................................99 
10.8.1.1 Primary Efficacy Analysis ..............................................................................99 
10.8.1.2 Secondary Efficacy Analyses ........................................................................99 
10.8.2 Determination of Sample Size .............................................................................100 

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10.8.3 Randomization.....................................................................................................101 
10.9 Stopping Rules to Suspend Vaccination....................................................................102 
10.9.1 Phase I.................................................................................................................102 
10.9.2 Phase II................................................................................................................102 
10.10 Criteria for Proceeding to Phase II.............................................................................102 
10.11 Data and Safety Monitoring Board.............................................................................103 
10.12 Early Reporting to DSMB and IRBs...........................................................................104 
10.13 Changes in the Conduct of the Study or Planned Analyses......................................104 
11. Policies and Practices.....................................................................................................105 
11.1 Compensation, Insurance, and Indemnity .................................................................105 
11.2 Study Material Accountability.....................................................................................106 
11.3 Case Report Forms ...................................................................................................106 
11.4 Data Clarification Forms ............................................................................................106 
11.5 Study Monitoring........................................................................................................107 
11.6 Retention of Records.................................................................................................107 
11.7 Use of Information and Publication............................................................................107 
11.8 Statement of Obligations ...........................................................................................108 
11.8.1 Sponsor and Monitor ...........................................................................................108 
11.8.2 Clinical Investigators............................................................................................109 
12. Questions and Answers on Study Plan, Policies, Ethics.............................................111 
13. List of Associated Documents .......................................................................................118 
14. References........................................................................................................................118 
 
5. 
Study and Events Timeline 
0
3
6
9
12
15
18
21
24
27
------------------------------------------------------------------------------------
STUDY PREPARATION                                                               
Finalize protocol, forms, and documentation • 1m
IRB submission and other approval processes • 2m
Initial DSMB meeting / conf call  • 1d
Procurement of equipment and supplies • 2m
Training of onsite personnel • 2m
------------------------------------------------------------------------------------
PHASE I  (n=48)                                                                            
Recruitment @15/week, 1st blood draw, 1st INF dose • 1m
1-month A.E. followup until day 28 • 2m
2nd blood draw; 2nd INF dose • 1m
1-month A.E. followup after dose 2 until day 56 • 2m
3rd blood draw, "insurance" 3rd INF dose • 1m
Send phase I specimens in separate aliquot shipments • 2w
Blinded serologic assays on phase I bloods • 2m
Blinded phase I data summary for DSMB use • 2w
Unblinded DSMB assessment of safety • 2w
------------------------------------------------------------------------------------
DECISION POINT FOR PROCEEDING TO PHASE II • 2w     
If YES, continue to phase II.  If NO, finish striped steps here.
Unblinded phase I data analysis and prepare reports • 4m
Present/publish phase I results (conferences/articles) • 4m
------------------------------------------------------------------------------------
PHASE II  (n=402)                                                                         
Recruitment @15/week, 1st blood draw, 1st INF dose • 6.5m
1-month A.E. folllowup until day 28 • 7.5m
2nd blood draw; 2nd INF dose • 6.5m
1-month A.E. followup after dose 2 until day 56 • 7.5m
3rd blood draw, "insurance" 3rd INF dose • 6.5m
Send phase II specimens in separate aliquot shipments • 2w
"Bonus" 4th booster INF dose • 6.5m
------------------------------------------------------------------------------------
ASSAYS, ANALYSIS, PRESENTATIONS, PUBLICATIONS   
Blinded laboratory assays of phase II specimens • 2m
Analyze phase I and II data and prepare reports • 4.5m
Present results at conferences and publish articles • 3m
------------------------------------------------------------------------------------
Months since all approvals received
Figure 1.  Study and events timeline. 

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6. 
List of Abbreviations and Definition of Termsc 
AE 
 
Adverse event 
CDC 
 
Centers for Disease Control and Prevention 
CPMP  
Committee for Proprietary Medicinal Products, of the EMEA 
CONABIOS 
Consejo Nacional de Bioética en Salud [National Council on Bioethics in Health, 
an organ of SESPAS] 
CRF 
 
Case report form 
DCF 
 
Data clarification form 
DSMB  
Data Safety Monitoring Board 
DTPw  
Diphtheria, tetanus, pertussis (whole cell) combination vaccine 
EC 
 
ethics committee (generic term for institutional review board; see IRB) 
EIA 
 
Enzyme immunoassay 
EMEA  
European Medicines Agency (formerly know as the European Agency for the 
Evaluation of Medicinal Products) 
FDA 
 
Food and Drug Administration 
FDI 
 
Fundación Dominicana de Infectología  [Dominican Infectious Disease 
Foundation]  
GAVI  
Global Alliance for Vaccines and Immunizations  
GSK 
 
GlaxoSmithKline, vaccine manufacturer 
GCP 
 
Good Clinical Practice; also cGMP: current Good Clinical Practice 
GLP 
 
Good Laboratory Practice; also cGLP: current Good Laboratory Practice 
GMT 
 
Geometric mean titer 
HIRRC  
Hospital Infantil [Children’s Hospital] Dr. Robert Reid Cabral 
HI 
 
Hemagglutination inhibition [assay] 
ICH 
 
International Conference on Harmonization 
IM 
 
Intramuscular 
IND 
 
Investigational New Drug 
IRB 
 
Institutional review board 
ISO 
 
Immunization Safety Office, unit of OCSO 
JI 
 
Jet injector, jet injection, or jet-injected 
JI-IM 
 
jet-injected intramuscular administration 
JI-SC 
 
jet-injected subcutaneous administration 
MEA  
Measles virus vaccine 
MEN  
Meningococcal (Neisseria meningitidis) vaccine, not otherwise specified 
MENps-AC 
Meningococcal (Neisseria meningitidis) vaccine, polysaccharide, serogroups A and 
C 
MMR  
Measles, mumps, rubella virus combination vaccine 
NIP 
 
National Immunization Program, unit of CDC 
N-S 
 
Needle-syringe 
NS-IM  
needle-syringe intramuscular administration 
OCSO  
Office of the Chief Science Officer, CDC 
OPS 
 
Organización Panamericana de la Salud [PAHO]  
PAHO  
Pan American Health Organization [OPS]  
PI 
 
Principal Investigator (U.S. or Dominican) 
POLOPV 
Poliomyelitis vaccine, live attenuated oral “Sabin” type 
Sn-Pa  
Sanofi-Pasteur, vaccine manufacturer subsidiary of sanofi-aventis Group 
                                                 
c  Vaccine abbreviations in this table and elsewhere in the document conform to the recommendations of the 
Vaccine Identification Standards Initiative (http://www.cdc.gov/nip/visi/prototypes/vaxabbrev.htm)  

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SAR 
 
MEA, measles (sarampión in Spanish) virus vaccine 
SRP 
 
Abbreviation in Spanish for sarampión (measles), rubéola (rubella), paperas 
(mumps) combination virus vaccine, which in English is MMR (measles, 
mumps, rubella)  
SC 
 
Subcutaneous, or seroconversion, depending on context 
SESPAS 
Secretaría de Estado de Salud Pública y Asistencia Social [ministry of health 
equivalent] 
SI 
 
Senior Investigator (U.S. or Dominican)  
SP 
 
Seroprotection 
 
 
7. 
Ethics 
7.1 
Institutional Review Boards or Independent Ethics Committees 
The principal investigators will provide the institutional review board (IRB)/ethics 
committee (EC) of both the Fundación Dominicana de Infectología (FDI), serving the 
Hospital Infantil Dr. Robert Reid Cabral (HIRRC) [national children’s hospital], and of the 
Centers for Disease Control and Prevention (CDC) with all appropriate material, including 
the informed consent document.   
 
The trial will not be initiated until appropriate approvals of the protocol, the informed 
consent document, and all recruiting materials are obtained in writing from the IRB/ECs of 
both the FDI and the CDC.  In addition, before implementation, the study will be submitted 
for requisite approval by the Consejo Nacional de Bioética en Salud (CONABIOS), the 
ethical review body of the Secretaría del Estado de Salud Pública y Asistencia Social 
(SESPAS) [ministry of health equivalent].  In anticipation of supplementary funding for the 
study, the Ethics Review Committee of the World Health Organization (WHO), Geneva, 
Switzerland, will also review the documentation (logged as RPC170).   
 
Appropriate reports on the progress of the study will be made to both the FDI and CDC 
IRBs (and to authorized others so requesting) by the principal investigators in accordance 
with applicable governmental regulations and in agreement with policy established by the 
sponsor.  
 
The IRB #1 of the Fundación Dominicana de Infectología, Inc. (parent organization 
IORG0004207) was registered as no. IRB00004986 with the U.S. Office for Human 
Research Protections on 8 August 2005 (renewal scheduled 8 August 2008), under a 
Federalwide Assurance no. FWA00008924 approved on 11 August 2005 (expiration 11 
August 2008).    
 
IRB nos. 1-A, 2-B, 3-C, 6-G, and 7-S of the Centers for Disease Control and Prevention 
(CDC) (parent organization IORG 0000112) were re-registered as IRB nos. IRB00000183, -
0184, -0185, -0188, and -2724, respectively, on 27 February 2004 (expiration 27 February 
2007), under FWA no. FWA00001413, approved on 6 November 2001 (expiration 5 April 
2008).  
 

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7.2 
Ethical Conduct of the Study 
This study will be conducted in accordance with the Declaration of Helsinki and good 
clinical practice (GCP) guidelines of the International Conference on Harmonization.  
Specifically, this study is based on adequately performed laboratory and animal 
experimentation (by others); will be conducted under a protocol reviewed by an IRB or EC; 
will be conducted by scientifically and medically qualified persons; the benefits of the study 
are in proportion to its risks; the rights and welfare of the participants will be respected; the 
physicians conducting the study do not find the hazards to outweigh the potential benefits; 
and each participant’s parent or legal guardian will give his or her informed consent.  
 
7.3 
Participant Information and Consent 
A properly executed, written, informed consent, in compliance with GCP according to ICH 
guidelines, will be obtained from the parent or legal guardian of each participant minor prior 
to entering the participant into the trial.  The consent form was pre-tested for understanding 
among an informal sample of parents bringing children for care at the HIRRC, and was 
revised accordingly before finalization.  A copy of the informed consent document will be 
submitted by the investigator to the IRB/ECs for review and approval prior to the start of the 
study.  The investigators will provide a copy of the signed informed consent form to each 
participant’s parent or guardian, and will maintain a copy in the participant’s record file.  A 
social worker will observe the informed consent process, talk privately with a voluntary 
sample of parents, and advise the investigators of any needed improvement in the process 
(see section 10.6.4). 
 
The informed consent document will explain and provide for a separate written signature for 
permission to maintain indefinite storage of leftover blood or swab specimens not needed in 
the study assays for future testing (except for HIV and human genetics).  Parents may 
decline such signature without affecting their participation in the study.   
 
7.4 
Obligation to Participants for Related Adverse Consequences  
Any participants suffering medical harm as a consequence of participation in the study shall 
receive from the HIRRC regular or intensive medical care for such harm, as needed and as 
normally available from the HIRRC for its other patients, without limitation as to the 
duration of such care, and without charge for such care to the participant or to his/her family 
(see section 11).   
 
7.5 
Insurance  
Prior to the start of participant enrollment and vaccination, an insurance policy will be 
purchased by the World Health Organization and put into effect on behalf of the 
collaborating institutions conducting this study (HIRRC, FDI, CDC, and WHO) to cover 
liability for harm to participants which may result as a direct consequence of participating in 
this study (see details in section 11.1).  
 

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7.6 
Level of Risk for Research on Children 
In accordance with the Code of Federal Regulations (45 CFR 46.405), the investigators 
suggest that this research should be classified as “greater than minimal risk” because all 
vaccination carries some risk for usually mild and self-limiting local (e.g., swelling, 
soreness) and systemic (e.g., fever, irritability) reactions.  However, there is “the prospect of 
direct benefit to the individual subjects” because of the likely protection that will be 
conferred on participants from the known morbidity and mortality of influenza disease as a 
result of trial participation.   
 
Thus, under 46 CFR 46 Subpart D, this minimal risk is justified by the anticipated benefit to 
participants, and such anticipated benefit is at least as favorable as any alternative 
approaches to preventing influenza, of which there are none in these circumstances.  
Although it is not possible to solicit the assent of children of the target ages, the informed 
permission of their parents/guardians will be a requirement of enrollment.   
 
7.7 
Regulatory Oversight of Investigational Products 
The study will not proceed until written authorization for use in the trial of the Vaxigrip 
Vaccine in its vial packaging and “off-label” route and dose, and of the Biojector® 2000 jet 
injector with investigational intradermal spacer, is received from the national pharmaceutical 
regulatory authority of the Dominican Republic, its General Directorate of Drugs and 
Pharmacies (Dirección General de Drogas y Farmacias), a unit of its ministry of health, the 
Secretaría de Estado de Salud Pública y Asistencia Social (Secretariat of State of Public 
Health and Social Assistance, abbreviated SESPAS).  FDA regulatory oversight is precluded 
(see section 12.4). 
 
 
8. 
Investigators and Study Administrative Structure 
The trial will be overseen by staff of the Immunization Safety Office (ISO), Office of the 
Chief Science Officer, U.S. Centers for Disease Control and Prevention (CDC), and the 
National Center for Immunization and Respiratory Diseases (NCIRD), CDC.  It will be 
jointly planned and clinically administered by employees or representatives of the 
Fundación Dominicana de Infectología (FDI) and the affiliated Hospital Infantil Dr. 
Robert Reid Cabral (HIRRC) [national children’s hospital] of the Dominican Republic.  
Technical and financial input is contributed by the World Health Organization and the 
Program for Appropriate Technology in Health. 
 
Employees or representatives of the manufacturer of the jet injector and investigational 
spacer to be used, Bioject, Inc. and the vaccine manufacturer, sanofi pasteur SA, will 
assist with provision of supplies, equipment, and/or related training and consultation.  In 
collaboration with HIRRC, staff of CDC will perform verification of source 
documentation for each participant.  HIRRC and CDC will be responsible for the timely 
reporting of serious adverse events (SAEs) to the necessary authorities and the 
corresponding manufacturers.   
 

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8.1 
U.S. Investigators 
8.1.1 
U.S. Principal Investigator, Sponsor Representative 
 
 
Bruce G. Weniger, M.D., M.P.H. 
 
 
Assistant Chief for Vaccine Development 
 
 
Immunization Safety Office (ISO) 
 
 
Office of the Chief Science Officer (OCSO) 
 
 
Centers for Disease Control and Prevention (CDC) 
 
 
1600 Clifton Road (D-26), Atlanta, GA 30333 
 
 
 
Tel:  [+1] (404) 639-8779 
 
 
 
Fax:  [+1] (404) 639-8834 
 
 
 
Cell:  [+1] (678) 478-1101 
 
 
 
Email:  bgw2@cdc.gov     http://www.cdc.gov/nip/dev 
 
8.1.2 
U.S. Co-Investigators d 
 
 
Pedro L. Moro, M.D., M.P.H. 
 
 
ORISE Fellow 
 
 
ISO, OCSO, CDC 
 
 
1600 Clifton Road (D-26), Atlanta, GA 30333 
 
 
 
Tel:  [+1] (404) 639-8946 
 
 
 
Fax:  [+1] (404) 639-8834 
 
 
 
Email:  psm9@cdc.gov 
 
 
 
Carolyn B. Bridges, M.D. 
 
 
Medical Epidemiologist 
 
 
National Center for Immunization and Respir. Diseases, CDC 
 
 
1600 Clifton Road (A-47), Atlanta, GA 30333 
 
 
 
Tel:  [+1] (404) 639-8689 
 
 
 
Mobile:  [+1] (404) 964-8691 
 
 
 
Email:  ctb1@cdc.gov 
 
8.1.3 
U.S. Senior Investigator 
 
 
John K. Iskander, M.D., M.P.H. 
 
 
Acting Co-Director 
 
 
ISO, OCSO, CDC 
 
 
1600 Clifton Road (D-26), Atlanta, GA  30333 
 
 
 
Tel:  [+1] (404) 639-8889 
 
 
 
Fax:  [+1] (404) 639-8834 
 
 
 
Email:  jxi0@cdc.gov 
 
                                                 
d Pursuant to CDC IRB rules, the roles of the CDC investigators in this study are anticipated as follows:  1. Study 
design and protocol/forms development (BGW, PLM, CBB).  2. Negotiation and coordination with HIRRC and 
other partners and collaborators within and outside CDC (BGW, PLM, CBB).  3. Data analysis and writeup of 
results (BGW, PLM).  4. General oversight and supervision of project director (JKI).   

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8.2 
Dominican Investigators 
8.2.1 
Dominican Principal Investigator 
 
 
Virgen Gómez, M.D. 
 
 
Director of Followup Clinic 
 
 
Department of Infectious Diseases  
 
 
Hospital Infantil Dr. Robert Reid Cabral (HIRRC) 
 
 
Av. Abraham Lincoln No. 2, Centro de los Héroes 
 
 
Santo Domingo, Distrito Nacional, Dominican Republic 
 
 
 
Tel:  [+1] (809) 532-5872 
 
 
 
Mobile:  [+1] (809) 449-2913 
 
 
 
Fax:  [+1] (809) 532-2564 
 
 
 
Email:  virgen.gomez@verizon.net.do  
 
8.2.2 
Dominican Co-investigators 
 
Josefina Fernández, M.D. 
 
 
Director of Research 
 
 
Department of Infectious Diseases 
 
 
HIRRC 
 
 
 
Tel:  [+1] (809) 532-5872 
 
 
 
Mobile:  [+1] (809) 223-5129 
 
 
 
Fax:  [+1] (809) 532-2564 
 
 
 
Email:  infectologia1@verizon.net.do    infectologia01@yahoo.es  
 
 
 
Jacqueline Sánchez, M.D. 
 
 
Directora, Investigaciones Microbiológicas 
 
 
HIRRC 
 
 
 
Tel:  [+1] (809) 532-5872 
 
 
 
Cell:  [+1] (809) 696-2448 
 
 
 
Fax:  [+1] (809) 532-2564 
 
 
 
Email:  infectologia2@verizon.net.do   microdei10@hotmail.com   
 
 
 
Glenny Gúzman, M.D. 
 
 
Investigadora Médica 
 
 
HIRRC 
Tel:  [+1] (809) 532-5872    Cell:  [+1] (809) 258-0667 
 
 
 
Fax:  [+1] (809) 532-2564 
 
 
 
C. Sarah Mota T., M.D. 
 
 
Investigadora Médica 
 
 
HIRRC 
Tel:  [+1] (809) 532-5872     Cell:  [+1] (809) 697-4976 
 
 
 
Fax:  [+1] (809) 532-2564 
 

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8.2.3 
Dominican Senior Investigator 
 
 
Jesús M. Feris-Iglesias, M.D. 
 
 
Director, Fundación Dominicana de Infectología 
 
 
Chief, Department of Infectious Diseases 
 
 
HIRRC  
 
 
 
Tel:  [+1] (809) 532-5872 (morning), 685-2552 (afternoon) 
 
 
 
Mobiles:  (809) 222-4516, (809) 732-0820 
 
 
 
Fax:  [+1] (809) 532-2564 
 
 
 
Email:  infectologia@verizon.net.do    http://www.dei-rd.org 
 
8.3 
World Health Organization e 
 
 
Martin Friede, Ph.D. 
 
 
Initiative for Vaccine Research  
 
 
World Health Organization  
 
 
20, avenue Appia  
 
 
CH-12211, Geneva 27 Switzerland  
 
 
 
Tel:  [+41] (22) 791-4398  
 
 
 
Fax:  [+41] (22) 791-4860 
 
 
 
E-mail:  friedem@who.int      http://www.who.int/vaccine_research/en/ 
 
8.4 
Program for Appropriate Technology in Health f 
 
 
Darin Lee Zehrung 
 
 
Technical Officer 
 
 
PATH 
 
 
1455 NW Leary Way 
 
 
Seattle, WA 98107 USA 
 
 
 
Tel:  [+1] (206) 285-3500 
 
 
 
Fax:  [+1] (206) 285-6619 
 
 
 
E-mail:  dzehrun@path.org     http://www.path.org 
 
8.5 
Biojector® 2000 Injector Manufacturer 
 
 
Richard Stout, M.D. 
 
 
Vice President, Clinical Affairs 
 
 
Bioject, Inc.  
 
 
20245 S.W. 95th Avenue, Tualatin, OR  97062 
 
 
 
Tel:  [+1] (503) 691-4130 
                                                 
e  WHO’s participation includes (1) scientific and technical input in study design, (2) travel funding to assist CDC 
staff in international planning and oversight, (3) purchase on the study’s behalf of liability insurance for the 
participating institutions (section 7.5), and (4) contracting upon competitive tender for laboratory services to 
perform the serologic assays and virus identification needed for the study (section 10.6.12.1). 
 
f  PATH is providing technical consultation and funding up to $30,000 to the Dominican collaborators for 
equipment and supplies needed for the study.  

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Fax:  [+1] (503) 692-6698 
 
 
 
Email:  rstout@bioject.com     http://www.bioject.com 
 
8.6 
Vaxigrip® Influenza Vaccine Manufacturer 
 
 
Olivier Raynaud, M.D. 
 
 
Vice President, Africa – French Overseas Depts. and Terrs. – Caribbean & Indian 
Ocean 
 
 
sanofi pasteur international 
 
 
1, Avenue Pont Pasteur, 69637 Lyon, Cedex 07 - France 
 
 
 
Tel:  [+33] (4) 37 37 76 64 
 
 
 
Fax:  [+33] (4) _ _  _ _  _ _  _ _ 
 
 
 
Email:  Olivier.Raynaud@sanofipasteur.com     http://sanofipasteur.com 
 
8.7 
Consultants 
 
 
Dr. Jon Andrus  
 
 
Immunization Unit 
 
 
Pan American Health Organization 
 
 
525 23rd St. N.W., Washington, D.C. 20037  
 
 
 
Tel:  [+1] 202-974-3745 
 
 
 
Email:  andrusjo@paho.org 
 
 
 
Dr. Raúl Montesano 
 
 
Organización Panamericana de la Salud 
 
 
Edif. OPS/OMS – Cruz Roja, 2da Planta, Plaza de la Salud, C/Pepillo Salcedo 
 
 
Recta Final, Ensanche La Fe, Santo Domingo, Dominican Republic  
 
 
 
Tel:  [+1] 809-562-1519, ext. 31 
 
 
 
Fax:  [+1] 809-544-0322 
 
 
 
Mobile:  [+1] 809-723-3638  
 
 
 
Email:  montesano@dor.ops-oms.org 
 
 
 
Dr. Ciro de Quadros 
 
 
International Programs / Public Affairs 
 
 
Albert B. Sabin Vaccine Foundation 
 
 
1718 Connecticut Avenue, NW, Suite 700, Washington, DC 20009-1162 
 
 
 
Tel:  [+1] 202-265-6515 
 
 
 
Email:  ciro.dequadros@sabin.org 
 
 
 
Orin S. Levine, PhD 
 
 
Director, GAVI’s Pneumococcal Vaccines Accelerated Development and 
Introduction Plan 
 
 
Department Of International Health  
 
 
Bloomberg School of Public Health  
 
 
Johns Hopkins University 
 
 
615 N. Wolfe Street, Baltimore, MD  21205 

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Tel: [+1] (443) 287-2019 
 
 
 
Fax: [+1] (410) 614-1419 
 
 
 
Email:  olevine@jhsph.edu 
 
 
 
Dr. David M. Salisbury 
 
 
Director of Immunization, Department of Health 
 
 
Wellington House, Room 510 
 
 
133 - 155 Waterloo Road 
 
 
London SE1 8UG, United Kingdom 
 
 
 
Tel:  [+44] (20) 7972-4377 
 
 
 
Fax:  [+44] (20) 7972-3989 
 
 
 
Email:  David.Salisbury@dh.gsi.gov.uk 
 
 
 
Mr. John Lloyd 
 
 
Children’s Vaccine Program 
 
 
Program for Appropriate Technology in Health (PATH)  
 
 
La Ferme Le Fleutron, F-01220  Divonne-les-Bains  FRANCE 
 
 
 
Tel: [+33] (450) 20-30-91 
 
 
 
Fax: [+33] (870) 133-1363 
 
 
 
Email:  jlloyd@path.org 
 
 
 
Robert T. Chen, M.D. 
 
 
National Center for HIV, STD, and TB Prevention 
 
 
1600 Clifton Road (E-04), Atlanta, GA  30333 
 
 
 
Tel: [+1] (404) 639-8648 
 
 
 
Fax: [+1] (404) 639-8114 
 
 
 
Email:  rtc1@cdc.gov 
 
 
8.8 
Data and Safety Monitoring Board 
 
 
José R. Puig, M.D. 
 
 
Pediatrician, Infectious Disease Specialist 
 
 
Centro de Pediatría y Especialidades 
 
 
Avenida Independencia 504 (send mail c/o DEI/HIRRC address)  
 
 
Santo Domingo, Dominican Republic 
 
 
 
Tel: [+1] (809) 685-2353 
 
 
 
Tel: [+1] (809) 565-3428 (residence) 
 
 
 
Maria Deloria Knoll, Ph.D. (DSMB Chair) 
Director of Vaccine Research for GAVI's Pneumococcal ADIP 
Johns Hopkins University 
Bloomberg School of Public Health 
Department of International Health 
615 North Wolfe Street, Room E8005 
Baltimore, Maryland 21205  USA 

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Work: 443-287-0834 
Fax: 410-614-1419 
Email:  mknoll@jhsph.edu 
 
 
 
Professor David Goldblatt, M.D. 
 
 
Professor of Vaccinology and Immunology 
 
 
Consultant in Paediatric Immunology 
 
 
Director of Clinical Research and Development 
 
 
Institute of Child Health, University College London  
 
 
 
and Great Ormond Street Hospital for Children NHS Trust 
 
 
Immunobiology Unit,  Institute of Child Health 
 
 
30 Guilford Street, London WC1N 1EH 
 
 
United Kingdom 
 
 
 
Direct Tel:  [+44] (207) 813-8491 
 
 
 
Secretary Tel:  [+44] (207) 905-2215 
 
 
 
Fax:  [+44] (207) 813-8494 
 
 
 
Hospital Switchboard (via Aircall):  [+44] (207) 405 9200 
 
 
 
Email:  d.goldblatt@ich.ucl.ac.uk 
 
 
 
Keith P. Klugman. M.D. 
 
 
Professor of Infectious Diseases 
 
 
Department of International Health 
 
 
The Rollins School of Public Health, Emory University 
 
 
1518 Clifton Road, N.E  - Room 720 
 
 
Atlanta, GA  30322  USA 
 
 
 
Tel:  [+1] 404-712-9001 
 
 
 
Fax:  [+1] 404-727-4590 
 
 
 
Email:  kklugma@sph.emory.edu 
 
 
 
Janet Englund, MD 
 
 
Pediatric Infectious Disease 
 
 
Children's Hospital and Regional Medical Center 
 
 
4800 Sand Point Way NE (#R5441) 
 
 
Seattle, WA  98105 
 
 
 
Tel:  [+1] 206-987-2239 
 
 
 
Fax:  [+1] 206-987-3890 
 
 
 
Email:  janet.englund@seattlechildrens.org 
 
 
9. 
Introduction and Background 
9.1 
Pandemic Preparedness – Utility of Intradermal Jet Injection 
Were a pandemic of high-mortality influenza to occur, developing countries will be 
challenged in protecting their most vulnerable populations with the limited amounts of 
vaccine that would likely be available or affordable.  Administering reduced doses of 

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various antigens via the intradermal (ID) route have often found immune responses to be 
equivalent to full doses administered into conventional intramuscular (IM) or subcutaneous 
(SC) target tissues (e.g., for rabies,Briggs2000, Wilde2005  hepatitis B,Wahl1987, Bryan1992  and 
influenza,Halperin1979  among other vaccines).  One hypothesis explains this phenomenon by 
the skin’s rich endowment with antigen-presenting dendritic (Langerhans) cells, which upon 
activation migrate to deeper lymphoid tissues for the next steps in the immune 
response.Goldsby2003   
 
Such dose-sparing with ID-administered influenza vaccine (INF) may be a useful strategy to 
protect greater proportions of susceptible populations with scarce antigen.Avison1973  For 
example, reduction of the standard dose of INF from 0.25 mL (for young children) and from 
0.5 mL (for adults) to the usual intradermal volume of 0.1 mL can extend the protection of 
vaccine to 2½ and 5 times as many people, respectively, who would otherwise remain 
vulnerable to influenza morbidity and mortality.   
 
 
INF vaccination in the face of a pandemic threat will likely require mass campaigns in 
which a limited number of trained health workers would need rapidly to vaccinate large 
populations in limited periods of time.  ID vaccination with needle-syringe (N-S) by the 
traditional Mantoux method (see Figure 2), as used for PPD tuberculin application, would 
severely constrain mass campaigns because of the difficulty and tediousness of this 
technique, which requires practiced health workers and time.  Needle-free jet injectors, 
however, have a history of rapidly and easily administering tens of millions of doses of ID 
vaccines, primarily for smallpox (see Figure 3), but also BCG, using specialized intradermal 
nozzles.  Use of jet injectors for vaccination reduces the dangers and drawbacks of needle-
syringe injection (see section 9.2), including intentional or inadvertent unsterile reuse, 
needle-stick injuries to health workers, and the unsafe disposal of sharps waste.   
Figure 2.  Intradermal (ID) injection by Mantoux method with needle-syringe 

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9.2 
Dangers of Unsafe Needle-syringe Injections 
Only recently has the magnitude of unsafe injection practices in developing countries been 
widely recognized.Holding1998  The World Health Organization (WHO) estimates that up to 
half of all injections in the world are unsafe because the needle or syringe has been 
improperly reused without sterilization.Simonsen1999, Kane1999 
 
Transmission of blood borne pathogens such as human immunodeficiency virus (HIV) and 
hepatitis B virus to patients, healthcare workers and community members can occur from 
unsterile injections, accidental needle sticks, and improper “recycling” of needles and 
syringes.Aylward1995a  A study which modeled unsafe injections found that one nonsterile reuse 
of each clean needle and syringe would result in 980 new cases of hepatitis B for every 
100,000 fully immunized infants in areas of high hepatitis B prevalence.Aylward1995b  This rate 
increased to 3740 cases of hepatitis B per 100,000 if each sterilized or new needle was 
reused just four times.Simonsen1999  For HIV, an unsterile needle reused four times in areas 
where the HIV prevalence is 20% was estimated to cause up to 190 new cases of HIV 
infection per 100,000 fully immunized patients.Aylward1995b   
Figure 3.  Intradermal smallpox vaccination by jet injection,  
simultaneous with subcutaneous measles vaccination 

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Causes of improper use of needles and syringes include (1) inadequate training, knowledge, 
and motivation of health staff, (2) frequent shortages of supplies or fuel and the disrepair of 
equipment to sterilize needles and syringes, (3) inadequate disposal policies and facilities, 
and (4) a black market of recycled syringes used in the informal medical sector.  To address 
the problem WHO and the United Nations Children’s Fund (UNICEF) recommend the use 
of auto-disabling disposable syringes that cannot be reused.WHO1997  But the additional cost, 
compared to conventional syringes, has been a barrier to their use.   
 
