Research Paper
Research paper
Human Vaccines 6:9, 721-724; September 2010; © 2010 Landes Bioscience
Immunogenicity, safety and tolerance of a purified duck embryo vaccine (PDEV, VAXIRAB) for rabies post-exposure prophylaxis Results of a multicentric study in India
Bangalore Jayakrishna Mahendra,1 Shampur Narayan Madhusudana,2,* Gadey Sampath,3 Soma Subhra Datta,4 Doddabele Hanumanthaiah Ashwathnarayana,5 Gonibeedu Manjunath Venkatesh,5 Mysore Kalappa Sudarshan,5 Gangaboraiah Bilugumba5 and Manjula Shamanna6 Department of Community Medicine; Mandya Institute of Medical Sciences (MIMS); Mandya, India; 2Deptartment of Neurovirology; National Institute of Mental Health and Neurosciences (NIMHANS); Bangalore, INDIA; 3Institute of Preventive Medicine; Hyderabad, India; 4Pasteur Institute; Kolkata, India; 5Deptartment of Community Medicine; Kempegowda Institute of Medical Sciences (KIMS); Bangalore, India; 6Zydus Health Care Ltd; Ahmadabad, India
1
Key words: rabies, vaccines, post-exposure therapy, purified duck embryo vaccine Abbreviations: APCRI, Association for Prevention and Control of Rabies in India; PDEV, purified duck embryo vaccine; PCEC, purified chick embryo cell vaccine; PVRV, purified vero cell rabies vaccine; WHO, World Health Organization; RFFIT, rapid fluorescent focus inhibition test; RVNA, rabies virus neutralizing antibody; RIG, rabies immunoglobulin; ERIG, equine rabies imunoglobulin; NIMHANS, National Institute of Mental Health and Neurosciences; KIMS, Kempegowda Institute of Medical Sciences; IPM, Institute of Preventive Medicine
Rabies continues to be a major public health problem in India. Nearly 17 million people are getting exposed to this disease every year. Therefore, the need for effective post-exposure prophylaxis with safe and potent modern rabies vaccines continues to exist. Purified duck embryo vaccine (PDEV) was introduced in this country to meet the everincreasing need for modern rabies vaccines. In this study we have assessed the safety, imunogenicity and tolerance of an indigenously manufactured PDEV in people exposed to dog and other animal bites. One hundred and fifty people (5–59 years) with WHO category II or III animal bites were vaccinated with PDEV using the Essen Intramuscular regimen and rabies immunoglobluin (RIG) was administered to category III exposures. Their blood samples were analyzed for rabies virus-neutralizing antibody response (RVNA) by Rapid Fluorescent Focus Inhibition Test (RFFIT) on day 0, 14, 30, 90, 180 and 365. Adverse effects to vaccines were monitored during the course of vaccination. There was 100% sero-conversion from day 14 onwards with adequate RVNA titers (>=0.5 IU/mL) up to day 365. The incidence of side effects was minimal and self-limiting. Hence it can be concluded that indigenously manufactured PDEV (Vaxirab) is a safe and immunogenic vaccine and can safely be used for post-exposure prophylaxis.
