Immunogenicity and safety after one dose of adjuvanted …...1 Immunogenicity and safety after one...

Elsevier Editorial System(tm) for The Lancet Manuscript Draft Manuscript Number: Title: Immunogenicity and safety after one dose of adjuvanted whole-virion, adjuvanted split-virion and non-adjuvanted split-virion influenza A （H1N1）vaccines: a double-blind, randomized and controlled trial Article Type: Article (Randomised Controlled Trial) Corresponding Author: Mr. Weidong Yin, MBA Corresponding Author's Institution: Sinovac Biotech Co., Ltd First Author: Jiang Wu, MSc Order of Authors: Jiang Wu, MSc ; Wei Li, MSc; Hua-Qing Wang, Msc; Jiang-Ting Chen, BSc; Min Lu, PhD; Ji-Chen Zhou, BSc; Xiao-Feng Liang, MSc; Han-Hua Fang, BSc; Yan Liu, MSc; Li-Ying Liu, BSc; Xu Wang, BSc; Wu-Li Zhang, BSc; Xiao-Mei Zhang, BSc; Li-Fei Song, MSc; Yuan-Zheng Qiu, PhD; Chang-Gui Li, MSc; Jun-Zhi Wang, PhD; Yu Wang, PhD; Weidong Yin, MBA Abstract: Background A novel swine-origin influenza A (H1N1) virus had caused a pandemic influenza. The vaccines against the virus is undergoing clinical evaluation in many countries. Herein we report the results of immunogenicity and safety after one dose of adjuvanted whole-virion, adjuvanted split-virion and non-adjuvanted split-virion influenza A (H1N1) vaccines. Methods A double-blind, randomized and controlled trial was conducted in 1614 participants including 101 elders (≥61 years), 706 adults (18-60 years), 403 adolescents (12-17 years) and 404 children (3-11 years). The elders were assigned to receive 1 dose of 10-μg, adjuvanted whole-virion vaccine. The adults, adolescents and children were randomly assigned to receive 2 doses of the study vaccines 21 days apart. The adults received 5-30 μg of the three formulations of the study vaccines or placebo. The adolescents and children received 7*5-30 μg of adjuvanted or non-adjuvanted split-virion vaccines. Blood samples were collected for haemagglutination-inhibition (HI) assay. Adverse events were recorded after vaccination. The trial is registered with the ClinicalTrials.gov number NCT00956111. Findings All formulations were well tolerated with no serious adverse events. Most local and systemic reactions were mild or moderate. Before vaccination, some degree of preexisting immunity was found in participants more than 12 years. One dose of all the three formulations of the study vaccines induced satisfactory HI response complying with the licensure criteria set out by European Union in participants aged 3-60 years. The dose-dependent relationship was generally found. The highest immune response was observed in adults, adolescents and children after one dose of 30-μg non-adjuvanted split-virion vaccine with HI geometric mean titer (GMT) of 113-601, seroconversion rate of 95-98% and seroprotection rate of 96-100%. An unexpected finding was that aluminium adjuvant did not boost the immune response. One dose of 15-μg non-adjuvanted split-virion vaccine induced immune response with HI GMT of 79-475, seroconversion rate of 81-98% and seroprotection rate of 81-98% in adults, adolescents and children, which was better than that induced by 15-μg adjuvanted split-virion vaccine. Interpretation All the investigated three formulations of influenza A (H1N1) vaccine were well tolerated and immunogenic. One dose vaccination could induce satisfactory immune response in population aged 3-60 years. In the view of antigen sparing, the 15-μg non-adjuvanted split-virion vaccine is applicable for mass immunization.

1

Immunogenicity and safety after one dose of adjuvanted whole-virion,

adjuvanted split-virion and non-adjuvanted split-virion influenza A

（H1N1）vaccines: a double-blind, randomized and controled trial

Jiang Wu1, Wei Li

2, Hua-Qing Wang

3, Jiang-Ting Chen

4, Min Lu

1, Ji-Chen Zhou

5, Xiao-Feng Liang

3,

Han-Hua Fang2, Yan Liu

4, Li-Ying Liu

5, Xu Wang

4, Wu-Li Zhang

5, Xiao-Mei Zhang

4, Li-Fei Song

4,

Yuan-Zheng Qiu4, Chang-Gui Li

2, Jun-Zhi Wang

2, Yu Wang

3, Wei-Dong Yin

4†

1 Beijing Centers for Disease Control and Prevention, Beijing, China

2 National Institute for the Control of Pharmaceuticals and Biological Products, Beijing, China

3 Chinese Centers for Disease Control and Prevention

4 Sinovac Biotech Co. Ltd, Beijing, China

5 Huairou Center for Disease Control and Prevention, Beijing, China

† Corresponding authors:

Wei-Dong Yin

Tel: +86-10-82890878 Fax: +86-10-62966910 Email: [email protected]

Manuscript

2

Summary

Background A novel swine-origin influenza A (H1N1) virus had caused a pandemic influenza. The

vaccines against the virus is undergoing clinical evaluation in many countries. Herein we report the results

of immunogenicity and safety after one dose of adjuvanted whole-virion, adjuvanted split-virion and

non-adjuvanted split-virion influenza A (H1N1) vaccines.

Methods A double-blind, randomized and controled trial was conducted in 1614 participants including

101 elders (≥61 years), 706 adults (18–60 years), 403 adolescents (12–17 years) and 404 children (3–11

years). The elders were assigned to receive 1 dose of 10-μg, adjuvanted whole-virion vaccine. The adults,

adolescents and children were randomly assigned to receive 2 doses of the study vaccines 21 days apart.

The adults received 5–30 μg of the three formulations of the study vaccines or placebo. The adolescents

and children received 7·5–30 μg of adjuvanted or non-adjuvanted split-virion vaccines. Blood samples

were collected for hemagglutination-inhibition (HI) assay. Adverse events were recorded after vaccination.

The trial is registered with the ClinicalTrials.gov number NCT00956111.

Findings All formulations were well tolerated with no serious adverse events. Most local and systemic

reactions were mild or moderate. Before vaccination, some degree of preexisting immunity was found in

participants more than 12 years. One dose of all the three formulations of the study vaccines induced

satisfactory HI response complying with the licensure criteria set out by European Union in participants

aged 3–60 years. The dose-dependent relationship was generally found. The highest immune response was

observed in adults, adolescents and children after one dose of 30-μg non-adjuvanted split-virion vaccine

with HI geometric mean titer (GMT) of 113–601, seroconversion rate of 95–98% and seroprotection rate

of 96–100%. An unexpected finding was that aluminum adjuvant did not boost the immune response. One

dose of 15-μg non-adjuvanted split-virion vaccine induced immune response with HI GMT of 79–475,

seroconversion rate of 81–98% and seroprotection rate of 81–98% in adults, adolescents and children,

which was better than that induced by 15-μg adjuvanted split-virion vaccine.

Interpretation All the investigated three formulations of influenza A (H1N1) vaccine were well

3

tolerated and immunogenic. One dose vaccination could induce satisfactory immune response in

population aged 3–60 years. In the view of antigen sparing, the 15-μg non-adjuvanted split-virion vaccine

is applicable for mass immunization.

(Summary word count: 382)

4

Introduction

A novel swine-origin influenza A (H1N1) virus emerging in North America had caused a pandemic

influenza as declared by the World Health Organization (WHO) on June 11, 2009.1 As of August 13, 2009,

more than 170 countries and regions worldwide reported over 180,000 cases, 1799 of which were fatal.2

The rapid spread of the virus and high morbidity caused by it has drawn wide attention from international

community. So far vaccine is considered as the most effective means to reduce the morbidity and mortality

after infection, especial for those who have underlying medical conditions.

