Protective efficacy and correlates of immunity of immunodominant recombinant Babesia microti antigens

Abstract

Babesia microti, an intraerythrocytic apicomplexan parasite, is the primary causative agent of human babesiosis and an emerging threat to public health in the United States and elsewhere. An effective vaccine against B. microti would reduce disease severity in acute babesiosis patients and shorten the parasitemic period in asymptomatic individuals, thereby minimizing the risk of transfusion-transmitted babesiosis. Here we report on immunogenicity, protective efficacy, and correlates of immunity following immunization with four immunodominant recombinantly produced B. microti antigens-Serine Reactive Antigen 1 (SERA1), Maltese Cross Form Related Protein 1 (MCFRP1), Piroplasm β-Strand Domain 1 (PiβS1), and Babesia microti Alpha Helical Cell Surface Protein 1 (BAHCS1)-delivered subcutaneously in Montanide ISA 51/CpG adjuvant in three doses to BALB/c mice. Following B. microti parasite challenge, BAHCS1 led to the highest reduction in peak parasitemia (67.8%), followed by SERA1 (44.8%) and MCFRP1 (41.9%); PiβS1 (27.6%) had minimal protective effect. All four B. microti antigens induced high ELISA total IgG and each isotype; however, antibody levels did not directly correlate with anti-parasitic activity in mice. Increased prechallenge levels of some cell populations including follicular helper T cells (T_FH) and memory B cells, along with a set of six cytokines [IL-1α, IL-2, IL-3, IL-6, IL-12(p40), and G-CSF] that belong to both innate and adaptive immune responses, were generally associated with protective immunity. Our results indicate that mechanisms driving recombinant B. microti antigen-induced immunity are complex and multifactorial. We think that BAHCS1 warrants further evaluation in preclinical studies.

Keywords: Babesia microti; immune correlates; recombinant vaccine.

Fig 1

The course of parasitemia following intravenous challenge with Babesia microti parasites. Five female BALB/c mice per group were subcutaneously immunized with a 100 µL emulsion consisting of a 1:1 ratio of recombinant B. microti antigen (SERA1, MCFRP1, BAHCS1, or PiβS1) and an adjuvant (Montanide ISA 51/CpG) or adjuvant alone as indicated on each graph. Mice were immunized subcutaneously with 50 µg of recombinant antigen and boosted twice with 25 µg of recombinant protein delivered at 3 week intervals by the same route. Two weeks after the third immunization, mice were intravenously injected with 5,000 B. microti parasites. Unimmunized control mice were injected with PBS on the same schedule. Parasitemia was monitored every other day by Giemsa-stained blood smears produced from tail blood until the mouse was aparasitemic for two consecutive smears. Parasitemias shown are the mean + SD of all five mice in an immunization group. Two-way (immunization group ×day p.i.) analysis of variance (ANOVA) was used to distinguish the time course of infection of immunized and unimmunized mice; resulting P-values are shown. Protective efficacy of each vaccine was primarily quantified by the reduction in parasitemia in terms of reduced peak parasitemia and reduction in overall parasite burden throughout the course of infection [quantified by the area under the parasitemia curve (AUC)], which were compared by t-test. Other metrics considered for evaluating vaccine efficacy include date of onset and clearance of parasitemia, overall duration of parasitemia, and date of peak parasitemia.

Fig 2

Splenic antibody-secreting and memory B cell populations induced by immunization of female BALB/c (n = 5) mice with three subcutaneous doses of recombinant Babesia microti antigens (SERA1, MCFRP1, PiβS1, or BAHCS1 plus Montanide ISA 51/CpG adjuvant) prior to challenge infection. Statistical significance (by one-way ANOVA) is indicated by *. (a) Antibody secreting cell populations as a proportion of all live splenic lymphocytes (top) and total count per spleen (bottom). (b) Memory B cells and subpopulations defined by co-expression of CD73, CD80, and/or PD-L2 as a proportion of all live splenic lymphocytes (top) and total count per spleen (bottom).

