Modular platforms for the assembly of self-adjuvanting lipopeptide-based vaccines for use in an out-bred population
Introduction
Recent years have seen efforts directed to the development of subunit vaccines for diseases where the use of attenuated or inactivated pathogens are not appropriate due to the potential risk of reversion to virulence, the presence of deleterious sequences or the limited availability of the antigens [1], [2]. One such strategy is to use peptide-based vaccine constructs containing target epitopes representing the minimal immunogenic region of an antigen allowing for precise direction of induced immune responses [1], [2]. Both free [3] and lipidated short peptides [4] were used successfully to raise virus-specific cytotoxic T lymphocyte responses by in vivo priming 30 years ago shortly after the first studies of peptide vaccine use in humans to treat cancer were reported [5]. Since then numerous studies of peptide-based vaccines have been carried out and have been reviewed [6], [7], [8], [9]. ClinicalTrials.gov, a website maintained by the National Institutes of Health lists 603 peptide vaccine-related clinical studies (October 2019) with 17 reaching Phase 3/4. Because of their relative small size, peptides lend themselves conveniently to chemical modification such as the attachment of carbohydrate antigens, lipid moieties and other functional groups leading us and others to develop the use of synthetic lipopeptides as self-adjuvanting vaccines [10]. Candidate lipopeptide vaccines that have been used in our studies typically comprise of the following three components: (i) a target epitope which can recognised by B or CD8+ T cells; (ii) a CD4+ T helper cell epitope (TH) to harness T helper cell-mediated responses important for initiating and maintaining immune responses to the target epitope and (iii) the lipid moiety S-[2,3-bis(palmitoyloxy)propyl]cysteine (Pam2Cys, or P2C in the figures), an agonist for Toll-like receptor-2 (TLR2) as a built-in adjuvant. Engagement of these lipopeptides by TLR2 on the dendritic cells results in their maturation, improved antigen uptake and processing leading to the induction of effective immune responses [11]. We have demonstrated that such lipopeptides, depending on the target epitope, are capable of inducing both humoral and cellular immunity in animals [10], [12], [13], [14], [15], [16], [17] and even mucosal responses if administered intranasally [10], [18].
Conventionally, these constructs are assembled in toto whereby the defined target and TH epitopes are synthesized contiguously but separated by an intervening lysine residue before attachment of Pam2Cys to its ε-amino group [10]. More recently, we have established a modular approach in which a TH epitope and Pam2Cys are first assembled as a module, which can then be coupled to a separately prepared target epitope [19] (Fig. 1A). This “TH modular approach”, enables the attachment of a conformational target epitope sequence i.e. a B cell epitope, that would otherwise be unlikely to fold correctly when assembled as a contiguous sequence. We have used this approach to build a candidate vaccine based on the heat-stable enterotoxin (ST) from enterotoxigenic Escherichia coli, which contains three-disulphide bridges in its tertiary structure. Antibodies to the ST-antigen elicited by this construct cross-react and neutralise the native toxin [19], [20].
In this study, we describe two novel approaches that extend this modular concept of assembling lipopeptide-based vaccines. The first approach is termed a “target modular approach” whereby the target epitope and Pam2Cys is now assembled as a module which can be coupled to a separately prepared TH epitope in a form as single peptide or a carrier protein (Fig. 1B). The TH epitope we have used here is a peptide determinant derived from the light chain of hemagglutinin of influenza virus (TFLU) that is recognised by CD4+ T cells in BALB/c mice [21]. Alternatively, a carrier protein was used to provide the source of TH epitopes overcoming the need to identify and use epitopes that are specific to an individual’s MHC Class II haplotype thereby potentially allowing this approach to be used in the outbred population. Here, we have used diphtheria tetanus toxoid (DT) which is not only a vaccine in itself [22] but also utilised as a protein carrier for carbohydrate- and peptide-conjugate human [23] and veterinary vaccines [24]. The target epitope is luteinizing hormone-releasing hormone (LHRH), a peptide hormone secreted by the hypothalamus to initiate a cascade of endocrine events that regulate reproduction in mammals. The sequence of this hormone is relatively conserved in all mammals and there are a multitude of studies which have shown that antibodies elicited against this hormone can effectively inhibit the reproductive capability of male and female mammals [10], [19].
The second approach is termed a “sequential ligation modular approach” and describes a potentially more flexible strategy in which each of the three components of a self-adjuvanting peptide vaccine are separately prepared before being assembled in a one-pot procedure. Both the TH epitope/carrier protein and the target epitope each carry a functional group that is chemically orthogonal to each other but complimentary to one of two functional groups that are incorporated into Pam2Cys (Fig. 1C). pH-mediated sequential chemoselective ligations of the lipid module with the target epitope followed by the TH epitope/carrier protein would then lead to the formation of the final vaccine construct.
Herein, we present the strategies used in the assembly of these constructs using these two approaches. We describe results of structural-functional experiments which allowed us to optimize the chemical linkages and positions of components as well as the progress of chemical reactions monitoring the progress of assembling these constructs. The ability of these constructs to induce antigen specific antibody responses without extraneous adjuvants were then evaluated in four different strains of mice including both conventional laboratory and humanised transgenic mouse strains to demonstrate the potential utility of this approach in the preparation of vaccines for use in an outbred population.
Section snippets
The general procedures of solid phase synthesis, cleavage, purification and mass analysis of peptides and lipopeptides
The synthesis of the target epitope LHRH (LHRH1-10; HWSYGLRPG), the influenza virus hemagglutinin derived IAb-restricted TH epitope TFLU (HA166-180; ALNNRFQIKGVELKS) and the ovalbumin derived IAd-restricted TH epitope TOVA (OVA329-339-; ISQAVHAAHAEINEAGR) were performed using Fmoc/tert-butyl strategy on a TentaGel S Ram resin (Rapp Polymere, Tuebingen, Germany) in a microwave-assisted peptide synthesizer (CEM, Liberty peptide synthesizer, Matthews, NC). Couplings were carried out using 5-fold
Evaluation of different chemical linkages and configurations for the assembly of LHRH-lipopeptide based vaccines using the target modular approach
To assemble a module comprised of the target epitope and Pam2Cys that is suitable to be conjugated to a single peptide or a carrier protein, a lysine residue was introduced to the N-terminus of LHRH to which Pam2Cys was coupled to its side chain following two serine residues as spacers. A thiol functional group was then introduced in the lipidated target epitope by coupling a cysteine to its N-terminus to form the lipidated target epitope module Cys-P2C-LHRH (1, Fig. 2). This module adapts a
Discussion
Conjugate vaccines currently licensed for human and veterinary use such as those against Haemophilus influenzae type B [38], various serotypes of Meningococcal and Pneumococcal species [39] and LHRH [24] comprise of a target antigen in the form of either carbohydrate or peptide conjugated to a carrier protein. The efficacy of these vaccines in an outbred population relies on the carrier protein to provide a source of epitopes that can be presented by different MHC class II haplotypes to
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
This work was supported by Program Grant 567122 from the National Health and Medical Research Council of Australia.
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