MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY Volume 33, Number 2, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/mab.2013.0087
Efficient Generation of Monoclonal Antibodies Against Major Structural Proteins of Rabies Virus with Suckling Mouse Brain Antigen Jinyang Zhang,1,3* Xizhen Ruan,1,2* Jie Zan,1,2 Xiaojuan Zheng,1,2 Yan Yan,1,2 Min Liao,1,2 and Jiyong Zhou1,2
The rabies virus is a neurotropic virus that causes fatal disease in humans and animals. However, not all commercial antibodies against rabies virus (RABV) structural proteins are generally available, and production of high-quality monoclonal antibodies (MAbs) requires high purification of virus particles and special facilities and is time-consuming. By using RABV-infected suckling mouse brain as antigens in this study, 11 hybridoma cells secreting MAbs against RABV were obtained, which showed strong reactivity with RABV-infected Vero cells in immunofluorescence assay. Among the 11 MAbs, three MAbs (1B11, 1C8, and 8H12) showed a neutralizing effect to RABV, while MAb 4B7 recognized the recombinant nucleoprotein (N) of RABV expressed in Vero cells; seven MAbs (1H3, 3H7, 4E7, 4G3, 5A10, 6C9, and 7B3) reacted specifically with phosphoprotein (P) of RABV. The MAbs developed in this study will be useful in establishing a diagnostic test and study on the interactions between RABV and its host.
proteins expressed in Escherichia coli.(9) This process is time-consuming and requires complex equipment, such as ultracentrifuge and gene transfer materials. In this study, we attempt to directly use the suspension of RABV-infected suckling mouse brain as immunogen to prepare MAbs against RABV. The BALB/c mice were immunized with RABVinfected suckling mouse brain suspension, and then the splenic cells of the immunized mouse were fused with SP 2/0 cells. Finally 11 hybridoma cell lines producing MAbs against RABV were successfully obtained, including one MAb against N protein, three neutralizing antibodies, and seven anti-phosphoprotein antibodies. Here we report an efficient MAb-producing strategy with suckling mouse brain as antigen for the development of anti-rabies MAbs.
Introduction
R
abies is a deadly neuroviral disease that kills over 55,000 individuals worldwide every year. The etiological agent is rabies virus (RABV). RABV, a non-segmented, negative-strand RNA virus, is a member of the Rhabdoviridae family. The viral genome encodes five structural proteins: the glycoprotein (G), nucleoprotein (N), matrix protein (M), phosphoprotein (P), and RNA-dependent RNA polymerase (L). The most widely used analytical techniques for detection of rabies antigens are fluorescent antibody test (FAT), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry. However, all of these methods depend strongly upon the specific antibodies against RABV.(1,2) Not all commercial monoclonal antibodies (MAbs) specific for RABV structural proteins are generally available. To investigate the role of RABV structural proteins in the process of host-virus interaction and to develop various diagnostic techniques, it is necessary to develop different types of antibodies against viral proteins of RABV. Since the first MAb against RABV was produced in 1978,(3) numerous MAbs have been developed including human MAbs to RABV.(4–10) However, most of the antigens used to prepare MAb were from purified virus or recombinant
Materials and Methods Virus strains and cells
RABV strain ERA and Vero cells were maintained in our laboratory. Vero cells were propagated in minimal essential medium supplemented with 10% fetal calf serum, 100,000 U/L penicillin, and 100,000 mg/L streptomycin. The mouse neuroblastoma N2a cells were kindly provided by Prof. Xiaofeng Guo (South China Agricultural University, Guangzhou, P.R.
1
Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou, P.R. China. State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University, Hangzhou, P.R. China. 3 Current address, Research Center of Molecular Medicine of Yunnan Province, Kunming University of Science and Technology, Kunming, P.R. China. *These authors contributed equally to this work. 2
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Table 1. Characterization of MAbs to RABV Using Different Methods MAbs
Isotype
Target protein
Neutralization activity
pCI-P reactivity
pCI-N reactivity
Titer ( · 104)
Western blot
IFA
1B11 1C8 8H12 4B7 1H3 3H7 4E7 4G3 5A10 6C9 7B3
IgG2b/k IgG1/l IgM/k IgG2b/k IgG2b/k IgA/k IgG2a/k IgG2a/l IgG1/k IgG2a/k IgG1/k
ND ND ND N P P P P P P P
+ + + -
+ + + + + + +
+ -
3.2 10.24 6.4 1.6 5.12 25.6 20.48 5.12 40.96 40.96 12.8
+ + + + +
+ + + + + + + + + + +
ND, not detected. + , positive reaction; - , negative reaction; N, nucleoprotein; P, phosphoprotein.
China). SP2/0 myeloma cell line was routinely maintained in our laboratory. The recombinant viral protein N was prepared according to the method described previously.(11) Virus propagation and preparation
The N2a cells were cultured in DMEM medium (Gibco/ Invitrogen, Carlsbad, CA). When the cells reached approximately 90% confluence in the culture flask, RABV virus was inoculated at a multiplicity of infection (MOI) of 0.1. After being cultured 3–4 days in DMEM supplemented with 2% fetal calf serum, the infected cells were freeze-thawed three times. The cellular lysates were centrifuged at 12,000 g for 15 min to remove the cell debris. The virus suspension could be used as seed virus for mouse inoculation or for further purification. For further purification of the virus, the cell supernatant was inactivated with b-propiolactone and then ultra-centrifugated at 180,000 g for 2 h at 4C to collect the virus pellet. The virus pellet was diluted in the 0.01 M PBS and loaded onto AKTA SuperdexTM 200 at 0.2 mL/min, and the first fraction containing the majority of the intact virions was collected. The purified virus was aliquoted and stored at - 80C, which was used as antigen to characterize MAbs and to confirm the specificity of MAbs for RABV.
