Polymorphisms in Plasmodium vivax Circumsporozoite Protein (CSP) Influence Parasite Burden and Cytokine Balance in a Pre-Amazon Endemic Area from Brazil

Abstract

Mechanisms involved in severe P. vivax malaria remain unclear. Parasite polymorphisms, parasite load and host cytokine profile may influence the course of infection. In this study, we investigated the influence of circumsporozoite protein (CSP) polymorphisms on parasite load and cytokine profile in patients with vivax malaria. A cross-sectional study was carried out in three cities: São Luís, Cedral and Buriticupu, Maranhão state, Brazil, areas of high prevalence of P. vivax. Interleukin (IL)-2, IL-4, IL-10, IL-6, IL-17, tumor necrosis factor alpha (TNF-α, interferon gamma (IFN-γ and transforming growth factor beta (TGF-β were quantified in blood plasma of patients and in supernatants from peripheral blood mononuclear cell (PBMC) cultures. Furthermore, the levels of cytokines and parasite load were correlated with VK210, VK247 and P. vivax-like CSP variants. Patients infected with P. vivax showed increased IL-10 and IL-6 levels, which correlated with the parasite load, however, in multiple comparisons, only IL-10 kept this association. A regulatory cytokine profile prevailed in plasma, while an inflammatory profile prevailed in PBMC culture supernatants and these patterns were related to CSP polymorphisms. VK247 infected patients showed higher parasitaemia and IL-6 concentrations, which were not associated to IL-10 anti-inflammatory effect. By contrast, in VK210 patients, these two cytokines showed a strong positive correlation and the parasite load was lower. Patients with the VK210 variant showed a regulatory cytokine profile in plasma, while those infected with the VK247 variant have a predominantly inflammatory cytokine profile and higher parasite loads, which altogether may result in more complications in infection. In conclusion, we propose that CSP polymorphisms is associated to the increase of non-regulated inflammatory immune responses, which in turn may be associated with the outcome of infection.

Fig 1. Cytokine profile in the plasma of patients infected with P. vivax.

Cytokines were measured by CBA. A: Interleukin 6; B: Interleukin 10; C: Transforming growth factor beta; D: Interferon gamma. The bar represents the median of the group, n = 25 infected patients and 9 healthy patients (A,B,D) and n = 24 infected patients and 9 healthy patients (C). P values were determined by nonparametric Mann-Whitney U tests (*p<0.001).

Fig 2. Correlation between parasite density and the cytokine profile.

The x axis indicates Log Parasites/μL (A and B) or Log IL-6 (pg/mL) (C); the y axis indicates Log Interleukin 6 (pg/mL)(A), Log Interleukin 10 (pg/mL) (B) and (C). Each point represents the relative parasitaemia and cytokine responses of a single patient (A and B) or represents the relative IL-10 and IL-6 (C) responses of a single patient, n = 25 infected patients. The correlations were determined by Spearman’s correlation tests.

Fig 3. Cytokine profile and parasite density in P. vivax circumsporozoite protein (CSP) genotypes.

Cytokines were measured by CBA and parasite density was determined by real-time PCR. A: Parasite density (Log parasites/μL); B: Interleukin 6 (pg/mL); C: Interleukin 10 (pg/mL); The bar represents the median of the group, n = 25 patients. P values were determined by nonparametric Mann-Whitney U tests (*p≤0.05). In correlations analyses, the x axis indicates Log IL-10 patient concentrations (pg/mL) in VK247 (D) or VK210 (E); the y axis indicates Log IL-6 patient concentrations (pg/mL) in VK247 (D) or VK210 (E). Each point represents the relative IL-10 and IL-6 responses of a single patient. Correlations were determined by Spearman’s correlation tests.

Fig 4. Cytokine profile in supernatant of patients infected with P. vivax.

Cytokines were measured by CBA. A: Interleukin 6 (pg/mL); B: Interleukin 10 (pg/mL); C: Tumor necrosis factor alpha (pg/mL). The bar represents the median of the group, n = 25 patients and 9 healthy group. P values were determined by nonparametric Mann-Whitney U tests (*p≤0.05). In E, F, and G, each point represents the relative plasma/supernatant IL-6 (E), IL-10 (F), and TNF-α (G) responses of a single patient. In D, the bars represent the median of each cytokine in the plasma or supernatant.