Feasibility, optimisation and scale-up studies for the production of P. falciparum putative vaccine candidates for a malaria microarray-based antigenicity screening
Author(s)
Mazzoleni, Giorgio
Type
Thesis
Abstract
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.
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.
Date Issued
2011-10
Date Awarded
2012-03
Advisor
Crisanti, Andrea
Creator
Mazzoleni, Giorgio
Publisher Department
Division of Molecular Biosciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)