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Conjugation Reaction with 8-arm PEG Markedly Improves the Immunogenicity of Mycobacterium Tuberculosis CFP10-TB10.4 Fusion Protein Xiaowei Sun, Weili Yu, Quanhai Pang, and Tao Hu Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 16 May 2017 Downloaded from http://pubs.acs.org on May 16, 2017
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Bioconjugate Chemistry
Conjugation Reaction with 8-arm PEG Markedly Improves the Immunogenicity of Mycobacterium Tuberculosis CFP10-TB10.4 Fusion Protein
Xiaowei Sun1,2,#, Weili Yu2,#, Quanhai Pang1,*, Tao Hu2,*
1
College of Animal Science, Shanxi Agricultural University, Taigu 030801, Shanxi Province, China
2
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China
Running title: Conjugation reaction with 8-arm PEG
#
The two authors contribute equally to the work.
* To whom the correspondence should be addressed.
Tao Hu, State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences No. 1 Bei-Er-Tiao Street, Haidian District, Beijing 100190, China. E-mail:
[email protected]. Tel: +86-10-62555217. Fax: +86-10-62551813.
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ABSTRACT Mycobacterium tuberculosis (Mtb) is a serious fatal pathogen responsible for tuberculosis (TB). Effective vaccination is highly desired for immunoprotection against Mtb infection. CFP10 and TB10.4 are two important immunodominant Mtb-secreted protein antigens, which suffer from poor immunogenicity. Thus, an antigen delivery system and adjuvants are needed to improve the immunogenicity of the two proteins. A CFP10-TB10.4 fusion protein (CT) was used as the antigen in the present study. Conjugation of 4-6 CT molecules in one entity with 8-arm polyethylene glycol (PEG) acted as an antigen delivery system. Aluminum-loxoribine mixture (A-L) and poly(I:C) functioned as the adjuvants. As compared with CT, the polymerized CT (CT-PEG) elicited significantly higher CT-specific IgG titers, higher Th1- and Th2-type cytokines and higher percentages of CD4+ IFN-γ+ and CD4+ IL-4+ cells in BALB/c mice. The presence of A-L and poly(I:C) could both increase the immune response to CT-PEG. Conjugation reaction with 8-arm PEG showed a predominant driving force to improve the immunogenicity of CT. Pharmacokinetic study in SD rats revealed that conjugation reaction with 8-arm PEG prolonged the systemic circulation of CT and exposure to the immune system. CT-PEG with A-L showed no apparent toxicity to organs, whereas CT-PEG with poly(I:C) displayed some toxicity to organs. Thus, an effective and safe vaccine against Mtb infection could be rationally designed by conjugation reaction of Mtb-secreted protein antigen with 8-arm PEG and subsequent addition of A-L.
Keywords: Mycobacterium tuberculosis, conjugation reaction, polyethylene glycol, antigen delivery, adjuvant, subunit vaccine
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INTRODUCTION Mycobacterium tuberculosis (Mtb) is a serious fatal pathogen that gives rise to tuberculosis (TB), one of the most leading infectious diseases in the world.1 Due to the increased antibiotic resistance of Mtb, effective vaccination is highly desired to fight against the TB epidemic and decrease the current disease burden.2-3 As the only approved vaccine against Mtb, BCG vaccine consists of attenuated Mycobacterium bovis and leads to efficient immunoprotection in infants.4 However, BCG vaccine fails to protect adolescent or adults against pulmonary TB and in some cases harmful to immunodeficient individuals.4 As compared with traditional vaccines based on attenuated or inactivated whole pathogens, protein-based subunit vaccine is potentially safer without any virulence factors and easier to produce.5-6 Mtb-secreted protein antigens are important virulence determinants, and of potential to replace or boost BCG vaccine.7-8 However, the poor immunogenicity of protein antigens severely limits their immunoprotection against Mtb infection. Thus, antigen delivery systems and exogenous immune activating components are urgently needed to improve the immunogenicity of protein antigens.9-10 Antigen delivery systems have been used to improve the immunogenicity of protein antigens.11 For example, nanoparticles loaded with antigens show high immunogenicity in mice.12 However, toxicity and mass production of nanoparticles still remain challenges for development of nanoparticle-based vaccine.13 Conjugation of antigen with adjuvant acts as another delivery system to improve the immunogenicity of antigen, which can insure that both antigen and adjuvant reach the APCs simultaneously.14-15 Alternatively, self-conjugation of antigens can possibly act as an antigen delivery system. For example, a series of homologous oligomerized 3
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protein domains were shown to augment both B and T cell responses in mice.16 Polyethylene glycol (PEG) is a linear biocompatible and hydrophilic polymer that has been approved by FDA for human use.17 PEG has been used as a spacer arm between meningococcal polysaccharide (PS) and carrier protein to elongate their spatial distance. Thus, PEG decreased the steric shielding effect of carrier protein on PS and improved the PS-specific immunogenicity of meningococcal conjugate vaccine.17 Although self-conjugation of antigen has been carried out for antigen delivery, conjugation reaction with multi-functional PEG is seldom used to improve the immunogenicity of antigens. As exogenous immune activating components, adjuvants have been used to improve the cellular and humoral immune response to protein antigens.18 In particular, cellular immunity has been considered as a paradigm to play a predominant role in protection against intracellular Mtb infection.19 As a synthetic dsRNA adjuvant, polyriboinosinic polyribocytidylic acid (poly(I:C)) can boost a strong cellular immunity via TLR3.20 However, some evidences suggest that humoral immunity can have a protective role against Mtb at various stages of infection.21 For example, protective antibodies could promote ingestion by phagocytic cells and increase intracellular killing through FcR-mediated phagocytosis.21 Such antibodies could also modify the intensity of the inflammatory response and activate complement to promote phagocytosis and inflammation. Thus, successful vaccines against intracellular Mtb infections could be achieved by induction of mixed cellular and humoral immunity. Aluminum salt is a widely utilized adjuvant to stimulate humoral immunity.22 7-Allyl-7,8-dihydro-8-oxo-guanosine (i.e., loxoribine) is a synthetic Toll like receptor 7 (TLR7a) that activates immune cells via TLR7.23 Agonists of TLR7a can initiate a cascade of immune responses to impact the magnitude and the 4
