Fig 1.
Summary diagram of experimental results.
Fig 2.
Classification of the two hundred and ten P. vivax blood-stage proteins expressed using the wheat germ cell-free (WGCF) method based on predicted subcellular functions or locations.
Table 1.
Characteristics of patient clinical serum samples.
Fig 3.
Immunoreactivity profiles of P. vivax proteins.
(A) Reactions for the 11 proteins assayed with the sera from patients with acute P. vivax infection, from 5-year recovery of P. vivax-infected patients (5-y-RI), 12-year recovery of P. vivax-infected patients (12-y-RI) and 30-year recovery of P. vivax-infected patients (30-y-RI). The color bar 0–100 means the normalized MFI values of protein-serum reactions. (B) Proportion of positive reactions for paired proteins tested with sera from 5/12/30-y-RI. The color bar 0–100 means the sensitivity of two combined proteins reacting with the sera from 5/12/30-y-RI.
Fig 4.
Humoral immune responses in PvMSP1-42 or PvGAMA-immunized mice.
(A) Western blot analysis of PvMSP1-42 and PvGAMA probed with AVM serum (lanes 1–7) and healthy individual serum (lanes 8–10). (B) Subcellular localization of PvMSP1-42 and PvGAMA proteins in P. vivax positive blood smears. The nuclei were visualized using DAPI (blue). Bars represent 5 μm. (C) Antibody titers of sera obtained from PvMSP1-42-and PvGAMA or PBS immunized mice. Blood samples were collected on days 13, 27, and 43. (D) Series of two-fold dilutions (from 1:125 to 1:16,000) of sera collected on day-43 and assayed with PvMSP1-42 and PvGAMA proteins; the linearity and replicability of the data are shown in the graph. *p< 0.05, **p< 0.01, ***p< 0.001. Unpaired Student’s t-test. Error bars, mean ± SEM.
Fig 5.
Number of ASCs in the spleen and bone marrow of immunized mice.
(A). The diagrams present the frequency of plasmablasts (CD138+B220+) (on the left) and plasma cells (CD138+B220−) (right) detected by FACS in the spleen and bone marrow of PvMSP1-42-, PvGAMA-, and PBS-immunized mice. The graphs present histograms of CD93 expression in CD138+B220+ B cells (B) and CD138+B220- B cells (C) detected by FACS in the spleen and bone marrow of PvMSP1-42-, PvGAMA-, and PBS-immunized mice. The graph on the right summarizes the data. MFI, mean fluorescence intensity. (D) Representative B cell ELISpot data (left) and the number of PvMSP1-42 or PvGAMA-specific IgG-producing ASCs and total IgG in total cell suspensions in the spleen (right) prepared from individual mice. (E) Representative B cell ELISpot data (left) and the number of PvMSP1-42 or PvGAMA-specific IgG-producing ASCs and total IgG in total cell suspensions in the bone marrow (right), which were prepared from individual mice. Results are expressed as the mean ± SEM (n = 4) from one representative experiment out of three with similar results. *p< 0.05, **p< 0.01, ***p< 0.001.
Fig 6.
Frequency of cells related to the formation of the B cell memory response in PvMSP1-42- or PvGAMA-immunized mice.
(A) Histograms present the frequency of CD73+CD80+ MBCs (left) and CD73-CD80- naïve B cells (right) on B220+ pre-gated B cells of PvMSP1-42-, PvGAMA- and PBS-immunized mice. (B) Histograms present the frequency of splenic GC B cells (GL7+Fas+) on B220+ pre-gated B cells (left) and of GC Tfh cells (CXCR5+PD-1high) on CD4+ CD44+ pre-gated T cells (right) of PvMSP1-42-, PvGAMA- and PBS-immunized mice. Results are expressed as the mean ± SEM (n = 4) from one representative experiment out of three with similar results. *p< 0.05, **p< 0.01, ***p< 0.001.
Fig 7.
Schematic figure describing the changes in the number of cells associated with B cell memory formation after protein immunization.
The quantity of both GC B cells and GC Tfh cells present within the spleens of PvMSP1-42-immunized mice were higher than those found within PvGAMA protein. In contrast, there were noticeably more CD80+CD73+ MBCs observed within the splenic tissue. Conversely, fewer CD73−CD80− MBCs were detected in the spleen. It is worth mentioning that CD80+CD73+ IgG1 MBC subpopulations could generate PBs, which could give rise to a limited population size consisting primarily of GC B cells. Furthermore, an increased abundance pertaining to both plasma cell populations residing within splenic tissues as well as bone marrow compartments could be observed following administration with PvMSP1-42.