RESEARCH LETTER
Development of a neutralizing mouse–pig chimeric antibody with therapeutic potential against Haemophilus parasuis in Pichia pastoris Zheng Chai, Fang Fu, Fucheng Jiang, Huabin Tian, Zhuo Wang, Nan Zheng, Xueyun Zhang, Xiangling Wang & Xi Li State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
Correspondence: Xi Li, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences No. 427 Maduan St., Nangang District, Harbin 150001, China. Tel.: +86 189 4606 6129; fax: +86 451 5199 7166; e-mail: [email protected] Received 2 March 2014; revised 20 March 2014; accepted 31 March 2014. Final version published online 16 April 2014. DOI: 10.1111/1574-6968.12437
MICROBIOLOGY LETTERS
Editor: Jan-Ingmar Flock Keywords chimeric antibody; Haemophilus parasuis; immunotherapy; Pichia pastoris.
Abstract Haemophilus parasuis is one of the most important bacterial diseases of pigs worldwide. The lack of a vaccine against a broad spectrum of strains and the limitation of antimicrobial susceptibility hamper the control of disease. In this study, we cloned the constant regions of gamma heavy chains and kappa light chain of pig lymphocytes in frame with the variable regions of heavy and light chains of mouse monoclonal antibody 1D8, which reacts with all 15 serotypes of H. parasuis and has neutralizing activity. The constructed mouse–pig chimeric antibody was expressed in Pichia pastoris. Results demonstrated that the expressed chimeric antibody inhibited the growth of H. parasuis in vitro. Furthermore, the experiments in mice showed that chimeric antibody increased survival rate of the mice compared with that of the control group (P < 0.05). Importantly, the chimeric antibody partially protected piglets against H. parasuis infection according to the clinical lesion scores and PCR results of H. parasuis in the tissues from piglets of the chimeric antibody-inoculated group and the PBS group. In summary, our results demonstrated that the mouse–pig chimeric antibody could be a therapeutic candidate to prevent the H. parasuis infection and control the prevalence of disease.
Introduction Haemophilus parasuis (H. parasuis) is a Gram-negative, nonhemolytic, NAD-dependent bacterium. To date, 15 serovars of H. parasuis have been described, but c. 15% to 41% of the field isolates are untypeable (Rapp-Gabrielson & Gabrielson, 1992; Oliveira & Pijoan, 2004). In China, the 15 serovars and diverse genotypes of H. parasuis were widely distributed (Zhang et al., 2012b). Haemophilus parasuis is the etiological agent of Gl€asser’s disease in swine, pathologically characterized by fibrinous polyserositis, meningitis, polyarthritis, and arthritis syndrome (Nielsen, 1993). In recent years, H. parasuis infection has been considered as a major cause of nursery mortality in swine herd (Oliveira & Pijoan, 2004). The previous studies have been reported that Gl€asser’s disease can be successfully treated with antimicrobials. However, many H. parasuis isolates were susceptible to antimicrobial agents routinely used for treatment (Aarestrup FEMS Microbiol Lett 354 (2014) 85–91
et al., 2004; de la Fuente et al., 2007). Bacterins have been used to prevent Gl€asser’s disease. However, limited crossprotection among strains has complicated the control of Gl€asser’s disease (Nielsen, 1993). Some virulence factors of H. parasuis have been reported (Sack & Baltes, 2009; Costa-Hurtado et al., 2012; Zhang et al., 2012a; Zhou et al., 2012); however, little is known about protective antigens. Therefore, the development of novel therapeutic methods is necessary. We have previously reported that the neutralizing monoclonal antibody (MAb) 1D8 can react with all 15 serotypes of H. parasuis and protect mice from both homologous and heterologous challenges with H. parasuis (Tian et al., 2011). It might be a potential therapy method to protect swine against H. parasuis. However, direct application of murine MAbs in other species may be problematic. Antibodies from different animal species may not interact with Fc receptors and/or complement, which lead to a lack of appropriate down-stream ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Z. Chai et al.
86
functions and rapid clearance from the body (Tabrizi et al., 2006; Goodchild et al., 2011). An attempt to overcome this disadvantage is to generate chimeric MAbs that contain swine Fc domains and retain targeting specificity by incorporating portions of the murine variable regions. The production of chimeric antibodies using recombinant DNA technologies for therapy and prophylaxis in human has been used widely in the last years (Johnson et al., 1997; Zeitlin et al., 1998; Oliphant et al., 2005; Hu et al., 2007). Furthermore, recombinant antibodies have been developed for the control of infectious diseases in animals (Jar et al., 2009; Pyo et al., 2009). In most humanized antibody cases, chimeric antibodies are generated in the mammalian expression system (Xiong et al., 2005; Kim et al., 2011). However, the yield of transient expression in this expression system is usually low. Meanwhile, product cost plays an important role in the development of animal biologics. In this study, we grafted heavy- and light-chain variable domains (VH and VL) of murine MAb 1D8 onto the swine immunoglobulin gamma (IgG1a) heavy- and lightchain constant regions (CH and CL), respectively. The mouse–pig chimeric antibody were generated in Pichia pastoris (P. pastoris) expression system. The chimeric antibodies retained neutralizing activity in vitro and showed the therapeutic efficacy in mice and pigs with H. parasuis infection.
Materials and methods Ethics statement
This study was carried out in accordance with animal ethics guidelines and approved protocols. All animal studies were approved by the Animal Ethics Committee of Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (SYXK (H) 2006-032). Cloning of variable regions of murine kappa and gamma chains and constant regions of porcine kappa and gamma chains
The hybridoma 1D8 cell line was kept in our laboratory, which produced a murine immunoglobulin of IgG2b/ kappa isotype (Tian et al., 2011). RNA was separately extracted from murine hybridoma 1D8 or swine lymphocytes and then was used to prime cDNA synthesis (Promega). PCR amplification was performed using the primer pairs (Supporting Information, Table S1) for the variable regions (VH and VL) of the MAb 1D8 and the constant region (CH and CL) of porcine IgG. The fragments H and L chains were separately generated and verified by sequencing. ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Construction and expression of recombinant chimeric antibody in P. pastoris
The (Gly)3-Ser linker was designed between the H chain and L chain. The H-L gene fragment was ligated into the pPIC9K vector. The recombinant plasmid was linearized with SalI and then transformed into P. pastoris GS115 cells using a Gene Pulser (Bio-Rad; conditions: 1.5 kV, 25 lF, and 200Ω). His+ transformants were selected and then inoculated in 5-mL buffered complex glycerol medium. The thalli were resuspended in 20-mL buffered complex methanol medium and induction performed for 48 h. Methanol was added to a final concentration of 0.5% (v/v) at 24 h intervals. The supernatant was harvested. Western blot and Dot blot analysis
The culture supernatant of the recombinant yeast strain was subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. For Western blot, proteins in the gel were transferred to nitrocellulose membrane that was then blocked. The membrane was incubated with a horseradish peroxidase-conjugated goat antiporcine IgG antibody (whole molecule) (Sigma) and then incubated with 3, 3-diaminobenzidine tetrahydrochloride as a substrate to visualize the reaction result. The dot blot procedure was performed as Western blot described. Protein denaturation reagents were omitted during sample preparation to preserve the native conformation. Bactericidal assay in vitro
The bactericidal assay was performed as previously described (Gatto et al., 2002; Tian et al., 2011). The highly virulent H. parasuis HLJ-018 strain used in this study was kept in our laboratory (Tian et al., 2011). The log-phase H. parasuis were diluted to c. 2.4 9 103 CFU mL 1 and mixed with recombinant protein (20 lg), Mab 1D8 (20 lg), and PBS, respectively. The mixtures were incubated for 30 min at 37 °C and then plated onto the TSA agar plates. After growth overnight, colony numbers were measured. This experiment was performed three times.
