Discussion
Vaccine adjuvants are represented by different classes of compounds that display adjuvant activity in pre-clinical models. Among them, microbial products, mineral salts, emulsions, microparticles, nucleic acids, small molecules, saponins, and liposomes, exert their function by diverse and often, poorly characterized mechanisms of action. However, only a few of them have been licensed for human use, while the vast majority failed to demonstrate an unacceptable safety profile.
An efficient adjuvant should have minimal toxicity and evoke a powerful humoral or/and cellular immune response against the specific antigen (Kohn et al., 1969). Our previous studies indicated that the extracts and components of a Morus alba L fruit water extract were safe (Chang, Kim, Lee, Park, & Kim, 2016), immunostimulatory (Chang et al., 2015; S. B. Kim, Chang, Hwang, Kim, & Lee, 2014; S. B. Kim et al., 2013; X. Y. Yang et al., 2009), and had indirect anti-cancer activity by enhancing the immune responses mediated by TLR4 signaling. TLR4 is expressed on macrophages, dendritic cells, B-cells, T-cells, and endothelial cells. The role of TLR as an M. alba L. receptor was clearly demonstrated in our previous study (Chang et al., 2015). The binding of TLR4 to M. alba L. activated signaling pathways, including MAPKs and NF-κB. The activation of MAPKs is required for the induction of nitric oxide (NO) as it controls the activation of NF-κB. In this study, the role of TLR4 as an M. alba L receptor was confirmed in macrophages (Chang et al., 2015; X. Y. Yang et al., 2009).
The immune adjuvant property of Morus alba L. was investigated using Morus alba L. as a weak antigen in a Balb/c mice animal model. OVA is a well-known model antigen with weak immunogenic properties (Roy et al., 2014; Wusiman et al., 2019). In the present study, Morus alba L. was assessed for its potential adjuvant properties. This study showed that Morus alba L. increased serum antibody titers up to those induced by CT, the most powerful modern adjuvant, used as a positive control (Terrinoni, Holmgren, Lebens, & Larena, 2019). To characterized the nature of the immune responses enhanced byMorus alba L., we analyzed the serum IgG subclass patterns. In the absence of cytokine secretion data, IgG isotypes are commonly used as an indirect means of determining the Th-bias of induced immune responses. It is generally accepted that an IgG1 response is induced with help from Th2 cells, whereas Th1 activity leads to the additional production of IgG2a Abs. OVA alone was found to predominantly enhance specific IgG1 Abs. Consistent with previous reports, oral immunization with OVA and CT induced robust IgG1 and low IgG2a Ab responses.
The co-administration of OVA with Morus alba L. was used to increase levels of both IgG1 and IgG2a. The data obtained indicated thatMorus alba L. stimulated the humoral response without effecting the IgG1/IgG2a titer ratios. Dual IgG1 and IgG2a-potentiating activity has been reported for QS-21, polyacryl starch microparticles, and a pectic polysaccharide of Lemnan minor L. (Cribbs et al., 2003; Popov et al., 2006; Rydell & Sjoholm, 2004). The strong systemic cellular response demonstrated by the IgG2a titers showed that the Th1 profiles were increased by Morus alba L. Antibody responses were detected in mice immunized with OVA and Morus albaL. However, Morus alba L. failed to change serum OVA-specific IgE titers.
Macrophages, which function as antigen-presenting cells for T-cells, have evolved a set of non-clonal pattern recognition receptors that bind carbohydrate polymers. Morus alba L. may, therefore, stimulate macrophages by mimicking natural danger signals provided by bacteria and viruses (Ghorpade, Holla, Sinha, Alagesan, & Balaji, 2013; Medvedev, 2013). CT has previously been shown to enhance the cell surface expression of MHC antigens, co-stimulatory molecules, and chemokine receptors on macrophages and to affect the secretion of cytokines, such as IL-12 and TNF-α (Roy et al., 2014). We also found a strong upregulation of co-stimulatory molecules, MHCII, CD80, and CD86. Cytokines have pivotal functions in regulating immune responses. Th2 lymphocyte cytokines, such as IL- 4, IL-5, and IL-10 can augment IgG1 production, while IL-12, TNF-α, and IFN-γ produced by Th1 lymphocytes can improve IgG2a production (Cribbs et al., 2003; Jones et al., 2002; Tuo, Palmer, McGuire, Zhu, & Brown, 2000). The higher level of all IgG subclasses may be explained by increased IL-4, IL-12 and IFN-γ production, suggesting that Morus alba L. can enhance both Th1 and Th2 immune responses. Bacterial toxins, such as CT are potentially too toxic for use in humans. In contrast, Morus alba L. has been administered to humans by a variety of routes without toxic effects.
Mucosal tissues are the main entry portal of many pathogens, including influenza, and the mucosal immune system provides the first line of defense against infections, apart from innate immunity (Akter et al., 2019; Rydell & Sjoholm, 2004). Immunization with Morus alba L. in the vaccinated group increased IgM and IgG more than vaccination only.
The immune reaction to inactivated influenza virus is characterized by IgG2a and IgG1 responses. Particulate adjuvants, such as alum or M59, may stimulate immune responses to IgG1, while some TLR agonists may stimulate IgG2a responses (Goff et al., 2017; Jian et al., 2013; Yanase et al., 2014). In comparisons between Morus alba L., alum, and non-adjuvant vaccines conducted in the present study, significant differences in IgG-subtypes were observed. Morus alba L. induced higher levels of IgG1 and IgG2a antibodies than HA vaccines. In contrast, alum increased IgG1 antibody levels similar to those of Morus alba L. but did not elicit an appropriate IgG2a response. Indeed, alum attenuated the IgG2a response induced by HA vaccines.
Influenza virus vaccines are largely effective only against a matched strain, necessitating accurate prediction of the upcoming epidemic strains during annual vaccine reformulation. By enhancing vaccine immunogenicity and improving the quality and persistence of the immune response, adjuvants may provide long-lasting efficacy against drifted strains (Huber et al., 2006; Joshi et al., 2009). Indeed, whenMorus alba L. was administered with the HA vaccine, it provided a protective immune response against a lethal challenge with the H1N1 virus.
In the last decade, experimental adjuvants for viral vaccines have proliferated. While many have demonstrated efficacy in animal models, few have been able to move through the regulatory hurdles because of safety and reactogenicity concerns (Kuznetsova & Persiyanova, 2019). Morus alba L., with a variety of antigens, induced rapid and cross-reactive humoral and cellular immunity to elicit broad protection. Morus alba L., which has increased antigenicity and favorable safety, has confirmed its potential as an oral adjuvant for a variety of influenza virus vaccines, as well as an immunopotentiator.
Author contributions: B.Y.C, and S.Y.K. designed the experiments. B.Y.C, and S.Y.K. performed the experiments. B.Y.C, and S.Y.K. drafted the manuscript; and all the authors revised, edited, and approved the final version of the manuscript.
Acknowledgments: This work was supported by Wonkwangs University in 2020.
Conflict of interest: The authors declare no conflicts of interest.
Declaration of transparency and scientific rigour: This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design & Analysis, and Animal Experimentation and as recommended by funding agencies, publishers and other organizations engaged with supporting research.