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Prompt and Robust Humoral Immunity Elicited by a Conjugated Chimeric Malaria Antigen with a Truncated Flagellin Fangxia Guo, Yong Dong Liu, Chun Zhang, Qi Wang, Lianyan Wang, Yuhui Gao, Jingxiu Bi, Heng Wang, and Zhiguo Su Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00320 • Publication Date (Web): 10 Aug 2017 Downloaded from http://pubs.acs.org on August 11, 2017
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Table of contents graphic 85x51mm (300 x 300 DPI)
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Prompt and Robust Humoral Immunity Elicited by a
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Conjugated Chimeric Malaria Antigen with a Truncated
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Flagellin
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Fangxia Guo†,‡ , Yongdong Liu†,*, Chun Zhang†, Qi Wang†,‡, Lianyan Wang†, Yuhui Gao§, Jingxiu Bi┴, Heng Wang§, Zhiguo Su†,*
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†
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Academy of Sciences, Beijing 100190, PR China
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‡
University of Chinese Academy of Sciences, Beijing 10049, PR China
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§
Molecular Parasitology Laboratory, Peking Union Medical College, Chinese Academy of Medical
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Sciences, Institute of Basic Medical Sciences, Beijing 100005, PR China
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┴
School of Chemical Engineering, The University of Adelaide, Adelaide SA, 5005, Australia
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∗
Corresponding authors at National Key Laboratory of Bioengineering, Institute of Process
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Engineering, Chinese Academy of Sciences, Beijing 100190, PR China. Tel.: +86 10 82545028/+86 10
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62561817; E-mail addresses:
[email protected] (Y. Liu),
[email protected] (Z. Su).
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese
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Abstract
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As one of the pathogen-associated molecular patterns (PAMPs), flagellin is recently
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utilized as a potent adjuvant for many subunit vaccines. In this study, a truncated flagellin
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(tFL) with deletion of the hypervariable regions was adopted as a carrier-adjuvant by
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chemical conjugation with a chimeric malaria antigen M.RCAg-1 (M312) via a
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hetero-bifunctional PEG linker. After boosting immunization in mice without any extra
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adjuvants, the M312-PEG-tFL conjugates elicited 100-1,000 times higher M312-specific
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antibody titers than M312, and 10-100 times higher than the physical mixture of M312 and
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tFL. The elicited specific antibodies could recognize the native parasites and the
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immune-fluorescence assay (IFA) titer was 2,100 for M312-P5k-tFL, which was about 7
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times higher than M312. Furthermore, the IFA titers of the conjugates were comparable with
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the positive control of complete Freund’s adjuvant (CFA). Compared with M312, the
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M312-PEG-tFL conjugates enhanced the proliferation index, lymphocyte activation and
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memory T cell generation. IgG subclasses of sera and cytokines analysis of splenocytes
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showed that conjugation with tFL could slightly trigger the Th1 polarization, while the
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antigen alone predominantly induced a Th2-biased immune response. Furthermore, a more
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efficient innate immune response was provoked by the M312-PEG-tFL conjugates, as
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determined by the detection of antigen-specific TNF-α secretion by splenocytes. Our results
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indicated that tFL mainly retained the function as an agonist of TLR5. Conjugation of antigen
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to tFL could induce strong humoral and moderate cellular immune responses. Thus,
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conjugation of antigen to tFL as a potent carrier-adjuvant is an effective strategy to develop a
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promising protein-based vaccine.
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Key Words: malaria vaccine; flagellin; TLR5; adjuvant; conjugate vaccine; immunostimulant
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INTRODUCTION
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Protein or peptide-based subunit vaccines are considered as the new generation vaccine
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species with the merits of safety and cost-effective preparation.1 To reduce the burden of
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malaria, numerous protein-based vaccine candidates have been successfully produced; some
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progressed to clinical testing.2 However, like many other subunit vaccines, the protein-based
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malaria vaccines have largely yielded disappointing outcomes in clinical investigations,
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mainly due to the weak immunogenicity.3,4 So it is urgently required to enhance the
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immunogenicity of malaria subunit vaccines via additional immunostimulant that is capable
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of eliciting much stronger immune responses to prevent parasites from rapidly invading or
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developing.5
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Developing a conjugate vaccine is an effective strategy to enhance the immunogenicity
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of protein-based malaria antigens by combination of the antigen and immunostimulant (also
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called as carrier protein or adjuvant) as an entity through genetic fusion or chemical
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conjugation.6,7 Actually, the carrier protein diphtheria toxoid (DT), which is widely used for
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polysaccharide conjugate vaccines,8,9 was adopted to overcome the incapability of inducing
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specific antibodies to recognize the recombinant Plasmodium yoelii merozoite surface protein
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1 (PyMSP-1).10 Other carriers such as outer-membrane protein complex (OMPC) of Neisseria
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meningitidis serogroup B and Pseudomonas aeruginosa exoprotein A (EPA) were
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demonstrated the ability to enhance the specific antibody responses for several malaria
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subunit vaccines.11,12 Plasmodium falciparum 25 kDa (Pfs25) is a leading malaria
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transmission-blocking protein vaccine candidate. Conjugates consisting of Pfs25 via covalent 4 / 27
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linkage with OMPC or rEPA have been investigated extensively.13,14 Coupling OMPC with
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Pfs25 could enhance 1,000 times more Pfs25-specific antibodies than Pfs25 alone.11 Recently,
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the conjugate Pfs25-rEPA formulated with Alhydrogel has progressed in Phase 1 trial to
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assess its safety and immunogenicity.15 However, high dose and multiple vaccinations are
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needed to induce sufficient Pfs25-specific antibodies because the antibodies still declined to
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the baseline after vaccinated one year.16 The results suggest that a more immunogenic vaccine
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will still be needed to efficiently struggle against malaria through exploring alternate antigens
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or alternate carrier-adjuvants.
