Safety and Immunogenicity of purified chick embryo cell rabies vaccine (VaxiRab N) administered intradermally as post exposure prophylaxis

Abstract

The affordability to rabies vaccine for intramuscular administration in post exposure prophylaxis is a major constraint. Therefore, in countries, where there are financial constraints, World Health Organization recommends intradermal rabies vaccination that reduces the quantity and cost of vaccination. This study was done to evaluate the safety and immunogenicity of indigenously developed rabies vaccine (VaxiRab N) in comparison to a WHO recommended rabies vaccine (Rabipur) with demonstrated efficacy when administered by intradermal route using updated Thai Red Cross regimen. Eighty-six dog bite cases were randomly given either VaxiRab N (n = 43) or Rabipur (n = 43) as post exposure prophylaxis. The rabies virus neutralizing antibody concentrations on days 14, 28, 90, and 180 were tested by modified rapid fluorescent focus inhibition test. The geometric mean RVNA concentration of both the groups were compared using t- test and was found that, P value > 0.05 on all days, thus showing no significant difference between the 2 groups. The adverse drug events were also compared using Z-test and was found to be not statistically significant (Z = 1.476, P = 0.139). In conclusion, VaxiRab N was found to be safe and effective in post exposure prophylaxis by intradermal route and was similar to the WHO recommended rabies vaccine (Rabipur) of demonstrated efficacy.

Keywords: :

• rabies
• rabies vaccines
• purified chick embryo cell vaccine
• post exposure rabies prophylaxis
• intradermal vaccination

Introduction

Rabies is a fatal encephalitis that occurs in >100 countries throughout the world. It is transmitted to humans and other animals through close contact with saliva from infected animals i.e., bite, scratches, licks on broken skin, and mucous membranes. Although a number of animals serve as vectors for transmission, dogs are the main source of human infections and poses a potential threat to >3.3 billion people worldwide.¹ Timely and correct post exposure prophylaxis (PEP) for these animal bite victims is necessary to prevent rabies. Proper wound management and simultaneous administration of rabies immunoglobulins (RIG) in all category III exposures combined with prompt administration of potent cell culture or purified embryonated egg vaccines (CCEEVs) is usually effective in preventing rabies, even after high-risk exposure.²

Since their development, more than 4 decades ago, CCEEVs have proved to be safe and effective in preventing rabies. These vaccines are intended for both pre- and post-exposure prophylaxis and have been administered to millions of people worldwide.³ But, the affordability to CCEEVs for intramuscular administration during PEP is a major constraint in developing countries of Asia and Africa. Therefore, World Health Organization (WHO) recommends intradermal rabies vaccination for these countries to reduce the quantity and cost of vaccination.

In India, animal bites in humans are a major public health problem and an estimated 17.4 million animal bites occur annually which accounts to an incidence of 1.7%.^4,5 Considering the large number of animal bite cases in the country and huge demand for modern rabies vaccines, there is a need to indigenously develop anti rabies vaccine which can be administered by both intramuscular and intradermal route. In this regard, M/s. Cadila Healthcare Ltd. has developed and produced a new purified chick embryo cell rabies vaccine (PCECV-PM) using Pittman Moore strain (VaxiRab N) which can be used both by intramuscular and intradermal route. The vaccine has already been approved by Drug Controller General of India (DCGI), the National drug authority for use by both intramuscular and intradermal route and available in the market. The PCECV-PM has already been proven to be safe and immunogenic by both intramuscular and intradermal route as pre exposure prophylaxis and simulated post exposure prophylaxis by clinical trials and is recommended for rabies prophylaxis.^6,7

World Health Organization (WHO) recommends that, when new rabies vaccine is produced and approved, their immunogenicity has to be evaluated by comparing the rabies virus neutralizing antibody (RVNA) titers induced by the new vaccine being tested with those induced by a vaccine of demonstrated efficacy.¹ Therefore, this study was done to evaluate the safety and immunogenicity of an indigenously developed PCECV-PM (VaxiRab N) in comparison to a WHO recommended PCECV Flury-LEP (Rabipur) when administered by intradermal route .

Results

Eighty-six subjects bitten by suspect rabid dog were included in the study and were randomized into 2 groups viz., 43 in PCECV-PM group and 43 in PCECV-Flury LEP group. None of the study subjects had taken any pre- or post-exposure prophylaxis in the past nor had any animal bites.

