Potent Antigen-Adjuvant Delivery System by Conjugation of

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A potent antigen-adjuvant delivery system by conjugation of Mycobacterium tuberculosis Ag85B-HspX fusion protein with arabinogalactan-poly(I:C) conjugate Qingrui Huang, Weili Yu, and Tao Hu Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00116 • Publication Date (Web): 22 Mar 2016 Downloaded from http://pubs.acs.org on March 24, 2016

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Bioconjugate Chemistry

A potent antigen-adjuvant delivery system by conjugation of Mycobacterium tuberculosis Ag85B-HspX fusion protein with arabinogalactan-poly(I:C) conjugate

Qingrui Huang 1, 2, #, Weili Yu 1, 2, #, Tao Hu1,*

1

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China

2

University of Chinese Academy of Sciences, Beijing 100190, China

#

The authors contribute equally to the work

Running title: Antigen-adjuvant delivery system

* To whom the correspondence should be addressed. Tao Hu, National 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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Table of contents

1200000

AH-specific IgG titers

Poly(I:C) AG Conjugation

Ag85B-HspX (AH)

900000

600000

300000

AH-AG-P

0

AH

AH-AG

AH-P AH-AG-P

AH-specific IgG titers

400000 60

IFN-γ (ng/ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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40

20

0

AH

AH-AG

AH-P

AH AH-AG AH-P AH-AG-P

300000

200000

100000

Second dose

0 0

AH-AG-P

60

120

180

240

Time (h)

2


300

360

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ABSTRACT Protein-based vaccine is promising to improve or replace Mycobacterium bovis BCG vaccine for its specificity, safety and easy production. However, protein-based vaccine calls for potent adjuvants and improved delivery systems to protect against Mycobacterium tuberculosis. Poly(I:C) is one of the most potent pathogen-associated molecular patterns that signals primarily via TLR3. Arabinogalactan (AG) is a biocompatible polysaccharide that can increase splenocyte proliferation and stimulate macrophages. The AG-poly(I:C) conjugate (AG-P) showed an adjuvant potency through a synergistic interaction of AG and poly(I:C). Ag85B and HspX are two important virulent protein antigens of Mycobacterium tuberculosis and Ag85B-HspX fusion protein (AH) was prepared. An antigen-adjuvant delivery system (AH-AG-P) was developed by conjugation of AH with AG-P to insure that both AH and AG-P reach the APCs simultaneously. AH-AG-P elicited high AH-specific IgG titers and stimulated lymphocyte proliferation. AH-AG-P provoked the secretion of Th1-type cytokines (TNF-α, IFN-γ and IL-2) and Th2-type cytokines (IL-4 and IL-10). Pharmacokinetics revealed that conjugation with AG-P could prolong the serum exposure of AH to the immune system. Pharmacodynamics suggested that conjugation with AG-P led to a rapid and intense production of AH-specific IgG. Accordingly, conjugation with AG-P could promote a robust cellular and humoral immune response to AH. Thus, conjugation of AH with a potent adjuvant AG-P is an effective strategy to develop an efficacious protein-based vaccine against Mycobacterium tuberculosis.

Keywords: Mycobacterium tuberculosis; poly(I:C); arabinogalactan; adjuvant

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INTRODUCTION Mycobacterium tuberculosis is a major causative agent of tuberculosis (TB), a serious intracellular infection disease

in the

world.1 The emergence of

multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has remarkably increased the difficulty in TB treatment.2-3 Vaccination is an efficient strategy to deal with these severe situations. Mycobacterium bovis BCG is the only available vaccine that has been used for more than ninety years. However, BCG has poor protection and can not prevent the reactivation of dormant M. tuberculosis.4 Alternatively, protein-based vaccine is promising to improve or replace BCG vaccine for its specificity and safety.5 However, it often suffers from poor immunogenicity due to lack of ‘danger signals’ or pathogen-associated molecular patterns (PAMPs) to activate the immune system.6 Thus, protein-based vaccine calls for potent adjuvants and improved delivery systems to protect against M. tuberculosis. Recently, 2-methoxyethoxy-8-oxo-9-(4-carboxy benzyl)adenine (1V209, a TLR7 ligand) was conjugated with dextran (a non-adjuvant polysaccharide).7 Dextran can act as a carrier to improve water solubility and potency of 1V209.7 In addition, a TLR7 agonist was conjugated with an antigen from Streptococcus pneumoniae to increase the anti-antigen response.8 A conjugate consisting of two different adjuvants is another strategy to obtain a potent adjuvant. For example, two Toll-like receptor (TLR) ligands (CpG and lipoteichoic acid) were conjugated to obtain stronger immune cell stimulation relative to a lipoteichoic acid-CpG mixture.9 Polyriboinosinic-polyribocytidylic acid (poly(I:C)) is a mismatched synthetic double-stranded RNA with one strand being a polymer of inosinic acid, the other a polymer of cytidylic acid. Poly(I:C) is one of the most potent PAMPs that signals primarily via TLR3.10 Recent studies suggest that poly(I:C) is crucial to enhance the

