Ovalbumin lipid core peptide vaccines and their CD4 + and ...

Abstract

The lipid core peptide (LCP) system has successfully been used in development of peptide-based vaccines against cancer and infectious diseases (such as group A streptococcal infection). CD8+ T cells are important targets for vaccines, however developing a vaccine that activates long-lasting immunity has proven challenging. The ability of LCP vaccines to activate antigen-specific CD8+ and/or CD4+ T cell responses was tested using compounds that contained two or four copies of OVA257–264 and/or OVA323–339 peptides conjugated to LCP, which are recognised by OTI (CD8+ specific) and OTII (CD4+ specific) T cells, respectively. The LCP–ovalbumin vaccines developed in this study were synthesised in 30% yields and showed no significant haemolytic effect on red blood cells (below 4% haemolysis when tested with compounds at up to 100 μM concentrations). Promising in vivo data in mice suggested that this LCP–ovalbumin vaccine system could act as a novel and potent vehicle for the stimulation of robust antigen-specific CD8+ T cell responses.

Introduction

Many peptides have been identified as potential new drug and vaccine targets for treatment and/or prevention of a variety of diseases. However, for the majority of peptide compounds, progression into the clinic is hampered by their unfavourable physico-chemical properties e.g. their rapid degradation in the body, problematic delivery and/or their poor inherent immunogenicity. Thus, there is a strong need for a new delivery system which overcomes all those issues [1].

A lipid core peptide (LCP) delivery system based on lipoamino acids, α-amino acids with long alkyl side chains, was designed and tested in variety of disease models [1]. Antigenic peptides that contained LCP elicited strong antibody production without the use of conventional adjuvants [2]. The self-adjuvanting characteristic of this LCP system is advantageous, because many strong adjuvants used in animal models are toxic for humans and the currently available aluminium-based adjuvants used in humans are generally weak and unstable.

The ability of the immune system to identify and eliminate tumour cells is established in the literature [3]. Immunotherapeutic strategies for cancer treatment require the generation of anti-tumour CD8+ T cells because the number of CD8+ T cells often correlates with a positive prognosis [4]. The CD8+ T cell response also has a central role in the host response to intracellular (viral) infections. Many research groups have focused on the production of therapeutic and prophylactic vaccines that elicit T cell responses, particularly a strong anti-tumour CD8+ T cell response. Several preclinical and clinical trials showed promising results, yet the U.S. Food and Drug Administration has only approved a few cancer vaccines for human use [5], [6].

Vaccine-induced immunity also includes antigen-specific CD4+ T cells, which can influence surrounding T cells that have unrelated antigen specificities [7]. For example, parallel expansion of CD4+ T cells against tetanus toxoid was previously found during influenza virus infection [8]. Although CD8+ T cells are the principal effector cells for fighting cancer, the CD4+ T cells may help to induce, maintain and recall CD8+ T cell responses [9]. Ovalbumin peptides are often used as a model for studying vaccine applications [10], [11]. We included two ovalbumin peptides to induce a CD8+ T cell response and possibly a helper CD4+ T cell response (Fig. 1). The OVA257–264 (OVA1) peptide has been shown to induce a strong cytolytic CD8+ T cell response [12], while OVA323–339 (OVA2) promotes a CD4+ T cell response [13]. Although OVA is not a vaccine antigen candidate, the availability of OVA-specific CD4+ and CD8+ T cells, allowed us to measure the ability of the LCP system to activate antigen-specific T cell responses, thereby providing proof-of-principal for the utility of this system to stimulate self-adjuvanting activity of bona fide vaccine antigen candidates.

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Section snippets

Materials

Solvents and reagents for peptide synthesis including dimethylformamide (DMF), dichloromethane (DCM), methanol, trifluoroacetic acid (TFA) and N,N-diisopropylethylamine (DIPEA), were purchased from Auspep (Melbourne, VIC, Australia). (O-Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) and di-tert-butyl dicarbonate (Boc2O) were supplied by GL Biochem Ltd. (Shanghai, China). Nα-tert-Butyloxycarbonyl (Boc)- and 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acids,

Synthesis of LCP–ovalbumin vaccines

The desired ovalbumin peptides were synthesised from Fmoc- and Boc-protected amino acids via conventional SPPS with MBHA resins. Two ovalbumin peptide fragments, OVA257–264 peptide (SIINFEKL, OVA1) and OVA323–339 peptide (ISQAVHAAHAEINEAGR OVA2) were synthesised in over 50% purified yields and used as a control peptides for in vitro and in vivo studies.

Vaccine candidates with a built-in adjuvant comprised of four copies of OVA1 or two copies of the OVA1 and two copies of OVA2, which were

Discussion

We aimed to investigate the T cell activation properties of vaccine constructs that contained both OVA1 and OVA2 peptides incorporated in the same complexes and coupled to the LCP system. With the exception of the HAAH peptide sequence in OVA2 and the final cleavage of Boc-synthesised compounds from the resin, standard SPPS protocols were used [18]. Manual synthesis of the HAAH sequence was necessary to avoid racemisation of the histidine. The Boc-chemistry strategy was chosen for the synthesis

Acknowledgements

We acknowledge the Australian Research Council for their support of this work with the Discovery Project Grant DP1092829, Professorial Research Fellowship to I.T. (DP110100212), and an Australian Postdoctoral Fellowship to P.S. (DP1092829). We thank Abdul Kader Shabbir for help with haemolytic assay, which was performed with the approval of the University of Queensland Ethics Committee (No. 2009000661). All institutional and national guidelines for the care and use of laboratory animals were

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