Up to now, clinical evaluation of influenza A (H1N1) vaccine is ongoing in China, Australia, USA, Austria

and European Union.3 None of these trials has been completed. As registered in ClinicalTrials.gov, the

vaccines to be evaluated included whole-virion and split-virion vaccines, adjuvanted and non-adjuvanted

vaccines, egg-derived and cell-derived vaccines, all of which were monovalent vaccine. The dosage of the

investigated vaccines ranged from 3·75 μg to 30 μg, depending on the adjuvant and vaccine type. The

study population covered infants, children, adolescents, adults and elders. Two-dose vaccination schedule

was adopted by all these trials, most of which were 21-day interval. Based on the experience with H5N1

vaccine trials which showed that two doses were necessary to induce satisfactory immune response in

immunologically naïve population, 4–7

it is little expected that one dose could elicit satisfactory immune

response.

To evaluate the immunogenicity and safety of influenza A (H1N1) vaccines manufactured by Sinovac

Biotech (Beijing, China), a double-blind, randomized and controled trial was conducted in elders, adults,

adolescents and children. The study design is two-dose vaccination and participants will be followed up

for at least 42 days. After participants received one dose and completed the visit of day 21, however, as

required by Chinese Centers for Disease Control and Prevention (CDC) and reviewed by Data Security

Monitor Board and experts committee, the investigators and sponsor decided to do code-breaking in order

to have preliminary evaluation of the investigated vaccines. Herein we reported the results after one dose

of the study vaccines. The results after two doses will be presented in additional report when the entire

trial is completed.

5

Methods

Participants

From July 15 to 25, 2009, a total of 1614 participants were enrolled from healthy male or non-pregnant

female volunteers aged 3-75 years in Huairou District, Beijing, China. Eligible participants were clinically

healthy as determined by history talking and physical examination. The main exclusion criteria were:

current febrile illness as determined by over 37·0℃ axillary temperature on the day of vaccination; cases or

cured cases of influenza A (H1N1) virus or those close contacting with cases; any acute diseases; allergic

history to vaccines and eggs; history of hematologic, hepatic, renal, cardiac or respiratory diseases;

immunodeficiency; treatment with cytotoxic or immunosuppressive drugs within the past 6 months;

receipt of blood, blood-derived products or any other vaccines within the past 3 months; currently attended

or planed to attend any other clinical trials; unable to comply with the visit schedule. Woman volunteers

who were of pregnancy or lactation or planned to be pregnant in 60 days were excluded.

The trial was registered with the ClinicalTrials.gov number NCT00956111 and approved by the Chinese

regulatory authority State Food and Drug Administration (SFDA) and Chinese CDC. All relevant

documents were approved by the ethical review committee of Beijing Centers for Disease Control and

Prevention. All participants gave their written informed consent.

Procedures

The vaccine strain X-179A was used in this study for vaccine production which was a reassortant between

A/California/07/2009 and A/PR/8/34 prepared by New York Medical College using classical reassortment

technology. The strain was recommended by WHO for the development of influenza A (H1N1) vaccine.8

The vaccines used in this study were produced by Sinovac Biotech (Beijing, China) on a pilot scale in

embryonated hens’ eggs as previously reported.6 Three formulations of influenza A (H1N1) vaccine were

used: aluminum-adjuvanted whole-virion vaccine, aluminmium-adjuvanted split-virion vaccine and

non-adjuvanted split-virion vaccine, which were prepared in vial as 20 μg/mL/vial (lot W-20090702), 30

μg/mL/vial (lot S-20090702) and 60 μg/mL/vial (lot 20090704), respectively. Each vial contained 2 or 4

doses. The two adjuvanted vaccines were formulated with aluminum hydroxide to contain aluminum of

6

0·3–0·6 mg/mL. The placebo control was phosphate buffer saline (lot E-R090630-110). All vaccines and

placebo were free of preservative. The vaccines or placebo were given at two-dose schedule (day 0, 21) or

one-dose schedule. The vaccines or placebo given at two-dose schedule (for adults, adolescents and

children) were subjected to stratification and randomization. A randomization list was generated by a

statistician who was not involved in the rest of the trial. Then the randomized vaccines were blindly

labeled with a sequential number according to the randomization list. The vaccines given at one-dose

schedule (for elders) were openly labelled. The vaccines were stored and transported at 2℃ to 8℃.

The trial profile is shown in Figure 1. The eligible participants were stratified by age: elders (≥61 years),

adults (18–60 years), adolescent (12–17 years) and children (3–11 years). The elders were assigned to

receive one dose of openly-labelled, 10-μg, adjuvanted whole-virion vaccine. The adults, adolescents and

children were randomly assigned to receive two doses of blind-labelled vaccines or placebo 21 days apart.

The adults received 2 doses of 5 or 10 μg of adjuvanted, whole-virion vaccine, or 7·5 or 15 μg of

adjuvanted, split-virion vaccine, or 15 or 30 μg of non-adjuvanted, split-virion vaccine, or placebo. The

adolescents and children received 2 doses of 7·5 or 15 μg of adjuvanted, split-virion vaccine, or 15 or 30

μg of non-adjuvanted, split-virion vaccine. Participants received either 0·5 or 0·25 mL per dose, depending

on the random allocation. The vaccines were administered intramuscularly into the deltoid muscle by a

nurse who did not participated in the safety observation and immunogenic assessment.

Participants were kept to observe adverse events for 30 minutes after vaccination. Diary cards were used

to record the presence and intensity of adverse events. In the next three days following vaccination, any

local adverse events (pain, erythema, swelling, induration, rash and itching) at the injection site and

systemic adverse events (allergic reactions, headache, dizziness, fatigue, angina, nausea, vomit,

inappetence, abdominal pain, diarrhea, myalgia, arthralgia, cough, fever) would be recorded on the diary

cards. The diameters of any erythema, swelling, induration and rash were determined by investigators, and

the daily axillary temperatures were measured by volunteers themselves. The adverse events recorded by

participants were reviewed by investigators. All adverse events were graded by a standard scale.9,10

Serum samples for the assessment of humoral immune response were collected on day 0, 14, 21 and 35 for

elders, on day 0, 14, 21, 35 and 42 for adults and adolescents, on day 0, 21 and 42 for children. All serum

samples were assayed by hemagglutination-inhibition (HI) methods against the homologous X-179A strain.

7

The immunogenic assays were blindly performed. HI assays were done in accordance with established

procedures using turkey erythrocytes as reported previously.6

Briefly, serum samples were treated with

receptor-destroying enzyme (cholera filtrate, Sigma) at 36℃ for 16 h before titration in order to remove

non-specific inhibitors of agglutination. Samples were tested in two-fold dilution starting with 1:10

dilution. The titers were expressed as the reciprocal of the highest dilution that showed complete inhibition

of haemagglutination. All samples were assayed in duplicate and double-checked by at least two persons.

For the purpose of calculation, HI titers below 1:10 were assigned a value of 1:5.

Statistical analysis

Safety and immunogenicity were the co-primary objectives. The incidence of adverse events was based on

the most severe response, expressed in terms of the number and proportion of individuals who had adverse

events. The safety data were summarized in the full-analysis set.

