Immunogenicity and Safety of Quadrivalent Influenza Vaccine In 6 to 35 Months Population: a Randomized, Double-blind, Parallel-group Ⅱ Phase of Clinical Trials
Phase: PHASE2
Conditions: Influenza
Sponsor: Shanghai Institute Of Biological Products

 
CLINICAL TRAIL PROTOCOL 
 
 
Protocol Title: Immunogenicity and Safety of Quadrivalent Influenza 
Vaccine In 6 to 35 months population: a Randomized, Double-blind, 
Parallel-group Ⅱ phase of clinical trials 
 
 
 
 
 
 
 
 
 
Collaborators: Henan Center for Disease Control and Prevention 
 
Sponsor: Shanghai Institute of Biological Products 
 
 
 
 
 
 
Document Date: January 1, 2020 
 
 

BACKGROUD INFORMATION  
Since 1978, influenza A (H1N1) influenza A (H3N2) and influenza B virus are commonly 
circling every season. Then, the trivalent influenza vaccine had been developed against the three 
kinds of influenza virus (H1N1 and H3N2 and B). According to the result of global influenza 
surveillance, the World Health Organization has been to predict the next season's pandemic strains 
for more than 30 years. WHO recommended vaccine components containing two subtypes of A 
influenza virus (H1N1 and H3N2) and a subtype of B influenza virus (B/Victoria lineage or 
B/Yamagata lineage ). However, since the 2001-2002 influenza season, two strains of influenza B 
have been circulating together. This made it difficult to predict the dominant strain of influenza B 
in the next influenza season. Several studies demonstrated that in 7 of the past 13 seasons, major 
cycling subtype of B virus strains was not included in the vaccine, which significantly reduces the 
immunological effects of the influenza vaccine. Unincluded subtype B influenza virus caused a 
potential threat to human beings. To expand the broad protection of the seasonal influenza vaccine 
and improve the protective effect of the vaccine, many influenza vaccine manufacturers had actively 
carried out the development of the quadrivalent influenza vaccine, which has become an important 
direction for the upgrading of influenza vaccine. 
 
At the same time, several studies showed that the influenza vaccine induces a poor immune 
response in young children. Unvaccinated children below 3 years older were induced by vaccine 
weaker than unvaccinated children above 3 years older younger or vaccinated children below 3 
years older, especially for the B subtype influenza virus. For 6 ~ 35 months old children were 
recommended vaccination dosage of 0.25 ml antigen of influenza inactivated vaccines. This 
recommendation was based on the observation in the 1970s of increased adverse reactivity in 
younger children with an adult’s dosage of whole-virus inactivated influenza vaccines, especially 
the fever. but at present in the seasonal flu vaccine has been without the use of an inactivated virus. 
The safety and immunogenicity of 0.5ml and 0.25ml dosage antigen of vaccine were evaluated in 
6~35 months children in a large number of clinical trials. The results showed that the high dosage 
of the split vaccine was well tolerated and could induce a strong immune response. Hence, in 2016, 
the FDA approved the adult dosage of the influenza vaccine for children above 6 months. Besides, 
many countries such as Canada, Brazil, Mexico, Finland, and Britain approved dosage of 15μg and 

7.5 μg antigen for children below 3 years. 
 
Given the situation of children-using the influenza vaccine, we carried out the clinical trial of 
the quadrivalent influenza vaccine suitable for children aged 6 to 35 months, which is of great 
significance for children to prevent influenza in China. 
 
STUDY OBJECT 
To evaluate the immunogenicity and safety of 2 doses of quadrivalent influenza virus split 
vaccine (0.25ml/ dose and 0.5ml/ dose) in healthy population aged 6-35 month., so as to provide a 
data support for phase III clinical trials. 
 
STUDY DESIGN 
A randomized, double-blind, parallel control design was set to enroll 1,980 healthy subjects 
aged 6-35 months. Subjects were randomly inoculated with experimental vaccine 1, experimental 
vaccine 2, control vaccine 1 or control vaccine 2 in a ratio of 2:2:1:1 for safety and immunogenicity 
evaluation. The inoculation time was 0 and 28 days. 
Immunogenicity observation 
⚫ 
All subjects were required to take bloody samples for 2 times, each time about 3.0ml, for 
the detection of influenza virus HI antibody: 
⚫ 
First time: before the first dose; 
⚫ 
Second dose: 28 (+10) days after the second dose. 
⚫ 
After 30 days of safety observation and 28 days of immunogenicity test results after 2 
doses of immunization, uncovered blind and the immunogenicity results were statistically 
analyzed. 
Safety observation: 
⚫ 
All adverse events were collected at 0-7 days after immunization, all adverse events at 0-
28/30 days, and all serious adverse events (SAE) within 6 months (180 days) after 2 doses 
of vaccine immunization. 
Age 
Group 
Vaccine 
Number 
Inoculation
（day） 
Serum 
collection
(day) 
Safety observation 

