Vaccination is the most effective way to prevent and control diseases. Although the safety of vaccines has increased with recombinant vaccine technology, these vaccines generally show weak immunogenic properties (Mchugh et al., 2015; Petrovsky & Aguilar, 2004). Although it has been studied for years for the administration of weak immunogen vaccines in a single dose, it has not been advanced in practice. Vaccine adjuvants used to increase the immunogenicity of the antigen have gained importance again, especially during the Covid-19 pandemic.
It is known that the polymers in the vaccine formulation increase its stability by binding to the antigen and serve as both carrier and adjuvant (Mustafaev, 2004). Many plant and microbial polysaccharides have been investigated for their adjuvant potential. Various dextran derivatives have been shown to have immunological properties (Bauer et al., 2010). It has been reported that lentinan increases macrophage activity against influenza virus and simultaneously induces cytokine production and cytotoxic T cell production (Vannucci et al., 2017). The adjuvant potency of different inulin isoforms has been reported both in vitro and in female Balb/c mice. It has been stated that delta inulin is the most immunologically effective derivative in activating the complement system, regulating chemokine production and cell surface protein expression by binding to monocytes. It has been reported to convert to potent immune adjuvant activity that enhances humoral and cellular responses to antigens (Cooper & Petrovsky, 2011). In addition, among the biopolymers, mannan (Sheng et al., 2006), chitosan (Vasiliev, 2014) and poly(γ-glutamic acid) (Seth et al., 2015) are reported to have adjuvant potential as alternatives to aluminum salts.
In this study, the vaccine adjuvant potential of the microfibrillar structure of BC, which has unique properties such as biocompatibility, high purity, physical and chemical stability, was investigated (Keskin et al., 2017; Pertile et al., 2010; Piatkowski et al., 2011; Martínez-Sanz et al., 2011; Zaborowska et al., 2010). It has been known that physicochemical properties such as size, hydrophobicity, surface charge, shape and composition of the adjuvants play an important role in the adjuvant effect (Bastola & Lee, 2019; Kreuter & Haenzel, 1978). Apart from other physicochemical properties, the water-insoluble adjuvants have a higher cellular uptake, thus triggering a higher immune response (Woodard, 1989).
One of the properties of BC is that it is in the form of water-insoluble micro fibrils that makes it a good candidate for vaccine adjuvant. There are no studies in the literature investigating the vaccine adjuvant potential of BC. Hydrolysis processes on cellulose samples are carried out with sulfuric acid, which cause the amorphous areas of the material to break into fibrils by controlled hydrolysis. Although plant-derived cellulose and BC have the same chemical structure, they have different structural and mechanical properties. BC shows finer fibril structure, higher water holding capacity and higher crystallinity (M. Iguchi; et al., 2000; Wan et al., 2009). In addition, plant-derived cellulose is combined with lignin and hemicellulose structures, while BC has chemically pure structure. In our study, after the hydrolysis process, it was observed that micro fibrils with similar size were obtained by hydrolysis with 2.5M H2SO4 in the study by De Oliveira et al (De Oliveira et al., 2011).
SEM images and analysis showed that after hydrolysis, the BC fibrils were 0.57-4.072-8 µm in size and needle-shaped (Fig. 3d). It is known that needle-like particles can result in inflammasome activation thus better induce immune response compare with spherical particles (Donaldson et al., 2010). Among needle-like particles, larger particles with micro meter size are typically more capable of activating inflammasomes (Caicedo et al., 2013).
The physical absorption of the antigen has advantages compared to other conjugation procedures. In fact, the conjugation method may cause modification of peptide epitope in addition to complications with regard to the reproducibility of conjugate structures (Kazzaz et al., 2000; Singh et al., 2004). Due to the high water absorption capacity of BC, simple mixing method was used which is frequently used in polymer adjuvants as a conjugation method (Kreuter et al., 1988). The spectrum of the BCmfs-BSA conjugate in the protein-specific amide-I and amide-II bands (Guo et al., 2019) and associated with the cellulose chain backbone (Abderrahim et al., 2015) is an indication that the conjugate contains both cellulose and model antigen (Fig. 4).
The immunostimulatory effects of the BCmfs-BSA conjugates was measured with TNF-α and IL-6 ELISA kit on the U937 human macrophage cell line. Conjugates containing different BSA concentrations (500, 250 and 100 µg/ml) were administered to cells and all were found to induce TNF-α secretion in cells. When TNF-α levels were examined, while there was no significant difference between the sample containing BCmfs and the negative control, BCmfs-BSA conjugate had significantly higher (3,7 times) TNF-α level than BSA alone. Furthermore, the BCmfs-BSA conjugate had a higher (1.78 times) TNF-α level than the alum-BSA conjugate (Fig. 7a). The reason of the higher TNF-α secretion induced by BCmfs in comparison to the alum group containing the same amount of BSA is thought to be due to the higher cellular uptake. Physicochemical properties of particles such as size and shape (Zheng & Yu, 2016), surface charge (He et al., 2010), surface hydrophobicity/hydrophilicity (Kreuter et al., 1988) and solubility/insolubility (Woodard, 1989) directly affect the cellular uptake mechanism. Studies have shown that filamentous particles with a higher aspect ratio have a higher cellular uptake potential than spherical particles with the same size (Dasgupta et al., 2014). The limited number of binding sites of spherical particles allows limited interaction with target cell receptors, while filamentous particles have a higher surface area that facilitates their interaction with the cell surface; thus filamentous, elongated particles are more effective in adhesion to cells than spherical particles (Agarwal et al., 2013). Woodard et al. have shown that water-insoluble molecules induce a higher immune response by increasing cellular uptake (Woodard, 1989). Due to the higher aspect ratio and filamentous structure of BCmfs compared to alum adjuvant, it was thought that the cellular uptake was higher and therefore higher TNF-α level was achieved.
Depending on their mode of action, adjuvants are used to stimulate the Th1 and/or Th2 mediated immune response. The production of IL-2, TNF- α, and IFN-γ are characteristics of the Th1 immune response, which is mediated by Th1 cells. In addition, production of cytotoxic T lymphocytes (CTL) and cell-mediated inflammatory reactions requires a Th1 immune response. Th2 cells are characterized with secretion of IL-4, IL-5, IL-6, IL-10, and IL-13 cytokines, which regulate B cell activation and antibody production. While the Th1 response is essential for protective immunity against intracellular pathogens, including as viruses, bacteria, and protozoa, as well as against cancer cells, Th2 immunity is effective for a defense against most bacteria as well as against some viral infections (Cox & Coulter, 1997). The most commonly used adjuvants such as alum, stimulate the Th2 immune response and is ineffective against intracellular pathogens. Studies have shown that IL-6 secretion supports Th2 differentiation, inhibits Th1 polarization and IFN-γ production by inducing the expression of IL-4 during the activation of CD4 + T cells (Diehl & Rincón, 2002). When IL-6 levels were examined in this study, no statistically significant results were obtained between the groups except the positive control group (LPS), and it was shown that the BCmfs did not induce the IL-6 production and appear to induce Th1 immune response (Fig. 7b).
In this study, BC micro fibrils were shown to be phagocytosed by macrophage cells and increased cellular uptake of target antigen (Fig. 8) Consistent with these results, increase TNF-α cytokine level with restricted IL-6 production results also showed that BCmfs enhanced the immune response to the antigen BSA. These results are the first to report that BCmfs have the potential as an alternative to alum adjuvants.