The immunologic endpoints were based on HI licensure criteria set out by the European Union Committee

for Medicinal Products for Human Use (CHMP) including geometric mean titer (GMT),

post-to-pre-vaccination GMT ratio, seroconversion rate and seroprotection rate.11,12

Seroconversion rate is

defined as the percentage of vaccinees who have a titer before vaccination of less than 1:10 and a titer after

vaccination of 1:40 or more, or a titer before vaccination of 1:10 or more and at least a fourfold increase

after vaccination. HI titer≥1:40 is considered as seroprotection in seasonal influenza vaccine, which is

temporally adopted for influenza A (H1N1) vaccine in this study. The antibody titers were transformed

into logarithmic scale for the calculation of GMT. The titer distributions were described with reverse

cumulative distribution curves. The immunogenic data were summarized in the per-protocol set.

The results of immunogenicity and safety were summarized with point estimates and two-sided 95%CI.

Pearson’s chi-square test or Fisher’s exact test were used to compare groups when relevant. The statistical

analysis was conducted by an independent statistician. Statistical analysis was done by intension to

treatment. The significance level was 0·05 (two-sided).

Role of the funding source

This study was funded by a research grant from Sinovac Biotech which had a role in study design, trial

monitoring, interpretation of data and writing of the report. The sponsor had no role in data collection. The

corresponding authors had full access to all the data and had final responsibility for the decision to submit

8

for publication.

Results

A total of 1614 participants were enrolled and stratified by age including 101 elders, 706 adults, 403

adolescents and 404 children. The adults were randomly allocated into 7 groups with 100–102 per group.

The adolescents were randomly allocated into 4 groups with 100–102 per group. The children were

randomly allocated into 4 groups with 100–102 per group. All participants received the first dose and

completed the safety observation, and one elder was excluded by investigator due to protocol violation.

Then 1613 participants were included in safety analysis. Eight and 31 participants dropped out on day 14

and 21, respectively. Then 1613, 1201 (404 children not attended the visit) and 1582 participants were

included in immunogenicity analysis on day 0, 14 and 21, respectively. The demographic details of the

study population are summarized in Table 1 and 2. In term of age and sex, statistical differences were not

found in the vaccination groups of adults (P=0·964 and P=0·837), of adolescents (P=0·904 and P=0·438),

of children (P=0·479 and P=1·000).

All vaccine formulations were well tolerated without immediate allergic reactions or serious adverse

events. The adverse reactions are shown in Table 3 and 4. A total of 189 participants (incidence of 11·7%)

reported adverse reactions, and the incidences of mild, moderate and severe reactions were 9·1%, 2·5%

and 0·1%, respectively. Pain (5·1%) at the injection site was the most common local reaction. The main

systemic reactions were myalgia (1·9%), headache (1·7%), fatigue (1·7%) and fever (1·6%). In adults, the

incidence of adverse reactions in participants receiving placebo was statistically similar to that in

participants receiving the study vaccines (7% vs 13·2%, P=0·08). The incidences of adverse reactions were

not statistically different in the 6 groups of adults receiving the study vaccines (P=0·249), in the 4 groups

of adolescents (P=0·991), and in the 4 groups of children (P=0·262).

The results of HI assays are summarized in Table 5, 6 and Figure 2. Before vaccination, 80 out of 1613

participants (5·0%) included in immunogenic analysis showed seroprotective antibody (HI≥1:40). The

pre-vaccination seroprotection rate in the 4 age strata ranged from 0·2% to 10·2% with the adolescents

having the highest and the children having the lowest. After receiving one dose of the study vaccines, HI

antibody responses were significantly induced in adults, adolescents and children. The GMTs, GMT ratios,

9

seroconversion rates and seroprotection rates in the 3 age strata were 44–601, 8–66, 64–98% and

64–100% on day 21, respectively. However, the immune response was lower in elders where the GMT,

GMT ratio, seroconvertion rate and seroprotection rate were 41, 5, 50% and 56% on day 14, respectively.

The dose-dependent relationship was generally found in the three formulations of the study vaccines. The

highest immune response was observed in adults, adolescents and children after one dose of 30-μg

non-adjuvanted split-virion vaccine with HI GMT of 113–601, seroconversion rate of 95–98% and

seroprotection rate of 96–100%. One dose of 15-μg non-adjuvanted split-virion vaccine also induced high

immune response with HI GMT of 79–475, seroconversion rate of 81–98% and seroprotection rate of

81–98% in adults, adolescents and children, which was better than that induced by 15-μg adjuvanted

split-virion vaccine (P

10

might be applicable.

An unexpected finding of this trial is that influenza A (H1N1) vaccine is of good immunogenicity. Except

for in elders (≥61 years), one dose of the investigated influenza A (H1N1) vaccines induced satisfactory

immune response complying with all the three licensure criteria (GMT ratio, seroconversion rate and

seroprotection rate) set out by EU CHMP in population aged 3-60 years. The immune response induced by

influenza A (H1N1) vaccine is similar to that of seasonal influenza vaccine. In adults, all the three

formulations (each containing two dosages) induced immune response meeting the three criteria 14 and 21

days after one dose. In adolescents, all the two formulations (each containing two dosages) induced

immune response meeting the three criteria 14 and 21 days after one dose. In children, except for 7.5-μg

adjuvanted split-virion vaccine, the 15-μg adjuvanted split-virion vaccine and 15- and 30-μg

non-adjuvanted split-virion vaccine induced immune response meeting the three criteria 21 days after one

dose. In elders, although the seroprotection rate after one dose of 10-μg adjuvanted whole-virion vaccine

did not comply with the EU CHMP criteria, it is very close to it.

Another unexpected finding is that aluminum adjuvant dose not boost the immune response. However, the

aluminum-adjuvanted vaccines elicited lower immune response than its non-adjuvanted counterparts did,

which was found in children, adolescents and adults. This phenomena was also found in previous trial with

H5N1 vaccine.5 In view of the sustained release endowed by adjuvant, a longer term may be needed to

observe the effect of adjuvant on immune response. At the current stage, it is too early to draw a

conclusion about the adjuvant effect.

Before vaccination, some degree of preexisting immunity against the novel influenza A (H1N1) virus was

found in the study population more than 12 years, which agreed with our previous study.14

The satisfactory

immune response after one dose vaccination may largely attribute to the preexisting immunity. In children

aged 3–11 years, the immune response after one dose was lower than that in adolescents and adults, which

may largely attribute to the absence of preexisting immunity. Although preexisting immunity was found in

elders, the immune response in elders did not comply with the licensure criteria after one dose, which was

probable due to the effect of age on immune response. Then the preexisting immunity and age would be

the two critical factors affecting the immune response after vaccination.

The safety data showed that the three formulations of the study vaccines were well tolerated without

11

serious adverse reactions. Most of the adverse reactions were mild or moderate. The profile of adverse

reactions was similar to that of seasonal influenza vaccines. Although the incidence of adverse reactions in

participants receiving vaccines was slightly higher than that in participants receiving placebo, the

difference was not statistically significant.

Based on the immunogenicity and safety results in this study and the considerations for antigen sparing

and production capacity, we can conclude that 15-μg non-adjuvanted split-virion vaccine is applicable for

the immunization of population aged 3–60 years. For adults aged 18–60 years, 5-μg adjuvanted

whole-virion vaccine and 7.5-μg adjuvanted split-virion vaccine are also applicable. For adolescents,

7.5-μg adjuvanted split-virion vaccine is also applicable. What formulation and dosage is applicable for

the immunization in elders needs further study.