method.(12) After subcloning procedure, stable antibodyproducing clones were obtained. Prior to commencement of the study, all experimental protocols that involved animals had received approval by the Scientific and Ethical Committee of the Zhejiang University (P.R. China). Determination of MAb neutralizing capacity
The neutralizing activity of the MAbs was evaluated by the fluorescent focus inhibition test as previously described with some modifications.(13) The supernatant of each MAb was made two-fold serial dilutions and mixed with equal amounts of ERA virus suspension (100 TCID50), and then the mixtures were incubated in 5% CO2 at 37C for 1.5 h. The SP2/0 cell culture supernatant was also mixed with equal amounts of virus as a negative control. After incubation, the mixtures were added to 96-well plates seeded with Vero cell monolayers; then the cells were cultured in 5% CO2 at 37C for 24– 48 h, fixed and stained with anti-RABV N MAb previously prepared in our laboratory(14) for 2 h. The plate was observed under an inverted fluorescence microscope for IFA analysis. Calculation of virus-neutralizing antibody titers was carried out according to standard methodology.(15) Screening of anti-RABV N antibodies
Preparation of immunogen and mouse monoclonal hybridoma cell lines
Three-day-old BALB/c mice were inoculated with virus suspension by intracranial injection. At 7 days post-inoculation, the brain mass was harvested. The brain mass was ground to suspension in MEM media, and centrifuged at 2500 g for 15 min at 4C. The supernatant was inactivated by ultraviolet radiation, as an immunogen, and then injected in 6- to 8-weekold female BALB/c mice. For the first immunization, equal volume of immunogen and Freund’s complete adjuvant (SigmaAldrich, St. Louis, MO) was hypodermically injected into mice. The equal dose of antigen in incomplete Freund’s adjuvant (Sigma-Aldrich) was injected into mice on days 14 and 28 after primary immunization, respectively. The equal antigen suspension mixed with saline solution was injected into mice on day 42 for the boost. The mouse immune response was detected by immunofluorescence assay (IFA). The mouse splenocytes were harvested and fused with myeloma cells SP2/0 using 50% (v/v) PEG4000 according to the conventional
The antibodies were screened by indirect ELISA with recombinant protein N expressed in E. coli as coating antigen. The procedures were carried out as described previously(16–18) with some modifications. The 96-well plates were coated with 2 mg/mL of protein N and blocked with 5% skimmed milk. Then supernatants of all MAbs were added to the plates to incubate for 2 h and were subsequently incubated with horseradish peroxidase-labeled goat anti-mouse IgG at 1:104 for 1 h. After washing five times, tetramethylbenzidine substrate solution was added, followed by termination of the reaction with 2 M H2SO4. Absorbances of each well were measured at 450 nm. Immunodot assay and Western blot analysis
Five mL purified virus, normal cell supernatant, and virusand mock-infected Vero cell lysates were dotted onto the nitrocellulose membranes (AP Biotech, Little Chalfont, United Kingdom), respectively. The cell lysates were prepared
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FIG. 1. Reactivity of anti-RABV MAbs in RABV-infected Vero cells (320x). Cells were infected with RABV ERA strain. At 48 h post-infection, cells were fixed with ice-cold acetone/methanol (1/1) and signals were detected by MAbs followed by FITC-coupled goat anti-mouse secondary antibody. Mock-infected cells were used as negative control. by urea lysis buffer as described by Zheng and colleagues.(19) After air-drying, sample-blotted membranes were blocked with 5% skim milk and then incubated with MAbs at the ratio of 1:104. The color reactions were developed with substrate 3-amino-9-ethylcarbazole (AEC) staining system (Santa Cruz Biotechnology, Santa Cruz, CA) according to the manufacturer’s instructions. SDS-PAGE and Western blot analysis were performed as described previously,(20) with some modifications. Briefly, the purified virus or cell lysates were fractionated by 10% SDS-polyacrylamide gel electrophoresis and electrotransferred to a supported nitrocellulose membrane (AP Biotech). The membranes were blocked with 5% skim milk and incubated with MAbs and horseradish peroxidaseconjugated goat anti-mouse IgG. Finally the blots were incubated with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Rockford, IL).
Plasmid construction and transfections
The complete P gene and N gene of RABV strain ERA (GenBank: EF206707) were amplified by PCR using specific primer pairs: P1: 5¢-GCCGAATTCATGAGCAAGAT CTTTGTC-3¢ and P2: 5¢-GACGTCGACTTAGCAA GATG TATAGCG-3¢; and N1: 5¢-GAACTCGAGATGGATGCCG ACAAGATT-3¢ and N2: 5¢-GCTCTAGATTATGAGTCAC TCGAATACGTT-3¢, respectively, and cloned into the pCIneo vector using the EcoR I/Sal I and Xho I/Xba I restriction sites within the multicloning site. The resultant plasmids were confirmed by sequence determination. One day prior to transfection, N2a cells were seeded into 96-well plates (Corning, Corning, NY). Then, according to the manufacturer’s instructions, the resultant vectors were transfected by Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) and expressed transiently in the N2a cells. N2a cells transfected with the pCI-neo vector without inserted DNA were used as negative controls. Two days later, cells were washed with phosphate-buffered saline and fixed with ice-cold acetone/ methanol (1/1) for subsequent use in IFA. Results Generation of monoclonal antibody to RABV
FIG. 2. Western blot analysis of five MAbs with purified RABV. Lane 1 reacted with mouse MAb 4B7; lane 2 reacted with MAb 3H7; lane 3 reacted with MAb 4E7; lane 4 reacted with MAb 6C9; lane 5 reacted with MAb 7B3.