The Global Alliance for Vaccines and Immunization (GAVI), created with the initiative and 
financial support of the Gates Foundation and others, has focused attention on vaccination 
technology.  Among its priorities are knowledge, methods, and products to reduce the 
dangers of re-use of injection equipment and to ensure proper management of “sharps” 
waste.Birmingham2000, Jacobs2001 
 
9.3 
Needle-free Jet Injection Technology 
9.3.1 
Description and Clinical History 
 
Needle-free jet injection offers one potential solution to the dangers and drawbacks of 
using needles and syringes to administer vaccines.  Jet injectors (once referred to as “jet 
guns”) use high pressure to deliver a fine stream of liquid medication or vaccines 
through the skin.  Such devices have been used by patients themselves, by 
immunization clinics, and in mass vaccination campaigns since the late 1940’s and 
early 1950s successfully to administer a wide variety of medications (e.g., anesthetics, 
antibiotics, corticosteroids, hormones, immunodiagnostics, immunomodulators, and 
vitamins) as well as vaccines. Reis1998; NIP2005a  At least 15 million doses of measles 
vaccine were administered by jet injectors equipped with subcutaneous nozzles from 
1967-1969 alone (along with 92 million smallpox vaccine doses via intradermal nozzles 
in the opposite arm) in West Africa’s smallpox eradication program (Figure 3).Millar1969, 
Fenner1988  In Brazil, tens of millions of doses of smallpox vaccine in the 1960s,Veronesi1966 
and tens of millions more measles vaccine doses in the 1990sdeQuadros1998 were given by 
jet injectors in Brazil’s successful campaigns against those diseases.   
 
The fluid injected by jet injectors is generally distributed conically, following paths of 
least resistance into either the subcutaneous (SC) tissues, or further into intramuscular 
(IM) tissue.  The site of deposition where most of the dose is delivered – SC, IM, or 
intradermal (ID) – depends on such variables as the power of the device, its orifice size, 
shape, distance and angle relative to the skin, the viscosity of the fluid, the angle of 
injection relative to the muscle fascia plane, the skin thickness injected, and other 
factors.Bennett1971, Schramm2002  Even while using the same power source, orifice, and 
perpendicular injection as for IM or SC delivery, ID injection can be achieved by 
creating a gap with tubing or a spacer inserted between the nozzle orifice and the skin, 
thus reducing the force of the jet stream (see section 9.6.3).Meyer1964, Kalabus1967, Dull1968, 
Zsigmond1999a, Zsigmond1999b, Sugibayashi2000, Med-E-Jet2006 

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9.3.2 
MUNJIs vs. DCJIs 
Safety concerns arose over multi-use-nozzle jet injectors (MUNJIs), example 
illustrated in Figure 3, which use the same nozzle to inject consecutive patients without 
intervening sterilization.  A hepatitis B outbreak in the mid 1980s caused by one 
MUNJI,CDC1986, WHO1986, Canter1990 as well as other publishedKremer1970, Darlow1970, Brink1985, 
Zachoval1988, Lukin1997, Weintraub1998, Hoffman2001, Souto2001  and unpublisheddeSouzaBrito1994 studies of 
this and other devices, indicated blood and tissue fluid containing pathogenic agents 
could be transmitted among patients.  This led to discontinuation and recommendations 
against their use in public health, CDC2002, WHO1997 and market removal in 1997 of the 
most common device, the Ped-O-Jet®.NIP2005a  
 
Since the 1990s, a new generation of safer disposable-cartridge jet injectors (DCJIs) 
have appeared.  DCJIs avoid the inherently unsafe design of MUNJIs, since the 
disposable cartridge has its own sterile orifice and nozzle and is discarded between 
patients.  One such DCJI device is the Biojector® 2000 
(http://www.bioject.com/biojector2000.html), which has been studied in a number of 
clinical trials (see section 10.4.3), and is licensed in the U.S., Europe, and elsewhere for 
either subcutaneous or intramuscular injection, depending on cartridge orifice size.  (A 
disposable investigational spacer applied to the nozzle of its disposable cartridge is to 
be studied by this protocol, and is described in detail in section 10.4.3.4).   
 
9.3.3 
Jet Injectors in Children 
 
Needle-free injections have been studied and used before in pediatric populations.  The 
Biojector® 2000 is used routinely for immunization of infants, toddlers, and older 
children in a number of county health department clinics in the U.S. 
(http://www.bioject.com/biojector2000.html).  For example, the public health clinics of 
Cobb County, a suburban jurisdiction just outside of Atlanta, Georgia, has been using 
jet injectors for several years for all routine childhood immunizations, including 
vaccines for diphtheria-tetanus-pertussis (DTP), Haemophilus influenzae type B (HIB), 
and hepatitis B (HBV) (personal communications: Richard Stout, Bioject, Inc., 1999 
and Jan Smith, Cobb County Immunization Program, 1999).Dodson1997   
 
As a result of the abandonment of MUNJIs in the late 1990s as described in section 9.3 
above, since 2000 the U.S. Navy and Coast Guard have used the Biojector® 2000 to 
administer vaccines to both military recruits at basic training sites, as well as to 
pediatric and adult dependents at regional health facilities.  In the year from October 
2003 to October 2004, nearly half a million Biojector cartridges were thus used by the 
military (personal communication, Kurt Lynam, Bioject, Inc., 2004).  
 
Another DCJI device, the INJEX® 50 (http://www.injex.com/products/injex30.asp), has 
been studied in the administration of MMR vaccine to teenagers and found to produce 
satisfactory immune responses and no significant difference in pain score compared to 

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control needle injections.Sarno2000  The INJEX®, however, is not used for routine 
vaccination in the United States because it currently lacks capability for intramuscular 
injection, which are recommended for several common vaccines.  
 
9.3.4 
Immunogenicity of Jet-injected Vaccines 
A large body of clinical literature documents jet injector immunogenicity, which is 
usually equal to or better than that induced by conventional needle and syringe for a 
variety of conventional inactivated and live vaccines.Reis1998; NIP2005b  Vaccines that have 
been successfully administered via jet injection include typhoid,ParentduChatelet1997  
cholera,McBean1972, PhillipinesCholeraCommittee1973  bacille Calmette-Guérin (BCG),Parker1948  
typhoid-diphtheria (Td),Wegmann1976  whole cell diphtheria-tetanus-pertussis 
(DTP),Stanfield1972, Ruben1973, ParentduChatelet1997, Rossier1998  measles,Meyer1964, Ruben1973  
meningococcal A and C,Rey1989  smallpox,Meyer1964, Veronesi1966, Ruben1973, Fenner1988  yellow 
fever,Meyer1964, Ruben1973  hepatitis A,ParentduChatelet1997, Fisch1996  hepatitis B,Lemon1983, Whittle1987, 
Mathei1997  influenza,Davies1969, Spiegel1994a, ParentduChatelet1997  plague,Lipson1958  polio,Lipson1958 and 
tetanus.Veronesi1966, Rey1967, Rey1973, ParentduChatelet1997, Schlumberger1999  
 
The reported increased immunogenicity via jet injector may result because injection 
inevitably leaves a small amount of vaccine in the skin, which is richly endowed with 
dendritic (Langerhans) cells which have important roles in processing and presenting 
antigens in the immune system.ParentduChatelet1997  Various studies have suggested this 
improved immunogenicity may allow lower doses of vaccine to be administered.  For 
example, Hendrickse, et al demonstrated adequate levels of protective antibodies 
against measles after administration SC of a reduced dosage with the most widespread 
device, the Ped-O-Jet®.Hendrickse1968, Macintosh1977  
 
9.3.4.1 
Non-adjuvanted Vaccines Delivered by Jet Injection 
9.3.4.1.1 
Influenza Vaccines 
Since the adaptation of needle-free jet injectors for mass vaccination in the 
early 1950s, there has been a long and well-documented history for 
comparable immunogenicity (to needle-syringes) and tolerable reactogenicity 
in their use for the administration of influenza vaccines.Anderson1958, Clark1965, 
Wright1968, Davies1969, Vibes1971, Ivannikov1980, Spiegel1994a, ParentduChatelet1997  During the 
swine influenza mass campaign of 1976-1977 in the U.S., a substantial 
proportion of the approximately 80 million doses administered that season 
were delivered by jet injectors (CDC, unpublished data).Ehrengut1977 
 
9.3.4.1.2 
Measles Vaccines 
Studies have well documented the immune response of the hundreds of 
millions of doses of measles vaccine administered by jet injectors, 
particularly  in mass campaigns in Africa and South America (see section 
9.3.1).Meyer1964, Kalabus1967, Ruben1973, Kok1983, deQuadros1998  
 

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9.3.4.1.3 
Meningococcal Vaccines 
Traditional meningococcal polysaccharide (MENps) vaccines have been 
extensively administered by needle-free jet injection technology.AmatoNeto1974, 
Artenstein1971, Binkin1982; Chippaux1998, Gotschlich1972a, Gotschlich1972b, Greenwood1980, 
Mohammed1981, Mohammed1984, Rey1989, Spiegel1994b, Taunay1974, Taunay1978  Most of these 
citations reported on the use of monovalent serogroup A polysaccharide 
vaccine, which more commonly causes serious epidemics in the so-called 
“meningitis belt” of western Sub-Saharan Africa.  Amato Neto et al, both 
Taunay et al reports. and Rey et al. describe the administration of 
meningococcal C vaccine by this method.   
 
9.3.5 
Reactogenicity of Jet-injected IM and SC Vaccines 
The medical literature reports varying results in studies regarding the pain and 
reactogenicity of needle-free injectors compared to needles to deliver intramuscular 
(IM) and subcutaneous (SC) injections.  Insulin and other non-irritating drugs and non-
adjuvanted vaccines generally result in either reduced or equivalent pain for jet injectors 
compared to needles,Hingson1947, Hughes1949, Anderson1958, Kutscher1962, Meyer1964, Greenberg1995  but not 
always.Jackson2001   
 
Vaccines with irritating adjuvants like aluminum salts usually result in somewhat higher 
frequencies of local reactions (e.g., edema, erythema, tenderness) when jet injected, but 
this has not generally been of a magnitude sufficient to compromise clinical tolerance 
and safety.Barrett1962, Lenz1966, Edwards1974, Agafonov1974, Agafonov1978, Hoke1992, Hoke1995, 
ParentduChatelet1997  The irritation probably results from the residual vaccine remaining in 
the skin and superficial subcutaneous tissues, even if most of the dose administered is 
deposited more deeply.  (Most modern influenza vaccines, including Vaxigrip®, have no 
adjuvant.)  
 
9.3.5.1 
Alum-adjuvanted Vaccines Delivered by Jet Injection 
In addition to some of the papers mentioned in preceding sections, a number of 
reports have been published on the administration by jet injection of the following 
alum-adjuvanted vaccines by either the IM or SC routes.  In general, these papers 
reported somewhat increased -- but tolerable -- rates of local reactions compared to 
needle-syringe administration.  Such reactions are usually mild and resolve within 
days without treatment.   
 
• Diphtheria-Tetanus-Pertussis (whole-cell) (DTPw)Stanfield1972, Ruben1973, 
ParentduChâtelet1997 
• Hepatitis A (HAV)Hoke1992, Hoke1995, Parent du Châtelet1997, Williams2000 
• Hepatitis B (HBV)Lemon1983, Whittle1987, Mathei1997 
• Tetanus (TET)Veronesi1966, Rey1967, Rey1973, Parent du Châtelet1997, Schlumberger1999 
• Tetanus-Diphtheria (Td)Wegman1976 
• Tetanus-Diphtheria-polio (Td-POLIPV)Barrett1962 

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• Typhoid (TYD)Agafonov1974, Edwards1974, Agafonov1978 
 
9.3.5.1.1 
Diphtheria-Tetanus-Pertussis (whole cell) Vaccines 
Parent du Châtelet et al administered three monthly injections of 
DTPw(PMSV) to African infants recruited at age 2-to-3 months, using 
prefilled disposable jet injection cartridges (Imule®) and an investigational 
Mini-Imojet® needle-free jet injector (JI), and compared it to similar controls 
receiving the same vaccine and doses by needle-syringe (N-S).Parent du 
Châtelet1997  Comparing the JI and N-S groups, frequencies of delayed reactions 
for pain were 34.2% and 27.4%, respectively (not significant [NS]), 68.3% 
and 51.2% for induration (p<0.05), 1.2% and 2.4% for adenopathy (NS), and 
4.9% and 2.4% for fever (NS). 
 
9.3.5.1.2 
Hepatitis A Vaccines 
Local redness was reported by significantly more soldiers (23%) who 
received their first dose of Merck HAV vaccine IM by Ped-O-Jet® injector, 
compared to 3% among those vaccinated IM by needle-syringe.Hoke1995  After 
the second dose, the significantly-different jet injector/needle-syringe 
frequencies were 14%/0% for redness, and 8%/0% for swelling.  After the 
third dose, only redness occurred with significantly increased frequency 
among the jet injector recipients (18%) versus the needle-syringe group 
(5%).  Nevertheless, the investigators concluded that “the jet injector is a 
highly satisfactory means for mass inoculation of military recruits with 
hepatitis A vaccine”.Hoke1992   
 
The Pasteur-Mérieux (now Sanofi-Pasteur) HAV vaccine was administered 
SC by Mini-Imojet® jet injector to 48 adults, and compared to needle-syringe 
injections by the IM (n=50) and SC (n=49) routes.Parent du Châtelet1997  Relative 
frequencies of side effects for SC jet injector, IM needle, and SC needle were 
35% / 13% / 26%, respectively, for delayed pain (p=0.05).  No significant 
difference in proportions were found between the three injection groups for 
delayed erythema (9% / 0% / 13%, respectively), induration (2% / 0% / 0%), 
hematoma (7% / 0% / 7%), adenopathy (0% / 2% / 2%), and fever (11% / 9% 
/ 9%).   
 
A Biojector® 2000 needle-free jet injector (JI), similar to that proposed to be 
used in this study, was used to administer two doses IM of GlaxoSmithKline 
HAV vaccine, which was also administered IM by needle-syringe 
(N-S).Williams2000  There was no significant difference in the frequencies of 
systemic side effects between the two study groups.  But local reactions were 
more frequent in the JI group: redness after 58 (20%) of 289 doses 
(compared to 6 [2%] of 288 N-S doses); swelling after 57 (20%) doses (N-S: 
9 [3%]); and bruising after 75 (26%) doses (N-S: 7 [2%]).   
 

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9.3.5.1.3 
Hepatitis B Vaccines 
Three doses of Merck HBV vaccine were administered SC in unblinded 
fashion by Ped-O-Jet® injector above the deltoid insertion region of the 
non-dominant arms of 19 volunteer adults.  These injections were compared 
with simultaneous saline injection by needle-syringe (N-S) into the dominant 
arms.Lemon1983  Induration of >5 mm was reported after 9 (16%) of 57 jet 
injections, compared to only 1 (2%) of placebo N-S control injections.  
Erythema of >5 mm reported in 5 (9%) of jet injections, compared to none 
from N-S placebo.  Firm, indurated, painless nodules from 5-to-10 mm in 
diameter appeared one or two days after 9 (16% of 57) jet injections.  All 
eventually disappeared, at times leaving a pigmented macule.   
 
A Biojector® 2000 device was used to administer GlaxoSmithKline HBV 
vaccine IM to 47 randomly selected adult volunteers, and to 50 vaccinees by 
needle-syringe.Matheï1997  Local side effects of soreness, redness, or swelling 
were reported significantly more frequently (126 adverse effects) after 93 jet 
injections (doses 1 and 2) than after 98 needle-syringe injections (49 adverse 
effects).   
 
9.3.5.1.4 
Tetanus and Diphtheria Vaccines 
Reactogenicity at 3 days to a monovalent tetanus toxoid vaccine adsorbed on 
aluminum hydroxide (Tetavax®, Pasteur Mérieux Sérums et Vaccins, now 
Sanofi Pasteur) was examined in 213 African adults vaccinated with the 
investigational Imule® disposable cartridge and Mini-Imojet® needle-free 
injector, without the use of a control group receiving needle-syringe injection 
to compare adverse reactions.ParentduChâtelet1997  Frequencies of AE were 
reported for pain (68.5%), fever (35.9%), induration (26.0%), erythema 
(7.7%), and hematoma (0%).   
 
Brazilian investigators administered via Press-O-Jet™ needle-free injector a 
dose volume of 1.0 mL of tetanus toxoid precipitated on alum to 300 adults 
and 350 children in São Paulo, and reported “no disagreeable 
reactions”.Veronesi1966  
 
An aluminum-phosphate-adsorbed bivalent Td vaccine was administered via 
needle-free Dermo-Jet® injector in a 0.1 mL intradermal dose to 19 adults, 
and found by the investigators to be “well-tolerated” and equivalent to an SC 
injection.Wegmann1976    
 
Barrett studied immunization of high-school students with combination 
Td-POLIPV vaccine using the American Hypospray® jet injector, finding 12 
percent to have a transient local erythema of “3+ severity”, defined as a 
reddened area of 80 square millimeters, with accompanying induration, heat, 
and tenderness.Barrett1962   
 

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9.3.5.1.5 
Typhoid Vaccines 
Agafonov reported on typhoid vaccine via jet injection with a Soviet jet 
injector, finding the frequency of systemic reactions to the typhoid vaccine 
was 6.9%, while local reactions were 82% - 93%.Agafonov1978  Comparing jet 
injection with needle-syringe for other typhoid and typhoid-paratyphoid 
vaccines (TYDAKD and TYD-PTDTAB), Edwards found 82 - 88 percent of 
jet injection vaccinees had one or more local reactions (pain, erythema, heat, 
swelling, tenderness, induration, or nodes), compared to only 24 percent of 
needle-syringe recipients.Edwards1974  By 72 hours, only 8 percent of the 
jet-injected TYDAKD vaccinees and none of the TYD-PTDTAB vaccinees 
still had such local reactions.   
 
9.4 
Intradermal Route of Vaccination 
9.4.1 
Vaccines other than Influenza 
The intradermal (ID) route has been described in the literature for at least 16 different 
vaccine types.  There are numerous reports, of course, for those vaccines in which the 
ID route is the normal one, such as BCG,Chambon1970a, Chambon1970b, Chambon1970c, Collas1973, 
Carnus1973, Carnus1974, Fillastre1970  smallpox,Fenner1988  and combined BCG-smallpox vaccine. 
Vaughan1972, Vaughan1973  The intradermal route has also been studied with good results for 
killed vaccines such as typhoidTufts1931  and rabies,Briggs2000  the latter of which has been 
used widely for dose-sparing purposes in the developing world.Wilde2005  Generally good 
results have been reported for hepatitis B,Bryan1990  but not always,Coberly1994  while mixed 
results have been reported for cholera,McBean1972  hepatitis A,Brindle1994, Pancharoen2003, 
Pancharoen2005  and measles.Calafiore1966, Kok1983, deMoraes1994  Other vaccines studied rarely by 
this route include polio,Salk1953a, 1953b  meningococcal A, diphtheria-tetanus-pertussis, 
tetanus-diphtheria, tick-borne encephalitis, and Rift Valley fever).  
 
In addition, intradermal jet injection has been used to administer immunomodulators 
like interferon,Nathan1986 as well as tuberculin (purified protein derivative, PPD) for 
tuberculosis skin testing among patients of all ages.Bettag1967, Brólio1976, Cockburn1965, 
DePartearroyo1966, Dull1968, Hendrix1966, Luby1968, Marsallon1972, Morse1967, Neumann1973, Wijsmuller1975  
 
9.4.2 
Influenza by Intradermal Needle Injection 
There is a substantial literature, since the 1930s, starting with Thomas Francis (of Salk 
polio vaccine trial fame),Francis1937  documenting the equivalence and occasionally 
improved immunogenicity of intradermal influenza vaccination by needle-syringe 
compared to larger doses by the subcutaneous and intramuscular routes. Bruyn1947, 
VanGelder1947, Weller1948, Bruyn1949a, Bruyn1949b, Edwards1958, Hilleman1958, Kirkham1958, Sanger1959, Stille1959, 
Beasley1960, Saslaw1964, Clark1965, Tauraso1969, Marks1971, Brown1977, Halperin1979, Spiegel1994a, Belshe2004, 
Kenney2004,   On the other hand, a few studies found ID influenza responses less then IM or 
SC on some or all of the antigens that were studied.Boger1957, Saslaw1963, Phillips1970, Sigel1975, 
Hutchinson1977, Herbert1979  
 

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9.4.3 
Reduced-dose Influenza Vaccination by Different Routes 
When the same reduced dosage of influenza antigen was compared via the intradermal 
versus IM or SC routes, there were conflicting results from clinical trials.  Bruyn et al 
found GMTs in children receiving 0.2 mL intradermally of INF to be higher than those 
receiving the same dose SC,Bruyn1949a  as did Davies et al.Davies1969  and Tauraso et 
alTauraso1969  administering 0.1 mL by both routes.  Stille et al also found greater ID 
responses when administering small antigen masses, but conversely, SC responses 
exceeded ID ones when delivering larger doses.Stille1959  On the other hand, McCarroll et 
al,McCarroll1958  studying hospital employees 18 to 65 years of age, and Klein et al,Klein1961  
studying infants 2 months to 5 years of age, both found no difference in responses 
between the ID and SC routes.   
 
Regarding systemic reactions, among 101 infants from 2 months to 2 years of age 
receiving 0.1 mL of influenza vaccine in the Klein et al study, febrile reactions were 
reported among 34.7% (17/49) in the intradermal group and only 19.2% (10/52) in the 
subcutaneous group getting the same reduced dose.Klein1961  Similarly, local reactions of 
small areas of erythema and induration with 2 to 3 days of slight tenderness and itching 
were described for “all” intradermal participants (ages 2 month to 5 years, n = 96), 
while only 2 of 94 children vaccinated subcutaneously had local pain and induration. 
 
9.4.4 
Influenza by Intradermal Needle-free Jet Injection 
The use of needle-free jet injectors to administer influenza vaccine intradermally (ID) 
has been studied and documented since the 1940s.Parker1948  Two studies of jet injected 
ID influenza vaccination in particular were performed.  Davies et al studied monovalent 
A2/Australia/54 and found significantly higher GMTs for 0.1 mL administered ID by JI 
(114) than for 0.5 mL by the SC route (75.8).Davies1969  Although there was no 
statistically significant difference between the proportion with a 4-fold titer rise in the JI 
ID group (58%) versus the full-dose SC one (40%).  On the other hand, Payler et al 
studied a trivalent product, administering 1.0 mL SC compared to 0.15 mL ID by JI, 
and found a trend to SC to be superior, but the difference did not reach statistical 
significance.Payler1974   
 
9.5 
Influenza Disease Burden 
In 2004, recognizing the morbidity and mortality of influenza disease in young children, the 
Advisory Committee on Immunization Practices (ACIP) of the CDC first recommended 
universal vaccination of all children from the ages of 6 through 23 months, healthy and 
otherwise,CDC2004  and continues to recommend so in 2005.CDC2005  Similarly, the Pan 
American Health Organization encourages countries to introduce yearly seasonal influenza 
vaccination in routine programs for children from 6-23 months of age.PAHO2005   
 
Influenza causes both epidemic and pandemic disease with the 1918-19 pandemic as the 
most devastating, with an estimated 40-50 million deaths worldwide.Nicholson2003  In the 
United States, influenza is associated with an average of 36,000 underlying respiratory and 
circulatory deaths annually and more than 200,000 hospitalizations.Thompson2003   

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Each year between 5% and 20% of the population is infected with influenza.Monto1986  Based 
on the 1980 population, among persons age <20 years, the annual burden of influenza in the 
United States includes an average of:  
•  13.8 to 16.0 million excess influenza-related illnesses 
•  152.0 to 176.4 million excess influenza-related illness days 
•  47.1 to 54.7 million excess influenza-related excess bed and restricted activity 
days.  
 
Among persons age >20 years, the annual burden of influenza in the United States is on 
average:  
•  4.1 to 4.4 million excess influenza-related illnesses 
•  65.7 to 70.6 million excess influenza related illness days 
•  16.6 to 17.9 million excess influenza-related excess bed and restricted activity 
days Sullivan1993 
 
About 90% of influenza-associated deaths occur among persons aged 65 years and older.  
The influenza-attributable mortality rate for persons aged 85 years and up is significantly 
higher as compared to persons 65 to 69 years.Thompson 2003   
 
Table 1 
Estimated Annual Influenza Associated Mortality 
1990-91 Through 1998-99 Seasons 1 
Age Group 
Rate  
per 100,00 person-years 
<1 
0.6 
1-4 
0.4 
5-49 
0.5 
50-64 
7.5 
>65 
98.3 
Total 
13.8 
1 Underlying respiratory and circulatory deaths 
Source:  Unpublished, CDC 
(http://inside.nip.cdc.gov/divisions/isd/irl/dis_ep_burd.asp) 
 
 
Influenza A (H3N2) viruses are associated with the highest annual rates of influenza-
associated hospitalizations. Persons aged 65 and up have the highest rates of influenza-
associated hospitalization followed by children younger than 5.  
 
On average 94,735 primary pneumonia and influenza hospitalizations, 133,900 any listed 
pneumonia and influenza hospitalizations, and  226,054 primary respiratory and circulatory 
hospitalizations per year occur in the US.Thompson 2004   
 

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Table 2 
Age-Specific Annual Average Numbers and Rates of  
Influenza-Associated Hospitalizations 1 
Age Groups 
Number 
Rate 
<5 
20,031 
107.9 
5-49 
34,867 
  20.8 
50-64 
29,447 
  83.8 
65-69 
18,301 
189.7 
70-74 
26,501 
321.2 
75-79 
27,516 
431.1 
80-84 
28,578 
686.1 
>85 
40,813 
1194.9 
Total 
226,054 
88.4 
1 Respiratory and circulatory hospitalizations, primary 
Source: Unpublished, CDC 
(http://inside.nip.cdc.gov/divisions/isd/irl/dis_ep_burd.asp) 
ACIP 2005 RecommendationsCDC2005 
 
9.5.1 
Epidemiology of Influenza in Dominican Republic and Caribbean 
The epidemiology of influenza in the Dominican Republic is atypical in that this 
Caribbean island experiences transmission more-or-less year round as a consequence of 
virus introductions from tourists escaping winters in North America from October 
through March, and winters in South America from April through September (personal 
communication, Dr. Jesús Feris Iglesias, 2005).  There is scant quantitative data 
available, however, on influenza incidence from the Dominican Republic or from its 
neighbor, Haiti, on the island of Hispaniola. 
 
General health indicators for the Dominican Republic are summarized by 
PAHO.PAHO2004  The estimated infant (0-12 months) mortality rate between 1995 and 
2000 was 40 per 1,000 live births (l.b.).  In 1999, the leading cause of morbidity in such 
infants was acute respiratory infection (668.8 per 1,000 l.b.), followed by acute 
diarrheal diseases (329.3), and parasitoses (138.5).  In children 1-4 years, respiratory 
infections were also the leading cause of illness at 221.2 cases per 1,000 population, 
followed by acute diarrheal diseases (69.4).  The immunization infrastructure performed 
well, with the proportion of infants less than one year of age immunized with POLOPV, 
DTP, and TUBBCG measured at 87%, 72%, and 96%, respectively.  Influenza vaccine is 
not a routine antigen included in the public immunization program in the Dominican 
Republic. 
 
Puerto Rico, another island about 100 km to the east, does have somewhat more 
epidemiologic data on influenza, which may be relevant to the Dominican Republic 
because of geographic, climatic, and cultural similarities.  Among Puerto Rican children 
less than 6 years of age, the rate of influenza incidence in the selected years 1987, 1989, 
1992, 1994, and 1996 was 8.1, 38.1, 12.7, 6.6, and 2.4, respectively, per 100 persons, 
suggesting substantial morbidity and great year-to-year variation.PuertoRico2005   

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Among the English-speaking Caribbean members of the Caribbean Epidemiology 
Center (CAREC), which does not include the Dominican Republic, acute respiratory 
infections decreased over time, but still remain a leading cause of death in children 1-4 
years of age, with an age-adjusted rate of 30 per 100,000 in 1980 and 5 per 100,000 in 
1995. Holder2000  In 2000 and 2001, a total of 71,201 and 60,236 influenza cases, 
respectively, were reported from the 20 CAREC member countries.   
 
On Jamaica, an island 400 km to the southwest of the Dominican Republic, a study of 
etiologic agents of acute respiratory infections determined the proportion in 83 
malnourished children.Christie1990  Influenza virus was isolated from 14% (12 of 83), 
second only to parainfluenza viruses (18%), and more frequent than adenovirus (12%), 
RSV in 8%, and Mycoplasma in 8%.  During the 1918-19 influenza pandemic, the virus 
was noted to have swept through the Caribbean and resulted in an estimated 100,000 
deaths.Killingray1994   
 
9.6 
Objectives and Rationale for Study 
9.6.1 
Study Objectives 
This study intends to demonstrate a dose-sparing intradermal (ID) method to allow 
larger numbers of young children to be protected when supplies of influenza vaccine are 
limited, and to prove the principle that needle-free jet injection can obviate the cost, 
time, expertise, and difficulty administering ID injections by the Mantoux needle-
syringe method.  DCJIs thus also avoid this and other drawbacks and dangers of needle-
syringe injections, such as safe sharps disposal, unsterile reuse, and needlestick injury.   
 
9.6.2 
Rationale 
 
This study will provide new clinical data on the degree of safety and immunogenicity of 
protecting young children from influenza by a needle-free reduced-dose route.  It would 
provide information essential for developing country public health officials, 
immunization programs, and clinicians who must make difficult policy decisions as 
they face likely vaccine shortages in the inevitable pandemic of influenza of the future.   
 
There is already a large body of data on the immunogenicity and reactogenicity of 
intradermal administration of INF vaccine by needle-syringe (see sections 9.4.2 and 
9.4.3), and prior studies of this route using needle-free jet injection in adultsDavies1969  
and in schoolchildrenPayler1974  (see section 9.4.4).  But there is none in the intended age 
group of 6-to-24 month old children.  This study will fill that gap for an age group at 
high risk for morbidity and death from influenza disease.   
 
9.6.3 
Intended / Potential Use of Study Findings 
Proving the concept of dose-sparing by intradermal (ID) injection could provide a 
useful strategy for public health policymakers in developing countries to protect their 

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populations against pandemic influenza threats for which vaccine supplies are likely to 
be in short supply.  Demonstrating that needle-free jet injectors can accomplish this ID 
delivery quickly and easily without the risks of needlestick accidents can provide a 
valuable logistical tool for mass campaigns.   
 
