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Clinical and Vaccine Immunology, December 2006, p. 1367-1372, Vol. 13, No. 12
1071-412X/06/$08.00+0     doi:10.1128/CVI.00273-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Development and Characterization of a Stable Vero Cell Line Constitutively Expressing Peste des Petits Ruminants Virus (PPRV) Hemagglutinin Protein and Its Potential Use as Antigen in Enzyme-Linked Immunosorbent Assay for Serosurveillance of PPRV{triangledown}

V. Balamurugan,* A. Sen, P. Saravanan, T. J. Rasool, M. P. Yadav, S. K. Bandyopadhyay, and R. K. Singh

National Morbillivirus Referral Laboratory, Division of Virology, Indian Veterinary Research Institute, Mukteswar-263 138, Nainital District, Uttaranchal, India

Received 13 June 2006/ Returned for modification 15 September 2006/ Accepted 27 September 2006


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ABSTRACT
 
We developed and characterized a stable Vero cell line constitutively expressing Peste des petits ruminants virus (PPRV) hemagglutinin (H) protein and assessed its potential use as diagnostic antigen in enzyme-linked immunosorbent assay (ELISA). PPRV H gene of the vaccine strain (Sungri-96) was amplified by reverse transcription (RT)-PCR, cloned into a eukaryotic expression vector (pTarget), and subsequently transfected and expressed in Vero cells. A stable Vero cell line was developed after 20 repeated passages by using G418 antibiotic selection pressure (400 to 600 µg/ml). The integration of PPRV H gene in the Vero cell genome and its genomic transcription were confirmed by PCR and RT-PCR assays, respectively, and the 70-kDa PPRV H protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The recombinant protein reacted specifically with PPRV anti-H neutralizing monoclonal and polyclonal antibody in competitive, sandwich, and indirect ELISA, respectively, indicating that the native form of the protein was expressed. Evaluation of the protein in competitive ELISA and indirect ELISA vis a vis whole virus was done using 306 and 146 goat field serum samples, respectively; comparable results were obtained with high degrees of relative diagnostic specificity (93.53% and 100%, respectively) and sensitivity (99.04% and 79.16%, respectively). This study shows that the PPRV H protein could be a sustainable source of safe antigen in countries of nonendemicity without the need to handle infectious virus for serodiagnosis.


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TEXT
 
Peste des petits ruminants (PPR) is an acute highly contagious and economically important viral disease of small ruminants, especially goats and sheep, and is characterized by severe pyrexia, oculonasal discharges, necrotizing and erosive stomatitis, enteritis, and pneumonia (10, 11), with morbidity and mortality rates as high as 100% and 90%, respectively (1). The causative agent, Peste des petits ruminants virus (PPRV) (genus Morbillivirus, family Paramyxoviridae), has a single-stranded, negative-sense RNA genome encoding eight proteins in the order 3' N-P/C/V-M-F-H-L 5' (12) and is antigenically closely related to rinderpest virus (RPV) (12). PPRV was first described in the Ivory Coast in West Africa (11), and it subsequently spread widely across sub-Saharan Africa, the Arabian Peninsula, the Middle East (18), southwest Asia, and India and its neighboring countries (30). In India, the disease was first reported in 1987 from Tamil Nadu (29). During ensuing years, the disease took an epidemic form, causing serious economic losses throughout the country (20, 22).

Many countries are at present pursuing serosurveillance and monitoring for RPV under the Global Rinderpest Eradication Programme. In countries where both RPV and PPRV are prevalent, independent surveillance programs for RPV and PPRV are to be implemented. Serological methods for detection of antibodies in infected animals appropriately support diagnosis. Virus neutralization test and enzyme-linked immunosorbent assay (ELISA) are the commonly employed diagnostic techniques for testing serum samples. Antibody detection methods employing monoclonal antibody (MAb) raised against either hemagglutinin (H) protein (3, 26) or nucleoprotein (N) (19) have been earlier described, with two such commercial sources available in the world (2, 19).