In IFA, the serum from the mouse immunized with RABVinfected suckling mouse brain antigen reacted well with the RABV-infected Vero cells but displayed no reaction with mock-infected Vero cells (data not shown); therefore the IFA is the optional test for anti-RABV MAbs screening. In all, 288 hybridoma clones were screened in RABV-infected Vero cells by IFA, and more than 120 showed positive reactions. However, after three subclonings, only 11 hybridoma clones persistently secreted antibodies reacting with RABV-infected Vero cells
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immunodot assay, every MAb could react with the RABV antigen effectively (Fig. 3, column 1) and none of these MAbs reacted with the normal Vero cell supernatant (Fig. 3, col 2) and mock-infected cells lysis sample (Fig. 3, col 4). Moreover, not all of the MAbs reacted with the virus-infected cell sample treated by urea lysis buffer (Fig. 3, col 3). No reaction was observed for MAbs 1B11 and 8H12. These results indicating that MAbs 1B11 and 8H12 might recognize the conformational epitopes but not the linear epitopes, which are relatively vulnerable so that lysis buffer is sufficient to destroy the conformation epitopes recognized by MAbs 1B11 and 8H12. Identification of MAbs against N protein of RABV
In the first screening, approximately one third of clones (19 out of 62 tested) positively reacted with viral protein N. The fluorescent antibody staining showed that the viral protein N was mainly focused in the Negri body-like structures, which was consistent with previous report.(14,21) Finally, only one MAb 4B7 was found to react with recombinant viral protein N expressed in E. coli in indirect ELISA, which also recognized the N protein of RABV sample in Western blotting (Fig. 2), indicating that 4B7 recognized the linear epitope. To further characterize the MAb against N protein, we constructed the eukaryotic expression vector pCI-N and transfected into the Vero cells. As shown in Figure 4, MAb 4B7 showed positive reaction with pCI-N transfected cells by IFA, while no signals were observed for pCI-neo transfected cells. Neutralizing activity of MAbs
The neutralizing activity of MAbs to RABV was evaluated by fluorescent focus inhibition test in 96-well cell plates. The MAbs 1B11, 1C8, and 8H12 were capable of neutralizing the infectivity of RABV strain ERA in Vero cells (Fig. 5), whose neutralizing titers were 1:256, 1:128, and 1:256, respectively. As negative control, the SP2/0 cell culture supernatant and the medium showed no inhibitory activity against the replication of RABV by fluorescent focus inhibition assay. The data showed that the MAbs 1B11, 1C8, and 8H12 have a neutralizing activity to RABV strain ERA.
FIG. 3. Reactivity of MAbs with antigen by immunodot blotting. Solutions containing virus or control were spotted in a volume of 10 mL onto nitrocellulose. The dots were then subjected to different MAb for immunostaining. Column 1, purified rabies virus preparation with gel chromatography; 2, multiple freeze-thawed normal Vero cell supernatant; 3, total protein of virus infected cells lysed by lysis buffer; 4, total protein mock-infected cells lysates. (Fig. 1). The characterization of the 11 MAbs specifically reacting with RABV antigens is summarized in Table 1. Data also demonstrated that fluorescent signals stained by different MAbs in RABV-infected Vero cells are different (Fig. 1). Reactivity of MAbs with RABV-encoding proteins in Western blot and immunodot analyses
Western blot analysis was carried out to identify the specificity of MAbs to RABV proteins. The results showed that only MAbs 3H7, 4B7, 4E7, 6C9, and 7B3 could react with RABV proteins in a Western blot analysis (Fig. 2) while in the
Identification of monoclonal antibodies to P protein of RABV
The full length of P gene of RABV is 605 bp. For directional cloning of the P gene into the pCI-neo mammalian expression vector, restriction enzyme sites EcoR I and Sal I were incorporated into two primers designed to amplify the full length of P gene. The P gene was amplified successfully and digested with EcoR I and Sal I restriction enzymes and inserted to the pCI-neo plasmid. The pCI-neo-P recombinant expression vector was verified by PCR and restriction enzyme digestion (data not shown). The pCI-neo-P was introduced to N2a cells for transient P protein expression. In addition to 3H7, 4E7, 6C9, and 7B3, three conformational antibodies 1H3, 4G3, and 5A10 showed strong reaction with P protein in transfected cells (Fig. 6). Discussion
Monoclonal antibodies are valuable tools for characterizing antigenic variation and specific epitopes. The MAbs recognizing the conformational epitopes are especially
