Poster Presentation 8th Australasian Vaccines & Immunotherapeutics Development Meeting 2020

Polymerized Amino Acids as a Self-Adjuvanting Delivery System for Vaccine against Group A Streptococcus (#102)

Mariusz Skwarczynski 1 , Istvan Toth 1 2 3
  1. School of Chemistry and Molecular Biosciences, The University of Queensland , St Lucia, QLD 4072, Australia
  2. School of Pharmacy, University of Queensland, Brisbane, QLD, Australia
  3. Institute of Molecular Bioscience, The University of Queensland, St Lucia, Queensland 4072, Australia

Introduction

Vaccines based on the whole pathogen as well as protein-based vaccines are able to deliver protection against variety of pathogens; however, they are often not fully safe and may induce undesirable immune responses (e.g. inflammation, allergy, etc) [1]. Moreover some vaccines must use even smaller component than protein to avoid triggering autoimmune responses, for example all modern vaccines against Group A Streptococcus (GAS) that have entered clinical are based on peptide antigens [2]. Thus, peptide-based vaccine can solve above problems; however, they need powerfully adjuvants (immunostimulators), which are usually bacteria derived and are associated with some toxicity [3, 4].

Previously we have demonstrated that hydrophobic dendritic poly(tert-butyl acrylate) can be conjugated to a variety of peptides epitopes, including GAS-derived peptides, self-assembled to form particles, which can induce potent humoral and cellular immune responses [5-7]. However, these polymers were not biodegradable, had undefined stereochemistry and number of unit repeats. This typical variability of polymer structure may affect in vitro and in vivo efficacy of vaccine and therefore might not be suitable for clinical trials.

Thus we have designed fully-defined and biodegradable polymers compose of natural hydrophobic amino acids (HAA) [8]. Polymer produced based on this system are fully defined and biodegradable to non-toxic natural amino acids. For purpose of this study we selected conserved B-cell epitope derived from GAS M-protein (J8, QAEDKVKQSREAKKQVEKALKQLEDKVQ), and a universal T-helper epitope (PADRE, AKFVAAWTLKAAA) which were incorporated into pHAA-based system (see Figure 1).

Materials and methods

All the vaccine candidates were synthesized by Boc-solid-phase peptide synthesis (SPPS) method. Particle following simple self-assembly in water/PBS were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Their secondary structure were measured by circular dichroism (CD). Cytotoxicity against SW620 (human colon carcinoma) and HEK293 (human embryonic kidney) were examined MTT assay. Antigen presenting cells (APCs) maturation was examined in spleen cells isolated from naïve C57BL/6 mice using CD40 and MHCII markers. Vaccination was performed subcutaneously in C57BL/6 female mice. The mice were challenged intranasally with the M1 GAS strain one month after immunization. ELISA assay was used to measure antibody titers. An opsonization (bactericidal) assay was performed with immunized mice serum against variety of clinical GAS isolates.

Results

The pHAA, J8 and PADRE were conjugated on the resin using the standard Boc-SPPS method. Produced conjugates were self-assemble in PBS to form chain-like aggregates of the nanoparticles (CLAN) as determined was visible on TEM images, with each nanoparticle size around 20 nm. Conjugate bearing polyleucine adopted helical conformation required for J8 epitope to generated conformation antibody against GAS M protein. All the examined conjugate were non-toxic to human cells lines and not adverse effects during in vivo study was observed. Polyleucine conjugate was the most efficient in stimulation of APCs maturation. The endotoxin level in this formulation was negligible. All conjugates elicited significant J8-specific IgG titers after the final immunization. Polyleucine conjugate induced significantly stronger responses than complete Freund’s adjuvant (CFA), and all other conjugates, in both serum and saliva. Antibodies produced by mice immunized with the conjugated were able to opsonize all tested GAS clinical isolates. Furthermore, only polyleucine-based conjugate greatly reduce bacteria burden in Nasal Associated Lymphoid Tissue (NALT; a murine functional homolog to human tonsils) and the spleen, as well as in nasal shedding, and throat swabs. The powerful but toxic CFA adjuvant was less effective. Moreover, the conjugate did not induce inflammatory responses, which are often related to undesired side effects when classical adjuvants are incorporated in the vaccines. 

Conclusions

The discovery of potent and safe self-adjuvanting delivery system for poorly immunogenic antigens is one of the major challenges in vaccine development. Here we demonstrated that conjugation of peptide antigen to polymerized hydrophoic amino acids can form nanoparticles, which can induce strong humoral immune responses. This new polymeric system is fully-defined, has no chain or stereochemistry variability, and biodegradable to non-toxic natural amino acids. It can be customized to adopt the polymer to selected antigen, for example to produce nanoparticle of desired size. Therefore, the system is expected to find application in the vast variety of vaccines against infectious diseases.

Acknowledgements

We thank the facilities, and the scientific and technical assistance, of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, The University of Queensland. We also acknowledge AD Paterson and P Harris (The University of Queensland Centre for Clinical Research) for providing Streptococcus isolates, GC 2 203, D3840 and D2612. This work was supported by the National Health and Medical Research Council [NHMRC Program Grant APP1132975 and NHMRC Project Grant APP1099999]. We also thank Guangzu Zhao, Jennifer C. Boer, Ashwini Kumar Giddam, Manisha Pandey, Mohini A. Shibu, Reshma J. Nevagi, Michael R. Batzloff, James W. Wells, Robert J. Capon, Victoria Ozberk; Armira Azuar; Jazmina Gonzalez Cruz; Zeinab G. Khalil, Waleed M. Hussein, Magdalena Plebanski, Michael F. Good for their contributions in this work.

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