Introduction Rabies is a fatal but preventable disease. It is still a significant health problem in India and other developing countries despite the availability of safe and potent vaccines. As per a WHO estimate, worldwide about 50,000 people die of rabies each year mostly in Asia, Africa and South America.1 A recent WHOsponsored multicentric survey conducted by the Association for Prevention and Control of Rabies in India (APCRI) revealed that about 20,000 humans die of rabies every year in India, and an astounding 17 million people are bitten by dogs and other animals.2 Thus there is a great demand for rabies vaccines, and
following the complete stopping of nerve tissue derived Semple vaccine in 2004, even government-run antirabies clinics are administering modern rabies vaccines. Two types of modern vaccines are approved by WHO for post-exposure prophylaxis Viz: cell culture vaccines (CCV) and purified duck embryo vaccine (PDEV).3 Though PDEV is in strict sense not a CCV (infected duck embryos are homogenized and viruses are purified by zonal centrifugation and inactivated with BPL), it is considered on par with any CCV by the WHO. Two WHO-approved CCVs Viz: purified chick embryo cell vaccine (PCEC, Rabipur) and purified Vero cell rabies vaccine (PDEV, Verorab) have been available in India for the past two decades. The PDEV was developed by Gluck et al.4 in 1985 and was originally manufactured by Berna
*Correspondence to: Shampur Narayan Madhusudana; Email:
[email protected] Submitted: 02/17/10; Revised: 04/01/10; Accepted: 04/30/10 Previously published online: www.landesbioscience.com/journals/vaccines/article/12216 DOI: 10.4161/hv.6.9.12216 www.landesbioscience.com
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Table 1. Demographic profile of subjects recruited Parameter
Number
Percentage
Subjects Total:
Table 2. Rabies virus neutralizing sntibody (RVNAb) titers (IU/ml) following administration of VaxiRab (n = 150) Day
150
Adult males
72
47.9
Adult Females
40
26.6
Children =0.5 IU/mL) from day 14 through day 365 (Table 2). We compared the GMTs of subjects who had concomitant RIG administration (n = 85) with those taking vaccine alone (n = 65).
Figure 1. The RVNA titers (GMT) observed in subjects after administration of Vaxirab alone (n = 85) or in combination with ERIG (n = 65). There was no statistical difference between the groups. 722
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There was no statistical difference between titers in subjects who were administered RIGs and those who were not. Both groups of subjects had protective titers from day 14 through to day 365 (Fig. 1). Subjects were observed for 30 minutes after vaccine administration for any hypersensitivity reaction. All observed or volunteered adverse reactions to the study drugs, regardless of treatment group or suspected causal relationship, were recorded. The reactions were graded on a 4 point scale, 0—None (absence of symptoms), 1—Mild (presence of mild symptoms), 2—Moderate (symptoms which have an impact on normal activities) and 3—Severe (symptoms which prevent daily activities). A total of 700 doses of Vaxirab was administered to 150 patients of whom 47 (6.7%) reported adverse reactions. The number of adverse events was 87 (12.4%) of these 67 were local and 20 systemic reactions. All the reactions were mild to moderate and did not require additional medication (Table 3). Discussion Dog and other animal bites leading to risk of developing rabies will continue to occur in most developing countries and there will be ever-increasing demand for safe and potent rabies vaccines. In India, which reports the largest number of human rabies deaths as well as dog bites, the nerve tissue-derived Semple vaccine was the mainstay of post-exposure treatment in all government run anti rabies centers until 2005. Since then, modern CCVs produced both indigenously and also imported have replaced the Semple vaccine. Keeping in view the huge demand for modern vaccines, indigenous production and introduction of PDEV into the Indian market is a welcome step, which may go a long way in alleviating vaccine shortage not only in government centers but also in private practice. Two of the WHO-approved vaccines viz: Rabipur (which is produced in India) and Verorab have excellent safety and immunogenecity records and have been administered to millions of exposed people both in developed and developing countries. The other WHO-approved vaccine is the PDEV, which had a limited production and supply till the year 2000. However, a large scale production of this vaccine began in the year 2001 in India after the transfer of technology from the parent company Berna Biotech, Switzerland. As reported earlier, it was comparable in all aspects with the original counterpart, Lyssavac N.8 One of the main objectives of the present study was to show that indigenously produced PDEV is immunogeneic and safe in the population exposed to the risk of rabies. The study was multicentric and open label and included people from different parts of urban and rural India who belonged to different age groups. All the subjects were vaccinated using the Essen regimen that was first approved by WHO in 1975 when HDCV was introduced and six doses of vaccines were administered IM on days 0, 3, 7, 14, 28 and 90. This regimen was named “Essen” as a tribute to the contribution of Ernest Kuwert of the Essen Institute of Medical Virology and Immunology, West Germany.9 The booster dose on day 90 was omitted in 1992 after a WHO expert committee meeting.10
