To determine the optimum combination, concentration, and formulation of synthetic peptides and adjuvants to induce protective CTL responses against the Plasmodium yoelii circumsporozoite protein (PyCSP), BALB/c mice were immunized with linear and multiple antigen peptides (MAP) including PyCSP CTL and Th epitopes in Montanide's ISA51, Lipofectin, and Lipofectamine. An H-2K(d)-restricted PyCSP CTL epitope, SYVPSAEQI (amino acids (aa) 280-288), recognized by protective CTL clones, was included in the following peptides: a 9-aa linear peptide (SYVPSAEQI; PyCSP9), a 20-aa linear peptide (aa 280-299; SYVPSAEQILEFVKQISSL; PyCSP20), a MAP containing four branches of PyCSP20 (MAP(280-299)), and a linear peptide and a MAP(MAP(280-299)p2p30) in which PyCSP20 was colinearly synthesized with two universal Th epitopes from tetanus toxin (p2p30). A MAP containing the PyCSP Th epitope (aa 57-70; KIYNRNIVNRLLGD) was included in some experiments. The highest specific lytic activity against peptide-pulsed target cells was obtained with splenocytes from mice immunized with three doses at 3-wk intervals of MAP(280-299)p2p30 in Lipofectin or Lipofectamine. Forty percent of the mice immunized with MAP(280-299)p2p30 and Lipofectin were protected against sporozoite challenge. Immunization with CTL and Th epitopes co-linearly synthesized in a MAP induced significantly better CTL than did immunization with the same sequence as a linear peptide, or immunization with a mixture of two individual MAPs, one with the CTL epitope and the second with the Th epitope.

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