A clinical trial to assess the immunogenicity and safety of Inactivated Influenza Vaccine (Whole Virion) IP (Pandemic Influenza (H1N1) 2009 Monovalent Vaccine; VaxiFlu-S TM ) in healthy Indian adult populationAH Kubavat1, R Mittal1, PM Patel2, DH Jarsaniya2, PR Pawar1
1 Clinical Research & Regulatory Affairs Department, Cadila Healthcare Ltd., Ahmedabad, India
2 Vaccine Department, Cadila Healthcare Ltd., Ahmedabad, India
Correspondence Address: Source of Support: Cadila Healthcare Ltd. Satellite Cross Roads, Sarkhej-Gandhinagar Highway, Ahmedabad - 380 015. Gujarat, India, Conflict of Interest: None DOI: 10.4103/0022-3859.81860
Source of Support: Cadila Healthcare Ltd. Satellite Cross Roads, Sarkhej-Gandhinagar Highway, Ahmedabad - 380 015. Gujarat, India, Conflict of Interest: None
Background : The pandemic of H1N1 2009 influenza has spread world over and low degree of virus transmission has continued in several regions of India. Aims : To assess the immunogenicity and safety of Pandemic Influenza (H1N1) 2009 Monovalent Vaccine in healthy adult Indian population. Settings and Design : Prospective, open label, multicentric, phase 2/3 clinical trial. Materials and Methods : Healthy adult Indian subjects belonging to either 18-59 years or ≥60 years age groups were enrolled and administered a single 0.5 ml (≥15 mcg of hemagglutinin antigen) dose of vaccine in the deltoid muscle. Anti-hemagglutinin antibody titer was assessed at baseline and 21 (±2) days after vaccination by Hemagglutination Inhibition (HI) test. Safety assessments were done for a period of 42 days. Statistical Analysis Used : Percentages of appropriate population with 95% confidence intervals calculated, log transformation of the data to calculate Geometric Mean Titers (GMTs) and chi-square test and student's t-test applied for significance testing. Results : 182/198 and 53/63 volunteers in age groups of 18-59 years and ≥60 years, respectively, achieved an HI titer ≥1 : 40 at Day 21 (91.9% [95% confidence interval: 88.1-95.7%] and 84.1% [75.1-93.2%]; P=0.072). Further, 171/198 and 50/63 volunteers in the respective age groups achieved seroconversion/four-fold increase in titer at Day 21 (86.4% [81.6-91.1%] and 79.4% [69.4-89.4%]; P=0.179). A significant rise of 22.6-fold [18.0-28.4] and 10.5-fold [7.4-15.0] was noted in GMT in the respective age groups (P<0.001 for both groups as compared to baseline). Nine vaccine-related adverse events were reported (3.4% incidence [1.2-5.6%]), which were of low severity only. Conclusions : Pandemic Influenza (H1N1) 2009 Monovalent Vaccine produces excellent immunogenic response with a good tolerability profile in adult Indian population.
Keywords: Adults, clinical trial, H1N1, inactivated, India, influenza, intra-muscular, pandemic, vaccine
Novel H1N1 influenza A (also referred to as "swine flu") is caused by a new influenza virus first detected in Mexico in March 2009. This virus, which can be transmitted from person-to-person,  has spread worldwide to more than 214 countries and overseas territories,  and is presently co-circulating with seasonal influenza viruses. , Limited transmission of oseltamivir-resistant H1N1 2009 viruses has also been reported. 
The highest number of cases of H1N1 2009 influenza A infection were recorded in India around the months of September to December 2009,  and currently several regions of India are still reported to be one of the most active areas of H1N1 transmission in Asia.  As of 3 rd October 2010, a total of 44,687 confirmed cases of H1N1 infection and 2575 deaths have been reported in the country. 
The new H1N1 virus was isolated, modified to prepare influenza vaccines and distributed by World Health Organization (WHO) and other government agencies to vaccine manufacturers to begin vaccine production. Manufacturing vaccine is a complex multi-step process and candidate vaccines need to be tested in clinical trials to establish their immunogenicity and safety.
The present study was conducted to assess the immunogenicity and safety of Inactivated Influenza Vaccine (Whole Virion) IP (Pandemic Influenza (H1N1) 2009 Monovalent Vaccine; VaxiFlu-S TM ) given intramuscularly in healthy adult Indian population.