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FIG. 4. Characterization of anti-N RABV MAb by immunofluorescence assay. Vero cells were transfected with pCI-N eukaryotic expression plasmid. At 48 h post-transfection, cells were fixed with ice-cold acetone/methanol (1/1), reacted with MAb 4B7, followed by goat anti-mouse IgG labeled with FITC. important for understanding virus maturation and export as well as biological processes. The intrinsic quality of immunogen is one of the critical factors for antibody production.(22) Antibody development is time-consuming work, when there is a need to produce a series of antibodies. In MAb development, if the antigen was not purified sufficiently before immunization, it is very difficult to screen a good cell line without a highly purified detecting antigen. The method we used to prepare the immunogen in this study is not only an efficient way to induce a strong immune response, but also could achieve the goal of producing several kinds of MAbs by one cell fusion. As one of the key structural proteins, protein N of RABV is a super-antigen and possesses adjuvant properties.(23) In our experiment, even 5 months after the third immunization with suckling mouse brain suspension, the anti-N antibody titer in mouse serum was still 1:1600 (data not shown), and at the first screening there were many positive clones. However, we had very limited MAb cell lines against N after the third
subcloning in this experiment, most likely because it is only a probability event in the process of subcloning and screening. Thus this antigen could be effectively used to prepare the antibody against the protein N. The panel of MAbs we produced may recognize different epitopes; their characteristics were summarized in Table 1. Some of the MAbs were proven to recognize linear epitopes (3H7, 4B7, 4E7, 6C9, and 7B3) as shown in Figure 2, and others were specific for conformation-dependent epitopes (1B11, 1C8, 1H3, 4G3, 5A10, and 8H12) as shown in Figure 6; especially for MAbs 1B11 and 8H12, the epitope is much more vulnerable. The lysis buffer containing urea may disrupt the epitopes’ conformation of the protein in infected cells, which was recognized by 1B11 and 8H12. In this experiment, we found MAbs 1B11, 1C8, and 8H12 could significantly inhibit the virus infection, and all of them recognized the conformational epitopes. We suspect that these MAbs may react with G of RABV. However we failed to characterize them by eukaryotic expressing G recombinant
FIG. 5. Inhibition of rabies virus infection by neutralizing MAbs in Vero cells (320x). Vero cells were infected with the antibody-virus mixtures. After incubation for 48 h, the infectivity of the virus in the mixture was evaluated, as described in the Materials and Methods section. Supernatant of the SP2/0 culture was used as a control.
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FIG. 6. IFA showing that MAbs 1H3, 3H7, 4E7, 4G3, 5A10, 6C9, and 7B3 reacted specifically to phosphoprotein of RABV (320 · ). The nuclei of the cells were stained with DAPI.
protein. For this to occur, the expression condition or gene sequence might need to be optimized. Conclusion
The brain suspension of RABV-infected suckling mice as immunizing antigen for preparation of MAb to RABV was evaluated in this study. The antigen presents a similar immunogenicity to that of recombinant protein expressed in E. coli. The strategy for MAb production is rapid and efficient, and it is beneficial to the production of conformational antibodies. This approach may be an ideal strategy for highthroughput MAb production.
3.
4.
5.
Acknowledgments
This work was supported by the National Special Fund for Public Welfare Industry (project no. 201103032), the National High-Tech Program of China (2012AA101303), and the National Key Technologies Research and Development Program of China (2010BAD04B01).
6.
7. Author Disclosure Statement
The authors have no financial interests to disclose. References
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antibodies to G5, a linear neutralization epitope on glycoprotein of rabies virus, by phage display technology. Microbiol Immunol 2008;52:89–93. Wiktor TJ, and Koprowski H: Monoclonal antibodies against rabies virus produced by somatic cell hybridization: detection of antigenic variants. Proc Natl Acad Sci USA 1978;75:3938–3942. Nadin-Davis SA, Fehlner-Gardiner C, Sheen M, and Wandeler AI: Characterization of a panel of anti-phosphoprotein monoclonal antibodies generated against the raccoon strain of rabies virus. Virus Res 2010;152:126–136. Zanluca C, Aires LRD, Mueller PP, dos Santos VV, Carrieri ML, Pinto AR, and Zanetti CR: Novel monoclonal antibodies that bind to wild and fixed rabies virus strains. J Virol Methods 2011;175:66–73. Champion JM, Kean RB, Rupprecht CE, Notkins AL, Koprowski H, Dietzschold B, and Hooper DC: The development of monoclonal human rabies virus-neutralizing antibodies as a substitute for pooled human immune globulin in the prophylactic treatment of rabies virus exposure. J Immunol Methods 2000;235:81–90. Dorfman N, Dietzschold B, Kajiyama W, Fu ZF, Koprowski H, and Notkins AL: Development of human monoclonal-antibodies to rabies. Hybridoma 1994;13: 397–402. Lafon M, Edelman L, Bouvet JP, Lafage M, and Montchatre E: Human monoclonal-antibodies specific for the rabies virus glycoprotein and N protein. J Gen Virol 1990;71:1689–1696. Wang WJ, Li X, Wang LH, Shan H, Cao L, Yu PC, Tang Q, and Liang GD: Preparation and identification of antirabies virus monoclonal antibodies. Virol Sinica 2012;27: 172–178.
