Rabies Vaccine Potency Research Articles

Potency, immunogenicity, and efficacy of rabies vaccine: In vitro and in vivo approach.

Rabies, a potentially lethal virus, affects more than 150 countries. Although the rabies vaccine and immunoglobulin have been available since 1908, Bangladesh is new to vaccine manufacturing. We checked the quality of the local manufacturing rabies vaccine for substandard. The potency and immunogenicity of 20 vaccines were analyzed by three in vivo and in vitro methods from March 2020 to May 2023. Single radial immunodiffusion, fluorescent antibody virus neutralization, and national institutes of health tests were carried out to evaluate the vaccine's efficacy to provide sufficient protection against the rabies virus. The potency of the rabies vaccine was determined by the in vitro SRID method by measuring glycoprotein content. An average of 16 articles from each batch was calculated. The minimum and maximum average mean values of the 20 batches were 5.058 and 5.346, respectively. The variance was calculated at 0.00566. We found a coefficient of variation (CV) between 9.36% and 14.80%. The 100% sample was satisfactory, as these samples had a potency of over 2.5 IU/mL. To observe immunogenicity, we applied the FAVN method for determining antibody titers. An average of 16 articles from every batch were counted to quantify antibody titers. The mean quantity of antibody titers ranged from 2.389 to 3.3875. The CV was slightly lower because of the dispersion of the data. At last, we performed an in vivo method, the NIH test method, to determine potency based on mortality rate. We found a mean value of 4.777 IU/SHD with a standard deviation of 1.13 IU/SHD. All 20 batches were found 100% satisfactory in the NIH test. The study implies that the rabies human vaccines manufactured in Bangladesh are potent enough to provide sufficient immunogenicity. Our research is warranted testimony for healthcare providers who work to extirpate rabies.

A New Antibody Binding Test for Potency Assessment of the Human Rabies Vaccine.

This study established a new protocol of the antibody binding test to evaluate the potency of the rabies vaccine containing the final bulk and the product. The principle of this experiment is to combine rabies vaccine with quantitative anti-rabies virus neutralizing antibody. After combination, the remaining rabies vaccine is combined with the quantitative fluorescent labeled rabies virus. After this, observe the remaining fluorescent labeled rabies virus, calculate the fluorescence area with fluorescence observation equipment, then calculate the potency of rabies vaccine by Reed and Muench method. The test results of many batches of rabies vaccine final bulk and finished products showed that the potency detected by this method was consistent with that of National Institute of Health method.

An ELISA-based antigenicity test of rabies recombinant glycoprotein cannot predict its protective potency in vivo

The potency of human and veterinary rabies vaccines is measured based on the National Institute of Health (NIH) potency test that is laborious, time-consuming, variable, and requires sacrifice of large numbers of mice. ELISA-based methods quantifying rabies glycoprotein (rGP) are being developed as potential alternatives to the NIH potency test for release of rabies vaccines. The aim of the current study was focused on the evaluation of in vitro- and in vivo-based assays in order to assess their concurrence for adequate and reliable assessment of immunogenicity and protective potency of a plant-derived recombinant rGP. The recombinant rGP of strain ERA.KK was engineered, expressed and purified from Nicotiana benthamiana plants. The recombinant rGP excluded the transmembrane and intracytoplasmic domains. It was purified by chromatography (≥90%) from the plant biomass, characterized, and mainly presented as high molecular weight forms, most likely soluble aggregates, of the rGP ectodomain. It was well-recognized and quantified by an ELISA, which utilizes two mouse monoclonal antibodies, D1-25 and 1112-1, and which should only recognize the native trimeric form of the rGP. However, in mice, the recombinant rGP did not induce the production of anti-rabies virus neutralizing antibodies and did not confer protection after intracerebral viral challenge. Similar immunogenicity was observed in guinea pigs and rabbits. Our results demonstrate that use of the ELISA method described here is not predictive of performance in vivo. These data highlight the critical need to develop in vitro potency assays that reliably define the antigen content that can induce a protective response.

Evaluation and Correlation of Rabies Vaccine Potency Using the National Institute of Health, Rapid Focus Fluorescent Inhibition, and Passive Hemagglutination Tests.

