Elsevier

Biomaterials

Volume 35, Issue 29, September 2014, Pages 8427-8438
Biomaterials

Branched-linear and agglomerate protein polymers as vaccine platforms

https://doi.org/10.1016/j.biomaterials.2014.06.021Get rights and content

Abstract

Many viral structural proteins and their truncated domains share a common feature of homotypic interaction forming dimers, trimers, and/or oligomers with various valences. We reported previously a simple strategy for construction of linear and network polymers through the dimerization feature of viral proteins for vaccine development. In this study, technologies were developed to produce more sophisticated polyvalent complexes through both the dimerization and oligomerization natures of viral antigens. As proof of concept, branched-linear and agglomerate polymers were made via fusions of the dimeric glutathione-s-transferase (GST) with either a tetrameric hepatitis E virus (HEV) protruding protein or a 24-meric norovirus (NoV) protruding protein. Furthermore, a monomeric antigen, either the M2e epitope of influenza A virus or the VP8* antigen of rotavirus, was inserted and displayed by the polymer platform. All resulting polymers were easily produced in Escherichia coli at high yields. Immunization of mice showed that the polymer vaccines induced significantly higher specific humoral and T cell responses than those induced by the dimeric antigens. Additional evidence in supporting use of polymer vaccines included the significantly higher neutralization activity and protective immunity of the polymer vaccines against the corresponding viruses than those of the dimer vaccines. Thus, our technology for production of polymers containing different viral antigens offers a strategy for vaccine development against infectious pathogens and their associated diseases.

Introduction

Biomaterials have emerged as a promising and important field that may have wide applications in healthcare of twenty-first century. One such application is development of recombinant antigen complexes as non-replicating subunit vaccines to control and prevent infectious diseases that lead to hundreds of thousand deaths worldwide each year. Through various technologies, small antigens or epitopes of infectious pathogens having low immunogenicity can assemble into large polyvalent complexes with high immunogenicity that can be further developed into effective vaccines. Production of these recombinant protein-based subunit vaccines does not contain an infectious agent and therefore, such non-replicating vaccines may be safer than those developed from a conventional vaccine strategy that relies on cultured viruses. Several subunit vaccines have been commercially available for human use [1], including the VLP vaccines against hepatitis B virus (HBV) [2], [3], human papilloma virus (HPV) [4], [5], [6], [7], and hepatitis E virus (HEV) [8], [9]. Furthermore, two other subunit vaccines have been developed for use in domestic pigs against porcine circovirus type 2 (PCV2) infection and diseases [10], [11]. In addition, numerous reports in the literatures have indicated that many other subunit vaccines are under clinical and preclinical evaluations (reviewed in Refs. [1], [12]). Hence, recombinant protein-based subunit vaccines denote a strategy to combat infectious diseases.

Many neutralizing antigens and epitopes, usually identified on large surface proteins of some pathogens, have been defined as the result of extensive characterizations of these pathogens. These defined antigens and epitopes have generally low immunogenic capability due to their small sizes and low valence. Thus, improvement of their immunogenicity is the key to turn the antigens and epitopes into effective vaccines, which can be achieved by a conjugation of the antigens/epitopes to a large polyvalent platform, such as a virus-like particles (VLPs) (reviewed in Refs. [1], [12]). In our previous studies [13], [14], two types of polyvalent complexes, linear and network, were developed to turn the small dimeric viral antigens into large, polyvalent complexes with significantly improved immunogenicity. These polymers can also function as vaccine platforms for presentation of monomeric antigens [14]. Thus, the polyvalent complexes offer a strategy for subunit vaccine development.

Based on the principle of the linear and network polymer formation [13], [14], two more sophisticated polymers were designed and constructed through introduction of oligomeric proteins into the basic fusion protein unit (Fig. 1). Branched-linear polymers assemble when a dimeric protein is fused with a dimeric and tetrameric protein (Fig. 1A), while agglomerate polymers form when a dimeric protein is fused with a multimeric protein (Fig. 1B). The immunogenicity of the two polymers should be improved owing to their significantly increased valency and complexity compared with those of dimers or oligomers. In addition, a monomeric antigen can be inserted into the polymers through a surface loop of a polymer component (Fig. 1C) for increased immunogenicity.

The feasibility of the proposed polymer formations will be examined using the dimeric glutathione S-transferase (GST) of Schistosoma japonicum [15], the modified protruding (P) domain of HEV (forming dimers and tetramers [16], and this report), and the modified P domain of norovirus (NoV, forming 24mer) [17], [18] as models. Additionally, the monomeric M2e epitope of influenza A virus (IAV) [19] and the VP8* antigen of rotavirus (RV) [20] will be used to examine the capability of the polymers as vaccine platforms. Except for GST that is also a commonly used tag for protein purification; the other four are well-defined neutralizing antigens and epitopes of the corresponding viral pathogens that cause significant morbidity and mortality worldwide.

