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The Potential of Synthetic Lipid-Based Peptide Vaccines
by Dr. Brendon Chua

Department of Microbiology & Immunology
University of Melbourne, Australia




After clean water and good hygiene, vaccination is the most successful and economically effective strategy to significantly reduce mortality and morbidity rates caused by infectious diseases. At least 26 infectious diseases are now preventable through the use of vaccines and it is estimated that immunization saves the lives of 3 million children a year (1).


Advances made in the field of immunology over the last few years have produced insights into the mechanisms governing the initiation and maintenance of immune responses against pathogens. This has resulted in the advent of new technologies and manufacturing processes that now allow us to rationally design novel prophylactic and therapeutic vaccines.


Amongst these technologies is the utilization of synthetic peptides. The prospect of making totally synthetic vaccines based on peptides is mainly due to the awareness of the role that short peptide sequences play in immune recognition. Upon encounter of a pathogen such as bacteria or a parasite, it is captured and degraded by specialised antigen presenting cells of the immune system into short peptide sequences known as epitopes. These epitopes are then presented to effector T cells and if the correct cellular signals are given, this can trigger off a cascade of events that eventually culminates in the induction of an immune response.


The concept of using synthetic peptides, as a basis for vaccine design is simple: if the epitope that is recognised by an antibody or a T cell is known, then a vaccine can be designed around that epitope. Past literature is filled with studies that have shown that vaccination of peptide epitopes, administered in adjuvant can elicit both antibody and cellular-mediated immune responses that can induce protective immunity against a repertoire of targeted pathogens that have included viruses, bacteria, parasites, as well as tumours and even self-hormones.

    Furthermore, the use of synthetic peptides as vaccines offers many advantages over conventional vaccines:
  • Unlike live or attenuated vaccines, their composition does not contain materials from biological sources and there is therefore no need to handle potentially infectious or bio-hazardous material.
  • Issues pertaining to the possibility of virulence due to reversion and re-assortment associated with the use of these vaccines as well as concerns of genetic integration or recombination associated with the use of DNA-based vaccines are irrelevant.
  • Synthetic peptide vaccine preparations lend themselves to chemical definition using analytical techniques such as liquid chromatography and mass spectrometry, thereby lending themselves to robust quality control procedures.
  • Economically, the large-scale production of these vaccines is not restricted by expensive and labour intensive equipment, thus facilitating the manufacturing process to result in a commercially attractive product.
  • Logistical advantages are also apparent; peptides can be freeze-dried in a lyophilised form circumventing the requirement of “cold-chain” facilities necessary for their storage and transport. This attribute in particular is an important consideration when aiming to vaccinate populations in developing countries that lack the infrastructure to accommodate temperature sensitive preparations.


Despite these many advantages and examples of proof of principle in many models, however, synthetic peptides are usually poorly immunogenic. This shortcoming is true of most soluble proteins in general due to their size and susceptibility to degradation in serum. It is this poor immunogenicity that dictates the requirement of a co-administered adjuvant in order to attract the attention of the immune system. Although several adjuvants are available for use in animals, so far only squalene oil water emulsions and aluminium-based salt adjuvants have been approved for human use. Even so, the use of these adjuvants is limited by the range of immune responses they can induce (2, 3). It is not surprising therefore that many researchers are focused on developing other ways to enhance peptide immunogenicity.


Of particular interest to many groups are palmitic acid-based adjuvants ranging from palmitic acid itself to the somewhat more sophisticated derivatives dipalmitoyl-S-glyceryl-cysteine (Pam2Cys) and tripalmitoyl-S-glyceryl-cysteine (Pam3Cys). These simple lipid moieties have been shown to be effective in adjuvanting epitope-based vaccines and importantly, do not exhibit the harmful side effects that are commonly associated with many other adjuvant formulations and appear to hold promise as candidates to achieve this objective.


Initial reports that the immunogenicity of peptides could be improved by the incorporation of lipids appeared in the 1980’s beginning with the acylation of a peptide derived from hepatitis B surface antigen with palmitic acid groups (4). Following this, Pam3Cys, a synthetic version of a lipid known as Braun’s lipoprotein which is found in the cell wall of Gram negative bacteria (5), was discovered to enhance the immunogenicity of T cell epitopes derived from influenza virus and shown to be capable of inducing T cell responses (6). This lipid group moiety has since been studied extensively and although highly immunogenic and effective at adjuvanting peptide epitopes, lipopeptides containing this lipid group can also be quite difficult to dissolve in solution making accurate dosing difficult. This is mainly due to the hydrophobic structure of the lipid, which comprises of three palmitic acid groups that are bound in an ester linkage to a cysteine residue. Other factors that affect solubility also include the position of the lipid in relation to the peptide epitope itself and the type of spacer that is used to distance the lipid group from the structure (7, 8). Some investigations have focused on using the structurally similar derivative, Pam2Cys, which has one less palmitic acid group. This lipid is a synthetic analogue of MALP-2 (macrophage activating lipopeptide-2), a lipid derived from the cytoplasmic membrane of Mycoplasma fermentans (9). Effective immune responses, both antibody and/or cell-mediated can also be successfully induced through the use of lipopeptides containing this lipid moiety (10). Furthermore, such formulations have the added advantage that solubility is improved.


