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MODEL
Microbes and Infection xx (2014) 1e10 www.elsevier.com/locate/micinf
Original article
FbaA- and M protein-based multi-epitope vaccine elicits strong protective immune responses against group A streptococcus in mouse model Cuiqing Ma a,1, Zheng Liu a,1, Wenjian Li a, Xuesong Qian a, Song Zhang c, Xue Gao a, Shibo Jiang b,*, Lin Wei a,** b
a Department of Immunology, Basic Medical College, Hebei Medical University, Shijiazhuang 050017, China Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Shanghai Medical College and Institute of Medical Microbiology, Fudan University, Shanghai 200032, China c Third Hospital of Hebei Medical University, Shijiazhuang 050000, China
Received 10 January 2014; accepted 20 March 2014
Abstract We report the construction of a recombinant multivalent vaccine against group A streptococcus (GAS), designated F7M5. It contains seven predominant epitopes of FbaA identified by phage display technology, five non-tissue cross-reactive M protein fragments expressed on four selected serotypes prevalent in China, a Trojan antigen (TA) and a poly-alanine DR epitope (PADRE). BALB/c mice were immunized subcutaneously with F7M5 formulated with Freund’s adjuvant, using recombinant FbaA and M protein in parallel as control. Using enzyme-linked immunosorbent assay (ELISA), mouse immune sera were assayed for IgG titers, IgG subclasses, and binding of F7M5 with M1GAS. Results indicated that the multivalent vaccine was highly immunogenic and elicited a balanced IgG1/IgG2a response. We also tested the reactivity of F7M5 to antistreptolysin O (ASO) antibodies in sera of GAS-infected patients and found a 95.8% positive rate, indicating that the epitopes of the vaccine were widely expressed in the prevalent serotypes of GAS. More importantly, the F7M5 vaccine elicited strong protective immune responses against lethal-dose challenge with a survival rate of 90%, but induced no cross-reactions or pathological lesions in mouse model, suggesting that F7M5 can be further developed as an effective and safe anti-GAS vaccine. Ó 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Group A streptococcus (GAS); Epitope; Vaccine; FbaA; M protein
1. Introduction Group A streptococcus (GAS), a common Gram-positive bacterium, is capable of causing serious invasive infection and autoimmune diseases. In developing countries, acute rheumatic fever (ARF) remains rampant and can be followed by rheumatic heart disease, causing hundreds of thousands of deaths every year [1,2]. Epidemiological investigation found
* Corresponding author. Tel.: þ86 21 54237673; fax: þ86 21 54237465. ** Corresponding author. Tel./fax: þ86 311 86261191. E-mail addresses: [email protected] (S. Jiang), [email protected] (L. Wei). 1 These authors contributed equally to this work.
50% of children aged 8e13 years infected by GAS in five cities of China, even higher at 70%w80% in the countryside [3]. Efforts to develop an effective vaccine for GAS had focused on the M protein, however, its type specificity and the cross-reactivity of some common epitopes with human tissues impels researchers to look to other GAS surface proteins [4e8]. In our previous research, FbaA, a novel fibronectinbinding protein [9], has shown effective immunogenicity and immune protection against GAS [10], making it a potentially successful vaccine candidate against GAS. Particularly, an immunodominant epitope, Fba96-118 [11], and six linear epitopes were identified by monoclonal antibody FbaAmab2 and phage display technology. Based on an epidemiological investigation in China, we selected four GAS serotypes, M1,
http://dx.doi.org/10.1016/j.micinf.2014.03.006 1286-4579/Ó 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Ma C, et al., FbaA- and M protein-based multi-epitope vaccine elicits strong protective immune responses against group A streptococcus in mouse model, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.03.006
2
C. Ma et al. / Microbes and Infection xx (2014) 1e10
