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Immunogenicity and safety study of Indirab: A Vero cell based chromatographically purified human rabies vaccine
Vaccine 28 (2010) 4086–4090
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Immunogenicity and safety study of Indirab: A Vero cell based chromatographically purified human rabies vaccine Gadey Sampath a , Shampur Narayan Madhusudana b,∗ , Mysore Kalappa Sudarshan c , Doddabele Hanumanthaiah Ashwathnarayana c , Bangalore Jayakrishna Mahendra d , Thankappan P. Ullas b , Krishna Mohan e , Santhosh Kumar Madhusudhan e , Haradanahalli Shankariah Ravish c a
Institute of Preventive Medicine, Hyderabad, India Dept. of Neurovirology, National Institute of Mental Health & Neurosciences (NIMHANS), Hosur Road, Bangalore, India c Dept. of Community Medicine, Kempegowda Institute of Medical Sciences (KIMS), Bangalore, India d Dept. of Community Medicine, Mandya Institute of Medical Sciences, Mandya, India e Bharat Biotech International Ltd., Hyderabad, India b
a r t i c l e
i n f o
Article history: Received 20 August 2009 Received in revised form 6 March 2010 Accepted 26 March 2010 Available online 18 April 2010 Keywords: Rabies Rabies vaccines Purified Vero cell rabies vaccine Chromatographically purified Vero cell rabies vaccine
a b s t r a c t A chromatographically purified Vero cell rabies vaccine, Indirab manufactured by Bharat Biotech International Limited, Hyderabad, India was subjected to safety and immunogenicity studies by both intramuscular and intradermal routes of administration in parallel with a reference vaccine, Verorab. A Pre-exposure study was undertaken in 239 subjects by intramuscular (IM) route (Study I), Post-exposure study in 188 patients by intramuscular route (Study II) and Simulated post-exposure study in 134 subjects by intradermal (ID) route (Study III). All subjects in these studies were administered with either the test or the reference vaccine as per WHO approved intramuscular and intradermal regimens. The blood samples were collected on days 0, 14 and 35 in case of Study 1, and days 0, 14, 28 and 90 in case of studies II and III. In all studies both vaccine groups had adequate antibody titers (>0.5 IU/mL) on all days tested post-vaccination and there was no significant difference in the titers observed (p > 0.05). Some side effects like pain, induration, itching and fever were noted in both vaccine groups in all studies. Both vaccines were well tolerated. Hence it can be concluded that Indirab is as safe and immunogenic as Verorab when administered by both intramuscular and intradermal routes. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Rabies is a fatal viral encephalitis which still continues to be a major public health and veterinary problem in many parts of the world particularly the developing nations of Asia and Africa. As per a WHO estimate around 55,000 people die of rabies every year globally [1]. India reports the highest number of human rabies deaths and animal bites. As per a recent WHO sponsored multi-centric study conducted in India by the Association for Prevention and Control of Rabies in India (APCRI), about 20,000 human rabies deaths are reported and around 17 million animal bites occur annually [2]. Till recently all the animal bite victims who attended government run antirabies clinics and hospitals were administered with nerve tissue derived Semple vaccine. Use of cell culture vaccines (CCVs) were restricted to a small segment of population who could afford their cost. However following the order of Supreme
∗ Corresponding author. Tel.: +91 80 26995129; fax: +91 80 26564830. E-mail address: [email protected] (S.N. Madhusudana). 0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2010.03.064
Court of India, the use of Semple vaccine was completely banned in 2005 and all the animal bite victims attending the government run clinics are also being administered with CCVs. Consequently the demand for CCVs has increased considerably over the past few years. Modern CCVs such as Purified Chick Embryo Cell (PCEC) vaccine and Purified Vero cell Rabies Vaccine (PVRV) are all being manufactured in India in the private and public sectors for many years but the quantity produced is not sufficient to meet the required demand. Thus there is scope for increasing the production capacities of these vaccines and also produce newer vaccines in sufficient quantities to meet the huge demand for rabies vaccine in this country. In this context the production and supply of a new PVRV (Indirab) which is derived from Pitman-Moore strain and purified chromatographically is a welcome step. The other brands of PVRV produced in India and France are presently purified by zonal centrifugation. Efforts were made earlier by Lang et al. [3] to purify PVRV by chromatographic method, and a few batches were tested for potency and immunogenicity by pre-exposure vaccination of healthy volunteers. A post-exposure clinical trial was also conducted in Philippines using this CPVRV with good results
G. Sampath et al. / Vaccine 28 (2010) 4086–4090
[4]. However, commercialization of this product was not materialized and presently Indirab is the only chromatographically purified PVRV available commercially in the world. This vaccine is produced in India by Bharat Biotech International Ltd., Hyderabad using disposable cell factories with automated cell factory manipulator (ACFM) and WHO approved current Good Manufacturing Practices (cGMP). The vaccine was certified for human use by the Drugs Controller General of India (DCGI) after undergoing clinical trials in 3 phases, viz. Pre-exposure studies in healthy volunteers, Postexposure studies by standard intramuscular route and Simulated post-exposure studies by intradermal routes. A WHO approved vaccine viz. Purified Vero cell Rabies Vaccine (PVRV, Verorab) was used as reference vaccine to evaluate this new vaccine in terms of safety, tolerability and immunogenicity. This paper gives an overview of these clinical trials. The results of these trials clearly indicate that this new chromatographically purified rabies vaccine produced indigenously is safe, well tolerated and as immunogenic as the reference vaccine used in these studies. 2. Materials and methods The protocols of these clinical studies were approved by Drugs Controller General of India (DCGI) and Institutional Ethics Committee (IEC) of the respective study centers. Written informed consent was obtained from all the subjects before enrollment into the respective study. 2.1. Study design All these were blinded, randomized, active controlled, multicenter, comparative (parallel), Phase III studies. These studies were performed under out-patient department conditions in subjects enrolled as per the inclusion and exclusion criteria. The inclusion criteria were healthy subjects, not previously exposed to rabies and not taken any rabies vaccine in the past, willingness to participate in the study and their availability for the entire study period in Study 1 and Study 3. The Study 1 (Pre-exposure intramuscular) was conducted in four centers, Study 2 (Postexposure intramuscular) in two centers and Study 3 (Simulated post-exposure Intradermal) in two centers. The test and reference vaccines in intradermal (ID) studies were administered by trained staff from Institute of Preventive Medicine (IPM), Hyderabad, India and Kempegowda Institute of Medical Sciences (KIMS), Bangalore, India under the supervision of Principal Investigators. In each center, the subjects received either the test vaccine or the reference vaccine as per the randomization codes generated. A commercially available WHO pre-qualified vaccine Verorab procured from open market was used in all studies as a reference vaccine. Subjects were randomly assigned to one of the groups as per the codes generated by the computer programme Minitab 14 version. The group assignment was not known to the subject, investigator and the laboratory staff performing the serologic assay. 2.1.1. Study 1 (Pre-exposure intramuscular) A total of 239 healthy volunteers participated in the study; 180 were given Indirab vaccine (Batch No. 62AN4001, potency 5.84 IU/dose) and 59 subjects were given Verorab vaccine (Batch No. Y0659, potency >2.5 IU/dose). Both test and reference vaccines were reconstituted with 0.5 ml diluent provided along with the vaccine vial. Vaccine was administered intramuscularly on days 0, 7 and 28 and blood samples of 5 ml were collected on days 0, 28 and 35. We were able to collect blood samples at the end of 1 year from 18 subjects who had taken Indirab and 12 subjects who had taken Verorab. The study population was between 10 and 65 years of age.
