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.