Fig 1.
Schematic diagram for cDNA construction of vaccine vectors.
Based on the reverse genetic operating system of the vaccine vector RABV SRV9, which contains the exogenous gene expression component, foreign genes were cloned between the P and M genes of full-length SRV9 using BsiW I and Pme I restriction digestion sites. (A) ZI-D: full-length prM-E gene of ZIKV. (B) ZI-E: full-length prM-E of ZIKV with the TM domain replaced by the TM-CD of RABV. (C) ZI-F: full-length prM-E of ZIKV with the signal sequence and TM domain replaced by the corresponding region of RABV.
Fig 2.
Identification of recombinant viruses.
(A-B) Confocal microscopy analysis (6000×). After infection with ZI-D, ZI-E, ZI-F and RABV SRV9 control for 48 h, NA cells were permeabilized (A) or nonpermeabilized (B) and immunostained with antibodies against RABV G protein (red fluorescence) and ZIKV E protein (green fluorescence). Blue indicates DAPI-stained nuclei. Scale bars represent 10 μm. (C) Sucrose density gradient centrifugation-purified virions were analyzed by SDS-PAGE. (D) Detection of ZIKV E protein in sucrose-purified virions by WB. (E) Electron microscopy (EM) and dual-label immunogold electron microscopy (IEM) detection of recombinant viruses. 10 nm gold particles (blue arrow) were used to label RABV G, and 18 nm gold particles (red arrow) were used to label flavivirus E. Scale bars represent 200 nm.
Fig 3.
Growth curves of the recombinant virus.
(A-B) BHK suspension cells were infected with ZI-D or ZI-E at an MOI of 0.1, 0.5 or 1. On days 1, 2, 3 and 4 after infection, samples were obtained, and the RABV titers were determined in NA cells. (C) RABV titer comparison of the recombinant virus with RABV SRV9 at an MOI of 0.5. The tests were repeated three times.
Fig 4.
(A) Immunization strategy. BALB/c mice (n = 9) were immunized IM with ZI-D or ZI-E (20 μg) mixed with a complex adjuvant of ISA 201 VG and poly(I:C). The mice were boosted twice at 2-week intervals. Mice that received PBS were used as controls. Blood samples (n = 6/group) were collected on days 0, 14, 28, 42 and 70. On the 35th day, mouse spleens (n = 3/group) were collected. (B) ZIKV E-specific IgG titers were assessed by indirect ELISA with the purified E protein and are displayed as the end-point dilution titers. (C) IgG2a/IgG1 ratios at week 6 as assessed by indirect ELISA with the purified E protein. (D) ZIKV NAb titer at week 6 as determined by a standard PRNT50. (E) RABV NAb titer at week 6 as determined by a FAVN test.
Fig 5.
One week after the last immunization, mouse spleens (n = 3/group) were collected, and splenocytes were restimulated with purified ZIKV E protein. (A) The proliferative index was detected using a CCK-8 assay. (B) The levels of IFN-γ and IL-4 secreted by splenocytes were quantified by ELISpot assay. (C) Cytokine levels in splenocyte culture supernatants were measured by MSD. Each sample was repeated three times. The data are expressed as the mean ± SD for each group. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
Fig 6.
One week after the last immunization, mouse splenocytes (n = 3/group) were collected and restimulated with purified ZIKV E protein. The activation of lymphocytes, including CD4+ T cells (A-B), CD8+ T cells (C-D) and B cells (E-F), was evaluated 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.
Fig 7.
One week after the last immunization, mouse splenocytes (n = 3/group) were collected and restimulated with purified ZIKV E protein. The proportions of TCMs (CD44+CD62L+) among CD4+ (A-B) and CD8+ T (C-D) cells were evaluated by flow cytometry. (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.