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This broad age range was particularly included to assess the safety and immunogenicity in young children as well as elderly people. 2.1.2. Study 2 (Post-exposure Intramuscular) A total of 188 (aged 5–55 years) cases participated in the study, out of which 141 subjects were given Indirab vaccine (Batch No. 62AN4002, potency 6.08 IU/dose) and 47 subjects were given Verorab vaccine (Batch No. Z0502, potency >2.5 IU/dose). Majority of animal bite victims in India belong to this age group. Category III patients were administered rabies immunoglobulin (Equine rabies Immunoglobulin, Equirab, Bharat Serums and Vaccines) as per WHO recommended dose. Both test and reference vaccines were reconstituted with 0.5 ml diluent. Vaccine was administered intramuscularly on days 0, 3, 7, 14 and 28 and blood samples of 5 ml were collected on days 0, 14, 28 and 90. 2.1.3. Study 3 (Simulated post-exposure intradermal) A total of 134 healthy volunteers (aged 18–55 years) participated in the study, 68 were given Indirab vaccine (Batch No. 62AN7005, potency 5.7 IU/dose) and 66 subjects were given Verorab vaccine (Batch No. A0920, potency >2.5 IU/dose). Both test and reference vaccines were reconstituted with 0.5 ml diluent. Vaccine was administered ID as per updated Thai Red Cross Regimen (2-2-2-0-2) on days 0, 3, 7 and 28 and blood samples of 5 ml were collected on day 0, 14, 28 and 90. 2.2. Estimation of Rabies Virus Neutralizing Antibody (RVNA) titers The blood samples were centrifuged after collection, serum separated and transferred into 2 coded cryovial aliquots and stored at −20 ◦ C until transferring to testing laboratory, Department of Neurovirology, National Institute of Mental Health and Neurosciences (NIMHANS) Bangalore, India which is WHO collaborating centre for reference and research on rabies. Serum samples were tested by Rapid-Fluorescent-Focus Inhibition Test (RFFIT) as per WHO recommendation with some modifications [5]. The test was performed using 96 well tissue culture plates (Nunc, USA) and the cell line used was BHK 21 (ATCC CCL 10). The virus used was CVS 11 (obtained from Central Research Institute, Kasauli, India) and adapted to grow in BHK 21. We used 50 FFD50 of the virus as challenge dose. The highest dilution of the serum samples which reduced fluorescent foci in 50% of the cells was taken as the endpoint. The titers were expressed in IU/mL in comparison to an in-house reference serum calibrated against 2nd international reference serum having a potency of 30 IU/mL. This was procured from National Institute of Biological Standards, UK. 2.2.1. Statistical analysis Geometric mean titers (GMT) and 95% confidence intervals (CI) were computed by taking the exponent (log 10) of the mean and of the lower and upper limits of the 95% confidence intervals of log 10 transformed titers. The safety end points (percentage of subjects and 95% confidence intervals) were solicited and unsolicited adverse events. We used Mann-Whitney Test to compare the values between the vaccine groups. A p value 0.04 or less was considered to indicate statistical significance. Data analysis was done by using MINITAB software (version 14). 3. Results The demographic profile of the subjects recruited in the 3 studies is given in Table 1. The purpose of the Study I was to assess the safety and immunogenicity of this new vaccine in healthy volunteers. It can be seen from Table 2 that all subjects in both vaccine groups attained more than adequate levels of RVNA titers by day
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Table 1 Demographic profile of subjects recruited in the studies. Study number
Parameter
Test group mean
Range
Reference group mean
Range
I
Age (years) Weight (kg) Height (cm) Sex (M/F)
21.3 51.2 164
13–59 27–88 158–182
20.9 50.5 167
15–54 33–86 155–180
II
Age (years) Weight (kg) Height (cm) Sex (M/F)
25.5 55 164
III
Age (years) Weight (kg) Height (cm) Sex (M/F)
27.6 62 167
141/39
Vaccine type
N
GMT
15–43 35–70 157–181
20.2 56.3 164
19–39 45–92 155–185
26.57 59.83 161
18–39 36–81 155–188
89/52
36/11 19–47 45–88 152–188
67/1
