| Popis: |
Malaria is a tropical parasitic disease spread worldwide by protozoan parasites of the genus Plasmodium. Attempts to eradicate or control the disease have largely failed and the weapon-of-choice in combating malaria, a vaccine, still eludes us. Now that the Plasmodium falciparum (Pf ) genome has been revealed, it is essential to search for new potential antigens. On that basis, my project aims to select and produce putative vaccine candidates that together with protein microarray techniques will unravel possible correlates of protection against the human malaria parasite Pf, responsible for the majority (90%) of deaths from malaria. My PhD project forms part of FightMal a consortium-based study that first use bioinformatic analysis to identify those proteins within the Pf proteome that are known or anticipated to be either anchored on the parasite/infected host cell surface or secreted. Such in silico analysis produced 3 ‘candidate lists’ of interest, one for each of the 3 types of protein chips planned for production: i) current vaccine candidates (n.25), ii) putative immune targets (n.260), iii) variant surface antigens, VSA (n.74). Candidates belonging to CHIP 2 were cloned and expressed (in vivo) using customized high-throughput platforms. To date, 394 (88%) Chip 2 DNA targets have been successfully amplified by PCR, 325 (82%) of these have been inserted into the expression plasmid and sequenced. A total of 269 constructs resulted suitable for heterologous expression. Due to the complexity of soluble expression of plasmodial proteins a number of different expression conditions were tested. Both the strain of the E. coli host and the composition of the expression medium resulted crucial variables for satisfactory results. Via a multi-step optimisation phase we increased the initial number of soluble candidates from 18% to 44%, which eventually led to a 6-fold increase in the number of targets available for purification. During an early evaluation study, 77% (10/13) of the recombinant proteins produced in large-scale were purified using embedded affinity tags yielding a ≥90% purity level. Preliminary results, obtained spotting 40 in vitro expressed Pf candidates, successfully demonstrated the potentials of the protein microarray technology as an efficient serum-based multiplex assay capable of identifying new antigens. Using an optimised protocol, over 7000 antigen-antibody interactions were evaluated. 92% of the Pf protein panel was recognised by both total and cytophilic IgGs, with 6 novel candidates resulting as antigenic as some of the antigens evaluated in vaccine clinical trials for malaria. It is envisaged that my PhD will deliver multi-level technical know-how, which could improve the production and evaluation of novel plasmodial vaccine candidates. The final Pf microarray will be employed to screen clinical samples collected from protected and non-protected children enrolled in a longitudinal, case-control study in an area of Uganda where malaria is endemic. It is expected that the FightMal project overall will eventually generate novel immunological data and information pertaining to both the complexity of the plasmodial proteome and variability of the naturally-induced immune response against the malaria parasite. |