Treatment of chimeric antibody in mice
For the protective efficacy in mice, eighteen 6-week-old BALB/c mice were randomly assigned to three groups of six each. All of the mice were challenge with a lethal dose of H. parasuis HLJ-018 (c. 3.0 9 1010 CFU) by intraperitoneal (IP) injection. One hour later, the mice were inoculated by IP injection in a 500 lL volume with the FEMS Microbiol Lett 354 (2014) 85–91
A mouse–pig chimeric antibody against Haemophilus parasuis
supernatant of recombinant yeast cells (25 lg/mouse), MAb 1D8 (25 lg/mouse), or PBS. The survival rates of mice were analyzed. Treatment of chimeric antibody in piglets
The piglets were obtained from a farm with a standard health status and no reported Gl€asser’s disease outbreaks. All of the piglets were negative for the detection of Actinobacillus pleuropneumoniae, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, and porcine circovirus. Eighteen large white piglets (33 days old) were randomly assigned to three groups of six each. All animals were intranasally inoculates with 2 9 1010 CFU/animal of H. parasuis HLJ 018. After 1 h, the recombinant protein (220 lg/piglet), MAb 1D8 (220 lg/piglet), or PBS was intravenously inoculated. Rectal temperatures were recorded once a day during the whole experiment. All piglets were euthanized after 7 days postchallenge. A lesion score was calculated as the sum of individual lesions/signs (lack of lesion = 0; presence of lesion = 1): pleuritis, peritonitis, arthritis, meningitis, and pericarditis. The heart, liver, spleen, lung, kidney, inguinal lymph node tissues, and pericardial fluid were removed from each euthanized piglet for re-isolating bacteria. Randomly selected bacterial isolates recovered from different tissues of all the animals were confirmed as H. parasuis by PCR (Oliveira et al., 2001). Statistical analysis
The GRAPHPAD PRISM version 5.01 was used for statistical analysis. Survival curve was analyzed with the log-rank test. P < 0.05 was defined as a significant difference.
Results
87
spliced into the whole H and L chains of chimeric antibody using overlapping PCR (Fig. 1b). Expression of chimeric antibodies in baculovirus and P. pastoris system
The constructed vector was integrated into the His4 locus of P. pastoris GS115 by electroporation. The recombinant yeast strain was selected from the His+Mut+ Pichia transformants and induced with 0.5% methanol in shake flask culture. The chimeric antibody proteins were obtained from the supernatants of culture media after 48 h of methanol induction. The recombinant protein using P. pastoris expression system accumulated up to 55 lg mL 1. The expression of chimeric antibodies was confirmed by Western bolt (Fig. 2a) and dot blot (Fig. 2b). The expressed H and L chains with molecular weight of about 50 kDa and 25 kDa were observed in the cultural supernatant of the yeast cells (Fig. 2a). In contrast, no similar band (Fig. 2a) or spot (Fig. 2b) appeared in negative control strain GS115. Assessment of chimeric antibody in bacteria activity assay
To ensure that the chimeric antibodies have the ability to inhibit the growth of H. parasuis HLJ-018 strain, the bactericidal activity of chimeric antibody proteins was assessed by the differences of colony numbers between experimental and control groups (Fig. 3). The colony numbers of H. parasuis that were treated with chimeric antibody or MAb 1D8 were reduced significantly compared with those of PBS-inoculated group (P < 0.01). Moreover, there was no significant difference between chimeric antibody and MAb 1D8. The results suggested that chimeric antibodies as well as MAb 1D8 can effectively inhibit the growth of H. parasuis in vitro.
Cloning of mouse–pig chimeric antibody
Each of the genes encoding VH and VL of murine MAb 1D8 was amplified from hybridoma MAb 1D8. The genes of swine IgG constant regions (CH and CL) were separately amplified from the cDNA of lymphocytes that were isolated from swine peripheral blood mononuclear cells. As shown in Fig. 1a, the fragments of VH, VL, CH, and CL were obtained, respectively. The sequences of the VH, VL, and CL were identical to the gene sequence reported at GenBank (KF561241, KF561242 and KF561240, respectively). Porcine constant gamma chain showed 100% identity with previously reported gamma (GenBank accession number U03781). The variable regions from murine MAb 1D8 and constant regions from swine IgG1a were
FEMS Microbiol Lett 354 (2014) 85–91
Treatment of chimeric antibodies against H. parasuis in mice
To determine the therapeutic efficacy of chimeric antibodies in vivo, all the mice were challenged with a lethal dose of H. parasuis HLJ-018 strain (c. 3.0 9 1010 CFU/mouse). After 1 h, chimeric antibodies, MAb 1D8, or PBS was intraperitoneally inoculated into BALB/c mice. The result is shown in Fig. 4. After 72 h, the survival rate of the recombinant protein group was 16.67%. There is no significant difference in the survival rate between the groups of recombinant protein and MAb 1D8 (P = 0.0719). However, there is a significant difference between the groups of recombinant protein and PBS (P < 0.05, P = 0.0422).
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Z. Chai et al.
88
(a)
(b)
Fig. 1. Product of nucleotide sequence of mouse–pig chimeric antibody. (a) RT-PCR products of VH, VL, CL, and CH genes were generated from hybridoma 1D8 cells and swine PMBC lymphocytes and were analyzed by agarose gel electrophoresis. (b) PCR products of H and L chain.
(a)
(b)
Fig. 3. Bactericidal activity of mouse–pig chimeric antibody in vitro. Haemophilus parasuis was mixed with recombinant protein, Mab 1D8, and PBS, respectively. The mixtures were plated onto the TSA agar plates. After growth overnight, colony numbers were measured. Asterisk indicated a statistically significant difference compared with the PBS control. ns indicated no statistically significant difference.
Fig. 2. Expression of the recombinant chimeric antibody protein in Pichia pastoris. (a) Western blot analysis. (b) Dot blot analysis. A 10 lL culture supernatant of the recombinant P. pastoris strain induced for 48 h was separated by SDS-PAGE and blotted onto a nitrocellulose membrane. The second antibody was detected using antiporcine IgG (H + L). The supernatant of GS115 strain after 48 h of induction was negative control.
