A subdominant CD8(+) cytotoxic T lymphocyte (CTL) epitope from the Plasmodium yoelii circumsporozoite protein induces CTLs that eliminate infected hepatocytes from culture

Abstract

Previous studies indicated that the Plasmodium yoelii circumsporozoite protein (PyCSP) 57-70 region elicits T cells capable of eliminating infected hepatocytes in vitro. Herein, we report that the PyCSP58-67 sequence contains an H-2(d) binding motif, which binds purified K(d) molecules in vitro with low affinity (3, 267 nM) and encodes an H-2(d)-restricted cytotoxic T lymphocyte (CTL) epitope. Immunization of BALB/c mice with three doses of a multiple antigen peptide (MAP) construct containing four branches of amino acids 57 to 70 linked to a lysine-glycine core [MAP4(PyCSP57-70)] and Lipofectin as the adjuvant induced both T-cell proliferation and a peptide-specific CTL response that was PyCSP59-67 specific, H-2(d) restricted, and CD8(+) T cell dependent. Immunization with either DNA encoding the PyCSP or irradiated sporozoites demonstrated that this CTL epitope is subdominant since it is not recognized in the context of whole CSP immunization. The biological relevance of this CTL response was underlined by the demonstration that it could mediate genetically restricted, CD8(+)- and nitric-oxide-dependent elimination of infected hepatocytes in vitro, as well as partial protection of BALB/c mice against sporozoite challenge. These findings indicate that subdominant epitopes with low major histocompatibility complex affinity can be used to engineer epitope-based vaccines and have implications for the selection of epitopes for subunit-based vaccines.

FIG. 1

Peptides from the PyCSP that were used as immunogens and in the in vitro assays.

FIG. 2

Definition of the CTL epitope. Mice were immunized two times with 40 μg of MAP4(PyCSP57–70) plus 15 μg of Lipofectin. Results shown are those of the CTL assay done with splenocytes removed 14 days after the second immunization. Target P815 cells were coated with 1 μM PyCSP59–70, PyCSP57–70, PyCSP57–66, PyCSP58–67, PyCSP59–68, PyCSP60–69, or PyCSP61–70 or the irrelevant peptides, PfSSP2-15 and PfSSP2-9. E:T Ratio, effector/target cell ratio.

FIG. 3

Depletion of CD8⁺ (−CD8) or CD4⁺ (−CD4) T cells from splenocyte cultures of mice immunized three times with 40 μg of MAP4(PyCSP57–70) and 15 μg of Lipofectin. Effectors were stimulated with 2.5 μM PyCSP59–70. Lysis of target P815 cells coated with 2.5 μM PyCSP59–70 is shown. E:T Ratio, effector/target cell ratio.

FIG. 4

(A) Immunization with PyCSP DNA or irradiated P. yoelii sporozoites did not induce CTLs specific for PyCSP58–67; however, immunization with MAP4(PyCSP57–70) induced high levels of CTLs specific PyCSP58–67. Effector cells were stimulated for 6 days with 1 μM PyCSP58–67; target cells were coated with 1 μM PyCSP58–67. (B) In the same experiment, immunization with PyCSP DNA or irradiated P. yoelii sporozoites induced CTLs against PyCSP280–288; the dominant CD8⁺ CTL epitope. Effector cells were stimulated for 6 days with 2.5 μM PyCSP280–296; target cells were coated with 0.025 μM PyCSP280–288. E:T Ratio, effector/target cell ratio.

FIG. 5

Immunization with PyCSP DNA induces low levels of proliferation against PyCSP57–70. Mice were immunized three times with either 40 μg of MAP4(PyCSP57–70) and Lipofectin, 40 μg of MAPp2p30 and Lipofectin, or 40 μg of PyCSP DNA and PBS. Results shown are those of the lymphocyte proliferation assay done with splenocytes removed 14 days after the last immunization.