Determinants of Malaria Protective Immunity in Mice Immunized with Live Sporozoites during Trimethoprim-Sulfamethoxazole Prophylaxis

Abstract

HIV and malaria geographically overlap. Trimethoprim-sulfamethoxazole (TMP-SMX) is a drug widely used in HIV-exposed uninfected and infected children in malaria-endemic areas, and is known to have antimalarial effects. Further study in terms of antimalarial impact and effect on development of malaria-specific immunity is therefore essential. Using rodent malaria models, we previously showed that repeated Plasmodium exposure during TMP-SMX administration, or chemoprophylaxis vaccination (CVac), induces CD8 T-cell-dependent preerythrocytic immunity. However, humoral immune responses have been shown to be important in models of preerythrocytic immunity. Herein, we demonstrate that antibody-mediated responses contribute to protective immunity induced by CVac immune sera using TMP-SMX in models of homologous, but not heterologous, parasite species. Clinical studies must account for potential anti-Plasmodium antibody induced during TMP-SMX prophylaxis.

Figure 1.

Antibody response generated by TMP-SMX-CVac are protective against homologous sporozoite infection. (A) Chemoprophylaxis vaccination (CVac) sera incubation with Plasmodium yoelii sporozoites (SPZs) results in reduced liver-stage parasite burden as measured by qPCR after in vitro infection. Shown is liver stage burden after in vitro sporozoite neutralization assay (SNA), with experiments conducted in quadruplicate, normalized to parasite burden estimated in SNA using naive mouse serum. DC = drug Control; TMP-SMX = trimethoprim-sulfamethoxazole-CVac immunized. P < 0.03 determined by Mann-Whitney test. Sporozoite neutralization assays (SNAs) demonstrated that incubation of P. yoelii SPZs with CVac sera resulted in significantly reduced liver-stage parasite burden in vitro, with reduction from median of 80% to median of 62% (A, P = 0.03, Mann–Whitney test). However, incubation of P. berghei SPZs with CVac sera did not reduce liver-stage parasite burden (data not shown). (B) CVac sera preincubation with P. yoelii SPZ delays but does not prevent patency. Shown herein are Kaplan-Meier plots representing data from in vivo SNAs, representative of two experiments with 5–10 mice per condition for each species of parasite. Preincubation of P. yoelii SPZs with CVac sera resulted in a significant delay in P. yoelii patency (day of detection of parasites in blood) compared with DC sera preincubation (P = 0.031, log-rank/Mantel–Cox test). By contrast, patency of heterologous P. berghei parasites was not significantly delayed (data not shown). Preincubation with sera from naive or DC mice with either parasite species did not affect mean days to patency compared with infection only controls (B for P. yoelii, data not shown for P. berghei). This figure appears in color at www.ajtmh.org.

Figure 2.

Antibody generated by TMP-SMX-CVac recognizes antigens from sporozoites (SPZ) and liver stages (exoerytrocytic forms, EEFs) of homologous parasites, but only EEFs of heterologous parasites. GFP-expressing P. yoelii (A: SPZ and B: EEFs) and P. berghei (C: SPZ and D: EEFs) SPZ and EEFs were immunostained with either serum (at a dilution of 1:400) from control/nave or TMP-SMX-CVac immunized mice and antibody recognition was detected using Alexa-594 conjugated anti-mouse IgG (red signal). EEF images were acquired 48h post-SPZ infection of either HepG2 (P. berghei) or HepG2:CD81 (P. yoelii). DAPI was used to stain the nucleus of both parasite and host cells. Chemoprophylaxis vaccination (CVac)/trimethoprim–sulfamethoxazole (TMP-SMX) mouse sera recognizes Plasmodium yoelii SPZs and exoerythrocytic forms EEFs (A and B), but only Plasmodium berghei EEFs (C and D). DC: Drug control; Trimethoprim-Sulfamethoxazole-CVac immunized; DIC = differential interference contrast image.