For control of PPR, the most effective homologous vaccines reported are tissue culture-adapted PPRV vaccines (10, 27). India was provisionally free from RP since March 1998, and the World Organization for Animal Health declared India free from RP from May 2006. PPRV, however, is still widespread and endemic to the area. Recently, we developed a MAb (anti-H protein of PPRV) (31)-based competitive ELISA (c-ELISA) kit (32) and polyclonal antibody-based indirect ELISA (I-ELISA) (4) using tissue culture PPRV (27) as an antigen for detection of antibodies to PPRV for serosurveillance and seromonitoring throughout the country.

Bulk viral antigen production using cell culture systems needs sophisticated infrastructure, which in turn adds to the overall cost of production in addition to the risk of virus handling and a possible accidental release into environment. Thus, a recombinant antigen-based diagnostic assay would be of immense value during the final stages of disease eradication as well as for serosurveillance of PPRV in countries free from the disease. The conventional viral antigen shows batch-to-batch variations in yield/titers during passages in cell culture, whereas the yield remains more or less constant in the case of expressed protein.

Several approaches have been attempted to develop recombinant antigen-based diagnostic ELISAs as an alternative to conventional ones. Production of recombinant H or N protein of either PPRV (14, 33) or RPV (13, 24) in an insect (baculovirus) cell system has been tried, and the proteins were successfully used as a coating antigen in ELISA for serodiagnosis. Even though the production level is very high, there is one drawback with the insect system in terms of glycosylation of a protein which is biochemically different from that in mammalian cells, which could subsequently lead to improper presentation of epitopes in the target protein. Mammalian systems are the ideal choice for production of biologically active/therapeutic proteins, as these are capable of correctly glycosylating the protein at their proper sites. In this direction, Seth and Shaila (28) transiently expressed HN of the PPRV and H of RPV under the control of cytomegalovirus (CMV) promoter in CV-1 mammalian cells; these were found to be biologically active in possessing hemadsorption and neuraminidase activities. In view of the above facts, this study was undertaken to develop a stable Vero cell line constitutively expressing PPRV H protein and assess its future potential use as a diagnostic antigen in ELISA for diagnosis of PPRV.

Vero cells (CCL-81) between the 130th and 150th passage levels were propagated in Eagle's minimum essential medium supplemented with 10% fetal bovine serum (HyClone, Utah) and used for development of a stable cell line constitutively expressing PPRV H protein. The PPRV vaccine (Sungri 96 isolate attenuated, 60th passage) (27) was used as a virus source for amplification of H gene and raising hyperimmune serum (HIS) in rabbits. pTargeT Mammalian Expression vector (Promega Corporation, Madison, WI) was used for cloning, sequencing, and expression of PPRV H protein in the Vero cell system. The oligonucleotide primers used in the study are shown in Table 1.


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  		TABLE 1. Oligonucleotide primers used in the study

The HIS against PPRV was raised in adult rabbits for Western blotting and standardization of a polyclonal antibody-based sandwich ELISA (s-ELISA). Partially purified viral antigen was prepared using sucrose density gradient according to methods described previously (32). This antigen ({approx}750 µg/ml) was blended with an equal volume of Freund's complete adjuvant, and 1 ml was injected intramuscularly (i.m.) at two different sites in three healthy rabbits, free of PPRV antibodies. After 2 weeks, the second inoculum was prepared with Freund's incomplete adjuvant and administered i.m. Two weeks later, a second booster was given at 1 ml i.m. to each animal. Ten days after the last injection, blood was collected from the immunized rabbit, and serum was separated and stored in small aliquots at –20°C until further use.