In conclusion, this study found that all the investigated three formulations of influenza A (H1N1) vaccine

were well tolerated and immunogenic. One dose vaccination could induce satisfactory immune response in

population aged 3–60 years. In the view of antigen sparing, the 15-μg non-adjuvanted split-virion vaccine

is applicable for mass immunization. These results in this study would be helpful for the ongoing clinical

trials of influenza A (H1N1) vaccines produced by other manufacturers and it also provided public health

authorities with the feasibility to deploy vaccine ahead of the planned schedule in order to control

influenza A (H1N1) as earlier as possible.

(Summary word count: 2955)

Contributors

All authors played their roles in the study. J Wu was the principal investigator leading the clinical team

assisted by M Lu, JC Zhou, LY Liu and WL Zhang. J Wu, JT Chen, WD Yin, HQ Wang, XF Liang and Y

Wang jointly designed the protocol with expert advice. XM Zhang was responsible for the production of

the study vaccine. X Wang and YZ Qiu were responsible for trial monitoring. M Lu and Y Liu did the data

analysis. HH Fang, CG Li, JZ Wang, W Li and LF Song were responsible for the laboratory assay.

Conflict of interest statement

WD Yin, XM Zhang, JT Chen, Y Liu, X Wang, YZ Qiu and LF Song are employed by Sinovac Biotech. J

Wu had ever received research funding from Sinovac Biotech. All other authors declare no conflict of

12

interest.

Acknowledgments

We appreciate the US Centers for Disease Control and Prevention and the UK National Institute for

Biological Standards and Control for the kindly providing the vaccine strains and reference standards. We

also thank YD Zheng from Peking University Health Science Center for the help of statistical analysis, X

Zhong and YD Zhou from Sinova Biotech for the help of manuscript writing.

References

1. World Health Organization. DG Statement following the meeting of the Emergency Committee.

http://www.who.int/csr/disease/swineflu/4th_meeting_ihr/en/index.html (accessed August 5, 2009)

2. World Health Organization. Laboratory-confirmed cases of pandemic (H1N1) 2009 as officially

reported to WHO by States Parties to the IHR (2005) as of 13 August 2009.

http://www.who.int/csr/don/2009_08_19/en/index.htmlon (accessed August 20, 2009)

3. ClinicalTrials.gov. H1N1 vaccine trial. http://clinicaltrials.gov/ct2/results?term= H1N1+vaccine+trial

(accessed August 20, 2009)

4. Treanor JJ, Campbell JD, Zangwill KM, Rowe T, Wolff M. Safety and immunogenicity of an

inactivated subvirion influenza A (H5N1) vaccine. N Engl J Med 2006; 354: 1343–51.

5. Bresson JL, Perronne C, Launay O, et al. Safety and immunogenicity of an inactivated split-virion

influenza A/Vietnam/1194/2004 (H5N1) Vaccine: phaseⅠrandomized trial. Lancet 2006; 367: 1657–64.

6. Lin J, Zhang J, Dong X, et al. Safety and immunogenicity of an inactivated adjuvanted whole-virion

influenza A (H5N1) vaccine: a phase I randomised controled trial. Lancet 2006; 368: 991–97.

7. Leroux-Roels I, Borkowski A, Vanwolleghem T, et al. Antigen sparing and cross-reactive immunity

with an adjuvanted rH5N1 prototype pandemic influenza vaccine: a randomised controled trial. Lancet

2007; 370: 580–89.

8. World Health Organization. Availability of a candidate reassortant vaccine virus for the novel

influenza A (H1N1) vaccine development X-179A.

http://www.who.int/csr/resources/publications/swineflu/candidates_X-179a/en/ (accessed August 20,

http://www.who.int/csr/disease/swineflu/4th_meeting_ihr/%20en/index.html

13

2009)

9. Division of Microbiology and Infectious Diseases. Adult toxicity table.

http://www3.niaid.nih.gov/LabsAndResources/resources/DMIDClinRsrch/toxtables.htm (accessed

August 20, 2009)

10. Division of Microbiology and Infectious Diseases. Pediatric toxicity table.

http://www3.niaid.nih.gov/LabsAndResources/resources/DMIDClinRsrch/toxtables.htm (accessed

August 20, 2009)

11. European Committee for Proprietary Medicinal Products. Note for guidance on harmonisation of

requirements for influenza vaccines (CPMP/BWP/214/96). European Agency for the Evaluation of

Medicinal Products, 1997.

12. European Committee for Proprietary Medicinal Products. Guideline on dossier structure and content

for pandemic influenza vaccine marketing authorisation application (CPMP/VEG/4717/03). European

Agency for the Evaluation of Medicinal Products, 2004.

13. Katz J, Hancock K, Veguilla V, et al. Serum Cross-Reactive Antibody Response to a Novel Inluenza A

(H1N1) Virus After Vaccination with Seasonal Influenza Vaccine. MMWR 2009;58: 521-4.

14. Fang HH, Wu J, Zhang ZL, Jiang FC, Chen JT. Preexisting immunity and cross-reactive immunity to

a novel influenza A (H1N1) virus in Chinese population. Lancet 2009 (submitted)

14

Table 1 Demographic characteristics of the study population by age

Elders

(n=100)

Adults

(n=706)

Adolescents

(n= 403)

Children

(n=404)

Total

(n=1613)

Age (y)

Mean±SD 66·4±4·2 41·6±10·0 14·3±1·5 7·3±2·6 27·7±19·3

Median(range) 65·0

(61–75)

43·0

(18–60)

14·0

(12–17)

8·0

(3–11)

17·5

(3–75)

Male/Female 1·30 0·69 0·88 1·00 0·84

15

Table 2 Demographic characteristics of the study population by vaccine type and dose

Vaccine type and dose

Adjuvanted whole–virion

Adjuvanted split–virion

Non–adjuvanted

split–virion

Placebo

(n=100) 5 μg

(n=101)

10 μg

(n=102)

7·5 μg

(n=101)

15 μg

(n=100)

15 μg

(n= 101)

30 μg

(n= 101)

Adults

Age (y)

Mean±SD 41·3±10·0 41·1±9·9 41·1±10·2 41·7±10·1 41·6±10·3 41·9±9·6 42·0±9·8

Median (range) 42·0

(18–60)

42·0

(18–60)

42·0

(18–59)

43·0

(18–59)

42·0

(18–60)

43·0

(19–60)

44·0

(18–60)

Male/Female 0·74 0·65 0·58 0·69 0·63 0·71 0·89

Adolescents

Age (y) / / /

Mean±SD / / 14·3±1·7 14·4±1·6 14·3±1·5 14·3±1·5 /

Median (range) / /

14·0

(12–17)

14·0

(12–17)

14·0

(12–17)

14·0

(12–17) /

Male/Female / / 1·04 0·85 0·68 1·00 /

Children

Age (y) / / /

Mean±SD / / 7·7±2·3 7·3±2·6 7·1±2·6 7·2±2·8 /

Median (range) / /

8·0

(3–11)

8·0

(3–11)

7·0

(3–11)

8·0

(3–11) /

Male/Female / / 1·08 0·85 0·96 1·13 /

16

Table 3 Participants with adverse reactions after one dose by age

Elders (n=100) Adults (n=706 )

Adolescents

(n= 403) Children (n=404 ) Total (n=1613)

Total 1·0 (1) 12·3 (87) 10·7 (43) 14·4 (58) 11·7 (189)

Mild 1·0 (1) 10·1 (71) 9·4 (38) 9·2 (37) 9·1 (147)

Moderate 0·0 (0) 2·1 (15) 1·0 (4) 5·2 (21) 2·5 (40)

