Assembly of synthetic peptide vaccines by chemoselective ligation of epitopes: influence of different chemical linkages and epitope orientations on biological activity

doi:10.1016/S0264-410X(01)00152-9

Vaccine

Volume 19, Issues 28–29, 16 July 2001, Pages 3843-3852

https://doi.org/10.1016/S0264-410X(01)00152-9 Get rights and content

Abstract

In this paper, we describe the assembly of synthetic peptide vaccines composed of a T helper cell epitope and a B cell epitope that were synthesized separately and then attached using three different chemoselective ligation methods: oxime bond formation, thioether bond formation and disulfide bond formation. The resulting vaccines were tested in animals to investigate their efficacy. We found that thioether bond formation gave the highest yield of material and that the chemistry involved did not adversely affect immunogenicity and biological activity of the peptide vaccine. Ligation of epitopes by oxime bond formation did not diminish biological activity either, but the yields of peptide vaccine were lower than when thioether bond formation was used. The vaccines in which a disulfide bond was used to attach the two epitopes resulted in the lowest yield and produced vaccines that also generated a weaker immune response with sub-optimal biological activity. Connecting the T helper epitope via its N-terminus or its C-terminus to the N-terminus of the B cell epitope had little influence on resulting immunogenicity and biological activity.

Introduction

Chemoselective ligation has been used to synthesise complex molecules, such as peptide dendrimers [1], [2], [3], [4], [5], with defined structure. One of the popular applications of such branched peptides is their use as immunogens and synthetic vaccines [6], [7], [8], [9]. Often, in these peptide dendrimers, a T helper cell epitope and a B cell epitope are synthesised as a linear tandem sequence, which is then conjugated to a core matrix or template to form the dendrimer structure. These peptide dendrimers have been reported to be more immunogenic than their corresponding monomeric peptide immunogens [6], [8]. Several chemoselective methods using the formation of thioether bonds [10], oxime bonds [11], disulfide bonds [12], thioester bonds [13], thiazolidine bonds [14], and hydrazone bonds [15], etc. have also been used to construct peptide dendrimers. Each method results in different linkages being created at the conjugation site, and several reports concerning the reaction rates and the different chemical stabilities of some of these linkages have also been published [1], [16]. Although most peptide dendrimers have been reported to be good immunogens, there has been no direct comparison of the influence of these different linkages on the biological activity of peptide dendrimers.

Careful study of the influence of the different linkages used to generate these constructs on their stability and subsequent biological activity not only gives an indication of the best way of obtaining the highest yield and purity of product but also provides knowledge on the quality of the immune response that is elicited. Such studies could lead to an understanding of the best way of presenting immunogens to the immune system. In this paper, we describe the synthesis of a peptide-based immunogen assembled by chemoselective conjugation of a T helper cell epitope and a B cell epitope using either oxime bond formation, thioether bond formation or disulfide bond formation. As a B cell epitope, we chose luteinising hormone releasing hormone (LHRH), which has the sequence EHWSYGLRPG, abbreviated here as B. LHRH is secreted by the hypothalamus and initiates a cascade of endocrine events that are involved in reproduction. Furthermore, antibodies directed to LHRH have been shown [17], [18] to prevent reproduction in males and females. Another peptide from the light chain (HA2) of influenza virus hemagglutinin, which has the sequence GALNNRFQIKGVELKS, abbreviated as T [19], was chosen as the T helper cell epitope. In a previous study from this laboratory [20], it was shown that inoculation of mice with a linear tandem format (TB) of these two epitopes generated strong antibody responses and also induced sterility in mice.

Because the synthesis of peptides becomes problematic as peptide length increases, we investigated the feasibility of synthesising the individual epitopes separately followed by conjugation using chemoselective ligation. This approach not only has the potential of allowing different epitopes to be mixed and matched but also allows for an ‘off the shelf’ approach in which different immunogenic modules can be ligated in various orientations to provide the highest yield and the most immunogenic vaccine. In this paper, a series of constructs assembled using different linkages and orientations were synthesised and used to inoculate mice. The anti-LHRH antibody titre in mouse sera was measured and mating experiments also conducted to investigate the biological activities of these peptide vaccines.

Section snippets

Chemicals

Unless otherwise stated, chemicals were of analytical grade or its equivalent. Dichloromethane (DCM), N,N′-dimethylformamide (DMF), piperidine, trifluoracetic acid (TFA), O′benzotriazole-N,N,N′,N′-tetra methyl-uronium-hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt), diisopropylethylamine (DIPEA) and diisopropylcarbodiimide (DICI) were obtained from Auspep Pty Ltd (Melbourne, Australia) and Fluka (Switzerland). Aminooxyacetic acid (AoA), phenol and triisopropylsilane (TIPS) were from

Synthesis of the peptide constructs

A summary of the total yields and mass-spectrometric analyses of the various peptide vaccines is shown in Table 1. The total yields were obtained by taking into consideration the yields at each step leading to the final product. In all cases, the experimentally detected masses of the peptides were close to the theoretical masses indicating fidelity of the synthetic procedures.

Discussion

A number of chemoselective ligation methods such as formation of thioether, disulfide, oxime, hydrazone bonds and thiazolidine ring formation have been used to synthesise large and complex peptide vaccines. Studies on the stability of the oxime, thiazolidine and hydrazone bonds at various pH values and at room temperature [1], [16] have shown that each of these three different linkages is stable in the physiological pH range. It is also known that thioether and disulfide bonds are stable under

Acknowledgements

This work was supported by grant 980664 from the National Health and Medical Research Council of Australia and from the Cooperative Research Centre for Vaccine Technology.

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Assembly of synthetic peptide vaccines by chemoselective ligation of epitopes: influence of different chemical linkages and epitope orientations on biological activity

Abstract

Introduction

Section snippets

Chemicals

Synthesis of the peptide constructs

Discussion

Acknowledgements

Curr. Opin. Biotechnol.

Curr. Opin. Biotechnol.

Mol. Immunol.

Vaccine

Vaccine

Vaccine

Mol. Immunol.

Anal. Chim. Acta

Unprotected peptides as building blocks for the synthesis of peptide dendrimers with oxime, hydrazone, and thiazolidine linkages

J. Am. Chem. Soc.

Peptide synthesis using unprotected peptides through orthogonal coupling methods

Proc. Natl. Acad. Sci. USA

Orthogonal ligation strategies for peptide and protein

Biopolymers

One-Pot synthesis of antigen-bearing, lysine-based cluster mannosides using two orthogonal chemoselective ligation reactions

Angew. Chem. Int. Ed.

A rational design of synthetic peptide vaccine with a built-in adjuvant. A modular approach for unambiguity

Int. J. Pept. Prot. Res.

Facile synthesis of homogeneous artificial proteins

J. Am. Chem. Soc.

A new synthetic functionalized antigen carrier

Int. J. Pept. Prot. Res.

Constructing proteins by dovetailing unprotected synthetic peptides: backbone-engineered HIV protease

Science