Viral RNA was extracted from purified antigen by using TRI-REAGENT (Sigma), with necessary modifications described previously (6, 9). Reverse transcription (RT) for first-strand cDNA synthesis was performed on 1 to 5 µg of total RNA using Moloney murine leukemia virus reverse transcriptase (Promega Corporation, Madison, WI) and random hexamers at 37°C for 1 h, and subsequent PCR amplification was carried out using 5 µl of the RT product. The detailed conditions standardized for different primers used in PCR are listed in Table 2. The amplicon was purified using AuPrep GELX kit (Life Technologies India Pvt Ltd., New Delhi, India) as per the manufacturer's protocol, cloned under the control of CMV promoter into pTargeT vector, and characterized by colony PCR and restriction enzyme (RE) analysis. Finally the sequence determination was carried out using the ABI PRISM 3100 (AME Bioscience, Toroed, Norway) automated DNA sequencer to confirm the frame and specificity of insert.


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  		TABLE 2. PCR conditions standardized for different set of primers

Endotoxin-free plasmid DNA (pTargeT plus PPRV H clone no. 8) was extracted by using the QIAGEN endotoxin-free plasmid extraction kit and transfected into Vero cells using Transfast reagent (Promega Corporation, Madison, WI) as per the protocol with some modifications. Briefly, 2 µg endotoxin-free plasmid was mixed with 4 µg of Transfast in serum-free Opti-Mem-I media (Gibco-BRL, New York) and incubated at 37°C for 15 min. After incubation, the mixture was added at 1 ml/well to a preformed 70% monolayer of Vero cells in six-well plates along with the appropriate control. After incubation for 4 h at 37°C, 1 ml of maintenance media containing 2% fetal bovine serum was added. After maintaining the cells in nonselective medium for 1 to 2 days posttransfection, the cells were placed in selective medium containing the drug G418 (neomycin sulfate, 600 µg/ml of media) until the cells of the control well (nontransfected) died completely. Individual foci from the transfected well were selected, subcultured, and transferred to other plates/flasks for further propagation. The process was repeated several times and for various passage levels until a stable (Vero/PPRV H) cell line was obtained.

For ascertaining integration of the PPRV H gene, genomic DNA was isolated from both normal and stable Vero cells using the AuPrep GENbt DNA extraction kit and subjected to PCR amplification. For determination of genomic transcription of the PPRV H gene in stable cells, total RNA was extracted from the cells as described earlier and used in RT-PCR for amplification using PPRV H gene internal primers (Table 1). Then the stable cells were propagated in growth medium, and after attaining a confluent monolayer, the cells were harvested by trypsinization, centrifuged at 3,000 x g for 5 min, and resuspended in phosphate-buffered saline containing 1 mM phenylmethylsulfonyl fluoride. The cell suspension was sonicated on ice for 30 to 60 s in a sonicator (Sonics; Cell Vibra) at an amplitude of 30% with a 9.9-s pulse and mixed with 2x SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) sample buffer for characterization of protein. Normal Vero cells were also processed in a similar way and included as negative controls. These cell lysates were analyzed by 12% SDS-PAGE under denaturing conditions (17). A duplicate gel was transblotted onto a nitrocellulose membrane for immunodetection as per the method of Burnette et al. (5) with modifications. The recombinant PPRV H protein on the blot was detected by incubation with PPRV HIS (1:200 dilution) followed by an anti-rabbit antibody horseradish peroxidase conjugate at 1:2,000 (Sigma), and the reaction was developed with 3,3'-diamino benzidine as a chromogen with urea as the substrate (Fast tab; Sigma).

Expressed protein (sonicated Vero/PPRV H cell lysate) along with normal Vero cell lysate was tested for its suitability as antigen in (i) c-ELISA (32), (ii) I-ELISA (4), and (iii) recently standardized s-ELISA (unpublished data). This s-ELISA uses rabbit anti-PPRV antibody and goat anti-PPRV antibody as coating and tracing antibodies, respectively. One 25-cm2 flask of stable cell lysate was used to coat 20 wells (50 µl/well) in ELISA. The amount of H protein in 1 ml of lysate was estimated by anti-H protein MAb-based s-ELISA, which is equivalent to the amount of H protein present in the 1 ml of PPRV vaccine (titer of 105.5 50% tissue culture infective doses [TCID50]/ml). Once it gave desirable reactivity, the recombinant protein was further evaluated by ELISA using a total of 452 (306 for c-ELISA and 146 for I-ELISA) field goat serum samples obtained from diverse geographical locations in the country.