6-35 
Month 
A1 
Experimental Vaccine 1 
660 
0、28
（+10） 
0，56
（+10） 
1. Adverse events within 
28/30 days after each 
dose of immunization 
were collected; 
2. Serious adverse events 
(SAE) were collected 
within 6 months after the 
first dose of inoculation 
and after the whole 
course of immunization. 
A2 
Experimental Vaccine 2 
330 
B1 
Control Vaccine 1 
330 
B2 
Control Vaccine 2 
660 
Total 
 
 
1980 
 
 
 
 
STUDY ENDPOINT 
Immunogenicity 
(1) 28 days after receiving two doses of vaccine in subjects aged 6-35 months, seroconversion 
rate of HI antibodies against any subtype of influenza virus in each group. 
(2) 28 days after receiving two doses of vaccine in subjects aged 6-35 months, seroprotection 
rate of HI antibodies against any subtype of influenza virus in each group. 
(3) 28 days after receiving two doses of vaccine in subjects aged 6-35 months, GMT and GMI 
of HI antibodies against any subtype of influenza virus in each group. 
 
Safety 
(1) The proportion of all adverse reactions/events in each group through 30 days after the 
second dose. 
(2) The proportion of serious adverse events (SAE) within 6 months after inoculation in each 
group. 
 
SAFETY OBSERVATION 
Systemic adverse reactions: Fever, Diarrhea, constipation, difficulty swallowing, anorexia, 
vomiting, nausea, muscle pain (not the vaccination site), arthritis, joint pain, headache, new 
convulsions, cough and difficulty breathing, the vaccination site itching (no skin damage), 
abnormal skin and mucosa, insomnia, irritability, or inhibiting, acute allergic reaction to pain, 
fatigue, lack of power, not the vaccination site. 
 

Local adverse reactions: pain, induration, swelling, rash, redness, itching 
 
IMMUNOGENICITY OBSERVATION 
Seroconversion: 1:10 is the lowest serum dilution. Prevaccination HI titer < 1:10 and 
postvaccination titer ≥ 1:40 or a prevaccination titer ≥ 1:10 and at least a 4-fold increase in the 
postvaccination titer 
 
Seroprotection: Postvaccination titer ≥ 1:40 is considered to have antibody protection. 
 
GMT: geometric mean titer. 
 
GMI: GMT increase fold. 
 
EVALUATION CRITERIA OF SAFETY 
The safety evaluation of each group will be divided into three stages. From the first dose 
inoculation to day 7 after inoculation; From the first dose inoculation to day 21 after inoculation; 
From the second dose inoculation to day 30 after the second dose inoculation. The first stage is the 
basis for the follow-up group of the same vaccine, and will be carried out under blind. The latter 
two stages are the basis for evaluation of vaccine safety, and will be carried out under unblind. If 
each evaluation is not found level 4 local and systemic adverse reactions associated with vaccine 
and the total incidence rate of level 3 of local and systemic adverse reactions and abnormal 
laboratory test which associated with the vaccination is lower than 15%, that the vaccine will be 
acceptable safety. 
 
EVALUATION CRITERIA OF IMMUNOGENICITY 
If seroconversion rate is higher than 40%, the antibody protection rate higher than 70%, GMI 
more than 2.5 times, the vaccine will be acceptable immunogenicity. 
 
STUDY STATUS 
Record Verification: January 2020 
Study Start: April 2020 
Primary Completion: August 2020 
Study Completion: March 2021 
 
ELIGIBILITY 
Inclusion Criteria 

Healthy infants aged 6-35 months. 
Volunteer legal guardian or client informed consent, voluntarily participate in and sign 
informed consent. 
The volunteer legal guardian or client has the ability (non-illiterate) to understand the study 
procedures, to use a thermometer, scale, and fill in a diary card as required, and be able to complete 
the clinical study in compliance with the clinical trial protocol. 
 
