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1 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 splitvirion 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 NCT 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 nonadjuvanted split-virion vaccine with HI geometric mean titer (GMT) of , seroconversion rate of 95-98% and seroprotection rate of %. 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 , 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.

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3 Manuscript 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 Wu 1, Wei Li 2, Hua-Qing Wang 3, Jiang-Ting Chen 4, Min Lu 1, Ji-Chen Zhou 5, Xiao-Feng Liang 3, Han-Hua Fang 2, Yan Liu 4, Li-Ying Liu 5, Xu Wang 4, Wu-Li Zhang 5, Xiao-Mei Zhang 4, Li-Fei Song 4, Yuan-Zheng Qiu 4, 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: Fax: yinweidong@sinovac.com 1

4 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 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 NCT 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 , seroconversion rate of 95 98% and seroprotection rate of %. 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 , 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 2

5 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) 3

6 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, 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. 4

7 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 NCT 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 ), 30 μg/ml/vial (lot S ) and 60 μg/ml/vial (lot ), respectively. Each vial contained 2 or 4 doses. The two adjuvanted vaccines were formulated with aluminum hydroxide to contain aluminum of 5

8 mg/ml. The placebo control was phosphate buffer saline (lot E-R ). 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. 6

9 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 7

10 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 per group. The adolescents were randomly allocated into 4 groups with per group. The children were randomly allocated into 4 groups with 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, 8

11 seroconversion rates and seroprotection rates in the 3 age strata were , 8 66, 64 98% and % 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 , seroconversion rate of 95 98% and seroprotection rate of %. One dose of 15-μg non-adjuvanted split-virion vaccine also induced high immune response with HI GMT of , 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<0 01). Meanwhile, the placebo group in adult showed no immunogenicity compared to vaccine groups. Discussion Originated from North American, the novel influenza A (H1N1) virus has quickly spread worldwide, resulting in the WHO s declaration of human influenza pandemic on June 11, In the last decade, the world had been preparing against a potential human influenza pandemic. Much attention and investment had been drawn for the preparedness of potential pandemic including preparation and distribution of vaccine strain, quality control of vaccine, clinical evaluation and licensure criteria, and so on, which made the world better prepared against an influenza pandemic than ever. In recent years, many prototype pandemic influenza vaccine (H5N1) had been clinically evaluated and those results had been well documented, 4 7 which were helpful for the current clinical evaluation of influenza A (H1N1) vaccine. Since the novel influenza A (H1N1) virus spread so quickly and caused high morbidity, it is generally believed that the population are immunologically naïve to the virus. Previous experiences with H5N1 vaccine trials showed that two doses were necessary to induce satisfactory immune response in immunologically naïve population. 4 7 Therefore, it seems reasonable to adopt two-dose vaccination schedule in clinical evaluation of influenza A (H1N1) vaccine which is used in all the current trials. 3 It was reported that, however, some degree of detectable preexisting immunity was found in U.S. population over 18 years and in Chinese population over 12 years, 13,14 suggesting that an alternative vaccination schedule 9

12 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 10

13 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 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 11

14 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. (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 (accessed August 20, 2009) 3. ClinicalTrials.gov. H1N1 vaccine trial. 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: 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: 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: 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: World Health Organization. Availability of a candidate reassortant vaccine virus for the novel influenza A (H1N1) vaccine development X-179A. (accessed August 20, 12

15 2009) 9. Division of Microbiology and Infectious Diseases. Adult toxicity table. (accessed August 20, 2009) 10. Division of Microbiology and Infectious Diseases. Pediatric toxicity table. (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, 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, 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: 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) 13

16 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± ± ± ± ±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

17 Table 2 Demographic characteristics of the study population by vaccine type and dose Vaccine type and dose Adjuvanted whole virion 5 μg (n=101) 10 μg (n=102) Adjuvanted split virion 7 5 μg (n=101) 15 μg (n=100) Non adjuvanted split virion 15 μg (n= 101) 30 μg (n= 101) Placebo (n=100) Age (y) Mean±SD 41 3± ± ± ± ± ± ±9 8 Adults 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 Age (y) / / / Mean±SD / / 14 3± ± ± ±1 5 / Adolescents Median (range) / / (12 17) (12 17) (12 17) (12 17) / Male/Female / / / Age (y) / / / Mean±SD / / 7 7± ± ± ±2 8 / Children Median (range) / / (3 11) (3 11) (3 11) (3 11) / Male/Female / / / 15

18 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 16

19 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 N 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) Adults 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) N / / / 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) / Adolescents 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) / N / / / 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) / Children 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 17