Table 2 Rabies virus neutralizing antibody titers observed after pre-exposure immunization with Indirab and Verorab (Study I). Day
41/18
95% CI LB
p value
66/0
Table 4 Rabies virus neutralizing antibody titers (IU/mL) observed after intradermal vaccination with Indirab and Verorab in healthy volunteers (Study III). Day
Vaccine type
N
GMT
UB
95% CI
p value
LB
UB
Day 0
Indirab Verorab
177 57
0.02 0.03
0.01 0.01
0.04 0.04
0.4050
Day 14
Indirab Verorab
67 63
3.36 3.26
3.26 3.14
3.96 3.76
0.4923
Day 14
Indirab Verorab
174 56
6.1 8.3
5.19 5.59
7.17 11.03
0.1929
Day 28
Indirab Verorab
67 63
7.83 7.70
7.57 7.43
8.51 8.36
0.936
Day 35
Indirab Verorab
174 56
18.8 18.5
17.24 16.79
20.45 20.34
0.3127
Day 90
Indirab Verorab
67 63
3.62 3.57
3.43 3.43
4.12 4.06
0.7590
Day 365
Indirab Verorab
18 12
3.5 3.8
2.9 3.2
4.5 4.9
0.3412
14 which increased substantially on day 35. Though the geometric mean titer (GMT) on day 14 in the Verorab group was higher, it was not statistically significant (p > 0.05). Subjects who have the positive serum antibody titers at baseline (3 from Indirab group and 2 from Verorab group) were excluded from analysis. In some of the subjects, whose serum was tested at 1 year period, adequate antibody titers were seen in both test and reference groups. There were 5 dropouts in the study after the first dose of vaccination, 4 in the test group and 1 in the reference group. However, on telephonic enquiry, it was ascertained that the dropout was not due to any side effect of the vaccine. In the Study II, we administered either Indirab (n = 141) or Verorab (n = 47) to subjects bitten by suspect rabid animal and having either category II or III exposure. Six patients (4 in the Indirab group and 2 in the Verorab group) were lost to follow up after the 3rd dose of vaccine. Efforts to trace these patients were not successful. It can be seen that all subjects tested for RVNA had sero-
converted in both the vaccine groups by day 14. More than adequate levels of RVNA titers were observed on days 28 and 90 and there was no significant difference in the titers on all days tested (Table 3). The purpose of Study III was to show the immunogenicity of Indirab when administered by WHO approved ID schedule of vaccination in healthy volunteers. Indirab was administered to 68 subjects and Verorab to 66 subjects. There was one dropout in test group and 3 dropouts in reference group. The RVNA titers observed on different days in the two groups are depicted in Table 4. The results show that Indirab is as immunogenic as Verorab even when administered by ID route of vaccination. The incidence of side effects was negligible, did not differ significantly between the two groups and did not require medication in all three studies (Table 5). 4. Discussion Vero cells were approved for the production of human vaccines in 1987 [6], and following this inactivated polio and rabies vaccines were produced for human use [7]. A potential concern in
Table 3 Rabies virus neutralizing antibody titers (IU/mL) after post-exposure vaccination with Indirab and Verorab (Study II). Day
Vaccine type
N
GMT
95% CI LB
% sero conversion
p value
UB
Day 14
Indirab Indirab + RIG Verorab Verorab + RIG
107 30 35 10
4.94 5.70 5.10 5.05
4.55 4.96 4.38 3.1
5.33 6.44 5.82 6.17
100 100 100 100
0.62 0.31 4.5 0.31
Day 28
Indirab Indirab + RIG Verorab Verorab + RIG
107 30 35 10
10.86 11.79 11.2 11.0
9.31 10.53 10.03 9.01
11.55 13.16 12.37 12.9
100 100 100 100
0.36 0.37 0.36 0.42
Day 90
Indirab Indirab + RIG Verorab Verorab + RIG
107 30 35 10
8.16 8.46 7.83 8.5
7.52 7.5 6.63 7.4
8.80 9.42 9.03 9.6
100 100 100 100
0.52 0.63 0.53 0.62
G. Sampath et al. / Vaccine 28 (2010) 4086–4090
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Table 5 Side effects observed with Indirab and Verorab in the three studies. Study
Vaccine type
Side effects observed
p value (total side effects)
Pain
Swelling
Fever
I
Indirab (n = 180) Verorab (n = 59)
5 2
2 1
2 1
0 0
0 0
0.68
II
Indirab (n = 141) Verorab (n = 47)
3 1
3 1
3 2
0 0
0 0
0.89
III
Indirab (n = 68) Verorab (n = 66)
2 2
0 2
1 2
19 21
2 2
0.78
Itching
Redness
Study I = pre-exposure intramuscular. Study II = post-exposure intramuscular. Study III = simulated post-exposure intradermal.
