A dual-adjuvanting strategy for peptide-based subunit vaccines against group A Streptococcus: Lipidation and polyelectrolyte complexes

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Abstract

In order to improve the immunogenicity of peptide-based vaccines against group A Streptococcus (GAS), lipid moieties (C16 lipoamino acid and cholic acid) were conjugated with peptide antigen (P25-J8) and further modified with α-poly(glutamic acid) (α-PGA). Thus, positively charged lipopeptide vaccine candidates LCP-1 (P25-K(J8)-SS-C16-C16) and LCP-2 (P25-K(J8)-SS-K(cholic acid)) were synthesized. Negatively charged LCP-3 (P25-K(PGA-J8)-SS-K(cholic acid)) was also produced by attaching α-PGA to the J8 N-terminus of LCP-2. Polyelectrolyte complex (PEC) nanoparticles were formulated with heparin and/or trimethyl chitosan (TMC) for delivery of the lipopeptide vaccine candidates. The ability of the antigen-loaded nanoparticles to induce humoral immune responses was examined in outbred female Swiss mice following intranasal immunization. The antibodies produced were opsonic against all clinical GAS isolates tested.

Introduction

Group A Streptococcus (GAS) is a gram-positive bacterium that typically invades the human throat and skin. GAS can cause multiple diseases, such as strep throat, post-streptococcal glomerulonephritis, rheumatic heart disease (RHD) and rheumatic fever.1, 2. The most pressing global concern of GAS infection is the substantial mortality from RHD. RHD is caused by valvular damage, commonly mitral valve incompetence, resulting from an abnormal (auto)immune response to GAS infection.3. It was estimated that there were 33.4 million (95% uncertainty interval) cases of RHD worldwide in 2015, and 319,400 (95% uncertainty interval) deaths from the disease.4. Therefore, prophylaxis, early diagnosis and effective treatment of GAS infections are critical. At present, antibiotic therapy, especially injected penicillin, is the most effective and safe clinical treatment for GAS infections.5, 6. However, GAS can be transmitted easily through the respiratory route, which makes reinfection very common and difficult to control, especially in rural areas. GAS infection is an increasing health problem, despite the emergence of antibiotics.1, 7. Consequently, there is an urgent need for an effective, affordable and safe vaccine against GAS.

Peptide-based subunit vaccines that can be chemically synthesized have many advantages over traditional vaccines: they do not revert to an infectious state; are less likely to cause allergic and autoimmune responses; and are relatively easy to produce in large-scale.8, 9. Self-adjuvanting lipid core peptide (LCP) systems, for example, peptide antigens conjugated with immunostimulatory lipids, have emerged as a promising approach for vaccine development.10. To date, LCP systems have been used in the delivery of vaccines against a variety of pathogens, including GAS, hookworm, Schistosoma, respiratory syncytial virus, and malaria.11, 12, 13, 14, 15, 16, 17. Lipidation of peptide antigens can improve immunogenicity by increasing cellular uptake and/or the activation of Toll-like receptors (TLRs).18, 19 The adjuvanting activity of lipidic 2-amino-d,l-hexadecanoic acid (C16) has been studied in many peptide antigen-based subunit vaccines.20, 21, 22. Recently, we found that cholic acid conjugated to GAS antigen induced stronger humoral immune responses in inbred mice following intranasal immunization than C16-adjuvanted antigen.23 Cholic acid is a biodegradable, non-toxic, human bile acid with potential to regulate innate immunity.24, 25

Intranasal administration of vaccines provides opportunities for self-administration, reduced dose compared to oral vaccination, improved patient compliance, and the elimination of injection-related side-effects.26, 27 However, antigens delivery via the nasal pathway suffer from rapid clearance by nasal mucociliary movement, poor absorption through tight junctions between epithelial cells and keratinized epithelium, and instability due to the presence of nasal enzymes.28 Hence, nasal vaccines must be administered with the help of special delivery systems that provide extended nasal residence of the antigen and facilitate permeability through the epithelium. A variety of intranasal delivery strategies have been examined to improve the efficacy of mucosal vaccinations.27, 29, 30, 31