The performance of the recombinant antigen-based ELISA was compared with c-ELISA/I-ELISA. Diagnostic sensitivity and specificity of ELISA was calculated from goat serum samples using a two-sided contingency table described earlier (15) in correlation with both c-ELISA according to the methods described by Singh and colleagues (32). The proportions of positive and negative samples detected, out of the known actual positive and negative samples, were taken as the sensitivity and specificity of the assay, respectively.

Eradication of the disease depends on rapid and accurate diagnosis of infection and the implementation of prompt control measures. For effective epidemiological surveys, a rapid serological test like ELISA is very suitable for open bench work, especially under field conditions wherein the equipment infrastructure is poor, for the detection of antibodies to PPRV. Surface proteins of morbilliviruses are epidemiologically important because they are the proteins most exposed to the environment and, therefore, the main target of the host immune system (16). The H protein is the most important target for neutralizing antibodies and is, therefore, thought to be subject to increased immunological pressure (25). Moreover, Renukaradhya et al. (23), upon mapping of B-cell epitopic sites on the H protein of PPRV with MAbs, showed that this protein possesses major neutralizing immunodominant epitopes. To meet the increasing demand of diagnostic reagents in the near future, it is possible for the laboratory to supply a safe, potent, and cost-effective antigen based on recombinant protein(s), which is vital for the efficient control of the disease, especially in countries of nonendemicity. Safe, potent, cost-effective noninfectious recombinant antigen-based diagnostics and prophylactics would be of immense value during the last stages of disease eradication in countries of endemicity, like India, or in countries of nonendemicity without handling infectious virus.

To generate a continuous source of recombinant PPRV H protein, it was necessary to express PPRV H protein constitutively in a eukaryotic cell line. This was achieved by employing the expression vector cassette of PPRV H gene under a CMV promoter-DNA construct with integration into the Vero cell genome. The sequence coding for PPRV H protein was amplified by RT-PCR, and the size of the amplified product (1,869 bp) and its nested PCR amplicon (718 bp) were in agreement with the reported sizes of the PPRV H gene (8, 21). The amplicon cloned into the pTargeT vector was screened by colony PCR using T7 (Vector specific) and PPRV H (NotI) R His (virus specific) primers, which resulted in amplification of a 1,926-bp product, confirming the presence of an insert in the proper orientation in 3 of 7 colonies screened (data not shown). One of the clones after RE analysis of the plasmid DNA was selected for sequence analysis, which revealed that the gene is specific and inserted in the proper frame.

After 10 days of transfection, there were a few live cells scattered throughout the transfected well (Fig. 1). Selected clones/foci were passaged many times (>20 passages) to develop a stable cell line. Further, transfected cells were single cell cloned by limiting dilution under G418 selection that led to selection of only those clones that were stable transformants for subsequent propagation. It is also possible that the plasmid remains episomal in the cell, which enables the transfectant to survive under selection pressure. In such cases of the insert remaining as an extrachromosomal entity, it would also be possible for the selected clone to survive under G418 selection, albeit only transiently. Hence, repeated passaging of the stable cell line was done for the characterization of constitutionally expressing PPRV H protein. Integration of the PPRV H gene into the genome of stable cells was confirmed by PCR amplification (Fig. 2A) of the 1,869-bp PPRV H gene (lane 1) from genomic DNA extracted from the stable cells even after 6 to 9 months of preservation in LN2 and repeated passaging after revival, with no amplification in Vero cells (Fig. 2A, lane 2). Since the plasmid integrates randomly into the cellular genome under selection pressure, it does not always ensure the expression of the desired gene products. However, in this case, we encountered the soluble form of the expressed protein, as it conspicuously reacted in ELISA.


