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In this study, we have investigated the extent of natural polymorphism in the CD8+ cytotoxic T lymphocyte (CTL) determinant (amino acids 368-390) of circumsporozoite (CS) protein of Plasmodium falciparum field isolates from a holoendemic region of Kenya, and determined how this variation affects the CTL reactivities in clinically immune adults and binding specificities to human histocompatibility leukocyte antigen (HLA)-B35. Among the eight variant sequences that were found in this region, four were new and not seen in parasites from other geographical regions. When synthetic peptides corresponding to the eight variants were used to test the presence of CTL response in different donors, a different spectrum of CTL reactivity to these variants was noticed. While CTL from some donors recognized the P1 sequence (the most prevalent type of sequence) but not P8 (another major variant), other donors showed a reverse pattern of reactivity. Although none of the donors was able to recognize all the variants, CTL responses to all the eight variant sequences were found in this population. An octamer peptide with P1 sequence KPKDELDY in this polymorphic determinant was known to bind HLA-B35. When we tested the effect of natural variation in this octamer sequence on HLA-B35 binding, it became evident that SP13 with D --> N substitution retained its binding specificity to HLA-B35. On the other hand, the SP12 octamer sequence which had two substitutions did not bind HLA-B35. The most interesting finding was the observation that a D --> G substitution at position 374 rescued the binding ability of SP14, which otherwise could not bind to this HLA molecule due to E --> Q amino acid substitution at position 372. To our knowledge, this is the first demonstration showing that a natural polymorphism can rescue the binding specificity to an HLA-class I molecule that was lost due to another natural amino acid substitution. Altogether, these results demonstrate that natural polymorphism in the CS protein affects both the CTL reactivity and the ability to bind to HLA-B35.

Studies in The Gambia have provided indirect evidence that cytotoxic T lymphocytes (CTL) play a protective role against malaria in humans and recently, using allele-specific HLA class I peptide motifs, several peptide epitopes for CTL in four pre-erythrocytic Plasmodium falciparum antigens have been identified in naturally exposed Gambians. However, CTL levels were low, suggesting that boosting these low levels by immunization might provide substantial protection. In the Kilombero valley of Tanzania, malaria transmission is holoendemic and 300 times more intense than in The Gambia. We report here that several of the epitopes identified in The Gambia are also recognized in naturally exposed, partially immune Tanzanian adults and that levels of CTL are similar to or slightly higher than in Gambian subjects, despite the much higher inoculation rate. We report a new HLA-A2.1-restricted epitope from the thrombospondin-related anonymous protein (TRAP) and we demonstrate that peptide epitopes in TRAP are naturally processed for recognition by CTL from naturally exposed humans. The common allele of a variable HLA-B7-restricted epitope in the circumsporozoite protein behaved as an altered peptide ligand (APL) with respect to CTL cognate for a rarer allelic variant of this epitope, suggesting that APL antagonism may occur in natural CTL responses to P. falciparum. The moderate levels of CTL observed, even in this area of intense malaria transmission, points to the need to assess candidate vaccines aimed at increasing CTL levels.

Various protocols were developed and compared for eliciting specific cytotoxic T lymphocyte (CTL) cell lines from the unselected human peripheral blood mononuclear cells of naive donors. Interleukin-7 and CD4+ T cells primed in vitro by keyhole limpet hemocyanin were shown to act together in the generation of these responses. Primary responses were consistently induced with a variety of different HLA class I-binding malarial peptides. Primary CTL responses could be induced from unselected CD8+ and from CD45RA+CD8+ T cells. The CTL lines derived from these naive donors were CD8+ and demonstrated a high level of HLA class I-restricted killing for > 3 months after priming in vitro. They were also able to recognize and kill targets infected with a recombinant vaccinia virus containing the full-length antigen. In addition, this same protocol enhanced up to fourfold the levels of secondary CTL responses induced. The optimal method presented for naive cytotoxic T cell stimulation is simple, rapid and generally applicable and should provide a useful tool for both basic research and human therapy.