Rabies is a generally fatal and incurable zoonotic disease caused by rabies virus and is usually transmitted to humans through the bite of a rabid dog . Human and animal vaccination remains the most successful public health intervention. Since the emergence of inactivated rabies brain-tissue vaccines (Fermi, Hempt and Semple types) in the early 20th century, inactivated vaccines have been the most commonly used anti-rabies biologics for humans and domestic animals . The potency of an inactivated rabies vaccine, primarily determined by the amount and concentration of virus antigen, is an indispensable evaluation index in the production process . A number of methods, such as the NIH test or single-dose mouse protection test (a modified protocol of the NIH method) [18, 24], Rabies peripheral challenge (RPC) test , Habel test  and single radial immunodiffusion test , have been established to assay the potency. Of these, the NIH test and the single-dose mouse protection test recommended by the World Health Organization (WHO) and the European Pharmacopoeia (EP) respectively are the most commonly used methods for quality control of inactivated rabies vaccines [18, 24].
However, in the NIH test, single-dose mouse protection test or RPC test, hundreds of mice are required for challenge with rabies virus strain CVS two or four weeks after immunization, and results are acquired over a whole test period of 30-45 days. In addition, poor precision, significant variability and ethical animal welfare problems in both tests have been widely reported [1, 2, 23]. Therefore, in order to establish a suitable alternative method to reduce animal distress and number, bear less experimental uncertainties, and be faster and easier to perform, some in vitro techniques have been developed such as ELISA and serological potency assay methods [4, 8, 9, 11, 14-17, 19-22].
Enzyme-linked immunosorbent assays (ELISAs) based on rabies-related polyclonal or monoclonal antibodies are designed to assess rabies virus glycoprotein content (the only protective antigen) for determination of the potency of inactivated vaccines, but they reflect only the reactivity of antigens in vitro and are inadequate to evaluate the immunogenicity in vivo.
For improving vaccine production efficiency, it is important to have a way to rapidly determine whether the potency of a batch of vaccine satisfies the requirements, e.g., ≥1 IU/dose, or ≥2 IU/dose or ≥3 IU/dose, whereas it is not absolutely necessary to know the exact value of the potency of a product.
Serological potency assays can compensate for the deficiency of ELISAs by quantifying the titers of rabies-neutralizing antibody in serum samples in the vaccinated animals. A modified rapid fluorescent focus inhibition test (mRFFIT) has been used for potency assaying of inactivated rabies vaccines [14, 15], and a good correlation between the results of the challenge test and the mean titers determined by the mRFFIT has been obtained. In the mRFFIT, the potency of test vaccine was determined by comparing mean values of serum antibody titers of the test and reference vaccines. The mean value is generally used when data (i.e., serum antibody titers) follows a normal distribution. However, the data cannot always be analyzed by a parametric method of statistics in practice, especially when the variations of antibody titer among individuals is high, which actually happens. So the method can not be reliably performed in practical vaccine production.
Here we report a method based on the median or interquartile range of antibody titers determined by fluorescent antibody virus neutralization (FAVN) test to semi-quantitatively assay the potency of inactivated rabies vaccines through intramuscular immunization of mice, using fewer animals and a shorter time than the standard methods.
The 4th batch of the Biological Reference Preparation (BRP) with an assigned titer of 11 international units per vial (IU/vial) was provided by the European Directorate for the Quality of Medicines (EDQM, Strasbourg, France). Reference vaccines were stored at -20 ℃ and reconstituted with 1 mL deionized water for injection. All reference dilutions indicated in the text were prepared with phosphate buffered saline (PBS) from these stock solutions (Table 1).
Table 1. Vaccines for mice immunization
As shown in Table 1, test vaccines were inactivated rabies vaccines for veterinary use from 4 different manufacturers, including Nobivac® Rabies (Batch No. A154A01, Intervet International B.V), RabvacTM3 (Batch No. 1215337A, Fort Dodge Animal Health), Rabisin-R (Batch No. L369409, Merial SAS. France) and Rabigen®mono (Batch No. 33XZ, Virbac Laboratories).
Rabies challenge virus standard CVS-24, a fixed laboratory strain, was propagated in mouse brains, and vials containing CVS suspensions were stored at -80 ℃. The intracerebral (I.C.) LD50/0.03mL of the virus seed was 104.50 in mice. Dilutions of CVS-24 were made in distilled water supplemented with 2% horse serum. CVS-11 virus was grown in BHK-21 cells and used in the neutralization test to monitor the neutralizing antibody titer. The TCID50/mL of the virus seed was 107.25 in BHK-21 cells.
The standard anti-rabies serum was obtained from Agence Fran aise de Sécurité Sanitaire des Aliments (AFSSA, Nancy, France). A negative control serum was prepared from unvaccinated mice.
The test was performed according to the standard operating procedure (SOP) of the EP . In brief, SPF out-bred mice, strain Kunming, at the age of 4 weeks, were selected at random and placed into groups of 10 animals. Four five-fold dilutions of reference and test vaccines were freshly prepared. On day 0, each group was injected intraperitoneally with 0.5 mL of a vaccine dilution. At the same time, the challenge dose (mouse intracerebral LD50) of CVS-24 suspension was measured in control mice and calculated using the Spearman-K rber formula. On the 14th day after vaccination, each treated mouse was injected intracerebrally with 0.03 mL of a CVS-24 suspension containing approximately 50 times of mouse LD50. Mice with clear rabies symptoms were euthanized to avoid unnecessary suffering. The number of mice dying of rabies between the 5th and 14th day after challenge was recorded. The relative potency of the test vaccines was calculated by probit analysis using CombiStats software (EDQM).