Figure 1
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  		FIG. 1. Selection of transfected Vero cell foci after 2 weeks posttransfection under the selection pressure of 600 µg/ml of G418 antibiotic.


Figure 2
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  		FIG. 2. Agarose gel electrophoresis of PCR (A) and RT-PCR (B and C) products from Vero and Vero/PPRV H using different primer sets. (A) PPR H (NotI) F and PPR H (NotI) R His primers. Lane 1, stable (Vero/PPRV H) Vero cell genomic DNA; lane 2, normal Vero cell genomic DNA. (B) BA1 and BA2 primers. Lane 1, stable Vero cell RNA; lane 2, normal Vero cell RNA. (C) Internal PPRV H gene primers. Lanes 1 and 5, stable Vero cell RNA; lanes 2 and 4, normal Vero cell RNA; lane 3, positive control (pTarget cloned plasmid); lanes M, 100-bp plus DNA ladder (MBI Fermentas).

The genomic transcription of the PPRV H gene in a stable cell line was also determined by RT-PCR (Fig. 2C) using 3' end gene-specific internal primers. The presence of amplicons of the desired size, i.e., 718 bp for ppr-hfr3 and ppr-hre4 (lane 5) and 347 bp for ppr-hfr1 and ppr-hre2 (lane 1) (which were absent in normal Vero cells) (Fig. 2C, lanes 2 and 4), confirms the expression of the cloned H gene in stable cells unequivocally with stable integration of plasmid DNA into the Vero cell genome, leading to constitutive expression of the PPRV H protein. To confirm isolation of RNA in both stable and normal Vero cell lines, conserved ß-actin gene (housekeeping gene) primers (BA1 and BA2) (7) were also included in the assay, which amplified a specific 275-bp product (Fig. 2B).

Expressed protein (sonicated Vero/PPRV H cell lysate) reacted in I-ELISA (4), c-ELISA (32) and s-ELISA (our unpublished data) that gave an indication that the protein expressed was the PPRV-specific H protein. The mean reactivity of cell lysate of recombinant protein vis-à-vis whole virus in terms of optical density at 492 nm were found to be 0.27 versus 0.56, 0.28 versus 0.61, and 0.418 versus 0.76 when tested by I-ELISA, c-ELISA, and s-ELISA, respectively. Further, the expression of protein in sonicated cell lysate was confirmed by SDS-PAGE and Western blotting (Fig. 3). Upon SDS-PAGE analysis, in addition to many individual separated bands, an intense band at a position equivalent to 70 kDa appeared in the stable cell lysate (Fig. 3A, lane 1), with no such protein band in the normal Vero cell lysate (lane 2), indicating expression of PPRV H protein in stable cells. The calculated size of the PPRV H protein along with the His tag at the C-terminal end comes to around 69.98 kDa, which is in agreement with the mobility of the additional protein band observed in SDS-PAGE, indicating that the 70-kDa protein is the product from the cloned PPRV H gene (8).


Figure 3
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  		FIG. 3. Characterization of expressed recombinant PPRV H protein. (A) SDS-PAGE analysis of expressed PPRV H protein (denatured condition). lane M: mark 12 unstained standard protein molecular mass marker (Invitrogen); lane 1, stable (Vero/PPRV H) cell lysate proteins; lane 2, normal Vero cell lysate proteins. (B) Western blot analysis of expressed PPRVH protein. Lane M, Blue Ranger prestained protein molecular mass marker mix (Pierce); lane 1, stable (Vero/PPRV H) cell lysate proteins; lane 2, normal Vero cell lysate proteins.

SDS-PAGE-resolved protein bands were transferred to a nitrocellulose membrane and immunodetected with antiserum specific to PPRV (Fig. 3B). On Western blot analysis using rabbit HIS, 3 to 4 bands reacted in both stable and normal Vero cell lysates, but one additional band of about 70 kDa was observed only in the stable cell lysate (lane 1), which further confirmed that the 70-kDa protein is PPRV specific. These three nonspecific protein bands observed in both stable and normal Vero cell lysates could be due to the reaction of HIS with Vero cell proteins because HIS was raised in rabbits against partially purified PPRV grown in Vero cells.

