USEFULNESS OF RECOMBINANT RHDV ANTIGEN AND VLPs BASED HYPERIMMUNE SERA IN ELISA FOR RHD DIAGNOSIS

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1 Bull Vet Inst Pulawy 51, , 2007 USEFULNESS OF RECOMBINANT RHDV ANTIGEN AND VLPs BASED HYPERIMMUNE SERA IN ELISA FOR RHD DIAGNOSIS ANDRZEJ FITZNER, AND BEATA GROMADZKA 1 Department of Foot-and-Mouth Disease, National Veterinary Research Institute, Zduńska Wola, Poland, andrzejf@piwzp.invar.net.pl 1 Department of Molecular Virology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Gdańsk, Poland Received for publication October 17, 2007 Abstract A recombinant capsid protein of RHDV (rvp60) expressed in the baculovirus and polyclonal antibodies against purified virus-like particles (VLPs) produced in rabbits and guinea pigs were standardised as diagnostic reagents for the development of indirect sandwich ELISA to detect RHD virus and for the liquid-phase blocking ELISA (LPB ELISA) standardisation to identify and titrate specific RHDV antibodies. KGM HA positive and BLA HA nonhaemagglutinating RHDV strains and 130 liver homogenates from experimentally infected rabbits were tested by the new indirect sandwich ELISA and compared to HA and Mab ELISA. LPB ELISA was used to detect RHDV antibodies in serum samples taken from unvaccinated and vaccinated rabbits. The developed ELISAs were sensitive, specific, and gave reproducible results as compared with reference tests. Key words: rabbit, rabbit haemorrhagic disease, virion-like structures, diagnostic reagents, ELISA. ELISA methods, using polyclonal antisera against purified native RHDV or monoclonal antibodies (1, 9, 14). Moreover, in RHD diagnosis, the immunofluorescence and immunoelectron microscopy techniques can be used. In rabbits that survived RHDV infection as well as in those vaccinated with inactivated vaccine, strong serological response is developed. It was found that all RHD isolates belong to one serotype. The detection and titration of specific antibodies arising from natural infection or from immunisation are performed with the haemagglutination inhibition test (HI) and ELISA, in which polyclonal antisera or monoclonal antibodies (Mabs) were used (1, 3, 5, 9, 15). The aim of the present study was to apply diagnostic reagents involving recombinant capsid protein (rvp60) produced in baculovirus expression system in the form of virus-like particles (VLPs) and highly specific polyclonal antibodies against VLPs in ELISAs for virological and serological diagnosis of RHD. Rabbit haemorrhagic disease (RHD) is an acute, fatal, and highly contagious viral disease of domestic rabbits, which since 1984 has been the cause of significant losses all over the world. The aetiological agent is a calicivirus, which possess the ability to haemagglutinate human red blood cells type 0 (1, 13). In routine laboratory diagnosis of RHD the HA, HI, and ELISA are commonly used tests for antigen detection and antibody assessment. The RHD virus (RHDV) as many others caliciviruses cannot be propagated in vitro. The main source of virus antigen used for diagnostic purposes and vaccine production are liver homogenates from infected rabbits (1). Due to the fact that nonhemagglutinating RHDV strains exist (2, 4, 12), and HA negative samples are recorded in rabbits showing a chronic form of the disease (more than 3 days of duration), the more precise and sensitive techniques widely used for viral antigen detection are various Material and Methods Recombinant RHD virus antigen. The Polish RHDV SGM strain with haemagglutinating activity, isolated in 1988, was used for nucleic acid isolation, reverse transcription, and for PCR amplification of vp60 gene. RNA was isolated from the liver of infected rabbits who died before 48 h p.i. (8). The genomic fragment of 1,796 kb encoding full-length of RHDV structural protein was cloned end expressed in baculovirus system. A recombinant baculovirus containing the vp60 gene was used to transfect insect cells of the Spodoptera frugiperda, line Sf9 (7, 10). Insect cell medium harvested 5 d after the infection was the source of recombinant protein (rvp60) with haemagglutinating activity, which self-assembled into virion-like structures (VLPs). Three consecutive

