Armamentarium of Cryoprotectants in Peptide Vaccines: Mechanistic Insight, Challenges, Opportunities and Future Prospects

Abstract

Vaccines are designed to leverage the immune system and produce long-lasting protection against specific diseases. Peptide vaccines are regarded as safe and effective way of circumventing problems such as mild allergic reactions associated with conventional vaccines. The biggest challenges associated with formulation of peptide vaccines are stability issues and conformational changes which lead to destruction of their activity when exposed to lyophilization process that may act as stressors. Lyophilization process is aimed at removal of water which involves freezing, primary drying and secondary drying. To safeguard the peptide molecules from such stresses, cryoprotectants are used to offer them viability and structural stability. This paper is an attempt to understand the physicochemical properties of peptide vaccines, mechanism of cryoprotection under the shed of water replacement, water substitution theory and cation-pi interaction theory of amino acids which aims at shielding the peptide from external environment by formation of hydrogen bonds, covalent bonds or cation-pi interaction between cryoprotectant and peptide followed by selection criteria of cryoprotectants and their utility in peptide vaccines development along with challenges and opportunities.

Keywords: Cryoprotectants; Lyophilization; Peptides and proteins; Synthetic peptide vaccine.

Fig. 1

Synthetic peptide vaccines exhibited several lucrative advantages over the conventional vaccines besides few limitations like low immunogenicity and instability

Fig. 2

Peptides are recognized by the pattern recognition receptors (PPRs, Toll like receptors; TLRs) in order to elicit the immune response. Peptide molecules are then identified by antigen presenting cells (APCs). Further, they presented to the major histocompatibility complex (MHC) proteins and trigger the T-helper or CD4+ cells and lead to activation of cellular immunity or humoral immunity

Fig. 3

Physicochemical properties like solubility, pH, isoelectric point influence the stability of peptide. For example, 1 out of 5 amino acids should be charged to obtain sufficient solubility in aqueous medium. On the other hand, at pH below isoelectric point, the net charge is positive and above isoelectric point, it is negative. Any alteration in pH equivalent to the isoelectric point drastically change the physicochemical properties

Fig. 4

Summary of the challenges faced in synthetic peptide vaccine development like scale-up issue, high production cost, low immune response, stability of peptides etc.

Fig. 5

Chemical structures of commonly used cryoprotectants lie trehalose and sucrose

Fig. 6

Mechanism of action of cryoprotectants under a Water replacement theory. Small sugar molecules like trehalose possess better flexibility than larger and rigid sugars. If the sugars have a greater number of hydrogen bond forming groups and are small in size then they can easily enter the grooves of the peptide molecules and can decrease the free volume by filling all the voids. b Water substitution theory. The oxygen molecules of trehalose from their glycosidic bonds interact with the surrounding water molecules and thus structure them around trehalose rather than around the peptide molecules. This leads to reduction in the stress caused due to dehydration of the preparation. It effectively reduces the amount of water available around peptides for freezing and thus reduces the freeze and drying induced stress. c Cation-pi interaction theory of amino acids. Arginine increases the stability of proteins through electrostatic interactions and also through cation-pi type of interactions. Arginine interacts with the hydrophobic portions of the proteins (through its –CH₂ groups) and augments the stability

Fig. 7

Simulation and prediction representation of hydrogen bonding (black) capacity of cryoprotectants (yellow) in water (blue) and between cryoprotectants (green and yellow) and protein using molecular dynamics. Surface tension of water is affected by the hydrogen bond formation between molecules of water and cryoprotectant. Moreover, the cluster formation of cryoprotectants also restricts water molecules to come together, thereby affecting nucleation. Avoidance of water crystal formation is an important factor during freeze drying (Color figure online)