M3, M6 and M18, which are known to be the predominant cause and are either currently, or have historically been, associated with ARF in China [12]. Here we constructed a recombinant multivalent vaccine, designated F7M5, which contains the seven predominant epitopes of FbaA and five non-tissue cross-reactive M protein fragments expressed on the above four serotypes, as well as a Trojan antigen (TA) and a poly-alanine DR epitope (PADRE) that were expected to promote the delivery of exogenous antigens into the intracellular compartments of antigen presenting cells (APCs) and to balance Th lymphocyte responses, respectively [13e16]. Our results demonstrated that F7M5 could provide comparable levels of protection against bacterial challenge with no induction of cross-reactivity in vivo, suggesting its ability to feasibly prevent GAS infections across a broad spectrum of strains in China. 2. Materials and methods 2.1. Ethics statement This study was approved by the Institutional Ethics Committee of Hebei Medical University (Ethics statement number: 2012019) and was granted exempt status because the leftover blood from clinics were de-identified and would normally have been discarded. Therefore, informed consent of the study subject was not required. 2.2. Materials Streptococcal strain GAS SF370 M1 was stored at 80 C and routinely grown at 37 C in Todd Hewitt Broth supplemented with 0.5% yeast extract (THB; Difco, MI). Protein concentrations were determined by MicroBSA and Bradford protein assay kit (Thermo Fisher Scientific, USA). DNA concentrations were determined spectrophotometrically. 2.3. Preparation and characterization of multivalent vaccine Polyclonal antibodies against FbaA were prepared [10], purified and used to screen peptides and mimotopes of FbaA protein with phage display technology, as previously described [11], seven epitopes of FbaA, including the epitope specific for FbaAmab2 [11], had been screened and linked with five glycines by PCR, termed F7. Synchronously, four gene segments from the M-protein N-terminus of each serotype and their conserved sequence (J14) had been selected to link to F7 in the order of 1-3-6-18-J14 by PCR (termed F7M5). Additionally, TA and PADRE were designed (Fig. 1a) at the N-terminus of F7M5. PCR products were digested with BamHI and HindIII, and ligated with BamHI-/HindIII-restricted pET28a. All constructs were verified by DNA sequencing. Primers used were listed in Table S1. Recombinant plasmid pET28a/F7M5-transformed Escherichia coli BL21 was induced with IPTG (0.1 mM, Merck) to express recombinant protein F7M5, which was purified by
Fig. 1. Design of FbaA- and M protein-based multi-epitope vaccine against group A streptococcus. (a) Schematic representation of the construction of recombinant multivalent vaccine F7M5. (b) Sequences of the FbaA epitopes and M protein fragment, as well as TA and PADRE in multivalent vaccine F7M5.
HisTrap affinity columns, according to the manufacturer’s instructions (GE Healthcare). FbaA protein and M protein, as control proteins, were purified as previously described [10]. 2.4. Animal immunizations Female BALB/c mice, aged 5e6 weeks, were purchased from Beijing Laboratory Animal Center, housed and manipulated according to the Care and Use of Laboratory Animals (Beijing), and kept under specific pathogen-free conditions. 20 mice of each group were immunized at 0, 2, and 4 weeks with 20 mg per dose of F7M5, FbaA or M protein formulated with complete Freund’s adjuvant (FA, Sigma) for priming and incomplete FA for boost. All immunizations were performed via subcutaneous route. Serum samples were collected before the first immunization and on day 10 after each immunization. Half of the mice in each group were sacrificed to detect humoral and cell-mediated immune response, and the remaining mice were challenged with GAS. 2.5. ELISA Enzyme-linked immunosorbent assay (ELISA) was performed to measure IgG antibody titers, the IgG subclasses, or the positive rate of antistreptolysin O (ASO)-positive patient sera when F7M5 was coated on 96-well plates as a diagnostic antigen. Briefly, 96-well plates were coated with purified F7M5, FbaA or M protein (10 mg/ml) in carbonate buffer at 4 C overnight and then blocked with 3% bovine serum albumin (BSA) in PBST. Serum samples were initially diluted 1:100 in 2% BSA in PBST and transferred into coatedblocked plates. Upon removing the unbound antibody, the plates were incubated with 1:5000e10,000 dilution of HRPlabeled second antibody (IgG, IgG1 or IgG2a) (Bio-Rad).