Severe 0·0 (0) 0·1 (1) 0·2 (1) 0·0 (0) 0·1 (2)

Local reactions 0·0 (0) 6·4 (45) 5·0 (20) 5·9 (24) 5·5 (89)

Pain 0·0 (0) 5·9 (42) 4·7 (19) 5·4 (22) 5·1 (83)

Erythema 0·0 (0) 0·0 (0) 0·0 (0) 0·2 (1) 0·1 (1)

Swelling 0·0 (0) 0·0 (0) 0·0 (0) 0·2 (1) 0·1 (1)

Induration 0·0 (0) 0·4 (3) 0·0 (0) 0·0 (0) 0·2 (3)

Itching 0·0 (0) 0·3 (2) 1·0 (4) 1·0 (4) 0·6 (10)

Others 0·0 (0) 0·1 (1) 0·0 (0) 0·0 (0) 0·1 (1)

Systemic reactions 1·0 (1) 7·9 (56) 7·7 (31) 10·9 (44) 8·2 (132)

Fever* 0·0 (0) 0·8 (6) 1·7 (7) 3·2 (13) 1·6 (26)

Allergy 0·0 (0) 0·6 (4) 1·0 (4) 0·7 (3) 0·7 (11)

Headache 0·0 (0) 2·3 (16) 1·7 (7) 1·0 (4) 1·7 (27)

Dizziness 0·0 (0) 1·3 (9) 1·7 (7) 1·7 (7) 1·4 (23)

Fatigue 1·0 (1) 2·1 (15) 1·5 (6) 1·2 (5) 1·7 (27)

Angina 0·0 (0) 0·7 (5) 1·2 (5) 0·2 (1) 0·7 (11)

Nausea 0·0 (0) 0·8 (6) 2·0 (8) 1·0 (4) 1·1 (18)

Vomit 0·0 (0) 0·1 (1) 0·0 (0) 0·0 (0) 0·1 (1)

Inappetence 0·0 (0) 0·3 (2) 0·2 (1) 0·7 (3) 0·4 (6)

Abdominal pain 0·0 (0) 0·6 (4) 1·2 (5) 0·5 (2) 0·7 (11)

Diarrhea 0·0 (0) 0·7 (5) 0·0 (0) 0·5 (2) 0·4 (7)

Myalgia 0·0 (0) 2·4 (17) 1·7 (7) 1·5 (6) 1·9 (30)

Arthralgia 0·0 (0) 0·8 (6) 0·2 (1) 0·2 (1) 0·5 (8)

Cough 0·0 (0) 0·3 (2) 0·5 (2) 1·7 (7) 0·7 (11)

Activity level 0·0 (0) 0·3 (2) 0·2 (1) 0·2 (1) 0·2 (4)

Others 0·0 (0) 0·3 (2) 0·7 (3) 0·0 (0) 0·3 (5)

Data are proportion (number) of participants with adverse reactions

17

Table 4 Participants with adverse reactions after one dose by vaccine type and dose

Vaccine type and dose

Adjuvanted

whole-virion

Adjuvanted

split-virion

Non-adjuvanted

split-virion

Placebo

5 μg 10 μg 7·5 μg 15 μg 15 μg 30 μg

Adults

N 101 102 101 100 101 101 100

Total 12·9 (13) 19·6 (20) 14·9 (15) 12·0 (12) 7·9 (8) 11·9 (12) 7·0 (7)

Mild 9·9 (10) 17·8 (18) 12·9(13) 12·0 (12) 5·0 (5) 7·9 (8) 5·0 (5)

Moderate 2·0 (2) 2·0 (2) 2·1(2) 0·0 (0) 3·0 (3) 4·0 (4) 2·0 (2)

Severe 1·0 (1) 0·0 (0) 0·0 (0) 0·0 (0) 0·0 (0) 0·0 (0) 0·0 (0)

Local reactions 6·9 (7) 9·8 (10) 5·9 (6) 7·0 (7) 3·0 (3) 7·9 (8) 4·0 (4) Systemic reactions 9·9 (10) 11·8 (12) 10·9 (11) 6·0 (6) 6·9 (7) 5·9 (6) 4·0 (4)

Adolescents

N / / 100 100 101 102 /

Total / / 11·0 (11) 11·0 (11) 10·9 (11) 9·8 (10) /

Mild / / 9·0 (9) 10·0 (10) 9·9 (10) 8·8 (9) /

Moderate / / 2·0 (2) 1·0 (1) 1·0 (1) 0·0 (0) /

Severe / / 0·0 (0) 0·0 (0) 0·0 (0) 1·0 (1) /

Local reactions / / 7·0 (7) 6·0 (6) 5·0 (5) 2·0 (2) /

Systemic reactions / / 5·0 (5) 8·0 (8) 9·9 (10) 7·8 (8) /

Children

N / / 102 100 100 102 /

Total / / 14·7 (15) 13·0 (13) 10·0(10) 19·6 (20) /

Mild / / 11·8 (12) 9·0 (9) 7·0 (7) 8·8 (9) /

Moderate / / 2·9 (3) 4·0 (4) 3·0 (3) 10·8 (11) /

Severe / / 0·0 (0) 0·0 (0) 0·0 (0) 0·0 (0) /

Local reactions / / 6·9 (7) 8·0 (8) 4·0 (4) 4·9 (5) /

Systemic reactions / / 7·8 (8) 10·0 (10) 7·0 (7) 18·6 (19) /

Data are proportion (number) of participants with adverse reactions

18

Table 5 Hemagglutination-inhibition antibody response after one dose in adults and elders

Adults (18–60 y) Elders(≥61 y)

Placebo Whole-virion+Al Split-virion+Al Split-virion Whole-virion+Al

5 μg 10 μg 7·5 μg 15 μg 15 μg 30 μg 10 μg

Before vaccination

N 100 101 102 101 100 101 101 100

GMT 7·0

(6·2–7·8)

6·5

(5·8–7·3)

6·8

(6·1–7·7)

7·0

(6·1–8·1)

6·8

(6·1–7·7)

6·6

(5·8–7·6)

7·4

(6·4–8·6)

8·6

(7·3–10·1)

Seroprotection (%)

4·0

(1·3–10·5)

5·9

(2·4–13·0)

5·9

(2·4–12·9)

4·0

(1·3–10·4)

3·0

(0·8–9·2)

3·0

(0·8–9·1)

5·9

(2·4–13·0)

6·0

(2·5–13·1)

Day 14

N 100 99 102 101 100 100 101 100

GMT 8·0

(6·8–9·5)

73·6

(55·1–98·1)

127·9

(96·8–168·9)

77·3

(60·0–99·5)

142·2

(107·9–187·4)

256·3

(199·0–330·2)

357·1

(277·3–459·9)

40·8

(31·9–52·4)

GMT ratio

1·1

(1·0–1·3)

11·5

(8·7–15·3)

18·7

(14·0–25·0)

11·0

(8·5–14·3)

20·8

(15·5–27·9)

39·4

(30·2–51·3)

48·3

(37·2–62·7)

4·8

(3·7–6·1)

Seroconversion (%)

3·0

(0·8–9·2)

70·7

(60·6–79·4)

84·3

(75·5–90·7)

73·3

(63·4–81·5)

86·0

(77·3–92·1)

95·0

(88·2–98·5)

95·0

(88·3–98·5)

50·0

(39·9–60·2)

Seroprotection (%)

9·0

(4·5–16·9)

72·7

(62·7–81·1)

86·3

(77·7–92·2)

77·2

(67·6–84·9)