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immune response against intracellular pathogens, including a strong Th1-type immune response characterized by high IgG titers, high IFN-γ titers and T cell proliferation.11 Arabinogalactan (AG) is a biocompatible and mass-producible natural polysaccharide.12 AG can increase splenocytes proliferation, stimulate macrophages and enhance the relevant cytokine releases.13 Thus, a potent adjuvant could be achieved by conjugation of AG and poly(I:C). An efficacious antigen-adjuvant delivery system is important to achieve a strong immune response to the antigen. Typically, antigen was delivered by mixing with adjuvants. However, simple mixing allows activating antigen presenting cells (APCs) that do not present the delivered antigen and potentially induce autoimmune responses.14-15 In addition, high adjuvant dose is required to maintain a satisfactory immune response to the antigen and leads to unwanted side effect. Nanoparticles loaded with antigen and adjuvant are very immunogenic in mice.16 However, toxicity and mass-production of nanoparticles still remain challenges.17 Alternatively, an antigen-adjuvant conjugate can insure that both antigen and adjuvant reach the APC simultaneously, achieving a stronger immune response than an antigen-adjuvant mixture.18 In addition, an antigen-adjuvant conjugate can minimize the side effects of adjuvant by decreasing the adjuvant dose and ensure the activation of APCs that have taken up the delivered antigen. However, the conjugate comprising two different adjuvants has seldom been used for covalent conjugation of protein antigens. Protein antigens of M. tuberculosis with a relatively strong immunogenicity are important for protein-based vaccine.6 Fusion proteins typically appear to evoke a more potent immune response than individual protein antigen or a mixture of two proteins.19 Ag85B is an early protein secreted by replicating M. tuberculosis and can induce a strong Th1-type immune response.20 HspX is an important dominant antigen

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secreted by non-replicating bacilli and can induce a strong immune response in latently infected individuals.21 Thus, an Ag85B-HspX fusion protein has been expressed and used as a multi-stage vaccine against M. tuberculosis.21 In the present study, an Ag85B-HspX fusion protein (AH) was genetically expressed and used as an antigen protein. An AG-poly(I:C) conjugate (AG-P) was prepared to obtain a potent adjuvant. An antigen-adjuvant delivery system (AH-AG-P) was developed by covalent conjugation of AH with AG-P. The immunological properties of AH-AG-P were measured for evaluating the adjuvant potency of AG-P. The pharmacokinetic and pharmacodynamic properties of AH-AG-P were measured for evaluating the delivery potency of AH-AG-P. Our study was expected to pave a foundation for developing an efficacious vaccine against M. tuberculosis.

RESULTS Purification and characterization of AH. As revealed by SDS-PAGE analysis (Fig. 1a), the fusion protein AH was predominantly present in the form of inclusion body (Lane 3). The inclusion body was subsequently dissolved and renaturated, followed by purification using a Q Sepharose HP column (2.6 cm×15 cm) (Fig. 1b). Two major peaks were observed and the fractions corresponding to Peak 2 were pooled and concentrated. As shown in the inset (Fig. 1b), AH showed a single band corresponding to an apparent Mw of ∼51 kDa that indicated a high purity of AH. MALDI-TOF analysis indicated that AH had an Mw of 51730.5 Da, which was close to the theoretical Mw of AH (51725.9 Da). Size exclusion chromatography. AH and its derivatives were analyzed by a Superose 6 column (1 cm×30 cm). As shown in Fig. 1c, AH was eluted as a single and symmetric peak at 28.5 min. In contrast, AH-AG and AH-P were both eluted as a

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single peak at 18.9 min and 16.9 min, respectively. The two peaks were left-shifted as compared with that of AH, due to the increased hydrodynamic volume of AH by conjugation of AG or poly(I:C). AH-AG-P was eluted with an elution peak at 15.0 min, indicating the highest hydrodynamic volume among the four samples. DLS analysis. DLS analysis was used to measure the molecular radii of AH and its derivatives. The molecular radii of AH-AG (7.1 nm) and AH-P (8.5 nm) were higher than that of AH (4.7 nm) (P

Potent Antigen-Adjuvant Delivery System by Conjugation of

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