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10. Turki I, Hammami A, Kharmachi H, and Mousli M: Engineering of a recombinant trivalent single-chain variable fragment antibody directed against rabies virus glycoprotein G with improved neutralizing potency. Mol Immunol 2013;57:66–73. 11. Cai YQ MYP, Shen HG, Guo JQ, and Zhou JY: High level expression of nucleoprotein gene of rabies virus in Escherichia coli. Vet Sci China 2007;37:145–149. 12. Ko¨hler G, and Milstein C: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:495–497. 13. Khawplod P, Inoue K, Shoji Y, Wilde H, Ubol S, Nishizono A, Kurane I, and Morimoto K: A novel rapid fluorescent focus inhibition test for rabies virus using a recombinant rabies virus visualizing a green fluorescent protein. J Virol Methods 2005;125:35–40. 14. Zhang J, Wu X, Zan J, Wu Y, Ye C, Ruan X, and Zhou J: Cellular chaperonin CCTgamma contributes to rabies virus replication during infection. J Virol 2013;87:7608–7621. 15. Smith JS, Yager PA, and Baer GM: A rapid fluorescent focus inhibition test (RFFIT) for determining rabies virusneutralizing antibody. In: Laboratory Techniques in Rabies, 4th ed. Meslin F-X, Kaplan MM, and Koprowski H, Eds. World Health Organization, Geneva, 1996, pp. 181–191. 16. Zhang J, Li P, Zhang W, Zhang Q, Ding X, Chen X, Wu W, and Zhang X: Production and characterization of monoclonal antibodies against aflatoxin g(1). Hybridoma 2009;28:67–70. 17. Zhang X, Ma G, Li Y, Jiang X, He J, and Zhou J: Characterization of monoclonal antibody against replicationassociated protein of porcine circovirus. DNA Cell Biol 2009;28:23–29. 18. Zheng X, Hong L, Li Y, Guo J, Zhang G, and Zhou J: In vitro expression and monoclonal antibody of RNA-dependent RNA polymerase for infectious bursal disease virus. DNA Cell Biol 2006;25:646–653.
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19. Zheng X, Hong L, Shi L, Guo J, Sun Z, and Zhou J: Proteomics analysis of host cells infected with infectious bursal disease virus. Mol Cell Proteomics 2008;7: 612–625. 20. Zhou JY, Shang SB, Gong H, Chen QX, Wu JX, Shen HG, Chen TF, and Guo JQ: In vitro expression, monoclonal antibody and bioactivity for capsid protein of porcine circovirus type II without nuclear localization signal. J Biotechnol 2005;118:201–211. 21. Blondel D, Lahaye X, Vidy A, Pomier C, Obiang L, Harper F, and Gaudin Y: Functional characterization of Negri bodies (nbs) in rabies virus-infected cells: evidence that NBS are sites of viral transcription and replication. J Virol 2009;83:7948–7958. 22. Pata S, Tayapiwatana C, and Kasinrerk W: Three different immunogen preparation strategies for production of CD4 monoclonal antibodies. Hybridoma 2009;28:159–165. 23. Astoul E, Lafage M, and Lafon M: Rabies superantigen as a Vbeta T-dependent adjuvant. J Exp Med 1996;183:1623– 1631.
Address correspondence to: Jiyong Zhou Key Laboratory of Animal Virology of Ministry of Agriculture Zhejiang University 388 Yuhangtang Road Hangzhou 310058 P.R. China E-mail: [email protected] [email protected] Received: December 3, 2013 Accepted: January 16, 2014
Efficient Generation of Monoclonal Antibodies Against Major Structural Proteins of Rabies Virus with Suckling Mouse Brain Antigen Jinyang Zhang,1,3* Xizhen Ruan,1,2* Jie Zan,1,2 Xiaojuan Zheng,1,2 Yan Yan,1,2 Min Liao,1,2 and Jiyong Zhou1,2
The rabies virus is a neurotropic virus that causes fatal disease in humans and animals. However, not all commercial antibodies against rabies virus (RABV) structural proteins are generally available, and production of high-quality monoclonal antibodies (MAbs) requires high purification of virus particles and special facilities and is time-consuming. By using RABV-infected suckling mouse brain as antigens in this study, 11 hybridoma cells secreting MAbs against RABV were obtained, which showed strong reactivity with RABV-infected Vero cells in immunofluorescence assay. Among the 11 MAbs, three MAbs (1B11, 1C8, and 8H12) showed a neutralizing effect to RABV, while MAb 4B7 recognized the recombinant nucleoprotein (N) of RABV expressed in Vero cells; seven MAbs (1H3, 3H7, 4E7, 4G3, 5A10, 6C9, and 7B3) reacted specifically with phosphoprotein (P) of RABV. The MAbs developed in this study will be useful in establishing a diagnostic test and study on the interactions between RABV and its host.
proteins expressed in Escherichia coli.(9) This process is time-consuming and requires complex equipment, such as ultracentrifuge and gene transfer materials. In this study, we attempt to directly use the suspension of RABV-infected suckling mouse brain as immunogen to prepare MAbs against RABV. The BALB/c mice were immunized with RABVinfected suckling mouse brain suspension, and then the splenic cells of the immunized mouse were fused with SP 2/0 cells. Finally 11 hybridoma cell lines producing MAbs against RABV were successfully obtained, including one MAb against N protein, three neutralizing antibodies, and seven anti-phosphoprotein antibodies. Here we report an efficient MAb-producing strategy with suckling mouse brain as antigen for the development of anti-rabies MAbs.
Introduction
R
abies is a deadly neuroviral disease that kills over 55,000 individuals worldwide every year. The etiological agent is rabies virus (RABV). RABV, a non-segmented, negative-strand RNA virus, is a member of the Rhabdoviridae family. The viral genome encodes five structural proteins: the glycoprotein (G), nucleoprotein (N), matrix protein (M), phosphoprotein (P), and RNA-dependent RNA polymerase (L). The most widely used analytical techniques for detection of rabies antigens are fluorescent antibody test (FAT), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry. However, all of these methods depend strongly upon the specific antibodies against RABV.(1,2) Not all commercial monoclonal antibodies (MAbs) specific for RABV structural proteins are generally available. To investigate the role of RABV structural proteins in the process of host-virus interaction and to develop various diagnostic techniques, it is necessary to develop different types of antibodies against viral proteins of RABV. Since the first MAb against RABV was produced in 1978,(3) numerous MAbs have been developed including human MAbs to RABV.(4–10) However, most of the antigens used to prepare MAb were from purified virus or recombinant
Materials and Methods Virus strains and cells
RABV strain ERA and Vero cells were maintained in our laboratory. Vero cells were propagated in minimal essential medium supplemented with 10% fetal calf serum, 100,000 U/L penicillin, and 100,000 mg/L streptomycin. The mouse neuroblastoma N2a cells were kindly provided by Prof. Xiaofeng Guo (South China Agricultural University, Guangzhou, P.R.
