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.1999 Jul;67(7):3290-6.
doi: 10.1128/IAI.67.7.3290-3296.1999.

Cytotoxic T-lymphocyte epitopes fused to anthrax toxin induce protective antiviral immunity

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Cytotoxic T-lymphocyte epitopes fused to anthrax toxin induce protective antiviral immunity

A M Doling et al. Infect Immun.1999 Jul.

Abstract

We have investigated the use of the protective antigen (PA) and lethal factor (LF) components of anthrax toxin as a system for in vivo delivery of cytotoxic T-lymphocyte (CTL) epitopes. During intoxication, PA directs the translocation of LF into the cytoplasm of mammalian cells. Here we demonstrate that antiviral immunity can be induced in BALB/c mice immunized with PA plus a fusion protein containing the N-terminal 255 amino acids of LF (LFn) and an epitope from the nucleoprotein (NP) of lymphocytic choriomeningitis virus. We also demonstrate that BALB/c mice immunized with a single LFn fusion protein containing NP and listeriolysin O protein epitopes in tandem mount a CTL response against both pathogens. Furthermore, we show that NP-specific CTL are primed in both BALB/c and C57BL/6 mice when the mice are immunized with a single fusion containing two epitopes, one presented by Ld and one presented by Db. The data presented here demonstrate the versatility of the anthrax toxin delivery system and indicate that this system may be used as a general approach to vaccinate outbred populations against a variety of pathogens.

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Figures

FIG. 1
FIG. 1
CTL-mediated lysis of peptide coated EL-4 or P815 cells. Mice were injected i.p. with individual LFn fusion proteins plus PA. After in vitro stimulation, samples were assayed for their ability to lyse51Cr-labeled EL-4 or P815 cells coated with the cognate peptide (closed circles) or not coated (open circles). EL-4 cells (H-2b) were used as targets for cultures from C57BL/6 mice. P815 cells (H-2d) were used as targets for cultures from BALB/c mice. The cultures tested in these assays were splenocytes from individual mice immunized with LFn-NP118–126 (A), LFn-NP396–404 (B), LFn-LLO91–99-NP118–126 (C), or LFn-NP118–126-NP396–404 (D). Targeting was evaluated by51Cr release. Threefold effector dilutions are shown. Similar levels of lysis were observed for each of three replicates.
FIG. 2
FIG. 2
CTL-mediated lysis of peptide coated EL-4 or P815 cells. Mice were injected i.p. with LFn-NP118–404 plus PA or LFn-NP118–404 alone. After in vitro stimulation, samples were assayed for their ability to lyse51Cr-labeled EL-4 or P815 cells coated with the cognate peptide (closed circles) or not coated (open circles). The cultures tested in this assay were splenocytes from individual mice immunized with LFn-NP118–404 plus PA (A) or LFn-NP118–404 in the absence of PA (B). Targeting was evaluated by measuring51Cr release. Threefold effector dilutions are shown. Similar levels of lysis were observed for each of three replicates.
FIG. 3
FIG. 3
Protection of immunized BALB/c mice after challenge with LCMV Armstrong. Groups of mice were vaccinated with the LFn fusion proteins shown below the graph. Each of the fusion proteins were mixed with PA prior to injection. Control groups were given injections of phosphate-buffered saline. Mice were challenged i.p. with LCMV Armstrong. The number of LCMV PFU per milligram of lung and spleen samples is shown. For all constructs tested,P < 0.05. Significance was calculated by using Wilcoxon’s rank sum analysis.
FIG. 4
FIG. 4
Protection of immunized BALB/c mice against LCMV clone 13. Mice were vaccinated with LFn-NP118–126 plus PA (closed circles). Control groups were injected with the haplotype-mismatched fusion protein LFn-NP396–404 plus PA (open circles). All mice were challenged i.v. with LCMV clone 13. The number of LCMV PFU per milliliter of serum is shown at 7 and 14 days after challenge. The graph contains data pooled from two experiments. The asterisks represent mice that died during the bleeding procedure.P < 0.05 for days 7 and 14. Significance was calculated by using Wilcoxon’s rank sum analysis.
FIG. 5
FIG. 5
Enhanced recall response against LCMV Armstrong challenge in immunized BALB/c mice. Immunized and control mice were challenged with LCMV Armstrong. Three days after challenge the animals were sacrificed, and pooled spleen cells from the groups were directly assayed for their ability to target51Cr-labeled BALB/c clone 7 cells treated with NP118–126 peptide (closed circles) or not treated (open circles). (A) The immunized group was vaccinated with LFn-NP118–126 mixed with PA. (B) The control group was injected with LFn-NP396–404 mixed with PA.
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References

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