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. 2001 Jun;69(6):3845-52.
doi: 10.1128/IAI.69.6.3845-3952.2001.

Human antibodies against Plasmodium falciparum liver-stage antigen 3 cross-react with Plasmodium yoelii preerythrocytic-stage epitopes and inhibit sporozoite invasion in vitro and in vivo

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Human antibodies against Plasmodium falciparum liver-stage antigen 3 cross-react with Plasmodium yoelii preerythrocytic-stage epitopes and inhibit sporozoite invasion in vitro and in vivo

K Brahimi et al. Infect Immun. 2001 Jun.

Abstract

The Plasmodium falciparum liver-stage antigen 3 (LSA3), a recently identified preerythrocytic antigen, induces protection against malaria in chimpanzees. Using antibodies from individuals with hyperimmunity to malaria affinity purified on recombinant or synthetic polypeptides of LSA3, we identified four non-cross-reactive B-cell epitopes in Plasmodium yoelii preerythrocytic stages. On sporozoites the P. yoelii protein detected has a molecular mass similar to that of LSA3. T-cell epitopes cross-reacting with P. yoelii were also demonstrated using peripheral blood lymphocytes from LSA3-immunized chimpanzees. In contrast, no cross-reactive epitopes were found in Plasmodium berghei. LSA3-specific human antibodies exerted up to 100% inhibition of in vitro invasion of P. yoelii sporozoites into mouse hepatocytes. This strong in vitro activity was reproduced in vivo by passive transfer of LSA3 antibodies. These results indicate that the homologous epitopes may be biologically functional and suggest that P. yoelii could be used as a model to assess the antisporozoite activity of anti-LSA3 antibodies.

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Figures

FIG. 1
FIG. 1
Location of the various LSA3 peptides and recombinant proteins. R1, R2, and R3 represent repeat regions. DG729, NN, and PC sequences were expressed as either GST-fused or β-Gal-fused recombinant proteins (see Materials and Methods).
FIG. 2
FIG. 2
Ab reactivity on sporozoite and liver stages. (A) Western blot analysis of 17XNL sporozoites extracts with human Abs immunopurified on the recombinant protein GST-PC (LSA3-Cterm) (lane 1) or human Abs immunopurified on the recombinant protein β-Gal-DG671 (SALSA antigen) used as control (lane 2). Masses are shown in kilodaltons. ∗, reactivity with a 205-kDa polypeptide. IFAT was performed with human anti-β-Gal-DG729 (LSA3-Nterm) immunopurified Abs on P. yoelii sporozoites isolated from salivary glands of infected Anopheles stephensi mosquitoes (B) and on a cryosection of P. yoelii liver schizont (44-h development) obtained in C3H/Hej mice (C). (D) A direct immunofluorescence assay was performed on the suspension injected into mice (Table 3), showing the detachment of the sporozoite membrane induced by anti-LSA3 antibodies upon live P. yoelii sporozoites. White bars, 10 μm.
FIG. 3
FIG. 3
T-cell responses to P. yoelii sporozoite extract. Antisporozoite proliferative responses of lymphocytes from chimpanzee Gerda sampled 4 weeks after the third immunizing dose with lipopeptide LSA3-NRII. PBMC from Gerda were prepared and were incubated with freeze-thawed P. yoelii or P. berghei sporozoites at concentrations ranging from 50 to 5,000 sporozoites per 2 ×ばつ 105 cells (given as x axis). Experimental and control wells were run in triplicate. The results are presented as stimulation indices calculated as the ratio of geometric mean counts per minute from Ag-stimulated cultures to the value from cultures without Ag. The mean background counts per minute of responses in control cultures without Ag was 2,659 for Gerda.
FIG. 4
FIG. 4
In vitro inhibition of hepatocyte invasion by P. yoelii sporozoites. The inhibitory effect of human anti-LSA3 Abs was evaluated upon P. yoelii or P. berghei sporozoite invasion into mice hepatocytes. Abs were affinity purified on the recombinant protein β-Gal-DG729 (LSA3-Nterm) or β-Gal-DG671 (SALSA). Anti-SALSA Abs (α-SALSA) were used as control for human Abs (α-LSA3). The anti-CS MAb of P. berghei was used as control for the P. berghei sporozoite invasion. The human Abs were tested at a concentration corresponding to their IFAT end point titer (1/2). The anti-CS MAb of P. berghei was used at a dilution of 5 ×ばつ 10−4 (IFAT end point titer of 10−6). All samples were tested in duplicate. The number of liver schizonts was counted in each well, and the percentage of inhibition was calculated compared to that for control wells to which no Abs were added. The numbers of P. yoelii and P. berghei liver schizonts in control wells without Abs were 101.5 ± 15.45 and 110.5 ± 21.70, respectively.

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