Rabies vaccine preparations are quantitatively assayed for potency using the in-vivo challenge National Institute of Health (NIH), the main test that consumes a high number of animals, takes a long time, and has wide variability. The Rapid focus fluorescent inhibition (RFFIT) and the passive hemagglutination (PHA) tests, the two serologically based tests, were also used for such purpose. In this study, we aimed to evaluate and correlate the potency of the NIH, RFFIT, and PHA tests according to the World Health Organization (WHO) validity criteria, aiming to validate the use of RFFIT or PHA test as a substitute to the NIH test for determining the potency of commercially available Rabies vaccine preparations. The results showed that, the three tests can be successfully used; however, a higher correlation between RFFIT and NIH than PHA and NIH was recorded (Pearson correlation = 1). The potency of rabies vaccine preparations using NIH, RFFIT, and PHA were 3.73, 3.51, and 4.50, respectively. NIH is the main test for the determination of vaccine potency carried out by conducting 25 experiments and consuming about 5,000 mice compared to 1,200 mice used with RFFIT and 1,000 mice used with PHA test. Taken together, we concluded that (i) in some tested preparations, both RFFIT and PHA tests gave comparable results, and they can be used interchangeably; (ii) RFFIT could successfully replace NIH test, but not PHA; (iii) RFFIT and PHA tests are faster, more accurate, more economic, and more sensitive than NIH; nevertheless, PHA needs further investigations; and (iv) both RFFIT and NIH tests complement and reinforce each other as they provide a comprehensive picture of the product potency.

Safety and immunogenicity of SURE RAB™, an inactivated cell culture derived rabies vaccine: A comparative, phase III clinical trial

IntroductionRabies is a viral zoonotic disease widespread across the world. In India, various cell culture vaccines are available for pre and post exposure prophylaxis (PEP) but are not sufficient to meet the rising demand. The present study evaluated the safety and immunogenicity of Rabies vaccine Human I.P. (Brand name: SURE RAB™) in PEP and demonstrated its non-inferiority to already approved rabies vaccine (Brand name: VERORAB). Material and methodsIt was a phase-III randomized, open label, comparative, single centre clinical trial in post exposure subjects. Safety and immunogenicity were evaluated at Day 0, 14 and 45 ± 7 post vaccination. Day 14 serum samples were analyzed by Enzyme Linked Immunesorbent assay (IgG ELISA, Bio-Rad) and Day 0, 14 and 45 ± 7 serum were tested by Rapid Fluorescent Focus Inhibition Test (RFFIT). Paired t-test was applied to compare the results of Rabies virus neutralizing antibody (RVNA). The severity of adverse reactions was measured on a scale of excellent, good, fair and bad; p-value (p < 0.05) was considered as statistically significant. ResultsAll the subjects achieved a protective titer value between 0.5 and 9.0 IU/ml by Day 14 tested by ELISA and significant rise in the antibody titer in all the groups when tested after 45 days. Statistically significant p-value (p < 0.001) observed with RFFIT test indicated biological potency of rabies vaccine. Adverse events and safety was comparable statistically between three groups (p ​= ​0.886) and Group I ​+ ​II combined versus Group III (p = 0.495). ConclusionThe study results conclusively demonstrate that SURE RAB™ is comparable to VERORAB in terms of safety and immunogenicity and can be used for PEP in rabies.

Development of competitive inhibition ELISA as an effective potency test to analyze human rabies vaccines and assessment of the antigenic epitope of rabies glycoprotein