1
Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou, P.R. China. State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University, Hangzhou, P.R. China. 3 Current address, Research Center of Molecular Medicine of Yunnan Province, Kunming University of Science and Technology, Kunming, P.R. China. *These authors contributed equally to this work. 2
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Table 1. Characterization of MAbs to RABV Using Different Methods MAbs
Isotype
Target protein
Neutralization activity
pCI-P reactivity
pCI-N reactivity
Titer ( · 104)
Western blot
IFA
1B11 1C8 8H12 4B7 1H3 3H7 4E7 4G3 5A10 6C9 7B3
IgG2b/k IgG1/l IgM/k IgG2b/k IgG2b/k IgA/k IgG2a/k IgG2a/l IgG1/k IgG2a/k IgG1/k
ND ND ND N P P P P P P P
+ + + -
+ + + + + + +
+ -
3.2 10.24 6.4 1.6 5.12 25.6 20.48 5.12 40.96 40.96 12.8
+ + + + +
+ + + + + + + + + + +
ND, not detected. + , positive reaction; - , negative reaction; N, nucleoprotein; P, phosphoprotein.
China). SP2/0 myeloma cell line was routinely maintained in our laboratory. The recombinant viral protein N was prepared according to the method described previously.(11) Virus propagation and preparation
The N2a cells were cultured in DMEM medium (Gibco/ Invitrogen, Carlsbad, CA). When the cells reached approximately 90% confluence in the culture flask, RABV virus was inoculated at a multiplicity of infection (MOI) of 0.1. After being cultured 3–4 days in DMEM supplemented with 2% fetal calf serum, the infected cells were freeze-thawed three times. The cellular lysates were centrifuged at 12,000 g for 15 min to remove the cell debris. The virus suspension could be used as seed virus for mouse inoculation or for further purification. For further purification of the virus, the cell supernatant was inactivated with b-propiolactone and then ultra-centrifugated at 180,000 g for 2 h at 4C to collect the virus pellet. The virus pellet was diluted in the 0.01 M PBS and loaded onto AKTA SuperdexTM 200 at 0.2 mL/min, and the first fraction containing the majority of the intact virions was collected. The purified virus was aliquoted and stored at - 80C, which was used as antigen to characterize MAbs and to confirm the specificity of MAbs for RABV.
method.(12) After subcloning procedure, stable antibodyproducing clones were obtained. Prior to commencement of the study, all experimental protocols that involved animals had received approval by the Scientific and Ethical Committee of the Zhejiang University (P.R. China). Determination of MAb neutralizing capacity
The neutralizing activity of the MAbs was evaluated by the fluorescent focus inhibition test as previously described with some modifications.(13) The supernatant of each MAb was made two-fold serial dilutions and mixed with equal amounts of ERA virus suspension (100 TCID50), and then the mixtures were incubated in 5% CO2 at 37C for 1.5 h. The SP2/0 cell culture supernatant was also mixed with equal amounts of virus as a negative control. After incubation, the mixtures were added to 96-well plates seeded with Vero cell monolayers; then the cells were cultured in 5% CO2 at 37C for 24– 48 h, fixed and stained with anti-RABV N MAb previously prepared in our laboratory(14) for 2 h. The plate was observed under an inverted fluorescence microscope for IFA analysis. Calculation of virus-neutralizing antibody titers was carried out according to standard methodology.(15) Screening of anti-RABV N antibodies
Preparation of immunogen and mouse monoclonal hybridoma cell lines
Three-day-old BALB/c mice were inoculated with virus suspension by intracranial injection. At 7 days post-inoculation, the brain mass was harvested. The brain mass was ground to suspension in MEM media, and centrifuged at 2500 g for 15 min at 4C. The supernatant was inactivated by ultraviolet radiation, as an immunogen, and then injected in 6- to 8-weekold female BALB/c mice. For the first immunization, equal volume of immunogen and Freund’s complete adjuvant (SigmaAldrich, St. Louis, MO) was hypodermically injected into mice. The equal dose of antigen in incomplete Freund’s adjuvant (Sigma-Aldrich) was injected into mice on days 14 and 28 after primary immunization, respectively. The equal antigen suspension mixed with saline solution was injected into mice on day 42 for the boost. The mouse immune response was detected by immunofluorescence assay (IFA). The mouse splenocytes were harvested and fused with myeloma cells SP2/0 using 50% (v/v) PEG4000 according to the conventional
The antibodies were screened by indirect ELISA with recombinant protein N expressed in E. coli as coating antigen. The procedures were carried out as described previously(16–18) with some modifications. The 96-well plates were coated with 2 mg/mL of protein N and blocked with 5% skimmed milk. Then supernatants of all MAbs were added to the plates to incubate for 2 h and were subsequently incubated with horseradish peroxidase-labeled goat anti-mouse IgG at 1:104 for 1 h. After washing five times, tetramethylbenzidine substrate solution was added, followed by termination of the reaction with 2 M H2SO4. Absorbances of each well were measured at 450 nm. Immunodot assay and Western blot analysis
Five mL purified virus, normal cell supernatant, and virusand mock-infected Vero cell lysates were dotted onto the nitrocellulose membranes (AP Biotech, Little Chalfont, United Kingdom), respectively. The cell lysates were prepared
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FIG. 1. Reactivity of anti-RABV MAbs in RABV-infected Vero cells (320x). Cells were infected with RABV ERA strain. At 48 h post-infection, cells were fixed with ice-cold acetone/methanol (1/1) and signals were detected by MAbs followed by FITC-coupled goat anti-mouse secondary antibody. Mock-infected cells were used as negative control. by urea lysis buffer as described by Zheng and colleagues.(19) After air-drying, sample-blotted membranes were blocked with 5% skim milk and then incubated with MAbs at the ratio of 1:104. The color reactions were developed with substrate 3-amino-9-ethylcarbazole (AEC) staining system (Santa Cruz Biotechnology, Santa Cruz, CA) according to the manufacturer’s instructions. SDS-PAGE and Western blot analysis were performed as described previously,(20) with some modifications. Briefly, the purified virus or cell lysates were fractionated by 10% SDS-polyacrylamide gel electrophoresis and electrotransferred to a supported nitrocellulose membrane (AP Biotech). The membranes were blocked with 5% skim milk and incubated with MAbs and horseradish peroxidaseconjugated goat anti-mouse IgG. Finally the blots were incubated with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Rockford, IL).