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Table 3. Side effects observed after administration of vaxirab Local
No. of events
Percentage
Pain
25
13%
Erythema
07
3.3%
Swelling or induration
08
4.6%
Fever
10
5.3%
Giddiness
01
0.6%
Malaise
01
0.6%
Systemic
Myalgia
05
3.3%
Headache
02
1.3%
Arthralgia
01
0.6%
Results of immunogenecity as revealed by RFFIT showed that by day 14, all the subjects developed protective titers. The titers were maintained throughout the study period of one year. We compared RVNA titers induced by this vaccine with other vaccines used in India. For this purpose, we used the data from a meta-analysis of the immunogenecity of selected vaccines that was reported recently by Sudrashan et al.11 We observed that the RVNA titers were comparable to those obtained with other commonly used vaccines such as PCEC, PVRV and HDCV. As per the WHO recommendation, all category III cases require administration of RIG. Though in India RIG usage is generally very low (about 3% of category III exposures), all our subjects in category III received RIG as per the study protocol. There is a theoretical concern that concomitant administration of RIG may suppress active immune response to the vaccine. In our study we did observe some differences in the titers among the two groups on all time points, but the difference was not statistically significant (p > 0.15). Few adverse reactions were observed with all three vaccines and local pain at the injection site was the most commonly observed ADR. However, these minimal ADRs did not result in noncompliance of the patients, there were no dropouts in the study and the subjects were available for observation throughout the study period. Adverse events have also been reported with the use of other vaccines like PCEC (Rabipur)12 and PVRV (Verorab and Abhayrab).13,14 When we compared the incidence of these events, there was no apparent difference between the Vaxirab and other vaccines. All the subjects were alive and healthy at the end of the observation period. As the rabid status of the biting animal was not confirmed in our study, we cannot really comment on the true efficacy of the vaccine, but as true efficacy studies in human population are difficult to conduct, immunogeneicty of the vaccine is used as a criteria to evaluate the performance of the vaccine. To conclude, this study has clearly shown that PDEV (Vaxirab) is immunogenic, safe and well-tolerated and can be used as an alternative to cell culture vaccines. It is hoped that the production and utility of this vaccine will overcome the shortage of modern WHO-approved vaccines in Asian countries and will continue to benefit thousands of people exposed to rabies by dog and other animal bites.
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Materials and Methods Subjects. People exposed to rabies through dog and other animal bites were recruited for the study at three different centers: The Kempegowda Institute of Medical Sciences (KIMS), Bangalore; The Institute of Preventive Medicine (IPM), Hyderabad both located in southern part of India; Pasteur Institute, Kolkata located in the eastern part of the country. A total of 150 subjects were recruited. The inclusion criteria were willingness to participate in the study, to be in the age group of 5–59 years, willingness to give blood samples on stipulated dates and availability for follow up 6 months post-vaccination. The exclusion criteria were history of previous rabies vaccination or any animal bite, pregnancy, concomitant illness or on any medication, simultaneous participation in any other study and subjects known to be HIV positive as these subjects may be immunocompromised and we neither had facility nor funds to assess their immune status. The study was started simultaneously at all centers and conducted for a period of one year from 2006 to 2007. The study protocol was approved by a human ethics committee constituted by the respective study centers. Vaccine and regimen. The vaccine used was Vaxirab (Batch No. BD 101 with a potency of 8.9 per dose). It was administered intramuscularly in to the deltoid muscle on days 0, 3, 7, 14 and 28 as per the WHO-recommended Essen regimen.3 Side effects both solicited and unsolicited were recorded on each visit of the patient to clinic. Rabies immunoglobulin (ERIG) was administered to all category III exposures as per the WHO-recommended procedure. The ERIG used in the study was obtained from open market (Equirab with a potency of 300 IU/mL). Estimation of rabies virus-neutralizing antibody titers (RVNA). About 5 mL of venous blood was collected from each References 1.
