Virus Genes DOI 10.1007/s11262-015-1169-x
A recombinant canine distemper virus expressing a modified rabies virus glycoprotein induces immune responses in mice Zhili Li • Jigui Wang • Daoli Yuan • Shuang Wang • Jiazeng Sun • Bao Yi • Qiang Hou • Yaping Mao • Weiquan Liu
Received: 5 October 2014 / Accepted: 8 January 2015 Ó Springer Science+Business Media New York 2015
Abstract Canine distemper virus (CDV) and rabies virus (RV) are two important pathogens of the dog. CDV, a member of the morbillivirus genus, has shown promise as an expression vector. The glycoprotein from RV is a main contributor to protective immunity and capable of eliciting the production of virus-neutralizing antibodies. In this study, we recovered an attenuated strain of canine distemper virus and constructed a recombinant virus, rCDVRV-G, expressing a modified (R333Q) rabies virus glycoprotein (RV-G) of RV Flury strain LEP. RV-G expression by the recombinant viruses was confirmed. Furthermore, G was proved to be incorporated into the surface of CDV particles. While replication of the recombinant virus was slightly reduced compared with the parental CDV, it stably expressed the RV-G over ten serial passages. Inoculation of mice induced specific neutralizing antibodies against both RV-G and CDV. Therefore, the rCDV-RV-G has the potential as a vaccine that may be used to control rabies virus infection in dogs and other animals. Keywords Canine distemper virus Rabies virus Reverse genetics Canine distemper virus-based live recombinant rabies vaccine Edited by Zhen F. Fu.
Electronic supplementary material The online version of this article (doi:10.1007/s11262-015-1169-x) contains supplementary material, which is available to authorized users. Z. Li J. Wang D. Yuan S. Wang J. Sun B. Yi Q. Hou Y. Mao W. Liu (&) State Key Laboratory of Agrobiotechnology, Department of Biochemistry and Molecular Biology, College of Biological Sciences, China Agricultural University, No. 2 Yuanmingyuan West Road, Haidian District, Beijing 100193, China e-mail:
[email protected] Introduction Canine distemper virus (CDV) is an enveloped, singlestranded, negative-sense RNA virus of the Morbillivirus genus within the family Paramyxoviridae. The CDV genome contains 15,690 nucleotides with a short 30 leader region and six genes encoding structural nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin (H), and large polymerase (L) proteins organized 30 -N-PM-F-H-L-50 , with a short 50 trailer region. Canine distemper (CD) is a highly contagious disease and is transmitted by aerosols/droplets causing an acute disease characterized by generalized rash, respiratory and gastrointestinal signs, and immunosuppression, with occasional neurological complications [2, 30]. It infects most members of the order Carnivora, wild and domestic, but with different susceptibilities and mortalities [31]. Infection can be controlled only by vaccination [4]. Many members of the nonsegmented negative-strand RNA viruses (NNSV) have been developed as vaccine vectors [3, 13, 15] since several features make them attractive for this purpose. NNSVs replicate only in the cytoplasm and therefore have no chance to integrate with the host genome. Their genes are nonoverlapping, each having its own transcriptional signal, and proteins are synthesized separately. This modular organization facilitates insertion of exogenous genes [20]. Previous studies have demonstrated that NNSVs can accommodate several foreign genes with an aggregate length of 4–5 kb [21, 22]. Vaccine vectors based on NNSVs have been shown to stably express exogenous genes for more than ten passages [9]. In addition to having the above advantages, CDV replicates well in African green monkey kidney (Vero) cells, a suitable substrate for vaccine production, and targets the immune system, meaning that CDV vectors can deliver their
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antigens directly to antigen-presenting cells [25]. Therefore, CDV has properties highly advantageous to a live vaccine vector. In the present study, we investigated whether immunization of mice with a CDV-based live recombinant rabies vaccine could elicit protective neutralizing antibody against CDV and rabies viral antigen as a bivalent vaccine.