This prospective, open label, multicentric, single treatment arm, clinical study was undertaken from 28 th February to 28 th April 2010. Healthy adult volunteers of either gender of more than 18 years of age were enrolled in the study, who were likely to be available for all visits during the follow-up period and willing to sign informed consent. Female subjects were required not to be pregnant or lactating at the time of enrolment and not planning pregnancy during the study period.
Inactivated Influenza Vaccine (Whole Virion) IP (Pandemic Influenza (H1N1) 2009 Monovalent Vaccine; VaxiFlu-S TM ) has been developed by M/s Cadila Healthcare Ltd. in liaison with Indian Regulatory Authorities. The vaccine is prepared from influenza virus propagated in the allantoic fluid of hen's eggs. Following harvest, the virus is purified on sucrose density gradient using a continuous zonal ultra-centrifugation. The purified virus is inactivated with b-propiolactone and suspended in a phosphate-buffered isotonic solution. Each 0.5 ml dose of the final vaccine contains purified 15 mcg Hemagglutinin Antigen of A/California/7/2009 H1N1 Influenza NIBRG-121xp virus.
Subjects with a history of anaphylaxis or serious reactions to any other vaccine, hypersensitivity to influenza viral proteins, egg proteins, neomycin, polymyxin, or products containing mercury were not eligible for enrolment in the study. Subjects known to be suffering from diseases that can affect immune competence e.g. diabetes, immunodeficiency disorders or HIV infection were excluded from the study. Subjects having acute respiratory pathology or infections requiring systemic antibiotic or antiviral therapy during the preceding seven days or having suspected/confirmed pandemic influenza H1N1 2009 infection could not participate in the study. Subjects having received seasonal influenza vaccine within the previous six months or any other vaccine or investigational medicinal product in the preceding three months were also excluded. Subjects with continuing history of alcohol and/or drug abuse were also not eligible for enrolment.
Subjects were not permitted to take any antiviral medications e.g. amantadine, rimantadine, oseltamivir, zanamivir, etc. for a period of two weeks after the administration of the vaccine. Administration of any other vaccine was not permitted during the entire period of the study i.e. 42 days. Further, use of medications that can affect the immune function of the body i.e. immunostimulants (e.g. interferons) or immunosuppressants (e.g. corticosteroids, cytotoxic drugs or antimetabolites, etc.), blood products or immunoglobulins was also not permitted concomitantly or in the preceding three months.
This open label, multicentric, phase 2/3 clinical study was approved by the Drugs Controller General of India (DCGI) and Institutional/Independent Ethics Committees (ECs) of each of the six participating centers. The present study was conducted in compliance with the Good Clinical Practice (GCP) Guidelines issued by the Central Drugs Standard Control Organization, Ministry of Health, Government of India and the ethical principles of Declaration of Helsinki. EC notifications as per GCP Guidelines and Ethical Guidelines for Biomedical Research on Human Subjects, issued by Indian Council of Medical Research (ICMR) were followed during the conduct of the study. The clinical trial was registered with Clinical Trials Registry - India (CTRI; http://www.ctri.in ) and written informed consent was obtained from each of the participating subjects. Healthy adult subjects of either sex and in the age group of either 18-59 years or more than 60 years were planned to be enrolled in this open labelled, phase 2/3 clinical trial. All the subjects were evaluated as per the Inclusion and Exclusion Criteria and underwent a thorough general physical and systemic examination at screening visit. Eligible subjects were administered a single 0.5 ml intramuscular (I.M.) dose of the vaccine containing ≥15 mcg of hemagglutinin antigen in the deltoid muscle. Vaccine of three different batches, which had been approved by Central Drugs Laboratory, Kasauli, was used in the trial. Two aliquots of blood samples for each subject were collected at baseline prior to vaccination and 21 (±2) days after vaccination. One aliquot was estimated for anti-hemagglutinin antibody titer by Hemagglutination Inhibition (HI) Test at Vaccine Bioanalytics Facility of M/s Cadila Healthcare Ltd., Ahmadabad, and the other aliquot was preserved for future reference.