Exclusion Criteria 
The underarm body temperature on the day of enrollment was > 37.0℃. 
Have been suffering from influenza within the previous 3 months (confirmed by either 
clinical, serological or microbiological methods). 
Any previous influenza vaccination (registered or experimental) within 6 months or any 
planned influenza vaccination during the study period. 
Allergic to any component of the vaccine, a history of allergic reactions to eggs or 
gentamicin sulfate. 
A history of severe allergy to any vaccine or drug. 
Preterm birth (delivered before 37 weeks of gestation), low birth weight baby (birth weight < 
2300g for girls, <2500g for boys). 
Dystocia, asphyxia rescue, nervous system damage history; 
Congenital malformations or developmental disorders, genetic defects, severe malnutrition, 
etc. 
Acute illness, severe chronic illness or acute attack of chronic disease on the day of 
vaccination; 
A history of live attenuated vaccination within 14 days prior to vaccination and a history of 
other vaccinations within 7 days prior to vaccination; 
Patients who received immunoenhancement or inhibitor therapy within 3 months (continued 
oral or intravenous administration for more than 14 days); 
Congenital or acquired immune deficiency, HIV infection, lymphoma, leukemia or other 
autoimmune diseases; 
History of asthma, past two years of instability requiring emergency treatment, 
hospitalization, intubation, oral or intravenous administration of corticosteroids; 
Have received blood or blood-related products; 
A history of convulsion, epilepsy, encephalopathy, guillain-barre syndrome, a history of 
mental illness or a family history; 
A history of abnormal coagulation function (such as coagulation factor deficiency, 

coagulation disease); 
Planning to relocate before the end of the study or to leave for an extended period during the 
scheduled study visit; 
Participating in or planning to participate in other clinical trials in the near future; 
The investigators determined that any conditions were inappropriate to participate in the 
clinical trial. 
 
DATA STATISTICS 
Statistics analysis of immunogenicity  
The prevaccination and postvaccination geometric mean of antibody and its 95% confidence 
interval (confidence interval, CI) will be described. The postvaccination serum conversion rate 
and protection rate of antibody and their 95% CI will be described. 
 
Comparisons will be conducted to evaluate differences in response between study groups using a 
2

 test or Fisher's exact test to compare to the difference of serum conversion rate and 
protection rate. Statistical significance will be considered at a level of α = 0.05 and all tests will be 
2-sided. 
 
If serum HI antibody conversion rate is more than 40%; protection rate more than 70%; GMI more 
than 2.5 folds, the immunogenicity will reach the design requirements.  
 
Statistics analysis of safety 
After the first dose inoculation and the second dose inoculation, the number of adverse reactions 
(rate), number of cases and adverse reaction grade were recorded. 
The number of adverse events after inoculation will be described. 
 
Comparisons will be conducted to evaluate differences in response between study groups using a 
2

 test or Fisher's exact test to compare to the difference of adverse events rate. Statistical 
significance will be considered at a level of α = 0.05 and all tests will be 2-sided. 
 
Comparison of compliance 
The rate of expulsion and the rate of drug combination will be described. 
 
Comparisons will be conducted to evaluate differences in response between study groups using a 
2

 test or Fisher's exact test to compare to the difference of the rate of expulsion and the rate of 
drug combination. Statistical significance will be considered at a level of α = 0.05 and all tests will 

be 2-sided. 

APPENDIX Ⅰ. GRADING STANDARDS FOR SEVERITY OF SAFETY INFORMATION 
The Event of clinical response and laboratory abnormalities after vaccination is judged by the China State Food and Drug Administration on "Guidelines for the 
classification of adverse events in vaccine clinical trials ". 
Grading of Local Adverse Events 
Local 
Adverse 
Reactions 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potential Life Threatened 
(Grade 4) 
Pain 
Resistance or withdrawal after 
contact or touch 
Crying 
after 
touching 
or 
touching, but soothing 
There is no comfort in continual 
crying 
Require 
emergency 
or 
hospitalization 
Induration 
Diameter < 2.5 cm 
Diameter ≥2.5cm, and area < 
50% of the inoculated limb 
(anatomically, the limb where 
the inoculated site is located, 
such as the upper arm or thigh) 
Area ≥ 50% of inoculated limb 
or 
ulceration 
or 
secondary 
infection or phlebitis or wound 
drainage 
Abscesses, 
exfoliating 
dermatitis, and necrosis of the 
dermis or deep tissue 
Redness 
Diameter < 2.5 cm 
Diameter ≥2.5cm, and area < 
50% of the inoculated limb 
(anatomically, the limb where 
the inoculated site is located, 
such as the upper arm or thigh) 
Area ≥ 50% of inoculated limb 
or 
ulceration 
or 
secondary 
infection or phlebitis or wound 
drainage 
Abscesses, 
exfoliating 
dermatitis, and necrosis of the 
dermis or deep tissue 