Fig 3

Simple linear regression of prechallenge antibody secreting and memory B cell count induced in female BALB/c mice (n = 5) by immunization with three subcutaneous doses of recombinant Babesia microti antigens (SERA1, MCFRP1, PiβS1, or BAHCS1 plus Montanide ISA 51/CpG adjuvant) prior to challenge infection. Data are shown as reduction in parasitemia with respect to immunized vs unimmunized control mice [both total parasite burden (AUC) and peak parasitemia] for populations for which the slope of the line of best fit differed significantly from 0 (P < 0.05) for at least one regression model. (a) Total cells per spleen vs reduction in parasitemia. (b) Cell count as a proportion of live, singlet lymphocytes vs reduction in parasitemia. Regression models with significantly nonzero slopes are indicated by filled error bands surrounding the line of best fit.

Fig 4

Splenic effector CD4⁺ T cell populations induced by immunization of female BALB/c mice (n = 5) by three subcutaneous doses with recombinant Babesia microti antigens (SERA1, MCFRP1, PiβS1, or BAHCS1 plus Montanide ISA 51/CpG adjuvant) prior to challenge infection. Statistical significance (by t test) is indicated by *. (a) Effector T cell populations as a proportion of all splenic CD4⁺ T cells (top) and total count per spleen (bottom). (b) Median fluorescence intensity (MFI) of flow cytometric signal for three intracellular functional cytokines (IFNγ, IL-2, and TNFα) in CD4⁺ T cells. (c) Ratio of T-bet^-GATA3⁺ T_H2 cells to T-bet⁺GATA3^- T_H1 cells in each mouse.

Fig 5

Splenic effector CD4⁺ T cell populations induced by immunization of female BALB/c mice (n = 5) with three subcutaneous doses of recombinant Babesia microti antigens (SERA1, MCFRP1, PiβS1, or BAHCS1 plus Montanide ISA 51/CpG adjuvant) prior to challenge infection. Statistical significance (by t test) is indicated by *. (a) Memory and total effector CD4⁺ T cell populations as a proportion of all splenic CD4⁺ T cells (top) and total count per spleen (bottom). (b) Memory and effector CD8⁺ T cell populations as a proportion of all splenic CD8⁺ T cells (top) and total count per spleen (bottom).

Fig 6

Simple linear regression of prechallenge effector T cell count and effector cytokines induced in BALB/c mice (n = 5) by immunization with three subcutaneous doses of recombinant Babesia microti antigens (SERA1, MCFRP1, PiβS1, or BAHCS1 plus Montanide ISA 51/CpG adjuvant) prior to challenge infection. Data are shown as reduction in parasitemia with respect to immunized vs unimmunized control mice [both total parasite burden (AUC) and peak parasitemia] for populations for which the slope of the line of best fit differed significantly from 0 (P < 0.05) for at least one regression model. (a) Total cells per spleen vs reduction in parasitemia. (b) Cell count as a proportion of CD4⁺ T cells vs reduction in parasitemia. Regression models with significantly nonzero slopes are indicated by filled error bands surrounding the line of best fit.

Fig 7

Simple linear regression of prechallenge cytokine serum concentration in female BALB/c mice (n = 5) induced by immunization mice with three subcutaneous doses of recombinant Babesia microti antigens (SERA1, MCFRP1, PiβS1, or BAHCS1 plus Montanide ISA 51/CpG adjuvant) prior to challenge infection. Data are shown as reduction in parasitemia with respect to immunized vs unimmunized control mice [both total parasite burden (AUC) and peak parasitemia] for cytokines for which the slope of the line of best fit differed significantly from 0 (P < 0.05). Of the 23 cytokines detected by the Bio-Plex Pro Mouse Cytokine 23-plex Assay kit, seven exhibited a significant linear correlation between prechallenge serum concentration and reduction in peak parasitemia and/or reduction in area under the parasite curve; five cytokines exhibited significant linear correlations between prechallenge serum concentration and both parasite reduction metrics. Regression models with significantly nonzero slopes are indicated by filled error bands surrounding the line of best fit.