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persistence of the immune response. Numerous studies have demonstrated that these molecules augment human DC maturation and enhance cellular and humoral adaptive immunity.24 Aluminum-loxoribine mixture (A-L) and poly(I:C) are expected to combine with a proper antigen delivery system to improve the immunogenicity of Mtb-secreted protein antigens. CFP10 and TB10.4 are two important immunodominant protein antigens secreted by Mtb, which belong to the ESAT6 protein family.25-26 Mtb-secreted antigen fusion proteins typically appear to ensure broad immunological coverage in humans than individual protein antigen or a mixture of two antigens.27 Thus, a CFP10-TB10.4 fusion protein (CT) was used as the Mtb-secreted antigen in the present study. Conjugation reaction of CT with 8-arm PEG N-hydroxysuccinimide ester (8-arm PEG) could act as an antigen delivery system for CT. A-L and poly(I:C) were physically mixed with the polymerized CT (CT-PEG) to function as adjuvants, respectively. Our study demonstrated that CT-PEG stimulated a robust humoral and cellular immune response to CT in BALB/c mice. The presence of adjuvants further enhanced the immune response. Thus, an effective vaccine against Mtb infection was expected to be designed by conjugation reaction of protein antigen with 8-arm PEG and subsequent addition of adjuvants.
RESULTS Purification of CT-PEG. CT-PEG was purified from the reaction mixture by a Superdex 200 column (2.6 cm × 60 cm), based on size exclusion chromatography (SEC). As shown in Fig. 1a, two well-separated elution peaks corresponding to CT-PEG and PEG were observed, indicating good separation of CT-PEG from free PEG. The larger peak corresponding to PEG was due to the presence of NHS moieties 5
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in 8-arm PEG. The peak corresponding to CT-PEG was fractionated as the arrow indicated in the inset (Fig. 1a). SDS-PAGE. As shown in Fig. 1b, CT showed a single electrophoresis band corresponding to ~26.0 kDa (Lane 2, the right gel). In contrast, CT-PEG displayed an apparent disperse band with much slower mobility. The disperse band corresponded to an apparent Mw distribution of 90-200 kDa, along with major Mw distribution of 110-170 kDa (Lane 3, the left gel). The iodine stain of CT-PEG (Lane 3, the middle gel) indicated that CT was successfully conjugated with 8-arm PEG. Thus, CT-PEG was obtained by conjugation of 4-6 CT molecules with one PEG (10 kDa). Moreover, free CT and free PEG reagent were absent in CT-PEG (Lane 3). 400
a
b 16
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kDa 1
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Elution time (min)
66.4
CT-PEG
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0 0
Iodine stain
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kDa
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PEG
CT-PEG
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30
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94.0 66.2 43.0 31.0 20.1 14.4
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Elution time (min)
Figure 1 Purification and analysis of CT-PEG. CT-PEG was purified from the reaction mixture by a Superdex 200 column (2.6 cm × 60 cm) (a). The peak corresponding to CT-PEG was fractionated as indicated in the inset (Fig. 1a). SDS-PAGE analysis of CT-PEG (b) was conducted using a 4-14% gradient polyacrylamide gel (left) and 12% polyacrylamide gel (right), respectively. The middle gel was stained with iodine for PEG detection, followed by Coomassie blue R-250 for protein detection. Lanes 1 and 5, marker; Lane 2, CT; Lane 3, CT-PEG; Lane 4, PEG. Reverse Phase HPLC. The reverse phase HPLC pattern of CT showed a single symmetric elution peak. In contrast, CT-PEG was eluted as three elution peaks that 6
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were later than CT (Fig. 2). The three peaks suggested that there were three major components in CT-PEG. In addition, the elution peak corresponding to CT was completely disappeared for CT-PEG, indicating that free CT was absent in CT-PEG.
Absorbance at 210 nm
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CT
CT-PEG
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Time (min)
Figure 2 Reverse performance HPLC analysis of CT-PEG. RP-HPLC analysis was carried out using a Proteonavi C4 column (0.46 cm × 25 cm). The column was eluted by a linear gradient of 35%-75% acetonitrile containing 0.1% trifluoroacetic acid for 50 min at a flow rate of 0.5 ml/min.
SEC Analysis. CT-PEG was analyzed by an analytical Superdex 200 column (1 cm × 30 cm). As shown in Fig. 3a, CT was eluted as a single symmetric peak at 30.4 min, indicating high purity of CT. CT-PEG was eluted as an asymmetric broad peak at 20.1 min that was earlier than CT. The earlier elution of CT-PEG indicated that the molecular size of CT-PEG was higher than that of CT, due to the conjugation reaction of CT with 8-arm PEG. The asymmetric broad peak reflected the heterogeneity of CT-PEG. The apparent Mw of CT was calculated to be ~140 kDa, indicating that the average of five CT molecules were polymerized with one 8-arm PEG. DLS Analysis. DLS analysis was used to measure the molecular radii of CT and its derivatives. The molecular radius of CT-PEG (6.7±0.3 nm) were higher than that 7
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of CT (3.0±0.2 nm) (P