Materials and methods Cells and viruses Vero cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) with 10 % fetal bovine serum (FBS, Gibco). Baby hamster kidney (BHK-21) cells and the stably transfected cell line BHK-T7 were maintained in the same medium with 5 % FBS, and 1 % penicillin– streptomycin (Gibco). An attenuated strain of canine distemper virus, CDV-L, was isolated from a mink by our laboratory [10]. CDV-L and the recombinant virus, rCDVRV-G, were propagated in Vero cells. RV Flury strain LEP and CVS-11 were kindly provided by Professor Rongliang Hu (Academy of Military Medical Sciences, Changchun). All experiments with RV were conducted in a biosafety level 3 laboratory facility at the Changchun Institute of Veterinary Science. Construction of an infectious virus clone CDV-L RNA was isolated from infected Vero cells using a QIAamp RNeasyÒ Mini Kit (Qiagen). Amplicons covering the entire viral genome were obtained from six reverse transcription PCRs (FL1–FL6) using CDV-specific oligonucleotides. A full-length cDNA clone of CDV-L was generated mainly by two steps using the In-Fusion PCR Cloning Kit (Clontech) to form plasmid p(?)CDV. The plasmid pBluescript II SK(?) (Stratagene) was chosen as the backbone and modified by replacing the multiple cloning sites and adjacent T3/T7 promoters by a very short polylinker carrying the restriction sites for BssHII, SnaBI, and MluI through PCR amplification (Fig. 1a). The T7 promoter sequence was fused to the leader region of the CDV antigenome by insertion directly into the forward primer. The hepatitis d-ribozyme sequence followed by the T7 termination signal was amplified from p(?)MV [18] (a kind gift from Dr. Agata Fazzio) and attached to the trailer region of the CDV antigenome by overlap extension PCR. The In-Fusion assembly on plasmid p(?)CDV was performed as follows. First, the modified vector was linearized by double digestion with BssHII and SnaBI, and PCR products of FL1, FL2, FL3 were inserted by one In-Fusion reaction to form an intermediate plasmid. Second, the
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intermediate plasmid was linearized with SnaBI and MluI, and FL4, FL5, FL6 were inserted by another In-Fusion reaction leading to the full-length antigenomic CDV cDNA clone p(?)CDV. Three helper vectors, pEMC-N, pEMC-P, and pEMC-L, expressing the CDV nucleocapsid (N), phosphoprotein (P), and large protein (L) were constructed. The coding sequence of the L gene of measles virus (MV) was excised from pEMC-La [18] (a kind gift from Dr. Agata Fazzio) and replaced by CDV N, P, and L genes individually. Genes of N and P were amplified by RT-PCR from CDV RNA, but the L gene was amplified directly from the fulllength CDV cDNA clone by PCR. A plasmid, p(?)CDV-RV-G, was constructed containing an additional transcription unit (ATU) for the G protein of the LEP-Flury strain of rabies virus. The fulllength G gene (1,575 nucleotides) was amplified by RTPCR from LEP-Flury genomic RNA using a forward primer containing the CDV intergenic trinucleotide, synthetic gene start (GS) and 50 UTR sequences of M gene, and the Kozak consensus sequence and a reverse primer containing synthetic 30 UTR and gene end (GE) sequences of P gene. The R333Q mutation in G protein was made by site-directed mutagenesis. The ATU was inserted in the P-M intergenic region at nucleotide position 3432 of the CDV genome. Cloning strategy for plasmid p(?)CDV-RV-G was similar to that for p(?)CDV. PCR products for FL1–FL4 fragments were inserted into the modified pBluscript II SK(?) vector by one In-Fusion reaction. The lengths of the cloned genomes respected the rule of six [11]. Generation of stable cell lines Plasmid pcDNA3.0-T7 expressing T7 RNA polymerase was constructed. BHK-21 cells at 80 % confluence in one 35-mm well were transfected with 4 lg pcDNA3.0T7. One day later, the cells were trypsinized and diluted to 1:1,000. Selection was initiated by addition of 0.6 mg/ ml G418 (Merck). After 2 weeks, cells were trypsinized and incubated in 96-well cell culture plates at a dilution giving 1 cell/100 ll/well. Upon formation of a clone, cells of the clone were trypsinized and transferred to a 35-mm well for screening. To monitor the functionality of T7 RNA polymerase, plasmid pEMC–EGFP was generated in which expression of the enhanced green fluorescent protein (EGFP) was under control of the T7 promoter; i.e., fluorescence was observable only in T7 RNA polymerase-positive cells. Levels of T7 RNA polymerase activity were determined by direct visualization of GFP expression using an inverted fluorescence microscope (Nikon) at low magnification (49). A highexpressing cell line, named BHK-T7, was selected.