Hematological and biochemical investigations like complete hemogram, liver and renal function tests were done at the screening visit and at 21 days after vaccination. Unsolicited clinical adverse events were recorded for a period of 42 days starting from the day of vaccination; along with their nature, intensity, remedial action taken, relationship to the vaccine and outcome. All the enrolled subjects were specifically monitored for local reactions like pain, tenderness, erythema/redness, swelling, induration and ecchymosis; and systemic reactions like fever, shivering, malaise, nausea/vomiting, diarrhea, headache, fatigue or myalgia (along with their severity grade) and for the development of any neurological abnormality for the entire study duration as per the regulatory requirements. At the end of the study, the investigators ranked the global tolerability of the vaccine on a four-point rating scale.
The following immunogenicity criteria were assessed in both the age groups of vaccine recipients as per WHO and European Medicines Agency (EMEA) Guidelines: , 1) Number of seroconversions or significant increase in HI titer; 2) Fold increase in Geometric Mean Titer (GMT); 3) Proportion of subjects achieving an HI titre ≥1:40. Seroconversion is considered as a post-vaccination titer ≥1:40 from a pre-vaccination titer <1:10 and significant increase in antibody titre is considered as at least a four-fold increase from pre-vaccination titer ≥1:10.
The data was analysed for demographics, immunogenicity and safety using standard statistical tests. For qualitative data, statistical analysis describes counts and percentages of respective population. 95% confidence intervals (95% CI) were calculated for each of the qualitative proportions with assumption of normal distribution of data and chi-square test was used for significance testing. Log transformation of the data was carried out to obtain approximate normal distribution of antibody titer values for the calculation of GMTs and 95% CIs, and student's t-test was applied to the transformed data for significance testing of GMT values and fold increases in GMT.
A total of 336 healthy adult volunteers were screened and 269 volunteers were found to be eligible for enrolment in the study. Of the enrolled 269 healthy volunteers, 203 subjects were between the age of 18 to 59 years and 66 subjects were more than 60 years of age. At the end of study, 261 volunteers (198 of 18-59 years of age and 63 of ≥60 years of age) were eligible for immunogenicity analysis while 265 volunteers (202 of 18-59 years of age and 63 of ≥60 years of age) were eligible for safety analysis. Flow diagram of the subjects enrolled in the study is shown in [Figure 1]. The detailed demographic profile and baseline characteristics of the enrolled volunteers are shown in [Table 1].
At baseline a similar proportion of subjects i.e. 134/198 (67.7% [95% CI: 61.2-74.2%]) and 43/63 (68.3% [95% CI: 56.8-79.7%]) in the age group of 18-59 years and ≥60 years, respectively, were seronegative with HI titers of <1:10 (P=0.932, chi-square test). Moreover, baseline antibody titer of ≥1:40 was reported in 28/198 (14.1% [95% CI: 9.3-19.0%]) and 7/63 (11.1% [95% CI: 3.4-18.9%]) subjects, which was also not significantly different in both the above respective age groups (P=0.539, chi-square test) [Table 2].
Further, 182/198 (91.9% [95% CI: 88.1-95.7%]) and 53/63 (84.1% [95% CI: 75.1-93.2%]) healthy adult volunteers in the age group of 18-59 years and ≥60 years, respectively, achieved a post-vaccination HI titre ≥1:40 at Day 21 (irrespective of baseline titer, P=0.072, chi-square test) [Table 2] and [Figure 2]. Seroconversion or significant (at least four-fold) increase in anti-hemagglutinin antibody titer was noted in 171/198 subjects (86.4% [95% CI: 81.6-91.1%]) in the age group of 18-59 years and 50/63 subjects (79.4% [95% CI: 69.4-89.4%]) in the age group of ≥60 years at 21 (±2) days after vaccination [Table 2] and [Figure 3]. Thus, a similar degree of seroconversion or significant rise in antibody titer was noted in both the age groups studied (P=0.179, chi-square test).