90·0

(82·0–95·1)

96·0

(89·5–99·1)

97·0

(90·9–99·7)

56·0

(45·7–65·9)

Day 21

N 100 99 102 101 100 100 101 99

GMT 7·1

(6·3–8·1)

87·6

(65·4–117·3)

155·7

(117·0–207·1)

86·9

(66·3–113·8)

155·6

(116·5–207·8)

294·5

(221·9–390·7)

374·7

(289·9–484·3)

37·6

(29·1–48·6)

GMT ratio

1·0

(0·9–1·10

13·0

(10·2–18·0)

22·8

(16·8–30·9)

12·4

(9·5–16·3)

22·8

(16·8–31·0)

44·3

(33·0–59·5)

50·7

(39·2–65·5)

4·5

(3·5–5·8)

Seroconversion (%)

1·0

(0·0–6·3)

73·7

(63·8–82·0)

88·2

(80·0–93·7)

72·3

(62·3–80·6)

87·0

(78·4–92·9)

95·0

(88·2–98·5)

97·0

(90·7–99·7)

47·5

(37·4–57·8)

Seroprotection (%)

6·0

(2·5–13·1)

76·8

(67·0–84·6)

91·2

(83·5–95·9)

77·2

(67·6–84·9)

90·0

(82·0–95·1)

97·0

(90·8–99·7)

99·0

(93·8–100·0)

53·5

(43·3–63·6)

19

Table 6 Hemagglutination-inhibition antibody response after one dose in children and adolescents

Children (3–11 y) Adolescents (12–17 y)

Split-virion+Al Split-virion Split-virion+Al Split-virion

7·5 μg 15 μg 15 μg 30 μg 7·5 μg 15 μg 15 μg 30 μg

Before vaccination

N 102 100 100 102 100 100 101 102

GMT 5·3

（5·0–5·5）

5·4

(5·1–5·6)

5·2

(5·0–5·4)

5·6

(5·2–6·0)

8·6

(7·3–10·1)

8·6

(7·1–10·5)

8·9

(7·5–10·6)

9·1

(7·5–11·0)

Seroprotection (%)

0·0

（0·0–4·5）

0·0

(0·0–4·6)

0·0

(0·0–4·6)

1·0

(0·0–6·1)

11·0

(5·9–19·3)

9·0

(4·5–16·9)

11·9

(6·6–20·2)

8·8

(4·4–16·9)

Day 14

N / / / / 99 100 100 99

GMT / / / /

84·6

(66·6–107·6)

107·8

(84·0–138·3)

320·0

(248·0–412·9)

406·0

(319·3–516·3)

GMT ratio

/ / / /

9·9

(7·8–12·7)

12·5

(9·6–16·3)

35·8

(27·7–46·1)

44·5

(33·6–58·8)

Seroconversion (%)

/ / / /

76·8

(67·0–84·6)

78·0

(68·4–85·6)

98·0

(92·3–100·0)

97·0

(90·8–99·7)

Seroprotection (%)

/ / / /

84·8

(75·9–91·2)

85·0

(76·1–91·3)

99·0

(93·8–100·0)

99·0

(93·7–100·0)

Day 21

N 96 96 97 97 97 98 100 99

GMT 43·6

（35·4–53·8）

56·2

(46·4–67·7)

78·9

(63·4–98·1)

112·7

93·3–136·2

131·9

(101·5–171·4)

150·1

(115·8–194·6)

475·0

(359·7–627·3)

600·9

(458·3–787·9)

GMT ratio

8·3

(6·8–10·1)

10·5

(8·8–12·7)

15·1

(12·2–18·8)

20·1

(16·5–24·5)

15·3

(11·9–19·7)

17·3

(13·2–22·7)

53·1

(40·7–69·2)

65·8

(49·4–87·7)

Seroconversion (%)

63·5

(53·0–73·1)

81·3

(71·7–88·4)

81·4

(72·0–88·5)

94·8

(87·8–98·5)

87·6

(79·0–93·4)

82·7

(73·4–89·5)

98·0

(92·3–100·0)

98·0

(90·8–99·7)

Seroprotection (%)

63·5

(53·0–73·1)

81·3

(71·7–88·4)

81·4

(72·0–88·5)

95·9

(89·2–99·1)

92·8

(85·2–97·1)

87·8

(79·2–93·5)

98·0

(92·3–100·0)

100·0

(95·3–100·0)

20

Figure legend

Figure 1: Trial profile

Figure 2: Reverse cumulative distribution curve of hemagglutinatination-inhibition titers against

recombinant A/California/07/2009-A/PR/8/34 strain (X-179A) in elders (A), adults (B), adolescents (C)

and children (D) 21 days after the first dose.

Par

tici

pan

ts (

%)

21

Figure 1

703 donated samples and received

the 2nd dose on day 21

100 in placebo

99 in 5-μg whole-virion +Al


101 in 7.5-μg split-virion +Al

100 in 15-μg split-virion +Al

100 in 15-μg split-virion


100 donated

samples on day

21













Safety assess

Safety assess

Safety assess

Assessed for the immunogenicity on day 35 and

42

genicity

Assessed for the immuno-

genicity on day 35

Assessed for the immuno-

genicity on day 42

100 received placebo

101 received 5-μg whole-virion +Al

102 received 10-μg whole-virion +Al

101 received 7.5-μg split-virion +Al

100 received 15-μg split-virion +Al

101 received 15-μg split-virion


100 received 10-μg

whole-virion +Al.

One was excluded









1614 eligible participants stratified by age

101 elders 403 adolescents randomly assigned 404 children randomly

assigned

706 adults randomly

assigned

Safety assess Safety assess Safety assess Safety assess

703 donated samples on day 14

100 in placebo







100 donated

samples on day

14

398 donated samples on day 14





22

El der s

0

1 0

20

30

40

50

60

70

80

90

1 00

5 1 0 20 40 80 1 60 320 640 1 280 2560

1 0μg W+Al

Adul t s

0

1 0

20

30

40

50

60

70

80

90

1 00

5 1 0 20 40 80 1 60 320 640 1 280 2560

5μg W+Al

1 0μg W+Al

7. 5μg S+Al

1 5μg S+Al

1 5μg S

30μg S

pl acebo

Adol escent s

0

1 0

20

30

40

50

60

70

80

90

1 00

5 1 0 20 40 80 1 60 320 640 1 280 2560

7. 5μg S+Al

1 5μg S+Al

1 5μg S

30μg S

Chi l dr en

0

1 0

20

30

40

50

60

70

80

90

1 00

5 1 0 20 40 80 1 60 320 640 1 280 2560

7. 5μg S+Al

1 5μg S+Al

1 5μg S

30μg S

Par

tici

pan

ts (

%)

Par

tici

pan

ts (

%)

Par

tici

pan

ts (

%)

Par

tici

pan

ts (

%)

Titer Titer

Titer Titer

A B

C D

Figure 2

1

The clinical trial of adjuvanted whole-virion, adjuvanted split-virion and non-adjuvanted

split-virion influenza A （H1N1）vaccines: a double-blind, randomized and controlled trial

Summary

A single center, double-blind, randomized and controlled clinical trial is to be conducted in heathy people aged

over 3-years to evaluate the safety and immunogenicity of adjuvanted whole-virion, adjuvanted split-virion

and non-adjuvanted split-virion influenza A （H1N1）vaccines. The vaccine investigated is producted in

embryonated hen's eggs using the H1N1 reference strain X-179A which is a reassortant between

A/California/07/2009 and A/PR/8/34 prepared by New York Medical College using classical reassortment

technology. Total 1600 volunteers aged over 3-years old will be enrolled, the eligible participants were

stratified by age: elders (≥61 years), adults (18–60 years), adolescent (12–17 years) and children (3–11 years).