Treatment efficacy of chimeric antibodies against H. parasuis in piglets
To characterize the therapeutic potential of mouse–pig chimeric antibody in pigs, all of the piglets were challenged with high dose of H. parasuis HLJ-018 strain. The animals treated with MAb 1D8 or PBS showed high temperatures at 1 day postchallenged (dpc.). The highest temperatures were obtained from 2 to 4 dpc. (Fig. 5). A similar tendency was observed for the group chimeric antibody (Fig. 5). However, there were significant differences in comparison with the piglets that were treated with MAb 1D8 or PBS ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Fig. 4. Survival of mice treated with mouse–pig chimeric antibody against Haemophilus parasuis HLJ-018 challenge. Kaplan–Meier survival curves of Balb/c mice are shown. All of the mice were challenged with a lethal dose of H. parasuis HLJ-018 (c. 3.0 9 1010 CFU) via intraperitoneal injection. One hour later, the mice received recombinant chimeric antibody, MAb 1D8, or PBS intraperitoneally.
FEMS Microbiol Lett 354 (2014) 85–91
A mouse–pig chimeric antibody against Haemophilus parasuis
89
Fig. 5. Rectal temperatures in piglets at different intervals after challenge with Haemophilus parasuis.
from 2 to 4 dpc. (P < 0.05). The results demonstrated that all the piglets that were inoculated with chimeric antibody showed the improved clinical performance. All the PBS control piglets and some of those inoculating chimeric antibodies or MAb 1D8 showed the characteristic inflammatory caused by H. parasuis (Table 1). Mild or moderate fibrinous polyserositis in the pericardial, pleural, and peritoneal cavities was observed in all of the piglets (Table 1). However, some piglets from MAb 1D8 or PBS group further presented fibrinous polyarthritis and mild meningitis (Table 1). Furthermore, pure cultures were recovered from heart, liver, spleen, lung, kidney, lymph node tissues, and pericardial fluid of most of PBS control piglets, while the decreased isolation rates were found for chimeric antibody group. In addition, all of the positive cultures were confirmed by PCR (Table 1).
Discussion Haemophilus parasuis is one of the most serious porcine respiratory diseases and causes large economic losses in the pig industry (Oliveira & Pijoan, 2004). The limited cross-protection of traditional vaccines and antimicrobial susceptibility hampers control of the disease. In this study, mouse–pig chimeric antibody were expressed in P. pastoris, maintained the inhibiting bacterium activity in vitro and showed its therapeutic efficacy against H. parasuis challenge in mice and piglets.
We previously found that a neutralized MAb 1D8 could reduce the colony numbers and prevalence of H. parasuis in various organs in mice that were challenged by H. parasuis (Tian et al., 2011). To limit or avoid possible side effects linked to the murine origin of the immunoglobulin, a cloning strategy replacing the constant regions of mice by the swine counterparts produces a mouse–pig chimeric antibody. Heterologous expression of mouse–pig chimeric antibody has been reported in Escherichia coli as well as in baculovirus (Jar et al., 2009; Pyo et al., 2009). Escherichia coli expression system is simple and low costs; however, the expressed proteins may lack important post-translational modifications. Moreover, as recombinant protein in baculovirus system was generated in eukaryotic cell, its cost was very expensive. The lytic baculovirus cycle of Sf9 cells releases intracellular proteases that not only may alter the stability of secreted functional antibodies, but also could interfere with functional antibody qualification (Prigent et al., 2010). Pichia pastoris as a eukaryote has the advantages of higher eukaryotic expression systems and is easier and less expensive to use than other eukaryotic expression systems. In P. pastoris system, the a-factor secretion signal attached upstream of chimeric antibody. The expressed chimeric antibody was secreted into the medium. The advantage of secretory expression is that P. pastoris secretes very few native proteins and its culture medium is almost protein-free, and therefore, the secreted chimeric antibody should comprise the majority of the total protein in the medium (Cregg et al., 1987; Romanos, 1995). Therefore, we used P. pastoris expression systems to produce mouse–pig chimeric antibody for production and application of chimeric antibody in animals. Western blot showed that the mouse–pig chimeric antibody was expressed in P. pastoris (Fig. 2a). Dot blot indicated that the constant region of chimeric antibody from swine was biological activite as chimeric antibody protein without denaturation reacted with anti-pig secondary antibody (Fig. 2b). Furthermore, chimeric antibodies significantly inhibited the bacterial growth in vitro (Fig. 3), which suggested that expression of variable
Table 1. Numbers of piglets showing the lesions and recovery of Haemophilus parasuis after challenge No. of different lesions attributable to H. parasuis
No. of piglets positive for H. parasuis
Group (n)
Pleuritis
Peritonitis
Arthritis
Meningitis
Pericarditis
Heart
Liver
Spleen
Lung
Kidney
Lymph node tissues
Pericardial fluid
Recombinant protein (6) MAb 1D8 (6) PBS (6)
4/6
3/6
0/6*
0/6*,†
3/6*
4/6
1/6*
0/6*,†
3/6
1/6
2/6
2/6*
4/6 6/6
4/6 5/6
1/6 3/6
3/6 5/6
5/6 6/6
4/6 6/6
2/6 5/6
2/6 6/6
3/6 5/6
3/6 3/6
3/6 5/6
4/6 6/6
*Significant difference compared with the PBS group (P < 0.05). Significant difference compared with the MAb 1D8 (P < 0.05).
†
FEMS Microbiol Lett 354 (2014) 85–91
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Z. Chai et al.
90
regions for murine MAb 1D8 grafted with pig IgG antibodies does not impair the neutralization of the paratope in vitro. To our knowledge, there was no data to show the therapeutic effect of mouse–pig chimeric antibody in animals, although the mouse–pig chimeric antibody have been reported (Jar et al., 2009; Pyo et al., 2009). In this study, our findings showed that mouse–pig chimeric antibody partially protected mice against lethal challenge with H. parasuis HLJ-018, suggesting that variable region of chimeric antibody has biologic activity in vivo (Fig. 4). Furthermore, the piglets were used to determine the therapeutic efficacy of mouse–pig chimeric antibody. Unfortunately, all of the piglets were survival, including the PBS control group. Because it is difficult to adjust a dose to reproduce the disease, limitations of the disease model used should be noticed (Olvera et al., 2011). However, results of clinical performance and lesion analysis showed that the chimeric antibody provided partial protection against H. parasuis infection (Fig. 5 and Table 1). On the one hand, the rectal temperatures of the piglets that were inoculated with MAb 1D8 or PBS were significantly higher than those of piglets with chimeric antibody treatment after H. parasuis infection (P < 0.01), indicating that the chimeric antibody can alleviate the clinical manifestations of infected piglets On the other hand, the lesion records and bacterial isolations from tissues showed that the piglets with chimeric antibody treatment were partially protected against H. parasuis infection compared with the results of PBS control piglets. In further study, we would determine the therapeutic efficacy of the chimeric antibody against multiserotype strains of H. parasuis in vivo. Meanwhile, we would attempt to enhance the yields of chimeric antibody protein in P. pastoris by means of increasing chimeric antibody copy numbers or producing chimeric antibody in P. pastoris bioreactor. This study described a novel H. parasuis prophylactic by expressing mouse–pig chimeric antibody in P. pastoris. The results demonstrated that the chimeric antibody was capable to inhibit bacterial growth in vitro and showed significantly therapeutic efficacy in the piglets with H. parasuis infection, which represents a potentially preventive and therapeutic tool in animals. Furthermore, chimeric antibody could be used alone or combined with other vaccination strategy to provide instant immunity in the face of an outbreak and before the establishment of effective adaptive immune response of the swine.