The age and sex distribution of both the groups were similar. The mean age ± standard deviation of the study subjects being 34.4 ± 12.3 y and 33.8 ± 11.8 y in PCECV-PM and PCECV-Flury LEP group respectively. Each group consisted of 29 (67.5%) males and 14 (32.5%) females. The biting animal was dog in all the study subjects. None of the dogs were available for follow up due to logistical reasons. Most of the dog bites were unprovoked and were on the limbs in both the groups as depicted in Table 1, the majority of them being category III bites i.e., 81.3% and 86.0% among PCECV-PM and PCECV-Flury LEP group respectively.

Table 1. Characteristics of the study population:

Characteristics	VaxiRab N (PCECV-PM) group (n = 43)	Rabipur (PCECV-Flury LEP) Group (n = 43)
Age range (mean ± S.D)	18 – 55 y (34.4 ± 12.3)	18 – 55 y (33.8 ± 11.8)
Sex: Male Female	29 (67.5%) 14 (32.5%)	29 (67.5%) 14 (32.5%)
Biting animal: Stray Pet	Dog (100%) 25 (58.2%) 18 (41.8%)	Dog (100%) 23 (53.5%) 20 (46.5%)
Provocation: Unprovoked Provoked	27 (63.8%) 16 (36.2%)	26 (60.5%) 17 (39.5%)
Site of bite: Lower limb Upper limb Trunk Head and Neck	23 (53.5%) 12 (27.8%) 6 (14.0%) 2 (4.7%)	24 (55.9%) 12 (27.8%) 5 (11.6%) 2 (4.7%)
Immediate wound wash: Yes No	28 (65.2%) 15 (34.8%)	22 (51.5%) 21 (48.5%)
Categorization of wound: Cat II Cat III	08 (18.7%) 35 (81.3%)	06 (14.0%) 37 (86.0%)

All the bite victims were given thorough wound wash in the hospital and all category III exposures were given total quantity of required equine rabies immunoglobulin (ERIG: Equirab, manufactured by Bharat Serums and Vaccines Ltd.) locally, into and around the wound/s. Intradermal rabies vaccination was given for all the subjects.

The incidence of adverse drug events (ADEs) was calculated based on number of ADEs reported divided by total number of ID doses given and it was found to be 7.7% in PCECV-PM group and 9.8% among PCECV-Flury LEP group of subjects. All the ADEs were local in nature and there were no systemic reactions. The number of local ADEs between the 2 study group of vaccinees was compared using Z-test and was found to be not statistically significant (Z = 1.476, P = 0.139). All the ADEs were mild in both the groups and resolved without any complications. The common ADEs were erythema at the site of injection, itching at the site of injection, pain at the site of injection and induration at the site of injection in both the groups as shown in Table 2.

Table 2. Adverse drug events among the study groups

Adverse drug events	VaxiRab N (PCECV-PM) (n = 42)	Rabipur (PCECV-Flury LEP (n = 43)
Erythema	12 (3.6%)	11 (3.2%)
Itching	9 (2.6%)	16 (4.6%)
Pain	3 (0.9%)	3 (0.8%)
Induration	2 (0.6%)	4 (1.2%)
Total	26/336* (7.7%)	34/344** (9.8%)
Z value	1.476
P value	0.139

* 42 subjects X 4 doses X 2 sites **43 subjects X 4 doses X 2 sites.

The RVNA response was tested among all the subjects in both the groups on days 14, 28, 90, and 180 and all the study subjects had adequate RVNA concentration of ≥0.5 IU/mL from day 14 onwards till day 180. The geometric mean RVNA concentration (GMC) were 13.29 IU/mL, 11.77 IU/mL, 9.73IU/mL, and 8.12IU/mL in PCECV-PM group and 13.73IU/mL, 11.38 IU/mL, 9.71IU/mL, and 8.27 IU/mL in PCECV- Flury LEP group on days 14, 28, 90, and 180 respectively as shown in Table 3. The geometric mean concentration of both the groups were compared using t- test for independent sample means and was found that, P value > 0.05 on all days, thus showing no significant difference between the 2 groups. All the study subjects in both the groups were healthy and alive after 1 y of completing PEP.