Plasmid construction and transfections
The complete P gene and N gene of RABV strain ERA (GenBank: EF206707) were amplified by PCR using specific primer pairs: P1: 5¢-GCCGAATTCATGAGCAAGAT CTTTGTC-3¢ and P2: 5¢-GACGTCGACTTAGCAA GATG TATAGCG-3¢; and N1: 5¢-GAACTCGAGATGGATGCCG ACAAGATT-3¢ and N2: 5¢-GCTCTAGATTATGAGTCAC TCGAATACGTT-3¢, respectively, and cloned into the pCIneo vector using the EcoR I/Sal I and Xho I/Xba I restriction sites within the multicloning site. The resultant plasmids were confirmed by sequence determination. One day prior to transfection, N2a cells were seeded into 96-well plates (Corning, Corning, NY). Then, according to the manufacturer’s instructions, the resultant vectors were transfected by Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) and expressed transiently in the N2a cells. N2a cells transfected with the pCI-neo vector without inserted DNA were used as negative controls. Two days later, cells were washed with phosphate-buffered saline and fixed with ice-cold acetone/ methanol (1/1) for subsequent use in IFA. Results Generation of monoclonal antibody to RABV
FIG. 2. Western blot analysis of five MAbs with purified RABV. Lane 1 reacted with mouse MAb 4B7; lane 2 reacted with MAb 3H7; lane 3 reacted with MAb 4E7; lane 4 reacted with MAb 6C9; lane 5 reacted with MAb 7B3.
In IFA, the serum from the mouse immunized with RABVinfected suckling mouse brain antigen reacted well with the RABV-infected Vero cells but displayed no reaction with mock-infected Vero cells (data not shown); therefore the IFA is the optional test for anti-RABV MAbs screening. In all, 288 hybridoma clones were screened in RABV-infected Vero cells by IFA, and more than 120 showed positive reactions. However, after three subclonings, only 11 hybridoma clones persistently secreted antibodies reacting with RABV-infected Vero cells
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immunodot assay, every MAb could react with the RABV antigen effectively (Fig. 3, column 1) and none of these MAbs reacted with the normal Vero cell supernatant (Fig. 3, col 2) and mock-infected cells lysis sample (Fig. 3, col 4). Moreover, not all of the MAbs reacted with the virus-infected cell sample treated by urea lysis buffer (Fig. 3, col 3). No reaction was observed for MAbs 1B11 and 8H12. These results indicating that MAbs 1B11 and 8H12 might recognize the conformational epitopes but not the linear epitopes, which are relatively vulnerable so that lysis buffer is sufficient to destroy the conformation epitopes recognized by MAbs 1B11 and 8H12. Identification of MAbs against N protein of RABV
In the first screening, approximately one third of clones (19 out of 62 tested) positively reacted with viral protein N. The fluorescent antibody staining showed that the viral protein N was mainly focused in the Negri body-like structures, which was consistent with previous report.(14,21) Finally, only one MAb 4B7 was found to react with recombinant viral protein N expressed in E. coli in indirect ELISA, which also recognized the N protein of RABV sample in Western blotting (Fig. 2), indicating that 4B7 recognized the linear epitope. To further characterize the MAb against N protein, we constructed the eukaryotic expression vector pCI-N and transfected into the Vero cells. As shown in Figure 4, MAb 4B7 showed positive reaction with pCI-N transfected cells by IFA, while no signals were observed for pCI-neo transfected cells. Neutralizing activity of MAbs
The neutralizing activity of MAbs to RABV was evaluated by fluorescent focus inhibition test in 96-well cell plates. The MAbs 1B11, 1C8, and 8H12 were capable of neutralizing the infectivity of RABV strain ERA in Vero cells (Fig. 5), whose neutralizing titers were 1:256, 1:128, and 1:256, respectively. As negative control, the SP2/0 cell culture supernatant and the medium showed no inhibitory activity against the replication of RABV by fluorescent focus inhibition assay. The data showed that the MAbs 1B11, 1C8, and 8H12 have a neutralizing activity to RABV strain ERA.
