Immunization with a lymphocytic choriomeningitis virus peptide mixed with heat shock protein 70 results in protective antiviral immunity and specific cytotoxic T lymphocytes

Abstract

Heat shock proteins (hsp's) isolated from murine cancer cells can elicit protective immunity and specific cytotoxic T lymphocytes (CTLs) by channeling tumor-derived peptides bound to hsp's to the major histocompatibility class I antigen presentation pathway. Here we have investigated if hsp70 can be used in a novel peptide vaccine for the induction of protective antiviral immunity and memory CTLs. A CTL epitope from the well-defined lymphocytic choriomeningitis virus (LCMV) system was mixed with recombinant hsp70 in vitro under conditions that optimize peptide binding to hsp70. Mice were immunized with the hsp70-peptide mixture and challenged with LCMV. Virus titers were reduced 10-100-fold in these mice compared to control mice. Immunization with the hsp70-peptide mixture resulted in the development of CTL memory cells that could be reactivated during LCMV infection, and that in a 51Cr-release assay could lyse cells pulsed with the same peptide, but not cells pulsed with another LCMV peptide. These results show that hsp70 can be used with CTL epitopes to induce efficient protective antiviral immunity and the generation of peptide-specific CTLs. The results also demonstrate the usefulness of hsp70 as an alternative to adjuvants and DNA vectors for the delivery of CTL epitopes to antigen-presenting cells.

Figure 1

In vivo resistance to challenge with LCMV. Mice immunized with 8mer, rhsp70, or 8mer mixed with BSA have high virus titers after challenge with LCMV, whereas mice immunized with rhsp70-mixed 8mer have 8–100-fold lower virus titers. (a) Experiment 1, day 3 after infection with LCMV (n = 3). (b) Experiment 1, day 5 after infection (n = 2). (c) Experiment 2, day 3 (n = 3). (d) Experiment 2, day 5 (n = 2). (e) Experiment 3, day 5 (n = 3 or 4) N.D. = not done. Bars show the standard deviation.

Figure 2

Immunization with 13mer alone gives resistance to LCMV challenge. Mice immunized with the 13mer alone had lower viral titers than did buffer controls, with or without hsp70 or BSA. (a) Experiment 1, day 3 after infection with LCMV (n = 3). (b) Experiment 1, day 5 after infection (n = 2). (c) Experiment 2, day 3 (n = 3). (d) Experiment 2, day 5 (n = 2) (e) Experiment 3, day 5 (n = 3 or 4) N.D. = not done. Bars show the standard deviation.

Figure 3

Immunization with hsp70-mixed peptide elicits peptide-specific CTLs 5 d after challenge with LCMV. (a) Immunization with rhsp70-mixed 8mer or 13mer, but not 8mer or 13mer alone or hsp70 alone, primes for 8mer-specific CTLs. Target: RMA-S pulsed with 8mer. (b) Immunization with hsp70-mixed 8mer does not prime for NP-specific CTLs. Target: RMA-S pulsed with NP. BSA cannot replace hsp70. (c) Neither BSA nor BSA with peptide prime for 8mer-specific CTLs. Target: RMA-S pulsed with 8mer. (d) Immunization with hsp70-mixed or BSA-mixed 8mer does not prime for NP-specific CTL. Target: RMA-S pulsed with NP peptide. The killing of nonpulsed RMA-S cells has been subtracted from the killing of the peptide-pulsed cells in all four panels.

Figure 3

Immunization with hsp70-mixed peptide elicits peptide-specific CTLs 5 d after challenge with LCMV. (a) Immunization with rhsp70-mixed 8mer or 13mer, but not 8mer or 13mer alone or hsp70 alone, primes for 8mer-specific CTLs. Target: RMA-S pulsed with 8mer. (b) Immunization with hsp70-mixed 8mer does not prime for NP-specific CTLs. Target: RMA-S pulsed with NP. BSA cannot replace hsp70. (c) Neither BSA nor BSA with peptide prime for 8mer-specific CTLs. Target: RMA-S pulsed with 8mer. (d) Immunization with hsp70-mixed or BSA-mixed 8mer does not prime for NP-specific CTL. Target: RMA-S pulsed with NP peptide. The killing of nonpulsed RMA-S cells has been subtracted from the killing of the peptide-pulsed cells in all four panels.

Figure 4

Immunization with hsp70-mixed 13mer primes for LCMV-specific CTL activity 5 d after challenge with the virus. Immunization with hsp70-mixed bound 13mer, but not 13mer alone, hsp70 alone, or peptide mixed with BSA primes for CTL activity detected on LCMV-infected targets. Target: LCMV-infected cells. The killing of noninfected MC57G cells has been subtracted from the killing of the infected cells.