Importantly, delivery systems can also protect antigens from degradation and elimination from the body. Delivery systems often have self-adjuvanting properties, such as mucoadhesivity, improved membrane permeation, and specialized targeting of surface receptors on antigen presenting cells (APCs), such as dendritic cells (DCs).31 Nanoparticles with a size of 50–200 nm are most effectively taken up and internalized by DCs; nanoparticles with a size of 10–100 nm can enter lymphatic systems on their own.32 In addition, nanoparticles, especially cationic nanoparticles, have been widely used to deliver antigens because they can interact with ionic cell membranes, thereby enhancing antigen uptake.33 Due to these unique advantages, the application of nano-delivery systems is considered to be a promising strategy for vaccine development, especially for subunit vaccines.34 Polyelectrolyte complexes (PECs) are usually nano-sized particles composed of oppositely charged polyelectrolytes. They have been widely used as adjuvant/delivery platforms for protein/peptide-based subunit vaccines.35 Previously, we found that PECs, especially when containing anionic polymer heparin and positively charged mucoadhesive trimethyl chitosan (TMC), could function as a promising platform for peptide-based subunit vaccine delivery for intranasal administration.36 We also demonstrated that conjugation of α-poly(glutamic acid) (α-PGA) to peptide antigen introduced a net negative charge to the conjugate, allowing it to directly form a complex with cationic TMC.37, 38, 39 α-PGA is a biodegradable, non-immunogenic, and highly anionic polymer, clinically proven to be safe for systemic administration in humans.40

Herein, we utilized a double-adjuvanting strategy to improve the efficacy of a peptide-based vaccine. LCP-1 (Fig. 1) designed based on B-cell epitope J8 (QAEDKVKQSREAKKQVEKALKQLEDKVQ) derived from GAS M protein, universal T-helper epitope P25 (KLIPNASLIENCTKAEL), and immunostimulatory lipid moiety C16 was selected, as a previously identified lead vaccine candidate.41 Two new vaccine candidates LCP-2 (P25-K(J8)-SS-K(cholic acid)) and LCP-3 (P25-K(PGA-J8)-SS-K(cholic acid)) were constructed (Fig. 1). LCP-1, LCP-2 and LCP-3 were formulated into PECs with heparin and/or TMC to further improve the immunogenicity of the GAS antigen. The ability of the PEC nanoparticles to induce humoral responses was evaluated in outbred mice following intranasal administration. The opsonic activity of the generated antibodies was examined against GAS clinical isolates.

Section snippets

Materials

Protected l-amino acids, rink amide p-methylbenzhydrylamine (MBHA) resin, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluoro- phosphate (HATU), and triisopropylsilane (TIS) were purchased from Novabiochem (Hohenbrunn, Germany) and Mimotopes (Melbourne, Australia). Peptide synthesis-grade dichloromethane (DCM), N,N-diisopropylethylamine (DIPEA), and trifluoroacetic acid (TFA) were purchased from Merck (Hohenbrunn, Germany). 1,2-ethanethiol (EDT) was purchased

Synthesis of vaccine candidates

All lipopeptides (Fig. 1) were synthesized on p-MBHA rink-amide resin (substitution degree: 0.59 mmol/g, 0.1 mmol, 0.17 g) using microwave-assisted Fmoc-solid phase peptide synthesis (SPPS). LCP-1 was synthesized as previously reported.36 For the synthesis of LCP-2 and LCP-3, Fmoc-protected amino acids (0.84 mmol, 4.2 equiv.) were preactivated with HATU (0.80 mmol, 4.0 equiv.) and DIPEA (1.04 mmol, 5.2 equiv.) for 1–2 min before being adding to the resin. Coupling of amino acids was conducted

Synthesis of vaccine candidates

Solid-phase peptide synthesis was used to produce the lipopeptide vaccine candidates LCP-1, LCP-2 and LCP-3 (Fig. 1). To synthesize LCP-2 (P25-K(J8)-SS-K(cholic acid)) and LCP-3 (P25-K(PGA-J8)-SS-K(cholic acid)), lysine with quasi-orthogonal amino protecting groups was introduced to allow site-selective modification of the branching moiety. The use of Mtt and ivDde for lysine ɛ-amino protection has proven to be a reliable method in the synthesis of branched peptides in SPPS.44, 45 The groups

Conclusion

Among tested PEC nanoparticles, P3 (LCP-3/TMC), bearing peptide antigens P25-J8, cholic acid, PGA and TMC, induced the strongest immune responses in outbred mice, and the IgG antibodies generated were opsonic against the tested GAS clinical isolates. The opsonic activity of antibodies induced by P3 was comparable to that of the positive control (LCP-1 adjuvanted with CTB). Furthermore, P3 was the simplest formulation to produce, as it was based on only two components, LCP-3 and TMC. P3 can be

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 the National Health and Medical Research Council [NHMRC Program Grant APP1132975]. The authors acknowledge the support of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, The University of Queensland.

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