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Fig. 1 Schematic representation of p(?)CDV and p(?)CDV-RV-G. Cloning strategy and the full-length cDNA clone. a The multiple cloning sites of pBS SK II and adjacent T3/T7 promoters were replaced by a very short polylinker-containing restriction sites for BssHII, SnaBI, and MluI through PCR amplification. b A full-length cDNA clone of CDV was generated mainly by two steps using the In-
Fusion method to form plasmid p(?)CDV. c Construction of plasmids p(?)CDV-RV-G in which the additional transcription unit (ATU) for RV-G was inserted. PCR products for F1–F4 fragments were inserted to the modified pBS SK II vector by one In-Fusion reaction. T7 T7 RNA polymerase promoter, d hepatitis delta virus ribozyme, Tu T7 RNA polymerase terminator
During the whole selection process, cells were grown in DMEM/5 % FBS with 0.6 mg/ml G418, with medium changes every second day.
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Virus rescue Transfection was carried out using FuGENEÒ HD Transfection Reagent (Promega) and BHK-T7 cells at about 50 % confluence in 35-mm plates. 10 ll FuGENEÒ HD Transfection Reagent was diluted in 100 ll of OptiMEM (Invitrogen), then 4 lg plasmids p(?)CDV or p(?)CDVRV-G together with three other helper plasmids (1 lg pEMC-N, 0.5 lg pEMC-P, and 1 lg pEMC-L) were then added, followed by incubation for 15 min at room temperature. The mixture was added dropwise to the cells. Two days later, the cells were trypsinized and divided among two 75-cm2 flasks. The syncytia were presented in cultures in a further 2–3 days. Then the cultures were maintained in DMEM/5 % FBS for 4 days after which time the virus was harvested. Infected cells were lysed by freezing and thawing, and clarified supernatants were used to infect monolayers of Vero cells either to grow virus stocks or to obtain total RNA for analysis.
Vero cells in 6-well plates were infected with CDV or rCDV-RV-G at a multiplicity of infection (MOI) of 0.01 for 1 h. The inocula were then removed and replaced with 2 ml DMEM/2 % FBS. Total virus (i.e., both cell-associated and supernatant) was collected daily for 5 days. The virus titers were determined by endpoint dilution assays to calculate 50 % tissue culture infectious dose (TCID50) values. Vero cells in 96-well cell culture plates were infected with the virus stocks in serial dilution (1:10-1 to 1:10-6). After 1 h, the inoculating mixture was removed and replaced with 200 ll DMEM/2 % FBS. Cultures were observed for cytopathic effects (CPE) at 5 days postinoculation (dpi). Indirect immunofluorescence assay Vero cells were grown in 24-well plates and infected with CDV or rCDV-RV-G at a MOI of 0.01. At 2 dpi, the cells were washed 39 with PBS and fixed in 4 % formaldehyde for 10 min at 4 °C. Cells were blocked in PBS containing 1 % (wt/vol) bovine serum albumin (BSA) at 37 °C for 30 min, then incubated for 1 h with mouse anti-RABV-G
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antibody (a kind gift from Dr. Rongliang Hu) at a dilution of 1:100 or mouse anti-CDV-N antibody (a kind gift from Dr. Yongshan Wang, Institute of veterinary medicine, JAAS) at a dilution of 1:100 and for another 1 h with fluorescein isothiocyanate (FITC)-conjugated goat antimouse antibody (MBL) at 1:5,000 dilution. Fluorescence was observed with a confocal laser scanning microscope (Nikon TE2000-E).
approximately 1 and 2 weeks later by the same route using double the first dose. Two weeks after the last dose was administered, blood was collected separately from all mice by the retro-orbital route. All animal experiments were performed following protocols approved by the State Key Laboratory of Agrobiotechnology Animal Study Proposal of China Agricultural University. VNA assay
Western blotting Vero cells were infected with CDV or rCDV-RV-G at a MOI of 0.01. Total virus was harvested at 4 days postinfection, freeze-thawed at -80 °C, and clarified by centrifugation at 4,0009g for 15 min. Supernatants were mixed with an equal volume of 29 SDS loading buffer [100 mM Tris–HCl (pH 6.8), 20 % glycerol, 4 % SDS, 200 mM dithiothreitol (DTT), 0.1 % bromophenol blue], heated at 95 °C for 10 min, and resolved by sodium dodecyl sulfate-12 % polyacrylamide gel (SDS-PAGE) electrophoresis. The gels were then transferred onto polyvinylidene difluoride (PVDF) membranes and incubated with mouse anti-RV-G antibody or mouse anti-CDVN antibody followed by goat anti-mouse secondary antibody labeled with horseradish peroxidase (HRP). Target proteins were visualized by an enhanced chemiluminescence (ECL, Thermo) Western blot detection system.