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Agonists for toll-like receptors (TLRs) have been widely investigated as potent adjuvants
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especially for the development of subunit vaccines, with function of inducing DCs maturation
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and upregulating costimulatory molecules required for initiation of adaptive immunity.17,18 As
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a pathogen-associated molecular pattern (PAMP), flagellin is the only agonist of toll-like
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receptor 5 (TLR5).19,20 Pattern recognition of TLR5 initiates signal transduction pathways that
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manipulate innate immunity and further instruct the antigen-specific adaptive immunity.21 The
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ability of flagellin to induce both cellular and humoral immunity as a potent adjuvant has
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been proven for several subunit vaccines and even inactivated virus vaccines through physical
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mixture or genetic fusion with the antigens.22 Conjugation is another strategy to take full
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advantages of flagellin as an effective adjuvant.23,24 But data on flagellin-based malaria
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conjugate vaccines is lacking. Recently, a short peptide EXP153 from Plasmodium
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falciparum exported protein-1 (PfEPX-1) was conjugated with recombinant Salmonella
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flagellin (rFliC) to demonstrate the carrier–adjuvant activity.25,26 However, conjugation of
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flagellin to a larger protein-based antigen would be more challenging due to the possible 5 / 27
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disturbance on the active site by steric hindrance.
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For a conjugate malaria vaccine, the antigen is the crucial aspect to provide the
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protection profile.27,28 Because of the complicated life cycle of Plasmodium falciparum, it is
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believed that multi-epitope antigens should provide a wider range of protection than
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single-epitope antigens.29,30 In our previous study, a multi-epitope chimeric antigen named
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M.RCAg-1(hereafter referred to as M312), containing eleven T-cell and B-cell epitope
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antigens of Plasmodium falciparum, has been produced in E.coli and been proven to be
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effective in animal models with CFA.31,32 However, the antigen-specific IgG titers induced by
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M312/Alhydrogel were low, indicating that a more potent formulation is required for inducing
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higher antibody responses.32
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The Salmonella flagellin with full length is composed of highly conserved N/C regions
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crucial for TLR5 agonist activity and the hypervariable regions.33,34 It is reported that the
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hypervariable regions of full length flagellin would induce high reactogenicity and possible
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inflammatory injury that might restrict its clinical usage.35 Here, a truncated form of
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Salmonella flagellin (tFL) with deletion of the hypervariable regions was constructed and
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expressed in E.coli, aiming to strengthen the antigen-specific immune responses by
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conjugating of M312 to tFL through a 2 kDa or 5 kDa PEG linker. After immunization in
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mice, significantly increased antigen-specific antibody titers, splenocytes proliferation index,
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as well as IFA titers were induced by the M312-PEG-tFL conjugates in absence of any other
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adjuvants. Our results indicated this truncated flagellin retained its function as the agonist of
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TLR5, which could profoundly and rapidly enhance the adaptive immune responses for the
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protein-based malaria antigens as a carrier-adjuvant. 6 / 27
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RESULTS
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Expression and Purification of the M312 and tFL in E.coli
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The M312 with a purity above 95% was largely obtained briefly according to the
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previous protocol.36 tFL was expressed as inclusion bodies in E. coli. After refolding and a
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two-step chromatographic purification, tFL was obtained with purity of 95% and molecular
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weight of about 31 kDa (as shown in Figure S1).
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Preparation of the M312-PEG-tFL Conjugates
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Figure 1. Preparation and purification of the conjugates. (A) Schematic representation of M312-PEG-tFL. (B) Q
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FF chromatographic profile of reaction 1 products. (C) Size exchange chromatographic profile of reaction 2
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products. (D) SDS-PAGE analysis. Lane 1: tFL; lane 2: M312; lane 3: The eluted peak for Q FF; lane 4: The
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mixture after reaction 2; lane 5: The final conjugate of M312-P2k-tFL collected from Superdex 200; lane 6: The
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final conjugate for M312-P5k-tFL collected from Superdex 200.
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There are free cysteines exited in M312 protein,32 so a heterobifunctional PEG linker of
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SC-PEG-MAL with the length of 2 kDa and 5 kDa were respectively used to conjugate of tFL
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to M312 (Figure 1A). Firstly, the PEG linker was connected to tFL through the functional
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group of succinimidyl carbonate. The tFL-PEG-MAL conjugates were purified using an anion
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exchange chromatography to remove the extra PEG reagents (Figure 1B). Then
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tFL-PEG-MALs were reacted with M312, and the resulted M312-PEG-tFL conjugates were
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purified using a Superdex 200 media (Figure 1C). As shown in Figure 1D, the final
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conjugates contained two main bands in the gel for both 2 kDa and 5 kDa PEG linkages.
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M312-PEG-tFL Conjugates Elicited Prompt and Robust Antibody Responses
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M312-specific Antibodies
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The sera IgG responses to M312 were determined in BALB/c mice immunized
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subcutaneously with M312, the physical mixture of M312 and tFL, M312-P2k-tFL, and
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M312-P5k-tFL, respectively. All the samples were injected in the absence of any adjuvants
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except the positive control of vaccinating M312 with CFA. As expected, the mice treated by
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the two conjugates exhibited high anti-M312 IgG titers following vaccination, while injection
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with M312 alone induced low IgG titers. On day 24 (Figure 2A), the anti-M312 IgG titers for
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the two conjugates were about 1,000 times higher than M312 alone (for M312-P2k-tFL, p