Although the particular intradermal spacer to be utilized in this study is a proprietary 
design of the jet injector manufacturer, the concept of creating a gap between a jet 
injector nozzle and the skin to achieve intradermal delivery by weakening a standard 
perpendicular jet stream may be considered prior art and thus no longer patent-
protectable.  It has been long described in the scientific literature and applied in the field 
with various jet injectors (section 9.3.1).Meyer1964, Kalabus1967, Dull1968, Zsigmond1999a, 
Zsigmond1999b, Sugibayashi2000, Med-E-Jet2006  This precedent provides “freedom to operate” for 
various manufacturers to adapt and study their devices in achieving ID delivery.  Thus, 
the findings of this study may spur further competitive research and development to 
pursue a safer, needle-free administration route for influenza and perhaps other 
antigens.   
 
9.6.4 
Research Questions 
 
1. Will the investigational study arm of children receiving two doses of 0.1 mL of 
influenza vaccine administered ID demonstrate non-inferiority to the immune 
responses achieved by the control study arm of children receiving two 
conventional, full-doses of 0.25 mL administered IM by needle-syringe? 
(Primary endpoint: proportion of participants achieving inverse titer of ≥40 on 
hemagglutination inhibition assay for each virus strain in the vaccine.)  
 
2. What will be the safety profile of jet-injected, reduced-dose, intradermal 
influenza vaccination (erythema, induration, limb swelling, bleeding, and other 
local and systemic reactions) delivered by DCJI using an investigational spacer 
for ID delivery?   
 
3. Will an investigational study arm of children receiving two reduced doses of 0.1 
mL of vaccine administered IM by needle-syringe demonstrate non-inferiority to 
the immune responses achieved by the control arm of children receiving 
conventional, full-dose 0.25 mL administered by the same method in the same 
target tissue (obviating any need for intradermal delivery to achieve dose 
sparing)?  
 
10. Investigational Plan 
10.1 
Overall Study Design and Plan 
The study is a randomized, double-blinded, controlled, phases I and II clinical vaccine trial 
of safety and immunogenicity, among children enrolled at ≥9 -to- <24 months of age.  It will 
compare using a non-inferiority statistical model two standard doses in this age group of 

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0.25 mL of a commercial, trivalent, inactivated, split-virus product administered 
intramuscularly (IM) one month apart by conventional needle-syringe injection with two 
reduced doses of 0.1 mL, administered by either the intradermal (ID) route via needle-free 
jet injector or by the IM route with needle-syringe.  The ID route will utilize an 
investigational ID spacer on a U.S.-licensed needle-free jet injector. 
 
Healthy participants will be recruited from among eligible patients and their siblings 
attending immunization, outpatient, or inpatient clinics and wards of a large, public tertiary 
care children’s hospital.  Participants will be randomly assigned to receive two doses of 
Sanofi-Pasteur Vaxigrip® influenza vaccine by one of the three study arms.  At the 
conclusion of the study, at the time of collection of the final blood specimen, participants in 
the two investigational groups that received reduced doses will receive a third “insurance” 
dose of the influenza vaccine via the conventional route, method, and dose, to ensure 
adequate protection.  Six months after this study “graduation”, all participants will receive a 
fourth “bonus” booster dose for protection during the following influenza season. 
 
10.1.1 Study Arms 
The three study arms are:  
 
• Group “IM-NS-0.25” (controls)  -  two full 0.25 mL doses administered 
intramuscularly (IM) by standard needle-syringe (NS)  
 
• Group “ID-JI-0.1” (investigational)  -  two reduced 0.1 mL doses administered 
intradermally (ID) by needle-free jet injector (JI) 
 
• Group “IM-NS-0.1” (investigational)  -  two reduced 0.1 mL doses administered 
intramuscularly (IM) by needle-syringe (NS) 
 
10.1.2 Phases 
The studies will be conducted in two phases:  
 
Phase I.  48 children assigned randomly into 3 study arms of 16 each.   
 
Phase II.  Upon analysis of adverse events and clearance by the DSMB, an additional 
402 infants of the same age range will similarly be enrolled into the same three study 
arms of 134 participants each.   
 
Total participants in phase I and II = 450 (150 in each of three study arms).   
 
If phase II is not pursued for whatever reason, sera from phase I will be assayed as 
described below, after which the study group codes will be unblinded for analysis of 
phase I by the investigators.  
 

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10.1.3 Endpoints 
Study endpoints to be measured or determined are the following:  
 
10.1.3.1 Primary Immunologic 
The primary immunologic endpoint of the study will be the titers measured by 
hemagglutination inhibition on serum drawn on the days of vaccine dose 1, 
vaccine dose 2, and one month after dose 2.  See section 10.5.1 for definitions of 
immunogenicity variables seroconversion, seroprotection, mean geometric 
increase, etc.    
 
10.1.3.2 Primary Safety 
Primary safety endpoints are local and systemic reactions manifested for each of 
the following prompted adverse events during the intervals specified (see section 
10.5.2.2).   
 
Immediately after injection: 
Local at injection site 
•  Drop of blood on skin 
•  Flow of blood on skin 
•  Wetness (not blood) on skin 
•  Other, may be entered and specified 
 
≥30 -to- ≤60 minutes after injection: 
Local at injection site 
•  Erythema, largest diameter 
•  Swelling, largest diameter 
•  Induration, largest diameter 
•  Other, may be entered and specified 
 
Systemic 
•  Fever, via axillary temperature 
•  Sleepiness 
•  Irritability 
•  Seizures 
•  Anaphylactic shock 
•  Other, may be entered and specified 
 
0-2 days and 3-7 days after injection: 
Local at injection site 
•  Tenderness, 0 to 4 scale 
•  Redness, largest diameter 
•  Swelling, largest diameter 
•  Hematoma, largest diameter 
•  Midpoint circumference of limb (compared to baseline) 

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•  Entire limb swelling 
•  Other, may be entered and specified 
 
Systemic 
•  Fever, via axillary temperature 
•  Antipyretic medication given 
•  Change in eating habits, loss of appetite 
•  Sleepiness 
•  Irritability 
•  Unusual or inconsolable crying 
•  Vomiting 
•  Diarrhea 
•  Seizures 
•  Other, may be entered and specified 
 
8-28 days after injection: 
Local at injection site or systemic 
•  Unprompted adverse events solicited on diary card and reported 
by any means 
 
10.2 
Physical Facilities 
10.2.1 Description 
Clinical aspects of the study will take place at the Hospital Infantil Dr. Robert Reid 
Cabral (HIRRC), Avenida Abraham Lincoln No. 2, Centro de los Heroes, in the city of 
Santo Domingo [Distrito Nacional], capital of the Dominican Republic.  This is the 
national children’s hospital, which is a component of the Secretaría del Estado de Salud 
Pública y Asistencia Social (SESPAS, equivalent to a ministry of health).  HIRRC 
serves as a major primary, secondary, and tertiary treatment facility and also provides 
public health outpatient immunization services for patients both from within its 
catchment area in and around the capital district, as well as other parts of the country.  It 
has prior experience in the conduct of infectious disease surveillanceGomez1998, Gomez2000, 
Terrero1998, DEI1995, Rondón1998, FDI2001 and of clinical vaccine trials.Fernandez1999, Fernandez2000a, 
Fernandez2000b  
 
The HIRRC has 350 inpatient beds and admits approximately 1,000 pediatric inpatients 
per month (12,000/year) for stays averaging seven days.  The most common causes for 
admission are pneumonia and septicemia.  Since 1963, the HIRRC has served as a 
formal residency training facility in the fields of pediatrics, pediatric infectious diseases, 
pediatric cardiology, and pediatric surgery.   
 
The HIRRC Department of Infectious Diseases (DEI in Spanish), which will serve as 
the base for the proposed study, has 42 beds in its dedicated ward, and is served by a 
Chief of Service and five pediatric residents.   
 

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The HIRRC laboratory possesses refrigerators to maintain the cold chain for the INF 
vaccine, and a –70º C freezer for storage of sera.  Both will be backed up with a >20 
kilo-watt emergency power generator to be installed and tested onsite prior to 
commencement of the study, under financing arranged under contract with CDC.  The 
HIRRC laboratory has trained personnel and equipment to centrifuge blood samples, 
and prepare and ship aliquots of sera. 
 
10.2.2 Participant Recruitment 
Eligible participants for this study will be recruited from among children present in the 
hospital’s Healthy Children and Vaccination Service, known informally as the 
immunization clinic (Centro de Vacunas).   
 
The HIRRC immunization clinic is  located in a satellite building on the ground level 
just steps from the main outpatient building, and draws a heterogeneous patient 
population because it is known for having a consistent supply of vaccines.  Many 
parents bring children long distances to receive immunization services in this popular 
facility.  A review of those receiving measles vaccine at the immunization clinic (when 
the recommended age was 9 months) in the full six months from February through July 
2003, identified a total of 718 patients of age ≥9 -to- <12 months in which both date of 
birth and age of vaccination were recorded in the medical record (personal 
communication, Dr. Virgen Gómez, 2003).  This represents an average of 120 patients 
per month, or 28 per week, in an age range that is only one-sixth of the target age range 
for recruitment into this study of ≥6 –to- <24 months.   
 
Parents of potential participants in the waiting room of the Centro de Vacunas will be 
solicited for interest in the study and invited to learn more at the Centro del Estudio 
[Study Center].  See section 10.6.2. 
 
10.2.3 Study Center 
A suite of two rooms (office and injection room) is renovated on the 2nd floor of the 
main outpatient building for use as the planned research study center (Centro del 
Estudio) for this trial.  Chairs in a waiting area are located immediately outside the 
suite.  The physician investigator’s office where medical history and clinical 
examinations will be performed is located about 15 meters down the hall.  In these 
rooms, the orientation, interview, and explanation of the informed consent will be 
conducted with the parents, and the child either recruited at that time, dismissed as 
ineligible, or scheduled to return at a later date upon reaching eligibility.  Vaccinations 
and return followup visits will occur in the Study Center. 
 

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10.3 
Selection of Study Population 
10.3.1 Inclusion Criteria 
Participants enrolled in the study will be those who meet the following criteria from 
among infants presenting at the Study Center according to the recruitment methods 
described in section 10.2:  
 
a. Age from ≥6 -to- <24 months (not having reached 2nd birthday).  (The date of first 
study vaccination may be the same day of the 6th month after the child’s day 
and month of birth, or a later day, but no earlier.  Maximum age may not 
include the child’s 2nd birthday, nor a later day.  For example, a child born on 
1 January 2005 may only be vaccinated for dose 1 from 1 June 2005 through 
31 December 2006.  A child born on 31 May 2005 may only be enrolled from 
1 December 2005 (not 30 November) through 30 May 2007.) 
b. Born after a full-term pregnancy of gestational age of ≥37 weeks, and a birth weight 
of ≥2.5 Kg (≥5 pounds, 8 ounces)  
c. History of prior or first attendance as a patient, or as a sibling of a patient, seeking 
routine immunization or other clinical care at the HIRRC 
d. The accompanying parent(s) or legal guardian(s) provide written informed consent 
after the nature of the study has been explained, and agree to bring the infant 
back to the clinic for all visits scheduled in the study 
e. Up-to-date for routine doses of vaccines officially recommended for the 
participant’s age in the Dominican Republic to prevent tuberculosis, polio, 
diphtheria, tetanus, pertussis, hepatitis B, and Haemophilus influenzae B  (See 
exclusion criterion below for any vaccination received within 30 days prior to 
first study vaccination.) 
f. In good health, as determined by medical history and physical examination collected 
in accordance with the Case Report Form (CRF), and by the clinical judgment 
of the investigators.  
10.3.2 Exclusion Criteria 
To be excluded are infants WHOSE PARENT(S)/LEGAL GUARDIAN(S) … :  
 
a.  are unable or unwilling to give written informed consent for their infant to 
participate in the study 
b.  cannot be contacted by telephone (family’s own or a neighbor’s) by the study nurse 
if necessary for surveillance of adverse events if scheduled followup return 
appointments are not fulfilled 
c.  are unable to complete the diary form for adverse events, to measure and record 
temperature with the TraxIt™ skin-appliqué thermometer, to measure the 
maximal diameter of local reactions or limb circumference, or have difficulty 
reading or understanding written instructions, or other factors which indicate 
exclusion in the judgment of the study staff 
 

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Also to be excluded are INFANTS who … :  
 
d.  have fever (by parental report or by rectal temperature ≥38.5° C or axillary ≥38.0° 
C) currently or within the past 3 days, or who are currently suffering from an 
acute or chronic infectious disease (including known HIV)  
e.  have had an acute or chronic infection requiring systemic antimicrobial therapy 
(antibiotic or antiviral) or other prescribed treatment within the past 21 days. 
This includes any underlying illness that may limit their response to 
vaccination, such as those receiving intravenous immunoglobulin for 
agammaglobulinemia, or systemic steroid therapy  
f.  are malnourished, defined by weight less than two standard deviations below the 
median weight for their age  
g.  are allergic to eggs, or have a history of any anaphylactic shock, asthma, urticaria, 
or other allergic reaction after previous vaccinations, or have allergy or 
hypersensitivity to any component of the study vaccine 
h.  have ever received previously any influenza vaccine 
i.  have received within the prior 28 days, or for whom there is the indication to 
receive in the next 56 days, any non-study vaccination or investigational agent 
outside of the study (This would exclude infants “behind schedule” and 
needing to receive on the day of first interview protection against tuberculosis, 
polio, diphtheria, tetanus, pertussis, hepatitis B, Haemophilus influenzae type 
b, measles, mumps, or rubella.  See section 10.4.7 for the effect on recruited 
age ranges)  
j.  have a known bleeding diathesis, or any condition that may be associated with a 
prolonged bleeding time  
k.  have currently any serious confirmed or suspected disease, such as metabolic, 
cardiac, or autoimmune disease, or diabetes  
l.  have a history of epilepsy or a seizure disorder, or neurodevelopmental disorders 
such as autism 
m.  have a genetic anomaly or known cytogenic disorder (e.g., Down’s syndrome)  
n.  have leukemia, lymphoma, or any other cancer/neoplasm  
o.  have known or suspected immune dysfunction, including HIV infection, or receives 
now or in the past immunosuppressive therapy, including the use of steroids 
associated with the suppression of the hypothalamic-pituitary-adrenal (HPA) 
axis (systemic corticosteroids, e.g., 1 mg/Kg/day of prednisone or its 
equivalent, or the chronic use of inhaled high-potency corticosteroids, e.g., 
800 µg per day of budesonida or 750 µg per day of fluticasone)  
p.  have ever received blood, blood products, or parenteral preparations of 
immunoglobulin 
q.  have any other serious disease (e.g., with signs of cardiac or renal failure), 
including progressive neurologic disease  
r.  have any condition which, in the opinion of the investigator, may interfere in the 
evaluation of the objectives of the study 

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10.3.3 Removal of Participants from Therapy or Assessment 
The parent(s) or legal guardian(s) of infant participants may withdraw consent for 
his/her/their child to participate in the study at any time without prejudice.  At any time, 
the investigator may withdraw a participant if, in his or her clinical judgment, it is in the 
best interest of the participant or if the participant or parent(s) cannot comply with the 
protocol.  Whenever possible, the tests and evaluations listed for the final visit should 
be carried out.  The Dominican and U.S. Senior Investigators should be notified in a 
timely manner to discuss all study withdrawals.  
 
10.4 
Treatments 
10.4.1 Treatments Administered 
All recruited participants will receive the same influenza vaccine (INF), but in different 
antigen volumes, tissue compartments, or means of injection.   
 
• Group “IM-NS-0.25” (controls)  -  two full 0.25 mL vaccine doses administered 
one month apart intramuscularly (IM) by sterile needle of 22-25 gauge, with a 
minimum length of 25mm (1-inch), attached to a sterile disposable needle.    
 
• Group “ID-JI-0.1” (investigational)  -  two reduced 0.1 mL vaccine doses 
administered one month apart intradermally (ID) by sterile, disposable needle-free 
Biojector® Syringe [cartridge] #2 and Biojector® 2000 jet injector, fitted with an 
investigational spacer on the cartridge nozzle creating a gap of 2 cm between 
nozzle orifice and skin. 
 
• Group “IM-NS-0.1” (investigational)  -  two reduced 0.1 mL vaccine doses 
administered one month apart intramuscularly (IM) by sterile needle of 22-25 
gauge, with a minimum length of 25mm (1-inch), attached to a sterile disposable 
needle intramuscularly (IM) by needle-syringe (NS)  
 
One month after the 2nd dose of INF vaccine 
described above, participants in the two 
investigational groups (ID-JI-0.1 and IM-NS-0.1) 
that received reduced doses will receive a 3rd 
“insurance” dose of 0.25 mL of the same vaccine 
intramuscularly (IM) by sterile needle-syringe.  
Controls (IM-NS-0.25) who already received the 
standard two full doses will receive a mock 
injection to avoid extravaccination.  Six months 
later, all groups three groups will return for a 
“bonus” booster dose. 
 
10.4.2 Identity of Investigational Vaccine 
Figure 4.  Vaxigrip® vaccine 5 mL vial 
(bilingual English-French label) 

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(Vaxigrip®, Sanofi-Pasteur) 
The Vaxigrip® brand of Inactivated Influenza Vaccine Trivalent Types A and B (Split 
Virion) to be used in this study (see Figure 4) is manufactured in Val De Reuil, France, 
by Sanofi-Pasteur (headquartered in Lyon, France and initially known as Institut 
Mérieux, then as Pasteur Mérieux Sérums et Vaccins, Pasteur-Mérieux-Connaught, and 
later Aventis Pasteur).  The Vaxigrip® brand of influenza vaccine was first marketed in 
France in 1968. Saliou2005  Current formulations were developed in the 1980s and first 
marketed in 1995.  It uses octoxynol-9 (Triton® X-100) detergent to “split” the virus 
particles and render them less reactogenic, a technique that previously required an ether 
compound that was dangerous to handle in manufacturing. Lina2000  
 
Vaxigrip® is currently licensed for sale and routine use of its prefilled syringe 
packaging format by the Dominican Republic national pharmaceutical regulatory 
authority, the General Directorate of Drugs and Pharmacies (Dirección General de 
Drogas y Farmacias), a unit of the Secretariat of State of Public Health and Social 
Assistance (SESPAS [ministry of health]).  However, the Vaxigrip® vaccine packaged 
in standard, multidose 5 mL vials to be used in this study are not specifically registered 
in the Dominican Republic, and thus will be imported and administered under special 
investigational authorization from the national regulatory authority.  
 
10.4.2.1 Product Characteristics 
As described in its Canadian product [insert] monograph: AventisPasteur2005    
 
“VAXIGRIP® [Inactivated Influenza Vaccine Trivalent Types A and B 
(Split Virion)] for intramuscular use, is a sterile suspension prepared 
from influenza viruses propagated in chicken embryos.  Following 
incubation, the virus-containing fluids are collected and clarified and the 
viruses are concentrated then purified by zonal centrifugation using a 
sucrose density gradient.  Subsequent stages consist of treatment with 
octoxynol-9 (Triton® X-100) to obtain split antigens, then inactivation 
using formaldehyde solution. The split-antigen is suspended in sodium 
phosphate-buffered, isotonic sodium chloride solution. The type and 
amount of viral antigens contained in VAXIGRIP® conform to the 
current requirements of the World Health Organization (WHO).” WHO2005 
 
The study will commence with the 2006 Southern Hemisphere formulation of 
Vaxigrip® INF vaccine, the content of which is summarized in Table 3.  This 
formulation becomes available in early 2006 and will expire on 31 December of 
2006.  It is anticipated that a proportion of vaccinations will occur after that date 
(see timeline in section 5), requiring use of either or both of the next available 
formulations (2006-2007 Northern Hemisphere and 2007 Southern Hemisphere).  
Their content will not be known until after the study begins.  If there is a change of 
strains between formulations used, each participant will receive all his or her doses 
from the same unexpired formulation.   
 

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As described in section 9.5.1, the biseasonal epidemiology of influenza in the 
Dominican Republic justifies use of either Northern or Southern hemisphere 
formulations.  If strains change between seasonal formulations used in the study, 
serologic assays with appropriate antigens will be segregated accordingly.   
 
Table 3  Vaxigrip® INF vaccine content, by study arm 
Study Arm 
2006 Southern Hemisphere formulation 
“ID-JI-0.1” 
“IM-NS-0.1” 
“IM-NS-0.25” 
(control) 
Hemagglutinin antigen 
 
A/New Caledonia/20/99 (H1N1) [IVR-116]:  
 
A/New York/55/2004 (H3N2) [NYMC X-157]  
 
 
(A/California/7/2004-like):  
 
B/Victoria/2/87 lineage (B/Malaysia/2506/2004-like):  
 
3 µg 
 
3 µg 
3 µg 
 
7.5 µg 
 
7.5 µg 
7.5 µg 
Sodium-phosphate buffered, isotonic sodium chloride solution 
up to 0.1 mL 
up to 0.25 mL 
Formaldehyde 
≤6 µg 
≤15 µg 
Thimerosal preservative 
0.4 µg 
1 µg 
Triton® X-100, sucrose, neomycin 
Trace 
trace 
 
10.4.2.2 Storage 
As per the manufacturer’s recommendations, Aventis-Pasteur2004  the vaccine should be 
stored at +2° C to +8° C when not being used.  It must NOT be frozen.  Open only 
one vial at a time.  The doses for all three study arms should be filled from the 
same vial until empty.  When not vaccinating participants, return the vial to the 
cold box carrier or vaccine refrigerator.  At the end of the clinic day, return the vial 
from the cold box carrier to the vaccine refrigerator for overnight storage.  Dispose 
of any vial, whether empty or not, which has been entered and in use for 7 days, or 
for which there is any other evidence or suspicion that it has been compromised.   
 
10.4.2.3 Directions for Vaccine Administration 
Preparation. Aventis-Pasteur2004   
•  Inspect the vaccine vial for extraneous particulate matter and/or discoloration 
before use.  If these conditions exist, the product should not be administered.  
Do not remove either the vial stopper or the metal seal holding it in place.  
Aseptic technique must be used for withdrawal of each dose.   
•  Remove the aluminum tab covering the vial stopper, and swab the stopper with 
alcohol.   
•  Open and attach to the vaccine vial a sterile, disposable multidose vial adaptor 
(e.g., SmartSite® vial access device - 
http://www.alarismed.com/na/products/smartsite_compon.asp) by pushing its 
plastic spike through the vial stopper.   
•  Write onto the vial label the date when the vial adaptor is attached.   
 
Needle and syringe administration for study groups IM-NS-0.1 and IM-NS-0.25 
•  Politely request the parent(s) and any other personnel to remain blinded by study 
protocol to leave the injection room.  Confirm the participant has been 
assigned to the intramuscular, needle-syringe, reduced-dose study arm (IM-

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NS-0.1) or to the intramuscular, needle-syringe, full-dose study arm (IM-NS-
0.25).   
•  Swab the vial adaptor port valve with alcohol. 
•  Gently shake the vial to uniformly distribute the suspension before withdrawing 
each dose.   
•  Holding the vial upside down, insert upward into the port the Luer tip of a 
sterile, 1mL capacity tuberculin-style or universal vaccination syringe.   
•  Draw downward on the syringe plunger until the correct volume of either 0.1 
mL or 0.25 mL shows on the syringe (according to the participant’s assigned 
study group), then withdraw an additional amount (~0.1 mL) to account for 
wastespace in the syringe tip and needle hub.   
•  Maintaining the syringe pointing upward, remove it from the vial adapter and 
attach a sterile 22-25 gauge needle with a minimum length of 25mm (1-
inch).   
•  Still holding the syringe upward, pull down on the plunger until at least 0.5 
mL of air enters the syringe above the vaccine.   
•  Tap the syringe barrel until all vaccine liquid has moved to the bottom of the 
barrel towards the plunger and all air bubbles have risen to the top towards 
the needle.   
•  Slowly push up on the plunger until the air is expelled from the syringe and 
needle and the plunger seal reaches the appropriate dose mark on the barrel 
(0.25 or 0.1 mL, according to participant group).   
•  For children aged >1 year, insert the needle intramuscularly to its hub at a 90º 
angle into the participant’s vastus lateralis muscle on the anterolateral 
portion of the mid-to-upper LEFT thigh.  For children ≥1 year of age, 
administer into the deltoid muscle of the LEFT arm.   
•  After the injection, observe 5 seconds for any leakage of liquid or blood on the 
skin. 
•  Note observations on the case report form, along with vaccine lot number and 
clock time. 
•  Cover the injection site with an adhesive bandage (to be removed in 30-60 
minutes for inspection).   
•  Safely dispose of the needle-syringe before the parent enters the room.   
•  Return the vaccine vial to the cold box or refrigerator unless the next 
participant will be vaccinated shortly.   
 
Jet injection administration for study group ID-JI-0.1 
•  Politely request the parent(s) and any other personnel to remain blinded by study 
protocol to leave the injection room.    Confirm the participant has been 
assigned to the intradermal, jet-injected, reduced-dose study group (ID-JI-
0.1).   
•  Gently shake the vial to uniformly distribute the suspension before withdrawing 
each dose.   
•  Swab the vial adaptor port valve with alcohol. 
•  Insert into the port the Luer tip of a sterile Biojector® cartridge (#2/green).   

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•  Holding the vial upside down, draw Vaxigrip® vaccine into the cartridge until 
the correct volume of 0.1 mL shows on the cartridge.  Then withdraw an 
additional amount (~0.1 mL) to account for wastespace in the cartridge 
nozzle.   
•  Maintaining the cartridge pointing upward, remove it from the vial adapter.  
•  Still holding the cartridge pointing upward, pull down on the plunger until at 
least 0.5 mL of air enters the cartridge above the vaccine.   
•  Tap the cartridge barrel until all vaccine liquid has moved to the bottom of the 
barrel towards the plunger and all air bubbles have risen to the top towards 
the nozzle.   
•  Slowly push up on the plunger until the air is expelled from the cartridge and 
the top edge of the orange plunger seal reaches the 0.1 mL dose mark on the 
barrel.   
•  Attach a sterile, disposable 2-centimeter spacer onto the cartridge nozzle.   
•  Verify from the indicator gauge on the Biojector® 2000 jet injector that it is 
in the green range indicating sufficient pressure of CO2 gas for the injection.  
If not, release any remaining residual pressure by slowly unscrewing the 
CO2 canister knob, and replace the canister with a new one.  Then repeat 
this step. 
•  Keeping one’s fingers away from the trigger, insert the cartridge into the jet 
injector front end and rotate all the way clockwise until the indicator 
window shows green.   
•  When ready to inject children aged <1 year, hold the spacer, cartridge, and 
device firmly against the vastus lateralis muscle of the anterolateral aspect 
of the mid-to-upper LEFT thigh.  Stabilize the back of the infant’s thigh 
with one’s other hand. While maintaining a 90º angle between the axis of 
the cartridge and the thigh, pull and immediately release the injector 
activator lever and maintain the relative positions of the injector and thigh 
for one full second to be sure the injection is over.  For children ≥1 year of 
age, follow the above procedure to administer the dose into the deltoid muscle 
of the LEFT arm.   
•  After the injection, observe 5 seconds for any leakage of liquid or blood on the 
skin. 
•  Note observations on the case report form, along with vaccine lot number and 
clock time. 
•  Cover the injection site with an adhesive bandage (to be removed in 30-60 
minutes for inspection).   
•  Safely dispose of the cartridge and spacer before the parent enters the room.   
•  Return the vaccine vial to the cold box or refrigerator unless the next 
participant will be vaccinated shortly.   
 
10.4.2.4 Vaccine Immunogenicity Profile 
Substantial clinical experience in use of the current “Triton® X-100 split” 
formulation of Vaxigrip® influenza vaccine has accumulated since the early 1990s 
when it replaced the earlier “Tween-ether split” product, first introduced in France 

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in 1968. Lina2000, Saliou2005  Hundreds of millions of doses of this current Vaxigrip® 
formulation have been distributed and used worldwide over many seasons with, of 
course, the annual modifications of virus strain composition.   
 
The immunogenicity of Vaxigrip® has been demonstrated in clinical trials in adults 
from age 18 to 60 years, in the elderly over 60 years, and in children from 6-36 
months and from 3-10 years.  Annual studies in adults are performed to verify the 
immunogenicity of annual changes in strain composition (data on file at Aventis 
Pasteur S.A., Lyon France AventisPasteur2005)  In the unpublished Annual Study (see 
Figure 5), and in Study 3 (35), Lina2000 a single dose was given and antibody titers 
were assessed immediately before vaccination and then 21 days later.  In Study 2 
(36) Gonzalez2000 antibody titers were assessed immediately before the first dose and 
27-33 days following the second vaccine dose.  
 
 
The results of these studies are quoted verbatim from the product monograph 
AventisPasteur2005:  
 
“The efficacy of influenza vaccine is assessed using a surrogate for 
protection defined as the immune response elicited by the vaccine 
(hemagglutination inhibition).  In the annual studies, the serologic responses 
of both adult age groups to all antigens must meet the assessment criteria as 
defined in the European Requirements for Influenza Vaccines (i.e., for 
subjects 18-60 years – at least one of seroconversion or significant increase 
in antihemagglutinin antibody titre in >40%, mean GMT increase >2.5, 
proportion of subjects achieving HI titre or seroprotection >70%, and for 
Figure 5.   Table 3 from the Vaxigrip® monograph AventisPasteur2005 
references:  (13) = “data on file”,  (35) = Lina2000,  (36) = Gonzalez2000 

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subjects >60 years at least one of seroconversion or significant increase in 
antihemagglutinin antibody titre in >30%, mean GMT increase >2.0, 
proportion of subjects achieving HI titre >60%.) CPMP1997  Elderly subjects 
generally respond less well to influenza vaccines than young healthy adults, 
and those with chronic debilitating medical conditions generally respond 
less well than healthy subjects of similar age. Fukuda2004    
 
“The results in children met the criteria defined for young adults; no criteria 
for children have been set.”   
 
Gonzalez, et al, studied two 0.25 mL doses of Vaxigrip® administered one month 
apart to 67 children from 6 months to 3 years (36 months) of age in an uncontrolled 
study. Gonzalez2000  Seroconversion (see definition in section 10.5.1.2) was achieved 
by 83.7% of the children to the A/H3N2 strain, by 81.6% to the A/H1N1 strain, and 
by 61.2% to the B strain.  Seroprotection (see definition in section 10.5.1.3) rates 
were 91.8%, 81.6%, and 93.9%, respectively.  The geometric mean increases 
(section 10.5.1.3) were 9.8, 13.3, and 4.1, respectively.   
 
In a recent unpublished poster presentation, Saliou2005  manufacturer representatives 
summarized the data from manufacturer trials designated GRT04, GRT11 and 
GRT51 among children from 6 months to 3 years of age.  Precise seroconversion 
and seroprotection rates were estimated from histogram bars without value labels 
by interpolating from the scale.  The mean seroconversion rates were estimated at 
84% for A/H3N2, 88% for A/H1N1, and 54% for B.   
 
Among a pediatric population older than the one to be studied here, Lina, et al 
reported a study of 42 children from 8 to 10 years of age, finding seroconversion 
rates of 57% (A/H3N2), 76% (A/H1N1), and 29% (B) three weeks after a single 
0.5 mL dose of Vaxigrip. Lina2000  Seroprotection was 83%, 93%, and 100%, 
respectively.  The geometric mean increase was 6.4, 10.1, and 2.3, respectively.   
 