We hypothesize that fusion of the dimeric GST with the dimeric/tetrameric HEV P protein will lead to formation of branched-linear polymers, while fusion of GST with the multimeric NoV P protein will form agglomerate complexes. Monomeric M2e epitope or VP8* antigen will be displayed well by the agglomerate complexes. The polymers will assemble spontaneously when the fusion proteins are produced and purified from Escherichia coli. Most importantly, we anticipate that the resulting polymers will induce strong humoral and cellular immune responses, neutralize virus replication in cell culture, and protect vaccinated animals against virus challenge and thus are promising vaccines against the four viruses. Data from this study will prove the concept of the branched-linear and agglomerate polymers as vaccines and vaccine platforms that may find broad applications in biomedicine.

Section snippets

Plasmid constructs

The plasmid constructs for expression of recombinant fusion proteins were created through the glutathione S-transferase (GST)-gene fusion system (GE Healthcare Life Sciences) using the vector pGEX-4T-1. For expression of the fusion protein of GST with a modified P protein of HEV (GST-HEV P+), a plasmid was constructed by inserting the partial P1 and P2 domain-encoding sequences (residue 452 to 617, GenBank AC#: DQ079627) of a genotype 3 HEV [16], [21] into the pGEX-4T-1 plasmid between BamH I

Production of branched-linear polymers

In a previous study we showed that linear complexes formed spontaneously through intermolecular dimerization when two dimeric proteins were fused together [14]. Based on this principle, we anticipated that branched-linear complexes would form when one of the two dimeric components form tetramers (Fig. 1A). To test this hypothesis, dimeric GST [15] was fused with a modified HEV P protein [16]. We noted that, when a cysteine-containing peptide CDCRGDCFC was linked to the C-terminus of a truncated

Discussion

In a previous study we proposed and verified a simple strategy to turn small dimeric proteins into linear or network polymers for improved immunogenicity [14]. The polymers assembled through intermolecular dimerization of two to three dimeric proteins that were fused together by recombinant DNA technique. In this study, we further developed a technology to produce more sophisticated polymers by introducing an oligomeric protein into the basic units of fusion proteins (Fig. 1). As a proof of

Conclusion

We have proposed an innovative technology and proven its feasibility to turn small, low immunogenic antigens into large polymers with significantly enhanced immunogenicity and functionality. Two polymers, the branched-linear and agglomerate, representing a simple and a very complex polymer, have been made as proofs of the feasibility of our technology. The polymers were easily constructed in the bacterial system and were able to induce significantly higher humoral and cellular immune responses

Acknowledgments

The research described in this article was supported by the National Institute of Health, the National Institute of Allergy and Infectious Diseases (R21 AI092434 to M.T. and R01AI089634 and P01 HD13021 to X.J.) and by an Institutional Clinical and Translational Science Award (NIH/NCRR 8UL1TR000077-04) to M.T. Support was also from the Innovation Fund of Cincinnati Children's Hospital Medical Center to M.T.

References (40)

  • I. Ahmad et al.

    Molecular virology of hepatitis E virus

    Virus Res

    (2011)
  • M. Tan et al.

    Norovirus P particle as a platform for antigen presentation

    Procedia Vaccinol

    (2011)
  • M. Tan et al.

    Terminal modifications of norovirus P domain resulted in a new type of subviral particles, the small P particles

    Virology

    (2011)
  • X.S. Chen et al.

    Papillomavirus capsid protein expression in Escherichia coli: purification and assembly of HPV11 and HPV16 L1

    J Mol Biol

    (2001)
  • J.Z. Bereszczak et al.

    Structure, stability and dynamics of norovirus P domain derived protein complexes studied by native mass spectrometry

    J Struct Biol

    (2012)
  • W.J. McAleer et al.

    Human hepatitis B vaccine from recombinant yeast

    Nature

    (1984)
  • F.E. Andre et al.

    Summary of clinical findings on Engerix-B, a genetically engineered yeast derived hepatitis B vaccine

    Postgrad Med J

    (1987)
  • R. Kirnbauer et al.

    Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic

    Proc Natl Acad Sci U S A

    (1992)
  • A. Proffitt

    First HEV vaccine approved

    Nat Biotechnol

    (2012)
  • P.C. Wu et al.

    Characterization of porcine circovirus type 2 (PCV2) capsid particle assembly and its application to virus-like particle vaccine development

    Appl Microbiol Biotechnol

    (2012)
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    Present address: Animal Disease Diagnostic Laboratory, Ohio Department of Agriculture, 8995 East Main Street, Reynoldsburg, OH 43068, USA.

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