20 Table 5 Hemagglutination-inhibition antibody response after one dose in adults and elders Placebo Adults (18 60 y) Elders( 61 y) 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 GMT 7 0 ( ) 6 5 ( ) 6 8 ( ) 7 0 ( ) 6 8 ( ) 6 6 ( ) 7 4 ( ) 8 6 ( ) Seroprotection (%) 4 0 ( ) 5 9 ( ) 5 9 ( ) 4 0 ( ) 3 0 ( ) 3 0 ( ) 5 9 ( ) 6 0 ( ) Day 14 N GMT 8 0 ( ) 73 6 ( ) ( ) 77 3 ( ) ( ) ( ) ( ) 40 8 ( ) GMT ratio 1 1 ( ) 11 5 ( ) 18 7 ( ) 11 0 ( ) 20 8 ( ) 39 4 ( ) 48 3 ( ) 4 8 ( ) Seroconversion (%) 3 0 ( ) 70 7 ( ) 84 3 ( ) 73 3 ( ) 86 0 ( ) 95 0 ( ) 95 0 ( ) 50 0 ( ) Seroprotection (%) 9 0 ( ) 72 7 ( ) 86 3 ( ) 77 2 ( ) 90 0 ( ) 96 0 ( ) 97 0 ( ) 56 0 ( ) Day 21 N GMT 7 1 ( ) 87 6 ( ) ( ) 86 9 ( ) ( ) ( ) ( ) 37 6 ( ) GMT ratio 1 0 ( ( ) 22 8 ( ) 12 4 ( ) 22 8 ( ) 44 3 ( ) 50 7 ( ) 4 5 ( ) Seroconversion (%) 1 0 ( ) 73 7 ( ) 88 2 ( ) 72 3 ( ) 87 0 ( ) 95 0 ( ) 97 0 ( ) 47 5 ( ) Seroprotection (%) 6 0 ( ) 76 8 ( ) 91 2 ( ) 77 2 ( ) 90 0 ( ) 97 0 ( ) 99 0 ( ) 53 5 ( ) 18

21 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 GMT 5 3 ( ) 5 4 ( ) 5 2 ( ) 5 6 ( ) 8 6 ( ) 8 6 ( ) 8 9 ( ) 9 1 ( ) Seroprotection (%) 0 0 ( ) 0 0 ( ) 0 0 ( ) 1 0 ( ) 11 0 ( ) 9 0 ( ) 11 9 ( ) 8 8 ( ) Day 14 N / / / / GMT / / / / ( ) ( ) ( ) ( ) GMT ratio / / / / ( ) ( ) ( ) ( ) Seroconversion (%) / / / / ( ) ( ) ( ) ( ) Seroprotection (%) / / / / ( ) ( ) ( ) ( ) Day 21 N GMT 43 6 ( ) 56 2 ( ) 78 9 ( ) ( ) ( ) ( ) ( ) GMT ratio 8 3 ( ) 10 5 ( ) 15 1 ( ) 20 1 ( ) 15 3 ( ) 17 3 ( ) 53 1 ( ) 65 8 ( ) Seroconversion (%) ( ) Seroprotection (%) 63 5 ( ) ( ) 81 3 ( ) ( ) 81 4 ( ) ( ) 95 9 ( ) ( ) 92 8 ( ) ( ) 87 8 ( ) ( ) 98 0 ( ) ( ) ( ) 19

22 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. 20

23 1614 eligible participants stratified by age 101 elders 706 adults randomly 403 adolescents randomly assigned 404 children randomly 100 received 10-μg 100 received placebo 100 received 7.5-μg split-virion +Al 102 received 7.5-μg split-virion +Al whole-virion +Al. One was excluded 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 102 received 30-μg split-virion 100 received 15-μg split-virion +Al 100 received 15-μg split-virion 102 received 30-μg split-virion 100 received 15-μg split-virion +Al 101 received 15-μg split-virion 101 received 30-μg split-virion Safety assess Safety assess Safety assess Safety assess 100 donated samples on day 703 donated samples on day in placebo 99 in 5-μg whole-virion +Al 398 donated samples on day in 7.5-μg split-virion +Al 100 in 15-μg split-virion +Al 102 in 10-μg whole-virion +Al 100 in 15-μg split-virion 101 in 7.5-μg split-virion +Al 99 in 30-μg split-virion 100 in 15-μg split-virion +Al 100 in 15-μg split-virion 101 in 30-μg split-virion 100 donated samples on day 703 donated samples and received the 2nd dose on day in placebo 99 in 5-μg whole-virion +Al 102 in 10-μg whole-virion +Al 101 in 7.5-μg split-virion +Al 100 in 15-μg split-virion +Al 394 donated samples and received the 2nd dose on day in 7.5-μg split-virion +Al 98 in 15-μg split-virion +Al 100 in 15-μg split-virion 99 in 30-μg split-virion 386 donated samples and received the 2nd dose on day in 7.5-μg split-virion +Al 96 in 15-μg split-virion +Al 97 in 15-μg split-virion 97 in 30-μg split-virion 100 in 15-μg split-virion 101 in 30-μg split-virion Safety assess Safety assess Safety assess Assessed for the immuno- genicity on day 35 Assessed for the immunogenicity on day 35 and Assessed for the immuno- genicity on day 42 Figure 1 21

24 Participants (%) Participants (%) Participants (%) Participants (%) A B El der s 10μg W+Al Adul t s 5μg W+Al 10μg W+Al 7. 5μg S+Al 15μg S+Al 15μg S 30μg S pl acebo Titer Titer C D Adol escent s 7. 5μg S+Al 15μg S+Al 15μg S 30μg S Chi l dr en 7. 5μg S+Al 15μg S+Al 15μg S 30μg S Figure 2 Titer Titer 22

25 23

26 * Protocol 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, 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, 1

27 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: wj81732@yahoo.com.cn Sponsors Sinovac Biotech Co., Ltd, Beijing, China 2

28 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 3

29 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, 4

30 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 10μg 7.5μg 15μg 15μg 30μg +Al +Al +Al +Al Elders Adults Adolescents Children Total 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. 5