Four-week-old Kunming mice were purchased from Changchun H & N Animal Breeding Center for Medicine (China) and divided in a randomized manner into seven groups (10 mice/group); i.e., Vac 1, Vac 2, Vac 3, Vac 4, Reference 1, Reference 2 and Reference 3 (Table 1) in the serological potency assay. According to prior experience [14, 15, 28], each mouse was injected with 0.05 mL of vaccine in the upper hind leg using a 29G * 1/2″ insulin syringe (BD Ultra-FineTM, batch 0116106, USA), and peripheral serum samples were collected from the retro-orbital plexus on the 14th day after treatment .
To demonstrate the vaccine potency assayed by the serological method in mice, unvaccinated beagles (3–4 months old) were divided into 7 groups (6 dogs /group); i.e. Vac 1–4 and reference 1–3 (Table 1). Each dog was inoculated with one dose of vaccine in the triceps brachii muscle of the right front leg, and serum samples were collected from the right front leg vein on the 14th day after vaccination.
All the mice and dogs used in this study were treated in accordance with DIRECTIVE 2010/63/EU OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 22 September 2010 on the protection of animals used for scientific purposes.
The antibody titers of serum samples were measured by the FAVN test performed according to the method described by Cliquet et al. . Briefly, 3-fold serial dilutions of both the serum samples and the controls were prepared in microplates. Each serum dilution was tested in quadruplicate. 50 μL (100 TCID50) of challenge rabies virus CVS-11 was also added to each well. After 1 h incubation at 37℃ in a 5% CO2 humidified incubator, 50 μL of BHK-21 cell suspension containing 4×105 cells/mL was added to each well and the plates were incubated for 48 h at 37℃. After fixing the cells in 80% cold acetone for 30 min at room temperature, the plates were stained by adding FITC-conjugated anti-rabies monoclonal antibody (prepared in our laboratory), incubating at 37℃ for 30 min and then washing with PBS-Tween 20. The plates were examined by fluorescence microscopy (Olympus Corp., Tokyo, Japan). Presence or absence of fluorescent foci in the cells was recorded. Endpoints were calculated as the inverse of the highest dilution showing no fluorescence, using the Spearman-K rber formula. A standard anti-rabies serum (AFSSA, Nancy, France) was included as a positive control in all assays, and data were calculated as IU/mL of rabies virus-neutralizing antibody.
The median (Md) and interquartile range (IQR) of serum antibody titers of each group were calculated using the SPSS 16.0 program (SPSS Inc., Chicago, IL, USA) for Windows by a non-parametric method . By comparing the Md of the reference vaccines with that of the test vaccine, the potency of test vaccine can be semi-quantitatively determined within a range; e.g. > 2 IU/dose. However, when the Md of test vaccine is equal to that of the reference, the IQRs should be compared to determine the potency of the test vaccine in which, when the IQR of the test vaccine is larger than that of the reference, the potency of the test vaccine should be considered smaller than that of the reference, rather than vice versa. The conformity between the single-dose mouse protection test, the serological potency assay and immune efficacy in dogs was assessed by comparing with quality standards of test vaccines.
Reference and test vaccines
Challenge virus and standard serum
Mouse potency test
Animals and immunization
FAVN test for potency testing of vaccines
7 groups (10 mice / group) were respectively immunized with different vaccines, and then the antibody titers of serum samples were measured by the FAVN test. As shown in Table 2, on the 14th day after vaccination, the median (20.58 IU/mL) of rabies-neutralizing antibody titers of group Vac 1 exceeded reference 3 (15.64 IU/mL), and the medians of group Vac 2, Vac 3 and Vac 4 were semiquantitated as being between reference 2 (3.01 IU/mL) and reference 3 (15.64 IU/mL). As a result, the potencies of the four test vaccines were in accord with their required qualities.
Table 2. Antibody responses to different vaccines in serological potency assay
To evaluate the accuracy and repeatability of the serological potency assay-based FAVN, repeat tests (3 times) and a single-dose mouse protection test were performed. As shown in Table 3, all results of repeat tests by the serological potency assay and of the single-dose mouse protection test accorded with the required qualities of test vaccines.
Table 3. Conformity between single-dose mouse protection test and serological potency assay
The antibody titers of serum samples assayed by FAVN test on 14th day after vaccination are shown in Table 4, and the potency of test vaccines was evaluated by comparison of the protective efficacy of the test and reference vaccines in dogs. The median titer (6.15 IU/mL) of the rabies-neutralizing antibody of the group Vac 1 was equal to that of the group reference 3; however, the IQR (7.34 IU/mL) of Vac 1 was less than that (8.20 IU/mL) of reference 3; i.e., the protective efficacy of Vac 1 in dogs was higher than that of reference 3 (6.15 IU/mL). The protective efficacy of Vac 2, Vac 3 and Vac 4 in dogs was estimated to be between reference 2 (1.32 IU/mL) and reference 3 (6.15 IU/mL); i.e., the protective efficacy of the test vaccines in dogs was in line with their required potency.
Table 4. Humoral immune response in dogs vaccinated with different potency vaccines