Linkage of exogenous T-cell epitopes to the 19-kilodalton region of Plasmodium yoelii merozoite surface protein 1 (MSP1(19)) can enhance protective immunity against malaria and modulate the immunoglobulin subclass response to MSP1(19)

Abstract

The degree of protection against Plasmodium yoelii asexual blood stages induced by immunization of mice with the 19-kDa region of merozoite surface protein 1 (MSP1(19)) is H-2 dependent. As a strategy to improve the protection, mouse strains with disparate H-2 haplotypes were immunized with glutathione S-transferase (GST)-MSP1(19) proteins including either a universal T-cell epitope from tetanus toxin (P2) or an I-A(k)-restricted T-cell epitope (P8) from Plasmodium falciparum Pf332. In H-2(k) mice which are poorly protected following immunization with GST-MSP1(19), GST-P2-MSP1(19) significantly improved the protection. In mice partially (H-2(k/b)) or well protected by GST-MSP1(19) (H-2(d) and H-2(b)), P2 did not further increase the protection. However, the protection of H-2(k/b) mice and to some extent H-2(k) mice was improved by immunization with GST-P8-MSP1(19). The magnitudes of immunoglobulin G1 (IgG1) and IgG2a responses in mice immunized with the GST-MSP1(19) variants correlated with low peak parasitemia, indicating a protective capacity of these IgG subclasses. In H-2(k) mice immunized with GST-P2-MSP1(19), both IgG1 and IgG2a responses were significantly enhanced. The epitope P2 appeared to have a general ability to modulate the IgG subclass response since all four mouse strains displayed elevated IgG2a and/or IgG2b levels after immunization with GST-P2-MSP1(19). In contrast, GST-P8-MSP1(19) induced a slight enhancement of IgG responses in H-2(k/b) and H-2(k) mice without any major shift in IgG subclass patterns. The ability to improve the protective immunity elicited by P. yoelii MSP1(19) may have implications for improvement of human vaccines based on P. falciparum MSP1(19).

FIG. 1

Parasite challenge of mice immunized with GST-MSP1₉ with or without additional T-cell epitopes. Two weeks after the third injection of immunogen, groups of five mice were inoculated intravenously with 10⁴ P. yoelii YM-infected RBC. Parasitemia was monitored daily from day 3. B10.BR, B10.A(4R), and C57BL/6 mice were either naive or were immunized with GST-MSP1₁₉, GST-P2-MSP1₁₉, GST-P8-MSP1₁₉, or a mixture of GST and MAP-P2; B10.D2 mice were immunized with GST-MSP1₁₉, GST-P2-MSP1₁₉, or GST/MAP-P2. All groups were analyzed in parallel except for the C57BL/6 mice immunized with GST-P8-MSP1₁₉, which were analyzed in a separate experiment where C57BL/6 immunized with GST-MSP1₁₉ or GST-P8-MSP1₁₉ were found to be similarly protected (maximal parasitemias for the GST-MSP1₁₉ group were <0.001 [two mice], 0.22, 0.44, and 1.7% and for the GST-P8-MSP1₁₉ group were <0.001, 0.16, 0.18, 0.65, and 17%).

FIG. 2

MSP1₁₉-specific IgG subclass responses in mice immunized with GST-MSP1₁₉ with or without additional T-cell epitopes as measured by ELISA. Groups of five B10.A(4R), B10.BR, C57BL/6, and B10.D2 mice were immunized with GST-MSP1₁₉, GST-P2-MSP1₁₉, or GST-P8-MSP1₁₉ [B10.A(4R) and B10.BR mice only]. The bars display mean reactivities plus standard deviations of sera analyzed for reactivity with MSP1₁₉ at dilutions of 1:2¹⁸ (IgG1), 1:2¹² (IgG2a), 1:2¹⁷ (IgG2b), or 1:2¹⁰ (IgG3). No significant reactivity was displayed by sera from GST-immunized or naive mice at these dilutions (not shown). Abs₄₉₂, absorbance at 492 nm.

FIG. 3

GST-specific IgG subclass responses in B10.BR mice immunized with GST fusion proteins, as determined by ELISA. Groups of five mice were immunized with GST/MAP-P2, GST-MSP1₁₉, GST-P2-MSP1₁₉, or GST-P8-MSP1₁₉. The bars display reactivities of pooled sera analyzed for reactivity with GST at dilutions of 1:2¹⁵ (IgG1 and IgG2b) or 1:2¹⁰ (IgG2a and IgG3). Sera from naive mice did not react significantly at these dilutions (not shown). Abs₄₉₂, absorbance at 492 nm.

FIG. 4

Comparison of MSP1₁₉-specific IgG subclass responses in B10.A(4R) mice immunized with GST-MSP1₁₉ or MSP1₁₉ as measured by ELISA. Groups of five mice were immunized s.c. with either GST-MSP1₁₉ or GST-free MSP1₁₉. The bars display mean reactivities of sera analyzed for reactivity with MSP1₁₉ at dilutions of 1:2¹⁹ (IgG1), 1:2¹⁴ (IgG2a), 1:2¹⁸ (IgG2b), or 1:2¹³ (IgG3). Abs₄₉₂, absorbance at 492 nm.

FIG. 5

Relationship between the MSP1₁₉-specific IgG titers of antisera to GST-MSP1₁₉ variants and the capacity to inhibit the binding of MAb F5 or MAb B10 to MSP1₁₉. The MSP1₁₉-specific titers of sera from B10.A(4R), B10.BR, C57BL/6, and B10.D2 mice immunized with GST-MSP1₁₉, GST-P2-MSP1₁₉, or GST-P8-MSP1₁₉ [B10.A(4R) and B10.BR mice]) were determined by ELISA. To establish the capacity of the sera to inhibit the binding of MAb to MSP1₁₉, ELISA plates were incubated first with serum diluted 1:600 and subsequently with biotinylated MAb F5 (A) or MAb B10 (B). The IgG titers of individual sera are plotted against the level of competition obtained with the corresponding sera. Open circles, mice that died following challenge infection. The lines display the association between IgG titer and inhibition of MAb F5 (r = 0.52; n = 50; P ≤ 0.0001) (A) and MAb B10 (r = 0.66; n = 50; P ≤ 0.0001) (B). The binding of biotinylated MAb could be inhibited by adding an excess of nonlabeled homologous but not heterologous MAb (not shown).