approving Vero cells for vaccine production was the presence of cellular DNA, which can theoretically lead to genetic transformation and tumorigenicity in the vaccinees. However, after a series of meetings coordinated by the World Health Organization (WHO) and based on substantial experimental evidence, Vero cells were approved for vaccine production provided the final product met all the WHO requirements including the DNA concentration less than 10 ng/dose [6]. Purified Vero cell Rabies Vaccines (PVRV) were first approved for human use in 1992 [8]. The first PVRV to be available commercially was Verorab produced by Pasteur Merieux in 1989. This vaccine was made using Pitman Moore strain of rabies virus and the purification step involved zonal centrifugation [7]. The vaccine was found to be safe and immunogenic in several clinical studies, found to be safe in children and pregnant women and was used in several Asian countries both by IM and ID routes of administration [9–12]. Though the WHO has accepted 10 ng/dose of cellular DNA as a safe upper limit [13], the upper limit of DNA as per European and American pharmacopoeia is 100 pg/dose. In order to further reduce the cellular DNA content to less than 100 pg/dose, purification of the vaccine was done by column chromatography by Lang et al. with the PVRV produced by Pasteur Merieux Ltd., France. The immunogenicity and safety profile of this new chromatographically purified Vero cell rabies vaccine (CPRV) was assessed in both healthy volunteers and in post-exposure clinical trials [4,5]. However, this vaccine was never produced in large scale and was never commercially sold in the international market. Recently an Indian Biotech Company, Bharat Biotech International Ltd., has indigenously produced this CPRV for commercial purpose. The vaccine is produced using disposable cell factories with automated cell factory manipulator (ACFM). The vaccine has been cleared by the Central Drugs Laboratory of the National Quality Control Authority and all batches of vaccine produced so far have residual cellular DNA content ranging from 60 to 70 pg/dose which is far less than the requirement of American and European pharmacopoeia. This chromatographically purified PVRV underwent three clinical trials. The first study involved assessing safety and immunogenicity in healthy volunteers. The performance of the vaccine Indirab in terms of safety, tolerance and immunogenicity was as good as the reference vaccine Verorab. In the second study the vaccine was administered to people bitten by suspect rabid animal as a post-exposure vaccination. In all people whose blood was tested for RVNA, 100% seroconversion occurred by day 14 and more than adequate levels of antibodies persisted till day 90 (Table 3). These people were alive and quite healthy after 1 year observation period. As we obtained very good antibody titers after the conventional IM schedule of vaccination, we wanted to know if Indirab vaccine is equally immunogenic when administered by the intradermal (ID) route. As per WHO requirement and also as per national guidelines, new vaccines are approved for ID use only after generating sufficient clinical data on their immunogenicity when administered by that route. This prompted us to undertake a simulated post-exposure study in adult healthy volunteers.
We used the newly approved 2-2-2-0-2 (Updated Thai Red Cross Regimen) for this study, which is recommended by both WHO and Government of India [14]. The results of this study clearly indicate that, Indirab is equally safe and immunogenic as the reference vaccine when administered by ID route (Table 4). The side effects observed in all the three studies were only minimal and self-limiting. The incidence of side effects did not vary between the two groups of vaccines. To conclude, in a country like India, where the burden of animal bites and rabies deaths ranks number one in the world, there is always a huge demand for safe and potent rabies vaccines both in public and private sectors. This new vaccine, Indirab has proved to be safe and immunogenic by both IM and ID routes. In addition, this is the only PVRV that is purified by chromatographic technique, reducing the cellular DNA content to less than 100 pg/dose. Considering this fact, we are of the view that this vaccine may serve as a better option along with the presently available PVRV both in Indian market as well as in international market. In an official notification, the government of India has already cleared this vaccine for use in India and for export purpose by both IM and ID routes. The vaccine is now undergoing Post Marketing Surveillance for both IM and ID routes. Preliminary results indicate that the vaccine is well tolerated, side effects are minimal and antibody assay on random blood samples collected from different vaccination centers across the country has shown more than adequate antibody response, thus building up confidence in using this vaccine in the general population.
References [1] World Health Organization. World survey of rabies No 35 for the year 1999. Geneva: WHO/CDS/CSR/EPH/2002.10; 2002. [2] Sudarshan MK, Madhusudana SN, Mahendra BJ, Rao NS, Ashwathnarayana DH, Abdul Rahman S, et al. Assessing burden of human rabies in India: results of a national multi-center epidemiological survey. Int J Infect Dis 2007;11(1):29–35. [3] Lang J, Cetre JC, Picot N, Lanta M, Briantais P, Vital S, et al. Immunogenicity and safety in adults of a new chromatographically purified vero-cell rabies vaccine (CPRV): a randomized double blind trial with purified vero cell rabies vaccine (PVRV). Biologicals 1998;26:295–308. [4] Quiambao BP, Lang J, Vital S, Montalban CG, Le Mener V, Wood SC, et al. Immunogenicity and effectiveness of post-exposure rabies prophylaxis with a new chromatographically purified vero-cell rabies vaccine (CPRV): a two stage randomized clinical trial in the Philippines. Acta Trop 2000;75:39–52. [5] Smith JS, Yager PA, Baer GM. A rapid fluorescent focus inhibition test (RFFIT) for determining rabies-neutralizing antibody. In: Meslin FX, Koprowsky H, Kaplan MM, editors. Laboratory techniques in rabies. 4th ed. Geneva: WHO; 1996. p. 181–92. [6] World Health Organization. WHO expert committee on biological standardization 37th report. WHO Tech Rep Ser 760. Geneva: WHO; 1987. [7] Montagnon BJ. Polio and rabies vaccines produced in continuous cell lines. A reality for vero cell line. Dev Biol Stand 1989;70:27–47. [8] World Health Organization. WHO expert committee on rabies: 8th report. WHO Tech Rep Ser 824. Geneva: WHO; 1992. [9] Chutivongse S, Supich C, Wilde H. Acceptability and efficacy of purified vero-cell rabies vaccine in Thai children. Asia Pac J Public Health 1988;2:179–85. [10] Sudarshan MK, Madhusudana SN, Mahendra BJ. Post-exposure prophylaxis of purified vero cell rabies vaccine (PVRV) during pregnancy: safety and immunogenicity study. J Commun Dis 1999;31(4):229–36.
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[11] Sampath G, Reddy SV, Prasada Rao ML, Rao YU, Palaniappan C. An immunogenicity study of a newly introduced vero cell rabies vaccine (Abhayrab) manufactured in India. Vaccine 2000;23:897–900. [12] Briggs DJ, Banzhoff A, Nicolay U, Sirikwin S, Dumavibhat B, Tongswas S, et al. Antibody response in patients after post-exposure rabies vaccination with small intradermal doses of purified chick embryo cell vaccine or purified verocell rabies vaccine. Bull World Health Org 2000;78:683–7.
[13] World Health Organization. Requirements for rabies vaccines for human use produced in continuous cell lines (amendment 1992) WHO expert committee on biological Standardization. In: 43rd report WHO Tech rep Ser 840. Geneva: WHO; 1992. [14] World Health Organization. Expert consultation on rabies 2004. WHO Tech Rep Ser, 931. Geneva: WHO; 2005.
