Oral immunization with a recombinant malaria protein induces conformational antibodies and protects mice against lethal malaria

Abstract

The increasing death toll from malaria, due to the decreasing effectiveness of current prophylactic and therapeutic regimens, has sparked a search for alternative methods of control, such as vaccines. Although several single proteins have shown some promise as subunit vaccines against sexual blood stages in experimental systems, it is clear that multicomponent vaccines are required. Many logistic difficulties make such an approach prohibitively expensive. In an effort to try to overcome some of these issues, we examined the possibility of oral immunization as a route for inducing host protective immunity. We report here that oral feeding of a malaria protein induced serum antibody levels similar to those induced by intraperitoneal immunization with Freund's adjuvant. Further, responses to conformational epitopes were induced. In the rodent challenge system, significant levels of protection to lethal challenge with malaria were induced in mice. The protective efficacy was highly correlated with antibody levels, which depended on the antigen dosage and required cholera toxin subunit B as an oral adjuvant. These findings offer new approaches to the development of a malaria vaccine and provide justification for the investigation of transgenic plants as a means of vaccine delivery.

FIG. 1.

Characteristics of antibodies raised in mice by oral immunization with 25 μg of EcMSP4 and 10 μg of CTB. (A) Antibody level. (B) Epitope specificity. (C) Sensitivity of reduction. (D) Isotype distribution. All sera were measured at a 1:10,000 dilution. In panel A, the dotted line shows the mean antibody level induced by intraperitoneal immunization with the corresponding protein emulsified in Freund's adjuvant; in panels B, C, and D, the results are represented as the averages for six individually tested serum samples per group, and the error bars indicate the standard errors of the means. In panel C, antibody reactivities to nonreduced MSP4D (NR) and reduced and alkylated MSP4D (RA) are compared by using a Wilcoxon matched-pair test.

FIG. 2.

Characteristics of antibodies raised in mice by oral immunization with 25 μg of EcMSP4/5 and 10 μg of CTB. (A) Antibody level. (B) Epitope specificity. (C) Sensitivity of reduction. (D) Isotype distribution. All sera were measured at a 1:10,000 dilution. In panel A, the dotted line shows the mean antibody level induced by intraperitoneal immunization with the corresponding protein emulsified in Freund's adjuvant; in panels B, C, and D, the results are represented as the averages for six individually tested serum samples per group, and the error bars indicate the standard errors of the means. In panel C, antibody reactivities to nonreduced MSP4/5C (NR) and reduced and alkylated MSP4/5C (RA) are compared by using a Wilcoxon matched-pair test.

FIG. 3.

Blood-stage parasitemia of mice orally immunized with 10 μg of CTB (A) or 25 μg of EcMSP4/5 in the presence of 10 μg of CTB (B) prior to challenge with P. yoelii YM. The survival rate (number of surviving mice/total number of mice) is shown at the top of each graph.

FIG. 4.

Blood-stage parasitemia of mice orally immunized with different amounts of EcMSP4/5 with or without CTB prior to challenge with P. yoelii YM. The amounts of EcMSP4/5 and/or CTB and the survival rate (number of surviving mice/total number of mice) are shown at the top of each graph.

FIG. 5.

Scatter diagram showing the correlation between PyMSP4/5-specific antibody levels (OD) in prechallenge sera and peak levels of parasitemia in 31 mice orally immunized with different amounts of EcMSP4/5 with or without CTB. (A) Antibodies to full-length PyMSP4/5. (B, C, and D) Antibodies to MSP4/5A, MSP4/5B, and MSP4/5C, respectively. The sera were measured at dilutions of 1:10,000 against full-length PyMSP4/5 and 1:1,000 against the three PyMSP4/5 fragments. Circled points indicate mice that survived parasite challenge. Spearman's rank correlation coefficient (r_s) and the associated P value are shown at the top of each graph.