Please cite this article in press as: Ma C, et al., FbaA- and M protein-based multi-epitope vaccine elicits strong protective immune responses against group A streptococcus in mouse model, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.03.006
C. Ma et al. / Microbes and Infection xx (2014) 1e10
Positives were indicated by a fold increase in absorbance at 450 nm between samples and negative control, and the antibody titers were expressed as the reciprocal of the last sample dilution, giving an absorbance at least two-fold higher than that of negative control, with an optical density 0.15. We also used ELISA to measure the cytokine levels in the supernatants of cultured splenocytes from the immunized mice with an ELISA kit obtained from eBioscience (San Diego, CA) according to the manufacturer’s instruction. 2.6. Real-time PCR analysis The mouse splenocytes were harvested after the immunized animals were sacrificed, followed by isolation of total RNA using the RNeasy (Takara). RT-PCR analysis was performed using oligo (dT) and random primers at an annealing temperature of 60 C and with 35 amplification cycles following the manufacturer’s instructions. Each sample was assayed three times. Primer pairs were as follows: IL-4: 50 -GGAGATGGATGTGCCAAACG-30 (sense), and 50 -GCACCTTGGAAGCC CTACA G-30 (antisense); IL-6: 50 - AAG GAG TGG CTA AGG ACC AA -30 (sense), and 50 - GTT TGC CGA GTA GAT CTC AAA -30 (antisense); IFN-g: 50 -AGCAACAGCAAGGCGAAAAA-30 (sense) and 50 -TGGTGGACCACTCGGATG A-30 (antisense); b-actin: 50 -TACCCA GGCTTAGCTGACAGG-30 (sense) and 50 -ACTTGCGGTGCACGATGGA-30 (antisense).
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2.9. Challenge with GAS The immunized animals were intraperitoneally (i.p.) challenged on day 10 after the last immunization with a lethal dose of M1GAS (5 107 CFU per mouse). Animals were monitored on a daily basis and euthanized when they exhibited defined humane endpoints. 2.10. HE staining and immunohistochemistry The mouse hearts and kidneys were separated from the sacrificed mice, and fixed in 4% buffered paraformaldehyde and routinely processed for hematoxylin-eosin (HE) staining. The pathological examinations were performed with a transmission electron microscope (Nikon, Japan). 2.11. Statistics Unpaired t-test was applied to natural logarithm values of serum ELISA titers to determine the differences between the immunization groups. ManneWhitney t-test was used to determine the differences in protection level. The resulting Pvalues of
MODEL
Microbes and Infection xx (2014) 1e10 www.elsevier.com/locate/micinf
Original article
FbaA- and M protein-based multi-epitope vaccine elicits strong protective immune responses against group A streptococcus in mouse model Cuiqing Ma a,1, Zheng Liu a,1, Wenjian Li a, Xuesong Qian a, Song Zhang c, Xue Gao a, Shibo Jiang b,*, Lin Wei a,** b
a Department of Immunology, Basic Medical College, Hebei Medical University, Shijiazhuang 050017, China Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Shanghai Medical College and Institute of Medical Microbiology, Fudan University, Shanghai 200032, China c Third Hospital of Hebei Medical University, Shijiazhuang 050000, China
Received 10 January 2014; accepted 20 March 2014
Abstract We report the construction of a recombinant multivalent vaccine against group A streptococcus (GAS), designated F7M5. It contains seven predominant epitopes of FbaA identified by phage display technology, five non-tissue cross-reactive M protein fragments expressed on four selected serotypes prevalent in China, a Trojan antigen (TA) and a poly-alanine DR epitope (PADRE). BALB/c mice were immunized subcutaneously with F7M5 formulated with Freund’s adjuvant, using recombinant FbaA and M protein in parallel as control. Using enzyme-linked immunosorbent assay (ELISA), mouse immune sera were assayed for IgG titers, IgG subclasses, and binding of F7M5 with M1GAS. Results indicated that the multivalent vaccine was highly immunogenic and elicited a balanced IgG1/IgG2a response. We also tested the reactivity of F7M5 to antistreptolysin O (ASO) antibodies in sera of GAS-infected patients and found a 95.8% positive rate, indicating that the epitopes of the vaccine were widely expressed in the prevalent serotypes of GAS. More importantly, the F7M5 vaccine elicited strong protective immune responses against lethal-dose challenge with a survival rate of 90%, but induced no cross-reactions or pathological lesions in mouse model, suggesting that F7M5 can be further developed as an effective and safe anti-GAS vaccine. Ó 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Group A streptococcus (GAS); Epitope; Vaccine; FbaA; M protein
1. Introduction Group A streptococcus (GAS), a common Gram-positive bacterium, is capable of causing serious invasive infection and autoimmune diseases. In developing countries, acute rheumatic fever (ARF) remains rampant and can be followed by rheumatic heart disease, causing hundreds of thousands of deaths every year [1,2]. Epidemiological investigation found
* Corresponding author. Tel.: þ86 21 54237673; fax: þ86 21 54237465. ** Corresponding author. Tel./fax: þ86 311 86261191. E-mail addresses: [email protected] (S. Jiang), [email protected] (L. Wei). 1 These authors contributed equally to this work.