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Immunogenicity and safety study of Indirab: A Vero cell based chromatographically purified human rabies vaccine Gadey Sampath a , Shampur Narayan Madhusudana b,∗ , Mysore Kalappa Sudarshan c , Doddabele Hanumanthaiah Ashwathnarayana c , Bangalore Jayakrishna Mahendra d , Thankappan P. Ullas b , Krishna Mohan e , Santhosh Kumar Madhusudhan e , Haradanahalli Shankariah Ravish c a
Institute of Preventive Medicine, Hyderabad, India Dept. of Neurovirology, National Institute of Mental Health & Neurosciences (NIMHANS), Hosur Road, Bangalore, India c Dept. of Community Medicine, Kempegowda Institute of Medical Sciences (KIMS), Bangalore, India d Dept. of Community Medicine, Mandya Institute of Medical Sciences, Mandya, India e Bharat Biotech International Ltd., Hyderabad, India b
a r t i c l e
i n f o
Article history: Received 20 August 2009 Received in revised form 6 March 2010 Accepted 26 March 2010 Available online 18 April 2010 Keywords: Rabies Rabies vaccines Purified Vero cell rabies vaccine Chromatographically purified Vero cell rabies vaccine
a b s t r a c t A chromatographically purified Vero cell rabies vaccine, Indirab manufactured by Bharat Biotech International Limited, Hyderabad, India was subjected to safety and immunogenicity studies by both intramuscular and intradermal routes of administration in parallel with a reference vaccine, Verorab. A Pre-exposure study was undertaken in 239 subjects by intramuscular (IM) route (Study I), Post-exposure study in 188 patients by intramuscular route (Study II) and Simulated post-exposure study in 134 subjects by intradermal (ID) route (Study III). All subjects in these studies were administered with either the test or the reference vaccine as per WHO approved intramuscular and intradermal regimens. The blood samples were collected on days 0, 14 and 35 in case of Study 1, and days 0, 14, 28 and 90 in case of studies II and III. In all studies both vaccine groups had adequate antibody titers (>0.5 IU/mL) on all days tested post-vaccination and there was no significant difference in the titers observed (p > 0.05). Some side effects like pain, induration, itching and fever were noted in both vaccine groups in all studies. Both vaccines were well tolerated. Hence it can be concluded that Indirab is as safe and immunogenic as Verorab when administered by both intramuscular and intradermal routes. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Rabies is a fatal viral encephalitis which still continues to be a major public health and veterinary problem in many parts of the world particularly the developing nations of Asia and Africa. As per a WHO estimate around 55,000 people die of rabies every year globally [1]. India reports the highest number of human rabies deaths and animal bites. As per a recent WHO sponsored multi-centric study conducted in India by the Association for Prevention and Control of Rabies in India (APCRI), about 20,000 human rabies deaths are reported and around 17 million animal bites occur annually [2]. Till recently all the animal bite victims who attended government run antirabies clinics and hospitals were administered with nerve tissue derived Semple vaccine. Use of cell culture vaccines (CCVs) were restricted to a small segment of population who could afford their cost. However following the order of Supreme
∗ Corresponding author. Tel.: +91 80 26995129; fax: +91 80 26564830. E-mail address: [email protected] (S.N. Madhusudana). 0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2010.03.064
Court of India, the use of Semple vaccine was completely banned in 2005 and all the animal bite victims attending the government run clinics are also being administered with CCVs. Consequently the demand for CCVs has increased considerably over the past few years. Modern CCVs such as Purified Chick Embryo Cell (PCEC) vaccine and Purified Vero cell Rabies Vaccine (PVRV) are all being manufactured in India in the private and public sectors for many years but the quantity produced is not sufficient to meet the required demand. Thus there is scope for increasing the production capacities of these vaccines and also produce newer vaccines in sufficient quantities to meet the huge demand for rabies vaccine in this country. In this context the production and supply of a new PVRV (Indirab) which is derived from Pitman-Moore strain and purified chromatographically is a welcome step. The other brands of PVRV produced in India and France are presently purified by zonal centrifugation. Efforts were made earlier by Lang et al. [3] to purify PVRV by chromatographic method, and a few batches were tested for potency and immunogenicity by pre-exposure vaccination of healthy volunteers. A post-exposure clinical trial was also conducted in Philippines using this CPVRV with good results
G. Sampath et al. / Vaccine 28 (2010) 4086–4090
[4]. However, commercialization of this product was not materialized and presently Indirab is the only chromatographically purified PVRV available commercially in the world. This vaccine is produced in India by Bharat Biotech International Ltd., Hyderabad using disposable cell factories with automated cell factory manipulator (ACFM) and WHO approved current Good Manufacturing Practices (cGMP). The vaccine was certified for human use by the Drugs Controller General of India (DCGI) after undergoing clinical trials in 3 phases, viz. Pre-exposure studies in healthy volunteers, Postexposure studies by standard intramuscular route and Simulated post-exposure studies by intradermal routes. A WHO approved vaccine viz. Purified Vero cell Rabies Vaccine (PVRV, Verorab) was used as reference vaccine to evaluate this new vaccine in terms of safety, tolerability and immunogenicity. This paper gives an overview of these clinical trials. The results of these trials clearly indicate that this new chromatographically purified rabies vaccine produced indigenously is safe, well tolerated and as immunogenic as the reference vaccine used in these studies. 2. Materials and methods The protocols of these clinical studies were approved by Drugs Controller General of India (DCGI) and Institutional Ethics Committee (IEC) of the respective study centers. Written informed consent was obtained from all the subjects before enrollment into the respective study. 2.1. Study design All these were blinded, randomized, active controlled, multicenter, comparative (parallel), Phase III studies. These studies were performed under out-patient department conditions in subjects enrolled as per the inclusion and exclusion criteria. The inclusion criteria were healthy subjects, not previously exposed to rabies and not taken any rabies vaccine in the past, willingness to participate in the study and their availability for the entire study period in Study 1 and Study 3. The Study 1 (Pre-exposure intramuscular) was conducted in four centers, Study 2 (Postexposure intramuscular) in two centers and Study 3 (Simulated post-exposure Intradermal) in two centers. The test and reference vaccines in intradermal (ID) studies were administered by trained staff from Institute of Preventive Medicine (IPM), Hyderabad, India and Kempegowda Institute of Medical Sciences (KIMS), Bangalore, India under the supervision of Principal Investigators. In each center, the subjects received either the test vaccine or the reference vaccine as per the randomization codes generated. A commercially available WHO pre-qualified vaccine Verorab procured from open market was used in all studies as a reference vaccine. Subjects were randomly assigned to one of the groups as per the codes generated by the computer programme Minitab 14 version. The group assignment was not known to the subject, investigator and the laboratory staff performing the serologic assay. 2.1.1. Study 1 (Pre-exposure intramuscular) A total of 239 healthy volunteers participated in the study; 180 were given Indirab vaccine (Batch No. 62AN4001, potency 5.84 IU/dose) and 59 subjects were given Verorab vaccine (Batch No. Y0659, potency >2.5 IU/dose). Both test and reference vaccines were reconstituted with 0.5 ml diluent provided along with the vaccine vial. Vaccine was administered intramuscularly on days 0, 7 and 28 and blood samples of 5 ml were collected on days 0, 28 and 35. We were able to collect blood samples at the end of 1 year from 18 subjects who had taken Indirab and 12 subjects who had taken Verorab. The study population was between 10 and 65 years of age.