Averaging the 2-dose seroconversion rates in age groups of 6-36 months, similar to 
those to be studied here (6-23+ months), from the Gonzalez et al study and the 
three studies reported by Saliou et al, the mean SC was 85.2% (A/H3N2), 86.4% 
(A/H1N1), and 56.0% (B).   
 
10.4.2.5 Vaccine Efficacy 
Phase III field efficacy trials of influenza vaccine to measure protection against 
influenza-like symptoms or disease are rare.  In one unblinded trial in China among 
80 Vaxigrip recipients and 88 observed controls, Jianping et al reported reduction 
of reported symptoms by 84.8% over the 6-month followup period in children 3-6 
years of age Jianping1999   
 

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10.4.2.6 Vaccine Safety Profile 
Clinical trials and post-marketing surveillance are the source for knowledge 
concerning anticipated adverse events (AEs) following administration of Vaxigrip.  
The overall summary of AEs are quoted here from the product monograph 
AventisPasteur2005 :  
 
“The most frequent side effect of influenza vaccination is soreness at the 
vaccination site.  These local reactions generally are mild and rarely 
interfere with the person’s ability to conduct usual daily activities.  (3) 
[CDC2004]  Local redness, swelling, induration and bruising have also been 
reported. (13) [Data on file at Aventis Pasteur SA]   
 
“Fever, malaise, myalgia, arthralgia, lymphadenopathy, headache, 
shivering, sweating, fatigue (13) [Data on file at Aventis Pasteur SA] and other systemic 
symptoms can occur following vaccination with inactivated influenza 
vaccine and most often affect persons who have had no exposure to the 
influenza virus antigens in the vaccine (e.g., young children). (3) [CDC2004] 
(14) Schiefele1990  These reactions usually disappear within 1 - 2 days without 
treatment.   
 
“Placebo-controlled trials suggest that among elderly persons and healthy 
young adults administration of split-virus influenza vaccine is not associated 
with higher rates of systemic symptoms (e.g., fever, malaise, myalgia and 
headache) when compared with placebo injections. (3) [CDC2004] (15) Nichol1996  
Prophylactic acetaminophen may decrease the frequency of some side 
effects in adults. (2) (16) Aoki1993 ”  
 
In the Gonzalez et al clinical trial of Vaxigrip among children 6 months to 3 years 
of age, the only immediate reactions found were 9 cases of injection site macules or 
papules after the 2nd immunization (of 57, 16%), which was deemed allergic in 
nature. Gonzalez2000  Delayed local reaction(s) occurred among 9% (5 of 57), 
including reddened papules of <3cm diameter, and 1 child with severe limb pain 
preventing voluntary movement.  Thirty systemic events within 3 days after an 
injection were observed in 28% (16) of 57 patients.  These comprised 19 events of 
rhinitis and cough, 5 fevers (38.5° – 38.9° C).  Systemic events between 4 days and 
30 after vaccination were reported after 30 1st injections and 37 2nd injections, but 
were deemed common childhood symptoms and not described.  One patient with a 
history of bronchiolitis experienced 2 serious adverse events of bronchospasm 
occurring 25 days and 2 days after doses 1 and 2, respectively, which were deemed 
unrelated to Vaxigrip. 
 
In the Saliou et al meta-analysis of published and unpublished Vaxigrip studies, 
safety results from 6 trials (including Gonzalez et al) were pooled into a single 
database. Saliou2005  Among 337 subjects in the age range of 6 to 36 months, 
frequencies of non-severe AEs were 19% fever, 8.3% pain, 8.3% erythema, 2.7% 
induration, and 4.2% ecchymosis.  Severe AE frequencies were 2.1% fever, 0.3% 
pain, 0% erythema, 0.3% induration, and 0% ecchymosis.  

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Other very rare AEs reported by the manufacturer AventisPasteur2005 as a result of post 
marketing surveillance include: 
 
•  Blood and lymphatic system disorders (transient thrombocytopenia, 
lymphadenopathy)  
•  Immune system disorders (allergic reactions: pruritis, rash erythematous 
urticaria, dyspnea, angioneurotic edema, or anaphylactic shock) 
•  Nervous system disorders (paraesthesia, Guillain-Barre Syndrome [GBS], 
neuritis, neuralgia, convulsions, encephalomyelitis) 
•  Vascular disorders (vasculitis, with transient renal involvement in certain 
cases) 
 
In Canada, a newly-described oculorespiratory syndrome (ORS) following 
influenza vaccination, consisting of early onset (2-24 hours) of bilateral red eyes, 
and/or cough, wheezing, chest tightness and other respiratory symptoms, was 
recognized in 2000-2001 and subsequent seasons. Canada2000, Canada2001a, Canada2001b  The 
syndrome was generally mild and self-limited.  Children less than 9 years of age 
constituted about 1 percent of cases, which clustered in females (75%) and healthy 
adults 40-59 years of age. Boulianne2001  Of the 960 reports satisfying an ORS 
definition, 96% followed Fluviral® influenza vaccine, manufactured by Biochem 
Pharma (subsequently Shire Biologicals), which distributed 4 million doses in 
Canada. Canada2001b  Only 2% of ORS cases occurred following either the Vaxigrip or 
Fluzone® products of Aventis Pasteur, which distributed 9 million doses of its two 
vaccines, manufactured in France and the USA, respectively.  A 2001 survey 
among 642 diabetic children and their siblings who had been vaccinated in a 
province using only the Fluviral product in the 2000 season identified 83 (13%) 
who satisfied the case ORS definition, and 1% who required hospitalization. 
Skowronski2005   
 
Studies at multiple laboratories detected an unusually large frequency of aggregates 
of unsplit virus (up to one-third of total virus) in the Fluviral product, which had 
recently undergone a change in manufacturing technique. Skowronski2003  Aggregates 
were nearly absent in the prior 1999 Fluviral product, as well as in the Fluzone and 
Vaxigrip products, the latter of which was measured to have a maximum of only 
2% unsplit virus.  A retrospective cohort study in Québec province suggested the 
frequency of ORS was equally around 5 percent for recipients of both Fluviral and 
Vaxigrip products. DeSerres2003  Since the 2000-2001 season, incidence has declined 
steadily from 46.6 reported cases per 100,000 doses distributed, to 34.2, 20.6, and 
9, in the 2001-, 2002-, and 2003- seasons, respectively. DeSerres2005    
 
In a manufacturer-independent side-by-side trial of Vaxigrip and Fluarix® 
(SmithKline Beecham Biologicals [now GlaxoSmithKline], Rixensart, Belgium) 
conducted among 18-60 year old adults in 1996-97 in Czechoslovakia, redness at 
the injection site occurred more frequently (28%) in the Vaxigrip group (n=100) 
than in the Fluzone one (13%, n=99). Beran1998  Frequencies of other local reactions 

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for Vaxigrip vs. Fluarix were 15% / 7%, respectively, for swelling and 50% / 36% 
for pain (not statistically significant).  Systemic reaction rates for Vaxigrip vs. 
Fluarix were 18% / 11% for headache, 16% / 12% for myalgia, 2% / 1% for 
shivering, and 13% / 6% for malaise.  (Seroconversion and seroprotection rates 
were generally 10 to 30 percentage points higher for Vaxigrip than for Fluarix, and 
geometric mean titers were generally fifty percent or more higher, as well.  Both 
vaccines satisfied EMEA requirements for Immunogenicity.CPMP1997)   
 
10.4.2.7 Guillain-Barré Sequelae of Influenza Vaccination 
Guillain-Barré syndrome (GBS) is a neurologic disorder that occurs overall at a 
rate of about 10-20 cases per million population in the U.S.Ropper1992  More rarely it 
may follow influenza vaccination.   
 
Schonberger et al described the epidemiology of GBS following the “swine” 
influenza mass campaign of 1976 in the U.S., which measured a vaccine-
attributable risk of GBS of just under 10 cases per million vaccinated.  They found 
the period of increased risk following the swine vaccine strain was concentrated 
within 5 weeks after vaccination, although lasting for approximately 9 or 10 weeks. 
 
Haber, et al found much lower rates for routine seasonal influenza vaccination by 
analyzing reports to the national Vaccine Adverse Events Reporting System 
between 1990 and 2003.Haber2004  The highest frequency was 1.7 cases of GBS per 
million vaccinees in the 1993-1994 season, and the lowest rate of 0.4 cases per 
million in 2002-2003.  The median interval after vaccination for onset of GBS was 
13 days, with about 85% occurring within 4 weeks and about 95% within 8 weeks.   
 
Lasky et al identified patients with GBS in four states from hospital discharge data 
from the 1992-93 and 1993-94 seasons and contacted them and their providers for 
vaccination history.Lasky1998  Their adjusted relative risk for GBS among those 
receiving influenza vaccine was 1.7 times higher (p=0.04) than those who had not 
been vaccinated.  This risk corresponded to slightly more than one additional case 
of GBS per million persons vaccinated.  In 9 of 19 vaccine associated cases where 
dates were known, the onset of GBS was in the second week after vaccination, all 
between day 9 and day 12.   
 
At a rate of one per million, the probability of experiencing GBS in this study of 
450 children is 0.00045, or about one-twentieth of one percent.  This is much less 
than the risk of serious illness and death in this susceptible age group from 
influenza disease if they remain unvaccinated, according to ACIP:CDC2005  
 
“Cases of GBS after influenza infection have been reported, but no epidemiologic studies 
have documented such an association ([ref]209,210). Substantial evidence exists that 
multiple infectious illnesses, most notably Campylobacter jejuni, and upper respiratory 
tract infections are associated with GBS (203,211--213).  

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“Even if GBS were a true side effect of vaccination in the years after 1976, the estimated 
risk for GBS of approximately 1 additional case/1 million persons vaccinated is 
substantially less than the risk for severe influenza, which can be prevented by 
vaccination among all age groups, especially persons aged >65 years and those who 
have medical indications for influenza vaccination (Table 1) (see Hospitalizations and 
Deaths from Influenza). The potential benefits of influenza vaccination in preventing 
serious illness, hospitalization, and death substantially outweigh the possible risks for 
experiencing vaccine-associated GBS. The average case fatality ratio for GBS is 6% and 
increases with age (203,214). No evidence indicates that the case fatality ratio for GBS 
differs among vaccinated persons and those not vaccinated.”  
 
10.4.3 Identity of Injection Device (Biojector® 2000, Bioject, Inc.) 
The jet injector to be used for delivery of influenza vaccine in the intradermal arm (ID-
JI-0.1) of this study is an American-made, hand-held, reusable device powered by 
standard CO2 cartridges (see Figure 6).  It was introduced into the United States in the 
mid-1990s and is now cleared for marketing and use in humans in the U.S. (FDA 
numbers K920631 and K960373),FDA1997  Canada, the English-speaking countries of the 
European Union, China, India, Bahrain, Jordan, Korea, Syria, and the United Arab 
Emirates (2005, personal communication, Susan Frank, Bioject, Inc.).  (See: 
http://www.bioject.com/biojector2000.html)   
 
It uses sterile, disposable cartridges (“syringes”), for which different versions are 
available for patients of varying size and weight.  Subcutaneous or intramuscular 
injections are achieved by varying the diameter of the orifice in the nozzle of the 
cartridges (the larger the diameter, the deeper the injection).  The no. 2 cartridge (green, 
0.10 mm orifice diameter) is for subcutaneous injection for patients of all sizes. 
Bioject1997, NIP2005b  Cartridge nos. 3 (brown, 0.15 mm), 4 (blue, 0.20 mm), 5 (silver, 0.25 
mm), and 7 (red, 0.36 mm) are for intramuscular injections for infants, toddlers, adults 
and “large adults”, respectively.  (An investigational spacer applied to the nozzle of its 
no. 2 disposable cartridge is to be studied by this protocol, and is described in detail in 
section 10.4.3.4.)   
 
Figure 6.  Biojector® 2000 needle-free jet injector. 

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10.4.3.1 Preclinical and Veterinary Studies in Animals 
Various published bench, preclinical, and veterinary studies of the Biojector system 
have been conducted both prior to and subsequent to initial device licensure for humans.  
These include studies in mice,Davis1994, Cartier2000, Singh2001, Trimble2003, Brave2005, Choi2005  
rats,Davis1994  guinea pigs,Davis1994, Ledwith2000, Manam2000  pigs,Babiuk2003  rabbits,Davis1994, 
Aguiar2001, Hartikka2001 dogs,Smith1998 rhesus macaque monkeys,Barouch1998, Barouch2000a, 
Barouch2000b, Rogers2001, Amara2001, Amara2002, O’Neill2002, Rogers2002, Raviprakash2003, Sadagopal2005  
cynomolgus macaque monkeys,Sullivan2000, Rao2005  aotus monkeys,Gramzinski1998 and 
chimpanzees.Davis1996   
 
Many of the postlicensure studies using the Biojector involved the delivery of 
investigational DNA vaccines.Mumper2003, Trimble2003  See section 10.4.3.5 for additional 
details on some of these animal studies that included the intradermal spacer.  Analysis 
by two high performance liquid chromatography methods of 18 different proteins 
ejected by a similar investigational jet injector (Iject™, also from the Bioject company) 
that shares a similar performance profile to the Biojector found no damage to the 
proteins nor the formation of aggregates.Benedek2005   
 
As-yet unpublished animal research using the Biojector includes the following, among 
other collaborations between Bioject, Inc. and the U.S. FDA, the NIH, Imclone 
Systems, Emory University, Georgetown University, Johns Hopkins, and the 
Universities of Kentucky, Massachusetts, Michigan, and Puerto Rico (source: Bioject, 
Inc., 2005):   
•  Antex Biologicals, Inc. (now Bioport): DNA vaccines for chronic bacterial 
infection 
•  Auburn University: DNA vaccines for canine/feline parvovirus 
•  Corixa, Inc. and Oregon Regional Primate Research Center: adjuvanted DNA 
vaccines in rhesus macaques is enhanced by Biojector delivery or microsphere 
encapsulation (tuberculosis antigen IM and ID) Evans2001, Evans2002, Mossman2003 
•  Genencor International: experimental animal models for hepatitis A and C, and 
human papillomavirus vaccines 
•  Genetronics, Inc., and Veterinary Infectious Disease Organization, University of 
Saskatchewan:  DNA vaccination in pigs with DNA plasmid for bovine 
herpes virusBaizer2001, Babiuk2003 
•  Genzyme, Inc.:  Intradermal administration into guinea pigs of recombinant 
adenovirus as cancer vaccine modelPerricone2000 
•  Iowa State University:  DNA vaccines for swine  
•  Memorial Sloan Kettering Cancer Center:  DNA melanoma vaccine study in dogs 
(http://www.mskcc.org/mskcc/html/13199.cfm) 
•  Universities of Toronto and Wisconsin:  multiantigen HIV vaccines in primates 
 
10.4.3.2 Clinical Studies of Licensed Drugs and Vaccines 
Human trials using the Biojector and its standard intramuscular or subcutaneous 
cartridges have demonstrated satisfactory immune responses and safety for vaccination 

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with licensed vaccines against influenza,Jackson2001  yellow fever,Jackson1993  and hepatitis 
A.Williams2000  In one study, alum-adjuvanted hepatitis B vaccine produced about twice 
the frequency of local reactions by Biojector IM than control needle-syringe IM, but 
comparable seroconversion rates.Matheï1997  In a study of apprehension among 6th grade 
children receiving hepatitis B by Biojector IM on one dose and needle-syringe on the 
other, pre- and post-injection anxiety scores were similar; however 61% of subjects 
preferred the Biojector (p=0.08).Polillio1997  Other studies have described effective 
Biojector use in administering anesthesia, both local Gerbert1996, Florentine1997, Koenig2004  and 
general,Greenberg1995, Baer1996, Bennett1998, Phero1998, Fine2004  as well as for heparin therapy.Baer1996  
 
10.4.3.3 Clinical Studies of Investigational Drugs and Vaccines 
Published reports have documented use of the Biojector system in administering 
investigational drugs and vaccines such as (1) carcinoembryonic antigen in recombinant 
viral vectors to cancer patients,Marshall1999, Marshall2000, Conry1999  (2) malaria 
vaccines,Wang2001, Epstein2002  and (3) HIV/AIDSBoyer2000  (see section 10.4.3.6 for 
additional details on some of these and other studies that included the investigational ID 
spacer).   
 
As described in the first two paragraphs of section 9.3.3, the Biojector system is in use 
by a number of immunization providers since its introduction in the mid-1990s.  
Bioject, Inc. reported that about one million injections per year are performed with the 
device, as estimated from cartridge sales (personal communication, Kurt Lynam, 2000).  
The U.S. Navy uses the regular system for routine use among adult and pediatric 
dependents of sailors, and also speeds up mass vaccination of naval recruits by 
attaching large-volume nitrogen tanks to avoid replacing empty CO2 cartridges every 10 
to 15 injections (see Figure 7).  The system is also used in a number of public health 
(see Figure 8) and pediatric settings (see Figure 9) around the United States.    
 

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Figure 7.  Use of  Biojector system in U.S. military, 2003-2004.  Source: Bioject, Inc. 
Figure 8.  Use of Biojector system in public health, 2003-2004.  Source: Bioject, 
Inc. 

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10.4.3.4 Investigational Spacer 
This investigational spacer to be used to administer intradermal injections is a high 
density polypropylene cylindrical tube that attaches by friction fit to the front of the 
Biojector no. 2 (subcutaneous) cartridge (see Figure 10).  
 
 
Figure 10.  Investigational intradermal spacer (top)  
fitted to no. 2 Biojector cartridge (by itself, bottom). 
Figure 9.  Use of Biojector system in general pediatrics, 2003-2004. 
Source: Bioject, Inc. 

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The spacer creates a gap of 2 cm between the skin and the orifice in the nozzle of 
the cartridge, weakening the stream enough to achieve the desired intradermal 
delivery (see Figure 11).  This variation of jet injection practice has been 
demonstrated by others since at least the 1970s and was discussed in detail earlier 
in sections 9.3.1 and 9.6.3.   
 
 
10.4.3.5 Preclinical Studies of the Biojector ID Spacer 
A number of animal studies with the Biojector spacer have been performed to 
demonstrate safety and/or efficacy of the investigational spacer used to administer 
intradermal injections with the Biojector cartridge and injector.   
 
10.4.3.5.1 Mice, University of Kentucky 
BALB/C mice were immunized by subcutaneous (SC) needle-syringe (N-S) 
injection or intradermally (ID) by the Biojector system and spacer with three 
doses of nanoparticles coated with plasmid DNA (pDNA) expressing β-
galactosidase, or the pDNA alone, or the protein alone with alum. Cui2003  For 
pDNA alone, there was no difference in IgG titers to the antigen between the 
two routes of administration.  But for the particle pDNA, ID jet injection 
generated 20-fold higher antibody titers than SC by N-S.   
 
10.4.3.5.2 Mice, Rotavirus Antigen, Cincinnati Children’s 
Hospital 
The rotavirus vaccine antigen VP6 was administered by various routes to 
mice at the Cincinnati Children’s Hospital to explore non-oral routes of 
vaccination to avoid gastrointestinal side effects of the vaccine.Choi2005  
Figure 11.  Artist's rendition of dose distribution for Biojector needle-free jet injections in human 
tissues.  Intradermal cartridge on right reflects manufacturer's intention to incorporate a spacer 
into the nozzle of a future product, rather than as a separate attachment which now exists.  Source: 
Bioject, Inc. 

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Groups of the BALB/c subjects were given the antigen, with or without a 
modified heat-labile E. coli toxin as an adjuvant, by (1) skin patch, (2) 
Biojector intradermal injection followed by skin patch with adjuvant only 
over the injection site, or (3) intranasal instillation.   
 
Only the intranasal route produced significant reductions in fecal shedding 
following oral rotavirus challenge.  The Biojector ID route, however, with or 
without adjuvant, produced from 35 to 100 times as much specific IgG 
immunoglobulin as did the comparable patch vaccination, and also exceeded 
the antibodies generated by the intranasal route.  The authors speculated that 
the inability of the Biojector ID method to induce protection was because the 
strongly hydrophobic VP6 antigen was not effectively reaching the 
epidermis.  Needle-syringe ID and SC delivery did not have this problem, 
although only the SC route produced protection from shedding.   
 
10.4.3.5.3 Pigs, Hepatitis B Vaccine, University of Saskatchewan 
Researchers at the Vaccine and Infectious Disease Organization and 
collaborators at the University of Saskatchewan studied vaccination with a 
DNA plasmid encoding for hepatitis B surface antigen (HBsAg) and a 
commercial human recombinant hepatitis B vaccine 
(GlaxoSmithKline).Babiuk2003  The DNA plasmid antigen was administered 
intradermally on the abdomen of 4-to-6-week old outbred pigs, using either 
Biojector with spacer or a conventional ID needle injection, both with and 
without followup electroporation of the injection site (60 ms electrical pulses 
of 60-80 volts, believed to permeabilize cell membranes and improve 
uptake).  Two additional groups of pigs received conventional liquid vaccine 
containing hepatitis B surface antigen (HBsAg) by either ID Biojector (2 
injections totalling 0.5 mL per animal) or IM needle injection (0.5 mL).  All 
six groups were boosted at week 8 with the conventional vaccine by IM 
needle, except the group which had been primed with this same antigen by 
ID Biojector received the booster by the same method and route as before.    
 
By the time of the 8-week booster, most of the conventional vaccine 
recipients (4/5 for Biojector ID, 5/5 for needle ID) had developed antibody 
responses ≥10 IU and OD ≥2 standard deviations over background for both 
the ELISA and AUSAB® assays.  On the other hand, only the Biojector DNA 
plasmid group with (2/5) or without (1/5) electroporation achieved a response 
on both assay, while none of the ID needle groups did so.  Two weeks after 
the booster, all electroporation DNA plasmid groups (Biojector ID and 
needle ID), as well as the groups receiving only the conventional vaccine 
achieved 100% immune response (5/5 or 4/4) on both assays.  The DNA 
plasmid groups without electroporation -- both Biojector ID and needle ID -- 
had 60% (3/5) responses to the ELISA assay, while the former’s response 
was 80% (4/5) and the latter’s 100% (5/5) to the AUSAB assay.   
 

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The authors did not comment on observable local abdominal reactions 
around the injection sites.  Histological examination macrophage infiltration 
in the dermis and epidermis attributable to the electroporation voltage.   
 
10.4.3.5.4 Mice, guinea pigs, monkeys, HIV Vaccine, Emory 
University and CDC 
In pre-primate preparatory studies at the Yerkes National Primate Research 
Center, Emory University, Balb/c mice and Hartley guinea pigs were injected 
with modified vaccinia Ankara expressing HIV (MVA/HIV) antigens by 
either Biojector intradermally (ID) or needle-syringe intramuscularly (IM). 
Wyatt2004  Both routes were found to be equivalent for raising neutralizing 
antibody.  The authors did not note any difficulties with intradermal jet 
injection in the experimental animals.   
 
Twenty-four rhesus macaque monkeys at CDC’s Lawrenceville, GA animal 
facility received two priming doses of DNA vaccine by Biojector 2000 by 
either the ID (with spacer) or IM routes (without spacer). Amara2001, Amara2002, 
Buge2003  They were then boosted with a single MVA/HIV dose by needle-
syringe ID or IM.  The ID delivery of DNA priming was about 10 times more 
effective than IM for generating gag antibody.  At comparable dose levels, 
the ID groups achieved higher ELISPOT assay results and proliferative 
responses than the IM groups. Amara2001 
 
Following intrarectal challenge of pathogenic virus administered 7 months 
after vaccination, animals in both routes of delivery showed effective control 
of virus.  Gp120 protein administered in some animals alongside the second 
prime and the boost doses 2 and 3 did not improve protection. Buge2003  Two 
hundred weeks post-challenge, 22 of 23 animals had successfully controlled 
the viremia of their challenge strain, demonstrating long-term immune 
control as a result of vaccination. Sadagopal2005   
 
10.4.3.5.5 Rhesus Macaques, DNA HIV Vaccine, University of 
Puerto Rico 
At the Caribbean Primate Research Center in San Juan, six groups of five 
rhesus macaque monkeys each were vaccinated three times at 13-week 
intervals with a 2 mL solution of DNA expressing various HIV antigens with 
or without various cytokine genes as adjuvant.O’Neill2002  Except for a seventh 
naïve control group, on each vaccination day each animal received two IM 
injections of 0.5 mL each in the triceps muscle by Biojector, plus 10 ID 
injections of 0.1 mL each on the thigh by Biojector with spacer.   
 
At 39 weeks after the first priming dose, all animals except the naïve controls 
were boosted with a virus-like particle expressing simian immunodeficiency 
virus (SIV) antigens, in the presence (groups 1, 3, 5) or absence (groups 2, 4, 

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6) of recombinant IL-12 cytokine as adjuvant.  At 47 weeks, all animals were 
challenged intrarectally with the highly pathogenic SIVsmE660 virus strain.   
 
All but one monkey became infected by the challenge virus, as evidenced by 
plasma viral RNA.  Compared to animals receiving the antigens only, 
however, monkeys administered as well the cytokine adjuvants showed 
complete protection from clinical disease for at least 14 months (including 
the uninfected subject).  Antigen-only groups evidenced clinical illness and 
lower survival.  Vaccinated animals demonstrated significantly lower peak 
plasma viral loads than the controls.  The authors did not comment on local 
reactions to the Biojector IM and ID injections, if any.   
 
10.4.3.5.6 Infant Rhesus Macaques, DNA Measles Vaccine, 
University of Maryland 
Preclinical studies in infant rhesus macaque monkeys were conducted at the 
Center for Vaccine Development of the University of Maryland, Baltimore, 
as preparation towards planned human studies of an experimental DNA-
construct measles vaccine that might ultimately protect human infants as 
young as 3 months of age (Karen Kotloff, personal communication, 2005).  
Current measles vaccines are not generally used in children younger than 9 
months of age because of interference from transplacental maternal antibody 
which lowers efficacy.   
 
The vaccination regimen was administered at the Johns Hopkins University 
Rhesus colony to six monkeys less than 2 months of age as a model for 
young human infants.  Under suitable anesthesia of both mother and infant, 
each infant was vaccinated with the DNA measles vaccine in two intradermal 
doses of 0.1 mL each at different body sites on both days 0 and 28, using the 
Biojector system and intradermal spacer (see Figure 12 and Figure13).  All 
were boosted at around day 56 with a conventional, live attenuated, measles 
vaccine administered in an aerosol via pulmonary inhalation. 

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Regarding safety, the researchers noted that “all vaccine doses were well 
tolerated”.  “No adverse events or complications were observed and the 
young animals showed a steady increase in weight throughout the 
experiment.”   (Normal weight gain in this animal model is a good indicator 
for the absence of illness, discomfort, or complications.)   
 
Figure 12.  Intradermal injection of infant rhesus monkey using Biojector 
spacer.  Source: Center for Vaccine Development, Univ. of Md. 
Figure13.  Desired visible wheal resulting from Biojector intradermal 
injection in rhesus macaque monkey.  Source: Center for Vaccine 
Development, Univ. of Md. 

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Regarding immune response, all monkeys developed good to excellent 
plaque reduction neutralization antibody titers following either the first or 
second intradermal vaccinations, which were further strengthened after the 
aerosol booster.  Upon challenge on day 220 with wild type measles virus, all 
subjects were protected from viremia and symptomatic illness, except for 
signs of rash in one and a few spots in another. 
 
10.4.3.5.7 Rabbits, Monkeys, Malaria Vaccine, Naval Medical 
Research Center 
As preparatory work in advance of planned human trials of a DNA plasmid 
falciparum malaria circumsporozoite vaccine (see section 10.4.3.6.2), Navy 
researchers compared administration on day 0, weeks 4 and 8, and month 7 
by three routes to groups of albino rabbits.Aguiar 2001  These routes were (1) 0.5 
mL (500 μg) vaccinated IM by needle-syringe (N-S), (2) 0.5 mL IM by 
standard no. 2 Biojector needle-free cartridge, (3) 0.35 mL IM by standard 
no. 2 Biojector needle-free cartridge plus three simultaneous doses of 0.05 
mL ID by Biojector cartridge with intradermal spacer, and (4) unvaccinated 
controls.  All groups 1 to 4 also received at month 11 a boost by IM needle-
syringe with ALVAC canarypox vaccine expressing the same protein as the 
DNA prime.   
 
After four doses, the IM Biojector group developed indirect fluorescence 
antibody test (IFAT) geometric mean titers (GMTs) to sporozoite antigen that 
were 3-fold higher than GMTs of the N-S group, and GMTs of the IM/ID 
Biojector group were 8-fold higher.  After the canarypox boost, GMTs were 
18-fold and 16-fold higher in the Biojector groups, respectively, than in the 
N-S group.  The researchers did not report any monitoring of local adverse 
effects of the injections.   
 
The Navy also studied rhesus macaque monkeys undergoing malaria 
vaccination with the Biojector used for both IM and ID injections.Rogers2001, 
Rogers2002  Three groups of four monkeys each received three doses of a 
mixture of four DNA plasmids encoding multiple Plasmodium knowlesi 
antigens, followed by recombinant canarypox boosting.  The groups 
comprised (1) IM via N-S, (2) IM via Biojector, and mixed IM and ID 
injections, all with the Biojector.  In addition, there were tour unimmunized 
controls.  All immunization routes induced antibodies against multiple 
antigens.  Following P. knowlesi challenge, all controls were infected with 
overwhelming parasitemia requiring life-saving treatment, while 2 of 11 
immunized monkeys achieved “sterile” protection, and the remaining 7 
resolved their parasitemias spontaneously.   
 

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10.4.3.5.8 Mice, HIV Vaccine, Karolinska Institute, Sweden 
A DNA vaccine encoding nine HIV protein antigens was administered IM or 
ID with novel adjuvant to BALB/c mice by Biojector system, inducing strong 
HIV env and gag specific cellular and humoral immune responses. Brave2005  A 
subsequent GMP-produced lot of this vaccine intended for human use was 
again administered to mice ID by Biojector and IM with GM-CSF adjuvant 
by needle-syringe, with control animals receiving placebo by both routes.  
Splenocyte interferon γ was produced in both group, although higher in the 
adjuvanted IM group.  No mice showed toxic effects on health status, 
hematology, clinical chemistry, or histopathology.   
 
The authors concluded that this vaccine delivered intradermally by jet 
injector was safe and efficacious in inducing high levels of specific CD8+ T 
cells and antibodies.  The vaccine was subsequently approved by the Swedish 
Medicinal Products Agency for a clinical phase I trial, which began 
vaccination in February 2005 (see section 10.4.3.6.6).   
 
10.4.3.6 Clinical Studies of the Biojector ID Spacer 
A number of completed and ongoing human trials using the investigational 
Biojector spacer have been conducted and found its use safe and tolerable (see 
Figure 15 and Figure 14).  These studies were approved for human subjects by the 
national regulatory authorities of the United States (FDA), the United Kingdom 
(Medicines Control Agency), Sweden (personal communication, Richard Stout, 
Biojector, Inc., 2006), Finland (National Agency for Medicines), and South Africa 
(Medicines Control Council).  Copies of confidential documentation from these 
agencies are available via the manufacturer upon request by ethical review 
committees.  
 