50% of children aged 8e13 years infected by GAS in five cities of China, even higher at 70%w80% in the countryside [3]. Efforts to develop an effective vaccine for GAS had focused on the M protein, however, its type specificity and the cross-reactivity of some common epitopes with human tissues impels researchers to look to other GAS surface proteins [4e8]. In our previous research, FbaA, a novel fibronectinbinding protein [9], has shown effective immunogenicity and immune protection against GAS [10], making it a potentially successful vaccine candidate against GAS. Particularly, an immunodominant epitope, Fba96-118 [11], and six linear epitopes were identified by monoclonal antibody FbaAmab2 and phage display technology. Based on an epidemiological investigation in China, we selected four GAS serotypes, M1,
http://dx.doi.org/10.1016/j.micinf.2014.03.006 1286-4579/Ó 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Ma C, et al., FbaA- and M protein-based multi-epitope vaccine elicits strong protective immune responses against group A streptococcus in mouse model, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.03.006
2
C. Ma et al. / Microbes and Infection xx (2014) 1e10
M3, M6 and M18, which are known to be the predominant cause and are either currently, or have historically been, associated with ARF in China [12]. Here we constructed a recombinant multivalent vaccine, designated F7M5, which contains the seven predominant epitopes of FbaA and five non-tissue cross-reactive M protein fragments expressed on the above four serotypes, as well as a Trojan antigen (TA) and a poly-alanine DR epitope (PADRE) that were expected to promote the delivery of exogenous antigens into the intracellular compartments of antigen presenting cells (APCs) and to balance Th lymphocyte responses, respectively [13e16]. Our results demonstrated that F7M5 could provide comparable levels of protection against bacterial challenge with no induction of cross-reactivity in vivo, suggesting its ability to feasibly prevent GAS infections across a broad spectrum of strains in China. 2. Materials and methods 2.1. Ethics statement This study was approved by the Institutional Ethics Committee of Hebei Medical University (Ethics statement number: 2012019) and was granted exempt status because the leftover blood from clinics were de-identified and would normally have been discarded. Therefore, informed consent of the study subject was not required. 2.2. Materials Streptococcal strain GAS SF370 M1 was stored at 80 C and routinely grown at 37 C in Todd Hewitt Broth supplemented with 0.5% yeast extract (THB; Difco, MI). Protein concentrations were determined by MicroBSA and Bradford protein assay kit (Thermo Fisher Scientific, USA). DNA concentrations were determined spectrophotometrically. 2.3. Preparation and characterization of multivalent vaccine Polyclonal antibodies against FbaA were prepared [10], purified and used to screen peptides and mimotopes of FbaA protein with phage display technology, as previously described [11], seven epitopes of FbaA, including the epitope specific for FbaAmab2 [11], had been screened and linked with five glycines by PCR, termed F7. Synchronously, four gene segments from the M-protein N-terminus of each serotype and their conserved sequence (J14) had been selected to link to F7 in the order of 1-3-6-18-J14 by PCR (termed F7M5). Additionally, TA and PADRE were designed (Fig. 1a) at the N-terminus of F7M5. PCR products were digested with BamHI and HindIII, and ligated with BamHI-/HindIII-restricted pET28a. All constructs were verified by DNA sequencing. Primers used were listed in Table S1. Recombinant plasmid pET28a/F7M5-transformed Escherichia coli BL21 was induced with IPTG (0.1 mM, Merck) to express recombinant protein F7M5, which was purified by
Fig. 1. Design of FbaA- and M protein-based multi-epitope vaccine against group A streptococcus. (a) Schematic representation of the construction of recombinant multivalent vaccine F7M5. (b) Sequences of the FbaA epitopes and M protein fragment, as well as TA and PADRE in multivalent vaccine F7M5.