The potency of all modern tissue culture human rabies vaccines is measured based on the National Institute of Health (NIH) potency test that is laborious, time-consuming, involves large test variations and requires sacrifice of large number of animals. To circumvent these limitations, several researchers and WHO expert working groups have discussed development of alternative in vitro methods to replace the NIH potency test. Although several immunochemical methods have been proposed to quantify rabies glycoprotein (G-protein) using multiple murine monoclonal antibodies, we report an In vitro competitive inhibition ELISA (CIA) method based on the use of a neutralizing rabies glycoprotein site III directed novel therapeutic human rabies monoclonal antibody (RAB1) that shows equivalence to the mice NIH potency test in recognition of neutralization site of the glycoprotein. In vitro potency testing of WHO 7th International Standard for rabies vaccine (IS) by CIA using RAB1 and In-house reference standard (IHRS) as a standard to assess its suitability for the assessment of validation parameters showed accurate and precise values with <15% coefficient variance. The method was validated using 5PL standard curve with linearity r2 > 0.98 and LLOQ of 0.125 IU/mL indicating sensitivity of the method. The method was found to be precise, robust and accurate to quantitate intact rabies glycoprotein in final vaccine and showed a strong correlation (Pearson's r = 0.81) with the NIH potency values of licensed Vero cell rabies vaccine. The CIA test using RAB1 was able to accurately quantitate degradation of rabies vaccine and assess loss in antigenicity of lyophilized and reconstituted liquid rabies vaccine under thermal stress conditions. The method was able to differentiate between potent and reduced potency vaccine samples. The new in vitro competitive inhibition ELISA method using RAB1 thus can be a valid alternative to the NIH test.

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency.

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future. The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Possibility of Immediate Introduction of a Single-Dose Antibody Induction Test as a Refinement of the NIH Test for Inactivated Rabies Vaccine Potency Determination.

Antibody induction test (AIT) is a promising candidate as a refinement of the troublesome National institutes of Health (NIH) test in the sense of animal welfare 3R approach for determination of potency of inactivated rabies vaccines for veterinary and human use. In this study, we initially try to develop AIT as a suitable alternative to NIH test, to achieve a reduction of test duration and diminish animal suffering by omitting intracerebral CVS infection and measuring humoral immunity upon vaccination. Designs of both multi-dose and single-dose AIT were examined. Biological reference preparation, batch 5 with assigned titer of 10 IU/vial, was taken as both standard and test vaccine. Six consecutive AITs were performed and eight pools of sera in each AIT were tested in triplicate by rapid fluorescent focus inhibition test. We estimated the upper detection limit and calculated test variability for individual dilutions. For multi-dose AIT, we estimated the dose-response function and performed calculations of final test results and statistical validity parameters for both linear and sigmoidal model using CombiStats program. Sigmoidal 4-parameter dose-response model was found optimal. Presented design of multi-dose AIT showed a satisfactory detection limit for testing of inactivated rabies vaccines for both veterinary and human use. However, due to nonconformity of obtained results with statistical validity criteria, we concluded that the presented model of multi-dose AIT was unsuitable for introduction in routine practice. However, we concluded that there was a realistic option for introduction of two versions of single-dose AIT. The first version would be with two standard vaccine controls and could be introduced immediately, while the second version would include testing of the sample only and rely on comparison of the induced rabies antibody level with absolute cut-off limits set in advance.

A novel ELISA for quantification of glycoprotein in human rabies vaccines using a clinically proven virus neutralizing human monoclonal antibody

ABSTRACT Global efforts on the replacement of the in vivo rabies vaccine potency test (NIH method) with in vitro methods for quantification of immunodominant glycoprotein (GP) in rabies vaccine have made significant progress. We report here, a sandwich ELISA method based on the use of a neutralizing rabies GP site III directed human monoclonal antibody (RAB-1) and a polyclonal GP specific antibody recognizing the intact form of viral GP. The method was shown to be robust, specific, linear, precise and accurate in the quantification of intact GP in vaccine samples. The assay was able to differentiate between potent and sub-potent vaccine samples. The assay was shown to be linear over the range of 0.07–2.25 IU/mL with LOD and LLOQ values of 0.035 and 0.070 IU/mL, respectively. The assay was able to quantify the GP content of rabies vaccines derived from rabies vaccine strains, e.g., Pittman-Moore, Pasteur and Flury LEP with acceptable precision (CV < 20%) and also showed good agreement with NIH potency estimates. The binding kinetics of RAB-1 with intact and modified vaccine samples were also characterized using biolayer interferometry (BLI). The developed method may be used as an alternative to the NIH method in quality control testing of human rabies vaccines.

