Peptide-Based Drug Design

248 Chua et al.
vaccines, including peptide epitope-based vaccines, is the weak immunogenicity
that they display in the absence of adjuvant. Although many experimental
adjuvants are available, only oil-in-water emulsions, aluminum-based
salt adjuvants, and virosomes are currently licensed for use in humans, while
surfactant-based and microbial-derived formulations are presently still at the
stage of preclinical and clinical trial testing (19,20).
An effective way to enhance the immunogenicity of peptide epitopes is
to incorporate lipid groups that have self-adjuvanting properties into the
peptide structure. Of particular interest to many groups are palmitic acid–based
adjuvants ranging from palmitic acid itself (21,22) to the somewhat more sophisticated
derivatives dipalmitoyl-S-glyceryl cysteine (Pam2Cys) (1,4,23–25) and
tripalmitoyl-S-glyceryl cysteine (Pam3Cys) (26–28). These simple lipid moieties
have been shown to be effective in adjuvanting epitope-based vaccines and,
importantly, do not exhibit the harmful side effects commonly associated with
many other adjuvant formulations (29).
Lipopeptides bearing Pam2Cys in particular have been shown to be able
to induce effective Th1- and Th2-type immune responses as demonstrated by
the generation of both short- and long-term protective cytotoxic T lymphocytes
and induction of antibody. Pam2Cys is a synthetic analogue of MALP-2
(macrophage-activating lipopeptide-2) derived from the cytoplasmic membrane
of Mycoplasma fermentans (24). The potent immunogenicity of lipopeptides
bearing Pam2Cys can be explained by their ability to activate dendritic cells
(4,23,25), key cells that are responsible for initiating immune responses. This
event occurs through a mechanism consistent with signal transduction via Tolllike
receptor-2 (TLR-2) (4), one of many receptors involved in the first line of
host defense against pathogenic threats.
This chapter describes in detail the techniques involved in the synthesis of a
Pam2Cys-based lipopeptide vaccine that can induce the production of specific
antibodies in vaccinated mice. As an example, we have used the sequence for
luteinizing hormone-releasing hormone (LHRH) as the target antigen, but this
can be substituted with other B-cell epitopes depending on the requirements of
the investigator. LHRH is a 10-amino-acid peptide hormone that is secreted by
the hypothalamus to initiate a cascade of events that regulates gametogenesis and
reproductive function. It does this by stimulating the synthesis and secretion of
follicle-stimulating hormone and luteinizing hormone from the anterior pituitary
(30). The LHRH sequence is an ideal immunocontraceptive target because its
sequence, HWSYGLRPG, is conserved in all mammals. Such a vaccine could
be of benefit as an alternative to surgical castration in companion animals and
particularly in the livestock industry (31). Although its use in humans is limited
because of the behavioral problems associated with the suppression of sex
hormone production, it may nonetheless still prove useful as a supplementary
treatment for the control of hormone-dependent malignancies (32,33).