ABSTRACT
Bacillus anthrax lethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8-9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro. These results lay the foundation for future clinical vaccine studies.
Subject(s)
AIDS Vaccines/immunology , Antigens, Bacterial , Bacterial Toxins/genetics , CD8-Positive T-Lymphocytes/immunology , Cytotoxicity, Immunologic , HIV Antigens/immunology , AIDS Vaccines/genetics , Animals , Antigen Presentation , Cytosol/immunology , Cytotoxicity Tests, Immunologic , Drug Carriers , HIV Antigens/genetics , Macrophages , Mice , Mice, Inbred BALB C , Protein Engineering , Rabbits , Recombinant Fusion Proteins/immunology , VaccinationABSTRACT
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.
Subject(s)
Antigens, Bacterial , Antigens, Viral/immunology , Bacterial Toxins/immunology , Cytotoxicity, Immunologic , Lymphocytic choriomeningitis virus/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Antigen Presentation , Antigens, Viral/genetics , Antiviral Agents/immunology , Bacterial Toxins/genetics , Epitopes/genetics , Epitopes/immunology , Immunotherapy/methods , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Recombinant Fusion Proteins/immunology , Virus Diseases/immunologyABSTRACT
We reported earlier that a nontoxic form of anthrax toxin was capable of delivering a cytotoxic T-lymphocyte (CTL) epitope in vivo, such that a specific CTL response was primed against the epitope. The epitope, of bacterial origin, was fused to an N-terminal fragment (LFn) from the lethal-factor component of the toxin, and the fusion protein was injected, together with the protective antigen (PA) component, into BALB/c mice. Here we report that PA plus LFn is capable of delivering a different epitope--OVA(257-264) from ovalbumin. Delivery was accomplished in a different mouse haplotype, H-2Kb and occurred in vitro as well as in vivo. An OVA(257-264)-specific CTL clone, GA-4, recognized EL-4 cells treated in vitro with PA plus as little as 30 fmol of the LFn-OVA(257-264) fusion protein. PA mutants attenuated in toxin self-assembly or translocation were inactive, implying that the role of PA in epitope delivery is the same as that in toxin action. Also, we showed that OVA(257-264)-specific CTL could be induced to proliferate by incubation with splenocytes treated with PA plus LFn-OVA(257-264). These findings imply that PA-LFn may serve as a general delivery vehicle for CTL epitopes in vivo and as a safe, efficient tool for the ex vivo expansion of patient-derived CTL for use in adoptive immunotherapy.