Acknowledgements We thank Dr Chunlai Wang for providing experimental suggestions. This study was supported by Special Fund ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
for Agro-scientific Research in the Public Interest (201203056).
References Aarestrup FM, Seyfarth AM & Angen O (2004) Antimicrobial susceptibility of Haemophilus parasuis and Histophilus somni from pigs and cattle in Denmark. Vet Microbiol 101: 143– 146. Costa-Hurtado M, Ballester M, Galofre-Mila N, Darji A & Aragon V (2012) VtaA8 and VtaA9 from Haemophilus parasuis delay phagocytosis by alveolar macrophages. Vet Res 43: 57. Cregg JM, Tschopp JF, Stillman C et al. (1987) High-level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris. Nat Biotech 5: 479–485. de la Fuente AJ, Tucker AW, Navas J, Blanco M, Morris SJ & Gutierrez-Martin CB (2007) Antimicrobial susceptibility patterns of Haemophilus parasuis from pigs in the United Kingdom and Spain. Vet Microbiol 120: 184–191. Gatto NT, Dabo SM, Hancock RE & Confer AW (2002) Characterization of, and immune responses of mice to, the purified OmpA-equivalent outer membrane protein of Pasteurella multocida serotype A:3 (Omp28). Vet Microbiol 87: 221–235. Goodchild SA, O’Brien LM, Steven J et al. (2011) A humanised murine monoclonal antibody with broad serogroup specificity protects mice from challenge with Venezuelan equine encephalitis virus. Antiviral Res 90: 1–8. Hu W-G, Chau D, Wu J, Jager S & Nagata LP (2007) Humanization and mammalian expression of a murine monoclonal antibody against Venezuelan equine encephalitis virus. Vaccine 25: 3210–3214. Jar AM, Osorio FA & L opez OJ (2009) Mouse 9 pig chimeric antibodies expressed in Baculovirus retain the same properties of their parent antibodies. Biotechnol Prog 25: 516–523. Johnson S, Oliver C, Prince GA et al. (1997) Development of a humanized monoclonal antibody (MEDI-493) with potent in vitro and in vivo activity against respiratory syncytial virus. J Infect Dis 176: 1215–1224. Kim H-Y, Tsai S, Lo S-C, Wear DJ & Izadjoo MJ (2011) Production and characterization of chimeric monoclonal antibodies against Burkholderia pseudomallei and B. malleiUsing the DHFR expression system. PLoS One 6: e19867. Nielsen R (1993) Pathogenicity and immunity studies of Haemophilus parasuis serotypes. Acta Vet Scand 34: 193–198. Oliphant T, Engle M, Nybakken GE et al. (2005) Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus. Nat Med 11: 522–530. Oliveira S & Pijoan C (2004) Haemophilus parasuis: new trends on diagnosis, epidemiology and control. Vet Microbiol 99: 1–12. Oliveira S, Galina L & Pijoan C (2001) Development of a PCR Test to Diagnose Haemophilus Parasuis Infections. J Vet Diagn Invest 13: 495–501.
FEMS Microbiol Lett 354 (2014) 85–91
A mouse–pig chimeric antibody against Haemophilus parasuis
Olvera A, Pina S, Perez-Sim o M, Arag on V, Segales J & Bensaid A (2011) Immunogenicity and protection against Haemophilus parasuis infection after vaccination with recombinant virulence associated trimeric autotransporters (VtaA). Vaccine 29: 2797–2802. Prigent J, Mazuet C, Boquet D et al. (2010) Production and characterisation of a neutralising chimeric antibody against botulinum neurotoxin A. PLoS One 5: e13245. Pyo H-M, Kim I-J, Kim S-H, Kim H-S, Cho S-D, Cho I-S & Hyun B-H (2009) Escherichia coli expressing single-chain Fv on the cell surface as a potential prophylactic of porcine epidemic diarrhea virus. Vaccine 27: 2030–2036. Rapp-Gabrielson VJ & Gabrielson DA (1992) Prevalence of Haemophilus parasuis serovars among isolates from swine. Am J Vet Res 53: 659–664. Romanos M (1995) Advances in the use of Pichia pastoris for high-level gene expression. Curr Opin Biotechnol 6: 527–533. Sack M & Baltes N (2009) Identification of novel potential virulence-associated factors in Haemophilus parasuis. Vet Microbiol 136: 382–386. Tabrizi MA, Tseng C-ML & Roskos LK (2006) Elimination mechanisms of therapeutic monoclonal antibodies. Drug Discovery To 11: 81–88. Tian H, Fu F, Li X et al. (2011) Identification of the immunogenic outer membrane protein a antigen of Haemophilus parasuis by a proteomics approach and passive immunization with monoclonal antibodies in mice. Clin Vaccine Immunol 18: 1695–1701.
FEMS Microbiol Lett 354 (2014) 85–91
91
Xiong K-H, Liang Q-C, Xiong H, Zou C-X, Gao G-D, Zhao Z-W & Zhang H (2005) Expression of chimeric antibody in mammalian cells using dicistronic expression vector. Biotechnol Lett 27: 1713–1717. Zeitlin L, Olmsted SS, Moench TR et al. (1998) A humanized monoclonal antibody produced in transgenic plants for immunoprotection of the vagina against genital herpes. Nat Biotech 16: 1361–1364. Zhang B, Feng S, Xu C et al. (2012a) Serum resistance in Haemophilus parasuisSC096 strain requires outer membrane protein P2 expression. FEMS Microbiol Lett 326: 109–115. Zhang J, Xu C, Guo L et al. (2012b) Prevalence and characterization of genotypic diversity of Haemophilus parasuis isolates from southern China. Can J Vet Res 76: 224–229. Zhou M, Zhang Q, Zhao J & Jin M (2012) Haemophilus parasuis encodes two functional cytolethal distending toxins: CdtC contains an atypical cholesterol recognition/interaction region. PLoS One 7: e32580.
Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Sequence of primers used in the generation of PCR fragments.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Development of a neutralizing mouse–pig chimeric antibody with therapeutic potential against Haemophilus parasuis in Pichia pastoris Zheng Chai, Fang Fu, Fucheng Jiang, Huabin Tian, Zhuo Wang, Nan Zheng, Xueyun Zhang, Xiangling Wang & Xi Li State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
Correspondence: Xi Li, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences No. 427 Maduan St., Nangang District, Harbin 150001, China. Tel.: +86 189 4606 6129; fax: +86 451 5199 7166; e-mail: [email protected] Received 2 March 2014; revised 20 March 2014; accepted 31 March 2014. Final version published online 16 April 2014. DOI: 10.1111/1574-6968.12437
MICROBIOLOGY LETTERS
Editor: Jan-Ingmar Flock Keywords chimeric antibody; Haemophilus parasuis; immunotherapy; Pichia pastoris.
Abstract Haemophilus parasuis is one of the most important bacterial diseases of pigs worldwide. The lack of a vaccine against a broad spectrum of strains and the limitation of antimicrobial susceptibility hamper the control of disease. In this study, we cloned the constant regions of gamma heavy chains and kappa light chain of pig lymphocytes in frame with the variable regions of heavy and light chains of mouse monoclonal antibody 1D8, which reacts with all 15 serotypes of H. parasuis and has neutralizing activity. The constructed mouse–pig chimeric antibody was expressed in Pichia pastoris. Results demonstrated that the expressed chimeric antibody inhibited the growth of H. parasuis in vitro. Furthermore, the experiments in mice showed that chimeric antibody increased survival rate of the mice compared with that of the control group (P < 0.05). Importantly, the chimeric antibody partially protected piglets against H. parasuis infection according to the clinical lesion scores and PCR results of H. parasuis in the tissues from piglets of the chimeric antibody-inoculated group and the PBS group. In summary, our results demonstrated that the mouse–pig chimeric antibody could be a therapeutic candidate to prevent the H. parasuis infection and control the prevalence of disease.
Introduction Haemophilus parasuis (H. parasuis) is a Gram-negative, nonhemolytic, NAD-dependent bacterium. To date, 15 serovars of H. parasuis have been described, but c. 15% to 41% of the field isolates are untypeable (Rapp-Gabrielson & Gabrielson, 1992; Oliveira & Pijoan, 2004). In China, the 15 serovars and diverse genotypes of H. parasuis were widely distributed (Zhang et al., 2012b). Haemophilus parasuis is the etiological agent of Gl€asser’s disease in swine, pathologically characterized by fibrinous polyserositis, meningitis, polyarthritis, and arthritis syndrome (Nielsen, 1993). In recent years, H. parasuis infection has been considered as a major cause of nursery mortality in swine herd (Oliveira & Pijoan, 2004). The previous studies have been reported that Gl€asser’s disease can be successfully treated with antimicrobials. However, many H. parasuis isolates were susceptible to antimicrobial agents routinely used for treatment (Aarestrup FEMS Microbiol Lett 354 (2014) 85–91
et al., 2004; de la Fuente et al., 2007). Bacterins have been used to prevent Gl€asser’s disease. However, limited crossprotection among strains has complicated the control of Gl€asser’s disease (Nielsen, 1993). Some virulence factors of H. parasuis have been reported (Sack & Baltes, 2009; Costa-Hurtado et al., 2012; Zhang et al., 2012a; Zhou et al., 2012); however, little is known about protective antigens. Therefore, the development of novel therapeutic methods is necessary. We have previously reported that the neutralizing monoclonal antibody (MAb) 1D8 can react with all 15 serotypes of H. parasuis and protect mice from both homologous and heterologous challenges with H. parasuis (Tian et al., 2011). It might be a potential therapy method to protect swine against H. parasuis. However, direct application of murine MAbs in other species may be problematic. Antibodies from different animal species may not interact with Fc receptors and/or complement, which lead to a lack of appropriate down-stream ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Z. Chai et al.
86
functions and rapid clearance from the body (Tabrizi et al., 2006; Goodchild et al., 2011). An attempt to overcome this disadvantage is to generate chimeric MAbs that contain swine Fc domains and retain targeting specificity by incorporating portions of the murine variable regions. The production of chimeric antibodies using recombinant DNA technologies for therapy and prophylaxis in human has been used widely in the last years (Johnson et al., 1997; Zeitlin et al., 1998; Oliphant et al., 2005; Hu et al., 2007). Furthermore, recombinant antibodies have been developed for the control of infectious diseases in animals (Jar et al., 2009; Pyo et al., 2009). In most humanized antibody cases, chimeric antibodies are generated in the mammalian expression system (Xiong et al., 2005; Kim et al., 2011). However, the yield of transient expression in this expression system is usually low. Meanwhile, product cost plays an important role in the development of animal biologics. In this study, we grafted heavy- and light-chain variable domains (VH and VL) of murine MAb 1D8 onto the swine immunoglobulin gamma (IgG1a) heavy- and lightchain constant regions (CH and CL), respectively. The mouse–pig chimeric antibody were generated in Pichia pastoris (P. pastoris) expression system. The chimeric antibodies retained neutralizing activity in vitro and showed the therapeutic efficacy in mice and pigs with H. parasuis infection.
Materials and methods Ethics statement
This study was carried out in accordance with animal ethics guidelines and approved protocols. All animal studies were approved by the Animal Ethics Committee of Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (SYXK (H) 2006-032). Cloning of variable regions of murine kappa and gamma chains and constant regions of porcine kappa and gamma chains
The hybridoma 1D8 cell line was kept in our laboratory, which produced a murine immunoglobulin of IgG2b/ kappa isotype (Tian et al., 2011). RNA was separately extracted from murine hybridoma 1D8 or swine lymphocytes and then was used to prime cDNA synthesis (Promega). PCR amplification was performed using the primer pairs (Supporting Information, Table S1) for the variable regions (VH and VL) of the MAb 1D8 and the constant region (CH and CL) of porcine IgG. The fragments H and L chains were separately generated and verified by sequencing. ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Construction and expression of recombinant chimeric antibody in P. pastoris
The (Gly)3-Ser linker was designed between the H chain and L chain. The H-L gene fragment was ligated into the pPIC9K vector. The recombinant plasmid was linearized with SalI and then transformed into P. pastoris GS115 cells using a Gene Pulser (Bio-Rad; conditions: 1.5 kV, 25 lF, and 200Ω). His+ transformants were selected and then inoculated in 5-mL buffered complex glycerol medium. The thalli were resuspended in 20-mL buffered complex methanol medium and induction performed for 48 h. Methanol was added to a final concentration of 0.5% (v/v) at 24 h intervals. The supernatant was harvested. Western blot and Dot blot analysis
The culture supernatant of the recombinant yeast strain was subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. For Western blot, proteins in the gel were transferred to nitrocellulose membrane that was then blocked. The membrane was incubated with a horseradish peroxidase-conjugated goat antiporcine IgG antibody (whole molecule) (Sigma) and then incubated with 3, 3-diaminobenzidine tetrahydrochloride as a substrate to visualize the reaction result. The dot blot procedure was performed as Western blot described. Protein denaturation reagents were omitted during sample preparation to preserve the native conformation. Bactericidal assay in vitro
The bactericidal assay was performed as previously described (Gatto et al., 2002; Tian et al., 2011). The highly virulent H. parasuis HLJ-018 strain used in this study was kept in our laboratory (Tian et al., 2011). The log-phase H. parasuis were diluted to c. 2.4 9 103 CFU mL 1 and mixed with recombinant protein (20 lg), Mab 1D8 (20 lg), and PBS, respectively. The mixtures were incubated for 30 min at 37 °C and then plated onto the TSA agar plates. After growth overnight, colony numbers were measured. This experiment was performed three times.