Sera were tested for the presence of neutralizing antibodies (VNA) by the rapid fluorescence inhibition test as described previously [23], and titers of RV-NAs were expressed in international units (IU/ml) as compared with a World Health Organization anti-RV antibody standard. CDV NA assessment was performed in Vero cells. Twofold dilutions of serum samples were preincubated with 100 TCID50 CDV for 1 h and then added to Vero cells in 96-well plates. Cultures were observed for CPE at 7 dpi. Endpoints were taken as the inverse of the highest dilution showing CPE, and titers of CDV NAs were calculated by the method of Reed and Muench [19].
Results Generation of recombinant CDV expressing the G protein of RV Flury strain LEP
Immuno-electron microscopy To examine incorporation of the RV G protein into rCDV-RV-G particles, CDV or rCDV-RV-G virions were adsorbed onto carbon-coated copper grids for 30 min. Grids were washed three times with PBS, then floated on a drop of RV G-specific mouse monoclonal antibody diluted to 1:100 in 1 % BSA/PBS for 30 min. After 39 washing with PBS, grids were incubated with goat antimouse IgG coupled to 10-nm gold particles (Sigma) diluted to 1:10 in 1 % BSA/PBS for 30 min. Grids were again 39 washed with PBS, stained with 1 % phosphotungstic acid, and examined using a JEOL 1230 transmission electron microscope. Animal studies To assess the immunogenicity of the recombinant viruses in mice, a three-dose vaccination schedule was performed. Three-week-old Kunming mice were divided into three groups of ten animals each. Two groups were injected intramuscularly (i.m.) with 100 ll 5 9 104 TCID50 CDV or rCDV-RV-G; the third group received 100 ll DMEM i.m. The second and third doses were administered
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Infectious recombinant CDV from a full-length cDNA clone p(?)CDV was rescued by a conventional T7 RNA polymerase-based system (Fig. 1b). A modified R333Q gene of RV LEP strain was inserted between the P and M genes of CDV, the recombinant virus, rCDV-RV-G, was recovered (Fig. 1c), and the insertion of the G gene was confirmed by RT-PCR analysis and sequencing. Expression of the LEP G protein was detected by immunostaining-infected Vero cells at 48 h postinoculation (hpi). Cells infected with rCDV-RV-G were stained by mouse monoclonal antibody against RV G, while CDVinfected cells were not stained (Fig. 2a). RV G expression by the recombinant viruses was also confirmed by Western blotting. Extracts of rCDV-RV-G-infected cells but not CDV-infected cells were stained with mouse monoclonal antibody against RV G (Fig. 2b). To see whether expressing a foreign viral glycoprotein altered the replication property of the CDV vector virus, the growth of recombinant virus rCDV-RV-G and CDV was analyzed in Vero cells. Results showed that the growth of rCDV-RV-G was slightly delayed compared to parental CDV and produced lower levels of infectious virus. CDV reached a peak titer of 105 TCID50/ml at 72 hpi, whereas
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Fig. 2 Expression of G protein from the 10th passage of recombinant rCDV-RV-G. a Immunofluorescence analysis of RV-G protein expression. Vero cells were infected with CDV or rCDV-RV-G, RV G expression was identified by IFA using mouse anti-RV-G
antibody, CDV N expression was identified using mouse anti-CDV-N antibody. b RV-G expression in rCDV-RV-G-infected Vero cells by Western blotting using mouse anti-RV-G antibody with CDV-infected cells as a negative control
rCDV-RV-G produced approximately half a log lower by 96 hpi (Fig. 3). To determine the stability of the G gene in the recombinant viruses, rCDV-RV-G was serially passaged 10 times in Vero cells. RT-PCR analysis and sequencing confirmed the presence of the G gene in the genome of CDV vector at the 10th passage. Stable expression of the protein was further confirmed by immunofluorescence and Western blotting. Identification of RV G incorporation into the surface of rCDV-RV-G particles
Fig. 3 Growth characterization of rCDV-RV-G and CDV in Vero cells. Growth characterization of rCDV-RV-G and CDV. Vero cells were infected with CDV or rCDV-RV-G at a MOI of 0.01. Viruses were collected every 24 h over a period of 5 days. Titers were determined in Vero cells expressed as TCID50
RV G is an envelope protein with the potential to be incorporated into the budding virus particle. By immunoelectron microscopy using RV G-specific antibody, RV G was found to be evenly distributed on the surface of rCDVRV-G. No G was detected in CDV particles (Fig. 4).