The elders are assigned to receive one dose of openly-labelled, 10-μg, adjuvanted whole-virion vaccine. The

adults, adolescents and children are randomly assigned to receive two doses of blind-labelled vaccines or

placebo 21 days apart. The adults will receive 2 doses of 5 or 10 μg of adjuvanted, whole-virion vaccine, or 7·5

or 15 μg of adjuvanted, split-virion vaccine, or 15 or 30 μg of non-adjuvanted, split-virion vaccine, or placebo.

The adolescents and children will receive 2 doses of 7·5 or 15 μg of adjuvanted, split-virion vaccine, or 15 or

30 μg of non-adjuvanted, split-virion vaccine. Serum samples for the assessment of humoral immune response

are collected on day 0, 14, 21 and 35 for elders, day 0, 14, 21, 35 and 42 for adults and adolescents, and day 0,

21 and 42 for children, and then will be assayed by haemagglutination-inhibition (HI) methods against the

homologous X-179A strain. Local and systematic adverse reactions will be recorded during the study.

Backgroud

A novel swine-origin influenza A (H1N1) virus emerging in North America had caused a pandemic influenza

as declared by the World Health Organization (WHO) on June 11, 2009. As of July 3, 2009, more than 120

countries and regions worldwide reported around 90,000 cases, 382 of which were fatal. The rapid spread of

the virus and high morbidity caused by it has drawn wide attention from international community. So far

vaccine is considered as the most effective means to reduce the morbidity and mortality after infection,

* Protocol

2

especial for those who have underlying medical conditions.

Up to now, clinical evaluation of influenza A (H1N1) vaccine is ongoing in China, Australia, USA, Austria and

European Union. None of these trials has been completed. As registered in ClinicalTrials.gov, the vaccines to

be evaluated included whole-virion and split-virion vaccines, adjuvanted and non-adjuvanted vaccines,

egg-derived and cell-derived vaccines, all of which were monovalent vaccine. The dosage of the investigated

vaccines ranged from 3·75 μg to 30 μg, depending on the adjuvant and vaccine type. The study population

covered infants, children, adolescents, adults and elders. Two-dose vaccination schedule was adopted by all

these trials, most of which were 21-day interval. Based on the experience with H5N1 vaccine trials which

showed that two doses were necessary to induce satisfactory immune response in immunologically naïve

population, a double-blind, randomized and controlled trial was conducted in elders, adults, adolescents and

children to evaluate the immunogenicity and safety of influenza A (H1N1) vaccines manufactured by Sinovac

Biotech (Beijing, China)

Aims

The purpose is to evaluate immunogenicity and safety of influenza A （H1N1）vaccines in elders, adults,

adolescent and children and provide a basis to determine the best type and dose of vaccine and immunization

procedures.

General Informations

Principal Investigator

Jiang Wu, associate professor, Beijing Centers for Diseases Control and Prevention, Beijing, China.

Main Center

Beijing Centers for Diseases Control and Prevention

Contact Details

Jiang Wu

Tel and fax: 86-10-64407107

Email: [email protected]

Sponsors

Sinovac Biotech Co., Ltd, Beijing, China

mailto:[email protected]

3

Intending start date

July 2009

Intending finishing date

September 2009

Design

The trial is double blind, randomized and controlled study.

Inclusion Criteria

Healthy male or female aged 3 and older

Be able to show legal identity card for the sake of recruitment

Volunteers or their guardians are able to understand and sign the informed consent.

Exclusion Criteria

Cases, cured cases and close contact of influenza A (H1N1) virus

Women of pregnancy, lactation or about to be pregnant in 60 days

Subject that has a medical history of any of the following: allergic history, or allergic to any ingredient

of vaccine, such as egg, egg protein, etc

Serious adverse reactions to vaccines such as anaphylaxis, hives, respiratory difficulty, angioedema,

or abdominal pain

Autoimmune disease or immunodeficiency

Asthma that is unstable or required emergent care, hospitalization or intubation during the past two

years or that required the use of oral or intravenous corticosteroids

Diabetes mellitus (type I or II), with the exception of gestational diabetes

History of thyroidectomy or thyroid disease that required medication within the past 12 months

Serious angioedema episodes within the previous 3 years or requiring medication in the previous two

years

Bleeding disorder diagnosed by a doctor (e.g. factor deficiency, coagulopathy, or platelet disorder

requiring special precautions) or significant bruising or bleeding difficulties with IM injections or

blood draws

4

Active malignancy or treated malignancy for which there is not reasonable assurance of sustained cure

or malignancy that is likely to recur during the period of study

Seizure disorder other than:

1. Febrile seizures under the age of two years old

2. Seizures secondary to alcohol withdrawal more than 3 years ago, or

3. A singular seizure not requiring treatment within the last 3 years

Asplenia, functional asplenia or any condition resulting in the absence or removal o the spleen

Guillain-Barre Syndrome

Any history of immunosuppressive medications or cytotoxic medications or inhaled corticosteroids

within the past six months (with the exception of corticosteroid nasal spray for allergic rhinitis or

topical corticosteroids for an acute uncomplicated dermatitis)

History of any blood products or seasonal influenza vaccine administration within 3 months before the

dosing

Administration of any other investigational research agents within 30 days before the dosing

Administration of any live attenuated vaccine within 30 days before the dosing

Administration of subunit or inactivated vaccines, e.g., pneumococcal vaccine, or allergy treatment

with antigen injections, within 14 days before the dosing

Be receiving anti-TB prophylaxis or therapy currently

Axillary temperature > 37.0 centigrade at the time of dosing

Psychiatric condition that precludes compliance with the protocol:

1. Past or present psychoses

2. Past or present bipolar disorder requiring therapy that has not been well controlled on

medication for the past two years

3. Disorder requiring lithium

4. Suicidal ideation occurring within five years prior to enrollment

Any medical, psychiatric, social condition, occupational reason or other responsibility that, in the

judgment of the investigator, is a contraindication to protocol participation or impairs a volunteer's

ability to give informed consent.

Intervention

The inactivated, aluminum-adjuvanted, whole-virion influenza (H1N1) vaccine is prepared on a pilot scale by

following procedures: the virus seed X-179A strain is grown in in embryonated hens’ eggs, harvested,

5

inactivated, purified and formulated with aluminum hydroxide. A 1.0 mL-dose contained 20 μg H1N1

haemagglutinin antigen, without preservative. Non-pathogenicity of the reassortant had been confirmed by the

National Institute for the Control of Pharmaceuticals and Biological Products (NIBSC) by use of various

animal model.

The aluminum-adjuvanted, split-virion influenza (H1N1) vaccine is prepared on a pilot scale by following

procedures: the virus seed X-179A strain is grown in in embryonated hens’ eggs, harvested, inactivated, splited,

purified and formulated with aluminum hydroxide. A 1.0 mL-dose contained 30 μg H1N1 haemagglutinin

antigen, without preservative. Non-pathogenicity of the reassortant had been confirmed by NIBSC by use of

various animal model.

The non-adjuvanted, split-virion influenza (H1N1) vaccine is prepared on a pilot scale by following procedures:

the virus seed X-179A strain is grown in in embryonated hens’ eggs, harvested, inactivated, splited and

purified. A 1.0 mL-dose contained 60 μg H1N1 haemagglutinin antigen, without preservative.