A similar GMTs of 14.2 [95% CI: 12.6-16.1] and 13.3 [95% CI: 11.1-16.0] were reported in the volunteers in both the age groups comprising of 18-59 years and ≥60 years, respectively, at baseline (P=0.543, student's t-test after log transformation); which increased to 322.2 (95% CI: 261.4-397.2) and 140.2 (95% CI: 98.6-199.3), respectively, 21 days after the vaccination. However, post-vaccination GMTs were significantly different in both the age groups (P=0.0001, student's t-test after log transformation). Thus, a significant rise of 22.6-fold (95% CI: 18.0-28.4) and 10.5-fold (95% CI: 7.4-15.0) was noted in GMT as compared to baseline in the age groups of 18-59 years and ≥60 years, respectively (P<0.001 for both as compared to baseline, student's t-test after log transformation). Again the increase in GMT was significantly different across both the age groups (P=0.0005, student's t-test after log transformation).
The result of assessment of immunogenicity criteria for the age groups of 18-59 years and ≥60 years is summarized in [Table 3].
Further, the effect of baseline serostatus on the immune response to H1N1 vaccination was analysed. In both the age groups studied, subjects who were seronegative at baseline (with HI titer <1:10) had numerically lower GMT values after vaccination than seropositive subjects (with baseline titer ≥1:10), which did not differ significantly in each age group (P=0.229 for younger age group and P=0.168 for elder age group) [Table 4]. However, subjects of 18-59 years of age, who were seronegative at baseline had a significantly higher factor increases in the GMT as compared to seropositive individuals in the same age group (P=0.0001), while this factor increase in GMT was not significantly higher in similar subjects ≥60 years of age (P=0.392).
No "serious", "severe" or "unexpected" adverse event was reported in any of the healthy adult volunteers enrolled in either of the age groups in the study. None of the volunteers discontinued the study due to adverse event during the entire course of the study. None of the vaccinated volunteers reported any neurological abnormality during the course of study at any of the study centers. Further, no clinically significant alteration was reported in any of the routine hematological and biochemical investigations as estimated at baseline and at 21 days in any of the healthy adult volunteers during the entire course of the study.
A total of 12 adverse events were reported by 11 healthy adult volunteers (ten volunteers in the age group of 18-59 years and one volunteer in the age group of ≥60 years) [Table 5]. Thus, the incidence of adverse drug events for total number of administered vaccine doses was 4.5% [95% CI: 2.0-7.0%], while subject adverse event rate was 4.2% [95% CI: 1.7-6.6%] in the study. Nine of these 12 adverse events [3.4%; 95% CI: 1.2-5.6%] were rated to have a "possible" association with the vaccine administration, while three events (one event each of stomach upset, myalgia and fever) had a "remote" association. The local reaction of pain and systemic reactions of fatigue, malaise, fever and myalgia reported in the study were of "Grade 1" severity (no interference with routine activity) only. All these adverse events settled completely with/without symptomatic treatment during the course of study. At the end of the study, 257/265 [97.0%; 95% CI: 94.9-99.0%] healthy adult volunteers eligible for tolerability assessment in the study were rated by the investigators to have an "excellent" tolerability to the vaccine as per the four-point global assessment of tolerability scale [Figure 4].
The present study was carried out to assess the immunogenicity and safety of Inactivated Influenza Vaccine (Whole Virion) IP (Pandemic Influenza (H1N1) 2009 Monovalent Vaccine; VaxiFlu-S TM manufactured by M/s. Cadila Healthcare Ltd.) given intramuscularly in Indian adult population. The study has demonstrated that lower limits of 95% confidence intervals for each of the immunogenicity criteria are well above the WHO and EMEA requirements. Local/systemic adverse events reported in the study were of the severity which did not interfere with the daily activities of the participants. Thus, the results indicate that the Pandemic Influenza H1N1 2009 (Whole Virion) Monovalent Vaccine produces an adequate immunogenic response as per internationally established standards for Influenza Vaccines and has a fairly acceptable safety profile.
Several influenza vaccines have been developed and are currently in use for the prevention of H1N1 2009 Influenza A; these include inactivated I.M. (adjuvanted and unadjuvanted) vaccines and live attenuated intranasal vaccines. Adjuvanted inactivated vaccines generally produce a better immune response with lesser amount of antigen per dose; albeit with an increased incidence of local and systemic reactions.  Further, unadjuvanted inactivated vaccines currently available internationally are "whole virion" vaccines and "subvirion" (split virion/surface antigen) vaccines. While the "whole virion" vaccines are generally considered to be more immunogenic than "subvirion" vaccines in naοve populations;  the latter, which are prepared by disruption of viral lipid envelop by a solvent and purification to reduce the amount of other viral proteins, are believed to be better tolerated.  These "unadjuvanted subvirion" inactivated vaccines produce a good immunologic response by a single I.M. dose of 15 μg antigen with good tolerability, which is also the recommended dose for adult population.  Similarly, our study with an "unadjuvanted whole virion" vaccine prepared as per the Indian Pharmacopoeia standards,  has shown that this vaccine preparation produces an excellent immunogenic response with a single I.M. dose of 15 μg antigen and also has a good tolerability profile.