Potency test to discriminate between differentially over-inactivated rabies vaccines: Agreement between the NIH assay and a G-protein based ELISA

The NIH assay is used to assess the potency of rabies vaccine and is currently a key measure required for vaccine release. As this test involves immunization of mice and subsequent viral challenge, efforts are being made to develop alternative analytical methods that do not rely on animal testing. Sanofi Pasteur has reported the development of a G-protein specific ELISA assay that has shown agreement with the NIH test. In this study we have generated several non-conform vaccine lots by an excessive inactivation with β-propiolactone (BPL) and assessed the capacity of both tests to detect the corresponding consequences. Excessive BPL inactivation causes G-protein unfolding, altering in turn viral morphology and the continuity of the G-protein layer in the viral particle. Both the NIH and the ELISA tests were able to monitor the consequences of excessive inactivation in a similar manner. Of note, the experimental error of the ELISA test was well below that of the NIH test. These results increase the prospect that the ELISA test could be considered a suitable candidate for the replacement of the NIH test.

Development and pre-validation of a quantitative multi-dose serological assay for potency testing of inactivated rabies vaccines for human use

It is mandatory to ensure the quality of biological products used in the prevention of rabies, a zoonosis with nearly 100% lethality. Fifteen million people receive post-exposure prophylaxis yearly. The vaccine batches are assessed by the National Institutes of Health (NIH) test which has several disadvantages such as significant variability and animal welfare issues. The estimation of immunogenicity based on titration of neutralizing antibodies (NA) is not applied to the human vaccine yet. Despite this, a satisfactory concentration of NA (0.5 IU/ml) can be used as a predictor of the clinical efficacy and for estimating rabies vaccine potency. The objective of this study was to develop and pre-validate a Serological Potency Test (SPT) using the modified Rapid Fluorescent Focus Inhibition Test (mRFFIT) to determine the potency of rabies vaccines for human use, demonstrating its relevance and reliability. The results show good agreement between the potencies determined by the SPT and the NIH test. The assay was able to distinguish between potent and sub-potent lots of vaccines. The results demonstrated that SPT is a viable candidate for validation and inclusion in pharmacopeias as a reduction and refinement for the NIH test.

Comparison of immunogenicity, efficacy and transcriptome changes of inactivated rabies virus vaccine with different adjuvants

Adjuvants have been proven to be very effective in enhancement of immune response of many antigens. However, few studies involved head-to-head comparison of their potentials in inactive rabies virus vaccine. In this study, we investigated two types of aluminum adjuvants and five other adjuvants (BLPs, AS02, AS03, MF59 and Poly I:C) on their capacity in enhancing the efficacy of rabies vaccine. The differences in immunogenicity and potency of rabies vaccines with different adjuvants were evaluated by immunizations in ICR mice. Compared with other adjuvants, nano-sized aluminum induced earlier and more vigorous production of rabies virus neutralizing antibodies and facilitated a more effective protection in the challenge test. Based on these results, to comprehensively and systematically explore the role of adjuvants in rabies vaccine immunization, blood samples from four groups were chosen to perform mRNA sequencing. The differentially expressed genes (DEGs) of groups were identified, both gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted for these DEGs. The results showed that there were significant differences in mRNA expression between the mice after immunization with different adjuvants, but the two aluminum adjuvant vaccines induced similar gene expression. Moreover, the data of enrichment analysis indicated that adjuvants were more advantageous in activating the pathways associated with antigen processing, presentation and initial immunity. These results revealed that adjuvants can be used as an enhancer in rabies vaccination, and nano-sized aluminum may be a candidate adjuvant for the development of more effective rabies vaccines. And these data also provide a basis for understanding the mechanisms underlying adjuvants enhancement of the immune response.

A time-resolved fluoroimmunoassay to assay the rabies virus glycoprotein: application for estimation of human rabies vaccine potency

Replacement of the in vivo rabies vaccine potency test (NIH test) by in vitro methods had been discussed by several researcher including WHO expert working groups. In this paper, a time-resolved fluoroimmunoassay (TRFIA) for the assay of rabies virus glycoprotein in rabies vaccine was first established to estimate the rabies vaccine potency by using specific monoclonal antibody that only recognized the native, trimeric and immunogenic form of rabies virus glycoprotein. Potency of the rabies virus glycoprotein was assayed with satisfactory performance under optimal conditions, and the method demonstrated satisfactory results when applied in practical samples. The correlation coefficient of potency values obtained from the present TRFIA and ELISA was 0.912, and 0.903 for those from the present TRFIA and NIH test. These preliminary results confirmed that this TRFIA can replace ELISA with higher performance, and could be a promising replacement of the NIH test. Based upon these results, the present TRFIA seemed to be a convenient tool for evaluating rabies vaccine potency and its products at different stages accordingly.