Figure 14.  Intradermal wheal visible on skin after use 
of  Biojector® 2000 intradermal spacer. 
Figure 15.  Biojector® 2000 injector with 
investigational spacer on cartridge nozzle 
administering intradermal injection. 

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10.4.3.6.1 Local anesthesia in adults, University of Illinois 
Anesthesiologists at the University of Illinois Medical Center in Chicago 
used intradermal spacers on two different jet injectors, including the 
Biojector 2000, to anesthetize with lidocaine the dorsum skin of the hand of 
25 consenting adult patients each prior to usually painful intravenous 
catheterization. Zsigmond1999a, Zsigmond1999b  Another 25 patients received control 
anesthesia administered intradermally by 25-gauge needle-syringe.  Patients 
applying visual analog scales (VAS) for pain reported zero pain during the 
lidocaine injections as well as subsequent cannulations in much higher 
proportions for both jet injectors (≥84%) compared with the control needle-
syringe injections (≤24%).   
 
 
10.4.3.6.2 Malaria DNA vaccine in adults, U.S. Navy 
Among 21 human volunteers, investigators at the U.S. Naval Medical 
Research Center carefully described the safety and tolerability of a malaria 
DNA vaccine delivered either IM by needle-syringe, IM alone by Biojector 
2000, and both IM and ID by Biojector 2000 using the investigational 
spacer.Wang2001, Epstein2002  In this trial, five subjects in the third study arm 
received three “identical” ID injections by Biojector in one arm (with ~1 cm 
between each bleb), and a standard IM injection by Biojector in the other arm 
at each of three visits.  Because of their proximity, the blebs were treated as a 
single immunization site and the IM site as a separate immunization site in 
the assessment of local reactogenicity.   
 
The investigators found that:  
• Subjects receiving the Biojector injection (IM or ID) preferred this mode 
of administration relative to their recall of previous needle injected 
vaccinations when questioned at each immunization time point (26 
preferring Bioject versus 3 preferring needle injection).   
• At 6 months, all volunteers indicated that they would choose Biojector 
over needle and syringe for future immunization.  Reported reasons for 
preferring the Biojector included ease of administration, sterility of 
single-use applicators, and less pain associated with injection.  However, 
this was a nonblinded, nonplacebo-controlled comparison and there was 
no comparison group that received an ID injection by needle and syringe.   
• Of the 172 AEs reported by Biojector recipients that were thought to be 
definitely or probably related to immunization, all were localized to the 
injection site except for 3 mild constitutional symptoms.   
• Of these 172 AEs, 169 were mild and 3 were moderate (all 3 moderate 
AEs were in the IM group).  Two of the mild reactions following ID 
injection lasted 10 days or more (bruising). 

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Regarding immune response to the DNA vaccine, none of the vaccinees, 
even from the IM needle-syringe control study arm, developed specific 
antibodies to the malaria antigens, despite positive results from prior animal 
studies in rabbits and monkeys (see section 10.4.3.5.7).   
 
10.4.3.6.3 Influenza in the Frail Elderly, Eastern VA Medical 
Center 
Biedenbender and colleagues implemented an elegant double-blind study 
design in comparing the 2001-2002 season influenza vaccine administered 
IM by N-S (0.5 mL, n = 44) versus ID by Biojector (0.1 mL, n = 45) among 
randomized frail nursing home patients who received a simultaneous 
injection of placebo by the other method and volume into the opposing 
arm.Biedenbender2002  This design permitted each subject to compare the 
perceived pain between the two injections using visual analog and verbal 
pain scales.   
 
The mean age of the subjects receiving vaccine IM by N-S was 83 years, and 
81 years for those vaccinated ID by Biojector (minimum inclusion criteria 
age was 60 years).  Immune responses were significantly poorer in the ID-
vaccinated group, whose hemagglutination inhibition GMTs on day 28 were 
50.7, 14.1, and 23.3 for A/Panama, A/ New Caledonia, and B/Victoria, 
respectively.  These were roughly half the GMTs of 100.5, 28,8, and 52.4, 
respectively among the IM-vaccinated group.  Of the IM-vaccinated group, 
42 of 44 (95%) demonstrated either a 4-fold rise in titer or an HI titer of ≥40 
to all three virus antigens, compared to 31 of 45 (69%) of the ID-vaccinated 
group.   
 
Local reaction frequency rates were not reported.  But redness at the vaccine 
injection site was noted to be significantly more frequent on days 1 (p=0.035) 
and 2 (p=0.009) among those vaccinated ID than IM, while similar redness 
frequencies were reported for the ID and IM placebo injections.  The other 
two local reactions monitored – arm swelling and regional adenopathy – 
were similar between the two vaccination routes.  The pain scores reported 
immediately after vaccination was similar between the ID and IM 
vaccination groups.  But when each patient compared the immediate (day 0) 
pain between his or her two injections, ID injections had less pain than IM 
ones on both the visual (p<0.001) and verbal (p=0.02) pain scales, but there 
was no intrapatient difference by days 1 and 2.  The researchers indicated no 
skin tears or other local trauma from the vaccinations.   
 
Systemic symptoms such as headache, lethargy, nausea, vomiting, malaise, 
fever, and rash were reported in fewer than 7 percent of each study group, 
and none were significantly more common in one than the other.  Outcomes 
in the 60 day followup period included myocardial infarction, leg gangrene, 
hypotension, respiratory distress, rectal bleeding, and death, however these 

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were deemed by the investigators as unrelated to the study and not associated 
statistically with either study arm. 
 
10.4.3.6.4 Plasmid DNA Lymphoma Vaccine, Stanford University 
Excised neoplastic peripheral lymph node(s) of 12 adult patients with B-cell 
lymphomas were used to create patient-specific DNA plasmids expressing 
each patient’s tumor-specific variable region of a chimeric immunoglobulin 
molecule.Timmerman2002  These were administered as vaccines to the patients 
after completion of chemotherapy in three series of vaccinations, each with 
three monthly doses.  The first series used IM needle injections in escalating 
dose amounts of antigen.  Beginning 17 months later for the second series, 
and 14 months thereafter for the third series, the highest dose in series 1 
(1800 µg) was administered by Biojector system, of which 80% was by IM 
cartridge (0.8 mL in one posterior upper arm) and 20% in two adjacent ID-
spacer injections of 0.1 mL in the opposite arm.  In series 3, GM-CSF 
plasmids were also injected.  The researchers carefully observed local and 
systemic reactions for one hour, and patients used a diary card to record 
subsequent reactions.   
 
After vaccination series 2, 9 of 12 patients demonstrated immune responses, 
either humoral (6), T cell (6) or both (3).  After series 3, 7 of the 9 responders 
to series 2 continued to display the measured immune responses, and there 
was one new responder.   
 
The incidence and severity of both the series 2 Biojector IM injections were 
indistinguishable from that experienced after the needle-syringe IM 
injections of series 1.  Similarly, the series 3 injections were 
indistinguishable from the series 2 for local reactions.  The authors wrote, 
“No appreciable local reactions were observed at the sites of ID inoculation” 
and “No systemic toxicity was noted”.    
 
10.4.3.6.5 Colorectal Cancer Vaccine, Oxford Biomedica, UK 
In a trial among patients with colorectal cancer (Duke’s stage D), the 
Biojector system with intradermal spacer was used for six patients receiving 
three doses (1x108 pfu in 0.1 mL at 0, 4, and 8 weeks) of a modified vaccinia 
Ankara (MVA) vector expressing tumor-associated antigen 5T4 (TroVax®), 
and compared with 16 patients receiving the same antigen intramuscularly by 
needle-syringe in dose escalation (5x107 to 5x108 pfu).Harrop2003, HarropMs, 
Reinis2004  If clinical or immunological response was detected, two more doses 
at 14 and 20 weeks were offered.   
 
Most patients showed proliferative responses to the protein or peptide 5T4 
antigen, although responses tended to be greater and less transient in the IM 
than ID groups.  The most commonly reported treatment-related adverse 
events were non serious, local ones relating to the injection site.  There was a 

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notably higher incidence of injection site erythema in the intradermal 
injection group (3 of 6, 50%).   
 
10.4.3.6.6 Adult Volunteers, HIV vaccine, Karolinska Institute 
At the Karolinska Institute in Stockholm, Sweden in February, 2005, the first 
human volunteers began receiving vaccination in an ongoing HIV vaccine 
trial using a multigene DNA vaccine (animal studies described earlier in 
section 10.4.3.5.8).Brave2005   
 
10.4.4 Method of Assigning Participants to Treatment Groups 
For each phase of the study (I and II), an independent, outside party (e.g., a member of 
the Data and Safety Monitoring Board - DSMB) will determine in advance the injection 
method to be used for each participant identification (ID) number to be assigned in the 
study: 
 
 
•  IM-NS-0.25 (control) 
(intramuscular, needle-syringe, full dose) 
 
•  IM-NS-0.1  
 
(intramuscular, needle-syringe, reduced dose) 
 
•  ID-JI-0.1 
 
 
(intradermal, jet injector, reduced dose) 
 
The outside party shall provide copies of these treatment group assignments for each 
study ID number to at least two [other] members of the DSMB.  These records shall 
remain confidentially in their custody in separate locations outside of the Dominican 
Republic and released only at the time and in the manner as described in this protocol. 
 
A study nurse will assign permanent participant ID numbers consecutively to each 
patient in the chronological order in which the corresponding informed consent form is 
signed and he or she is enrolled.   
 
10.4.4.1 Equal Allocation by Sextuplet Blocks 
Treatment group assignments will be made by generating a series of “sextuplet” 
blocks of 6.  For each block, the independent party will assign in random order two 
injections from each of the three study arms to a randomly selected position (1st, 
2nd, 3rd, 4th, 5th, or 6th) within the block.  Thus, there will be 36 (3! x 3!) possible 
orders for any given block of six, as illustrated in Table 4.   
 
Table 4.  Examples of random sextuplet blocks for allocating samples 
Sequence Position 
 
1st 
2nd 
3rd 
4th 
5th 
6th 
A. 
IM-NS-0.25 
IM-NS-0.25 
IM-NS-0.1 
IM-NS-0.1 
ID-JI-0.1 
ID-JI-0.1 
B. 
IM-NS-0.25 
IM-NS-0.1 
IM-NS-0.25 
IM-NS-0.1 
ID-JI-0.1 
ID-JI-0.1 
C. 
ID-JI-0.1 
IM-NS-0.1 
IM-NS-0.1 
ID-JI-0.1 
IM-NS-0.25 
IM-NS-0.25 
D. 
IM-NS-0.1 
IM-NS-0.25 
IM-NS-0.25 
ID-JI-0.1 
ID-JI-0.1 
IM-NS-0.1 
Sample 
 
Blocks 
etc. 
 
 
 
 
 
 

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For combined phases I and II, a total of 80 blocks will be prepared (480 
assignments), 30 more than the target sample size (n=450), in case of dropouts or 
other contingency.  This system will provide for equal or nearly equal sizes of each 
study group throughout the enrollment process.   
 
10.4.4.2 Avoiding Block Counting 
To prevent “block counting” to predict the route of vaccination for a child, the first 
block will be split randomly into two parts (1+5, 2+4, 3+3, 4+2, or 5+1), with its 
first segment applicable to the initial participant ID numbers, and its remainder 
applied to the final ID numbers.  
 
For example, if block 1 were randomly determined to follow sample sequence A in 
Table 4 above, and is then randomly split into “1 + 5”, then participants would be 
assigned their injection route as follows: 
 
      Last 3 digits are  
Participant ID no.* ↓            
 
Vaccination Group 
 CDC-ISO-4785-001   
 
IM-NS-0.25  (as per block 1, example A) 
 CDC-ISO-4785-002 – 475 
 
(in accordance with blocks 2 through 79) 
 CDC-ISO-4785-476  
 
IM-NS-0.25  (as per block 1, example A) 
 CDC-ISO-4785-477  
 
IM-NS-0.1  (as per block 1, example A) 
 CDC-ISO-4785-478  
 
IM-NS-0.1  (as per block 1, example A) 
 CDC-ISO-4785-479  
 
ID-JI-0.1  (as per block 1, example A) 
 CDC-ISO-4785-480  
 
ID-JI-0.1  (as per block 1, example A) 
 
 
Similarly, if block 1 were randomized to follow sequence D in Table 4 above, and 
is randomly split 4+2, then participants would be vaccinated as follows: 
 
Participant ID no.* ↓            
 
Vaccination Group 
 CDC-ISO-4785-001   
 
IM-NS-0.1  (as per block 1, example D) 
 CDC-ISO-4785-002  
 
IM-NS-0.25  (as per block 1, example D) 
 CDC-ISO-4785-003  
 
IM-NS-0.25  (as per block 1, example D) 
 CDC-ISO-4785-004  
 
ID-JI-0.1  (as per block 1, example D) 
 CDC-ISO-4785-005 – 478 
 
(in accordance with blocks 2 through 79) 
 CDC-ISO-4785-479  
 
ID-JI-0.1  (as per block 1, example D) 
 CDC-ISO-4785-480  
 
IM-NS-0.1  (as per block 1, example D) 
 
 
10.4.4.3 Allocation Group Randomization Cards 
The study investigators shall provide the outside, independent party with sufficient 
numbers of allocation group randomization cards, samples of which are pictured in 
Figure 16 through Figure 19 below. 

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The text of the mock injection card to avoid the extravaccination of a 3rd 
“insurance” dose on day 56 (Figure 19, section) translates as follows in English:  
 
Do NOT administer vaccine to this child. 
Apply a bandage even though there is no injection 
• Keep the child in the injection room por the time it usually takes to 
give an injection. 
• Do not inform the parents or doctors that no injection was given. 
• Revealing this “mock injection” can compromised the blinding of the 
study. 
• Destroy this card before inviting the parents to enter or yourself 
leaving the room.  
Apply a bandage in the place where an 
injection would be given: in the left thigh for 
children <12 months of age.  In the left deltoid 
for children 12 months and older. 
 
 
 
 
 
Figure 16.  Randomization card for “IM-NS-0.25” group.  
 
 

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Figure 17.  Randomization card for “IM-NS-0.1” group. 
 
 
 
Figure 18.  Randomization card for "ID-JI-0.1" group. 
 

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Figure 19.  Card for mock 3rd “insurance” injection for group “IM-NS-0.25". 
 
 
The outside, independent party who randomly determined the treatment group 
assignments, as described above, shall insert the cards in accordance with the 
randomization results into corresponding participant-ID-numbered allocation 
group randomization envelopes (see below).  A peel-off “confirmation sticker” 
with the participant’s study ID number and dose (1 = “A”, 2 = “B”, 3 = “C”) will 
be affixed to the face of each card for attachment to the CRF to verify accuracy in 
administering the correct type of injection.   
 
10.4.4.4 Allocation Group Randomization Envelopes 
The outside, independent party shall prepare four (quadruplicate) sets of allocation 
group randomization envelopes.  The envelopes shall be sealed, opaque, and not 
permit visualization of its contents upon holding up to the light.  For each set, 
consecutive participant ID numbers shall be written, printed, or stickered clearly on 
the outside of each envelope, e.g., “CDC-ISO-4785 – 001”, “CDC-ISO-4785 – 
002”, etc.   
 
Three sets of envelopes, the GREEN, YELLOW and RED sets shall be provided to 
the Dominican Principal Investigator for use in the study injection room for the 
FIRST, SECOND, and THIRD study injections, respectively.  They shall be 
marked on their covers “1 - Primera”, “2 - Segunda”, and “3 - Tercera”, 

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respectively.  (Envelopes for the RED set for the “insurance” dose will contain the 
IM full-dose randomization card shown in Figure 16 for the two reduced-dose 
groups IM-NS-0.1 and ID-JI-0.1.  RED envelopes for the full-dose group, IM-NS-
0.25, having already received the standard 2-dose regimen, will contain a card 
indicating a mock injection, as shown in Figure 19.).   
 
A fourth set of envelopes, the BLUE set, similarly identified on their outside by the 
participant’s study ID number, shall be kept sealed in a secure location (e.g., locked 
safe) in the custody of the director of the HIRRC.  The individual BLUE envelope 
for a specific participant shall be opened only upon the application of the 
Dominican Principal or Senior Investigators in the event of a serious adverse 
reaction for which treatment will depend upon knowledge of the vaccination 
method (IM-NS-0.25, IM-NS-0.1, or ID-JI-0.1) received by that participant.   
 
The allocation group randomization envelope shall be opened by the unblinded 
injection nurse only when she and the other unblinded study staff (holding 
assistant) are all in the injection room with the participant of corresponding ID 
number, and his or her parents have left the room temporarily.  
 
After preparing the proper injection equipment with the proper dose of vaccine, the 
two staff shall confirm that the delivery device is the same as indicated on the card, 
its peel-off “confirmation sticker” shall be affixed to the designated box on the 
corresponding vaccination page in the CRF (parts 7, 11, or 15), and then the card 
shall be destroyed and disposed in a locked trash receptacle in the same cubicle.  
After the vaccination, the device used to administer it shall be removed from view 
and the participant parent invited into the cubicle to sit down and console the child.   
 
10.4.5 Timing of Doses for Each Participant 
Upon obtaining informed consent from participants, all participants will receive a dose 
of influenza vaccine at day 0 (zero) by one of the three dosages and routes of 
administration (see section 10.1.1).  After followup return visits in person to assess 
adverse reactions scheduled on days 2 and 7, each participant will return for a second 
dose of vaccine on day 28 (4 weeks), which will be followed again with return visits for 
adverse reactions on days 30 and 35.  A final visit on day 56 (8 weeks) will be 
scheduled for the third and final blood draw and an “insurance” dose of full-strength 
vaccine administered by conventional needle-syringe.  The days listed above for 
followup visits and actions have various margins of acceptable deviation, as listed in 
Table 6 in section 10.6.1.   
 
10.4.6 Blinding 
The study will be observer-blinded.  The study investigators, plus the study clinical staff 
who will assess and record adverse reactions, the laboratory personnel who will process 
and perform serological assays on the specimens, as well as the participant’s parent(s) 
will all be unaware of the study group into which the participant is assigned.  Only the 

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unblinded injection nurse and holding assistant will observe the method of injection.  
None of these unblinded staff will be involved in assessing or recording adverse events 
in the Case Report or other forms.  Parents will be required to remain outside the 
injection room during the 1 to 2 minutes necessary to carry out the vaccination.  The 
allocation group randomization cards (see 10.4.4.3) will be destroyed and discarded at 
the time when the device is filled for the associated participant.  No written record of 
vaccination dose or route will be maintained in the study clinic.  
 
10.4.6.1 Premature “Breaking” of the Allocation Code 
The coding list maintained by the independent outside party and other member(s) 
of the DSMB (see section 10.4.4) will not be divulged to anyone outside the 
DSMB, except at the conclusion of the clinical and laboratory aspects of each phase 
of the study, or on an emergency basis upon request from the U.S. or Dominican 
Principal Investigators.   
 
In case of emergency requiring immediate knowledge of the method of influenza 
vaccination for a particular study participant in order to provide appropriate 
treatment or care, and upon request by the Dominican Principal or Senior 
Investigator, the blue envelope corresponding to the child’s study identification 
number shall be produced by the hospital director and opened.  Such an occurrence 
shall be reported to the U.S. Principal Investigator.   
 
10.4.6.2 Separation of Parent from Child during Injection  
As the careful, credible assessment of adverse events are important for scientific 
credibility, which determines its usefulness for public health policymakers, it is 
necessary to ensure the parents are blinded to the treatment group of their child 
until after the study is completed.  Observations and reports by the parents are a 
significant component of the study.  If parents are aware of the vaccination method 
used, their reports my be subconsciously biased, and they may inadvertently 
unblind the investigators through a casual comment or question, further 
undermining objectivity of the study findings.   
 
Unlike other vaccine trials where the investigational product and the control 
product or placebo are both administered in a syringe, in this study the method of 
administration itself is under investigation and not easily concealed.  The 
psychological discomfort of both child and parent that may be caused by the brief 
1-to-2 minute separation are deemed an unfortunate but necessary requirement for 
the study, for which the parents will be thoroughly informed in advance and their 
voluntary consent required to participate.  Alternative blinding methods that might 
have permitted the parent to remain in the injection room with the child were 
considered, but deemed unsatisfactory:  
 
•  Multiple injections on each visit.  This method has been used in prior studies by 
others, in which every patient gets injected on every visit with both methods of 

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injection, only one of which contains the drug of interest, and the other saline 
placebo.  In our study, this would require administration of a needle-syringe IM 
(intramuscular) injection and a jet injector ID injection for every injection visit.  An 
unblinded pharmacist or other health worker outside the parent’s and investigator’s 
view would fill only one of these with the influenza vaccine, according to the 
randomization group, and the other device with a placebo of similar appearance.  
 
Although perfectly controlled and thus valid from a scientific perspective, this 
method of blinding was rejected as causing twice as much psychological and 
physical discomfort than otherwise needed to satisfactorily answer the research 
questions.   
 
•  Blindfolding and earmuffing the parent in the injection room.  Blindfolding 
the parent, who could be visible to the child in the room, would not be sufficient as 
a blinding method, as the jet injector makes a “poof” of noise.  Masking that sound 
with electrically-powered, noise-cancelling headphones playing masking music 
into the parent’s ears was considered.  Alternatively, a simultaneous jet injector 
could be fired into the air by a third unblinded health worker, so that all parents 
heard the noise, regardless of study group allocation.   
 
Blindfolding (with or without earmuffing) was rejected as demeaning to the parent 
and potentially frightening to the child.  Simultaneous firing of a jet injector for 
those receiving needle injections was rejected as (1) labor-intensive, (2) potentially 
ineffective for blinding if needle injection (and patient recoil) and jet firing are not 
exactly simultaneous, (3) time-consuming, and (4) prone to medical error, such as 
inadvertent confusion of a jet cartridge filled with tap water for a mock injection 
into the air with a cartridge to be used to administer actual vaccine to a child in the 
ID-JI-0.1 group.   
 
Standard practice in the Dominican Republic is for parents to be absent during 
certain pediatric procedures, particularly delicate or painful ones, such as 
phlebotomy and tympanocentesis, for which informed-consent otitis media studies 
are now being performed with parents absent from the treatment room.   
 
Beyond the need for blinding, other reasons to avoid the parent holding the child 
during the injection have been provided by the U.S. Naval Medical Research 
Center (NMRC) pediatrician, IRB member, and principal investigator of a prior 
study of the Biojector with investigational ID spacer, Dr. Judith Epstein.Epstein2002   
Since the sound of the injector might cause the parent to release firm hold on the 
child, it was suggested that the child should be restrained instead by a trained nurse 
aware of the manufacturer’s instructions that the limb and injection not move in 
relation to each other during the 1/3 second that the injection requires, to avoid any 
risk of laceration.   
 
Other informed-consent trials with parents excluded from the vaccination room 
have been performed when the investigational vaccine could not otherwise be 

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blinded.  These included a recent trial of the intranasal FluMist® influenza vaccine 
squirted into the nose, compared to an injected vaccine.King2006  Other influenza 
vaccine studies have been done in children without parents present for the 
vaccination, and with such absence duly consented in advance.Khan1996, Rudenko1993, 
Tanaka1993 
 
The consent form advises the parents of the need to be separated from the child, 
just outside the door of the injection room, for the estimated 1 to 2 minutes to 
prepare and administer each of the first three study injections.  The consent form 
permits them the opportunity freely to withdraw consent in advance of the study 
(and thus not to enroll), or to withdraw at any time during the study.   
 
Phlebotomy.  The presence or absence of the parents during the required three 
blood collections in this study would not affect blinding of the study.  In keeping 
with common pediatric practice in the Dominican Republic and in most U.S. 
medical facilities, the parents will be requested, for their own peace of mind, to 
remain outside the room where phlebotomy and similar uncomfortable or 
frightening diagnostic (e.g., lumbar puncture) or therapeutic (e.g., tympanocentesis, 
surgery) treatments are performed.  However, in this study, if the parents request to 
be present during phlebotomy, they will be permitted to do so, and this option is 
mentioned in the consent form. 
 
10.4.6.3 Caching of Immediate Injection Site Observations by Nurses 
To prevent unblinding of the physician investigators to the method of injection 
used, the immediate reactions observed by the unblinded injection nurses at the 
injection site (e.g., drop or flow of blood or wetness) for investigational doses 1 
(Part 8, questions 1 to 8) and 2 (Part 11, questions 1 to 8) will be noted on separate 
colored pages (green 12enf and yellow 16enf, respectively) of the Case Report 
Form.  For reasons of convenience, onto these removable colored pages will be 
moved other data items also completed by the nurses for dose 1 on page 11 (Part 7, 
questions 7 to 11) and for dose 2 on page 15 (Part 11, questions 7 to 11).  For the 
injection, the nurses will separate and complete these pages and place them in a 
closed file in the injection room.  After the investigational period of observation 
after dose 2, and after the physician investigator has entered all related assessments 
into the C.R.F. for a particular participant, these colored pages will be retrieved for 
insertion back into the rest of the C.R.F. and copied for forwarding to the CDC 
investigators for data entry and analysis. 
 
10.4.7 Prior and Concomitant Therapy - Other Vaccinations 
No other routine or investigational vaccines will be administered within 28 days before 
or after either of the study’s investigational INF doses 1 or 2.  As per the study 
exclusion criteria (see paragraph i. in section 10.3.2), otherwise eligible participants will 
not be recruited if they have received in the prior 28 days any routinely recommended 
vaccinations of the current Dominican Republic public immunization policy, or are due 

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to receive them in the following 56 days.  The recommended Dominican immunization 
schedule indicates vaccines at 6 months (POLOPV, DTPw-HBV-HIB), 12 months 
(MMR), and 18 months (POLOPV, DTPw-HBV-HIB) of age.SESPAS   
 
This restriction thus excludes infants “behind schedule” and needing to receive on the 
day of first interview protection against tuberculosis, polio, diphtheria, tetanus, 
pertussis, hepatitis B, Haemophilus influenzae type b, measles, mumps, or rubella.  It 
also creates three gaps in the intended ≥ 6 -to- <24 months age range of recruited 
participants for this study.  The first gap would be ages <7 months (unless the 
participant received his or her third POLOPV and DTPw-HBV-HIB doses at ≤5 
months); the second gap from >10 -to- <13 months; and the third gap from >16 -to- <19 
months.   
 
If, in the opinion of the Dominican investigators, a child may benefit by immediately 
receiving another vaccine during the 28-day-period after either of the study’s influenza 
vaccine doses (e.g., because of exposure to meningitis or another vaccine-preventable 
disease), such other vaccines should be administered by the appropriate authority and 
duly noted on the Case Report Form, part 20.   
 
10.4.8 Treatment Compliance and Participant Incentives 
Adherence of study participants to the vaccination and visit schedule will be supervised 
by the study nurses and senior clinical staff.  A total of 8 visits are planned, on days 0, 
2, 7, 28 (4 weeks), 30, 35, 56 (8 weeks), and 8 months (see Table 5 in section 10.6.1 for 
details).   
 
Parents will be encouraged to return with their infant, diary form, and related items for 
scheduled clinic visits through the provision of modest incentives, which may include: 
 
 
•  round trip taxi fares from and to home 
 
•  voucher for lunch or snack in the hospital snack bar 
 
•  folder and pencils for storing and marking take-home materials (diary form, 
measuring tape and template, adhesive thermometers)  
 
For those children who do not return to the clinic as scheduled, study health workers 
will collect the information by telephone, reschedule the visit, and/or make home visits 
on the days following the missed scheduled visit.   
 

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10.5 
Efficacy and Safety Variables 
10.5.1 Efficacy 
10.5.1.1 Immunogenicity 
In this trial, the surrogate for measuring actual field efficacy (protection from actual 
disease through natural exposure to wild virus) will be the immune responses 
generally considered predictive of such protection.   
 
10.5.1.2 Primary Efficacy Variable 
Seroconversion (SC) is the primary efficacy variable.  It is defined as the 
development of an inverse hemagglutination inhibition (HI) titer of ≥40 on 
followup serum collected one month after the second vaccine dose among 
initially-seronegative participants (undetectable inverse titer of <8 on baseline day 
0); OR, for participants with a detectable baseline titer, the development of a 
followup HI titer which rises ≥4-fold.  (Usually analyzed as a proportion among 
participants.)   
 
10.5.1.3 Secondary Efficacy Variables 
Secondary efficacy variables are:   
 
Seroprotection (SP), defined as the development of an inverse HI titer of ≥40 on 
followup serum collected one month after the second vaccine dose, regardless of 
evidence of immunity at baseline.  (Usually analyzed as a proportion among 
participants.) 
 
Mean geometric increase, defined as the ratio of the geometric mean titers 
(GMT) of all participants on followup HI assay divided by the GMT of HI assay 
on the same participants at baseline (excludes participants with baseline titers lost 
to followup).  (For example, a mean baseline inverse GMT of 18 and followup of 
81 would have a mean geometric increase of 4.5.) 
 
Dose 1 seroconversion, defined according to the seroconversion criteria above 
one month after only the first dose of influenza vaccine, before receiving dose 2. 
 
Dose 1 seroprotection, defined according to the seroprotection criteria above one 
month after only the first dose of influenza vaccine, before receiving dose 2. 
 
Dose 1 mean geometric increase, defined according to the mean geometric 
increase calculation above after only the first dose of influenza vaccine, before 
receiving dose 2. 
 

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10.5.1.4 Subsequent Specimen Storage and Laboratory Assay(s) 
Upon separate written signature in the informed consent form, leftover serum not 
needed for study assays may be retained in indefinite long-term storage.  Such 
consent may be provided or declined without affecting consent for participation in 
the study.   In accordance with the informed consent form, and at the discretion of 
the sponsor and upon identification of required resources and interest, any such 
serum remaining from the study after completion of the testing described above 
may be tested by other laboratories by additional assays for purposes of comparison 
and correlation with the results obtained in the laboratory testing described herein.   
 
10.5.2 Safety 
Participants will be observed and followed prospectively after both dose 1 and dose 2 of 
the investigational INF vaccinations for the occurrence, frequency, and severity of 
immediate and delayed local and systemic reactions and other adverse events.   
 
10.5.2.1 Local and Systemic Reactions 
Reactogenicity will be assessed from clinical staff and parental observation, 
physical examination, and measurement of immediate and delayed local reactions 
at the sites of injection (i.e., drop or flow of blood, tenderness, erythema, swelling, 
hematoma, induration, warmth, nodule, crying upon movement of limb, limb 
swelling, limb circumference at midpoint) and of immediate and delayed systemic 
reactions (i.e., anaphylaxis, fever, sleepiness, irritability, unusual or inconsolable 
crying, vomiting, diarrhea, convulsions, change in eating habits,), among non-
prompted other adverse events to be solicited.   
 