The potent immunogenicity of Pam2Cys- and Pam3Cys-based lipopeptides can be explained by their ability to activate specialized antigen-presenting cells of the immune system known as dendritic cells by triggering surface receptors known as Toll-like receptors (TLRs). These cells play a critical role in immune response induction because in addition to being adept at capturing pathogens through the expression of specific receptors, they are also able to provide critical signals required for the recruitment and activation of downstream effector cells.


It is clear that the adjuvanting properties of such lipids are due to their ability to be recognised by TLRs; Pam2Cys has been shown to induce signalling through TLR-2 and 6 (9) whilst Pam3Cys can signal through TLR-2 and 1 (12). This signalling requires the involvement of the adaptor molecule MyD88 and results in the eventual activation of transcription factors including NF-κB (13). In DCs, this leads to an up-regulation of MHC and costimulatory molecules and secretion of pro-inflammatory chemokines (14). The ability of these lipids to be recognized by TLRs is a reflection of the evolvement of vertebrate immune systems to recognize the signatures of microorganisms such as these lipid structures. It is however, still unclear how lipids with one or two isolated palmitic acid residues, as opposed to when in the context of an acylated Cys, exert their biological effect because these lipid components are only distantly related to those derived from bacterial lipopeptides and proteins. There is some suggestion that these lipids anchoring themselves within the cell membrane to peptide epitopes direct access to the interior of the cell.


The discovery of TLRs and the realization of their significance in the recognition of pathogens has not only expanded our understanding of how innate immunity is critically linked to the adaptive immune response through the dendritic cell but has also prompted an exploration into the use of their ligands as vaccine adjuvants. As such, current investigations have extended to other related lipid groups such as stearic, lauric and even octanoic acid, focusing on the relationship between the structure and length of the lipid moiety and its biological function (7). Such studies will hopefully enable to gain further insights into how the immune system functions to detect pathogens as well as to provide vaccinologists with more options to design and formulate future vaccines.








References
  1. Andre F. E. (2003) Vaccinology: past achievements, present roadblocks and future promises. Vaccine 21 (7-8): 593-5.

  2. Mesa C. and Fernandez L. E. (2004) Challenges facing adjuvants for cancer immunotherapy. Immunol Cell Biol 82 (6): 644-50.

  3. Brewer J. M. (2006) (How) do aluminium adjuvants work? Immunol Lett 102 (1): 10-5.

  4. Hopp TP. Immunogenicity of a synthetic HBsAg peptide: enhancement by conjugation to a fatty acid carrier. Mol Immunol 1984;21(1):13-6.

  5. Weismuller KH, Bessler WG, Jung G. Synthesis of the mitogenic S-(2,3-bis(palmitoloxy)propyl)-N-palmitolyl pentapeptide from the Escherichia coli lipoprotein. Hoppe Seyler's Z Physiol Chem 1983;364:593.

  6. Deres K, Schild H, Wiesmuller KH, Jung G, Rammensee HG. In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine. Nature 1989;342(6249):561-4.

  7. Chua BY, Zeng WG, Lau YF, Jackson DC. Comparison of lipopeptide-based immunocontraceptive vaccines containing different lipid groups Vaccine, 2007, 25 (1): 92-101.

  8. Zeng, W., Ghosh, S., Lau, Y.F., Brown, L.E. and Jackson, D.C. Highly Immunogenic and Totally Synthetic Lipopeptides as Self-Adjuvanting Immunocontraceptive Vaccines. J. Immunol. 2002. 169: 4905-12.

  9. Muhlradt PF, Kiess M, Meyer H, Sussmuth R, Jung G. Isolation, structure elucidation, and synthesis of a macrophage stimulatory lipopeptide from Mycoplasma fermentans acting at picomolar concentration. J Exp Med 1997;185(11):1951-8.

  10. Jackson, D.C., Lau, Y.F., Le, T. et al. A totally synthetic vaccine of generic structure that targets Toll-like receptor 2 on dendritic cells and promotes antibody or cytotoxic T cell responses. Proc Natl Acad Sci U S A 2004, 101(43), 15440-15445.

  11. Lee, J.Y., Zhao, L., Youn, H.S. et al. Saturated fatty acid activates but polyunsaturated fatty acid inhibits Toll-like receptor 2 dimerized with Toll-like receptor 6 or 1. J Biol Chem 2004, 279(17), 16971-16979.

  12. Takeuchi, O., Sato, S., Horiuchi, T. et al. Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J Immunol 2002, 169(1), 10-14.

  13. Medzhitov, R. Toll-like receptors and innate immunity. Nat Rev Immunol 2001, 1(2), 135-145.

  14. Kaisho, T. & Akira, S. Toll-like receptors as adjuvant receptors. Biochim Biophys Acta 2002, 1589(1), 1-13.





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