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This broad age range was particularly included to assess the safety and immunogenicity in young children as well as elderly people. 2.1.2. Study 2 (Post-exposure Intramuscular) A total of 188 (aged 5–55 years) cases participated in the study, out of which 141 subjects were given Indirab vaccine (Batch No. 62AN4002, potency 6.08 IU/dose) and 47 subjects were given Verorab vaccine (Batch No. Z0502, potency >2.5 IU/dose). Majority of animal bite victims in India belong to this age group. Category III patients were administered rabies immunoglobulin (Equine rabies Immunoglobulin, Equirab, Bharat Serums and Vaccines) as per WHO recommended dose. Both test and reference vaccines were reconstituted with 0.5 ml diluent. Vaccine was administered intramuscularly on days 0, 3, 7, 14 and 28 and blood samples of 5 ml were collected on days 0, 14, 28 and 90. 2.1.3. Study 3 (Simulated post-exposure intradermal) A total of 134 healthy volunteers (aged 18–55 years) participated in the study, 68 were given Indirab vaccine (Batch No. 62AN7005, potency 5.7 IU/dose) and 66 subjects were given Verorab vaccine (Batch No. A0920, potency >2.5 IU/dose). Both test and reference vaccines were reconstituted with 0.5 ml diluent. Vaccine was administered ID as per updated Thai Red Cross Regimen (2-2-2-0-2) on days 0, 3, 7 and 28 and blood samples of 5 ml were collected on day 0, 14, 28 and 90. 2.2. Estimation of Rabies Virus Neutralizing Antibody (RVNA) titers The blood samples were centrifuged after collection, serum separated and transferred into 2 coded cryovial aliquots and stored at −20 ◦ C until transferring to testing laboratory, Department of Neurovirology, National Institute of Mental Health and Neurosciences (NIMHANS) Bangalore, India which is WHO collaborating centre for reference and research on rabies. Serum samples were tested by Rapid-Fluorescent-Focus Inhibition Test (RFFIT) as per WHO recommendation with some modifications [5]. The test was performed using 96 well tissue culture plates (Nunc, USA) and the cell line used was BHK 21 (ATCC CCL 10). The virus used was CVS 11 (obtained from Central Research Institute, Kasauli, India) and adapted to grow in BHK 21. We used 50 FFD50 of the virus as challenge dose. The highest dilution of the serum samples which reduced fluorescent foci in 50% of the cells was taken as the endpoint. The titers were expressed in IU/mL in comparison to an in-house reference serum calibrated against 2nd international reference serum having a potency of 30 IU/mL. This was procured from National Institute of Biological Standards, UK. 2.2.1. Statistical analysis Geometric mean titers (GMT) and 95% confidence intervals (CI) were computed by taking the exponent (log 10) of the mean and of the lower and upper limits of the 95% confidence intervals of log 10 transformed titers. The safety end points (percentage of subjects and 95% confidence intervals) were solicited and unsolicited adverse events. We used Mann-Whitney Test to compare the values between the vaccine groups. A p value 0.04 or less was considered to indicate statistical significance. Data analysis was done by using MINITAB software (version 14). 3. Results The demographic profile of the subjects recruited in the 3 studies is given in Table 1. The purpose of the Study I was to assess the safety and immunogenicity of this new vaccine in healthy volunteers. It can be seen from Table 2 that all subjects in both vaccine groups attained more than adequate levels of RVNA titers by day
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Table 1 Demographic profile of subjects recruited in the studies. Study number
Parameter
Test group mean
Range
Reference group mean
Range
I
Age (years) Weight (kg) Height (cm) Sex (M/F)
21.3 51.2 164
13–59 27–88 158–182
20.9 50.5 167
15–54 33–86 155–180
II
Age (years) Weight (kg) Height (cm) Sex (M/F)
25.5 55 164
III
Age (years) Weight (kg) Height (cm) Sex (M/F)
27.6 62 167
141/39
Vaccine type
N
GMT
15–43 35–70 157–181
20.2 56.3 164
19–39 45–92 155–185
26.57 59.83 161
18–39 36–81 155–188
89/52
36/11 19–47 45–88 152–188
67/1