Table 3. Geometric mean RVNA concentration

Day of blood sample	Vaccine	No. of subjects	GMC (IU/mL)	GSD (IU/mL)	SE (GMC)	95% CI		t value	P value
						Lower bound	Upper bound
14	VaxiRab N	42	13.29	1.28	1.04	12.29	14.36	0.813	0.418
	Rabipur	43	13.73	1.08	1.01	13.39	14.07
28	VaxiRab N	42	11.77	1.15	1.02	11.26	12.31	1.095	0.277
	Rabipur	43	11.38	1.15	1.02	10.91	11.88
90	VaxiRab N	42	9.73	1.16	1.03	9.25	10.23	0.066	0.948
	Rabipur	43	9.71	1.15	1.02	9.29	10.14
180	VaxiRab N	42	8.12	1.20	1.03	7.65	8.61	0.514	0.608
	Rabipur	43	8.27	1.16	1.02	7.89	8.68

GMC, Geometric Mean Concentration; GSD, Geometric Standard Deviation; SE, Standard Error; CI, Confidence Interval.

Discussion

India is highly endemic for rabies and has the largest number of animal bites in the world. Cell culture vaccines are the main stay of PEP for animal bite victims. Intradermal administration of cell culture rabies vaccines offers an equally safe and immunogenic alternative to intramuscular rabies vaccination and requires less volume of vaccine and is recommended by WHO in resource constraint countries. Thus, intradermal rabies vaccination reduces the direct cost of vaccine by 60–80% compared with standard intramuscular vaccination.¹ Intradermal rabies vaccination has been implemented in several Government sector hospitals in India, which largely benefits the lower socioeconomic group of people. Increased urbanization and ineffective dog population control measures have resulted in increased incidence of dog bites. Therefore, the demand for a safe and potent cell culture vaccine that can be administered by cost effective intradermal route is ever increasing. In this context, production and availability of this new vaccine may play a significant role for preventing rabies in India.

The new PCECV-PM has been produced after successful adaptation of Pittman-Moore strain of rabies virus. The genetic stability of the strain was maintained during the adaptation procedure.⁸ The Pittman-Moore strain is well documented for safety and immunogenicity and has been genetically characterized and found to have 100% homology with original Pasteur virus strain. The first ever cell culture vaccine for human use, the human diploid cell, vaccine (HDCV) was produced using this strain.⁹ The first PCECV, Rabipur was developed using Flury-LEP strain which was already adapted to grow in chick embryo by Barth.¹⁰ Rabipur has been found to be efficacious in preventing rabies in exposed individuals both by conventional IM and the ID routes of vaccination and has been recommended by WHO.¹¹ Recent studies based on genetic characterization of different vaccine strains have shown close homology between Flury LEP and Pittman-Moore strain.¹²

The present study showed that, the PCECV-PM was safe and immunogenic for post exposure prophylaxis and was similar to PCECV-Flury LEP of demonstrated efficacy. The number of local adverse drug events between the 2 study group of vaccinees was compared using Z-test and was found to be not statistically significant (Z = 1.476, P = 0.139) and all the ADEs in both the study groups were mild which resolved without any complication. Similarly, GMCs of both the groups were compared using t-test for independent sample means. It was found that P value was >0.05 on all days 14, 28, 90, and 180, thus showing no significant difference between the GMCs of PCECV-PM and PCECV-Flury LEP group when administered intradermally using Updated TRC regimen.

To conclude, the present study showed that, the study vaccine PCECV-PM was found to be safe and effective in post exposure prophylaxis by intradermal route using Updated TRC regimen (2-2-2-0-2) and was similar to the WHO recommended PCECV-Flury LEP of demonstrated efficacy. Therefore, this PCECV-PM along with already approved vaccines will have an important role in effective post exposure prophylaxis by intradermal route not only in India but also in other Asian and African countries which still accounts for large number of human rabies deaths.

Materials and Methods

The study was conducted at anti rabies clinic, Kempegowda Institute of Medical Sciences (KIMS) Hospital and Research Centre, Bangalore, India. The study was initiated, following clearance from the institutional ethics committee and registered in clinical trial registry, India (CTRI) with the registration number CTRI/2012/06/002720. The study was conducted in accordance with ICH- GCP guidelines.

Enrolment of subjects

This study was a randomized (1:1), active controlled, parallel assigned, open label, phase IV clinical trial. The random numbers were generated from computer software, SPSS 11.0 Version. The subject, who fulfilled the inclusion and exclusion criteria, was asked to read and understand the study information sheet provided in their own language and if subject agreed to participate in the study, written informed consent was taken and was enrolled in the study. Thorough and detailed enquiry was done among all the study subjects to rule out taking any rabies vaccine either as pre exposure prophylaxis (PrEP) or PEP and history of any animal bite in the past. Similarly, any concomitant medical conditions/treatments were ruled out.

