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1.
Figure 7

Figure 7. Immunofluorescence of P. vivax parasites and anti-MSP-3 antibodies.. From: Antigenicity and Immunogenicity of Plasmodium vivax Merozoite Surface Protein-3.

Immunofluorescence patterns in the sera of mice immunized with recombinant PvMSP-3α and PvMSP-3β (FP-3) on acetone-fixed P. vivax-infected erythrocytes (Pv-iE). The smears were incubated with pooled antisera (1∶100) from mice immunized with: (A) PBS emulsified in adjuvant, (B) PvMSP-3α, or (C) PvMSP-3β in Freund’s Adjuvant. Antibody binding was detected with secondary Alexa 568-labeled antibody (red) and nuclei were visualized by DAPI staining (blue). BF, bright field.

Amanda R. Bitencourt, et al. PLoS One. 2013;8(2):e56061.
2.
Figure 2

Figure 2. Reactivity against recombinant PvMSP-3 and PvMSP119 proteins in 220 sera from individuals with patent P. vivax malaria infection.. From: Antigenicity and Immunogenicity of Plasmodium vivax Merozoite Surface Protein-3.

Each panel represents the reactivity index of serum samples against the indicated recombinant proteins. The serum samples were tested at a 1∶100 dilution, as described in Figure 1B. Symbols represent the IR IgG antibodies against recombinant MSP proteins in the sera of P. vivax-infected individuals. The values of the Spearman correlation coefficient (r) and p values are shown in each panel.

Amanda R. Bitencourt, et al. PLoS One. 2013;8(2):e56061.
3.
Figure 6

Figure 6. Serum IgG isotype responses in mice after immunization with PvMSP-3β in the presence of adjuvant.. From: Antigenicity and Immunogenicity of Plasmodium vivax Merozoite Surface Protein-3.

BALB/c mice were immunized with recombinant PvMSP-3β in the presence of adjuvant as described in Figure 5. PvMSP-3β-specific IgG1 and IgG2a antibody titers in the sera of immunized mice were analyzed by ELISA 2 weeks after the third dose. Results are the means of IgG1/IgG2a ± SEM for 6 mice per group. All groups were compared by one-way ANOVA and Tukey’s test for multiple comparisons.

Amanda R. Bitencourt, et al. PLoS One. 2013;8(2):e56061.
4.
Figure 3

Figure 3. IgG antibody response in C57BL/6 wild-type (WT) and TLR4 KO mice after immunization with MSP-3 in the absence of adjuvant.. From: Antigenicity and Immunogenicity of Plasmodium vivax Merozoite Surface Protein-3.

Groups of 5 mice were immunized 3 times (s.c.) with 10 µg of PvMSP-3α, PvMSP-3β (FP-1), PvMSP-3β (FP-2), or PvMSP-3β (FP-3) and antibody titers to homologous PvMSP-3 were determined after each dose. Results are expressed as the means of antibody titers (log10) ± SEM and were compared by one-way ANOVA followed by Tukey’s test for multiple comparisons. After the third dose, non-significant differences between groups of immunized mice (C57BL/6 WT vs. TKR4 KO) are denoted on the graph as “n.s.” Significant difference between 2 groups of mice immunized with 3 doses of PvMSP-3β (FP-2) are denoted on the graph (*p<0.05).

Amanda R. Bitencourt, et al. PLoS One. 2013;8(2):e56061.
5.
Figure 4

Figure 4. IgG anti-PvMSP-3α in mice immunized with various adjuvant formulations.. From: Antigenicity and Immunogenicity of Plasmodium vivax Merozoite Surface Protein-3.

Groups of 6 female BALB/c mice were immunized 3 times (s.c.) with 10 µg of protein in the following adjuvant formulations: Alum, FliC, CpG ODN 1826, Quil A, TiterMax, or IFA. Anti-PvMSP-3α in the sera of immunized mice was analyzed by ELISA 2 weeks after the first (A), second (B), and third (C) immunizing dose. Results are expressed as mean IgG antibody titers (log10) ± SEM and were compared by one-way ANOVA followed by Tukey’s test for multiple comparisons. Significant differences are noted on the graph: *p<0.05; **p<0.01; ***p<0.001. Non-significant (n.s.) differences are indicated (p>0.05). Data representative of 2 independent experiments.

Amanda R. Bitencourt, et al. PLoS One. 2013;8(2):e56061.
6.
Figure 5

Figure 5. IgG anti-PvMSP-3β and IgG subclass profiles in mice immunized in the presence of adjuvant.. From: Antigenicity and Immunogenicity of Plasmodium vivax Merozoite Surface Protein-3.

Groups of 6 females BALB/c were immunized 3 times (s.c.) with 10 µg of protein in the presence of the following adjuvant formulations: Alum, FliC, CpG ODN 1826, Quil A, TiterMax, or IFA. The adjuvants Alum, FliC, and CpG ODN 1826 were also tested in combination (Alum plus CpG ODN 1826 and FliC+CpG ODN 1826). Anti-PvMSP-3β in the sera of immunized mice was analyzed by ELISA 2 weeks after the first (A), second (B), and third (C) doses. Results are expressed as mean IgG antibody titers (log10) ± SEM and were compared by one-way ANOVA followed by Tukey’s test for multiple comparisons. Significant differences are noted on the graph: *p<0.05; **p<0.01; ***p<0.001. Non-significant (n.s.) differences are indicated (p>0.05). Data representative of 2 independent experiments.

Amanda R. Bitencourt, et al. PLoS One. 2013;8(2):e56061.
7.
Figure 1

Figure 1. Human antibody response to recombinant PvMSP-3α, PvMSP-3β, and PvMSP119 proteins during patent P. vivax infection.. From: Antigenicity and Immunogenicity of Plasmodium vivax Merozoite Surface Protein-3.

A) The bars express the percent response for each of the analyzed proteins. Sera from 220 individuals were analyzed for the presence of specific IgG antibodies by ELISA and tested at a 1∶100 dilution in duplicate. The cutoff proteins obtained from the PvMSP-3α, PvMSP-3β (FP-1), PvMSP-3β (FP-2), PvMSP-3β (FP-3), and PvMSP119 were 0.179, 0.715, 0.195, 0.396, and 0.185, respectively. B) Comparison of individual IR IgG antibodies to MSPs in sera of individuals infected by P. vivax. The line indicates the limit of positivity (IR = 1). IR: Index of reactivity (mean absorbance of test serum/cutoff). C) Comparative analysis of the IgG antibody response against MSP proteins and the frequency of previous episodes of vivax malaria. We analyzed 213 serum samples from individuals who reported the number of previous episodes of malaria for the presence of specific IgG antibodies by ELISA. All sera were tested in duplicate at 1∶100 dilution. *: the percentage of responders with statistically significant correlation to the frequency of previous malaria episodes.

Amanda R. Bitencourt, et al. PLoS One. 2013;8(2):e56061.

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