Table 2 Rabies virus neutralizing antibody titers observed after pre-exposure immunization with Indirab and Verorab (Study I). Day
41/18
95% CI LB
p value
66/0
Table 4 Rabies virus neutralizing antibody titers (IU/mL) observed after intradermal vaccination with Indirab and Verorab in healthy volunteers (Study III). Day
Vaccine type
N
GMT
UB
95% CI
p value
LB
UB
Day 0
Indirab Verorab
177 57
0.02 0.03
0.01 0.01
0.04 0.04
0.4050
Day 14
Indirab Verorab
67 63
3.36 3.26
3.26 3.14
3.96 3.76
0.4923
Day 14
Indirab Verorab
174 56
6.1 8.3
5.19 5.59
7.17 11.03
0.1929
Day 28
Indirab Verorab
67 63
7.83 7.70
7.57 7.43
8.51 8.36
0.936
Day 35
Indirab Verorab
174 56
18.8 18.5
17.24 16.79
20.45 20.34
0.3127
Day 90
Indirab Verorab
67 63
3.62 3.57
3.43 3.43
4.12 4.06
0.7590
Day 365
Indirab Verorab
18 12
3.5 3.8
2.9 3.2
4.5 4.9
0.3412
14 which increased substantially on day 35. Though the geometric mean titer (GMT) on day 14 in the Verorab group was higher, it was not statistically significant (p > 0.05). Subjects who have the positive serum antibody titers at baseline (3 from Indirab group and 2 from Verorab group) were excluded from analysis. In some of the subjects, whose serum was tested at 1 year period, adequate antibody titers were seen in both test and reference groups. There were 5 dropouts in the study after the first dose of vaccination, 4 in the test group and 1 in the reference group. However, on telephonic enquiry, it was ascertained that the dropout was not due to any side effect of the vaccine. In the Study II, we administered either Indirab (n = 141) or Verorab (n = 47) to subjects bitten by suspect rabid animal and having either category II or III exposure. Six patients (4 in the Indirab group and 2 in the Verorab group) were lost to follow up after the 3rd dose of vaccine. Efforts to trace these patients were not successful. It can be seen that all subjects tested for RVNA had sero-
converted in both the vaccine groups by day 14. More than adequate levels of RVNA titers were observed on days 28 and 90 and there was no significant difference in the titers on all days tested (Table 3). The purpose of Study III was to show the immunogenicity of Indirab when administered by WHO approved ID schedule of vaccination in healthy volunteers. Indirab was administered to 68 subjects and Verorab to 66 subjects. There was one dropout in test group and 3 dropouts in reference group. The RVNA titers observed on different days in the two groups are depicted in Table 4. The results show that Indirab is as immunogenic as Verorab even when administered by ID route of vaccination. The incidence of side effects was negligible, did not differ significantly between the two groups and did not require medication in all three studies (Table 5). 4. Discussion Vero cells were approved for the production of human vaccines in 1987 [6], and following this inactivated polio and rabies vaccines were produced for human use [7]. A potential concern in
Table 3 Rabies virus neutralizing antibody titers (IU/mL) after post-exposure vaccination with Indirab and Verorab (Study II). Day
Vaccine type
N
GMT
95% CI LB
% sero conversion
p value
UB
Day 14
Indirab Indirab + RIG Verorab Verorab + RIG
107 30 35 10
4.94 5.70 5.10 5.05
4.55 4.96 4.38 3.1
5.33 6.44 5.82 6.17
100 100 100 100
0.62 0.31 4.5 0.31
Day 28
Indirab Indirab + RIG Verorab Verorab + RIG
107 30 35 10
10.86 11.79 11.2 11.0
9.31 10.53 10.03 9.01
11.55 13.16 12.37 12.9
100 100 100 100
0.36 0.37 0.36 0.42
Day 90
Indirab Indirab + RIG Verorab Verorab + RIG
107 30 35 10
8.16 8.46 7.83 8.5
7.52 7.5 6.63 7.4
8.80 9.42 9.03 9.6
100 100 100 100
0.52 0.63 0.53 0.62
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Table 5 Side effects observed with Indirab and Verorab in the three studies. Study
Vaccine type
Side effects observed
p value (total side effects)
Pain
Swelling
Fever
I
Indirab (n = 180) Verorab (n = 59)
5 2
2 1
2 1
0 0
0 0
0.68
II
Indirab (n = 141) Verorab (n = 47)
3 1
3 1
3 2
0 0
0 0
0.89
III
Indirab (n = 68) Verorab (n = 66)
2 2
0 2
1 2
19 21
2 2
0.78
Itching
Redness
Study I = pre-exposure intramuscular. Study II = post-exposure intramuscular. Study III = simulated post-exposure intradermal.