Temperature.  Axillary temperatures will be measured and recorded by nursing 
staff and parents from reusable, disposable, 2-day-wearable adhesive thermometers 
(TraxIt® McKenzie2003, MedicalIndicators2004 
http://www.medicalindicators.com/html/traxit.html) at clinic visits and at home.  
The TraxIt® will be applied by the nurse in one axilla on day 0, and replaced by the 
parent with a new one in the opposite axilla on days 2, 4, and 6.  Parents will be 
trained upon recruitment in daily reading of the thermometer from days 0 through 
7, and to record the temperatures, as well as any use of analgesic/antipyretic 
medication and other items prompted on the diary card.  An additional TraxIt™ 
will be provided to the parents for use to record temperatures, as needed for 
suspected fever or illness which may occur on days 9 through 27 after each 
investigational vaccination.  
 
Local Reaction Size Measurements.  Parents will be trained upon recruitment in 
(1) using supplied plastic template cards to measure the largest diameters of any 
erythema and induration for the vaccine injection site in the left thigh (age <12 
months) or left deltoid (age ≥12 months), (2) using supplied [infant head] 
circumference tapes to measure the circumference of the thigh or upper arm at its 

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midpoint, and (3) filling out diary cards to record these and other prompted and 
unprompted local and systemic reactions.  During clinic visit interviews and 
telephone followup or home visits, as needed, nurses will assist parents in 
completing the diary cards.   
 
Photography of Injection Site.  On days 2 and 7 after vaccination, the injection 
site will be photographed in color with a digital camera.  The field of view will 
include a scale to assess reaction sizes, and a label with the participant’s 
identification number, initials, and the current date.  In case of unexpected or 
unusual local reaction during the 30-60 minute period after vaccination, such as 
frank bleeding or laceration, the injection nurses will notify the physician 
investigator, who will photograph it (after first initiating medical treatment, if 
necessary, and subsequently entering details in the adverse events section of the 
Case Report Form).  Any visible local reactions persisting on day 28 after 
vaccination will be photographed, as well.  In addition, upon recruitment and 
signed consent, a digital photo will be taken of the mother and participant for 
pasting into the Case Report Form to assist in preventing entries into the wrong 
form.  For privacy protection, this photo will be removed before the CRF is copied 
for archival purposes at the study site and then mailed to the sponsor.  
 
10.5.2.2 Safety Variables and Monitoring 
10.5.2.2.1 Definitions 
Definitions and classifications for specific adverse events to be reported from 
this study will conform, as much as practicable, with those in draft development 
or finalized by the Brighton Collaboration (http://brightoncollaboration.org).   
 
•  An adverse event (AE) is defined as any untoward medical occurrence in 
a clinical investigation subject administered a pharmaceutical product, 
at any dose, or placebo.  It does not necessarily require a causal 
relationship with this treatment.  An AE can therefore be any 
unfavorable and unintended sign (including abnormal laboratory 
findings), symptom, or disease temporally associated with the use of a 
medicinal product, whether or not considered related to the medicinal 
product.  This definition includes intercurrent illnesses or injuries, and 
exacerbation of pre-existing conditions.   
 
•  An expected AE is one previously known or anticipated as a result of 
(a) the interventions and interactions used in the research,  (b) the 
collection of identifiable private information for the research;  (c) 
an underlying disease, disorder, or condition of the human subject; 
and/or  (d) other circumstances unrelated to the research or any 
underlying disease, disorder, or condition of the subject.  
 
•  An unanticipated AE is an unexpected occurrence or frequency of a type 
not previously known or commonly identified to result from (a) the 

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interventions and interactions used in the research; (b) the collection 
of identifiable private information under the research; (c) an 
underlying disease, disorder, or condition of the human subject; 
and/or (d) other circumstances unrelated to the research or to any 
underlying disease, disorder, or condition of the subject.  
 
•  An unanticipated problem that does not result in an AE is an 
occurrence that otherwise may have increased the risk of physical, 
emotional, social, or legal harm to the participant, when no actual 
harm was detected.  Examples include (1) breaches in 
confidentiality if participant identifying information is improperly 
disseminated, lost, or stolen; (2) administration of an expired, 
recalled, or improper dose (too little, too much) of either 
investigational or routine medication; (3) exposure of the 
participant or family member to an active tuberculosis patient while 
in the study waiting room.   
 
10.5.2.2.2 Severity 
The severity of events reported on the AE Case Report Form (CRF) will be 
classified by the investigators as follows:  
 
•  Mild:   An adverse experience that the subject tolerates easily, causing a 
minimum of uncomfortability that does not interfere with 
daily activities.  
•  Moderate:   An adverse experience of sufficient uncomfortability to 
interfere with normal daily activities.  
•  Severe:   An adverse experience that prevents normal daily activities.  In a 
young child, such adverse effect might, for example, impede 
attendance at school/pre-school/day care, and/or cause 
parents/teachers to seek medical attention.  
•  Life-threatening:   An adverse experience with immediate risk of death.  
 
10.5.2.2.3 Seriousness 
A Serious Adverse Event is defined as any untoward medical occurrence 
which… : 
•  Results in death.  
•  Is life threatening (i.e., the subject was, in the opinion of the investigator, 
at immediate risk of death from the event as it occurred).  
•  Requires or prolongs inpatient hospitalization.  
•  Results in persistent or significant disability or incapacity, (i.e., the event 
causes a substantial disruption of a person’s ability to conduct normal 
life functions).  
•  Is a congenital anomaly or birth defect.  
•  Is an important and significant medical event that, based upon appropriate 
medical judgment, may jeopardize the subject and may require 

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medical or surgical intervention to prevent one of the other outcomes 
defining serious. 1 
_____________ 
1.  Medical and scientific judgment should be exercised in deciding whether an adverse 
event not satisfying any of the first five bullets above is "important and significant" 
and thus should be classified as a Serious Adverse Event (SAE) and reported by 
expedited method.   Examples of such events that would usually be considered 
important and significant include (a) allergic bronchospasm requiring intensive 
treatment in an emergency room or at home, (b) bleeding disorders or (c) convulsions 
that may not result in inpatient hospitalization, (d) the development of drug 
dependency or drug abuse, or (e) a diagnosis of cancer.   Examples of medical events 
that would usually NOT be considered SAEs, if deemed unrelated to the vaccine, 
which resolve upon standard treatment with drugs or other therapies, and which do 
not result in any of the outcomes described in the first five bullets of the SAE 
definition above, would usually include common acute childhood conditions such as 
(f) otitis media, (g) upper respiratory infection, (h) influenza and similar illness, (i) 
mild reactive airway disease or asthma, (j) diarrhea, (k) impetigo, and (l) intestinal 
parasitosis.  All such events not considered SAEs would still be reported routinely as 
adverse events (AEs) on the Case Report Form. 
 
It should be noted that a severe Adverse Event need not be serious in nature 
and that a serious Adverse Event need not, by definition, be severe.  
 
10.5.2.2.4 Relationship to Vaccine 
The relationship of the study treatment to an AE will be determined by the 
investigator based on the following definitions:  
 
•  Not related  -  The AE was definitely not caused by administration of 
the study vaccine(s).  
 
•  Possibly Related  -  The administration of the study vaccine(s) and the 
adverse experience are reasonably related in time AND the 
adverse experience could equally be explained by causes 
other than exposure to the vaccine(s).   
 
•  Probably Related  -  The study treatment and the AE occurrence were 
reasonably related in time, and the AE was more likely 
explained by exposure to the study product than by other 
causes.  
 
10.5.2.2.5 Reporting 
Any serious AE, whether or not considered study related, will be reported 
immediately (within 24 hours) to the U.S. PI.  The individuals to be contacted at 
CDC for notification of a serious AE are the U.S. PI and co-investigator 
identified in sections 8.1.1 and 8.1.2 above, who will in turn notify the U.S. IRB 
and provide courtesy notification to the vaccine manufacturer.   
 

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Any serious or unanticipated AE must also be reported to the Dominican IRB 
in a timely manner.  Adequate documentation must be provided to CDC 
showing that the IRB has been properly notified.  Any medication or other 
therapeutic measures used to treat the event will be recorded on the appropriate 
case report form (CRF) page(s) in addition to the outcome of the AE.   
 
10.5.2.2.6 Premature Discontinuation of Participants; Followup 
A genuine effort must be made to determine the reason(s) why a subject fails 
to return for the scheduled visits.  If the subject’s parent/guardian is 
unreachable by telephone, a study staff member should be sent to find him or 
her and encourage contact with the clinic.  This information should be 
recorded in the appropriate section(s) of the Case Report Form, including Part 
20 – Comments.  
 
AEs will be monitored until resolution or, if the AE is determined to be 
chronic, a cause is identified.  If an AE remains unresolved at the conclusion 
of the study, a clinical assessment will be made by the investigator whether 
continued follow-up of the AE is warranted.  
 
10.5.2.3 Determination of Followup Period for Adverse Events 
For influenza vaccination, 28 days of followup is relatively conservative and 
would catch all but the rarest of complications.  The common local and system 
reactions following influenza vaccination occur within the first two days.CDC2005 
The day 2 visit was included particularly to permit investigator observation of 
such reactions.  Nearly all would occur by the day 7 visit, and certainly by the 
day 28 visit.   
 
As noted in the informed consent form, a very, very rare complication of 
influenza vaccination is the nervous system Guillain-Barré syndrome.  Section 
10.4.2.7 discusses the risk of GBS in more detail and its usual window of onset 
for it after influenza vaccination. 
 
The 28 days of followup after the each investigational doses 1 and 2 would pick 
up the great majority of this extremely rare condition.Schonberger1979, Haber2004, 
Lasky1998  Moreover, with the “bonus” 4th influenza vaccination 6 months after 
the “insurance” dose 3, virtually all GBS which might rarely occur as a result of 
the first 2 or 3 doses of the study would come to the investigator’s attention.   
 
The Completion Certificate and Bonus Vaccination Reminder for the parents 
upon their child’s graduation from the study (on day 56 at the time of the 3rd 
“insurance” dose) reminds them to bring the child back 6 months later for the 
“bonus” dose for next influenza season, as well as to contact the doctors for any 
unusual illness or condition which may occur in the child before that revisit.   
 

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10.5.3 Appropriateness of measurements 
The measures of efficacy to be used in this study are standard, i.e., widely used and 
generally recognized as reliable, accurate, and relevant (able to discriminate between 
effective and ineffective agents).  The measures of safety used in this study are routine 
clinical and laboratory procedures  
 
10.5.4 Pharmacokinetics 
No pharmacokinetic studies are intended in this project, as they are not relevant to most 
vaccine research.   
 
10.6 
Study Procedures 
10.6.1 Time and Events Table 
The sequence and chronology for conducting clinical and diagnostic activities is 
summarized in Table 5.  Allowable variations from the planned schedule of followup 
visits are detailed in Table 6.   
 
Table 5.  Time and events  
days* 
Procedure /  Test 
0 
2 
7 
28 
30 
35 
56 56d + 6
months
SCREENING – recruitment, informed consent, medical 
history  
9 
 
 
 
 
 
 
 
EXAM – weight, height, limb circumference, 
temperature, local/systemic reactions 
9 
9 
9 
9 
9 
9 
9 
 
BLOOD SAMPLE for hemagglutination inhibition 
serologies 
9 
 
 
9 
 
 
9 
 
VACCINATION with INF vaccine by blinded dose and 
route 
9 
 
 
9 
 
 
 
 
PHOTOGRAPH of INF injection site, closeup with 
scale 
‡ 
9 
9 
‡ 
9 
9 
 
 
REVIEW and discuss parent diaries; collect completed 
ones 
 
9 
9 
9 
9 
9 
9 
 
“Insurance” VACCINATION 0.25 mL INF IM by N-S† 
 
 
 
 
 
 
9 
 
“Bonus” booster VACCINATION 0.25 mL INF IM by 
N-S; inquiry on intervening adverse events 
 
 
 
 
 
 
 
9 
*  See next table for acceptable windows for scheduled visit and followup dates. 
†  Groups IM-NS-0.1 and ID-JI-0.1 receive full dose.  Group IM-NS-0.25 receives mock injection.  
‡  Photography only if any injury or persistent local reaction is visible to be photographed.   
 
 
Table 6.  Acceptable windows for followup visits 
 
Intended day 
since 1st 
Day to be 
scheduled 
Permitted 
variation 
Earliest 
permitted 
Latest permitted day 
Latest 
weeks 

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Visit 
no. 
vaccination 
since last 
vaccination
from 
scheduled 
day 
day since 
1st vacc. 
since  
1st vacc. 
since  
2nd vacc. 
since 1st 
vacc. 
1 
“Day 0” 
0 
 
 
 
 
 
2 
“Day 2” 
+2 
+/- 1 day 
1 
3 
 
0.4 
3 
“Day 7” 
+7 
+/- 1 day 
6 
8 
 
1.1 
4 
“Day 28” 
+28 
+  14 days 
28 
42 
 
6.0 
5 
“Day 30” 
real 2nd-
dose day 
+2 
+/- 1 day 
29 
45 * 
3 
6.4 * 
6 
“Day 35” 
real 2nd-
dose day 
+7 
+/- 1 day 
34 
50 * 
8 
7.1 * 
7 
“Day 56” 
real 2nd-
dose day 
+28 
+  28 days 
56 
98 * 
56 
14 * 
8 
8 months 
+6 months 
+3 months 
8 months 
98 +  
9 months 
56 +  
9 months 
53 * 
* Assumes 2nd vaccination at latest permissible day (42) since 1st vaccination. 
 
10.6.2 Training of Study Personnel 
Training for Dominican study personnel will be conducted in two stages lasting one 
week each.  In addition, U.S. investigators and trainers from the jet injector 
manufacturer will be present during relevant periods of this training.  Dominican study 
staff include 5 registered nurses, 3 auxiliary nurses, 3 physicians, one resident in 
pediatrics, one data/files manager, one laboratory professional, one laboratory 
technician, and 3 auxiliary nurses.  
 
10.6.2.1 Stage 1: Logistics and Practice 
Monday in the first training week will be devoted to study logistics, making sure 
that all the necessary equipment and materials are in place and functioning, and 
telecommunications are working.  Principal and senior investigators and co-
investigators will inspect each physical location to verify all study materials and 
equipment all are in place, each is functional, and ready to start the study (See 
Table 7).   
 
Table 7.  Required Study Supplies and Equipment 
1.  Lockable entry door with “Vaccination Study Center” 
identification sign viewable from hallway 
2.  Sufficient electrical outlets to supply below equipment 
3.  Lockable Filing cabinet 
4.  4 chairs 
5.  1 desk 
 
Study Center Office 
6.  Direct line and/or PBX extension telephone 

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7.  Laptop computer, including cables, charger 
8.  Printer/Scanner/Copier/Fax 
9.  Color photo printer, and cables 
10.  Digital camera, including spare memory chip, batteries, 
charger, cables 
11.  Registration card photo cutter and laminator and 
supplies 
12.  Regular waste disposal container 
13.  Individual participant files by ID number for storing 
relevant active and completed forms (i.e., green, yellow, and 
red randomization envelopes with attached blood specimen 
peeloff numbering stickers, signed Consent, 0-7 and 7-28 
Diaries doses 1 and 2, CRF, Participant Identification, 
Serious Adverse Events, Data Clarification) 
14.  Daily appointment book and/or wallboard for logging 
and tracking followup appointments 
15.  Study files: Spanish Protocol, English Protocol 
16.  Study files: Study Staff Responsibilities, Signature 
Registry and Log 
17.  Study files:  Current and completed Enrollment 
Log/Participant Inscription sheets 
18.  Supply of blank Serious Adverse Events forms 
19.  Supply of blank Appreciation/Bonus Dose Reminder 
sheets 
20.  Supply of blank Participant Registration cards 
21.  Supply of blank Appointment Reminder slips 
22.  Blank copier paper 
23.  Blank fax transmission cover sheets 
24.  Supply of TraxIt skin-applique thermometers 
25.  Regular thermometers 
26.  General office supplies, including pens, pencils, paper 
clips, rubber bands, blank CD-ROMs 
 
 
Waiting Area 
27.  At least 10 chairs available 
 
 
28.  Emergency resuscitation equipment, including 
epinephrine and 1cc syringes, ventilatory bag&mask of 
appropriate pediatric sizes for manual respiration  
29.  Insulated cold box with wet ice containing day’s supply 
of opened and spare vaccine vials, and virus specimen 
collection kits (return contents to Microbiology refrigerator 
at end of day) 
30.  Writing table 
31.  Exam table 
 
Study Center Injection 
Room 
32.  2 stools or chairs 

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33.  Restraint board 
34.  Regular waste disposal container 
35.  Medical waste disposal container 
36.  Sharps waste disposal box 
37.  Examination gloves, various sizes 
38.  Alcohol, alcohol wipes 
39.  Gauze 
40.  skin adhesive tape 
41.  Latex-free bandages (e.g., Band-Aids®) for covering 
injection site 
42.  Butterfly needles, various gauges 
43.  Blood collection tubes, various sizes, red top for serum 
44.  Waterproof marker pens for specimen vessels 
45.  Sterile Alaris SmartSite® vial adaptors multi-use access 
ports for vaccine vials 
46.  Syringes, 3-5 mL 
47.  Syringes, 10 mL 
48.  Syringes, 1 mL, tuberculin/vaccination type  
49. Biojector devices and spare CO2 cartridges, instructions 
50.  Sterile Biojector jet injector cartridges 
51.  Identification stickers for injection site photography 
52.  Shredder for destroying randomization cards 
 
53.  Virus collection specimen peel-off numbering stickers 
 
 
54.  Scale 
55.  Height/length measuring board 
56.  Furniture 
57.  Week’s supply of blank Consent - Additional 
Information handouts 
58.  Week’s supply of blank Informed Consent Forms 
59.  Week’s supply of blank Participant Identification 
Forms 
60.  Week’s supply of blank Enrollment Log / Participant 
Inscription sheets 
61.  Week’s supply of blank Case Report Forms 
62.  Week’s supply of blank Parent Diary Forms Days 0-7 
63.  Week’s supply of blank Parent Diary Forms Days 7-28 
64.  Week’s supply of blank Data Clarification Forms 
65.  Week’s supply of blank Serious Adverse Event Forms 
66.  Week’s supply of adhesive thermometers 
67.  Week’s supply of circumference tapes 
68.  Supply of skin-measuring ruler circle templates 
69.  Supply of TraxIt skin-applique thermometers 
70.  Regular thermometers 
 
Clinical Investigations 
Consultant’s Office 
71.  Identification stickers for injection site photography 

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72.  Week’s supply of parent take-home notebook/satchel 
 
 
 
Hospital Director 
Office 
73.  Sealed blue envelopes for emergency opening opening 
containing study group allocations for each participant ID 
number. 
 
 
74.  Freezer at -70° C for serum and virus specimen storage 
– protected by emergency backup generator 
75.  Refrigerator at 2-8° C for storage of vaccine (and 
unused virus collection kits, and influenza rapid assay test 
kits, if needed) – protected by emergency backup generator 
76.  15 kW backup generator for above items with automatic 
switching and start control equipment. 
77.  Sufficient fuel in backup generator tank for 72 hours 
operation 
78.  Centrifuge for serum 
79.  Study vaccine supply in above refrigerator 
80.  Study virus collection kits supply in above refrigerator ( 
if indicated) 
81.  Study rapid influenza test kits supply in above 
refrigerator, if indicated 
82.  Designated space in -70° C  freezer for serum cryovials 
and virus collection specimens 
83.  Pipettors and pipette tips 
84.  Cryovial tubes and holding racks 
85.  Storage/freezing boxes for cryovial tubes 
86.  Cold packs for shipments  
 
Microbiology 
Laboratory 
87.  Miscellaneous laboratory supplies and equipment 
 
 
88.  Study supplies in reserve and excess which do not fit 
into above rooms 
 
Study Storage Room 
89. Cold shipping containers for overseas transport of 
specimens 
 
 
90.  FDI IRB files and documents 
Dept. of Infectious 
Disease / Fundación 
Dominicana de 
Infectología office 
91.  Study administrative files, contracts, etc. 
 
 
 
Stage 1 Monday will also be devoted to general orientation of the physician 
investigators and study nurses for the study and its timelines, processes, and forms, 
in lecture style with handouts, questions and answers, and discussion.  Repeat 
morning and afternoon sessions may be used for staff on shift duty during the other 
session.   

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Stage 1 Tuesday and Wednesday will be devoted to training the physician 
investigators who will administer the informed consent, do clinical assessments, 
and complete the CRF and other data forms.  It will also include the training of the 
nurses who will administer the blinded injections and manage the flow of 
participants, paper, and specimens.     
 
With social worker observation, physicians will explain the informed consent to 
one another in pairs, with one serving as mock parent.  Upon signatures, the mock 
parent will later complete each of the 0-7 and 7-28 day diary forms.  Later, the pair 
will reunite to continue mock revisits on day 2, day 7, and day 28,  The completed 
mock Diary forms will be used for physician-“parent” interview and data entry into 
mock CRFs.  Afterwards, all investigators and mock parents will review the 
process in an interactive session seeking discussion, questions, answers, and 
suggestions.   
 
The study nurses will undergo training by principal/senior investigators in the 
blinding process and related security issues.  This will include the process of 
opening the random allocation cards, preparing the injection equipment, shredding 
the card, and administering the injections.   
 
Representatives of the Biojector manufacturer will demonstrate proper jet injector 
technique, which will be practiced by study nurses.  Proper needle-injection 
technique for children above and below 12 months will also be taught.   
 
On Phase 1 Wednesday, laboratory personnel will conduct a session for 
physicians and nurses regarding laboratory procedures for the collection, labeling, 
processing and handling of blood specimens from all participants, and 
nasopharyngeal swabs from those with influenza-like illness.  Demonstrations and 
training will occur in collecting nasopharyngeal swabs, placing them into swab 
containers and virus collection tubes, labeling with mock sticker, and transporting 
to the laboratory for processing and placement into the freezer.   
 
Phase 1 Thursday and Friday will be devoted to ensuring no gaps in the flow of 
parents, patients, forms, supplies, specimens, and data.  The sponsor 
representatives, physician investigators, and study nurses will “walk-through” the 
entire physical and administrative process with mock parents using participant 
number “000” to work out the patient flow and paperwork entries from ground 
floor immunization clinic to 2nd floor study center rooms.   
 
The first walk-through will be for a mock day 0 recruitment from the immunization 
clinic for consent and vaccination with dose 1.  Walk throughs will be repeated for 
a mock day 2 visit, then a day 7 visit, then a day 28 visit, then a day 56 visit, then a 
day 56 +6 months visit.  Investigators will observe the process.  Unofficial samples 
of all study forms will be completely filled out as if real, including opening of 
mock random allocation envelopes.  Mock injections will be made into the air, 

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mock blood tubes labeled with mock stickers will be brought to lab, etc.  Real 
photographs and printed out of mock Parent/Child for registration card and CRF, 
and of injection site reactions.  
 
Volunteers for the day 0 walk-through will be sought from among parents waiting 
in the immunization clinic (or among other hospital staff or volunteers, if engaging 
parents are not possible).   Volunteer parents will be compensated for their time.  
The vaccination or procedures for which they came to the clinic will be completed 
first.   
 
Volunteer parents and children for the mock walk-through will be directed to the 
study clinic where the the study will be explained and they will read and consider 
the informed consent and additional information handouts, as if in a real situation.  
After a mock signing, all other relevant study forms will be filled out (e.g., CRF, 
Diary dates, etc.).  Parents will be trained in taking measurements and filling out 
the diary forms.  That will be the extent of participation by these parent volunteers 
in the study walk-through.  Unless obviously ineligible, they should be invited to 
return in the future for actual recruitment into the study. 
 
The rest of the simulation exercise will use three hospital staff, who will be given 
baby dolls to represent study participants.  They will “arrive” at the study center 
and be directed to wait outside, as would actual participants.  They will be invited 
to enter for processing, photographs, and issuance of mock registration cards and 
next appointment reminders.  They will wait outside until called in for blood 
collection and vaccination.   
 
The study nurses will take custody of the dolls for mock venipuncture and mock 
“vaccination” according to mock randomization envelopes.  One of the dolls will 
be presumed to have influenza-like illness and undergo mock nasopharyngeal 
swabbing and specimen collection, as well.  Specimens will be labeled with mock 
stickers and brought to the laboratory for mock processing.  Laboratory personnel 
will simulate processing them (labeling, separation, aliquoting) and storing in the -
70° C freezer.  The steps in the simulation will be timed.   
 
Week 1 Friday afternoon will feature a debriefing session by sponsors and 
investigators to assess readiness and preparedness.  If positive, training phase 2 will 
begin the following Monday, with or without using the intervening Saturday 
and/or Sunday to resolve any weak points.  If readiness and preparedness are not 
sufficient, training phase 1 will be continued into the next week.   
 
10.6.2.2 Phase 2: Pilot Actual Recruitment and Study Initiation 
On training phase 2, on a Monday and Tuesday, the actual study will begin in a 
slow-motion pilot with recruitment at a reduced rate.  Only one volunteer 
parent/participant will be recruited into the study each morning and each afternoon 
of Monday and Tuesday only, with actual blood collection, blinded vaccinations, 

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laboratory processing and all other study procedures.  At any time when conditions 
dictate, the investigators may suspend further recruitment until readiness resumes.  
These four pilot phase 2 participants will return for their 2 day visits on 
Wednesday and Thursday, respectively, and for their remaining visits in future 
weeks, as per protocol.   
 
Extra time will be spent assessing the parent diaries for correctness of completion 
and revising the parent-training process to overcome any problems detected.  Each 
afternoon of the training phase 2 week, the investigators and other study personnel 
will meet to discuss problems and develop solutions.  On Friday, a decision will be 
made whether and how much to increase the recruitment rate for the following 
week, and to add the remaining vaccination day of Wednesdays.   
 
10.6.3 Screening and Recruitment 
Recruitment and screening of participants will occur in the following steps.  There will 
be no posters or public announcements.   
 
A.  The nurses who staff the Healthy Children and Vaccination Service [immunization 
clinic] of the hospital will identify eligible children among those in their waiting room 
by examining their visitor log and questioning parents.  Eligibles will be patients or 
their accompanying siblings who are age 6 to 24 months and up-to-date with the 
national immunization schedule.  (These nurses, in alternating morning and afternoon 
shifts, will serve as the study nurses who will handle logistics in the study center and 
will be trained to administer unblinded the study vaccinations.) 
 
B.  Parent(s) or guardian(s) (hereafter referred to by the singular “parent” but including 
the plural) of potential eligibles will be invited to consider participation of their child in 
an influenza vaccination study.  The parent will be informed that non-interest or non-
participation will not in any way affect the services they came to receive in the clinic.   
 
C.  If the parent expresses interest in the study, or asks technical questions that the 
nurses cannot easily answer, the parent and child will be escorted or directed to the 
Clinical Investigations Consultant’s office, room 33, second floor.   
 
D.  There, the investigator physician will fully explain the study verbally, as detailed in 
the study information document, which the parent will be requested to read, as well. 
 
E.  If participation is declined, the parent will be thanked for their time and interest and 
escorted or directed with the child back to the ground floor Healthy Children and 
Vaccination Service [immunization clinic], where they will be attended to, if needed, or 
resume their original position in the queue.  
 
F.  If participation is desired, the investigator will interview the parent to determine that 
the child fulfills all the inclusion criteria and none of the exclusion criteria for 
participation.  If not currently eligible due to age or recent vaccination, a return date 

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will be selected for their return, and the parent and child escorted or directed back to the 
immunization clinic, where they will resume, if needed, their original position in the 
queue.  If permanently ineligible, this will be explained to the parent, who will be 
thanked for her time and interest  and escorted or directed back to the immunization 
clinic, where they will resume, if needed, their original position in the queue. 
 
G.  To ensure compliance with the inclusion and exclusion criteria of the protocol and 
Part 4 of the Case Report Form (CRF), duplicate verifications will be required.  First, 
the physician investigator who originally collects the demographic and clinical history 
in CRF Parts 2 and 3 will certify eligibility in question 30 of the CRF Part 4.  Second, a 
different physician investigator will review the data to independently verify and certify 
eligibility of the child for the study in question 31, and duly sign in field 32. 
 
10.6.4 Informed Consent Process 
The informed consent process will occur in the following steps: 
 
A.  The investigator will inform parents that participation requires their written consent 
and the content of the consent form will be explained verbally.  The parents will be 
requested also to read the consent form themselves, as well.  If they agree to participate, 
they are requested to sign and date it.   
 
B.  Parents will be given the opportunity separately to consent or decline long-term 
storage of leftover, unused specimens for future testing (sections 7.3 and 10.5.1.4).  
Declining long-term storage and future testing shall not bar participation in the study if 
otherwise consented. 
 
C.  Parents who do not wish to participate in the study or to sign its consent form will be 
thanked for their time and interest and escorted or directed back to the Healthy Children 
and Vaccination Service [immunization clinic], where they will be attended to, if 
needed, or resume their original position in the queue.   
 
10.6.5 Monitoring of Informed Consent Process 
A social worker from the office of social work at the HIRRC will silently observe the 
first two weeks (or longer, as needed) of the informed consent process, in order to 
identify any difficulties or misunderstandings.  He or she will request voluntary 
informal interviews with a convenience sample of parents upon their completion of day 
0 activities, or upon their return on days 2 or 7, to explore the parents’s comprehension 
of the informed consent, and any lingering questions.  The social worker will meet three 
times weekly during this period, or more frequently as needed, with the Dominican 
Investigators to provide feedback on any gaps or weaknesses perceived in the informed 
consent process, so that its verbal explanations and other processes may promptly be 
revised or improved, if necessary.   
 

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10.6.6 Parent Training Procedure 
Both in the Dominican Republic and the U.S., the filling out of diaries by parents to 
record vaccination-related observations at home are a common and accepted procedure.  
In the years 2002 through 2006 alone, the HIRRC itself collaborated in three multi-
center studies with international pharmaceutical manufacturers on studies meeting 
current good clinical practice (cGCP).  These required Dominican parents to record 
information daily at home about their child’s illness following treatment for recent onset 
of middle ear infection (acute otitis media).  In the United States, controlled, clinical 
trials of influenza vaccine have used parental diary cards to record information at 
home.Edwards1994, Belshe1998, Belshe2000 
 
Parent training will occur, as follows: 
A.  While the physician investigator is conducting the physical examinations required in 
the CRF, he or she will explain in the parents presence how to perform those methods 
which they will repeat at home and record in the Diary form.   
 
B.  Before handing the Diary form to the parent, the investigator (or study nurses 
earlier) will prefill fields in the diary form for participant initials and ID number, study 
center telephone numbers, and the dates and days of the week at the top of Diary 
columns for recording reactions. 
 
C.  Using sample forms, the investigator will show and practice with the parents how to 
complete the Diary form.  On the Day 7 visit, similar filling out and training will occur 
for the 7-28 day Diary form. 
 
D.  Specific skills the investigators and/or nurses will teach the parents include correct 
application, reading, and removal of the skin appliqué thermometer, use of the tape 
measure for limb circumference, and reading local reaction sizes using the plastic 
ruler/template.   
 
