Discussion
The GEM surface display system has the following advantages. (1) Simple
purification of a foreign protein by low-speed centrifugation is
convenient and efficient. (2) Lactococcus lactis (L.
lactis ) is globally recognized as a safe probiotic
[20,
21]. After treatment, GEM has no safety
risk as a carrier due to the absence of nucleic acids and proteins. (3)
The displayed protein can effectively stimulate a stronger immune
response [22]. Therefore this system
has been studied and applied in many vaccine studies
[15, 21,
23]. Natural PA contains three LysMs,
and serine, threonine and aspartic acid are used as the interval
sequences between the repeat sequences
[24]. Multiple LysMs might form
multivalent domains, thus increasing the anchoring activity of PA, but
the anchoring activity of PA containing four LysMs was significantly
reduced [13,
24]. Based on our previous research
[25], we chose PA3 (containing three
LysMs) as the carrier protein and an 8-amino acid sequence as the
linker.
Chang [26] indicated that only the
construct efficiently secreting prM/M and E antigens had the capacity to
stimulate high-titered neutralizing antibodies in plasmid-vaccinated
mice. Therefore, in this study, we replaced the signal peptide of ZIKV
with the corresponding sequence of JEV or gp67 to realize secretory
expression of target proteins. Davis
[27] replaced the signal peptide of
prM-E from West Nile virus (WNV) with the signal peptide of JEV, and the
prediction results showed that raw cleavage site (C), signal peptide
(S), and combined cleavage site (Y) scores significantly increased.
Besides, the recombinant DNA vaccine could secret high levels of target
proteins. Dowd [28] exchanged the
signal sequence of prM-E with the analogous region of JEV to improve
expression. In addition, we initially replaced the ST-TM region of ZIKV
with the corresponding JEV region (data not shown). After fusion of
prM-E with PA3, the target protein was hardly secreted into the
supernatant and could be detected in only the sonicated supernatant.
Moreover, the fusion protein with the ST-TM region could not bind to the
surface of GEM (as detected by TEM, showing a smooth GEM surface, data
not shown). The ST-TM region likely affected the correct expression or
function of the PA3 anchor protein. Therefore, we further deleted the
ST-TM region, and the fusion protein was secreted into the supernatant
and successfully bound to the surface of GEM. In ZIKV DNA and virus-like
particle (VLP) vaccine construction strategies, the ST-TM region is
beneficial for protein expression and packaging
[28-30]. However, in this study, when
the target protein was co-expressed with exogenous protein PA3, deletion
of the ST-TM region was beneficial for the binding of the target protein
to GEM.
ZI-△-PA-GEM stimulated animals to produce specific antibodies against
the ZIKV-E protein for at least 8 weeks. The PRNT50value of neutralizing antibody titer was above 1:10. There is only one
serotype of ZIKV [31], and
neutralizing antibody titers >10 have been found to
correlate with protective efficacy [29,
32, 33].
Our results indicated that ZI-△-PA-GEM could induce potent E-specific
IgG antibodies, as well as neutralizing antibody responses. In addition,
the antibody subtype was Th2 biased, which might be advantageous for
protection in some ways and has been reported for vaccines
[25,
34-37].
As a surface molecule of B cells, CD40 is essential for the B cells
isotype switching [38,
39]. The coreceptor CD19 plays an
essential role in mediating the spreading of B cells and enhancing
signaling through the B cell receptor (BCR) in response to
membrane-bound antigen [40]. In this
study, in the early stage after the first immunization, ZI-△-PA-GEM
stimulated B cell activation, with an increase in
CD19+ CD40+ double-positive cells.
Activated B cells can produce plasma cells that generate high-affinity
antibodies and memory cells, thus protecting the body against infectious
diseases [41]. Moreover, ZI-△-PA-GEM
recruited and/or activated DCs in secondary lymphoid organs and
significantly stimulate the expression of MHC II molecules, with an
increase in CD11c+ MHC II+double-positive cells. CD11c is a sign of DC maturation, and DCs are the
most professional antigen-presenting cells (APCs) and the only APCs that
can stimulate the initial T cell response
[42]. In addition, DCs are an
important factor regulating the differentiation of immature cells into
Th1 or Th2 cells. MHC II molecules are important for presenting
antigens on the surface of DCs [43,
44], mainly exogenous antigens, and
play multiple roles in inducing protective immunity after vaccination
[45]. The presentation of antigen
peptides on the MHC II molecules of DCs can induce initial
CD4+ T cell activation
[43].
Functional regulation of Th subsets and the cytokine environment is
beneficial to protective immunity
[46]. ZI-△-PA-GEM induced significant
cytokine secretion, including IFN-γ, IL-4, IL-6 and IL-10. Th1 cells
produce important cytokines for the production of cytotoxic T cells and
complement-fixing antibodies. Th2 cells produce a series of cytokines,
that support antibody production
[47]. The cytokines induced by
vaccines are synergistic and rarely work alone, which complicates the
interpretation of immune correlations
[48]. The differential cytokine
levels between the immunized and control groups may contribute to
protection against flaviviruses [49].
Moreover, cytokines affect the differentiation of memory T cells
[46]. There are two subpopulations of
memory cells: effector memory T cells (migrate mainly to peripheral
tissues) and TCMs (localize to the lymph nodes, blood and spleen)
[46]. Here, the proportion of TCMs
among splenic lymphocytes stimulated by ZI-△-PA-GEM was significantly
higher, which was beneficial for rapid stimulation of the immune
response during antigen stimulation.
High levels of T cell activation were also detected, especially with
ZI-△-PA-GEM. Although there was no clear final conclusion concerning
whether the cell-mediated immune responses against ZIKV contributed to
protection, the results of a previous study support that T cells play an
important role in protecting against ZIKV
[3,
50]. CD8+ T cells
limit the spread of viruses by recognizing and killing infected cells or
secreting specific antiviral cytokines, and CD4+ T
cells contribute to cytokine production and support the generation and
maintenance of antibody and CD8+ T cell responses
[49].
In this study, potent B cell/DC activation, cytokine responses, specific
splenocytes capable of proliferation, and T cell responses, together
with the production of specific antibodies, are likely to jointly
contribute to protection in mice. Taken together, our data indicate that
ZI-△-PA-GEM is capable of inducing humoral and cellular immune responses
in mice that may protect against ZIKV infection.