The expressed protein reacted well with PPRV neutralizing MAbs in c-ELISA and polyclonal goat PPRV serum in I-ELISA and s-ELISA, indicating that the epitopes in the expressed protein are well recognized by these antibodies, and confirmed that the native form of PPRV H glycoprotein was expressed in mammalian system. This is concurrent with the earlier report of biologically active expressed PPRV HN protein in CV-1 mammalian cells (28). Similarly, the membrane-bound form of recombinant PPRV H protein expressed in baculovirus induced both humoral and cell-mediated immune responses against PPRV in immunized goats and antibodies generated in immunized animals could neutralize both PPRV and RPV in vitro (33).

Further evaluation of the protein was carried out by c-ELISA using 306 goat serum samples received from the field, which were tested simultaneously using both whole-virus and recombinant H antigen. The performance of the recombinant antigen-based assay was very much comparable to that of whole-virus particles in a two-sided contingency table (Table 3). Of 306 goat serum samples tested, 105 samples were found to be positive by recombinant c-ELISA and compared very well with c-ELISA (32), with a high degree of specificity (93.53%) and sensitivity (99.04%). Similarly, 146 goat serum samples were tested by I-ELISA, and 76 serum samples were found positive for PPRV antibodies, with a high degree of relative diagnostic specificity (100%) and sensitivity (79.16%) (Table 4). The low sensitivity of I-ELISA is better explained by the quote "diagnostic sensitivity of a test is inversely proportional to reduction in the specificity" (15). The recombinant N protein of the virulent Kabete O strain of RPV (13) and Nigeria 1/75 PPRV (14) expressed in baculovirus have been used as coating antigens in ELISA to distinguish vaccinated animals from those infected with RPV and for serodiagnosis of PPRV. Renukaradhya et al. (24) also expressed the recombinant H protein of RPV in insect cells and used the same for serosurveillance of RPV in MAb-based c-ELISA.


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  		TABLE 3. Relative specificity and sensitivity of recombinant H antigen vis-à-vis whole-virus antigen-based c-ELISA based on 306 field goat serum samplesa


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  		TABLE 4. Relative specificity and sensitivity of recombinant H protein vis-à-vis whole-virus antigen-based I-ELISA based on 146 field goat serum samplesa

Conclusively, the stable Vero cell line expressing PPRV H protein could be a continuous source of viral antigen, which could be used in the diagnosis of PPRV and can replace the conventional cell culture infectious antigen in ELISA, albeit after further extensive validation. This recombinant H protein will be of much use in developing PPRV diagnostics in the future, as the safe, potent, noninfectious antigen may replace the present day infectious whole-virus antigen, which may not be desirable during the post-PPRV eradication phase. Further, the potential to elicit a protective immune response by recombinant H protein can be confirmed by immunizing laboratory animal models (rabbit) and natural hosts (sheep and goats) with this protein. New vistas of studies on the potential role of this protein in protection from PPRV in natural hosts, in addition to its diagnostic potential, will open up if antibodies from these immunized animals can neutralize the whole virus.


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ACKNOWLEDGMENTS
 
We thank the director of the Indian Veterinary Research Institute for providing all the facilities and the staff of the National Morbillivirus Referral Laboratory for their help.


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FOOTNOTES
 
* Corresponding author. Mailing address: National Morbillivirus Referral Laboratory, Division of Virology, Indian Veterinary Research Institute, Mukteswar, Nainital Distt. Uttaranchal 263 138, India. Phone: 91 5942 286348. Fax: 91 5942 286347. E-mail: balavirol{at}rediffmail.com. Back

{triangledown} Published ahead of print on 18 October 2006. Back


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Clinical and Vaccine Immunology, December 2006, p. 1367-1372, Vol. 13, No. 12
1071-412X/06/$08.00+0     doi:10.1128/CVI.00273-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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