World Health Organization. World survey of rabies No 35 for the year 1999. WHO/CDS/CSR/EPH/2002.10. Geneva. 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; 11:29-35. 3. World Health Organization. Expert consultation on rabies. WHO Tech Rep Ser 931 Geneva WHO, 2005. 4. Gluck R, Matthieu JM, Wegman A, Mean F. Absence of myelin basic protein in an improved purified duck embryo vaccine. Neurochem Pathol 1986; 4:69-75. 5. Wegmann A, Gluck R, Keller H, Matthieu JM. A highly efficient, highly purified duck embryo rabies vaccine. Schweiz Med Wochenchr 1985; 115:1779-81. 6. Kamoltham T, Singhsa J, Promsaranee U, Sonthon P, Mathean P, Thinyounyong W. Elimination of human rabies in a canine endemic province in Thailand: Five year Programme. Bull World Health Organ 2003; 81:375-7.
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subject on day 0 (before vaccination) and on days 14, 28, 90, 180 and 365 following vaccination. The serum was separated and stored at -20°C before sending in dry ice to the department of Neurovirology, National Institute of Mental health and Neurosciences (NIMHANS), Bangalore which is a WHO collaborating center for reference and research on rabies. The serum samples were tested for RVNA by rapid fluorescent focus inhibition test (RFFIT) at the above center. The methodology used was as recommended by WHO with some modifications.15 The cell line used was BHK 21 (ATCC CCL10) and the challenge virus used was CVS 13 adapted to grow in BHK 21 cells. The test was performed using 96-well tissue culture plates (Nunc, USA). The dose of the CVS (obtained from Central Research Institute, Kasauli, India as a mouse brain homogenate and adapted to grow in BHK 21 cells) used was 50 FFD50. The plates were read using a Nikon inverted fluorescence microscope (Eclipse TS 100). The 50% neutralizing titers of the serum samples were converted to international units in comparison to titers observed with a in house reference rabies immunoglobulin (RIG) calibrated against second international reference RIG prepared at National Institute of Biologicals, UK and having a potency of 30 IU per mL. This product was obtained through WHO. Adverse reactions. All subjects were closely interrogated and physically examined on each of their visit for any adverse reaction to the vaccines. Reactions if any were recorded. Statistical analysis. The Geometric mean titers (GMT) of the antibody titers were calculated along with Geometric standard deviation (GSD), standard error (SE) and 95% confidence interval. The difference in titers observed after vaccination in subjects with or without administration of RIG was statistically analyzed using a Student’s t-test.
7.
Wild H, Tipkong P, Khawplod P. Economic issues in post exposure rabies Treatment J Travel Med 1999; 6:238-42. 8. Mahendra BJ, Madhusudana SN, Ashwathnarayana DH, Sampath G, Datta S, Sudarshan MK, et al. A comparative study on the immunogenecity, safety and tolerance of purified duck embryo vaccine (PDEV) manufactured in India (Vaxirab) and Switzerland (Lyssavac N): A randomized simulated post exposure study in healthy volunteers. Vaccine 2007; 25:8405-9. 9. World Health Organization. Expert committee on rabies 8th report. Tech Rep Ser 824 Geneva, WHO 1992. 10. Vodopija I, Clark HF. Human Vaccination against rabies. In Baer GM, (ed). The Natural History of Rabies. 2nd Ed. Boca Raton, CRC Press 1991; 571-95. 11. Sudarshan MK, Mahendra BJ, Madhusudana SN, Ashwathnarayana DH, Sanjay TV, Gangaboraiah B, et al. Hum Vaccin 2005; 1:187-90.
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12. Sehgal S, Bhattcharya D, Bharadwaj M. Ten year longitudinal study of the efficacy and safety of purified chick embryo vaccine for pre and post exposure of rabies in Indian population. J Commun Dis 1995; 27:1536-43. 13. Sehgal S, Bhattacharya D, Bharadwaj M. Clinical evaluation of purified vero cell rabies vaccine (PVRV) in patients bitten by rabid animals in India. J Commun Dis 1994; 26:139-46. 14. Sampath G, Reddy SV, Rao MLP, Rao YU, Palaniappan C. An immunogenicity study of a newly introduced purified vero cell rabies vaccine (Abhayrab) manufactured in in India. Vaccine 2005; 23:897-900. 15. 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, (eds). Laboratory Techniques in Rabies, 4th ed. Geneva, WHO 1996; 181-2.
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