G-protein based ELISA as a potency test for rabies vaccines

The NIH test is currently used to assess the potency of rabies vaccine, a key criterion for vaccine release. This test is based on mice immunization followed by intracerebral viral challenge. As part of global efforts to reduce animal experimentation and in the framework of the development of Sanofi Pasteur next generation, highly-purified vaccine, produced without any material of human or animal origin, we developed an ELISA as an alternative to the NIH test. This ELISA is based on monoclonal antibodies recognizing specifically the native form of the viral G-protein, the major antigen that induces neutralizing antibody response to rabies virus. We show here that our ELISA is able to distinguish between potent and different types of sub-potent vaccine lots. Satisfactory agreement was observed between the ELISA and the NIH test in the determination of the vaccine titer and their capacity to discern conform from non-conform batches. Our ELISA meets the criteria for a stability-indicating assay and has been successfully used to develop the new generation of rabies vaccine candidates. After an EPAA international pre-collaborative study, this ELISA was selected as the assay of choice for the EDQM collaborative study aimed at replacing the rabies vaccine NIH in vivo potency test.

Replacement of in vivo human rabies vaccine potency testing by in vitro glycoprotein quantification using ELISA – Results of an international collaborative study

Three different ELISAs quantifying rabies glycoprotein were evaluated as in vitro alternatives to the National Institutes of Health (NIH) in vivo potency test for batch release of human rabies vaccines. The evaluation was carried out as an international collaborative study supported by the European Partnership for Alternatives to Animal Testing (EPAA). This pre-validation study, the results of which are presented in this paper, compared three different ELISA designs, assessing their within- and between-laboratory precision. One of the ELISA designs was proposed to the European Directorate for the Quality of Medicines & HealthCare (EDQM) and accepted for an international collaborative study under the umbrella of the Biological Standardisation Programme.

Reduction of animal suffering in rabies vaccine potency testing by introduction of humane endpoints

Potency controls of inactivated rabies vaccines for human use are confirmed by the National Institutes of Health challenge test in which lethal infection with severe neurological symptoms should be observed in approximately half of the mice inoculated with the rabies virus. Weight loss, decreased body temperature, and the presence of rabies-associated neurological signs have been proposed as humane endpoints. The potential for reduction of animal suffering by introducing humane endpoints in the potency test for inactivated rabies vaccine for human use was investigated. The clinical signs were scored and body weight was monitored. The average times to death following inoculation were 10.49 and 10.99 days post-inoculation (dpi) by the potency and challenge control tests, respectively, whereas the average times to showing Score-2 signs (paralysis, trembling, and coma) were 6.26 and 6.55 dpi, respectively. Body weight loss of more than 15% appeared at 5.82 and 6.42 dpi. The data provided here support the introduction of obvious neuronal signs combined with a body weight loss of ≥15% as a humane endpoint to reduce the time of animal suffering by approximately 4 days.

Green synthesis and evaluation of silver nanoparticles as adjuvant in rabies veterinary vaccine.

