1. IntroductionProtein drugs and therapeutics such as vaccines are increasing steadily and grabbing attention among drug developers due to their selectivity, specificity, and biocompatibility. The activity of protein pharmaceuticals depends on its structure, and hence any alterations in the structure or conformational change are responsible for the difference in the three-dimensional (3D) structure of the protein, which can lead to unexpected results including protein instability and other adverse effects such asallergic reactions, etc. Proteins consist of multiple polypeptide chains termed protein subunits, which may be monomeric, dimeric, or multimeric. These complex protein structures are stabilized by various interactions such ashydrogen bonds, disulfide bonds, hydrophobic interactions, electrostatic interactions, and van der Waals forces [1]. Although these biological interactions are essential to managing the 3D structure of the proteins, therapeutic/prophylactic proteins such asvaccines, when exposed to different environmental changes including temperature, pH, mechanical agitation, etc., are prone to lose their activity; thus, the storage and transportation become challenging. This can be harmful or even fatal for patients who take the medications and can also increase costs because of the requirement of a cold chain [2].In this present study, we have characterized the recombinant LcrV protein of Yersinia pestis for its stability. Earlier, LcrV has been characterized as a potential vaccine candidate in formulation with the capsular antigen (F1) against the plague in a mouse model [3]. Plague—a zoonotic infection—remains to be one of the fatal diseases caused by a Gram-negative bacterium called Yersinia pestis. The plague has been considered as a re-emerging infectious disease by the World Health Organization (WHO) with possibilities of being used as a bioweapon [4], like the pneumonic form of the plague transmits from human to human [5]. The Centre for Disease Control (CDC) [6] and Stanford University report [7] have categorized Y. pestis as a biosafety level-3 pathogen, and around 5000 human plague cases are reported every year globally [8]. Essential antibiotics and supportive care are the available medical aids to cure the plague if diagnosed at the earliest, as no commercial vaccines are available against the plague to date. Sub-unit recombinant vaccines are under trial, targeting the two virulence protein factors F1 and LcrV of Y. pestis to boost immunity against the infection [9]. These proteins suffer rapid instability when exposed to a temperature above 4–8 °C, i.e., they deactivate at stressful conditions, leading to loss of potency of the vaccine, and hence remain challenging. The general reason proposed for the instability of the proteins is the transition from a folded to unfolded state in unfavorable conditions. The unfolding of proteins (and of protein vaccines) leads to alterations in the protein structure and subsequent aggregation of partially denatured proteins, causing unfavorable thermodynamic interactions [10]. Chemical instability owing to unwanted reactions such as hydrolysis, oxidation, deamination, and breakage or formation of disulfide bonds can also result in loss of vaccine potency. All these destabilizing processes are influenced by factors such as pH, buffer, salts, etc., and are accelerated by fluctuations in temperature.To overcome these shortcomings, various stabilization methods viz. low-temperature storage, freeze-drying, and the addition of various excipients such asosmolytes, additional amino acids, salts, sugars, and polymers are used [11]. A proper understanding of the interactions between proteins and these excipients is essential to designing an optimal formulation. Protein drug formulations are in combat due to the lack of a good understanding of the protein’s structure and its conformational characteristics. Though the four classes of protein structures (primary, secondary, tertiary, and quaternary structure) are widely studied, protein structure prediction in formulations remains challenging. Among different stabilization methods, polymeric materials that are capable of stabilizing biomolecules at room temperature and to agitation are of significant interest. Various kinds of polymers have been shown to stabilize proteins. Stabilization is generally due to one or more properties of polymers, such assurface activity, steric hindrance of protein-protein interaction, preferential exclusion, and increased viscosity that limit the protein’s structural movement [12]. The use of polymers as stabilizers appears to be one of the most efficient methods because of their solubilizing property and safety for parenteral administration.

We aim to briefly cover the behavior of our target protein LcrV in the formulation, mainly the protein–polymer interaction using feasible analytical tools to analyze the structural stability of the protein in the formulation. This work also investigates the biological activity of the formulated protein stored at room temperature (30 ± 2 °C) for a reasonable period. The mechanisms involved in stabilization by the naturally occurring biocompatible polymer dextran and sodium chloride in improving the stability and shelf life of LcrV antigen, a promising vaccine candidate against the plague, have also been investigated.

4. ConclusionsIn this work, the LcrV protein, a highly thermally unstable vaccine candidate for thr plague, has been stabilized using biocompatible polysaccharide dextran at room temperature (30 ± 2 °C) for around three weeks. The stability of LcrV based on protein–polymer interaction was analyzed using various analytical tools such asUV–Visible spectrophotometer, fluorescence spectrophotometer, Fourier transform infrared spectrometer, particle size distribution, gel electrophoresis, transmission electron microscopy, differential scanning calorimetry, and circular dichroism. Based on the UV analysis, a peak at 280 nm corresponding to the aromatic amino acid was obtained in the formulation with hypochromicity observed after a few hours, without change in adsorption maxima, representing the masking or molecular crowding of LcrV by the stabilizers, to provide stability. Based on the fluorescence spec, the emission maxima wavelengths (λmax) and the ratio of the intensity at 345 nm gave an indication of the compactness of the protein in formulations. The FTIR analysis gave a clear background on the possible molecular interactions between LcrV and the stabilizers in enhancing the protein’s secondary structure stability. Next, from the particle size analysis, it was clear that the formulation showed a 100% size distribution (by intensity) with mono dispersion, proving the molecular crowding of the protein by the polymer and the co-excipient, thereby enhancing stability. Based on gel electrophoresis, the formulation showed stability for up to three weeks, beyond which degradation was observed. TEM analysis was performed to ensure the presence of protein in the formulation, as masking of the protein by the stabilizers was observed in all the analyses. The TEM proved the presence of protein (LcrV) in the formulation. Finally, DSC (for melting and enthalpy change) and circular dichroism (for the secondary structure compactness) were performed in the third week to determine the thermal stability of the protein in the formulation and the results ensured the protein stability. Apart from the structural stability, the biological activity of the formulated LcrV stored at room temperature (30 ± 2 °C) for three weeks, in comparison to native LcrV (stored at 4–8 °C) was also performed invivoby ELISA. A significant difference in humoral immunity (IgG endpoint titer) of the formulation (PD1) was observed when compared to the adjuvanted LcrV. It was remarkable to note that the biocompatible polymer dextran had given nearly the same or improved humoral immune response (IgG titer) when compared to alum adjuvanted LcrV. This gives considerable hope to the adjuvant effect of the dextran [55], which has to be explored further.