Treatment of chimeric antibody in mice
For the protective efficacy in mice, eighteen 6-week-old BALB/c mice were randomly assigned to three groups of six each. All of the mice were challenge with a lethal dose of H. parasuis HLJ-018 (c. 3.0 9 1010 CFU) by intraperitoneal (IP) injection. One hour later, the mice were inoculated by IP injection in a 500 lL volume with the FEMS Microbiol Lett 354 (2014) 85–91
A mouse–pig chimeric antibody against Haemophilus parasuis
supernatant of recombinant yeast cells (25 lg/mouse), MAb 1D8 (25 lg/mouse), or PBS. The survival rates of mice were analyzed. Treatment of chimeric antibody in piglets
The piglets were obtained from a farm with a standard health status and no reported Gl€asser’s disease outbreaks. All of the piglets were negative for the detection of Actinobacillus pleuropneumoniae, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, and porcine circovirus. Eighteen large white piglets (33 days old) were randomly assigned to three groups of six each. All animals were intranasally inoculates with 2 9 1010 CFU/animal of H. parasuis HLJ 018. After 1 h, the recombinant protein (220 lg/piglet), MAb 1D8 (220 lg/piglet), or PBS was intravenously inoculated. Rectal temperatures were recorded once a day during the whole experiment. All piglets were euthanized after 7 days postchallenge. A lesion score was calculated as the sum of individual lesions/signs (lack of lesion = 0; presence of lesion = 1): pleuritis, peritonitis, arthritis, meningitis, and pericarditis. The heart, liver, spleen, lung, kidney, inguinal lymph node tissues, and pericardial fluid were removed from each euthanized piglet for re-isolating bacteria. Randomly selected bacterial isolates recovered from different tissues of all the animals were confirmed as H. parasuis by PCR (Oliveira et al., 2001). Statistical analysis
The GRAPHPAD PRISM version 5.01 was used for statistical analysis. Survival curve was analyzed with the log-rank test. P < 0.05 was defined as a significant difference.
Results
87
spliced into the whole H and L chains of chimeric antibody using overlapping PCR (Fig. 1b). Expression of chimeric antibodies in baculovirus and P. pastoris system
The constructed vector was integrated into the His4 locus of P. pastoris GS115 by electroporation. The recombinant yeast strain was selected from the His+Mut+ Pichia transformants and induced with 0.5% methanol in shake flask culture. The chimeric antibody proteins were obtained from the supernatants of culture media after 48 h of methanol induction. The recombinant protein using P. pastoris expression system accumulated up to 55 lg mL 1. The expression of chimeric antibodies was confirmed by Western bolt (Fig. 2a) and dot blot (Fig. 2b). The expressed H and L chains with molecular weight of about 50 kDa and 25 kDa were observed in the cultural supernatant of the yeast cells (Fig. 2a). In contrast, no similar band (Fig. 2a) or spot (Fig. 2b) appeared in negative control strain GS115. Assessment of chimeric antibody in bacteria activity assay
To ensure that the chimeric antibodies have the ability to inhibit the growth of H. parasuis HLJ-018 strain, the bactericidal activity of chimeric antibody proteins was assessed by the differences of colony numbers between experimental and control groups (Fig. 3). The colony numbers of H. parasuis that were treated with chimeric antibody or MAb 1D8 were reduced significantly compared with those of PBS-inoculated group (P < 0.01). Moreover, there was no significant difference between chimeric antibody and MAb 1D8. The results suggested that chimeric antibodies as well as MAb 1D8 can effectively inhibit the growth of H. parasuis in vitro.
Cloning of mouse–pig chimeric antibody
Each of the genes encoding VH and VL of murine MAb 1D8 was amplified from hybridoma MAb 1D8. The genes of swine IgG constant regions (CH and CL) were separately amplified from the cDNA of lymphocytes that were isolated from swine peripheral blood mononuclear cells. As shown in Fig. 1a, the fragments of VH, VL, CH, and CL were obtained, respectively. The sequences of the VH, VL, and CL were identical to the gene sequence reported at GenBank (KF561241, KF561242 and KF561240, respectively). Porcine constant gamma chain showed 100% identity with previously reported gamma (GenBank accession number U03781). The variable regions from murine MAb 1D8 and constant regions from swine IgG1a were
FEMS Microbiol Lett 354 (2014) 85–91
Treatment of chimeric antibodies against H. parasuis in mice
To determine the therapeutic efficacy of chimeric antibodies in vivo, all the mice were challenged with a lethal dose of H. parasuis HLJ-018 strain (c. 3.0 9 1010 CFU/mouse). After 1 h, chimeric antibodies, MAb 1D8, or PBS was intraperitoneally inoculated into BALB/c mice. The result is shown in Fig. 4. After 72 h, the survival rate of the recombinant protein group was 16.67%. There is no significant difference in the survival rate between the groups of recombinant protein and MAb 1D8 (P = 0.0719). However, there is a significant difference between the groups of recombinant protein and PBS (P < 0.05, P = 0.0422).
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Z. Chai et al.
88
(a)
(b)
Fig. 1. Product of nucleotide sequence of mouse–pig chimeric antibody. (a) RT-PCR products of VH, VL, CL, and CH genes were generated from hybridoma 1D8 cells and swine PMBC lymphocytes and were analyzed by agarose gel electrophoresis. (b) PCR products of H and L chain.
(a)
(b)
Fig. 3. Bactericidal activity of mouse–pig chimeric antibody in vitro. Haemophilus parasuis was mixed with recombinant protein, Mab 1D8, and PBS, respectively. The mixtures were plated onto the TSA agar plates. After growth overnight, colony numbers were measured. Asterisk indicated a statistically significant difference compared with the PBS control. ns indicated no statistically significant difference.
Fig. 2. Expression of the recombinant chimeric antibody protein in Pichia pastoris. (a) Western blot analysis. (b) Dot blot analysis. A 10 lL culture supernatant of the recombinant P. pastoris strain induced for 48 h was separated by SDS-PAGE and blotted onto a nitrocellulose membrane. The second antibody was detected using antiporcine IgG (H + L). The supernatant of GS115 strain after 48 h of induction was negative control.