Our study excluded patients who may have received seasonal trivalent influenza vaccine within six months of enrolment, to avoid potential interference in the immunogenic response to the study vaccine. However, earlier studies with subvirion vaccines have demonstrated that seasonal trivalent inactivated influenza vaccines (with/without adjuvant) do not interfere with the immunogenic response or alter the safety profile of pandemic monovalent inactivated adjuvanted influenza vaccines, whether given concomitantly or subsequently. ,
Since, the study design of our clinical trial did not include a placebo control group, there is a possibility of ongoing virus circulation affecting the immunogenicity assessments of the pandemic vaccine. However, it may be noted that none of the volunteers enrolled in the study reported any adverse event suggestive of influenza infection during the course of study. Further, current international scientific and regulatory guidelines , do not emphasize requirement of placebo controlled trials to establish the immunogenicity of a candidate pandemic vaccine, from ethical perspective in potentially urgent pandemic situation. Moreover, a study by Talaat et al reported that seroconversion (0-2.6%), fold-increase in GMT (0.95-1.1 fold) and change in proportion of subjects with ≥1:40 titer at 21 days as compared to baseline (0.1-0.5%) was negligible in the placebo arm in all the age groups.  Still, considering the possibility of subclinical viral infections, placebo-controlled trials can clarify with certainty the effect of this potential confounding factor during clinical trials with pandemic influenza vaccines.
In our study, 14.1 and 11.1% subjects in the age groups of 18-59 years and ≥60 years, respectively, had baseline HI titers of ≥1:40. Though optimum subject selection criteria were used for the enrolment of the volunteers, these findings may be due to an earlier exposure to H1N1 2009 influenza virus leading to subclinical illness only, cross reacting antibodies from past seasonal influenza infections/vaccination or other unknown reasons.
Assessment of the immunogenicity criteria in the two age groups studied by us showed that the GMT values, factor increase in GMT and the proportions of subjects achieving a particular GMT value is lower in the elderly subjects of ≥60 years of age as compared to the younger subjects of 18-59 years of age. These findings are anticipated as well as documented and hence the immunogenicity criteria requirements are also lower for the elderly population.  However, it is noteworthy in the study that a single dose of the vaccine did produce the required immune response with good tolerability in the elderly subjects. Some earlier studies suggested the possibility of sufficient but delayed immune response in the elderly;  however, rapid decline in antibody titers as compared to the younger population is also reported.  An immunogenicity assessment scheduled further later in the course of study could have confirmed a delayed response leading to further increase in HI titers in this age group. Presently development of newer strategies to enhance the immune response in the elderly,  including the use of increased antigen content in the vaccine, , are advocated to improve the protection of this vulnerable population against influenza.
Other published studies comparing multiple doses of adjuvanted or unadjuvanted inactivated H1N1 2009 vaccines have shown a similar degree of immune response with a single dose of 15 μg unadjuvanted formulations corresponding to that used in our study. The proportion of subjects with seroconversion/significant rise in HI titer were reportedly higher in our study at 21 days after single-dose administration as compared to the study conducted by Greenberg et al with "split virion" unadjuvanted vaccine (for young; 86.4 vs. 77.6% and for elderly subjects; 79.4 vs. 71.0%); so also was the factor increase in GMT (for young; 22.6-fold vs. 15.1-fold and for elderly; 10.5-fold vs. 9.4-fold). However, the proportion of subjects with a post-vaccination titer ≥1:40, irrespective of baseline titers was reported to be lower in our study (for young; 91.9 vs. 96.6% and for elderly; 84.1 vs. 93.5%). 