2 476 samples of the recombinant antigen were tested. The medium was mixed with glycerol (1:1), analysed by HA and Mab ELISA and then stored at -18ºC prior to use in LPB ELISA. The same antigen was purified by sucrose density gradient (60-10%) as described previously (10), examined by HA and Mab ELISA, and used to immunise laboratory animals. Production of hyperimmune antiserum. Specific polyclonal antibodies against recombinant VP60 (VLPs) were produced in rabbits and guinea pigs. Three seronegative guinea pigs and four rabbits were immunised twice with VLPs containing µg of recombinant purified viral protein VP60 (fractions number 6 and 7 were used - Table 1), mixed with Freund s complete adjuvant, and boosted 3 weeks later with the same dose in Freund s incomplete adjuvant. The animals were bled 2 weeks after the second injection. One rabbit and one guinea pig were maintained as non-immunised controls. The sera were collected separately, analysed by HI / Mab ELISA and stored at - 18ºC until use. The titre of specific anti-vlps sera ranged from to , both in guinea pigs and rabbits. The hyperimmune serum from rabbits and guinea pigs were assayed in developed ELISAs as trapping and detector antibodies, respectively. Mab ELISA. Commercially available ELISA kits with monoclonal antibodies for virological and serological diagnosis of RHD, purchased from the OIE Reference Laboratory for RHD (Istituto Zooprofilattico Sperimentale, IZS, Brescia Italy), were used as reference tests for RHDV and RHDV antibodies assessment. The assays were performed according to the instructions of manufacturer. HA/HI. Haemagglutination assay (HA) for antigen detection and haemagglutination inhibition (HI) assay for antibodies titration using 0.75% human erythrocytes 0 were applied as described previously (9). RHDV samples. RHDV HA positive strain from laboratory collection (KGM), RHDV HA negative Polish strain (BLA), and 130 liver homogenates collected from rabbits experimentally infected with 100 LD 50 of KGM strain used for seed virus production, were assayed by HA test, Mab ELISA, and developed indirect sandwich ELISA. The tissues were collected up to 5 days and tested as 20% liver homogenates in 0.85% saline buffer. Negative and positive serum samples collection. One hundred and twenty rabbits from a commercial rabbitry and 68 rabbits purchased from small farms were examined by HI and Mab ELISA. Out of these, three RHDV positive sera were detected among rabbits from a small farm. The rabbits free from RHDV antibodies were used for routine vaccine control, including vaccination and challenge. A total of 345 serum samples taken before vaccination ( 0 ) and two weeks after vaccination were tested by the developed LPB-ELISA and compared to HI and Mab ELISA results. The highly positive (HI 2560), and negative (H<10) rabbit control sera were included. ELISA procedures. An indirect sandwich ELISA to determine the presence of RHDV antigen and liquid-phase blocking ELISA (LPBE) for serological assay of specific antibodies for RHDV were developed. The immunoassays protocols described previously in our laboratory for RHD diagnosis (9) were adopted for new diagnostic reagents. In LPB ELISA, recombinant antigen (VLPs) was used. The optimal concentrations of the produced reagents (trapping and detecting antibodies, recombinant antigen) were determined by the chequerboard titration according to Hamblin (11). The immunoplates coated with rabbit serum against VLPs RHDV (trapping antibody) were kept overnight at 5ºC and after washing were used directly for the test or stored at -18 C. For RHDV antigen detection, the indirect sandwich ELISA protocol was used. The samples were tested at dilutions 1:5 and 1:30. The positive virus antigen (liver homogenate) was trapped by the rabbit RHDV VLPs antibodies. At the next stage, the specific guinea pig RHDV VLPs antibodies reacted with the trapped antigen. The fixed guinea pig antibodies were detected by commercial rabbit antiguinea pig immunoglobulin (IgG) conjugated with horseradish peroxidase (1:1000). After the addition of orthophenylenediamine and H 2 O 2 solution, a colour developed. The reaction was stopped after 5 min with 1.25 M H 2 SO 4. The intensity of the reaction was measured with the microplate reader at 492 nm to indicate the presence of antigen in the test sample. The positive and negative controls were included. The positive samples were classified as an absorbance twofold greater than the negative control (at dilution 1/5). In LPB ELISA procedure, the sera were tested at a final dilution of 1:20 (spot test) or were titrated. The test sera, positive and negative control sera, reacted with recombinant antigen (RHDV VLPs) at previously determined dose in U-bottomed plates (liquid-phase) and then were transferred to immunoplate (MaxiSorb) coated with rabbit antiserum. In the next two stages, guinea pig anti VLPs serum and anti-guinea pig IgG peroxidase conjugate were added. As described above for the enzymatic reaction, the substrate and chromogen were added and the plates were incubated for 15 min at room temperature. The optical densities (OD) were read at 492 nm. A positive reaction was that in which the OD was reduced more than 50% in comparison with that of controls without serum. Antibody titres were expressed as the final dilution of serum giving 50 % of the mean OD value recorded in negative control serum or antigen control. The negative and positive control serum samples were included in each test.