Treatment efficacy of chimeric antibodies against H. parasuis in piglets
To characterize the therapeutic potential of mouse–pig chimeric antibody in pigs, all of the piglets were challenged with high dose of H. parasuis HLJ-018 strain. The animals treated with MAb 1D8 or PBS showed high temperatures at 1 day postchallenged (dpc.). The highest temperatures were obtained from 2 to 4 dpc. (Fig. 5). A similar tendency was observed for the group chimeric antibody (Fig. 5). However, there were significant differences in comparison with the piglets that were treated with MAb 1D8 or PBS ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Fig. 4. Survival of mice treated with mouse–pig chimeric antibody against Haemophilus parasuis HLJ-018 challenge. Kaplan–Meier survival curves of Balb/c mice are shown. All of the mice were challenged with a lethal dose of H. parasuis HLJ-018 (c. 3.0 9 1010 CFU) via intraperitoneal injection. One hour later, the mice received recombinant chimeric antibody, MAb 1D8, or PBS intraperitoneally.
FEMS Microbiol Lett 354 (2014) 85–91
A mouse–pig chimeric antibody against Haemophilus parasuis
89
Fig. 5. Rectal temperatures in piglets at different intervals after challenge with Haemophilus parasuis.
from 2 to 4 dpc. (P < 0.05). The results demonstrated that all the piglets that were inoculated with chimeric antibody showed the improved clinical performance. All the PBS control piglets and some of those inoculating chimeric antibodies or MAb 1D8 showed the characteristic inflammatory caused by H. parasuis (Table 1). Mild or moderate fibrinous polyserositis in the pericardial, pleural, and peritoneal cavities was observed in all of the piglets (Table 1). However, some piglets from MAb 1D8 or PBS group further presented fibrinous polyarthritis and mild meningitis (Table 1). Furthermore, pure cultures were recovered from heart, liver, spleen, lung, kidney, lymph node tissues, and pericardial fluid of most of PBS control piglets, while the decreased isolation rates were found for chimeric antibody group. In addition, all of the positive cultures were confirmed by PCR (Table 1).
Discussion Haemophilus parasuis is one of the most serious porcine respiratory diseases and causes large economic losses in the pig industry (Oliveira & Pijoan, 2004). The limited cross-protection of traditional vaccines and antimicrobial susceptibility hampers control of the disease. In this study, mouse–pig chimeric antibody were expressed in P. pastoris, maintained the inhibiting bacterium activity in vitro and showed its therapeutic efficacy against H. parasuis challenge in mice and piglets.
We previously found that a neutralized MAb 1D8 could reduce the colony numbers and prevalence of H. parasuis in various organs in mice that were challenged by H. parasuis (Tian et al., 2011). To limit or avoid possible side effects linked to the murine origin of the immunoglobulin, a cloning strategy replacing the constant regions of mice by the swine counterparts produces a mouse–pig chimeric antibody. Heterologous expression of mouse–pig chimeric antibody has been reported in Escherichia coli as well as in baculovirus (Jar et al., 2009; Pyo et al., 2009). Escherichia coli expression system is simple and low costs; however, the expressed proteins may lack important post-translational modifications. Moreover, as recombinant protein in baculovirus system was generated in eukaryotic cell, its cost was very expensive. The lytic baculovirus cycle of Sf9 cells releases intracellular proteases that not only may alter the stability of secreted functional antibodies, but also could interfere with functional antibody qualification (Prigent et al., 2010). Pichia pastoris as a eukaryote has the advantages of higher eukaryotic expression systems and is easier and less expensive to use than other eukaryotic expression systems. In P. pastoris system, the a-factor secretion signal attached upstream of chimeric antibody. The expressed chimeric antibody was secreted into the medium. The advantage of secretory expression is that P. pastoris secretes very few native proteins and its culture medium is almost protein-free, and therefore, the secreted chimeric antibody should comprise the majority of the total protein in the medium (Cregg et al., 1987; Romanos, 1995). Therefore, we used P. pastoris expression systems to produce mouse–pig chimeric antibody for production and application of chimeric antibody in animals. Western blot showed that the mouse–pig chimeric antibody was expressed in P. pastoris (Fig. 2a). Dot blot indicated that the constant region of chimeric antibody from swine was biological activite as chimeric antibody protein without denaturation reacted with anti-pig secondary antibody (Fig. 2b). Furthermore, chimeric antibodies significantly inhibited the bacterial growth in vitro (Fig. 3), which suggested that expression of variable
Table 1. Numbers of piglets showing the lesions and recovery of Haemophilus parasuis after challenge No. of different lesions attributable to H. parasuis
No. of piglets positive for H. parasuis
Group (n)
Pleuritis
Peritonitis
Arthritis
Meningitis
Pericarditis
Heart
Liver
Spleen
Lung
Kidney
Lymph node tissues
Pericardial fluid
Recombinant protein (6) MAb 1D8 (6) PBS (6)
4/6
3/6
0/6*
0/6*,†
3/6*
4/6
1/6*
0/6*,†
3/6
1/6
2/6
2/6*
4/6 6/6
4/6 5/6
1/6 3/6
3/6 5/6
5/6 6/6
4/6 6/6
2/6 5/6
2/6 6/6
3/6 5/6
3/6 3/6
3/6 5/6
4/6 6/6
*Significant difference compared with the PBS group (P < 0.05). Significant difference compared with the MAb 1D8 (P < 0.05).
†
FEMS Microbiol Lett 354 (2014) 85–91
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Z. Chai et al.
90
regions for murine MAb 1D8 grafted with pig IgG antibodies does not impair the neutralization of the paratope in vitro. To our knowledge, there was no data to show the therapeutic effect of mouse–pig chimeric antibody in animals, although the mouse–pig chimeric antibody have been reported (Jar et al., 2009; Pyo et al., 2009). In this study, our findings showed that mouse–pig chimeric antibody partially protected mice against lethal challenge with H. parasuis HLJ-018, suggesting that variable region of chimeric antibody has biologic activity in vivo (Fig. 4). Furthermore, the piglets were used to determine the therapeutic efficacy of mouse–pig chimeric antibody. Unfortunately, all of the piglets were survival, including the PBS control group. Because it is difficult to adjust a dose to reproduce the disease, limitations of the disease model used should be noticed (Olvera et al., 2011). However, results of clinical performance and lesion analysis showed that the chimeric antibody provided partial protection against H. parasuis infection (Fig. 5 and Table 1). On the one hand, the rectal temperatures of the piglets that were inoculated with MAb 1D8 or PBS were significantly higher than those of piglets with chimeric antibody treatment after H. parasuis infection (P < 0.01), indicating that the chimeric antibody can alleviate the clinical manifestations of infected piglets On the other hand, the lesion records and bacterial isolations from tissues showed that the piglets with chimeric antibody treatment were partially protected against H. parasuis infection compared with the results of PBS control piglets. In further study, we would determine the therapeutic efficacy of the chimeric antibody against multiserotype strains of H. parasuis in vivo. Meanwhile, we would attempt to enhance the yields of chimeric antibody protein in P. pastoris by means of increasing chimeric antibody copy numbers or producing chimeric antibody in P. pastoris bioreactor. This study described a novel H. parasuis prophylactic by expressing mouse–pig chimeric antibody in P. pastoris. The results demonstrated that the chimeric antibody was capable to inhibit bacterial growth in vitro and showed significantly therapeutic efficacy in the piglets with H. parasuis infection, which represents a potentially preventive and therapeutic tool in animals. Furthermore, chimeric antibody could be used alone or combined with other vaccination strategy to provide instant immunity in the face of an outbreak and before the establishment of effective adaptive immune response of the swine.