approving Vero cells for vaccine production was the presence of cellular DNA, which can theoretically lead to genetic transformation and tumorigenicity in the vaccinees. However, after a series of meetings coordinated by the World Health Organization (WHO) and based on substantial experimental evidence, Vero cells were approved for vaccine production provided the final product met all the WHO requirements including the DNA concentration less than 10 ng/dose [6]. Purified Vero cell Rabies Vaccines (PVRV) were first approved for human use in 1992 [8]. The first PVRV to be available commercially was Verorab produced by Pasteur Merieux in 1989. This vaccine was made using Pitman Moore strain of rabies virus and the purification step involved zonal centrifugation [7]. The vaccine was found to be safe and immunogenic in several clinical studies, found to be safe in children and pregnant women and was used in several Asian countries both by IM and ID routes of administration [9–12]. Though the WHO has accepted 10 ng/dose of cellular DNA as a safe upper limit [13], the upper limit of DNA as per European and American pharmacopoeia is 100 pg/dose. In order to further reduce the cellular DNA content to less than 100 pg/dose, purification of the vaccine was done by column chromatography by Lang et al. with the PVRV produced by Pasteur Merieux Ltd., France. The immunogenicity and safety profile of this new chromatographically purified Vero cell rabies vaccine (CPRV) was assessed in both healthy volunteers and in post-exposure clinical trials [4,5]. However, this vaccine was never produced in large scale and was never commercially sold in the international market. Recently an Indian Biotech Company, Bharat Biotech International Ltd., has indigenously produced this CPRV for commercial purpose. The vaccine is produced using disposable cell factories with automated cell factory manipulator (ACFM). The vaccine has been cleared by the Central Drugs Laboratory of the National Quality Control Authority and all batches of vaccine produced so far have residual cellular DNA content ranging from 60 to 70 pg/dose which is far less than the requirement of American and European pharmacopoeia. This chromatographically purified PVRV underwent three clinical trials. The first study involved assessing safety and immunogenicity in healthy volunteers. The performance of the vaccine Indirab in terms of safety, tolerance and immunogenicity was as good as the reference vaccine Verorab. In the second study the vaccine was administered to people bitten by suspect rabid animal as a post-exposure vaccination. In all people whose blood was tested for RVNA, 100% seroconversion occurred by day 14 and more than adequate levels of antibodies persisted till day 90 (Table 3). These people were alive and quite healthy after 1 year observation period. As we obtained very good antibody titers after the conventional IM schedule of vaccination, we wanted to know if Indirab vaccine is equally immunogenic when administered by the intradermal (ID) route. As per WHO requirement and also as per national guidelines, new vaccines are approved for ID use only after generating sufficient clinical data on their immunogenicity when administered by that route. This prompted us to undertake a simulated post-exposure study in adult healthy volunteers.
We used the newly approved 2-2-2-0-2 (Updated Thai Red Cross Regimen) for this study, which is recommended by both WHO and Government of India [14]. The results of this study clearly indicate that, Indirab is equally safe and immunogenic as the reference vaccine when administered by ID route (Table 4). The side effects observed in all the three studies were only minimal and self-limiting. The incidence of side effects did not vary between the two groups of vaccines. To conclude, in a country like India, where the burden of animal bites and rabies deaths ranks number one in the world, there is always a huge demand for safe and potent rabies vaccines both in public and private sectors. This new vaccine, Indirab has proved to be safe and immunogenic by both IM and ID routes. In addition, this is the only PVRV that is purified by chromatographic technique, reducing the cellular DNA content to less than 100 pg/dose. Considering this fact, we are of the view that this vaccine may serve as a better option along with the presently available PVRV both in Indian market as well as in international market. In an official notification, the government of India has already cleared this vaccine for use in India and for export purpose by both IM and ID routes. The vaccine is now undergoing Post Marketing Surveillance for both IM and ID routes. Preliminary results indicate that the vaccine is well tolerated, side effects are minimal and antibody assay on random blood samples collected from different vaccination centers across the country has shown more than adequate antibody response, thus building up confidence in using this vaccine in the general population.
References [1] World Health Organization. World survey of rabies No 35 for the year 1999. Geneva: WHO/CDS/CSR/EPH/2002.10; 2002. [2] Sudarshan MK, Madhusudana SN, Mahendra BJ, Rao NS, Ashwathnarayana DH, Abdul Rahman S, et al. Assessing burden of human rabies in India: results of a national multi-center epidemiological survey. Int J Infect Dis 2007;11(1):29–35. [3] Lang J, Cetre JC, Picot N, Lanta M, Briantais P, Vital S, et al. Immunogenicity and safety in adults of a new chromatographically purified vero-cell rabies vaccine (CPRV): a randomized double blind trial with purified vero cell rabies vaccine (PVRV). Biologicals 1998;26:295–308. [4] Quiambao BP, Lang J, Vital S, Montalban CG, Le Mener V, Wood SC, et al. Immunogenicity and effectiveness of post-exposure rabies prophylaxis with a new chromatographically purified vero-cell rabies vaccine (CPRV): a two stage randomized clinical trial in the Philippines. Acta Trop 2000;75:39–52. [5] Smith JS, Yager PA, Baer GM. A rapid fluorescent focus inhibition test (RFFIT) for determining rabies-neutralizing antibody. In: Meslin FX, Koprowsky H, Kaplan MM, editors. Laboratory techniques in rabies. 4th ed. Geneva: WHO; 1996. p. 181–92. [6] World Health Organization. WHO expert committee on biological standardization 37th report. WHO Tech Rep Ser 760. Geneva: WHO; 1987. [7] Montagnon BJ. Polio and rabies vaccines produced in continuous cell lines. A reality for vero cell line. Dev Biol Stand 1989;70:27–47. [8] World Health Organization. WHO expert committee on rabies: 8th report. WHO Tech Rep Ser 824. Geneva: WHO; 1992. [9] Chutivongse S, Supich C, Wilde H. Acceptability and efficacy of purified vero-cell rabies vaccine in Thai children. Asia Pac J Public Health 1988;2:179–85. [10] Sudarshan MK, Madhusudana SN, Mahendra BJ. Post-exposure prophylaxis of purified vero cell rabies vaccine (PVRV) during pregnancy: safety and immunogenicity study. J Commun Dis 1999;31(4):229–36.
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[11] Sampath G, Reddy SV, Prasada Rao ML, Rao YU, Palaniappan C. An immunogenicity study of a newly introduced vero cell rabies vaccine (Abhayrab) manufactured in India. Vaccine 2000;23:897–900. [12] Briggs DJ, Banzhoff A, Nicolay U, Sirikwin S, Dumavibhat B, Tongswas S, et al. Antibody response in patients after post-exposure rabies vaccination with small intradermal doses of purified chick embryo cell vaccine or purified verocell rabies vaccine. Bull World Health Org 2000;78:683–7.
[13] World Health Organization. Requirements for rabies vaccines for human use produced in continuous cell lines (amendment 1992) WHO expert committee on biological Standardization. In: 43rd report WHO Tech rep Ser 840. Geneva: WHO; 1992. [14] World Health Organization. Expert consultation on rabies 2004. WHO Tech Rep Ser, 931. Geneva: WHO; 2005.