3 477 Table 1 Characteristics of the purified VLPs (recombinant RHDV VP60) examined by HA and ELISA VLPs fractions purified in sucrose density gradient (10-60%) HA titre Mab ELISA OD 490 nm 1:5 1: * * Positive control RHDV (+) Negative control RHDV (-) < * - VLPs fractions used for immunisation. Results The HA and ELISA examination of three supernatant samples from transfected insect cells (Sf9), demonstrated the presence of very high HA titre ( ) of recombinant antigen (rvp60) and confirmed our earlier data (7). The recombinant protein was antigenic in Mab ELISA with OD values above 1.5. The VLPs fractions number 6 and 7, purified in sucrose density gradient, had the highest HA/ELISA titres (Table 1) and stimulated hyperimmune specific sera production. Rabbit and guinea pig polyclonal sera assayed by HI and Mab ELISA demonstrated the presence of highly positive specific antibodies, ranging from to , which reacted with native RHD virus (Table 2). Table 2 Hyperimmune sera production the antibody titres in sera of rabbits and guinea pigs immunised with purified rvp60 (VLPs) Animal Antibody titres HI Mab ELISA Rabbit Rabbit Rabbit Rabbit Guinea Pig Guinea Pig Guinea Pig Development of ELISA for RHDV antigen and anti rvp60 RHDV antibody detection. All the rabbit and guinea pig antisera tested as a trapping and detector antibodies reacted with native RHDV antigen in the developed ELISA procedure. Fig. 1 shows the titration curves of VLPs antigen (HA titre ) in indirect sandwich ELISA against hyperimmune sera from rabbit No. 4 and guinea pig No. 2. For LPB ELISA, the recombinant antigen was used at optimal dilution 1:6000, the trapping rabbit antiserum at the dilution 1:16000, and the guinea pig antiserum at the dilution 1:8000. The comparative virological diagnosis shown specific antigen detection by the developed ELISA in all Mab ELISA positive samples, HA positive samples, as well as in HA negative BLA strain. The samples tested at 1:5 dilution presented a high OD absorbance in the range of The OD negative samples and negative control gave OD values The results of antigen detection by a new ELISA and Mab ELISA of 15 examined samples with different HA titres presented as OD percentage at dilution 1/30 in comparison to positive control at dilution 1:5 are shown in figure 2. In all the analysed samples, a similar decrease in OD values at dilution 1/30 in both ELISA methods was found. Comparative studies of 130 liver homogenates of infected rabbits demonstrated that all positive samples in HA and Mab ELISA were also positive in the developed ELISA. The samples with the highest HA titres had also high concentration of RHDV antigen, as it was determined by OD inhibition at 1:5 and 1:30 dilution (data not shown). It was found that all positives samples in HA and Mab ELISA were also positive in the developed indirect sandwich ELISA method. The samples with low HA titre also demonstrated decrease in OD values at 1:5 and 1:30 dilutions respectively. A total of 380 sera from unvaccinated and vaccinated rabbits, collected before and 14 days after immunisation were examined by HI, Mab ELISA, and LPB ELISA as a spot test and titration tests. All 185 serum samples taken at the day 0 of the experiment, as well as 84 samples taken from controls two weeks later, found negative by Mab ELISA and HI, were also negative by LPB ELISA (titre < 10). The specific antibodies to RHDV were detected by LPB ELISA in three Mab ELISA seropositive samples and in all the 101 serum samples taken from vaccinated animals two weeks after immunisation (Table 3).