Rash 
Swelling 
<15 mm and does not affect 
activity 
15-30 mm or  affect activity 
>30 mm or limit daily activity 
Gangrene 
Itching 
Itching at the site of inoculation 
was relieved by itself or within 
48 hours after treatment 
The site of inoculation was itchy, 
which did not relieve within 48 
hours after treatment 
Affect daily life 
N/A 

 
Grading of Systemic Adverse Events 
Systemic 
Adverse 
Reactions 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potential Life Threatened 
(Grade 4) 
Fever 
37.5-38.0℃ 
38.0-39.5℃ 
≥39.5℃ 
 
Diarrhea 
Mild or transient, 3 ~ 4 stools 
per day or mild diarrhea lasting 
for less than 1 weeks 
Moderate or persistent, 5~7 
times per day, or more than 1 
week diarrhea 
> 7 times of water stool per day, 
or blood diarrhea, orthostatic 
hypotension 
Hypotension shock requiring 
hospitalization 
Constipation 
Need stool softener and diet 
adjustment 
Need a laxative 
Stubborn constipation needs to 
be dredge manually or use 
enema 
Toxic megacolon or ileus 
Dysphagia 
Slight discomfort in swallowing 
Diet restricted 
Diet and conversation are very 
limited; Cannot eat solid food 
No liquid food; Need intravenous 
nutrition 
Anorexia 
Decreased appetite, but not 
reduced food intake 
Decreased appetite, decreased 
food intake, but no significant 
weight loss 
Decreased appetite and 
significantly decreased body 
weight 
Need for intervention(E.g., 
gastric feeding, parenteral 
nutrition) 
Vomiting 
1~2/24 hours with no impact on 
activities 
3~5/24 hours or restricted 
activities 
>6 times within 24 hours or 
intravenous rehydration is 
required 
Hospitalize or other routes of 
nutrition are required because of 
hypotensive shock. 

Nausea 
Transient (<24 hours) or 
intermittent and basically normal 
food intake 
Reduced food intake due to 
persistent nausea (24~48 hours) 
Persistent nausea causes almost 
no food intake (>48 hours) or 
requires intravenous fluids 
A threat to life(E.g. hypotensive 
shock) 
muscle pain 
(Non-
inoculated 
site) 
Do not affect daily activities 
Slightly affect daily activities 
Severe muscle pain that severely 
affects daily activities 
Emergency or hospitalize 
Arthritis 
Mild pain with inflammation, 
erythema or joint swelling; But 
without hindrance to function 
Moderate pain with 
inflammation, erythema or joint 
swelling; Hinder function, but do 
not affect daily activities 
Severe pain with inflammation, 
erythema or joint swelling; 
Impact on daily activities 
Permanent and/or incapacitated 
joint injury 
Arthralgia 
Mild pain, no impairment of 
function 
Moderate pain; Painkillers and/or 
pain are required to interfere with 
function, but do not affect daily 
activities 
Severe pain; The need for 
painkillers and/or pain affects 
daily activities 
Impotence pain 
Headache 
No effect on daily activities, no 
treatment required 
Having a temporary effect on 
daily activities and may require 
treatment or intervention. 
Seriously affecting daily 
activities, requiring treatment or 
intervention 
Stubbornness, emergency or 
hospitalize 
New 
convulsions 
Duration of convulsions < 5 
minutes, and status after 
convulsions <24 hours 
Duration of convulsions ≥5~< 
20 minutes, and status after 
convulsions <24 hours 
Duration of convulsions ≥ 20 
minutes or status after 
convulsions >24 hours 
Prolonged and multiple 
convulsions (e.g., status 

convulsions) or difficult to 
control (e.g., intractable epilepsy) 
Cough 
Transient, no treatment required 
Keep coughing 
Coughing, uncontrollable 
treatment 
Emergency or hospitalize 
Difficulty in 
breathing 
Moving difficulty in breathing 
Normal activity difficulty in 
breathing 
Rest time difficulty in breathing 
Difficulty in breathing, need 
oxygen therapy, hospitalize or 
assisted breathing 
Pruritus at 
non-inoculated 
site (no skin 
damage) 
Slightly itchy, not affecting or 
slightly affecting everyday life 
Itching affects everyday life 
Pruritus makes daily life 
impossible 
NA 
Abnormal 
mucous 
membrane of 
skin 
Erythema/pruritus/color Change 
Diffuse rash/maculopapular 
rash/dry/detritus 
Blister/exudate/desquamate/ulcer 
Exfoliative dermatitis involving 
mucosa, or erythema multiforme, 
or suspected Stevens-Johnsons 
syndrome 
Insomnia * 
Mild difficulty in falling asleep, 
not affecting or slightly affecting 
daily life 
Moderate difficulty in falling 
asleep, affecting everyday life 
Severe sleep difficulty, severe 
impact on daily life, need to be 
treated or hospitalize 
NA 
To provoke or 
inhibit. 
Mild irritability or mild inhibition Irritable or sleepy. 
Unable to soothe or react poorly 
NA 

