Design, synthesis and efficacy of Pam2Cys-based vaccine delivery modules
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2013
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Melbourne Authors
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Department of Microbiology and Immunology
Microbiology & Immunology
Microbiology & Immunology
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Masters Research thesis
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Prasetyoputri, A. (2013). Design, synthesis and efficacy of Pam2Cys-based vaccine delivery modules. Masters Research thesis, Department of Microbiology and Immunology, The University of Melbourne.
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© 2013 Anggia Prasetyoputri
Abstract
Sub-unit vaccines including protein-based and peptide-based structures are generally weak immunogens and require the addition of adjuvant to enhance their immunogenicity. Despite the many types of potential adjuvants available their use is often hampered by issues of toxicity and adverse side effects. An alternative approach in constructing more immunogenic vaccines is to utilize a delivery system or module that facilitates direct targeting to the all-important antigen-presenting cell (APC) – the dendritic cells (DCs) – to subsequently activate the adaptive immune system leading to potent humoral and/or cellular immune responses.
In this study we have designed simple and versatile delivery modules that are applicable to a variety of antigens including peptides and proteins, which consist of Pam2Cys and nitriloacetic acid (NTA) with varying numbers of NTA molecules. This synthetic analogue of MALP-2 (macrophage activating lipopeptide-2) derived from the cytoplasmic membrane of Mycoplasma fermentans has proven to have impressive adjuvanting properties owing to its ability to target and stimulate DC maturation via Toll-like receptor (TLR) 2 binding, resulting in potent immunogenicity of Pam2Cys-containing lipopeptides and also proteins. Recent studies also provided evidence that delivery of antigen to DC could occur via electrostatic association without covalent coupling between antigen and the Pam2Cys moiety, hence we hypothesized that association between the NTA portion of the Pam2Cys-based module and the His-tag coupled to the antigen can facilitate delivery of antigen to DC and subsequently activate their maturation. Here we elucidated and optimized the conditions for association between Pam2Cys-NTA modules and His-tag peptides as well as investigated their efficacy in experimental animals.
The results showed that Pam2Cys-Lys-fan-NTA4 precipitated in the presence of nickel ions without any His-tag peptide. This may be due to self-aggregation of multiple NTA molecules present; therefore this module is not suitable for analysis of association between Pam2Cys-NTA modules and His-tag peptides. Pam2Cys-PEG-NTA (under development as “Monokelp”) was able to form complexes with His-tag peptides and the concentrations of each component for optimal association were 20nmoles of His-tag peptide, 60nmoles of Pam2Cys-PEG-NTA and 160nmoles of NiSO4. Results from in vivo studies in mice showed that Pam2Cys-PEG-NTA is potentially capable of delivering His-tagged peptide because antibody was induced in the group of mice receiving Pam2Cys-PEG-NTA, 6His-tagged antigen and NiSO4. However, further studies are needed to improve the design to ensure strong affinity between His-tagged antigen and the Pam2Cys-NTA module, as well as studies to investigate the survival of peptide-module complexes following inoculation as the immune response was not as potent as expected. The feasibility of the Pam2Cys-NTA module in delivering His-tagged antigen to elicit cellular responses remains to be elucidated. Using this technology to deliver His-tagged proteins rather than peptides will also establish the versatility of Pam2Cys-NTA module. The experimental work described in this thesis has highlighted the feasibility of incorporating Pam2Cys into a delivery module that is easy to manufacture and potentially immunogenic.
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Pam2Cys, vaccine delivery, peptide, immune response