BackgroundGreen synthesis of nanoparticles by plant extracts plays a significant role in different applications. Recently, several studies were conducted on the use of nanoparticles as adjuvant. The main aim of this study was to evaluate green synthesized silver nanoparticles (AgNPs) as adjuvant in rabies veterinary vaccine and compare the results with the existing commercially available alum adjuvant.Materials and methodsIn the current study, AgNPs were prepared by the reduction of aqueous silver nitrate by leaf extract of Eucalyptus procera. The formation of AgNPs was confirmed by ultraviolet (UV)–visible spectrophotometer, scanning electron microscopy, dynamic light scattering, and X-ray diffraction analysis. Then, different amounts of AgNPs (200 µg, 400 µg, 600 µg, and 800 µg) were added to 1 mL of inactivated rabies virus. The loaded vaccines (0.5 mL) were injected intraperitoneally into six Naval Medical Research Institute mice in each group on days 1 and 7. On the 15th day, the mice were intracerebrally challenged with 0.03 mL of challenge rabies virus (challenge virus strain-11, 20 lethal dose [20 LD50]), and after the latency period of rabies disease in mice (5 days), the mice were monitored for 21 days. Neutralizing antibodies against rabies virus were also investigated using the rapid fluorescent focus inhibition test method. The National Institutes of Health test was performed to determine the potency of optimum concentration of AgNPs as adjuvant. In vitro toxicity of AgNPs was assessed in L929 cell line using MTT assay. In addition, in vivo toxicity of AgNPs and AgNPs-loaded vaccine was investigated according to the European Pharmacopeia 8.0.ResultsAgNPs were successfully synthesized, and the identity was confirmed by UV–visible spectrophotometry and X-ray diffraction analysis. The prepared AgNPs were spherical in shape, with an average size of 60 nm and a negative zeta potential of −14 mV as determined by dynamic light scattering technique. The highest percentage of viability was observed at 15 mg/kg and 20 mg/kg of AgNPs-loaded vaccine concentrations after injecting into the mice. The calculated potencies for alum-containing vaccine and AgNPs-loaded vaccine (dose 15 mg/kg) were 1.897 and 1.303, respectively. MTT assay demonstrated that alum at the concentration of 10 mg/mL was toxic, but AgNPs were not toxic. The in vivo toxicity also elucidated the safety of AgNPs and AgNPs-loaded vaccine in mice and dogs, respectively.ConclusionIn the current study, for the first time, the adjuvanticity effect of green synthesized AgNPs on veterinary rabies vaccine potency with no in vivo toxicity was elucidated according to the European Pharmacopeia 8.0.

Comparative evaluation for safety & potency of inactivated Cell Culture Rabies Vaccines from four Indian manufacturers

Rabies is a fatal but preventable disease. Various cell culture rabies vaccines (CCRV) are recommended by World Health Organization (WHO) for pre- exposure prophylaxis and post-exposure therapeutic application. In the present study, we have evaluated seventy batches of inactivated CCRV, for safety and potency by test for Virus Inactivation and National Institute of Health (NIH) potency test respectively in Swiss albino mice, produced by four manufacturers of India, using different cell substrates, rabies fixed virus strains and inactivation methods. For a single batch evaluation, 0.03ml of undiluted vaccine sample was injected intracerebrally into each of 10 mice weighting between 12-15gms in Virus Inactivation test. In NIH potency test, three fivefold dilutions each of reference and test vaccines were used for immunization of mice. A group of 16 mice were immunized with each dilution on day 0 and 7, followed by a challenge dose of 5-50 LD50 (Median Lethal dose) of Rabies Challenge Virus Standard (CVS) on day 14. The mice were observed for 14 days after challenge dose for the symptoms of fixed rabies virus. The ED50 (50 % effective dose) of reference and test vaccine was calculated by Reed and Muench formula and the relative potency is calculated through comparison with the reference vaccine. The relative potency of the all the four rabies vaccines were above the recommended potency of 2.5IU/single human dose. All the vaccines tested were found to be safe & potent irrespective of the strain of rabies virus, substrate and inactivation process used in production.

Replacing the NIH test for rabies vaccine potency testing: A synopsis of drivers and barriers

Approximately 70% of animal use is utilized to demonstrate quality control of vaccines. Especially rabies vaccine potency testing, using the NIH challenge test, involves objections in terms of scientific relevance, animal welfare concern and costs. Several 3R models have been proposed to refine, reduce or replace this test. Some are formally incorporated into regulatory requirements, but actual regulatory acceptance and use by industry lags behind, raising the question concerning which factors influence this process. This question is answered by a combination of literature review, interviews and a survey among 50 rabies vaccine experts. The findings are analyzed using the multilevel perspective on technology transition, which distinguishes 3 levels of factors influencing innovation acceptance. At the micro level (where 3R models are developed and validated) the dis-advantages of, and fractional experience with, 3R models, scarce data sharing and demanding validation processes exist. The meso level (existing regulatory regime) encloses the barriers of the ‘gold standard’, the lack of harmonization and the driving force of legislation stimulating 3Rs use. The macro level (the societal context) combines risk aversion and increased concern for animal welfare. Regulatory acceptance and use of 3R models requires dedicated stakeholder communication, cooperation and coordination at all three levels.