On the delivery of blood stage malaria DNA vaccine using magnetic nanoparticles
thesisposted on 08.02.2017 by Nawwab Al-Deen, Fatin Muhammed
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Abstract Biomedical nanotechnology is revolutionizing the approach to many infectious diseases by providing rapid and simple therapeutic and diagnostic agents. About half of the world’s population is at risk of infection with malaria, while treatment and control have become more difficult. Traditional protein-based malaria vaccines elicit only antibody-mediated (humoral) immune responses and are often expensive because they rely on costly manufacturing methods that are not usually available in countries where the disease is endemic. Thus, there is an urgent need to develop a rapid, effective and inexpensive vaccine that might be affordable in low-resource areas. DNA vaccines have emerged as a potential new strategy against malaria because of their low manufacturing cost and ability to induce both humoral and cellular immune responses against antigens encoded by recombinant DNA. However, the efficiency of delivery of the DNA vaccines is often observed to be low. The use of superparamagnetic nanoparticles (SPIONs) vectors to deliver the malaria gene via magnetofection could help improve the efficacy of gene delivery with a low dose and site specific in vivo applications. Here, magnetofection was used to enhance the delivery of malaria DNA vaccine encoding Plasmodium yoelii merozoite surface protein MSP119 (VR1020-PyMSP119) that plays a critical role in Plasmodium immunity. The plasmid DNA (pDNA) containing membrane associated 19-kDa carboxyl-terminal fragment of merozoite surface protein1 (PyMSP119) was conjugated with polyethyleneimine (PEI) - coated superparamagnetic nanoparticles (SPIONs) with different molar ratios of PEI nitrogen to DNA phosphate (N/P). The effects of SPIONs/PEI complexation pH on the properties of the resulting particles are reported, including their ability to condense DNA and express gene in eukaryotic cells in vitro. SPIONs/PEI complexes under acidic pH conditions showed a better binding capability with VR1020-PyMSP119 than those at neutral conditions, despite the negligible differences in size and surface charge of complexes. The transfection efficiency of magnetic nanoparticles as a carrier for malaria DNA vaccines in vitro as indicated via PyMSP119 expression was significantly enhanced under the application of an external magnetic field, while the cytotoxicity was comparable to the benchmark non-viral reagent (Lipofectamine 2000). Subsequently, magnetofection also showed higher serum antibody titers against PyMSP119 with intraperitoneal and intramuscular injections via in vivo mouse study, compared to subcutaneous and intradermal injections. Robust IgG2a and IgG1 responses were observed for intraperitoneal administration, which could be due to the physiology of peritoneum as a major reservoir of macrophages and dendritic cells. Heterologous DNA prime followed by a single protein boost with recombinant EcPyMSP119 protein vaccination regime was also tested. The regime showed enhanced IgG2a, IgG1, and IgG2b responses, indicating that the induction of appropriate memory immunity that can be elicited by proteins on recall. The assembly order of different quaternary magnetic gene vector configurations comprising SPIONs, PEI, hyaluronic acid (HA), and pDNA (VR1020-PyMSP119) was also investigated in this thesis. The impacts of different cell media on the particle stability in terms of complex size, surface charge, stability, and ability to bind and release DNA were studied. Generally, all vectors showed a relatively small size in water, whereas a higher degree of aggregation was observed immediately after transferring to high-ionic strength media such as 150 mM NaCl buffer and RPMI 1640 culture media. However, the pre-addition of HA to DNA prior adding them to SPIONs/PEI complexes effectively reduced the extent of aggregation in serum-free RPMI, particularly at high HA : PEI % charge ratio. This study demonstrated that structurally well-deﬁned magnetic gene carriers could be designed to improve malaria DNA vaccine delivery systems, for in vitro and in vivo applications. Non-viral polymeric DNA vaccine carriers are likely to be more immunogenic if they can be efficiently taken up by potent antigen-presenting cells (APCs) such as dendritic cells (DCs). Efficient DC targeting vaccines require high efficiency for binding and up-take of the vaccine by cells, followed by DC activation and maturation. Since the pre-addition of HA to DNA prior to SPION/PEI configuration vectors, in particular, displayed the desired degree of stability in terms of narrow size distributions and high stability in RPMI cell media, these vectors was used to transfect dendritic cells that were problematic in order to transfect through HA receptor-mediated endocytosis. The effects of magnetic fields on the transfection and maturation of DCs in vitro by these vectors were investigated using different molecular weights of the HA and % charge ratios of HA: PEI. Among the vectors tested, complexes containing high molecular weight of HA with % high charge ratio of HA : PEI under an external magnetic field yielded a better DC transfection/ maturation than others. This phenomenon was attributed to gene complex stability and transfection efficiency, possibly due to long molecular chains and the higher mucoadhesive properties of high molecular weight HA that enhanced HA ligands accessibility to the DC cell receptors and promoted the multivalent binding to the receptors. Insights gained should improve the design of more effective DNA vaccine delivery systems.