Table 1.
Mouse immunization strategies.
Fig 1.
SDS-PAGE and Western blot analysis of APN1.
(A) SDS-PAGE analysis of purified APN1. Lane M: Molecular weight protein marker (Fermentas, 116–14.4 kDa), lanes 1,2: E. coli BL21(DE3)-pET23a 3,4: E. coli BL21(DE3)-pET23a-APN1; lanes 1,3: Before induction; lanes 2,4: 16h after induction with IPTG. Lanes 5–7: Purified APN1 (10 μl/well was loaded). (B) SDS-PAGE analysis of desalted purified APN1. Lane M: Molecular weight protein marker (Fermentas, 116–14.4 kDa), Lanes 1–6: Desalted purified APN1. 20 μl of each sample from the desalting process was loaded in each well. (C) Western blot analysis of APN1 protein with anti-His tag mAb. Lanes 1 and 2: Before induction and 16h after induction of E. coli BL21(DE3)- pET23a-APN1, Lane 3: 16h after induction of E. coli BL21(DE3)-pET23a as negative control and Lane M: Molecular weight protein marker (Fermentas, 116–14.4 kDa) stained with Ponceau S and marked with a pencil (10 μl/well was loaded).
Fig 2.
Evaluation of the level of anti-APN1 IgG antibody among vaccinated mouse groups at different time points by an ELISA.
Mouse groups were immunized subcutaneously with recombinant APN1 alone or formulated with different adjuvants (CpG, MPL, QS21, and CMQ [CpG/MPL/QS21]). Control groups received 1×PBS alone or with adjuvants. Anti-APN1 IgG antibody levels were compared at different time points on days 10 (n = 9), 24 (n = 9), 38 (n = 9), and 180 (n = 4) after the first immunization in each mouse group. There was a significant difference in total IgG antibody levels between different immunization time points (days 10, 24, and 38) in each vaccine group (Adjusted P <0.05, paired-sample t-test). Comparing the anti-APN1 IgG antibodies in vaccinated mouse groups on day 38 revealed a significant difference (P < 0.0001, one-way ANOVA test), and the highest level of anti-APN1 IgG antibodies was observed in mice receiving APN1/CMQ.
Fig 3.
Analysis of the profile and persistence of anti-APN1 IgG subclasses.
The profile of anti-APN1 IgG subclasses among vaccinated mouse groups. Pre-immune sera were used as negative controls to determine the ELISA cut-offs. The bars and error bars show the mean OD450 values and standard deviations (SD) for 9 individual mice (day 38) and 4 individual mice (day 180) in each group, respectively. The significant difference in the level of anti-APN1 IgG subclasses was observed in vaccine groups (P < 0.05, one-way ANOVA). The table shows multiple comparisons of means for anti-APN-1 subclasses among the non-adjuvanted (G1) and adjuvanted (G 2–5) vaccine groups on days 38 after the first immunization, which was performed using the Bonferroni post hoc test.
Fig 4.
Analysis of the avidity of anti-APN1 IgG, IgG2a, and IgG2b antibodies.
The avidity index (AI) was calculated as the portion of the OD value of urea-treated serum samples to that of untreated samples multiplied by 100. AI values <30%, 30 to 50%, and >50% correspond to low, intermediate, and high avidity antibodies, respectively. The highest AI of anti-APN1 IgG, IgG2a, and IgG2b antibodies was observed in vaccine group 5 (APN-1/CMQ [CpG/MPL/QS21]) on day 38 after the first immunization. The table shows multiple comparisons of means for the avidity indices of anti-APN-1 subclasses among the non-adjuvanted (G1) and adjuvanted (G 2–5) vaccine groups on days 38 after the first immunization, which was performed using the Bonferroni post hoc test.
Fig 5.
Evaluation of end-point titers of anti-APN1 IgG, IgG1, IgG2a, and IgG2b antibodies among vaccine groups.
For titration, 1:800 to 1:1,638,400 dilutions of mouse sera from different vaccine groups (groups 1 to 5) were analyzed using an ELISA. The titer was calculated as the last dilution of test sera in which OD450 nm values were above that cut-off. Among all the vaccine groups (groups 1 to 5), the highest endpoint titers of anti-APN1 total IgG, IgG1, IgG2a, and IgG2b were detected in mouse group 5 receiving APN1/CMQ (CpG/MPL/QS21) on day 38 after the first immunization. The cut-off values were calculated as the mean OD450nm of the 20 NMS plus three standard deviations (SD). The cut-off values are as follows: IgG: 0.17, IgG1: 0.132, IgG2a: 0.138, and IgG2b: 0.16. The cut-offs are shown with gray lines.
Fig 6.
Assessment of IFN-γ (A), TNF (B), IL-10 (C), and IL-4 (D) production in vaccine groups (G 1–5) and control groups (G 6–10) on days 38 and 180 after primary immunization. For immunized mice receiving APN1 with different adjuvants alone or in combination, the mean IFN- γ responses in the presence of ConA (as the positive control) and no antigen (as the negative control) were 1426 and 6 pg/ml, respectively. In addition, the mean TNF response in the presence of ConA (as the positive control) and no antigen (as the negative control) were 161 and 30 pg/ml, respectively. Besides, IL-10 responses in the presence of ConA (as the positive control) and no antigen (as the negative control) were 374 and 31 pg/ml, respectively. Moreover, IL-4 responses in the presence of ConA (as the positive control) and no antigen (as the negative control) were 48.497 and 28.37 pg/ml. On day 180 after primary immunization, the levels of all cytokines were significantly reduced in all the vaccine mouse groups (P < 0.05, Independent-samples t-test).
Fig 7.
Inhibition of P. falciparum NF54 parasite development by anti-rAPN1 polyclonal antibodies in An. stephensi mosquitoes.
Pooled mouse sera (n = 9) from different vaccine groups (groups 1 to 5) collected on day 38 after the first immunization were admixed with mature P. falciparum NF54 cultured gametocytes and fed to An. stephensi mosquitoes (n = 50/cup) in standard membrane feeding assay (SMFA). Pooled normal mouse serum (NMS) (n = 30 randomly selected from 160 female BALB/c mice before immunization) was used as the negative control. On days 8–10 after feeding, the mosquitoes’ midguts were dissected and oocysts counted, which revealed the successful development of P. falciparum oocysts in An. stephensi mosquitoes were recorded. Two separate membrane feeds were done using serum from each vaccine group (groups 1 to 5), and oocyst counts were pooled for statistical analysis. The dots represent the numbers of oocysts in individual mosquitoes, and the vertical lines indicate the arithmetic means of oocyst counts, respectively. The prevalence of infected mosquitoes in the vaccine groups (groups 1 to 5) and the control group (NMS), range of oocyst numbers, mean number of oocysts, and inhibition percent relative to the NMS control group are indicated in the table. The statistical differences in infection prevalence between each test group and control group were analyzed using Fisher’s exact test and the adjusted P values were presented. The Kruskal-Wallis H test yielded a value of 20.66, with an asymptotic significance of <0.0009, Followed by multiple comparisons with the Bonferroni-Dunn’s correction test. P < 0.05 was considered statistically significant and shown in bold. *: TRA = 100 (1-Mean number of oocyst in the test group/mean number of oocyst in the control group).