All the three immunogenicity criteria results i.e. seroconversion/significant titre increase (86.4 vs. 52.0%), ≥1:40 titre (91.9 vs. 63.0%) as well as GMT factor increase (22.6-fold vs. 13.5-fold) were reported to be higher in our study after single vaccination at 21 days' measurements, as compared to another study conducted by Clark et al. This is despite the fact that our study had enrolled subjects of 18-59 years of age while the study by Clark et al. had enrolled only young participants of 18 to 50 years of age in the unadjuvanted "surface antigen" vaccine study arm similar to our study. 
Further, a placebo-controlled clinical trial conducted by Talaat et al evaluated 15 μg unadjuvanted "split virion" vaccine in age groups of 18-65 (young), 65-75 and ≥75 years (elderly). Again, seroconversion results were comparable or higher in our study in comparison to this study (for young; 86.4 vs. 88.3% and for elderly subjects; 79.4 vs. 50 to 62%) along with GMT factor increase (for young; 22.6-fold vs. 21.3-fold and for elderly; 10.5-fold vs. 4.5 to 5.9-fold), while subject proportions with post-vaccination titer ≥1:40 was lower in our study (for young; 91.9 vs. 96.4% and for elderly; 84.1 vs. 90 to 93.8%). 
Thus, Inactivated Influenza Vaccine (Whole Virion) IP (Pandemic Influenza (H1N1) 2009 Monovalent Vaccine; VaxiFlu-S TM ) has immunogenicity comparable to other unadjuvanted inactivated vaccine formulations studied and approved internationally. Direct safety comparisons of the results of our study could not be made with these published studies ,, due to the differences in the methods of adverse events' recording in terms of solicitation.
The present study did not include pediatric population for the assessment of immunogenicity and safety of H1N1 2009 influenza vaccine. Though, unadjuvanted inactivated influenza vaccines are known to be safe and immunogenic with requirement of additional doses in pediatric population, a clinical trial can definitely establish response to the current vaccine in children. Further, the effect of vaccine for the prevention of H1N1 2009 infection and safety in general population needs to be assessed in effectiveness evaluation studies and post-marketing safety surveillance studies particularly for rare neurological adverse events like Guillain-Barre Syndrome. In fact, post-approval surveillance and a prospective clinical study are presently ongoing to further elucidate the safety profile of the vaccine.
In conclusion, the results of this open label, multicentric clinical trial demonstrate that Inactivated Influenza Vaccine (Whole Virion) IP (Pandemic Influenza (H1N1) 2009 Monovalent Vaccine; VaxiFlu-S TM ) given as a single I.M. injection in the deltoid muscle (0.5 ml dose containing ≥15 mcg of hemagglutinin antigen) in adult Indian population produces an excellent immunogenic response. Though the immunogenic response in elderly persons of ≥60 years age is generally lower than younger individuals as is anticipated, adequate antibody titers are achieved with a single dose. Moreover, the H1N1 vaccine has a good tolerability profile. Based on these findings, this Pandemic Influenza (H1N1) 2009 Monovalent Vaccine is suitable for use in adult Indian population for the prevention of H1N1 2009 Influenza A infection.
We acknowledge the H1N1 2009 vaccine study group: Dr. Praveen Garg, Consultant Physician, Shashwat Hospital & Research Centre, Ahmedabad; Dr. Puneet Rijhwani, Associate Professor, Department of Medicine, Mahatma Gandhi National Institute of Medical Sciences, Jaipur; Dr. Vikas G. Pai, Consultant Physician, Pai Clinic & Diagnostic Centre, Pune; Dr. Raj Bhagat, Honorary Physician - L.G. Hospital, Associate Professor - Dept. of Medicine, A.M.C. Medical Education Trust, Dr. Bhagat's Allergy, Asthma Clinic & Resp. Care Centre, Ahmedabad; Dr. V. Shankar, Consultant Physician, Standard Laboratory and Polyclinic, Bangalore and Dr. Dinesh V. Kamath, Consultant Physician & Diabetologist, Sudeep Diabetes Care Centre, Bangalore.
We also acknowledge the following other personnel: Dr. Reinhard Glueck, Dr. Rajendra Jani, Dr. Manjula Shamanna, Mr. Rakesh Kshatriya, Mr. Abhijeet Srirao, all from Cadila Healthcare Ltd and Ms. Gargi Ladia, Ashish Pathology Laboratory, A-103/104, Aasthan Complex, Opp. Polytechnic, Ahmedabad.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]