Acknowledgements We thank Dr Chunlai Wang for providing experimental suggestions. This study was supported by Special Fund ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
for Agro-scientific Research in the Public Interest (201203056).
References Aarestrup FM, Seyfarth AM & Angen O (2004) Antimicrobial susceptibility of Haemophilus parasuis and Histophilus somni from pigs and cattle in Denmark. Vet Microbiol 101: 143– 146. Costa-Hurtado M, Ballester M, Galofre-Mila N, Darji A & Aragon V (2012) VtaA8 and VtaA9 from Haemophilus parasuis delay phagocytosis by alveolar macrophages. Vet Res 43: 57. Cregg JM, Tschopp JF, Stillman C et al. (1987) High-level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris. Nat Biotech 5: 479–485. de la Fuente AJ, Tucker AW, Navas J, Blanco M, Morris SJ & Gutierrez-Martin CB (2007) Antimicrobial susceptibility patterns of Haemophilus parasuis from pigs in the United Kingdom and Spain. Vet Microbiol 120: 184–191. Gatto NT, Dabo SM, Hancock RE & Confer AW (2002) Characterization of, and immune responses of mice to, the purified OmpA-equivalent outer membrane protein of Pasteurella multocida serotype A:3 (Omp28). Vet Microbiol 87: 221–235. Goodchild SA, O’Brien LM, Steven J et al. (2011) A humanised murine monoclonal antibody with broad serogroup specificity protects mice from challenge with Venezuelan equine encephalitis virus. Antiviral Res 90: 1–8. Hu W-G, Chau D, Wu J, Jager S & Nagata LP (2007) Humanization and mammalian expression of a murine monoclonal antibody against Venezuelan equine encephalitis virus. Vaccine 25: 3210–3214. Jar AM, Osorio FA & L opez OJ (2009) Mouse 9 pig chimeric antibodies expressed in Baculovirus retain the same properties of their parent antibodies. Biotechnol Prog 25: 516–523. Johnson S, Oliver C, Prince GA et al. (1997) Development of a humanized monoclonal antibody (MEDI-493) with potent in vitro and in vivo activity against respiratory syncytial virus. J Infect Dis 176: 1215–1224. Kim H-Y, Tsai S, Lo S-C, Wear DJ & Izadjoo MJ (2011) Production and characterization of chimeric monoclonal antibodies against Burkholderia pseudomallei and B. malleiUsing the DHFR expression system. PLoS One 6: e19867. Nielsen R (1993) Pathogenicity and immunity studies of Haemophilus parasuis serotypes. Acta Vet Scand 34: 193–198. Oliphant T, Engle M, Nybakken GE et al. (2005) Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus. Nat Med 11: 522–530. Oliveira S & Pijoan C (2004) Haemophilus parasuis: new trends on diagnosis, epidemiology and control. Vet Microbiol 99: 1–12. Oliveira S, Galina L & Pijoan C (2001) Development of a PCR Test to Diagnose Haemophilus Parasuis Infections. J Vet Diagn Invest 13: 495–501.
FEMS Microbiol Lett 354 (2014) 85–91
A mouse–pig chimeric antibody against Haemophilus parasuis
Olvera A, Pina S, Perez-Sim o M, Arag on V, Segales J & Bensaid A (2011) Immunogenicity and protection against Haemophilus parasuis infection after vaccination with recombinant virulence associated trimeric autotransporters (VtaA). Vaccine 29: 2797–2802. Prigent J, Mazuet C, Boquet D et al. (2010) Production and characterisation of a neutralising chimeric antibody against botulinum neurotoxin A. PLoS One 5: e13245. Pyo H-M, Kim I-J, Kim S-H, Kim H-S, Cho S-D, Cho I-S & Hyun B-H (2009) Escherichia coli expressing single-chain Fv on the cell surface as a potential prophylactic of porcine epidemic diarrhea virus. Vaccine 27: 2030–2036. Rapp-Gabrielson VJ & Gabrielson DA (1992) Prevalence of Haemophilus parasuis serovars among isolates from swine. Am J Vet Res 53: 659–664. Romanos M (1995) Advances in the use of Pichia pastoris for high-level gene expression. Curr Opin Biotechnol 6: 527–533. Sack M & Baltes N (2009) Identification of novel potential virulence-associated factors in Haemophilus parasuis. Vet Microbiol 136: 382–386. Tabrizi MA, Tseng C-ML & Roskos LK (2006) Elimination mechanisms of therapeutic monoclonal antibodies. Drug Discovery To 11: 81–88. Tian H, Fu F, Li X et al. (2011) Identification of the immunogenic outer membrane protein a antigen of Haemophilus parasuis by a proteomics approach and passive immunization with monoclonal antibodies in mice. Clin Vaccine Immunol 18: 1695–1701.
FEMS Microbiol Lett 354 (2014) 85–91
91
Xiong K-H, Liang Q-C, Xiong H, Zou C-X, Gao G-D, Zhao Z-W & Zhang H (2005) Expression of chimeric antibody in mammalian cells using dicistronic expression vector. Biotechnol Lett 27: 1713–1717. Zeitlin L, Olmsted SS, Moench TR et al. (1998) A humanized monoclonal antibody produced in transgenic plants for immunoprotection of the vagina against genital herpes. Nat Biotech 16: 1361–1364. Zhang B, Feng S, Xu C et al. (2012a) Serum resistance in Haemophilus parasuisSC096 strain requires outer membrane protein P2 expression. FEMS Microbiol Lett 326: 109–115. Zhang J, Xu C, Guo L et al. (2012b) Prevalence and characterization of genotypic diversity of Haemophilus parasuis isolates from southern China. Can J Vet Res 76: 224–229. Zhou M, Zhang Q, Zhao J & Jin M (2012) Haemophilus parasuis encodes two functional cytolethal distending toxins: CdtC contains an atypical cholesterol recognition/interaction region. PLoS One 7: e32580.
Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Sequence of primers used in the generation of PCR fragments.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
