Acquired Clinical Immunity to Malaria in Nonhuman Primates Coinfected with Schistosoma and Plasmodium Parasites

Abstract

Naturally acquired immunity to malaria develops over several years and can be compromised by concomitant infections. This study explored the influence of chronic schistosomiasis on clinical outcome and immunity to repeated malaria infection. Two groups of baboons (n = 8 each), were infected with Schistosoma mansoni cercariae to establish chronic infections. One of the two groups was treated with praziquantel (PZQ) to eliminate schistosome infection. The two groups plus a new malaria control group (n = 8) were inoculated three times with Plasmodium knowlesi parasites at 1-month intervals. Clinical data and IgG, IgG1, memory T-cell, and monocyte levels were recorded. After three P. knowlesi infections, we observed (i) reduced clinical symptoms in all groups with each subsequent infection, (ii) increased IgG and IgG1 levels in the malaria control (Pk-only) group, (iii) increased IgG, IgG1, CD14⁺, and CD14^- CD16⁺ levels in the Schistosoma-treated (Schisto/PZQ+Pk) group, and (iv) significantly lower IgG and IgG1 levels compared to those of the Pk-only group, reduced CD4⁺ CD45RO⁺ levels, and increased levels of CD14^- CD16⁺ cells in the coinfected (Schisto+Pk) group. Chronic S. mansoni infection does not compromise establishment of clinical immunity after multiple malaria infections, with nonclassical monocytes seeming to play a role. Failure to develop robust antibody and memory T cells may have a long-term impact on acquired immunity to malaria infection.

Keywords: acquired immunity; coinfection; malaria; schistosomiasis.

FIG 1

S. mansoni and P. knowlesi infection and treatment protocol. Blue arrows indicate S. mansoni infection and red arrows P. knowlesi infection. Praziquantel (PZQ) for schistosomiasis treatment was administered at weeks 14, 15, and 16 postinfection. For phase I, the first, second, and third P. knowlesi infections were at weeks 23, 32, and 37, respectively, while phase II infections were at weeks 42, 50, and 55. The end point for phase 1 was at week 40 and that for phase II at 57 weeks post schistosome infection. After each malaria infection, animals were treated with antimalarial drug (blue triangle). Blood for serum was collected (red triangles) before schistosome infection (week 0), before malaria infection (weeks 20 and 40 for phases I and II, respectively) and after three malaria infections (weeks 40 and 57). PBMC isolation was done before malaria infection and after three malaria infections.

FIG 2

Increased mortality in phase II experiment. Five animals succumbed to malaria infection during the experimental period. (A) Percent parasitemia of these animals that died compared to those that survived is. (B) Survival outcome of animals during the two experimental phases. Differences in parasite density were analyzed using the Welch two-sample t test. Animal survival was analyzed using the log-rank test.

FIG 3

Reduced morbidity after three P. knowlesi infections. Animals were infected three times with malaria parasites, indicated as first (1MI), second (2MI), and third (3MI) infections. The bar graphs (A to F) represent proportions of animals with clinical symptoms of severe malaria in the Schisto+Pk (I), Schisto/PZQ+Pk (II), and Pk-only (III) groups. Statistical analysis within groups to identify differences in clinical severity after three malaria infections was done using chi-square analysis.

FIG 4

Reduced parasitemia after three P. knowlesi infections. Individual % parasitemia is presented as parasites/10,000 RBCs (log₁₀). Daily parasitemia was recorded from days 3 to 12 after iRBC/10,000 malaria infection, shown on the x axis. The graphs represent parasitemia in the Schisto+Pk (A, B, and C), Schisto/PZQ+Pk (E, F, and G), and Pk (I, J, and K) at first (1MI), second (2MI), and third (3MI) infections. Area under the curve (AUC) during 1MI, 2MI, and 3MI up to the time of treatment is shown for Schisto+PK (D), Schisto/PZQ+Pk (H), and Pk-only (L), with the median shown. Differences in parasite density were measured as AUC was analyzed using Kruskall-Wallis with Dunn’s post hoc analysis and multiple comparisons adjusted by the Holm method.

FIG 5

Lower levels of IgG and IgG1 antibody responses in coinfected animals after three P. knowlesi infections. The graphs represent IgG (A) and IgG1 (B) antibody responses against malaria before schistosome and malaria infections (BL), after first malaria infection (1MI), and after three malaria infections (3MI). Antibody levels are presented as optical density (OD) measured at a wavelength of 450 nm. Kruskall-Wallis with Dunn’s post hoc analysis and multiple comparisons adjusted by the Holm method was done for the following data: between-group data at IgG-3MI, IgG1-BL, and IgG1-1MI and within-group data for IgG-Schisto/PZQ+PK, IgG-Pk-only, and IgG1 in all three groups. Between-group data for IgG-BL, IgG-1MI, and IgG1-3MI and within-group IgG-Schisto+Pk data were analyzed using one-way ANOVA with Tukey’s multiple comparisons of means.

FIG 6

Decreased levels of memory T cells in coinfected animals after three P. knowlesi infections. Memory T cells were characterized as the percentages of CD3⁺ cells expressing CD4⁺ CD45RO⁺ (A) and CD4⁻ CD45RO⁺ (B). Comparisons were done between the Schisto+Pk (black circles), Schisto/PZQ+Pk (red squares), and Pk-only groups (blue triangles) before malaria infection (BMI) and after three malaria infections (3MI). Between-group data were analyzed by one-way ANOVA with Tukey’s multiple comparisons. Within-group data were analyzed by two-sample t test.

FIG 7

Increased CD14 and CD16 expression in PBMCs of coinfected animals after three P. knowlesi infections. CD3⁻ CD14⁺ (A) and CD3⁻ CD14⁻ CD16⁺ (B) were expressed as percentage of total lymphocytes. Comparisons were made between the Schisto+Pk (black circles), Schisto/PZQ+Pk (red squares), and Pk-only groups (blue triangles) before malaria infection (BMI) and after three malaria infections (3MI). Differences between the three groups were analyzed using Kruskal-Wallis with Dunn’s post hoc analysis and multiple comparisons adjusted by the Holm method at 3MI-CD14, 1MI-CD16, and 3MI-CD16. Differences between the three groups at BMI-CD14 was measured by one-way ANOVA with Tukey’s multiple comparison. Within-group data were analyzed as follows: CD14⁻ Schisto+Pk, CD16⁻ Schisto+Pk, and CD16⁻ Schisto/PZQ+Pk by Wilcoxon rank sum test and CD16⁻ Schisto/PZQ+PK, CD14⁻ Pk-only, and CD16⁻ Pk-only by two-sample t test.