Immunogenicity of PvCyRPA, PvCelTOS and Pvs25 chimeric recombinant protein of Plasmodium vivax in murine model

Abstract

In the Americas, P. vivax is the predominant causative species of malaria, a debilitating and economically significant disease. Due to the complexity of the malaria parasite life cycle, a vaccine formulation with multiple antigens expressed in various parasite stages may represent an effective approach. Based on this, we previously designed and constructed a chimeric recombinant protein, PvRMC-1, composed by PvCyRPA, PvCelTOS, and Pvs25 epitopes. This chimeric protein was strongly recognized by naturally acquired antibodies from exposed population in the Brazilian Amazon. However, there was no investigation about the induced immune response of PvRMC-1. Therefore, in this work, we evaluated the immunogenicity of this chimeric antigen formulated in three distinct adjuvants: Stimune, AddaVax or Aluminum hydroxide (Al(OH)3) in BALB/c mice. Our results suggested that the chimeric protein PvRMC-1 were capable to generate humoral and cellular responses across all three formulations. Antibodies recognized full-length PvRMC-1 and linear B-cell epitopes from PvCyRPA, PvCelTOS, and Pvs25 individually. Moreover, mice's splenocytes were activated, producing IFN-γ in response to PvCelTOS and PvCyRPA peptide epitopes, affirming T-cell epitopes in the antigen. While aluminum hydroxide showed notable cellular response, Stimune and Addavax induced a more comprehensive immune response, encompassing both cellular and humoral components. Thus, our findings indicate that PvRMC-1 would be a promising multistage vaccine candidate that could advance to further preclinical studies.

Keywords: BALB/c; immunization; immunogenicity; in silico simulation; multistage chimeric protein; plasmodium vivax; vaccine.

Figure 1

Kinetics with immunization, blood collection, and euthanasia phases. (A) BALB/c mice were separated into groups and described above according to the numbering, maintained throughout the study. Each symbol is identified below with the captions. (B) BALB/c mice (n = 8 per group) were immunized intramuscularly with PvRMC-1 formulated with three adjuvants: Al(OH)3, AddaVax, and Stimune. PBS was used as a control. ELISA was performed to calculate antibody titers at all time points. Throughout the follow-up time, a continuous increase in response was observed, even in the most diluted serum samples. The group immunized with the adjuvant Stimune exhibited the most prominent response, followed by AddaVax, Antigen + PBS, and lastly Al(OH)3.

Figure 2

IgG isotype mean Optical Density (OD) magnitude compared to the control group. (A) Enzyme-linked immunosorbent Assay (ELISA) was performed to calculate antibody titers in Day 19 sera. The mean OD for each subclass was calculated and compared inside each IgG subclass group. IgG1 and IgG3 Stimune were higher than the control group. The same response was observed for IgG2a and IgG2b of its adjuvant. IgG2b AddaVax performed the same scenario. (B) On Day 62, Stimune IgG1, IgG2a, and IgG3 continued higher than the control, as observed for IgG2a of AddaVax In this point of kinects, IgG1 Al(OH)3 was also higher than the control group. The P values were calculated by two-way ANOVA with the Kruskal-Wallis test (* P ≤ 0.05 ** P ≤ 0.01). (C) The complete scenario of the mean OD values for IgG subclasses for each immunized group on the two ELISA assay days. In D19 the variations of OD means and Standard Deviation (SD) values for the control group PBS (IgG1 = 0.067 SD = 0.007; IgG2a =0.075; DP = 0.008; IgG2b = 0.051; DP = 0.004; IgG3 = 0.059; DP = 0.005, antigen alone (IgG1 = 0.503; SD = 0.153; IgG2a = 0.385; DP = 0.261; IgG2b = 0.193. DP = 0.079; IgG3 = 0.089; DP = 0.035), AddaVax (IgG1 = 1.058; SD = 0.278; IgG2a = 0.723; DP = 0.208; IgG2b = 0.449; DP = 0.219; IgG3 = 0.096; DP = 0.015), Stimune (IgG1 = 1.775; SD = 0.004; IgG2a = 0.508; DP = 0.404; IgG2b = 1.046; DP = 0.372; IgG3 = 0.306; DP = 0.097), and Al(OH)3 (IgG1 = 0.526; DP = 0.346; IgG2 = 0.249; DP = 0.131; IgG2b = 0.186; DP = 0.086; IgG3 = 0.103; DP =0.042) are presented, as well as for D62 for PBS (IgG1 = 0.091; SD = 0.021; IgG2a = 0.067; DP = 0.004; IgG2b = 0.050; DP = 0.003; IgG3 = 0.057; DP = 0.003) antigen alone (IgG1 = 2.062; SD = 0.060; IgG2a = 1.727; DP = 0.004; IgG2b = 1.276; DP = 0.589; IgG3 = 0.309; DP = 0.348), AddaVax (IgG1 = 2.126; SD = 0.066; IgG2a = 2.011; DP = 0.114; IgG2b = 1.639; DP = 0.289; IgG3 = 0.403; SD = 0.294), Stimune (IgG1 = 2.211; SD = 0.058; IgG2a = 2.015; DP = 0.114; IgG2b = 1.960; DP= 0.123; IgG3 = 0.489; DP = 0.326) and Al(OH)3 (IgG1 = 2.251; DP = 0.071; IgG2a = 1.432; DP = 0.692; IgG2b = 0.936; DP = 0.192; IgG3 = 0.179; DP = 0.082).

Figure 3

The magnitude of B-cell peptides OD for each group immunized. Enzyme-linked immunosorbent assay (ELISA) was performed with sera isolated from BALB/c immunized in D62. To calculate the cut-off value, we used the mean OD adjuvanted-group/OD mean PBS. The numbers on the Reactivity Index (RI) bar represent how much each group was higher when compared to the control group (PBS). (**p<0.01 and ****p<0.0001).

Figure 4

Cellular immune response scenario. Enzyme-linked immunosorbent spot (ELISpot) was performed in Day 62 splenocyte cells to evaluate after the complete vaccine scheme. (A) Number of spots stimulated for each T-cell peptide tested in all immunized groups. (B) Heatmap of spots mean for each immunized group. The closer to the yellow shade on the intensity bar, the higher the mean found. Stimune and Al(OH)3 showed the highest intensities, especially for Pool 2 of PvCelTOS. AddaVax demonstrated a lower intensity, often close to the blue shade observed for the control group (PBS).

Figure 5

Immune simulation prediction: (A) Antigen and immunoglobulins predicted dynamics, with high antibody titers reached only after the third immunization (IgM+IgG). (B) B lymphocytes: total count and sub-divided. Memory B cells were predicted to reach the peak after the third dose and non-memory B-Cells decay along the kinetics (C) B lymphocyte population per entity-state also predict the active B-cell being maintained after 160 days. The CD4 T-helper lymphocytes count also shows a high peak of memory T-cells (D) and active T-cells (E) and after the third dose. The plot shows total and memory counts. D0/D20/D40 = first, second, and thirst doses.