Eighty-six dog bite cases with category II and III bites were enrolled and randomly given either PCECV-PM or PCECV-Flury LEP (43 subjects in each group) as post exposure prophylaxis along with wound wash and equine rabies immunoglobulin (ERIG) in all category III bites. Both the vaccines were purchased from the market available batch. The vaccine was administered intradermally using Updated Thai Red Cross regimen i.e., 2 doses of 0.1 mL vaccine given intradermally over both the deltoid muscle on days 0, 3, 7, and 28.

Vaccines

PCECV-PM (VaxiRab N), produced and marketed by Zydus Health Care Ltd. (market Batch No. CK101, mfg. date Nov 2011 and exp. date Oct 2013; potency 7.04 IU per IM dose) and PCECV-Flury LEP (Rabipur), manufactured by Novartis Vaccines (market Batch No. 2307, mfg. date 10/2011, exp. date 09/15; potency ≥ 2.5 IU per IM dose)

Assessment of safety

Following vaccination, all the subjects were observed for half an hour for possible immediate local/ systemic adverse drug reactions (ADRs). At the end of half an hour, reactogenicity was recorded, only if the subject spontaneously reported a problem to a question on general wellbeing i.e., unaided recall. The subjects were given a follow up card to indicate if they had any late adverse events and was recorded in the subsequent visits i.e., on day 3, 7, 14, 28, and 90.

Assessment of immunogenicity

Blood samples were drawn from all the subjects on day 0 to confirm the absence of RVNA. Subsequently, blood samples were collected on days 14, 28, 90, and 180 for estimation of rabies virus neutralizing antibody (RVNA). An amount of 5 mL of venous blood was drawn from each patient under aseptic precautions and the sera were separated and tested for RVNA by modified rapid fluorescent focus inhibition test (RFFIT) at the Department of Neurovirology, National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore, India which is a WHO collaborating center for reference and research on rabies.

Estimation of rabies virus neutralizing antibody (RVNA)

Modified RFFIT was done as per WHO recommended procedure. The cell line used was BHK 21 (ATCC CCL 10) and 96 well tissue culture plates (Sigma) and BHK21 adapted CVS 11 strain of rabies virus. The reference serum used was an in house serum calibrated against 2nd international reference standard having a titer of 30 IU/mL (obtained from National Institute of Biological Standards). Briefly, doubling dilutions of serum samples and reference serum (after heat inactivation at 56 °C for 30 min in a water bath) in duplicate were made in 96 well plates using IMDM (Sigma Cat No.17633). To each 100 uL of serum dilution 100 uL of CVS (100 FFD ₅₀) was added and the plate to was incubated at 37° C for 1 h. A confluent monolayer of BHK 21 cells were trypsinized and re- suspended in 10 mL of IMDM with 10% FCS (Sigma, cat No. F2442). Cell control and virus controls were also included. To each well of the 96 well plate 100 uL of cell suspension was added and the plate was incubated at 37° C in a CO2 incubator (Sanyo). After 24 h the cells were fixed in cold acetone for 30 min and stained by direct FAT using commercially available rabies N conjugate (Light Diagnostics, Cat No. F199). The plates were then observed under an inverted fluorescence microscope (Nikon Eclipse). The highest dilution of serum showing 50% inhibition of fluorescence foci was taken as end point dilution. The titer was converted to IU/mL in comparison with reference serum.

All information pertaining to the study subjects were recorded in a separate case record form which contained detailed information on socio demographic profile, relevant past and present medical history, anthropometry, physical and systemic examination findings, details of PEP provided, dates of vaccination and blood withdrawal, adverse drug reactions (ADR), treatment of ADRs, and RFFIT results.

All the study subjects were followed up for 1 y to know their survival status.

All the biting dogs could not be traced or caught for laboratory examination due to logistical difficulties.

Statistical analysis

The data was analyzed statistically by computing percentages, geometric mean concentration (GMC), range, geometric standard deviation (GSD), standard error (SE), 95% confidence interval (CI) for GMC, t test and P value. The difference in proportion of ADRs between the vaccine groups was assessed by Z test.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

10.4161/hv.29403