E.  Parents will be required to demonstrate actual use of these devices, and proper 
recording of the results in the form, in order to verify their understanding.  Mistakes 
will require additional explanation until the parents can complete the form correctly, or 
the investigator determines that the parents are unable to do so and are excused from the 
study.   
 
F.  Parents will be provided with a study registration card with a photograph of the 
parent and child (to reduce the chance of mistaken identity on later visits).  They will 
also receive an appointment card on which the days and hours of their next appointment 
will be written.  Both cards will contain the telephone numbers to be contacted at any 
hour if any questions or concerns arise.  
 
G.  Parents will be provided with a take-home, closable notebook and/or satchel to carry 
their registration and appointment cards, the ruler/template and circumference tape, 
additional skin thermometers for replacing every two days, a signed copy of the 
informed consent and its accompanying additional information handout, plus the 

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parental Diary form to be used until day 7 (on that day, it will be replaced with another 
for the period from day 7 to 28).  
 
H.  The importance will be explained to the parents for their correctly completing the 
Diary form each day, and bringing the child back on days 2 and 7 and 28 after 
vaccination to be evaluated by the doctor.   
 
I.  After completion of the parent training and related forms, the study staff will 
undertake the venipuncture.   
 
10.6.7 Venipuncture Procedure 
For the required blood collections, the physician, after proper hand washing and 
restraint of the child, with assistance from study nurses, will locate the vein, clean the 
area with 70% isopropyl alcohol, allow it to dry, and collect the blood sample into 
sterile vacuum tube.  Parents are free to remain present for the blood collection. 
 
10.6.8 Vaccination Procedure 
First, the injection nurses close the “injection cubicle” while the participant and parent 
remain outside.  They open the allocation group randomization envelope (see section 
10.4.4.3) for that child’s dose (green for dose 1, yellow for dose 2, red for “insurance” 
dose 3).  One injection nurse then shows the card to the other to confirm the dosage and 
method.  Following the details described in section 10.4.2.3 above, the nurse duly 
prepares the syringe or jet injector cartridge, covers its tip to maintain cleanliness, and 
sets it down upon a clean surface and covers it.   
 
The child is then brought into the “injection cubicle” and the parents are requested to 
remain outside.  The INF vaccine is administered in the dose and route (ID or IM) 
specified by the randomization card into the anterolateral portion of the left thigh for 
children less than 12 months of age, or to the left deltoid muscle for children 12 months 
and older. 
 
Immediately after the vaccination, the injection nurse will observe for several seconds 
the injection site for any wheal or bleb and any drops of blood or clear liquid, apply a 
band-aid, and duly note the information in the CRF, along with the time of injection.  
The allocation card is then destroyed and its pieces disposed into a paper shredder, and 
any remaining injection supplies or equipment removed from view.   
 
The parent(s) and child are then reunited for holding and consoling (in the waiting area) 
during the subsequent minimum of 30 minutes of observation before reexamination of 
the injection site by the injection nurse (to avoid unblinding of the physican investigator 
from any temporary skin dimpling from the injector flange).  During the post-
vaccination waiting period, the parent/guardian will receive a final briefing and further 
training for aspects covered in section 10.6.4 above and reminded when to return next to 
the study center.   

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At least 30 minutes following vaccination, the injection nurse will note any immediate 
or early local adverse events and complete the remaining data in parts 7, 11, or 15 of 
Case Report Form (CRF) (“Medical Exam and [1st/2nd/3rd] Vaccination”) and questions 
1 through 8 of Parts 8 and 12 of the CRF (“Local Immediate Reactions”).  In the event 
of an unexpected immediate local reaction – such as frank bleeding or laceration – the 
physician will be notified to institute needed medical care, take a photograph, and make 
appropriate entries in the adverse event section of the CRF.  In all cases, the physician 
investigator will assess questions 10 through 15 of CRF parts 8 and 12 (“Immediate 
Systemic Reactions”).  The parent and participant will then be discharged from the 
study center. 
 
10.6.9 Rapid Diagnosis and Virus Culture for Influenza-Like Illness 
It is useful to know the strains of Influenza A virus circulating in the community during 
the study, as well as to know which antibody responses in participants may be the result 
of infection with wild virus and not induced by the vaccine.  Any participants who 
develop influenza-like illness (ILI) during the course of the study, either during their 
individual phase of active surveillance, as well as after they have “graduated” from the 
trial, shall have nasal and pharyngeal specimens collected for immediate rapid diagnosis 
of influenza A versus B.  Simultaneous specimens placed in virus culture transport 
tubes will be quickly frozen and stored at -70°C.  Frozen specimens from participants 
testing positive for influenza A will be shipped on dry ice to a laboratory for virus 
culture and typing.   
 
The criteria for defining influenza-like illness that will prompt influenza rapid test and 
virus culture are listed in Table  8.  
 
Table  8.  Definition of “Influenza-Like Illness”  
 
A.  Temperature ≥38.3° C 
And 
B.  2 or more symptoms of  
 
B1.  Cough 
 
B2.  Sore throat 
 
B3.  Rhinitis or runny nose 
 
B4.  General malaise 
And 
C.  No other obvious explanation for the fever 
And 
D.  The symptoms have persisted 4 days or less 
 
10.6.10 Specimen Preparation, Labeling, Storage, and Shipping 
For use in the study, the “CDC and ATSDR Specimen Packaging, Inventory and 
Repository” (CASPIR) facility for receiving, processing, and shipping medical 

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specimens (http://www.cdc.gov/od/ads/caspir/), will prepare preprinted sets of peel-off, 
freezer safe, specimen labels meeting its guidelines.  Each label will contain its own 
unique CASPIR-assigned serial no. and barcode.   
 
10.6.10.1 
Serum Specimens 
For collection of serum for antibody assay, sets of 12 adjacent labels will also be 
labeled with identical barcoding and printing of the study designation code + study 
number + participant identification number, followed by the character “A” to link 
and identify blood collection tubes and aliquots from the first blood draw on day 0.  
Similarly, 12 labels for that participant will be printed with “B” to link and identify 
specimens from the second blood draw on nominal day 28, as well as another 12 
labels printed with “C” for the third blood draw on nominal day 56.   
 
Each label within each series of 12 (A, B, and C) will also be numbered and 
barcoded sequentially with “aliquot-01”, “aliquot-02”, and so on up to “aliquot-
12”.  The highest aliquot number(s) will be affixed to the appropriate box on the 
CRF for that blood specimen, and onto other documents as needed.   
 
The 5 mL specimens of whole blood to be collected on day 0, day 28, and day 56 
for each participant will be prepared into serum the same day as drawn.  The 
DEI/HIRRC laboratory will divide them into sequential cryovial tubes to be labeled 
starting with “aliquot-01”, “aliquot-02”, etc.  Each tube will contain only 300 µL 
(0.3 mL) volume until the serum is used up or aliquot-08 is reached, which shall 
contain the remainder of serum available.  If less than 600 µL total serum is 
available, it should be divided into only two aliquots.  Serum should be stored at -
70º C (with emergency power generator backup).  Storage as warm as -18º C is 
acceptable in case of emergency.   
 
At periodic intervals determined by the investigators, one half of the total aliquots 
from each participant for each draw will be consolidated into a subset for shipment 
on a Monday or Tuesday only, during non-holiday weeks, by overseas dry-ice air 
freight service to the CASPIRg  for interim and long-term storage and eventual 
transshipment to the study assay laboratory (see section 10.6.12).  Shipments from 
the Dominican Republic will be arranged and handled by experienced international 
medical specimen shipment companies, e.g., OCASA 
(universalpack@verizon.net.do), World Courier 
(http://worldcourier.com/us/indexsz.html) or Quick International 
(http://www.quickintl.com/).  
 
While the first subset of aliquots is being shipped, the remainder shall remain at 
HIRRC in case of damage or loss in shipment of the first set.  After the first set is 
received successfully at the specimen storage facility, the remaining aliquots will 
                                                 
g  Shipment delivery address: CDC CASPIR, 602 Webb Gin House Road, Lawrenceville, Georgia 30045-5427  
USA, Tel: +1 770-339-5950, Fax: +1 770-339-5943, Contact person:  Robert J. Davidson, rum8@cdc.gov.   

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then be shipped.  Similar procedures should be followed for phase II specimens, 
except interim shipments may be made at the discretion of the study investigators.  
Upon the direction of the U.S. Principal Investigator, CASPIR will transfer 
specified aliquot(s) to the designated laboratory for serologic assays.   
 
Serum remaining from the study after testing shall be kept in long-term storage by 
the CASPIR specimen repository and by the testing laboratory.  They may be 
transferred elsewhere upon request by the CDC or HIRRC investigators for 
additional testing or permanent storage, in accordance with the provisions of the 
informed consent.  Such specimens shall remain the joint property of CDC and 
HIRRC, and should not be disposed or tested without their mutual consent.   
 
10.6.10.2 
Virus Culture Specimens 
CASPIR will also prepare sets of 8 duplicate-numbered stickers for use in linking 
and identifying nasal and pharyngeal specimens collected into virus culture 
transport tubes and kits for rapid influenza testing.  One label from each series will 
be inserted into the appropriate box on the CRF.  These specimens will be collected 
only when participants develop influenza-like illness, in order to determine which 
strains of influenza A may be circulating during the study period.   
 
10.6.11 Data Coding, Handling, and Analysis 
As detailed in section 10.4.4 above, the blinding codes will be maintained in three 
secure locations by the independent party and other member(s) of the Data Safety 
Monitoring Board, as well as kept under seal by the HIRRC hospital director (in the 
BLUE envelopes, see 10.4.4.4).  Codes will not be shared (“broken”) with the study 
investigators (except as described in 10.4.4) until all clinical aspects of the study are 
completed and missing data obtained, and the serologic assays, performed blind, are 
completed and results shared.   
 
Case Report Forms, Parent Diary, and other data recording sheets and forms from the 
study will be duplicated, with the originals kept in the HIRRC and complete copies 
furnished and maintained for analysis and preservation by the CDC.  Data entry and 
analysis will be performed at CDC, with sharing of programming and data files with 
HIRRC (and with other parties at the discretion of the principal investigators once 
blinded laboratory assays are completed and results provided to CDC and HIRRC).  
 
Once a CRF and its related forms are completed, verified, collected, and copied by the 
Dominican Principal Investigator, no additional information may be added to the 
originals.  Any additions or corrections to the data of the CRF and related forms shall be 
made by means of the Data Clarification Form (DCF).  This form (sections 11.4 and 13) 
shall be used to track inquiries to the clinical investigators from the sponsor (CDC) 
about entries in data fields of the Case Report Form, and/or to explain any subsequent 
changes in the data by the clinical investigators at HIRRC.   
 

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10.6.12 Laboratory Assays 
10.6.12.1 
Serology 
Serologic assays for the study will be performed by a facility selected through 
WHO tender from among qualified research level institutions.   For example, 
invitations will be extended to the Laboratory for Specialized Clinical Studies, 
Division of Infectious Diseases, Cincinnati Children’s Hospital Medical Center, 
Cincinnati, Ohio, USA, as well as to the National Institute for Biological Standards 
and Control, Potters Bar, Hertfordshire, UK.  
 
Hemagglutination inhibition assays (HI) against viral strains similar to those 
included in the vaccine studied will be performed according to current Good 
Laboratory Practices (cGLP) and standard assay protocol. Palmer1975, Lennette1995  
Neutralization and/or other assays, may also be performed as valid indications arise 
and resources permit.    
 
Subject to agreement for justifiable deviations, specimens will be handled and 
tested as follows:  Upon arrival in the serology laboratory, sera will be stored at -
20°C or colder until analyzed.  Specimens will be treated at a dilution of 1:4 with 
receptor-destroying enzyme (RDE, Vibrio cholerae abnormal type 558 strain RDE; 
Denka Seiken Co., Ogdensburg, N.Y. and Tokyo, Japan, or equivalent) to eliminate 
nonspecific inhibitors, and then incubated for 16-18 hours in a 37°C H2O bath.  
This will be followed by a second incubation for 30 minutes in H2O bath at 56°C in 
order to inactivate the RDE.   
 
After RDE inactivation, the HI process proceeds by diluting serum already at 1:4 in 
serial 2-fold steps (4, 8, 16, 32, 64, 128, 256, 512…) until a final dilution of 
1:1024.  Each specimen is separately diluted 1:4 and 1:8 for use as serum controls 
on each plate in which the specimen is tested.  In addition to the serum controls, a 
red blood cell (RBC) control is also included on the plate for each specimen 
analyzed.  At this point, 0.025 mL is transferred to duplicate rows of one test plate 
for each virus antigen to be analyzed.  This yields four specimens per test plate 
with nine usable test dilutions, two serum controls and one RBC control in each of 
the duplicate rows.   
 
The prepared, specified virus strain antigens are added to the nine test dilutions of 
every specimen and 0.025 ml of phosphate-buffered saline (PBS) is added to the 
serum and RBC controls.  Each day assays are performed, the antigen is prepared 
to contain 4HA units/0.025ml.  Virus strain antigens may be supplied from external 
reference laboratories, or prepared on site by the laboratory.   
 
The antigen-serum mixtures are incubated at room temperature for 30 minutes.  
Next, 0.050 mL of a 0.5% chicken RBC solution is added to each well.  Turkey 
RBCs may be used if chicken RBCs are not compatible with a specific virus 
antigen.  Upon adding the RBC solution, the plates are incubated for 30 minutes at 

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room temperature, after which they are tilted and read.  The titer is the reciprocal of 
the last dilution that shows complete inhibition of agglutination.   
 
10.6.12.2 
Rapid Test for Influenza Illness 
The Microbiology Laboratory of the HIRRC will be supplied with rapid diagnostic 
tests capable of distinguishing influenza A from B (e.g., Remel Xpect® Flu A&B, 
Quidel QuickVue® Influenza A+B, Becton Dickinson Directigen™ Flu A+B, or 
equivalent).  Nasopharyngeal and/or oropharyngeal specimens will be collected 
from participants with Influenza-like Illness (section 10.6.9) using swabs, 
collection kits, and instructions provided or indicated by the test involved.  
Participants who develop influenza-like illness multiple times during the study 
period may be tested multiple times as well.  Specimens will be promptly assayed 
according to the manufacturer’s instructions. 
 
10.6.12.3 
Influenza Virus Culture 
The Microbiology Laboratory of the HIRRC will be supplied with virus culture 
collection and transport tubes with Hank’s medium (e.g., BD Cellmatics™ Viral 
Transport Pack, Remel MicroTest™ M4®, or equivalent).  These contain swabs 
with Dacron® tips and aluminum or plastic shafts to avoid damage to viruses 
present from calcium alginate or cotton tips and wooden shafts.  Nasal and throat 
samples will be combined and stored at -70°C until it is determined by positive 
findings for influenza A to ship them to a specialist virology laboratory for culture 
and serotyping.  The laboratory to perform such cultures will be determined by 
competitive tender by WHO among qualified reference laboratories.   
 
10.6.13 Termination Visit 
The final study visit will occur at ~ 56 days after dose 1 of INF vaccine, which should 
be approximately 28 days after dose 2.  At this time, the 3rd blood draw and a 3rd 
(“insurance”) dose of INF will be administered.  If the participant does not return as 
scheduled for this visit, followup will be performed by home visit by study personnel.   
 
10.7 
Data Quality Assurance 
10.7.1 Clinical Procedures 
Personnel of CDC will visit the study site prior to initiation of the study to review with 
the site personnel information about the investigational agent, protocol requirements, 
randomization procedures, case report forms (CRFs), monitoring requirements, needle-
free injection techniques, and reporting of serious adverse events.  
 

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10.7.2 Good Clinical Practices Monitoring 
At visits during and after the study, the site will be monitored by CDC for compliance 
with current good clinical practices (cGCP) and other requirements, including accurate 
and complete recording of data on CRFs, source documents, and drug accountability 
records.  
 
10.7.3 Data Handling 
Case report form data will be entered in duplicate at CDC, using EPI-INFO (CDC, 
Atlanta, GA), Microsoft Access® (Redmond, WA), and/or equivalent software.  
Analysis will be performed by EPI-INFO, SAS™ (SAS Institute, Cary, NC), or 
equivalent software.  Analyses will be performed by a predefined analysis plan.  
 
10.8 
Statistical Methods and Determination of Sample Size 
10.8.1 Statistical and Analytical Plans 
10.8.1.1 Primary Efficacy Analysis 
The primary analysis of efficacy will be comparison of the proportion of 
participants within each investigational route of administration – ID-JI-0.1  and IM-
NS-0.1 – achieving seroconversion one month after the second dose of vaccine to 
each vaccine strain versus the control needle-syringe full-dose route (IM-NS-0.25).  
As defined in section 10.5.1.2, the criteria for seroconversion to a vaccine strain 
will be a hemagglutination inhibition (HI) assay titer of 1:40 or greater for those 
with initial undetectable titers, or an increase of 4-fold or greater from a measurable 
initial titer to final titer.   
 
Each reduced-dose investigational study arm, ID-JI-0.1  and IM-NS-0.1, will be 
considered an acceptable alternative method of vaccination if there is no more than 
a twelve percent decrement in the proportion of participants seroconverting 
compared to that of the full-dose, IM, needle-syringe control vaccination 
comparator (IM-NS-0.25) (non-inferiority model ).FDA1998, Chow2002 
 
10.8.1.2 Secondary Efficacy Analyses 
Secondary efficacy analyses will compare the three study arms on the following: 
 
a.  proportion seroconverting one month after the first dose of vaccine, but before 
the second dose 
b.  proportions achieving seroprotection one month after both the first and second 
doses of vaccine.  Seroprotection is defined as HI titers of ≥1:40, 
including among those pre-immune participants with detectable titers 
before vaccination. 

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c.  geometric mean titers (GMTs) of HI assays performed one month after both 
doses 1 and 2. 
d.  satisfying the clinical criteria of the European guidelines for annual vaccine 
registration for adults aged 18 to 60 (there are none for children), CPMP1997 
that antibody responses to at least one strain of the vaccine achieve: 
 
•  >40% seroconversion among participants 
 
•  >2.5 increase in inverse geometric mean titer from pre-vaccination to 
final titers (e.g., from inverse GMT of 8 initially to final of ≥20) 
 
•  >70% seroprotection among participants 
e.  proportion with prompted and unsolicited adverse events following 
immunization. 
10.8.2 Determination of Sample Size 
The total participants of the combined phases I and II (N=450), results from 150 
participants in each of three study arms.  This number results from rounding up, in 
anticipation of an estimated 5% loss-to-followup, from a calculated 142 participants 
(non-inferiority model FDA1998, Chow2002) or 139 participants (superiority model) required 
per arm, derived using PASS 2002™ software (Number Cruncher Statistical Systems, 
Kaysville, UT, http://www.ncss.com),Hintze2005  as follows: 
 
The full-dose, standard IM route control arm of the study (IM-NS-0.25) is assumed to 
achieve the mean seroconversion rate against the A/H1N1 strain (86.4%) demonstrated 
in 1 published Gonzales2000 (76%) and 3 unpublished Saliou2005 recent clinical trials (~88%, 
~79%, ~97%) of Vaxigrip® influenza vaccine (Sanofi-Pasteur, Lyon, France) 
conducted among a similar age range (6-35 months) of children as those to be studied in 
this trial (6-24 months).  (Immunogenicity for at least one strain satisfies European 
Committee for Proprietary Medicinal Product requirements for European registration. 
CPMP1997)  This rate is somewhat lower but consistent with the 91% seroconversion 
reported after use of a different influenza vaccine, Fluzone®, manufactured by the same 
company at its Swiftwater, PA, USA plant. SanofiPasteur2004, Hoberman2003   
 
Based on prior literature indicating equivalent serologic responses between standard, 
full-dose, needle-syringe influenza vaccination and (1) reduced-dose, needle-syringe 
intradermal (ID) vaccination (see section 9.4.2), as well as (2) reduced-dose jet injected 
(JI) ID vaccination (see section 9.4.4), the same 86.4% seroconversion rate is postulated 
for the investigational intradermal study arm.  
 
There is a paucity of and some conflicting results among clinical trials of reduced-dose 
influenza vaccination comparing the same antigen dosage by the ID versus IM or SC 
routes (see section 9.4.3).  Dose-sparing trials without an ID arm showed a small 
decrement in response,Treanor2002  so reduced dosage via the standard IM route is 
suggested as a useful strategy in time of shortage.Treanor2004  Others have called for 
careful clinical trials to confirm the effect of the ID route.LaMontagne2004  To fulfill this call 

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and clarify the issue, the reduced-dose IM arm is included in this trial with the same 
statistical assumptions as per the ID arm.   
 
Applying a non-inferiority model using a score test for two independent 
proportions,Farrington1990 a margin around these expected seroconversion rates (0.864) of 
twelve percent is selected, i.e., 0.104.  This corresponds to an acceptable 
seroconversion rate as low as 0.760 (76.0%) in the investigational study arms, to 
demonstrate that the investigational arms are not worse than the control arm by more 
than this amount.  (Decrements of up to 15% are suggested for non-inferiority trials in 
which the standard treatment rate is between 80% and 90%.Chow2003)  Model parameters 
were set at:  
target α  = 0.05 
Power = 0.80  (β = 0.2) 
Standard group proportion (P2) = 0.864  (from prior studies) 
Actual experimental group proportion (P1.1) = 0.864  (anticipated) 
Equivalent experimental group proportion (P1.0) = 0.760  (lowest 
acceptable) 
Equivalent margin difference (D0) = -0.104  (acceptable twelve percent 
decrement) 
Actual margin difference (D1) = -0.104  (acceptable twelve percent 
decrement) 
Experimental group sample size (N1) = 142 
Standard (control) group sample size (N2) = 142 
 
The null hypothesis Ho is that the seroconversion rate for the treatment arm is at least 
12% lower than for the standard arm, and that under the alternative hypothesis Ha, the 
difference in seroconversion rates between the treatment and standard arms is less than 
12% (i.e., the treatment is not inferior to standard).  If the study rejects the null 
hypothesis Ho and accepts the alternative Ha, we may conclude that the treatment is not 
inferior to standard, with only a 0.05 probability of reaching this conclusion due to 
random error.    
 
Applying an alternative superiority model for two independent proportions (null case) 
using a one-sided Z test with pooled variance, Chow2003 a sample size of 139 or greater in 
each group will detect with 80% power (β = 0.2) a superiority of ten percent or greater 
(seroconversion ≥95%) for either of the investigational study arms over the control arm 
(assumed 86.4%).  As for the non-inferiority model, above, the significance level of the 
test was targeted at α = 0.05.   
 
10.8.3 Randomization 
As detailed in section 10.4.4, participants will be allocated to one of the three treatment 
groups (ID-JI-0.1, IM-NS-0.1, IM-NS-0.25) by a random allocation method not subject 
to observer choice or whim.  Participants will be enrolled in blocks of six, each 
containing two members of each of the three treatment groups, in random order.  One 

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block will be randomly split between the first and last enrollees in the study, to prevent 
block counting.   
 
10.9 
Stopping Rules to Suspend Vaccination 
10.9.1 
Phase I 
During phase I (n=48), upon the occurrence of Serious Adverse Events in three (3 
[6%]) or more enrolled vaccinees, or incidents of entire-limb swellingRennels2000 or other 
severe local reactions at the INF injection site in five (5 [10%]) or more vaccinees, 
which, in the opinion of any of the Principal or Senior Dominican or U.S. Investigators 
are possibly-related or probably-related to the investigational vaccination (see section 
10.5.2.2 for definitions).   
 
Further INF vaccination for all participants will be suspended immediately until review 
of the data by the DSMB.  Upon analysis using unblinded access to the study group 
code allocations, the DSMB shall provide either its clearance to resume vaccination, 
with restrictions, if any, as appropriate, or its denial to proceed.  (The IRBs will be 
contemporaneously informed of any such suspension, as well as the outcome of the 
DSMB review, and the investigators will adhere to any resulting IRB guidance.) 
 
10.9.2 
Phase II 
In accordance with the same criteria and procedures as described for Phase I in section 
10.9.1 above, in phase II (n=402), all vaccination shall be suspended, upon the 
occurrence of possibly-related or probably-related Serious Adverse Events in sixteen 
(16 [4%]) or more vaccinees, or possibly-related or probably-related entire-limb 
swelling or other severe local reactions at the INF injection site in twenty (20 [5%]) or 
more vaccinees.   
 
10.10 Criteria for Proceeding to Phase II 
Upon unblinded analysis by the DSMB (see section 10.11) of the results of the phase I 
study, the observed adverse reactions will be tallied by the DSMB for each of the study arms 
(control IM-NS-0.25, investigational IM-NS-0.1 and ID-JI-0.1).  Either or both 
investigational study arm(s) for which an observed numbers of reactions exceeds the number 
observed in the IM-NS-0.25 control arm by the following degree for any reactions listed will 
be eliminated from the phase II study.  If both investigational arms are thus eliminated, the 
study will not proceed to phase II. 
 
 
 
 
 
 
 
 
 
 
             Excess  =  
 
 
 
 
 
 
 
 
 
no. in investigational arm 
 
 
 
 
 
 
 
 
 
   less no. in control arm    
Serious Adverse Reaction, possibly- or probably-related 
≥3 
Fever >40º C, possibly- or probably-related 
≥4 
Injection site whole-limb swelling 
≥4 

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Injection site induration >5 cm in diameter 
≥5 
Injection site erythema >5 cm in diameter 
≥5 
Injection site pain level 3 (cries upon moving limb) 
≥5 
 
For example, if four (4) children in the IM-NS-0.25 control arm experience induration of >5 
cm, eight (8) do so from the IM-NS-0.1 arm, and nine (9) from the ID-JI-0.1 arm, then the 
IM-NS-0.1 study arm may proceed to phase II, but the ID-JI-0.1 arm will be dropped from 
phase II.   
 
Based upon this analysis and any other relevant information, the DSMB will decide whether 
or not the study should proceed to phase II as designed, should be modified and then 
proceed, or should be terminated.  This decision shall be duly informed to the investigators, 
without revealing unblinded information nor study-group-specific analyses.  Regardless of a 
DSMB decision permitting phase II, the study sponsor may elect for any reason not to 
proceed, and to terminate the study after phase I.   
 
10.11 Data and Safety Monitoring Board 
A Data and Safety Monitoring Board (DSMB) will be appointed in advance of the study (see 
section 8.8).  Its members will certify no financial or related interests in any of the 
organizations conducting or cooperating with the study: CDC, HIRRC, WHO, PATH, or 
Bioject.  Any disclosed exceptions to the certification shall be considered by the DSMB 
prior to material discussions so that they may be reflected in the minutes along with the 
DSMB’s determination how to handle such exceptions.  The group expertise will include the 
fields of vaccine science, immunization, pediatrics, public health, statistics, and clinical 
medical research.  Statistical expertise will be provided by Maria Deloria Knoll, PhD, 
DSMB Chair, who will be assisted, as needed, by fellow faculty and programmers of the 
Johns Hopkins University School of Public Health. 
 
In advance of implementation of the study, members of the DSMB will each be provided 
with drafts of key study documents (protocol, consent form, case report form, diary forms, 
etc.) for an opportunity to review them and suggest changes on the basis of their scientific 
expertise and personal experience.  One or more of the DSMB members will assist in 
preparing the allocation cards and envelopes used to randomize consecutive participant ID 
numbers into the three study arms.  During the blinded period of the trial the DSMB will 
maintain in at least three separate locations the coding list to identify these allocations by 
study ID number. 
 
The DSMB shall meet formally by telephone conference call at least four times:  (1) prior to 
initiation of the study, (2) during the phase I enrollment period, (3) upon being provided 
with blinded clinical data from the completion of the phase I clinical aspects, and (3) upon 
being provided with unblinded analysis following the completion of clinical and laboratory 
analysis from phase II.  In addition, the DSMB may meet for any requested ad hoc meetings 
as described above, or upon its own initiative for cause.   
 

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Upon the incidence of SAEs which trigger the criteria for suspending vaccination, as per 
section 10.9, the DSMB will analyze the situation in unblinded fashion using the coding 
sheets, and make its recommendation to the investigators whether or not vaccination may 
resume or whether the study -- or one of its investigational study arms -- should be 
terminated.   
 
At the conclusion of phase I, the DSMB will be provided by the investigators with adverse 
events and other results from the phase, linked to study ID number.  As per section 10.10, 
the DSMB will link this data with the study allocation group information from the coding 
sheets and analyze them for purposes of applying the rules for proceeding to phase II, and so 
inform the investigators of their conclusions.   
 
Upon the completion of the blinded clinical and serologic aspects of the study, after either a 
termination following phase I or upon completion of phase II, upon request the DSMB will 
furnish the investigators the coding lists so that unblinded analysis of the results may occur.   
 
10.12 Early Reporting to DSMB and IRBs 
The conjunction of certain adverse events (AEs) and unanticipated problems will be 
reported promptly as they occur to the DSMB and both IRBs (see definitions for terms in 
section 10.5.2.2)  These shall include:  
 
•  Any serious AE (SAE) that is also an unanticipated AE, and is probably-related or 
possibly-related to participation in the research.   
•  A series of serious AEs that are expected in some participants, but in the judgment of 
the investigators are occurring at a significantly higher frequency or severity than 
expected.  
•  Certain unanticipated AEs, regardless of severity, that might alter the IRB’s analysis of 
the risk versus potential benefit of the research and, as a result, might warrant 
substantive changes in the research protocol such as the informed consent process 
and form, exclusion criteria, monitoring methods, or termination of enrollment.   
•  Unanticipated problems that do not involve adverse events, but which may have 
increased the risk of past or future harm to participants  
In the event of any such occurrences, the Dominican investigators will document the 
situation in writing and notify promptly by telephone, fax, or email both the FDI IRB and 
the U.S. investigators, who shall notify the CDC IRB and the DSMB members, and at their 
discretion, the vaccine and jet injector manufacturers. 
 
10.13 Changes in the Conduct of the Study or Planned Analyses 
Only the sponsor (CDC, represented by the U.S. Principal and Senior Investigators) may 
modify the protocol.  Amendments to the protocol will be made only upon consultation and 
agreement between the sponsor and the Department of Infectious Diseases of the HIRRC 
(represented by the Dominican Principal and Senior Investigators), with consultation from 

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the other cooperating institutions.  The only exception is when a clinical investigator 
considers that a participant’s safety is compromised without immediate action.  In such 
circumstances, immediate approval of the chairman of the FDI Institutional Review Board 
(IRB) or ethical committee (EC) must be sought, and the investigator should inform the 
sponsor and the full IRBs or ECs within 5 working days after the emergency occurred.   
 
All amendments that have an impact on participant risk or the study objectives, or require 
revision of the informed consent document, must be notified to the DSMB (see section 
10.11) and receive approval from both the CDC and FDI IRBs or ECs prior to their 
implementation.  The IRBs or ECs will also be routinely notified of any minor or 
administrative changes in the protocol.   The DSMB shall also be informed and advised on 
major study modifications affecting scientific integrity or participant safety, such as 
increasing sample size.   
 