HisTrap affinity columns, according to the manufacturer’s instructions (GE Healthcare). FbaA protein and M protein, as control proteins, were purified as previously described [10]. 2.4. Animal immunizations Female BALB/c mice, aged 5e6 weeks, were purchased from Beijing Laboratory Animal Center, housed and manipulated according to the Care and Use of Laboratory Animals (Beijing), and kept under specific pathogen-free conditions. 20 mice of each group were immunized at 0, 2, and 4 weeks with 20 mg per dose of F7M5, FbaA or M protein formulated with complete Freund’s adjuvant (FA, Sigma) for priming and incomplete FA for boost. All immunizations were performed via subcutaneous route. Serum samples were collected before the first immunization and on day 10 after each immunization. Half of the mice in each group were sacrificed to detect humoral and cell-mediated immune response, and the remaining mice were challenged with GAS. 2.5. ELISA Enzyme-linked immunosorbent assay (ELISA) was performed to measure IgG antibody titers, the IgG subclasses, or the positive rate of antistreptolysin O (ASO)-positive patient sera when F7M5 was coated on 96-well plates as a diagnostic antigen. Briefly, 96-well plates were coated with purified F7M5, FbaA or M protein (10 mg/ml) in carbonate buffer at 4 C overnight and then blocked with 3% bovine serum albumin (BSA) in PBST. Serum samples were initially diluted 1:100 in 2% BSA in PBST and transferred into coatedblocked plates. Upon removing the unbound antibody, the plates were incubated with 1:5000e10,000 dilution of HRPlabeled second antibody (IgG, IgG1 or IgG2a) (Bio-Rad).
Please cite this article in press as: Ma C, et al., FbaA- and M protein-based multi-epitope vaccine elicits strong protective immune responses against group A streptococcus in mouse model, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.03.006
C. Ma et al. / Microbes and Infection xx (2014) 1e10
Positives were indicated by a fold increase in absorbance at 450 nm between samples and negative control, and the antibody titers were expressed as the reciprocal of the last sample dilution, giving an absorbance at least two-fold higher than that of negative control, with an optical density 0.15. We also used ELISA to measure the cytokine levels in the supernatants of cultured splenocytes from the immunized mice with an ELISA kit obtained from eBioscience (San Diego, CA) according to the manufacturer’s instruction. 2.6. Real-time PCR analysis The mouse splenocytes were harvested after the immunized animals were sacrificed, followed by isolation of total RNA using the RNeasy (Takara). RT-PCR analysis was performed using oligo (dT) and random primers at an annealing temperature of 60 C and with 35 amplification cycles following the manufacturer’s instructions. Each sample was assayed three times. Primer pairs were as follows: IL-4: 50 -GGAGATGGATGTGCCAAACG-30 (sense), and 50 -GCACCTTGGAAGCC CTACA G-30 (antisense); IL-6: 50 - AAG GAG TGG CTA AGG ACC AA -30 (sense), and 50 - GTT TGC CGA GTA GAT CTC AAA -30 (antisense); IFN-g: 50 -AGCAACAGCAAGGCGAAAAA-30 (sense) and 50 -TGGTGGACCACTCGGATG A-30 (antisense); b-actin: 50 -TACCCA GGCTTAGCTGACAGG-30 (sense) and 50 -ACTTGCGGTGCACGATGGA-30 (antisense).
3
2.9. Challenge with GAS The immunized animals were intraperitoneally (i.p.) challenged on day 10 after the last immunization with a lethal dose of M1GAS (5 107 CFU per mouse). Animals were monitored on a daily basis and euthanized when they exhibited defined humane endpoints. 2.10. HE staining and immunohistochemistry The mouse hearts and kidneys were separated from the sacrificed mice, and fixed in 4% buffered paraformaldehyde and routinely processed for hematoxylin-eosin (HE) staining. The pathological examinations were performed with a transmission electron microscope (Nikon, Japan). 2.11. Statistics Unpaired t-test was applied to natural logarithm values of serum ELISA titers to determine the differences between the immunization groups. ManneWhitney t-test was used to determine the differences in protection level. The resulting Pvalues of