Acute allergic 
reaction ** 
Local urticaria (blister), no 
treatment required 
Local urticaria, requiring 
treatment or mild angioedema, no 
treatment required 
Extensive urticaria or 
angioedema requiring treatment 
or mild bronchospasm 
Anaphylactic shock or life-
threatening bronchospasm or 
laryngeal edema 
Fatigue, 
fatigue 
Do not affect daily activities 
Affect normal daily activities 
Seriously affect daily activities, 
unable to work 
Emergency or hospitalize 
Pain at non-
inoculated site 
#(Location 
identified at 
the time of 
reporting) 
Mild pain, not affecting or 
slightly affecting daily life 
Pain affects everyday life 
Pain does not work in everyday 
life 
Disabling pain, loss of basic self-
care 

 
Grading of Blood Biochemical 
Serum 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potential Life 
Threatened 
(Grade 4) 
Elevated liver function 
caused by AST ALT, 
influence factors 
1.25-2.5 *ULN 
2.6-5*ULN 
5.1-10*ULN 
>10*ULN 
Creatinine 
1.1-1.5*ULN 
1.6-3.0*ULN 
3.1-6*ULN 
>6*ULN 
Urea nitrogen (BUN) 
1.25-2.5 *ULN 
2.6-5*ULN 
5.1-10*ULN 
>10*ULN 
Bilirubin: elevation 
caused by factors, but 
functional 
examination is normal 
1.1-1.5*ULN 
1.6-2.0*ULN 
2.0-3.0*ULN 
>3.0*ULN 
Bilirubin: the increase 
caused by the factors 
associated with the 
increase of liver 
function test index 
1.1-1.25*ULN 
1.26-1.5*ULN 
1.51-1.75*ULN 
>1.75*ULN 
 
Grading of Blood Routine 
Detection indicators 
Level 1 
Level 2 
Level 3 
Level 4 
Liver function (ALT, 
AST increased) 
1.25~<2.5 ×ULN 
2.5~<5.0×ULN 
5.0~<10×ULN 
≥10×ULN 
Total bilirubin 
increased 
(Mg/dL; Μmol/L ） 
1.1~<1.6×ULN 
1.6~<2.6×ULN 
2.6~5.0×ULN 
≥5.0×ULN 
 
Grading of Urine Routine 
Test Indicators/Grades 
Level 1 
Level 2 
Level 3 
Level 4 
WBC (109/L) 
11~<13 
13~<15 
15~<30 
≥30 
WBC (109/L) 
2.000~2.499 
1.500~1.999 
1.000~1.499 
<1.000 
Lymphocyte Decrease (LY, 
109/L) 
0.75~1.00 
0.5~0.749 
0.25~0.49 
<0.25 

Neutropenia (ANC, 109/L) 
0.800~1.000 
0.600~0.799 
0.400~0.599 
<0.400 
Eosinophils (Eos, 109/L) 
0.65~1.5 
1.51~5.0 
>5.0 
High 
eosinophil 
syndrome 
Thrombocytopenia (PLT, 
109/L) 
NA 
50~75 
25~49 
<25 
Low hemoglobin (g/dL) 
9.5~10.4 
8.5~<9.5 
6.5~<8.5 
<6.5 
 
 
 

APPENDIX Ⅱ. THE RELATIONSHIP BETWEEN ADVERSE 
EVENTS AND TRIAL VACCINES 
 
Absolutely unrelated: Because of other factors lead to adverse events, there is 
evidence that adverse events are caused by other causes, not related to vaccination. 
 
Possible unrelated: Adverse events probable be caused by other factors, such as the 
clinical status of the subjects, other treatment or concomitant medication, inconsistent 
with the known adverse reactions of vaccination. 
 
Quite possible related: Adverse events are consistent with known information of 
vaccine, and there is a causal relationship with the vaccine, not by other factors, such 
as the clinical status of the subjects, or other treatment with medication. 
 
Possible related: Adverse events are consistent with known information of vaccine, 
and there is a causal relationship with the vaccine, not by other factors, such as the 
clinical status of the subjects, or other treatment with medication. 
 
Related: Adverse events are consistent with known information of vaccine, and there 
is a causal relationship with the vaccine, not by other factors, such as the clinical status 
of the subjects, or other treatment with medication. In addition, adverse events will be 
repeated when subjects are tested with the vaccine.