4 478 Table 3 Comparative analysis of RHDV antibody detection in serum from unvaccinated and vaccinated rabbits Rabbits Assay Unvaccinated Vaccinated Found free from antibodies Controls at day 14 2 weeks after vaccination at day 0 before vaccination Positive/tested Specificity (%) Positive/tested Specificity (%) Positive/tested Sensitivity (%) HI 0/ / / Mab 0/ / / ELISA LPB ELISA 0/ / / Table 4 Antibody titres in the sera from vaccinated rabbits assayed by LPB ELISA, Mab ELISA, and HI two weeks after immunisation Test LPBE Mab ELISA HI Titre > > > > > > Percentage ,5 3 OD ELISA (490 nm) 2,5 2 1,5 1 0,5 0 1,3 1,6 1,9 2,2 2,5 2,8 3,1 3,4 3,7 3,9 4,3 4,6 Reciprocal log 10 recombinant antigen dilution trapping antiserum, dil. 1/500 trapping antiserum dil. 1/16000 trapping antiserum dil. 1/4000 trapping antiserum dil. 1/32000 Fig. 1. Estimation of recombinant antigen dose. Titration against different trapping antiserum dilutions.

5 479 OD ELISA - % of positive control at 1:5 dil % 100.0% 80.0% 60.0% 40.0% 20.0% 0.0% 1 BLA HA neg. strain Positive control Negative control HA titre tested samples New ELISA Mab ELISA HA Fig. 2. RHDV antigen detection in liver homogenates of infected rabbits - comparison of new indirect sandwich ELISA, Mab ELISA, and HA. All the vaccinated rabbits survived 10 d observation period after the challenge and the mortality rate among controls animals was higher than 90%. Antibody titres detected by LPB ELISA as well as by Mab ELISA and HI test ranged from 10 to The serum titres from the immunised rabbits showed low (10-40), medium (80-320), and high values (Table 4), with predominance of medium titres. This is in accordance with general observations for vaccinated rabbits (1). The antibody titres recorded by both ELISA were similar two weeks after vaccination. Five serum samples after challenge were highly positive in all the tests, demonstrating the presence of specific antibodies in the titre range of to Discussion RHDV as many other caliciviruses cannot be propagated in vitro. Due to the lack of convenient system for RHD virus propagation, the only source of viral antigen used in laboratory diagnostics and vaccine production is the tissue obtained from infected rabbits, mainly the liver, which contains the highest concentration of the viral particles. In the presented paper, it was demonstrated that recombinant capsid protein (rvp60) of RHDV can be used as the source of non-infectious antigen for the production of sera and in LPB ELISA. The presence of recombinant RHDV VP60 protein, which selfassembled into VLPs was confirmed by IPMA, western blot, and EM (7, 10). These VLPs were present in insect cell medium in large quantities, demonstrating very strong haemaglutinating profile in HA, and antigenic activity in the ELISA. Recombinant RHDV antigen (rvp60) induced a specific antibody production, which reacted with RHD virus present in the tissues of infected rabbits. The ELISAs are widely used in diagnosis of many animal contagious diseases. The indirect sandwich ELISA and LPB ELISA protocols are successfully employed in foot-and-mouth disease diagnostics (6, 11). Comparison of different diagnostic techniques, including HA, HI, and Mab ELISA for the detecting of RHD virus and RHDV specific antibodies, indicated a positive correlation of the developed ELISAs. It was found that in the new ELISA a recombinant structural capsid protein VP60 and monospecific polyclonal hyperimmune anti VLPs sera can be used in RHD laboratory diagnosis. Hyperimmune sera used in virological test enable also the detecting of non-haemagglutinating RHDV strain. The liver homogenates from experimentally infected rabbits as well as HA positive reference strain KGM and HA and negative BLA strain were evidently discriminated from negative controls by the developed indirect sandwich ELISA. The specificity of the LPB ELISA was also demonstrated using sera taken from unvaccinated rabbits, which were used for test of vaccine safety and potency. Furthermore, we have observed that recombinant RHDV antigen mixed with glycerol and stored frozen (-18ºC) conserved its haemagglutinating and antigenic properties and can be

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