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Figure Lgends
Figure 1. Expression of the fusion protein prM-E-△-PA3 in the
baculovirus expression system. (A) Schematic diagram of the fusion
protein prM-E-△-PA3 fragment. “△” represents a deletion of the
stem-transmembrane (ST-TM) region. ZIKV prM-E gene: the prM-E gene of
ZIKV (accession: KX601168.1; does not contain the start codon). ZI-△-PA
gene: the sequences from the N-terminus to the C-terminus were prM-E-△
of ZIKV, a linker and protein anchor 3 (PA3). ZI-JE-△-PA gene: the
signal peptide of ZI-△-PA gene was replaced with the signal peptide of
JEV. ZI-GP-△-PA gene: the signal peptide of ZI-△-PA gene was replaced
with the gp67 signal peptide. (B) IFA detection of the ZIKV E protein
(100×). Recombinant baculovirus-infected Sf9 cells were subjected to IFA
for the detection of ZIKV E protein expression. Uninfected Sf9 cells
were used as a cell control. Positive results showed bright green
fluorescence, and negative results showed red due to Evans blue. (C) WB
detection of the ZIKV E protein in baculovirus-infected cells. The
proteins were detected in the supernatant or the sonicated supernatant
of infected cells. M represents the protein marker, and the lines in the
figure correspond to 100, 70 and 55 kDa.
Figure 2. Investigation of fusion-GEM-complexes binding. (A)
Combination diagram of the prM-E-△-PA3 protein binding to GEM. (B)
Electron microscopy observation of fusion-GEM-complexes. The scale bars
represent 200 nm. GEM was used as the control. (C) IFA detection of ZIKV
E protein in fusion-GEM-complexes (1000×). (D) WB detection of the ZIKV
E protein in fusion-GEM-complexes. M represents the protein marker.
Figure 3. Efficacy of the immunogen in BALB/c mice. (A)
Experimental scheme. BALB/c mice (n=18 mice/group) were immunized with
ZI-△-PA-GEM, ZI-JE-△-PA-GEM or ZI-GP-△-PA (10 µg) mixed with a complex
adjuvant of ISA201 VG and poly(I:C) by intramuscular injection. Mice
injected with PBS combined with adjuvant were used as the control. On
the 3rd, 6th and
9th days after the first immunization, lymphocytes
from the inguinal lymph nodes of the mice (3 mice/group/day) were
analyzed. Nine mice were boosted twice at 2-week intervals, and sera
were collected on days 0, 28, 42, 56 and 70. On the
35th day, splenic lymphocytes from the mice (n=3
mice/group) were analyzed. (B) ZIKV-specific IgG titers in the serum
(dilution 128-fold) were measured by ELISA at the indicated time points,
displayed as the OD450 ratio of sample/control. (C) ZIKV
neutralizing antibody titers detected at week 6 by a standard 50%
plaque reduction neutralization test (PRNT50). (D) The
ratios of IgG2a/IgG1 at week 6 were determined by ELISA.
Figure 4.Detection of B cell and DC
activation early after immunization. On the 3rd,
6th and 9th days after the first
immunization, lymphocytes from the inguinal lymph nodes of the mice (3
mice/group/day) were analyzed by flow cytometry. (A) B cell activation,
displayed as CD19+ CD40+. (B)
Representative flow cytometric plots of lymphocytes of B cell activation
on D6. (C) DC activation, displayed as CD11c+MHCII+. (D) Representative flow cytometric plots of
lymphocytes of DC activation on D6. The data are expressed as the mean
±SD for each group. *p<0.05; **p<0.01;
***p<0.001.
Figure 5. Splenocyte proliferation analysis. On the
35th day after immunization, splenic lymphocytes from
the mice (3 mice/group) were harvested and stimulated with purified ZIKV
E protein. (A) After ex vivo culture for 44 h, the proliferative
index was determined using a CCK-8 assay. (B) After culture for 24 h,
the levels of IFN-γ and IL-4 secreted by the splenocytes were quantified
by the ELISpot assay. (C) After culture for 72 h, the cytokine levels in
splenocyte culture supernatants were measured by MesoScale Discovery
(MSD). The data are expressed as the mean ±SD for each group.
*p<0.05; **p<0.01; ***p<0.001;
****p<0.0001.
Figure 6. Lymphocyte activation analysis.On the 35th day
after immunization, splenic lymphocytes from the mice (3 mice/group)
were stimulated with purified ZIKV E protein and cultured ex vivofor 72 h. The activation of CD4 T cells (A-B), CD8 T cells (C-D) and B
cells (E-F) was measured by flow cytometry. (A, C, E) Representative
flow cytometric plots of lymphocytes from each group. (B, D, F)
Percentages of the indicated lymphocytes. The data are expressed as the
mean ±SD for each group. *p<0.05; **p<0.01;
***p<0.001.
Figure 7. Central memory T cells (TCMs) analysis. On the
35th day after immunization, splenic lymphocytes from
the mice (3 mice/group) were stimulated with purified ZIKV E protein and
cultured ex vivo for 72 h. The proportion of TCMs among
CD4+ T (A-B) or CD8+ (C-D) T cells
was evaluated by the presence of CD44 and CD62L surface markers. (A, C)
Representative flow cytometric plots of lymphocytes from each group. (B,
D) Percentages of the indicated lymphocytes. The data are expressed as
the mean ±SD for each group. *p<0.05; **p<0.01.