Non-pathogenicity of the reassortant had been confirmed by NIBSC by use of various animal model.

The reference strain was prepared by the UK National Institute for Biological Standards and Control (NIBSC),

and recommended as suitable for use as a prototype influenza vaccine strain by the European Union

Committee for Medicinal Products for Human Use (CHMP).

The study vaccine was produced by Sinovac Biotech Co., Ltd. and certified and released by NICPBP.

Treatment groups as follows:

Age Whole-virion

vaccine Split-virion vaccine Placebo Total

5μg

+Al

10μg

+Al

7.5μg

+Al

15μg 15μg

+Al

30μg

Elders 100 100

Adults 100 100

100 100 100 100

100 700

Adolescents 100 100 100 100 400

Children 100 100 100 100 400

Total 100 200 300 300 300 300 100 1600

The elders receive one dose and the adults, adolescent and children receive two doses apart 21 days. The

vaccines are administered intramuscularly into the deltoid muscle by nurse who will not participated in the

safety observation and immunogenic assessment.

6

Randomization

Those vaccines administered at 0,21 days regimen are subjected to randomization. A randomization list will be

generated at Sinovac Biotech by a statistician who is not involved in the rest of the trial. A randomized block

design is used, with a block size of a 1:1:1:1:1:1 ratio in adults, a 1:1:1:1 ratio in adolescents and children to

ensure that balance between vaccine types and dosages is maintained. Then the randomized vaccines of the

three formulations are blind labelled with a sequential number according to the randomization list. The blind

labelled vaccines are the same in appearance and the random code (A001-A700 for adults, N001-N400 for

adolescent and C001-C400 for children) is the only sign to identify the vaccines. The 10-μg, adjuvanted

whole-virion vaccine used for elders are openly labelled.

Allocation

The 700 adults, 400 adolescents and 400 children are randomly assigned (as seen in the table above) to receive

two doses of blind-labelled vaccine 21 days apart. The 100 elders are assigned to receive one doses of

openly-labelled 10-μg adjuvanted whole-virion vaccine.

Endpoints

The primary immunological endpoint is seroconversion rate of haemaggultination-inhibition (HI) antibody 14

days or 21 days after the first or the second dose. The vaccinees are defined as seroconversion who have a HI

titer before vaccination of less than 1:10 and a titer after vaccination of 1:40 or more, or a titer before

vaccination of 1:10 or more and at least a four fold increase after vaccination.

The secondary immunological endpoints include post-to-pre-vaccination geometric mean titer (GMT) ratio and

post-vaccination seroprotection rate (≥1:40, deemed to be the seroprotective threshold for seasonal influenza

vaccines).

Adverse events and reactions

Participants should be kept to observe adverse events for 30 minutes after receipt of every dose. Diary cards

will be used to record the presence and intensity of adverse events. For the next three days following

vaccination, any local adverse events (pain, erythema, swelling, induration, rash and itching) at the injection

site and systematic adverse events (allergic reactions, headache, dizziness, fatigue, angina, nausea, vomit,

inappetence, abdominal pain, diarrhea, myalgia, arthralgia, cough, fever) will be recorded by participants on

the first three diary cards. From the fourth day to the next visit, the adverse events will be recorded by

participants on the fourth dairy cards. The diameters of any erythema, swelling, induration and rash at the

7

injection site should be determined by investigators, and daily axillary temperature will be measured by

volunteers. The adverse events recorded by participants should be reviewed by members of the study staff. All

adverse events are graded by Adult Toxicity Table released by Division of Microbiology and Infectious

Diseases, USA.

The presence of solicited and unsolicited adverse events and any serious adverse events are described in terms

of the incidence, intensity and relation to vaccination. The incidence of adverse events is based on the most

severe response, and expressed in terms of the number and proportion of individuals who had adverse events in

each group.

Serious adverse event (SAE) is any untoward medical occurrence that at any dose:

- results in death

- is life-threatening

- requires inpatient hospitalization or prolongation of existing hospitalization

- results in persistent or significant disability/incapacity

- or is a congenital anomaly/birth defect.

Investigators should report any SAE in 24 hours by telephone, fax or email to SFDA, sponsors, monitor, Data

Safety Monitoring Committee (DSMB) and ethical review committee of Beijing Centers for Diseases Control

and Prevention.

Immunological assay

Serum samples for the assessment of humoral immune response were collected on day 0, 14, 21 and 35 for

elders, day 0, 14, 21, 35 and 42 for adults and adolescents, and day 0, 21 and 42 for children. Serum sample

will be isolated under sterile conditions. Hemagglutination-inhibition (HI) antibody titers and

microneutralization antibody titers against the vaccine seed virus (X-179A) will be measured. The

immunogenic assays will be done under blinded conditions, i.e, the laboratory staffs do not know the treatment

groups and bleeding times. HI assays are to be done in accordance with established procedures with turkey

erythrocytes. Before titration, all serum samples will be treated with receptor-destroying enzyme (cholera

filtrate, Sigma) at 36℃ for 16 h to remove non-specific inhibitors of agglutination, then incubated at 56℃ to

inactivate the enzyme. Samples are tested in two-fold dilution starting with an initial dilution of 1 in 10. The

serum titers are expressed as the reciprocal of the highest dilution that shows complete inhibition of

8

hemagglutination. All samples will be assayed in duplicate and double-checked by at least two persons. For the

purpose of calculation, HI titres below 1:10 are assigned a value of 1:5.

Statistical analysis

Safety and immunogenicity were the co-primary objectives. The incidence of adverse events was based on the

most severe response, expressed in terms of the number and proportion of individuals who had adverse events.

The safety data were summarized in the full-analysis set.

The immunologic endpoints were based on HI licensure criteria set out by the European Union Committee for

Medicinal Products for Human Use (CHMP) including geometric mean titre (GMT), post-to-pre-vaccination

GMT ratio, seroconversion rate and seroprotection rate. Seroconversion rate is defined as the percentage of

vaccinees who have a titre before vaccination of less than 1:10 and a titre after vaccination of 1:40 or more, or

a titre before vaccination of 1:10 or more and at least a fourfold increase after vaccination. HI titre≥1:40 is

considered as seroprotection in seasonal influenza vaccine, which is temporally adopted for influenza A (H1N1)

vaccine in this study. The antibody titres were transformed into logarithmic scale for the calculation of GMT.

The titre distributions were described with reverse cumulative distribution curves. The immunogenic data were

summarized in the per-protocol set.

The results of immunogenicity and safety are summarized with point estimates and two-sided 95%CI. The

antibody titers are transformed into logarithmic scale for the calculation of GMT and 95%CI. Statistical

analysis is done by intension to treatment (ITT). Immunogenic data are summarized in the per-protocol

population. Pearson’s chi-square test or Fisher’s exact test are used to compare groups when relevant.

Spearman’s correlation coefficient is used to assess the correlation between HI titer and neutralization titer. The

significance level was 0·05 (two-sided).

Ethical issues

The trial was registered with the ClinicalTrials.gov number NCT00956111 and approved by the Chinese

regulatory authority State Food and Drug Administration (SFDA) and Chinese CDC. All relevant documents

were approved by the ethical review committee of Beijing Centers for Disease Control and Prevention. All

participants provided their written informed consent.

Immunogenicity and safety after one dose of adjuvanted …...1 Immunogenicity and safety after one...

Documents

Transcript of Immunogenicity and safety after one dose of adjuvanted …...1 Immunogenicity and safety after one...