11. Policies and Practices 
11.1 
Compensation, Insurance, and Indemnity 
Study participant parents/guardians will receive monetary reimbursement to cover estimated 
transportation costs from home to the hospital for the initial recruitment trip and all follow-
up visits related to the study.  They will also receive a voucher for each visit entitling them 
to a snack lunch at the hospital snack bar to compensate for the extra time away from home 
required by the study.  They may also receive a waterproof folder or carrying case for 
storing the participant diary, the thermometers, the circumference tape, the local reaction 
measurement template, and any other study materials provided to them.   
 
The study participants will be entitled to necessary outpatient and inpatient medical care at 
the HIRRC similar to that available to other patients at this public institution, and at no 
charge for any adverse results or consequences of their participation in the study (see section 
7.4).   
 
In addition, an insurance policy will be purchased after all approvals are received from the 
IRB and other necessary parties to cover liability of HIRRC, CDC, and WHO for harm or 
damages resulting to participants from involvement in the study.  The coverage will be 
purchased by the WHO from a reputable international insurer of clinical research 
worldwide, such as Biomedic-Insure (http://www.biomedic-insure.com/indexus.htm, 
Vannes, France), or equivalent company.  Coverage shall be approximately €800,000 per 
protocol and a sublimit of about €160,000 per participant, equivalent as of 23 February 2006 
to US$953,754 (RD pesos 32.8m) per protocol and US$190,737 (RD pesos 6.57m) per 
participant.   
 
A summary of the above compensation, entitlement, and insurance will be mentioned in the 
informed consent form, but the policy amounts will NOT be disclosed, and the participant’s 
parents will specifically be informed that coverage is for liability of the insureds to the 
participants, rather than direct coverage for health claims of the participants.   
 

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11.2 
Study Material Accountability 
The study investigators at the HIRRC must maintain accurate records of dates and quantities 
of product(s) received, to whom dispensed (subject-by-subject accounting), and accounts of 
any product accidentally or deliberately destroyed.  The investigator must retain all unused 
or expired study supplies until confirmation of the accountability data by the sponsor.  
 
11.3 
Case Report Forms 
Case report forms will be provided for each participant.  Correction to data on CRFs may be 
made only by putting a line through the incorrect data and writing the correct values, 
allowing the original text to remain legible.  Each correction must be initialed and dated by 
the person making the change.  If corrections are made after review and signature by the 
HIRRC investigator, he or she must be made aware of the changes and document this 
awareness.   
 
It is expected that the CRF will serve as the official medical record into which most data 
about the participant for this study is primarily or exclusively entered.  It is the policy of this 
study that any other secondary data which is copied from other records into the CRF must be 
verifiable to the source data.  This necessitates access by the sponsor, CDC, to all original 
recordings, laboratory reports, and participant’s records.  The HIRRC investigators agree to 
allow access to participant records and other sources for all study data, which will be 
maintained in confidence by CDC.  The participants (or their legal representatives) must 
also allow access to the participant’s medical records, and they will be informed of this and 
will be signing their agreement when giving informed consent.  
 
11.4 
Data Clarification Forms 
Investigators and collaborators may have questions about the Case Report Form data after 
copies of the CRF data are transmitted from the study site to the sponsor.  These questions 
will be submitted and answered by documenting them with Data Clarification Forms 
(DCFs).  There are two types of DCF inquiries, internal and external: 
 
Internal DCF:  A query involving matters OTHER than safety, adverse events, and primary 
endpoint data, for which the resolution is self-evident.  It can be answered without changing 
the meaning of the data (such as moving data incorrectly entered into one box on the form 
into another, correct box).  It can be answered using logical numeric flow (for example, if 
the serial reporting number of an adverse event is missing, the next number in sequence can 
be assigned).  Any of the U.S. (Sponsor) or Dominican investigators can create and answer 
an internal query.  The U.S. Investigators will send the Dominican investigators a copy of 
each internal DCF completed by the Sponsor.  The Dominican investigators will do the same 
for the U.S. Investigators on internal DCFs completed by the Dominican Investigators.  The 
recipients will review the internal DCFs received, and if he/she disagrees, he/she will notify 
the other party, who will then generate a new DCF documenting the investigator’s 
correction. 
 

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External DCF:  A query from the U.S. Investigator (Sponsor) that requires information from 
the Dominican Principal Investigator, particularly on key subjects such as safety (e.g. the 
relationship of an adverse event to vaccination) or data on a primary endpoint (immune 
response, specimen identity).  The Dominican investigators will provide the answer on an 
external DCF, sign it, and return it to the Sponsor.   
 
11.5 
Study Monitoring 
In satisfaction of common regulatory requirements and current good clinical practice 
(cGCP), all aspects of the study will be monitored by designated investigators of the 
sponsor, CDC.  For such monitoring purposes, the HIRRC investigators agree to allow 
access to the clinical supplies, dispensing, storage areas, and the clinical files of the study 
participants, and, if requested, agree to assist in such monitoring.  
 
In certain circumstances, a secondary audit may be conducted by the sponsor, CDC, or its 
designee.  The HIRRC investigators will be informed if this is to take place and advised as 
to the nature of the audit.   
 
11.6 
Retention of Records 
As required by applicable regulations, HIRRC investigators must retain all study records in 
a secure and safe facility.  The HIRRC investigators must consult a representative of the 
sponsor, CDC, before intended disposal of any study records, and must notify the CDC of 
any change in the location, disposition, or custody of the study files.  The period of record 
retention should be consistent with the record retention policies of the Dominican Republic, 
or the Committee for Proprietary Medicinal Products (CPMP) of the European Agency for 
the Evaluation of Medicinal Products, whichever is longer.  The CPMP requires retention 
for the maximal period of time permitted by the institution, but not less than 15 years.  
HIRRC shall destroy the Participant Identification Form containing confidential information 
at the end of any applicable retention periods, or after 15 years, whichever is later.  CDC 
shall retain for 15 years copies of those anonymous records furnished to it by HIRRC for 
data entry and analysis purposes.   
 
11.7 
Use of Information and Publication 
The principal parties to and collaborators with the study -- HIRRC, CDC, WHO, PATH, 
Bioject, and the serology laboratory -- recognize the importance of communicating medical 
research results and therefore each encourages publication of the results of this study in 
reputable scientific journals and at seminars or conferences.   
 
All parties agree to furnish the others with a copy of any proposed public presentations or 
publication related to the study.  The comments thereon shall be replied to the submitting 
party without undue delay, and not later than within 60 days.  If comments or suggestions by 
any of the parties are not accepted, the senior author of the abstract or manuscript and 
representatives of the other parties shall promptly meet and endeavor to agree mutually on 
the final wording and disposition of the publication.  In the event such agreement is not 

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achieved, the author(s) of any abstract, presentation, or publication shall duly disclose 
therein the objections of the other party(ies).  No participant in this research may be listed as 
an author or co-author on a publication or conference abstract presentation without their 
prior consent.  The same procedure and policy shall apply to publication or presentation of 
the results of prematurely discontinued and any other noncompleted aspects of this study.   
 
Due regard shall be given to the legitimate interests of Bioject and any other manufacturer 
whose products are used in the study.  Such interests include technical accuracy, interpretive 
conclusions, manuscript co-authorship, obtaining patent protection, coordinating and 
maintaining the proprietary nature of submissions to health authorities, coordinating with 
other ongoing studies in the same field, and protecting confidential data furnished in 
furtherance of the study.  
 
Unpublished results from investigations shall not be made available to any outside party not 
otherwise involved in the study by the investigating team without similar use of the 
presentations and publications procedure as outlined previously.  Neither Bioject nor any 
other manufacturer whose products are used in the study shall quote from publications by 
investigators in its scientific information or promotional material without full 
acknowledgment of the source (i.e., author and reference).  
 
It is intended and understood by the Dominican investigators at HIRRC and the U.S. 
investigators at CDC that the information generated in this study may be used by the 
manufacturer(s) of product(s) used therein in connection with the development of its product 
and therefore may be disclosed to government agencies in various countries.  To allow for 
the use of information derived from the study, within three months after the conclusion of 
the study and its data analyses, upon request, the HIRRC and CDC investigators will 
provide such manufacturer(s) with copies or summaries of clinical data and records, except 
confidential Participant Identification Form information.   
 
No manufacturer furnishing products used in this study may make any claims to the public, 
in writing or in oral presentations, nor may it cite in its advertising nor product labeling, any 
statement suggesting or implying that HIRRC, CDC, WHO, or PATH in any way endorse 
the products.  However, a manufacturer may state that it collaborated with these 
organizations in experimental use of its products.  It may cite published results of the study.  
It may use unpublished results for regulatory or commercial purposes other than publication 
or advertising.  
 
Specimens of serum, extracts, or isolates provided to laboratories under this protocol shall 
not be transferred to other parties without an express written Materials Transfer Agreement 
executed by HIRRC and CDC.  
 
11.8 
Statement of Obligations 
11.8.1 Sponsor and Monitor 
The sponsor (CDC) has already, or will:  

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A. Conduct one or more prestudy visits to: 
1. 
Establish the acceptability of the facility and record this in a written report 
(memorandum or form). 
2. 
Discuss the proposed trial with the clinical investigators at the HIRRC, 
supply the case report form, draft protocol, and other documents for 
review and approval.  
3. 
Discuss with the investigator regulatory requirements with respect to 
informed consent, IRB or EC approval of the trial, the protocol, 
including protocol amendments, and informed consent changes.  
 
B. Conduct periodic site visits to: 
1. 
Assure adherence to the protocol.  
2. 
Review case report forms and medical records for accuracy and 
completeness of information.  
3. 
Examine pharmacy records for documentation of quantity and date of 
receipt of investigational supplies, dispensation and accountability data 
for administration to each Participant, loss of materials, contamination, 
and unused supplies.  
4. 
Record and report (summarize) observations on the progress of the trial 
and continued acceptability of the facilities in a site visit report. 
5. 
Review investigator files for required documents, e.g., protocols, protocol 
amendments, IRB or EC approvals (protocols, amendments, informed 
consent, etc), IRB charter and membership, and communications to and 
from the IRB or EC and the investigator.  
 
11.8.2 Clinical Investigators 
A. Institutional Review Board or Ethics Committee.  The Dominican Principal 
and Senior Investigators must assure the sponsor or monitor that the IRB or 
EC of the Fundación Dominicana de Infectología (FDI): 
 
1. 
Meets regulations as defined in 21 CFR Part 56 of the U.S. Code and other 
applicable requirements.  
2. 
Has authority delegated by the parent institution, which authority is found 
in the by-laws of the IRB or EC, its operating guidelines or its charter, 
to approve or disapprove clinical trials and protocols, including 
informed consent forms and other documents (protocol amendments, 
information to be supplied to participants concerning informed consent, 
etc).  
3. 
Complies with proper personnel makeup of an IRB or EC. 
4. 
Convenes meetings using acceptable rules of order for making decisions, 
recording such decisions and implementing them. 
5. 
Files contain (a) documentation of its decisions such as are found in IRB 
or EC minutes and correspondence, (b) written guidelines or bylaws 
governing IRB or EC functions, (c) protocols, (d) protocol information 

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to be supplied to the subject, (f) correspondence between the IRB or EC 
and the investigator (consent changes, protocol amendments, etc). 
 
B. Informed Consent of Human Subjects.  The Dominican Principal and Senior 
Investigators must assure the sponsor or monitor that the informed consent 
form for participant subjects: 
 
1. 
Meets regulations as defined in 21 CFR Part 50 of U.S. Code for Informed 
Consent, and other applicable requirements. 
2. 
Has been approved by the FDI IRB or EC, including, when required, 
information to be given to the participant regarding the trial he or she is 
enrolled in. 
a. Informed consent includes the basic elements and any additional 
elements necessary. 
b. The participant and a study site representative sign the form and 
the participant is given a copy. 
 
C. Storage and Dispensing of Study Supplies.  The Dominican Principal and 
Senior Investigators (or pharmacist) must demonstrate to the sponsor or 
monitor that: 
 
1. 
Adequate and accurate written records show receipt and disposition of all 
study supplies, including dates, serial or lot numbers, quantities 
received, and each quantity dispensed, administered, or used, with 
identification of each participant.  
2. 
Purpose and reasons are given in written records for study material 
disposal, e.g., the amount contaminated, broken, or lost, and the 
quantity returned to the sponsor. 
 
D. Case Report Forms. The Dominican Principal and Senior Investigators must 
assure the sponsor that: 
 
1. 
Case Report Forms, when completed, accurately reflect the medical 
records on each participant.  
2. 
Case report forms and medical records will be accessible to the sponsor or 
other authorized inspectors during site visits.  
 
E. Files and Records.  (See section 11.6.)  The Dominican Principal and Senior 
Investigators must assure the quality, integrity, and content of his or her 
files that will be inspected by the sponsor, and any other authorized 
inspectors.  The files must contain, as a minimum: 
 
1. 
Correspondence to and from the IRB or EC and the investigator.  
2. 
Documents that include:  
a. IRB/EC-approved protocols. 
b. IRB/EC-approved protocol amendments. 

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c. IRB/EC-approved informed consent and information supplied to 
the participant. 
d. IRB/EC charter, membership, and qualifications. 
 
3. 
Clinical supplies: 
a. Record of receipt, date and quantity, batch or lot number. 
b. Disposition dates and quantity administered to each participant. 
c. Inventory records. 
 
 
12. Questions and Answers on Study Plan, Policies, Ethics 
 
12.1 Why is this study being done in infants from 6 to 24 months of age, rather than first 
in adults or older children?  
There is already a substantial literature documenting the clinical immune response 
and safety for the intradermal administration of inactivated influenza vaccine to 
adults (see sections 9.4.2 to 9.4.4), and in some studies to older childrenBeasley1960,  
or to children of unspecified ageBruyn1947, Sigel1957  .  But there is littleBruyn1949b, 
Klein1961 or no data on intradermal influenza vaccination in the age group of infants 
6 months to 2 years who are particularly susceptible to serious morbidity and 
mortality from this disease, and who are now recommended for routine universal 
vaccination in some developed countries.   
 
The investigational spacer to be studied in this trial has already been subjected to 
a number of well-conducted studies on adults without serious adverse 
consequences (see section 10.4.3.6).  Thus, it is reasonable now to study young 
children in order to gather essential data for policymaking on how such 
participants may be protected in the event of pandemic.  This study is phased with 
conservative stopping rules that can quickly respond to any clinical outcomes 
suggesting untoward risks to these children.   
 
12.2 Why is this study being done in the Dominican Republic and not in the United 
States or Europe, where its sponsor and principal parties are based?  
 
This kind of study could and should very well be conducted in the U.S. or Europe, 
as the questions it poses are relevant to those geographic areas as well.  However, 
the collaboration supporting this study benefits from the technical scientific 
and/or financial contributions of WHO and PATH, organizations with primary 
missions to seek answers to the health problems of the developing world.  This 
global perspective is also within the mission of CDC, which recognizes that 
national health and security depends on the health and security of all nations.  The 
selection of the HIRRC as a partner in this study resulted from its excellent track 
record of clinical trials of vaccines and antimicrobials (see section 10.2.1).   
 

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12.3 Why is the study planning to use Vaxigrip® instead of an influenza vaccine licensed 
in the U.S.A.?  
 
The brand of influenza vaccine to use in this study was selected by a process of 
elimination from among the seven major products on the international market, of 
which three are now sold in the U.S.   
 
One priority was to minimize the number of “investigational” aspects of the study 
by using an influenza vaccine already licensed and marketed in the Dominican 
Republic.  There are three:  (1) Vaxigrip (in prefilled syringes), (2) Fluarix® 
(GSK, manufactured in Dresden, Germany), and (3) Agrippal® S1 (Chiron, 
manufactured in Siena, Italy).  Well after planning for the study began, Fluarix 
was licensed in the U.S., but worldwide it is only available in prefilled glass 
syringes of 0.5 mL.  Thus, using Fluarix would be wasteful for administering the 
0.25 mL and 0.1 mL doses for the two needle-syringe study arms.  Also, it would 
be impractical to transfer 0.1 mL from the glass Fluarix syringe into the male tip 
of the Biojector cartridge, requiring an expensive, disposable Luer adapter (and 
would still waste 80 percent of the vaccine left in the syringe).  Agrippal S1 is 
not licensed in the U.S., and its prefilled syringes would produce the same 
obstacles as Fluarix.  Agrippal S1 also comes in single-dose vials without 
preservative, which means they cannot be used to fill multiple syringes.   
 
Neither (4) Fluzone® (Sanofi-Pasteur, manufactured in Swiftwater, PA), (5) 
Fluvirin® (Chiron, manufactured in Liverpool, UK), (6) Begrivac™ (Chiron, 
manufactured in Marburg, Germany), nor (7) Fluad® (Chiron, manufactured in 
Siena, Italy) are licensed for use in the Dominican Republic.  Moreover, Fluvirin 
is not licensed for use in ages under 4 years.  Begrivac and Fluad are also 
adjuvanted products, which may increase reactogenicity when administered into 
the skin.   
 
12.4 Why is the study not being carried out under Investigational New Drug (IND) 
application to the FDA/CBER because of the non-labeled ID route and reduced 
dosage?   
 
The original intention for this study was to seek such regulatory oversight by 
FDA, as an indicator of the most rigorous standards of research quality, even 
though it is not technically required for studies outside the U.S. for vaccines not 
anticipated to seek U.S. licensure.  However, consultations with Loris D McVittie 
(mcvittie@cber.fda.gov) of the Center for Biologics Evaluation and Research of 
FDA indicated that the agency would be unable to accept such a filing.  This is 
because FDA has no documentation on file for the chemistry, manufacturing, and 
controls (“CMC”) of Vaxigrip.  Such information is very extensive and expensive 
to assemble, and is proprietary to the manufacturer.  In the absence of any plans to 
bring this product into the U.S. market, Sanofi-Pasteur would not do so for this 
study.   

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The planned importation of multidose vials of Vaxigrip into the Dominican 
Republic for investigational use in this study will require the authorization of the 
national pharmaceutical regulatory authority of the Dominican Republic, the 
Dirección General de Drogas y Farmacias of the SESPAS [Ministry of Health].  
In addition, such studies in its facilities must be approved by appropriate officials.  
Thus, SESPAS has both regulatory oversight and political authority over this 
study, and will be exercising it accordingly.  
 
Before implementation, this study will undergo and require consideration and 
approval not only from the IRBs of CDC and the FDI, but also from appropriate 
review bodies of the Dominican Republic Ministry of Health (SESPAS), the 
World Health Organization (WHO), and the Program for Appropriate Technology 
in Health (PATH).  Such reviews will inevitably apply rigorous ethical, scientific, 
and policy scrutiny that will more than compensate for the absence of FDA 
oversight via the IND process.  
 
12.5 How will one know the amount of vaccine actually delivered into the skin by the 
intradermal spacer, versus going into deeper tissues, or dripping out onto the skin?  
 
It is very difficult for any vaccination study to document the amount of vaccine 
that actually enters the intended target tissue, whether skin (ID), muscle (IM), or 
fat (SC).  Indeed, there is evidence that a substantial proportion of intended IM 
injections likely end up in the fat.  Health workers have been found to 
underestimate the fat pad thickness in selecting needle length, or do not insert 
enough of it.Cockshott1982, Poland1997    
 
Proving the site of deposition or measuring the quantity of antigen reaching 
various tissue compartments would require contrast-imaging, biopsy, and similar 
expensive and invasive procedures.  Regulatory agencies consider these 
unnecessary in most routine clinical trials because noninvasive measures and 
outcomes are available to document that the vaccine achieves its purpose.  For ID 
injections, the presence of a wheal immediately after injection, and the absence of 
clear liquid drops on the skin, suggest that the dose went neither too deep nor too 
shallow.  Both these variables will be collected in this study by observation 
immediately after vaccine doses 1 and 2 and noted on the Case Report Form.   
 
In general for almost all vaccine trials, the immune response is empirical evidence 
of success.  If antigen is placed into the wrong compartment in some participants, 
or partly escapes through the injection site, this would be an academic issue if 
participants are otherwise adequately protected from disease.  If not, the 
immediate observations of the injection site may provide clues to any failure to 
effectively immunize, and provide a basis for further studies to elucidate the 
problem, if warranted.   
 

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12.6 Will the local side effects of intradermal injection be more painful for participants 
than needle-syringe?   
 
The first studies of the Biojector with intradermal spacer were conducted by 
anesthesiologist professor Elemer Zsigmond at the University of Illinois (see 0), 
and suggested that ID injection was less painful than using a needle to administer 
lidocaine.Zsigmond1999a, Zsigmond1999b  Other clinical studies have demonstrated 
favorable safety and tolerability.   
 
Subjects in a Navy vaccine trial who received Biojector ID injections in one arm 
and Biojector IM in the other (see 10.4.3.6.2), reported overwhelming preference 
(26 vs. 3) for either Biojector route over their prior experience with needle-
syringe (but no control needle injections were tested).   
 
In a study of infants like this one, it is difficult to assess pain.  However, parents 
and staff will be assessing local pain daily on the diary form and followup visit, 
by the participant’s reaction when the limb is touched or moved.   
 
12.7 Why is there no study arm of children receiving the reduced vaccine dose 
intradermally using needle-syringe?  
 
Although it would be of intellectual interest to compared two different methods of 
intradermal delivery -- needle-syringe (N-S) and jet injector (JI) --  such a 
comparison was precluded for two reasons.  
 
First, adding another study arm would increase the cost of the trial by about one-
third, and would extend its duration by that same degree.  Resources are 
insufficient to do so, and delaying the results is deemed inadvisable because of its 
potential importance for pandemic response.  Second, the Mantoux method of 
needle-syringe injection into the skin is considered impractical for widespread use 
for reasons of speed, logistics, occupational injuries, and waste disposal burdens 
(see section 9.1).  Thus the data would not be actionable.   
 
12.8 Why was the Biojector system picked for use in this study, rather than other 
simpler, more economical jet injectors?  
 
It is quite true that there are other needle-free jet injectors on the market or in 
development that are less complex in their construction than the Biojector, cost 
less to purchase and to buy their disposable cartridges, do not require CO2 
canisters which can be hard to procure in developing countries, and other reasons 
that might make them more suitable for use in developing countries than the 
Biojector.   
 
Despite these disadvantages, however, the Biojector is the only one among them 
for which substantial data has been accumulated in bench, animal, and clinical 

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trials for the investigational intradermal spacer (see section 10.4.3).  To try to 
study intradermal delivery of any of these other jet injectors would require 
substantial additional resources and delay in conducting such prior studies in 
order ethically to justify the type of study intended here.   
 
This study can be considered a proof-of-principle trial.  If needle-free jet injection 
is demonstrated a safe and effective means of dose-sparing for influenza 
vaccination, it would serve to encourage other jet injector developers and 
manufacturers to pursue this route.  The general concept of creating a gap 
between the orifice and the skin at the nozzle of a jet injector in order to weaken 
its stream to achieve intradermal injection is an old one (see section 9.6.3) and no 
longer under patent protection.  Thus, other manufacturers would have freedom to 
adapt their own devices with gaps of appropriate length.  This would help to 
produce a competitive marketplace for safer, simpler, and swifter intradermal 
delivery by jet injection.   
 
12.9 What is the justification for giving the third “insurance” dose earlier than the 
routine annual revaccination?  What are the risks, particularly for the control 
group which has already received the recommended two full doses? 
It would be ideal if we could know immediately the results of the final blood draw 
on day 56 (8 weeks), so that we might avoid the inconvenience and expense of a 
third dose that day in those children who already have protective levels of 
antibody against influenza.  But that is not possible.   
 
Another option considered, but not chosen, would be to break the code of random 
allocation at the time of the final blood draw and vaccinate only those who had 
received the investigational low-dose regimens by ID or IM.  These two groups 
might be at higher risk of not being protected than the control group which 
received the standard regimen for age and would not be indicated for 
revaccination until the next season.  But it was decided that breaking the code so 
soon would severely compromise the study design.  It may bias ascertainment for 
any delayed adverse events that may occur in the weeks and months after the 
active surveillance phase ends on day 56.  
 
The primary reason for vaccinating on day 56 is to ensure that all participants are 
protected promptly against influenza, to which they might be exposed in the 
months ahead while waiting for antibody levels are being determined.   
 
Extravaccination is the term used for the administration of vaccine antigens, 
earlier than otherwise indicated, for which a patient may already be immune as a 
consequence of prior immunizations or natural immunity.  The Advisory 
Committee on Immunization Practices (ACIP) of CDC, the American Academy 
of Pediatrics (AAP), and the American Academy of Family Physicians (AAFP) 
have made recommendations on this subject in their joint statement on 
combination vaccines (nominal pages 5-6, .pdf file pages 19-20 of 30: 
http://www.cdc.gov/mmwr/PDF/rr/rr4805.pdf). CDC1999, AAP1999, AAFP1999    

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To avoid missed opportunities to vaccinate in the era of newer combination 
vaccine, it sometimes becomes necessary to give additional doses of some 
antigens because they were combined with others indicated for the child at the 
time, and monovalent products may not be available.  The statement 
recommended that giving non-indicated antigens might be justified when 
“potential benefits to the child outweigh the risk of adverse events associated with 
the extra antigen(s)”.   
 
ACIP/CDC, AAP, and AAFP pointed out that “an extra dose of many live virus 
vaccines and Hib or HepB vaccines has not been found to be harmful.  However, 
the risk of adverse reactions might increase when extra doses are administered 
earlier than the recommended interval for certain vaccines (e.g., tetanus toxoid 
vaccines and pneumococcal polysaccharide vaccine).”   They went on to write 
that:  
 
“Inactivated Vaccines 
“When inactivated (killed) or subunit vaccines (which are often adsorbed to 
aluminum-salt adjuvants) are administered, the reactogenicity of the 
vaccine must be considered in balancing the benefits and risks of extra 
doses. Because clinical experience suggests low reactogenicity, an extra 
dose of Hib or HepB vaccine may be administered as part of a combination 
vaccine to complete a vaccination series for another component of the 
combination. Administration of extra doses of tetanus toxoid-containing 
vaccines earlier than the recommended intervals can increase the risk of 
hypersensitivity reactions.”  CDC1999 
 
The statement did not comment on extravaccination with influenza vaccine.  The 
investigators for the present study are unaware of any clinical data on giving a 
third dose of influenza vaccine one month after completion of the recommended 
two-dose primary series.  Unlike tetanus-toxoid and hepatitis B vaccines, 
however, the inactivated Vaxigrip influenza vaccine does not contain any 
irritating adjuvants.  It is also “split” to reduce reactogenicity compared to a 
whole-virus vaccine.  Annual influenza vaccinations are now routinely 
recommended for all infants in the U.S. in the age group to be studied here.  This 
means that some infants and young children completing their primary series at the 
end of the influenza immunization program period might be revaccinated with 
their next, third dose as early as 6 months later, at the beginning of the next 
period, which would be in full compliance with ACIP/CDC recommendations.  
 
Given this context, the researchers believe that the benefits of ensuring prompt 
protection from influenza for all the study participants and avoiding loss-to-
followup by accelerating the next permissible third dose from 6 months later to 1 
month later outweighs the anticipated routine side effects of the “insurance” dose.  
 

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12.10  Why is the final blood draw (day 56) conducted at the same time as the 3rd 
“insurance” dose of vaccine is received by the reduced-dose participants, rather 
than later?  Why is serology not performed after receipt of this 3rd dose?   
 
The 3rd dose of vaccine provided on day 56 to participants in the reduced-dose 
study arms of the trial is not provided to answer any research question of the 
study.  Instead it brings the total dose of vaccine administered to the IM-NS-0.1 
and ID-JI-0.1 study groups to 0.45 mL, essentially equivalent to the standard 0.5 
mL administered over two doses to the control IM-ND-0.25 group.  (It may also 
be more immunogenic than the standard regimen, as it was delivered in 3 doses 
over a longer interval.)   Giving the 3rd “insurance” dose is a safer, quicker, more 
reliable, and less discomfiting means to ensure that the IM-NS-0.1 and ID-JI-0.1 
children are protected against influenza, which is the major benefit for his or her 
participation in the study.   
 
The alternative to the insurance dose would be to wait several months, or longer, 
for the serologic assays results to come in.  These tests will be batched and run at 
the end of each phase to minimize dissimilarities in assay conditions.  In such a 
scenario -- not pursued here -- those with inadequate immune responses would 
need to be called back for another dose.  In the meantime, while waiting for 
serologic results, or waiting for notified parents to bring children back for 
revaccination, some of the reduced-dose children, having received only 0.2 mL of 
total vaccine (without the insurance dose), might have been unprotected all along, 
exposed to influenza, and become sick unnecessarily.   
 
A key scientific question of the study is whether two intradermal doses at 0 and 
28 days will protect the child.  Thus the day 56 blood draw is a necessity, 
regardless of whether further vaccine doses (“insurance” and “bonus”) are given 
for ethical reasons.  Since three or more doses in a primary influenza vaccination 
series would not be a practical public health strategy for the general population -- 
as two pediatric doses are the accepted standard -- there is little or no scientific 
justification to document the antibody responses after two investigational doses 
plus the 3rd insurance dose.   
 
Drawing blood from a young child’s vein for diagnostic purposes is a 
discomfiting procedure for patient, parent, and physician alike.  Although the 
results of a 4th blood draw in this study after the 3rd “insurance” dose might satisfy 
intellectual curiosity, without any valid scientific reason it would be unethical to 
subject the children to it.  It would again, take some months for serologic assays 
to be performed and to call back any patients unprotected even after the insurance 
dose.  Giving another dose of vaccine is less painful, less costly, and less 
logistically complicated than phlebotomy and overseas laboratory assays.   
 
No routine vaccinations, particularly influenza, provide 100% seroconversion 
rates in all ages.  Except in rare circumstances, such as proving rabies immunity 

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among veterinarians, standard clinical practice avoids the pain and societal 
expense of serologic assays to prove a vaccine has worked.   
 
This is the reason for our providng a 4th “bonus” dose, 6 months after the final 
blood draw, on ethical grounds to provide protection against the next season’s 
influenza for all children, including those whose response to the prior doses may 
have been suboptimal.  For “annual” recommended revaccinations, six months is 
the standard minimum interval for a patient vaccinated at the tail end of one 
Northern Hemisphere season (March 30) and at the beginning of the next 
(October 1).  
 
 
13. List of Associated Documents 
• 
Protocol – Spanish version 
• 
Informed Consent Form - Spanish 
• 
Informed Consent Form - Additional Information - Spanish 
• 
Informed Consent Form - English translation 
• 
Informed Consent Form - Additional Information - English translation 
• 
Participant Identification Form (PIF) 
• 
Enrollment Log /  Participant Inscription Form 
• 
Study Staff Responsibilities and Signatures Registry and Log 
• 
Case Report Form (CRF) 
• 
Parent’s Diary Form, Vaccination 1, Days 0 – 7 
• 
Parent’s Diary Form, Vaccination 1, Days 7 – 28 
• 
Parent’s Diary Form, Vaccination 2, Days 0 – 7 
• 
Parent’s Diary Form, Vaccination 2, Days 7 – 28 
• 
Serious Adverse Event Form (SAE) 
• 
Data Clarification Form 
• 
Appreciation Certificate and Bonus Vaccination Reminder Form 
 
 
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