FIG. 3. Reactivity of MAbs with antigen by immunodot blotting. Solutions containing virus or control were spotted in a volume of 10 mL onto nitrocellulose. The dots were then subjected to different MAb for immunostaining. Column 1, purified rabies virus preparation with gel chromatography; 2, multiple freeze-thawed normal Vero cell supernatant; 3, total protein of virus infected cells lysed by lysis buffer; 4, total protein mock-infected cells lysates. (Fig. 1). The characterization of the 11 MAbs specifically reacting with RABV antigens is summarized in Table 1. Data also demonstrated that fluorescent signals stained by different MAbs in RABV-infected Vero cells are different (Fig. 1). Reactivity of MAbs with RABV-encoding proteins in Western blot and immunodot analyses
Western blot analysis was carried out to identify the specificity of MAbs to RABV proteins. The results showed that only MAbs 3H7, 4B7, 4E7, 6C9, and 7B3 could react with RABV proteins in a Western blot analysis (Fig. 2) while in the
Identification of monoclonal antibodies to P protein of RABV
The full length of P gene of RABV is 605 bp. For directional cloning of the P gene into the pCI-neo mammalian expression vector, restriction enzyme sites EcoR I and Sal I were incorporated into two primers designed to amplify the full length of P gene. The P gene was amplified successfully and digested with EcoR I and Sal I restriction enzymes and inserted to the pCI-neo plasmid. The pCI-neo-P recombinant expression vector was verified by PCR and restriction enzyme digestion (data not shown). The pCI-neo-P was introduced to N2a cells for transient P protein expression. In addition to 3H7, 4E7, 6C9, and 7B3, three conformational antibodies 1H3, 4G3, and 5A10 showed strong reaction with P protein in transfected cells (Fig. 6). Discussion
Monoclonal antibodies are valuable tools for characterizing antigenic variation and specific epitopes. The MAbs recognizing the conformational epitopes are especially
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FIG. 4. Characterization of anti-N RABV MAb by immunofluorescence assay. Vero cells were transfected with pCI-N eukaryotic expression plasmid. At 48 h post-transfection, cells were fixed with ice-cold acetone/methanol (1/1), reacted with MAb 4B7, followed by goat anti-mouse IgG labeled with FITC. important for understanding virus maturation and export as well as biological processes. The intrinsic quality of immunogen is one of the critical factors for antibody production.(22) Antibody development is time-consuming work, when there is a need to produce a series of antibodies. In MAb development, if the antigen was not purified sufficiently before immunization, it is very difficult to screen a good cell line without a highly purified detecting antigen. The method we used to prepare the immunogen in this study is not only an efficient way to induce a strong immune response, but also could achieve the goal of producing several kinds of MAbs by one cell fusion. As one of the key structural proteins, protein N of RABV is a super-antigen and possesses adjuvant properties.(23) In our experiment, even 5 months after the third immunization with suckling mouse brain suspension, the anti-N antibody titer in mouse serum was still 1:1600 (data not shown), and at the first screening there were many positive clones. However, we had very limited MAb cell lines against N after the third
subcloning in this experiment, most likely because it is only a probability event in the process of subcloning and screening. Thus this antigen could be effectively used to prepare the antibody against the protein N. The panel of MAbs we produced may recognize different epitopes; their characteristics were summarized in Table 1. Some of the MAbs were proven to recognize linear epitopes (3H7, 4B7, 4E7, 6C9, and 7B3) as shown in Figure 2, and others were specific for conformation-dependent epitopes (1B11, 1C8, 1H3, 4G3, 5A10, and 8H12) as shown in Figure 6; especially for MAbs 1B11 and 8H12, the epitope is much more vulnerable. The lysis buffer containing urea may disrupt the epitopes’ conformation of the protein in infected cells, which was recognized by 1B11 and 8H12. In this experiment, we found MAbs 1B11, 1C8, and 8H12 could significantly inhibit the virus infection, and all of them recognized the conformational epitopes. We suspect that these MAbs may react with G of RABV. However we failed to characterize them by eukaryotic expressing G recombinant
FIG. 5. Inhibition of rabies virus infection by neutralizing MAbs in Vero cells (320x). Vero cells were infected with the antibody-virus mixtures. After incubation for 48 h, the infectivity of the virus in the mixture was evaluated, as described in the Materials and Methods section. Supernatant of the SP2/0 culture was used as a control.
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FIG. 6. IFA showing that MAbs 1H3, 3H7, 4E7, 4G3, 5A10, 6C9, and 7B3 reacted specifically to phosphoprotein of RABV (320 · ). The nuclei of the cells were stained with DAPI.
protein. For this to occur, the expression condition or gene sequence might need to be optimized. Conclusion
The brain suspension of RABV-infected suckling mice as immunizing antigen for preparation of MAb to RABV was evaluated in this study. The antigen presents a similar immunogenicity to that of recombinant protein expressed in E. coli. The strategy for MAb production is rapid and efficient, and it is beneficial to the production of conformational antibodies. This approach may be an ideal strategy for highthroughput MAb production.
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Acknowledgments
This work was supported by the National Special Fund for Public Welfare Industry (project no. 201103032), the National High-Tech Program of China (2012AA101303), and the National Key Technologies Research and Development Program of China (2010BAD04B01).
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7. Author Disclosure Statement
The authors have no financial interests to disclose. References
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Address correspondence to: Jiyong Zhou Key Laboratory of Animal Virology of Ministry of Agriculture Zhejiang University 388 Yuhangtang Road Hangzhou 310058 P.R. China E-mail: [email protected] [email protected] Received: December 3, 2013 Accepted: January 16, 2014