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Fig. 4 Incorporation of RV G into recombinant virus particles. Negative staining followed incubation with a mouse monoclonal antibody against RV G and a goat anti-mouse IgG conjugated with colloidal gold. Pictures were taken at a magnification of 40.0 K
Induction of anti-RV-neutralizing antibodies The immunogenicity of recombinant viruses was examined in mice. Following the three-dose vaccination series, mouse sera were collected for CDV and RV NA assays. Mice that received CDV or rCDV-RV-G displayed specific anti-CDV antibodies at a dilution of 1:8 (Fig. 5a). The mean titer of RV NA to the RV virus CVS-11 induced by rCDV-RV-G was 10.5 IU/ml (Fig. 5b), whereas no RV NA was detected in mice immunized with CDV. Neither was detected in the DMEM group.
Discussion In this study, we established a rescue system for a recombinant CDV, rCDV-RV-G. The full-length cDNA was generated using the In-Fusion PCR Cloning System. In-Fusion technology allows for insertion of multiple PCR products into a linearized vector seamlessly by a one-step In-Fusion reaction, providing that the ends of the insert share 15 bp of
Fig. 5 Immunogenicity of rCDV-RV-G in mice. Groups of 10 3-week-old mice were injected i.m. with 100 ll 5 9 104 TCID50 of CDV or rCDV-RV-G. The second and third doses were administered approximately 1 and 2 weeks later by the same route using double the
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homology with the ends of the vector [32]. This method makes the cloning fast and straightforward. Mostly, the PCR products do not need any restriction digestion and no changes are introduced, thereby maintaining the integrity of the entire CDV genome. This strategy would be also helpful to manipulate the genome of CDV like inserting any other fragments at any sites of the full genome of CDV. To develop this rCDV as a vector supporting expression of other genes, we generated recombinant CDV expressing the G protein of RV Flury strains LEP. G is the only envelope protein in rabies virus. It induces virus-neutralizing antibody and protects animals against subsequent RV infection [1, 5, 26]. As G is the major antigen of RV, various vaccines expressing G alone such as DNA-based rabies vaccines and live recombinant vectors have successfully elicited strong protective immune responses [6, 8, 28, 29]. A modified G may induce a stronger immune reaction: administration of a DNA vaccine expressing ERA G with R333Q resulted in a higher VNA than the unmodified G [16]. One important factor involved in the elevated immune response may be apoptosis which has been reported to contribute to the induction of immune responses [14]. G protein isolated from attenuated rabies viruses has been found to induce apoptosis [17]. Therefore, we incorporated the same R333Q substitution and evaluated its immunogenicity. After a three-dose vaccination, the recombinant viruses induced a strong neutralizing antibody response to RV. The mean titer of RV NA in rCDV-RV-G-vaccinated mice was 10.5 IU/ml, significantly exceeding the minimal acceptable protective level of antibody (0.5 IU/ml), and thereby providing evidence of effective vaccination. During the past decade, there are some successful rabies vaccine candidates based on recombinant viruses expressing
first dose. Two weeks after the third vaccination, blood was collected for assay of NA to CDV (a) and RV (b). RV NA titer was converted to international units (IU) by comparison with a WHO standard serum
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RV G [6, 8]. CDV also has shown its potential as an effective vaccine vector for RV [27]. We got the similar results. Furthermore, we firstly found out that the G protein was incorporated in rCDV-RV-G particles. This is probably an important factor for the protective level of RV-G antibody. Insertion of the G into the CDV envelope, mimicking the existing way of G in RV, may retain the immunogenicity to the maximal degree, not only the conserved neutralization epitopes in monomeric G proteins, but also the conformation needed to induce the high-affinity neutralizing antibodies [24]. It has been reported that insertion of foreign genes can have a negative impact on the growth of the virus vector [7, 12]. We found the same phenomenon in our study: the growth of CDV expressing the RV glycoproteins was slightly delayed. The parental virus CDV reached a peak titer by 72 hpi, while rCDV-RV-G required 96 hpi to attain this. Nevertheless, slower replication of the CDV vector did not appear to have any significant influence on its immunogenicity as CDV and rCDV-RV-G elicited similar NA titers against CDV in vaccinated mice. We developed a replication-competent CDV vector for delivering membrane-bound RV G. Safety is a major concern for a live vector vaccine. No clinical symptom was observed in the group of mice inoculated with rCDVRV-G, which demonstrated that expression of RV-G in the CDV vector did not result in an increase of virulence. The fact that immunizing the mice with rCDV-RV-G results in the production of antibodies against RV demonstrates the potential of rCDV-RV-G as a vaccine that may be used to control rabies virus infection in dogs and other animals. Acknowledgments We thank Dr. Agata Fazzio for the generous donation of plasmid vectors. This work was supported by 863 Project (2011AA10A213).
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